EGCG Inhibits TNFα-Induced NFκB Activation in Nicotine Treated Epithelium

Objectives: To determine if EGCG inhibits TNFα-induced phosphorylation and activation of NFκB in nicotine-treated cultured human oral epithelial cells. Methods: Commercially available human oral epithelial cells (ScienCell, Carlsbad, CA) were treated for 24 hours with 0.1 mM nicotine, then pre-treated or not with 10 μg/ml EGCG. Cells were additionally treated with 10 μg/ml TNFα for 10, 20, or 30 minutes. The cells were then processed in situ to determine levels of NFκB serine468 and serine536 phosphorylation as well as total levels of NFκB (FACE NFkB p65 profiler in-cell phosphorylation ELISA, Active Motif, Carlsbad, CA). Data were calculated as relative percentages of total cellular NFκB phosphorylation. Whole-cell extracts of similarly treated cells subsequently were assayed for NFκB subunit activation (TransAM NFkB Family ELISA). Statistical analysis was completed using ANOVA and Tukey post-test with probability set at p < 0.05. Results: TNFα-induced stimulation of NFκB phosphorylation and activation was similar in nicotine- and non-nicotine-treated cells. Serine468 levels were increased in cells treated with nicotine, EGCG and TNFα compared to cells treated with nicotine and TNFα. In contrast, at 30 minutes of TNFα stimulation, serine536 phosphorylation was reduced in cells treated with EGCG. Of the five NFκB family members analyzed (RelA/p65, p50, c-Rel, p52, and RelB), only p65 and p50 showed effects of EGCG treatment. EGCG significantly inhibited the activation of p65 and p50 in nicotine- and TNFα-treated oral epithelial cells. Conclusions: EGCG appears to suppress the activation of the p65/p50 heterodimer of NFκB in nicotine and TNFα-stimulated cells. Suppression of NFκB may explain, at least in part, why EGCG can inhibit the secretion of pro-inflammatory interleukins such as IL-8. Continued examination of the cellular function of EGCG may lead to its use as a natural inhibitor of oral inflammation in patients who use nicotine.