双硫仑抑制非小细胞肺癌细胞上皮间质转化机制研究

Study on the Mechanism of Disulfiram Inhibiting Epithelial-mesenchymal Transition in Non-small Cell Lung Cancer Cells

目的研究双硫仑(DSF)对转化细胞生长因子β_(1)(TGF-β_(1))诱导A549细胞的上皮间质转化(EMT)的抑制作用及其可能的作用机制。方法构建EMT模型,采用MTT实验检测细胞活性,通过细胞划痕、基质粘附、内皮细胞粘附、transwell小室实验测定DSF对细胞迁移、粘附和侵袭能力的作用。Western blot分析双硫仑对EMT模型中E-cadherin、Vimentin蛋白表达水平的影响;qPCR检测DSF对Vimentin、E-cadherin以及EMT相关干细胞标志基因乙醛脱氢酶-1(ALDH-1)、性别决定区Y框蛋白2(SOX2)和Octamer转录因子4(OCT4)等的mRNA表达水平的影响。结果DSF可呈现剂量依赖性抑制肿瘤细胞的EMT过程,IC_(50)=80μM。与对照组相比,DSF对TGF-β_(1)诱导的A549发生EMT后迁移、粘附、侵袭能力的增强具有明显的抑制作用;DSF显著降低Vimentin的表达,促进E-cadherin的表达,且能显著降低干细胞标志基因ALDH-1、SOX2和OCT4的mRNA表达水平。结论DSF能够逆转TGF-β_(1)诱导的A549细胞的EMT过程,从而抑制细胞的侵袭和迁移。其发挥作用的机制可能与下调肿瘤细胞干性相关基因ALDH-1、SOX2和OCT4有关。

OBJECTIVE To study the inhibitory effect of Disulfiram(DSF) on TGF-β_(1)induced Epithelial-mesenchymal transition(EMT) in A549 cells and its possible mechanism.METHODS EMT model was established, cell activity was detected by MTT assay, and the effects of DSF on cell migration, adhesion and invasion were determined by cell scratch, matrix adhesion, endothelial cell adhesion and Transwell chamber assay.Western blot analysis of the effects of DSF on the protein expression levels of E-cadherin and Vimentin in EMT model;qPCR was used to detect the effects of DSF on the mRNA expression levels of Vimentin, E-cadherin and EMT-related stem cell marker genes Acet aldehyde dehydrogenase-1(ALDH-1),Sex-determined Y-frame protein 2(SOX2) and Octamer transcription factor 4(OCT4).RESULTS DSF inhibited the EMT process of tumor cells in a dose-dependent manner with IC_(50)=80 μM.Compared with the control group, DSF significantly inhibited the enhancement of migration, adhesion and invasion of TGF-β_(1)-induced A549 after EMT.DSF significantly reduced the expression of Vimentin, promoted the expression of E-cadherin, and significantly reduced the mRNA expression levels of stem cell marker genes ALDH-1,SOX2 and OCT4.RESULTS DSF can reverse the EMT process induced by TGF-β_(1)in A549 cells and inhibit the invasion and migration of A549 cells.The mechanism of its action may be related to downregulation of stem cell related genes ALDH-1,SOX2 and OCT4.