人口腔鳞癌细胞中姜黄素靶向蛋白的确立  

Identification of curcumin binding proteins in oral cancer cells

作　　者：葛化冰[1] 宿颖[2] 陈新 张辛燕[2] GE Hua-bing;SU Ying;CHEN Xin;ZHANG Xinyan(Beijing Institute of Dental Research,Capital Medical University School of Stomatology,Beijing 100050,China)

机构地区：[1]首都医科大学口腔医学院王府井部口腔综合科,北京100050 [2]首都医科大学口腔医学院王府井部口腔医学研究所,北京100050 [3]常州大学制药与生命科学学院

出　　处：《北京口腔医学》2020年第3期136-140,共5页Beijing Journal of Stomatology

基　　金：国家自然科学基金(81772868);北京市自然科学基金(7202057)。

摘　　要：目的确定口腔癌细胞中姜黄素的靶向蛋白。方法利用姜黄素-生物素亲和素pull-down技术和液相色谱和串联质谱分析筛选分离口腔癌细胞中姜黄素靶向蛋白,采用姜黄素-生物素亲和技术和Western Blotting鉴定靶向蛋白和姜黄素的结合,生物学功能检测姜黄素对其潜在的靶向蛋白在口腔鳞癌细胞中的作用,建立姜黄素与靶向蛋白相互作用的计算机模型,并进一步阐明它与靶向蛋白结合位点。结果根据姜黄素潜在的功能位点和暴露蛋白结合位点设计和合成姜黄素-生物素共轭化合物。采用生物素-亲和素pull-down技术和液相色谱和串联质谱分析(LC-MS/MS)分离和鉴定姜黄素在口腔癌细胞中的结合蛋白,采用MASCOT搜索引擎检索数据库NCBInr,一共筛选到69个姜黄素结合蛋白,其中一些和肿瘤的发生密切相关。利用生物素-亲和素pull-down技术结合Western blotting进一步验证Annexin A2和CDKN2B为姜黄素在口腔鳞癌细胞内的结合蛋白。进一步功能检测证实姜黄素处理口腔鳞癌细胞,导致其潜在的靶向蛋白Annexin A2,CDKN2B表达增高,并呈一定的剂量依赖关系。计算机模型对接证实和明确了姜黄素和Annexin A2和CDKN2B的结合位点。结论利用生物素亲和素技术和液相色谱和串联质谱分析筛选和验证两个新的姜黄素在口腔鳞癌细胞中的靶向蛋白Annexin A2和CDKN2B。Objective To identify the potential protein targets of curcumin in oral cancer cells.Methods The curcumin-biotin conjugate was chemically synthesized.Potential curcumin binding proteins were pulled down by streptavidin technique.Determination of curcumin-binding proteins was performed by liquid chromatography coupled with tandem mass spectrometry(LC-MS/MS).Proteins were identified with an online search engine,MASCOT Peptide Mass Fingerprint.Further binding assay was also performed by treating oral cancer cells with curcumin-biotin and curcumin and biotin.After being pulled down with streptavidin,Annexin A2 and CDKN2B were detected by Western blotting.The biological effect of curcumin on Annexin A2 and CDKN2B was detected by immunoflurenscence.Cumputer modeling was used to dock the bind sites of the target proteins.Results Curcumin-binding proteins were eluted with streptavidin resin from curcumin-biotin treated OSCC cells,separated by polyacrylamide gel electrophoresis,and identified by liquid chromatography coupled with tandem mass spectrometry(LC-MS/MS).Tentative curcumin-binding proteins were identified on the basis of their mass fingerprinting.69 potential curcumin binding proteins were selected.Curcumin-binding properties of two of these proteins,Annexin A2 and CDKN2B,were studied further in vitro and the biological consequences of this binding was evaluated.Binding assay with Western blotting and computer modeling docking confirmed binding of Annexin A2 and CDKN2B.Curcumin binding increased the protein expression of Annexin A2 and CDKN2B in a concentrationdependent manner.Computer modeling identified the bind sites of the two target proteins.Conclusion This study confirmed Annexin A2 and CDKN2B as new curcumin-binding proteins in oral squamous cell carcinoma cells.

关 键 词：姜黄素 靶向蛋白 口腔鳞癌细胞 

分 类 号：R285.5[医药卫生—中药学]