雷帕霉素增强RSL3对睾丸癌细胞I-10的增殖、侵袭与迁移的抑制作用

Rapamycin enhances inhibitory effect of RSL3 on proliferation,invasion and migration of testicular cancer I-10 cells in vitro

目的探究雷帕霉素增强RSL3对睾丸癌细胞I-10的增殖、侵袭与迁移能力的抑制作用。方法MTT法检测RSL3(TypeII)及合用雷帕霉素(RAPA)作用后I-10细胞的存活率。将RSL3(0~16μmol/L)分别以及联合16μmol/L雷帕霉素作用于睾丸癌细胞I-10,采用MTT法检测I-10细胞增殖抑制情况,利用集落克隆形成实验检测细胞增殖能力,采用划痕实验和Transwell小室检测I-10细胞的迁移与侵袭能力,采用流式细胞术检测I-10细胞的脂质活性氧水平,采用GSH和MDA检测试剂盒检测细胞中GSH和MDA含量,利用Westernblot检测细胞GPX4蛋白表达。结果随着RSL3浓度的提高,其对I-10细胞的毒性作用逐渐增强。在合用雷帕霉素后,对细胞的毒性进步一增强。与对照组相比,单用RSL3组I-10细胞集落形成数减少(P<0.05)、划痕愈合率降低(P<0.01)、侵袭和迁移细胞数减少(P<0.05)、脂质活性氧水平升高(P<0.0001)、GSH含量降低(P<0.01)、MDA含量升高(P<0.05)、GPX4蛋白表达水平降低(P<0.01);与单用RSL3组相比,雷帕霉素联用RSL3组集落形成数、侵袭和迁移细胞数进一步减少(P<0.05),划痕愈合率、GSH含量、GPX4蛋白表达水平进一步降低(P<0.01,P<0.05),脂质活性氧水平、MDA含量进一步升高(P<0.05,P<0.01)。结论雷帕霉素增强RSL3对睾丸癌细胞I-10的增殖、侵袭与迁移的抑制作用,其机制与其能够增强RSL3诱导的铁死亡有关。

Objective To investigate the effect of rapamycin for enhancing the inhibitory effect of RSL3 on proliferation,invasion and migration of testicular cancer I-10 cells in vitro.Methods I-10 cells treated with 0-16μmol/L RSL3(Type II)either alone or in combination with 16μmol/L rapamycin(RAPA)were examined for changes in proliferation using MTT assay and colony-forming assay,and the changes in cell migration and invasion abilities were detected with wounding-healing assay and Transwell assay.The changes in the levels of lipid reactive oxygen species in the treated cells were detected using flow cytometry.GSH and MDA contents in the cells were detected using commercial detection kits,and GPX4 protein expression level was determined with Western blotting.Results The cytotoxic effect of RSL3 increased dose-dependently in I-10 cells,and the combined treatment with rapamycin further enhanced its cytotoxicity.Treatment of I-10 cells with RSL3 alone significantly decreased cell colony numbers(P<0.05),wounding-healing rates(P<0.01),and invasion and migration cell numbers(P<0.05),increased lipid reactive oxygen species level and MDA content(P<0.05),and lowered GSH content and expression level of GPX4 protein in the cells(P<0.01).The inhibitory effects of RSL3 were significantly enhanced by co-treatment of the cells with rapamycin(P<0.05 or 0.01).Conclusion Rapamycin enhances the inhibitory effect of RSL3 on proliferation,invasion and migration of I-10 cells by enhancing RSL3-mediated cell ferroptosis.