EGFR酪氨酸激酶抑制剂HS-10296诱导三阴乳腺癌MDA-MB-231细胞发生自噬和凋亡

EGFR tyrosine kinase inhibitor HS-10296 induces autophagy and apoptosis in triplenegative breast cancer MDA-MB-231 cells

目的探讨表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKT)HS-10296对三阴乳腺癌MDA-MB-231细胞的增殖抑制作用及潜在分子机制。方法HS-10296处理MDA-MB-231细胞24、48、72 h。CCK-8法检测MDA-MB-231细胞存活率;集落克隆法检测HS-10296对细胞的增殖抑制作用;JC-1与流式细胞术检测细胞凋亡情况;电镜观察细胞超微结构;自噬抑制剂氯喹预处理后分为不含药物的对照组、单用氯喹组、单用HS-10296(4、6μmol/L)组和两者联合组,CCK-8法检测MDA-MB-231细胞对HS-10296的敏感性变化;Western blot分析HS-10296对MDA-MB-231细胞凋亡和自噬相关蛋白以及EGFR信号通路的影响。结果CCK-8和集落克隆结果显示,HS-10296对三阴乳腺癌MDA-MB-231细胞具有增殖抑制作用(P<0.01),24、48、72 h的IC50值分别为8.393、2.777、2.016μmol/L。JC-1与流式细胞术显示HS-10296可诱导细胞发生凋亡,8μmol/L HS-10296处理后,细胞凋亡率为(21.63±2.97)%。电镜观察到经HS-10296处理后,细胞内出现自噬囊泡,使用氯喹预处理细胞后,观察到抑制自噬可增强HS-10296诱导的细胞死亡。Western blot显示经HS-10296处理后凋亡相关蛋白Caspase-3出现活化形式,并对其底物PARP进行剪切。自噬相关蛋白轻链3B表达高于对照组(P<0.01),同时,HS-10296可抑制细胞内EGFR及AKT蛋白的磷酸化。结论HS-10296可通过抑制EGFR/PI3K/AKT信号通路,抑制MDA-MB-231细胞增殖,诱导细胞发生自噬和凋亡。

Objective To investigate the inhibitory effect of epidermal growth factor receptor tyrosine kinase inhibitor(EGFRTKI)HS-10296 on the proliferation of triple-negative breast cancer(TNBC)MDA-MB-231 cells and explore the possible molecular mechanism.Methods MDA-MB-231 cells were treated with HS-10296 for 24,48,or 72 h,and CCK-8 assay was used to assess the changes in the cell viability.The inhibitory effect of HS-10296 on cell proliferation was determined by clonogenic assay.JC-1 and flow cytometry were employed for analyzing the cell apoptosis,and the ultrastructure of the cells was observed under electron microscope.After pretreatment with autophagy inhibitor chloroquine(CQ),MDA-MB-231 cells were divided into control group,CQ treatment group,HS-10296(4 and 6μmol/L)treatment groups and combined treatment groups,and the sensitivity of the treated cells to HS-10296 was determined using CCK-8 assay.The effects of HS-10296 on EGFR pathway and apoptosis-and autophagy-related proteins in MDA-MB-231 cells were investigated using Western blotting.Results HS-10296 significantly inhibited the proliferation of MDA-MB-231 cells with IC50 values at 24,48 and 72 h of 8.393,2.777 and 2.016μmol/L,respectively.JC-1 and flow cytometry showed that HS-10296 induced obvious apoptosis of MDA-MB-231 cells,which showed an apoptosis rate of(21.63±2.97)% following treatment with 8μmol/L HS-10296.Autophagy vesicles were observed in the cells treated with HS-10296 under electron microscope.In MDA-MB-231 cells pretreated with CQ,inhibition of autophagy significantly enhanced HS-10296-induced cell death.Western blotting showed that the apoptosis-related protein caspase-3 was activated after HS-10296 treatment to cut its substrate PARP.The expression of autophagy-related protein light chain 3B(LC3B)was significantly enhanced after HS-10296 treatment(P<0.01),which also resulted in inhibited phosphorylation of EGFR and AKT proteins in the cells.Conclusion HS-10296 can inhibit the proliferation and induce autophagy and apoptosis of MDA-MB-231 cells by inhibiting the EGFR/PI3K/AKT signaling pathway.