蛇床子素联合TRAIL诱导人卵巢癌细胞的凋亡及其相关机制

Osthole combined with TRAIL in inducing apoptosis of human ovarian cancer cells and its related mechanisms

目的探讨蛇床子素联合TRAIL诱导人卵巢癌细胞的凋亡及其相关机制。方法人卵巢癌细胞株SKOV-3与OVCAR-3各分为四组,分别为蛇床子素组(OS组),TRAIL组(TR组),蛇床子素联合TRAIL组(OT组),空白对照组(Blank组)。各组分别以MTT法检测细胞活力,流式细胞术检测细胞凋亡,ELISA检测caspase-3、caspase-9活化和细胞色素C表达相对水平,Western blot检测凋亡酶激活因子(Apaf-1)相对表达水平。免疫共沉淀法检测Apaf-1和caspase-9前体的相互作用,通过转染Apaf-1siRNA探究Apaf-1对卵巢癌细胞凋亡的影响。结果与空白对照组相比,另外三组卵巢癌细胞活性更低,凋亡率更高,其中蛇床子素联合TRAIL组卵巢癌活性抑制与凋亡促进相对于蛇床子素组和TRAIL组更为显著;TRAIL组中Apaf-1相对水平较空白对照组无明显差异,而蛇床子素联合TRAIL组中Apaf-1高表达;TRAIL组细胞色素C相对水平呈高表达,蛇床子素组与空白对照组细胞色素C、caspase-9前体蛋白表达相对水平一致,而联合TRAIL后,caspase-9前体蛋白表达相对水平显著提高。Apaf-1与caspase-9前体蛋白存在相互作用,Apaf-1通过此机制影响卵巢癌细胞凋亡。结论蛇床子素联合TRAIL可降低卵巢癌细胞活性,进而促使细胞凋亡,原因与其诱导卵巢癌细胞中细胞色素C的分泌及上调Apaf-1表达,促进caspase-9的活化表达有关,其具有较高卵巢癌治疗潜力,可为后续卵巢癌临床治疗提供重要参考。

Objective To investigate the apoptosis of human ovarian cancer cells induced by osthole combined with TRAIL and its related mechanism.Methods Human ovarian cancer cell lines SKOV-3and OVCAR-3were divided into four groups,namely osthole group(OS group),TRAIL group(TR group),osthole combined with TRAIL group(OT group),and blank control group(Blank group).Cell viability was detected by MTT method,cell apoptosis was detected by flow cytometry,caspase-3,caspase-9activation and cytochrome C expression were detected by ELISA,and apoptosis enzyme activation factor(Apaf-1)expression was detected by Western blot.The interaction between Apaf-1and caspase-9precursor was detected by immunoprecipitation.The effect of Apaf-1on apoptosis of ovarian cancer cells was investigated by transfection of Apaf-1siRNA.Results Compared with the blank control group,the activity of ovarian cancer cells in the other three groups was lower and the apoptosis rate was higher.The activity inhibition and apoptosis promotion of ovarian cancer in osthole combined with TRAIL group were more obvious than those in osthole group and TRAIL group.The relative level of Apaf-1in TRAIL group was not significantly different from that in the blank control group,while the expression of Apaf-1in osthole combined with TRAIL group was high.The relative expression level of cytochrome C in TRAIL group was high,and the relative expression levels of cytochrome C and caspase-9precursor protein in osthole group were consistent with those in blank control group.After combined with TRAIL,the relative expression level of caspase-9precursor protein was significantly increased.Apaf-1interacts with caspase-9precursor protein,and Apaf-1affects apoptosis of ovarian cancer cells through this mechanism.Conclusion Osthole combined with TRAIL in the treatment of ovarian cancer can reduce cell activity and promote cell apoptosis,which is related to inducing the secretion of cytochromic C in ovarian cancer cells,up-regulating the expression of APAF-1and promoting the activation expression of caspase-9.Osthole combined with TRAIL has high therapeutic potential for ovarian cancer and can be used as a follow-up ovarian cancer.