双氢青蒿素和索拉非尼诱导未分化甲状腺癌铁死亡的协同机制

Synergistic mechanisms of ferroptosis in anaplastic thyroid cancer induced by dihydroartemisinin and sorafenib

目的探究双氢青蒿素(dihydroartemisinin, DHA)和索拉非尼(sorafenib, SOR)诱导未分化甲状腺癌(anaplastic thyroid cancer, ATC)细胞发生铁死亡的协同作用及分子机制。方法以细胞计数试剂盒(CCK-8)和流式细胞仪检测DHA和SOR对ATC细胞CAL-62增殖及铁死亡的影响;实时荧光定量PCR和蛋白印迹检测铁死亡相关基因谷胱甘肽过氧化酶4(GPX4)、溶质载体家族7成员11基因(SCL7A11)、脂氧合酶-15(LOX-15)及p53表达水平的变化;对应的检测试剂盒检测铁死亡中间代谢产物乳酸脱氢酶(LDH)、谷胱甘肽(GSH)、丙二醛、亚铁离子(Fe2+)、一氧化氮(NO)、活性氧簇(ROS)水平;建立裸鼠肿瘤模型分析DHA和SOR对ATC在体内的抑制作用。结果与对照组相比, DHA、SOR和DHA+SOR处理均呈剂量依赖性抑制细胞增殖(P<0.001), LDH、Fe2+、丙二醛及ROS含量明显增多, GSH活性明显降低(P<0.001), 其作用均被硫酸亚铁(FeSO4)促进, 被铁抑素-1逆转。与对照组及单独用药组比较, DHA+SOR组15-LOX-2及p53表达上调, GPX4和SCL7A11水平降低(P<0.001), 15-LOX-1蛋白含量差异无统计学意义;此外, DHA+SOR组NO的含量显著降低(P<0.001)。DHA和SOR在体抑制ATC肿瘤生长。结论 DHA与SOR协同作用上调15-LOX-2基因的表达, 抑制NO的合成, 从而诱导ATC细胞发生铁死亡。

Objective To investigate the synergistic effects and molecular mechanisms of dihydroartemisinin(DHA)and sorafenib(SOR)in inducing ferroptosis in anaplastic thyroid cancer(ATC)cells.Methods CCK-8 and flow cytometry assays were performed to detect the effects of DHA and SOR on the proliferation and ferroptosis of ATC cells(CAL-62).Real-time fluorescence quantitative PCR and Western blotting assays were performed to detect the expressions of ferroptosis-related genes glutathione peroxidase 4(GPX4),solute carrier family 7 member 11 gene(SCL7A11),lipoxygenase-15(LOX-15),and p53.The levels of iron death intermediate metabolites including lactate dehydrogenase(LDH),glutathione(GSH),malondialdehyde(MDA),ferrous ion(Fe2+),nitric oxide(NO),and reactive oxygen species(ROS)were measured by corresponding assay kits.The corresponding inhibition of DHA and SOR on ATC in vivo was analyzed in a tumor model in nude mice.Results Compared with the control group,DHA,SOR,and DHA+SOR treatment significantly inhibited cell proliferation and apoptosis in a dose-dependent manner(P<0.001),with increased LDH,Fe2+,MDA,and ROS contents and reduced GSH activity(P<0.001),which were promoted by ferrous sulfate(FeSO4)and reversed by ferroptosis inhibitor-1.Compared with the control group and the drug monotherapy group,15-LOX-2 and p53 expressions were upregulated in DHA+SOR group while GPX4 and SCL7A11 expressions were decreased(P<0.001),without significant difference in 15-LOX-1 protein content.In addition,NO level was significantly increased in DHA+SOR group(P<0.001).DHA and SOR inhibited tumor growth of ATC in vivo.Conclusion DHA and SOR synergistically induced ferroptosis via upregulating the expression of 15-LOX-2 gene and inhibiting NO synthesis in ATC cells.