熊果酸对乳腺癌细胞凋亡影响及其机制

目的：探讨熊果酸对乳腺癌细胞系MDA-MB-231细胞凋亡的影响及其机制。方法：体外培养MDAMB-231细胞,用不同浓度的熊果酸处理24、48、72 h,MTT法测定其对MDA-MB-231细胞增殖的影响;倒置显微镜下观察细胞的形态学变化;熊果酸（浓度分别为20、40、80μmol/L）处理MDA-MB-231细胞48 h后,分别采用流式细胞仪检测细胞凋亡和周期,分光光度计检测Casepase-3、Casepase-9相对活性,western blot法检测p16、p27基因表达情况。结果：熊果酸（浓度10~100μmol/L）能抑制MDA-MB-231细胞的增殖,呈剂量和时间依赖性;倒置显微镜下可观察到典型的细胞凋亡形态;熊果酸（浓度分别为20、40、80μmol/L）作用MDA-MB-231细胞48 h后,细胞凋亡率分别为11.5%、21.4%、36.6%,对照组凋亡率仅为2.2%;细胞周期结果显示熊果酸能抑制细胞的G1期行进和G1/S转换,S期细胞比例降低;Casepase-3、Casepase-9的相对活性显著增加;p16、p27的表达升高,呈剂量依赖性。结论：熊果酸能抑制MDA-MB-231细胞的增殖并诱导其凋亡,其作用机制可能与上调p16、p27表达阻滞细胞周期并增加细胞Caspase-3、Caspase-9激活线粒体凋亡途径有关。熊果酸是一种前景广阔的抗肿瘤药物。

Effects of Ursolic Acid on Cell Apoptosis in MDA-MB-231 Cells and Its Mechanisms

Objective ： To explore the effect of Ursolic acid on cell apoptosis in MDA-MB-231 cells and discuss its possible mechanisms. Methods： MDA-MB-231Cells were Cultured in vitro. After treatment by Ursolic acid at different concentrations respectively at different time points, the cell survival was determined by the MTT method. The changes of cell morphology were observed by inverted microscope. Apoptosis was detected by flow cytometry. The relative activity of Caspase-3 and Caspase-9 was monitored by spectrophotometer. The changes of protein expression of p16 and p27 were detected by Western blot. Results ： From the data of MTT, the cell proliferation of MDA-MB-231 cells was inhibited by Ursolic acid in a dose-dependent and time-dependent manner. Typical apoptosis morphology of MDA-MB-231 cells was observed by inverted microscope. Flow cytometry assays showed that Ursolic acid significantly induced apoptosis in MDA-MB-231 cells. After treated with Ursolic acid, the apoptosis rate of MDA-MB-231ce｜ls was 11.5%, 21.4% and 36.6% respectively, which showed an obvious concentration-effect relationship. Flow eytometry assays demonstrated that Ursolic acid blocked the cell cycle at the G1 to S transition, and Ursolic acid markedly decreased the percentage of S-phase cells. The relative activity of Caspase-3 of Ursolic acid group was 0.195 ± 0.02,0.251±0.04 and 0.317 ± 0.03 respectively. And the relative activity of Caspase-9 was 0.178 ± 0.02,0.235 ± 0.03 and 0.303± 0.04. The data of Western blot showed that Ursolic acid upregulated p16 and p27 in a dose-dependent manner. Conclusion ： Ursolic acid can inhibit the proliferation of MDA-MB-231 ceils and induce apoptosis, and the mechanism of Ursolic acid on apoptosis may be related to the up regulation of p16 and p27 expressions, and blocking the cell cycle, as well as the increase of relative activity of Caspase-3 and Caspase-9. Ursolic acid may be a promising antitumor agent for mammary cancer treatment.