金诺芬逆转非小细胞肺癌奥希替尼获得性耐药的作用

Auranofin reverses acquired resistance to osimertinib in non-small cell lung cancer

目的探讨金诺芬(auranofin,ANF)逆转非小细胞肺癌(non-small cell cancer,NSCLC)细胞奥希替尼(osimertinib,Osi)获得性耐药的作用。方法利用Osi敏感性NSCLC细胞株H1975和PC9,建立Osi获得性耐药的NSCLC细胞株H1975/OR和PC9/OR;通过CCK-8法在FDA批准的1470种药物分子库中高通量筛选Osi获得性耐药的逆转剂,Compusyn软件计算Osi与ANF联用时的药物联合指数;CCK-8、EdU细胞增殖、流式细胞术、Transwell实验检测Osi、ANF及两者联用对Osi获得性耐药NSCLC细胞生长增殖、凋亡、迁移和侵袭的影响;RNA-seq检测联合用药与单独用药后耐药株中差异变化的mRNA,目的基因和蛋白表达量用qRT-PCR和Western blot进行验证。结果与Osi敏感性H1975和PC9细胞相比,H1975/OR和PC9/OR在Osi处理后生长增殖能力明显升高,凋亡率明显降低,其耐药指数分别为70.31和136.99;在FDA批准的1470种药物分子库中,只有ANF可同时增强Osi对两种耐药细胞株的杀伤作用;1μmol·L^(-1)ANF与2μmol·L^(-1)Osi联用时,对NSCLC细胞Osi获得性耐药的逆转作用最为显著,可明显抑制耐药细胞株的生长增殖与侵袭、迁移能力,提高细胞凋亡率;联合用药后HSPB8和LC3-II/LC3-I上调导致自噬增多。结论ANF与Osi联用增强Osi对NSCLC耐药细胞的敏感性,其机制与HSPB8的上调导致自噬增多有关。

Aim To investigate the role of auranofin in reversing acquired resistance to osimertinib in non-small cell lung cancer.Methods Osimertinib-sensitive NSCLC cell lines H1975 and PC9 were used to establish osimertinib-resistant NSCLC cell lines H1975/OR and PC9/OR.An FDA approved library of 1470 FDA drugs was used to high-throughput screen the reversal agents of acquired resistant of osimertinib by CCK-8.Compusyn was used to calculate the combination index of osimertinib and auranofin to determine the optimal dose of drug combination.CCK-8,EdU,flow cytometry and Transwell experiments were used to detect osimertinib,auranofin and the combination drug effect on proliferation,apoptosis,migration and invasion of osimertinib acquired NSCLC cell lines.RNA-sequencing was applied to screen differentially expressed mRNAs in osimertinib treatment alone and osimertinib combined with auranofin treatment group.qRT-PCR and western blot were employed to validate the selected gene expression and protein expression.Results Compared with osimertinib sensitive cell lines H1975 and PC9,H1975/OR and PC9/OR showed significantly higher cell viability and lower apoptosis rate after osimertinib treatment.The resistance index was 70.31 and 136.99,respectively.In FDA approved 1470 drug library,only auranofin could enhance the sensitivity of osimertinib in H1975/OR and PC9/OR.When 1μmol·L^(-1)auranofin plus 2μmol·L^(-1)osimertinib,the effect to enhance osimertinib sensitivity was most significant,which significantly inhibited the growth,proliferation,migration,and invasion ability of resistant cells,and increased the apoptosis rate of resistant cells.Combination treatment upregulated HSPB8 expression and increased LC3-II/LC3-I levels to induce autophagy.Conclusions The combination of auranofin and osimertinib significantly overcomes the acquired resistance to osimertinib in NSCLC cells.Mechanistically,the combination of auranofin and osimertinib increases the expression of HSPB8 and induces autophagy in osimertinib-resistant cells.