Enterococcus faecalis co-cultured with oral cancer cells exhibits higher virulence and promotes cancer cell survival, proliferation, and migration: an in vitro study

<b>Introduction.</b> <i>Enterococcus faecalis</i> is a common pathogen associated with many oral diseases and is often isolated from oral cancer patients. However, limited information is available on its key virulence gene expression in oral cancer cell microenvironment and cancer cell behaviour in co-culture studies.<b>Hypothesis.</b> <i>E. faecalis</i> overexpresses virulence genes when co-cultured with oral cancer cells and possibly alters the tumour microenvironment, promoting oral cancer proliferation and survival.<b>Aim.</b> To investigate altered virulence gene expression in <i>E. faecalis</i> and oral cancer cell behaviour using <i>in vitro</i> co-culture experiments.<b>Methodology.</b> Cal27 cells were co-cultured with <i>E. faecalis</i> and assessed for their cell proliferation, apoptosis, migration and clonogenicity using standard cell culture assays. The levels of reactive oxygen species (ROS) and inflammatory cytokines, along with proliferative, angiogenic and apoptotic biomarker expressions, were also assessed. <i>E. faecalis</i> adherence to cancer cells was demonstrated by the gentamicin protection assay. Real time-PCR was used to analyse the expression of virulence genes.<b>Results.</b> Co-culture of Cal27 cells with <i>E. faecalis</i> showed significantly higher cell proliferation, migration and clonogenicity compared to the control (<i>P</i><0.01). A significant increase in the levels of ROS and inflammatory cytokines and overexpression of Ki67, vascular endothelial growth factor, extracellular signal-regulated kinase 1/2, phosphoinositide 3 kinase and Akt was observed in the co-culture group. <i>E. faecalis</i> also downregulated <i>p53</i> and <i>Bax</i> genes while upregulated <i>Bcl-2</i>. The virulence genes <i>GelE</i>, <i>Asa</i> and <i>Ace</i> were overexpressed in <i>E. faecalis</i> co-cultured with Cal27 cells.<b>Conclusion.</b> The results from this study indicate the possible risks of <i>E. faecalis</i> infection in oral cancer. An effective antibiotic strategy against <i>E. faecalis</i> to prevent complications associated with oral diseases, including cancer, is needed.