Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2006 Molecular Characterization of Tea Catechin Treated Human Prostate Cancer Cell Lines Yewseok Suh Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES MOLECULAR CHARACTERIZATION OF TEA CATECHIN TREATED HUMAN PROSTATE CANCER CELL LINES By YEWSEOK SUH A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy Degree Awarded: Summer Semester, 2006 The members of the Committee approve the dissertation of Yewseok Suh defended on May 12, 2006. Qing-Xiang Amy Sang Professor Directing Dissertation Thomas C.S. Keller III Outside Committee Member Joseph B. Schlenoff Committee Member Hong Li Committee Member Approved: Naresh Dalal, Chair, Department of Chemistry and Biochemistry Joseph Travis, Dean, College of Arts and Sciences The Office of Graduate Studies has verified and approved the above named committee members. ii This dissertation is dedicated to my parents for their endless love and encouragement, to my lovely wife Inok Park for her support, and to our charming daughter, Tae-won. iii ACKNOWLEDGEMENTS I would like to express my gratitude to my major professor for her support throughout the research. Also thanks to all my committee members, Dr. Thomas C. S. Keller III, Dr. Hong Li, and Dr. Joseph B. Schlenoff, for their support, advice, and guidance. Special thanks to our lab members especially, Ziad Sahab and Robert G. Newcomer and former lab member Douglas R. Hurst for their helpful and critical discussions. I would like to thank all my family members including my sister Sun-young, brother Seung-bum, and my uncle Dr. Jeong-hun Suh for their support and encouragement. It has been a pleasure and privilege to work in Dr. Sang’s lab for the past couple of years. iv TABLE OF CONTENTS List of Tables viii List of Figures ix List of Abbreviations xi Abstract xv 1. INTRODUCTION 1 Cancer 1 Cancer Statistics 1 Prostate Cancer 2 Apoptosis 3 Green Tea and EGCG 6 EGCG and Cancer 8 Figures and Tables 11 2. MATERIALS AND METHODS 17 Reagents and Cell Culture 17 Tea Polyphenol Treatment and Cell Lysis 17 Nuclear and Cytoplasmic Extraction of Cells 18 Determination of Protein Concentration by BCA Assay 19 SDS-PAGE and Western Blotting 19 Cell Proliferation Assay 20 Cell Death Detection Assay 21 DNA Fragmentation Assay 22 Morphological Analysis of Apoptotic Cell Nucleus 23 Cell Adhesion Assay 23 Anchorage-Independent Growth Assay 24 GeneChip Microarray Analysis 24 Total RNA Preparation 25 Reverse Transcription - Polymerase Chain Reaction 26 v Quantitative Measurements of proMMP1 and MMP3 by ELISA 27 Statistic Analysis of the Data 28 Figures and Tables 30 3. EFFECTS OF EGCG ON THE GROWTH AND APOTOSIS OF PROSTATE CANCER CELL LINES 34 Prostate Cancer and EGCG 34 R1881 Suppresses the Growth of ARCaP Cells but Facilitates LNCaP Cell Growth 36 EGCG Represses the Growth and Induces Detachment and Membrane Blebbing in Cultured LNCaP Cells but Not in ARCaP Cells 36 EGCG Induces Apoptosis in LNCaP Cells but Not in ARCaP Cells 37 EGCG Induces Chromatin Condensation and Nuclei Fragmentation in LNCaP Cells but Not in ARCaP Cells 38 EGCG Induces Caspase 3 Activation, PARP Cleavage and p53 Increase Only in LNCaP Cells Leading to Apoptosis 39 EGCG Triggers Activation of NF-κB in ARCaP Cells but Induces Inactivation in LNCaP Cells 40 EGCG Decreases the Ratio of Bax/Bcl-2 in ARCaP Cells but Increases the Ratio in LNCaP Cells 41 EGCG Does Not Change the Level of p21/CIP1/WAF1 and CDKs in ARCaP Cells but the Level is Affected in LNCaP Cells in Favor of Apoptosis 42 EGCG Affects the Activation of MAPKs towards Cell Survival in ARCaP Cells but Favors Cell Death in LNCaP Cells 44 EGCG Confers Stronger ECM Adhesion via Collagen in ARCaP Cells whereas the Adhesion to ECM Proteins is Significantly Weakened in LNCaP Cells 46 Tea Catechin EGCG Renders ARCaP Cells Capable of Facilitated Anchorage-Independent Growth 49 Figures 51 vi 4. SELECTIVE CHANGES IN GENE EXPRESSION IN EGCG TREATED PROSTATE CANCER CELLS 79 GeneChip Microarray Analysis 79 ADAMs and MMPs 81 Effects of Tea Catechins in Growth of LNCaP and ARCaP Cells 82 Effects of Tea Catechins in MMP1 and MMP3 Expression by RT-PCR 83 Effects of Tea Catechins in proMMP1 and MMP3 Expression by ELISA 84 The Effect of EGCG on the Expression of MMPs 86 The Role of MMPs and ECM in Programmed Cell Death 86 Figures and Tables 90 5. CONCLUSIONS AND FUTURE DIRECTIONS 103 EGCG and Prostate Cancer Chemoprevention 103 EGR1 in Apoptosis 104 Possible Roles of GADD45 and Topoisomerase 105 Current and Future Studies 106 Figures and Tables 108 APPENDIX 110 REFERENCES 157 BIOGRAPHICAL SKETCH 171 vii LIST OF TABLES 1. The composition of tea leaf 15 2. The information of primers used in PCR 32 3. The sequences of primers used in PCR 33 4. Changes of expression levels of adhesion-related proteins in LNCaP cells by GeneChip assay 90 5. Combinations of integrin subunits and their respective ligands 91 6. Changes of expression levels of ADAMs by GeneChip assay in LNCaP cells 92 7. Changes of expression levels of cell cycle-related proteins by GeneChip assay 108 8. The effect of R1881 on the growth of human prostate cancer cell lines 110 9. The effect of EGCG on the growth of LNCaP prostate cancer cell line 111 10. The effect of EGCG on the growth of ARCaP prostate cancer cell line 112 11. The ‘Enrichment Factor’ of ARCaP and LNCaP cells upon EGCG treatment measured by Cell Death Detection Assay PLUS 113 12. The ratio of Bax to Bcl-2 upon EGCG treatment 114 13. The effect of etoposide on the growth of ARCaP cells 115 14. Relative adherence of ARCaP cells to ECM proteins 116 15. Relative adherence of LNCaP cells to ECM proteins 117 16. The changes in relative adherence after EGCG treatment 118 17. The number of colonies formed in agar plate after EGCG treatment 119 18. GeneChip assay of ARCaP cells 120 19. GeneChip assay of LNCaP cells 130 20. The effect of tea catechins on the growth of LNCaP cells 151 21. The effect of tea catechins on the growth of ARCaP cells 152 22. The effect of tea catechins on the production of proMMP1 in LNCaP cells 153 23. The effect of tea catechins on the production of proMMP1 in ARCaP cells 154 24. The effect of tea catechins on the production of total MMP3 in LNCaP cells 155 25. The effect of tea catechins on the production of total MMP3 in ARCaP cells 156 viii LIST OF FIGURES 1. Estimated new cases and deaths by ten leading cancer types, by sex, US, 2006 11 2. Pictures of prostate and the model of prostate cancer progression 12 3. A picture of a cell undergoing apoptosis 13 4. The overview of apoptosis 14 5. Structures of basic flavonoid and major tea catechins 16 6. Schematic representation of Cell Death Detection ELISA PLUS assay 30 7. The schematic diagram of GeneChip assay 31 8. The effect of R1881 on the growth of ARCaP cells 51 9. The effect of green tea polyphenol EGCG on the growth of LNCaP human prostate cancer cells 52 10. The effect of green tea polyphenol EGCG on the growth of ARCaP human prostate cancer cells 53 11. Morphological changes upon EGCG addition in LNCaP cells 54 12. Morphological changes upon EGCG addition in ARCaP cells 55 13. The extent of apoptosis determined by Cell Death Detection Assay PLUS 56 14. The extent of apoptosis determined by DNA fragmentation assay 57 15. The extent of ARCaP cell apoptosis determined by Hoechst staining 58 16. The extent of LNCaP cell apoptosis determined by Hoechst staining 59 17. Immunoblot results with caspase 3 and PARP specific antibodies 60 18. Immunoblot results with p53 specific antibody 61 19. Immunoblot results with p65, phospho-p65, and Iκ-Bα specific antibodies 62 20. The role of Bcl-2 family members in mitochondrial outer membrane permeabilization during apoptosis 63 21. Immunoblot results with Bax, Bcl-2, and Bcl-XL specific antibodies 64 22. The changes of Bax/Bcl-2 ratio upon EGCG treatment 65 23. The role of p21/CIP1/WAF1 in cell cycle progression and apoptosis 66 24. Immunoblot results with p21, CDK2, CDK4, and CDK6 specific antibodies 67 25. The effects of etoposide on growth of ARCaP cells 68 26. The effects of etoposide on apoptosis of ARCaP cells 69 ix 27. Immunoblot results of etoposide treated ARCaP cell lysates with caspase 3, PARP, and p21 specific antibodies 70 28. Immunoblot results with Akt, p38, and JNK specific antibodies 71 29. Cell detachment assay of ARCaP cells 72 30. Cell detachment assay of LNCaP cells 73 31. Extracellular matrix adhesion assay of ARCaP cells 74 32. Extracellular matrix adhesion assay of LNCaP cells 75 33. The anchorage-independent growth assay of LNCaP cells 76 34. The anchorage-independent growth assay of ARCaP cells 77 35. The number of colonies formed in anchorage-independent growth assay 78 36. Changes of expression levels of MMPs by GeneChip assay in LNCaP cells 93 37. The effect of tea catechins on the growth of LNCaP cells 94 38. The effect of tea catechins on the growth of ARCaP cells 95 39. The effect of tea catechins on the expression of MMP1 and MMP3 mRNA in LNCaP cells 96 40. The effect of tea catechins on the expression of MMP1 and MMP3 mRNA in ARCaP cells 97 41.