促凋亡蛋白Bax和自噬相关蛋白LC3-Ⅱ参与穿心莲内酯对结肠癌细胞的抑制作用  

Pro-apoptotic Protein Bax and Autophagy-related Protein LC3-Ⅱ Contribute to Inhibition of Andrographolide to Colon Cancer Cells

作　　者：李静[1] 何亮 杨俐萍[1] 赵文静[1] 钱红燕[1] 刘焕良[1] 邵晶晶[1] 王勇军 

机构地区：[1]南通大学附属肿瘤医院肿瘤研究所中心实验室,江苏南通226361

出　　处：《肿瘤学杂志》2017年第12期1085-1092,共8页Journal of Chinese Oncology

基　　金：江苏省创新创业人才基金(苏人才20013-41);南通市市级科技计划项目(MS12015116)

摘　　要：[目的]观察穿心莲内酯(Andrographolide,Andro)对不同分化结肠癌细胞克隆形成、迁移及凋亡的影响,检测凋亡蛋白Bax和自噬蛋白LC3-Ⅱ表达改变,探讨其分子机制。[方法]不同浓度Andro分别作用人结肠癌细胞株Caco-2(高分化)和Lovo(低分化)24h后,MTT法检测药物细胞毒性;划痕愈合、transwell观察细胞迁移能力;检测细胞克隆形成能力;流式细胞术检测细胞凋亡;Western blot检测Bax、LC3-Ⅱ及cleaved-caspase-3的蛋白表达水平。[结果 ](1)Andro以时间和浓度依赖的方式抑制结肠癌细胞生长,Lovo细胞对Andro的敏感性高于Caco-2细胞。(2)Andro明显抑制Caco-2和Lovo细胞的迁移及克隆形成能力,Lovo细胞的效应更为显著。(3)Andro增加Bax和LC3-Ⅱ表达,且在Lovo细胞中表现更明显。(4)凋亡酶caspase-3的激活参与了Andro诱导Caco-2和Lovo细胞的凋亡。[结论]低分化结肠癌细胞Lovo对Andro的敏感性明显高于高分化Caco-2细胞,Andro通过增加Bax的表达,激活caspase-3介导凋亡信号通路,同时通过增强LC3-Ⅱ表达,促进自噬过程,最终抑制结肠癌细胞生长。[Objective] To observe the effects of Andrographolide（Andro） on cellular clone formation,migration and apoptosis of different differentiated colon cancer cells,and to detect the expression of pro-apoptotic protein Bax and autophagy-related protein LC3-Ⅱ. [Methods] Human colon cancer cell lines Caco-2（well-differentiated） and Lovo（poorly differentiated） were treated with different concentrations of Andro for24 h. MTT assay was used to detect Andro cytotoxicity. Wound healing and transwell assays were used to observe cell migration. Clone formation assay was used to detect cellular cloning ability. Flow cytometry was used for analysis of apoptosis. The expression of Bax,LC3-Ⅱ and cleaved-caspase-3 protein was detected by Western blot. [Results]（1） Andro inhibited the growth of colon cancer cells in a time-and concentration-dependent manner. Lovo cells were more sensitive to Andro treatment than Caco-2 cells.（2） Andro significantly inhibited the cell abilities for migration and clone formation of Caco-2 and Lovo cells,especially to Lovo cells.（3） Andro induced the expression of Bax and LC3-Ⅱ,which were more in poorly differentiated Lovo than Caco-2 cells.（4） Andro induced Caco-2 and Lovo cells apoptosis by activating caspase-3. [Conclusion] The sensitivity of poorly differentiated colon cancer cell line Lovo to Andro is significantly higher than that of well-differentiated colon cancer cell line Caco-2. Andro inhibits colon cancer development through increasing Bax expression that activates caspase-3 apoptotic signaling pathway,meanwhile through increasing LC3-Ⅱ expression that induces autophagy process.

关 键 词：穿心莲内酯 结肠肿瘤 克隆形成 细胞迁移 凋亡 自噬 

分 类 号：R735.3[医药卫生—肿瘤]