中药植物圆叶紫胆(Celastrus orbiculatus Thunb.)在中国已广泛使用了数千年，其茎的乙酸乙酯提取物(Celastrus orbiculatus Thunb. Extract, COE)在多个临床前研究中被报告具有抗肿瘤和抗炎作用。但COE对非小细胞肺癌(NSCLC)的抗癌活性及其潜在机制尚未完全了解。本研究旨在探究COE在NSCLC细胞上的抗肿瘤效应，并从Hippo信号通路、YAP核转位及活性氧(ROS)生成角度探讨其分子机制。通过CCK-8、克隆形成、流式细胞术和β-半乳糖苷酶染色等测定，确定COE对NSCLC细胞系增殖、细胞周期、凋亡、干性和衰老的影响。通过Western blotting研究COE对Hippo信号通路的影响。采用免疫荧光检测分析YAP在细胞内的表达和分布，采用DCFH-DA探针结合流式细胞术检测COE处理后NSCLC细胞内总ROS水平。建立异种移植肿瘤模型，并采用动物活体成像系统分析COE对Hippo-YAP信号通路在体内的影响。本研究表明COE能够显著抑制NSCLC的活性，主要通过抑制增殖、细胞周期阻滞、促进凋亡、促进衰老以及降低干性。COE能够强烈激活Hippo信号通路，抑制YAP表达和核保留。COE激活的Hippo信号通路与ROS介导的MOB1磷酸化有关。本研究揭示了COE通过激活Hippo信号通路和抑制YAP核转位来抑制NSCLC，其中ROS可能在MOB1蛋白的磷酸化中发挥作用。版权所有 ©2023 Elsevier GmbH出版。

Celastrus orbiculatus Thunb. is a medicinal plant that has been widely used for thousands of years in China, and the ethyl acetate extract (Celastrus orbiculatus Thunb. Extract, COE) from its stem was reported to exert antitumor and anti-inflammatory effects in various preclinical studies. However, the anti-non-small-cell lung cancer activity of COE and its potential mechanism are not yet fully understood.To investigate the antitumor effects of COE on non-small-cell lung cancer (NSCLC) cells and explore its molecular mechanism from the perspective of Hippo signaling, YAP nuclear translocation, and reactive oxygen species (ROS) generation.The effects of COE on proliferation, cell cycle arrest, apoptosis, stemness, and senescence in NSCLC cell lines were determined by CCK-8, clone formation, flow cytometry, and β-galactosidase staining assays. The effects of COE on Hippo signaling were investigated by Western blotting. The intracellular expression and distribution of YAP were analyzed by immunofluorescence assay. DCFH-DA probe combined with flow cytometry was used to detect intracellular total ROS levels in NSCLC cells after COE treatment. Xenograft tumor model was established, and the animal living image system was employed to analyze the effects of COE on the Hippo-YAP signaling in vivo.COE significantly inhibited NSCLC activity in vitro and in vivo, mainly by proliferation inhibition, cycle arrest, apoptosis promotion, senescence promotion, and stemness downregulation. COE strongly activated Hippo signaling and inhibited YAP expression and nuclear retention. Activation of Hippo signaling induced by COE was associated with ROS-mediated phosphorylation of MOB1.This study demonstrated that COE inhibited NSCLC through activating Hippo signaling and suppressing YAP nuclear translocation, in which ROS may play a role in the phosphorylation of the MOB1 protein.Copyright © 2023. Published by Elsevier GmbH.