Breast cancer is the most common cancer among females and it is the leading cause of death in mid-age women. Epidemiological studies indicate that Asian women have a lower incidence of breast cancer compared with their counterparts in the West, which soy consumption has been suggested as a contributory factor. Soy and soy-based food contain a rich amount of phytoestrogens, which are suggested to be protective against cancer. Cyclooxygenase (COX) is the rate-limiting enzyme for the conversion of arachidonic acid to prostaglandins. Recent studies have revealed that up-regulation of cyclooxygenase-2 (COX-2), an isoform of COX, plays an important role in tumorigenesis and metastasis. COX-2 may facilitate carcinogenesis in a number of means, may include altering cell proliferation and apoptosis, enhancing angiogenesis and suppressing immune surveillance. Clinical examinations of breast cancer specimens indicated that COX-2 is overexpressed. In the present study, we investigated the effect of flavonoids on COX-2 expression in human breast cells. / Our results showed that daidzein and its metabolite eqoul, genistein, butein, isoliquiritigenin (ILN) and apigenin could inhibit phorbol 12-myristate 13-acetate (PMA)-induced COX-2 expression in breast cells MCF-7 and MCF-10A. The inhibitory effects were in concentration-dependent manners. Real-time PCR and western blot analysis showed that the flavonoids suppressed the induced mRNA and protein expression. Suppression could be observed in concentration as low as 0.1 muM. Luciferase reporter assay indicated that the inhibition was at the gene transactivation level. Further investigation using truncated hCOX-2 promoter plasmids revealed that the AP-1 site (-67/-61) and cyclic AMP response element (CRE) site (-59/-53) on hCOX-2 promoter were responsible for the suppression. Electrophoretic mobility shift assay results further confirmed that the flavonoids acted through inhibiting AP-1/CREB DNA binding to suppress the expression. / To examine the possible upstream signal transduction pathways involved, inhibitors of protein kinase A (PKA), protein kinase (PKC) and mitogen activated protein kinase (MAPK) were employed. Reporter gene assay revealed a possible involvement of ERK1/2 MAP kinase in AP-1 and/or CRE activation of hCOX-2 promoter. Taken together, these results suggested that the phytochemicals down-regulated PMA-induced COX-2 expression by counteracting AP-1 and CRE sites via the modulation of MAPK pathway. The findings might have significant implications in the chemopreventive and chemotherapeutic applications of flavonoids in breast cancer. / Lau, Tak Yi. / "December 2007." / Adviser: Lai Kwok Leung. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4734. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 142-164). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

by Wong Che-cheuk, Dobe. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 98-112). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstracts --- p.ii / List of Abbreviation --- p.vi / Table of Contents --- p.vii / Chapter 1. --- Introduction / Chapter 1.1 --- Stages of Cancer Development --- p.1 / Chapter 1.2 --- Comparison of Breast Cancer in Hong Kong & the United States --- p.2 / Chapter 1.2.1 --- Statistics of Breast Cancer in the United States --- p.2 / Chapter 1.2.2 --- Statistics of Breast Cancer in Hong Kong --- p.2 / Chapter 1.3 --- Factors for Breast Cancer --- p.6 / Chapter 1.3.1 --- Genetic Factor --- p.6 / Chapter 1.3.2 --- Hormonal Factor --- p.7 / Chapter 1.3.3 --- Genetic Bias --- p.9 / Chapter 1.3.4 --- Influence of Diet --- p.10 / Chapter 1.3.5 --- Obesity --- p.14 / Chapter 1.3.6 --- Xenoestrogen --- p.14 / Chapter 1.4 --- Hormonal Therapy in Breast Cancer --- p.15 / Chapter 1.4.1 --- Antiestrogen --- p.15 / Chapter 1.4.2 --- Progestin Antagonist --- p.19 / Chapter 1.4.3 --- Aromatase Inhibitor --- p.20 / Chapter 1.4.4 --- Gonadotropin Releasing Hormone (GnRH) Analogue --- p.23 / Chapter 1.5 --- Metabolism of Estrogen --- p.25 / Chapter 1.6 --- Substance with Chemopreventive Properties towards Breast Cancer --- p.29 / Chapter 1.7 --- Aryl Hydrocarbon Receptor --- p.33 / Chapter 1.8 --- Cytochrome P450s --- p.34 / Chapter 1.9 --- Yuehchukene --- p.36 / Chapter 1.10 --- Objectives of the Present Study --- p.38 / Chapter 2. --- Materials and Methods / Chapter 2.1 --- Animals --- p.40 / Chapter 2.2 --- Animal Treatment --- p.40 / Chapter 2.3 --- Preparation of Crude Microsomal Fraction --- p.41 / Chapter 2.4 --- Protein Assay --- p.41 / Chapter 2.5 --- Ethoxyresorufm-O-deethylase Assay --- p.41 / Chapter 2.6 --- Methoxyresorufin-O-deethylase Assay --- p.42 / Chapter 2.7 --- Estradiol-2-hydroxylase Assay --- p.42 / Chapter 2.8 --- Progesterone Hydroxylase Assay --- p.43 / Chapter 2.9 --- Hepatic Aromatase Activity Assay --- p.43 / Chapter 2.10 --- Inhibition of Ethoxyresorufm-O-deethylase and Estradiol-2-hydroxylase --- p.44 / Chapter 2.11 --- Free Radicals Scavenging Assay --- p.44 / Chapter 2.12 --- Chemicals --- p.45 / Chapter 3. --- Result / Chapter 3.1 --- Optimization of Condition --- p.47 / Chapter 3.1.1 --- Dosage --- p.47 / Chapter 3.1.2 --- Time for Sacrifice --- p.47 / Chapter 3.2 --- "Effect of Isoprenyl Compounds on the Body Weight, Liver Weight and Hepatic Microsomal Protein Content" --- p.50 / Chapter 3.3 --- Hepatic Enzyme Activities --- p.54 / Chapter 3.3.1 --- Ethoxyresorufm-O-deethylase --- p.54 / Chapter 3.3.2 --- Methoxyresorufm-O-deethylase --- p.57 / Chapter 3.3.3 --- Estradiol-2-hydroxylase --- p.60 / Chapter 3.3.4 --- Progesterone Hydroxylase --- p.62 / Chapter 3.3.5 --- Aromatase --- p.65 / Chapter 3.4 --- Effect of Inhibitors in Ethoxyresorufin-O-deethylase and Estradiol-2-hydroxylase Activity --- p.65 / Chapter 3.5 --- Free Radical Scavenging Activity --- p.72 / Chapter 4. --- Discussion --- p.77 / Chapter 5. --- Conclusion --- p.95 / Chapter 6. --- References --- p.98 / Chapter 7. --- Appendix --- p.113

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

Luk Wing Yan Vivien. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 207-239). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Abstract --- p.iii / 摘要 --- p.vi / Contents --- p.viii / List of Figures --- p.xiv / List of Table --- p.xxv / Abbreviations --- p.xxv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Ganoderma spp --- p.1 / Chapter 1.2 --- Bioactive components of Ganoderma spp --- p.3 / Chapter 1.2.1 --- Lingzhi polysaccharide --- p.3 / Chapter 1.2.2 --- Terpenes --- p.4 / Chapter 1.3 --- Ganoderma spp. as Chinese traditional medicine --- p.5 / Chapter 1.4 --- Artificial hybridisation of Ganoderma luciudm and G. tsugae --- p.6 / Chapter 1.4.1 --- Protoplast isolation and fusion of Ganoderma tsugae and G. lucidum --- p.8 / Chapter 1.5 --- Breast Cancer --- p.8 / Chapter 1.5.1 --- Anti-tumor effects of natural substances against breast cancer cell MCF-7 --- p.9 / Chapter 1.5.2 --- Anti-tumor effects of natural substances against breast cancer cell MDA-MB-231 --- p.11 / Chapter 1.5.3 --- Anti-proliferation of cancer --- p.12 / Chapter 1.5.3.1 --- Cell cycle arrest --- p.12 / Chapter 1.5.3.2 --- Cell death --- p.13 / Chapter 1.5.4 --- Anti-proliferation assays --- p.17 / Chapter 1.5.4.1 --- MTT assay --- p.17 / Chapter 1.5.4.2 --- Trypan blue cell viability assay --- p.18 / Chapter 1.5.4.3 --- BrdU assay --- p.18 / Chapter 1.6 --- Endocrine system and hormones --- p.19 / Chapter 1.6.1 --- Estrogen --- p.23 / Chapter 1.6.2 --- Estrogen receptors --- p.24 / Chapter 1.6.3 --- Estrogen action --- p.29 / Chapter 1.6.4 --- Estrogenicity assays --- p.32 / Chapter 1.6.4.1 --- Recombinant yeast assay --- p.33 / Chapter 1.6.4.2 --- E-screen assay --- p.35 / Chapter 1.6.4.3 --- Estrogen receptor competitor binding assay --- p.36 / Chapter 1.6.4.4 --- Endogenous estrogen-regulated gene expression assay --- p.39 / Chapter 1.6.4.4.1 --- Transforming growth factor --- p.39 / Chapter 1.6.4.4.2 --- Monoamine oxidase A --- p.40 / Chapter 1.6.4.4.3 --- pS2 --- p.40 / Chapter 1.6.4.5 --- Uterotrophic assay --- p.41 / Chapter 1.6.4.6 --- Comparison of in vitro and in vivo assay --- p.42 / Chapter 1.7 --- Aim of study --- p.45 / Chapter 1.7.1 --- Objectives --- p.45 / Chapter Chapter 2 --- Materials and Methods --- p.47 / Chapter 2.1 --- Fungal culture --- p.47 / Chapter 2.2 --- Artificial hybridization of Ganoderma tsugae and G. lucidum --- p.47 / Chapter 2.2.1 --- Protoplast isolation of Ganoderma tsugae and G. lucidum --- p.47 / Chapter 2.2.2 --- Protoplast fusion of Ganoderma tsugae and G. lucidum --- p.48 / Chapter 2.3 --- Screening and selection of hybrid ´Ø --- p.49 / Chapter 2.3.1 --- Temperature screening --- p.49 / Chapter 2.3.2 --- DNA fingerprint by Arbitarily-primed polymerase chain reaction --- p.49 / Chapter 2.3.2.1 --- Extraction of genomic DNA --- p.49 / Chapter 2.3.2.2 --- Arbitrarily-primed polymerase chain reaction --- p.50 / Chapter 2.3.2.3 --- Gel electrophoresis --- p.51 / Chapter 2.4 --- Confirmation test --- p.51 / Chapter 2.4.1 --- Somatic incompatibility test --- p.51 / Chapter 2.4.2 --- DNA fingerprinting by specific polymerase chain reaction --- p.52 / Chapter 2.4.2.1 --- Specific Polymerase Chain Reaction (PCR) --- p.52 / Chapter 2.4.2.2 --- Purification of PCR products --- p.52 / Chapter 2.4.2.3 --- Cycle-sequencing --- p.53 / Chapter 2.3.2.4 --- Sequencing --- p.54 / Chapter 2.3.2.5 --- Sequence analysis --- p.54 / Chapter 2.5 --- Characterization of the selected hybrid --- p.56 / Chapter 2.5.1 --- Scanning electron microscopy (SEM) --- p.56 / Chapter 2.5.1.1 --- Preparation of specimens for scanning electron microscopy --- p.56 / Chapter 2.5.1.2 --- "Cytological studies of pileus, stipe and spores of G. lucidum, G. tsugae and hybrid" --- p.57 / Chapter 2.5.2 --- Temperature effect --- p.57 / Chapter 2.5.3 --- Submerged fermentation --- p.57 / Chapter 2.5.4 --- Fruiting test --- p.58 / Chapter 2.6 --- "Bioactive components of G. lucidum, G. tsugae and hybrid" --- p.58 / Chapter 2.6.1 --- Sample preparation --- p.58 / Chapter 2 6.2 --- Lingzhi polysaccharide --- p.59 / Chapter 2.6.3 --- Terpenes --- p.59 / Chapter 2.7 --- Effect of extracts against breast cancer cell lines --- p.60 / Chapter 2.7.1 --- Cell culture --- p.60 / Chapter 2.7.2 --- Lingzhi Extract preparation --- p.61 / Chapter 2.7.3 --- Optimization of cell density --- p.61 / Chapter 2.7.3.1 --- MTT assay --- p.61 / Chapter 2 7.3.2 --- Trypan blue cell viability assay --- p.62 / Chapter 2.7.3.3 --- BrdU assay --- p.62 / Chapter 2.7.3.4 --- Growth curve of MCF-7 --- p.63 / Chapter 2.7.3.5 --- Growth curve of MDA-MB-231 --- p.64 / Chapter 2.7.4 --- Anti-proliferative effect of extracts on MCF-7 cells --- p.69 / Chapter 2.7.4.1 --- MTT assay --- p.69 / Chapter 2 7.4.2 --- Trypan blue cell viability assay --- p.69 / Chapter 2.7.4.3 --- BrdU assay --- p.70 / Chapter 2.7.5 --- Study of cultured medium effect of biomass and pileus extracts on MCF-7 cells --- p.71 / Chapter 2.7.5.1 --- Cultured medium effect ofbiomass and pileus extracts --- p.71 / Chapter 2.7.6 --- mRNA expression assay (RT-PCR) --- p.71 / Chapter 2.7.6.1 --- Effect of extract on gene expression --- p.71 / Chapter 2.7.6.2 --- Time effect of extract on gene expression --- p.72 / Chapter 2.7.6.3 --- Isolation of RNA --- p.72 / Chapter 2.7.6.4 --- Quantification and qualification of DNA and RNA by spectrophotometry --- p.73 / Chapter 2.7.6.5 --- First strand cDNA synthesis --- p.73 / Chapter 2.7.6.6 --- Amplification of cDNA --- p.74 / Chapter 2.7.7 --- Effect of biomass and pileus lingzhi polysacchandes and terpenes on MCF-7 cells --- p.75 / Chapter 2.7.7.1 --- Effect of reconstitution of lingzhi polysacchande and terpenes on MCF-7 cells --- p.75 / Chapter 2.7.8 --- Effect of biomass and pileus extracts on MDA-MB-231 cells --- p.76 / Chapter 2.8 --- Estrogenicigy assay --- p.76 / Chapter 2 8.1 --- E-screen test --- p.76 / Chapter 2.8.2 --- Estrogen receptor competitor binding assay --- p.77 / Chapter 2.8.3 --- pS2 mRNA expression assay --- p.78 / Chapter 2.9 --- DNA microarray analysis --- p.79 / Chapter 2.9.1 --- mRNA purification --- p.79 / Chapter 2.9.2 --- RT and LPR (Linear Polymerase Reaction) labeling --- p.80 / Chapter 2 9.3 --- pre-hybridization --- p.81 / Chapter 2.9.4 --- Hybridization --- p.82 / Chapter 2.9.5 --- Detection --- p.82 / Chapter 2.9.6 --- Image acquisition and analysis --- p.83 / Chapter Chapter 3 --- Result --- p.84 / Chapter 3.1 --- Artificial hybndization of Ganoderma tsugae and G. lucidum --- p.84 / Chapter 3.1.1 --- protoplast isomation and fusion of Ganoderma tsugae and G. lucidum --- p.84 / Chapter 3.2 --- Screening and selection of hybrid --- p.84 / Chapter 3.2.1 --- Temperature screening --- p.84 / Chapter 3.2.2 --- DNA fingerprint by Arbitrarily-primed polymerase chain reaction --- p.86 / Chapter 3.3 --- Confirmation tests --- p.88 / Chapter 3.3.1 --- Somatic incompatibility test --- p.88 / Chapter 3.3.2 --- DNA fingerprinting by specific polymerase chain reaction --- p.90 / Chapter 3.4 --- Characterization of selected hybrid --- p.100 / Chapter 3.4.1 --- Scanning electron micscropy --- p.100 / Chapter 3.4.2 --- Temperature effect --- p.103 / Chapter 3.4.3 --- Submerged fermentation --- p.105 / Chapter 3.4.4 --- Fruiting test --- p.107 / Chapter 3.5 --- "Bioactive components of G. lucidum, G. tsugae and hybrid" --- p.109 / Chapter 3.5.1 --- Lingzhi polysaccharide --- p.109 / Chapter 3.5.2 --- Terpenes --- p.109 / Chapter 3.6 --- Effect of extracts against breast cancer cell lines --- p.119 / Chapter 3.6.1 --- Anti-proliferative effect of extracts on MCF-7 cells --- p.119 / Chapter 3.6.2 --- Study of medium effect of biomass and pileus extracts on MCF-7 cells --- p.139 / Chapter 3.6.3 --- mRNA expression assay (RT-PCR) --- p.143 / Chapter 3.6.4 --- Effect of biomass and pileus lingzhi polysaccharides and terpenes on MCF-7 cells --- p.150 / Chapter 3.6.5 --- Effect of biomass and pileus extracts on MDA-MB231- cells --- p.159 / Chapter 3.7 --- Estrogenicity assay --- p.166 / Chapter 3.7.1 --- E-screen assay on biomass and pileus extracts --- p.166 / Chapter 3.7.2 --- E-screen assay on biomass and pileus terpenes and lingzhi polysaccharide --- p.166 / Chapter 3.7.3 --- Estrogen receptor competitor binding assay --- p.169 / Chapter 3.7.4 --- pS2 mRNA expression assay --- p.175 / Chapter 3.8 --- DNA microarray analysis --- p.177 / Chapter Chapter 4 --- Discussion --- p.184 / Chapter 4.1 --- Artificial hybridization of Ganoderma tsugae and G. lucidum --- p.184 / Chapter 4.1.1 --- Protoplast isolation and fusion of Ganoderma tsugae and G. luciudm --- p.184 / Chapter 4.1.2 --- Screening and selection of hybrid --- p.184 / Chapter 4.1.3 --- Characterization of the selected hybrid --- p.185 / Chapter 4.1.4 --- "Nature of hybrid, mutant and variant" --- p.189 / Chapter 4.2 --- Effect of extracts against breast cancer cell lines --- p.190 / Chapter 4.2.1 --- Anti-proliferative effect of extracts on MCF-7 cells --- p.190 / Chapter 4.2.2 --- Study of effect of cultured medium of biomass and pileus extracts on MCF-7 cells --- p.193 / Chapter 4.2.3 --- Effect of biomass and pileus extracts on MDA-MB231- cells --- p.194 / Chapter 4.2.4 --- mRNA expression assay (RT-PCR) --- p.195 / Chapter 4.3 --- Estrogenicity --- p.198 / Chapter 4.3.1 --- E-screen assay --- p.198 / Chapter 4.3.2 --- Estrogen receptor competitor binding assay --- p.199 / Chapter 4.3.3 --- pS2 mRNA expression assay --- p.200 / Chapter 4.3.4 --- Ganoderma spp. As hormonal therapy --- p.201 / Chapter 4.4 --- DNA microarray analysis --- p.201 / Chapter 4.5 --- Further investigation --- p.204 / Chapter Chapter 5 --- Conclusion --- p.205 / Chapter Chapter 6 --- Reference --- p.207

Kong Ka-lai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 126-149). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract (Chinese Version) --- p.vi / Table of Contents --- p.ix / List of Figures/Table --- p.xiii / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- An Overview on Cancer --- p.1 / Chapter 1.2 --- Biological Effects of Chlorophyllin --- p.7 / Chapter 1.2.1 --- CHL as Photosensitizer --- p.7 / Chapter 1.2.2 --- CHL as Antioxidant --- p.8 / Chapter 1.2.3 --- CHL as Anticarcinogenic Agent --- p.9 / Chapter 1.3 --- Regulation of Cell Cycle --- p.13 / Chapter 1.3.1 --- Cell-Cycle Checkpoints --- p.13 / Chapter 1.3.2 --- Cell-Cycle Regulatory Proteins --- p.15 / Chapter 1.4 --- Regulation of Mitogen-Activated Protein Kinase (MAPK) Signaling Cascade --- p.21 / Chapter 1.5 --- Programmed Cell Death (or Apoptosis) --- p.27 / Chapter 1.5.1 --- Regulation of Caspase-Dependent Apoptosis --- p.28 / Chapter 1.5.2 --- Regulation of Caspase-Independent Cell Death --- p.32 / Chapter 1.5.3 --- Bcl-2 Family Proteins in Modulation of Cell Death --- p.32 / Chapter 1.6 --- In Vivo Antitumor Screening System --- p.37 / Chapter 1.7 --- Aims of the Present Study --- p.38 / Chapter Chapter 2 --- In Vitro Studies of the Anticancer Effect of Chlorophyllin / Chapter 2.1 --- Introduction --- p.39 / Chapter 2.1.1 --- DNA-Flow Cytometric Analysis --- p.51 / Chapter 2.1.2 --- Western Blot Analysis --- p.54 / Chapter 2.2 --- Materials and Methods --- p.56 / Chapter 2.2.1 --- Maintenance of Cell Lines --- p.56 / Chapter 2.2.2 --- Cytotoxic and Cytostatic Effects on the Cancer Cells --- p.56 / Chapter 2.2.3 --- DNA-Flow Cytometric Analysis --- p.60 / Chapter 2.2.4 --- Western Blot Analysis --- p.61 / Chapter 2.2.5 --- JC-1 Mitochondrial Potential Sensor --- p.64 / Chapter 2.2.6 --- Caspase Inhibitors --- p.65 / Chapter 2.2.7 --- Statistical Analysis --- p.66 / Chapter 2.2.8 --- Densitometric Analysis --- p.66 / Chapter 2.3 --- Results --- p.67 / Chapter 2.3.1 --- Effects of CHL on the Growth of Human Cancer Cells by MTT Assay --- p.67 / Chapter 2.3.2 --- Effect of CHL on the Proliferation of MCF-7 Cells by Chemi-BrdU Incorporation --- p.69 / Chapter 2.3.3 --- Effect of CHL on Cell Cycle of MCF-7 Cells --- p.71 / Chapter 2.3.4 --- Effect of CHL on the Cyclin D1 Expression in MCF-7 Cells --- p.74 / Chapter 2.3.5 --- Effects of CHL on JNK and c-Jun Expressions and Their Phosphorylations in MCF-7 Cells --- p.76 / Chapter 2.3.6 --- Effect of CHL on DNA fragmentation in MCF-7 Cells --- p.78 / Chapter 2.3.7 --- Effect of CHL on Mitochondrial Membrane Potential of MCF-7 Cells --- p.80 / Chapter 2.3.8 --- Effects of CHL on the PARP Expression and Cleavage in MCF-7 Cells --- p.83 / Chapter 2.3.9 --- "Effects of CHL on Bcl-2, Bcl-xL and Bad Expressions in MCF-7 Cells" --- p.85 / Chapter 2.3.10 --- Effects of CHL on Caspase Activations in MCF-7 Cells --- p.88 / Chapter 2.3.11 --- Effects of Caspase Inhibitors on the CHL-Induced Apoptosis in MCF-7 Cells --- p.90 / Chapter 2.4 --- Discussion --- p.93 / Chapter Chapter 3 --- In Vivo Studies of the Anticancer Effect of Chlorophyllin / Chapter 3.1 --- Introduction --- p.104 / Chapter 3.2 --- Materials and Methods --- p.106 / Chapter 3.2.1 --- Transplantation of MCF-7 Cells into the Nude Mice and Treatment --- p.106 / Chapter 3.2.2 --- Western Blot Analysis --- p.107 / Chapter 3.2.3 --- Statistical Analysis --- p.107 / Chapter 3.3 --- Results --- p.108 / Chapter 3.3.1 --- In Vivo Antitumor Activity of CHL --- p.108 / Chapter 3.3.2 --- In Vivo Effects of CHL on Cyclin D1 and Bcl-2 Expressions in MCF-7 Solid Tumor --- p.111 / Chapter 3.4 --- Discussion --- p.113 / Chapter Chapter 4 --- General Discussion --- p.115 / References --- p.126

乳腺癌是女性最常見的腫瘤之一，多發生在女性絶經後，並具有雌激素依賴性。芳香化酶(CYP19)是雌激素生物合成過程中的關鍵酶，而芳香化酶抑製劑(AI)則被用於替代治療雌激素依賴性的乳腺癌。然而，AI在降低雌激素水平的同時能夠引起骨質酥鬆。此項研究的目的是找尋AI替代物。 / 黃酮類化合物是一種多酚化合物，廣泛分佈于植物中。我們先前的研究發現二氢黄酮陈皮素能夠抑制芳香化酶的生物活性，并且抑制芳香化酶高表達的乳腺癌生長。在本研究中，我們發現陳皮素在抑制腫瘤生長的同時能夠降低来曲唑引起的骨質流失。木犀草素是另外一種黄酮类化合物，它同樣能夠抑制芳香化酶的活性并減少骨流失。而與陳皮素不同的是，它能夠抑制芳香化酶的表達。在芳香化酶高表達的乳腺癌細胞(MCF-7 aro)中，木犀草素抑制芳香化酶活性的IC50是3 μM。在MCF-7 細胞中，5 μM的木犀草素能夠抑制CYP19 mRNA 的表達，螢光素酶報告實驗顯示木犀草素是通過作用于啟動子I.3和II來抑制CYP19的表達。蛋白印跡實驗表明木犀草素抑制CYP19表達的分子機制可能通過調節JNK信號通路進而減少AP-1的活性來實現。動物實驗結果顯示木犀草素能夠抑制MCF-7aro腫瘤的生長并改善來曲唑引起的骨流失。 / 環氧化酶(COX)是花生四烯酸轉化為前列腺素途徑中的一種關鍵酶。研究發現COX-2在乳腺癌組織中廣泛表達。本實驗研究了COX抑製劑在裸鼠動物模型中對乳腺癌腫瘤的作用機制。研究結果表明塞來昔布和阿司匹林在不影響血液中雌激素水平的情況下抑制乳腺癌腫瘤的生長。蛋白印迹實驗顯示這兩種藥物能夠降低腫瘤中COX-2，Cyclin A和Bcl-xL的表達。miR-98, miR-222和miR-145也能夠被塞來昔布和阿司匹林影響。 / 本研究表明陳皮素，木犀草素及COX抑制劑有潛力成為替代AI的化學治療藥物或共同治療藥物。 / Breast cancer is one of the most prevalent cancers affecting women. The majority of breast tumor growth occurred in the post-menopausal period are estrogen dependent. Aromatase (CYP19) catalyzes the rate-limiting step in the synthetic reaction of estrogen and aromatase inhibitors (AIs) are contemporary treatment for estrogen-positive breast cancer. However, estrogen-lowering drugs may promote osteoporosis. Our objective of this study further identified some alternatives for AIs. / Flavonoids are polyphenolic compounds that are ubiquitously distributed in plants. We have previously found that the flavanone hesperetin can inhibit the activity of aromatase and suppress aromatase-expressing breast tumor growth. In this project, we investigated the potential interaction between hesperetin and the AI letrozole in a mouse model. Our results showed that hesperetin could inhibit the tumor growth and reduce bone loss induced by letrozole. Similarly, another flavonoid luteolin also inhibited aromatase and prevented bone deterioration as observed in this project. In cells stably transfected with CYP19 (MCF-7aro), luteolin inhibited the aromatase activity with an IC50 value of 3μM. In addition, 5μM luteolin significantly reduced CYP19 mRNA expression in MCF-7 cells. Luciferase reporter assay revealed that luteolin could suppress CYP19 transcription at promoter regions I.3 and II. Western analysis illustrated that JNK signaling pathway was involved and deactivation of AP-1 could be the underlying molecular mechanism. Subsequently, we examined the effect in vivo. Our results showed that luteolin could inhibit the MCF-7aro tumor growth and improved bone loss induced by letrozole. / Cyclooxygenase (COX) is an enzyme responsible for the conversion of arachidonic acid into prostaglandins. It is over-expressed in breast cancer tissue and an increased expression of COX-2 was also observed in the xenograft model employed in this project. In the last study we evaluated the importance of COX-2 in breast tumor growth in this model. Our data showed that celecoxib and aspirin could significantly suppress the tumor growth without changing the plasma estrogen level. Western analysis illustrated that COX-2, Cyclin A, Bcl-xL and ER were reduced in celecoxib- and aspirin- treated tumor samples and miR-98, miR-222 and miR-145 were altered by celecoxib or aspirin. / After all, this project demonstrated that hesperetin, luteolin and COX-inhibitors could be potential chemopreventive or co-therapeutic agents. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Fengjuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 131-148). / Abstract also in Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.II / 摘要 --- p.IV / LIST OF ABBREVIATIONS --- p.V / TABLE OF CONTENTS --- p.VII / CHAPTER 1 --- p.1 / GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Types of Breast Cancer --- p.3 / Chapter 1.2 --- Nuclear Receptor Signaling Pathways in Breast Cancer --- p.5 / Chapter 1.3 --- Estrogen and Breast Cancer --- p.7 / Chapter 1.4 --- Estrogen and Bone Health --- p.8 / Chapter 1.5 --- Estrogen Biosynthesis and Aromatase --- p.10 / Chapter 1.6 --- Tissue Specific Promoter for Aromatase Expression --- p.13 / Chapter 1.7 --- Nuclear Receptors and Aromatase Promoter Regulation --- p.15 / Chapter 1.8 --- Signaling Pathway and Aromatase Expression --- p.17 / Chapter 1.9 --- Cell Cycle in Breast Cancer --- p.20 / Chapter 1.10 --- Cell Apoptosis --- p.23 / Chapter 1.11 --- Treatment of breast cancer --- p.25 / Chapter 1.12 --- Phytoestrogens --- p.29 / Chapter 1.13 --- Aim of My Study --- p.32 / CHAPTER 2 --- p.33 / MATERIALS AND METHODS --- p.33 / Chapter 2.1 --- Chemicals and Materials --- p.33 / Chapter 2.1.1 --- Chemicals --- p.33 / Chapter 2.1.2 --- Plasmids --- p.33 / Chapter 2.2 --- Cell Culture --- p.33 / Chapter 2.3 --- Aromatase Activity Assay --- p.34 / Chapter 2.4 --- Quantitative Real Time PCR --- p.36 / Chapter 2.4.1 --- RNA Isolation and cDNA Synthesis --- p.36 / Chapter 2.4.2 --- Quantitative Real Time PCR Assay --- p.37 / Chapter 2.4.3 --- MiRNA Quantitative Real Time PCR Assay --- p.38 / Chapter 2.5 --- Western Blot --- p.39 / Chapter 2.6 --- Measurement of Promoter Activity --- p.41 / Chapter 2.6.1 --- Plasmid Preparation --- p.41 / Chapter 2.6.2 --- Transient Transfection and Dual-Luciferase Assay --- p.42 / Chapter 2.7 --- Electrophoretic Mobility Shift Assay (EMSA) --- p.43 / Chapter 2.7.1 --- Nuclear protein extraction --- p.43 / Chapter 2.7.2 --- Electrophorectic Mobility Shift Assay --- p.44 / Chapter 2.8 --- Animal Experiment Design --- p.45 / Chapter 2.8.1 --- Animal Model for Hesperetin Study --- p.45 / Chapter 2.8.2 --- Animal Model for Luteolin Study --- p.46 / Chapter 2.8.3 --- Animal Model for Cycooxygenase Inhibitors Study --- p.48 / Chapter 2.8.4 --- Serum Estradiol Determination --- p.49 / Chapter 2.8.5 --- Analysis of serum lipoproteins --- p.49 / Chapter 2.8.6 --- Bone Image Acquisition and Region of Interest Selection --- p.50 / Chapter 2.9 --- Statistical Analysis --- p.50 / CHAPTER 3 --- p.51 / The citrus flavonone hesperetin prevents letrozole- induced bone loss in a mouse model of breast cancer --- p.51 / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Results --- p.54 / Chapter 3.2.1 --- Murine Body Weight and Liver Weight --- p.54 / Chapter 3.2.2 --- Effect of Hesperetin and Letrozole on Xenograft Growth in Ovariectomized Mice --- p.55 / Chapter 3.2.3 --- Hesperetin Reduced Plasma Estradiol Concentration --- p.58 / Chapter 3.2.4 --- PS2 mRNA Expression in Tumor --- p.59 / Chapter 3.2.5 --- Uterine Wet Weight --- p.60 / Chapter 3.2.6 --- Hesperetin Prevent Bone Deterioration Induced by Letrozole --- p.61 / Chapter 3.3 --- DISCUSSION --- p.63 / CHAPTER 4 --- p.66 / dIETARY FLAVONOID LUTEOLIN ON cyp19 transcription in the breast cancer cells mcf-7 --- p.66 / Chapter 4.1 --- Introduction --- p.66 / Chapter 4.2 --- Results --- p.68 / Chapter 4.2.1 --- Inhibitory Effect of Luteolin on Aromatase Activity --- p.68 / Chapter 4.2.2 --- Luteolin Reduced Aromatase mRNA Expression in MCF-7 Cells --- p.70 / Chapter 4.2.3 --- Effect of Luteolin on Promoter I.3/II Activity of CYP19 in MCF-7 Cells --- p.71 / Chapter 4.2.4 --- The Effect of Luteolin on Truncation CYP19 Gene Reporter Assay --- p.72 / Chapter 4.2.5 --- Luteolin Reduced AP-1 Binding in Promoter I.3/II DNA Fragment --- p.74 / Chapter 4.2.6 --- Inhibitory Effect of Luteolin on Protein Kinase Signaling --- p.76 / Chapter 4.3 --- Discussion --- p.78 / CHAPTER 5 --- p.83 / interaction OF LUTEOLIN and letrozole in a postmenopausal breast cancer model --- p.83 / Chapter 5.1 --- Introduction --- p.83 / Chapter 5.2 --- Results --- p.86 / Chapter 5.2.1 --- Luteolin and letrozole treatment had no effect on mouse body weight and liver weight --- p.86 / Chapter 5.2.2 --- Effect of luteolin and Letrozole on Xenograft Growth in Ovariectomized Mice --- p.88 / Chapter 5.2.3 --- Luteolin reduced plasma estradiol concentration --- p.91 / Chapter 5.2.4 --- Luteolin Counteracted Uterine Weight Reduction under Letrozole Treatment --- p.92 / Chapter 5.2.5 --- Luteolin Prevented Bone Deterioration Induced by Letrozole --- p.93 / Chapter 5.2.6 --- The Effect of Luteolin on Plasma TC and TG --- p.95 / Chapter 5.2.7 --- Luteolin Increased HDL Level and Reduced the Ratio of LDL/HDL --- p.97 / Chapter 5.2.8 --- Effect of Luteolin on Cell Cycle and Apoptotic Protein Expression --- p.99 / Chapter 5.3 --- DISCUSSION --- p.104 / CHAPTER 6 --- p.107 / cyclooxygenase inhibitors suppresse breast tumor growth in NUDE MICE --- p.107 / Chapter 6.1 --- Introduction --- p.107 / Chapter 6.2 --- Results --- p.109 / Chapter 6.2.1 --- Celecoxib and aspirin treatment had no effect on mouse body weight and liver weight --- p.109 / Chapter 6.2.2 --- Effect of celecoxib and aspirin on Xenograft Growth in Ovariectomized Mice --- p.111 / Chapter 6.2.3 --- Celecoxib and aspirin had no effect on plasma estradiol concentration --- p.113 / Chapter 6.2.4 --- Celecoxib and Aspirin Had no Effect on Uterine Weight --- p.114 / Chapter 6.2.5 --- Protein expression of COX-2, Cell cycle-related and cell Apoptotic Genes --- p.115 / Chapter 6.2.6 --- Detection of Related miRNA Expression Level in Tumors --- p.118 / Chapter 6.2.7 --- c-Myc mRNA Expression Level were Regulated in Tumors --- p.121 / Chapter 6.3 --- DISCUSSION --- p.124 / CHAPTER 7 --- p.127 / SUMMARY --- p.127 / REFERENCE --- p.131

Zhou, Linli. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 108-118). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Abbreviations --- p.v / List of Figures --- p.vii / List of Tables --- p.ix / Table of Contents --- p.x / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Breast Cancer --- p.1 / Chapter 1.1.1 --- Introduction to Breast Cancer --- p.1 / Chapter 1.1.2 --- Types of Breast Cancer --- p.3 / Chapter 1.1.3 --- Epidemiologic Risk Factors and Etiology --- p.4 / Chapter 1.2 --- Estrogen and Breast Cancer --- p.7 / Chapter 1.3 --- Estrogen Receptor --- p.9 / Chapter 1.4 --- Current Treatment of Breast Cancer --- p.10 / Chapter 1.4.1 --- Chemotherapy --- p.10 / Chapter 1.4.2 --- Hormonal (Anti-Estrogen) Therapy --- p.11 / Chapter 1.4.2.1 --- Tamoxifen and Other Anti-estrogens --- p.12 / Chapter 1.4.2.2 --- Disadvantages of Tamoxifen --- p.13 / Chapter 1.5 --- Arsenic Trioxide --- p.14 / Chapter 1.5.1 --- The Characteristics of Arsenic Trioxide (AS2O3) --- p.14 / Chapter 1.5.2 --- The Medical use of Arsenic Trioxide (As2O3) --- p.16 / Chapter 1.5.3 --- Arsenic Trioxide (As2O3) in treating Acute Promyelocytic Leukemia (APL) --- p.17 / Chapter 1.5.3.1 --- Acute Promyelocytic Leukemia (APL) --- p.17 / Chapter 1.5.3.2 --- All-trans Retinoic Acid (ATRA) Treatment of APL --- p.18 / Chapter 1.5.3.3 --- Clinical Trial of the Arsenic Trioxide on APL --- p.19 / Chapter 1.5.3.4 --- In vitro and in vivo Study of Arsenic Trioxide (As2O3) in treating APL --- p.19 / Chapter 1.5.3.5 --- Common Side Effects of Arsenic Trioxide (As2O3) on APL --- p.21 / Chapter 1.5.4 --- Anti-cancer effect of Arsenic Trioxide on other cancers --- p.23 / Chapter 1.6 --- Aim of Study --- p.24 / Chapter Chapter 2 --- Materials and Methods --- p.26 / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- Cell Lines and Culture Medium --- p.27 / Chapter 2.1.1.1 --- Cell Lines --- p.27 / Chapter 2.1.1.2 --- Culture Medium --- p.27 / Chapter 2.1.2 --- Chemicals --- p.28 / Chapter 2.1.3 --- Buffers and Reagents --- p.29 / Chapter 2.1.4 --- Reagents for MTT Assay --- p.30 / Chapter 2.1.5 --- Reagents for DNA Fragmentation --- p.31 / Chapter 2.1.5.1 --- Reagents for DNA Extraction --- p.31 / Chapter 2.1.5.2 --- Reagents for Gel Electrophoresis --- p.31 / Chapter 2.1.6 --- Reagents for Western Blotting --- p.32 / Chapter 2.1.6.1 --- Reagents for Protein Extraction --- p.32 / Chapter 2.1.6.2 --- Reagents for SDS-PAGE --- p.33 / Chapter 2.1.7 --- Reagents for Flow Cytometry --- p.36 / Chapter 2.1.8 --- In Vivo Study --- p.37 / Chapter 2.2 --- Methods --- p.38 / Chapter 2.2.1 --- Cell Treatment --- p.38 / Chapter 2.2.2 --- Trypan Blue Exclusion Assay --- p.38 / Chapter 2.2.3 --- MTT Assay --- p.38 / Chapter 2.2.4 --- Detection of DNA Fragmentation --- p.39 / Chapter 2.2.5 --- Flow Cytometry --- p.40 / Chapter 2.2.5.1 --- Detection of Cell Cycle Pattern with PI --- p.40 / Chapter 2.2.5.2 --- Detection of Apoptosis with Annexin V-PI --- p.40 / Chapter 2.2.6 --- Western Blot Analysis --- p.41 / Chapter 2.2.6.1 --- Protein Extraction --- p.41 / Chapter 2.2.6.2 --- Protein Concentration Determination --- p.41 / Chapter 2.2.6.3 --- Western Blotting --- p.42 / Chapter 2.2.7 --- In Vivo Study --- p.44 / Chapter 2.2.7.1 --- Animal Model --- p.44 / Chapter 2.2.7.2 --- Treatment Schedule --- p.44 / Chapter 2.2.7.3 --- Toxicity of Arsenic Trioxide --- p.45 / Chapter Chapter 3 --- Anti-Proliferation Effect of As2O3 on MDA-MB-231 cells --- p.47 / Chapter 3.1 --- Study the Anti-proliferation Effect of As2O3 on MDA-MB-231 Cells by MTT Assay --- p.48 / Chapter 3.2 --- Comparsion Anti-proliferation Effect of AS2O3 on MDA-MB-231 Cells to that of Tamoxifen --- p.50 / Chapter 3.3 --- "Study Toxicity of AS2O3 on Normal Breast Cells Line, 184B5" --- p.52 / Chapter 3.4 --- Summary --- p.54 / Chapter Chapter 4 --- Mechanism of Growth Inhibition Effect of As2O3 on MDA-MB-231 cells --- p.56 / Chapter 4.1 --- Cell Cycle Analysis of As2O3 Treated MDA-MB-231 Cells --- p.57 / Chapter 4.2 --- Detection of DNA Fragmentation --- p.60 / Chapter 4.3 --- Detection of Apoptosis Induced by AS2O3 on MDA-MB-231 Cells by Flow Cytometry --- p.62 / Chapter 4.4 --- Regulation of Apoptotic Related Protein by As2O3 on MDA-MB-231 Cells --- p.64 / Chapter 4.4.1 --- Expression Level of Bcl-2 and Bax Protein --- p.66 / Chapter 4.4.2 --- Expression Level of Cytochrome C --- p.69 / Chapter 4.4.3 --- Expression Level of Caspase9 --- p.71 / Chapter 4.4.4 --- Expression Level of FasL --- p.73 / Chapter 4.4.5 --- Expression Level of Caspase8 --- p.75 / Chapter 4.4.6 --- Expression Level of Caspase3 --- p.77 / Chapter 4.4.7 --- Expression Level of Poly (ADP-ribose) Polymerase (PARP) --- p.79 / Chapter 4.4.8 --- Expression Level of p53 --- p.81 / Chapter 4.5 --- Regulation of Cell Cycle Related Protein by AS2O3 on MDA-MB-231 Cells --- p.83 / Chapter 4.5.1 --- Expression Level of Cyclin B --- p.84 / Chapter 4.5.2 --- Expression Level of Cyclin E --- p.86 / Chapter 4.6 --- Summary --- p.88 / Chapter Chapter 5 --- In Vivo Study of Anti-tumor Effect of As2O3 --- p.89 / Chapter 5.1 --- Anti-tumor Effect of AS2O3 on Tumor Bearing Nude Mice --- p.90 / Chapter 5.2 --- Toxic Effect of AS2O3 on Normal Tissues --- p.93 / Chapter 5.3 --- Summary --- p.98 / Chapter Chapter 6 --- Discussion --- p.99 / Chapter 6.1 --- Anti-tumor Effect of AS2O3 on Breast Cancer --- p.100 / Chapter 6.2 --- Induction of Apoptosis and Cell Cycle arrest by AS2O3 --- p.101 / Chapter 6.3 --- Side Effect of AS2O3 on Breast Cancer Treatment --- p.103 / Chapter Chapter 7 --- Future Perspectives --- p.105 / Chapter 7.1 --- Future Perspectives --- p.106 / References --- p.108

Focal adhesion kinase (FAK), a non-receptor kinase, is a key regulator of integrin and focal adhesion signaling required for cancer cell survival, cell migration, and cell invasion. Amplification/Overexpression of FAK occurs in a wide variety of human cancers, supporting a role in carcinogenesis. Moreover, preclinical studies using cancer models where FAK is genetically inhibited indicate that this kinase is a potential therapeutic target to interfere with cancer progression. However, very little progress has been made in the identification of chemical inhibitors for potential therapeutic applications, in contrast to other kinases. Herein, I report optimization of the high-throughput in vitro Glo kinase assay for screening inhibitors of FAK kinase activity. Screening a large library of small molecule chemicals using these assays identified at least twenty FAK inhibitors, including a new FAK inhibitor developed by Pfizer and undergoing human clinical trials, and the non-specific kinase inhibitor staurosporine. Molecular studies of selective FAK inhibitors are undergoing in my host laboratory. In addition to this in vitro assay, I established similar assays to examine FAK kinase and adapter function in intact cells. The latter consists of ErbB-transformed cells deficient in FAK, and their matched cells where wild-type or kinase-dead FAK was restored. Biological characterization of these models revealed that both FAK kinase and adaptor activities cooperate for the regulation of cell migration, cell invasion, and tumor formation.

No description available.

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