Selective Elimination of Malignant Melanoma Using the Novel Anti-Tumor Agents, OSW-1 and PEITC

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The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences

10-2014

Selective Elimination of Malignant Melanoma Using the Novel Anti-tumor Agents, OSW-1 AND PEITC

Kausar Begam Riaz Ahmed

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Recommended Citation Riaz Ahmed, Kausar Begam, "Selective Elimination of Malignant Melanoma Using the Novel Anti-tumor Agents, OSW-1 AND PEITC" (2014). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 515. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/515

This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. SELECTIVE ELIMINATION OF MALIGNANT MELANOMA USING THE NOVEL

ANTI-TUMOR AGENTS, OSW-1 AND PEITC

Kausar Begam Riaz Ahmed, B.Tech., M.S.

APPROVED:

______Peng Huang, M.D., Ph.D. (Advisory Professor)

______Michael Davies, M.D., Ph.D.

______Varsha Gandhi, Ph.D.

______Elizabeth Grimm, Ph.D.

______Zahid Siddik, Ph.D.

APPROVED:

______Dean, The University of Texas Graduate School of Biomedical Sciences at Houston

Selective Elimination of Malignant Melanoma Using the Novel Anti-tumor Agents,

OSW-1 and PEITC

DISSERTATION

Presented to the Faculty of

The University of Texas

Health Science Center at Houston

and

The University of Texas

MD Anderson Cancer Center

Graduate School of Biomedical Sciences

in Partial Fulfillment

of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Kausar Begam Riaz Ahmed, B. Tech., M.S.

Houston, Texas

August, 2014

Acknowledgements

I would like to thank my mentor, Dr. Peng Huang for his guidance and support during the course of my PhD study. I would also like to thank my PhD committee members Drs.

Varsha Gandhi, Elizabeth Grimm, Michael Davies, Zahid Siddik, Nancy Poindexter,

Sendurai Mani, David McConkey and Kevin Kim for their constructive feedback and scientific guidance throughout these years. I am grateful to all my colleagues in Dr. Huang’s lab for technical assistance and insightful discussions, which were instrumental in the completion of this work. I am immensely thankful to my parents and siblings who supported me in all possible ways during graduate school and always encouraged me to dream big and work hard.

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Selective Elimination of Malignant Melanoma Using the Novel Anti-tumor Agents,

OSW-1 and PEITC

Kausar Begam Riaz Ahmed, B. Tech., M.S.

(Advisory Professor: Peng Huang, M.D., Ph.D.)

Metastatic melanoma is amongst the most refractory of cancers. Drug resistance and lack of therapeutic selectivity are two main challenges to successful melanoma therapy.

Herein, we investigated the mechanims of anticancer activity and therapeutic selectivity of two novel agents, 3β, 16 β, 17 α-trihydroxycholest-5-en-22-one 16-O-[2-O-4-

methoxybenzoyl-β-D-xylopyranosyl]- [1 →3]-2-O-acetyl-α-L-arabinopyranoside (OSW-1) and β-Phenylethyl Isothiocyanate (PEITC) in melanoma.

OSW-1 inhibited melanoma cell viability at nanomolar concentrations with minimal toxicity to normal melanocytes. Mechanistic studies revealed that OSW-1 suppressed

Disialoganglioside 3 Synthase (GD3S) gene expression in melanoma cells, leading to inhibition of gangliosides GD3 and GD2. GD3 is an abundantly expressed melanoma cell surface antigen with pivotal roles in cancer progression and invasion. OSW-1 promoted interaction between GD3 and mitochondrial voltage-dependent gating protein 1 (VDAC1).

Subsequent VDAC1 activation and autophagic cell death was observed. Downregulation of

VDAC1 ameliorated the cytotoxic effect of OSW-1 in melanoma, indicating that VDAC1 was critical to mediating OSW-1’s activity. Studies with OSW-1 resistant melanoma cells revealed that elevated ganglioside expression may form the mechanistic basis for the selectivity of OSW-1 towards melanoma.

Melanoma cells are characterized by mitochondrial dysfunction and intrinsic oxidative stress compared to normal melanocytes. Reactive oxygen species (ROS) activate

pro-oncogenic signaling pathways that promote cancer progression and metastasis. However, an excessive overload of ROS, beyond the cellular tolerance threshold may trigger oxidative damage and cell death. The natural product PEITC induced apoptosis in malignant melanoma. Biochemical studies revealed that PEITC depleted cellular glutathione leading to

ROS accumulation, mitochondrial damage and cell death. Glutathione inhibition was crucial to mediating PEITC’s activity since the glutathione precursor, N-Acetyl Cysteine reversed

ROS elevation and PEITC induced death. Normal melanocytes with a much lower basal ROS capacity were resistant to PEITC. Drug combination effect of PEITC with Carmustine and

Temozolomide was synergistic, suggesting that redox modulating strategies may be effective in attenuating chemoresistance to these agents.

My study outlines novel therapeutic strategies to effectively eliminate malignant melanomas. The intrinsic differences in ganglioside expression and redox status between normal and cancer cells may be exploited to selectively kill melanoma cells using OSW-1 and PEITC, thus making these compounds worthy of further pre-clinical and clinical investigation.

Table of Contents

Approval sheet …………………………………………………………………………. i

Title page ……………………………………………………………………………….. ii

Acknowledgements ……………………………………………………………………... iii

Abstract ………………………………………………………………………………….. iv

Table of contents ………………………………………………………………………… vi

List of illustrations ……………………………………………………………………….. x

List of abbreviations ……………………………………………………………………... xiv

Chapter 1: Introduction ...... 1

1.1 Metabolic and biochemical alterations in melanoma ...... 1

1.1.1 Dysregulated lipid metabolism: role of gangliosides...... 1

1.1.2 Mitochondrial dysfunction ...... 5

1.1.3 Redox homeostasis and oxidative stress ...... 6

1.2 Purpose and significance of this study ...... 9

1.2.1 Rationale for this study ...... 9

1.2.2 Background information on OSW-1 ...... 11

1.2.3 Background information on PEITC ...... 16

1.3 Research hypothesis and specific aims ...... 20

Chapter 2: Materials and Methods ...... 22

2.1 Chemicals and reagents...... 22

2.2 Cell culture ...... 22

2.3 MTT assay for cell viability ...... 23

2.4 Colony formation assay ...... 23

2.5 Mitochondrial membrane potential measurement by Rhodamine 123 staining ..... 24

2.6 Annexin-V/PI analysis for apoptosis ...... 24

2.7 Electron microscopy ...... 24

2.8 Immunoblotting...... 25

2.9 Detection of ganglioside expression by flow cytometry...... 25

2.10 Lipid raft / ganglioside enriched microdomain (GEM) isolation ...... 26

2.11 Real time RT-PCR ...... 26

2.12 Immunofluorescence staining and confocal microscopy ...... 27

2.13 Cytosolic calcium detection ...... 28

2.14 Detection of cellular free radical expression by staining with the fluorescent dyes

DCFDA, DAF-FM, Dihyroethidium and mitoSOX mitochondrial superoxide indicator28

2.15 siRNA transfection...... 28

2.16 Glutathione assay ...... 29

2.17 Reverse phase protein array analysis ...... 29

2.18 Statistical analysis ...... 30

Chapter 3: The anti-tumor agent, OSW-1, effectively eliminates melanoma cells through ganglioside GD3 and mitochondrial VDAC1 dependent mechanisms ...... 31

3.1 Introduction ...... 31

3.2 Results ...... 32

vii

3.2.1 Cytotoxic effect of OSW-1 in melanoma ...... 32

3.2.2 OSW-1 triggered cell death in melanoma is associated with autophagy ...... 37

3.2.3 Normal melanocytes are resistant to OSW-1 induced cytotoxicity ...... 48

3.2.4 OSW-1 inhibits ganglioside GD3/GD2 expression in melanoma cells ...... 52

3.2.5 OSW-1 promotes interaction of ganglioside GD3 with VDAC1 in the

mitochondria ...... 61

3.2.6 OSW-1 eliminates melanoma cells through mitochondrial VDAC1 dependent

mechanisms ...... 66

3.2.7 VDAC1 is a critical player in mediating the cytotoxic effect of OSW-1 ...... 78

3.2.8 Characterization of the mechanisms underlying the therapeutic selectivity of

OSW-1 ...... 82

3.3 Discussion ...... 89

Chapter 4: PEITC effectively eliminates melanoma cells through redox mechanisms94

4.1: Introduction ...... 94

4.2 Results ...... 95

4.2.1 Redox status of melanoma cells ...... 95

4.2.2 The cytotoxic effect of PEITC in melanoma ...... 98

4.2.3 PEITC exhibits minimal toxicity in normal melanocytes ...... 106

4.2.4 The ROS mediated mechanisms of cell death by PEITC ...... 111

4.2.5 Combination of Temozolomide / BCNU with PEITC attenuates drug resistance to

these agents in melanoma ...... 119

viii

4.3 Discussion ...... 127

Chapter 5: Summary and future directions ...... 132

5.1 Summary ...... 132

5.2 Future directions ...... 133

5.2.1 Mechanisms of selectivity of OSW-1 and PEITC against malignant melanoma

cells ...... 133

5.2.2 Characterization of the direct molecular target for OSW-1: SREBP and GD3S

regulatory signaling pathways ...... 137

5.2.3 Oxidative stress based strategies to combat drug resistance in melanoma using

PEITC ...... 139

References ...... 142

Vita ...... 176

List of Illustrations

Chapter 1: Introduction ………………………………………………………………… 1

Figure 1.1: Convergence of growth factor receptor-mediated and integrin mediated adhesion signaling pathways under GD3 expression in melanoma……………………. 4

Figure 1.2: Altered redox dynamics between normal and cancer cells………………… 8

Figure1.3: Structure of OSW-1. …………………………………………………………... 14

Figure 1.4: Structure of PEITC………………………………………………………….. 18

Chapter 2: Materials and Methods (no illustrations)…………………………………... 22

Chapter 3: The anti-tumor agent, OSW-1, effectively eliminates melanoma cells 31 through ganglioside GD3 and mitochondrial VDAC1 dependent mechanisms

Figure 3.1: Effect of OSW-1 on melanoma cell viability ………………………………. 33

Figure 3.2: OSW-1 inhibits colony formation in melanoma cells…………………….. 34

Figure 3.3: Effect of OSW-1 on mitochondrial membrane potential ( Ψ m) in melanoma

cells……………………………………………………………………………………….. 36

Figure 3.4: OSW-1 promotes autophagic vacuole formation in melanoma cells……… 39

Figure 3.5: Effect of OSW-1 on the autophagy marker, LC3B expression……………. 40

Figure 3.6: Autophagy induction is crucial to cell death induction by OSW-1……….. 41

Figure 3.7: Cytotoxic effect of OSW-1 ± Chloroquine drug combination in melanoma

cells……………………………………………………………………………………….. 44

Figure 3.8: Cytotoxic effect of OSW-1 ± Rapamycin drug combination in melanoma

cells………………………………………………………………………………………… 46

Figure 3.9: Effect of OSW-1 on mitochondrial membrane potential in normal melanocytes vs. melanoma cells…………………………………………………………. 49

Figure 3.10: Selective killing of melanoma cells by OSW-1…………………………… 50

Figure 3.11: Effect of OSW-1 on ganglioside expression in melanoma……………….. 54

Figure 3.12: Effect of OSW-1 on ganglioside GD2 expression in melanoma cells……. 56

Figure 3.13: OSW-1 represses gene expression of GD3S, FASN and HMGCR enzymes... 57

Figure 3.14: Effect of OSW-1 on GD2 Synthase gene expression……………………… 58

Figure 3.15: Schematics of OSW-1 mediated modulation of ganglioside expression…. 59

Figure 3.16: Combination of OSW-1 with Triptolide partially reverses the OSW-1

induced mitochondrial membrane potential loss in melanoma………………………… 60

Figure 3.17: Effect of OSW-1 on GD3 and VDAC1 co-expression in lipid rafts………. 63

Figure 3.18: OSW-1 promotes co-localization of VDAC1 with ganglioside GD3…….. 64

Figure 3.19: Correlation between OSW-1 induced mitochondrial membrane potential loss and ganglioside GD2 inhibition……………………………………………………………. 65

Figure 3.20: OSW-1 induces VDAC1 upregulation in melanoma cells………………… 68

Figure 3.21: Effect of cycloheximide on OSW-1 induced VDAC1 upregulation in melanoma cells…………………………………………………………………………….. 69

Figure 3.22: Effect of Triptolide on VDAC1 expression in melanoma cells…………… 70

Figure 3.23: Induction of mitochondrial superoxide release by OSW-1 in melanoma… 71

Figure 3.24: OSW-1 induces cytosolic calcium elevation in melanoma cells………….. 72

Figure 3.25: Combination of OSW-1 with Cyclosporin A (CsA) causes elevated cell death effect in melanoma cells…………………………………………………………… 74

Figure 3.26: Effect of OSW-1 and Cyclosporin A drug combination on VDAC1 expression and calcium homeostasis…………………………………………………….. 75

Figure 3.27: OSW-1 treatment causes elevated expression of phosphorylated ERK1/2 in 76 melanoma. ………………………………………………………………………………..

Figure 3.28: MEF VDAC1-/- cells are resistant to the cytotoxic effects of OSW-1….. 79

Figure 3.29: Down regulation of VDAC1 in A375SM cells confers resistance to OSW-1. 80

Figure 3.30: Combination of OSW-1 with the VDAC1 inhibitor, DIDS, indicates that

VDAC1 does not regulate ganglioside expression……………………………………… 81

Figure 3.31: The OSW-1 resistant WM35 cell model………………………………….. 83

Figure 3.32: OSW-1 resistant WM35 DW cells have a decreased ganglioside GD3/GD2

expression profile compared to the parental WM35 melanoma cells…………………… 84

Figure 3.33: OSW-1 resistant WM35 DW cells have an altered free radical expression profile compared to the parental WM35 melanoma cells……………………………….. 85

Figure 3.34: Differential protein expression between WM35 vs. WM35 R vs. WM35 DW cells…………………………………………………………………………………………. 87

Figure 3.35: Differential expression of lipogenesis and mitochondria signaling proteins in

WM35 vs. WM35 R vs. WM35 DW cells……………………………………………….. 88

Chapter 4: PEITC effectively eliminates melanoma cells through redox mechanisms. 94

Figure 4.1: Basal free radical expression profile in melanoma cells……………………. 97

Figure 4.2: Effect of PEITC on melanoma cell viability………………………………… 99

Figure 4.3: PEITC induced cytotoxicity is through apoptosis in melanoma cells…….. 100

Figure 4.4: PEITC induces mitochondrial membrane potential loss in melanoma cells. 103

Figure 4.5: Correlation between cellular basal ROS levels and cell death induced by

PEITC……………………………………………………………………………………… 104

Figure 4.6: Effect of PEITC on JNK and p38 expression in melanoma………………… 105

Figure 4.7: Preferential killing of melanoma cells by PEITC…………………………. 108

Figure 4.8: Induction of ROS accumulation in melanoma cells and normal melanocytes 109 by PEITC…………………………………………………………………………………..

xii

Figure 4.9: Comparison of ROS expression between melanoma cells and normal

melanocytes………………………………………………………………………………. 110

Figure 4.10: Effective killing of melanoma cells through ROS induced damage……… 112

Figure 4.11: Correlation between cell death and PEITC induced ROS stress…………. 114

Figure 4.12: PEITC causes depletion of cellular glutathione………………………….. 116

Figure 4.13: N-Acetyl Cysteine (NAC) reverses the inhibitory effect of PEITC………. 117

Figure 4.14: Effect of PEITC ± TMZ/BCNU treatment on melanoma cell viability…. 121

Figure 4.15: Combination of Temozolomide / BCNU with PEITC inhibits colony

formation in melanoma cells……………………………………………………………. 122

Figure 4.16: Combination index analysis of PEITC ± TMZ/BCNU treatment………. 123

Figure 4.17: Redox mechanisms mediate the combinatorial cell death effect of TMZ/

BCNU ± PEITC treatment in melanoma………………………………………………… 125

Chapter 5: Summary and future directions (no illustrations)……………………….. 132

xiii

List of Abbreviations

ADM Adriamycin

APP Amyloid Precursor Protein

BSO L-Buthionine (S, R)-Sulphoximine

CDDP cis -Diamminedichloroplatinum(II) or cisplatin

ChIP Chromatin Immunoprecipation

CM-H2-DCFDA 5, 6-Chloromethyl-2’7’-Dichlorodihydrofluorescein Diacetate

CsA Cyclosporin A

DAF-FM 4-Amino-5-Methylamino-2',7’-Difluorofluorescein

DCF Dichlorohydrofluorescein

DMSO Dimethyl Sulfoxide

ER Endoplasmic Reticulum

FAK Focal Adhesion Kinase

FASN Fatty Acid Synthase

FCCP p-Trifluoromethoxyphenyl Hydrazone

GCS γ-Glutamyl Cysteinyl Synthetase

GD Disialoganglioside

GD3S Disialoganglioside 3 Synthase

GM Monosialoganglioside

GNE UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase

GPX Glutathione Peroxidase

GST Glutathione S-Transferase

GS-X Glutathione Efflux Pump

xiv

GR Glutathione Reductase

HEt Hydroethidium

HMGCR 3-Hydroxy-3-Methylglutaryl-CoA Reductase

iNOS Inducible Nitric Oxide Synthase

i.p. Intraperitoneal

JNK c-Jun N-terminal Kinase

MEF Mouse Embryonic Fibroblasts

MMP Mitochondrial Membrane Potential

MTP Mitochondrial Transition Pore

MTX Methotrexate

NAC N-Acetyl Cysteine

NCX1 Sodium-Calcium Exchanger 1

NF-κB Nuclear Factor κB

OSBP Oxysterol Binding Protein

ORP4L OSBP-related Protein 4L

OSW-1 3β, 16 β, 17 α-trihydroxycholest-5-en-22-one 16-O-[2-O-4-

methoxybenzoyl-β-D-xylopyranosyl]- [1 →3]-2-O-acetyl-α-L-

arabinopyranoside p450scc Cholesterol side chain cleavage cytochrome P-450

PBS Phosphate Buffered Saline

PGC1 α Peroxisome Proliferator-activated Receptor Gamma Coactivator 1 α

PEITC β-Phenylethyl Isothiocyanate

PI Propidium Iodide

ROS Reactive Oxygen Species

RPPA Reverse Phase Protein Array

RT-PCR Reverse Transcriptase-Polymerase Chain Reaction

SAR Structure Activity Relationship

SREBP Steroid Regulatory Element Binding Protein

UV Ultraviolet

VDAC1 Voltage Dependent Anion Channel 1

VEGF Vascular Endothelial Growth Factor

TAX Taxol

xvi

Chapter 1: Introduction

1.1 Metabolic and biochemical alterations in melanoma

1.1.1 Dysregulated lipid metabolism: role of gangliosides.

Altered metabolism is a hallmark of cancer cells (1, 2). In addition to exhibiting the

warburg effect phenotype, aggressive cancers are also characterized by increased de novo

lipogenesis which provides membrane building blocks for rapid proliferation, post-

translational protein modifications and cell signaling (3). The expression of critical lipogenic

enzymes such as fatty acid synthase (FASN), HMG Co-A reductase (HMGCR), acetyl-coA

carboxylase (ACC) is upregulated in human cancers, including malignant melanoma (4, 5).

Increased expression of the key fatty acid synthesis enzyme, FASN is correlated with

poor prognosis in breast cancer and melanoma (6, 7). Elevated fatty acid synthesis has been

associated with a high proliferative index in prostate cancers (8). Aberrant fatty acid

biosynthesis induced by FASN overexpression has been known to induce a cancer-like

phenotype in non-cancer epithelial cells (9). Further, oncogenic signaling pathways also

activated FASN leading to increased fatty acid biosynthesis. Yamauchi et al . (2011) demonstrated that the PI3K-Akt-mTORC1 oncogenic signaling in melanoma cells activates the steroid regulatory element binding protein (SREBP), which is the master transcription factor for lipid metabolism (10). SREBP, in turn, activates the downstream HMGCR enzyme leading to increased cholesterol biosynthesis. Inhibition of the PI3K-Akt-mTORC1-SREBP signaling axis resulted in reduced cholesterol levels in the cell membrane lipid rafts.

Importantly, inhibition of this SREBP mediated cholesterol biosynthesis using pharmacological inhibitors suppressed Akt activation in lipid rafts and attenuated tumor

growth (10). Ganglioside GD3 positively regulated the lipogenic phenotype in melanoma cells by mediating Akt activation and thus the downstream cholesterol biosynthesis.

Gangliosides are sialic acid containing glycosphingolipids that are ubiquitously expressed in human cancers and regulate signals transduced at the membrane microdomains

(11). In general, while disialic acid containing gangliosides (GD3, GD2, GD1) promote tumor cell proliferation, invasion and motility and are considered to be pro-oncogenic (11,

12), monosialic acid containing gangliosides (GM3, GM2, GM1) are tumor suppressive and inhibit cell growth and metastasis (11, 13, 14). In melanoma, elevated levels of gangliosides

GD3, GD2, GM3 and GM2 were reported (15). Normal melanocytes exhibited high expression of ganglioside GM3 and minimal levels of GD3 (16). This differential ganglioside expression was due to a very low expression of the GD3 synthesis regulating enzyme GD3 synthase in normal melanocytes (16). Using GD3-negative mutant of SK-MEL-28 cells,

Furukawa et al . (2011), demonstrated that GD3 expression contributed to increased phosphorylation of adaptor molecules, p130Cas, paxillin, and focal adhesion kinase (FAK).

Further, higher localization of the Src kinase, Yes, to the lipid rafts in the cell membrane of

GD3-positive SK-MEL-28 cells was observed. Also, enhanced integrin function and a higher localization of integrin to the ganglioside enriched microdomains (GEM)/lipid rafts in the

GD3-positive SK-MEL-28 cells, where it clusters with GD3 on the rafts, was observed. (17-

19). Thus ganglioside GD3 acts as a point of convergence for growth factor receptor- mediated signaling and integrin mediated adhesion signaling, leading to activation of enhanced malignant signals and cancer phenotypes (figure 1.1) (18, 20). More significantly, injection of anti-GD3 monoclonal antibody suppressed tumor progression in melanoma tissues and patients making it an important target for melanoma therapy (21, 22).

The ganglioside GD2 is overexpressed in breast and lung cancers. Battula et al. reported that GD2 is a stem cell marker in human breast cancer and may contribute to c-Met activation and chemoresistance in these cells (23, 24). Anti-GD2 antibody treatment of small cell lung cancer (SCLC) cells led to dephosphorylation of FAK, activation of p38 MAPK and finally cell death by anoikis (25).

Majority of gangliosides are constituted in the lipid rafts, where they interact with the receptors and other downstream proteins. It was observed that overexpression of the lipid raft-resident protein, caveolin-1, led to the dispersion of GD3 from the lipid raft domains

(26). Further, the fine structure in the ceramide group of GD3 expressed in lipid raft was reported to be different from GD3 located in non-raft cell fractions (26). Further, a translocation of GD3 to the mitochondria during cell death signaling, where it regulates microtubule polymerization and apoptotic induction, has also been reported (27). Further studies need to be undertaken to shed light on the complex roles of GD3 in melanoma biology.

Figure 1.1:

Figure 1.1: Convergence of growth factor receptor-mediated and integrin mediated adhesion signaling pathways under GD3 expression in melanoma.

Reprinted by permission of Oxford University Press on behalf of the Japanese Biochemical

Society.

(11): Furukawa K, Hamamura K, Ohkawa Y, Ohmi Y. Disialyl gangliosides enhance tumor phenotypes with differential modalities. Glycoconjugate journal. 2012;29(8-9):579-84.

1.1.2 Mitochondrial dysfunction

Mitochondria are dynamic cellular organelles which act as functional sites for

oxidative phosphorylation, fatty acid oxidation, tricarboxylic acid cycle and execution of

apoptosis (28, 29). Cancer cells are characterized by Warburg effect, wherein the tumor cells

rely on glycolysis rather than oxidative phosphorylation for their energy and metabolic needs

due to defects in their mitochondrial respiratory machinery, decreased oxidation of NADH-

linked substrates or mitochondrial mutations. Increase in reactive oxygen species (ROS), due

to various causes such as defects in electron transport, reduced ROS scavenging,

environmental or oncogene induced stress etc., is a characteristic feature of cancer cell

mitochondria (28). Enhanced ROS contributes to stabilization of hypoxia inducible factor-α, loss of p16, further oxidative damage and metastasis of cancer cells. In addition, mutations in mitochondrial DNA have also been observed. For instance, Tan et al . (2002) identified

somatic mutations in the D-loop region, 16sRNA, ND2 and ATPase 6 genes of mitochondrial

DNA in breast cancer patients (29, 30). In melanoma, a comprehensive analysis by Poetsch

et al. (2004) discovered mitochondrial instability in the D-loop region in 13% of nodular

primary cancers and 20% metastatic melanomas (31). Interestingly, the pro-oncogenic

mutant BRAF, BRAF V600E localized to mitochondria in thyroid cancer cells leading to

inhibition of oxidative phosphorylation, increased glycolytic activity and a high glucose

uptake (32).

Mitochondrial autophagy or mitophagy is also activated in cancer cells as a cellular

response mechanism to nutrient depletion (28). Various studies indicate that mitophagy is

preceded by mitochondrial fragmentation and membrane permeabilization. The

mitochondrial transition pore protein, voltage dependent anion channel 1 (VDAC1) has been

reported to induce apoptotic and autophagic cell death in cells (33-35). Recent studies by De

Pinto et al. (2007) and Tomasello et al (2009) demonstrated that overexpression of human

(h)VDAC1 caused depolarization of the inner membrane and triggered mitochondrial permeability transition (MPT), which may be followed by autophagy (33, 36). In cancer cells, the hypoxia regulated BNIP3/NIX proteins have been recognized as key players in mediating mitochondrial autophagy (28, 37).

1.1.3 Redox homeostasis and oxidative stress

A unique feature of melanoma biology is the free radical generation by aberrant melanosomes (38). Reactive oxygen species play important roles in melanoma development, immune response, metabolism, and metastasis (38). Loss of PTEN and decreased PTEN activity is observed in about 20% of melanomas (39). Activation of Akt signaling can promote ROS generation by inducing the expression of the ROS generating enzyme NOX4 in melanoma cells and mice (40). Also, Akt may inhibit the activity of various pro-apoptotic proteins such as BAD and forkhead transcription factor (FOXO) thus stabilizing the cells with mitochondrial damage and potentiating ROS production. Further, MAPK which are activated in melanoma due to BRAF V600E activating mutation also modulate ROS generation in cells (41, 42). Further loss of function of the tumor suppressors, p53 and p16 may also contribute to ROS stress phenotype (43). Chronic oxidative stress in combination with ultraviolet (UV) exposure may lead to malignant transformation of cells (38, 44, 45).

ROS can activate the transcription factors, NF-κB and AP-1 thus promoting melanoma progression (46, 47). ROS generated by hypoxic mitochondria and NADPH oxidases may stabilize hypoxia inducible factor 1-α (HIF1-α) and potentially block PTEN mediated inhibition of Akt signaling leading to activation of downstream anti-apoptotic proteins

including Bcl-1 and Bcl-xL (48). Thus ROS plays pivotal roles in melanoma transformation and metastasis.

Reactive oxygen species function as a doubled-edged sword. Intrinsically high ROS levels in melanoma cells promote tumor development. However, if the ROS stress in melanoma is at a level that is beyond the cell’s toxicity tolerance threshold, then it may trigger cell death. Normal cells which have lower basal ROS stress levels and a reserve antioxidant capacity will be resistant to this ROS accretion since their antioxidants will be mobilized to neutralize the ROS increase and prevent it from reaching the cell-death threshold. The pro-oncogenic signaling in melanoma cells triggers increased ROS production and elevates the antioxidant capacity of the cells so as to neutralize the high level of free radicals being generated. Eventually, this results in a shift of redox dynamics and a high dependence of cancer on antioxidants to sustain the ROS levels within the cell’s toxicity threshold (49). Hence cancer cells are more vulnerable to redox modulatory or pro-oxidant based therapeutic strategies that by either scavenging the antioxidants or instigating excessive ROS production (for instance through inhibition of oxidative phosphorylation) may drive the ROS levels above the toxicity tolerance threshold of the cell leading to massive cell death (49). This therapeutic strategy is illustrated in figure 2.

This therapeutic strategy is relatively novel and may find great utility in inducing cell death as well as combating drug resistance to other conventional melanoma chemotherapeutic agents. Currently, Elesclomal, a pro-oxidant compound that utilizes a similar mechanism of action is in clinical trials for melanoma (50-52).

Figure 1.2:

Figure 1.2: Altered redox dynamics between normal and cancer cells.

(49): Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nature reviews Drug discovery. 2009;8(7):579-

91.

1.2 Purpose and significance of this study

The purpose of this PhD dissertation was to characterize novel therapeutic strategies based on altered lipid metabolism and reactive oxygen species mediated stress in melanoma cells to selectively eliminate malignant melanomas using the novel anticancer agents, OSW-1 and PEITC.

The rationale for this research design, study hypothesis and specific aims have been described in the sections below.

1.2.1 Rationale for this study

Selective elimination of malignant melanomas by OSW-1:

The ganglioside GD3 is highly expressed in a majority of melanoma cell lines and tissues (53, 54) and mediates cell proliferation and metastasis (17, 19). Hamamura et al.

(2005) demonstrated that GD3 enhanced cell growth and invasion through FAK, p130cas and paxicillin signaling in melanoma cells (17). Enhanced integrin cluster formation in lipid rafts on the melanoma cell membrane was observed under GD3 expression suggesting that it contributed to adhesion signaling in malignant melanoma (19). Thus GD3 acts as a convergence point for growth factor and integrin-mediated adhesion signaling in melanoma cells (18, 20). Further, GD3 expression induced cholesterol biosynthesis, lipogenic phenotype and activation of Akt signaling in melanoma (10). Significantly, only trace amounts of GD3 were detected in normal melanocytes as compared to melanoma cells (16), thus highlighting its potential as a surface antigen that can be targeted to selectively eliminate melanoma cells with limited toxicity to the surrounding normal melanocytes. Currently, GD3 is being investigated in melanoma immunotherapy (21, 22, 55).

Recent studies identified that Triptolide, which bears structural similarities with

OSW-1, inhibited ganglioside GD3 and GD2 expression in melanoma cells (56). Yokoyama

et al. (2005), discovered that OSW-1 encapsulation in GM3 liposomes led to melanoma cell killing (57). These results suggested that OSW-1 might potentially inhibit ganglioside expression in melanoma cells. Previous studies from our lab have demonstrated that OSW-1 induces cytotoxicity through mitochondrial dependent mechanisms in leukemia cells (58,

59). Ganglioside GD3 translocates to mitochondria during cell death and through its interactions with the raft-like mitochondrial microdomains may have a regulatory function in inducing mitochondrial membrane permeabilization and apoptosis (27, 60).

The rationale for my research study was that OSW-1, because of its similarity to

Triptolide and higher affinity towards GM3 may have an inhibitory effect on ganglioside

(GM3, GD3, GD2) expression. Inhibition of ganglioside GD3, which plays important roles in melanoma proliferation and invasion, would promote melanoma cell death. Further, because of the melanoma cell specific expression of GD3, cell death due to GD3 inhibition by OSW-

1 may be selective towards cancer cells. It was essential to investigate the effect of GD3 in mitochondrial signaling since OSW-1 was previously to act through mitochondria dependent mechanisms.

Effective elimination of melanoma by PEITC:

Increased ROS generation and altered redox status is observed in cancer cells

compared to normal cells. Recent studies have suggested that this differential redox biology

may be exploited to selectively target malignant cells, overcome drug resistance and improve

the therapeutic activity of current clinically available anticancer agents (61).

Melanoma cells are under a higher intrinsic oxidative stress compared to normal

melanocytes due to oncogene activation and aberrant melanosome function (62). This

elevated ROS promotes melanoma oncogenic transformation, invasion, metastasis and

metabolism. However, if the ROS stress in cells is pushed beyond the cells’ tolerance limit

either by using pro-oxidant compounds or by abrogating the cellular antioxidant capacity, it

would lead to massive toxicity and cell death.

The anticancer agent PEITC causes ROS accumulation in cancer cells either through

depletion of the antioxidant glutathione or by inhibiting oxidative phosphorylation (63, 64).

In my research, I postulated that melanoma cells would be at a higher intrinsic oxidative

stress than normal melanocytes, making them vulnerable to ROS stress based therapeutic

strategies. PEITC, because of its ability to potentiate further ROS increase, will effectively

eliminate melanoma cells. Normal melanocytes, which maintain basal redox homeostasis,

will be able to withstand the exogenous ROS stress induced by PEITC to a certain extent,

and thus would be less sensitive to PEITC or other redox modulating agents. Further, these

principles of redox modulation by PEITC may be utilized in attenuating drug resistance to

currently used anticancer agents such as Temozolomide and Carmustine.

1.2.2 Background information on OSW-1

The natural product, 3 β, 16 β, 17 α-trihydroxycholest-5-en-22-one 16-O-[2-O-4- methoxybenzoyl-β-D-xylopyranosyl]- [1 →3]-2-O-acetyl-α-L-arabinopyranoside (OSW-1), an acylated disaccharide cholestane saponin molecule was originally isolated as a main constituent of the bulbs of Ornithogalum saundersiae (Liliaceae) (65-67). It is highly cytotoxic against several human cancer cell lines with half maximal inhibitory concentrations in the nanomolar range (58, 59, 68). It exhibited significantly higher inhibitory effect than

many clinically used anticancer agents including etoposide, adriamycin (ADM), taxol

(TAX), cisplatin (CDDP), mitomycin c and methotrexate (MTX) in leukemia, pulmonary adenocarcinoma, mouse mastrocarcinoma, pancreatic and brain cancers (59, 67).

Importantly, adriamycin-resistant and camptothecin-resistant p388 leukemia as well as fludarabine refractory primary leukemia cells from chronic lymphocytic leukemia (CLL) patients were sensitive to OSW-1 suggesting that it had a unique mechanism of action that was different from these clinically used agents (59, 67). In vivo evaluation showed that it was extremely potent against mouse p388 tumors with a single 0.01 mg/kg dose increasing life span by 59% (67). Importantly, OSW-1 was therapeutically selective towards malignant cells with half-maximal inhibitory concentrations (IC 50 ) 40- to 150-fold higher in normal cells than cancer cells (59).

An in vitro screen against the National Cancer Institute (NCI)-60 cell line panel indicated that OSW-1 exhibited significant toxicity with an IC50 of 0.78 nM (67). Melanoma cells were specifically sensitive to OSW-1. Further Pearson’s correlation analysis using

NCI’s COMPARE algorithm generated a Pearson’s correlation co-efficient between 0.6 and

0.83 for OSW-1 vs. cephalostatin 1 suggesting that they had related mechanisms of action and shared cellular targets (67, 69). Structure activity relationship (SAR) analysis by Guo et al., identified that OSW-1 was analogous to one-half of the cephalostatin molecule with C22- oxocarbenium ions playing a common role in the bioactivity of both OSW-1 and cephalostatin (68). Structural studies also revealed that the acetyl (Ac) and the 4- methoxybenzoyl (MBz) groups on the disaccharide moiety of OSW-1 were essential as their removal diminished its cytotoxicity by 1000 fold (67, 70). In addition, the steroidal aglycone was critical to OSW-1’s potent cytotoxicity since SAR analysis indicated that substituting the

aglycone group on the steroidal backbone of OSW-1 with a totally disparate synthetic aglycone, while still retaining the disaccharide moiety, resulted in the compound not exhibiting any cytotoxic effects even at a concentration of 10 µM (66, 68, 71, 72). Thus both the disaccharide and aglycone groups are important to the antineoplastic activity of OSW-1.

The first chemical synthesis of OSW-1 was reported by Deng et al . (1999), with an overall yield of 6% and 14 steps (73). Later, Yu et al . (2002), published an improvised synthesis procedure which reduced the total chemical synthesis steps to 10 and improved the overall yield to 28% (74). These findings inspired several studies on the synthesis and in vitro cytotoxic activity of OSW-1 analogues. Of specific interest to this dissertation, is the synthesis of biotinylated OSW-1 which is 10-times less toxic to cells than OSW-1 and may be used as a tool to identify the cellular interacting partners of OSW-1 (75).

Figure1.3: Structure of OSW-1.

Chemical formula: C 47 H68 O15

Molecular weight: 873.03

(69): Burgett AW, Poulsen TB, Wangkanont K, Anderson DR, Kikuchi C, Shimada K,

Okubo S, Fortner KC, Mimaki Y, Kuroda M, Murphy JP, Schwalb DJ, Petrella EC, Cornella-

Taracido I, Schirle M, Tallarico JA, Shair MD. Natural products reveal cancer cell dependence on oxysterol-binding proteins. Nature chemical biology. 2011;7(9):639-47.

Despite numerous studies on the chemical synthesis, bioactivity and structure activity

relationship (SAR) of OSW-1 and its analogues, the mechanisms underlying the antitumor

effect of this inhibitor are still not completely understood (67, 73-79). Tamura et al ., demonstrated that OSW-1 directly inhibited gene expression of the ovarian steroidal limiting enzyme, cholesterol side chain cleavage cytochrome P-450 (p450scc), leading to suppression of ovarian estradiol secretion and granulosa cells proliferation in the ovary (80). Mechanistic studies from our lab revealed that OSW-1 inhibited the sodium-calcium exchanger 1 (NCX1) on the plasma membrane of leukemia cells, leading to cellular calcium overload, cytochrome c release and mitochondria dependent apoptosis (58). The mitochondrial respiration deficient human leukemia cells (HL60 C6F) were less sensitive to OSW-1 than the parental HL60 cells, suggesting that OSW-1 mediated cell death was through mitochondria dependent mechanisms. Burgett et al . (2011), identified the oxysterol binding protein (OSBP) and its closest paralog, OSBP-related protein 4L (ORP4L) as cellular binding targets of OSW-1 and cephalostatin, thus implying a role for these compounds in signal transduction, lipid transport and lipid metabolism (69). OSW-1 also induced necroptotic death in hepatocellular

carcinoma through inhibition of cell invasiveness, angiogenesis, cell polarity and adhesion

signaling, in addition to promoting mitochondria dependent apoptosis (81). Recently Yamada

et al., identified endoplasmic reticulum (ER), golgi apparatus and to a lesser extent

mitochondria as putative subcellular sites of OSW-1 localization (82).

Atomic force microscopy studies by Yokoyama et al . (2005), identified that OSW-1 distributed in the ganglioside GM3-phospholipid membrane monolayer in cells (83). Further, a strong attractive interaction between OSW-1 and GM3 was observed which seemed to be

related to the potent activity of OSW-1 against cancer cells. Specifically, treatment of B16-

F0 melanoma cells with OSW-1 encapsulated in ganglioside GM3 liposomes led to a

complete inhibition of cell viability within 48 h (57). Interestingly, Triptolide, a structural

analogue of OSW-1, suppressed the gene expression of GD3 synthase (GD3S), the rate

limiting enzyme for GD3 biosynthesis, in melanoma and breast cancer cells (24, 56).

Gangliosides are glycosphingolipids containing sialic acids which have been reported to be

overexpressed in carcinomas of the lung, breast, neuroblastoma and melanoma (24, 53, 54,

84-86). Ganglioside, GD3 is overexpressed in melanoma and is a well characterized

therapeutic target (21).

These research findings describe a role for OSW-1 in ganglioside biology and

mitochondria dependent cell signaling. Through my PhD research, I sought to investigate the

effect of OSW-1 on ganglioside GM3/GD3 expression and mechanisms of mitochondria

dependent cell death in melanoma cells. Further, I conducted mechanistic studies to delineate

the molecular mechanisms underlying the therapeutic selectivity of OSW-1 towards

melanoma cells. The results and discussion in Chapter 3 focus on the important outcomes of

my research with the antitumor agent OSW-1 in melanoma.

1.2.3 Background information on PEITC

The natural product β-Phenylethyl Isothiocyanate (PEITC) is an active metabolite of

cruciferous vegetables such as broccoli, cauliflower and watercress (87). It exhibits

chemopreventive properties by inhibiting the activity of the transcription factor, Nuclear

Factor κB (NF-κB), which plays an important role in inflammation, cancer progression and

survival. NF-κB is regulated by the protein I κBα, which sequesters NF-κB in the cytosol thus preventing its nuclear translocation and activation of downstream pro-inflammatory and

oncogenic genes (88). I κBα is a well characterized target of PEITC in prostate cancer (89).

PEITC stabilizes I κBα expression thus inhibiting NF-κB activation, nuclear translocation of

p65, and promotes suppression of NF-κB mediated VEGF, cyclin D1, Bcl-xL, iNOS and

COX-2 protein expression (89-91).

Numerous studies have reported that PEITC induces apoptosis in cancer cells, both in

in vitro and in vivo settings (63, 87, 92-94) . This apoptosis induction by PEITC was

characterized by G2-M cell cycle arrest, inhibition of the anti-apoptotic proteins, Bcl-xL,

Bcl-2 and Mcl1 and caspases 8 and 9 cleavage in prostate cancer and leukemia cells (95). Mi

et al., identified tubulin as an important binding target of PEITC in A549 lung cancer cells

(96). This study demonstrated that the covalent binding of PEITC and other isothiocyanates

to the tubulin proteins leads to disruption of microtubule polymerization, cell cycle arrest,

down regulation of cyclins A, D and E and cell death (96, 97). Interestingly, apoptosis

initiation in HT-29 colon cancer cells by PEITC was dependent upon activation of the MAP

kinase, JNK. Consequently SP600125, an anthrapyrazolone inhibitor of JNK suppressed apoptosis by PEITC (98).

Figure 1.4: Structure of PEITC

Chemical formula: C 9H9NS

Molecular weight: 163.24

Reprinted with permission from (99): Moon YJ, Brazeau DA, Morris ME. Dietary phenethyl isothiocyanate alters gene expression in human breast cancer cells. Evidence-based complementary and alternative medicine : 2011.

PEITC mediated cytotoxicity in cancer cells was unique because of two observations:

(a) PEITC treatment in cells led to excessive reactive oxygen species (ROS) generation (63,

64, 94, 100-102). Recent studies have attributed this ROS production to two mechanisms – glutathione depletion and inhibition of oxidative phosphorylation by PEITC. This ROS stress was of functional significance and correlated with cell death in cancer cells. In addition, the

ROS induction by PEITC was instrumental in ameliorating drug resistance to clinically used anticancer agents such as Gleevec, Vorinostat and Fludarabine in leukemia cells.

(b) The cell death induced by PEITC was limited towards malignant cells with little cytotoxicity against normal cells (63, 64, 94, 103). Previous studies in our lab demonstrated that this preferential PEITC-cytotoxicity was due to altered redox biology between normal and cancer cells. Mitochondrial respiration deficient Rho-0 prostate cancer cells were also resistant to the antiproliferative activity of PEITC suggesting that the therapeutic selectivity of PEITC relies on mitochondria dependent mechanisms.

My PhD dissertation was focused on investigating the mechanisms of cytotoxicity and therapeutic selectivity of PEITC against melanoma cells. I had been particularly interested in examining if the redox differences between normal melanocytes and melanoma cells could be used as a therapeutic basis to preferentially eliminate melanoma cells. Further, mechanistic studies were undertaken to characterize the utility of PEITC to combat drug resistance to clinically used melanoma drugs such as Temozolomide and Carmustine. The results from my study on the anticancer activity of PEITC in melanoma have been discussed in chapter 4.

1.3 Research hypothesis and specific aims

The central research hypothesis for my PhD dissertation was - alterations in reactive oxygen species (ROS) and lipid metabolism in melanoma cells, as compared to normal melanocytes, may serve as a biochemical basis to preferentially kill malignant melanoma cells using proper pharmacological agents such as OSW-1 and PEITC.

To address this hypothesis I identified the following two specific aims:

1. To investigate altered lipid metabolism as the biological basis to preferentially

eliminate melanoma cells using OSW-1.

2. To investigate ROS stress as a biochemical basis to selectively kill melanoma cells

using PEITC.

In order to effectively achieve both of these specific aims, each specific aim was further divided into three sub aims. Experiments were designed to investigate each of these sub aims. The specific aims and the corresponding sub aims have been listed below:

Specific aim 1: To investigate altered lipid metabolism as the biological basis to preferentially eliminate melanoma cells using OSW-1.

a) To determine the effect of OSW-1 on ganglioside expression in melanoma cells.

b) To investigate the effect of OSW-1 on mitochondrial functionality in melanomas.

c) To characterize the molecular mechanisms underlying the therapeutic selectivity of

OSW-1 towards melanoma cells.

Specific aim 2: To investigate ROS stress as a biochemical basis to selectively kill melanoma cells using PEITC.

a) To determine the antiproliferative effect of PEITC in melanoma cells.

b) To characterize the underlying redox mechanisms that are pivotal to PEITC’s activity

in melanoma cells.

c) To elucidate therapeutic strategies to combat drug resistance in melanoma using

PEITC.

The following chapters describe the project design, results obtained, key conclusions from my study and the future directions of my research.

Chapter 2: Materials and Methods

2.1 Chemicals and reagents

The compound OSW-1 was kindly provided by Dr. Zhendong Jin from the University of

Iowa (Iowa City, Iowa). Β-Phenylethyl Isothiocyanate (PEITC) was purchased from Sigma-

Aldrich (St. Louis, MO). The original stock solutions of OSW-1 and PEITC were prepared in

DMSO. For all experiments, OSW-1 and PEITC were diluted in the media at a concentration of 0.1% or less of the solvent DMSO. The fluorescent dyes Rhodamine 123, calcium green-1

AM, mitotracker red CMXRos, Dihydroethidium and mitoSOX red mitochondrial superoxide indicator were purchased from Life Technologies (Grand Island, NY). Annexin

V-FITC and the 10X binding buffer were purchased from BD Pharmingen (San Jose, CA). 3-

(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetra-zolium bromide (MTT), Giemsa stain, 4,4'- diisothiocyano stilbene-2,2'-disulfonic acid (DIDS), Cyclosporin A (CsA), 3-Methyladenine

(3-MA), Chloroquine (CQ), Triptolide, and propidium iodide (PI) were purchased from

Sigma-Aldrich (St. Louis, MO).

2.2 Cell culture

The melanoma cell lines A375P, A2058, WM115 were purchased from American Type

Culture Collection (ATCC, Manassas, VA). A375SM, WM35, WM46, MEWO, MEL624,

SK-MEL-2, UCSD-354L, and Hs294T cell lines were purchased from the characterized cell line core at University of Texas MD Anderson Cancer Center (Houston, TX); these cell lines were validated at the core facility by STR finger printing. The normal human epithelial melanocytes (NHEM) were purchased from Promocell (Heidelberg, Germany). NHEM cells were cultured in M254CF medium (Life Technologies, Long Island, NY) supplemented with

PMA-free human melanocyte growth supplement-2 (Life Technologies, Long Island, NY)

and were grown at 37 ºC, 5% CO 2 in a cell culture incubator. MEF VDAC1-/- cells were a

generous gift of Dr. William Craigen (Baylor College of Medicine, Houston, TX) and were

cultured in DMEM medium supplemented with 10% heat inactivated fetal bovine serum

(FBS) and sodium pyruvate. A375P, A375SM, A2058 cells cultured in DMEM medium

(Mediatech, Manassas, VA) supplemented with 10% heat inactivated FBS were grown in a

cell culture incubator with 5% CO 2 at 37 ºC in humidified air. WM35, WM46, WM115,

MEWO, MEL624, SK-MEL-2, UCSD-354L, and Hs294T cells were grown in RPMI 1640 medium (Mediatech, Manassas, VA) supplemented with 10% heat inactivated FBS in a cell culture incubator with 5% CO 2 at 37 ºC in humidified air.

2.3 MTT assay for cell viability

Cell growth inhibition induced by OSW-1 and PEITC was assayed by performing the MTT

assay as previously described (104). Briefly, melanoma cells, seeded in 96-well plates, were

treated with log-scale serial diluted concentrations of OSW-1 and PEITC. After 72 h of

incubation, 50 l of MTT reagent per well was added for 4h. The cell media was then

aspirated, formazan precipitates dissolved in DMSO and absorbance at 570 nm was

measured in a Multiskan MK3 microplate-reader (Thermo Labsystem, Franklin, MA). The

50% inhibitory concentration (IC 50 ) values were then computed using GraphPad Prism

(GraphPad Software, Inc. La Jolla, CA).

2.4 Colony formation assay

The colony formation assay was performed as described previously (105). Briefly, melanoma cells (5000 cells/well) were seeded in a 6-well plate and treated with varying concentrations of inhibitors for 2 weeks. The cells were then fixed with methanol/acetic acid

(10:1) solution, and stained with Giemsa (Sigma-Aldrich, St. Louis, MO). The cells were

photographed and the colonies counted.

2.5 Mitochondrial membrane potential measurement by Rhodamine 123 staining

Rhodamine 123 assay was used to monitor the integrity of mitochondria following drug

treatments as previously described (106, 107). Melanoma cells or normal melanocytes were

treated with OSW-1, PEITC or other inhibitors for the indicated time points and then

incubated in 0.5 µM rhodamine 123 dye during the last 1 h of drug treatment. The cells were then dissociated, washed twice with PBS and Ψ m measured using a BD Biosciences

FACSCalibur flow cytometer (Mountain View, CA). Data analysis was performed using

FlowJo (TreeStar Inc., Ashland, OR).

2.6 Annexin-V/PI analysis for apoptosis

Cell death by apoptosis/necrosis was measured by flow cytometric analysis of cells dual stained for annexin V-FITC and propidium iodide (108). Briefly, melanoma cells grown in 6- well plates were treated with inhibitors for the indicated time points, harvested and stained with annexin V-FITC for 15 minutes, washed and then stained for propidium iodide. Cell death was measured using a BD Biosciences FACSCalibur flow cytometer (Mountain View,

CA) and the results were analyzed using the FlowJo software (TreeStar Inc., Ashland, OR).

2.7 Electron microscopy

The electron microscopy experiment was performed at the high resolution electron

microscopy facility (core grant CA16672) at the MD Anderson cancer center, as per the

core’s prescribed protocol. Briefly, melanoma cell samples were fixed with a solution

containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3,

for 1 h. After fixation, the samples were washed and treated with 0.1% Millipore-filtered

cacodylate buffered tannic acid, post-fixed with 1% buffered osmium tetroxide for 30 min, and stained en bloc with 1% Millipore-filtered uranyl acetate. The samples were dehydrated in increasing concentrations of ethanol, infiltrated, and embedded in LX-112 medium. The samples were polymerized in a 70 ºC oven for 2 days. Ultrathin sections were cut in a Leica

Ultracut microtome (Leica, Deerfield, IL), stained with uranyl acetate and lead citrate in a

Leica EM Stainer, and examined in a JEM 1010 transmission electron microscope (JEOL,

USA, Inc., Peabody, MA) at an accelerating voltage of 80 kV. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques Corp, Danvers, MA).

2.8 Immunoblotting

Immunoblotting was performed to assess protein expression in drug treated and control cell lysates (109). Proteins were separated on 10-15% SDS-PAGE gels, transferred onto nitrocellulose membranes and immunoblotted using the following commercially available antibodies: β-actin (Calbiochem, San Diego, CA), LC3B (Novus Biologicals, Littleton, CO),

VDAC1 (Abcam, Cambridge, MA), phosphorylated and total ERK1/2, and Caveolin (Cell

Signaling Technology, Inc., Danvers, MA). Membranes were then incubated with appropriate horseradish peroxidase conjugated secondary antibodies and the signal visualized using supersignal enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL).

2.9 Detection of ganglioside expression by flow cytometry

Assessment of ganglioside expression changes following drug treatment was performed by flow cytometry as previously reported (24). Melanoma cells were treated with OSW-1 or other inhibitors for the indicated time durations, harvested, washed twice in PBS and stained for 30 min, 4 ºC, with the following commercially available antibodies: PE-anti human ganglioside GD2 (Biolegend, San Diego, CA), disialoganglioside GD3 (BD Biosciences, San

Jose, CA) and anti GM3 (NeuAc) antibody (Cosmo Bio USA, Inc., Carlsbad, CA). Samples stained with PE-conjugated GD2 antibody were washed and directly assayed using the BD

Biosciences FACSCalibur flow cytometer (Mountain View, CA). Samples probed with GM3 and GD3 antibodies were washed, stained with a FITC-conjugated secondary antibody for another 30 min, 4 ºC and the GM3/GD3 expression was measured by flow cytometry. All data analysis was performed using FlowJo (TreeStar Inc., Ashland, OR).

2.10 Lipid raft / ganglioside enriched microdomain (GEM) isolation

The lipid raft fractions were prepared using a detergent extraction protocol described by

Mitsuda et al (110). Two T-175 flasks of 80-90% melanoma cells were used for each preparation. Briefly, cells were harvested, washed twice in ice-cold PBS and suspended in 1 mL of TNE buffer (1% Triton X-100, 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM

EGTA). The cells were then dounce homogenized 24 times, mixed with equal volume of

80% sucrose and placed at the bottom of ultra-clear centrifuge tubes (Beckman Coulter,

Brea, CA). 2 mL of 30% sucrose solution in TNE buffer without Triton X-100 and 1 mL of

5% sucrose solution in TNE buffer without Triton X-100 were carefully laid on top of the preparation. The samples were then centrifuged at 105,000 × g in a Beckman Coulter Optima

XPN-100 ultracentrifuge (Brea, CA) for 16 h at 4°C. From the top of the gradient, 9 fractions of 0.5 mL sample were collected and assayed by SDS-PAGE or dot blot.

2.11 Real time RT-PCR

To analyze mRNA expression of GD3S, GD2S, FASN and HMGCR, real time RT-PCR was performed as previously described (111). Briefly, total RNA was extracted from melanoma cells using the RNeasy mini kit (Qiagen, Valencia, CA). 2 µg of RNA was subjected to reverse transcription with random hexamer primers utilizing the revertaid first strand cDNA

synthesis kit (Thermo Scientific, Pittsburg, PA). Real time PCR was performed on an

Applied Biosystems Viia7 machine using SYBR green with the following cDNA primers:

FASN forward: 5’ – CGCTCGGCATGGCTATCT-3’; FASN reverse: 5’-

CTCGTTGAAGAACGCATCCA-3’; HMGCR forward: 5’-GGCCCAGTTGTGCGTCTT-

3’; HMGCR reverse: 5’-CGAGCCAGGCTTTCACTTCT-3’; GD3S forward: 5’-

TGTGGTCCAGAAAGACATTTGTGGACA-3’; GD3S reverse: 5’-

TGGAGTGAGGTATCTTCACATGGGTCC-3’; GD2S forward: 5’-

GACAAGCCAGAGCGCGTTA-3’ and GD2S reverse: 5’-

TACTTGAGACACGGCCAGGTT-3’. All experiments were performed with three biological replicates and three technical replicates and the results have been reported as the mean of the biological replicates plus or minus standard error.

2.12 Immunofluorescence staining and confocal microscopy

Immunofluorescence staining and confocal microscopic analysis were performed based on previously reported protocols (10, 112). Melanoma cells were cultured on sterilized glass coverslips, treated with OSW-1 and incubated in 50 nM mitotracker red for 25 min. Cells were washed twice with PBS, fixed with 3.7% paraformaldehyde in medium at 37 °C for

10 min, permeabilized using 0.3% Triton X-100 for 10 minutes, washed and then blocked with 4% FBS for 1 h. The samples were incubated overnight with the disialoganglioside GD3

(BD Biosciences, San Jose, CA) and/ or VDAC1 (Proteintech, Chicago, IL) antibodies, washed and incubated in FITC- and rhodamine red X-conjugated secondary antibodies

(Jackson Immunoresearch Laboratories, Inc., West grove, PA) for 1 h. The samples were then washed and mounted onto glass slides using the Ultracruz mounting medium (Santa

Cruz Biotechnology, Dallas, TX). Images were taken by NIKON Eclipse TE2000 confocal microscope and analyzed using the Nikon EZ-C1 software.

2.13 Cytosolic calcium detection

Cytosolic calcium levels were measured as previously described (109). Briefly, melanoma cells were treated with OSW-1 or other inhibitors for the indicated time durations, harvested, washed twice in PBS and stained with calcium green fluorescent dye for 30 min at 4 ºC.

Cytosolic calcium levels were detected in the FL-1 channel of the BD Biosciences

FACSCalibur flow cytometer (Mountain View, CA).

2.14 Detection of cellular free radical expression by staining with the fluorescent dyes

DCFDA, DAF-FM, Dihyroethidium and mitoSOX mitochondrial superoxide indicator

Flow cytometric measurements using the CM-H2-DCFDA, DAF-FM or Dihydroethidium fluorescent probes was performed to detect free radical levels in cells (63, 113). Briefly, melanoma cells were treated with OSW-1, PEITC or other inhibitors for the indicated time points and then incubated in 1 µM mitosox red or CM-H2-DCFDA or DAF-FM or

Dihydroethidium during the last 1 h of drug treatment. The cells were then dissociated, washed twice with PBS and the free radical expression levels were measured using a BD

Biosciences FACSCalibur flow cytometer (Mountain View, CA). Data analysis was performed using FlowJo (TreeStar Inc., Ashland, OR).

2.15 siRNA transfection

Tranfection of VDAC1 and control siRNAs was performed as previously reported (114).

Briefly, A375SM melanoma cells were plated in a 6-well plate and cultured until 50% confluency, at which point, they were transfected with 50 nM silencer select siVDAC1 (ID: s14769) or the negative control siRNA (Life Technologies, Long Island, NY) by the

Lipofectamine RNAiMAX reagent (Life Technologies, Long Island, NY). Western blot was performed to determine VDAC1 expression 48 h post-transfection.

2.16 Glutathione assay

A glutathione assay kit (Cayman Chemicals, Ann Arbor, MI) was used to detect cellular glutathione levels (63, 94, 113). Cell extracts were obtained by sonication (power setting - 4,

10 s 3X) and deproteination as per the manufacturer’s guidelines. The reaction product of glutathionylated 5, 5’-dithiobis-(2-nitrobenzoic acid) was detected spectrophotometrically using the Multiskan MK3 microplate-reader (Thermo Labsystem, Franklin, MA) at 450 nm.

A standard curve for glutathione was generated and the glutathione concentration in each sample was calculated. The Bicinchoninic Acid (BCA) assay was performed to determine sample protein concentration and to normalize the glutathione concentration across samples.

GraphPad Prism (GraphPad Software, Inc. La Jolla, CA) was used to perform all statistical analysis.

2.17 Reverse phase protein array (RPPA) analysis

The RPPA experiments were performed at the functional proteomics reverse phase protein array core (NCI # CA16672) at the MD Anderson cancer center, following the core’s experimental protocols (115). Briefly, melanoma cells lysates were extracted in the following lysis buffer: 1% Triton X-100, 50mM HEPES, pH 7.4, 150mM NaCl, 1.5mM MgCl 2, 1mM

EGTA, 100mM NaF, 10mM Na pyrophosphate, 1mM Na 3VO 4, 10% glycerol, containing freshly added protease and phosphatase inhibitors. Cellular proteins were denatured by 1%

SDS (with beta-mercaptoethanol) and diluted in five 2-fold serial dilutions in dilution buffer

(lysis buffer containing 1% SDS). Serial diluted lysates were arrayed on nitrocellulose-coated slides (Grace Biolab) by Aushon 2470 Arrayer (Aushon BioSystems). Sample slides were

probed with 217 primary antibodies and the corresponding biotin-conjugated secondary antibody. The signal obtained was amplified using a Dako Cytomation–catalyzed system

(Dako) and visualized by DAB colorimetric reaction. The slides were scanned, analyzed, and quantified using a customerized-software Microvigene (VigeneTech Inc.) to generate spot intensity. Each dilution curve was fitted with a logistic model (“Supercurve Fitting” developed by the Department of Bioinformatics and Computational Biology in MD

Anderson Cancer Center, “http://bioinformatics.mdanderson.org/OOMPA”). The protein concentration of each set of slides was then normalized by median polish, which was corrected across samples by the linear expression values using the median expression levels of all antibody experiments to calculate a loading correction factor for each sample. Heat maps were generated in Cluster 3.0 (http://www.eisenlab.org/eisen/) as a hierarchical cluster using Pearson Correlation and a center metric. The resulting heatmap was visualized in

Treeview (http://www.eisenlab.org/eisen/).

2.18 Statistical analysis

Statistical analysis was computed using GraphPad Prism version 6 (La Jolla, CA). Data were graphed as mean ± standard error (SEM) for experiments performed in triplicates unless noted differently. A standard student’s two tailed t-test was performed to determine statistically significant difference between samples at a 95% confidence interval. In all cases, **** denotes p < 0.0001, *** denotes p < 0.001, ** denotes p < 0.01 and * denotes p<

0.05.

Chapter 3: The anti-tumor agent, OSW-1, effectively eliminates melanoma cells through ganglioside GD3 and mitochondrial VDAC1 dependent mechanisms

3.1 Introduction

The natural product OSW-1 is a highly potent anticancer agent. Previous studies both

from our lab and others demonstrated that OSW-1 inhibited cancer cell viability at sub-

nanomolar concentrations with minimal toxicity towards normal cells (67, 81). Further, it

was effective in eliminating various cancer cells including leukemia, glioblastoma and

pancreatic cancers that were resistant to other clinically used agents such as taxol, adriamycin

and fludarabine (59, 67).

Previous research efforts from our lab revealed that OSW-1 inhibited the sodium-

calcium exchanger 1 (NCX1) on the cell membrane of HL-60 leukemia cells, leading to

cytosolic calcium elevations, caspase 3 activation and mitochondria dependent apoptosis

(58). However, the mechanisms of OSW-1 induced cytotoxicity in solid tumors still remain

to be elucidated. Recent studies demonstrated that steroidal compounds such as Estradiol and

Triptolide inhibited ganglioside 3 synthase (GD3S) expression in cancer cells, thus causing

cell death (56, 116). Further, a strong interaction between OSW-1 and GM3 gangliosides in

melanoma cells has been noted (83).

In this study, we investigated the effect of OSW-1 on ganglioside expression in melanoma.

Effect of OSW-1 on the ganglioside synthesis regulatory enzyme, GD3 synthase (GD3S) was

also examined. Our results indicate that OSW-1 suppresses GD3 synthase gene expression in

melanoma cells, leading to inhibition of gangliosides, GD3, GD2 and subsequent cell death

by autophagy. Further, OSW-1 promoted interaction between GD3 and mitochondrial

VDAC1 in melanoma cells. Significantly, OSW-1 upregulated VDAC1 expression in

melanoma, leading to loss of calcium homeostasis and cell death. These OSW-1 induced alterations in ganglioside and VDAC1 expression play a critical role in mediating the selectivity of OSW-1 to malignant melanoma cells.

3.2 Results

3.2.1 Cytotoxic effect of OSW-1 in melanoma

Previous findings, both from our lab and others, demonstrate that OSW-1 inhibits cell viability in multiple tumor cell lines including Leukemia, brain, colorectal, hepatic and pancreatic cancers at nanomolar concentrations (59, 69). To investigate the effect of OSW-1 on melanoma cell proliferation, we first performed an MTT assay with multiple melanoma cell lines. As illustrated in figure 3.1, OSW-1 is highly potent in inhibiting melanoma cell growth with an average half-maximal inhibitory concentration (IC50 ) of 0.178 nM. A subsequent colony formation assay of WM35 cells treated with increasing concentrations of

OSW-1 indicated that only 36% of cells formed colonies at a concentration of 0.1 nM, with a complete inhibition of colony formation at higher concentrations (figure 3.2 A, B). Because many of the metastatic melanoma cells did not form colonies on 6-well plates, we adapted the MTT assay for long term (figure 3.2 C, D). Melanoma cells were incubated in log-scale increasing concentrations of OSW-1 for 8 days. MTT assay was performed in these cells to elucidate the effect of OSW-1 on cell proliferation (figure 3.2 C). As indicated in figure 3.2

D, OSW-1 inhibited melanoma cell growth at sub-nanomolar concentrations with an average

IC 50 of 0.058 nM.

Figure 3.1:

Cell line IC value Mean IC ± SEM B 50 50 (nM) (nM) A375P 0.164 ± 0.045

A375SM 0.105 ± 0.024

HS294T 0.027 ± 0.007

MEL624 0.072 ± 0.010 0.178 ± 0.043 SK-MEL-2 0.421±0.218

UCSD-354L 0.218 ± 0.050

WM35 0.156 ± 0.024 WM46 0.097 ± 0.001

MEWO 0.343 ± 0.068

Figure 3.1: Effect of OSW-1 on melanoma cell viability

(A) Melanoma cells were treated with log-scale serial diluted concentrations of OSW-1 for

72 h, and the antiproliferative effect was determined by performing the MTT assay. The cell viability curves were plotted. Each data point represents mean ± SEM of three independent measurements. (B) The 50% cell inhibitory concentration (IC 50 ) ± SEM for each cell line is indicated.

Figure 3.2:

A D B M S

O-1

0.1 nM OSW

****

-1

0. 3 nM OSW

-1

1nM OSW

C D

Figure 3.2: OSW-1 inhibits colony formation in melanoma cells.

(A) and (B) WM35 cells treated with 0.1, 0.3 and 1 nM OSW-1 for 2 weeks were fixed, stained with Giemsa and the number of colonies formed were counted. **** denotes p <

0.0001. (C) Melanoma cells were treated with log-scale serial diluted concentrations of

OSW-1 for 8 days, and the antiproliferative effect of OSW-1 was determined by performing the MTT assay. The cell viability curves were plotted. Each data point represents mean ±

SEM of three independent measurements. (D) The 50% cell inhibitory concentration (IC 50 ) of each cell line in the long-term MTT assay is indicated.

Figure 3.3:

P=0.42 P=0.05 * ***

** * * **

**** **** ****

Figure 3.3: Effect of OSW-1 on mitochondrial membrane potential (Ψ m) in melanoma cells.

Melanoma cells were treated with either DMSO control or 1 nM OSW-1 for 72 h and the effect of OSW-1 on mitochondrial membrane potential was determined by rhodamine 123 staining and flow cytometric analysis. OSW-1 induced collapse of membrane potential in melanoma cells with an average DMSO vs. OSW-1 p-value = 0.007. **** denotes p <

0.0001, *** denotes p < 0.001, ** denotes p < 0.01 and * denotes p< 0.05.

Mitochondrial dysfunction has been implicated in cell death signaling in cells.

Maintenance of proper mitochondrial membrane potential ( Ψ m) is required for cellular homeostasis and normal functioning of cells as it drives ATP synthesis and oxidative phosphorylation (117). However, an acute induction of mitochondrial membrane permeabilization (MMP) due to calcium overload, oxidative stress, inhibitors such as etoposide, arsenite, lonidamine etc. can lead to cell death (118-122). Hence we sought to investigate the effect of OSW-1 on mitochondrial transmembrane potential ( Ψ m) across a panel of eleven melanoma cell lines (figure 3.3). OSW-1 caused an average decrease in mitochondrial membrane potential in melanoma cells by 48%, as compared to the DMSO treated control cells (control vs. OSW-1, p-value = 0.007, n = 11). Interestingly, SK-MEL-2 cells, which have a slightly higher IC 50 (0.421 nM, figure 3.1 B) compared to other melanoma cells, also exhibited decreased sensitivity to OSW-1 in the mitochondrial membrane potential measurement assay, thus pointing towards the heterogeneity of drug response to OSW-1 in melanomas.

3.2.2 OSW-1 triggered cell death in melanoma is associated with autophagy

Autophagy is an evolutionarily conserved lysosomal degradation pathway that is essential for cellular homeostasis and response to stress conditions (123, 124). Previous studies have classified autophagy as a non-apoptotic cell death characterized by enhanced autophagic flux and accumulation of autophagic vacuoles in cells (125, 126). Transmission electron microscopy results showed formation of numerous autophagic vacuoles in WM35 cells treated with OSW-1 (figure 3.4 A). This autophagic vacuole formation increased with time with about a 9- and 21-fold increase in the observed number of autophagic vacuole per cell at 24 h and 48 h of 1 nM OSW-1 treatment (figure 3.4 B). The autophagosome-

associated lipidated form of LC3B (LC3B-II) can be differentiated from the cytosolic free isoform (LC3B-I) by immunoblotting (127). Consistent with the morphological findings, a time dependent increased expression of the lipidated LC3B (LC3B-II) isoform was observed in cells treated with OSW-1 (figure 3.5).

Figure 3.4:

A DMSO OSW-1 24 h OSW-1 48 h

Figure 3.4: OSW-1 promotes autophagic vacuole formation in melanoma cells.

(A) Transmission electron microscopy images of WM35 cells treated with 1 nM OSW-1 for

24 h and 48 h. The black arrows indicate the autophagic vacuole formation induced by OSW-

1. (B) The number of autophagic vacuoles formed per cells for each samples were counted

and the mean ± SEM of three independent measurements are plotted as bar graphs. **

denotes p < 0.01.

Figure 3.5:

OSW-1

24 h 12 h 48 h DMSO

LC3B-I

LC3B-II

β-Actin

Figure 3.5: Effect of OSW-1 on the autophagy marker, LC3B expression.

WM35 cells were treated with 1 nM OSW-1 for 12, 24 and 48 h and immunoblotting was performed to examine lipidation of the autophagy marker protein LC3B.

Figure 3.6:

Figure 3.6: Autophagy induction is crucial to cell death induction by OSW-1.

(A) WT and Atg5-/- MEF cells were treated with varying concentrations of OSW -1 for 72 h

and the cytotoxic effect was determined by the MTT assay, as described in materials and

methods. Percent cell su rvival corresponding to each OSW -1 concentration was plotted. (B)

WM35 cells preincubated with 1mM 3 -methyl adenine for 1 h were treated 1nM OSW -1 for

48 h. Effect of OSW-1 ± 3-MA combination on cell viability was evaluated by annexin -V/PI

analysis and is represented as bar graphs normalized to the DMSO control. ** denotes p <

0.01.

The autophagy related protein 5, Atg5, is an essential autophagy protein, which conjugates with Atg12 to form a multimeric structure that is crucial for autophagosome formation (128, 129). Hence, we employed Atg5 knock out (Atg5-/-) MEF cells to characterize the role of autophagy in mediating the cell inhibitory effect of OSW-1. MTT results from WT and Atg5-/- MEF cells treated with log-scale incremental concentrations of

OSW-1 for 72 h demonstrated that the Atg5-/- cells exhibited decreased sensitivity to OSW-1

(figure 3.6 A). 1 nM OSW-1 effectively inhibited cell viability by approximately 80% in

MEF WT cells while an approximate 75% of MEF Atg5-/- cells were still viable at this concentration. Nevertheless, higher concentrations of OSW-1 killed both MEF WT and

Atg5-/- cells. In complementation to this observation, the autophagy inhibitor, 3-Methyl

Adenine (3-MA), partially rescued WM35 cells from the cytotoxic effect of OSW-1 (130,

131). As shown in figure 3.6 B, OSW-1 treatment for 48 h abolished cell survival to 30%.

However, combination of OSW-1 with 3-MA reversed cell survival to 60.76%. These findings not only delineate the significance of autophagy in OSW-1 induced elimination of melanoma cells, but also allude to the fact that there might also be simultaneous alternate cell death mechanisms facilitating OSW-1’s activity.

Interestingly, combination of OSW-1 with the lysosomotrophic agent Chloroquine led to rapid apoptotic cell death. Chloroquine inhibits late phase autophagy by preventing the fusion of the autophagosomes to lysosomes (132). Because of this mechanism of action, chloroquine has found utility in sensitizing multiple cancer cells including hepatocellular and cervical cancers to radiation and drugs like cisplatin and oxaliplatin (133, 134). Combination of OSW-1 with chloroquine produced approximately 48% (normalized to control) increased cell death in WM35 cells as compared to OSW-1 treatment alone (figure 3.7 A). To evaluate

the type of drug interaction, we performed combination index (CI) analysis of different

combination regimens of OSW-1 (0.1, 0.3 and 1 nM, 48 h) and chloroquine (5, 10 and 20

M, 48 h) in melanoma cells (135, 136). As described in figure 3.7 B, the combination regimen between OSW-1 and chloroquine was synergistic (CI <1) for seven of the nine combination treatments with 1 nM OSW-1 and 5 M chloroquine showing the strongest effect (CI = 0.111). Interestingly, an antagonistic combination effect (CI >1) was observed on combining 20 M chloroquine with 1 nM OSW-1 in WM35 cells.

Figure 3.7:

96% 93%

DMSO 10 M CQ

56% 10%

1nM OSW-1 OSW-1 + CQ

B Chloroquine OSW-1 Combination (µM) (nM) Index (CI)

5 0.1 1.067 5 0.3 0.494

5 1 0.111

10 0.1 0.768

10 0.3 0.533

10 1 0.184 20 0.1 0.714

20 0.3 1.039 20 1 1.349

Figure 3.7: Cytotoxic effect of OSW-1 ± Chloroquine drug combination in melanoma cells.

(A) 1 nM OSW-1 was added to WM35 cells pretreated with 10 µM chloroquine for 20 min.

After 72 h incubation, cell viability was determined by annexin v/PI staining and flow cytometric analysis. The percentage of viable cells (negative for annexin V and PI) is indicated. (B) WM35 cells were treated with the indicated concentrations of OSW-1 ± CQ drug combination for 48 h and combination index analysis was performed using Compusyn software. The combination index (CI) values were computed. The following quantitative definitions, based on the Chou-Talalay theorem, were employed to define combination effect: CI < 1 - synergism, CI = 1 - additive effect and CI > 1 - antagonism.

Figure 3.8:

A OSW-1 + B OSW-1 Rapamycin **

Rapamycin

DMSO

Figure 3.8: Cytotoxic effect of OSW-1 ± Rapamycin drug combination in melanoma cells.

(A) A375SM cells were treated with 1 nM OSW-1 ± 100 nM Rapamycin for 48 h and the effect on mitochondrial membrane potential was determined by rhodamine 123 analysis. (B)

Bar graphs represent the mitochondrial membrane potential changes induced by A375SM and WM35 cells treated with 1 nM OSW-1 ± 100 nM Rapamycin for 48 h. (C) Effect of 1 nM OSW-1 ± 100 nM Rapamycin 48 h treatment on the viability of A375SM and WM35 cells was determined by annexin-V/PI analysis and is represented as bar graphs. ** denotes p

< 0.01.

The mammalian target of rapamycin (mTOR) serves as a central regulator of

autophagy by inhibiting phosphorylation of the autophagy proteins Atg13 and Unc51-like

kinase (ULK), and preventing autophagosome formation (137-139). The antibiotic,

Rapamycin inhibits mTOR thus acting as a potent autophagy inducer in multiple cell lines

(140, 141). In our study, we found that rapamycin ameliorated the cytotoxic effect of OSW-1

in melanoma cells (figure 3.8). A375SM and WM35 cells were co-treated with 1 nM OSW-1

and 100 nM rapamycin for 48 h and the effect on mitochondrial transmembrane potential

(Ψ m) and cell death was assessed through flow cytometry by staning the cells with rhodamine 123 (figure 3.8 A, B) and annexin-FITC/PI (figure 3.8 C). As illustrated in figure

3.8 A and B, rapamycin partially reversed the mitochondrial membrane permeabilization induced by OSW-1. 1 nM OSW-1 caused a loss of membrane potential by 56.33% and

60.3% in A375SM and WM35 cells. However, rapamycin + OSW-1 treatment led to a reversal of this effect to 16.2% and 34.07% in A375SM and WM35 cells. Similarly, rapamycin also partially rescued A375SM and WM35 cells from the antiproliferation effect of OSW-1 (figure 3.8 C). These drug combination (OSW ± chloroquine/rapamycin) results elucidate the dual roles of autophagy in maintaining cellular homeostasis wherein though autophagy might start-off as a cell protective mechanism, sustained autophagy mediated by

OSW-1 leads to melanoma cell killing.

3.2.3 Normal melanocytes are resistant to OSW-1 induced cytotoxicity

The promising antineoplastic effect of OSW-1 against melanoma cells led us to examine the effect of this compound in normal melanocytes. As shown in figure 3.9, OSW-

1induced a time dependent loss of mitochondrial potential in A375SM melanoma cells, while normal melanocytes were resistant to this effect of OSW-1. 1 nM OSW-1, 72 h treatment, did

not trigger significant mitochondrial membrane potential loss in normal melanocytes (17.4%;

figure 3.9 A) compared to melanoma cells (n = 11, 52%; figure 3.3).

Figure 3.9:

A B OSW-1 OSW-1 OSW-1 48 h 72h 48 h

OSW-1 DMSO 72h DMSO

NHEM A375SM

Figure 3.9: Effect of OSW-1 on mitochondrial membrane potential in normal

melanocytes vs. melanoma cells.

Mitochondrial membrane potential in normal melanocytes and A375SM cells treated with 1

nM OSW-1 for 48 h and 72 h was evaluated by rhodamine 123 staining as described in

materials and methods.

Figure 3.10:

A 37 5S 90.0 % 6.87 % 3.86 % 3.51 % M

N H E 81.2 % 80.6 % 76.7 % 78.8 % M

DMSO OSW-1 48 h OSW-1 72h OSW-1 96 h

Figure 3.10: Selective killing of melanoma cells by OSW-1.

(A) Melanoma cells and normal melanocytes were treated with 0.5, 1 and 2 nM OSW-1 for

72 h and the effect on cell viability was determined by annexin V/PI analysis. Each data point represents the mean of three independent measurements. (B) Comparison of viability of

A375SM cells and normal melanocytes treated with 1 nM OSW-1 for indicated time duration. Cell viability was determined by annexin V-FITC/PI staining. The numbers indicate the percentage of viable cells (negative for annexin V and PI).

We designed a follow-up time course experiment with 1 nM OSW-1 and performed

an annexin-V/PI staining assay to determine the effect of OSW-1 on cell death. Normal

melanocytes were resistant to the cytotoxic effect of OSW-1; 97.04% of normal melanocytes

were still viable after OSW-1 treatment for 96 h as compared to only 3.9% of A375SM

melanoma cells (figure 3.10 B). Figure 3.10 A represents the cell inhibitory effect of OSW-1

against multiple melanoma cell lines (n = 7) and normal melanocytes treated with increasing

concentrations of OSW-1 for 72 h. The scatter plot while delineating the heterogeneous

response of melanoma cells to OSW-1, also highlights the therapeutic selectivity of OSW-1

towards melanoma cells (1 nM OSW-1, average melanoma (n = 7) cell viability = 46.88%)

with limited toxicity to normal melanocytes (1 nM OSW-1, average cell viability = 93.30%).

3.2.4 OSW-1 inhibits ganglioside GD3/GD2 expression in melanoma cells

Recent studies by Kwon et al and Battula et al demonstrated that Triptolide, a diterpenoid triepoxide, inhibits ganglioside GD3 and GD2 expression by repressing GD3 synthase (ST8Sia I) expression (24, 56). Since Triptolide bears structural similarity to OSW-

1, we hypothesized that OSW-1 would inhibit gangliosides GD3 and GD2 expression by suppressing GD3 synthase (GD3S) expression. To test this hypothesis we assessed alterations in ganglioside expression after OSW-1 treatment of melanoma cells (figure 3.11). Flow cytometric analysis of WM35 and A375SM treated with OSW-1 and stained with ganglioside antibodies revealed that OSW-1 caused a moderate decrease in ganglioside GD3 (figure 3.11

A, B) and a more striking inhibition of GD2 (figure 3.11 C, D). This inhibition of ganglioside expression was time dependent, with GD2 inhibition starting as early as 3 h post OSW-1 treatment (figure 3.11 C, D). Figure 3.12 depicts the effect of OSW-1 on GD2 expression against a panel of melanoma cell lines. On average, a 24 h OSW-1 treatment of melanoma

cells reduced GD2 expression to 66.86% (DMSO vs. OSW-1, p-value = 0.0029). Further, this GD3 and GD2 inhibition correlated with an accumulation of ganglioside GM3 (figure

3.11 E, F). A 24 h 1nM OSW-1 treatment led to an increased expression of ganglioside GM3 by 178.2% and 127.4% in A375SM and WM35 melanoma cells (figure 3.11 F).

Figure 3.11:

A GD3 B * * 24 h ** ** 12 h 3 h 6 h DMSO

C GD2 D 12 * 24 h 3 h * * ** 6 h DMSO **

E GM3 F * P = 0.06 24 h

DMSO

Figure 3.11: Effect of OSW-1 on ganglioside expression in melanoma.

(A), (C) and (E) A75SM cells treated with 1 nM OSW-1 for the indicated time points were analyzed for ganglioside GD3, GD2 and GM3 expression by flow cytometry as described in materials and methods. (B), (D) and (F) GD3, GD2 and GM3 ganglioside expression in

A375SM and WM35 cells treated with 1 nM OSW-1 for the indicated time points was determined by flow cytometric measurements. Bar graphs represent mean ± SEM of percent ganglioside (GD3/GD2/GM3) inhibition from three independent experimental measurements.

** denotes p < 0.01 and * denotes p< 0.05.

Figure 3.12:

* P = 0.73

P=0.08 * P = 0.07 ** ** *** *** *** ***

Figure 3.12: Effect of OSW-1 on ganglioside GD2 expression in melanoma cells.

Melanoma cells were treated with either DMSO control or 1 nM OSW-1 for 24 h and the effect of OSW-1 on ganglioside GD2 expression was quantified. Bar graphs represent mean

± SEM from three independent experiments. OSW-1 induced inhibited GD2 expression in melanoma cells with an average DMSO vs. OSW-1 p-value = 0.0029. **** denotes p <

0.0001, *** denotes p < 0.001, ** denotes p < 0.01 and * denotes p< 0.05.

Figure 3.13:

**** **** *** **** ****

Figure 3.13: OSW-1 represses gene expression of GD3S, FASN and HMGCR enzymes.

Real time PCR was performed in A375SM cells treated with 1 nM OSW-1 for 12h and 24 h.

The effect of OSW-1 on the expression of GD3S, FASN and HMGCR genes was determined. Each experiment was performed with three technical replicates. The bar graphs represent the mean result from three biological replicates. **** denotes p < 0.0001, *** denotes p < 0.001, ** denotes p < 0.01 and * denotes p < 0.05.

Figure 3.14:

Figure 3.14: Effect of OSW-1 on GD2 Synthase gene expression.

Real time PCR was performed in A375SM cells treated with 1 nM OSW-1 for 12h and 24 h.

The effect of OSW-1 on the expression of GD2S gene was determined. * denotes p < 0.05.

Figure 3.15:

OSW-1

GD3 Synthase GD3 GM3

GD2 Synthase

GD2

Figure 3.15: Schematics of OSW-1 mediated modulation of ganglioside expression.

The model summarizes the effect of OSW-1 on ganglioside expression. OSW-1 suppresses the expression of GD3 synthase gene leading to inhibition of GD3, GD2 and accumulation of ganglioside GM3.

Figure 3.16:

OSW-1 TPL + OSW-1

TPL

DMSO

Figure 3.16: Combination of OSW-1 with Triptolide partially reverses the OSW-1 induced mitochondrial membrane potential loss in melanoma.

A375SM cells were treated with 1nM OSW-1 ± 20 nM Triptolide for 48 h and the effect on mitochondrial membrane potential was determined by rhodamine 123 analyses.

As shown in figure 3.15, ganglioside GD3 is produced from its precursor GM3 through a reaction catalyzed the enzyme GD3 synthase. Results from real-time PCR, performed to ascertain the effect of OSW-1 on GD3S mRNA expression, indicated that

OSW-1 represses GD3S gene expression in a time dependent fashion (figure 3.13).

Expression of GD2 synthase (GD2S) gene though initially suppressed by OSW-1, increased after 24 h of OSW-1 treatment (figure 3.14). Interestingly, combination of Triptolide with

OSW-1 partially reversed the mitochondrial membrane collapse induced by OSW-1 (figure

3.16), suggesting that both OSW-1 and Triptolide elicit their antiproliferation activity probably by acting on similar cellular targets. Yamauchi et al., (2011) demonstrated that elevated GD3 levels correlated with increased expression of key lipogenic enzymes in melanoma cell lines (10). Based on this, we hypothesized that inhibition of GD3 by OSW-1 would lead to suppressed expression of enzymes mediating the lipogenesis pathway.

Accordingly, 1 nM OSW-1 treatment for 24 h caused a 3.24- and 5.73-fold decrease in fatty

acid Synthase (FASN) and 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR)

expression in melanoma cells (figure 3.13). Thus we conclude that OSW-1 suppresses GD3S

expression in melanoma leading to inhibition of gangliosides GD3 and GD2 and the

downstream lipogenic enzymes, FASN and HMGCR.

3.2.5 OSW-1 promotes interaction of ganglioside GD3 with VDAC1 in the mitochondria

Raft-like microdomains on the mitochondrial membrane are enriched with gangliosides GD3 and GM3, and act as potential catalytic sites for autophagic and apoptotic reactions (60, 142, 143). Activation of cell death receptor signaling via CD95/Fas or tumor

necrosis factor-α (TNF-α) promotes intracellular redistribution and vesicular transport of

ganglioside GD3 to mitochondria, wherein GD3 promotes apoptosis by acting on the

mitochondrial transition pore and causing cytochrome c release (144-146). Garofalo et al .

(2005) also demonstrated that during CD95/Fas mediated apoptosis GD3 interacts with voltage dependent anion channel 1 (VDAC1) and the fission protein hFis1 in the mitochondrial lipid microdomains of lymphoblastoid CEM cells (143). Furthermore, prior results from our lab suggest that OSW-1 has a mitochondrial mechanism of action (59).

Hence we investigated the effect of OSW-1 on mitochondrial targeting of GD3. To characterize the distribution of GD3 and VDAC1 in the membrane microdomains, we isolated lipid rafts fractions by performing a linear 5-80% sucrose gradient of the DMSO control and OSW-1 treated A375SM cell lysates (figure 3.17). Majority of the lipid raft resident protein caveolin-1 was recovered in fractions 2, 3 and 4. Ganglioside GD3 was also concentrated in fractions 2, 3 (highest expression) and 4 of the DMSO control sample.

Nevertheless, OSW-1 administration promoted redistribution of GD3 expression to fractions

2, 3, 4, and 5. More significantly, OSW-1 stimulated elevated VDAC1 expression in the corresponding fractions (2, 3, 4, and 5) whereas a diminished expression of VDAC1 was observed in these caveolin enriched lipid raft fractions of the control sample. To confirm these results, A375SM cells were treated with OSW-1 for 24 h and the cellular co- localization of VDAC1 and GD3 was examined by confocal microscopy. As shown in figure

3.18, OSW-1 potentiated increased co-localization of ganglioside GD3 with VDAC1in the mitochondria, thus corroborating previous findings that cell death signaling triggered intracellular redistribution and mitochondrial trafficking of GD3 (143, 145).

Figure 3.17:

1 2 3 4 5 6 7 8 9

VDAC1 DMSO Caveolin-1

GD3

1 2 3 4 5 6 7 8 9

VDAC1 OSW-1 Caveolin-1

GD3

Figure 3.17: Effect of OSW-1 on GD3 and VDAC1 co-expression in lipid rafts.

Glycosphingolipid enriched microdomain/lipid raft fractions from DMSO and 1 nM OSW-1

24 h treated A375SM samples were prepared and equal amounts of the fractions were subjected to an immunoblot for VDAC1 and the lipid raft resident protein caveolin-1.

Simultaneously, a dot blot was performed to determine GD3 expression in the lipid raft fractions. OSW-1 potentiates redistribution of GD3 and VDAC1 expression in lipid rafts.

Figure 3.18:

VDAC1 GD3 Merge

DMSO

OSW - 1

Figure 3.18: OSW-1 promotes co-localization of VDAC1 with ganglioside GD3.

A375SM cells were grown on coverslips and treated with 1 nM OSW-1 for 24 h. The cells were stained with VDAC1 and GD3 antibodies as per the procedure described in materials and methods and confocal microscopic images were taken.

Figure 3.19:

r =0.7100 p = 0.0144

Figure 3.19: Correlation between OSW-1 induced mitochondrial membrane potential loss and ganglioside GD2 inhibition.

The fold change in mitochondrial membrane potential induced by OSW-1 was plotted as a function of fold change in GD2 expression. Linear regression analysis was performed and a

Pearson’s correlation coefficient of 0.7100 with a p value of 0.0144 was obtained.

The relationship between inhibition of ganglioside GD3/GD2 expression and

sensitivity of melanoma cells to OSW-1 was evaluated quantitatively by plotting fold change

in mitochondrial membrane potential ( Ψ m) as a function of ganglioside GD2 expression change upon OSW-1 treatment (figure 3.19). Our results indicate that there was a positive correlation between these two factors (r = 0.7100, p = 0.0144), thus suggesting that ganglioside inhibition sensitizes melanoma cells to the cytotoxic effect of OSW-1.

3.2.6 OSW-1 eliminates melanoma cells through mitochondrial VDAC1 dependent mechanisms

OSW-1 stimulated increased interaction between GD3 and mitochondrial VDAC1

(figure 3.18). VDAC1 plays an important role in the transport of adenine nucelotides, Ca 2+ , and other ions and metabolites across the mitochondria in addition to mediating apoptotic and autophagic signaling in mitochondria (147-152). Therefore, we studied the role of

VDAC1 in OSW-1 induced cell death. Towards this end, we first examined melanoma cells for VDAC1 expression after OSW-1 treatment. VDAC1 expression was markedly increased by OSW-1 in A375SM and WM35 cells but was slightly decreased in normal melanocytes which were resistant to OSW-1 (figure 3.20 A, B). On average, OSW-1 incubation for 36 h triggered a 4.8- and 3.2-fold increase in VDAC1 expression in A375SM and WM35 cells while a 1.7-fold decrease was observed in normal melanocytes (figure 3.20 C). Further, the de novo protein synthesis inhibitor, cycloheximide reduced this VDAC1 elevation in melanoma cells, indicating that OSW-1 caused a translational upregulation of VDAC1

(figure 3.21). Further, cycloheximide treatment for 2 h resulted in inhibition of 50% of

VDAC1 expression compared to the untreated control suggesting that the half-life of VDAC1 was around 2 h (figure 3.21). Significantly, Triptolide, which represses GD3 synthase, also

led to VDAC1 induction by 1.4-fold in A375SM cells (figure 3.22). Collectively, these data indicate that GD3S inhibition by OSW-1 may lead to VDAC1 activation and this VDAC1 increase positively correlates with cellular sensitivity to OSW-1 in melanoma cells.

Figure 3.20:

A OSW-1 B A375SM WM35 NHEM

-1 -1 DMSO 0.5 12 24 36 h -1 DMSO DMSO OSW OSW A VDAC1 DMSO OSW 37 5S VDAC1 M β-Actin β-Actin

VDAC1 W M 35 β-

N VDAC1 H E β-Actin M

C *

* *

Figure 3.20: OSW-1 induces VDAC1 upregulation in melanoma cells.

Immunoblots of A375SM, WM35 and NHEM cells treated with 1 nM OSW-1 for 0.5, 12, 24

and 36 h (A) and 24 h only (B). (C) Densitometric measurement of the VDAC1 band

intensity relative to β-actin is shown. The bar graphs represent mean ± SEM of normalized

VDAC1 expression from three independent immunoblot assessments. * denotes p < 0.05.

Figure 3.21:

1 nM OSW-1 (24 h) + D M 2 M Cycloheximide S 0 0.5 1 2 4 8h O VDAC1

β-Actin

D M 2 M Cycloheximide

S 0 0.5 1 2 4 O

VDAC1

β-Actin

Figure 3.21: Effect of cycloheximide on OSW-1 induced VDAC1 upregulation in melanoma cells.

A375SM cells were pre-incubated in 1 nM OSW-1 for 24 h, followed by 2 µM cycloheximide treatments for 0.5, 1, 2, 4 and 8 h. Simultaneously A375SM cells were treated with 2 µM cycloheximide as a single agent for 0.5, 1, 2, 4 and 8 h. Immunoblot was performed to examine changes in VDAC1 expression.

Figure 3.22:

T DMSO P L

VDAC1

1 1.44

β-Actin

Figure 3.22: Effect of Triptolide on VDAC1 expression in melanoma cells.

A375SM cells treated with 20 nM Triptolide for 24 h and VDAC1 protein expression was detected by immunoblot analysis.

Figure 3.23:

*** **** **** *

Figure 3.23: Induction of mitochondrial superoxide release by OSW-1 in melanoma.

A375SM and WM35 cells treated with 1 nM OSW-1 for the indicated time points were probed with the fluorescent mitochondrial superoxide indicator, mitosox red and the mitochondrial superoxide release was determined by flow cytometric analysis. **** denotes p < 0.0001, *** denotes p < 0.001, ** denotes p < 0.01 and * denotes p < 0.05.

Figure 3.24:

A B * * 24 h * * 16 h

8 h DMSO

Figure 3.24: OSW-1 induces cytosolic calcium elevation in melanoma cells.

A375SM and WM35 cells were treated with OSW-1 for the indicated time points and the changes in cytosolic calcium levels were quantified using the calcium green fluorescent probe. (A) A representative histogram of the calcium changes induced by OSW-1. (B) Bar graph indicates the mean ± SEM of percent calcium increase in A375SM and WM35 cells from three independent experiments. * denotes p < 0.05.

Apart from VDAC1 induction, OSW-1 also stimulated mitochondrial superoxide release (figure 3.23) and increase of cytosolic calcium (figure 3.24) in a time-dependent fashion. The immunosuppressant cyclosporin A (CsA) inhibits cyclophilin D, a mitochondrial matrix peptidyl-prolyl isomerase known to modulate opening of the mitochondrial transition pore (153, 154). In addition, CsA, through pharmacological

inhibition of the serine/threonine phosphatase, calcineurin, promotes cell death in melanoma

and other solid tumors (155-157). Therefore it was conceivable to evaluate the effect of CsA

on OSW-1 mediated cytotoxicity in melanoma. Pre-incubation of WM35 cells with CsA

increased the antiproliferation effect of 0.3 nM OSW-1 from 22.0% to 73.3% (figure 3.25 A).

Similarly, combination of OSW-1 with CsA caused a 10.25-fold decrease in mitochondrial

membrane potential as compared to the DMSO control while 0.3 nM OSW-1 single agent

treatment caused a slight increase by 1.8 fold (figure 3.25 B). Interestingly, CsA+OSW-1

incubation for 24h promoted incremental VDAC1 expression in WM35 cells (figure 3.26 A).

This VDAC1 induction by CsA+OSW-1 combination was slightly higher than the effect of

OSW-1 as a single agent, while CsA by itself did not cause affect VDAC1. Further, pre-

incubation of CsA with OSW-1 potentiated higher cytosolic calcium release when compared

to the effect of CsA/OSW-1 as a single agent (figure 3.26 B, C). The cytosolic Ca 2+ levels in

WM35 cells treated with CsA+OSW-1 for 24 h increased to 229.5% while OSW-1 or CsA

alone caused Ca 2+ elevation to 200.7% and 156.2% respectively (figure 3.26 C). These

results, together with our previous findings, demonstrate that the mitochondrial outer

membrane protein, VDAC1 and other mitochondria dependent mechanisms including

superoxide release and Ca 2+ elevation play an important in leukemia cells executing the anticancer activity of OSW-1 (58, 59).

Figure 3.25:

96.5 % 91.4 % 78.0 % 26.7 %

DMSO 10 M CsA 0.3 nM OSW-1 OSW-1 + CsA

B CsA + OSW-1 CsA

OSW-1

DMSO

Figure 3.25: Combination of OSW-1 with Cyclosporin A (CsA) causes elevated cell

death effect in melanoma cells.

(A) Melanoma cells treated with the indicated concentrations of OSW-1 and CsA for 48 h,

were analyzed for cell death by annexin V/PI staining. The numbers denote percent cell

viability (annexin V negative/PI negative) for each sample. (B) Mitochondrial membrane

potential loss in cells treated with 0.3 nM OSW-1 ± 10 µM CsA for 48 h was detected by

rhodamine 123 staining.

Figure 3.26:

A Cs Cs A A O + S D O W M S -1 S W -1 O VDAC1

β-Actin

B C

* *** * CsA + * OSW-1 * * CsA

OSW-1 DMSO

Figure 3.26: Effect of OSW-1 and Cyclosporin A (CsA) drug combination on VDAC1 expression and calcium homeostasis.

(A) Immunoblot was performed in WM35 cells treated with 1 nM OSW-1 ± 2 µM CsA for

24 h. (B) and (C) Cytosolic calcium changes in A375SM and WM35 cells treated with 1 nM

OSW-1 ± 3 µM CsA for 24 h was determined. Figure (B) denotes a representative histogram of the calcium changes induced by OSW-1 in A375SM cells.

Figure 3.27:

A OSW-1 OSW-1 DMSO 0.5 12 24 36 DMSO 0.5 12 24 36 A p-ERK 1/2 p-ERK 1/2 37 5S W ERK 1/2 M ERK 1/2 M 35

β-Actin β-

B C

OSW-1 + U0126 U0126

OSW-1

DMSO

Figure 3.27: OSW-1 treatment causes elevated expression of phosphorylated ERK1/2 in melanoma.

(A) A375SM and WM35 cells were treated with 1 nM OSW-1 for the indicated time points and immunoblot was performed for total and phosphorylated-ERK1/2 proteins. (B) and (C)

A375SM cells pre-incubated in 5 µM U0126 for 1 h were treated with 1 nM OSW-1 for 48 h.

Mitochondrial membrane potential and cell viability were determined by rhodamine 123 (B) and annexin-V/PI analysis (C). Bar graphs represent the mean ± SEM measurements of three individual experiments.

Tringali et al . identified that ganglioside GM3 accumulation in K562 cells drove cell differentiation towards megakaryocytes and increased ERK1/2 phosphorylation through protein kinase C dependent cell signaling (158). Recent reports also indicate a positive correlation between GM3 accumulation and ERK1/2 activation in the brain astrocytes from a sandhoff disease mouse model (159). Further, overexpression of GD3 synthase gene inhibited PDGF-mediated ERK1/2 phosphorylation, thereby modulating cell proliferation and cell cycle progression in vascular smooth muscle cells (VSMC) cells (160). OSW-1 induced a time dependent increase in phospho-ERK1/2 expression in A375SM and WM35 cells (figure 3.27 A). We hypothesized that if ERK1/2 activation was crucial to the antitumor activity of OSW-1, then pre-incubation of melanoma cells with the MEK1/2 inhibitor, U0126 will rescue the cells from the cytotoxic effect of OSW-1. U0126 did not reverse the mitochondrial membrane potential loss triggered by OSW-1 (figure 3.27 B). Moreover, combination of OSW-1 with U0126 led to incremental inhibition of cell viability in melanoma cells (figure 3.27 C). U0126+OSW-1 combination produced 31.70% increased cell death in A375SM cells than OSW-1 single agent treatment (figure 3.27 C). Based on our results, we conclude that ERK1/2 activation is not a critical mechanism in mediating OSW-

1’s anticancer effect, and is plausibly a downstream effect of the modulation of ganglioside expression pattern by OSW-1.

3.2.7 VDAC1 is a critical player in mediating the cytotoxic effect of OSW-1

To determine if VDAC1 induction is of functional significance to OSW-1 induced

cytotoxicity, we used VDAC1 knock out (VDAC1-/-) mouse embryonic fibroblast (MEF)

cells. We performed a72 h MTT assay with MEF WT and VDAC1-/- cells treated with log

scale serial diluted concentrations of OSW-1 (figure 3.28 A). MEF VDAC1-/- cells were

significantly resistant to OSW-1 with a half-maximal inhibitory concentration approximately

44 times higher than the WT MEF cells (figure 3.28 A). Likewise MEF VDAC1-/- cells were

resistant to the loss of mitochondrial membrane potential induced by OSW-1, whereas

incubation of MEF WT cells with 1, 3 and 10 nM OSW-1 incubation for 72 h resulted a

mitochondrial collapse to 41.85%, 29.50% and 16.85% respectively (figure 3.28 B).

To characterize the role of VDAC1 in melanoma cell sensitivity to OSW-1, we

performed siRNA mediated knock down of VDAC1 expression in A375SM cells (figure 3.29

A). As shown in figure 3.29 B and C, OSW-1 induced mitochondrial membrane potential

(Ψ m) loss was substantially attenuated in siVDAC1 transfected A375SM cells. Further, a 48 h MTT assay in scrambled control and siVDAC1 transfected A375SM cells demonstrated that the siVDAC1 cells were 4-times less sensitive to OSW-1 compared to the scrambled control cells (figure 3.29 D). Interestingly the degree of resistance to OSW-1 observed in siVDAC1 transfected A375SM cells was lower than the MEF VDAC1-/- cells ((figure 3.29

D, figure 3.28 A). This may be because of the transient nature of VDAC1 knockdown and the need for A375SM cells to get acclimatized to this down regulation of VDAC1. Also, pre-

incubation of A375SM cells with the VDAC inhibitor, DIDS, did not rescue the cells from

GD2 ganglioside inhibition triggered by OSW-1 (figure 3.30), thus indicating that VDAC1

does not have a regulatory function in the ganglioside expression change stimulated by

OSW-1. Collectively, our data strongly suggest that VDAC1 plays a crucial role in mediating

OSW-1 induced cytotoxicity in melanoma cells.

Figure 3.28:

* * **

Figure 3.28: MEF VDAC1 -/- cells are resistant to the cytotoxic effects of OSW-1.

(A) MEF WT and VDAC1-/- cells were treated with log scale serial diluted concentrations of

OSW-1 for 72 h and the MTT assay was performed. IC 50 values were determined. (B)

Mitochondrial membrane potential change induced by 72 h treatment of 1, 3 and 10 nM

OSW-1 in MEF WT and VDAC1 -/- cells was determined by rhodamine 123 analysis. **

denotes p < 0.01 and * denotes p < 0.05.

Figure 3.29:

A B

- +

siVDAC1 OSW-1 OSW-1

VDAC1 DMSO DMSO β-Actin

Scrambled siVDAC

C D

p = 0.13

****

Figure 3.29: Down regulation of VDAC1 in A375SM cells confers resistance to OSW-1.

(A) Immunoblot for VDAC1 in A375SM cells transfected with siVDAC1 and negative

control. (B) and (C) Mitochondrial membrane potential analysis of siVDAC1 and negative

control transfected A375SM cells treated with 1 nM OSW -1 for 48 h. (D) A375SM WT and

VDAC1-/- cells were treated with log scale serial diluted concentrations of OSW -1 for 48 h

and the MTT assay was performed. IC 50 values were determined. *** denotes p < 0.001 .

Figure 3.30:

Figure 3.30: Combination of OSW-1 with the VDAC1 inhibitor, DIDS, indicates that

VDAC1 does not regulate ganglioside expression.

A375SM cells were treated with 1 nM OSW-1 ± 200 µM DIDS for 24 h and ganglioside

GD2 expression was determined by flow cytometry.

3.2.8 Characterization of the mechanisms underlying the therapeutic selectivity of

OSW-1

Therapeutic selectivity is a major concern while developing an anticancer agent. Our early findings indicate that OSW-1 is highly selective towards melanoma cells with limited toxicity in normal melanocytes (figure 3.9, 3.10). Hence we sought to delineate the mechanisms causal this selectivity of OSW-1 for melanoma cells. Towards this end, we first cultured WM35 cells in RPMI media supplemented with increasing concentrations of OSW-

1 for a period of 2-3 months to generate “OSW-1 resistant” WM35 cells (WM35R). An aliquot of these cells were then returned back to RPMI media without OSW-1 (WM35 drug withdrawn, WM35DW). These WM35DW cells continued to display the OSW-1 resistant phenotype, despite being cultured in non-OSW-1 growth media, and thus represent the truly

OSW-1 resistant melanoma cells. A preliminary MTT assay indicated that WM35DW and

WM35R cells were about 680 and 332 times more resistant to OSW-1 than the parental

WM35 cells (figure 3.31). Henceforth, we used this WM35 vs. WM35DW vs. WM35R cell model for our further study.

Figure 3.31:

Figure 3.31: The OSW-1 resistant WM35 cell model .

Parental WM35 and the OSW -1 resistant WM35 R and WM35 DW melanoma cells were treated with log scale serial diluted concentrations of OSW -1 for 72 h and the MTT assay was performed to determine the antiproli ferative effect of OSW-1 in these cells . The IC 50 values for OSW-1 in each cell sample were determined.

Figure 3.32:

A p = 0.08

B *

Figure 3.32: OSW-1 resistant WM35 DW cells have a decreased ganglioside GD3/GD2 expression profile compared to the parental WM35 melanoma cells.

The basal GD3 (A) and GD2 (B) expression in WM35, WM35 DW and WM35 R cells was determined by flow cytometry as described in materials and methods. * denotes p < 0.05.

Figure 3.33:

OSW-1

* DMSO

WM35 WM35 WM35 WM35 DW WM35 DW WM35 DW

Reactive Oxygen Species Superoxide Nitric Oxide

Figure 3.33: OSW-1 resistant WM35 DW cells have an altered free radical expression

profile compared to the parental WM35 melanoma cells.

(A) Basal mitochondrial superoxide levels were evaluated using the mitosox red fluorescent

indicator as described in materials and methods. Bar graphs represent mean ±SEM from three

independent experiments. (B) Basal level expression of reactive oxygen species, superoxides

and nitric oxide was determined by flow cytometric analysis of cells stained for DCFDA,

Dihydroethidium and DAF-FM fluorescent probes. * denotes p < 0.05.

A comparison of the basal GD3 and GD2 ganglioside expression indicated that

WM35DW cells have a significantly decreased GD3 (p = 0.08) and GD2 (p = 0.02) expression as compared to the parental WM35 cells (figure 3.32 A, B). But no significant change in ganglioside GM3 expression between the two cell samples was observed. Further, the basal mitochondrial superoxide levels were significantly reduced in WM35DW than the parental WM35 cells (figure 3.33 A). This correlated with decreased expression of reactive oxygen species, cytosolic superoxides and nitric oxide in the WM35DW vs. WM35 cells

(figure 3.33 B). Based on these results, we postulate that the basal ganglioside GD3/GD2 expression profile plays a critical role in governing the response of melanoma cells to the antitumor effect of OSW-1.

To explore other potential markers of sensitivity to OSW-1, we performed reverse phase protein array (RPPA) of 217 total- and phospho-proteins (figure 3.34). Of specific interest to us were lipogenesis (figure 3.35 A, B), and mitochondria (figure 3.35 A, C) signaling proteins, whose expression was statistically significantly different (p < 0.05) between WM35 vs. WM35DW vs. WM35 R cells. RPPA analysis results demonstrate that

VDAC1 (porin), Bcl-2, Mcl-1, GSK3 and PKC-α expression was reduced in WM35DW and

WM35R compared to the parental WM35 cells (figure 3.35 A, C). However cyclophilin F and phosphorylated NF-κB levels were higher in the resistant cells. Among the lipogenesis proteins, fatty acid synthase (FASN) expression was significantly increased, while caveolin-

1, and focal adhesion kinase (FAK) expression was decreased in the WM35DW and WM35R resistant cells than the parental WM35 cells (figure 3.35 A, B). Interestingly, expression levels of cell survival and proliferation proteins like PI3K, Akt and phospho-MAPK and –

MEK was lower in the resistant WM35DW and WM35R cells than the parental WM35 cells

(figure 3.34). (Log 2) Figure 3.34: -0.8955 2.0779

Figure 3.34: Differential protein expression in WM35 vs. WM35 R vs. WM35 DW cells .

Heat map generated based on the RPPA analysis of WM35 vs. WM35 DW vs. WM35 R cells for the basal expression of the proteins indicated.

Figure 3.35:

B C

Figure 3.35: Differential expression of lipogenesis and mitochondria signaling proteins

in WM35 vs. WM35 R vs. WM35 DW cells .

RPPA analysis was performed in WM35 vs. WM35 DW vs. WM35 R cells. The expression

of proteins from the lipogensis (figures A, B) and mitochondrial signaling (figure A, C)

pathways that differed significantly in WM35 vs. WM35 DW vs. WM35 R cells is indicate d

as a heat map and bar graphs.

3.3 Discussion

In this study we demonstrated that OSW-1 effectively eliminates melanoma cells by causing inhibition of gangliosides GD3/GD2 and upregulation of mitochondrial VDAC1 leading to autophagic cell death.

Burgett et al identified the lipid metabolism proteins, OSBP and ORP4L as cellular binding targets of OSW-1(69). Prior findings from our lab demonstrated that OSW-1 functions by disrupting cellular calcium homeostasis through inhibition of the Na +/Ca 2+ exchanger 1 (NCX1) leading to apoptotic cell death in leukemia cells (58). More recently,

Yamada et al ., employed a fluorescent analog to investigate the cellular internalization and localization of OSW-1 in HeLa cells (82). Microscopic imaging studies revealed that OSW-1 rapidly internalized and distributed primarily in the intracellular membranes of the endoplasmic reticulum (ER) and Golgi apparatus and to a lesser extent in the mitochondria

(82). Cumulatively, these findings suggest that a novel target for OSW-1 other than

NCX1/OSBP may exist in these subcellular sites and merits further investigation.

Gangliosides are constituents of the glycosphingolipid-enriched microdomains (GEM or rafts) in the cell membrane, where they play a pivotal role in mediating cell proliferation, invasion and adhesion signaling (17, 19). Previous investigations provide evidence that ganglioside synthesis occurs intracellularly in the golgi complex and the ‘mitochondria- associated membrane’ (MAM) compartment of the endoplasmic reticulum through a glycosylation process regulated by glycosyltransferases, including GD3 synthase, a resident

Golgi protein (161, 162). Specifically, the ganglioside GD3 plays a paradigmatic role, wherein, in contrast to its cell proliferative function, it also acts as a cell death effector

through its intracellular translocation to the mitochondria during stress conditions, leading to cytochrome c release, ROS generation and cell killing (143, 144, 146).

Our results demonstrate that OSW-1 inhibits ganglioside GD3 in melanoma cells by repressing gene expression of the enzyme, GD3 synthase (ST8SIA1 gene), which catalyzes the biosynthesis of ganglioside GD3 from its precursor GM3 by addition of a second sialic acid residue (163, 164). Consequently we observed a moderate decrease in ganglioside GD3, a more significant inhibition of its downstream product, GD2 and an accumulation of the precursor GM3 (figure 3.11). Importantly, this inhibition of ganglioside GD2 correlated with the cell death effect (represented by MMP) of OSW-1 in melanoma cells (figure 3.19).

Nevertheless, it is unresolved if GD3S is a direct target for OSW-1 in melanoma.

Interestingly, the OSW-1 structural analog, Triptolide, induced down regulation of human

GD3 synthase (hST8SIA1) gene expression through NF-κB activation in melanoma cells

(56). In addition, the transcription factor TWIST and GNE kinase are also known to positively regulate GD3S expression (165, 166). Therefore, it might be important to examine the effect of OSW-1 on signaling transduction proteins that are upstream of GD3 synthase.

Another key protein that may potentially be a cellular interacting target of OSW-1 is the steroid regulatory element binding protein (SREBP), which acts as a master transcription factor regulating cholesterol and fatty acid biosynthesis (167). Prior structure-activity relationship studies demonstrated that the steroid nucleus in OSW-1’s structure was critical to its potent cytotoxic effect (66, 72). The SREBP protein, because of its ability to bind to sterol regulatory elements (168), may form a putative binding partner of OSW-1. Further,

OSW-1 treatment inhibits expression of lipogenesis genes, FASN and HMGCR, which are downstream targets of SREBP. Cumulatively, these results point towards the possibility of

SREBP being a direct target of OSW-1 in melanoma cells. My future studies will be directed towards characterizing the molecular binding target(s) of OSW-1, with a strong focus on

SREBP. Please refer to chapter 5(summary and future directions) for a detailed description of my experimental plans for this aspect of the OSW-1 future study.VDAC1 is a multifunctional channel protein involved in mitochondria dependent cell death, redox metabolism, and cancer cell signaling. It has been implicated in cell death induction by stimuli such as cisplatin, curcumin, myostatin, UV irradiation and oxidative stress through either homo- oligomerization or interaction with Bcl-2 family proteins, adenine nucleotide translocase

(ANT), regulation of Ca 2+ and ATP flux, or ROS detoxification (169, 170). Our original results demonstrated that OSW-1 induced interaction between ganglioside GD3 and VDAC1 in the mitochondrial membranes (figure 3.17, 3.18). Hence we characterized the effect of

OSW-1 on VDAC1 expression and VDAC1 dependent cell death signaling in melanoma.

Our data indicated that OSW-1 caused VDAC1 upregulation, increased cytosolic calcium and mitochondrial superoxide release in melanoma cells (figures 3.20, 3.23, 3.24).

Interestingly, Triptolide, which inhibits GD3S, also activated VDAC1expression (figure

3.22). Nevertheless, combination of OSW-1 with the VDAC inhibitor, DIDS, did not affect

OSW-1 mediated ganglioside GD3/GD2 inhibition (figure 3.30), thus suggesting that GD3S might regulate VDAC1 expression but not vice versa. Significantly, combination of OSW-1 with the immunosuppressant drug, cyclosporin A, not only upregulated VDAC1 expression but also led to elevated cell death (figure 3. 26 A, 3.25). Considering that combination therapy with cyclosporine has been known to improve clinical efficacy in advanced malignancies, we propose that this OSW-1+CsA treatment strategy may improve the therapeutic benefit of OSW-1 in melanoma and thus merits further investigation (171, 172).

Finally, knock out or knock down of VDAC1 ameliorated the cytotoxic effect of OSW-1 in melanoma (figure 3.28, 3.29), thus corroborating our findings that VDAC1 and mitochondria dependent cell death mechanisms are crucial to the antineoplastic activity of OSW-1.

Increased expression of gangliosides GD3 and GM3 in melanoma cell lines and tissues, and a correlation between GD3 expression levels and the GD3 synthase enzymatic activity in cells has been reported (53, 54, 173). This is obvious in the comparison between melanoma cells and normal melanocytes wherein the melanocytes exhibit extremely low

GD3 synthase activity and GD3 cell surface expression (16). Based on this information, we postulated that differential ganglioside expression profile may form a basis for the therapeutic selectivity of OSW-1 towards melanoma cells with limited toxicity to normal melanocytes (figure 3.9, 3.10). To test this hypothesis, we compared the basal ganglioside expression pattern between the OSW-1 resistant WM35DW and the parental WM35 cells

(figure 3.32). Our results indicate WM35DW cells have a reduced GD3 and GD2 expression than the parental WM35 cells. A decreased free radical expression profile was also observed in the resistant WM35DW cells (figure 3.33). RPPA analysis revealed altered expression of multiple lipid metabolism proteins including FASN, YAP, FAK and caveolin-1 in WM35 vs.

WM35DW cells (figure 3.35 A, B). Differential expression of mitochondrial signaling proteins such as VDAC1, cyclophilin and various interacting partners of VDAC1 such as

Bcl-2 family members, and GSK3 was also observed (figure 3.35 A, C). Further investigation into the molecular mechanisms regulating GD3 synthase activity and the cell signaling regulating VDAC1-GD3 interaction may provide deeper insights into the therapeutic basis for the selective elimination of melanoma cells by OSW-1.

In summary, our study delineates the mechanisms essential to the highly potent anticancer activity of OSW-1 in melanoma cells. Our results demonstrate that OSW-1 induces autophagic cell death in melanoma by repressing GD3 synthase gene expression, leading to inhibition of gangliosides GD3, GD2 and accumulation of GM3. Ganglioside GD3 then interacts with the mitochondrial VDAC1 leading to mitochondrial membrane potential collapse, aberrant calcium signaling and cell death. VDAC1 is a critical player in mediating

OSW-1’s activity as its down regulation confers resistance to OSW-1. A significant characteristic of OSW-1 induced cytotoxicity is its therapeutic selectivity towards melanoma cells. Our investigation reveals that elevated ganglioside expression in melanoma may form the basis for the increased sensitivity of these cells towards OSW-1. To our knowledge, this is the first study to demonstrate the antitumor effect of OSW-1 on ganglioside expression and

VDAC1 mediated mitochondrial signaling in melanoma. The role of OSW-1 as an inhibitor of GD3 synthase gene expression is crucial, since in melanoma ganglioside GD3 is a well characterized therapeutic target. Therefore, further research needs to be undertaken to shed light on the mechanisms regulating this GD3 synthase inhibition by OSW-1.

Chapter 4: PEITC effectively eliminates melanoma cells through redox mechanisms

4.1: Introduction

PEITC is a highly active metabolite of cruciferous vegetables with potent chemopreventive and chemotherapeutic properties. It inhibits expression of pro-oncogenic proteins such as Bcl-2, Mcl1 and Bcl-xL, triggers cell cycle arrest, DNA and mitochondrial damage leading to caspase activation, cytochrome c release and apoptosis (95, 98).

PEITC exhibits a unique mechanism of cell death – it induces excessive ROS accumulation in cancer cells leading to massive cytotoxicity (63, 64, 94). This ROS induced toxicity by PEITC is specific to tumor cells; normal melanocytes with a lower basal oxidative stress phenotype and reserve antioxidant capacity are resistant to PEITC and other pro- oxidant compounds (63).

In this dissertation, I investigated the mechanisms of cytotoxic action and therapeutic selectivity of PEITC in melanoma cells. I aimed to characterize the differences in redox status between the normal cells and cancer cells and use this as a mechanistic basis to specifically eliminate melanoma cells. My results demonstrate the PEITC depletes cellular glutathione in melanoma cells leading to ROS stress, loss of mitochondrial membrane potential and cell death through apoptosis. Further, a positive correlation between the basal cellular ROS expression levels and sensitivity to PEITC was observed. Accordingly, normal melanocytes which have a much lower intrinsic oxidative stress compared to melanoma cells are resistant to PEITC. The glutathione depletion mediated by PEITC is an early event which occurs much before cell death and is crucial to the cytotoxic effect of PEITC in melanoma.

Due to this distinctive mechanism of cell death, PEITC is effective in attenuating drug resistance to clinically used melanoma agents.

4.2 Results 4.2.1 Redox status of melanoma cells

A characteristic feature of melanoma biology is the presence of aberrant melanosomes in melanoma cells that generate free radicals (174). Chronic oxidative stress potentiated by oncogenic Akt signaling or loss of tumor suppressors such as p53 and p16, in combination with exposure to ultraviolet radiations (UV), UVA and UVB, may lead to oncogenic transformation of dysplastic nevi and development of an intrinsic drug resistant phenotype in melanoma cells (44, 62, 175-177). On the other hand, excessive reactive oxygen species (ROS) accumulation in melanoma cells leads to massive toxicity and cell death, thus making it a unique therapeutic target. Hence, I hypothesized that melanoma cells are under elevated intrinsic oxidative stress and can be preferentially eliminated by pharmacological agents such as PEITC which modulate the cellular redox homeostasis.

To investigate this hypothesis, we first characterized the basal redox status of melanoma cells. The intracellular free radical expression levels were determined across a panel of melanoma cells by flow cytometric analysis using the fluorescent reagents, CM-H2-

DCFDA, DAF-FM and dihydroethidium. 5-,6-Chloromethyl-2’7’-

Dichlorodihydrofluorescein Diacetate (CM-H2-DCFDA) is an indicator of hydroxyl radicals, peroxides and other ROS molecules in cells. DCFDA passively diffuses into the cells, where its acetate groups are cleaved by intracellular esterases and the compound is subsequently oxidized to yield the fluorescent 2’7’-Dichlorofluorescein (DCF), which can be detected by flow cytometry (178). 4-Amino-5-Methylamino-2', 7’-Difluorofluorescein (DAF-FM) detects nitric oxide (NO) in cells. The non-fluorescent DAF-FM reacts with intracellular NO and is converted into the fluorescent benzotriazole molecule which can be quantified (179).

Dihydroethidium (Het) is a blue fluorescent dye which is oxidized by superoxides to form

oxiethidium, which then intercalates with nucleic acids to emit a bright red fluorescence that can be detected by flow cytometry or microscopy (180).

Our results indicated that Hs294T (BRAF WT, metastatic) melanoma cells had the highest expression of basal ROS and nitric oxide (figure 4.1 A, B, D). Superoxide levels were elevated across the melanoma cells evaluated in this study. Overall, SK-MEL-2 (BRAF

WT, NRAS Q617R, metastatic) cells contained relatively higher free radical expression among the melanoma cells tested (figure 4.1). The A375SM (BRAF V600E, NRAS WT, highly metastatic) cells had a moderately decreased free radical expression profile compared to the other melanoma cells (figure 4.1). Hence, SK-MEL-2 and A375SM cells were used as representative cell line models in the follow-up mechanistic studies.

Figure 4.1:

A B MEWO A375SM WM46 WM35 WM46 MEL624 WM35 MEWO MEL624 UCSD-354L SK-MEL-2 SK-MEL-2 A375SM UCSD-354L Hs294T Hs294T

D - C Basal ROS Basal NO Basal O 2 HS294T Cell Lines (mean (mean (mean count) count) count) MEL624 SK-MEL-2 A375SM A375SM 33.250 29.750 88.700 WM4 WM35 WM35 38.150 37.050 101.350

UCSD- SK-MEL-2 184.900 54.200 150.450 MEW

WM46 19.100 58.050 157.000

UCSD-354L 53.950 53.800 130.000

HS294T 419.000 133.600 58.000

MEL624 39.550 80.300 169.000

MEWO 39.200 30.400 86.700

Figure 4.1: Basal free radical expression profile in melanoma cells.

(A), (B), (C) Melanoma cells were plated in 6-well plates for 24 h prior to the measurement

of cellular reactive oxygen species, nitric oxide and superoxides using the fluorescent probes

DCFDA, DAF-FM and Dihydroethidium by flow cytometry. (D) Mean count of the basal

reactive oxygen species, nitric oxide and superoxide expression levels in melanoma cells.

4.2.2 The cytotoxic effect of PEITC in melanoma

Earlier studies from our lab demonstrated that the natural product, PEITC was effective in inhibiting leukemia cells including fludarabine resistant chronic lymphocytic leukemia and Gleevec-resistant chronic myelogenous leukemia (63, 102). To investigate the effect of PEITC on melanoma cell viability, I performed an MTT assay across a panel of cell lines treated with varying concentrations of PEITC for 72 h. As illustrated in figure 4.2,

PEITC was cytotoxic to melanoma cells with an average half-maximal inhibitory concentration (IC 50 ) of 2.21 µM. Interestingly, WM46 cells which have the lowest levels of basal ROS expression amongst all the melanoma cells tested (figure 4.1D), also have the highest IC 50 value (4.10 µM; figure 4.2B).

Biochemical analysis by annexin V-FITC/propidium iodide staining indicated that

PEITC induced apoptosis in melanoma cells in a time dependent fashion (figure 4.3A, B).

PEITC induced cell death was detectable at 12 h and reached over 70% by 24 h in A375SM and SK-MEL-2 cells (figure 4.3A). The bar graphs in figure 4.3B represent the heterogeneity of melanoma cell response to PEITC. On average, a 24 h PEITC treatment caused 55.73%

(DMSO vs. PEITC 24h, p-value < 0.0001) cell death in melanoma cells. Significantly, combination of PEITC with the pan caspase inhibitor, Z-VAD-FMK, partially rescued melanoma cells from the cytotoxic effect of PEITC (figure 4.3C). Caspases are a group of intracellular proteases that play a pivotal role during apoptosis by catalyzing the disassembly of cells into membrane-enclosed vesicles (apoptotic bodies), ultimately leading to death

(181). Treatment of SK-MEL-2 cells with 5 µM PEITC induced 71.74% cell killing.

However, co-treatment of cells with 20 µM Z-VAD-FMK and 5 µM PEITC reduced cell

death to 30.23% (figure 4.3C). These results delineate the role of caspase activation and

apoptosis in mediating the inhibitory activity of PEITC.

Figure 4.2:

Cell line IC Value Mean ± SEM 50 B (µM) (µM)

A375SM 2.42 ± 0.179

WM35 1.52 ± 0.327

SK-MEL-2 2.70 ± 0.418 2.206 ± 0.362 WM46 4.10 ± 0.618

UCSD354L 2.18 ± 0.219

HS294T 1.00 ± 0.140 MEL624 2.67 ± 0.295

MEWO 1.06 ± 0.241

Figure 4.2: Effect of PEITC on melanoma cell viability

Melanoma cells were treated with log-scale serial diluted concentrations of PEITC for 72 h,

and the cytotoxic effect was determined by performing the MTT assay. The cell viability

curves were plotted. Each data point represents mean ± SEM for three independent

measurements. (B) The 50% cell inhibitory concentrations (IC 50 ) of PEITC for each cell line

are indicated.

Figure 4.3: A 10 µM PEITC

DMSO 6 h 12 h 24 h

Pr A375SM op 91.8% 84.4% 71% idi 23% u m io di SK-MEL-2de

94.2% 75.5% 60.7% 21.3%

Annexin V-FITC

* P=0.180 P=0.057 P=0.069 * * P=0.088 * * P=0.173 P=0.198 P=0.242 P=0.201 * ** P=0.128 * * **** ** * P=0.065 * ***

* * ** **** **

***

91.3% 93.4% 25.8% 63.7%

DMSO 20 µM Z-VAD-FMK 5 µM PEITC PEITC + Z-VAD-FMK

100

Figure 4.3: PEITC induced cytotoxicity is through apoptosis in melanoma cells

(A) SK-MEl-2 and A375SM cells were treated with 10 µM PEITC for the indicated time points and PEITC induced cell death induced was detected by annexin V-FITC/PI staining and flow cytometric analysis as described in materials and methods. (B) Percent cell viability of melanoma cells treated with 10 µM PEITC for the indicated time points. Bar graphs represent mean ± SEM of three independent measurements. (C) SK-MEL-2 cells incubated in 20 µM Z-VAD-FMK for 1 h were treated with 5 µM PEITC for 24 h and cell death was measured by flow cytometric analysis (annexin V-FITC/PI staining). **** denotes p <

0.0001, *** denotes p < 0.001, ** denotes p < 0.01 and * denotes p< 0.05.

101

Mitochondrial dysfunction constitutes an important event in the induction of cell death. Opening of the mitochondrial transition pore leading to depolarization of membrane potential, release of apoptogenic proteins and uncoupling of oxidative phosphorylation have been linked to apoptosis stimulation by agents such as tert-Butylhydroperoxide, the calcium ionophore A23187, selenite, sulforaphane etc. (182-186). To test if PEITC caused mitochondrial membrane damage in melanoma, I treated the cells with 5 µM PEITC for varying time points and the effect on mitochondrial membrane integrity was assessed by flow cytometric analysis of cells labeled with Rhodamine 123, a lipophilic, cationic fluorescent dye that is readily taken up by active mitochondria. Depolarization of the mitochondrial membrane, therefore, decreases the mitochondrial rhodamine 123 signal, which can be quantified (187). My results indicate that PEITC potentiated a time-dependent mitochondrial membrane potential collapse in SK-MEL-2 and A375SM cells (figure 4.4). Mitochondrial membrane permeabilization was first observed at 16 h and by 24 h there was a massive (over

90%) loss of membrane potential in PEITC treated cells as compared to the DMSO treated controls (DMSO vs. PEITC 24h, p-value < 0.0001).

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Figure 4.4:

24 h 6 24 h 16 h 16 h

6 h DMSO DMSO

SK-MEL-2 A375SM

Figure 4.4: PEITC induces mitochondrial membrane potential loss in melanoma cells.

A375SM and SK-MEL-2 cells were treated with 5 µM PEITC for the indicated time points and the effect on mitochondrial membrane potential was evaluated by flow cytometric analysis of Rhodamine 123 labelled cells.

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Figure 4.5:

100 r = 0.8068 P = 0.0155 80

40 % Cell% death 20

0 0 100 200 300 400 500 Cellular ROS

Figure 4.5: Correlation between cellular basal ROS levels and cell death induced by

PEITC.

Cellular intrinsic ROS expression levels were measured by performing the DCFDA assay.

Cell death was detected by annexin V-FITC/PI assay in melanoma cells treated with 10 µM

PEITC for 24 h. Linear regression analysis between these two parameters was performed using the mean ± SEM measurements of three independent experiments.

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Figure 4.6:

PEITC

DMSO 6 h 12 h 24 h Phospho-JNK

JNK

Phospho-p38

p38

β-Actin

Figure 4.6: Effect of PEITC on JNK and p38 expression in melanoma.

SK-MEL-2 cells were treated with 5 µM PEITC for the indicated time points and the effect

on JNK and p38 kinase expression was assessed by western blotting.

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Quantitative evaluation of the relationship between basal cellular ROS levels and

PEITC induced cell death revealed a strong correlation between these two parameters (r =

0.8068, p-value = 0.0155) (figure 4.5). ROS have been known to promote apoptotic signaling

by mediating the activation of pro-apoptotic JNK and ASK1 kinases, leading to upregulation

of TNF α, Fas-L and Bak, release of cytochrome C, and mitochondrial translocation of Bax and Bad apoptotic proteins (188-191). Interestingly, western blot analysis indicated that

PEITC stimulated a time-dependent increase in phosphorylated-JNK expression in SK-

MEL2 cells (figure 4.6). However, no significant change was observed in the expression of total JNK as well as total and phosphorylated p38 MAP kinase (figure 4.6). Collectively these results indicate that PEITC executes its anticancer effect through apoptosis and ROS mediated mechanisms in melanoma cells. Of significance is the observation that the basal

ROS status of melanoma cells is proportional to its sensitivity to PEITC.

4.2.3 PEITC exhibits minimal toxicity in normal melanocytes

The promising cytotoxic activity of PEITC in melanoma cells led us to investigate the effect of this compound on normal melanocyte cell viability. As shown in figure 4.7, normal melanocytes were significantly less sensitive to PEITC compared to melanoma cells.

Annexin-FITC/PI analysis demonstrated that a 24 h PEITC treatment caused 60% cell death in melanoma cells (n = 9), while 94.73% of normal melanocytes were still viable at this time point (figure 4.7B). Interestingly, PEITC stimulated massive cytotoxicity in HS294T (figure

4.7A) and SK-MEL-2 (figure 4.3A) melanoma cells, which have a relatively higher ROS capacity. Previous research findings have shown that PEITC mediated cell death is characterized by ROS production either though depletion of the glutathione antioxidant system or inhibition of oxidative phosphorylation in leukemia, ovarian and prostate cancers

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(63, 64, 113). This led me to speculate that the anticancer effect of PEITC relied on disrupting on the cellular redox homeostasis leading to ROS accumulation and subsequent toxicity. As expected, incubation of SK-MEL-2 cells with 5 µM PEITC for 8 h resulted in substantial ROS accumulation, whereas only a slight increase in ROS levels was observed in normal melanocytes (figure 4.8). These results suggest that distinct redox regulatory mechanisms exist between melanoma cells and normal melanocytes and these may form the basis for their differential response to PEITC.

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Figure 4.7: DMSO PEITC 24 A

NHEM 90.2% 84.1%

HS294T

94.0% 9.63%

B **

Figure 4.7: Preferential killing of melanoma cells by PEITC

(A) Cell death induced by 10 µM PEITC in melanoma cells and normal melanocytes was determined by flow cytometric analysis of annexin V-FITC/PI stained cells. Representative dot blots are shown here. (B) Bar graphs illustrate the quantitative comparison of PEITC triggered cell death (normalized to DMSO control) in melanoma (n = 9) and normal melanocyte cells at 24 h treatment. Mean ± SEM of cell death measurements from three independent experiments of each melanoma cell line and normal melanocytes is denoted here. ** denotes p < 0.01.

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Figure 4.8:

NHEM SK-MEL-2

8 h 8 h

DMSO DMSO

Figure 4.8: Induction of ROS accumulation in melanoma cells and normal melanocytes by PEITC.

SK-MEL-2 cells and normal melanocytes were treated with 5 µM PEITC for 8 h and ROS levels were detected by flow cytometric analysis of cells labelled with 1 µM DCFDA.

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Figure 4.9:

* A B

SK-MEL-2

NHEM

C D **

SK-MEL-2

NHEM

Figure 4.9: Comparison of ROS expression between melanoma cells and normal

melanocytes.

(A) and (C) Flow cytometric analysis to detect ROS (DCFDA) and specifically

superoxides (Dihydroethidium) was performed in SK-MEL-2 and normal melanocytes

(NHEM) stained with 1 µM DCFDA and Dihydroethidium. (B) and (D) Bar graphs

represent the mean ± SEM of ROS and superoxide measurements from three independent

experiments of each melanoma cell line (n = 8) and normal melanocytes. ** denotes p <

0.01 and * denotes p< 0.05.

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The preferential ROS accumulation in melanoma cells treated with PEITC suggested that these cells were under increased intrinsic oxidative stress thus making them more vulnerable to further free radical elevation and redox disturbance by PEITC. To evaluate this observation, I compared the basal ROS and specifically superoxide expression levels between melanoma cells and normal melanocytes. As illustrated in figure 4.9 A and B, melanoma cells exhibit increased ROS expression compared to normal melanocytes (P-value

< 0.05). SK-MEL-2 cells had a 2.3 fold increased intrinsic ROS expression compared to normal melanocytes (figure 4.9A). Importantly, the intrinsic superoxide levels were 3-fold higher (p-value < 0.01) in melanoma cells than the normal melanocytes (figure 4.9D). A representative histogram indicating the differential superoxide expression in melanoma cells is shown in figure 4.9C. Based on these results, I concluded that the higher intrinsic oxidative stress observed in melanoma cells was important in modulating their sensitivity to PEITC.

4.2.4 The ROS mediated mechanisms of cell death by PEITC

Treatment of SK-MEL-2 and A375SM cells with 5 µM PEITC produced a time dependent increase in cellular ROS levels (figure 4.10B). A 6 h PEITC incubation of SK-

MEL-2 cells increased cellular ROS levels by 3.2-fold (figure 4.10A). In contrast, PEITC (10

µM, 8 h) did not induce any significant ROS change in normal melanocytes (figure 4.8), suggesting that this ROS elevation rendered the melanoma cells susceptible to PEITC’s cytotoxic activity. Interestingly, flow cytometric analysis of melanoma cells stained with

Dihydroethidium indicated that PEITC treatment did not generate any significant change in the cellular superoxides (O 2¯ ) (figure 4.10D), although the intrinsic superoxide expression was significantly increased in melanoma cells compared to normal melanocytes (figure 4.9

C, D). PEITC stimulated a time dependent increase in nitric oxide levels (figure 4.10C).

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Figure 4.10:

A B ** ** * * 6 h ** *** P=0.162 4 h P=0.321 2 h 1 h

DMSO

C D 6 h

4 h 4 h 2 h 6 h 1 h 2 h

1 h DMSO DMSO

Figure 4.10: Effective killing of melanoma cells through ROS induced damage

(A), (C) and (D): SK-MEL-2 cells were treated with 5µM PEITC for the indicated time points and alterations in ROS, nitric oxide and superoxide expression in cells was assessed by DCFDA, DAF-FM and dihydroethidium staining. (B) A375SM and SK-MEL-2 cells were treated with 10µM PEITC for a varying time course and DCFDA assay was performed. *** denotes p < 0.001, ** denotes p < 0.01 and * denotes p< 0.05.

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Cumulatively, these results suggest that PEITC induced ROS may include other molecules such as hydrogen peroxide, hydroxyl radical and nitric oxide but not superoxides.

This data corroborates with previous findings from our lab in oncogenic Ras transformed ovarian epithelial cells (T72 Ras) where PEITC induced ROS and NO generation, without any significant changes in O 2¯ (113). Further, the relationship between ROS accretion and

PEITC mediated toxicity was quantitatively evaluated by plotting PEITC induced cell death as a function of ROS increase (figure 4.11). A positive correlation (r = 0.7357, p-value <

0.05) between these two factors was observed.

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Figure 4.11:

r = 0.7357 P = 0.0153

Figure 4.11: Correlation between cell death and PEITC induced ROS stress

Cell death was detected by annexin V-FITC/PI assay in melanoma cells treated with 10 µM

PEITC for 24 h. ROS expression levels in PEITC treated melanoma cells were measured by

DCFDA assay. Cell death was plotted as a function of ROS fold change and linear regression analysis was performed.

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Since PEITC triggered cell death was characterized by ROS accumulation in cells, I next investigated the potential cause for this ROS stimulation by PEITC. Glutathione is a major antioxidant which has been shown to promote metastatic melanoma cell survival in the hepatic microvasculature by protecting the cells against oxidative stress induced damage

(192). Importantly, glutathione depletion either by pharmacological intervention using L-

Buthionine (S, R)-Sulphoximine (BSO) or by dietary modulation using an L-glutamine enriched diet reduced melanoma tumor growth and sensitized cells to combination therapy

(192, 193). Further, studies by Trachootham et al., and Lam et al., indicated that PEITC scavenges glutathione in leukemia and breast cancer cells leading to massive cytotoxicity

(93, 113). I postulated that the high ROS capacity in malignant melanoma cells would make them dependent upon the glutathione antioxidant system to maintain redox balance in cells, and therefore depletion of glutathione by PEITC would disrupt the redox homeostasis resulting in excessive ROS accumulation, beyond the cell’s tolerance threshold, and subsequent cell death. To examine this hypothesis, I treated SK-MEL-2 and A375SM cells with 5 µM PEITC for 3 and 6 h and detected the cellular glutathione levels (figure 4.12).

PEITC induced inhibition of glutathione was detected at 3 h and by 6 h about 60% of cellular glutathione was depleted. It is important to note that PEITC treatment exhibits limited cytotoxicity at these time durations (figure 4.3B), suggesting that this glutathione depletion may be a primary event that potentiates ROS accumulation and consequent cell death. To verify this possibility, I treated SK-MEL-2 and A375SM cells pre-incubated in the glutathione precursor, N-Acetyl Cysteine (NAC) with PEITC (figure 4.13). As shown in figure 4.13 A and B, NAC reverses the ROS elevation caused by PEITC. More importantly, combination of NAC with PEITC completely abrogates the cytotoxic effect of PEITC in SK-

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MEL-2 cells (figure 4.13C). Hence glutathione depletion is an important mechanism in

PEITC induced ROS increase and cell death.

Figure 4.12:

P=0.189

** **

Figure 4.12: PEITC causes depletion of cellular glutathione.

A375SM and SK-MEL-2 cells were treated with 5 µM PEITC for the indicated time points and cellular glutathione levels were detected by spectrophotometric analysis as described in materials and methods. ** denotes a statistical significance of p < 0.01 between the DMSO control and PEITC treated samples in each of the respective cell lines .

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Figure 4.13:

A B ***

PEITC+NAC ****

NAC

PEITC DMSO

92.1% 49.0%

DMSO 1 mM NAC

90.8% 87.2%

5 M PEITC NAC+PEITC

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Figure 4.13: N-Acetyl Cysteine (NAC) reverses the inhibitory effect of PEITC

(A) SK-MEL-2 cells incubated in 1 mM NAC for 2 h were treated with 5 µM PEITC for 6 h and ROS were detected by the DCFDA assay. (B) Bar graphs denote the mean ± SEM of three independent measurements of ROS in A375SM and SK-MEL-2 cells treated with

PEITC ± NAC. (C) Annexin V-FITC/PI assay was performed to detect the effect on cell death in SK-MEL-2 cells treated with PEITC ± NAC for 24 h. **** denotes p < 0.0001 and

*** denotes p < 0.001.

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4.2.5 Combination of Temozolomide / BCNU with PEITC attenuates drug resistance to

these agents in melanoma

Malignant melanomas are characterized by significant drug resistance to currently

used chemotherapeutic agents such as Temozolomide (TMZ) and 1,3-bis-(2-chloroethyl)-1-

nitrosourea (BCNU). Interestingly, BCNU has been shown to inhibit glutathione reductase

activity in erythrocytes from patients as well as in mouse liver, lung and heart tissues (194,

195). Further, Kohsaka et al . demonstrated that combination of TMZ with the glutathione synthesis inhibitor, BSO, potentiated the toxic effect of Temozolomide in TMZ resistant glioblastoma cells (196). Based on this information, I postulated that combination of PEITC with either BCNU or TMZ would cause incremental oxidative stress due to an enhanced depletion of cellular glutathione. This would lead to significant toxicity in melanoma cells and would be of therapeutic benefit. Towards this end, I first performed annexin V-FITC/PI assay to examine the effect of PEITC ± TMZ/BCNU combination on cell death in melanoma cells (figure 4.14). The results indicate that supplementation of PEITC with either BCNU or

TMZ promoted significantly higher inhibitory effect than that of any (PEITC/TMZ/BCNU) single agent treatment. The cytotoxic effect of this drug combination was evaluated by performing the colony formation assay. As illustrated in figure 4.15, PEITC, BCNU or TMZ as a single agent moderately inhibited colony formation (figure 4.15 A, C). However, combination of TMZ/BCNU with PEITC caused a striking decrease in the number of colonies formed. For instance, PEITC+BCNU treatment potentiated 3.5-fold decrease in the number of colonies as compared to PEITC single agent treatment (figure 4.15B).

Furthermore, combination index (CI) analysis using the Chou-Talalay algorithm was performed to characterize the type of drug interaction (figure 4.16). SK-MEL-2 cells were

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incubated with varying combination regimens of PEITC (5 µM, 10 µM and 20 µM; last 24 h) and BCNU (50 µM, 100 µM and 200 µM; 48 h) or TMZ (0.3 mM, 0.6 mM and 1 mM; 48 h).

As described in figure 14.6B, the combination of PEITC with TMZ was synergistic (CI<1.0).

Similarly, BCNU and PEITC combination index analysis mostly produced synergistic and additive (CI=1) end points (figure 4.16D).

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Figure 4.14:

A B P=0.113 P=0.231 * * P=0.157 ** * **** * * *** * ** *** ** *** ** ** **** ****

Figure 4.14: Effect of PEITC ± TMZ/BCNU treatment on melanoma cell viability

SK-MEL-2 and A375SM cells were incubated in varying concentrations of BCNU and TMZ

for 48 h. During the last 24 h of treatment 5 µM PEITC was added and the effect of the drug

combination on cell death was determined by annexin V-FITC/propidium iodide staining.

Bar graphs represent the percentage of viable cells in each drug treatment sample as a

function of the DMSO treated control cell viability. **** denotes p < 0.0001, *** denotes p

< 0.001, ** denotes p < 0.01 and * denotes p< 0.05.

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Figure 4.15:

A B

* **

***

DMSO M

M M PEIT C 5 50 BCN U PEIT C+B CNU

C D

DMSO M

PEIT C 5 0.3 mM TMZ PEIT C+ TMZ

Figure 4.15: Combination of Temozolomide / BCNU with PEITC inhibits colony formation in melanoma cells

(A) and (C) A375P cells were treated with the indicated concentrations of PEITC and

BCNU/TMZ for 2 weeks and the effect of the drug combination on colony formation was determined by Giemsa staining. (B) and (D) The colonies formed were counted manually and have been represented as bar graphs. *** denotes p < 0.001, ** denotes p < 0.01 and * denotes p< 0.05.

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Figure 4.16:

A B

50.5% 90.2%

DMSO 0.3 mM TMZ

43.4% 61.8%

5 M PEITC TMZ+PEITC C D

DMSO 50 M BCNU

5 M PEITC BCNU+PEITC

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Figure 4.16: Combination index analysis of PEITC ± TMZ/BCNU treatment

(A ) and (C) Representative dot blots indicating cell death induced by TMZ ± PEITC and

BCNU ± PEITC 48 h treatment in SK-MEL-2 cells. PEITC was added during the last 24 h of treatment. (B) and (D) SK-MEL-2 cells were treated with the indicated concentrations of

PEITC ± TMZ/BCNU for 48 h, and combination index (CI) analysis was performed using

Compusyn software. The following quantitative definitions, based on the Chou-Talalay theorem, were employed to define combination effect: CI < 1 represents synergism, CI = 1 represents an additive effect, and CI > 1 represents antagonism.

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Figure 4.17:

A B C PEITC+TMZ PEITC+TMZ PEITC+TMZ

TMZ TMZ DMSO TMZ PEITC PEITC DMSO DMSO PEITC

D P=0.621 *

Figure 4.17: Redox mechanisms mediate the combinatorial cell death effect of TMZ/

BCNU ± PEITC treatment in melanoma.

(A) Melanoma cells were treated with 0.5 mM TMZ for 24 h. 5 µM PEITC was added during

the last 6 h of treatment. ROS, nitric oxide and mitochondrial superoxides were measured by

DCFDA, DAF-FM and mitosox red staining and flow cytometric analysis. (B) 5 µM PEITC

was added during the last 3h of a 24 h 100 µM BCNU or 0.5mM TMZ treatment. Bar graphs

represent mean ± SEM of cellular glutathione measurements for the indicated drug

concentrations in three independent experiments. * denotes p< 0.05.

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To delineate the mechanisms that are pivotal to this increased cytotoxicity of

PEITC+TMZ/BCNU drug combination in melanoma cells, we analyzed for changes in redox expression in cells treated with PEITC ± TMZ/BCNU. Co-treatment of PEITC (5 µM, 6 h) with TMZ (0.5 mM, 24 h) led to increased ROS and nitric oxide generation as compared to the effect of PEITC as a single agent (figure 4.17A, B). Also, Temozolomide, by itself, did not induce any significant changes in ROS. Further, this combination (PEITC+TMZ) also stimulated a slightly increased generation of mitochondrial superoxides as detected by the mitosox red indicator (figure 4.17C). Importantly, this ROS increase was preceded by glutathione depletion in SK-MEL-2 cells. Addition of 5 µM PEITC to melanoma cells pre- incubated in BCNU or TMZ led to inhibited cellular glutathione. The glutathione depletion was higher in combinatorial treatment of PEITC+BCNU/TMZ as compared to a single agent.

Thus, glutathione depletion forms a primary event in attenuating drug resistance to TMZ or

BCNU in melanoma using the redox modulating agent, PEITC.

In conclusion, the natural product PEITC exhibits significant anticancer activity in melanoma. Its inhibitory effects in melanoma are mediated by ROS accumulation and disabling of the glutathione antioxidant system.

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4.3 Discussion

Reactive oxygen species have been long known to promote melanoma development, metabolism and metastasis (38). However, the utility of ROS as a therapeutic target is yet to be characterized. Earlier studies in our lab revealed that pharmacologically induced ROS accumulation effectively eliminated cancer cells (61, 113). In this study, I demonstrate that the redox modulatory agent, PEITC selectively kills melanoma cells based on their differential intrinsic oxidative stress status as compared to normal melanocytes. Melanoma cells are under an elevated ROS stress and therefore, are dependent upon antioxidants to maintain redox balance. This makes them vulnerable to therapeutic agents that impair the cellular antioxidant system, leading to ROS accumulation and massive cytotoxicity. Normal melanocytes, on the other hand, are less susceptible to this strategy since they have a lower basal ROS expression and so any additional oxidative stress induced by pharmacological agents is still within the cells’ tolerance threshold. This biochemical difference between normal and cancer cells may be used to selectively eliminate melanomas.

The key conclusions from this study are: (a) PEITC is highly cytotoxic to melanoma cells with limited toxicity in normal melanocytes (figure 4.7). (b) Melanoma cells are under higher intrinsic oxidative stress compared to normal melanocytes (figure 4.9). Basal ROS expression levels of melanoma cells positively correlate with their sensitivity to PEITC

(figure 4.5). (c) The elevated oxidative stress renders melanomas sensitive to PEITC which depletes the antioxidant glutathione, leading to severe ROS accumulation, mitochondrial membrane damage, activation of JNK kinase and rapid apoptotic cell death (figure 4.3, 4.4,

4.6, 4.10, 4.12). In contrast, PEITC causes modest ROS increase in normal melanocytes, which is not significant enough to promote cell death (figure 4.8). Consequently, these cells

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are resistant to PEITC. (d) Inhibition of cellular glutathione by PEITC is a critical event since the glutathione precursor, NAC, reverses ROS accumulation as well as PEITC mediated cell death (figure 4.13). Furthermore, glutathione depletion can be detected much earlier than cell death suggesting that it is a primary event that instigates ROS elevation and PEITC induced toxicity in melanoma (figure 4.12). (e) Combination of PEITC with BCNU/TMZ ameliorates drug resistance to these agents in melanoma (figure 4.14, 4.16). Higher glutathione depletion,

ROS accumulation and consequent cell death is observed in the samples treated with

PEITC+BCNU/TMZ as compared to single agent treated samples (figure 4.17).

Trachootham et al . and Xu et al . demonstrated that PEITC disabled glutathione in leukemia cells by two mechanisms: conjugating with glutathione, thus promoting its export from the cells and inhibiting Glutathione Peroxidase (GPX) activity (100, 113). Interestingly, incubation of cells with 5 – 10 µM PEITC caused inhibition of glutathione in the mM range

(113). A possible explanation for this stochiometric discrepancy may be that PEITC is concentrated at higher concentrations in cells. Indeed, incubation of the oncogenic T72Ras cells with 5 - 10 µM PEITC for 2 h led to intracellular concentrations of 0.25 – 0.59 mM.

Subsequent biochemical analysis indicated that 0.5 mM PEITC suppressed GPX activity by over 90% in a cell-free enzyme assay. These data suggest that PEITC at µM concentrations, used in cell culture assays, may deplete the cellular glutathione pool and GPX enzyme activity significantly leading to ROS accumulation and cytotoxicity. PEITC mediated inhibition of oxidative phosphorylation and mitochondrial damage leading to ROS generation in leukemia and prostate cancer has also been reported (64, 94). Furthermore, PEITC potentiated activation of JNK kinase in DU145 prostate cancer cells (197). Interestingly, this

PEITC-induced apoptosis was attenuated by pharmacological inhibition of JNKs with

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SP600125 suggesting that JNK activation was critical to PEITC’s activity in these cells

(197). Stress induced activation of ERK1/2 was also observed. JNK proteins play a crucial

role in regulating cytochrome C release and apoptosis induction following UV irradiation in

MEF cells (198). JNK kinases have also been reported to activate apoptotic signaling by

modulating the expression of pro-apoptotic and anti-apoptotic mitochondrial proteins (188,

199, 200). PEITC causes a time dependent increased JNK phosphorylation in melanoma cells

(figure 4.6). Considering that PEITC triggers apoptosis through mitochondrial membrane

permeabilization and caspase activation (figure 4.3, 4.4), it is plausible that JNK proteins

may be instrumental in mediating this cell death process. Further studies, using either

inhibitors or siRNAs to downregulate JNK expression need to be undertaken to delineate the

role of JNK in effecting PEITC’s cytotoxic activity in melanoma cells.

PEITC potentiated ROS accumulation in cells was detected by DCFDA and DAF-FM

fluorescent probes but not by HEt suggesting that the free radicals activated by PEITC were

mostly peroxides, hydroxyl radicals and nitric oxide but not superoxides (figure 4.10).

Nevertheless, the basal superoxide expression in melanoma cells is significantly higher than

normal melanocytes (figure 4.9C, D). This may be because of the rapid conversion of

superoxides to hydrogen peroxide, peroxynitrites etc (201, 202).

A recent study by Vazquez et al . (2013), demonstrated that the transcriptional coactivator, PGC1 α, promoted increased mitochondrial energy metabolism and ROS detoxification capacities in melanoma (203). Consequently, depletion of PGC1 α in these cells sensitized them to ROS-inducing drugs such as PEITC and Piperlongumine (203).

These results suggest that redox modulation therapies might be especially useful for the subset of melanomas which are PGC1 α negative. Further, combining PGC1 α inhibition with

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ROS accretion (using PEITC or other ROS inducing agents) might be another strategy to

increase drug sensitivity in PGC1 α positive tumors. The mutant BRAF inhibitor,

Vemurafenib promoted mitochondrial respiration and elevated ROS stress in BRAF V600E

melanoma cells irrespective of their PGC1 α status (204). Further, vemurafenib-resistant cells, generated by treating the parental A375, SKMEL28 and WM9 melanoma cells with increasing concentrations of vemurafenib, exhibited high mitochondrial activity and intrinsic oxidative stress compared to the parental cells (204). Importantly, the vemurafenib-resistant cells were more sensitive to pro-oxidative drugs such as Elesclomal and PEITC than the parental cells (204). Thus redox mediated cell death strategies find increasing utility in melanoma chemotherapy either as a single agent or in combination with currently used clinical melanoma drugs including vemurafenib.

Recent studies reported that activation of oncogenes such as K-Ras and Akt in an

inducible system led to an elevated intrinsic oxidative stress phenotype in cells (112, 205).

Hence, cancer cells with these specific oncogene activations may be especially susceptible to

ROS inducing agents. Nogueira et al., through in vitro and in vivo studies demonstrated that

using PEITC (as a single agent or in combination with Rapamycin) in cancer cells with Akt

activation may constitute a strategy to selectively inhibit these cells (205). This therapeutic

scheme may be of significant utility in melanomas where 20% tumors exhibit PTEN loss

leading to increased basal Akt activation (39).

My results indicate that combination of PEITC with either BCNU or TMZ had a

synergistic cell death effect (figure 4.16). The cytotoxicity of this drug combination was

mediated by redox mechanisms wherein PEITC + TMZ treatment led to significantly higher

glutathione depletion and ROS accumulation compared to that of a single agent treatment of

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either PEITC or TMZ (figure 4.17). Glutathione efflux pumps (GS-X) play a major role in resistance to various chemotherapeutic inhibitors such as cisplatin, temozolomide, carmustine and other alkylating agents, daunorubicin and natural products (206, 207). The drug resistance mediated by GS-X pumps directly correlates with the cellular glutathione or glutathione S- transferase (GST) levels and involves two mechanisms (a) formation of GSH-

S drug conjugate (b) exclusion of this GSH-S drug conjugate from the cell by the GS-X export carrier pump (208, 209). Incidentally, the glutathione synthesis inhibitor, BSO has been reported to partially reverse this GS-X pump induced drug resistance to alkylating agents in lung tumor cell lines (210). Previous reports have indicated that PEITC conjugates with glutathione causing its export from the cell (100). Hence, I propose that PEITC mediated export of glutathione by GS-X pumps would inhibit the exclusion of BCNU and

TMZ from the cells. This would sensitize the cells to the DNA alkylating effects of

Temozolomide/BCNU, trigger ROS elevation and subsequently cause cell death. Further investigation needs to be undertaken to shed light on the mechanisms that direct the synergistic combinatorial effect of PEITC+TMZ/BCNU in melanoma. These studies may constitute the future perspectives of this project.

In conclusion, my research on the anticancer effects of PEITC in melanoma cells presents a novel strategy to selectively kill melanoma cells based on the differential redox biology between normal melanocytes and melanoma cells. PEITC scavenges glutathione antioxidant system leading to ROS accumulation and cytotoxicity in melanomas. This unique mechanism of action of PEITC and other glutathione inhibitors such as BSO can be used to combat resistance to other chemotherapeutic agents such as Temozolomide, BCNU and

Vemurafenib.

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Chapter 5: Summary and future directions

5.1 Summary

My PhD dissertation outlines two novel therapeutic strategies to effectively eliminate melanoma cells with limited toxicity to normal melanocytes.

The saponin molecule, OSW-1 is highly cytotoxic to melanoma cells with half- maximal inhibitory concentrations in the sub-nanomolar range. It induces autophagic cell death in melanoma cells by repressing the gene expression of GD3 synthase enzyme, leading to inhibition of gangliosides GD3, GD2 and accumulation of GM3. Ganglioside GD3 is overexpressed in melanoma cells and is a well characterized therapeutic target. OSW-1 also promotes co-localization of ganglioside GD3 with the mitochondrial voltage dependent anion channel 1 (VDAC1) protein. This causes VDAC1 upregulation, mitochondrial membrane permeabilization, superoxide release and loss of calcium homeostasis leading to cell death.

The elevated GD3 and GD2 expression in melanoma cells may be the basis of therapeutic selectivity of OSW-1 against melanoma cells since the normal melanocytes as well as the

OSW-1 resistant, WM35 DW cells exhibit a decreased basal GD3 and GD2 expression.

Further studies need to be undertaken to identify the direct cellular binding target of OSW-1.

Studies also need to focus on delineating the mechanisms of selectivity of OSW-1.

The natural product PEITC relies on the redox differences between normal and cancer cells to selectively eliminate cancer cells. In my study, I first characterized the basal ROS and RNOS expression status of melanoma cells and normal melanocytes. I observed that the sensitivity of melanoma cells to PEITC correlated with the basal ROS expression status of the cells. Normal melanocytes with reduced intrinsic oxidative stress were resistant to

PEITC. PEITC induced ROS accumulation and glutathione depletion in melanoma cells.

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Glutathione depletion occurred much before cell death. Supplementing the cells with the

glutathione precursor, NAC, reversed the ROS accumulation and apoptosis mediated by

PEITC. This indicates that the glutathione depletion is an important mechanism to PEITC

induced cytotoxicity. Further, drug combination of PEITC with TMZ or BCNU ameliorates

the chemoresistance to these agents. Future studies will focus on investigating oxidative

stress based strategies to combat drug resistance in melanoma.

5.2 Future directions

5.2.1 Mechanisms of selectivity of OSW-1 and PEITC against malignant melanoma cells

Mechanisms of selectivity of OSW-1 towards melanoma cells:

An important feature of OSW-1 mediated cell death was its selectivity against

melanoma cells with minimal toxicity to normal melanocytes. I generated OSW-1 resistant

WM35 (WM35 DW) cells (figure 3.31) to characterize the mechanisms underlying this

therapeutic selectivity of OSW-1. My results indicate that WM35DW cells have a reduced

basal ganglioside GD3 and GD2 expression profile compared to the parent WM35 cells

(figure 3.32). This ganglioside expression pattern is similar to normal melanocytes which

express only trace amounts of the pro-oncogenic ganglioside GD3 but prominent levels of

the tumor suppressive ganglioside GM3 (16). OSW-1 inhibits GD3 by repressing gene

expression of the enzyme GD3 synthase. Previous reports demonstrated an elevated

expression of GD3 Synthase (GD3S) in melanoma compared to other cancers (173). Further,

the initial NCI-60 cell line panel in vitro screening identified that melanoma cells (exhibit ganglioside GD3 overexpression) were more sensitive to OSW-1 than the other cell lines tested (67). Hence, I speculate that the differential ganglioside GD3 expression in normal

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melanocytes compared to melanoma cells may form the basis for the selectivity of OSW-1 against malignant melanoma cells.

In order to test this possibility, I would perform ganglioside GD3 overexpression experiments in normal melanocytes. I hypothesize that if GD3 mediated signaling is critical to executing the cytotoxic activity of OSW-1, then overexpressing GD3 in normal melanocytes by transfecting them with the GD3S gene would sensitize these cells to OSW-1.

For this purpose, GD3 deficient SK-Mel-28 cells (referred as N1) as well as the GD3 synthase overexpressing G5 and G11 cell clones that were established from N1 cells would serve as valuable cell models (17, 211). If the GD3 expression status of the cell governs its sensitivity to OSW-1, then the N1 cells would be resistant to OSW-1 whereas the G5 and

G11 SK-MEL-28 cells would exhibit increased sensitivity to OSW-1. I hope to initiate collaborations to acquire these cells and test them for the cytotoxic effect of OSW-1.

VDAC1 was a critical effector molecule in mediating the cell death effect of OSW-1.

RPPA results showed that the OSW-1 resistant WM35 DW cells exhibited a lower basal expression of VDAC 1 and VDAC1 interacting proteins such as Bcl-2 and GSK-3 (figure

3.35 A, C). Further the VDAC1 knock out MEF cells (MEF VDAC1-/-) and siVDAC1 transfected A375SM melanoma cells were resistant to OSW-1 (figure 3.28, 3.29). Hence, I think it is important to compare the basal VDAC1 expression levels across melanoma cell lines and the normal melanocytes and investigate if there is a correlation between VDAC1 expression status and sensitivity to OSW-1. It would be worthwhile to perform VDAC1 transfection experiments in normal melanocytes to examine if induced expression of VDAC1 would sensitize these cells to OSW-1.

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Finally OSW-1 mediated cell death in melanoma cells is through autophagy.

Inhibition of autophagy by using pharmacological inhibitors or down regulating the expression of autophagy genes abrogates OSW-1 induced cell death (figure 3.6). Hence, I would investigate normal melanocytes and melanoma cells to ascertain if differences in intrinsic autophagy flux (as indicated by expression of p62, Beclin-1, Atg5, Atg 8) and mitochondrial mass between the cells contributes to their sensitivity to OSW-1.

Mechanisms of melanoma cell selectivity of PEITC:

PEITC inhibited prostate and leukemia cancer growth by disrupting mitochondrial oxidative phosphorylation (OXPHOS) leading to ATP depletion and ROS production (64,

94). Further, ROS generation in was an early, upstream event that contributed to DNA fragmentation, collapse of mitochondrial membrane potential, cytochrome c release and apoptosis. Interestingly, this ROS production and other downstream events such as caspase activation and mitochondrial membrane damage were not observed in the OXPHOS deficient

Rho-0 prostate cancer cells, indicating that oxidative stress were a crucial mechanism regulating the selectivity circuitry of PEITC (64). Similar results were observed in my study wherein ROS generation was observed much before cell death and normal melanocytes were resistant to free radical induction by PEITC. This ROS accumulation in melanoma cells was partly due to glutathione depletion. My next step would be to investigate the molecular mechanisms that promote ROS elevation in melanoma cells. Towards this end, my focus will be two-fold: investigate effect of PEITC on (a) mechanisms regulating glutathione biosynthesis and metabolism (b) mitochondrial respiration and the respiratory complex activity.

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(a) Glutathione synthesis is a 2-step process: γ-glutamyl cysteinyl synthetase (GCS) enzyme first catalyzes the generation of γ-glutamyl cysteine from cysteine and glutamate. Second, the glutathione synthetase enzyme combines γ-glutamyl cysteine and glycine to produce glutathione (GSH) (212). The availability of cysteine is the rate limiting step in this process

(212, 213). The balance between reduced (GSH) and oxidized (GSSG) forms of glutathione in the cell is maintained by the enzymes glutathione peroxidase (GPX) and glutathione reductase (GR). Previous research from our lab indicated that PEITC inhibited glutathione peroxidase activity thus potentiating hydrogen peroxide accumulation in cells (113). First I would perform enzyme activity assays to determine if PEITC treatment abrogates the activity of GPX, GR, GCS and glutathione synthetase enzymes. The fact that supplementation of

NAC reverses the cell death effect of PEITC (figure 4.13C) suggests that PEITC may not be inhibiting the activity of enzymes that catalyze glutathione biosynthesis (i.e. GCS and glutathione synthetase). After having identified the putative target enzyme modulated by

PEITC, I would then examine the effect of PEITC on this target enzyme expression and activity in melanoma cells vs. normal melanocytes.

(b) To examine the effect of PEITC on oxidative phosphorylation, I would employ the

Seahorse Bioscience XF24 extracellular flux analyzer to compare oxygen consumption rate, and reserve respiration capacity in normal melanocytes and melanoma cells (both basal levels and PEITC induced changes). I would then use metabolic inhibitors such as oligomycin, 2-

Deoxy glucose, rotenone and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) to determine the effect of PEITC on each of the mitochondrial respiratory complex activity.

Simultaneously, I would perform western blots to test the effect of PEITC on mitochondrial respiratory complex (es) expression in both normal melanocytes and melanoma cells.

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I believe that this two-fold approach to characterizing the mechanisms that regulate

PEITC induced ROS accumulation in melanoma cells vs. normal melanocytes will provide insights into the selectivity of PEITC towards melanoma cells.

5.2.2 Characterization of the direct molecular target for OSW-1: SREBP and GD3S regulatory signaling pathways

My PhD dissertation research demonstrates that OSW-1 represses the gene expression of GD3 synthase enzyme in melanoma cells. However, it remains unclear if GD3 Synthase is the direct target of OSW-1. The fact that OSW-1 inhibits GD3S gene expression points towards the possibility that OSW-1 may potentially interact/bind with a protein (such as a transcription factor) that is upstream of GD3 synthase signaling pathway and regulates its expression. Recent studies demonstrated that GD3 expression in melanoma cells correlated with activation of the steroid regulatory element binding protein (SREBP) leading to increased expression of HMGCR and elevated cholesterol biosynthesis (10). Burgett et al .

(2011), through affinity chromatography and quantitative mass spectrometry approaches in

HeLa-S3 cell lysate treated with the OSW-1 affinity reagent (prepared by covalently linking

OSW-1 analogue to sepharose) identified the oxysterol binding protein (OSBP) as a cellular binding target of OSW-1 (69). However, the authors in this study did not explore the functional significance (such as effect on downstream proteins, signaling pathways etc.) of this interaction between OSW-1 and OSBP; nor did they prove that this interaction was imperative to mediating the cytotoxic effect of OSW-1. More importantly, shRNA mediated knockdown of OSBP expression did not exhibit a cytotoxicity profile similar to OSW-1 treatment of cells, suggesting that there may be other molecular binding targets for OSW-1.

Yamada et al . (2014), using fluorescent OSW-1 demonstrated that the putative binding

137

targets of OSW-1 could be located in the ER and golgi apparatus since the fluorescent OSW-

1 molecule localized with high affinity to these organelles in the cell (82). Based on these

studies and my own research results I postulate that OSW-1 potentially interacts with a

ganglioside or lipid signaling protein which is primarily located in the ER/golgi apparatus of

the cell.

SREBP is a master transcription factor regulating cholesterol and fatty acid

biosynthesis (167). The premature and mature forms of SREBP reside in the ER and golgi

apparatus, before being cleaved by serine proteases and transported to the nucleus (214).

Further, SREBP expression correlates with GD3 expression status of the cell (10). Hence, I

plan to investigate if OSW-1 directly interacts with SREBP in melanoma cells. Below I

outline the experimental approaches I would employ to test this hypothesis.

(a) I would perform Chromatin Immunoprecipation (ChIP) for SREBP binding DNA

elements in the control and OSW-1 treated cells. I would then perform real time-PCR

experiment with the ChIP output DNA product to detect the expression of SREBP target

genes including FASN and HMGCR. If OSW-1 directly interacted with SREBP, then the

expression of FASN and HMGCR genes would be suppressed in the OSW-1 treated samples.

(b) Biotinylated OSW-1 (75) will be used for this experimental design. Streptavidin magnetic beads (Solulink, San Diego, CA) may be used to isolate proteins that bind to biotinylated

OSW-1 in an experimental design similar to affinity chromatography. Gel electrophoresis and quantitative mass spectrometry will then be performed with the product protein sample from the streptavidin beads-biotinylated OSW-1 pull down assay to identify the interaction partners of OSW-1, including SREBP-1.

138

The second approach of using the streptavidin beads-biotinylated OSW-1 pull down assay (similar design as affinity chromatography) will be extremely useful in identifying the interaction protein partners of OSW-1. It would also be important to determine the effect of

OSW-1 on the expression of GD3 synthase expression regulatory proteins such as TWIST,

UDP-GlcNAc 2-epimerase/ManNAc 6-kinase (GNE), and amyloid precursor protein (APP) by immunoprecipitation, streptavidin beads-biotinylated OSW-1 pull down assay and western blotting (165, 166, 215).

5.2.3 Oxidative stress based strategies to combat drug resistance in melanoma using

PEITC

My results demonstrate that combination of PEITC with either TMZ or BCNU induces a synergistic drug cytotoxic effect. This increased cell death due to

PEITC+TMZ/BCNU co-treatment in melanoma cells was characterized by enhanced depletion of glutathione and consequent ROS accumulation in cells. Prior studies have shown

BCNU inhibits glutathione reductase in blood cells (194). My results indicate that treatment of melanoma cells with 100 µM BCNU or 0.5 mM TMZ for 24 h causes modest glutathione inhibition. However, supplementation of PEITC (5 µM) during the last 3 h triggered significant glutathione depletion. Glutathione impairment occurred much earlier than cell death implying that it is an important mechanism regulating the anticancer effect of PEITC ±

BCNU/TMZ drug combination in melanoma cells. I postulate that PEITC would promote increased cytotoxicity in TMZ/BCNU treated cells due to its ability to conjugate with and export GSH out of the cell thus limiting drug resistance to TMZ/BCNU by Glutathione efflux pumps. This in combination with inhibition of GR by BCNU may lead to a higher cell death.

139

To test this possibility, I would examine intracellular as well as culture media concentrations of glutathione and PEITC after PEITC ± TMZ/BCNU treatments in melanoma cells.

Furthermore, I would also investigate the effect of TMZ/BCNU/PEITC treatment on

GPX/GR enzyme activity in melanoma cells.

ROS generation and ERK1/2-mediated protective autophagy has been reported to cause resistance to Temozolomide in glioblastoma cells (216, 217). I propose that combination of PEITC with TMZ/BCNU would push the ROS generation triggered by these alkylating agents beyond the cell’s tolerance levels leading to massive toxicity. Figure 4.17

A, B and C describes that PEITC + BCNU/TMZ treatment causes elevated ROS, nitric oxide and mitochondrial superoxide generation. Of specific interest is the induction mitochondrial superoxide by this drug combination. Inhibition of oxidative phosphorylation may cause superoxide accumulation in the mitochondria. Hence I would perform western blot as well as use Seahorse Bioscience XF24 extracellular flux analyzer to investigate the effect of PEITC

+ TMZ/BCNU drug combination on mitochondrial respiration and oxygen consumption.

Finally, I would test the efficacy of this PEITC + TMZ/BCNU drug combination in vivo using athymic nude mice. Khor et al., demonstrated that i.p. injections of PEITC (2.5 µmol,

3X per week) and curcumin (3 µmol, 3X per week) in human PC-3 prostate xenografts in immunodeficient mice significantly reduced tumor growth (218). I propose to test the effect of PEITC ± TMZ/BCNU in immunodeficient mice inoculated with A375SM cells with optimized drug dosage and treatment times.

Recent studies have indicated that the clinical mutant BRAF inhibitor, vemurafenib, causes ROS stress in melanoma cells (204). Combination of vemurafenib with ROS inducing agents might be a worthwhile strategy to eliminate these tumors. Important PEITC may be

140

effective in killing vemurafenib resistant tumors since these are characterized by elevated intrinsic oxidative stress compared to the vemurafenib sensitive melanomas.

Both OSW-1 and PEITC induced cell death is characterized by elevations in ROS levels and mitochondria dependent mechanisms of action. Hence it will be worthwhile to characterize the combinatorial drug effect of OSW-1 and PEITC in melanoma cells. In this study, the effect of OSW-1 + PEITC combination on various molecular markers such as glutathione, mitochondrial VDAC1, gangliosides GD3 and GD2, nitric oxide and superoxides will be examined. It may be crucial to be compare and correlate the basal expression and drug induced changes of these markers in a particular cell line to its sensitivity to OSW-1+PEITC treatment. This type of analysis will be of clinical significance as it may lead to the identification of biomarkers that would predict patient response to this combinatorial OSW-1+PEITC drug treatment strategy.

Therapeutic selectivity is an important characteristic of any successful pharmacological agent. In this dissertation, I elucidate the mechanisms of anticancer activity and therapeutic selectivity of two novel inhibitors, OSW-1 and PEITC. OSW-1 exerts its antiproliferative effect by inhibiting ganglioside GD3 expression and causing VDAC1 activation, subsequently leading to acute autophagy. PEITC relies on the differential redox biology between the cancer cells and normal melanocytes to preferentially eliminate melanoma cells. Both OSW-1 and PEITC employ relatively novel therapeutic strategies to effectively eliminate malignant melanomas and warrant further testing in pre-clinical and clinical settings.

141

References

1. Cantor JR, Sabatini DM. Cancer cell metabolism: one hallmark, many faces. Cancer discovery. 2012;2(10):881-98. Epub 2012/09/27.

2. Hsu PP, Sabatini DM. Cancer cell metabolism: Warburg and beyond. Cell.

2008;134(5):703-7. Epub 2008/09/09.

3. Cairns RA, Harris IS, Mak TW. Regulation of cancer cell metabolism. Nature reviews Cancer. 2011;11(2):85-95. Epub 2011/01/25.

4. Menendez JA, Lupu R. Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis. Nature reviews Cancer. 2007;7(10):763-77. Epub 2007/09/21.

5. Swinnen JV, Brusselmans K, Verhoeven G. Increased lipogenesis in cancer cells: new players, novel targets. Current opinion in clinical nutrition and metabolic care.

2006;9(4):358-65. Epub 2006/06/17.

6. Innocenzi D, Alo PL, Balzani A, Sebastiani V, Silipo V, La Torre G, Ricciardi G,

Bosman C, Calvieri S. Fatty acid synthase expression in melanoma. Journal of cutaneous pathology. 2003;30(1):23-8. Epub 2003/01/22.

7. Alo PL, Visca P, Marci A, Mangoni A, Botti C, Di Tondo U. Expression of fatty acid synthase (FAS) as a predictor of recurrence in stage I breast carcinoma patients. Cancer.

1996;77(3):474-82. Epub 1996/02/01.

8. Swinnen JV, Roskams T, Joniau S, Van Poppel H, Oyen R, Baert L, Heyns W,

Verhoeven G. Overexpression of fatty acid synthase is an early and common event in the development of prostate cancer. International journal of cancer Journal international du cancer. 2002;98(1):19-22. Epub 2002/02/22.

142

9. Vazquez-Martin A, Colomer R, Brunet J, Lupu R, Menendez JA. Overexpression of fatty acid synthase gene activates HER1/HER2 tyrosine kinase receptors in human breast epithelial cells. Cell proliferation. 2008;41(1):59-85. Epub 2008/01/24.

10. Yamauchi Y, Furukawa K, Hamamura K. Positive feedback loop between PI3K-Akt- mTORC1 signaling and the lipogenic pathway boosts Akt signaling: induction of the lipogenic pathway by a melanoma antigen. Cancer research. 2011;71(14):4989-97. Epub

2011/06/03.

11. Furukawa K, Hamamura K, Ohkawa Y, Ohmi Y. Disialyl gangliosides enhance tumor phenotypes with differential modalities. Glycoconjugate journal. 2012;29(8-9):579-84.

Epub 2012/07/06.

12. Furukawa K, Hamamura K, Nakashima H. Molecules in the signaling pathway activated by gangliosides can be targets of therapeutics for malignant melanomas.

Proteomics. 2008;8(16):3312-6. Epub 2008/08/12.

13. Dong Y, Ikeda K, Hamamura K, Zhang Q, Kondo Y, Matsumoto Y, Ohmi Y,

Yamauchi Y, Furukawa K, Taguchi R. GM1 / GD1b / GA1 synthase expression results in the reduced cancer phenotypes with modulation of composition and raft-localization of gangliosides in a melanoma cell line. Cancer science. 2010;101(9):2039-47. Epub

2010/07/03.

14. Zhang Q, Furukawa K, Chen HH, Sakakibara T, Urano T. Metastatic potential of mouse Lewis lung cancer cells is regulated via ganglioside GM1 by modulating the matrix metalloprotease-9 localization in lipid rafts. The Journal of biological chemistry.

2006;281(26):18145-55. Epub 2006/04/26.

143

15. Tsuchida T, Saxton RE, Morton DL, Irie RF. Gangliosides of human melanoma.

Journal of the National Cancer Institute. 1987;78(1):45-54. Epub 1987/01/01.

16. Thampoe IJ, Furukawa K, Vellve E, Lloyd KO. Sialyltransferase levels and ganglioside expression in melanoma and other cultured human cancer cells. Cancer research.

1989;49(22):6258-64. Epub 1989/11/15.

17. Hamamura K, Furukawa K, Hayashi T, Hattori T, Nakano J, Nakashima H, Okuda T,

Mizutani H, Hattori H, Ueda M, Urano T, Lloyd KO. Ganglioside GD3 promotes cell growth and invasion through p130Cas and paxillin in malignant melanoma cells. Proceedings of the

National Academy of Sciences of the United States of America. 2005;102(31):11041-6. Epub

2005/07/26.

18. Furukawa K, Kambe M, Miyata M, Ohkawa Y, Tajima O. Ganglioside GD3 induces convergence and synergism of adhesion and hepatocyte growth factor/Met signals in melanomas. Cancer science. 2014;105(1):52-63. Epub 2014/01/01.

19. Ohkawa Y, Miyazaki S, Hamamura K, Kambe M, Miyata M, Tajima O, Ohmi Y,

Yamauchi Y, Furukawa K. Ganglioside GD3 enhances adhesion signals and augments malignant properties of melanoma cells by recruiting integrins to glycolipid-enriched microdomains. The Journal of biological chemistry. 2010;285(35):27213-23. Epub

2010/06/29.

20. Furukawa K, Ohkawa Y, Yamauchi Y, Hamamura K, Ohmi Y. Fine tuning of cell signals by glycosylation. Journal of biochemistry. 2012;151(6):573-8. Epub 2012/05/25.

21. Houghton AN, Mintzer D, Cordon-Cardo C, Welt S, Fliegel B, Vadhan S,

Carswell E, Melamed MR, Oettgen HF, Old LJ. Mouse monoclonal IgG3 antibody detecting

GD3 ganglioside: a phase I trial in patients with malignant melanoma. Proceedings of the

144

National Academy of Sciences of the United States of America. 1985;82(4):1242-6. Epub

1985/02/01.

22. McCaffery M, Yao TJ, Williams L, Livingston PO, Houghton AN, Chapman

PB. Immunization of melanoma patients with BEC2 anti-idiotypic monoclonal antibody that mimics GD3 ganglioside: enhanced immunogenicity when combined with adjuvant. Clinical cancer research : an official journal of the American Association for Cancer Research.

1996;2(4):679-86. Epub 1996/04/01.

23. Cazet A, Lefebvre J, Adriaenssens E, Julien S, Bobowski M, Grigoriadis A,

Tutt A, Tulasne D, Le Bourhis X, Delannoy P. GD(3) synthase expression enhances proliferation and tumor growth of MDA-MB-231 breast cancer cells through c-Met activation. Molecular cancer research : MCR. 2010;8(11):1526-35. Epub 2010/10/05.

24. Battula VL, Shi Y, Evans KW, Wang RY, Spaeth EL, Jacamo RO, Guerra R,

Sahin AA, Marini FC, Hortobagyi G, Mani SA, Andreeff M. Ganglioside GD2 identifies breast cancer stem cells and promotes tumorigenesis. The Journal of clinical investigation.

2012;122(6):2066-78. Epub 2012/05/16.

25. Aixinjueluo W, Furukawa K, Zhang Q, Hamamura K, Tokuda N, Yoshida S,

Ueda R. Mechanisms for the apoptosis of small cell lung cancer cells induced by anti-GD2 monoclonal antibodies: roles of anoikis. The Journal of biological chemistry.

2005;280(33):29828-36. Epub 2005/06/01.

26. Nakashima H, Hamamura K, Houjou T, Taguchi R, Yamamoto N, Mitsudo K,

Tohnai I, Ueda M, Urano T, Furukawa K. Overexpression of caveolin-1 in a human melanoma cell line results in dispersion of ganglioside GD3 from lipid rafts and alteration of

145

leading edges, leading to attenuation of malignant properties. Cancer science.

2007;98(4):512-20. Epub 2007/02/08.

27. Sorice M, Garofalo T, Misasi R, Manganelli V, Vona R, Malorni W.

Ganglioside GD3 as a raft component in cell death regulation. Anti-cancer agents in medicinal chemistry. 2012;12(4):376-82. Epub 2011/05/11.

28. Boland ML, Chourasia AH, Macleod KF. Mitochondrial dysfunction in cancer.

Frontiers in oncology. 2013;3:292. Epub 2013/12/19.

29. Carew JS, Huang P. Mitochondrial defects in cancer. Molecular cancer.

2002;1:9. Epub 2003/01/07.

30. Tan DJ, Bai RK, Wong LJ. Comprehensive scanning of somatic mitochondrial

DNA mutations in breast cancer. Cancer research. 2002;62(4):972-6. Epub 2002/02/28.

31. Poetsch M, Petersmann A, Lignitz E, Kleist B. Relationship between

mitochondrial DNA instability, mitochondrial DNA large deletions, and nuclear

microsatellite instability in head and neck squamous cell carcinomas. Diagnostic molecular

pathology : the American journal of surgical pathology, part B. 2004;13(1):26-32. Epub

2004/05/28.

32. Lee MH, Lee SE, Kim DW, Ryu MJ, Kim SJ, Kim YK, Park JH, Kweon GR,

Kim JM, Lee JU, De Falco V, Jo YS, Shong M. Mitochondrial localization and regulation of

BRAFV600E in thyroid cancer: a clinically used RAF inhibitor is unable to block the

mitochondrial activities of BRAFV600E. The Journal of clinical endocrinology and

metabolism. 2011;96(1):E19-30. Epub 2010/10/12.

33. Tomasello F, Messina A, Lartigue L, Schembri L, Medina C, Reina S,

Thoraval D, Crouzet M, Ichas F, De Pinto V, De Giorgi F. Outer membrane VDAC1 controls

146

permeability transition of the inner mitochondrial membrane in cellulo during stress-induced apoptosis. Cell research. 2009;19(12):1363-76. Epub 2009/08/12.

34. Ghosh T, Pandey N, Maitra A, Brahmachari SK, Pillai B. A role for voltage- dependent anion channel Vdac1 in polyglutamine-mediated neuronal cell death. PloS one.

2007;2(11):e1170. Epub 2007/11/15.

35. Lu AJ, Dong CW, Du CS, Zhang QY. Characterization and expression analysis of Paralichthys olivaceus voltage-dependent anion channel (VDAC) gene in response to virus infection. Fish & shellfish immunology. 2007;23(3):601-13. Epub 2007/05/01.

36. De Pinto V, Tomasello F, Messina A, Guarino F, Benz R, La Mendola D,

Magri A, Milardi D, Pappalardo G. Determination of the conformation of the human VDAC1

N-terminal peptide, a protein moiety essential for the functional properties of the pore.

Chembiochem : a European journal of chemical biology. 2007;8(7):744-56. Epub

2007/03/28.

37. Zhang J, Ney PA. Role of BNIP3 and NIX in cell death, autophagy, and mitophagy. Cell death and differentiation. 2009;16(7):939-46. Epub 2009/02/21.

38. Wittgen HG, van Kempen LC. Reactive oxygen species in melanoma and its therapeutic implications. Melanoma research. 2007;17(6):400-9. Epub 2007/11/10.

39. Tsao H, Zhang X, Benoit E, Haluska FG. Identification of PTEN/MMAC1 alterations in uncultured melanomas and melanoma cell lines. Oncogene. 1998;16(26):3397-

402. Epub 1998/08/06.

40. Govindarajan B, Sligh JE, Vincent BJ, Li M, Canter JA, Nickoloff BJ,

Rodenburg RJ, Smeitink JA, Oberley L, Zhang Y, Slingerland J, Arnold RS, Lambeth JD,

Cohen C, Hilenski L, Griendling K, Martinez-Diez M, Cuezva JM, Arbiser JL.

147

Overexpression of Akt converts radial growth melanoma to vertical growth melanoma. The

Journal of clinical investigation. 2007;117(3):719-29. Epub 2007/02/24.

41. Satyamoorthy K, Li G, Gerrero MR, Brose MS, Volpe P, Weber BL, Van Belle

P, Elder DE, Herlyn M. Constitutive mitogen-activated protein kinase activation in

melanoma is mediated by both BRAF mutations and autocrine growth factor stimulation.

Cancer research. 2003;63(4):756-9. Epub 2003/02/20.

42. Smalley KS. A pivotal role for ERK in the oncogenic behaviour of malignant

melanoma? International journal of cancer Journal international du cancer. 2003;104(5):527-

32. Epub 2003/02/21.

43. Sekulic A, Haluska P, Jr., Miller AJ, Genebriera De Lamo J, Ejadi S, Pulido JS,

Salomao DR, Thorland EC, Vile RG, Swanson DL, Pockaj BA, Laman SD, Pittelkow MR,

Markovic SN. Malignant melanoma in the 21st century: the emerging molecular landscape.

Mayo Clinic proceedings. 2008;83(7):825-46. Epub 2008/07/11.

44. Meyskens FL, Jr., Farmer P, Fruehauf JP. Redox regulation in human melanocytes and melanoma. Pigment cell research / sponsored by the European Society for

Pigment Cell Research and the International Pigment Cell Society. 2001;14(3):148-54. Epub

2001/07/04.

45. McEligot AJ, Yang S, Meyskens FL, Jr. Redox regulation by intrinsic species and extrinsic nutrients in normal and cancer cells. Annual review of nutrition. 2005;25:261-

95. Epub 2005/07/14.

46. Ueda Y, Richmond A. NF-kappaB activation in melanoma. Pigment cell research / sponsored by the European Society for Pigment Cell Research and the

International Pigment Cell Society. 2006;19(2):112-24. Epub 2006/03/10.

148

47. Xanthoudakis S, Miao G, Wang F, Pan YC, Curran T. Redox activation of Fos-Jun

DNA binding activity is mediated by a DNA repair enzyme. The EMBO journal.

1992;11(9):3323-35. Epub 1992/09/01.

48. Fruehauf JP, Meyskens FL, Jr. Reactive oxygen species: a breath of life or death?

Clinical cancer research : an official journal of the American Association for Cancer

Research. 2007;13(3):789-94. Epub 2007/02/10.

49. Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nature reviews Drug discovery. 2009;8(7):579-

91. Epub 2009/05/30.

50. Berkenblit A, Eder JP, Jr., Ryan DP, Seiden MV, Tatsuta N, Sherman ML, Dahl TA,

Dezube BJ, Supko JG. Phase I clinical trial of STA-4783 in combination with paclitaxel in patients with refractory solid tumors. Clinical cancer research : an official journal of the

American Association for Cancer Research. 2007;13(2 Pt 1):584-90. Epub 2007/01/27.

51. Kirshner JR, He S, Balasubramanyam V, Kepros J, Yang CY, Zhang M, Du Z,

Barsoum J, Bertin J. Elesclomol induces cancer cell apoptosis through oxidative stress.

Molecular cancer therapeutics. 2008;7(8):2319-27. Epub 2008/08/30.

52. Gehrmann M. Drug evaluation: STA-4783--enhancing taxane efficacy by induction of Hsp70. Curr Opin Investig Drugs. 2006;7(6):574-80. Epub 2006/06/21.

53. Dippold WG, Lloyd KO, Li LT, Ikeda H, Oettgen HF, Old LJ. Cell surface antigens of human malignant melanoma: definition of six antigenic systems with mouse monoclonal antibodies. Proceedings of the National Academy of Sciences of the United States of

America. 1980;77(10):6114-8. Epub 1980/10/01.

149

54. Pukel CS, Lloyd KO, Travassos LR, Dippold WG, Oettgen HF, Old LJ. GD3, a prominent ganglioside of human melanoma. Detection and characterisation by mouse monoclonal antibody. The Journal of experimental medicine. 1982;155(4):1133-47. Epub

1982/04/01.

55. Chapman PB, Wu D, Ragupathi G, Lu S, Williams L, Hwu WJ, Johnson D,

Livingston PO. Sequential immunization of melanoma patients with GD3 ganglioside vaccine and anti-idiotypic monoclonal antibody that mimics GD3 ganglioside. Clinical cancer research : an official journal of the American Association for Cancer Research.

2004;10(14):4717-23. Epub 2004/07/23.

56. Kwon HY, Kim SJ, Kim CH, Son SW, Kim KS, Lee JH, Do SI, Lee YC. Triptolide downregulates human GD3 synthase (hST8Sia I) gene expression in SK-MEL-2 human melanoma cells. Experimental & molecular medicine. 2010;42(12):849-55. Epub

2010/11/13.

57. Yokoyama S. TT, and Sashida Y. Stability of an antitumor OSW-1 encapsulated into

Ganglioside GM3 liposomes, and accumulation of the OSW-1/GM3 liposomes on the melanoma cells. Material technology. 2005;23(2):85-9.

58. Garcia-Prieto C, Riaz Ahmed KB, Chen Z, Zhou Y, Hammoudi N, Kang Y, Lou C,

Mei Y, Jin Z, Huang P. Effective killing of leukemia cells by the natural product OSW-1 through disruption of cellular calcium homeostasis. The Journal of biological chemistry.

2013;288(5):3240-50. Epub 2012/12/20.

150

59. Zhou Y, Garcia-Prieto C, Carney DA, Xu RH, Pelicano H, Kang Y, Yu W, Lou C,

Kondo S, Liu J, Harris DM, Estrov Z, Keating MJ, Jin Z, Huang P. OSW-1: a natural compound with potent anticancer activity and a novel mechanism of action. Journal of the

National Cancer Institute. 2005;97(23):1781-5. Epub 2005/12/08.

60. Malorni W, Giammarioli AM, Garofalo T, Sorice M. Dynamics of lipid raft components during lymphocyte apoptosis: the paradigmatic role of GD3. Apoptosis : an international journal on programmed cell death. 2007;12(5):941-9. Epub 2007/04/25.

61. Pelicano H, Feng L, Zhou Y, Carew JS, Hileman EO, Plunkett W, Keating MJ,

Huang P. Inhibition of mitochondrial respiration: a novel strategy to enhance drug-induced apoptosis in human leukemia cells by a reactive oxygen species-mediated mechanism. The

Journal of biological chemistry. 2003;278(39):37832-9. Epub 2003/07/11.

62. Fruehauf JP, Trapp V. Reactive oxygen species: an Achilles' heel of melanoma?

Expert review of anticancer therapy. 2008;8(11):1751-7. Epub 2008/11/06.

63. Trachootham D, Zhang H, Zhang W, Feng L, Du M, Zhou Y, Chen Z, Pelicano H,

Plunkett W, Wierda WG, Keating MJ, Huang P. Effective elimination of fludarabine- resistant CLL cells by PEITC through a redox-mediated mechanism. Blood.

2008;112(5):1912-22. Epub 2008/06/25.

64. Xiao D, Powolny AA, Moura MB, Kelley EE, Bommareddy A, Kim SH, Hahm ER,

Normolle D, Van Houten B, Singh SV. Phenethyl isothiocyanate inhibits oxidative phosphorylation to trigger reactive oxygen species-mediated death of human prostate cancer cells. The Journal of biological chemistry. 2010;285(34):26558-69. Epub 2010/06/24.

151

65. Kubo S MY, Terao M, Sashida Y, Nikaido T, and Ohmoto T. Acylated cholestane glycosides from the bulbs of ornithogalum saundersiae. Phytochemistry. 1992;31(11):3969-

73.

66. Zheng D, Guan Y, Chen X, Xu Y, Lei P. Synthesis of cholestane saponins as mimics of OSW-1 and their cytotoxic activities. Bioorg Med Chem Lett. Epub 2011/05/03.

67. Mimaki Y KM, Kameyama A, Sashida Y, Hirano T and Oka K. Cholestane glycosides with potent cytostatic activities on various tumor cells from Ornithogalum saundersiae bulbs Bioorg Med Chem Lett. 1997;7(5). Epub 633-636.

68. Guo C, LaCour TG, Fuchs PL. On the relationship of OSW-1 to the cephalostatins.

Bioorg Med Chem Lett. 1999;9(3):419-24. Epub 1999/03/26.

69. Burgett AW, Poulsen TB, Wangkanont K, Anderson DR, Kikuchi C, Shimada K,

Okubo S, Fortner KC, Mimaki Y, Kuroda M, Murphy JP, Schwalb DJ, Petrella EC, Cornella-

Taracido I, Schirle M, Tallarico JA, Shair MD. Natural products reveal cancer cell dependence on oxysterol-binding proteins. Nature chemical biology. 2011;7(9):639-47. Epub

2011/08/09.

70. Kuroda M, Mimaki Y, Yokosuka A, Sashida Y, Beutler JA. Cytotoxic cholestane glycosides from the bulbs of Ornithogalum saundersiae. Journal of natural products.

2001;64(1):88-91. Epub 2001/02/15.

71. Ma X, B. Yu, Y. Hui, D. Xiao, and J. Ding. Synthesis of glycosides bearing the disaccharide of OSW-1 or its 14-linked analogue and their antitumor activities. Carbohydrate research. 2000;329:495-505.

152

72. Ma X, Yu B, Hui Y, Miao Z, Ding J. Synthesis of steroidal glycosides bearing the disaccharide moiety of OSW-1 and their antitumor activities. Carbohydrate research.

2001;334(2):159-64. Epub 2001/08/15.

73. Deng S, Yu B, Lou Y, Hui Y. First Total Synthesis of an Exceptionally Potent

Antitumor Saponin, OSW-1. The Journal of organic chemistry. 1999;64(1):202-8. Epub

2001/10/25.

74. Yu W, Jin Z. Total synthesis of the anticancer natural product OSW-1. Journal of the

American Chemical Society. 2002;124(23):6576-83. Epub 2002/06/06.

75. Kang Y, Lou C, Ahmed KB, Huang P, Jin Z. Synthesis of biotinylated OSW-1.

Bioorganic & medicinal chemistry letters. 2009;19(17):5166-8. Epub 2009/07/31.

76. Guan Y, Zheng D, Zhou L, Wang H, Yan Z, Wang N, Chang H, She P, Lei P.

Synthesis of 5(6)-dihydro-OSW-1 analogs bearing three kinds of disaccharides linking at 15- hydroxy and their antitumor activities. Bioorg Med Chem Lett.21(10):2921-4. Epub

2011/04/13.

77. Guan YY, Song C, Lei PS. Synthesis of three OSW-1 analogs with maltose side chains bearing different protection groups. Journal of Asian natural products research.

2014;16(1):43-52. Epub 2013/12/10.

78. Yu W, Jin Z. A new strategy for the stereoselective introduction of steroid side chain via alpha-alkoxy vinyl cuprates: total synthesis of a highly potent antitumor natural product

OSW-1. Journal of the American Chemical Society. 2001;123(14):3369-70. Epub

2001/07/18.

153

79. Tschamber T, Adam S, Matsuya Y, Masuda S, Ohsawa N, Maruyama S, Kamoshita

K, Nemoto H, Eustache J. OSW-1 analogues: modification of the carbohydrate moiety.

Bioorg Med Chem Lett. 2007;17(18):5101-6. Epub 2007/07/28.

80. Tamura K, Honda H, Mimaki Y, Sashida Y, Kogo H. Inhibitory effect of a new

steroidal saponin, OSW-1, on ovarian functions in rats. British journal of pharmacology.

1997;121(8):1796-802. Epub 1997/08/01.

81. Jin J, Jin X, Qian C, Ruan Y, Jiang H. Signaling network of OSW1induced apoptosis

and necroptosis in hepatocellular carcinoma. Molecular medicine reports. 2013;7(5):1646-50.

Epub 2013/03/19.

82. Yamada R, Takeshita T, Hiraizumi M, Shinohe D, Ohta Y, Sakurai K. Fluorescent

analog of OSW-1 and its cellular localization. Bioorg Med Chem Lett. 2014;24(7):1839-42.

Epub 2014/03/13.

83. Yokoyama S, Ohtsuka I., Takeda T., and Sashida Y. Distribution of an antitumor

natural product OSW-1 in ganglioside GM3-phospholipid membranes. material technology.

2005;23(1):54-8.

84. Cheung NK, Saarinen UM, Neely JE, Landmeier B, Donovan D, Coccia PF.

Monoclonal antibodies to a glycolipid antigen on human neuroblastoma cells. Cancer research. 1985;45(6):2642-9. Epub 1985/06/01.

85. Modak S, Cheung NK. Disialoganglioside directed immunotherapy of neuroblastoma.

Cancer investigation. 2007;25(1):67-77. Epub 2007/03/17.

86. Yoshida S, Fukumoto S, Kawaguchi H, Sato S, Ueda R, Furukawa K. Ganglioside

G(D2) in small cell lung cancer cell lines: enhancement of cell proliferation and mediation of

apoptosis. Cancer research. 2001;61(10):4244-52. Epub 2001/05/19.

154

87. Yu R, Mandlekar S, Harvey KJ, Ucker DS, Kong AN. Chemopreventive isothiocyanates induce apoptosis and caspase-3-like protease activity. Cancer research.

1998;58(3):402-8. Epub 1998/02/11.

88. Jacobs MD, Harrison SC. Structure of an IkappaBalpha/NF-kappaB complex. Cell.

1998;95(6):749-58. Epub 1998/12/29.

89. Xu C, Shen G, Chen C, Gelinas C, Kong AN. Suppression of NF-kappaB and NF- kappaB-regulated gene expression by sulforaphane and PEITC through IkappaBalpha, IKK pathway in human prostate cancer PC-3 cells. Oncogene. 2005;24(28):4486-95. Epub

2005/04/28.

90. Rose P, Won YK, Ong CN, Whiteman M. Beta-phenylethyl and 8- methylsulphinyloctyl isothiocyanates, constituents of watercress, suppress LPS induced production of nitric oxide and prostaglandin E2 in RAW 264.7 macrophages. Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society. 2005;12(4):237-43. Epub

2005/05/27.

91. Jeong WS, Kim IW, Hu R, Kong AN. Modulatory properties of various natural chemopreventive agents on the activation of NF-kappaB signaling pathway. Pharmaceutical research. 2004;21(4):661-70. Epub 2004/05/14.

92. Zhang Y, Tang L, Gonzalez V. Selected isothiocyanates rapidly induce growth inhibition of cancer cells. Molecular cancer therapeutics. 2003;2(10):1045-52. Epub

2003/10/28.

93. Lam YSaO-A, Richard. Enzyme induction and cytotoxicity of phenethyl isothiocyanate and its glutathione conjugate towards breast cancer cells. Pure and Applied

Chemical Sciences. 2013;1(2):63-73.

155

94. Chen G, Chen Z, Hu Y, Huang P. Inhibition of mitochondrial respiration and rapid depletion of mitochondrial glutathione by beta-phenethyl isothiocyanate: mechanisms for anti-leukemia activity. Antioxidants & redox signaling. 2011;15(12):2911-21. Epub

2011/08/11.

95. Xiao D, Johnson CS, Trump DL, Singh SV. Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-

M-phase cell cycle arrest in PC-3 human prostate cancer cells. Molecular cancer therapeutics.

2004;3(5):567-75. Epub 2004/05/14.

96. Mi L, Xiao Z, Hood BL, Dakshanamurthy S, Wang X, Govind S, Conrads TP,

Veenstra TD, Chung FL. Covalent binding to tubulin by isothiocyanates. A mechanism of cell growth arrest and apoptosis. The Journal of biological chemistry. 2008;283(32):22136-

46. Epub 2008/06/06.

97. Mi L, Chung FL. Binding to protein by isothiocyanates: a potential mechanism for apoptosis induction in human non small lung cancer cells. Nutrition and cancer. 2008;60

Suppl 1:12-20. Epub 2008/11/15.

98. Hu R, Kim BR, Chen C, Hebbar V, Kong AN. The roles of JNK and apoptotic signaling pathways in PEITC-mediated responses in human HT-29 colon adenocarcinoma cells. Carcinogenesis. 2003;24(8):1361-7. Epub 2003/06/24.

99. Moon YJ, Brazeau DA, Morris ME. Dietary phenethyl isothiocyanate alters gene expression in human breast cancer cells. Evidence-based complementary and alternative medicine : eCAM. 2011;2011. Epub 2010/10/19.

156

100. Xu K, Thornalley PJ. Involvement of glutathione metabolism in the cytotoxicity of the phenethyl isothiocyanate and its cysteine conjugate to human leukaemia cells in vitro.

Biochemical pharmacology. 2001;61(2):165-77. Epub 2001/02/13.

101. Hu Y, Lu W, Chen G, Zhang H, Jia Y, Wei Y, Yang H, Zhang W, Fiskus W, Bhalla

K, Keating M, Huang P, Garcia-Manero G. Overcoming resistance to histone deacetylase inhibitors in human leukemia with the redox modulating compound beta-phenylethyl isothiocyanate. Blood. 2010;116(15):2732-41. Epub 2010/06/23.

102. Zhang H, Trachootham D, Lu W, Carew J, Giles FJ, Keating MJ, Arlinghaus RB,

Huang P. Effective killing of Gleevec-resistant CML cells with T315I mutation by a natural compound PEITC through redox-mediated mechanism. Leukemia. 2008;22(6):1191-9. Epub

2008/04/04.

103. Xu K, Thornalley PJ. Studies on the mechanism of the inhibition of human leukaemia cell growth by dietary isothiocyanates and their cysteine adducts in vitro. Biochemical pharmacology. 2000;60(2):221-31. Epub 2000/05/29.

104. Xu RH, Pelicano H, Zhou Y, Carew JS, Feng L, Bhalla KN, Keating MJ, Huang P.

Inhibition of glycolysis in cancer cells: a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia. Cancer research.

2005;65(2):613-21. Epub 2005/02/08.

105. Pelicano H, Xu RH, Du M, Feng L, Sasaki R, Carew JS, Hu Y, Ramdas L, Hu L,

Keating MJ, Zhang W, Plunkett W, Huang P. Mitochondrial respiration defects in cancer cells cause activation of Akt survival pathway through a redox-mediated mechanism. The

Journal of cell biology. 2006;175(6):913-23. Epub 2006/12/13.

157

106. Johnson LV, Walsh ML, Chen LB. Localization of mitochondria in living cells with rhodamine 123. Proceedings of the National Academy of Sciences of the United States of

America. 1980;77(2):990-4. Epub 1980/02/01.

107. Mone AP, Huang P, Pelicano H, Cheney CM, Green JM, Tso JY, Johnson AJ,

Jefferson S, Lin TS, Byrd JC. Hu1D10 induces apoptosis concurrent with activation of the

AKT survival pathway in human chronic lymphocytic leukemia cells. Blood.

2004;103(5):1846-54. Epub 2003/11/25.

108. Luo Y, Yang C, Lu W, Xie R, Jin C, Huang P, Wang F, McKeehan WL. Metabolic regulator betaKlotho interacts with fibroblast growth factor receptor 4 (FGFR4) to induce apoptosis and inhibit tumor cell proliferation. The Journal of biological chemistry.

2010;285(39):30069-78. Epub 2010/07/27.

109. Nawrocki ST, Carew JS, Pino MS, Highshaw RA, Andtbacka RH, Dunner K, Jr., Pal

A, Bornmann WG, Chiao PJ, Huang P, Xiong H, Abbruzzese JL, McConkey DJ. Aggresome disruption: a novel strategy to enhance bortezomib-induced apoptosis in pancreatic cancer cells. Cancer research. 2006;66(7):3773-81. Epub 2006/04/06.

110. Mitsuda T, Furukawa K, Fukumoto S, Miyazaki H, Urano T. Overexpression of ganglioside GM1 results in the dispersion of platelet-derived growth factor receptor from glycolipid-enriched microdomains and in the suppression of cell growth signals. The Journal of biological chemistry. 2002;277(13):11239-46. Epub 2002/01/10.

111. Taube JH, Malouf GG, Lu E, Sphyris N, Vijay V, Ramachandran PP, Ueno KR, Gaur

S, Nicoloso MS, Rossi S, Herschkowitz JI, Rosen JM, Issa JP, Calin GA, Chang JT, Mani

SA. Epigenetic silencing of microRNA-203 is required for EMT and cancer stem cell properties. Scientific reports. 2013;3:2687. Epub 2013/09/21.

158

112. Hu Y, Lu W, Chen G, Wang P, Chen Z, Zhou Y, Ogasawara M, Trachootham D,

Feng L, Pelicano H, Chiao PJ, Keating MJ, Garcia-Manero G, Huang P. K-ras(G12V) transformation leads to mitochondrial dysfunction and a metabolic switch from oxidative phosphorylation to glycolysis. Cell research. 2012;22(2):399-412. Epub 2011/08/31.

113. Trachootham D, Zhou Y, Zhang H, Demizu Y, Chen Z, Pelicano H, Chiao PJ,

Achanta G, Arlinghaus RB, Liu J, Huang P. Selective killing of oncogenically transformed cells through a ROS-mediated mechanism by beta-phenylethyl isothiocyanate. Cancer cell.

2006;10(3):241-52. Epub 2006/09/09.

114. Maldonado EN, Sheldon KL, DeHart DN, Patnaik J, Manevich Y, Townsend DM,

Bezrukov SM, Rostovtseva TK, Lemasters JJ. Voltage-dependent anion channels modulate mitochondrial metabolism in cancer cells: regulation by free tubulin and erastin. The Journal of biological chemistry. 2013;288(17):11920-9. Epub 2013/03/09.

115. Tibes R, Qiu Y, Lu Y, Hennessy B, Andreeff M, Mills GB, Kornblau SM. Reverse phase protein array: validation of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells. Molecular cancer therapeutics.

2006;5(10):2512-21. Epub 2006/10/17.

116. Bobowski M, Vincent A, Steenackers A, Colomb F, Van Seuningen I, Julien S,

Delannoy P. Estradiol represses the G(D3) synthase gene ST8SIA1 expression in human breast cancer cells by preventing NFkappaB binding to ST8SIA1 promoter. PloS one.

2013;8(4):e62559. Epub 2013/04/30.

117. Geoffrey M Cooper RMH. The Cell: A Molecular Approach. Sixth ed: Sinauer

Associates, Inc.; 2013. 832 p.

159

118. Haworth RA, Hunter DR. The Ca2+-induced membrane transition in mitochondria.

II. Nature of the Ca2+ trigger site. Archives of biochemistry and biophysics.

1979;195(2):460-7. Epub 1979/07/01.

119. Beatrice MC, Stiers DL, Pfeiffer DR. Increased permeability of mitochondria during

Ca2+ release induced by t-butyl hydroperoxide or oxalacetate. the effect of ruthenium red.

The Journal of biological chemistry. 1982;257(12):7161-71. Epub 1982/06/25.

120. Custodio JB, Cardoso CM, Madeira VM, Almeida LM. Mitochondrial permeability transition induced by the anticancer drug etoposide. Toxicology in vitro : an international journal published in association with BIBRA. 2001;15(4-5):265-70. Epub 2001/09/22.

121. Larochette N, Decaudin D, Jacotot E, Brenner C, Marzo I, Susin SA, Zamzami N,

Xie Z, Reed J, Kroemer G. Arsenite induces apoptosis via a direct effect on the mitochondrial permeability transition pore. Experimental cell research. 1999;249(2):413-21.

Epub 1999/06/15.

122. Ravagnan L, Marzo I, Costantini P, Susin SA, Zamzami N, Petit PX, Hirsch F,

Goulbern M, Poupon MF, Miccoli L, Xie Z, Reed JC, Kroemer G. Lonidamine triggers apoptosis via a direct, Bcl-2-inhibited effect on the mitochondrial permeability transition pore. Oncogene. 1999;18(16):2537-46. Epub 1999/06/03.

123. Kroemer G, Marino G, Levine B. Autophagy and the integrated stress response.

Molecular cell. 2010;40(2):280-93. Epub 2010/10/23.

124. Nakahira K, Cloonan SM, Mizumura K, Choi AM, Ryter SW. Autophagy: a crucial moderator of redox balance, inflammation, and apoptosis in lung disease. Antioxidants & redox signaling. 2014;20(3):474-94. Epub 2013/07/25.

160

125. Shen HM, Codogno P. Autophagic cell death: Loch Ness monster or endangered species? Autophagy. 2011;7(5):457-65. Epub 2010/12/15.

126. Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH,

Blagosklonny MV, El-Deiry WS, Golstein P, Green DR, Hengartner M, Knight RA, Kumar

S, Lipton SA, Malorni W, Nunez G, Peter ME, Tschopp J, Yuan J, Piacentini M,

Zhivotovsky B, Melino G. Classification of cell death: recommendations of the

Nomenclature Committee on Cell Death 2009. Cell death and differentiation. 2009;16(1):3-

11. Epub 2008/10/11.

127. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E,

Ohsumi Y, Yoshimori T. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. The EMBO journal. 2000;19(21):5720-8. Epub

2000/11/04.

128. Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K,

Tokuhisa T, Ohsumi Y, Yoshimori T. Dissection of autophagosome formation using Apg5- deficient mouse embryonic stem cells. The Journal of cell biology. 2001;152(4):657-68.

Epub 2001/03/27.

129. Kuma A, Mizushima N, Ishihara N, Ohsumi Y. Formation of the approximately 350- kDa Apg12-Apg5.Apg16 multimeric complex, mediated by Apg16 oligomerization, is essential for autophagy in yeast. The Journal of biological chemistry. 2002;277(21):18619-

25. Epub 2002/03/19.

161

130. Petiot A, Ogier-Denis E, Blommaart EF, Meijer AJ, Codogno P. Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. The Journal of biological chemistry. 2000;275(2):992-8.

Epub 2000/01/08.

131. Seglen PO, Gordon PB. 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proceedings of the National Academy of

Sciences of the United States of America. 1982;79(6):1889-92. Epub 1982/03/01.

132. Deretic V. Autophagosome and Phagosome New Jersey: Humana Press; 2008. 455 p.

133. Xu Y, Yu H, Qin H, Kang J, Yu C, Zhong J, Su J, Li H, Sun L. Inhibition of autophagy enhances cisplatin cytotoxicity through endoplasmic reticulum stress in human cervical cancer cells. Cancer letters. 2012;314(2):232-43. Epub 2011/10/25.

134. Ding ZB, Hui B, Shi YH, Zhou J, Peng YF, Gu CY, Yang H, Shi GM, Ke AW, Wang

XY, Song K, Dai Z, Shen YH, Fan J. Autophagy activation in hepatocellular carcinoma contributes to the tolerance of oxaliplatin via reactive oxygen species modulation. Clinical cancer research : an official journal of the American Association for Cancer Research.

2011;17(19):6229-38. Epub 2011/08/10.

135. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacological reviews.

2006;58(3):621-81. Epub 2006/09/14.

136. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Advances in enzyme regulation. 1984;22:27-

55. Epub 1984/01/01.

162

137. Stephan JS, Yeh YY, Ramachandran V, Deminoff SJ, Herman PK. The Tor and PKA signaling pathways independently target the Atg1/Atg13 protein kinase complex to control autophagy. Proceedings of the National Academy of Sciences of the United States of

America. 2009;106(40):17049-54. Epub 2009/10/07.

138. Hosokawa N, Hara T, Kaizuka T, Kishi C, Takamura A, Miura Y, Iemura S, Natsume

T, Takehana K, Yamada N, Guan JL, Oshiro N, Mizushima N. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Molecular biology of the cell. 2009;20(7):1981-91. Epub 2009/02/13.

139. Jung CH, Jun CB, Ro SH, Kim YM, Otto NM, Cao J, Kundu M, Kim DH. ULK-

Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Molecular biology of the cell. 2009;20(7):1992-2003. Epub 2009/02/20.

140. Paglin S, Lee NY, Nakar C, Fitzgerald M, Plotkin J, Deuel B, Hackett N, McMahill

M, Sphicas E, Lampen N, Yahalom J. Rapamycin-sensitive pathway regulates mitochondrial membrane potential, autophagy, and survival in irradiated MCF-7 cells. Cancer research.

2005;65(23):11061-70. Epub 2005/12/03.

141. Takeuchi H, Kondo Y, Fujiwara K, Kanzawa T, Aoki H, Mills GB, Kondo S.

Synergistic augmentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors. Cancer research. 2005;65(8):3336-

46. Epub 2005/04/19.

142. Hwang J, Lee S, Lee JT, Kwon TK, Kim DR, Kim H, Park HC, Suk K. Gangliosides induce autophagic cell death in astrocytes. British journal of pharmacology.

2010;159(3):586-603. Epub 2010/01/14.

163

143. Garofalo T, Giammarioli AM, Misasi R, Tinari A, Manganelli V, Gambardella L,

Pavan A, Malorni W, Sorice M. Lipid microdomains contribute to apoptosis-associated modifications of mitochondria in T cells. Cell death and differentiation. 2005;12(11):1378-

89. Epub 2005/06/11.

144. Garcia-Ruiz C, Colell A, Morales A, Calvo M, Enrich C, Fernandez-Checa JC.

Trafficking of ganglioside GD3 to mitochondria by tumor necrosis factor-alpha. The Journal of biological chemistry. 2002;277(39):36443-8. Epub 2002/07/16.

145. Rippo MR, Malisan F, Ravagnan L, Tomassini B, Condo I, Costantini P, Susin SA,

Rufini A, Todaro M, Kroemer G, Testi R. GD3 ganglioside directly targets mitochondria in a bcl-2-controlled fashion. FASEB journal : official publication of the Federation of American

Societies for Experimental Biology. 2000;14(13):2047-54. Epub 2000/10/12.

146. Garcia-Ruiz C, Colell A, Paris R, Fernandez-Checa JC. Direct interaction of GD3 ganglioside with mitochondria generates reactive oxygen species followed by mitochondrial permeability transition, cytochrome c release, and caspase activation. FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

2000;14(7):847-58. Epub 2000/04/27.

147. Hodge T, Colombini M. Regulation of metabolite flux through voltage-gating of

VDAC channels. The Journal of membrane biology. 1997;157(3):271-9. Epub 1997/06/01.

148. Rostovtseva T, Colombini M. ATP flux is controlled by a voltage-gated channel from the mitochondrial outer membrane. The Journal of biological chemistry.

1996;271(45):28006-8. Epub 1996/11/08.

164

149. Gincel D, Zaid H, Shoshan-Barmatz V. Calcium binding and translocation by the voltage-dependent anion channel: a possible regulatory mechanism in mitochondrial function. The Biochemical journal. 2001;358(Pt 1):147-55. Epub 2001/08/04.

150. Zaid H, Abu-Hamad S, Israelson A, Nathan I, Shoshan-Barmatz V. The voltage- dependent anion channel-1 modulates apoptotic cell death. Cell death and differentiation.

2005;12(7):751-60. Epub 2005/04/09.

151. Abu-Hamad S, Sivan S, Shoshan-Barmatz V. The expression level of the voltage- dependent anion channel controls life and death of the cell. Proceedings of the National

Academy of Sciences of the United States of America. 2006;103(15):5787-92. Epub

2006/04/06.

152. Geisler S, Holmstrom KM, Skujat D, Fiesel FC, Rothfuss OC, Kahle PJ, Springer W.

PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nature cell biology. 2010;12(2):119-31. Epub 2010/01/26.

153. Halestrap AP, McStay GP, Clarke SJ. The permeability transition pore complex: another view. Biochimie. 2002;84(2-3):153-66. Epub 2002/05/23.

154. Crompton M, Ellinger H, Costi A. Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. The

Biochemical journal. 1988;255(1):357-60. Epub 1988/10/01.

155. Ciechomska I, Legat M, Golab J, Wesolowska A, Kurzaj Z, Mackiewicz A,

Kaminska B. Cyclosporine A and its non-immunosuppressive derivative NIM811 induce apoptosis of malignant melanoma cells in in vitro and in vivo studies. International journal of cancer Journal international du cancer. 2005;117(1):59-67. Epub 2005/05/10.

165

156. Eckstein LA, Van Quill KR, Bui SK, Uusitalo MS, O'Brien JM. Cyclosporin a inhibits calcineurin/nuclear factor of activated T-cells signaling and induces apoptosis in retinoblastoma cells. Investigative ophthalmology & visual science. 2005;46(3):782-90.

Epub 2005/02/25.

157. Munoz M, Rosso M, Gonzalez A, Saenz J, Covenas R. The broad-spectrum antitumor action of cyclosporin A is due to its tachykinin receptor antagonist pharmacological profile.

Peptides. 2010;31(9):1643-8. Epub 2010/06/15.

158. Tringali C, Lupo B, Cirillo F, Papini N, Anastasia L, Lamorte G, Colombi P,

Bresciani R, Monti E, Tettamanti G, Venerando B. Silencing of membrane-associated sialidase Neu3 diminishes apoptosis resistance and triggers megakaryocytic differentiation of chronic myeloid leukemic cells K562 through the increase of ganglioside GM3. Cell death and differentiation. 2009;16(1):164-74. Epub 2008/09/30.

159. Kawashima N, Tsuji D, Okuda T, Itoh K, Nakayama K. Mechanism of abnormal growth in astrocytes derived from a mouse model of GM2 gangliosidosis. Journal of neurochemistry. 2009;111(4):1031-41. Epub 2009/09/22.

160. Moon SK, Kim HM, Lee YC, Kim CH. Disialoganglioside (GD3) synthase gene expression suppresses vascular smooth muscle cell responses via the inhibition of ERK1/2 phosphorylation, cell cycle progression, and matrix metalloproteinase-9 expression. The

Journal of biological chemistry. 2004;279(32):33063-70. Epub 2004/06/04.

161. Maccioni HJ, Daniotti JL, Martina JA. Organization of ganglioside synthesis in the

Golgi apparatus. Biochimica et biophysica acta. 1999;1437(2):101-18. Epub 1999/03/05.

166

162. Ardail D, Popa I, Bodennec J, Louisot P, Schmitt D, Portoukalian J. The mitochondria-associated endoplasmic-reticulum subcompartment (MAM fraction) of rat liver contains highly active sphingolipid-specific glycosyltransferases. The Biochemical journal.

2003;371(Pt 3):1013-9. Epub 2003/02/13.

163. Nara K, Watanabe Y, Kawashima I, Tai T, Nagai Y, Sanai Y. Acceptor substrate specificity of a cloned GD3 synthase that catalyzes the biosynthesis of both GD3 and

GD1c/GT1a/GQ1b. European journal of biochemistry / FEBS. 1996;238(3):647-52. Epub

1996/06/15.

164. Nakayama J, Fukuda MN, Hirabayashi Y, Kanamori A, Sasaki K, Nishi T, Fukuda

M. Expression cloning of a human GT3 synthase. GD3 AND GT3 are synthesized by a single enzyme. The Journal of biological chemistry. 1996;271(7):3684-91. Epub 1996/02/16.

165. Wang Z, Sun Z, Li AV, Yarema KJ. Roles for UDP-GlcNAc 2-epimerase/ManNAc

6-kinase outside of sialic acid biosynthesis: modulation of sialyltransferase and BiP expression, GM3 and GD3 biosynthesis, proliferation, and apoptosis, and ERK1/2 phosphorylation. The Journal of biological chemistry. 2006;281(37):27016-28. Epub

2006/07/19.

166. Liang YJ, Ding Y, Levery SB, Lobaton M, Handa K, Hakomori SI. Differential expression profiles of glycosphingolipids in human breast cancer stem cells vs. cancer non- stem cells. Proceedings of the National Academy of Sciences of the United States of

America. 2013;110(13):4968-73. Epub 2013/03/13.

167. Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. The Journal of clinical investigation.

2002;109(9):1125-31. Epub 2002/05/08.

167

168. Parraga A, Bellsolell L, Ferre-D'Amare AR, Burley SK. Co-crystal structure of sterol regulatory element binding protein 1a at 2.3 A resolution. Structure. 1998;6(5):661-72. Epub

1998/06/23.

169. Shoshan-Barmatz V, De Pinto V, Zweckstetter M, Raviv Z, Keinan N, Arbel N.

VDAC, a multi-functional mitochondrial protein regulating cell life and death. Molecular aspects of medicine. 2010;31(3):227-85. Epub 2010/03/30.

170. Keinan N, Tyomkin D, Shoshan-Barmatz V. Oligomerization of the mitochondrial protein voltage-dependent anion channel is coupled to the induction of apoptosis. Molecular and cellular biology. 2010;30(24):5698-709. Epub 2010/10/13.

171. Britten CD, Baker SD, Denis LJ, Johnson T, Drengler R, Siu LL, Duchin K, Kuhn J,

Rowinsky EK. Oral paclitaxel and concurrent cyclosporin A: targeting clinically relevant systemic exposure to paclitaxel. Clinical cancer research : an official journal of the American

Association for Cancer Research. 2000;6(9):3459-68. Epub 2000/09/22.

172. Qadir M, O'Loughlin KL, Fricke SM, Williamson NA, Greco WR, Minderman H,

Baer MR. Cyclosporin A is a broad-spectrum multidrug resistance modulator. Clinical cancer research : an official journal of the American Association for Cancer Research.

2005;11(6):2320-6. Epub 2005/03/25.

173. Yamashiro S, Okada M, Haraguchi M, Furukawa K, Lloyd KO, Shiku H. Expression of alpha 2,8-sialyltransferase (GD3 synthase) gene in human cancer cell lines: high level expression in melanomas and up-regulation in activated T lymphocytes. Glycoconjugate journal. 1995;12(6):894-900. Epub 1995/12/01.

168

174. Pavel S, van Nieuwpoort F, van der Meulen H, Out C, Pizinger K, Cetkovska P, Smit

NP, Koerten HK. Disturbed melanin synthesis and chronic oxidative stress in dysplastic

naevi. Eur J Cancer. 2004;40(9):1423-30. Epub 2004/06/05.

175. Jenkins NC, Liu T, Cassidy P, Leachman SA, Boucher KM, Goodson AG,

Samadashwily G, Grossman D. The p16(INK4A) tumor suppressor regulates cellular

oxidative stress. Oncogene. 2011;30(3):265-74. Epub 2010/09/15.

176. Gidanian S, Mentelle M, Meyskens FL, Jr., Farmer PJ. Melanosomal damage in

normal human melanocytes induced by UVB and metal uptake--a basis for the pro-oxidant

state of melanoma. Photochemistry and photobiology. 2008;84(3):556-64. Epub 2008/03/12.

177. Chandel NS, Vander Heiden MG, Thompson CB, Schumacker PT. Redox regulation of p53 during hypoxia. Oncogene. 2000;19(34):3840-8. Epub 2000/08/22.

178. Eruslanov E, Kusmartsev S. Identification of ROS using oxidized DCFDA and flow- cytometry. Methods Mol Biol. 2010;594:57-72. Epub 2010/01/15.

179. McQuade LE, Lippard SJ. Fluorescent probes to investigate nitric oxide and other reactive nitrogen species in biology (truncated form: fluorescent probes of reactive nitrogen species). Current opinion in chemical biology. 2010;14(1):43-9. Epub 2009/11/21.

180. Navarro-Antolin J, Lopez-Munoz MJ, Klatt P, Soria J, Michel T, Lamas S. Formation of peroxynitrite in vascular endothelial cells exposed to cyclosporine A. FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

2001;15(7):1291-3. Epub 2001/05/10.

181. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science.

1998;281(5381):1312-6. Epub 1998/08/28.

169

182. Jo GH, Kim GY, Kim WJ, Park KY, Choi YH. Sulforaphane induces apoptosis in

T24 human urinary bladder cancer cells through a reactive oxygen species-mediated mitochondrial pathway: The involvement of endoplasmic reticulum stress and the Nrf2 signaling pathway. International journal of oncology. 2014. Epub 2014/07/06.

183. Nieminen AL, Saylor AK, Tesfai SA, Herman B, Lemasters JJ. Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t- butylhydroperoxide. The Biochemical journal. 1995;307 ( Pt 1):99-106. Epub 1995/04/01.

184. Shoshan-Barmatz V, Keinan N, Abu-Hamad S, Tyomkin D, Aram L. Apoptosis is regulated by the VDAC1 N-terminal region and by VDAC oligomerization: release of cytochrome c, AIF and Smac/Diablo. Biochimica et biophysica acta. 2010;1797(6-7):1281-

91. Epub 2010/03/11.

185. Qian T, Herman B, Lemasters JJ. The mitochondrial permeability transition mediates both necrotic and apoptotic death of hepatocytes exposed to Br-A23187. Toxicology and applied pharmacology. 1999;154(2):117-25. Epub 1999/02/02.

186. Ly JD, Grubb DR, Lawen A. The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update. Apoptosis : an international journal on programmed cell death.

2003;8(2):115-28. Epub 2003/05/27.

187. Scaduto RC, Jr., Grotyohann LW. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophysical journal. 1999;76(1 Pt 1):469-77. Epub

1999/01/06.

188. Dhanasekaran DN, Reddy EP. JNK signaling in apoptosis. Oncogene.

2008;27(48):6245-51. Epub 2008/10/22.

170

189. Matsuzawa A, Ichijo H. Redox control of cell fate by MAP kinase: physiological roles of ASK1-MAP kinase pathway in stress signaling. Biochimica et biophysica acta.

2008;1780(11):1325-36. Epub 2008/01/22.

190. Fan M, Goodwin ME, Birrer MJ, Chambers TC. The c-Jun NH(2)-terminal protein kinase/AP-1 pathway is required for efficient apoptosis induced by vinblastine. Cancer research. 2001;61(11):4450-8. Epub 2001/06/05.

191. Kharbanda S, Saxena S, Yoshida K, Pandey P, Kaneki M, Wang Q, Cheng K, Chen

YN, Campbell A, Sudha T, Yuan ZM, Narula J, Weichselbaum R, Nalin C, Kufe D.

Translocation of SAPK/JNK to mitochondria and interaction with Bcl-x(L) in response to

DNA damage. The Journal of biological chemistry. 2000;275(1):322-7. Epub 2000/01/05.

192. Anasagasti MJ, Martin JJ, Mendoza L, Obrador E, Estrela JM, McCuskey RS, Vidal-

Vanaclocha F. Glutathione protects metastatic melanoma cells against oxidative stress in the murine hepatic microvasculature. Hepatology. 1998;27(5):1249-56. Epub 1998/05/15.

193. Mena S, Benlloch M, Ortega A, Carretero J, Obrador E, Asensi M, Petschen I, Brown

BD, Estrela JM. Bcl-2 and glutathione depletion sensitizes B16 melanoma to combination therapy and eliminates metastatic disease. Clinical cancer research : an official journal of the

American Association for Cancer Research. 2007;13(9):2658-66. Epub 2007/05/03.

194. Frischer H, Ahmad T. Severe generalized glutathione reductase deficiency after antitumor chemotherapy with BCNU" [1,3-bis(chloroethyl)-1-nitrosourea]. The Journal of laboratory and clinical medicine. 1977;89(5):1080-91. Epub 1977/05/01.

195. Kehrer JP. The effect of BCNU (carmustine) on tissue glutathione reductase activity.

Toxicology letters. 1983;17(1-2):63-8. Epub 1983/06/01.

171

196. Kohsaka S, Takahashi K, Wang L, Tanino M, Kimura T, Nishihara H, Tanaka S.

Inhibition of GSH synthesis potentiates temozolomide-induced bystander effect in glioblastoma. Cancer letters. 2013;331(1):68-75. Epub 2012/12/19.

197. Xiao D, Choi S, Lee YJ, Singh SV. Role of mitogen-activated protein kinases in phenethyl isothiocyanate-induced apoptosis in human prostate cancer cells. Molecular carcinogenesis. 2005;43(3):130-40. Epub 2005/05/10.

198. Tournier C, Hess P, Yang DD, Xu J, Turner TK, Nimnual A, Bar-Sagi D, Jones SN,

Flavell RA, Davis RJ. Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science. 2000;288(5467):870-4. Epub 2000/05/08.

199. Aoki H, Kang PM, Hampe J, Yoshimura K, Noma T, Matsuzaki M, Izumo S. Direct activation of mitochondrial apoptosis machinery by c-Jun N-terminal kinase in adult cardiac myocytes. The Journal of biological chemistry. 2002;277(12):10244-50. Epub 2002/01/12.

200. Schroeter H, Boyd CS, Ahmed R, Spencer JP, Duncan RF, Rice-Evans C, Cadenas E. c-Jun N-terminal kinase (JNK)-mediated modulation of brain mitochondria function: new target proteins for JNK signalling in mitochondrion-dependent apoptosis. The Biochemical journal. 2003;372(Pt 2):359-69. Epub 2003/03/05.

201. Radi R. Peroxynitrite, a stealthy biological oxidant. The Journal of biological chemistry. 2013;288(37):26464-72. Epub 2013/07/19.

202. Buettner GR. Superoxide dismutase in redox biology: the roles of superoxide and hydrogen peroxide. Anti-cancer agents in medicinal chemistry. 2011;11(4):341-6. Epub

2011/04/02.

172

203. Vazquez F, Lim JH, Chim H, Bhalla K, Girnun G, Pierce K, Clish CB, Granter SR,

Widlund HR, Spiegelman BM, Puigserver P. PGC1alpha expression defines a subset of human melanoma tumors with increased mitochondrial capacity and resistance to oxidative stress. Cancer cell. 2013;23(3):287-301. Epub 2013/02/19.

204. Corazao-Rozas P, Guerreschi P, Jendoubi M, Andre F, Jonneaux A, Scalbert C,

Garcon G, Malet-Martino M, Balayssac S, Rocchi S, Savina A, Formstecher P, Mortier L,

Kluza J, Marchetti P. Mitochondrial oxidative stress is the Achille's heel of melanoma cells resistant to Braf-mutant inhibitor. Oncotarget. 2013;4(11):1986-98. Epub 2013/10/29.

205. Nogueira V, Park Y, Chen CC, Xu PZ, Chen ML, Tonic I, Unterman T, Hay N. Akt determines replicative senescence and oxidative or oncogenic premature senescence and sensitizes cells to oxidative apoptosis. Cancer cell. 2008;14(6):458-70. Epub 2008/12/09.

206. Lorico A, Rappa G, Flavell RA, Sartorelli AC. Double knockout of the MRP gene leads to increased drug sensitivity in vitro. Cancer research. 1996;56(23):5351-5. Epub

1996/12/01.

207. Rappa G, Lorico A, Flavell RA, Sartorelli AC. Evidence that the multidrug resistance protein (MRP) functions as a co-transporter of glutathione and natural product toxins. Cancer research. 1997;57(23):5232-7. Epub 1997/12/11.

208. Muller M, Meijer C, Zaman GJ, Borst P, Scheper RJ, Mulder NH, de Vries EG,

Jansen PL. Overexpression of the gene encoding the multidrug resistance-associated protein results in increased ATP-dependent glutathione S-conjugate transport. Proceedings of the

National Academy of Sciences of the United States of America. 1994;91(26):13033-7. Epub

1994/12/20.

173

209. Ishikawa T. The ATP-dependent glutathione S-conjugate export pump. Trends in biochemical sciences. 1992;17(11):463-8. Epub 1992/11/01.

210. Versantvoort CH, Broxterman HJ, Bagrij T, Scheper RJ, Twentyman PR. Regulation by glutathione of drug transport in multidrug-resistant human lung tumour cell lines overexpressing multidrug resistance-associated protein. British journal of cancer.

1995;72(1):82-9. Epub 1995/07/01.

211. Nakano J, Raj BK, Asagami C, Lloyd KO. Human melanoma cell lines deficient in

GD3 ganglioside expression exhibit altered growth and tumorigenic characteristics. The

Journal of investigative dermatology. 1996;107(4):543-8. Epub 1996/10/01.

212. Wu G, Fang YZ, Yang S, Lupton JR, Turner ND. Glutathione metabolism and its implications for health. The Journal of nutrition. 2004;134(3):489-92. Epub 2004/02/28.

213. Lu SC. Regulation of glutathione synthesis. Current topics in cellular regulation.

2000;36:95-116. Epub 2000/06/08.

214. Goldstein JL, DeBose-Boyd RA, Brown MS. Protein sensors for membrane sterols.

Cell. 2006;124(1):35-46. Epub 2006/01/18.

215. Grimm MO, Zinser EG, Grosgen S, Hundsdorfer B, Rothhaar TL, Burg VK, Kaestner

L, Bayer TA, Lipp P, Muller U, Grimm HS, Hartmann T. Amyloid precursor protein (APP) mediated regulation of ganglioside homeostasis linking Alzheimer's disease pathology with ganglioside metabolism. PloS one. 2012;7(3):e34095. Epub 2012/04/04.

216. Lin CJ, Lee CC, Shih YL, Lin TY, Wang SH, Lin YF, Shih CM. Resveratrol enhances the therapeutic effect of temozolomide against malignant glioma in vitro and in vivo by inhibiting autophagy. Free radical biology & medicine. 2012;52(2):377-91. Epub

2011/11/19.

174

217. Silber JR, Bobola MS, Blank A, Schoeler KD, Haroldson PD, Huynh MB, Kolstoe

DD. The apurinic/apyrimidinic endonuclease activity of Ape1/Ref-1 contributes to human glioma cell resistance to alkylating agents and is elevated by oxidative stress. Clinical cancer research : an official journal of the American Association for Cancer Research.

2002;8(9):3008-18. Epub 2002/09/17.

218. Khor TO, Keum YS, Lin W, Kim JH, Hu R, Shen G, Xu C, Gopalakrishnan A, Reddy

B, Zheng X, Conney AH, Kong AN. Combined inhibitory effects of curcumin and phenethyl isothiocyanate on the growth of human PC-3 prostate xenografts in immunodeficient mice.

Cancer research. 2006;66(2):613-21. Epub 2006/01/21.

175

Vita

Kausar Begam Riaz Ahmed was born in Chennai, India on November 15, 1984, the daughter of Rahimunissa Riaz Ahmed and Riaz Ahmed. After completing her high school at Holy

Family Convent Higher Secondary School, Chennai, India in 2002, she entered Anna

University in Chennai, India. She received the degree of Bachelor of Technology with a major in industrial biotechnology from Anna University in May, 2006. She then travelled to the United States of America to pursue a Masters in Biotechnology at the University of Utah.

In August of 2008 she entered The University of Texas Graduate School of Biomedical

Sciences at Houston.

Permanent address:

No.1, Swami Malai Nagar,

Old Pallavaram, Chennai – 600 117,

India.

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