H_2O_2氧化刺激对紫杉醇耐药和敏感肺癌细胞的不同影响

The different reactions of chemo-resistant and chemo-sensitive lung adenocarcinoma cells to redox stimulation of H_2O_2

目的:探讨人肺腺癌细胞株A549及紫杉醇(paclitaxel)耐药细胞株A549/Taxol对于H2O2氧化刺激的不同反应性及可能的作用机制。方法:用不同浓度的H2O2处理A549及A549/Taxol细胞后,采用细胞计数试剂盒-8(cell counting kit-8,CCK-8)检测细胞存活率,并计算半数细胞抑制浓度(half inhibitory concentration,IC50)值;采用荧光探针氯甲基-2’,7’-二氯二氢荧光素二乙酯[5-(and-6)-chloromethyl-2’,7’-dichlorouorescein diacetateacctyl ester,CM-H2DCFDA]染色法检测细胞内活性氧(reactive oxygen species,ROS)水平;四甲基罗丹明甲酯(tetramethylrhodamine methyl ester perchlorate,TMRM)染色法检测细胞线粒体膜电位水平;AnnexinⅤ-FITC/PI双染法检测细胞凋亡率;实时荧光定量-PCR法检测细胞内超氧化物歧化酶1(superoxide dismutase1,SOD1)、SOD2、过氧化氢酶(catalase,CAT)、γ-谷氨酰半胱氨酸链接酶(glutamate-cysteineligase,GCL)、谷胱甘肽合成酶(glutathione synthetase,GSS)和谷胱甘肽还原酶(glutathione reductase,GSR)mRNA表达的变化。结果:耐药细胞株A549/Taxol对H2O2的IC50值高于亲本细胞株A549;采用低毒浓度H2O2(50和100μmol/L)作用2h后,A549/Taxol细胞中的ROS水平增高幅度显著低于A549细胞,A549/Taxol细胞的线粒体膜电位保持相对稳定而A549细胞显著增高,且A549/Taxol细胞的凋亡发生率显著低于A549细胞。A549/Taxol细胞中抗氧化系统相关基因SOD1、SOD2、CAT、GCL、GSS和GSRmRNA的表达量高于A549细胞;在H2O2作用后,A549细胞中上述基因表达量显著增高,而A549/Taxol细胞中仅GSRmRNA表达上调。结论:紫杉醇耐药细胞对氧化刺激的耐受能力显著高于敏感细胞株,其机制可能与耐药细胞自身氧化-还原平衡系统的适应性变化有关。

Objective： To investigate the different reactions of paclitaxel-resistant subline A549/Taxol and its parental sensitive cell line A549 to redox stimulation of H202 and to explore its possible mechanism. Methods： Both A549/Taxol and A549 cell lines were treated with different concentrations of H202. The survival rate of the cells was determined by CCK-8 （cell counting kit-8） assay and the ICso （half inhibitory concentration） value of H202 was calculated. The change of intracellular ROS （reactive oxygen species） level was detected by using CM-H2DCFDA [5-（and-6）-chloromethyl-2＇,7＇-dichlorodihydrofluorescein diacetate acetyl ester] staining assay. The mitochondrial membrane potential level was examined by TMRM （tetramethylrhodamine methyl ester） staining assay. The apoptosis rate was examined by AnnexinY-FITC/ PI double staining. The changes of mRNA levels of anti-redox-related genes including SOD1 （superoxide dismutase 1）, SOD2, CAT （catalase）, GCL （glutamate-cysteine ligase）, GSS （glutathione synthetase） and GSR （glutathione reductase） were detected by real-time fluorescence-based quantitative PCR. Results： The ICs0 value of A549/Taxol cells was significantly lower than that of A549 cells after redox stimulation of H202. After short-term 2 h treatment with low dose of H202 （50 and 100 IJmol/L）, the increasing degree of ROS level in A549 cells was significantly higher than that in A549/Taxol cells, the mitochondrial membranepotential of A549/Taxol cells remained relatively stable while that of A549 cells rised significantly, and the apoptosis rate of A549/Taxol cells was significantly lower than that of A549 cells. The mRNA expression levels of anti-redox-related genes including SOD1, SOD2, CAT, GCL, GSS and GSR were higher in A549/ Taxol cells than those in A549 cells. The expression levels of these genes were increased significantly in A549 cells, but in A549/Taxol cells, only the expression level of GSR mRNA was increased slightly after redox stimulation of H202. Conclusion： The resistant ability to redox stimulation for paclitaxel-resistant A549/Taxol cells was significantly stronger than of parental sensitive A549 cells. This mechanism may be associated with the adaptive balancinq of oxidation-reduction system in A549/Taxol cells.