Li B, Pan S T, Qiu J X
Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang 330000, China.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2017 Jul 9;52(7):421-426. doi: 10.3760/cma.j.issn.1002-0098.2017.07.006.
To study the effect of plumbagin on epithelial-mesenchymal transition (EMT) and underlying mechanisms in human tongue squamous cell carcinoma (TSCC) cells. Methyl thiazolyl tetrazolium assay was apllied to examine the proliferation inhibition effect and half maximal inhibitory concentration (IC(50)) 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). After the treatment of plumbagin for 12, 24, and 48 h, the IC(50) of TSCC cells 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], 0.05) in comparison to control group (204±6), as well as the cells mobility ([18.2±2.3]%, 0.05) in comparison to control group ([49.3±1.2]%). 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 (0.05), and vimentin, Slug, p-p38MAPK/p38MAPK were down-regulated in plumbagin group (0.05). In comparison to plumbagin group, the expression of E-cadherin was down-regulated (0.05), and vimentin, Slug, p-p38MAPK/p38MAPK were up-regulated in GSH combined group (0.05). Treatment of cells with p38MAPK activator could decrease the expression of E-cadherin significantly (0.05) and increase the expression of vimentin (0.05) and Slug (0.05) in comparison to plumbagin group. Treatment of cells with p38MAPK inhibitor could increase the expression of E-cadherin significantly (0.05) and decrease the expression of vimentin (0.05) and Slug (0.05) in comparison to plumbagin group. Plumbagin inhibits EMT via ROS/p38MAPK-mediated pathway in human TSCC cells.
研究白花丹醌对人舌鳞状细胞癌（TSCC）细胞上皮-间质转化（EMT）的影响及其潜在机制。采用甲基噻唑基四氮唑法检测白花丹醌（0.1、1.0、5.0、10.0、20.0 μmol/L）在12、24、48小时对TSCC细胞的增殖抑制作用及半数抑制浓度（IC50）。使用Transwell实验计数跨膜细胞数量，并进行划痕实验检测细胞迁移能力。应用流式细胞术检测对照组、白花丹醌组（1.0 μmol/L，24小时）和谷胱甘肽（GSH）+白花丹醌组细胞内活性氧（ROS）水平。通过蛋白质印迹法检测E-钙黏蛋白、波形蛋白、锌指蛋白Slug、p38丝裂原活化蛋白激酶（p38MAPK）和磷酸化p38丝裂原活化蛋白激酶（p-p38MAPK）蛋白的表达。白花丹醌处理12、24和48小时后，TSCC细胞的IC50分别为10.3、3.1、1.5 μmol/L。1.0 μmol/L白花丹醌处理24小时后，与对照组（204±6）相比，跨膜细胞数量显著减少（[50±13]，P<0.05），与对照组（[49.3±1.2]%）相比，细胞迁移能力也显著降低（[18.2±2.3]%，P<0.05）。与对照组（2.32±0.52）相比，白花丹醌组ROS水平升高（902.20±10.69），而与白花丹醌组相比，GSH联合组ROS水平降低（2.18±0.15）。与对照组相比，白花丹醌组E-钙黏蛋白表达上调（P<0.05），波形蛋白、Slug、p-p38MAPK/p38MAPK表达下调（P<0.05）。与白花丹醌组相比，GSH联合组E-钙黏蛋白表达下调（P<0.05），波形蛋白、Slug、p-p38MAPK/p38MAPK表达上调（P<0.05）。与白花丹醌组相比，用p38MAPK激活剂处理细胞可显著降低E-钙黏蛋白表达（P<0.05），增加波形蛋白表达（P<0.05）和Slug表达（P<0.05）。与白花丹醌组相比，用p38MAPK抑制剂处理细胞可显著增加E-钙黏蛋白表达（P<0.05），降低波形蛋白表达（P<0.05）和Slug表达（P<0.05）。白花丹醌通过ROS/p38MAPK介导的途径抑制人TSCC细胞的EMT。
