Evn-50对人乳腺癌MCF-7耐三苯氧胺细胞株生长和凋亡的影响

目的:通过研究黄荆子的乙酸乙酯提取物Evn-50在体外对人乳腺癌MCF-7和耐三苯氧胺细胞株(MCF-7/TAM-R)的生长和凋亡的影响,探索Evn-50在逆转乳腺癌耐药中的作用及其可能机制。方法:构建MCF-7/TAM-R细胞系,分别用DMSO、5μmol/L TAM、5μmol/L TAM+50μg/ml Evn-50和50μg/ml Evn-50处理细胞。然后,采用MTT法测定细胞存活率,PI单染流式细胞术检测细胞凋亡率及细胞周期分布情况,免疫蛋白印记法观察Evn-50和TAM单独或联合作用时的机制。结果:Evn-50单药或与TAM联合均可以降低人乳腺癌MCF-7和三苯氧胺耐药细胞株MCF-7/TAM-R的存活率,两药联合作用更为明显,同单用TAM组相比差异有统计学意义(P<0.01)。Evn-50单药或与TAM联合应用对两株细胞的凋亡均有明显影响,且随着作用时间的延长细胞凋亡逐渐增多,其中以72 h时细胞凋亡率和G2期细胞比例最为明显(P<0.01)。TAM和Evn-50联合处理无论是对MCF-7细胞还是对三苯氧胺耐药细胞MCF-7/TAM-R,均能明显下调p-AKT(Ser473)和p-MAPK44/42(Thr202/Tyr204)蛋白水平。结论:Evn-50能够抑制MCF-7和MCF-7/TAM-R细胞株的生长并诱导细胞凋亡,尤其在Evn-50与TAM联合处理时更为明显。其作用机制可能与AKT和MAPK信号通路的下调相关,但需要进一步研究证实。

Effect of Evn-50 on cell growth and apoptosis in tamoxifen-resistance human breast cancer cell line MCF-7/TAM-R

Objective: To investigate the effect of Evn-50 extracted from Vitex negundo on human breast cancer cell line MCF-7 and MCF-7/TAM-R cells in vitro.Methods: MCF-7 and tamoxifen-resistant MCF-7/TAM-R cells were treated with Evn-50,tamoxifen or combination of Evn-50 and tamoxifen.Cell proliferation inhibition rates were determined by MTT assay.The apoptosis rate and the change of cell cycle were detected by PI staining flow cytometry.Protein expression of phospho-MAPK 44/42(Thr202/Tyr204),MAPK P44/42,phospho-AKT(Ser473) and AKT were detected with Western blotting.Results: The viability of MCF-7 cells was decreased in combination group [(28.65±11.43)%] and Evn-50 group [(53.02±15.14)%] compared with TAM group(P<0.01).The cell viability of MCF-7/TAM-R in combination group [(42.11±14.30)%] was significantly lower than that in TAM group [(92.18±13.16)%](P<0.01).The cell apoptosis rate was dependent on the time of treatment in all groups,the effects on apoptosis and G2/M phase cells were most prominent at 72 h(P<0.01).Western blotting revealed that protein levels of phosphorylated AKT and p-MAPK44/42 decreased,while the expression of total AKT and MAPK44/42 was stable.In MCF-7/TAM-R cells,the expression of phosphorylation of AKT and MAPK44/42 protein was not changed in Evn-50 or TAM alone group,but significantly inhibited in the combination group at 72 h.Conclusions: Evn-50 can inhibit cell growth and induce apoptosis in MCF-7 and MCF-7/TAM-R cells,it can reverse tamoxifen-resistance of MCF-7/TAM-R cells.The mechanisms may be related to the down-regulation of phosphorylated ERK1/2 in MAPK signal pathway and phosphorylated AKT in AKT signal pathway.