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目的自噬是细胞内的一种溶酶体降解过程,通过降解损伤的蛋白以及衰老的细胞器来维持细胞稳态,也是肿瘤治疗耐药性产生的原因之一。本实验探讨自噬抑制剂联合吉非替尼(gefitinib,GE)对人三阴性乳腺癌(triple-negative breast cancer,TNBC)MDA-MB-468细胞敏感性的影响。方法常规复苏、传代培养MDA-MB-468细胞,待细胞处于对数生长期后分别以GE单药或联合自噬抑制剂3-methyladenine(3MA)或联合bafilomycin A1(BAF)处理细胞。MTT比色法检测细胞相对增殖率,克隆形成实验检测细胞存活率,流式细胞术检测细胞凋亡,蛋白质印迹法检测自噬相关蛋白的表达。结果自噬抑制剂明显增加GE的细胞毒性,MTT比色法检测GE联合3MA、BAF处理MDA-MB-468细胞48h后半抑制率(IC50)分别为(4.1±0.2)和(3.8±0.3)μmol/L,显著低于单用GE组的(7.0±0.2)μmol/L,Z=-2.611,P<0.017。克隆形成实验显示,GE与自噬抑制剂联合3MA、BAF克隆形成率分别为(29.85±5.54)%和(33.58±6.31)%,显著低于单用GE组(63.46±8.32)%,Z=-2.309,P<0.017。细胞凋亡的研究显示,自噬抑制可促进细胞凋亡,GE联合3MA、BAF后,凋亡率分别为(23.37±2.71)%、(18.71±2.81)%,高于单用GE组(12.43±3.18)%,Z=-2.309,P<0.017。蛋白质印迹法检测结果显示,GE处理诱导自噬增加,自噬抑制剂联合GE后,MDA-MB-468细胞Cleaved Caspase-9和Cleaved Caspase-3蛋白的活性增加。结论自噬抑制可增加MDA-MB-468细胞对GE的敏感性,自噬抑制剂联合GE可能通过启动Caspase级联反应促进MDA-MB-468细胞凋亡。
Aim Autophagy is a lysosomal degradation process in cells. It is one of the reasons for the drug resistance of tumors to be treated by degrading damaged proteins and aging organelles. This study investigated the effect of autophagy inhibitor combined with gefitinib (GE) on the sensitivity of human triple-negative breast cancer (TNBC) MDA-MB-468 cells. Methods After routine resuscitation, MDA-MB-468 cells were subcultured. Cells were treated with 3-methyladenine (3MA) or bafilomycin A1 (BAF) combined with GE alone or in combination with autophagy after the cells were in logarithmic growth phase. Cell proliferation was detected by MTT colorimetric assay. Cell viability was detected by colony formation assay. Apoptosis was detected by flow cytometry. Expression of autophagy-related protein was detected by Western blotting. Results The autophagy inhibitor significantly increased the cytotoxicity of GE. The IC50 values of GE combined with 3MA and BAF treated MDA-MB-468 cells 48h were (4.1 ± 0.2) and (3.8 ± 0.3) μmol / L, significantly lower than 7.0 ± 0.2 μmol / L for GE alone group, Z = -2.611, P <0.017. Clone formation experiments showed that the formation rates of GE and autophagy inhibitor combined with 3MA and BAF were (29.85 ± 5.54)% and (33.58 ± 6.31)%, respectively, which were significantly lower than that of GE alone group (63.46 ± 8.32)% and Z = -2.309, P <0.017. Apoptotic studies have shown that autophagy inhibition can promote apoptosis. After GE combined with 3MA and BAF, the apoptotic rates were (23.37 ± 2.71)% and (18.71 ± 2.81)%, respectively, which were higher than those of GE group (12.43 ± 3.18)%, Z = -2.309, P <0.017. Western blotting showed that GE treatment induced an increase in autophagy, and increased the activities of Cleaved Caspase-9 and Cleaved Caspase-3 in MDA-MB-468 cells after autophagy inhibitor combined with GE. Conclusion The autophagy inhibition may increase the sensitivity of MDA-MB-468 cells to GE. The autophagy inhibitor combined with GE may promote the apoptosis of MDA-MB-468 cells by activating caspase cascade.