Ying Liu, Zu-An Zhu, Qing-Hua Liu, Qing-Yan Kong, Lei Liu, Tao Cui, Yong-Ping Wu
Department of Pathology, Xuzhou Medical College, Xuzhou, China.
Tumour Biol. 2014 Sep;35(9):9171-7. doi: 10.1007/s13277-014-1719-1. Epub 2014 Jun 12.
To investigate the role of RAD001 in the reversing of drug resistance of SGC7901/DDP, we cultured SGC7901/DDP cells with different groups of drugs (RAD001, cisplatin (DDP) alone, or the combination of RAD001 and DDP); after that, we detected the drug sensitivity, cell apoptosis, and levels of P-gp, MRP1, and survivin in the cells of SGC7901/DDP by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphe-nyltetrazolium bromide) assay, flow cytometry, immunohistochemical analysis, and Western blot analysis. There was no significant difference between DDP 2.5-mg/L group and negative control group. When the cells were pretreated with RAD001 2.5, 5 nmol/L, the proliferation of SGC7901/DDP cells was inhibited by DDP 2.5 mg/L significantly, compared to negative control group, DDP 2.5-mg/L group, and RAD001 2.5, 5-nmol/L group, respectively (P < 0.05); there were significant differences between combination groups (P < 0.05). DDP 2.5 mg/L and RAD001 2.5 nmol/L did not induce apoptosis of SGC7901/DDP cells alone (P > 0.05). When SGC7901/DDP cells were pretreated with RAD001 2.5 nmol/L, DDP 2.5 mg/L increased the apoptosis rate significantly compared to groups of control and DDP 2.5 mg/L alone (P < 0.05). Immunohistochemical staining (Table 5, Fig. 2) and Western blot analysis (Fig. 3) indicated that when SGC7901/DDP cells were pretreated with RAD001 2.5 nmol/L, the expression of P-gp, MRP1, and survivin decreased by different degrees. Our results have confirmed that RAD001 in combination with DDP could overcome chemoresistance of SGC7901/DDP cells by decreasing the levels of P-gp, MRP1, and survivin through the mTOR pathway.
为研究RAD001在逆转SGC7901/DDP耐药性中的作用，我们用不同组药物（RAD001、单独顺铂（DDP）或RAD001与DDP联合）培养SGC7901/DDP细胞；之后，我们通过MTT（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐）法、流式细胞术、免疫组织化学分析和蛋白质印迹分析检测SGC7901/DDP细胞的药物敏感性、细胞凋亡以及P-糖蛋白、多药耐药相关蛋白1（MRP1）和生存素的水平。2.5 mg/L DDP组与阴性对照组之间无显著差异。当细胞用2.5、5 nmol/L RAD001预处理时，与阴性对照组、2.5 mg/L DDP组及2.5、5 nmol/L RAD001组相比，2.5 mg/L DDP对SGC7901/DDP细胞增殖的抑制作用显著增强（P < 0.05）；联合组之间存在显著差异（P < 0.05）。2.5 mg/L DDP和2.5 nmol/L RAD001单独处理均未诱导SGC7901/DDP细胞凋亡（P > 0.05）。当SGC7901/DDP细胞用2.5 nmol/L RAD001预处理时，与对照组和单独2.5 mg/L DDP组相比，2.5 mg/L DDP显著提高了凋亡率（P < 0.05）。免疫组织化学染色（表5，图2）和蛋白质印迹分析（图3）表明，当SGC7901/DDP细胞用2.5 nmol/L RAD001预处理时，P-糖蛋白、MRP1和生存素的表达不同程度降低。我们的结果证实，RAD001与DDP联合可通过mTOR途径降低P-糖蛋白、MRP1和生存素水平，从而克服SGC7901/DDP细胞的化疗耐药性。
