Molecular, biological studies on the mechanism of neck metastasis from nasopharyngeal carcinoma

鼻咽癌是一种具有明显地理和人群差异的上皮性肿瘤，血清学研究发现鼻咽癌与EB病毒(EBV)相关，鼻咽癌细胞中EB病毒DNA和EB编码的RNA的检测证实了这种关系。而鼻咽癌是一种高转移性肿瘤，其与EBV的相关性已被证实。潜伏膜蛋白1(LMP1)是一种在潜伏感染中表达的EBV膜蛋白，被认为是EBV癌蛋白。基质金属蛋白酶9(MMP9)是基质金属蛋白酶家族的一员，它降解细胞外基质的主要成分IV型胶原，被认为是肿瘤侵袭和转移的关键。尽管有报道称MMP9在多种癌症中都有表达，但目前还没有关于鼻咽癌的报道。我们在体外实验中发现LMP1可诱导MMP9的表达，提示EB病毒LMP1参与…转移的可能机制。MMP9诱导鼻咽癌的发生发展采用免疫组织化学方法检测LMP1、MMP9蛋白的表达，并对二者之间的关系进行统计学分析。我们还分析了这些蛋白与临床特征的关系。结果表明，LMP1和MMP9蛋白主要分布在癌巢中。MMP9表达与LMP1表达呈显著正相关。此外，我们还证明LMP1通过激活核因子κB和激活蛋白(AP)-1来增强MMP9的表达。因此，我们测试了LMP1对MMP9的上调是否与肿瘤细胞体外侵袭力的增强有关。此外，还检测了阿司匹林和水杨酸钠是否能降低肿瘤的侵袭力，以及LMP1是否能增强裸鼠移植瘤中MMP9的表达。与载体转染组相比，稳定表达LMP1的CS3A细胞MMP9表达增加，并表现出更强的侵袭能力。阿司匹林和水杨酸钠处理抑制了表达LMP1的C33A细胞的侵袭性，并在酶谱分析中抑制了LMP1诱导的MMP9的表达，在CAT报告实验中抑制了LMP1诱导的MMP9启动子的活性。阿司匹林对NF-κB活性的抑制作用可能与抑制I-κB活性有关。最后，在裸鼠体内生长的稳定表达LMP1的C3SA细胞的肿瘤与载体感染的C33A细胞的肿瘤相比，MMP9的水平升高。这种增强被用阿司匹林治疗的小鼠抑制。这些结果表明，阿司匹林可能通过抑制基质金属蛋白酶-9来抑制表达LMP1的EBV相关肿瘤的侵袭和转移。较少

Nasopharyngeal carcinoma(NPC), an epithelial tumor which is characterized by marked geographic and population differences in incidence, is found to be associated with Epstein-Barr virus(EBV) by serologic evidence, and the relationship was confirmed by the detection of EBVDNA and EB-encoded RNAs in NPC cells. While, NPC is highly metastatic carcinoma whose consistent associated with EBV has been established. Latent membrane protein 1(LMP1), an EBV membrane protein expressed in latent infection is considered to be the EBV oncoprotein. Matrix metalloproteinase 9(MMP9), one of the MMP families, degrades Type IV collagen, a major 4 component of extracellural matrix and is believed to be crucial for cancer invasion and metastasis. Although MMP9 is reported to be expressed in a variety of cancers, no reports concerning NPC have been published. We have shown that LMP1 induces MMP9 in vitro cell line, which suggests the possibility of mechanism in which LMP1 of EBV contributes to the metastasis … More and tumorgenesis of NPC by the induction of MMP9. Then the expression of LMP1 and MMP9 were immunohistochemically examined, and the relation of these proteins was statistically analyzed. We also analyzed the association of these proteins with clinical features. As results, both LMP1 and MMP9 proteins were predominantly immunolocalized in cancer nests. The expression of MMP9 showed a significant positive correlation with the expression of LMP1. Also, the expression of MMP9 correlated with lymphnode metastasis.We also demonstrated that LMP1 enhances MMP9 expression by activation of nuclear factor(NF)κB and activator protein(AP)-1. We therefore tested whether up-regulation of MMP9 by LMP1 could be correlated with enhanced invasiveness of tumor cells in vitro. Whether aspirin and sodium salicylate could reduce invasiveness and whether LMP1 could enhance MMP9 expression in tumors grown in nude mice were also tested. CS3A cells stably expressing LMP1 had increased expression of MMP9 and showed greater invasion through reconstituted basement membrane compared with vector-transfected C33A cells. Treatment with aspirin and sodium salicylate inhibited invasiveness of the LMP1-expressing C33A cells and suppressed both the LMP1-induced MMP9 expewssion in zymographic analyses and LMP1-induced MMP9 promoter activity in CAT reporter assays. The inhibitory effect of aspirin on NF-κB activity was attributable to the inhibition of I-κB kinase activity. Finally, tumors derived from vector-transfected C3SA cells stably expressing LMP1 grown in nude mice showed enhanced MMP9 levels compared with tumors derived from vector-trancfected C33A cells. This enhancement was inhibited by treatment of the mice with aspirin. These results suggest that aspirin may be able to suppress invasion and metastasis of EBV-associated tumors that express LMP1 by suppression of MMP-9. Less