葫芦素E对人结肠癌细胞Caco-2增殖与迁移的影响及其作用机制.docVIP

DOI：10.16016/j.1000-5404. 201411205 葫芦素E对人结肠癌细胞Caco-2增殖与迁移的影响及其作用机制 李 丽，随何欢，黄家君（400016重庆，重庆医科大学药学院药理学教研室，重庆市生物化学与分子药理学重点实验室） [摘要] 目的 研究葫芦素E（CuE）对人结肠癌细胞Caco-2增殖与迁移的影响，并探讨CuE作用的分子机制。方法 用0.001、0.01、0.1、1、10μmol/L的CuE分别处理培养的Caco-2细胞，CCK-8法检测细胞增殖能力，采用划痕实验及Transwell小室细胞跨膜实验检测细胞迁移能力，荧光法检测细胞F-actin与G-actin相对含量，激光共聚焦显微镜观察细胞骨架F-actin，Western blot分别检测cofilin、p-cofilin、LIMK2及p-LIMK2蛋白表达。结果 CCK-8法、划痕实验与Transwell跨膜实验显示：0.1、1及10μmol/L的CuE对Caco-2细胞的增殖与迁移均具有明显的抑制作用（P0.05），并呈现出剂量与时间依赖性；荧光法表明0.1μmol/L的CuE能增加Caco-2细胞G-actin含量（P0.05），引起F-actin/G-actin的动态平衡失调，并在0.1μmol/L的CuE处理1、2、6、12、24h后，对细胞骨架F-actin有明显的破坏作用，Western blot检测显示，不同浓度CuE处理Caco-2细胞后，cofilin、p-cofilin及p-LIMK2的蛋白表达明显降低（P0.05），而LIMK2表达与对照组比较无明显差异。结论 CuE可能通过抑制LIMK/cofilin信号通路而损害细胞骨架actin，从而导致结肠癌Caco-2细胞的增殖与迁移受到抑制。 [关键词] 葫芦素E；Caco-2细胞；增殖；迁移；F-肌动蛋白；G-肌动蛋白；LIM激酶2；cofilin [中图法分类号] [suppr文献标志码] A [通信作者] 黄家君，电话：（023E-mail: huangjiajun1964@163.com Cucurbitacin E inhibits proliferation and migration in human colonic cancer cells Caco-2 by suppressing LIMK/cofilin Li Li, Sui Hehuan, Huang Jiajun ( Department of Pharmacology, Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology, College of Pharmacy, Chongqing Medical University, Chongqing, 400016, China) [Abstract] Objective To determine the effect of cucurbitacin E (CuE) on the proliferation and migration of human colonic cancer Caco-2 cells, and to determine the underlying molecular mechanism in the action. Methods The cultured Caco-2 cells were treated with CuE at 0.001, 0.01, 0.1, 1 and 10 μmol/L respectively. CCK-8 assay was used to detect the proliferation of Caco-2 cells. The migration of Caco-2 was analyzed by both the scratch assay and transwell chamber assay. Fluorescence assay was employed to determine the relative contents of both F-actin and G-actin. Laser confocal microscopy was used to observe the cytoskeleton F-actin. Western blot assay was used to detect the protein expression of cofilin, phosphorylated c