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 by 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 ii 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