Caffeic acid phenethyl ester inhibits nitric oxide synthase gene expression and enzyme activity

doi:10.1016/S0304-3835(01)00787-X

Cancer Letters

Volume 175, Issue 1, 10 January 2002, Pages 53-61

https://doi.org/10.1016/S0304-3835(01)00787-X Get rights and content

Abstract

Since nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been known to be involved in inflammatory and autoimmune-mediated tissue destruction, modulation of NO synthesis or action represents a new approach to the treatment of inflammatory and autoimmune diseases. Caffeic acid phenethyl ester (CAPE), an active component of honeybee propolis, has been identified to show anti-inflammatory, anti-viral and anti-cancer activities. The present study, therefore, examined effects of CAPE on iNOS expression and activity of iNOS enzyme itself. Treatment of RAW 264.7 cells with CAPE significantly inhibited NO production and iNOS protein expression induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-γ). CAPE also inhibited iNOS mRNA expression and nuclear factor-kappa B (NF-κB) binding activity in a concentration-dependent manner. Furthermore, transfection of RAW 264.7 cells with iNOS promoter linked to a chloramphenicol acetyltransferase reporter gene, revealed that CAPE inhibited the iNOS promoter activity induced by LPS plus IFN-γ through the NF-κB sites of the iNOS promoter. In addition, CAPE directly interfered with the catalytic activity of murine recombinant iNOS enzyme. These results suggest that CAPE may exert its anti-inflammatory effect by inhibiting the iNOS gene expression at the transcriptional level through the suppression of NF-κB activation, and by directly inhibiting the catalytic activity of iNOS.

Introduction

Nitric oxide (NO) is produced from l-arginine by nitric oxide synthase (NOS), a family of ubiquitous enzymes. Inducible NOS (iNOS) is expressed only after exposure to specific stimulants such as cytokines, bacterial lipopolysaccharide (LPS), and calcium ionophore in some cells. iNOS is not detectable in healthy tissues, but is expressed after immunological challenge or injury. iNOS, under pathologic conditions, can be expressed in most tissues including neurons, astrocytes, and endothelial cells [1]. NO formation is increased during autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis), and several classic inflammatory symptoms (erythema and vascular leakiness) are reversed by NOS inhibitors [1], [2]. Because cells cannot sequester and regulate the local concentration of NO, regulation of NO synthesis is the key to eliciting its biological activity.

The murine iNOS promoter contains various transcription-factor binding sites, including nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) sites involved in induction of other genes by cytokines or LPS [3]. Analysis of the transcriptional activity of iNOS promoter using deletional mutants revealed the essential role of two NF-κB binding motifs in the control of iNOS expression [3], [4]. Bacterial LPS and cytokines are key mediators in the inflammatory response and have been shown to activate NF-κB that is critical for the inducible expression of multiple genes involved in inflammation [5], [6]. NF-κB is, therefore, an obvious target for new types of anti-inflammatory treatment [7].

Caffeic acid phenethyl ester (CAPE) is an active component of propolis obtained from honeybee hives. It has anti-mitogenic, anti-carcinogenic, anti-inflammatory and immunomodulatory activities in diverse systems [8], [9], [10]. It has been reported that CAPE displays antioxidant activity and inhibits lipoxygenase activities, protein tyrosine kinase, and NF-κB activation [11], [12], [13], [14], [15]. The possibility that CAPE exhibits biological effects by inhibiting the release of arachidonic acid from cell membranes and the enzyme activities of cyclooxygenase (COX)-1 and COX-2 was also suggested [16], [17]. However, there has been no report systematically investigating effects of CAPE on the NO-producing system in RAW 264.7 cells stimulated by LPS plus interferon-gamma (IFN-γ). The present study, therefore, examined effects of CAPE on iNOS expression and activity of the iNOS enzyme itself to further elucidate the chemopreventive and anti-inflammatory mechanisms of CAPE.

Section snippets

Chemicals

Murine IFN-γ and rabbit anti-murine iNOS polyclonal antibody were purchased from Chemicon (Temecula, CA). Poly(dI-dC) poly(dI-dC) and dNTP were purchased from Pharmacia LKB Biotechnology (Piscataway, NJ). Quantities of 1-deoxydichloroacetyl-1-[¹⁴C]chloramphenicol and [α-³²P]dCTP were purchased from Dupont New England Nuclear Corp. (Boston, MA). Peroxidase-labeled anti-rabbit IgG and Supersignal^® were purchased from Pierce (Rockford, IL). Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine

Effect of CAPE on the LPS plus IFN-γ-induced iNOS expression

The effect of CAPE on the LPS plus IFN-γ-induced NO production was first examined in RAW 264.7 macrophages (Fig. 1). The accumulated nitrite, estimated by the Griess method, in the culture medium was used as an index for NO synthesis from these cells. After treatment with LPS plus IFN-γ for 24 h, nitrite concentration markedly increased about 11-fold (∽20 μM). When cells were treated with various concentrations of CAPE, nitrite production induced by LPS plus IFN-γ was significantly inhibited at

Discussion

Caffeic acid phenethyl ester (CAPE), an active component of propolis, has been shown to possess anti-inflammatory, immunomodulatory, anti-carcinogenic, and antioxidant properties [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [24], [25]. These diverse effects of CAPE might be attributed to its ability to pass through the cell membranes more easily than caffeic acid [25]. The precise mechanisms by which CAPE exerts anti-inflammatory effects on target cells are still largely unknown.

Acknowledgements

This research was supported by a grant (PF002103-01) from Plant Diversity Research Center of 21^st Century Frontier Research Program funded by Ministry of Science and Technology of Korean Government.

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Caffeic acid phenethyl ester inhibits nitric oxide synthase gene expression and enzyme activity

Abstract

Introduction

Section snippets

Chemicals

Effect of CAPE on the LPS plus IFN-γ-induced iNOS expression

Discussion

Acknowledgements

Cell

Cell

Cell

Adv. Immunol.

J. Ethnopharmacol.

FEBS Lett.

Prostaglandins Leukot. Essent. Fatty Acids

Biochem. Biophys. Res. Commun.

Anal. Biochem.

J. Biol. Chem.

Cell

Cell

Cell

Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation

Proc. Natl. Acad. Sci. U.S.A.

Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide

Proc. Natl. Acad. Sci. U.S.A

Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide

J. Exp. Med.

Hydroxylated aromatic inhibitors of HIV-1 integrase

J. Med. Chem.

Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis

Experientia

Lipophilic derivatives of caffeic acid as lipoxygenase inhibitors with antioxidant properties

Bioorg. Khim.