進階搜尋


 
系統識別號 U0026-0812200911313550
論文名稱(中文) 地方品種Ipomoea batatas Lam.抗癌活性之研究
論文名稱(英文) The anticancer activities of a local variety of Ipomoea batatas Lam.
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 93
學期 2
出版年 94
研究生(中文) 楊博文
研究生(英文) Po-Wen Yang
電子信箱 sselesujanas@yahoo.com.tw
學號 s2692409
學位類別 碩士
語文別 中文
論文頁數 79頁
口試委員 口試委員-洪正路
指導教授-李益謙
口試委員-鄭瑞棠
中文關鍵字 轉移  血管新生  癌症 
英文關鍵字 anticancer  metastasis  angiogenesis 
學科別分類
中文摘要   許多植物中皆含有具抗癌作用的成分,如太平洋紫杉中的taxol、喜樹中的camptothecin。本實驗室自坊間收集一種宣稱具有抗癌活性特殊品種的Ipomoea batatas Lam.,進一步萃取其成分來進行抗癌活性分析。我們取Ipomoea batatas Lam.葉片甲醇萃取物,再利用水與乙酸乙酯分離,取得乙酸乙酯萃取物,簡稱IB-EAE(Ipomoea batatas ethyl acetate extract)。由離體癌細胞增生試驗,發現IB-EAE對於許多癌細胞皆具有抑制生長的作用。並且由活體實驗,發現IB-EAE能夠抑制小鼠黑色素癌細胞B16F10、小鼠肺癌細胞LLC與小鼠肝癌細胞ML-1 4a原位腫瘤的生長。接著我們進一步探討IB-EAE抑制腫瘤生長的機制。
  
  利用流式細胞儀(FACScan)與BrdU嵌入試驗,發現IB-EAE抑制癌細胞增生之原因,主要是藉由抑制細胞由G1期進入S期,使細胞週期停止。另外觀察IB-EAE對於腫瘤血管新生(angiogenesis)的影響。由離體外,發現IB-EAE能夠抑制牛大動脈內皮細胞BAEC移行的能力。於ex vivo與semi in vivo,發現IB-EAE能夠抑制主動脈微脈管的形成與雞胚胎絨毛膜的血管新生。最後由活體試驗,發現IB-EAE能夠抑制腫瘤組織中血色素(hemoglobin)的含量。由以上結果,顯示IB-EAE具有抑制血管新生的活性。最後觀察IB-EAE對於腫瘤轉移(metastasis)之影響。由離體外癒合試驗與癌細胞移行試驗,發現IB-EAE能夠抑制高轉移性的B16F10細胞與PC-3細胞移行的能力。由酶譜電泳分析(gelatin zymography),發現IB-EAE能夠抑制PC-3細胞分泌MMP-9,並且能夠降低活體腫瘤組織間質液內MMP-9的活性,由此顯示IB-EAE可能具有抑制腫瘤轉移的活性。

英文摘要   Many anticancer drugs are derived from nature, such as taxol and camptothecin. A specific variety of Ipomoea batatas Lam obtained from local market is tested for its anticancer activity. The leaves were extracted with methanol and then partitioned with water and ethyl acetate. The ethyl acetate extract (IB-EAE)is found to exert significant cytotoxicity against several cancer cell lines tested in vitro in a dose-dependent manner. The anticancer activity of IB-EAE was investigated further in vivo using mouse bearing transplanted tumor cells. IB-EAE significantly inhibits the growths of tumors originating from B16F10 melanoma, LLC carcinoma and ML1-4a hepatoma.
  
  Flow cytometry assay and BrdU incorporation assay show that the inhibitory activity of cell proliferation is due to cell cycle arrest because IB-EAE inhibits the progression of cells from G1 phase into S phase. Besides, we found IB-EAE inhibits the migration activity of BAEC in vitro, and the microvessels formation of aortic ring ex vivo as well as the blood vessel formation in a semi in vivo chicken chorioallantonic membrane assay. Finally, by measuring total hemoglobin content in the tumor tissues, we demonstrate that IB-EAE does possess anti-angiogenic activity. We also found IB-EAE inhibits the migrations of PC-3 human prostate cancer cells and B16F10 mouse melanoma cells by either the Boyden chamber assay or the wound healing assay. In a gelatin zymography assay for matrix metalloproteinase-9 (MMP-9), IB-EAE inhibits the secretion of MMP-9 form PC-3 cells and the total MMP-9 activity in the B16F10 tumors, suggesting the extract possesses an interesting anti-metastatic principle.

論文目次 考試合格證明
致謝
目錄........................................I
圖目錄......................................III
表目錄......................................IV
中文摘要....................................V
英文摘要....................................VI
縮寫表......................................VII

第一章 研究背景.............................1

第二章 實驗材料.............................11
 第一節 細胞株.............................11
 第二節 試劑...............................11
 第三節 葉片乙酸乙酯萃取物(IB-EAE)之配製.13

第三章 實驗方法.............................14
 第一節 細胞培養...........................14
 第二節 離體細胞增生試驗...................15
 第三節 細胞週期之分析.....................17
 第四節 BrdU嵌合試驗.......................18
 第五節 離體合併藥物分析...................20
 第六節 癌細胞誘發皮下原位腫瘤模式.........21
 第七節 內皮細胞移行試驗...................22
 第八節 主動脈環試驗.......................24
 第九節 雞胚胎絨毛膜血管新生試驗...........25
 第十節 腫瘤組織血色素之測定...............26
 第十一節 離體癒合試驗.....................27
 第十二節 博登細胞移行試驗.................27
 第十三節 酶譜電泳分析.....................29

第四章 實驗結果.............................32

第五章 討論.................................41

參考文獻.....................................46
圖表.........................................55
作者簡歷.....................................80
參考文獻 Adams, J.M. and Cory, S. Apoptosomes: engines for
 caspase activation. Curr. Opin. Cell Biol. 14,  
 715-720 (2002).
Ashkenazi, A. and Dixit, V.M. Death receptors:  
 signaling and modulation. Science 281,  
 1305-1308 (1998).
Benelli, R. and Albini, A. In vitro models of  
 angiogenesis: the use of Matrigel. Int. J.  
 Biol. Markers 14, 243-246 (1999).
Borgne, A. and Meijer, L. Sequential   
 dephosphorylation of p34(cdc2) on Thr-14 and  
 Tyr-15 at the prophase/metaphase transition. J.
 Biol. Chem. 271, 27847-27854 (1996).
Carlson, B.A., Dubay, M.M., Sausville, E.A.,  
 Brizuela, L. and Worland, P.J.Flavopiridol
 induces G1 arrest with inhibition of
 cyclin-dependent kinase (CDK)2 and CDK4 in
 human breast carcinoma cells. Cancer Res. 56,
 2973-2978 (1996).
Carmeliet, P. and Jain, K.R. Angiogenesis in cancer
 and other diseases. Nature 407, 249-257 (2000).
Cohen, O., Feinstein, E. and Kimchi, A. DAP-kinase
 is a Ca2+/calmodulin-dependent, cytoskeletal-
 associated protein kinase, with cell death- 
 inducing functions that depend on its catalytic
 activity. EMBO J. 16,998-1008 (1997).
Coqueret, O. New roles for p21 and p27 cell-cycle
 inhibitors: a function for each cell compartment?
 Trends Cell Biol. 13, 65-70 (2003).
Dhanikula, A.B. and Panchagnula, R. Localized
 paclitaxel delivery. Int. J. Pharm. 183, 85-100
 (1999).
Dosquet, C., Coudert, M.C., Lepage, E., Cabane, J.
 and Richard, F. Are angiogenic factors,
 cytokines, and soluble adhesion molecules
 prognostic factors in patients with renal cell
 carcinoma? Cancer Res. 3, 2451-2458 (1997).
Fattaey, A. and Booher, R.N. Myt1: a Wee1-type
 kinase that phosphorylates Cdc2 on residue
 Thr14. Prog. Cell Cycle Res. 3, 233-240 (1997).
Fidler, I.J. Critical factors in the biology of  
 human cancer metastasis: twenty-eighth G.H.A.  
 Clowes memorial award lecture. Cancer Res. 50,
 6130-6138 (1990).
Folkman, J. Tumor angiogenesis: therapeutic  
 implications. N. Engl. J. Med. 285, 1182-1186
 (1971).
Folkman, J. and Shing, Y. Angiogenesis. J. Biol.
 Chem. 267, 10931-10934 (1992).
Fong, T.A., Shawver, L.K., Sun, L., Tang, C., App,
 H., Powell, T.J., Kim, Y.H., Schreck, R., Wang,
 X., Risau, W., Ullrich, A., Hirth, K.P. and
 McMahon, G. SU5416 is a potent and selective
 inhibitor of the vascular endothelial growth  
 factor receptor (Flk-1/KDR) that inhibits
 tyrosine kinase catalysis, tumor vascularization,
 and growth of multiple tumor types. Cancer Res.
 59, 99-106 (1999).
Fuji, N., Ueda, Y., Fujiwara, H., Toh, T.,
 Yoshimura, T. and Yamagishi, H. Antitumor effect
 of alpha-galactosylceramide (KRN7000) on
 spontaneous hepatic metastases requires
 endogenous interleukin 12 in the liver. Clin.
 Cancer Res. 6, 3380-3387 (2000).
Gingras, D., Boivin, D., Deckers, C., Gendron, S.,
 Barthomeuf, C., Beliveau, R. Neovastat-a novel
 antiangiogenic drug for cancer therapy.
 Anticancer Drugs 14, 91-96 (2003).
Gullino, P.M. Angiogenesis and oncogenesis. J.
 Natl. Cancer Inst. 61, 639-643 (1978).
Halin, S., Wikstrom, P., Rudolfsson, S.H., Stattin,
 P., Doll, J.A., Crawford, S.E. and Bergh, A.
 Decreased pigment epithelium- derived factor is
 associated with metastatic phenotype in human and
 rat prostate tumors. Cancer Res. 64, 5664-5671
 (2004).
Hartwell, L.H. and Weinert, T.A. Checkpoints:
 controls that ensure the order of cell cycle
 events. Science 246, 629-634 (1989).
Hu, D.E., Hiley, C.R., Smither, R.L., Gresman, G.A.
 and Fan, T.P.D. Correlation of 133Xe clearance,
 Blood flow and histology in the rat sponge model
 for angiogenesis. Lab Invest. 72, 601-610 (1995).
Huang, P.C. Nutritive value of sweet potato. In R.
 L., Villareal and T.D. Griggs (eds), Proc. 1st
 Symp. Sweet potato. AVRDC, Taiwan, pp.35-36
 (1982).
Huang, C., Liu, J., Haudenschild, C.C. and Zhan, X.
 The role of tyrosine phosphorylation of cortactin
 in the locomotion of endothelial cells. J. Biol.
 Chem. 273, 25770-25776 (1998).
Islam, M.S., Yoshimoto, M., Terahara, N. and
 Yamakawa, O. Anthocyanin compositions in
 sweetpotato (Ipomoea batatas L.) leaves. Biosci.
 Biotechnol. Biochem. 66, 2483-2486 (2002).
Itoh, T., Tanioka, M., Yoshida, H., Yoshioka, T.,
 Nishimoto, H. and Itohara, S. Reduced 
 angiogenesis and tumor progression in gelatinase
 A-deficient mice. Cancer Res. 58, 1048-1051
 (1998).
Itoh, T., Tanioka, M., Matsuda, H., Nishimoto, H.,
 Yoshioka, T.,Suzuki, R. and Uehira, M.
 Experimental metastasis is suppressed in
 MMP-9-deficient mice. Clin. Exp. Metastasis 17,
 177-181 (1999).
Jeffrey, P.D., Tong, L.and Pavletich, N.P.
 Structural basis of inhibition of CDK-cyclin
 complexes by INK4 inhibitors. Genes Dev. 14,
 3115-3125 (2000).
Juarez, J.C., Guan, X., Shipulina, N.V., Plunkett,
 M.L., Parry, G.C., Shaw, D.E., Zhang, J.C.,
 Rabbani, S.A., McCrae, K.R., Mazar, A.P., Morgan,
 W.T. and Donate.  F. Histidine-Proline- rich
 Glycoprotein Has Potent Antiangiogenic Activity
 Mediated through the Histidine-Proline-rich
 Domain. Cancer Res. 62, 5344-5350  (2002).
Juliano, R.L. Signal transduction by cell adhesion
 receptors and the cytoskeleton: functions of
 integrins, cadherins, selectins, and  
 immunoglobulin-superfamily members. Annu. Rev.
 Pharmacol. Toxicol. 42, 283-323 (2002).
Kerbel, R.S. Tumor angiogenesis: past, present and
 the near future. Carcinogenesis 21, 505-515
 (2000).
Kimura, Y. and Okuda, H. Effects of naturally
 occurring stilbene glucosides from  medicinal
 plants and wine, on tumour growth and lung
 metastasis in Lewis lung carcinoma-bearing mice.
 J. Pharma. Pharmacol. 52, 1287-1295 (2000).
Koshkina, N.V., Kleinerman, E.S., Waidrep, C., Jia,
 S.F., Worth, L.L., Gilbert, B.E. and Knight, V.
 9-Nitrocamptothecin liposome aerosol treatment of
 melanoma and osteosarcoma lung metastases in
 mice. Clin. Cancer Res. 6, 2876-2880 (2000).
Kragh, M., Quistorff, B., Lund, E.L. and
 Kristjansen, P.E. Quantitative estimates of
 vascularity in solid tumors by non-invasive
 near-infrared spectroscopy. Neoplasia 3, 324-330
 (2001).
Krude, T., Jackson, M., Pines, J., and Laskey, R.A.
 Cyclin/ Cdk-dependent initiation of DNA
 replication in a human cell-free system. Cell 88,
 109-119 (1997).
Li, D., Williams, J.I. and Pietras, R.J. Squalamine
 and cisplatin block angiogenesis and growth of
 human ovarian cancer cells with or without HER-2
 gene overexpression. Oncogene 21, 2805-2814  
 (2002).
Lundberg, A.S. and Weinberg, R.A. Functional
 inactivation of the retinoblastoma protein
 requires sequential modification by at least two
 distinct cyclin-Cdk complexes. Mol. Cell. Biol.
 18, 753-761 (1998).
Malinda, K.M., Nomizu, M., Chung, M., Delgado, M.,
 Kuratomi, Y., Yamada, Y., Kleinman, H.K. and
 Ponce, M.L. Identification of laminin alpha1 and
 beta1 chain peptides active for endothelial cell
 adhesion, tube formation, and aortic
 sprouting. FASEB J. 13, 53-62 (1999).
Maskos, K. and Bode, W. Structural basis of matrix
 metalloproteinases and tissue inhibitors of
 metalloproteinases. Mol. Biotechnol. 25, 241-266
 (2003).
Matsuoka, S., Huang, M. and Elledge, S.J. Linkage
 of ATM to cell cycle regulation  by the Chk2
 protein kinase. Science 282, 1893 -1897 (1998).
McCawley, L.J.and Matrisian, L.M. Matrix
 metalloproteinases, multifunctional  
 contributors to tumor progression. Mol. Med.
 Today 6, 149-156 (2000).
McNamara, D.A., Harmey,J.H., Walsh, T.N., Redmond,
 H.P. and Bouchier-Hayes, D.J. Significance of
 angiogenesis in cancer therapy. Br. J. Surg. 85,
 1044-1055 (1998).
Michalides, R.J. Cell cycle r
 egulators: mechanisms and their role in
 aetiology,  prognosis,  and treatment of
 cancer. J. Clin. Pathol. 52, 555-568 (1999).
Michalopoulos, G. and Pitot, H.C. Primary culture
 of parenchymal liver cells on  collagen  
 membranes. Morphological and biochemical
 observations. Exp. Cell Res.  94, 70-78 (1975).
Murakami. K., Matsuura, T., Sano, M., Hashimoto,
 A., Yonekura, K., Sakukawa, R.,  Yamada, Y. and
 Saiki, I,4-[3,5-Bis(trimethylsilyl)benzamido]
 benzoic acid (TAC-101) inhibits the intrahepatic
 spread of hepatocellular carcinoma and prolongs
 the life-span of tumor-bearing animals. Clin.
 Exp. Metastasis 16, 633-643 (1998).
Nguyen, M., Arkell, J. and Jackson, C.J. Thrombin
 rapidly and efficiently activates gelatinase A in
 human microvascular endothelial cells via a
 mechanism independent of active MT1-MMP. Lab.
 Invest. 79, 467-475 (1999).
Nguyen, M., Arkell, J. and Jackson, C.J.
 Three-dimensional collagen matrices induce
 delayed but sustained activation of gelatinase A
 in human endothelial cells via MT1-MMP. Inter. J.
 Biochem. Cell Biol. 32, 621-631 (2000).
Nguyen, M., Arkell, J. and Jackson, C.J. Human
 endothelial gelatinases and angiogenesis. Int. J.
 Biochem. Cell Biol. 33, 960-970 (2001).
Ohtani, K. Implication of transcription factor E2F
 in regulation of DNA replication. Front. Biosci.
 4, 793-804 (1999).
Olofsson, B., Pajusola, K., Kaipainen, A., von
 Euler, G., Joukov, V., Saksela, O., Orpana, A.,
 Pettersson, R.F., Alitalo, K. and Eriksson, U.
 Vascular endothelial growth factor B, a novel
 growt factor for endothelial cells. Proc.
 Natl. Acad. Sci. 93, 2576-2581 (1996).
O'Reilly, M.S., Holmgren, L., Shing, Y., Chen, C.,
 Rosenthal, R.A., Moses, M., Lane, W.S., Cao,Y,,
 Sage, E.H. and Folkman, J.. Angiostatin: a novel
 angiogenesis inhibitor that mediates the
 suppression of metastases by a Lewis
 lung carcinoma. Cell 79, 3 15-328 (1994).
O'Reilly, M.S., Boehm, T., Shing, Y., Fukai, N.,
 Vasios, G., Lane, W.S., Flynn, E., Birkhead,
 J.R., Olsen, B.R. and Folkman, J. Endostatin: an
 endogenous inhibitor of angiogenesis and tumor
 growth. Cell 88,277-285 (1997).
Overall, C.M. and Lopez-Otin, C. Strategies for MMP
 inhibition in cancer:innovations for the  
 post-trial era. Nat. Rev. Cancer 2,657-672
 (2002).
Owa, T., Yoshino, H., Yoshimatsu, K. and Nagasu,
 T., Cell cycle regulation in the G1 phase: a
 promising target for the development of new
 chemotherapeutic anticancer agents. Curr. Med.
 Chem. 8, 1487-1503 (2001).
Owen-Schaub, L.B., van Golen, K.L., Hill, L.L. and
 Price, J.E. Fas and Fas ligand interactions
 suppress melanoma lung metastasis. J. Exp. Med.
 188, 1717-1723 (1998).
Price, L.S. and Collard, J.G. Regulation of the
 cytoskeleton by Rho-family GTPases:implications
 for tumour cell invasion. Semin. Cancer Biol.
 11, 167-173 (2001).
Purcell, A.E., Swaisgood, H.E. and Pope, D.T.
 Protein and amino acid content of sweetpotato
 cultivars. J. Amer. Soc. Hort. Sci. 97, 30-33
 (1972).
Roche-Nagle, G., Connolly, E.M., Eng, M., 
 Bouchier-Hayes, D.J. and Harmey, J.H.
 Antimetastatic activity of a cyclooxygenase-2
 inhibitor. Br. J. Cancer 91, 359-365 (2004).
Sancar, A., Lindsey-Boltz, L.A., Unsal-Kacmaz, K.
 and Linn, S. Molecular mechanisms of mammalian
 DNA repair and the DNA damage checkpoints. Annu.
 Rev. Biochem. 73, 39-85 (2004).
Saraste, A. and Pulkki, K. Morphologic and
 biochemical hallmarks of apoptosis. Cardiol. Res.
 45, 528-537 (2000).
Shimozono, H., Kobori, M., Shinmoto, H. and
 Tsushida, T. Suppression of the melanogenesis of
 mouse melanoma B16 cells by sweetpotato extract.
 Nippon Shokuhin Kagaku Kaishi. 43, 313-317
 (1996).
Suda, I., Furuta, S., Nishiba, Y., Yamakawa, O.,
 Matsugano, K. and Sugita, K. Reduction of liver
 injury induced by carbon tetrachloride in rats  
 administered purple-colored sweetpotato juice.
 Nippon Shokuhin Kagaku Kogaku Kaishi 44, 315-318
 (1997).
Supuran, C.T. Indisulam: an anticancer sulfonamide
 in clinical development. Expert Opin. Investig.
 Drugs 12, 283-287 (2003).
Takeda, K., Hayakawa, Y., Smyth, M.J., Kayagaki,
 N., Yamaguchi. N, Kakuta, S., Iwakura, Y.,
 Yagita, H. and Okumura, K. Involvement of tumor
 necrosis factor-related apoptosis-inducing ligand
 in surveillance of tumor metastasis by liver
 natural killer cells. Nature Med. 7, 94-100
 (2001).
Taylor, W.R. and Stark, G.R. Regulation of the G2/M
 transition by p53. Oncogene 20, 1803-1815 (2001).
van Moorsel, C.J., Pinedo, H.M., Smid, K., Comijn,
 E.M., Voorn, D.A., Veerman, G., Lakerveld, B.,  
 Van der Vijgh, W.J., Giaccone, G., Postmus, P.E.
 and Peters, G.J. Schedule-dependent  
 pharmacodynamic effects of gemcitabine and
 cisplatin in mice bearing Lewis lung murine
 non-small cell lung tumours. Eur. J. Cancer 36,
 2420-2429 (2000).
Vermes, I., Haanen, C. and Reutelingsperger, C.
 Flow cytometry of apoptotic cell death. J.
 Immumol. Methods 243, 167-190 (2000).
Will, H., Atkinson, S.J., Butler, G.S., Smith, B.
 and Murphy, G. The soluble catalytic domain of
 membrane type 1 matrix metalloproteinase cleaves
 the pro-peptide of pro-gelatinase A and initiates
 autoproteolytic activation. J. Biol.Chem. 271,
 17119-17123 (1996).
Wong, C.W., Lee, A., Shientag, L., Yu, J., Dong,
 Y., Kao, G., Al-Mehdi, A.B., Bernhard, E.J. and
 Muschel, R.J. Apoptosis: an early event in
 metastatic inefficiency. Cancer Res. 61, 333-338
 (2001).
Yanase, T., Tamura, M., Fujita, K., Kodama, S. and
 Tanaka, K. Inhibitory effect of angioge nesis
 inhibitor TNP-470 on tumor growth and metastasis
 of human cell lines in vitro and in vivo. Cancer
 Res. 53, 2566-2570 (1993).
Yoshimoto, M., Okuno, S., Yoshinaga, M., Yamakawa,
 O., Yamaguchi, M. and Yamada, J. Antimutagenicity
 of sweetpotato (Ipomoea batatas) roots. Biosci.
 Biotechnol. Biochem. 63, 537-541 (1999).
Zimmermann, C., Bonzon, C., and Green, D.R. The
 machinery of programmed cell death. Pharmacol.
 Ther. 92, 57-70 (2001).
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2008-07-14起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2008-07-14起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw