自噬抑制剂可增强三阴性乳腺癌细胞系MDA—MB-468和MDA-MB-231对吉非替尼的敏感性

Effect of autophagy inhibitor combined with EGFR inhibitor on triple-negative breast cancer MDA- MB-468 and MDA-MB-231 cells

目的观察自噬抑制剂能否增强三阴性乳腺癌（TNBC）细胞系MDA—MB-468和MDA-MB-231对表皮生长因子受体（EGFR）抑制剂吉非替尼的敏感性。方法以吉非替尼单药或联合自噬抑制剂3-甲基腺嘌呤（3-MA）或巴弗洛霉素A1（BAF）处理MDA—MB．468和MDA．MB．231细胞，以及雌激素受体阳性的MCF-7细胞，采用四甲基偶氮唑蓝（MTT）法检测细胞增殖，流式细胞术检测细胞凋亡，Westernblot法检测自噬标记蛋白以及凋亡通路相关蛋白的表达。结果吉非替尼对吉非替尼＋3-MA组和吉非替尼＋BAF组MDA-MB-468细胞的半数抑制浓度（IC50）分别为（4．1±0．2）μmol／L和（3．8±0．3）μmol／L，均明显低于吉非替尼组MDA-MB-468细胞[（7．0±0．2）μmol,／L，均P〈0．05]。吉非替尼对吉非替尼＋3．MA组和吉非替尼＋BAF组MDA-MB-231细胞的IC50分别为（9．7±0．1）／xmol／L和（7．7±0．2）μmoL／L，均明显低于吉非替尼组MDA-MB-231细胞[（14．7±0．1）μmol／L，均P〈0．05]。吉非替尼组、吉非替尼＋3．MA组、吉非替尼＋BAF组MDA-MB-468细胞的凋亡率分别（12．43±3．18）％、（23．37±2．71）％和（18．71±2．81）％，吉非替尼联合自噬抑制剂3-MA或BAF时，MDA-MB-468细胞的凋亡率均明显高于单用吉非替尼时的凋亡率（均P〈0．05）。吉非替尼组、吉非替尼＋3-MA组、吉非替尼＋BAF组MDA-MB-231细胞的凋亡率分别为（12．15±1．82）％、（16．94±2．19）％和（33．83±5．92）％，吉非替尼联合自噬抑制剂3-MA或BAF时，MDA-MB-231细胞的凋亡率均明显高于单用吉非替尼时的凋亡率（均P〈0．05）。吉非替尼联合3-MA或BAF对MCF．7细胞的凋亡无明显影响（P〉0．05）。吉非替尼联合3-MA或BAF后，MDA．MB．468和MDA．MB．231细胞中p-PTEN的表达明显增加，LC3和p-Akt蛋白表达明显下调，cleavedaspa$e-9和cleavedaspase-3蛋白的活性增加。结论自噬抑制剂可能通过激活PTEN／P13K／Akt通路相关蛋白的表达增强MDA—MB-468和MDA-MB-231细胞对吉非替尼的敏感性；自噬抑制剂联合吉非替尼可能通过启动caspase级联反应促进MDA-MB-468和MDA-MB-231细胞凋亡。

Objective To investigate the effect of combined administration of autophagy inhibitor 3- methyladenine/bafilomyein A1 and EGFR inhibitor gefitinib on triple-negative breast cancer MDA-MB-468, MDA-MB-231 ceils and estrogen receptor-positive MCF-7 cells. Methods All the cells were treated with 3- methyladenine/bafilomycin A1 and/or gefitinib. The effect of autophagy inhibitor and gefitinib on the cell growth was evaluated by MTT assay. Cell apoptosis was detected by flow cytometry. Western blot analysis was used to determine the alteration of autophagy-related protein （ such as LC3） and apoptosis-related proteins （such as caspase-3 and caspase-9）. Results MTT assay showed that the ICs0 in the GE＋3-MA and GE＋ BAF groups were （4.1±0.2） μmol/L and （3.8±0.3）μmol/L, significantly lower than that of the gefitinib alone group [ （7.0±0.2）μmol/L] in MDA-MB-468 cells （P〈0.05）. Similarly, the ICs0 in the GE＋3-MA and GE＋BAF groups were （9.7±0.1）μmol/L and （7.7±0.2） p, mol/L, significantly lower than that of the gefitinib alone group [ （ 14.7± 0.1 ）μmol/L] in MDA-MB231 cells （P〈0.05）. The flow cytometry assay revealed that the apoptosis rates of MDA-MB-468 cells in GE, GE＋3-MA and GE＋BAF groups were （12.43± 3.18） %, （23.37±2.71） % and （18.71±2.81） %, respectively. The apoptosis rates of MDA-MB-231 cells of the GE, GE＋3-MA and GE＋BAF groups were （ 12.15± 1.82） %, （16.94±2.19） % and （33.83±5.92） %, significantly higher than that of the gefitinib alone group （All P〈0.05）. The apoptosis rates of the MCF-7 cells were not changed significantly among the three groups （P〉0.05）. Western blot data showed that the expression levels of LC3 and p-Akt were decreased in the combined groups than that of the gefitinib alone group, while the p-PTEN, caspase-3 and caspase-9 were increased. Conclusions Autophagy inhibitor may enhance the sensitivity to gefitinib in MDA-MB-468 and MDA-MB-231 cells by activation of the PTEN/ P13K/Akt pathway. Apoptosis in MDA-MB-468 and MDA-MB-231 cells might be enhanced by the combination treatment through caspase cascade.