Investigations into the effects of traditional chinese medicinal herbs used in the treatment of human breast cancer

INVESTIGATIONS INTO THE EFFECTS OF TRADITIONAL CHINESE MEDICINAL HERBS USED IN THE TREATMENT OF HUMAN BREAST CANCER AGNES SLATER B.A (ELTE University, Budapest) M.B.B.S (Beijing Medical University, Beijing) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY FACULTY OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2005 ii ACKNOWLEDGEMENTS I would like to express my sincere appreciation and gratitude to the following: - my supervisor, Assoc Prof Benny Kwong Huat Tan, for his invaluable guidance, patience and constant encouragement - our Senior Laboratory Officer, Ms Annie Hsu, for her assistance, expertise and kindness - Head and Staff of Department of Pharmacology for making the laboratory facilities available for my use and for their cooperation - Dr C.S.L Fones, Dr Teo Eng Kiat, Dr Ruth Kiew, Mr Jonathan Ward, Prof Chong Siew Meng for the willingness to share their expertise - National University of Singapore for providing me with the scholarship - my family and friends for their moral support Without the above it would not have been possible to complete this project iii TABLE OF CONTENTS ACKNOWLEDGEMENT Page ii TABLE OF CONTENTS iii LIST OF TABLE viii LIST OF FIGURES ix LIST OF ILLUSTRATIONS xi LIST OF ABBREVIATIONS xii LIST OF PUBLICATIONS AND CONFERENCE PAPERS xv SUMMARY xvi CHAPTER ONE: GENERAL INTRODUCTION 1.1 1.2 Herbs used in TCM for cancer treatment 1.3 Breast cancer: significance in modern day society 20 1.4 Traditional Chinese Medicine (TCM) Rationale and purpose of this study 22 CHAPTER TWO: MATERIALS AND METHODS 2.1 Materials 24 25 2.1.1 25 2.1.2 Animals 27 2.1.3 Chemicals and reagents 27 2.1.4 Kits 28 2.1.5 2.2 Cell lines Facilities 28 Methods 29 2.2.1 Quality-of-life (QOL) study ………………………… 29 2.2.1.1.QOL study design……………… …………………………… 29 iv 2.2.1.2.Scoring procedures for the EORTC QLQ-30 and BR-23 questionnaires……….………………………………………… 2.2.2 41 Extraction of herbs .………… 41 2.2.2.1 Preparation of water and ethanolic extract of Astragalus membranaceus, Curcuma aromatica, Scutellaria barbata and Solanum nigrum 42 2.2.2.2 Fractionation of the water boiled extract of Astragalus membranaceus ……… 42 2.2.2.3 Characterization of the ethanolic and water extract of Solanum nigrum……… 2.2.3 Breast cancer cell culture 2.2.4 43 43 Cell viability assays: MTT test and trypan blue exclusion examination 44 2.2.5 Lymphocyte activation assay 44 2.2.6 Assay of cytokines: IL-2, IL-6 and TNF-α 45 2.2.7 In-vitro cytotoxicity assay 46 2.2.8 In-vitro analysis to determine the type of cell death 46 2.2.8.1 DNA fragmentation ELISA assay (Fig.1) 46 2.2.8.2 Cellular DNA fragmentation ELISA assay (Fig.2) 48 2.2.9 Animal handling 49 2.2.9.1 Housing and maintenance 49 2.2.9.2 Mice gavage 50 2.2.9.3 Blood collection by intracardiac puncture 50 2.2.9.4 Intraperitoneal (i.p.) injection 50 2.2.9.5 Acute toxicity testing 50 v 2.2.10 Breast tumor model in BALB/c nude mice 51 2.2.10.1.Tumor xenografts…………………………………………… 51 2.2.10.2.In-vivo treatment protocols……………………………… 51 2.2.11 52 2.2.12 Histopathological analysis of tumor tissue sections Statistical analysis 52 CHAPTER THREE: RESULTS AND DISCUSSION 3.1 Quality-of-life (QOL) study in breast cancer patients 53 54 3.1.1 Aims and experimental approach 54 3.1.2 Results 55 3.1.3 Discussion 68 Extraction and characterization of Solanum nigrum 71 3.2 3.2.1 Aims and experimental approach 71 3.2.2 Results 72 3.2.3 Discussion 74 3.3 Results of in-vitro immunomodulatory studies 74 3.3.1 Aims and experimental approach .… 74 3.3.2 Results 75 3.3.2.1.Effect of ethanolic extract of Astragalus membranaceus and Curcuma aromatica…………………… 75 3.3.2.2.Effect of the fractions of Astragalus membranaceus water extract………………………… 76 Discussion 80 Cytokine studies from mice serum 80 3.3.3 3.4 3.4.1 Aims and experimental approach 80 vi 3.4.2 81 3.4.3 3.5 Results Discussion 81 In-vitro cytotoxicity studies 82 3.5.1 Aims and experimental approach …………………… 82 3.5.2 Results 82 3.5.2.1 Results of cytotoxicity assays of Solanum nigrum and Scutellaria barbata in MCF-7 human breast cancer cell lines … 82 3.5.2.2 Results of cytotoxicity assays of Solanum nigrum and in MCF-7, MDA-MB-231, T-47D and ZR-75-1 human breast cancer cell lines 85 3.5.2.3 Results of DNA fragmentation in MCF-7 cells treated with Solanum nigrum……………………………………………………… 86 3.5.2.4 Results of cellular DNA fragmentation in MDA-MB-231 cells treated with Solanum nigrum…………………………………… 3.5.3 3.6 Discussion…………………………………………………………… In-vivo cytotoxicity studies…………………………………… 88 92 92 3.6.1 Aims and experimental approach……………………………… 92 3.6.2 Results………………………………………………… 93 3.6.2.1 Acute toxicity test of Solanum nigrum in normal and nude BALB/c mice 93 3.6.2.2 Anti-tumor activity of Solanum nigrum on MCF-7 tumor-bearing oophorectomized BALB/c nude mice……… 94 vii 3.6.2.3 Estrogen-induced uterine cancer in oophorectomized BALB/c nude mice………………………………………… 97 3.6.2.4 Anti-tumor activity of Solanum nigrum on MDA-MB-231 tumor-bearing oophorectomized BALB/c nude mice……… 99 3.6.2.4.1 Results of therapeutic treatment with Solanum nigrum 99 3.6.2.4.2 Results of prophylactic treatment with Solanum nigrum 101 3.6.2.5 Histopathological examination of Solanum nigrum induced apoptosis in MDA-MB-231 cells 3.6.3 Discussion CHAPTER FOUR: SUMMARY AND DISCUSSION 102 102 104 4.1 105 4.2 Summary of results Overall discussion 108 CHAPTER FIVE: CONCLUSION AND FUTURE STUDIES 109 5.1 110 5.2 Conclusion Future studies 110 BIBLIOGRAPHY 111 viii LIST OF TABLES Page Table Conventional treatment of selected patients………………… 30 Table Composition of EORTC QLQ-C30 (version 3.0) 36 Table Composition of EORTC breast cancer module QLQ-BR23………… 38 Table Effect of different concentrations of 80% ethanol extract of Astragalus membranaceus and Curcuma aromatica on BALB/c mice lymphocyte proliferation……………………………………………………………… 75 Table Cytokine concentration after AM treatment in BALB/c mice…………… 81 ix LIST OF FIGURES Page Figure Cell death detection ELISAPLUS 47 Figure Cellular DNA fragmentaion ELISA………………………………… 49 Figure QLQ-30 scores for breast cancer patients receiving radio-, chemo-, hormonal and herbal TCM therapy………………………………… 55 Figure QLQ-30 scores for breast cancer patient receiving radio-, hormonal and herbal TCM therapy 57 Figure QLQ-30 scores for breast cancer patients receiving hormonal and herbal TCM therapy………………………………………………… 59 Figure QLQ-30 scores for breast cancer patient receiving only herbal TCM Therapy……………………………………………………………… 61 Figure QLQ-30 reference scores compared to the baseline scores of breast cancer patients in Singapore receiving herbal TCM therapy………… 63 Figure BR-23 scores of breast cancer patients at baseline and after 11-13 weeks of TCM treatment……………………………………………… 65 Figure Single item score distribution of breast cancer patients at baseline and after 11-13 weeks of TCM………………………………………… 67 Figure 10 SN water and ethanolic extract UV spectophotometer scan……… 72 Figure 11 HPLC chromatogram of SN water extract…………………………… 73 Figure 12 Immunomodulatory effect of the ethanolic extract of AM on mouse lymphocytes………………………………………………………… 77 Figure 13 Immunomodulatory effect of the aqueous extract of AM on mouse splenocytes………………………………………………………… 77 Figure 14 Immunomodulatory effect of butanolic fraction of aqueous extract of AM on mouse splenocytes……………………………………… 78 Figure 15 Immunomodulatory effect of ethyl acetate fraction of aqueous extract of AM on mouse splenocytes………………………………… 78 Figure 16 Immunomodulatory effect of remaining water fraction of aqueous Extract of AM on mouse splenocytes………………………………… 79 Figure 17 Comparative cytotoxic effect of Solanum nigrum and Scutellaria barbata on MCF-7 cells using MTT-tetrazolium assay …………………………… 83 x Figure 18 Comparative cytotoxic effect of Solanum nigrum and Scutellaria barbata on MCF-7 cells (log scale)… ……………………………… 84 Figure 19 Cytotoxic effect of the ethanol extract of SN on MCF-7, MDA-MB-231, T-47D and ZR-75-1 cell lines………………………………………… 85 Figure 20 DNA fragmentation in MCF-7 cells (SN 4h)……………………… 86 Figure 21 DNA fragmentation in MCF-7 cells (SN 8h)………………… ……… 86 Figure 22 DNA fragmentation in MCF-7 cells (SN 12h)………………… …… 87 Figure 23 DNA fragmentation in MCF-7 cells (SN 24h)………………… … … 87 Figure 24 Cytotoxic effect and cellular DNA fragmentation of the ethanol extract of SN on MDA-MB-231 after 2h incubation …………………………… 88 Figure 25 Cytotoxic effect and cellular DNA fragmentation of the ethanol extract of SN on MDA-MB-231 after 4h incubation …………………………… 89 Figure 26 Cytotoxic effect and cellular DNA fragmentation of the ethanol extract of SN on MDA-MB-231 after 6h incubation …………………………… 89 Figure 27 Acute toxicity test of Solanum nigrum (water extract) in Swiss albino mice……………………………………………………………………………… 93 Figure 28 Mortality of 50 beta-estradiol implanted nude mice over time……… 97 Figure 29 Prophylactic treatment of Solanum nigrum (ethanolic extract) on MDA-MB-231 implanted nude mice…………………………………………… 101 112 randomized, placebo-controlled, double-blind clinical trial Scandinavian Journal of Gastroenterology 40: 936-943 Brown H (1986) The deadliest delusion: unproven methods in management of cancer Women Health 11(3-4): 165-178 Burstein HJ, Gelber S, Guadagnoli E, Weeks CJ (1999) Use of alternative medicine by women with early-stage breast cancer The New England Journal of Medicine 340: 1733-1739 Cai YZ, Luo QO, Sun M, Corke H (2004) Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer Life Sciences 74: 2157-2184 Cameron DA, Ritchie AA, Langdon S, Anderson TJ, Miller WR (1997) Tamoxifen induced apoptosis in ZR-75 breast cancer xenografts antedates tumour regression Breast Cancer Research and Treatment 45: 99-107 Cha YY, Lee EO, Lee HJ, Park YD, Ko SG, Kim SG, Kim DH, Kim HM, Kang IC, Kim SH (2004) Methylene chloride fraction of Scutellaria barbata induces apoptosis in human U937 leukemia cells via the mitochondrial signaling pathway Clinica Chimica Acta 348: 41-48 Cham BE, Meares HM (1987) Glykoalkaloids from Solanum sodomaeum are effective in the treatment of skin cancers in man Cancer Letters 36: 111-118 Chang RS, Yeung HW (1988) Inhibition of growth of human immunodeficiency virus in vitro by crude extracts of Chinese medicinal herbs Antiviral Research 9: 163-175 Cheng JH, Chang G, Wu Wy (2001) A controlled clinical study between hepatic arterial infusion with embolized curcuma aromatica oil and chemical drugs in treating primary liver cancer Zhongguo Zhong Xi Yi Ji He Za Zhi 21: 165-167 Cheng SK (2005) The integrative use of TCM and Western medicine: Case studies on liver cancer HealthWorlds Asia International Medical Congress 2005, Singapore Chiang HC, Tseng TH, Wang CJ, Chen CF, Kan WS (1991) experimental antitumor agents from Solanum indicum L Anticancer Research 11: 1911-1917 Chu DT, Wong WL, Mavligit GM (1988a) Immunotherapy with Chinese medicinal herbs I Immune restoration of local xenogeneic graft-versus-host reaction in cancer patients by fractionated Astragalus membranaceus in vitro Journal of Clinical and Laboratory Immunology 25: 119-123 Chu DT, Wong WL, Mavligit GM (1988b) Immunotherapy with Chinese medicinal herbs II Reversal of cyclophosphamide-induced immune suppression by administration of fractionated Astragalus membranaceus in vivo Journal of Clinical and Laboratory Immunology 25: 125-129 113 Chu DT, Lepe-Zuniga J, Wong WL, LaPushin R, Mavligit GM (1990) Fractionated extract of Astragalus membranaceus, a Chinese medicinal herb, potentiates LAK cell cytotoxicity generated by a low dose of recombinant interleukin-2 Zhong Xi Yi Jie He Za Zhi 10: 34-36 Cui Y, Shu XO, Gao YT, Cai H, Wen WQ, Ruan ZX, Jin F, Zheng W (2004) The long-term impact of medical and socio-demographic factors on the quality of life of breast cancer survivors among Chinese women Breast Cancer Research and Treatment 87: 135-147 Dafni A, Yaniv Z (1994) Solanaceae as medicinal plants in Israel Journal of Ethnopharmacology 44: 11-18 Deng YJ (2002) Brief introduction to application of dosage of Chinese herbs Journal of Traditional Chinese Medicine 22(2): 155-156 Dewick PM (1997) Medicinal natural products A biosynthetic approach John Wiley & Sons Ltd., England Dharmananda S (1997) Oriental perspectives on cancer and its treatment (http://www.itmonline.org/arts/cancer.htm ) Di Gianni LM, Garber JE, Winer EP (2000) Complementary and alternative medicine use among women with breast cancer Journal of Clinical Oncology 20: 34s-38s Desagher S., Martinou JC (2000) Mitochondria as the central control point of apoptosis Cell Biology 10: 369-377 Dobs A (2001) Modernization of TCM Traditional Chinese Medicine (TCM) Research Symposium 2001 Singapore Duvoix a, Blasius R, Delhalle S, Schnekenburger M, Morceau F, Henry E, Dicato M, Diederich M (2005) Chemopreventive and therapeutic effects of curcumin Cancer Letters 223: 181-190 Efferth T (2005) Mechanistic perspectives for 1,2,4-trioxanes in anti-cancer therapy Drug Resistance Updates 8: 85-97 Eisenberg DM, Kessler RC, Foster C, Norlock FE, Calkins DR, Delbanco TL (1993) Unconventional medicine in the United States Prevalence, costs, and patterns of use New England Journal of Medicine 328(4): 246–252 Eltayeb EA, Al-Ansari AS, Roddick JG (1997) Changes in the steroidal alkaloid solasodine during development of Solanum nigrum and Solanum incanum Phytochemistry 46: 489-494 114 Ernst E, Resch KL, Mills S, Hill R, Mitchell A, Willoughby M (1995) Complementary medicine – a definition British Journal of General Practice 45: 506 Ernst E, De Smet P (1996) Risks associated with complementary therapies Meyler’s side effects of drugs, Elsevier Science B.V, Germany Ernst E, Cassileth BR (1998) The Prevalence of Complementary/Alternative Medicine In Cancer Cancer 83: 777-782 Fayers PM, Weeden S, Curran D on behalf of the EORTC (1998) Quality of Life Study Group EORTC QLQ-C30 Reference Values, EORTC Brussels Fayers PM, Aaronson NK, Bjordal K, Curran D, Groenvold M on behalf of the EORTC Quality of Life Study Group (1999) The EORTC QLQ-30 Scoring Manual (2nd Edition) European Organization for research and treatment of Cancer Brussels Fayers PM (2001) Interpreting quality of life data: population based reference data for the EORTC QLQ-C30 European Journal of Cancer 37: 1331-1334 Fetrow CW, Avila JR (2001) Professional’s Handbook of Complementary & Alternative medicine (2nd Edition) Springhouse, USA Fung KW, Lau Y, Fielding R, Or A, Yip AW (2001) The impact of mastectomy, breast-conserving treatment and immediate breast reconstruction on the quality of life of Chinese women Australian and New Zealand Journal of Surgery 71: 202-206 Gangemi D (2003) Evaluation of fractionated Astragalus membranaceus constituents: “Inflammatory and anti-inflammatory cytokine induction profiles National Nutraceutical Center, Clemson University, Clemson, SC, USA Gee JMW, Robertson JFR, Ellis IO, Willsher P, McClelland RA, Hoyle HB, Kyme SR, Finlay P, Blamey RW, Nicholson RI (1994) Immunocytochemical localization of Bcl-2 protein in human breast cancers and its relationship to a series of prognostic markers and response to endocrine therapy International Journal of Cancer 59: 619-628 Gerlier D, Thomasset N (1986) Use of MTT colorimetric assay to measure cell activation Journal of Immunological Methods 94: 57-63 Giveon SM, Lieberman N, Klang S, Kahan E (2003) A survey of primary care physicians’ perceptions of their patients’ use of complementary medicine Complementary Therapies in Medicine 11: 254-260 Goel A, Boland CR, Chauhan DP (2001) Specific inhibition of cyclooxygenase2 (COX-2) expression by dietary curcumin in HT-29 human colon cancer cells Cancer Letters 172: 111-118 115 Goodman SM, Hobbs JJ (1988) The ethnobotany of the Egyptian eastern desert: A comparison of common plant usage between two culturally distinct Bedouin groups Journal of Ethnopharmacology 23: 73-89 Gottardis MM, Robinson SP, Jordan VC (1988) Estradiol-stimulated growth of MCF-7 tumors implanted in athymic mice: a model to study the tumoristatic action of Tamoxifen Journal of Steroid Biochemistry 30: 311-314 Govindarajan VS (1980) Turmeric—chemistry, technology and quality, CRC Critical Reviews in Food Science and Nutrition 12: 199–301 Grosvernor PW, Supriono A, Gray DO (1995) Medicinal plants from Riau province, Sumatra, Indonesia Part 2: antibacterial and antifungal activity Journal of Ethnopharmacology 45: 97-111 Gupta S, Cohen JJ (1996) Mechanism of lymphocyte activation and immune regulation VI Cell cycle and Programmed Cell Death in the Immune system Plenum Press, USA Hawkins RA, Arends MJ, Ritchie AA, Langdon S, Miller WR (2000) Tamoxifen increases apoptosis but does not influence markers of proliferation in an MCF-7 xenograft model of breast cancer The Breast 9: 96-106 Health Science Authorities (2002) Singapore (www.hsa.gov.sg) He ZQ, Wang BQ (1990) Isolation and identification of chemical constituents of Astragalus root Acta Pharmaceutica Sinica 25: 694-698 Hengartner MO (2000) The biochemistry of apoptosis Nature 407: 770-776 Heo KS, Lee SJ, Ko JH, Lim K, Lim KT (2004) Glycoprotein isolated from Solanum nigrum L inhibits the DNA-binding activities of NF-κB and AP-1, and increases the production of nitric oxide in TPA-stimulated MCF-7 cells Toxicology in Vitro 18: 755-763 Hirotani M, Zhou Y, Lui HK, Furuya T (1994) Astragalosides from hairy root cultures of Astragalus membranaceus Phytochemistry 36: 665-670 Ho C, Lun KC, Ng CH (1980) The Role of Chinese Traditional Medical Practice as a Form of Health Care in Singapore American Journal of Chinese Medicine VIII: 26-36 Ho GH, Phang BH, Ng ISL, Law HY, Soo KC, Ng EH (2000) Novel Germline BRCA1 Mutations Detected in Women in Singapore who Developed Breast Carcinoma befor the Age of 36 Years Cancer 89: 811-816 Ho NK (2001) Understanding Traditional Chinese Medicine – A Doctor’s Viewpoint Singapore Medical Journal 42: 487-492 116 Hong SH, Jeong HJ, Chung HS, Kim HR, Chae HJ, Shin T, Seo Y, Kim HM (2005) An herbal formula, Herbkines, enhances cytokines production from immune cells Journal of Ethnopharmacology 98: 149-155 Hsu SH, Li YP, Lin WL, Liu LF, Chang LC, Lin CC, Lin CN on behalf of Ministry of Health Singapore (1995) Report by the Committee on Traditional Chinese Medicine in Singapore, Singapore National Printers, Singapore Hu K, Kobayashi H, Dong A, Jing Y, Iwasaki S, Yao X (1999) Antineoplastic agents III: Steroidal glycosides from Solanum nigrum Planta Medica 65(1): 3538 Huang MT, Ma W, Yen P, Xic JG, Han J, Frenkel K (1997) Inhibitory effect of low doses of curcumin topical application on 12-0-tetradecanoylphorbol-13acetate-induced tumour promotion and oxidized DNA bases in mouse epidermis Carcinogenesis 18: 83-88 Ikeda T, Ando J, Miyazano A, Zhu XH, Tsumagari H, Nohara T, Yokomizo K, Uyeda M (2000) Anit-herpes virus activity of Solanum steroidal glycosides Biological & Pharmaceutical Bulletin 23: 363-364 Imaginis (2005) General Information on Breast Cancer The Breast Health Resource (http://imaginis.cowww.singaporecancersociety.org.sg/c_breastcancer.htmm/breasthealth /statistics.asp#1) Inano H, Onoda M, Inafuku N, Kubota M, Kamada Y, Osawa T, Kobayashi H, Wakabayashi K (2000) Potent preventive action of curcumin on radiationinduced initiation of mammary tumorigenesis in rats Carcinogenesis 12: 18351841 Jamison JM, Gilloteaux J, Taper HS, Calderon PB, Summers JL (2002) Autoschizis: a novel cell death Biochemical Pharmacology 63: 1773-1783 Jayaprakasha GK, Rao LJM, Sakariah KK (2005) Chemistry and biological activities of C longa Trends in Food Science & Technology 16: 533-548 Jellin JM (2005) Natural medicines in the Clinical management of Breast Cancer Natural Medicines Comprehensive database (http://naturaldatabase.com) Jiao Y, Wen J, Yu x (1999) Influence of flavonoid of Astragalus membranaceus’s stem and leaves on the function of cell mediated immunity in mice Zhong Xi Yi Jie He Za Zhi 19: 356-358 Kaasa A, Bjordal K, Aaronson N, Moum T, Wist E, Hagen S (1995) The EORTC Core Quality of Life Questionnaire (QLQ-C30): Validity and Reliability When Analyzed With Patients Treated With Palliative Radiotherapy European Journal of Cancer 31A: 2260-2263 117 Kaegi E (1998) Unconventional therapies for cancer: 1.Essiac Journal of Canadian Medical Association 158(7): 897-902 Kaufmann SH, Hengartner MO (2001) Programmed cell death: alive and well in the new millennium TRENDS in Cell Biology 11: 526-534 Keys JD (1997) Chinese Herbs Their Botany, Chemistry and Pharmacodynamics Charles E Tuttle Company, Japan Kim C, Ha H, Kim JS, Kim YT, Kwon SC, Park SW (2003) Induction of growth hormone by the roots of Astragalus membranaceus in pituitary cell culture Archives of Pharmacol Research 26: 34-39 Kitai E, Vinker S, Sandiuk A, Hornik O, Zelcer C,Gaver A (1998) Use of complementary and alternative medicine among primary care patients Family Practice 15(5): 411–414 Kojima H, Yanai T, Toyota A (1998) Essential Oil Constituents from Japanese and Indian Curcuma aromatica Rhizomes Planta Medica 64: 380-381 Kresina TK (1998) Immune Modulating Agents Marcel Dekker, Inc USA Kumar PV, Shashidhara S, Kumar MM, Sridhara BY (2001) Cytoprotective role of Solanum nigrum against gentamicin-induced kidney cell (Vero cells) damage in vitro Fitoterapia 72: 481-486 Kuo KW, Lin CN (2001) United States Patent # 6214803 ( http://www.uspto.gov/patft) Kuo SHM (2000) Anticancer activity evaluation of the solanum glycoalkaloid solamargine Triggering apoptosis in human hepatoma cells Biochem Pharmacol 60(12): 1865-1873 Latoxan Data Sheet (2005) L6096 Solasodine Valence, France (http://www.latoxan.com/HTML/00000088.html ) Lee SJ, Lim KT (2003) Antioxidative effects of glycoprotein isolated from Solanum nigrum Linne on oxygen radicals and its cytotoxic effects on the MCF-7 cell Journal of Food Science 68: 466-470 Lee SJ, Ko JH, Lim K, Lim KT (2005) 150 kDa glycoprotein isolated from Solanum nigrum Linne enhances activities of detoxicant enzymes and lowers plasmic cholesterol in mouse Pharmacological Research 51: 399-408 Lee YS, Han OH, Park CW, Yang CH, Jeon TW, Yoo WK, Kim SH, Kim HJ (2005) Pro-inflammatory cytokine gene expression and nitric oxide regulation of aqueous extracted Astragali radix in RAW 264.7 macrophage cells Journal of Ethnopharmacology 100: 289-294 118 Levinson W, Jawetz E (1996) Medical Microbiology & Immunology Appleton & Lange, Stamford, USA : 324-327 Li WL, Zheng HC, Bukuru J, De Kimpe N (2004) Natural medicines used in the traditional Chinese medical system for therapy of diabetes mellitus Journal of Ethnopharmacology 92: 1-21 Lin CC, Lin WC, Yang SR, Shieh DH (1995) Anti-inflammatory and hepatoprotective effects of Solanum alatum American Journal of Chinese Medicine 23: 65-69 Liu QY (2002) Effect of polyunsaturated fatty acids and antioxidants on Doxorubicin treatment: studies in both cancer cellcultures and tumor bearing mice PhD Thesis, National University of Singapore, Singapore Locher CP, Burch MT, Mower HF, Berestecky J, Davis H, van Poel B, Lasure A, Vanden Berghe DA, Vlietinck AJ (1995) Anti-microbial activity and anti-complement activity of extracts obtained from selected Hawaiian medicinal plants Journal of Ethnopharmacology 49: 23-32 Lorke D (1983) A New Approach to Practical Acute Toxicity Testing Archives of Toxicology 54: 275-287 Luo YM, Qin Z, Hong Z, Zhang XM, Ding D, Fu JH, Zhang WD, Chen J (2004) Astragaloside IV protects ischemic brain injury in a murine model of transient focal ischemia Neuroscience Letters 363: 218-223 Lund TD, Lephant ED (2001) Dietary soy phytoestrogens produce anxiolytic effects in the elevated plus-maze Brain Research 913: 180-184 Ma XF, Tu PF, Chen YJ, Zhang TY, Wei Y, Ito Y (2003) Preparative isolation and purification of two isoflavones from Astragalus membranaceus Bge.var.mongholicus (bge.)Hsiao by high-speed counter-current chromatography Journal of Chromatography 992: 193-197 Mao SP, Cheng KL, Zhou YF(2004) Modulatory effect of Astragalus membranaceus on Th1/Th2 cytokine in patients with herpes simplex keratitis Zhongguo Zhong Xi Yi Jie He Za Zhi 24:121-123 Mazumder A, Raghavan K, Weinstein J, Kohn KW, Pommer Y (1995) Inhibition of human immunodeficiency virus type-1 integrase by curcumin Biochemical Pharmacology 49: 1165-1170 Mimnaugh EG, Fairchild CR, Fruehauf JP, Sinha BK (1991) Biochemical and pharmacological characterization of MCF-7 drug-sensitive and AdrR multidrugresistant human breast tumor xenografts in athymic nude mice Biochemical Pharmacology 42: 391-402 119 Nakamura A, Nagai K, Suzuki S, Ando K, Tamura G (1986) A novel method of screening for immunomodulating substances, establishment of an assay system and its application to culture broths of microorganisms Journal of Antibiotics 39: 1148-1154 National Center for Complementary and Alternative Medicine (2000) NCCAM Strategic Plan 2000-2005, ( http//:nccam.nih.gov) USA Navarro DdF, de Souza RA, Neto RA, Golin V, Niero R, Yunes RA, Monache FD, VC Filho (2002) Phytochemical analysis and analgesic properties of Curcuma zedoaria grown in Brazil Phytomedicine 9: 427-432 Nelson W, DeWeese T (2001) Effects of CAM Interventions on Oxidative Damage in Cancer Cells Traditional Chinese Medicine (TCM) Research Symposium 2001 Singapore Ng EH, Gao F, Ji CY, Ho GH, Soo KC (1997) Risk factors for breast carcinoma in Singaporean Chinese women: the role of central obesity Cancer 80: 725-731 Niederau C, Göpfert E (1999) The effect of chelidonium- and turmeric root extract on upper abdominal pain due to functional disorder of the biliary system Results fro a placebo-controlled double-blind study Medizinische Klinik 94: 425430 Niu HM, Liu JX (2003) Advances in TCM treatment for metastasis of tumors Journal of Traditional Chinese Medicine 23(2): 151-157 Oltvai NZ, Milliman CL, Korsmeyer SJ (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death Cell 74: 609-619 Osborne CK, Hobbs K, Clark GM (1985) Effects of estrogens and antiestrogens on growth of human breast cancer cells in athymic nude mice Cancer Research 45: 584 Osoba D, Rodrigues G, Myles J, Zee B, Pater J (1998) Interpreting the significance of changes in health-related quality-of-life scores Journal of Clinical Oncology 16: 139-144 Paltiel O, Avitzour M, Peretz T, Cherny N, Kaduri L, Pfeffer RM, Wagner N, Soskolne V (2001) Determinants of the Use of Complementary Therapies by patients With Cancer Journal of Clinical Oncology 19: 2439-2448 Park WH, Kim CH, Lee YC, Kim CH (2004) Anti-inflammatory effects of a traditional Korean formulation, Silsisangami, consisting of seven medicinal herbs: effect on hemolysis, neutrophil function, and gene expression of iNOS and COX2 Vascular Phramacology 42: 7-15 Parslow TG, Stites DP, Terr AI, Imboden JB (2003) Medical Immunology The McGraw-Hill Companies, Inc., Singapore: 148-157 120 Patwardhan B, Gautam M (2005) Botanical immunodrugs: scope and opportunities Drug Discovery Today 10: 495-502 Peirce A (1999) The American Pharmaceutical Association Practical Guide to Natural Medicines Stonesong Press, USA Perlman AI, Eisenberg DM, Panush RS (1999) Talking with patients about alternative and complementary medicine Rheumatic Diseases Clinics of North America 25(4): 815–822 Perez GRM, Perez LJA, Garcia DLM, Sossa MH (1998) Neuropharmacological activity of Solanum nigrum fruit Journal of Ethnopharmacology 62: 43-48 Personal communications with Teo Eng Kiat (2001) Singapore Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I et al (2005) Trastuzumab after Adjuvant Chemotherapy in HER2Positive Breats Cancer The New England Journal of Medicine 353: 1659-1672 Polyak K (2001) On the birth of breast cancer Biochimica et Biophysica Acta 1552: 1-13 Powell CB, Fun P, Jackson J, Dall’Era J, Lewkowicz D, cohen I, SmithMcCune K (2003) Aqueous extract of herba Scutellaria barbatae, a Chinese herb used for ovarian cancer, induces apoptosis of ovarian cancer cell lines Gynecologic Oncology 91: 332-340 Purchase IFH, Tustin RC, van Rensburg SJ (1975) Biological testing of food grown in Transkei Food and Cosmetic Toxicology 13: 639-644 Rang HP, Dale MM, Ritter JM, Moore PK (2003) Pharmacology Churchill Livingstone, United Kingdom Read BE (1977) Chinese Materia Medica, Southern Material Press, Taiwan Reed JC (1994) Bcl-2 and the regulation of programmed cell death Journal of Cell Biology 124: 1-6 Reed JC(2001) Apoptosis-regulating proteins as targets for drug discovery TRENDS in Moleculer Medicine 7: 314-319 Reid DP (1993) Chinese Herbal Medicine, CFW Publication Limited, Hong Kong Renehan AG, Booth C, Potten CS (2005) What is apoptosis and why is important? British Medical Journal 322: 1536-1538 121 Richardson MA, Sanders T, Palmer JL, Greisinger A, Singletary SE (2000) Complementary / Alternative Medicine Use in a Comprehensive Cancer Center and the Implications for Oncology Journal of Clinical Oncology 18: 2505-2514 Riondel J, Jacrot , Picot F, Beriel , Moriquand C, Potier P (1986) Therapeutic response to taxol of six human tumors xenografted into nude mice Cancer Chemotherapy and Pharmacology 24: 148-154 Roche Diagnostics GmbH (2005a) Roche Applied Diagnostics Catalog no: 1920685 (http://www.roche-applied-science.com/sis/apoptosis/ ) Roche Diagnostics GmbH (2005b) Roche Applied Diagnostics Catalog no: 1585045 (http://www.roche-applied-science.com/sis/apoptosis/ ) Sabourini JC, Martin A, Baruch J, True JB, Gompel A, Poitout P (1994) Bcl2 expression in normal breast tissue during the menstrual cycle International Journal of Canccer 59: 1-6 Sammon AM (1992) A case-contol study of diet and social factors in cancer of the esophagus in Transkei Cancer 69: 860-865 Sato T, Hanada M, Bodrug S, Irie S, Iwama N, Boise LH, Reed JC (1994) Interaction among members of the Bcl-2 protein family analyzed with a yeast twohybrid system Proceedings of the National Academy of Sciencse of the United States of America 91: 9238-9242 Schnaubelt K (2005) Essential oil therapy according to traditional Chinese medical concepts International Journal of Aromatherapy 15: 98-105 Schwarz A, Felippe EC, Bernardi MM, Spinosa HS (2005) Impaired female sexual behavior of rat offspring exposed to Solanum lycocarpum unripe fruits during gestation and lactation: lack of hormonal and fertility alterations Pharmacology, Biochemistry and Behavior 81: 928-934 Schwartzman RA, Cidlowski JA (1993) Apoptosis: the biochemistry and molecular biology of programmed cell death Endocrine Reviews 14: 133-151 Selvam C, Jachak SM, Thilagavathi R ,Chakraborti AK (2005) Design, synthesis, biological evaluation and molecular docking of curcumin analogues as antioxidant, cyclooxygenase inhibitory and anti-inflammatory agents Bioorganic & Medicinal Chemistry Letters 15: 1793–1797 Selvam R, Subramanian L, Gayathri R, Angayarkanni N (1995) The antioxidant activity of turmeric (Curcuma Longa) Journal of Ethnopharmacology 47:59-67 Seto A, Kusaka C, Nakazato S (1992) Detection of extraordinary large biomagnetic field strength from human hand International Journal of Acupuncture and Electro-Therapeutics Research 17:75-94 122 Shan BE, Yoshida Y, Sugiura T, Yamashita U (1999) Stimulating activity of Chinese medicinal herbs on human lymphocytes in vitro International Journal of Immunopharmacology 21: 149-159 Shao BM, XuW, Dai H, Tu PF, Li ZJ, Gao XM (2004) A study on the immune receptors for polysaccharides from the roots of Astragalus membranaceus, a Chinese medicinal herb Biochemical and Biophysical Research Communications 320: 1103-1111 Shaw D, Leon C, Kolev S, Murray V (1997) Traditional remedies and food supplements A 5-year toxicological study 1991-1995 Drug Safety 17(5): 342356 Seow A, Duffy SW, McGee MA, Lee HP (1996) Breast cancer in Singapore: trends in incidence 1968-1992 International Journal of Epidemiology 25: 40-45 Simpson PB (2003) Family beliefs about diet and traditional Chinese medicine for Hong Kong women with breast cancer Oncology Nursing Forum 30: 834-840 Singapore Cancer Society (2002) Breast Cancer (www.singaporecancersociety.org.sg/c_breastcancer.htm ) Singapore Department of Statistics (2000) Census of Population (http://www.singstat.gov.sg ) Singapore Smith M, Boon HS (1999) Counseling cancer patients about herbal medicine Patient Education and Counseling 38: 109-120 Son YO, Kim J, Lim JC, Chung Y, Chung GH, Lee JC ( 2003) Ripe fruits of Solanum nigrum L inhibits cell growth and induces apoptosis in MCF-7 cells Food and Chemical Toxicology 41: 1421-1428 Song ZW, Steller H (1999) Death by design: mechanism and control of apoptosis TCB, TIBS, TIG Millennium issue M: 49-52 Sprangers MAG, Groenvold M, Arraras JI (1996) The EORTC breast cancerspecific quality-of-life questionnaire module: first results from a three-country field study Journal of Clinical Oncology 14: 2756-68 Srivastava KC, Mustafa T (1989) Spices: Antiplatelet activity and prostanoid metabolism Prostaglandins, Leukotrienes and Essential Fatty Acids 38: 255-266 Sultana S, Pervaiz S, Iqbal M, Athar M (1995) Crude extracts of hepatoprotective plants, Solanum nigrum and Cichorium intybus inhibit free radical-mediated DNA damage Journal of Ethnopharmacology 45: 189-192 Swamy SMK, Tan BKH (2000) Cytotoxic and immunopotentiating effects of ethanolic extract of Nigella sativa L seeds Journal of Ethnopharmacology 70: 1-7 123 Swee YP (2005) Management of cancer in TCM HealthWorlds Asia International Medical Congress 2005, Singapore Tan BKH, Slater A (2003) Traditional Chinese Medicines in breast cancer: clinical and experimental data International Journal of Molecular Medicine S1(12): 68 Tang JL (2001) Clinical Trials in Traditional Chinese Medicine: Opportunities and Challenges Traditional Healing Systems: Negotiating Science & Technology Challenges, Singapore Teledyne (2003) Effective Organic Compound Purification, Teledyne Isco Inc., Lincoln, USA The Cochrane Collaboration (1993) Cochrane Complementary Medicine Field, (http://www.cochranelibrary.com) USA Tomayko MM, Reynolds CP (1989) Determination of subcutaneous tumor size in athymic (nude) mice Cancer Chemotherapy and Pharmacology 24: 148-154 Wang Y, Qian XJ, Hadley HR, Lau BH (1992) Phytochemicals potentiate interleukin-2 generated lymphokine-atcivated killer cell cytotoxicity against murine renal cell carcinoma Molecular Biotherapy 4: 143-146 Wang TTY, Phang JM (1995) Effects of Estrogen on Apoptotic Pathways in Human Breast Cancer Cell Line MCF-7 Cancer Research 55: 2487-2489 Weir DM, Herzenberg LA, Herzenberg LA, Blackwell C (1996a) The Lymphoid system Blackwell Science, Inc., USA Weir DM, Herzenberg LA, Herzenberg LA, Blackwell C (1996b) The Integrated Immune System Blackwell Science, Inc., USA Wong KT, Tan BKH (1996) In vitro cytotoxicity and immunomodulating property of Rhaphidophora korthalsii Journal of Ethnopharmacology 523: 53-57 Wong MK, Koh LL (1985) Mercury, lead, and other Heavy metals in Chinese Medicines Biological Trace Element Research 10: 91-97 Wong R, Sagar CM, Sagar SM (2001) Integration of Chinese medicine into supportive cancer care: A modern role for an ancient tradition Cancer Treatment Reviews (27): 235-246 World Health Organization (2005) International Agency for Research on Cancer (http://www-dep.iarc.fr/ ) Wu RT, Chiang HC, Fu WC, Chien KY, Chung YM, Horng LY (1990) Formosanin C, an immunomodulatory with antitumor activity Int Journal of Immunopharmacology 12: 777-786 124 Wu T, Bligh A, Gu LH, Wang ZT, Liu HO, Cheng XM, Brandford-White CJ, Hu ZB (2005) Simultaneous determination of six isoflavonoids in commercial Radix Astragali by HPLC-UV Fitoterapia 76: 157-165 Wu W, Deng R, Ou Y (2000) Therapeutic efficacy of microshere-entrapped Curcuma aromatica oil infused via hepatic artery against transplanted hepatoma in rats Zhonghua Gan Zang Bing Za Zhi 8(1): 24-26 Wu WY, Yu EX (2001) Advances in the researches on the blocking effect of Chinese drugs on tumors Journal of Traditional Chinese Medicine 21(3): 236240 Xia Q, Zhao KJ, Huang ZG, Zhang P, Dong TTX, Li SP, Tsim KWK (2005) Molecular genetic and chemical assessment of Rhizoma Curcumae in China Journal of Agricultural and Food Chemistry 53: 6019-6026 Xiao HB, Krucker M, Albert K, Liang XM (2004) Determination and identification of isoflavonoids in Radix Astragali by matrix solid-phase dispersion extraction and high-performance liquid chromatography with photodiode array and mass spectrometric detection Journal of Chromatography 1032: 117-124 Yang HL, Chen CS, Chang WH, Lu FJ, Lai YC, Chen CC, Hseu TH, Kuo CT, Hseu YC (2005) Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by ANtrodia camphorate Cancer Letters In Press Yang FQ, Li SP, Chen Y, Lao SC, Wang YT, Dong TTX, Tsim KWK (2005) Identification and quantitation of eleven sesquiterpenes in thre species of Curcuma rhizomes by pressurized liquid extraction and gas chromatography-mass spectrometry Journal of Pharmaceutical and Biomedical Analysis 39: 552-558 Yang YZ, Jin PY, Guo Q, Wu WZ, Pu SY, Chen HZ, Yang JH, Wang KQ, Shi JY, Gong ZX (1990) Treatment of experimental Coxsackie B-3 viral myocarditis with Astragalus membranaceus in mice Chinese Medical Journal 103: 14-18 Yang ZG, Sun HX, Fang WH (2005) Haemolytic activities and adjuvant effect of Astragalus membranaceus saponins (AMS) on the immune response to ovalbumin in mice Vaccine 23: 5196-5203 Yano H, Mizoguchi A, Fukuda K (1994) The herbal medicine Sho-saiko-to inhibits proliferation of cancer cell lines by inducing apoptosis and arrest at the G0/G1 stage Cancer Research 54(2): 448 Yao XJ, Wainberg MA, Parniak MA (1992) Mechanism of inhibition of HIV-1 infection in vitro by purified extract of Prunella vulgaris Virology 187: 56-62 Yap SP, Shen P, Butler MS, Gong YH, Loy CJ, Yong EL (2005) New estrogenic prenylflavone from Epimedium brevicornum inhibits the growth of breast cancer cells Planta Medica 71:114-119 125 Yen GC, Chen HY, Peng HH (2001) Evaluation of the cytotoxicity, mutagenecity and antimutagenicity of emerging edible plants Food Chemical and Toxicology 39: 1045-1053 Yesilada E, Bedir E, Çaliş I, Takaishi Y, Ohmoto Y (2005) Effets of triterpene from Astragalus species on in-vitro cytokine release Journal of Ethnopharmacology 96: 71-77 Yin XL, Zhou JB, JieCF, Xing DM, Zhang Y (2004) Anticancer activity and mechanism of Scutellaria barbata extract on human lung cancer cell line A549 Life Sciences 75: 2233-2244 Yoshida Y, Wang MQ, Liu JN, Shan BE, Yamashita U (1997) Immunomodulating activity of Chinese medicinal herbs and Oldenlandia diffusa in particular Int Journal of Immunopharmacology 19(7): 359-70 Yu CLM, Fielding R, Chan CL, Tse VK, Choi PHK, Lau WH, Choy DTK, O SK, Lee AWM, Sham JST (2000) Measuring quality of life of Chinese cancer patients: a validation of the Chinese version of the Functional Assessment of Cancer Therapy-General (FACT-G) scale Cancer 88: 1715-1727 Yuan R, Lin Y (2000) Traditional Chinese medicine: an approach to scientific proof and clinical validation Pharmacology and Therapeutics 86: 191-198 Yuan WL, Chen HZ, Yang YZ, Yang XY, Lin YS, Jin PY, Zhou TS (1990) Effect of astragalus membranaceus on electric activities of cultured rat beating heart cells infected with Coxsackie B-2 virus Chinese Medical Journal 103: 177182 Zhang CZ, Wang SX, Zhang Y, Chen JP, Liang XM (2005) Invitro estrogenic activities of Chinese medicinal plants traditionally used for the management of menopausal symptoms Journal of Ethnopharmacology 98: 295-300 Zhang WD, Zhang C, Liu RH, Li HL, Zhang JT, Mao C, Chen CL (2005) Quantitative determination of Astragaloside IV, a natural product with cardioprotective activity, inplasma, urine and other biological samples by HPLC coupled with tandem mass spectrometry Journal of Chromatography 822: 170-177 Zhang WJ, Hufnagl P, Binder BR, Wojta J (2003) Antiinflammatory activity of astragaloside IV is mediated by inhibition of NF-kappaβ activation and adhesion molecule expression Thrombosis and Haemostasis 90: 904-914 Zhao KS, Mancini C, Doria G (1990) Enhancement of the immune response in mice by Astragalus membranaceus extracts Immunopharmacology 20: 225-234 Zhao KW, Kong HY (1993) Effect of Astragalan on secretion of tumor necrosis factors in human peripheral blood mononuclear cells Zhongguo Zhong Xi Yi Jiehe Zazhi 13(5): 263-265 126 Zhao ZZ (2004) An Illustrated Chinese Materia Medica in Hong Kong Chong Hwa Book Co., (H.K.) Ltd, Hong Kong Zheng Z, Song C, Liu D, Hu Z (1998) Determination of isoflavonoid in the hairy root cultures of Astragalus membranaceus by HPLC Acta Pharmaceutica Sinica 33: 148-151 Zhou YQ, Sun HX (2001) Chinese Herbal Enema for Treatment of Tumors at the Middle and Late stage Traditional Chinese Medicine 21(4): 256-258 ... clinical trial designs very challenging 1.2 Herbs used in TCM for cancer treatment In TCM, cancer is viewed as the result of imbalance of the whole body-mind network by taking into account the. .. anti-inflammatory, anti-infectious and diuretic effects The combination of these herbs might explain their efficacy in improving the quality -of- life (QOL) in cancer patients (Read, 1977) 10 The effects. .. evaluate the effectiveness of the combined use of Western and traditional medicine in the treatment of disease states like breast cancer 1.4 Rationale and purpose of this study Breast carcinoma