Hsp90抑制剂减弱肝癌耐药细胞株对索拉非尼诱导铁死亡的耐受性  

Inhibition of Hsp90 attenuates the ferroptosis tolerance of hepatocellular carcinoma cell line to Sorafenib

作　　者：林智才 陈燕丽 苏小清 刘俊 许丽贞 LIN Zhicai;CHEN Yanli;SU Xiaoqing;LIU Jun;XU Lizhen(Chenggong Hospital Affiliated to Xiamen University,Fujian Xiamen 361001,China)

机构地区：[1]厦门大学附属成功医院,福建厦门361001

出　　处：《现代肿瘤医学》2022年第21期3862-3868,共7页Journal of Modern Oncology

基　　金：福建省厦门市医疗卫生项目(指导性项目)基金(编号:3502720189012)。

摘　　要：目的:检测热休克蛋白90(heat shock protein 90,Hsp90)抑制剂协同索拉非尼(Sorafenib)对肝癌耐药细胞的作用机制,并研究其对Sorafenib诱导肝癌耐药细胞铁死亡的增敏效应。方法:MTT法检测格尔德霉素(Geldanamycin)联用Sorafenib在抑制HepG2/Sorafenib细胞增殖中的联合效应;流式细胞术检测HepG2/Sorafenib及HepG2细胞株在Sorafenib作用下,细胞内活性氧(reactive oxygen species,ROS)水平的变化,以及Geldanamycin对细胞内ROS的协同诱导效应;FerroOrange染色法检测HepG2/Sorafenib及HepG2细胞株在Sorafenib作用下,细胞内二价铁离子水平变化;RT-PCR及Western blot实验检测Geldanamycin对铁死亡相关基因SLC7A11及GPX4等转录和表达的调节作用。结果:Geldanamycin可增强HepG2/Sorafenib耐药细胞株对Sorafenib的敏感性,经30 nmol/L浓度的Geldanamycin预处理后,Sorafenib的半数抑制率IC_(50)由原来的(2.6±0.1)×10^(3) nmol/L降为(37.2±6.9)nmol/L,变化差异显著(P<0.01)。当Sorafenib浓度高于32 nmol/L后,二者的联用指数CI小于0.2,表明Geldanamycin与Sorafenib联用处理HepG2/Sorafenib耐药细胞株具有强烈的协同效应;HepG2/Sorafenib耐药细胞株经30 nmol/L Geldanamycin预处理后,其细胞内的ROS水平可重新被Sorafenib诱导,相同浓度下与无Geldanamycin处理组比较,增强了4.2倍(P<0.01);30 nmol/L Geldanamycin可显著增强Sorafenib诱导HepG2/Sorafenib细胞内二价铁离子水平提高,提示其协同Sorafenib促细胞铁死亡作用;Geldanamycin能通过Akt-Nrf2途径下调SLC7A11蛋白的转录,并影响GPX4蛋白的表达。结论:抑制Hsp90能减弱肝癌耐药细胞株对Sorafenib诱导铁死亡的耐受性,其机制可能是通过阻断Akt/Nrf2介导的SLC7A11转录下调实现的。Objective:Detection of heat shock protein 90(Hsp90)inhibitors in synergy with Sorafenib mechanism of action in liver cancer resistant cells,and sensitizing effect on Sorafenib-induced ferroptosis in HepG2/Sorafenib cell line.Methods:MTT was used to detect the combined effect of Geldanamycin and Sorafenib on the inhibition of HepG2/Sorafenib proliferation.Flow cytometry was employed to monitor the reactive oxygen species(ROS)induced via Sorafenib in HepG2/Sorafenib and HepG2 cell lines,and synergistic effect of Geldanamycin to Sorafenib during the induction process.Felevel of HepG2/Sorafenib and HepG2 cell lines were monitored by FerroOrange staining to indicate a process of ferroptosis.RT-PCR and Western blot assay were respectively utilized to probe the transcription and expression of SLC7 A11 and GPX4 in HepG2/Sorafenib cell line.Results:Geldanamycin is able to sensitize HepG2/Sorafenib cell line to Sorafenib,enabling a decrease of IC_(50) of Sorafenib from(2.6±0.1)×10^(3) nmol/L to(37.2±6.9)nmol/L(P<0.01),after pretreatment of 30 nmol/L Geldanamycin.The combine index(CI)was less than 0.2 while the concentration of Sorafenib was higher than 32 nmol/L,indicating strong synergies of Geldanamycin and Sorafenib to HepG2/Sorafenib cell line.In addition,Geldanamycin could elicit the ROS of HepG2/Sorafenib,which was not able to be induced through Sorafenib alone,and the ROS level was elevated to 4.2 times comparing with the assay group without Geldanamycin(P<0.01).The Feaccumulation was significantly enhanced by Geldanamycin in HepG2/Sorafenib cell line,which simultaneously treated with Sorafenib.Finally,Geldanamycin can down-regulate the transcription of SLC7 A11 protein through the Akt-Nrf2 pathway,in turn affecting the expression of GPX4 protein.Conclusion:Inhibition of Hsp90 can attenuate the tolerance of HepG2/Sorafenib cell line to Sorafenib-induced ferroptosis,by blocking Akt/Nrf2 axis-mediated transcription of SLC7 A11.

关 键 词：HSP90 索拉非尼 活性氧 铁死亡 

分 类 号：R735.7[医药卫生—肿瘤]