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Abstract：
Glioblastoma (GBM) is the most prevalent and advanced malignant primary brain tumor in adults. GBM frequently harbors epidermal growth factor receptor (EGFR) wild-type (EGFRwt) gene amplification and/or EGFRvIII activating mutation. EGFR-driven GBM relies on the thioredoxin (Trx) and/or glutathione (GSH) antioxidant systems to withstand the excessive production of reactive oxygen species (ROS). The impact of EGFRwt or EGFRvIII overexpression on the response to a Trx/GSH co-targeting strategy is unknown. In this study, we investigated Trx/GSH co-targeting in the context of EGFR overexpression in GBM. Auranofin is a thioredoxin reductase (TrxR) inhibitor, FDA-approved for rheumatoid arthritis. L-buthionine-sulfoximine (L-BSO) inhibits GSH synthesis by targeting the glutamate-cysteine ligase catalytic (GCLC) enzyme subunit. We analyzed the mechanisms of cytotoxicity of auranofin and the interaction between auranofin and L-BSO in U87MG, U87/EGFRwt, and U87/EGFRvIII GBM isogenic GBM cell lines. ROS-dependent effects were assessed using the antioxidant N-acetylsteine. We show that auranofin decreased TrxR1 activity and increased ROS. Auranofin decreased cell vitality and colony formation and increased protein polyubiquitination through ROS-dependent mechanisms, suggesting the role of ROS in auranofin-induced cytotoxicity in the three cell lines. ROS-dependent PARP-1 cleavage was associated with EGFRvIII downregulation in U87/EGFRvIII cells. Remarkably, the auranofin and L-BSO combination induced the significant depletion of intracellular GSH and synergistic cytotoxicity regardless of EGFR overexpression. Nevertheless, molecular mechanisms associated with cytotoxicity were modulated to a different extent among the three cell lines. U87/EGFRvIII exhibited the most prominent ROS increase, P-AKT(Ser-473), and AKT decrease along with drastic EGFRvIII downregulation. U87/EGFRwt and U87/EGFRvIII displayed lower basal intracellular GSH levels and synergistic ROS-dependent DNA damage compared to U87MG cells. Our study provides evidence for ROS-dependent synergistic cytotoxicity of auranofin and L-BSO combination in GBM in vitro. Unraveling the sensitivity of EGFR-overexpressing cells to auranofin alone, and synergistic auranofin and L-BSO combination, supports the rationale to repurpose this promising pro-oxidant treatment strategy in GBM.
摘要：
胶质母细胞瘤（GBM）是成人中最常见和晚期的恶性原发性脑肿瘤。GBM经常携带表皮生长因子受体(EGFR)野生型(EGFRwt)基因扩增和/或EGFRvIII激活突变。EGFR驱动的GBM依赖于硫氧还蛋白（Trx）和/或谷胱甘肽（GSH）抗氧化剂系统来抵抗活性氧（ROS）的过量产生。EGFRwt或EGFRvIII过表达对对Trx/GSH共靶向策略的反应的影响是未知的。在这项研究中,我们在GBM中EGFR过度表达的背景下研究了Trx/GSH共靶向。Auranofin是一种硫氧还蛋白还原酶（TrxR）抑制剂，FDA批准用于类风湿性关节炎。L-丁硫氨酸-磺基肟（L-BSO）通过靶向谷氨酸-半胱氨酸连接酶催化（GCLC）酶亚基来抑制GSH合成。我们分析了金诺芬的细胞毒性机制以及金诺芬与U87MG中L-BSO的相互作用，U87/EGFRwt,和U87/EGFRvIIIGBM等基因GBM细胞系。使用抗氧化剂N-乙酰半胱氨酸评估ROS依赖性作用。我们表明，金诺芬降低了TrxR1活性，增加了ROS。Auranofin通过ROS依赖性机制降低细胞活力和集落形成并增加蛋白质聚泛素化，提示ROS在金诺芬诱导的三种细胞系中的细胞毒性中的作用。ROS依赖性PARP-1裂解与U87/EGFRvIII细胞中EGFRvIII下调相关。值得注意的是,金诺芬和L-BSO联合诱导了细胞内GSH的显着消耗和协同细胞毒性，而与EGFR的过度表达无关。然而，与细胞毒性相关的分子机制在三种细胞系中受到不同程度的调节。U87/EGFRvIII表现出最显著的ROS增加，P-AKT(Ser-473),AKT随着EGFRvIII的大幅下调而下降。与U87MG细胞相比，U87/EGFRwt和U87/EGFRvIII显示较低的基础细胞内GSH水平和协同的ROS依赖性DNA损伤。我们的研究提供了体外GBM中金诺芬和L-BSO组合的ROS依赖性协同细胞毒性的证据。揭示EGFR过表达细胞对单独的金诺芬的敏感性，和协同金诺芬和L-BSO组合，支持在GBM中重新应用这种有前途的促氧化剂治疗策略的理由。
