Laboratory of Immunology, Department of Biological Science, College of Science, Sungkyunkwan University, Suwon, Korea.
Laboratory of Immunology, Department of Biological Science, College of Science, Sungkyunkwan University, Suwon, Korea,
Cell Physiol Biochem. 2020 Oct 14;54(5):1026-1040. doi: 10.33594/000000285.
BACKGROUND/AIMS: Fractionated ionizing radiation (FIR) is an anti-cancer protocol widely applied for the treatment of diverse types of cancers to reduce damage to normal cells. However, cancer cells receiving multiple irradiations at low doses during FIR, often develop resistance to the therapy exhibiting malignant features including epithelial to mesenchymal transition (EMT). The present study has been performed to elucidate the mechanism of FIR-induced EMT signaling pathways and to identify a molecular target for radioresistance modulated by suppressors of cytokine signaling (SOCS)1.
Colorectal cancer cell lines received FIR with a daily dose of 2 Gy for 3 days. Generation of intracellular reactive oxygen species (ROS) and its role in EMT signaling induced by FIR were analyzed in SOCS1 over-expressing and knock-down cells. ROS were measured by DCF fluorescence using flow cytometry. Expression levels of EMT markers and signaling molecules were analyzed by Western blotting and confocal microscopy.
FIR induced ROS and changes in EMT markers including down-regulation of E-cadherin with up-regulation of Twist and Snail. Pretreatment of anti-oxidant N-acetyl cysteine (NAC) abrogated the FIR-induced ROS generation and EMT response. Mechanistic studies indicated that the FIR-induced ROS-mediated EMT signaling proceeded through Akt→Src→Erk pathways. In accordance with the anti-ROS function, SOCS1 blocked the FIR-induced EMT and the associated signaling pathways through thioredoxin (Trx1) up-regulation. This is evidenced by the observation that Trx1 ablation in SOCS1 over-expressing cells negated the inhibitory action of SOCS1 by restoring the FIR-induced ROS and EMT markers. In addition, we have obtained data supporting that the FIR-induced ROS is derived from functional mitochondria and NADPH oxidases (Nox), which are both down-regulated by SOCS1.
The results demonstrate that ROS signal acts as a mediator of the FIR-induced EMT. The data also suggest a potential anti-tumor function of SOCS1 by blocking the FIR therapy-induced resistance through the counter-regulation of ROS generating and scavenging systems.
背景/目的：分次电离辐射（FIR）是一种广泛应用于治疗多种类型癌症的抗癌方案，旨在减少对正常细胞的损伤。然而，在 FIR 中，癌细胞多次接受低剂量照射后，通常会对治疗产生耐药性，表现出包括上皮间质转化（EMT）在内的恶性特征。本研究旨在阐明 FIR 诱导的 EMT 信号通路的机制，并确定受细胞因子信号抑制物（SOCS）1 调节的放射抵抗的分子靶点。
用每天 2 Gy 的剂量对结直肠癌细胞系进行 FIR，连续 3 天。在 SOCS1 过表达和敲低细胞中分析由 FIR 诱导的细胞内活性氧（ROS）的产生及其在 EMT 信号通路中的作用。通过流式细胞术用 DCF 荧光法测量 ROS 水平。通过 Western blot 和共聚焦显微镜分析 EMT 标志物和信号分子的表达水平。
FIR 诱导了 ROS 的产生，并改变了 EMT 标志物，包括下调 E-钙粘蛋白，而上调 Twist 和 Snail。抗氧化剂 N-乙酰半胱氨酸（NAC）预处理可阻断 FIR 诱导的 ROS 产生和 EMT 反应。机制研究表明，FIR 诱导的 ROS 介导的 EMT 信号通路通过 Akt→Src→Erk 途径进行。与抗氧化功能一致，SOCS1 通过上调硫氧还蛋白（Trx1）阻断了 FIR 诱导的 EMT 及其相关信号通路。这一点可以从以下观察结果中得到证实，即 SOCS1 过表达细胞中 Trx1 的缺失通过恢复 FIR 诱导的 ROS 和 EMT 标志物，否定了 SOCS1 的抑制作用。此外，我们获得的数据支持 FIR 诱导的 ROS 来源于功能线粒体和 NADPH 氧化酶（Nox），SOCS1 可下调这两种酶。
研究结果表明，ROS 信号作为 FIR 诱导的 EMT 的介导物。数据还表明，SOCS1 通过拮抗氧化生成和清除系统，阻断 FIR 治疗诱导的耐药性，从而发挥潜在的抗肿瘤功能。
