BNIP3-mediated mitophagy is involved in multiple physiological and pathological processes, such as limiting the glycolytic shift in radioresistant cancer, promoting the differentiation of cardiac progenitor cells and rescuing renal cells from ischemic injury [10C12]

BNIP3-mediated mitophagy is involved in multiple physiological and pathological processes, such as limiting the glycolytic shift in radioresistant cancer, promoting the differentiation of cardiac progenitor cells and rescuing renal cells from ischemic injury [10C12]. dysfunction, Docebenone but also upregulated FOXO3a expression. Knockdown of FOXO3a aggravated TMZ-induced DNA DSBs and mitochondrial damage, as well as glioma cell death. TMZ treatment not only upregulated BNIP3 and activated autophagy, but also triggered mitophagy Docebenone by prompting BNIP3 translocation to mitochondria and reinforcing BNIP3 interaction with Docebenone LC3BII. Inhibition of mitophagy by knocking down BNIP3 with SiRNA or blocking Docebenone autophagy with 3MA or bafilomycin A1 exacerbated mitochondrial superoxide and intracellular ROS accumulation. Moreover, FOXO3a knockdown inhibited TMZ-induced BNIP3 upregulation and autophagy activation. In addition, we showed that treatment with TMZ (100?mgkg?1d?1, ip) for 12 days in C6 cell xenograft mice markedly inhibited tumor growth accompanied by inducing FOXO3a upregulation, oxidative stress and BNIP3-mediated mitophagy in tumor tissues. These results demonstrate that FOXO3a attenuates temozolomide-induced DNA double strand breaks in human glioma cells via promoting BNIP3-mediated mitophagy. [9]. Although autophagy confers protection to glioma cells against TMZ-induced death [5, 6], it remains unclear whether this protection depends on the removal of damaged mitochondria. Priming damaged mitochondria is a key step leading to mitophagy, although mitochondrial fragmentation following the loss of the mitochondrial membrane potential often precedes mitophagy. In addition to NIX and FUNDC1, BNIP3 (Bcl-2/adenovirus E1B 19-kDa-interacting protein 3) is a receptor that primes damaged mitochondria for autophagic removal [8]. BNIP3-mediated mitophagy is involved in multiple physiological and pathological processes, such as limiting the glycolytic shift in radioresistant cancer, promoting the differentiation of cardiac progenitor cells and rescuing renal cells from ischemic injury [10C12]. Endothelial monocyte-activating polypeptide-II was found to enhance TMZ toxicity in glioma stem cells by inducing BNIP3-mediated mitophagy [13], but it remains elusive whether TMZ alone could trigger mitophagy in glioma cells. FOXO3a (forkhead box transcription factor 3a) is a member of the FOX family. As a transcription factor, FOXO3a regulates multiple cellular functions, such as cell proliferation, differentiation, invasive migration and cell death [14]. A clinical study showed that FOXO3a expression is upregulated with glioblastoma progression and could be used to predict poor patient prognosis [15]. Experimental data showed that FOXO3a Docebenone promoted glioma cell resistance to TMZ by causing nuclear accumulation of -catenin [16]. However, FOXO3a plays dual roles in the regulation of autophagy in cancer cells. FOXO3A not only regulates doxorubicin-induced protective autophagy in hepatocellular carcinoma Met cells [17] but also contributes to brazilin-induced lethal autophagy in osteosarcoma cells [18]. Notably, it was reported to participate in regulating tunicamycin-induced BNIP3 upregulation in cardiomyocytes [19]. Although these previous studies suggested that autophagy activation and BNIP3 expression are both regulated by FOXO3a, it is necessary to clarify whether FOXO3a contributes to BNIP3-mediated mitophagy. Therefore, in this study, we investigated the role of FOXO3a in TMZ-induced glioma cell death and its relationship with BNIP3-mediated mitophagy. Materials and Methods Reagents Temozolomide (TMZ), glutathione (GSH), metalloporphyrin Mn(III) tetrakis(4-benzoic acid)porphyrin (MnTBAP), and anti-LC3B antibodies were purchased from Sigma (St. Louis, MO). TMZ was dissolved in DMSO to a storage concentration of 50?mmol/L. Primary antibodies against the following proteins were purchased from Abcam (Cambridge, MA): FOXO3a, BNIP3, ATG5, p62 (SQSTM1), AIF, TOMM20, COXIV, VDAC1, phospho-H2AX at Ser139, HIF- and H2A. Anti–Actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Other reagents were purchased from Sigma (St. Louis, MO). Glioma cell lines and culture Human U87, U251, T98G, and LN18 glioma cells.