| Background:Gliomas are the most common primary tumors in the brain,representing 81%of central nervous system(CNS)malignancies.They usually originate from glial or precursor cells and develop into astrocytoma,oligodendroglioma,ependymoma,or oligoastrocytoma.Glioblastoma,which accounts for 50%of all gliomas,is the deadliest type of primary brain tumor,characterized by aggressiveness,recurrence,treatment resistance and poor clinical prognosis.Despite current progress in understanding the biology of these tumors and advances in surgery,chemotherapy and radiotherapy,continues to have a very poor prognosis,with and only 4.3%of patients reaching 5-year survival.Ferroptosis is a nonapoptotic,oxidative cell death and identified as a potential anticancer mechanism in recent years.Erastin acts as a ferroptosis activator and shows a potential role in tumor treatment.Tumor microenvironment usually shows a certain degree of hypoxia and its contributed much to radiotherapy resistance and chemoresistance of cancer,but the relationship between hypoxia and erastin resistance has not been explained.Objective:The purpose of this study is to explore the relationship between hypoxia and erastin resistance,elaborate its potential molecular mechanism through experiments in vivo and in vitro,and provide a new idea for the treatment of glioblastoma.Methods:The correlation between HIF-1α and GPX4 expression was detected in data from the TCGA database and was determined by ChIP-qPCR assay.Lipid peroxides level was analyzed by Malondialdehyde(MDA)assay and flow cytometry using C11-BODIPY dye.Western blot were used to explore and verify the regulatory relationship between PI3K,Akt,HIF-1α and GPX4.The effect of combination of PX-478 and erastin in glioblastoma was determined by Cell Counting Kit-8(CCK-8)assay,Clone formation assay,TUNEL assays and flow cytometry.A subcutaneous xenograft and orthotopic xenograft model was established to test our findings in vivo.Results:1.Hypoxia for at least 16 hours significantly suppressed erastin-induced ferroptosis in U87 and U251 cells and the time of cell culture in the hypoxia condition was determined for the follow-up experiment.2.GPX4 acted as a potential effector molecular of hypoxia and hypoxia enhanced the resistance to erastin via increasing GPX4 expression.And PI3K/AKT/HIF-la pathway involved in the up-regulation of GPX4 induced by hypoxia.AKT inhibitor MK-2206 and HIF-1α inhibitor PX-478 could significantly reverse the effect.3.The expression of HIF-1α was positively correlated with GPX4,and HIF-la can indeed bind to the GPX4 promoter region-131 bp to 1 bp and promoted GPX4 transcription.Therefore,Hypoxia enhances the resistance to ferroptosis induced by erastin by up-regulating GPX4 via PI3K/AKT/HIF-1α axis in U87 and U251 cells.4.HIF-1α inhibitorPX-478 could induce a higher lipid peroxidation level by decreasing GPX4 expression in U87 and U251 cells but cannot induced cell death directly,and it could significantly enhance the tumor cell killing effect of erastin.In vivo,combination of PX-478 and erastin had a coordinated intensification effect on anticancer activity uncovered by subcutaneous xenograft and orthotopic xenograft mouse model.Conclusions:Hypoxia enhanced glioblastoma resistance to erastin-induced ferroptosis by activating PI3K/AKT/HIF-1α pathway and promoting GPX4 expression in a transcriptional regulation way.Combination therapy of PX-478 and erastin has synergistic effect on the killing of glioblastoma in vivo and in vitro,which may be a potential strategy against glioblastoma. |
