臺灣博碩士論文加值系統

近年來，由本土黑殭菌(Metarhizium anisopliae)所產生之黑殭菌素(Destruxins)的研究，已有很大的成果。由近幾年的研究得知，Destruxins在醫學研究方面，具有抗病毒、抗腫瘤的效能。因而，利用人類血癌細胞K562來測試DB對此細胞株是否具有抗癌活性。而目前用來篩選有效抗癌藥的重要指標，都是藉由細胞週期停滯、誘導細胞凋亡與細胞分化來選擇。DB對人類血癌細胞細胞株生長增殖之作用，以trypan blue染色計數，經不同濃度DB處理K562血癌細胞，分別培養24、48、72 hr後，可抑制細胞增殖率的作用，其IC50為6.75�b0.06 μg/mL。DB會造成K562血癌細胞之細胞週期停滯在G0/G1期，且經由Western blot分析結果顯示DB的生長抑制作用，可能與減少cyclin A及增加cyclin E之蛋白表現量有關。經不同濃度DB處理不同類型癌細胞株：兩種肝癌細胞(Huh7、HepG2)及一種喉癌細胞(A549)，也觀察到有抑制細胞增生的作用。由以上結果顯示，DB抑制癌細胞增殖的作用，可能與抑制細胞週期進行有關，而同時對於其它癌細胞也有抑制其增生的現象。

Metarhizium anisopliae isolated locally could produce a family of cyclic peptide toxins, destruxins (DTXs), which demonstrated various insecticidal activities, and has received a great attention. Indeed, destruxins exhibit a wide variety of biological activities, but are best known for their insecticidal and phytotoxic activities. The great interest in destruxins derives from their potential role as anti-viral and anti-tumor activities. The indicator for the anti-tumor drug screening was deduced by means of cell cycle arrest, as well as induction of apoptosis and cell proliferation. In this study, the anti-tumor effects of destruxin B (DB) on human leukemia cell line K562 were investigated. These were accomplished by examining the cell growth, cell viability and cell cycle perturbation using cytometric methodology with various concentrations of DB together with different time exposure. It was observed that DB suppressed the growth and proliferation of K-562 cells after 24, 48, and 72 hrs treatment, as examined by trypan blue staining. The proliferation of K562 cells were found to be concealed not only dosage dependent but time-dependently. Accordingly, the IC50 for DB was found to be 6.75�b0.06 �慊/mL under experimental conditions. Flow cytometry results suggested that the anti-proliferation of K562 cell caused by DB was arrest the cell cycling at G1 phase. Furthermore, the diminished in cyclin A expression level alone with the increased in the cyclin E, as shown from western blot, was associated with inhibition of cell growth. Similar results were also found in Huh7 (hepatoma), HepG2 (hepatoma), and A549 (carcinoma of larynx) cell lines. Morphological features of treated cells revealed that the cytotoxicity (or inhibition of cell proliferation) was due to induction of cell cycle arrest but not apoptosis. This was mediated by inactivation of caspase 3. In conclusion, the DB exhibited the ability to repress the proliferation of K562 and others cell lines.

中文摘要.............................................................................................................Ⅰ
英文摘要.............................................................................................................Ⅱ
目錄.....................................................................................................................Ⅳ
圖目錄.................................................................................................................Ⅶ

第一章 緒論……………………………………………………………….......1
1-1 癌症-白血病...............................................................................................1
1-2 細胞週期與癌症的關係............................................................................5
1-3 細胞凋亡與癌症的關係............................................................................9
1-4 黑殭菌素..................................................................................................14
1-5 研究背景與動機......................................................................................17
第二章 材料與方法………………………………………………………….19
2-1 實驗材料..................................................................................................19
2-1-1 化學試劑........................................................................................ 19
2-1-2 抗體.................................................................................................19
2-1-3 儀器.................................................................................................20
2-2 DB樣品配製………................................................................................21
2-3 人類血癌細胞株的培養..........................................................................21
2-4 細胞增殖率(proliferation)之分析...........................................................21
2-4-1 Trypan blue染色法........................................................................21
2-4-2 MTs呈色法....................................................................................22
2-5 細胞型態(morphology)之分析................................................................22
2-5-1 Liu’s stain染色法...........................................................................22
2-5-2 TMB染色法...................................................................................23
2-5-3 添加EDTA法.................................................................................23
2-6 細胞週期(cell cycle)之分析....................................................................23
2-7 Annexin V-FITC/PI雙染法之分析.........................................................24
2-8 西方墨點(western blot)分析...................................................................24
第三章 結果………………………………………………………………….26
3-1 DB之結構式..........................................................................................26
3-2 DB對人類血癌細胞株K562增殖率的影響.......................................26
3-3 DB對人類血癌細胞株K562細胞週期的影響...................................27
3-4 DB對人類血癌細胞株K562細胞凋亡的影響...................................27
3-5 DB對人類血癌細胞株K562細胞週期相關蛋白表現的影響……...32
3-6 DB對人類血癌細胞株K562細胞型態的影響...................................36
3-6-1 DAB2、phospho-p42/44及FAK之蛋白的表現情形………….36
3-6-2添加EDTA在K562細胞上其細胞聚集(aggregation)的影響....37
3-6-3 DB對不同血癌細胞株(U937、HL-60、HEL)之聚集的影響....37
3-7 DB對人類血癌細胞株K562細胞分化的影響……………………...45
3-8 DB對其它不同類型的腫瘤細胞株之增殖率的影響………………..45
第四章 討論………………………………………………………………….47
第五章 結論與未來展望…………………………………………………….54
參考文獻……………………………………………………………………...56

王斐編譯 (2001) 醫學細胞分子生物學，合記圖書出版社，107-113。

李權益、高數嫚 (1997) 分子醫學: 愛滋病、癌症、老年癡呆症，合記圖書出版社。

吳明賢、林肇堂 (1996) 細胞凋亡與消化系統疾病，當代醫學，49-57。

葉哲豪 (2002) 黑殭菌素的回收與純化之探討。朝陽科技大學應用化學系研究所碩士論文。

瞿中和、王喜忠、丁明孝 (2001) 細胞分子生物學，九州圖書文物有限公司，
440-503。

Adachi, K., Nanba, H. and Kuroda, H. (1987) Potentiation of host mediated antitumor activity in mice by ��-glucan obtained from Grifola frondosa (Maitake). Chemical & Pharmaceutical Bulletin, 35, 262-270.

Alessandro, S. and Weinstein, J. B. (2000) Mutilple functions of p27Kip1 and its alterations in tumor cells: a review. Journal of cellular physiology, 183, 18-27.

Bamford, M., Walkinshaw, G. and Brown, R. (2000) Therapeutic applications of apoptosis research. Experimental Cell Research, 256, 1-11.

Bandani, A.R., Amiri, B., Butt, T.M. and Gordon-Weeks, R. (2001) Effects of efrapeptin and destruxin, metabolites of entomogenous fungi, on the hydrolytic activity of a vacuolar type ATPase identified on the brush border membrane vesicles of Galleria mellonella midgut and on plant membrane bound hydrolytic enzymes. Biochimica et Biophysica Acta, 1510, 367-377.

Bergh, B. G., Ehinger, M., Olofsson, T., Baldetorp, B., Johnsson, E., Brycke, H., Lindgren, G., Olsson, I. and Gullberg, U. (1997) Altered expression of the retinoblastoma tumor-suppressor gene in leukemic cell lines inhibits induction of differentiation but not G1-accumulation. Blood, 89, 2938-2950.

Birchmeier, W. and Behrens, J. (1994) Cadherin expression in carcinomas role in the formation of cell junctions and the prevention of invasiveness. Biochimica et Biophysica Acta, 1198, 11-26.

Blomberg, I. and Hoffmann, I. (1999) Ectopic expression of Cdc25A accelerates the G1/S transition and leads to premature activation of cyclin E and cyclin A-dependent kinases. Molecular & Cellular Biology, 19, 6183-6194.

Cai, P., Smith, D., Katz, B., Pearce, C., Venables, D. and Houck, D. (1998) Destruxin-A4 chlorohydrin, a novel destruxin from fungus OS-F68576: isolation, structure determination, and biological activity as an inducer of erythropoietin. Journal of Natural Products, 61, 290-293.

Chen, H. C., Yeh, S. F., Ong, G. T., Wu, S. H., Sun, C. M. and Chou, C. K. (1995) The novel desmethyldestruxin B2, from Metarhizium anisopliae, that suppresses hepatitis B virus surface antigen production in human hepatoma cells. Journal of Natural Products, 58, 527-531.

Chen, H. C., Chou, C. K., Sun, C. M. and Yeh, S. F. (1997) Suppressive effects of destruxin B on hepatitis B virus surface antigen gene expression in human hepatoma cells. Antiviral Research, 34, 137-144.

Chen, J. W., Liu, B. L. and Tzeng, Y. M. (1999) Purification and quantification of destruxins A and B from Metarhizium anisopliae. Journal of Chromatography, 830, 115-125.

Crans, H. N. and Sakamoto, K. M. (2001) Transcription factors and translocations in lymphoid and myeloid leukemia. Leukemia, 15, 313-331.

Dipietrantonio, A., Hsieh, T. C. and Wu, J. M. (1996) Differential effects of retinoic acid (RA) and N-(4-Hydroxyphenyl) retinamide (4-HPR) on cell growth, induction of differentiation, and changes in p34cdc2, Bcl-2, and actin expression in human promyelocytic HL-60 leukemia cells. Biochemical and Biophysical Research Communications, 224, 837-842.

Fang, F., Gertraud, O., Nobumoto, W., Tony, H. and Erkki, R. (1996) Dependence of cyclin E-CDK2 kinase activity on cell anchorage. Science, 271, 499-502.

Grabel, L. B., Singer, M. S., Martin, G. R. and Rosen, S. D. (1983) Teratocarcinoma stem cell adhesion: The role of divalent cations and a cell surface lectin. The Journal of Cell Biology, 96, 1532-1537.

Hasinoff, B. B., Abram, M. E., Barnabe, N., Khelifa, T., Allan, W. P. and Yalowich, J. C. (2001) The catalytic DNA topoisomerase II inhibitor dexrazoxane (ICRF-187) induces differentiation and apoptosis in human leukemia K562 cells. Molecular Pharmacology, 59, 453-461.

Hengartner, M. O. (2000) The biochemistry of apoptosis. Nature, 407, 770-776.

Huang, C. L., Cheng, J. C., Liao, C. H., Stern, A., Hsieh, J. T., Wang, C. H., Hsu,
H. L. and Tseng, C. P. (2004) Disabled-2 is a negative regulator of integrin alpha Ⅱbbeta 3-mediated fibrinogen adhesion and cell signaling. Joumal of Biological Chemistry. (in pross)

Itoigawa, M., Kashiwada, Y., Ito, C., Furukawa, H., Tachibana, Y., Bastow, K. F. and Lee, K. H. (2000) Anticancer agents. 203 carbazole alkaloid murrayaquinone A and related synthetic carbazolequinones as cytotoxic agents. Journal of Natural Products, 63, 893-897.

John, F. R. K. and Harmon, B. V. (1994) Apoptosis: Its significance in cancer and cancer therapy. Cancer, 73, 2013-2026.

Kantarjian, H. M. and Simth, T. L. (1995) Prolonged survival in chronic myelogenous leukemia after cytogenetic response to interferon-alpha therapy. Annals of intemal medicine, 122, 254-261.

Kasinrerk, W., Tokrasinwit, N. and Phunpae, P. (1999) CD147 monoclonal antibodies induce homotypic cell aggregation of monocytic cell line U937 via LFA-1/ICAM-1 pathway. Immunology, 96, 184-192.

Kerr, J.F.R., Wyllie, A.H. and Currie, A.R. (1972) Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer, 26, 239-257.

Kim, N. (1996) Viewpoint: putting the cell cycle in order. Science, 274, 1643-1645.

Kobayashi, T., Ikeno, S., Hosokawa, N., Uehara, Y., Hori, M. and Tsuchiya, K. (2004) Destruxin E, a cyclodepsipeptide antibiotic, reduces cyclin D1 levels and inhibits anchorage-independent growth of v-Ki-ras-expressed pMAM-ras-REF cells. Biological & pharmaceutical bulletin, 27, 587-590.

Kodaira, Y. (1961) Toxic substances to insects produced by Aspergillus ochraceus and Oospera destructo. Agricultural and Biological Chemistry, 25, 261-262.

Kodaira, Y., (1962) Studies of the new toxic substances to insects destruxin A & B produced by Oospera destructor. Agricultural and Biological Chemistry, 26, 36-42.

Koeffler, H. P. and Golde, G. W. (1980) Human myeloid leukemia cell lines: a review. Blood, 56, 344-350.

Lania, L., Majello, B. and Napolitano, G. (1999) Transcriptional control by cell-cycle regulators. Journal of Cellular Physiology, 179, 134-141.

Latta, R. K., Grondin, A., Jarrell, H. C., Nicholls, G. R. and Berube, L. R. (1999) Differential expression of nonagglutinating fimbriae and MR/P pili in swarming colonies of Proteus mirabilis. Journal of Bacteriology, 181, 3220-3225.

Mauro, M. J. and Druker, B. J. (2001) Chronic myelogenous leukemia. Current Opinion in Oncology, 13, 3-7.

McCloskey, D. E., Kaufmann, S. H., Prestigiacomo, L. J. and Davidson, N. E. (1996) Paclitaxol induces programmed cell death in MDA-MB-468 human breast cancer cells. Clinical Cancer Research, 847, 847-854.

McDonald, T. A., Holland, N. T., Skibola, C., Duramad, P. and Smith, M. T. (2001) Hypothesis: phenol and hydroquinone derived mainly from diet and gastrointestinal flora activity are causal factors in leukemia. Leukemia, 15, 10-20.

Morel, E., PaÏs, M., Turpin, M. and Guyot, M. (1983) Cytotoxicity of cyclodepsipeptides on murine lymphocytes and on L1210 leukemia cells. Biomedicine & Pharmacotherapy, 37, 184-185.

Nicoletti, I., Migliorati, G., Pagliacci, M.C., Grignani, F. and Riccardi, C. (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. Journal of Immunological Methods, 139, 271-270.

Odier, F., Vey, A. and Bureau, J. (1992) In vitro effect of fungal cyclodepsipeptides on leukemic cells:study of destruxins A, B and E. Biology of the Cell, 74, 267-271.

Ohmori, T., Tamura, K., Tsuru, S. and Nomoto, K. (1986) Antitumor activity of protein-bound polysaccharide from Cordyceps ophioglossoides in mice. Japanese Journal of Cancer Research, 77, 1256-1263.

Park, J. I., Choi, H. S., Jeong, J. S., Han, J. Y. and Kim, I. H. (2001) Involvement of p38 kinase in hydroxyurea-induced differentiation of K562 cells. Cell Growth & Differentiation, 12, 481-486.

Powell, B. L. (2001) Acute progranulocytic leukemia. Current Opinion in Oncology, 13, 8-13.

Shah, M. A. and Schwartz, G. K. (2001) Cell cycle-mediated drug resistance: an emerging concept in cancer therapy. Clinical Cancer Research, 7, 2168-2181.

Sieleck, T. M., Boylan, T. F. and Traninor, G. L. (2000) Cyclin-dependent kinase inhibitors: useful targets in cell cycle regulation. Journal of Medicinal Chemistry, 43, 1-18.

Tannoch, V. J., Hind, P. W. and Tasi, L. H. (2000) Cell cycle control. Advances in Experimental Medicine & Biology, 465, 127-140.

Tseng, C. P., Ely, B. D., Li, Y., Pong, R. C. and Hsieh, J. T. (1998) Regulation of rat DOC-2 gene during castration-induced rat ventral prostate degeneration and its growth inhibitory function in human prostatic carcinoma cells. Endocrinology, 139, 3542-3553.

Tseng, C. P., Huang, C. H., Tseng, C. C., Lin, M. H., Hsieh, J. T. and Tseng, C. H. (2001) Induction of disabled-2 gene during megakaryocyte differentiation of k562 cells. Biochemical and Biophysical Research Communications, 285, 129-135.

Tseng, C. P., Huang, C. L., Huang, C. H., Cheng, J.C., Stern, A., Tseng, C. H. and Chiu, D. T. (2003) Disabled-2 small interfering RNA modulates cellular adhesivefunction and MAPK activity during megakaryocytic differentiation of K562 cells. FEBS Letters, 541, 21-27.

Vey, A., Matha, V. and Dumas, C. (2002) Effects of the peptide mycotoxin destruxin E on insect haemocytes and on dynamics and efficiency of the multicellular immune reaction. Journal of Invertebrate Pathology, 80, 177-187.

Wani, M. C., Taylor, H. L., Wall, M. E., Coggen, P. and McPhail, A. T. (1971) Plant anticancer agents. VI. The isolation and structure of taxol, a novel antileukemic and anticancer agent from Taxus brevifolia. Journal of the American Chemical Society, 93, 2325-2327.

Willingham, M. C. (1999) Review: Cytochemical methods for the detection of apoptosis. The Journal of Histochemistry and Cytochemistry, 47, 1101-1109.

Yeh, S. F., Pan, W., Ong, G. T., Chiou, A. J., Chuang, C. C., Chiou, S. H. and Wu, S. H. (1996) Study of structure-activity correlation in destruxins, a class of cyclodepsipeptides possessing suppressive effect on the generation of hepatitis B virus surface antigen in human hepatoma cells. Biochemical and Biophysical Research Communications, 229, 65-72.