The Epstein–Barr virus (EBV) is a human herpesvirus associated with various cancers. The number of reports that describe infection of EBV in oral squamous carcinoma cells is increasing. However, there is no available in vitro model to study the possible role of EBV in the development of oral squamous cell carcinoma. Herein, we report establishment of a latent EBV infection of well-differentiated HSC1 cells and poorly differentiated SCC25 cells. Viral copy numbers per cell in EBV-infected HSC1 and SCC25 cells are 2 and 5, respectively. Although the EBV copy number was small, spontaneous viral replication was observed in EBV-infected HSC1 cells. Contrarily, infectious viral production was not observed in EBV-infected SCC25 cells, despite containing larger number of EBV genomes. Chemical activation of cells induced expression of viral lytic BZLF1 gene in EBV-infected HSC1 cells, but not in EBV-infected SCC25 cells. EBV infection activated proliferation and migration of HSC1 cells. However, EBV-infection activated migration but not proliferation in SCC25 cells. In conclusion, EBV can infect squamous cells and establish latent infection, but promotion of cell proliferation and of lytic EBV replication may vary depending on stages of cell differentiation. Our model can be used to study the role of EBV in the development of EBV-associated oral squamous cell carcinoma.
Peroxiredoxin 2 (PRDX2) is upregulated in various cancers including oral squamous cell carcinoma (OSCC). It is a known tumor promoter in some cancers, but its role in OSCC is unclear. This study aimed to investigate the effect of arecoline, an alkaloid of the betel nut, and human papillomavirus type 16 (HPV16) E6/E7 oncoproteins on induction of PRDX2 expression, and also the effects of PRDX2 overexpression in oral cell lines. Levels of PRDX2 protein were determined using western blot analysis of samples of exfoliated normal oral cells (n = 75) and oral lesion cells from OSCC cases (n = 75). Some OSCC cases were positive for HPV infection and some patients had a history of betel quid chewing. To explore the level of PRDX2 by western blot, the proteins were extracted from oral cell lines that were treated with arecoline or retroviruses containing HPV16 E6 gene and HPV16 E6/E7 expressing vector. For analysis of PRDX2 functions, cell proliferation, cell-cycle progression, apoptosis and migration was compared between oral cells overexpressing PRDX2 and cells with PRDX2-knockdown. PRDX2 expression levels tended to be higher in OSCC samples that were positive for HPV infection and had history of betel quid chewing. Arecoline treatment in vitro at low concentrations and overexpression of HPV16 E6 or E6/E7 in oral cells induced PRDX2 overexpression. Interestingly, in oral cells, PRDX2 promoted cell proliferation, cell-cycle progression (G2/M phase), cell migration and inhibited apoptosis. Upregulation of PRDX2 in oral cells was induced by arecoline and HPV16 oncoproteins and promoted growth of OSCC cells.
Andrographolide is the principal bioactive chemical constituent of Andrographis paniculata and exhibits activity against several viruses, including Epstein–Barr virus (EBV). However, the particular mechanism by which andrographolide exerts an anti-EBV effect in EBV-associated gastric cancer (EBVaGC) cells remains unclear. We investigated the molecular mechanism by which andrographolide inhibits lytic reactivation of EBV in EBVaGC cells (AGS-EBV cell line) using proteomics and bioinformatics approaches. An andrographolide treatment altered EBV protein-expression patterns in AGS-EBV cells by suppressing the expression of EBV lytic protein. Interestingly cellular transcription factors (TFs), activators for EBV lytic reactivation, such as MEF2D and SP1, were significantly abolished in AGS-EBV cells treated with andrographolide and sodium butyrate (NaB) compared with NaB-treated cells. In contrast, the suppressors of EBV lytic reactivation, such as EZH2 and HDAC6, were significantly up-regulated in cells treated with both andrographolide and NaB compared with NaB treatment alone. In addition, bioinformatics predicted that HDAC6 could interact directly with MEF2D and SP1. Furthermore, andrographolide significantly induced cell cytotoxicity and apoptosis of AGS-EBV cells by induction of apoptosis-related protein expression. Our results suggest that andrographolide inhibits EBV lytic reactivation by inhibition of host TFs, partially through the interaction of HDAC6 with TFs, and induces apoptosis of EBVaGC cells.
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