柠檬苦素通过靶向Skp2蛋白抑制非小细胞肺癌细胞增殖  

Inhibitory effects of limonin on the proliferation of non-small cell lung cancer cells via targeting S-phase kinase-associated protein 2

作　　者：朱嘉贤 张梦婷 李盛青[1] 刘永强 Zhu Jiaxian;Zhang Mengting;Li Shengqing;Liu Yongqiang(Research Center of Chinese Herbal Resource Science and Engineering,School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine,Guangzhou 510006,Guangdong,China;Key Laboratory of Chinese Medicinal Resource of Lingnan(Guangzhou University of Chinese Medicine),Ministry of Education,Guangzhou 510006,Guangdong,China;Dongguan Institute of Guangzhou University of Chinese Medicine,Dongguan 523808,Guangdong,China)

机构地区：[1]广州中医药大学中药学院,中药资源科学与工程研究中心,广东广州510006 [2]广州中医药大学,岭南中药资源教育部重点实验室,广东广州510006 [3]东莞广州中医药大学研究院,广东东莞523808

出　　处：《生物医学转化》2024年第4期96-105,共10页Biomedical Transformation

基　　金：广东省基础与应用基础研究基金项目(2022A1515011881;2021B1515140052)。

摘　　要：目的 探讨柠檬苦素抑制非小细胞肺癌(NSCLC)细胞增殖能力及其分子作用机制。方法 首先通过MTT实验检测柠檬苦素对NSCLC细胞活性抑制能力;随后分别采用流式细胞技术检测细胞周期与凋亡、平板克隆形成实验检测低密度细胞增殖、细胞基质胶3D成球实验评价干细胞特性。接着通过Western blotting分析柠檬苦素抑制NSCLC细胞增殖的分子机制;通过细胞热位移测定(CETSA)证明柠檬苦素与靶蛋白结合能力;通过分子对接实验模拟柠檬苦素与靶蛋白相互作用位点。最后,通过小分子干扰RNA (siRNA)与S期激酶相关蛋白2 (Skp2)过表达细胞株构建,验证柠檬苦素通过作用Skp2发挥抑制NSCLC增殖的分子机制。结果 柠檬苦素通过诱导细胞周期G0/G1期阻滞以剂量依赖性方式抑制NSCLC细胞增殖,同时显著减弱NSCLC干细胞样特性。柠檬苦素结合Skp2蛋白并下调其蛋白水平,从而促进Skp2底物p27累积。Skp2特异siRNA干扰其表达后显著增强柠檬苦素抑制NSCLC细胞活性能力,而Skp2过表达拮抗柠檬苦素对NSCLC细胞活性抑制能力,进一步验证了柠檬苦素通过靶向Skp2抑制NSCLC细胞增殖的分子机制。结论 柠檬苦素通过靶向Skp2升高抑癌蛋白p27水平,从而抑制NSCLC细胞增殖,为柠檬苦素作为抗肿瘤药物研发提供了重要的理论依据。Objective Aims to investigate the inhibitory effects of limonin on the proliferation of non-small cell lung cancer(NSCLC) cells and its molecular mechanisms.Methods To investigate the inhibitory effects of limonin on NSCLC cell activity,the MTT assay was employed.Then,flow cytometry was used to detect the cell cycle and apoptosis,while colony formation assay assessed the low-density proliferation ability of NSCLC cells.The matrigel 3D pelletization assay was performed to evaluate stem cell properties.Further,Western blotting was used to analyze the underlying molecular mechanisms of limonin inhibiting NSCLC cell proliferation.Cell thermal shift assay(CETSA) was used to demonstrate the binding capability of limonin with target proteins,and molecular docking was employed to predict the interaction sites between limonin and target proteins.Finally,the study utilized small interfering RNA(siRNA) and S-phase kinase-associated protein 2(Skp2) overexpressing cell lines to validate the molecular mechanisms of limonin inhibiting NSCLC cell proliferation.Results Limonin inhibited NSCLC cell proliferation in a dose-dependent manner by inducing cell cycle G0/G1 phase arrest while significantly attenuating NSCLC stem cell-like properties.Mechanistically,limonin was bound to the Skp2 protein and induced its downregulation,thus promoting the accumulation of its substrate protein p27.Skp2 knockdown by using Skp2-specific siRNA markedly enhanced the inhibitory effects of limonin on NSCLC cell activity.At the same time,Skp2 overexpression attenuated the capability of limonin to inhibit NSCLC cell activity,further validating the underlying molecular mechanisms of limonin inhibiting NSCLC cell proliferation by targeting Skp2.Conclusion Limonin inhibits NSCLC cell proliferation by targeting Skp2 protein to increase the level of the tumor suppressor protein p27,providing crucial theoretical support for the development of limonin as an antitumor drug.

关 键 词：柠檬苦素 非小细胞肺癌 S期激酶相关蛋白2 细胞增殖 

分 类 号：R734.2[医药卫生—肿瘤] R285.5[医药卫生—临床医学]