南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (07): 981-987.doi: 10.12122/j.issn.1673-4254.2020.07.10

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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

葛贤明，周 巧，张语涵，周文静，伍 宇，甄 诚，张梦晓，范方田，陈刚胜，赵军军，刘 浩   

  • 出版日期:2020-07-20 发布日期:2020-07-20
  • 基金资助:

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

  

  • Online:2020-07-20 Published:2020-07-20

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摘要： 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、6 µmol/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、2.016 µmol/L。JC-1与流式细胞术显示HS-10296可诱导细胞发生凋亡，8 µmol/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。

关键词: 三阴乳腺癌, 表皮生长因子受体, 自噬, 凋亡

Abstract: 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 6 µmol/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 2.016 μmol/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 8 μmol/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

Key words: triple-negative breast cancer, epidermal growth factor receptor, autophagy, apoptosis