大黄素通过非半胱氨酰天冬氨酸特异性蛋白酶依赖途径诱导前列腺癌细胞凋亡的研究

Emodin induces apoptosis of prostate cancer cells via cysteinyl aspartate-specific protease-independent pathway

目的 观察大黄素对前列腺癌细胞凋亡及凋亡诱导因子（AIF）和核酸内切酶G（Endo G）表达的影响,探讨大黄素的非半胱氨酰天冬氨酸特异性蛋白酶（Caspase）依赖细胞凋亡途径.方法 实验分空白对照组、大黄素组、Caspase抑制剂干预组、大黄素＋Caspase抑制剂组,噻唑蓝（MTT）法筛选大黄素的干预条件;流式细胞术检测干预后前列腺癌细胞凋亡率;实时定量聚合酶链反应（Real-time PCR）及Western blot法检测AIF、Endo G mRNA及蛋白表达.结果 EM浓度在20 μmol/L以上能显著抑制前列腺癌细胞株的增殖,其作用呈剂量与时间依赖性.当EM浓度为160 μmol/L时,48、96 h细胞抑制率分别为（59.76 ±3.90）％和（71.77±3.50）％.EM能显著增加前列腺癌细胞凋亡率,与对照组相比差异有统计学意义[（22.54±2.32）％比（4.54±1.26）％,P＜0.05];与EM单独干预比较,EM＋ Caspase-3抑制剂干预后凋亡率虽然有所下降,但仍显著高于空白对照组[（15.65±1.84）％比（4.54±1.26）％,P＜0.05].Real-time PCR及Western blot检测结果显示,与空白对照组比较,EM及EM＋ Caspase抑制剂均可显著增加AIF、EndoG mRNA及蛋白表达（P＜0.05）.结论 大黄素可上调前列腺癌细胞AIF、Endo G表达,通过非Caspase依赖通路而发挥促凋亡的作用.

Objective To investigate the effects of emodin on apoptosis and the expression of apoptosis inducing factor （AIF） and endonuclease G （Endo G） in prostate cancer cells and the mechanism.Methods The prostate cancer cells cultured in vitro were divided into 4 groups：blank control group,emodin group,cysteinyl aspartate-specific protease （Caspase） inhibitor group and emodin combined with caspase inhibitor group.Methyl thiazol tetrazolium （MTT） colorimetric assay was used to measure the cell viability.The apoptosis of the cells was measured by flow cytometry.The expression of AIF and Endo G mRNA and protents was detected by real-time quantitative polymerase chain reaction （Real-time PCR） and Western blotting,respectively.Results MTT results showed that the cell growth was inhibited by emodin in a dose-dependent and time-dependent manner when the concentration was upper than 20 μmol/L （P ＜ 0.05）.The inhibiting rate in prostate cancer cells after 48 and 96 hours were （59.76 ± 3.90） ％ and （71.77 ± 3.50）％ when the Emodin concentration was 160 μmol/L.Emodin significantly increased the apoptosis rate in prostate cancer cells compared with blank control group [（22.54 ± 2.32） ％ vs.（4.54 ± 1.26）％,P ＜ 0.05].Emodin combined with caspase inhibitor can also significantly increase the apoptosis rate in prostate cancer cells compared with blank control group [（15.65 ± 1.84） ％ vs.（4.54 ± 1.26） ％,P ＜ 0.05].Compared with blank control group,emodin,with or without caspase inhibitor,significantly upregulated the expressions of AIF and Endo G mRNA and protein （P ＜ 0.05）.Conclusion Emodin could induce apoptosis of prostate cancer cells via caspase-independent AIF/Endo G pathway.