Periodontal pathogens promote epithelial-mesenchymal transition in oral squamous carcinoma cells in vitro

Abstract

Epithelial-mesenchymal transition (EMT) is potentially involved in increasing metastasis of oral squamous cell carcinoma (OSCC). Periodontal pathogens are well-known for their ability to induce intense immune responses and here we investigated whether they are involved in inducing EMT. Cultures of OSCC cell line (H400) were treated separately with heat-killed periodontal pathogens F. nucleatum, or P. gingivalis or E. coli LPS for 8 d. EMT-associated features were assayed using sq-PCR and PCR-arrays, for EMT-related markers, and ELISAs for TGF-β1, TNF-α, and EGF. The migratory ability of cells was investigated using scratch and transwell migration assays. E-cadherin and vimentin expression was assessed using immunofluorescence while Snail activation was detected with immunocytochemistry. In addition, the integrity of the cultured epithelial layer was investigated using transepithelial electrical resistance (TEER). PCR data showed significant upregulation after 1, 5, and 8 d in transcription of mesenchymal markers and downregulation of epithelial ones compared with unstimulated controls, which were confirmed by immunofluorescence. Periodontal pathogens also caused a significant increase in level of all cytokines investigated which could be involved in EMT-induction and Snail activation. Exposure of cells to the bacteria increased migration and the rate of wound closure. Downregulation of epithelial markers also resulted in a significant decrease in impedance resistance of cell monolayers to passage of electrical current. These results suggested that EMT was likely induced in OSCC cells in response to stimulation by periodontal pathogens.

Role of Porphyromonas gingivalis in oral squamous cell carcinoma development: A systematic review

“…Abdulkareem et al also observed an increase of MMP2, 3 and 9, vimentin, and transcription factors, Twist and Snail, in combination with a downregulation of E‐cadherin in OSCC cells (H400 line) infected with P gingivalis . Moreover, transcriptional levels of EGF and TNF‐α were also high.…”

“…Studies were classified into three main groups according to oral specimen origins. In the first one, three studies (18.75%) used epithelial cells from human samples (Table ); in the second, three studies (18.75%) evaluated murine models (Table ); and in the last one, 11 studies (62.5%) were developed in pre‐established cell lines (Table ).…”

“…Alterations of these cell pathways reduce epithelial expression of cancerous cells. These changes appear in healthy gingival epithelial cells, both from human biopsies and from pre‐established cell lines, as well as in OSCC cells infected with P gingivalis . Sztukowska et al were the only authors to obtain these results in an in vivo OSCC and healthy in vitro.…”

“…In a second one (promotion), tissue growth and cancer could develop throughout overexpression of receptors and growth factors, cytokines, extracellular matrix destruction, angiogenesis, etc In the last one (progression), malignant cells invade local or distant tissues, while molecular alterations continue to carry on. Steps of oral carcinogenesis in which P gingivalis play an important role (Figure ) are epithelial‐mesenchymal transition of oral epithelial cells, neoplastic cell proliferation, and tumor cell invasiveness …”

“…Epithelial‐mesenchymal transformation (EMT) determines a change of oral epithelial cells into a mesenchymal phenotype, and a loss of their adhesion properties . In this sense, P gingivalis may become an activation factor for ZEB1, or Snail/ Slug/ Twist transcription factors . Alterations of these cell pathways reduce epithelial expression of cancerous cells.…”