高压氧增强人肺癌A549/Gem耐药细胞对吉西他滨的敏感性及其作用机制

Effects of hyperbaric oxygen on enhancing the drug sensitivity of gemcitabine-resistant human lung cancer A549 cell lines to gemcitabine

目的:探讨高压氧(HBO)对人肺癌A549吉西他滨(Gem)耐药细胞的逆转作用及其作用机制。方法:采用体外低浓度梯度递增法构建Gem耐药A549细胞株(A549/Gem)模型。根据实验设计将A549/Gem耐药细胞株分为耐药对照组、高压氧组(HBO组)、Gem组及HBO联合Gem组(HBO+Gem组),处理方式分别为:不做处理,0.2 MPa HBO处理3 h,10.0μmol/L Gem处理24 h,0.2 MPa HBO处理3 h后用10.0μmol/L Gem处理24 h。CCK-8法检测A549/Gem耐药细胞的增殖能力,流式细胞术检测A549/Gem耐药细胞的凋亡水平,Western blotting实验检测A549/Gem耐药细胞凋亡蛋白、耐药蛋白及通道蛋白色素框同源蛋白6(CBX6)表达量的变化。结果:与其他3组比较,HBO+Gem组A549/Gem耐药细胞增殖能力明显受到抑制(P<0.01)。HBO+Gem组A549/Gem耐药细胞凋亡率(32.4%)明显高于对照组(1.08%)、HBO组(5.42%)和Gem组(9.28%),差异均有统计学意义(P<0.01)。与其他3组相比,HBO+Gem组凋亡蛋白Bcl-2表达量明显降低,Bad、caspase-3表达量明显升高,差异均有统计学意义(P<0.01);HBO+Gem组耐药P糖蛋白(P-gp)、多药耐药相关蛋白(MRP)表达量明显降低,差异均有统计学意义(P<0.01);HBO+Gem处理明显上调A549/Gem耐药细胞CBX6的表达(P<0.01)。结论:HBO联合Gem能够显著降低A549/Gem耐药细胞对Gem的耐药性,其主要作用机制是通过调控A549/Gem耐药细胞通道蛋白、耐药蛋白和凋亡相关蛋白的表达,促进A549/Gem耐药细胞凋亡而实现的。

Objective To study the reversal effect and the mechanism of hyperbaric oxygen(HBO)on gemcitabine(Gem)-resistance of human lung cancer A549 cell lines.Methods The A549 cell lines were exposed to stepwise escalating concentration of Gem in vitro until the Gem-resistant A549(A549/Gem)cell lines were developed.The CCK-8 and flow cytometry were used to detect the effect of HBO on the proliferation and apoptosis of A549/GEM cells,respectively,and western blot was used to detect the expression levels of apoptosis proteins,drug-resistant proteins,and the chromobox protein homolog 6(CBX6)in A549/GEM cells.Results Compared with those in the control,HBO,and Gem groups,the proliferation of A549/GEM cells in the HBO+GEM group was significantly inhibited(P<0.01).The apoptosis ratio in the HBO+GEM group was 32.4%,which was much higher than those in the control(1.08%),HBO(5.42%),and GEM groups(9.28%),with statistically significant differences(P<0.01).Western blot results showed that,in the HBO+GEM group,the expression levels of apoptosis proteins,such as Bcl-2,significantly decreased,and the expression levels of Bad and Caspase-3 significantly increased,and all the differences were statistically significant(P<0.01).In the HBO+GEM group,the expression levels of drug-resistant P-glycoprotein(P-gp)and MRP-related protein(MRP)in the A549/GEM cells significantly decreased,and the differences were statistically significant(P<0.01).The expression level of CBX6 in the HBO+GEM group was significantly up-regulated.Conclusion The combination of HBO and Gem can significantly reduce the resistance of A549/GEM cells to Gem,and its main mechanism is to up-regulate the expression level of channel proteins and down-regulate the expression level of drug-resistant proteins,thus regulating the expressions of apoptosis-related proteins and promoting the apoptosis of A549/GEM cells.