地高辛对三阴性乳腺癌MDA-MB-231细胞凋亡作用的研究

Effect of Digoxin on apoptosis of MDA-MB-231 cells in triple negative breast cancer

目的探讨地高辛对体外培养的三阴性乳腺癌MDA-MB-231细胞凋亡的影响,并揭示其发生的可能机制。方法体外培养乳腺癌MDA-MB-231细胞,分为对照组和给药组(25、50、100、200 nmol/L地高辛)。CCK-8法检测不同浓度的地高辛、不同作用时间(24、48、72 h)对MDA-MB-231细胞活力的影响。流式细胞术定量考察不同浓度的地高辛对细胞凋亡的作用及对线粒体膜电位的影响。Western blot检测MDA-MB-231细胞中Bax、Bcl-2和P62、LC3B蛋白的表达情况。透射电镜观察不同浓度的地高辛对MDA-MB-231细胞自噬体产生的影响。结果CCK-8结果提示,与对照组比较,在不同时间点,给药组使MDA-MB-231细胞活力降低,差异有统计学意义(P<0.05或P<0.01)。Annexin V/PI双染结果提示,与对照组比较,给药组使细胞凋亡率增加,差异有统计学意义(P<0.05或P<0.01)。JC-1染色结果提示,与对照组比较,50、100、200 nmol/L地高辛使MDA-MB-231细胞线粒体膜电位电位降低,JC-1所占比例减少,差异有统计学意义(P<0.05或P<0.01)。透视射电镜结果提示,与对照组比较,给药组细胞形态发生凋亡样改变,给药组可见自噬体的形成。Western blot结果提示,与对照组比较,给药组上调促凋亡因子Bax,下调抗凋亡因子Bcl-2的表达,并上调自噬标志性蛋白LC3B表达,差异有统计学意义(P<0.05或P<0.01);与对照组比较,50、100、200 nmol/L地高辛使P62表达下降,差异有统计学意义(P<0.05或P<0.01)。结论地高辛抑制MDA-MB-321细胞的活力,促进凋亡,诱导自噬的发生,可能对三阴性乳腺癌有潜在作用。

Objective To investigate the effect of Digoxin on apoptosis of MDA-MB-231 cells of triple negative breast cancer in vitro,and to reveal its possible mechanism.Methods Breast cancer MDA-MB-231 cells were cultured in vitro and divided into control group and drug administration group(25,50,100,200 nmol/L Digoxin).CCK-8 method was used to detect the effects of Digoxin at different concentrations on the activity of MDA-MB-231 cells at different action time(24,48,72 h).The effects of Digoxin at different concentrations on apoptosis and mitochondrial membrane potential were quantitatively investigated by flow cytometry.Expression of Bax,Bcl-2,P62 and LC3B in MDA-MB-231 cells was detected by Western blot.The effects of Digoxin at different concentrations on autophagosomes in MDA-MB-231 cells were observed by transmission electron microscopy.Results CCK-8 results indicated that,compared with the control group,the activity of MDA-MB-231 cells decreased in the drug administration group at different time,with statistically significant differences(P<0.05 or P<0.01).Annexin V/PI double staining results indicated that compared with the control group,the apoptosis rate of the treated group increased with a statistically signifi-cant difference(P<0.05 or P<0.01).The results of JC-1 staining indicated that compared with the control group,50,100 and 200 nmol/L of Digoxin reduced the mitochondrial membrane potential of MDA-MB-231 cells,and the proportion of JC-1 decreased,with sta-tistically significant differences(P<0.05 or P<0.01).The results of fluoroscopy indicated that,compared with the control group,the cell morphology of the drug administra-tion group underwent apoptotic changes,and the formation of autophagosomes was observed in the drug administration group.Western blot results indicated that,compared with the control group,the administration group up-regulated the expression of pro-apoptotic factor Bax,down-regulated the expression of anti-apoptotic factor Bcl-2,and up-regulated the expression of autophagy marker protein LC3B,with statistically significant differences(P<0.05 or P<0.01);com-pared with the control group,50,100 and 200 nmol/L of Digoxin decreased the expression of P62,with statistically sig-nificant differences(P<0.05 or P<0.01).Conclusion Digoxin inhibits the activity of MDA-MB-231 cells,promotes apoptosis,induces autophagy,and may have a potential effect on triple negative breast cancer.