Targeting Epstein-Barr virus oncoprotein LMP1-mediated high oxidative stress suppresses EBV lytic reactivation and sensitizes tumors to radiation therapy.
Targeting Epstein-Barr virus oncoprotein LMP1-mediated high oxidative stress suppresses EBV lytic reactivation and sensitizes tumors to radiation therapy.
复制标题
靶向 Epstein-Barr 病毒癌蛋白 LMP1 介导的高氧化应激可抑制 EBV 溶解再激活并使肿瘤对放射治疗敏感。
作者：
Hu J;Li Y;Li H;Shi F;Xie L;Zhao L;Tang M;Luo X;Jia W;Fan J;Zhou J;Gao Q;Qiu S;Wu W;Zhang X;Liao W;Bode AM;Cao Y
产生氧化应激是宿主细胞防御病原微生物感染的关键机制。放射抵抗是肿瘤放射治疗中的一个关键问题。EB病毒（Epstein-Barr virus，EBV）是一种致癌病毒，其再活化在EBV相关肿瘤的发生中起重要作用。本研究旨在探讨氧化应激、EB病毒再活化与放射抵抗的内在联系和调控机制，寻找新的分子分型模型和治疗策略，以提高放射治疗的疗效。方法：以ROS、NADP+/NADPH、GSSG/GSH为指标，检测细胞的氧化应激。8-OHdG是一个可靠的氧化应激指标，以评估患者的氧化应激。使用ELISA方法检测血清中的浓度，并使用IHC检测活检组织中的浓度。进行qPCR阵列以评估必需氧化应激基因的表达。使用qPCR、Western印迹和IHC来测量体外和体内EBV再活化的水平。用Rta-IgG ELISA试剂盒和EBVDNA检测试剂盒检测鼻咽癌患者血清中EB病毒的再活化情况。NPC肿瘤组织微阵列被用来研究氧化应激和EBV再激活的预后作用。通过集落形成测定评价辐射抗性。用NAC、放射或NAC和放射的组合处理异种移植物。用EBVDNA检测试剂盒检测肿瘤组织中的EBVDNA载量。分别采用8-OHdG法、EAD法和TUNEL法检测肿瘤组织中的氧化应激、EBV再活化和细胞凋亡率。结果：EB病毒可诱导机体产生高水平的氧化应激，促进其活化，从而产生辐射抗性。基本上，EBV导致NPC细胞经历“氧化还原重置”过程，通过增加ROS产生酶NOX 2和细胞主抗氧化调节剂Nrf 2的表达，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。此外，EBV编码的驱动蛋白LMP 1通过产生ROS促进EBV再活化。此外，高氧化应激和EBV再激活与放疗后患者的总生存率低呈正相关，并与NPC患者的复发和临床分期显著相关。通过使用FDA批准的抗氧化剂药物NAC降低氧化应激，肿瘤对辐射的敏感性增加。8-OHdG和EBVDNA可作为判断鼻咽癌预后的双重指标。结论：氧化应激介导EBV再激活并导致辐射抗性。靶向氧化应激可以为具有辐射抗性的癌症患者提供治疗益处。在临床上，我们首次建立了以8-OHdG和EBVDNA水平为基础的鼻咽癌分子分型模型。这些双重标志物可以识别出预后不良的高风险患者，但这些患者可能受益于活性氧阻断和放射治疗的序贯治疗，这为鼻咽癌的精确治疗提供了新的视角。
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. Methods: 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. Results: 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. Conclusions: 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.