Antitumor Activities of Polysaccharides Extracted from Sclerotium of Polyporus umbellatus CHENG Chiu-man A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Philosophy in Biology © The Chinese University of Hong Kong September 2004 The Chinese University of Hong Kong holds the copyright of this thesis. Any person(s) intending to use a part or whole of the materials in the thesis in a proposed publication must seek copyright release from the Dean of the Graduate School. 统系fi?書因 g[T 1 •麗 jij UNIVERSITYy^/J N^XLIBRARY SYSTEWy^^ Thesis Committee Professor Vincent E. C. Ooi (Supervisor) Professor Peter C. K. Cheung (Supervisor) Professor Paul P. H. But (Internal Examiner) Professor Y. S. Wong (Internal Examiner) Professor W. N. Leung (External Examiner) Acknowledgements I am deeply grateful to a number of people for their assistance and support for the fulfillment of this thesis. First of all, I am extremely grateful to my supervisor, Professor Vincent E. C. Ooi，who offered me an opportunity to study such an interesting research topic by providing me with a hospitable working environment. I also appreciate him for clarifying concepts of developing drugs from natural products and giving me valuable advice on my thesis writing. I am also deeply grateful to my supervisor, Professor Peter C. K. Cheung and Dr. M. Zhang for highlighting the concepts of polysaccharide characterization, especially extraction techniques as a part of my research project. Their professional comments and suggestions during the phase of the thesis writing also made my work perfectly completed. My gratitude is also extended to the members of my thesis committee, Professor Paul P. H. But, Professor Y. S. Wong and Professor W. N. Leung for their valuable advice and time on my thesis. I am also grateful to Dr. Linda S. M. Ooi for her sincere care and sharing, helpful guidance and professional suggestions on my research project, especially on SDS-PAGE technique. I am sincerely grateful to all the staff of the Department of Biology and LASEC, especially the members of Team A for assistance in my 2-year research. I would like to thank Mr. K. H. Man, a laboratory assistant of Department of Bio logy, and Ms. L. Y. Lee for their generous sharing their animal handling skills with me. Special thanks are extended to my lab technician, Ms S. N. Lim for her technical I support in HPLC and GC work, her encouragement and sincere help when I got into trouble in the experiments. I would like to thank Dr. Lawrence C. M. Chiu for introducing me to the novel technique. A "thank you" never seems to be enough for my friends and lab mates who have supported me in their unique personal ways along the route of this research. Thus I am grateful to and deeply appreciative to the following people: I am indebted to Ms. Elaine, Y. L. Wong for her inspiring sharing and guidance in the use of flow cytometry, together with her sincere assistance and friendship. Without her, I do believe that it was hard for me to overcome the difficulties in my study. I am deeply grateful to Ms. W. S. Ho who usually gives me spiritual and practical support, especially when I was in difficult moments. She is a good listener to share my ups and downs in my research. Her humor and encouragement always keeps me in a good mood to work. I would also like to thank Ms. H. Wang for her truly sharing, regard and supports. I never forget that she stayed and worked with me in laboratory 283 for many working nights. I would also like to express my sincere thanks to my lab mates, especially Ms. Mandy M. Y. To, Ms. Anita W. Y. Li, Mr. Dickson T. S. Ho and Ms. Y. L. Li for their enthusiastic help and emotional supports. Finally, I am deeply indebted to my family particularly my love, Mr. J. Q. Chen for their love, patience and constant supports throughout the two-year study. Without their encouragement and sharing, I definitely would have not enough energy to complete my research work and thesis. II Abstract In the 21 St Century, cancer becomes the second largest cause of death in people of various ages and racial backgrounds. Modern investigations have been focused on the use of immunotherapy to target and remove cancer cells by identifying substances like immunopotentiators and biological response modulators (BRM) that can prevent carcinogenesis and induce carcinostasis. Among various types of BRM, natural products like mushroom polysaccharides have become more and more popular and acceptable because they are natural and harmless to the hosts in general. In the present study, polysaccharides extracted from the sclerotium of Polyporus umbellatus were selected to evaluate their antitumor potentials both in vivo and in vitro. Immunomodulatory effect induced by the polysaccharides was also assessed by using ELISA and mouse cytokine array in the study. Two fractions, PU60a and PU60b, separated by ion exchange chromatography from 60% ethanol-precipitated hot-water crude extract of P. umbellatus, were identified as p-glucans with heteroglycan chains. Fraction PU60a is regarded as a group of homogenous neutral polysaccharides while PU60b is a group of heterogenous acidic polysaccharides with various molecular weights ranged from 19.7 to 607.0 kDa. In the S-180 tumor model using BALB/c mice, PU60b exhibited the highest tumor inhibition ratio (89%) at a dose of 5 mg/kg (i.p). However, no remarkable proliferation inhibition of the cancer cell lines S-180, HL-60, HepG2, MCF-7 and Colon 201 was observed with all fractions isolated from hot-water crude extract (PUw). Hence, the possibility of direct cytotoxicity of PU60b on tumor cells was excluded. Other than that, fraction PU60b could inhibit tumor growth effectively in III BALB/c mice but mediated no tumor regression in athymic nude mice which are genetically defective in immune defense system in the body. It was implied that PU60b was not metabolized to tumor-sensitive toxic substances. Based on the result, the fraction PU60b most likely mediated tumor regression in vivo by the activation of host immune effector mechanism. In the assessment of TNF-a production using ELISA after administration of PU60b, the amount of TNF-a generated by LSP-primed mice on Day 10 was larger than that on Day 0; PU60b-treated mice on Day 10 with LPS stimulation generate a 3-fold increase in the production of TNF-a when compared with the control group. The results reflected that fraction PU60b exhibited immunostimulating effect which might last long in the hosts. Among 32 types of cytokines detected on membranes of mouse cytokine array (Ray Biotech), intensities of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) and macrophage inflammatory protein-2 (MIP-2) in PU60b-treated healthy mice serum were high. These studies could support that TIMP-1 and MIP-2 were probably involved in antitumor activity causing the production of a powerful cascade of chemokines and cytokines in the tumor mass and stimulating an efficient antitumor immunity. IV 摘要 踏入二十一世紀，癌症成爲了全球人類中第二大的致命疾病。現代的癌症治 療研究漸漸重視免疫療法的運用。這些研究主要集中檢驗、應用一些能夠預防 癌症以及抑制癌症的物質如免疫增強劑和生物反應調節劑（BRM)在衆多的� 生 物反應調節劑中，天然產物例如燕類多糖要算是較爲人所能接受，因爲它們是 來自天然的無害物質。 本課題研究的對象是屬多孔菌科的緒苍菌核(Polyporus umbellatus sclewtium).利用體外和體内抗癌作用實驗測試，對從豬等菌核中所提取的緒苳 多糖作出評估。此外，本研究亦透過酵素連結免疫吸附分析（ELISA)以及鼠 類細胞因子微陣列技術（mouse cytokine array)探討多糖於免疫調節功能上可能 扮演或參與的角色。 本試驗主要以熱水提取及60%酒精沉澱的藉苳菌核粗糖（PIJ60),以及離子 交換管柱層析萃取分離PU60a和PU60b兩部位多糖。兩部位多糖經測定同屬附 帶雜多糖的3 -葡聚糖，但不同的是PlJ60a是較純化的中性多糖，而PU60b則 是包含不同分子量的酸性多糖類，其分子量介乎19.7至607.0 kDa之間。 在小鼠肉瘤S-180接種動物模型中，豬茶多糖PU60a及PU60b均顯示出一 定程度的抗腫瘤作用。相比下，PU60b的抗腫瘤的功效較爲顯著，達到百分之 八十九（5mg/kg X10天)。然而，所有水溶性緒苳多糖於體外抗腫瘤測試中未 V 能有效地抑制各種癌細胞株，包括S-180, HL-60, HepG2, MCF-7與及Colon 201 之繁殖，這大抵反映出緒苳多糖的抗腫瘤機制並不是出於直接殺死癌細胞。此 夕卜，PU60a能夠明顯地抑制接種在BALB/C小鼠身上的肉瘤生長，但未能令失 去免疫系統的裸鼠經接種在身上的腫瘤受抑制並消失，這可見PU60b的抗腫瘤 作用不能基於產生一些對癌細胞有毒的物質，而是與免疫細胞產生的關係。綜 合以上實驗結果，PU60b極可能通過激發體内免疫效應細胞而發揮出抗腫瘤的 作用。 在酵素連結免疫吸附分析（ELISA)下，豬苳多糖PU60b能夠持久地提升 小鼠血清的腫瘤壞死因子（TNF-a)濃度。在脂多糖組（LPS)的刺激下，於 觀察期第十天抽取的小鼠血清所產生之TNF-a濃度高於對照組，以及觀察期第 一天所產生的濃度3倍之多。在小鼠細胞因數微陣列技術中，金屬蛋白酵組織 抑制因數-1 (TIMP-1)和巨n筵細胞炎性蛋白-2 (MIP-2)所呈現的濃度頗高。在 相關的研究指出，TIMP-1與MIP-2會牽引其他細胞因子，共同建立抗腫瘤的免 疫力。 VI Contents ACKNOWLEDGEMENTS I ABSTRACT Ill m m V LIST OF FIGURES XII LIST OF TABLES XV LIST OF ABBREVIATIONS XVII CHAPTER 1 LITERATURE REVIEW 1 1.1 General Introduction 1 1.2 Cancer Treatment Modalities 3 1.3 Cancer Immunotherapy 4 1.3.1 Immunomodulation 4 1.3.2 Conventional immunotherapy 5 1.3.2.1 Active immunotherapy 5 1.3.2.2 Passive immunotherapy 5 1.3.3 Novel immunotherapy g 1.3.3.1 Natural Products from Chinese Medicinal Herbs as BRMs 9 1.4 Antitumor polysaccharides from Mushrooms 10 1.4.1 Composition of mushroom antitumor polysaccharides 10 VII 1.4.1.1 p-glucan 10 1.4.1.2 Heteroglucan 11 1.4.1.3 Glycan and polysaccharide-protein complexes 11 1.4.2 Antitumor polysaccharides from Polyporaceae mushrooms 14 1.4.2.1 Lentinan from Lentinus edodes 16 1.4.2.2 Maitake from Grifola frondosa 17 1.4.2.3 Polysaccharides from Ganoderma lucidum 18 1.4.3 Extraction and purification of Polysaccharides from Mushrooms 19 1.4.4 Chemical modification 20 1.5 Polyporus umbellatus (Zhuling) 20 1.5.1.1 Nutritional Contents 21 1.5.1.2 Medicinal properties 21 1.6 The Objective of the Present Project 23 CHAPTER 2 MATERIALS AND METHODS 25 2.1 Extraction 25 2.1.1 Hot-water extraction of crude Polyporus umbellatus polysaccharides 25 2.1.2 Cold-alkaline extraction of crude Polyporus umbellatus polysaccharides 26 2.2 Purification of crude Polyporus umbellatus polysaccharides 29 2.2.1 Preparation of DEAE-cellulose ion exchange column 29 2.2.2 Fractionation of hot-water soluble crude