Generating oxidative stress is a critical mechanism by which host cells defend against infection by pathogenic microorganisms. Radiation resistance is a critical problem in radiotherapy against cancer. Epstein-Barr virus (EBV) is a cancer-causing virus and its reactivation plays an important role in the development of EBV-related tumors. This study aimed to explore the inner relationship and regulatory mechanism among oxidative stress, EBV reactivation, and radioresistance and to identify new molecular subtyping models and treatment strategies to improve the therapeutic effects of radiotherapy./nMethods: ROS, NADP⁺/NADPH, and GSSG/GSH were detected to evaluate the oxidative stress of cells. 8-OHdG is a reliable oxidative stress marker to evaluate the oxidative stress in patients. Its concentration in serum was detected using an ELISA method and in biopsies was detected using IHC. qPCR array was performed to evaluate the expression of essential oxidative stress genes. qPCR, Western blot, and IHC were used to measure the level of EBV reactivation in vitro and in vivo. A Rta-IgG ELISA kit and EBV DNA detection kit were used to analyze the reactivation of EBV in serum from NPC patients. NPC tumor tissue microarrays was used to investigate the prognostic role of oxidative stress and EBV reactivation. Radiation resistance was evaluated by a colony formation assay. Xenografts were treated with NAC, radiation, or a combination of NAC and radiation. EBV DNA load of tumor tissue was evaluated using an EBV DNA detection kit. Oxidative stress, EBV reactivation, and the apoptosis rate in tumor tissues were detected by using 8-OHdG, EAD, and TUNEL assays, respectively./nResults: We found that EBV can induce high oxidative stress, which promotes its reactivation and thus leads to radioresistance. Basically, EBV caused NPC cells to undergo a process of 'Redox Resetting' to acquire a new redox status with higher levels of ROS accumulation and stronger antioxidant systems by increasing the expression of the ROS-producing enzyme, NOX2, and the cellular master antioxidant regulator, Nrf2. Also, EBV encoded driving protein LMP1 promotes EBV reactivation through production of ROS. Furthermore, high oxidative stress and EBV reactivation were positively associated with poor overall survival of patients following radiation therapy and were significant related to NPC patients' recurrence and clinical stage. By decreasing oxidative stress using an FDA approved antioxidant drug, NAC, sensitivity of tumors to radiation was increased. Additionally, 8-OHdG and EBV DNA could be dual prognostic markers for NPC patients./nConclusions: Oxidative stress mediates EBV reactivation and leads to radioresistance. Targeting oxidative stress can provide therapeutic benefits to cancer patients with radiation resistance. Clinically, we, for the first time, generated a molecular subtyping model for NPC relying on 8-OHdG and EBV DNA level. These dual markers could identify patients who are at a high risk of poor outcomes but who might benefit from the sequential therapy of reactive oxygen blockade followed by radiation therapy, which provides novel perspectives for the precise treatment of NPC.

中文翻译：

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靶向 Epstein-Barr 病毒癌蛋白 LMP1 介导的高氧化应激抑制 EBV 裂解再激活并使肿瘤对放射治疗敏感

产生氧化应激是宿主细胞抵御病原微生物感染的关键机制。抗辐射性是癌症放射治疗中的一个关键问题。爱泼斯坦-巴尔病毒 (EBV) 是一种致癌病毒，其再激活在 EBV 相关肿瘤的发展中起重要作用。本研究旨在探讨氧化应激、EBV 再激活和放射抗性之间的内在关系和调控机制，并确定新的分子亚型模型和治疗策略，以提高放射治疗的疗效。/n方法： ROS、NADP ⁺检测/NADPH和GSSG/GSH以评估细胞的氧化应激。8-OHdG 是评估患者氧化应激的可靠氧化应激标志物。使用 ELISA 方法检测其在血清中的浓度，并使用 IHC 检测活组织检查中的浓度。进行qPCR阵列以评估必需氧化应激基因的表达。qPCR、蛋白质印迹和 IHC 用于测量体外和体内EBV 再激活水平. 使用 Rta-IgG ELISA 试剂盒和 EBV DNA 检测试剂盒分析鼻咽癌患者血清中 EBV 的再激活。NPC 肿瘤组织微阵列用于研究氧化应激和 EBV 再激活的预后作用。通过集落形成测定评估辐射抗性。用 NAC、辐射或 NAC 和辐射的组合处理异种移植物。使用 EBV DNA 检测试剂盒评估肿瘤组织的 EBV DNA 载量。分别使用 8-OHdG、EAD 和 TUNEL 测定法检测肿瘤组织中的氧化应激、EBV 再激活和凋亡率。/n结果：我们发现 EBV 可以诱导高氧化应激，从而促进其再激活，从而导致放射抗性。基本上，EBV 通过增加产生 ROS 的酶 NOX2 和细胞主要抗氧化调节剂的表达，使 NPC 细胞经历“氧化还原重置”过程，从而获得具有更高水平 ROS 积累和更强抗氧化系统的新氧化还原状态, NRF2。此外，EBV 编码的驱动蛋白 LMP1 通过产生 ROS 促进 EBV 再激活。此外，高氧化应激和 EBV 再激活与放射治疗后患者的总体生存率低呈正相关，并且与 NPC 患者的复发和临床分期显着相关。通过使用 FDA 批准的抗氧化药物 NAC 减少氧化应激，肿瘤对辐射的敏感性增加。此外，8-OHdG 和 EBV DNA 可能是 NPC 患者的双重预后标志物。/n结论：氧化应激介导 EBV 再激活并导致放射抗性。靶向氧化应激可以为具有抗辐射性的癌症患者提供治疗益处。在临床上，我们首次根据 8-OHdG 和 EBV DNA 水平为 NPC 生成了分子亚型模型。这些双重标志物可以识别出预后不良风险高但可能受益于活性氧阻断序贯治疗和放射治疗的患者，这为鼻咽癌的精准治疗提供了新的视角。