姜黄素-生物素偶联物的合成和姜黄素结合蛋白的分离

›› 2019, Vol. 10 ›› Issue (4): 185-190.

姜黄素-生物素偶联物的合成和姜黄素结合蛋白的分离

葛化冰¹,宿颖¹,陈新²,张辛燕¹

1. 首都医科大学附属北京口腔医院
2. 常州大学制药与生命科学学院

收稿日期:2019-08-19 修回日期:2019-11-27 出版日期:2019-12-25 发布日期:2020-01-06
通讯作者: 张辛燕 E-mail:xinyanzhangzh@126.com
基金资助:
国家自然科学基金

Synthesis of curcumin-biotin conjugate and identification of curcumin-binding proteins in oral cancer cells

Received:2019-08-19 Revised:2019-11-27 Online:2019-12-25 Published:2020-01-06
Supported by:
National Natural Science Foundation of China

摘要/Abstract

摘要： 目的：利用姜黄素-生物素亲和素技术分离鉴定口腔鳞癌细胞中潜在的姜黄素靶向蛋白。方法：利用化学合成的方法合成姜黄素-生物素偶联物，利用姜黄素-生物素与链霉亲和素的结合放大技术和聚丙烯酰胺凝胶电泳分离口腔鳞癌细胞中潜在的姜黄素结合蛋白，液相质谱和串联质谱分析(LC-MS/MS)筛选分离口腔癌细胞中姜黄素靶向蛋白。姜黄素-生物素亲和素结合实验和Western Blotting进一步鉴定姜黄素结合蛋白。结果：合成的姜黄素-生物素偶联物与姜黄素对比并没有显著影响其活性，且化学性质稳定。姜黄素-生物素-链霉亲和素的结合放大技术和聚丙烯酰胺凝胶电泳成功地分离出潜在的姜黄素结合蛋白，液相色谱和串联质谱分析鉴定出167个潜在的姜黄素结合蛋白，其中一些和肿瘤的发生密切相关。进一步的蛋白结合实验和Western blotting验证Annexin A2和CDKN2B为姜黄素在口腔鳞癌细胞内的结合蛋白，并呈一定的剂量依赖关系。结论：Annexin A2和CDKN2B可能是姜黄素在口腔鳞癌细胞潜在的靶向蛋白。

关键词: 姜黄素, 蛋白靶点, 口腔癌

Abstract: Objective: The goal of this study was to identify the potential curcumin binding proteins in oral cancer cells by biotin-streptavidin pull down technique. Methods: A curcumin-biotin conjugate was chemically synthesized and its activity was measured by MTT assay. Curcumin-binding proteins were eluted with streptavidin resin from curcumin-biotin treated oral cancer cells, separated by polyacrylamide gel electrophoresis, and identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Curcumin-binding properties of two of these proteins, Annexin A2 and CDKN2B, were studied further by streptavidin binding assay and western blotting with recombinant humum proteins. Results: Curcumin-biotin conjugate was successfully chemically synthesized and its activity was not affected by MTT assay. The potential curcumin binding proteins were eluted with streptavidin resin and separated by SDS-PAGE electrophoresis, and then analyzed by LC-MS/MS. 167 potential curcumin binding proteins were identifed with MASCOT. Binding assay with western blotting confirmed the binding of Annexin A2 and CDKN2B with curcumin in a dose-dependent manner. Conclusions: In summary, this study confirmed Annexin A2 and CDKN2B as new curcumin-binding proteins in oral squamous carcinoma cells.

Key words: Curcumin, Protein targets, Oral cancer

中图分类号:

R739.8

葛化冰宿颖陈新张辛燕. 姜黄素-生物素偶联物的合成和姜黄素结合蛋白的分离[J]. , 2019, 10(4): 185-190.

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