RSL3 Induces Autophagic Death In Glioma Cells Via Causing Glycolysis Dysfunction

Background:Therapies for glioma mainly consist of surgical excision combined with radiotherapy and chemotherapy.However,the prognosis is still poor on account that glioma is of high malignant degree and resistance to apoptosis.Thus,new clue for glioma therapy is needed urgently.Ferroptosis is a newly discovered form of non-apoptotic cell death characterized by excessive accumulation of iron-dependent lipid reactive oxygen species.Triggering ferroptosis in tumor cells has been regarded as a promising strategy for cancer treatment.Recent studies have reported that autophagy played an important role in ferroptosis.RSL3 is a small molecular compound who triggers ferroptosis by limiting GPX4 activity,it has been reported to induce various cancer cell death.However,report of RSL3 on glioma cell is rare.The specific mechanism of RSL3 on glioma cells and its relationship to autophagy has not been elaborated yet.Glycolysis is the main energy source of tumor cells and triggering glycolysis dysfunction is one of the strategies to kill tumor cells.Although numerous studies have explored the mechanism of RSL3 in ferroptosis-related pathways,it is unclear whether RSL3 affects glycolysis function in tumor cells.Exploring the specific mechanism of RSL3 on glioma cells is conducive to provide theoretical foundation for its clinical application.Objective:Rat C6 glioma cells and human U373,U87,U251 glioma cells and xenograft nude mouse models were established to investigate whether RSL3 could induce autophagic death and its mechanism.Methods:1.MTT assay detects the inhibitory rate of RSL3 on proliferation of glioma cells;LDH release test was used to detect the toxicity of RSL3 on tumor cells and the killing effect of RSL3 after added inhibitor.2.Western blotting was used to detect the autophagy-related protein levels of the samples of RSL3 time-gradient and the samples of RSL3+Inhibitor.3.Fluorescence microscopy was used to observed the morphological characteristics of U87 cells transfected with RFP-GFP-LC3 lentivirus.4.ATP assay kit and pyruvate assay kit were used to detect the ATP and pyruvate levels in glioma cells and xenograft tissue respectively.5.C6 glioma was xenografted in Balb/c nude mice and RSL3 was injected intraperitoneally to observe the drug killing effect on C6 glioma.The levels of autophagy-related protein and the contents of ATP and pyruvate in tumor tissues were detected.Results:1.RSL3 inhibited the survival rate and induced the death of glioma in a dose-dependent manner.2.RSL3 increased the protein levels of ATG5,ATG12,beclin-1,LC3 B and decreased the level of p62.3.Fluorescence microscopy showed that autophagosomes in U87 cells transfected with RFP-GFP-LC3 lentivirus increased significantly after RSL3 treatment and autolysosomes were detected.4.RSL3-induced cell death could be alleviated by pretreatment of 3-MA,baf-A1 and knockdown of ATG5 with siRNA.Pretreatment of 3-MA and knockdown of ATG5 inhibited the changes of autophagy-related protein levels induced by RSL3.Protein levels of LC3 B and p62 increased after baf-A1 pretreatment.5.The ATP and pyruvate contents were decreased in RSL3-treated cells.RSL3 decreased the protein levels of HKII,PFKP and PKM2.6.RSL3 increased the protein levels of AMPK,p-AMPK,ULK1,p-ULK1.Pretreatment of PAS alleviated the cell death as well as the changes of autophagy-related protein levels caused by RSL3.7.The tumor volume of RSL3-treated nude mice was significantly reduced compared with that in control group.The changes of autophagy-related protein levels,ATP and pyruvate contents in tumor tissues treated by RSL3 were consistent with those in vitro experiments.Conclusion1.RSL3 inhibits the survival rate and induces the death of glioma.2.RSL3 induces autophagic death in glioma cells.3.RSL3 induces autophagic death in glioma cells via causing glycolysis dysfunction.