摘要
目的:比较顺铂压力下人肺腺癌亲本A549细胞与获得性顺铂耐药表型的A549/DDP细胞自噬水平,探寻肿瘤细胞获得性耐药表型重塑的分子机制。方法:不同浓度顺铂(cisplatin,DDP)干预亲本A549细胞及获得性顺铂耐药表型的A549/DDP细胞,应用CCK-8法比较两株细胞耐药指数、免疫印迹法比较自噬相关蛋白(Beclin 1、LC3Ⅱ及p62)表达水平;应用10μmol/L顺铂(相当于亲本A549细胞半数抑制浓度,即A549 IC 50值)干预A549及A549/DDP细胞,构建细胞应激状态,CCK-8法比较两株细胞生存活力,免疫印迹法比较自噬相关蛋白(Beclin 1、LC3Ⅱ及p62)、凋亡相关蛋白(Bcl-2、Bax、Cleaved Caspase-3)、转录因子FOXO3a及其磷酸化蛋白(p-FOXO3a)表达水平,免疫荧光法观察FOXO3a亚细胞定位,并利用自噬晚期抑制剂巴弗洛霉素A1(Baf A1)干预A549及A549/DDP细胞,进一步验证顺铂压力下细胞自噬激活情况;利用蛋白激酶抑制剂干预PI3K/Akt信号通路,免疫印迹法检测顺铂压力下A549及A549/DDP细胞Akt、p-Akt及自噬相关蛋白表达水平,以探寻肿瘤细胞获得性耐药表型重塑的分子机制。结果:与亲本A549细胞相比,获得性顺铂耐药表型的A549/DDP细胞具有更强的顺铂耐药性及较高的基础自噬水平。在10μmol/L顺铂压力下,A549/DDP细胞存活率明显高于A549细胞;A549细胞中,顺铂通过抑制PI3K/Akt/FOXO3a信号通路,上调转录因子FOXO3a表达水平,下调Bcl-2表达、促进Bax及Cleaved Caspase-3活性片段表达增加,诱导细胞凋亡;A549/DDP细胞中,相同浓度的顺铂通过抑制PI3K/Akt/FOXO3a信号通路,抑制FOXO3a磷酸化,上调Beclin 1及LC3Ⅱ表达、下调p62表达,促进细胞保护性自噬,使耐药细胞获得生存活性。结论:顺铂诱导亲本A549细胞凋亡可能与抑制PI3K/Akt/FOXO3a信号通路,上调转录因子FOXO3a表达有关;A549/DDP细胞顺铂耐药表型的获得可能与顺铂抑制PI3K/Akt/FOXO3a信号通路,抑制FOXO3a磷酸化,上调细胞保护性自
AIM:To investigate the mechanism of acquired resistance remodeling to DDP(named cisplatin)by comparing the level of autophagy between the parental and DDP-resistant cells.METHODS:Human lung adenocarcinoma A549(parental cells)and A549/DDP cells(DDP-resistant cells)were treated with different concentrations of DDP,the drug resistance index(RI)was determined by CCK-8 assay and the autophagy associated proteins,like Beclin 1,LC3Ⅱand p62 were measured by Western blot.A549 and A549/DDP cells were treated with 10μmol/L DDP(about IC 50 of A549 cells),the cell viability was determined by CCK-8 assay,the autophagy and apoptosis associated proteins(including Beclin 1,LC3Ⅱ,p62,Bcl-2,Bax and cleaved-caspase 3)were measured by Western blot,and the activity of transcription factor FOXO3a and its subcellular localization were detected by Western blot and laser confocal scanning microscopy.Finally,the autophagy inhibitor Baflomycin A1(Baf A1)and the protein kinase inhibitor of PI3K/Akt signaling pathway were co-treated with DDP respectively to test the mechanism of drug resistance.RESULTS:Compared with the parental A549 cells,the acquired resistant A549/DDP cells showed DDP-resistance and higher level of basal autophagy.More survival count of A549/DDP cells than that of A549 cells in the same environment stress of 10μmol/L DDP.10μmol/L DDP induced A549 cells apoptosis by down-regulated Bcl-2,and increased Bax and cleaved-caspase 3,which followed the inhibition of PI3K/Akt signaling pathway and up-regulated the expression level of transcription factor FOXO3a.While the same concentration of DDP activated A549/DDP cells autophagy by up-regulated Beclin 1 and LC3Ⅱ,down-regulated p62,which followed the inhibition of PI3K/Akt signaling pathway and inhibited the phosphorylation of FOXO3a.CONCLUSION:DDP induces apoptosis by up-regulating the transcription factor FOXO3a via inhibiting the PI3K/Akt/FOXO3a signaling pathway in A549 cells,while activating autophagy by inducing the phosphorylation of FOXO3a via inhibiting the PI3K/Ak
作者
周欢
徐铭
李波
刘春英
王淳
ZHOU Huan;XU Ming;LI Bo;LIU Chunying;WANG Chun(College of Integrated Chinese and Western Medical,Liaoning University of Traditional Chinese Medicine,Shenyang 110847,Liaoning,China;Chaoyang Central Hospital,Chaoyang 122000,Liaoning,China;School of Life Sciences,China Medical University,Shenyang 110122,Liaoning,China)
出处
《中国临床药理学与治疗学》
CAS
CSCD
2022年第9期961-970,共10页
Chinese Journal of Clinical Pharmacology and Therapeutics
基金
国家自然科学基金项目(82174254,81973735)。
