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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 生物化學研究所 > 依資料類型分類 > 碩博士論文 >  蘋婆豆莢萃取物誘導三陰性乳癌細胞凋亡的分子機制

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152337

標題: 蘋婆豆莢萃取物誘導三陰性乳癌細胞凋亡的分子機制
The extracts of Sterculia Nobililis induced G1 phase arrest and apoptosis in triple-negative breast cancer cells
作者: 葉于弘
Ye, Yu-Hung
Contributors: 高振益
生物化學研究所
關鍵字: 蘋婆;三陰性乳癌
Sterculia Nobililis;triple-negative breast cancer cells
日期: 2012
Issue Date: 2013-11-07 13:21:23 (UTC+8)
Publisher: 生物化學研究所
摘要: 三陰性乳癌(Triple Negative Breast Cancer,TNBC)約佔乳癌病患的10-20%,此類型癌細胞的動情激素接受體(ER)、黃體激素接受體(PR)及Her-2/neu接受體很少,因此不能使用荷爾蒙的治療方式且癒後狀況差。蘋婆(Sterculia nobililis)又稱鳳眼果,是梧桐科常綠喬木,原生於台灣及中國大陸南部,其種子可以食用。藉由細胞活性測試發現蘋婆豆莢殼萃取物(EPSN)具有毒殺癌細胞的能力,而在針對不同類型的癌細胞中觀察到BT-20 (IC50:30-40 μg/mL)和MBA-MD-231 (IC50: 20-30 μg/mL)三陰性乳癌的細胞株效果最為顯著。在BT-20加入蘋婆豆莢殼乙醯醋酸層提取物(EPSN)之後可明顯地觀察到細胞生長週期被迫停留在G1期。進一步利用雙染(Double stain)實驗得到細胞在EPSN處理24小時後有細胞凋亡(apoptosis)訊號的增加。
藉由西方點墨法釐清細胞週期停留與細胞凋亡路徑,當以EPSN處理細胞之後可觀察到調控G1期的調控蛋白Cyclin E及Cyclin D1會被降解,同時p53和PARP也隨著濃度增加而被啟動,推估蘋婆豆莢提取物會導致DNA的受損引發p53被表達,而p53會活化下游的p21,p21進一步導致cyclin E的降解,致細胞週期停滯。另一方面,雙染的實驗結果顯示細胞在EPSN使用40μg/ml的濃度處理時有細胞凋亡的早期訊號,因此以40 μg/ml EPSN處理細胞在36、48小時可以觀察到caspase-8、3有被剪切的片段出現,但與MTT的實驗結果在24小時就有著細胞死亡的現象相比較,可能有其他的凋亡機制,推論EPSN可能藉由活化細胞凋亡因子AIF的表達,而進入核內將DNA片斷化,引發細胞快速凋亡。實驗結果也顯示當以40 μg/mL EPSN處理細胞時,將細胞依6、12、18、24小時處理之後,發現在12小時處理後AIF有被明顯的表達,因此推論EPSN在早期利用AIF來引發BT-20細胞的凋亡。
Triple negative breast cancer (TNBC), defined as lacking of estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor-2 (HER-2), has been recognized as a risk of disease recurrence and poorer prognosis. Therefore, it does not use hormone drugs for treatment. Sterculia nobililis belongs to Sterculiaceae evergreen trees and a native in Taiwan. The fruit of Sterculia nobililis is eadible and related no relative studied to showany anticancer activity. However we found that the shell of pod of Sterculia nobililis (EPSN) has effect to inhibit the growth of TNBC cell lines with MTT assay [BT-20 (IC50:30-40μg/mL) and MBA-MD-231 (IC50: 20-30μg/mL)]. EPSN can arrest cancer cell growth at G1 phase in cell cycle test. Double stain data showed that the apoptosis signal was expressed in BT-20 cell after treatment with 40 μg/mL of EPSN for 48 hours. The cyclin E and Cyclin D1 were decreased after BT-20 been treated with EPSN. We also observed that p53 expression were increased. On the other hand, apoptosis signal protein caspase-3,8 and PARP had been cleaved when cell treated with 40μg/ml of EPSN at 36 hour. However, the apoptosis of BT-20 cell had appeared less than 24hrs after EPSN treatment. Apoptosis induce factor(AIF), indepent of caspase-related apoptosis pathway, can enter into nuclear for DNA fragmentation and induce cell apoptosis with time intervals at 12, 18 and 24 hours for BT-20 cell treatment with 40μg/mL of EPSN. The data showed that the concentration of AIF was overexpressed with western blotting. AIF might be the major factor to cause the apoptosis of BT-20 at early stage when cancer cells were treated with EPSN.
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