臺灣博碩士論文加值系統

靈芝自古即被”神農本草經 ”與”本草綱目”列為上品中草藥，具有延年益壽功能。近年來亦有相關文獻報導靈芝具有抗老化與抗腫瘤效果。另外先前的研究指出腫瘤初始形成及免疫耐受性的產生與吲哚胺 2,3-雙氧化酶 (IDO) 蛋白有很大的相關性，IDO藉由催化必需胺基酸色胺酸而抑制了對色胺酸短缺極為敏感的T細胞增生，造成T細胞生長受到抑制。此外，第二型人類表皮生長因子受體 (HER2) 與癌症的惡性度息息相關，包括腫瘤的增生、轉移、侵襲能力，造成癌症臨床預後效果不佳的狀況。因此，本論文探討靈芝是否可藉由抑制HER2誘導產生的IDO進而抑制癌細胞的免疫逃脫現象。結果發現靈芝乙醇萃取物在HER2高度表現細胞株中具有濃度依賴性的抑制HER2的表現，此種抑制現象在RNA及蛋白質的層次上皆有相同的效果。HER2受抑制後，進而使IDO基因及其蛋白表現也受到抑制。綜合以上結果顯示靈芝乙醇萃取物具有抗癌效果，同時藉由抑制IDO的基因表現，顯示也可能具有增強免疫能力的作用，是一個極具有開發潛力的中草藥。

Lingzhi has been classified as one of the best grade of traditional Chinese medicines (TCMs) by the Chinese ancient medical encyclopedia, ”Shen-Nong-Ben-Cao-Jing” and “Ben-Cao-Gang-Mu”. Recent reports have shown that Lingzhi, including Ganoderma tsugae, has anti-tumor and antioxidative properties. On the other hand, the protein indoleamide 2,3-dioxygenase (IDO) has been demonstrated to be closely involved in tumor formation and immune tolerance. IDO catalyzes the metabolism of the essential amino acid “tryptophan” resulting in blocking T cell proliferation. While the aberrant expression of human epithelial growth factor receptor 2 (HER2; ErbB2) is known to be associated with cancer malignancy, including tumor progression, tumor metastasis, and invasive abilities, leading to poor clinical outcomes. In this study, we reveal that the Ganoderma tsugae ethanol extract (GTEE) can be used to down-regulate the expression of HER2 in HER2-overexpression cancer cell lines. Furthermore, the differential suppression of HER2 is in response to the inhibition of IDO along with a dose-dependent increase of GTEE. These results provide that GTEE may be a potential TCM for blocking tumor immune escape and enhancing anti-tumor effects.

中文摘要 I
英文摘要 II
誌謝 III
目錄 V
研究緣起 1
第一章 文獻回顧 3
第一節 吲哚胺 2,3-雙氧化酶 (Indoleamine 2,3-dioxygenase; IDO) 3
一、 IDO蛋白質的功能與結構 3
二、 IDO的免疫耐受性與癌症之關係 4
第二節 人類表皮生長因子接受器第二型 5
一、HER2的功能與結構 5
二、HER2的訊息傳遞路徑 6
第三節 靈芝 7
一、 靈芝概述 7
二、 靈芝醫療相關研究 8
三、 實驗用松杉靈芝(Ganoderma tsugae)來源 10
第二章 材料與方法 11
第一節 藥品與試劑 11
第二節 主要器材與儀器 14
第三節 細胞株 15
第四節 實驗方法 15
壹、細胞培養 15
貳、靈芝萃取 18
参、西方點墨法 (Western blotting) 19
肆、核醣核酸的萃取 (Isolation of total RNA) 21
伍、DNA轉染 (Transfection) 23
陸、冷光測試啟動子活性 (Luciferase assay) 23
柒、細胞存活分析 (MTT assay) 24
捌、統計分析 (Statistical Analysis) 25
第三章 結果 26
第一節 靈芝乙醇萃取物對SKOV-3卵巢癌細胞株的影響 26
1、 靈芝乙醇萃取物對SKOV-3卵巢癌細胞株的半數抑制
濃度 (IC50) 26
2、 靈芝乙醇萃取物對SKOV-3卵巢癌細胞及MCF7/HER2乳癌細胞株形態的影響 26
第二節 靈芝乙醇萃取物對SKOV-3卵巢癌細胞及MCF7/HER2
乳癌細胞中的HER2蛋白質表現量的影響 27
第三節 靈芝乙醇萃取物對SKOV-3卵巢癌細胞及MCF7/HER2
乳癌細胞中的HER2蛋白質表現量的影響 27
第四節 靈芝乙醇萃取物對SKOV-3卵巢癌細胞及MCF7/HER2
乳癌細胞株中IDO mRNA表現量的影響 28
第五節 靈芝乙醇萃取物對SKOV-3卵巢癌細胞株中蛋白質穩
定度的影響 28
第六節 靈芝乙醇萃取物對SKBR-3及MCF7/HER2乳癌細胞
株啟動子活性影響 29
第七節 靈芝乙醇萃取物是藉由何種路徑影響卵巢癌細胞SKOV-3及乳癌細胞MCF7/HER2中的HER2及IDO 30
第四章 討論 31
第五章 結論 36
第六章 參考文獻 37
第七章 圖表 42

Bao, X. F., Liu, C.P., Fang, J.N., Li, X. Y. (2001). Structural and immunological studies of a major polysaccharide from spores of Ganoderma lucidum (Fr.) Karst. Carbohydr. Res. 332,67-74.

Brandacher G, Perathoner A, Ladurner R, Schneeberger S, Obrist P, Winkler C, Werner ER, Werner-Felmayer G, Weiss HG, Gobel G. (2006).Prognostic value of indoleamine 2,3-dioxygenase expression in colorectal cancer: effect on tumor-infiltrating T cells. Clin Cancer Res,12 (4):1144–1151.

Cheng, C.W., Shieh, P.C., Lin, Y.C., Chen, Y.J., Lin, Y.H., Kuo, D.H., Liu, J.Y., Kao, J.Y., Kao, M.C., and Way, T.D. (2010). "Indoleamine 2,3-dioxygenase, an immunomodulatory protein, is suppressed by (-)-epigallocatechin-3-gallate via blocking of gamma-interferon-induced JAK-PKC-delta-STAT1 signaling in human oral cancer cells." J Agric Food Chem 58(2): 887-94.

Chen, W.Q., J.W. Huang, L.Q Luo, S.H. Luo and H. Yang. (2005a). Study of Ganoderma polysaccharides modulating the blood sugar and lipid in experimental rats. Chin. J. Gerontol. 25,957–958.

Dong, Z., Huang, C., and Ma, W.Y. (1999). PI-3 kinase in signal transduction, cell transformation, and as a target for chemoprevention of cancer. Anticancer Res 19, 3743-3747.

Efimov, I., Basran, J., Thackray, S.J., Handa, S., Mowat, C.G., and Raven, E.L. (2011). "Structure and reaction mechanism in the heme dioxygenases." Biochemistry 50(14): 2717-24.

Friberg, M., Jennings, R., Alsarraj, M., Dessureault, S., Cantor, A., Extermann, M., Mellor A.L., Munn, D.H., and Antonia, S.J. (2002). Indoleamine 2,3-dioxygenase contributes to tumor cell evasion of T cell-mediated rejection. Int J Cancer 101, 151-155.

Gao, J.J., Min, B.S., Ahn, E.M., Nakamura, N., Lee, H.K., and Hattori, M. (2002). New triterpene aldehydes, lucialdehydes A-C, from Ganoderma lucidum and their cytotoxicity against murine and human tumor cells. Chem. Pharm. Bull. 50: 837-840.

Gao, Y., Zhou, S., Jiang, W., Huang, M., and Dai, X. (2003). Effects of gaboply (a ganoderma lucidum polysaccharide extract) on the immune functions in advanced-stage cancer patients. Immunological Investigations 32, 201-215.

Gershtein ES, Ermilova VD, Kuz’mina ZV, Kuzlinskii NE, Letiagin VP. (1996).Expression of epidermal growth factor receptors and their ligands in malignant tumors of the breast. Vestn Ross Akad Med Nauk 15–19.

Hellyer, N.J., Kim, M.S., and Koland, J.G. (2001). Hergulin-dependent activation of phosphoinositide 3-kinase and Akt via the ErbB2/ErbB3 co-receptor. J Biol Chem 276, 42153-42161.

Hsu RH 許瑞祥, 1993, 靈芝概論, 萬年出版社

Klebanoff CA, Khong HT, Antony PA, Palmer DC, Restifo NP. (2005). Sinks, suppressors and antigen presenters: how lymphodepletion enhances T cell-mediated tumor immunotherapy. Trends Immunol 26:111–7.

S.C. Hsu, C.C. Ou, J.W. Li, T.C. Chuang, H.P. Kuo, J.Y. Liu, C.S. Chen, S.C. Lin, C.H. Su, M.C. Kao. (2008). Ganoderma tsugae extracts inhibit colorectal cancer cell growth via G2/M cell cycle arrest, J. Ethnopharmacol. 120, 394–401.

Sugimoto, H., Oda, S., Otsuki, T., Hino, T., Yoshida, T., and Shiro, Y. (2006). Crystal structure of human indoleamine 2,3-dioxygenase: catalytic mechanism of O2 incorporation by a heme-containing dioxygenase. Proc Natl Acad Sci U S A 103, 2611-2616.

Hsu SC, Ou CC, Li JW, Chuang TC, Kuo HP, Liu JY, Chen CS, Lin SC, Su CH, Kao MC. (2008). Ganoderma tsugae extracts inhibit colorectal cancer cell growth via G(2)/M cell cycle arrest. J Ethnopharmacol 120(3):394-401.

Karni, R., Jove, R., and Levitzki, A. (1999). Inhibition of pp60c-Src reduces Bcl-XL expression and reverses the transformed phenotype of cells overexpressing EGF and HER-2 receptors. Oncogene 18(33): 4654-62.

Kassis, J., Moellinger, J., Lo, H., Greenberg, N.M., Kim, H.G., and Wells, A. (1999). A role for phospholipase C-gamma-mediated signaling in tumor cell invasion. Clin Cancer Res 5(8): 2251-60.

King, N.J., and Thomas, S.R. (2007). Molecules in focus: indoleamine 2,3-dioxygenase. Int J Biochem Cell Biol 39. 2167-2172.

Liu, Y.W., Gao, J.L., Guan, J., Qian, Z.M., Feng, K., and Li, S.P. (2009). Evaluation of antiproliferative activities and action mechanisms of extracts from two species of Ganoderma on tumor cell lines. J Agric Food Chem 57(8): 3087-93.

Mellor AL, Sivakumar J, Chandler P, Smith K, Molina H, Mao D, Munn DH. (2001). Prevention of T cell-driven complement activation and inflammation by tryptophan catabolism during pregnancy. Nat Immunol 2(1):64-68.

Mellor, A.L., and Munn, D.H. (2004). IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat Rev Immunol 4, 762-744.

S. Menard, S.M. Pupa, M. Campiglio, E. (2003). Tagliabue, Biologic and therapeutic roleof HER2 in cancer. Oncogene 22,6570-6578.

Miyazaki, T. and M. Nishijima (1981). "Studies on fungal polysaccharides. XXVII. Structural examination of a water-soluble, antitumor polysaccharide of Ganoderma lucidum." Chem Pharm Bull (Tokyo) 29(12): 3611-6

Najfeld, V., Menninger, J., Muhleman, D., Comings, D.E., and Gupta, S.L. (1993). Localization of indoleamine 2,3-dioxygenase gene (INDO) to chromosome 8p12-->p11 by fluorescent in situ hybridization. Cytogenet Cell Genet 64, 231-232.

Ohno, N., Miura, N. N., Sugawara, N., Tokunaka, K., Kirigaya, N., and Yadomae, T. (1998). Immunomodulation by hot water and ethanol extracts of Ganoderma lucidum. Pharm. PHARMACOL. Lett. 8,174-177.

Orabona, C., Pallotta, M.T., Volpi, C., Fallarino, F., Vacca, C., Bianchi, R., Belladonna, M.L., Fioretti, M.C., Grohmann, U., and Puccetti, P. (2008). SOCS3 drives proteasomal degradation of indoleamine 2,3-dioxygenase (IDO) and antagonizes IDO-dependent tolerogenesis. Proc. Natl. Acad. Sci. U. S. A. 105:20828–20833.

Ou, C.C., Hsu, s.c., Hsieh, Y.H., Tsou, W.L., Chuang, T.C., Liu, J.Y., and Kao, M.C.(2008). Downregulation of HER2 by RIG1 involves the PI3K/Akt pathway in ovarian cancer cells. Carcinogenesis 29, 299-306.

Pallotta, M.T., Orabona, C., Volpi, C., Grohmann, U., Puccetti, P., and Fallarino, F. (2010) Proteasomal degradation of indoleamine 2,3-dioxygenase in CD8 dendritic cells is mediated by suppressor of cytokine signaling 3 (SOCS3). Int. J. Tryptophan Res. 3:91–7.

Rodriguez, P.C., Zea, A.H., and Ochoa, A.C. (2003). Mechanisms of tumor evasion from the immune response. Cancer chemother Biol Response Modif 21, 351-364.

Shimizu, A., Yano, T., Saito, Y., and Inada, Y. (1985). Isolation of an inhibitior of platelet aggregation from a fungus, Ganoderma lucidum. Chemical & Pharmaceutical Bulletin 33, 3012-3015.

Sivaraman, V.S., Wang, H., Nuovo, G.J., and Malbon, C.C. (1997). Hyperexpression of mitogen-activated protein kinase in human breast cancer. J Clin Invest 99, 1478-1483.

Sliva, D., Labarrere, C., Slivova, V., Sedlak, M., Lloyd, F.P., Jr., and Ho, N.W. (2002). Ganoderma lucidum suppresses motility of highly invasive breast and prostate cancer cells. Biochemical and Biophysical Research Communications 298, 603-612.

Stanley, G., Harvey, K., Slivova, V., Jiang, J., and Sliva, D. (2005). Ganoderma lucidum suppresses angiogenesis through the inhibition of secretion of VEGF and TGF-beta1 from prostate cancer cells. Biochem Biophys Res Commun 330(1): 46-52.

Sugimoto, H., Oda, S., Otsuki, T., Hino, T., Yoshida, T., and Shiro, Y. (2006). Crystal structure of human indoleamine 2,3-dioxygenase: catalytic mechanism of O2 incorporation by a heme-containing dioxygenase. Proc Natl Acad Sci U S A 103, 2611-2616.


Suzuki,s., s. Tone, O. Takikawa, T. Kubo, I. Kohno, and Y. Minatogawa. (2001). Expression of indoleamine 2,3-dioxygenase, and tryptophan 2,3-dioxygenase in early concepti. Biochem. J. 355:425.

Uyttenhove, C., Pilotte, L., Theate, I., Stroobant, V., Colau, D., Parmentier, N., Boon, T., and Van den Eynde, B.J. (2003). Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat Med 9, 1269-1274.

Van der Hem, L. G., VAN DER Vlie, J.A., Kino, K., Hoitsma, A.J., and Tax, W.J. (1996). Ling-Zhi-8: a fungal protein with immunomodulatory effects. Transplantation Proceedings 28, 958-959.

Wang, S.Y., Hsu, M.L., Hsu, H.C., Tzeng, C.H., Lee, S.S., Shiao, M.S., and Ho, C.K. (1997). The anti-tumor effect of Ganoderma lucidum is mediated by cytokines released from activated macrophages and T lymphocytes. International Journal of Cancer 70, 699-705.

Wasser, S.P., and Weis, A.L. (1999). Therapeutic effects of substances occurring in higher Basidiomycetes mushrooms: a modern perspective. Critical Reviews in Immunology 19, 65-96.

Williams MD, Roberts D, Blumenschein GR Jr, Temam S, Kies MS, Rosenthal DI, Weber RS, El-Naggar AK. (2007). Differential expression of hormonal and growth factor receptors in salivary duct carcinomas: biologic significance and potential role in therapeutic stratification of patients. Am J Surg Pathol 31:1645–1652.

Won, S.J., Lin, M.T., and Wu. (1992). Ganoderma tsugae mycelium enhances splenic natural killer cell activity and serum interferon production in mice. JapaneseJournal of Pharmacology 59, 171-176.

Yarden, Y., and Sliwkowski, M.X. (2001). Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2, 127-137.

Yang, C.S., and Wang, Z.Y. (1993). Tea and cancer. J Natl Cancer Inst 85, 1038-1049.
Yarden, Y. (2001). Biology of HER2 and its importance in breast cancer. Oncology 61 Suppl 2, 1-13.

Yu, D., and Hung, M.C. (2000b). Role of erbB2 in breast cancer chemosensitivity. Bioessays 22, 673-680.

Yu, Y.H., Kuo, H.P., Hsieh, H.H., Li, J.W., Hsu, W.H., Chen, S.J., Su, M.H., Liu, S.H., Cheng, Y.C., Chen, C.Y., Kao, M.C. (2012). Ganoderma tsugae Induces S Phase Arrest and Apoptosis in Doxorubicin-Resistant Lung Adenocarcinoma H23/0.3 Cells via Modulation of the PI3K/Akt Signaling Pathway. Evid Based Complement Alternat Med 371286.

Yuan, G., Qian, L., Shi, M., Lu, F., Li, D., Hu, M., Yu, M., Yu,M., Shen, B., and Guo, N. (2008). HER2-dependent MMP-7 expression is mediated by activated STAT3. Cell Signal 20, 1284-1291

Zarich, N., Oliva, J.L., Martinez, N., Jorge, R., Ballester, A., Gutierrez-Eisman, S., Garcia-Vargas, S., and Rojas, J.M. (2006). Grb2 is a negative modulator of the intrinsic Ras-GEF activity of hSosl. Mol Biol Cell 17, 3591-3597.

鄭傑文. 2011. 以吲哚 2,3-雙氧化酶作為標靶的癌症免疫逃脫之研究. 國立中興大學生物化學研究所博士學位論文.

郭漢鵬. 2011. 松杉靈芝萃取物抑制神經膠原致癌基因表現之研究. 國防醫學院生物化學研究所碩士論文.