Xu Yingyi, Zhang Na, Chen Cheng, Xu Xinke, Luo Ailing, Yan Yaping, Lu Yanhua, Liu Jianhua, Ou Xinxu, Tan Yonghong, Liang Yufeng, Chen Lihe, Song Xingrong, Liu Xiaoping
Department of Anaesthesiology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, China.
Department of Neurosurgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, China.
Front Oncol. 2022 Mar 17;12:859621. doi: 10.3389/fonc.2022.859621. eCollection 2022.
To clarify the function and mechanisms of sevoflurane (Sev) on ferroptosis in glioma cells.
Different concentrations of Sev were used to treat glioma cells U87 and U251. Ferroptosis inducer Erastin was used to incubate glioma cells combined with Sev and ATF4 siRNA transfection treatment. CCK-8 assay and colorimetric assay were performed to analyze cell viability and Fe concentration, respectively. The releases of reactive oxygen species (ROS) were determined by flow cytometry analysis. Transcriptional sequencing was used to screen the differential genes affected by Sev in U251 cells. The mRNA and protein expression of ferroptosis-associated genes was detected by qRT-PCR and Western blotting.
Sev could suppress cell viability, increase ROS levels and Fe concentration, downregulate the protein expression levels of GPX4, and upregulate transferrin, ferritin, and Beclin-1 in a dose-dependent manner in U87 and U251 cells. The expression of ferroptosis and mitophagy-related gene activating transcription factor 4 (ATF4) was identified to be enhanced by Sev analyzed by transcriptional sequencing. ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1), which is involved in ferroptosis, is a downstream gene of ATF4. Inhibition of ATF4 could interrupt the expression of CHAC1 induced by Sev in U87 and U251 cells. Ferroptosis inducer Erastin treatment obviously inhibited the cell viability, elevated the Fe concentration, and promoted ROS generation in U87 and U251 cells. The protein level of ATF4 and CHAC1 was increased in Erastin-treated U87 and U251 cells. Moreover, the interruption of Sev-induced ferroptosis and CHAC1 activating induced by ATF4 suppression could be reversed by Erastin.
In summary, this study suggested that Sev exposure-induced ferroptosis by the ATF4-CHAC1 pathway in glioma cells.
阐明七氟醚（Sev）对胶质瘤细胞铁死亡的作用及机制。
采用不同浓度的Sev处理胶质瘤细胞U87和U251。使用铁死亡诱导剂Erastin与Sev联合孵育胶质瘤细胞，并进行ATF4小干扰RNA转染处理。分别采用CCK-8法和比色法分析细胞活力和铁浓度。通过流式细胞术分析测定活性氧（ROS）的释放。利用转录组测序筛选Sev在U251细胞中影响的差异基因。通过qRT-PCR和蛋白质免疫印迹法检测铁死亡相关基因的mRNA和蛋白质表达。
Sev可剂量依赖性地抑制U87和U251细胞的活力，提高ROS水平和铁浓度，下调GPX4的蛋白质表达水平，并上调转铁蛋白、铁蛋白和Beclin-1。通过转录组测序分析发现，Sev可增强铁死亡和线粒体自噬相关基因激活转录因子4（ATF4）的表达。参与铁死亡的ChaC谷胱甘肽特异性γ-谷氨酰环转移酶1（CHAC1）是ATF4的下游基因。抑制ATF4可阻断Sev诱导的U87和U251细胞中CHAC1的表达。铁死亡诱导剂Erastin处理明显抑制U87和U251细胞的活力，提高铁浓度，并促进ROS生成。在Erastin处理的U87和U251细胞中，ATF4和CHAC1的蛋白质水平升高。此外，Erastin可逆转ATF4抑制所诱导的Sev诱导的铁死亡和CHAC1激活的中断。
综上所述本研究提示，Sev暴露通过ATF4-CHAC1途径诱导胶质瘤细胞发生铁死亡。
