臺灣博碩士論文加值系統

本篇研究利用蘋婆果莢乙酸乙酯精萃物(Ethyl acetate extract shell of Pod Sterculia Nobililis，簡稱為EPSN)針對人類三陰性乳癌細胞株BT-20與MDA-MB-231進行細胞毒殺實驗。藉由細胞存活率測試(MTT assay)，經EPSN處理48小時的IC50，BT-20為40-50 μg/mL，和前人研究結果相類似(30-40 μg/mL)。由前人研究得知BT-20經EPSN處理後可能是藉由細胞凋亡誘導因子(Apoptosis-inducing factor，AIF)經凋亡蛋白酶非依賴型路徑(caspase-independent pathway)來調控細胞凋亡。
本篇研究調整EPSN處理細胞的時間並於收取細胞蛋白時分離細胞質與粒線體兩部分，經西方墨點法分析發現BT-20與MDA-MB-231皆於加入EPSN 2小時後，AIF在細胞質中有過度表現(overexpression)的情形，進一步利用Real-time PCR觀察BT-20經EPSN處理3小時後的AIF mRNA表現量，發現AIF mRNA有明顯上升趨勢，由此可得知蘋婆果莢乙酸乙酯精萃物在短時間內即具有誘導三陰性乳癌細胞凋亡的能力。

In previous studies at our laboratory, we found that the Ethyl acetate extract, isolated from shell of pod of Sterculia nobililis (EPSN), can induce the apoptosis of human triple-negative breast cancer cell line BT-20 through the regulation of apoptosis-inducing factor (AIF) in caspase-independent pathway. In this study, we used EPSN to treat BT-20 cells for cytotoxicity test (48 Hr IC50：40-50 μg/mL) and to investigate how does AIF regulate the mechanism of apoptosis in cancer cells.
The results of Western blotting analysis revealed that, in both BT-20 and MDA-MB-231 cell lines, AIF was started to overexpress in cytosol compartment after 2 hours treatment of EPSN. We further used Real-time PCR to observe the mRNA expression level AIF in BT-20 after the treatment of EPSN. The data showed that there was indeed a signicant increment expressed in mRNA level of AIF after 3 hours treatment of EPSN. These results demonstrated that EPSN can induce apoptosis at the early stage in triple-negative breast cancer cells with the mRNA and protein overexpressions of AIF leading to the cell death through the caspase-independent pathway.

誌謝.......................................................I
中文摘要..................................................III
Abstract..................................................IV
目錄.......................................................V
圖表目錄.................................................VIII
第一章 緒論.................................................1
一、癌症....................................................1
(一) 乳癌簡介與分期..........................................2
(二) 乳癌成因...............................................3
(三) 病理分類...............................................4
(四) 三陰性乳癌(Triple negative breast cancer，TNBC)及其治療策略.........................................................5
二、蘋婆....................................................7
(一) 生理介紹...............................................7
(二) 相關文獻探討............................................8
三、細胞週期(cell cycle)....................................10
(一) 定義及分期.............................................10
(二) 細胞週期之調控..........................................11
四、細胞凋亡(apoptosis).....................................13
(一) 凋亡蛋白酶依賴型(caspase-dependent)途徑 ..................13
1. 外源性路徑(Extrinsic pathway)...........................14
2. 內源性路徑(Intrinsic pathway)...........................15
(二) 凋亡蛋白酶非依賴型(caspase-independent)途徑..............16
五、細胞凋亡誘導因子(Apoptosis-inducing factor，AIF)..........17
(一) AIF簡介及其異構蛋白(isoform)............................17
(二) AIF其他生理功能........................................18
第二章 研究動機.............................................19
一、本實驗室蘋婆研究文獻回顧...................................19
二、研究動機................................................23
第三章 材料與方法............................................24
一、實驗材料................................................24
(一) 萃取物材料.............................................24
(二) 細胞株................................................24
(三) 藥品與試劑.............................................24
(四) 抗體..................................................26
1. 一級抗體(primary antibody)..............................26
2. 二級抗體(secondary antibody)............................26
(五) 主要儀器與耗材..........................................26
二、實驗用溶液配方...........................................29
三、實驗方法................................................32
1. 蘋婆果莢乙酸乙酯(Ethyl acetate)萃取.......................32
2. 細胞培養與繼代...........................................32
3. 細胞冷凍保存.............................................33
4. 冷凍細胞活化.............................................33
5. 細胞存活率測試(MTT assay)................................33
6. 蛋白質萃取..............................................34
A. 全細胞蛋白質萃取.........................................34
B. 細胞質與粒線體蛋白質之分離.................................35
7. 蛋白質濃度測定(定量)與蛋白質樣品製備.........................36
8. 蛋白質電泳分析(SDS-PAGE).................................37
9. 西方墨點法(Western Blotting)............................37
10. 細胞RNA萃取(RNA extraction)............................39
11. RNA反轉錄(reverse transcription).......................40
12. 即時定量聚合酶鏈鎖反應(Real-time Quantitative Polymerase Chain Reaction，簡稱Real-time PCR或qPCR)...................41
第四章 實驗結果.............................................44
一、驗證蘋婆果莢乙酸乙酯精萃物對三陰性乳癌細胞株BT-20的毒殺能力.....44
二、探討蘋婆果莢乙酸乙酯精萃物對三陰性乳癌細胞株BT-20的凋亡蛋白酶依賴型(caspase-dependent)細胞凋亡路徑.............................46
三、探討蘋婆果莢乙酸乙酯精萃物對三陰性乳癌細胞株的凋亡蛋白酶非依賴型(caspase-independent)細胞凋亡路徑...........................49
四、探討蘋婆果莢乙酸乙酯精萃物對三陰性乳癌細胞株BT-20所誘導的AIF過度表現(overexpression)是否在mRNA層級(level)即產生影響..............55
第五章 討論與結論............................................58
參考文獻...................................................62



圖表目錄
附圖 1、台灣地區民國99及100年國人十大死因統計圖 ...................1
附圖 2、台灣地區民國99及100年十大癌症死亡率統計圖.................2
附圖 3、蘋婆花序.............................................7
附圖 4、蘋婆果實.............................................8
附圖 5、細胞週期相關調控因子之綜觀..............................10
附圖 6、不同Cyclin在細胞週期中表現量變化圖......................12
附圖 7、細胞凋亡的外源性及內源性路徑............................14
附圖 8、蘋婆果莢乙酸乙酯精萃物(EPSN)對三陰性乳癌細胞株MDA-MB-231及BT-20之毒性測試 19
附圖 9、蘋婆果莢乙酸乙酯精萃物對三陰性乳癌細胞株BT-20之細胞週期影響........................................................20
附圖 10、蘋婆果莢乙酸乙酯精萃物對三陰性乳癌細胞株BT-20作用24小時，細胞週期調控蛋白(Cyclin E及Cyclin D1)之影響......................21
附圖 11、蘋婆果莢乙酸乙酯精萃物誘導三陰性乳癌細胞株BT-20死亡方式之偵測........................................................22
圖 1、蘋婆果莢乙酸乙酯精萃物對三陰性乳癌細胞株BT-20之毒性測試.......45
圖 2、蘋婆果莢乙酸乙酯精萃物對三陰性乳癌細胞株BT-20在不同時間點條件下凋亡蛋白酶家族(caspase)的影響..................................47
圖 3、蘋婆果莢乙酸乙酯精萃物對三陰性乳癌細胞株BT-20在不同時間點條件下DNA修補蛋白(PARP)的影響.....................................48
圖 4、蘋婆果莢乙酸乙酯精萃物對三陰性乳癌細胞株BT-20在不同濃度及不同時間點條件下，細胞凋亡誘導因子(AIF)於總量蛋白中的影響.................50
圖 5、蘋婆果莢乙酸乙酯精萃物對三陰性乳癌細胞株BT-20在不同濃度下處理24小時之細胞凋亡誘導因子(AIF)於細胞質蛋白及粒線體蛋白質的影響..........51
圖 6、蘋婆果莢乙酸乙酯精萃物對三陰性乳癌細胞株BT-20以含50 μg/mL劑量EPSN的培養液處理細胞至不同時間點之細胞凋亡誘導因子(AIF)於細胞質蛋白及粒線體蛋白質的影響.............................................52
圖 7、蘋婆果莢乙酸乙酯精萃物對三陰性乳癌細胞株BT-20以含50 μg/mL劑量EPSN的培養液處理細胞至不同時間點之細胞凋亡誘導因子(AIF)於細胞質蛋白及粒線體蛋白質的影響.............................................53
圖 8、蘋婆果莢乙酸乙酯精萃物對三陰性乳癌細胞株MDA-MB-231以含40 μg/mL劑量EPSN的培養液處理細胞至不同時間點之細胞凋亡誘導因子(AIF)於細胞質蛋白及粒線體蛋白質的影響.........................................53
圖 9、蘋婆果莢乙酸乙酯精萃物對三陰性乳癌細胞株BT-20以含50 μg/mL劑量EPSN的培養液處理細胞至不同時間點之細胞凋亡誘導因子(AIF) mRNA level的影響......................................................56
圖 10、蘋婆果莢乙酸乙酯精萃物對三陰性乳癌細胞株BT-20以含50 μg/mL劑量EPSN的培養液處理細胞至不同時間點之細胞凋亡誘導因子(AIF) mRNA level的影響......................................................57
表 1、乳癌亞型之受體表現.......................................5
表 2、Real-time PCR上機專用的96 well部分區域示意圖.............43
附錄 1、Real-time PCR實驗所使用的Primer序列...................61

臺灣行政院衛生署，2012年死亡人數及十大死因統計資料

陳文恆 (2005) AIF基因表現與非小細胞肺癌抗藥性之研究，碩士論文，國立中興大學生物醫學研究所

黃智洋 (2007) AIF核內運輸與hHR23A之研究，碩士論文，國立中興大學生物醫學研究所

林如璠 (2008) PTPγ在台灣女性乳癌的高度表現，碩士論文，國立中興大學生物醫學研究所

夏鵬飛 (2009) 掌葉蘋婆的化學成分及藥理活性研究，博士論文，北京協和醫學院，中國醫學科學院

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. The EMBO journal 22, 4385-4399.

Arpino, G., Bardou, V.J., Clark, G.M., and Elledge, R.M. (2004). Infiltrating lobular carcinoma of the breast: tumor characteristics and clinical outcome. Breast Cancer Res 6, R149-R156.

Ashkenazi, A. (2002). Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nature Reviews Cancer 2, 420-430.

Bras, M., Queenan, B., and Susin, S. (2005). Programmed cell death via mitochondria: different modes of dying. Biochemistry (Moscow) 70, 231-239.

Brennan, M., WILCKEN, N., FRENCH, J., UNG, O., and BOYAGES, J. (2005). Management of early breast cancer: the current approach. Australian family physician 34, 755-760.

Bucher, N., and Britten, C. (2008). G2 checkpoint abrogation and checkpoint kinase-1 targeting in the treatment of cancer. British journal of cancer 98, 523-528.

Cande, C., Cecconi, F., Dessen, P., and Kroemer, G. (2002). Apoptosis-inducing factor (AIF): key to the conserved caspase-independent pathways of cell death? Journal of cell science 115, 4727-4734.

Cande, C., Vahsen, N., Kouranti, I., Schmitt, E., Daugas, E., Spahr, C., Luban, J., Kroemer, R.T., Giordanetto, F., and Garrido, C. (2004). AIF and cyclophilin A cooperate in apoptosis-associated chromatinolysis. Oncogene 23, 1514-1521.

Chao, W., Shen, Y., Li, L., Zhao, H., Meiler, S.E., Cook, S.A., and Rosenzweig, A. (2005). Fas-associated death-domain protein inhibits TNF-α mediated NF-κB activation in cardiomyocytes. American Journal of Physiology-Heart and Circulatory Physiology 289, H2073-H2080.

Csikasz-Nagy, A. (2009). Computational systems biology of the cell cycle. Briefings in bioinformatics 10, 424-434.

Cunningham, D., Humblet, Y., Siena, S., Khayat, D., Bleiberg, H., Santoro, A., Bets, D., Mueser, M., Harstrick, A., and Verslype, C. (2004). Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. New England Journal of Medicine 351, 337-345.

Delettre, C., Yuste, V.J., Moubarak, R.S., Bras, M., Lesbordes-Brion, J.-C., Petres, S., Bellalou, J., and Susin, S.A. (2006a). AIFsh, a novel apoptosis-inducing factor (AIF) pro-apoptotic isoform with potential pathological relevance in human cancer. Journal of Biological Chemistry 281, 6413-6427.

Delettre, C., Yuste, V.J., Moubarak, R.S., Bras, M., Robert, N., and Susin, S.A. (2006b). Identification and characterization of AIFsh2, a mitochondrial apoptosis-inducing factor (AIF) isoform with NADH oxidase activity. Journal of Biological Chemistry 281, 18507-18518.

Dent, R., Trudeau, M., Pritchard, K.I., Hanna, W.M., Kahn, H.K., Sawka, C.A., Lickley, L.A., Rawlinson, E., Sun, P., and Narod, S.A. (2007). Triple-negative breast cancer: clinical features and patterns of recurrence. Clinical Cancer Research 13, 4429-4434.

Fackenthal, J.D., and Olopade, O.I. (2007). Breast cancer risk associated with BRCA1 and BRCA2 in diverse populations. Nature Reviews Cancer 7, 937-948.

Feng, X., Zhou, Y., Proctor, A.M., Hopkins, M.M., Liu, M., and Koh, D.W. (2012). Silencing of Apoptosis-Inducing factor and poly (ADP-ribose) glycohydrolase reveals novel roles in breast cancer cell death after chemotherapy. Mol Cancer 11, 48.

Green, D.R., and Reed, J.C. (1998). Mitochondria and apoptosis. Science-AAAS-Weekly Paper Edition 281, 1309-1311.

Hartwell, L.H., and Kastan, M.B. (1994). Cell cycle control and cancer. Science 266, 1821-1828.

Israels, E., and Israels, L. (2000). The cell cycle. The oncologist 5, 510-513.

Kohler, C., Orrenius, S., and Zhivotovsky, B. (2002). Evaluation of caspase activity in apoptotic cells. Journal of immunological methods 265, 97-110.

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.

Kong, N., Fotouhi, N., Wovkulich, P.M., and Roberts, J. (2003). Cell cycle inhibitors for the treatment of cancer. Drugs of the Future 28, 881-896.


LOEFFLER, M., DAUGAS, E., SUSIN, S.A., ZAMZAMI, N., METIVIER, D., NIEMINEN, A.-L., BROTHERS, G., PENNINGER, J.M., and KROEMER, G. (2001). Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor. The FASEB Journal 15, 758-767.

Lukas, J., Bartkova, J., and Bartek, J. (1996). Convergence of mitogenic signalling cascades from diverse classes of receptors at the cyclin D-cyclin-dependent kinase-pRb-controlled G1 checkpoint. Molecular and cellular biology 16, 6917-6925.

Lv, B., Shi, T., Wang, X., Song, Q., Zhang, Y., Shen, Y., Ma, D., and Lou, Y. (2006). Overexpression of the novel human gene, nuclear apoptosis-inducing factor 1, induces apoptosis. The international journal of biochemistry & cell biology 38, 671-683.

Malumbres, M., and Barbacid, M. (2009). Cell cycle, CDKs and cancer: a changing paradigm. Nature Reviews Cancer 9, 153-166.

Martin, L., Price, A., Kaiser, A., Shaikh, A., and Liu, Z. (2000). Mechanisms for neuronal degeneration in amyotrophic lateral sclerosis and in models of motor neuron death (Review). International journal of molecular medicine 5, 3-16.

Mohapatra, S., Chu, B., Zhao, X., and Pledger, W. (2005). Accumulation of p53 and reductions in XIAP abundance promote the apoptosis of prostate cancer cells. Cancer research 65, 7717-7723.

MURAKAMI, H., and NURSE, P. (2000). DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts. Biochem J 349, 1-12.

Murray, A.W., and Kirschner, M.W. (1989). Cyclin synthesis drives the early embryonic cell cycle. Nature 339, 275-280.

Musacchio, A., and Salmon, E.D. (2007). The spindle-assembly checkpoint in space and time. Nature reviews Molecular cell biology 8, 379-393.


Ohiro, Y., Garkavtsev, I., Kobayashi, S., Sreekumar, K.R., Nantz, R., Higashikubo, B.T., Duffy, S.L., Higashikubo, R., Usheva, A., and Gius, D. (2002). A novel p53-inducible apoptogenic gene, PRG3, encodes a homologue of the apoptosis-inducing factor (AIF). FEBS letters 524, 163-171.

Osborne, C., Wilson, P., and Tripathy, D. (2004). Oncogenes and tumor suppressor genes in breast cancer: potential diagnostic and therapeutic applications. The oncologist 9, 361-377.

Otera, H., Ohsakaya, S., Nagaura, Z.-I., Ishihara, N., and Mihara, K. (2005). Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space. The EMBO journal 24, 1375-1386.

Pao, W., Miller, V., Zakowski, M., Doherty, J., Politi, K., Sarkaria, I., Singh, B., Heelan, R., Rusch, V., and Fulton, L. (2004). EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib. Proceedings of the National Academy of Sciences of the United States of America 101, 13306-13311.

Peitsh, M.C., Muuller, C., and Tschopp, J. (1993). DNA fragmentation during apoptosis is caused by frequent single-strand cuts. Nucleic acids research 21, 4206-4209.

Polster, B.M., Basanez, G., Etxebarria, A., Hardwick, J.M., and Nicholls, D.G. (2005). Calpain I induces cleavage and release of apoptosis-inducing factor from isolated mitochondria. Journal of Biological Chemistry 280, 6447-6454.

Rakha, E.A., Reis-Filho, J.S., and Ellis, I.O. (2008). Basal-like breast cancer: a critical review. Journal of Clinical Oncology 26, 2568-2581.

Ravagnan, L., Gurbuxani, S., Susin, S.A., Maisse, C., Daugas, E., Zamzami, N., Mak, T., Jaattela, M., Penninger, J.M., and Garrido, C. (2001). Heat-shock protein 70 antagonizes apoptosis-inducing factor. Nature Cell Biology 3, 839-843.

Rebecca, S., and Carol, B. (2000). Concepts in oncology therapeutics, seconds edition: 2-18.

Sorlie, T., Perou, C.M., Tibshirani, R., Aas, T., Geisler, S., Johnsen, H., Hastie, T., Eisen, M.B., van de Rijn, M., and Jeffrey, S.S. (2001). Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proceedings of the National Academy of Sciences 98, 10869-10874.

Sorlie, T., Tibshirani, R., Parker, J., Hastie, T., Marron, J., Nobel, A., Deng, S., Johnsen, H., Pesich, R., and Geisler, S. (2003). Repeated observation of breast tumor subtypes in independent gene expression data sets. Proceedings of the National Academy of Sciences 100, 8418-8423.

Seidman, J.D., Schnaper, L.A., and Aisner, S.C. (1995). Relationship of the size of the invasive component of the primary breast carcinoma to axillary lymph node metastasis. Cancer 75, 65-71.

Sneige, N., Zachariah, S., Fanning, T., Dekmezian, R., and Ordonez, N. (1989). Fine-needle aspiration cytology of metastatic neoplasms in the breast. American journal of clinical pathology 92, 27.

Susin, S.A., Lorenzo, H.K., Zamzami, N., Marzo, I., Snow, B.E., Brothers, G.M., Mangion, J., Jacotot, E., Costantini, P., and Loeffler, M. (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.

Tutt, A., Robson, M., Garber, J.E., Domchek, S.M., Audeh, M.W., Weitzel, J.N., Friedlander, M., Arun, B., Loman, N., and Schmutzler, R.K. (2010). Oral poly (ADP-ribose) polymerase inhibitor olaparib in patients with< i> BRCA1 or< i> BRCA2 mutations and advanced breast cancer: a proof-of-concept trial. The Lancet 376, 235-244.

Utah, A. (1989). Relation of tumor size, lymph node status, and survival in 24,740 breast cancer cases. Cancer 63, 181-187.

Vahsen, N., Cande, C., Dupaigne, P., Giordanetto, F., Kroemer, R., Herker, E., Scholz, S., Modjtahedi, N., Madeo, F., and Le Cam, E. (2005). Physical interaction of apoptosis-inducing factor with DNA and RNA. Oncogene 25, 1763-1774.

Wu, M., Xu, L.-G., Li, X., Zhai, Z., and Shu, H.-B. (2002). AMID, an apoptosis-inducing factor-homologous mitochondrion-associated protein, induces caspase-independent apoptosis. Journal of Biological Chemistry 277, 25617-25623.

Xie, Q., Lin, T., Zhang, Y., Zheng, J., and Bonanno, J.A. (2005). Molecular cloning and characterization of a human AIF-like gene with ability to induce apoptosis. Journal of Biological Chemistry 280, 19673-19681.