A study of the biological activities of cordyceps militaris and the action mechanisms of the anti-tumor effect of cordycepin.

by Lee Kin Ming.Thesis (M.Phil.)--Chinese University of Hong Kong, 2003.Includes bibliographical references (leaves 214-225).Abstracts in English and Chinese.ACKNOWLEDGEMENTS --- p.iABBREVIATIONS --- p.iiABSTRACT --- p.viiABSTRACT IN CHINESE --- p.ixLIST OF FIGURES --- p.xiLIST OF TABLES --- p.xvCONTENTS --- p.xviChapter CHAPTER 1: --- INTRODUCTION --- p.1Chapter 1.1 --- Cordyceps --- p.2Chapter 1.1.1 --- Pharmacological Functions of Cordyceps --- p.5Chapter 1.1.1.1 --- Anti-tumor Activities --- p.5Chapter 1.1.1.2 --- Immunomodulatory Activities --- p.7Chapter 1.1.1.3 --- Hepatic Functions --- p.9Chapter 1.1.1.4 --- Cardiovascular Functions --- p.10Chapter 1.1.1.5 --- Renal Functions --- p.10Chapter 1.2 --- Biological Activities of Cordycepin --- p.12Chapter 1.2.1 --- Inhibition of RNA Synthesis --- p.12Chapter 1.2.2 --- Disruption of Microtubule Network --- p.12Chapter 1.2.3 --- Inhibition of Nucleic Acid Methylation --- p.13Chapter 1.2.4 --- Enhancement of Cell Differentiation --- p.13Chapter 1.2.5 --- Anti-tumor Activity --- p.13Chapter 1.2.6 --- Anti-fungal Activity --- p.14Chapter 1.3 --- Hepatocellular Carcinoma --- p.16Chapter 1.3.1 --- Incidence and Risk Factor of Hepatocellular Carcinoma --- p.16Chapter 1.3.2 --- Treatment of Hepatocellular Carcinoma --- p.16Chapter 1.3.2.1 --- Hepatic Resection --- p.16Chapter 1.3.2.2 --- Liver Transplantation --- p.17Chapter 1.3.2.3 --- Non-surgical Therapeutic Modalities for Hepatocellular Carcinoma --- p.17Chapter 1.3.3 --- Human Hepatocellular Carcinoma Cell Lines --- p.20Chapter 1.3.3.1 --- Human Hepatocellular Carcinoma Cell Line HepG2 --- p.20Chapter 1.3.3.2 --- Multidrug Resistant Human Hepatocellular Carcinoma Cell Line R-HepG2 --- p.20Chapter 1.4 --- Multidrug Resistance of Tumor Cells --- p.22Chapter 1.4.1 --- Multidrug Resistance Mediated by P-Glycoprotein --- p.22Chapter 1.4.1.1 --- Location and Structure of P-Glycoprotein --- p.22Chapter 1.4.1.2 --- Substrates of P-Glycoprotein --- p.23Chapter 1.4.1.3 --- Mechanism of Action of P-Glycoprotein --- p.23Chapter 1.4.2 --- Reversal of Multidrug Resistance by Chemosensitizers --- p.24Chapter 1.5 --- LeukemiaChapter 1.5.1 --- Acute Myeloid Leukemia --- p.28Chapter 1.5.2 --- Acute Promyelocytic Leukemia and Treatment --- p.28Chapter 1.5.3 --- Human Promyelocytic Leukemia Cell Lines --- p.30Chapter 1.5.3.1 --- HL-60 --- p.30Chapter 1.5.3.2 --- NB-4 --- p.30Chapter 1.6 --- Objectives of Study --- p.33Chapter 1.6.1 --- Study of Biological Activities of Cordyceps militaris --- p.33Chapter 1.6.2 --- Study of Anti-tumor Activity of Cordycepin --- p.33Chapter CHAPTER 2: --- MATERIALS AND METHODS --- p.34Chapter 2.1 --- Materials --- p.35Chapter 2.1.1 --- Animal --- p.35Chapter 2.1.2 --- Cell Culture --- p.35Chapter 2.1.2.1 --- Cell Lines --- p.35Chapter 2.1.2.2 --- Cell Culture Media --- p.37Chapter 2.1.2.3 --- Buffers and other Reagents --- p.38Chapter 2.1.3 --- Reagents and Buffers for Different Assays --- p.40Chapter 2.1.3.1 --- Reagents and Buffers for Flow Cytometry --- p.40Chapter 2.1.3.2 --- Reagents and Buffers for DNA Fragmentation Assay --- p.40Chapter 2.1.3.3 --- Reagents and Buffers for Western Blot Analysis --- p.42Chapter 2.1.3.4 --- Reagents and Buffers for Caspases Activities --- p.46Chapter 2.1.3.5 --- Reagents and Buffers for Cell Surface Marker (CD3，CD4 and CD8) Staining --- p.48Chapter 2.1.3.6 --- Reagents and Buffers for Cytokine Determination --- p.49Chapter 2.2 --- Methods --- p.50Chapter 2.2.1 --- Preparation of Water Extract of Cordyceps militaris --- p.50Chapter 2.2.2 --- MTT Assay --- p.50Chapter 2.2.3 --- In Vivo Anti-tumor Study --- p.51Chapter 2.2.4 --- Preparation of Splenic Lymphocytes --- p.51Chapter 2.2.5 --- Lymphoproliferation Test --- p.51Chapter 2.2.6 --- "Cell Surface Marker (CD3, CD4 and CD8) Staining" --- p.52Chapter 2.2.7 --- Measurement of Cytokine Production by ELISA --- p.53Chapter 2.2.8 --- In Vivo Study of the Toxicity of WECM --- p.54Chapter 2.2.9 --- Cell Cycle Analysis --- p.55Chapter 2.2.10 --- DNA Fragmentation Assay --- p.56Chapter 2.2.11 --- Cell Morphology Study --- p.57Chapter 2.2.12 --- Detection of Apoptotic Cells with Annexin V-FITC/PI --- p.57Chapter 2.2.13 --- Detection of Mitochondrial Membrane Potential by JC-1 Fluorescent Dye --- p.58Chapter 2.2.14 --- Simultaneous Detection of Mitochondrial Membrane Potential and Intracellular Hydrogen Peroxide --- p.58Chapter 2.2.15 --- Western Blot Analysis --- p.59Chapter 2.2.15.1 --- Total Protein Extraction --- p.59Chapter 2.2.15.2 --- Determination of Protein Amount --- p.59Chapter 2.2.15.3 --- SDS Polyacrylamide Gel Electrophoresis --- p.60Chapter 2.2.15.4 --- Electroblotting of Protein --- p.61Chapter 2.2.15.5 --- Probing of Proteins with Antibodies --- p.61Chapter 2.2.15.6 --- Enhanced Chemiluminescence (ECL) Assay --- p.64Chapter 2.2.15.7 --- Extraction of Cytosolic Protein --- p.64Chapter 2.2.16 --- Determination of Caspases Enzymatic Activity --- p.65Chapter 2.2.16.1 --- Extraction of Proteins --- p.65Chapter 2.2.16.2 --- Determination of Caspase-3 Activity --- p.65Chapter 2.2.16.3 --- Determination of Caspase-8 Activity --- p.66Chapter 2.2.16.4 --- Determination of Caspase-9 Activity --- p.67Chapter 2.2.17 --- Hemolysis Assay --- p.69Chapter 2.2.18 --- Measurement of Intracellular Doxorubicin Accumulation --- p.69Chapter CHAPTER 3: --- ANTI-TUMOR AND IMMUNO- MODULATORY EFFECTS OF cordyceps militaris --- p.71Chapter 3.1 --- In Vitro Anti-tumor Study of Water Extract of Cordyceps militaris (WECM) --- p.72Chapter 3.2 --- In Vitro Study of Immunomodulatory Effect of WECM --- p.78Chapter 3.3 --- In Vivo Anti-tumor Study of WECM --- p.80Chapter 3.4 --- Anti-tumor Effect of WECM Mediated by Stimulating T-cell Proliferation --- p.83Chapter 3.5 --- Toxicity Studies of WECM --- p.92Chapter CHAPTER 4: --- ANTI-PROLIFERATIVE EFFECT OF THE ACTIVE COMPONENTS OF cordyceps militaris --- p.97Chapter 4.1 --- "Anti-proliferative Study of D-mannitol, Adenosine and Cordycepin (3'deoxyadenosine)" --- p.98Chapter 4.2 --- Anti-proliferative Study of Doxorubicin --- p.105Chapter 4.3 --- Accumulation of Doxorubicin in HepG2 and R-HepG2 Cells --- p.109Chapter 4.4 --- Cytotoxicity Study of Cordycepin and Doxorubicin on Normal Liver Cells --- p.114Chapter 4.5 --- Hemolytic Study of Cordycepin --- p.116Chapter CHAPTER 5: --- MECHANISTIC STUDY OF CORDYCEPIN IN THE INDUCTION OF APOPTOSIS IN LEUKEMIA CELLS --- p.118Chapter 5.1 --- Cell Cycle Analysis of Leukemia Cells --- p.119Chapter 5.2 --- Hallmarks of Apoptosis --- p.123Chapter 5.2.1 --- Induction of Phosphatidylserine Externalization in Leukemia Cells by Cordycepin --- p.123Chapter 5.2.2 --- Induction of DNA Fragmentation in Leukemia Cells by Cordycepin --- p.127Chapter 5.2.3 --- Morphological Changes in Leukemia Cells Induced by Cordycepin --- p.130Chapter 5.2.4 --- Caspase-3 Activation in Leukemia Cells Induced by Cordycepin --- p.133Chapter 5.3 --- Study of the Underlying Mechanisms of Cordycepin-induced Apoptosis in Leukemia Cells --- p.140Chapter 5.3.1 --- Induction of Mitochondrial Membrane Depolarization in Leukemia Cells --- p.140Chapter 5.3.2 --- Elevation of Intracellular Hydrogen Peroxide Level in Cordycepin-treated Leukemia Cells --- p.144Chapter 5.3.3 --- Induction of Cytochrome c Release from Mitochondria of Leukemia Cells --- p.148Chapter 5.3.4 --- Caspase-9 Activation in Leukemia Cells Induced by Cordycepin --- p.150Chapter 5.3.5 --- Involvement of Bcl-2 Family Members in Cordycepin-induced Apoptosis --- p.153Chapter 5.3.6 --- Involvement of Death Receptor Pathway in Cordycepin-induced Apoptosis in Leukemia Cells --- p.159Chapter CHAPTER 6: --- MECHANISTIC STUDY OF CORDYCEPIN IN THE INDUCTION OF CELL CYCLE ARREST IN HEPATOCELLULAR CARCINOMA CELLS --- p.164Chapter 6.1 --- Cell Cycle Analysis of Hepatocellular Carcinoma Cells --- p.165Chapter 6.2 --- Expression of Cell Cycle Regulatory Proteins in Cordycepin-treated Hepatocellular Carcinoma Cells --- p.170Chapter 6.3 --- Increased Expression of p21 in Cordycepin-treated Hepatocellular Carcinoma Cells --- p.176Chapter 6.4 --- Involvement of p53 in G2/M Phase Arrest of the Cell Cycle in Hepatocellular Carcinoma Cells --- p.178Chapter 6.5 --- Induction of Apoptosis in Cordycepin-treated R-HepG2 cells --- p.180Chapter CHAPTER 7: --- DISCUSSION --- p.185Chapter 7.1 --- In Vitro and In Vivo Studies in the Biological Activities of WECM --- p.186Chapter 7.2 --- Induction of Apoptosis in Leukemia Cells by Cordycepin --- p.192Chapter 7.3 --- Induction of Cell Cycle Arrest in Hepatocellular Carcinoma Cells by Cordycepin --- p.202Chapter CHAPTER 8: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.210REFERENCES --- p.21