A Novel Resveratrol Analog: Its Cell Cycle Inhibitory, Pro-Apoptotic and Anti-Inflammatory Activities on Human Tumor Cells

A NOVEL RESVERATROL ANALOG : ITS CELL CYCLE INHIBITORY, PRO-APOPTOTIC AND ANTI-INFLAMMATORY ACTIVITIES ON HUMAN TUMOR CELLS

A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Boren Lin

May 2006

Dissertation written by

Boren Lin

B.S., Tunghai University, 1996

M.S., Kent State University, 2003

Ph. D., Kent State University, 2006

Approved by

Dr. Chun-che Tsai , Chair, Doctoral Dissertation Committee

Dr. Bryan R. G. Williams , Co-chair, Doctoral Dissertation Committee

Dr. Johnnie W. Baker , Members, Doctoral Dissertation Committee

Dr. James L. Blank ,

Dr. Bansidhar Datta ,

Dr. Gail C. Fraizer ,

Accepted by

Dr. Robert V. Dorman , Director, School of Biomedical Sciences

Dr. John R. Stalvey , Dean, College of Arts and Sciences

TABLE OF CONTENTS

LIST OF FIGURES……………………………………………………………….………v

LIST OF TABLES……………………………………………………………………….vii

ACKNOWLEDGEMENTS….………………………………………………………….viii

I INTRODUCTION….………………………………………………….1

Background and Significance……………………………………………………..1

Specific Aims………………………………………………………………………12

II MATERIALS AND METHODS.…………………………………………….16

Cell Culture and Compounds…….……………….…………………………….….16

MTT Cell Viability Assay………………………………………………………….16

Trypan Blue Exclusive Assay……………………………………………………...18

Flow Cytometry for Cell Cycle Analysis……………..……………....……………19

DNA Fragmentation Assay……………………………………………...…………23

Caspase-3 Activity Assay………………………………...……….….…….………24

Annexin V-FITC Staining Assay…………………………………..…...….………28

NF-kappa B p65 Activity Assay……………………………………..………….…29

COX Enzyme Inhibition Assay……………………………………..….…..………30

Free Radical Scavenging Assay……………………………………..…………….33

iii Fluorescent DCF Assay……………….……………………………..…………….35

Cancer-related Gene Microarray………………………………………….……….36

III RESULTS ……………………....……………………………..…………..40

Inhibition of Cancer Cell Proliferation by the Compounds…………..…….40

Cell Cycle Analysis of Cancer Cells Treated with Resveratrol and KST201……...51

DNA Fragmentation Induced by Resveratrol and KST201 in DU145…………….53

Quantification of Proteolytic Activity of Caspase-3 in Resveratrol- and KST201-

treated DU145 cells…………………...………………………………………...…58

Morphological Changes and Phospholipid Flip-flop on Cell Membranes Induced

by Resveratrol and KST201 in DU145…...……………………………………….60

Quantification of DNA-binding Activity of NF-kappa B p65 in Resveratrol- and

KST201-treated DU145 Cells…………………….……………………………….78

Measurement of Inhibitory Activity against COX Enzymes..…………………….81

Antioxidant Activity of Resveratrol and KST201 and the Effect of Hydrogen

Peroxide on DU145 Cells…………………...…………………………………….84

Cancer-related Gene Expression in Resveratrol- and KST201-treated DU145 ….89

IV DISCUSSION……………………………………………..………………………112

V REFERENCES………..…..………….…………………………..…….………..133

iv LIST OF FIGURES

Fig. 1: Resveratrol Shares Structural Similarity with Flavonoids and Hormones.....5

Fig. 2: The Model Used for New Anticancer Agent Design………………………..13

Fig. 3: Structural Basis for the Design of Novel Resveratrol Analogs….………14

Fig. 4: Chemical Reaction to Convert Purple MTT Formazan from MTT (3-(4,

5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide)…………………………..18

Fig. 5: Chemical Structure of Trypan Blue…………..…………………………19

Fig. 6: Illustration of Flow Cytometer and Chemical Structure of PI (Propidium Iodide)

Used for DNA Labeling………………………………………………………….20

Fig. 7: The Cell Cycle……………………………..………………..…….…….…21

Fig. 8: Principle of the Enzyme-linked Immunosorbent Assay (ELISA) for DNA

Fragmentation Study……………………………………………………………………23

Fig. 9: Death Receptor Pathway of Apoptosis Signaling……………..…….…25

Fig. 10: Mitochondrial Pathway of Apoptosis Signaling……………....……………...26

Fig. 11 Principle of the Enzyme-linked Immunosorbent Assay (ELISA) for caspase-3

Activity Study………………………………………………………………………27

Fig. 12: Principle of the Enzyme-linked Immunosorbent Assay (ELISA) for NF-kappa B p65 Activity Study……………………………………………………………………30

Fig. 13: Principle of the Enzyme-linked Immunosorbent Assay (ELISA) for COX enzyme

Activity Study…………………………………………………………….32

Fig. 14: Chemical Structure of Reagents Used in Free Radical Scavenging Assays...34

v Fig. 15: Chemical Reaction to Convert fluorescent DCF (2'7'-dichlorofluorescein) from

DCFH-DA (2',7'-dichlorofluorescein diacetate)…………………………………..……36

Fig. 16: Principle of the cDNA Microarray Techniqu………………………………37

Fig. 17A-C. SCI Changes of the Compounds on Cancer Cells..………...………45-47

Fig. 18A-C. Inhibitory Activity of Resveratrol and KST201 on Cell Proliferation.48-50

Fig. 19: Live Cells Counting Using Trypan Blue Staining……………………….…52

Fig. 20: Cell Cycle Analysis by Flow Cytometry on resveratrol-treated DU145……54

Fig. 21: Cell Cycle Analysis by Flow Cytometry on KST201-treated DU145……55

Fig. 22:. Cell Cycle Analysis by Flow Cytometry on KST201-treated MDAH…….56

Fig. 23: Cell Cycle Analysis by Flow Cytometry on KST201-treated T24…………57

Fig. 24: Quantification of DNA Fragmentation induced in DU145…………..……59

Fig. 25A-B: Quantification of Caspase-3 Activity KST201-treated DU145………61-62

Fig. 26: Quantification of Caspase-3 Activity Resveratrol-treated DU145…………63

Fig. 27A-B: Images of Untreated DU145…………………………………………64-65

Fig. 28A-B: Images of Resveratrol-treated DU145……….………………………66-67

Fig. 29A-D: Images of KST201-treated DU145……………………………..……68-71

Fig. 30A-C: A Closer Look of Annexin V-FITC Stained KST201-treated DU145..72-74

Fig. 31: Images of Resveratrol-treated DU145 (40x)…………………………………75

Fig. 32A-B: Images of KST201-treated DU145 (40x)……………………...……76-77

Fig. 33A-B: Quantification of DNA-binding Activity of NF-kappa B………..…79-80

Fig. 34A-B: Measurement of Inhibitory Activity against COX Enzymes………82-83

Fig. 35A-B: Free Radical Scavenging Activity of Resveratrol and KST201….85-86

vi Fig. 36: Measurement of Fluorescent DCF in DU145………………………..……87

Fig. 37: Effect of Hydrogen Peroxide on Anti-proliferatory Activity of Resveratrol and

KST201………………………………………………………………………………..…88

Fig. 38: Non-selective COX Inhibitor Flurbiprofen and COX2 Inhibitor SC55…118

Fig. 39: Illustration of the Catalytic Channel of Cyclooxygenase in the Presence of

Flurbiprofen…………………………………………………………………..……..119

Fig. 40: Regulation of NF-kappa B Signaling Pathway by Resveratrol and KST201.127

Fig. 41: Illustration of Cell Survival/Cell Death Status in Normal Cells, Cancer Cells and

Resveratrol- and KST201-treated Cells…………………………………....………129

vii LIST OF TABLES

Table 1: Effects of Resveratrol on Different Cell Signaling Pathway…………6

Table 2: Cytotoxicity of Testing Compounds on DU145 and MHRF…………42

Table 3: Cytotoxicity of Testing Compounds on MDAH and MHRF…………..43

Table 4: Cytotoxicity of Testing Compounds on T24 and MHRF………………44

Table 5: Cancer-related Gene Expression of Resveratrol-treated DU145.……90-92

Table 6: Cancer-related Gene Expression of KST201-treated DU145………..98-102

Table 7. Distribution of Differentially Expressed Genes among Categories of Biological

Processes and Molecular Functions…………………………..……………………..103

Table 8: Cancer-related Genes Significantly Regulated by Both Resveratrol and KST201 in DU145…………………………………………………………………………...….104

viii ACKNOWLEDGEMENTS

I would like to express my sincere thanks to:

Drs. Chun-che Tsai and Bryan R. G. Williams, my advisors who have enlightened and

guided me to gain the knowledge I need to achieve my goal.

Dr. James M. Jamison and Deborah R. Neal, my masters who instructed me on all

techniques used in the laboratory and assisted me in solving all the problems.

Drs. Johnnie W. Baker, James L. Blank, Bansidhar Datta and Gail C. Fraizer, my

committee members who have given me invaluable suggestions for my search work.

Drs. Thomas S. Alexander, Pieter W. Faber, Hans G. Folkesson and Michael A. Model,

who have kindly offered me instruments and technical support for many key experiments.

Faculty and staff in Chemistry and Biomedical Sciences at Kent State University, who have been patient, friendly and always ready for help.

My parents Ming Fong Lin and Ya Lan Hsih, who are always supporting me both morally

and financially.

My wife Ling Kuo and son Gene, who are always tolerant of my impatience and

disregard.

LIFE WOULD NOT BE THAT WONDERFUL WITHOUT YOU.

ix 1

INTRODUCTION

Background and Significance

Carcinogenesis is a multistage process controlled by a variety of factors which cause damage to the genetic material or other intracellular components of the cell. Heredity, lifestyle, viruses, external stress and stimuli, hormones and immunology have been implicated in carcinogenesis directly or indirectly (53, 135, 145). Most cancers derive from a single abnormal cell which has undergone an initial mutation. The single primary tumor acquires further changes of basic behavior allowing its progeny to be able to disregard signals which regulate cell proliferation, cell differentiation or cell death, and moreover to become invasive and metastatic (53, 135, 140). The latent period before diagnosis of cancer is relatively long since multiple mutations have to be accumulated.

Mutations in genes can result in overproduced, under-produced or dysfunctional proteins, which become critical in the process of cancer formation if these proteins play important roles in normal cell proliferation (143, 145). Normal cell proliferation is tightly controlled by the signaling network via growth factors, cytokines and other signaling molecules.

Proto-oncogenes encode proteins involved in these cascades of signal transduction. When mutated through interaction with carcinogens, they become oncogenetic. Guanine nucleotide binding protein ras, transcription factor fos, jun and myc were examples which are mutant in many cancers (143, 148). Oncogenes overproduce their protein products activating their downstream molecules without restriction, and thus the mechanisms of 2

cell proliferation can be altered leading to the abnormal cell growth. On the other hand, tumor suppressor genes serve as brakes for carcinogenesis (142, 143, 148, 149). They encode proteins responsible for the induction of cell death and prevention of inappropriate cell growth. For example, mutations often inactivate or down-regulate the

tumor suppressor p53 which functions as a regulator of cell growth, DNA repair and

apoptosis, thus contributes to uncontrolled cell proliferation (149). The activation of

oncogenes and the inactivation of tumor suppressor genes have been both observed in

human cancers.

Carcinogenesis involves three stages: initiation, promotion and progression. Initiation

occurs because of random or carcinogen-induced DNA damage (53, 135, 145). Usually

the mechanisms responsible for DNA repair protect the cell from the initial mutation;

however, if the DNA damage is located at the gene that regulates DNA repair or cell proliferation, the risk of becoming transformed can be increased. A population of mutated cells is formed during tumor promotion by continuous and unrestrained stimulation of

cell growth. Hormones and reactive oxygen species are substances involved in human

cancer promotion (53, 135, 145). Further growth and expansion of the tumor cells over

normal cells is observed during the stages of progression. Genes in tumor cells are

considered less stable and more sensitive to additional mutations. Mutations of oncogenes,

tumor suppressor genes, and DNA mismatch-repair genes are further accumulated.

Eventually, proteins responsible for cell adhesion, migration, invasion and metastasis are

abnormally altered leading to the formation of secondary tumors growing at new sites of

the body because of the spread of primary tumor cells through the circulatory system (53, 3

135, 140, 143, 145).

Oxidative stress and reactive oxygen species (ROS) increase due to the increase in

oxidant generation and/or the decrease in antioxidant protection. Numerous biological

processes can be influenced by ROS that result in variations of the redox conditions in

the cell. Such variations may cause changes in signal transduction cascades upstream of

nuclear transcription factors (92). ROS are free radicals or molecules containing oxygen

atoms that produce free radicals such as hydroxyl radicals, superoxide, hydrogen

peroxide and peroxynitrite. They are generated by cellular respiration, ionizing radiation of biological molecules, cytochrome P450 metabolism of xenobiotic compounds and phagocytic cells like neutrophils and macrophages, as part of the cellular defense system

(1, 64, 92, 93, 144). The main damage to the cell results from the ROS-induced alteration of macromolecules such as polyunsaturated fatty acids in membrane lipids, essential

proteins and DNA. In addition, ROS serve as secondary messengers for various

physiological and pathological stimuli, such as inflammatory cytokines, angiotensin,

growth factors, ionizing radiation and others (1), and thus induce MAPK family (54),

ERK5 or BMK1 (big MAP kinase) (2) and protein kinase C (PKC) (28). ROS may

interact with signaling molecules which are differently sensitive to oxidation according to

their content of critical cysteine residues, to their conformation or to the intensity of the oxidative stress (119). Therefore, ROS can regulate protein phosphorylation by

modifying the phosphorylation/dephosphorylation cascade and play a role in modulating

several biochemical events that control cell growth, differentiation and apoptosis.

Evidence has been published suggesting that ROS at low concentration promote 4

mutations, chromosomal aberrations or carcinogenesis, whereas it leads to cytotoxicity

and cell death at high concentrations (1, 64, 92, 93, 144).

One of the strategies to prevent or attenuate cancer formation is to block one or several

steps in this multistage process. For instance, some potential anticancer agents either

inhibit abnormal cell proliferation or trigger cell death by interacting with key

components in signaling pathways responsible for regulation of cell behaviors (49, 56,

106). Phytochemicals, responding to injury and infection as part of the defense system in plants, have been studied for decades. Many of them exert multiple effects which are beneficial to human health. A non-flavonoid polyphenol resveratrol

(3,5,4'-trihydroxy-trans-stilbene) (Fig. 1) is a typical example. It has diverse bioactivities

(3, 39) (Table 1) including antioxidant activity, modulation of lipid and lipoprotein

metabolism, anti-platelet aggregation, vasorelaxing activity, anticancer activity,

estrogenic activity, etc. These contribute to the prevention of development and

progression of cardiovascular disease and cancer (3, 9, 35, 39, 49, 56, 106, 115).

Resveratrol, an active ingredient in several folk medicines in Asia used to treat

inflammatory and allergic diseases, is produced by a variety of plants, such as grapes,

peanuts and mulberries in response to fungal infection, wounding and irradiation with

ultraviolet light. Fresh grape skin contains 50-100 mg resveratrol per gram, and red wine,

containing 10 µM resveratrol on average, is the most well-known source of resveratrol.

The chemical structure of resveratrol consists of two aromatic rings linked by a styrene

double bond with two hydroxyl groups at the 3 and 5 positions of one ring and one

hydroxyl group at the 4’ position of the other ring. It is less soluble in water then in 5

Resveratrol Piceid, a Glucoside Analog of Resveratrol

Diethylstilbestrol, a Synthetic Estrogen Estradiol, an Estrogen

Testosterone, an Androgen Apigenin, a Flavone

Epigallocatechin, a Flavan-3-ol Quercetin, a Flavonol

Fig. 1. Resveratrol Shares Structural Similarity with Flavonoids and Hormones 6

Effects of Resveratrol on Different Cell Signaling Pathways

Activation of adenyl-cyclase pathway Modulation of metabolism of carcinogens

Activation of ceramide pathway Modulation of NO/NOS pathway

Activation of p53 pathway Radioprotective and radiosensitive

Antioxidant effects Regulation of Egr-1 pathway

Chemosensitization Suppression of adhesion molecules

Effects on bone cells Suppression of androgen receptors

Suppression of angiogenesis, invasion and Effects on normal cells metastasis

Estrogenic/antiestrogenic effects Suppression of cell cycle proteins

Immunomodulatory effects Suppression of COX2 and lipooxygenase

Suppression of growth factors and associated Induction of cellular differentiation kinases Suppression of inflammatory cytokines and Inhibition of AP-1 signaling pathway inflammation

Inhibition of MAPK pathway Suppression of mutagenesis

Inhibition of mitochondrial pathway Suppression of protein kinases

Inhibition of NF-kappa B signaling pathway Suppression of PSA

Inhibition of Rb/E2FDP pathway Suppression of tranformation

Inhibition of the expression of cytochrome Up-regulation of Fas pathway p450

Inhibition of tubulin polymerazation pathway

Table 1. Effects of Resveratrol on Different Cell Signaling Pathway

ethanol and dimethyl sulfoxide (DMSO) with a melting point of 253-255°C and

molecular weight of 228.25. The trans-isomer was shown more stable compared to the

cis-isomer (3).

The anticancer potential of resveratrol was first published in 1997 when it was found to

inhibit dimethylbenzanthracene (DMBA)-induced pre-neoplastic lesion formation in mouse mammary organ culture, and to reduce the incidence and multiplicity of

DMBA/12-tetradecanoylphorbol-13-acetate (TPA)-induced papillomas in the mouse skin.

Significant efforts have been made over a decade trying to demonstrate the mechanisms

involved in its chemopreventive and chemotherapeutic properties. A variety of models

and approaches were utilized generating numerous evidence that promised resveratrol to

be an anticancer candidate. Mechanisms modulated by resveratrol included cell survival,

cell division, cell death, DNA synthesis, inflammation and phase II detoxification, to

name a few (3, 9, 35, 39, 49, 56, 106, 115).

Biotransformation is a process converting xenobiotics (lipophilic pro-carcinogens) to

more hydrophilic compounds in order to be excretable from the cell. Two steps were

involved: oxidation by phase I enzymes such as cytochrome P450 monooxygenase and

conjugation with polar groups by phase II enzymes such as glutathione-S –transferase and

NAD(P)H:quinone oxidoreductase. Nevertheless, highly reactive radicals which might

damage DNA were generated during the process. Chemoprevention of resveratrol

includes inhibition of phase I enzymes and activation of phase II enzymes which block

the initiation of carcinogenesis (3, 9, 35, 39, 49, 56).

Resveratrol was reported to inhibit cell proliferation in a variety of cell models and result 8

in apoptotic cell death by modulating numerous key mediators of cell cycle and survival

signaling. Depending on the concentrations, resveratrol switched cells between reversible

cell cycle arrest and irreversible apoptosis. Cyclin-dependent kinases and their activators

cyclins and inhibitors control the cell cycle machinery. Responding to stress, p53 serves

as a tumor suppressor by activating cell cycle inhibitor p21 and pro-apoptotic Bcl-2

family members and caspases. Treatment with resveratrol caused blockage of cell cycle

in the G0/G1 phase, G1/S transition, S phase or G2/M phase cell line-dependently through

suppression of cyclins and their corresponding kinases, induction of p53 and cell cycle

inhibitors, or inhibition of DNA synthesis (3, 9, 35, 49, 56, 106, 115). Cells undergoing

DNA synthesis are more sensitive to apoptosis-inducing agents; therefore, resveratrol-induced S-phase blockage may lead to apoptosis. The Bcl2 family members include pro- and anti-apoptotic proteins. Pro-apoptotic Bcl-2 members activate caspases leading to apoptosis, whereas anti-apoptotic Bcl-2 proteins inhibit their activity through heterodimerization with them. Resveratrol was able to cause up-regulation of pro-apoptotic Bcl-2 members and down-regulation of anti-apoptotic Bcl-2 members (3, 9,

35, 49, 56, 106, 115), which then released cytochrome c from mitochondria and initiated the apoptotic signal transduction. NF-kappa B and AP-1 are transcription factors involved in stress responses and cell proliferation through production of cell survival signals.

Resveratrol was shown to inhibit NF-kappa B and AP-1 signaling pathway as well as their upstream kinases and downstream targets including inducible cyclooxygenase-2, inducible nitric oxide synthase and matrix metalloprotease-9. These effects then led to anti-proliferation and induction of cell death (3, 9, 35, 49, 56, 106, 115). 9

Abnormal stimulation of hormone may favor cell proliferation leading to carcinogenesis.

Selective estrogen receptor modulators (SERMs) have been used to prevent development of hormone-dependent cancers. Resveratrol was considered a phytoestrogen because of its chemical structure resembling to estrogens. Although with lower affinity comparing to estradiol, a principal female sex hormone, resveratrol can compete with estrogens for their receptors and activate hormone receptor-mediated gene transcription. However, resveratrol also exerted an anti-estrogen action and inhibited hormone-induced carcinogenesis (3, 39). The agonistic or antagonistic hormonal activity of resveratrol depends on the intake concentration, tissue-specific expression of estrogen receptors, cofactors present for DNA binding and different gene promoters (3, 39).

According to the statistics provided by Prostate Cancer Foundation, prostate cancer is the most common non-skin cancer in United States, and in 2006, over 232,000 men will be diagnosed with prostate cancer, and over 30,000 men will die from it. Japanese men develop a higher incidence of prostate cancer after their migration to United States suggesting that environmental factors such as diet and lifestyle are determinants to prostate carcinogenesis. Prostate carcinoma is hormone dependent initially but eventually progresses to a hormone-independent phenotype after androgen deprivation therapy (7,

27, 45, 53, 135). Various cell lines have been developed corresponding to different stages of prostate cancer. For instance, LNCaP cells (hormone-sensitive prostate cells with wide-type p53) are often used to mimic the initial stage of prostate carcinoma, PC-3 cells

(hormone-independent cells possessing dysfunctional androgen receptors and p53-null) and DU145 cells (hormone-independent cells lacking androgen receptors with mutant p53) 10

resemble the advanced stage of prostate carcinoma. The anti-carcinogenesis activity of

resveratrol against human prostate cancer has been reported using both

hormone-dependent and hormone-independent models in vitro (97).

Mitchell and colleagues showed that resveratrol repressed different classes of androgen

up-regulated genes at the protein or mRNA level, which might be attributable to the

reduction in androgen receptor (AR) content. Thus resveratrol may inhibit

androgen-stimulated cell growth and gene expression in hormone-dependent prostate

cancer cells LNCaP (82). Moreover, it was demonstrated that overexpression of c-Jun

induced by resveratrol inhibited the expression and function of AR (141). Resveratrol

effects on AR are complex, as at low concentrations, resveratrol activated AR-stumilated

gene expression through the Raf-MEK-ERK kinase pathway, whereas it repressed the

AR-dependent reporter gene activity at high concentrations (44). Additionally, Hsieh’s

group reported that resveratrol decreased expression of prostate-specific antigen (PSA) in

LNCaP cells AR-independently (52).

A variety of phenolic phytoalexins have been tested against prostate cancer cells. Kampa

and colleagues reported that resveratrol, prepared from wine extract, was the most potent

cell growth inhibitor on hormone-independent prostate cancer DU145 cells, whereas

flavonoids (catechin, epicatechin and quercetin) were preferentially more effective on

LNCaP and hormone-independent PC-3 cells. Their anti-proliferatory action correlated

with the decreased nitric oxide production (59). Resveratrol blocked cell cycle progression at G1/S transition in all androgen-independent cell lines, but not in the androgen-responsive

LNCaP cells (51). Resveratrol induced LNCaP cells but not DU145 to enter S phase, but at 11

concentrations above 15 μM, it blocked the progression through S phase via the inhibitory effect on DNA synthesis (71). Evidence indicated that the cell cycle blockage of resveratrol-treated DU145 was associated with the inhibition of cyclin D and cyclin-dependent kinase (Cdk) 4 expression along with the induction of p53 and Cdk inhibitor p21. Furthermore, cyclin E and Cdk 2 were also inhibited but not at their transcription levels (62). These findings indicated that resveratrol regulated cell cycle on both hormone-dependent and hormone-independent prostate cancer cells.

Resveratrol induced apoptosis in LNCaP cells; however, it was inhibited by epidermal

growth factor (EGF) via a PKC-alpha-mediated mechanism (109). In PC-3 cells,

resveratrol suppressed the EGF receptor-dependent ERK1/2 activation pathway stimulated by EGF and phorbol ester and inhibited PKC-alpha suggesting that resveratrol may

manipulate the phosphorylation during signal transduction mediated by kinases (117). Lin

and colleagues showed that resveratrol induced apoptosis in DU145 cells through the

activation of MAPK leading to phosphorylation of p53 (75). The pro-apoptotic molecule

Bax, but not anti-apoptotic Bcl-2 and Bcl-xL, was shown up-regulated at both protein and

mRNA levels, and caspase-3 and caspase-9 were induced as well (62). Using the same cell

line, cell viability was reduced and membrane breakdown was increased by resveratrol at

the concentrations of 50 and 100 μM (22). In addition, at high concentration (100 or 200

μM), resveratrol exerted its anticancer activity through necrosis instead of apoptosis (107).

Especially for drug candidates targeting multiple signaling pathways, microarray is a

powerful technique which can be utilized to investigate alterations of gene expression

patterns in treated cells (19, 23). The global effects of resveratrol on gene expression of 12

LNCaP have been studied using microarray (57, 86, 87). Resveratrol produced expression

changes in a number of important genes in the androgen pathway as well as genes

regulating cell cycle and proliferation in LNCaP. Narayanan and colleagues showed that

48-hour resveratrol treated-LNCaP cells resulted in down-regulation of PSA, AR

co-activator ARA24 and NF-kappa B p65. Altered expression of these genes was

associated with the activation of p53-responsive genes such as p53, PIG-7, p21, p300/CBP

and Apaf-1 (86, 87).

Animal studies of chemopreventive and therapeutic effects of resveratrol against skin,

colon, breast, liver, lung and stomach cancers have been reported using mice and rats;

however, in vivo experimental data on prostate cancer to date is limited (3).

Specific Aims

We have developed a model to design new anticancer agents derived from natural

products or FDA-approved compounds (Fig. 2). Several approaches including data

mining through literature and investigations using molecular or cellular biological

methods are applied. Experimental results which can be used to interpret

structure-activity relationships of a group of structurally related compounds help to

identify the essential chemical structures necessary to induce or suppress key

mechanisms. Furthermore, this approach allows us to create novel derivatives of the

original compound with altered activities. Once modified compounds are designed and

synthesized, sequential experiments to examine their various properties will be carried

out. Our goal is to discover pathways which can be regulated by these novel compounds. 13

Structure-activity Relationship Studies

Drug Design and Synthesis

Cell Viability and Cytotoxicity Studies

Single Cellular Gene Expression Profile Event-based Studies

Molecular Analysis Mechanistic Studies

Fig. 2. The Model Used for New Anticancer Agent Design

Fig. 3. Structural Basis for the Design of Novel Resveratrol Analogs

Resveratrol has been considered a chemopreventive and therapeutic candidate against carcinogenesis because of its potential to modulate multiple molecular pathways associated with the development and progression of cancer. However, significant toxicity to normal cells has also been reported (3). Structure-activity relationship studies of resveratrol and its analogs revealed the functional groups and chemical structures essential to their bioactivities. Based on these findings, by altering the positions of hydroxyl groups or substituting them for other types of functional groups, or by changing the chemical structure of the linkage between the two aromatic rings (Fig. 3), one can synthesize new compounds with an increase or decrease of certain properties. This concept has been applied to the design and development of novel compounds with better anticancer activity and less side effects on normal cells using resveratrol as a lead compound for comparison. KSA and KST series were synthesized, and each of them contains different numbers of hydroxyl groups at varied positions on two aromatic rings, and the double-bond linkage was also modified. 15

The specific aims of this study were to demonstrate multiple bioactivities of newly

synthesized resveratrol analogs and to reveal possible mechanisms to fight cancer. The hypothesis was that analogs sharing similar chemical structures might respond to similar targets and trigger similar signal transductions, and structural changes on derivatives

might increase or decrease certain known bioactivities of the original compound.

Techniques used to demonstrate their activities of cell growth inhibition,

anti-inflammation and cell death induction are listed below:

1. To investigate the cell viability and cell cycle of cancer cells in the presence of the

compounds, the MTT cell viability assay and flow cytometry were used.

2. To demonstrate apoptosis induced by the compounds in cancer cells, DNA fragments

and activated caspase-3 were quantified. Apoptotic cells were visualized by

fluorescence microscopy using annexin V staining method.

3. To demonstrate the regulatory activity of cell survival signaling of the compounds,

inhibition of NF-kappa B was investigated.

4. To demonstrate the anti-inflammatory activity of the compounds, the enzymatic

activities of cyclooxygenase (COX) isomers in the presence of the compounds were

measured.

5. To measure the antioxidant/oxidant activity of the compounds, scavenging activity

against free radicals and inhibition of hydrogen peroxide were demonstrated.

6. To identify the genes differentially expressed in cancer cells treated by the

compounds, the cDNA microarray was used to the generate expression profiles.

MATERIALS AND METHODS

Cell Culture and Compounds

DU145 (American Type Culture Collection, Manassas, VA), an adherent, epithelial and

metastatic prostate carcinoma, was maintained in McCoy’s 5A medium (Gibco, Grand

Island, NY) with 10% fetal bovine serum at 37°C and in an atmosphere containing 5% CO2.

Ovarian cancer MDAH cells, bladder cancer T24 cells and foreskin fibroblast MHRF

(American Type Culture Collection, Manassas, VA) were maintained under identical conditions as DU145 cells except MHRF cells which were grown in Minimum Essential

Medium (Gibco, Grand Island, NY) with 10% fetal bovine serum. Resveratrol

(Sigma-Aldrich, St. Louis, MO) and its novel analogs KSA and KST series, synthesized in Dr. Tsai’s laboratory, were dissolved in absolute ethanol, and fresh serial dilutions were prepared in the medium used to grow the cells before each experiment. Since all compounds used in this experiment have been shown light sensitive, preparation and treatment were carried out in the dark.

MTT Cell Viability Assay

An ideal colorimetric assay for studies of cell survival and proliferation utilizes a colorless substrate which can be modified to a colored product by live cells, but not dead cells or tissue culture medium. MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium 17

bromide) is a water-soluble tetrazolium salt with a yellow color when prepared in aqueous

solution. Dissolved MTT is reduced to an insoluble purple formazan by cleavage of the

tetrazolium ring by mitochondrial dehydrogenase enzymes (Fig. 4) (17, 33, 48, 50, 84).

The precipitate can be dissolved in DMSO or other solvents. The increase in cell number

corresponds to the increased amount of formazan and optical density detected by a

multiple scanning spectrophotometric plate reader. A large number of samples can be

measured with high precision and consistency simultaneously on a 96-well plate.

5,000 cells per well of a 96-well plate were incubated for 24 hours at 37°C and 5% CO2 to

allow attachment and spreading before treatment. Resveratrol, KSA or KST compounds were dissolved in the culture medium used to grow the cells to a series of two-fold dilutions and then added to the plate followed by 24-, 48- and 72-hour incubation at 37°C and 5%

CO2. To make the working solution, 50 mg MTT (Sigma-Aldrich, St. Louis, MO) was

dissolved in 10 ml PBS plus 40 ml Minimum Essential Medium (Gibco, Grand Island, NY)

without fetal bovine serum. MTT solution was then added into each well, the plates were

incubated for four hours at 37°C and 5% CO2, and the medium was replaced by DMSO in

each well. The optical density of each well was obtained from the Bio-Tek microplate reader (Bio-Tek, Winooski, VT) at a wavelength of 570 nm. The dose-response curve was obtained , and based on the method of linear regression, the line of best fit was determined for the calculation of the cytotoxic dose causing 50% reduction of cell viability (CD50), and the selective cytotoxicity index (SCI) which was the ratio of CD50 of

the normal cell line to CD50 of the cancer cell line. Parallel experiments were carried out

to investigate the effect of oxidants, using hydrogen peroxide, in presence of an 18

endogenous antioxidant enzyme catalase (2 mg/ml) (Sigma-Aldrich, St. Louis, MO) involved in the catalytic conversion of hydrogen peroxide to oxygen and water.

Fig. 4. Chemical Reaction to Convert Purple MTT Formazan from MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide)

Trypan Blue Exclusive Assay

Trypan blue (Fig. 5) stains dead cells depending on loss of membrane integrity. It is normally excluded by intact cell membranes but if membranes are sufficiently damaged, trypan blue is able to enter the cells and binds to intracellular proteins. Dead cells can be recognized under microscope as they are stained blue. The cell viability can be determined by counting non-stained cells.

2×106 prostate cancer DU145 cells were grown in a 60 mm tissue culture dish for 24 hours at 37°C and 5% CO2. After 24-hour resveratrol or KST201 treatment, cells were detached using typsin-EDTA (Gibco, Grand Island, NY), collected and re-suspended in the medium used to grow the cells. The cell suspension was then diluted to 1:5 in Trypan

Blue Stain (Invitrogen, Carlsbad, CA) and counted in a hemocytometer using a microscope. A non-treated sample was prepared as a control. The percentage of cell 19

viability (or cell death) was calculated by comparing the sum of non-stained cells counted

in five separate squares of the hemocytometer from treated samples with the sum from

the untreated control.

Fig. 5. Chemical Structure of Trypan Blue

Flow Cytometry for Cell Cycle Analysis

Flow cytometry measures characteristics of a group of cells passing through a sensing point using laser as a light source which then produces scattered light and emitted fluorescence. Physical characteristics including cell size, shape, internal complexity and cell components labeled by fluorophore can be detected (Fig. 6). Cell cycle analysis can be obtained by flow cytometry via measuring the amount of DNA in cells and then

calculating the percentage of cells in different subpopulations. Samples for flow cytometry

are prepared in a solution which ruptures cell membranes and releases the nuclei. The

nuclei are then stained with propidium iodide (PI) (Fig. 6) which intercalates into the major

groove of double-stranded DNA and forms a highly fluorescent complex that can be

excited at 488 nm with emission of 600 nm (131). While passing through the system, the

fluorescent complex producs fluorescence, and the intensity measured by the detector is proportional to the amount of DNA in the cell. Since in different (G0/G1, S or G2/M) phases, 20

propidium iodide

Fig. 6. Illustration of Flow Cytometer and Chemical Structure of PI (Propidium Iodide) Used for DNA Labeling 21

Fig. 7. The Cell Cycle. The DNA diploid is represented by the alphabetical letter X.

the cell contains different amount of DNA (Fig. 7), the percentage of cells in each

subpopulation can be determined by the flow cytometric histogram. Moreover, apoptotic

cells are represented by a sub G0/G1 population seen to the left of the G0/G1 peak (72).

6 10 cells were grown in a 35 mm tissue culture dish for 24 hours at 37°C and 5% CO2 and

treated with resveratrol or KST201 for another 24 hours. A detergent trypsin method was

utilized to dissolve cell membrane, stabilize nuclei and label DNA with fluorescent dye

(131). Cells were washed in cold PBS three times and transferred to a centrifuge tube.

Stock detergent (1 g trisodium citrate, 1 ml NP40 and 60.5 g Tris in 1 l distilled water at pH

7.6), solution A (3 mg trypsin in 100 ml stock detergent at pH 7.6), solution B (50 mg

trypsin inhibitor and 10 mg RNase A in 100 ml stock detergent at pH 7.6) and solution C

(41.6 mg propidium iodide and 116 mg spermine tetrahydrochloride in 100 ml stock

detergent) were prepared for cell membrane rupture and DNA staining. Solution A and B

were added into the cell pellet sequentially and allowed to incubate for 10 and 10 minutes

at room temperature, respectively. Ice-cold solution C was then added, and the mixture

was placed in the dark for 15 minutes on ice. After centrifuge, half of supernatant was discarded, and propidium iodide-stained nuclei were re-suspended in the solution. DNA ploidy and cell cycle were analyzed using the Becton Dickinson FACSCalibur and

CellQuest (Becton Dickinson, Mountain View, CA) carried out in the Molecular Pathology

Research Laboratory in Summa Health System, Akron, OH.

DNA Fragmentation Assay

The concept is based on the sandwich-enzyme-immunoassay-principle using antibodies

specifically against DNA and histones, respectively, for quantitative determination of

mono- and oligonucleosomes (histone-associated DNA-fragments) in the cytoplasmatic

portion of cell lysates after apoptosis is induced. Endonucleases activated during cell

death cleave DNA at internucleosomal linker region, generating histone-associated

DNA-fragments. The enrichment of DNA fragment in the cytoplasm of apoptotic cells is due to the fact that DNA fragments released to the cytoplasm cannot further cross the plasma membranes since they remain intact in apoptotic cells (6, 129).

Fig. 8. Principle of the Enzyme-linked Immunosorbent Assay (ELISA) for DNA Fragmentation Study

Materials utilized to quantify apoptotic DNA fragmentation were provided by a cell death

detection kit (Roche, Indianapolis, IN) (Fig. 8). 104 prostate cancer DU145 cells were

grown in a 96-well culture plate at 5% CO2 and 37ºC followed by 24-hour resveratrol- or 24

KST201-treatment at various concentrations. The lysis buffer was directly added into

each well, and the supernatant containing cytoplasmic DNA fragment was collected after

centrifuge, which separated cell nuclei containing un-fragmented DNA from the

cytoplasmic portion of the sample. The supernatant was then transferred to a

streptavidin-coated 96-well plate, and a mixture of anti-histone-biotin and

anti-DNA-peroxidase was added. The former bound to the histone of the nucleosomes

and immobilized onto the surface of the well through biotinylation with streptavidin.

Simultaneously, the latter bound to the DNA of the nucleosomes, and thus formed a fixed

nucleosomes-peroxidase complex. Unbound antibodies were removed by a washstep, and

ABTS was added as a substrate. The peroxidase activity, which was proportional to the amount of nucleosomes-peroxidase complex, of each well was measured colorimetrically at 405 nm using the Multiskan Ascent plate reader (Thermo, Waltham, MA).

Caspase-3 Activity Assay

Caspase-3 exists in the cytoplasm as an inactive precursor. Once the apoptotic process has been initiated, it is activated through proteolytic cleavage by other caspases in a cascade

(Fig.s 9-10). Caspase-3 functions as an executor which regulates various components responsible for morphological changes including cell shrinkage, membrane blebbing, nuclear condensation and DNA fragmentation and eventually leads to cell death (4, 6, 26).

2×107 prostate cancer DU145 cells were grown in a 60 mm tissue culture dish for 24 hours

at 37°C and 5% CO2 and treated with resveratrol or KST201. After washand centrifuge,

the pellet was collected and incubated with dithiothreitol (DTT) lysis buffer on ice. The 25

Fig. 9. Death Receptor Pathway of Apoptosis Signaling 26

Fig. 10. Mitochondrial Pathway of Apoptosis Signaling

cell lysate was then transferred to a centrifuge tube for measurement of proteolytic

cleavage by caspase-3 utilizing a fluorometric immunosorbent enzyme assay kit (Roche,

Indianapolis, IN) (Fig. 11). The antibody against caspase-3 was prepared in the coating

buffer, added to a 96-well plate and immobilized on the surface of each well followed by

washsteps. Compound-treated cell lysates and the untreated control were added to the plate and incubated for one hour at 37°C. Any caspase-3 present in the samples was captured by the coated antibody, and it cleaved its substrate Ac-DEVD-AFC, when added into the wells. Proteolytic cleavage then produced free fluorescent AFC measured by the

Fluoroskan Ascent FL plate reader (Thermo, Waltham, MA) with a filter set of excitation/emission of 390 nm/590 nm. The developed fluorescent intensity was proportional to the concentration of AFC determined by the standard curve, generated by different dilutions of AFC standard included in the kit, and the activated caspase-3 in the sample was then quantified.

Fig. 11. Principle of the Enzyme-linked Immunosorbent Assay (ELISA) for Caspase-3 Activity Study 28

Annexin V-FITC Staining Assay

Phosphatidyl serine (PS) flips from the inside to the outside of the plasma membrane

during apoptosis (6, 129). This change can be detected by fluorescein-conjugated annexin

V which binds to PS. During the process of necrosis, the plasma membrane loses its

integrity, and the annexin V can also bind to the inner face, so PI which is excluded from

intact cells is added to distinguish apoptotic cells with intact plasma membrane and those

undergoing necrosis with leaky membrane. The type of cell death induced by resveratrol

and KST201 treatment can be determined by the fluorescence microscopy with dual

filters.

5×105 prostate cancer DU145 cells were grown in a 35 mm tissue culture dish with

coverslips for 24 hours at 37°C and 5% CO2 followed by 24-hour treatment of resveratrol or KST201 at various concentrations. The staining solution was prepared by adding 25 ul

Annexin V and 50 ul PI in 1 ml binding buffer (Becton Dickinson, Mountain View, CA)

and added onto the coverslips after a washstep. Cells were allowed to incubate for 15

minutes at room temperature in the dark and fixed with 2% formaldehyde. The coverslip

was then transfer onto a glass slide and visualized with a dual filter set for FITC and

rhodamine using the FluoView 500 confocal laser scanning imaging system (Olympus,

Tokyo, Japan) in the Imaging and Visualization Center at Kent State University, Kent,

OH, and the Lumam research fluorescence microscope (LOMO, St. Petersburg, Russia).

NF-kappa B p65 Activity Assay

The NF-kappa B family members are transcription factors playing multiple roles in the

regulation of immune and inflammatory responses, developmental processes and diseases

such as cancer. NF-kappa B and its inhibitor I kappa B form an inactive complex in

cytoplasm. Activation occurs by phosphorylation, ubiquitination and degradation of I

kappa B, which then releases active NF-kappa B into the nucleus where it regulates the

expression of target genes leading to a variety of cellular behaviors (60, 77, 138).

An enzyme-linked immunosorbent assay kit (Pierce, Rockford, IL) (Fig. 12) was used to detect the active forms of NF-kappa B p65 by measuring its binding to a consensus DNA

sequence (122). 3×106 prostate cancer DU145 cells was grown in a 60 mm tissue culture

dish for 24 hours at 37°C and 5% CO2 and then were treated with resveratrol or KST201.

Untreated cells were prepared under the same conditions as control. Cells were collected by scraping and incubated in ice-cold RIPA lysis buffer containing 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail. After centrifuge, the supernatant was transferred to a streptavidin-coated 96-well plate with the bound NF-kappa B biotinylated-consensus sequence that allowed the active p65 in the sample to bind. To ensure the signal specificity, competitive DNA fragments containing a NF-kappa B binding site used to prevent the binding of NF-kappa B to the consensus sequence were provided. A specific primary antibody against p65 and a secondary HRP conjugated antibody were added into each well in sequence, and thus an immobilized p65-HRP complex was formed. Unbound antibodies were removed by washafter incubating steps. A mixture of luminol/enhancer and peroxide solution was added into each well as a substrate 30

for HRP. The chemiluminescent signal, proportional to the relative amount of active p65 in

the sample, then was detected using the Fluoroskan Ascent FL plate reader (Thermo,

Waltham, MA).

Fig. 12. Principle of the Enzyme-linked Immunosorbent Assay (ELISA) for NF-kappa B p65 Activity Study

COX Enzyme Inhibition Assay

COX enzymes perform dual functions, cyclooxygenase and peroxidase, converting prostaglandin H2 (PGH2) from arachidonic acid; therefore, it is essential to the prostaglandin (PG) biosynthesis. One of the two isomers COX1 is constitutively expressed in a variety of cell types and responsible for cellular homeostasis; on the other hand, COX2 is inducible via extracellular stimuli including phorbol esters, lipopolysaccharides and cytokines under inflammatory conditions (63, 93). Accordingly,

COX2 serves as a target for non-steroidal anti-inflammatory drugs (NSAID) (138, 146).

To measure the effect of COX inhibitors, the approach that quantifies PG production in 31

COX enzyme/substrate/inhibitor mixture has been utilized.

PGH2 can be further reduced to PGF2-alpha by SnCl2, and the amount of PGF2-alpha is proportional to the COX enzymatic activity. By measuring PGF2-alpha, an enzyme

immunoassay kit (Cayman, Ann Arbor, MI) (Fig. 13) was used to determine the

inhibitory activity of resveratrol and KST201 against COX1 and COX2. The solutions of

resveratrol or KST201 were prepared in ethanol at various concentrations, and 20 μl of

each was mixed with 10 μl of heme and 10 μl of COX-1 or COX-2 in 950 μl of reaction

buffer. Reaction was initiated by adding arachidonic acid into the mixture, and enzyme

catalysis was stopped by adding 1 M hydrochloride. Saturated SnCl2 solution was used to

generate PGF2-alpha from PGH2. 1:2000 dilution of the sample was then transferred to a

mouse anti-rabbit IgG coated 96-well plate with a solution containing PG Tracer and PG

Antiserum. The free PG competed with acetylcholinesterase(AChE)-linked PG tracer for

the binding site of PG antiserum which bound to the IgG and formed an immobilized

complex on the surface of the well. After 18-hour incubation at room temperature and

five wash steps, Ellman’s reagent containing a substrate for AChE was added, and the

reaction produced a color that can be detected at 405 nm of wavelength. The intensity

was proportional to the amount of PG tracer bound to the well, which is inversely

proportional to the amount of free PG present in the sample.

Plate are pre-coated with Step 1. Incubation with tracer mouse monocloned antibody antiserum, and either standard and blocked with a proprietary or unknown sample formulation of proteins

Step 2. Wash to remove all Step 3. Develop the well with Unbound reagents Ellman’s Reagent Mouse Monoclonal Antibody

Blocking Proteins

Acetylcholinesterase linked to PG (Tracer)

Specific Antibody to PG

Free PG

Fig. 13. Principle of the Enzyme-linked Immunosorbent Assay (ELISA) for COX enzyme Activity Study

Free Radical Scavenging Assay

A variety of methods were used to determine free radical scavenging of resveratrol in solutions. Two approaches were chosen: the scavenging of radical cation ABTS

(2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate)) mixed with the peroxy-radical generator AAPH (2,2'-azobis(2- amidinopropane) dihydrochloride) (Fig. 14), and the

scavenging of stable radical DPPH (2,2-diphenyl-1-picrylhydrazyl) (Fig. 14) (104). Both

ABTS/AAPH and DPPH radicals in solution produce color which can be reduced by free

radical scavengers and detected using a spectrophotometer.

For ABTS free radical scavenging assay, the ABTS (Sigma-Aldrich, St. Louis,

MO)/AAPH (Cayman, Ann Arbor, MI) mixture was prepare at the final concentration of

2.5 mM AAPH and 1.0 mM ABTS in PBS and incubated in 68°C water bath at dark until

the absorbance developed to 0.65 at 734 nm. Serial concentrations of ethanolic

resveratrol or KST201 were then diluted to 1:50 in the ABTS free radical solutions

followed by 10-minute incubation in 37°C water bath in the dark. The absorbance was

measured by a spectrophotometer at 734 nm. For DPPH free radical scavenging assay, 50

μM DPPH (Sigma-Aldrich, St. Louis, MO) solution was prepared in ethanol. Serial

concentrations of ethanolic resveratrol or KST201 and absolute ethanol were then diluted

to 1:3 in the DPPH free radical solutions followed by 30-minute incubation at room

temperature in the dark. The absorbance was measured by the Spectronic 601 UV/Vis

spectrophotometer (Milton Roy, Rochester, NY) at 517 nm. For both assays, the pure

solvent without the testing compound was added into the free radical solution serving as a

control. 34

AAPH (2,2'-azobis(2- amidinopropane) dihydrochloride)

ABTS (2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate))

DPPH (2,2-diphenyl-1-picrylhydrazyl)

Fig. 14. Chemical Structure of Reagents Used in Free Radical Scavenging Assays

Fluorescent DCF Assay

DCFH-DA (2',7'-dichlorofluorescein diacetate) is a non-fluorescent molecule permeable

to the cell membrane. Its acetate group can be removed by cytosolic esterases generating

DCFH (2'7'-dichlorofluorescin) which become impermeable to the cell membrane, and

the further oxidation converts DCFH to fluorescent DCF (2'7'-dichlorofluorescein) by

intracellular oxidants. This chain reaction occurring in live cells has been used as an

indicator for reactive oxygen species, especially for hydrogen peroxide (Fig. 15) (73).

5,000 prostate cancer DU145 cells were grown in a FluoroNunc 96-Well white plate

(Fisher, Pittsburgh, PA) allowed to incubate for 24 hours at 37°C and 5% CO2. DCFH-DA

(Molecular Probe, Eugene, OR) was dissolved in DMSO and diluted to 100 μM in the medium used to grow the cells. After discarding the medium, DCFH-DA solution was added into each well followed by 30-minunte incubation at 37°C and 5% CO2. Serial concentrations of resveratrol or KST201 were prepared in the medium, and cells were treated with these testing compounds for three hours at 37°C and 5% CO2.

To ensure that the reaction was specifically catalyzed by hydrogen peroxide, parallel experiments for co-treatment with 2 mg/ml catalase, which inhibited the reaction by

depleting hydrogen peroxide, were carried out as comparison. Fluorescent intensity of each sample was measured using the Fluoroskan Ascent FL plate reader at excitation of

488 nm and emission of 530 nm

Hydrolysis Oxidation DCFH-DA DCFH DCF

Fig. 15. Chemical Reaction to Convert fluorescent DCF (2'7'-dichlorofluorescein) from DCFH-DA (2',7'-dichlorofluorescein diacetate)

Cancer-related Gene Microarray

In the biological systems, numerous of genes and their products function in a complicated

and orchestrated way. Studies on single gene may not be able to demonstrate the whole

picture of gene function. DNA microarray has been developed to investigate the complete

genome on a single chip, which enables the assessment of the relative gene expression

levels of thousands of genes simultaneously, thus one can discover the regulatory mechanisms involved under certain conditions. (19, 23). To prepare the matrix for the array, cDNA probes are fixed on a glass slide on which each spot contains numerous identical DNA. cDNAs representing mRNA pools from test and reference cells are labeled with two different fluorescent dyes, cy5 (red) and cy3 (green), respectively. During

hybridization, fluorescently tagged targets hybridized to their complementary sequences

spotted on the glass slide. The relative expression levels of the genes are estimated by

measuring the fluorescence intensities and colors at each spot. (Fig. 16). 37

Fig. 16. Principle of the cDNA Microarray Technique 38

The clone set of cancer-related genes used in this study contained 1800 genes implicated in

metastasis or tumor development in general from the literature and Affymetrix cancer

G110 array (http://www.affymetrix.com/analysis/index.affx), 1809 genes involved in

kidney development (golgi.ana.ed.ac.uk/kidhome.html) or showing more than three-fold variation of expression among four primary Wilms tumors and fetal kidney in the previous

Affymetrix GeneChip experiments (74), 1400 potential downstream targets of p53 transcriptional regulation, 950 genes with AU-rich elements in their 3’ and/or 5’ UTR and

1245 cytokine responsive genes, genes for zinc finger proteins or genes implicated in apoptosis (http://geacf.cwru.edu/geacf/geaaspottedtechnology.shtml). 106 prostate cancer

DU145 cells were grown in a 35 mm tissue culture dish, treated with resveratrol or

KST201 for 12 hours at 37°C and 5% CO2 and then collected as starting material. The

preparation of fluorescent cDNA, the hybridization and evaluation of gene expression

levels were carried out in the cDNA Spotted Array Core Facility at Cleveland Clinic,

Cleveland, OH. Each expression microarray also contained control cDNAs serve as

controls for hybridization, washand staining.

The expression level was represented by the ratio of the fluorescence intensity of the test

sample (treated cells) over the reference sample (control cells). The expression levels were

considered significant if the ratio was lower than 0.5 (two-fold down-regulated) or higher

than two (two-fold up-regulated). Functional classifications of each gene-coded protein

were obtained from Onto-Express (http://vortex.cs.wayne.edu/projects.htm), EASEonline

(http://apps1.niaid.nih.gov/david/), Online Mendelian Inheritance in Man (OMIM,

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM) and GeneCards 39

(http://www.genecards.org/).

RESULTS

Inhibition of Cancer Cell Proliferation by the Compounds

All resveratrol analogs were tested to evaluate their inhibitory activity against proliferation of various cell lines using the MTT cell viability assay.

Hormone-independent prostate cancer cell line DU145, bladder cancer cell line T24, ovarian cancer cell line MDAH and normal fibroblast MHRF were treated with resveratrol, KSA and KST series on 96-well tissue culture plates at serial concentrations to generate the dose-response curves and CD50, a quantitative measurement representing the efficiency of the inhibitory property to cell proliferation. The results indicated that

24-hour treatment with resveratrol and most of the KSA and KST compounds performed anti-proliferatory activity (CD50 lower then 100 μM), and CD50 of many compounds dropped dramatically after 48 and 72-hour treatment indicating the sufficient time required for the best performance (Tables 2-4). DU145 cell proliferation was inhibited by

KST201 and KST401 remarkably. Resveratrol, KST201 and KST401 showed significant inhibitory activity of cell proliferation on MDAH, and KST201 was the most effective compound amount them. On T24 cells, the inhibitory activity of resveratrol, KST213 and

KST401 were significant after the first 24 hour treatment, but KST201 was showed to be the most active compound after 72 hour treatment. In KSA series, KSA1201 was more effective compared to resveratrol and other KSA members on all three cancer cell lines.

Resveratrol and other compounds were found toxic to normal cells. For instance, 41

treatment of resveratrol, KST213 or KST 401 was able to cause decrease of MHRF cell

viability at low concentrations implying a cytotoxic effect on normal cells. An optimal

anticancer candidate has to reduce cancer cell viability effectively but not to block normal

cell proliferation; in other words, it should be toxic to cancer cells but non-toxic to

normal cells. The CSI value, CD50 on normal cells divided by CD50 on cancer cells,

represented the selectivity of an anticancer candidate against cancer cells. The higher

ratio for a particular compound indicated a relatively higher dose requirement to block

the normal cell growth versus the cancer cell growth (Tables 2-4). Data have shown

KST201 was the most selective compound compared to all other compounds tested in this study. Moreover, KST201 was unique since its CSI on all three cell lines increased time-dependently which was not showed by other compounds (Fig. 17A-C). A possible explanation might be that KST201 further metabolized to be more toxic in cancer cells but even less toxic to normal cells after a certain period of time, although no evidence has yet been brought out.

Resveratrol and KST201 suppressed proliferation of all three cell lines both

dose-dependently and time-dependently (Fig. 18A-C). On DU145 cells, detectable effects

were able to be observed at all concentrations of resveratrol at three different time points.

After 24-hour treatment, cell viability decreased to 71% at the concentration of 50 µM,

55% at 100 µM and 18% at 200 µM. Elongation of treatment time was able to elevate the

effect, 49% cell viability after 48-hour treatment and 20% cell viability after 72-hour

treatment at the concentration of 50 µM, for instance. KST201, at 50µM causing reduction

of cell viability to 55% after 24 hours, 17% after 48 hour, and 3% after 72 hours, was 42

Cytotoxicity of Testing Compounds on Fibroblast and Cancer Cells

DU145 MHRF

CD50 SD CD50 SD CSI 24hrs Resveratrol 92.2 2.9 154.8 16.5 1.68

KSA1201 16.5 0.7 51.8 2.6 3.14 KSA1301 90.2 1.1 358.5 21.0 3.97 KSA1302 175.5 3.6 177.0 4.9 1.01

KST201 57.3 0.8 298.8 3.5 5.21 KST213 74.9 0.4 74.5 0.3 0.99 KST301 185.0 2.8 235.3 15.3 1.27 KST401 57.0 3.0 59.3 1.8 1.04

48hrs Resveratrol 58.2 0.4 50.9 0.6 0.87

KSA1201 10.7 0.1 39.6 1.8 3.70 KSA1301 75.5 0.1 330.2 6.0 4.37 KSA1302 157.5 0.6 169.4 14.9 1.08

KST201 16.8 0.6 194.1 9.6 11.55 KST213 62.3 0.7 62.3 0.4 1.00 KST301 176.6 5.1 198.4 6.7 1.12 KST401 31.5 1.2 28.4 2.7 0.90

72hrs Resveratrol 27.9 0.7 32.0 1.1 1.15

KSA1201 6.6 0.3 46.0 2.5 6.97 KSA1301 75.0 0.1 241.7 7.0 3.22 KSA1302 150.7 0.6 167.5 6.5 1.11

KST201 9.4 0.4 153.2 4.4 16.30 KST213 50.5 0.5 52.0 1 1.03 KST301 102.5 3.9 174.9 3.2 1.71 KST401 17.0 0.2 17.0 1.3 1.00

Table 2. Cytotoxicity of the Compounds on DU145 and MHRF. MTT cell viability assay described previously was used to investigate resveratrol-, KSA and KST compound-inhibited cell proliferation. DU145 were treated at 200, 100, 50, 25, 12.5 and 6.25 µM for 24, 48 and 72 hours. CD50 and CSI were calculated based on dose-response curves. The anti-proliferatory activity was shown dose- and time-dependent. The results represent mean ± SD of triplicate determinations. 43

Cytotoxicity of Testing Compounds on Fibroblast and Cancer Cells

MDAH MHRF

CD50 SD CD50 SD CSI 24hrs Resveratrol 36.9 2.5 154.8 16.5 4.20

KSA1201 34.8 1.7 51.8 2.6 1.49 KSA1301 307.2 9.5 358.5 21.0 1.17 KSA1302 177.0 4.9

KST201 34.6 0.4 298.8 3.5 8.64 KST213 75.7 0.1 74.5 0.3 0.98 KST301 180.8 4.2 235.3 15.3 1.30 KST401 18.5 0.4 59.3 1.8 3.21

48hrs Resveratrol 23.0 0.1 50.9 0.6 2.21

KSA1201 31.3 0.2 39.6 1.8 1.27 KSA1301 207.8 11.3 330.2 6.0 1.59 KSA1302 169.4 14.9

KST201 5.1 0.1 194.1 9.6 38.06 KST213 48.6 3.4 62.3 0.4 1.28 KST301 151.2 3.4 198.4 6.7 1.31 KST401 15.5 0.2 28.4 2.7 1.83

72hrs Resveratrol 14.6 0.1 32.0 1.1 2.19

KSA1201 6.2 0.4 46.0 2.5 7.42 KSA1301 128.0 1.9 241.7 7.0 1.89 KSA1302 167.5 6.5

KST201 3.3 0.1 153.2 4.4 46.42 KST213 49.3 1.5 52.0 1 1.05 KST301 119.0 1.9 174.9 3.2 1.47 KST401 14.5 0.3 17.0 1.3 1.17

Table 3. Cytotoxicity of the Compounds on MDAH and MHRF. MTT cell viability assay described previously was used to investigate resveratrol-, KSA and KST compound-inhibited cell proliferation. MDAH were treated at 200, 100, 50, 25, 12.5 and 6.25 µM for 24, 48 and 72 hours. CD50 and CSI were calculated based on dose-response curves. The anti-proliferatory activity was shown dose- and time-dependent. The results represent mean ± SD of triplicate determinations. 44

Cytotoxicity of Testing Compounds on Fibroblast and Cancer Cells

T24 MHRF

CD50 SD CD50 SD CSI 24hrs Resveratrol 241.1 1.0 154.8 16.5 0.64

KSA1201 32.8 1.8 51.8 2.6 1.58 KSA1301 373.9 14.7 358.5 21.0 0.96 KSA1302 177.0 4.9

KST201 82.6 1.6 298.8 3.5 3.62 KST213 40.8 0.3 74.5 0.3 1.83 KST301 263.9 10.3 235.3 15.3 0.89 KST401 29.0 0.9 59.3 1.8 2.04

48hrs Resveratrol 156.5 0.1 50.9 0.6 0.33

KSA1201 18.0 3.0 39.6 1.8 2.20 KSA1301 264.7 1.4 330.2 6.0 1.25 KSA1302 169.4 14.9

KST201 31.1 0.8 194.1 9.6 6.24 KST213 37.8 0.1 62.3 0.4 1.65 KST301 152.8 0.8 198.4 6.7 1.30 KST401 14.4 0.4 28.4 2.7 1.97

72hrs Resveratrol 22.6 0.3 32.0 1.1 1.42

KSA1201 8.7 0.7 46.0 2.5 5.29 KSA1301 215.0 2.9 241.7 7.0 1.12 KSA1302 167.5 6.5

KST201 8.5 0.3 153.2 4.4 18.02 KST213 37.6 0.0 52.0 1 1.38 KST301 124.2 3.6 174.9 3.2 1.41 KST401 12.5 0.1 17.0 1.3 1.36

Table 4. Cytotoxicity of the Compounds on T24 and MHRF. MTT cell viability assay described previously was used to investigate resveratrol-, KSA and KST compound-inhibited cell proliferation. T24 were treated at 200, 100, 50, 25, 12.5 and 6.25 µM for 24, 48 and 72 hours. CD50 and CSI were calculated based on dose-response curves. The anti-proliferatory activity was shown dose- and time-dependent. The results represent mean ± SD of triplicate determinations. 45

Selective Index of Cytotoxicity on (MHRF vs DU145)

20.00 Resveratrol KSA1201 16.00 KSA1301 KSA1302 KST201 12.00 KST213 KST301 KST401

CSI value CSI 8.00

4.00

0.00 24hrs 48hrs 72hrs

Fig. 17A. CSI Changes of the Compounds on Cancer Cells. The time-CSI value curves indicated that only KST201 performed increased selectivity to DU145 cells time-dependently. These diagrams were plotted based on Table 2.

Selective Index of Cytotoxicity on (MHRF vs MDAH)

50.00 Resveratrol KSA1201 40.00 KSA1301 KST201

30.00 KST213 KST301 KST401 CSI value CSI 20.00

10.00

0.00 24hrs 48hrs 72hrs

Fig. 17B. CSI Changes of the Compounds on Cancer Cells. The time-CSI value curves indicated that only KST201 performed increased selectivity to MDAH cells time-dependently. These diagrams were plotted based on Table 2.

Selective Index of Cytotoxicity (MHRF vs T24)

20.00 Resveratrol KSA1201 16.00 KSA1301 KST201

12.00 KST213 KST301 KST401 CSI value 8.00

4.00

0.00 24hrs 48hrs 72hrs

Fig. 17C. CSI Changes of the Compounds on Cancer Cells. The time-CSI value curves indicated that only KST201 performed increased selectivity to T24 cells time-dependently. These diagrams were plotted based on Table 2.

MTT Cell Viability Assay on DU145 125% Res/24hrs

Res/48hrs 100% Res/72hrs

KST201/24hrs 75% KST201/48hrs

KST201/72hrs

Cell viability Cell 50%

25%

0% 0 50 100 Conc. (uM) 150 200 250

Fig. 18A. Inhibitory Activity of Resveratrol and KST201 on Cell Proliferation. MTT cell viability assay described previously was used to investigate resveratrol- and KST201-suppressed cell proliferation on DU145. Cells were treated at 200, 100, 50, 25, 12.5 and 6.25 µM for 24, 48 and 72 hours. The anti-proliferatory activity was shown dose- and time-dependent. The results represent mean ± SD of triplicate determinations.

MTT Cell Viability Assay on MDAH 125% Res/24hrs

Res/48hrs 100% Res/72hrs

KST201/24hrs 75% KST201/48hrs

KST201/72hrs

Cell viability Cell 50%

25%

0% 0 50 100 Conc. (uM) 150 200 250

Fig. 18B. Inhibitory Activity of Resveratrol and KST201 on Cell Proliferation. MTT cell viability assay described previously was used to investigate resveratrol- and KST201-suppressed cell proliferation on MDAH. Cells were treated at 200, 100, 50, 25, 12.5 and 6.25 µM for 24, 48 and 72 hours. The anti-proliferatory activity was shown dose- and time-dependent. The results represent mean ± SD of triplicate determinations.

MTT Cell Viability Assay on T24 125% Res/24hrs

Res/48hrs 100% Res/72hrs

KST201/24hrs 75% KST201/48hrs

KST201/72hrs

Cell viability Cell 50%

25%

0% 0 50 100 Conc. (uM) 150 200 250

Fig. 18C. Inhibitory Activity of Resveratrol and KST201 on Cell Proliferation. MTT cell viability assay described previously was used to investigate resveratrol- and KST201-suppressed cell proliferation on T24. Cells were treated at 200, 100, 50, 25, 12.5 and 6.25 µM for 24, 48 and 72 hours. The anti-proliferatory activity was shown dose- and time-dependent. The results represent mean ± SD of triplicate determinations.

more effective compared to resveratrol against DU145 cells. The dose-response curves

revealed a nearly linear correlation of concentration against cell viability at 24-hour

treatment ranging from 25 to 200 µM suggesting an ideal range of concentration which can be applied to the following experiments in this study. The results suggested an orchestration of cell cycle arrest, apoptosis and necrosis, which were not fully elucidated, since the MTT cell viability assay alone provided little information of actual mechanisms involved.

Inhibitory activity of cell proliferation performed by resveratrol and KST201 was further

confirmed by trypan blue exclusive assay (Fig. 19). Unstained cells were counted under

microscope after 24-hour treatment at various concentrations of both compounds.

KST201, again, was more effective at DU145 cell killing compared to resveratrol.

However, since this method depended on loss of integrity of cell membrane which was retained in the early stage of apoptotic cells, cells undergoing this type of death might not

be identified.

Cell Cycle Analysis of Cancer Cells Treated with Resveratrol and KST201

Prostate cancer DU145 cells treated with resveratrol showed an accumulation of cells in

the G0/G1 phase of the cell cycle and a significant decrease in G2/M cells, as compared with control cells. A sub G0/G1 population was present suggesting that certain amount of

cells underwent apoptosis after 24-hour treatment. Results indicated that resveratrol

inhibited cell proliferation through cell cycle blockage mainly at the concentration of

0.5-fold CD50 (about 50 μM), and, at CD50 (about 100 μM), apoptosis was induced 52

Trypan Blue Exclusive Assay on DU145 80% RES KST201 60%

40% % Cell Death

20%

0% 25 50 Conc. (uM) 100

Fig. 19. Live Cells Counting Using Trypan Blue Staining. DU145 cells were treated with both resveratrol and KST201 at 100, 50 and 25 µM for 24 hours. Unstained cells representing live cells were counted on a hemocytometer under microscope. Compared to untreated control, the percentage of dead cells was determined. The results represent mean ± SD of triplicate determinations.

playing a role in anti-proliferation (Fig. 20). The similar phenomenon was observed on

KST201-treated DU145 cells (Fig. 21). At the concentration of 0.25-fold CD50, which was around 15 μM and more then three times lower then 0.5-fold CD50 resveratrol,

KST201 was able to cause blockage in the G0/G1 phase of the cell cycle. Apoptotic peaks were present when the cells were treated at 0.5-fold CD50 (about 30 μM) and CD50 (about

60 μM). Compared to resveratrol, KST201 suppressed cell cycle and induced apoptosis at

relatively lower concentration. Different patterns were shown in the flow cytometric

histogram generated from KST201-treated MDAH and T24 cells (Fig. 22-23). An

accumulation of cells in the S phase of MDAH and T24 indicating the interruption during

the process of DNA synthesis by KST201 at 0.25-fold CD50, i.e. 5 μM for MDAH and 20

μM for T24. Moreover, at CD50 (about 80 μM), KST201 was able to push T24 cells

undergoing apoptosis. Since the doses of treatment used for this study were based on the

corresponding CD50, which varied, of individual cell lines, the results generated from different cell lines were not comparable.

DNA Fragmentation Induced by Resveratrol and KST201 in DU145

Both resveratrol and KST201 induced DNA fragmentation, one of the main features of

apoptotic cell, in prostate cancer DU145 cells. The relative amount of DNA fragments

detected in the sample was represented by the enrichment factor, an index calculated from

the colorimetric intensity of the treated sample divided by the intensity of the untreated

sample. Resveratrol-treated DU145 cells produced approximately four times of

cytoplasmic DNA fragments at 50 μM, eight times at 100 μM and 16 times at 200 μM 54

G0/G1

G2/M

S SubG0/G1

Fig. 20. Cell Cycle Analysis by Flow Cytometry on resveratrol-treated DU145. PI-stained nuclei were collected using a detergent trypsin method described previously. Upper: DU145 control (G0/G1: 45.71%, S: 39.76%, G2/M: 14.53%) Middle: DU145 treated with resveratrol at 0.5-fold CD50 (46.1 μM) for 24 hours (G0/G1: 70.03%, S: 29.84%, G2/M: 0.13%) Lower: DU145 treated with resveratrol at CD50 (92.2 μM) for 24 hours (G0/G1: 76.18%, S: 18.42%, G2/M: 5.4%) 55

Fig. 21. Cell Cycle Analysis by Flow Cytometry on KST201-treated DU145. PI-stained nuclei were collected using a detergent trypsin method described previously. Upper left: DU145 control (G0/G1: 45.71%, S: 39.76%, G2/M: 14.53%) Upper right: DU145 treated with KST201 at 0.25-fold CD50 (14.33 μM) for 24 hours (G0/G1: 72.47%, S: 27.53%, G2/M: 0.00%) Lower left: DU145 treated with KST201 at 0.5-fold CD50 (28.7 μM) for 24 hours (G0/G1: 76.80%, S: 19.29%, G2/M: 3.91%) Lower right: DU145 treated with KST201 at CD50 (57.3 μM) for 24 hours (G0/G1: 77.65%, S: 17.89%, G2/M: 4.46%)

Fig. 22. Cell Cycle Analysis by Flow Cytometry on KST201-treated MDAH. PI-stained nuclei were collected using a detergent trypsin method described previously. Upper: : MDAH control (G0/G1: 54.45%, S: 35.80%, G2/M: 9.76%) Lower: MDAH treated with KST201 at 0.25-fold CD50 (5.38 μM) for 24 hours (G0/G1: 29.63%, S: 70.37%, G2/M: 0.00%)

Fig. 23. Cell Cycle Analysis by Flow Cytometry on KST201-treated T24. PI-stained nuclei were collected using a detergent trypsin method described previously. Upper: T24 control (G0/G1: 45.73%, S: 6.51%, G2/M: 47.77%) Middle: T24 treated with KST201 at 0.25-fold CD50 (20.65 μM) for 24 hours (G0/G1: 22.72%, S: 57.89%, G2/M: 19.39%) Lower: T24 treated with KST201 at CD50 (82.60 μM) for 24 hours (G0/G1:52.50%, S: 47.19%, G2/M: 0.31%) 58

compared to untreated cell (Fig. 24). At the concentration of 25 μM, KST201 induced six times of DNA fragments in DU145 cells compared to untreated cells, and at the same concentrations of resveratrol, KST201-treated cells were able to produce nine times and

12 times of DNA fragments at 50 and 100 μM, respectively. However, when cells were treated at as high concentration as 200 μM, the amount of cytoplasmic DNA fragments in

KST201-treated DU145 cells decreased markedly. It was possible that the type of cell death might be switched from apoptosis to necrosis. Since the necrotic cell membrane was damaged, the cytoplasmic DNA fragments were released into extracellullar environment and not be able to detected by this method. Technically, the same concept can be applied to measure necrotic DNA fragments by collecting the medium after treatment; however, the excess of the compounds present in the medium might have interfering effect.

Quantification of Proteolytic Activity of Caspase-3 in Resveratrol- and

KST201-treated DU145 cells

Activated caspase-3 functions as an executor responsible for morphological changes of

apoptotic cells including cell shrinkage, membrane blebbing, nuclear condensation and

DNA fragmentation. Using Ac-DEVD-AFC as a substrate which produced fluorescent

AFC, the activation of caspase-3 induced by resveratrol or KST201 can be quantified by

measuring the fluorescent intensity after specifically capturing the caspase-3 in the

resveratrol- or KST201-treated cell lysate by the immobilized antibody against caspase-3.

In DU145 cells, the proteolytic activity of caspase-3 was activated significantly after

24-hour treatment of KST201 at the concentration of its CD50. The result indicated that 59

Measurement of Cytoplasmic DNA Fragment in DU145

20 KST201 RES 15

10 Enrichment Factor Enrichment 5

0 5 25 50 100 200 Conc. (uM)

Fig. 24. Quantification of DNA Fragmentation induced in DU145. The assay described previously was used to investigate resveratrol- and KST201-induced apoptosis. DU145 was treated at 200, 100, 50, 25 and 5 µM for 24 hours. The amount of cytoplamsic DNA fragments produced after treatment was determined colorimetrically using ABTS as a substrate for oligouncleosome-peroxidase complex immobilized on the well surface. DU145 was shown undergoing apoptosis and releasing DNA fragment into cytoplasm induced after treatment. The results represent mean ± SD of triplicate determinations.

induction of apoptosis by KST201 was associated with activation of capase-3 (Fig. 25A).

KST201 could effectively activate caspase-3 at 0.5-fold CD50 after 24-hour treatment;

however, increasing time or concentration of treatment to 48 hours or 2-fold CD50, respectively, resulted in the loss of caspase-3 activity (Fig. 25A-B). It seemed that the majority of cells passed the early stage of apoptosis rapidly and were undergoing necrosis, and degradation of caspase-3 occurred. Also, an increase of caspase-3 activity was observed comparing the untreated DU145 cells with 24-hour-treated cells (Fig. 26).

Morphological Changes and Phospholipid Flip-flop on Cell Membranes Induced by

Resveratrol and KST201 in DU145

Changes on morphology of 100 μM resveratrol- and 50 and 100 μM KST201-treated

DU145 cells were visualized under the confocal light microscope (Fig. 27A, 28A, 29A,

30A). Both compounds induced elongated shape of cells with long processes as intercellular bridges for communication with adjacent cells. Bubble-like blebs on cell membranes and nuclear condensation were also observed. Flip-flop of PS is another feature present in apoptotic cells. Cells were stained using annexin V-FITC and PI, and when visualized under fluorescence microscope, apoptotic cells are annexin V positive and PI negative, necrotic cells are both annexin V and PI positive, and live cells are both annexin V and PI negative. Compared to untreated cells, annexin V was showed bound to cell surface indicating translocation of PS on resveratrol- and KST201-treated DU145 cells (Fig. 27B, 28B 29B, 30B-C). Apoptosis but not necrosis was induced since PI staining was not detectable in the presence of annexin V-FITC staining; however, when 61

Activation of Caspase-3 in KST201-treated DU145

6 AFC Conc. 4

0 w/o treat 0.5xCD50 CD50 2xCD50 Post. Ctl Conc. (uM)

Fig. 25A. Measurement of Caspase-3 Activity KST201-treated DU145. Caspase-3 activity assay described previously was used to investigate KST201-induced apoptosis. DU145 was treated at 0.5-fold CD50, CD50 and 2-fold CD50 of KST201 for 24 hours. Activity of proteolytic cleavage was determined fluorometrically using Ac-DEVD-AFC as a substrate for activated caspase-3 immobilized on the well surface. The results represent mean ± SD of triplicate determinations. Camptothecin-induced apoptosis in U937 cells was shown as positive control.

Activation of Caspase-3 in KST201-treated DU145

6 AFC Conc. 4

0 w/o treat 12hr 24hr 48hr Post. Ctl

Fig. 25B. Measurement of Caspase-3 Activity KST201-treated DU145. Caspase-3 activity assay described previously was used to investigate KST201-induced apoptosis. DU145 was treated at CD50 of KST201 for 12, 24 and 48 hours. Activity of proteolytic cleavage was determined fluorometrically using Ac-DEVD-AFC as a substrate for activated caspase-3 immobilized on the well surface. The results represent mean ± SD of triplicate determinations. Camptothecin-induced apoptosis in U937 cells was shown as positive control.

Activation of Caspase-3 in Resveratrol-treated DU145

6 AFC Conc. 4

0 w/o treat Res Post. Ctl

Fig. 26. Measurement of Caspase-3 Activity Resveratrol-treated DU145. Caspase-3 activity assay described previously was used to investigate resveratrol-induced apoptosis. DU145 was treated at CD50, of resveratrol for 24 hours. Activity of proteolytic cleavage was determined fluorometrically using Ac-DEVD-AFC as a substrate for activated caspase-3 immobilized on the well surface. The results represent mean ± SD of triplicate determinations. Camptothecin-induced apoptosis in U937 cells was shown as positive control.

Fig. 27A. Images of Untreated DU145. Confocal light microscopy at 20× of magnification

Fig. 27B. Images of Untreated DU145. Cells were visualized under confocal fluorescence microscope at 20× of magnification. Normal cells: annexin and PI negative; apoptotic cells: annexin positive and PI negative; necrotic cells: annexin and PI positive 66

Fig. 28A. Images of Resveratrol-treated DU145. Cells were treated with 100 μM resveratrol for 24 hours. Confocal light microscopy at 20× of magnification 67

Fig. 28B. Images of Resveratrol-treated DU145. Cells were treated with 100 μM resveratrol for 24 hours, stained with annexin V-FITC/PI and fixed on a coverslip before visualization under confocal fluorescence microscope at 20× of magnification. Normal cells: annexin and PI negative; apoptotic cells: annexin positive and PI negative; necrotic cells: annexin and PI positive

Fig. 29A. Images of KST201-treated DU145. Cells were treated with 50 μM resveratrol for 24 hours. Confocal light microscopy at 20× of magnification

Fig. 29B. Images of KST201-treated DU145. Cells were treated with 50 μM resveratrol for 24 hours, stained with annexin V-FITC/PI and fixed on a coverslip before visualization under confocal fluorescence microscope at 20× of magnification. Normal cells: annexin and PI negative; apoptotic cells: annexin positive and PI negative; necrotic cells: annexin and PI positive 70

Fig. 29C. Images of KST201-treated DU145. Cells were treated with 100 μM resveratrol for 24 hours. Confocal light microscopy at 20× of magnification

Fig. 29D. Images of KST201-treated DU145. Cells were treated with 100 μM resveratrol for 24 hours, stained with annexin V-FITC/PI and fixed on a coverslip before visualization under confocal fluorescence microscope at 20× of magnification. Normal cells: annexin and PI negative; apoptotic cells: annexin positive and PI negative; necrotic cells: annexin and PI positive 72

Fig. 30A. A Closer Look of Annexin V-FITC Stained KST201-treated DU145. Cells were treated with 50 μM KST201 for 24 hours, stained with annexin V-FITC/PI and fixed on a coverslip before visualization. Confocal light microscopy at 60× of magnification.

Fig. 30B. A Closer Look of Annexin V-FITC Stained KST201-treated DU145. Cells were treated with 50 μM KST201 for 24 hours, stained with annexin V-FITC/PI and fixed on a coverslip before visualization. Confocal fluorescent microscopy at 60× of magnification. Normal cells: annexin and PI negative; apoptotic cells: annexin positive and PI negative; necrotic cells: annexin and PI positive

Fig. 30C. A Closer Look of Annexin V-FITC Stained KST201-treated DU145. Cells were treated with 50 μM KST201 for 24 hours, stained with annexin V-FITC/PI and fixed on a coverslip before visualization. Superimposition of Fig. 30A and B 75

Fig. 31. Images of Resveratrol-treated DU145 (40x). Cells were treated with 200 μM resveratrol for 24 hours, stained with annexin V-FITC/PI and fixed on a coverslip before visualization. Normal cells: annexin and PI negative; apoptotic cells: annexin positive and PI negative; necrotic cells: annexin and PI positive 76

Fig. 32A. Images of KST201-treated DU145 (40x). Cells were treated with 100 μM of KST201 for 24 hours, stained with annexin V-FITC/PI and fixed on a coverslip before visualization at 40× of magnification Normal cells: annexin and PI negative; apoptotic cells: annexin positive and PI negative; necrotic cells: annexin and PI positive 77

Fig. 32B. Images of KST201-treated DU145 (40x). Cells were treated with 200 μM of KST201 for 24 hours, stained with annexin V-FITC/PI and fixed on a coverslip before visualization at 40× of magnification. Normal cells: annexin and PI negative; apoptotic cells: annexin positive and PI negative; necrotic cells: annexin and PI positive

higher concentrations were introduced, a portion of cells were showing both annexin V and PI positive representing the occurrence of necrosis after treatment (Fig. 31-32A-B).

None of quantitative data was collected using this method.

Quantification of DNA-binding Activity of NF-kappa B p65 in Resveratrol- and

KST201-treated DU145 Cells

Cell lysates for DNA binding study of NF-kappa B p65 were prepared from prostate cancer

DU145 cells treated with resveratrol or KST201 at different concentrations and time points.

DNA sequences containing p65-binding site were fixed onto the surface of the 96-well plate allowing activated p65 to interact. After washing, the amount of bound p65 was detected and quantified by the chemiluminescent sandwich enzyme-linked immunosorbent method. NF-kappa B was constitutively activated in DU145 cells, and the basal level was measured for normalization of data obtained from treated cells (Fig. 33A-B). The results revealed that resveratrol slightly influenced the DNA binding activity, which approximately decreased to 80% of its basal activity, after 24-hour treatment at 5, 25 and

50 μM. Remarkably, the DNA binding activity of p65 was suppressed to 55.3% and 40.2% of its basal activity by resveratrol after 24- and 12-hour treatment, respectively, at 100 μM.

After 24-hour treatment, KST201 was more effective in terms of inhibitory activity against NF-kappa B at the concentration of 50 and 100 μM compared to resveratrol.

Distinct from resveratrol, 12-hour treatment of KST201 was not sufficient to cause significant reduction (at least 50%) of DNA-binding activity of NF-kappa B.

NF-kB p65 Activity in Treated DU145 Cells

1.5 T201

RES

T201 w/ inhibitor (0 & 1.0 100 uM) RES w/ inhibitor (0 & 100 uM)

0.5 DNA Binding Activity

0.0 0 5 25 50 100 Conc. (uM)

Fig. 33A. Quantification of DNA-binding Activity of NF-kappa B. NF-kappa B p65 activity assay described previously was used to investigate NF-kappa B inhibitory activity of resveratrol and KST201. DU145 was treated at 5, 25, 50 and 100 µM for 24 hours. DNA-binding activity of active p65 was determined by the intensity of chemiluminescence produced by DNA-p65-HRP complex fixed on the well surface. The results represent mean ± SD of triplicate determinations. An inhibitor for p65-DNA binding was added to confirm the specificity of this assay.

NF-kB p65 Activity in Treated DU145 Cells

1.5 KST201

RES

KST201 w/ inhibitor 1.0 (untreat. & 24 hrs) RES w/ inhibitor (untreat. & 24 hrs)

0.5 DNA Binding Activity

0.0 untreat. 6 hrs 12 hrs 24 hrs Time (at 100 uM)

Fig. 33B. Quantification of DNA-binding Activity of NF-kappa B. NF-kappa B p65 activity assay described previously was used to investigate NF-kappa B inhibitory activity of resveratrol and KST201. DU145 was treated at 100 µM for 6, 12 and 24 hours. DNA-binding activity of active p65 was determined by the intensity of chemiluminescence produced by DNA-p65-HRP complex fixed on the well surface. The results represent mean ± SD of triplicate determinations. An inhibitor for p65-DNA binding was added to confirm the specificity of this assay.

Measurement of Inhibitory Activity against COX Enzymes

COX catalyzes the biosynthesis of PGH2 from arachidonic acid by its cyclooxygenase

and peroxidase activities at separate active sites. The assay determined the inhibitory effect on both isomers COX1 and COX2 by comparing the amount of the final stable product PGF2-alpha, generated from PGH2 by SnCl2, produced in the presence or absence

of resveratrol or KST201. The reaction for PGF2-alpha synthesis were prepared by mixing testing solutions (resveratrol or KST201), COX1 or COX2, heme, arachidonic acid and saturated SnCl2 solution and then transferring to a 96-well plate followed by

colorimetric development using Ellman’s reagent. The intensity of color was inversely

proportional to the amount of free prostaglandins quantitatively representing the COX

enzymatic activity, which has been influenced by resveratrol or KST201.

Results indicated resveratrol and KST201 decreased both COX1 and COX2 ability to

catalyze the prostaglandin precursor (Fig. 34A-B). After treatment of resveratrol ranging

from 10 μM up to 200 μM, the enzymatic activity of both COX isomers was significantly

decreased: from 5% of initial activity down to complete inhibition of COX1; from 31%

down to 6% of initial activity of COX2. KST201 showed less anti-COX activity

compared to resveratrol, but still performed approximately 50% inhibition on both

enzymes.

Inhibitory Activity on COX1

1.2

RES KST201 1.0

0.8

0.6

COX Activity 0.4

0.2

0.0 0 10 25 50 100 200 Conc. (uM)

Fig. 34A. Measurement of Inhibitory Activity against COX Enzymes. COX enzyme activity assay described previously was used to investigate inhibitory activity of resveratrol and KST201 on PG biosynthesis. 10, 25, 50, 100 and 200 µM of resveratrol and KST201 were tested in the presence of COX1. COX activity was quantified by measuring the amount of final product PGF2-alpha which competed with AChE-linked PGs for binding to its antibody fixed on the well surface. The color developed by adding substrate for AChE and was reversely proportional to the amount of PGF2-alpha and COX activity. The results represent mean ± SD of triplicate determinations.

Inhibitory Activity on COX2

1.2

1.0 RES KST201

0.8

0.6

COX Activity 0.4

0.2

0.0 0 10 25 50 100 200 Conc. (uM)

Fig. 34B. Measurement of Inhibitory Activity against COX Enzymes. COX enzyme activity assay described previously was used to investigate inhibitory activity of resveratrol and KST201 on PG biosynthesis. 10, 25, 50, 100 and 200 µM of resveratrol and KST201 were tested in the presence of COX2. COX activity was quantified by measuring the amount of final product PGF2-alpha which competed with AChE-linked PGs for binding to its antibody fixed on the well surface. The color developed by adding substrate for AChE and was reversely proportional to the amount of PGF2-alpha and COX activity. The results represent mean ± SD of triplicate determinations.

Antioxidant Activity of Resveratrol and KST201 and the Effect of Hydrogen

Peroxide on DU145 Cells

Elevated oxidative stress was observed in prostate carcinogenesis. Resveratrol has been shown to be an antioxidant agent against ROS. Its free radical scavenging was investigated in ABTS/AAPH and DPPH solutions which contained stable free radicals

producing blue-green and dark purple color respectively. Decrease of free radicals was

measured spectrophotometrically. Serial dilutions of resveratrol and KST201 were tested,

and the solvents used to dissolved the compounds served as controls for normalization.

Resveratrol attenuated the colorimetric intensity of both free radical solutions

dose-dependently. The results confirmed the antioxidant activity of resveratrol; however,

KST201 was shown to be a less effective free radical scavenger in both assays (Fig.

35A-B). Hydrogen peroxide is one of the ROS responsible for oxidative stress. It

oxidizes DCFH, which can be generated from the hydrolysis of cell-membrane permeable

non-fluorescent DCFH-DA, to impermeable fluorescent DCF. This chain reaction has

been applied to measure the presence of hydrogen peroxide in living cells. The

DCFH-DA solution was added into DU145 cell culture prior to the compounds, and the

fluorescent intensity was detected using a plate reader. Unsurprisingly, resveratrol caused

decrease of fluorescent DCF possibly through blocking the oxidation of DCFH mediated

by hydrogen peroxide or accelerating the metabolism of hydrogen peroxide (Fig. 36).

KST201, on the other hand, showed insignificant antioxidant activity at lower

concentration (25 μM) but acted as a pro-oxidant agent enhancing the production of DCF

at higher concentrations in DU145 cells. Specificity of hydrogen peroxide was confirmed 85

ABTS Radical Scavenging Assay

125% RES KST201 100%

75%

50% Absorbance

25%

0% 0 50 100conc (uM) 150 200 250

Fig. 35A. Free Radical Scavenging Activity of Resveratrol and KST201. ABTS/AAPH free radical solution producing blue-green color described previously was prepared to investigate antioxidant activity of resveratrol and KST201. 6.25, 12.5, 25, 50, 100 and 200 µM of resveratrol and KST201 were tested, and the pure solvent used to dissolve the compounds served as a control. The absorbance detected by a spectrophotometer was proportional to the amount of free radicals in the solution. The results represent mean ± SD of triplicate determinations.

DPPH Radical Scavenging Assay

125% RES KST201 100%

75%

50% Absorbance

25%

0% 0 50 100 conc (uM) 150 200 250

Fig. 35B. Free Radical Scavenging Activity of Resveratrol and KST201. DPPH free radical solution producing dark purple color described previously was prepared to investigate antioxidant activity of resveratrol and KST201. 6.25, 12.5, 25, 50, 100 and 200 µM of resveratrol and KST201 were tested, and the pure solvent used to dissolve the compounds served as a control. The absorbance detected by a spectrophotometer was proportional to the amount of free radicals in the solution. The results represent mean ± SD of triplicate determinations.

Fluorescent DCF Measurement in DU145 Cells

100 Res KST201 Res w/ CAT (50 & 100uM) KST201 w/ CAT (50 & 100uM)

Fluorescence Intensity 25

0 0 25 conc (uM) 50 100

Fig. 36. Measurement of Fluorescent DCF in DU145. The fluorescent DCF assay described previously was used to investigate the effect of resveratrol and KST201 on hydrogen peroxide-mediated oxidation. 25, 50 and 100 µM of resveratrol and KST201 were added followed by introduction of DCFH-DA solution. The fluorescent DCF due to the oxidation via hydrogen peroxide was detected using a plate reader. The results represent mean ± SD of triplicate determinations.

Effect of Hydrogen Peroxide on Cell Viability of DU145 125% Res/24hrs

100% KST201/24hrs

Res/24hrs w/ 75% CAT KST201/24hrs w/ CAT 50% Cell viability Cell

25%

0% 0 50 100 Conc. (uM) 150 200 250

Fig. 37. Effect of Hydrogen Peroxide on Anti-proliferatory Activity of Resveratrol and KST201. MTT cell viability assay described previously was used to investigate resveratrol- and KST201-suppressed cell proliferation on DU145 in the presence of 2 mg/ml catalase. Cells were treated at 200, 100, 50 and 25 µM for 24 hours. The anti-proliferatory activity was shown dose- and time-dependent. The results represent mean ± SD of triplicate determinations.

using catalase in a parallel experiment. Moreover, the effect of hydrogen peroxide in

resveratrol- and KST201-treated cells was revealed by depletion of hydrogen peroxide

using excess of catalase. Inhibition of cell proliferation was not affected by

resveratrol-treated cells but markedly decreased in KST201-treated cells suggesting a

different mechanism involved (Fig. 37).

Cancer-related Gene Expression in Resveratrol- and KST201-treated DU145

Gene expression profile of resveratrol-treated DU145 (Table 5) showed up-regulation of

BIRC3. Baculovirus inhibitors of apoptosis (IAPs) act in insect cells to prevent cell death.

Three human IAP homologs, MIHC (mammalian IAP homolog C), MIHB and MIHA all

contain three BIR (baculovirus IAP repeat) domains in the N-terminal and have shown to

inhibit apoptosis triggered by serum deprivation or by free radical (116, 127).

TRAF1, TNFAIP3 and CSNK1G2, induced by resveratrol, perform regulatory functions in

TNF/TNFR signaling pathway. TRAF1 is an inducible TRAF member and interacts with

TNF receptors, other TRAF members and numerous cytoplasmic proteins directly or

indirectly. After caspase cleavage, it has been reported to suppress NF-kappa B activation

(14). TNFAIP3 expression is activated by TNF dramatically in all tissues. It is a

cytoplasmic zinc finger protein that inhibits NF kappa B activity and apoptosis (113).

Casein kinase (CSNK) members, serine/threonine kinases, have been reported to

phosphorylate the insulin receptor and inhibit its tyrosine kinase activity and constitutively

associate and phosphorylate TNF receptor to negatively regulate TNF signaling (12).

Cancer-related Gene Expression of Resveratrol-treated DU145 Cells

Genbank No. Gene Descriprion Synonyms Fold-change SD

AA456321 insulin-like growth factor 1 (somatomedin c) IGF1 0.36 0.12

AA419164 retinoic acid receptor, beta RARB 0.39 0.11

AA629262 polo-like kinase 1 (drosophila) PLK1 0.40 0.02

cytochrome p450, family 1, subfamily b, AA448157 CYP1B1 0.45 0.03 polypeptide 1

AA479199 nidogen 2 (osteonidogen) NID2 0.46 0.08

W93717 discs, large homolog 7 (drosophila) DLG7 0.47 0.03

T54121 transcribed locus 2.00 0.05

nuclear factor of activated t-cells, cytoplasmic, AA664145 NFATC1 2.06 0.39 calcineurin-dependent 1

AA418990 kiaa0265 protein KIAA0265 2.07 0.21

AA460981 golgi autoantigen, golgin subfamily a, 4 GOLGA4 2.08 1.10

W86876 talin 2 TLN2 2.10 0.21

H37761 nuclear receptor subfamily 4, group a, member 3 NR4A3 2.10 0.53

Continued

AA476272 tumor necrosis factor, alpha-induced protein 3 TNFAIP3 2.11 0.01

H48706 baculoviral iap repeat-containing 3 BIRC3 2.13 0.23

epidermal growth factor receptor pathway H13623 EPS8 2.15 0.20 substrate 8

N93505 transmembrane 4 superfamily member 2 TM4SF2 2.18 0.99

AA670279 collapsin response mediator protein 1 CRMP1 2.25 0.76

AA436227 casein kinase 1, gamma 2 CSNK1G2 2.27 0.22

H21041 activating transcription factor 3 ATF3 2.34 0.09

R80217 prostaglandin-endoperoxide synthase 2 PTGS2 2.35 0.28

R71725 tnf receptor-associated factor 1 TRAF1 2.36 0.18

AA857098 collagen, type v, alpha 2 COL5A2 2.59 1.13

phosphatidylinositol-4-phosphate 5-kinase, type i, R39069 PIP5K1B 2.60 0.42 beta

AA680300 endothelial pas domain protein 1 EPAS1 2.61 1.23

AA135813 chromosome 14 open reading frame 32 C14orf32 2.65 0.36

AA598794 connective tissue growth factor CTGF 2.68 0.25

AA894927 asparagine synthetase ASNS 2.85 0.33

Continued

AI341604 leucine rich repeat containing 17 LRRC17 2.93 0.66

AA464970 phospholipase c, beta 2 PLCB2 3.04 1.60

AA777187 cysteine-rich, angiogenic inducer, 61 CYR61 3.37 0.32

AA707531 similar to kiaa0752 protein 3.98 1.49

AA598496 iq motif containing gtpase activating protein 1 IQGAP1 4.12 0.97

H24206 rab40a, member ras oncogene family RAB40A 7.43 7.08

AA706022 keratin 1 (epidermolytic hyperkeratosis) KRT1 10.07 9.50

H11003 endothelin 1 EDN1 10.27 6.12

Table 5. Cancer-related Gene Expression of Resveratrol-treated DU145. DU145 was treated at 50 µM of resveratrol for 12 hours. The intensity of cy5-labeled test and cy3-labeled reference were detected, and cy5/cy3 ratio was calculated representing the changes of mRNA content after treatment. In this table, only ratios higher than two as well as ratios lower than 0.5 were present. The results represent mean ± SD of triplicate determinations.

Expression of kinase PIP5K1B gene was shown to be up-regulated. Type I PIP kinases

phosphorylates phosphatidylinositol 4-phosphate to generate phosphatidylinositol

4,5-bisphosphate (PIP2). The phosphatidylinositol-3-kinase (PI3K) then converts PIP2 into

phosphatidylinositol-3,4,5-trisphosphate (PIP3) which recruits protein kinase B (Akt) to

the cell membrane. Akt then can be phosphorylated and activated by

phosphoinositide-dependent kinase-1 (PDK1). The role of PI3K/Akt signaling pathway is

blocking apoptotic cell death in response to extracellular stimuli (20, 90). DiPaolo and

colleagues demonstrated that TLN2 interacted with PIP5K1C and increased the local

production of PIP2 (34). This gene was up-regulated by resveratrol as well.

Transmembrane 4 superfamily (TM4SF) proteins are integral to cell membrane, and a

subgroup of TM4SF proteins, including TM4SF2 which was up-regulated by resveratrol,

may modulate PI-dependent signaling by recruiting PI4K to specific locations on the

membrane (139).

T cell receptor (TCR)-induced apoptosis of thymocytes is mediated by calcium-dependent

expression of the steroid receptors Nur77 and NR4A3, members of nuclear receptor

superfamily 4 functioning as ligand-activated transcription factors that regulate

reproduction, development, and general metabolism (134). Mashima and colleagues

reported that ATF3 accelerated the drug-induced apoptosis and enhanced caspase

activation by controlling the upstream signaling of apoptosis through repressing

CRE-dependent gene expression of cell survival factors (78). Both NR4A3 and AIF3 gene were up-regulated by the treatment of resveratrol.

PTGS2 (or COX2) is the key enzyme responsible for the synthesis of prostaglandins (PG)

from arachidonic acid serving as one of the downstream inflammatory mediators of NF

kappa B signaling pathway. PGs stimulate cell proliferation and angiogenesis and inhibit immune surveillance. Overexpression of COX2 has been shown to induce oxidative stress

leading to prostate cancer development and progression (63, 93). Its role involved in

carcinogenesis was further revealed by the studies of selective COX2 inhibitors (145)

which induced apoptosis and prevented tumor angiogenesis by targeting Akt and ERK2

signaling pathways and by down-regulating potent angiogenic vascular endothelial growth factor (VEFG). Subbarayan and colleagues reported that the basal COX2 mRNA and

protein levels were high in normal prostate epithelial cell and low in prostate carcinoma cell

lines (PC-3, LNCaP and DU145), which was contrary to other types of tumor and their adjacent normal tissue (118). In this study, treatment of resveratrol induced gene expression of COX2.

The cytochrome P450 proteins are monooxygenases which determine the rate of conversion of carcinogens from pro-carcinogens as well as the metabolism of hormones involved in diseases of the prostate and generate reactive oxygen species as its byproducts

(144). CYP1B1 was consistently expressed in human prostate tumor (38), and was negatively regulated by resveratrol shown in this study.

EPS8 and IQGAP1 were both significantly up-regulated after a 12-hour treatment of resveratrol in DU145 cells. ESP8 has been shown to form a Rac-specific guanine nucleotide exchange factor complex with ABI1 and SOS1, and the complex mediated positive regulation of Rac activity leading to production of intracellular reactive oxygen 95

species and downstream kinase cascades (42). The actin-binding scaffold protein IQGAP1,

providing linkage between Rac and Cdc42 and the cytoskeleton, directly activated these

GTPases. Furthermore, overexpression of IQGAP1 was reported to increase cell migration

and invasion (11, 79). Rab GTPases, the largest family of the Ras superfamily, regulate

membrane traffic pathways by recognizing specific locations on the intracellular

membrane and recruiting downstream effectors (18). RAB40A is a member of this family

and was induced by resveratrol.

The insulin-like growth factor (IGF) signaling efficiently induces cell growth,

differentiation, and survival. CYR61 and CTGF, members of insulin-like growth factor

(IGF) binding protein (IGFBP), promote cell growth, migration, adhesion, survival as well

as angiogenesis (10, 13, 16). These genes were induced by resveratrol at transcription level.

EDN1 enhances the mitogenic effects of IGF, platelet-derived growth factor, basic

fibroblast growth factor and epidermal growth factor in vitro and induces prostate cancer

proliferation directly (89). High level of circulating IGF1 is associated with an increased

risk of prostate cancer (135). Interestingly, EDN1 was induced while IGF1 was shown to

be down-regulated after resveratrol-treatment.

PLK1 and DLG7 were suppressed at their transcription level by resveratrol. PLK1,

positively regulating cell proliferation and found overexpressed in prostate cancer, is a

serine/threonine kinase required for passage through mitosis playing a key role in the

regulation of cell cycle (99). Tsou and colleagues reported that hepatoma up-regulated

protein (HURP or DLG7) accumulated at the spindle poles during mitosis as a potential

cell cycle regulator involved in the carcinogenesis of human cells (123). 96

Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are hormone-activated transcription factors that repress or activate their target genes by RAR/RXR heterodimers.

RARB was reported as a tumor suppressor gene since selective loss or down-regulation of

RARB gene expression has been observed in prostate cancer as well as other cancers (85).

It was shown to be down-regulated by resveratrol in DU145. PLCB2, a member of

phospholipase C (PLC) which was induced in this study, catalyzes hydrolysis of PIP2 generating second messengers the soluble 1,4,5-inositol trisphosphate (IP3) and the membrane-associated 1,2-diacylglycerol (DAG). IP3 is important for the release of calcium from the intracellular reserves, and DAG is involved in the activation of protein kinase C (PKC) which positively regulates cell proliferation.

EPAS1 regulates transcription of the genes encoding VEGF responsible for angiogenesis and tumor growth (137). It was up-regulated by resveratrol. Nidogens, connecting the laminin and collagen IV networks, stabilize basement membrane of cells and control cell adhesion and migration (65). NID2 was shown down-regulated after 12-hour treatment of resveratrol in DU145. However, another extracellular matrix gene COL5A2 was up-regulated, conversely. CRMP1 characterized as an invasion-suppressing gene (110) was up-regulated

KRT1 is the member of the type II intermediate filaments which are principal structural units and associated proteins of cytoplasmic cytoskeleton. It serves as a biomarker in tumor as it has been reported highly expressed in newborn mouse skin but gradually suppressed while the carcinogenesis was initiated and promoted (103). KRT1 gene expression was up-regulated after treatment of resveratrol. Another target for diagnostics and monoclonal 97

antibody therapy, prostate-specific membrane antigen (PSMA), is highly produced in

hormone-refractory prostate cancer cells. The transcription factor NFATC1, which was induced by resveratrol, mediated PSMA expression through the cooperation of AP3 (55).

Asparagine is required for the production of protein in cells. It can be produced within normal cells by ASNS which is often not present in tumor, especially in lymphocyte leukemia (101). Resveratrol induced gene expression of ASNS in DU145.

KST201 was able to cause significant alterations of 72 cancer-related genes (Table 6),

which were more than two times compared with resveratrol-treated cells (35 genes), at

transcription level in prostate cancer DU145 cells. Expression of these genes regulates

various biological processes and molecular functions (Table 7). 20 genes were at least

two-fold up-regulated or down-regulated by both two compounds (Table 8); in other

words, 57% of resveratrol-regulated genes can be induced or suppressed by KST201.

Moreover, KST201 targeted an even broader spectrum of signaling molecules altered at

the level of transcription. Similar to resveratrol-treated cells, in KST201-treated cells,

molecules involved in TNF/TNFR signaling, PI3K/Akt signaling, IGF signaling and cell

invasion and mitosis were positively or negatively regulated potentially breaking the balance between cell death and survival. In addition to genes which were significantly regulated by both resveratrol and KST201 and discussed previously, the gene expression patterns specifically altered by KST201 were described in the following sections.

KST201 resulted in alterations of a few genes whose products have been considered

inducer or suppressor of apoptosis. Kuribara and colleagues showed that expression of

NFIL3 was regulated by oncogenic Ras mutants through both the Raf/MAPK and PI3K 98

Cancer-related Gene Expression of KST201-treated DU145 Cells

Genbank No. Gene Descriprion Synonyms Fold-change SD

AA456321 insulin-like growth factor 1 (somatomedin c) IGF1 0.28 0.04

AA629262 polo-like kinase 1 (drosophila) PLK1 0.28 0.01

W93717 discs, large homolog 7 (drosophila) DLG7 0.37 0.06

R10284 hyaluronan-mediated motility receptor (rhamm) HMMR 0.40 0.03

AA479199 nidogen 2 (osteonidogen) NID2 0.42 0.07

H24956 ret proto-oncogene RET 0.44 0.06

AA701455 centromere protein f, 350/400ka (mitosin) CENPF 0.45 0.03

met proto-oncogene (hepatocyte growth factor AA410591 MET 0.45 0.15 receptor)

AA419164 retinoic acid receptor, beta RARB 0.48 0.05

AI620493 erythropoietin EPO 0.49 0.12

karyopherin alpha 2 (rag cohort 1, importin AA676460 KPNA2 0.49 0.07 alpha 1)

nuclear receptor subfamily 3, group c, member 1 N30428 NR3C1 2.01 0.54 (glucocorticoid receptor)

Continued

AA400720 ccaat/enhancer binding protein zeta CEBPZ 2.02 1.21

H37761 nuclear receptor subfamily 4, group a, member 3 NR4A3 2.02 0.09

AA490498 kiaa0261 KIAA0261 2.02 1.08

bone morphogenetic protein 7 (osteogenic protein W73473 BMP7 2.03 0.91 1)

AA490721 splicing factor, arginine/serine-rich 9 SFRS9 2.03 0.38

transforming growth factor, beta receptor iii H62473 TGFBR3 2.05 0.99 (betaglycan, 300kda)

W96099 retinoid x receptor, gamma RXRG 2.05 0.22

AA057204 interleukin 2 receptor, beta IL2RB 2.06 0.90

AA102068 heat shock transcription factor 4 HSF4 2.07 0.69

R85545 suppressor of ty 6 homolog (s. cerevisiae) SUPT6H 2.08 1.93

AA443746 general transcription factor iib GTF2B 2.09 0.19

N98485 forkhead box f2 FOXF2 2.10 0.90

nadh dehydrogenase (ubiquinone) fe-s protein 8, AA127014 NDUFS8 2.10 0.71 23kda protein kinase, camp-dependent, regulatory, type AA630507 PRKAR1A 2.12 1.17 i, alpha (tissue specific extinguisher 1)

AA450363 phosphatidylinositol glycan, class f PIGF 2.12 1.95

Continued

100

W23690 adenylate kinase 1 AK1 2.13 1.14

AI277208 homeo box d11 HOXD11 2.14 1.16

R14663 diphtheria toxin receptor DTR 2.21 0.38

AI571806 cadherin 15, m-cadherin (myotubule) CDH15 2.23 0.86

AA029963 ataxin 2-like ATXN2L 2.26 0.16

H87351 serine/threonine kinase 19 STK19 2.26 0.32

H26183 ccaat/enhancer binding protein (c/ebp), beta CEBPB 2.28 0.22

AA633811 nuclear factor, interleukin 3 regulated NFIL3 2.29 0.34

AA430744 enhancer of zeste homolog 2 (drosophila) EZH2 2.30 0.88

H48122 breast cancer 2, early onset BRCA2 2.36 1.36

AA291749 estrogen receptor 1 ESR1 2.37 1.01

phosphatidylinositol-4-phosphate 5-kinase, type i, R39069 PIP5K1B 2.38 0.35 beta potassium inwardly-rectifying channel, subfamily AI636094 KCNJ1 2.43 0.75 j, member 1

R71725 tnf receptor-associated factor 1 TRAF1 2.45 0.40

AA857098 collagen, type v, alpha 2 COL5A2 2.45 0.01

Continued

101

potassium voltage-gated channel, kqt-like H51461 KCNQ2 2.48 1.00 subfamily, member 2

H30546 collagen, type ix, alpha 2 COL9A2 2.50 0.40

R80217 prostaglandin-endoperoxide synthase 2 PTGS2 2.51 0.59

AA460981 golgi autoantigen, golgin subfamily a, 4 GOLGA4 2.54 1.02

H29897 phospholipase c, beta 4 PLCB4 2.54 1.31

AA718910 mad1 mitotic arrest deficient-like 1 (yeast) MAD1L1 2.55 1.24

swi/snf related, matrix associated, actin dependent AA598648 SMARCA4 2.55 1.07 regulator of chromatin, subfamily a, member 4

H66116 angiotensin ii receptor, type 1 AGTR1 2.57 2.35

AI459073 frizzled homolog 9 (drosophila) FZD9 2.58 0.17

AA894927 asparagine synthetase ASNS 2.59 0.32

AA135813 chromosome 14 open reading frame 32 C14orf32 2.60 0.88

AI375353 serum/glucocorticoid regulated kinase SGK 2.62 0.36

H05800 mcf.2 cell line derived transforming sequence MCF2 2.62 0.09

T54121 transcribed locus 2.65 0.51

AA070437 smoothened homolog (drosophila) SMO 2.68 2.21

Continued

102

AA099568 uridine phosphorylase 1 UPP1 2.70 0.69

AA476272 tumor necrosis factor, alpha-induced protein 3 TNFAIP3 2.85 0.29

W96155 v-jun sarcoma virus 17 oncogene homolog (avian) JUN 2.98 0.77

AA598794 connective tissue growth factor CTGF 3.27 0.18

AA707531 similar to kiaa0752 protein 3.31 1.12

AA418813 splicing factor, arginine/serine-rich 7, 35kda SFRS7 3.32 1.81

H11003 endothelin 1 EDN1 3.49 0.98

AA456028 rab geranylgeranyltransferase, beta subunit RABGGTB 3.64 2.06

AA447835 small proline-rich protein 1b (cornifin) SPRR1B 3.66 2.61

AA703169 5-hydroxytryptamine (serotonin) receptor 3a HTR3A 4.19 3.33

H21041 activating transcription factor 3 ATF3 4.90 1.96

AA777187 cysteine-rich, angiogenic inducer, 61 CYR61 4.95 0.72

AA598496 iq motif containing gtpase activating protein 1 IQGAP1 4.95 2.07

AI341604 leucine rich repeat containing 17 LRRC17 5.27 2.40

AA180742 tubulin, alpha 1 (testis specific) TUBA1 11.95 11.89

Table 6. Cancer-related Gene Expression of KST201-treated DU145. DU145 was treated at 50 µM of KSt201 for 12 hours. The intensity of cy5-labeled test and cy3-labeled reference were detected, and cy5/cy3 ratio was calculated representing the changes of mRNA content after treatment. In this table, only ratios higher than two as well as ratios lower than 0.5 were present. The results represent mean ± SD of triplicate determinations.

103

Distribution of Expressed Genes in Treated DU145 Cells

Numbers of Genes Functional Categories Resveratrol KST201

Physiological process 21 53

Binding 15 43

Cellular process 18 41

Signal transducer activity 4 17

Catalytic activity 8 14

Development 6 14

Transcription regulator activity 4 14

Enzyme regulator activity 0 5

Regulation of biological process 3 4

Structural molecule activity 0 4

Transporter activity 0 4

Table 7. Distribution of Differentially Expressed Genes among Categories of Biological Processes and Molecular Functions

104

Cancer-related Genes Significantly Regulated by Both Resveratrol and KST201 Resveratrol Treatment KST201 Treatment Genbank No. Synonyms Fold-change SD Fold-change SD AA456321 IGF1 0.36 0.12 0.28 0.04 AA419164 RARB 0.39 0.11 0.48 0.05 AA629262 PLK1 0.40 0.02 0.28 0.01 AA479199 NID2 0.46 0.08 0.42 0.07 W93717 DLG7 0.47 0.03 0.37 0.06 AA460981 GOLGA4 2.08 1.10 2.54 1.02 H37761 NR4A3 2.10 0.53 2.02 0.09 AA476272 TNFAIP3 2.11 0.01 2.85 0.29 H21041 ATF3 2.34 0.09 4.90 1.96 R80217 PTGS2 2.35 0.28 2.51 0.59 R71725 TRAF1 2.36 0.18 2.45 0.40 AA857098 COL5A2 2.59 1.13 2.45 0.01 R39069 PIP5K1B 2.60 0.42 2.38 0.35 AA135813 C14orf32 2.65 0.36 2.60 0.88 AA598794 CTGF 2.68 0.25 2.68 0.25 AA894927 ASNS 2.85 0.33 2.59 0.32 AI341604 LRRC17 2.93 0.66 5.27 2.40 AA777187 CYR61 3.37 0.32 4.95 0.72 AA598496 IQGAP1 4.12 0.97 4.95 2.07 H11003 EDN1 10.27 6.12 3.49 0.98

Table 8. Cancer-related Genes Significantly Regulated by Both Resveratrol and KST201 in DU145 105

signaling pathways and performed anti-apoptosis without affecting Bcl-xL expression.

CEBPB is a CCAAT/enhancer binding proteins. Suppression of CEBPB expression by siRNA resulted in spontaneous apoptosis in a metastatic Wilms tumor suggesting its cell survival effect. Exposure to extreme conditions induces the heat shock response, characterized by increased expression of heat shock proteins (HSPs) regulating cellular homeostasis and promoting survival. Under severe stress, apoptosis can be activated; therefore HSPs maintain balance between cell survival and death. Heat shock transcription factors (HSFs) are responsible for HSP expression. HSF4 is the member of

HSF family. NFIL3, CEBPB and HSF4 were potential anti-apoptotic genes induced by

KST201 as well as pro-apoptotic JUN. JUN and FOS proteins form transcription factor

AP1 involved in cellular proliferation, transformation and death. The growth-promoting activity of c-JUN is mediated by suppression of tumor suppressors such as p53 and up-regulation of positive cell cycle regulators; however, phosphorylated c-JUN is able to induce apoptosis via Jun N-terminal kinase (JNK) signaling pathway. In addition, KCNJ1 and KCNQ2, controlling potassium ion transport through their potassium channel activity, were shown to be up-regulated. Decreases in potassium ion appear to promote critical events during the early phases of cell death including proteolytic cleavage of pro-caspsase-3 and enhanced endonuclease activation (69, 74, 95, 108, 88, 91).

Various genes encoding positive proliferatory signaling regulators and molecules involved in cell cycle machinery were suppressed in KST201-treated DU145. RET proto-oncogene encoding is a tyrosine kinase growth factor receptor responsible for the growth of both benign and neoplastic prostate epithelial cells. MET, another tyrosine 106

kinase of oncogenes, and its ligand hepatocyte growth factor were shown expressed in

several types of malignant tumor cells including prostate cancer cells. EPO protein is a

hematopoietic cytokine that regulates the production of red blood cells. In clinical specimens of primary prostate tumors cancer and LNCaP and PC-3 cell lines, abundant expression of EPO and its receptor were found. These three genes encoding potential positive proliferatory regulators were down-regulated by KST201 in DU145 cells.

Moreover, several genes producing molecules involved in cell cycle machinery were suppressed as well. Import of proteins into the nucleus requires nuclear localization sequence (NLS)-dependent docking of the substrate at the nuclear envelope followed by

translocation through the nuclear pore. KPNA2 was shown to bind to the NLS motif of its

substrate and promote docking of import substrates to the nuclear envelope. Moreover,

yeast KPNA2 interacted with multiple components required for mitosis in the nucleus. In

cancers, HMMR was reported to affect centrosomal structure and spindle integrity

potentially modulating apoptotic and cell cycle. CENPF encodes a protein associating

with the centromere-kinetochore complex in late G2 phase of cell cycle, and this

association is maintained through early anaphase (30, 68, 5, 132, 70, 80, 147).

Simultaneously several genes encoding proteins functioning as negative regulator of cell

proliferation were up-regulated by KST201. BRCA2 functions as a tumor suppressor

gene encoding a product which is increased during the progression of cell cycle

performing growth inhibitory activity. TGF-beta members, normally mediating growth

inhibition, initiate their cellular action by binding to serine/threonine kinase receptors.

TGF-beta 1 binds directly to the transforming growth factor-beta receptor (TGFBR) 2 107

while binding of TGF-beta 2 to TGFBR2 requires co-expression of TGFBR1 or TGFBR3.

Miyazaki and colleagues reported that BMP7, a member of transforming growth factor-beta superfamily, inhibited the cell proliferation of PC-3 and DU-145 through the

G1 phase blockage of cell cycle and up-regulation of the Cdk inhibitor p21 leading to

decrease of Cdk2 activity. MAD1L1 plays a role in cell cycle control and tumor

suppression through its mitotic check point function preventing the onset of anaphase

until all chromosome are properly aligned at the metaphase. RARs and RXRs mediate

hormone-activated gene transcription through RAR/RXR heterodimers. RXRG, a member

of the RXR family of nuclear receptors, involved in mediating the anti-proliferatory

effect of retinoic acid. SMARCA4 (or BRG1) protein is the catalytic subunit of the

SWI/SNF chromatin-remodelling complex and influences transcriptional regulation by

disrupting the interaction between histone and DNA contact ATP-dependently. It has been

demonstrated that binding to the retinoblastoma (RB) tumor suppressor protein was

required for RB-mediated cell cycle inhibition. SPRR1B was shown responsible for the

entry into G0 phase withdrawing cells from the proliferatory state possibly in concert with

other proteins. In addition, CEBPZ, one of CCAAT/enhancer binding proteins inducible

by a variety of cellular stresses, such as nutrient deprivation and DNA damage has been

show causing growth arrest in many cell types (98, 25, 102, 83, 124, 85, 21, 29).

Glucocorticoid receptor (GR or NR3C1) encoding protein acts as a transcription factor when binds to its ligand glucocorticoid, a hormone predominantly affecting the metabolism of carbohydrates. Glucocorticoids have been shown to interfere with the

transcriptional activity of several transcription factors including NF-kappa B, thus may 108

inhibit survival signaling (31). SGK encodes a serum-and glucocorticoid-induced

serine/threonine protein kinase and is the transcriptional target of the activated GR. In

breast cancer cells, SGK acted as an anti-apoptotic kinase (81). Both NR3C1 and SGK

were up-regulated by KST201 in DU145 cells

KST201 similar to resveratrol caused up-regulation of various genes which encode

proteins tend to contributing cell proliferation through multiple signaling pathways. Fu

and colleagues reported that DTR behaved as an effecter of JUN-induced oncogenic

transformation. The coupling between the signaling cascades of ESR1 and ESR2 and the

IGF1 receptor (IGF-1R) was investigated in Kahlert’s laboratory. The ligand bound ESR1

required for rapid activation of the IGF-1R signaling cascade was demonstrated. cAMP,

through the activation of cAMP-dependent protein kinase (PKA), regulates various

cellular functions including metabolism, cell proliferation and differentiation, and gene

expression. The inactive PKA is a tetramer composed of two regulatory and two catalytic

subunits. The protein encoded by PRKARIA is one of the regulatory subunits. HOXD11

encodes a transcription factor which has been reported activating IGFBP1 by interacting

its promoter. Varambally and colleagues reported that EZH2 was over-expressed in

hormone-refractory metastatic prostate cancer, and suppression of EZH2 by siRNA

caused inhibition of cell proliferation. Many human carcinomas constitutively express

mRNA and protein of interleukin-2 (IL-2) and its receptor IL-2 alpha, beta and gamma

(IL2RA, IL2RB and IL2RG) chains controlling cell cycle progression. FOXF2 belongs to

human forkhead-box (FOX) transcription factor gene family which consists of at least 43

members. Over-expressed FOX genes result in congenital disorders, diabetes mellitus, or 109

carcinogenesis. PLCB4 converts PIP2 to second messengers the soluble IP3 and the

membrane-associated DAG. IP3 is responsible for the release of calcium and DAG is

involved in the activation of PKC. Angiotensin II was shown to enhance the proliferation

of prostate cancer LNCaP and DU145 cells through AGTR1 (angioteinsin II

receptor)-mediated activation of MAPK and STAT3 phosphorylation. Wnt

proto-oncogenes and Wnt signaling play an important role in embryogenesis and cancer.

FZD9 is a frizzled gene encoding a receptor for Wnt proteins. SMO signaling triggers a

cascade of intracellular events. Mutant transmembrane protein Patched or SMO induce ligand-independent Hedgehog signaling pathway leading to human tumors such as basal cell carcinoma. Furthermore, HTR3A encodes a receptor for 5-hydroxytryptamine (5HT).

Its proliferatory effect was revealed by investigating the activity of 5HT antagonist which

caused growth inhibition in a variety of tumor cells including prostate, lung and colonic

carcinoma (40, 58, 105, 43, 128, 100, 61, 126, 15, 120, 113).

In addition to IQGAP1, two genes involved in GTPase signaling were up-regulated by

KST201 in DU145 cells. Activation of Rab GTPases is regulated by the cycle of

reversible attachment to membrane mediated through geranylgeranylation of two

C-terminal cysteines by RABGGTB and Rab escort protein, and by replacement of GDP

by GTP (47). MCF2 is a member of GDP/GTP exchange factors that modulate the

activity of Rho GTPases family (67).

Several genes encoding proteins involved in RNA synthesis as well as post-transcription

processes were induced in KST201-treated DU145. GTF2B encoding protein is one of

the ubiquitous factors required for transcription initiation by RNA polymerase II. 110

SUPT6H was reported to regulate mRNA synthesis through its interaction with histones

and RNA polymerase II elongation. SFRS7 and SFRS9 are members of

arginine/serine-rich splicing factor family required for early spliceosome assembly and 5'

splice site selection. AK1 is responsible for catalyzing the reversible transfer of

phosphate group among adinine nucleotides regulating the adenine nucleotide

composition within cells. STK19 encodes a serine/threonine kinase predominantly

localized in nucleus. It possibly functions as a transcriptional regulator through

phosphorylation. UPP1 gene product is an uridine phosphorylase responsible for the

reversible phosphorolysis of uridine to uracil tightly regulating the concentration of

uridine, a pyrimidine nucleoside essential for the synthesis of RNA and bio-membranes

in plasma and tissues. (24, 36, 41, 46, 96)

Formation of ROS in electron transport (ETC) increases oxidative stress. In the first step

of ETC, NDUFS8 is the complex I NADH dehydrogenase, an electron carrier is

responsible for conservation of mitochondrial energy. Coupling with vitamin E to restore

the antioxidant activity, its reduced form functions as a powerful antioxidant against

oxidative stress (37). NDUFS8 was up-regulated by KST201.

Many proteins are anchored to the cell membrane by glycosylphosphatidylinositol (GPI).

PIGF has been shown involved in GPI-anchor biosynthesis. CDH15 is a member of the

cadherin superfamily of genes, encoding calcium-dependent intercellular adhesion

glycoproteins essential for the control of morphogenetic processes, specifically

myogenesis. COL9A2 protein is one of the three alpha chains of type IX collagen. In

KST201-treated DU145 cells, TUBA1, the main constituent of microtubules, was shown 111

to be up-regulated which was consistent with the presence of intercellular processes connecting neighbor cells (112, 111, 130). The transcription level of all these genes was found significantly increased in KST201-treated DU145 cells.

112

DISCUSSION

The major goal of this study was to discover and demonstrate cellular behaviors of cancer

cells treated with potential anticancer agents. The MTT cell viability assay provided an

overall view on cell proliferation (17, 33, 48, 50, 84) without information of mechanisms

involved. Results gathered from this experiment were helpful to sieve out optimal

compounds of interest from a group of chemical structural-related analogs and to direct

the following research design. Inhibition of cell proliferation can be achieved by

induction of cell death and blockage of cell division. Cell cycle analysis using flow

cytometry was an idea method which simultaneously was able to quantify cell population

in different phases of cell cycle as well as cells undergoing apoptosis (72, 131) after

treated with these potential anticancer agents. Several other methods were commonly

utilized to investigate apoptosis quantitatively and qualitatively. DNA fragmentation

assay and annexin V-FITC/PI staining were based on features of apoptotic cells, and the

assay for caspase-3 activity detected the presence of a key player of apoptosis (4, 6, 26,

129). Since NF-kappa B plays a crucial role in the balance of cell death/cell survival (60,

77, 138), regulation of this transcription factor has been considered one of the possible strategies to fight cancer. Inhibition of COX enzymes has been reported contributory to anticancer activity, since both COX isomers, especially inducible COX2 responding to inflammation, produce PGs and ROS involved in carcinogenesis (63, 93, 138, 146). It was likely that these potential anticancer agents acted as NF-kappa B regulators and COX 113

inhibitors. Oxidative stress has been implicated to the signal transduction network and shown involved in all stages of cancer formation. The balance of antioxidant/oxidant is extremely important for cells to retain controlled on cell proliferation and to prevent damage on intracellular components (64, 92, 93, 144), for instance, hydrogen peroxide has been reported to activate tyrosine kinases, MAPKs, PKC, EGF receptor, protein phosphatases, potassium channels and AP-1 and NF-kappa B. These potential anticancer agents might be able to maintain the cellular environment in a non-proliferatory reduced state or to enhance a severe cytotoxic oxidative state eventually leading to cell death.

Free radical scavenging assay and investigation of hydrogen peroxide effect revealed the role of these potential anticancer agents in the redox reactions in cancer cells.

Gene expression profile provides information of individual genes in genome at

transcription level under certain conditions, for instance, after introduction of extracellular

stimuli or stress. Responding to the stimuli or stress, some genes might be activated whereas some might be suppressed. Theoretically the total content of their encoding proteins in the system can be altered, and these changes further affect their target signaling pathways. Nevertheless, a single cellular event has been considered a balance among the signal transduction network, and complexity of crosstalk between conflictive signaling components makes it difficult to identify the exact players driving the biological reactions.

Therefore, gene expression profile alone may not be sufficient to predict the destiny of a treated cell, especially a compound regulating multiple mechanisms is present. In addition, post-transcriptional as well as post-translational processes are critical for newly synthesized proteins in order to be fully functional; in other words, a highly expressed 114

protein may not be effective until properly modified, for instance, phosphorylated or proteolytic cleavaged. Data obtained from microarray lack the quantitative measurement of activated proteins and the intensity of their activities. However, the transcriptional pattern of genes may imply key components and possible mechanisms when correlating

with the evidence collected to support the present of cellular behaviors of interest by other methods.

Resveratrol was composed of two phenolic rings with two hydroxy groups on one ring and

one hydroxy group on the other. These functional groups favor resveratrol’s diffusion in

both hydrophilic and lipophilic environments and interactions with a variety of

biomolecules including DNA, proteins and lipids via redox reaction or hydrogen bonding

for instance. Moreover, the chemical structure of resveratrol resembles some steroid

hormones to compete with their receptors and regulate target genes. Therefore, it was not

surprising that resveratrol was able to interact with a broad spectrum of intracellular targets,

and KST201 with similar structural characteristics may perform duplicate effects under

the same conditions.

Results of MTT cell viability assay have demonstrated the anti-proliferatory activity of

resveratrol and several KSA and KST compounds on all three cell lines tested. This effect

was shown in a dose- and time-dependent manner. In addition to resveratrol, KST201,

KST213, KST401 and KSA1201 were able to cause 50% decrease of cell viability at

concentration lower then 100 μM for 24-hour treatment on all three cell lines, and the

activity was more significant after 48 and 72-hour treatment. However, except KST201,

all other compounds were shown to be toxic based on their CD50 values against normal 115

MHRF cells. In other words, if used as anticancer drugs, these compounds might target

not only the cancerous tissue but also the healthy tissue while being delivered in the body.

Therefore, CSI value, CD50 on normal cells divided by CD50 on cancer cells, representing

the selectivity against cancer cells became essential for evaluation of optimal anticancer

agents. KST201 was shown performing the highest CSI value in all cell lines at all time

points of investigation. It reduced cancer cell viability effectively but without blocking

normal cell proliferation; thus, it should be toxic to cancer cells but non-toxic to normal

cells. Moreover, KST201 was unique since its CSI values on DU145, MDAH and T24

cells increased time-dependently which was not observed on other compounds. It turned

to be more selective against cancer cells when longer treatment time was allowed. Up to a

certain period of treatment time, the dosage of KST201 used to inhibit cancer cell

proliferation can be dramatically decreased but still effective with even less side effect to

normal tissue. More work has to be done to explain this phenomenon, and it seemed that

metabolism of KST201 in cancer and normal cells might be relevant.

To study how resveratrol and KST201 regulate cell cycle machinery, PI-stained

resveratrol- and KST201-teated cell nuclei were detected using a flow cytometer. In

DU145 cells, agreeing with literature (62), resveratrol at its CD50 resulted in cell cycle

arrest and apoptosis indicated by the accumulation of G0/G1 cell population and

appearance of the sub G0/G1 peak, respectively. If half of CD50 was used, the amount of

apoptotic cells induced by resveratrol was markedly less that when treated at its CD50.

Together with results from MTT assay, the flow cytometric histogram revealed that at

CD50, which cell viability of treated DU145 cells was decreased to 50% of untreated 116

control, resveratrol exerted its anti-proliferatory action through blocking cell cycle and

inducing cell apoptosis, and at lower concentration it mostly inhibited cell cycle.

Dissimilarly, KST201 acted as a strong apoptosis inducer at both half CD50 and CD50.

Although KST201 inhibited cell proliferation in both MDAH and T24 cells as well, at low concentration it tended to cause interruption of DNA synthesis indicated by the increased population in the S phase of the cell cycle.

Apoptosis induced in both resveratrol- and KST201-treated DU145 cells was further confirmed by a series of experiments typically and commonly utilized to investigate this type of cell death. Morphological changes including intercellular processes for contact with neighboring cells, chromatin condense to the nucleus periphery, DNA fragmentation, membrane blebbing and rearrangement of phospholipids on cell membrane were mediated through the proteolytic activity of caspase-3 (4, 6, 26, 129). These features of apoptotic cells were observed by cytoplasmic DNA fragmentation assay and fluorescence microscopy, and activation of caspase-3 was shown correlated with resveratrol- and

KST201-induced apoptosis. Both compounds exerted pro-apoptotic activity, and as expected, KST201 was more effective than resveratrol. At certain condition, for instance, introducing much higher concentrations for treatment on DU145 cells, both compounds were able to push apoptotic cells undergoing necrosis, and KST201-treated cells seemed to be more susceptible to necrotic cell death.

NF-kappa B is a multiple-target transcription factor regulating gene expression leading to a variety of cellular behaviors (60, 77, 138). Resveratrol has been reported to suppress activation of NF-kappa B and cell survival signaling in numerous cell types contributing 117

its anticancer activity (3). Both resveratrol and KST201 inhibited DNA-binding activity of NF-kappa B p65. Interestingly, resveratrol was able to performed more then 50% blockage of its binding activity after 12-hour treatment at 100 µM, whereas 24 hours was required for 100 µM KST201 to achieve 50% blockage. Possibly it was because of different signaling molecules these two compounds targeting or different manners these two compounds exerting to modulate NF-kappa B signaling pathway.

Testing the potential anticancer agents for their COX inhibitory activity was one of the interests of this study since COX2 inhibitors were shown to fight cancer by inducing apoptosis (63, 93, 138, 144). Both resveratrol and KST201 inhibited COX1 by remarkable preference although COX2 activity producing PGs was blocked as well.

Resveratrol showed selective inhibitory activity on COX1, which was consistent with published data reported by other researchers. Since resveratrol is not a carboxylic acid as are non-selective or COX1 inhibitors, and does not contain a sulphonamide or sulphone group as do COX2 inhibitors, this small molecule with two aromatic rings and three hydroxyl groups hardly fits the catalytic channel of cyclooxygenase active site to completely block the access of its substrates (Fig. 38-39). However, as long as hydrogen bonding is present between the compound and the polar arginine 120, which is half way down the channel, the cyclooxygenase activity can be partially blocked. On the other hand, COX inhibition can occur by interference of its peroxidase activity. Enzymatic activity at cyclooxygenase active site is initiated by reduction of oxygen-derived free radicals at peroxidase site, and peroxide intermediate PGG2 generated at cyclooxygenase active site serves as substrate for peroxidase site to continue activating cyclooxygenase 118

active site. Resveratrol is well known as an antioxidant and free radical scavenger, it can

interrupt the redox at peroxidase site, which is responsible for initiation of cyclooxygenase activity and synthesis of stable precursor PGH2. Since much higher

peroxide level is required for COX1 than for COX2 to initiate cyclooxygenase active site, resveratrol performed preferential inhibition to COX1. However, KST201, which was

proved not an antioxidant, performed its anti-COX activity through distinct mechanism.

COX1 is important for maintenance of the normal lining of the stomach and intestine,

and blocking this enzyme can lead to ulcers. Therefore, KST201 with less inhibitory

activity than resveratrol against COX1 might cause less side effect in the gastrointestinal

tract.

Fig. 38. Non-selective COX Inhibitor Flurbiprofen and COX2 inhibitor SC558 119

Fig. 39. Illustration of the Catalytic Channel of Cyclooxygenase in the Presence of Flurbiprofen

120

Although both resveratrol and KST201 caused resembling cellular events, mechanisms in

which KST201 was involved might not be the same as resveratrol. Multiple actions

performed by resveratrol were mediated by its antioxidant activity (6). Using stable free

radical system, ABTS/AAPH and DPPH solutions, resveratrol was shown to attenuate the amount of free radicals in both solutions dose-dependently. While the antioxidant activity of resveratrol was confirmed, KST201 acted as a less effective free radical scavenger. In addition, in hydrogen peroxide-oxidized fluorescent DCF assay, resveratrol again caused decrease of DCF production possibly through blocking the oxidation process mediated by hydrogen peroxide or accelerating the metabolism of hydrogen peroxide. KST201, however, showed pro-oxidant activity in DU145 cells. Moreover, a parallel experiment of

MTT assay in the presence of catalase used to deplete hydrogen peroxide indicated that inhibition of cell proliferation was not affected in resveratrol-treated cells but markedly decreased in KST201-treated cells suggesting that the anticancer activity of KST201 was mediated through hydrogen peroxide.

After 12 hour-treatment of 50 µM on DU145 cells, resveratrol and KST201 preformed comprehensive regulatory effects on gene expression including genes responsible for

apoptosis, cell proliferation, cell cycle, angiogenesis, cell migration as well as

detoxification, which involved in different stages of carcinogenesis. KST201 regulated 72

cancer-related genes, whereas resveratrol caused alteration of 35 genes at transcription

level in prostate cancer DU145 cells. Twenty genes were at least two-fold up-regulated or

down-regulated by both two compounds; in other words, 57% of resveratrol-regulated

genes can be induced or suppressed by KST201. 121

Since numerous signaling pathways leading to target gene expression are able to be

modulated by ROS (64, 92, 93), resveratrol regulates these pathways by serving as a

switch for the balance between antioxidant and oxidant via its antioxidant activity.

CYP1B1 encoding protein responding to cellular detoxification converts carcinogens from

pro-carcinogens through monooxygenase activity and generates reactive oxygen species as

its byproducts (144). CYP1B1 consistently expressed in human prostate tumor was

negatively regulated by resveratrol, shown in DU145 gene expression profile, contributing

to the decrease of DNA damage and the moderation of oxidative stress. This gene was not

affected in KST201-treated DU145 cells. Instead, NDUFS8, encoding complex I NADH dehydrogenase, was up-regulated by KST201.

One of the death receptor-mediated apoptotic pathways is initiated through the interaction

of TNF and its receptor TNFR. The intracellular death domain of the receptor recruits the

adapter molecule FADD, which further recruits and activates caspase-8 leading to

activation of caspase-3 and induction of mitochondrial damages (4, 6, 26, 129). On the

other hand, TNF/TNFR binding has been shown to stimulate NF-kappa B activity that

produces survival signals under certain circumstances. Cells tend to undergo apoptosis in

response to TNF when the NF-kappa B signaling pathway is blocked. DU145 but not

LNCaP was reported to be highly sensitive to this TNF-related apoptosis-inducing ligand

(TRAIL)-induced cell death. TRAF1, TNFAIP3 and CSNK1G2 encoding proteins perform

regulatory functions in TNF/TNFR-related signal transduction. They were shown

significantly expressed after treatment with resveratrol. In KST201-treated cells, TRAF1

and TNFAIP3 but not CSNK1G2 was induced. TRAF1 protein suppressed NF-kappa B 122

activation in the presence of caspase enzymes, and both TNFAIP3 and CSNK1G2 proteins

negatively regulate NF-kappa B activation and apoptosis. It was consistent with the

decreased p65-DNA binding demonstrated by NF-kappa B activity assay in resveratrol and

KST201 treated cells. Contradictorily, a downstream NF-kappa B-inducing protein COX2

was up-regulated by the treatment of both compounds. Resveratrol and KST201 might be

able to activate signaling molecules which act as both NF-kappa B inducers or suppressors.

Activation of NF-kappa B remained possibly for a short period of time allowing a few

target genes to be turned on, and the system eventually favored anti-NF-kappa B due to

continuing expression of NF-kappa B suppressing genes. In addition, resveratrol inhibited

NF-kappa B through other mechanisms such as blocking kinases upstream in the signaling cascade (6), which has not yet be investigated in KST201-treated cells.

TM4SF2, PIP5K1B and TLN2 protein products involved in PI3K/Akt signaling pathway

leading to the inhibition of apoptosis. Moreover, overexpression of COX2 has been

reported to induce Akt against apoptotic cell death. Significant up-regulation of these

genes in resveratrol-treated DU145 cells implied that, through the activation of Akt, might perform inhibitory effect on apoptosis. Additionally, BIRC3, an inhibitor of apoptosis through IAP-caspase interaction, was induced. Meanwhile two pro-apoptotic genes,

NR4A3 and ATF3, were significantly up-regulated by resveratrol. The overall effect of resveratrol on DU145 was actually pro-apoptotic according to other experiments. Together, the alterations of relevant genes at transcription level observed from cDNA microarray and previous findings indicated that most likely the inhibition of NF-kappa B signaling pathway was the determinant driving the system toward apoptotic cell death and 123

overwhelmed the activation of PI3K/Akt signaling pathway counteracting the pro-apoptotic effect. In KST201-treated cells, only PIP5K1B was up-regulated which seemed to be less effective on Akt activity. Moreover, both pro-apoptotic NR4A3 and

ATF3 but not BIRC3 gene were induced. Other apoptosis-related genes including potential anti-apoptotic NFIL3, CEBPB, HSF4 and SGK, and possible pro-apoptotic JUN,

KCNJ1 and KCNQ2 were up-regulated revealing diverse molecules were targeted by

KST201 in terms of apoptosis.

Cell cycle analysis of resveratrol- and KST201 treated DU145 indicated the interruption in

the G0/G1 phase leading to cell cycle arrest. PLK1 and DLG7 protein products were

responsible for this effect since both proteins were shown essential for cells to enter mitosis

and suppressed at their transcription level by resveratrol and KST201. Moreover, KPNA2,

HMMR and CENPF encoding proteins essential for cell cycle machinery were

down-regulated, and MAD1L1 and SPRR1B encoding proteins, playing roles in mitotic

check point and entry of G0 phase respectively, were up-regulated in KST201-treated

DU145 cells.

Several gene products relevant to cell proliferation were found to be up-regulated in resveratrol-treated DU145 cells including EPS8, IQGAP1 and RAB40A involved in

GTP/GDP exchange of GTPase signaling, and PLCB2 for PKC activation. Moreover,

RARB, a tumor suppressor gene which was down-regulated. CYR61, CTGF and EDN1 encoding proteins playing roles in IGF signaling were all induced; however, IGF1 was suppressed.

Various genes encoding positive regulators of cell proliferation were suppressed in 124

KST201-treated DU145 including IGF1, proto-oncogene RET and MET, and cytokine

EPO. Simultaneously, several genes encoding proteins functioning as negative regulator

of cell proliferation were up-regulated including tumor suppressor gene BRCA2, BMP7

and TGFBR3 involved in TGF-beta signaling, RXRG belonging to the RXR family of

genes, SMARCA4 required for RB-mediated cell cycle inhibition, CEBPZ protein

product causing growth arrest in response to DNA damage as well as NR3C1 protein

product inhibiting survival signaling.

Similar to resveratrol, KST201 caused up-regulation of genes which encode proteins

contributing to cell proliferation through multiple signaling pathways. These were

HOXD11, ESR1, EDN1, CTGF and CYR61 involved in IGF signaling, DTR encoding an

effecter of JUN-induced oncogenic transformation, PRKARIA encoding a regulatory

subunits of PKA, HTR3A and EZH2 and AGTR1 encoding proteins responsible for cell

proliferation, IL-2 receptor gene IL2RB, transcription factor gene FOXF2, PLCB4 encoding protein activating PKC, FZD9 involved in Wnt signaling, SMO involved in

Hedgehog signaling as well as IQGAP1, RABGGTB and MCF2 which are members of

GDP/GTP exchange factor genes.

Several genes encoding proteins involved in RNA synthesis as well as post-transcription

processes and were induced in KST201-treated but not resveratrol-treated DU145 cells

including GTF2B, SUPT6H, SFRS7 and SFRS9, AK1, STK19 and UPP1.

It was obvious that treatment with KST201 at 50 μM for 12 hours might regulate more

diverse cell proliferation- or apoptosis-related signaling pathways by altering gene

expression of their key components. Surprisingly, none of cyclins, cyclin-dependent 125

kinases (cdk), cdk inhibitors, caspases or Bcl-2 proteins was shown activated or suppressed at transcription level as other laboratories have reported using the same cell line (62), notwithstanding the anti-proliferatory activity of resveratrol was reproduced. The time point for sample preparation and data collection might be critical.

Resveratrol interacted diverse signaling molecules causing changes at gene transcription in both hormone-sensitive LNCaP (57) and hormone-independent DU145 cells, yet the discrepancy of molecular targets was manifested. In resveratrol-treated LNCaP, activation of p53-responsive genes was demonstrated using cDNA microarray, and it was correlated with resveratrol-induced apoptosis; however, based on the results of this study, pro-apoptotic effect was mediated through other signaling pathways in resveratrol-treated

DU145. Since AR mediates proliferation and differentiation in the prostate (7, 27, 45), and p53 is responsible for genomic stability and apoptosis, both of them are crucial in prostate carcinogenesis. ARs are normally expressed in LNCaP but not in DU145 cells, and LNCaP produces wild-type p53 whereas DU145 express mutant p53. However, even in the absence of key components, such as p53 and AR, resveratrol still was able to yield equivalent cellular behaviors. According to experimental results generated from this study as well as published data collected from literature, resveratrol might perform a wide range of potential therapeutic applications against prostate cancer due to its pro-apoptotic and cell growth inhibitory activity in both cell lines representing earlier androgen-dependent stage and advanced androgen-independent stage of prostate tumor.

NF-kappa B regulates inflammatory and immune responses and cell proliferation by increasing the expression of specific genes. These genes encode cytokines and 126

chemokines, proteins responsible for immune recognition, antigen presentation and

neutrophil adhesion and migration, enzymes involved in inflammatory process including inducible nitric oxide synthase (iNOS) and COX2, as well as anti-apoptotic molecules

including inhibitors of apoptosis, TNF receptor–associated factors and anti-apoptotic

Bcl-2 family members (138). Cytokine IL-1beta and TNF-alpha can be induced by

NF-kappa B and establish a positive feedback to amplify the inflammatory response via

directly activation of the NF-kappa B pathway. iNOS produces nitric oxide (NO), and

COX2 generates prostaglandins causing increased blood flow and temperature, redness,

swelling and pain. Survival signals generated by the NF-kappa B pathway modulate

proliferation and differentiation of both B- and T-lymphocytes. Constitutive activation of

the NF-kappa B responding to inflammatory stimuli was implicated to breast, ovarian, prostate, and colon cancer formation due to up-regulation of its downstream targets which can induce cell proliferation and prevent cell death. Therefore, inhibition of prolonged

activation of the NF-kappa B was considered one of the therapeutic strategies in the

cancer treatment. Phyto-polyphenols such as myricetin, quercetin and resveratrol have been reported suppressing the NF-kappa B pathway (138). The inhibition of NF-kappa B activity mediated by resveratrol was confirmed in this study. Moreover, its inhibitory effect on COX enzymes was demonstrated although with higher selectivity on COX1.

KST201 performed inhibitory effects on NF-kappa B and COX enzymes suggesting that both compounds might be able to negatively regulate inflammatory process (Fig. 40).

Resveratrol has been shown to exert its anticancer activity by producing the reduced environment in cancer cells which are usually oxidative and favoring proliferation. By 127

Resveratrol KST201

Resveratrol

KST201???

Resveratrol

KST201

Resveratrol KST201

Fig. 40. Regulation of NF-kappa B Signaling Pathway by Resveratrol and KST201. Resveratrol and KST201 inhibited activation of NF-kappa B and COX2 activity; however, COX2 gene expression was up-regulated. The mechanism of KST201-mediated blockage of this pathway is not known yet.

128

decreasing ROS and attenuating oxidative stress, a variety of signaling pathways leading

to cell growth are able to be blocked. Cell survival signaling, for instance, induced via

NF-kappa B pathway can be inhibited switching the status of cell growth toward cell

death. On the other hand, part of the anticancer activity mediated by KST201 seemed to

be performed via different patterns. Although inhibition of NF-kappa B was observed as

well, treatment with KST201 tended to produce more ROS, especially hydrogen peroxide,

in the cell. If oxidative stress in a cell reaches an overwhelming level, it pushes the cell

undergoing apoptosis as well as necrosis. The balance between cell death and cell

survival is maintained tightly in normal cells. Inclining to either side causes diseases such

as cancer or neuronal diseases because of uncontrolled cell proliferation or

over-degradation of cells. Cancer cells are able to escape from cell growth restriction and apoptosis by constitutively producing cell survival signals and retaining the oxidative state (Fig. 41). Excess of resveratrol shifted the oxidative state back to reduced state and inhibited survival signals leading to cell death, whereas in addition to survival signal

blockage, KST201 elevated oxidative stress more severely thus pushed cells toward death.

Although both resveratrol and KST201 at the concentrations ranging from 25-200 μM led to similar cellular events, these two compounds triggered not quite the same signaling pathways. Details of mechanisms involved in the pro-apoptotic and anti-proliferatory actions of KST201 were not available so far. Flavonoids and resveratrol analogs were considered sources of antioxidant agents, and antioxidant activity was often correlated to chemoprevention and therapeutics against cancer; however, KST201 was shown not a member of these polyphenols suggesting a novel chemical structural basis used for the 129

Apoptosis

Cell Survival Cell Death

KST201

Cell Survival Cell Death Cell Survival Cell Death

Normal Cell Cancer Cell

Resveratrol

Apoptosis

Cell Surviv al Cell Death

Fig. 41. Illustration of Cell Survival/Cell Death Status in Normal Cells, Cancer Cells and Resveratrol- and KST201-treated Cells. 130

design of anticancer agents.

Results obtained from experiments described in this study demonstrated the potential

anticancer activity of novel resveratrol analog KST201. KST201 exerted significant

anti-proliferatory activity against cancer cells and was the most selective compound

based on its CSI value which increased in a time-dependent manner. KST201 caused

prostate cancer DU145 cell cycle arrest in the G0/G1 and ovarian cancer MDAH and

bladder cancer T24 in the S phase. All three cell lines (DU145, MDAH and T24)

produced the subG0/G1 cell population indicating the presence of DNA fragmentation

which was further confirmed by another assay quantifying cytoplasmic DNA fragments

in DU145 cells. Changes of cell shape and nucleus, membrane blebbing, elongated processes as well as phospholipids PS translocation on the cell membrane were observed

in KST201-treated DU145 cells. These findings, together with the activation of caspase-3, manifested the occurrence of apoptotic cell death. NF-kappa B signaling pathway was

blocked by KST201 in DU145 cells, and the anti-proliferation was mediated by hydrogen

peroxide. KST201 has been considered as a pro-oxidant that performed less activity

compared to resveratrol on COX enzyme inhibition; however, it may prevent gastric ulcer

caused by COX1 suppression. In DU145 cells, KST201 regulated more cancer-related genes than resveratrol. These genes encode proteins playing roles in cell proliferation, cell cycle, cell death, cell migration and invasion, etc. Many resveratrol-regulated genes can be affected by KST201 indicating the possibility that signaling pathways targeted by these two compounds may overlap. TRAF1 encoding a pro-apoptotic protein through the

TNF signaling pathway was up-regulated, and IGF1 encoding the growth factor inducing 131

proliferatory IGF signaling pathway was down-regulated by both compounds. PLK1 protein product involved in cell cycle was suppressed, and ATF3 protein product inducing apoptosis was activated. These important genes relevant to cell proliferation and cell death were significantly affected by resveratrol and KST201 on DU145 cells.

Many questions remain unanswered because of the limitations of techniques used in this study; therefore, solving these problems could be a future project. The MTT cell viability assay and flow cytometry provide little information of key components and mechanisms involved. Other methods suitable to detect cell cycle modulating proteins such as western blotting can be used to further explore the mechanisms in detail. For further study on the activation of caspase-3, cleavage of its downstream targets such as the poly (ADP-ribose) polymerase (PARP) can be evidence for caspase-3 activity. Signaling molecules affected by KST201 in NF-kappa B pathway can further be elucidated by investigating the activation of NF-kappa B-inducing kinase, phosphorylation and degradation of I kappa B, and translocation of NF-kappa B between the cytoplasm and nucleus. The COX activity assay used in this study might not be able to fully reproduce actual biological system in cancer cells, since it utilized ovine COX enzymes in a buffer with essential elements to initiate the chemical reaction in test tubes. However, the concept of this method can be applied using cell lysates prepared from cells constitutively expressed COX1 or COX2.

More efforts have to be made to demonstrate other pharmacological properties of

KST201. Experimental data of absorption after intake, transport in the blood stream, metabolism and excretion, which are not available yet, are important for evaluation of its practical use. Since most assay were carried out in cell lines which can only mimic 132

specific stage of cancers, studies using proper animal models, for instance, mice with gene mutations gradually developing prostate cancer, might be necessary in order to confirm the anticancer activity and toxicity of KST201 in a more complicated biological

system. Another topic of interest is to develop a combination therapy containing KST201

and one or more compounds that synergistically enhance its activity. Since carcinogenesis

is a multi-stage process, combination of several inhibitors targeting different pathways

corresponding to different stages might perform better overall inhibitory activity against

cancer formation. Moreover, co-treatment with certain compounds which can attenuate

the metabolism of the drug and/or restore its activity helps to maintain the concentration

level of the drug in the system. Therefore, the dosage used to achieve its anticancer effect

can be lowered that might decrease the toxicity to normal tissue; furthermore, the

effective concentration in the body can be maintained during a relatively longer period of time. The optimal form of KST201 in terms of drug delivery and metabolism can be obtained by investigating the modification of functional groups on the aromatic rings of the compound. Another follow-up research project will focus on the synthesis and examination of the next generation of compounds derived from KST201 to study the structural-activity relationship of its analogs against cancer. These will be critical for

further design of more effective and less toxic compounds, for combination therapy, and

for clinical studies.

133

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