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/orEGFRvIIIactivating 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.
胶质母细胞瘤（GBM）是成人中最常见且恶性程度最高的原发性脑肿瘤。GBM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依赖硫氧还蛋白（Trx）和/或谷胱甘肽（GSH）抗氧化系统来应对过量产生的活性氧（ROS）。EGFRwt或EGFRvIII过表达对Trx/GSH协同靶向治疗策略反应的影响尚不明确。本研究探讨了在GBM中EGFR过表达背景下Trx/GSH协同靶向的作用。金诺芬是一种硫氧还蛋白还原酶（TrxR）抑制剂，已获FDA批准用于治疗类风湿关节炎。L-丁硫氨酸-亚砜亚胺（L-BSO）通过靶向谷氨酸-半胱氨酸连接酶催化（GCLC）亚基抑制GSH合成。我们在U87MG、U87/EGFRwt和U87/EGFRvIII三种GBM同源细胞系中分析了金诺芬的细胞毒性机制及其与L-BSO的相互作用，并使用抗氧化剂N-乙酰半胱氨酸评估了ROS依赖性效应。研究发现，金诺芬可降低TrxR1活性并增加ROS水平。通过ROS依赖机制，金诺芬降低了细胞活力和集落形成能力，并增加了蛋白质多聚泛素化，表明ROS在金诺芬诱导的三种细胞系细胞毒性中发挥关键作用。在U87/EGFRvIII细胞中，ROS依赖的PARP-1切割与EGFRvIII下调相关。值得注意的是，无论EGFR是否过表达，金诺芬与L-BSO联用均能显著耗竭细胞内GSH并产生协同细胞毒性。然而，三种细胞系中与细胞毒性相关的分子机制调节程度存在差异：U87/EGFRvIII细胞表现出最显著的ROS增加、P-AKT(Ser-473)和AKT降低，以及EGFRvIII的急剧下调；与U87MG细胞相比，U87/EGFRwt和U87/EGFRvIII细胞的基础细胞内GSH水平更低，且表现出协同的ROS依赖性DNA损伤。本研究为金诺芬与L-BSO联用在GBM体外模型中产生ROS依赖性协同细胞毒性提供了证据。揭示EGFR过表达细胞对金诺芬单药及金诺芬-L-BSO联合治疗的敏感性，为在GBM中重新定位这一具有前景的促氧化治疗策略提供了理论依据。
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