本研究為探討一般公認具有滋養補氣血之人蔘（Panax ginseng CA Meyer）其中主要的抗癌成分—人蔘皂Rh2(Ginsenoside Rh2, G-Rh2) —是藉由何種抗癌分子機制來達成預防與治療癌症的效果。首先，分別測試G-Rh2以及含有1% G-Rh2之中草藥複方—Sabonin—是否對癌細胞的生長具有抑制效果。我們發現Sabonin對於NIH 3T3 /ras 7-4及SW480細胞的生長，具有劑量遞增性的抑制效果。而G-Rh2對於A549、NIH 3T3 /ras 7-4、B16F10細胞的生長，亦具有劑量遞增性的抑制效果。但在相同劑量下，G-Rh2對NIH 3T3及PC-3細胞的生長較不具抑制效果，顯示G-Rh2對癌細胞的生長抑制效果，是具有選擇性的。由流式細胞分析儀測試的結果得知，G-Rh2可使A549、B16F10細胞之細胞週期先停頓在per G0/G1 phase，再進一步促進細胞發生凋亡。我們接著測試G-Rh2對血管新生是否具有抑制效果。已知腫瘤的轉移有賴於血管新生。腫瘤周圍新血管的生長，是提供癌細胞進入血液循環及轉移到遠端位置的良好管道。在細胞移行分析(cell migration assay)實驗中，發現G-Rh2對BAEC及PC-3細胞的移行能力具有劑量遞增性的抑制效果。在雞胚胎絨毛膜血管形成試驗(chorioallantoic membrane assay)中，無法觀察出G-Rh2對血管新生的影響，但 Sabonin萃取液卻可以抑制雞胚胎血管新生。Sabonin亦可以抑制由 NIH 3T3 /ras 7-4細胞誘發的Balb/c小鼠皮下腫瘤。然而，利用C57/BL6小鼠尾靜脈注射黑色素癌細胞B16F10細胞的肺臟轉移模式，發現Sabonin並不具有抑制癌細胞轉移的能力。若以預防的角度來探討，長期於飼料中混合0.1% Sabonin粉末投予B6C3F1小鼠，確實可以預防由致癌劑2-acetylaminofluorence誘導的自發性原位肝癌產生，故再次證實了Sabonin可以抑制體內腫瘤的成長。未來仍需更進一步釐清G-Rh2在癌症發展的各個過程中參與的調控機制，及Sabonin中個別成份在抗癌上扮演的角色，以暸解G-Rh2及Sabonin的個別成份，在腫瘤抑制上更詳細的分子藥理學作用。

The anticancer activity of a pure ginsenoside from Panax ginseng C.A. Meyer, G-Rh2, was investigated. We first tested the cytotoxicities of G-Rh2 and a mixture format termed “Sabonin” that contains 1% G-Rh2 and other herbal ingredients on a variety of cancer cell lines. Sabonin exhibits a dose-dependent growth inhibitions on NIH3T3/ras and SW 480 cells. G-Rh2 exhibits a dose-dependent growth inhibitions of A549, NIH3T3/ras and B16F10 cells. However, the same dose has little effect on NIH3T3 wild-type and PC-3 cells, suggesting discriminatory inhibitions of ginsenoside. Cell flowcytometry assay shows that G-Rh2 arrests cells at the per-G0/G1 phase, and later leads to apoptosis of B16F10 and A549 cells. We then tested the antiangiogenesis effect of G-Rh2. Tumor metastasis is angiogenesis dependent. The new blood vessels embedded in a tumor provide a gateway for tumor cells to enter the circulation and to metastasize to distants sites. Cell migration assay using Boyden chamber shows that G-Rh2 inhibits the movements of bovine aortic endothelial and PC-3 cells. However, in the semi in vivo chicken chorioallantonic membrane (CAM) assay, Sabonin does but G-Rh2 does not inhibit vascular formation. Sabonin when fed orally to Balb/c mouse inhibits strongly the growth of tumor induced by the subcutaneous transplantation of NIH3T3/ras cells into the testing animal. However, Sabonin does not reduce the numbers of tumor colonies induced by the tail vein injection of B16F10 cells into C57BL/6 mouse in the experimental metastasis assay. Feeding food containing 0.1% Sabonin to B6C3F1 mouse inhibits strongly the growth of hepatoma induced by the 2-acetylaminofluorence injected intraperitoneally, suggesting again the effectiveness of Sabonin in inhibiting tumor growth in vivo.

Ausprunk, D. H. and J. Folkman (1977). "Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis." Microvasc Res 14(1): 53-65.

Bae, E., Han, M. J., Park, S. Y., Kim, D. H. (2002). "Metabolism of 20(S)-and 20(R)-ginsenoside Rg3 by human intestinal bacteria and its relation to in vitro biological activities." Biol Pharm Bull 25(1):58-63.

Border, W. A., and Noble, N. A. (1994). "Transforming growth factor b in tissue fibrosis." N Engl J Med 331(19): 1286-1292.

Byun, B. H., Shin, I., Yoon, Y. S., Kim, S. I., Joe, C. O. (1997).
"Modulation of protein kinase C activity in NIH 3T3 cells by plant glycosides from Panax ginseng." Planta Medica 63(5): 389-92.

Chunzi, Z., Hongshan, Yu., Yongming, Bao., Lijia, An., and Fengxie, Jin. (2001). "Purification and Characterization of
Ginsenoside-B-Glucosidase for Ginseng." Chem Pharm Bull 49(7):795-798.

Darzynkiewicz, Z., S. Bruno, et al. (1992). "Features of apoptotic cells measured by flow cytometry." Cytometry 13(8): 795-808. Eguchi, Y., S. Shimizu, et al. (1997). "Intracellular ATP levels determine cell death fate by apoptosis or necrosis." Cancer Res 57(10):1835-40.

Ellis, L. M. and I. J. Fidler (1996). "Angiogenesis and metastasis." Eur J Cancer 32A(14): 2451-60.
Farber, E. (1984). "Cellular biochemistry of the stepwise development of cancer with chemicals: G. H. A. Cloves Memorial Lecture." Cancer
Res 44(12pt1): 5463-74.

Folkman, J. (1971). "Tumor angiogenesis: therapeutic implications." N Engl J Med 285(21): 1182-6.

Folkman, J. (1984). "What is the role of endothelial cells in angiogenesis?" Lab Invest 51(6): 601-4.

Folkman, J. and C. Haudenschild (1980). "Angiogenesis in vitro." Nature 288(5791): 551-6.

Folkman, J. and Y. Shing (1992). "Angiogenesis." J Biol Chem 267(16):10931-4.

Gimbrone, M. A., Jr., S. B. Leapman, et al. (1972). "Tumor dormancy in vivo by prevention of neovascularization." J Exp Med 136(2):261-76.

Grunberger, D., and Weinstein, I. B. (1971). "Modifications of ribonucleic acid by chemical carcinogens." J Biol Chem 246(4):1123-8.

Hogan, M. E., Dattagupta, N., and Whitlock, J. P. (1981).
"Carcinogen-induced alteration of DNA structure." J Biol Chem 256(9): 4504-13.

Hortobagyi, G. N. (1999). "Recent progress in the clinical development of docetaxel (Taxotere)." Semin Oncol 26(3 Suppl 9): 32-6.

Hyun-Eui, K., Jae, H., Oh, Seung., Ki, Lee., Young, J. Oh. (1999)."Ginsenoside RH-2 induces apoptotic cell death in rat C6 glioma via a reactive oxygen- and caspase-dependent but
Bcl-X(L)-independent pathway." Life Sciences 65(3): PL33-40.

Jacobson, M. D., M. Weil, et al. (1997). "Programmed cell death in animal development." Cell 88(3): 347-54.

Jeong, A. P., Kwang, Youl. Lee., Young, J. Oh., Kyu-Won, Kim., Seung,Ki. Lee. (1997). "Activation of caspase-3 protease via a 51
Bcl-2-insensitive pathway during the process of ginsenoside
Rh2-induced apoptosis." Cancer Letters 121(1): 73-81.

Jin-Hua, Z., Tatsuya, Takeshita., Isao, Kitagawa. (1995). "Suppression of the formation of sister chromatid exchanges by low concentrations of ginsenoside Rh2 in human blood lymphocytes. " Cancer Res 55(6): 1221-3.

Kenyon, B. M., E. E. Voest., Chen, C. C., Flynn, E., Folkman, J., D’Amato, R. J. (1996). "A model of angiogenesis in the mouse cornea." Invest Ophthalmol Vis Sci 37(8): 1625-32.

Kim, Y. S., Jin, S. H., Lee, Y. H., Park, J. D., Kim, S. I. (2000)."Differential expression of protein kinase C subtypes during ginsenoside Rh2-lnduced apoptosis in SK-N-BE(2) and C6Bu-1 cells." Arch of Pharm Res 23(5): 518-24.

Korsmeyer, S. J. (1992). "Bcl-2: an antidote to programmed cell death." Cancer Surv 15: 105-18.

Kriek, E., Miller, J. A., Juhl, U., and Miller, E. C. (1967). "8,-(N-2-fluorenylacetamido)guanosine, and arylamidation reaction product of guanosine and the carcinogen N-acetoxy-N-2-fluorenylacetamide in neutral solution." Biochemistry 6(1): 177-82.

Kwang, Y. L., Jeong, Ae. Park., Eunah, Chung., You, Hui. Lee., ShinIl, Kim., Seung, Ki. Lee. (1996). "Ginsenoside-Rh2 blocks the cell cycle of SK-HEP-1 cells at the G1/S boundary by selectively inducing the protein expression of p27kip1." Cancer Letters 110(1-2): 193-200.

Miller, E. C., and Miller, J. A. (1981). "Searches for ultimate chemical carcinogens and their reactions with cellular macromolecular." 52 Cancer 47(10): 2327-45.

Nguyen, M., Y. Shing, et al. (1994). "Quantitation of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane." Microvasc Res 47(1): 31-40.

Odashima, S., Ohta, T., Kohno, H., Matsuda, T., Kitagawa, I., Abe, H., and Arichi, S. (1985). "Control of phenotypic expression of cultured B16 melanoma cells by plant glycosides." Cancer Res 45(6): 2781-4.

Oh, M., Choi, Y. H., Choi, S., Chung, H., Kim, K., Kim, SI., Kim, DK., Kim, ND. (1999). "Anti-proliferating effects of ginsenoside Rh2 on MCF-7 human breast cancer cells." Int J Onco 14(5): 869-75.

O'Reilly, M. S., L. Holmgren, et al. (1994). "Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma." Cell 79(2): 315-28.

Ormerod, M. G. (1998). "The study of apoptotic cells by flow cytometry."Leukemia 12(7): 1013-25.

Ota, T., Kohno, H., Maeda, M., Tanino, M., Odashima, S. (1993)."Involvement of peanut agglutinin-binding sugar chains in experimental metastasis of B16 melanoma cells." Onco Res 5(6-7):235-43.

Ota, T., Maeda, M., Odashima, S. (1991). "Mechanism of action of ginsenoside Rh2: uptake and metabolism of ginsenoside Rh2 by cultured B16 melanoma cells." J Pharm Sci 80(12): 1141-6.

Peraino, C., Fry, R. J. M., Staffeldt, E., and Kisielski, W. E. (1973). "Effects of varying the exposure to phenobarbital on its enhancement of 2-Acetylaminofluorence-induces hepatic tumorigenesis in the rat." Cancer Res 33(11): 2701-5. 53

Ramsden, J. D. (2000). "Angiogenesis in the thyroid gland." J Endocrinol 166(3): 475-80.

Rastinejad, F., P. J. Polverini, et al. (1989). "Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene." Cell 56(3): 345-55.

Takahide, O., Kohzaburo, Fujikawa-yamamoto., Zhi-ping, Zong., Masakazu, Yamazaki., Shizuo, Odashima., Isao, Kitagawa., Hiroko, Abe., and Shigeru, Arichi. (1987). "Plant-glycoside modulation of cell surface related to control of differentiation in cultured B16 melanoma cells." Cancer Res 47(14): 3863-7.

Takahide, O., Masayo, Maeda., Shizuo, Odashima., Jun, Ninomiya-Tsuji.,and Masaaki, Tatsuka., (1997). "G1 phase-specific suppression of the Cdk2 activity by ginsenoside Rh2 in cultured murine cells." Life Sci 60(2): PL39-44.

Tode, T., Kikuchi, Y., Kita, T., Hirata, J., Imaizumi, E., Nagata, I. (1993)."Inhibitory effects by oral administration of ginsenoside Rh2 on the growth of human ovarian cancer cells in nude mice." J Cancer Res & Cli Onco 120(1-2): 24-6.

Van Engeland, M., L. J. Nieland., Ramaekers, F. C., Schutte, B., Reutelingsperger C. P. (1998). "Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure." Cytometry 31(1): 1-9.

Vermes, I., C. Haanen., Reutelingsperger, C. (2000). "Flow cytometry of apoptotic cell death." J Immunol Methods 243(1-2): 167-90.

Wands, J. R., and Okuda, K. (1999). "Foreword. Semin." Semin. Liver Dis. 19: 233-234.

Warren, B. A. and P. Shubik (1966). "The growth of the blood supply to melanoma transplants in the hamster cheek pouch." Lab Invest 15(2): 464-78.

Xiaoguang, C., Hongyan, Liu., Xiaohong, Lei., Zhaodi, Fu., Yan, Li.,Lihua, Tao., and Rui, Han. (1998). "Cancer chemopreventive and therapeutic activities of red ginseng." journal of Ethnopharmacology 60(1): 71-78.

Ying, H. J., Kye, Jin. Yoo., You, Hui. Lee., Seung, Ki. Lee. (2000). "Caspase 3-mediated cleavage of p21WAF1/CIP1 associated with the cyclin A-cyclin-dependent kinase 2 complex is a prerequisite for apoptosis in SK-HEP-1 cells." J Bio l Chem 275(39): 30256-63.

Youl-Nam, L., Ho-Young, Lee., You Mie, Lee., Hae-young, Chung., Shin-Il, Kim., Seung-Ki, Lee., Byung Chae, Park., Kyu-Wom, Kim. (1998). "Involvement of glucocorticoid receptor in the induction of differentiation by ginsenosides in F9 teratocarcinoma cells. " J Steroid Bio l & Mol Bio l 67(2): 105-11.

Young, S. K., Dong, Seon. Kim., Shinll, kim(1998). "Ginsenoside Rh2 and Rh3 induce differentiation of HL-60 cells into granulocytes: modulation of protein kinase C isoforms during differentiation by ginsenoside Rh2." Int J Biol and Cell Bio l 30(3): 327-338.

Zetter, B. R. (1998). "Angiogenesis and tumor metastasis." Annu Rev Med 49: 407-24.