EP联合方案耐药小细胞肺癌细胞株的构建及机制探讨

Construction and mechanism of a small cell lung cancer cell line resistant to EP chemotherapy scheme

目的构建依托泊苷(etoposide,VP-16)联合顺铂(cisplatin,DDP)化疗方案(EP联合方案)耐药的小细胞肺癌(small cell lung cancer,SCLC)细胞株H446/EP,并进行耐药特性鉴定及机制探讨。方法以药物浓度递增法,使用VP-16联合DDP处理NCI-H446细胞构建H446/EP细胞株。以H446/EP与NCI-H446细胞为研究对象,MTT法检测细胞活力,计算H446/EP细胞耐药指数(resistance index,RI);细胞克隆实验、Incucyte细胞增殖(无标记)法检测细胞增殖能力;转录组学测序后,对2种细胞差异表达基因进行富集分析;Western blot检测细胞耐药、DNA损伤修复(repair of DNA damage,DDR)、自噬标志分子的蛋白表达量。结果MTT结果显示,H446/EP细胞对VP-16、DDP与DOX的RI分别为6.14、3.43与1.96;细胞克隆实验与Incucyte细胞增殖(无标记)法结果显示,H446/EP细胞增殖能力显著高于NCI-H446细胞(P<0.01);转录组学测序、通路富集分析显示差异表达基因在肿瘤化疗耐药、DDR、自噬相关通路富集;Western blot结果显示,相比NCI-H446细胞,H446/EP细胞MRP1、BCRP、RAD51、γ-H2AX及LC3-II/LC3-I蛋白表达量显著升高,p62蛋白表达量显著降低(P<0.05)。结论EP联合方案耐药的SCLC细胞株H446/EP构建成功,增殖能力增强;细胞外排转运蛋白表达量增加、DDR及自噬水平升高可能是SCLC对EP联合方案产生耐药的机制。

ObjectiveTo construct the etoposide(VP-16)combined with cisplatin(DDP)chemotherapy scheme(EP chemotherapy scheme)resistant small cell lung cancer(SCLC)cell line H446/EP and to identify the drug resistance characteristics and explore the mechanism.MethodsNCI-H446 cells were treated with increasing concentrations of VP-16 and DDP to construct an H446/EP cell line.H446/EP and NCI-H446 cells were used as the research objects.The cell viability was detected by MTT assay,and the resistance index(RI)of H446/EP cells was calculated.Cell cloning assay and Incucyte cell proliferation(label-free)assay were used to detect cell proliferation ability.Transcriptome sequencing was performed to analyze the enrichment of differentially expressed genes(DEGs)in the 2 cell lines.Western blotting was applied to detect the protein expression levels of drug resistance,DNA damage repair(DDR),and autophagy markers.ResultsMTT assay showed that the resistance index(RI)of H446/EP cells to VP-16,DDP,and DOX were 6.14,3.43,and 1.96,respectively.The results of cell cloning assay and Incucyte cell proliferation assay indicated that the proliferation ability was significantly higher in the H446/EP cells than the NCI-H446 cells(P<0.01).Transcriptome sequencing and pathway enrichment analysis displayed that the DEGs between H446/EP and NCI-H446 cells were enriched in tumor chemoresistance,DDR,and autophagy pathways.Western blot results showed the expression levels of MRP1,BCRP,RAD51,γ-H2AX,and LC3-II/LC3-I were significantly increased,and that of p62 was obviously decreased in the H446/EP cells when compared with the NCI-H446 cells(P<0.05).ConclusionAn EP chemotherapy-resistant H446/EP cell line is successfully constructed.Stronger proliferation ability,increased expression of efflux transporters,and enhanced DDR and autophagy may be the mechanisms of the resistance of SCLC to EP chemotherapy scheme.