川楝素通过Fas/FasL信号通路诱导人卵巢癌细胞凋亡

Apoptosis of Ovarian Cancer Cells Induced by Toosendanin through Fas/FasL Signaling Pathway

目的探讨川楝素(TSN)对人卵巢癌细胞凋亡的影响及可能机制。方法对数生长期的CAOV-3和ES-2细胞,用不同浓度TSN(0、100、250、500、1000 nmol/L)处理,采用CCK-8法在不同时间点(24、48、72、96 h)检测细胞增殖抑制率。另将细胞分为阴性对照组、TSN组(500 nmol/L TSN)、TSN+z-DEVE-FMK(Caspase-3抑制剂)组(500 nmol/L TSN+20μmol/L z-DEVE-FMK)、TSN+z-IETD-FMK(Caspase-8抑制剂)组(500 nmol/L TSN+20μmol/L z-IETD-FMK)及紫杉醇(TAX)阳性对照组(5 mg/L TAX)。采用比色法检测Caspase-3、Caspase-8的活性,EdU法检测细胞增殖率,Western Blot法检测Fas、FasL蛋白的表达。结果 TSN能明显抑制CAOV-3和ES-2细胞的增殖,并呈剂量和时间依赖性。TSN孵育72 h后CAOV-3和ES-2细胞增殖抑制率分别为53.46%和60.68%。与阴性对照组比较, TSN组CAOV-3和ES-2细胞Caspase-3、Caspase-8活性显著升高(P<0.05)。与TSN组比较,TSN+z-DEVE-FMK组和TSN+z-IETD-FMK组CAOV-3和ES-2细胞Caspase-3、Caspase-8活性下降,导致TSN对两种细胞的增殖抑制率下降(P<0.05)。TSN作用后可使ES-2细胞中Fas、FasL蛋白表达增加(P<0.05),但该作用可被z-IETD-FMK逆转(P<0.05)。结论川楝素能诱导人卵巢癌细胞凋亡,其机制可能与Fas/FasL信号通路相关。

Objective To observe the effect of Toosendanin(TSN)on the apoptosis of human ovarian cancer cells and to reveal its potential mechanism.Methods CAOV-3 and ES-2 cells in the logarithmic phase were treated with TSN of different concentrations(0,100,250,500,1000 nmol/L).Subsequently,CCK-8 method was applied to detect cell proliferation inhibition rate at different time point(24 h,48 h,72 h,96 h).Cells were divided into negative control group,TSN group(500 nmol/L TSN),TSN+z-DEVE-FMK group(500 nmol/L TSN+20μmol/L z-DEVE-FMK),TSN+z-IETD-FMK group(500 nmol/L TSN+20μmol/L z-IETD-FMK)and taxol(TAX)positive control group(5 mg/L TAX).MTT colorimetric method was used to measure the activity of Caspase-3 and Caspase-8,EdU assay was performed to determine the cell proliferation rate,and Western Blot was used to measure the expression of Fas and FasL proteins.Results TSN could significantly inhibit proliferation of CAOV-3 and ES-2 cells in a dose-and time-dependent manner.The inhibition rates of proliferation of CAOV-3 and ES-2 cells in TSN group(72 h)were 53.46%and 60.68%,respectively.Compared with the negative control group,the activity of Caspase-3 and Caspase-8 in CAOV-3 and ES-2 cells increased significantly(P<0.05)after TSN treatment.However,the effect was reversed by the inhibitors of Caspase-3(z-DEVE-FMK)and Caspase-8(z-IETD-FMK),which resulted in the decrease of the inhibition rate of TSN on the proliferation of both cells(P<0.05).In addition,TSN could increase the expression of Fas and FasL proteins in ES-2 cells notably,which was reversed by z-IETD-FMK(P<0.05).Conclusion The apoptosis rate of human ovarian cancer cells could be induced by toosendanin,the mechanism might be related to Fas/FasL signal pathway.