二去水卫矛醇对肺癌NCI-H460细胞DNA拓扑异构酶Ⅱ的抑制作用  

Inhibitory effect of dianhydrogalactitol on DNA TopoⅡ in NCI-H460 cells

作　　者：黄银妹[1] 刘华钢 苏桂玉 李英杰[4] 王小洁[1] 蒋霞[5] 

机构地区：[1]广西医科大学药学院,广西南宁530021 [2]广西食品药品监督管理局,广西南宁530022 [3]广西中医药大学药学院,广西南宁530001 [4]暨南大学药学院中药及天然药物研究所,广东广州510632 [5]广西医科大学第一附属医院临床药学部,广西南宁530021

出　　处：《中国药理学通报》2016年第11期1601-1607,共7页Chinese Pharmacological Bulletin

基　　金：广西科学研究与技术开发计划项目(No桂科能12237007);广西自然科学基金资助项目(No2015GXNSFAA139194)

摘　　要：目的评价二去水卫矛醇(dianhydrogalactitol,DAG)在肺癌NCI-H460细胞上的抗肿瘤作用,探讨其抗肿瘤作用机制。方法采用CCK-8法、细胞克隆形成实验,评价DAG对NCI-H460细胞的增殖抑制作用。显微拍照观察细胞形态改变;Hoechst 33342检测细胞核染色质的变化。Real time PCR法检测拓扑异构酶Ⅱ(TopoⅡ)mRNA的表达水平。Western blot法检测TopoⅡ蛋白表达水平。另外,应用计算机模拟分子对接技术来预测DAG与TopoⅡ的相互作用。结果 CCK-8法检测结果显示,DAG对NCI-H460细胞的体外抗肿瘤活性明显。细胞克隆形成实验结果表明,DAG能持续抑制肿瘤细胞的增殖。Hoechst 33342检测细胞凋亡发现细胞核染色质发生明显改变。Real time PCR和Western blot检测结果显示TopoⅡ mRNA和蛋白表达量降低。计算机模拟分子对接显示DAG与TopoⅡ有相互结合作用。结论DAG能明显抑制NCI-H460细胞的增值,作用机制研究表明DAG能降低TopoⅡ mRNA和蛋白水平,并与TopoⅡ结合,最终可能导致DNA双链断裂,引起细胞死亡。Aim To evaluate the antitumor activity of dianhydrogalactitol( DAG) in vitro,and further clarify its underlying mechanisms. Methods The inhibitory effect of DAG in NCI-H460 cells was detected by CCK-8 assay and colony formation assay. The morphology of cells treated with DAG was observed under optical microscope. Nuclear morphology was captured by fluorescence microscopy after Hoechst 33342 staining. Realtime PCR was used to analyze the expression level of topoisomerase Ⅱ( Topo Ⅱ) mRNA. The protein expression level of Topo Ⅱ was detected by Western blot. Additionally,molecular docking approaches were used to predict the interaction between DAG and TopoⅡ. Results DAG exhibited potent antitumor activity in NCI-H460 cells,and inhibited cell proliferation persistently. DAG obviously induced nuclear morphological changes of NCI-H460 cells. Furthermore,DAG could down-regulate the mRNA and protein expression level of Topo Ⅱ detected by Real-time PCR analysis and Western blot, respectively. Molecular docking predicted that DAG could bind to Topo Ⅱ. Conclusion DAG can significantly inhibit the proliferation of NCI-H460 cells,and its underlying mechanisms may involve the down-regulation of Topo Ⅱ mRNA and direct binding to Topo Ⅱ,leading to cancer cell death.

关 键 词：二去水卫矛醇 肺癌 NCI-H460细胞 拓扑异构酶Ⅱ 拓扑异构酶Ⅱ抑制剂 分子对接 

分 类 号：R282.71[医药卫生—中药学] R329.24[医药卫生—中医学]