摘要
目的基于中药活性分子开发靶向泛素-蛋白酶体系统(UPS)的抗肿瘤小分子。方法建立稳定表达UbG76V-GFP融合蛋白的细胞系来筛选靶向UPS的新型小分子抑制剂。通过Suc-LLVY-AMC、Z-LLE-AMC和Boc-LRR-AMC底物检测薯蓣皂苷对20S蛋白酶体水解酶活性的影响,使用Ub-AMC底物评价其对细胞内去泛素化酶活性的作用。采用Western blot检测薯蓣皂苷对细胞内泛素化水平的影响。CCK-8和克隆形成实验检测薯蓣皂苷对肿瘤细胞增殖的抑制作用。结果通过UbG76V-GFP报告系统筛选发现薯蓣皂苷是新型的UPS抑制剂,可以抑制细胞内去泛素化酶活性,增强细胞内泛素化水平,抑制肿瘤细胞增殖,减少克隆形成。结论薯蓣皂苷靶向泛素-蛋白酶体可显著抑制肿瘤细胞增殖。
Objective To explore the antitumor small molecules targeting the ubiquitin–proteasome system(UPS)on the basis of active molecules from traditional Chinese medicine.Methods UbG76V-GFP stably expressing cell line was constructed to screen novel small molecule inhibitors targeting UPS.The fluorogenic substrates of Suc-LLVY-AMC,Z-LLE-AMC,and Boc-LRR-AMC were used to assess the effect of dioscin on the 20S proteasome hydrolase activity.The Ub-AMC substrate was used to evaluate the effect of dioscin on the intracellular deubiquitinating enzyme activity.Western blot was used to detect the effect of dioscin on intracellular ubiquitination levels.CCK-8 and colony formation assays were used to detect the inhibitory effect of dioscin on the tumor cell proliferation.Results Dioscin is a UPS inhibitor discovered through the UbG76V-GFP reporter system.It enhances intracellular ubiquitination and inhibits tumor cell proliferation and colony formation by targeting deubiquitinating enzymes.Conclusion Dioscin could significantly inhibit tumor cell proliferation by targeting ubiquitin–proteasome.
作者
李欣茹
王嘉琦
柯细松
周香莲
LI Xinru;WANG Jiaqi;KE Xisong;ZHOU Xianglian(Center for Chemical Biology,Institute of Interdisciplinary Integrative Medicine Research,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)
出处
《肿瘤防治研究》
CAS
2023年第6期567-572,共6页
Cancer Research on Prevention and Treatment
基金
国家自然科学基金面上项目(82173845)。