DNA-PKcs 介导多药耐药恶性胶质瘤细胞化疗抗性及分子机制

摘要

Objective:To obesrve the influence of DNA-PKcs in the chemoresistance of multi-drug resistance malignant glioma cells,and to explore its molecuIar mechanism in chemoresistance.Methods:siRNA was used to construct the DNA-PKcs knockdown human glioma U251 cell line;Western blotting method was used to detect the expressions of DNA-PKcs in U251 cells (U251 cells), doxorubicin (ADM)resistant U251 cells (U251/ADM cells),DNA-PKcs knockdown and ADM resistant U251 cells (U251/ADM/siDNA-PKcs cells).CCK8 method was used to detect the cell proliferation activity in three groups;Western blotting method was used to detect the expressions of MDR1, pNF-κB/p6, total Akt, pAkt/T308 and pAKT/S473 in the cells in three groups. Results:The expression level of DNA-PKcs in U251/ADM cells was significantly higher than those in U251 cells and U251/ADM/siDNA-PKcs cells (P < 0.01 ).The IC50 values of doxorubicin (ADM),paclitaxel (PTX), gemcitabine (GEM)in U251/ADM/siDNA-PKcs cells were significantly lower than that in U251/ADM cells (P <0.05);the expression levels of pAKT/S473,pNF-κB/p65,and MDR1 in U251/ADM/siDNA-PKcs cells were significantly lower than those in U251/ADM cells (P <0.01),but the total Akt and pAkt/T308 had no significant differences between two groups (P >0.05).Conclusion:DNA-PKcs can significantly enhance the chemoresistance of multi-drug resistance malignant glioma cells,the underlying mechanism is related to up-regulation of pAKT/S473,pNF-κB/p65 and MDR1 expressions.%目的：观察 DNA 依赖的蛋白激酶的催化亚单位（DNA-PKcs）对多药耐药的恶性胶质瘤细胞化疗抗性的影响，探讨其介导化疗抗性的分子机制。方法：采用 siRNA 技术构建 DNA-PKcs 基因表达沉默的人胶质瘤U251细胞株；采用 Western blotting 法检测 U251细胞（U251细胞）、多柔比星（ADM）耐药的 U251细胞（U251／ADM 细胞）和 DNA-PKcs 基因表达沉默的多柔比星耐药的 U251细胞（U251／ADM／siDNA-PKcs 细胞）中 DNA-PKcs 蛋白表达水平；采用 CCK8法检测3组细胞增殖活性；采用 Western blotting 法检测3组细胞中多药耐药性1（MDR1）、报告基因质粒 pNF-κB／p6、总 Akt 蛋白、pAkt／T308和 pAKT／S473蛋白表达水平。结果：U251／ADM细胞 DNA-PKcs 蛋白表达水平 明 显 高 于 U251细胞 和 U251／ADM／siDNA-PKcs 细胞（P ＜0.01）；ADM、紫杉醇（PTX）和吉西他滨（GEM）对 U251／ADM／siDNA-PKcs 细胞的半数抑制浓度（IC50）值较 U251／ADM 细胞明显降低（P ＜0.01）；U251／ADM／SIDNA-PKcs 细胞中 pAKT／S473、 pNF-κB／p65和MDR1蛋白表达水平均明显低于 U251／ADM 细胞（P ＜0.05），而两者总 Akt 和 pAkt／T308蛋白表达水平比较差异无统计学意义（P ＞0.05）。结论：DNA-PKcs 能明显增强多重耐药的恶性胶质瘤细胞化疗抗性，其作用机制与 pAKT／S473和 pNF-κB／p65表达上调，诱导 MDR1表达有关。