Abstract
Abstract Epstein-Barr virus (EBV) is one of the etiological factors in the carcinogenesis of nasopharyngeal carcinoma. EBV genome can be detected in almost all NPC biopsies of NPC patients. The presence of several EBV latent gene products is correlated with the carcinogenesis of NPC. On the other hand, more and more studies have indicated that EBV lytic gene products, such as BLLF1 and BZLF1(Zta) were also detected in NPC tissues. EBV lytic proteins had been found to express in NPC biopsies. In NPC patients, the titer of anti-EBV lytic antibodies, including VCA, EAD, BGLF5, BALF5, BHRF1, BXLF1, BRLF1(Rta) and BZLF1(Zta), were found to increase during the progression of this disease. These results suggested that induction of EBV into lytic cycle (“the EBV reactivation”) is present in NPC patients. Our previous study has indicated that EBV lytic products can induce host cell DNA damage and genome instability, which may lead to the carcinogenesis of NPC. Since there is only little evidence that indicates EBV reactivation occurs in vivo, the need for establishment of a specific procedure for detecting EBV reactivation is important for elucidating the relationship between EBV reactivation and NPC carcinogenesis. In this study, several anti-EBV lytic antibodies were used for detection of EBV lytic gene, including Zta, Rta and EAD. In addition, an RNA in situ hybridization assay was used to detect Zta and Rta mRNA in cell and NPC tumor xenografts. These studies may provide ways for studying the relation between EBV reactivation and NPC carcinogenesis. Previously, our laboratory had screened several compounds that can effectively inhibit EBV reactivation including (-)-epigallocatechin-3-gallate (EGCG), which is in accordance with the characteristics of chemopreventive agents. Therefore we chose EGCG to study its chemopreventive ability on NPC in this study. EGCG is the major polyphenol compound in green tea. It has been reported that EGCG can inhibit the proliferation of human breast, lung, gastric and hepatic cancer cells in vitro. The main goal of this study was to investigate whether EGCG can inhibit proliferation, mobility and induce apoptosis of human nasopharyngeal carcinoma cells. Cytotoxicity assay revealed that EGCG can inhibit cell growth at concentration greater than 25μM. By migration and invasiveness assay, treatment of EGCG could significantly reduce NPC cells mobility, migration and invasion. In addition, gelatin zymography indicated that EGCG can reduce the activity of MMP-2 and MMP-9. The expression of cell adhesion molecules, E-cadherin and β-catenin, was increased upon EGCG trement, as evaluated by western blotting. EGCG treatment was also found to increase the cytosolic accumulation of β-catenin and NF-κB, while reducing it’s nuclear levels. The expression level of tropomyosins was increased after EGCG treatment in NPC cells. Moreover, the levels of cleaved-caspase-3 and annexin-V-positive cells were increased after high concentration of EGCG indicating the apoptosis-inducing ability of EGCG. By spheroid formation assay, EGCG was found to effectively inhibit tumor spheroid formation even at lower concentrations. Finally in an animal experiment, administration of EGCG could markedly reduce the growth of NPC tumors in SCID mice. Additionally, we examined whether EGCG can inhibit EBV reactivation and EBV reactivation induced apoptosis by IHC. However, because of individual differences in mice, the result is not significant. Taken together, these results indicate that EGCG can effectively inhibit cell growth, migration and invasion, and induce apoptosis of NPC cells. However, whether EGCG can inhibit EBV reactivation remains to be examined in the future. Key words：EGCG, Epstein–Barr virus, human nasopharyngeal carcinoma (NPC), cells adhesion, apoptosis, spheroid formation, cell mobility, western blot, immunohistochemistry, immunofluorescence stain, QuantiGene viewRNA ISH Tissue Assay.