Many antioxidants, including cysteine, metal chelators, dithiocarbamates, quinone derivatives, vitamin E, vitamin C and
α-lipoic acid, suppress activation of NF-
κB in response to diverse stimuli (
Meyer et al., 1993;
Bowie & O'Neill, 2000a,
2000b).
In this study, we have demonstrated that CAPE is significantly more effective than the antioxidants vitamin C and NAC in inhibiting NF-κB DNA-binding activity in response to H. pylori. Inhibition of NF-κB activation by CAPE required concentrations in the micromolar range, whereas inhibition by vitamin C and NAC required relatively high concentrations in the millimolar range, 10 and 20 mm, respectively. CAPE (10 μg ml−1; ∼88 μm) is 227 times more potent as an inhibitor of NF-κB compared to vitamin C or NAC. We have also shown that, although vitamin C and NAC inhibit NF-
κB, neither were effective inhibitors of AP-1 DNA binding. This observation is particularly interesting in that CAPE also inhibited AP-1 activation. Therefore, it is unlikely that the potent inhibitory effect of CAPE on NF-
κB and AP-1 DNA-binding activity is solely due to its antioxidant properties.
The exact mechanism whereby CAPE inhibits NF-
κB activation is not known. In this study, we found that pretreatment of gastric epithelial cells with CAPE upregulated I
κB-
α levels and prevented nuclear translocation of NF-
κB/p65 in
H. pylori-treated AGS cells. NF-
κB presents in an inactive state in the cytosol bound to the inhibitory I
κB protein.
H. pylori infection of gastric epithelial cells results in phosphorylation and degradation of the I
κB, thus allowing nuclear translocation of NF-
κB. Our data demonstrated that control gastric cells (AGS) had high levels of I
κB-
α and no active p65. When AGS cells pretreated with CAPE, there was upregulation of I
κB-
α levels, which subsequently prevented p65 from translocation to the nucleus. Treatment of AGS cells with
H. pylorior cytokines was associated with lower levels of I
κB-
α in the cytosol, as shown by immunoblotting, and increased levels of p65 in the nucleus, as shown by immunofluorescence. NF-
κB activation involves phosphorylation and degradation of I
κB, and protein kinase C has been shown to be involved in NF-
κB activation by a variety of agents (
Domínguez et al., 1993;
Kopp & Ghosh, 1995;
Barens & Karin, 1997). The inhibition of I
κB-
αdegradation appears to be the initial step in NF-
κB activation. Consistent with this,
Bowie & O'Neill (2000b) have demonstrated that treatment of the endothelial cells ECV304 with vitamin C blocked IL-1- and TNF-mediated degradation and phosphorylation of I
κB-
α, due to inhibition of IKKinase (IKK) activation. The inhibition of TNF-induced IKK activation was mediated by p38 MAPK, as treatment of cells with vitamin C led to a rapid and sustained activation of p38 MAPK.