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

Article abstract of DOI:10.1016/j.ejphar.2017.07.006

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 monocy

Full text of DOI:10.1016/j.ejphar.2017.07.006

European Journal of Pharmacology 810 (2017) 149–155
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
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
Jae B. Park⁎, Thomas T.Y. Wang
Diet, Genomics, and Immunology Laboratory, BHNRC, ARS, USDA, Bldg. 307C, Rm. 131, Beltsville, MD 20705, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
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
Methyl (E)-(3-(3,4-dihydroxyphenyl)acryloyl)
tryptophanate (MHAT)
Monocyte chemotactic protein-1 (MCP-1)
p38
ATF-2
NF-κB
THP-1
<
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 transcrip-
tional 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.
1. Introduction
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
Monocyte chemotactic factor-1 (MCP-1) is a small but potent chemo-
tactic 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
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
⁎
Corresponding author.
E-mail address: jae.park@ars.usda.gov (J.B. Park).
http://dx.doi.org/10.1016/j.ejphar.2017.07.006
Received 20 April 2017; Received in revised form 23 June 2017; Accepted 3 July 2017
Available online 06 July 2017
0014-2999/ © 2017 Published by Elsevier B.V.

J.B. Park, T.T.Y. Wang
European Journal of Pharmacology 810 (2017) 149–155
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).
followed by LPS treatment (10 ng/ml) for 4 h. Cell growth was
analyzed using the sulforhodamine B assay as described previously
(Vichai et al., 2006).
2.7. Western blot of phospho-ATF-2
2. Materials and methods
For the Western blot of phospho-ATF-2, the samples were prepared
using the PMA-differentiated THP-1 cells treated with MHAT (0, 20,
40 µM) followed by the treatment with LPS (10 ng/ml) for 0.5 h. The
Western blots were generated using Novex 4–12% Tris-Glycine Mini
Gels and a XCell II™ Blot Module kit (Life Technologies, Cambridge,
MA, USA) with ATF-2 (Catalog no. 9226), phospho-ATF-2 (Catalog no.
5112) and alpha-tubulin (Catalog no. 2144) antibodies (Cell Signaling,
Danvers, MA, USA). LI-COR IRDye 800CW anti-rabbit IgG (Catalog
no. 926-32213) (Lincoln, NE, USA) was used as a second antibody. For
the blot, the amounts of protein in the samples were determined using
the Bio-Rad protein assay kit (Hercules, CA, USA), and the protein
bands in the Western blot were quantified using an Odyssey CLx
imaging system (Lincoln, NE, USA).
2.1. Materials
Tryptophan, cinnamic acid, dichloromethane, N,N-dimethylforma-
mide, 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)
2.2. Chemical synthesis
The synthesis of MHAT was performed using a method described
previously (Park, 2012). Briefly, caffeic acid was dissolved in dimethyl
sulfoxide (DMSO) and converted to the symmetrical anhydride with
1,3-diisopropylcarbodiimide (DIC). Next, tryptophan-O-methyl was
synthesized using tryptophan and methanol and added to the reaction
mixture. The reaction mixture was incubated at room temperature with
a gentle stirring for 12 h. The synthesized products were recovered and
purified by HPLC (Waters, Milford, MA) as described previously (Park,
2012). MHAT was prepared in ethanol for use in experiments.
2.8. Binding assay of NF-κB (p65)
The binding of NF-ĸB (p65) to its cognate element site was
measured using the NF-ĸB (p65) Transcription Factor Assay kit
according to the manufacturer's protocol (Cayman Chemical, Ann
Arbor, Michigan). The NF-ĸB (human p65) transcription factor assay
is a convenient and sensitive method for detecting specific transcrip-
tion factor DNA binding activity in nuclear extracts. A specific double
stranded DNA (dsDNA) sequence with the NF-κB response element is
coated onto the wells of a 96-well plate. The nuclear extracts with NF-
κB (p65) were prepared using differentiated THP-1 cells treated with
MHAT (0, 1, 10, 20 µM) followed by LPS treatment (10 ng/ml) for
10 h. The samples were incubated overnight at 4 °C in the wells,
allowing the NF-κB (p65) in the extracts to bind to its cognate site. The
bound NF-κB (p65) transcriptional factors were detected using a
specific primary antibody against NF-κB (p65) followed by a secondary
antibody conjugated to horseradish peroxidase (HRP), which provides
a sensitive colorimetric readout at 450 nm.
2.3. p38 MAP kinase assay
Measurement of p38 MAP kinase activity was carried out by using
CycLex p38 assay/inhibitor screening kit-1 (MBL International,
Woburn, MA), which uses anti-phospho-ATF2 Thr71 polyclonal anti-
body (PPT-08) and peroxidase coupled anti-rabbit IgG antibody as the
reporter molecule in a 96-well ELISA format. The assay method
involves the incubation of the p38 sample with the substrate and the
tested compound in the presence of Mn2+ and ATP, and the assay was
carried out according to the manufacturer's protocol.
2.9. NF-κB reporter assay
2.4. Molecular docking
The NF-κB signal transduction pathway was monitored using the
NF-κB reporter (Luc)-HEK293 cell line (Signosis, Inc., Santa Clara,
CA), which contains a firefly luciferase gene driven by four copies of the
NF-κB response element located upstream of the minimal TATA
promoter. For the study, the HEK293 cells were pre-incubated with
MHAT (0, 10, 20, 40 µM) for 1 h followed by treatment with TNF-
alpha (10 ng/ml). The treatment led to the binding of endogenous NF-
kB transcription factors to the DNA response elements and induced
transcription of the luciferase reporter gene, which monitors NF-κB
signal transduction. Luciferase activity was measured using a micro-
plate luminometer (Berthold Technologies, Bad Wildbad, Germany).
Molecular docking studies were performed using the algorithm-
based docking program ICM-pro (MolSoft, San Diego, CA). The
proposed binding to the active site pocket of p38 MAP kinase was
determined by the best ranked scoring function of ICM-pro, which also
represents the conformational structures with the most favorable free
binding energy (ΔGbinding).
2.5. Cell culture
THP-1 cells were purchased from the ATCC (Manassas, VA). Cells
were grown in RPMI 1640 medium with L-glutamine containing 10%
FBS, 100 units/ml penicillin, and 100 units/ml streptomycin. THP-1
cells were maintained at 37 °C in a humidified atmosphere of 5% CO2.
THP-1 cells were induced to differentiate into macrophages by
incubation with PMA (25 ng/ml) for 48 h. The differentiated cells (2
× 106 cells/well) were treated with several concentrations of MHAT (0–
40 µM) followed by the treatment with LPS (10 ng/ml) (Huang et al.,
2012) and incubated for specified times at each experiment. Also, the
media were saved for quantifying MCP-1 and RANTES proteins.
2.10. RNA isolation and real time RT-PCR quantitation of COX-2 and
MCP-1 mRNA
Total RNA was isolated from the differentiated THP-1 cells treated
with various concentrations of MHAT (0, 5, 20 and 40 µM) followed by
LPS treatment (10 ng/ml) for 4 h using TRIzol™ reagent (Invitrogen,
Carlsbad, CA). Complementary DNA (cDNA) was synthesized from
1 µg of the total RNA using the AffinityScript Multiple Temperature
cDNA Synthesis Kit (Agilent Technologies, Inc., Santa Clara, CA)
according to the manufacturer's protocol. Real time semi-quantitative
PCR was carried out using the TaqMan® Fast Universal PCR Master
Mix (2X) (Applied Biosystems, Foster City, CA) and a ViiA7 Real-Time
PCR Detection System (Applied Biosystems, Foster City, CA) following
2.6. Cell growth assay
The differentiated THP-1 cells (5 × 105 cells/well) plated in 6-well
plates were treated with 0, 5, 20, and 40 µM of MHAT for 10 min
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J.B. Park, T.T.Y. Wang
European Journal of Pharmacology 810 (2017) 149–155
the manufacturer's protocol. Human TaqMan probes and primers were
purchased from Applied Biosystems (Foster City, CA) using inventoried
TaqMan gene expression assays: TATA box binding protein (assay ID:
Hs00427620_m1), MCP-1/CCL2 (assay ID: Hs00234140_m1)
(Takahashi et al., 2007) and COX-2/PTGS2 (assay ID:
Hs00153133_m1) (For more information; http://www.thermofisher.
com).
2.11. Determination of MCP-1 and RANTES
MCP-1 and RANTES levels in the media of the differentiated THP-1
cells treated with various concentrations of MHAT (0–40 µM) followed
by LPS treatment (10 ng/ml) for 9 h were determined using the
Human Quantikine MCP-1 Elisa kit and the RANTES Elisa kit from
R & D systems (Minneapolis, MN) according to the manufacturer's
protocol.
Fig. 2. The inhibition of p38 MAP kinase and determination of IC50. The inhibition of
p38 by MHAT was determined at the concentrations between 0 and 50µM. Data points
are shown as the means
S.D. (n = 4). The P value was calculated using one-way
2.12. Statistical analysis
ANOVA with the Holm-Sidak method, and the asterisks (*) denote significant differences
(P < 0.05) from the vehicle control.
All statistical analyses were performed with SigmaPlot 11.0
(Chicago, IL). The P value was calculated using one-way ANOVA with
the Holm-Sidak method, and P < 0.05 was considered as statistically
significant. Data points in all figures were represented as the means
S.D.
based docking program ICM-pro as described in Section 2. The docking
experiments were performed with MHAT and SB203580 (a known p38
MAP kinase inhibitor) using the experimentally determined crystal
structures of p38 MAP kinase complexes (14 complexes, Table 1).
Briefly, the p38 MAP kinase complex was divided into ligand and protein,
and the ligands (MHAT and SB203580) were docked into the protein
complexes. The docked complexes with the lowest energy and outcome
scoring values were compared (Table 1). In Fig. 3, the lowest energy
position of MHAT docked into the p38 MAP kinase protein (PDB_ID;
3HV5) was presented as a putative binding mode. Overall, MHAT
exhibited higher scores than those of SB203580, suggesting that MHAT
may inhibit p38 MAP kinase.
Often, cell death induces massive gene deregulation, which could
complicate efforts to delineate specific gene regulation after treatment
with inhibitors. Therefore, we performed a cell growth assay to ensure
that MHAT is not cytotoxic to the differentiated THP-1 cells at the
concentrations used in this experiment (Fig. 4). At concentrations
between 0 and 40 μM, MHAT was found to inhibit p38 MAP kinase
greatly but was considered non-cytotoxic (Fig. 4), although a slight
decrease in cell attachment was found at 40 µM. In accordance with
this result, other cell death assays (e.g., MTT, LDH) also showed no
significant cell death (data not shown here). Next, the effect of MHAT
on the phosphorylation of ATF-2 was investigated because the activa-
tion of p38 MAP kinase played an important role in the expression of
MCP-1 (van Dam and Castellazzi, 2001) and because p38 MAP kinase
can modulate transcriptional up-regulation of MCP-1 by the phosphor-
ylation of several transcription factors such as activating transcription
factor-2 (ATF-2) (van Dam and Castellazzi, 2001). As shown in Fig. 5,
phosphorylated ATF-2 was significantly reduced in the differentiated
THP-1 cells by the treatment with MHAT (20 and 40 µM). These data
3. 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 (E)-(3-(3,4-dihydroxyphenyl)acryloyl)tryptophanate (Fig. 1). The
p38 MAP kinase pathway is critically involved in the production of several
inflammatory chemokines including MCP-1 (Waas et al., 2001; Liang
et al., 2015; Genovese et al., 2011; MacNee et al., 2013; Yang et al., 2014;
Lomas et al., 2012; Bison et al., 2011; Elkhawad et al., 2012). Also, several
studies suggested that the inhibition of p38 MAP kinase often led to the
suppression of MCP-1 expression in humans (Genovese et al., 2011;
MacNee et al., 2013; Lomas et al., 2012; Bison et al., 2011; Elkhawad
et al., 2012). Therefore, we investigated the potential effects of MHAT on
p38 MAP kinase activity to find a candidate compound for inhibiting
MCP-1 expression. As shown in Fig. 2, MHAT was able to inhibit p38
MAP kinase significantly, and the calculated IC50 was approximately
12 μM. Since MHAT showed relatively strong p38 MAP kinase inhibition,
a qualitative molecular docking was performed with MHAT against
available p38 MAP kinase complexes to confirm the observed p38 MAP
kinase inhibition. This docking study was performed using the algorithm-
Fig. 1. Chemical structure of MHAT. The chemical synthesis was performed as described in Section 2. The chemical structure was verified using NMR.
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European Journal of Pharmacology 810 (2017) 149–155
Table 1
p38 MAPK docking scores of MHAT and SB203580. Docking scores and energy values were obtained using a docking program ICM-pro as described in Section 2. Average data
represents the means
S.D. (n = 14).
MHAT
SB203580
p38 (PDB_ID)
Score
ΔE (Kcal/mol)
Score
ΔE (Kcal/mol)
3HV5
1W83
1W84
2PUU
3BV2
3GC9
3GCU
3D7Z
3DS6
3QUD
3L8S
− 39.2
− 31.7
− 28.4
− 25.9
− 27.8
− 32.6
− 36.7
− 29.7
− 31.2
− 18.4
− 27.2
− 26.1
− 27.9
− 28.7
− 29.3
− 47.1
− 39.4
− 37.5
− 35.4
− 38.3
− 39.1
− 46.6
− 38.8
− 44.2
− 29.4
− 39.7
− 38.2
− 37.4
− 36.7
− 34.4
− 24.7
− 22.3
− 21.5
− 5.9
− 24.1
− 24.3
− 16.0
− 21.8
− 16.0
− 19.3
− 17.0
− 16.0
− 25.3
− 20.6.1
− 42.3
− 37.1
− 32.1
− 29.5
− 12.2
− 36.3
− 35.5
− 17.0
− 32.9
− 27.6
− 30.2
− 30.5
− 25.1
− 34.5
4R3C
4KIP
4DLI
Average
4.9a
− 39.1
4.5b
6.5
− 30.2
7.9
a
P < 0.05 MHAT vs SB203580 Score (paired t-test; t = 6.482 df = 13).
P < 0.05 MHAT vs SB203580 ΔE (paired t-test; t = 4.552 df = 13).
b
suggest that MHAT treatment could suppress the phosphorylation of
ATF-2 possibly via inhibiting p38 MAP kinase.
The NF-κB/Rel family of transcription factors is comprised of several
structurally related proteins that form homodimers and heterodimers (e.g.,
p50/p105, p52/p100, and RelA/p65) (Tai et al., 2013; Perkins, 2007). Some
studies indicated that LPS treatment could lead to the binding of p65/p65, c-
Rel/p65 and p50/p65 to their cognate DNA sites, which eventually increased
MCP-1 mRNA in THP-1 cells (Tai et al., 2013). Therefore, we prepared the
nuclear extracts of the differentiated THP-1 cells treated with MHAT (0, 1,
20, 40 µM) as described in Section 2 and investigated whether the binding of
nuclear NF-κB p65 to the NF-κB response element could be affected by the
treatment with MHAT. As shown in Fig. 6, MHAT treatment blocked the
binding of NF-κB (p65) to the NF-κB response element when compared to
the control, suggesting that the treatment could inhibit NF-κB (p65)
phosphorylation. The reduction of NF-κB transcriptional activity by MHAT
was also investigated using the NF-κB reporter (Luc)-HEK293 cell line,
which is designed for monitoring nuclear factor Kappa B (NF-κB) signal
transduction pathways (Pessara and Koch, 1990). As shown in Fig. 7, MHAT
treatment (20 and 40 µM) lowered relative luminescent signals from the
reporter gene by 28–40% (P < 0.05). Furthermore, potential effect of
MHAT on COX-2 mRNA expression was investigated, because NF-κB is
critically involved in the expression of COX-2 mRNA (Surh et al., 2001;
Jobin et al., 1998). As expected, MHAT significantly inhibited COX-2 mRNA
Fig. 4. Effects of MHAT on the growth of differentiated THP-1 cells. Cells were treated
with several concentrations of MHAT (0, 5, 20, 40 µM), and cell growth was determined
at 4 h. Data are presented as the means
S.D. (n = 3). The P value was calculated using
one-way ANOVA with the Holm-Sidak method, and the asterisks (*) indicate statistically
significant differences (P < 0.05) compared to the control.
expression (Fig. 8), suggesting that MHAT could inhibit the transcriptional
activity of NF-κB stimulated by LPS.
Since MHAT was able to inhibit p38 MAP kinase, ATF-2 phosphor-
ylation and NF-κB transcriptional activity, the dose-dependent effects
of MHAT on the expression of MCP-1 mRNA were investigated in
differentiated THP-1 cells as described in Section 2. As shown in Fig. 9,
Fig. 3. In silico analysis of MHAT binding to p38. The MHAT in p38 complex (3HV5) is shown with two potential hydrogen bonds denoted by two green dots (E71) and (D168).
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European Journal of Pharmacology 810 (2017) 149–155
Fig. 7. Effects of MHAT on HEK293 cells containing the NF-κB reporter gene. The
samples were prepared by treating HEK293 cells containing the NF-κB reporter gene
with several concentrations of MHAT (0, 10, 20, 40 µM) followed by TNF-alpha (10 ng/
ml) for 9 h. Data points represent the means
S.D. (n = 4). The P value was calculated
using one-way ANOVA with the Holm-Sidak method, and the asterisks (*) indicate
significant differences (P < 0.05) compared to the control.
Fig. 5. Effects of MHAT on ATF-2 phosphorylation. The samples were prepared using
differentiated THP-1 cells treated with MHAT (0, 20, 40 µM) followed by LPS treatment
(0.01 µg/ml) for 0.5 h. For the phospho-ATF-2, ATF-2 control and alpha-tubulin blots,
the data are presented as values of the means
S.D. (n = 3) and each LPS data are
presented as 100% relative blot signal. The P value was calculated using one-way ANOVA
with the Holm-Sidak method, and the asterisks (*) indicate significant differences (P <
0.05) compared to the LPS control.
Fig. 8. Effects of MHAT on LPS-induction of COX-2 mRNA in differentiated THP-1
cells. The differentiated THP-1 cells were treated with 0, 5, 20 and 40 µM MHAT
followed by treatment with LPS (0.01 µg/ml). RNA was isolated at 4 h, and gene
expression of COX-2 was determined using real-time PCR. The percentage inhibition is
expressed as the means
S.D. (n = 3). Data were analyzed using one-way ANOVA with
the Holm-Sidak method as described in Section 2. The asterisks (*) denote significant
differences (P < 0.05) compared to the vehicle control.
Fig. 6. Effects of MHAT on NF-κB (p65) phosphorylation. The samples were prepared
using differentiated THP-1 cells treated with MHAT (0, 1, 20, 40 µM) followed by
treatment with LPS (0.01 µg/ml) for 10 h. Data points represent the means
S.D. (n =
4). The P value was calculated using one-way ANOVA with the Holm-Sidak method and
the asterisks (*) denote significant differences (P < 0.05) for all groups (except group 2
vs. group 3, group vs. group 4, and group 4 vs. group 5).
Fig. 9. Effects of MHAT on LPS-induction of MCP-1 mRNA in differentiated THP-1
cells. The differentiated THP-1 cells were treated with 0, 5, 20 and 40 µM MHAT
followed by treatment with LPS (0.01 µg/ml). RNA was isolated at 4 h, and gene
expression of MCP-1 was determined using real-time PCR. The percentage inhibition is
the treatment with MHAT decreased MCP-1 mRNA expression in a
dose-dependent manner. These data clearly suggest that MHAT can
suppress MCP-1 mRNA expression significantly at the concentrations
of 20 and 40 μM. Since MHAT could reduce the expression of MCP-1
mRNA, we investigated its effects on the expression of MCP-1 protein
in the same THP-1 cells. As in previous experiments, the differentiated
cells were treated with MHAT (0, 10, 20, 30, 40 μM) and followed by
LPS treatment as described in Section 2. As expected, the MHAT
treatment significantly inhibited the production of MCP-1 protein by
10–70% (P < 0.05) in the THP-1 cells (Fig. 10). Since SB203580 is a
expressed as the means
S.D. (n = 3)(S.D. was too small to mark). Data were analyzed
using one-way ANOVA with the Holm-Sidak method as described in Section 2. The
asterisks (*) denote significant differences (P < 0.05) compared to the vehicle control.
well-known p38 inhibitor that can suppress LPS-induced MCP-1
expression (Perkins, 2007), the inhibitory effects of MHAT and
SB203580 on the expression of MCP-1 protein were compared at 20
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European Journal of Pharmacology 810 (2017) 149–155
and 40 μM. As shown in Fig. 11, MHAT was found to be a marginally
better inhibitor of MCP-1 protein than SB203580. To further sub-
stantiate the purported inhibition of p38 kinase and NF-κB, we
investigated the effect of MHAT on another chemokine, RANTES,
which is also regulated significantly by both p38 kinase and NF-κB
(Hirano et al., 2003). As expected, MHAT treatment (10–40 μM)
inhibited RANTES expression by 15–65% (P < 0.05) in the differ-
entiated THP-1 cells (Fig. 12). These data suggest that MHAT can
suppress p38 MAP kinase, ATF-2 phosphorylation, and NF-κB, thereby
inhibiting MCP-1 and RANTES production in LPS-stimulated differ-
entiated THP cells.
4. Discussion
Fig. 10. Effects of MHAT on MCP-1 protein expression in differentiated THP-1 cells.
The samples were prepared using differentiated THP-1 cells treated with MHAT (0, 10,
20, 30, 40 µM) followed by LPS treatment (0.01 µg/ml) for 9 h as described in Section 2.
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 inflamma-
tory 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 including osmotic shock, inflammatory
cytokines, LPS, UV light and growth factors (Yang et al., 2014;
Saklatvala, 2004; Lee et al., 1999; Sheryanna et al., 2007).
Particularly, some p38 MAP kinase isoforms (α and β) play critical
roles in the production of chemokines and cytokines (Lin et al., 2014;
Ruster and Wolf, 2008; Yang et al., 2014; Takaishi et al., 2003).
Interestingly, several p38 MAPK inhibitors have been demonstrated to
inhibit the production of inflammatory cytokines/chemokines such as
interleukin-1, TNF-alpha and MCP-1. Although the observed inhibitory
effects are likely from the combined effects at the transcriptional and
translational levels rather than the p38 MAPK pathway alone, several
studies suggest that p38 MAPK inhibitors may be proficient in reducing
plasma MCP-1 expression in humans (Liang et al., 2015; Genovese
et al., 2011; MacNee et al., 2013), thereby providing protective effects
in inflammatory cardiovascular disease (Lomas et al., 2012; Bison
et al., 2011). Therefore, in this study, the potential effects of MHAT on
MCP-1 expression were investigated in differentiated THP-1 cells in
order to explore a candidate for MCP-1 inhibitor. As shown in Table 1,
the docking data showed that the sum of ΔE value of MHAT binding
(ΔE = − 39.1) was lower than that of the p38 inhibitor SB203580 (ΔE =
− 31.7), suggesting that MHAT can bind to the putative binding site
better than SB203580. Also, as shown in Fig. 3, the contours of the
putative binding site were more accessible to MHAT in the tested p38
enzyme complex (3HV5) than SB203580 because MHAT has a less
bulky structure than SB203580. As a consequence, the proposed two
hydrogen bonds ((E71) and (D168) in Fig. 3) is very unlikely to be
formed with SB203508 due to its structural conformation. However, in
vitro p38 assay data showed that the IC50 of p38 MAP kinase inhibition
by MHAT was approximately 12 μM, which showed that it was less
effective than SB203580 (Fehr et al., 2015) and not in line with the
docking data. Therefore, we reasonably thought that SB203580 may
inhibit MCP-1 production stronger than MHAT in the cells. However,
in differentiated THP-1 cells, the inhibition of MCP-1 protein produc-
tion by MHAT was marginally better than that of SB203580, suggesting
that MHAT may inhibit more than p38 MAP kinase with regard to
MCP-1 inhibition. This inference was in fact supported by the finding
that MHAT could inhibit NF-κB transcriptional activity. Like p38 MAP
kinase, NF-κB is also significantly involved in the synthesis of MCP-1 in
cells. The NF-κB transcription factors are composed of several proteins,
which are present as homodimers and heterodimers (e.g., p50/p105,
p52/p100, and RelA/p65) (Tai et al., 2013; Perkins, 2007). After the
phosphorylation of IKB by IKK, these transcription factors move into
the nucleus and bind to their cognate binding elements on DNA,
regulating the expression of target genes (Perkins, 2007). Some studies
showed that LPS treatment could lead to the binding of p65/p65, c-
Data points represent the means
S.D. (n = 5). The P value was calculated using one-
way ANOVA with the Holm-Sidak method, and the asterisks (*) indicate significant
differences (P < 0.05; F (5, 24) = 259.5) for all groups (except group 2 vs. group 3 and
group 5 vs. group 6).
Fig. 11. Comparison of the effects of MHAT and SB203580 on MCP-1 protein
expression in differentiated THP-1 cells. The samples were prepared using differentiated
THP-1 cells treated with two concentrations of MHAT and SB203580 (20 and 40 µM)
followed by treatment with LPS (0.01 µg/ml) for 9 h as described in Section 2. Data
points represent the means
S.D. (n = 4). The P value was calculated using one-way
ANOVA with the Holm-Sidak method, and the asterisks (*) denote significant differences
(P < 0.05; F (3, 16) = 198.7) for all groups.
Fig. 12. Effects of MHAT on RANTES expression in differentiated THP-1 cells. The
samples were prepared using differentiated THP-1 cells treated with MHAT (0, 10, 20,
30, 40 µM) followed by treatment with LPS (0.01 µg/ml) for 9 h as described in Section
2. Data points represent the means
S.D. (n = 5). The P value was calculated using one-
way ANOVA with the Holm-Sidak method, and the asterisks (*) denote significant
differences (P < 0.05; F (5, 24) = 188.3) for all groups (except group 4 vs. group 5 and
group 5 vs. group 6).
154

J.B. Park, T.T.Y. Wang
European Journal of Pharmacology 810 (2017) 149–155
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Conflict of interest statement
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There are no disclosures to make about financial, consulting, and
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Appendix A. Supporting information
Supplementary data associated with this article can be found in the
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