Department of Radiotherapy, Policlinico Umberto I, "Sapienza" University of Rome, Rome, Italy.
Division of Biotechnology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.
J Cancer Res Clin Oncol. 2019 Apr;145(4):881-893. doi: 10.1007/s00432-019-02851-0. Epub 2019 Jan 30.
Tumor cells generally exhibit higher levels of reactive oxygen species (ROS), however, when stressed, tumor cells can undergo a process of 'Redox Resetting' to acquire a new redox balance with stronger antioxidant systems that enable cancer cells to become resistant to radiation therapy (RT). Here, we describe how RT affects the oxidant/antioxidant balance in human embryonal (RD) and alveolar (RH30) rhabdomyosarcoma (RMS) cell lines, investigating on the molecular mechanisms involved.
Radiations were delivered using an x-6 MV photon linear accelerator and their effects were assessed by vitality and clonogenic assays. The expression of specific antioxidant-enzymes, such as Superoxide Dismutases (SODs), Catalase (CAT) and Glutathione Peroxidases 4 (GPx4), miRNAs (miR-22, -126, -210, -375, -146a, -34a) and the transcription factor NRF2 was analyzed by quantitative polymerase chain reaction (q-PCR) and western blotting. RNA interference experiments were performed to evaluate the role of NRF2.
Doses of RT higher than 2 Gy significantly affected RMS clonogenic ability by increasing ROS production. RMS rapidly and efficiently brought back ROS levels by up-regulating the gene expression of antioxidant enzymes, miRNAs as well as of NRF2. Silencing of NRF2 restrained the RMS ability to counteract RT-induced ROS accumulation, antioxidant enzyme and miRNA expression and was able to increase the abundance of γ-H2AX, a biomarker of DNA damage, in RT-treated cells.
Taken together, our data suggest the strategic role of oxidant/antioxidant balance in restraining the therapeutic efficiency of RT in RMS treatment and identify NRF2 as a new potential molecular target whose inhibition might represent a novel radiosensitizing therapeutic strategy for RMS clinical management.
肿瘤细胞通常表现出更高水平的活性氧（ROS），然而，当受到压力时，肿瘤细胞可以经历一个“氧化还原重置”的过程，获得一个新的氧化还原平衡，具有更强的抗氧化系统，使癌细胞对放射治疗（RT）产生抗性。在这里，我们描述了 RT 如何影响人胚（RD）和肺泡（RH30）横纹肌肉瘤（RMS）细胞系中的氧化剂/抗氧化剂平衡，研究了涉及的分子机制。
使用 X-6 MV 光子线性加速器进行辐射，通过活力和克隆形成测定评估其作用。通过定量聚合酶链反应（q-PCR）和 Western 印迹分析特定抗氧化酶（如超氧化物歧化酶（SODs）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶 4（GPx4））、miRNA（miR-22、-126、-210、-375、-146a、-34a）和转录因子 NRF2 的表达。进行 RNA 干扰实验以评估 NRF2 的作用。
RT 剂量高于 2 Gy 会显著通过增加 ROS 产生来影响 RMS 的集落形成能力。RMS 通过上调抗氧化酶、miRNA 以及 NRF2 的基因表达，迅速而有效地将 ROS 水平恢复正常。沉默 NRF2 抑制了 RMS 对抗 RT 诱导的 ROS 积累、抗氧化酶和 miRNA 表达的能力，并能够增加 RT 处理细胞中 DNA 损伤标志物 γ-H2AX 的丰度。
综上所述，我们的数据表明氧化还原平衡在抑制 RMS 治疗中 RT 的治疗效率方面具有战略作用，并确定 NRF2 为新的潜在分子靶点，其抑制可能代表 RMS 临床管理的一种新的放射增敏治疗策略。
