We sought to determine if alcohol also stimulates mRNA expression of Snail. We first assessed the ability of alcohol to stimulate mRNA expression from a Snail reporter plasmid. Caco-2 cells were transfected with a PGL3 luciferase reporter containing the Snail promoter (gift of A.G. Herreros) (Barbera et al., 2004). As seen in Figure 4A, after 8h alcohol stimulated a 2-fold increase in Snail promoter activity compared to control (no alcohol) cells (p ≤ .05, t-test). Because Snail is known to repress E-cadherin mRNA expression we also determined the effect of alcohol on an E-cadherin reporter (gift of L. Larue)(Julien et al., 2007). Recent studies show E-cadherin mRNA expression is suppressed at 24–48h during EMT (Billottet et al., 2008), so we assessed the effects of alcohol on E-cadherin reporter activity at 24h and 48h after alcohol stimulation.. Figure 4A shows that alcohol stimulation resulted in significant inhibition of E-cadherin reporter activity at 24h/48h (50%/48%; p ≤ .05, t-test) consistent with activation of Snail nuclear activity by alcohol.
