Perspective: Contribution of Epstein-Barr virus (EBV) Reactivation to the Carcinogenicity of Nasopharyngeal Cancer Cells.

Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma derived from the epithelium of the post-nasal cavity, with a unique geographic and ethnic distribution. Epstein–Barr virus (EBV) is an etiological agent of NPC, but how it contributes to carcinogenesis is not completely clear. Although it is thought that EBV latency participates in the development of NPC, increasing evidence reveals that the lytic cycle also plays an important role in the carcinogenic process. In this review, we summarize our recent studies on how EBV reactivation causes genomic instability and accelerates tumorigenesis in epithelial cells. The roles of three lytic genes, namely, BRLF1, BGLF5 and BALF3, in this process are also introduced. Moreover, blocking EBV reactivation using natural compounds may help delay the progression of NPC tumorigenesis. These studies provide a new insight into NPC carcinogenesis and raise the possibility that inhibition of EBV reactivation may be a novel approach to prevent the relapse of NPC.

Epstein-Barr virus BRLF1 induces genomic instability and progressive malignancy in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is a serious health problem in China and Southeast Asia. Relapse is the major cause of mortality, but mechanisms of relapse are mysterious. Epstein-Barr virus (EBV) reactivation and host genomic instability (GI) have correlated with NPC development. Previously, we reported that lytic early genes DNase and BALF3 induce genetic alterations and progressive malignancy in NPC cells, implying lytic proteins may be required for NPC relapse. In this study, we show that immediate early gene BRLF1 induces chromosome mis-segregation and genomic instability in the NPC cells. Similar phenomenon was also demonstrated in 293 and zebrafish embryonic cells. BRLF1 nuclear localization signal (NLS) mutant still induced genomic instability and inhibitor experiments revealed that BRLF1 interferes with chromosome segregation and induces genomic instability by activating Erk signaling. Furthermore, the chromosome aberrations and tumorigenic features of NPC cells were significantly increased with the rounds of BRLF1 expression, and these cells developed into larger tumor nodules in mice. Therefore, BRLF1 may be the important factor contributing to NPC relapse and targeting BRLF1 may benefit patients.

Inhibition of Epstein-Barr virus reactivation by the flavonoid apigenin.

BACKGROUND: Lytic reactivation of EBV has been reported to play an important role in human diseases, including NPC carcinogenesis. Inhibition of EBV reactivation is considered to be of great benefit in the treatment of virus-associated diseases. For this purpose, we screened for inhibitory compounds and found that apigenin, a flavonoid, seemed to have the ability to inhibit EBV reactivation. METHODS: We performed western blotting, immunofluorescence and luciferase analyses to determine whether apigenin has anti-EBV activity. RESULTS: Apigenin inhibited expression of the EBV lytic proteins, Zta, Rta, EAD and DNase in epithelial and B cells. It also reduced the number of EBV-reactivating cells detectable by immunofluorescence analysis. In addition, apigenin has been found to reduce dramatically the production of EBV virions. Luciferase reporter analysis was performed to determine the mechanism by which apigenin inhibits EBV reactivation: apigenin suppressed the activity of the immediate-early (IE) gene Zta and Rta promoters, suggesting it can block initiation of the EBV lytic cycle. CONCLUSION: Taken together, apigenin inhibits EBV reactivation by suppressing the promoter activities of two viral IE genes, suggesting apigenin is a potential dietary compound for prevention of EBV reactivation.

Luteolin inhibits Epstein-Barr virus lytic reactivation by repressing the promoter activities of immediate-early genes.

The lytic reactivation of Epstein-Barr virus (EBV) has been reported to be strongly associated with several human diseases, including nasopharyngeal carcinoma (NPC). Inhibition of the EBV lytic cycle has been shown to be of great benefit in the treatment of EBV-associated diseases. The administration of dietary compounds is safer and more convenient than other approaches to preventing EBV reactivation. We screened several dietary compounds for their ability to inhibit EBV reactivation in NPC cells. Among them, the flavonoid luteolin showed significant inhibition of EBV reactivation. Luteolin inhibited protein expression from EBV lytic genes in EBV-positive epithelial and B cell lines. It also reduced the numbers of EBV-reactivating cells detected by immunofluorescence analysis and reduced the production of virion. Furthermore, luteolin reduced the activities of the promoters of the immediate-early genes Zta (Zp) and Rta (Rp) and also inhibited Sp1-luc activity, suggesting that disruption of Sp1 binding is involved in the inhibitory mechanism. CHIP analysis revealed that luteolin suppressed the activities of Zp and Rp by deregulating Sp1 binding. Taken together, luteolin inhibits EBV reactivation by repressing the promoter activities of Zp and Rp, suggesting luteolin is a potential dietary compound for prevention of virus infection.

EBV reactivation as a target of luteolin to repress NPC tumorigenesis.

Nasopharyngeal carcinoma (NPC) is a malignancy derived from the epithelial cells of the nasopharynx. Although a combination of radiotherapy with chemotherapy is effective for therapy, relapse and metastasis after remission remain major causes of mortality. Epstein-Barr virus (EBV) is believed to be one of causes of NPC development. We demonstrated previously that EBV reactivation is important for the carcinogenesis of NPC. We sought, therefore, to determine whether EBV reactivation can be a target for retardation of relapse of NPC. After screening, we found luteolin is able to inhibit EBV reactivation. It inhibited EBV lytic protein expression and repressed the promoter activities of two major immediate-early genes, Zta and Rta. Furthermore, luteolin was shown to reduce genomic instability induced by recurrent EBV reactivation in NPC cells. EBV reactivation-induced NPC cell proliferation and migration, as well as matrigel invasiveness, were also repressed by luteolin treatment. Tumorigenicity in mice, induced by EBV reactivation, was decreased profoundly following luteolin administration. Together, these results suggest that inhibition of EBV reactivation is a novel approach to prevent the relapse of NPC.

EGCG inhibits proliferation, invasiveness and tumor growth by up-regulation of adhesion molecules, suppression of gelatinases activity, and induction of apoptosis in nasopharyngeal carcinoma cells.

(-)-Epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, has been shown to inhibit the proliferation of a variety of tumor cells. Epidemiological studies have shown that drinking green tea can reduce the incidence of nasopharyngeal carcinoma (NPC), yet the underlying mechanism is not well understood. In this study, the inhibitory effect of EGCG was tested on a set of Epstein Barr virus-negative and -positive NPC cell lines. Treatment with EGCG inhibited the proliferation of NPC cells but did not affect the growth of a non-malignant nasopharyngeal cell line, NP460hTert. Moreover, EGCG treated cells had reduced migration and invasive properties. The expression of the cell adhesion molecules E-cadherin and ß-catenin was found to be up-regulated by EGCG treatment, while the down-regulation of matrix metalloproteinases (MMP)-2 and MMP-9 were found to be mediated by suppression of extracellular signal-regulated kinase (ERK) phosphorylation and AP-1 and Sp1 transactivation. Spheroid formation by NPC cells in suspension was significantly inhibited by EGCG. Oral administration of EGCG was capable of suppressing tumor growth in xenografted mice bearing NPC tumors. Treatment with EGCG was found to elevate the expression of p53 and p21, and eventually led to apoptosis of NPC cells via caspase 3 activation. The nuclear translocation of NF-κB and ß-catenin was also suppressed by EGCG treatment. These results indicate that EGCG can inhibit the proliferation and invasiveness, and induce apoptosis, of NPC cells, making it a promising agent for chemoprevention or adjuvant therapy of NPC.

Reactive oxygen species mediate Epstein-Barr virus reactivation by N-methyl-N'-nitro-N-nitrosoguanidine.

N-nitroso compounds (NOCs) and Epstein-Barr virus (EBV) reactivation have been suggested to play a role in the development of nasopharyngeal carcinoma (NPC). Although chemicals have been shown to be a risk factor contributing to the carcinogenesis of NPC, the underlying mechanism is not fully understood. We demonstrated recently that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) enhances the genomic instability and tumorigenicity of NPC cells via induction of EBV reactivation. However, the mechanisms that trigger EBV reactivation from latency remain unclear. Here, we address the role of ROS in induction of EBV reactivation under MNNG treatment. EBV reactivation was induced in over 70% of EBV-positive NA cells and the promoter of Rta (Rp) was activated after MNNG treatment. Inhibitor experiments revealed ATM, p38 MAPK and JNK were activated by ROS and involved in MNNG-induced EBV reactivation. Significantly, ROS scavengers N-acetyl-L-cysteine (NAC), catalase and reduced glutathione inhibited EBV reactivation under MNNG and H2O2 treatment, suggesting ROS mediate EBV reactivation. The p53 was essential for EBV reactivation and the Rp activation by MNNG. Moreover, the p53 was phosphorylated, translocated into nucleus, and bound to Rp following ROS stimulation. The results suggest ROS play an important role in initiation of EBV reactivation by MNNG through a p53-dependent mechanism. Our findings demonstrate novel signaling mechanisms used by NOCs to induce EBV reactivation and provide a novel insight into NOCs link the EBV reactivation in the contribution to the development of NPC. Notably, this study indicates that antioxidants might be effective for inhibiting N-nitroso compound-induced EBV reactivation and therefore could be promising preventive and therapeutic agents for EBV reactivation-associated malignancies.

Microtubule depolymerization activates the Epstein-Barr virus lytic cycle through protein kinase C pathways in nasopharyngeal carcinoma cells.

Elevated levels of antibodies against Epstein-Barr virus (EBV) and the presence of viral DNA in plasma are reliable biomarkers for the diagnosis of nasopharyngeal carcinoma (NPC) in high-prevalence areas, such as South-East Asia. The presence of these viral markers in the circulation suggests that a minimal level of virus reactivation may have occurred in an infected individual, although the underlying mechanism of reactivation remains to be elucidated. Here, we showed that treatment with nocodazole, which provokes the depolymerization of microtubules, induces the expression of two EBV lytic cycle proteins, Zta and EA-D, in EBV-positive NPC cells. This effect was independent of mitotic arrest, as viral reactivation was not abolished in cells synchronized at interphase. Notably, the induction of Zta by nocodazole was mediated by transcriptional upregulation via protein kinase C (PKC). Pre-treatment with inhibitors for PKC or its downstream signalling partners p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) abolished the nocodazole-mediated induction of Zta and EA-D. Interestingly, the effect of nocodazole, as well as colchicine and vinblastine, on lytic gene expression occurred only in NPC epithelial cells but not in cells derived from lymphocytes. These results establish a novel role of microtubule integrity in controlling the EBV life cycle through PKC and its downstream pathways, which represents a tissue-specific mechanism for controlling the life-cycle switch of EBV.

Inhibition of Epstein-Barr virus reactivation in nasopharyngeal carcinoma cells by dietary sulforaphane.

Epstein-Barr virus (EBV) has been associated with several human malignancies including nasopharyngeal carcinoma (NPC). Reactivation of latent EBV has been considered to contribute to the carcinogenesis of NPC. Blocking the EBV lytic cycle has been shown effective in the treatment of EBV-associated diseases. We have searched for natural dietary compounds inhibiting EBV reactivation in NPC cells. Among them, sulforaphane (SFN) was found to be effective in the inhibition of EBV reactivation in latent EBV-positive NPC cells, NA and HA. SFN is a histone deacetylase (HDAC) inhibitor and has been recognized as an antioxidant and antitumor compound for chemoprevention. However, its antiviral effect is less well elucidated. In this study, after determination of the cytotoxicity of SFN on various epithelial cells, we showed that SFN treatment inhibits EBV reactivation, rather than induction, by detection of EBV lytic gene expression in EBV-positive NPC cells. We also determined that the number of cells supporting the EBV lytic cycle is decreased using immunofluorescence and flow cytometric analysis. Moreover, we have found that this inhibitory effect decreases virus production. To elucidate the inhibitory mechanism of SFN on the EBV lytic cycle, luciferase reporter assays were carried out on the Zta and Rta promoters. The results show that SFN inhibits transactivation activity of the EBV immediate-early gene Rta but not Zta. Together, our results suggest that SFN has the capability to inhibit EBV lytic cycle and the potential to be taken as a dietary compound for prevention of EBV reactivation.

The synergistic effect of chemical carcinogens enhances Epstein-Barr virus reactivation and tumor progression of nasopharyngeal carcinoma cells.

Seroepidemiological studies imply a correlation between Epstein-Barr virus (EBV) reactivation and the development of nasopharyngeal carcinoma (NPC). N-nitroso compounds, phorbols, and butyrates are chemicals found in food and herb samples collected from NPC high-risk areas. These chemicals have been reported to be risk factors contributing to the development of NPC, however, the underlying mechanism is not fully understood. We have demonstrated previously that low dose N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.1 µg/ml) had a synergistic effect with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate (SB) in enhancing EBV reactivation and genome instability in NPC cells harboring EBV. Considering that residents in NPC high-risk areas may contact regularly with these chemical carcinogens, it is vital to elucidate the relation between chemicals and EBV and their contributions to the carcinogenesis of NPC. In this study, we constructed a cell culture model to show that genome instability, alterations of cancer hallmark gene expression, and tumorigenicity were increased after recurrent EBV reactivation in NPC cells following combined treatment of TPA/SB and MNNG. NPC cells latently infected with EBV, NA, and the corresponding EBV-negative cell, NPC-TW01, were periodically treated with MNNG, TPA/SB, or TPA/SB combined with MNNG. With chemically-induced recurrent reactivation of EBV, the degree of genome instability was significantly enhanced in NA cells treated with a combination of TPA/SB and MNNG than those treated individually. The Matrigel invasiveness, as well as the tumorigenicity in mouse, was also enhanced in NA cells after recurrent EBV reactivation. Expression profile analysis by microarray indicates that many carcinogenesis-related genes were altered after recurrent EBV reactivation, and several aberrations observed in cell lines correspond to alterations in NPC lesions. These results indicate that cooperation between chemical carcinogens can enhance the reactivation of EBV and, over recurrent reactivations, lead to alteration of cancer hallmark gene expression with resultant enhancement of tumorigenesis in NPC.

Epstein-Barr virus Rta-mediated transactivation of p21 and 14-3-3σ arrests cells at the G1/S transition by reducing cyclin E/CDK2 activity.

Many herpesviral immediate-early proteins promote their robust lytic phase replications by hijacking the cell cycle machinery. Previously, lytic replication of Epstein-Barr virus (EBV) was found to be concurrent with host cell cycle arrest. In this study, we showed that ectopic expression of EBV immediate-early protein Rta in HEp-2 cells resulted in increased G1/S population, hypophosphorylation of pRb and decreased incorporation of 5-bromo-2'-deoxyuridine. In addition, EBV Rta transcriptionally upregulates the expressions of p21 and 14-3-3σ in HEp-2 cells, 293 cells and nasopharyngeal carcinoma TW01 cells. Although p21 and 14-3-3σ are known targets for p53, Rta-mediated p21 and 14-3-3σ transactivation can be detected in the absence of p53. In addition, results from luciferase reporter assays indicated that direct binding of Rta to either promoter sequences is not required for activation. On the other hand, a special class of Sp1-responsive elements was involved in Rta-mediated transcriptional activation on both promoters. Finally, Rta-induced p21 expression diminished the activity of CDK2/cyclin E complex, and, Rta-induced 14-3-3σ expression sequestered CDK1 and CDK2 in the cytoplasm. Based on these results, we hypothesize that through the disruption of CDK1 and CDK2 activities, EBV Rta might contribute to cell cycle arrest in EBV-infected epithelial cells during viral reactivation.

N-methyl-N'-nitro-N-nitrosoguanidine induces and cooperates with 12-O-tetradecanoylphorbol-1,3-acetate/sodium butyrate to enhance Epstein-Barr virus reactivation and genome instability in nasopharyngeal carcinoma cells.

Seroepidemiological studies implicate a correlation between Epstein-Barr virus (EBV) reactivation and the development of nasopharyngeal carcinoma (NPC). Moreover, N-nitroso compounds are known chemical carcinogens in preserved foodstuffs and cigarettes and have been implicated as risk factors contributing to the development of NPC. Here, NPC cell lines latently infected with EBV, NA and HA, and the corresponding EBV-negative NPC cell lines, NPC-TW01 and HONE-1, were used as the model system in this study. We demonstrate that the reactivation of EBV increases with increasing concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MNNG at a single non-toxic concentration (0.1µg/ml) did not induce discernible reactivation of EBV, but repeated treatment with this concentration of MNNG significantly induced viral reactivation. Furthermore, low dose MNNG (0.1µg/ml) had a synergistic effect with 12-O-tetradecanoylphorbol-1,3-acetate (TPA)/sodium butyrate (SB) (10ng/ml and 0.75mM, respectively) on EBV reactivation. Through promoter activity assay, MNNG was found to enhance the transcriptional activity of Rta on Rta and Zta promoters. Using siZta to block EBV reactivation, the concomitant induction of genome instability was diminished indicating that reactivation is critical for enhancing genome instability. Co-treatment with TPA/SB and MNNG markedly increased the levels of γH2AX and ROS formation in NPC cells, which may be responsible for the increase of genome instability. Our findings offer a possible mechanism by which N-nitroso compounds induce reactivation of EBV and contribute to malignant progression by enhancing genome instability in NPC cells.