EGCG对三阴乳腺癌细胞MDA-MB-231的增殖抑制作用及机制

The inhibitory action of EGCG on the proliferation in the triple-negative breast cancer cell MDA-MB-231 and its mechanism

目的探讨表没食子儿茶素没食子酸酯[(-)-epigallo-catechin-3-gallate,EGCG]对三阴乳腺癌细胞MDA-MB-231增殖和凋亡的影响及其可能作用机制。方法通过CCK-8实验观察不同浓度EGCG(10、20、40、80、160 mg.L-1)对MDA-MB-231细胞增殖的影响;Hoechst33258染色法观察EGCG对MDA-MB-231细胞凋亡的影响;JC-1法测定细胞线粒体膜电位;caspase-3活性检测试剂盒检测caspase-3活性的变化;Western blot法检测葡萄糖调节蛋白78(glucose reg-ulatd protein 78,GRP78)和caspase-3蛋白表达变化。结果EGCG能明显抑制MDA-MB-231细胞增殖,且随EGCG浓度的增加和作用时间的延长而增强,EGCG作用MDA-MB-231细胞12、24、48 h的IC50分别为69.1、40.4、29.4 mg.L-1。EGCG作用MDA-MB-231细胞24 h后,细胞出现体积变小、染色质聚集、细胞核边缘化等典型的凋亡形态学改变,且随EGCG浓度的增加,MDA-MB-231细胞凋亡率逐渐增加,线粒体膜电位明显降低,caspase-3活性明显增强,West-ern blot结果显示EGCG可抑制GRP78蛋白表达,增强活性caspase-3蛋白表达。结论 EGCG能够促进MDA-MB-231凋亡,其机制可能与内质网应激(Endoplasmic reticulumstress,ERS)引起的caspase-3激活有关。

Aim To study the effects of EGCG [ （-）- epigallocatechin-3-gallate ] on the proliferation and apoptosis in the triple-negative breast cancer cell MDAMB-231 and the possible mechanism. Methods MDA-MB-231cells were treated with different concentrations of EGCG（ 10,20,40,80,160 mg.L-1 ） and then the proliferation of MDA-MB-231 cells was detec- ted by CCK-8 assay. The morphological change of apoptosis of MDA- MB-231 cells was observed under fluorescence microscope through Hoechst-33258 staining. The mitochondrial membrane potential was measured by JC-1assay. The caspase-3 activity was measured by caspase-3 activity assay kit, Western blot assay was used to analyse the change of protein expression of GRP78 （glucose regulatd protein 78 ） and activated caspase-3. ,Results EGCG significantly inhibited the proliferation of MDA-MB-231 cells in a time and concentration dependent manner. The IC50 of EGCG stim-ulating the cells 12,24,48 h was 69. 1,40. 4, 29.4mg.L-1 respectively. After treatment with different coneentrations of EGCG for 24h, MDA-MB-231 cells showed the signs of the typical apoptosis morphology changes such as shrinkage, chromatin aggregation and nucleus marginalization and so on. Moreover, with increase of the concentration of EGCG, the apoptotic rate gradually increased, the mitochondrial membrane potential decreased, and the activated caspase-3 increased. Protein expression of GRP78 was inhibited and that of activated caspase-3 was increased. Conclusion EGCG can inhibit the proliferation and induce the apoptosis of MDA-MB-231 cells significantly, which might be related to the ERS （ endoplasmie retieulum stress） pathway inducing caspase-3 activated.