LMP1诱导鼻咽癌细胞发生TRAIL抵抗的体外实验

 目的探讨EBV膜潜伏蛋白1(latent membrane protein 1，LMP1)基因过表达后对肿瘤坏死因子相关的凋亡诱导配体(TNF related apoptosis inducing ligand，TRAIL)凋亡诱导作用和凋亡信号传导的影响。方法利用脂质体介导的pGL6 LMP1上调LMP1低表达的TRAIL抵抗性鼻咽癌细胞CNE 1中LMP1的表达；通过MTT比色法、流式细胞术观察LMP1过表达后对CNE 1细胞TRAIL敏感性的影响；流式细胞术、Western blot、线粒体膜电位检测观察LMP1过表达后对死亡受体表达、细胞内外凋亡信号通路激活、线粒体膜电位改变的影响。结果死亡受体荧光标记后流式细胞术检测发现CNE 1和CNE 1 LMP1之间细胞膜蛋白死亡受体4（death receptor 4,DR4），死亡受体5（death receptor,DR5）表达差异无统计学意义（P>0.05）。Western blot结果显示TRAIL(100 ng/ml)分别作用2、4、6、12 h后，CNE 1细胞中caspase 8 p43/p41，p18亚单位蛋白和caspase 3 p17，p10亚单位蛋白表达明显高于CNE 1 LMP1细胞，而Bcl 2相互作用域死亡激动蛋白的截短形式（truncated form of Bid,tBid）和caspase 9 p35亚单位蛋白表达无明显改变，且两个细胞株间也无明显区别。JC 1线粒体膜电位检测法结果显示TRAIL干预后，线粒体膜电位无明显改变，且两个细胞株间也无明显区别。结论LMP1过表达抑制TRAIL对鼻咽癌细胞的凋亡诱导作用和其细胞外凋亡信号的传导，提示LMP1是通过抑制细胞外信号通路激活诱导鼻咽癌细胞发生TRAIL抵抗。

In vitro study of LMP1 induced TRAIL resistance in nasopharyngeal carcinoma cells

 ObjectiveTo observe the effect of latent membrane protein 1 (LMP1) over expression on TNF related apoptosis inducing ligand(TRAIL) induced apoptosis and TRAIL activated apoptotic pathway.MethodsLMP1 expression in CNE 1 which was TRAIL resistant LMP1 low expressive nasopharyngeal carcinoma (NPC) cell was up regulated by liposome mediated pGL6 LMP1. The TRAIL sensitivities of CNE 1 LMP1 and CNE 1 were measured by MTT and flow cytometry. The expression of death receptor (DR), activation of apoptotic pathway and mitochondrial transmembrane potential were detected by flow cytometry, Western blot and JC 1 assessment, respectively.ResultsFlow cytometry showed no significant differences in expressions of DR4 and DR5 between CNE 1 and CNE 1 LMP1 cell lines (all P>0.05). Moreover, after TRAIL (100 ng/ml) administration for 2, 4, 6, 12 hours, the expressions of caspase 8 p43/p41, tathmin/oncoprotein 18p18 and caspase 3 p17, p10 in CNE 1 were higher than those in CNE 1 LMP1. However, there were no significant differences in expressions of tBid and caspase 9 p35 between this two cell lines. JC 1 assessment showed that TRAIL administration could not affect the mitochondrial transmembrane potential and there was no significant difference in mitochondrial transmembrane potential between CNE 1 and CNE 1 LMP1.ConclusionLMP1 over expression can inhibit apoptosis and extracellular apoptotic pathway, which indicates LMP1 may induce TRAIL resistance in NPC cell through inhibiting extracellular apoptotic pathway.