by Chow Ka Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 203-221). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract in Chinese --- p.iv / List of Abbreviations --- p.vi / Table of Contents --- p.xi / List of Figures --- p.xviii / List of Tables --- p.xxii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- The Characteristics of Arsenic Trioxide (AS2O3) --- p.2 / Chapter 1.2 --- The Therapeutic Applications of Arsenic Trioxide (As203) --- p.5 / Chapter 1.3 --- Acute Promyelocytic Leukemia (APL) --- p.6 / Chapter 1.3.1 --- Pathologies of APL --- p.7 / Chapter 1.3.2 --- All Trans Retinoic Acid (ATRA) Treatment of APL Patients --- p.7 / Chapter 1.3.3 --- Clinical Trials of Arsenic Trioxide (As203) on APL Patients --- p.9 / Chapter 1.3.4 --- In Vitro and In Vivo Studies of Arsenic Trioxide (As203) in the Treatment of APL --- p.10 / Chapter 1.3.4.1 --- Induction of Apoptosis --- p.11 / Chapter 1.3.4.2 --- Induction of Cell Differentiation --- p.11 / Chapter 1.3.5 --- General Toxicity and Side Effects of Arsenic Trioxide (AS2O3) on APL Patients --- p.12 / Chapter 1.4 --- Effects of Arsenic Trioxide (As203) on Other Primary Cancer Cells and Cancer Cell Lines --- p.12 / Chapter 1.5 --- Epidemiology of Breast Cancer --- p.14 / Chapter 1.6 --- Classification of Breast Cancer --- p.17 / Chapter 1.7 --- Etiology of Breast Cancer --- p.17 / Chapter 1.8 --- Hormones and Breast Cancer --- p.18 / Chapter 1.9 --- Estrogen Receptors (ER) --- p.20 / Chapter 1.9.1 --- Structures of Estrogen Receptors (ER) --- p.21 / Chapter 1.9.2 --- Estrogen Receptors (ER) Mediated Signaling Pathway --- p.22 / Chapter 1.9.2.1 --- Ligand Dependent Pathway --- p.22 / Chapter 1.9.2.2 --- Ligand Independent Pathway --- p.22 / Chapter 1.9.2.3 --- Estrogen Response Element (ERE)-Independent Pathway --- p.23 / Chapter 1.9.2.4 --- Non-Genomic Pathway --- p.23 / Chapter 1.9.3 --- Estrogen Receptors (ER) Regulated Gene Expression --- p.25 / Chapter 1.10 --- Current Therapy of Breast Cancer --- p.26 / Chapter 1.10.1 --- Hormonal Therapy (Anti-Estrogenicity) --- p.26 / Chapter 1.10.1.1 --- Tamoxifen --- p.26 / Chapter 1.10.1.2 --- Other Pure Anti-Estrogens --- p.28 / Chapter 1.10.2 --- Regulation of Estrogen Receptors (ER) and Transcription Coregulators --- p.29 / Chapter 1.10.3 --- Apoptosis Induction --- p.29 / Chapter 1.11 --- Aims of Study --- p.30 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.32 / Chapter 2.1 --- Materials --- p.33 / Chapter 2.1.1 --- Cell Lines and Culture Media --- p.33 / Chapter 2.1.1.1 --- Cell Lines --- p.33 / Chapter 2.1.1.2 --- Culture Media --- p.34 / Chapter 2.1.2 --- Chemicals --- p.35 / Chapter 2.1.3 --- Reagents and Buffers --- p.36 / Chapter 2.1.3.1 --- Reagents for MTT Assay --- p.36 / Chapter 2.1.3.2 --- Reagents for [methyl-3H] Thymidine Incorporation into DNA --- p.37 / Chapter 2.1.3.3 --- Reagents for Trypan Blue Exclusion Assay --- p.37 / Chapter 2.1.3.4 --- Reagents and Buffers for Western Blot Analysis --- p.37 / Chapter 2.1.3.5 --- Reagents and Buffers for Flow Cytometry --- p.40 / Chapter 2.1.3.6 --- Reagents and Buffers Reverse Transcription Polymerase Chain Reaction (RT-PCR) --- p.40 / Chapter 2.1.3.7 --- Reagents for Transfection and Luciferase Reporter Assay --- p.41 / Chapter 2.1.3.8 --- Reagents and Buffers for In Vivo Studies --- p.42 / Chapter 2.2 --- Methods --- p.42 / Chapter 2.2.1 --- In Vitro Studies --- p.42 / Chapter 2.2.1.1 --- Cell Treatment --- p.42 / Chapter 2.2.1.2 --- Drug Preparation --- p.43 / Chapter 2.2.1.3 --- MTT Assay --- p.43 / Chapter 2.2.14 --- Trypan Blue Exclusion Assay --- p.44 / Chapter 2.2.1.5 --- [methyl-3H] Thymidine Incorporation into DNA --- p.45 / Chapter 2.2.1.6 --- Detection of DNA Fragmentation --- p.45 / Chapter 2.2.1.7 --- ERα Competitive Binding Assay --- p.47 / Chapter 2.2.1.8 --- Cell Cycle Analysis by Flow Cytometry with Propidium Iodide (PI) Staining --- p.48 / Chapter 2.2.1.9 --- Cell Cycle Analysis by Flow Cytometry with Annexin V-PI Staining --- p.48 / Chapter 2.2.1.10 --- Cell Cycle Analysis by Flow Cytometry with JC-1 Staining --- p.49 / Chapter 2.2.1.11 --- Cell Cycle Analysis by Flow Cytometry with Hydroethidine (HE) Staining --- p.50 / Chapter 2.2.1.12 --- Western Blot Analysis of Proteins --- p.50 / Chapter 2.2.1.13 --- Assessment of the Transcriptional Activity of ERα --- p.55 / Chapter 3.2.1.14 --- Reverse Transcription Polymerase Chain Reaction (RT-PCR) --- p.57 / Chapter 2.2.2 --- In Vivo Studies --- p.61 / Chapter 2.2.2.1 --- Animal Models --- p.61 / Chapter 2.2.2.2 --- Treatment Schedules --- p.61 / Chapter 2.2.2.3 --- Sacrifice of Nude Mice --- p.61 / Chapter 2.2.2.4 --- Enzymatic Assays --- p.62 / Chapter 2.2.2.4.1 --- Aspartate Transaminase (AST) --- p.63 / Chapter 2.2.2.4.2 --- Alanine Transaminase (ALT) --- p.64 / Chapter 2.2.2.4.3 --- Creatine Kinase (CK) --- p.65 / Chapter 2.2.2.4.4 --- Lactate Dehydrogenase (LDH) --- p.66 / Chapter CHAPTER 3 --- "Effects of Arsenic Trioxide (As203) on Human Breast Adenocarcinoma Cell Line, MCF-7 Cell Line" --- p.68 / Chapter 3.1 --- Introduction --- p.69 / Chapter 3.2 --- Effect of As203 on Cell Survival of MCF-7 cells by MTT Assay --- p.70 / Chapter 3.3 --- Cytotoxicity of As203 on MCF-7 Cells by Trypan Blue Exclusion Assay --- p.72 / Chapter 3.4 --- Effect of As203 on DNA Synthesis and Cell Proliferation of MCF-7 cells by [methyl-3H] Thymidine Incorporation into DNA --- p.76 / Chapter 3.5 --- Comparison of Cytotoxicity of AS2O3 on MCF-7 Cells with that of Tamoxifen --- p.79 / Chapter 3.6 --- Summary --- p.82 / Chapter CHAPTER 4 --- Effects of Arsenic Trioxide (As203) on 17β Estradiol Stimulated MCF-7 cells --- p.83 / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.2 --- Effect of 17β estradiol on Cell Viability of MCF-7 Cells by MTT Assay --- p.86 / Chapter 4.3 --- Effect of As203 and 17β Estradiol on Cell Survival of MCF-7 Cells by MTT Assay --- p.88 / Chapter 4.4 --- Cytotoxicity of As203 on 17β Estradiol Stimulated MCF-7 cells by Cell Number Counting with Hemacytometer --- p.92 / Chapter 4.5 --- Growth Inhibitory Effect of As203 on 17β Estradiol stimulated MCF-7 cells by [methyl-3H] Thymidine Incorporation into DNA --- p.94 / Chapter 4.6 --- "Effect of As203 on Cell Survival of Hormone Independent Breast Cancer Cell Line, MDA-MB-231 Cells" --- p.96 / Chapter 4.7 --- Summary --- p.100 / Chapter CHAPTER 5 --- Effects of Arsenic Trioxide (As203) on Normal Cells --- p.102 / Chapter 5.1 --- Introduction --- p.103 / Chapter 5.2 --- "Effect of As203 on Normal Human Fibroblast Cell Line, Hs68" --- p.104 / Chapter 5.3 --- Effects of As203 on the Normal Cells of Nude Mice --- p.106 / Chapter 5.3.1 --- Effect of AS2O3 on Aspartate Transaminase (AST) Activity of Nude Mice --- p.107 / Chapter 5.3.2 --- Effect of As203 on Alanine Transaminase (ALT) Activity of Nude Mice --- p.109 / Chapter 5.3.3 --- Effect of As203 on Creatine Kinase (CK) Activity of Nude Mice TABLE OF CONTENTS --- p.111 / Chapter 5.3.4 --- Effect of As203 on Lactate Dehydrogenase (LDH) Activity of Nude Mice --- p.113 / Chapter 5.4 --- Summary --- p.115 / Chapter CHAPTER 6 --- Action Mechanisms underlying the Survival Inhibitory Effects of Arsenic Trioxide (As203) on MCF-7 cells --- p.116 / Chapter 6.1 --- Introduction --- p.117 / Chapter 6.2 --- Detection of Apoptosis --- p.119 / Chapter 6.2.1 --- Detection of DNA Fragmentation --- p.119 / Chapter 6.2.2 --- Phosphatidylserine (PS) Externalization Detected by Flow Cytometry with Annexin V-PI Staining --- p.124 / Chapter 6.2.2.1 --- The Principle --- p.124 / Chapter 6.2.2.2 --- PS Externalization upon AS2O3 Treatment --- p.126 / Chapter 6.3 --- Analysis of Cell Cycle Distribution of MCF-7 Cells --- p.130 / Chapter 6.3.1 --- The Principle --- p.130 / Chapter 6.3.2 --- Regulation of Cell Cycle Distribution of MCF-7 Cells upon As2O3 Treatment --- p.131 / Chapter 6.4 --- The Action Mechanisms Underlying As203 Induced Apoptosis or Cell Cycle Arrest --- p.137 / Chapter 6.4.1 --- Effect of As203 on Mitochondrial Membrane Potential of MCF-7 Cells --- p.137 / Chapter 6.4.2 --- Regulation of Free Oxidative Species (ROS) Production in MCF-7 Cells upon AS2O3 Treatment --- p.140 / Chapter 6.4.2.1 --- Analysis of Superoxide Production in MCF-7 Cells upon AS2O3 Treatment by Flow Cytometry with Hydroethidine (HE) Staining --- p.140 / Chapter 6.4.2.2 --- Effect of As203 on Cell Survival of MCF-7 Cells Co-treated with N-Acteyl-L-Cysteine (NAC) by MTT Assay --- p.143 / Chapter 6.4.3 --- Regulation of Bcl-2 Protein Level in MCF-7 Cells upon As2O3 Treatment --- p.145 / Chapter 6.4.4 --- Regulation of p53 Protein Level in MCF-7 Cells upon AS2O3 Treatment --- p.147 / Chapter 6.5 --- Summary --- p.149 / Chapter CHAPTER 7 --- Effects of Arsenic Trioxide (As203) on Estrogen Receptor a (ERα) Mediated Signaling Pathway in MCF-7 cells --- p.150 / Chapter 7.1 --- Introduction --- p.151 / Chapter 7.2 --- Effect of As203 on Estrogen Binding to Estrogen Receptor a (ERα) by ERα Competitive Binding Assay --- p.152 / Chapter 7.3 --- Regulation of Estrogen Receptor a (ERα) mRNA Level upon As2O3 Treatment by RT-PCR --- p.156 / Chapter 7.4 --- Regulation of Estrogen Receptor a (ERα) Protein Level upon As2O3 Treatment --- p.159 / Chapter 7.5 --- Regulation of Estrogen Receptor a (ERα) Transcriptional Activity upon AS2O3 treatment --- p.161 / Chapter 7.6 --- "Regulation of Estrogen Target Gene, c-myc, Protein Level upon As2O3 Treatment" --- p.164 / Chapter 7.7 --- Effects of As203 on Cell Cycle Distribution of MCF-7 Cells under Estrogens Stimulation --- p.167 / Chapter 7.8 --- Summary --- p.173 / Chapter CHAPTER 8 --- Discussion --- p.174 / Chapter 8.1 --- The Anti-Tumor Effects of As203 on MCF-7 Cells --- p.175 / Chapter 8.2 --- Cytotoxicity of As203 on MCF-7 Cells --- p.175 / Chapter 8.2.1 --- Induction of Apoptosis in MCF-7 Cells upon As2〇3 Treatment --- p.176 / Chapter 8.2.2 --- Action Mechanisms Underlying the Induction of Apoptosis by As2〇3 --- p.178 / Chapter 8.3 --- Growth Inhibition of As203 on MCF-7 Cells --- p.182 / Chapter 8.3.1 --- Cell Cycle Regulation of MCF-7 Cells upon As203 Treatment --- p.182 / Chapter 8.4 --- Growth Inhibitory Effects of As203 on Estrogen Stimulated MCF-7 Cells --- p.186 / Chapter 8.4.1 --- Regulation of Estrogen Receptor a (ERα) Signaling Pathway in MCF-7 cells upon as2o3 Treatment --- p.188 / Chapter 8.5 --- Cross Talk of ERα Signaling Pathway and Apoptosis in Mediating the Anti-Tumor Effects of As203 on MCF-7 Cells --- p.195 / Chapter 8.6 --- Toxicity of AS2O3 towards Normal Tissues --- p.197 / Chapter CHAPTER 9 --- Conclusion and Future Perspectives --- p.200 / Chapter 9.1 --- Conclusion --- p.200 / Chapter 9.2 --- Future Perspectives --- p.202 / References --- p.203

Po Lai See. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 142-152). / Abstracts in English and Chinese. / TITLE PAGE --- p.p.1 / ACKNOWLEGDEMENTS --- p.p.2 / ABSTRACT --- p.p.3 / 摘要 --- p.p.6 / TABLE OF CONTENTS --- p.p.9 / LIST OF FIGURES AND TABLES --- p.p.16 / Chapter CHAPTER 1 --- GENERAL INTRODUCTION / Chapter 1.1 --- Estrogen and Estrogen Receptors and its Action --- p.p.18 / Chapter 1.1.1 --- Estrogen --- p.p.19 / Chapter 1.1.2 --- Estrogen Receptors --- p.p.19 / Chapter 1.1.3 --- Structural Differences between ERa and ERp --- p.p.21 / Chapter 1.1.4 --- Functional Differences --- p.p.22 / Chapter 1.1.5 --- Effects of Selective Estrogen Receptor Modulators --- p.p.22 / Chapter 1.1.6 --- Estrogen works --- p.p.23 / Chapter 1.1.7 --- Estrogen Receptors and Breast Cancer --- p.p.24 / Chapter 1.2 --- Flavonoids： Properties and Biological Activities --- p.p.25 / Chapter 1.2.1 --- Chemical Structure and Classification of flavonoids --- p.p.25 / Chapter 1.2.2 --- Biological Properties and Action Mechanism of Flavonoids… --- p.p.27 / Chapter 1.2.3 --- Flavonoids and breast cancer prevention --- p.p.27 / Chapter 1.3 --- Aims and Scopes of Investigation --- p.p.29 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Chemicals --- p.p.30 / Chapter 2.1.1 --- Flavonoids --- p.p.30 / Chapter 2.1.2 --- Plasmids --- p.p.30 / Chapter 2.2 --- Mammalian cell culture --- p.p.31 / Chapter 2.2.1 --- Maintenance of cells --- p.p.31 / Chapter 2.2.2 --- Preparation of cell stock --- p.p.32 / Chapter 2.2.3 --- Cell recovery from liquid nitrogen stock --- p.p.32 / Chapter 2.3 --- Identification of estrogenic activity in flavonoids --- p.p.33 / Chapter 2.3.1 --- Steady Glo Luciferase Assay --- p.p.33 / Chapter 2.3.2 --- The Biorad Protein Assay kit (a modified Bradford method). --- p.p.33 / Chapter 2.4 --- Viability Assay --- p.p.34 / Chapter 2.5 --- ERE Luciferase reporter gene assay --- p.p.35 / Chapter 2.5.1 --- Transient transfect ion of cell using lipofectamine PLUS reagent --- p.p.36 / Chapter 2.5.2 --- Dual Luciferase Assay --- p.p.37 / Chapter 2.6 --- ERα competitive binding ASSAY --- p.p.37 / Chapter 2.7 --- Apoptotic death assay --- p.p.38 / Chapter 2.8 --- Semi-quantitative RT-PCR Assay --- p.p.40 / Chapter 2.8.1 --- "Isolation of RNA using TRIzol® Reagent (Life Technology,USA) " --- p.p.40 / Chapter 2.8.2 --- Quantitation of RNA --- p.p.41 / Chapter 2.8.3 --- First strand cDNA synthesis --- p.p.41 / Chapter 2.8.4 --- PCR reactions --- p.p.43 / Chapter 2.9 --- Flow Cytometry Analysis --- p.p.43 / Chapter 2.10 --- Total triglyceride and cholesterol measurement --- p.p.44 / Chapter 2.10.1 --- Determination of the total cholesterol --- p.p.45 / Chapter 2.10.2 --- Determination of the total triglyceride --- p.p.46 / Chapter 2.11 --- Manipulation of DNA and RNA --- p.p.46 / Chapter 2.11.1 --- Transformation of DH5α --- p.p.46 / Chapter 2.11.2 --- Mini preparation of plasmid DNA --- p.p.47 / Chapter 2.11.3 --- Preparation of plasmid DNA using QIAGEN-tip 100 midi-prep kit --- p.p.48 / Chapter 2.11.4 --- Preparation of plasmid DNA using QIAGEN-tip 10000 Giga-prep kit --- p.p.49 / Chapter 2.11.5 --- Ethanol preparation of DNA and RNA --- p.p.50 / Chapter 2.11.6 --- Agarose gel electrophoresis of DNA --- p.p.51 / Chapter 2.12 --- Statistical methods --- p.p.52 / Chapter CHAPTER 3 --- Estrogenic and antiproliferative activities on MCF-7 breast cancer cells by flavonoids / Chapter 3.1 --- Introduction --- p.p.53 / Chapter 3.2 --- Results --- p.p.56 / Screening of phytoestrogens for estrogenic activities on MELN cells --- p.p.56 / Cell proliferation activity of phytoestrogens on MCF-7 and MDA-MA231 cells --- p.p.59 / Estrogenic and antiestrogenic activity of phytoestrogens on ERα or erβ transfected hepg2 cells --- p.p.64 / Chapter 3.3 --- Discussion --- p.p.73 / Chapter Chapter 4 --- interaction of baicalein with estrogen receptors / Chapter 4.1 --- Introduction --- p.p.76 / Chapter 4.2 --- Results --- p.p.78 / Estrogen receptor competition assay --- p.p.78 / ERE-Luciferase gene reporter assay --- p.p.82 / Chapter 4.3 --- Discussion --- p.p.88 / Chapter Chapter 5 --- baicalein and genistein display differential actions on er transactivation / Chapter 5.1 --- Introduction --- p.p.90 / Chapter 5.2 --- Results --- p.p.92 / Estrogenic and antiestrogenic activities of genistein and baicalein on ER transactivation --- p.p.92 / Chapter 5.3 --- Discussion --- p.p.105 / Chapter CHAPTER 6 --- APOPTOTIC EFFECTS OF BAICALEIN ON MCF-7 AND MDA-MB-231 CELL LINES / Chapter 6.1 --- Introduction --- p.p.107 / Chapter 6.2 --- Results --- p.p.111 / ER POSITIVE MCF-7 AND ER NEGATIVE MDA-MB-231 cell death assay --- p.p.111 / "Bcl-2, Bax and PS2 mRNA expression " --- p.p.116 / Arrest at sub G1 phase of MCF-7 by baicalein --- p.p.124 / Chapter 6.3 --- Discussion --- p.p.127 / Chapter CHAPTER 7 --- BAICALEIN CAN REDUCE INTRACELLULAR cholesterol and triglceride / Chapter 5.1 --- Introduction --- p.p.129 / Chapter 5.2 --- Results --- p.p.130 / Baicalein has beneficial effect on lipid metabolism --- p.p.130 / Chapter 5.3 --- Discussion --- p.p.139 / Chapter chapter 8 --- Summary --- p.p.140 / BIBLIOGRAPHY --- p.p.142 / APPENDIX 1 ABBREVIATIONS --- p.p.153 / APPENDIX 2 PRIMER LISTS --- p.p.156 / APPENDIX 3 REAGENTS AND BUFFERS --- p.p.157