顺铂杀伤舌鳞状细胞癌中铁死亡机制研究  

Mechanism of ferroptosis in cisplatin killing tongue squamous cell carcinoma

作　　者：程瑞[1] 钟兆铭 李春燕 齐潇 王学敏 秦汝佳 孙瑞梅[1] 李国萍[1] 李磊[1] 孙传政[1] CHENG Rui;ZHONG Zhaoming;LI Chunyan;QI Xiao;WANG Xuemin;QIN Rujia;SUN Ruimei;Li Guoping;LI Lei;SUN Chuanzheng(Department of Head and Neck Surgery SectionⅡ,Third Affiliated Hospital of Kunming Medical University,Kunming650118,China)

机构地区：[1]昆明医科大学第三附属医院头颈外二科,云南昆明650118

出　　处：《中华肿瘤防治杂志》2023年第8期461-468,共8页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81960543;81260402)。

摘　　要：目的探讨铁死亡在顺铂(DDP)杀伤舌鳞状细胞癌(TSCC)中的作用,以及通过调控铁死亡增加TSCC细胞DDP化疗敏感性的方法与机制。方法实验随机分为空白对照组、DDP组、DDP+铁死亡诱导剂Erastin、DDP+铁死亡抑制剂Liproxstatin-1和DDP+铁死亡抑制剂Fer-1组,每组各3个重复样本,每24 h追加同样等量药物,共24~72 h。应用MTT比色法及细胞内脂质氧化实验分别检测DDP、铁死亡诱导剂Erastin以及DDP联合铁死亡抑制剂(Liproxstatin-1和Fer-1)对TSCC细胞增殖抑制作用及脂质氧化水平,探讨DDP杀伤肿瘤与铁死亡的关系;蛋白质印迹法、实时荧光定量PCR(RT-qPCR)及免疫荧光实验分别检测GPX4、Nrf2和Keap1等蛋白表达水平、GPX4 mRNA表达水平及Nrf2蛋白细胞内定位变化,探讨DDP诱导铁死亡发生的分子机制。结果脂质氧化实验显示,DDP组TSCC细胞内脂质氧化水平(1.701±0.148)明显升高,与空白对照组(1.000±0.000)比较,差异有统计学意义,F=67.775,P<0.001。DDP+Liproxstatin-1组(0.747±0.085)和DDP+Fer-1组(0.954±0.062)与空白对照组比较,差异无统计学意义,F值分别为26.701和1.667,P值分别为0.088和0.921。蛋白质印迹法结果显示,DDP可抑制TSCC细胞GPX4蛋白表达,且具有浓度和时间依赖性。免疫荧光实验显示,DDP诱导TSCC细胞Nrf2蛋白从细胞质移位入细胞核,而铁死亡抑制剂Liproxstatin-1可明显抑制Nrf2蛋白核移位。RT-qPCR分析显示,DDP组CAL27细胞内GPX4 mRNA表达(0.565±0.076)与空白对照组(1.000±0.000)相比,差异有统计学意义,F=98.734,P<0.001。结论DDP抑制TSCC细胞GPX4酶的表达而诱发铁死亡;同时,DDP亦可通过Keap1-Nrf2信号通路,促进胞质中的转录因子Nrf2核移位激活抗氧化基因而诱导铁死亡;铁死亡诱导剂Erastin可增强DDP对TSCC细胞的杀伤毒性。Objective To investigate the role of ferroptosis in the killing of tongue squamous cell carcinoma(TSCC)by cisplatin(DDP),and the method and mechanism of sensitization of TSCC cells to DDP chemotherapy by regulating ferroptosis.Methods The experiment was randomly divided into blank control group,DDP group,DDP combined ferroptosis inducer Erastin group and DDP combined ferroptosis inhibitor(Liproxstatin-1,Fer-1)group with three replicates in each group.The same amount of drug was added to each group every 24hfor a total of 24-72h.MTT and malondialdehyde assays were respectively used to detect the growth inhibition of cisplatin,ferroptosis inducer Erastin,combination of cisplatin and ferroptosis inhibitors(Liproxstatin-1,Fer-1)and the lipid oxidation level in TSCC,and explore the relationship between cisplatin killing tumor and ferroptosis.Western blot,real-time PCR and immunofluorescence were respectively performed for examining the GPX4,Nrf2and Keap1protein expression,the GPX4mRNA expression and the localization of Nrf2protein after cisplatin treatment in TSCC,and exploring the molecular mechanism of cisplatin induced ferroptosis.Results Malondialdehyde experiment showed that the level of lipid oxidation in TSCC cells in DDP group(1.701±0.148)was significantly higher than that in blank control group(1.000±0.000),and the difference was statistically significant(F=67.775,P<0.001).There was no significant difference in lipid oxidation level between DDP+Liproxstatin-1 group(0.747±0.085)and DDP+Fer-1group(0.954±0.062)compared with blank control group,Fvalues were 26.701 and 1.667,Pvalues were 0.088and 0.921,respectively.Western blot showed that DDP inhibited the decrease of GPX4 protein expression in a concentration and time-dependent manner in TSCC cells.Immunofluorescence assay showed that DDP induced Nrf2protein translocation from cytoplasm into nucleus of TSCC cells,and ferroptosis inhibitor Liproxstatin-1inhibited Nrf2protein nuclear translocation significantly.RT-qPCR analysis showed that GPX4mRNA expression

关 键 词：舌鳞癌 顺铂 铁死亡 Erastin 抗氧化 

分 类 号：R739.86[医药卫生—肿瘤]