依维莫司联合RSL3诱导肺腺癌细胞铁死亡的机制研究

Mechanism of everolimus combined with RSL3 inducing ferroptosis in lung adenocarcinoma cells

目的探讨依维莫司(EVE)联合铁死亡诱导剂(RSL3)诱导肺腺癌细胞铁死亡的作用及机制。方法人肺腺癌细胞系PC9和H1299分为对照组(药物浓度为0)、不同浓度EVE组、不同浓度RSL3组和EVE+RSL3组,加入相应药物培养。检测铁抑制素1(Fer-1)效果时再加入Fer-1。采用噻唑蓝溴化四唑法检测各组细胞存活率,采用丙二醛(MDA)检测法、铁比色分析法分别检测细胞内MDA、亚铁离子(Fe2+)相对水平;采用Western blot法检测磷酸化哺乳动物雷帕霉素靶蛋白(pmTOR)及谷胱甘肽过氧化物酶(GPX)4相对表达量。结果相比对照组,不同浓度EVE组细胞存活率无明显变化(P>0.05);相比RSL3组,EVE+RSL3组细胞存活率显著降低(P<0.01)。相比未加Fer-1的EVE+RSL3组,加入Fer-1后EVE+RSL3组细胞存活率显著升高(P<0.05)。相比其他3组,EVE+RSL3组细胞内Fe2+和MDA相对水平均显著升高(均P<0.05),p-mTOR和GPX4蛋白相对表达量均显著降低(均P<0.05);相比RSL3组,EVE+RSL3组p-mTOR和GPX4蛋白相对表达量均显著降低(均P<0.01)。结论EVE联合RSL3可能通过mTOR/GPX4通路诱导肺腺癌细胞发生铁死亡。

Objective To investigate the effect and mechanism of everolimus(EVE)combined with RSL3 on ferroptosis in lung adenocarcinoma cells.Methods Human lung adenocarcinoma H1299 and PC9 cells were divided into control group,EVE group,RSL3 group and EVE+RSL3 group.Cells were treated with ferrostatin-1(Fer-1)and cell survival rate was detected by MTT assay.The levels of malondialdehyde(MDA)and ferrous ion in cells were detected by MDA detection method and iron colorimetric analysis method,respectively.The expression of phosphorylated mammalian target of rapamycin(p-mTOR)and glutathione peroxidase 4(GPX4)proteins were detected by Western blot.Results Compared with control group,EVE group with various concentrations alone had not significant effects on cell proliferation(P>0.05).Compared with RSL3 group,the cell survival rate in EVE+RSL3 group was significantly decreased(P<0.01).Compared with EVE+RSL3 group without Fer-1,the cell survival rate in EVE+RSL3 group with Fer-1 was significantly increased(P<0.05).Compared with control group,EVE group and RSL3 group,the levels of Fe2+and MDA in EVE+RSL3 group were both significantly increased(both P<0.05),while the levels of p-mTOR and GPX4 protein were both significantly decreased(both P<0.05).Compared with the RSL3 group,levels of p-mTOR and GPX4 protein in EVE+RSL3 group were both significantly decreased(both P<0.01).Conclusion Everolimus combined with RSL3 may induce ferroptosis in lung adenocarcinoma cells through the mTOR/GPX4 pathway.