白花丹素对乳腺癌细胞凋亡和自噬的影响

Effects of Plumbagin on Apoptosis and Autophagy of Breast Cancer Cells

目的观察白花丹素对乳腺癌细胞凋亡及自噬的影响,探讨其可能的作用机制。方法采用不同浓度白花丹素作用于乳腺癌MCF-7和BT549细胞,CCK-8法检测细胞存活率,DCFH-DA荧光探针检测细胞内活性氧(ROS)水平,JC-1荧光探针检测线粒体膜电位(MMP),流式细胞术检测细胞凋亡情况,免疫荧光检测细胞内LC3B蛋白表达,Western blot检测细胞内Bax、Bcl-2、LC3B、AMPK和p-AMPK蛋白表达。结果不同浓度白花丹素可抑制乳腺癌MCF-7和BT549细胞生长,抑制细胞克隆形成,其IC50分别为15.9μmol/L和13.51μmol/L;16、32μmol/L白花丹素处理后,MCF-7细胞MMP降低,ROS水平升高,Bcl-2蛋白表达降低,Bax、LC3BⅡ、p-AMPK蛋白表达升高,差异均有统计学意义(P<0.05,P<0.01)。使用AMPK抑制剂(10μmol/L Dorsomorphin)或自噬抑制剂(5 mmol/L 3-MA)与白花丹素(32μmol/L)同时处理MCF-7细胞后,LC3BⅡ、p-AMPK蛋白表达较单独使用白花丹素(32μmol/L)时降低(P<0.01,P<0.05);使用溶酶体抑制剂(10μmol/L氯喹)与白花丹素(32μmol/L)同时处理MCF-7细胞后,LC3BⅡ、p-AMPK蛋白表达较单独使用白花丹素(32μmol/L)时升高(P<0.01)。结论白花丹素可能通过激活AMPK通路促进乳腺癌细胞自噬,从而诱导细胞凋亡。

Objective To investigate the effects of plumbagin on apoptosis and autophagy of breast cancer cells;To explore its possible mechanism.Methods Breast cancer MCF-7 and BT549 cells were treated with different concentrations of plumbagin,CCK-8 was used to detect the survival rate.The production of intracellular reactive oxygen species(ROS)was detected by DCF-DA probe.Mitochondrial membrane potential(MMP)was detected with JC-1 probe.Cell apoptosis was detected by flow cytometry.The expression of LC3 B protein in cells was detected by immunofluorescence.The proteins expression of Bax,Bcl-2,AMPK and p-AMPK in cells were detected by Western blot.Results Different concentrations of plumbagin inhibited the growth of breast cancer MCF-7 and BT549 cells with IC50 of 15.9μmol/L and 13.51μmol/L,respectively,and inhibited cell clone formation.After 16 and 32μmol/L plumbagin treatment,MCF-7 cells MMP decreased,ROS level increased,Bcl-2 protein expression decreased,the expression of Bax,LC3 BⅡ,p-AMPK protein increased,with statistical significance(P<0.05,P<0.01).After using AMPK inhibitor(10μmol/L Dorsomorphin)or autophagy inhibitor(5 mmol/L 3-MA)and plumbagin(32μmol/L)to treat MCF-7 cells simultaneously,LC3 BⅡ,p-AMPK protein expression was lower than when plumbagin(32μmol/L)was used alone(P<0.01,P<0.05);after simultaneously treating MCF-7 cells with lysosomal inhibitor(10μmol/L chloroquine)and plumbagin(32μmol/L),the proteins expression of LC3 BⅡand p-AMPK were higher than those of plumbagin(32μmol/L)alone(P<0.01).Conclusion Plumbagin may promote autophagy of breast cancer cells by activating the AMPK pathway,thereby inducing cell apoptosis.