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Targeting cellular redox modulations for pancreatic cancer treatment

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Targeting cellular redox modulations for pancreatic cancer treatment

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TARGETING CELLULAR REDOX MODULATIONS FOR
PANCREATIC CANCER TREATMENT

by

Divya Pathania

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSPHY
(PHARMACEUTICAL SCIENCES)

August 2013

Copyright 2013 Divya Pathania
ii

“The woods are lovely, dark, and deep,
But I have promises to keep,
And miles to go before I sleep,
And miles to go before I sleep.”
-Robert Frost

iii
DEDICATION
I dedicate this work and all my accomplishments at USC and in my life to my
beloved nani (Mrs. Premlata Singh) and badepapa (Mr. R.C. Singh) (maternal
grandparents) who are my inspiration. They have served as a source of unconditional and
eternal support, motivation, encouragement, strength and love.

iv
ACKNOWLEDGMENTS
I am grateful to my destiny which gave me an opportunity to be a part of one the
esteemed institutions, University of Southern California. I cannot thank enough the
Almighty for always providing me with the strength and focus to move ahead in life. I
have a profound gratitude towards my loving nani (Mrs. Premlata Singh) and badepapa
(Mr. R.C. Singh) for inculcating deep values in me and always encouraging me to follow
my dreams and my heart. I am extremely thankful to my parents (Mrs. Malika Pathania
and Lt. Col. (Retd.) Ramesh Pathania), my sister (Rupa Pathania), and my boyfriend
(Anshul Kumar) for being my support system, having an undying faith in me and
encouraging me to achieve my aspirations all these years. I would also like to
acknowledge some beautiful souls who touched my life and supported me in their own
special way without even speaking my language, my beloved pets (Snoopy, Bubble and
my rescue pups).
I am immensely thankful to my mentor and my guide, Dr. Nouri Neamati for
taking me under his erudite guidance. My experience in his lab has facilitated my growth
as a scientist as well as an individual. I feel lucky to have his expert supervision and to be
a part of his brilliant and successful research team. I am grateful to him for believing in
me and providing me with great opportunities at USC. I have deep gratitude towards my
committee members, Dr. Enrique Cadenas, Dr. Ian S. Haworth and Dr. Bangyan Stiles
for their expert guidance, kind support and valuable feedback throughout my journey as a
graduate student. I am also sincerely thankful to Dr. Roger F. Duncan who served on my
qualifying exam committee and has helped me tremendously throughout my experience
v
at USC. I thank Dr. Mario Sechi and Dr. Bogdan Z. Olenyuk for kindly synthesizing the
compounds that are mentioned herein. I am also immensely grateful to Dr. Enrique
Cadenas’ lab for providing me several reagents used for various assays in this study.
I would like to extend my gratitude to my labmates for making my stay at USC a
memorable experience. I am lucky to be part of a scholarly, intellectual, educated and
caring team. My lab buddies made it a home away from home for me. I am thankful to
Dr. Yumna Shabaik for always being there for me and listening to me. Helen Ha is one
of the sweetest persons that I have ever known in my life and am thankful to her for being
my friend. I am grateful to Kavya Ramkumar for always making me smile. I am grateful
to Dr. Melissa Millard for all her help and support during my stay at USC. I am thankful
to Dr. Shili Xu for his inspiring ideas. I am thankful to Yuting Kuang for her friendship
and her help in lab especially towards the end of my graduate studies, and am also
thankful to Si Li for her suggestions and help. I also thank my other labmates, Rasha Al-
Safi, Dr. Erik Serrao, Dr. Tino Sanchez, Dr. Shuzo Tamura, Dr. Bikash Debnath, Dr.
Xuefei Cao, Dr. Roppei Yamada, Dr. Hiroyuki Otake, Dr. Srinivas Odde and Dr.
Rambabu Gundla. I am also thankful to the students that I have supervised in the lab who
helped me develop my mentoring skills (Alis Bogumian, Mariam Sahakyan, Mary Fouad,
Shadi Doroudgar, Nazanin Ghasemiannik and Yasaman Aletomeh).
I reserve a special thanks for my two great friends, Malini S. Iyer and Niyati S.
Jhaveri. They are two wonderful people who joined USC with me and have always been
there for me and are my family in US. Last but not the least, I am thankful to USC and
vi
especially USC School of Pharmacy for providing me with a place to flourish as a
scientist and an individual.
vii
ABSTRACT
Altered cellular bioenergetics and oxidative stress are emerging hallmarks of most
cancers including pancreatic cancer. Elevated levels of intrinsic reactive oxygen species
(ROS) in tumors make them more susceptible to exogenously induced oxidative stress.
Excessive oxidative insults overwhelm their adaptive antioxidant capacity and trigger
ROS-mediated cell death. Recently, we have discovered two novel classes of compounds,
triphenylphosphoniums and quinazoline-5,8-diones that exert their cytotoxic effects by
modulating ROS-mediated signaling (This dissertation mainly focuses on
quinazolinediones).
Compound 3a was identified through a medium throughput screen of ~1000
highly diverse in-house compounds and chemotherapeutic agents for their ability to alter
cellular bioenergetics. Further structural optimizations led to the discovery of a more
potent analogue, 3b that displayed anti-proliferative activities in low micromolar range in
both drug-sensitive and drug-resistant cancer cells. Treatment with 3b causes Akt
activation resulting in increased cellular oxygen consumption and oxidative stress in
pancreatic cancer cells. Moreover, oxidative stress induced by 3b promoted activation of
stress kinases (p38/JNK) resulting in cancer cell death. Treatment with antioxidants was
able to reduce cell death confirming ROS-mediated cytotoxicity. Since our compounds
exert Akt-dependent ROS-mediated cell death, they may provide potential therapeutic
options for chemoresistant and Akt-overexpressing cancers.
Pancreatic cancer is a complex disease characterized by alterations in several key
regulators of signaling pathways. Increased expression and activity of Src and FAK have
viii
been observed in pancreatic cancer and linked to its inherent and acquired
chemoresistance. Sustained Src inhibition leads to reactivation of survival pathways
regulated by STAT3 resulting in resistant forms of cancer. Therefore, targeting the
Src/FAK axis could provide an important strategy for the treatment of pancreatic cancer.
Mechanistic evaluation of compound 3b revealed that it potently decreases Src/FAK and
STAT3 phosphorylation leading to inhibition of cancer cell migration and angiogenesis.
Therefore, we named our lead compound 3b, sarkostat
TM
. Furthermore, sarkostat arrests
cell cycle progression and causes apoptotic cell death in cancer cells in low micromolar
range. Additionally, sarkostat overcomes the limitations of Src inhibitors by decreasing
STAT3 activity.
Simultaneous studies with triphenylphosphoniums (TPs) revealed their OCR
decreasing effects in cancer cell lines. Interestingly, TPs also induced oxidative stress and
activated stress kinases leading to apoptosis. Moreover, TPs inhibited Src/FAK complex
and decreased cell migration and invasion in pancreatic cancer cells.
Lastly, we have studied one of the bystander effects of ROS generating therapy,
chemotherapy induced peripheral neuropathy. It is attributed to the oxidative stress and
mitochondrial dysfunction resulting from chemotherapeutic agents. We have designed
and synthesized novel redox modulators tagged to triphenylphosphonium moiety. Our
novel mitochondrial targeted redox modulators exert protective antioxidant effects
without interfering with the anticancer effects of chemodrugs. In conclusion, we have
discovered and characterized promising agents with unique mechanisms that show great
potential as a therapy for pancreatic cancer.
ix
PREFACE
The overall goal of the research project presented in this dissertation is to develop
novel small molecule redox modulating anticancer agents. We have identified two novel
classes of compounds, triphenylphosphoniums and quinazoline-5,8-diones that alter
cellular bioenergetics and redox status resulting in cancer cell death.
In chapter 1, I have provided a brief review on the rationale for exploiting reactive
oxygen species (ROS)-mediated anticancer therapeutic approach. I have discussed the
causes and outcomes of oxidative stress in cells, and the anticancer therapeutic
approaches exploiting the redox perturbations in cancer cells.
Chapter 2 has the detailed description of all the materials and methods used in this
study. The synthesis and characterization of the novel quinazoline-5,8-diones were
conducted by our collaborators in Italy, Dr. Mario Sechi, Dr. Michele Palomba, Dr.
Vanna Sanna, Dr. Francesco Berrettini, Dr. Angela Sias and Dr. Alessia Cosseddu.
In chapter 3, I describe the identification of quinazoline-5,8-diones and
triphenylphosphonium compounds using XF24 extracellular flux analyzer that measures
the changes in cellular oxygen consumption rate (OCR) and extracellular acidification
rate (ECAR) in real time. I also discuss the oxidative stress inducing potential of TPs and
quinazoline-5,8-diones (Dr. Yumna Shabaik kindly conducted the MitoSOX experiment
for TP 421).
In chapter 4, I discuss the cell death mechanisms of TPs and quinazoline-5,8-
diones. Oxidative stress results in activation of stress kinases’ signaling resulting in cell
death. Proteomics analysis, western blotting, MTT assays, colony formation assays, cell
x
cycle analysis and Annexin-V staining assays were utilized for delineating ROS-mediated
cell death mechanisms of TPs and compound 3b. (Dr. Yumna Shabaik graciously
performed the western blotting and cell cycle analysis experiments for TP compounds).
In chapter 5, I further characterize the anticancer mechanisms of TPs and
compound 3b, lead compound for quinazoline-5,8-diones. Besides inducing ROS
production, the anticancer effects of TPs and compound 3b include inhibition of Src/FAK
complex regulated cellular signaling events. This results in decreased cell migration and
invasion in cancer cells as depicted by western blotting, cell migration assays, wound
closure (scratch) assays and in vitro tube formation assays. (Dr. Yumna Shabaik kindly
performed the western blotting, cell migration and wound closure assays for TP
compounds. Dr. Melissa Millard graciously conducted the in vitro tube formation assay
for compound 3b).
Chapter 6 focuses on one of the other roles of ROS in cancer cells. ROS are the
central players in Chemotherapy Induced Peripheral Neuropathy (CIPN) that is observed
with treatment with several chemotherapeutic agents. Oxidative stress and mitochondrial
dysfunctional are the driving force for CIPN. Therefore, we designed mitochondrial
targeted redox and/or energy modulators for prevention and/or treatment of CIPN. This
study was conceptualized in collaboration with Dr. Enrique Cadenas and Dr. Bogdan Z.
Olenyuk. John Gallagher efficiently synthesized the compounds for this part of the study.
Chapter 7 summarizes the findings of this research study and focuses on the
future perspectives of these compounds in the current field of redox-modulated
therapeutics.
xi
TABLE OF CONTENTS
EPIGRAPH ......................................................................................................................... ii

DEDICATION ................................................................................................................... iii

ACKNOWLEDGMENTS ................................................................................................. iv

ABSTRACT ...................................................................................................................... vii

PREFACE .......................................................................................................................... ix

LIST OF TABLES .......................................................................................................... xvii

LIST OF FIGURES ......................................................................................................... xix

LIST OF SCHEMES ...................................................................................................... xxv

CHAPTER ONE: INTRODUCTION ................................................................................. 1

1.1. Altered cellular bioenergetics and oxidative stress ...................................................... 1

1.2. Redox regulation in cells ............................................................................................. 3

1.3. Dual role of ROS: the double edged sword ................................................................. 5

1.4 Evidence for higher ROS in cancer cells ...................................................................... 8

1.5. Mechanisms resulting in increased ROS in cancer cells ........................................... 11

1.6. Outcomes of oxidative stress in cancer cells ............................................................. 13

1.7. Strategies for redox targeted anticancer therapies ..................................................... 15

1.8. Oxidative stress in pancreatic cancer ......................................................................... 23

1.8.1. Pancreatic cancer ................................................................................................ 23

1.8.2. Rationale for developing ROS-mediated anticancer therapy for pancreatic cancer
....................................................................................................................................... 24

1.9. Src/FAK complex as a therapeutic target for pancreatic cancer ................................ 30

xii
1.9.1. Src/FAK in pancreatic cancer ............................................................................. 30

1.9.2. Redox regulation of Src ...................................................................................... 35

1.9.3. Challenges with current Src inhibitors ................................................................ 37

1.10. Hypothesis of this study ........................................................................................... 38

CHAPTER TWO: MATERIALS AND METHODS ....................................................... 40

2.1. Cell Culture ................................................................................................................ 40

2.2. Compounds ................................................................................................................ 41

2.3. Measurement of cellular oxygen consumption .......................................................... 42

2.3.1. Assay medium ..................................................................................................... 43

2.4. Superoxide detection .................................................................................................. 43

2.5. Flow cytometric analyses of superoxide .................................................................... 44

2.6. Thiol detection ........................................................................................................... 44

2.7. Cytotoxicity assay ...................................................................................................... 45

2.8. Colony formation assay ............................................................................................. 46

2.9. Cell cycle analysis ...................................................................................................... 47

2.10. Western blotting analyses ........................................................................................ 47

2.11. Annexin V-FITC apoptosis assay ............................................................................ 48

2.12. siRNA Transfection ................................................................................................. 49

2.13. Kinexus Antibody Microarray ................................................................................. 49

2.14. Ingenuity Pathway Analysis .................................................................................... 49

2.15. Immunofluorescence ................................................................................................ 50

2.16. In vitro wound healing (scratch) assay .................................................................... 50

xiii
2.17. In vitro migration assay ........................................................................................... 51

2.18. Imaging .................................................................................................................... 52

2.19. 3-dimensional endothelial cell tube formation (angiogenesis) assay ...................... 52

2.20. In vitro kinase activity assay .................................................................................... 53

2.21. Statistics ................................................................................................................... 53

2.22. Chemistry ................................................................................................................. 53

2.22.1. Reagents and equipment ................................................................................... 53

2.22.2. General procedure for preparation of compounds and intermediates ............... 54

2.22.3. X-Ray Crystallography ..................................................................................... 61

CHAPTER THREE: IDENTIFICATION OF NOVEL REDOX MODULATING
SMALL MOLECULE COMPOUNDS ............................................................................ 67

3.1. Quinazoline-5,8-diones and TPs alter bioenergetics ................................................ 68

3.1.1. Identification of bioenergetics modulating classes of compounds, quinazoline-
5,8-diones and TPs ........................................................................................................ 68
3.1.2. Structural optimizations of compound 3a led to discovery of more potent
analogues ....................................................................................................................... 70

3.1.3. Compound 3b exerts dose dependent increase in OCR in MIA PaCa-2 pancreatic
cancer cells .................................................................................................................... 72

3.1.4. Cell signaling machinery is essential for compound 3b’s activity ..................... 73

3.1.5. Mitochondrial inhibitors do not abolish the OCR increasing effects of compound
3b .................................................................................................................................. 74

3.1.6. Effect of metabolic inhibitor on the effects of 3b on cellular OCR .................... 76

xiv
3.2. TP compounds and quinazolinediones inhibit cancer cell proliferation .................... 77

3.3. TP compounds and quinazoline-5,8-diones induce oxidative stress in cancer cells .. 81

3.3.1. Superoxide production as assessed by cytochrome c assay ................................ 82

3.3.2 Evaluation of mitochondrial superoxide production ........................................... 85

3.3.3. Effect of compound 3b on cellular antioxidant content ...................................... 87

3.4. TPs and quinazoline-5,8-diones induce rapid and sustained inhibition of cancer cell
proliferation ....................................................................................................................... 90

3.5. Antioxidants overcome the cytotoxic effects of compound 3b and TP 421 .............. 94

3.6. Quinazoline-5,8-diones promote Akt-directed ROS-mediated cell death in cancer
cells ................................................................................................................................... 96

3.7. Discussion and conclusions ..................................................................................... 101

CHAPTER FOUR: DETERMINING THE CELL DEATH MECHANISMS INDUCED
BY TPs AND QUINAZOLINE-5,8-DIONES ............................................................... 106

4.1. Quinazoline-5,8-diones inhibit cell proliferation in a panel of cancer cell lines of
different origin ................................................................................................................ 107

4.2. TPs induce broad spectrum cytotoxic effects .......................................................... 112

4.3. Quinazoline-5,8-diones and TPs result in DNA damage and activation of stress
kinases in pancreatic cancer cells ................................................................................... 114

4.4. Quinazoline-5,8-diones and TPs result in apoptotic cell death in pancreatic cancer
cells ................................................................................................................................. 117

4.5. Quinazoline-5,8-diones and TPs cause inhibition of cell cycle progression ........... 120

4.6. Discussion and conclusions ..................................................................................... 122

xv
CHAPTER FIVE: MECHANISTIC EVALUATION OF SARKOSTAT IN
PANCREATIC CANCER .............................................................................................. 124

5.1. Sarkostat affects several critical cell-signaling pathways governing cell migration
and invasion .................................................................................................................... 125

5.2. Sarkostat decreases activating phosphorylation of Src and FAK in pancreatic cancer
cells ................................................................................................................................. 128

5.3. Sarkostat treatment produces changes in cell morphology, inhibits cell migration and
blocks tube formation ..................................................................................................... 132

5.4. Sarkostat can be beneficial in resistant forms of cancer .......................................... 141

5.5. Sarkostat exerts ROS-mediated effects .................................................................... 143

5.6. TP 421 decreases Src/FAK phosphorylation and inhibits cell migration ................ 148

5.7. Discussion and conclusions ..................................................................................... 150

CHAPTER SIX: NOVEL MITOCHONDRIAL-TARGETED SMALL MOLECULES
AS THERAPIES FOR CHEMOTHERAPY INDUCED PERIPHERAL NEUROPATHY
......................................................................................................................................... 155

6.1 Introduction ............................................................................................................... 155

6.1.1. Background ....................................................................................................... 155

6.1.2. Rationale ........................................................................................................... 156

6.1.3.Mitochondrial targeting of novel redox and energy modulating
triphenylphosphonium (TP) conjugates ...................................................................... 157

6.2. Synthesis of mitochondrial-targeted redox modulators ........................................... 160

6.3. Mito-LA and Mito-NAC do not interfere with the anticancer effects of cisplatin .. 162

xvi
6.4. Mito-LA does not affect the respiratory capacity of cancer cells ............................ 164

6.5.Mito-LA and Mito-NAC decrease cisplatin induced mitochondrial superoxide
production ....................................................................................................................... 166

6.6. Discussion and conclusions ..................................................................................... 168

CHAPTER SEVEN: CONCLUDING REMARKS AND FUTURE PERSPECTIVES 171

7.1. Concluding remarks ................................................................................................. 171

7.2. Future perspectives .................................................................................................. 176

7.2.1. Triphenylphosphoniums ................................................................................... 176

7.2.2. Quinazoline-5,8-diones ..................................................................................... 176

7.2.3. Chemotherapy induced peripheral neuropathy ................................................. 176

7.2.4. Newer avenues for our novel redox modulating compounds .......................... 177

BIBLIOGRAPHY ........................................................................................................... 182

xvii
LIST OF TABLES
Table 1.1. Biomarkers of cellular oxidative stress. ............................................................. 9

Table 1.2. Reactive pharmacophores for redox based anticancer therapy. ....................... 16

Table 1.3. Molecular targets for anticancer redox therapy................................................ 19

Table 1.4. Functional targets for anticancer redox therapy............................................... 22

Table 2.1. Crystal data and structure refinement for 3a.................................................... 63

Table 2.2. Most relevant angles [°] for 3a......................................................................... 64

Table 2.3. Most relevant bond lengths [Å] for 3a. ............................................................ 65

Table 2.4. Elemental analyses for compounds 3a-f and 4................................................. 66

Table 3.1. Druglikeliness of compounds 3a-f, 4 and TPs

................................................ 71

Table 3.2. IC
50
of TP compounds in pancreatic cancer cells............................................ 78

Table 3.3. IC
50
of compounds 3a-f and 4 in pancreatic cancer cell lines......................... 80

Table 3.4. Rates of compound 3b induced superoxide production in the presence of
increasing concentrations of superoxide dismutase (SOD)............................................... 83

Table 3.5. Rates of superoxide production for increasing concentrations of 3b............... 83

Table 3.6. Rates of superoxide production for compound 3b and TP 187........................ 85

Table 3.7. TP 421 induces rapid and sustained inhibition of cancer cell proliferation ..... 93

Table 3.8. Fold change of proteins belonging to Akt pathway in response to compound 3b
treatment (Kinexus proteomic analysis)............................................................................ 98

Table 4.1. Cytotoxicity of compounds 3a-f and 4 in a panel of cancer cell lines. .......... 109

Table 4.2. Activity of compound 3b in resistant pancreatic cancer cells........................ 111

xviii
Table 4.3. IC
50
of structural analogues of TP 187, TP 197 and TP 421 in a panel of cancer
cell lines .......................................................................................................................... 113

Table 4.4. Percent distribution of DNA content per cell cycle phase of MIA PaCa-2
pancreatic cancer cell line in response to treatment with TP compounds....................... 121

Table 5.1. Antibody microarray results of Src/FAK signaling pathway......................... 126

Table 6.1. TPP conjugates and their expected effects on mitochondria. ........................ 161

Table 7.1. Summary of experiments for assessing neuroprotective effects. ................... 177

xix
LIST OF FIGURES
Figure 1.1. Hallmarks of cancer...........................................................................................2

Figure 1.2. Cells maintain a state of redox homeostasis by maintaining a balance between
ROS generation and degradation......................................................................................... 4

Figure 1.3. Rationale for ROS-mediated cancer therapy..................................................... 6

Figure 1.4. ROS exert concentration dependent effects in cells.......................................... 7

Figure 1.5. Evidence of oxidative stress in cancer cells.................................................... 10

Figure 1.6. Mechanisms resulting in increased ROS in cancer cells................................. 12

Figure 1.7. Outcomes of oxidative stress........................................................................... 14

Figure 1.8. Oxidative stress causing genetic alterations.................................................... 29

Figure 1.9. Structure and activation of Src........................................................................ 32

Figure 1.10. Multi-domain structure of FAK. ................................................................... 34

Figure 1.11. Regulation of FAK activity through phosphorylations of its various tyrosine
residues.............................................................................................................................. 34

Figure 1.12. Cysteines in redox regulation of proteins...................................................... 36

Figure 1.13. Redox regulation of Src kinase. .................................................................... 37

Figure 2.1. Perspective view and atomic numbering of 3a crystal structure..................... 62

Figure 3.1. Compounds 3a-f and 4 induce significant increase in cellular oxygen
consumption....................................................................................................................... 68

Figure 3.2. TP compounds decrease cellular oxygen consumption rate in MDA-MB-435
cancer cells......................................................................................................................... 69

xx
Figure 3.3. Quinazoline-5,8-diones and triphenylphosphoniums have opposing effects on
cellular oxygen consumption rate of MDA-MB-435 cells................................................ 70

Figure 3.4. Compounds 3a-f and 4 induce significant increase in cellular oxygen
consumption....................................................................................................................... 72

Figure 3.5. Compound 3b exerted a dose dependent increase in OCR in MIA PaCa-2
cells.................................................................................................................................... 73

Figure 3.6. Compound 3b does not exert any effect in the absence of cells..................... 74

Figure 3.7. Effect of mitochondrial inhibitors on OCR inducing effects of compound 3b.
........................................................................................................................................... 75

Figure 3.8. Effect of mitochondrial inhibitors on OCR inducing effects of compound 3b.
........................................................................................................................................... 76

Figure 3.9. Effect of inhibitors of metabolism and NAD(P)H oxidase on OCR inducing
effects of compound 3b..................................................................................................... 77

Figure 3.10. Compounds 3a-3c induce immediate and significant increase in superoxide
production in MIA PaCa-2 pancreatic cancer cells........................................................... 84

Figure 3.11. The phenomenon of ROS-induced ROS-release........................................... 85

Figure 3.12. Compound 3b induces mitochondrial superoxide production in a dose- and
time-dependent manner in pancreatic cancer cells............................................................ 86

Figure 3.13. TP 421 promotes mitochondrial superoxide production in MIA PaCa-2
pancreatic cancer cells....................................................................................................... 87

Figure 3.14. Compound 3b depletes cellular thiols in MIA PaCa-2 pancreatic cancer
cells.................................................................................................................................... 88

xxi
Figure 3.15. Compounds 3b depletes cellular antioxidants in pancreatic cancer cells. .... 89

Figure 3.16. Treatment with compound 3b results in rapid inhibition of cancer cell
proliferation. ...................................................................................................................... 91

Figure 3.17. Compounds 3a-f, 4 induce rapid and sustained inhibition of cell proliferation
in MIA PaCa-2 pancreatic cancer cells..............................................................................92

Figure 3.18. Effect of shorter exposure times for TP 421 treatment on its cytotoxicity in
MIA PaCa-2 pancreatic cancer cells. ................................................................................ 93

Figure 3.19. Treatment with compound 3b causes ROS-mediated cell death in pancreatic
cancer cells......................................................................................................................... 95

Figure 3.20. TP 421 results in ROS-mediated cell death in PANC-1 pancreatic cancer
cells.................................................................................................................................... 96

Figure 3.21. Compound 3b promotes Akt-directed increase in cellular oxidative stress. 99

Figure 3.22. Compound 3b induces ROS-mediated cell death in cancer cells................ 100

Figure 3.23. TP 197 does not influence Akt phosphorylation......................................... 101

Figure 4.1. Compound 3b inhibits cell proliferation in (A) HCT 116 p53
+/+
, (B) MDA-
MB-435 and (C) HEY cells............................................................................................. 110

Figure 4.2. Compound 3b exerts decreased toxicity in HFF-1 primary fibroblasts as
compared to immortalized fibroblasts and cancer cells................................................... 111

Figure 4.3. Potent structural analogues of TP 187, TP 197 and TP 421..........................112

Figure 4.4. Compound 3b activates stress kinases and induces cell death in pancreatic
cancer cells....................................................................................................................... 115

xxii
Figure 4.5. TP 421 activates stress kinases and induces cell death in pancreatic cancer
cells.................................................................................................................................. 116

Figure 4.6. Compound 3b induces cell death in pancreatic cancer cells......................... 119

Figure 4.7. Compound 3b activates stress induced cellular signaling pathways resulting in
cell death.......................................................................................................................... 120

Figure 4.8. Compound 3b induces cell cycle arrest in pancreatic cancer cells............... 122

Figure 5.1. Sarkostat affects critical signaling pathways governing cell migration and
invasion............................................................................................................................ 127

Figure 5.2. Sarkostat decreases phosphorylation of signaling proteins involved in cell
migration in MIA PaCa-2 pancreatic cancer cells........................................................... 129

Figure 5.3. Treatment with sarkostat decreases phosphorylation of FAK, Src and STAT3
in PANC-1 pancreatic cancer cells.................................................................................. 130

Figure 5.4. Sarkostat causes rapid decrease in p-Src in MIA PaCa-2 pancreatic cancer
cells.................................................................................................................................. 131

Figure 5.5. Sarkostat causes rapid change in morphology of cancer cells...................... 132

Figure 5.6. Sarkostat causes rapid change in morphology of cancer cells but not of normal
cells.................................................................................................................................. 133

Figure 5.7. Sarkostat inhibits cell migration.................................................................... 134

Figure 5.8. Sarkostat inhibits cell migration in MIA PaCa-2 pancreatic cancer cells..... 135

Figure 5.9. Sarkostat inhibits cell migration in ASPC-1 pancreatic cancer cells............ 136

Figure 5.10. Sarkostat inhibits cell migration in BxPC-3 pancreatic cancer cells...........137

Figure 5.11. Sarkostat inhibits cell migration in PANC-1 pancreatic cancer cells. ........ 138

xxiii
Figure 5.12. Sarkostat inhibits cell migration in PC-3 prostate cancer cells................... 139

Figure 5.13. Sarkostat inhibits angiogenesis. .................................................................. 140

Figure 5.14. Sarkostat shows additive/synergistic effect with gemcitabine.................... 142

Figure 5.15. Sarkostat overcomes drawbacks of current Src inhibitors used as
chemotherapeutic options for pancreatic cancer.............................................................. 143

Figure 5.16. Effects of sarkostat on STAT3 are independent of its effects on Src and vice
versa ................................................................................................................................ 144

Figure 5.17. Sarkostat does not cause any significant inhibition in activity of oncogenic
kinases in vitro................................................................................................................. 145

Figure 5.18. Sarkostat exhibits ROS-mediated effects on cell signaling. ....................... 147

Figure 5.19. TP 421 decreases Src/FAK phosphorylation and cell migration in pancreatic
cancer cells...................................................................................................................... 149

Figure 6.1. Hypothesis of this study................................................................................ 157

Figure 6.2. Triphenylphosphonium moiety directs compounds to mitochondria............ 160

Figure 6.3. Mito-LA does not interfere with the anticancer effects of cisplatin..............163

Figure 6.4. Mito-NAC does not interfere with the anticancer effects of cisplatin. ......... 164

Figure 6.5. Mito-LA does not affect the respiratory capacity of cancer cells................. 165

Figure 6.6. Mito-LA decreases cisplatin induced mitochondrial superoxide production
more potently than lipoic acid. ........................................................................................ 167

Figure 6.7. Mito-NAC decreases cisplatin induced mitochondrial superoxide production.
......................................................................................................................................... 168

xxiv
Figure 7.1. Compound 3b activates stress kinases and induces cell death in pancreatic
cancer cells....................................................................................................................... 173

Figure 7.2. Proposed mechanism of action of sarkostat (3b).......................................... 175

Figure 7.3. Two approaches for drug repurposing. ......................................................... 179

Figure 7.4. Drug repurposing for sarkostat...................................................................... 180

xxv
LIST OF SCHEMES
Scheme 2.1. Preparation of lead compound (3b) and its analogues (3a,c-f).................... 55

Scheme 2.2. Preparation of 4............................................................................................. 56

Scheme 2.3. Preparation of key synthone 1....................................................................... 56

Scheme 6.1. Preparation of R-α-lipoic acid-TPP conjugate (Mito-LA).......................... 161

Scheme 6.2. Preparation of TPP conjugated N-acetylcysteine (Mito-NAC)................... 162

1
CHAPTER ONE:
INTRODUCTION
1.1. Altered cellular bioenergetics and oxidative stress
Normal cells evolve through an integrated sequence of events into a
heterogeneous population of cancer cells (Floor et al., 2012). Most cancer cells are
characterized by distinctive features including sustained proliferative signaling, evading
growth suppressors, resisting cell death, enabling replicative immortality, inducing
angiogenesis and activating invasion and metastasis. Recently, two new hallmarks have
been added to this list, deregulating cellular energetics and avoiding immune destruction
(Hanahan and Weinberg, 2011) (Figure 1.1).
Altered cellular bioenergetics is an emerging hallmark of cancer cells. Otto
Warburg was first to identify the sweet tooth of cancer (Warburg, 1956). He suggested
that cancer cells rely on aerobic glycolysis for energy. He proposed that glycolytic
dependency of cancer cells was due to defective oxidative phosphorylation (OXPHOS) in
cancer. However, it is now known that defective OXPHOS does not result in Warburg
effect and most forms of cancer have functional mitochondria. Cancer cells get addicted
to aerobic glycolysis due to invasive and adaptive benefits. The increased glycolytic
dependency of cancer cells is due to mitochondrial defects and malfunctions, nuclear
DNA mutations, abnormal expression of metabolic enzymes (fumarate hydratase and
succinate dehydrogenase), adaptation to the tumor microenvironment (HIF-1α), and
2
disruption in oncogenic or tumor suppressor signaling (Cadenas, 1995; Kim and Dang,
2006).

Aerobic glycolysis, glutamine dependent anaplerosis, de novo lipid and nucleotide
biosynthesis and oxidative stress are some of the key bioenergetic alterations observed in
cancer cells (Pathania et al., 2009). Changes in cellular metabolism results in redox
Figure 1.1. Hallmarks of cancer.
Cancer cells enjoy distinct characteristics which differentiate them from
normal cells. These include uncontrolled proliferation, evading cell death,
escaping growth suppressors, angiogenesis, invasion and metastasis,
deregulated cellular energetics, and avoiding immune destruction
[Adapted from (Hanahan and Weinberg, 2011)].

3
dysregulation in cells. Cancer cells are known to be under persistent oxidative stress
(Kawanishi et al., 2006; Szatrowski and Nathan, 1991; Toyokuni et al., 1995). Therefore,
oxidative stress has become an important hallmark of most cancer cells including
pancreatic cancer (Kodydkova et al., 2013).
1.2. Redox regulation in cells
Reactive oxygen species (ROS) including free radicals (superoxide anion,
hydroxyl radical) and non-radical species (hydrogen peroxide) are highly reactive and
can be detrimental when present at high concentration. ROS are generated as a byproduct
of various cellular processes which mostly comprise of cell metabolism. Several complex
mechanisms aid in regulating redox homeostasis by maintaining a delicate balance
between ROS generation and degradation. ROS are produced in biological systems by
various enzymatic (e.g. NADPH oxidase) and non-enzymatic (mitochondrial electron
transport chain) processes. To maintain ROS below their detrimental levels, several
antioxidant enzymes (superoxide dismutase, glutathione peroxidase, peroxiredoxins,
glutaredoxin and catalase) and non-enzymatic systems (glutathione, ascorbic acid,
tocopherol, thioredoxin) act as ROS scavengers (Weinberg and Chandel, 2009) (Figure
1.2).

4

Figure 1.2. Cells maintain a state of redox homeostasis by maintaining a
balance between ROS generation and degradation.
ROS are produced by enzymatic and non-enzymatic (mitochondria, endoplasmic
reticulum) sources in the cell. Oxygen accepts electrons resulting in free radical
superoxide (O
2

-
) production, which in the presence of superoxide dismutase
(SOD) and hydrogen ions forms another reactive species, hydrogen peroxide
(H
2
O
2
). Hydrogen peroxide can undergo Fenton reaction or Haber-Weiss reaction
generating highly reactive hydroxyl radical (OH) or can be broken down into
water by the action of catalase. Glutathione (GSH) and thioredoxin (Trx) also
serve as cellular redox buffering systems. (GSH, glutathione reduced; GSSG,
glutathione oxidized; Trx(O), thioredoxin oxidized; Trx(R), thioredoxin reduced;
TrxRd, thioredoxin reductase; GPx, glutathione peroxidase; GSHRd, glutathione
reductase).

5
1.3. Dual role of ROS: the double edged sword
ROS play an important role as second messengers in cell signaling and regulate a
myriad of signal transduction pathways and cell cycle events. They can affect cellular
signaling pathways by acting upon the transcriptional regulation or by direct oxidative
modifications of signaling proteins (Shackelford et al., 2000; Storz, 2005). ROS have also
been implicated in various diseases including cancer, neurodegenerative diseases,
diabetes, cardiac disorders and mitochondrial diseases. Therefore, maintaining a balance
between ROS generation and degradation is essential for normal cell proliferation,
growth and survival (Boonstra and Post, 2004b). Increased synthesis and/or decreased
elimination of ROS result in oxidative stress leading to deleterious effects on cellular
proteins, lipids and nucleic acids. Moreover, ROS regulate several processes associated
with cancer development and tumor invasiveness (Behrend et al., 2003; Kang and
Hamasaki, 2003). Increased ROS levels are known to facilitate tumor initiation and
progression (Wu, 2006) by inducing DNA damage leading to oncogenic transformation
(Jackson and Loeb, 2001). Therefore, several therapeutic strategies focus on the use of
antioxidants as cancer preventive agents (N. I. H., 2012).
In addition to their tumor promoting actions, ROS exert a critical effect on cell
migration by activating specific genes to promote epithelial-mesenchymal like transition
and metastasis. Furthermore, cancer cells are known to develop adaptive responses to
increased ROS levels to cope with the hazardous effects of oxidative stress (Perry et al.,
2000; Schneider and Kulesz-Martin, 2004). These include the activation of redox
sensitive transcription factors that increase the expression of endogenous antioxidants,
6
promote survival pathways, induce chemoresistance and alter caspase activation
(Pelicano et al., 2004; Trachootham et al., 2009). Despite this adaptive mechanism,
higher basal ROS levels make cancer cells more susceptible to exogenously induced
ROS-mediated cell death. Excessive supply of exogenous oxidative insults will
overwhelm the adaptive capacity of cancer cells and promote cell death (Kong et al.,
2000a; Kong and Lillehei, 1998; Trachootham et al., 2009). Increasing ROS production
or decreasing ROS scavenging has shown therapeutic potential for selectively targeting
tumor cells that are under persistent oxidative stress (Figure 1.3).
Figure 1.3. Rationale for ROS-mediated cancer therapy.
Normal cells regulate cellular redox homeostasis by maintaining a balance
between ROS generation and scavenging. Increased ROS levels promote tumor
initiation, progression and metastasis. Cancer cells are known to develop adaptive
responses to increased ROS levels to protect themselves from the hazardous
effects of oxidative stress. However, higher basal ROS levels make cancer cells
more susceptible to exogenously induced ROS-mediated cell death. Excessive
supply of exogenous oxidative insults will overwhelm the adaptive capacity of
cancer cells, promote cell death that can be exploited as a therapeutic approach.
(prod., production; degrad., degradation).

7
This suggests a “two-faced” or dual role of ROS in cancer (Kong et al., 2000b;
Valko et al., 2007). The specificity of the action of ROS depends on the concentration,
localization and extent of ROS exposure at the target site. At lower levels they act as
tumor promoters, whereas in excessive amount they cause cell death (Figure 1.4).
Furthermore, ROS-mediated anticancer therapy aids in overcoming resistance associated
with other antineoplastic agents (Wartenberg et al., 2005).

Figure 1.4. ROS exert concentration dependent effects in cells.
At lower concentrations ROS play an important role in regulating cellular
signaling events in normal cells. At higher concentrations, ROS result in
increased cell proliferation, tumorigenesis, drug-resistance and activation of
several pathways leading to cancer. However, ROS increased beyond this level
can result in cell death and can be used as a therapeutic approach for targeting
cancer cells with higher basal ROS.

8
1.4 Evidence for higher ROS in cancer cells
Persistent oxidative stress in cancer cells is evident due to their increased ROS
production, enhanced accumulation and excretion of ROS-mediated reaction products,
and overexpression of antioxidants in response to increased ROS in cancer cells (Figure
1.5) (Pelicano et al., 2004; Toyokuni et al., 1995). Cancer cells have relatively higher
basal ROS content as compared their normal counterparts. For example, hepatic cancer
cells display higher rates of mitochondrial superoxide generation as compared to normal
liver cells indicating that the tumorigenic changes in cancer cells might be aiding in ROS
generation (Konstantinov et al., 1987). Constitutive generation of hydrogen peroxide in
several human cancer lines have been reported (Szatrowski and Nathan, 1991). Studies
have demonstrated that oncogenic signals from c-myc and ras stimulate superoxide
generation in cancer cells (Hlavata et al., 2003; Vafa et al., 2002). Furthermore, primary
leukemia cells isolated from patients with different types of leukemia displayed a
significant increase in ROS content in the cancer cells as compared to normal
lymphocytes (Devi et al., 2000; Hileman et al., 2001; Zhou et al., 2003).
Increased cellular ROS react with nucleic acids, lipids and proteins resulting in
accumulation of their reaction products in tissues and their excretion in plasma or urine.
Increase in DNA oxidative adducts (8-oxo-G, 8-oxo-dG) and lipid peroxidation products
have been detected in several forms of cancer (Bras et al., 1999; Devi et al., 2000; Jaruga
et al., 1994; Okamoto et al., 1994; Olinski et al., 1992; Toyokuni, 1998; Toyokuni et al.,
1995; Wu et al., 2004). Furthermore, presence of 8-hydroxydeoxyguanosine in urine
serves as a marker for increased oxidative stress in cancer (Wu et al., 2004) (Table 1.1).
9

Table 1.1. Biomarkers of cellular oxidative stress.

Lastly, increased expression of antioxidant system in cancer cells as an adaptive
response serves as a valid proof for excessive oxidative stress (Storey, 1996). Escalated
levels of SOD have been reported in several forms of cancer including adenocarcinomas
of stomach, squamous cell carcinomas of the esophagus (Janssen et al., 2000), colorectal
adenomas and carcinomas (Janssen et al., 1999), breast cancer tissue from patients
(Punnonen et al., 1994), ovarian carcinomas and malignant brain tumors (Cobbs et al.,
1996; Ishikawa et al., 1990), leukemia (Devi et al., 2000; Nishiura et al., 1992) etc.
Furthermore, increased expression of SOD and catalase have been reported in chronic
lymphocytic leukemia cells and ovarian cancer cells (Hileman et al., 2004). Moreover,
significant increase in the activities of SOD, glutathione peroxidase (GPx), and
Target Biomarker
Lipid Malondialdehyde
4-hydroxynonenal
Hydroxypropanodeoxyguanosines (HO-PdGs)
Exocyclic etheno DNA adducts (etheno-dA, -dC, -dG)
Isoprostanes
Proteins 2-pyrrolidone
Bityrosine crosslinks
Carbonyl groups
Oxidative scissions
Amino acid radicals
Sugars Carboxymethyl-lysine
Pentosidine
DNA 8-oxo-2’-deoxyguanosine (8-oxo-dG)
8-oxo-2’-deoxyadenosine (8-oxo-dA)
4,6-diamino-5-formamidopyrimidine (FapyAde)
PAH- DNA adducts (PAH, polyaromatic hydrocarbons)
Oxidative DNA clusters
Adapted from (Ogino and Wang, 2007; Ziech et al., 2010).
10
glutathione-S-transferase (GST) was observed in the mitochondria of colorectal cancer
tissues as compared to normal tissues of the same subjects (Kanbagli et al., 2000).
However, antioxidant expression is not always increased in cancer cells. For instance,
decreased SOD activity was reported in H6 hepatoma cells in mice and in colorectal
carcinomas (Oberley et al., 1978; Van Driel et al., 1997). Furthermore, there is a decrease
in SOD activity in the cancer cell lines as compared to normal cells (Marklund et al.,
1982). Moreover, in some forms of cancer, SOD expression and activity is unaltered
(Jung et al., 1997; Preuss et al., 2000) suggesting that cancer cells have complex redox
biology.

Figure 1.5. Evidence of oxidative stress in cancer cells.
Persistent oxidative stress in cancer cells is evident due to their
increased ROS production, enhanced accumulation and
excretion of ROS-mediated reaction products, and altered
antioxidant system observed in several forms of cancer.

11
1.5. Mechanisms resulting in increased ROS in cancer cells
Increased cellular ROS content in cancer cells can be attributed to various
mechanisms (Figure 1.6). Oncogenic stimulation plays an important role in inducing
oxidative stress in cancer cells. For instance, c-myc induces ROS generation resulting in
DNA damage (Vafa et al., 2002). Oncogenic RAS2 has been linked to increased ROS
production and thus oxidative protein damage (Hlavata et al., 2003). Furthermore, src or
ras promote ROS generation by membrane-associated NADPH oxidase (Irani et al.,
1997; Schimmel and Bauer, 2002; Suh et al., 1999).
Secondly, close proximity to ROS generating electron transport chain and
comparatively less protection, makes mitochondrial DNA more prone to mutations than
nuclear DNA. Several forms of cancer exhibit mutations in their mitochondrial DNA
(Augenlicht and Heerdt, 2001; Carew and Huang, 2002; Carew et al., 2003; Copeland et
al., 2002; Fliss et al., 2000; Kang and Hamasaki, 2003). Mitochondrial DNA mutations
can influence the functions of the 13 components of the respiration complexes encoded
by it. This results in defective mitochondrial respiratory chain. Compromised
mitochondrial respiratory chain would further result in increased ROS production due to
increased leakage of electrons from the respiratory chain (Carew et al., 2003).
Furthermore, increased energy demands of cancer cells can stress the mitochondrial
respiratory chain resulting in its further damage and thus increased ROS production.
Moreover, hypoxia and angiogenesis induce ROS production in cancer cells
(Brown and Bicknell, 2001; Denko et al., 2003). In certain cases, thymidine
phosphorylase mediates ROS production in cancer cells. Thymidine phosphorylase
12
breaks down thymidine to thymine and 2-deoxy-D-ribose-1-phosphate. The latter acts as
a powerful reducing sugar that glycates proteins leading to ROS production (Amadori
reaction) (Brown et al., 2000). Additionally, excessive ROS production has been linked
to malignant transformation of chronic inflammation (Hussain et al., 2003). Lastly,
decreased expression or activity of antioxidant systems can also cause ROS
accumulation. For instance, some forms of cancer have decreased expression of SOD
resulting in oxidative stress due to decreased elimination of free radicals (Oberley et al.,
1978; Van Driel et al., 1997).

Figure 1.6. Mechanisms resulting in increased ROS in cancer cells.
Increased cellular oxidative stress in cancer cells can result from oncogenic
stimulations, mutations in mitochondrial DNA and perturbations in cellular
antioxidant system.

13
1.6. Outcomes of oxidative stress in cancer cells
Oxidative stress can result in different outcomes in cells depending on the specific
ROS type, its level and duration of action, and on the genetic makeup of the cells
(Davies, 1999). Cellular oxidative stress can cause an adaptive response, increased cell
proliferation, cell injury or cell death, DNA damage and genetic instability, or altered
drug sensitivity (Figure 1.7) (Pelicano et al., 2004).
The first response of cells to oxidative stress is to initiate an adaptive response by
activation of cellular redox buffering systems (glutathione and thioredoxin) and/ or
upregulation of antioxidant enzymes (SOD, catalase, peroxidases etc.) (Holmgren, 1985,
2000; Sasada et al., 1996; Schafer and Buettner, 2001). However, cancer cells already
have several alterations resulting in adaptation to their persistent oxidative stress. Hence,
increased ROS stress by exogenous insults exhausts their ROS-buffering capacity leading
to ROS-mediated cell death that can be exploited as a therapeutic approach (Figure 1.3)
(Trachootham et al., 2009).
ROS can act as second messengers and regulate several cell signaling pathways
governing cell proliferation. Increased cell proliferation under the influence of ROS can
result in cancer development (Behrend et al., 2003). Enhanced ROS levels in cells can
result in DNA damage resulting in genetic instability. These damages in DNA can further
contribute to mutations in cancer (Jackson and Loeb, 2001). Moreover, excessive
oxidative stress can damage nucleic acids, proteins and lipids compromising cellular
functions and leading to cell death (Hensley et al., 2000).
14
Lastly, increased oxidative stress leads to altered drug sensitivity. Sustained
oxidative stress in cancer cells makes them more susceptible to ROS-mediated cell death.
For instance, selective killing of leukemia cells versus normal lymphocytes or of ovarian
cancer cells versus normal cells by 2-methoxyestradiol (2-ME) occurs due to ROS
accumulation resulting from SOD inhibition (Hileman et al., 2004; Huang et al., 2000;
Mooberry, 2003; Zhou et al., 2003). Furthermore, ROS-mediated anticancer therapy
helps in overcoming resistance associated with other antineoplastic drugs. Expression of
multidrug resistant transporter (p- glycoprotein) is dependent on intracellular ROS levels.
For example, DU-145NOX1 (prostate cancer cell line over-expressing NADPH oxidase
1) has higher intracellular ROS and lower expression of P-gp as compared to DU-145
(Wartenberg et al., 2005). However, increased ROS levels can also result in inducing
chemoresistance to anticancer agents. For instance, leukemia cells from CLL patients
having higher intracellular levels of ROS are resistant to treatment with fludarabine and
cyclophosphamide (Carew et al., 2003).

Figure 1.7. Outcomes of oxidative stress.
Increased ROS levels in cells can result in
various outcomes. Cells can either try and
adapt to oxidative stress, undergo cell injury
resulting in cell death, face DNA damage
causing genetic instability, increase their
proliferation or experience alterations in
their drug sensitivity.

15
1.7. Strategies for redox targeted anticancer therapies
Developing compounds that exploit the high basal ROS levels uniquely present in
cancer cells is an innovative and promising approach in drug discovery (Kong et al.,
2000a; Trachootham et al., 2009). ROS inducers, antioxidant inhibitors, agents
decreasing cellular oxidative-buffering capacity, or a combination of these, can produce
exogenous oxidative stress in cancer cells. Moreover, several chemotherapeutic agents
and ionizing radiations induce oxidative stress. Normal cells have much lower ROS
levels as compared to cancer cells, and enjoy a higher antioxidant and oxidative-buffering
reserve to deal with exogenous ROS insults. Therefore, it has been shown that these
agents are not significantly toxic to normal cells (Schumacker, 2006).
Majority of the anticancer drug development focuses on the development of
neocytotoxics, agents that manifest their antineoplastic effects due to their preferential
selectivity for rapidly dividing cancer cells. Similarly, redox targeted therapies are
designed for choosing cancer cells over normal cells. These drugs can be broadly
classified based on their reactive pharmacophore or targets (molecular or functional)
(Wondrak, 2009). A concise classification of the redox targeted therapies is illustrated in
the following tables (Table 1.2, Table 1.3 and Table 1.4). Emerging studies have revealed
that several known anticancer agents with established targets and mechanisms also exert
redox directed effects in cancer cells (example: bleomycin, bortezomib, cisplatin,
anthracyclines, etoposide, gemtuzumab containing calicheamicin, taxanes etc.) (Montero
and Jassem, 2011; Pelicano et al., 2004).
16
Table 1.2. Reactive pharmacophores for redox based anticancer therapy.
Reactive
pharmacophore
Agents Mechanisms
Arsenicals • Arsenic trioxide
• Darinaparsin
(ZIO 101)
• Covalent crosslinking of
vicinal thiols and oxidation of
cysteine residues in GSH and
proteins
(Jing et al., 1999; Pelicano et
al., 2003; Quintas-Cardama et
al., 2008; Wang and Chen,
2008)
Organic
endoperoxides
• Artemisinins • Intracellular prodrug
activation leading to
formation of prooxidant
reactive species triggered by
redox-active iron ions
(Efferth, 2006, 2007; Kaiser
et al., 2007; Singh and Lai,
2004)
Metal Chelators • Disulfiram
• Triapine
• Desferal
• Disulfiram induces oxidation
of cellular glutathione, DNA
fragmentation and oxidative
stress-induced apoptosis.
• Iron chelators (Triapine and
Desferal) cause iron depletion
and stimulation of iron-
dependent free radical
damage
(Bernhardt et al., 2008;
Burkitt et al., 1998; Cen et al.,
2004; Kalinowski and
Richardson, 2007)
Redox cyclers • Quinones
(anthracylines,
geldanamycin,
menadione)
• Motexafin gadolinium
• β-Lapachone
(ARQ 501)
• Acetaminophen and
O-acetylsalicylic acid
• 3,7-
Diaminophenothiazini
• Most of the antitumor
quinones undergo redox
cycling. Quinone reductases
catalyze one electron or two
electron transfer reactions
resulting in the formation of
semiquinone and
hydroquinone, respectively.
NADPH cytochrome P450
reductase is an example of
one electron transfer
17
-um redox dyes
• 2-(Phenyltelluryl)-3-
methyl-
[1,4]napthoquinone

catalyzing quinone reductase.
DT-diaphorase (NAD(P)H
quinone oxidoreductase)
catalyzes two electron
reduction of quinones to
hydroquinones. Semiquinone
reacts with oxygen, and
generates superoxide and the
parent quinone. Superoxide
anion acts as a propagating
species in the autoxidation of
hydroquinones. Redox
cycling of quinones is
accompanied by consumption
of oxygen, oxidation of
NAD(P)H, and formation of
reactive oxygen species (Bey
et al., 2007; Brunmark and
Cadenas, 1989; Cadenas,
1995; Chen et al., 2007; Fry
et al., 2005; Fukuyo et al.,
2008; Giles et al., 2003; Goin
et al., 1995; Kirszberg et al.,
2005; Magda and Miller,
2006; Vad et al., 2009; Vad et
al., 2008; Verrax et al., 2006;
Wondrak, 2007; Wu and Sun,
1999)
Di- and
polysulfides
• Calicheamicin γ
1
1

• Varacin
• Leinamycin
• Diallyldisulfide and
diallyltrisulfide
• Superoxide generation occurs
due to reaction of
polysulfides with cellular
thiols like glutathione
• This occurs in a quasi-
catalytic redox cycle that
produces ROS until reducing
thiols that drive redox cycling
are depleted
(Chatterji and Gates, 1998;
Chatterji et al., 2005; Jacob,
2006; Powolny and Singh,
2008)
Isothiocyanate
organosulfur
agents
• Sulforaphane
• β-Phenylethyl-
• Prooxidant effects include
ROS formation,
mitochondrial functional
18

isothiocyanate
(PEITC)
• Benzylisothiocyanate
• 6-
methylsulfinylhexyl-
isothiocyanate
impairment, oxidation of
mitochondrial peroxiredoxin
3, adduction of cysteine thiols
in crucial target proteins (β-
tubulin, Keap1), and
adduction, export and
depletion of cellular
glutathione
• Benzylisothiocyanate inhibits
mitochondrial complex III
leading to ROS formation
(Brown et al., 2008; Hong et
al., 2005; Kim et al., 2006;
Mi et al., 2008; Sahu et al.,
2009b; Xiao et al., 2008)
Electrophilic
Michael
acceptors

• Parthenolide
• Curcumin
• Dimethylfumarate
• Cinnamaldehyde
• Inuviscolide
• Neratinib
• Peltinib
• PMX464
• Michael acceptors target
thiol-group containing
reaction partners through
covalent adduction
(thioalkylation)
• Michael acceptor-induced
redox alterations result in
glutathione adduction and
ROS formation
• Michael acceptor-dependent
redox modulation can occur
by covalent adduction of
critical thiol residues in target
proteins (NFkB signaling or
thioredoxin redox system)
(Cabello et al., 2009; Garcia-
Pineres et al., 2001; Loewe et
al., 2006; Rozenblat et al.,
2008; Siwak et al., 2005;
Wissner and Mansour, 2008;
Zhang et al., 2009)
Sacrificial
antioxidants
• L-Ascorbate • Prooxidant effects due to
H
2
O
2
formation through
autoxidation
(Chen et al., 2005; Chen et
al., 2008; Wells et al., 1995)
Adapted from (Wondrak, 2009).
19
Table 1.3. Molecular targets for anticancer redox therapy.
Target Agents Mechanisms
Superoxide dismutase • Triethylenetetramine
• ATN-224
• 2-Methoxyestradiol
• Diethyldithiocarbamate
• Increase oxidative
stress by inhibiting
SOD
(Brown et al., 2009;
Golab et al., 2003;
Huang et al., 2000;
Juarez et al., 2006;
Wambi-Kiesse and
Katusic, 1999;
Wood et al., 2001)
• M40403
• Mangafodipir
• cis-FeMPy2P2P
• MnTBAP
• TEMPO
• Tetrathiomolybdate
(ATN-224)
• SOD mimetics
inhibit ROS-
directed
tumorigenesis and
cancer progression
(Alexandre et al.,
2006; Cabello et al.,
2007; Juarez et al.,
2008; Laurent et al.,
2005; Samlowski et
al., 2003; Suy et al.,
2005; Weydert et
al., 2006)
Catalase inhibitor • Aminotriazol • Increases cellular
hydrogen peroxide
content
(Jing et al., 1999)
Glutathione • NOV-002
• Imexon
• L-Buthionine-S,R-
sulfoximine
• PABA/NO
• Ascorbic acid
• Diethylmaleate
• Arsenic trioxide (GPx)
• Mercaptosuccinic acid
(GPx)
• Ethacrynic acid, TLK199
• Prooxidant action
by decreasing
cellular reduced
glutathione (GSH)
content or
mimicking oxidized
glutathione (GSSG)
(Dai et al., 1999;
Davison et al.,
2003; Dvorakova et
al., 2000;
Dvorakova et al.,
2001; Griffith,
1982; Hamilton and
20
Batist, 2005; Jing et
al., 1999;
Matsuzaki et al.,
2002; Miyajima et
al., 1997; O'Dwyer
et al., 1996;
Saavedra et al.,
2006; Townsend et
al., 2008)

Thioredoxin • PX-12
• PMX464
• PX-916
• Chaetocin
• Gliotoxin
• Inactivation of
thioredoxin redox
buffer system
(Kirkpatrick et al.,
1997; Mukherjee et
al., 2007; Powis et
al., 2006; Tibodeau
et al., 2009; Waring
et al., 1995)
Nrf2/Keap1-ARE • HO-1:
Zinc protoporphyrin IX
• NQO1: Dicoumarol and
ES936
• Nrf2-target genes
encode antioxidant
proteins and
enzymes and Keap1
is the physiological
inhibitor of Nrf2.
• HO-1 (Heme
oxygenase-1) and
NQO1 (NADPH
Quinone
Oxidoreductase 1)
are target genes of
Nrf2
(Cullen et al.,
2003a; Fang et al.,
2004; Nolan et al.,
2007;
Thimmulappa et al.,
2002)
APE/Ref1
(Apurinic/apyrimidinic
endonuclease/redox
effector factor-1)
• E3330
• PNRI-299 and resveratrol
• Lucanthone and
CRT0044876

• APE facilitates
DNA binding of
redox-sensitive
transcription factors
and is an important
21
DNA repair enzyme
(Luo et al., 2008;
Luo and Kelley,
2004; Nguyen et
al., 2003; Yang et
al., 2005)
Cdc25 phosphatase • NSC67121
• F-NSC67121
• Indolyldihydroxyquinones
• Cell division cycle
25 (Cdc25)
phosphatases are
redox-sensitive
activators of cyclin-
dependent kinases
that regulate
mammalian cell
cycle progression.
Cdc25
phosphatases are
upregulated in
several forms of
cancer
(Garuti et al., 2008;
Kar et al., 2006;
Lavecchia et al.,
2006; Rudolph,
2007)
Zinc finger
transcription factors
• DIBA
• BITA
• Zinc-coordinated
cysteine thiolates of
zinc fingers are
sensitive to
oxidative
inactivation by
small molecule
redox therapeutics
(Casini et al., 2002;
Wang et al., 2006;
Wang et al., 2004)
Unknown target • Elesclomol (STA-4783) • Induces oxidative
stress
(Kirshner et al.,
2008)
Adapted from (Wondrak, 2009).

22
Table 1.4. Functional targets for anticancer redox therapy.
Target Agents Mechanisms
Glucose
metabolism
• 2-Deoxyglucose
• 3-Bromopyruvate
• Dichloroacetate
• Oxythiamine
• Glucose deprivation by
glycolytic inhibitors and
metabolic modulators
induces oxidative stress and
cytotoxic effects
(Aykin-Burns et al., 2009;
Kato et al., 2007; Mathupala
et al., 2006; Rais et al.,
1999; Spitz et al., 2000)
Mitochondria Mitochondrial respiration:
• Alpha-TOS,
• 3,3’-Diindolylmethane
(DIM)
• Bz-423
VDACs:
• Erastin
• RSL5
• Alterations in mitochondrial
respiration and oxidative
phosphorylation leads to
increased ROS production
resulting in cell death
(Gong et al., 2006; Neuzil et
al., 2007; Pathania et al.,
2009; Ralph and Neuzil,
2009; Simamura et al., 2008;
Xiao et al., 2008; Yagoda et
al., 2007; Yang and
Stockwell, 2008)
Tumor hypoxia Hypoxia-activated redox
agents:
• AQ4N
• PR-104
• Tirapazamine (TPZ)
HIF-1alpha:
• PX-478
• Activation of hypoxia-
selective prodrugs results in
the formation of cytotoxic
organic free radicals and
other reactive intermediates
which kill hypoxic cancer
cells. However, under
normoxia these agents get
inactivated due to oxygen-
dependent electron transfer
reactions.
• Hypoxia-inducible factor-
1alpha (HIF-1α): key
regulator of hypoxia-
associated gene expression
and is a major target for drug
discovery focusing on redox
and metabolic adaptations of
tumors
(Brown, 2007; Hwang et al.,
23

1.8. Oxidative stress in pancreatic cancer
1.8.1. Pancreatic cancer
Pancreatic cancer is the 4
th
leading cause of cancer death in United States in both
men and women. It has been estimated that in 2013 around 45,220 people will be
diagnosed with and 38,460 people will die due to pancreatic cancer. Late stage detection
makes its effective treatment difficult resulting in poor prognosis with only 6% of
patients living beyond 5 years from detection (N.I.H., 2013). These grim statistics clearly
echo the urgent need for development of agents with novel mechanisms of action to
combat pancreatic cancer. Gemcitabine and erlotinib are the two most commonly used
drugs for the treatment of pancreatic cancer.
Pancreatic cancer is characterized by several critical changes in its genotypic and
phenotypic makeup (Deer et al., 2010; Moore et al., 2001). Owing to these modifications
a complex network of signaling pathways is present in pancreatic cancer cells. For
decades scientists have been trying to develop potent anticancer therapies for pancreatic
cancer (Buchholz and Gress, 2009; He et al., 2008; Huang et al., 2011; Mackenzie and
McCollum, 2009). However, limited therapeutic success with the current treatment
options has forced scientists to explore newer avenues for pancreatic cancer treatment.
Redox alterations in pancreatic cancer cells has opened new doors for its treatment.
1999; Liu et al., 2008;
Wardman, 2001; Welsh et
al., 2004)
Adapted from (Wondrak, 2009).
24
1.8.2. Rationale for developing ROS-mediated anticancer therapy for pancreatic
cancer
Recent studies have shown that like most types of cancer, pancreatic cancer also
exhibits ROS stress that acts as a prosurvival and anti-apoptotic factor in pancreatic
cancer cells. Cellular oxidative stress in pancreatic cancer can be attributed to myriad
genetic alterations that contribute to increased production or decreased scavenging of
ROS (Figure 1.8). Decreased antioxidant capacity and marked oxidative stress has been
observed in both chronic pancreatitis and pancreatic cancer patients (Kodydkova et al.,
2013). This suggests that oxidative stress plays an important role in development of
pancreatic cancer. Unresolved inflammation causes oxidative stress resulting in
pancreatitis and eventually pancreatic cancer (Farrow and Evers, 2002; Gukovsky et al.,
2013). Researchers have demonstrated that inflammation plays an important role in
pancreatic carcinogenesis (Farrow and Evers, 2002; Farrow et al., 2004; Greer and
Whitcomb, 2009; Jackson and Evers, 2006; Raimondi et al., 2010). Additionally, hypoxia
has been shown to promote epithelial-mesenchymal transition and metastasis of
pancreatic cancer cells through ROS generation (Shimojo et al., 2013). Furthermore,
epidermal growth factor directed invasion in pancreatic cancer cells is dependent on
ROS-mediated secretion and activation of MMP-2 (matrix metalloproteinase-2) (Binker
et al., 2009).
Increased ROS in pancreatic cancer suggests the use of ROS-mediated therapeutic
strategy. Several drugs like capsaicin, eicosapentaenoic acid (EPA), docosahexaenoic
acid (DHA), sulforaphane, benzyl isothiocyanate (BITC), CDDO-Me, imexon and nitric
25
oxide donating aspirin (NO-ASA) have shown ROS mediated cytotoxic effects in
pancreatic cancer cell lines (Deeb et al., 2012; Fukui et al., 2013; Naumann et al., 2011;
Pramanik et al., 2011; Qanungo et al., 2005; Sahu et al., 2009a; Sheveleva et al., 2012;
Zhang et al., 2008a; Zhang et al., 2006a; Zhou et al., 2009b).
Several genetic alterations contribute to oxidative stress in pancreatic cancer cells.
The key genetic perturbations are as follows (Figure 1.8):
K-Ras: It plays a key role in several cellular signal transduction pathways. Most
forms of pancreatic cancer have mutated K-Ras resulting in altered genetic and
phenotypic pattern including genes involved in redox homeostasis. Thus, K-Ras
transformation in cells provides them with increased resistance to oxidative stress and
altered detoxification status (Recktenwald et al., 2008). Ras oncogene can upregulate the
expression of NOX1 (a homologue of catalytic subunit of superoxide producing enzyme,
NADPH oxidase) and increase ROS generation (Mitsushita et al., 2004a). Additionally,
oncogenic transformation of cells with H-Ras results in their selective killing through
ROS-mediated mechanism (Trachootham et al., 2006). Furthermore, K-Ras mutation
sensitizes pancreatic cancer cells to PKC (protein kinase C) induced apoptosis via ROS-
driven mechanisms (Shen et al., 2012).
TP53INP1 (Tumor protein p53-induced nuclear protein 1): TP53INP1 is a p53
target gene and is one of the key regulators of p53-mediated response to cellular
oxidative stress. Its expression is decreased in pancreatic cancer progression during the
pancreatic intraepithelial neoplasia (PanIN) stage. Homozygous deletion of TP53INP1
generates cellular stress that aids in PanIN progression resulting in pancreatic cancer.
26
TP53INP1 inactivation correlates with increase cell migration. Moreover, oxidative stress
induced by TP53INP1 inactivation cooperates with K-Ras mutations in causing
pancreatic cancer (Al Saati et al., 2013; Seux et al., 2011).
Nrf2: Nuclear factor erythroid 2-related factor (Nrf2)/ Kelch-like-ECH-associated
protein 1 (Keap1) system is an important sensor and helps to adapt to chemical or
oxidative stress. Nrf2 is a transcriptional regulator of several genes that govern phase
II/III drug metabolism and defense against oxidative stress. Nrf2 itself is regulated
mainly by Keap1 which directs it to proteasomal degradation. Oxidative or chemical
stress perturbs Nrf2/Keap1 interaction leading to Nrf2 stabilization and nuclear
translocation. Nuclear Nrf2 interacts with antioxidant response elements in the promoter
regions of several genes coding for phase 2 detoxifying enzymes (e.g. glutathione-S-
transferase and NAD(P)H quinone oxidoreductase), antioxidant proteins (e.g. glutathione
synthesizing enzymes) and transporters (e.g. ABCC2, ABCC3, ABCG2 and x
c
-
subunit).
Increased Nrf2 expression is reported in pancreatic cancer cell lines and ductal
adenocarcinomas. This indicates enhanced capacity of these cells to withstand stress and
resist chemotherapeutics. Moreover, Nrf2 stimulates cell proliferation in pancreatic
adenocarcinomas (Lister et al., 2011). Studies have demonstrated that oncogene-induced
Nrf2 transcription promotes ROS detoxification and tumorigenesis (DeNicola et al.,
2011; Konig et al., 2005).
PALB2/FANCN: PALB2 competes with Nrf2 for Keap1 binding. It shares a
highly conserved ETGE-type Keap1 binding motif with Nrf2 structure. It promotes Nrf2
nuclear accumulation and function, and aids in lowering cellular oxidative stress (Ma et
27
al., 2012). Exomic sequencing studies have reported PALB2 as a pancreatic cancer
susceptibility gene (Jones et al., 2009). Furthermore, a study performed by Slater et al.
shows that PALB2 mutations have been linked to European familial pancreatic cancer
families (Slater et al., 2010).
APE1 (Ref-1): The dual functions of AP endonuclease 1 can be credited to its
redox domain and DNA repair domain. APE1 is overexpressed in several forms of cancer
including pancreatic cancer. Moreover, APE1 levels are upregulated further with
gemcitabine treatment, and APE1 inhibition sensitizes pancreatic cancer cells to
gemcitabine treatment. Inhibition of APE1 results in ROS-mediated decreased pancreatic
cancer cell growth and migration (Lau et al., 2004; Luo et al., 2008; Zou and Maitra,
2008).
Antioxidants: Pancreatic cancer enjoys complex alterations in cellular redox
signal transduction pathways. Increased Nrf2 expression in pancreatic cancer cells helps
them adapt to the persistent oxidative stress conditions. Several Nrf2 target gene products
aid in survival of cancer cells under oxidative stress conditions including glutathione-S-
transferase (Trachte et al., 2002). Studies have demonstrated a link between decreased
MnSOD expression and activity with the increased rates of tumor cell proliferation in
pancreatic cancer cell lines (Cullen et al., 2003b). Antioxidants like N-acetyl cysteine and
superoxide dismutase inhibit the proliferation and invasiveness of pancreatic cancer
(Mezencev et al., 2013; Teoh et al., 2007). However, upregulation of MnSOD expression
has been shown to aid in development of resistance to oxidative stress inducing
anticancer agents and to ionizing radiations (Zhou and Du, 2012).
28
NAD(P)H oxidase (NOX): NAD(P)H oxidase is a ROS producing
multicomponent enzyme with gp91
phox
and p22
phox
catalytic subunits that together form
an integral complex called flavocytochrome b
55
. NOX are mostly phagocytic enzymes.
However, various non-phagocytic forms of NOX have been reported.

Several biological
processes have been reported to increase NOX4 activation in pancreatic cancer cells.
Extracellular matrix stimulates ROS production in pancreatic cancer cells via NOX
(Edderkaoui et al., 2005). Insulin-like growth factor I and FBS (fetal bovine serum)
activate NADPH oxidase by transcriptional upregulation of p22 (phox) subunit due to
activation of NF-κB by Akt kinase. Up-regulation of p22 (phox) results in increased
NOX4-p22 (phox) complex formation and in turn activation of NADPH oxidase
(Edderkaoui et al., 2011). Activation of NOX4 mediates the prosurvival effects of ROS
by inhibiting protein tyrosine phosphatases resulting in enhanced and sustained
phosphorylation of survival signals like JAK2 (Lee et al., 2007). Moreover, growth
factors stimulated ROS production from NOX protects pancreatic cancer cells from
apoptosis (Vaquero et al., 2004). Additionally, NOX mediated ROS production is
important for transmitting cell survival signals through Akt-ASK1 pathway in pancreatic
cancer cells (Mochizuki et al., 2006). Furthermore, NOX plays an important role in
supporting aerobic glycolysis in cancer cells with mitochondrial dysfunction (Lu et al.,
2012).
NAD(P)H quinone oxidoreductase (NQO1): Pancreatic cancer cells have elevated
expression of NAD(P)H:quinone oxidoreductase (NQO1), which detoxifies quinones.
NQO1 reduces most quinones directly to hydroquinones, thus bypassing the highly
29
reactive semi-quinone intermediate (Awadallah et al., 2008; Colucci et al., 2008; Lewis et
al., 2005a; Lyn-Cook et al., 2006). Coumarin- (e.g.,Dicumarol) and indolequinone- (e.g.
ES936) based small molecule compounds inhibit NQO1 and demonstrate oxidative stress
mediated cytotoxic effects in pancreatic cancer (Colucci et al., 2007; Cullen et al., 2003a;
Dehn et al., 2006a; Lewis et al., 2004; Nolan et al., 2009; Nolan et al., 2007; Reigan et
al., 2007a). However, certain agents like streptonigrin, deoxynyboquinone and β-
Lapachone require NQO1 mediated activation for exerting their cytotoxic effects (Cone
et al., 1976a; Huang et al., 2012; Lewis et al., 2005a; Li et al., 2011; Ough et al., 2005;
Reinicke et al., 2005). Moreover, hydroquinone conversion of 17-AAG by NQO1 results
in a molecule that binds Hsp90 with a greater affinity as compared to the parent
compound, thus resulting in improved efficacy (Siegel et al., 2011).
K-Ras mutations increase oxidative stress through
alterations in NOX. Decreased MnSOD results in
increased ROS accumulation. Nrf2, PALB2,
TP53INP1 and APE1 changes help cancer cells to
adapt to increased oxidative stress. Nrf2 and NQO1 aid
in developing chemoresistance.

Figure 1.8. Oxidative stress causing genetic
alterations.
30
1.9. Src/FAK complex as a therapeutic target for pancreatic cancer
1.9.1. Src/FAK in pancreatic cancer
Pancreatic cancer is characterized by myriad major mutations in genes of several
signal transduction pathways including Src tyrosine kinase signaling. Src kinase is
overactivated in several forms of cancer where it plays an important role in regulation of
cell adhesion, motility, invasion, proliferation, survival and angiogenesis (Hilbig, 2008;
Kim et al., 2009; Lutz et al., 1998; Summy and Gallick, 2003). Src expression and
activation increases from normal pancreas to pancreatitis to pancreatic adenocarcinoma in
accordance with its role in cancer progression and metastasis (Hilbig, 2008; Lutz et al.,
1998; Summy and Gallick, 2003). Recent studies have successfully validated Src and p-
Src as novel biomarkers for pancreatic cancer (Yokoi et al., 2011).
Increased expression, phosphorylation and activity of Src have been reported in
gemcitabine resistant pancreatic cancer cells (Duxbury et al., 2004a, b). Src inhibition
effectively slows the growth of human pancreatic cancer xenografts in mice
(Rajeshkumar et al., 2009), suppresses invasiveness (Ito et al., 2003), and reverses 5-
fluorouracil and gemcitabine resistance (Duxbury et al., 2004a, b; Ischenko et al., 2008).
Combined use of Src inhibitors, pyrazolopyrimidine PP2 or AZM475271, and
gemcitabine results in decreased tumor growth and inhibition of metastatic spread in
orthotopic xenografts of pancreatic cancer (Duxbury et al., 2004a; Yezhelyev et al.,
2004). Additionally, Src inhibition by PP2 can augment sensitivity to 5-FU in 5-FU-
resistant pancreatic cancer cells (Ischenko et al., 2008).
31
One of the most important interactions of Src is with FAK (Focal adhesion
kinase). Activation of FAK increases the adhesion and invasion of pancreatic cancer cells
(Sawai et al., 2005). Moreover, intrinsic chemoresistance to gemcitabine has been linked
to constitutive and laminin-induced phosphorylation of FAK in pancreatic cancer cells
(Huanwen et al., 2009). Expression and activation of Src and FAK kinases have been
linked to clinicopathological characteristics of pancreatic ductal adenocarcinoma
(Chatzizacharias et al., 2010; Lutz et al., 1998).
Src/FAK act as a complex and regulate a myriad of cellular events including cell
proliferation, survival, adhesion, migration and invasion linked to disease progression
and metastasis (Bolos et al., 2010; Summy and Gallick, 2003). Therefore, targeting
pathways leading to inhibition of Src/FAK complex and its downstream effects is an
attractive strategy for developing anticancer therapies.
Src (steroid receptor coactivator) is a non-receptor tyrosine kinase. Src activation
is controlled by regulating the activities of various kinases and phosphatases.
Structurally, Src is a multi-domain protein comprising of seven regions: SH4 (SH: Src
homology) domain, unique domain, SH3 domain, SH2-linker, SH2 domain, SH1
(catalytic domain) and C-terminal negative regulatory domain. Each domain has a unique
function which is required for maintenance of its structure and function. Myristoylation
sequence located on the SH4 domain (N-terminal) is responsible for membrane
localization of Src. The SH3 domain mediates protein-protein interactions by binding to
proline-rich peptide sites. SH2 domain is required for binding to phosphorylated tyrosine
sites, and the SH1 domain consists of the catalytic kinase domain and autophosphorylated
32
site of Src Y419. The SH2- linker is present between SH2 and SH1. It can interact with
SH3 and regulate Src activity. The C-terminal of Src consists of negative regulatory
region consisting of Y530. Activation and inactivation of Src is mainly regulated by its
phosphorylation/ dephosphorylation at Y419 and Y530. This process is accompanied by
structural changes in the protein. In an inactivated state Y530 is phosphorylated by CSK
(C-terminal Src kinase) and SH2 domain of Src recognizes and binds to it, resulting in an
inactive closed state of Src. Activation of Src requires dephosphorylation of Y530 by
protein tyrosine phosphatases (PTPs) resulting in an active open conformation. Lastly,
autophosphorylation of Src at Y419 results in complete activation (Kim et al., 2009)
(Figure 1.9).

Src is a multi-domain protein whose activation is tightly
regulated by phosphorylation and dephosphorylation
processes. Phosphorylation at C-terminal Y530 results in its
interaction with SH2 domain causing closing of Src in an
inactive conformation (left). Activation of Src involves
dephosphorylation at Y530, opening of the structure and
phosphorylation at Y419 (Kim et al., 2009).

Figure 1.9. Structure and activation of Src.
33
FAK is also a multi-domain protein. Its contains an N-terminal FERM (protein
4.1, ezrin, radixin and moesin homology) domain, a kinase domain, proline-rich (PRR1-
3) domains and a C-terminal focal adhesion targeting (FAT) domain. The FERM domain
is responsible for its interactions with receptor tyrosine kinases (epidermal growth factor
receptor, platelet-derived growth factor receptor), non-receptor tyrosine kinases (ETK
tyrosine kinase), actin and adaptor proteins (ezrin). The FAT domain mediates the
interactions with integrin-associated proteins such as talin and paxillin and recruits FAK
to focal contacts. The FERM domain mediates its interaction with ubiquitin related
modifier SUMO (small ubiquitin-related modifier) at Lys152. Sumolyation results in
increased activity and nuclear import of FAK. FAT domain binds to guanine nucleotide-
exchange factors (GEFs) such as p190 RhoGEF for activation of Rho GTPases. The
three proline-rich regions (PRR1–3) mediate the interactions with SH3 domain
containing proteins such as p130Cas (aids in cell migration through activation of Rac at
membrane extensions), the GTPase regulator associated with FAK (GRAF) and the Arf-
GTPase-activating protein (GAP) ASAP1 (Figure 1.10).
FAK is also a non-receptor tyrosine kinase whose activity is regulated by its
various phospho sites. FAK can get phosphorylated at multiple sites including
autophosphorylation at Y397 that acts as a docking site for SH2 containing proteins
including Src family kinases, phospholipase Cγ (PLCγ), suppressor of cytokine signaling
(SOCS), growth-factor-receptor-bound protein 7 (GRB7), the Shc adaptor protein, p120
RasGAP and the p85 subunit of phosphatidylinositol 3-kinase (PI3K).

34

(McLean et al., 2005).

Figure 1.11. Regulation of FAK activity through
phosphorylations of its various tyrosine residues.

FAK consists of N-terminal FERM domain, a kinase domain, three proline
rich domains (PRR1-3) and a C-terminal FAT domain (Mitra et al., 2005).

Figure 1.10. Multi-domain structure of FAK.

35
Association of Src with FAK results in full activation of Src. Next, activated Src
phosphorylates FAK at Y576/577 (catalytic domain), Y925 (docking site for growth-
factor-receptor-bound protein 2, mediates RAS-MAPK signaling, and Src-induced
epithelial mesenchymal transition), and Y407. Phosphorylation at Y576/577 is essential
for complete enzymatic activity of FAK. The binding of FAK-family interacting protein
of 200 kDa (FIP200) to the kinase region can inhibit FAK’s catalytic activity. FAK can
also get phosphorylated at Y861 that regulates tumor vasculature by governing the
interaction of FAK with integrins, and controls FAK’s interaction with p130CAS
promoting cell invasion (Figure 1.11) (McLean et al., 2005; Mitra et al., 2005).
1.9.2. Redox regulation of Src
ROS play an important role as physiological regulators of several signal
transduction pathways. ROS can act as second messengers or induce covalent
modifications of specific cysteine residues in redox-sensitive target proteins. ROS
regulated inactivation of protein tyrosine phosphatases (PTPs) results in activation of
protein tyrosine kinases (PTKs) due to sustained presence of phosphotyrosine (p-Tyr)
(Ostman et al., 2011).
Moreover, redox perturbations of cysteine residues results in alterations of
biological activities of the target proteins. For instance, Src is fully active when present in
the reduced form. However, oxidized Src retains only 8-25% activity. Decrease in Src
activity results from oxidation of a specific cysteine residue (Cys277) present in the
catalytic domain. This causes Src homo-dimerization through disulfide linkage. The
sulfenic form (SOH) is readily reversible. However, the higher states of oxidation are
36
mostly irreversible modifications (Figure 1.12) (Finkel, 2011; Kemble and Sun, 2009).
But, cysteine oxidation has also been linked to Src activation. ROS-mediated oxidation of
Cys245 and Cys487 results in the formation of intramolecular disulfide bridge leading to
Src activation (Figure 1.13). (Giannoni et al., 2010). These conflicting reports make it
difficult to ascertain the ultimate fate of Src when exposed to oxidising conditions. Study
conducted by Tang et al. suggested that the redox regulation of a target protein is
governed by its cellular localisation and concentration of oxidising species. They suggest
that Src kinases located in cytoplasmic compartments like endosomes undergo activation
whereas the ones at focal adhesion complexes or plasma membrane get inactivated due to
ROS (Tang et al., 2005). Thus, there are several reports confirming activation or
inactivation of Src and FAK under the influence of oxidative stress (Basuroy et al., 2010;
Corcoran and Cotter, 2013; Cunnick et al., 1998; Kemble and Sun, 2009; Song et al.,
2010; Tang et al., 2005).

Cysteine residues in target
proteins can be covalently
modified by oxidative stress
(Finkel, 2011).

Figure 1.12. Cysteines in redox
regulation of proteins.
37

1.9.3. Challenges with current Src inhibitors
A major limitation of pancreatic cancer therapy is the development of resistance
to currently available therapies including gemcitabine. Continuous efforts to develop
drugs for late stage and highly resistant pancreatic cancer led to the evaluation of
dasatinib, a potent Src inhibitor, in clinical trials for metastatic and locally advanced
There are two contrasting models for redox regulation of Src
kinase. Redox activation (left): According to this model following
dephosphorylation and phosphorylation at Y530 and Y419
respectively, oxidation of Cys245 of the SH2 domain and Cys487
of the catalytic domain results in intramolecular disulfide bond
and an increase in kinase activity. Inactivation model (right):
Oxidation of Cys277 on different Src molecules results in
formation of an inactive dimer due to formation of intermolecular
disulfide bond (Corcoran and Cotter, 2013).

Figure 1.13. Redox regulation of Src kinase.
38
pancreatic cancer (ClinicalTrials.gov, 2013a, b, c, d). Studies have shown that dasatinib
inhibits cell proliferation, migration, invasion, and anchorage independent growth
resulting in tumor growth reduction in vivo. However, dasatinib treatment leads to
resistance due to lack of inhibition of STAT3 and MAPK signaling (Nagaraj et al., 2010).
STAT3 reactivation occurs upon Src inhibition in several forms of cancer resulting in
resistance to Src inhibitors (Byers et al., 2009; Sen et al., 2009). Therefore, there is a need
to develop safe and potent Src inhibitors that can overcome the resistance to current Src
inhibitors.
ROS generating agents have been reported to inhibit STAT3 activation. For
example, the natural product manumycin inhibits STAT3 by elevating intracellular ROS
in glioma cells (Dixit et al., 2009). Similarly, phenethyl isothiocyanate inhibits STAT3
activation in prostate cancer cells by generation of ROS (Gong et al., 2009). STAT3
deletion itself can sensitize cancer cells to oxidative stress (Barry et al., 2009). Therefore,
using a combination of Src inhibitor and an oxidative stress inducer might help overcome
the STAT-3 mediated resistance crisis faced by current Src inhibitors.
1.10. Hypothesis of this study
Most forms of cancer are under persistent oxidative stress including pancreatic
cancer. Increasing ROS production or impairing ROS scavenging will overwhelm their
antioxidant capacity resulting in cell death. Pancreatic cancer is a complex disease with
mutations in several signaling cascade proteins. Src kinase is one such proteins whose
expression and activity is linked to aggressiveness of pancreatic cancer. Moreover, Src
and oxidative stress serve as important biomarkers of pancreatic cancer. Furthermore, Src
39
kinase is a redox regulated tyrosine kinase. Activation of Src has been linked to resistant
forms of pancreatic cancer. Therefore, Src inhibitors are being developed for pancreatic
cancer treatment. However, resistance to current Src inhibitors develop due to activation
of STAT3 or MAPK signaling. This necessitates using Src inhibitor in conjunction with a
STAT3 inhibitor.
We propose to develop a single agent multi-targeted approach for treatment of
pancreatic cancer. Our novel small molecule compounds increase ROS production
resulting in ROS-mediated cell death. Moreover, they inhibit Src/FAK complex and
STAT3. We hypothesize that single agent multi-targeted approach will overcome the
drawbacks of currently available therapies. Furthermore, we hypothesize that Src/FAK
and STAT3 inhibition by our novel compounds is redox regulated.

40
CHAPTER TWO:
MATERIALS AND METHODS
2.1. Cell Culture
Pancreatic cancer (MIA PaCa-2, PANC-1 and BxPC-3), breast cancer (T-47D,
MDA-MB-231, MDA-MB-435 and MCF7), lung cancer (NCI-H460 and NCI-H1299),
and prostate cancer (PC-3) cell lines were purchased from the American Type Cell
Culture (Manassas, VA). HCT116 p53
+/+
and HCT116 p53
-/-
cells were kindly provided
by Dr. Bert Vogelstein (The Sidney Kimmel Comprehensive Cancer Center, Baltimore,
MD). Human ovarian carcinoma cell line (HEY) naturally resistant to cisplatin (CDDP)
was kindly provided by Dr. Louis Dubeau (USC Norris Cancer Center, Los Angeles, CA)
(Buick et al., 1985; Hamaguchi et al., 1993). OVCAR-8 (ovarian cancer) and multi-drug
resistant NCI/ADR-RES cells were obtained from the Developmental Therapeutics
Program, NCI (Bethesda, MD). Dr. Carla Grandori (Fred Hutchinson Cancer Research
Center, Seattle, WA) kindly provided the human foreskin fibroblast cell lines (HFF-1,
HFF-eMYC and HFF-iMYC) (Wang et al., 2011). Pancreatic cancer cell line (ASPC-1)
was kindly given by Dr. Alan L. Epstein (Keck School of Medicine, University of
Southern California, Los Angeles, CA). Resistant cell lines, MIA PaCa-2-GR
(gemcitabine resistant) and MIA PaCa-2-GTR (gemcitabine and erlotinb resistant) were
kindly provided by Dr. Sarkar (Department of Pathology, Wayne State University School
of Medicine, Detroit, MI) (Soubani et al., 2012). Human umbilical vein endothelial cells
(HUV-EC-C) were purchased from the American Type Cell Culture (Manassas, VA).
41
All cell lines used for experiments were maintained in culture under 35 (15 for
HFFs and 6 for HUV-EC-Cs) passages and tested regularly for mycoplasma
contamination using Plasmo Test
TM
(InvivoGen, San Diego, CA). Cell lines were
maintained in the appropriate growth media (DMEM [Cellgro, Mediatech, Manassas,
VA] for MDA-MB-435, MDA-MB-231, MCF7, PANC-1, MIA PaCa-2, PC-3; RPMI-
1640 [(Cellgro, Mediatech, Manassas, VA] for ASPC-1, BxPC-3, HEY, NCI/ADR-RES,
NCI-H1299, NCI-H460, T47-D, OVCAR-8, HFF-1 and HCT116 cell lines) containing
10% heat-inactivated fetal bovine serum (Gemini-Bioproducts, West Sacramento, CA) at
37 ºC in a humidified atmosphere of 5% CO
2
. HUV-EC-Cs were cultured in complete
medium comprising of F-12K medium (Bioreagent and Cell Culture Core, Norris
Comprehensive Cancer Center, University of Southern California, Los Angeles, CA), 0.1
mg/mL heparin, 0.03-0.05 mg/mL endothelial cell growth supplement (ECGS) (BD
Bioscience, San Jose, CA) and 10 % FBS. MIA Paca-2-GR and MIA PaCa-2-GTR were
maintained in DMEM (with 10% FBS) supplemented with 200 nM gemcitabine, and 200
nM gemcitabine and 2 µM erlotinib (every other week), respectively.
For subculture and experiments cells were washed with 1x DPBS (Cellgro,
Mediatech, Manassas, VA), detached using 0.025% Trypsin-EDTA (Cellgro, Mediatech,
Manassas, VA), collected in growth media and centrifuged. All experiments were
performed in growth media using sub-confluent cells in the exponential growth phase.
2.2. Compounds
Stock solutions of all compounds were prepared in dimethylsulfoxide (DMSO)
(EMD Chemicals, Gibbstown, NJ) and stored at -20 °C. Further dilutions were made
42
fresh in DPBS or cell-culture media. Gemcitabine hydrochloride and erlotinib
hydrochloride were purchased from LKT Laboratories (St. Paul, MN) and LC
Laboratories (Woburn, MA), respectively. Dasatinib monohydrate was purchased from
LKT Laboratories (St. Paul, MN). Resveratrol, N-acetylcysteine, cell permeable
glutathione (glutathione reduced ethyl ester) and sulphoraphane were bought from
Sigma-Aldrich (St. Louis, MO). Sodium lipoate was purchased from GeroNova Research
Inc. (Richmond, CA). Control samples in all the experiments were treated with vehicle
(0.1% DMSO).
2.3. Measurement of cellular oxygen consumption
Cellular oxygen consumption rate was analyzed by using XF 24 Extracellular
Flux Analyzer (Seahorse Bioscience, Billerica, MA) as previously described (Millard et
al., 2010). Briefly, the assays were performed in a disposable sensor cartridge containing
24 pairs of fluorescent biosensors coupled to a fiberoptic waveguide, and a 24-well XF24
cell culture microplate.
The biosensor cartridge was prepared by hydrating with XF24 calibrant solution
(Seahorse Bioscience, Billerica, MA), and then incubating overnight at 37 °C in CO
2
-free
incubator. Simultaneously, cells were seeded (MDA-MB-435 120,000 cells/well; MIA
PaCa-2 60,000 cells/well) in 100 µL culture medium in each well of the XF 24 cell
microplate except A1, B4, C3 and D6 control wells. The cells were allowed to adhere for
6 h and then 150 µL of culture media was added to each well. The plate was then
incubated overnight at 37 °C in the presence of 5 % CO
2
.
43
At the start of the experiment, the assay medium was warmed to 37 °C, and the
pH was adjusted to 7.4. Culture medium was removed from the XF24 cell microplate,
and the cells were washed using assay medium. After that, 600 µL of the assay medium
was added to each well and the XF24 cell microplate was incubated at 37 °C for 1 h in
CO
2
-free incubator. Concurrently, dilutions of all the compounds were prepared in assay
media. 60 µL of the dilutions were added to the injection ports of the biosensor cartridge,
and it was then maintained at 37 °C without CO
2
supplementation. Calibration and assay
measurements were then performed at 37 °C using XF24.
2.3.1. Assay medium
DMEM base (8.3 g, Sigma-Aldrich, St. Louis, MO) and sodium chloride (1.85 g,
Sigma-Aldrich, St. Louis, MO) were dissolved separately in 500 mL distilled water. The
two solutions were then combined together and 20 mL of this combined solution was
replaced with 10 mL of 100x GlutaMax-1 (Gibco, Invitrogen, Carlsbad, CA) and 10 mL
of 100 mM sodium pyruvate (Sigma-Aldrich, St. Louis, MO). The complete medium was
then warmed to 37 °C, and its pH was adjusted to 7.4 using 5 M sodium hydroxide
(Sigma-Aldrich, St. Louis, MO). Finally, it was sterilized by filtration and stored at 4 °C
for future use. Temperature and pH were again adjusted to 37 °C and 7.4 respectively on
the day of the assay.
2.4. Superoxide detection
Superoxide production was quantified by measuring the rate of reduction of
ferricytochrome c to ferrocytochrome c (Azzi et al., 1975). Briefly, cells were seeded in a
96-well plate at a density of 20,000 cells per well and allowed to adhere overnight. The
44
next day, cells were treated with compounds with or without superoxide dismutase
(Sigma-Aldrich, St. Louis, MO). 125 µM of partially acetylated cytochrome c (Sigma-
Aldrich, St. Louis) was added right before starting the measurements. The absorbance
was measured every 5 min for 3 h at 550 nm. The initial rate was calculated by analyzing
the change in absorbance with time.
2.5. Flow cytometric analyses of superoxide
250,000 cells (MIA PaCa-2, PANC-1 and BxPC-3) were treated with 5 µM of
compound 3b for increasing time periods (10 min, 1 h, 4 h and 8 h) or with increasing
concentrations (2.5 µM, 5 µM, 10 µM and 20 µM) for 24 h prior to incubation with 5 µM
MitoSOX Red mitochondrial superoxide indicator (Invitrogen, Carlsbad, CA) at 37 ºC for
10 min. Cells were trypsinized, washed thrice with Hank’s Balanced Salt Solution
(HBSS, Cellgro, Mediatech, Manassas, VA) to remove residual MitoSOX before
resuspending in 1 mL 1x DPBS. Fluorescence intensity corresponding to oxidation of
MitoSOX Red by superoxide radicals was recorded for emission wavelengths between
562-588 nm in response to excitation with the 488 nm Sapphire™ argon-ion laser of the
BD LSR II flow cytometer (BD Biosciences, San Jose, CA).
2.6. Thiol detection
Microscopy: BxPC-3 (20,000 cells/well) and MIA PaCa-2 (20,000 cells/well)
cells were seeded in a 96-well clear bottom black plate and allowed to adhere overnight.
The next day cells were treated with increasing concentrations of compound 3b for 4 h.
Cells were then washed with 1xDPBS and stained with 1 µM CellTracker™ Green
CMFDA (Invitrogen, Carlsbad, CA) for 45 min at 37 °C. Following which cells were
45
incubated in fresh culture media for 30 min at 37 °C, washed with 1x DPBS,
counterstained with DRAQ5
®
(Cell Signaling Technology, Danvers, MA). Cells were
then fixed with 3.7% formaldehyde in 1x DPBS at 37 °C for 10 min, and then imaged
using BD Pathway 435 High-Content Bioimager (BD Biosciences, San Jose, CA) at 10x
magnification.
Gels. MIA PaCa-2 (500,000 cells/ 60 mm dish) were allowed to adhere overnight.
Next day cells were treated with 3b (5 µM) for 2 h and then with 500 nM of
CellTracker
TM
Green CMFDA. Cells were then lysed using RIPA lysis buffer (1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) in the presence of protease
inhibitor (SIGMAFAST™ protease inhibitor cocktail tablet, EDTA-free, Sigma-Aldrich,
St. Louis, MO) and phosphatase inhibitor (sodium orthovanadate, VWR International,
Radnor, PA). Cell lysates were sonicated and centrifuged at 12000 rpm for 10 min at 4
°C. Protein concentration of the whole cell lysates was measured using BCA protein
assay and equal amounts of total protein were resolved on a 15% polyacrylamide via
SDS−PAGE. Gels were then scanned for CellTracker
TM
Green CMFDA’s fluorescence
using Typhoon 8610 imager (GE Healthcare Lifesciences, Pittsburgh, PA). Gels were
then stained with SYPRO Ruby (Bio-Rad Laboratories, Hercules, CA) and again scanned
using Typhoon 8610 imager (GE Healthcare Lifesciences, Pittsburgh, PA) to check for
protein loading.
2.7. Cytotoxicity assay
Cytotoxicity was assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay as previously described (Carmichael et al.,
46
1987). Briefly, cells were seeded in 96-well tissue culture treated plates and allowed to
adhere overnight. Cells were subsequently treated with compounds or vehicle for
required amount of time. After 72 h, MTT (0.3 mg/mL) was added to each well. Cells
were incubated with MTT for 3 h at 37 °C. After removal of the supernatant, DMSO was
added and the absorbance was read at 570 nm. All assays were done in triplicate. Percent
cytotoxicity was calculated by comparing the absorbance from drug treated wells to that
of the control wells using the following formula: % cytotoxicity = 100 x (1 - [Abs (drug
treated)/Abs (control)]). The IC
50
was determined for each compound from a plot of log
(drug concentration) versus percentage of cell kill.
2.8. Colony formation assay
Colony formation assay was performed as previously described (Munshi et al.,
2005). Briefly, cells were seeded in 96 well tissue culture plates at a density of 200 cells
per well (or in 6 well plates at a density of 250 cells per well) in growth media and
allowed to adhere overnight. Cells were subsequently treated with different
concentrations of compounds at various times. Following treatment, cells were washed
with 1x DPBS and incubated in growth media for a period of 7-10 days, allowing
sufficient time for colonies to form in control wells. To visualize the extent of colony
formation, cells were fixed and stained in a 0.25% solution of crystal violet containing
10% formalin and 80% methanol. Excess stain was removed through multiple washes in
distilled water and allowed to air dry. Stained plates were imaged using ChemiDoc™
XRS+ Imaging system (Bio-Rad Laboratories, Hercules, CA) and analyzed with Quantity
One software (Bio-Rad Laboratories, Hercules, CA). Quantification of colonies was done
47
by dissolving colonies in 2% SDS (sodium dodecyl sulfate, VWR International, Radnor,
PA) solution by 2 h shaking on a platform rocker and then measuring the optical density
at 570 nm. Calculations were performed as mentioned for MTT assay.
2.9. Cell cycle analysis
Sub-confluent cells were seeded in 60 mm tissue culture dishes at a density of
2×10
5
cells/plate in growth media and allowed to adhere overnight. The following day
cells were treated with sarkostat or dasatinib or DMSO as vehicle control. Upon
completion of treatment, cells were detached with trypsin, and both media and cells were
collected by centrifugation. Cells were washed and resuspended in 1x DPBS, and then
fixed in 70% ethanol overnight at −20°C. For determination of DNA content, fixed cells
were washed with 1x DPBS and then treated with 100 µg/mL RNase A (Sigma-Aldrich,
St. Louis, MO) and stained with propidium iodide (50 µg/mL, Sigma-Aldrich). DNA
content of the samples was analyzed by flow cytometry using the BD LSR II (BD
Biosciences, San Jose, CA) equipped with a 488 nM Sapphire™ argon-ion laser and PE
emission detector.
2.10. Western blotting analyses
Cells were seeded and allowed to adhere overnight. After the desired treatment,
cells were washed with 1x DPBS and lysed using RIPA lysis buffer (1% Nonidet P-40,
0.5% sodium deoxycholate, 0.1% SDS) in the presence of protease inhibitor
(SIGMAFAST™ protease inhibitor cocktail tablet, EDTA-free, Sigma-Aldrich, St.
Louis, MO) and phosphatase inhibitor (sodium orthovanadate, VWR International,
Radnor, PA). Cell lysates were then sonicated and centrifuged at 12000 rpm for 10 min at
48
4 °C. Protein concentration of the whole cell lysates was measured using BCA protein
assay and equal amounts of total protein were resolved on a 10% polyacrylamide via
SDS−PAGE. The separated proteins were electroblotted onto nitrocellulose membrane
and blocked in 5% milk in TBST (Tris-Buffered Saline with Tween® 20) for 1 h at room
temperature. The membrane was probed with primary antibodies to JNK, β-Tubulin
(Santa Cruz Biotechnology Inc., Santa Cruz, CA), to p-p38, p38, p-JNK, GAPDH, p-Akt,
Akt, p-FoxO3a, FoxO3a, p-H2AX, H2AX, c-Jun, p-c-Jun, FasL, Fas, cleaved caspase-8,
cleaved caspase-3, Src, p-Src, STAT3, p-STAT3, FAK, p-FAK, Smad, p-Smad, p-
p130CAS, p-paxillin, Bcl-2, survivin, Bax and PARP (Cell Signaling Technology,
Danvers, MA), MnSOD and catalase (Sigma-Aldrich, St. Louis, MO) at 4 °C overnight.
Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology Inc.,
Santa Cruz, CA) in combination with SuperSignal West Dura (ThermoFisher Scientific,
Waltham, MA) were used to visualize proteins of interest with a ChemiDoc™ XRS+
Imaging system (Bio-Rad Laboratories, Hercules, CA) and analyzed with Quantity One
software (Bio-Rad Laboratories, Hercules, CA).
2.11. Annexin V-FITC apoptosis assay
MIA PaCa-2 cells (2.5 x 10
5
cells/ 60 mm dish) were seeded and allowed to
adhere overnight. After the indicated treatments both floating and attached cells were
collected, stained with the Annexin V-FITC apoptosis detection kit (BioVision, Milpitas,
CA) according to the manufacturer’s protocol. The resulting fluorescence was measured
by a LSR II flow cytometer (BD Biosciences, San Jose, CA).

49
2.12. siRNA Transfection
Sub-confluent MIA PaCa-2 cells (500,000 cells/dish) were transfected in 35 mm
dishes with 80 nM STAT3, Src, or control siRNA for 24 h according to the
manufacturer’s protocol (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Cells were
treated with sarkostat (5 µM, 4 h), lysed and then blotted for desired proteins as described
above.
2.13. Kinexus Antibody Microarray
Mia PaCa-2 cells were treated with sarkostat (1 µM) for 24 h. Upon completion of
treatment, cells were washed with ice cold DPBS and lysed in 200 µL of lysis buffer (20
mM MOPS, pH 7.0, 2 mM EGTA, 5 mM EDTA, 30 mM sodium fluoride, 60 mM β-
glycerophosphate, pH 7.2, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate,
1 mM phenylmethylsulfonylfluoride, 3 mM benzamidine, 5 µM pepstatin A, 10 µM
leupeptin, 1% Triton X-100, 1 mM dithiothreitol). The cell lysates were further sonicated
on ice and the homogenates were cleared by centrifugation at 90,000x g for 30 min at
4°C, and the supernatants were collected. Protein concentrations were measured using
the BCA protein assay. Whole cell lysates in a final volume of 250 µL were submitted to
Kinexus for a 628-antibody microarray analysis using the Kinex™ KAM-1.1 Antibody
Microarray (Kinexus Bioinformatics Corp, British Columbia, Canada).
2.14. Ingenuity Pathway Analysis
The Kinexus™ antibody microarray results were analyzed using the Ingenuity
Pathway Analysis (IPA) software to determine the potential intracellular signaling
proteins or pathways affected by treatment with sarkostat. The significantly up- or down-
50
regulated pan-specific or phospho- proteins with their Swiss-Prot accession numbers and
the ratio changes were uploaded as a Microsoft Excel spreadsheet file to the IPA server.
Implicated signaling pathways were identified by core analysis.
2.15. Immunofluorescence
Sub-confluent MIA PaCa-2 cells (15000 cells/well) were seeded in a 384-well
plate and allowed to adhere overnight. Cells were then treated with sarkostat (5 µM) for
increasing time periods (0.5 h, 1 h and 4 h), washed, fixed with 4% formaldehyde for 15
min, and rinsed with DPBS three times (5 min each). Wells were blocked with blocking
buffer (1x DPBS, 5% normal goat serum, 0.3% Triton
™
X-100) for 60 min, incubated
with primary antibodies diluted in antibody dilution buffer (1x DPBS, 1% BSA, 0.3%
Triton
™
X-100) overnight at 4 °C, and rinsed three times with DPBS (5 min each). Cells
were then incubated with Cy3-conjugated secondary antibody diluted in antibody dilution
buffer for 1 h at room temperature in the dark, rinsed with DPBS three times (5 min
each), and imaged using BD Pathway 435 High-Content Bioimager (BD Biosciences,
San Jose, CA) using 10x objective.
2.16. In vitro wound healing (scratch) assay
For BxPC-3, MIA PaCa-2 and PC-3 cells: Sub-confluent cells (70,000/well)
were plated in a 96-well plate, allowed to adhere overnight, and then serum starved for 24
h. Wounds were made with a 200 µL pipette tip. Cells were treated with sarkostat (0 µM,
0.05 µM, 0.1 µM, 0.5 µM, 1 µM, 2.5 µM and 5 µM) in medium containing 10% FBS.
Negative control wells received serum free medium. After 24 h, cells were fixed with
100% methanol for 10 min and stained with giemsa nuclear stain (10% giemsa, 10%
51
methanol, 80% distilled water) for 30 min at room temperature and washed with distilled
water. Stained cells were imaged using BD Pathway 435 High-Content Bioimager (BD
Biosciences, San Jose, CA) using 4x objective.
For ASPC-1 and PANC-1 cells (Li et al., 2004): 96-well plates were pre-coated
with collagen (45 µg/mL dissolved in 0.2 N sterile filtered acetic acid) overnight at 4°C,
followed by BSA (bovine serum albumin) blocking (2 mg/mL in DPBS for 1 h) at room
temperature. Sub-confluent cells were seeded in serum free medium and allowed to
adhere overnight. Wounds and treatments were performed using the protocol described
above.
2.17. In vitro migration assay
Overnight serum starved cells (MIA PaCa-2, 75,000 cells/ well) were plated in
serum free medium on the top chamber of the 24-well plate cell culture inserts fitted with
transparent 8 µm sized pores PET membranes (BD Biosciences) and allowed to lightly
adhere overnight. Next day, cells were treated with sarkostat (0.1 µM and 1 µM) in serum
free medium in the top chamber. Cells were stimulated to migrate by adding fresh
medium with 10 % FBS to the lower chamber for 24 h. Negative control wells received
serum free medium in the lower chamber. Cells that did not migrate and remained
adherent to the topside of the membrane after 24 h were lightly scrapped off using a Q-
tip. Cells that migrated to the bottom side of the membrane were fixed with 100%
methanol for 10 min, stained with Giemsa nuclear stain for 30 min at room temperature,
and washed with deionized water. Images from representative fields of the stained
membranes were captured using Nikon inverted microscope with a 10x objective.
52
2.18. Imaging
Sub-confluent cells were seeded in a 60 mm dish (MIA PaCa-2, 500,000
cells/well) or a 384-well plate (MIA PaCa-2, 5000 cells/well; HFF-1, 8000 cells/well)
and allowed to adhere overnight. MIA PaCa-2 cells in 60 mm dish were treated with 5
µM sarkostat for increasing time periods (10 min, 30 min, 1 h, 4 h, 8 h, 12 h, or 24 h) and
imaged live using Nikon inverted microscope with a 10x objective. Cells in 384-well
plates were treated with 5 µM or 10 µM of sarkostat for 4 h, fixed with 100% methanol
for 10 min and stained with giemsa nuclear stain (10% giemsa, 10% methanol, 80%
distilled water) for 30 min at room temperature and washed with distilled water. Stained
cells were imaged using BD Pathway 435 High-Content Bioimager (BD Biosciences, San
Jose, CA) with 10x objective.
2.19. 3-dimensional endothelial cell tube formation (angiogenesis) assay
Growth-factor reduced basement membrane extract (BME) (Trevigen,
Gaithersburg, MD) was thawed overnight at 4 ºC on ice. The next day, 30 µL ice-cold
BME was added to the wells of a chilled, 384-well black-walled imaging plate. The
plates were then incubated at 37 ºC for thirty minutes to allow the BME to solidify.
Vehicle (DMSO), sulphorafane and sarkostat were prepared as serial dilutions at 2x final
concentration in Endothelial Cell Basal Medium (EBM-2) (Lonza, Allendale, NJ) at
concentrations ranging over three logs. Next, 25 µL of each dilution was added to the
respective BME-coated wells. Sub-confluent cultures of human umbilical vein
endothelial cells (HUVEC) were collected with 0.25% trypsin/EDTA and re-suspended
in 2x endothelial cell growth medium (EGM-2) consisting of EBM-2, 4 % FBS and 2x
53
concentration of the growth factors and supplements contained within the Endothelial
Growth Medium SingleQuots Kit (Lonza, Allendale, NJ) to give a cell density of 2.0 x
10
5
cells/ mL. To each well containing treatment or vehicle, 25 µL of cell suspension was
added to give a final volume of 50 µL and 1x concentrations of media and compound.
Cells were incubated at 37 ºC for 6-8 h to allow sufficient time for tube formation. When
tubular networks had formed in the control wells, 5 µL of EBM-2 containing 10x calcien
AM (final concentration 6 µM) (VWR International, Radnor, PA) was added to each
well. Imaging was performed on a BD-Pathway 435 high-content Bioimager (BD
Bioscience, San Jose, CA) equipped with calcien AM filter and 4x objective.
2.20. In vitro kinase activity assay
Sarkostat was tested in a panel of 91 oncogenic kinases in an in vitro
KinaseProfiler
TM
assay from EMD Millipore (Billerica, MA). The assay directly
measures the catalytic incorporation of phosphate onto kinase substrates. Sarkostat was
tested at 10 µM. ATP concentration used for the assay was the Km value for each kinase
and 10 µM ATP was used for kinases for which the Km was not known.
2.21. Statistics
Where indicated, p-values were calculated using the student’s t-test. p-values less
than 0.05 were considered to be statistically significant.
2.22. Chemistry
2.22.1. Reagents and equipment
Anhydrous solvents and all the reagents were purchased from Aldrich, Merck or
Carlo Erba. All reactions involving air-or moisture-sensitive compounds were performed
54
under nitrogen atmosphere using oven-dried glassware and syringes for transferring the
solutions. Melting points (mp) were determined using an Electrothermal or a Köfler
apparatus. Infrared (IR) spectra were recorded in potassium bromide (KBr) with a Perkin-
Elmer 781 IR spectrophotometer and are expressed in v (cm
-1
). Nuclear magnetic
resonance (
1
H-NMR,
13
C-NMR) spectra were determined in deuterated chloroform
(CDCl
3
), deuterated dimethyl sulfoxide (DMSO-d
6
) or CDCl
3
/DMSO-d
6
(3:1 ratio) on a
Varian XL-200 (200 MHz) spectrometer. Chemical shifts (δ scale) are reported in parts
per million (ppm) downfield from tetramethylsilane (TMS) used as an internal standard.
The assignment of exchangeable protons (OH and NH) was confirmed by the addition of
deuterated water (D
2
O). Electron ionization mass spectra (70 eV) were recorded on a
Hewlett-Packard 5989 Mass Engine Spectrometer. Analytical thin-layer chromatography
(TLC) was done on Merck silica gel F-254 plates. For flash chromatography Merck silica
gel 60 was used with a particle size 0.040-0.063 mm (230-400 mesh ASTM). Elemental
analyses were performed on a Perkin-Elmer 2400 instrument at Laboratorio di
Microanalisi, Dipartimento di Chimica and Pharmacia, Università di Sassari (Italy) (all
values are given as percentages, and the results were within ± 0.4% of the theoretical
values).
2.22.2. General procedure for preparation of compounds and intermediates
A solution of quinazoline-5,8-dione (1, 1.0 mM for 3a, 0.57 mM for 3b-d, 0.62
mM for 3e-f), cerium chloride (CeCl
3
·7H
2
O) (1.1 eq), and aminoacylbenzene (2a-f, 1.1
eq) in absolute ethanol (15 mL for 3a, 10 mL for 3b-f), pre-saturated with oxygen, was
stirred at room temperature (rt) for 5 h. After 5 h, most of the ethanol was removed under
55
vacuum, and water was added, followed by extraction of the mixture with chloroform
(CHCl
3
). The organic layers were dried over sodium sulfate (Na
2
SO
4
) and concentrated
to dryness. The crude product was purified by flash chromatography (eluent: ethyl
acetate) to give the desired compound.
The synthesis of compounds 3a-f and 4 (Scheme 2.1 and Scheme 2.2) was carried
out using Bracher's methodology (Bracher, 1989). Regioselective substitution of
quinazoline-5,8-dione (1) with appropriate aminoacylbenzene (2a-f) in the presence of
Ce(III) ions gave 3a-f. The acid-catalyzed cyclization of 3a led to the formation of the
condensed product 4.

Reagents and conditions: (i) CeCl
3
·7H
2
O, abs. EtOH, O
2
, rt, 5 h.

Scheme 2.1. Preparation of lead compound (3b) and its analogues
(3a,c-f).
56

Scheme 2.3 illustrates the synthesis of the key synthone 1 from readily available
dimethoxybenzaldehyde 5. Nitration of compound 5 with a molar excess of silica gel
supported nitric acid, under simple magnetic stirring or by ultrasonic agitation, afforded
the 3,6-dimethoxy-2-nitrobenzaldehyde (6). The latter was converted to diformamido-
derivative 7, which was then cyclized to give dimethoxyquinazoline 8. Final oxidation by
cerium ammonium nitrate resulted in the production of quinazoline-5,8-dione 1.

It has been reported that amines attack 6- and 7-positions of the isostere quinolin-
5,8-dione, and the regioselectivity depends on the charge density at these positions.
Reagents and conditions: (i) glacial CH
3
COOH-
conc. H
2
SO
4
(10:1), 90 °C, 30 min.

Scheme 2.2. Preparation of 4.
Reagents and conditions: (i) Method A. SiO
2
·HNO
3
, CH
2
Cl
2
, rt, 12 h, then flash
chromatography; Method B. SiO
2
·HNO
3
, CH
2
Cl
2
, ultrasonic bath, 3 h, then flash
chromatography; (ii) H
2
NCHO, HCl (g) from 40 °C to 80 °C, 1 h; (iii) glacial
CH
3
COOH, Zn, 0 °C for 2 h, then rt for 4 h; (iv) (NH
4
)
2
Ce(NO
3
)
6
, CH
3
CN/ H
2
O,
rt, 1.5 h.

Scheme 2.3. Preparation of key synthone 1.
57
Additionally, the acid medium and Ce
3+
ions increase 6-amino derivative formation
(Pratt, 1962). We performed X-ray crystallographic studies to prove the regiochemistry
of coupling between the aminoacetophenone 2a and the quinone 1 in 6-position of the
quinazolinedione ring (Figure 2.1, Table 2.1, Table 2.2 and Table 2.3).
6-[(2-acetylphenyl)amino]quinazoline-5,8-dione (3a). Red-brown solid. Yield:
56%; mp 206-209 °C. IR (KBr): v cm
-1
3315, 1685.
1
H NMR (CDCl
3
): δ 11.40 (s, 1H,
NH), 9.67 (s, 1H, H
4
), 9.54 (s, 1H, H
2
), 8.01 (d, 1H, Ar-H), 7.64 (d, 2H, Ar-H), 7.32-7.24
(m, 1H, Ar-H), 6.99 (s, 1H, H
7
), 2.71 (s, 3H, CH
3
).
13
C NMR (CDCl
3
): δ 201.5, 181.2,
180.3, 163.5, 156.7, 143.6, 139.1, 134.3, 132.4, 126.1, 124.1, 123.4, 121.0, 107.2, 28.5.

MS: m/z 293 (M
+
). Anal. (C
16
H
11
N
3
O
3
) C, H, N.
6-[(3-acetylphenyl)amino]quinazoline-5,8-dione (3b). Red-brown solid. Yield:
67%; mp 215-218 °C. IR (KBr): v cm
-1
3315, 1685.
1
H NMR (CDCl
3
): δ 9.68 (s, 1H,
H
4
), 9.52 (s, 1H, H
2
), 7.87 (d, 1H, Ar-H), 7.67-7-54 (m, 3H, Ar-H), 6.62 (s, 1H, H
7
), 2.65
(s, 3H, CH
3
). MS: m/z 293 (M
+
). Anal. (C
16
H
11
N
3
O
3
) C, H, N.
6-[(4-acetylphenyl)amino]quinazoline-5,8-dione (3c). Red-brown solid. Yield:
67%; mp 228-230 °C. IR (KBr): v cm
-1
3315, 1685.
1
H NMR (CDCl
3
): δ 9.69 (s, 1H,
H
4
), 9.53 (s, 1H, H
2
), 8.06 (d, 2H, Ar-H), 7.39 (d, 2H, Ar-H), 6.83 (s, 1H, H
7
), 2.63 (s,
3H, CH
3
). MS: m/z 293 (M
+
). Anal. (C
16
H
11
N
3
O
3
) C, H, N.
6-{[2-(phenylcarbonyl)phenyl]amino}quinazoline-5,8-dione (3d). Red-brown
solid. Yield: 77%; mp 167-170 °C. IR (KBr): v cm
-1
3320, 1680.
1
H NMR (CDCl
3
): δ
10.46 (bs, 1H, NH), 9.66 (s, 1H, H
4
), 9.51 (s, 1H, H
2
), 7.83-7.50 (m, 9H, Ar-H), 6.92 (s,
1H, H
7
). MS: m/z 355 (M
+
). Anal. (C
21
H
13
N
3
O
3
) C, H, N.
58
6-{[3-(phenylcarbonyl)phenyl]amino}quinazoline-5,8-dione (3e). Red-brown
solid. Yield: 82%; mp 196-199 °C. IR (KBr): v cm
-1
3320, 1680.
1
H NMR (CDCl
3
): δ
10.40 (bs, 1H, NH), 9.67 (s, 1H, H
4
), 9.51 (s, 1H, H
2
), 7.82 (d, 1H, Ar-H), 7.73-7.50 (m,
8H, Ar-H), 6.67 (s, 1H, H
7
). MS: m/z 355 (M
+
). Anal. (C
21
H
13
N
3
O
3
) C, H, N.
6-{[4-(phenylcarbonyl)phenyl]amino}quinazoline-5,8-dione (3f). Red-brown
solid. Yield: 73%; mp 233-236 °C. IR (KBr): v cm
-1
3320, 1680.
1
H NMR (CDCl
3
): δ
10.42 (bs, 1H, NH), 9.69 (s, 1H, H
4
), 9.53 (d, 1H, H
2
), 7.93 (d, 1H, Ar-H), 7.80 (d, 1H,
Ar-H), 7.64-7.49 (m, 6H, Ar-H), 7.42 (d, 1H, Ar-H)ò. MS: m/z 355 (M
+
). Anal.
(C
21
H
13
N
3
O
3
) C, H, N.
2.22.2.1 Synthesis of 11-Methylpyrimido[4,5-b]acridine-5,12-dione (4)
A solution of 3a (0.3 g, 1.02 mM) in glacial acetic acid and concentrated sulfuric
acid (CH
3
COOH-H
2
SO
4
, 10:1, 33 mL) was heated at 90 °C for 30 min. After 30 min, the
reaction media was cooled to room temperature and diluted with water. The mixture was
neutralized with a saturated solution of sodium bicarbonate (NaHCO
3
), and then
extracted with dichloromethane (CH
2
Cl
2
). The organic layers were dried over Na
2
SO
4

and concentrated to dryness. The crude brown product was purified by flash
chromatography (eluent: dichloromethane and methanol, CH
2
Cl
2
/CH
3
OH 9.5: 0.5) to
give the desired compound as a yellow-brown solid (Scheme 2.2). Yield: 25%; mp =
>280 °C; IR (KBr): ν cm
-1
= 1685.
1
H-NMR (CDCl
3
): δ 9.84 (s, 1H, H
4
), 9.78 (s, 1H, H
2
),
8.47 (m, 2H, Ar-H), 8.01 (m, 1H, Ar-H), 7.86 (m, 1H, Ar-H), 3.35 (s, 3H, CH
3
). MS: m/z
275 (M
+
). Anal. (C
16
H
9
N
3
O
2
) C, H, N.

59
2.22.2.2. 3,6-Dimethoxy-2-nitrobenzaldehyde (6) (Thummel et al., 1993)
Preparation of silica gel supported nitric acid. A mixture of silicon dioxide (SiO
2
,
60 g, 60-200 mesh) in 8 N nitric acid (HNO
3
, 140 mL) was magnetically agitated for 2 h
at 25 °C. After filtration, the impregnated SiO
2
was left to dry in air and stored in an
airtight bottle. The HNO
3
content was estimated to be 16-20% (by weight) by titration of
an aqueous suspension of the reagent with 0.1 N sodium hydroxide (NaOH).
Method A: A solution of 5 (8.2 g, 49 mM, 1 eq) in CH
2
Cl
2
(330 mL) containing 8
N HNO
3
·SiO
2
(164 g, 10 eq, 230-400 mesh) was subjected to magnetic stirring for 15 h.
Method B: The same amount of 5, CH
2
Cl
2
and HNO
3
·SiO
2
, were subjected to agitation
for 3 h using an ultrasonic bath.
The solutions (obtained from Method A and B) were then filtered and washed
with CH
2
Cl
2
(2x, 50 mL). The organic solutions were dried over Na
2
SO
4
and
concentrated to dryness. Evaporation of the solvent gave a yellow material, which was
purified by flash chromatography (eluting with petroleum ether/EtOAc 1:1) to give first
the regioisomer 2,5-dimethoxy-4-nitrobenzaldehyde (Thummel et al., 1993) and then (by
further elution with petroleum ether /EtOAc 3:7) gave the desired compound 6. Yield:
58% (from Method A) and 50% (from Method B); mp 165-167 °C (lit. mp 163-165 °C
and 159 °C). IR (KBr) ν cm
-1
= 2892, 1684, 1528, 1350.
1
H NMR (DMSO-d
6
): δ 10.25 (s,
1H, CHO), 7.70 (d, 1H, H
4
), 7.48 (d, 1H, H
5
), 3.95 (s, 3H, OCH
3
), 3.86 (s, 3H, OCH
3
).

[
l
H NMR (CDCl
3
): δ 10.36 (s, 1H, CHO), 7.28 (d, 1H, H
4
o H
5
), 7.04 (d, 1H, H
4
o H
5
),
3.95 (s, 3H, OCH
3
), 3.87 (s, 3H, OCH
3
)].
1
MS: m/z 211 (M
+
).
60
2.22.2.3. N,N'-[(3,6-dimethoxy-2-nitrophenyl)methanediyl]diformamide (7)
(Malesani et al., 1970; Malesani et al., 1968)
Solution of 2 (3.6 g, 17.1 mM) in formamide (H
2
NCHO, 110 mL), heated to 40
°C, was exposed to dry HCl gas (1 h) until the temperature was 80 °C. Then, the solution
was cooled to rt, and water/ice was added. Yellow colored precipitates were formed,
which after filtration and drying resulted in the desired compound. Yield: 83%; mp 254-
255 °C (lit. mp 254 °C). IR (KBr): ν cm
-1
= 3328, 1670.
1
H NMR (DMSO-d
6
): δ 8.68 (d,
2H, NH), 7.92 (s, 2H, CHO), 7.28 (s, 2H, H
4
and

H
5
), 6.77 (t, 1H, CH), 3.87 (s, 3H,
OCH
3
), 3.81 (s, 3H, OCH
3
). MS: m/z 283 (M
+
).
2.22.2..4. 5,8-dimethoxyquinazoline (8) (Malesani et al., 1970; Malesani et al.,
1968)
Zinc powder (6.24 g) was added to a suspension of 7 (1.9 g, 6.71 mM) in ice (25
g), and glacial acetic acid (8.8 mL) under constant magnetic stirring. The reaction
mixture was stirred for 2 h in ice bath, and for 4 h at rt, by monitoring with TLC (eluents:
CHCl
3
/CH
3
OH 9:1). Then, the reaction mixture was dropped (through filter paper) on
cooled 50% NaOH (35 mL). The yellow colored suspension thus formed was left without
stirring for 1 h, and then filtered to give the desired compound. Yield: 94%; mp 116-119
°C (lit. mp 119 °C). IR (KBr): ν cm
-1
= 3005, 1616, 1567.
1
H NMR (DMSO-d
6
): δ 9.64
(s, 1H, H
2
), 9.28 (s, 1H, H
4
), 7.38 (d, 1H, H
7
), 7.08 (d, 1H, H
6
), 3.98 (s, 3H, OCH
3
), 3.94
(s, 3H, OCH
3
). MS: m/z 190 (M
+
).

61
2.22.2.5. Quinazoline-5,8-dione (1) (Malesani et al., 1970; Malesani et al., 1968)
Ceric ammonium nitrate [(NH
4
)
2
Ce(NO
3
)
6
(13.85 g, 25.5 mM, 4 eq.) in water (15
mL)] was added dropwise to a constantly stirred solution of 5,8-dimethoxyquinazoline 8 [
(1.2 g, 6.3 mM) in acetonitrile (CH
3
CN,15 mL)]. After stirring for an additional 1.5 h (by
monitoring with TLC; CHCl
3
/CH
3
OH 9.5:0.5), the reaction mixture was extracted with
CH
2
Cl
2
, (3x, 100 mL). The organic layer extract was then washed with water, dried (with
anhydrous Na
2
SO
4
), and then evaporated. The oily residue was triturated with petroleum
ether yielding the quinone as brown irregular prisms. Yield: 34%; mp > 320 °C (lit. mp >
350 °C); IR (KBr) ν cm
-1
= 1685, 1670 cm
-1
;
1
H NMR (DMSO-d
6
) δ = 9.68 (s, 1H, H
4
),
9.43 (s, 1H, H
2
), 7.30 (d, 1H,

H
7
), 7.16 (d, 1H, H
6
); MS: m/z 160 (M
+
).
2.22.3. X-Ray Crystallography
Crystals of 3a suitable for X-ray diffraction were obtained by slow evaporation of
a CHCl
3
/CH
3
OH solution (50/50 v/v) of the compound. Single crystal X-ray diffraction
analysis was carried out at room temperature with a computer-controlled Siemens P4
diffractometer using MoKa (l = 0.71073 Å). No crystal decay was observed. The
intensity data were processed with a peak-profile analysis procedure and corrected for
Lorentz and polarization effects. The phase problem was solved by direct methods, using
SIR 92 (Burla et al., 2003). Full-matrix least-squares refinements were carried out with
SHELXL 97 (Sheldrick, 1997) implemented in the WinGX package (Farrugia, 1999).
Analyses of the extinction and intensity statistics indicate that 3a crystallizes in space
group P2
1
/n, with one independent molecule in the asymmetric unit. Anisotropic thermal
displacement parameters were refined for all non H atoms. All H atoms were
62
distinguished by Fourier maps and refined isotropically. The Cambridge Crystallographic
Database (Bruno et al., 2002) software and PARST97 (Nardelli, 1995) were used for
analyzing and drawing the molecular structures. Detailed analyses are listed in Table 2.1,
Table 2.2 and Table 2.3, and perspective view and atomic numbering of 3a crystal
structure is shown in Figure 2.1. Elemental analyses of the compounds are listed in Table
2.4.

Figure 2.1. Perspective view and atomic numbering of 3a crystal structure.

63
Table 2.1. Crystal data and structure refinement for 3a.

Characteristic Value
Empirical formula
C
16
H
11
N
3
O
3

Formula weight 293.277
Temperature 293(2) K
Wavelength 0.71073 Å
Crystal system Monoclinic
Space group P21/n
Unit cell dimensions (Å, °) a = 10.106(1) Å
b = 5.399(1) Å β= 99.33(1) Å
c = 24.424(3) Å
Volume
1315,303 (42) Å
3

Z 4
Absorption coefficient 0.953 mm
-1

Theta range for data
collection
2° - 50°
Reflections collected 3679
Independent reflections 2324 [R(int) = 0.0266]
Refinement method Full-matrix least-squares on F2
Data / restraints / parameters 2324 / 0 / 243
Goodness-of-fit on F
2

1.074
Final R indices [I>4s (I)] R1 = 0.0457, wR2 = 0.1308
R indices (all data) R1 = 0.0765
64
Table 2.2. Most relevant angles [°] for 3a.

Angle
a
(deg) Angle
a
(deg)
C(008)- N(002)- C(012) 132.9(3) C(021)- C(014)- H(14) 112.6(17)
C(008)- N(002)- H(2) 114.4(17) N(005)- C(015)- C(007) 122.1(3)
C(012)- N(002)- H(2) 112.7(17) N(005)- C(015)- H(15) 120.3(15)
C(015)- N(005)- C(022) 115.0(3) C(007)- C(015)- H(15) 117.6(15)
C(018)- N(006)- C(022) 115.1(3) C(011)- C(016)- H(16A) 115.8(17)
C(010)- C(007)- C(015) 121.7(3) C(011)- C(016)- H(16C) 108.6(18)
C(010)- C(007)- C(018) 120.8(2) C(011)- C(016)- H(16B) 110.9(20)
C(015)- C(007)- C(018) 117.4(2) H(16A)- C(016)- H(16C) 111.8(24)
N(002)- C(008)- C(010) 110.6(2) H(16A)- C(016)- H(16B) 102.8(25)
N(002)- C(008)- C(014) 130.5(3) H(16C)- C(016)- H(16B) 106.5(28)
C(010)- C(008)- C(014) 118.9(2) C(013)- C(017)- C(019) 120.4(3)
C(011)- C(009)- C(012) 122.8(2) C(013)- C(017)- H(17) 118.7(14)
C(011)- C(009)- C(020) 119.3(2) C(019)- C(017)- H(17) 120.9(14)
C(012)- C(009)- C(020) 117.9(2) N(006)- C(018)- C(007) 121.8(2)
O(003)- C(010)- C(007) 121.1(2) N(006)- C(018)- C(021) 118.3(2)
O(003)- C(010)- C(008) 120.7(2) C(007)- C(018)- C(021) 119.8(2)
C(007)- C(010)- C(008) 118.2(2) C(017)- C(019)- C(020) 119.1(3)
O(001)- C(011)- C(009) 121.7(3) C(017)- C(019)- H(19) 122.7(16)
O(001)- C(011)- C(016) 118.1(3) C(020)- C(019)- H(19) 118.2(16)
C(009)- C(011)- C(016) 120.2(3) C(009)- C(020)- C(019) 122.6(3)
N(002)- C(012)- C(009) 118.7(2) C(009)- C(020)- H(20) 116.0(14)
N(002)- C(012)- C(013) 122.7(2) C(019)- C(020)- H(20) 121.4(14)
C(009)- C(012)- C(013) 118.6(2) O(004)- C(021)- C(014) 122.2(3)
C(012)- C(013)- C(017) 121.5(3) O(004)- C(021)- C(018) 119.6(2)
C(012)- C(013)- H(13) 120.5(16) C(014)- C(021)- C(018) 118.2(3)
C(017)- C(013)- H(13) 118.0(16) N(005)- C(022)- N(006 128.5(3)
C(008)- C(014)- C(021) 123.7(3) N(005)- C(022)- H(22) 114.7(14)
C(008)- C(014)- H(14) 123.7(17) N(006)- C(022)- H(22) 116.8(14)
a
in parentheses are the su values
65

Table 2.3. Most relevant bond lengths [Å] for 3a.

Bond
Length
a

Bond
Length
a

O(001)- C(011) 1.221(3) C(011)- C(016) 1.498(4)
N(002)- C(008) 1.362(3) C(012)- C(013) 1.388(4)
N(002)- C(012) 1.395(3) C(013)- C(017) 1.384(4)
N(002)- H(2) 0.88(3) C(013)- H(13) 0.96(3)
O(003)- C(010) 1.213(3) C(014)- C(021) 1.439(4)
O(004)- C(021) 1.222(3) C(014)- H(14) 0.91(3)
N(005)- C(015) 1.331(4) C(015)- H(15) 0.95(3)
N(005)- C(022) 1.337(4) C(016)- H(16A) 1.01(3)
N(006)- C(018) 1.344(3) C(016)- H(16C) 0.97(4)
N(006)- C(022) 1.319(4) C(016)- H(16B) 1.02(4)
C(007)- C(010) 1.481(3) C(017)- C(019) 1.376(4)
C(007)- C(015) 1.382(4) C(017)- H(17) 0.99(3)
C(007)- C(018) 1.378(4) C(018)- C(021) 1.495(4)
C(008)- C(010) 1.502(4) C(019)- C(020) 1.370(4)
C(008)- C(014) 1.354(4) C(019)- H(19) 0.96(3)
C(009)- C(011) 1.481(4) C(020)- H(20) 1.01(3)
C(009)- C(012) 1.422(3) C(022)- H(22) 1.01(3)
C(009)- C(020) 1.401(4)
a
in parentheses are the su values.
66
Table 2.4. Elemental analyses for compounds 3a-f and 4.

Compound Formula Theoretical Experimental
C% H% N% C% H% N%
3a
C
16
H
11
N
3
O
3
65.53 3.78 14.33 65.23 3.82 14.58
3b
C
16
H
11
N
3
O
3
65.53 3.78 14.33 65.44 3.68 14.12
3c
C
16
H
11
N
3
O
3
65.53 3.78 14.33 65.77 4.05 14.41
3d
C
21
H
13
N
3
O
3
70.98 3.69 11.83 71.10 3.88 11.80
3e
C
21
H
13
N
3
O
3
70.98 3.69 11.83 70.83 3.63 11.71
3f
C
21
H
13
N
3
O
3
70.98 3.69 11.83 70.71 3.90 11.99
4
C
16
H
9
N
3
O
2
69.81 3.30 15.27 69.65 3.41 15.13
67
CHAPTER THREE:
IDENTIFICATION OF NOVEL REDOX MODULATING
SMALL MOLECULE COMPOUNDS
The novel compounds described herein that target the altered cellular
bioenergetics/ redox homeostasis characteristic of cancer cells were identified in a
medium throughput screen using XF24 Extracellular Flux Analyzer (Seahorse
Bioscience, Billerica, MD). XF24 measures the rates of cellular oxygen consumption and
extracellular acidification in real time. Through our screening we identified two classes
of potential anticancer agents, triphenylphosphoniums (TPs) (Millard et al., 2010;
Shabaik et al., 2013) and quinazoline-5,8-diones (3a-f,4). Mitochondrial targeted
triphenylphosphonium salts decrease the cellular oxygen consumption rate. Interesting,
our other novel class of compounds, quinazolinediones exert the highest level of reported
increase in cellular oxygen consumption rate (OCR) in cancer cells resulting in ROS
production and exogenous oxidative stress. Despite having differing effects on the
cellular oxygen consumption rate both the classes of compounds induce oxidative stress.

68
3.1. Quinazoline-5,8-diones and TPs alter bioenergetics
3.1.1. Identification of bioenergetics modulating classes of compounds,
quinazoline-5,8-diones and TPs
Compound 3a was discovered in a random medium throughput screen of a highly
diverse in-house library of ~1000 drug-like small-molecule compounds for their effects
on cellular bioenergetics. 3a resulted in immediate (first measurement was taken at 9 min
after compound addition), and significant increase in cellular OCR in cancer cells.
Furthermore, 3a promoted 3-fold increase in cellular OCR as compared to resveratrol and
lipoic acid (Figure 3.1). Resveratrol and lipoic acid are compounds that are being
investigated for their anticancer potential. They are also known to enhance oxygen
consumption in cells (Baur et al., 2006; Hagen et al., 1999; Jang et al., 1997; Kim et al.,
2012; Zhou et al., 2011).

Compound 3a was identified by a
medium throughput screen of ~1000
highly diverse in-house compounds and
chemotherapeutic agents using XF24
extracellular flux analyzer. 3a (5 µM)
induced 3-fold increase in cellular
oxygen consumption in MDA-MB-435
cells when compared to resveratrol (50
µM) and lipoic acid (100 µM).
Figure 3.1. Compounds 3a-f and 4
induce significant increase in cellular
oxygen consumption.
69
Triphenylphosphonium is the other class of bioenergetics altering compounds
studied by us. Treatment of MDA-MB-435 cancer cells resulted in an immediate and
sustained decrease in cellular oxygen consumption (Figure 3.2) (Millard et al., 2010;
Shabaik et al., 2013). It is worthwhile to note that the two classes compounds discovered
by our group have opposite effects on cellular oxygen consumption (Figure 3.3).
[Detailed studies on TP compounds have been published in (Millard et al., 2010)].

Treatment with TP compounds resulted in a decrease in OCR.
Furthermore, decrease in OCR was not affected by addition of inhibitor of
ATP synthase, uncoupling agent or complex I inhibitor. [OCR was
measured using XF 24 Extracellular Flux Analyzer. Port A: TP compound
(5 µM) or DMEM or DMSO, Port B: Oligomycin (0.005 mg/mL), Port C:
FCCP (1 µM) Port D: Rotenone (1 µM)] (Millard et al., 2010).

Figure 3.2. TP compounds decrease cellular oxygen consumption rate
in MDA-MB-435 cancer cells.
70

3.1.2. Structural optimizations of compound 3a led to discovery of more potent
analogues
Further structural optimization of 3a led to a series of analogues with varying
degrees of effect on cellular OCR (Synthesis, NMR and X-ray crystallographic studies
are described in chapter 2). All these analogues exhibit favorable drug like properties
(Table 3.1). The analogues were designed with a bulky benzophenone or a smaller
Treatment with TP 197 (5 µM, blue) decreases
OCR whereas treatment with a quinazoline-5,8-
dione (3b, 5 µM, pink) increases OCR. (Port A: TP
compound or compound 3b or DMEM. Yellow
represents DMEM control).
Figure 3.3. Quinazoline-5,8-diones and
triphenylphosphoniums have opposing effects
on cellular oxygen consumption rate of MDA-
MB-435 cells.
71
acetophenone group attached to the N on the 6-position of quinazoline-5,8-dione, or by
ring closure at that position. Compounds with closed ring system (4) or bulkier groups
(3d, 3f) exerted less effect on OCR. Moreover, meta substitution was most effective
substitution (compound 3b and 3e). Compound 3e even with a bulkier benzophenone
group (also present in 3d, 3f) showed similar activity as compound 3b. Compound 3b
was the most potent analogue of 3a, which resulted in 11-fold increase in cellular OCR
(Figure 3.4). Therefore, we used compound 3b as a representative of this class of
compounds for further studies.
Table 3.1. Druglikeliness of compounds 3a-f, 4 and TPs
a

Compound Mol. wt.
b
miLogP
c
HBD
d
HBA
e
No. of rotatable
bonds
f

3a 293.28 1.83 1 6 3
3b 293.28 1.85 1 6 3
3c 293.28 1.88 1 6 3
3d 355.35 3.39 1 6 4
3e 355.35 3.41 1 6 4
3f 355.35 3.43 1 6 4
4 275.27 2.17 0 5 0
TP 187 303.37 3.97 0 0 4
TP 197 532.43 6.96 0 3 7
TP 421 520.63 6.43 0 3 9
a
Druglikeliness of a compound is defined by its ability to follow Lipinski’s rule of
five. These properties were calculated using www.molinspiration.com.
b
Mol. wt. should be less than 500 daltons.
c
miLogP refers to Log P calculated by molinspiration (mi). Log P is the measure
of lipophilicity of the compound and should be less than 5.
d
HBD refers to number of hydrogen bond donors in the molecule. HBD should be
less than 5.
e
HBA refers to number of hydrogen bond acceptors in the molecule. HBA should
be less than 10.
f
No. of rotatable bonds should be less than 10.
72

3.1.3. Compound 3b exerts dose dependent increase in OCR in MIA PaCa-2
pancreatic cancer cells
Treatment of MIA PaCa-2 pancreatic cancer cells with increasing concentrations
of compound 3b resulted in a dose dependent increase in cellular OCR. MIA PaCa-2
cells were treated with 0.5 µM, 5 µM and 50 µM of compound 3b. There was no change
in OCR at the lowest concentration of compound 3b. However, a significant increase in
cellular OCR was observed with 5 µM of 3b. The increase in OCR was even higher with
the highest concentration tested (50 µM 3b) (Figure 3.5).

(A) Structural optimization of compound 3a led to discovery of 6 analogues.
Compound 3b (5 µM) was the most potent analogue in increasing OCR in MDA-MB-
435 cells. (B) Fold change increase in OCR was most significant for compound 3b.
(Compounds added in Port A: 3a-f, 5 µM; 4, 5 µM; Resveratrol, 50 µM; Lipoic acid
(sodium lipoate) 100 µM; DMEM)
Figure 3.4. Compounds 3a-f and 4 induce significant increase in cellular oxygen
consumption.
73

3.1.4. Cell signaling machinery is essential for compound 3b’s activity
In order to eliminate the chances of getting false positive results the experiment
was repeated in the absence of cells. The OCR inducing effects were not seen in the
absence of cells (Figure 3.6), suggesting that the cell signaling machinery is crucial for
3b and its analogues to exert their effects. Furthermore, it hints that the effects seen are as
a result of biological process and not merely due to chemical reactions of redox active
compounds.

(A). Increase in cellular OCR upon treatment with compound 3b is proportional
to the concentration of compound used. (B) Quantification of data shown in (A) in
terms of fold change (Port A: 3b at 0.5 µM, 5 µM or 50 µM).

Figure 3.5. Compound 3b exerted a dose dependent increase in OCR in MIA
PaCa-2 cells.
74

3.1.5. Mitochondrial inhibitors do not abolish the OCR increasing effects of
compound 3b
As mentioned in chapter one, mitochondria and cell metabolism play a vital role
in regulating cellular bioenergetics and redox status. Therefore, we assessed for any
changes in OCR increasing efficacy of compound 3b in presence of mitochondrial
inhibitors (Figure 3.7 and Figure 3.8).
Bioenergetic evaluation of compound 3b was
performed in the presence and absence of MDA-MB-
435 cancer cells. There was no signal detected for the
reaction of compound 3b and components of the assay
medium (DMEM). (Port A: DMEM or compound 3b).
Figure 3.6. Compound 3b does not exert any effect in
the absence of cells.
75
Compound 3b was able to increase cellular OCR in cells pretreated with
oligomycin (ATP synthase inhibitor) or FCCP ( mitochondrial uncoupler) (Figure 3.7).
However, the change in OCR right after the compound 3b addition was slightly lower or
higher in the cells pretreated with oligomycin or FCCP, respectively as compared to cells
pretreated with medium control (DMEM). This difference can be attributed to the change
in the OCR right before compound 3b addition [The change due to oligomycin (decrease)
or FCCP (increase) treatment] (Figure 3.7)

Similar results were obtained with pretreatment with inhibitors of mitochondrial
complexes (Figure 3.8). Pretreatment with antimycin A (complex III inhibitor) or
rotenone (complex I inhibitor) did not completely abolish the effects of compound 3b on
OCR.
Pre-treatment of MIA PaCa-2 pancreatic cancer cells with mitochondrial
inhibitors (ATP synthase inhibitor: Oligomycin, 0.005 mg/ mL; Uncoupler:
FCCP, 1 µM), did not completely abolish the effect of compound 3b on cellular
OCR.

Figure 3.7. Effect of mitochondrial inhibitors on OCR inducing effects of
compound 3b.
76

3.1.6. Effect of metabolic inhibitor on the effects of 3b on cellular OCR
Pretreatment with a metabolic inhibitor (Hexokinase inhibitor: 2-deoxyglucose)
resulted in decrease in 3b’s effects on OCR (Figure 3.9A). Hexokinase converts glucose
to glucose-6-phosphate. The latter can be used in further steps of glycolysis. Glucose-6-
phosphate can also act as a substrate for pentose phosphate pathway (PPP) for generating
cellular reductive reserve (NADPH) through the oxidative phase of PPP (Pathania et al.,
2009). Therefore, it is possible that compound 3b might be targeting an enzyme which
requires NADPH as a cofactor. Hence, we pretreated cells with an NAD(P)H oxidase
inhibitor. [NAD(P)H oxidase is over expressed in several forms of cancer including
pancreatic cancer (Lu et al., 2012)]. However, no change in activity of compound 3b
was observed in the presence of apocynin [NAD(P)H oxidase inhibitor] (Figure 3.9B).

Pre-treatment of MIA PaCa-2 pancreatic cancer cells with mitochondrial
inhibitors (Complex III inhibitor: Antimycin A, 0.002 mg/ mL; Complex I
inhibitor: Rotenone, 1 µM), did not completely abolish the effect of compound 3b
on cellular OCR.

Figure 3.8. Effect of mitochondrial inhibitors on OCR inducing effects of
compound 3b.
77

3.2. TP compounds and quinazolinediones inhibit cancer cell
proliferation
TP compounds decreased cellular OCR (Figure 3.2). This suggests they might be
resulting in cell death due lack of oxygen consumption in cells. As expected, treatment
with TP 187, TP 197 and TP 421 resulted in decreased cell proliferation in cancer cells
(Table 3.2) (Millard et al., 2010; Shabaik et al., 2013).

Pre-treatment of MIA PaCa-2 pancreatic cancer cells with mitochondrial
inhibitors (2-deoxyglucose (2-DG): Hexokinase inhibitor, 100 mM; Apocynin:
NAD(P)H oxidase inhibitor, 10 µM), slightly reduced or did not influence the
effect of compound 3b on cellular OCR, respectively.

Figure 3.9. Effect of inhibitors of metabolism and NAD(P)H oxidase on OCR
inducing effects of compound 3b.
78
Table 3.2. IC
50
of TP compounds in pancreatic cancer cells (Shabaik et al., 2013).
Compound Structure MIA PaCa-2 PANC-1
TP 187
P

0.06±0.4 0.8±0.1
TP 197
P
O
N
Cl
Cl
N

0.2±0.05 0.6±0.01
TP 421
P
O O N

0.5±0.3 1.1±0.4
IC
50
is defined as drug concentration causing a 50% decrease in cell population
using MTT assay. Values with standard deviation are from at least three
independent experiments. Each experiment was generated from an average of
three independent wells. The concentrations of DMSO used in the experiments
were not cytotoxic to cells.

However, compound 3b and analogues resulted in increased cellular oxygen
consumption. Increased oxygen consumption could be due to improved respiratory
capacity of cancer cells. Nonetheless, the increased oxygen consumed by the cells might
get converted to ROS and result in ROS-mediated cell death. Therefore, we wanted to see
if our novel quinazoline-5,8-diones exert pro-survival or pro-death effects in cancer cell
lines.
79
Cytotoxicity of compounds 3a-f and 4 was tested in a panel of pancreatic cancer
cell lines by MTT assay (Table 3.3). All the compounds showed IC
50
values in the low
micromolar range in all cell lines. However, lower potencies were observed for
compounds 3d in BxPC-3, MIA PaCa-2 and PANC-1, 3e in MIA PaCa-2 and BxPC-3,
and 3f in BxPC-3 cells, suggesting that addition of a bulkier group (3d, 3e, 3f) decreased
their potency. More importantly, all our novel compounds are more potent than erlotinib,
one of the standard clinically used drugs for pancreatic cancer [Erlotinib, an EGFR
inhibitor acts a multikinase inhibitor in pancreatic ductal adenocarcinomas, and in
combination with gemcitabine was approved in 2005 for treatment of pancreatic cancer
(Conradt et al., 2011)]. Moreover, these compounds are more potent than resveratrol and
lipoic acid that are known inducers of OCR and are being developed as anticancer agents
(Table 3.3) (Baur et al., 2006; Hagen et al., 1999; Jang et al., 1997; Kim et al., 2012;
Zhou et al., 2011).

80
Table 3.3. IC
50
of compounds 3a-f and 4 in pancreatic cancer cell lines.
IC
50
(µM)
a

Compound Structure BxPC-3 MIA PaCa-2 PANC-1
3a
N
N
O
O
H
N
O

2.7±0.3 1.9±0.1 2.2±0.3
3b
N
N
O
O
H
N
O

2.4±0.1 2.3±0.2 2.0±0.2
3c
N
N
O
O
H
N
O

2.2±0.2 1.7±0.4 1.9±0.3
3d
N
N
O
O
H
N
O

6.5±0.1 3.4±0.2 4.1±0.8
3e
N
N
O
O
H
N
O

3.1±0.9 3.1±0.4 2.1±0.4
3f
N
N
O
O
H
N
O

7.8±0.3 1.6±0.2 1.8±0.3
4
N
N
O
O
N

2.0±0.0 2.3±0.5 2.3±0.5
81
Gemcitabine
N
N
NH
2
O
O
OH
HO
F
F

0.026±0.003 0.042±0.018 2.8±1.7
Erlotinib
N
N O
O
O
O
HN
C
CH

>10 >10 >10
Resveratrol
HO
OH
OH

>10 >10 >10
Sodium
lipoate
(lipoic acid)
S
S
O
O
Na

>10 >10 >10
a
IC
50
is defined as drug concentration causing a 50% decrease in cell population
using MTT assay. Values with standard deviation are from at least three
independent experiments. Each experiment was generated from an average of
three independent wells. The concentrations of DMSO used in the experiments
were not cytotoxic to cells.

3.3. TP compounds and quinazoline-5,8-diones induce oxidative stress in
cancer cells
Our novel quinazolinediones significantly increased cellular OCR and potently
inhibited cancer cell proliferation. Increased oxygen consumption by the treated cells
might increase cellular ROS levels resulting in ROS-mediated cell death. To test this
hypothesis we evaluated the ROS producing capacity of these compounds. We also
evaluated the ROS generating power of TP compounds.
82
3.3.1. Superoxide production as assessed by cytochrome c assay
Superoxide production upon treatment with these compounds was analyzed using
the partially-acetylated cytochrome c assay. This assay is based on the ability of
superoxide to reduce the partially-acetylated cytochrome c (Azzi et al., 1975). Addition
of compound 3b and its closest analogues (3a, 3c) resulted in immediate (first
measurement at 5 min after compound addition) and sustained (lasting 3 h) increase in
superoxide production (Figure 3.10A, B, C). The initial rate of superoxide generation was
highest in cells treated with compound 3b, the most potent analogue, corroborating the
results obtained from XF24’s screening (Figure 3.10D).
Further confirmation of superoxide generation was achieved by using different
concentrations of SOD to quench the signal for superoxide generation. Increased
inhibition in superoxide signal was observed with increased SOD concentrations in MIA
PaCa-2 pancreatic cancer cells treated with compound 3b (Table 3.4). Superoxide
generation was induced in MIA PaCa-2 pancreatic cancer cells by treatment with
compound 3b (5 µM) and an increasing range of amounts of SOD were used to curb the
superoxide production. At lowest level (0.2 U) SOD exhibited slight antioxidant effect.
The antioxidant effect was more pronounced in cells treated with 2 U of SOD. Complete
abrogation of superoxide production was seen at the highest concentration of SOD (20
U). These results confirmed superoxide production in MIA PaCa-2 due to compound 3b.

83
Table 3.4. Rates of compound 3b induced superoxide production in the presence
of increasing concentrations of superoxide dismutase (SOD).

Moreover, compound 3b exerted dose dependent increase in superoxide
generation which tends to saturate at higher concentration. MIA PaCa-2 cells were
exposed to increasing concentrations of compound 3b (0.5 µM, 5 µM and 10 µM). Rate
of superoxide generation increased with an increase in concentration of compound 3b.
However, rate of superoxide production for 5 µM and 10 µM were almost identical
suggesting a saturating effect which is indicative of enzyme kinetics. This suggests
enzyme kinetics and hence enzymatic generation of superoxide production (Table 3.5).

Table 3.5. Rates of superoxide production for increasing concentrations of 3b.

Condition Rate of superoxide production (L mole
-1
sec
-1
)
Control 0.9 x 10
-3

3b (5 µM) 8.0 x 10
-3

3b (5 µM) + SOD (0.2 U) 6.2 x 10
-3

3b (5 µM) + SOD ( 2 U) 2.1 x 10
-3

3b (5 µM) + SOD ( 20 U) 0.7 x 10
-3

Data is generated using cytochrome c based superoxide generation assay.
Condition Rate of superoxide production (L mole
-1
sec
-1
)
0 µM (Control) 1.3 x 10
-3

0.5 µM 2.4 x 10
-3

5 µM 4.0 x 10
-3

10 µM 3.3 x 10
-3

Data is generated using cytochrome c based superoxide generation assay.
84

In order to determine the superoxide generation capacity of TP compounds, TP
187 was used a representative compound. Treatment with TP 187 did not result in
significant increase in superoxide production as compared to compound 3b (Table 3.6).
This suggests that either TP compounds do not produce significant amount of superoxide
or superoxide produced by TPs is not detected by the cytochrome c based superoxide
detection method. Therefore, TP compounds and compound 3b were tested in another
superoxide detection assay. Since TP compounds are directed to mitochondria we used
Compounds 3a (A), 3b (B), and 3c (C) cause significant increase in superoxide
production. Cells treated with compounds 3a-c (blue); Cells treated with
compounds 3a-c in presence of superoxide dismutase (green); Control cells
treated with partially acetylated cytochrome c (yellow). (D) Rate of superoxide
generation calculated from the curves shown in A, B and C. (3a-c, 5 µM; SOD,
20 U; partially acetylated cytochrome c, 125 µM. * indicates p<0.05).

Figure 3.10. Compounds 3a-3c induce immediate and significant increase in
superoxide production in MIA PaCa-2 pancreatic cancer cells.
85
MitoSOX (a mitochondrial targeted probe that is specific for mitochondrial superoxide)
to analyze the mitochondrial superoxide production.
Table 3.6. Rates of superoxide production for compound 3b and TP 187.
Condition Rate of superoxide production (L mole
-1
sec
-1
)
Control 1.1 x 10
-3

TP 187 (5 µM) 2.1 x 10
-3

Compound 3b (5 µM) 8.0 x 10
-3

Data is generated using cytochrome c based superoxide generation assay.

3.3.2 Evaluation of mitochondrial superoxide production
Mitochondria are the major source of cellular ROS production and are implicated
in the process of “ROS-induced ROS release” (Jezek and Hlavatá, 2005). ROS produced
by a mitochondrion or any other cellular source are capable of stimulating ROS
production in another mitochondrion initiating a chain reaction of ROS generation
(Figure 3.11) (Zorov et al.). Therefore, we investigated the ability of 3b to generate
mitochondrial ROS using MitoSOX Red, a mitochondrial targeted probe that undergoes
oxidation in the presence of superoxide giving rise to a fluorescent product. Treatment
with 3b resulted in a significant increase in mitochondrial superoxide in a time- and dose-
dependent manner in all three pancreatic cancer cell lines tested (Figure 3.12).
ROS generated by mitochondria or any other cellular
species are capable of triggering ROS production in
another mitochondrion. This starts a cascade of ROS
generating steps resulting in oxidative stress (Zorov et
al., 2006).
Figure 3.11. The phenomenon of ROS-induced
ROS-release.
86

TP compounds mentioned herein were also tested for their mitochondrial
superoxide generating capacity. It was found that TP compounds promote immediate and
sustained mitochondrial superoxide generation (Figure 3.13) (Millard et al., 2010;
Shabaik et al., 2013)

(A) BxPC-3, MIA PaCa-2 and PANC-1 cells were treated with a range of
concentrations of compound 3b (2.5 µM, 5 µM, 10 µM and 20 µM).
Mitochondrial superoxide production was measured after 24 h by flow cytometry
using MitoSOX Red, a fluorogenic indicator of mitochondrial superoxide
production. Compound 3b exhibited a dose dependent increase of superoxide
production in all the three cell lines. (B) BxPC-3, MIA PaCa-2 and PANC-1 cells
were treated with 5 µM of compound 3b for increasing times (10 min, 1 h, 4 h
and 8 h) and mitochondrial superoxide production was analyzed by MitoSOX Red
using flow cytometer. Compound 3b induced mitochondrial superoxide
production in a time dependent manner.

Figure 3.12. Compound 3b induces mitochondrial superoxide production in a
dose- and time-dependent manner in pancreatic cancer cells.
87

3.3.3. Effect of compound 3b on cellular antioxidant content
Depletion of cellular antioxidants’ pool can make cancer cells more susceptible to
oxidative stress induced cell death (Trachootham et al., 2009). Therefore, we tested the
effect of 3b on the levels of cellular antioxidants. As expected, treatment with 3b
depleted cellular thiols (Figure 3.14 and Figure 3.15). MIA PaCa-2 cells were treated
with Cell Tracker Green
TM
CMFDA and compound 3b. CMFDA binds to cellular thiols
(glutathione) and its signal intensity is directly proportional to the amount of cellular
thiols. Cells treated with compound 3b (5 µM) exhibited decreased signal intensity for
CMFDA (Figure 3.14). Moreover, similar results were obtained in microscopy
experiments using CMFDA. Cells treated with compound 3b (5 µM, 2.5 µM and 1 µM)
for 4 h and then exposed to Cell Tracker Green
TM
CMFDA for ~1 h exhibited decreased
MIA PaCa-2 cells were treated with increasing concentrations of TP 421 for 4 h
(A) or 24 h (B). Mitochondrial superoxide production was measured after
indicated time by flow cytometry using MitoSOX Red, a fluorogenic indicator of
mitochondrial superoxide production. TP 421 resulted in increased superoxide
production which was sustained for 24 h (Shabaik et al., 2013).

Figure 3.13. TP 421 promotes mitochondrial superoxide production in MIA
PaCa-2 pancreatic cancer cells.
88
signal intensity as compared to control, suggesting depletion of cellular thiols (Figure
3.15).

Cells were pretreated with 500 nM
of Cell Tracker Green
TM
CMFDA
and were then treated with 3b (5
µM) for 2 h. Signal intensity
reflects cellular thiol content.
SYPRO Ruby staining of the gels
shown on left was done to ensure
even loading of proteins for the
control and treated samples.

Figure 3.14. Compound 3b
depletes cellular thiols in MIA
PaCa-2 pancreatic cancer cells.
89

BxPC-3 and MIA PaCa-2
cells were treated with
increasing concentrations of
compound 3b (1 µM, 2.5 µM
and 5 µM) for 4 h. Treatment
of (A) BxPC-3 and (B) MIA
PaCa-2 cells with compound
3b resulted in decrease in
cellular glutathione content in
a dose dependent manner
(CellTracker Green
TM

CMFDA stains for cellular
glutathione, DRAQ5
®
is the
nuclear stain).

Figure 3.15. Compounds 3b
depletes cellular antioxidants
in pancreatic cancer cells.
90
3.4. TPs and quinazoline-5,8-diones induce rapid and sustained
inhibition of cancer cell proliferation
Compounds 3a-3f and 4 increased cellular oxygen consumption and superoxide
production in pancreatic cancer cells within minutes (Figure 3.3, Figure 3.4, Figure 3.5,
Figure 3.10 and Figure 3.12). Therefore, we investigated their cytotoxic potential using
shorter drug treatments in the colony formation and the MTT assays. In agreement with
above results, 10 min treatments with 3b followed by removal of drug, was sufficient to
inhibit cell proliferation in BxPC-3, MIA Paca-2 and PANC-1 cells, indicating that 3b
has a rapid and irreversible mechanism of action (Figure 3.16). Furthermore, 3b was most
potent in MIA Paca-2 cells with an IC
50
of 8.7±0.6 µM with just 30 min of drug exposure
as determined by MTT assay (Figure 3.16B). Therefore, we used MIA Paca-2 cells for all
our further studies. Similar results were observed for all analogues indicating that these
compounds have immediate and sustained inhibitory effects on cancer cell proliferation
(Figure 3.17). Compounds 3a-3c are more potent than compounds 3d-3f and 4 supporting
the results obtained in OCR experiments.

91

Colony formation and MTT assays were performed using compound 3b in BxPC-
3 (A), MIA PaCa-2 (B) and PANC-1 (C) cells. Cells were treated with a range of
concentrations (1 µM, 5 µM and 10 µM) for increasing time periods (10 min, 30
min, 1 h, 4 h, 8 h, 12 h, 24 h, 48 h and 72 h), washed with 1x DPBS and allowed
to grow into colonies in drug-free media for 5 days (MIA PaCa-2 cells), 7 days
(PANC-1 cells) and 10 days (BxPC-3 cells), or incubated for 72 h MTT assay
after the wash. The colonies obtained were stained using crystal violet and then
imaged using VersaDoc imaging platform. (Curves represent the data generated
from MTT assay. Inset tables show the IC
50
values from MTT assay performed
for a total of 72h).

Figure 3.16. Treatment with compound 3b results in rapid inhibition of
cancer cell proliferation.
92

Similar experiments were performed for TP 421, one of the lead compounds of
TP class. MIA PaCa-2 cells were incubated with increasing concentrations of TP 421 for
0.25 h, 0.5 h, 1 h or 5 h, followed by incubation in drug-free media for 24 h, 48 h or 72 h.
Simultaneously, the cells were exposed to a continuous exposure of TP 421 for 24 h, 48 h
or 72 h. Cell viability was assessed by MTT based cytotoxicity assay. The IC
50
of TP 421
decreased with increased treatment time as well as the total duration of incubation time.
However, TP 421 was able to induce 50% cytotoxicity even with a very short exposure of
Colony formation assay was performed for compounds 3a-3f, 4. Cells were
treated with compounds (0.1 µM, 1 µM, 5 µM and 10 µM) for increasing times
(15 min, 30 min, 1 h, 24 h), washed with 1x DPBS and allowed to grow in drug-
free media for 5 days. Cells were stained with crystal violet and imaged using
VersaDoc imager (Bio-Rad Laboratories, Hercules, CA).

Figure 3.17. Compounds 3a-f, 4 induce rapid and sustained inhibition of cell
proliferation in MIA PaCa-2 pancreatic cancer cells.
93
15 min TP 421 (20 µM) suggesting a very rapid initial drug effect (Table 3.7 and Figure
3.18) (Shabaik et al., 2013).
Table 3.7. TP 421 induces rapid and sustained inhibition of cancer cell
proliferation (Shabaik et al., 2013)

Exposure (h) Incubation time (h)
24 48 72
IC
50
(µM)
Continuous 2.6 0.8 0.9
5 15 8 6
1 20 16 10
0.5 >20 16 11.5
0.25 >20 20 14
MIA PaCa-2 pancreatic cancer cells were treated with increasing concentrations
of TP 421. Cells were either washed after 0.25 h and incubated in drug free
medium or continuously exposed to TP 421 for indicated times. Dose response
survival curves were plotted for comparing continuous exposure (closed squares)
to 0.25 h drug exposure followed by a media wash out (open squares) and further
incubation. Total incubation time is indicated in parentheses. The data are mean ±
SD from three independent experiments (Shabaik et al., 2013).
Figure 3.18. Effect of shorter exposure times for TP 421 treatment on its
cytotoxicity in MIA PaCa-2 pancreatic cancer cells.
94
3.5. Antioxidants overcome the cytotoxic effects of compound 3b and TP
421
On the basis of the data presented thus far we hypothesize that the cytotoxic
effects of quinazolinediones and TPs on cancer cells is mediated by the production of
ROS. To test this hypothesis we treated pancreatic cancer cells with compound 3b or TP
421 in the presence or absence of antioxidants (N-acetylcysteine, NAC or glutathione
reduced ethyl ester, cell permeable glutathione, GSH).
Cells were pretreated with 0 mM, 1 mM or 5 mM of NAC or GSH for 2 h
followed by treatment with compounds 3b at 5 µM for 72 h. Cells treated with compound
3b in the presence of an antioxidant showed increased survival in a dose dependent
manner as compared to cells treated with compound 3b alone, confirming a ROS-
mediated cell death mechanism for these compounds (Figure 3.19).
Similar experiments were conducted for TPs. Pancreatic cancer cells were
pretreated with NAC (15 mM) for 2 h and then treated with increasing concentrations of
TP 421 for 24 h. NAC exerted protection to cells from TP 421 induced cytotoxicity by up
to 35% (Figure 3.20). Therefore, suggesting ROS-mediated cell death mechanism for TP
421 as well (Shabaik et al., 2013).

95

Treatment with antioxidants (A) Glutathione (GSH, cell permeable analogue) or
(B) N-acetylcysteine (NAC) increases survival of pancreatic cancer cells treated
with 3b. X-axis represents the concentration of antioxidant in mM (0, 1 and 5).
Blue bars represent % survival in cells treated with antioxidant alone. Red bars
represent cells pre-treated with the antioxidant for 2 h and then treated with
compound 3b (5 µM) for a total of 72 h. Results are obtained from MTT based
cytotoxicity assay.

Figure 3.19. Treatment with compound 3b causes ROS-mediated cell death
in pancreatic cancer cells.
96

3.6. Quinazoline-5,8-diones promote Akt-directed ROS-mediated cell
death in cancer cells
Previously, it has been shown that Akt increases intracellular ROS by increasing
oxygen consumption and decreasing ROS scavenging (Nogueira et al., 2008). Similarly,
compound 3b increases cellular OCR (Figure 3.3, Figure 3.4 and Figure 3.5) and induces
cellular superoxide generation (Figure 3.10 and Figure 3.12). Therefore, we postulated
PANC-1 cells were treated with N-acetyl cysteine (NAC; 15 mM) for 2 h,
followed by increasing concentrations of TP 421. NAC exerted a protective effect
on cells exposed to TP421. *, ** and *** indicate p<0.05, p<0.005 and p<0.001
respectively (Shabaik et al., 2013).
Figure 3.20. TP 421 results in ROS-mediated cell death in PANC-1
pancreatic cancer cells.
97
that the ROS-mediated cell death by compound 3b maybe due to Akt activation. To test
this hypothesis, MIA PaCa-2 pancreatic cancer cells were treated with compound 3b (5
µM) at various time points. We observed a robust phosphorylation of Akt at S473 and
T308 as early as 10 minutes. Activated Akt in turn phosphorylated its downstream target,
FoxO3a (forkhead box O3a). However, phosphorylation of FoxO3a by Akt results in its
inactivation and thus decreased expression of its target antioxidant enzymes [Manganese
superoxide dismutase (MnSOD) and catalase] (Figure 3.21). Mechanistically, compound
3b causes a rapid increase in ROS upon treatment and a subsequent decrease of
antioxidants’ expression in cells between 8-12 h (Figure 3.21) suggesting that compound
3b promotes Akt-directed oxidative stress in MIA PaCa-2 cells (Figure 3.22).
Furthermore, proteomic studies were performed in order to investigate the
mechanism of action of these compounds. Proteomic analysis was conducted using
Kinex
TM
Antibody Microarray service from Kinexus Bionformatics Corporation.
PI3K/Akt pathway was one of the key pathways that gets activated with compound 3b
treatment (Table 3.8).
Similar experiments were performed for finding the effects of TP compounds on
Akt phosphorylation. Treatment with TP 197, a lead representative from TP class did not
affect Akt phosphorylation indicating that the mechanism of action of TP class of
compounds is independent of Akt phosphorylation (Figure 3.23).

98

Table 3.8. Fold change of proteins belonging to Akt pathway in
response to compound 3b treatment (Kinexus proteomic analysis)

Protein Fold change
p-Akt (S473) 2.96
p-Akt (T308) 2.73
PDK 1 1.85
99

Treatment with compound 3b resulted in activation of
Akt by increased phosphorylation at S473 and T308.
Activated Akt phosphorylates FoxO3a (S253) resulting
in its inactivation leading to decreased expression of its
downstream target antioxidant enzymes manganese
superoxide dismutase (MnSOD) and catalase. Thus, 3b
decreased the antioxidant content of cells. GAPDH was
used as a loading control. Western blots were repeated at
least thrice for each condition. Representative blots are
shown in here. Quantification is done using ImageJ
software. Total proteins were normalized to loading
control. phospho-proteins were normalized to their
loading control and then to their respective total protein
content.

Figure 3.21. Compound 3b promotes Akt-directed
increase in cellular oxidative stress.
100

Treatment with compound 3b resulted in Akt-directed increase in cellular oxygen
consumption, ROS production and decreased antioxidants’ expression (MnSOD
and catalase). Increased oxidative stress induced by 3b overwhelms the adaptive
capacity of cancer cells to survive at relatively higher basal ROS. Finally,
increased cellular ROS resulted in activation of stress kinases and induction of
cell death in cancer cells.
Figure 3.22. Compound 3b induces ROS-mediated cell death in cancer cells.
101

3.7. Discussion and conclusions
Quinazoline-5,8-diones and TP compounds have a rapid and sustained mechanism
of action. Quinazoline-5,8-diones and TPs induce rapid increase or decrease in cellular
OCR, respectively (Figure 3.2, Figure 3.3, Figure 3.4 and Figure 3.5). Both the classes of
compounds promote significant increase in cellular superoxide generation (Figure 3.10,
Figure 3.12, Figure 3.13, Table 3.4, Table 3.5). Furthermore, 3b depletes cellular thiols
(Figure 3.14 and Figure 3.15) and aggravates cellular oxidative stress. A short exposure
to the compound 3b or TP 421 is adequate to induce cell death in cancer cells with
sustained effect (Figure 3.16, Figure 3.17, Figure 3.18 and Table 3.7).
Akt is a pro-survival kinase that is upregulated in several forms of human cancers.
Hyperactive Akt inhibits apoptosis induced by numerous stimuli. However, Akt is unable
to inhibit ROS-mediated cell death, and in fact, Akt aids in ROS-directed cell death by
inducing cellular oxygen consumption, promoting ROS generation, and impairing ROS
degeneration. Akt mediates decreased ROS scavenging by phosphorylating the
p-Akt (S473)
TP197 (5 µM) - 10 min 0.5 h 1 h
Akt
Treatment of MIA PaCa-2 pancreatic cancer cells with TP
197 (5 µM) did not affect Akt phosphorylation for up to 1 h
(The blots are from one time experiment).
Figure 3.23. TP 197 does not influence Akt
phosphorylation.
102
transcription factor FoxO causing its inactivation. This leads to decreased expression of
its target antioxidant enzymes (MnSOD and catalase). Therefore, hyperactivated Akt
sensitizes cancer cells to ROS-directed cell death (Nogueira et al., 2008). Parthenolide, a
sesquiterpene lactone, has been shown to induce Akt-directed ROS-mediated cell death in
prostrate cancer cells (Sun et al., 2010). Compound 3b increases Akt phosphorylation
causing a decrease in FoxO3a activity that in turn reduces the cellular pool of
antioxidants resulting in excessive oxidative stress in cancer cells (Figure 3.21 and Figure
3.22). However, TP 197 does not influence Akt phosphorylation (Figure 3.23). Since TP
compounds are mitochondrial targeted therefore their target might be a mitochondrial
protein.
Due to limited therapeutic options for pancreatic cancer we plan to develop these
compounds for pancreatic cancer treatment. Recent studies have shown that like most
types of cancer, pancreatic cancer also exhibits increased oxidative stress (MacMillan-
Crow et al., 2000). Increased ROS in pancreatic cancer suggests the usefulness of ROS-
mediated therapeutic strategy in pancreatic cancer. Most forms of pancreatic cancer
express over-activated mutated Ras. Ras oncogene can upregulate the expression of
NOX1 (a homologue of catalytic subunit of superoxide producing enzyme, NADPH
oxidase) and increase ROS generation (Mitsushita et al., 2004b). Therefore, these
compounds might be acting through Ras. However, since quinazoline-5,8-diones have
similar activities in Ras wt (BxPC-3) and mutated (MIA PaCa-2 and PANC-1) forms of
cancer (Table 3.3), NOX1 does not seem a probable target for these compounds.
103
Additionally, pancreatic cancer cells have elevated expression of
NAD(P)H:quinone oxidoreductase (NQO1), which detoxifies quinones. NQO1 reduces
most quinones directly to hydroquinones, thus bypassing the highly reactive semi-
quinone intermediate (Lewis et al., 2005b). Dicumarol and ES936 inhibit NQO1 and
demonstrate cytotoxic effects in pancreatic cancer (Dehn et al., 2006b; Reigan et al.,
2007b). However, certain agents like streptonigrin require NQO1 mediated activation for
exerting cytotoxic effects (Cone et al., 1976b; Lewis et al., 2005b). Since these
compounds have a quinone moiety, there is possibility that NQO1 might be activating
them like streptonigrin. Several other drugs like capsaicin, benzyl isothiocyanate and
nitric oxide donating aspirin (NO-ASA) have shown ROS mediated cytotoxic effects in
pancreatic cancer cell lines (Zhang et al., 2008b; Zhang et al., 2006b; Zhou et al., 2009a).
Moreover, pancreatic cancer cells are known to express activated Akt, which could aid in
achieving Akt-directed ROS-mediated cell death in pancreatic cancer cells (Fahy et al.,
2003). All these reports suggest that ROS mediated treatment is a rational approach for
pancreatic cancer.
Lastly, these compounds have a quinone moiety in their structures, and most
antitumor quinones undergo redox cycling (Redox cycling: Quinone reductases catalyze
one electron or two electron transfer reactions resulting in the formation of semiquinone
and hydroquinone, respectively. Semiquinone reacts with oxygen, and generates
superoxide and the parent quinone. Superoxide anion acts as a propagating species in the
autoxidation of hydroquinones. NADPH cytochrome P450 reductase and NAD(P)H
quinone oxidoreductase are examples of one and two electron transfer catalyzing quinone
104
reductases, respectively). Redox cycling of quinones is accompanied by consumption of
oxygen, oxidation of NAD(P)H and formation of reactive oxygen species (Cadenas,
1995). It is plausible that these compounds might be undergoing redox cycling resulting
in increased superoxide generation and oxygen consumption. Therefore, they may be
acted upon by one of the quinone reductases.
Dissimilar mechanism of action of TPs and quinazoline-5,8-diones can be
deciphered from the difference in superoxide detected in cytochrome c based assay
(Table 3.6). Cytochrome c reacts with superoxide resulting in increased absorbance that
is an indicator of increase in superoxide production. Since, 3b seems to be more potent
than TP187 in superoxide production as detected from cytochrome c assay, we thought
that may be TPs do not produce significant superoxide. However, TP compounds
significantly increased mitochondrial superoxide production (Figure 3.13). This
mitochondrial superoxide can not cross cell membrane of MIA PaCa-2 cells, is rapidly
converted to hydrogen peroxide and thus remains undetected by cytochrome c. However,
increase in superoxide production by 3b was detected using both cytochrome c and
MitoSOX (Figure 3.10 and Figure 3.12). This suggests that major superoxide production
in TP treated cells is mitochondrial whereas compound 3b treated cells can induce both
mitochondrial and non-mitochondrial superoxide production.
In conclusion, we have discovered novel classes of compounds displaying unique
mechanisms of action. Compound 3b and its analogues exert significant increase in OCR
in cells leading to ROS generation and cell death. This effect can be overcome by pre-
treatment with antioxidants, validating the role of ROS in promoting cytotoxicity of 3b.
105
Additionally, TPs cause significant decrease in cellular OCR with a concomitant increase
in ROS production resulting in cell death. Antioxidants can protect the cells from
cytotoxic effects of TPs indicating ROS-mediated cell death mechanism. Therefore, with
the discovery of compound 3b and TPs we have provided compelling evidence in support
of a hypothesis previously proposed by the Huang Lab “Targeting cancer cells by ROS-
mediated mechanisms: a radical therapeutic approach?” (Trachootham et al., 2009).
High ROS levels could represent the Achilles’ heel of cancer cells. Even though
cancer cells have adapted to survive in an oxidative stress environment, exogenous
oxidative insults can overwhelm the adaptive antioxidant capacity of cancer cells and
trigger ROS-mediated cell death. Our novel class of quinazolinediones and TPs takes
advantage of this weakness, and by exacerbating superoxide generation and diminishing
the antioxidant capacity of cancer cells they tip the balance towards activation of stress
kinases that ultimately lead to cell death.

106
CHAPTER FOUR:
DETERMINING THE CELL DEATH MECHANISMS
INDUCED BY TPs AND QUINAZOLINE-5,8-DIONES
TPs and quinazoline-5,8-diones discussed in the previous chapter have shown to
exhibit ROS-dependent cell death effects. This chapter will focus on the cell death
mechanisms promoted by TPs and quinazoline-5,8-diones. More emphasis is on
discussing the effects of quinazoline-5,8-diones. Cell death mechanism of TPs have been
published in (Millard et al., 2010; Shabaik et al., 2013). Herein, we study the effects of
TPs or quinazoline-5,8-diones on cellular stress kinases, DNA damage, apoptosis and cell
cycle regulation.

107
4.1. Quinazoline-5,8-diones inhibit cell proliferation in a panel of cancer
cell lines of different origin
All the seven of our novel quinazoline-5,8-diones were tested in a panel of cancer
cells of different origin. These compounds inhibit cell proliferation in breast, colon,
prostrate, ovarian and lung cancer cell lines (Table 4.1). The inhibition of cell
proliferation obtained by MTT assay was confirmed by colony formation assay (Figure
4.1). Interestingly, these compounds exert similar activity in HCT 116 p53
+/+
and HCT
116 p53
-/-
, suggesting that their mode of action might be independent of p53 expression.
More importantly, all compounds were potent in adriamycin-resistant NCI/ADR-RES
cells overexpressing MDR-1 (Scudiero et al., 1998) and in HEY cells that are inherently
resistant to cisplatin (Buick et al., 1985; Hamaguchi et al., 1993). Therefore, compound
3b and its analogues have great potential for treating resistant forms of cancer and
warrant further investigation.
Furthermore, cytotoxic potential of lead compound 3b was compared with that of
gemcitabine in normal fibroblasts. The extent of cytotoxic effects exerted by compound
3b in normal fibroblasts HFF-1 was similar to the toxicity induced by gemcitabine in
HFF-1 cells. Compound 3b was comparatively less cytotoxic to HFF-1 primary human
foreskin fibroblasts and HFF-eMYC (overexpressing cMYC but not immortalized cells)
as compared to their immortalized counterpart (HFF-iMYC), and cancer cells. This
suggests that these compounds will be potentially non-toxic to normal cells in vivo.
(Figure 4.2). Additionally, compound 3b inhibited cell proliferation in a mouse cancer
stem line (Ding et al., 2010; Rountree et al., 2009), and in triple negative MDA-MB-231
108
breast cancer cell line, enriched in cells with cancer stem cell markers and resistant to
chemotherapy, with an IC
50
of 2.4 µM and 2.2 µM, respectively (Hwang-Verslues et al.,
2009). Furthermore, the cytotoxic effects of lead compound 3b were analyzed in
gemcitabine, and gemcitabine and erlotinib resistant MIA PaCa-2 pancreatic cancer cells.
ROS have been shown to aid in overcoming chemoresistance. Compound 3b is active in
chemoresistant NCI/ADR-RES and HEY cells. Therefore, we expect compound 3b to be
active in chemoresistant forms MIA PaCa-2 pancreatic cancer cells. As expected,
compound 3b was active in both the resistant cell lines (Table 4.2). Therefore, compound
3b has an added advantage of targeting a wide range of cancer cell types including
resistant forms.

109
Table 4.1. Cytotoxicity of compounds 3a-f and 4 in a panel of cancer cell lines.
Cell Lines
IC
50
(µM)
a

3a 3b 3c 3d 3e 3f 4
Breast Cancer
T-47D 2.4±1.8 0.4±0.04 0.5±0.1 5.6; 8 1.7±1.1 3.4±2.2 5±1.0
MDA-MB-231 8.9 2.2 1.8 >10 2.5 7 2.8
MCF7 0.8; 1.8 2; 3 2.2; 3 6.9; 3.9 5; 3 >10 2.4; 3.1
MDA-MB-435 1.9±0.2 2.0±0.3 2.1±0.2 5.6±2.7 1.4±0.2 7.6±0.4 2.9±0.5
Colon Cancer
HCT 116 p53
+/+
4.6±0.2 2.2±0.3 2.4±0.7 6.9±0.9 2.3±0.1 >10 2.4±0.4
HCT 116 p53
-/-
2.0±0.2 1.7±0.5 2.3±0 4.9±2.7 4.6±2.6 7.1 1.9±0.2
Prostate
Cancer

PC-3 1.8; 2.2 2.2; 1.8 2.8; 4 4.2 2.3; 3.1 9.2 3.2; 3.2
Ovarian
Cancer

OVCAR-8 9.7; 5 2.2; 1.7 2.2; 2 >10 3.5 4 6; 4
HEY 1.6±0.2 2.4±0.2 2.6±0.2 >10 4.5 10 1.9±0.1
NCI/ADR-RES 4 0.8 0.52 4 2.2 1.1 4
Lung Cancer
NCI-H460 1.8; 2 2.5; 2.7 2.5; 3.5 9.2; 5 5.2; 3.8 >10 4; 3.1
NCI-H1299 1.9; 2.2 1.8; 0.8 0.21;
0.3
4.8; 4 2.2; 3 2.2; 3.8 1.9; 3.7
a
IC
50
is defined as drug concentration causing a 50% decrease in cell population
using MTT assay. Values with standard deviation are from at least three
independent experiments. Each experiment was generated from an average of
three independent wells. The concentrations of DMSO used in the experiments
were not cytotoxic to cells.

110

Colony formation assay was performed in HCT
116 p53
+/+
, MDA-MB-435 and HEY cells treated
with a range of concentrations of compound 3b
(0.1 µM, 1 µM, 2 µM and 5 µM) for 24h. The
colonies were stained after 10 days and imaged
using VersaDoc imager (Bio-Rad Laboratories,
Hercules, CA).

Figure 4.1. Compound 3b inhibits cell
proliferation in (A) HCT 116 p53
+/+
, (B) MDA-
MB-435 and (C) HEY cells.
111

Table 4.2. Activity of compound 3b in resistant pancreatic cancer cells.

Cell Line Resistance Compound IC
50
(µM)
Mito-GR Gemcitabine Compound 3b 2.7
Erlotinib >10
Gemcitabine >10
Mito-GTR Erlotinib and
Gemcitabine
Compound 3b 2.3
Erlotinib >10
Gemcitabine >10
Cells were treated with compound 3b (10 µM) or gemcitabine (10
µM) for 1 h, washed with 1x DPBS and incubated with drug-free
media for 72 h. Values represent the data obtained from MTT
assay performed for a total of 72 h.

Figure 4.2. Compound 3b exerts decreased toxicity in HFF-1
primary fibroblasts as compared to immortalized fibroblasts
and cancer cells.
112
4.2. TPs induce broad spectrum cytotoxic effects
700 structural analogues of TP 187, TP 197 and TP 421 were designed and
screened for cytotoxic effects in HCT 116 p53
+/+
colon carcinoma cell line. Compounds
that exerted more than 50% cytotoxicity at 10 µM were further tested in a panel of cancer
cell lines in MTT based cytotoxicity assay and colony formation assay (Table 4.3). The
most active compounds (having IC
50
values ≤5 µM in MTT assay) are reported in Figure
4.3. Most of the compounds displayed a good correlation between the results obtained in
the two assays. Not all TP compounds induced cell death. This suggests that TP moiety
by itself does not confer cytotoxic properties to cancer cells (Millard et al., 2010).

Figure 4.3. Potent structural analogues of TP 187, TP 197 and TP 421
(Millard et al., 2010).

113

Table 4.3. IC
50
of structural analogues of TP 187, TP 197 and TP 421 in a panel of
cancer cell lines (Millard et al., 2010).

Compound
IC
50
(µM)
MCF-7 MDA-
MB435
PC3 HCT116
p53+/+
HCT116
p53-/-
MTT CFA MTT CFA MTT CFA MTT CFA MTT CFA
TP 783 2.8 2.2 1.6 3.8 2.8 3 2 1.4 2.7 1.8
TP 831 2.8 3.5 3.4 4.1 3 1.1 2.8 1.6 2.7 3.8
TP 738 3.2 2.8 3.8 2.2 2.8 1 2.8 2.8 3.2 3.1
TP 791 4 3 4 2.9 3.8 3 3.5 5 5.8 >10
TP 734 4.2 2.4 4.2 10 3.4 4 5 >10 6 >10
TP 728 3.8 1.1 3.4 2.8 3.7 3.1 3.1 2.9 3.1 2.3
TP 794 3 2.4 2.8 3.5 3 3.1 2.8 3.2 2.6 3
TP 752 5 3.2 8.4 >10 5 4 4 6 5 5
TP 749 2.6 >10 2.5 >10 2.2 8.5 3.1 >10 3.1 4
TP 759 3.1 0.8 3.8 4 3 2.9 1.8 3 3.1 2.8
TP 790 0.8 3.2 0.78 4.1 3 3.2 2.8 6 3.2 3
TP 744 0.65 0.3 2.5 2 3 3.4 1.5 3.1 1.8 3.9
TP 726 0.5 0.15 0.6 2.1 0.5 3.2 0.4 2.4 0.58 2.8
TP 824 0.3 0.38 0.6 1.5 0.42 0.22 0.3 0.6 0.4 0.48
TP 760 0.5 0.28 0.98 0.18 3 0.31 0.31 0.6 0.42 0.78
TP 737 0.5 0.31 1 1.1 0.58 0.6 1.8 3.2 2.5 3
TP 736 5 2.8 5 4 4 3.4 3.8 3 4 3
TP 822 1 2.8 1.5 6 2.8 2 3.5 2.8 2.9 3.2
TP 772 0.65 0.7 5 3.2 1.6 0.6 0.9 2.8 3.1 3
TP 731 0.45 0.2 0.5 0.35 0.4 0.28 0.25 0.28 0.35 0.32
TP 768 0.45 1.1 0.5 3.2 3 0.8 0.99 3.1 2.1 3.8
TP 781 3.2 2.2 4 3.1 5 2.4 1.8 4 3.5 3.8
TP 821 0.55 1.1 1.8 7 3.8 1.8 2.4 3.1 3.8 2.2
TP 769 0.7 0.8 0.5 0.45 1 0.39 0.4 2.8 1.9 2.2
TP 801 3.2 2.4 6 4.1 4 2.8 2.8 3 2.8 3
TP 765 2.4 1.1 >10 3 4.1 2.8 2 3 4 3.8
TP 826 0.58 0.9 0.6 4 0.8 0.25 0.56 2 1.2 1.8
TP 764 7 6 6 >10 8 >10 9 >10 >10 >10
TP 825 1.2 4.5 4 5.1 5 2.2 3.5 3.1 3.9 3
TP 740 2 0.9 2.1 2 1.2 0.7 0.72 2.6 2.8 3
TP 785 2.7 2.2 3.4 3.8 2.8 3.5 2.8 2.8 3 2.3
TP 835 3 3 3.6 3 2 0.7 2.5 1.2 2.2 3.2
TP 812 3.1 1.5 3.2 3.5 3.1 >10 2.8 2.9 3.8 3.7

114
4.3. Quinazoline-5,8-diones and TPs result in DNA damage and
activation of stress kinases in pancreatic cancer cells
The stress response caused by 3b is supported by the early activation (10 min) of
stress induced protein kinases, p38, and JNK and its downstream target c-Jun (Figure
4.4A). Activated p38 and JNK have been implicated in induction of cell death resulting
from various forms of stress including oxidative stress (Chang and Karin, 2001; Cobb,
1999; Davis, 2000; Tournier et al., 2000; Verheij et al., 1996). DNA-damage was
observed by a robust and sustained phosphorylation of histone H2AX at S139, a key
marker for DNA double strand break (Figure 4.4B). DNA double strand breaks can lead
to apoptotic cell death (Roos and Kaina, 2006).
TPs have also shown to increase ROS production resulting in oxidative stress.
Therefore, we tested the effect of TP 421 on cellular stress pathways. As expected,
treatment with TP 421 resulted in activation of stress kinases (p38 and JNK) and DNA
damage (Figure 4.5) (Shabaik et al., 2013).

115

(A) 3b treatment resulted in increased phosphorylation of stress kinases p38
(T180/Y182) and JNK (T183/Y185). Activated pJNK phosphorylates its
downstream target c-Jun (S73). (B) Furthermore, treatment with compound 3b
also resulted in phosphorylation of H2AX (S139) a marker of DNA damage. (C)
Oxidative stress results in increased expression of FASL, activation of FAS
receptor and its downstream caspases (D, E). (F) Proposed mechanism: compound
3b induced ROS-directed Fas-mediated apoptotic cell death in pancreatic cancer
cell. Western blots were repeated at least thrice for each condition. Representative
blots are shown in here. Quantification is done using ImageJ software. Total
proteins were normalized to loading control. phospho-proteins were normalized to
their loading control and then to their respective total protein content.

Figure 4.4. Compound 3b activates stress kinases and induces cell death in
pancreatic cancer cells.
116

(A) TP 421 treatment resulted in increased phosphorylation of stress kinases p38
(T180/Y182) and JNK (T183/Y185). Activated pJNK phosphorylates its
downstream target c-Jun (S73). (B) Furthermore, treatment with compound TP
421 also resulted in phosphorylation of H2AX (S139), a marker of DNA damage.
(D) TP 421 treatment altered several apoptosis regulating proteins. It decreases
the expression of pro-survival proteins Bcl-2 and survivin, and induced PARP and
caspase cleavage (Shabaik et al., 2013).

Figure 4.5. TP 421 activates stress kinases and induces cell death in
pancreatic cancer cells.
117
4.4. Quinazoline-5,8-diones and TPs result in apoptotic cell death in
pancreatic cancer cells
Increased ROS generation by 3b resulted in immediate and sustained activation of
stress kinases’ pathways (p38, JNK) that have been implicated in induction of cell death
(Figure 4.4A) (Chang and Karin, 2001; Lee et al., 2010; Shen and Liu, 2006). Moreover,
3b promoted H2AX phosphorylation in pancreatic cancer cells (Figure 4.4B). H2AX is a
marker for DNA damage and can be phosphorylated by JNK and p38, or under DNA
damaging or oxidative stress conditions (Li et al., 2006; Lu et al., 2008; Lu et al., 2006).
DNA damage can in turn lead to apoptosis (Roos and Kaina, 2006). Furthermore, ROS
can lead to increased expression of FasL (Bauer et al., 1998; Suzuki et al., 2006).
Additionally, activation of p38 and JNK by ROS has also been implicated in Fas/FasL
mediated apoptosis (Chandra et al., 2000; Circu and Aw, 2010; Liu et al., 2009).
Treatment with compound 3b resulted in increased expression and activation of Fas/FasL
and the downstream caspases (caspase 8 and caspase 3) (Figure 4.4C, D, E). Moreover,
Fas can further activate JNK (Wilson et al., 1996) and can intensify the cell death effect.
Additionally, activated JNK can also migrate to mitochondria and induce mitochondrial
apoptosis (Circu and Aw, 2010) and further aid in cell death initiated by ROS.
Furthermore, several other key apoptosis regulating proteins were altered under
the effect of compound 3b (Figure 4.6). Compound 3b decreases the expression of major
cell survival proteins including Bcl-2 and survivin after treatment of MIA PaCa-2 cells
with 5 µM of 3b for at least 12 h (Figure 4.6A, B, C). Additionally, compound 3b
increases expression of pro-apoptotic protein Bax (Figure 4.6B). Compound 3b also
118
results in PARP (poly ADP ribose polymerase) cleavage in MIA PaCa-2 pancreatic
cancer cells, which is another marker for apoptosis (Figure 4.6D). Apoptotic activity of
compound 3b was further confirmed by Annexin-V/PI staining. Sarkostat resulted in dose
dependent increase in apoptotic population of MIA PaCa-2 cells (Figure 4.6E). Lastly,
proteomics analysis revealed that treatment with compound 3b results in alterations of
key oxidative stress regulating pathways and apoptosis (Figure 4.7).
Similar results were obtained with TP 421 treatment of MIA PaCa-2 pancreatic
cancer cells. TP 421 exposure resulted in activation of apoptotic machinery in cells
resulting in cell death (Figure 4.5).

119

Treatment with 3b resulted in decreased expression of cell survival proteins Bcl-2
(A and B) and survivin (C), increased expression of pro-apoptotic Bax (B) and
cleavage of PARP (D) in a dose-and time dependent manner. (E) Apoptotic cell
death induced by 3b in MIA PaCa-2 cells was further confirmed by AnnexinV
assay. Control and 3b treated MIA PaCa-2 cells were stained with Annexin V-
FITC and PI and analyzed by flow cytometry. Annexin V-negative and PI-
negative cells were viable. Annexin V-positive and PI-negative were in early
stages of apoptosis, and cells positive for both Annexin-V and PI represented cell
death by late stages of apoptosis and necrosis. 5 µM of 3b was used for time
course experiments in panels B and D.

Figure 4.6. Compound 3b induces cell death in pancreatic cancer cells.
120

Treatment with compound 3b (1 µM, 24 h) resulted in activation of stress kinases
(SAPK/JNK, p38 MAPK) and apoptotic signaling as depicted from the Kinexus
proteomics microarray experiment.
Figure 4.7. Compound 3b activates stress induced cellular signaling
pathways resulting in cell death.
121
4.5. Quinazoline-5,8-diones and TPs cause inhibition of cell cycle
progression
ROS are known to regulate several cellular signaling mechanisms including cell
cycle events (Boonstra and Post, 2004a; Shackelford et al., 2000). Therefore, we
investigated the effects of 3b and TP 421 on cell cycle progression in pancreatic cancer
cells. 3b resulted in arresting MIA PaCa-2 and PANC-1 pancreatic cancer cells in S
phase of cell cycle in a dose- and time-dependent manner (Figure 4.8). Treatment with
TP 187, TP 197 and TP 421 resulted in G0/G1 arrest in cell cycle progression of
pancreatic cancer cells (Table 4.4).

Table 4.4. Percent distribution of DNA content per cell cycle phase of MIA
PaCa-2 pancreatic cancer cell line in response to treatment with TP compounds
(Shabaik et al., 2013).

24 h
Sample G0/G1 S G2/M
Control 66.9 29.0 4.1
2 µM TP 187 83.9 16.1 0.0
2 µM TP 197 87.8 9.3 2.9
2 µM TP 421 92.0 8.0 0.0
122

4.6. Discussion and conclusions
Quinazoline-5,8-diones and TPs induce oxidative stress in cancer cells resulting
in cell death (Chapter 3). Increase in oxidative stress can result in activation of stress
kinase signaling and DNA damage which can ultimately cause cell death (Li et al., 2006;
Liu et al., 2009; Lu et al., 2008; Lu et al., 2006; Shen and Liu, 2006). As expected,
treatment with compound 3b (lead compound for quinazoline-5,8-diones) or TP 421 (one
(A,B). Exposure of MIA PaCa-2 cells to compound 3b resulted in inhibition
of their cell cycle progression in S phase in a dose- and time-dependent
manner. (C,D). Exposure of PANC-1 cells to compound 3b resulted in
inhibition of their cell cycle progression in S phase. Dasatinib was used as a
positive control for inducing cell cycle arrest in pancreatic cancer cells

Figure 4.8. Compound 3b induces cell cycle arrest in pancreatic cancer
cells.
123
of the lead compounds for triphenylphosphoniums) resulted in activation of stress
kinases. Moreover, treatment with these compounds caused DNA damage. These effects
eventually led to apoptotic cell death in pancreatic cancer cells (Figure 4.4, Figure 4.5
and Figure 4.6). Additionally, these compounds are active in broad range of cancer cell
lines including resistant forms of cancer (Table 4.1, Table 4.2 and Table 4.3). ROS
generation have been linked to overcoming chemoresistance (Wartenberg et al., 2005).
Reactive oxygen species regulate several cellular signal transduction events
including cell cycle progression (Boonstra and Post, 2004a). Since both TPs and
compound 3b induced ROS generation we tested their effects on cell cycle progression.
Treatment with compound 3b resulted in S phase arrest whereas TPs inhibited cell cycle
progression in G0/G1 phase (Figure 4.8 and Table 4.4). The difference in activity can be
attributed to the difference in site of ROS generation. TP compounds promote
predominantly mitochondrial ROS generation whereas compound 3b seems to induce
both cellular and mitochondrial ROS synthesis (Chapter 3).
In conclusion, our novel classes of compounds, quinazoline-5,8-diones and
triphenylphosphoniums exert ROS-mediated anticancer effects in pancreatic cancer cells.
The increase in cellular ROS is the driving force for oxidative stress which results in
activation of stress signaling and DNA damage causing apoptotic cell death in pancreatic
cancer cells. Moreover, ROS generation also inhibits cell cycle progression in cancer
cells treated with TPs or compound 3b.
124
CHAPTER FIVE:
MECHANISTIC EVALUATION OF SARKOSTAT IN
PANCREATIC CANCER
Further mechanistic evaluation was performed on the lead compound 3b that was
identified from the screen for bioenergetics/redox modulating agents. Protein microarray
technique was used to decipher the major signaling pathways influenced by the treatment
with compound 3b. Through the proteomics approach we found that in addition to redox
regulating pathways, compound 3b alters the activation of critical signaling proteins
involved in cell proliferation, invasion and metastasis. Src/FAK complex was the
epicenter of these changes.
Pancreatic cancer is a multifaceted disorder with complex perturbations in several
key regulators of signal transduction pathways. Enhanced Src/FAK expression and
consequent activity have been reported in pancreatic cancer and linked to its inherent and
acquired chemoresistance. Therefore, targeting the Src/FAK axis could provide an
important strategy for the treatment of pancreatic cancer. However, sustained Src
inhibition leads to reactivation of survival pathways regulated by STAT3 resulting in
resistant forms of cancer. Herein, we show that compound 3b (sarkostat
TM
) decreases
Src/FAK and STAT3 phosphorylation leading to inhibition of cancer cell migration and
angiogenesis. Additionally, sarkostat overcomes the limitations of Src inhibitors by
decreasing STAT3 activity.
125
5.1. Sarkostat affects several critical cell-signaling pathways governing
cell migration and invasion
In order to analyze the effects of sarkostat on cellular signal transduction
pathways, we performed a Kinex
TM
(Kinexus, Vancouver, Canada) antibody microarray
proteomic analysis. The results obtained from the antibody microarray were further
analyzed by ingenuity pathway analysis (IPA), and the top canonical pathways were
ranked in the order of significance (Figure 5.1). Treatment of MIA PaCa-2 pancreatic
cancer cells with sarkostat (1 µM for 24 h) affected critical cell-signaling pathways
involving FAK/Src complex, which play an important role in regulation of cell adhesion,
motility, invasion, proliferation, survival and angiogenesis (Kim et al., 2009) (Figure
5.1). The results obtained from IPA that depict the effects of sarkostat treatment on
signaling proteins of Src/FAK complex are listed in table 5.1.

126
Table 5.1. Antibody microarray results of Src/FAK signaling pathway.
Protein Site % CFC
(% change from
control)
Function
Src Pan-specific -15.9
FAK Y397 -9.5 Autophosphorylation
site
FAK Y577 -17.9 Activation loop
FAK Y576 -43.5 Activation loop
Paxillin 1 Pan-specific -16.9
Paxillin 1 Y31 -18.0 Phosphorylated by active
Src/FAK complex
Paxillin 1 Y118 -4.3 Phosphorylated by active
Src/FAK complex
Paxillin 1 Y118 -3.4 Phosphorylated by active
Src/FAK complex
127

MIA PaCa-2 cells were treated
with sarkostat (1 µM, 24 h),
lysed following Kinexus’
protocol, and screened for 378
pan-specific (protein
abundance) and 273 phospho-
site-specific antibodies.
Control and treated cell lysates
were labeled with fluorescent
dye and analyzed separately on
same chip spotted with array of
antibodies. Quantitative
analysis of the fluorescence
intensity signal for each target
protein was performed for each
sample, and fold-change ratios
of treated-to-control were
reported. This data was further
analyzed by using Ingenuity
Pathway Analysis (IPA)
software and the plot
displaying top canonical
pathways affected by sarkostat
treatment was generated. –log
p: statistical significance, and
the pathways are arranged in
the order of their statistical
significance. Ratio: number of
signaling proteins affected by
the treatment to the total
number of signaling proteins in
that pathway.

Figure 5.1. Sarkostat affects
critical signaling pathways
governing cell migration and
invasion.
128
5.2. Sarkostat decreases activating phosphorylation of Src and FAK in
pancreatic cancer cells
In order to confirm the results obtained from Kinex
TM
antibody microarray
proteomic analysis, we analyzed the effect of sarkostat on Src and FAK phosphorylation
by western blotting. As expected, treatment of pancreatic cancer cells (MIA PaCa-2 and
PANC-1) with sarkostat resulted in decreased activating phosphorylation of FAK at
Y576/Y577, Y397, Y861 and Y925 in a dose and time- dependent manner (Figure 5.2A,
F, G and Figure 5.3 A, D, E). Additionally, treatment with sarkostat resulted in decreased
phosphorylation of Src at Y416 in dose-and time-dependent manner (Y416 is the
activating phosphorylation of Src) (Figure 5.2B, F, G and Figure 5.3B, D, E). Decrease in
Src phosphorylation was further confirmed by immunofluorescence (Figure 5.4).
Autophosphorylation of FAK at Y397 results in its activation and creates a docking site
for Src. Once activated by FAK, Src can further phosphorylate FAK at various sites
resulting in a feedback activation loop (Bolos et al., 2010). Together Src and FAK
function as a protein complex for regulation of several cellular signal transduction
pathways through their downstream targets.
Src activation promotes the interaction and phosphorylation of target proteins,
paxillin and CAS family proteins that also bind FAK. Activation and phosphorylation of
p130CAS and paxillin influences focal adhesion turnover supporting cell migration.
Treatment with sarkostat resulted in decreased phosphorylation of both p130 CAS and
paxillin suggesting inhibition of cell migration (Figure 5.3D, E). Moreover, sarkostat
treatment also resulted in decreased phosphorylation of Smad1/5/8 that are known to be
129
hyper-phosphorylated in precursor lesions of pancreatic ductal adenocarcinoma (Mohri et
al., 2012) and influence cancer metastasis (Katsuno et al., 2008) (Figure 5.2E).

Treatment of MIA PaCa-2 cells with sarkostat (5 µM) results in inhibition of
activating phosphorylations of FAK (A) Src (B), STAT3 (C), p130CAS and
paxillin (D), and Smads 1/5/8 (E) in a time-dependent manner. Furthermore,
sarkostat treatment causes a dose-dependent decrease in Src, FAK and STAT3
phosphorylation (F and G). Schematic representation of the effects of sarkostat on
select cell-signaling pathways (H).

Figure 5.2. Sarkostat decreases phosphorylation of signaling proteins
involved in cell migration in MIA PaCa-2 pancreatic cancer cells.
130

Treatment of PANC-1 cells with sarkostat (5 µM) inhibits activating
phosphorylations of FAK (A) Src (B) and STAT3 (C) in a time-dependent
manner. (D and E). Sarkostat treatment results in a dose-dependent decrease in
Src, FAK and STAT3 phosphorylation.

Figure 5.3. Treatment with sarkostat decreases phosphorylation of FAK, Src
and STAT3 in PANC-1 pancreatic cancer cells.
131

Sarkostat (5 µM) results in decreasing phosphorylation of p-Src (A) in a
time-dependent manner without affecting the expression of total Src (B).
Cells were fixed with 0.4% formaldehyde and stained using primary
antibodies for p-Src and Src followed by incubation with respective
secondary antibodies and then counter-stained with DAPI nuclear dye. Cells
were imaged using BD Pathway 435 High-Content Bioimager (BD
Biosciences, San Jose, CA) using 10x objective.

Figure 5.4. Sarkostat causes rapid decrease in p-Src in MIA PaCa-2
pancreatic cancer cells.
132
5.3. Sarkostat treatment produces changes in cell morphology, inhibits
cell migration and blocks tube formation
Sarkostat blocks several pathways that support metastasis in pancreatic cancer
cells (Figure 5.2, Figure 5.3 and Figure 5.4). As expected, sarkostat treatment resulted in
morphological changes of pancreatic cancer cells within 4 h (Figure 5.5 and Figure 5.6),
while similar treatment did not induce significant morphological changes in HFF-1 cells
(normal human foreskin fibroblasts) (Figure 5.6).
5 µM sarkostat resulted in irreversible loss of adhesion and rounding up of
MIA PaCa-2 pancreatic cancer cells within 4 h of treatment. Cells were
imaged live using Nikon microscope with 10x objective.

Figure 5.5. Sarkostat causes rapid change in morphology of cancer cells.
133

Because cell migration precedes metastasis, we tested the ability of sarkostat to
inhibit cell migration in the Boyden chamber. Decreased migration of Mia-PaCa-2 cells
was observed in the presence of 1 µM sarkostat (Figure 5.7A). Inhibition of migration
was confirmed in the wound healing assay and this difference was not due to cytotoxicity
as determined in cells treated in parallel (Figure 5.7B). Sarkostat decreased the ability of
a panel of serum-starved pancreatic (Mia-PaCa-2, PANC-1, BxPC-3 and ASPC-1) and
5 µM and 10 µM sarkostat resulted in loss of adhesion and rounding up of
MIA PaCa-2 pancreatic cancer cells within 4 h of treatment. However, no
change in morphology was observed in normal HFF-1 fibroblasts even at 10
µM treatment with sarkostat. Cells were imaged after fixing with methanol and
staining with giemsa using BD Pathway 435 High-Content Bioimager (BD
Biosciences, San Jose, CA) using 10x objective.

Figure 5.6. Sarkostat causes rapid change in morphology of cancer cells
but not of normal cells.
134
prostate (PC-3) cancer cells to close the wound when stimulated with 10% FBS (Figure
5.8, Figure 5.9, Figure 5.10, Figure 5.11 and Figure 5.12). These results clearly indicate
that sarkostat inhibits cell migration in cancer cells. Furthermore, Src/FAK complex is
known to play an important role in regulating angiogenesis (Kim et al., 2009). Sarkostat
potently inhibited tube formation in the HUVECs suggesting its potential to hinder
angiogenesis (Figure 5.13).

(A) 24 h treatment with sarkostat (1 µM) resulted in decreased migration of
serum-starved MIA PaCa-2 cells through the membrane of Boyden chamber in
the presence of 10% FBS stimulation. Cells were imaged after fixing with
methanol and staining with giemsa using Nikon microscope with 10x objective
(B). Cell viability as determined by MTT assay of MIA PaCa-2 cells treated with
sarkostat concentrations that are used in cell migration assays.
Figure 5.7. Sarkostat inhibits cell migration.
135

Treatment with sarkostat for 24 h inhibited closure of wounds stimulated by 10%
FBS in serum-starved MIA PaCa-2 cells in a dose-dependent manner. No FBS
was used as a positive control for the inhibition of wound closure. Cells were
imaged after fixing with methanol and staining with giemsa using BD Pathway
435 High-Content Bioimager using 4x objective.

Figure 5.8. Sarkostat inhibits cell migration in MIA PaCa-2 pancreatic
cancer cells.

136

Treatment with sarkostat for 24 h inhibited closure of wounds stimulated by 10%
FBS in serum-starved ASPC-1 cells in a dose-dependent manner. No FBS was
used as a positive control for the inhibition of wound closure. Cells were imaged
after fixing with methanol and staining with giemsa using BD Pathway 435 High-
Content Bioimager using 4x objective.

Figure 5.9. Sarkostat inhibits cell migration in ASPC-1 pancreatic cancer
cells.

137

Treatment with sarkostat for 24 h inhibited closure of wounds stimulated by 10%
FBS in serum-starved BxPC-3 cells in a dose-dependent manner. No FBS was
used as a positive control for the inhibition of wound closure. Cells were imaged
after fixing with methanol and staining with giemsa using BD Pathway 435 High-
Content Bioimager using 4x objective.

Figure 5.10. Sarkostat inhibits cell migration in BxPC-3 pancreatic cancer
cells.

138

Treatment with sarkostat for 24 h inhibited closure of wounds stimulated by 10%
FBS in serum-starved PANC-1 cells in a dose-dependent manner. No FBS was
used as a positive control for the inhibition of wound closure. Cells were imaged
after fixing with methanol and staining with giemsa using BD Pathway 435 High-
Content Bioimager using 4x objective.

Figure 5.11. Sarkostat inhibits cell migration in PANC-1 pancreatic cancer
cells.

139

Treatment with sarkostat for 24 h inhibited closure of wounds stimulated by 10%
FBS in serum-starved PC-3 cells in a dose-dependent manner. No FBS was used
as a positive control for the inhibition of wound closure. Cells were imaged after
fixing with methanol and staining with giemsa using BD Pathway 435 High-
Content Bioimager using 4x objective.

Figure 5.12. Sarkostat inhibits cell migration in PC-3 prostate cancer cells.
140

Sarkostat inhibits tube formation in HUVECs in a dose-dependent manner.
Sulforaphane was used as a positive control for inhibiting tube formation in
HUVECs. Vehicle (DMSO), sulphorafane and sarkostat were prepared as serial
dilutions at 2x final concentration in Endothelial Cell Basal Medium (EBM-2)
at concentrations ranging over three logs, and 25 µL of each dilution was added
to the respective BME-coated wells in a 384-well plate. 2.0 x 10
5
cells/ mL of
sub-confluent HUVECs were prepared in 2x endothelial cell growth medium
(EGM-2) consisting of EBM-2, 4 % FBS and 2x concentration of the growth
factors and supplements contained within the Endothelial Growth Medium
SingleQuots Kit. To each well containing treatment or vehicle, 25 µL of cell
suspension was added to give a final volume of 50 µL and 1x concentrations of
media and compound. Cells were incubated at 37º C for 6-8 h to allow
sufficient time for tube formation. When tubular networks had formed in the
control wells, 5 µL of EBM-2 containing 10x calcien AM (final concentration 6
µM) was added to each well. Imaging was performed on a BD-Pathway 435
high-content bioimager equipped with calcien AM filter and 4x objective.
Absence of tubular networks indicate drug activity.

Figure 5.13. Sarkostat inhibits angiogenesis.

141
5.4. Sarkostat can be beneficial in resistant forms of cancer
Increased Src and FAK phosphorylation are associated with the inherent and
acquired resistance to gemcitabine in pancreatic cancer cells (Duxbury et al., 2004a)
(Zhou et al., 2011). Sarkostat treatment decreases both Src and FAK phosphorylation
(Figure 5.2, Figure 5.3 and Figure 5.4) and could be synergistic with gemcitabine.
Combined treatment of pancreatic cancer cells with sarkostat and gemcitabine at
concentrations at least 10x lower than the IC
50
value for each drug resulted in additive
inhibition of cancer cell proliferation as shown in MTT assay (Figure 5.14) (IC
50
for
gemcitabine is 0.042 ± 0.018 µM and for sarkostat it is 2.3 ± 0.2 µM in MIA PaCa-2
pancreatic cancer cells for 72 h MTT assay).
Inability to inhibit STAT3 phosphorylation is one of the reasons for resistance to
Src inhibitor, dasatinib therapy (Nagaraj et al., 2010). However, sarkostat can lead to a
significant decrease of STAT3 phosphorylation in a dose- and time-dependent manner
(Figure 5.2C, G, and Figure 5.3 C, E). Moreover, it can exert its effects in the presence
and absence of dasatinib (Figure 5.15). MIA PaCa-2 cells treated with sarkostat can
decrease pSTAT3 within 30 min of drug exposure and pSrc in 4 h. However, dasatinib
can decrease only pSrc and not pSTAT3 even after 12 h of drug exposure. Furthermore,
sarkostat when used in combination with dasatinib was able to decrease pSTAT3, this
will be advantageous in overcoming pSTAT3 mediated dasatinib resistance. This further
validates its potential to be used in resistant and non-resistant forms of cancer (Figure
5.15).
142

Gemcitabine (0.001 µM)
Sarkostat (0.1 µM)
Sequential treatment of MIA PaCa-2 cells with sarkostat and
gemcitabine at doses below their IC
50
resulted in higher
inhibition in cell proliferation as compared to individual
treatment. Cells were either pretreated with sarkostat (24 h)
followed by treatment with gemcitabine. Total duration for MTT
assay was 72 h.

Figure 5.14. Sarkostat shows additive/synergistic effect with
gemcitabine.
143

Unlike dasatinib, sarkostat can cause robust decrease in phosphorylation
of Src as well as STAT3. Cells were treated with increasing concentrations
of dasatinib (2 samples for each concentration) for 12 h. After this, one of
these sets was treated with sarkostat (5 µM) for 4 h. A third set of cells
was treated with sarkostat (5 µM) for 0.5 h to 4 h. In cells treated only
with dasatinib, there is clear inhibition of Src phosphorylation but no
effect on p-STAT3. However, when sarkostat was added to these cells, we
observed a complete inhibition of STAT3 phosphorylation.

Figure 5.15. Sarkostat overcomes drawbacks of current Src inhibitors
used as chemotherapeutic options for pancreatic cancer.
144
5.5. Sarkostat exerts ROS-mediated effects
STAT3 is one of the downstream targets of Src and is directly activated by Src.
Sarkostat induces a robust decrease in Src and STAT3 phosphorylation. In order to
determine if the decrease in STAT3 phosphorylation by sarkostat is only dependent on its
effects on Src, we knocked down Src and checked the effects of sarkostat on STAT3
phosphorylation. In MIA PaCa-2 cells, STAT3 is activated even in the absence of Src
(Figure 5.16A). Interestingly, sarkostat was able to decrease STAT3 phosphorylation in
Src knocked down MIA PaCa-2 pancreatic cancer cells (Figure 5.16A). Moreover,
sarkostat was able to decrease Src phosphorylation in STAT3 knocked down cells (Figure
5.16B) suggesting lack of feedback loop from STAT3 to Src. In a cell-free in vitro kinase
activity assay of 91 oncogenic kinases, sarkostat at 10 µM caused only <25% inhibition
or stimulation of the tested targets (Figure 5.17). These data suggest that the effect of
sarkostat on Src and STAT3 activation is mechanistically different from dasatinib.

(A). Sarkostat decreases
STAT3 phosphorylation in Src
knocked down MIA PaCa-2
cells (B). Sarkostat decreases
Src phosphorylation in STAT3
knocked down MIA PaCa-2
cells.
Figure 5.16. Effects of
sarkostat on STAT3 are
independent of its effects on
Src and vice versa.
145

We have previously shown that sarkostat can cause significant and rapid increase
in cellular content of reactive oxygen species. ROS are known to act as essential second
messengers in cells and can influence a myriad of signal transduction pathways. They are
known to inhibit Src/FAK signaling (Cunnick et al., 1998; Kemble and Sun, 2009; Tang
et al., 2005). Therefore, we analyzed the effects of sarkostat on Src, FAK and STAT3 in
the presence of antioxidants N-acetylcysteine (NAC) or glutathione (GSH). Cells were
Sarkostat (10 µM) does not significantly inhibit the activity of oncogenic kinases
in a kinase profiling in vitro substrate assay. Kinase Profiler
TM
assay was
performed by EMD Millipore, Billerica, MA. Concentration of ATP used in the
assay was Km for each kinase. 10 µM ATP was used for kinases for which Km
was not known.

Figure 5.17. Sarkostat does not cause any significant inhibition in activity of
oncogenic kinases in vitro.
146
either pretreated with an antioxidant (p) or the antioxidant was added together (t) with
sarkostat. Sarkostat was not able to decrease phosphorylation of Src, FAK or STAT3 in
MIA PaCa-2 cells pretreated or simultaneously treated with the antioxidants. However,
dasatinib was able to exert its effects on Src phosphorylation in the presence of
antioxidants (Figure 5.18). This suggests that sarkostat exerts ROS-mediated effects on
Src/FAK/STAT3 signaling in pancreatic cancer cells and its effects are distinctly
different from those of dasatinib.

147

Sequential or concomitant treatment with sarkostat in the
presence of antioxidant (NAC, N-acetylcysteine or GSH,
glutathione) blocked the ability of sarkostat to inhibit Src,
FAK or STAT3 phosphorylation. In contrast, NAC and
GSH treatment did not affect the ability of dasatinib to
inhibit Src phosphorylation. Cells were either pretreated
(p) for 2 h with an antioxidant (10 mM), washed with 1x
DPBS and then exposed to sarkostat (5 µM, 4 h) or
dasatinib (200 nM, 12 h), or were treated together (t) with
antioxidant (10 mM) and sarkostat (5 µM, 4 h) or
dasatinib (200 nM, 12h).

Figure 5.18. Sarkostat exhibits ROS-mediated effects
on cell signaling.
148
5.6. TP 421 decreases Src/FAK phosphorylation and inhibits cell
migration
Simultaneous studies with TP 421 showed that TP 421 decreases activating
phosphorylation of Src (Y416) (Figure 5.19A). Furthermore, it decreased FAK
phosphorylation at Y576 and Y861 that are downstream targets of activated Src (Figure
5.19B). Additionally, there was decrease in phosphorylation of p130Cas and paxillin
(Figure 5.19C). Moreover, there was also de-phosphoryaltion of Smads 1, 5 and 8 due to
TP 421 treatment (Figure 5.19D).
Furthermore, TP 421 inhibited cell migration in MIA PaCa-2 cells in a Boyden
Chamber assay. FBS was used to stimulate cell migration (Figure 5.19E). (This
difference was not due to cytotoxicity. Cells treated in parallel showed approximately
30% cell kill at 5 µM of TP 421). Lastly, TP 421 treatment potently inhibited wound
closure in PANC-1 pancreatic cancer cells (Figure 5.19 F) (Shabaik et al., 2013).

149

(A) MIA PaCa-2 cells treated with 5 µM TP 421 resulted in increase in non-
phospho Y416 (activating phosphorylation) in Src. (B) MIA PaCa-2 and BxPC-3
cells treated with 5 µM TP 421 resulted in decreased phosphorylation of FAK.
(C) TP 421 (5 µM) treatment caused decrease in phosphorylation of p130Cas and
paxillin proteins that are downstream of Src/FAK complex. (D) 5 µM TP 421
treatment decreases the phosphorylation of Smad1/5/8 in MIA PaCa-2 cells. (E)
TP 421 treatments resulted in decreased cell migration in serum starved MIA
PaCa-2 cells in a Boyden Chamber setup. (F) TP 421 inhibited wound closure in
PANC-1 cells (Shabaik et al., 2013).

Figure 5.19. TP 421 decreases Src/FAK phosphorylation and cell migration
in pancreatic cancer cells.
150
5.7. Discussion and conclusions
Pancreatic cancer is one of the deadliest and most aggressive diseases with
relatively poor prognosis and limited therapeutic options for most patients (N.I.H., 2013).
Therefore, there is a pressing need for more effective treatment strategies for this disease.
Expression and activation of Src and FAK kinases have been linked to
clinicopathological characteristics of pancreatic ductal adenocarcinoma (Chatzizacharias
et al., 2010; Lutz et al., 1998). Src/FAK act as a complex and regulate a myriad of
cellular events including cell proliferation, survival, adhesion, migration and invasion
linked to disease progression and metastasis (Bolos et al., 2010; Summy and Gallick,
2003). Therefore, targeting pathways leading to inhibition of Src/FAK complex and its
downstream effects is an attractive strategy for developing anticancer therapies.
Treatment of pancreatic cancer cells with sarkostat resulted in a robust decrease in
phosphorylation of Src/FAK complex at various phospho sites (Figure 5.2, Figures 5.3
and Figure 5.4). Sarkostat decreases phosphorylation of FAK at multiple sites including
autophosphorylated Y397 that acts as a docking site for Src (Figure 5.2A, F, G and
Figure 5.3A, D, E). Association of Src with FAK results in full activation of Src. In turn,
activated Src phosphorylates FAK at Y576/577 (catalytic domain) and Y925 (docking
site for growth-factor-receptor-bound protein 2, mediates RAS-MAPK signaling, and
Src-induced epithelial mesenchymal transition) (McLean et al., 2005). Sarkostat
decreases Src phosphorylation at Y416 (activating phosphorylation) and Src-directed
FAK phosphorylations at Y576/577 and Y925 (Figure 5.2A, B, F, G and Figure 5.3B, D,
E). Moreover, sarkostat decreases phosphorylation of FAK-Y861 (Figure 5.2A, F and
151
Figure 5.3D) that influences tumor vasculature by governing the interaction of FAK with
integrins and regulates FAK’s interaction with p130CAS that promotes cell invasion
(McLean et al., 2005). Decrease in FAK-861 phosphorylation by sarkostat results in
decreased phosphorylation of its substrate p130CAS (Figure 5.3D). Sarkostat also
decreases phosphorylation of paxillin (Figure 5.3D), a substrate of the Src/FAK complex
that acts as an adaptor for several proteins involved in cell adhesion (Bolos et al., 2010).
Furthermore, sarkostat causes a potent decrease in phosphorylation of Smads without
affecting total Smad levels in cells (Figure 5.3E). Smads play an important role in
governing epithelial mesenchymal transition and thus regulate cancer invasiveness
(Katsuno et al., 2008; Mohri et al., 2012).
As a result of the decrease in Src/FAK phosphorylation, morphological changes
were observed in cancer cells (Figure 5.5). These morphological changes were not
observed in normal fibroblasts suggesting that sarkostat exerts selective action on cancer
cells (Figure 5.6). Several studies have shown that cells lacking FAK expression migrate
poorly as compared to normal fibroblasts (Ilic et al., 1995; Owen et al., 1999; Sieg et al.,
2000). Sarkostat decreases Src/FAK activation (Figure 5.2, Figures 5.3 and Figure 5.4)
and hence inhibit cell migration in pancreatic cancer cells (Figure 5.7A, Figure 5.8,
Figure 5.9, Figure 5.10 and Figure 5.11). In addition, sarkostat inhibits tube formation in
HUVECs suggesting its potential to inhibit angiogenesis (Figure 5.13).
A major limitation of pancreatic cancer therapy is the development of resistance to
currently available therapies including gemcitabine. Src expression and activation
increases from normal pancreas to pancreatitis to pancreatic adenocarcinoma in
152
accordance with its role in cancer progression and metastasis (Hilbig, 2008; Lutz et al.,
1998; Summy and Gallick, 2003). Decreasing Src/FAK signaling has been shown to
restore sensitivity to gemcitabine and 5-fluorouracil in pancreatic cancer cells. (Duxbury
et al., 2004a, b; Ischenko et al., 2008). Our results demonstrate that sarkostat shows
additive effects with gemcitabine in pancreatic cancer cells at concentrations lower than
their respective IC
50
values (Figure 5.14). Therefore, sarkostat might have an added
benefit of overcoming resistance to gemcitabine.
Src inhibitors like dasatinib are being developed for treatment of pancreatic
cancer but they have limited single agent efficacy in clinical settings (ClinicalTrials.gov,
2013a, b, c, d). Recent studies have shown that dasatinib- resistant pancreatic cancer cells
have activated STAT3 (Nagaraj et al., 2010). Additionally, STAT3 reactivation has been
linked with sustained Src inhibition in other types of cancer (Sen et al., 2009). Our
studies demonstrate that sarkostat can inhibit both STAT3 and Src phosphorylation, and
activation of STAT3 is also inhibited in the absence of Src (Figure 5.2, Figure 5.3, Figure
5.4 and Figure 5.16A). Therefore, sarkostat treatment might be beneficial in resistant
forms of pancreatic cancer exhibiting STAT3 activation. In addition to the effects on
pancreatic cancer cells, sarkostat inhibits tube formation in HUVEC cells (Figure 5.13)
suggesting a possible effect in blockade of tumor angiogenesis. This two-pronged effect
in tumor and endothelial cells could result in improved efficacy by a single agent.
Sustained Src inhibition results in STAT3 activation by induction of a
homeostatic feedback loop leading to Src inhibitor resistance (Sen et al., 2009).
Conversely, complete inhibition of p-STAT3 results in Src activation as a result of
153
induction of homoeostatic feedback loop (Byers et al., 2009). More importantly,
simultaneous blockade of Src and STAT3 activation has been shown to be a promising
therapeutic approach for several forms of cancer including pancreatic cancer (Nagaraj et
al., 2011). Our novel inhibitor, sarkostat, decreases STAT3 phosphorylation in Src
knockdown cells and decreases Src phosphorylation in STAT3 knockdown cells (Figure
5.16). Targeting three oncoproteins (Src/FAK and STAT3) and potentially overcoming
chemoresistance by a single agent may lead to improved treatment for pancreatic cancer.
Our data demonstrates that the inhibition of the Src/FAK, and STAT3 by
sarkostat is mediated through ROS production and not due to direct inhibition of kinase
activity (Figure 5.17 and Figure 5.18). ROS regulate several cellular events by governing
various signal transduction pathways. For example, the natural product manumycin
inhibits STAT3 by elevating intracellular ROS in glioma cells (Dixit et al., 2009).
Similarly, phenethyl isothiocyanate inhibits STAT3 activation in prostate cancer cells by
generation of ROS (Gong et al., 2009). STAT3 deletion itself can sensitize cancer cells to
oxidative stress (Barry et al., 2009). Additionally, inactivation of Src family tyrosine
kinases by ROS has also been reported (Cunnick et al., 1998; Kemble and Sun, 2009;
Tang et al., 2005). Previously, we have reported that sarkostat treatment causes
immediate and significant increase in cellular ROS production. In this study, we
demonstrate that treatment with sarkostat in the presence of antioxidants did not result in
decreased STAT3, Src or FAK phosphorylation (Figure 5.18) suggesting that generation
of ROS is essential for sarkostat activity. Additionally, TP compounds can also decrease
Src/FAK pathway and inhibit cell migration and invasion (Figure 5.19).
154
In conclusion, we have discovered a novel small molecule compound that exerts
ROS-mediated decrease in Src/FAK and STAT3 phosphorylation resulting in efficient
inhibition of cell proliferation, adhesion and migration, ultimately leading to pancreatic
cancer cell death by apoptosis. Although drugs that individually and selectively inhibit
Src, FAK or STAT3 are under development, when used as single agent they tend to
develop resistance. For example, sustained inhibition of Src by dasatinib results in
resistance mediated by STAT3 activation. Discovery of a single agent blocking the
activation all these three critical signaling pathways simultaneously will significantly
contribute to efficient treatment and overcome resistance. Therefore, sarkostat can serve
not only as a unique pharmacological probe but can also be developed into an effective
therapeutic option for pancreatic cancer as a single agent or in combination with
chemotherapy.

155
CHAPTER SIX:
NOVEL MITOCHONDRIAL-TARGETED SMALL
MOLECULES AS THERAPIES FOR CHEMOTHERAPY
INDUCED PERIPHERAL NEUROPATHY
6.1 Introduction
6.1.1 Background
Chemotherapy-induced peripheral neuropathy (CIPN) is the key dose-limiting
toxicity of clinically used antineoplastic agents. The occurrence of CIPN ranges from
10% to 100% depending on the chemotherapeutic agent being used (Balayssac et al.;
Wilkes, 2007). The major neurotoxic chemotherapeutics include platinum compounds,
taxanes, vinca alkaloids, epothilones, bortezomib and thalidomide. These drugs are
widely used to treat several forms of cancer. But, incidence of CIPN restricts their
therapeutic utility. Therefore, there is an unmet urgent need of neuroprotective strategies
for preventing and treating CIPN. However, there is not enough clinical data to validate
the use of currently available neuroprotectants for CIPN (Wolf et al., 2008). This limits
the neuroprotective therapeutic options to treatment modifications or therapy
discontinuation of the chemodrugs. However, in case of platinum compounds the
neuropathic symptoms exacerbate months after the treatment withdrawal. Pre-existent
neuropathy due to diabetes or tumor itself, and combined use of these neurotoxic
chemotherapeutics further complicates the situation. Hence, there is an unmet urgent
need to develop effective ways for prophylaxis and treatment of CIPN.
156
6.1.2. Rationale
Mitochondrial dysfunction and the ensuing oxidative stress have been implicated
in CIPN resulting from several drugs including taxanes, platinum compounds and
bortezomib. Therefore, redox and/or energy modulators selectively targeting
mitochondria will provide efficient treatment and prophylactic options for CIPN (Figure
6.1). This hypothesis is based on the studies that show:
A. Platinum drugs can induce oxidative stress and mitochondrial dysregulation.
B. Excessive oxidative stress and altered mitochondrial function results in neuronal
apoptosis.
C. Paclitaxel treatment induces swollen and vacuolated axonal mitochondria in A-fibers
and C-fibers (Flatters and Bennett, 2006).
D. Bortezomib promotes oxidative stress and mitochondrial dysfunction, leading to
neuronal apoptosis and neurotoxicity (McDonald and Windebank, 2002; Zhang et al.,
2007).
E. Bortezomib can trigger mitochondrial damage, and dysregulation of neutrophins by
activating mitochondrion-mediated apoptosis or inhibition of nerve growth factor-
directed neuronal survival (Landowski et al., 2005; Montagut et al., 2006).
F. Several antioxidants and mitochondrial protectants have showed neuroprotective
potential (Ames and Liu, 2004; De Grandis, 2007; Fidanboylu et al.; Jin et al., 2008;
Wolf et al., 2008).

157

6.1.3. Mitochondrial targeting of novel redox and energy modulating
triphenylphosphonium (TP) conjugates
Several chemotherapeutic agents including platinum compounds, taxanes and
bortezomib alter mitochondrial functions in peripheral neurons and result in CIPN.
Mitochondrial dysfunction and oxidative stress have been implicated in most
neurodegenerative diseases. Platinum compounds have been reported to induce oxidative
stress and alter mitochondrial functions. Increased oxidative stress and dysregulated
mitochondrial function promote neuronal apoptosis. Moreover, paclitaxel increases the
Oxidative stress and mitochondrial dysfunction have been implicated in most
neurodegenerative diseases. (Left). Several chemotherapeutic agents can induce
oxidative stress and alter mitochondrial functions. Increased oxidative stress and
dysregulated mitochondrial function promote neuronal apoptosis and in turn
produce neurotoxicity. Mitochondria are the major site of oxidative stress and
play a crucial role in apoptosis. Therefore, mitochondrially targeted novel
energy/redox modulating agents will offer neuroprotective effects in
chemotherapy induced peripheral neuropathy (Right). These agents will decrease
oxidative stress, restore mitochondrial function and basal reactive oxygen species
production in neurons.
Figure 6.1. Hypothesis of this study.
158
appearance of swollen and vacuolated axonal mitochondria in A-fibers and C-fibers
(Flatters and Bennett, 2006).
Furthermore, bortezomib can induce oxidative stress and mitochondrial
dysfunction, thereby triggering neuronal apoptosis and neurotoxicity (McDonald and
Windebank, 2002; Zhang et al., 2007). Bortezomib can also result in dysregulation of
neutrophins. Bortezomib induces neuronal cell death by activating mitochondria-
mediated apoptosis or inhibiting nerve growth factor-directed neuronal survival
(Landowski et al., 2005; Montagut et al., 2006). Various protective agents such as lipoic
acid, acetyl-L-carnitine, N-acetyl cysteine, and vitamin E have been evaluated for
prophylaxis or treatment of CIPN (De Grandis, 2007; Fidanboylu et al.). Studies have
demonstrated that acetyl-L-carnitine prevents the occurrence of paclitaxel induced
increase in swollen and vacuolated C-fiber mitochondria (Ames and Liu, 2004; Jin et al.,
2008). Peripheral neuropathy can be controlled by decreasing activity of enzymes
involved in DNA base excision repair, oxidative damage repair and redox regulation
(Jiang et al., 2008). Since mitochondria are effective cellular sources of oxidants and play
a crucial role in apoptosis, it is imperative to develop neuroprotective strategies targeting
mitochondria.
Using triphenylphosphonium (TPP or TP) salts to target small molecules to
mitochondria seems a promising approach. The presence of the lipophilic TPP moiety
increases the uptake and localization of the attached cargo to the mitochondria. This
occurs mainly due to the presence of an electrochemical potential across the
mitochondrial membrane. The mitochondrial electron transport chain generates a proton
159
gradient that is used to produce ATP by the ATP synthase machinery. Therefore, there is
a negative potential of 150-160 mV (negative inside) across inner mitochondrial
membrane. This, in combination with the plasma membrane potential of 30-60mV
(negative inside), directs the cations to mitochondria (Figure 6.2).
Despite the fact that many charged molecules poorly cross cell membranes
without the aid of transporter proteins, the charge distribution across the large lipophilic
surface of the phosphonium ion significantly lowers activation energy of desolvation,
thereby facilitating the passage of the TP conjugate across lipid membranes. The high
negative membrane potential of mitochondria is an important driving force that directs
the accumulation of the TPP salts within mitochondria (Ross et al., 2005).
TPP compounds undergo membrane potential-driven rapid uptake in
mitochondria within minutes. The uptake of TPP cations into mitochondria is self-
limiting due to their equilibration between the extracellular environment and the
mitochondria. As the levels of the TPP conjugates decrease in the extracellular fluid due
to excretion, the compounds redistribute from the mitochondria and cytoplasm into the
extracellular fluid and are excreted out. This prevents extensive accumulation of
lipophilic cations within mitochondria in vivo and thus decreases the incidence of
unacceptable toxicity. Furthermore, studies have revealed that TPP administration is safe
in humans and does not exert deleterious effects during long-term use, and thus provide
an efficient way to deliver drugs to mitochondria in vivo (Porteous et al., 2010;
Rodriguez-Cuenca et al., 2010; Smith et al., 2011; Smith et al., 2003).

160

6.2. Synthesis of mitochondrial-targeted redox modulators
CIPN is the key dose limiting side effect of a significant number of
chemotherapeutic agents. Mitochondrial-targeted energy or redox modulators can exert
protective/prophylactic effects for CIPN by altering mitochondrial functions and
maintaining cellular redox balance (Table 6.1).
(A).Cell membranes and inner mitochondrial membranes have a negative charge
of 30-60mV and 150-160mV respectively which direct the TPP cations to
mitochondria. (B). TP 421 is a coumarin containing fluorescent probe designed by
our lab to study mitochondrial translocation of TPP cations. (C). TP 421 (blue)
co-localizes with mitotracker red (red) in cells suggesting its mitochondrial
accumulation (Millard et al., 2010; Murphy, 2008; Shabaik et al., 2013).
Figure 6.2. Triphenylphosphonium moiety directs compounds to
mitochondria.
161

Table 6.1. TPP conjugates and their expected effects on mitochondria.

Lipoic acid-TPP conjugate (Mito-LA) was synthesized from the commercially
available (R)-α-lipoic acid and the readily made (3-aminopropyl) triphenylphosphonium
bromide hydrobromide in one step as outlined in Scheme 6.1.

Synthesis of Mito-NAC was achieved from the commercially available N-Boc-S-
Tritylcysteine (1) via an amide coupling step with the readily made (3-aminopropyl)
triphenylphosphonium bromide hydrobromide (TPP-Amine, 2) to give the intermediate 3.
Removal of Boc group in 3 and subsequent acetylation results in the formation of the
tritylated compound 4. Deprotection of the trityl group under acidic conditions yields
Mito-NAC (Scheme 6.2).

Cargo Conjugate Redox active Energy-modulating
N-acetyl
cysteine
Mito-NAC •
Lipoic acid Mito-LA • •
1. HBTU,DIEA,DMF
2. TPP-Amine,DMF
N
H
PPh
3
Br
O
OH
S
S
O
S
S
HBr‡NH
2
PPh
3
Br
HBr‡NH
2
Br PPh
3 +
Acetonitrile, reflux, 16 h
TPP-Amine
46%
80%
Lipoic Acid
Mito-LA
Scheme 6.1. Preparation of R-α-lipoic acid-TPP conjugate (Mito-LA).
162

6.3. Mito-LA and Mito-NAC do not interfere with the anticancer effects
of cisplatin
Mito-LA and Mito-NAC were designed to overcome the serious side effect of
CIPN associated with several chemodrugs. Therefore, they should abrogate the
undesirable side effects without affecting the chemotherapeutic potency of the
chemodrugs. Hence, we tested the efficacy of cisplatin (representative chemodrug) in the
presence of Mito-LA and Mito-NAC.
24 h or 4 h pre-treatment with Mito-LA does not affect the anticancer effects of
cisplatin. OVCAR8 ovarian cancer cells were treated with Mito-LA (20 µM) for 24 h or 4
h followed by subsequent treatment with cisplatin at increasing concentrations. Mito-LA
does not diminish the anticancer effects of cisplatin. Moreover, Mito-LA does not induce
any cytotoxic effects on its own (Figure 6.3). Mito-NAC also does not induce any
cytotoxic effects. Furthermore, Mito-NAC does not interfere with the anticancer effects
of cisplatin in OVCAR8 ovarian cancer cells Pretreatment of OVCAR-8 cells with Mito-
BocHN
H
N PPh
3
O
Ph
3
CS
Br
BocHN
OH
O
Ph
3
CS
H
3
N PPh
3
Br Br
DCM, rt
+
HBtU, DIPEA
AcNH
H
N PPh
3
O
Ph
3
CS
Br
AcHN
H
N PPh
3
O
HS
Br
1) TFA, r.t.
2) Ac
2
O/Pyridine
33% HBr/AcOH, r.t.
Mito-NAC
1
2: TPP-Amine
3
4
Scheme 6.2. Preparation of TPP conjugated N-acetylcysteine (Mito-NAC).
163
NAC (10 µM, 25 µM, 50 µM or 100 µM) for 4 h did not decrease the anticancer effects
of cisplatin on the cells (Figure 6.4).

B.
A.
Cells were pretreated with Mito-LA (20 µM) for 4 h or 24 h, followed by
treatment with increasing concentrations of cisplatin. Total treatment time was 72
h. Mito-LA does not exert any toxicity (green bars). X-axis shows cisplatin
concentrations.

Figure 6.3. Mito-LA does not interfere with the anticancer effects of cisplatin.
164

6.4. Mito-LA does not affect the respiratory capacity of cancer cells
Treatment with Mito-LA does not result in any change in the respiratory capacity
of cancer cells. Cellular oxygen consumption rate was measured in OVCAR8 ovarian
cancer cells either after pre-treating them with Mito-LA (20 µM) overnight or by
measuring the effects in real time for 2 h. Mito-LA did not influence the cellular oxygen
consumption. Moreover, cisplatin (36 µM) did not affect the cellular OCR by itself or
after pretreatment with Mito-LA. Furthermore, mitochondrial inhibitors (oligomycin,
FCCP, rotenone) were able to alter OCR in cells treated with Mito-LA or cisplatin or
Mito-NAC (µM)
Cells were pretreated with Mito-NAC (10, 25, 50 and 100 µM) for 4 h,
followed by treatment with increasing concentrations of cisplatin (10, 25 and
50 µM). Total treatment time was 72 h. Mito-NAC does not exert any
toxicity. X-axis shows cisplatin concentrations.

Figure 6.4. Mito-NAC does not interfere with the anticancer effects of
cisplatin.
165
both, suggesting responsive and functional mitochondria in cells treated with Mito-LA,
cisplatin or both (Figure 6.5).

A.
B.
Cancer cells pretreated with Mito-LA (20 µM) overnight or in real time for
2 h did not change the cellular oxygen consumption. Moreover, cisplatin
(36 µM) did not affect the cellular OCR by itself or after pretreatment with
Mito-LA. (oligomycin, 0.005 mg/mL; FCCP, 1 µM; rotenone, 1 µM). 1-
Basal, 2-DMEM/ Drug, 3-Oligomycin, 4-FCCP, 5-Rotenone

Figure 6.5. Mito-LA does not affect the respiratory capacity of cancer
cells.
166
6.5. Mito-LA and Mito-NAC decrease cisplatin induced mitochondrial
superoxide production
Cisplatin increases mitochondrial oxidative stress by increasing mitochondrial
superoxide generation. 2 h treatment with Mito-LA or Mito-NAC decreases cisplatin
induced mitochondrial superoxide production in OVCAR8 ovarian cancer cells
(pretreated with cisplatin for 24 h) more potently than their respective parent moieties
(Figure 6.6 and Figure 6.7). 100 µM of Mito-LA was sufficient to inhibit the oxidative
stress induced by 25 and 100 µM of cisplatin (Figure 6.6A). However, 1 mM of lipoic
acid was required to have similar effects in OVCAR8 cells (Figure 6.8B). Similarly, 100
µM of Mito-NAC was better than NAC (100 µM) in decreasing the oxidative stress
induced by cisplatin treatment (Figure 6.7). This suggests that mitochdonrial targeting of
redox and/or energy modulators is more effective in decreasing mitochondrial oxidative
stress as compared to non-targeted counterparts.

167

1-Control
2-Cisplatin (25 µM)
3-Cisplatin (50 µM)
4-Lipoic Acid (100 µM)
5-Cisplatin (25 µM) + Lipoic Acid (100 µM)
6-Cisplatin (50 µM) + Lipoic Acid (100 µM)
7-Lipoic Acid (1 mM)
8-Cisplatin (25 µM) +Lipoic Acid (1 mM)
9-Cisplatin (50 µM) + Lipoic Acid (1 mM)
B.
1-Control
2-Mito-LA (100 µM)
3-Cisplatin (25 µM)
4-Cisplatin (25 µM) + Mito-LA (100 µM)
5-Cisplatin (50 µM)
6-Cisplatin (50 µM) + Mito-LA (100 µM)
A.
Mitochondrial superoxide production was evaluated by measuring MitoSOX
red fluorescence by flow cytometry. MitoSOX red is a probe that becomes
fluorescent after being specifically oxidized by mitochondrial superoxide.

Figure 6.6. Mito-LA decreases cisplatin induced mitochondrial
superoxide production more potently than lipoic acid.

168

6.6. Discussion and conclusions
Incidence of CIPN in cancer patients can range from 10% - 100%. Neuropathic
pain can occur due to several reasons including the tumor itself, chemotherapy or any
pre-existing condition. Tumor can grow in or around a nerve leading to nerve
compression through mechanical distortion, exposure to inflammatory
1-Control
2-Cisplatin (50 µM)
3-Mito-NAC (10 µM)
4-Mito-NAC (25 µM)
5-Mito-NAC (100 µM)
6-Mito-NAC (10 µM) + Cisplatin (50 µM)
7-Mito-NAC (25 µM) + Cisplatin (50 µM)
8-Mito-NAC (100 µM) + Cisplatin (50 µM)
9-NAC (10 µM)
10-NAC (25 µM)
11-NAC(100 µM)
12-NAC (10 µM) + Cisplatin (50 µM)
13-NAC(25 µM) + Cisplatin (50 µM)
14-NAC(100 µM) + Cisplatin (50 µM)
Mitochondrial superoxide production was evaluated by
measuring MitoSOX red fluorescence by flow cytometry.

Figure 6.7. Mito-NAC decreases cisplatin induced
mitochondrial superoxide production.

169
microenvironment, and loss of blood supply resulting in ischemia. Moreover, anticancer
therapeutics, mainly chemotherapy can induce or exacerbate pre-existent neuropathy.
Therefore, there is an unmet clinical need to develop protective agents to improve the
quality of life of patients suffering from CIPN.
We have synthesized and evaluated the protective capacity of novel
mitochondrial targeted redox and energy modulators using triphenylphosphine (TPP)
conjugates. Our results indicate that Mito-LA and Mito-NAC (Mito-Lipoic acid or Mito-
N-acetyl cysteine with lipoic acid or N-acetyl cysteine, respectively conjugated to TPP
for selective delivery to mitochondria), does not interfere with the anticancer effects of
chemotherapeutics like cisplatin. Moreover, these compounds are not cytotoxic.
However, they decrease the oxidative stress induced by chemodrugs. Additionally, these
compounds are more potent than their parent counterparts in decreasing the
chemotherapy induced oxidative stress.
This study provides an innovative strategy for treatment and/or prevention of
CIPN that mainly occurs due to mitochondrial dysfunction and oxidative stress.
Therefore, targeting the redox or energy modulators at the source of trouble seems to be a
rational approach. Addition of TPP helps in targeting these compounds to mitochondria.
TPP conjugation does not affect the activity of these agents as they still retain their
antioxidant effects. We have tested a large collection of TPP compounds for their
cytotoxicity and effects on mitochondria. Only a few TPP compounds tested by us were
cytotoxic, suggesting that addition of TPP does not confer cytotoxic properties to a
compound (Millard et al., 2010).
170
The redox-active compound NAC exerts its antioxidant activity by one-electron
transfers and reacts with a variety of free radicals. Delayed treatment with N-acetyl
cysteine has shown to protect Schwann cells without compromising the anti-myeloma
activity of bortezomib (Nakano et al., 2011). Use of N-acetyl cysteine has been shown to
exert protective effects in initial clinical trials (Pachman et al., 2011).
The energy-modulating compound mentioned herein, lipoic acid can also display
modest free-radical quenching activity. However, its major function is as a cofactor of the
pyruvate dehydrogenase and a-ketoglutarate dehydrogenase complexes. Lipoic acid, due
to its ability to undergo intramolecular disulfide bond formation can serve both as redox-
and energy-modulating compound.
The results presented herein are only in cancer cells. The neuroprotective
evaluation of these compounds is required for taking them forward for CIPN therapy
(details are mentioned in future perspectives).

171
CHAPTER SEVEN:
CONCLUDING REMARKS AND FUTURE
PERSPECTIVES
7.1. Concluding remarks
Pancreatic cancer is a complex disorder characterized by oxidative stress and
alterations in signaling proteins of several signal transduction pathways including
Src/FAK mediated cell signaling. We have identified two novel classes of compounds
that exert differing effects on cellular bioenergetics of cancer cells. Quinazoline-5,8-
diones induce a significant increase in cellular oxygen consumption rate with a
concomitant increase in superoxide production. While the other class of compounds,
triphenylphosphoniums decrease cellular oxygen consumption, it also increased the
superoxide content in the cancer cells. Triphenylphosphonium salts are selectively
targeted to mitochondria (Murphy, 2008). This selectivity might be the cause of
dissimilar nature of ROS production by TPs and 3b. Superoxide produced by compound
3b and its analogues can be detected by both cytochrome c and MitoSOX based assays
indicating both cellular and mitochondrial superoxide generation. However, for TP
compounds significant increase in superoxide production was assessed by MitoSOX
assay but not cytochrome c assay. This suggests that TP compounds mainly induce
superoxide generation in the mitochondria which is their site of localization.
Our discovery of the novel quinazoline-5,8-diones serve as an example of
exploiting Akt-directed ROS-mediated cell death mechanisms. Akt is a pro-survival
172
kinase that is upregulated in several forms of human cancers. Hyperactive Akt inhibits
apoptosis induced by numerous stimuli. However, Akt is unable to inhibit ROS-mediated
cell death. Furthermore, Akt aids in ROS-directed cell death by inducing cellular oxygen
consumption, promoting ROS generation, and impairing ROS degeneration. Akt mediates
decreased ROS scavenging by phosphorylating the transcription factor FoxO causing its
inactivation. This leads to decreased expression of its target antioxidant enzymes
(MnSOD and catalase). Therefore, hyperactivated Akt sensitizes cancer cells to ROS-
directed cell death (Nogueira et al., 2008). Parthenolide, a sesquiterpene lactone, has been
shown to induce Akt-directed ROS-mediated cell death in prostrate cancer cells (Sun et
al., 2010). Compound 3b increases Akt phosphorylation causing a decrease in FoxO3a
activity that in turn reduces the cellular pool of antioxidants resulting in excessive
oxidative stress in cancer cells. The mechanism of action of TP compounds seems to be
independent of Akt. Since the site of targeting of TP compounds is mitochondria they
might be targeting one of the complexes in mitochondrial respiratory chain and impairing
its function with resultant increase in mitochondrial superoxide generation.
Both TPs and quinazoline-5,8-diones activate the stress kinase pathways resulting
in switching on the apoptotic machinery for cell death. Compound 3b activates stress
signaling, induces DNA damage and activates FAS-mediated apoptotic cell death (Figure
7.1).

173

Anticancer mechanistic studies with compound 3b (sarkostat) revealed that
besides inducing oxidative stress it also inhibits activity of Src/FAK complex. Inhibition
of Src/FAK resulted in decrease in cell migration and invasion. A major limitation of
pancreatic cancer therapy is the development of resistance to currently available therapies
including gemcitabine. Src expression and activation increases from normal pancreas to
pancreatitis to pancreatic adenocarcinoma in accordance with its role in cancer
progression and metastasis (Hilbig, 2008; Lutz et al., 1998; Summy and Gallick, 2003).
Decreasing Src/FAK signaling has been shown to restore sensitivity to gemcitabine and
Compound 3b increases cellular oxygen consumption and oxidative stress
through Akt-directed mechanisms. Oxidative stress results in activation of stress
kinases (JNK and p38), DNA damage, increased expression of FASL, activation
of FAS receptor and its downstream caspases resulting in cancer cell death.
Figure 7.1. Compound 3b activates stress kinases and induces cell death in
pancreatic cancer cells.
174
5-fluorouracil in pancreatic cancer cells. (Duxbury et al., 2004a, b; Ischenko et al., 2008).
Treatment of compound 3b with gemcitabine has shown slight additive benefit indicating
that Src/FAK inhibition increases sensitization of pancreatic cancer towards gemcitabine.
Moreover, it is also active in gemcitabine, and gemcitabine and erlotinib resistant MIA
PaCa-2 pancreatic cancer cells.
Src inhibitors like dasatinib are being developed for treatment of pancreatic
cancer but they have limited single agent efficacy in clinical settings (ClinicalTrials.gov,
2013a, b, c, d). Recent studies have shown that dasatinib- resistant pancreatic cancer cells
have activated STAT3 (Nagaraj et al., 2010). Additionally, STAT3 reactivation has been
linked with sustained Src inhibition in other types of cancer (Sen et al., 2009). Our
studies demonstrate that compound 3b can inhibit both STAT3 and Src phosphorylation,
and activation of STAT3 is also inhibited in the absence of Src. Therefore, compound 3b
treatment might be beneficial in resistant forms of pancreatic cancer exhibiting STAT3
activation. In addition to the effects on pancreatic cancer cells, sarkostat inhibits tube
formation in HUVECs suggesting a possible effect in blockade of tumor angiogenesis.
This dual effect in tumor and endothelial cells could result in improved efficacy by a
single agent.
Furthermore, our data demonstrates that the inhibition of the Src/FAK, and
STAT3 by compound 3b is mediated through ROS production and not due to direct
inhibition of kinase activity (Figure 7.2). Targeting three oncoproteins (Src/FAK and
STAT3) and potentially overcoming chemoresistance by a single agent may lead to
improved treatment for pancreatic cancer.
175

ROS have also been implicated in chemotherapy induced peripheral neuropathy
(CIPN). Mitochondrial dysfunction and oxidative stress are the driving force behind
development of CIPN. We have designed novel mitochondrial targeted redox and/ or
energy modulators that exert antioxidant effects at the site of damage i.e. mitochondria.
These agents can have major impact in treatment and prophylaxis of CIPN. These agents
effectively and specifically deliver the protective cargo to mitochondria, which is a major
damage site for CIPN. Our initial studies with Mito-LA and Mito-NAC have shown to
decrease oxidative stress induced by chemotherapeutic agents like cisplatin. However, we
still need to assess the neuroprotective effects of these compounds (discussed under
future perspectives).
Sustained Src-inhibition by dasatinib results in STAT-3 reactivation and induces
resistance to Src inhibitors. Sarkostat through ROS induction causes inhibition of
Src, FAK and STAT3 activation leading to decreased cell proliferation, adhesion
and migration followed by apoptotic cell death.

Figure 7.2. Proposed mechanism of action of sarkostat (3b).
176
In conclusion, we have identified novel classes of compounds,
triphenylphosphoniums and quinazoline-5,8-diones that increase cellular oxidative stress,
activate stress kinase pathways and causes DNA damage ultimately leading to cancer cell
death. Moreover, these compounds arrest cell cycle progression in pancreatic cancer
cells. Additionally, these compounds inhibit the activity of Src/FAK complex resulting in
decreased cell migration and invasion. We have also studied CIPN, one of the bystander
effects of ROS in chemotherapy. We propose use of novel mitochondrial targeted redox
and/or energy modulators for effective treatment of CIPN.
7.2. Future perspectives
7.2.1. Triphenylphosphoniums
TP compounds are mitochondrial targeted and hence their target in all probability
should be a mitochondrial protein. DARTS (drug affinity responsive target stability)
assay could be utilized for identifying the exact molecular target of TP compounds.
DARTS assay is based on the principle that binding of the drug to its target protein
protects the latter from proteolytic damage (Lomenick et al., 2009).
7.2.2. Quinazoline-5,8-diones
The exact target identification of sarkostat (3b) and its analogues can be
performed by using DARTS assay. Moreover, activity assays can be used to further
validate the target.
7.2.3. Chemotherapy induced peripheral neuropathy
We have designed mitochondrial targeted novel redox and/or energy modulating
agents for prophylaxis and/or treatment of chemotherapy induced peripheral neuropathy.
177
We have assessed the antioxidant capacity of these compounds. Moreover, these
compounds are not cytotoxic and do not interfere with the anticancer effects of
chemotherapeutic agents. However, these compounds should be assessed for their
neuroprotective effects (Table 7.1)

Table 7.1. Summary of experiments for assessing neuroprotective effects.
Assay Rationale/Method Potential outcome
Axon
outgrowth
Neurons will be pretreated with
protective agents for 3-4 h, and then
treated with chemotherapeutic agent for
further 24 h. Following which, cells will
be fixed and stained. Analysis will be
done with respect to four parameters:
Cell number (decrease in cell number is
an indicator of toxicity)
Neurite number (chemicals disrupting
molecular signaling during neuronal
differentiation may interrupt neurite
initiation)
Neurite length (neurite elaboration or
axonal degeneration may result in
changes in neurite length)
% Cells with neurites (it is an indication
of effect on neurite outgrowth)
Little or no effect on
cell number

No decrease in %
cells with neurites as
compared to control
cells
AnnexinV
assay
Neurons pretreated with TP compounds
followed by chemotherapeutic agents
will be assessed in AnnexinV assay
No cytotoxic effects
in neurons

7.2.4. Newer avenues for our novel redox modulating compounds
Redox modulation is an important characteristic of several diseases. One such
example is provided in our study which is mentioned herein. Oxidative stress in cells
results in the transformation of normal cells to malignant cancer cells. Due to higher
basal oxidative stress levels in cancer cells they become more prone to exogenous
178
oxidative insults that mediate anticancer effects. TPs and novel quinaoline-5,8-diones
tend to exploit this characteristic feature of cancer cells and exert their anticancer effects.
Rationally designed analogues of these compounds can be used for treatment or
prophylaxis of several other redox regulated disorders.
Development of multimodal drugs can alleviate symptoms of multiple diseases. It
is very attractive to develop one drug for several diseases. This provides several
advantages. It reduces the drug development time and is also cost effective. Drug proved
safe in one preclinical study will not have to undergo the similar studies when it will be
developed for other indications.
Drug repurposing/repositioning refers to the development of novel uses of known
drugs for new indications. Drug repurposing can be divided into two categories. The first
class involves compounds which have multiple targets. In this case usually the ‘off-
target’ effects of a drug leads to its development for a new disease. Hence, undesirable
side effects of a compound in one case can offer beneficial effects in some other diseased
condition. This phenomenon is referred to as “known compound-new target’ drug
development approach. For example, thalidomide exerted undesirable side effects in
children of pregnant women who were taking it for nausea and vomiting. It was later
discovered that thalidomide has immuno-modulatory effects, and is now marketed as an
anti-leprotic drug by Celgene. The other category of drug repurposing includes
compounds which are beneficial in different indications by exerting same mechanism of
action. This is known as “known target-new indication” or “known mechanism-new
indication”. Merck’s finasteride (Proscar) is an example of this category of repurposing
179
approach. It was originally prescribed for treating prostate enlargement. However, its side
effect of preventing male baldness led to its development for the treatment of hair loss in
men (Figure 7.3) (Grau, 2005).

We can develop analogues of sarkostat for different ailments. We have shown that
sarkostat results in tremendous increase in cellular oxygen consumption. Additionally,
increased cellular oxygen consumption is an indicator of aerobic and metabolic capacity
of cells. Drugs like resveratrol improve cellular aerobic and metabolic capacity, and are
useful for preventing aging and obesity (Kim et al., 2007; Murase et al., 2009).
Furthermore, resveratrol protects from heart diseases, neuro-degeneration, insulin
resistance and cancer (Dirks Naylor, 2009; Kim et al., 2007; Liu et al., 2007). Resveratrol
and lipoic acid have both pro- and anti-oxidant effects (de la Lastra and Villegas, 2007;
Moini et al., 2002). Therefore, analogues of sarkostat can be developed to treat diseased
conditions like diabetes, obesity, cardiovascular disorders, neuronal disorders, cancer and
aging (Figure 7.4).

(Adapted from CombinatoRx)
Figure 7.3. Two approaches for drug repurposing.
180

Furthermore, we can develop non-toxic Mito-sarkostat compound that could
specifically increase mitochondrial oxygen consumption and could improve
mitochondrial functioning.
Secondly, we can use oxidative stress inducers TPs and sarkostat in combination
with other novel therapeutic approaches like photodynamic therapy or
chemophototherapy. Furthermore, these compounds can be tested for their photodynamic
We have shown that sarkostat results in tremendous increases in cellular oxygen
consumption. Additionally, increased cellular oxygen consumption is an indicator
of aerobic and metabolic capacity of cells. Drugs like resveratrol improve cellular
aerobic and metabolic capacity, and are useful for preventing aging, and obesity
(Kim et al., 2007; Murase et al., 2009). Furthermore, resveratrol protects from
heart diseases, neuro-degeneration, insulin resistance and cancer (Dirks Naylor,
2009; Kim et al., 2007; Liu et al., 2007). Resveratrol and lipoic acid have both
pro- and anti-oxidant effects (de la Lastra and Villegas, 2007; Moini et al., 2002).
Therefore, analogues of sarkostat can be developed to treat diseased conditions
like diabetes, obesity, cardiovascular disorders, neuronal disorders, cancer and
aging.
Figure 7.4. Drug repurposing for sarkostat.
181
potential. (Photodynamic therapy refers to using a drug that acts a photosensitizer or
photosensitizing agent at a particular wavelength of light. When such compounds are
exposed to their specific wavelength they produce reactive oxygen species).
Another possible area of exploration for these compounds could be assessing their
by-stander effect. For example, recent studies have demonstrated that paclitaxel activates
plasma membrane localized NAD(P)H oxidase and produces reactive oxygen species.
These ROS are majorly located extracellularly while the intracellular ROS content
remains unaltered. Increased extracellular ROS by paclitaxel resulted in lethal damage to
the bystander cancer cells that were not exposed to paclitaxel. Similar effects have been
seen with vincristine and taxotere treatment (Alexandre et al., 2007). Therefore, it is
possible that our compounds could exert similar bystander effects. Both TPs and
sarkostat increase intracellular superoxide content. Hence, it would be beneficial if they
would exert bystander effects to neighboring cancer cells. However, such phenomenon
could be hazardous if they are exerted against normal cells. This would result in toxic
side effects. Therefore, careful tweaking of the phenomenon would be required before
actually using it in practice.
In conclusion, we can prepare analogues of these compounds and use them for
other ailments or explore their photodynamic or bystander effect potential. In this
dissertation I have provided my work which focuses on redox regulation. It mainly
touches upon the aspect of exploiting ROS-directed cell death for anticancer therapy by
inducing oxidative stress. It briefly discusses the chemotherapy induced ROS-directed
side effects of chemotherapy, CIPN and our novel approach to combat it.
182
BIBLIOGRAPHY
Al Saati, T., Clerc, P., Hanoun, N., Peuget, S., Lulka, H., Gigoux, V., Capilla, F.,
Beluchon, B., Couvelard, A., Selves, J., et al. (2013). Oxidative Stress Induced by
Inactivation of TP53INP1 Cooperates with KrasG12D to Initiate and Promote Pancreatic
Carcinogenesis in the Murine Pancreas. Am J Pathol 182, 1996-2004.
Alexandre, J., Hu, Y., Lu, W., Pelicano, H., and Huang, P. (2007). Novel action of
paclitaxel against cancer cells: bystander effect mediated by reactive oxygen species.
Cancer Res 67, 3512-3517.
Alexandre, J., Nicco, C., Chereau, C., Laurent, A., Weill, B., Goldwasser, F., and
Batteux, F. (2006). Improvement of the therapeutic index of anticancer drugs by the
superoxide dismutase mimic mangafodipir. Journal of the National Cancer Institute 98,
236-244.
Ames, B., and Liu, J. (2004). Delaying the mitochondrial decay of aging with
acetylcarnitine. Ann N Y Acad Sci 1033, 108-116.
Augenlicht, L.H., and Heerdt, B.G. (2001). Mitochondria: integrators in tumorigenesis?
Nat Genet 28, 104-105.
183
Awadallah, N.S., Dehn, D., Shah, R.J., Russell Nash, S., Chen, Y.K., Ross, D., Bentz,
J.S., and Shroyer, K.R. (2008). NQO1 expression in pancreatic cancer and its potential
use as a biomarker. Appl Immunohistochem Mol Morphol 16, 24-31.
Aykin-Burns, N., Ahmad, I.M., Zhu, Y., Oberley, L.W., and Spitz, D.R. (2009).
Increased levels of superoxide and H2O2 mediate the differential susceptibility of cancer
cells versus normal cells to glucose deprivation. Biochem J 418, 29-37.
Azzi, A., Montecucco, C., and Richter, C. (1975). The use of acetylated ferricytochrome
c for the detection of superoxide radicals produced in biological membranes. Biochem
Biophys Res Commun 65, 597-603.
Balayssac, D., Ferrier, J., Descoeur, J., Ling, B., Pezet, D., Eschalier, A., and Authier, N.
Chemotherapy-induced peripheral neuropathies: from clinical relevance to preclinical
evidence. Expert Opin Drug Saf 10, 407-417.
Barry, S.P., Townsend, P.A., McCormick, J., Knight, R.A., Scarabelli, T.M., Latchman,
D.S., and Stephanou, A. (2009). STAT3 deletion sensitizes cells to oxidative stress.
Biochem Biophys Res Commun 385, 324-329.
Basuroy, S., Dunagan, M., Sheth, P., Seth, A., and Rao, R.K. (2010). Hydrogen peroxide
activates focal adhesion kinase and c-Src by a phosphatidylinositol 3 kinase-dependent
184
mechanism and promotes cell migration in Caco-2 cell monolayers. Am J Physiol
Gastrointest Liver Physiol 299, G186-195.
Baur, J.A., Pearson, K.J., Price, N.L., Jamieson, H.A., Lerin, C., Kalra, A., Prabhu, V.V.,
Allard, J.S., Lopez-Lluch, G., Lewis, K., et al. (2006). Resveratrol improves health and
survival of mice on a high-calorie diet. Nature 444, 337-342.
Behrend, L., Henderson, G., and Zwacka, R. (2003). Reactive oxygen species in
oncogenic transformation. Biochem Soc Trans 31, 1441-1444.
Bernhardt, P.V., Wilson, G.J., Sharpe, P.C., Kalinowski, D.S., and Richardson, D.R.
(2008). Tuning the antiproliferative activity of biologically active iron chelators:
characterization of the coordination chemistry and biological efficacy of 2-acetylpyridine
and 2-benzoylpyridine hydrazone ligands. J Biol Inorg Chem 13, 107-119.
Bey, E.A., Bentle, M.S., Reinicke, K.E., Dong, Y., Yang, C.R., Girard, L., Minna, J.D.,
Bornmann, W.G., Gao, J., and Boothman, D.A. (2007). An NQO1- and PARP-1-
mediated cell death pathway induced in non-small-cell lung cancer cells by beta-
lapachone. Proc Natl Acad Sci U S A 104, 11832-11837.
Binker, M.G., Binker-Cosen, A.A., Richards, D., Oliver, B., and Cosen-Binker, L.I.
(2009). EGF promotes invasion by PANC-1 cells through Rac1/ROS-dependent secretion
and activation of MMP-2. Biochem Biophys Res Commun 379, 445-450.
185
Bolos, V., Gasent, J.M., Lopez-Tarruella, S., and Grande, E. (2010). The dual kinase
complex FAK-Src as a promising therapeutic target in cancer. OncoTargets and therapy
3, 83-97.
Boonstra, J., and Post, J. (2004a). Molecular events associated with reactive oxygen
species and cell cycle progression in mammalian cells. Gene 337, 1-13.
Boonstra, J., and Post, J.A. (2004b). Molecular events associated with reactive oxygen
species and cell cycle progression in mammalian cells. Gene 337, 1-13.
Bracher, F. (1989). Total synthesis of the pentacyclic alkaloid ascididemin. Heterocycles
29, 2093–2095.
Bras, A., Sanches, R., Cristovao, L., Fidalgo, P., Chagas, C., Mexia, J., Leitao, N., and
Rueff, J. (1999). Oxidative stress in familial adenomatous polyposis. Eur J Cancer Prev 8,
305-310.
Brown, D.P., Chin-Sinex, H., Nie, B., Mendonca, M.S., and Wang, M. (2009). Targeting
superoxide dismutase 1 to overcome cisplatin resistance in human ovarian cancer. Cancer
Chemother Pharmacol 63, 723-730.
Brown, J.M. (2007). Tumor hypoxia in cancer therapy. Methods Enzymol 435, 297-321.
186
Brown, K.K., Eriksson, S.E., Arner, E.S., and Hampton, M.B. (2008). Mitochondrial
peroxiredoxin 3 is rapidly oxidized in cells treated with isothiocyanates. Free Radic Biol
Med 45, 494-502.
Brown, N.S., and Bicknell, R. (2001). Hypoxia and oxidative stress in breast cancer.
Oxidative stress: its effects on the growth, metastatic potential and response to therapy of
breast cancer. Breast Cancer Res 3, 323-327.
Brown, N.S., Jones, A., Fujiyama, C., Harris, A.L., and Bicknell, R. (2000). Thymidine
phosphorylase induces carcinoma cell oxidative stress and promotes secretion of
angiogenic factors. Cancer Res 60, 6298-6302.
Brunmark, A., and Cadenas, E. (1989). Redox and addition chemistry of quinoid
compounds and its biological implications. Free Radic Biol Med 7, 435-477.
Bruno, I., Cole, J., Edgington, P., Kessler, M., Macrae, C., McCabe, P., Pearson, J., and
Taylor, R. (2002). New software for searching the Cambridge Structural Database and
visualizing crystal structures. Acta Crystallogr B 58, 389-397.
Buchholz, M., and Gress, T.M. (2009). Molecular changes in pancreatic cancer. Expert
Rev Anticancer Ther 9, 1487-1497.
187
Buick, R., Pullano, R., and Trent, J. (1985). Comparative properties of five human
ovarian adenocarcinoma cell lines. Cancer Res 45, 3668-3676.
Burkitt, M.J., Bishop, H.S., Milne, L., Tsang, S.Y., Provan, G.J., Nobel, C.S., Orrenius,
S., and Slater, A.F. (1998). Dithiocarbamate toxicity toward thymocytes involves their
copper-catalyzed conversion to thiuram disulfides, which oxidize glutathione in a redox
cycle without the release of reactive oxygen species. Archives of biochemistry and
biophysics 353, 73-84.
Burla, M.C., Camalli, M., Carrozzini, B., Cascarano, G.L., Giacovazzo, C., Polidori, G.,
and Spagna, R. (2003). SIR2002: the program. J Appl Cryst 36, 1103.
Byers, L.A., Sen, B., Saigal, B., Diao, L., Wang, J., Nanjundan, M., Cascone, T., Mills,
G.B., Heymach, J.V., and Johnson, F.M. (2009). Reciprocal regulation of c-Src and
STAT3 in non-small cell lung cancer. Clin Cancer Res 15, 6852-6861.
Cabello, C.M., Bair, W.B., 3rd, Lamore, S.D., Ley, S., Bause, A.S., Azimian, S., and
Wondrak, G.T. (2009). The cinnamon-derived Michael acceptor cinnamic aldehyde
impairs melanoma cell proliferation, invasiveness, and tumor growth. Free Radic Biol
Med 46, 220-231.
Cabello, C.M., Bair, W.B., 3rd, and Wondrak, G.T. (2007). Experimental therapeutics:
targeting the redox Achilles heel of cancer. Curr Opin Investig Drugs 8, 1022-1037.
188
Cadenas, E. (1995). Antioxidant and prooxidant functions of DT-diaphorase in quinone
metabolism. Biochemical pharmacology 49, 127-140.
Carew, J.S., and Huang, P. (2002). Mitochondrial defects in cancer. Mol Cancer 1, 9.
Carew, J.S., Zhou, Y., Albitar, M., Carew, J.D., Keating, M.J., and Huang, P. (2003).
Mitochondrial DNA mutations in primary leukemia cells after chemotherapy: clinical
significance and therapeutic implications. Leukemia 17, 1437-1447.
Carmichael, J., DeGraff, W., Gazdar, A., Minna, J., and Mitchell, J. (1987). Evaluation of
a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity
testing. Cancer Res 47, 936-942.
Casini, A., Scozzafava, A., and Supuran, C.T. (2002). Cysteine-modifying agents: a
possible approach for effective anticancer and antiviral drugs. Environ Health Perspect
110 Suppl 5, 801-806.
Cen, D., Brayton, D., Shahandeh, B., Meyskens, F.L., Jr., and Farmer, P.J. (2004).
Disulfiram facilitates intracellular Cu uptake and induces apoptosis in human melanoma
cells. J Med Chem 47, 6914-6920.
Chang, L., and Karin, M. (2001). Mammalian MAP kinase signalling cascades. Nature
410, 37-40.
189
Chatterji, T., and Gates, K.S. (1998). DNA cleavage by 7-methylbenzopentathiepin: a
simple analog of the antitumor antibiotic varacin. Bioorg Med Chem Lett 8, 535-538.
Chatterji, T., Keerthi, K., and Gates, K.S. (2005). Generation of reactive oxygen species
by a persulfide (BnSSH). Bioorg Med Chem Lett 15, 3921-3924.
Chatzizacharias, N.A., Giaginis, C., Zizi-Serbetzoglou, D., Kouraklis, G.P., Karatzas, G.,
and Theocharis, S.E. (2010). Evaluation of the clinical significance of focal adhesion
kinase and SRC expression in human pancreatic ductal adenocarcinoma. Pancreas 39,
930-936.
Chen, Q., Espey, M.G., Krishna, M.C., Mitchell, J.B., Corpe, C.P., Buettner, G.R.,
Shacter, E., and Levine, M. (2005). Pharmacologic ascorbic acid concentrations
selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues.
Proc Natl Acad Sci U S A 102, 13604-13609.
Chen, Q., Espey, M.G., Sun, A.Y., Pooput, C., Kirk, K.L., Krishna, M.C., Khosh, D.B.,
Drisko, J., and Levine, M. (2008). Pharmacologic doses of ascorbate act as a prooxidant
and decrease growth of aggressive tumor xenografts in mice. Proc Natl Acad Sci U S A
105, 11105-11109.
190
Chen, Y., Jungsuwadee, P., Vore, M., Butterfield, D.A., and St Clair, D.K. (2007).
Collateral damage in cancer chemotherapy: oxidative stress in nontargeted tissues. Mol
Interv 7, 147-156.
ClinicalTrials.gov (2013a). Dasatinib Added to Gemcitabine for Subjects With Locally-
advanced Pancreatic Cancer (LAPC) (N.I.H).
ClinicalTrials.gov (2013b). Dasatinib in Treating Patients With Metastatic Pancreatic
Cancer (N.I.H).
ClinicalTrials.gov (2013c). Gemcitabine Hydrochloride, Dasatinib, and Erlotinib
Hydrochloride in Treating Patients With Metastatic Pancreatic Cancer That Cannot Be
Removed By Surgery (N.I.H).
ClinicalTrials.gov (2013d). Phase II Study of 5-FU, Oxaliplatin Plus Dasatinib in
Metastatic Pancreatic Adenocarcinoma (FOLFOX-D) (N.I.H.).
Cobb, M.H. (1999). MAP kinase pathways. Prog Biophys Mol Biol 71, 479-500.
Cobbs, C.S., Levi, D.S., Aldape, K., and Israel, M.A. (1996). Manganese superoxide
dismutase expression in human central nervous system tumors. Cancer Res 56, 3192-
3195.
191
Colucci, M.A., Moody, C.J., and Couch, G.D. (2008). Natural and synthetic quinones and
their reduction by the quinone reductase enzyme NQO1: from synthetic organic
chemistry to compounds with anticancer potential. Org Biomol Chem 6, 637-656.
Colucci, M.A., Reigan, P., Siegel, D., Chilloux, A., Ross, D., and Moody, C.J. (2007).
Synthesis and evaluation of 3-aryloxymethyl-1,2-dimethylindole-4,7-diones as
mechanism-based inhibitors of NAD(P)H:quinone oxidoreductase 1 (NQO1) activity. J
Med Chem 50, 5780-5789.
Cone, R., Hasan, S., Lown, J., and Morgan, A. (1976a). The mechanism of the
degradation of DNA by streptonigrin. Can J Biochem 54, 219-223.
Cone, R., Hasan, S.K., Lown, J.W., and Morgan, A.R. (1976b). The mechanism of the
degradation of DNA by streptonigrin. Canadian journal of biochemistry 54, 219-223.
Conradt, L., Godl, K., Schaab, C., Tebbe, A., Eser, S., Diersch, S., Michalski, C.W.,
Kleeff, J., Schnieke, A., Schmid, R.M., et al. (2011). Disclosure of erlotinib as a
multikinase inhibitor in pancreatic ductal adenocarcinoma. Neoplasia 13, 1026-1034.
Copeland, W.C., Wachsman, J.T., Johnson, F.M., and Penta, J.S. (2002). Mitochondrial
DNA alterations in cancer. Cancer Invest 20, 557-569.
192
Corcoran, A., and Cotter, T.G. (2013). Redox regulation of protein kinases. FEBS J 280,
1944-1965.
Cullen, J., Hinkhouse, M., Grady, M., Gaut, A., Liu, J., Zhang, Y., Weydert, C., Domann,
F., and Oberley, L. (2003a). Dicumarol inhibition of NADPH:quinone oxidoreductase
induces growth inhibition of pancreatic cancer via a superoxide-mediated mechanism.
Cancer Res 63, 5513-5520.
Cullen, J.J., Weydert, C., Hinkhouse, M.M., Ritchie, J., Domann, F.E., Spitz, D., and
Oberley, L.W. (2003b). The role of manganese superoxide dismutase in the growth of
pancreatic adenocarcinoma. Cancer Res 63, 1297-1303.
Cunnick, J.M., Dorsey, J.F., Standley, T., Turkson, J., Kraker, A.J., Fry, D.W., Jove, R.,
and Wu, J. (1998). Role of tyrosine kinase activity of epidermal growth factor receptor in
the lysophosphatidic acid-stimulated mitogen-activated protein kinase pathway. J Biol
Chem 273, 14468-14475.
Dai, J., Weinberg, R.S., Waxman, S., and Jing, Y. (1999). Malignant cells can be
sensitized to undergo growth inhibition and apoptosis by arsenic trioxide through
modulation of the glutathione redox system. Blood 93, 268-277.
Davies, K.J. (1999). The broad spectrum of responses to oxidants in proliferating cells: a
new paradigm for oxidative stress. IUBMB Life 48, 41-47.
193
Davis, R.J. (2000). Signal transduction by the JNK group of MAP kinases. Cell 103, 239-
252.
Davison, K., Cote, S., Mader, S., and Miller, W.H. (2003). Glutathione depletion
overcomes resistance to arsenic trioxide in arsenic-resistant cell lines. Leukemia 17, 931-
940.
De Grandis, D. (2007). Acetyl-L-carnitine for the treatment of chemotherapy-induced
peripheral neuropathy: a short review. CNS Drugs 21 Suppl 1, 39-43; discussion 45-36.
de la Lastra, C., and Villegas, I. (2007). Resveratrol as an antioxidant and pro-oxidant
agent: mechanisms and clinical implications. Biochem Soc Trans 35, 1156-1160.
Deeb, D., Gao, X., Liu, Y.B., and Gautam, S.C. (2012). Inhibition of cell proliferation
and induction of apoptosis by CDDO-Me in pancreatic cancer cells is ROS-dependent. J
Exp Ther Oncol 10, 51-64.
Deer, E.L., Gonzalez-Hernandez, J., Coursen, J.D., Shea, J.E., Ngatia, J., Scaife, C.L.,
Firpo, M.A., and Mulvihill, S.J. (2010). Phenotype and genotype of pancreatic cancer cell
lines. Pancreas 39, 425-435.
Dehn, D., Siegel, D., Zafar, K., Reigan, P., Swann, E., Moody, C., and Ross, D. (2006a).
5-Methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione, a mechanism-
194
based inhibitor of NAD(P)H:quinone oxidoreductase 1, exhibits activity against human
pancreatic cancer in vitro and in vivo. Mol Cancer Ther 5, 1702-1709.
Dehn, D.L., Siegel, D., Zafar, K.S., Reigan, P., Swann, E., Moody, C.J., and Ross, D.
(2006b). 5-Methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione, a
mechanism-based inhibitor of NAD(P)H:quinone oxidoreductase 1, exhibits activity
against human pancreatic cancer in vitro and in vivo. Molecular cancer therapeutics 5,
1702-1709.
DeNicola, G.M., Karreth, F.A., Humpton, T.J., Gopinathan, A., Wei, C., Frese, K.,
Mangal, D., Yu, K.H., Yeo, C.J., Calhoun, E.S., et al. (2011). Oncogene-induced Nrf2
transcription promotes ROS detoxification and tumorigenesis. Nature 475, 106-109.
Denko, N.C., Fontana, L.A., Hudson, K.M., Sutphin, P.D., Raychaudhuri, S., Altman, R.,
and Giaccia, A.J. (2003). Investigating hypoxic tumor physiology through gene
expression patterns. Oncogene 22, 5907-5914.
Devi, G.S., Prasad, M.H., Saraswathi, I., Raghu, D., Rao, D.N., and Reddy, P.P. (2000).
Free radicals antioxidant enzymes and lipid peroxidation in different types of leukemias.
Clin Chim Acta 293, 53-62.
195
Ding, W., You, H., Dang, H., LeBlanc, F., Galicia, V., Lu, S.C., Stiles, B., and Rountree,
C.B. (2010). Epithelial-to-mesenchymal transition of murine liver tumor cells promotes
invasion. Hepatology 52, 945-953.
Dirks Naylor, A. (2009). Cellular effects of resveratrol in skeletal muscle. Life Sci 84,
637-640.
Dixit, D., Sharma, V., Ghosh, S., Koul, N., Mishra, P.K., and Sen, E. (2009). Manumycin
inhibits STAT3, telomerase activity, and growth of glioma cells by elevating intracellular
reactive oxygen species generation. Free Radic Biol Med 47, 364-374.
Duxbury, M.S., Ito, H., Zinner, M.J., Ashley, S.W., and Whang, E.E. (2004a). Inhibition
of SRC tyrosine kinase impairs inherent and acquired gemcitabine resistance in human
pancreatic adenocarcinoma cells. Clin Cancer Res 10, 2307-2318.
Duxbury, M.S., Ito, H., Zinner, M.J., Ashley, S.W., and Whang, E.E. (2004b). siRNA
directed against c-Src enhances pancreatic adenocarcinoma cell gemcitabine
chemosensitivity. J Am Coll Surg 198, 953-959.
Dvorakova, K., Payne, C.M., Tome, M.E., Briehl, M.M., McClure, T., and Dorr, R.T.
(2000). Induction of oxidative stress and apoptosis in myeloma cells by the aziridine-
containing agent imexon. Biochemical pharmacology 60, 749-758.
196
Dvorakova, K., Waltmire, C.N., Payne, C.M., Tome, M.E., Briehl, M.M., and Dorr, R.T.
(2001). Induction of mitochondrial changes in myeloma cells by imexon. Blood 97,
3544-3551.
Edderkaoui, M., Hong, P., Vaquero, E.C., Lee, J.K., Fischer, L., Friess, H., Buchler,
M.W., Lerch, M.M., Pandol, S.J., and Gukovskaya, A.S. (2005). Extracellular matrix
stimulates reactive oxygen species production and increases pancreatic cancer cell
survival through 5-lipoxygenase and NADPH oxidase. Am J Physiol Gastrointest Liver
Physiol 289, G1137-1147.
Edderkaoui, M., Nitsche, C., Zheng, L., Pandol, S.J., Gukovsky, I., and Gukovskaya,
A.S. (2011). NADPH oxidase activation in pancreatic cancer cells is mediated through
Akt-dependent up-regulation of p22phox. J Biol Chem 286, 7779-7787.
Efferth, T. (2006). Molecular pharmacology and pharmacogenomics of artemisinin and
its derivatives in cancer cells. Curr Drug Targets 7, 407-421.
Efferth, T. (2007). Willmar Schwabe Award 2006: antiplasmodial and antitumor activity
of artemisinin--from bench to bedside. Planta Med 73, 299-309.
Fahy, B.N., Schlieman, M., Virudachalam, S., and Bold, R.J. (2003). AKT inhibition is
associated with chemosensitisation in the pancreatic cancer cell line MIA-PaCa-2. British
journal of cancer 89, 391-397.
197
Fang, J., Sawa, T., Akaike, T., Greish, K., and Maeda, H. (2004). Enhancement of
chemotherapeutic response of tumor cells by a heme oxygenase inhibitor, pegylated zinc
protoporphyrin. Int J Cancer 109, 1-8.
Farrow, B., and Evers, B.M. (2002). Inflammation and the development of pancreatic
cancer. Surg Oncol 10, 153-169.
Farrow, B., Sugiyama, Y., Chen, A., Uffort, E., Nealon, W., and Mark Evers, B. (2004).
Inflammatory mechanisms contributing to pancreatic cancer development. Ann Surg 239,
763-769; discussion 769-771.
Farrugia, L.J. (1999). WinGX suite for small-molecule single-crystal crystallography. J
Appl Cryst 32, 837-838.
Fidanboylu, M., Griffiths, L.A., and Flatters, S.J. Global inhibition of reactive oxygen
species (ROS) inhibits paclitaxel-induced painful peripheral neuropathy. PLoS One 6,
e25212.
Finkel, T. (2011). Signal transduction by reactive oxygen species. J Cell Biol 194, 7-15.
Flatters, S.J., and Bennett, G.J. (2006). Studies of peripheral sensory nerves in paclitaxel-
induced painful peripheral neuropathy: evidence for mitochondrial dysfunction. Pain 122,
245-257.
198
Fliss, M.S., Usadel, H., Caballero, O.L., Wu, L., Buta, M.R., Eleff, S.M., Jen, J., and
Sidransky, D. (2000). Facile detection of mitochondrial DNA mutations in tumors and
bodily fluids. Science 287, 2017-2019.
Floor, S.L., Dumont, J.E., Maenhaut, C., and Raspe, E. (2012). Hallmarks of cancer: of
all cancer cells, all the time? Trends in molecular medicine 18, 509-515.
Fry, F.H., Holme, A.L., Giles, N.M., Giles, G.I., Collins, C., Holt, K., Pariagh, S.,
Gelbrich, T., Hursthouse, M.B., Gutowski, N.J., et al. (2005). Multifunctional redox
catalysts as selective enhancers of oxidative stress. Org Biomol Chem 3, 2579-2587.
Fukui, M., Kang, K.S., Okada, K., and Zhu, B.T. (2013). EPA, an omega-3 fatty acid,
induces apoptosis in human pancreatic cancer cells: role of ROS accumulation, caspase-8
activation, and autophagy induction. J Cell Biochem 114, 192-203.
Fukuyo, Y., Inoue, M., Nakajima, T., Higashikubo, R., Horikoshi, N.T., Hunt, C.,
Usheva, A., Freeman, M.L., and Horikoshi, N. (2008). Oxidative stress plays a critical
role in inactivating mutant BRAF by geldanamycin derivatives. Cancer Res 68, 6324-
6330.
Garcia-Pineres, A.J., Castro, V., Mora, G., Schmidt, T.J., Strunck, E., Pahl, H.L., and
Merfort, I. (2001). Cysteine 38 in p65/NF-kappaB plays a crucial role in DNA binding
inhibition by sesquiterpene lactones. J Biol Chem 276, 39713-39720.
199
Garuti, L., Roberti, M., and Pizzirani, D. (2008). Synthetic small molecule Cdc25
phosphatases inhibitors. Curr Med Chem 15, 573-580.
Giannoni, E., Taddei, M.L., and Chiarugi, P. (2010). Src redox regulation: again in the
front line. Free Radic Biol Med 49, 516-527.
Giles, N.M., Gutowski, N.J., Giles, G.I., and Jacob, C. (2003). Redox catalysts as
sensitisers towards oxidative stress. FEBS Lett 535, 179-182.
Goin, J., Giulivi, C., Butler, J., and Cadenas, E. (1995). Enzymic- and thiol-mediated
activation of halogen-substituted diaziridinylbenzoquinones: redox transitions of the
semiquinone and semiquinone-thioether species. Free Radic Biol Med 18, 525-536.
Golab, J., Nowis, D., Skrzycki, M., Czeczot, H., Baranczyk-Kuzma, A., Wilczynski,
G.M., Makowski, M., Mroz, P., Kozar, K., Kaminski, R., et al. (2003). Antitumor effects
of photodynamic therapy are potentiated by 2-methoxyestradiol. A superoxide dismutase
inhibitor. J Biol Chem 278, 407-414.
Gong, A., He, M., Krishna Vanaja, D., Yin, P., Karnes, R.J., and Young, C.Y. (2009).
Phenethyl isothiocyanate inhibits STAT3 activation in prostate cancer cells. Mol Nutr
Food Res 53, 878-886.
200
Gong, Y., Sohn, H., Xue, L., Firestone, G.L., and Bjeldanes, L.F. (2006). 3,3'-
Diindolylmethane is a novel mitochondrial H(+)-ATP synthase inhibitor that can induce
p21(Cip1/Waf1) expression by induction of oxidative stress in human breast cancer cells.
Cancer Res 66, 4880-4887.
Grau, D., Serbedzija, George (2005). Innovative Strategies for Drug Repurposing. In
Drug Discovery & Development.
Greer, J.B., and Whitcomb, D.C. (2009). Inflammation and pancreatic cancer: an
evidence-based review. Curr Opin Pharmacol 9, 411-418.
Griffith, O.W. (1982). Mechanism of action, metabolism, and toxicity of buthionine
sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis. J Biol
Chem 257, 13704-13712.
Gukovsky, I., Li, N., Todoric, J., Gukovskaya, A., and Karin, M. (2013). Inflammation,
autophagy, and obesity: common features in the pathogenesis of pancreatitis and
pancreatic cancer. Gastroenterology 144, 1199-1209 e1194.
Hagen, T.M., Ingersoll, R.T., Lykkesfeldt, J., Liu, J., Wehr, C.M., Vinarsky, V.,
Bartholomew, J.C., and Ames, A.B. (1999). (R)-alpha-lipoic acid-supplemented old rats
have improved mitochondrial function, decreased oxidative damage, and increased
metabolic rate. FASEB J 13, 411-418.
201
Hamaguchi, K., Godwin, A., Yakushiji, M., O'Dwyer, P., Ozols, R., and Hamilton, T.
(1993). Cross-resistance to diverse drugs is associated with primary cisplatin resistance in
ovarian cancer cell lines. Cancer Res 53, 5225-5232.
Hamilton, D., and Batist, G. (2005). TLK-199 (Telik). IDrugs 8, 662-669.
Hanahan, D., and Weinberg, R.A. (2011). Hallmarks of cancer: the next generation. Cell
144, 646-674.
He, A.R., Lindenberg, A.P., and Marshall, J.L. (2008). Biologic therapies for advanced
pancreatic cancer. Expert Rev Anticancer Ther 8, 1331-1338.
Hensley, K., Robinson, K.A., Gabbita, S.P., Salsman, S., and Floyd, R.A. (2000).
Reactive oxygen species, cell signaling, and cell injury. Free Radic Biol Med 28, 1456-
1462.
Hilbig, A. (2008). Src kinase and pancreatic cancer. Recent Results Cancer Res 177, 179-
185.
Hileman, E.A., Achanta, G., and Huang, P. (2001). Superoxide dismutase: an emerging
target for cancer therapeutics. Expert Opin Ther Targets 5, 697-710.
202
Hileman, E.O., Liu, J., Albitar, M., Keating, M.J., and Huang, P. (2004). Intrinsic
oxidative stress in cancer cells: a biochemical basis for therapeutic selectivity. Cancer
Chemother Pharmacol 53, 209-219.
Hlavata, L., Aguilaniu, H., Pichova, A., and Nystrom, T. (2003). The oncogenic
RAS2(val19) mutation locks respiration, independently of PKA, in a mode prone to
generate ROS. EMBO J 22, 3337-3345.
Holmgren, A. (1985). Thioredoxin. Annu Rev Biochem 54, 237-271.
Holmgren, A. (2000). Antioxidant function of thioredoxin and glutaredoxin systems.
Antioxid Redox Signal 2, 811-820.
Hong, F., Freeman, M.L., and Liebler, D.C. (2005). Identification of sensor cysteines in
human Keap1 modified by the cancer chemopreventive agent sulforaphane. Chem Res
Toxicol 18, 1917-1926.
Huang, P., Feng, L., Oldham, E.A., Keating, M.J., and Plunkett, W. (2000). Superoxide
dismutase as a target for the selective killing of cancer cells. Nature 407, 390-395.
Huang, X., Dong, Y., Bey, E.A., Kilgore, J.A., Bair, J.S., Li, L.S., Patel, M., Parkinson,
E.I., Wang, Y., Williams, N.S., et al. (2012). An NQO1 substrate with potent antitumor
203
activity that selectively kills by PARP1-induced programmed necrosis. Cancer Res 72,
3038-3047.
Huang, Z.Q., Saluja, A.K., Dudeja, V., Vickers, S.M., and Buchsbaum, D.J. (2011).
Molecular targeted approaches for treatment of pancreatic cancer. Curr Pharm Des 17,
2221-2238.
Huanwen, W., Zhiyong, L., Xiaohua, S., Xinyu, R., Kai, W., and Tonghua, L. (2009).
Intrinsic chemoresistance to gemcitabine is associated with constitutive and laminin-
induced phosphorylation of FAK in pancreatic cancer cell lines. Mol Cancer 8, 125.
Hussain, S.P., Hofseth, L.J., and Harris, C.C. (2003). Radical causes of cancer. Nat Rev
Cancer 3, 276-285.
Hwang, J.T., Greenberg, M.M., Fuchs, T., and Gates, K.S. (1999). Reaction of the
hypoxia-selective antitumor agent tirapazamine with a C1'-radical in single-stranded and
double-stranded DNA: the drug and its metabolites can serve as surrogates for molecular
oxygen in radical-mediated DNA damage reactions. Biochemistry 38, 14248-14255.
Hwang-Verslues, W.W., Kuo, W.H., Chang, P.H., Pan, C.C., Wang, H.H., Tsai, S.T.,
Jeng, Y.M., Shew, J.Y., Kung, J.T., Chen, C.H., et al. (2009). Multiple lineages of human
breast cancer stem/progenitor cells identified by profiling with stem cell markers. PLoS
One 4, e8377.
204
Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S.,
Fujimoto, J., Okada, M., and Yamamoto, T. (1995). Reduced cell motility and enhanced
focal adhesion contact formation in cells from FAK-deficient mice. Nature 377, 539-544.
Irani, K., Xia, Y., Zweier, J., Sollott, S., Der, C., Fearon, E., Sundaresan, M., Finkel, T.,
and Goldschmidt-Clermont, P. (1997). Mitogenic signaling mediated by oxidants in Ras-
transformed fibroblasts. Science 275, 1649-1652.
Ischenko, I., Camaj, P., Seeliger, H., Kleespies, A., Guba, M., De Toni, E.N., Schwarz,
B., Graeb, C., Eichhorn, M.E., Jauch, K.W., et al. (2008). Inhibition of Src tyrosine
kinase reverts chemoresistance toward 5-fluorouracil in human pancreatic carcinoma
cells: an involvement of epidermal growth factor receptor signaling. Oncogene 27, 7212-
7222.
Ishikawa, M., Yaginuma, Y., Hayashi, H., Shimizu, T., Endo, Y., and Taniguchi, N.
(1990). Reactivity of a monoclonal antibody to manganese superoxide dismutase with
human ovarian carcinoma. Cancer Res 50, 2538-2542.
Ito, H., Gardner-Thorpe, J., Zinner, M.J., Ashley, S.W., and Whang, E.E. (2003).
Inhibition of tyrosine kinase Src suppresses pancreatic cancer invasiveness. Surgery 134,
221-226.
205
Jackson, A., and Loeb, L. (2001). The contribution of endogenous sources of DNA
damage to the multiple mutations in cancer. Mutat Res 477, 7-21.
Jackson, L., and Evers, B.M. (2006). Chronic inflammation and pathogenesis of GI and
pancreatic cancers. Cancer Treat Res 130, 39-65.
Jacob, C. (2006). A scent of therapy: pharmacological implications of natural products
containing redox-active sulfur atoms. Nat Prod Rep 23, 851-863.
Jang, M., Cai, L., Udeani, G.O., Slowing, K.V., Thomas, C.F., Beecher, C.W., Fong,
H.H., Farnsworth, N.R., Kinghorn, A.D., Mehta, R.G., et al. (1997). Cancer
chemopreventive activity of resveratrol, a natural product derived from grapes. Science
275, 218-220.
Janssen, A.M., Bosman, C.B., Kruidenier, L., Griffioen, G., Lamers, C.B., van Krieken,
J.H., van de Velde, C.J., and Verspaget, H.W. (1999). Superoxide dismutases in the
human colorectal cancer sequence. J Cancer Res Clin Oncol 125, 327-335.
Janssen, A.M., Bosman, C.B., van Duijn, W., Oostendorp-van de Ruit, M.M., Kubben,
F.J., Griffioen, G., Lamers, C.B., van Krieken, J.H., van de Velde, C.J., and Verspaget,
H.W. (2000). Superoxide dismutases in gastric and esophageal cancer and the prognostic
impact in gastric cancer. Clin Cancer Res 6, 3183-3192.
206
Jaruga, P., Zastawny, T.H., Skokowski, J., Dizdaroglu, M., and Olinski, R. (1994).
Oxidative DNA base damage and antioxidant enzyme activities in human lung cancer.
FEBS Lett 341, 59-64.
Jezek, P., and Hlavatá, L. (2005). Mitochondria in homeostasis of reactive oxygen
species in cell, tissues, and organism. Int J Biochem Cell Biol 37, 2478-2503.
Jiang, Y., Guo, C., Vasko, M.R., and Kelley, M.R. (2008). Implications of
apurinic/apyrimidinic endonuclease in reactive oxygen signaling response after cisplatin
treatment of dorsal root ganglion neurons. Cancer Res 68, 6425-6434.
Jin, H.W., Flatters, S.J., Xiao, W.H., Mulhern, H.L., and Bennett, G.J. (2008). Prevention
of paclitaxel-evoked painful peripheral neuropathy by acetyl-L-carnitine: effects on
axonal mitochondria, sensory nerve fiber terminal arbors, and cutaneous Langerhans
cells. Exp Neurol 210, 229-237.
Jing, Y., Dai, J., Chalmers-Redman, R.M., Tatton, W.G., and Waxman, S. (1999).
Arsenic trioxide selectively induces acute promyelocytic leukemia cell apoptosis via a
hydrogen peroxide-dependent pathway. Blood 94, 2102-2111.
Jones, S., Hruban, R.H., Kamiyama, M., Borges, M., Zhang, X., Parsons, D.W., Lin, J.C.,
Palmisano, E., Brune, K., Jaffee, E.M., et al. (2009). Exomic sequencing identifies
PALB2 as a pancreatic cancer susceptibility gene. Science 324, 217.
207
Juarez, J.C., Betancourt, O., Jr., Pirie-Shepherd, S.R., Guan, X., Price, M.L., Shaw, D.E.,
Mazar, A.P., and Donate, F. (2006). Copper binding by tetrathiomolybdate attenuates
angiogenesis and tumor cell proliferation through the inhibition of superoxide dismutase
1. Clin Cancer Res 12, 4974-4982.
Juarez, J.C., Manuia, M., Burnett, M.E., Betancourt, O., Boivin, B., Shaw, D.E., Tonks,
N.K., Mazar, A.P., and Donate, F. (2008). Superoxide dismutase 1 (SOD1) is essential
for H2O2-mediated oxidation and inactivation of phosphatases in growth factor
signaling. Proc Natl Acad Sci U S A 105, 7147-7152.
Jung, K., Seidel, B., Rudolph, B., Lein, M., Cronauer, M.V., Henke, W., Hampel, G.,
Schnorr, D., and Loening, S.A. (1997). Antioxidant enzymes in malignant prostate cell
lines and in primary cultured prostatic cells. Free Radic Biol Med 23, 127-133.
Kaiser, M., Wittlin, S., Nehrbass-Stuedli, A., Dong, Y., Wang, X., Hemphill, A., Matile,
H., Brun, R., and Vennerstrom, J.L. (2007). Peroxide bond-dependent antiplasmodial
specificity of artemisinin and OZ277 (RBx11160). Antimicrob Agents Chemother 51,
2991-2993.
Kalinowski, D.S., and Richardson, D.R. (2007). Future of toxicology--iron chelators and
differing modes of action and toxicity: the changing face of iron chelation therapy. Chem
Res Toxicol 20, 715-720.
208
Kanbagli, O., Ozdemirler, G., Bulut, T., Yamaner, S., Aykac-Toker, G., and Uysal, M.
(2000). Mitochondrial lipid peroxides and antioxidant enzymes in colorectal
adenocarcinoma tissues. Jpn J Cancer Res 91, 1258-1263.
Kang, D., and Hamasaki, N. (2003). Mitochondrial oxidative stress and mitochondrial
DNA. Clin Chem Lab Med 41, 1281-1288.
Kar, S., Wang, M., Yao, W., Michejda, C.J., and Carr, B.I. (2006). PM-20, a novel
inhibitor of Cdc25A, induces extracellular signal-regulated kinase 1/2 phosphorylation
and inhibits hepatocellular carcinoma growth in vitro and in vivo. Molecular cancer
therapeutics 5, 1511-1519.
Kato, M., Li, J., Chuang, J.L., and Chuang, D.T. (2007). Distinct structural mechanisms
for inhibition of pyruvate dehydrogenase kinase isoforms by AZD7545, dichloroacetate,
and radicicol. Structure 15, 992-1004.
Katsuno, Y., Hanyu, A., Kanda, H., Ishikawa, Y., Akiyama, F., Iwase, T., Ogata, E.,
Ehata, S., Miyazono, K., and Imamura, T. (2008). Bone morphogenetic protein signaling
enhances invasion and bone metastasis of breast cancer cells through Smad pathway.
Oncogene 27, 6322-6333.
209
Kawanishi, S., Hiraku, Y., Pinlaor, S., and Ma, N. (2006). Oxidative and nitrative DNA
damage in animals and patients with inflammatory diseases in relation to inflammation-
related carcinogenesis. Biol Chem 387, 365-372.
Kemble, D.J., and Sun, G. (2009). Direct and specific inactivation of protein tyrosine
kinases in the Src and FGFR families by reversible cysteine oxidation. Proc Natl Acad
Sci U S A 106, 5070-5075.
Kim, D., Nguyen, M., Dobbin, M., Fischer, A., Sananbenesi, F., Rodgers, J., Delalle, I.,
Baur, J., Sui, G., Armour, S., et al. (2007). SIRT1 deacetylase protects against
neurodegeneration in models for Alzheimer's disease and amyotrophic lateral sclerosis.
EMBO J 26, 3169-3179.
Kim, H., Kim, E.H., Eom, Y.W., Kim, W.H., Kwon, T.K., Lee, S.J., and Choi, K.S.
(2006). Sulforaphane sensitizes tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL)-resistant hepatoma cells to TRAIL-induced apoptosis through reactive oxygen
species-mediated up-regulation of DR5. Cancer Res 66, 1740-1750.
Kim, J.I., Cho, S.R., Lee, C.M., Park, E.S., Kim, K.N., Kim, H.C., and Lee, H.Y. (2012).
Induction of ER Stress-Mediated Apoptosis by α-Lipoic Acid in A549 Cell Lines. Korean
J Thorac Cardiovasc Surg 45, 1-10.
210
Kim, J.W., and Dang, C.V. (2006). Cancer's molecular sweet tooth and the Warburg
effect. Cancer Res 66, 8927-8930.
Kim, L.C., Song, L., and Haura, E.B. (2009). Src kinases as therapeutic targets for
cancer. Nat Rev Clin Oncol 6, 587-595.
Kirkpatrick, D.L., Ehrmantraut, G., Stettner, S., Kunkel, M., and Powis, G. (1997).
Redox active disulfides: the thioredoxin system as a drug target. Oncol Res 9, 351-356.
Kirshner, J.R., He, S., Balasubramanyam, V., Kepros, J., Yang, C.Y., Zhang, M., Du, Z.,
Barsoum, J., and Bertin, J. (2008). Elesclomol induces cancer cell apoptosis through
oxidative stress. Molecular cancer therapeutics 7, 2319-2327.
Kirszberg, C., Rumjanek, V.M., and Capella, M.A. (2005). Methylene blue is more toxic
to erythroleukemic cells than to normal peripheral blood mononuclear cells: a possible
use in chemotherapy. Cancer Chemother Pharmacol 56, 659-665.
Kodydkova, J., Vavrova, L., Stankova, B., Macasek, J., Krechler, T., and Zak, A. (2013).
Antioxidant status and oxidative stress markers in pancreatic cancer and chronic
pancreatitis. Pancreas 42, 614-621.
Kong, Q., Beel, J., and Lillehei, K. (2000a). A threshold concept for cancer therapy.
Medical hypotheses 55, 29-35.
211
Kong, Q., Beel, J.A., and Lillehei, K.O. (2000b). A threshold concept for cancer therapy.
Medical hypotheses 55, 29-35.
Kong, Q., and Lillehei, K.O. (1998). Antioxidant inhibitors for cancer therapy. Medical
hypotheses 51, 405-409.
Konig, J., Hartel, M., Nies, A.T., Martignoni, M.E., Guo, J., Buchler, M.W., Friess, H.,
and Keppler, D. (2005). Expression and localization of human multidrug resistance
protein (ABCC) family members in pancreatic carcinoma. Int J Cancer 115, 359-367.
Konstantinov, A.A., Peskin, A.V., Popova, E., Khomutov, G.B., and Ruuge, E.K. (1987).
Superoxide generation by the respiratory chain of tumor mitochondria. Biochim Biophys
Acta 894, 1-10.
Landowski, T.H., Megli, C.J., Nullmeyer, K.D., Lynch, R.M., and Dorr, R.T. (2005).
Mitochondrial-mediated disregulation of Ca2+ is a critical determinant of Velcade (PS-
341/bortezomib) cytotoxicity in myeloma cell lines. Cancer Res 65, 3828-3836.
Lau, J.P., Weatherdon, K.L., Skalski, V., and Hedley, D.W. (2004). Effects of
gemcitabine on APE/ref-1 endonuclease activity in pancreatic cancer cells, and the
therapeutic potential of antisense oligonucleotides. British journal of cancer 91, 1166-
1173.
212
Laurent, A., Nicco, C., Chereau, C., Goulvestre, C., Alexandre, J., Alves, A., Levy, E.,
Goldwasser, F., Panis, Y., Soubrane, O., et al. (2005). Controlling tumor growth by
modulating endogenous production of reactive oxygen species. Cancer Res 65, 948-956.
Lavecchia, A., Cosconati, S., Limongelli, V., and Novellino, E. (2006). Modeling of
Cdc25B dual specifity protein phosphatase inhibitors: docking of ligands and enzymatic
inhibition mechanism. ChemMedChem 1, 540-550.
Lee, J.K., Edderkaoui, M., Truong, P., Ohno, I., Jang, K.T., Berti, A., Pandol, S.J., and
Gukovskaya, A.S. (2007). NADPH oxidase promotes pancreatic cancer cell survival via
inhibiting JAK2 dephosphorylation by tyrosine phosphatases. Gastroenterology 133,
1637-1648.
Lewis, A., Ough, M., Hinkhouse, M., Tsao, M., Oberley, L., and Cullen, J. (2005a).
Targeting NAD(P)H:quinone oxidoreductase (NQO1) in pancreatic cancer. Mol Carcinog
43, 215-224.
Lewis, A., Ough, M., Li, L., Hinkhouse, M.M., Ritchie, J.M., Spitz, D.R., and Cullen, J.J.
(2004). Treatment of pancreatic cancer cells with dicumarol induces cytotoxicity and
oxidative stress. Clin Cancer Res 10, 4550-4558.
213
Lewis, A.M., Ough, M., Hinkhouse, M.M., Tsao, M.S., Oberley, L.W., and Cullen, J.J.
(2005b). Targeting NAD(P)H:quinone oxidoreductase (NQO1) in pancreatic cancer.
Molecular carcinogenesis 43, 215-224.
Li, L.S., Bey, E.A., Dong, Y., Meng, J., Patra, B., Yan, J., Xie, X.J., Brekken, R.A.,
Barnett, C.C., Bornmann, W.G., et al. (2011). Modulating endogenous NQO1 levels
identifies key regulatory mechanisms of action of beta-lapachone for pancreatic cancer
therapy. Clin Cancer Res 17, 275-285.
Li, W., Fan, J., Chen, M., Guan, S., Sawcer, D., Bokoch, G.M., and Woodley, D.T.
(2004). Mechanism of human dermal fibroblast migration driven by type I collagen and
platelet-derived growth factor-BB. Molecular biology of the cell 15, 294-309.
Li, Z., Yang, J., and Huang, H. (2006). Oxidative stress induces H2AX phosphorylation
in human spermatozoa. FEBS Lett 580, 6161-6168.
Lister, A., Nedjadi, T., Kitteringham, N.R., Campbell, F., Costello, E., Lloyd, B., Copple,
I.M., Williams, S., Owen, A., Neoptolemos, J.P., et al. (2011). Nrf2 is overexpressed in
pancreatic cancer: implications for cell proliferation and therapy. Mol Cancer 10, 37.
Liu, B., Zhang, X., Zhang, W., and Zhen, H. (2007). New enlightenment of French
Paradox: resveratrol's potential for cancer chemoprevention and anti-cancer therapy.
Cancer Biol Ther 6, 1833-1836.
214
Liu, S.C., Ahn, G.O., Kioi, M., Dorie, M.J., Patterson, A.V., and Brown, J.M. (2008).
Optimized clostridium-directed enzyme prodrug therapy improves the antitumor activity
of the novel DNA cross-linking agent PR-104. Cancer Res 68, 7995-8003.
Liu, W.H., Cheng, Y.C., and Chang, L.S. (2009). ROS-mediated p38alpha MAPK
activation and ERK inactivation responsible for upregulation of Fas and FasL and
autocrine Fas-mediated cell death in Taiwan cobra phospholipase A(2)-treated U937
cells. Journal of cellular physiology 219, 642-651.
Loewe, R., Valero, T., Kremling, S., Pratscher, B., Kunstfeld, R., Pehamberger, H., and
Petzelbauer, P. (2006). Dimethylfumarate impairs melanoma growth and metastasis.
Cancer Res 66, 11888-11896.
Lomenick, B., Hao, R., Jonai, N., Chin, R.M., Aghajan, M., Warburton, S., Wang, J.,
Wu, R.P., Gomez, F., Loo, J.A., et al. (2009). Target identification using drug affinity
responsive target stability (DARTS). Proc Natl Acad Sci U S A 106, 21984-21989.
Lu, C., Shi, Y., Wang, Z., Song, Z., Zhu, M., Cai, Q., and Chen, T. (2008). Serum
starvation induces H2AX phosphorylation to regulate apoptosis via p38 MAPK pathway.
FEBS Lett 582, 2703-2708.
215
Lu, C., Zhu, F., Cho, Y.Y., Tang, F., Zykova, T., Ma, W.Y., Bode, A.M., and Dong, Z.
(2006). Cell apoptosis: requirement of H2AX in DNA ladder formation, but not for the
activation of caspase-3. Mol Cell 23, 121-132.
Lu, W., Hu, Y., Chen, G., Chen, Z., Zhang, H., Wang, F., Feng, L., Pelicano, H., Wang,
H., Keating, M.J., et al. (2012). Novel role of NOX in supporting aerobic glycolysis in
cancer cells with mitochondrial dysfunction and as a potential target for cancer therapy.
PLoS Biol 10, e1001326.
Luo, M., Delaplane, S., Jiang, A., Reed, A., He, Y., Fishel, M., Nyland, R.L., 2nd, Borch,
R.F., Qiao, X., Georgiadis, M.M., et al. (2008). Role of the multifunctional DNA repair
and redox signaling protein Ape1/Ref-1 in cancer and endothelial cells: small-molecule
inhibition of the redox function of Ape1. Antioxid Redox Signal 10, 1853-1867.
Luo, M., and Kelley, M.R. (2004). Inhibition of the human apurinic/apyrimidinic
endonuclease (APE1) repair activity and sensitization of breast cancer cells to DNA
alkylating agents with lucanthone. Anticancer Res 24, 2127-2134.
Lutz, M.P., Esser, I.B., Flossmann-Kast, B.B., Vogelmann, R., Lührs, H., Friess, H.,
Büchler, M.W., and Adler, G. (1998). Overexpression and activation of the tyrosine
kinase Src in human pancreatic carcinoma. Biochem Biophys Res Commun 243, 503-
508.
216
Lyn-Cook, B.D., Yan-Sanders, Y., Moore, S., Taylor, S., Word, B., and Hammons, G.J.
(2006). Increased levels of NAD(P)H: quinone oxidoreductase 1 (NQO1) in pancreatic
tissues from smokers and pancreatic adenocarcinomas: A potential biomarker of early
damage in the pancreas. Cell Biol Toxicol 22, 73-80.
Ma, J., Cai, H., Wu, T., Sobhian, B., Huo, Y., Alcivar, A., Mehta, M., Cheung, K.L.,
Ganesan, S., Kong, A.N., et al. (2012). PALB2 interacts with KEAP1 to promote NRF2
nuclear accumulation and function. Mol Cell Biol 32, 1506-1517.
Mackenzie, R.P., and McCollum, A.D. (2009). Novel agents for the treatment of
adenocarcinoma of the pancreas. Expert Rev Anticancer Ther 9, 1473-1485.
MacMillan-Crow, L., Greendorfer, J., Vickers, S., and Thompson, J. (2000). Tyrosine
nitration of c-SRC tyrosine kinase in human pancreatic ductal adenocarcinoma. Arch
Biochem Biophys 377, 350-356.
Magda, D., and Miller, R.A. (2006). Motexafin gadolinium: a novel redox active drug for
cancer therapy. Semin Cancer Biol 16, 466-476.
Malesani, G., Marcolin, F., and Rodighiero, G. (1970). Synthesis of 5,8-
quinazolinedione. J Med Chem 13, 161-162.
217
Malesani, G., Pietrogrande, A., and Rodighiero, G. (1968). [5,8-dioxyquinazoline
derivates]. Farmaco Sci 23, 765-777.
Marklund, S.L., Westman, N.G., Lundgren, E., and Roos, G. (1982). Copper- and zinc-
containing superoxide dismutase, manganese-containing superoxide dismutase, catalase,
and glutathione peroxidase in normal and neoplastic human cell lines and normal human
tissues. Cancer Res 42, 1955-1961.
Mathupala, S.P., Ko, Y.H., and Pedersen, P.L. (2006). Hexokinase II: cancer's double-
edged sword acting as both facilitator and gatekeeper of malignancy when bound to
mitochondria. Oncogene 25, 4777-4786.
Matsuzaki, T., Sakanashi, M., Nakasone, J., Noguchi, K., Miyagi, K., Sakanashi, M.,
Kukita, I., Aniya, Y., and Sakanashi, M. (2002). Effects of glutathione S-transferase
inhibitors on nitroglycerin action in pig isolated coronary arteries. Clin Exp Pharmacol
Physiol 29, 1091-1095.
McDonald, E.S., and Windebank, A.J. (2002). Cisplatin-induced apoptosis of DRG
neurons involves bax redistribution and cytochrome c release but not fas receptor
signaling. Neurobiol Dis 9, 220-233.
218
McLean, G.W., Carragher, N.O., Avizienyte, E., Evans, J., Brunton, V.G., and Frame,
M.C. (2005). The role of focal-adhesion kinase in cancer - a new therapeutic opportunity.
Nat Rev Cancer 5, 505-515.
Mezencev, R., Wang, L., Xu, W., Kim, B., Sulchek, T.A., Daneker, G.W., and
McDonald, J.F. (2013). Molecular analysis of the inhibitory effect of N-acetyl-L-cysteine
on the proliferation and invasiveness of pancreatic cancer cells. Anticancer Drugs 24,
504-518.
Mi, L., Xiao, Z., Hood, B.L., Dakshanamurthy, S., Wang, X., Govind, S., Conrads, T.P.,
Veenstra, T.D., and Chung, F.L. (2008). Covalent binding to tubulin by isothiocyanates.
A mechanism of cell growth arrest and apoptosis. J Biol Chem 283, 22136-22146.
Millard, M., Pathania, D., Shabaik, Y., Taheri, L., Deng, J., and Neamati, N. (2010).
Preclinical evaluation of novel triphenylphosphonium salts with broad-spectrum activity.
PLoS One 5.
Mitra, S.K., Hanson, D.A., and Schlaepfer, D.D. (2005). Focal adhesion kinase: in
command and control of cell motility. Nat Rev Mol Cell Biol 6, 56-68.
Mitsushita, J., Lambeth, J., and Kamata, T. (2004a). The superoxide-generating oxidase
Nox1 is functionally required for Ras oncogene transformation. Cancer Res 64, 3580-
3585.
219
Mitsushita, J., Lambeth, J.D., and Kamata, T. (2004b). The superoxide-generating
oxidase Nox1 is functionally required for Ras oncogene transformation. Cancer Res 64,
3580-3585.
Miyajima, A., Nakashima, J., Yoshioka, K., Tachibana, M., Tazaki, H., and Murai, M.
(1997). Role of reactive oxygen species in cis-dichlorodiammineplatinum-induced
cytotoxicity on bladder cancer cells. British journal of cancer 76, 206-210.
Mochizuki, T., Furuta, S., Mitsushita, J., Shang, W.H., Ito, M., Yokoo, Y., Yamaura, M.,
Ishizone, S., Nakayama, J., Konagai, A., et al. (2006). Inhibition of NADPH oxidase 4
activates apoptosis via the AKT/apoptosis signal-regulating kinase 1 pathway in
pancreatic cancer PANC-1 cells. Oncogene 25, 3699-3707.
Mohri, D., Asaoka, Y., Ijichi, H., Miyabayashi, K., Kudo, Y., Seto, M., Ohta, M., Tada,
M., Tanaka, Y., Ikenoue, T., et al. (2012). Different subtypes of intraductal papillary
mucinous neoplasm in the pancreas have distinct pathways to pancreatic cancer
progression. J Gastroenterol 47, 203-213.
Moini, H., Packer, L., and Saris, N. (2002). Antioxidant and prooxidant activities of
alpha-lipoic acid and dihydrolipoic acid. Toxicol Appl Pharmacol 182, 84-90.
Montagut, C., Rovira, A., and Albanell, J. (2006). The proteasome: a novel target for
anticancer therapy. Clin Transl Oncol 8, 313-317.
220
Montero, A.J., and Jassem, J. (2011). Cellular redox pathways as a therapeutic target in
the treatment of cancer. Drugs 71, 1385-1396.
Mooberry, S.L. (2003). Mechanism of action of 2-methoxyestradiol: new developments.
Drug Resist Updat 6, 355-361.
Moore, P.S., Sipos, B., Orlandini, S., Sorio, C., Real, F.X., Lemoine, N.R., Gress, T.,
Bassi, C., Kloppel, G., Kalthoff, H., et al. (2001). Genetic profile of 22 pancreatic
carcinoma cell lines. Analysis of K-ras, p53, p16 and DPC4/Smad4. Virchows Arch 439,
798-802.
Mukherjee, A., Huber, K., Evans, H., Lakhani, N., and Martin, S. (2007). A cellular and
molecular investigation of the action of PMX464, a putative thioredoxin inhibitor, in
normal and colorectal cancer cell lines. Br J Pharmacol 151, 1167-1175.
Munshi, A., Hobbs, M., and Meyn, R. (2005). Clonogenic cell survival assay. Methods
Mol Med 110, 21-28.
Murase, T., Haramizu, S., Ota, N., and Hase, T. (2009). Suppression of the aging-
associated decline in physical performance by a combination of resveratrol intake and
habitual exercise in senescence-accelerated mice. Biogerontology 10, 423-434.
221
Murphy, M.P. (2008). Targeting lipophilic cations to mitochondria. Biochim Biophys
Acta 1777, 1028-1031.
N. I. H., N.C.I. (2012). Antioxidant and Cancer Prevention: Fact Sheet.
N.I.H., N.C.I. (2013). Pancreatic Cancer.
Nagaraj, N.S., Smith, J.J., Revetta, F., Washington, M.K., and Merchant, N.B. (2010).
Targeted inhibition of SRC kinase signaling attenuates pancreatic tumorigenesis.
Molecular cancer therapeutics 9, 2322-2332.
Nagaraj, N.S., Washington, M.K., and Merchant, N.B. (2011). Combined blockade of Src
kinase and epidermal growth factor receptor with gemcitabine overcomes STAT3-
mediated resistance of inhibition of pancreatic tumor growth. Clin Cancer Res 17, 483-
493.
Nakano, A., Abe, M., Oda, A., Amou, H., Hiasa, M., Nakamura, S., Miki, H., Harada, T.,
Fujii, S., Kagawa, K., et al. (2011). Delayed treatment with vitamin C and N-acetyl-L-
cysteine protects Schwann cells without compromising the anti-myeloma activity of
bortezomib. Int J Hematol 93, 727-735.
222
Nardelli, M. (1995). PARST95 - an update to PARST: a system of Fortran routines for
calculating molecular structure parameters from the results of crystal structure analyses. J
Appl Crystallogr 28, 659.
Naumann, P., Fortunato, F., Zentgraf, H., Buchler, M.W., Herr, I., and Werner, J. (2011).
Autophagy and cell death signaling following dietary sulforaphane act independently of
each other and require oxidative stress in pancreatic cancer. Int J Oncol 39, 101-109.
Neuzil, J., Tomasetti, M., Zhao, Y., Dong, L.F., Birringer, M., Wang, X.F., Low, P., Wu,
K., Salvatore, B.A., and Ralph, S.J. (2007). Vitamin E analogs, a novel group of
"mitocans," as anticancer agents: the importance of being redox-silent. Mol Pharmacol
71, 1185-1199.
Nguyen, C., Teo, J.L., Matsuda, A., Eguchi, M., Chi, E.Y., Henderson, W.R., Jr., and
Kahn, M. (2003). Chemogenomic identification of Ref-1/AP-1 as a therapeutic target for
asthma. Proc Natl Acad Sci U S A 100, 1169-1173.
Nishiura, T., Suzuki, K., Kawaguchi, T., Nakao, H., Kawamura, N., Taniguchi, M.,
Kanayama, Y., Yonezawa, T., Iizuka, S., and Taniguchi, N. (1992). Elevated serum
manganese superoxide dismutase in acute leukemias. Cancer letters 62, 211-215.
223
Nogueira, V., Park, Y., Chen, C., Xu, P., Chen, M., Tonic, I., Unterman, T., and Hay, N.
(2008). Akt determines replicative senescence and oxidative or oncogenic premature
senescence and sensitizes cells to oxidative apoptosis. Cancer Cell 14, 458-470.
Nolan, K.A., Doncaster, J.R., Dunstan, M.S., Scott, K.A., Frenkel, A.D., Siegel, D., Ross,
D., Barnes, J., Levy, C., Leys, D., et al. (2009). Synthesis and biological evaluation of
coumarin-based inhibitors of NAD(P)H: quinone oxidoreductase-1 (NQO1). J Med Chem
52, 7142-7156.
Nolan, K.A., Zhao, H., Faulder, P.F., Frenkel, A.D., Timson, D.J., Siegel, D., Ross, D.,
Burke, T.R., Jr., Stratford, I.J., and Bryce, R.A. (2007). Coumarin-based inhibitors of
human NAD(P)H:quinone oxidoreductase-1. Identification, structure-activity, off-target
effects and in vitro human pancreatic cancer toxicity. J Med Chem 50, 6316-6325.
O'Dwyer, P.J., Hamilton, T.C., LaCreta, F.P., Gallo, J.M., Kilpatrick, D., Halbherr, T.,
Brennan, J., Bookman, M.A., Hoffman, J., Young, R.C., et al. (1996). Phase I trial of
buthionine sulfoximine in combination with melphalan in patients with cancer. J Clin
Oncol 14, 249-256.
Oberley, L.W., Bize, I.B., Sahu, S.K., Leuthauser, S.W., and Gruber, H.E. (1978).
Superoxide dismutase activity of normal murine liver, regenerating liver, and H6
hepatoma. Journal of the National Cancer Institute 61, 375-379.
224
Ogino, K., and Wang, D.H. (2007). Biomarkers of oxidative/nitrosative stress: an
approach to disease prevention. Acta Med Okayama 61, 181-189.
Okamoto, K., Toyokuni, S., Uchida, K., Ogawa, O., Takenewa, J., Kakehi, Y., Kinoshita,
H., Hattori-Nakakuki, Y., Hiai, H., and Yoshida, O. (1994). Formation of 8-hydroxy-2'-
deoxyguanosine and 4-hydroxy-2-nonenal-modified proteins in human renal-cell
carcinoma. Int J Cancer 58, 825-829.
Olinski, R., Zastawny, T., Budzbon, J., Skokowski, J., Zegarski, W., and Dizdaroglu, M.
(1992). DNA base modifications in chromatin of human cancerous tissues. FEBS Lett
309, 193-198.
Ostman, A., Frijhoff, J., Sandin, A., and Bohmer, F.D. (2011). Regulation of protein
tyrosine phosphatases by reversible oxidation. J Biochem 150, 345-356.
Ough, M., Lewis, A., Bey, E.A., Gao, J., Ritchie, J.M., Bornmann, W., Boothman, D.A.,
Oberley, L.W., and Cullen, J.J. (2005). Efficacy of beta-lapachone in pancreatic cancer
treatment: exploiting the novel, therapeutic target NQO1. Cancer Biol Ther 4, 95-102.
Owen, J.D., Ruest, P.J., Fry, D.W., and Hanks, S.K. (1999). Induced focal adhesion
kinase (FAK) expression in FAK-null cells enhances cell spreading and migration
requiring both auto- and activation loop phosphorylation sites and inhibits adhesion-
dependent tyrosine phosphorylation of Pyk2. Mol Cell Biol 19, 4806-4818.
225
Pachman, D.R., Barton, D.L., Watson, J.C., and Loprinzi, C.L. (2011). Chemotherapy-
induced peripheral neuropathy: prevention and treatment. Clin Pharmacol Ther 90, 377-
387.
Pathania, D., Millard, M., and Neamati, N. (2009). Opportunities in discovery and
delivery of anticancer drugs targeting mitochondria and cancer cell metabolism. Adv
Drug Deliv Rev.
Pelicano, H., Carney, D., and Huang, P. (2004). ROS stress in cancer cells and
therapeutic implications. Drug Resist Updat 7, 97-110.
Pelicano, H., Feng, L., Zhou, Y., Carew, J.S., Hileman, E.O., Plunkett, W., Keating, M.J.,
and Huang, P. (2003). Inhibition of mitochondrial respiration: a novel strategy to enhance
drug-induced apoptosis in human leukemia cells by a reactive oxygen species-mediated
mechanism. J Biol Chem 278, 37832-37839.
Perry, G., Raina, A., Nunomura, A., Wataya, T., Sayre, L., and Smith, M. (2000). How
important is oxidative damage? Lessons from Alzheimer's disease. Free Radic Biol Med
28, 831-834.
Porteous, C.M., Logan, A., Evans, C., Ledgerwood, E.C., Menon, D.K., Aigbirhio, F.,
Smith, R.A., and Murphy, M.P. (2010). Rapid uptake of lipophilic triphenylphosphonium
226
cations by mitochondria in vivo following intravenous injection: implications for
mitochondria-specific therapies and probes. Biochim Biophys Acta 1800, 1009-1017.
Powis, G., Wipf, P., Lynch, S.M., Birmingham, A., and Kirkpatrick, D.L. (2006).
Molecular pharmacology and antitumor activity of palmarumycin-based inhibitors of
thioredoxin reductase. Molecular cancer therapeutics 5, 630-636.
Powolny, A.A., and Singh, S.V. (2008). Multitargeted prevention and therapy of cancer
by diallyl trisulfide and related Allium vegetable-derived organosulfur compounds.
Cancer letters 269, 305-314.
Pramanik, K.C., Boreddy, S.R., and Srivastava, S.K. (2011). Role of mitochondrial
electron transport chain complexes in capsaicin mediated oxidative stress leading to
apoptosis in pancreatic cancer cells. PLoS One 6, e20151.
Pratt, Y.T. (1962). Quinolinequinones. VI. Reactions with aromatic amines. J Org Chem
27, 3905–3910.
Preuss, M., Girnun, G.D., Darby, C.J., Khoo, N., Spector, A.A., and Robbins, M.E.
(2000). Role of antioxidant enzyme expression in the selective cytotoxic response of
glioma cells to gamma-linolenic acid supplementation. Free Radic Biol Med 28, 1143-
1156.
227
Punnonen, K., Ahotupa, M., Asaishi, K., Hyoty, M., Kudo, R., and Punnonen, R. (1994).
Antioxidant enzyme activities and oxidative stress in human breast cancer. J Cancer Res
Clin Oncol 120, 374-377.
Qanungo, S., Das, M., Haldar, S., and Basu, A. (2005). Epigallocatechin-3-gallate
induces mitochondrial membrane depolarization and caspase-dependent apoptosis in
pancreatic cancer cells. Carcinogenesis 26, 958-967.
Quintas-Cardama, A., Verstovsek, S., Freireich, E., Kantarjian, H., Chen, Y.W., and
Zingaro, R. (2008). Chemical and clinical development of darinaparsin, a novel organic
arsenic derivative. Anticancer Agents Med Chem 8, 904-909.
Raimondi, S., Lowenfels, A.B., Morselli-Labate, A.M., Maisonneuve, P., and Pezzilli, R.
(2010). Pancreatic cancer in chronic pancreatitis; aetiology, incidence, and early
detection. Best Pract Res Clin Gastroenterol 24, 349-358.
Rais, B., Comin, B., Puigjaner, J., Brandes, J.L., Creppy, E., Saboureau, D., Ennamany,
R., Lee, W.N., Boros, L.G., and Cascante, M. (1999). Oxythiamine and
dehydroepiandrosterone induce a G1 phase cycle arrest in Ehrlich's tumor cells through
inhibition of the pentose cycle. FEBS Lett 456, 113-118.
Rajeshkumar, N.V., Tan, A.C., De Oliveira, E., Womack, C., Wombwell, H., Morgan, S.,
Warren, M.V., Walker, J., Green, T.P., Jimeno, A., et al. (2009). Antitumor effects and
228
biomarkers of activity of AZD0530, a Src inhibitor, in pancreatic cancer. Clin Cancer Res
15, 4138-4146.
Ralph, S.J., and Neuzil, J. (2009). Mitochondria as targets for cancer therapy. Mol Nutr
Food Res 53, 9-28.
Recktenwald, C.V., Kellner, R., Lichtenfels, R., and Seliger, B. (2008). Altered
detoxification status and increased resistance to oxidative stress by K-ras transformation.
Cancer Res 68, 10086-10093.
Reigan, P., Colucci, M., Siegel, D., Chilloux, A., Moody, C., and Ross, D. (2007a).
Development of indolequinone mechanism-based inhibitors of NAD(P)H:quinone
oxidoreductase 1 (NQO1): NQO1 inhibition and growth inhibitory activity in human
pancreatic MIA PaCa-2 cancer cells. Biochemistry 46, 5941-5950.
Reigan, P., Colucci, M.A., Siegel, D., Chilloux, A., Moody, C.J., and Ross, D. (2007b).
Development of indolequinone mechanism-based inhibitors of NAD(P)H:quinone
oxidoreductase 1 (NQO1): NQO1 inhibition and growth inhibitory activity in human
pancreatic MIA PaCa-2 cancer cells. Biochemistry 46, 5941-5950.
Reinicke, K.E., Bey, E.A., Bentle, M.S., Pink, J.J., Ingalls, S.T., Hoppel, C.L., Misico,
R.I., Arzac, G.M., Burton, G., Bornmann, W.G., et al. (2005). Development of beta-
229
lapachone prodrugs for therapy against human cancer cells with elevated
NAD(P)H:quinone oxidoreductase 1 levels. Clin Cancer Res 11, 3055-3064.
Rodriguez-Cuenca, S., Cochemé, H.M., Logan, A., Abakumova, I., Prime, T.A., Rose,
C., Vidal-Puig, A., Smith, A.C., Rubinsztein, D.C., Fearnley, I.M., et al. (2010).
Consequences of long-term oral administration of the mitochondria-targeted antioxidant
MitoQ to wild-type mice. Free Radic Biol Med 48, 161-172.
Roos, W.P., and Kaina, B. (2006). DNA damage-induced cell death by apoptosis. Trends
in molecular medicine 12, 440-450.
Ross, M.F., Kelso, G.F., Blaikie, F.H., James, A.M., Cochemé, H.M., Filipovska, A., Da
Ros, T., Hurd, T.R., Smith, R.A., and Murphy, M.P. (2005). Lipophilic
triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical
biology. Biochemistry (Mosc) 70, 222-230.
Rountree, C.B., Ding, W., He, L., and Stiles, B. (2009). Expansion of CD133-expressing
liver cancer stem cells in liver-specific phosphatase and tensin homolog deleted on
chromosome 10-deleted mice. Stem Cells 27, 290-299.
Rozenblat, S., Grossman, S., Bergman, M., Gottlieb, H., Cohen, Y., and Dovrat, S.
(2008). Induction of G2/M arrest and apoptosis by sesquiterpene lactones in human
melanoma cell lines. Biochemical pharmacology 75, 369-382.
230
Rudolph, J. (2007). Cdc25 phosphatases: structure, specificity, and mechanism.
Biochemistry 46, 3595-3604.
Saavedra, J.E., Srinivasan, A., Buzard, G.S., Davies, K.M., Waterhouse, D.J., Inami, K.,
Wilde, T.C., Citro, M.L., Cuellar, M., Deschamps, J.R., et al. (2006). PABA/NO as an
anticancer lead: analogue synthesis, structure revision, solution chemistry, reactivity
toward glutathione, and in vitro activity. J Med Chem 49, 1157-1164.
Sahu, R., Zhang, R., Batra, S., Shi, Y., and Srivastava, S. (2009a). Benzyl isothiocyanate-
mediated generation of reactive oxygen species causes cell cycle arrest and induces
apoptosis via activation of MAPK in human pancreatic cancer cells. Carcinogenesis 30,
1744-1753.
Sahu, R.P., Zhang, R., Batra, S., Shi, Y., and Srivastava, S.K. (2009b). Benzyl
isothiocyanate-mediated generation of reactive oxygen species causes cell cycle arrest
and induces apoptosis via activation of MAPK in human pancreatic cancer cells.
Carcinogenesis 30, 1744-1753.
Samlowski, W.E., Petersen, R., Cuzzocrea, S., Macarthur, H., Burton, D., McGregor,
J.R., and Salvemini, D. (2003). A nonpeptidyl mimic of superoxide dismutase, M40403,
inhibits dose-limiting hypotension associated with interleukin-2 and increases its
antitumor effects. Nat Med 9, 750-755.
231
Sasada, T., Iwata, S., Sato, N., Kitaoka, Y., Hirota, K., Nakamura, K., Nishiyama, A.,
Taniguchi, Y., Takabayashi, A., and Yodoi, J. (1996). Redox control of resistance to cis-
diamminedichloroplatinum (II) (CDDP): protective effect of human thioredoxin against
CDDP-induced cytotoxicity. J Clin Invest 97, 2268-2276.
Sawai, H., Okada, Y., Funahashi, H., Matsuo, Y., Takahashi, H., Takeyama, H., and
Manabe, T. (2005). Activation of focal adhesion kinase enhances the adhesion and
invasion of pancreatic cancer cells via extracellular signal-regulated kinase-1/2 signaling
pathway activation. Mol Cancer 4, 37.
Schafer, F.Q., and Buettner, G.R. (2001). Redox environment of the cell as viewed
through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol
Med 30, 1191-1212.
Schimmel, M., and Bauer, G. (2002). Proapoptotic and redox state-related signaling of
reactive oxygen species generated by transformed fibroblasts. Oncogene 21, 5886-5896.
Schneider, B., and Kulesz-Martin, M. (2004). Destructive cycles: the role of genomic
instability and adaptation in carcinogenesis. Carcinogenesis 25, 2033-2044.
Schumacker, P.T. (2006). Reactive oxygen species in cancer cells: live by the sword, die
by the sword. Cancer Cell 10, 175-176.
232
Scudiero, D.A., Monks, A., and Sausville, E.A. (1998). Cell line designation change:
multidrug-resistant cell line in the NCI anticancer screen. Journal of the National Cancer
Institute 90, 862.
Sen, B., Saigal, B., Parikh, N., Gallick, G., and Johnson, F.M. (2009). Sustained Src
inhibition results in signal transducer and activator of transcription 3 (STAT3) activation
and cancer cell survival via altered Janus-activated kinase-STAT3 binding. Cancer Res
69, 1958-1965.
Seux, M., Peuget, S., Montero, M.P., Siret, C., Rigot, V., Clerc, P., Gigoux, V.,
Pellegrino, E., Pouyet, L., N'Guessan, P., et al. (2011). TP53INP1 decreases pancreatic
cancer cell migration by regulating SPARC expression. Oncogene 30, 3049-3061.
Shabaik, Y.H., Millard, M., and Neamati, N. (2013). Mechanistic evaluation of a novel
small molecule targeting mitochondria in pancreatic cancer cells. PLoS One 8, e54346.
Shackelford, R., Kaufmann, W., and Paules, R. (2000). Oxidative stress and cell cycle
checkpoint function. Free Radic Biol Med 28, 1387-1404.
Sheldrick, G.M. (1997). SHELXL97. Program for the Refinement of Crystal Structures.
University of Gottingen, Germany.
233
Shen, H.M., and Liu, Z.G. (2006). JNK signaling pathway is a key modulator in cell
death mediated by reactive oxygen and nitrogen species. Free Radic Biol Med 40, 928-
939.
Shen, L., Kim, S.H., and Chen, C.Y. (2012). Sensitization of human pancreatic cancer
cells harboring mutated K-ras to apoptosis. PLoS One 7, e40435.
Sheveleva, E.V., Landowski, T.H., Samulitis, B.K., Bartholomeusz, G., Powis, G., and
Dorr, R.T. (2012). Imexon induces an oxidative endoplasmic reticulum stress response in
pancreatic cancer cells. Mol Cancer Res 10, 392-400.
Shimojo, Y., Akimoto, M., Hisanaga, T., Tanaka, T., Tajima, Y., Honma, Y., and
Takenaga, K. (2013). Attenuation of reactive oxygen species by antioxidants suppresses
hypoxia-induced epithelial-mesenchymal transition and metastasis of pancreatic cancer
cells. Clin Exp Metastasis 30, 143-154.
Sieg, D.J., Hauck, C.R., Ilic, D., Klingbeil, C.K., Schaefer, E., Damsky, C.H., and
Schlaepfer, D.D. (2000). FAK integrates growth-factor and integrin signals to promote
cell migration. Nat Cell Biol 2, 249-256.
Siegel, D., Shieh, B., Yan, C., Kepa, J.K., and Ross, D. (2011). Role for
NAD(P)H:quinone oxidoreductase 1 and manganese-dependent superoxide dismutase in
234
17-(allylamino)-17-demethoxygeldanamycin-induced heat shock protein 90 inhibition in
pancreatic cancer cells. J Pharmacol Exp Ther 336, 874-880.
Simamura, E., Shimada, H., Hatta, T., and Hirai, K. (2008). Mitochondrial voltage-
dependent anion channels (VDACs) as novel pharmacological targets for anti-cancer
agents. J Bioenerg Biomembr 40, 213-217.
Singh, N.P., and Lai, H.C. (2004). Artemisinin induces apoptosis in human cancer cells.
Anticancer Res 24, 2277-2280.
Siwak, D.R., Shishodia, S., Aggarwal, B.B., and Kurzrock, R. (2005). Curcumin-induced
antiproliferative and proapoptotic effects in melanoma cells are associated with
suppression of IkappaB kinase and nuclear factor kappaB activity and are independent of
the B-Raf/mitogen-activated/extracellular signal-regulated protein kinase pathway and
the Akt pathway. Cancer 104, 879-890.
Slater, E.P., Langer, P., Niemczyk, E., Strauch, K., Butler, J., Habbe, N., Neoptolemos,
J.P., Greenhalf, W., and Bartsch, D.K. (2010). PALB2 mutations in European familial
pancreatic cancer families. Clin Genet 78, 490-494.
Smith, R.A., Hartley, R.C., and Murphy, M.P. (2011). Mitochondria-targeted small
molecule therapeutics and probes. Antioxid Redox Signal 15, 3021-3038.
235
Smith, R.A., Porteous, C.M., Gane, A.M., and Murphy, M.P. (2003). Delivery of
bioactive molecules to mitochondria in vivo. Proc Natl Acad Sci U S A 100, 5407-5412.
Song, H., Cha, M.J., Song, B.W., Kim, I.K., Chang, W., Lim, S., Choi, E.J., Ham, O.,
Lee, S.Y., Chung, N., et al. (2010). Reactive oxygen species inhibit adhesion of
mesenchymal stem cells implanted into ischemic myocardium via interference of focal
adhesion complex. Stem Cells 28, 555-563.
Soubani, O., Ali, A.S., Logna, F., Ali, S., Philip, P.A., and Sarkar, F.H. (2012). Re-
expression of miR-200 by novel approaches regulates the expression of PTEN and MT1-
MMP in pancreatic cancer. Carcinogenesis 33, 1563-1571.
Spitz, D.R., Sim, J.E., Ridnour, L.A., Galoforo, S.S., and Lee, Y.J. (2000). Glucose
deprivation-induced oxidative stress in human tumor cells. A fundamental defect in
metabolism? Ann N Y Acad Sci 899, 349-362.
Storey, K.B. (1996). Oxidative stress: animal adaptations in nature. Braz J Med Biol Res
29, 1715-1733.
Storz, P. (2005). Reactive oxygen species in tumor progression. Front Biosci 10, 1881-
1896.
236
Suh, Y.A., Arnold, R.S., Lassegue, B., Shi, J., Xu, X., Sorescu, D., Chung, A.B.,
Griendling, K.K., and Lambeth, J.D. (1999). Cell transformation by the superoxide-
generating oxidase Mox1. Nature 401, 79-82.
Summy, J.M., and Gallick, G.E. (2003). Src family kinases in tumor progression and
metastasis. Cancer Metastasis Rev 22, 337-358.
Sun, Y., St Clair, D.K., Xu, Y., Crooks, P.A., and St Clair, W.H. (2010). A NADPH
oxidase-dependent redox signaling pathway mediates the selective radiosensitization
effect of parthenolide in prostate cancer cells. Cancer Res 70, 2880-2890.
Suy, S., Mitchell, J.B., Samuni, A., Mueller, S., and Kasid, U. (2005). Nitroxide tempo, a
small molecule, induces apoptosis in prostate carcinoma cells and suppresses tumor
growth in athymic mice. Cancer 103, 1302-1313.
Szatrowski, T.P., and Nathan, C.F. (1991). Production of large amounts of hydrogen
peroxide by human tumor cells. Cancer Res 51, 794-798.
Tang, H., Hao, Q., Rutherford, S.A., Low, B., and Zhao, Z.J. (2005). Inactivation of SRC
family tyrosine kinases by reactive oxygen species in vivo. J Biol Chem 280, 23918-
23925.
237
Teoh, M.L., Sun, W., Smith, B.J., Oberley, L.W., and Cullen, J.J. (2007). Modulation of
reactive oxygen species in pancreatic cancer. Clin Cancer Res 13, 7441-7450.
Thimmulappa, R.K., Mai, K.H., Srisuma, S., Kensler, T.W., Yamamoto, M., and Biswal,
S. (2002). Identification of Nrf2-regulated genes induced by the chemopreventive agent
sulforaphane by oligonucleotide microarray. Cancer Res 62, 5196-5203.
Thummel, R.P., Chirayil, S., Hery, C., Lim, J.L., and Wang, T.L. (1993). Incorporation
of the quinoline-5,8-quinone moiety into polyaza cavities. J Org Chem 58, 1666–1671.
Tibodeau, J.D., Benson, L.M., Isham, C.R., Owen, W.G., and Bible, K.C. (2009). The
anticancer agent chaetocin is a competitive substrate and inhibitor of thioredoxin
reductase. Antioxid Redox Signal 11, 1097-1106.
Tournier, C., Hess, P., Yang, D.D., Xu, J., Turner, T.K., Nimnual, A., Bar-Sagi, D.,
Jones, S.N., Flavell, R.A., and Davis, R.J. (2000). Requirement of JNK for stress-induced
activation of the cytochrome c-mediated death pathway. Science 288, 870-874.
Townsend, D.M., He, L., Hutchens, S., Garrett, T.E., Pazoles, C.J., and Tew, K.D.
(2008). NOV-002, a glutathione disulfide mimetic, as a modulator of cellular redox
balance. Cancer Res 68, 2870-2877.
238
Toyokuni, S. (1998). Oxidative stress and cancer: the role of redox regulation. Biotherapy
11, 147-154.
Toyokuni, S., Okamoto, K., Yodoi, J., and Hiai, H. (1995). Persistent oxidative stress in
cancer. FEBS Lett 358, 1-3.
Trachootham, D., Alexandre, J., and Huang, P. (2009). Targeting cancer cells by ROS-
mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Discov 8, 579-591.
Trachootham, D., Zhou, Y., Zhang, H., Demizu, Y., Chen, Z., Pelicano, H., Chiao, P.J.,
Achanta, G., Arlinghaus, R.B., Liu, J., et al. (2006). Selective killing of oncogenically
transformed cells through a ROS-mediated mechanism by beta-phenylethyl
isothiocyanate. Cancer Cell 10, 241-252.
Trachte, A.L., Suthers, S.E., Lerner, M.R., Hanas, J.S., Jupe, E.R., Sienko, A.E., Adesina,
A.M., Lightfoot, S.A., Brackett, D.J., and Postier, R.G. (2002). Increased expression of
alpha-1-antitrypsin, glutathione S-transferase pi and vascular endothelial growth factor in
human pancreatic adenocarcinoma. Am J Surg 184, 642-647; discussion 647-648.
Vad, N.M., Yount, G., Moore, D., Weidanz, J., and Moridani, M.Y. (2009). Biochemical
mechanism of acetaminophen (APAP) induced toxicity in melanoma cell lines. J Pharm
Sci 98, 1409-1425.
239
Vad, N.M., Yount, G., and Moridani, M.Y. (2008). Biochemical mechanism of
acetylsalicylic acid (Aspirin) selective toxicity toward melanoma cell lines. Melanoma
Res 18, 386-399.
Vafa, O., Wade, M., Kern, S., Beeche, M., Pandita, T.K., Hampton, G.M., and Wahl,
G.M. (2002). c-Myc can induce DNA damage, increase reactive oxygen species, and
mitigate p53 function: a mechanism for oncogene-induced genetic instability. Mol Cell 9,
1031-1044.
Valko, M., Leibfritz, D., Moncol, J., Cronin, M., Mazur, M., and Telser, J. (2007). Free
radicals and antioxidants in normal physiological functions and human disease. Int J
Biochem Cell Biol 39, 44-84.
Van Driel, B.E., Lyon, H., Hoogenraad, D.C., Anten, S., Hansen, U., and Van Noorden,
C.J. (1997). Expression of CuZn- and Mn-superoxide dismutase in human colorectal
neoplasms. Free Radic Biol Med 23, 435-444.
Vaquero, E.C., Edderkaoui, M., Pandol, S.J., Gukovsky, I., and Gukovskaya, A.S.
(2004). Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in
pancreatic cancer cells. J Biol Chem 279, 34643-34654.
240
Verheij, M., Bose, R., Lin, X.H., Yao, B., Jarvis, W.D., Grant, S., Birrer, M.J., Szabo, E.,
Zon, L.I., Kyriakis, J.M., et al. (1996). Requirement for ceramide-initiated SAPK/JNK
signalling in stress-induced apoptosis. Nature 380, 75-79.
Verrax, J., Stockis, J., Tison, A., Taper, H.S., and Calderon, P.B. (2006). Oxidative stress
by ascorbate/menadione association kills K562 human chronic myelogenous leukaemia
cells and inhibits its tumour growth in nude mice. Biochemical pharmacology 72, 671-
680.
Wambi-Kiesse, C.O., and Katusic, Z.S. (1999). Inhibition of copper/zinc superoxide
dismutase impairs NO.-mediated endothelium-dependent relaxations. Am J Physiol 276,
H1043-1048.
Wang, L.H., Yang, X.Y., Zhang, X., An, P., Kim, H.J., Huang, J., Clarke, R., Osborne,
C.K., Inman, J.K., Appella, E., et al. (2006). Disruption of estrogen receptor DNA-
binding domain and related intramolecular communication restores tamoxifen sensitivity
in resistant breast cancer. Cancer Cell 10, 487-499.
Wang, L.H., Yang, X.Y., Zhang, X., Mihalic, K., Fan, Y.X., Xiao, W., Howard, O.M.,
Appella, E., Maynard, A.T., and Farrar, W.L. (2004). Suppression of breast cancer by
chemical modulation of vulnerable zinc fingers in estrogen receptor. Nat Med 10, 40-47.
241
Wang, M.L., Walsh, R., Robinson, K.L., Burchard, J., Bartz, S.R., Cleary, M., Galloway,
D.A., and Grandori, C. (2011). Gene expression signature of c-MYC-immortalized
human fibroblasts reveals loss of growth inhibitory response to TGFbeta. Cell Cycle 10,
2540-2548.
Wang, Z.Y., and Chen, Z. (2008). Acute promyelocytic leukemia: from highly fatal to
highly curable. Blood 111, 2505-2515.
Warburg, O. (1956). On the origin of cancer cells. Science 123, 309-314.
Wardman, P. (2001). Electron transfer and oxidative stress as key factors in the design of
drugs selectively active in hypoxia. Curr Med Chem 8, 739-761.
Waring, P., Sjaarda, A., and Lin, Q.H. (1995). Gliotoxin inactivates alcohol
dehydrogenase by either covalent modification or free radical damage mediated by redox
cycling. Biochemical pharmacology 49, 1195-1201.
Wartenberg, M., Hoffmann, E., Schwindt, H., Grünheck, F., Petros, J., Arnold, J.,
Hescheler, J., and Sauer, H. (2005). Reactive oxygen species-linked regulation of the
multidrug resistance transporter P-glycoprotein in Nox-1 overexpressing prostate tumor
spheroids. FEBS Lett 579, 4541-4549.
242
Weinberg, F., and Chandel, N. (2009). Reactive oxygen species-dependent signaling
regulates cancer. Cell Mol Life Sci 66, 3663-3673.
Wells, W.W., Rocque, P.A., Xu, D.P., Meyer, E.B., Charamella, L.J., and Dimitrov, N.V.
(1995). Ascorbic acid and cell survival of adriamycin resistant and sensitive MCF-7
breast tumor cells. Free Radic Biol Med 18, 699-708.
Welsh, S., Williams, R., Kirkpatrick, L., Paine-Murrieta, G., and Powis, G. (2004).
Antitumor activity and pharmacodynamic properties of PX-478, an inhibitor of hypoxia-
inducible factor-1alpha. Molecular cancer therapeutics 3, 233-244.
Weydert, C.J., Waugh, T.A., Ritchie, J.M., Iyer, K.S., Smith, J.L., Li, L., Spitz, D.R., and
Oberley, L.W. (2006). Overexpression of manganese or copper-zinc superoxide
dismutase inhibits breast cancer growth. Free Radic Biol Med 41, 226-237.
Wilkes, G. (2007). Peripheral neuropathy related to chemotherapy. Semin Oncol Nurs 23,
162-173.
Wissner, A., and Mansour, T.S. (2008). The development of HKI-272 and related
compounds for the treatment of cancer. Arch Pharm (Weinheim) 341, 465-477.
243
Wolf, S., Barton, D., Kottschade, L., Grothey, A., and Loprinzi, C. (2008).
Chemotherapy-induced peripheral neuropathy: prevention and treatment strategies. Eur J
Cancer 44, 1507-1515.
Wondrak, G.T. (2007). NQO1-activated phenothiazinium redox cyclers for the targeted
bioreductive induction of cancer cell apoptosis. Free Radic Biol Med 43, 178-190.
Wondrak, G.T. (2009). Redox-directed cancer therapeutics: molecular mechanisms and
opportunities. Antioxid Redox Signal 11, 3013-3069.
Wood, L., Leese, M.R., Leblond, B., Woo, L.W., Ganeshapillai, D., Purohit, A., Reed,
M.J., Potter, B.V., and Packham, G. (2001). Inhibition of superoxide dismutase by 2-
methoxyoestradiol analogues and oestrogen derivatives: structure-activity relationships.
Anticancer Drug Des 16, 209-215.
Wu, F.Y., and Sun, T.P. (1999). Vitamin K3 induces cell cycle arrest and cell death by
inhibiting Cdc25 phosphatase. Eur J Cancer 35, 1388-1393.
Wu, L.L., Chiou, C.C., Chang, P.Y., and Wu, J.T. (2004). Urinary 8-OHdG: a marker of
oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics. Clin
Chim Acta 339, 1-9.
244
Wu, W. (2006). The signaling mechanism of ROS in tumor progression. Cancer
Metastasis Rev 25, 695-705.
Xiao, D., Powolny, A.A., and Singh, S.V. (2008). Benzyl isothiocyanate targets
mitochondrial respiratory chain to trigger reactive oxygen species-dependent apoptosis in
human breast cancer cells. J Biol Chem 283, 30151-30163.
Yagoda, N., von Rechenberg, M., Zaganjor, E., Bauer, A.J., Yang, W.S., Fridman, D.J.,
Wolpaw, A.J., Smukste, I., Peltier, J.M., Boniface, J.J., et al. (2007). RAS-RAF-MEK-
dependent oxidative cell death involving voltage-dependent anion channels. Nature 447,
864-868.
Yang, S., Irani, K., Heffron, S.E., Jurnak, F., and Meyskens, F.L., Jr. (2005). Alterations
in the expression of the apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/Ref-
1) in human melanoma and identification of the therapeutic potential of resveratrol as an
APE/Ref-1 inhibitor. Molecular cancer therapeutics 4, 1923-1935.
Yang, W.S., and Stockwell, B.R. (2008). Synthetic lethal screening identifies compounds
activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer
cells. Chem Biol 15, 234-245.
Yezhelyev, M.V., Koehl, G., Guba, M., Brabletz, T., Jauch, K.W., Ryan, A., Barge, A.,
Green, T., Fennell, M., and Bruns, C.J. (2004). Inhibition of SRC tyrosine kinase as
245
treatment for human pancreatic cancer growing orthotopically in nude mice. Clin Cancer
Res 10, 8028-8036.
Yokoi, K., Hawke, D., Oborn, C.J., Jang, J.Y., Nishioka, Y., Fan, D., Kim, S.W., Kim,
S.J., and Fidler, I.J. (2011). Identification and validation of SRC and phospho-SRC
family proteins in circulating mononuclear cells as novel biomarkers for pancreatic
cancer. Transl Oncol 4, 83-91.
Zhang, H., Mizumachi, T., Carcel-Trullols, J., Li, L., Naito, A., Spencer, H.J., Spring,
P.M., Smoller, B.R., Watson, A.J., Margison, G.P., et al. (2007). Targeting human 8-
oxoguanine DNA glycosylase (hOGG1) to mitochondria enhances cisplatin cytotoxicity
in hepatoma cells. Carcinogenesis 28, 1629-1637.
Zhang, J., Yang, P.L., and Gray, N.S. (2009). Targeting cancer with small molecule
kinase inhibitors. Nat Rev Cancer 9, 28-39.
Zhang, R., Humphreys, I., Sahu, R., Shi, Y., and Srivastava, S. (2008a). In vitro and in
vivo induction of apoptosis by capsaicin in pancreatic cancer cells is mediated through
ROS generation and mitochondrial death pathway. Apoptosis 13, 1465-1478.
Zhang, R., Humphreys, I., Sahu, R.P., Shi, Y., and Srivastava, S.K. (2008b). In vitro and
in vivo induction of apoptosis by capsaicin in pancreatic cancer cells is mediated through
246
ROS generation and mitochondrial death pathway. Apoptosis : an international journal on
programmed cell death 13, 1465-1478.
Zhang, R., Loganathan, S., Humphreys, I., and Srivastava, S. (2006a). Benzyl
isothiocyanate-induced DNA damage causes G2/M cell cycle arrest and apoptosis in
human pancreatic cancer cells. J Nutr 136, 2728-2734.
Zhang, R., Loganathan, S., Humphreys, I., and Srivastava, S.K. (2006b). Benzyl
isothiocyanate-induced DNA damage causes G2/M cell cycle arrest and apoptosis in
human pancreatic cancer cells. The Journal of nutrition 136, 2728-2734.
Zhou, H., Huang, L., Sun, Y., and Rigas, B. (2009a). Nitric oxide-donating aspirin
inhibits the growth of pancreatic cancer cells through redox-dependent signaling. Cancer
letters 273, 292-299.
Zhou, H., Huang, L., Sun, Y., and Rigas, B. (2009b). Nitric oxide-donating aspirin
inhibits the growth of pancreatic cancer cells through redox-dependent signaling. Cancer
Lett 273, 292-299.
Zhou, J., and Du, Y. (2012). Acquisition of resistance of pancreatic cancer cells to 2-
methoxyestradiol is associated with the upregulation of manganese superoxide dismutase.
Mol Cancer Res 10, 768-777.
247
Zhou, J.H., Cheng, H.Y., Yu, Z.Q., He, D.W., Pan, Z., and Yang, D.T. (2011).
Resveratrol induces apoptosis in pancreatic cancer cells. Chinese medical journal 124,
1695-1699.
Zhou, Y., Hileman, E.O., Plunkett, W., Keating, M.J., and Huang, P. (2003). Free radical
stress in chronic lymphocytic leukemia cells and its role in cellular sensitivity to ROS-
generating anticancer agents. Blood 101, 4098-4104.
Ziech, D., Franco, R., Georgakilas, A.G., Georgakila, S., Malamou-Mitsi, V.,
Schoneveld, O., Pappa, A., and Panayiotidis, M.I. (2010). The role of reactive oxygen
species and oxidative stress in environmental carcinogenesis and biomarker development.
Chem Biol Interact 188, 334-339.
Zorov, D., Juhaszova, M., and Sollott, S. Mitochondrial ROS-induced ROS release: an
update and review. Biochim Biophys Acta 1757, 509-517.
Zorov, D.B., Juhaszova, M., and Sollott, S.J. (2006). Mitochondrial ROS-induced ROS
release: an update and review. Biochim Biophys Acta 1757, 509-517.
Zou, G.M., and Maitra, A. (2008). Small-molecule inhibitor of the AP endonuclease
1/REF-1 E3330 inhibits pancreatic cancer cell growth and migration. Molecular cancer
therapeutics 7, 2012-2021.

Abstract (if available)

Abstract Altered cellular bioenergetics and oxidative stress are emerging hallmarks of most cancers including pancreatic cancer. Elevated levels of intrinsic reactive oxygen species (ROS) in tumors make them more susceptible to exogenously induced oxidative stress. Excessive oxidative insults overwhelm their adaptive antioxidant capacity and trigger ROS-mediated cell death. Recently, we have discovered two novel classes of compounds, triphenylphosphoniums and quinazoline-5,8-diones that exert their cytotoxic effects by modulating ROS-mediated signaling (This dissertation mainly focuses on quinazolinediones). ❧ Compound 3a was identified through a medium throughput screen of ~1000 highly diverse in-house compounds and chemotherapeutic agents for their ability to alter cellular bioenergetics. Further structural optimizations led to the discovery of a more potent analogue, 3b that displayed anti-proliferative activities in low micromolar range in both drug-sensitive and drug-resistant cancer cells. Treatment with 3b causes Akt activation resulting in increased cellular oxygen consumption and oxidative stress in pancreatic cancer cells. Moreover, oxidative stress induced by 3b promoted activation of stress kinases (p38/JNK) resulting in cancer cell death. Treatment with antioxidants was able to reduce cell death confirming ROS-mediated cytotoxicity. Since our compounds exert Akt-dependent ROS-mediated cell death, they may provide potential therapeutic options for chemoresistant and Akt-overexpressing cancers. ❧ Pancreatic cancer is a complex disease characterized by alterations in several key regulators of signaling pathways. Increased expression and activity of Src and FAK have been observed in pancreatic cancer and linked to its inherent and acquired chemoresistance. Sustained Src inhibition leads to reactivation of survival pathways regulated by STAT3 resulting in resistant forms of cancer. Therefore, targeting the Src/FAK axis could provide an important strategy for the treatment of pancreatic cancer. Mechanistic evaluation of compound 3b revealed that it potently decreases Src/FAK and STAT3 phosphorylation leading to inhibition of cancer cell migration and angiogenesis. Therefore, we named our lead compound 3b, sarkostat™. Furthermore, sarkostat arrests cell cycle progression and causes apoptotic cell death in cancer cells in low micromolar range. Additionally, sarkostat overcomes the limitations of Src inhibitors by decreasing STAT3 activity. ❧ Simultaneous studies with triphenylphosphoniums (TPs) revealed their OCR decreasing effects in cancer cell lines. Interestingly, TPs also induced oxidative stress and activated stress kinases leading to apoptosis. Moreover, TPs inhibited Src/FAK complex and decreased cell migration and invasion in pancreatic cancer cells. ❧ Lastly, we have studied one of the bystander effects of ROS generating therapy, chemotherapy induced peripheral neuropathy. It is attributed to the oxidative stress and mitochondrial dysfunction resulting from chemotherapeutic agents. We have designed and synthesized novel redox modulators tagged to triphenylphosphonium moiety. Our novel mitochondrial targeted redox modulators exert protective antioxidant effects without interfering with the anticancer effects of chemodrugs. In conclusion, we have discovered and characterized promising agents with unique mechanisms that show great potential as a therapy for pancreatic cancer.

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Asset Metadata

Creator Pathania, Divya (author)

Core Title Targeting cellular redox modulations for pancreatic cancer treatment

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 08/04/2013

Defense Date 06/13/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cellular oxygen consumption rate,chemotherapy induced peripheral neuropathy,OAI-PMH Harvest,oxidative stress,pancreatic cancer,quinazolinediones,reactive oxygen species,small molecule anticancer drugs,triphenylphosphoniums

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Neamati, Nouri (committee chair), Cadenas, Enrique (committee member), Haworth, Ian S. (committee member), Stiles, Bangyan L. (committee member)

Creator Email divyapathania84@gmail.com,pathania@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-314353

Unique identifier UC11292728

Identifier etd-PathaniaDi-1945.pdf (filename),usctheses-c3-314353 (legacy record id)

Legacy Identifier etd-PathaniaDi-1945.pdf

Dmrecord 314353

Document Type Dissertation

Format application/pdf (imt)

Rights Pathania, Divya

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA