Li Qian. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 102-117). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / List of Abbreviations --- p.ix / List of tables and figures --- p.ix / Content --- p.x / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Traditional Chinese Medicine: Agrimony --- p.1 / Chapter 1.2 --- Hepatocellular carcinoma (HCC) and its risk factors --- p.4 / Chapter 1.3 --- Basic concepts relevant to cancer prevention --- p.6 / Chapter 1.3.1 --- Multistage process of carcinogenesis --- p.6 / Chapter 1.3.2 --- Chemical carcinogenesis --- p.7 / Chapter 1.3.3 --- Possible chemopreventive strategies --- p.8 / Chapter 1.3.4 --- Phase I and phase II systems in chemical carcinogenesis --- p.10 / Chapter Chapter 2 --- Materials and methods --- p.12 / Chapter 2.1 --- Preparation of aqueous extract of Agrimonia pilosa --- p.12 / Chapter 2.2 --- In vivo study --- p.13 / Chapter 2.2.1 --- Animal model for hepatocarcinogenesis --- p.13 / Chapter 2.2.1.1 --- Chemical carcinogens --- p.13 / Chapter 2.2.1.2 --- Animals --- p.16 / Chapter 2.2.1.3 --- Animal treatment and sacrifice --- p.17 / Chapter 2.2.2 --- Histological and immunohistochemical study --- p.20 / Chapter 2.2.3 --- Preparation of liver homogenates and microsomes from rat --- p.23 / Chapter 2.2.4 --- Determination of protein concentration --- p.24 / Chapter 2.2.5 --- COX-2 Activity Assay --- p.25 / Chapter 2.2.6 --- Cytochrome P450 2E1 Assay --- p.26 / Chapter 2.2.7 --- Spectrophotometry Assay for GST --- p.28 / Chapter 2.2.8 --- Isolation of total RNA from liver homogenate --- p.29 / Chapter 2.2.9 --- Semi-quantitative RT-PCR analysis --- p.32 / Chapter 2.3 --- In vitro study --- p.36 / Chapter 2.3.1 --- Cell cultures --- p.36 / Chapter 2.3.2 --- Cytotoxicity assay - Neutral Red Assay --- p.38 / Chapter 2.3.3 --- Cell cycle distribution analysis by flow cytometry --- p.39 / Chapter 2.3.4 --- DNA fragmentation --- p.40 / Chapter Chapter 3 --- Results --- p.43 / Chapter 3.1 --- In vivo study --- p.43 / Chapter 3.1.1 --- Body weight and relative liver weight --- p.43 / Chapter 3.1.2 --- Gross Morphological changes --- p.46 / Chapter 3.1.3 --- Hematoxylin & Eosin (H&E) staining for histological detection --- p.50 / Chapter 3.1.4 --- Effect of AP on DEN-CCl4-induced GST-P positive foci formation and GST-P mRNA expression --- p.60 / Chapter 3.1.5 --- Effects of AP on COX-2 --- p.72 / Chapter 3.1.6 --- Effects of AP on phase I and phase II enzymes --- p.76 / Chapter 3.2 --- In vitro study --- p.80 / Chapter 3.2.1 --- Effects of AP on proliferation of H4IIE cells detected by Neutral Red Assay --- p.80 / Chapter 3.2.2 --- Assessment of cell cycle distribution by flow cytometry --- p.82 / Chapter 3.2.3 --- DNA Fragmentation Assay --- p.88 / Chapter Chapter 4 --- Discussion --- p.90 / Chapter 4.1 --- In vivo study --- p.90 / Chapter 4.1.1 --- Morphological changes during the induction of hepatocarcinogenesis --- p.90 / Chapter 4.1.2 --- Effects of AP on GST-P foci and its mRNA --- p.91 / Chapter 4.1.3 --- Effects of AP on COX-2 enzyme activity and mRNA expression --- p.93 / Chapter 4.1.4 --- Modulation effects of AP on CYP2E1 and GST enzyme activity --- p.95 / Chapter 4.2 --- In vitro study: effects of AP on cancer cell proliferation --- p.97 / Chapter 4.3 --- Summary --- p.99 / References --- p.102

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

Lai Kin Ming Connie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 121-141). / Abstracts in English and Chinese. / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Introduction on growth cycle of mushroom --- p.1 / Chapter 1.2 --- Literature review of mushroom biological activities --- p.3 / Chapter 1.2.1 --- Various bioactivities of mushroom --- p.3 / Chapter 1.2.2 --- Components responsible for various bioactivities of mushrooms --- p.3 / Chapter 1.3 --- Mushroom polysaccharides and polysaccharide-protein complexes --- p.5 / Chapter 1.3.1 --- Polysaccharides important for antitumor effects --- p.5 / Chapter 1.3.2 --- Polysaccharide-protein complexes important for antitumor effects --- p.7 / Chapter 1.4 --- Structure-function relationship of antitumor activities of polysaccharides --- p.8 / Chapter 1.4.1 --- Effect of molecular mass --- p.8 / Chapter 1.4.2 --- Effect of linkages --- p.9 / Chapter 1.4.3 --- Effect of degree of branching --- p.9 / Chapter 1.4.4 --- Effect of conformation --- p.10 / Chapter 1.5 --- Immunomodulatory effects of mushroom polysaccharides and polysaccharide-protein complexes --- p.11 / Chapter 1.5.1 --- Immunomodulatory effects of polysaccharides --- p.11 / Chapter 1.5.1.1 --- Bioactive polysaccharides in Lentinus edodes --- p.11 / Chapter 1.5.1.2 --- Bioactive polysaccharides in Ganoderma lucidum --- p.12 / Chapter 1.5.2 --- Immunomodulatory effects of polysaccharide-protein complexes --- p.12 / Chapter 1.5.2.1 --- Bioactive polysaccharide-protein complexes in Trametes versicolor --- p.13 / Chapter 1.5.3 --- Immunotherapeutic effects of mushroom polysaccharides --- p.14 / Chapter 1.6 --- Cell cycle and apoptosis --- p.14 / Chapter 1.6.1 --- Introduction of cell cycle --- p.14 / Chapter 1.6.2 --- Cell cycle regulation --- p.15 / Chapter 1.6.3 --- Antitumor effects through apoptotic gene regulation --- p.17 / Chapter 1.7 --- Mushroom sclerotium with antitumor activity --- p.20 / Chapter 1.7.1 --- Literature review on Pleurotus tuber-regium --- p.20 / Chapter 1.7.2 --- Literature review on Poria cocos --- p.22 / Chapter 1.7.3 --- Literature review on Polyporus rhinocerus --- p.23 / Chapter 1.8 --- Objectives --- p.23 / Chapter Chapter 2. --- Materials and Methods --- p.25 / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Mushroom sclerotia --- p.25 / Chapter 2.1.2 --- Animal Model --- p.25 / Chapter 2.1.3 --- Cell lines --- p.27 / Chapter 2.2 --- Methods --- p.28 / Chapter 2.2.1 --- Extraction Scheme for mushroom sclerotia --- p.28 / Chapter 2.2.1.1 --- Hot water extraction only --- p.28 / Chapter 2.2.1.2 --- Sequential extraction scheme --- p.28 / Chapter 2.2.2 --- Measurement of monosaccharide profile --- p.31 / Chapter 2.2.2.1 --- Acid Depolymerisation --- p.31 / Chapter 2.2.2.2 --- Neutral Sugar Derivatization --- p.31 / Chapter 2.2.2.3 --- Gas Chromatography (GC) --- p.32 / Chapter 2.2.3 --- High Pressure Liquid Chromatography (HPLC) --- p.33 / Chapter 2.2.3.1 --- Size exclusion chromatography --- p.33 / Chapter 2.2.3.2 --- Anion exchange chromatography --- p.34 / Chapter 2.2.4 --- Linkage analysis by methylation --- p.34 / Chapter 2.2.4.1 --- Preparation of partially methylated polysaccharides --- p.34 / Chapter 2.2.4.2 --- Preparation of partially methylated alditol acetates (PMAAs) --- p.37 / Chapter 2.2.4.3 --- Gas chromatography-Mass spectrometry (GC-MS) analysis --- p.37 / Chapter 2.2.5 --- Determination of total sugar by phenol-sulphuric acid Method --- p.38 / Chapter 2.2.6 --- Determination of acidic sugars by measurement of uronic acid content --- p.39 / Chapter 2.2.7 --- Determination of protein content by Lowry-Folin method --- p.40 / Chapter 2.2.8 --- Chemical modification by carboxymethylation --- p.41 / Chapter 2.2.9 --- In vitro antitumor assay --- p.41 / Chapter 2.2.9.1 --- Trypan blue exclusion assay --- p.42 / Chapter 2.2.9.2 --- MTT Assay --- p.42 / Chapter 2.2.10 --- Cell cycle analysis by Flow Cytometry --- p.43 / Chapter 2.2.11 --- In vivo antitumor and immunomodulatory assay --- p.44 / Chapter 2.2.11.1 --- Measurement on tumor growth --- p.44 / Chapter 2.2.11.2 --- Blood sampling for immunostimulatory effects --- p.45 / Chapter 2.2.12 --- Mouse Cytokine Array --- p.45 / Chapter 2.2.13 --- Quantification of Mouse IL-13 by ELISA --- p.46 / Chapter 2.2.14 --- Enumeration of peritoneal cells --- p.47 / Chapter 2.2.15 --- Enumeration of splenocytes --- p.49 / Chapter 2.2.16 --- Statistical methods --- p.50 / Chapter Chapter 3. --- Results and Discussion --- p.51 / Chapter 3.1 --- Yield of crude mushroom sclerotial extracts --- p.51 / Chapter 3.2 --- Chemical composition of crude mushroom sclerotial extracts --- p.57 / Chapter 3.2.1 --- Total carbohydrate content --- p.57 / Chapter 3.2.2 --- Uronic acid content --- p.58 / Chapter 3.2.3 --- Soluble protein content --- p.58 / Chapter 3.3 --- Monosaccharide profiles of mushroom sclerotial extracts by GC --- p.60 / Chapter 3.4 --- Chromatographic analyses of mushroom sclerotial extracts --- p.65 / Chapter 3.4.1 --- Molecular weight profile by size exclusion chromatography (SEC) --- p.65 / Chapter 3.4.2 --- Charge distribution by ion exchange chromatography (IEC) --- p.73 / Chapter 3.5 --- Antitumor effects of mushroom sclerotial extracts from hot water extraction alone --- p.73 / Chapter 3.5.1 --- In vitro antiproliferative study by HL-60 --- p.73 / Chapter 3.5.2 --- In vitro antiproliferative study by MCF-7 --- p.74 / Chapter 3.5.3 --- In vivo antitumor study by BALB/c mice --- p.75 / Chapter 3.6 --- Antitumor effects of extracts from sequential extraction scheme --- p.76 / Chapter 3.6.1 --- In vitro antiproliferative study by HL-60 --- p.76 / Chapter 3.6.2 --- In vitro antiproliferative study by MCF-7 --- p.78 / Chapter 3.6.3 --- In vivo antitumor study by BALB/c mice --- p.80 / Chapter 3.7 --- Comparison of in vitro and in vivo activities of mushroom sclerotial extracts --- p.82 / Chapter 3.8 --- Dose-response relationship of hot water extract from PR on cancer cell lines --- p.85 / Chapter 3.8.1 --- In vitro dose-response antiproliferation of PR-W and PR-HWE on HL-60 --- p.85 / Chapter 3.8.2 --- In vitro dose-response antiproliferation of PR-W on K562 and S180 --- p.88 / Chapter 3.8.3 --- In vivo dose-response relationship of PR-W on S180 --- p.91 / Chapter 3.9 --- Flow cytometric analysis of PR-W on cancer cell lines --- p.92 / Chapter 3.9.1 --- Antiproliferative effect of PR-W on HL-60 --- p.92 / Chapter 3.9.2 --- Antiproliferative effect of PR-W on K562 --- p.95 / Chapter 3.9.3 --- Proposed mechanisms of cell cycle arrest by PR-W --- p.98 / Chapter 3.10 --- Host-mediated antitumor mechanism of PR-W --- p.100 / Chapter 3.10.1 --- Mouse cytokine array --- p.100 / Chapter 3.10.2 --- Quantification of IL-13 by ELISA --- p.105 / Chapter 3.10.3 --- Immunostimulatory effects of PR-W on mice --- p.109 / Chapter 3.11 --- Correlation between antitumor activity and structure of mushroom sclerotial extract from hot water extraction alone --- p.114 / Chapter Chapter 4. --- Conclusions and Future works --- p.118 / List of References --- p.121 / Related Publications --- p.142

神經母細胞瘤是一種交感神經系統的腫瘤。它是最常見的兒童顱外實體瘤。神經母細胞瘤約佔兒童腫瘤的8-10%，佔15%的兒童腫瘤死亡率。目前神經母細胞瘤的治療方法包括外科手術， 化學藥物治療， 放射治療， 幹細胞移植， 誘導分化治療和免疫治療。 然而，這些治療方法通常會導致許多無法避免的嚴重的副作用。因此，開發能高效抑制神經母細胞瘤但對正常細胞無明顯副作用的新型藥物顯得至關重要。最近，用來源於天然產物或中藥的化合物治療癌症引起了科學家的廣泛關注。靛玉紅-3’-肟（Indirubin-3’-oxime, I3M）和色胺酮（tryptanthrin）分別是從板藍根中分離得到的靛藍生物鹼和吲哚喹唑啉類生物鹼。據研究報導，這兩種生物鹼具有多種生物學和藥理學作用，包括抗菌，抗炎症和抗腫瘤作用。它們對體外的多種人腫瘤細胞具有抗腫瘤作用。然而，它們對人神經母細胞瘤的調節作用和作用機理仍不太清楚。在我的博士研究課題中，我們對板藍根生物鹼包括靛玉紅-3’-肟和色胺酮對人神經母細胞瘤的抗腫瘤活性和作用機制進行了研究和探討。 / 首先，我們研究了靛玉紅-3’-肟對人神經母細胞瘤的抗腫瘤活性和作用機制。實驗結果表明，靛玉紅-3’-肟能夠抑制人神經母細胞瘤LA-N-1, SH-SY5Y 和 SK-N-DZ細胞系的生長，並且其抑制效果呈時間和濃度依賴性。然而，靛玉紅-3’-肟對正常細胞無顯著的細胞毒性作用。對其生長抑制作用機制的研究結果表明靛玉紅-3’-肟能夠特異性地減少LA-N-1細胞系中線粒體的調節子ERR和 PGC-1的表達，從而導致線粒體生成減少，線粒體膜電位降低以及線粒體活性氧（ROS）增加。靛玉紅-3’-肟還增加週期蛋白依賴性蛋白激酶（CDK）抑制蛋白p27{U+1D37}{U+2071}{U+1D56}¹的蛋白水平並降低週期蛋白依賴性蛋白激酶2（CDK2）和細胞週期蛋白E（Cyclin E）的表達，從而導致細胞週期阻滯在G0/G1期。 另外，我們發現靛玉紅-3’-肟能減少SH-SY5Y細胞系的線粒體生成，增加細胞內活性氧的水準從而導致細胞週期停滯在G0/G1期和細胞凋亡。以上結果表明靛玉紅-3’-肟可能通過破壞線粒體的功能從而導致LA-N-1和SH-SY5Y細胞的細胞週期阻滯和誘導SH-SY5Y細胞的細胞凋亡來發揮其抗腫瘤的作用。 / 接著，我們對色胺酮對人神經母細胞瘤的抗腫瘤活性和作用機制進行了探討。我們研究的結果表明，色胺酮可以時間和濃度依賴性地抑制人神經母細胞瘤LA-N-1, SH-SY5Y 和 SK-N-DZ細胞系的生長，而對正常的細胞無顯著的細胞毒性。對色胺酮抑制人神經母細胞瘤生長的機制研究表明，色胺酮能顯著地降低細胞週期蛋白（Cyclin D1和 Cyclin D3）和週期蛋白依賴性蛋白激酶（CDK4和CDK6）的蛋白水平從而導致細胞週期停滯在G0/G1期。色胺酮可以激活半胱氨酸天冬氨酸蛋白酶8,9和3/7（caspase 8, caspase 9 和 caspase 3/7）從而誘導LA-N-1細胞凋亡。色胺酮還可以誘導LA-N-1細胞分化，表現為神經細胞分化的細胞形態，乙醯膽鹼酯酶活性的增加和多種細胞分化的分子標記的表達上調。另外，色胺酮還能降低LA-N-1細胞中N-myc的表達。有趣的是，通過RNA干擾技術降低N-myc的表達能誘導LA-N-1細胞的分化。總的來說，以上結果顯示色胺酮通過誘導細胞週期阻滯，細胞凋亡和細胞分化從而發揮其抗腫瘤的作用。它可能被開發為治療有N-myc基因擴增的高危的人神經母細胞瘤的潛在藥物。 / 此外，我們還研究了靛玉紅-3’-肟和色胺酮是否具有抗血管生成的作用。體外實驗的研究結果表明，靛玉紅-3’-肟和色胺酮能夠濃度依賴性地抑制人微血管上皮細胞 （HMEC-1細胞）的增殖，遷徙和血管生成，但對HMEC-1細胞卻沒有顯著的細胞毒性作用。此外，靛玉紅-3’-肟和色胺酮能顯著地抑制小鼠體內的基質膠栓（Matrigel plug）的血管生成。對它們抑制血管生成的機制的研究表明，靛玉紅-3’-肟能下調血管生成素1（Ang-1）和基質金屬蛋白酶2（MMP2）的表達，上調血管生成素2（Ang-2）的表達。靛玉紅-3’-肟能結合到血管內皮生長因數受體2（VEGFR2） 的ATP結合位點上從而抑制血管內皮生長因數受體2的磷酸化和下游的MEK/ERK和PI3K/AKT/GSK信號轉導通路。色胺酮同樣可以抑制多種血管生成因子（Ang-1,PDGFB 和MMP2）的表達。此外，它可以結合到血管內皮生長因數受體2 的ATP結合位點上從而抑制血管內皮生長因數受體2的磷酸化和血管內皮生長因數受體2介導的ERK1/2信號通路。以上的體外和體內實驗研究結果表明靛玉紅-3’-肟和色胺酮通過靶向血管內皮生長因數受體2介導的信號通路來發揮其抗血管生成的作用。它們可能被開發為治療血管生成相關疾病的潛在藥物。 / 總而言之，我們的研究結果表明靛玉紅-3’-肟和色胺酮通過誘導人神經母細胞瘤細胞的細胞週期阻滯，細胞凋亡或誘導神經細胞分化從而抑制人神經母細胞瘤細胞的生長。然而，它們對正常細胞無顯著的細胞毒性作用。此外，靛玉紅-3’-肟和色胺酮通過靶向血管內皮生長因數受體2介導的信號通路來發揮其抗血管生成的作用。未來的研究將進一步探討靛玉紅-3’-肟和色胺酮對人神經母細胞瘤細胞的分子作用機理。另外，通過人神經母細胞瘤細胞的裸鼠移植瘤動物模型可進一步去了解這些板藍根生物鹼在體內的抗腫瘤效果。 / Neuroblastoma, a tumor of the sympathetic nervous system, is the most common extracranial solid cancer in childhood. It accounts for 8% to 10% of all childhood cancers and for approximately 15% of cancer deaths in children. Current treatment modalities consist of surgery, chemotherapy, radiation therapy, stem cell transplantation, differentiation therapy and immunotherapy. However, these treatments often cause severe and inevitable side effects. It is important to develop novel drugs with higher efficacy on neuroblastoma cells and minimal side effects on normal cells. The use of new promising therapeutic compounds derived from natural products or Chinese herbs have attracted much attention of scientist as an alternative strategy in cancer treatment. Indirubin-3’-oxime (I3M) is an indigo alkaloid and tryptanthrin is an indoloquinazoline alkaloid which can be isolated from the dried roots of medicinal indigo plants known as Banlangen. These two alkaloids have been reported to possess various biological and pharmacological activities, such as anti-microbial, anti-inflammatory, and anti-tumor effects. They were found to exhibit potent anti-tumor activities on various types of human cancer cells in vitro. However, their modulatory effects on human neuroblastoma and the underlying mechanisms remain poorly understood. In my PhD project, the possible anti-tumor activities and action mechanisms of Banlangen alkaloids, including I3M and tryptanthrin, on human neuroblastoma cells were investigated. / Firstly, the anti-cancer effects of I3M on human neuroblastoma cells and the underlying mechanisms were investigated. I3M was found to inhibit the growth of the human neuroblastoma LA-N-1, SH-SY5Y and SK-N-DZ cells in a concentration- and time-dependent manner, but exhibited little, if any, direct cytotoxicity on normal cells. Mechanistic studies showed that I3M specifically decreased the expression of mitochondrial regulators ERRγ and PGC-1βand resulted in decreased mitochondrial mass and altered mitochondrial function characterized by reduction in mitochondrial membrane potential and elevation of reactive oxygen species (ROS) level in LA-N-1 cells. I3M also increased the level of cyclin-dependent kinase (CDK) inhibitor p27{U+1D37}{U+2071}{U+1D56}¹ and reduced the levels of CDK2 and cyclin E in LA-N-1 cells, leading to cell cycle arrest at the G0/G1 phase. In addition, I3M was also found to reduce the mitochondrial mass and increase the ROS level leading to cell cycle arrest at G0/G1 phase and apoptosis in SH-SY5Y cells. These results, when taken together, suggest that I3M might exert its anti-tumor activity by causing mitochondrial dysfunction which led to cell cycle arrest in LA-N-1 cells and resulted in cycle arrest and apoptosis in SH-SY5Y cells. / The anti-tumor effects and action mechanisms of tryptanthrin on the human neuroblastoma cells were also examined. Our results showed that tryptanthrin inhibited the growth of the human neuroblastoma LA-N-1, SH-SY5Y and SK-N-DZ cells in a concentration- and time-dependent manner, but exhibited little, if any, direct cytotoxicity on normal cells. Mechanistic studies indicated that tryptanthrin significantly reduced the protein levels of cyclin D1, cyclin D3, CDK4 and CDK6 leading to cell cycle arrest at G0/G1 phase. In addition, tryptanthrin activated caspase 8, caspase 9 and caspase 3/7 resulting in apoptosis of the human neuroblastoma LA-N-1 cells. Moreover, tryptanthrin induced neuronal differentiation of LA-N-1 cells, as assessed by morphological criteria, enhancement of acetylcholine esterase activity and up-regulation of various differentiation markers. Tryptanthrin treatment also led to the significant reduction of N-myc expression in LA-N-1 cells. Interestingly, down-regulating N-myc expression using siRNA induced neuronal differentiation of LA-N-1 cells. Collectively, these results indicate that tryptanthrin might exert its anti-tumor activity on the human neuroblastoma LA-N-1 cells by inducing cell cycle arrest, apoptosis and neuronal differentiation. It might be exploited as a potential therapeutic candidate for the treatment of high-risk neuroblastomas with N-myc-amplification. / Moreover, the anti-angiogenic activities of I3M and tryptanthrin were studied. Our results showed that I3M and tryptanthrin inhibited the proliferation, migration, and tube formation of the human microvascular endothelial HMEC-1 cells in vitro in a concentration-dependent manner but exhibited no significant cytotoxicity on these cells. Moreover, I3M and tryptanthrin markedly suppressed the in vivo angiogenesis in Matrigel plugs in mice. Mechanistic studies indicated that I3M down-regulated the expression of Ang-1 and MMP2 and up-regulated the expression of Ang-2. It also bound to the ATP-binding site of VEGFR2 and inhibited the phosphorylation of VEGFR2 leading to suppression of the down-stream MEK/ERK and PI3K/AKT/GSK signaling pathways in HMEC-1 cells. Similarly, tryptanthrin also reduced the expression of several angiogenic factors (Ang-1, PDGFB and MMP2) in HMEC-1 cells. In addition, tryptanthrin also bound to the ATP-binding site of VEGFR2 and suppressed the phosphorylation of VEGFR2 and VEGFR2-mediated ERK1/2 signaling pathway in HMEC-1 cells. Collectively, our results demonstrated that I3M and tryptanthrin exhibited anti-angiogenic activity both in vitro and in vivo by specifically targeting the VEGFR2-mediated signaling pathways and might be exploited as potential therapeutic candidates for the treatment of angiogenesis-related diseases. / In conclusion, our findings indicate that I3M and tryptanthrin might exert their growth-inhibitory effect on the human neuroblastoma cells by causing cell cycle arrest, inducing apoptosis or inducing neuronal differentiation. However, they exhibited minimal cytotoxicity towards the normal cells. Moreover, I3M and tryptanthrin were found to possess anti-angiogenic activities by targeting the VEGFR2-mediated signaling pathways. In the future, investigations should be focused on further elucidation of the molecular action mechanisms of I3M and tryptanthrin on human neuroblastoma cells and to test the anti-tumor efficacy of I3M and tryptanthrin in animal models, using human neuroblastoma xenografts in nude mice. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liao, Xuemei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 206-229). / Abstract also in Chinese. / Acknowledgments --- p.i / Abbreviations --- p.ii / Publications --- p.vi / Abstract --- p.vii / 摘要 --- p.xii / Table of Contents --- p.xvi / Chapter One / General Introduction --- p.1 / Chapter 1.1 --- Neuroblastoma --- p.2 / Chapter 1.1.1 --- Epidemiology of neuroblastoma --- p.2 / Chapter 1.1.2 --- Classification of neuroblastoma --- p.6 / Chapter 1.1.3 --- Clinical symptoms and diagnosis of neuroblastoma --- p.10 / Chapter 1.1.4 --- Molecular pathogenesis of neuroblastoma --- p.13 / Chapter 1.1.4.1 --- Genetic alterations in neuroblastoma --- p.13 / Chapter 1.1.4.2 --- Disruption of cell division cycle, apoptotic and signaling pathways --- p.16 / Chapter 1.1.5 --- Treatment strategies --- p.19 / Chapter 1.1.5.1 --- Low-risk neuroblastoma treatment strategy --- p.19 / Chapter 1.1.5.2 --- Intermediate-risk neuroblastoma treatment strategy --- p.20 / Chapter 1.1.5.3 --- High-risk neuroblastoma treatment strategy --- p.21 / Chapter 1.1.5.4 --- Side effects of treatment --- p.23 / Chapter 1.2 --- Banlangen alkaloids --- p.23 / Chapter 1.2.1 --- Overview of Banlangen alkaloids --- p.23 / Chapter 1.2.2 --- Biological and pharmacological effects of Banlangen alkaloids --- p.28 / Chapter 1.2.2.1 --- Anti-inflammatory activity --- p.28 / Chapter 1.2.2.2 --- Anti-microbial activity --- p.29 / Chapter 1.2.2.3 --- Anti-tumor activity --- p.30 / Chapter 1.2.2.4 --- Other biological activities --- p.32 / Chapter 1.2.3 --- Bioavailability of Banlangen alkaloids --- p.33 / Chapter 1.2.4 --- Toxicity of Banlangen alkaloids --- p.34 / Chapter 1.3 --- Aims and scope of this project --- p.36 / Chapter Two / Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Animals --- p.39 / Chapter 2.1.2 --- Cell lines --- p.39 / Chapter 2.1.3 --- Cell culture media --- p.41 / Chapter 2.1.4 --- Drugs and chemicals --- p.42 / Chapter 2.1.5 --- Reagents and buffers for cell culture --- p.44 / Chapter 2.1.6 --- General staining solutions --- p.47 / Chapter 2.1.7 --- Reagents and buffers for cell growth assays --- p.48 / Chapter 2.1.8 --- Reagents and buffers for flow cytometry --- p.48 / Chapter 2.1.9 --- Reagents and buffers for acetylcholine esterase activity assay --- p.50 / Chapter 2.1.10 --- Reagents and buffers for immunocytochemistry --- p.51 / Chapter 2.1.11 --- Reagents and buffers for total RNA extraction --- p.53 / Chapter 2.1.12 --- Reagents and buffers for reverse transcription --- p.54 / Chapter 2.1.13 --- Reagents for quantitative real-time polymerase chain reaction (qRT-PCR) --- p.56 / Chapter 2.1.14 --- Reagents and buffers for Western blotting --- p.59 / Chapter 2.1.15 --- Assay kits --- p.65 / Chapter 2.2 --- Methods --- p.68 / Chapter 2.2.1 --- Culture of cells --- p.68 / Chapter 2.2.2 --- MTT assay --- p.69 / Chapter 2.2.3 --- Cell proliferation assay --- p.70 / Chapter 2.2.4 --- Trypan blue exclusion test --- p.70 / Chapter 2.2.5 --- Cytotoxicity assay --- p.71 / Chapter 2.2.6 --- Colony-forming assay --- p.72 / Chapter 2.2.7 --- Cell cycle analysis --- p.72 / Chapter 2.2.8 --- Assessment of apoptosis --- p.73 / Chapter 2.2.9 --- Caspase activity determination --- p.74 / Chapter 2.2.10 --- Mitochondrial mass assay --- p.75 / Chapter 2.2.11 --- Reactive oxygen species (ROS) assay --- p.75 / Chapter 2.2.12 --- Mitochondrial membrane potential determination --- p.76 / Chapter 2.2.13 --- Morphological detection of cell differentiation --- p.76 / Chapter 2.2.14 --- Acetylcholine esterase activity determination --- p.77 / Chapter 2.2.15 --- Immunocytochemistry --- p.77 / Chapter 2.2.16 --- RNA interference --- p.78 / Chapter 2.2.17 --- Wound healing assay --- p.79 / Chapter 2.2.18 --- Tube formation assay --- p.79 / Chapter 2.2.19 --- In vivo Matrigel plug assay --- p.80 / Chapter 2.2.20 --- Phospho-VEGFR2 Sandwich ELISA assay --- p.80 / Chapter 2.2.21 --- Isolation of total cellular RNA --- p.81 / Chapter 2.2.22 --- Reverse transcription (RT) --- p.82 / Chapter 2.2.23 --- Quantitative real-time PCR --- p.83 / Chapter 2.2.24 --- Total protein extraction --- p.84 / Chapter 2.2.25 --- Protein concentration determination --- p.84 / Chapter 2.2.26 --- Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) --- p.85 / Chapter 2.2.27 --- Semi-dry Western blotting --- p.85 / Chapter 2.2.28 --- Enhanced chemiluminescence (ECL) assay --- p.87 / Chapter 2.2.29 --- Molecular docking --- p.87 / Chapter 2.2.30 --- Statistical analysis --- p.88 / Chapter Three / Modulatory effects and action mechanisms of indirubin-3'-oxime on human neuroblastoma cells --- p.89 / Chapter 3.1 --- Introduction --- p.90 / Chapter 3.2 --- Results --- p.94 / Chapter 3.2.1 --- Indirubin-3’-oxime inhibited the growth and colony formation of human neuroblastoma cells in vitro --- p.94 / Chapter 3.2.2 --- Indirubin-3’-oxime exhibited no significant cytotoxicity on normal cells --- p.101 / Chapter 3.2.3 --- Indirubin-3’-oxime induced G0/G1 cell cycle arrest in LA-N-1 cells --- p.103 / Chapter 3.2.4 --- Indirubin-3’-oxime caused mitochondrial dysfunction in LA-N-1 cells --- p.106 / Chapter 3.2.5 --- Indirubin-3’-oxime selectively reduced ERR γ and PGC-1β protein and mRNA levels in LA-N-1 cells --- p.111 / Chapter 3.2.6 --- Indirubin-3’-oxime induced cell cycle arrest at G0/G1 phase and apoptosis of SH-SY5Y cells --- p.113 / Chapter 3.2.7 --- Indirubin-3’-oxime reduced mitochondrial mass and elevated mitochondrial ROS level in SH-SY5Y cells --- p.115 / Chapter 3.2.8 --- Indirubin-3’-oxime increased the caspase 8, caspase 9 and caspase 3/7 activities in SH-SY5Y cells --- p.117 / Chapter 3.3 --- Discussion --- p.119 / Chapter Four / Modulatory effects and action mechanisms of tryptanthrin on human neuroblastoma cells --- p.125 / Chapter 4.1 --- Introduction --- p.126 / Chapter 4.2 --- Results --- p.129 / Chapter 4.2.1 --- Tryptanthrin inhibited the cell growth and colony formation of human neuroblastoma cells --- p.129 / Chapter 4.2.2 --- Tryptanthrin exhibited no significant cytotoxicity on normal cells --- p.136 / Chapter 4.2.3 --- Tryptanthrin induced cell cycle arrest at G0/G1 phase --- p.138 / Chapter 4.2.4 --- Tryptanthrin induced apoptosis of LA-N-1 cells --- p.140 / Chapter 4.2.5 --- Tryptanthrin induced morphological neuronal differentiation in LA-N-1 cells --- p.143 / Chapter 4.2.6 --- Tryptanthrin induced the expression of neuronal differentiation markers --- p.146 / Chapter 4.2.7 --- Tryptanthrin down-regulated the expression of N-myc in LA-N-1 cells --- p.149 / Chapter 4.3 --- Discussion --- p.152 / Chapter Five / Anti-angiogenesis effects and action mechanisms of indirubin-3'-oxime and tryptanthrin --- p.158 / Chapter 5.1 --- Introduction --- p.159 / Chapter 5.2 --- Results --- p.163 / Chapter 5.2.1 --- Indirubin-3’-oxime and tryptanthrin inhibited the proliferation of endothelial cells --- p.163 / Chapter 5.2.3 --- Indirubin-3’-oxime and tryptanthrin reduced the tube formation of endothelial cells --- p.168 / Chapter 5.2.4 --- Indirubin-3’-oxime and tryptanthrin blocked angiogenesis in the in vivo Matrigel plug model --- p.171 / Chapter 5.2.5 --- Indirubin-3’-oxime and tryptanthrin reduced the angiogenic gene expression in endothelial cells --- p.174 / Chapter 5.2.6 --- Indirubin-3’-oxime and tryptanthrin attenuated VEGFR2-mediated signaling pathways in endothelial cells --- p.176 / Chapter 5.2.7 --- Indirubin-3’-oxime bound to the ATP-binding site of VEGFR2 kinase domain --- p.181 / Chapter 5.2.8 --- Tryptanthrin bound to the ATP-binding site of VEGFR2 kinase domain --- p.182 / Chapter 5.3 --- Discussion --- p.184 / Chapter Six / Conclusions and future perspectives --- p.191 / References --- p.206

In this study, a novel compound was isolated and purified from the solid culture medium (potato dextrose agar) of shiitake 1358 strain through series of methods, such as ethanol precipitation, macroporous resin column separation, semi-preparative high performance liquid chromatography separation and preparative thin-layer chromatography separation. Analyzing spectra from fourier transform infra-red spectroscopy, gas chromatography-mass spectrometry, 1-dimension and 2-dimension nuclear magnetic resonance, the chemical structure of the novel compound was determined and named as 4-amino-5,6-dihydrobenzo[d]oxonine-2,7(1H,4H)-dione. It could inhibit the proliferation of HL-60 leukemia cells significantly and with an IC50 of 1.56 mug/ml (7.123 mumol/L) in the 72-hour treatment. From the results, it is suggested that this compound could activate the G2 phase checkpoint control of the cell cycle to arrest the cell cycle in G2 phase. In addition, it could suppress the replicative DNA synthesis to inhibit the proliferation of HL-60 leukemia cells. The more important is that this compound can induce the apoptosis of HL-60 leukemia cells significantly through intrinsic and extrinsic apoptotic pathways. The compound could induce intrinsic and extrinsic apoptosis through the regulation of the apoptosis-related proteins, such as Fas ligand, Bax, Bcl-2, Caspase 8, Caspase 9, and Caspase 3. For intrinsic pathway, the compound might upregulate Bax, downregulated Bcl-2, activated the Caspase 9, subsequently activated Capase 3, and ultimately led to cell death. For extrinsic pathway, the compound upregulated the Fas ligand, cleaved and activated Procaspase 8 to active Caspase 8, further cleaved and activated Procaspase 3 to active Caspase 3 to commit the cells to apoptosis. / Leukemia is a malignant cancer that involves the bone marrow and blood circulation systems. Leukemia results in the uncontrolled growth of abnormal (leukemic) white blood cells and may also invade other organs, including the liver, spleen, lymph nodes, testes, and brain. In 2007, about 44,240 new cases of leukemia were diagnosed and 21,790 patients died from all types of leukemias in USA. / Shiitake was first cultivated in China more than 800 years ago. It is the second most commonly cultivated edible mushrooms in the world nowadays. For a long time, shiitake has been valued for its unique taste and flavor and as a medicinal invigorant. According to ancient Chinese medicinal theory, consumption of shiitake was in favor of long life and good health. In China and Japan, shiitake has been used as both a food and a medicinal herb for thousands of years. It is the source of several well-studied preparations with proven pharmacological properties, especially the polysaccharide lentinan. Currently, most researches concentrate on the anticancer activities of the extracts from the fruiting body of shiitake, especially polysaccharides. Report about the anti-cancer effects of other components from the shiitake mushroom is scarce. The objectives of this investigations were: (1) to study the anticancer activities of brownish substances obtained during the solid medium culture of shiitake on specific cancer cell unes, especially HL60 cancer cell line; (2) to isolate and characterize the active compound(s) in the brown mushroom exudates; and (3) to propose the possible mechanism of actions, especially the function of the bcl-2 family genes and proteins. / by Guo, Yuming. / Adviser: Chung Hale Yin. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3314. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 188-199). / 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.

Different cucurbitacin derivatives have been compared for the gamma globin induction potential. Cucurbitacin D turned out to be the most potential inducer among the derivatives had been tested. Later I had screened more herbs for the gamma globin induction activities. One of the herbs showed a higher activity than Fructus Trichosanthis, which could be the potential candidate to isolate more potent inducer. In the toxicity study, cucurbitacin D only have a mild toxic effect on the normal cell lines and transgenic mice. Finally, the efficacy of cucurbitacin D was tested on a sickle cell anemia mouse model and demonstrated a significant induction of fetal haemoglobin production. Cucurbitacin D may be a potential drug candidate for treating beta globinopathies. / Thalassemia is a global disease. It was report in 2001 that there were 270 million people who carried the severe disease. Most of the cases were found in Africa and south-east Asia. China has a high incidence rate of 0.66% in 2001. In the past, the treatments of the disease were blood transfusion and bone marrow transplantation. However, many defects in such kinds of treatments were reported. The balance of relieving the syndrome of the disease and the adverse effects of the drugs was the consideration to the physician. The drug, hydroxyurea, can activate the gamma globin gene and produce hemoglobin F to replace the beta globin as an oxygen transporter is considered as an better treatment to ameliorate the syndrome. Safety and effectiveness in the long-term treatment using hydroxyurea are questionable. Cucurbatacin D purified from a Chinese herb demonstrates 2000 folds more potent than hydroxyurea. It can activate the gamma globin gene and produce hemoglobin F shown in ELISA and confocal microscopy. The fundamental work for drug development is carrying out through this project. In this project the biological property and toxicity were studied. / Liu, Shuk Ming. / Adviser: M.C. Tung. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 245-270). / 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.

Ren-Wang Jiang. / "July 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Leung Wai-hang. / "November 2002." / On t.p. "33" and "42" are subscripts following the word "MSP-1" in the title. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 162-171). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Tang Wan-yee. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 192-212). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.