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研究生:游雅琪
研究生(外文):Ya-Chi Yu
論文名稱:黑殭菌素(DestruxinB)對於人類急性和慢性淋巴癌之抗癌機制探討
論文名稱(外文):Study the Mechanisms of Apoptosis of Destruxin B Anti-tumor Effects on Human Acute and Chronic Lymphoma
指導教授:陳建和陳建和引用關係
指導教授(外文):Chien-Ho Chen
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗生物技術學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:70
中文關鍵詞:黑殭菌素B(Destruxin B)人類急性淋巴性白血病Molt-4人類非霍金氏淋巴癌Toledo細胞凋亡粒線體膜電位
外文關鍵詞:Destruxin Bthe Molt-4 human acute lymphoblastic leukemia cell linethe Toledo human non Hodgkin’s lymphoma cell linesApoptosismitochondrial membrane potential
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黑殭菌素(Destruxin)是由昆蟲寄生性真菌(entomogenous fungi)之一 (Matarhizium anisopliae)所分泌出的毒素,在已分離出的三十餘種中,本研究中採用的是由此一菌種分泌的二次代謝物Destruxin B(DB)作為抗腫瘤製劑。DB對昆蟲具有毒性且運用在生物農藥已有數年之久。在之前的研究中發現到在體外試驗或是活體試驗DB對於DBA/2老鼠L5178Y淋巴癌細胞有抑制的效果。本論文將探討由黑殭菌素B (Destruxin B)對人類急性淋巴性白血病Molt-4和人類非霍金氏淋巴癌Toledo細胞造成生長抑制或毒殺作用。由初期的實驗結果得知Molt-4細胞在高於5.05 uM DB作用下可以有效的抑制癌細胞生長;而Toledo細胞則在1.26 uM DB作用下明顯的抑制癌細胞生長。利用Annexin V染色及JC-1染色的方式更進一步地證實出Destruxin B會引起Molt-4、Toledo細胞死亡是經由粒線體膜電位改變引起細胞凋亡,而非壞死。Destruxin B作用在Molt-4和Toledo細胞48小時後,以西方點墨法的結果證實,AIF和Bax蛋白表現有增加的情形,Bid和Bcl-2蛋白表現量遞減,更能證實細胞是經由粒線體膜電位改變引起細胞凋亡而死;並且也觀察到FADD、caspase 8、caspase 3的活化,發現Destruxin B可導致細胞經由死亡接受器途徑而凋亡。綜合以上初步結果得知,Destruxin B可以抑制人類急性淋巴性白血病Molt-4和人類非霍金氏淋巴癌Toledo細胞的生長,其機制是經由死亡接受器途徑和粒線體途徑而使細胞走向凋亡。
Destruxins are second metabolic products form an entomogenous fungus, Mararhuzuim anisopliae. These substances are toxic to insect and have been used as an insecticide for decades. Over thirty analogues of destruxins have been isolated by different laboratories, the one used in this study is Destruxin B (DB). The previous study in our laboratory revealed that DB has potent anti-tumor activity in mouse L5178Y lymphoma cells in DBA/2 mice both in vitro and in vivo experiments. In this report, the Molt-4 human acute lymphoblastic leukemia cell line and the Toledo human non Hodgkin’s lymphoma cell lines was used as a subject to evaluate its anti-tumor activity and study the mechanism of the effect. The initial experimental results showed that DB possesses a potent growth suppression effect on Molt-4 when the doses of DB higher than 5.05 uM and Toledo obviously had suppression cell growth at 1.26 uM DB. Further Annexin V and JC-1 staining experimental results showed that Destruxin B caused Molt-4, Toledo cell death by mitochondrial membrane potential changed to induce apoptosis. From the Western blot experimental results found that the expression of AIF and Bax was increased and that decreased in Bid and Bcl-2 expression in Molt-4 and Toledo cells at 48 hours after DB treatment, thus confirming cell death via mitochondrial membrane potential changed to induce apoptosis. We also found the activation of FADD, caspase 8, caspase 3, thus confirming cell death may via death receptor pathway to induce apoptosis also. In conclusion, the mechanisms of cell apoptosis by DB treatment may be due to change mitochondrial membrane potential and the death receptor pathway.
中文摘要 I
Abstract III
目錄 V
圖目錄 VIII
第一章 緒論 1
一、白血病簡介 1
(一) 急性淋巴性白血病 2
(二) 慢性淋巴性白血病 3
(三) 急性骨髓性白血病 3
(四) 慢性骨髓性白血病 4
二、黑殭菌素簡介 5
三、細胞凋亡簡介 8
(一)細胞壞死 (Necrosis) 8
(二)細胞凋亡 (Apoptosis) 9
四、細胞凋亡的路徑與調控 10
(一) 死亡接受器途徑 (Death receptor pathway) 10
(二) 粒線體途徑 (mitochondrial pathway) 11
(三) 內質網途徑 (endoplasmic reticulum pathway) 12
(四) 細胞凋亡之相關調控蛋白 13
1. Caspase家族 13
2. BCL-2家族 14
第二章 材料與方法 18
一、實驗材料 18
二、常用溶液 20
三、實驗方法: 22
1. 細胞培養 22
2. 藥物配製 22
3. 細胞存活率計算 23
4. 流式細胞儀分析之PI-Annexin V-FITC 雙染色法 23
5. 流式細胞儀分析之粒線體膜電位檢測 25
6. 西方墨點法 (Western blot) 26
第三章 結果 30
一、黑殭菌素 (Destruxin B)對Molt-4和Toledo細胞生長之影響 30
二、黑殭菌素 (Destruxin B)可誘使Molt-4和Toledo細胞發生細胞凋亡 31
三、黑殭菌素 (Destruxin B)可誘導Molt-4和Toledo細胞粒線體膜電位降低 32
四、Destruxin B可誘使Molt-4和Toledo發生細胞凋亡之相關蛋白表現 33
第四章 討論 36
第五章 圖表 43
第六章 參考文獻及回顧 63
黎培琪 (2004) 黑殭菌素(Destruxin B)對DBA/2老鼠L5178Y淋巴癌細胞之抑制效果及其機制。私立台北醫學大學生物醫學技術研究所碩士論文。
古欣平 (2004) 黑殭菌素B(Destruxin B)抑制人類血癌細胞之探討。私立朝陽科技大學應用化學系碩士論文。
呂佩珊 (2005) 黑殭菌素(Destruxin B)對大腸癌細胞HT-29之抑制效果及其機制。私立台北醫學大學生物醫學技術研究所碩士論文。
陳拯裕 (2006) 黑殭菌素B對人類大腸癌及肝癌細胞株之抗癌機制與探討。私立朝陽科技大學應用化學系碩士論文。
Aarstad, K., Zimmer, T.L., and Laland, S.G. (1979). Replacement of phenylalanine in gramicidin S by other amino acids. FEBS Lett 103, 118-121.
Adachi, K., Nanba, H., and Kuroda, H. (1987). Potentiation of host-mediated antitumor activity in mice by beta-glucan obtained from Grifola frondosa (maitake). Chemical & pharmaceutical bulletin 35, 262-270.
Arends, M.J., Morris, R.G., and Wyllie, A.H. (1990). Apoptosis. The role of the endonuclease. The American journal of pathology 136, 593-608.
Armstrong, J.S. (2006). The role of the mitochondrial permeability transition in cell death. Mitochondrion 6, 225-234.
Arnoult, D., Gaume, B., Karbowski, M., Sharpe, J.C., Cecconi, F., and Youle, R.J. (2003). Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization. Embo J 22, 4385-4399.
Bamford, M., and Kendall, C.H. (2000). Intravascular tumour in intraoral pleomorphic adenomas. Histopathology 37, 85.
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.
Bedner, E., Li, X., Gorczyca, W., Melamed, M.R., and Darzynkiewicz, Z. (1999). Analysis of apoptosis by laser scanning cytometry. Cytometry 35, 181-195.
Bradfisch, G.A., and Harmer, S.L. (1990). omega-Conotoxin GVIA and nifedipine inhibit the depolarizing action of the fungal metabolite, destruxin B on muscle from the tobacco budworm (Heliothis virescens). Toxicon 28, 1249-1254.
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. J Nat Prod 61, 290-293.
Calmes, M., Cavelier-Frontin, F., Jacquier, R., Mercadier, J.L., Sabil, S., Verducci, J., Quiot, J.M., and Vey, A. (1993). Synthesis and biological activity of a destruxin analogue: D-Lac-6 destruxin E. Int J Pept Protein Res 41, 528-535.
Cande, C., Vahsen, N., Garrido, C., and Kroemer, G. (2004). Apoptosis-inducing factor (AIF): caspase-independent after all. Cell Death Differ 11, 591-595.
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 Res 34, 137-144.
Clement, M.V., Hirpara, J.L., and Pervaiz, S. (2003). Decrease in intracellular superoxide sensitizes Bcl-2-overexpressing tumor cells to receptor and drug-induced apoptosis independent of the mitochondria. Cell death and differentiation 10, 1273-1285.
Crans, H.N., and Sakamoto, K.M. (2001). Transcription factors and translocations in lymphoid and myeloid leukemia. Leukemia 15, 313-331.
Crompton, M., and Costi, A. (1988). Kinetic evidence for a heart mitochondrial pore activated by Ca2+, inorganic phosphate and oxidative stress. A potential mechanism for mitochondrial dysfunction during cellular Ca2+ overload. European journal of biochemistry / FEBS 178, 489-501.
Crompton, M., Costi, A., and Hayat, L. (1987). Evidence for the presence of a reversible Ca2+-dependent pore activated by oxidative stress in heart mitochondria. The Biochemical journal 245, 915-918.
Danial, N.N., and Korsmeyer, S.J. (2004). Cell death: critical control points. Cell 116, 205-219.
Dumas, C., Matha, V., Quiot, J.M., and Vey, A. (1996). Effects of destruxins, cyclic depsipeptide mycotoxins, on calcium balance and phosphorylation of intracellular proteins in lepidopteran cell lines. Comparative biochemistry and physiology 114, 213-219.
Eastman, A., and Rigas, J.R. (1999). Modulation of apoptosis signaling pathways and cell cycle regulation. Seminars in oncology 26, 7-16; discussion 41-12.
Escobar-Diaz, E., Lopez-Martin, E.M., Hernandez del Cerro, M., Puig-Kroger, A., Soto-Cerrato, V., Montaner, B., Giralt, E., Garcia-Marco, J.A., Perez-Tomas, R., and Garcia-Pardo, A. (2005). AT514, a cyclic depsipeptide from Serratia marcescens, induces apoptosis of B-chronic lymphocytic leukemia cells: interference with the Akt/NF-kappaB survival pathway. Leukemia 19, 572-579.
Fadok, V.A., Voelker, D.R., Campbell, P.A., Cohen, J.J., Bratton, D.L., and Henson, P.M. (1992). Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148, 2207-2216.
Gabay, C., Ben-Bassat, H., Schlesinger, M., and Laskov, R. (1999). Somatic mutations and intraclonal variations in the rearranged Vkappa genes of B-non-Hodgkin''s lymphoma cell lines. European journal of haematology 63, 180-191.
Glucksmann, A. (1965). Cell death in normal development. Archives de biologie 76, 419-437.
Halestrap, A.P. (2006). Calcium, mitochondria and reperfusion injury: a pore way to die. Biochemical Society transactions 34, 232-237.
Hsu, S.Y., and Hsueh, A.J. (2000). Tissue-specific Bcl-2 protein partners in apoptosis: An ovarian paradigm. Physiological reviews 80, 593-614.
Huang, D.C., Adams, J.M., and Cory, S. (1998). The conserved N-terminal BH4 domain of Bcl-2 homologues is essential for inhibition of apoptosis and interaction with CED-4. The EMBO journal 17, 1029-1039.
Jin, Z., and El-Deiry, W.S. (2005). Overview of cell death signaling pathways. Cancer biology & therapy 4, 139-163.
Kerr, J.F. (1965). A histochemical study of hypertrophy and ischaemic injury of rat liver with special reference to changes in lysosomes. The Journal of pathology and bacteriology 90, 419-435.
Kerr, J.F. (1971). Shrinkage necrosis: a distinct mode of cellular death. The Journal of pathology 105, 13-20.
Kerr, J.F., 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, T.H., Zhao, Y., Barber, M.J., Kuharsky, D.K., and Yin, X.M. (2000). Bid-induced cytochrome c release is mediated by a pathway independent of mitochondrial permeability transition pore and Bax. The Journal of biological chemistry 275, 39474-39481.
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. Biol Pharm Bull 27, 587-590.
Kumar, S. (2007). Caspase function in programmed cell death. Cell death and differentiation 14, 32-43.
Liu, C.M., Huang, S.S., and Tzeng, Y.M. (2004). Analysis of destruxins produced from Metarhizium anisopliae by capillary electrophoresis. Journal of chromatographic science 42, 140-144.
Liu, C.M., Huang, S.S., and Tzeng, Y.M. (2004) Purification and quantitative analysis of Destruxins from Metarhizium anisopliae by high-performance liquid chromatography. J. Liq. Chromatogr. & Rel. Technol. 27(6)999-1011.
Marzo, I., Brenner, C., Zamzami, N., Jurgensmeier, J.M., Susin, S.A., Vieira, H.L., Prevost, M.C., Xie, Z., Matsuyama, S., Reed, J.C., et al. (1998). Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science (New York, NY 281, 2027-2031.
Mayer, B., and Oberbauer, R. (2003). Mitochondrial regulation of apoptosis. News Physiol Sci 18, 89-94.
Morel, E., Pais, M., Turpin, M., and Guyot, M. (1983). Cytotoxicity of cyclodepsipeptides on murine lymphocytes and on L 1210 leukemia cells. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 37, 184-185.
Muroi, M., Shiragami, N., and Takatsuki, A. (1994). Destruxin B, a specific and readily reversible inhibitor of vacuolar-type H(+)-translocating ATPase. Biochemical and biophysical research communications 205, 1358-1365.
Nakagawa, H., Takami, M., Udagawa, N., Sawae, Y., Suda, K., Sasaki, T., Takahashi, N., Wachi, M., Nagai, K., and Woo, J.T. (2003). Destruxins, cyclodepsipeptides, block the formation of actin rings and prominent clear zones and ruffled borders in osteoclasts. Bone 33, 443-455.
Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B.A., and Yuan, J. (2000). Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 403, 98-103.
Odier, F., Vago, P., Quiot, J.M., Devauchelle, G., and Bureau, J.P. (1987). [Cytometric study of the effects of destruxin E on leukemic cells in mice]. Comptes rendus de l''Academie des sciences 305, 575-578.
Odier, F., Vey, A., and Bureau, J.P. (1992). In vitro effect of fungal cyclodepsipeptides on leukemic cells: study of destruxins A, B and E. Biol Cell 74, 267-271.
Pal, S., St Leger, R.J., and Wu, L.P. (2007). Fungal peptide Destruxin A plays a specific role in suppressing the innate immune response in Drosophila melanogaster. The Journal of biological chemistry 282, 8969-8977.
Pedras, M.S., Biesenthal, C.J., and Zaharia, I.L. (2000). Comparison of the phytotoxic activity of the phytotoxin destruxin B and four natural analogs. Plant Science 156, 185-192.
Pedras, M.S., Irina Zaharia, L., and Ward, D.E. (2002). The destruxins: synthesis, biosynthesis, biotransformation, and biological activity. Phytochemistry 59, 579-596.
Rostovtseva, T.K., Antonsson, B., Suzuki, M., Youle, R.J., Colombini, M., and Bezrukov, S.M. (2004). Bid, but not Bax, regulates VDAC channels. The Journal of biological chemistry 279, 13575-13583.
Salvioli, S., Ardizzoni, A., Franceschi, C., and Cossarizza, A. (1997). JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS letters 411, 77-82.
Saraste, A., and Pulkki, K. (2000). Morphologic and biochemical hallmarks of apoptosis. Cardiovascular research 45, 528-537.
Saunders, J.W., Jr. (1966). Death in embryonic systems. Science (New York, NY 154, 604-612.
Sharpe, J.C., Arnoult, D., and Youle, R.J. (2004). Control of mitochondrial permeability by Bcl-2 family members. Biochimica et biophysica acta 1644, 107-113.
Shimizu, S., Narita, M., and Tsujimoto, Y. (1999). Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature 399, 483-487.
Smiley, S.T., Reers, M., Mottola-Hartshorn, C., Lin, M., Chen, A., Smith, T.W., Steele, G.D., Jr., and Chen, L.B. (1991). Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proceedings of the National Academy of Sciences of the United States of America 88, 3671-3675.
Susin, S.A., Lorenzo, H.K., Zamzami, N., Marzo, I., Snow, B.E., Brothers, G.M., Mangion, J., Jacotot, E., Costantini, P., Loeffler, M., et al. (1999). Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397, 441-446.
Susin, S.A., Zamzami, N., Castedo, M., Hirsch, T., Marchetti, P., Macho, A., Daugas, E., Geuskens, M., and Kroemer, G. (1996). Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. The Journal of experimental medicine 184, 1331-1341.
Szegezdi, E., Logue, S.E., Gorman, A.M., and Samali, A. (2006). Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO reports 7, 880-885.
Vermes, I., Haanen, C., Steffens-Nakken, H., and Reutelingsperger, C. (1995). A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. Journal of immunological methods 184, 39-51.
Wyllie, A.H. (1980). Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555-556.
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.
Yoshimoto, Y., and Imoto, M. (2002). Induction of EGF-dependent apoptosis by vacuolar-type H(+)-ATPase inhibitors in A431 cells overexpressing the EGF receptor. Experimental cell research 279, 118-127.
Zoratti, M., and Szabo, I. (1995). The mitochondrial permeability transition. Biochimica et biophysica acta 1241, 139-176.
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