大蒜素对骨肉瘤获得性耐药细胞株的作用及其机制

Effects of diallyl trisulfide on osteosarcoma acquired drug-resistant cell line and its possible mechanism

目的探讨大蒜素(DATS)对骨肉瘤获得性耐药细胞株的作用及其机制。方法体外间歇刺激法建立对阿霉素(DOX)产生获得性耐药的骨肉瘤细胞株Saos-2/DOX,MTT法检测DOX对Saos-2及Saos-2/DOX细胞的半数抑制浓度(IC50),并计算Saos-2/DOX细胞的耐药指数(RI);MTT法筛选DATS处理Saos-2/DOX细胞的非细胞毒性浓度;DATS与DOX单独或联合干预Saos-2/DOX细胞24、48、72 h后,MTT法检测细胞生存率;不同浓度DATS(0、10、50、100μmol/L)处理Saos-2/DOX细胞48 h后,Western blotting法检测细胞中p ERK1/2、ERK1/2、p AKT1和AKT1蛋白表达。结果成功建立对DOX耐药的骨肉瘤细胞株Saos-2/DOX,细胞的RI值为6.94。DATS干预Saos-2/DOX细胞的非细胞毒性浓度为10μmol/L。DOX 100μg/L与DATS 10μmol/L联合处理Saos-2/DOX耐药细胞24 h后细胞存活率显著低于DATS 10μmol/L或DOX 100μg/L单独处理,且随处理时间延长逐渐降低(P均<0.05)。不同浓度DATS处理Saos-2/DOX细胞48 h后,ERK1/2、p AKT1和AKT1蛋白表达变化差异无统计学意义(P均>0.05),而p ERK1/2蛋白表达水平随DATS浓度增高显著增加(P均<0.05)。结论 DATS可能通过激活ERK1/2信号通路,逆转Saos-2/DOX细胞的获得性耐药。

Objective To observe the effects of diallyl trisulfide（ DATS） on the osteosarcoma acquired drug-resistance cell line,and to investigate its possible mechanism. Methods We established an osteosarcoma cell line Saos-2/DOX which acquired drug-resistance to doxorubicin（ DOX） in vitro by the pulse selection. MTT assay was applied to determine the half maximal inhibitory concentration（ IC50） of Doxorubicin（ DOX） on the Saos-2 and Saos-2/DOX cells,besides the resistance index（ RI） of Saos-2/DOX was calculated. Moreover,the DATS concentration treating Saos-2/DOX without cytotoxicity was selected by MTT assay. The cell viability was detected by MTT assay at 24,48,and 72 h after the treatment of DOX alone or in combination with DATS on the Saos-2/DOX cells. At 48 h after the treatment of DATS（ 0,10,50,and 100 μmol/L） on the Saos-2/DOX cells,the protein expression levels of p ERK1/2,ERK1/2,p AKT1,and AKT1 were detected by Western blotting. Results The DOX-resistant osteosarcoma cell line Saos-2/DOX was successfully established and the RI of Saos-2/DOX was 6. 94. The DATS concentration treating Saos-2/DOX cells without cytotoxicity was 10 μmol/L. The survival rate of Saos-2/DOX-resistant cells treated with DOX 100 μg/L and DATS 10 μmol/L for 24 h was significantly lower than that of DOXS 10 μmol/L or DOX 100 μg/L,and decreased over the treatment time（ all P〈0. 05）. At 48 h after treatment of different concentrations of DATS,the protein expression level of p ERK1/2 was significantly up-regulated,which was concentration dependent（ P〈0. 05）. However,no significant difference was found in the protein expression levels of ERK1/2,p AKT1,and AKT1（ all P〉0. 05）. Conclusion DATS could reverse the acquired drug-resistance of Saos-2/DOX cells by activating the ERK1/2 signal pathway.