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 polysaccharide (PU) 29

2.3 Characterization of Polyporus umbellatus Polysaccharides 30 2.3.1 Carbohydrate content determination 30 2.3.2 Gas chromatography (GC) 31 2.3.3 Protein content determination 32 2.3.4 Uronic acid content determination 33 2.3.5 High Performance Liquid Chromatography (HPLC) 34

VIII 2.4 In vivo Antineoplastic Assay 35 2.4.1 Animals 35 2.4.1.1 BALB/c mice 35 2.4.1.2 Athymic BALB/c mice 36 2.4.2 Maintenance of cell lines 36 2.4.2.1 Murine sarcoma 180 36 2.4.2.2 Human breast cancer cell line (MCF-7) 37 2.4.3 S-180 tumor inoculation model using BALB/c mice 37 2.4.4 MCF-7 tumor inoculation model using athymic nude mice 38 2.4.5 Assay of antineoplastic activity with S-180 38 2.4.6 Assay of antineoplastic activity with MCF-7 39 2.4.7 Body weight change 40

2.5 In vitro anti-proliferation assay 41 2.5.1 Cell lines 41 2.5.1.1 Maintenance of cell lines 41 2.5.2 Assay of anti-proliferation with cancer cell lines 42 2.5.2.1 Cytotoxicity assay on suspensions of cancer cells 42 2.5.2.2 Cytotoxicity assay on adhesive cancer cells 42 2.5.2.3 Cytotoxicity assay on normal cells 43 2.5.3 Trypan blue exclusion method 43 2.5.4 MTT assay 44

2.6 Cytokine determination 45 2.6.1 Treatment of mice 45

2.6.2 Enzyme-linked immunosorbent assay (ELISA) for TNF-a production 46 2.6.3 Analysis of mouse cytokine array 47 2.6.3.1 Process of blocking and incubation 47 2.6.3.2 Process of cytokine detection 48

2.7 Statistical Analysis 49

IX CHAPTER 3 RESULTS 50

3.1 Extraction of Polyporus umbellatus polysaccharides (PU) 50 3.1.1 Percentage of Yield in extraction of crude PU extracts 50 3.1.2 Percentage of yield in fractionation of PU fractions 51

3.2 Chemical characterization of PUw fractions 55 3.2.1 Carbohydrate and protein contents of PUw fractions 55 3.2.2 Relative content of monosaccharides and uronic acid in PUw fractions 55 3.2.3 Infrared spectra of PUw fractions 59 3.2.4 Molecular weight estimation of PUw fractions 59

3.3 In vitro anti-proliferative assay 64 3.3.1 Anti-proliferative effects of PU fractions on suspension cancer cell lines 64 3.3.2 Anti-proliferative effects of PU fractions on adhesive cancer cell lines 64 3.3.3 Anti-proliferative effects of PU fractions on normal cell lines 65

3.4 In vivo antineoplastic assay 73 3.4.1 S-180 tumor inoculation model using BALB/c mice 73 3.4.1.1 In vivo antineoplastic effect of crude extracts - PUw and PUal 73 3.4.1.2 In vivo antineoplastic effect of PUw 73 3.4.1.3 In vivo antineoplastic effect of PU60 and PU80 78 3.4.1.4 In vivo antineoplastic effect ofPU60a and b 81 3.4.2 MCF-7 tumor inoculation model using athymic nude mice 81 3.4.2.1 In vivo antineoplastic effect ofPU60b 81 3.4.3 Identification of cytokines in the serum from healthy BALB/c mice using mouse cytokine array system 89 3.4.4 Effect of PU60b on TNF-a generation in healthy BALB/c mice studied with ELISA..…93 3.4.5 Effect ofPU60b on TNF-a generation in S-180 tumor bearing mice studied with ELISA 93

X CHAPTER 4 DISCUSSION 96

4.1 Extraction and Isolation of polysaccharide fractions 96

4.2 Chemical characterization of PUw fractions 97

4.3 In vitro anti-proliferative effect of PU fractions 99

4.4 In vivo antineoplastic assay of PU fractions 101 4.4.1 S-180 solid tumor model using BALB/c mice 101 4.4.2 MCF-7 tumor in vivo model using athymic nude mice 104

4.5 Estimation of Immunomodulatory properties of PU60b 106 4.5.1 Identification of cytokines in serum from healthy BALB/c mice 106

CHAPTER 5 CONCLUSIONS 110

REFERENCES 112

XI List of Figures

Figure 1.1 The significance ofIL-2 on cellular interactions and immune activation for tumor rejection. 7 Figure 1.2 A flow chart of the investigation of immunopharmacological and antitumor activities of polysaccharides of Zhuling. 24 Figure 2.1 a) Sclerotium of Polyporus umbellatus in the wild environment. (Hu, 2001) 27 Figure 2.2 Flow chart illustrating the extraction and fractionation of Polyporus umbellatus polysaccharides. 28 Figure 3.1 Anion exchange chromatography of PU60 on a DEAE-cellulose column. 54 Figure 3.2 Infrared Spectra of (a) PU60 and (b) PU60b. 60 Figure 3.3 Standard curve ofHPLC indicating retention time of the four dextran standard with molecular weights of 25，80，270 and 670 kDa. 61 Figure 3.4 Gel permeation of (a) PUw and (b) PU60 by HPLC. 62 Figure 3.5 Gel permeation of (a) PU60a and (b) PU60b by HPLC. 63 Figure 3.6 The effects of PU fractions on proliferation of S-180 cell line after 72 h incubation. 66 Figure 3.7 The effects of PU fractions on proliferation of HL-60 cell line after 72 h incubation. 67 Figure 3.8 The effects of PU fractions on proliferation of MCF-7 cell line after 72 h incubation. Figure 3.9 The effects of PU fractions on proliferation of HepG2 cell line after 72

XII h incubation. 70 Figure 3.10 The effects of PU fractions on proliferation of Colon 201 cell line after 72 h incubation. 71 Figure 3.11 The effects of PU fractions on proliferation of normal cell line (Vero) after 72 h incubation. 72 Figure 3.12 The Effects of crude extracts (Pw and PAL) on Sarcoma 180 solid tumor in vivo. 75 Figure 3.13 Effects of hot-water crude extract PUw in different concentrations on Sarcoma 180 solid tumor in vivo. 76 Figure 3.14 The effects of polysaccharide fractions PU60a and PU60b in different concentrations on S-180 solid tumor in vivo. 83 Figure 3.15 S-180 solid tumors excised from BALB/c mice under treatment of PU60b at doses of 10/mg/kg and 5 mg/kg. 84 Figure 3.16 The effects of PU60b in different concentrations on MCF-7 tumor bearing athymic nude mice. 85 Figure 3.17 The process of in vivo assay with MCF-7 tumor-inoculating model. ••…87 Figure 3.18 Comparison of MCF-7 tumor size of the athymic nude mice with the treatment of PU60b at doses of 5 mg/kg and 10 mg/kg. 88 Figure 3.19 The images of the mouse cytokine array membranes on films were showed in the figures (a) Treatment group and (b) control group. 91 Figure 3.20 The diagrams revealed the expression of cytokines on the corresponding membranes. 92 Figure 3.21 The effect ofPU60b on serum level ofTNF-a in healthy BALB/c mice. 94 Figure 3.22 The effect ofPU60b on serum level ofTNF-a in S-180 tumor bearing

XIII BALB/cmice 95

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XIV List of Tables

Table 1.1 Biological response modifiers can be categorized into different groups..8 Table 1.2 Antitumor Effects of mushroom water extracts on sarcoma 180 bearing mice 15 Table 2.1 Schedule for in vivo antitumor assays 40 Table 3.1 Percentage yield ofPU fractions extracted from 700 g dried sclerotia of Polyporus umbellatus. 52 Table 3.2 Percentage yields of PU60 and PU80 after ethanol precipitation of 200 g PUw. 52 Table 3.3 Recovery of PU fractions after application of 80 mg PU60 to DEAE-cellulose column. 53 Table 3.4 Carbohydrate and Protein contents in PUw fractions. 57 Table 3.5 Proportions of different monosaccharides and uronic acid in the five PUw fractions determined by Gas Chromatography and colorimetric method. 58 Table 3.6 Percentage of inhibition on proliferation of S-180 66 Table 3.7 Percentage of inhibition on proliferation of HL-60 67 Table 3.8 The effects ofPU fractions on viability of S-180 and HL-60 cell lines after 72 h incubation. 68 Table 3.9 Percentage of inhibition on proliferation of MCF-7 69 Table 3.10 Percentage of inhibition on proliferation of HepG2 70 Table 3.11 Percentage of inhibition on proliferation of Colon 201 71 Table 3.12 Percentage of inhibition on proliferation ofVero cell 72

Table 3.13 The effect of PUAL and PUw on S-180 tumor bearing BALB/c mice 75

XV Table 3.14 The effects of PUw in different concentrations on S-180 tumor bearing BALB/c mice 76 Table 3.15 Effects of hot-water crude extract PUw in different concentrations on body weight of S-180 tumor bearing mice. 77 Table 3.16 The effects of polysaccharide fractions PU60 and PU80 in different concentrations on Sarcoma 180 solid tumor in vivo. 79 Table 3.17 The effects of polysaccharide fractions PU60 and PU80 in different concentrations on body weight of S-180 tumor bearing mice. 80 Table 3.18 The effects of PU60a and PU60b on S-180 tumor bearing BALB/c mice 83 Table 3.19 The effects of polysaccharide fractions PU60b in different concentrations on MCF-7 tumor bearing athymic nude mice. 85 Table 3.20 The effects of PU60b on body weight of S-180 tumor bearing BALB/c mice and MCF-7 tumor bearing athymic mice. 86

Table 3.21 Antibody list on the membrane of RayBioTM Mouse Cytokine Array. 90

XVI List of Abbreviations

ATCC American type culture collection DEAE-cellose Diethylaminoethyl cellulose DMEM Dulbecco's modified eagle's medium ELISA Enzyme linked immunosorbent assay GC Gas chromatography HPLC High performance liquid chromatography i.p. Intraperitoneal i.v. Intravenous IL Interleukin I FN Interferon LPS Lipopolysaccharides MTT 3-(4, 5,-dimethylthiazoI-2-yl)-2, 5-diphenyl tetrazolium bromide MIP-2 Macrophage inflammatory protein-2 PU Polyporus umbellatus s.c. Subcutaneous S.D. Standard deviation S.E.M. Standard error of the mean TIMP-1 Tissue inhibitor of matrix metalloproteinases-1 TNF Tumor necrosis factor Tpo Thrombopoietin VEGF Vascular endothelial growth factor

XVII Chapter 2 Literature Review

Chapter 1

Literature Review

1.1 General Introduction

Cancer, in the 21®^ century, becomes the second largest cause of death in people of various ages and racial backgrounds, which has led to global scientific awareness and plenty of research studies in the fight against the disease. Recent investigations have been carried out on the development of immunotherapy to target and remove cancer cells as well as searching for substances like immunopotentiators and biological response modulators (BRM) that can prevent carcinogenesis and induce carcinostasis (Wasser, 1999).

Since the 1960s，Japanese scientists have revealed that hot-water extracts from mushrooms belonging to the families of Phelinusceae, Tricholomataceae and especially Polyporaceae showed a remarkable host-mediated antitumor activity

-1- Chapter 1 Literature Review against sarcoma 180 grafted on animals (Mizuno, 1995). Most medicinal mushrooms with effects against cancers of the stomach, esophagus and lungs belong to the family Polyporaceae (Mizuno, 1995; Daba and Ezeronye, 2003). The active substances that have antitumor potentials were isolated from these mushrooms afterwards and identified as polysaccharides. Among several healing properties, immunomodulatory and antitumor effects are regarded as their most promising biopharmacological activities (Ooi and Liu, 2000). The significant antitumor activity of mushroom polysaccharides is mainly due to their ability to activate the host immune system rather than direct cytotoxicity (Wasser, 2002). Lentinan, the first thoroughly investigated antitumor polysaccharide extracted from the fruiting body or mycelium oi Lentinus edodes in 1969 by Chihara and coworkers of the National Cancer Institute of Japan (Hobbs, 1995) is a classical example. Instead of attacking cancer cells directly, it serves as a host-defence potentiator, which restores and even enhances the responsiveness of host cells to lymphocytokines, hormone and other biologically active substances like cytokines (Hobbs, 2000). Injection of lentinan in mice produced either 80 % reduction in tumor size or complete regression of tumor in the test animals while treatment of immunosuppressive agents such as 6-benzylthioguanosine or X-radiation with lentinan administration to mice showed reduction of its antitumor effect (cited by Hobbs, 1995). An intriguing conclusion has been drawn that an intact immune system with a functioning thymus gland is a requisite for the anticancer effect of lentinan (Maeda and Chihara, 1973; Dennert and Tucker, 1973; Yamada et al, 1981; cited by Hobbs, 1995). Another well-known antitumor p-glucan, schizophyllan isolated from Schizophyllum commune, is similar to lentinan in the biological activity and mechanism of antitumor action (Jong, 1991). It functions as a T-cell adjuvant and macrophage activator that enables to recover and

-2- Chapter 2 Literature Review even enhance cellular immunity in the tumor-bearing host but it is ineffective in the mice that were neonatally thymectomized and treated with antithymus globulin (Suzuki, 1982; Isamu, 1984; cited by Ooi and Liu, 2000).

Apart from antitumor activities, immunomodulation effects of mushroom polysaccharides also arouse people's attentions. Grifolan, p-D-glucans predominating Maitake, extracted from Grifola frondosa, enhances the activities of macrophages, nature killer (NK) cells and cytotoxic T cells by 1.4, 1.86 and 1.6 times respectively (Mori et al, 1987). Schizophyllan increases cellular immunity by restoring the activity of the suppressed killer-cell to normal level in mice with tumors (cited by Hobbs, 1995) and activates macrophages which lead to augmentation of T cell activities and increase sensitivity of cytotoxic LAK and NK cells to produce interleukin (IL) -2 (Shimizu et al” 1992).

In the present investigation, Polyporus umbellatus, a famous Chinese medicinal mushroom in the family polyporaceae’ was selected to study its antitumor potentials both in vitro and in vivo. In the subsequent sections, more information on this medicinal mushroom together with the relevant literature reviews on this project will be given in detail.

1.2 Cancer Treatment Modalities

Cancer treatment modalities, up to now, can be categorized into local therapies and systemic therapies. Surgery and radiotherapy are examples of local therapies while chemotherapy is an example of systemic therapies. Depending on the type of cancer, tumor discovered before metastasis occurs can normally be cured with localized

-3- Chapter 2 Literature Review treatment. Nevertheless, early tumor information is hardly detected for many forms of cancers and even small tumors may metastasize. Because of the undetectable metastasis, many modes of local therapies fail and in turn systemic therapies, chemotherapy, is widely used after localized treatment. Unfortunately, most of the chemotherapeutic agents developed in the past two decades are cytotoxins that are not specific only to cancer cells, but also to normal cells. In addition, those cytotoxic chemotherapeutic agents can cause self-limiting adverse reactions and even death in a host. Many chemotherapeutic agents are even carcinogenic, thus inducing secondary tumors due to immunosuppression (BoIk, 2001).

1.3 Cancer Immunotherapy

1.3.1 I mmuno modulation

The major task of the immune system of a host is to defend against the attack of foreign pathogens. Immune surveillance, first proposed by Ehrlich in 1909，plays a critical role in preventing tumor development by searching out and destroying newly transformed cells (BoIk, 2001). Tumor cells recognized as transformed self-cells are present as foreign pathogens in the body, which may trigger actions of the immune system. It is believed that the nature of immune response is primarily determined by the nature of cytokine (Rincon et al, 1997). Cytokines are a group of low-molecular-weight regulatory proteins or glycoproteins secreted by leukocytes and a variety of other cells involved in the immune system; exerting a variety of effects on lymphocytes and other immune cells. Cytokine binds to specific receptor on the membrane of target cells, eliciting both biochemical and morphological changes responsible for signal transduction, which leads to transformed pattern of gene expression in the target cells. Recent investigations have focused on the effect of

-4- Chapter 1 Literature Review cytokines on activation and differentiation of T cells into Thl or Th2 subset (Schultze et al, 1999). Some of these cytokines exhibit cross-regulatory effect, on one hand enhancing their generation and a type of clone production and on the other hand suppressing production of other type of cytokines (Scott and Welt, 1997; Schultze et al, 1999; Inoue et al.’ 2002).

1.3.2 Conventional immunotherapy

Conventional cancer immunotherapy is to either augment or supplement these natural defense actions. In general, this kind of treatment in human is more effective in patients with a relatively healthy immune system and a low tumor burden such as patient at an early stage of malignancy (Bolk, 2001). It can be classified into two overlapping categories: active and passive immunotherapy.

1.3.2.1 Active immunotherapy

Active immunotherapy refers to methods stimulating the host cellular or humoral antitumor immunity. This is often accomplished specifically by using tumor vaccines to generate an immune response to tumor-associated antigens, or by injection with non-tumor-specific antigens, such as microbial and fungal agents.

1.3.2.2 Passive immunotherapy

Passive immunotherapy refers to administration of agents like serum, immune cells from immunized animals and monoclonal antibodies together with cytokines that enable to increase immune activity in the host. Cytokines, IL-2 and tumor necrosis factor-alpha (TNF-a), are clinically used for cancer treatment. IL-2, a glycoprotein with molecular weight (MW) of 15 kDa, is predominately generated by activated T

-5- Chapter 2 Literature Review cells, especially CD4+ lymphocytes and CD8+ in small amounts (Goldsby, 2002). Various cytokines have been tested in combination with IL-2 to activate cytotoxic cells like lymphokine-activated killer cells (LAK) and also induce a lymphokine cascade with the induction of IFN-y, TNF-a, IL-1 and IL-6, which improves cytotoxic activities and clinical results in cancer patients (Bergmanna, 1994). Furthermore, the modulation of susceptibility of tumor cells through up-regulation of adhesion molecules, MHC class I/II antigens or tumor-associated antigens is also enhanced (Figure 1.1). TNF-a, released from macrophages and monocytes, is a growth factor for fibroblasts and is responsible for cachexia in cancer patients. In some instances, TNF-a may exhibit direct antitumor activity, killing some tumor cells, reducing the rate of proliferation of sparing normal cells as well as inhibiting tumor-induced angiogenesis by damaging the vascular endothelial cells in the vicinity of a tumor (Goldsby, 2002). In addition, TNF-a has a strong ability to increase the expression of epidermal growth factor receptor (EGFR) via the p55 TNF receptor (Schmiegel, 1997). In the presence of TNF-ot, tumors undergo visible hemorrhagic necrosis and regression as well.

-6- Chapter I Literature Review

Activation/t of Inductionx of Up-regulatio n autologous secondary of surface cytotoxic cells cytokines antigens on tumor cells and effector cells 今 IFN-Y 今 NK cells � TNF-a 今 MHC class l/ll 今 CTL 今 IL-6 今 Tumor-associated 今 IL-8 antigens � CSF 令 Adhesion (GM-CSF, molecules ll__3) 今 Other antigens or receptors

(Bergmanna, 1994)

Figure 1.1 The significance of IL-2 on cellular interactions and immune activation for tumor rejection.

-7- Chapter 2 Literature Review On the other hands, passive immunotherapy with biological response modifiers (BRM) in Table 1.1 may be more successful. Any agents or approaches that enhance interactions between tumors and host via modification of host response to tumors are regarded as BRM.

Table 1.1 Biological response modifiers can be categorized into different groups. Type of BRM Examples Effect cell Macrophages, NK cell, T cell clone, Tumor-infiltrating lymphocytes (TIL) Cytokine IL-1, IL-2, TNF-a, IFN-a, IFN-p, IFN-y Monoclonal antibody Monoclonal antibody to growth-promoting factor, Anti-T cell antibody, Anti-T suppressor cell antibody Natural product Lentinan, Schizophyllan and Krestin (PSK) Bacterial product Muramyl dipeptide, Endotoxin, Trehalase dimycolate Synthetic molecule Pyran copolymer, Malic anhydride-divinyl ether, Pyrimidine Hormone Thymosin, Thymulin, Thymopoietin (Wong, 1993; Hobbs, 1995; Goldsby, 2002)

1.3.3 Novel immunotherapy

The immune response of the tumor-bearing host towards the tumor involves complicating interactions among the effector cells, the cytokines and the corresponding secreted antibodies. Application of substances such as immunopotentiators and biological response modulators (BRM) to inhibit tumor growth is currently a novel and safe way to fight against cancer without harmful

-8- Chapter 2 Literature Review effects to the host.

1.3.3.1 Natural Products from Chinese Medicinal Herbs as BRMs

Traditional Chinese medicine (TCM) is mainly applied based on the folklore or daily experience accumulated by Chinese people from generations to generations. Chinese medicine is more patient-oriented than disease-oriented. Instead of direct killing of cancer cells, TCM treatments usually restore the equilibrium in the human body, mobilizing the internal ability of the body to resist disease (Boik, 1996). This is difference from the idea of western medicine that puts emphasis mainly on etiology and the treatment of local condition. Other than that, toxic side effects mentioned above after chemotherapy in malignant tumors are one of the disadvantages of western medicine.

Many Chinese traditional herbs like mushrooms were regarded as BRMs in cancer therapy. BRMs provide a different mode of action from the conventional chemotherapeutic agents because it is immunotherapeutic. Among the dominant immunologically active compounds, several high-molecular-weight glycoproteins, polysaccharides, nucleotides and proteins can be isolated from both edible and medicinal mushrooms. To evaluate the immuno-pharmacological potentials of mushroom bioactive substances, three levels of immunological tests should be applied (Leung, 1994): Lymphocytes count, lymphocyte morphology and determination of immunoglobulin level are included in the first level test; histochemical analysis of lymphoid organs, lymphocyte surface marker analysis and effector cells like NK and LAK cells medicated cytotoxicity are included in the second level; the measurement of enzymes such as adenosine deaminase, purine mucleoside phosphorylase, cytokines

-9- Chapter 2 Literature Review namely IL-2, TNF-a and INF, phagocyte studies together with genetic analysis such as Northern blotting of specific mRNA are the third level study.

1.4 Antitumor polysaccharides from Mushrooms

1.4.1 Composition of mushroom antitumor polysaccharides

Because of the high potential for structural variability that monosaccharides units in polysaccharide can interconnect at several points to form wide variety of branched or linear structures (Sharon and Lis, 1993), polysaccharides are a structurally diverse class of macromolecules which enable to offer the highest capacity for carrying biological information (Wasser, 2002) and provide the necessary flexibility to the precise regulatory mechanisms of various cell-cell interactions in higher organisms (Ooi and Liu, 2000; Daba and Ezeronye, 2003). Most of the clinical evidence stated that anti-tumor polysaccharides isolated from mushrooms like lentinan, krestin (PSK) and schizophyllan are mainly composed of p-(l—3)-linked D-glucans as the skeleton with various degree of branches substituted on the main chain through (1—6)-linkages.

1.4.1.1 p-glucan

A special group of p-(l—3)-linked polyglucose usually named "glucan" or "p-glucan" which produce D-glucose with acid hydrolysis (Kogan, 2000; Freimund, 2003). The main source of antitumor polysacchrides is related to cell walls which consist of polysaccharides like chitin, cellulose, p-(l—>3), (1—6)-glucans and a- (1—>3)-glucans or polysaccharide-protein complex. The homo- and hetero-glucans with p-(l—>3), p-(l->4) and p-(l->6) glucosidic linkages probably play a key role in some healthy properties of mushrooms such as enhancement of macrophage ftinction and host

-10- Chapter 1 Literature Review resistance to bacterial, fungal, parasitic and viral infections, activation of non-specific immune stimulation, reduction of blood glucose and blood cholesterol levels (Cheung, 1998; Manzi, 2000). Some mushroom antitumor polysaccharides which bind to protein residues are regarded as polysaccharide-protein complexes.

1.4.1.2 Heteroglucan

Besides the well-known antitumor p-(l->3)-glucan, a wide range of biologically active glucans with other composition have linear or branched molecules in a backbone with a- or p-linkage of glucose (Wasser, 2002). Heteroglucan contains side chains including glucuronic acid, xylose, galactose, mannose, arabinose or ribose in different combinations. Ganoderma lucidum contains various kinds of heteroglucans such as glucuronoglucan, xyloglucan, mannoglucan and xylomannoglucan (Mizuno, 1999).

1.4.1.3 Glycan and polysaccharide-protein complexes

Many polysaccharides isolated from mushrooms are either homoglycans or heteroglycans. Glycans, in general, are polysaccharides containing units other than glucose in their backbone. Based on their individual sugar components in the backbone, they are classified as flicans, galactans, xylans and mannans. Heteroglycans contain side chains linked with arabinose, mannose, fucose, galactose, xylose and glucuronic acid and glucose in different combinations (Wasser, 2002). Polysaccharide-protein complexes are referred to polysaccharides bound to protein residues. PSK isolated from Coriolus versicolor is a typical example. It is a p-(l—4)glucan-protein complex with p-(l —>6)-glucopyranosidic side chains

-II- Chapter 1 Literature Review including 25 - 38 % protein residues. The major constituent sugar is glucose with small amounts of mannose, fucose, xylose and galactose. The protein residues of PSK mainly composed of acidic amino acids such as aspartic acid and glutamic acid with small amounts of basic amino acids such as lysine and arginine (Ooi and Liu,

2000).

1.4.1.3.1 Immunomodulatory role of p-glucan

The immunomodulatory activities ofp-glucans are still far from being understood up to now. Due to the use of p-glucans derived from various fungal sources with different MW and chemical modifications, the literature referring to the biological effects of p-glucans is inconsistent and often contradictory. In fact, immunomodulatory effects of P-glucans are also influenced by their degree of branching, polymer lengths and tertiary structure. Since the 1980s, Agaricus blazei was recognized to be an immune system stimulant. The immunostimulating polysaccharides extracted from this mushroom could promote the body's natural defense mechanisms to fight against diseases such as virus infection and cancer. Their main components was p-(l">6)-; p-(l-»3)-gIucan, acidic (3-(1^6)-; a .(l_>4)_glucan, and acidic a -(l->3)-glucan (Mizuno, 1990). Unlike the well-known p.(l_>3)-glucan, the antitumor polysaccharides of Agaricus blazei contain glucan with a (3-(l->6)-link:ed backbone. Another antitumor polysaccharide of Agaricus blazei’ HM3-G is a proteoglucan with mainly glucose (> 90 %) which mediated activation of natural killer cells (Wasser, 2002).

Studies on large MW or particulate p-glucans like zymosan demonstrated that they may directly activate leukocytes, stimulating their phagocytic, cytotoxic and

-12- Chapter 1 Literature Review antimicrobial activities in vitro. These carbohydrates stimulate the production of cytokines and chemokines namely IL-lp, IL-6, IL-8 and TNF-a (Brown, 2003). In addition, stimulation of p-glucans with large MW also enhances the activities of macrophages to recognize and clear apoptotic cells via up-regulation of the PS receptors. On the contrary, intermediate or low MW p-glucans such as glucan phosphate has less cellular effects in vitro, but exhibit biological activities in vivo. A classical example is Lentinan [400 - 800 kDa (MW)] which can stimulates activities of macrophages, NK-cells, T and B lymphocytes. Its primary structures comprise a p-(l->3)-glucan including five p-(l-»3)-glucose residues in a linear linkage and two p-( 1 -»6)-gIucopyranoside branches in side chains which form a right-handed triple helical structure (Ooi and Liu, 2000).

1.4.1.3.2 Structure-Activity Relationship of p-glucan

A critical factor contributing to the characteristics of the antitumor and immunomodulatory effects of p-glucans is their ultrastructure (Yadomae, 1996; Wasser, 2002). Though it is difficult to correlate the structure and antitumor activity of complex polysaccharides, their relationships can be inferred. The above quoted examples showed that structural features such as p-(l-^3) linkages in the main chain and p-(1^6)- branched chain are needed for antitumor action, p-glucans containing mainly (l->6)-linkages in the main chain have less activity. It is known that soluble p-(l->3)-D-glucan may exist in three kinds of conformers: the single helix, triple helix and random coil (Ohno, 1987a and b; Ooi and Liu, 2000). Ohno (1995) believed that the antitumor activity of p-glucans obviously depends on the helical conformation. A triple-helical tertiary conformation of p-(l->3)-glucan is important for their immunostimulating activity (Wasser, 2002). Schizophyllan in form of triple helix

-II- Chapter 1 Literature Review

Structure exhibits pronounced antitumor activity while schizphyllan-OH in form of a single helix structure derived from the alkaline-treated schizophyllan diminishes its ability to inhibit tumor growth (Chihara, 1992). The same results which verify the correlation between antitumor activity and triple helix structure were obtained upon the investigation of lentinan (Maeda, 1988).

1.4.2 Antitumor polysaccharides from Polyporaceae mushrooms

In 1968, notable host-mediated antitumor potential of mushrooms against cancer like sarcoma 180 was discovered by Ikegawa et al. He claimed that most of the antitumor mushrooms mainly belong to the families of Polyporaceae and Tricholomataceae Table 1.2). In the following parts, the polysaccharides extracted from Lentinus edodes, Grifola frondosa and Ganoderma lucidum are selected to introduce their promising immunomodulatory and antitumor effects in detail.

-II- Chapter 1 Literature Review

Table 1.2 Antitumor Effects of mushroom water extracts on sarcoma 180 bearing mice Scientific Name Family Polysaccharides Tumor inhibition obtained (%) Coriolus versicolor Polyporaceae p-Glucan; 77.5 p-Glucan-protein Coriolus hirsutus Polyporaceae - 65.0 Coriolus pubesscens Polyporaceae - 59.5 Daedaleopsis tricolor Polyporaceae - 70.2 Favolus alveolarius Polyporaceae - 71.9 Ganoderma Polyporaceae P-Glucan 64.9 applanatum Ganoderma lucidum Polyporaceae p-Glucan; - Hetero-p-glucan Ganoderma tsugae Polyporaceae - 77.8 Grifola frondosa Polyporaceae p-Glucan; 86.6 Acidic p-Glucan Trametes dickinsii Polyporaceae - 80.1 Trametes gibbosa Polyporaceae - 49.2 Lentinus edodes Tricholomataceae p-Glucan 80.7 Flammulina velutipes Tricholomataceae p-GIucan-protein 81.1 Pleurotus ostreatus Tricholomataceae p-Glucan 75.3 Tricholoma matsutake Tricholomataceae - 91.8

(Mizuno et al” 1995;Hobbs, 1995)~

-II- Chapter 2 Literature Review 1.4.2.1 Lentinan from Lentinus edodes

Lentinan, produced from Lentinus edodes, is a p-(l—>3), (1—6)-D-glucan with effective antitumor and immunopotentiating activity. A right-handed triple helical structure of lentinan is formed by its primary structure, p-(l—>-3)-D-glucan consisting of five p-(l—>3)-glucose residues in a linear linkage and two P-(l—6)-glucopyranoside branches in side chains (Ooi and Liu, 2000). The prepared glucan are usually heterogeneous and lentinan has particularly large MW ranging from 400 to 1000 kDa. Its oral bioavailability is reportedly limited; thus, it has been routinely administered intravenously. Lentinan, indicated by Open-label clinical studies, may prolong life in cancer patients especially those with gastric or colorectal carcinomas (Fume, et al, 1981). Tumor responses and prolonged median survival time of patients with recurrent gastric cancer were also noted (Daba and Ezeronye, 2003). Lentinan has been satisfactorily proven to potentiate human immunity (Wasser, 1999). The most intriguing aspect of lentinan use in conjunction with other chemotherapeutic agents such as cyclophosphamide is its apparent ability to greatly reduce the debilitating effects of the chemotherapy such as nausea, pain, hair loss and lowered immune status.

Lentinan does not attack cancer cells directly, but exhibits the anti-tumor effect by activating various immune responses in the host. It can stimulate peripheral blood lymphocytes to increased lymphokine-activated killer (LAK) cell and natural killer activity mediated by interkeukin 2 both in vitro and in vivo (Arinaga, et al.’ 1992; Fujimoto, et al., 1992 and Tani, et al.，1993). Other than these, lentinan can also inhibit both prostaglandin synthesis, which decelerates T-cell differentiation in animals and humans (Aoki, 1984)，and suppressor T-cell activity in vivo. In addition,

-16- Chapter 1 Literature Review lentinan enables to increase the ratio of activated T helper cells and cytotoxic T cell in the spleens of gastric cancer patients with chemotherapy (Hobbs, 2000). Miyakoshi and Aoki (1984) revealed that lentinan enables to stimulate Ig production in human cells in vitro and increase level of interferon in the peripheral blood circulation of cancer patients.

1.4.2.2 Maitake from Grifola frondosa

Maitake, p-D-glucan isolated from Grifola frondosa, was reported to have strong antitumor activity in xenographs (Kurashiga, et al.’ 1997). Crude maitake powder and Maitake D-fraction have demonstrated their considerable inhibition of hepatic metastasis in a series of experiment using a mouse model: 81 % inhibition was observed by crude Maitake powder while 91 % inhibition was performed by D-fraction (Nanba, 1995). In 1997，Nanba discovered that significant symptomatic improvement or tumor regression in 11 out of 15 hepatocellular carcinomas with D-fraction plus Maitake. Combination of D-fraction plus Maitake with chemotherapy made the overall response rates increased by 12 to 28 %•

Another highly purified polysaccharide from Grifola frondosa, p-glucan (Grifron-D, GD) that is a p-(l-> 6) glucan branched with a P-(l->3) linkage has also been revealed to have remarkable immunomodulatory and antitumor effects on animal models (Nishida et al, 1988; Daba and Ezeronye, 2003). Fullerton, et al (2000) observed the cytotoxic effect of GD on human prostate cancer cells (PC 9) in vitro. He implied that oxidative stress is a possible action causing 95 % cell death in apoptosis. The Food and Drug Administration has approved Grifon-D for clinical trials under an investigational New Drug Application (IND) for patients with advanced cancer

-17- Chapter 2 Literature Review (Nanba, 1997).

1.4.2.3 Polysaccharides from Ganoderma lucidum

Ganoderma lucidum and other related Ganoderma species have been used extensively as "mushroom of immortality" in China and other Asian countries for thousands of years (Hobbs, 1995). However, pure p-glucan product derived from this particular mushroom has not been commercially available until now. It is because polysaccharides extracted from the mycelium and fruiting body of Ganoderma species such as G. applanatum, G lucidum and G. tsugae showed promising antitumor and immunostimulating activities with qualitative and quantitative difference in the average molecular weight, sugar composition and protein contents.

A p-D-glucan called G-1 (Mizuno et al” 1982; Wasser and Weis, 1999; Gao and Zhou, 2002) exhibits potent action against sarcoma 180 (Willard, 1990) while a polysaccharide-rich fraction of G. lucidum has demonstrated to be a polysaccharide-protein complex with the ability to perform mitogenicity and stimulate immune effector cells like macrophages, NK and T cells to generate more tumor necrosis factor (TNF-a) and a number of interleukins (Gao and Zhou, 2002). The sugar portion of the protein bound polysaccharides contains glucose, galactose, mannose, and traces of xylose and flicose while the protein portion is composed of 17 amino acids (Hobbs, 1995).

There is evidence that the P-D-glucans induce a biological response by binding to membrane complement receptor type 3 (CR3) consisting of the otm (CD lib) and P2 (CD 18) chains on immune effector cells such as macrophages (Battle et al, 1998;

-18- Chapter I Literature Review Mueller et al.’ 2000; Brown, 2003). The p-D-glucan binding site, a lectin domain of CR3, maps to a site C-terminal to the I-domain (Muller et al” 1996) and its distinct metal ion dependent adhesion site for the many protein ligands of CR3 like iC3b, ICAM-1 and fibrinogen (Diamond et al, 1993; Thornton et al, 1996). The ligand-receptor complex can be internalized (Muller et al,’ 1996). Though the intercellular events occurring after glucan receptor binding have not been fully understood, it is believed that p-D-glucan can help override the resistance of iC3b-opsonized tumor cells to the cytotoxic activation of phagocyte and NK cell CR3, allowing the effector mechanism of the complement system to function against tumor cells (Xia et al., 1999).

1.4.3 Extraction and purification of Polysaccharides from Mushrooms

There is consideration in deciding methods of polysaccharide extraction. As briefly mentioned in section 2.3.1.1.1, antitumor and immunnomodulatory effects of P-glucans are definitely affected by many factors such as their chemical composition, solubility in water, size of molecules, degrees and modes of branching together with binding modes of p-(l->6) to the main sugar chain of 3) (Mizuno, et al, 1995; Ooi and Liu, 2000). Some findings on this issue stated that the conformation of glucans used for determining biological activities is strongly dependent on the extraction and purification procedures (Yadomae and Ohno, 1996).

Based on various principles for isolating and purifying polysaccharides from mushrooms, a list of method is summarized as below (Wong, 1993): (1) Solubilty: i) fractional precipitation, ii) fractional solution, iii) distribution between immiscible solvents; (2) Ultracentrifugation: i) density gradient techniques; (3) Memebrane

-19- Chapter 2 Literature Review ultrafiltration; (4) Chromatography: i) adsorption, ii) ion-exchange, iii) partition chromatography; (5) Gel filtration.

1.4.4 Chemical modification

Many studies demonstrated that chemical modification of mushroom polysaccharides is a possible technique to improve their antitumor activity. Water-insoluble p-glucans extracted from Cordyceps ophioglossoides and Aurilcularia auricula are relevant examples which can be converted into water soluble ones by carboxymethylation, hydroxyethylation and Smith degradation etc. so that their biological activities become higher (Mizuno, 1995). Some mushroom polysaccharides like Pachyman of p-glucan isolated from sclerotium of Poria cocos fail to show antitiumor activity originally. After Smith degradation, treatment of urea and hydroxyethylation, pachyman was respectively converted into pachymaran, U-pachyman and hydroxyethylpachyman exhibiting strong antitumor activity (Chihara, 1970).

1.5 Polyporus umbellatus (Zhuling)

Polyporus umbellatus is a well-known Chinese medicinal Basidiomyceta fungus belonging to the order Polyporales，and family Polyporaceae (Hu, 2001). This mushroom is also referred to as Grifola umbellata (Pers.: Fries) or polyporus sclerotium. Sometimes it is named as Zhu ling in China and Choreimaitake in Japan (Stamets, 1993; Hobbs, 1995). It is mainly found in the temperate regions of China like Fu Jian, Si Chun, and Yun Nan provinces but infrequently occur throughout the deciduous woodlands of northeastern North America (Hu, 2001; Hobbs, 1995). Its grayish white circular fruiting body with central stems arising from underground is a

-20- Chapter 2 Literature Review gourmet mushroom known as "Hog Tuber Flowers" while its sclerotium elongating lumps with tuberous or branched ginger-like structures is a Chinese medicinal herb that has been reported in China to have anti-tumor and immunodulatory effect (Hu, 2001; Ooi, 2001; Zhang, 2001). One thing should be reminded that the similar appearance of P. umbellatus and Grifola fwndosa readily confuses amateur collectors. Grifola frondosa, macro scop ically appears to be a close relative to P. umbellatus, but the two mushrooms have biologically unique life cycle. Grifola fwndosa lacks the sclerotial stage in its life cycle and arises from a multiple forking base.

1.5.1.1 Nutritional Contents

The sclerotia of P. umbellatus with its woody texture have substantially more polysaccharides and less protein than the fleshy fruitbody. According to Hu (2001), 80 % of the whole nutrient components of P. umbellatus sclerotia is polysaccharide. Other nutrients isolated from this mushroom are proteins, ergosterol, a-hydroxy-tetracosanoic acid, and biotin. Its mineral contents (ppm) like calcium (23,400), iron (1,800), potassium (1,010) and sodium (103) were also measured by Suzuki et al. (1982). An interesting finding by Guo et al. (1992) was that 2-year-old P. umbellatus contains polysaccharides and ergosterol much more that those of 1 and 3-year-old P. umbellatus.

1.5.1.2 Medicinal properties

P- umbellatus (Zhuling) used in traditional medicine is considered to have a sweet and bland taste (Bensky and Gamble, 1993). Zhuling primarily serves as a diuretic; meanwhile, it also acts as a drug exhibiting liver protection, antitumor and immunostimulating effects (Hu, 2001; Zhang, 2001).

-21- Chapter 2 Literature Review 1.5.1.2.1 Diuretic effect

Diuretic effect is the most important use of Zhuling in TCM. It serves as a diuretic for promoting urination and leaving out dampness. Some problems arose by stagnance of dampness such as edema, scanty urine, painful urination and urinary tract infections, diarrhea, jaundice together with leucorrhea were treated by Zhuling (Yuan et al, 2004; Bensky and Gamble, 1993; Chang and But, 1986). The mechanism of this property is that Zhu ling increases urine output and elevates excretion of sodium and chloride by inhibiting active transport of chloride and blocking sodium reabsorption in the ascending loop ofHelene (Huang et al., 1985).

1.5.1.2.2 Antitumor and immunomodulatory activity

Zhu ling has been announced to possess potent anticancer and immunopotentiating properties in China for long time. However few scientific studies have been conducted. According to the limited studies in the 1980s, not only crude and refined hot-water extracts of Zhu ling (Miyazaki, 1979，Ueno，et al 1982, Chang and But, 1986 and Ying, et al.’ 1987), but also its ethanolic extract demonstrated remarkable antitumor effects against sarcoma 180，hepatoma and other types of experimentally-induced cancers in a number of in vitro and in vivo studies. It is believed that the extracts enable to promote the Ig M production and strengthen the phagocytic power of monocytes (Chang and But, 1986); moreover, it improves cellular immunity such as macrophage proliferation and activity in normal and liver-compromised mice (Zhang et al” 1991).

-22- Chapter 1 Literature Review 1.6 The Objective of the Present Project

In the present investigation, Polyporus umbellatus under the family Polyporaceae, was selected to study its antitumor potentials both in vitro and in vivo. The sclerotium of the mushroom possesses medicinal properties figured prominently in Chinese pharmacopeia. Numerous Chinese physicians administer extracts of sclerotia of Polyporus umbellatus in the treatment of lung cancer, cervix cancer, liver cancer leukemia and cancer of mammary glands (Bo and Yun, 1980). Nevertheless, the quality of the pharmalogical studies and instrumentation in China is much variable and their protocols are not as systematic as western ones accepted for scientific testing and evaluation. Hence, clarification of both in vitro and in vivo antitumor effects of extracts, especially its polysaccharides components, from sclerotia of Polyporus umbellatus is necessary.

Apart from this, polysaccharides of Polyporus umbellatus were noted to provide nonspecific immunostimulant or immunopotentiating action (Chang and But, 1986). A courageous hypothesis is made that immunomodulatory effects induced by Polyporus umbellatus polysaccharides may have correlation to tumor regression. To verify this hypothesis, enzyme-linked immunosorbent assay (ELISA) and mouse cytokine array were conducted to determine the effects of cytokines and T cells productions in animal models. The flow chart illustrating the investigation of immunopharmacological and antitumor activities of polysaccharides extracted from Polyporus umbellatus sclerotia (Figure 1.2).

-II- Chapter 1 Literature Review

一 Dried Sclerotium of Polyporus umbellatus

^ r Primary purification: (1) Hot-water/ alkaline extraction (2) Ethanol precipitation (3) DEAE gel filtration

J r Antitumor activity in vitro and in vivo

I I • I i I In vitro - In vitro + In vitro - In vitro + In vivo - In vivo - In vivo + In vivo +

1 r Direct cytotoxic ^ effect on tumor cells ^ r Immunotoxicological studies ^ r Immunostimulating studies

• Athymic nudemice model • ELISA • Mouse cytokine array

y r Host-mediated antitumor activities

Figure 1.2 A flow chart of the investigation of immunopharmacological and antitumor activities of polysaccharides of Zhuling.

-II- Chapter 1 Materials and Methods

Chapter 2

Materials and Methods

2.1 Extraction

2.1.1 Hot-water extraction of crude Polyporus umbellatus polysaccharides

Dried sclerotium of Polyporus umbellatus (Figure 2.1) was purchased from a Guang Zhou herbal dealer. The sample was washed and ground into small pieces with a blender before boiling three times in distilled water [sample: distilled water (w/v) = 1 ： 7] for two hours each time (Figure 2.2). After each sample boiling, the residue and the insoluble components in the water extract were isolated by filtration with gauze and centrifugation at 15,600 g for 30 minutes with the aid of Beckman J2-MI centrifuge and a JA-14 rotor. The clear supernatants of each crude sample extract were pooled and then concentrated by a rotary evaporator (BUCHI Rotavapor R-200). Absolute ethanol was gently added into the part of the concentrated hot-water crude extract until 80 % aqueous ethanol concentration with stirring overnight at 4�C,

-II- Chapter 2 Materials and Methods allowing precipitation of the polysaccharide (PU80). Meanwhile, absolute ethanol was added gently into the other portion of the concentrated hot-water crude extract until 60 % aqueous ethanol concentration with stirring overnight at 4 °C in order to obtain the relatively higher molecular weight polysaccharides (PU60). Both precipitates were collected by centrifligation (15,600 g, 30 min). The pellets were re-dissolved into distilled water and were placed into dialysis tubing (Spectra/Por®, MWCO: 6 一 8,000) for dialysis against distilled water at 4 °C for two days. The two pale brown freeze-dried products collected from the 60 % and 80 % ethanol precipitation were regarded as PU60 and PU 80，respectively.

2.1.2 Cold-alkaline extraction of crude Polyporus umbellatus polysaccharides

The residue remained after the hot-water extraction was soaked with 1 M sodium hydroxide solution [sample: sodium hydroxide solution (w/v) = 1:7] with stirring overnight at 4 °C. The cold alkaline crude extract was collected by centrifligation (15,600 g, 30 min) and neutralized with 1 M hydrochloric acid. The salt formed in the mixture was removed by centrifligation and the supernatant was dialyzed. The alkaline-soluble crude polysaccharides were obtained by 80 % ethanol precipitation.

-26- Chapter 1 Materials and Methods

m(a)

I ....‘rv.��.._.

..L . . 一d- (b)

Figure 2.1 a) Sclerotium of Polyporus umbellatus in the wild environment. (Hu, 2001) b) Dried sclerotium of Polyporus umbellatus purchased from Guang Zhou.

-II- Chapter 2 Materials and Methods

Dried scelerotium of Polyporus umbellatus (700 g) i Washed and ground with a blender i _^ Boiled with distilled water (v/v =1:7) for 2 hours each time

Repeated extraction Removed the residue by centrifugation ()(15,600 g, 30 min) Residue Supernatant i Added absolute ethanol into the supernatant with stirring (4 °C, overnight) to achieve a final aqueous ethanol concentration of 60 % or 80% i Collected precipitates by centrifugation (15,600 g’ 30 min) i Re-dissolved in distilled water i Placed into dialysis tubing for dialysis against distilled water (2 days) i Lyophilized i t>U80APU60 i Fractionated PU60 with DEAE-column i

Eluted with distilled water ••[=== > mm

Eluted with 0.15 MNaCl •• 丨 > f&M Figure 2.2 Flow chart illustrating the extraction and fractionation of Polyporus umbellatus polysaccharides.

-28- Chapter 1 Materials and Methods 2.2 Purification of crude Polyporus umbellatus polysaccharides

2.2.1 Preparation of DEAE-cellulose ion exchange column

Dried form of DEAE-cellulose resin (Sigma, USA) was soaked in distilled water overnight at 4 °C. The swollen resin was treated in double volumes of 0.4 N hydrochloric acid for 30 minutes. After discarding the hydrochloric acid solution, the acidic resin was washed with distilled water until its pH value was near 7.0. The resin was further soaked in double volumes of 0.4 N sodium hydroxide solution for 30 minutes. The sodium hydroxide solution was discarded and the alkaline resin was washed with distilled water again until its pH value was neutral. The prepared resin was degassed and packed into a vertical column (2.5 cm x 30 cm).

2.2.2 Fractionation of hot-water soluble crude polysaccharide (PU)

Crude PU60 polysaccharides were fractionated by anion exchange chromatography. Eighty milligrams of the freeze-dried PU60 was completely dissolved into distilled water, loaded onto the distilled-water equilibrated DEAE-cellulose anion exchange column (2.5 cm x 30 cm) and eluted at a flow rate of 30 ml/h. The fractions were collected at 6 ml/tube. The unbound polysaccharide eluted by washing the column with distilled water was firstly collected, designated as PU60a. The retained components were then eluted with a linear gradient of sodium chloride solution ranged from 0 to 1.0 M at a flow rate of 30ml/h. Carbohydrate content on each tube was assayed by the phenol-sulfuric method (Dubois et al, 1956). According to the elution profile of PU fraction on DEAE cellulose column shown in the result section, majority of the retained polysaccharides was eluted by washing the column with 0.15 M sodium

-II- Chapter 1 Materials and Methods chloride solution. The retained component then was collected, which was designated as PU60b. PU60b was dialyzed against distilled water at 4 °C to remove the salt before lyophilizing.

2.3 Characterization of Polyporus umbellatus Polysaccharides

2.3.1 Carbohydrate content determination

The amounts of polysaccharides in the all fractions of PU were determined by the phenol-sulfliric assay (Dubois et al” 1956; Shouji et al., 2000), which is a simple but universal biochemical assay for estimating the carbohydrate content. The principle of this method is that mixing sugar samples with phenol and concentrated sulfuric acid forms yellowish brown products with color intensity directly proportional to the quantity of carbohydrate content. Compared with a calibration curve made from sugar standard, the approximate amount of carbohydrate in the PU fractions could be determined.

Before the experiment, freeze-dried PU fractions were dissolved into distilled water. Aliquots of the samples (200 |al) were mixed with equal volume of 5 % phenol in distilled water and stood at room temperature for 10 minutes. Concentrated sulfuric acid (98.8 %, 1 ml) was rapidly squeezed to the surface of liquid and the mixtures were mixed well with a vortex-mixer and left undisturbed for 30 minutes for color development. The absorbance was measured by a spectrophotometer (Milton Roy Spectronic 3000) at a wavelength of 490 nm. Blank and standard curve, using distilled water and D-glucose ranged from 0 - 800 ^ig/ml respectively, were treated by the same procedures. The carbohydrate content in the PU fractions can be calculated

-II- Chapter 1 Materials and Methods by the following equation derived from the glucose standard curve:

� Glucose content m. sampl,e (/ig/ml, , ,) = Average absorbance at^ 45謝0 nm of sample + 0.014

2.3.2 Gas chromatography (GC)

Gas chromatography (GC) is commonly used to determine the composition of neutral monosaccharides like glucose, mannose, and galactose in sugar samples. In this study, fifteen milligrams of all the PU fractions were hydrolyzed with 0.7 ml of 12 M sulfuric acid at 35 °C for 1 hour with frequent gently shaking. The hydrolysates were then diluted to 2 M sulfuric acid with 3.5 ml distilled water and boiled in a water bath for 1 hour with continuous shaking. After cooling, the hydro lysates (3 ml) were used for formation of alditol acetates of neutral and amino sugars while the remaining hydro lysates were reserved for the assay of uronic acid determination (details in Section 3.3.4).

p-D-allose (1 mg/ml), functioned as an internal standard, was added into the hydrolysate of each fraction. 12 M ammonia (1.25 ml) was used to neutralize the acidic hydro lysates while octan-2-ol (5 |xl) was added as an anti-foaming agent. Reduction of the hydro lysates was performed by adding 0.2 ml freshly prepared sodium borontetrahydride (200mg/ml) incubated in 40 °C water bath for 30 minutes. To stop the reduction, glacial acetic acid (0.1 ml) was added to the reducing hydro lysate. Acetylation was achieved by adding acetic acid anhydride (2 ml) and 1-methylimidzole (0.3 ml). Followed by vortex-mixing at room temperature for 10 minutes before adding 5 ml distilled water to dissolve the excess acetic anhydride,

-II- Chapter 1 Materials and Methods dichloromethane (1 ml) was added to the mixtures and phase separation was allowed. The upper aqueous layer was removed while the lower organic layer was washed twice with 2 ml distilled water each time. About 200 mg of anhydrous sodium sulphate then removed any water left in the organic layer. The alditol acetate sugar products were then stored in a vial at -20 °C before use. Determination of derivative sugars was conducted with a HP6890 gas chromatography using an Alltech DB-225 capillary column [15 m (L) x 0.25 mm (I.D.), 0.25 |Lim film thickness]. The oven temperature program was set as follows: the initial and final temperature was at 180 °C and 220� Cwith a temperature increase rate at 4 °C/min. Helium was used as the carrier gas. Sample (2 |li1) was injected into the column and the neutral monosaccharides in the samples were detected by flame ionization. The amount of monosaccharides in each sample was presented as proportion by weight (w/w %) and calculated by the following equations:

Monosaccharide(%) = XxY(mg) ^ 100% (1) Sample weight (mg) of GC

X = Rf (average from replicates) x coefficient (2) where the coefficient of hexose 二 1.26; pentose = 1.232; deoxyhexose = 1.246

Rf — monosaccharide concentration in standard (mg/ml) x 1.5 mix the area of allose in standard the area of monosaccharide in standard x 1 mg/ml x 1 ml V 一 The area of monosaccharide in samplex 1 mg/ml x 1 ml Y " (J))

the area of allosein sample

2.3.3 Protein content determination Lowry-Folin method (Lowry et al, 1951; Sapan et al., 1999), which is based on the -II- Chapter 2 Materials and Methods principles of the Biuret and Folin-Ciocalteau reaction, is a rapid and accurate assay to determine the amount of protein existed in the fractions of PU.

About one miligram of freeze-dried crude hot-water PU extract, PU80, PU60 and PU60a and b were accurately weighed and dissolved into 1 ml distilled water before the experiment. Sodium hydroxide solution (2N, 0.1 ml) was mixed with 0.1 ml of each aqueous sample. The mixtures were hydrolyzed at 100 °C for 10 minutes in a boiling water bath. After cooling to room temperature, the hydrolysates were further mixed well with 1 ml of freshly prepared complex-forming reagent which was composed of 2 % NaaCO〕，1 % CUSO4.5H2O and 2 % sodium potassium tartrate in a volume ratio of 100:1:1 and left undisturbed for 10 minutes at room temperature. Before measuring the absorbance of the mixtures at wavelength 750 nm with a spectrometer (Milton Roy Spectronic 3000)，0.1 ml of Folin-Ciocalteu's phenol reagent (BDH) was added to the mixtures which were left at room temperature for 30 minutes. Blank and standard curve, using distilled water and bovine serum albumin (Sigma) ranged from 0 - 500 fig/ml respectively, were prepared and treated as the same procedures. The protein content in the PU fractions can be calculated by the following equation derived from the BSA standard curve:

Average absorbance at 750 nm of sample - 0.049 Protein content in sample ("g/ml) =

2.3.4 Uronic acid content determination

According to the Official Methods of Analysis 45.4.11 (AO AC, 1996), a colorimetric method is typically applied to determine the content of uronic acid in samples.

-33- Chapter 2 Materials and Methods Hydrolysate collected from acid depolymerization in section 2.3.2 was used for the determination. An aliquot of 300 \x\ of hydro lysate was mixed thoroughly with 300 |il of boric acid-sodium chloride solution composed of 2 % sodium chloride solution and 3 % boric acid in distilled water. Five millilitres of 2 M concentrated sulfuric acid was added to the mixtures with shaking. After incubation at 70 °C for 40 minutes, the mixtures were cooled down to room temperature and mixed well with 200 of 0.1 % dimethyphenol in glacial acetic acid with vortex-mixing for 5 minutes. The mixture was stood at room temperature for 15 minutes before measuring its absorbance against blank at wavelengths 400 nm and 450 nm. Subtracting absorbance at 400 nm from those at 450 nm was required to correct any interference from hexoses in samples. Blank and a standard curve using 2 M sulfuric acid and D-galacturonic acid monohydrate ranged from 0 to 800 |ag/ml were prepared and treated in the same manners. The amount of uronic acids expressed as a polysaccharide residue present in each sample was shown as proportion by weight (w/w %). The amount of uronic acids in the PU fractions can be calculated by the following equation derived from the D-galacturonic acid standard curve:

Average absorbance (450 - 400 nm) of sample - 0.050 Uronic acid content in sample ("g/ml) = ——— 0.006

2.3.5 High Performance Liquid Chromatography (HPLC)

Analytical High Performance Liquid Chromatography (HPLC) is a sophisticating technique to determine the purity and molecular weight of samples using very small amount of sample volume (|il). Freeze-dried PU fractions (2 %) were completely dissolved in 0.5 M sodium chloride solution, then filtered through a sterile filter with

-34- Chapter 1 Materials and Methods 0.22 |Lim pore size before loading to a HPLC apparatus (Hewlett Packard series 1100 HPLC system) equipped with a TSK gel™ column G5000PW [30 cm(L) x 7.5 cm (I.D.)，particle size: 17 \xm] (SUPELCO). After conditioning the system, the sterile-filtered samples were eluted using 0.5 M sodium chloride solution at a flow rate of 0.5 ml/min at 25 °C and detected with Refractive Index (HP 1047A RI Detector). Five dextran standards with molecular weights 25，80，270,670 and 788 kDa were used for constructing a calibration curve for the determination of the molecular weight of sample.

2.4 In vivo Antineoplastic Assay

2.4.1 Animals

Two species of mice, BALB/c and athymic BALB/c nude mice were used in this project to evaluate the potential of anti-tumor effects of the PU polysaccharides in vivo. Both of them were supplied by the Laboratory Animal Service Centre (LASEC) of The Chinese University of Hong Kong.

2.4.1.1 BALB/c mice

Seven to eight-week-old male BALB/c mice weighed between 20 and 25 g were housed eight per plastic cages with wood chip bedding in the Animal House of Department of Biology (CUHK) with a 12-h light and 12-h dark cycle at room temperature (25 土 2°C). They were allowed free access to standard laboratory diet and tap water and acclimated in the laboratory one week before experimentation.

-II- Chapter 1 Materials and Methods 2.4.1.2 Athymic BALB/c mice

Seven to eight-week-old male and female athymic BALB/c nude mice weighed between 19 and 24 g were housed in the LASEC and acclimated in the laboratory one week before experimentation. The animals were housed in micro isolator cages, six per cage, in a 12-h light/dark cycle at a temperature constantly kept at 25 °C. All stuffs for the athymic mice including cages, bedding, rodent food and water are totally autoclaved before used.

2.4.2 Maintenance of cell lines

A murine cell line S-180 and a human cell line MCF-7 were used to study the anti-tumor effects of all fractions of PU. Cell line S-180 was provided by American Type Culture Collection (ATCC) (Rockville, MD) while cell line MCF-7 was supplied from the Institute of Genetics, Fudan University, Shanghai.

2.4.2.1 Murine sarcoma 180

Ascitic-form Sarcoma 180 (S-180) was maintained in the peritoneal cavities of male BALB/c mice. The cells collected from mouse peritoneum were weekly passed to another mouse by the procedures as below.

S-180 cells in ascitic form were collected from a mouse sacrificed by cervical dislocation. The cells were immediately washed twice with 0.45 % PBS by the aid of centrifuge (514 g for 5 minutes) (Beckman, Allegra™ 6R centrifuge) and the cell

-II- Chapter 1 Materials and Methods pellet was re-suspended in sterile PBS. With the aid of 0.4 % Trypan blue solution, the number of viable cells was counted under a hemocytometer. Cell suspension with cell density, 2.5 x cells/ml, was prepared and 0.2 ml of the cell suspension which contained 5x10^ cells was intraperitoneally transferred to another mouse.

2.4.2.2 Human breast cancer cell line (MCF-7)

MCF-7 cells used in this study were cultured in estrogen-free RPMI 1640 medium (Sigma), supplemented with 0.2 % sodium bicarbonate (Sigma), 10 % heat-inactivated fetal bovine serum, 100 U/ml of penicillin, 100 |Lig/ml of streptomycin and 0.25 ^ig/ml amphotericin B (all from Gibco-BRL). The cells were kept in 175 cm^ sterile polystyrene tissue culture flask (Sarstedt) and maintained under a folly humidified atmosphere of 95 % room air and 5 % CO2 at 37 °C. After 72-hour incubation, the cells were immediately washed twice and re-suspended in RPMI-1640 medium. With the aid of trypan blue solution, the number of viable cells was counted under a hemocytometer. Cell suspension with cell density, 2.5 x 10^ cells/ml, was prepared and 0.2 ml of the cell suspension which contained 5 x 10^ cells was intraperitoneally transferred to the nude mice.

2.4.3 S-180 tumor inoculation model using BALB/c mice

S-180 cell suspension with cell density 2.5 x 10^ living cells/ml was prepared as in the section 2.4.2.1. Male BALB/c mice under anesthesia by diethyl ether were subcutaneously inoculated with 0.2 ml cell suspension into the dorsal area (Day 0) (Table 2.1). Five days were allowed for the development of solid tumor in the

-II- Chapter 1 Materials and Methods animals. At Day 5，i.e. five days after tumor inoculation, mice were examined carefully by hands and only those bearing solid tumors were selected and randomly grouped into 8 per group and caged separately for the use in the experiment.

2.4.4 MCF-7 tumor inoculation model using athymic nude mice

MCF-7 cell suspension with cell density 2.5 x 10^ living cells/ml in RPMI 1640 medium was prepared as in the Section 3.4.2.2. Athymic male and female BALB/c mice were subcutaneously inoculated with 0.2 ml cell suspension into the dorsal area (Day 0). Seven days were allowed for solid tumor development. At Day 7，i.e. seven days after tumor inoculation, mice were examined carefully by hand and only those bearing solid tumors were selected and randomly divided into groups of 8 and caged separately for the use in the experiment (Clarke, 2002; Liu, 2003)

2.4.5 Assay of antineoplastic activity with S-180

After verifying tumor development on mice (Day 5)，crude PU extracts both from hot water and cold sodium hydroxide solution, PU80, PU60, PU60a and PU60b were intraperitoneally injected into corresponding treatment groups of mice once daily for ten consecutive days while sterile PBS was injected, instead of samples, into the control groups.

Ten days after the last i.p. injection of sample (Day 24), the mice were sacrificed by cervical dislocation and their body weight was measured. Their tumors were excised and measured in terms of weight by an electronic balance and in terms of size using

-II- Chapter 2 Materials and Methods calipers. The tumor volume of each group of mice was determined using the following formula (Teicher, 2002; Brady, 2002; Collins, 2003). The anti-tumor effects of PU fractions on S-180 solid tumor was expressed as percentage of inhibition compared to the control calculated using the following formulae (Kawagishi, 1989):

, , 3� Long diameter x Short diameter 2 Tumor volume (mm )= —

Averagetumor volume of treatment group 1 , Percentageof tumor inhibition (%) = \ \- — : — fx 100% [ Averagetumor volume of control group

2.4.6 Assay of antineoplastic activity with MCF-7

The procedures of the anti-tumor assay with MCF-7 were similar to those described in details previously. After verifying tumor development on mice (Day 7)，PU 60b at doses of 10 and 5 mg/kg body weight were intraperitoneally injected into corresponding treatment groups of mice once daily for ten consecutive days while sterile PBS was injected, instead of samples, into the control groups. It was most important that all samples including PBS injected into peritoneal cavities of the inoculated nude mice should be sterile filtered with 0.22 iiim pore-size sterile filter before use. Ten days after the last i.p. injection of sample (Day 24)，the mice were sacrificed by cervical dislocation and their body weight was measured.

Their tumors were excised and measured in term of weight by an electronic balance and in term of size using calipers. The average tumor volume of each group of mice and percentage of inhibition compared to the control calculated using the above mentioned formulae.

-39- Chapter 1 Materials and Methods 2.4.7 Body weight change

In order to assess the side effect of PU fractions, change in body weight was used as the parameter. Body weight of each mouse before sample injection and before sacrificed was measured. Average change in body weight in each group was calculated by the following equation. The change in treatment group was compared to the group without tumor inoculation and treatment.

•“，。/、 Average (Final body weight - initial body weight)�,細’ , Percentage change in body weight (%) = ^ . . .. — x 100% Average Initial body weight

Table 2.1 Schedule for in vivo antitumor assays

Date , Experiment procedures

Day 0 i. Tumor inoculation of S-180/ MCF-7 cells onto the dorsal ‘ sides of the acclimatized mice ‘ (s.c) I i Day 5 to 14 (for S-180 model) ！ i. Tumor development verification Day 7 to 16 (for ]S4CF-7 nMi4el) ii. Body weight measurement of I the tumor bearing mice ! iii. Sample injections (i.p) Day 14 to 24 (for S-180 model) i. 10-day Observation Day可6 to 26 (for K^CF-7 model) Day25 (for S-180 model) i. Mice Sacrifice by cervical ‘ [DayTlfforMtF:卞 model) j dislocation ii. Body weight measurement i iii. Tumor excise iv. Tumor weight and size ...•• """ . '"""""T"'"""

-II- Chapter 1 Materials and Methods 2.5 In vitro anti-proliferation assay

2.5.1 Cell lines Five human cancer cell lines: human acute promyelocytic leukemia cell (HL-60), human hepatoblastoma cell (HepG2) and human breast cancer cell (MCF-7), human colon cancer cell (Colon 201) together with one mouse sarcoma cell (S-180) and one monkey kidney normal cells (Vero) were used in this study. Among them, HL-60, S-180, HepG2, Colon 201 and Vero were purchased from American Type Culture Collection (ATCC), while MCF-7 was obtained from the Institute of Genetics, Fudan University, Shanghai.

2.5.1.1 Maintenance of cell lines

To prevent unwanted bacterial contamination, 100 unit/ml of penicillin, 100 |ig/ml of streptomycin and 0.25 |ig/ml of amphotericin B (all from Gibco-BRL) were added to the culture media.

HL-60, HepG2 and MCF-7 cells were maintained in RPMI 1640 medium (Sigma) supplemented with 2 g/L sodium bicarbonate (Sigma) and 10 % heat-inactivated (60 °C for 60 minutes) fetal bovine serum (Gibco-BRL).

S-180 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco-BRL) supplemented with 10 % heat-inactivated fetal bovine serum and 1.5 g/L sodium bicarbonate.

-II- Chapter 1 Materials and Methods All of the cell lines were cultured in 25 cm^ sterile polystyrene tissue culture flask (Sarstedt) with 5 ml of freshly completed medium and incubated at fully humidified atmosphere at 37 °C, 5 % CO2 and 95 % room air. The subculture of the cell lines were performed three times a week.

2.5.2 Assay of anti-proliferation with cancer cell lines

Direct cytotoxicity of samples on cancer cell lines, one possible anti-proliferation way to control non-stop cell growth, was investigated by direct incubation of the cancer cell line with sample for 72 hours.

2.5.2.1 Cytotoxicity assay on suspensions of cancer cells

One hundred microliters of suspensions of HL-60 and S-180 with initial cell density of 5 X 10)4 cell/ml were seeded separately in the wells of a sterile 96-welI round bottom culture microplates (IWAKI，Japan) and incubated with PU fractions at the doses of 50, 100，200 and 400 |ag/ml for 72 hours. The cytotoxic effects of PU fractions on HL-60, K-562 and S-180 cells were assessed by the Trypan blue exclusion test.

2.5.2.2 Cytotoxicity assay on adhesive cancer cells

One hundred microliters of culture medium containing adhesive cells HepG2 and MCF-7 with initial cell density of 2 x lO'* cell/ml were seeded separately in the wells of a sterile 96-well flat bottom culture microplates (IWAKI, Japan) and acclimated

-II- Chapter 1 Materials and Methods for 24 hours before mixing with 100 \i\ ofPU fractions at the doses of 50，100，200 and 400 fxg/ml for 72-hour incubation. The cytotoxic effects of PU fractions on HepG2 and MCF-7 cells were measured by the MTT assay.

2.5.2.3 Cytotoxicity assay on normal cells

Vero cells were used as control to evaluate toxicity of PU fractions on normal cells. Because of the adhesive nature of Vero cells, the sample treatments on the cell were same as those described in the Section 2.5.2.2. The cell density after 72-hour incubation was measured by the MTT assay.

2.5.3 Trypan blue exclusion method

The number of viable cells of control and sample-treated cell suspensions after 72-hour incubation was counted with the aid of the trypan blue dye, which can stain only dead cells which have lost their cell membrane integrity but not viable cells. One hundred microliters of 0.4 % trypan blue in PBS (w/v) was mixed thoroughly using a pipette with equal volume of cell suspensions in each well of 96-well microplate for 1 minute. The cell suspensions were gently transferred to a counting chamber of a hemocytometer. Only cells lying within the four big corner squares were counted. The cell density and viability were calculated by the equations as belows:

广 Total number of cells in the four corner squares of 1 mm 2 Cell Density = x Dilution factor x 10 cells/ml

./�/�Numbe r of viable cells Viability (%) = X 100% Total number of cells -II- Chapter 3 Materials and Methods The value of IC50 of PU fractions on corresponding cell lines was expressed as the concentration that exerts 50 % inhibition on cell density compared with control after 72-hour incubation by the following equation:

Percentage of inhibition (o/o) = |l- Average cell density of treatment group ！乂⑴。。/。 [ Average cell density of control group

2.5.4 MTT assay

Conversion of soluble yellow tetrazolium salt (MTT) into a dark blue colored formazan product in the presence of mitochondrial dehydrogenases released from viable cells is the principle of this quantitative colorimetric assay. The number of viable adhesive cells is proportional to the amount of blue formazan product which is reflected by its color intensity measured using spectrophotometer. Freshly prepared MTT (20 |Lil, 5 mg/ml) [3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyl tetrazolium bromide] (Sigma) in filtered PBS was added to each well of control and

sample-treated cells and incubated at 37 °C，5 % CO2 for 5 hours. The medium was discarded and dark blue formazan crystals were remained at the bottom of the wells. Acidic isopropanol (150 was added to each well to dissolve the dark blue crystals completely by vigorous pipetting. The absorbance at a wavelength 570 nm of the solution was measured by microplate reader (SPECTRAmax 250).

The extent of cell proliferation was indicated as the average value of absorbance while anti-tumor effect was showed as percentage of inhibition of sample treated groups to control group by the following equation:

-44- Chapter 2 Materials and Methods

Percentage of inhibition �)= 1{ - Average P.P.570nm in treatment group •�/� [ Average O.D.570 nm in control group J

2.6 Cytokine determination

Cytokines, involved in most disease processes including cancer, play an important role in innate immunity, apoptosis and angiogenesis (Baraldi-Junkins, 2000). The interaction between cytokines and the cellular immune system is a dynamic process. In this study, mouse cytokines were evaluated by a traditional assay, ELISA and a novel array system, Mouse Cytokine Array.

2.6.1 Treatment of mice

Seven to eight-week-old BALB/c male mice weighing between 20 to 25 g were involved in the assays. The acclimated mice were randomly divided into groups of 10. PU60 and PU60a at the most potent dose of 10 mg/kg was introduced daily to the treatment groups for 10 consecutive days while sterile PBS was used instead for intraperitoneal injection into the control group. At the day of the last sample injection and 10 days after injection, 10 p,g of lipopolysaccharide (LPS) in 0.2 ml sterile PBS was injected intravenously (i.v.) into each mouse one hour before blood collection. Half of the treatment and control groups received LPS stimulation while the remaining half of treatment and control groups did not.

Mouse blood was collected by making a cut on the veins of mouse tails an hour after

-45- Chapter 4 Materials and Methods LPS treatment. The blood was left undisturbed at room temperature for an hour prior to centrifugation at 1000 g for 15 minutes (Jouan A-14 centrifuge). The blood pellet was discarded and the supernatant was collected as serum sample for TNF-a assay. The serum was stored at -20 °C until used.

2.6.2 Enzyme-linked immunosorbent assay (ELISA) for TNF-a production

The amount of TNF-a in the mouse sera was revealed with the aid of a sandwich ELISA kit, Mouse TNF-alpha ELISA Ready-SET-Go! (eBioscience). The experimental procedures suggested by the supplier were followed as note below:

One hundred micro liters/well of capture antibody (purified pre-titrated anti-mouse TNF-a) in coating buffer were coated on the surfaces of 96 wells of Nunc Maxisorp microplate. The capture-antibody containing microplate was sealed with parafilm and incubated overnight at 4 °C. The wells of the plate were aspirated and washed three times with 320 ^il/well of wash buffer which contains 0.05 % Tween 20 in sterile PBS. After removing any residual buffer from each well, the wells were blocked with 200 |Lil/well of assay diluent and incubated at room temperature for one hour. The wells were aspirated and washed three times after 1-hour blocking as the previous steps. Diluted mouse serum (100 |li1, 1:20) was added into each antigen-coated well as primary antibody. The plates were sealed and incubated at room temperature for 2 hours. The plates were aspirated and washed five times with wash buffer as the previous steps. One hundred micro liters/we 11 of detection antibody (biotin-conjugated pre-titrated anti-mouse TNF-a cocktail) in assay diluent were added into each well. The plate was further incubated at room temperature for

-46- Chapter 4 Materials and Methods an hour. After aspirating and washing five times as above, one hundred microliters /well of detection enzyme (pre-titrated Avidin-HRP) diluted in assay diluent was added into each well. The plates were incubated at room temperature. After 30 minutes, the wells were soaked in wash buffer for 1 minute prior to aspiration. This step was repeated for seven times. One-hundred micro liters/well of TMB (3，3’，5， 5'-tetramethylbenzidine) substrate solution was added to each well and the plate was kept in darkness for 15 minutes. The experiment was completed by adding 50 |al/well of stop solution (1 M Phosphoric Acid) to each well. The absorbance of each well at the wavelength 450 nm was determined by the microplate reader at a reference wavelength of 570 nm. A standard calibration curve was prepared in the same manners using recombinant mouse TNF-a which was diluted two-fold of the stock standards, 1000 pg/ml.

2.6.3 Analysis of mouse cytokine array

2.6.3.1 Process of blocking and incubation

Mouse Cytokine Array (RayBio™ Mouse Cytokine Array) is the first commercially available cytokine protein array system. The kit provides a simple array format which can accurately and simultaneously identify the expression profiles of multiple cytokines from mouse sera in several hours.

Each membrane with an antibody-printed side of RayBio™ Mouse Cytokine Array was placed separately into the provided eight-well tray. Two milliliters of blocking buffer provided by the kit was overlaid on the membranes which were then incubated

-47- Chapter 2 Materials and Methods overnight at 4 °C. The blocking buffer was gently discarded from the wells and one mililiter of serum sample diluted by blocking buffer (1:10) from the mice in treatment and control groups was separately added to the membranes and allowed to be incubated at room temperature for 2 hours. The serum samples were decanted from each well and the membranes were washed three times with 2 ml of provided wash buffer I with shaking. Each washing was last for 5 minutes. The membranes were further washed twice with 2 ml of provided wash buffer II with shaking. Each washing was also last for 5 minutes. One milliliter of 250-fold diluted biotin-conjugated antibodies was added to each membrane which was incubated at room temperature. After 2-hour incubation, the membranes were washed three times with wash buffer I and twice with wash buffer II as mentioned above. Finally, two milliliters of 1000-fold diluted HRP-conjugated streptavidin was added to each membrane and the membranes were further incubated at room temperature for 45 minutes. Before the detection process, the membranes were washed again following the above-mentioned washing steps.

2.6.3.2 Process of cytokine detection

Five-hundred microliters of provided detection buffer A and 500 |li1 of provided detection buffer B were mixed and added to the membrane. Incubation was allowed at room temperature for 5 minutes. Excess detection reagent was drained off by holding the membrane vertically with forceps. The membranes with protein sides facing upwards were gently dried with Kimwipe. The dried membranes were wrapped with Saranwrap and exposed to x-ray films (Fuji medical film) for 45 minutes before signal detection using film developer.

-48- Chapter 2 Materials and Methods 2.7 Statistical Analysis

For statistical analysis of the data, the Student's /-test was applied to compare the levels of treatment group with those of the control groups. The results were expressed either as mean 士 standard deviation (S.D.) or standard error of the mean (S.E.M.). The data were defined as significant and very significant if p<0.05 and p<0.01 respectively.

-49- Chapter 3 Results

Chapter 3

Results

3.1 Extraction of Polyporus umbellatus polysaccharides (PU)

3.1.1 Percentage of Yield in extraction of crude PU extracts

In the extraction with hot water, 176.95 g crude water extract (PUw) was isolated from 700 g dried sclerotia of Polyporus umbellatus. On average, 100 g dried sclerotia might produce 25 g PUw. In alkaline extraction, 239.64 g crude alkaline extract (PUAL) was obtained from 700 g of the cooked residue of ground Polyporus umbellatus sclerotia. On average, 100 g cooked residue might produce 34 g PUAL (Table 3.1).

-50- Chapter 4 Results 3.1.2 Percentage of yield in fractionation of PU fractions

From 200 g of powder-form PUw under 60 % and 80 % ethanol precipitation, 0.96 g PU60 and 2.28 g PU80 were respectively isolated (Table 3.2). For further fractionation of the crude PU60 polysaccharides by anion exchange chromatography, application of 80 mg PU60 resulted in two fractions, PU60a and PU60b. 0.319 mg unbound component (PU60a) on DEAE-cellulose was eluted with distilled water while 4.527 mg retained component (PU60b) on DEAE-cellulose was eluted with 0.15 M NaCl (Figure 3.1). Recovery of the PU60 fractions after anion exchange chromatography was 6.06 % (Table 3.3).

-51- Chapter 3 Results

Table 3.1 Percentage yield of PU fractions extracted from 700 g dried sclerotia of Polyporus umbellatus. Product Color Weight (g) Yield (%)' Hot-water soluble PU (PUw) Brown 176.95 ^

Alkaline soluble PU (PUAL) Dark Brown 239.64 34.2

�%Yield = (weight of the fraction obtained/ 700 g of the dried sclerotia of Polyporus umbellatus) xlOO%

Table 3.2 Percentage yields of PU60 and PU80 after ethanol precipitation of 200 g PUw. Product C^ Weight (g) Yield (%)" 'PU60 Pale Brown 048 #PU80 Brown 2.28 1.14 b % Yield = (weight of the fraction obtained/ 200 g of the freeze-dried PUw) x 100 % "PU60 fraction was obtained from ethanol precipitation up to 60 % final concentration. PU80 fraction was obtained from ethanol precipitation up to 80 % final concentration.

-52- Chapter 3 Results

Table 3.3 Recovery of PU fractions after application of 80 mg PU60 to DEAE-cellulose column. Product Color Weight (mg) Yield (%) PU60a Whit^ 0li9 040^ PU60b Very pale brown 4.527 5.66' Total — 4.846 6.06 d

‘% Yield =( weight of the fraction obtained/80 mg PU 60) x 100 % d Recovery of total fractions after fractionation = total weight of all fractions / 80 mg PU60) X100%

-53- Chapter 3 Results

3 � Distilled water 0 to 1 M NaCl ^ r 2.5 - \\

i 2 - 1 PU60b

I 1.5 - PU60a \ I 1 - A \ ‘�.5 - V. Q I I — ( I ‘ 一 > — |一 _ /N ^ I I 0 20 40 60 80 100 120 Number of Tubes (6 ml/tube) Figure 3.1 Anion exchange chromatography of PU60 on a DEAE-cellulose column.

80 mg of PU60 was applied to a 2.5 x 30 cm DEAE-cellulose anion exchange column. The flow rate was 30 ml/h and the fractionation was collected at 6 ml/tube. The column was eluted stepwise with distilled water to obtain the unbound component (PU60a) and with a salt gradient of NaCl (0 to 1M) to obtain the retained component (PU60b). The presence of carbohydrate was determined by the phenol-sulphuric assay at 490 nm.

-54- Chapter 3 Results 3.2 Chemical characterization of PUw fractions

3.2.1 Carbohydrate and protein contents of PUw fractions

Carbohydrate and protein contents of PUw fractions were determined by phenol sulfuric acid assay and Lowry method, respectively (Table 3.4). Percentage of carbohydrate content of hot-water crude extract was 77.09 while those of its two ethanol-precipitated polysaccharides PU60 and PU80 were 82.01 and 85.76 respectively. Among the two fractions of PU60, PU60b contained much more carbohydrate (90.66 %) than PU60a did (78.56 %).

According to the Lowry method, PUw contained 2.93 % of protein. The highest protein content among the five fractions is 15.85% (PU80) while the lowest protein content is 0.55 % (PU60a).

3.2.2 Relative content of monosaccharides and uronic acid in PUw fractions

The relative content of monosaccharides and uronic acid in the four PUw fractions were determined by gas chromatography and colorimetric carboazole assay respectively. As listed in Table 3.5, glucose was the chief monosaccharide existing in all samples. Among the ten kinds of monosaccharides, only fucose and galactosamine failed to be detected in the samples. The results demonstrated that PUw contained trace amount of arabinose, mannose, ribose, xylose and glucosamine; PU80 contained trace amount of arabinose, galactose, mannose, rhamnose and ribose. The monosaccharide compositions of PU60 and PU60b were almost the same. The only difference between them is their proportion. Besides glucose, PU60a

-55- Chapter 4 Results contained galactose, mannose, glucosamine, arabinose, and rhamnose in decreasing order but had no uronic acid. The colorimetric assay revealed that all tested samples contained very little uronic acid (< 2.5%) and even PUw contained no uronic acid at all.

-56- Chapter 3 Results

Table 3.4 Carbohydrate and Protein contents in PUw fractions.

Fractions Carbohydrate Content (%) Protein Content (%) PU^ 77.09 ± 0.3 2.93 士 0.4 PU8G 85.76 士 0.1 15.85 ±0.2 PU60 82.01 土 0.2 12.62 土 0.1 78.56 土 0.3 0.55 ±0.2 90.66 ±0.1 4.75 ± 0.0

Carbohydrate and Protein contents were respectively determined by the Phenol-sulfuric assay and Lowry method. The results were expressed as mean 土S.D.

M

-57- Chapter 3 Results

Table 3.5 Proportions of different monosaccharides and uronic acid in the five PUw fractions determined by Gas Chromatography and colorimetric method. Proportions ( % w/w f Monosaccharides ^^pu60 PU60a PU60b Galactose I ^ ^ 3M 1^52 Mannose 35.56 3.71 3.98 3.38 3.17 Fucose - - - - - Galactosamine - - - + - Glucose 49.54 93.17 92.29 32.31 90.41 Rhamnose - 1.39 1.63 0.39 1.44 Ribose 5.97 + + + - Arabinose + 1.71 2.07 0.42 1.63 Xylose 5.94 - + - + Glucosamine 2.99 — 0.01 2.86 1.83 Uronic acid - 2.38 2.1 - 1.92

a The results are expressed as relative proportions (% w/w) in each fraction “ + ” indicates that only trace amount (> 0.001 %) of monosaccharide was detected. “-“indicates that the particular monosaccharide was not detectable.

-58- Chapter 3 Results

3.2.3 Infrared spectra of PUw fractions

Fractions PUw, PU60 and PU60a were completely dehydrated in a desiccator. Dried fractions (10 mg) were used for the IR spectrum test at 40000 - 500 cm"^ by the KBr tablet method. The hot-water crude extract PUw had no characteristic absorption (data not shown). By contrast, PU60 and PU60b exhibited sharp IR absorption at 890 cm'^ indicating that p-pyran glycosidic bond was present in the two fractions (Figure 3.2).

3.2.4 Molecular weight estimation of PUw fractions

The molecular weights of the crude PUw, PU60, PU60a and PU60b were estimated by measuring the retention time of the sample compared with those of dextran standard (Figure 3.3) with the aid of HPLC. The corresponding chromatograms were shown in Figure 3.4 and Figure 3.5.

A salt peak appeared in each elution curve of PU fractions at a retention time of about 25.5 minute. The molecular weight of PUw was about 6.14 kDa. PU60 was a heterogenous polysaccharide fraction with a molecular weight ranging from 19.7 to 607.0 kDa and about 93 % of PU60 was about 19.7 kDa in size (Figure 3.4b). Similarly, PU60b was also a heterogenous polysaccharide fraction with a molecular weight ranging from 21.45 to 607 kDa. About 90.4 % of PU60b eluted at 22.87 minute was about 21.45 kDa in size (Figure 3.5b). PU60a was relatively homogenous showing in the Figure 3.5a in the form of a small peak. Its molecular weight was about 20.31 kDa.

-59- Chapter 3 Results

iv 1890 cm-' ‘ J I ‘ n-um^ II |.'||.||||\ —‘•."’ ‘ ^ MOO WOO 2000 1«0 two WO WmmmbtramKl (a)

890 cm'' ！ ( i I 1 i ‘‘ ••)•-‘ 3800 3000 3900 聊 聊 ,000 9W WMmuffltwcm-l (b)

Figure 3.2 Infrared Spectra of (a) PU60 and (b) PU60b.

-60- Chapter 3 Results

1000 -： \670

g “ \27\0 Q ; \ ^ \ C/3 \

100 -： I由 V\8 0 I \ ‘: \

10 -I ‘ 1 ‘ 1 ‘ 1 ‘ 1 ‘ 1 15 17 19 21 23 25 Retention time (min)

Figure 3.3 Standard curve of HPLC indicating retention time of the four dextran standard with molecular weights of 25，80，270 and 670 kDa.

-61- Chapter 3 Results

mAu 2 丨、 • (' I—‘ •‘ I— 1— r'"’"'— T—"""““‘—‘— B 10 mkt (a) PUw

Ii KfI ！

J yji

！ 案 3 -i SW I . 1 j. , ^^ Salt peak 1 . ,.TH*n.-iCf.X •^••v^ “�-..--•.i-..-J..[-i.l.i.mit»«.IJiillCUH 1 “ / ,.“..-?._.-.产各'..“r .._.,.�.., . ... •.. . .i， .....• . ；, .. s 10 、，.... •+ >..•.�•»...-.•�-”•...iSi . ^V^ >uim- . (b) PU60

Figure 3.4 Gel permeation of (a) PUw and (b) PU60 by HPLC. Molecular weight of PU60 fractions was determined by HPLC using dextran standards with molecular weights of 25, 80, 270 and 670 kDa. Freeze-dried PU fractions (2 %) were completely dissolved in 0.5 M sodium chloride solution and filtered through a sterile filter with 0.22 fim pore size before loading to a HPLC apparatus.

-62- Chapter 3 Results

_ ： 簽 � n as ！"

^jj. Salt peak — I i^t \:： I. g ^ ...... V : I 之笠 ^ ,小 • .-”''--.' “ -.-•>—*..《.••； M^^WAWIN^, ••K，"^. _^^ t < tt"•••_,•H- .•.••• > ‘_ _ 4 26 .. ac . m,i

1V ‘ iV 、 . .，- . •• ^^, , • •,-,...‘-"«.'.、• ... .''、，’ * • • (a) PU60a i “ ‘ t ” j \

i /. \ [ ； \ � y Salt peak r ... - • .� .••.”-�

2 ^ T .. . ^ ‘ ,.. I .-AW^nl-^.W- A-..

...• • • Ir ；ff- « » » 料 (b) PU60b

Figure 3.5 Gel permeation of (a) PU60a and (b) PU60b by HPLC.

Molecular weight of PU60 fractions was determined by HPLC using dextran standards with molecular weights of 25, 80’ 270 and 670 kDa. Freeze-dried PU fractions (2 %) were completely dissolved in 0.5 M sodium chloride solution and filtered through a sterile filter with 0.22 jim pore size before loading to a HPLC apparatus.

-63- Chapter 3 Results 3.3 In vitro anti-proliferative assay

3.3.1 Anti-proliferative effects of PU fractions on suspension cancer cell lines

The in vitro effects of crude cold-alkaline extract (PUAL), hot-water extract (PUw) and polysaccharide fractions, PU80 and PU60 on proliferation and viability of HL-60 cells and S-180 cells were evaluated by Trypan blue exclusion method. Macroscopically, PU fractions showed no remarkable anti-proliferative effect on both

S-180 (Figure 3.6) and HL-60 cells; on the contrary, all PU fractions (PUw, PUAL, PU80 and PU60) might enhance the proliferation of HL-60 cells ranging from 20.9% to 58.1% (Figure 3.7). The viability of S-180 cells under the fraction treatment was over 96%, which was higher than that of HL-60.

3.3.2 Anti-proliferative effects of PU fractions on adhesive cancer cell lines

The in vitro effects of the two crude extracts PUAL and PUw, together with crude polysaccharide fractions, PU80 and PU60 on proliferation of MCF-7, HepG2 and Colon 201 were evaluated by MTT assay. The results were expressed in the mean absorbance of wavelength at 570 nm 士 S.D.

Microscopically, all fractions had no consistent anti-proliferative effect on the three adhesive cancer cell lines. Both PUAL and PU80 exhibited weak anti-proliferative effect on MCF-7 cells (Figure 3.8) but appeared to promote cell proliferation on HepG2 and Colon 201 (Figure 3.9 and Figure 3.10). The highest inhibition ratios of PUAL and PU80 on MCF-7 cell density were 12.3 % (400 |ag/ml) and 12 % (400 |ig/ml) respectively. Both fractions might remarkably promote cell proliferation of

-64- Chapter 4 Results HepG2 and Colon 201 at each dose. On the other hand, both PUw and PU60 demonstrated fluctuated results when treated with MCF-7, HepG2 and Colon 201 at doses of 100 |ag/ml, 200 |Lig/ml and 400 |xg/ml (Figure 3.8，Figure 3.9 and Figure 3.10). In brief, PUw might slightly inhibit proliferation of MCF-7 at 200 |Lig/ml and 400 |Lig/ml while it might enhance HepG2 proliferation at the three doses, which was totally opposite to the effects of PU60 on HepG2.

3.3.3 Anti-proliferative effects of PU fractions on normal cell lines

The cytotoxicity of PU fractions was evaluated using a normal cell line, Vero cell, which is a normal monkey kidney cell line with the aid of MTT assay performed the same manner as mentioned above. The four PU fractions show no any significant cytotoxicity towards Vero cell line (Figure 3.11).

-65- Chapter 3 Results Table 3.6 Percentage of inhibition on proliferation of S-180

Fraction % of inhibition at different concentrations 100 )ig/ml 200 |ig/ml 400 ^ig/ml PUAL -7.33 -8.61 - 10.04 PUw - 1.64 + 24.27 +30.11 PU80 - 6.87 - 15.27 - 9.73 PU60 - 1.33 - 6.83 - 16.67

140

�'2� ： “‘

i 80 - ^f— —=1 *

目60 -

1 40 - +PUW U ‘ PU60 _ + PU80 20 - • PUAL

Q I I I I I 1 1 1 I

0 50 100 150 200 250 300 350 400 450 Concentration ()j,g/ml)

Figure 3.6 The effects of PU fractions on proliferation of S-180 cell line after 72 h incubation. With the aid of Trypan blue method, antiproliferative effects on S-180 cell line were evaluated after 72 h incubation with PU fractions at doses of 100 Jdg/ml, 200 jLLg/ml and 400 jug/ml respectively. The results were expressed as mean of cell density (l(f cells/ml) 土 S.D. of three replicates.

The Student's t-test was applied. The data were defined as significant and very significant difference from control group if p < 0.05�and* �p < 0.01 (**)•

-66- Chapter 3 Results Table 3.7 Percentage of inhibition on proliferation of HL-60

Fraction % of inhibition at different concentrations 100 i-ig/ml 200 [ig/m\ 400 |ig/ml PUAL - 3.24 + 16.21 +20.9 PUw + 17.38 +31.29 +35.08 PU80 + 12.15 + 17.06 +26.4 PU60 + 45.42 +53.4 +58.1

140 �

120 - —— * f ^^^^^^ �10 0 - -^^^^ J

i 80

•55 § 60 - T3 = +PUW �4 0 - -—PUAL 士 PU60 20 _ HI~PU80

Q I I I 1 1 1 1 1 1 0 50 100 150 200 250 300 350 400 450 Concentration (|j,g/ml)

Figure 3.7 The effects of PU fractions on proliferation of HL-60 cell line after 72 h incubation. With the aid of Trypan blue method, antiproliferative effects on HL-60 cell line were evaluated after 72 h incubation with PU fractions at doses of 100 jig/ml, 200 jlg/ml and 400 jdg/ml respectively. The results were expressed as mean of cell density (IG^ cells/ml) 士 S.D. of three replicates.

The Student's t-test was applied. The data were defined as significant and very significant difference from control group if p < 0.05 (*) and p < 0.01〈**〉.

-67- Chapter 3 Results

Table 3.8 The effects of PU fractions on viability of S-180 and HL-60 cell lines after 72 h incubation.

~Fractions Concentration Viability (o/o) (Hg/ml) S-180 HL-60

0 99.36 ±0.01 95.41 ±0.01

PUal 100 96.64 ±0.01 90.89 士 0.01

200 97.33 士 0.02 88.40 士 0.02

400 96.51 ±0.01 81.39 士 0.01

PUw 100 98.97 士 0.01 98.26 士 0.03

200 99.24 士 0.01 86.75 ±0.06

400 99.83 士 0.01 78.36 士 0.05 PU80 100 97.01 ±0.00 96.15 士 0.02

200 98.65 士 0.00 96.60 土 0.01

400 98.72 ±0.01 94.39 士 0.01 PU60 100 98.18 士 0.01 88.21 士 0.04

200 97.78 土 0.00 85.73 士 0.00

400 96.57 士 0.01 80.62 士 0.01

The results were expressed as mean of percentage of viability 土 S.D. of three replicates.

-68- Chapter 3 Results Table 3.9 Percentage of inhibition on proliferation of MCF-7

Fraction % of inhibition at different concentrations 100 |ig/ml 200 \xg/ml 400 [ig/ml PUAL -3.9 -9.7 -12.3 PUw +8.9 -8.3 -8.8 PU80 -8.5 -7.8 -12.0 PU60 -5.8 -0.9 +13.9

1.4 - * PUw

i 1.0 i* ts 0.8 - (U 0 1 0.6 - J +PUW ^ 0.4 - —PUAL + PU60 0.2 “ • PU80

0.0 ‘ ‘ ‘ ‘ ‘ 0 100 200 300 400 500 Concentration (jj,g/ml)

Figure 3.8 The effects of PU fractions on proliferation of MCF-7 cell line after 72 h incubation. With the aid of MTT assay, antiproliferative effects on MCF-7 cell line were evaluated after 72 h incubation with PU fractions at doses of 100 jUg/ml, 200 lig/ml and 400 /LLg/ml respectively. The results were expressed as mean of absorbance at 570 nm 土 S.D. of three replicates. The Student 's t-test was applied and the data were defined as significant and very significant difference from control group if p < 0.05�*)and p < 0.01 鬥.

-69- Chapter 4 Results Table 3.10 Percentage of inhibition on proliferation of HepG2

Fraction % of inhibition at different concentrations 100 |Lig/ml 200 iig/ml 400 ^ig/ml PUAL +42.2 +43.1 +35.0 PUw +5.0 +12.0 +4.5 PU80 +34.3 +17.3 +8.3 PU60 -3.6 -11.9 -15.0

1.2 - T X. S 工 …-- 一 J

1.0 _ ** PU80 一~*

£ y ^^^^^^^^ 干 *PU80

-^——- , I 0.6 - ^ o ；I 0.4 - +PUW + PUAL 0.2 - +PU60 hb~pu80

0.0 ‘ ‘ ‘ ‘ ‘ 0 100 200 300 400 500 Concentration (昭/ml)

Figure 3.9 The effects of PU fractions on proliferation of HepG2 cell line after 72 h incubation. With the aid of MTT assay, antiproliferative effects on HepGl cell line were evaluated after 72 h incubation with PU fractions at doses of 100 fig/ml, 200 flg/ml and 400 jlg/ml respectively. The results were expressed as mean of absorbance at 570 nm 土 S.D. of three replicates. The Student 's t-test was applied and the data were defined as significant and very significant difference from control group if p < 0.05 (*J and p < 0.01

-70- Chapter 3 Results Table 3.11 Percentage of inhibition on proliferation of Co Ion 201

Fraction % of inhibition at different concentrations 100 fig/ml 200 [ig/m\ 400 昭/ml PUAL +25.2 +62.2 PUw -16.9 +12.8 -5.2 PU80 +6.9 +46.3 +39.0 PU60 -6.2 +3.1 +25.5

0.7「

0.6 - I ^ ："* PUA,. Z …——..… ..1..— c 0.5 - 乂 I ——- ,0.4 - z z 1*PU60 2 I 0.3 - ^^^^^^ I I* PUw J 0.2 - +PUW < —PUAL 0.1 - +PU60 • PU80 0.0 ‘ ‘ ‘ ‘ ‘ ‘ ^ ‘ ‘ 0 50 100 150 200 250 300 350 400 450 Concentration (|ag/ml)

Figure 3.10 The effects of PU fractions on proliferation of Colon 201 cell line after 72 h incubation. With the aid of MTT assay, antiproliferative effects on Colon 201 cell line were evaluated after 72 h incubation with PU fractions at doses of 100 jig/ml, 200 jig/ml and 400 jig/ml respectively. The results were expressed as mean of absorbance at 570 nm 士 S.D. of three replicates.

The Student's t-test was applied and the data were defined as significant and very significant difference from control group ifp < 0.05 (*) andp< 0.01�**;•

-71- Chapter 3 Results Table 3.12 Percentage of inhibition on proliferation ofVero cell

Fraction % of inhibition at different concentrations 100 |ig/ml 200 )ig/ml 400 ^ig/ml PUAL +10 T6 ^ PUw +5.0 -0.5 -1.0 PU80 +1.4 +10.0 +5.0 PU60 -0.7 -2.0 +1.0

1.4 -

1.2 - T

1 1.0 2 0.8 — c0 1 0.6 - ：< -^PUW 0.4 _ -^PUAL + PU60 0.2 _ • PU8Q

0.0 ‘ ‘ ‘ ‘ ‘ 0 100 200 300 400 500 Concentration (jig/ml)

Figure 3.11 The effects of PU fractions on proliferation of normal cell line (Vero) after 72 h incubation. With the aid of MTT assay, cytotoxic effects on Vero cell line were evaluated after 72 h incubation with PU fractions at doses of 100 jlg/ml, 200 jLLg/ml and 400 }ig/ml respectively. The results were expressed as mean of absorbance at 570 nm ±S.D. of three replicates.

The Student 's t-test was applied and the data were defined as significant and very significant difference from control group if p < 0.05 (*) and p < 0.01 r*;.

-72- Chapter 4 Results 3.4 In vivo antineoplastic assay

3.4.1 S-180 tumor inoculation model using BALB/c mice

3.4.1.1 In vivo antineoplastic effect of crude extracts - PUw and PUAL

The hot-water crude extract (PUw) and cold-alkaline crude extract (PUAL) were used in the preliminary in vivo antineoplastic assay using Sarcoma 180 tumor bearing BALB/c mice. One-hundred milligrams/kg of PUw and PUAL were intraperitoneally injected into the tumor bearing BALB/c mice respectively for 10 consecutive days. Antineoplastic effects on S-180 solid tumor in mice were expressed as percentage of inhibition to the control group. In shown in Figure 3.12, hot-water extract PUw had antineoplastic effect (76 %) higher than that of PUAL (68%) and only PUW-treatment group showed complete tumor regression (14 %). Mortality of the three groups of mice was low, but comparatively, PUAL treatment group had the highest mortality among the three groups.

3.4.1.2 In vivo antineoplastic effect of PUw

This assay was done in order to determine the effective range of dose of PUw. Antineoplastic effects on S-180 solid tumor in mice were expressed as percentage of the control level. Figure 3.13 showed that PUw exhibited dose-dependent effect only at the low doses ranging from 12.5 mg/kg to 50 mg/kg. The most potent dose was 100 mg/kg which significantly exhibited 79 % inhibition on tumor growth while a dose of 12.5 mg/kg might inhibit tumor growth by 69 %.

Initial and final body weights of the tested mice were measured and the record was

-73- Chapter 4 Results showed in Table 3.15. All mice in the treatment groups did not reduce their body weight in the end of experiment. The percentage of body weight gain of the treatment groups with doses of 12.5 mg/kg and 50 mg/kg respectively were higher than that of control group.

-74- Chapter 3 Results

Table 3.13 The effect of PUAL and PUw on S-180 tumor bearing BALB/c mice

Treatmentn Average Tumor weight (g) Mortality, 士 S.E.M. J Regression Control 7 0.921 ±0.204 2 0 PUAL 7 0.295 士 0.098 3 0 PUw 7 0.218 士 0.043 1 1 “The tumors were excised and weighed 10 days after 10-day observation and the average tumor weight was expressed in the unit (g) 士 S.E.M.

78 � 氺

“- -^m 羞:：• • 64 BBHI^ 1 ^ J PUw PUAL Fraction (100 mg/kg)

Figure 3.12 The Effects of crude extracts (Pw and PAL) on Sarcoma 180 solid tumor in vivo. S-180 tumor bearing mice were induced by subcutaneous injection of 5 x J(f cells (suspending in 0.2 ml sterile PBS) into the dorsal side of male BALB/c mice. 5 days after tumor inoculation, PBS, PUw and PUAL at the dose of 100 mg/kg were daily injected intraperitoneally into the tumor bearing mice for 10 consecutive days. Antineoplastic effects on S-180 solid tumor in mice were expressed as percentage of inhibition to the control group. Statistically significant values were respectively indicated with * and **forp <0.05 andp <0.01.

-75- Chapter 3 Results Table 3.14 The effects of PUw in different concentrations on S-180 tumor bearing BALB/c mice Complete b * t iTumor Treatments Mortality Tumor AverageTumor Regression weight (g) 土 S.E.M. rati�（o/。） "Control PBS 2/8 0/8 0.921 ±0.198 0 PUw 12.5mg/kg iii 0/8 0.283 ±0.100 ^ 25 mg/kg 1/8 0/8 0.338 ±0.153 63 50 mg/kg 2/8 0/8 0.393 ±0.094 57 100 mg/kg 0/8 1/8 0.194 士 0.051 79 “The row expressed the results of control group for PUw treatment assay. ''The average tumor weight was expressed in the unit (g) ± S.E.M. c Tumor inhibition ratio = (average tumor weight of treatment group- that of control group)/ average tumor weight of control group x 100 %

100�• Co 90 - • �o - • M 70 - ••

I1 50 1n1 * * B i i B PBS 12.5 25 50 100 Concentration (mg/kg)

Figure 3.13 Effects of hot-water crude extract PUw in different concentrations on Sarcoma 180 solid tumor in vivo. PUwat doses of 100 mg/kg, 50 mg/kg and 25 mg/kg and 10 mg/kg were daily i.p. injected into the tumor bearing BALB/c mice for 10 consecutive days and PBS was used as control. Antineoplastic effects on S-180 solid tumor in mice were expressed as percentage of control level (Average tumor weight of treatment group/Average Tumor weight of control x 100%). Statistically significant values were respectively indicated with * and **forp <0.05 andp <0.01.

-76- Chapter 2 Results

Table 3.15 Effects of hot-water crude extract PUw in different concentrations on body weight of S-180 tumor bearing mice. "Average Body Weight (g) 士 S.D. Treatments ————： Initial Final Control PBS 22.18 ± 1.35 23.88 ±1.76 12.5mg/kg 22.88 ± 1.46 25.11 ±1.06 25 mg/kg 23.16 ± 1.76 23.81 土 1.95 50 mg/kg 23.54 土 1.23 25.41 士 1.49 100 mg/kg 23.64 ± 1.35 24.19 ±1.26

S-180 tumor bearing mice were induced by subcutaneous injection of 5 x / 10^ cells (suspending in 0.2 ml sterile PBS) into the dorsal side of male BALB/c mice. 5 days after tumor inoculation, PBS and PU60 at doses of 100 mg/kg, 50 mg/kg, 25 mg/kg and 12.5 mg/kg were daily injected intraperitoneally into the tumor bearing mice for 10 consecutive days. The average tumor weight was expressed in the unit (g) 士 S.D. The initial body weight of mice was measured after tumor development verification (i.e. the first day of sample injection) and the final body weight was measured after the enucleation of the tumor from the mice.

-77- Chapter 3 Results

3.4.1.3 In vivo antineoplastic effect of PU60 and PU80

Doses of 100 mg/kg, 50 mg/kg and 10 mg/kg of PU60 and PU80 were respectively injected intraperitoneally into the mice. Between the two fractions, PU60 exhibited tumor inhibition effect stronger than PU80 did on the whole (Table 3.16). Both PU60 and PU80 had no obvious dose-dependent effect on tumor inhibition. The mice treated with 100 mg/kg PU60 showed the highest tumor inhibition percentage (80 %) when compared with the control groups. The second highest tumor inhibition effect (77 %) was not due to the dose of 50 mg/kg but 10 mg/kg (Table 3.16). On the other hands, the dose giving the highest tumor inhibition percentage of PU80 was 10 mg/kg (76 %) but the antineoplastic effect of PU80 declined when the concentrations ofPUSO were increased.

As expected, all mice in the treatment groups did not reduce their body weight in the process of experiment. The percentage of body weight gain of the treatment groups was higher than that of the control group.

-78- Chapter 3 Results

Table 3.16 The effects of polysaccharide fractions PU60 and PU80 in different concentrations on Sarcoma 180 solid tumor in vivo. Complete ® Average Tumor ''Tumor Treatments Mortality Tumor volume (mm^) 土 inhibition Regression S.E.M. ratio (%) 'ControlPBS 3/10 351.06 土 29.10 0 PU60 10 mg/kg 1/7 0/7 82.10 土 12.60 77 * 50 mg/kg 0/7 0/7 88.50 土 17.30 75 * 100 mg/kg 1/7 1/7 70.78 ± 17.70 80 * bControl？0/8 0/8 494.82 ±29.40 0 PU80 10 mg/kg 0/8 0/8 119.06 土37.86 76 50 mg/kg 0/8 0/8 202.13 土 61.32 59 100 mg/kg 0/8 0/8 177.70 ±75.23 64

PU80 and PU60 at doses of 100 mg/kg, 50 mg/kg and 10 mg/kg were daily injected (i.p.) into the tumor bearing BALB/c mice for 10 consecutive days and PBS was used as control.

Antineoplastic effects on S-180 solid tumor in mice were expressed as percentage of tumor inhibition. Statistically significant values were respectively indicated with * and ** for p < 0.05 andp <0.01.

° The item expressed the results of control group for PU60 treatment assay, b The item expressed the results of control group for PU80 treatment assay, c The tumors were excised and measured with a caliper 10 days after 10-day treatment and the average tumor volume was expressed in the unit (mm^) 士 S.E.M. d % Tumor inhibition ratio (%) = [1- (Average tumor volume of treatment group/Average tumor volume of control group)] X100%

-79- Chapter 3 Results

Table 3.17 The effects of polysaccharide fractions PU60 and PU80 in different concentrations on body weight of S-180 tumor bearing mice. ^Average Body Weight (g) 士 S.D. Treatments Initial Final Control PBS 23.91 ± 1.38 25.08 ± 1.90 PU60 lOmg/kg 24.32 土 0.44 25.67 ±0.72 50 mg/kg 24.94 土 1.39 25.86 土 1.90 100 mg/kg 25.09 ± 1.31 26.52 ±1.69 Control PBS 24.59 ± 1.45 24.82 ±1.80 PU80 10 mg/kg 24.52 ±0.52 25.50 ±1.00 50 mg/kg 24.55 士 1.10 24.89 ±1.09 100 mg/kg 24.15 土 0.62 24.69 士 0.60

PU80 and PU60 at doses of 100 mg/kg, 50 mg/kg and 10 mg/kg were daily injected intraperitoneally into the tumor bearing mice for 10 consecutive days and PBS was used as control. The average tumor weight was expressed in the unit (g) 士 S.D. The initial body weight of mice was measured after tumor development verification (i.e. the first day of sample injection) and the final body weight was measured after the enucleation of the tumor from the mice.

-80- Chapter 3 Results

3.4.1.4 In vivo antineoplastic effect of PU60a and b

After screening out the PU80, PU60 was further fractionated with the aid of DEAE column, producing PU60a and b. The dose of the two fractions was scaled down to 10 mg/kg and 5 mg/kg in the assay. Interestingly, the antineoplastic effect of PU60b still exists and even stronger than that of PU60. PU60b showed the highest tumor inhibition ratio (89%) at a dose of 5 mg/kg (Figure 3.14 and Figure 3.15) but the tumor inhibition ratio declined when the concentration of PU60b was doubled. The antineoplastic effect of PU60a was much weaker than that of PU60b. The highest tumor inhibition ratio ofPU60a was 48 % (5 mg/kg) and it further decreased to 11 % when the concentration of PU60a was doubled (Figure 3.14). No complete tumor regression was recorded in the whole assay.

3.4.2 MCF-7 tumor inoculation model using athymic nude mice

3.4.2.1 In vivo antineoplastic effect of PU60b

Because the most potent dose ofPU60b in S-180 tumor bearing BALB/c mice was 5 mg/kg, another set of experiment was performed by using athymic nude mice which were inoculated with MCF-7 tumors at the back before the treatment (Figure 3.17). Two doses of PU60b，10 mg/kg and 5 mg/kg, were respectively administered into the mice. The results were shown in Table 3.19 and Figure 3.18. The antineoplastic effect of PU60b was greatly lower in the athymic nude mice when compared with that on S-180 tumor bearing BALB/c mice. The MCF-7 tumor on athymic nude mice was reduced in size by 18 % at a dose of 5 mg/kg and 13 % at a dose of 10 mg/kg. Two sexes of mice were no mortality, no tumor regression and decrease in

-81- Chapter 3 Results body weight in the process of the experiments (Table 3.20)，which was different from the response in S-180 tumor bearing mice.

«

-82- Chapter 3 Results Table 3.18 The effects of PU60a and PU60b on S-180 tumor bearing BALB/c mice

Complete Average Tumor *^umor Treatments Mortality Tumor volume (iW) 土 inhibition Regression S.E.M. ratio (%) a Control^ 1/8 0/8 151.14 土 38.01 0 PU60a 5mg/kg 0/6 0/6 78.79 ±22.62 48 lOmg/kg 1/6 0/6 134.83 ±19.35 11 bControlPBS 3/8 0/8 350.00 ± 67.27 0 PU60b 5mg/kg 0/6 0/6 38.86 土 9.01 89** lOmg/kg 1/6 0/6 66.81 ± 14.02 81** “The item expressed the results of control group for PU60a treatment assay. The item expressed the results of control group for PU60b treatment assay. c The tumors were excised and measured with a caliper 10 days after 10-day treatment and the average tumor volume was expressed in the unit (mrn) 士 S.E.M. 丄 Tumor inhibition ratio (%) = (average tumor volume of treatment group - that of control group)/average tumor volume of control group X100%

120 � • PU60a 丨 PU6Qb

S 100 - j 80 - • • 60 - 40 - • • • **

”-• I I •一 PBS 5 10 Concentration (mg/kg) Figure 3.14 The effects of polysaccharide fractions PU60a and PU60b in different concentrations on S-180 solid tumor in vivo. PU60a and PU60b at doses of 10 mg/kg and 5 mg/kg were daily injected intraperitoneally into the tumor bearing BALB/c mice for 10 consecutive days and PBS was used as control Antineoplastic effects on S-180 solid tumor in mice were expressed as percentage of control level (Average tumor volume of treatment gwup/Avefcige Tumor volume of control x 100%). Statistically significant values were respectively indicated with * and **forp <0.05 andp <0.01.

-83- Chapter 2 Results

Ip— Contro1l 曲

PU60b (10 mg/kg) …

PU60b (5 mg/kg) ； : ‘

Figure 3.15 S-180 solid tumors excised from BALB/c mice under treatment of PU60b at doses of 10/mg/kg and 5 mg/kg.

PBS was used to treat the control group.

-84- Chapter 4 Results Table 3.19 The effects of polysaccharide fractions PU60b in different concentrations on MCF-7 tumor bearing athymic nude mice. Complete ° Average Tumor ''Tumor Treatments Mortality Tumor volume (mm�）土 inhibition Regression S.E.M. ratio (%) a ControlPBS 0/8 86.25 土 8.66 0 bpU60b 5 mg/kg 0/6 0/6 70.42 ±9.88 18 10 mg/kg 0/6 0/6 74.83 士 12.50 13 a&b The items expressed the control and treatment groups involving both male and female athymic mice where equal number of male and female mice were used in each group, c The tumors were excised and measured with a caliper 10 days after 10-day treatment and the average tumor volume was expressed in the unit (mm� S.E.M.)± “Tumor inhibition ratio (%) = (average tumor volume of treatment group - that of control group)/average tumor volume of control group x 100%.

120 - I ^ ——

g 80 -

§ 60 - f 40 I (£ 20 - ‘ ‘ 0 5 10 Concentration (mg/kg)

Figure 3.16 The effects of PU60b in different concentrations on MCF-7 tumor bearing athymic nude mice. PU60b at doses of 10 mg/kg and 5 mg/kg were daily injected intraperitoneally into the tumor bearing athymic mice for 10 consecutive days and PBS was used as control Antineoplastic effects on MCF-7 solid tumor on mice were expressed as percentage of control level (Average tumor volume of treatment group/Average Tumor volume of control x 100%). Statistically significant values were respectively indicated with * and **for p <0.05 and p <0.01.

-85- Chapter 3 Results

Table 3.20 The effects of PU60b on body weight of S-180 tumor bearing BALB/c mice and MCF-7 tumor bearing athymic mice.

a Average Body Weight (g) 士 S.D. "Percentage Treatments ^^~： change in Initial Final Body weight (0/0) S-180 tumor bearing BALB/c mice Control PBS 24.36 ± 0.94 25.14 ±1.10 +3.2 PU60b 5 mg/kg 25.04 ± 1.64 25.92 士 1.01 +3.5 10 mg/kg 24.61 ±0.80 25.01 土0.83 +1.6 CMCF-7 tumor bearing athymic mice Control PBS 21.00 ±2.26 20.20 土 2.17 — 0.8 PU60b 5 mg/kg 19.85 ± 1.90 19.42 土 1.98 -0.4 10 mg/kg 19.68 土 1.54 19.05 ±1.59 — 0.6

PU60b at doses of 10 mg/kg and 5 mg/kg were daily injected (Up.) into the tumor bearing mice for 10 consecutive days and PBS was used as control. a The average tumor weight was expressed in the unit (g) 士 S� D.The initial body weight of mice was measured after tumor development verification (i.e. the first day of sample injection) and the final body weight was measured after the enucleation of the tumor from the mice. b Percentage change in body weight = (average final body weight - average initial body weight)/ average initial body weight x 100 %. c All groups of MCF'7 tumor bearing athymic nude mice involved both male andfemale mice with equal number of mice.

-86- Chapter 3 Results

1(a) 圔 IHIHHiii^HHBn (b)

^^HHHBSNii (d) Figure 3.17 The process of in vivo assay with MCF-7 tumor-inoculating model. Tumor verification with (a) naked eyes and (b) by hand; (c) Intraperitoneal injection and (d) observation of tumor size during the treatment.

-87- Chapter 3 Results

^HjjjH cti • I Mj m till •jjH cti jm fA.o

Figure 3.18 Comparison of MCF-7 tumor size of the athymic nude mice with the treatment of PU60b at doses of 5 mg/kg and 10 mg/kg.

-88- Chapter 4 Results 3.4.3 Identification of cytokines in the serum from healthy BALB/c mice using mouse cytokine array system

Identification of cytokines in the serum from the healthy BALB/c mice which received 10-day consecutive treatment with a dose of 10 mg/kg PU60b was performed using a mouse cytokine array system (Ray Biotech). Thirty two mouse cytokine antibodies were coated on the membrane of the array system in a sequence as shown on Table 3.21. The images of the mouse cytokine array membranes probed separately with the sera from the control and treatment groups of mice were displayed on Figure 3.20. Comparing the relative expression levels between the two images of membranes, the serum from the control group of mice contained 21 types of cytokines out of the 32 cytokines while that from the treatment group contained 15 types. There were 15 cytokines that both sera contained. Among the expressed cytokines, the expression levels of KC，TIMP-1 and MIP-2 were remarkably higher than the others. Cytokines that only existed in the control group serum were IL-6，Eotaxin, GCSF, Leptin, Tpo and VEGF (Figure 3.20).

-89- Chapter 3 Results

Table 3.21 Antibody list on the membrane ofRayBio™ Mouse Cytokine Array.

A B C D E F G H I J K L 1 Pos Pos Neg Neg 6Ckine CTACK Eotaxin GCSF GM-CSF IL-2 IL-3 IL-4 2 Pos ~P^~~ Neg Neg 6Ckine CTACK Eotaxin" GCSF "GM-CSF IL-2— IL-3 IL-4 3 IL-5 IL-9 IL-10 IL-12p70 IL-13 “ IL-17 IFN-y KC Leptin MCP-1 4 _JLj_~lL-6 IL-9 IL-10 IL-12 IL-12p70 IL-13 “ IL-17 IFN-y KC — Leptin "MCP-1 5 MIP-2 MIP-3(3 RANTES SCF sTNFri" TARC TIMP-1 TNF-OT Tpo ~VEGF 6 JVlCP^^jp^ MIP-2 MIP-3P "^NTES SCF~~ sTNFri" TARC "LLMP-L TNF-CT Tpo ~YEG¥ 7 Blank "l^ank Blank Blank~ Blank Blank Blank Blank Blank Blank Pos 8 Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Pos (Ray Biotech, 2003)

tNote: Pos - positive control; Neg - negative control. All other cytokine short forms on the table are based on the standard abbreviations.

-90- Chapter 3 Results

gbi^ • • 1

(a) (b)

• • (c) (d)

Figure 3.19 The images of the mouse cytokine array membranes on films were showed in the figures (a) Treatment group and (b) control group.

The images referring to the treatment group and control group in the alternative style were respectively present as (c) and (d). Circle X encloses the spots of MIP-2; circle Y encloses the spots of KC and circle Z encloses the spots of TIMP-1.

-91- Chapter 3 Results

關圖 ======三I 三三三三三三| (treatment) (control) fe^M VAM

(combined)

Control Treatment

Control + Treatment Positive control of membrane

Figure 3.20 The diagrams revealed the expression of cytokines on the corresponding membranes.

-92- Chapter 3 Results 3.4.4 Effect of PU60b on TNF-a generation in healthy BALB/c mice studied with ELISA

TNF-a level in the serum of mice was evaluated with the aid of ELISA. In the present study, the mice in the experiment were first administered with PU60b at doses of 10 mg/kg and 5 mg/kg for 10 consecutive days. After that, the blood of mice was respectively collected on Day 0 and Day 10 of the observation period. Half of the control and treatment groups were stimulated by LPS one hour before blood collection while the rest of the groups were injected with PBS instead. The results were shown in the Figure 3.21. Obviously, the serum levels of TNF-a of LSP-primed mice were much higher than those of mice without LPS stimulation. The amount of TNF-a generated by LPS-primed mice on Day 10 was much larger than that on Day 0; the big difference was significantly obvious especially in those from the PU60b treatment groups. Mice injected with 10 mg/kg of PU60b might generate TNF-a on Day 10 triple in amounts of TNF-a on Day 0 while there was no marked increase as shown in the control group.

3.4.5 Effect of PU60b on TNF-a generation in S-180 tumor bearing mice studied with ELISA

This assay was done in the same manner as the above-mentioned experiment, except the animal model. S-180 tumor bearing mice were used instead. Similarly, the serum levels of TNF-a of LPS-primed mice were much higher than those of mice without LPS stimulation. However, the level of TNF-a of the control elevated as much as those of the treatment groups after LPS stimulation. Such a response was not the same in the healthy mice.

-93- Chapter 3 Results

9000 � • Control 8000 h • 10 mg/kg ^ f 7000�l�5mg^g| •

？_「 H 5000 L • g 4000 L • • g 3000 I- • ** ^H U 2000 P 1000 H^l ^^H 01 man —a • •L I Day 0 Day 10 Day 0 Day 10 I (No LPS) (LPS) Day

Figure 3.21 The effect of PU60b on serum level of TNF-a in healthy BALB/c mice. Level of TNF-a in the serum collected from the healthy BALB/c mice of the control and treatment groups with or without LPS stimulation was determined by ELISA using recombinant TNF-a as a standard. The blood of mice was collected on Day 0 of the observation period (The last day of i.p. injection) and Day 10 of observation period (The last day of observation) respectively. The results are expressed as mean (pg/ml) 士 S.D. (n=4). The Student 's t-test was applied. The data were defined as significant and very significant difference from the control group if p < 0.05 (*) andp <0.01〔**义

-94- Chapter 3 Results

9000� • Control • 10 mg/kg 8000 - • 5 mg/kg ^T ** r 7000 6000

4000

2000

1000

0 ^ 丁 丁 ^ No LPS LPS Treatment

Figure 3.22 The effect of PU60b on serum level of TNF-a in S-180 tumor bearing BALB/c mice Level of TNF-a in the serum collected from the tumor bearing mice of the control and treatment groups with or without LPS stimulation was determined by ELISA using recombinant TNF-a as a standard. The blood of mice was collected on Day 10 of the observation period. The results are expressed as mean (pg/ml) 土 S.D. (n=4).

The Student 's t-test was applied. The data were defined as significant and very significant difference from the control group if p < 0.05 (*) andp <0.01 (*V-

-95- Chapter 4 Discussion

Chapter 4

Discussion

4.1 Extraction and Isolation of polysaccharide fractions

Polyporus umbellatus sclerotium, a mass of hyphae compacted underground into rigid structures for survival in adverse conditions, was a target source of bioactive polysaccharides in the present study. To conserve the intact structure of polysaccharides extracted from the sclerotia, only hot water and cold alkali were used as solvents of extraction. However, the yields of hot-water extract (PUw) and cold-alkali extract (PUAL) were low (25.3% and 34.2%). Comparatively, the yield of cold-alkali extract ofPleuwtus tuber-regium was almost doubly higher (60 %) (Deng, 2000). It may be due to the additional step done in the extraction process that the powered sclerotia of Pleurotus tuber-regium were pre-treated with 80 % ethanol three times so that small molecular weight substances were removed and the tissues of sclerotium were soften, which allow the removal of alkaline soluble polysaccharides

-96- Chapter 4 Discussion readily.

Water-soluble crude polysaccharides were isolated from hot-water extract PUw by ethanol precipitation after pre-treatment with 60 % and 80 % ethanol. It was a predictable result that the yield ofPUSO (1.14 %) was higher than that of 60 % (0.48 %). The higher concentration of ethanol could precipitate almost all water-soluble polysaccharides in PUw while PU60 only precipitated certain portion of polysaccharides with relatively larger size. Because of the more potent bioactivity of fraction PU60 in the preliminary in vivo assay, PU80 was screened out and the work of polysaccharide isolation mainly focused on PU60.

PU60 was further separated into two main fractions (PU60a and PU60b) after elution on the DEAE cellulose column. PU60b was considered to be a major polysaccharide constituent for PUw (Figure 3.1). Fraction PU60a was regarded as neutral polysaccharides as they eluted with distilled water and no acidic sugar was detected in this fraction. Based on the ionic charge, polysaccharides eluted with sodium chloride solution from anion exchange chromatography are regarded as acidic polysaccharides. This was in agreement with the results from the sugar composition analysis for PU60b which was eluted with 0.15 M sodium chloride solution as galacturonic acid was detected, which indicated that a large proportion of PU60 belongs acidic polysaccharides.

4.2 Chemical characterization of PUw fractions

PU80 contained the most abundant protein content (w/w) (15.9 %) among all fractions

-97- Chapter 4 Discussion of PUw and PU60 contained proteins (12.6 %) slightly less than that of PU80. Surprisingly, the protein contents of PU60a and b which derived from PU60 were dramatically lower than that of PU60，which might be due to the fact that protein molecules were trapped in the DEAE-cellulose column. PU80 and PU60 shared some similarities in terms of types of monosaccharide compositions. Both fractions were rich in D-glucose with trace amounts of D-mannose, galacturonic acid, D-arabinose and D-galactose in decreasing order. Little difference in xylose and glucosamine contents between them was observed. This outcome indicated that the fractions PU60 and PU80 most likely contained same types of polysaccharide(s). However, the sugar composition of PU60 and PU80 was inconsistent with that in the polysaccharide-component analysis by Zhu (1988). Polysaccharides extracted from fermented Polyporus umbellatus contained mainly D-mannose, D-galactose and D-glucose at the ratio 20:4:1. The difference between the wild strain and the cultivated Polyporus umbellatus in monosaccharide composition implies that cultivation environments may affect the sugar compositions of the mushroom. On the other hand, PU60a and PU60b also contained D-glucose with trace amounts of mannose, glucosamine and xylose, rhamnose, galactose together with arabinose in decreasing order. Significant difference in contents of glucose and galacturonic acid between PU60a and PU60b was found. PU60b contained D-glucose (90.4 %) much more than that of PU60a (32.2 %)，which was inconsistent with the results of phenol-sulfuric acid assay. It was probably due to the strong hydrophilic ability of PU60a. Water in air was absorbed in the dried PU60a during the process of hydrolysis and then diluting the sugar composition of this fraction.

According to Figure 3.2，the hot-water crude extract PUw had no characteristic

-98- Chapter 4 Discussion absorption shown in the infrared spectra. This is probably due to existence of impurities and low concentration of polysaccharides in the crude extract, thus interfering with the measurement of resonance of functional bonding in the crude extract. By contrast, PU60 and PU60a exhibited sharp IR absorption at 890 cm\ indicating that the major glucosidic bond of these polysaccharide fractions was the P-form and hence they were identified as p-glucans with heteroglycan chains.

As illustrated in the HPLC chromatograms of PUw fractions (Figure 3.4 and Figure 3.5), PU60 and PU60b were present as asymmetric peaks, indicating that both fractions were probably heterogenous polysaccharides with various molecular weights ranged from 19.7 to 607.0 kDa. 93 % of PU60 and 90.4 % of PU60b were eluted using HPLC between retention time 17 and 23 minute. PU60a was a relatively homogenous polysaccharide showing in Figure 3.4 in the form of a small symmetric peak with 20.3 kDa in size.

4.3 In vitro anti-proliferative effect of PU fractions

There were not many successful cases of solely use of mushroom polysaccharides reported for studying in vitro antitumor effects on cancer cell lines as a result of the fact that polysaccharides from mushrooms do not attack cancer cells directly, but rather produce their antitumor effects by activating different immune responses in the host (Wasser, 2002). Instead, concurrent use of mushroom polysaccharides with antibodies like IgG or cytokines like TNF-a, INF-y and IL-2 in vitro were comparatively more common in recent research. In the present study, in vitro assay were used as preliminary estimation on anti-proliferative potentials of PU fractions as

-99- Chapter 4 Discussion usual. Research on in vitro anti-proliferative effects of Polyporus umbellatus polysaccharides by Yadomae (1979) demonstrated that the purified hot-water extracts ofPolyporus umbellatus demonstrated a mitogenic effect in vitro. Mitogenic effect is an effect in stimulating cellular multiplication (Hobbs, 1995). On the whole, mitogenic effect of hot-water crude extract (PUw) on cancer cells was also observed in the present study with high viability. Cell density of cell lines S-180, HL-60, HepG2 and Colon 201 was increased up to 35.1% under the 72-hour treatment. A trend of dose-dependent increase in cell density was found in HL-60 cells.

Interestingly, the mitogenic effect of fraction PUw on the above cancer cell lines did not consistently exhibit under the treatment of another three fractions PUAL, PU80 and PU60. The highest increase percentage of cell density was 62.2 % (Colon 201 cells treated with PUAL at a dose of 200 ng/ml). By contrast, proliferation of cell lines S-180and MCF-7 was inhibited when treated with the three fractions (Figure 3.6 and Figure 3.8). Despite limited cell proliferation inhibition, anti-proliferative effect of PU60 was the highest among the three fractions. Such a contradictory outcome was also found in mushroom heteropolysaccharides wc-PCMO, wb-PCMl and ab-PCMl isolated from Poria cocos (Jin, 2003). These fractions of Poria cocos enhanced cell proliferation of HepG2 cells but showed inhibition on HL-60 cell proliferation. The reason of this phenomenon is still unknown. Perhaps various types of cells possess different extent of sensitivity to the polysaccharides and present distinct cell surface receptors for generating cell signaling. To further identify the ability of direct cytotoxicity of PU fractions towards cell lines, normal kidney cells (Vero) was used as the control. Vero cells did not demonstrate considerable change in cell density

(Figure 3.10) and viability (data now shown) after 72-hour treatment of the PUw, PUAL

-100- Chapter 4 Discussion and PU60, indicating that the various PU fractions had no direct cytotoxicity on normal cells and they were safe to use in the animal models. Only PU80 exhibited mitogenic effect on Vero cells and the most potent dose was 200)ig/ml.

Similar to lentinan from Lentinus edodes (Chihara, 1992) and GLPP, a purified polysaccharide isolated from Ganoderma lucidum (Cao, 2004)，PU fractions failed to attack cancer cell lines directly, which revealed that Polyporus umbellatus polysaccharides were not ideal for use as an antiproliferative agent. Thus animal models were set up as the next step to determine in vivo antitumor activity of polysaccharide fractions in this present study.

4.4 In vivo antineoplastic assay of PU fractions

4.4.1 S-180 solid tumor model using BALB/c mice

In vivo preliminary antitumor tests were performed to estimate which crude extract,

PUw or PUAL. was worthy of fiirther fractionation. According to the review of Ooi and Liu (2000)，the preliminary determination of antineoplastic activity relied on several essential factors such as bioassay system, strain of mice, type of tumor and route of drug administration (Zhang, 2002). Ascitic Sarcoma 180 was selected so that an allogeneic tumor could be developed while BALB/c mice was chosen because this strain of mice is so commonly used in in vivo antitumor assay of mushrooms and hence facilitated comparison among them. In view of low yield and high solubility of the PU fractions, intraperitoneal injection was applied as less amount of sample was used in each dose when compared with the dose for oral administration and the soluble fractions might be directly absorbed into blood stream by diffusion via intraperitoneal

-101- Chapter 4 Discussion injection.

Hot-water and cold-alkali crude extracts at a dose of 100 mg/kg were administered into S-180 tumor bearing mice in the 20-clay preliminary study. Both crude extracts could effectively inhibit the tumor growth at the dosage (Figure 3.12). Fraction PUw retarded tumor growth by 76 %，which was slightly higher than PUAL did (68 %). About 14 % tumor bearing mice of PUw treatment group showed complete tumor regression while PUAL-treated mice showed no complete tumor regression. In the history of mushroom research, hot-water extracts from edible mushrooms belonging family Polyporaceae also showed remarkable antineoplastic activity on S-180 solid tumor. Ganoderma tsugae, Coriolus versicolor and Favolus alveolarius are good examples among them (Mizuno, 1995). Their tumor inhibition ratios are up to 77 %. Another mushroom in the same family, Trametes dickinsii, could exhibit tumor inhibition even by 80 % (Mizuno, 1995). Based on the distinguishable results between the two groups in addition to a fact that higher mortality was observed in the record of PUAL-treated groups (42.8 %)，fraction PUAL was neglected and the focus was placed on the PUw fraction in the following assay.

The following assay was done in order to determine the range of effective dose of PUw. As a dose of 100 mg/kg PUw had shown to exhibit such a notable tumor inhibition ratio, the dosage of the fraction was scaled down to 50 mg/kg, 25 mg/kg and 12.5 mg/kg. The result showed that the lower doses the treated mice took, the more potent the antineoplastic effect of PUw exhibited. Nevertheless, the most potent dose was found to be 100 mg/kg on the whole which significantly exhibited 79 % inhibition on tumor growth while a dose of 12.5 mg/kg might inhibit tumor

-102- Chapter 4 Discussion growth by 69 % only. Because of this abnormal phenomenon, the same range of dose with little amendment was repeated in the assay with fraction PU60 and PU80. The similar trend was found in fraction PU80 (Table 3.16): the ratio of tumor inhibition was high at a dose of 100 mg/kg and then decreased to 59 % at a dose of 50 mg/kg and rebounded to 76 % when the dose was 5-fold diluted. On the other hand, the tumor inhibition ratios at 75 to 80 % of fraction PU60 were quite similar at doses ranged from 10 mg/kg, 50 mg/kg and 100 mg/kg. As discussed in Section 4.2， fractions PU60 and PU80 most likely shared the same types of polysaccharide(s); the different outcome between the two assays of similar polysaccharide fractions reflected a possibility that PU60 fraction contained sugar compositions with more antineoplastic properties. Relatively, PU80 included extra sugar components that lowered the concentration of the effective monosaccharides at the fixed dose and its antineoplastic nature was distracted.

Change in body weight was measured in the duration of experiment. All mice in treatment groups did not reduce their body weight in the end of experiment. The percentage of body weight gain of PU60-treated mice was greater than those of the corresponding control group and PU80-treated mice. It basically indicated that they had no effect on body weight change of cancer objects as chemotherapeutic drugs do.

After screening out the PU80 fraction, PU60 was further fractionated with the aid of DEAE column, producing PU60a and b. As the tumor inhibition ratios of fraction PU60 showed steadily around 75 to 80 % at the doses ranged from 10 mg/kg, 50 mg/kg and 100 mg/kg, the doses of the two fractions used was fiirther scaled down to 10 mg/kg and 5 mg/kg in the assay. An inspiring outcome was that the

-103- Chapter 4 Discussion antineoplastic effect of PU60b was still prominent and was stronger than that of PU60. PU60b showed the highest tumor inhibition ratio (89%) at a dose of 5 mg/kg but the tumor inhibition effect slightly declined when the concentration of PU60b was doubled. On the other hand, the antineoplastic effect of PU60a was much lower than that ofPU60b. The highest tumor inhibition ratio of PU60a was 48 % (5 mg/kg) and it further decreased to 11 % when the concentration of PU60a was doubled. Obviously, acidic polysaccharides of Polyporus umbellatus (PU60b) exerted profound inhibition effect on tumor growth much better than the neutral polysaccharide (PU60a) did. This evidence was also found in the mushroom Tricholoma sp. (Liu, 1995). Acidic polysaccharide fraction A-2 of Tricholoma sp. (strain STC20-T1) showed much potent antineoplastic effect on both BALB/c and ICR mice when compared with its neutral polysaccharide fraction A-1. Despite that, there are differences in monosaccharide composition and protein content between the two effective acidic polysaccharides. PU60b contained mainly D-glucose (90.4%) with little protein (4.8 %) while A-2 included mainly galactose (49.3 %) with protein (30.1 %).

4.4.2 MCF-7 tumor in vivo model using athymic nude mice

Nude mice can be used to not only answer many questions in immunology, but support growth of many types of cells and tumors especially those of mouse origin due to their immunodeficiency. The main characteristics of an athymic nude mouse are the absence of the thymus and absence of hair due to impaired keratinization resulting in breakage of the hairs within the follicles (Corbett, 2002). There is a general agreement that the hairless condition and the failure of thymus development are due to some basic

-104- Chapter 4 Discussion defect in the embryonic ectoderm, and the first detectable abnormalities in the development of the nude thymus are in the ectodermal component occurring during the formation of a cervical vesicle on day 11 of embryonic life. The absence of functioning T cells prevents nude mice from rejecting not only allografts, but they cannot even reject xenografts (Clarke, 2002); that is, grafts of tissue from another species. T cells are white blood cells produced by the thymus gland and sent out to patrol the body in search of viruses and other invaders. T cells can differentiate into a surface marker called CD4+ T helper cells that regulate the activity of antibody-producing B cells and of other T cells such as CD8+ cytotoxic T cells which is responsible for cell-mediated immune responses and killing of virus-infected or malignant cells (Goldsby, 2002).

PU60 mediated a significant tumor inhibition of S-180 solid tumor in BALB/c model. Its in vivo antineoplastic effect might be due to direct toxicity, tumor-sensitive toxic metabolic derivatives such as NO and/or immunostimulatory properties of PU60b. The possibility of direct toxicity of PU60b was excluded because all fractions of PUw failed to show antiproliferative effect on cancer cells in this study. Other than that, fraction PU60b was able to inhibit tumor growth effectively in BALB/c mice but mediated no tumor regression in athymic mice, which implied that the fraction were not metabolized to tumor-sensitive toxic substances. Hence, fraction PU60b most likely mediates tumor regression in vivo by the activation of host immune effector mechanisms.

-105- Chapter 4 Discussion 4.5 Estimation of Immunomodulatory properties of PU60b

4.5.1 Identification of cytokines in serum from healthy BALB/c mice

The ability of a tumor to escape from immunological control may depend on a balance between the effectiveness of the immune system and a variety of factors promoting escape of tumor cells from destruction by effector components of the immune system. Mushroom polysaccharides are claimed to modulate the immune system and potentially exert antitumor effects (Borchers, 1999). With the aid of RayBio™ Mouse Cytokine Array, outline of serum cytokine production in PU60b-treated healthy mice was revealed on the membrane. RayBio™ Mouse Cytokine Array is a cytokine protein array system which can be used to rapidly and accurately identify the expression profiles of multiple cytokines. In fact, all cell functions, including cell differentiation，cell proliferation and cell death together with maintenance of health status and development of disease are regulated by gene expression and signaling pathways. New techniques such as cDNA microarrays facilitate people to analyze the global gene expression. However, almost all cell functions are regulated by proteins, which is not enough to be studied only by DNA and RNA solely. Gygi (1999) stated that a disparity between the relative expression levels of mRNA and their corresponding proteins. Thus, it is indispensable to analyze the protein profile. Currently, two-dimensional polyacrylamide SDS PAGE and mass spectrometry are the mainstream approach to analyzing multiple protein expression levels (Anderson, 2000; Legrain, 2000). Nevertheless, the requirement of sophisticated devices and the lack of quantitative measurements greatly limit their broad application. Considering that the cytokine protein array system can perform simultaneous detection of 32 types of cytokines, it was suitable for estimating the immunomodulatory properties ofPU60b.

-106- Chapter 4 Discussion Surprisingly, cytokines IL-6, Eotaxin, GCSF, Leptin, Tpo and VEGF existed only in the serum of control group. It was probably that PU60b down-regulated the expression of these kinds of cytokines. Despite that, Yang et al. (2004) demonstrated that the immunosuppression properties of tumor cell suspension S-180 such as declining of splenocyte proliferation, IL-2 production and natural killer cells activity were counteracted with polysaccharide extracted from P. umbellatus (PUPS) as a result of the down-regulating the synthesis and/or secretion of immunosuppressive substance by SI 80 cells. There were 15 types of cytokines commonly expressed in both sera. Among the expressed cytokines, the expression levels of KC, TIMP-1 and MIP-2 were higher than the others. Among them, tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) and macrophage inflammatory protein-2 (MIP-2) are involved in antitumor activity (Sun, 2000; Brand, 2002; Ikenaka, 2003).

Intratumoral injection of AdCMVmCD40L, an adenoviral vector, causes antitumor effect in the animal model associated with the intratumoral production of IL-12 and IFN-y and with an increased intratumoral expression of chemokines such as MIP- la, MIP-lp, MIP-2 and eotaxin (Sun, 2000). The induction of the antitumor agent leads a powerful cascade of chemokines and cytokines in the tumor mass and stimulate an efficient antitumor immunity. Matrix metalloproteinases (MMPs) are crucial enzymes involved in the degradation of the extracellular matrix for the invasive behavior of primary tumors and their metastases (Rigg, 2001; Brand，2002). TIMPs exert an anti-invasive effect by blocking MMP activity (Miyata et al.’ 2004). It has been shown that overexpression of TIMPs in tumors of various origins leads to reduced tumor growth and formation of metastases (Brand, 2002).

-107- Chapter 4 Discussion In the present study, both healthy and tumor bearing mice were used to investigate whether the polysaccharides from Polyporus umbellatus sclerotium were able to elevate TNF-a generation by using ELISA. The level of TNF-a is a parameter to evaluate the immunostimulating ability of polysaccharides on peritoneal macrophage proliferation as this cytokine, mainly produced by macrophages (Inoue, 2002), is one of the commonly used in tumor immunotherapy (Kuninaka et al., 2000; Terlikowski, 2002). Obviously, the serum level of TNF-a of LPS-primed healthy mice was much higher than that of mice without LPS stimulation. The amount of TNF-a generated by LPS-primed mice on Day 10 was much larger than that on Day 0; the big difference was significant especially in those from the PU60b treatment groups. PU60b-treated mice with LPS stimulation on Day 10 might generate a 3-fold increase in the production of TNF-a when compared with control group and that on Day 0. The results reflected that fraction PU60b exhibited immunostimulating effect which might last long in the host. More of acquired macrophages, T and B lymphocytes were produced and circulated in the blood. When the mice stimulated with LPS, the acquired immune cells secreted cytokines like TNF-a to protect the host. Similar outcome was also found in D-fraction (from Grifola fwndosa) treated mice (Inoue, 2002). D-fraction-treated mice exhibited a 2.5-fold increase in TNF-a production compared to the control.

When repeating the same assay with the sera collected from tumor-bearing mice, notable different in the level of TNF-a was also observed. However, an odd outcome was found in the control group. Perhaps, tumor cells in mice interfered with their immunity. In fact, immune system in tumor bearing mice maybe more complicated than that of healthy mice. The relationship between tumor cells and host immune

-108- Chapter 4 Discussion system was not clearly understood so fat. Further study on this issue is necessary to explain h.

-109- Chapter 5 Conclusions

Chapter 5

Conclusions

In the present study, two mushroom polysaccharide fractions, PU60a and PU60b, have been isolated from the sclerotium of Polyporus umbellatus. Both of them 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. PU60b exhibited the highest tumor inhibition ratio (89%) at a dose of 5 mg/kg in the S-180 tumor model using BALB/c mice. However, no remarkable proliferation inhibition was found in vitro assay. Hence, the possibility of direct cytotoxicity of PU60b on tumor cells was excluded. In the assessment of TNF-a production using ELISA, PU60b-treated mice on Day 10 with LPS stimulation could generate 3-fold increase in the production of TNF-a when compared with the control group and that on Day 0. The result reflected that the immunostimulation of the PU60b had a longer lasting effect in the hosts. In the assay of cytokines detected on -110- Chapter 5 Conclusions membranes of mouse cytokine array, 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.

Based on the conclusions of the experiment, some ftirther investigations are proposed. In the present study, the yield of PU60b extracted from the sclerotium was rather low; the extraction method can be modified to elevate the yield of the polysaccharide fraction by using enzymes such as chitinase that enhance the breakage of fungal cell wall to release the p-glucans. Other than that, the potent antineoplastic effects of PU60b will be further studied by mainly focusing on the role of TNF-a, TIMP-1 and MIP-2 with the aid of transgenic mice models and flow cytometry.

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-123- Chapter 3 Results

Table 3.21 Antibody list on the membrane of RayBio™ Mouse Cytokine Array.

ABC D E F G H I J K L 1 p� pn, N«a 6Ckine CTACK Eotaxin GCSF GM-CSF IL-2 IL-3 IL-4 2 —^ ^ 6Ckine CTACK Eotaxin GCSF GM-CSF IL-2 IL-3 IL-4 3 "iiTs iL^nirfe iLUT" IL-12p70 IL-13 IL-17 IFN-y KC Leptin MCP-1 4 "ITs O O~~nTiF" IL-12 IL-12p70 IL-13 IL-17 IFN-y KC Leptin MCP-1 5 Mrp-C MTP-lr. Mipl^j^-sp RANTES SCF sTNFri TARC TIMP-1 TNF-a Tpo VEGF 6 MCP.S MIP.I" Ml^ MIP-3B "RANTES SCF sTNFri TARC TIMP-1 TNF-g Tpo VEGF 7 Blank~~Rwik""^!^ Blank Blank Blank Blank Blank Blank Blank Blank Pos 8 Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Pos (Ray Biotech, 2003) tNote: Pos - positive control; Neg - negative control. All other cytokine short forms on the table are based on the standard abbreviations.

-90- Chapters Results

3 r Distilled water OtolMNaCl

2.5 f ‘ ‘

I I PU60b I \ I 1.5 - \

0 L-- ‘ 0 20 40 60 80 100 120 Number of Tubes (6 ml/tube)

Figure 3.1 Anion exchange chromatography of PU60 on a DEAE-cellulose column.

SO mg of PU60 was applied to a 2.5 x 30 cm DEAE-cellulose anion exchange column. The flow rale was 30 ml/h and the fracLation was collected at 6 ml/tube. The column was eluted stepwise with distilkd^ater to obtam the unbound component (PU60a) and with a salt gradient o/NaCl (0 to 1 M) to obtain the retained component (PU60b). The presence of carbohydrate was determined by the phenol-sulphuric assay at 490 nm.

-54- I ‘ • -

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