金雀异黄素调控Egr1/PTEN信号通路诱导卵巢癌SKOV3细胞凋亡的实验研究

Genistein induce apoptosis via Egr1/PTEN pathway in human ovarian cancer cell line SKOV3

目的通过金雀异黄素(genistein)诱导人卵巢癌细胞SKOV3细胞的凋亡,探讨即刻早期反应基因(Egr1)/细胞骨架蛋白同源在10号染色体有缺失的磷酸酶(PTEN)信号通路在金雀异黄素诱导SKOV3细胞凋亡过程中的作用。方法应用磺酰罗丹明B(SRB)染色法测定经金雀异黄素处理后的SKOV3卵巢癌细胞生长抑制率,组蛋白DNA酶联免疫吸附测定(ELISA)法检测细胞凋亡,采用游离硝基苯胺(p NA)法检测细胞内半胱氨酸天冬氨酸特异性蛋白酶3(Caspase-3)的蛋白含量,实时荧光定量PCR(Real-time PCR)测定细胞内Bcl-2相关X蛋白基因(bax)和B淋巴细胞瘤-2基因(bcl-2)的基因表达变化,蛋白质印迹法(Western blotting)分析Egr1、PTEN、蛋白激酶B(AKT)、磷酸化蛋白激酶B(p-Akt)蛋白的表达变化。结果金雀异黄素显著抑制卵巢癌细胞SKOV3的增殖(P<0.05),并呈浓度依赖性。金雀异黄素处理24 h后,处理浓度高于40μmol/L时,与对照组相比,调亡指数、Caspase-3活性、bax的相对表达及细胞中Egr1蛋白和PTEN蛋白的表达量均显著增加(P<0.05),Bcl-2的相对表达量及AKT蛋白的磷酸化水平p-Akt则显著降低(P<0.05)。当同时给予50 nmol/L PTEN抑制剂Bpv后,p-Akt表达明显增加,细胞凋亡程度受到明显抑制。结论金雀异黄素可能通过上调Egr1/PTEN信号通路的表达抑制PI3K/Akt信号通路的激活诱导SKOV3细胞凋亡,进一步影响了调亡相关基因Bcl-2、Bax及Caspase-3的表达。

Objective To investigate the effect of genistein on apoptosis in ovarian cancer cell SKOV3, and explore the role of Egr1/PTEN signaling pathway in this process. Method Cell growth inhibition was determined by SRB assay, apoptosis determined by histone DNA ELISA, p NA assay was applied to determine intracellular Caspase-3 protein activity, the m RNA levels of bax and bcl-2 were determined by real-time PCR, the expression of Egr1, PTEN, AKT and p-Akt were detected by Western blotting analysis. Result Genistein induced apoptosis of SKOV3 in a dose-and time-dependent manner.It increased the expression of Egr1 and suppressed the phosphorylation levels of AKT, meanwhile giving the PTEN inhibitor Bpv（phen） partially reversed the genistein-induced apoptosis in SKOV3. Conclusion Genistein can induce the apoptosis of SKOV3 cell, which might be mediated by inhibiting the activation of PI3K/Akt signaling pathway via upregulation the expression of Egr1/PTEN, and further affect the apoptosis-related genes Bcl-2, Bax and Caspase-3 expression.