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Development of novel small molecules targeting mitochondrial and oxidative stress signaling pathways for pancreatic cancer therapy

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Development of novel small molecules targeting mitochondrial and oxidative stress signaling pathways for pancreatic cancer therapy

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DEVELOPMENT OF NOVEL SMALL MOLECULES TARGETING
MITOCHONDRIAL AND OXIDATIVE STRESS SIGNALING PATHWAYS FOR
PANCREATIC CANCER THERAPY

by

Yumna Hosam Shabaik

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 PHILOSOPHY
(PHARMACEUTICAL SCIENCES)

May 2013

Copyright 2013 Yumna Hosam Shabaik
ii

“It always seems impossible until it's done.”

~ Nelson Mandela

iii

DEDICATION
I dedicate this work to my loving parents for whom no amount of gratitude and
appreciation can ever suffice.
iv

ACKNOWLEDGMENTS
I would like to acknowledge God first and foremost for granting me the resources and the
strength to achieve my goals. Secondly and most importantly, I would like to
acknowledge my beloved parents, Dr. Hosam E. Shabaik and Mrs. Nadia Eltantawy. I
love you more than words can express and I’m ever grateful for your endless love and
encouragement.
I would like to sincerely thank my mentor Dr. Nouri Neamati for welcoming me in to his
dynamic and successful research group back when I was a first year graduate student. I
have learnt a lot during my time as his student.
I would also like to extend my thanks and appreciation to my committee members Dr.
Roger Duncan, Dr. Bangyan Stiles, Dr. Julio Camarero and Dr. Ebrahim Zandi for their
kind guidance and support during various stages of my research.
I would like to thank all of my friends and colleagues in the Neamati lab, past and
present, for making the lab a wonderful second home. I am grateful to Melissa Millard,
Adrian Esqueda, Dr. Tino Sanchez and Dr. Francesca Aiello for their collaboration on
various studies included in this dissertation.
A special thank you is reserved for my friends Kavya Ramkumar, Divya Pathania, Helen
Ha, Rasha Alsafi and Natasha Sharma and for my brothers Kareem, Omar, Ahmed and
Mohammed for their love and support.
v

TABLE OF CONTENTS
Epigraph ii
Dedication iii
Acknowledgments iv
List of Tables vii
List of Figures viii
Abstract xi
Preface xiii
Chapter One: Introduction 1
1.1 Pancreatic cancer 1
1.2 Current treatment options and challenges 2
1.3 “Autophagy addiction” and mitochondrial dependence 3
1.4 Overview of autophagy 5
1.5 Targeting mitochondria in cancer 6
1.6 Using delocalized lipophilic cations to achieve mitochondrial targeting 7
1.7 A novel class of triphenylphosphonium salts 11

Chapter Two: Anti-proliferative properties of TP421 and selectivity for cancer
versus normal 12
2.1 Materials and Methods 12
2.2 Cytotoxicity of TP421and closely related analogs 15
2.3 Selectivity of TP421for cancer over normal cell lines 20
2.4 Effects of TP421 and analogs on cell cycle progression 22
2.5 Discussion and summary 25

Chapter Three: Sub-cellular localization of TP421 and biological consequences 27
3.1 Materials and Methods 27
3.2 Mitochondrial localization of TP421 in cancer cells 31
3.3 Effect of TP421 on mitochondrial respiration and cellular
bioenergetics 35
3.4 ROS accumulation 37
3.5 ROS mediated DNA damage 40
3.6 ROS induced cell death 42
3.7 Discussion and summary 44

vi

Chapter Four: Evaluation of TP421 mechanism of action 48
4.1 Materials and Methods 48
4.2 Proteomics analysis of TP421 treatment 53
4.3 Activation of MAPKs 58
4.4 Induction of apoptosis 59
4.5 Inhibition of autophagy 61
4.6 Inhibition of migration 66
4.7 Discussion and summary 72

Chapter Five: Identification of novel small molecule inhibitors of APE1
mediated DNA repair as sensitizers to DNA damaging chemotherapies 75
5.1 Introduction 75
5.1.1 Background 75
5.1.2 Rationale 78
5.2 Materials and Methods 81
5.3 Combination treatment with a mitochondria targeted agent and an
APE1 inhibitor 84
5.4 Specific inhibition of APE1 by 3-carbamoylbenzoic acid
derivatives 85
5.5 Cytotoxicity of 3-carbamoylbenzoic acid derivatives in
combination with MMS or 5-FdUrd 98
5.6 Discussion and summary 102

Chapter Six: Conclusions 104
6.1 Summary and significance 104
6.2 Future studies 105

Bibliography 107

vii

LIST OF TABLES

Table 2.1: Phenotype and genotype of cell lines used 15

Table 2.2: TP421 IC50 values in pancreatic cancer cell lines 16

Table 2.3: TP421 IC50 values by duration of drug exposure and length
of incubation 18

Table 2.4: Percent distribution of DNA content per cell cycle phase of
pancreatic cancer cell lines 24

Table 4.1: Antibody microarray results of proteins involved in
FAK/Src mediated cell migration signaling 67

Table 5.1: Inhibition of APE1 endonuclease activity by compounds
F325-FM262 90

Table 5.2: Inhibition of APE1 endonuclease activity by compounds
F326D-F350D 92

Table 5.3: Inhibition of APE1 endonuclease activity by F-1, F-2, F-3,
F-5, F-6 and F-7 93

Table 5.4: Inhibition of APE1 endonuclease activity by compounds
F3330bis – F-4 94

Table 5.5: Compounds exhibiting no activity against APE1
endonuclease activity 95

viii

LIST OF FIGURES

Figure 1.1: The % survival (5-year) of pancreatic cancer 1

Figure 1.2: Mitochondrial uptake of TPP cations 8

Figure 1.3: Examples of TPP conjugated molecules studied for
anticancer properties 10

Figure 1.4: Structures of TP compounds 11

Figure 2.1: Colony formation of pancreatic cancer cells treated with
TP421 17

Figure 2.2: Cell line survival curves for long and short TP421 exposure
durations 19

Figure 2.3: TP421 inhibits growth of MIA PaCa-2 cells but not HFF-1 20

Figure 2.4: TP421 cytotoxicity is selective for cancer cells 21

Figure 2.5: TP421 arrests pancreatic cancer cells in G0/G1 phase of the
cell cycle 23

Figure 3.1: Fluorescence of TP421 detected in PANC-1 cells 31

Figure 3.2: Accumulation and retention of TP421 in mitochondria 32

Figure 3.3: Effect of mitochondrial polarization on TP421 accumulation
and cytotoxicity 34

Figure 3.4: TP421 decreases mitochondrial respiration 36

Figure 3.5: TP421 causes accumulation of H
2
O
2
in BxPC-3 but not in
MIA PACa-2 cells 38

Figure 3.6: TP421 causes accumulation of O
2
-
in BxPC-3 and MIA
PACa-2 cells 39

Figure 3.7: Effect of TP421 on H2A.X phosphorylation up to 8 h 40

Figure 3.8: Effect of TP421 on H2A.X phosphorylation for prolonged
period of time 41
ix

Figure 3.9: Effect of NAC and IM-54 pretreatment on TP421
cytotoxicity 43

Figure 4.1: IPA statistical ranking of canonical signaling pathways
altered by TP421 55

Figure 4.2: The top ranked molecular mechanisms of cancer pathway 56

Figure 4.3: The second ranking apoptosis pathway identified by IPA 57

Figure 4.4: Effect of TP421 on MAPK signaling pathways 59

Figure 4.5: Apoptosis activation by TP421 60

Figure 4.6: TP421 affects autophagy in pancreatic cancer cells 62

Figure 4.7: LC3B puncta formation in TP421 treated MIA PaCa-2 cells 63

Figure 4.8: LysoTracker Red staining of TP421 treated MIA PaCa-2
cells 65

Figure 4.9: TP421 decreases signaling via Src-FAK 68

Figure 4.10: TP421 inhibits cell migration 69

Figure 4.11: TP421 inhibits wound healing 70

Figure 4.12: TP421 decreases phosphorylation of Stat3 in a time and
dose dependent manner 71

Figure 5.1: Sequence of events in BER 76

Figure 5.2: Schematic illustration of the effects of ROS on APE1 in
cells 80

Figure 5.3: TP421 combined with an APE1 inhibitor achieves
synergistic cell killing 84

Figure 5.4: Synthesized compounds with descriptive pharmacophore
properties 85

Figure 5.5: Endonuclease activity of APE1 86
x

Figure 5.6: Representative IN gel 87

Figure 5.7: Representative APE1 gel 88

Figure 5.8: H630 cells treated with MMS and F332D in combination 98

Figure 5.9: Combination treatments of F332D and several genotoxic
agents 99

Figure 5.10: Effect of combination treatment of 5-FdUrd and select
APE1 inhibitors on HT-29 cell survival 101

xi

ABSTRACT
Pancreatic cancer is one of the deadliest cancers with a 5-year survival rate of 6%.
Therapeutic options against this disease are limited and there is a critical unmet need for
safe and efficacious treatments. Cancer cell metabolism and mitochondria provide
unexplored targets for this disease. Here-in we describe the identification of a novel class
of triphenylphosphonium salts, TP compounds, which target the mitochondria of cancer
cells and display broad- spectrum anticancer properties. We examined the ability of our
prototypical compound TP421 to inhibit the growth of pancreatic cancer cells and further
investigated the molecular mechanisms by which it exerts its anticancer effects. TP421
showed sub-micromolar IC50 values in all the pancreatic cancer cell lines tested using
MTT and colony formation assays. TP421 localized predominantly to mitochondria and
induced G0/G1 arrest, ROS accumulation, and activation of several stress regulated
kinases. Multiple caspases and PARP-1 cleavage were observed indicating an apoptotic
response while LC3B-II and p62 were accumulated indicating inhibition of autophagy.
Furthermore, TP421 induced de-phosphorylation of key signaling molecules involved in
FAK mediated adhesion that correlated with inhibition of cell migration.
We also report the identification of a novel class of inhibitors of the essential
base excision repair enzyme apurinic/apyrimidinic (AP) endonuclease (APE1). APE1 is a
multi-faceted protein with an essential role in the base excision repair (BER) pathway. To
protect cell genomes from potentially mutagenic or cytotoxic base damage arising from
various exogenous or endogenous sources, APE1 nicks the DNA backbone 5’ to AP sites
generated primarily by lesion-specific glycosylases which remove damaged bases. Its
xii

implication in tumor development, progression and resistance has been confirmed in
multiple cancers making it a viable target of intense investigation. Here we have designed
and synthesized different classes of small molecule selective inhibitors of APE1’s
catalytic endonuclease function containing a 3-carbamoylbenzoic scaffold. Further
structural modifications have been made with the aim of increasing activity and
cytotoxicity of these inhibitors. Several of our compounds exhibited low micro-molar
potencies towards inhibiting APE1’s catalytic endonuclease function in vitro and thus
represent novel classes of APE1 inhibitors worthy of further development.
xiii

PREFACE

The overarching goal of the research project detailed in this dissertation is to
develop potent small molecules based on novel therapeutic approaches for use against
highly refractory cancers. To that end we have identified a novel class of small
molecules, designated TP compounds, which target the mitochondria of cancer cells and
exhibit sub-micromolar potency against pancreatic cancer.
In chapter 2, I provide a thorough analysis of the anti-proliferative properties of
TP421, the prototype and lead compound for this class of mitochondrial-targeted agents. I
also examine the ability of TP421 to selectively decrease viability of cancer cells over
normal cells.
In chapter 3, I studied the mitochondrial accumulation of TP421 and its
implication on cellular viability, bioenergetics and redox state. The fluorescent
microscopy and the bioenergetics portions of the study were kindly conducted by my
friends and colleagues in the lab, Melissa Millard and Divya Pathania, respectively. A
portion of the work presented in this chapter has been published in the journal PLoS One
under the title “Preclinical evaluation of novel triphenylphosphonium salts with broad-
spectrum activity”.
In chapter 4, I aimed to characterize the mechanism of action of my compound by
utilizing antibody microarray experimentation to achieve a global perspective of
proteomic changes occurring in response to TP421 treatment. The results of the
microarray were scrutinized using the powerful IPA software to identify and rank lead
xiv

signaling pathways affected by TP421. I followed up the microarray results with more
robust molecular biology techniques including western blotting, immunofluorescent
microscopy and cell migration assays to confirm TP421’s effect on key regulators in the
identified signaling pathways and to explore other pancreatic cancer-relevant pathways
including autophagy. Immunofluorescent microscopy presented in this chapter was also
skillfully conducted by Melissa. A version of the work presented in this chapter and
chapters 2 and 3 has been published in the journal PLoS One under the title “Mechanistic
evaluation of a novel small molecule targeting mitochondria in pancreatic cancer cells”.
Finally, in chapter 5, I explore the development of inhibitors targeting the DNA
repair function of the essential cellular enzyme APE1. This chapter is of particular
interest as APE1 has emerged as a key mediator of tumor resistance to many currently
utilized DNA-interacting chemotherapies and as such APE1 inhibitors are being heavily
researched with aim of using them to sensitize cells to classical chemotherapy regimens.
My interest is two-fold in this regard, as I propose the use of such repair inhibitors as
adjuvant therapy to mitochondrialy targeted anticancer agents. The mechanism of these
agents often includes generation of mitochondrial reactive oxygen species (ROS) in
cancer cells and thus the cytotoxicity of such treatments could benefit from the inhibition
of proteins involved in ameliorating damage inflicted by accumulating ROS. This
includes the repair of oxidative base damage and SSB repair of both nuclear and
mitochondrial DNA accomplished by APE1. A version of the work presented in this
chapter was published in the journal ChemMedChem under the title “Design and
synthesis of 3-carbamoylbenzoic acid derivatives as inhibitors of human
xv

apurinic/apyrimidinic endonuclease 1 (APE1)”. This study was carried out in
collaboration with Dr. Francesca Aiello who synthesized the compounds. Additionally,
the radiolabeled enzyme inhibition experiments were conducted by Adrian Esqueda, M.S.
and Tino W. Sanchez, Ph.D.
1

CHAPTER ONE: INTRODUCTION
1.1 Pancreatic cancer
Pancreatic cancer is currently the fourth leading cause of cancer related deaths in
the United States. According to the American Cancer Society, the overall 5-year survival
rate is 6% and the incidence rate is on the rise while those for other major cancers are
declining. Furthermore, the survival rate has not appreciably improved in over 30
years (Howlander et al., 2011). In contrast, the 5-year survival rate for cancers of
the brain and leukemia, which have yearly incidence rates similar to pancreatic
cancer, have steadily improved over the same period (Figure 1.1). With the estimated
43,920 new cases and 37,390 deaths for 2012, there is a clear unmet need for
development of effective treatments to improve the survival of diagnosed patients (ACS,
2012).

Figure 1.1 The % survival (5 year) by
year of diagnosis for pancreatic cancer
and cancers with similar yearly
incidence rates.
2

1.2 Current treatment options and challenges
A minority 20% of diagnosed patients with localized, non-metastatic disease
undergo surgical resection with adjuvant therapy and have a more favorable 5-year
survival rate of 20-25% (Neoptolemos et al., 2010). However, the majority of pancreatic
cancer patients are diagnosed at later stages of the disease progression, with significant
local and distant metastases, and are therefore not eligible for surgical resection. For
those patients, the only FDA approved treatment available is gemcitabine, a pyrimidine
analog, which is often combined with erlotinib, a kinase inhibitor, resulting in a modest
improvement in survival (1-year survival rates of 17% versus 23%) (Moore et al., 2007;
Senderowicz et al., 2007). However, since the approval of erlotinib as an adjuvant
therapy in 2005, no new drugs have been approved for treating PDAC. Countless other
studies have been conducted in attempts to achieve statistically significant clinical benefit
by combining gemcitabine with other chemo- or biologic therapies but benefits proved
minimal and have failed to lead to FDA approvals (Warsame and Grothey, 2012).
Gemcitabine targets ribonucleotide reductase causing depletion of
deoxyribonucleotide triphosphates (dNTPs) and its active metabolite, gemcitabine
triphosphate, is also incorporated into DNA leading to masked chain termination and a
halt in DNA synthesis (Plunkett et al., 1995). On the other hand erlotinib, inhibits
epidermal growth factor receptor (EGFR) signaling and has also been documented to be a
multi-kinase inhibitor (Conradt et al., 2011).
The pathway for gemcitabine activity is sufficiently complicated requiring
nucleoside uptake transporters and intracellular phosphorylation which contributes to the
3

low rate of response in patients and the increasing development of chemo-resistance
(Hung et al., 2012). The observed resistance can be intrinsic or acquired during therapy.
For instance, loss of cell surface expression and activity of the human concentrative
nucleoside transporter 1 (hCNT1) occurs frequently in pancreatic cancer and has been
directly correlated with gemcitabine resistance (Bhutia et al., 2011). Furthermore,
acquired gemcitabine chemoresistance has been linked to mutational deactivation and
reduced expression of deoxycytidine kinase (dCK), the enzyme responsible for
phosphorylating and hence activating gemcitabine in cells (Nakano et al., 2007; Saiki et
al., 2012).
Similarly, the use of erlotinib to treat pancreatic cancer patients is faced with
several challenges. EGFR receptor in pancreatic cancer is often over-expressed and is
associated with advanced disease progression and poor clinical outcome (Xiong and
Abbruzzese, 2002). However, response to erlotinib seems to be only marginally affected
by EGFR expression in pancreatic cancer cell lines (Conradt et al., 2011). Positive co-
expression of ErbB3 seems more important for predicting response to erlotinib (Frolov et
al., 2007) but this receptor’s role in pancreatic cancer tumorgenesis and the clinical
significance of its expression in pancreatic adenocarcinoma remain poorly understood
(Liles et al., 2010).
1.3 “Autophagy addiction” and mitochondria dependence
A model for pancreatic cancer progression, arising from histologically well-
defined precursor lesions, was proposed more than a decade ago (Hruban et al., 2000).
Combined with a set of chronologic oncogenic mutations and inactivation of tumor
4

suppressor genes, these advances shed light on the disease’s etiology and provide a
platform for development of novel targeted therapeutics.
Among the earliest molecular changes underlying pancreatic cancer is a
constitutively activating k-ras mutation that occurs in nearly 100% of cases and is
considered the initiating mutation (Hezel et al., 2006). During transformation, k-ras
signaling drives excessive cell proliferation and promotes survival. It has been proposed
that mitochondrial energy production is essential in supporting ras-transformed cells that
become heavily reliant on autophagy, a state referred to as “autophagy addiction”, to
maintain a healthy pool of mitochondria and sufficient citric acid cycle intermediates to
support oxidative phosphorylation (OXPHOS) (Guo et al., 2011; Yang et al., 2011).
Notably, in pancreatic cancer cell lines and patient samples, the basal level of autophagy
is elevated as compared to normal cells or cells from other tumor cell lines and is
correlated with poorer clinical outcomes (Fujii et al., 2008; Yang et al., 2011). This
phenotype, characteristic of ras-transformed cells, makes them uniquely susceptible to
disruption of mitochondrial respiration and autophagy. In fact, pharmacological
inhibition as well as silencing of key autophagy genes has successfully resulted in
reduction of mitochondrial oxygen consumption and intracellular ATP levels leading to
profound inhibition of pancreatic cancer growth both in vitro and in vivo (Yang et al.,
2011). Therefore, inhibition of autophagy and mitochondrial targeting could provide a
new approach for treating PDACs that are usually highly refractory to available
chemotherapies.

5

1.4 Overview of autophagy
Autophagy has recently garnered wide interest with respect to cancer research. It
is a highly conserved and regulated process by which damaged or long lived cellular
content is sequestered and degraded via lysosomal hydrolases. Autophagy begins with the
formation of a double membrane structure serving as an isolation membrane (a.k.a
phagophore) which envelops cytosolic content as it extends. Eventually completely
enclosing material, the outer membrane, of now fully formed autophagosomes, fuse with
lysosomes and deliver their contents to the hydrolases for degradation and subsequent
recycling (Kondo et al., 2005).
Originally identified as a cell survival mechanism of content recycling during
periods of nutrient deprivation, autophagy can also contribute to cell death via excessive
degradation of organelles and proteins. Because of these seemingly disparate roles of
autophagy in promoting both cell survival and cell death, multiple evidences exist
supporting either its inhibition or induction as strategies for anticancer therapy. It is
suggested that the contribution of autophagy to cell fate might vary depending on the
tumor type with certain cancers being more reliant on the process for survival as
compared to others (Kimmelman, 2011). Therefore, it follows that the appropriate course
of therapeutic modulation would be differentially determined by the specific cancer being
treated. In the case of pancreatic cancer, autophagy plays a key role in sustaining tumor
proliferation and its inhibition leads to profound reduction of pancreatic cancer growth
both in vitro as well as in vivo (Yang et al., 2011).
6

Autophagy plays an important role in regulating mitochondria under basal
conditions and during cellular stress. In fact, mitophagy – a specific autophagic pathway
that selectively delivers mitochondria, while sparing other organelles and cytosolic
content, to the lysosomal compartment for degradation – has been described (Kim et al.,
2007). For example, mitophagy is responsible for total mitochondrial clearance during
reticulocyte cell development and is accomplished using autophagy machinery (Zhang
and Ney, 2010). Aside from its role in cellular homeostasis, mitophagy may be initiated
in part by loss of mitochondrial membrane potential (Tolkovsky, 2009). Mitochondria
that become dysfunctional and can no longer generate ATP, can become troublesome
sources of damaging reactive oxygen species (ROS). In order to circumvent initiation of
apoptosis, cells attempt to rescue the situation by eliminating damaged mitochondria via
targeted autophagic elimination of dysfunctional mitochondria, however if the damage is
extensive, apoptosis ensues (Hirota et al., 2012). On the other hand, excessive
degradation of mitochondria by autophagy may be detrimental to cell survival as it
depletes cells of their sources for ATP. Although the role of mitophagy in tumor
progression is unclear, targeted disruption of mitochondrial energy production and
induction of mitophagy has been proposed as a novel mechanism for anticancer therapy
(Gargini et al., 2011). However, inhibition of mitophagy may also be an attractive option
if it occurs in concert with induction of excessive mitochondrial damage.
1.5 Targeting mitochondria in cancer
There has been a recent surge in interest for targeting cancer cell mitochondria
following the recognition of their altered bioenergetic status as a contributor to cancer
7

pathogenesis (Gogvadze, 2011). The use of mitochondrial targeted agents for anticancer
therapies presents an added benefit of directly acting upon the main regulator of
programmed cell death within the cell and entirely bypassing the upstream signaling
cascades that are often undermined (Fulda and Kroemer, 2011). Furthermore, a popular
but belief in cancer biology that mitochondria are inherently defective in generating ATP
and that the majority of the cell’s energy is supplied via aerobic glycolysis, a phenotype
known as the Warburg effect, has been disproven (Koppenol et al., 2011). While an
increase in aerobic glycolysis does indeed occur in most solid tumors, mitochondria have
been shown to contribute a significant fraction – and in some cases the majority – of total
cellular ATP content in various cancer types (Zu and Guppy, 2004). In such cancer cells,
use of mitochondrial OXPHOS inhibitors strongly inhibit cell proliferation as compared
to glycolytic inhibitors which have no effect (Rodriguez-Enriquez et al., 2006). These
indications support the strategy of targeting mitochondria to disrupt ATP supply as a
viable mechanism to achieve anticancer outcomes.
1.6 Using delocalized lipophilic cations to achieve mitochondrial targeting
Targeting mitochondria has emerged as a new ideal for anticancer therapy aided
in part by the knowledge of achieving precise delivery of drugs to the organelle using
delocalized lipohilic cations (DLCs) as a targeting moiety.
DLCs can easily traverse hydrophobic biological membranes due to their charge
distribution over a large lipophilic surface area. Once inside the cells, DLCs accumulate
almost entirely in mitochondria due to the large (negative inside) membrane potential
across mitochondrial inner membranes (Figure 1.2) (Murphy, 2008). It has been well
8

documented that mitochondria of malignant cells exhibit an even higher transmembrane
potential as compared to non-malignant cells with differences in enzyme activities,
electron carriers and membrane lipid structure as potential underlying causes (Modica-
Napolitano and Aprille, 2001). Exploiting this unique attribute, delocalized lipophilic
cationic molecules can preferentially accumulate in tumor cell mitochondria over normal
tissue driven by the increased transmembrane potential and provides an advantageous
tumor selectivity during therapy.

Figure 1.2 Mitochondrial uptake of TPP cations is higher in cancer cells. The plasma and
mitochondrial membrane potentials contribute to the accumulation of TPP cations. The larger
mitochondrial membrane potential observed in cancer cells allows TPP to become more
highly concentrated within their mitochondria as compared to normal cells. Adapted from
(Murphy, 2008).
9

Conjugation of triphenylphosphonium (TPP) cations, a type of DLC, to a variety
of chemical probes and drugs is widely used to achieve this type of specific and selective
targeting to the mitochondria (Biasutto et al., 2008; Biswas et al., 2012b; Burns et al.,
1995; Cocheme et al., 2007; Dong et al., 2011b). Perhaps the best understood TPP
conjugate extensively studied is MitoVES, a vitamin E analog with potent cytotoxic
activity (Figure 1.3). MitoVES targets the proximal ubiquitin binding site of complex II
in the electron transport chain and exhibits far enhanced apoptotic and anti-angiogenic
activity over that of the parental un-tagged compound (Dong et al., 2011a; Dong et al.,
2011b; Rohlena et al., 2011). Two other examples are MitoQ and Mito-CP
11
which act in
an antioxidant capacity to retard proliferation of various cancer cells in vitro with good
selectivity over non-cancerous cells (Cheng et al., 2011; Cocheme et al., 2007; Rao et al.,
2010).
Alternatively, drug delivery carriers have been modified with TPP cations and
utilized to target cargo to mitochondria (Biswas et al., 2012a; Biswas et al., 2012b; Malhi
et al., 2012; Wang et al., 2011). Two examples are the surface conjugation of STPP and
TPP-PEG-200-PE on to lipososmes (Figure 1.3). When these carriers were loaded with
model chemotherapeutic drugs including paclitaxel or doxorubicin, efficacy was
enhanced over non-targeted carriers. These studies further demonstrate that delivery to
the mitochondria can improve apoptotic and cytotoxic drug properties and provide a
secondary modality for designing mitochondrial-targeted therapies.

10

Figure 1.3 Examples of TPP conjugated molecules studied for anticancer properties. MitoVES,
MitoQ and Mito-CP 11 exhibit cytotoxic activity while STPP and TPP-PEG-200-PE are used as
conjugation materials for targeting carriers loaded with cytotoxic cargo to the mitochondria.
11

1.7 A novel class of triphenylphosphonium salts
Through our routine screening of small molecule chemical libraries we identified
a novel and potently cytotoxic class of compounds defined by the presence of the TPP
moiety. Several compounds belonging to this class, which we designated as TP
compounds, showed impressive cytotoxicity against a panel of pancreatic cancer cell
lines and therefore warranted further development. The prototypical analog, TP421,
represented the lead compound and was further tested in vitro to characterize its
biological activity. The results of this testing are the main focus of this dissertation. The
structures of TP421 and two other members of the TP class of compounds which we
utilized in our studies are shown below in Figure 1.4.

Figure 1.4 Structures of the lead compound TP421 and two of its analogs.
12

CHAPTER TWO: ANTI-PROLIFERATVE PROPERTIES OF TP421 AND
SELECTVITY FOR CANCER VERSUS NORMAL

2.1 Materials and Methods
Cell lines and culture reagents
MIA PaCa-2, PANC-1, BxPC-3 and HPAC pancreatic cancer cell lines were purchased
from the American Type Culture Collection (ATCC; Manassas, VA). MEF atg3 +/+ and
atg3 -/- murine cell lines were a gift from Masaaki Komatsu (Juntendo University School
of Medicine, Bunkyo-ku, Tokyo) (Sou et al., 2008). HFF-1 normal fibroblast cell line
was provided by Dr. Carla Grandori (Fred Hutchinson Cancer Research Center, Seattle,
WA). All cell lines used for experimentation were maintained in culture under 35
passages and tested regularly for mycoplasma contamination using PlasmoTest™
(InvivoGen, San Diego, CA). MIA PaCa-2 and PANC-1 cells were maintained in DMEM
supplemented with 10% fetal bovine serum (FBS; Gemini-Bioproducts, West
Sacramento, CA). BxPC-3, MEF atg3 +/+ and atg3 -/- cells were maintained in RPMI
supplemented with 10% FBS. HPAC cells were maintained in 1:1 mixture of DMEM and
Ham's F12 medium supplemented with 5 % FBS. Cells were grown at 37°C in a
humidified atmosphere of 5% CO2. For all experiments, cells in exponential growth
phase were rinsed with DPBS without calcium and magnesium, briefly trypsinized in a
small volume of 0.25% trypsin-EDTA solution (Sigma-Aldrich, St. Louis, MO), re-
suspended in complete culture media, manually counted and seeded on to sterile plates
and allowed to adhere overnight before treating.
13

Compounds
All compounds were stored as concentrated DMSO stock frozen at -20 °C. Dilutions
were prepared in DPBS, without calcium and magnesium, and re-used for a maximum of
3 freeze-thaw cycles. Compounds were purchased from LKT laboratories Inc. (St. Paul,
MN), Sigma-Aldrich Corp. (Saint Louis, MO) and Asinex Ltd. (Moscow, Russia).
Cytotoxicity assay
Cytotoxicity was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) colorimetric assay. Briefly, cells were seeded in 96-well tissue culture
treated plates at a density of 2,000 cells per well and allowed to adhere overnight. The
next day, cells were treated with drugs as described for the indicated time periods. At the
end of the treatment course, MTT solution was added to each well at a final concentration
of 0.3 mg/mL. Cells were then further incubated with MTT for 3–4 hours at 37°C to
allow viable cells to take up and reduce the dye. Finally, supernatant was removed from
the wells, DMSO was added to solubilize the converted dye and the absorbance was read
at 570 nm. % Cytotoxicities were determined by comparing the absorbance from drug
treated wells to that of the control untreated wells using the following formula: %
cytotoxicity = 100 * ( 1 - [ Abs(drug treated)/Abs(control untreated) ] ). IC
50
values were
determined from a plot of log drug concentration versus % cytotoxicity.
Colony formation assay
Cells were seeded in 6-well plates (500 - 1000 cells per well depending on the cell line)
and allowed to adhere overnight. The next day, drugs were added at various
14

concentrations to each of the wells for 24 h following which media was replaced with
drug free media. Cells were further incubated for 7 – 14 days to allow colonies to form.
At the end of the incubation period, wells were washed with DPBS, fixed and stained
with a crystal violet solution (0.05% crystal violet, 0.74% formaldehyde and 36%
methanol) for 30 minutes and rinsed in tap water.
Cell cycle determination
Sub-confluent cells seeded in 60 mm dishes were treated continuously with TP421 at
various concentrations and time points. At the end of the treatment, cells were collected
via brief trypsinization, spun down and rinsed with DPBS. Cells were then fixed in 70%
ethanol at - 20°C for at least 4 h. For determining DNA content, samples were spun down
and re-suspended in DPBS containing propidium iodide (50 µg/mL final concentration)
and RNase A (100 µg/mL final concentration) and analyzed by flow cytometry using the
BD LSR II (BD Biosciences, San Jose, CA) equipped with a PE emission detector.
15

2.2 Cytotoxicity of TP421 and closely related analogs
In our initial report on the anticancer activity of this class of compounds we
described their potent cytotoxicity against cancer cell lines of varying lineages (Millard et
al., 2010). Here in, we tested the ability of one of the lead compounds identified form our
initial screen, TP421, to induce cytotoxicity and inhibit cell proliferation in a panel of
pancreatic cancer cell lines. Key properties of the cell lines we used are summarized in
Table 2.1 modified from Deer et al., 2010. Using MTT assay, we evaluated the effect of
72 h continuous exposure to escalating doses of TP421, TP187, and TP197 on the growth
of MIA PaCa-2, BxPC-3, PANC-1 and HPAC cell lines. The results revealed potent
cytotoxicity, with TP421 IC
50
values in the sub-micromolar range in all of the tested cell
lines irrespective of their differences in differentiation state or genetic mutational
background (Table 2.2). Remarkably, the TPP moiety in this compound is essential for
activity as 7-diethylamino-4-methylcoumarin, a structurally identical analog of TP421
lacking the TPP moiety, did not produce any detectable cytotoxicity in the cell lines
tested.
Table 2.1 Key phenotype and genotype features of cell lines used in our studies
Cell line Differentiation
Doubling
time (h)
Commonly mutated genes
K-ras TP53 p16 Smad4
MIA
PaCa-2
poor 40 mutant mutant
homozygous
deletion
wild-type
BxPC-3 moderate – poor 48 - 60
wild-
type
mutant wild-type
homozygous
deletion
PANC-1 poor 52 mutant mutant
homozygous
deletion
wild-type
HPAC moderate 41 mutant wild-type mutant wild-type
16

Table 2.2 IC 50 values (µM) for TP421 and structural analogs in a panel of pancreatic cancer
cell lines
Compound Structure
MIA
PaCa-2
BxPC-3 PANC-1 HPAC
TP187

0.6 ±
0.4
N/A 0.8 ± 0.1
0.9 ±
0.4
TP197

0.2 ±
0.05
N/A
0.6 ±
0.01
1.2 ±
0.7
TP421

0.5 ±
0.3
0.8 ±
0.07
1.1 ± 0.4
0.4 ±
0.02
7-Diethylamino-
4-
methylcoumarin

> 20 > 10 > 10 N/A
Gemcitabine

0.07 ±
0.05
0.04 ±
0.02
> 20 N/A
Erlotinib

23.75 ±
7.4
70 ±
31.4
165 ±
30.5
N/A

We further examined the effect of 24 h exposure to TP421 on the colony forming
capability of MIA PaCa-2 cells as compared to gemcitabine and erlotinib (Figure 2.1).
The results confirmed the ability of TP421 to inhibit cell proliferation in vitro to levels
comparable with gemcitabine and far more potently than erlotinib.
17

Figure 2.1 TP421 significantly inhibits colony formation of pancreatic cancer cells. Effect of 24
h drug exposure on colony forming ability of (A) MIA PaCa-2 and (B) BxPC-3. Images are
representative of three independent experiments.

To further characterize the ability of TP421 to elicit its cytotoxic properties, we
tested the effect of short-term exposure on its IC
50
in cells. We incubated cells in the
presence or absence of a range of concentrations of TP421 for 0.25, 0.5, 1 or 5 h,
followed by incubation in drug-free media for 24, 48 and 72 h. In parallel, we treated
cells with a continuous exposure to TP421 for 24, 48 and 72 h. At the end of incubations
we assessed cell viability via MTT. As expected, the IC
50
of TP421 had an inverse
correlation with the treatment time as well as the total duration of incubation time, as
18

shown in Table 2.3. Unexpectedly however, we observed that with very short exposure
times, as short as 15 min, TP421 was capable of achieving 50% cytotoxicity at 20 µM
when cells were incubated for at least 48 h post initial treatment indicating a very rapid
drug effect. Interestingly, we found that for PANC-1 cells treated in this manner, all of
the shorter drug exposure times greatly reduced the cytotoxicity of TP421 such that even
a 5 h treatment could not produce 50% cytotoxicity after 72 h incubation. The survival
curves comparing continuous and 0.25 h exposure times in the three cell lines are shown
in Figure 2.2.

Table 2.3 IC 50 values (µM) for TP421 in pancreatic cancer cell lines by duration of drug
exposure and length of incubation before addition of MTT reagent
Incubation time (h)
Cell line TP421 exposure (h) 24 48 72
MIA PaCa-2 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
PANC-1 Continuous 4 1.7 1.2
5 >20 >20 >20
1 >20 >20 >20
0.5 >20 >20 >20
0.25 >20 >20 >20
BxPC-3 Continuous 5 2.9 2.1
5 >20 18 5
1 >20 18 14
0.5 >20 20 14
0.25 >20 20 14

19

Figure 2.2 Effect of shorter exposure time on the cytotoxicity of TP421 in pancreatic cancer
cells. Dose response survival curves for cell lines treated with TP421 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. TP421 concentrations are plotted on a
Log base 10 scale.

20

2.3 Selectivity of TP421 for cancer over normal cell lines
Following the assumption that TP421 would selectively accumulate in cancer
cells driven by the higher transmembrane potential, we compared the effect of TP421 on
the growth characteristics of the normal fibroblast cell line HFF-1 against the pancreatic
cancer cell lines. Treating MIA PaCa-2 and HFF-1 cells with a range of doses of TP421
for 72 h revealed distinctly higher sensitivity of the pancreatic cancer cell line towards
the drug (Figure 2.3).

Figure 2.3 TP421 inhibits growth of MIA PaCa-2 cells but not HFF-1. (A) Growth of normal
HFF-1 cells is unaffected while the pancreatic cancer MIA PaCa-2 cells show extensive death
following 72 h exposure to escalating doses of TP421.
The preferential cell killing of cancer cells was further confirmed by measuring
the percentage of dead cells accumulated by TP421 treatment which was significantly
higher in the pancreatic cancer cells as compared to HFF-1 (Figure 2.4A). Further,
utilizing the alamar blue indicator dye, we observed a 4-fold difference in the inhibition
of MIA PaCa-2 cell proliferation as compared to HFF-1 cells following 72 h incubation
period with TP421 at the indicated doses. (Figure 2.5B).
21

Figure 2.4 TP421 cytotoxicity is selective for cancer cells. (A) TP421 induces greater cell death
in pancreatic cancer cell lines as compared to HFF-1 cells as measured by trypan blue
exclusion. (B) Proliferation of MIA PaCa-2 but not HFF-1 is greatly inhibited by TP421 in the
alamar blue assay. mean ± SD are plotted and *, **, *** and **** indicate p-value < 0.05, p <
0.01, p < 0.001 and p < 0.00005 respectively.
22

2.4 Effects of TP421 and analogs on cell cycle progression
In order to elucidate the mechanism by which TP421 inhibits cell proliferation in
the colony formation assay, we evaluated its effect on cell cycle kinetics. MIA PaCa-2
(Figure 2.6), PANC-1, BxPC-3, and HPAC (Table 2.4) cells were treated with two
concentrations of TP421 for increasing durations of time and DNA content was analyzed
using flow cytometry. Despite minor differences in the sensitivity of the cell lines to
TP421, G
0
/G
1
arrest was observed in all the cell lines. In MIA PaCa-2 cells, the most
sensitive to TP421, 78% of the cells were arrested in G
0
/G
1
as early as 24 h following
treatment with 1 µM TP421. Interestingly, the cell cycle distribution for MIA PaCa-2
cells treated with the two other analogs, TP187 and TP197, also resembled TP421 with
significant and early arrest in G
0
/G
1
(Table 2.4). This result differs from the G
2
/M and S-
phase arrest induced by these compounds in non-pancreatic cancer cell lines (Millard et
al., 2010), indicating a potential tumor-type specific effect.

23

Figure 2.5 TP421 arrests pancreatic cancer cells in G0/G1 phase of the cell cycle. The effect of
TP421 treatment on the cell cycle distribution of MIA PACa-2 cells was examined in a dose and
time-dependent manner. Cells were untreated or treated with 0.1 and 1 µM TP421 for 24, 48
and 72 h. Histograms depicted are representative of three independent experiments.
24

25
2.5 Discussion and summary
We have shown that the novel small molecule TP421 exhibits potent anti-
proliferative properties in various pancreatic cancer cell lines utilized in our study. The
potent cell cycle arrest in G0/G1 phase induced by TP421 was independent of p53
mutations and p16 expression status as the cell lines used had variable mutations and or
deletions of these genes. Similarly, TP421 induced cytotoxicity with equal potency in all
of the cell lines we tested. By comparison, gemcitabine and erlotinib exhibited varying
efficacies depending on cell line. Notably, PANC-1 cells are reported to be resistant to
gemcitabine (Tang et al., 2011), which we also observed in our MTT results, but in
comparison TP421 retained all its potency in this cell line and its IC50 was not affected.
This may indicate a potential future clinical advantage for treating diverse patient
populations with TP421. In fact, Collisson et. al. recently proposed that PDAC
stratification into three subtypes, classical, quasimesenchymal and exocrine-like
subtypes, can be used to select specific therapy based on a differential response observed
between the subtypes. Seemingly, this system which also classifies pancreatic cancer
cells lines into the three subtypes provides a good correlation with response to
gemcitabine and erlotinib. The quasimesenchymal subtype is reportedly more sensitive to
gemcitabine than the classical subtype and vice versa for erlotinib. Two of the three
defined subtypes (classical and quasimesenchymal, but not exocrine-like) are represented
among the commonly used pancreatic cancer cell lines, including MIA PaCa-2, PANC-1
(classical) and HPAC (quasimesenchymal) which we utilized in our study and as
discussed, TP421 was similarly effective in the cell lines of both subtypes. Furthermore,
26

the ability of TP421 to induce cytotoxicity following very short treatment durations
demonstrates its capacity to rapidly accumulate within cells thereby affecting cell
survival over prolonged time and supports its prospective clinical efficacy where it can be
dosed intermittently and at lower concentrations. More importantly, we observed that
TP421 was not potently cytotoxic to the normal fibroblast cell line HFF-1 and that the
TPP moeity was essential to the efficacy of TP421. As described in the introduction, TPP
molecules accumulate in the mitochondria of cancer cells to a greater degree than non-
cancer. Therefore our results may be directly related to the ability of TP421 to target the
mitochondria which is the focus of the following chapter.

27

CHAPTER THREE: SUB-CELLULAR LOCALIZATION OF TP421 AND
BIOLOGICAL CONSEQUENCES

3.1 Materials and Methods
Fluorescent detection of TP421 in cells
Sub-confluent PANC-1 cells grown in 60 mm cell culture dishes were collected via brief
trypsinization, spun down and resuspended in DPBS. Cellular fluorescence was then
monitored before and immediately after addition of TP421 to the cell suspension using a
BD LSR II flow cytometer (BD Biosciences, San Jose, CA) equipped with a 355 nm
Lightwave Solid State laser and a DAPI emission detector.
Fluorescent microscopy of mitochondrial localization
PANC-1 cells were seeded in double chambered cover glass (Nalge Nunc International,
Rochester, NY) at a density of 50,000 cells / chamber and allowed overnight to adhere.
The following day, the cells were treated with 2 µM TP421 for time periods up to 72 h.
Prior to imaging, cells were stained for 15 minutes at 37 ºC in humidified atmosphere
containing 5 % CO
2
using 200 nM Mitotracker Red CMXRos live cell mitochondrial
stain (Life Technologies, Grand Island, NY) prepared as a 10x solution in warmed HBSS
in order to visualize mitochondria. Live cells were visualized using a Nikon DAIPHOT
300 inverted microscope (Nikon Instruments, Melville, NY) equipped with DAPI and
Cy3 filter blocks, 10 x eye piece and 100 x / 1.3 Nikon oil immersion lens and super high
pressure mercury lamp. To prevent photobleaching, sample exposure to light was
minimized during image acquisition by engaging neutral density (ND2 and ND4) filters
28

to limit the intensity of light reaching the specimen. Images were captured using a
Photometrics CoolSNAP 9 CCD camera (Roper Scientific, Ottobrun, Germany) and
processed using Q-capture Pro v 5.1.1.14 imaging software (Q imaging corporation,
Surrey, BC, Canada).
Oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR)
Cellular bioenergetics were measured using Seahorse Bioscience XF24 Extracellular
Flux Analyzer. Optimal cell plating density for MDA-MB-435 cells used in this assay
was determined to be 120,000 cells per well in a 24-well XF24 cell culture microplate.
Assay setup was done according to manufacturer guidelines. Working dilutions of the
stock compounds were prepared in assay media before addition to cells. Assay
measurements were recorded at short intervals over a treatment period of seven hours.
Bioenergetics assay medium
The bioenergetics assay medium was prepared by dissolving DMEM base (8.3 g/L,
Sigma, St. Louis, MO) in 500 mL distilled water. 1.85 g of sodium chloride (Sigma, St.
Louis, MO) was dissolved separately in 500 mL of distilled water. Solutions of sodium
chloride and DMEM base were then combined and 20 mL of this combined solution was
replaced with 10 mL of 100x GlutaMax-1 (Gibco), and 10 mL of 100 mM sodium
pyruvate (Sigma-Aldrich, St. Louis, MO). The media was then warmed to 37°C. The pH
of the media was adjusted to 7.4 using 5 M sodium hydroxide (Sigma, St. Louis, MO).
Finally, the media was sterilized by filtration and stored at 4°C for future use at which
time the temperature and pH of the media were again adjusted to 37°C and 7.4,
respectively on the day of the assay.
29

Measurement of hydrogen peroxide levels
The generation of hydrogen peroxide was measured using an Amplex Red enzyme assay.
Cells seeded in 96-well clear bottom black plates were treated for 4 h with the desired
concentration of drug in phenol red-free DMEM medium supplemented with 10% FBS.
At the end of treatment, cells were washed and lysed in DPBS buffer containing 50 µM
Amplex Red (Molecular Probes) and 0.1 units/mL horseradish peroxidase (HRP; Sigma,
St. Louis, MO) and incubated for 30 min at 37°C, protected from light. Following
incubation, fluorescence intensity of each well was determined using a BioTek Synergy
H1 Hybrid Multi-Mode Microplate Reader at 540/590 ex/em wavelengths.
Measurement of superoxide anion levels
Sub-confluent MIA PaCa-2 and BxPC-3 cells were treated with the TP421 at the
indicated dose and time-points. At the end of treatment, cells were stained with 5 µM
MitoSOX Red for 10 min at 37 °C, protected from light. The cells were then collected by
trypsinization and briefly spun down to remove the excess stain containing media. The
pelleted cells were re-suspended in DPBS and the fluorescence intensity was measured
using BD LSR II flow cytometer (BD Biosciences, San Jose, CA) equipped with a PE
emission detector.
Quantification of levels of DNA damage by monitoring p-H2A.X phosphorylation
Cells treated with TP421 were lysed and probed for H2A.X S139 phosphorylation level
by western blotting, described in detail in chapter 4. Membranes were developed using
Super Signal West Dura chemiluminescent substrate (ThermoFisher Scientific, Waltham,
30

MA) and imaged on ChemiDoc™ XRS+ system (Bio-Rad Laboratories, Hercules, CA).
p-H2A.X band intensities were quantified by image densitometry using ImageJ software
and normalized to loading control.
Cytotoxicity assay
Cytotoxicity for drug treatments was determined using the MTT colorimetric assay as
previously described in chapter 2.
31

3.2 Mitochondrial localization of TP421 in cancer cells
TP421 belongs to a class of compounds containing a TPP cation known to
accumulate in mitochondria (Murphy, 2008). In order to confirm mitochondrial
localization, we treated cells with TP421 and stained with a mitochondrial marker to
assess the degree of co-localization. PANC-1 cells were treated with 2 µM TP421 for
various periods of time. Immediately before imaging, cells were stained with 200 nM
MitoTracker Red (MTR) dye to label the mitochondria. Because TP421 is inherently
fluorescent (Figure 3.1), we were able to image the fluorescence of our compound and
overlay the images with MTR signal to determine the degree of co-localization. We
observed significant co-localization of the fluorescent signals for TP421 and MTR by 4 h
and persisting for up to 72 h (Figure 3.2) indicating that TP421 indeed accumulated in
mitochondria and was retained for a prolonged period of time.

Figure 3.1 Fluorescence of TP421 is detected in PANC-1 cells treated with TP421 by flow
cytometry using a 355 nm excitation laser and a DAPI emission detector.
32

Figure 3.2 Accumulation and retention of TP421 in mitochondria. PANC-1 cells treated with
TP421 and stained with MitoTracker Red (MTR) reveal extensive co-localization of TP421 and
mitochondrial marker dye.
33

As secondary verification of mitochondrial localization we sought to determine
the role of the mitochondrial transmembrane potential in the accumulation of TP421 in
cells. Using the uncoupler carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone
(FCCP), an agent commonly used to collapse the mitochondrial transmembrane potential,
we examined the effect on the pattern of TP421 fluorescence. PANC-1 cells were
pretreated for 30 min with 10 µM FCCP followed by a 15 min incubation with TP421.
Control cells received no FCCP and were exposed to 15 min TP421 only. PANC-1 cells
were imaged immediately following exposure to TP421. As expected, in the presence of
FCCP, we observed a distinctly diffuse TP421 fluorescence (Figure 3.3A) that did not
resemble mitochondrial localization. This confirmed that under normal conditions TP421
accumulated within mitochondria driven by the transmembrane potential. Since we had
also previously observed that 7-diethylamino-4-methylcoumarin was not cytotoxic to
cells, we further explored the relationship between mitochondrial localization and TP421
cytotoxicity. We pretreated MIA PaCa-2 cells for 30 min with 10 µM FCCP then added
TP421 for an additional 30 min. At the end of the treatment duration, we washed the cells
and replaced the medium to remove the excess drug and incubated the cells for 24, 48
and 72 h. Finally, MTT was added to measure cell viability. In the presence of FCCP the
cells were significantly protected from TP421 cytotoxicity (Figure 3.3B) indicating that
localization to mitochondria was directly responsible for the drug action.

34

Figure 3.3 Effect of mitochondrial polarization on TP421 accumulation and cytotoxicity. (A)
PANC-1 cells pretreated with FCCP show non-mitochondrial TP421 localization. (B) Reduced
cytotoxicity of TP421 in MIA PaCa-2 cells pretreated with FCCP is seen at 24, 48 and 72 h. The
data are mean ± SD from three independent experiments. * indicates p < 0.01.

35

3.3 Effect of TP421 on mitochondrial respiration and cellular bioenergetics
Having confirmed that our compound accumulates in mitochondria and that its
cytotoxicity is dependent on this, we sought to examine the effect of TP421 treatment on
mitochondrial function. Mitochondria are the main energy source for cells, requiring
adequate oxygen supply for the process of oxidative phosphorylation to generate ATP.
To test if TP421 resulted in impaired mitochondrial respiration, we measured the change
in mitochondrial oxygen consumption rate (OCR) in response to TP421 treatment using
an XF24 Extracellular Flux Analyzer. Data collected in short intervals over a period of 7
h showed that TP421 treatment significantly decreased oxygen consumption by
mitochondria in MDA-MB-435 cells (Figure 3.4A). The reduction in OCR induced by
TP421 was unaffected by subsequent sequential additions of oligomycin (ATP synthase
inhibitor), FCCP (uncoupler) or rotenone (complex I inhibitor) to the cells. Control cells
treated with media alone or media with DMSO exhibited normal baseline oxygen
consumption that was responsive to addition of the various ETC inhibitors.
In addition to monitoring the OCR, we also examined the effect of TP421 on the
extra-cellular acidification rate (ECAR), an indicator the of the level of glycolysis in
cells. Since TP421 could effectively inhibit mitochondrial respiration we expected that
there would be a compensatory increase in glycolysis to meet the energy demands of the
cells. Indeed, we observed a 50% increase in ECAR following addition of TP421 but
which tended to decrease with time to near basal levels (Figure 3.4B). This suggests that
the up-regulation in glycolysis in response to TP421-induced mitochondrial dysfunction
36

may be a short-termed adaptive response that would be incapable of sustaining cellular
energy supply for prolonged periods.

Figure 3.4 TP421 decreases mitochondrial respiration. MDA-MB-435 cells treated with 5 µM
TP421 (green) show lower oxygen consumption rate (A) and higher extra-cellular
acidification rate (B) as compared to the controls, DMEM (red) and DMSO (yellow). Port A: test
compound, Port B: oligomycin (5 ng/mL), Port C: FCCP (1 µM), Port D: rotenone (1 µM)
37

3.4 ROS accumulation
TP compounds cause superoxide (O
2
-
) accumulation in MDA-MB-435 cells
(Millard et al., 2010). However, as compared to O
2
-
, hydrogen peroxide (H
2
O
2
)

is a
relatively more stable and membrane-permeable ROS that can cause lipid, protein, and
DNA damage and participates in signal transduction (Bartz and Piantadosi, 2010).
Therefore, we examined the level of H
2
O
2
in pancreatic cancer cells following TP421
treatment. MIA PaCa-2 and BxPC-3 cells were treated with increasing doses of TP421
for 4 hours followed by a brief incubation with 50 µM Amplex Red in the presence of 0.1
units/mL HRP. In the presence of HRP, Amplex Red reagent reacts with H
2
O
2
in a 1:1
stoichiometry to produce highly fluorescent resorufin. The resulting fluorescence
intensity was measured at excitation and emission wavelengths of 540 nm and 590 nm,
respectively. As expected, treatment resulted in a 2.5-fold increase in H
2
O
2
levels in
BxPC-3 at the highest concentration of TP421 with a good correlation between H
2
O
2

levels and dose of TP421 used (Figure 3.5A). However, no increase in fluorescence could
be detected in MIA PaCa-2 cells at any of the doses of TP421 tested (Figure 3.5B).
To determine if the lack of H
2
O
2
accumulation in MIA PaCa-2 cells was due to an
absence of O
2
-
generation, we used a MitoSOX red assay to measure the level of
mitochondrial O
2
-
in MIA PaCa-2 and compared it to BxPC-3 cells. Cells were treated
with increasing doses of TP421 for 4 h. TP421 induced a similar 3 - 5 fold increase in O
2
-

level in both cell lines (Figure 3.6A) and those levels remained high at 24 h (Figure
3.6B). This suggests that the lack of H
2
O
2
accumulation observed in MIA PaCa-2 cells
was not due to deficient O
2
-
production in these cells.
38

Figure 3.5 TP421 causes accumulation of H 2O 2 in BxPC-3 (A) but not in MIA PACa-2 (B) cells.
* indicates p < 0.05.
39

Figure 3.6 TP421 causes accumulation of O 2
-
in BxPC-3 and MIA PACa-2 at 4 h post-treatment
(A) and is sustained up to 24 h (B). *, ** and *** indicate p < 0.05, p < 0.005 and p < 0.0005
respectively.
40

3.5 ROS mediated DNA damage
ROS cause various forms of DNA damage which include single and double strand
breaks, oxidative base damage and DNA-protein crosslinking (Slupphaug et al., 2003).
Having shown that TP421 can cause the accumulation of various forms of ROS
originating from mitochondria, we examined whether this correlated with DNA damage
in the nucleus. Using western blotting, BxPC-3 and MIA PaCa-2 cells treated with TP421
were examined for phosphorylated histone H2A.X (S139), a commonly used marker of
DNA double strand breaks. First, we examined the levels in a time dependent manner in
the MIA PaCa-2 cell line at earlier time points. There was a slight increase in pH2A.X
seen beginning at 6 to 8 h but did not reach substantial levels (Figure 3.7). Next, we
examined longer treatment durations and with a range of TP421 doses in MIA PaCa-2
and BxPC-3. There was a significant induction of phosphorylation in both cell lines
which occurred earlier in the MIA PaCa-2 cell line than BxPC-3 with a concentration of
at least 2.5 µM TP421 required in both (Figure 3.8).

41

Figure 3.7 Effect of 5 µM TP421 treatment on H2A.X phosphorylation in MIA PaCa-2 cells
treated upto 8 h.

Figure 3.8 Effect of TP421 treatment on DNA damage. BxPC-3 (A) and MIA PaCa-2 (B) cells
treated with increasing concentrations of TP421 for prolonged period of time were probed for
the DNA damage marker phospho-H2A.X (S139).

42

3.6 ROS induced cell death
As excessive ROS is detrimental to cell survival (Chandra et al., 2000), we
examined the potential protective role of an antioxidant in cells exposed to TP421.
PANC-1 cells were pretreated with 15 mM N-acetyl-L- cysteine (NAC) for 2 h prior to
addition of TP421. Following a 24 h incubation period the extent of cytotoxicity was
determined by MTT. Antioxidant pretreatment could protect cells from TP421 induced
cytotoxicity by up to 35% and similar results were observed in MIA PaCa-2 and BxPC-3
cells (Figure 3.9A).
ROS accumulation can lead to necrosis (Zong and Thompson, 2006) and ATP
depletion due to failure of mitochondrial energy production can also switch the mode of
cell death from apoptosis to necrosis (Chandra et al., 2000). Since we observed inhibition
of mitochondrial oxygen consumption, we questioned if necrosis may be occurring in
cells treated with TP421 and mediated by the associated accumulation of ROS. Similarly
as above, we pretreated cells for 1 h with IM-54 (10 µM), an inhibitor that has been
shown to block oxidative stress induced necrosis (Katoh et al., 2005; Sodeoka and Dodo,
2010). Following addition of TP421 for 24 we assessed the amount of cell death by MTT
(Figure 3.9B). While the results revealed a slight protection, the difference was minor
leading us to conclude that ROS induced necrosis was not significantly contributing to
TP421 cytotoxicity.

43

Figure 3.9 (A) Antioxidant pretreatment protects pancreatic cancer cell lines from TP421
cytotoxicity. (B) IM-54 has negligible effect on the cytotoxicity of TP421. *, ** and *** indicate
p < 0.05, p < 0.005 and p < 0.001 respectively.
44

3.6 Discussion and summary
TP421 localization to mitochondria was confirmed and an associated inhibition of
mitochondria respiration was noted. When accumulation of TP421 in mitochondria was
hindered by the use of the uncoupler FCCP to depolarize the mitochondria, the cells were
protected from TP421 cytotoxicity demonstrating that localization at the mitochondria is
critical for inducing potent cytotoxicity. This is further supported by results in the
previous chapter where the structurally identical 7-diethylamino-4-methylcoumarin
lacking the TPP moiety was non-cytotoxic to cells. Our results are in line with reported
findings that specific targeting of cytotoxic compounds to the mitochondria, via TPP
conjugation, can greatly enhance potency (Dong et al., 2011a; Dong et al., 2011b).
Lipophilic cations, including TPP, do not impart cytotoxicity as evidenced by their
development as tumor targeted PET tracers and targeting moieties for mitochondria
protective antioxidants (Madar et al., 2007; Murphy and Smith, 2007). In light of this, our
results validate that localization to the mitochondria is responsible for the cytotoxic
action of TP421 and suggest that its preferential accumulation within tumor mitochondria
will aid its clinical usefulness as a safe and effective anti-neoplastic agent.
To better understand the mechanism by which TP421 acts to cause cytotoxicity,
we extended our previous findings regarding mitochondrial respiratory dysfunction
induced by TP421 by examining the levels of ROS in the mitochondria and cytosol. O
2
-
is
mostly generated at complexes I and III of the OXPHOS chain as a consequence of
electron leakage and inhibitors of these complexes can also cause large amounts of O
2
-
to
be produced (Brand, 2010). Therefore increase in mitochondrial O
2
-
levels can be
45

indicative of inhibition of one of these sites along the OXPHOS chain however other
enzyme reactions can also release O
2
-
in mitochondria but are poorly characterized.
While not examined in this study, we speculate that TP421 may target one of these
complexes supported by the findings that TP421 inhibits mitochondrial oxygen
consumption and induces prolonged O
2
-
accumulation. TP421 produced a sustained
increase of mitochondrial O
2
-
in both MIA PaCa-2 and BxPC-3 cell lines but, only the
latter showed a concomitant increase in H
2
O
2
levels. Under normal cellular homeostatic
conditions O
2
-
is rapidly converted to H
2
O
2
by the action of superoxide dismutases (SOD)
that are present in the mitochondria (MnSOD), cytosol (CuZnSOD) and extracellularly
(EcSOD). However, cancer cells frequently have aberrant antioxidant mechanisms
including diminished SOD expression which might account for the discrepancy in H
2
O
2

accumulation we observed (Oberley and Buettner, 1979; Oberley and Oberley, 1988).
However, as both BxPC-3 and MIA PaCa-2 cells are reported to have similar levels of
SOD enzyme activities it does not explain the apparent impaired conversion of O
2
-
to
H
2
O
2
in the latter cell line (Cullen et al., 2003). Another possibility could be that MIA
PaCa-2 exhibit a stronger inducible antioxidant response as compared to BxPC-3 leading
to more efficient H
2
O
2
detoxification. During oxidative stress the transcription factor NF-
E2-related factor 2 (Nrf2) translocates to the nucleus and binds to antioxidant response
elements (ARE) to activate transcription of target genes involved in reducing intracellular
ROS (Lau et al., 2008). Several pancreatic cancer cell lines including MIA PaCa-2 have
documented Nrf2 overexpression (Lister et al., 2011) and intriguingly Nrf2 maybe up-
regulated in these cells by mutant k-ras which has been demonstrated to increase Nrf2
46

transcription and lead to an elevated antioxidant program (DeNicola et al., 2011). Given
that BxPC-3 is a wildtype k-ras cell line while MIA PaCa-2 harbors an activating
mutation in codon 12 of this gene, a variable Nrf2 response might explain the difference
in the H
2
O
2
levels we detected (Berrozpe et al., 1994).
Using p-H2A.X (S139) as a marker for DSBs, we attempted to correlate the ROS
findings with damage to nuclear DNA. Interestingly, levels of this marker were not
increased until 48 h post exposure to TP421 even though we could detect H
2
O
2
much
earlier in these cells. By comparison, MIA PaCa-2 had a much larger induction of p-
H2A.X and at an earlier time point. Since these results do not correlate with our H
2
O
2
results we are faced with two possibilities. One is that MIA PaCa-2 may be preferentially
accumulating a different species of ROS, other than H
2
O
2
and which may be causing
nuclear DSBs, or secondly, that the p-H2A.X levels are indicating a late apoptotic
response. In fact, phosphorylation of H2A.X at S139 is required for DNA fragmentation
during apoptosis (Cleaver, 2011). This second option seems more likely due to the fact
that phosphorylation increased significantly in both cell lines well after we could detect
increased ROS (at 4 h). As ROS induced DSBs would cause immediate increase in
pH2A.X, this apparent lag between both events suggests that H2A.X phosphorylation
may be occurring in response to apoptosis initiation.
The protective effects of NAC on cells treated with TP421 implicate ROS as a
direct mediator for TP421 cytotoxicity but also reveal that this role is limited and that
other mechanisms not involving ROS must also be occurring. Furthermore, we have ruled
out oxidative stress induced necrosis as means of cell death following TP421 treatment.
47

This is in spite of confirmed H
2
O
2
accumulation in at least one of the pancreatic cancer
cell lines (i.e. BxPC-3) and suggests that apoptosis is the major mode of cell death in
response to TP421 treatment. We explore this possibility as well as consider other effects
of TP421 on essential cancer promoting signaling pathways in the next chapter.
48

CHAPTER FOUR: EVALUATION OF TP421 MECHANISM OF ACTION
4.1 Materials and Methods
Kinexus Antibody Microarray
MIA PaCa-2 cells were treated with 5 µM TP421 for 24 h. Control cells were treated
with vehicle DMSO. At the end of treatment, cells were rinsed twice with DPBS and then
ice-cold lysis buffer was added. The cells were scraped in the buffer and transferred to 15
mL tubes. The lysate was sonicated on ice to shear nuclear DNA and then ultra-
centrifuged at 90,000 g for 30 min at 4°C. The supernatant was aliquoted into clean 1.5
mL microcentrifuge tubes and protein concentration was assessed by the BCA protein
assay. Lysate samples were then analyzed by the Kinex
TM
KAM-1.1 Antibody
Microarray (Kinexus Bioinformatics Corp).
IPA microarray data analysis
Results of the Kinex
TM
antibody microarray were analyzed using the Ingenuity Pathway
Analysis (IPA) software to identify potential intracellular signaling pathways affected by
TP421 treatment. An excel spreadsheet file was uploaded to IPA servers containing
protein ID and associated ratio changes and subjected to a core analysis.
Cell lysates and western blotting analysis
Adherent cell grown in tissue culture treated dishes and exposed to drug at various doses
and time points were collected for analysis by western blotting. Briefly, drug containing
media was discarded, the cells were rinsed with DPBS, and lysed on ice by addition of a
small volume of SDS-containing cell lysis buffer for 15 min. Lysates were collected in
49

eppendorf tubes, sonicated on ice to shear DNA, and quantified using the RC DC protein
assay (Bio-Rad Laboratories, Hercules, CA). Alternatively, cells were lysed using the
RIPA buffer using the same steps followed by protein quantification using the BCA
protein assay. Twenty-five to thirty-five micrograms of cell lysate was loaded in each
lane and subjected to SDS-PAGE and subsequently transferred to Immun-Blot
polyvinylidene fluoride membranes (PVDF; Bio-Rad Laboratories, Hercules, CA).
Following transfer membranes were stained with an amido black irreversible protein
staining solution to visualize even loading. Then membranes were blocked in 5% non-fat
milk in TBST, washed, incubated in primary antibody diluted in either 5% BSA (for Cell
Signaling antibodies) or 5% milk (for Santa Cruz antibodies) overnight at 4°C, washed,
incubated in the appropriate secondary antibody diluted in 5% milk and finally washed
before imaging. All antibodies were purchased from either Cell Signaling Technology
Inc. (Danvers, MA) or Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Protein
detection was carried out using Super Signal West Dura chemiluminescent substrate
(ThermoFisher Scientific, Waltham, MA) and imaged on ChemiDoc™ XRS+ system
(Bio-Rad Laboratories, Hercules, CA). For all western blot data, images are
representative blots chosen from at least two independent experiments.
Immunofluorescent staining of LC3B
MIA PaCa-2 cells were seeded on glass coverslips (VWR International, Radnor, PA) at a
density of 50,000 cells and allowed overnight to adhere. The following day, cells were
treated with 2 µM TP421 in the presence or absence of 5 µM rapamycin or 10 µM
chloroquine for 18 h. At the end of treatment, media was removed and cells were washed
50

with 500 µL 1x PBS prior to fixation with 3.7% formaldehyde for 15 m at RT. Fixed
cells were washed with 500 µL 1x PBS prior and subsequent to permeablization with ice-
cold acetone for 5 minutes at -20C. Coverslips were blocked for 30 min with 1 % bovine
serum albumin (BSA) in PBS to inhibit non-specific antibody binding prior to incubation
overnight at 4°C with antibody raised against LC3B (Cell Signaling Technology,
Beverly, MA) diluted 1:1000 in 1% BSA/PBS. Antibody was removed and coverslips
were washed 500 with µL 1x PBS with gentle agitation. Goat-anti-rabbit Cy5 conjugated
and Goat-anti-mouse Cy3-conjugated antibodies (GE Healthcare Lifesciences,
Pittsburgh, PA) were diluted 1:200 in 1% BSA/PBS and incubated with coverslips for 2
h. Coverslips were again washed with 500 µL 1x PBS with gentle agitation, air-dried and
mounted on pre-cleaned glass slides (Fisher Scientific, Wlatham, MA) using Prolong
Gold anti-fade mounting media (Life Technologies, Grand Island, NY). Images were
obtained using a Lieca SP2 scanning confocal microscope (Leica Microsystems,
Heidelberg, Germany) equipped with 488 nm argon and 633 nM krypton lasers (laser
power was kept to a minimum < 50% of total) and Leica Confocal software v 2.61 (Leica
Microsystems, Heidelberg, Germany).
Fluorescent microscopy of lysosomal localization
Cell handling and imaging was done exactly as described for mitochondrial localization
described in chapter 3. 50 nM of Lysotracker Red DND 99 live cell organelle stain (Life
Technologies, Grand Island, NY) was used to label the lysosomal compartments.

51

In vitro migration assay
Migration of MIA PaCa-2 cells was assayed using 24-well plate cell culture inserts fitted
with transparent PET membranes having 8 µm sized pores (BD Biosciences, San Jose,
CA). 7.5 x 10
4
serum starved MIA PaCa-2 cells were plated in the top chamber in serum
free media and allowed to lightly adhere overnight. On the next day, cells were
stimulated to migrate by adding 10% FBS medium to the lower chamber for 24 h.
Negative control wells received 1% serum medium instead. TP421 treated samples,
received 10% FBS medium in the lower chamber and compound in serum free medium in
the top chamber. After 24 h, those cells which did not migrate and remained adherent to
the top side of the membrane were lightly scrapped off using a Q-tip. The cells which did
migrate to the bottom side of the membrane were stained using Giemsa nuclear stain for
30 minutes at room temperature and washed with deionized water. Images of the stained
membranes were captured from representative fields using Nikon inverted microscope
using a 10X objective.
Wound healing assay
Tissue culture treated 96-well plates were coated with collagen dissolved in 0.2 N acetic
acid (sterile filtered) to a final concentration of 45 µg/mL. The collagen solution was left
in the wells at 4°C overnight. The next day, the collagen was removed, the wells were
washed twice with PBS and then blocked with 2 mg/ml BSA (dissolved in PBS) for 1 h
at room temperature. After blocking, the BSA was removed and the wells were again
washed once with PBS. PANC-1 cells were then seeded at a density of 35,000 cells/well
52

in full serum media and allowed to adhere overnight. Media was then changed to serum-
free media on the second day, and cells were further incubated overnight. Scratches were
made using the edge of a p-200 tip, wells were washed once with PBS. Treated and un-
treated control wells then received 10% FBS containing media and drug was added to
indicated wells. Negative control wells received serum-free media. Wounds were allowed
24 h to close at the end of which, wells were rinsed with DPBS, cells were fixed in 100%
methanol for 10 min and then stained with giemsa nuclear stain for 30 min. Finally, wells
were washed with tap water and wallowed to dry. Wounds were imaged with a 4X
objective.
53

4.2 Proteomics analysis of TP421 treatment
In an effort to understand the mechanism of action of our compound, we sought to
examine its global effect on signaling proteins in MIA PaCa-2 cells. To that end, we
employed the use of the Kinex™ KAM-1.1 Antibody Microarray (Kinexus
Bioinformatics Corp). This antibody array is capable of detecting changes in either the
phosphorylation status or the total abundance of over 400 well characterized proteins
representing a fraction of the total cellular proteome. Specifically, our samples were
screened against 378 pan-specific (measuring total protein abundance) and 273 phospho-
site-specific antibodies.
MIA PaCa-2 cells were treated with either vehicle control (DMSO) or TP421 (5
µM) for 24 hours and then lysed and sent for analysis. The data obtained was
subsequently analyzed using the commercially available software package, IPA
®

(Ingenuity Systems, Inc.) to expose protein-protein interaction networks, which are
generated by connecting the proteins showing significant changes to TP421 treatment and
relating them in biological context. In this way, the experimental data can be scrutinized
for relationships, mechanisms, functions, and pathways of relevance regarding the
mechanism of action of our compound.
Based on the identity of the proteins affected by TP421, we used IPA to generate
a list of the likeliest signaling pathways targeted by our drug. An abridged version of this
list is displayed in the chart in Figure 4.1 illustrating the top ranked pathways in
decreasing statistical significance. Highlighted in green and red are the percentage of
total pathway molecules for which total abundance and/or phosphorylation sites were up-
54

regulated (red) or down-regulated (green) by TP421 treatment. Several of these altered
pathways are directly relevant to cancer biology as they involve cell proliferation (HGF,
IGF-1 and EGF growth factor signaling), cell motility (FAK and integrin signaling), cell
cycle control signaling cascades (cyclins and cell cycle regulation) and cell death
(apoptosis, death receptor and 14-3-3 mediated signaling).
The top two ranking pathways are illustrated in Figures 4.2 and 4.3 below which
highlight the affected proteins within the context of the signaling pathways to which they
belong.
55

56

Figure 4.2. The top ranked molecular mechanisms of cancer pathway provides an overview of
the effects of TP421 treatment on molecules within key signaling pathways implicated in
cancer cell proliferation, survival and apoptosis.

57

Figure 4.3 The second ranking apoptosis pathway identified by IPA contains several
molecules significantly altered by TP421 treatment and implicates this mode of cell death as a
likely mechanism for TP421 induced cytotoxicity in pancreatic cancer cells.
58

4.3 Activation of MAPKs
Modulation of several MAPKs including JNK, p-38 and ERK were identified in
the proteomics screen suggesting TP421 elicits a cellular stress response involving these
pathways. Therefore we took a closer look at the effect on the phosphorylation status of
key residues on these and other proteins in their pathways to deduce the level of signaling
activation induced by our compound. MIA PaCa-2 and BxPC-3 cells were treated with 5
µM TP421 for 1-8 h and kinase activation was assessed by western blotting. MIA PaCa-2
cells had significant and sustained phosphorylation of c-Jun at S73 but only a transient
increase in the phosphorylation of the upstream kinase, JNK1/2 (Figure 4.4A). Similarly,
MIA PaCa-2 and BxPC-3 cells treated with TP421 exhibited enhanced phosphorylation
of the p-38 kinase that increased over time (Figure 4.4B). Finally, we examined the
activation state of the Erk1/2 kinase and its upstream kinase MEK1/2. We observed a
sustained increase in the activating phosphorylation of both kinases that appeared as early
as 1-2 h and persisted for at least 8 h (Figure 4.4C).
59

Figure 4.4 Effect of TP421 on MAPK signaling pathways. (A) MIA PaCa-2 cells treated with
TP421 at increasing time points were probed for the phosphorylation of JNK1/2 and c-Jun. (B)
TP421 treated MIA PaCa-2 and BxPC-3 cells were probed for p-38 phosphorylation. Even
loading for MIA PaCa-2 was verified using amido black total protein staining of membrane
following transfer (D). (C) MIA PaCa-2 cells treated with TP421 were probed for
phosphorylation of MEK1/2 and Erk1/2.

4.4 Induction of apoptosis
The apoptotic pathway was highly ranked by IPA prompting us to examine its
activation via western blotting. TP421 induced apoptosis as demonstrated by the cleavage
of caspases-8 and 7, and PARP-1 as early as 8 h post treatment (Figure 4.5A). Extensive
60

resistance to chemotherapy-induced apoptosis is a complicating factor in treating
pancreatic cancer and has been linked to cellular overexpression of Bcl-2 and other
apoptosis inhibitors (Sheikh et al., 2010). Therefore we also examined the effect of
TP421 treatment on the levels of Bcl-2 and survivin, a potent inhibitor of apoptosis.
TP421 treatment at the indicated times could cause significant reduction of the total
cellular level of both proteins (Figure 4.5B).

61

Figure 4.5 Apoptosis activation by TP421. (A) MIA PaCa-2 cells treated with TP421 were
probed for caspase activation and PARP cleavage. (B) The effect of TP421 treatment on Bcl-2
and survivin levels was analyzed in MIA PaCa-2 cells.

4.5 Inhibition of autophagy
Having established the direct effect of TP421 on mitochondria and because of the
reliance of pancreatic cancer on autophagy to maintain a healthy mitochondrial pool, we
investigated the effects of TP421 on autophagy. Whole cell lysates from MIA PaCa-2
and BxPC-3 cells treated with TP421 for 24 and 48 h were analyzed for the markers of
autophagy LC3B-I/II, p62, and Beclin-1. As a control, cells were treated with the late
stage autophagy inhibitor chloroquine that inhibits fusion of phagosomes and lysosomes
thereby accumulating lipidated LC3B-II in cells. TP421 treatment in both cell lines
caused a dose and time dependent increase in the lower migrating band of LC3B, the
lipidated form that associates with autophagosome membranes (Figure 4.6). This can
indicate an increase in autophagic flux or inhibition of autophagy and therefore
accumulation of autophagosome bound LC3B-II. In order to differentiate between these
two scenarios, we examined the levels of p62, a ubiquitin binding protein that is
selectively degraded during autophagy and its levels increase only if autophagy is
inhibited (Klionsky et al., 2008). Interestingly, TP421 treatment caused a robust
accumulation of p62 in MIA PaCa-2 cells, suggesting that the increase in lipidated LC3B
was indicative of inhibition of autophagy. Accordingly, the levels of Beclin-1, a protein
upstream of both LC3B and p62 in the autophagy pathway that contributes to the
62

initiation of autophagosome formation, decreased with TP421 treatment in a time and
dose-dependent manner and therefore correlated with a decrease in autophagy. On the
other hand, p62 levels in BxPC-3 cells decreased by 48 h following TP421 treatment
indicating a possible induction of autophagy in these cells, albeit only moderately.

Figure 4.6 TP421 affects autophagy in pancreatic cancer cells. MIA PaCa-2 (A) and BxPC-3 (B)
cells were treated with 0.5 and 2.5 µM TP421 or 10 µM chloroquine (CQ) for 24 and 48 h and
probed for Beclin1, p62, and LC3B-I/II.
63

To further confirm inhibition of autophagy in MIA PaCa-2 cells, we fixed and
stained cells treated with TP421 alone or in combination with an autophagy inducer (i.e.
rapamycin) or autophagy inhibitor (i.e. chloroquine) and observed the level of puncta
staining with LC3B antibody (Figure 4.7). TP421 alone could induce accumulation of
LC3B puncta and this was further increased in the presence of rapamycin confirming
TP421’s inhibition of autophagy in these cells. Cells treated with TP421 and chloroquine
resembled chloroquine only treated cells.

Figure 4.7 LC3B puncta formation in MIA PaCa-2 cells treated with TP421 for 18 h alone or in
combination with rapamycin or chloroquine.
To assess if TP421’s effect on autophagy could be modulated in part by a fraction
of its intracellular pool localizing at or interacting with lysosomes, we imaged MIA
PaCa-2 cells treated with 2 µM TP421 for increasing durations of time and observed its
64

location relative to the lysosome specific LysoTracker red dye. Interestingly, TP421 was
not found to localize to lysosomal compartments even as late as 72 h post treatment
(Figure 4.8).
65

Figure 4.8 MIA PaCa-2 cells treated with TP421 for increasing durations of time and co-
stained with LysoTracker Red (LTR).
66

4.6 Inhibition of cell migration
Of the proteins affected by TP421 and identified in the proteomics screen, several
belong to the Src kinase – focal adhesion kinase (FAK) pathway. Src/FAK signaling
plays an important role in mediating cell motility but is also involved in regulating cell
survival, proliferation and differentiation (Lim et al., 2008b). Furthermore, Src levels
have been shown to be elevated in many cancers including pancreatic cancer (Nagaraj et
al., 2010). This observation prompted investigations of Src and FAK inhibitors that
showed anticancer activity as well as ability to inhibit migration, invasion and anchorage-
independent survival of pancreatic cancer cells (Hochwald et al., 2009; Ischenko et al.,
2007; Nagaraj et al., 2010).
We observed that multiple sites on FAK were de-phosphorylated following
TP421 treatment including the main auto-phosphorylation site Y397 which provides the
SH2 binding site for active Src (Lim et al., 2008b) (Table 4.1). Furthermore,
phosphorylation of the negative regulatory site (Y529) on Src was significantly increased
indicating a reduction in kinase activity and this was accompanied by reduced
phosphorylation of FAK residues Y576/577 which are substrates for Src following its
binding to FAK (Lim et al., 2008b). In contrast, phosphorylation at FAK residue S843
was increased which in fact correlates with reduced Y397 phosphorylation (Jacamo et al.,
2007) and further indicates inhibition of Src/FAK signaling by TP421.

67

Table 4.1 Antibody microarray results of proteins involved in FAK/Src mediated cell
migration signaling
Protein Antibody Specificity % CFC Biological effect or function Reference
FAK Pan-specific -10
Initiates signaling at focal adhesions for cell
motility
(Lim et al., 2008b)
Y397 -23
Auto-phosphorylation site of activated FAK.
Creates SH2 binding site for active Src
(Lim et al., 2008b)
Y576 -48 Phosphorylated by activated/recruited Src (Lim et al., 2008b)
Y577 -29 Phosphorylated by activated/recruited Src (Lim et al., 2008b)
S843 26
Phosphorylation at S843 is associated with
decreased phosphorylation at Y397
(Jacamo et al., 2007)
Paxillin Pan-specific 4
Recruited to focal adhesions and acts as a
scaffolding protein.
(Lim et al., 2008b)
Y31 -46 Phosphorylated by active FAK/Src complex
(Deakin and Turner,
2008; Richardson et
al., 1997)
Y118 -22 Phosphorylated by active FAK/Src complex
(Deakin and Turner,
2008; Richardson et
al., 1997)
Src Y529 56
Phosphorylation at Y529 decreases Src kinase
activity
(Young et al., 2002)

Accordingly, we sought to confirm the effect of TP421 on key phosphorylation
events in Src-FAK signaling via western blotting. TP421 treatment caused a decrease in
phosphorylation of Src at residue Y416 which is the activating phosphorylation for Src
activity (Figure 4.9A). However, no change was detected at the negative regulatory
phosphorylation site, residue Y527 of Src. There was a corresponding decrease in
phosphorylation of FAK at residues S576 and Y861 that are downstream of activated Src
and are directly phosphorylated by it (Figure 4.9B). We also observed a decrease in
phosphorylation of p130Cas and paxillin (Figure 4.9C) that are members of a complex of
focal adhesion-associated proteins and are phosphorylated in response to activated Src
and FAK (Lim et al., 2008b).
68

Figure 4.9 TP421 decreases signaling via Src-FAK. (A) MIA PaCa-2 cells treated with 5 µM
TP421 for indicated time and probed for de-activating phosphorylation (Y527) and the
activating phosphorylation (Y416) of Src. (B) MIA PaCa-2 and BxPC-3 cells were treated with 5
µM TP421 and probed for phosphorylation of FAK. (C) Effect of 5 µM TP421 treatment on
phosphorylation status of p130Cas and Paxillin proteins downstream of Src activation.

69

Because we found a robust decrease in the phosphorylation of FAK, Src and
downstream targets, we proceeded to determine if TP421 could inhibit the migration of
MIA PaCa-2 cells in a boyden chamber assay when stimulated with FBS. We treated
serum-starved cells that were seeded in the top chamber with 1 or 5 µM TP421 and added
10% FBS medium in the bottom chamber. The cells were allowed 24 h to migrate. In the
well treated with 5 µM TP421, very few cells were able to migrate to the bottom chamber
as compared to the stimulated un-treated control cells (Figure 4.10A). This difference
was not due to cytotoxicity as cells treated in parallel and analyzed via MTT showed
approximately 70% viability at a 5 µM TP421 dose (Figure 4.10B).

Figure 4.10 TP421 inhibits cell migration. (A) Effect of 24 h TP421 treatment of serum starved
MIA PaCa-2 cells on their ability to migrate through a Boyden Chamber setup. (B) Cell viability
of MIA PaCa-2 cells treated with identical conditions as in (A).
70

We further examined the effect of TP421 on migration of PANC-1 cells using a
scratch assay. PANC-1 cells were seeded onto collagen coated 96-well plates. Following
attachment and serum starvation, scratches were made and the cells were allowed 24 h to
migrate into the wounded area. As compared to the untreated, FBS-stimulated control,
cells treated with TP421 were significantly inhibited from migrating into the denuded
area of the wound (Figure 4.11).

Figure 4.11 TP421 inhibits PANC-1 migration in a scratch assay.

A role for signal transducer and activator of transcription 3 (Stat3) in promoting
pancreatic cancer invasiveness and metastasis has been previously documented (Li et al.,
2011; Wei et al., 2003). Specifically, Stat3 has been found to localize to focal adhesions
by interacting with FAK and paxillin and is a substrate for phosphorylation by activated
Src (Silver et al., 2004). Furthermore, Stat3 is frequently constitutively activated in
71

pancreatic tumors by phosphorylation at Y705 (Scholz et al., 2003). As TP421 treatment
could cause decreased phosphorylation of both FAK and Src and could further inhibit
migration of MIA PaCa-2 and PANC-1, we examined if Stat3 phosphorylation was also
affected. Indeed, following TP421 treatment, phosphorylation of Stat3 was significantly
decreased in a time and dose dependent manner (Figure 4.12) and this inhibition may be
contributing to TP421’s ability to impede cell migration.

Figure 4.12 TP421 decreases phosphorylation of Stat3 in a time and dose-dependent manner.
(A) MIA PaCa-2 and BxPC-3 were exposed to 5 µM TP421 for indicated time and probed for
activating phosphorylation (Y705) of Stat3. (B) MIA PaCa-2 cells were treated with increasing
concentrations of TP421 for 4 h.

72

4.7 Discussion and summary
TP421 treatment produced sustained activation of JNK, p38 and Erk1/2 pathways.
Recent reports indicate that mitochondrial pools of each of these kinases exist and are
sensitive to mitochondrially generated O
2
-
(Horbinski and Chu, 2005; Kulich et al., 2007;
Lim et al., 2008a). Interestingly, activation of JNK by elevated superoxide in cells may
play a role in further amplifying the ROS produced at sites within mitochondria
(Chambers and LoGrasso, 2011). It has also been suggested that mitochondrial activation
of Erk1/2 in response to oxidative stress can suppress mitochondrial respiration and ATP
production (Nowak et al., 2006) thereby providing a plausible link between the TP421
induced Erk1/2 activation and mitochondrial dysfunction we observe. Furthermore,
prolonged robust activation of ERK1/2 is known to induce cell cycle arrest in the G1
phase (Meloche and Pouyssegur, 2007) which correlates well with our previous findings
in chapter 1 regarding arrest of pancreatic cancer cell lines in the G
0
/G
1
phase by TP421.
Activation of caspase-7 followed by PARP-1 cleavage also occurred after TP421
treatment confirming the role for TP421 in affecting the apoptosis pathway as identified
by IPA. Intriguingly, caspase-8 which is not a member of the intrinsic apoptotic pathway
was also activated which was difficult to reconcile with the knowledge that TP421
localizes exclusively to the mitochondria. However this may be explained by the fact that
caspase-8 has been shown to be activated by O
2
-
oxidative stress (Madesh et al., 2009)
and prolonged ERK1/2 activation (Cagnol et al., 2006) both of which are observed in our
treated cells, albeit the former requires a preceding activation of caspase-2. Furthermore,
apoptosis was accompanied by significant reduction in the protein levels of the apoptosis
73

inhibitors Bcl-2 and survivin. This diminution in survivin is possibly related to the strong
activation of p38 we observed. It has been previously reported that p38 activation can
cause reduction in survivin levels and that this effect could be prevented by a specific p38
inhibitor (Hsiao et al., 2007; Liu et al., 2010). As survivin expression has been associated
with poor prognostic outcome and resistance to chemotherapy there is interest in
targeting its degradation as a novel treatment for pancreatic cancer (Liu and Wang,
2011). Bcl-2 overexpression is also another common resistance mechanism precluding
gemcitabine efficacy in pancreatic cancer (Bold et al., 1999). Therefore, it is promising
that TP421 induces decreased Bcl-2 and survivin levels and provides a rationale for
future exploration of combination treatment with gemcitabine.
In addition to induction of apoptosis, it was observed that TP421 could inhibit
autophagy in pancreatic cancer cells as observed by LC3B-II and p62 accumulation. This
is particularly appealing because of the important role autophagy has been shown to play
in supporting pancreatic cancer survival and proliferation and suggests that TP421 could
be especially effective for treating these tumors in patients. Interestingly, caspase 7 has
recently been shown to affect autophagy regulation via its action on TDP-43, a protein
responsible for the maintenance of mRNA levels of a protein (atg7) required for
formation of autophagosomes (Cassel et al., 2012). Apparently, following caspase 7
mediated cleavage of TDP-43, atg7 protein levels decrease and LC3B-II is accumulated,
indicating inhibition of autophagy (Bose et al., 2011; Cassel et al., 2012). Considering
that TP421 causes casapse-7 activation, it is an intriguing possibility that TP421 might
achieve inhibition of autophagy via caspase-7 mediated degradation of TDP-43. In fact,
74

there is a lot of functional cross-talk between proteins regulating apoptosis and autophagy
such that the two effects of TP421 we observe; induction of apoptosis and inhibition of
autophagy, may be more intimately related than we expect (Eisenberg-Lerner et al.,
2009).
It is worthwhile to note that p62 levels, which were accumulated in response to
TP421 inhibition of autophagy, can also be increased in cells undergoing oxidative stress
independently of autophagy modulation. ROS disruption of a KEAP1-Nrf2 complex
stabilizes Nrf2 levels allowing it to initiate its transcriptional oxidative stress response
program which includes induction of the p62 gene (Nezis and Stenmark, 2012).
Alternatively, accumulated p62 levels due to autophagy interruption can disrupt KEAP1-
Nrf2 complex via p62 competitive binding to KEAP1(Lau et al., 2010). The resulting
Nrf2 activation would further increase p62 levels (Lau et al., 2010).
Finally, TP421 could inhibit the migration of MIA PaCa-2 and PANC-1 cells in
vitro, which may be mediated by its ability to cause de-phosphorylation of multiple
proteins involved in adhesion and motility signaling including FAK, Src and p130Cas.
Frequently in PDAC, FAK is activated and its levels are negatively correlated with
survival (Chatzizacharias et al., 2010). FAK phosphorylation has also been identified as a
cause for chemoresistance to gemcitabine (Huanwen et al., 2009), supporting its
inhibition as a means to overcoming resistance to cell death.
75

CHAPTER FIVE: IDENTIFICATION OF NOVEL SMALL MOLECULE
INHIBITORS OF APE1 MEDIATED DNA REPAIR AS SENSITIZERS TO DNA
DAMAGING CHEMOTHERAPIES
5.1 Introduction
5.1.1 Background
Repairing DNA damage is a critical process to ensure the survival of the cell and
maintenance of the integrity of the genetic code. Multiple repair pathways exist, each
specialized at recognizing and repairing a different type of damage. The base excision
repair pathway (BER) of which AP endonuclease 1 (APE1) is a member, is responsible
for replacing damaged bases.
DNA bases are susceptible to damaging modifications inflicted by various
endogenous and exogenous sources which, when acted upon by glycosylases, produce
apurinic/apyrimidinic (AP) sites that are potentially cytotoxic and mutagenic if left
unrepaired. Alternatively, AP sites can be generated by the spontaneous hydrolysis of
labile N-glycosidic bonds (Lindahl, 1993). Regardless of the source of damage, all abasic
sites, which must be repaired in order for cells to survive, are repaired by the BER
pathway. APE1 has the important role of recognizing AP sites in the genome, and
incising the DNA backbone immediately 5’ to the abasic sites, producing a 3’ hydroxyl
and a 5’ abasic deoxyribose phosphate, thereby facilitating their repair by an ensuing
succession of BER enzymes (Figure 5.1).

76

Figure 5.1 Sequence of events in BER. Damaged bases (red) are excised by DNA glycosylases
prompting APE1 to recognized the apurinic/apyrimidinic sites and cleave the DNA backbone
5’ to the excised base. The resulting gap is filled in and nick ligated by DNA polymerases and
DNA ligases, respectively, to complete the repair of the DNA.

While this endonuclease function is the predominant role for APE1 in cells, it also
exhibits weaker 3’ – 5’ exonuclease (Wilson et al., 1995), 3’ phosphatase (Chen et al.,
1991) and 3’ phosphodiesterase (Suh et al., 1997) activites. APE1 catalyzes all of these
reactions via its single DNA binding site located in the c-terminal domain of the protein
77

(Mol et al., 2000). In addition to its DNA repair activities, APE1 also functions as a
reduction-oxidation (redox) factor owing to an active cysteine residue located in a
domain that is separate from its DNA processing capabilities (Walker et al., 1993). By
way of this redox domain, APE1 is capable of activating various transcription factors
including but not limited to tumor protein 53 (p53), activator protein 1 (AP-1) and
nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), thereby
contributing to various growth signaling pathways (Kelley et al., 2011; Tell et al., 2009).
Moreover, APE1 has been shown to participate in RNA metabolism as evidenced by its
interactions with nucleophosmin (NPM1), its ability to cleave abasic RNA and its
regulation of c-Myc mRNA levels (Barnes et al., 2009; Vascotto et al., 2009).
Oxidative base damage induced by ROS is also repaired via the BER pathway
which begins by the recognition and excision of an oxidized base by one of several
substrate specific glycosylases. However, in contrast to other glycosylases, the oxidative-
damage specific glycosylases comprised of OGG1, NTH1, NEIL1, NEIL2 and NEIL3,
have an associated AP lyase activity which cleaves the DNA backbone 3’ to the abasic
site leaving behind 3’ blocked termini unrecognizable by DNA polymerases (Hegde et
al., 2012). Additionally, ROS can directly produce 3’ blocked ends by attacking and
fragmenting deoxyribose residues in DNA (Breen and Murphy, 1995). Nonetheless, all 3’
blocked ends must be cleaned to allow repair to continue. APE1 via its 3’
phosphodiesterase activity can correct blocks generated by glycosylases (OGG1 and
NTH1) and ROS, thus allowing the gap to be filled and ligated, completing repair of the
damage (Izumi et al., 2000).
78

As the foremost repair pathway responsible for removal and replacement of
damaged bases, the BER pathway has garnered great interest for pharmacological
inhibition. In particular, APE1 has emerged as an attractive therapeutic target for anti-
cancer drug development as demonstrated by studies that link its overexpression with
resistance to radio- and chemotherapy (Al-Attar et al., 2010; Bapat et al., 2009).
Although APE1 is considered to be a ubiquitously expressed protein with an estimated
350,000 – 7,000,000 copies per cell, its expression in cancer cells has been shown to be
even higher than surrounding tissue (Demple et al., 1991). Additionally, siRNA
knockdown of APE1 in cancer cells, which has the effect of potentiating the cell killing
ability of cytotoxic agents, has further confirmed its protective role in cancer against a
variety of DNA damaging agents (Fishel et al., 2008; Jiang et al., 2010). Therapeutics
targeting APE1 could reduce oncogenic cell advantage to evade apoptosis and sensitize
cancers to both radiation and chemotherapy (Fung and Demple, 2011; McNeill et al.,
2009). For instance, radio-resistance in glioma cell lines, which is associated with higher
expression of APE1, can be reversed following chemical inhibition of APE1 as can
cisplatin resistance in non-small cell lung cancer (Naidu et al., 2010; Wang et al., 2009).
As a result of the previous studies providing ample support for the inhibition of
APE1 as a means of complimenting current chemotherapeutic regimens, several
chemically diverse inhibitors have been identified. However, a clinical candidate is yet to
be realized and as such, we are working towards further identifying potent small
molecule inhibitors targeting the DNA repair function of APE1.

79

5.1.2 Rationale
Cell death is initiated in cells following various stimuli and damage including in
response to DNA damage exceeding the cell’s repair capacity. Many classically
employed anticancer drugs are DNA interacting agents which induce cell death by
introducing damage into DNA strands beyond the cell’s ability to repair them. As such,
combining these drugs with inhibitors targeting repair pathways induces a synergistic cell
killing effect. However the benefit of combining repair inhibitors could also be extended
to drugs which may indirectly induce DNA damage such as via ROS generation.
Most mitochondrialy targeted drugs have been observed to induce a significant
increase in ROS levels via various mechanisms. The accumulation of such toxic ROS
contribute to the cytotoxicity of these agents as ROS indiscriminately damage proteins,
lipids and DNA. As a means of augmenting the cell death induced by this class of
compounds, DNA repair inhibitors can be combined during treatment to overwhelm the
cellular response to oxidative stress. Being that mitochondria are the source of this ROS,
the mitochondrial DNA (mtDNA) would be particularly in need of intact BER for
correcting oxidative base damage. Specifically, targeting APE1 in combination with
mitochondrial agents should achieve a synergistic outcome. Evidence from recent studies
points to a protective role for APE1 against ROS as the latter has been shown to up-
regulate the expression level and enzymatic activity of APE1, indicating an adaptive
response to the genotoxicty of ROS (Ramana et al., 1998). Indeed, when cells were pre-
exposed to low-dose sub-lethal levels of ROS they became transiently resistant to
treatment with several genotoxic agents including H
2
O
2
and MMS further supporting the
80

important role of up-regulated APE1 in modulating cell survival following initial insult
(Ramana et al., 1998). Oxidative stress has also been linked to the translocation of APE1
to mitochondria possibly as a survival mechanism to repair the vulnerable mitochondrial
mtDNA following oxidative damage (Li et al., 2012). In fact, the targeted over-
expression of APE1 in mitochondria enhances mtDNA repair capacity and protects the
cells from H
2
O
2
induced cell death (Li et al., 2008). The relationship of ROS and APE1
levels and function in cells is illustrated schematically in Figure 5.2.
Given that the localization of APE1 to the mitochondria helps protect cells from
initiation of apoptosis following oxidative stress induced DNA damage we sought to
develop novel inhibitors of APE1 to serve as valuable adjuvant therapies to mitochondrial
targeted treatments.

Figure 5.2 Schematic illustration of the effects of ROS on APE1 in cells. Oxidative stress
resulting from accumulating ROS induces increased expression of APE1, up-regulated
enzymatic activity and is associated with translocation to the mitochondria. Repair of mtDNA
damage caused by increased ROS is repaired by APE1.
81

5.2 Materials and Methods
In Vitro APE1 assay
The extent of the endonuclease activity of APE1 was determined by diluting the test
compounds in DMSO and incubating the diluted compounds with recombinant APE1 at a
final concentration of 0.05 nM in the reaction buffer (50 mM NaCl, 1 mM HEPES, pH
7.5, 50 µM EDTA, 50 µM dithiothreitol, 10% glycerol (w/v), 7.5 mM MnCl
2
, 0.1
mg/mL bovine serum albumin, 10 mM 2-mercaptoethanol, 10% DMSO, and 25 mM
MOPS, pH 7.2) at 37ºC for 30 minutes. Thereafter, 200 nM of the 5’-end 32P-labeled
linear oligonucleotide substrate containing the AP site was added and incubated for an
additional 15 minutes. The reactions were quenched by the addition of an equal volume
(8 µL) of loading dye (98% deionized formamide, 10 mM EDTA, 0.025% xylene cyanol,
and 0.025% bromophenol blue). A 10 µL aliquot was electrophoresed on a denaturing
20% polyacrylamide gel. The gels were dried and exposed in a PhosphorImager cassette
and analyzed using a Typhoon 8610 Variable Mode Imager (Amersham Biosciences).
In vitro HIV-1 integrase assay
To determine the extent of 3'-processing and strand transfer, wild-type recombinant IN
was pre-incubated at a final concentration of 200 nM with the inhibitor in reaction buffer
(50 mM NaCl, 1 mM HEPES, pH 7.5, 50 µM EDTA, 50 µM dithiothreitol, 10% glycerol
(w/v), 7.5 mM MnCl
2
, 0.1 mg/ml bovine serum albumin, 10 mM 2-mercaptoethanol,
10% dimethyl sulfoxide, and 25 mM MOPS, pH 7.2) at 30
º
C for 30 min. Then, 20 nM
of the 5'-end
32
P-labeled linear oligonucleotide substrate was added, and the incubation
was continued for an additional one hour. Reactions were then quenched by the addition
82

of an equal volume (16 µl) of loading dye (98% deionized formamide, 10 mM EDTA,
0.025% xylene cyanol and 0.025% bromophenol blue). An aliquot (5 - 10 µl) was
electrophoresed on denaturing 20% polyacrylamide gels (0.09 M tris-borate pH 8.3, 2
mM EDTA, 20% acrylamide, 8M urea). Gels were dried, exposed in a PhosphorImager
cassette, and analyzed using a Typhoon 8610 Variable Mode Imager (Amersham
Biosciences) and quantitated using ImageQuant 5.2.
Quantification of APE1 and IN inhibition
Percent inhibition (% I) was calculated using equation: % I = 100 X [1 - (D - C)/(N -
C)], where C, N, and D are the fractions of 26-mer substrate cleaved to 13-mer products
by APE1 for DNA alone, DNA plus APE1, and DNA plus APE1 plus the test compound,
respectively. For IN inhibition, C, N, and D are the fractions of 21-mer substrate
converted to 19-mer (3'-processing product) or strand transfer products for DNA alone,
DNA plus IN, and DNA plus IN plus drug, respectively. The IC
50
values were
determined by plotting the logarithm of drug concentration versus percent inhibition to
obtain the concentration that produced 50% inhibition.
Cell culture
H630 and HT-29 colon cancer cells were obtained from the National Cancer Institute,
Bethesda, MD and were maintained as adherent monolayer cultures in Dulbecco’s
Modified Eagle Media (DMEM) and RPMI-1640 supplemented with 10% fetal bovine
serum (FBS; Gemini-Bioproducts, West Sacramento, CA), respectively. Cells were
grown at 37°C in a humidified atmosphere of 5% CO
2
. For all experiments, cells in
exponential growth phase were washed with PBS, briefly trypsinized in a small volume
83

of 0.25% trypsin-EDTA solution (Sigma-Aldrich, St. Louis, MO), re-suspended in
culture media and spun at 1,200 r.p.m. for 5 min. Pelleted cells were re-suspended in
complete growth medium, counted and plated in sterile plates and allowed to adhere
overnight before treating.
Cell viability assay
Cell viability was assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay as previously described. (Millard et al., 2010) Cells were seeded in
96-well tissue culture treated dishes at a density of 4,000 cells/well and allowed to attach
overnight. Cells were subsequently treated with a continuous exposure to a range of
concentrations of drugs for 72 hours. At the end of exposure, an MTT solution was added
to each well at a final concentration of 0.3 mg/mL MTT and further incubated for 3–4
hours at 37°C. After removal of the supernatant, DMSO was added and the absorbance of
the solubilized dye was read at 570 nm. % Cytotoxicity for each drug concentration was
determined using the following formula: % cytotoxicity = 100 * ( 1 - [ Abs
(drug)
/Abs
(control)

] ). Where possible, IC
50
values were determined from the plot of % cytotoxicity versus
logarithm of drug concentration.
Colony formation assay
Colony forming ability of cells in response to indicated drug treatment was carried out as
previously described in chapter 1. Plates were scanned in the Odyssey® Infrared Imaging
System (Licor, Lincoln, NE) using the 700 channel laser. Cytotoxicities were calculated
as a ratio of control as described above.
84

5.3 Combination treatment with a mitochondrial targeted agent and an APE1
inhibitor
In order to ascertain the value of combining an APE1 inhibitor with a
mitochondrialy targeted agent, we treated cells in combination to examine if they resulted
in a potentiation of cytotoxicity. We used TP421, the mitochondrial agent and ICM-1, an
APE1 DNA repair inhibitor previously identified in our lab. ICM-1 inhibits APE1 in an
in vitro assay with an IC50 of 33.5 ± 2 µg/mL.
When we exposed MIA PaCa-2 cells to TP421 in the presence of ICM-1, the
cytotoxicity of TP421 was greatly enhanced over single agent treatment (Figure 5.3).

Figure 5.3 TP421 combined with an APE1 inhibitor achieves synergistic cell killing. MIA PaCa-
2 cells were treated with 0.5 µM TP421 in the presence or absence of 4 µg/mL ICM-1 and
incubated for 72 h. At the end of treatment, cytotoxicity was determined using MTT.

85

5.4 Specific inhibition of APE1 by 3-carbamoylbenzoic acid derivatives
We had previously developed a three-dimensional interaction-based
pharmacophore model highlighting structural features important for inhibition of APE1
DNA processing capability (Zawahir et al., 2009). The key features of this model are
represented in Figure 5.4 and are based on the interactions retrieved from the co-crystal
structure of APE1 in complex with its substrate DNA bearing an abasic site (Mol et al.,
2000).

Figure 5.4 Synthesized compounds with the descriptive pharmacophore properties;
hydrophobic, negative ionizable, and linker moieties.

Utilizing this model, a series of novel 3-carbamoylbenzoic acid derivatives were
synthesized and their inhibition of recombinant APE1’s endonuclease activity was
86

determined using an in vitro enzymatic assay with a radiolabeled DNA substrate shown
schematically in Figure 5.5.

Figure 5.5 Endonuclease activity of APE1. During the base excision repair (BER) pathway,
APE1 cleaves the phosphodiester backbone 5' to the AP site. The 26-mer oligomer used in this
assay was radiolabeled with
32
P at the 5' end containing the AP site. The AP site in the 26-mer
oligomer was tetrahydrofuran.
A similar radiolabeled DNA assay was conducted on recombinant HIV-1
integrase (IN) to observe the degree of selectivity of our compounds for inhibiting APE1
over another DNA processing enzyme. In a fashion similar to APE1, IN catalytically
cleaves the phosphodiester backbone of virally encoded DNA 5’ to its recognition site
leaving a 3’ hydroxyl group. Furthermore, IN requires a divalent metal cation for
catalysis, similarly to APE1, making it a suitable counter screening target for establishing
the specificity of our compounds. Therefore, we assayed our compounds for their ability
to inhibit IN endonuclease and polynucleotidyl transferase activities as indicated by 3’-
87

processing and strand-transfer efficiencies respectively. Where possible, the selectivity
index of our agents for APE1 over IN were reported. The results of the APE1 and IN
assays is listed in the following tables discussed below. To examine if the in vitro
inhibition of APE1 by our compounds had biological consequences, we tested their
cytotoxicity in a colon cancer cell line using the MTT and colony formation assays and
the data are included in the tables alongside the enzyme inhibition IC50s. Representative
APE1 and IN gels are shown in Figures 5.6 and 5.7 below.

Figure 5.6 A representative gel showing inhibition of purified IN by select APE1 inhibitors.
The 21mer band corresponds to the unprocessed substrate, the 19mer is the product of the 3’-
processing reaction and the STP bands are produced once the 19mer is incorporated into the
21mer substrate as products of the strand transfer reaction. Lane 13: DNA alone; Lane 14:
DNA and IN without compounds; Lanes 1-12 and 15-26: DNA with IN and select compounds at
varying concentrations, 100, 33 and 11 µM. Final IN concentration is 200 nM.
88

Figure 5.7 A representative gel showing inhibition of purified APE1 by select compounds.
Lane 17: DNA alone; Lanes 18 and 19: DNA and APE1 without compounds; Lanes 1-16 and 20-
27: DNA with APE1 and select compounds at varying concentrations, 100, 33, 11, and 3.7 µM.
Final APE1 concentration is 0.05 nM.
The first series of compounds we synthesized and tested are listed in Table 5.1.
F325-F327 structurally varied only in the number and position of the methoxy group on
the phenyl ring. These compounds were selectively active for APE1 with IC
50
values
comprised between 18±8 and 21±7 μM. Halogenated compounds F328-F330 were
generated by the replacement of methoxy groups with chlorine atoms. Interestingly,
compound F328 having the chlorine atom in the para position did not show any
inhibition property, while compound F329 and F330 had IC
50
= 16±6 and 18±12 μM,
respectively. Compounds, F331, with a fluorine in the para position, and F332, with a
fluorine in the meta position, inhibited APE1 endonuclease activity with IC
50
= 26±2 and
27±16 μM, respectively. However, F333 containing two fluorine atoms did not show
89

inhibition properties. Apart from F350 which weakly inhibited IN endonuclease and
strand transfer activity with IC
50
values near 500 µM, all of the compounds showed no
activity against IN and displayed an APE1 selectivity greater than 35-fold.
90

Table 5.1 Inhibition of APE1 endonuclease activity by compounds F325-FM262
Compounds
HIV-1 IN APE1 IC
50
SI
[c]

3P
[a]
ST
[b]
MTT CFA
F325

>1000

>1000

21   

>30

>30

47.6
F326

>1000

>1000

19   

>30

>30

52.6
F327

>1000

>1000

18 8

>30

>30

55.5
F328

>1000

>1000

>100

>30

>30

1
F329

<1000

1000

16   

>30

>30

62.5
F330

>1000

>1000

18    

>30

>30

55.5
F331

>1000

>1000

26 ± 2

>30

>30

38.4
F332

>1000

>1000

27    

>30

>30

37
F333

1000

>1000

>100

>30

>30

1
F350

505

460

31    

>30

>30

16
[a] 3’-Processing. [b] Strand Transfer. [c] Selectivity Index: fold difference in IC 50 values of
the inhibitors for APE1 endonuclease catalysis versus IN endonuclease 3’-processing. All
IC 50 values listed are in μM.
91

Sequentially replacing the methoxy groups in compounds F326 and F327 with
hydroxyl groups giving rise to de-protected compounds F326D-F327DD produced a
negligible to profound reduction in their ability to inhibit APE1. F327DD saw a reduction
in activity from 18±8 μM to >100 μM as compared to F327. On the other hand, these
modifications mostly improved IN inhibition (Table 5.2) compared to counterparts in
Table 5.1 with the result of decreased selectivity indexes ranging from 0.52 to 26.3.
De-protected compounds F328D-F333D also with methoxy to hydroxyl
substitutions between the carbonyl moeities, had a slight to substantial decrease in
activities with the exception of F328D and F333D which had increased activity and
lowered IC50 values to 44±37 μM and 39±22 μM (respectively) as compared to their
methoxy containing counterparts in Table 5.1. Following the same trend as the previous
compounds in Table 5.2, F328D and F333D also had improved IN inhibition but still
retained modest selectivity for APE1.
Compound F327DD with two hydroxy groups in the meta and para positions of
the appended phenyl ring displayed no inhibitory properties against APE1 endonuclease
activity, whereas compound F326DD with a single hydroxy substituent at the meta
position exhibited weak inhibition with an IC
50
of 40±31 μM. Compound F350D
containing an additional phenyl group had an IC
50
of 33±24 μM comparable to that of
compound F350. Interestingly, these modifications improved the compounds’ ability to
inhibit IN and resulted in a reduction in their selectivity but were still able to retain
modest selectivity indexes favoring APE1, with the exception of F327DD which had
abolished APE1 inhibition but was active against IN.
92

Table 5.2 Inhibition of APE1 endonuclease activity by compounds F326D-F350D
[a] 3’-Processing. [b] Strand Transfer. [c] Selectivity Index: fold difference in IC 50 values of the
inhibitors for APE1 endonuclease catalysis versus IN endonuclease 3’-processing. All IC 50
values listed are in μM.
Compound
HIV-1 IN IC
50
APE1 Cell culture SI
[c]

3P
[a]
ST
[b]
MTT CFA
F326D

>1000 >1000 38 30 >30 >30 26.3
F326DD

280 195 40 31 >30 >30 7
F327D

61 99 19 2 >30 >30 3.2
F327DD

52 ±30 17 ± 9 >100 >30 >30 0.52
F328D

>333
>333,
100
44     >30 >30 7.5
F329D

>100 95 ± 8 33     >30 >30 3
F330D

>100 82 ±25 28     >30 >30 3.5
F331D

>100 70 ±14 27 9 >30 >30 3.7
F332D

>1000 >1000 >100 >30 >30 10
F333D

>333 98 ± 3 39     >30 >30 8.5
F350D

95 ±6 35 ±14 33     >30 >30 2.8
93

Pyridine derivatives F-1, F-2, and ester derivative F-3, designed as analogues of
compounds F329 and F332, did not show activity. Similarly, compounds F-5, F-6, and F-
7, based on a 8-hydroxyquinoline scaffold, were devoid of any significant activity (Table
5.3). These non-active compounds also did not inhibit IN.

Table 5.3 Inhibition of APE1 endonuclease activity by F-1, F-2, F-3, F-5, F-6 and F-7.
Compound
HIV-1 IN APE1 Cell culture SI
[c]

3P
[a]
ST
[b]
MTT CFA
F-1

>100 >100 >100
<20
(55%)
>30 NA
F-2

>100 >100 100 20 >30 > 1
F-3

>100 >100 100 >20
<30
(72%)
> 1
F-5

>100 >100 >100 >20
<30
(54%)
NA
F-6

>100 >100 >100 20 >30 NA
F-7

>100 >100 70
<20
(57%)
>30 > 1.4

[a] 3’-Processing. [b] Strand Transfer. [c] Selectivity Index: fold difference in IC 50
values of the inhibitors for APE1 endonuclease catalysis versus IN endonuclease 3’-
processing. All IC 50 values listed are in μM.
94

Interestingly, activity of symmetric diamide derivatives listed in Table 5.4 (IC50
ranging from 25±3 to 66±5 μM) resulted in comparable activity to that of mono-
substituted analogues, with a concomitant drop in selectivity of APE1 over IN.

Table 5.4 Inhibition of APE1 endonuclease activity by compounds F330bis – F-4.
Compound
HIV-1 IN APE1 Cell culture SI
[c]

3P
[a]
ST
[b]
MTT CFA
F330bis

666 395 27 5 >30 >30 24.7
F330bisD

195

105 ±8 66 5 >30 >30 3.0
F328bisD

150 170 28 4 > 30 >30 5.4
F329bisD

140 98 ± 4 41 22 >30 >30 3.4
F350bisD

105 37 25 3 >30 >30 4.2
F-4

>100 >100 >100 >20
<30
(53%)
NA

[a] 3’-Processing. [b] Strand Transfer. [c] Selectivity Index: fold difference in IC 50 values
of the inhibitors for APE1 endonuclease catalysis versus IN endonuclease 3’-processing.
All IC 50 values listed are in μM.

95

Finally, derivatives F-M260, F260D, F-M260S, F-M262, F262D, F-M262S,
CF34Ph, CF33Cl, CF33F, CN4Ph, and tetrazole derivative T3F (Table 5.5) were devoid
of activity against both APE1 and IN.

Table 5.5 Compounds exhibiting no activity against APE1 endonuclease activity.
Compound
HIV-1 IN APE1 Cell culture
3P
[a]
ST
[b]
MTT CFA
F-M260

>100 >100 >100 > 30 >30
F260D

410

250

>100

>30

>30
F-M260S

590 560 >100 >30 >30
F-M262

>1000 >1000 >100 >30 >30
F262D

600 510 >100 >30 >30
F-M262S

490 333 NT >30 >30
96

[a] 3’-Processing. [b] Strand Transfer. All IC 50 values listed are in μM.

Although many of the aforementioned 3-carbamoylbenzoic acid derivatives
effectively inhibited APE1 catalytic endonuclease function in vitro, cell proliferation was
largely unaffected in H630 cells at the highest dose tested for all of the derivatives.
5.5 Cytotoxicity of 3-carbamoylbenzoic acid derivatives in combination with MMS
or 5-FdUrd
While our compounds did not exhibit single agent cytotoxicity in cell culture, we
were interested in examining their effect on the proliferative capability of H630 cells as
CF
3
4Ph

1000 1000 >100 >30 >30
CF
3
3Cl

>1000 >1000 >100 >30 >30
CF
3
3F

>1000 >1000 >100 >30 >30
CN4Ph

1000 1000 >100 >30 >30
T3F

>1000 >1000 NT >30 >30
97

part of a combination regimen with methyl methanesulfonate (MMS). The alkylating
agent, MMS, is a commonly used laboratory agent for analyzing AP endonuclease
inhibition (Luo and Kelley, 2004; Simeonov et al., 2009). MMS forms alkylated bases
which are repaired by BER (Wyatt and Pittman, 2006) and as such, compounds which
can inhibit APE1 in cells should exhibit a sensitizing effect to the cytotoxicity produced
by MMS treatment. We therefore treated cells with a range of concentrations of MMS
alone, or in combination with a single dose (50 µM) of five of our compounds; F328,
F329, F330, F329D and F332D. Pairs of drugs in each combination were added
simultaneously to cells and incubated together for a total duration of 72 hours at the end
of which the cell viability was determined using MTT. Remarkably, only F332D, an non-
active derivative, showed a statistically significant potentiation of MMS cytotoxicity
(Figure 5.8). While this may indicate that F332D may be achieving some inhibition of
APE1 in cells, it is not presently clear how it accomplishes this.

98

Figure 5.8 H630 cells treated with MMS in the presence of F332D achieve greater cytotoxicity
indicating a synergistic outcome. *. ** and *** indicate p < 0.05, p < 0.01 and p < 0.005.
Interestingly, F332D also potentiated the observed cytotoxicity of 5-Flurouracil
(5-FU) when combined simultaneously in culture. It is known that 5-FU gets
incorporated in to the DNA of treated cells at significant levels and that BER proteins are
capable of detecting and removing the mis-incorporated base so that DNA can be
repaired (Fischer et al., 2007). This potentiation was specific to the BER-dependent DNA
lesions caused by 5-FU, as the combination of F332D with other genotoxic agents did not
produce a similar synergistic outcome on cell viability (Figure 5.9). Consequently, our
results indicate that F332D may be clinically useful as an adjuvant therapy with 5-FU.

99

Figure 5.9 Combination treatments of F332D with various genotoxic agents reveals selectivity
for potentiating 5-FU cytotoxicity.
100

Although a role for BER in repairing 5-FU damage has been documented, other
reports suggest that intact BER is more critical for surviving 5-Fluorodeoxyuridine (5-
FdUrd) treatment instead (Geng et al., 2011). 5-FU damages RNA and DNA while 5-
FdUrd, the phosphorylated form of 5-FU, only produces metabolites that damage DNA
(Geng et al., 2011). Accordingly, we sought to examine the effect of combined treatment
of our APE1 inhibitors with 5-FdUrd on the cell survival of HT-29 colon cancer cells.
We tested F328, F329, F330, F332, F328D, F329D and F332D to attempt to correlate
synergistic activity with APE1 inhibition. Of these compounds, F330, F328D and F332D
showed enhanced potentiation of 5-FdUrd induced cytotoxicity (Figure 5.10).

101

Figure 5.10 Effect of combination treatment with 5-FdUrd and select APE1 inhibitors on cell
survival as measured by colony formation in the HT-29 cell line. Cells were pre-treated with a
single concentration of 5-FdUrd [0.1 µM] for 24 h followed by addition of indicated inhibitor
[50 µM] for additional 24 h. Drug containing media was then replaced with fresh media and
colonies were allowed to form. % Survival indicates fraction of surviving colonies as
compared to untreated control.
102

5.6 Discussion and summary
A new class of APE1 inhibitors, based on a previously reported pharmacophore
model have been identified. Among the newly synthesized compounds, derivatives F326,
F327, F329 and F330, having a 3-benzylcarbamoyl-2-methoxybenzoic acid structure,
resulted in the most active and selective inhibition of APE1, showing IC50 values less
than 20 µM and with no activity against HIV1-IN at the highest concentrations tested.
The replacement of the methoxy group for a hydroxy group at position 2 (compound
F237D) did not result in a significant change in terms of activity, while such an alteration
considerably affected selectivity. Notably, some activity and selectivity was retained by
several dicarbamoyl derivatives, while the replacement of the carbocyclic aromatic ring
together with the carboxylic function for a pyridine or quinoline system proved
detrimental for activity. Thus, 3-benzylcarbamoyl-2-methoxybenzoic acid structure,
common to the active derivatives, represents a lead scaffold to be further developed with
the aim of finding more potent and selective APE1 inhibitors.
We identified three derivatives which exhibited noteworthy potentiation of
various DNA interacting drugs for which BER can repair damage. F328D and F330 were
among the most active analogs tested in the in vitro APE1 assay whereas F332D exhibits
very weak activity. Yet, F332D was able to specifically potentiate the cytotoxicity
produced by all the agents it was combined with that are repaired by BER. It is not
presently clear how F332D achieves this potentiation necessitating future studies to better
understand its mode of action.
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As APE1 has a single DNA active site, it has been shown that compounds
interfering with this site’s ability to bind and process DNA inhibit all APE1’s enzymatic
activities simultaneously (Madhusudan et al., 2005). Therefore we expect that the active
compounds we identified via our endonuclease assay will also inhibit the 3’
phosphodiesterase activity of APE1 which is particularly important for repairing
oxidative base damage resulting from ROS. The effect of APE1 inhibition by ICM-1 on
the cytotoxicity of TP421 in MIA PaCa-2 cells is an encouraging indication that this
combination modality could be a powerfully synergistic therapy. As such, the compounds
we identified using our pharmacophore model represent a new class of APE1 DNA repair
inhibitors that may be clinically relevant as combination treatments with certain
genotoxic agents and possibly also with mitochondrial targeted agents in as much as
those agents can cause mitochondrial and/or nuclear oxidative DNA damage.
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CHAPTER SIX: CONCLUSIONS
6.1 Summary and significance
Pancreatic cancer is a devastating disease with high mortality and limited
treatment options. TP421 which belongs to a novel class of small molecules, targets
mitochondria in pancreatic cancer cells and displays favorable anti-proliferative
properties making it a promising candidate for further development. Notably, our
compound exhibited highly selective in vitro cytotoxicity towards multiple pancreatic
cancer cell lines over a non-transformed epithelial cell line. This selectivity may be a
result of the known difference between mitochondrial membrane potentials of cancer and
non-cancer cells, which is the driving force for TP421 accumulation at its site of action
(Murphy, 2008). TP421 accumulated in mitochondria and mediated multiple effects
including cell cycle arrest, oxidative stress, apoptosis and inhibition of cell migration.
These manifold consequences of TP421 treatment including inhibition of mitochondrial
respiration and autophagy highlight the utility of this class of compounds at affecting
essential cancer cell processes.
We have preliminary evidence that mitochondrial targeted agents that accumulate
ROS could be synergistic with APE1 inhibitors. To the best of our knowledge, this has
not been shown before and could prove to be a clinically effective treatment option for
pancreatic cancer patients in the future. Using an interaction based pharmacophore model
derived from APE1 bound to its substrate DNA, we identified a novel class of inhibitors
with good activity and selectivity against APE1. These compounds add to the growing
105

pool of selective inhibitors of APE1 discovered to date and represent promising leads
towards development of clinically useful chemotherapies targeting APE1.

6.2 Future studies
The prospective clinical usefulness of our novel class of mitochondrial-targeted
agents for treating pancreatic cancer merits their further development. Towards that end,
more studies are required to identify the target of TP421 within the mitochondria. Our
evidence so far indicates that the target may be a protein within the electron transport
chain, most likely complex I or complex III as they are the main sources of mitochondrial
superoxide. Identifying if either of these proteins may be the direct site of TP421 binding
and/or action can facilitate the structural improvement of this class of compounds
towards increasing their potency. Also, examining the effects of TP421 on cellular ATP
levels and mitochondrial membrane permeability transition will be important towards
determining the extent of mitochondrial dysfunction induced by TP421.
The nature of the autophagy inhibition by TP421 also merits further attention.
Major unresolved questions regarding this property of TP421 include how it achieves
inhibition from a signaling standpoint and whether this inhibition is unique to pancreatic
cancer cell lines over other cancer types. Since pancreatic cancer has been described as
“autophagy-addicted” it is promising that TP421 can interfere with this critical aspect of
pancreatic cancer biology. Dysfunctional mitochondria that produce increased ROS and
become damaged at the level of their DNA are cleared by selective autophagy (a.k.a.
mitophagy) and it is quite intriguing that TP421 may be preventing this process.
106

Although mitophagy is a selective form of autophagy and is essentially carried out by the
same machinery, the signaling events, regulation and induction of mitophagy differ from
other forms of autophagy. Key proteins modulating mitophagy have only been very
recently identified and include stabilization of the levels of a mitochondrial ser/thr kinase,
PINK1, on the mitochondrial outer membrane and the subsequent translocation of a
cytosolic E3 ubiquitin ligase, Parkin, to the surface of mitochondria to initiate
degradation (Springer and Kahle, 2011). This area of mitochondrial dynamics and
regulation should be investigated as it could be an interesting function of TP421.
Finally, with increasing interest in targeting mitochondria for treating cancer, the
use of such agents could be nearing a clinical reality. The potential synergistic action of
APE1 inhibitors in combination with ROS-inducing mitochondrial agents is worth
exploring further.
107

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Abstract (if available)

Abstract Pancreatic cancer is one of the deadliest cancers with a 5-year survival rate of 6%. Therapeutic options against this disease are limited and there is a critical unmet need for safe and efficacious treatments. Cancer cell metabolism and mitochondria provide unexplored targets for this disease. Here-in we describe the identification of a novel class of triphenylphosphonium salts, TP compounds, which target the mitochondria of cancer cells and display broad- spectrum anticancer properties. We examined the ability of our prototypical compound TP421 to inhibit the growth of pancreatic cancer cells and further investigated the molecular mechanisms by which it exerts its anticancer effects. TP421 showed sub-micromolar IC50 values in all the pancreatic cancer cell lines tested using MTT and colony formation assays. TP421 localized predominantly to mitochondria and induced G0/G1 arrest, ROS accumulation, and activation of several stress regulated kinases. Multiple caspases and PARP-1 cleavage were observed indicating an apoptotic response while LC3B-II and p62 were accumulated indicating inhibition of autophagy. Furthermore, TP421 induced de-phosphorylation of key signaling molecules involved in FAK mediated adhesion that correlated with inhibition of cell migration. ❧ We also report the identification of a novel class of inhibitors of the essential base excision repair enzyme apurinic/apyrimidinic (AP) endonuclease (APE1). APE1 is a multi-faceted protein with an essential role in the base excision repair (BER) pathway. To protect cell genomes from potentially mutagenic or cytotoxic base damage arising from various exogenous or endogenous sources, APE1 nicks the DNA backbone 5’ to AP sites generated primarily by lesion-specific glycosylases which remove damaged bases. Its implication in tumor development, progression and resistance has been confirmed in multiple cancers making it a viable target of intense investigation. Here we have designed and synthesized different classes of small molecule selective inhibitors of APE1’s catalytic endonuclease function containing a 3-carbamoylbenzoic scaffold. Further structural modifications have been made with the aim of increasing activity and cytotoxicity of these inhibitors. Several of our compounds exhibited low micro-molar potencies towards inhibiting APE1’s catalytic endonuclease function in vitro and thus represent novel classes of APE1 inhibitors worthy of further development.

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Asset Metadata

Creator Shabaik, Yumna Hosam (author)

Core Title Development of novel small molecules targeting mitochondrial and oxidative stress signaling pathways for pancreatic cancer therapy

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmacy / Pharmaceutical Sciences

Publication Date 02/26/2013

Defense Date 12/17/2012

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

Tag cancer,mitochondria,OAI-PMH Harvest,small molecule,triphenylphosphonium cation

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Neamati, Nouri (committee chair), Camarero, Julio A. (committee member), Duncan, Roger (committee member), Stiles, Bangyan L. (committee member), Zandi, Ebrahim (committee member)

Creator Email shabaik@usc.edu,yshabaik@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-221777

Unique identifier UC11293162

Identifier usctheses-c3-221777 (legacy record id)

Legacy Identifier etd-ShabaikYum-1449.pdf

Dmrecord 221777

Document Type Dissertation

Rights Shabaik, Yumna Hosam

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...

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