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 |
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引用本文: | 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.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[J].南方医科大学学报,2020,40(7):981-987. |
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作者姓名: | 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 |
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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。
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关 键 词: | 三阴乳腺癌 表皮生长因子受体 自噬 凋亡 |
EGFR tyrosine kinase inhibitor HS-10296 induces autophagy and apoptosis in triplenegative breast cancer MDA-MB-231 cells |
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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 |
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Keywords: | triple-negative breast cancer epidermal growth factor receptor autophagy apoptosis |
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