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

Introduction. Enterococcus faecalis 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. Hypothesis. E. faecalis overexpresses virulence genes when co-cultured with oral cancer cells and possibly alters the tumour microenvironment, promoting oral cancer proliferation and survival. Aim. To investigate altered virulence gene expression in E. faecalis and oral cancer cell behaviour using in vitro co-culture experiments. Methodology. Cal27 cells were co-cultured with E. faecalis 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. E. faecalis adherence to cancer cells was demonstrated by the gentamicin protection assay. Real time-PCR was used to analyse the expression of virulence genes. Results. Co-culture of Cal27 cells with E. faecalis showed significantly higher cell proliferation, migration and clonogenicity compared to the control ( P <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. E. faecalis also downregulated p53 and Bax genes while upregulated Bcl-2 . The virulence genes GelE , Asa and Ace were overexpressed in E. faecalis co-cultured with Cal27 cells. Conclusion. The results from this study indicate the possible risks of E. faecalis infection in oral cancer. An effective antibiotic strategy against E. faecalis to prevent complications associated with oral diseases, including cancer, is needed.