Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Lung Cancer. 2017 Nov;113:72-78. doi: 10.1016/j.lungcan.2017.09.008. Epub 2017 Sep 14.
The regulation of redox balance in cancer cells is an important factor in tumor development and chemoresistance, with oncogene activation having been shown to induce the generation of reactive oxygen species (ROS). Activating mutations of the epidermal growth factor receptor gene (EGFR) are oncogenic drivers in non-small cell lung cancer (NSCLC), but it has remained unknown whether ligand-independent EGFR signaling conferred by EGFR mutation triggers ROS generation in NSCLC cells.
HEK293T cells were transfected with an expression vector for mutant EGFR. The expression of CD44 variant (CD44v) isoforms in NSCLC cell lines was evaluated by flow cytometry. Cells were depleted of CD44v by RNA interference and assayed for ROS and glutathione (GSH) levels. The effect of CD44v on cisplatin sensitivity was evaluated in vitro with the MTS assay.
EGFR signaling due to EGFR mutation increased ROS levels in transfected HEK293T cells. The expression of CD44v isoforms was found to be inversely correlated with basal ROS levels in EGFR mutation-positive NSCLC cell lines. Knockdown of CD44v induced depletion of intracellular GSH and increased ROS levels in EGFR-mutated NSCLC cells that express CD44v at a high level (CD44v). In addition, depletion of GSH by treatment with buthionine-[S, R]-sulfoximine induced marked accumulation of ROS and enhanced the cytotoxicity of cisplatin in CD44vEGFR-mutated NSCLC cells but not in corresponding CD44v cells. This enhancement of cisplatin cytotoxicity by GSH depletion was prevented by treatment with the antioxidant N-acetyl-l-cysteine. Knockdown of CD44v also enhanced cisplatin cytotoxicity in CD44vEGFR mutation-positive NSCLC cells but not in CD44v cells.
Our results thus implicate CD44v in redox adaptation and as a potential target for treatment in CD44vEGFR-mutated NSCLC cells.
癌细胞中氧化还原平衡的调节是肿瘤发展和化疗耐药的一个重要因素，已有研究表明癌基因的激活会诱导活性氧（ROS）的产生。表皮生长因子受体基因（EGFR）的激活突变是非小细胞肺癌（NSCLC）的致癌驱动因素，但尚不清楚 EGFR 突变引起的配体非依赖性 EGFR 信号是否会触发 NSCLC 细胞中 ROS 的产生。
用表达载体转染 HEK293T 细胞，通过流式细胞术评估 NSCLC 细胞系中 CD44 变体（CD44v）同工型的表达。通过 RNA 干扰耗尽 CD44v，并检测 ROS 和谷胱甘肽（GSH）水平。用 MTS 测定法评估 CD44v 对顺铂敏感性的影响。
EGFR 突变引起的 EGFR 信号增加了转染的 HEK293T 细胞中的 ROS 水平。发现 CD44v 同工型的表达与 EGFR 突变阳性 NSCLC 细胞系中的基础 ROS 水平呈负相关。在高水平表达 CD44v（CD44v）的 EGFR 突变型 NSCLC 细胞中，CD44v 的敲低诱导细胞内 GSH 的耗竭，并增加 ROS 水平。此外，用丁硫氨酸-[S，R]-亚砜亚胺处理耗尽 GSH 会导致 ROS 的显著积累，并增强 CD44vEGFR 突变型 NSCLC 细胞中顺铂的细胞毒性，但对相应的 CD44v 细胞则不然。抗氧化剂 N-乙酰-L-半胱氨酸处理可防止 GSH 耗竭增强顺铂的细胞毒性。CD44v 的敲低也增强了 CD44vEGFR 突变阳性 NSCLC 细胞中顺铂的细胞毒性，但对 CD44v 细胞则不然。
我们的结果表明 CD44v 参与了氧化还原适应，并可能成为 CD44vEGFR 突变型 NSCLC 细胞治疗的潜在靶点。
