魚藤素誘發人類非小細胞肺癌細胞株 NCI-H460 之凋亡機制

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Title:	魚藤素誘發人類非小細胞肺癌細胞株 NCI-H460 之凋亡機制 The mechanism of deguelin-induced apoptosis in human non-small cell lung cancer (NCI-H460)
Authors:	徐于絜
Contributors:	生物科技學系碩士班
Keywords:	魚藤素;人類非小細胞肺癌 deguelin;human non-small cell lung cancer
Date:	2011-06-29
Issue Date:	2011-10-17 16:21:33 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	魚藤素是從魚藤屬植物中所萃取出的一種天然化合物。在過去的文獻中已證實魚藤素可以透過細胞凋亡之機制誘導人類乳癌及血癌細胞死亡並且抑制癌細胞增生。然而，尚未有研究指出魚藤素對於人類非小細胞肺癌之抗癌相關機轉，因此，本研究的目的主要是在探討魚藤素對人類非小細胞肺癌 NCI-H460 細胞株之凋亡分子機制。首先，觀察不同魚藤素濃度對非小細胞肺癌 NCI-H460 細胞株型態的改變；利用流式細胞儀和 Trypan blue 染色分析存活率和細胞生長抑制情形；Annexin V, DAPI 染色, 彗星試驗和DNA 膠體電泳檢測細胞凋亡之現象；運用流式細胞儀檢測細胞內粒腺體膜電位及鈣離子濃度之改變, 西方墨點法, 凋亡蛋白酶活性檢測, AKT 激酶活性分析和免疫螢光染色法探討細胞內訊息傳遞路徑。根據研究結果顯示，魚藤素能有效抑制人類非小細胞肺癌NCI-H460 細胞株的增生，造成細胞死亡而降低細胞存活率；魚藤素可誘發細胞凋亡和 DNA 受損，致使磷脂絲胺酸蛋白外翻, DNA 斷裂和染色質濃縮形成凋亡小體；西方墨點和免疫螢光染色法證實，魚藤素能抑制 p-AKT 蛋白表現，促進 BAD 的活化，並且使之從細胞質轉移至粒腺體外膜上，進而影響促細胞凋亡相關蛋白 (Bid, Bax, Bak) 和抑制抗凋亡蛋白 (Bcl-2, Bcl-X) 的表現，致使粒腺體膜電位下降，釋出 AIF 和 cytochrome c，cytochrome c, Apaf-1 和caspase-9 結合形成凋亡體，活化caspase-3，最後誘發細胞凋亡；AIF 轉移至細胞核中造成DNA 受損。總觀以上結果，我們認為魚藤素會引起人類非小細胞肺癌NCI-H460 細胞株之粒腺體功能喪失並誘導細胞經由 AKT 和Bad 相關粒腺體內在路徑走向細胞凋亡。 Deguelin, is a natural product, isolated from Derris trifoliata Lour. It was reported that deguelin inhibited cell proliferation and caused cell death in human leukemia and breast cancer cells through the induction of apoptosis. Therefore, we investigate deguelin whether or not affect human lung cancer cells in vitro . First, we observed the change of cell morphology and percentage of cell viability after various doses of deguelin treatment, flow cytometric analysis and trypan blue staining were used to detect the cell viability and the cell growth inhibition of NCI-H460 cells. Second, we used annexin V, DAPI staining, comet assay and DNA gel electrophoresis to determined the phenomenon of apoptosis. Finally, we investigated signal transduction by examining the intracellular change of mitochondria membrane potential (Δψm) and Ca2+ levels, western blotting, caspase activity, AKT kinase assay and immunofluoresence were used in NCI-H460 cell line. Based on these results, we found that deguelin greatly suppressed cell proliferation and decreased cell viability in time- and dose-dependent manners. Deguelin induced cell apoptosis and DNA damage including chromatin condensation, the formation of apoptotic bodies, and translocation of phosphatidylserine (PS) of the plasma membrane. Western blotting and immunofluoresence proved that deguelin inhibited p-AKT protein expressions, resulting in activation of Bad and then translocate from cytosol to mitochondria where it binds to Bcl-2 and Bcl-X, followed by promoted the influence of Bcl-2 family proteins. There is a decrease in mitochondria membrane potential (Δψm), causing release of cytochrome c and AIF. Cytochrome c combined with apaf-1 and caspase-9 to an apoptosome , activated caspase-3, leading to apoptosis. Then, AIF translocated into nuclear causing DNA damage. Overall, we suggest that the major mechanism of deguelin-induced apoptosis in NCI-H460 is through AKT and Bad-releated signaling pathway.
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