Compounds from wild mushrooms with antitumor potential

For thousands of years medicine and natural products have been closely linked through the use of traditional medicines and natural poisons. Mushrooms have an established history of use in traditional oriental medicine, where most medicinal mushroom preparations are regarded as a tonic, that is, they have beneficial health effects without known negative side-effects and can be moderately used on a regular basis without harm. Mushrooms comprise a vast and yet largely untapped source of powerful new pharmaceutical products. In particular, and most importantly for modern medicine, they represent an unlimited source of compounds which are modulators of tumour cell growth. Furthermore, they may have potential as functional foods and sources of novel molecules. We will review the compounds with antitumor potential identified so far in mushrooms, including low-molecular-weight (LMW, e.g. quinones, cerebrosides, isoflavones, catechols, amines, triacylglycerols, sesquiterpenes, steroids, organic germanium and selenium) and high-molecular-weight compounds (HMW, e.g. homo and heteroglucans, glycans, glycoproteins, glycopeptides, proteoglycans, proteins and RNA-protein complexes)

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

Antioxidants of Edible Mushrooms

Oxidative stress caused by an imbalanced metabolism and an excess of reactive oxygen species (ROS) lead to a range of health disorders in humans. Our endogenous antioxidant defense mechanisms and our dietary intake of antioxidants potentially regulate our oxidative homeostasis. Numerous synthetic antioxidants can effectively improve defense mechanisms, but because of their adverse toxic effects under certain conditions, preference is given to natural compounds. Consequently, the requirements for natural, alternative sources of antioxidant foods identified in edible mushrooms, as well as the mechanistic action involved in their antioxidant properties, have increased rapidly. Chemical composition and antioxidant potential of mushrooms have been intensively studied. Edible mushrooms might be used directly in enhancement of antioxidant defenses through dietary supplementation to reduce the level of oxidative stress. Wild or cultivated, they have been related to significant antioxidant properties due to their bioactive compounds, such as polyphenols, polysaccharides, vitamins, carotenoids and minerals. Antioxidant and health benefits, observed in edible mushrooms, seem an additional reason for their traditional use as a popular delicacy food. This review discusses the consumption of edible mushrooms as a powerful instrument in maintaining health, longevity and life quality

Fibrinolysis and Thrombolysis

This book familiarizes the reader with some recent trends in the theory and practice of thrombolysis. It covers the field of fibrinolysis from the standpoint of basic scientists and clinicians and delivers the state-of-the-art information on the biochemistry and pharmacology of fibrinolysis, as well as related novel methodological and diagnostic tools in the field. An introductory chapter summarizes the basic molecular mechanisms in fibrinolysis (plasminogen, its endogenous activators and their inhibitors, plasmin and its inhibitors). Recent developments in our understanding of fibrin formation are described in the context of its impact on fibrinolysis. The discussion of neutrophil extracellular traps in the modulation of fibrin assembly and the consequences regarding plasminogen activation and plasmin action addresses a novel aspect of fibrinolysis

The anti-tumor activities of steroid saponin HK18 on human hepatocellular carcinoma cell line HepG2 and multidrug resistant human hepatocellular carcinoma cell line R-HepG2 and its action mechanisms.

by Cheung Yuen-Nei.Thesis (M.Phil.)--Chinese University of Hong Kong, 2002.Includes bibliographical references (leaves 194-208).Abstracts in English and Chinese.Acknowledgement --- p.iAbstract --- p.ii摘要 --- p.ivContents --- p.viList of Figures --- p.xiiList of Tables --- p.xvAbbreviations --- p.xviChapter Chapter 1 --- Introduction --- p.1Chapter 1 --- Introduction --- p.2Chapter 1.1 --- Characteristic of Saponins --- p.3Chapter 1.1.1 --- Occurrence of Saponins --- p.3Chapter 1.1.2 --- General Properties of Saponins --- p.3Chapter 1.1.2.1 --- Emulsifying Agents --- p.3Chapter 1.2.2.2 --- Forming Complex with Cholesterol --- p.4Chapter 1.1.2.3 --- Hemolytic Property --- p.4Chapter 1.1.3 --- Structure of Saponins --- p.5Chapter 1.1.3.1 --- Categories of Saponins --- p.5Chapter 1.1.3.1.1 --- Triterpene Saponins --- p.5Chapter 1.1.3.1.2 --- Steroid Saponins --- p.5Chapter 1.1.3.2 --- Monodesmosidic and Bidesmosidic Saponins --- p.7Chapter 1.1.4 --- Biological and Pharmacological Properties of Saponins --- p.9Chapter 1.1.4.1 --- Anti-microbial Activity --- p.9Chapter 1.1.4.1.1 --- Anti-fungal Activities --- p.9Chapter 1.1.4.1.2 --- Anti-bacterial Activities --- p.10Chapter 1.1.4.1.3 --- Anti-viral Activities --- p.10Chapter 1.1.4.2 --- Insecticidal Activity --- p.10Chapter 1.1.4.3 --- Molluscicidal Activity --- p.10Chapter 1.1.4.4 --- Hypocholesterolemic Activity --- p.11Chapter 1.1.4.5 --- Anti-ulcer Activity --- p.11Chapter 1.1.4.6 --- Contraceptive Activity --- p.12Chapter 1.1.4.7 --- Immunomodulatory Activities --- p.12Chapter 1.1.4.7.1 --- Direct Immunostimulation --- p.12Chapter 1.1.4.7.2 --- Acting as Immuno-adjuvants --- p.13Chapter 1.1.4.8 --- Anti-tumor Activity --- p.14Chapter 1.1.4.8.1 --- Anti-carcinogenesis --- p.15Chapter 1.1.4.8.2 --- Suppression of Tumor Growth --- p.16Chapter 1.1.5 --- Anti-tumor Activity of Steroid Saponins --- p.18Chapter 1.1.5.1 --- Diosgenin Steroid Saponin --- p.18Chapter 1.1.5.2 --- Hong Kong Compounds --- p.18Chapter 1.1.5.3 --- Hong Kong18 --- p.21Chapter 1.2 --- Human Hepatocellular Carcinoma (HCC) --- p.24Chapter 1.2.1 --- The Incidence of Liver Cancer --- p.24Chapter 1.2.2 --- Classification of Liver Cancer --- p.24Chapter 1.2.3 --- Human Hepatocellular Carcinoma Cell Lines --- p.25Chapter 1.2.3.1 --- Human Hepatocellular Carcinoma Cell Line HepG2 --- p.25Chapter 1.2.3.2 --- Multidrug Resistant Human Hepatocellular Carcinoma Cell Line R-HepG2 --- p.27Chapter 1.2.3.2.1 --- Mechanisms of Multidrug Resistance --- p.28Chapter 1.2.3.2.2 --- Structure and Characteristics of P-glycoprotein --- p.29Chapter 1.2.3.2.3 --- Methods in Dealing with P-glycoprotein Over-expressed MDR Cells --- p.31Chapter 1.3 --- Objectives of the Project --- p.32Chapter 1.3.1 --- Study of the Anti-tumor Activities of Hong Kong 18 on Human Hepatocellular Carcinoma Cell Line HepG2 and Unravel the Underlying Mechanisms --- p.32Chapter 1.3.2 --- Study of the Anti-tumor Activities of Hong Kong 18on Multidrug Resistant Human Hepatocellular Carcinoma Cell Line R-HepG2 and Unravel the Underlying Mechanisms --- p.32Chapter Chapter 2 --- Materials and Methods --- p.33Chapter 2.1 --- Materials --- p.34Chapter 2.1.1 --- Cell Culture --- p.34Chapter 2.1.1.1 --- Cell Lines --- p.34Chapter 2.1.1.2 --- Culture Media --- p.35Chapter 2.1.2 --- Reagents and Buffers --- p.36Chapter 2.1.2.1 --- Phosphate Buffered Saline (PBS) --- p.36Chapter 2.1.2.2 --- Reagents and Buffers for DNA Fragmentation --- p.36Chapter 2.1.2.3 --- Reagents and Buffers for Western Analysis --- p.37Chapter 2.1.2.4 --- Reagents and Buffer for Caspases Activities --- p.39Chapter 2.1.2.5 --- Fluorescent Dyes used for Flow Cytometry --- p.39Chapter 2.1.3 --- Chemicals --- p.39Chapter 2.2 --- Methods --- p.46Chapter 2.2.1 --- MTT Assay --- p.46Chapter 2.2.2 --- Determination of Cell Viability --- p.46Chapter 2.2.3 --- Purification of Macrophages from balb/c Mice --- p.47Chapter 2.2.4 --- Hemolysis Assay --- p.47Chapter 2.2.5 --- In vivo Studies of the Toxicity of HK18 --- p.48Chapter 2.2.6 --- DNA Fragmentation Assay --- p.50Chapter 2.2.7 --- Detection of Apoptotic and Necrotic / Late Apoptotic Cells Death by Flow Cytometry with Annexin V-FITC / PI --- p.51Chapter 2.2.8 --- Detection of Mitochondrial Membrane Potential by JC-1 Fluorescent Dye --- p.52Chapter 2.2.9 --- Detection of Intracellular Ca Level by Flow Cytometry with Fluo-3 Fluorescent Dye --- p.52Chapter 2.2.10 --- Detection of Intracellular Hydrogen Peroxide Level by Flow Cytometry with DCF Fluorescence Dye --- p.53Chapter 2.2.11 --- Simultaneous Detection of Mitochondrial Membrane Potential and Intracellular Ca2+ or Mitochondrial Membrane Potential and Intracellular Hydrogen Peroxide --- p.54Chapter 2.2.12 --- Western Analysis --- p.55Chapter 2.2.12.1 --- Total Protein Extraction --- p.55Chapter 2.2.12.2 --- Extraction of Cytosolic Proteins --- p.59Chapter 2.2.13 --- Determination of Caspases Enzymatic Activity --- p.63Chapter 2.2.14 --- Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) --- p.67Chapter 2.2.14.1 --- RNA Extraction by TRIzol Reagent --- p.67Chapter 2.2.14.2 --- Reverse Transcription --- p.68Chapter 2.2.14.3 --- Polymerase Chain Reaction --- p.68Chapter 2.3 --- Statistic Analysis --- p.71Chapter Chapter 3 --- Cytotoxicity of HK18 --- p.72Chapter 3.1 --- Cytotoxicity of HK18 on HepG2 Cells --- p.73Chapter 3.1.1 --- Study of the Cytotoxic Activity of HK18 on HepG2 Cells by MTT Assay --- p.73Chapter 3.1.2 --- Study of the Cytotoxic Activity of HK18 on HepG2 Cells by Tryphan Blue Exclusion Assay --- p.76Chapter 3.2 --- Cytotoxicity of HK18 on R-HepG2 Cells --- p.78Chapter 3.2.1 --- Study of the Cytotoxic Activity of HK18 on R-HepG2 Cells by MTT Assay --- p.78Chapter 3.2.2 --- Study of the Cytotoxic Activity of HK18 on R-HepG2 Cells by Tryphan Blue Exclusion Assay --- p.81Chapter 3.3 --- Cytotoxicity of HK18 on Macrophages --- p.83Chapter 3.4 --- Hemolytic Activity of HK18 --- p.85Chapter 3.5 --- In vivo Study of the Toxicity of HK18 --- p.87Chapter Chapter 4 --- Mechanistic Study of HK18 on HepG2 Cells --- p.90Chapter 4.1 --- Hallmarks of Apoptosis Induced by HK18 on HepG2 Cells --- p.91Chapter 4.1.1 --- Induction of Phosphatidylserine Externalization by HK18 on HepG2 Cells --- p.91Chapter 4.1.2 --- Induction of DNA Fragmentation by HK18 of HepG2 Cells --- p.97Chapter 4.2 --- Study of the Underlying Mechanisms of HK18 Induced Apoptosis in HepG2 Cells --- p.99Chapter 4.2.1 --- The Role of Mitochondria in HK18 Induced Apoptosis of HepG2 Cells --- p.99Chapter 4.2.1.1 --- HK18 Induced Mitochondrial Membrane Depolarization in HepG2 Cells --- p.101Chapter 4.2.1.2 --- Addition of Bongkrekic Acid Reduced HK18 Cytotoxicity on HepG2 Cells --- p.105Chapter 4.2.1.3 --- Elevation of Intracellular Hydrogen Peroxide Level in HK18 Treated HepG2 Cells --- p.108Chapter 4.2.1.4 --- Elevation of Intracellular Ca2+ Level in HK18 Treated HepG2 Cells --- p.114Chapter 4.2.1.5 --- HK18 Induced Cytochrome c and AIF Released from Mitochondria of HepG2 Cells --- p.120Chapter 4.3 --- Downstream Biochemical Changes Induced by HK18 on HepG2 Cells --- p.123Chapter 4.3.1 --- Activation of Caspase 3 of HepG2 Cells by HK18 as Demonstrated by Western Blot --- p.123Chapter 4.3.2 --- Induction of Caspases Activities of HepG2 Cells by HK18 as Demonstrated by Enzymatic Activity Assays --- p.125Chapter 4.4 --- Down-regulation of Anti-apoptotic Bcl-2 Family Members of HepG2 Cells by HK18 --- p.129Chapter Chapter 5 --- Mechanistic Study of HK18 on R-HepG2 Cells --- p.133Chapter 5.1 --- Hallmarks of Apoptosis Induced by HK18 on R-HepG2 Cells --- p.134Chapter 5.1.1 --- Induction of Phosphatidylserine Externalization by HK18 on R-HepG2 Cells --- p.134Chapter 5.1.2 --- Induction of DNA Fragmentation by HK18 of R-HepG2 Cells --- p.137Chapter 5.2 --- Study of the Underlying Mechanisms of HK18 Induced Apoptosis in R-HepG2 Cells --- p.139Chapter 5.2.1 --- The Role of Mitochondria in HK18 Induced Apoptosis of R-HepG2 Cells --- p.139Chapter 5.2.1.1 --- HK18 Induced Mitochondrial Membrane Depolarization in R-HepG2 Cells --- p.139Chapter 5.2.1.2 --- Addition of Bongkrekic Acid Reduced HK18 Cytotoxicity on R-HepG2 Cells --- p.142Chapter 5.2.1.3 --- Elevation of Intracellular Hydrogen Peroxide Level in HK18 Treated R-HepG2 Cells --- p.144Chapter 5.2.1.4 --- Elevation of Intracellular Ca2+ Level in HK18 Treated R-HepG2 Cells --- p.146Chapter 5.3 --- Downstream Biochemical Changes Induced by HK18 on R-HepG2 Cells --- p.148Chapter 5.3.1 --- Activation of Caspase 3 of R-HepG2 Cells by HK18 as Demonstrated by Western Blot --- p.148Chapter 5.3.2 --- Induction of Caspases Activation of R-HepG2 Cells by HK18 as Demonstrated by Enzymatic Activity Assays --- p.150Chapter 5.4 --- Down-regulation of the Anti-apoptotic Bcl-2 Protein of R-HepG2 Cells by HK18 --- p.154Chapter 5.5 --- HK18 was Not a Substrate of P-glycoprotein Contents --- p.156Chapter Chapter 6 --- Discussion --- p.158Chapter 6.1 --- Cytotoxicity of HK18 --- p.159Chapter 6.1.1 --- HK18 was Cytotoxic to the Human Hepatocellular Carcinoma Cell Line HepG2 and Multidrug Resistant Human Hepatocellular Carcinoma Cell Line R-HepG2 --- p.159Chapter 6.1.2 --- Study of the Toxicity of HK18 --- p.160Chapter 6.2 --- Mechanistic Studies of the Cytotoxic Effects of HK18 on HepG2 Cells --- p.161Chapter 6.2.1 --- Apoptotic Cell Death Induction of HK18 on HepG2 Cells --- p.161Chapter 6.2.2 --- Studies of the Underlying Mechanisms of HK18 Induced Apoptosis of HepG2 Cells --- p.162Chapter 6.3 --- Mechanistic Studies of the Cytotoxic Effects of HK18 on R-HepG2 Cells --- p.181Chapter 6.3.1 --- Apoptotic Cell Death Induction of HK18 on R-HepG2 Cells --- p.181Chapter 6.3.2 --- Studies of the Underlying Mechanisms of HK18 Induced Apoptosis of HepG2 Cells --- p.181Chapter Chapter 7 --- Future Perspectives --- p.190Chapter Chapter 8 --- References --- p.19

Characterising the functional role of rhizosphere fungi in Miscanthus giganteus bioenergy cropping systems

The rhizosphere has a rich fungal microbiome, including parasites, commensals and mutualists. An important group in the rhizosphere are assumed to be the arbuscular mycorrhizal fungi (AMF), which live in symbiosis with around 80% of plant species. AMF have been shown to increase plant yield, biomass, disease resistance, and shoot P. Plants exchange carbon in the form of sugars for nutrients assimilated by AMF. There is little known about AMF in association with Miscanthus giganteus, a productive bioenergy crop grown in the UK and abroad. Work was carried out to characterise the abundance, organisation, importance, function and stability over space and time of rhizosphere fungi and AMF in M. giganteus roots. Field samples from Lincolnshire were analysed using staining and molecular techniques, including small subunit rRNA gene terminal restriction fragment length polymorphism, clone libraries and amplicon pyrosequencing, and meta-transcriptomics. M. giganteus was also grown in a number of pot experiments, with various treatments including fungal inoculations and fungicide application. A number of fungal phyla were found in the roots, particularly Ascomycota, the composition of which shifted over time and exhibited diurnal patterns of activity. Fungi enhanced plant growth by a third, and were functionally active in the roots in the meta-transcriptome. AMF communities were found at much lower relative abundances in roots, and inoculation with AMF did not enhance M. giganteus growth. The work highlights the importance of the whole root mycobiome to plant growth and health, and the relatively small role Glomeromycota play in M. giganteus comparison with other fungi. The work also demonstrated the dynamic nature of fungal activity over hours, months, and years, and the complex interactions the fungal community has with environmental variables