雷帕霉素和顺铂对Hep-2细胞DNA切除修复交叉互补基因1表达的协同作用

Relationship between ERCC-1 Expression and Combined Effect of mTOR Inhibitor Rapamycin and Cisplatin on Survival of Hep-2 Cells In Vitro

【目的】探讨mTOR抑制剂雷帕霉素和顺铂对喉癌Hep-2细胞协同作用的机制。【方法】Hep-2细胞在雷帕霉素单药浓度为5、20μmol/L;顺铂单药浓度为3、12μmol/L;联合用药浓度为雷帕霉素5μmol/L联合顺铂3μmol/L中分别培养,检测Hep-2细胞AKT,mTOR,S6K和ERCC1(DNA切除修复交叉互补基因1)蛋白分别在3,6,12,24,48h的表达情况。【结果】雷帕霉素单药干预Hep-2细胞12h后,p-mTOR、S6K及ERCC-1表达表达下调,与对照组比较显著性,AKT蛋白表达在48h增加,与对照组比较差异有统计学意义;顺铂单药干预Hep-2细胞时,ERCC-1表达12h后增加,与对照组比较差异有统计学意义,p-mTOR、AKT和S6K蛋白表达差异无统计学意义;联合用药时,AKT蛋白表达在48h增加,与对照组比较差异有统计学意义,p-mTOR、S6K、在12h后表达下降,与对照组比较差异有统计学意义,ERCC-1的表达与对照组比较没有差异有统计学意义。【结论】雷帕霉素和顺铂联合应用时,呈协同作用,这可能与雷帕霉素能诱导肿瘤细胞凋亡及影响ERCC-1的表达有关。

[Objective] To investigate the cytotoxic effect and mechanism of action of cisplatin and the mTOR inhibitor rapamycin on laryngeal cancer（Hep2） cells..[Methods] Hep-2 cells were cultured in the presence of different concentrations of rapamycin,cisplatin or the two combined.A subset of cells was treated with rapamycin only at concentrations of 5 and 20 μmol/L.A further subset of cells was treated with cisplatin only at concentrations of 3 and 12 μmol/L.A final subset of cells was treated with media containing 5 μmol/L rapamycin and 3 μmol/L cisplatin combined.Western blot was used to determine expression of proteins p-Akt,p-mTOR,S6K,and ERCC-1 in 3,6,12,24,and 48 h.[Results] mTOR,S6K and ERCC-1 protein levels significantly decreased after treating with rapamycin 12 h.AKT protein levels significantly increase after treating with rapamycin 48 h.ERCC-1 protein levels significantly increase after treating with cisplatin 12 h.No changes in AKT,p-mTOR and S6K protein expression were apparent for treating with cisplatin.S6K and p-mTOR protein levels significantly decreased after treating with combined drug 12 h.AKT protein levels significantly increase after 48 h.No changes in ERCC-1 protein expression were apparent for this treatment group.[Conclusions] The synergistic effect of rapamycin and cisplatin improves their cytotoxicity on Hep2 cells.The expression of ERCC-1 influencing by rapamycin might be related to this.