The Protective Effect of Antioxidants in Areca Nut Extract-Induced Oral Carcinogenesis

Oral submucous fibrosis (OSF) is the premalignant disorder associated with fibrosis and epithelial atrophy. Areca Nut (AN) is the most significant risk factors for OSF. However, the molecular mechanism behind AN induced OSF remains unclear, and there exists no effective treatment for the malignant disorder. We aimed to investigate whether AN-extract causes epithelial-mesenchymal transition (EMT) in oral keratinocytes, and evaluated the therapeutic potential of antioxidants. The HPV16 E6/E7-transfected immortalized human oral keratinocytes (IHOK) were employed in the present study. For the preparation of AN-extract, dried AN was dissolved in distilled water overnight. The solution was centrifuged and the supernatant was collected for further use. For the determination of change in cytokine levels, ELISA was performed. To investigate EMT-related protein expression and phenotype, immunoblot and immunofluorescence were performed. Among tumor-promoting cytokines (Gro-α, IL-6 and IL-8), IL-6 was remarkably increased by AN in IHOK. AN-extract induced EMT phenotypes, such as cell elongation, up-regulation of vimentin and snail. After treatment with neutralizing antibody of IL-6, AN-induced snail expression was reduced remarkably. Collectively, AN-extract induced IL-6 expression and mediated EMT. The use of antioxidants (EGCG, glutathione and NAC) significantly reduced IL-6 expression in AN-treated IHOK. Also, AN-decreased E-cadherin and increased vimentin were reversed by antioxidants, indicating that the effectiveness of antioxidants in inhibiting IL-6-induced EMT by AN. AN promotes EMT and antioxidants interrupt AN-induced-EMT in oral keratinocytes. Consequently, it is proposed that antioxidants could prevent AN-induced carcinogenesis and function as a prototype for developing therapeutic interventions of OSF. .