FOXO3a protects glioma cells against temozolomide-induced DNA double strand breaks via promotion of BNIP3-mediated mitophagy

Chuan He,Guang-Fan Chi,Xuan-Zhong Wang,Shi-Peng Liang,Chong-Cheng Wang,Peng-Fei Ge,Shan Lu,Tian-Fei Luo,Lei Wang,Mei-Hua Piao,Zhen-Chuan Wang

doi:10.1038/s41401-021-00663-y

Abstract

FOXO3a (forkhead box transcription factor 3a) is involved in regulating multiple biological processes in cancer cells. BNIP3 (Bcl-2/adenovirus E1B 19-kDa-interacting protein 3) is a receptor accounting for priming damaged mitochondria for autophagic removal. In this study we investigated the role of FOXO3a in regulating the sensitivity of glioma cells to temozolomide (TMZ) and its relationship with BNIP3-mediated mitophagy. We showed that TMZ dosage-dependently inhibited the viability of human U87, U251, T98G, LN18 and rat C6 glioma cells with IC50 values of 135.75, 128.26, 142.65, 155.73 and 111.60 μM, respectively. In U87 and U251 cells, TMZ (200 μM) induced DNA double strand breaks (DSBs) and nuclear translocation of apoptosis inducing factor (AIF), which was accompanied by BNIP3-mediated mitophagy and FOXO3a accumulation in nucleus. TMZ treatment induced intracellular ROS accumulation in U87 and U251 cells via enhancing mitochondrial superoxide, which not only contributed to DNA DSBs and exacerbated mitochondrial dysfunction, 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 by prompting BNIP3 translocation to mitochondria and reinforcing BNIP3 interaction with LC3BII. Inhibition of mitophagy by knocking down BNIP3 with SiRNA or blocking 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 mg·kg−1·d−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.

Highlights

Glioma is the most common primary malignant brain tumor with a high modality and rate of recurrence [1]
TMZ-induced DNA double strand breaks (DSBs) and mitochondrial damage in glioma cells To investigate the toxic effect of TMZ on glioma cells, human U87, U251, T98G and LN18 glioma cells and rat C6 glioma cells were treated with TMZ at the indicated concentrations for 72 h, and cellular viabilities were determined by the MTT assay
In summary, we found in this study that TMZ inhibited glioma cell growth and induced DNA DSBs and nuclear translocation of apoptosis inducing factor (AIF) in vitro and in vivo, which was accompanied by BNIP3-mediated mitophagy and FOXO3a upregulation

Summary

Introduction

Glioma is the most common primary malignant brain tumor with a high modality and rate of recurrence [1]. As a widely used oral alkylating agent, TMZ is more effective than any other chemotherapeutic drug in prolonging the median survival of glioma patients [2, 3]. Multiple factors, such as upregulated expression of MGMT and multidrug resistance proteins, were reported to limit the toxicity of TMZ in glioma cells [3, 4], emerging evidence has revealed that autophagy plays a crucial role in protecting glioma cells against TMZ treatment [5, 6]. During the course of mitophagy, the mitochondria destined for removal are engulfed into autophagosomes to form mitophagosomes. 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

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