University of Southern California Dissertations and Theses

Inhibition of tumor cell growth by mefloquine via multimechanistic effects involving increased cellular stress, inhibition of autophagy, and impairment of cellular energy metabolism

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Inhibition of tumor cell growth by mefloquine via multimechanistic effects involving increased cellular stress, inhibition of autophagy, and impairment of cellular energy metabolism

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Content Inhibition of Tumor Cell Growth by Mefloquine via
Multimechanistic Effects Involving Increased Cellular
Stress, Inhibition of Autophagy, and Impairment of
Cellular Energy Metabolism
by
Natasha Sharma

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 Natasha Sharma
i

Dedication
I would like to dedicate my dissertation to my dad, who prior to his passing away in
2009, still is and will always be my inspiration. He has always taught me to work hard
and not to be scared of failures. His support and trust in me, has always been a strong
motivation, which has encouraged me throughout my life to fulfill my dreams.

ii

Acknowledgements
I would like to thank my parents for their tremendous support, encouragement and trust
in my abilities. I am grateful to my husband for his unconditional support, encouragement
and motivation, which helped me achieve my dreams. I would like to thank my sister,
brother and brother in law, who have always been there to help me move forward. I am
thankful to my mother and father in law for supporting my decision and letting me fulfill
my dreams.
I am extremely thankful to my PI and mentor Dr. Stan G. Louie for his support, guidance
and his trust in my abilities, for the past 4 years. He gave me the time I needed when I
was distressed and motivated me to accomplish my goals, which I am very grateful for.
I would also like to thank Dr. Axel H. Schönthal for being a great mentor for all these
years. His constructive criticism and encouragement enabled me to understand the minute
details of the subject and become a better researcher. I extend my gratitude to my
committee members Dr. Wei-Chiang Shen, Dr. Roger Duncan and Dr. Bangyan Stiles,
for their support, guidance and feedback for all these years that I have spent at USC,
School of Pharmacy. In addition, I would like to thank Dr. Florence M. Hofman and Dr.
Thomas C. Chen for their positive feedback, guidance and support.
I would like to thank all my friends, especially Leena Ali, Simmy Thomas, Neeti Verma,
Yumna Shabaik, Rasha Al Safi, Neeraj Agicha, Sejal Parakh, Dhwani Dagli, Rehan
Mulla, Sonal Ghura and Vani Yadav for their unconditional support and help, without
them achieving my goal was impossible.
iii

Last but not least, I would like to thank my colleagues, for their constant support and hard
work that helped me throughout the course of my studies: Dr. Simmy Thomas, Dr. Jared
Russell, Dr. Nick Mordwinkin, Dr. Hee-Yeon Cho, Dr. Dezheng Dong, Dr. Weijun
Wang, Dr. Encouse Golden, Dr. Fabienne Agasse, Dr. Marcela Salazar, Dr. Raquel
Ferreira, Sachin Jadhav, Niyati Jhaveri, Manasi Barath, Nidhi Sharda, Puneet Agarwal,
Ashish Anshu and Tiago Santos.

iv

Table of Contents

Dedication i

Acknowledgements ii

List of Figures ix

Abbreviations xi

Abstract xvi

Chapter 1: Introduction to autophagy and its role in cancer
1.1. Autophagy 1
1.2. Role of autophagy 3
1.3. Autophagy in cancer 3
1.3.1. Autophagy and tumor suppression 4
1.3.2. Autophagy enables tumor progression 6
1.4. Induction of autophagy as an adaptation strategy 7
1.4.1. Autophagy and chemotherapeutic stress 7
1.4.2. Autophagy mitigates endoplasmic reticulum stress 8
1.4.3. Induction of autophagy in response to sphingolipids 9
1.5. Autophagy and anticancer therapy 11
1.5.1. Targeting autophagy by pharmacological means 12
1.6. Hypothesis 13
1.7. Outline of the dissertation 14

Chapter 2: Inhibition of autophagy and induction of anticancer effects by mefloquine
2.1. Introduction 16
2.2. Materials and methods 20
2.2.1. Materials 20
2.2.2. Cell lines and culturing 20
v

2.2.3. MTT assay 21
2.2.4. Colony formation assay 21
2.2.5. Western blot analysis 21
2.2.6. siRNA transfection 22
2.2.7. Quantitative reverse transcriptase polymerase chain reaction 23
2.2.8. Cell death ELISA 23
2.2.9. Statistical analysis 23
2.3. Results 24
2.3.1. Differential cytotoxicity of lysosomotropic agents 24
2.3.2. Mefloquine is effective in multidrug resistant cancer cells 27
2.3.3. Mefloquine induces apoptosis 29
2.3.4. Mefloquine inhibits autophagy 32
2.3.5. Mefloquine chemosensitizes towards paclitaxel 36
2.4. Discussion 37
2.5. Conclusion 42

Chapter 3: Effect of mefloquine on endoplasmic reticulum stress and ubiquitin
proteasome system
3.1. Introduction 43
3.2. Materials and methods 48
3.2.1. Materials 48
3.2.2. Cell lines and culturing 48
3.2.3. Colony formation assay 48
3.2.4. Western blot analysis 49
3.2.5. siRNA transfection 49
3.2.6. MTT assay 49
3.2.7. Proteasome activity measurement 49
3.3. Results 50
vi

3.3.1. Mefloquine causes accumulation of polyubiquitinated proteins but
does not inhibit proteasome activity
51
3.3.2. Mefloquine triggers ER stress 53
3.3.3. Mefloquine enhances cytotoxic effects of ER stress aggravators 55
3.4. Discussion 56
3.5. Conclusion 58

Chapter 4: Effect of mefloquine on sphingolipid levels and the processes affected by
sphingosine-1-phosphate
4.1. Introduction 59
4.2. Materials and methods 63
4.2.1. Materials 63
4.2.2. Cell lines and culturing 64
4.2.3. MTT assay 64
4.2.4. Extraction of sphingolipids 64
4.2.5. LCMS analysis of sphingolipids 66
4.2.6. Wound healing assay/Scratch assay 67
4.2.7. Migration assay 67
4.2.8. Invasion assay 67
4.2.9. Sphingosine kinase 1 (Sphk 1) inhibition assay 68
4.3. Results 68
4.3.1. Mefloquine reduces sphingosine-1-phosphate levels in glioma cells 68
4.3.2. Mefloquine increases ceramide and sphingosine levels in glioma
cells
69
4.3.3. Mefloquine reduces Sphk1 activity 70
4.3.4. Mefloquine reduces proliferation of glioma cells 71
4.3.5. Reduction in migration of glioma cells by mefloquine 72
4.3.6. Mefloquine reduces invasion of glioma cells 74
vii

4.4. Discussion 75
4.5. Conclusion 77

Chapter 5: Reduced glucose uptake and impaired ATP synthase activity by mefloquine
results in death of Glioblastoma Multiforme
5.1. Introduction 78
5.2. Materials and methods 82
5.2.1. Materials 82
5.2.2. Cell lines and culturing 83
5.2.3. MTT assay 83
5.2.4. Colony formation assay 83
5.2.5. Western blot analysis 84
5.2.6. Glucose uptake assay 84
5.2.7. LDH release assay 85
5.2.8. Isolation of mitochondria from mouse liver 85
5.2.9. NADH dehydrogenase/complex I activity assay 86
5.2.10. Succinate dehydrogenase/complex II activity assay 86
5.2.11. ATP synthase/complex V activity assay 87
5.2.12. Measurement of mitochondrial respiration 87
5.2.13. Statistical analysis 88
5.3. Results 88
5.3.1. Mefloquine reduces cell viability of glioma 88
5.3.2. Mefloquine is effective in temozolomide resistant glioma 89
5.3.3. Mefloquine induces necrosis 91
5.3.4. Mefloquine reduces GLUT 3 expression and glucose uptake 93
5.3.5. 2-deoxy-D-glucose (2DG) potentiates mefloquine induced
cytotoxicity
96
5.3.6. Methyl pyruvate can partially protect glioma cells from MQ induced 97
viii

cytotoxicity
5.3.7. Mefloquine reduces mitochondrial respiration 98
5.3.8. Mefloquine reduces ATP synthase activity 99
5.3.9. PTEN sensitizes glioma cells to mefloquine treatment 101
5.4. Discussion 104
5.5. Conclusion 109

Chapter 6: Conclusion and future directions
6.1. Conclusion 110
6.2. Future directions 112
6.2.1 Determination of MQ’s effect on inflammation 113
6.2.2. To study the effect of GRP78 induction by MQ on tumor progression 114
6.2.3. Determination of MQ’s effect on angiogenesis and metastasis 115
6.2.4. Determination of MQ’s effect on amino acid transporters 115

Comprehensive Bibliography 117

ix

List of Figures
Figure 1. Process of autophagy 2
Figure 2. Role of autophagy in tumor suppression 5
Figure 3. Role of autophagy in tumor progression 7
Figure 4. Induction of autophay by different mechanism(s) 11
Figure 5. Structure of Mefloquine 14
Figure 6. Cytotoxic and antiproliferative effects of MQ 26
Figure 7. Cytotoxic effects of MQ on multidrug resistant cells 28
Figure 8. Induction of apoptosis by MQ 31
Figure 9. Inhibition of autophagy by MQ 34
Figure 10. Altered chemosensitivity to MQ after Beclin 1 knockdown 36
Figure 11. Increased chemosensitivity by MQ treatment 37
Figure 12. Role of GRP 78 and CHOP in cancer and for anticancer effects 46
Figure 13. Accumulation of ubiquitinated proteins and proteasome activity 52
Figure 14. Induction of ER stress by MQ 54
Figure 15. Mefloquine enhances cytotoxic effects of ERS aggravators 56
Figure 16. Diagram illustrating biosynthetic and metabolic pathways of
sphingolipids
60
Figure 17. Diagram representing opposite role of S1P and ceramide in
cancer and for anticancer effects
63
Figure 18. Decrease in S1P levels by MQ 69
x

Figure 19. Increase in ceramide and sphingosine levels by MQ 70
Figure 20. Effect of MQ on Sphk activity 71
Figure 21. Mefloquine reduces proliferation of glioma cells 72
Figure 22. Reduced migration of glioma cells by MQ 73
Figure 23. Mefloquine prevents closure of wound 74
Figure 24. Mefloquine reduces invasiveness of glioma 75
Figure 25. Cytotoxic and antiproliferative effects of MQ 89
Figure 26. Cytotoxic effects of MQ on TMZ resistant glioma cell lines 91
Figure 27. Induction of necrosis by MQ 93
Figure 28. Effect of MQ on GLUT 3 levels 95
Figure 29. MQ treatment reduces glucose uptake in glioma cells 96
Figure 30. 2DG potentiates MQ induced cytotoxicity 97
Figure 31. Partial protection of MQ treated cells by MP 98
Figure 32. MQ reduces mitochondrial respiration 99
Figure 33. MQ reduces ATP synthase activity 101
Figure 34. PTEN sensitizes cells to MQ 103
Figure 35. Schematic representation of MQ’s mechanism of action 112
Figure 36. Effect of MQ on tumor growth 114
Figure 37. Proposed effects of autophagy inhibition by MQ 116

xi

Abbreviations

2-NBDG 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose
3-MA 3-Methyladenine
AMC 7-amido-4-methylcoumarin
AML Acute Myeloid Leukemia
ANOVA Analysis of Variance
ATCC American Type Culture Collection
ATF6 Activation Transcription Factor 6
ATP Adeninosine Triphosphate
AV Autophagosome
BCA Bicinchoninic acid
BECN1

Beclin 1
BSA Bovine Serum Albumin
cDNA Complementary Deoxyribonucleic Acid
CFA Colony Formation Assay
CHOP CCAAT/enhancer binding protein homologous transcription factor
COX-2 Cyclooxygenase-2
CQ Chloroquine
DCIP Dichlorophenolindophenol
DMC 2,5-dimethyl-celecoxib
DMSO Dimethylsulphoxide
xii

DNA

Deoxyribonucleic Acid
DOX Doxorubicin
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetic Acid
EGTA Ethylene Glycol Tetraacetic Acid
ELISA Enzyme-Linked Immunosorbent Assay
ER Endoplasmic Reticulum
ERAD Endoplasmic Reticulum Associated Degradation
ERS Endoplamsic Reticulum Stress
ESI Electrospray Ionization
FBS Fetal Bovine Serum
FDA Food and Drug Administration
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
GFP Green Fluorescent Protein
GLUT Glucose Transporter
GRP 78 Glucose regulated protein 78
HCl

Hydrochloric Acid
HCQ Hydroxychloroquine
HIF-1α Hypoxia Inducible Factor - 1α
HIV Human Immunodeficiency Virus
HPLC High Performance Liquid Chromatography
IC50

Half Maximal Inhibitory Concentration
xiii

IL Interleukin
INT Iodonitrotetrazolium
iPTEN Inducible Phosphatase and Tensin homolog
IRE Inositol Requiring Kinase
JNK c-Jun N-Terminal Kinase
KCl Potassium Chloride
LC 3

Microtubule Associated Protein Light Chain 3
LCMS Liquid Chromatography Mass Spectrometry
LDH Lactate Dehydrogenase
MDR Multidrug Resistance
MgCl
2
Magnesium Chloride
MP Methyl Pyruvate
MQ Mefloquine
MRP 2 Multidrug Resistance-associated Protein 2
MRP 4 Multidrug Resistance-associated Protein 4
mTOR Mammalian Target of Rapamycin
MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)
Na
2
HPO
4
Disodium hydrogen phosphate
NADH Nicotineamide Adenine Dinucleotide
NF-ƙB

Nuclear Factor Kappa B
NFV Nelfinavir
OCR Oxygen Consumption Rate
xiv

OXPHOS Oxidative Phosphorylation
p53 Protein 53
PARP Poly (ADP ribose) Polymerase
PBS Phosphate Buffered Saline
PERK Protein Kinase RNA-like Endoplasmic Reticulum Kinase
Pgp P-Glycoprotein
PI3K Phosphoinositide 3-kinase
PTEN Phosphatase and Tensin Homolog
qRT-PCR Quantitative Real Time Polymerase Chain Reaction
Rb Retinoblastoma
RNA Ribonucleic Acid
ROS Reactive Oxygen Species
S1P Sphingosine-1-phosphate
SCID Severe Combined Immunodeficiency
SD Standard Deviation
siRNA Small Interfering Ribonucleic Acid
SMase Sphingomyelinase
Sphk Sphingosine Kinase
TCA Tricarboxylic Acid
Tg Thapsigargin
TME Tumor Microenvironment
TNBC Triple Negative Breast Cancer
xv

UPR

Unfolded Protein Response
UPS Ubiquitin Proteasome System
WB Western Blot
xvi

ABSTRACT
The ability of autophagy to support cancer cells under the conditions of metabolic,
chemotherapeutic and endoplasmic reticulum stress (ERS) has led to its emergence as an
important target for anticancer therapy. The antimalarial drug chloroquine (CQ) is able to
inhibit autophagy and therefore is being considered for cancer therapeutics. However, the
relatively low potency of CQ prompted us to investigate whether other FDA approved
lysosomotropic agents such as mefloquine (MQ), levofloxacin and ciprofloxacin might
me more effective and hence potentially more useful. We hypothesized that out of
various drugs tested MQ, an antimalarial drug will be most effective as an anticancer
agent. This hypothesis stems from the facts that MQ owing to its ability to manipulate
lysosome will inhibit autophagy. Moreover, previous studies have shown the ability of
MQ to induce the markers of ERS in neurons and also to increase intracellular ceramide
level in malarial parasite, which are known to induce autophagy as a survival mechanism.
Thus, higher potency of MQ will arise from its ability to produce two-thronged effect. On
one hand MQ will increase cellular stress and on the other inhibit autophagy. We found
that MQ was most potent compound tested; it inhibited autophagy and caused cell death
in breast and glioma cancer cells. Furthermore, MQ triggered ERS, increased ceramide
and reduced glucose metabolism and ATP synthase activity. Altogether, our study
demonstrates superior potency of MQ, because of its ability to elevate cellular stress and
inhibit autophagy – the adaptive response to cellular stress.
1

CHAPTER 1
Introduction to Autophagy and Its Role in Cancer

1.1. Autophagy
The progressive transformation of normal cells to malignant one endows the cell with the
ability to survive and proliferate under the unfavorable conditions as is present in the
tumor microenvironment by initiating different processes or protective mechanisms.
Among various adaptive processes contributing to improved survival is autophagy, which
promotes cellular survival under stressful conditions such as low nutrients. Autophagy is
a genetically regulated and evolutionary conserved process (Amaravadi et al., 2011). It is
a lysosomal degradation process, which degrades the decaying organelles and damaged
and unwanted proteins (Kondo et al., 2005; Mathew et al., 2007).
Autophagy consist of several different steps, which are controlled by autophagy related
proteins (Atg) (Amaravadi et al., 2011), summarized in Fig. 1. During autophagy an
isolation membrane called as phagophore is formed around the cargo targeted for
degradation. The edges of the phagophore then fuse to form a double membrane vesicle
called as autophagosomes (AV). This is followed by fusion of the autophagosome with
the lysosomes to form autophagolysosome, where the material encapsulated in the vesicle
along with the inner membrane of the autophagosome is degraded by the lysosomal
hydrolases, as shown in Fig. 1 (Amaravadi et al., 2011; Fehrenbacher and Jäättelä, 2005;
Levine and Kroemer, 2008).
2

Figure 1: Process of Autophagy. During autophagy an isolated membrane called as phagophore is formed around the
targeted cargo to be degraded, which is associated with the adaptor proteins. This is followed by the formation of
autophagosome, which are characterized by the association of LC 3II with the membrane. Final step involves fusion of
autophagosome with the lysosome to form autophagolysosome.

The formation of AV can be categorized into four different steps, 1) induction, 2)
nucleation, 3) elongation and 4) completion (Janku et al., 2011; Levine and Kroemer,
2008). All these steps are tightly regulated by different Atg proteins. The protein complex
consisting of ULK 1 / 2, Atg 13, FIP 200 and Atg 20 initiate the induction step. This is
followed by vesicle nucleation, which utilizes a multi-protein complex that includes class
III phosphatidylinositol 3 kinase (PI3K) and beclin 1. The elongation of the vesicle
requires an ubiquitin like protein conjugation, which is mediated by Atg 3, Atg 5, Atg 7
and microtubule associated protein light chain 3 (LC3). During this process, a
cytoplasmic ubiquitin like protein LC3 I, is conjugated with phosphoethanolamine to
form LC3 II, which is specifically associated with the membranes of AV (Amaravadi et
al., 2011; O'Donovan et al., 2011). LC 3 II integration with the membranes of AV
recruits the cargo adaptor proteins such as p62, NIX, Nbr 1. These adaptor proteins, in
turn recruit components such as ubiquitinated proteins, a common substrate for p62, from
3

the cytoplasm to the AV for autophagic degradation (Pankiv et al., 2007). The final step
of completely encapsulating the cytoplasmic cargo, the completion phase is assisted by
Atg 10, Atg 12 and Atg 16L (Janku et al., 2011).
1.2. Role of Autophagy in Cellular Homeostasis
The main role of autophagy under physiological conditions is to assist in removing
unwanted proteins and damaged or decaying organelles. This mechanism helps to
maintain protein and organelle quality control by preventing their accumulation in the
cell. In addition to its basic role in protein and organelle turnover it also functions as a
stress response mainly towards nutrient starvation (Kondo et al., 2005). The recycling of
cellular components by autophagy serves as an alternative means of energy during
metabolic stress, which helps to maintain homeostasis and viability. In this regard,
autophagy provides building blocks for the generation of macromolecules. In addition to
its housekeeping functions autophagy is also utilized for clearance of pathogens and
apoptotic bodies (Colombo, 2007; Qu et al., 2007; Saka et al., 2007). Also, autophagy
plays an important role in several pathological conditions such as cancer, aging, infection
and neurodegenerative disorders (Levine and Kroemer, 2008).
1.3. Autophagy in Cancer
The role of autophagy in cancer is context dependent. It can function as a tumor
suppressor and prevent initiation of tumor development. Paradoxically, in established
4

tumors autophagy functions as a survival mechanism, which assists tumor growth and
proliferation under stressful conditions (Amaravadi et al., 2011; Kondo et al., 2005).
1.3.1. Autophagy and Tumor Suppression
The role of autophagy in preventing tumor initiation was shown in studies demonstrating
that mice with defective autophagy were highly prone to tumor development (Amaravadi
et al., 2011). Several tumor models such as breast, ovarian and prostate have shown to
carry allelic deletion of beclin 1 (BECN1 gene), which is an essential autophagic protein.
It is believed that such defects in beclin 1 supports tumorigenesis (Mathew et al., 2007;
Qu et al., 2003). This was supported by studies showing that the introduction of BECN1
into MCF 7 cells were able to induce autophagy and thus prevent tumorigenesis (Kondo
et al., 2005; Liang et al., 1999).
The tumor suppressive role of autophagy is believed to arise from its ability to clear the
cellular components. As accumulation of damaged cellular content, often in large
aggregates or inclusions owing to defective autophagy is associated with the production
of reactive oxygen species (ROS), activation of DNA damage response, cell damage,
proteotoxicity and eventually cell death, which is associated with chronic inflammatory
state (Amaravadi et al., 2011; Degenhardt et al., 2006; Mathew et al., 2009). Moreover,
defective autophagy can also result in necrotic cell death and cell lysis under metabolic
stress, which in turn stimulate innate immune response, attract inflammatory cells,
increase cytokine production and activate nuclear factor – kappa B (NF-ƙB) signaling
(Karin, 2006; White et al., 2010). Moreover, autophagy inhibition causes p62
5

accumulation, which is shown to activate NF-ƙB signaling. Also, accumulated p62
interacts with mammalian target of rapamycin (mTOR), RAPTOR and RAG proteins
leading to activation of PI3K/Akt/mTOR mediated oncogenic signaling (Mathew et al.,
2009; White, 2012b). This state of chronic tissue damage and inflammation can
ultimately give rise to mutations resulting in cancer development and promote tumor
progression, as shown in Fig. 2 (Mathew et al., 2007; White et al., 2010).

Figure 2: Role of autophagy in tumor suppression. (a) Process of autophagy can suppress the initiation of tumor
development by clearing damaged proteins and organelles from the cell. (b) Defective autophagy causes accumulation
of damaged organelle and proteins leading to generation of stress. This stress can give rise to mutations, cell death and
evoke an immune response, eventually leading to tumor development (modified from (White, 2012a)).

6

1.3.2. Autophagy Enables Tumor Progression
Autophagy facilitates survival during metabolic stress
Although autophagy suppresses tumor development, it can paradoxically also promote
tumor proliferation and survival in established cancers. It is considered to be an adaptive
mechanism in response to metabolic stress and hypoxia, arising from inadequate blood
and oxygen supply, vascular collapse and therapeutic intervention (Amaravadi et al.,
2011; Mathew et al., 2007; White and DiPaola, 2009). Autophagy accomplishes its tumor
promoting effects by degradation and intracellular recycling of cellular components for
maintenance of energy metabolism and by maintaining the functional pool of
mitochondria (Fig. 3) (Kondo et al., 2005; White, 2012a; White et al., 2010). In addition,
autophagy can also direct cells to the state of dormancy or quiescence in response to
metabolic crisis or hypoxia, especially in cells with defective apoptosis. These cells are
viable but quiescent and hence are less responsive towards radiotherapy and
chemotherapeutics, which readily targets rapidly dividing cells. Thus, autophagy favors
dormant or quiescent cells that tolerate cytotoxic treatment and survive only to resume
growth and proliferation when conditions are appropriate. The existence of these
quiescent cells is the fundamental barrier for the effective cancer therapy as they
eventually re-emerge and result in disease relapse (Fig. 3) (Amaravadi, 2008; Mathew et
al., 2007; Sosa et al., 2013; White, 2012a).
7

Figure 3: Role of autophagy in tumor progression. Rapid proliferation of cells in tumor results in generation of hostile
environment characterized by hypoxia, low blood and nutrient supply. These conditions result in generation of stress
and induction of autophagy, which enable tumor progression.
1.4. Induction of Autophagy as an Adaptation Strategy
1.4.1. Autophagy and Chemotherapeutic Stress
Normally autophagy is activated in responses to oxygen and nutrient deprivation;
however it can also be induced in response to chemotherapeutic-induced stress such as
treatments that damage DNA, proteins and organelles. Also, targeted cancer therapy has
been shown to stimulate autophagy by up-regulating signaling of starvation or growth
factor deprivation. Induction of autophagy has been observed with the use of rapamycin,
8

an inhibitor of mTOR (Hanahan and Weinberg, 2011; White, 2012a). Induction of
autophagy not only activates cellular mechanisms that improve tumor survival, but these
mechanisms can also enhance resistance towards chemotherapeutics (Amaravadi, 2008).
Furthermore, inhibition of autophagy can restore tumor cell chemosensitivity towards
alkylating agents such as temozolomide (Kanzawa et al., 2004; White, 2012a).
1.4.2. Autophagy Mitigates Endoplasmic Reticulum Stress (ERS)
The endoplasmic reticulum (ER) is an intracellular organelle responsible for proper
folding of newly synthesized proteins, lipid biosynthesis and calcium homeostasis (Tsai
and Weissman, 2010). Under normal physiological conditions, the protein load on ER is
much lower than its protein folding capacity. Paradoxically, in cancer owing to rapid
proliferation there is an excessive accumulation of mis-folded proteins and creation of
hostile environment characterized by hypoxia and hypoglycemia, leading to elevated
ERS (Schönthal, 2009).
ERS can be a “double-edged sword” with the potential to promote protective and pro-
apoptotic pathways. The predominance of the biological activity is dependent on the level
of ERS. Low to moderate ERS enhances GRP 78 (glucose regulated protein 78)
induction, which promotes cell survival under stressful conditions by up-regulating
unfolded protein response (UPR). It is a process, which acts to relieve the load on ER by
increasing ER folding capacity, reducing the level of protein synthesis and up-regulating
the components of ER associated degradation pathway (ERAD). The cellular responses
induced by UPR are mediated through activation of three important stress sensors: IRE 1
9

(α and ß isoforms), activating transcription factor 6 (ATF 6) and protein kinase RNA-like
ER kinase (PERK) (Hetz, 2012; Suh et al., 2012). In contrast, a greater increase in ERS
tilts the balance towards the pro-apoptotic effects mainly mediated through the induction
of CCAAT/enhancer binding protein homologous transcription factor (CHOP)
(Schönthal, 2009; Tsai and Weissman, 2010).
Induction of autophagy and activation of ubiquitin proteasome system (UPS) in response
to elevated ERS represents an adaptive response for survival under stressful conditions.
Autophagy-lysosome system and UPS constitute two major degradation systems. UPS is
mainly involved in the degradation of mis-folded and short lived proteins, whereas
autophagy helps to clear long lived and aggregated proteins (Amaravadi et al., 2011). The
crosstalk between UPS and autophagy aids in mitigating ERS by degrading the mis-
folded and damaged proteins. Studies have shown that drug induced ERS up-regulates
autophagy, which by clearing unwanted proteins reduces ERS and prevent cell death by
ERS aggravators (Schönthal, 2009). Furthermore, recent evidences highlight that
targeting either UPS or autophagy results in increased stress on ER eventually leading to
activation of pro-apoptotic arm of ERS. Thus, induction of autophagy in response to
elevated ERS appears to be a survival mechanism.
1.4.3. Induction of Autophagy in Response to Sphingolipids
Sphingolipids are a family of membrane lipids with important structural roles in
maintaining membrane fluidity and sub domain structures like lipid rafts, recent
evidences highlight their role as signaling molecules. In addition, they have been shown
10

to play important role in regulating cellular process including apoptosis, cell growth,
migration, adhesion and autophagy. Sphingosine-1-phosphate (S1P) and ceramide are
two sphingolipids that play important role in regulating cancer survival. S1P and
ceramide have opposing effects on cancer survival, where S1P favors cell survival and
proliferation in contrast to ceramide that promotes apoptosis and growth arrest (Young et
al., 2013). Thus, for effective anticancer therapy it is essential to increase the intracellular
levels of pro-apoptotic ceramide and reduce the levels of S1P (Barth et al., 2011).
Recently, studies have shown induction of autophagy in response to elevated ceramide
levels. Although the exact mechanism(s) as how ceramide initiate autophagy induction is
not well delineated. The induction of autophagy by ceramide could arise from its ability
to inhibit Akt/mTOR signaling. Inhibition of Akt/mTOR signaling can mimic a state of
growth factor deprivation (Hanahan and Weinberg, 2011; Janku et al., 2011; Young et al.,
2013). Moreover, studies have shown that ceramide can downregulate nutrient
transporters such as glucose transporters (GLUT), thereby creating a state of starvation
(Guenther et al., 2008). In addition, ceramide activates stress activated kinase JNK,
which activates a transcription factor c-Jun. This activation of c-Jun by ceramide
eventually results in increased expression of Beclin 1 and LC3, thereby assisting the
induction of autophagy (Li et al., 2009; Sun et al., 2011). Overall, induction of autophagy
by ceramide represents a survival mechanism, which can compensate for ceramide
induced cellular stress.
11

Therefore from understanding of the aforementioned findings, autophagy plays an
important role in established cancers. Thus, autophagy is an important target for the
development of new anticancer therapy and for improving the efficacy of existing radio
and chemotherapeutics.

Figure 4: Induction of autophagy by different mechanism(s): Autophagy is induced in response to different stress
stimuli such as ERS, UPS inhibition, chemotherapeutic stress, ceramide and metabolic stress.
1.5. Autophagy and Anticancer Therapy
Autophagy has emerged as an important survival mechanism of tumor cells and hence its
inhibition has gained immense attention for the development of new anticancer therapy.
12

The main goal of autophagy inhibition in cancer is to augment stress. Since autophagy is
mainly activated as a stress response to different stimuli including metabolic and
therapeutic stress. Based on this, several studies have focused on combining autophagy
inhibition with variety of stress stimuli, such as ERS aggravators, proteasome inhibitors,
DNA damaging agents and therapy aimed at targeting PI3K/AKt/mTOR (Bellodi et al.,
2009; Schönthal, 2009; Thomas et al., 2012; White, 2012a).
1.5.1. Targeting autophagy by pharmacological means
The critical role autophagy plays in cellular survival, and drug resistance has led its
inhibition as an important druggable target. To this end, several small molecule
inhibitors of autophagy are in development (Garber, 2011; White, 2012a). Currently
autophagy inhibition can be achieved by targeting the nucleation of AV using inhibitors
that block recruitment of class III PI3K (Vps 34) such as wortmannin and 3-methyl
adenine. Other way to target autophagy is by blocking the delivery and degradation of the
content of the AV to and by the lysosomes. Since AV moves along the microtubules, the
drugs that disrupt microtubules such as nocodazole, colchicine, vinca alkaloid and
taxanes may block the delivery of autophagic cargo to lysosome (Amaravadi et al., 2011).
Lysosomes are intracellular organelles, containing enzymes that are able to digest
degrading organelles, damaged proteins and dead or damaged cells. The lysosomal
enzymes are dependent on the acidic pH found in the lysosomes for their optimum
activity. Disruption of lysosomal pH has emerged as an effective way to inhibit
autophagy by preventing the degradation of the content of AV (Amaravadi et al., 2011;
13

Boya and Kroemer, 2008). Based on this rationale, several lysosomotropic agents (drugs
that accumulate in lysosomes owing to their physicochemical characteristics) such as
bafilomycin A1 and antimalarial agent hydroxychloroquine (HCQ) and chloroquine (CQ)
are being evaluated as autophagy inhibitors in clinic for different types of cancers (Boya
and Kroemer, 2008; Bristol et al., 2013; Janku et al., 2011).
The development of CQ and HCQ as anticancer agents has revealed their ability to block
autophagy and produce anticancer effects but have relatively low potency. This low
antitumor potency demands the use of higher doses to achieve effective autophagy
inhibition, which eventually give rise to side effects (Amaravadi et al., 2011; Sharma et
al., 2012). Moreover, a recent study has revealed ineffectiveness of CQ in an in vivo
breast cancer model further casting doubts pertaining to its therapeutic efficacy (Bristol et
al., 2013).
In summary, the critical role of autophagy in tumor survival, disease progression and
drug resistance, and the drawbacks associated with current therapy aimed at targeting
autophagy demands the discovery of novel autophagy inhibitors (Amaravadi et al., 2011;
Sotelo et al., 2006).
1.6. Hypothesis
We analyzed the anticancer effects of several FDA approved lysosomotropic agents such
as levofloxacin, ciprofloxacin and mefloquine (MQ). We hypothesized that out of various
drugs tested MQ (Fig 5) will be most effective as an anticancer agent. This hypothesis
14

arises from the fact that MQ increases transcription of ERS markers such as GRP 78 and
CHOP in neurons (Dow et al., 2003) and raises intracellular ceramide levels in
Plasmodium falciparum, the malarial parasite (Pankova-Kholmyansky and Flescher,
2006). Since elevated ERS and ceramide are known to induce autophagy as an adaptive
response, we hypothesize that higher potency of MQ will result from its two thronged
effect. On one hand MQ will generate cellular stress and on the other owing to its ability
to manipulate lysosomes it will inhibit autophagy, the survival mechanism in response to
aggravated cellular stress.

Figure 5: Structure of Mefloquine
1.7. Outline of the dissertation
This dissertation presents the studies involving identification and characterization of MQ
as a novel autophagy inhibitor with potential anticancer efficacy (Chapter 2). Since our
study indicates the ability of MQ to produce potent anticancer effects in nutrient rich cell
15

culture conditions, we decided to analyze its effects on various processes, which induce
autophagy as an adaptive response (Fig. 4). Firstly, we analyzed the effect of MQ on the
levels of ERS markers and the contribution of ERS to MQ’s anticancer effects (Chapter
3). We also studied the effect of MQ on UPS, the other degradation system in the cell
(Chapter 3). Secondly, we focused on studying the effect of MQ on intracellular levels of
key sphingolipids (S1P and ceramide) and also analyzed the effect of MQ on various
processes affected by S1P concentration (Chapter 4). Thirdly, we analyzed the effect of
MQ on glucose metabolism, which is shown to be altered by the elevated ceramide
levels. Finally, we analyzed the effect of MQ on mitochondrial respiration, a key source
of ATP production in the cell (Chapter 5).

16

Chapter 2:
Inhibition of Autophagy and Induction of Anticancer
Effects by Mefloquine

2.1. INTRODUCTION
Autophagy is a homeostatic, genetically controlled and evolutionary conserved process
(Kondo et al., 2005). It is a catabolic degradation process, which utilizes lysosomal
enzymes for the degradation of unwanted and damaged proteins, organelles and
macromolecules with subsequent recycling of the components. Although the main role of
autophagy is to maintain nutrient and energy homeostasis (Mathew et al., 2007; Yang and
Kimmelman, 2011) recent evidences highlight its role in several pathological conditions
such as cancer (Murrow and Debnath, 2012). As discussed in Chapter 1, autophagy has
dual role in cancer, acting both as a tumor suppressor and promoter (Amaravadi et al.,
2011; White and DiPaola, 2009; Yang et al., 2011b).
The tumor promoting role of autophagy stems from several line of evidences. A recent
study involving the analysis of 1400 tumor specimen from different tissues of origin
showed increased LC3B expression (both diffuse and punctate) in the cytoplasm. High
LC3B expression strongly correlates with tumor proliferation, progression, metastatic
potential, and poor patient outcome especially in case of breast cancer (Lazova et al.,
2012). In addition, high level of autophagy has been reported in the malignant melanoma
17

patients with superficial spreading type of the disease and also in dermis of the patients
with invasive melanoma (Lazova et al., 2010). Moreover, in melanoma patients high
autophagic flux is associated with more aggressive form of disease and poor patient
survival (Ma et al., 2011). Another study has reported elevated autophagic index in
pancreatic ductal adenocarcinoma (PDAC). This type of cancer has been shown to
depend on autophagy even in stress free environment, in contrast to previous findings
where studies have shown induction of autophagy as a stress response generated from
hypoxia or nutrient deprivation owing to poor blood supply (Janku et al., 2011; Yang and
Kimmelman, 2011; Yang et al., 2011a). Moreover, increased LC3 expression in
pancreatic cancer patients has been associated with poor outcome and shorter disease free
period (Fujii et al., 2008). Thus, the tumor promoting effects of autophagy are
independent of the tissue of origin. Moreover, induction of autophagy is usually
associated with the more aggressive and difficult to treat form of disease.
In addition, autophagy also contributes to the development of chemoresistance increasing
the risk of treatment failure (Hippert et al., 2006; White, 2012a). One striking example
was derived from a study involving Akt inhibition (Degtyarev et al., 2008). According to
this study Akt inhibitors failed to induce anticancer effects due to induction of autophagy.
Thus, induction of autophagy in response to drugs has emerged as an adaptive response,
which favor cell survival over death (Amaravadi et al., 2011).
This recognition of autophagy process as an “escape route”, has led its inhibition as an
important target for the development of novel therapies. To this end, CQ and its
18

derivative HCQ, are being evaluated in various preclinical cancer models and in clinical
trials to reveal their efficacy as an adjuvant to cancer therapeutic regimens. These studies
have revealed promising, albeit low antitumor activity (Amaravadi et al., 2011; Sotelo et
al., 2006). This low potency of CQ and HCQ demands the discovery of novel autophagy
inhibitors.
In an effort to identify and characterize more potent inhibitors of autophagy with potent
anticancer properties, we investigated several lysosomotropic agents that currently are in
clinical use for other purposes, such as levofloxacin, ciprofloxacin, and MQ (Boya et al.,
2003; Glaumann et al., 1992; Ouedraogo et al., 1999; Ouédraogo et al., 2000). We
hypothesized that these drugs owing to their ability to accumulate in lysosomes and alter
its environment will block the degradation of the content of AV, eventually leading to
autophagy inhibition.
Levofloxacin and ciprofloxacin are synthetic fluoroquinolones that are widely used as
broad-spectrum antibiotics (Fass, 1990; Fu et al., 1992). MQ is an FDA-approved drug
for the prophylaxis and treatment of malaria (Dow et al., 2003). MQ is known to inhibit
MDR1/Pgp (multidrug resistance protein 1/permeability glycoprotein) (Riffkin et al.,
1996; Wu et al., 2005) and because of which, when used in combination with vinblastine
or doxorubicin, MQ was shown to reverse the resistance of cancer cells to the latter
(Fujita et al., 2000; Riffkin et al., 1996).
For the purpose of this study, we analyzed the effect of MQ on various breast cancer cell
lines. The current treatment for breast cancer involves surgery, chemotherapy with the
19

hormone therapy and/or targeted therapy. The hormone therapy is used for estrogen and
progesterone receptor positive breast cancers. The targeted therapy focuses on
specifically attacking the cancer cells without harming the normal cells. Currently
monoclonal antibodies against HER-2 (Herceptin) and the tyrosine kinase inhibitor that
blocks the effect of HER2 (Lapatinib) are used as a targeted therapy for breast cancer.
But approximately 15% of globally diagnosed patients have triple negative breast cancer
(TNBC) i.e., they are estrogen, progesterone receptor negative, and do not overexpress
epidermal growth factor receptor (Her2/Neu) and hence are unresponsive to the current
chemotherapy (Arnedos et al., 2012; Thomas et al., 2012). The rapid development of
chemoresistance towards current therapy and lack of effective treatment for TNBC
demands paradigm-changing approaches resulting in curative response. Hence, we
studied the effect of MQ on both, hormone receptor positive T47D and TNBC, MDA-
MB-231, which are KRas mutated (Hollestelle et al., 2010). Several studies have shown
that Ras oncogene upregulates autophagy, which is required for tumor cell survival. The
induction of autophagy helps to ensure the energy balance in tumors with Ras mutation.
Since Ras mutated and triple negative breast cancers have poor prognosis and are
difficult to treat, several studies have suggested to exploit this autophagy addiction for the
development of anticancer therapy (Elgendy et al., 2011; Guo et al., 2011; Tate et al.,
2012; Yang et al., 2011a).
Here, we present results characterizing MQ as a potent inhibitor of autophagy and
inducer of cell death in both hormone receptor positive and negative cell lines, indicating
its potential usage for such difficult to treat breast cancers.
20

2.2. Materials and Methods
2.2.1. Materials
MQ, CQ, levofloxacin, ciprofloxacin and 3-methyl adenine (3-MA) were purchased from
Sigma-Aldrich (St. Louis, MO). MQ and ciprofloxacin were dissolved in DMSO to
produce a 100 mM and 50 mM stock solution, respectively. Levofloxacin and CQ were
dissolved in water to produce 100 mM stock solutions. 3-MA was dissolved in water by
heating to produce 200 mM stock solution. Paclitaxel (Taxol
®
) was obtained from the
pharmacy as a 10 mM stock solution. ZVAD-fmk was purchased from Promega
(Madison, WI) as a 20 mM stock solution. All agents were added to the cell culture
medium in a manner so that the final concentration of solvent (DMSO or water) was
considerably less than 1%.
2.2.2. Cell lines and culturing
Human breast cancer cell lines MDA-MB-231, MDA-MB-468 and T47D were obtained
from American Tissue Culture Collection (ATCC, Manassas, VA). MCF7 and
MCF7/Dox (doxorubicin-resistant variant of MCF7) were kindly provided by Dr.
Amadeo M. Parissenti (Cho et al., 2009; Guo et al., 2004). All breast cancer cell lines
were propagated in DMEM supplemented with 10% fetal bovine serum, 100 U/ml
penicillin, and 0.1 mg/ml of streptomycin, and incubated in a humidified atmosphere at
37°C and 5% CO
2
.

21

2.2.3. MTT assay
Cellular viability was assessed using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide) assays, where 5.0 x 10
3
cells were seeded per well and analyzed
as described in detail (Golden et al., 2009). In individual experiments, each treatment
condition was set up in triplicate, and each experiment was repeated from one to five
times independently.
2.2.4. Colony formation assay (CFA)
Cells were seeded into six-well plates at a density of 200 cells per well. After overnight
incubation, the cells were exposed to the drug treatment for 48 hours. Thereafter, the drug
was removed by replacing the medium with fresh growth medium and the cells were kept
in culture undisturbed for 12-14 days, during which time the surviving cells produced
colonies. The colonies were visualized by staining for 4 hours with 1% methylene blue
(in 100% methanol) (Golden et al., 2009) and were counted.
2.2.5. Western Blot analysis
Total cell lysates were prepared and analyzed by Western blot as described earlier
(Chuang et al., 2008). The antibodies against CHOP, LC 3B, cleaved caspase 7, and
PARP were obtained from Cell Signaling Technologies (Beverly, MA; Cat Nos. #2895,
#2775, #9491, #9532, respectively). The antibodies against actin, GRP78, beclin1 and
ubiquitin were purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA; Cat
22

Nos. sc-130656, sc-13968, sc-11427, sc-8017, respectively). The antibodies were used at
the dilution of 1:1,000 and 1:500, respectively. The secondary antibodies were coupled to
horseradish peroxidase and detected by chemiluminescence using Super Signal West Pico
and Femto (Thermo Scientific, Rockford, IL). The membranes were imaged using
FujiFilm LAS-4000 (Fujita et al., 2000).
2.2.6. siRNA transfection
The siRNA control (scrambled) and siRNA against Beclin 1 were purchased from Cell
Signaling Tech., (Beverly, MA; Cat Nos. # 6568 and # 6246) and used at a final
concentration of 20 nM. The siRNA against CHOP/GADD153 was purchased from
Ambion (Carlsbad, CA; Cat No. s3996) and used at a final concentration of 5 nM. All
siRNA transfections were performed using LipofectAMINE 2000 (LA-2000) purchased
from Invitrogen (Carlsbad, CA). The cells were plated at 30-50% confluence in a 6-well
plate. After an overnight incubation, the medium was changed to antibiotic-free medium.
For transfection, 5 µl of LA-2000 was mixed with 245 µl of Opti-MEM (Invitrogen) at
room temperature for 5 min and then was incubated with 250 µl of a mixture of siRNA
duplex and Opti-MEM for 20 min at room temperature. The cells were incubated with
500 µl of this mixture for 12 hours. After incubation, the medium was replaced with fresh
DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The
following day, the cells were seeded and harvested for CFA and western blot
respectively.

23

2.2.7. Quantitative reverse-transcriptase polymerase chain reaction
The gene expression for efflux transporters was quantified using quantitative reverse
transcriptase-polymerase chain reaction (qRT-PCR). Total RNA was isolated from cells
using RNeasy kit (Qiagen, Valencia, CA) and RNA concentration was measured using a
ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) at 260 and
280 nm. Following RNA quantification, cDNA was synthesized by using SuperScript
TM III First strand Synthesis SuperMix (Invitrogen, Foster City, CA). The mRNA
expression for MRP 2, MRP 4 and P-gp was determined by qRT-PCR using SYBR
Green ER (Invitrogen, Foster City, CA). GAPDH was used as the control housekeeping
primer for each sample.
2.2.8. Cell death ELISA
Cells (4x10
3
cells/well) were seeded in a 96-well plate. After complete adherence, the
cells were treated with drug for 30 hours and then analyzed for the presence of histone-
complexed DNA fragments using cell death detection ELISA kit (Roche Diagnostics,
Indianapolis, IN) according to the manufacturer’s instructions. The kit was used to
specifically quantify apoptotic cell death.
2.2.9. Statistical analysis
Data are presented as mean ± SD. Comparisons were made between different treatments
using two-way ANOVA, and a p-value of less than 0.05 was considered significant.
24

2.3. Results
2.3.1. Differential cytotoxicity of lysosomotropic agents
We compared the cytotoxic potential of four established lysosomotropic agents, namely
two antimalarial drugs, CQ and MQ, and two fluoroquinolones, levofloxacin and
ciprofloxacin. Human breast cancer cell lines with different genetic backgrounds and
representing different subtypes of this disease (Hollestelle et al., 2010) were used in our
experiments: we chose MCF7 (estrogen receptor positive), T47D (estrogen receptor
positive, p53 mutation), MDA-MB-231 (estrogen receptor, progesterone receptor and
Her2/Neu receptor negative, p53 mutation, p16 deletion, K-ras mutation), and MDA-MB-
468 (estrogen receptor, progesterone receptor and Her2/Neu receptor negative, p53
mutation, PTEN mutation, Rb mutation). The cells were treated with increasing
concentrations of each drug, and cell viability was assessed after 48 hours using the MTT
assay. As shown in Fig. 6A, levofloxacin and ciprofloxacin displayed very little
cytotoxicity (IC50 >100 µM); CQ was more potent, although cytotoxic effect appeared to
depend on the cell type and the IC50 varied considerably from 30 µM to >100 µM. In
comparison, MQ was the most cytotoxic compound with an IC50 in the range of 3-12
µM (Fig. 6 A, B).
The above short-term (48 hours) cytotoxicity assays were complemented by long-term
colony formation assays (CFAs), where we assessed the ability of individual cells to
survive 48 hours of drug treatment and spawn a colony of descendants within the
25

following 2 weeks. As shown in Fig. 6C, MQ displayed further increased anticancer
activity in this type of assay. Concentrations as low as 2.5 and 5.0 µM reduced colony
formation to below 25% in the case of T47D and MDA-MB-231 cells, respectively, and
10 µM MQ effectively abolished any colony formation. CQ also displayed higher
potency in this assay (Fig. 6D), but it was substantially less potent than MQ and required
40 µM to reduce colony formation to below 25%, and 80 µM to completely inhibit
colony formation in the two cell lines tested. Altogether, these data demonstrate potent
cytotoxic activity of MQ in the low micromolar range. Because MQ was substantially
more potent than the other lysosomotropic agents including CQ—and thus holding
potential for clinical use as an adjuvant to anticancer therapy—we decided to characterize
its cellular and molecular effects in greater detail.
26

Figure 6: Cytotoxic and antiproliferative effects of MQ. Different breast cancer cell lines were treated with increasing
concentrations of various lysosomotropic drugs and cell viability was analyzed. (A) Cells were treated with the
indicated concentrations of MQ, chloroquine, ciprofloxacin and levofloxacin for 48 hours, and cell viability was
determined by MTT assay. (B) Cells were treated with increasing concentrations of MQ for 48 hours, and cell viability
was determined by MTT assay. Shown is percent cell survival (mean ±SD, n≥3), where the value from untreated
control cells was set to 100%. (C & D) Cells were treated with MQ and CQ respectively, for 48 hours and cell survival
was determined by colony formation assay after an additional 12 days in culture without drug. Shown is percent colony
formation (mean ±SD, n≥2), where the number of colonies derived from untreated cells was set to 100%.
27

2.3.2. Mefloquine is effective in multidrug-resistant cancer cells
The development of treatment resistance is among the most pressing problems in cancer
therapy. We therefore investigated the effects of MQ on MCF7/DOX cells, which
represents a highly doxorubicin-resistant variant of MCF7 and thus serves as a model for
breast cancer having become unresponsive to therapy (Guo et al., 2004). We verified that
these cells indeed are unresponsive to treatment with doxorubicin (Fig. 7A), and that
resistance correlated with >100-fold overexpression of P-glycoprotein (Pgp), as
determined by qRT-PCR measurements of its mRNA levels (Fig. 7B) (Doublier et al.,
2012; Mechetner et al., 1998). Parental MCF7 and MCF7/DOX cells were both exposed
to increasing concentrations of MQ and cellular viability was assessed by MTT assay
after 48 hours. Remarkably, MQ was similarly effective in killing MCF7 and MCF7/Dox
cells (Fig. 7C), with IC50s of 5 µM and 11 µM, respectively, which were well within the
IC50 range of 3-12 µM, as determined from the other breast cancer cell lines (see Fig.
6A,B). Thus, the development of Pgp-mediated multidrug resistance, which generated the
unresponsiveness of MCF7/DOX cells to doxorubicin, a commonly employed
chemotherapeutic drug used in breast cancer therapy, did not lead to protection of cells
from MQ-induced cell death.
28

29

Figure 7: Cytotoxic effects of MQ on multidrug-resistant cells. Parental MCF7 and doxorubicin resistant variant
MCF7/DOX were used. (A) Cells were treated with increasing concentrations of doxorubicin for 48 hours. Thereafter,
cell survival was determined by MTT assay. (B) mRNA expression of three different transmembrane efflux pumps was
determined by quantitative RT-PCR. Shown are the relative expression levels of multidrug resistance-associated
proteins 2 and 4 (MRP2/ABCC2 and MRP4/ABCC4), as well as of P-glycoprotein (Pgp/ABCB1). (C) Cells were
treated with increasing concentrations of MQ for 48 hours, and cell survival was determined by MTT assay. Shown is
percent cell survival (mean ±SD, n≥3), where the value from untreated control cells was set to 100%.
2.3.3. Mefloquine induces apoptosis
We next sought to characterize the central molecular processes underlying MQ’s
cytotoxic effects. In order to determine the effect of MQ on cell death, we investigated
the activation of an effector caspase, i.e., effector caspase 7, and cleavage of a common
target of caspase activity, poly ADP-ribose polymerase (PARP) (Salvesen and Dixit,
1997). MDA-MB-231 and T47D cells were treated with increasing concentrations of MQ
and CQ, and cleaved caspase 7 and PARP were examined using Western blot analysis.
Because the above MTT assays had indicated very effective cell death after 48 hours of
drug treatment, we chose earlier time points to examine these proteins for our
investigation of the molecular processes leading to cell death. As shown in Fig. 8A,
exposure of cells to MQ (15 and 20 µM) and CQ (60 and 80 µM) for 16 hours resulted in
the appearance of a proteolytically cleaved (i.e., activated) fragment of caspase 7, and
cleavage of PARP, as indicated by the accumulation of a smaller molecular weight
fragment of this protein. The enhanced cleavage of caspase 7 and PARP were also
observed when the cells were treated with 10 µM MQ for longer times up to 36 hours
(Fig. 8B). To further verify ongoing apoptosis, we analyzed the amount of histone-
complexed DNA fragments (mono- and oligonucleosomes) after drug treatment with the
use of a cell death ELISA kit. As shown in Fig. 8C, MQ dose-dependently caused very
30

prominent accumulation of these DNA fragments, thus indicating the induction of
apoptotsis. The importance of caspase-mediated cell death was further analyzed with the
use of a pan-caspase inhibitor, ZVAD-fmk. Caspase inhibition resulted in increased cell
viability, from 12% to 40% in T47D cells, following treatment with 10 µM of MQ for 48
hours (Fig. 8D). A similar effect was observed in MDA-MB-231 cells (data not shown).
However, the observed rescue of cells from apoptosis in the presence of ZVAD-fmk was
incomplete and could not be improved with higher dosages of this pan-caspase inhibitor,
indicating that other mechanisms of cell death might participate as well.
31

Figure 8: Induction of apoptosis by MQ. Apoptotic cell death was analyzed in MDA-MB-231 and T47D cells after
32

drug treatment. The expression of markers of apoptosis was examined by Western blot following treatment of cells (A)
with increasing concentrations of MQ and CQ for 16 h, and (B) with 10 μM MQ for different time points. Antibodies
were specific for cleaved (i.e., activated) caspase 7 and for PARP (a target of the caspase cascade). To verify equal
loading, the blots were probed with an antibody to actin. (C) Cells were treated with increasing concentrations of MQ
for 30 hours and apoptosis was determined by cell death ELISA. Shown is the relative increase in apoptotic cell death
following drug treatment. (D) T47D cells were pretreated for 1 hour with pan-caspase inhibitor ZVAD-fmk, followed
by MQ addition, and cell death was analyzed after 48 hours by MTT assay. Shown is percent cell survival (mean ±SD,
n≥2), where the value from untreated control cells was set to 100%.
2.3.4 Mefloquine inhibits autophagy
Based on the ability of MQ to accumulate in lysosomes, we suspected that it might be a
potential inhibitor of autophagy. We therefore investigate whether MQ was able to
impinge on autophagy by analyzing two components of the autophagic process,
microtubule-associated protein light chain type 3 (LC3) and sequestosome 1
(p62/A170/SQSTM1) protein. Conversion of the cytosolic, unconjugated form of LC 3
(LC3-I) to the phosphatidylethanolamine-conjugated, membrane-bound form LC3-II is a
widely accepted marker for the formation of autophagosomes (O'Donovan et al., 2011).
In addition, p62 is an indicator as to whether the autophagic process is completed (i.e.,
degradation of p62) or whether autophagy is blocked at the stage of autophagosome
formation (i.e., accumulation of p62) (Rusten and Stenmark, 2010).
MDA-MB-231 and T47D cells were treated with MQ in a concentration- and time-
dependent fashion, and LC3-I/-II and p62 levels were analyzed by Western blot. As
shown in Fig. 9A, treatment with MQ resulted in an overall increase of LC3 expression
and prominent conversion of LC3-I to LC3-II. While the drug effects were strongest at 20
µM when cells were treated for 16 hours, they also became prominent at a lower
concentration (10 µM) when treatment was prolonged. Similarly, there was obvious
accumulation of p62, and these effects could be detected at concentrations as low as 5
33

µM MQ. In comparison, CQ increased the conversion of LC3-I/II and p62 accumulation
only at higher concentration (Fig. 9B).
The inhibition of autophagy by MQ was also verified at the cellular level using cells
stably transfected with a plasmid encoding LC3 fused to green fluorescent protein (GFP)
(Yang et al., 2010). In this case, treatment with MQ resulted in the appearance of the
typical green punctate staining, which is indicative of autophagosome formation (Fig.
9C). Combined with our data on p62, these results reveal that MQ arrests autophagy at
the stage of autophagosome formation, and thus that MQ is able to block autophagy
similar to chloroquine, but at much lower concentrations.
34

Figure 9: Inhibition of autophagy by MQ. Various markers of autophagy were analyzed in drug-treated MDA-MB-231
and T47D cells. (A) Cells were exposed to increasing concentrations of MQ for 16 hours (top panel) or to 10 µM MQ
for different times (bottom panel) or (B) with increasing concentrations of CQ for 16 hours, and cell lysates were
analyzed by Western blot for the conversion of LC3-I to LC3-II (a marker for the formation of autophagosomes) and
for the expression level of p62 (accumulation of which indicates inhibition of autophagy). Actin was used as a loading
control. (C) GFP-LC3-transfected cells were treated with 8 µM MQ or vehicle only (untreated) for 4 hours and visually
inspected via fluorescent microscopy. Representative photographs are shown, presenting the characteristic punctate
staining (examples marked by arrows), which is indicative of autophagosome formation, in MQ-treated cells only.
35

To further investigate inhibitory effects on autophagy in our cell system, we used siRNA
to knock down the expression of Beclin1, an essential autophagy protein that is necessary
for nucleation of autophagosomes (Hippert et al., 2006). The efficiency of siRNA-
mediated knock down was verified by Western blot, which indicated an approximate
85% reduction in Beclin1 protein, whereas a non-specific (scrambled) control siRNA had
no apparent effect (Fig. 10A). Our results demonstrated no significant effect on LC3-I/II
conversion following Beclin1 knockdown (Fig. 10A), in line with this protein’s role
during the early stages of autophagy, we suspected that Beclin1 knockdown would
potentiate MQ’s effects. Thus, we treated the siRNA-transfected cells with MQ, and
long-term cellular survival was analyzed by CFA. As shown in Fig. 10B, in the absence
of MQ treatment, siBeclin1 alone reduced cell survival by about 20%. However, when
siBeclin1-transfected cells were treated with MQ, cell survival was significantly (p<0.05)
further decreased. Together, these results suggest that combined inhibition of autophagy
by different approaches, targeting early and late stages, may lead to overall greater tumor
cell toxicity. In order to further confirm that autophagy inhibition results in reduced cell
viability of MDA-MB-231 and T47D cells, we analyzed the effect of 3-methyladenine
(3-MA), a phosphatidylinositol-3-kinase (PI3K) type-3 inhibitor. PI3K type 3 forms
essential complexes with Beclin1 to initiate the nucleation of autophagosome (Codogno
et al., 2012). As shown in Fig. 10C, the inhibition of autophagy by 3-MA resulted in a
dose-dependent reduction of cell viability, where T47D cells were more sensitive to 3-
MA than MDA-MB-231 cells.
36

Figure 10: Altered chemosensitivity to MQ after Beclin 1 knockdown. The effect of Beclin1 knockdown on cytotoxic
and antiproliferative effects of MQ were analyzed by colony formation assay. MDA-MB-231 cells were transfected
with siRNA directed at Beclin1 (siBeclin) or scrambled siRNA (siControl) or remained untransfected (untreated). (A)
After transfection, cell lysates were analyzed by Western blot to confirm downregulation of Beclin1 expression and to
study the effect of siBeclin1 on LC3 I/II conversion (marker for autophagosome). Actin served as the loading control.
(B) In parallel, transfected cells were treated with increasing concentrations of MQ for 48 hours. Thereafter, the drug
was removed and the cells were incubated in the absence of drug for an additional 12 days, after which time the number
of colonies formed were counted. Shown is the percent number of colonies (mean ±SD, n≥2), where the number of
colonies derived from untreated cells was set to 100%. The result presents increased sensitivity to MQ following
Beclin1 knockdown. (C) MDA-MB-231 and T47D cells were treated with increasing concentration of 3-MA for 48
hours and cell death was analyzed by MTT assay. Shown is percent cell survival (mean ±SD, n≥2), where the value
from untreated control cells was set to 100%.
2.3.5. Mefloquine chemosensitizes towards paclitaxel
There are indications that inhibition of autophagy may increase the chemosensitivity of
certain tumor cells (Amaravadi et al., 2011). We therefore investigated whether MQ
37

would be able to achieve this outcome in combination with paclitaxel (Taxol
®
), which
was selected as a representative agent from among several chemotherapeutic drugs
currently in clinical use for the treatment of malignant breast cancer. Moreover, recent
studies have shown its ability to induce autophagy as a protective mechanism (Xi et al.,
2011). Thus, we combined 25 nM of paclitaxel with increasing concentration of MQ and
analyzed tumor cell viability by MTT assay. As shown in Fig. 11, the addition of non-
cytotoxic dose (5 µM) of MQ to paclitaxel resulted in further reduced survival of triple-
negative MDA-MB-231 and T47D cells, thus indicating chemosensitizing properties of
MQ.
Figure 11: Increased chemosensitivity by MQ treatment. MDA-MB-231 and T47D cells were simultaneously treated
with 25 nM paclitaxel and increasing concentrations of MQ for 48 hours and cell survival was assessed by MTT assay.
Shown is percent cell survival (mean ±SD,n≥2), where survival of untreated cells was set to 100%.
2.4. Discussion
One of the major challenges facing cancer therapy is tumor resistance towards cytotoxic
chemotherapy. Among the various cellular processes contributing to this effect is
38

autophagy, which generally is induced in response to metabolic and therapeutic stress,
and has been associated with disease relapse (Janku et al., 2011; White and DiPaola,
2009). This has prompted the evaluation of specific autophagy inhibitors, such as the
antimalarial medications chloroquine (CQ) and hydroxychloroquine (HCQ), as potential
anticancer therapeutic agents (Amaravadi et al., 2011; Sasaki et al., 2010; Sotelo et al.,
2006). Several studies using CQ and HCQ as an adjunct to existing chemotherapeutic
agents are under preclinical and clinical evaluation (Fujita et al., 2000). However,
presumably due to their low potency, the use of these compounds has resulted in poor
anticancer outcomes. In addition, their low efficacy necessitates higher dosages, which in
turn pose the problem of increasingly severe side effects (Sotelo et al., 2006). Taken
together, these limitations highlight the pressing need for the discovery of novel
autophagy inhibitors exhibiting superior potency.
Lysosomotropic agents, such as CQ and HCQ, inhibit autophagy by virtue of being weak
bases that accumulate in lysosomes in protonated form and increase pH, thereby blunting
the activity of lysosomal enzymes and preventing complete degradation of
autophagolysosomal contents. Based on this mechanism of action, we reasoned other
lysosomotropic agents can potentially be more effective and possibly better suited for
cancer therapeutic purposes. And indeed, among the several agents we evaluated, we
discovered that MQ displayed substantially greater activity than the previously
established and widely used autophagy inhibitor CQ. In comparison to CQ, which
generally requires concentrations of 30 to above 100 µM to be effective (Fig. 6) (Sasaki
et al., 2010), our data show that MQ displays anticancer activity in breast cancer cell lines
39

at concentrations as low as 2.5 µM (Fig. 6C), and more generally in the range of 2.5 to 15
µM. Importantly, MQ displayed this pronounced cytotoxic activity in estrogen receptor
positive, triple-negative, and drug-resistant breast cancer cells, indicating that this agent
should be evaluated further for the inclusion in therapies aimed at these types of difficult-
to-treat cancer. It is interesting to note that MQ and CQ were considerably more potent in
colony formation assays (CFAs) as compared to MTT assays (Fig. 6). In both cases drug
exposure was the same (48 hours), although MTT assays determined cellular viability at
the end of drug treatment (i.e., after 48 hours), whereas CFAs measured how many of the
drug-exposed cells were able to spawn a colony within 2 weeks after the termination of
drug treatment. Our results further indicate the higher potency of MQ as compared to CQ.
The increased potency of MQ over time could be a consequence of its long intracellular
half-life and its ability to accumulate preferentially in lysosomes (Glaumann et al., 1992;
Mu et al., 1975). As CFAs are reasonably good predictors of in vivo activity of anticancer
agents, our results suggest the exciting possibility that MQ might become a more
effective addition to cancer therapeutic regimens in the future.
To further study the type of cell death induced by MQ, we analyzed its effect on various
markers of apoptosis. As shown in Fig. 8A, B and C, MQ induced apoptotic cell death.
However, the combination of MQ with a pan-caspase inhibitor (ZVAD-fmk) only
partially protected cells against MQ (Fig. 8D), indicating the existence of caspase-
independent cell death, as has been previously observed with CQ (Geng et al., 2010).
40

Based on the known lysosomotropic nature of MQ, we expected that this agent would
function as an inhibitor of autophagy. This assumption was confirmed by our verification
of several established autophagy markers in response to MQ treatment, such as the
appearance of prominent punctate staining of LC3-GFP-transfected cells, induction of
LC3 with conversion to its phosphatidylethanolamine-conjugated form (LC3-II), and
increased levels of p62/SQSTM1 (Fig. 9). The latter has been shown to represent readout
for blockage of the autophagic process in particular (Myeku and Figueiredo-Pereira,
2011; Rusten and Stenmark, 2010). Our observation of increased levels of p62 in
response to MQ treatment is particularly noteworthy, because the opposite, i.e., the
reduction of p62 levels, is being viewed as a tumor cell-protective mechanism that
supports cell survival under hypoxia; as well, it has been related to the development of
resistance to chemotherapeutic agents, such as cisplatin and 5-fluorouracil (Jaakkola and
Pursiheimo, 2009; Pursiheimo et al., 2009).
Beclin 1 protein assists in the nucleation of autophagosomes, and thus its knockdown
results in inhibition of autophagy (Hu et al., 2009). Although siRNA-mediated ablation of
Beclin 1 reduced overall survival of the MDA-MB-231 cell line (Fig. 10), it was less
potent than exposure of cells to moderate concentrations (5–10 µM) of MQ. It is unclear
why inhibition of autophagy by siBeclin was less cytotoxic than inhibition of autophagy
by MQ, although several possibilities exist to explain this discrepancy. For example,
knockdown of Beclin 1 expression may have been incomplete, as indicated by the
residual signal in our Western blot shown in Fig. 10. Or else, it may point to the
involvement of Beclin 1-independent autophagy (Scarlatti et al., 2008; Wong et al.,
41

2010), as supported by our observation that knockdown of Beclin1 had no effect on LC3-
I/II conversion or formation of autophagosomes (Fig. 10A). In addition, inhibition of
PI3K type 3 by 5 mM 3-MA, a concentration that suffices for autophagy inhibition,
diminished MDA-MB-231 cell viability by approximately 20%, which correlated with
the effects of siBeclin in these cells. Thus, our results suggests the possibility that even
complete ablation of nucleation of autophagosomes (by Beclin1 knockdown and PI3K
type 3 inhibition) would only partially block the entire autophagic process, whereas
inhibition by MQ would be more comprehensive. Alternatively, MQ may exert
additional, non-autophagy related effects that may result in cytotoxic outcomes.
MQ displayed striking ability to kill MCF/DOX cells (Fig. 7), which is an MCF7 variant
that has been selected for high resistance to doxorubicin, paclitaxel, and other
chemotherapeutics commonly used during breast cancer therapeutic regimens (Cho et al.,
2009; Guo et al., 2004). Multidrug-resistance of these cells is due to the overexpression
of drug efflux membrane transporters, such as P-glycoprotein (Pgp) (Fig. 7 and
(Mechetner et al., 1998) ), which is noteworthy because MQ is able to inhibit such
membrane pumps (Fujita et al., 2000; Riffkin et al., 1996). The presence of this feature
indicates the possibility that MQ may exert anticancer activity via a highly desirable
multi-thronged approach that includes drug efflux pump inhibition, stimulation of p62
expression, and autophagy blockade. Thus, MQ’s ability to lead to tumor cell
chemosensitization, as also shown in combination with paclitaxel (Fig. 11), may be based
on additional features of this agent, in addition to its ability to block autophagy.
42

2.5. Conclusion
In summary, our study introduces MQ as a potent anticancer agent that exerts its
pharmacologic activity by inhibiting autophagy and is substantially more potent than CQ.
Its efficacy in highly drug-resistant cells, in combination with its chemosensitizing
potential, makes this agent a promising candidate for further evaluation as part of cancer
therapeutic regimens. As MQ already is in clinical use for other indications, repositioning
MQ as an anticancer agent, in particular as an adjuvant for difficult-to-treat subtypes of
breast cancer, should be pursued.

43

Chapter 3:
Effect of Mefloquine on Endoplamic Reticulum Stress and
Ubiquitin Proteasome System

3.1. Introduction
Autophagy inhibition by MQ under stress free, nutrient rich culture conditions reduced
cell survival and proliferation. Moreover, the cytotoxic effects associated with MQ were
more potent than those demonstrated by other autophagy inhibitors such as chloroquine
(CQ), 3 MA and siBeclin (as demonstrated in chapter 2). The aforementioned
observations led us to hypothesize that higher potency of MQ stems from its effects
independent of autophagy inhibition and these effects result in increased dependence of
cells on autophagy for survival. Thus, we decided to analyze the effect of MQ on
different processes, which activate autophagy as a stress response. Out of various such
processes (as mentioned in chapter 1), we began our analysis by studying the effects of
MQ treatment on endoplasmic reticulum stress (ERS) response and ubiquitin proteasome
system (UPS).
The endoplasmic reticulum (ER) is a perinuclear organelle responsible for the synthesis,
folding and modification of several secretory and membrane proteins. It is also a major
44

storage site for intracellular calcium. In addition, ER is also the site of lipid biosynthesis
and gluconeogenesis (Borgese et al., 2006; Schröder and Kaufman, 2005).
The ER is responsible for protein synthesis and the quality control of numerous proteins
where failure to do so results in generation of stress on ER. Under physiological
conditions the protein load on ER is much lower than its folding capacity. On the other
hand, in cancer due to rapid proliferation of cells the protein load on ER exceeds its
folding capacity. Moreover rapid proliferation of cells, create an environment
characterized by poor blood and oxygen supply, ultimately giving rise to hypoxia and
hypoglycemia. This accumulation of mis-folded proteins and generation of hypoxia and
hypoglycemia, results in elevated stress on ER, eventually leading to the upregulation of
unfolded protein response (UPR) (Cao and Kaufman, 2012; Schonthal, 2012).
The UPR alleviates the load on ER through orchestrating various signaling events. The
activation of UPR results in reduced protein translation, increased transcription of folding
enzymes and upregulation of ER associated degradation pathway (Tsai and Weissman,
2010). Autophagy is also induced in response to elevated ERS, mainly to remove
damaged ER and aggregated proteins. The cellular responses induced by UPR are
mediated through three ER transmembrane associated proteins: inositol requiring kinase
1 (IRE 1), activation transcription factor 6 (ATF 6) and protein kinase RNA like ER
kinase (PERK) (Cao and Kaufman, 2012; Hetz, 2012). Overall, these mechanisms work
together to ensure proper functioning of ER and to re-establish homeostasis. The failure
to do so results in increased stress on ER, which finally leads to initiation of apoptosis
45

(Hetz, 2012). Thus, ERS has two arms pro-survival and pro-apoptotic and the
predominance of the biological activity depends on the extent of ERS.
The main regulators of the pro-survival and pro-apoptotic arms of UPR are ER resident
chaperone BiP also called as glucose regulated protein 78 (GRP 78) and CCAAT element
binding protein homologous protein (CHOP/GADD-153), respectively (Kardosh et al.,
2008). The GRP 78/ BiP, is a calcium binding chaperone with antiapoptotic/pro-survival
properties. Under physiological conditions, GRP 78 binds to PERK, IRE and ATF 6 and
keeps them in an inactive state. On the other hand, generation of ERS causes the
translocation of GRP 78 resulting in activation of the three key modulators and
generation of UPR. Studies have shown chronically increased expression of GRP 78 in
tumors when compared to normal tissues, further indicating high stress levels in tumors.
The induction of GRP 78 promotes tumor cell proliferation, survival, metastasis and
chemoresistance. Moreover, GRP 78 prevents induction of CHOP, when ERS is below
the threshold value and hence prevents cell death by apoptosis (Lee, 2007; Schönthal,
2009). Thus, there exists a rheostat between the two arms of ERS (Fig 12A) and
disruption of this balance has emerged as an important target for development of novel
therapies. The main focus of such therapies is to shift the balance towards the pro-
apoptotic arm of ERS, which is achieved by increasing stress on ER (Fig 12B).
46

Figure 12: Role of GRP 78 and CHOP in cancer and for anticancer effects. (A) Representation of the balance between
GRP 78 and CHOP in cancer and (B) shift in balance for anticancer therapy.
Amongst various ways of augmenting ERS is by blocking the degradation of mis-folded
and damaged proteins. The activation of ER associated degradation pathway (ERAD) is
an important adaptive response to mitigate ERS. The ERAD is mediated through UPS in
the cells. Proteins to be degraded are labeled with ubiquitin. The 26S proteasome
complex then degrades the ubquitinated proteins. Because of the ability of UPS to clear
47

protein load on ER, its inhibition results in elevated ERS. Thus, blocking of UPS has
emerged as a means to initiate ERS mediated cell death (Cho et al., 2009).
Autophagy is another cellular degradation system that assists in mitigating ERS by virtue
of clearing unfolded and aggregated proteins. Moreover, studies have shown a link
between the two degradation pathways, where inhibition of UPS results in induction of
autophagy as a compensatory mechanism (Ding et al., 2007b). Thus, UPS and autophagy,
work in conjunction to decrease the stress on ER by reducing protein load (Amaravadi et
al., 2011)
Further studies have shown the interdependence of autophagy with the ERS response
(Schonthal, 2012; Schönthal, 2009). For example, the accumulation of ER resident
chaperone proteins in response to defective autophagy suggests a role for autophagy in
lowering ER stress by enhancing lysosomal degradation of unfolded proteins (Mathew et
al., 2009). Also, autophagy is stimulated in response to ERS conversely there are
indications that inhibition of autophagy increases ERS (Ding et al., 2007a; Thomas et al.,
2012). Thus, the elevated autophagic process may support cancer cell survival by
providing a way to protect from proteotoxicity. However, inhibition of autophagy blocks
this alternative route, where the additional protein load may trigger pro-apoptotic
pathways and increase the susceptibility to cell death-inducing mechanisms, such as ER
stress.
Considering the important link between autophagy induction on one hand and elevated
ERS and inhibition of UPS on the other, we decided to evaluate the effects of MQ on
48

ERS and UPS. We hypothesized that the increased dependence of cells on autophagy
following MQ exposure arises from its ability to alter UPS and ERS.
3.2. Materials and Methods
3.2.1. Materials
MQ and nelfinavir were purchased from Sigma Aldrich (St Louis, MO) and Agouron
Pharmaceuticals, Inc (San Diego, CA), respectively. 2,5 dimethyl celecoxib (DMC) was
synthesized as previously described (Penning et al., 1997). MQ and DMC were dissolved
in DMSO to produce 100 mM stock. Nelfinavir was dissolved in ethanol to produce 100
mM stock solution.
3.2.2. Cell lines and culturing
Human breast cancer cell lines MDA-MB-231 and T47D were purchased from ATCC
(Manassas, VA). The cell lines were propagated in DMEM supplemented with 10% fetal
bovine serum, 100 U/ml penicillin, and 0.1 mg/ml of streptomycin, and incubated in a
humidified atmosphere at 37°C and 5% CO
2
.
3.2.3. Colony formation assay (CFA)
CFA was performed as previously described in chapter 2.

49

3.2.4. Western Blot analysis
Total cell lysates were prepared and analyzed by Western blot as described earlier in
chapter 2 (Chuang et al., 2008). The antibody against CHOP was obtained from Cell
Signaling Technologies (Beverly, MA; Cat Nos. #2895). The antibodies against actin,
GRP78 and ubiquitin were purchased from Santa Cruz Biotechnology Inc., (Santa Cruz,
CA; Cat Nos. sc-130656, sc-13968, sc-8017, respectively).
3.2.5. siRNA transfection
The siRNA control (scrambled) was purchased from Cell Signaling Tech., (Beverly, MA;
Cat Nos. # 6568) and used at a final concentration of 20 nM. The siRNA against
CHOP/GADD153 was purchased from Ambion (Carlsbad, CA; Cat No. s3996) and used
at a final concentration of 5 nM. The siRNA transfection was performed as previously
described in chapter 2.
3.2.6. MTT assay
The cell viability was analyzed using MTT assay, as previously described in chapter 2.
3.2.7. Proteasome activity measurement
Chymotrypsin-like activity of the 20S proteasome was analyzed using previously
described methods (Thomas et al., 2007). Briefly, cells were washed with buffer 1 (50
mM Tris/HCl pH 7.4, supplemented with 0.1 mM EDTA, 2 mM DTT, 5 mM MgCl
2
, 2
50

mM ATP). Thereafter, cells were suspended in buffer 2 (50 mM Tris/HCl pH 7.4,
supplemented with 0.1 mM EDTA, 20 mM KCl, 5 mM MgCl
2,
1 mM DTT, 0.03% SDS)
and lysed via multiple passaging (10x) through a syringe fitted with a 25-gauge needle.
After lysis, the protein concentration was determined using the bicinchoninic acid (BCA)
assay (Pierce, Rockford, IL), and 20 µg of protein was taken for further analysis. The
samples were diluted to 200 µl in a 96-well plate. Then, 1.6 µL of fluorogenic
proteasome substrate SucLLVY-AMC (Sigma-Aldrich; stock solution: 10 mM) for
chymotrypsin-like activity was added to each well (final concentration: 80 µM) to start
the reaction. Proteolytic activity was measured by monitoring the release of the
fluorescent 7-amido-4-methylcoumarin (AMC) at 37ºC using Spectra Max Gemini
Spectrofluorometer at the excitation and emission wavelengths of 360 and 460 nm,
respectively. Readings were taken every 15 min for an hour.
3.3. Results
3.3.1. Mefloquine causes accumulation of polyubiquitinated
proteins but does not inhibit proteasome activity
Autophagy, as well as the UPS, has critical roles in the elimination of proteins that are
marked for clearance via polyubiquitination, and p62 functions as a receptor to deliver
ubiquitinated proteins to the autophagosome (Korolchuk et al., 2009; Rusten and
Stenmark, 2010). Since we found increased levels of p62 protein following MQ treatment
(Fig. 9A, chapter 2), indicating that autophagy was inhibited, we predicted that MQ
51

should also lead to the accumulation of ubiquitinated proteins. As shown in Fig. 13A, this
appeared to be the case, i.e., MQ treatment of cells resulted in the accumulation of
ubiquitin-conjugated proteins. In order to exclude the possibility that the effect on
ubiquitinated protein accumulation might be due to the potential inhibition of the UPS,
we studied the effect of increasing doses of MQ on the 20S proteasome, where we
measured chymotrypsin-like activity (Fig. 13B). Up to 15 µM there was no effect on the
proteasomal activity when MDA-MB-231 cells were treated for 16 hours but there was
reduction in the activity at the toxic dose of 20 µM. We also studied the effect of 10 µM
of MQ on chymotrypsin like activity over time (Fig. 13C). While there was no inhibition
of proteasome activity in MDA-MB-231 cells, minor reduction of chymotrypsin-like
activity in T47D cells after 24 hours was observed (Fig. 13C). To further investigate this
effect, cells were treated with 10 µM MQ for up to 36 hours, at which time the
accumulation of ubiquitinated proteins was analyzed by Western blot. As shown in Fig.
13D, there was no obvious accumulation of ubiquitinated proteins in either cell line
tested, indicating the ability of UPS to effectively clear the proteins. Altogether, these
data indicate that MQ may only exert a weak, if any, inhibitory effect onto UPS, and
otherwise are consistent with inhibition of autophagy as a major mode of action for MQ.
52

53

Figure 13: Accumulation of ubiquitinated proteins and proteasome activity. MDA-MB-231 and T47D cells were
treated with the indicated concentrations of MQ for 16 hours, or with MQ 10 µM for the indicated times, and the effect
of drug treatment on ubiquitinated proteins and proteasome activity was determined. (A) Cell lysates were analyzed by
Western blot with an antibody specific to ubiquitin (i.e., recognizing ubiquitinated proteins). Equal loading was verified
by using an antibody against actin. (B&C) The chymotrypsin-like proteasome activity of the 20S proteasome was
determined by assessing the release of a fluorescent substrate 60 minutes after the addition of fluorescent probe (mean
± SD, n≥2). (D) Cell lysates were analyzed as in (A).
3.3.2. Mefloquine triggers ER stress
Based on the known interrelation of autophagy and ER stress (ERS), we reasoned that
inhibition of autophagy by MQ might result in ERS due to the increased accumulation of
mis-folded and aggregated protein. Thus, we determined the effect of MQ on the
expression levels of GRP78 and CHOP, which are markers for ERS. As shown in Fig.
14A, 20 µM MQ triggered ERS, as evident from the pronounced increase in the levels of
both proteins. However, lower concentrations of MQ (i.e., 5 and 10 µM) did not seem to
trigger ERS. As well, it is noteworthy that increased levels of CHOP, which represents
the major executor of the pro-apoptotic function of the ER stress response, were only
transient (Fig. 14A, right panel). Because elevated levels of CHOP are necessary for ERS
to initiate apoptosis (Rutkowski et al., 2006), this result indicates that MQ-induced ERS
may not participate in MQ-mediated cytotoxic effects.
To investigate this aspect further, we knocked down CHOP via transfection with specific
siRNA. Since CHOP is an inducible gene, knock down was analyzed after treating cells
with 1 µM of thapsigargin (Tg), a model inducer of ERS and potent stimulus for CHOP
expression (Ding et al., 2007a). As shown in Fig. 14B, siRNA-mediated knock down was
strikingly effective and prevented CHOP induction by Tg, as compared to control cells
transfected with scrambled siRNA. These cells were treated with increasing
concentrations of MQ and survival was analyzed by long-term CFA. If ER stress/CHOP
54

were involved in mediating the cytotoxic potency of MQ, we would expect that ablation
of CHOP expression would lead to increased cell survival after drug treatment, as has
been demonstrated under several other conditions (Cho et al., 2009; Cho et al., 2012;
Virrey et al., 2010). However, as shown in Fig. 14C, the knock down of CHOP did not
affect the outcome of MQ treatment, i.e., MQ exert similar cytotoxic potency whether or
not CHOP was knocked down. Thus, these results exclude ER stress from playing a
central role during MQ-induced cell death.
Figure 14: Induction of ER stress by mefloquine. The expression of ER stress markers in MDA-MB-231 cells in
response to MQ treatment was analyzed by Western blot. (A) Cells were treated with increasing concentrations of MQ
for 16 hours (left panel) or with 20 μM MQ for various time points (right panel). Cell lysates were analyzed by Western
blot using specific antibodies to GRP78 (a pro-survival ER stress marker) and CHOP (a pro-apoptotic ER stress
marker). To verify equal loading, the blots were also probed for actin. (B) MDA-MB-231 cells were transfected with 5
nM siRNA directed against CHOP (siCHOP) or with scrambled siRNA (siControl). Knockdown of CHOP expression
was confirmed by Western blot after treatment of cells with 1 µM thapsigargin (to trigger elevated CHOP levels). (C)
55

In parallel, transfected cells were treated with MQ for 48 h and cell survival was analyzed by colony formation assay
after keeping cells in culture for an additional 12 days without drug. Shown is the percent number of colonies (mean
±SD, n≥2), where the number of colonies in the control untreated cells was set to 100 %.

3.3.3. Mefloquine enhances cytotoxic effects of ER stress
aggravators
Previous studies have shown that inhibition of autophagy increases cytotoxic effects of
various ERS aggravators (Ding et al., 2007a). To this end, we also analyzed the effects of
MQ addition on the cell death induced by 2,5 dimethyl celecoxib (DMC) and NFV
(nelfinavir). DMC, is a structural analog of celecoxib but lacks COX 2 inhibition activity
and NFV, an HIV protease inhibitor. Both NFV and DMC can elevate ERS (Cho et al.,
2009; Kardosh et al., 2008; Kardosh et al., 2005). As shown in Fig 15, we combined MQ
with DMC 20 µM and various concentrations of NFV to analyze the effect of MQ on
cytotoxic effects of DMC and NFV. Our results show that low concentrations of MQ by
itself (2.5 and 5 µM) reduced cell viability by 20%. But when combined with DMC and
NFV, it greatly enhanced the cytotoxic effects. For example, dual treatment with DMC
and NFV reduced cell viability to around 55%, but addition of MQ (2.5, 5 and 10 µM)
further reduced the viability to 35%, 9% and 0% respectively (Thomas et al., 2012).
56

Figure 15: Autophagy inhibition by MQ enhances cytotoxic effects of ERS aggravators. MDA-MB-231 cells were
treated with increasing concentrations of MQ alone or in combination with DMC and NFV for 48 hours. The cell
viability was analyzed at the end of 48 hours by MTT assay. The result is presented as the (mean ± SD, n≥3)
percentage (%) cell viability, where cell viability of untreated cells is set to 100%. The “0” in the graph, represents no
cell survival.
3.4. Discussion
Previous studies have demonstrated MQ’s ability to disrupt calcium homeostasis and
increase the transcription of various ER stress-associated proteins such as GRP78 and
CHOP in rat neuroblastoma and human neurons at concentration around 80 µM (Dow,
2003; Dow et al., 2005; Dow et al., 2003). Combined with the known interdependence of
ER stress and autophagy (Rouschop et al., 2010; Yu et al., 2011), this led us to postulate
a key role for ERS in MQ-induced cytotoxicity. However, this does not appear to be the
case for the following reasons. Although we did confirm the induction of ERS markers,
GRP78 and CHOP, this effect required very high concentrations of MQ (20 µM).
Moreover, the elevation in CHOP levels was only transient (Fig. 14A). This latter
observation is noteworthy, because it had been demonstrated that high CHOP levels have
57

to be maintained in order for ERS to become cytotoxic (Rutkowski et al., 2006). Thus,
declining levels of CHOP expression, as observed in our experimental conditions, are not
consistent with ERS-induced cell death. As well, several reports (Cho et al., 2009) have
demonstrated previously that CHOP knockdown effectively protects cells from
undergoing death in those cases where ERS represents a key mediator of drug toxicity.
This is not the case in our experimental system, i.e., despite effective CHOP knockdown
there was no detectable protection of cells from MQ toxicity. Thus, combined our results
exclude ERS as a major component of MQ-induced cytotoxicity.
Recent studies have highlighted that induction of autophagy in response to elevated ERS,
helps to reduce the stress on ER. This has even led to emergence of autophagy inhibition
as an important pharmacological means of elevating ERS (Amaravadi et al., 2011; Ding
et al., 2007b; Schönthal, 2009). Based on this, we also analyzed the effect of MQ on the
cytotoxic effects of ERS aggravators such as DMC and NFV. DMC and NFV have
previously been shown to aggravate ERS and the dual combination of these drugs is
effective in killing different breast cancer cells, even difficult to treat TNBC (Cho et al.,
2009; Golden et al., 2009; Kardosh et al., 2008; Kardosh et al., 2005). Our results
demonstrate that addition of MQ with the dual combination of DMC and NFV
significantly increases their cytotoxic potential. Altogether, we demonstrate that although
MQ’s cytotoxic effects are not dependent on ERS but because of its ability to inhibit
autophagy it can enhance the activity of other ERS aggravators.
58

Since the autophagy-lysosome and ubiquitin-proteasome system are the two important
degradation systems of the cell and previous studies have reported the interdependence
between these two systems (Amaravadi et al., 2011; White and DiPaola, 2009), we
analyzed the effects of MQ on UPS. We did see the accumulation of ubiquitinated
proteins but there was no significant effect of MQ on proteasome activity. The slight
reduction in the proteasome activity by MQ 20 µM in MDA-MB-231 or by higher time
of exposure to MQ 10 µM in T47D cells might be a result of caspase activation (as
shown in Fig 8, chapter 2) as previous studies have also shown reduction in proteasome
activity following activation of caspases (Adrain et al., 2004). Moreover, the increased
accumulation of ubiquitinated proteins was most likely a result of autophagy inhibition
by MQ. Since p62 acts as an adaptor protein, which binds with the cargo of ubiquitinated
proteins and directs them to autophagosome for the degradation by lysosome, we
conclude that accumulation of ubiquitinated proteins was because of autophagy inhibition
(Korolchuk et al., 2009; Mathew et al., 2009; Pankiv et al., 2007).
3.5. Conclusion
In summary, although MQ is able to induce GRP78 and CHOP but its cytotoxicity is not
related to its effect on ERS. Moreover, the accumulation of ubiquitinated proteins, appear
to be caused by MQ-mediated effect on autophagy rather than UPS. Furthermore, from
the data presented in this chapter we conclude that because of MQ’s ability to inhibit
autophagy it can be used in combination with other ERS aggravators to augment stress on
endoplasmic reticulum.
59

Chapter 4:
Effect of Mefloquine on Sphingolipid Levels and the
Processes Affected by Sphingosine-1-phosphate
4.1. Introduction
After ruling out the involvement of ERS in MQ induced cytotoxicity, we decided to
analyze its effect on sphingolipids such as sphingosine-1-phosphate (S1P) and ceramide.
Studies have also shown that MQ alter sphingolipid metabolism, in particular, increase
the intracellular levels of ceramide in malarial parasite Plasmodium falciparum
(Pankova-Kholmyansky et al., 2003; Pankova-Kholmyansky and Flescher, 2006) and
studies have shown the ability of ceramide to induce autophagy as a stress response
(discussed in chapter 1) (Young et al., 2013). Thus, we hypothesized that MQ alters
sphingolipid levels, which makes autophagy an important survival mechanism.
Sphingolipids are membrane lipids with important structural role, in maintaining
membrane fluidity and lipid rafts. In addition to their role as membrane lipids, recent
evidences highlight their important role in cancer development and disease progression.
The two important sphingolipids, ceramide and S1P have been implicated as signaling
molecules, which are shown to play important role in cancer pathogenesis (Ogretmen and
Hannun, 2004; Selzner et al., 2001). Ceramide is produced either through de novo
60

synthesis or by sphingomyelinase (SMase) mediated hydrolysis of sphingomyelin.
Ceramide is further metabolized by ceramidase to form sphingosine, which can either be,
reversibly converted back to ceramide by ceramide synthase or phosphorylated by
sphingosine kinase (SphK) to form S1P. The S1P formed is dephosphorylated by S1P
phosphatase to form sphingosine or can be irreversible cleaved by S1P lyase to form
ethanolamine-1-phosphate and C16 fatty aldehyde (Fig. 16) (Ogretmen, 2006).
Figure 16: The diagram illustrating the biosynthetic and metabolic pathway of sphingolipids, modified from (Ogretmen,
2006).
Mounting evidence highlights the contrasting role played by ceramide and S1P in cancer
development and disease progression (Fig. 17) (Basu and Kolesnick, 1998; Ogretmen and
Hannun, 2004; Radin, 2003). The evidences such as, increased apoptosis in colon cancer
61

cell line SW403 with the use ceramidase inhibitors, B13 and D-MAPP, which result in
elevated ceramide levels (Selzner et al., 2001) and the reduction in tumor growth in vivo
by increasing concentration of ceramide in diet (Ogretmen and Hannun, 2004), indicate
that ceramide possess potent anticancer activity. In addition, the ability of several
chemotherapeutics agents such as daunorubicin, gemcitabine, vincristine, cisplatin and
etoposide, to elevate ceramide levels further illustrates the anticancer properties of
ceramide (Carpinteiro et al., 2008; Radin, 2003).
On the other hand, S1P supports cancer cell survival and disease progression (Pyne and
Pyne, 2010). The key enzyme Sphk, which catalyzes the formation of S1P, has been
shown to be elevated in different types of cancers. High Sphk levels are associated with
rise in S1P levels. Studies have shown that S1P and Sphk favor tumor cell motility and
growth of MCF 7 cells (Gao and Smith, 2011). Moreover, high expression of Sphk
endows the cells with the ability to grow in serum free conditions; and has resulted in
transformation of NIH3T3 cells in to fibrosarcoma (Pyne and Pyne, 2010; Xia et al.,
2000). In addition, S1P increases migration of gastric, breast tumor cells and invasion of
ovarian carcinoma and glioblastoma (Pyne and Pyne, 2010; Pyne et al., 2012a). Recent
studies have indicated the ability of S1P to activate the inflammatory signaling mediated
by NF-ƙB/ STAT3 activation, which in turn favors tumor and stromal cell proliferation
(Liang et al., 2013; Pyne and Pyne, 2013). Furthermore there are evidences, which
indicate the role of Sphk in the development of resistance of breast cancer cells to
tamoxifen, pancreatic cancer cells to gemcitabine and leukemia cells to imatinib (Pyne et
al., 2012a). Also, studies have implicated that high Sphk levels are associated with poor
62

prognosis and patient outcome in glioma and breast cancer patients (Paugh et al., 2008;
Pyne et al., 2012b).
The crucial role played by sphingolipids in cancer, has led to its emergence as an
important target for the development of novel therapies. The main focus of the drugs,
which are being developed to target sphingolipids is to disrupt the balance between the
pro-survival and the pro-apoptotic lipids (as shown in Fig 17) by 1) inhibiting the key
player Sphk; 2) forming drugs that mimics ceramide or antagonize S1P; and 3) that can
cause an increase in ceramide and a decrease in S1P levels (Ogretmen and Hannun,
2004).
As mentioned above, previous studies have shown increase in ceramide levels following
MQ treatment, thus we evaluated the effect of MQ on the intracellular levels of S1P,
sphingosine and ceramide in an in vitro glioma model. Glioblastoma is the most
aggressive form of brain tumor and its ability to migrate and invade into normal brain
tissue makes it difficult to treat (Jhaveri et al., 2011). Moreover, growing evidence
indicates the role of S1P in promoting glioma cell migration and invasion (Young et al.,
2009). Thus, we also analyzed the effects of MQ on the migratory and invasive potential
of temozolomide (TMZ) sensitive and resistant glioma cells.
63

Figure 17: Diagram representing opposite role of S1P and ceramide in cancer and for anticancer effects.
4.2. Materials and methods
4.2.1. Materials
Ceramide, sphingosine and S1P were purchased from Avanti Polar Lipids (Alabaster,
AL). The lipids were dissolved in 100 % methanol. Latanoprost free acid was obtained
from Sigma Aldrich (St Louis, MO).

64

4.2.2. Cell lines and culturing
Human glioma cell lines U251, T98G and LN229 were purchased from American Tissue
Culture Collection (ATCC, Manassas, VA). The TMZ resistant variants of U251 and
LN229, U251-R and LN229-R respectively, were generated in our laboratory by
culturing the parenteral cell lines with increasing concentrations of TMZ over time (Cho
et al., 2012; Jhaveri et al., 2011). All cell lines were propagated in DMEM supplemented
with 10% fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml of streptomycin, and
incubated in a humidified atmosphere at 37°C and 5% CO
2
.
4.2.3. MTT assay
The cellular proliferation was analyzed using MTT assay, as previously described in
chapter 2.
4.2.4. Extraction of sphingolipids
The samples were lyzed by homogenization with 300 µl of extraction buffer composed of
250 mM sucrose, 5 mM Hepes and 1 mM EGTA, pH7.4. An aliquot was taken for the
protein determination and for the extraction of sphingolipids. Ceramide and sphingosine
were extracted by using previously described Bligh and Dryer method (BLIGH and
DYER, 1959; Selzner et al., 2001) and sphingosine-1-phosphate by using Baker method
(Baker et al., 2001).
Ceramide and sphingosine extraction:
65

Briefly, for the extraction of ceramide and sphingosine, 200 µl of the sample suspension
was spiked with 100 µl of 500 ng/ml latanoprost free acid (internal standard) and
extracted with 750 µl of methanol:chloroform (2:1 v/v) mixture. The sample was shaken
intermittently for 1-2 hours following which the sample was centrifuged at 3000 rpm for
5 min and the supernatant was collected. The residue was further extracted with 950 µl of
the mixture of methanol:chloroform:water (2:1:0.8) for 30 min with intermittent shaking.
After 30 min, the sample was centrifuged at 3000 rpm for 5 min and the supernatant was
pooled with the previous one. Following this, 500 µl of chloroform and 500 µl of water
were added to the pooled supernatant and the sample was centrifuged at 1000 rpm for 10
min. The lower chloroform layer was collected and dried under nitrogen. The dried
samples were reconstituted with 150 µl of chloroform.
S1P extraction:
200 µl of the sample was spiked with 100 µl of 500 ng/ml latanoprost free acid (internal
standard), followed by the addition of 666 µl of the mixture of 40 mM disodium
hydrogen phosphate (Na
2
HPO
4
) and 30 mM citric acid, pH 4. The sphingosine-1-
phosphate was extracted by the addition of 1.8 ml of 1-butanol with intermittent shaking
for 1 hour. The sample was further extracted with 888 µl of water saturated butanol, with
shaking for 30 min. Following which the sample was centrifuged at 1000 rpm for 10 min
and the upper butanol phase was separated and dried under nitrogen. The dried samples
were reconstituted with 150 µl of the mixture of chloroform:methanol:water:28%
ammonium hydroxide (250:100:15:0.3, v/v).
66

4.2.5. LCMS analysis of sphingolipids
A 10 μl aliquot was injected into an Agilent 1100 HPLC system linked to an API3000
(Applied Biosystems, Foster City, CA). The analytes were separated by Hypurity C18
(ThermoScientific) column with acetonitrile and water with 0.1% formic acid for
ceramide and acetonitrile and water with 0.1% ammonium hydroxide for S1P, as a
mobile phase at a flow rate of 0.400 ml/min. The mobile phase gradient of 5%
acetonitrile, which linearly increased to 95% acetonitrile over 10 min and was maintained
till 13 min, subsequently, the mobile phase was decreased to 5% acetonitrile over 20 min.
Ceramide and sphingosine were quantified using positive ion and S-1-P by negative ion
electrospray ionization (ESI) mass spectrometry. The conditions of the mass
spectrophotometer were as follows: capillary voltage, 3.5 kV; cone voltage, 15 ~18 V;
desolvation temperature, 300°C; desolvation gas flow, 1000 l/h; source temperature,
120°C. The analytes were quantified using multiple reaction monitoring (MRM) of
transitions: m/z 342.2→264.3 (ceramide), m/z 285.9→268.3 (sphingosine) m/z
378.2→78.8 (S-1-P), and m/z 389.2→345.2 (internal standard, latanoprost free acid). The
calibration curves were constructed over the range of 1 ng/ml to 5000 ng/ml for LC-
MS/MS per injection, and the concentrations of the sphingolipids in the samples were
determined by comparing their ratios of peak areas to the calibration curves.

67

4.2.6. Wound healing assay / Scratch assay
Glioma cells at a density of 3 X 10
5
cells/well for U251 and 2.5 X 10
5
cells/well for
LN229 were seeded in a 6-well plate. After overnight incubation, the medium was
aspirated and a wound was made using sterilized pipet tip. Following which, the cells
were washed with PBS and fresh pre-warmed medium was added with or without 5 µM
of MQ and cells were incubated for 24 hours. During the course of 24 hours, pictures of
the wound were taken at different time points.
4.2.7. Migration assay
The migration assay was performed as previously described (Charalambous et al., 2005;
Jhaveri et al., 2011). Briefly, 1 X 10
4
cells/well were seeded in the upper chamber of
Boyden chamber (BD Biocoat, Bedford, MA). In the bottom chamber MQ 5 µM was
added to the DMEM containing 10% FBS. The cells were incubated for 6 hours in
humidified incubator at 37ºC and 5% CO
2
. Following incubation the cells on the
underside of the filter were stained and counted at 40x magnification. Ten fields were
counted per chamber. Each condition was set in duplicate and each experiment was
repeated twice.
4.2.8. Invasion assay
The invasion of glioma cells was tested using previously described invasion assay (Cho
et al., 2012). Briefly, glioma cells were treated with 10 µg/ml of mitomycin C, rinsed and
68

plated at the density of 5 X 10
4
cells in 0.5% FBS in the top chamber of the 8 micron pore
filter of the modified Boyden Chamber. The wells were previously coated with matrigel
(BD Biosciences). The bottom chamber contained DMEM with 10% FBS, with or
without 5 µM of MQ. The chambers were incubated for 18 hours in humidified incubator
at 37 ºC and 5% CO
2
. Following incubation, the cells on the underside of the filter were
stained and counted at 40x magnification. Ten fields were counted for each chamber.

4.2.9. Sphingosine kinase 1 (Sphk1) inhibition assay
The inhibition of Sphk1 following drug treatment was analyzed using sphingosine kinase
1 inhibitor screening assay kit (catalog no. 700430) from Cayman Chemicals (Ann Arbor,
MI) as per manufacturer’s instruction. Briefly, in each well of a 96-well plate, 50 µl of
the cofactor mixture, 50 µl of enzyme mixture and 10 µl of diluted Sphk1 were added. To
the above mixture 10 µl of the drug to be tested or the solvent was added, and the
reaction was initiated by addition of 20 µl of the substrate and ATP. The plate was read
after 10 min of incubation at room temperature using fluorescent plate reader set to an
excitation wavelength of 530-540 nm and emission wavelength between 580-590 nm.
4.3. Results
4.3.1. Mefloquine reduces S1P levels in glioma cells
The ability of MQ to alter sphingolipid levels in malarial parasite, led us to hypothesis
that MQ may also alter intracellular concentration of S1P in glioma cells. To this, end we
69

treated U251 and LN229 cells with different concentration of MQ for 24 hours and
analyzed its effect on the intracellular levels of S1P using LCMS. As shown in Fig 18, in
U251 and LN229 cells MQ 5 µM resulted in substantial decrease in S1P, which was
further reduced by MQ 10 µM.
Figure 18: Decrease in S1P levels by MQ. U251 and LN229 glioma cells were treated with different concentrations of
MQ for 24 hours and S1P levels were analyzed using LCMS.
4.3.2. Mefloquine increase ceramide and sphingosine levels in
glioma cells
After analyzing the effect of MQ on S1P levels, we decided to analyze its effect on
ceramide and sphingosine concentrations in temozolomide resistant glioma cells. T98 G
and temozolomide resistant U251 (U251-R) were treated with 15 µM MQ for various
time points, and the level of ceramide and sphingosine were quantified using LCMS. As
shown in Fig 19, MQ treatment resulted in substantial rise in ceramide levels by 24
hours, which was further increased by 48 hours. Furthermore, we also observed a time
dependent rise in sphingosine levels in both the cell lines tested.
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Figure 19: Increase in ceramide and sphingosine levels by MQ. T98G and U251-R cells were treated with MQ 15 µM
for different time points and ceramide levels were analyzed using LCMS. Shown is the mean ± SD, of ceramide levels
after normalizing to mg protein.
4.3.3. Mefloquine reduces Sphk1 activity
The ability of MQ to increase ceramide and sphingosine and decrease S1P level, led us to
hypothesize that MQ may be a Sphk inhibitor, since Sphk is the key enzyme regulating
the levels of ceramide and S1P and has emerged as an important target for the treatment
of cancer (Pyne et al., 2012a). Thus, we analyzed MQ’s effect on Sphk. To assess the
effect of MQ on the activity of Sphk, isolated enzyme was treated with increasing
concentrations of MQ in the presence of enzyme substrate and ATP. As shown in Fig 20,
MQ was effective in inhibiting Sphk activity with an IC50 around 5 µM.
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Figure 20: Effect of MQ on Sphk activity. The effect of MQ on the activity of Sphk analyzed after treating the isolated
enzyme in the presence of the substrate and ATP for 10 min. Shown is the percent inhibition (mean ± SD, n≥2).
4.3.4 Mefloquine reduces proliferation of glioma cells
Since S1P is the key regulator of cellular proliferation, we decided to analyze the effect
of MQ on glioma cell proliferation. U251 and LN229 cells were treated with increasing
concentration of MQ for 0, 24, 48, 72 and 96 hours and cellular proliferation was
analyzed using MTT assay. As shown in Fig 21, in both U251 and LN229 cells, MQ
treatment up to 5 µM resulted in substantial reduction in absorbance over time when
compared to untreated cells. But increasing concentration to 7.5 or 10 µM, shows
decrease in absorbance below the baseline, which is shown using the red line. Overall,
72

our data illustrate that lower concentrations of MQ can inhibit glioma cells proliferation
whereas high concentrations cause cell death, and MQ 5 µM is more effective in reducing
proliferation of U251 than LN229 cells.
Figure 21: Mefloquine reduces proliferation of glioma cells. U251 and LN229 cells were treated with increasing
concentrations of MQ for different time points and its effect on cell proliferation was analyzed using MTT assay.
Shown is the absorbance recorded at 490 nm (mean ± SD), where the absorbance at 0 h indicates the baseline
absorbance (represented using a red line) for each treatment, respectively.
4.3.5. Reduction in migration of glioma cells by mefloquine
The other important process regulated by S1P is cellular migration. Thus, we further
analyzed the effect of MQ on migration of glioma cells. We treated U251, U251-R,
LN229 and LN229-R cells with MQ 5 µM for 6 hours and determined its effect on
migration by counting cells on the underside of the filter of the Boyden Chamber. As
presented in Fig 22, our result demonstrates that MQ was effective in reducing the
migration of both TMZ sensitive and resistant glioma cells. Interestingly, we found that
TMZ resistant cells have higher potential for migration than TMZ sensitive U251 and
LN229 cells.
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Figure 22: Reduced migration of glioma cells by MQ. U251, U251-R, LN229 and LN229-R cells were treated with
MQ 5 µM for 6 hours and cellular migration was analyzed using Boyden Chamber by staining the underside of the
filter and counting stained cells using 40x magnification. Shown is the number of cells migrated/ field (mean ± SD,
n≥2).
The effect of MQ on the migration of glioma was further studied using wound healing/
scratch assay. Here, U251 cells were treated with MQ 5 µM for 24 hours and the ability
of cells to migrate and close the wound was monitored at different time points, starting
from 0 h. As shown in Fig 23, the untreated U251 cells were able to migrate and close the
wound completely by 24 hours whereas MQ 5 µM prevented the closure of wound in
U251 cells. Overall, our data shows that MQ is able to reduce migration of glioma cells.
74

Figure 23: Mefloquine prevents closure of wound. U251 cells were treated with MQ 5 µM and its effect on the ability
of cells to migrate was analyzed using wound healing/scratch assay. Shown are the pictures taken at 0, 10 and 24 hours
of the untreated and the MQ treated cells.
4.3.6. Mefloquine reduces invasion of glioma cells
The ability of glioma cells to invade in to other parts of the brain is one of the biggest
challenges for the effective treatment of glioblastoma. Since previous studies have shown
that S1P plays an important role in regulating invasion of glioma cells, we decided to
study the effect of MQ on invasion. To assess the effect of MQ on invasion, we used
modified Boyden Chambers. The U251, U251-R, LN229 and LN229-R cells were treated
with MQ 5 µM for 18 hours. Our data demonstrates that MQ (Fig. 24) is able to reduce
the invasive potential of both TMZ sensitive and resistant glioma cells.
75

Figure 24: Mefloquine reduces invasiveness of glioma. U251, U251-R, LN229 and LN229-R cells were treated with
MQ for 18 hours and invasion was analyzed using modified Boyden Chamber by staining and counting cells on the
underside of the filter. Shown is the mean of the number of cells/field that were able to pass through matrigel coated
filters (mean ± SD).
4.4. Discussion
In this study, we analyzed the effect of MQ on the bioactive sphingolipid, S1P and
ceramide. Our results show that MQ is able to increase ceramide in glioma cells (Fig 19),
which is consistent with the previous report, where a study has shown rise in ceramide
levels in malarial parasite by MQ (Pankova-Kholmyansky et al., 2003). As discussed in
chapter 1, ceramide is able to induce autophagy by downregulating nutrient transporters,
we have also studied MQ’s effects on glucose metabolism (presented in chapter 5).
76

Interestingly, we found that MQ was able to reduce the levels of pro-survival
sphingolipid, S1P in glioblastoma (Fig 18). Considering the ability of MQ to disrupt the
balance between the two important bioactive sphingolipids, we analyzed its effect on the
activity of Sphk, it has been shown to play important role in regulating sphingolipid
levels. Also, studies have also shown that its high expression is associated with poor
prognosis and patient outcome (Ogretmen, 2006; Ogretmen and Hannun, 2004). Our
results show that MQ is able to reduce Sphk activity (Fig 20). Hence, we conclude that
inhibition of Sphk by MQ is responsible for the rise in ceramide and sphingosine levels
and a decrease in S1P levels.
Moreover, previous studies have shown that bioactive sphingolipid, S1P regulates the
proliferation, migration and stimulates the invasiveness of cancer cells such as
glioblastoma (Pyne and Pyne, 2010). Glioblastoma is the most aggressive form of brain
tumor with poor patient prognosis. Its ability to invade in to normal parts of brain makes
it very difficult to treat (Van Brocklyn et al., 2003; Young et al., 2009; Young and Van
Brocklyn, 2007). Thus, we further analyzed the effect of MQ on the proliferation,
migration and invasion of glioma cells. We found that MQ concentrations up to 5 µM
very effectively reduced the proliferation of glioma cells but increasing concentrations
resulted in cytotoxicity. We also found that this non-cytotoxic dose of MQ was effective
in reducing the migration and invasion of various glioma cell lines tested.

77

4.5. Conclusion
In conclusion, MQ disrupts the crucial balance between the two critical sphingolipids,
ceramide and S1P by reducing Sphk activity. Moreover, it is effective in reducing
proliferation, migration and invasion of glioma cells. Thus, our results indicate that MQ
is a promising candidate and should be considered for anticancer therapy aimed at
targeting sphingolipid metabolism.

78

Chapter 5:
Reduced Glucose Uptake and Impaired ATP Synthase
Activity by Mefloquine Results in Death of
Glioblastoma Multiforme

5.1. Introduction
MQ treatment results in an increase in intracellular ceramide levels (as shown in Chapter
4); and previous studies have shown that ceramide induces autophagy under nutrient rich
culture conditions (Pattingre et al., 2009). The induction of autophagy by ceramide is
believed to arise from its ability to reduce nutrient transporters such as GLUT (glucose
transporters) in the mammalian cells, leading to nutrient limitation. Since autophagy
serves as a survival mechanism under nutrient deprivation, its inhibition can further
sensitize cells to ceramide induced cytotoxic effects. In contrast, providing supplements
from outside such as membrane permeable nutrient, methyl pyruvate (MP) helps to
mitigate ceramide induced cytotoxic effects (Guenther et al., 2008). Based on the effect
of MQ on ceramide levels, we hypothesized that MQ is able to induce metabolic stress in
the cells, which makes autophagy an important process for cell survival. As previous
studies have also reported that metabolic stress up-regulates autophagy - a catabolic
79

process, which helps cells to adapt to such conditions (Kuma et al., 2004; Lum et al.,
2005)
The oncogenic transformation is closely related to metabolic transformation. It is
believed that cancer cells undergo genetic mutations resulting in increased expression of
growth receptors and intracellular signaling pathways, which favor increased nucleotide
and fatty acid synthesis and reduce catabolic processes such as ß oxidation of fatty acids
(Romero-Garcia et al., 2011). These metabolic changes support cancer cell proliferation
and uncontrolled cell division by maintaining energy status, macromolecular synthesis
and redox status (Cairns et al., 2011; Muñoz-Pinedo et al., 2012).
Evidence for such relationship was first reported by Otto Warburg in 1926 (Muñoz-
Pinedo et al., 2012). The studies conducted by Warburg show increased dependence of
cancer cells on glycolysis for the production of energy even in the presence of oxygen
(Cheong et al., 2012; WARBURG, 1956). This effect is called as aerobic glycolysis or
Warburg effect. The process of glycolysis involves breaking down of a mole of glucose
in to two moles of pyruvate, ATP and one mole of NADH. The NADH serves as an
electron donor of the oxidative phosphorylation (OXPHOS), which generates three ATP
molecules per NADH oxidized by the mitochondrial electron transport chain. The
pyruvate generated by glycolysis is further metabolized by mitochondria through
tricaboxylic acid cycle (TCA), which results in generation of 18-fold more ATP than
glycolysis (Romero-Garcia et al., 2011). Although the energy generated by glycolysis is
fairly low when compared to mitochondrial respiration; but still cancer cells show high
80

dependence on glucose metabolism mainly because 1) generation of ATP by glycolysis is
a faster process and 2) glucose metabolism provides substrates for macromolecular
synthesis (DeBerardinis et al., 2008). According to Warburg, the increased glycolysis in
cancer results from the impairment of mitochondrial respiration (Romero-Garcia et al.,
2011). However, the role of mitochondria in cancer is controversial, as several studies
have shown the existence of functional mitochondria and increased dependence of tumor
cells on mitochondria (Jose and Rossignol, 2012).
Several lines of evidence have reported that the high level of glycolysis found in most of
the cancers is facilitated by the activation of oncogenic signaling pathways mediated by
PI3K/Akt/mTOR and hypoxia inducible factor 1α (HIF-1α) (Cheong et al., 2012; Ward
and Thompson, 2012). The constitutive activation of PI3K/Akt/mTOR pathway is found
in several cancers such as glioblastoma, prostrate and ovarian carcinoma (Edinger and
Thompson, 2002). Recent evidence indicate important role played by PI3K/Akt/mTOR
pathway in regulating glucose metabolism, along with its role in regulating tumor cell
proliferation, growth, invasion and metastasis (Foster et al., 2012). This pathway
increases glycolysis by upregulating nutrient transporters such as GLUT, thereby
increasing glucose uptake and various glycolytic enzymes such as hexokinase. The other
key signaling event that favor glycolysis is mediated by HIF-1α, which is upregulated
under hypoxia and acidosis, the two important characteristics of tumor
microenvironment. HIF-1α causes increased expression of GLUT and various enzymes of
glycolysis and hence favors aerobic glycolysis over mitochondrial respiration (Cheong et
al., 2012; DeBerardinis et al., 2008; Ward and Thompson, 2012).
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Since the discovery of the Warburg effect, immense cancer research has focused on
targeting glucose metabolism for both diagnostic and therapeutic purposes (Hsu and
Sabatini, 2008). To this end, several strategies have been exploited such as targeting
glucose uptake using silybin or the use of 2-deoxy-D-glucose (2DG), which blocks
glycolysis after forming D-glucose-6-phosphate (Cheong et al., 2012; Zhan et al., 2011).
The studies highlighting the relation between high GLUT expression and elevated
glycolysis such as in glioblastoma, has led to the emergence of GLUT as an important
target for the development of new anticancer therapies (Marotta et al., 2011; Muñoz-
Pinedo et al., 2012).
Malignant glioblastoma are the most aggressive form of brain tumors. Studies have
shown that these tumors have high potential for glycolysis and depend more on
glycolysis for ATP production than mitochondrial respiration (Beckner et al., 2005).
Moreover, a study utilizing 18 primary human brain tumors has shown that expression of
GLUT 3, an isoform of glucose transporter correlates with the histological grading of
brain tumors. According to this study as the tumor progresses from low grade
astrocytoma to glioblastoma there is a significant increase in the GLUT 3 expression on
the tumor cells and even on the blood vessels associated with the tumor (Nishioka et al.,
1992). Furthermore, increased expression of GLUT is associated with the development of
resistance of glioblastoma to temozolomide (TMZ), the current choice of drug for the
treatment of malignant brain tumors (Le Calvé et al., 2010). Based on the
aforementioned, we decided to investigate the effects of MQ on glucose metabolism in
glioblastoma multiforme.
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To this end, we have investigated whether MQ is able to inhibit the growth of glioma
expressing high levels of GLUT 3. We evaluated the effect of MQ on glioma because 1)
of its ability to cross blood brain barrier, which is one of the biggest challenges for the
development of anticancer therapy for glioblastoma (de Lagerie et al., 2009), 2)
important role played by glycolysis in glioma progression and 3) we propose that MQ,
due to its ability to increase intracellular ceramide will reduce nutrient transporter
expression such as GLUT3 in glioblastoma . Moreover, MQ is currently being explored
for its anticancer effects in a clinical trial for post radiation glioblastoma, in combination
with TMZ, metformin and memantine (Clinical Trials.gov, A service of US National
Institute of Health), which further makes it crucial to understand the mechanism of MQ’s
anticancer effects in glioblastoma.
5.2. Materials and methods
5.2.1. Materials
MQ, 2DG, MP, trichloroacetic acid, glycylglycine hydrochloride,
dichlorophenolindophenol soldium salt, sodium succinate, iodonitrotetrazolium (INT)
chloride, NADH, potassium phosphate, sucrose, EDTA, EGTA, HEPES were purchased
from Sigma-Aldrich (St. Louis, MO). TMZ (Merck) was obtained from the pharmacy.
MQ and TMZ were dissolved in DMSO to obtain 100 and 50 mM stock concentrations,
respectively. 2DG was dissolved in water to obtain 1 M stock solution. All agents were
83

added to the cell culture medium in a manner so that the final concentration of solvent
(DMSO or water) was considerably less than 1%.
5.2.2. Cell lines and culturing
Human glioma cell lines U251, U87, T98G and LN229 were purchased from American
Tissue Culture Collection (ATCC, Manassas, VA) (Cho et al., 2012). U87 iPTEN wild
type and mutant (C142S) were kindly provided by Dr. Johnson (USC, Los Angeles). The
TMZ resistant variants of U251 and LN229, U251-R and LN229-R respectively, were
generated in our laboratory by culturing the parenteral cell lines with increasing
concentrations of TMZ over time (Cho et al., 2012; Jhaveri et al., 2011). All cell lines
were propagated in DMEM supplemented with 10% fetal bovine serum, 100 U/ml
penicillin, and 0.1 mg/ml of streptomycin, and incubated in a humidified atmosphere at
37°C and 5% CO
2
.
5.2.3. MTT assay
Cellular viability was assessed using MTT, as previously described in chapter 2.
5.2.4. Colony formation assay
CFA was performed as previously described in chapter 2.

84

5.2.5. Western Blot (WB) analysis
Total cell lysates were prepared and analyzed by WB as described earlier in chapter 2.
The antibodies against cleaved caspase 7, PARP and PTEN were obtained from Cell
Signaling Technologies (Beverly, MA; Cat Nos. #9491, #9532 and #9552, respectively).
The antibodies against actin and GLUT 3 were purchased from Santa Cruz
Biotechnology Inc., (Santa Cruz, CA; Cat Nos. sc-130656, sc-74399, respectively).
5.2.6. Glucose uptake assay
Glucose uptake was analyzed using a kit from Cayman Chemicals (Cat no. 600470) as
per manufacturer’s instructions. Briefly 200,000 cells/well were seeded in a 6-well plate.
After overnight incubation the cells were treated with different concentrations of MQ for
24 hours. Thereafter, the cells were incubated with fresh growth medium containing 150
µg/ml of 2-NBDG (fluorescent labeled 2 deoxyglucose) for 30 min. At the end of the
incubation, the cells were harvested and centrifuged for five minutes at 400 x g at room
temperature. Following which the medium was aspirated and 1 ml of assay buffer was
added and cells were centrifuged again. Finally the cells were suspended in 500 µl of the
assay buffer and 1 X 10
4
cells were analyzed by flow cytometer using fluorescein channel
(excitation: 485 nm and emission: 535 nm respectively).

85

5.2.7. LDH release assay
The LDH release was monitored using a kit from G-Biosciences (St. Louis, MO) as per
manufacturer’s instructions. Briefly, 5000 cells/well were seeded in a 96-well plate. After
overnight incubation the cells were treated with different concentration of MQ for 24
hours. Following treatment the plate was centrifuged at 250 x g for 5 min. After
centrifugation 50 µl of the supernatant was transferred to fresh 96 well plate and 50 µl of
the substrate mix was added and the plate was incubated at 37ºC for 20 min. The reaction
was stopped by the addition of stop solution and absorbance was recorded at 490 nm.
5.2.8. Isolation of mitochondria from mouse liver
Mitochondria from mouse liver were isolated using differential centrifugation as
previously described (Garcia et al., 2010). Briefly, liver from mouse were excised,
washed and homogenized in isolation buffer, consisting of 250 mM sucrose, 20 mM
HEPES, 1 mM EDTA, 1 mM EGTA, 1%(w/v) BSA and protease inhibitor cocktail,
pH7.4 using dounce homogenizer. The homogensate was centrifuged at 1000 X g for 10
min at 4ºC. The pellet was removed and the centrifugation was repeated. Following this
the resulting supernatant was centrifuged at 9000 X g for 15 min at 4ºC. The resulting
mitochondrial pellet was washed with isolation buffer and the high speed centrifugation
was repeated. The final mitochondrial pellet was suspended in isolation buffer (without
BSA) and used for different assays.

86

5.2.9. NADH dehydrogenase/complex I activity assay
Activity of complex I was assayed by monitoring the change in absorbance of artificial
electron acceptor dichlorophenolindophenol (DCIP) in presence of NADH as previously
described (Janssen et al., 2007). Briefly, in each well of 96-well plate 23 µl of 0.2 M
glycylglycine hydrochloride (pH8.5), 13 µl of 0.6 mM DCIP and 6 µl of 6 mM NADH
(prepared in 0.2 M glycylglycine buffer) and 48 µl of water were added. To the above
mixture 50 µl of the drug to be tested was added. Finally, the reaction was started by the
addition of 50 µl of isolated mitochondria to each well. The complex I activity was
monitored by recording the change in absorbance over time at 600 nm for DCIP against
reagent blank.
5.2.10. Succinate dehydrogenase/complex II activity assay
Activity of complex II was assayed using previously described method (PENNINGTON,
1961). The activity was monitored using 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl
tetrazolium (INT) as the electron acceptor, which forms formazan crystals on reduction.
Briefly, in each well of 96-well plate 20 µl of 0.5M potassium phosphate (pH 7.4), 20 µl
of 1% (w/v in 100% ethanol) INT, 20 µl of 0.5 M sodium succinate, 20 µl of 0.25 M
sucrose and 60 µl of water were added. To the above mixture 50 µl of the drug to be
tested was added. The reaction was started by addition of 50 µl of isolated mitochondria.
The assay mixture was incubated at 37ºC for 15 min to allow the reaction to occur.
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Finally the reaction was arrested by the addition of 50 µl of 10% trichloroacetic acid in
each well and the absorbance was read at 490 nm against reagent blank.
5.2.11. ATP synthase/complex V activity assay
The activity of complex V was assayed using Complex V OXPHOS activity assay kit
supplied by Abcam (catalog no. ab109907) as per manufacturer’s instructions. Briefly, 50
µl of the solubilized bovine heart mitochondria was added to each well of the pre-coated
96 well plate. The plate was incubated at room temperature for 2 hours. Following
incubation each well was washed 2X with 300 µl of wash buffer. After washing 40 µl of
phospholipids was added to each well and the plate was incubated for 45 min at room
temperature. At the end of the incubation period the drugs to be tested were diluted in
complex V activity solution and 200 µl of the dilution was added in quadruplet to the pre-
coated 96 well plate. The activity of complex V was measured by monitoring the change
in absorbance at 340 nm at 30ºC over a period of 1 hour.
5.2.12. Measurement of mitochondrial respiration
The cellular oxygen consumption rate was measured using Seahorse XF Extracellular
Flux Analyzer (Seahorse Bioscience INC., North Billerica, MA) according to
manufacturer’s instructions. Briefly, 5 X 10
4
cells were seeded per well in to a special 24-
well plate. After overnight incubation, cells were treated with MQ for 6 hours. Following
treatment the cells were washed with medium and 600 µL of fresh medium without drug
was added and the oxygen consumption was continuously recorded for 30 min.
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5.2.13. Statistical analysis
Data are presented as mean ± SD. Comparisons between different treatments was made
using two-way ANOVA, and a p-value of less than 0.05 was considered significant.
5.3. Results
5.3.1. Mefloquine reduces cell viability of glioma
We tested the cytotoxic potential of MQ in an in vitro model of glioblastoma multiforme.
Human glioma cell lines of different genetic backgrounds were used in our experiment.
We chose U251 (p53 mutant, PTEN mutant), LN229 (p53 mutant, PTEN wild type) and
U87 (p53 wild type, PTEN mutant) (Shono et al., 2002; Zagzag et al., 2003)[Sanger
institute, UK database]. The cells were treated for 48 hours with increasing
concentrations of MQ and cell viability was determined using MTT assay. As shown in
Fig 25A, amongst all the cell lines tested LN229 was most sensitive to MQ treatment.
Overall, the IC50 of different cell lines was in the range of 12.5 to 15 µM.
The short-term MTT assay was complemented with a long term colony formation assay,
where the ability of an individual cell to form a colony following 48 hours of MQ
exposure was analyzed after incubating the cells in drug free culture medium for
additional 12 days. As presented in Fig 25B, MQ shows more potent anticancer activity
in such long term assays. We found that there was a gradual dose dependent reduction in
the ability of U251 cells to form colonies. MQ 5 µM resulted in 50% reduction in the
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ability of U251 cells to form colonies and 10 µM effectively abolished any colony
formation. On the other hand, in LN229 cells, MQ 1 µM led to 5-10% reduction in
colony formation, which was only marginally increased by MQ 7.5 µM. But increasing
concentration to MQ 10 µM resulted in complete inhibition of colony formation.
Figure 25: Cytotoxic and antiproliferative effects of MQ. Different glioblastoma cell lines were treated with increasing
concentrations of MQ and cell viability was analyzed. (A) Cells were treated with increasing concentration of MQ for
48 h and cell viability was determined by MTT assay. Shown is the percent cell viability (mean ± SD, n≥ 3), where the
value from the untreated control cells was set to 100%. (B) Cells were treated with MQ for 48 h and cell viability was
determined by colony formation assay after additional 12 days in culture without drugs. Shown is the percent number
of colonies formed (mean ± SD, n≥ 3), where the number of colonies formed by untreated cells was set to 100%.
5.3.2. Mefloquine is effective in temozolomide resistant glioma
Development of resistance to TMZ is one of the biggest problems in effective treatment
of glioma. We therefore investigated the effects of MQ on TMZ resistant U251 and
LN229 cells, generated in our laboratory and T98G cells (p53 mutant, PTEN wild type)
(Shono et al., 2002) (Sanger institute database, UK), which are inherently resistant to
TMZ (Cho et al., 2012; Kanzawa et al., 2003). The cell viability was analyzed using long
term MTT assay (Fig 26 A &C), where cells were treated with MQ or TMZ for 48 hours
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following which the medium was replaced with drug free medium and cytotoxicity was
analyzed after additional 96 hours of incubation. As shown in Fig 26A, MQ became more
potent in long term assay. The LN229 sensitive and resistant cells (LN229-R) respond
similarly to MQ, with the IC50 around 7.5 µM. Our result shows that U251 resistant
(U251-R) cells are significantly more sensitive to MQ than U251 sensitive cells. The
IC50 of U251-R is well below 5 µM and that of U251 sensitive cells is above 7.5 µM.
MQ was able to reduce the viability of T98G cells with the IC50 below 5 µM. The effect
of MQ on TMZ resistant cells was further confirmed by using CFA. Here, cells were
treated with MQ for 48 hours and the ability of individual cells to form colony was
analyzed at the end of additional 10-12 days of incubation in drug free culture medium.
As shown in Fig 26B, 2.5 µM of MQ reduced the colony formation below 50% in U251-
R cells and there was complete inhibition by 7.5 µM. This result further indicates the
increased sensitivity of U251-R cells to MQ than TMZ sensitive U251 cells (Fig 25 &
26). The LN229-R cells exhibited similar trend as the parental LN229 cells. In LN229-R
cells, MQ 1 µM reduced the colony formation by 5-10%, which increased to 20% by MQ
7.5 µM and was completely inhibited by MQ 10 µM. T98G cells appeared to be very
sensitive to MQ, with the IC50 below 2.5 µM in CFA (Fig 26B). We further verified that
LN229-R, U251-R and T98G cells are resistant to TMZ using long term MTT assay, as
shown in Fig 26C. We found that the IC50 of LN229 and U251 cells to TMZ is 20 µM
and > 20 µM, respectively (Fig 26C) and the IC50 of LN229-R, U251-R and T98G cells
to TMZ is above 90 µM. Overall our data indicate that MQ is more potent than TMZ and
also the cells, which are unresponsive to TMZ treatment, are sensitive to MQ.
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Figure 26: Cytotoxic effects of MQ on TMZ resistant glioma cell lines. TMZ resistant (-R) U251 and LN229 cells; and
T98G cells were treated with MQ and TMZ and cell viability was analyzed by MTT assay. (A & C) U251-R, LN229-R
and T98G cells were treated with increasing concentration of MQ or TMZ for 48 h and cell death was analyzed by
MTT assay after additional 4 days of culture in drug free medium. Shown is the percent cytotoxicity (mean ± SD, n≥
3), where the cell viability of untreated cells was set to 100%. (B) The effect of 48 hours of MQ treatment was analyzed
on the ability of U251-R, LN229-R and T98G cells to spawn a colony after additional 10 days of incubation in drug
free culture condition. Shown is the percent number of colonies formed, where the percent of untreated cells was set to
100 %.
5.3.3. Mefloquine induces necrosis
We next decided to analyze the type of cell death induced by MQ. In order to study the
effect of MQ on apoptosis, we investigated the activation of executioner caspase, caspase
7 and cleavage of a common substrate of effector caspases, poly ADP-ribose polymerase
(PARP) (Sharma et al., 2012). We treated U251 and LN229 cells with increasing
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concentration of MQ for 24 hours and the effect of drug treatment on cleavage of caspase
7 and PARP was analyzed by WB analysis. Our results indicate that there was a weak
cleavage of PARP as indicated by the accumulation of low molecular weight band and
very weak induction of cleaved caspase 7, activated form of caspase by MQ 20 µM in
U251 cells and by MQ 15 and 20 µM in LN229 cells (Fig 27A). The faint induction of
apoptotic markers further indicated that apoptosis may not be the main mechanism of cell
death induced by MQ in glioma cells. Thus, we decided to analyze the effect of MQ on
necrosis, the other type of cell death. To study the effect of MQ on necrosis, we treated
cells for 24 hours with different concentrations of MQ and analyzed the release of LDH
in to the medium as the marker of necrotic cell death. As presented in Fig 27B, there was
a dose dependent increase in percent cytotoxicity, measured by quantifying the release of
LDH in to the medium. Overall, our results show that MQ induced more cytotoxicity in
LN229 than U251 cells and MQ induces necrotic cell death.
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Figure 27: Induction of necrosis by MQ. Apoptosis and necrosis were analyzed after treating U251 and LN229 cells
with increasing concentrations of MQ for 24 h. (A) The effect of MQ on apoptotic markers was analyzed by Western
Blot analysis. Antibodies specific for cleaved caspase 7 (activated caspase) and PARP (target of activated caspase)
were used. The equal loading of samples was verified by probing for actin. (B) Necrosis was analyzed by monitoring
the release of LDH following MQ treatment. Shown is the percent cytotoxicity, which is directly proportional to the
amount of LDH release in to the medium by the cells.
5.3.4. Mefloquine reduces GLUT 3 expression and glucose uptake
We next sought to investigate the mechanism of MQ induced cell death. As mentioned
before MQ is able to increase intracellular ceramide levels, and previous studies have
indicated that ceramide can affect cell survival by reducing nutrient transporters such as
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GLUT (Guenther et al., 2008; Pankova-Kholmyansky et al., 2003). We decided to
investigate the effect of MQ on GLUT3, which is highly expressed in glioblastoma. To
this end, the basal levels of GLUT 3 were determined in different glioma cell lines, such
as T98G, U251, U251-R, LN229 and LN229-R, as shown in Fig 28A. Our data indicates
that T98G cells have the highest expression of GLUT 3, among all the glioma cell lines
tested. The LN229 and its TMZ resistant variant LN229-R have similar expression of
GLUT 3, whereas in case of U251 and U251-R, there was significantly higher expression
of GLUT 3 in U251-R cells. After determining the basal level of GLUT 3, we treated
T98G, U251, U251-R, LN229 and LN229-R cells with increasing concentrations of MQ
for 24 hours and analyzed the cell lysate for GLUT 3 levels by WB analysis. As shown in
Fig 28(B, C & D), MQ treatment resulted in a dose dependent reduction in GLUT 3
levels. While the drug effects were strongest at 20 µM in U251 cells, they became
evident even at lower concentrations (10 and 15 µM) in U251-R, LN229, LN229-R and
T98G cells.
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Figure 28: Effect of MQ on GLUT 3 level. (A) The basal level of GLUT 3 was analyzed in various glioma cell lines.
(B, C & D) T98G, U251, U251-R, LN229 and LN229-R cells were treated with increasing concentration of MQ for
24h. The effect of MQ on the expression of GLUT 3 was analyzed using WB analysis. Antibody specific for GLUT 3
(isoform of glucose transporter most abundant in glioma) was used. To verify the equal loading of the sample the blots
were probed for actin.
This reduction in GLUT 3 levels was further complemented with a more functional assay,
where we analyzed the effect of MQ on the uptake of glucose. We studied the effect of
MQ on glucose uptake by treating U251 and LN229 cells with increasing concentrations
of MQ for 24 hours and analyzing the uptake of fluorescently labeled analog of D-
glucose (2NBDG) using flow cytometer. As presented in Fig 29, in U251 cells there was
a slight reduction in uptake with lower concentrations (10 and 15 µM) and this reduction
became more significant with MQ 20 µM. On the other hand, in LN229 cells the uptake
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of glucose was more significantly reduced by MQ treatment. As shown in Fig 29 in
LN229 cells MQ 15 µM reduced the uptake well below 50%. Overall, our results indicate
that MQ can more strongly reduce GLUT 3 levels and glucose uptake in LN229 cells
than U251.

Figure 29: MQ treatment reduces glucose uptake in glioma cells. U251 and LN229 cells were treated with various
concentrations of MQ for 24 hours and glucose uptake was measured by analyzing the mean fluorescence of 10
4
events
using flow cytometer. The mean fluorescence recorded is directly proportional to the uptake of fluorescently labeled
2DG (2-NBDG). Shown is the mean fluorescence of the cells (mean ± SD, n≥ 2). (*) represents a p-value of less than
0.05
5.3.5. 2-deoxy-D-glucose (2DG) potentiates MQ induced
cytotoxicity
Since our data indicates the ability of MQ to alter glucose metabolism by reducing GLUT
3 levels and glucose uptake in glioma cell lines, we decided to analyze the effect of
another glycolysis inhibitor 2DG (Cheong et al., 2012) in combination with MQ. To this
end, we treated U251 and LN229 cells with 5 µM of MQ in combination with 7.5 mM of
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2DG for 48 hours and analyzed the ability of cells to form colony, after incubating cells
in drug free culture conditions for additional 10-12 days. As shown in Fig 30, the
exposure of cells to MQ alone resulted in 50% and 10% reduction in colony formation in
U251 and LN229 cells, respectively. Although 2DG alone had little or no effect on
reducing the ability of U251 and LN229 cells to spawn a colony, it significantly
potentiated the effects of MQ, resulting in 75% and 50% reduction in colony formation in
U251 and LN229 cells, respectively.
Figure 30: 2DG potentiates MQ induced cytotoxicity. U251 and LN229 cells were treated with either MQ alone or in
combination with 2DG for 48 hours. The cell viability was analyzed using CFA after allowing the cells to spawn
colony after additional 10-12 days of incubation in drug free culture conditions. Shown is the percent reduction in the
colony formation (mean ± SD), where the number of colonies formed by the untreated cells was set to 100%. (**)
represents a p-value of less than 0.005
5.3.6. Methyl pyruvate can partially protect glioma cells from MQ
induced cytotoxicity
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Previous studies have shown that methyl pyruvate (MP), an exogenously added cell
permeable nutrient can rescue cells from cell death induced by reduced glucose uptake
(Guenther et al., 2008). Thus, we also decided to investigate the effect of MP on MQ
induced cytotoxicity. We pretreated cells with 11 mM of MP (as previously used by
(Guenther et al., 2008)) for 2 hours followed by MQ addition and analyzed cell viability
by MTT assay at the end of 48 hours. As shown in Fig 31, MP had a very little effect on
the MQ induced cytotoxicity in U251 cells. But in LN229 cells, MP was able to rescue
around 50% of cells from cell death induced by 15 µM of MQ. Our results further
indicate inability of MP to protect either cell line from cell death induced by MQ 20 µM
(Fig 31). Even the higher concentration of MP (22mM) could not protect cells from MQ
induced cytotoxicity (data not shown). Overall, our data indicate that MP can only
partially protect cells from MQ induced cytotoxicity.
Figure 31: Partial protection of MQ treated cells by MP. The effect of MP on MQ mediated cytotoxicity was studied
using MTT assay. U251 and LN229 cells were pretreated with MP for 2 h, which was followed by the addition of MQ.
The cell viability was analyzed at the end of 48 h using MTT assay. Shown is the percent cell viability (mean ± SD, n≥
3), where cell viability of untreated cells was set to 100%.
5.3.7 Mefloquine reduces mitochondrial respiration
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MP in absence of glucose would feed pyruvate in to TCA cycle and would maintain
cellular homeostasis through mitochondrial respiration, which should protect cells from
altered glucose metabolism (Guenther et al., 2008). But as presented above, MP only
partially protected cells from MQ induced cytotoxicity. Thus, we predicted that MQ
might also have an effect on mitochondrial respiration. To this end, we analyzed the
effect of MQ on the mitochondrial respiration by measuring the oxygen consumption rate
(OCR) using Seahorse XF analyzer. U251 and LN229 cells were treated with MQ 15 µM
for 6 hours, following treatment the cells were analyzed for oxygen consumption rate. As
presented in Fig 32(A&B), MQ treatment substantially reduced the oxygen consumption
in both U251 and LN229 cells. Thus, our results indicate the ability of MQ to block
mitochondrial respiration, in addition to its effect on glucose metabolism.
Figure 32: MQ reduces mitochondrial respiration. U251 and LN229 cells were treated with MQ 15 µM for 6 hours.
After drug treatment the effect of MQ on OCR was analyzed over time for 30 min using Seahorse XF analyzer. Shown
is the mean OCR ± SD for different time points.
5.3.8 Mefloquine reduces ATP synthase’s activity
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Since our data indicate the ability of MQ to reduce mitochondrial respiration we next set
to determine the effect of MQ on various complexes of electron transport chain. We first
analyzed the effect of MQ on complex I (NADH dehydrogenase), the isolated
mitochondria was treated with increasing concentrations of MQ in the presence of NADH
and an artificial electron acceptor. The decrease in absorbance recorded at 600 nm
indicates the proper functioning of complex I. As shown in Fig 33A, MQ treatment does
not affect complex I activity, even the higher concentrations have same outcome as the
untreated cells. We also analyzed the effect of MQ on complex II (succinate
dehydrogenase). Here the conversion of INT to formazan was analyzed by recording the
absorbance at 490 nm. As shown in Fig 33B, there was no significant effect of MQ on
complex II activity except for MQ 20 µM. But the effect produced by MQ 20 µM was
similar to that of DMSO, indicating that the observed effect is non-specific. Finally, we
analyzed the effect of MQ on complex V (ATP synthase) activity. Here, the isolated
mitochondria were treated with increasing concentrations of MQ and the absorbance was
recorded at 340 nm. The decrease in absorbance indicates the proper functioning of
complex V, as was observed with control and DMSO treated mitochondria (Fig 33C).
Overall, our results indicate that MQ dose dependently reduces complex V activity. MQ
15 µM and MQ 20 µM result in 35% and 85% inhibition of complex V activity,
respectively.
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Figure 33: MQ reduces ATP synthase’s activity. The effect of MQ on various complexes of electron transport chain
was analyzed using isolated mitochondria. (A) The activity of complex I was analyzed after treating mitochondria
isolated from mouse liver with different concentrations of MQ in presence of NADH and DCIP, an artificial electron
acceptor. Shown is the mean absorbance recorded at 600 nm. (B) The effect of MQ on complex II was analyzed by the
formation of formazan in the presence of isolated mitochondria. Shown is the absorbance recorded at 490 nm. (C) The
activity of Complex V was monitored by treating isolated bovine heart mitochondria with increasing concentration of
MQ and recording absorbance at 340 nm. Shown is the mean absorbance recorded every min for 1h.
5.3.9. PTEN sensitizes glioma cells to mefloquine treatment
As mentioned above, PI3K/Akt pathway plays an important role in regulating tumor cell
metabolism; and studies have revealed constitutive activation of PI3K/Akt mediated
oncogenic signaling in glioblastoma. PTEN (phosphatase and tensin homolog) is the
negative regulator of PI3K/Akt pathway and mutation in PTEN is the common
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occurrence in glioma patients, which eventually result in activated PI3K/Akt pathway
(DeBerardinis et al., 2008; Edinger and Thompson, 2002). Thus, we decided to analyze
the effect of PI3K/Akt inhibition on MQ induced cytotoxic effects. To this end, we used
U87 glioma cells, which were engineered with either doxycycline inducible wild type
PTEN gene (U87 iPTEN wild type) or mutant PTEN gene (U87 iPTEN mutant
(C142S))(Woiwode et al., 2008). To analyze the effects of MQ on these cells, we
pretreated the cells with 1 µg/ml of doxycycline for 24 hours to induce PTEN. Following
which the cells were treated with MQ for additional 24 hours and its effect on cell death
were analyzed using WB (Fig 34A) or LDH release (Fig 34B). As shown in Fig 34A,
doxycycline treatment resulted in induction of PTEN in both mutant and wild type cells.
The cell lysate was further analyzed for apoptotic marker such as PARP cleavage, as
presented our results show no cleavage of PARP in mutant cells in presence or absence of
PTEN (left panel), although there was slight increase in total PARP levels following MQ
treatment. In contrast, in U87 iPTEN wild type cells, MQ (10 and 15 µM) treatment
resulted in slight cleavage of PARP, in cells where PTEN was induced (right panel). As
previously shown (Fig 27), apoptosis in not the main type of cell death induced by MQ,
we decided to analyze the effect of MQ on necrosis in U87 cells with inducible PTEN.
The cells were pretreated with doxycycline 1 µg/ml for 24 hours, following which cells
were treated with various concentrations of MQ for additional 24 hours. At the end of
drug treatment, the release of LDH was analyzed and quantified to determine the
percentage cytotoxicity. As shown in Fig 34B, in U87 iPTEN mutant cells (left panel),
although there was around 5% cytotoxicity induced by doxycycline treatment alone, in
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other words by the induction of mutant PTEN, but doxycycline had no effect on MQ
induced cytotoxicity. On the other hand, in U87 iPTEN wild type cells (right panel),
induction of wild type PTEN by doxycycline treatment alone resulted in 30%
cytotoxicity. Moreover, our results show an increase in the cytotoxic effects of 5, 10 and
15 µM of MQ by doxycycline. Overall, our data suggest that introduction of wild type
PTEN increases MQ mediated cytotoxicity in glioma cells.

Figure 34: PTEN sensitizes cells to MQ. U87 iPTEN mutant (C142S) and wild type cells were pretreated with
doxycycline 1 µg/ml for 24 hours to induce mutant or wild type PTEN, respectively. Following which, the cells were
treated with MQ for 24 hours and its effect on apoptosis and necrosis was analyzed. (A) The total cell lysate were
analyzed for the induction of PTEN, using antibody specific for PTEN and for the apoptotic marker, cleavage of PARP.
The blot was also probed for actin, used as a loading control. (B) The effect of PTEN induction on MQ induced
necrosis was analyzed by monitoring the release of LDH in to the medium. Shown is the percent cytotoxicity (mean ±
SD, n≥2), quantified from the amount of LDH released.
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5.4. Discussion
The transformation of normal human cell into malignant cell is commonly associated
with the changes in the genome, which alters the regulatory pathways that govern cellular
proliferation and death. Such genomic alterations during malignancy assist the cancer
cells to grow in the absence of growth signal, to evade programmed cell death, to become
insensitive to growth inhibitory signals, to gain the ability to sustain angiogenesis and to
invade and metastasize to other parts of the body (Hanahan and Weinberg, 2000). Recent
studies have also shown that such oncogenic transformations are closely associated with
changes in cellular metabolism, which assist the cancer cells to adapt to such changes
(Hanahan and Weinberg, 2011; Muñoz-Pinedo et al., 2012). Increased dependence of
cancer cells on glucose metabolism further indicate that metabolic alterations play an
important role in cancer development and disease progression (Cheong et al., 2012).
Considering the importance of cellular metabolism in cancer, we analyzed the effects of
MQ on glucose metabolism. Since previous studies have revealed MQ’s ability to
increase ceramide, which has been shown to affect glucose metabolism by
downregulating nutrient transporters, we hypothesized that MQ will also downregulate
nutrient transporters (Guenther et al., 2008; Pankova-Kholmyansky and Flescher, 2006).
For the purpose of this study, we used glioblastoma multiforme as an in vitro cancer
model since studies have shown increased dependence of glioma on glycolysis, which is
associated with high GLUT 3 levels (Beckner et al., 2005).
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Our study shows that MQ possess potent anticancer activity and is effective in wide
variety of glioma cell lines. It displayed pronounced activity in various TMZ resistant cell
lines including T98G, which is inherently resistant to TMZ and also in the resistant cell
lines, which were generated by culturing cells with increasing concentrations of TMZ
over time (Cho et al., 2012; Jhaveri et al., 2011). We found that MQ is considerably more
potent in long term assays, (Fig 25B, 26A & 26B) even when the time of exposure to MQ
was same. This increase in MQ’s potency can be attributed to its long intracellular half-
life, which results from its increased accumulation in lysososmes (Glaumann et al., 1992;
Mu et al., 1975; Sharma et al., 2012). Thus, our results indicate that MQ possess potent
anticancer activity in different cell lines, irrespective of their genetic background or their
sensitivity to TMZ. Since development of resistance to TMZ is one of the biggest
challenges for the effective treatment of glioma (Thomas et al., 2013), our study suggests
that MQ might be an exciting candidate to be considered for such cancers.
Interestingly, our study demonstrates that T98G and U251-R cells are most sensitivity to
MQ and this increased sensitivity correlates with the basal level of GLUT 3 in these cell
lines (Fig 28A). Moreover, we found that LN229 and LN229-R cells, which have similar
GLUT 3 levels exhibit no difference in their response to MQ treatment, whereas in case
of U251 and U251-R cells, the resistant cells show significantly higher sensitivity to MQ,
which also correlates with the higher expression of GLUT 3 in U251-R cells as compared
to U251 (Fig 28A, 26A & 26B). Overall, we present data that indicates the ability of MQ
to reduce GLUT 3 levels in wide array of glioma cell lines tested. Previous studies have
shown that long term treatment of glioma cells such as T98G with TMZ results in higher
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expression of GLUT3, which has been considered as one of the several mechanisms for
the development of resistance to TMZ. The increased sensitivity of U251-R and T98G
cells to MQ might be the result of their increased dependence on GLUT3, which arose
from the development of resistance to TMZ. Thus, based on the long term effects of TMZ
on GLUT3, targeting GLUT 3 in the cells, which are responsive to TMZ might delay the
development of resistance to TMZ (Le Calvé et al., 2010). Therefore, we decided to
further evaluate the effects of MQ in TMZ responsive, U251 and LN229 cells.
After analyzing the effect of MQ on GLUT 3 levels, we studied MQ’s effect on the
uptake of glucose. As presented in Fig 29, MQ reduced glucose uptake in dose dependent
manner for both U251 and LN229 cells. Moreover, the effect of MQ on glucose uptake
correlated well with its effect on GLUT 3 expression (Fig 28). Since the emergence of the
important role played by glucose metabolism in cancer, several studies have focused on
targeting it using different inhibitors. 2DG is a glycolysis inhibitor, which is being tested
in several clinical trials for its anticancer effects. The major drawback associated with the
use of 2DG is the potential toxicity, arising from the concentration needed to inhibit
glycolysis (Muñoz-Pinedo et al., 2012). Hence, we proposed that combination of MQ
with 2DG might increase the cytotoxic effects at therapeutically safe concentrations. Our
data indeed shows that combination of two drugs, targeting glucose metabolism result in
substantial reduction in colony formation in both U251 and LN229 cells (Fig 30).
Although MQ exhibits its effects in both U251 and LN229 cells but it is significantly
more potent in LN229 than U251 cells. The main difference between the LN229 and
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U251 cells lies in their PTEN status. The LN229 cells are PTEN wild type whereas U251
cells are PTEN mutated (Shono et al., 2002; Zagzag et al., 2003). The mutation in PTEN
or loss of PTEN is a common occurrence in glioma, which leads to constitutive activation
of PI3K/Akt pathway (Edinger and Thompson, 2002; Muñoz-Pinedo et al., 2012).
Previous studies have shown that Akt activation results in increased rate of glycolysis,
which is due to its ability to increase GLUT levels and hence glucose uptake and several
enzymes of glycolytic pathway (Buzzai et al., 2005; El Mjiyad et al., 2011; Elstrom et al.,
2004). Thus, this strong regulation of cellular metabolism by Akt might be responsible
for the reduced sensitivity of U251 cells to MQ treatment. Moreover, the presence of wild
type PTEN might also be the reason for preventing the increase in GLUT 3 levels in
LN229-R cells, in contrast to T98G and U251-R cells, which are PTEN mutated. To
further verify the involvement of PTEN in MQ induced cytotoxic effects, we used U87
cells engineered with inducible PTEN, wild or mutant gene. As shown in Fig 34, we
found that introduction of wild type PTEN increases the sensitivity of the cells to MQ.
Therefore, our data indicates that it might be important to use a PI3K/Akt inhibitor in
combination with MQ in PTEN mutated cancer cells to achieve its optimum anticancer
activity.
Reduced glucose levels do not correlate well with the patient outcome because glucose
depleted cells depend on glutamine, which helps cancer cells to overcome low or no
glucose condition. Glioma cells depend on glutamine and fatty acid oxidation for the
production of pyruvate and ATP (El Mjiyad et al., 2011). Also, it has been shown that
pyruvate provided from outside in the form of MP can protect glioma and other cancer
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cells from glucose deprivation and 2DG (Guenther et al., 2008; Yang et al., 2009). This
led us to postulate that MP should also protect cells from MQ induced cytotoxicity.
However, we found that MP can only partially protect cells from MQ (Fig 31). The
partial protection by MP can arise either because of autophagy inhibition or because of
mitochondrial impairment. Since MP cannot meet all the requirements of the cell under
altered glucose metabolism, the cells depend on autophagy (Guenther et al., 2008). Thus,
the partial protection by MP might be the result of autophagy inhibition, as MQ is a
known autophagy inhibitor (Sharma et al., 2012). To verify whether MQ has any effect
on mitochondria, we analyzed its effect on OCR and also various complexes of electron
transport chain. Surprisingly, we found that MQ reduces OCR and dose dependently
reduces complex V activity. Therefore, we conclude that the partial protection by MP
results from MQ’s effect on both autophagy and mitochondrial function. This ability of
MQ to target the two important metabolic pathways, namely glucose metabolism and
mitochondrial respiration, further indicates its advantage over drugs that can target one or
the other process. As previous study, has also shown that combination of 2DG and
metformin, which inhibits glycolysis and respiratory chain, respectively is more effective
than either drug alone (Cheong et al., 2011).
To further study the type of cell death induced by MQ, we analyzed the effect of MQ on
various markers of apoptosis and necrosis. We found that MQ caused very weak
induction of apoptotic markers (Fig 27A), which is in accordance with our previous study
where we have shown partial reversal of MQ induced cell death by pan caspase inhibitor
(Sharma et al., 2012). Moreover, our results show significant rise in LDH release by MQ
109

treatment, indicating involvement of necrosis (Fig 27B). The necrotic cell death could
rise because of the decrease in ATP levels. Since previous studies have shown that brain
tumors depend largely on glycolysis and reduction in glucose initiate necrotic cell death
(Hossmann et al., 1986). Furthermore, metabolic stress up-regulates autophagy, which
acts like a survival mechanism and help cancer cells to adapt to such conditions. Also,
studies have shown that targeting autophagy under metabolic stress can direct the cells to
necrotic cell death, which might also be the case with MQ, as it inhibits autophagy
(Sharma et al., 2012; Thomas et al., 2012). This induction of necrosis by MQ may be a
limiting factor in its anticancer effects. Since studies have shown that necrotic cell death
under metabolic stress and when autophagy is inhibited can result in an inflammatory
response, which may favor tumor growth (Degenhardt et al., 2006; Vakkila and Lotze,
2004).
5.5. Conclusion
In conclusion, our study introduces MQ as an agent, which can target both glycolysis and
mitochondrial respiration. MQ is effective in both TMZ sensitive and resistant glioma
cell lines. The initiation of necrotic cell death by MQ and its effect on MQ mediated
anticancer activity needs to be further evaluated.

110

Chapter 6:
Conclusion and Future Directions

6.1. Conclusion
The recognition of the ability of autophagy to support tumor cell survival and disease
progression under stressful conditions, has led to intense interest in development of
strategies to pharmacologically inhibit autophagy for better therapeutic outcome. This has
generated numerous clinical trials, which are aimed to test the ability of antimalarial drug
CQ and HCQ to improve efficacy of various chemotherapeutics that induce autophagy as
a survival mechanism (Amaravadi et al., 2011; Janku et al., 2011). Based on the role of
autophagy in cancer and the drawbacks associated with use of CQ and HCQ, we tested
several different FDA approved lysosomotropic agents.
Our study illustrates MQ as an inhibitor of autophagy with potent anticancer properties.
Interestingly, autophagy inhibition mediated by MQ under stress free culture conditions
was reduced tumor cell survival and proliferation. Moreover, the cytotoxic effects
associated with MQ were more potent than those demonstrated by other autophagy
inhibitors such as CQ, 3 MA and siBeclin under similar culture conditions. This led us to
hypothesize that MQ possess effects independent of autophagy inhibition but these
effects make autophagy induction an important process for cell survival. Thus, we studied
the effects of MQ on several such processes, as described in Chapter 1. Further
111

investigation lead to the discovery that MQ was capable of inducing ERS markers but the
cytotoxic effects of MQ were independent of ERS. Moreover, MQ treatment led to the
accumulation of ubiquitinated proteins but MQ had no effect on the proteasome activity.
After ruling out the involvement of ERS and UPS in mediating MQ-induced anticancer
effects, its effect on sphingolipids was evaluated. MQ was found to be able to disrupt the
balance between the pro-apoptotic and pro-survival sphingolipids. MQ treatment resulted
in the increase in ceramide and sphingosine, while reducing cellular S1P levels.
Furthermore, we found that MQ was able to reduce proliferation, migration and invasion
of glioma cells corresponding to reducing S1P levels (Van Brocklyn et al., 2003; Young
et al., 2009).
The rise in ceramide level is associated with down regulation of nutrient transporters
(Guenther et al., 2008). Since decrease in nutrient uptake would create a state of
metabolic stress, a common upregulator of autophagy (Amaravadi et al., 2011). We
hypothesized that the increased dependence of cells on autophagy following MQ
treatment is probably due to altered metabolism. Our results indeed, indicate that MQ
was able to reduce glucose uptake and impair mitochondrial respiration, the two
important processes regulating cellular energy production.
In conclusion, this thesis demonstrates that MQ, an FDA approved antimalarial drug
exhibits potent anticancer effects, which arise from its two-thronged effect. On one hand,
MQ is able to generate metabolic stress by downregulating GLUT 3 levels and by
reducing complex V activity and on the other hand, it is capable of blocking autophagy.
112

Furthermore, since low glucose and hypoxia upregulate ERS, we conclude that the
induction of ERS markers by MQ, is probably the response of cells to altered metabolism
(as summarized in Fig 35).
Figure 35: Schematic representation of mefloquine’s mechanism of action
6.2. Future Directions
Although our study reveals potent activity of MQ in an in vitro model of breast and
glioma cancer but it fail to illustrate any antitumor activity in in vivo model of glioma, as
shown in Fig 36. In this study, U251 glioma cells were implanted in the athymic nude
113

mice and were treated with different doses of MQ thrice a week. In contrast to our
findings, a recent study has shown that MQ is very effective in killing acute myeloid
leukemia (AML) cells and AML progenitors in cell culture and in mice (Sukhai et al.,
2013). In this study, the mouse and human leukemia cells were implanted in sublethally
irradiated NOD/SCID mice and MQ 50 mg/kg was given every day. Thus, MQ’s
anticancer effects might be cancer specific and may also depend on the state of immune
system of the in vivo model and hence it is important to reanalyze the effect of MQ in an
in vivo model of glioma using irradiated NOD/SCID mice. Moreover, to ensure MQ’s
safety and benefits for cancer patients further investigation is required.
Some of the future studies aiming at analyzing the effect of MQ on processes and
pathways that can cause treatment failure and poor disease outcome are important for
better understanding of MQ’s anticancer effects and are discussed below (Fig 37).
6.2.1 Determination of MQ’s effect on inflammation
It is important to analyze the effect of MQ on inflammation. Since our study has revealed
the ability of MQ to induce necrotic cell death and induction of necrosis is associated
with the generation of an inflammatory response. Necrotic cells release cytokines such as
IL 1 alpha, which can induce proliferation of the neighboring cells (Hanahan and
Weinberg, 2011) and hence support tumor progression. Thus, it becomes extremely
important to analyze the effect of MQ on inflammation and tumor progression in an
immune competent mouse model.
114

Figure 36: Effect of MQ on tumor growth. U251 glioma cells were implanted in athymic nude mice. After the
formation of palpable tumor the mice were treated with different concentrations of MQ or with vehicle (untreated) and
tumor volume was measured over time.
6.2.2. To study the effect of GRP78 induction by MQ on tumor
progression
We present data indicating the ability of MQ to increase GRP78 levels (Chapter 3),
which probably results from MQ’s effect on glucose metabolism. Since GRP78 has been
shown to play important role in cancer progression and development of chemoresistance
(Lee, 2007), it is important analyze the effects of MQ induced GRP78 on its efficacy in
an in vivo cancer model.

115

6.2.3. Determination of the effect of MQ on angiogenesis and
metastasis
Angiogenesis and metastasis are the two hallmarks of cancer, which support tumor
survival and disease progression (Hanahan and Weinberg, 2000). Stimulation of an
immune response and generation of metabolic stress, which might result from the ability
of MQ to induce necrosis and inhibit autophagy, will result in secretion of various
cytokines, attraction of immune cells to the site of tumor. These together can lead to
activation of angiogenesis and metastasis (Hanahan and Coussens, 2012). Although our
study reveals the ability of MQ to reduce migration and invasion of cells in an in vitro
model of glioma it is essential to analyze these effects in the presence of cells of tumor
microenvironment (TME). As TME plays an important role in deciding the fate of tumor
cells.
6.2.4. Determination of the effect of MQ on amino acid
transporters and uptake
Previous study has shown the ability of ceramide to downregulate amino acid transporters
(Guenther et al., 2008). Thus, it is essential to analyze whether MQ can affect amino acid
transporters in addition to its effect on GLUT.

116

Figure 37: Proposed effects of Autophagy inhibition by MQ. Inhibition of autophagy has emerged as an important
target for the development of novel therapeutics. On the contrary, this figure reveals the pathways and the processes
that can emerge in response to autophagy inhibition by MQ, which can ultimately result in treatment failure and poor
disease outcome.

117

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Abstract (if available)

Abstract The ability of autophagy to support cancer cells under the conditions of metabolic, chemotherapeutic and endoplasmic reticulum stress (ERS) has led to its emergence as an important target for anticancer therapy. The antimalarial drug chloroquine (CQ) is able to inhibit autophagy and therefore is being considered for cancer therapeutics. However, the relatively low potency of CQ prompted us to investigate whether other FDA approved lysosomotropic agents such as mefloquine (MQ), levofloxacin and ciprofloxacin might me more effective and hence potentially more useful. We hypothesized that out of various drugs tested MQ, an antimalarial drug will be most effective as an anticancer agent. This hypothesis stems from the facts that MQ owing to its ability to manipulate lysosome will inhibit autophagy. Moreover, previous studies have shown the ability of MQ to induce the markers of ERS in neurons and also to increase intracellular ceramide level in malarial parasite, which are known to induce autophagy as a survival mechanism. Thus, higher potency of MQ will arise from its ability to produce two-thronged effect. On one hand MQ will increase cellular stress and on the other inhibit autophagy. We found that MQ was most potent compound tested

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Asset Metadata

Creator Sharma, Natasha (author)

Core Title Inhibition of tumor cell growth by mefloquine via multimechanistic effects involving increased cellular stress, inhibition of autophagy, and impairment of cellular energy metabolism

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 11/06/2013

Defense Date 03/11/2013

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

Tag autophagy,cancer,mefloquine,metabolism,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Louie, Stan G. (committee chair), Duncan, Roger (committee member), Schönthal, Axel H. (committee member), Shen, Wei-Chiang (committee member), Stiles, Bangyan L. (committee member)

Creator Email natasha.dipsar@gmail.com,natashas@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-252066

Unique identifier UC11288315

Identifier etd-SharmaNata-1660.pdf (filename),usctheses-c3-252066 (legacy record id)

Legacy Identifier etd-SharmaNata-1660.pdf

Dmrecord 252066

Document Type Dissertation

Rights Sharma, Natasha

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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