我們實驗室自Canavalia Gladiata的地方品種，用不同濃度的硫酸胺沉澱法萃取，得到一具有抗癌功效的成分，將此萃取物命名為IC-1。體外試驗中發現，IC-1對多種癌細胞株具有良好的毒殺效果，並可抑制癌細胞與內皮細胞的移行能力。此外，動物實驗發現當植入NIH3T3/ras7-4細胞24小時後即投予IC-1，可以有效毒殺小鼠腹腔內的腫瘤細胞，並延長小鼠之存活天數。在體內肺癌轉移實驗中，IC-1不但能有效的抑制原位肺癌細胞的生長，並能抑制轉移至肺臟腫瘤的生長。
　　我對此結果進行更進一步的探討。首先，血球凝集法顯示IC-1具有凝集活性。再者，以流式細胞儀分析IC-1造成癌細胞死亡的方式，發現應是屬於細胞凋亡的表現，再以Hoechst細胞核染色法觀察細胞的變化，發現確實有細胞質濃縮或凋亡小體的表徵。此外，將IC-1與現今的化療藥物並用，發現其可輔助化療藥物的功效。另一方面，體外內皮細胞移行試驗也再度證明IC-1確實對此有所抑制，於是以zymography試驗發現IC-1對MMP2,9蛋白的表現隨劑量增加有明顯抑制的情形，顯示IC-1在抑制癌細胞侵襲方面有其作用。在動物試驗方面，以ML1-4a肝癌細胞進行原位癌模式的抗癌功效評估，發現IC-1確實能抑制癌症的生長。
　　由於之前實驗室也發現IC-1能增進體內免疫細胞活化，於是再度使用動物模式評估IC-1對免疫的促進作用。除了再度證實IC-1有此功效外，也發現IC-1能夠將cisplatin所引起的免疫抑制予以回復，並有更佳活化免疫細胞的趨勢。

　　Our lab had previous shown that cold water extract from a local variety of Canavalia gladiata, termed IC-1, possessed some interesting anticancer activities. In this investigation, I clarify and identify further the anticancer activities of IC-1. IC-1 shows in vitro cytotoxicities toward some cancer cell lines in a dose-dependent manner. Flow cytometry assay reveals that cancer cells death is due to apoptosis, which is supported by two more assays—DNA fragmentation and Hoechst 33258 nucleus staining. Gelatin zymography of matrix metalloproteinase 2 and 9, the two indicators of tumor invasion of basemembrane, indicates that IC-1 can inhibit both enzymes. Next I tested the antiangiogenesis of IC-1 using bovine aorta endothelial cell (BAEC) in a migration assay. The result indicates IC-1 can inhibit the blood endothelial cell migration. Furthermore, I tested the anticancer activity of IC-1 in vivo using ML1-4A hepatoma cells as primary tumor model. IC-1 treatment reduces tumor size by more than 50% as compare to the control, confirming further the previous observation made in this lab.
　　The immune boosting effect of IC-1 was confirmed and further analyzed in vivo. Injected intraperitoneally into Balb/c mouse, IC-1 was found to stimulate splenocytes proliferation, which was assayed by 3H-thymidine incorporation method. The proliferated cell population, identified by fluorescence activated cell sorter (FACS), was found to be B cells, T cells and nature killer cells. IC-1 also can stimulate the expression of cytokines IL-2 and IFN-g. In addition, IC-1 can reverse the immune impairment, including B cells, T cells and nature killer cells in the spleen, elicited by cisplatin.
　　Taken together, these results prove that IC-1 not only inhibit tumor growth but also activate immune responses, a dual effect that renders its anticancer activities.

1. 內政部統計處 (2004)．91年臺閩地區特定死因除外簡易生命表提要分析．Available at: http://www.moi.gov.tw/W3/stat/Life/T04-analysis.html
2. 行政院衛生署 (2004)．臺灣地區主要死因死亡率趨勢圖． Available at: http://www.doh.gov.tw/statistic/data/死因摘要/92年/主要死因死亡率趨勢圖.xls
3. de Mejia, E. G., Bradford, T., & Hasler. (2003). The anticarcinogenic potential of soybean lectin and lunasin. Nutrition Review 6, 239-246.
4. Messina, M. J. & Barnes, S. (1991). The role of soy products as cancer preventive agents. J Natl Cancer Inst 83, 541-546.
5. Su, S. J., Yeh, T.M., Lei, H. Y., & Chow, N. H. (2000). The potential of soybean foods as a chemoprevention approach for human urinary tract cancer. Clin Cancer Res 6, 230-236.
6. Abe, M., Suzuki, O., Tasaki, K., Tominaga, K., & Wakasa, H. (1996). Analysis of lectin binding properties on human Burkitt’s lymphoma cell lines that show high spontaneous metastasis to distant organs in SCID mice. Pathol Inter 46, 977-983.
7. Lis, H. & Sharon, N. (1981). Lectins in higher plants. Biochem Plants 6, 371-447.
8. Sharon, N. & Lis, H. (1995). Lectins. Proteins with a sweet tooth, functions in cell recognition. Essays Biochem 30, 59-75
9. Gold, E. R. & Balding, P. (1975). “Receptor specific protein, plant and animal lectin” Excerpta Medica, Amsterdam.
10. Kaasaki, T. & Ashwell, G. (1976). Chemical and physical properties of a hepatic membrance protein that specifically binds asilo-glycoproteins. J Biol Chem 251, 1296-1301.
11. Sampaio, A. H., Rogers, D. J., & Barwell, C. J. (1999). A new isolation procedure and further characterization of the lectin from the red marine alga Ptilota serrata. J Appl Phycol 10, 539-546.
12. Fugita, Y., Oishi, K., & Imahori, K. (1975). Purification and properties of an anti-B hemagglutinin produced by Streptomyces sp. Biochemistry 14, 4465-4470.
13. Nathan, S. (1987). Lectin agglutination of thermophilic Campylobactor species. J Microbial 15, 163-173.
14. Goldstein, I. J., Winter, H. C., Mo, H., Misaki, A., Van Damme, E. J., & Peumans, W.J. (2001). Carbohydrate binding properties of banana (Musa acuminata) lectin II. Binding of laminaribiose oligosaccharides and betaglucans containing beta1, 6-glucosyl end groups. Eur J Biochem 268, 2616-2619.
15. Barre, A., Van Damme, E. J., Peumans, W. J., & Rouge, P. (1996). Structure-function relationship of monocot mannose- binding lectins. Plant Physiol 112, 1531-1540.
16. Bourne, Y., Astoul, C. H., Zamboni, V., et al. (2003). Structural basis for the unusual carbohydrate-binding specificity of jacalin towards galactose and mannose. Biochem J 364, 173-180.
17. Unverzagt, C., Andre, S., Seifert, J., et al. (2002). Structure activity proles of complex biantennary glycans with core fucosylation and with/without additional alpha 2,3/alpha 2,6 sialylation, synthesis of neoglycoproteins and their properties in lectin assays, cell binding, and organ uptake. J Med Chem 45, 478-491.
18. Sparvoli, F., Gallo, A., Marinelli, D., Santucci, A., & Bollini, R. (1998). Novel lectin-related proteins are major components in Lima bean (Phaseolus lunatus L.) seeds. Biochimica et Biophysica (BBA) / Protein Stru & Mol Enzym 138, 311-323.
19. Rolfe, M., Parmar, A., Hoy, T. G., & Coakley, W. T. (2001). Erythrocyte agglutination by wheat germ agglutinin, ionic strength dependence of the contact seam topology. Mol Membr Biol 18, 169-176.
20. Lis H. Sharon N. (1986). Lectins as molecules and as tools. Annu Rev Biochem 55, 35-67.
21. Gollob, J.A., Li, J., Reinherz, E. L., & Ritz, J. (1995).CD2 regulates responsiveness of activated T cells to interleukin 12. J Exp Med 182, 721-31.
22. 黎光南等 (1990)．雲南中藥志Ⅰ．雲南科技出版社.
23. 江蘇新醫學院編 (1977)．中藥大辭典．上海科學技術出版社.
24. 董大成 (1998)．白鳳豆與靈芝．台北元氣齋.
25. Murakami, T., Kohno, K.; Kishi, A., Matsuda, H., & Yoshikawa, M. (2000). Medicinal Foodstuffs. XIX. Absolute Stereostructures of Canavalioside, a New Ent-Kaurane-Type Diterpene Glycoside, and Gladiatosides A1, A2, A3, B1, B2, B3, C1, and C2, New Acylated Flavonol Glycosides, from Sword Bean, the Seeds of Canavalia gladiata Chem Pharm Bul 48, 1673-80.
26. Hanahan, D. & Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57-70.
27. Fiakow, P. J. (1976). Clonal origin of human tumors. Biochim Biophys Acta 458, 283-321.
28. Evan, G. I. & Vousden, K. H. (2001). Proliferation, cell cycle and apoptosis. Nature 411, 342-348.
29. Ruoslahti, E. (1996). How cancer spreads. Sci Am, 275, 72-77
30. Aupperle, K. R., Boyle, D. L., Hendrix, M., Seftor, E. A., Zvaifler, N. J., Barbosa, M., et al. (1998). Regulation of synoviocytes proliferation, apoptosis, and invasion by the p53 tumor suppressor gene. Am J Pathol 152, 1091-1098.
31. Fields, M. L., Sokol, C. L., Eaton-Bassiri, A., Seo, S., Madaio, M. P., & Erikson, J. (2001). Fas/Fas ligand deficiency results in altered localization of anti–double-stranded DNA B cells and dendritic cells. J Immunol 167, 2370-2378.
32. Kerr, J. F., Wyllie, A. H., & Currie, A. R. (1972). Apoptosis, a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26, 239-257.
33. Vaux, D. L. & Flavell, R. A. (2000). Apoptosis genes and autoimmunity. Curr Opin Immunol 12, 719-724.
34. Schultz, D. R. & Harrington, W. J. (2003). Apoptosis, Programmed Cell Death at a Molecular Level. Semni Arthr Rheuma 32, 345-369.
35. Vermes, I., Haanen, C., & Reutelingsperger, C. (2000). Flow cytometry of apoptotic cell death. J Immumol Methods 243, 167-190.
36. Wyllie, A. H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555-556.
37. Duvall, E., Wyllie, A. H., & Morris, R. G. (1985). Macrophage recognition of cells undergoing programmed cell death (apoptosis). Immunology 56, 351-358.
38. Kerr, J. F. R., Wyllie, A. H., & Currie, A. R. (1972). Apoptosis, a basic biological phenomenon with wide ranging implications in tissue kinetics. Br J Cancer 26, 239-57.
39. Wyllie, A. H., Morris, R. G., Smith, A. L., & Dunlop, D. (1984). Chromatin cleavage in apoptosis, association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol 142, 67-77.
40. Sakahira, H., Enari, M., & Nagata, S. (1998). Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391, 96-99.
41. Dive, C., Gregory, C, D, Phipps, D. J., Evans, D. L., Milner, A. E., & Wyllie, A. H. (1992). Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry. Biochim Biophys Acta 1133, 275-285.
42. Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T., & Traganos, F. (1997). Cytometry in cell necrobiology, analysis of apoptosis and accidental cell death (necrosis). Cytometry 27, 1-20.
43. Tardif, A. L., Farrell, P. B., Trouern-Trend, V., Simkin, M. E., & Foote, R. H. (1998). Use of Hoechst 33342 stain to evaluate live fresh and frozen bull sperm by computer-assisted analysis. J Androl 19, 201-206.
44. Allen, R. T., Hunter III, W. J., & Agrawal, D. K. (1997). Morphological and biochemical characterization and analysis of apoptosis. J Pharmacol Toxicol Methods 37, 215-228.
45. Murata, S-I., Herman, P., Kalowicz, J. R. (2001). Texture analysis of fluorescence lifetime images of AT- and GC-rich regions in nuclei. J Histochem Cytochem 49, 1443-1451.
46. Belloc, F., Dumain, M. R., Jalloustre C, et al. (1994). A flow cytometric method using Hoechst 33342 and propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells. Cytometry 17, 59-64.
47. Lizard, G., Deckert, V., Dubrez, L., Moisant, M., Gambert, P., & Lagrost, L. (1996). Induction of apoptosis in endothelial cells treated with cholesterol oxides. Am J Pathol 148, 1625-1628.
48. Zhang, X. & Kiechle, F. L. (1997). Hoechst 33342-induced apoptosis in BC3H-1 myocytes. Ann Clin Lab Sci 27, 260-275.
49. Zhang, X., Kiechle, F. L. (1998). Hoechst 33342 induces apoptosis in hepatoma cells and inhibits topoisomerase I in vivo. J Clin Ligand Assay 21, 62-67
50. Coussens, L. M., Fingleton, B. and Matrisian, L. (2002). Matrix metalloproteinase inhibitors and cancer, trials and tribulations. Science 295, 2387-2392.
51. McCawley, L. J. & Matrisian, L. M. (2000). Matrix metalloproteinases, multifunctional contributors to tumor progression. Mol Med Today 6, 149-156.
52. Gross, J. & Lapiere, C. M. (1962). Collagenolytic activity in amphibian tissues, a tissue culture assay. Proc Natl Acad Sci 48, 1014-1022.
53. Singer, C. F., Kronsteiner, N., Marton, E., Kubista, M., Cullen, K. J., Hirtenlehner, K., Seifert, M., & Kubista, E. (2002). MMP-2 and MMP-9 expression in breast cancer-derived human fibroblast is differentially regulated by stromal-epithelial interaction. Breast Cancer Res Treat 72, 69-77.
54. Nagase, H. & Woessner, J. F. (1999). Matrix metalloproteinases. J Biol Chem 274, 21491-21494.
55. Benaud, C., Dickson, R. B., & Thompson, E. W. (1998). Roles of the matrix metalloproteinases in mammary gland development and cancer. Breast Cancer Res Treat 50, 97-116.
56. Massova, I., Kotra, L. P., Fridman, R., & Mobashery, S. (1998). Matrix metalloproteinases, structures, evolution, and diversification. FASEB J 12, 1075-1095.
57. Cawston, T. E. (1996). Metalloproteinase inhibitors and the prevention of connective tissue breakdown. Pharmacol Ther. 70, 163-182.
58. Woessner, J. F. (1991). Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 5, 2145-2154.
59. Himelstein, B. P., Canete-Soler, R., Bernhand, E. J., Dilks, D. W., & Muschel, R. J. (1995). Metalloproteinases in tumor progression, The contribution of MMP9. Invasion Metastasis 14, 246-258.
60. Stetler-Stevenson, W. G., Hewitt, R., & Corcoran, M. (1996). Matrix metalloproteinases and tumor invasion, from correlation and causality to the clinic. Semin Cancer Biol 7, 147-154.
61. Biswas, C., Zhang, Y., DeCastro, R., Guo, H., Nakamura, T., Kataoka, H., & Nabeshima, K. (1995). The human tumor cell-derived collagenase stimulatory factor (renamed EMMPRIN) is a member of the immunoglobulin superfamily. Cancer Res 55, 434-439.
62. Jackson, C. J. & Nguyen, M. (1997). Human microvascular endothelial cells differ from macrovascular endothelial cells in their expression of matrix metalloproteinases. Int J Biochem Cell Biol 29, 1167-1177.
63. Fridman, R., Toth, M., Pena, D., & Mobashery, S. (1995). Activation of progelatinase B (MMP-9) by gelatinase A (MMP-2). Cancer Res 55, 2548-2555.
64. Itoh, T., Tanioka, M., Yoshida, H., Yoshioka, T., Nishimoto, H., & Itohara, S. (1998). Reduced angiogenesis and tumor progression gelatinase A-deficient mice. Cancer Res 58, 1048-1051.
65. Seftor, R. E., Seftor, E. A., Koshikawa, N., et al. (2001). Cooperative interactions of laminin 5 gamma2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma.. Cancer Res 61, 6322-6327.
66. Stetler-Stevenson, W. G. (1996). Dynamics of matrix turnover during pathologic remodeling of the extracellular matrix. Am J Pathol 148, 1345-1350.
67. Dumas, V., Kanitakis, J., Charvat, S., Euvrard, S., Faure, M., & Claudy, A. (1999). Expression of basement membrane antigens and matrix metalloproteinases 2 and 9 in cutaneous basal and squamous cell carcinomas. Anticancer Res 19, 2929-2938.
68. Duffy, M. J., Maguire, T. M., Hill, A., McDermott, E., & O'Higgins, N. (2000). Metalloproteinases, role in breast carcinogenesis, invasion and metastasis. Breast Cancer Res 2, 252-257.
69. Folkman, J. & Shing, Y. (1992). Angiogenesis. J Biol Chem 267, 10931-10934.
70. Folkman, J. (1971). Tumor angiogenesis, therapeutic implications. N Engl J Med 285, 1182-1186.
71. Pluda, J. M. (1997). Tumor-associated angiogenesis, mechanisms, clinical implications, and therapeutic strategies. Semin Oncol 24, 203-218.
72. Hanahan, D. & Folkman, J. (1996). Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. J. Cell 86, 353–364.
73. Brower V. (1999). Tumor angiogenesis--new drugs on the block. Nat Biotechnol 17, 963-968.
74. Olofsson, B., Pajusola, K., Kaipainen, A., von Euler, G., Joukov, V., Saksela, O., Orpana, A., Pettersson, R. F., Alitalo, K., & Eriksson, U. (1996). Vascular endothelial growth factor B, a novel growth factor for endothelial cells. Proc Natl Acad Sci 93, 2576-2581.
75. McNamara, D. A., Harmey, J. H., Walsh, T. N., Redmond, H. P., & Bouchier-Hayes, D. J. (1998). Significance of angiogenesis in cancer therapy. Br J Surg 85, 1044-1055.
76. Zetter, B. R. (1998). Angiogenesis and tumor metastasis. Annu Rev Med 49, 407-424.
77. Dachs, G. U. & Chaplin, D. J. (1998). Microenvironmental control of gene expression, implications for tumor angiogenesis, progression, and metastasis. Semin Radiat Oncol 8, 208-216.