Abstract
BackgroundEpstein-Barr Virus (EBV) is associated with hematopoietic malignancies, such as Burkitt’s lymphoma, post-transplantation lymphoproliferative disorder, and diffuse large B-cell lymphoma. The current approach for EBV-associated lymphoma involves chemotherapy to eradicate cancer cells, however, normal cells may be injured and organ dysfunction may occur with currently employed regimens. This research is focused on employing arsenic trioxide (ATO) as EBV-specific cancer therapy takes advantage of the fact the EBV resides within the malignant cells.Methods and resultsOur research reveals that low ATO inhibits EBV gene expression and genome replication. EBV spontaneous reactivation starts as early as 6 h after re-suspending EBV-positive Mutu cells in RPMI media in the absence of ATO, however this does not occur in Mutu cells cultured with ATO. ATO’s inhibition of EBV spontaneous reactivation is dose dependent. The expression of the EBV immediate early gene Zta and early gene BMRF1 is blocked with low concentrations of ATO (0.5 nM – 2 nM) in EBV latency type I cells and EBV-infected PBMC cells. The combination of ATO and ganciclovir further diminishes EBV gene expression. ATO-mediated reduction of EBV gene expression can be rescued by co-treatment with the proteasome inhibitor MG132, indicating that ATO promotes ubiquitin conjugation and proteasomal degradation of EBV genes. Co-immunoprecipitation assays with antibodies against Zta pulls down more ubiquitin in ATO treated cell lysates. Furthermore, MG132 reverses the inhibitory effect of ATO on anti-IgM-, PMA- and TGF-β-mediated EBV reactivation. Thus, mechanistically ATO’s inhibition of EBV gene expression occurs via the ubiquitin pathway. Moreover, ATO treatment results in increased cell death in EBV-positive cells compared to EBV-negative cells, as demonstrated by both MTT and trypan blue assays. ATO-induced cell death in EBV-positive cells is dose dependent. ATO and ganciclovir in combination further enhances cell death specifically in EBV-positive cells.ConclusionATO-mediated inhibition of EBV lytic gene expression results in cell death selectively in EBV-positive lymphocytes, suggesting that ATO may potentially serve as a drug to treat EBV-related lymphomas in the clinical setting.
Highlights
Epstein-Barr Virus (EBV) is associated with hematopoietic malignancies, such as Burkitt’s lymphoma, post-transplantation lymphoproliferative disorder, and diffuse large B-cell lymphoma
In the work presented here we show that arsenic inhibits the expression of EBV lytic genes Zta, Rta and BMRF1, and promotes cell death in EBV-positive lymphoma cells
BMRF1 was not induced when the media contained arsenic trioxide (ATO), and the expression of Zta and Rta were lower compared with no treatment (NT) at day 1, 2 and 3 (Fig. 1a right)
Summary
Epstein-Barr Virus (EBV) is associated with hematopoietic malignancies, such as Burkitt’s lymphoma, post-transplantation lymphoproliferative disorder, and diffuse large B-cell lymphoma. Zta degrades the tumor suppressor p53 and inhibits its transcriptional function [22,23,24,25,26]; EBV lytic genes inhibit antiviral cytokines such as TNF-alpha, and stimulate synthesis of cellular cytokines, such as interleukin–10, −8, and −13, which serve as growth factors to promote cell cycling and thereby tumor cell proliferation [27,28,29]. Studies using EBV-positive lymphoma cells have demonstrated that loss of the EBV genome in Akata cells results in cell death [35,36,37]. These manuscripts imply that inhibition of EBV lytic reactivation may reduce the occurrence of cancer and suggest that antiviral therapy may be useful for treating EBV-related malignancies [38]
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