Zijian Li

DNAzymes targeted to EBV-encoded latent membrane protein-1 induce apoptosis and enhance radiosensitivity in nasopharyngeal carcinoma

Epstein-Barr virus (EBV) is involved in the carcinogenesis of several types of cancers such as nasopharyngeal carcinoma (NPC) and Burkitts lymphoma. The latent membrane protein (LMP1) encoded by EBV is expressed in the majority of EBV-associated human malignancies and has been suggested to be one of the major oncogenic factors in EBV-mediated carcinogenesis. Therefore, genetic manipulation of LMP1 expression may provide a novel strategy for the treatment of the EBV-associated human cancers. Deoxyribozymes (DNAzymes) are catalytic nucleic acids that bind and cleave a target RNA in a highly sequence-specific manner. We have designed several LMP1-specific DNAzymes and tested their effect on cell proliferation and apoptosis in LMP1-positive cells. Here, we show that active DNAzymes down-regulated the expression of the EBV oncoprotein LMP1 and inhibited cellular signal transduction pathways abnormally activated by LMP1. This down-regulation of the LMP1 expression was shown to be associated with a decrease in the level of antiapoptotic Bcl-2 and an increase in Caspase-3 and -9 activities in the nasopharyngeal carcinoma cell line CNE1-LMP1, which constitutively expresses the LMP1. When combined with radiation treatment, the DNAzymes significantly induced apoptosis in CNE1-LMP1 cells, leading to an increased radiosensitivity both in cells and in a xenograft NPC model in mice. The results suggest that LMP1 may represent a molecular target for DNAzymes and provide a basis for the use of the LMP1 DNAzymes as potential radiosensitizers for treatment of the EBV-associated carcinomas.

(-)-Epigallocatechin-3-gallate inhibition of Epstein–Barr virus spontaneous lytic infection involves ERK1/2 and PI3-K/Akt signaling in EBV-positive cells

Epstein-Barr virus (EBV) reactivation into the lytic cycle plays certain roles in the development of EBV-associated diseases, including nasopharyngeal carcinoma and lymphoma. In this study, we investigated the effects of the tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) on EBV spontaneous lytic infection and the mechanism(s) involved in EBV-positive cells. We found that EGCG could effectively inhibit the constitutive lytic infection of EBV at the DNA, gene transcription and protein levels by decreasing the phosphorylation and activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt. By using cellular signaling pathway-specific inhibitors, we also explored the signaling mechanisms underlying the inhibitory effects of EGCG on EBV spontaneous lytic infection in cell models. Results show that specific inhibitors of Mitogen-Activated Protein Kinase Kinase (MEK) (PD98059) and phosphatidylinositol 3-kinase [PI3-K (LY294002)] markedly downregulated gene transcription and expression of BZLF1 and BMRF1 indicating that the MEK/ERK1/2 and PI3-K/Akt pathways are involved in the EBV spontaneous lytic cycle cascade. Therefore, one of the mechanisms by which EGCG inhibits EBV spontaneous lytic infection appears to involve the suppression of the activation of MEK/ERK1/2 and PI3-K/Akt signaling.

Down-Regulation of EBV-LMP1 Radio-Sensitizes Nasal Pharyngeal Carcinoma Cells via NF-κB Regulated ATM Expression

Background The latent membrane protein 1 (LMP1) encoded by EBV is expressed in the majority of EBV-associated human malignancies and has been suggested to be one of the major oncogenic factors in EBV-mediated carcinogenesis. In previous studies we experimentally demonstrated that down-regulation of LMP1 expression by DNAzymes could increase radiosensitivity both in cells and in a xenograft NPC model in mice. Results In this study we explored the molecular mechanisms underlying the radiosensitization caused by the down-regulation of LMP1 in nasopharyngeal carcinoma. It was confirmed that LMP1 could up-regulate ATM expression in NPCs. Bioinformatic analysis of the ATM ptomoter region revealed three tentative binding sites for NF-κB. By using a specific inhibitor of NF-κB signaling and the dominant negative mutant of IkappaB, it was shown that the ATM expression in CNE1-LMP1 cells could be efficiently suppressed. Inhibition of LMP1 expression by the DNAzyme led to attenuation of the NF-κB DNA binding activity. We further showed that the silence of ATM expression by ATM-targeted siRNA could enhance the radiosensitivity in LMP1 positive NPC cells. Conclusions Together, our results indicate that ATM expression can be regulated by LMP1 via the NF-κB pathways through direct promoter binding, which resulted in the change of radiosensitivity in NPCs.

LMP1-augmented kappa intron enhancer activity contributes to upregulation expression of Ig kappa light chain via NF-kappaB and AP-1 pathways in nasopharyngeal carcinoma cells

BackgroundExpression of kappa gene is under the control of distinct cis-regulatory elements, including the kappa intron enhancer (iEκ) and the kappa 3 enhancer (3Eκ). The active enhancers and expression of immunoglobulin is generally considered to be restricted to B lymphocytes. However, accumulating evidence indicated that epithelial cancer cells, including nasopharyngeal carcinoma (NPC) cell lines, express immunoglobulins. The mechanisms underlying the expression of Igs in nonlymphoid cells remain unknown. On the basis of our previous finding that expression of kappa light chain in NPC cells can be upregulated by EBV-encoded latent membrane protein 1(LMP1) through the activation of NF-κB and AP-1 signaling pathways, we thus use NPC cells as model to further explore the molecular mechanisms of nonlymphoid cells expressing Ig kappa.ResultsIn this study, luciferase reporter plasmid containing human wild-type iEκ, and its derivative plasmids containing mutant binding sites for transcription factor NF-κB or AP-1 were constructed. Luciferase reporter assays demonstrate iEκ is active in Igκ-expressing NPC cells and LMP1 expression can upregulate the activity of iEκ in NPC cells. Mutation of the NF-κB or AP-1 site within and downstream the iEκ, inhibition of the NF-κB and AP-1 pathways by their respective chemical inhibitor Bay11-7082 and SP600125 as well as stable or transient expression of dominant-negative mutant of IκBα (DNMIκBα) or of c-Jun (TAM67) indicate that both sites are functional and LMP1-enhanced iEκ activity is partly regulated by these two sites. Gel shift assays show that LMP1 promotes NF-κB subunits p52 and p65 as well as AP-1 family members c-Jun and c-Fos binding to the κNF-κB and the κAP-1 motifs in vitro, respectively. Both chemical inhibitors and dominant negative mutants targeting for NF-κB and AP-1 pathways can attenuate the LMP1-enhanced bindings. Co-IP assays using nuclear extracts from HNE2-LMP1 cells reveal that p52 and p65, c-Jun and c-Fos proteins interact with each other at endogenous levels. ChIP assays further demonstrate p52 and p65 binding to the κB motif as well as c-Jun and c-Fos binding to the AP-1 motif of Ig kappa gene in vivo.ConclusionThese results suggest that human iEκ is active in Igκ-expressing NPC cells and LMP1-stimulated NF-κB and AP-1 activation results in an augmenting activation of the iEκ. LMP1 promotes the interactions of heterodimeric NF-κB (p52/p65) and heterodimeric AP-1 (c-Jun/c-Fos) transcription factors with the human iEκ enhancer region are important for the upregulation of kappa light chain in LMP1-positive nasopharyngeal carcinoma cells.

EBV-encoded LMP1 regulates Op18/stathmin signaling pathway by cdc2 mediation in nasopharyngeal carcinoma cells.

Oncoprotein 18/stathmin (Op18/stathmin) plays a crucial role in maintaining cell biological characteristics by regulating microtubule dynamics, especially entry into mitosis; phosphorylated Op18/stathmin promotes microtubule polymerization to form the mitotic spindle, which is essential for chromosome segregation and cell division. Cdc2 is a critical kinase in starting M phase events in cell‐cycle progression and is a positive regulator of the cell cycle. Latent membrane protein 1 (LMP1) is an Epstein‐Barr virus (EBV)‐encoded oncogenic protein that is able to induce carcinogenesis via various signaling pathways. This study focused on regulation by LMP1 of Op18/stathmin signaling in nasopharyngeal carcinoma (NPC) cells and showed that LMP1 regulates Op18/stathmin signaling through cdc2 mediation, LMP1 upregulates cdc2 kinase activity, and Op18/stathmin phosphorylation promotes the interaction of cdc2 with Op18/stathmin and microtubule polymerization during mitosis, and inhibition of LMP1 expression attenuates the interaction of cdc2 and Op18/stathmin and promotes microtubule depolymerization. These results reveal a new pathway via which LMP1 regulates Op18/stathmin signaling by cdc2 mediation; this new signaling pathway not only perfects the LMP1 regulation network but also elucidates the molecular mechanism of LMP1 that leads to carcinogenesis.

EBV encoded miR-BHRF1-1 potentiates viral lytic replication by downregulating host p53 in nasopharyngeal carcinoma

miRNAs (microRNAs) are a class of non-coding small RNAs. The Epstein-Barr-virus (EBV) encoded miR-BHRF1-1 is barely expressed in most nasopharyngeal carcinoma (NPC) cells with EBV latent infection. Here, we used a strategy of overexpression and inhibition of miR-BHRF1-1 and showed that miR-BHRF1-1 is involved in TPA-induced accumulation of EBV lytic proteins and viral copies in late lytic cycle. The data further suggested that the miR-BHRF1-1-potentiated induction of EBV lytic replication was accompanied by inhibiting p53 expression. Our results demonstrated that the EBV original pathogen miR-BHRF1-1 is involved in the control of EBV late lytic replication by directly targeting the host p53 gene.

Epstein–Barr virus‐encoded LMP1 triggers regulation of the ERK‐mediated Op18/stathmin signaling pathway in association with cell cycle

The MAPKs are activated by a variety of cellular stimuli to participate in a series of signaling cascades and mediate diverse intracellular responses. One potential target of the MAPKs is Op18/stathmin, a molecule that acts as an integrator of diverse cell signaling pathways and regulates the dynamics of microtubules, which are involved in modulating a variety of cellular processes, including cell cycle progression and cell growth. Our study focused on the regulation of the MAPK‐mediated Op18/stathmin signaling pathway, which is triggered by the Epstein–Barr virus‐encoded latent membrane protein 1 ( LMP1 ) oncogene in nasopharyngeal carcinoma cells. The results showed that the activity of MAPK, which was induced by LMP1, varied with cell cycle progression; LMP1 upregulated phosphorylation of ERK during the G1/S phase, but negatively regulated phosphorylation of ERK during the G2/M phase. We found that the regulation of Op18/stathmin signaling by LMP1 was mainly mediated through ERK. The inhibition of LMP1 expression attenuated the interaction of ERK with Op18/stathmin and promoted microtubule depolymerization. These findings indicate the existence of a new cell cycle‐associated signaling pathway in which LMP1 regulates ERK‐mediated Op18/stathmin signaling. (Cancer Sci 2012; 103: 993–999)

Aspirin induces lytic cytotoxicity in Epstein–Barr virus-positive cells

Epstein-Barr virus (EBV) infection in tumor cells is generally restricted to the latent forms of viral infection. Switching the latent form of viral infection into the lytic form may induce tumor cell death. High levels of nuclear factor (NF)-kappaB can inhibit EBV lytic replication, and aspirin has the ability to inhibit NF-kappaB activity. The aims of the current study were to determine the effects of aspirin on inducing EBV lytic infection, and thus to reveal the possibility of targeting EBV-positive cancer cells by aspirin. Our results showed that aspirin depleted NF-kappaB (p65) in the nucleus and reactivated EBV into lytic replication. Cells exhibited decreased viability in a dose- and time-dependent manner when incubated with aspirin. When ganciclovir was used in combination with aspirin to treat EBV-positive B95.8 cells and Raji cells, the cytotoxic effect of aspirin was amplified. We demonstrated that aspirin reduced the viability of EBV-positive B lymphocytes due to its ability to induce EBV lytic replication.

The activation of p53 mediated by Epstein-Barr virus latent membrane protein 1 in SV40 large T-antigen transformed cells

The Epstein‐Barr virus (EBV) encoded latent membrane protein 1 (LMP1) plays an important role in the carcinogenesis of nasopharyngeal carcinoma (NPC). The tumor suppressor p53 is an important transcription factor. The mutation of the p53 gene is the frequent alteration in most of tumors, but nearly 100% wild‐type p53 gene is found in NPC biopsy. Here, our study testified that SV40 T‐antigen transformed nasopharyngeal epithelial cells contained free, wild‐type p53. Moreover, LMP1 regulated p53 both at transcriptional and translational level. Furthermore, the mechanism of p53 accumulation mediated by LMP1 from post‐translational level‐phosphorylation and ubiquitination were determined. Therefore, the effects of EBV LMP1 on p53 may potentially contribute to EBV‐associated pathogenesis.