白花丹素对舌鳞状细胞癌细胞上皮间质转化的作用及机制研究

Effect of plumbagin on epithelial-mesenchymal transition and underlying mechanisms in human tongue squamous cell carcinoma cells

目的探讨白花丹素对舌鳞状细胞癌（tonguesquamouscellcarcinoma，TSCC）细胞上皮间质转化的作用及其可能机制，为白花丹素治疗TSCC提供理论依据。方法甲基噻唑基四唑法检测0．1、1．0、5．0、10．0、20．0μmol／L白花丹素对TSCC细胞增殖的抑制作用，并计算12、24、48h时白花丹素的半数抑制浓度（IC50）。0（对照）、0．1、0．5、1．0μmol／L白花丹素作用细胞24h后，Transwell实验计数穿膜细胞数量、划痕实验测量细胞迁移率。流式细胞仪检测空白对照组、白花丹素组（1．0μmol／L、24h）和谷胱甘肽联合组（活性氧清除剂谷胱甘肽＋白花丹素）细胞内活性氧水平；蛋白质印迹法检测3组上皮钙黏素、波形蛋白、锌指转录因子（Slug）、p38丝裂原活化蛋白激酶（p38mitogen-activatedproteinkinase，p38MAPK）和磷酸化p38MAPK（phospho—p38MAPK，p-p38MAPK）的蛋白表达。蛋白质印迹法分别检测空白对照组、白花丹素组、激动剂联合组（p38MAPK激动剂＋白花丹素）、抑制剂联合组（p38MAPK抑制剂＋白花丹素）上皮钙黏素、波形蛋白、S1ug的蛋白表达。结果白花丹素作用TSCC细胞12、24、48h的IC50分别为10．3、3．1、1．5μmol／L；1．0μmol／L白花丹素作用TSCC细胞24h后，穿膜细胞数量[（50±13）个]较对照[（204±6）个]显著下降（P〈0．05），细胞迁移率[（18．2±2.3）％]也较对照[（49．3±1．2）％1显著下降（P〈0．05）。与空白对照组（2．32±0．52）相比，白花丹素组活性氧（902．20±10．69）显著增加（P〈0．05）；而谷胱甘肽联合组活性氧（2．18±0．15）比白花丹素组显著下降（P〈0．05）。与空白对照组相比，白花丹素组上皮钙黏索显著增加（P〈0．05），波形蛋白、Slug、P—p38MAPK／p38MAPK显著减少（P〈0．05）；而谷胱甘肽联合组上皮钙黏素较白花丹素组显著减少（P〈0．05），波形蛋白、Slug、p-p38MAPK／p38MAPK均较白花丹素组显著增加（P〈0．05）。激动剂联合组上皮钙黏素较白花丹素组显著减少（P〈0．05），波形蛋白和Slug均较白花丹素组显著增加（P〈0．05）。抑制剂联合组上皮钙黏素较白花丹素组显著增加（P〈0．05），波形蛋白和Slug均较白花丹素组显著减少（P〈0．05）。结论白花丹素可抑制TSCC细胞的上皮间质转化，活性氧／p38MAPK信号通路参与了这一进程。

Objective To study the effect of plumbagin on epithelial-mesenchymal transition （EMT） and underlying mechanisms in human tongue squamous cell carcinoma （TSCC） cells. Methods Methyl thiazolyl tetrazolium assay was apllied to examine the proliferation inhibition effect and half maximal inhibitory concentration （IC50） of plumbagin （0.1, 1.0, 5.0, 10.0, 20.0 μmol/L） in 12, 24, 48 h in TSCC cells. Transwell assay was used to count the number of transmembrane cells and scratch test was performed to examine cells mobility. The flow cytometry was applied to measure intracellular reactive oxygen species （ROS） level in control group, plumbagin group （1.0 μmol/L, 24 h） and glutathione （GSH）＋plumbagin group. The expression of E-cadherin, vimentin, Slug, p38 mitogen activated protein kinases （p38MAPK） and phospho-p38MAPK （p-p38MAPK） proteins were determined by Western blotting. The expression of E-cadherin, vimentin and Slug were detected by Western blotting in control group, plumbagin group, activator combined group （p38MAPK activator ＋ plumbagin） and inhibitor combined group （p38MAPK inhibitor＋plumbagin）. Results After the treatment of plumbagin for 12, 24, and 48 h, the ICs0 of TSCC ceils were 10.3, 3.1, 1.5 μmol/L. After treated by 1.0 μmol/L plumbagin for 24 h, the number of transmembrane cells were significantly reduced （[50±13], P〈0.05） in comparison to control group （204±6）, as well as the cells mobility （[18.2 ± 2.31%, P〈0.05） in comparison to control group （[49.3 ± 1.21%）. Compared to control group （2.32±0.52）, the ROS level was increased in plumbagin group （902.20±10.69）, while compared to plumbagin group, the ROS level was reduced in GSH combined group （2.18±0.15）. In comparison to control group, the expression of E-cadherin was up-regulated （P〈0.05）, and vimentin, Slug, p-p38MAPK/p38MAPK were down-regulated in plumbagin group （P〈0.05）. In comparison to plumbagin group, the expression of E-cadherin was down-regulated （P〈0.05）, and vimentin, Slug, p-p38MAPK/p38MAPK were up-regulated in GSH combined group （P〈0.05）. Treatment of cells with p38MAPK activator could decrease the expression of E-cadherin significantly （P〈0.05） and increase the expression of vimentin （P〈0.05） and Slug （P〈0.05） in comparison to plumbagin group. Treatment of cells with p38MAPK inhibitor could increase the expression of E-cadherin significantly （P〈0.05） and decrease the expression of vimentin （P〈0.05） and Slug （P〈0.05） in comparison to plumbagin group. Conclusions Plumbagin inhibits EMT via ROS/p38MAPK-mediated pathway in human TSCC cells.