薯蓣皂苷靶向泛素-蛋白酶体的抗肿瘤活性研究

Dioscin Inhibits Ubiquitin-Proteasome System in Cancer Cells

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.