Phenethyl Isothiocyanate (PEITC)經由活性氧化物(ROS)與粒腺體依存路徑誘導人類惡性黑色素瘤細胞A375.S2細胞凋亡

PEITC Induces Apoptosis in Human Malignant Melanoma A375.S2 Cells through Reactive Oxygen Species and Mitochondria-dependent Pathways

徐敏軒(Min-Hsuan Hsu)

指導教授 ： 黃素華 ； 鍾景光

亞洲大學/健康學院/生物科技學系碩士班/碩士(2009年)

摘要

根據流行病學顯示，多攝取十字花科植物有助於降低罹癌的風險。十字花科的植物種類有很多，常見的包含有高麗菜、大白菜、水田芥以及花椰菜等，皆含有異硫氰酸鹽(Isothiocyanates)的成分。過去的研究顯示，多攝食異硫氰酸鹽這類的天然的化合物，有助於預防癌症，同時異硫氰酸鹽也可用來做為治療癌症的藥物。 PEITC是最常見的異硫氰酸鹽類的化合物之一，過去有許多癌症治療的相關研究都曾指出，PEITC能夠誘導人類前列腺癌、血癌、肝癌等癌細胞凋亡並且抑制其生長。然而，目前對於PEITC誘導人類皮膚癌細胞A375.S2細胞凋亡的相關路徑以及機制尚未明確，於是我們在本研究探討PEITC能否誘導A375.S2細胞凋亡。首先在A375.S2經不同濃度的PEITC加藥過後，利用MTT試驗於不同時間測定細胞活性，可以發現加藥濃度以及加藥時間對於細胞致死率具有正相關性。另外利用流式細胞儀，分析經PEITC加藥過後的細胞週期分布、活性氧化物表現量、細胞內鈣離子濃度、粒腺體膜電位變化以及caspase-3的活性。並利用DAPI染色、彗星試驗以及DNA電泳證明DNA損傷。結果顯示，PEITC會造成DNA損傷、染色質凝集、使細胞週期停滯於G2/M期，並誘導細胞內活性氧化物生成、鈣離子釋放，造成粒腺體膜電位下降以及活化caspase-3，最終導致人類惡性黑色素瘤細胞A375.S2細胞凋亡。

關鍵字

異硫氰酸鹽、phenethyl isothiocyanate (PEITC)、活性氧化物、鈣離子、粒腺體膜電位、caspase-3、細胞凋亡

並列摘要

According to epidemiological studies, dietary intake of cruciferous vegetables may be protective against the risk of cancer. Isothiocyanates (ITCs) are found in cruciferous vegetables, including broccoli, cabbage, watercress and cauliflower. Recent studies had been shown that ITCs are classes of naturally occurring chemopreventive and possibly chemotherapeutic agents. Phenethyl isothiocyanate (PEITC) is one of the most common researched ITCs. The recent studies reported that PEITC can induce cancer cell growth inhibition or apoptosis of human prostate, leukemia and hepatoma cancer cells. However, the molecular mechanism and signaling pathway of PEITC-induced apoptosis in human malignant melanoma A375.S2 cells are still not clear. In this study, apoptosis induced by PEITC in A375.S2 cells was investigated. PEITC induced cell death in time- and dose-dependent manners was found in MTT assay. Cell cycle analysis, reactive oxygen species (ROS) generation, intercellular Ca2+ detection, the change of level mitochondria membrane potential (ΔΨm) and caspase-3 activity in A375.S2 cells were detected by flow cytometry after exposure to PEITC. Chromatin condensation and DNA damage were determined by DAPI staining, Comet assay and DNA gel electrophoresis. The results showed that PEITC caused chromatin condensation, DNA damage, cell cycle G2/M phase arrest, promoted ROS generation, stimulated Ca2+ release, loss of ΔΨm and activated caspase-3 activity lead to cell apoptosis in human malignant melanoma A375.S2 cells.

並列關鍵字

isothiocyanate (ITC), phenethyl isothiocyanate (PEITC), reactive oxygen species (ROS), Ca2+, mitochondria membrane potential (ΔΨm), caspase-3, apoptosis

參考文獻

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