Martinez-Jaramillo Elvis, Jamali Fatemeh, Abdalbari Farah H, Abdulkarim Bassam, Jean-Claude Bertrand J, Telleria Carlos M, Sabri Siham
Experimental Pathology Unit, McGill University, Montréal, QC H3A 2B4, Canada.
Cancer Research Program, Research Institute of the McGill University Health Centre, Montréal, QC H4A 3J1, Canada.
Cancers (Basel). 2024 Jun 25;16(13):2319. doi: 10.3390/cancers16132319.
Glioblastoma (GBM) is the most prevalent and advanced malignant primary brain tumor in adults. GBM frequently harbors epidermal growth factor receptor (EGFR) wild-type () gene amplification and/or 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.
胶质母细胞瘤（GBM）是成人中最常见且最晚期的原发性恶性脑肿瘤。GBM常伴有表皮生长因子受体（EGFR）野生型（）基因扩增和/或激活突变。由EGFR驱动的GBM依赖硫氧还蛋白（Trx）和/或谷胱甘肽（GSH）抗氧化系统来抵御活性氧（ROS）的过量产生。EGFR野生型（EGFRwt）或EGFRvIII过表达对Trx/GSH共同靶向策略反应的影响尚不清楚。在本研究中，我们研究了在GBM中EGFR过表达情况下Trx/GSH的共同靶向作用。金诺芬是一种硫氧还蛋白还原酶（TrxR）抑制剂，已获美国食品药品监督管理局（FDA）批准用于治疗类风湿性关节炎。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）亚基来抑制GSH的合成。我们分析了金诺芬的细胞毒性机制以及金诺芬与L-BSO在U87MG、U87/EGFRwt和U87/EGFRvIII胶质母细胞瘤同基因细胞系中的相互作用。使用抗氧化剂N-乙酰半胱氨酸评估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依赖性机制降低了细胞活力和集落形成，并增加了蛋白质多聚泛素化，这表明ROS在金诺芬诱导的这三种细胞系的细胞毒性中发挥作用。在U87/EGFRvIII细胞中，ROS依赖性聚（ADP-核糖）聚合酶-1（PARP-1）裂解与EGFRvIII下调有关。值得注意的是，无论EGFR过表达情况如何，金诺芬与L-BSO联合使用均能显著耗尽细胞内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受到不同程度的调节。U87/EGFRvIII表现出最显著的ROS增加、磷酸化AKT（Ser-473）和AKT减少，同时伴有EGFRvIII的急剧下调。与U87MG细胞相比，U87/EGFRwt和U87/EGFRvIII显示出较低的基础细胞内GSH水平以及协同的ROS依赖性DNA损伤。我们的研究为金诺芬与L-BSO联合使用在体外GBM中产生ROS依赖性协同细胞毒性提供了证据。阐明EGFR过表达细胞对单独金诺芬以及金诺芬与L-BSO联合使用的敏感性，为在GBM中重新利用这种有前景的促氧化治疗策略提供了理论依据。
