青蒿素衍生物DHAARTS诱导肾癌细胞系786-0发生铁死亡的机制  

Mechanism of iron death induced by artemisinin derivative DHAARTS in 786-0 cell line

作　　者：黄成丽 林芳荣 李根 王忠玲 王雪娇[1] HUANG Chengli;LIN Fangrong;LI Gen;WANG Zhongling;WANG Xuejiao(Department of Outpatient, The First Affiliated Hospital of Hainan Medical University, Haikou 570145, China)

机构地区：[1]海南医学院第一附属医院门诊部,海南海口570145

出　　处：《西部医学》2022年第2期178-184,共7页Medical Journal of West China

摘　　要：目的探讨双氢青蒿素(DHA)/青蒿琥酯(ARTS)诱导肾癌细胞系786-0发生铁死亡的机制。方法将DHA/ARTS刺激肾癌细胞系786-0,确定DHA/ARTS所诱导的肾癌细胞死亡即为铁死亡;利用RT-qPCR、Western blot等实验技术,观察NCOA4和FTH1在DHA/ARTS作用后的变化;两种不同siRNA敲减NCOA4后,观察DHA/ARTS对786-0作用的变化;构建pmCherry-EGFP-FTH1质粒和荧光显色,过表达该质粒,观察DHA/ARTS作用后荧光颜色及亮度变化,判断FTH1的自噬降解情况;敲减NCOA4后再转染pmCherry-GFP-FTH1质粒,检测DHA/ARTS作用后荧光颜色及亮度变化,探索NCOA4调节FTH1的自噬降解。结果786-0细胞系在DHA和ARTS作用后,从显微镜观察和CCK-8检测,显示细胞发生了明显死亡,并且随着药物浓度的增大存活细胞数越少。而且由DHA和ARTS所产生的致死作用可以被铁死亡抑制剂DFO和Fer-1所抑制。DHA和ARTS作用下786-0细胞内ROS的荧光强度明显高于对照组,差异有统计学意义(q=2.894,2.342;P<0.05)。在铁死亡抑制剂DFO和Fer-1干预下,DHA+DFO组、DHA+Fer-1组、ARTS+DFO组、ARTS+Fer-1组786-0细胞内ROS的荧光强度明显低于DHA组和ARTS组(P<0.05)。从自噬结果来看,DHA组和ARTS组的细胞自噬率明显高于对照组(P<0.05),并且50μM的DHA和ARTS组细胞的自噬率明显高于20μM的DHA和ARTS组(P<0.05)。在siRNA-NCOA4沉默NCOA4表达后,细胞自噬率明显降低(P<0.05)。结论青蒿素衍生物DHA和ARTS可诱导786-0肾癌细胞系发生铁死亡,其致铁死亡的发生是通过NCOA4介导FTH1特异性自噬降解而实现的。Objective To investigate the mechanism of iron death induced by dihydroartemisinin(DHA)/artesunate(ARTS)in kidney cancer cell line 786-0.Methods After DHA/ARTS stimulated kidney cancer cell line 786-0,the renal cancer cell death induced by DHA/ARTS was determined as iron death.The changes of NCOA4 and FTH1 after DHA/ARTS were observed by RT-qPCR and Western blot.The effects of DHA/ARTS on 786-0 were observed after NCOA4 was knocked down by two different siRNA.The plasmid pmCherry-EGFP-FTH1 was constructed and overexpressed.The changes of fluorescence color and brightness after DHA/ARTS were observed to determine the autophagy degradation of FTH1.After NCOA4 was deleted,pmCherry-GFP-FTH1 plasmid was transfected.The fluorescence color and brightness of DHA/ARTS were detected,and the autophagy degradation of FTH1 was regulated by NCOA4.Results 786-0 cell line treated with DHA and ARTS,the cell death was observed by microscope and CCK-8,and the number of viable cells decreased with the increase of drug concentration.The lethal effects of DHA and ARTS could be inhibited by iron death inhibitors DFO and Fer-1.The fluorescence intensity of ROS in 786-0 cells under DHA and ARTS was significantly higher than that in control group,and the difference was statistically significant(2.894,2.342;P<0.05).The fluorescence intensity of ROS in 786-0 cells of DHA+DFO,DHA+Fer-1,ARTS+DFO,ARTS+Fer-1 group was significantly lower than that of DHA and ARTS group(P<0.05).The autophagy rates of DHA and ARTS groups were significantly higher than those of control group(P<0.05),and the autophagy rates of 50μM DHA and ARTS groups were significantly higher than those of 20μM DHA and ARTS groups(P<0.05).After NCOA4 expression was silenced by siRNA-NCOA4,autophagy rate was significantly decreased(P<0.05).Conclusion Artemisinin derivatives DHA and ARTS can induce iron death in 786-0 renal cancer cell lines,which is realized by NCOA4 mediated specific autophagy degradation of FTH1.

关 键 词：双氢青蒿素 青蒿琥酯 铁死亡 NCOA4 FTH1 

分 类 号：R737.11[医药卫生—肿瘤]