Baicalein induces apoptosis in human astrocytoma cells via a pro-oxidant mechanism.

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