自噬抑制联合吉非替尼对MDA-MB-468细胞敏感性的影响  

Effects of autophagy inhibitor combined with gefitinib on human breast cancer MDA-MB-468 cells

作　　者：刘兆芸 李景[3] 贺科文 马清华[2] 卓培英[2] 朱文尧[1,2] 王新昭[2] 于志勇[2] 

机构地区：[1]济南大学.山东省医学科学院医学与生命科学学院,山东济南250022 [2]山东大学附属山东省肿瘤医院外科一病区山东省医学科学院,山东济南250117 [3]济宁市第一人民医院北院区.济宁肿瘤医院外二科,山东济宁272123

出　　处：《中华肿瘤防治杂志》2016年第15期983-987,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：山东省自然科学基金(ZR2015HM055)

摘　　要：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。OBJECTIVE Autophagy participates in drug resistance in cancer treatment, our study aimed to explore the effects of gefitinib combining with autophagy inhibitior in breast cancer MDA-MB-468 cells. METHODS Human breast cancer MDA-MB-468 cells with exponential growth in routine culture were treated by gefitinib combined with 3-methyladenine（3MA） or bafilomycin A1 （BAF） or monotherapy. The growth inhibition rates of MDA MB-468 cells were evaluated by MTT. Cell proliferation were evaluated by elonogenic assay. Cell apoptosis were detected by flowcytometry. Western Blot was used to detect the alteration of autophagy-related protein and apoptosis related proteins. RE- SULTS Autophagy inhibitor enhances cytotoxicity of gefitinib to breast cancer cells MDA-MB-468. When combined with gefitinib and autophagy inhibitor 3MA or BAF, the IC50 were （4.1±0.2） μmol/L and （3.8±0.3） μmol/L, lower than that ingefitinib alone （7. 0±0. 2）μmol/L （Z =2. 611,P〈0. 017）. The cloning formation capacity in GE＋3MA （29.85 ± 5.54）% and GE＋BAF （33.58± 6.31） % were significantly lower than that in GE alone （63.46 ±8.32）%, Z=2. 309, P〈0. 017. Cell apoptosis assay revealed that the apoptosis rate of MDA MB-468 ceils were higher in GE＋MA （23.37±2.71）% and GE＋BAF （18.71±2.81）% than in monotherapy groups （12. 43±3.18）%, Z =-2.309, P〈0. 017. Wesern blot analysis revealed that gefitinib induced autophagy. When combined with autophagy inhititor and gefitinib, Cleaved Caspase-9 and Cleaved Caspase 3 were up-regulated. CONCLUSION Autophagy inhibitor may enhance the sensitivity of gefitinb in MDA-MB-468 cells through activation of caspase cascade.

关 键 词：乳腺肿瘤 自噬抑制 吉非替尼 MDA- MB-468细胞 

分 类 号：R737.9[医药卫生—肿瘤]