Elsevier

European Journal of Pharmacology

Volume 810, 5 September 2017, Pages 149-155
European Journal of Pharmacology

Molecular and cellular pharmacology
Methyl (E)-(3-(3,4-dihydroxyphenyl)acryloyl)tryptophanate can suppress MCP-1 expression by inhibiting p38 MAP kinase and NF-κB in LPS-stimulated differentiated THP-1 cells

https://doi.org/10.1016/j.ejphar.2017.07.006Get rights and content

Abstract

Methyl (E)-(3-(3,4-dihydroxyphenyl)acryloyl)tryptophanate (MHAT) is an O-methyl ester of javamide-II showing strong anti-inflammatory activity. Therefore, in this study, MHAT was chemically synthesized, and its effects on p38 MAP kinase, NF-κB, and monocyte chemotactic factor-1 (MCP-1) expression were investigated in LPS-stimulated differentiated THP-1 cells. MHAT inhibited p38 MAP kinase with an IC50 of 12 μM, and the inhibition was supported by an in silico model showing that its binding to p38 MAP kinase was stronger than that of SB203580. At the concentration of 20 μM, the p38 inhibition reduced ATF-2 phosphorylation by 55% (P < 0.05). Additionally, MHAT inhibited NF-κB (p65) phosphorylation by 30% (P < 0.05) at the same concentration, suggesting that MHAT was able to reduce NF-κB transcriptional activity. This supposition was confirmed by the NF-κB reporter assay, demonstrating that MHAT (20 μM) could suppress NF-κB transcriptional activity by 29% (P < 0.05) in the NF-κB reporter (Luc)-HEK293 cell line. As expected, the treatment with MHAT (5–40 μM) significantly inhibited MCP-1 mRNA expression by 9–73% (P < 0.05) and the production of MCP-1 protein by 10–70% (P < 0.05) in the THP-1 cells. Furthermore, MHAT was found to inhibit RANTES expression as well in the same THP-1 cells, supporting its purported inhibition of p38 MAP kinase and NF-κB. All these data suggest that MHAT is a potent compound that can inhibit MCP-1 production by suppressing p38 kinase/ATF-2 phosphorylation and NF-κB in the differentiated THP-1 cells.

Introduction

Monocyte chemotactic factor-1 (MCP-1) is a small but potent chemotactic factor belonging to the subfamily of CC chemokines (Deshmane et al., 2009, Leonard and Yoshimura, 1990). Its expression is reported to be ubiquitous in many different cell types and is often up-regulated by various stimuli including lipopolysaccharide (LPS), IL-1, TNF-alpha, and 2-O-tetradecanoylphorbol 13-acetate (TPA) (Kunkel et al., 1991, Martin et al., 1997). Because MCP-1 has a strong chemotactic activity on inflammatory cells, it can recruit blood monocytes into the vessel wall, thereby leading to systemic vascular inflammation commonly observed in cardiovascular disease (CVD) and other diseases (Fox and Kahn, 2005, Christodoulidis et al., 2014, Lin et al., 2014, Ruster and Wolf, 2008). Although the regulation of MCP-1 expression is considered complex and cell-specific, p38 MAP kinase and NF-κB are considered major regulatory molecules in the production of MCP-1 in cells (Sutcliffe et al., 2009, Yang et al., 2014, Saklatvala, 2004, Lee et al., 1999). Therefore, the inhibition of these molecules has been suggested as an effective means for suppressing MCP-1 production (Sheryanna et al., 2007, Wong et al., 2005).

Methyl (E)-(3-(3,4-dihydroxyphenyl)acryloyl)tryptophanate (MHAT) is an O-methyl ester of javamide-II which is a phenolic amide found in coffee (Park, 2016). During the course of our study, MHAT was found to have stronger anti-inflammatory activity than the parent chemical javamide-II, speculating its potential to inhibit the expression of inflammation-related molecules including MCP-1 in cells. However, there is currently little information about the capability of MHAT to inhibit p38 MAP kinase, NF-κB and MCP-1 expression in monocytic/macrophage-like cells. Therefore, in this study, MHAT was synthesized, and its potential effect on the expression of MCP-1 was investigated in LPS-stimulated differentiated THP-1 cells by examining its effects on p38 MAP kinase/ATF-2 phosphorylation and NF-κB phosphorylation/transcriptional activity. Additionally, the potential effect of MHAT on RANTES was investigated in the same cells because p38 MAP kinase and NF-κB are also significantly involved in the production of RANTES in the cells (Wong et al., 2005).

Section snippets

Materials

Tryptophan, cinnamic acid, dichloromethane, N,N-dimethylformamide, SB203580, Phorbol 12-myristate 13-acetate (PMA) and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). THP-1 cells were purchased from ATCC (Manassas, VA). Alpha-tubulin (Catalog no. 2144), ATF-2 (Catalog no. 9226) and phospho-ATF-2 (Catalog no. 5112) antibodies were purchased from Cell Signaling (Danvers, MA, USA)

Chemical synthesis

The synthesis of MHAT was performed using a method described previously (Park, 2012). Briefly,

Results

MHAT (methyl (E)-(3-(3,4-dihydroxyphenyl)acryloyl)tryptophanate) (Fig. 1) was synthesized using the method described in Section 2,and the product was purified by HPLC (Waters, Milford, MA) as described previously (Park, 2012). To confirm the identities of the synthesized products, the sample was analyzed using NMR spectroscopic methods as described in Section 2, (see Supplementary data for NMR data). Based on the NMR data, the structure of the product was determined as that of methyl

Discussion

In this study, MHAT was found to inhibit MCP-1 production in LPS-stimulated differentiated THP cells by inhibiting p38 kinase/ATF-2 phosphorylation as well as NF-κB. MCP-1 is a significant inflammatory chemokine involved in progressive chronic vascular/interstitial inflammation in CVD, chronic kidney disease and other diseases (Christodoulidis et al., 2014, Lin et al., 2014). There are several isoforms (p38α, β, γ and δ) of p38 MAP kinase that are activated by a variety of cellular stresses

Conflict of interest statement

There are no disclosures to make about financial, consulting, and personal relationships linked to this paper.

Acknowledgements

This study was funded by the USDA (Project no. 8040-51000-057-00D).

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