樟芝生長於台灣特有品種牛樟樹樹幹內，為台灣民間廣為流傳的天然藥物。一般用來當作解毒劑或治療高血壓、高血脂及抗癌。經實驗顯示樟芝富含三帖類、倍半帖類、胺基酸、蛋白質、糖類和固醇類、多醣體，具相當高的營養價值。另一方面，研究指出，從樟芝子實體體所萃取出來的取出來的固醇類物質zhankuic acid 對於小鼠血癌細胞P338具有毒殺效果。之後，又有科學家發現樟芝菌絲體水萃取物會毒殺人類血癌細胞HL-60，但對於培養的人類內皮細胞卻不具此活性。截至目前為止，對於樟芝抗腫瘤方面的研究侷限在體外實驗(in vitro)。因此，我們想要評估樟芝甲醇萃取液(ACME)在體內(in vivo)及體外(in vitro)，其整體的抗癌效果為何。
　　體外實驗方面，於一序列細胞毒殺試驗中，發現ACME對於許多癌細胞皆有毒殺效果，且具劑量正相關性。我們進一步利用流式細胞儀，以PI染色，觀察樟芝萃取液對細胞週期的影響，發現ACME會使腫瘤細胞週期停頓在Sub-G1 phase。之後，再以Hoechst 33258進行細胞核染色及DNA片段電泳分析，結果顯示ACME處理之後，癌細胞趨向程式死亡。體內實驗方面，我們發現背負ML1-4A腫瘤細胞的Balb/c小鼠腫瘤及背負LLC腫瘤細胞之C57BL/6小鼠的腫瘤皆不同層級的被ACME所抑制。在癌細胞移行(cell migration assay)實驗中，我們發現ACME對PC-3人類前列腺癌細胞移行能力具有抑制效果，進一步利用電泳酵素分析(Gelatin Zymography)發現ACME會降低PC-3分泌MMP-9但對MMP-2卻無顯著影響，推測其可能具抑制腫瘤細胞侵入的能力。關於血管新生方面，ACME可抑制BAEC（牛肺動脈內皮細胞）的移行，同時於雞胚胎絨毛膜試驗中高劑量ACME可顯著抑制雞胚胎血管新生。將ML1-4A原位腫瘤以CD31染色，發現血管無明顯被抑制。因此，推測ACME的抗腫瘤效果主要在於抑制癌細胞的生長而非血管的增生。

　　Antrodia camphorata, or ”chang-chih” in its folk name, is a tree fungus that has been used by Taiwanese indigenes for years for its anti-inflammatory activity. Several compounds have been identified from the fruiting body of Antrodia camphorate, such as triterpenoids, sesquiterpenoids, steroids, and polysaccharides. Previous researches indicated that zhankuic acid extracted from fruit body of A. camphorata exhibited cytotoxicity against P388 murine leukemia and aqueous extract of A. camphorata reduced the viability of HL-60 human leukemia cells. And yet no animal experiments and other aspects of anticancer activities of A. camphorata have been tested. My study aims at investigating the anticancer activity of a methanolic extract obtained from cultured mycelia of A. camphorate, termed ACME.
　　The extract shows markedly dose-dependent cytotoxicities against several cancer cell lines. The flow cytometry and DNA fragmentation assays demonstrate that the cytotoxicity is due to cell apoptosis. The anticancer activity of ACME was investigated further in vivo using mouse bearing transplanted tumor cells. ACME significantly inhibits the growths of tumors both in the Lewis lung carcinoma and ML1-4A hepatoma animal models. Besides, we found ACME inhibits the migration of human prostate cancer cells (PC-3) toward chemo-attractant and reduces MMP-9 but not MMP-2 secretion, suggesting ACME possesses an anti-metastatic principle. ACME also exhibits anti-angiogenesis activities in an endothelia cell migration assay and blood vessel formation in a semi in vivo chicken chorioallantonic membrane (CAM) assay. But no apparent differences are found in CD31 immunohistochemistry of ML1-4A primary tumors from either control animal or animal treated with ACME, indicating the tumor-inhibiting activity is not due to angiogenesis inhibition.
　　The active anticancer ingredient and its detailed molecular action mechanism in A. camphorate warrant further investigation.

1.行政院衛生署
2. Watanabe, N. Oncogene and tumor suppressor gene. Rinsho Byori - Japanese Journal of Clinical Pathology 123, 131-6 (2002).
3. Zimmermann, C., Bonzon, C., and Green, D. R. The machinery of programmed cell death. Pharmacology & Therapeutics 92, 57-70 (2001).
4. Kerr, J. F. R., Wyllie, A., and Currie, A. R. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26, 239-257 (1976).
5. Lee, J. K., Sayers, T. J., Back, T. C., Wigginton, J. M., and Wiltrout, R. H. Lack of FasL-mediated killing leads to in vivo tumor promotion in mouse Lewis lung cancer. Apoptosis 8, 151-60 (2003).
6. Jemmerson, R., LaPlante, B., and Treeful, A. Release of intact, monomeric cytochrome c from apoptotic and necrotic cells. Cell Death & Differentiation 9, 538-48 (2002).
7. Allen, R. T., Hunter, Ⅲ. W. J., and Agrawal, D.K. Morphological and biochemical analysis of apoptosis. J Pharmacol Toxical Methods 37, 215-228 (1997).
8. Liu, X., Zou, H., Slaughter, C., and Wang X. Cell 89, 175-184 (1997)
9. Engeland, M. V., Nieland, L. J. Ramaekers, F. C., Schutte, B., and Reutelingsperger, C. P. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 31, 1-9 (1998).
10. Darzynkiewicz, Z., and Targanos, F. Features of apoptoic cells measured by flow cytometry. Cytometry 13, 795-808 (1992).
11. Kaufmann, S.H., and Vaux, D.L. Alterations in the apoptotic machinery and their potential role in anticancer drug resistance. Oncogene 22, 7414-30 (2003).
12. Thornberry, N. A., and Lazebnik, Y. Caspases: Enemies Within. Science 281, 1312-1314 (1998).
13. Cory, S., Huang, D. C., and Adams, J. M. The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene 22, 8590-607 (2003).
14. Sun, J., Nam, S., Lee, C. S., Li, B., Coppola, D., Hamilton, A. D., Dou, Q. P., and Sebti, S. M. CEP1612, a Dipeptidyl Proteasome Inhibitor, Induces p21WAF1 and p27KIP1 Expression and Apoptosis and Inhibits the Growth of the Human Lung Adenocarcinoma A-549 in Nude Mice. Cancer Res 1, 1280-1284 (2001).
15. Green, D. R., and Reed, J. C. Mitocondria and Apoptosis. Science 281, 1309-1312 (1998).
16. Wang, X. The expandin role of mitochondria in apoptosis. Gene Dev 15, 2922-2933 (2001).
17. Folkman, J. Tumor angiogenesis: therapeutic implications. N Engl J Med 285, 1182-1186 (1971).
18. Hanahan, D., and Folkman, J. Patterns of emerging mechanisms of the angiogenic swith during tumerigenesis. Cell 86, 353-364 (1996).
19. Ausprunk, D. H., and Folkman, J. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvascular Res 14, 53-65 (1977).
20. Petersen, T. E., Martzen, M. R., Ichinose, A., and Davie, E.W. Characterization of the gene for human plasminogen, a key proenzyme in the fibrinolytic system. J Bio Chem 265, 6104-6111 (1990).
21. O'Reilly, M., Boehm, T., Shing, Y. Fukai, N., Vasios, G., Lane, Wu., Flynn, E. Birkhead, J., Olsen, B., and Folkman, J. Endostatin. An endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 277-285 (1997).
22. Good, D. J., Polverini, P. J., and Rastinejad, F. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci 87, 6624-6628 (1990).
23. Hamano, Y., Sugimoto, H., Soubasakos, M. A., Kieran, M., Olsen, B. R., Lawler, J., Sudhakar, A., and Kalluri, R. Thrombospondin-1 associated with tumor microenvironment contributes to low-dose cyclophosphamide-mediated endothelial cell apoptosis and tumor growth suppression. Cancer Res 64, 1570-4, (2004).
24. Chen, W., Fu, J., Liu, Q., Ruan, C., and Xiao, S. Retroviral endostatin gene transfer inhibits human colon cancer cell growth in vivo. Chinese Medical J 116, 1582-4 (2003).
25. Kerbel, R. S. A cancer therapy resistant to resistance. Nature 390, 335-6 (1997).
26. Zetter, B. R. Angiogensis and tumor metastasis. Annu Rev Med 49, 407-24 (1998).
27. Wu, S. H., Ryvarden, L., and Chang, T. T. Antrodia camphorata (‘niu-chang-chih’), new combination of a medicinal fungus in Taiwan. Botanical Bulletin of Academia Sinica 38, 273–275 (1997).
28. Tsai, Z. T., and Liaw, S. L. The Use and the Effect of Ganoderma. San Yun Press, Taichung, Taiwan, p. 116 (1985).
29. Chen, C. H., Yang, S. W., and Shen, Y. C. New steroid acids from Antrodia cinnamomea, a fungal parasite of Cinnamomum micranthum. Journal of Natural Product 58, 1655–1661 (1995).
30. Yang, S. W., Shen, Y. C., and Chen, C. H. Steroids and triterpenoids of Antrodia cinnamomea—a fungus parasitic on Cinnamomum micranthum. Phytochemistry 41, 1389–1392 (1996).
31. Chiang, H. C., Wu, D. P., Cherng, I. W., and Ueng, C. H. A sesquiterpene lactone, phenyl and biphenyl compounds from Antrodia cinnamomea. Phytochemistry 39, 613–616 (1995).
32. Lee, I. H., Huang, R. L., Chen, C. T., Chen, H. C., Hsu, W. C., and Lu, M. K. Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects. FEMS Microbiology Letters 209, 63-7 (2002).
33. Song, T. Y., and Yen, G. C. Protective effects of fermented filtrate from Antrodia camphorata in submerged culture against CCl4-induce hepatic toxicity in rats. Journal of Agricultural ＆Food Chemistry 51, 1571-1577 (2003).
34. Dai, Y. Y., Chuang, C. H., Tsai, C. C., Sio, H. M., Huang, S. C., Chen J. C., and Hu, M. L. The Protection of Anthrodia camphorata against Acute Hepatotoxicity of Alcohol in Rats. Journal of Food and Drug Analysis 11, 177-185 (2003).
35. Song, T. Y., and Yen, G. C. Antioxidant properties of Antrodia camphorata in submerged culture. Journal of Agricultural & Food Chemistry 50, 3322-7 (2002).
36. 曹巧吟，樟芝中免疫調節蛋白的純化與其生理活性之探討，國立台灣大學園藝學研究所碩士論文(2003)
37. 蔡敬民，樟芝菌絲體及發酵液對於血糖及血脂的影響，行政院國家科學委員會九十年度保健食品研究開發計畫，2001。
38. 楊新玲，樟芝對人類臍帶血管內皮細胞之影響及作用之探討，行政院國家科學委員會九十年度保健食品研究開發計畫，2001。
39. 劉俊仁，中草藥抗癌機制研究，第二部分: 樟芝活性多醣體之生物活性分析及其經由免疫調節抑制腫瘤生長之研究。國立台灣大學生物化學暨分子生物學研究所，2003。
40. Hseu, Y. C., Chang, W. C., Hseu, Y. T., Lee, C. Y., Yech, Y. J., Chen, P. C., Chen, J. Y., and Yang, H. L. Protection of oxidative damage by aqueous extract from Antrodia camphorata mycelia in normal human erythrocytes. Life Science 71, 469–482 (2002).
41. Ikekawa, T., Uehara, N., Maeda, Y., Nakanishi, M., and Fukuoka, F. Antitumor activity of aqueous extracts of edible mushrooms. Cancer Res 29, 734–735 (1969).
42. Lin, C. N., Tome, W. P., and Won, S. J. Novel cytoxic principles of Formosan Ganoderma lucidum. J. Nat. Prod. 54, 998—1002 (1991).
43. Min, B. S., Gao, J. J., Nakamura, N., and Hattori, M. Triterpenes from spores of Ganoderma lucidum and their cytotoxicity against Meth-A and LLC tumor cells, Chem Pharm Bull 48, 1026-1033 (2000).
44. Kimura, Y., Taniguchi, M., and Kimiye, K. Antitumor and Antimetastatic Effects on Liver of Triterpenoid Fractions of Ganoderma lucidum: Mechanism of Action and Isolation of an Active Substance. Anticancer Res 22, 3309-3318 (2002).
45. 張中姿，樟芝菌絲體之甲醇萃取部分對人類肝癌細胞株(HepG2)生長抑制作用的機轉探討，國立台灣大學生化學研究所，2002。
46. Lakka, S. S., Gondi, C. S., Yanamandra, N., Dinh, D. H., Olivero, W. C., Gujrati, M., and Rao, J. S. Synergistic down-regulation of urokinase plasminogen activator receptor and matrix metalloproteinase-9 in SNB19 glioblastoma cells efficiently inhibits glioma cell invasion, angiogenesis, and tumor growth. Cancer Res 63, 2454-61 (2003).
47. Devoogdt, N., Ghassabeh, G. H., Zhang, J., Brys, L., Baetselier, D. P., and Revents, H. Secretory leukocyte protease inhibitor promotes the tumorigenic and metastatic potential of cancer cells. Proc Natl Acad Sci 100, 5778-5782 (2003).
48. Koliakos, K. K., Sapountzi, Z., Papageorgiou, A., Trachana, V., Kotsinou, S., and Koliakos, G. Antiidiotypic antibodies carrying the "internal image" of peptide YIGSR inhibit spontaneous metastasis of Lewis lung carcinoma in mice. In Vivo 16, 511-8 (2002).
49. Hortobagyi, G. N. Recent progress in the clinical development of docetaxel (Taxotere). Semin Oncol 26, 32-6 (1999).
50. Grover, J. K., and Vats, K. Thalidomide: from teratogen to anti-angiogenic. Indian Journal of Cancer 38, 22-32 ( 2001).
51. Cherng, I. H., Wu D. P., and Chiang H. C. Triterpenoids from Antrodia cinnamomea. Phytochemistry 41, 263-267 (1996).