Liu, Xiaozhuo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 156-169). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.iv / Acknowledgments --- p.vi / List of Abbreviations --- p.vii / Table of Contents --- p.xiv / Chapter Chapter One: --- General Introduction / Chapter 1.1 --- Hematopoiesis and Leukemia --- p.1 / Chapter 1.1.1 --- An Overview on Hematopoiesis --- p.1 / Chapter 1.1.2 --- Leukemia --- p.6 / Chapter 1.1.2.1 --- An Overview of Leukemia --- p.6 / Chapter 1.1.2.2 --- Classification and Epidemiology of Leukemia --- p.8 / Chapter 1.1.2.3 --- Conventional Approaches to Leukemia Therapy --- p.12 / Chapter 1.1.2.4 --- Novel Approaches to Leukemia Therapy --- p.15 / Chapter 1.2 --- Sophoraflavanone G: A Bioactive Compound Isolated from Kushen --- p.18 / Chapter 1.2.1 --- An Overview of Kushen: A Traditional Chinese Medicine --- p.19 / Chapter 1.2.2 --- An Overview of Lavandulyl Flavanones --- p.22 / Chapter 1.2.3 --- Historical Development and Occurrence of Sophoraflavanone G --- p.24 / Chapter 1.2.4 --- Biological Activities of Sophoraflavanone G --- p.25 / Chapter 1.2.4.1 --- Anti-microbial and Insecticidal Activities --- p.25 / Chapter 1.2.4.2 --- Anti-tumor Activities --- p.26 / Chapter 1.2.4.3 --- Pharmacodynamics of Sophoraflavanone G --- p.27 / Chapter 1.3 --- Objectives and Scopes of the Present Study --- p.30 / Chapter Chapter Two: --- Materials and Methods / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Animals --- p.32 / Chapter 2.1.2 --- Cell lines --- p.32 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.34 / Chapter 2.1.4 --- Reagents and Buffers for Flow Cytometry --- p.37 / Chapter 2.1.5 --- Reagents for DNA Extraction --- p.39 / Chapter 2.1.6 --- Reagents for Measuring Caspase Activity --- p.40 / Chapter 2.1.7 --- "Reagents, Buffers and Materials for Western Blotting" --- p.43 / Chapter 2.2 --- Methods --- p.48 / Chapter 2.2.1 --- Extraction and Isolation of Sophoraflavanone G from Kushen --- p.48 / Chapter 2.2.2 --- Culture of Tumor Cell Lines --- p.49 / Chapter 2.2.3 --- "Isolation, Preparation and Culturing of Human Peripheral Blood Leukocytes and Murine Bone Marrow Cells" --- p.50 / Chapter 2.2.4 --- Assays for Anti-proliferation and Cytotoxicity --- p.51 / Chapter 2.2.5 --- Determination of Anti-leukemic Activity In Vivo (In Vivo Tumorigenicity Assay) --- p.52 / Chapter 2.2.6 --- Cell Cycle Analysis by Flow Cytometry --- p.53 / Chapter 2.2.7 --- Measurement of Apoptosis-induced Activities --- p.54 / Chapter 2.2.8 --- Protein Expression Study --- p.59 / Chapter 2.2.9 --- Assessment of Differentiation-associated Characteristics --- p.64 / Chapter 2.2.10 --- Statistical Analysis --- p.65 / Chapter Chapter Three: --- Studies on the Anti-proliferative Effect of Sophoraflavanone G on Human Myeloid Leukemia Cells / Chapter 3.1 --- Introduction --- p.66 / Chapter 3.2 --- Results --- p.69 / Chapter 3.2.1 --- Structure Identification of Sophoraflavanone G Isolated from Sophora flavescens --- p.69 / Chapter 3.2.2 --- Anti-proliferative Activity of Sophoraflavanone G on Various Myeloid Leukemia Cell Lines --- p.72 / Chapter 3.2.3 --- Effect of Sophoraflavanone G on the Viability of the Human Promyelocytic Leukemia HL-60 Cells --- p.80 / Chapter 3.2.4 --- Cytotoxic Effect of Sophoraflavanone G on Primary Normal Cells In Vitro --- p.83 / Chapter 3.2.5 --- Kinetic and Reversibility Studies of the Anti-proliferative Effect of Sophoraflavanone G on the Human Promyelocytic Leukemia HL-60 Cells --- p.85 / Chapter 3.2.6 --- Effect of Sophoraflavanone G on the In Vivo Tumorigenicity of the HL-60 Cells --- p.88 / Chapter 3.2.7 --- Effect of Sophoraflavanone G on the Cell Cycle Profile of the HL-60 cells In Vitro --- p.90 / Chapter 3.2.8 --- Effect of Sophoraflavanone G on the Expression of Cell Cycle-regulatory Proteins in the HL-60 Cells --- p.93 / Chapter 3.2.9 --- Anti-proliferative Effect of Sophoraflavanone G on Multidrug-resistant (MDR) Leukemia Cell Line HL-60/MX2 Cells --- p.95 / Chapter 3.3 --- Discussion --- p.101 / Chapter Chapter Four: --- Studies on the Apoptosis- and Differentiation-inducing Activities of Sophoraflavanone G on Human Myeloid Leukemia Cells / Chapter 4.1 --- Introduction --- p.109 / Chapter 4.2 --- Results --- p.114 / Chapter 4.2.1 --- Induction of DNA Fragmentation in the Human Promyelocytic Leukemia HL-60 Cells by Sophoraflavanone G --- p.114 / Chapter 4.2.2 --- Induction of Phosphatidylserine Externalization in the Human Promyelocytic Leukemia HL-60 Cells by Sophoraflavanone G as Detected by Annexin V-GFP and PI Double Staining Method --- p.116 / Chapter 4.2.3 --- Effects of Sophoraflavanone G on the Caspase Activities in the Human Promyelocytic Leukemia HL-60 Cells --- p.119 / Chapter 4.2.4 --- Induction of Mitochondrial Membrane Depolarization in the Human Promyelocytic Leukemia HL-60 Cells by Sophoraflavanone G --- p.124 / Chapter 4.2.5 --- Involvement of Bcl-2 Family Members in Sophoraflavanone G-induced Apoptosis in the Human Promyelocytic Leukemia HL-60 Cells --- p.128 / Chapter 4.2.6 --- Effects of Sophoraflavanone G on the Induction of Reactive Oxygen Species in the Human Promyelocytic Leukemia HL-60 Cells --- p.131 / Chapter 4.2.7 --- Effect of Sophoraflavanone G on the Intracellular Ca2+ Level in the Human Promyelocytic Leukemia HL-60 Cells --- p.134 / Chapter 4.2.8 --- Morphological Studies on the Sophoraflavanone G-treated Human Promyelocytic Leukemia HL-60 Cells --- p.136 / Chapter 4.2.9 --- Effect of Sophoraflavanone G on the NBT Reducing Activity of the Human Promyelocytic Leukemia HL-60 Cells --- p.138 / Chapter 4.3 --- Discussion --- p.140 / Chapter Chapter Five: --- Conclusions and Future Perspectives --- p.148 / References --- p.156

Yeung, Tak Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 181-197). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Acknowledgements --- p.vi / List of Publications --- p.vii / Presentation --- p.vii / List of Abbreviations --- p.viii / Abbreviations in Figures --- p.xiii / Abbreviations in Symbols --- p.xiv / List of Cell Lines Used in this Study --- p.xv / Table of Contents --- p.xvi / List of Figures --- p.xxv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cellular Redox State and Cancer Biology --- p.1 / Chapter 1.2 --- Reactive Oxygen Species (ROS) --- p.1 / Chapter 1.3 --- Regulation of Cellular Redox State by Intrinsic and Extrinsic Antioxidant Systems --- p.5 / Chapter 1.3.1 --- Intrinsic Antioxidant System --- p.6 / Chapter 1.3.2 --- Extrinsic Antioxidant System --- p.8 / Chapter 1.4 --- Glutathione --- p.9 / Chapter 1.4.1 --- General Information of Glutathione --- p.9 / Chapter 1.4.2 --- Functions of Glutathione --- p.12 / Chapter 1.4.2.1 --- As an Antioxidant and Free Radical Scavenger --- p.12 / Chapter 1.4.2.2 --- As a Detoxifier --- p.13 / Chapter 1.4.2.3 --- As a Regulator of Cell Signaling --- p.14 / Chapter 1.4.3 --- Synthesis of Glutathione --- p.15 / Chapter 1.4.4 --- Catabolism of Glutathione --- p.15 / Chapter 1.4.5 --- Transport and Uptake of Glutathione --- p.16 / Chapter 1.4.6 --- Glutathione in Cancer Biology --- p.18 / Chapter 1.4.6.1 --- "Role of Glutathione in the Regulation of Carcinogenesis, Growth and Apoptosis of Cancer Cells" --- p.18 / Chapter 1.4.6.1.1 --- Role of Glutathione in Carcinogenesis --- p.18 / Chapter 1.4.6.1.2 --- Role of Glutathione in the Growth of Cancer Cells --- p.20 / Chapter 1.4.6.1.3 --- Role of Glutathione in Apoptosis of Cancer Cells --- p.21 / Chapter 1.4.6.2 --- Role of Glutathione in the Regulation of Metastasis --- p.23 / Chapter 1.4.6.3 --- Role of Glutathione in Cancer Resistance and Therapy --- p.24 / Chapter 1.4.6.3.1 --- Role of Glutathione in Cancer Resistance --- p.24 / Chapter 1.4.6.3.2 --- Role of Glutathione in Cancer Therapy --- p.24 / Chapter 1.5 --- Aims of the Present Study --- p.25 / Chapter Chapter 2 --- In Vitro Study of Bαicαlein and Baicalin on Glutathione Depletion --- p.28 / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.1.1 --- Scutellaria bαicαlensis Georgi --- p.28 / Chapter 2.1.1.1 --- General Clinical Applications to Treat or Prevent Diseases --- p.28 / Chapter 2.1.1.2 --- As an Antioxidant and Free Radical Scavenger --- p.29 / Chapter 2.1.1.3 --- Long History for Treatment of Cancers with the Obscure Mechanism --- p.30 / Chapter 2.1.1.4 --- Major Components --- p.31 / Chapter 2.1.2 --- Baicalein and Baicalin --- p.32 / Chapter 2.1.2.1 --- General Clinical Applications to Treat or Prevent Diseases --- p.32 / Chapter 2.1.2.2 --- As an Antioxidant and Free Radical Scavenger --- p.33 / Chapter 2.1.3 --- Hypothesis: Baicalein and Baicalin Induce Cancer Cell Death Via Glutathione Depletion --- p.35 / Chapter 2.2 --- Materials and Methods --- p.36 / Chapter 2.2.1 --- Chemicals --- p.36 / Chapter 2.2.2 --- Buffers and Solutions --- p.36 / Chapter 2.2.3 --- Animals --- p.37 / Chapter 2.2.4 --- Preparation of Rat Brain Microsomes --- p.37 / Chapter 2.2.5 --- Glutathione Depletion Assay In Vitro and Thiol Depletion Assay in Rat Brain Microsomes --- p.38 / Chapter 2.2.6 --- Statistical Analysis --- p.39 / Chapter 2.3 --- Results --- p.40 / Chapter 2.3.1 --- Effects of Baicalein and Baicalin on Sulfhydryl Contents of Glutathione --- p.42 / Chapter 2.3.2 --- Effects of Baicalein and Baicalin on Sulfhydryl Contents of Rat Brain Microsomes --- p.42 / Chapter 2.4 --- Discussion --- p.44 / Chapter Chapter 3 --- Effects of Baicalein and Baicalin on Proliferation of Different Human Cancer and Normal Cells --- p.45 / Chapter 3.1 --- Introduction-Importance of Developing A Novel Compound Inducing Cancer Cells to Cell Death with the Least Side Effects on Normal Cells --- p.45 / Chapter 3.2 --- Materials and Methods --- p.46 / Chapter 3.2.1 --- Instruments --- p.46 / Chapter 3.2.2 --- Chemicals and Cell Culture Reagents --- p.46 / Chapter 3.2.3 --- Buffers --- p.46 / Chapter 3.2.4 --- Cell Lines --- p.47 / Chapter 3.2.5 --- Cell Culture --- p.48 / Chapter 3.2.6 --- Determination of Cell Proliferation by MTT Assay --- p.49 / Chapter 3.3 --- Results --- p.51 / Chapter 3.3.1 --- Anti-Proliferative Effects of Baicalein and Baicalin on Different Cancer Cell Lines --- p.51 / Chapter 3.3.2 --- Effects of Baicalein on Different Normal Cell Lines --- p.56 / Chapter 3.4 --- Discussion --- p.58 / Chapter 3.4.1 --- Anti-Proliferative Effects of Baicalein and Baicalin on Different Cancer Cell Lines --- p.58 / Chapter 3.4.2 --- Effects of Baicalein on Cell Proliferation on Different Human Normal Cell Lines --- p.60 / Chapter Chapter 4 --- Glutathione-Depleting Effects of Baicalein on Cell Proliferation of Different Cell Lines --- p.61 / Chapter 4.1 --- Introduction-Brain Tumors --- p.61 / Chapter 4.1.1 --- Types and Classifications of Brain Tumors --- p.61 / Chapter 4.1.2 --- "Incidence Time, Patient Survival Time and Rate for" --- p.65 / Chapter 4.1.3 --- Symptoms and Diagnostic Methods for Brain Tumors --- p.66 / Chapter 4.1.4 --- "Treatments, Side Effects and Difficulties of Treatments for Brain Tumors" --- p.67 / Chapter 4.1.5 --- Glutathione Levels in Brain Normal and Cancer Cells --- p.69 / Chapter 4.2 --- Materials and Methods --- p.70 / Chapter 4.2.1 --- Instruments --- p.70 / Chapter 4.2.2 --- Chemicals --- p.70 / Chapter 4.2.3 --- Buffers --- p.70 / Chapter 4.2.4 --- Determination of Cell Proliferation by MTT Assay --- p.70 / Chapter 4.2.5 --- Determination of Intracellular Glutathione Depletion by Fluorescent Dye CMAC --- p.71 / Chapter 4.2.6 --- Determination of Cellular Reduced Glutathione Levels by DTNB-Coupled Glutathione Reductase Recycling Assay --- p.73 / Chapter 4.3 --- Results --- p.75 / Chapter 4.3.1 --- Effects of Baicalein on Intracellular GSH Levels and Cell Proliferation for Different Cell Lines --- p.75 / Chapter 4.3.2 --- Basal Intracellular Glutathione in Different Cell Lines --- p.81 / Chapter 4.4 --- Discussion --- p.84 / Chapter 4.4.1 --- Intracellular Glutathione Depletion and Cell Death Induction Effects of Baicalein on Different Cell Lines --- p.84 / Chapter 4.4.2 --- Relationship between Basal Glutathione Levels and Drug Susceptibilities --- p.85 / Chapter Chapter 5 --- Effects of Baicalein on Apoptosis and Caspase Pathways --- p.88 / Chapter 5.1 --- Introduction-Modes of Cell Death --- p.88 / Chapter 5.1.1 --- Necrosis --- p.88 / Chapter 5.1.2 --- Apoptosis --- p.89 / Chapter 5.2 --- Materials and Methods --- p.92 / Chapter 5.2.1 --- Chemicals --- p.92 / Chapter 5.2.2 --- Buffers --- p.92 / Chapter 5.2.3 --- Determination of Change of Mitochondrial Membrane Potential by JC-1 --- p.93 / Chapter 5.2.4 --- Determination of Apoptosis by Annexin V-Propidium Iodide Staining --- p.94 / Chapter 5.2.5 --- Determination of Cell Cycle Arrest by Propidium Iodide Staining --- p.95 / Chapter 5.2.6 --- "Determination of Caspase-3, -8 and -9 Activities by Fluorescent-Labeled Peptides" --- p.96 / Chapter 5.2.7 --- Determination of DNA Fragmentation --- p.97 / Chapter 5.2.8 --- Terminal Deoxynucleotidyl Transferase Mediated dUTP End Labeling (TUNEL) Assay --- p.99 / Chapter 5.2.9 --- Flow Cytometry --- p.101 / Chapter 5.3 --- Results --- p.102 / Chapter 5.3.1 --- Effects of Baicalein on Mitochondrial Membrane Potential by JC-1 Staining --- p.102 / Chapter 5.3.2 --- Effects of Baicalein on Apoptosis and Necrosis by Annexin V-Propidium Iodide Staining --- p.104 / Chapter 5.3.3 --- Effects of Baicalein on Cell Cycle Arrest by Propidium Iodide Staining --- p.108 / Chapter 5.3.4 --- "Effects of Baicalein on Caspase-3, -8 and -9 Activities" --- p.110 / Chapter 5.3.5 --- Effeets of Baiealein on DNA Fragmentation --- p.115 / Chapter 5.3.6 --- Effects of Baicalein on TUNEL Assay --- p.117 / Chapter 5.4 --- Discussion --- p.120 / Chapter Chapter 6 --- Pro-Oxidant Role of Baicalein on Reactive Oxygen Species Generation --- p.122 / Chapter 6.1 --- Introduction --- p.122 / Chapter 6.2 --- Materials and Methods --- p.122 / Chapter 6.2.1 --- Chemicals --- p.122 / Chapter 6.2.2 --- Determination of Cellular Reactive Oxygen Species Generation by Fluorescent Dye cDCFDA --- p.123 / Chapter 6.2.3 --- Determination of Mitochondrial Reactive Oxygen Species Generation by Fluorescent Dye Rhl23 --- p.124 / Chapter 6.3 --- Results --- p.125 / Chapter 6.3.1 --- Effects of Baicalein on Cellular ROS Generation by Fluorescent Dye cDCFDA --- p.125 / Chapter 6.3.2 --- Effects of Baicalein on Mitochondrial ROS Generation by Fluorescent Dye Rhl23 --- p.129 / Chapter 6.4 --- Discussion --- p.132 / Chapter Chapter 7 --- The Anticancer Mechanistic Study of Baicalein --- p.133 / Chapter 7.1 --- Introduction --- p.133 / Chapter 7.2 --- Materials and Methods --- p.134 / Chapter 7.2.1 --- Chemicals --- p.134 / Chapter 7.2.2 --- Reversibility of Baicalein-Induced GSH Depletion and Cell Death by Different Antioxidant Treatments --- p.134 / Chapter 7.2.3 --- Reversibility of Baicalein-Induced Cellular ROS Generation --- p.136 / Chapter 7.2.4 --- Reversibility of Baicalein-Induced Apoptosis by Co-Treatment of Different Antioxidants and Caspase Inhibitors --- p.137 / Chapter 7.2.5 --- "Reversibility of Baicalein-Induced Caspase-3, -8 and -9 Activation by Co-Treatment of Different Antioxidants" --- p.138 / Chapter 7.3 --- Results --- p.139 / Chapter 7.3.1 --- Reversibility of Baicalein-Induced GSH Depletion and Cell Death by Different Antioxidant Treatments --- p.139 / Chapter 7.3.1.1 --- Pre-treatments --- p.139 / Chapter 7.3.1.2 --- Co-treatments --- p.141 / Chapter 7.3.1.3 --- Post-treatments --- p.144 / Chapter 7.3.2 --- Reversibility of Baicalein-Induced Cellular ROS Generation by Co-Treatment of Different Antioxidants --- p.147 / Chapter 7.3.3 --- Reversibility of Baicalein-Induced Apoptosis by Co-Treatment of Different Antioxidants and Caspase Inhibitors --- p.152 / Chapter 7.3.4 --- Reversibility of Baicalein-Induced Caspase-3 Activation by Co-Treatment of Different Antioxidants --- p.156 / Chapter 7.3.5 --- Reversibility of Baicalein-Induced Caspase-8 and -9 Activation by Co-Treatment of Different Antioxidants --- p.160 / Chapter 7.4 --- Discussion --- p.164 / Chapter 7.4.1 --- Reversibility of Baicalein-Induced GSH Depletion and Cell Death --- p.164 / Chapter 7.4.2 --- "Reversibility of Baicalein-Induced ROS Generation," --- p.167 / Chapter 7.5 --- Concluding Remarks --- p.168 / Chapter Chapter 8 --- General Discussion --- p.169 / Chapter 8.1 --- Drug Delivery to Brain --- p.169 / Chapter 8.2 --- Protective Roles of Baicalein on Brain Cells --- p.170 / Chapter 8.2.1 --- Actions Against Oxidative Stress --- p.170 / Chapter 8.2.2 --- Actions Against Other Neurotoxic Damages --- p.171 / Chapter 8.2.3 --- Actions Against Neuronal Diseases --- p.172 / Chapter 8.3 --- Anticancer Roles of Baicalein on Astrocytoma --- p.173 / Chapter 8.4 --- Implications on the Dual Roles of Baicalein: Antioxidant and Pro-oxidant --- p.175 / Chapter 8.5 --- Future Perspectives --- p.175 / Chapter 8.5.1 --- Effects of Baicalein on Antioxidant System --- p.175 / Chapter 8.5.2 --- Effects of Baicalein on GSH Synthesis --- p.176 / Chapter 8.5.3 --- In Vivo Studies on Cytotoxic Effects of Baicalein --- p.177 / Chapter 8.5.4 --- In Vivo Studies on Anti-Tumor Effects and In Vitro Studies on Anti-Metastasis Effects of Baicalein --- p.178 / Reference List --- p.181

Li, Wing Yan Kate. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 130-154). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / Abstract (Chinese Version) --- p.vi / Table of Contents --- p.viii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction / Chapter 1.1. --- Overview of cancer --- p.1 / Chapter 1.2. --- Apoptosis and cancer --- p.4 / Chapter 1.3. --- Roles and regulation of caspase-dependent apoptosis --- p.7 / Chapter 1.3.1. --- Extrinsic death receptor pathway --- p.8 / Chapter i. --- TNFR1 and TNFa --- p.13 / Chapter ii. --- CD95/Fas and CD95 Ligand/FasL --- p.14 / Chapter iii. --- "TRAIL-R1(DR4), TRAIL-R2 (DR5) and TRAIL" --- p.14 / Chapter 1.3.2. --- Intrinsic mitochondrial pathway --- p.16 / Chapter i. --- Bcl-2 family of proteins --- p.17 / Chapter ii. --- Reactive Oxygen Species (ROS) --- p.19 / Chapter 1.4. --- Phytochemicals from Traditional Chinese Medicine (TCM) as a source of new therapeutics --- p.22 / Chapter 1.5. --- Biological effects of baicalein --- p.25 / Chapter 1.5.1 --- Roles of baicalein as a lipoxygenase inhibitor --- p.28 / Chapter 1.5.2 --- Dual roles of baicalein as an antioxidant and prooxidant --- p.28 / Chapter 1.5.3 --- "Roles of baicalein as an anti-carcinogenic, anti-proliferative and anti-metastatic agent" --- p.29 / Chapter 1.6. --- Aims of current study --- p.30 / Chapter Chapter 2 --- Effects of Baicalein on Growth and Survival of Human Cancer Cells / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and Methods / Chapter 2.2.1 --- Cell culture --- p.35 / Chapter 2.2.2 --- Measurement of growth and survival of various cell lines --- p.36 / Chapter 2.2.3 --- Statistical analysis --- p.37 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Baicalein retards the growth and survival of human melanoma A375 and colorectal carcinoma Caco-2 --- p.37 / Chapter 2.3.2 --- Baicalein reduces the growth and survival of melanoma A375 but not in normal skin fibroblast Hs68 cells --- p.40 / Chapter 2.4 --- Discussion --- p.42 / Chapter Chapter 3 --- Effects of Baicalein on Cell Cycle and the Apoptosis in Human Melanoma A375 Cells / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Determination of cell cycle changes and quantification of apoptosis --- p.51 / Chapter 3.2.2 --- Immunoblotting --- p.52 / Chapter 3.2.3 --- Inhibition of caspase-8 by caspase-8 inhibitor --- p.54 / Chapter 3.2.4 --- Fluorometric measurement of caspase-3 activity --- p.54 / Chapter 3.2.5 --- Statistical analysis --- p.55 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Baicalein induces S-phase arrest in cell cycle and triggers apoptosis --- p.55 / Chapter 3.3.2 --- Baicalein induces proteolytic inactivation of PARP and activation of caspases --- p.59 / Chapter 3.3.3 --- Caspase-8 is the major initiator caspase eliciting the baicalein-induced apoptosis --- p.62 / Chapter 3.4 --- Discussion --- p.67 / Chapter Chapter 4 --- Effects of Baicalein on the Extrinsic Apoptotic Pathways in Human Melanoma A375 Cells / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1 --- Immunoblotting --- p.75 / Chapter 4.2.2 --- Determination of sub-lethal dose of exogenous TRAIL --- p.76 / Chapter 4.2.3 --- Determination of the combinatory effect of exogenous TRAIL and baicalein --- p.76 / Chapter 4.2.4 --- Statistical analysis --- p.77 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Baicalein upregulates the expressions of death receptor 4 (DR4) and death receptor 5 (DR5) --- p.77 / Chapter 4.3.2 --- Baicalein sensitizes the melanoma cells to sub-lethal dose of exogenous TRAIL --- p.80 / Chapter 4.4 --- Discussion --- p.84 / Chapter Chapter 5 --- Effects of Baicalein on the Extrinsic Apoptotic Pathways in Human Melanoma A375 Cells Cancer Cells / Chapter 5.1 --- Introduction --- p.88 / Chapter 5.2 --- Materials and Methods / Chapter 5.2.1 --- Analysis of mitochondrial membrane potential --- p.94 / Chapter 5.2.2 --- Fractionation of cell lysates into cytosolic and mitochondrial fractions for immunoblotting --- p.95 / Chapter 5.2.3 --- Immunoblotting --- p.95 / Chapter 5.2.4 --- Determination of cellular reactive oxygen species (ROS) production --- p.96 / Chapter 5.2.5 --- Verification of ROS generation via the addition of Trolox´ёØ --- p.96 / Chapter 5.2.6 --- Statistical analysis --- p.97 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Baicalein induces mitochondrial membrane depolarization --- p.97 / Chapter 5.3.2 --- Cytochrome c is released in the baicalein-induced mitochondrial membrane depolarization --- p.100 / Chapter 5.3.3 --- Baicalein does not elicit the intrinsic apoptotic pathway via modulation of some better-characterized Bcl-2 family proteins in A375 cells --- p.102 / Chapter 5.3.4 --- Baicalein induces ROS production --- p.105 / Chapter 5.3.5 --- Baicalein induces mitochondrial permeabilization via ROS-mediated mechanisms --- p.108 / Chapter 5.4 --- Discussion --- p.112 / Chapter Chapter 6 --- General Discussion --- p.119 / References --- p.130

Conclusion. The present results indicate that, baicalein with optimal dosage of 30 muM suppressed collagen deposition in AngII stimulated SHR CF cultures. In animal model of hypertension, high dose of baicalein feeding for 12 week showed optimal antifibrotic effect in hypertensive hearts. (Abstract shortened by UMI.) / For in-vivo study, comparing to control group, HW/BW (x1000) of SHR was significantly reduced in 12 weeks-high dose baicalein and (-0.78+/-0.23, p=0.014) 12 weeks-Valsartan group (-0.71+/-0.22, p=0.021), however, no significant change was observed in the LW/BW ratio. / In Blood pressure control, no effects on attenuation of SBP were observed after 4 weeks and 12 weeks daily administration of baicalein, only 12 weeks feeding of Valsartan significantly down-regulated the systolic blood pressure by -19.25+/-10.09 mmHg, p=0.049. / In the in-vivo study, SHR was used as a model of genetic hypertension. The objectives were: firstly, to determine the efficacy of baicalein in the prevention of myocardial fibrosis (interstitial fibrosis) in SHR, & compared with WKY rats as normal controls. Secondly, to determine if over-expression of pro-collagen I (and III, if any) gene in the ventricles could be normalized by baicalein. Thirdly, to determine if left ventricular hypertrophy in SHR is improved by baicalein. Furthermore, to determine if blood pressure and blood biochemistry parameters (plasma level of brain natriuretic peptides (BNP), and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) level could be alternated by baicalein. Besides, to determine the body weight (BW), heart weight to body ratio (HW/BW), liver weight to body weight ratio (LW/BW), serum AST and ALT level could be alternated by baicalein. Finally to evaluate by echocardiography if there are changes of ivss and ivsd in SHR after administration of baicalein. / Keywords. baicalein, wogonin, collagen, cardiac fibrosis, hypertension / Objectives. In the in-vitro study, cardiac fibroblast culture was prepared from neonatal SHR and WKY rats. The objectives were multi-fold: firstly, to determine over-expression of pro-collagen I mRNA (and III, if any) in cardiac fibroblasts cultures could be normalized by baicalein and wogonin after AngII activation. Secondly, to evaluate the efficacy of baicalein and wogonin on the suppression of total collagen protein production in cardiac fibroblasts cultures after AngII activation. Thirdly, to evaluate the mechanism (in protein level) of baicalein and wogonin on regulating collagen deposition in cardiac fibroblasts after AngII activation. Furthermore, to determine if there were any effects on cytotoxicity and membrane integrity of baicalein and wogonin towards cardiac fibroblasts cultures. Finally, to determine the optimal concentration of baicalein and wogonin for the above actions in-vitro. / Results. For in-vitro study, incubation of AngII resulted in significant up-regulation of COL-I and COL-III mRNA and total collagen protein production. Addition of either baicalein or wogonin significantly suppressed the mRNA synthesis and total collagen protein in CF with an optimal dosage of 30 muM. No effects on viability and membrane integrity were observed on baicalein and wogonin towards cardiac fibroblasts cultures. / Kong, Kam Chuen Ebenezer. / Advisers: Cheuk-Man Yu; Gabriel W. K. Yip. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0242. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 176-204). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Baicalein is known to be a potent antifungal agent and induces programmed cell death in Candida albicans. In the present study, we found that baicalein also inhibited the growth of C. krusei isolates. The minimal inhibitory concentrations of baicalein against eight C. krusei isolates were 1.35--2.70 microg/ml. One-hour exposure to baicalein elicited a consistent and moderate post-antifungal effect on the C. krusei isolates. Further flow cytometric study demonstrated a depolarization of mitochondrial membrane potential. However, both the levels of reactive oxygen species and DNA fragmentation were not significantly changed after baicalein treatment in C. krusei. It can be concluded that the antifungal activity of baicalein was mitochondria-dependent in both C. krusei and C. albicans, but the antifungal mechanism was different. Reactive oxygen species may not play a direct role and baicalein does not initiate programmed cell death or apoptosis in C. krusei. The structure-activity relationship study showed that the three hydroxyl groups in baicalein were essential for its antifungal potency. / Candidiasis has become a serious infection with very high mortality and morbidity in the world if not providing effective treatments. However, due to clinical limitation and resistance of the current antifungal agents, there is an urgent need to search for novel antifungals. In this study, after screening a compound library (n=400) for antifungal activity, three members (metergoline, purpurin and baicalein) were chosen for further study. Their antifungal characteristics and the antifungal mechanisms were investigated. / Metergoline, a serotonin receptor antagonist, was found to have potent antifungal activity against the intrinsically fluconazole-resistant human fungal pathogen Candida krusei. The minimal inhibitory concentration and minimal fungicidal concentration of metergoline against C. krusei were 4 microg/ml and 8 microg/ml respectively. Metergoline induced post-antifungal effect. Significant synergism was found in combination of metergoline with amphotericin B by a checkerboard assay, which may be due to the perturbation of cell permeability and increase in the intracellular accumulation of antifungal agents. Metergoline also inhibited extracellular phospholipase production in C. krusei. To gain insights into the mechanisms, intracellular changes that accompany apoptosis were examined by flow cytometry and spectrophotometry. The results showed an increase in the level of reactive oxygen species, depolarization of mitochondrial membrane potential, phosphatidylserine externalization, and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling in the metergoline-treated C. krusei . Taken together, we conclude that metergoline may promote apoptosis in C. krusei through reactive oxygen species production and perturbation in mitochondrial homeostasis, implying its antifungal potential to treat candidiasis. / The antifungal activity of purpurin, a natural red anthraquinone pigment in madder root (Rubia tinctorum L.), was evaluated against Candida isolates by a broth microdilution assay. The minimal inhibitory concentrations of purpurin against Candida species isolates were 1.28--5.12 microg/ml. Mechanistic studies indicated that purpurin inhibited energy-dependent efflux pumps of Candida isolates. Furthermore, purpurin demonstrated a depolarization of mitochondrial membrane potential, suggesting a possible linkage of the antifungal mechanism of purpurin to Candida apoptosis. / Kang, Kai. / Adviser: Fong Wing Ping. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 98-123). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.