Comparative evaluation of pro-oncogenic cytokines in current and past areca nut users

Objectives: Areca nut (AN) chewing is strongly associated with oral submucous fibrosis (OSMF) and oral cancer, which carry a high malignant transformation rate of up to 30% in Indian populations. The alkaloid arecoline in AN is known to induce pro-oncogenic cytokines that promote chronic inflammation, fibrosis, and carcinogenesis. This study aimed to determine and compare the oral secretory concentrations of HIF-1α, IL-6, TNF-α, and TGF-β among current areca nut users (CAN), past users (PAN), and controls (C). Methods: In this prospective, cross-sectional observational study, 80 CAN, 80 PAN (defined as abstinent for >1 month), and 21 age- and sex-matched controls (never users) were recruited after obtaining ethical clearance and informed consent (CTRI/2021/01/030290). Oral secretions were collected using sterile ophthalmic sponges pre-moistened with saline, placed on the buccal mucosa or lesion sites for 1 minute without friction. Samples were stored at −80 °C and analyzed using enzyme-linked immunosorbent assay (ELISA) for HIF-1α, IL-6, TNF-α, and TGF-β. Statistical comparisons were made using appropriate parametric and non-parametric tests, with significance set at p < 0.05. Results: HIF-1α levels were significantly higher in PAN compared to both CAN and controls (p < 0.001) and in CAN compared to controls (p < 0.01). TNF-α concentrations were also significantly elevated in both CAN and PAN relative to controls (p < 0.001). IL-6 and TGF-β levels did not show significant intergroup differences. Among subjects with OSMF, both HIF-1α and TNF-α were markedly elevated in CAN and PAN groups, suggesting persistent inflammation even after cessation of AN use. Conclusions: Pro-oncogenic cytokines HIF-1α and TNF-α remain significantly elevated in both current and past areca nut users, as well as in individuals with OSMF, indicating sustained inflammatory and carcinogenic potential even after habit cessation. These findings highlight the need for ongoing surveillance of AN users and suggest potential therapeutic avenues targeting HIF-1α and TNF-α pathways to prevent oral carcinogenesis.