Epithelial Expression of Claudin-1 and Vimentin by Areca Immunomodulatorty Effects

Objectives: Areca nut chewing is a major cause of oral squamous cell carcinoma (OSCC) in Taiwan. Epithelial-mesenchymal transition (EMT) is achieved by down-regulation of epithelial proteins like E-cadherin and claudin-1 and increased expression of mesenchymal proteins such as vimentin and α-smooth muscle actin (α-SMA). The study aims to investigate the immunomodulatory effects of ANE with/without other oral risk factors like nicotine (NT) and lipopolysaccharides (LPS) on the EMT of epithelial cells.
Methods: The study was approved by the Institutional Review Board of Taipei Veterans General Hospital. The oral tissue samples were retrieved from patients undergoing routine oral surgery with informed consent. The expression of EMT markers in tissues was examined using immunohistochemistry. Purified PBMC obtained from healthy volunteers were treated with 40 μg/ml ANE, 1μg/ml of LPS and/or 100 μg/ml of NT for 20 hours. The supernatants of PBMC were collected as conditioned media (CM). Oral epithelial cells were treated with CM or oral risk factors for 24 hours, the expression of vimentin and claudin-1 was investigated using western blot analysis or immunofluorescence staining. Cytokine production in supernatants of PBMC was detected using enzyme-linked immunosorbent assay.
Results: Histopathologic sections revealed that the expression of claudin-1, vimentin and α-SMA, but not E-cadherin, in oral cancer tissues was higher than in adjacent normal tissues. In epithelial experiments, ANE, LPS and/or NT increased the production of vimentin, but showed no significant effects on claudin-1. However, nearly all CM of risk factors-treated PBMC increased the expression of claudin-1 and vimentin, especially apparent in the ANE-containing groups. Moreover, the secretion of TNF-α and IFN-γ by PBMC was increased by ANE, but not LPS/NT.
Conclusions: Besides the direct effects, the altered immune cell function by risk factors, especially areca nut, might play a role in the EMT and collective invasion during areca-related oral carcinogenesis.