The anti-tumor effects of arsenic trioxide on human breast adenocarcinoma cell line, MCF-7.

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