Leung Pui-Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 226-266). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.iii / ABSTRACT --- p.viii / 撮要 --- p.xii / TABLE OF CONTENTS --- p.xvi / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis & Leukemia --- p.1 / Chapter 1.1.1 --- Introduction to Hematopoiesis and its Regulation --- p.1 / Chapter 1.1.2 --- Leukemia --- p.7 / Chapter 1.1.2.1 --- Classification and Epidemiology of Leukemia --- p.7 / Chapter 1.1.2.2 --- Conventional Approaches to Leukemia Therapy --- p.13 / Chapter 1.1.2.3 --- New Approaches to Leukemia Therapy --- p.16 / Chapter 1.2 --- Coumarins --- p.18 / Chapter 1.2.1 --- Introduction to Coumarins --- p.18 / Chapter 1.2.1.1 --- "Historical Development, Occurrence of Coumarins and their Functions in Plants" --- p.18 / Chapter 1.2.1.2 --- Beneficial Effects of Coumarins for Human Use --- p.20 / Chapter 1.2.2 --- Phytochemistry and Metabolism of Coumarins --- p.22 / Chapter 1.2.2.1 --- Chemical Structures of Coumarins --- p.22 / Chapter 1.2.2.2 --- Biosynthesis of Coumarins --- p.24 / Chapter 1.2.2.3 --- Toxicology of Coumarins --- p.24 / Chapter 1.2.2.4 --- Mode of Entry of Coumarins --- p.25 / Chapter 1.2.2.5 --- Metabolic Pathways of Coumarins --- p.27 / Chapter 1.2.3 --- Pharmacological Activities of Coumarins --- p.30 / Chapter 1.2.3.1 --- Anti-edema and Anti-inflammatory Activities --- p.30 / Chapter 1.2.3.2 --- Cardiovascular Protective Function --- p.32 / Chapter 1.2.3.3 --- Anti-tumor Activity --- p.33 / Chapter 1.2.3.3.1 --- Anti-tumor Activity In Vitro --- p.33 / Chapter 1.2.3.3.2 --- Anti-tumor Activity In Vivo and Clinical Applications --- p.34 / Chapter 1.2.3.4 --- Immunomodulatory Activity --- p.36 / Chapter 1.2.3.4.1 --- Immunological Considerations --- p.36 / Chapter 1.2.3.4.2 --- Immunomodulation In Vitro --- p.37 / Chapter 1.2.3.4.3 --- Immunomodulation In Vivo --- p.38 / Chapter 1.3 --- Aims and Scopes of This Investigation --- p.41 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.43 / Chapter 2.1.1 --- Animals --- p.43 / Chapter 2.1.2 --- Cell Lines --- p.43 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.44 / Chapter 2.1.4 --- Reagents for 3H-Thymidine Incorporation Assay --- p.49 / Chapter 2.1.5 --- Reagents and Buffers for Flow Cytometry --- p.49 / Chapter 2.1.6 --- Reagents for DNA Extraction --- p.52 / Chapter 2.1.7 --- Cell Death Detection ELISAplus Kit --- p.54 / Chapter 2.1.8 --- Reagents for Total RNA Isolation --- p.55 / Chapter 2.1.9 --- Reagents and Buffers for RT-PCR --- p.56 / Chapter 2.1.10 --- Reagents and Buffers for Gel Electrophoresis of Nucleic Aicds --- p.60 / Chapter 2.1.11 --- "Reagents, Buffers and Materials for Western Blot Analysis" --- p.61 / Chapter 2.1.12 --- Reagents for Measuring Caspase Activity --- p.67 / Chapter 2.1.13 --- Reagents for Neutral Red Assay --- p.70 / Chapter 2.2 --- Methods --- p.71 / Chapter 2.2.1 --- Culture of the Tumor Cell Lines --- p.11 / Chapter 2.2.2 --- Determination of Cell Proliferation by [3H]-TdR Incorporation Assay --- p.71 / Chapter 2.2.3 --- Determination of Cell Viability --- p.72 / Chapter 2.2.4 --- In Vivo Anti-tumor Study --- p.73 / Chapter 2.2.5 --- Analysis of Cell Cycle Profile/DNA Content by Flow Cytometry --- p.74 / Chapter 2.2.6 --- Measurement of Apoptosis --- p.74 / Chapter 2.2.7 --- "Islation ,Preparation and Culture of Mouse Peritoneal Macrophages" --- p.76 / Chapter 2.2.8 --- Gene Expression Study --- p.77 / Chapter 2.2.9 --- Protein Expression Study --- p.80 / Chapter 2.2.10 --- Determination of the Mitochondrial Membrane Potential --- p.84 / Chapter 2.2.11 --- Measurement of Caspase Activity --- p.84 / Chapter 2.2.12 --- In Vivo Macrophage Migration Assay --- p.85 / Chapter 2.2.13 --- Study of Endocytic Activity of Macrophages --- p.85 / Chapter 2.2.14 --- Determination of Nitric Oxide Production by Macrophages --- p.86 / Chapter 2.2.15 --- Study of Mitogenesis of Murine Splenocytes --- p.87 / Chapter 2.2.16 --- Analysis of T Cell Population and its Sub-populations --- p.88 / Chapter 2.2.17 --- Measurement of Lymphokine Activated Killer (LAK) Cell Activity by Neutral Red Assay --- p.90 / Chapter 2.2.18 --- Statistical Analysis --- p.91 / Chapter CHAPTER 3: --- STUDIES ON THE ANTI PROLIFERATIVE EFFECT OF COUMARINS ON MACROPHAGE-LIKE LEUKEMIA CELLS / Chapter 3.1 --- Introduction --- p.92 / Chapter 3.2 --- Results --- p.94 / Chapter 3.2.1 --- Anti-proliferative Effect of Coumarins on Various Macrophage-like Leukemia Cell Lines In Vitro --- p.94 / Chapter 3.2.2 --- Kinetic and Reversibility Studies of the Anti-proliferative Effect of Coumarins on Macrophage-like Leukemia PU5-1.8 Cells --- p.105 / Chapter 3.2.3 --- Cytotoxic Effect of Coumarins on Macrophage-like Leukemia PU5-1.8 Cells In Vitro --- p.110 / Chapter 3.2.4 --- Effect of Coumarins on the Cell Cycle Kinetics of the Macrophage-like Leukemia PU5-1.8 Cells In Vitro --- p.113 / Chapter 3.2.5 --- Effect of Coumarins on the Expression of Cell Cycle- and Growth-regulatory Genes in the Macrophage-like Leukemia PU5-1.8 Cells --- p.119 / Chapter 3.2.6 --- Effect of Coumarins on the Expression of Cell Cycle-regulatory Proteins in the Macrophage-like Leukemia PU5-1.8 Cells --- p.123 / Chapter 3.2.7 --- Effect of Coumarins on the In Vivo Tumorigenicity of the Macrophage-like Leukemia PU5-1.8 Cells --- p.127 / Chapter 3.2.8 --- Effect of Coumarins on the In Vivo Growth of the Macrophage- like Leukemia PU5-1.8 Cells in Syngeneic BALB/c Mice --- p.131 / Chapter 3.3 --- Discussion --- p.133 / Chapter CHAPTER 4: --- STUDIES ON THE APOPTOSIS-INDUCING EFFECT OF COUMARINS ON MACROPHAGE-LIKE LEUKEMIA PU5- CELLS --- p.1.8 / Chapter 4.1 --- Introduction --- p.143 / Chapter 4.2 --- Results --- p.152 / Chapter 4.2.1 --- Induction of Apoptosis in the Murine Macrophage-like Leukemia PU5-1.8 Cells by Coumarins --- p.152 / Chapter 4.2.2 --- Modulatory Effect of Coumarins on the Expression of Apoptosis-regulatory Genes in the Macrophage-like Leukemia PU5-1.8 Cells --- p.159 / Chapter 4.2.3 --- Modulatory Effect of Coumarins on the Expression of Apoptosis-regulatory Proteins in the Macrophage-like Leukemia PU5-1.8 Cells --- p.166 / Chapter 4.2.4 --- Effect of Coumarins on the Mitochondrial Membrane Depolarization of the Macrophage-like Leukemia PU5-1.8 Cells --- p.169 / Chapter 4.2.5 --- Effect of Coumarins on the Induction of Caspase Activity in the Macrophage-like Leukemia PU5-1.8 Cells --- p.172 / Chapter 4.2 --- Discussion --- p.177 / Chapter CHAPTER 5: --- STUDIES ON THE IMMUNOMODULATORY EFFECT OF COUMARINS ON MURINE MACROPHAGES AND OTHER IMMUNE CELLS / Chapter 5.1 --- Introduction --- p.184 / Chapter 5.2 --- Results --- p.187 / Chapter 5.2.1 --- Effect of Coumarins on the Viability of Macrophages In Vitro --- p.187 / Chapter 5.2.2 --- Effect of Coumarins on the In Vivo Migration of Macrophages --- p.190 / Chapter 5.2.3 --- Effect of Coumarins on the Endocytic Ability of Murine Macrophages --- p.192 / Chapter 5.2.4 --- Effect of Coumarins on the Nitric Oxide Production by Murine Macrophages --- p.194 / Chapter 5.2.5 --- Effect of Coumarins on the Viability of Murine Splenocytes --- p.199 / Chapter 5.2.6 --- Effect of Coumarins on the Mitogenesis of Murine Splenocytes --- p.201 / Chapter 5.2.7 --- Effect of Coumarins on T-cell Population and its Sub-populations --- p.205 / Chapter 5.2.8 --- Effect of Coumarins on the Induction of Murine LAK Activity --- p.208 / Chapter 5.3 --- Discussion --- p.210 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.218 / REFERENCES --- p.226

No direct stimulation of human peripheral blood mononuclear cells proliferation could be detected with incubation of various AB extracts including JAB80E70 in vitro. However, ex vivo assay performed using BALB/c mice orally fed with either water (control) or JAB80E70 for 28 days indicated for the first time that extract-treated groups significantly lowered the ex vivo mitogen-stimulated lymphocyte proliferation. / Our study started with the preparations of different AB extracts using various solvent systems with or without heating. Among all AB extracts tested, the extract JAB80E70 (extracted with 70% v/v ethanol at 80°C) showed the strongest selective suppression on the growth of human leukemia NB-4 and K-562 cells. The anti-leukemia effect of JAB80E70 was also confirmed in vivo using xenografted NB-4 bearing athymic nude mice model. / Phytochemical studies suggested that the selective anti-leukemia activity of JAB80E70-W-B-1 was retained in a relatively polar sub-fraction. One compound was isolated and identified as adenosine from JAB80E70-W-B-1. To the best of our knowledge, this is the first report of the presence of adenosine in AB, even though it exhibited no anti-leukemia activity in vitro. / The mushroom Agaricus blazei (AB) is traditionally used as a remedy against various cancers, infections and immune-related diseases. In the present study, the underlying mechanisms of the anti-tumor and immunomodulatory effects of AB extracts on human leukemia cells were systematically investigated. / With bioassay-guided fractionation, a sub-fraction (JAB80E70-W-B-1) with almost 5-fold improved selective cytotoxicity towards NB-4 cells was obtained. Both JAB80E70 and JAB80E70-W-B-1 could induce apoptosis characteristic DNA fragmentation and nucleosomes enrichment in NB-4 cells. An increase in the pro-apoptotic and a decrease in the anti-apoptotic Bcl-2 family proteins expression were observed in NB-4 cells treated with JAB80E70-W-B-1. JAB80E70-W-B-1 was also found to enhance activities of caspases 3, 8 and 9 in NB-4 cells. However, the activation of caspases 3 and 9 was not affected by the inhibition of caspase 8. Furthermore, JAB80E70-W-B-1 induced apoptosis in NB-4 cells was accompanied by a significant reduction in mitochondria membrane potential and telomerase activity. These results demonstrated that JAB80E70-W-B-1 induced apoptosis in NB-4 cells was dependent on caspase activity, and involved multiple molecular pathways. / Jiang, Jingjing. / Adviser: Lau Bik San Clara. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3454. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 224-250). / 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.