携带萤光素酶报告基因细胞筛选靶向RAC1的含氮取代甲酰胺蒽醌修饰物  

Study on RAC1 luciferase reporter gene cell model screening N-substituted formamide an⁃thraquinone modifiers

作　　者：翟利娜 李俊莹 李欣晓 赵玉华 侯华新[2] 黎丹戎[1,3] Zhai Lina;Li Junying;Li Xinxiao;Zhao Yuhua;Hou Huaxin;Li Danrong(Academy of Life Sciences,Guangxi Medical University,Nanning 530021,China;College of Pharmacy,Guangxi Medical University,Nanning 530021,China;School of Oncology,Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学生命科学院,南宁530021 [2]广西医科大学药学院,南宁530021 [3]广西医科大学肿瘤医学院,南宁530021

出　　处：《广西医科大学学报》2021年第5期847-852,共6页Journal of Guangxi Medical University

基　　金：国家自然科学基金资助项目(No.81360502);广西自然科学基金资助项目(No.2018GXNSFAA281064,No.2020GXNSFDA238016);区域性高发肿瘤省部共建重点实验室开放课题资助项目(No.GKE2018-09,No.GEK2019-23)。

摘　　要：目的:采用携带RAC1启动子基因片段及萤光素酶报告基因的肺癌细胞株A549-RAC1-Luc2,筛选靶向抑制RAC1表达的含氮取代甲酰胺蒽醌修饰物。方法:运用分子对接软件MOE分析含氮取代甲酰胺蒽醌修饰物与RAC1结合能力。采用MTT法检测目标化合物对肺癌A549细胞生长活性的抑制作用;以无细胞毒浓度的含氮取代甲酰胺蒽醌修饰物处理A549-RAC1-Luc2细胞后,采用萤光素酶报告基因检测试剂盒检测各组细胞萤光素酶活性变化,Western blotting法检测各组细胞RAC1蛋白的表达水平。结果:合成的系列含氮取代甲酰胺蒽醌修饰物4A、4B、4H、4L、4C、4D、4E均能抑制肺癌A549细胞增殖,其半数抑制浓度IC50均明显低于先导化合物Rhein(P<0.01);RAC1激活剂PMA和抑制剂NSC23766可调控A549-RAC1-Luc2细胞的萤光素酶活性(P<0.01),含氮取代甲酰胺蒽醌修饰物4D、4E能抑制细胞萤光素酶活性及RAC1蛋白的表达,且4D、4E处理后A549-RAC1-Luc2细胞中萤光素酶的表达量明显低于使用RAC1抑制剂NSC23766处理的细胞(P<0.01),且与RAC1结合亲和力最强、稳定性最高。结论:含RAC1启动子及萤光素酶报告基因的肺癌细胞模型可筛选靶向RAC1的抑制剂,含氮取代甲酰胺蒽醌修饰物4D、4E可能是潜在的RAC1抑制剂。Objective:Lung cancer cell line A549-RAC1-Luc2with luciferase reporter gene was used to screen N-substituted formamide anthraquinone modifiers targeting RAC1.Methods:The binding ability of N-substituted formamide anthraquinone modifiers to RAC1 was analyzed by molecular docking MOE software.The growth inhibitory activity of formamide anthraquinone modifiers on lung cancer A549 cells was detected by MTT assay.A549-RAC1-Luc2 cells containing RAC1 luciferase reporter gene were treated with a series of anthraquinone modifiers with no cytotoxic concentration.Luciferase reporter gene detection kit was used to detect the changes of luciferase activity in cells of each group.Western blotting was used to detect the expression of RAC1 protein.Results:The N-substituted formamide anthraquinone modifiers 4A,4B,4H,4L,4C,4D,4E had good growth inhibitory activity on lung cancer A549 cells,and their 50%inhibitory concentration(IC50)was significantly lower than that of the lead compound Rhein(P<0.01).RAC1 activator PMA and inhibitor NSC23766 could regulate luciferase activity in A549-RAC1-Luc2 cells(P<0.01).The N-substituted formamide anthraquinone modifiers 4D,4E could inhibit luciferase activity in A549-RAC1-Luc2 cells.The expression of luciferase in A549-RAC1-Luc2 cells treated with4D and 4E was significantly lower than thattreated with Rac1 inhibitor NSC23766(P<0.01),and modifiers 4D and 4E al-so had the strongest binding affinity and stability to RAC1.Conclusion:Luciferase reporter gene cell model can be used to screen RAC1-targeting inhibitors.Nitrogen-substituted formamide anthraquinone modifiers 4D and 4E might be potential inhibitors targeting RAC1.

关 键 词：RAC1 萤光素酶报告基因 分子对接 蒽醌修饰物 抗肿瘤活性 

分 类 号：R733.7[医药卫生—肿瘤]