Caffeic acid phenethyl ester modulates H pylori-induced nuclear factor-kappa B and activator protein-1 expression in gastric epithelial cells

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Abstract​

1. Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives (honeybee resin), has anti-inflammatory, anti-carcinogenic and anti-bacterial properties. This study was designed to investigate the anti-inflammatory effects of CAPE on Helicobacter pylori-induced NF-κB and AP-1 in the gastric epithelial cell line AGS.
2. Electrophoretic mobility shift assay was used to measure NF-κB- and AP-1-DNA binding activity. Western blotting was used to detect IκB-α and COX-2 expression in AGS cells cocultured with H. pylori. The antiproliferative effect of CAPE was measured by MTT assay.
3. Our results showed that caffeic phenethyl ester inhibits H. pylori-induced NF-κB and AP-1 DNA-binding activity in a dose (0.1–25 μg ml−1∼0.35–88 μm) and time- (15–240 min) dependent manner in AGS cells. Maximum inhibition by CAPE was observed at concentrations of 25 μg ml−1 (∼88 μm) CAPE prevented H. pylori- and cytokine-induced degradation of IκB-α protein.
4. Pretreatment of AGS cells with CAPE also blocked cytokine- and mitogen-induced NF-κB and AP-1 expression. Furthermore, CAPE suppressed H. pylori-induced cell proliferation and production of the cytokines TNF-α and IL-8. In addition, CAPE blocked H. pylori-induced COX-2 expression.
5. The inhibition of such transcription by CAPE could result in suppression of many genes during H. pylori-induced inflammation, and also provide new insights into the anti-cancer and anti-inflammatory properties of CAPE.

 

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Introduction​

Honey is a traditional remedy for dyspepsia and there have been a number of reports suggesting that honey can inhibit the growth of Helicobacter pylori (Ali et al., 1991; Osato et al., 1999), the causative agent of gastric ulcer and cancer (Graham, 1989; Nomura et al., 1991), and of other pathogenic organisms. However, the molecular mechanisms for its antibacterial effects are unclear. Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives (honeybee resin), has been reported to have anti-inflammatory, anti-carcinogenic and immunomodulatory properties (Grunberger et al., 1988).

CAPE has many biological and pharmacological effects, including antioxidant properties and tumour cell cytotoxicity. Various investigators have demonstrated an anti-inflammatory action for CAPE both in vitro and in vivo (Michaulart et al., 1999; Orban et al., 2000). CAPE has been reported to be a specific inhibitor of nuclear factor-kappa B (NF-κB), which may account for some of its anti-inflammatory properties (Natarajan et al., 1996; Fitzpatrick et al., 2001). NF-κB resides in the cytoplasm in an inactive form as a heterodimer consisting of p50 and p65 (RelA) subunits complexed to the inhibitory molecule IκB, which prevents the migration of the heterodimer to the nucleus. Following a range of stimuli in many cell types, NF-κB translocates to the nucleus and binds to its specific DNA site and subsequently upregulates gene expression (Kopp & Ghosh, 1995; Barens & Karin, 1997). CAPE also alters the redox state, induces apoptosis, suppresses lipid peroxidation and displays antioxidant activity (Kimura et al., 1985; Chiao et al., 1995; Laranjinha et al., 1995). CAPE has also been shown to inhibit the growth of different types of transformed cells (Guarini et al., 1992; Su et al., 1994; Burke et al., 1995).

 

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The transcription factor NF-κB plays a central role in regulating various host responses during the inflammatory process. Other transcription factors such as activator protein-1 (AP-1) also play an important role in the regulation of cellular functions, including proliferation and apoptosis, and may work in concert with NF-κB to elicit the inflammatory response during the infection. Direct contact between H. pylori and gastric epithelial cells induces NF-κB (Keates et al., 1997; Maeda et al., 2000), AP-1 (Meyer-ter-Vehn et al., 2000) and COX-2 expression (Kim et al., 2001) in gastric epithelial cells. The activation of such transcription factors by microbial pathogens including H. pylori determines the outcome of the cellular innate immune defence. Therefore, exploitation of the mechanisms causing activation represents an important field of potential therapeutic intervention that may be relevant to several inflammatory disease states.

The aim of our study was to examine the effect of CAPE on the transcription factors NF-κB and AP-1 activities during H. pylori infection of gastric epithelial cells. CAPE inhibited H. pylori-induced NF-κB, AP-1 and COX-2 expression in AGS cells. Furthermore, cytokine levels of tumour necrosis factor-α(TNF-α) and IL-8 were significantly reduced in CAPE-treated cells. Our findings demonstrate that CAPE modulates H. pylori-induced NF-κB and AP-1 activities in gastric epithelial cells and also downregulates the production of the cytokines.

 

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Discussion​

The pharmacologically active molecules in the propolis are flavanoids and phenolic acid and their esters. These compounds, including CAPE, have multiple effects on bacteria, viruses and fungi. There have been a number of reports suggesting that honey has an inhibitory effect on H. pylori in vitro (Ali et al., 1991; al Somal et al., 1994). CAPE is one of the major components of propolis and CAPE has a strong antioxidant activity higher than that for galangin (Russo et al., 2002). The precise mechanisms of physiological and pharmacological properties responsible for the anti-inflammatory effect of honey and its product CAPE are yet unclear. In this study, we have demonstrated for the first time that CAPE, a major component of honey, modulates H. pylori-induced NF-κB, AP-1 DNA-binding activity and COX-2 expression in gastric epithelial cells. In addition, CAPE reduced TNF-α and IL-8 levels and suppressed the proliferative response of AGS cells to H. pylori.

The regulation of gene expression by transcription factors such as NF-κB and AP-1 is fundamental to the phenotype of all cells engaged in inflammatory processes. NF-κB and AP-1 are responsible for the expression of wide range of cytokines, enzymes and cell adhesion molecules, and therefore play an important role in the pathogenesis of several diseases including H. pylori infection. The redox regulation of NF-κB and related proteins has received increased attention because this protein controls the inducible expression of a wide range of genes involved in inflammatory and immune responses (Kopp & Ghosh, 1995; Barens & Karin, 1997). Treatment of gastric epithelial cells with CAPE inhibited NF-κB, AP-1 and COX-2 activation by H. pylori. Consistent with this observation, Natarajan et al. (1996) have demonstrated that CAPE inhibits the activation of NF-κB induced by a wide variety of agents. CAPE was also shown to be a potent inhibitor of cytokine- and mitogen-induced NF-κB and AP-1 in AGS cells.

 

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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 IκB-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 IκB protein. H. pylori infection of gastric epithelial cells results in phosphorylation and degradation of the IκB, thus allowing nuclear translocation of NF-κB. Our data demonstrated that control gastric cells (AGS) had high levels of IκB-α and no active p65. When AGS cells pretreated with CAPE, there was upregulation of IκB-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 IκB-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 IκB, 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 IκB-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 IκB-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.

 

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In addition to many biological and pharmacological activities of CAPE in blocking NF-κB and AP-1 activation and cytokine production, we found that CAPE treatment (25 μg ml−1) also resulted in a decrease in cell number of AGS cells. In rat macrophage and colonic epithelial cell lines, it has been shown that CAPE is an effective inducer of apoptosis in macrophages, as opposed to colonic epithelial cells (Fitzpatrick et al., 2001). In this regard, other investigators have also reported that CAPE can preferentially induce apoptosis, depending on the cell type treated with this compound (Chiao et al., 1995). The reasons for a selective effect on apoptosis are not fully understood, but may be related to the inherent redox state of a particular cell type (Chiao et al., 1995; Orban et al., 2000). In one study, in HL-60 cells, CAPE rapidly entered cells and induced DNA fragmentation and morphological changes typical of apoptosis. Moreover, treatment with CAPE caused rapid activation of caspase-3, downregulation of Bcl-2 expression, and upregulation of Bax expression (Chen et al., 2001).

The NF-κB pathway is a key mediator of genes involved in cellular proliferation, apoptosis and cytokine production. NF-κB activates the expression of various genes involved in the host immune and inflammatory response, including cytokine genes such as IL-1β, IL-2, IL-6, IL-8 and TNF-α, adhesion molecule genes and genes coding for acute-phase proteins (Barens & Karin, 1997; Yamamoto & Gaynor, 2001). Colonization of gastric epithelial cells with H. pylori induces NF-κB (Keates et al., 1997; Münzenmaier et al., 1997) and results in increased production of the proinflammatory cytokines TNF-α, IL-1, IL-6 and IL-8 (Aihara et al., 1997; Yamaoka et al., 1997), all of which are regulated by NF-κB. Significantly, CAPE inhibited H. pylori-induced IL-8 and TNF-α production by AGS cells in addition to H. pylori-induced AGS proliferation. Taken together, these observations indicate a potentially novel therapeutic use of CAPE for the treatment of the effects of H. pyloriinfection. This is supported by the finding that CAPE inhibited H. pylori-induced COX-2 expression in AGS cells. H. pylori infection is known to be associated with increased levels of COX-2 expression in gastric epithelial cells (Kim et al., 2001). The inhibition of COX-2 expression by CAPE is likely to contribute, in part, to both its anti-inflammatory and potentially chemopreventive activity.

 

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In summary, the results presented in this study demonstrated that CAPE inhibited H. pylori-induced NF-κB, AP-1 and COX-2 expression in gastric epithelial cells. CAPE also inhibited cytokine-induced NF-κB and AP-1 expression. Our findings provide an insight into the molecular mechanisms for the anti-inflammatory and immunomodulatory activities of CAPE in relationship to H. pyloriinfection and other inflammatory disease states. Future studies are needed to clarify the molecular mechanisms by which CAPE inhibits H. pylori-mediated activation of NF-κB, AP-1 and COX-2 expression in gastric epithelial cells and to identify additional targets in gene regulation.