硒化合物与EGFR/MAPK信号传导相关性的初步研究

Preliminary study on the correlation of selenite,Se-Methylselenocystein and the EGFR/MAP kinase signal transduction in human esophageal carcinoma EC109 cell line

目的 :比较研究亚硒酸钠 (Na2 SeO3 )及硒 -甲基硒代半胱氨酸 (MSC )与EGFR/MAPK信号传导的相关性 ,初步探索这两种硒化合物可能的与MAPK信号传导相关的抗肿瘤作用机制。方法 :利用细胞形态学、丫啶橙 (acridineorange ,AO)荧光染色、免疫组化及免疫蛋白印迹等方法 ,观察分析Na2 SeO3和MSC在不同作用浓度与时间对人食管癌EC10 9细胞的细胞形态学改变以及EGFR、MAPK、act ERK1/2蛋白的表达。结果 :Na2 SeO3 及MSC这两种硒化合物对人食管癌EC10 9细胞EGFR及act ERK1/2蛋白表达均有低浓度 ( 1μg/mL)激活、高浓度抑制的作用 ;并均呈明显的时间和浓度的依赖性。但其区别表现在 :1)细胞形态学观察 :Na2 SeO3主要表现为细胞肿胀 ,出现胞质空泡及颗粒状物质增多等毒性改变 ,并随剂量增加毒性改变愈明显 ;MSC则主要呈现细胞固缩、凋亡小体形成 ,未见明显细胞毒性作用。 2 )MSC组对EC10 9细胞EGFR/act ERK1/2信号传导的抑制作用均明显高于相应浓度的Na2 SeO3组 ,P <0 0 1,但MSC 16μg/mL作用 2 4、48h时EGFR及act ERK1/2蛋白表达有很微弱回升。结论 :1)Na2 SeO3 和MSC对EC10 9细胞系细胞形态学影响与MAPK信号传导相关 ,且对EGFR/MAPK信号通路有抑制作用。 2 )与Na2 SeO3 比较 。

OBJECTIVE: To compare and investigate the anti-cancer effects of the selenite and Se-Methylselenocysteine (MSC) and the correlation of MAP kinase signal transduction. The anti-cancer mechanism of the selenium compound was also rudimently researched.METHODS:By using the cell morphology,acridine orange fluorescence staining,immunohistochemistry and Western blot, the expressions of epidermal growth factor receptor (EGFR),mitogen-actived protein kinase (MAPK) and activated-extracellular signal regulated kinase1/2 (act-ERK1/2) in human esophageal carcinoma EC109 cell line were observed and analysed at different concentrations and times of selenite and MSC.RESULTS:Immunohistochemistry and Western blot analyses indicated that phosphorylation of EGFR and ERK1/2 was actived in lower dose (1 μg/mL) and was inhibited in higher dose following by concentration and time dependent. In contrast, exposure of EC109 cell to MSC versus selenite appeared differentia: 1)cell morphology: selenite exposure caused cell swelling and cytotoxicity of dose-dependent such as extensive cytoplasmic vacuolization and increasing granule substance. MSC exposure presented cell shrinking with rugous membrane and nuclei condensation and apoptosis body was observed in cytoplasm.The preceding changes were more obvious with the increasing dosage and no obvious cytotoxicitic changes. 2)MSC exposure appeared stronger effects than corresponding concentration of selenite exposure in inhibited EGFR/act-ERK1/2 protein expression, P<0.01, but the expressions of EGFR and act-ERK1/2 protein appeared a high level again in MSC 16 μg/mL (24,48 h).CONCLUSIONS:Selenite and MSC can hinder the EGFR/MAPK signal passway.MSC has lower cytotoxicity and stronger effects on EC109 cell line than selenite.