Li Deqiang, Zhang Junlong, Yin Lijun, Jin Zhen, Chen Xuejun, Meng Xiangxue
Department of Anesthesiology, Tianjin Baodi Hospital, Baodi Clinical College of Tianjin Medical University, Tianjin 301800, P.R. China.
Department of Anesthesiology, The Second People's Hospital of Lianyungang, Lianyungang, Jiangsu 222000, P.R. China.
Exp Ther Med. 2021 Nov;22(5):1254. doi: 10.3892/etm.2021.10689. Epub 2021 Sep 3.
Etomidate (ETO) is a commonly used intravenous anesthetic that has been reported to exert a tumor suppressive effect in several types of cancer. The present study aimed to investigate the effect of ETO on cell proliferation and apoptosis in non-small cell lung cancer (NSCLC) cells and elucidate its potential mechanism of action. Therefore, Cell Counting Kit-8 assay was performed to evaluate the effect of different concentrations of ETO (0, 1, 2 or 3 µg/ml) on A549 cell viability. In addition, the possible interaction between ETO and WW domain containing E3 ubiquitin protein ligase 2 (WWP2) was predicted using the STITCH database. Additionally, a stable WWP2-overexpressing A549 cell line was constructed by transfecting A549 cells with the pcDNA3.1-WWP2 plasmid. Cell proliferation and apoptosis were assessed using colony formation and TUNEL assays, respectively. The mRNA and protein expression levels of the apoptosis-related proteins Bcl-2, Bax, caspase 3 and cleaved-caspase 3 were determined by reverse transcription-quantitative PCR and western blotting. In addition, the expression and phosphorylation levels of proliferation-associated genes (PCNA and Ki-67) and proteins in the PI3K/Akt pathway were analyzed by western blotting. The results showed that treatment with ETO attenuated the cell viability and proliferation of A549 cells. ETO also promoted cell apoptosis and decreased the expression of the anti-apoptotic protein Bcl-2, whilst increasing that of pro-apoptotic proteins Bax and cleaved caspase 3 in a dose-dependent manner. Furthermore, ETO was found to negatively regulate the expression of WWP2, such that WWP2 overexpression reversed the potentiating effects of ETO on cell apoptosis. In addition, ETO promoted the expression of PTEN and reduced the phosphorylation levels of the PI3K/AKT pathway-related proteins. These effects aforementioned could also be reversed by WWP2 overexpression. Therefore, data from the present study suggest that ETO can attenuate the progression of NSCLC through by the PI3K/AKT pathway, specifically by targeting WWP2. These findings may provide a novel target for the treatment of NSCLC.
依托咪酯（ETO）是一种常用的静脉麻醉剂，据报道它在几种类型的癌症中具有肿瘤抑制作用。本研究旨在探讨ETO对非小细胞肺癌（NSCLC）细胞增殖和凋亡的影响，并阐明其潜在的作用机制。因此，进行了细胞计数试剂盒-8检测，以评估不同浓度的ETO（0、1、2或3μg/ml）对A549细胞活力的影响。此外，使用STITCH数据库预测了ETO与含WW结构域的E3泛素蛋白连接酶2（WWP2）之间可能的相互作用。另外，通过用pcDNA3.1-WWP2质粒转染A549细胞构建了稳定的WWP2过表达A549细胞系。分别使用集落形成和TUNEL检测评估细胞增殖和凋亡。通过逆转录定量PCR和蛋白质印迹法测定凋亡相关蛋白Bcl-2、Bax、半胱天冬酶3和裂解的半胱天冬酶3的mRNA和蛋白质表达水平。此外，通过蛋白质印迹法分析PI3K/Akt途径中增殖相关基因（PCNA和Ki-67）和蛋白质的表达及磷酸化水平。结果表明，ETO处理减弱了A549细胞的活力和增殖。ETO还促进细胞凋亡，并以剂量依赖性方式降低抗凋亡蛋白Bcl-2的表达，同时增加促凋亡蛋白Bax和裂解的半胱天冬酶3的表达。此外，发现ETO负向调节WWP2的表达，使得WWP2过表达逆转了ETO对细胞凋亡的增强作用。另外，ETO促进PTEN的表达并降低PI3K/AKT途径相关蛋白的磷酸化水平。上述这些作用也可被WWP2过表达逆转。因此，本研究的数据表明，ETO可通过PI3K/AKT途径，特别是通过靶向WWP2来减弱NSCLC的进展。这些发现可能为NSCLC的治疗提供一个新的靶点。
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