维生素E琥珀酸酯诱导人胃腺癌细胞凋亡途径
Fas signaling transduction pathway of vitamin E succinate-induced apoptosis in human gastric adenocarcinoma cell
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摘要:目的 探讨维生素E琥珀酸酯(VES)诱导人胃腺癌SGC-7901细胞凋亡的死亡受体(Fas)信号转导途径.方法 人胃腺癌SGC-7901细胞经不同剂量VES(5, 10, 20μg/ml)处理, 同时做琥珀酸、维生素E和空白对照, 采用DAPI(4, 6-贰脒基-2-苯基吲哚)荧光染色法观察细胞凋亡情况, 用WesternBlot法检测Fas、带有死亡结构域的Fas相关蛋白(FADD)和天冬氨酸特异性半胱氨酸蛋白酶(caspase-8)蛋白表达情况, Fas和FADD反义寡聚核苷分别转染SGC-7901细胞后, 用荧光法检测caspase-8活性.结果 经VES处理后的细胞DAPI染色可见凋亡的形态学改变, 20μg/mlVES处理48h后的细胞凋亡率为89.6%;VES处理48h后Fas、FADD和caspase-8蛋白表达明显增加, 且呈剂量-效应关系; 阻断Fas可明显抑制FADD蛋白表达, Fas和FADD反义寡聚核苷转染细胞后caspase-8活性明显降低(P < 0.01), 其中阻断Fas的效果高于阻断FADD.结论 维生素E琥珀酸酯诱导人胃腺癌SGC-7901细胞凋亡过程中启动了Fas信号转导途径, VES启动Fas后, FADD将Fas和caspase-8联接起来, 活化caspases级联反应, 从而构成Fas/FADD/caspase-8的凋亡信号途径.Abstract:Objective To study the fas signaling transduction pathway of vitamin E succinate(VES)-induced apoptosis in human gastric adenocarcinoma cells.Methods Human gastric adeno carcinoma cells were treated with VES at 5, 10, 20 μg/ml, succinic acid, vitamin E and untreated control.Apoptotic morphology was observed by DAPI staining.Western Blot analysis was applied to measure the expression of fas, FADD and caspase-8 proteins.After the cells were transiently transfected with fas and FADD antisense oligonucleotides, respectively, caspase-8 activity was determined by flurometric method.Results The morphologically apoptotic changes were observed after VES treatment by DAPI staining.89.6% apoptosis occurred after 48h of 20 μg/ml VES treatment.The protein levels of fas, FADD and caspase-8 were evidently increased in a dose-dependent manner after 48h of VES treatment.The blockage of fas by transfection with fas antisense oligonucleotides obviously inhibited the expression of FADD protein.After SGC-7901 cells were transfected with fas and FADD antisense oligonucleotides, caspase-8 activity was obviously decreased(P < 0.01), whereas fas blocked more than FADD.Conclusion VES-induced apoptosis in human gastric adenocarcinoma SGC-7901 cells involved fas signaling transduction pathway including the interaction of fas, FADD and caspase-8.That was fas/FA DD/caspase-8 signaling transduction pathway.
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