Hepatocellular carcinoma (HCC), the major form of primary liver cancer, is a common cause of cancer-related death worldwide. Signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in the pathogenesis of HCC. Inhibition of STAT3 signaling has been proposed as a promising strategy for treating HCC. Due to the limitations of conventional therapeutics, increasing attention has been paid to complementary and alternative medicines (CAM) including traditional Chinese medicine (TCM) for the management of HCC. Antrodia camphorata (AC), a medicinal mushroom, is historically used for treating HCC. Pharmacological data showed that extracts and constituents of AC are able to inhibit STAT3 activation. Natural AC is scarce, cultured AC mycelia are becoming alternatives. AC mycelia have been demonstrated to possess anti-HCC properties. We hypothesize that inhibition of the STAT3 signaling pathway contributes to the anti-HCC mechanisms of AC mycelia. To test our hypothesis, we evaluated the safety and investigated the anti-HCC effects of the ethyl acetate fraction of an ethanolic extract of AC mycelia (EEAC); and we further explored the involvement of STAT3 signaling in EEAC's anti-HCC effects. Acute and repeated dose 28-day oral toxicity studies showed that EEAC had no toxicity in rats. The maximum tolerable dose for acute oral toxicity and the no-observed-adverse effects level for repeated dose 28-day oral toxicity of EEAC were higher than 5,000 mg/kg body weight and 1,000 mg/kg body weight, respectively, in rats. In cultured cells, EEAC is less toxic in normal liver-derived cells than in HCC cells. In HepG2 and SMMC-7721 cells, EEAC reduced viability, induced apoptosis, and retarded migration and invasion. In SMMC-7721 cell-bearing mice, EEAC significantly suppressed tumor growth. EEAC inhibited cell proliferation, induced apoptosis and suppressed angiogenesis in tumors. Mechanistic studies showed that EEAC downregulated protein levels of phosphorylated and total STAT3 and JAK2 (an upstream kinase of STAT3) in HCC cells and tumors. In cultured HCC cells, EEAC lowered the protein level of nuclear STAT3, decreased the transcriptional activity of STAT3, and downregulated protein levels of STAT3 targeted molecules. Over-activation of STAT3 in HCC cells diminished the cytotoxic effects of EEAC. STAT3 can be activated by receptor tyrosine kinases (RTKs). Phospho-RTK array assays showed that EEAC significantly inhibited the tyrosine phosphorylation of platelet-derived growth factor receptor-beta (PDGFR-β) in HepG2 cells. EEAC dose-dependently lowered mRNA levels of PDGF BB (a ligand of PDGFR-β) and protein levels of p-PDGFR-β and PDGFR-β in HCC cells. Activating PDGFR-β enhanced STAT3 activation, and inhibiting PDGFR-β blocked STAT3 activation in HCC cells. EEAC reversed PDGF BB induced STAT3 activation in HCC cells. Our data indicate that EEAC exerts anti-HCC effects, and inhibition of PDGFR-β/STAT3 signaling is, at least in part, responsible for these effects. In summary, we have demonstrated that EEAC exerts anti-HCC effects without significant toxicity in vitro and in vivo. We have also demonstrated that inhibition of PDGFR-β/STAT3 signaling contributes to the anti-HCC mechanisms of EEAC. Our findings provide a pharmacological basis for the development of EEAC as a modern anti-HCC agent and for the traditional use of AC in treating HCC. In addition, our data suggest that the PDGFR-β/STAT3 pathway plays a pathogenic role and presents a novel therapeutic target in HCC.

Lai, Tsz Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 121-136). / Abstracts in English and Chinese. / Acknowledgements / Abstract / 摘要 / Content / List of tables / List of figures / List of abbreviations / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Introduction of food market trends in Hong Kong and mushroom productivity in the world --- p.1 / Chapter 1.1.1 --- Agrocybe aegerita --- p.1 / Chapter 1.1.2 --- Pleurotus spp --- p.2 / Chapter 1.1.3 --- Pholiota nameko --- p.3 / Chapter 1.2 --- Objectives --- p.5 / Chapter Chapter 2: --- Chemical assays for in vitro antioxidative properties of mushroom extracts --- p.6 / Chapter 2.1 --- Introduction --- p.6 / Chapter 2.1.1 --- Reactive oxygen species (ROS) --- p.6 / Chapter 2.1.1.1 --- Definition of ROS --- p.6 / Chapter 2.1.1.2 --- Sources of ROS --- p.6 / Chapter 2.1.1.2.1 --- Endogenous sources of ROS --- p.6 / Chapter 2.1.1.2.2 --- Exogenous sources of ROS --- p.8 / Chapter 2.1.1.3 --- Damaging effects of ROS --- p.8 / Chapter 2.1.2 --- Antioxidants --- p.10 / Chapter 2.1.2.1 --- Mechanism of action --- p.10 / Chapter 2.1.2.2 --- Sources of antioxidants --- p.11 / Chapter 2.1.2.2.1 --- Dietary antioxidants --- p.11 / Chapter 2.1.2.2.2 --- Antioxidants in edible mushrooms --- p.12 / Chapter 2.1.2.2.3 --- Phenolic compounds in mushrooms --- p.13 / Chapter 2.2 --- Materials and Methods --- p.16 / Chapter 2.2.1 --- Materials --- p.16 / Chapter 2.2.1.1 --- Mushroom fruiting bodies --- p.16 / Chapter 2.2.2 --- Principles of Methods and Experimental Protocols --- p.17 / Chapter 2.2.2.1 --- Sample preparation --- p.17 / Chapter 2.2.2.2 --- Evaluation of antioxidant capacity --- p.18 / Chapter 2.2.2.2.1 --- DPPH radical scavenging activity --- p.18 / Chapter 2.2.2.2.2 --- Superoxide anion scavenging activity --- p.19 / Chapter 2.2.2.2.3 --- Hydroxyl radical scavenging activity --- p.20 / Chapter 2.2.2.2.4 --- Hydrogen peroxide scavenging activity --- p.22 / Chapter 2.2.2.3 --- Determination of phenolic compounds --- p.24 / Chapter 2.2.2.3.1 --- Total phenolic content --- p.24 / Chapter 2.2.2.3.2 --- Identification of phenolic acids --- p.25 / Chapter 2.2.3 --- Statistical analysis --- p.27 / Chapter 2.3 --- Results and Discussion --- p.28 / Chapter 2.3.1 --- Extraction yield --- p.28 / Chapter 2.3.2 --- Evaluation of antioxidant capacity --- p.29 / Chapter 2.3.2.1 --- DPPH radical scavenging activity --- p.29 / Chapter 2.3.2.2 --- Superoxide anion scavenging activity --- p.31 / Chapter 2.3.2.3 --- Hydroxyl radical scavenging activity --- p.33 / Chapter 2.3.2.4 --- Hydrogen peroxide scavenging activity --- p.35 / Chapter 2.3.2.5 --- Comparison of the effective concentrations (EC50) of mushroom water extracts in different antioxidant assays --- p.37 / Chapter 2.3.3 --- Determination of phenolic compounds --- p.38 / Chapter 2.3.3.1 --- Total phenolic content --- p.38 / Chapter 2.3.3.2 --- Identification of phenolic acids --- p.39 / Chapter 2.4 --- Summary --- p.45 / Chapter Chapter 3: --- Anti-angiogenic properties of the Aa water extract --- p.46 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.1.1 --- Angiogenesis --- p.46 / Chapter 3.1.1.1 --- Process of angiogenesis --- p.46 / Chapter 3.1.1.2 --- Regulations of angiogenesis --- p.47 / Chapter 3.1.1.2.1 --- Fibroblast growth factor (bFGF) --- p.47 / Chapter 3.1.1.2.2 --- Vascular endothelial growth factor (VEGF) --- p.48 / Chapter 3.1.2 --- Tumor angiogenesis --- p.49 / Chapter 3.1.2.1 --- ROS generation in tumor cells --- p.50 / Chapter 3.1.2.2 --- Hydrogen peroxide and VEGF --- p.51 / Chapter 3.1.2.3 --- Previous studies on tumor angiogenesis --- p.52 / Chapter 3.1.2.3.1 --- ROS and endothelial cells proliferation --- p.52 / Chapter 3.1.2.3.2 --- VEGF and endothelial cells functions --- p.53 / Chapter 3.1.3 --- Use of antioxidants in cancer treatment --- p.53 / Chapter 3.1.3.1 --- Antioxidant use of cancer therapy --- p.53 / Chapter 3.1.3.2 --- Antioxidant and endothelial cells functions --- p.54 / Chapter 3.1.3.3 --- Anti-angiogenic effects of polyphenols --- p.56 / Chapter 3.1.3.3.1 --- Phenolic acids --- p.56 / Chapter 3.1.3.3.2 --- Tea catechin --- p.57 / Chapter 3.1.3.3.3 --- Resveratrol --- p.57 / Chapter 3.1.3.3.4 --- Genistein --- p.58 / Chapter 3.2 --- Principles of Methods and Experimental Protocols --- p.60 / Chapter 3.2.1 --- Sample preparation --- p.60 / Chapter 3.2.2 --- Toxicity of the Aa water extract --- p.60 / Chapter 3.2.2.1 --- Limulus amebocyte lysate (LAL) test --- p.60 / Chapter 3.2.2.2 --- Toxicity towards normal cells --- p.61 / Chapter 3.2.2.2.1 --- Cell line and its subculture --- p.61 / Chapter 3.2.2.2.2 --- Colorimetric (MTT) assay --- p.62 / Chapter 3.2.3 --- Effect of the Aa water extract on cancer cells --- p.63 / Chapter 3.2.3.1 --- Cell line and its subculture --- p.63 / Chapter 3.2.3.2 --- Redox status --- p.63 / Chapter 3.2.3.3 --- VEGF secretion --- p.65 / Chapter 3.2.4 --- In vitro cell culture anti-angioenesis analysis --- p.66 / Chapter 3.2.4.1 --- Cell line and its subculture --- p.66 / Chapter 3.2.4.2 --- Endothelial cells proliferation --- p.67 / Chapter 3.2.4.3 --- Endothelial cells migration --- p.68 / Chapter 3.2.4.3.1 --- Wound healing assay --- p.68 / Chapter 3.2.4.3.2 --- Transwell culture insert assay --- p.69 / Chapter 3.2.4.4 --- Endothelial cells tubule formation --- p.71 / Chapter 3.2.5 --- In vitro organ culture anti-angiogenesis analysis --- p.72 / Chapter 3.2.5.1 --- Aortic ring assay --- p.72 / Chapter 3.2.6 --- Statistical analysis --- p.74 / Chapter 3.3 --- Results and Discussions --- p.75 / Chapter 3.3.1 --- Toxicity of the Aa water extract --- p.75 / Chapter 3.3.1.1 --- Limulus amebocyte lysate (LAL) test --- p.75 / Chapter 3.3.1.2 --- Toxicity towards normal cells --- p.75 / Chapter 3.3.2 --- Effect of the Aa water extract on cancer cells --- p.77 / Chapter 3.3.2.1 --- Redox status --- p.77 / Chapter 3.3.2.2 --- VEGF secretion --- p.79 / Chapter 3.3.2.3 --- Relationship between intracellular ROS and VEGF secretion detected --- p.80 / Chapter 3.3.3 --- Effect of the Aa water extract on angiogenesis --- p.82 / Chapter 3.3.3.1 --- Endothelial cells proliferation --- p.82 / Chapter 3.3.3.2 --- Endothelial cells migration --- p.84 / Chapter 3.3.3.2.1 --- Wound healing assay --- p.84 / Chapter 3.3.3.2.2 --- Transwell culture insert assay --- p.87 / Chapter 3.3.3.3 --- Endothelial cells tubule formation --- p.90 / Chapter 3.3.3.4 --- Aortic ring assay --- p.97 / Chapter 3.3.4 --- Effect of phenolic acids on endothelial cells --- p.101 / Chapter 3.3.4.1 --- Endothelial cells proliferation --- p.101 / Chapter 3.3.4.2 --- Endothelial cells migration --- p.102 / Chapter 3.3.4.2.1 --- Wound healing assay --- p.102 / Chapter 3.3.4.2.2 --- Transwell culture insert assay --- p.105 / Chapter 3.3.4.3 --- Endothelial cells tubule formation --- p.106 / Chapter 3.3.4.4 --- Aortic ring assay --- p.112 / Chapter 3.4 --- Summary --- p.116 / Chapter Chapter 4 --- Conclusions and future works --- p.118 / References --- p.121

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.

by Ng Tak Pan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 151-168). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / List of Tables --- p.xiii / Chapter Chapter One --- Literature Review / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Nutraceutical and Medicinal Properties of L. edodes --- p.4 / Chapter 1.2.1 --- Nutritional value --- p.4 / Chapter 1.2.2 --- Hypocholesterolaemic Effect --- p.5 / Chapter 1.2.3 --- Anti-tumor Effect --- p.5 / Chapter 1.2.4 --- Anti-viral Effect --- p.6 / Chapter 1.2.5 --- Immunopotentiating Effect --- p.6 / Chapter 1.3 --- Life cycle of L. edodes --- p.7 / Chapter 1.4 --- Environmental factors affecting mycelial growth and fruit body --- p.11 / Chapter 1.4.1 --- Nutrient requirement --- p.11 / Chapter 1.4.2 --- Physical and chemical factors --- p.12 / Chapter 1.5 --- Molecular studies on mushroom development --- p.15 / Chapter 1.5.1 --- Mating-type genes --- p.15 / Chapter 1.5.2 --- Hydrophobins --- p.19 / Chapter 1.5.3 --- Fruiting regulatory genes --- p.23 / Chapter 1.5.4 --- Molecular studies on fruit body development of I. edodes --- p.24 / Chapter 1.5.4.1 --- Identification of L. edodes genes --- p.24 / Chapter 1.5.4.2 --- Functional characterization of L. edodes genes --- p.27 / Chapter 1.5.4.3 --- Transformation in L. edodes --- p.28 / Chapter Chapter Two --- Expressed Sequence Tags (ESTs) of L. edodes / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.2 --- Materials and Methods --- p.33 / Chapter 2.2.1 --- Generation of expressed sequence tag --- p.33 / Chapter 2.2.1.1 --- Mushroom cultivation and RNA extraction --- p.33 / Chapter 2.2.1.2 --- Construction of primordium cDNA library --- p.34 / Chapter 2.2.1.3 --- Mass excision of pBK-CMV plasmid --- p.34 / Chapter 2.2.1.4 --- Random screening of mass excised cDNA clone --- p.38 / Chapter 2.2.1.5 --- Isolation of recombinant plasmid --- p.38 / Chapter 2.2.1.6 --- Generation of 3´ة end partially sequence --- p.39 / Chapter 2.2.1.7 --- Sequence analysis --- p.40 / Chapter 2.2.2 --- Reverse dot-blot Hybridization --- p.40 / Chapter 2.2.2.1 --- PCR amplification of cDNA clone --- p.40 / Chapter 2.2.2.2 --- Membrane preparation --- p.40 / Chapter 2.2.2.3 --- cDNA probe preparation --- p.41 / Chapter 2.2.2.4 --- Hybridization --- p.42 / Chapter 2.2.2.5 --- Stringent washing and autoradiography --- p.43 / Chapter 2.3 --- Results --- p.44 / Chapter 2.3.1 --- Construction of primordium cDNA library --- p.44 / Chapter 2.3.2 --- Screening of recombinant clone --- p.44 / Chapter 2.3.3 --- Isolation and reconfirmation of recombinant plasmid --- p.46 / Chapter 2.3.4 --- Generation of EST --- p.47 / Chapter 2.3.5 --- EST identity --- p.47 / Chapter 2.3.6 --- Reverse dot-blot hybridization --- p.56 / Chapter 2.3.7 --- Analysis of hybridization signal --- p.60 / Chapter 2.4 --- Discussion --- p.71 / Chapter Chapter Three --- Sequence Analysis and Transcriptional Profiling of Genes Encoding GTP-binding Proteins / Chapter 3.1 --- Introduction --- p.78 / Chapter 3.2 --- Materials and Methods --- p.82 / Chapter 3.2.1 --- Sequence manipulation --- p.82 / Chapter 3.2.2 --- Northern blot hybridization --- p.82 / Chapter 3.2.2.1 --- RNA fragmentation by formaldehyde gel electrophoresis --- p.82 / Chapter 3.2.2.2 --- RNA fixation by capillary method --- p.83 / Chapter 3.2.2.3 --- Probe preparation --- p.84 / Chapter 3.2.2.4 --- Hybridization --- p.85 / Chapter 3.2.2.5 --- Stringent washing and autoradiography --- p.85 / Chapter 3.2.3 --- Real-Time SYBR Green RT-PCR --- p.85 / Chapter 3.2.3.1 --- Primer design --- p.85 / Chapter 3.2.3.2 --- RT-PCR reaction --- p.86 / Chapter 3.3 --- Results --- p.88 / Chapter 3.3.1 --- Sequence manipulation --- p.88 / Chapter 3.3.2 --- Transcriptional analysis --- p.103 / Chapter 3.4 --- Discussion --- p.108 / Chapter 3.4.1 --- Heterotrimeric G proteins --- p.108 / Chapter 3.4.2 --- Ras-related protein Rab7 --- p.112 / Chapter 3.4.3 --- Developmentally regulated GTP-binding protein --- p.113 / Chapter Chapter Four --- Yeast Complementation and Over-expression tests of Le.Gβ1 and Le.Gγ1 / Chapter 4.1 --- Introduction --- p.115 / Chapter 4.2 --- Materials and Methods --- p.120 / Chapter 4.2.1 --- "Yeast strains, media and yeast vectors" --- p.120 / Chapter 4.2.2 --- Primer design --- p.121 / Chapter 4.2.3 --- RT-PCR Amplification of Le.Gβ1 and Le.Gγ1 --- p.121 / Chapter 4.2.4 --- Purification of PCR products --- p.122 / Chapter 4.2.5 --- Enzymatic digestion and purification --- p.122 / Chapter 4.2.6 --- Ligation and E. coli transformation --- p.122 / Chapter 4.2.7 --- PCR screening of E. coli transformants --- p.124 / Chapter 4.2.8 --- Plasmids extraction --- p.124 / Chapter 4.2.9 --- Yeast transformation --- p.124 / Chapter 4.2.10 --- Mating test --- p.125 / Chapter 4.3 --- Results --- p.129 / Chapter 4.3.1 --- Cloning of Le.Gβ1 and Le.Gγ1 --- p.129 / Chapter 4.3.2 --- Yeast transformation --- p.129 / Chapter 4.3.3 --- Mating test --- p.130 / Chapter 4.4 --- Discussion --- p.141 / Chapter Chapter Five --- General Discussion --- p.144 / References --- p.151