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 经常含有表皮生长因子受体 (EGFR) 野生型 (EGFRwt) 基因扩增和/或 EGFRvIII 激活突变。 EGFR 驱动的 GBM 依赖硫氧还蛋白 (Trx) 和/或谷胱甘肽 (GSH) 抗氧化系统来承受活性氧 (ROS) 的过量产生。 EGFRwt 或 EGFRvIII 过度表达对 Trx/GSH 联合靶向策略反应的影响尚不清楚。在这项研究中，我们研究了 GBM 中 EGFR 过度表达背景下的 Trx/GSH 联合靶向。 Auranofin 是一种硫氧还蛋白还原酶 (TrxR) 抑制剂，经 FDA 批准用于治疗类风湿关节炎。 L-丁硫氨酸-亚磺酰亚胺 (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和 L-BSO 在 U87MG、U87/EGFRwt 和 U87/EGFRvIII GBM 同基因 GBM 细胞系中的相互作用。使用抗氧化剂 N-乙酰斯坦评估 ROS 依赖性效应。我们发现金诺芬降低了 TrxR1 活性并增加了 ROS。金诺芬通过 ROS 依赖性机制降低细胞活力和集落形成，并增加蛋白质多泛素化，表明 ROS 在金诺芬诱导的三种细胞系细胞毒性中的作用。 U87/EGFRvIII 细胞中 ROS 依赖性 PARP-1 裂解与 EGFRvIII 下调相关。值得注意的是，无论 EGFR 过表达如何，金诺芬和 L-BSO 组合都会诱导细胞内 GSH 的显着消耗和协同细胞毒性。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 表现出最显着的 ROS 增加、P-AKT(Ser-473) 和 AKT 减少，同时 EGFRvIII 急剧下调。与 U87MG 细胞相比，U87/EGFRwt 和 U87/EGFRvIII 显示出较低的基础细胞内 GSH 水平和协同 ROS 依赖性 DNA 损伤。我们的研究为体外 GBM 中金诺芬和 L-BSO 组合的 ROS 依赖性协同细胞毒性提供了证据。阐明 EGFR 过表达细胞对单独使用金诺芬以及协同金诺芬和 L-BSO 组合的敏感性，支持了在 GBM 中重新利用这种有前景的促氧化治疗策略的基本原理。

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.