FOXO3a Protects Glioma Cells Against Temozolomide-induced DNA Double Strand Breaks Via Promotion Of BNIP3-mediated Mitophagy

Background:Glioma is the most common primary malignant brain tumor with higher modality and recurrence[1,2].The current standard of care for the newly diagnosed malignant glioma includes surgery as a first-line therapy,followed by radiotherapy and adjuvant chemotherapy[3].Temozolomide(TMZ)is a first-line chemotherapy agent,but it is often limited by the establishment of chemo-resistance[4].Accumulating evidence shows as well that autophagy also plays an essential role in decreasing glioma cells susceptible to TMZ treatment[9].Accumulating evidence shows as well that autophagy also plays an essential role in decreasing glioma cells susceptible to TMZ treatment [9-12].Inhibition of autophagy has been demonstrated to be an effective strategy to increase susceptibilities of glioma cells and glioma stem cells to TMZ.However,it remains elusive how autophagy protects glioma cells against TMZ treatment.Mitophagy is a selective form of autophagy,in which the mitochondria destined for removal are encapsulated by phagophore to form autophagosome and then degraded in autolysosome [19].As a part of mitochondrial quality control,mitophagy targets impaired or dysfunctional mitochondria for prompt elimination thereby preventing excessive ROS production and release of mitochondrial pro-death factors [14].Although autophagy enables glioma cells of TMZ-resistance,it remains unclear that whether the clearance of damaged mitochondria is involved in the protective effect of autophagy.As well as NIX and FUNDC1,BNIP3(Bcl-2/adenovirus E1 B 19-k Da-interacting protein 3)is an autophagy receptor accounting for priming mitochondria for autophagy machinery[13]. Forkhead box transcription factor 3a,FOXO3 a is a member of the FOX protein family.It regulated multiple cell process such as proliferation,differentiation,invasion,metastasis and death[16-18].However,it is still unclear whether FOXO3 a is involved in the upregulation of BNIP3-mediated mitophagy.Therefore,we investigated the role that FOXO3 a played in TMZ-induced cell death and the relationship between FOXO3 a and BNIP3-mediated mitophagy.Objective:In this study,we used human and rat glioma cells and BALB/C nude mice model of xenograft glioma to investigate the role of BNIP3 in TMZ-induced glioma cell death and the underlying mechanism.Methods:1.MTT was used to assay cell viability.2.LDH release assay was used to measure cell death induced by TMZ.3.si RNA was transfected to knock down protein expression to investigate their role in TMZ-induced glioma cell death.4.Immunofluorescence staining was used to observe the changes of protein expression in glioma cells through laser scanning confocal microscope.5.DCFH-DA probe was used to measure the ROS levels in glioma cells.6.Mito SOX probe was used to evaluate the mitochondrial superoxide levels in glioma cells.7.JC-1 probe was used to evaluate the mitochondrial potential in glioma cells.8.Mito Tracker probe was used to assay the changes of mitochondria in glioma cells.9.Western Blotting was used to detect the alterations of protein levels in glioma cells and tissues.10.Stub RFP-Sens GFP-LC3 B lentivirus was transfected to U87 cells to evaluate autophagy in glioma cells through laser scanning confocal microscope.11.The mice model of C6 xenograft glioma was used to investigate the effect of TMZ on glioma cells in vivo.MTT and LDH release was used to assay cell viability and death ratio.Results:1.TMZ inhibited glioma cell viability in a does-dependent manner in vitro,and inhibited glioma growth in vivo.2.TMZ induced DNA DSBs,AIF nuclei translocation and decreased mtiochondrial membrane potential in glioma cells.3.TMZ induced intracellular ROS accumulation in glioma cells.Pretreatment of GSH suppressed TMZ-induced DNA DSBs via inhibiting accumulation of intracellular ROS.TMZ also upregulated mitochondrial superoxide.Pretreatment of Mn TBAP inhibited mitochondrial superoxide,resulting in suppression of intracellular ROS accumulation and TMZ-induced DNA DSBs.4.TMZ activated autophagy in glioma cells.Inhibiting autophagy with 3MA,bafilomycin A1 or ATG5 si RNA reinforced TMZ-induced AIF nuclei translocation and intracellular ROS accumulation,thus enhanced the toxocity of TMZ against glioma cells.5.TMZ activated BNIP3-mediated mitophagy.Knockdown of BNIP3 reinforced TMZ-induced AIF nuclei translocation and intracellular accumulation,thus enhanced the toxicity of TMZ against glioma cells.6.Knockdown of FOXO3 a suppressed cyto-protective mitophagy induced by TMZ by inhibiting BNIP3 and LC3 B,reinforced the toxicity of TMZ against glioma cells.Conclusion:1.TMZ induced mitochondria damage via upregulation of oxidative stress,resulting in AIF nuclei translocation intracellular ROS accumulation,DNA DSBs,and glioma cell death.2.BNIP3-mediated mitophagy protects glioma cells against TMZ via inhibition of oxidative stress by removing of damaged mitochondria.3.Inhibition of BNIP3-mediated mitophagy regulated by FOXO3 a enhanced the toxicity of TMZ against glioma cells.