Accepted Manuscript Title: In vitro effect of caffeic acid phenethyl ester on matrix metalloproteinases (MMP-1 and MMP-9) and their inhibitor (TIMP-1) in lipopolysaccharide-activated human monocytes Author: Polyana das Grac¸as Figueiredo Vilela Jonatas Rafael de Oliveira Patr´ıcia Pimentel de Barros Mariella Vieira Pereira Le˜ao Luciane Dias de Oliveira Antonio Olavo Cardoso Jorge PII: DOI: Reference:
S0003-9969(15)00098-9 http://dx.doi.org/doi:10.1016/j.archoralbio.2015.04.009 AOB 3381
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
3-7-2014 24-3-2015 16-4-2015
Please cite this article as: Vilela PGF, Oliveira JR, Barros PP, Le˜ao MVP, Oliveira LD, Jorge AOC, In vitro effect of caffeic acid phenethyl ester on matrix metalloproteinases (MMP-1 and MMP-9) and their inhibitor (TIMP-1) in lipopolysaccharide-activated human monocytes, Archives of Oral Biology (2015), http://dx.doi.org/10.1016/j.archoralbio.2015.04.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
ip t
CAPE inhibited the expression, production and activity of MMP in human
cr
monocytes.
us
CAPE showed an important role in the balance between MMP and TIMP.
an
This study provides an additional evidence for a therapeutic potential of
Ac ce p
te
d
M
CAPE.
Page 1 of 30
Title In vitro effect of caffeic acid phenethyl ester on matrix metalloproteinases (MMP-1 and MMP-9) and their inhibitor (TIMP-1) in lipopolysaccharide-activated human
ip t
monocytes
Running Title
cr
The effect of CAPE in MMP expression
us
Authors
M
an
Polyana das Graças Figueiredo Vilela* Laboratory of Microbiology and Immunology. Department of Biosciences and Oral Diagnosis. Institute of Science and Technology - Univ. Estadual Paulista- UNESP. Address: Engenheiro Francisco José Longo, 777, Jardim São Dimas, CEP 12245-000, São José dos Campos, SP, Brazil. *Corresponding author. +1 201 9138230 [email protected]
te
d
Jonatas Rafael de Oliveira Laboratory of Microbiology and Immunology. Department of Biosciences and Oral Diagnosis. Institute of Science and Technology - Univ. Estadual Paulista- UNESP São José dos Campos, SP, Brazil. [email protected]
Ac ce p
Patrícia Pimentel de Barros Laboratory of Microbiology and Immunology. Department of Biosciences and Oral Diagnosis. Institute of Science and Technology - Univ. Estadual Paulista- UNESP. São José dos Campos, SP, Brazil. [email protected] Mariella Vieira Pereira Leão Bioscience Basic Institute, University of Taubaté. Taubaté, SP, Brazil. [email protected] Luciane Dias de Oliveira Laboratory of Biochemistry and Pharmacology. Department of Biosciences and Oral Diagnosis. Institute of Science and Technology - Univ. Estadual Paulista- UNESP São José dos Campos, SP, Brazil. [email protected] Antonio Olavo Cardoso Jorge Laboratory of Microbiology and Immunology. Department of Biosciences and Oral Diagnosis. Institute of Science and Technology - Univ. Estadual Paulista- UNESP. São José dos Campos, SP, Brazil. [email protected]
Page 2 of 30
Abstract
Objective: The role of matrix metalloproteinases (MMPs) in tissue degradation has
ip t
become evident in many diseases and great interest therefore exists in the pharmacological control of the activity of these enzymes. This study evaluated the
cr
effect of caffeic acid phenethyl ester (CAPE) on the production of MMPs and their inhibitor (TIMP) in monocytes activated by lipopolysaccharide (LPS). Design: The
us
human monocytic cell line (THP-1) was treated with non-cytotoxic concentrations of
an
CAPE (10 and 60 μM) combined with 1 μg/mL of LPS. The gene expression of MMP1, MMP-9 and TIMP-1 was evaluated by quantitative real-time polymerase chain
M
reaction. The protein secretion into the culture medium was assessed via enzymelinked immunosorbent assay and the gelatinolytic activity of MMP-9 by zymography.
d
Results: CAPE, especially at the highest concentration, down-regulated MMP-1 and
te
MMP-9 gene expression but up-regulated the gene expression of TIMP-1. Furthermore, CAPE reduced the secreted protein level of MMP-1 and MMP-9 as well
Ac ce p
as the gelatinolytic activity of MMP-9. Conclusion: CAPE was able to inhibit the gene expression, production and the activity of MMPs induced by LPS and also increased the gene expression of TIMP-1. The present observations suggest that CAPE exerted a positive effect on the regulatory mechanism between MMPs and TIMP, which is important for the control of different diseases.
Keywords: matrix metalloproteinases; lipopolysaccharide; monocytes; CAPE.
Page 3 of 30
Introduction
Studies have shown the cytotoxic effect and the activity of caffeic acid
its
ability
to
suppress
the
enzymatic
activity
or
expression
of
matrix
cr
metalloproteinases 9 (MMP-9).3,4
ip t
phenethyl ester (CAPE), an active component of propolis, against tumor cells,1,2 and
MMPs are involved in a variety of physiological processes associated with the
us
modulation and regulation of extracellular matrix (ECM) turnover.5-7 However, in
an
recent years it has become evident that these enzymes also play a role in tissue degradation in different diseases such as cancer and chronic inflammatory
M
processes.6-13 The MMPs have been also associated with periodontitis and apical periodontitis in which bacteria and their components induce an inflammatory response that leads to the excessive production and the activation of these enzymes
te
d
which, in turn, cause tissue destruction.13-16 In addition, it is also known that an imbalance between MMP and tissue
Ac ce p
inhibitors of metalloproteinase (TIMP) is responsible for tissue degradation.13 TIMP is the main endogenous inhibitors of MMP and comprise a group of four different proteins, TIMP-1, TIMP-2, TIMP-3 and TIMP-4.7 Knowledge of the factors that regulate the synthesis and secretion of MMPs
and TIMPs is important for the understanding of the pathogenesis of oral disease.13,17 Therefore, growing interest exists in the pharmacological control of the activity of these enzymes6 once the MMP inhibitors may be useful therapeutic agents for the prevention and treatment of different oral diseases.14 Since most of the studies have demonstrated the effects of CAPE in cancer cells and the main sources of MMPs in pathogenic processes are the cells of
Page 4 of 30
inflammatory line, the objective of the present study was to evaluate the effect of CAPE on the activity and production of MMPs and on the gene expression of MMP-1, MMP-9 and TIMP-1 in human monocytes (THP-1) activated by lipopolysaccharide
ip t
(LPS).
cr
Material and Methods
an
us
Cell culture
Cells of a human monocytic cell line (THP-1) were obtained from the cell bank
M
of the Paul Ehrlich Technical-Scientific Association (Associação Técnico Científica Paul Ehrlich - APABCAM, Rio de Janeiro, Brazil). The cells were cultured in RPMI-
d
1640 medium (Gibco, Life Technologies Corporation, Grand Island, NY, USA),
te
supplemented with 10% fetal bovine serum, 1% MEM vitamin solution, 1% sodium pyruvate, 1% MEM non-essential amino acid solution, 0.01% beta-mercaptoethanol,
Ac ce p
and 1% penicillin-streptomycin (all from Gibco, Life Technologies Corporation). The cells were maintained in an incubator at 37ºC with 5% CO2 and the culture medium was changed at 2-day intervals.
Determination of cell viability by the MTS assay
The cytotoxicity of CAPE (Sigma- Aldrich, St. Louis, MO, USA) was evaluated by the MTS assay. After exposure to different concentrations of CAPE (0, 10, 60, 100 and 200 μM), 5 x 104 cells were incubated for 2 h in CellTiter 96® AQueous solution (Promega, Madison, WI, USA). The absorbance was read in a microplate reader
Page 5 of 30
(Biotek®, Winooski, VT, USA) at 490 nm and converted into percentage of cell viability.
ip t
Experimental assays
cr
THP-1 cells (0.5 x 106 cells per well) were transferred to 6-well plates (TPP, Trasadingen, Switzerland) in RPMI medium not containing fetal bovine serum or
us
additives. The cells were treated with non-cytotoxic concentrations of CAPE
an
combined with 1 μg/mL of LPS (Escherichia coli 0055:B5; Sigma- Aldrich). The control group consisted of cells exposed only to 1 μg/mL of LPS (n=6 for each group,
M
2 independent experiments). The plates were maintained in an incubator at 37ºC with 5% CO2 for 24 h.
d
After incubation, the cell culture was collected and centrifuged (2400 x g for 5
te
min). The cell supernatant was collected and frozen at -20ºC for zymography and enzyme-linked immunosorbent assay (ELISA). The cell pellet was resuspended in
Ac ce p
Trizol® solution (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA), transferred to new tubes and frozen at -80ºC for subsequent RNA extraction and quantitative real-time polymerase chain reaction analysis (PCR).
RNA extraction, synthesis of complementary DNA (cDNA), and Quantitative Realtime PCR
The total RNA was extracted with Trizol® reagent according to manufacturer instructions. RNA (1 µg) was treated with DNAse (Deoxyribonuclease I kit, Amplification Grade; Invitrogen, Life Technologies Corporation) and then used for
Page 6 of 30
cDNA synthesis (SuperScript III, First-Strand Synthesis SuperMix, Invitrogen, Life Technologies Corporation). The resulting cDNA was amplified by real-time PCR (Platinum®SYBR Green, Invitrogen, Life Technologies Corporation) and quantified
ip t
using the Line-Gene K system (Bior Technology Hitech, Binjiang, Hangzhou, China). The sequences of the primers used were checked at Primer-Blast software from the
cr
National Center for Biotechnology Information nucleotide sequence database and is shown in Table 1.
us
The reactions were performed in triplicate and previously standardized based
an
on efficiency curves. Hypoxanthine phosphoribosyl transferase 1 (HPRT1) was used as housekeeping gene to normalize the PCR array data.
M
Relative RNA levels were presented as ratio relative to control cells after normalization for HPRT1 expression. The results were obtained as threshold cycle
d
(Ct) values. Delta delta Ct (∆∆Ct) method was performed to analyze RNA expression
te
levels by using the Line Gene K software.
Ac ce p
Enzyme-linked immunosorbent assay (ELISA)
The production of MMP-9 and MMP-1 was evaluated by ELISA according to
the recommendations of the manufacturers of the DuoSet® ELISA kits for human MMP-9 (R&D Systems, Minneapolis, MN, USA) and human MMP-1 (Sigma Chemical Co., St. Louis, MO, USA). Briefly, the plates were coated with anti-MMP capture antibody and kept overnight. The next day, the samples and standard curve were added and after incubation and the washed, the wells are incubated with biotinylated anti-MMP detection antibody. The reaction was developed with a chromogen solution. The absorbance was read in a microplate reader at 450 nm and the results
Page 7 of 30
were converted into concentrations (pg/mL) of MMP using the GraphPad Prism® 5 Program.
ip t
Zymography
cr
The gelatinolytic activity of MMP-9 was determined by zymography. First, total protein concentration in the cell supernatant was quantified by the method of Lowry
us
(Sigma Chemical Co.). Next, a known concentration of total protein in the samples
an
and of the MMP-9 standard (R&D Systems) was mixed with non-reducing sample buffer (62.5 mM Tris-HCl, 3% SDS, 10% glycerol, 0.25% bromophenol blue) and the
M
mixture was loaded onto 10% polyacrylamide gel containing 0.2% gelatin (Sigma Chemical Co.). After electrophoresis, the gel was incubated in a solution of 10 mM
d
Tris (pH 8.0) and 2.5% Triton X-100 (Sigma Chemical Co.) for 1 h at 37ºC. Next, the
te
gel was incubated with developing solution containing 50 mM Tris buffer (pH 8.8) and 5 mM CaCl2 for 20 h at 37ºC. The gel was stained with Coomassie blue R-250
Ac ce p
(Sigma Chemical Co.), followed by decolorization with a solution containing 40% methanol, 20% acetic acid and distilled water, which resulted in the formation of clear bands of lysis against a blue background, corresponding to enzymes with gelatinolytic activity. For an objective quantification, the optical intensities of the bands were measured with the Image J software.
Statistical analysis
The cell viability and ELISA results were analyzed statistically by analysis of variance (ANOVA) and Tukey’s test, and the quantitative real-time PCR results were
Page 8 of 30
performed using Kruskal-Wallis test followed by Dunn’s test. p values of 0.05 or less were considered statistically significant.
cr
Evaluation of the viability of THP-1 cells by the MTS assay
ip t
Results
us
Cell viability, expressed as the percentage in relation to the group showing
an
100% viability (0 μM CAPE), was 98.79% for cells treated with 10 μM CAPE, 74.83% for cells treated with 60 μM CAPE, and 71.47% for cells treated with 100 μM CAPE.
M
Compared with the other concentrations, only the concentration of 200 μM CAPE
d
was significantly cytotoxic to the cells (p < 0.05) (Fig. 1).
te
Effect of CAPE on the gene expression of MMP-1, MMP-9 and TIMP-1
Ac ce p
When compared to the control group, all concentrations of CAPE were able to
significantly inhibit (p < 0.05) the gene expression of MMP-1 induced by LPS (Fig. 2). With respect to MMP-9, only the concentration of 60 μM CAPE inhibited the expression of this enzyme (Fig. 3). Although all CAPE concentrations induced an increase in the expression of TIMP-1, this increase was only significant for the concentration of 60 μM (p < 0.05) (Fig. 4).
Effect of CAPE on the MMP-1 and MMP-9 production
Page 9 of 30
Analysis of MMP production in the cell supernatant by ELISA revealed a significant reduction (p < 0.05) in the production of MMP-1 after treatment with the two CAPE concentrations (10 and 60 μM) compared to the control group (Fig. 5).
ip t
There was also a reduction in the production of MMP-9; however, this reduction was only significant (p < 0.05) when a CAPE concentration of 60 μM was used (Fig. 6).
us
were compared (the B and the C groups) (Fig. 5 and 6)
cr
Statistically significant difference (p < 0.05) was observed when the treated groups
an
Effect of CAPE on MMP-9 activity
M
In the control group (A), the strong lysis bands are clearly visible corresponding to active MMP-9. After the treatment with CAPE the intensity of the
d
bands was changed. Mainly in the group treated with 60 μM of CAPE, weak lysis
te
bands are visible indicating that CAPE was able to reduce the MMP-9 activity in comparison with the control group (Fig. 7). To confirmed this analysis we have
Ac ce p
measured the intensities of the bands and observed a reduction of 64.17% of the band density in the group treated with 60 μM of CAPE and 30.22% in the group treated with 10 μM of CAPE, in relation to the density of the control group (A).
Discussion
The biological and pharmacological properties of propolis have gained attention in the scientific community,4 and its main active compound, CAPE, has been the subject of several studies.4, 18-20 The different properties of CAPE include its anticarcinogenic activity through the inhibition of the activity of gelatinases such as
Page 10 of 30
MMP-9. This enzyme plays an important role in extracellular matrix degradation during tumor growth and invasion3 and in pathological events that occur in the oral environment.13-16
ip t
MMPs are involved in tissue remodeling during both physiological and pathological processes. An increase in the expression of the MMPs is observed
cr
under different pathological condition such as inflammatory processes and tumor invasion.14 To gain knowledge of the mechanisms whereby CAPE alters the
us
production of MMPs in phagocytic cells involved in inflammatory processes,21 the
an
present study evaluated the in vitro effect of CAPE on the expression of MMP-1, MMP-9 and TIMP-1 in LPS-activated human monocytes (THP-1).
M
With respect to cell viability after exposure to CAPE, the results reported in the literature regarding the cytotoxic concentrations of this substance are divergent. Jin
d
et al. (2005)4 observed cytotoxicity in hepatocellular cell cultures when a CAPE
te
concentration higher than 40 μg/mL was used. However, the authors also found that a concentration 200 μg/mL of CAPE was not cytotoxic to primary mouse
Ac ce p
hepatocytes. In the present study, the CAPE concentrations used (10 and 60 μM) resulted in cell viability higher than 74%. These findings agree with Peng et al. (2012),20 who observed cell viability higher than 80% for all CAPE concentrations tested (0 to 40 μM) in both a human carcinoma cell line (SCC-9) and normal gingival fibroblasts.
The present study showed that CAPE was able to inhibit the gene expression,
production and the activity of MMP-9 in LPS-activated human monocytes. These results agree with Chung et al. (2004)22 who observed a significant reduction in the expression and activity of MMP-9 mediated by CAPE. The authors demonstrated that
Page 11 of 30
this reduction occurred through inhibition of the NF-kB pathway and blockade of the catalytic site of this MMP, respectively. The inhibition observed in the present study was only significant when CAPE
ip t
was used at the highest non-cytotoxic concentration (60 μM). This result corroborates the findings of Peng et al. (2012)20 who studied different CAPE concentrations (0, 5,
cr
10, 20 and 40 μM) and observed a significant reduction in the expression of gelatinase MMP-2 at the highest concentrations tested. Furthermore, Hwang et al.
an
cells (HT1080) in a dose-dependent manner.
us
(2006)3 showed that CAPE inhibited the expression of MMP-9 in human fibrosarcoma
In addition, the highest concentration of CAPE was able to enhance
M
significantly the gene expression of TIMP-1. Similarly, Peng et al. (2012)20 demonstrated an increase in the gene expression of the MMP inhibitor TIMP-2 and this increase was more expressive when a higher CAPE concentration (40 μM) was
te
d
used. On the other hand, another study3 observed the suppression of TIMP-2 after 24 h of treatment by CAPE. Although TIMP-2 is also an inhibitor of MMPs, the
Ac ce p
expression of this protein is regulated differently in vivo and in cell culture.23 TIMP-1 is considered to be the most important inhibitor of the mechanism of extracellular matrix degradation because it suppresses the activity of all MMPs, except for MTMMP.24
In agreement with other studies,3, 4, 18, 20, 22, 25 the present results showed that
CAPE was able to inhibit the activity of MMP-9. This inhibition was probably the result of reduced gene expression and consequent decreased production of this enzyme and because of the action of TIMP-1 which inhibited its activity. Studies4, 22 have shown that the addition of TIMP-1 significantly reduced the gelatinolytic activity of MMP-9. According to Hwang et al. (2006),3 TIMP-1 binds to the active site of
Page 12 of 30
MMP-9 and blocks its binding to the substrate, reducing the gelatinolytic activity of this MMP. With respect to MMP-1 that serves as an initiator of ECM breakdown13 and
ip t
has been detected in high level in teeth with apical periodontitis,16 the present study showed that CAPE was capable to inhibit the gene expression and the production of
cr
this enzyme in the human monocytic cell line THP-1. These results agree with Gencer et al. (2013)26 who also observed a significant reduction in the gene
us
expression of MMP-1 after treatment of human adenocarcinoma cells (MKN-45) with
an
CAPE. Since few studies have evaluated the effect of CAPE on the expression of MMP-1 and TIMP-1, the present results are important and contribute to the
M
discussion of future studies in this area.
In the present study especially the highest concentration of CAPE was able to
d
inhibit the gene expression, production and the activity of MMPs induced by LPS and
te
also increased the gene expression of TIMP-1. The results suggested that CAPE determined a positive correlation in the regulation between MMPs and TIMPs. This
Ac ce p
finding has important implications for the therapeutic potential of CAPE. The understanding of the mechanisms whereby CAPE interferes with the expression and activity of MMP and TIMP is fundamental for the development of in vivo studies in order to determine the ideal conditions (concentration, time, and indications) for the prevention and treatment of different diseases.
Acknowledgements
This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) through a PhD fellowship in Brazil with overseas training
Page 13 of 30
(PDEE/Grant 6608/10-8) and by Fundação de Amparo à Pesquisa de São Paulo (FAPESP) through a PhD fellowship (Grant 2010/18093-7).
He YJ, Liu BH, Xiang DB, Qiao ZY, Fu T, He YH. Inhibitory effect of caffeic
cr
1.
ip t
References
acid phenethyl ester on the growth of SW480 colorectal tumor cells involves beta-
us
catenin associated signaling pathway down-regulation. World J Gastroenterol
2.
an
2006;12(31):4981-5.
Wang D, Xiang DB, He YJ, Li ZP, Wu XH, Mou JH, et al. Effect of caffeic acid
M
phenethyl ester on proliferation and apoptosis of colorectal cancer cells in vitro. World J Gastroenterol 2005;11(26):4008-12.
Hwang HJ, Park HJ, Chung HJ, Min HY, Park EJ, Hong JY, et al. Inhibitory
d
3.
down-regulation of
te
effects of caffeic acid phenethyl ester on cancer cell metastasis mediated by the matrix
metalloproteinase
expression
in
human
HT1080
4.
Ac ce p
fibrosarcoma cells. J Nutr Biochem 2006;17(5):356-62. Jin UH, Chung TW, Kang SK, Suh SJ, Kim JK, Chung KH, et al. Caffeic acid
phenyl ester in propolis is a strong inhibitor of matrix metalloproteinase-9 and invasion inhibitor: isolation and identification. Clin Chim Acta 2005;362(1-2):57-64. 5.
Llano E, Pendás AM, Knäuper V, Sorsa T, Salo T, Salido E, et al. Identification
and structural and functional characterization of human enamelysin (MMP-20). Biochemistry 1997;36(49):15101-8. 6.
Sorsa T, Tjäderhane L, Salo T. Matrix metalloproteinases (MMPs) in oral
diseases. Oral Dis 2004;10(6):311-18.
Page 14 of 30
7.
Klein
T,
Bischoff
R.
Physiology
and
pathophysiology
of
matrix
metalloproteases. Amino Acids 2011;41(2):271-90. 8.
Huang JL, Wu SY, Xie XJ, Wang MX, Zhu S, Gu JR. Inhibiting effects of
differentiated THP-1 cells. Eur J Pharmacol 2011;670(1):304-10.
Chou YC, Sheu JR, Chung CL, Chen CY, Lin FL, Hsu MJ, et al. Nuclear-
cr
9.
ip t
Leflunomide metabolite on overexpression of CD147, MMP-2 and MMP-9 in PMA
targeted inhibition of NF-kappaB on MMP-9 production by N-2-(4-bromophenyl) ethyl
Chuang SY, Yang SH, Chen TY, Pang JH. Cilostazol inhibits matrix invasion
an
10.
us
caffeamide in human monocytic cells. Chem Biol Interact 2010;184(3):403-12.
and modulates the gene expressions of MMP-9 and TIMP-1 in PMA-differentiated
11.
M
THP-1 cells. Eur J Pharmacol 2011;670(2-3):419-26.
Lee WR, Chung CL, Hsiao CJ, Chou YC, Hsueh PJ, Yang PC, et al.
d
Suppression of matrix metalloproteinase-9 expression by andrographolide in human
4):270-7.
Desai A, Darland G, Bland JS, Tripp ML, Konda VR. META060 attenuates
Ac ce p
12.
te
monocytic THP-1 cells via inhibition of NF-κB activation. Phytomedicine 2012;19(3-
TNF-α-activated
inflammation,
endothelial-monocyte
interactions,
and
matrix
metalloproteinase-9 expression, and inhibits NF-κB and AP-1 in THP-1 monocytes. Atherosclerosis 2012;223(1):130-6. 13.
Wu Y, Zhu L, Wei H, Peng B. Regulation of matrix metalloproteinases, tissue
inhibitor of matrix metalloproteinase-1, and extracellular metalloproteinase inducer by interleukin-17 in human periodontal ligament fibroblasts. J Endod 2013;39(1):62-7. 14.
Hannas AR, Pereira JC, Granjeiro JM, Tjäderhane L. The role of matrix
metalloproteinases in the oral environment. Acta Odontol Scand 2007;65(1):1-13.
Page 15 of 30
15.
Itoh T, Nakamura H, Kishi J, Hayakawa T. The activation of matrix
metalloproteinases by a whole-cell extract from Prevotella nigrescens. J Endod 2009;35(1):55-9. Paula-Silva FW, da Silva LA, Kapila YL. Matrix metalloproteinase expression
ip t
16.
in teeth with apical periodontitis is differentially modulated by the modality of root
cr
canal treatment. J Endod 2010;36(2):231-7. 17.
Wisithphrom K, Windsor LJ. The effects of tumor necrosis factor-alpha,
us
interleukin-1beta, interleukin-6, and transforming growth factor-beta1 on pulp
an
fibroblast mediated collagen degradation. J Endod 2006;32(9):853-61. 18.
Lee KW, Kang NJ, Kim JH, Lee KM, Lee DE, Hur HJ, et al. Caffeic acid
M
phenethyl ester inhibits invasion and expression of matrix metalloproteinase in SKHep1 human hepatocellular carcinoma cells by targeting nuclear factor kappa B.
d
Genes Nutr 2008;2(4):319-22.
Jin UH, Song KH, Motomura M, Suzuki I, Gu YH, Kang YJ, et al. Caffeic acid
Genes
Nutr
te
19.
2008;2(4):319-22.phenethyl
ester
induces mitochondria-mediated
Ac ce p
apoptosis in human myeloid leukemia U937 cells. Mol Cell Biochem 2008;310(12):43-8. 20.
Peng CY, Yang HW, Chu YH, Chang YC, Hsieh MJ, Chou MY, et al. Caffeic
Acid phenethyl ester inhibits oral cancer cell metastasis by regulating matrix metalloproteinase-2 and the mitogen-activated protein kinase pathway. Evid Based Complement Alternat Med 2012;2012:732578. 21.
Melgarejo E, Medina MA, Sánchez-Jiménez F, Urdiales JL. Epigallocatechin
gallate reduces human monocyte mobility and adhesion in vitro. Br J Pharmacol 2009;158(7):1705-12.
Page 16 of 30
22.
Chung TW, Moon SK, Chang YC, Ko JH, Lee YC, Cho G, et al. Novel and
therapeutic effect of caffeic acid and caffeic acid phenyl ester on hepatocarcinoma cells: complete regression of hepatoma growth and metastasis by dual mechanism.
23.
ip t
FASEB J 2004;18(14):1670-81. Stetler-Stevenson WG, Brown PD, Onisto M, Levy AT, Liotta LA. Tissue
human tumor tissues. J Biol Chem 1990;265(23):13933-8.
Lee JH, Chung JH, Cho KH. The effects of epigallocatechin-3-gallate on
us
24.
cr
inhibitor of metalloproteinases-2 (TIMP-2) mRNA expression in tumor cell lines and
25.
an
extracellular matrix metabolism. J Dermatol Sci 2005;40(3):195-204.
Lin WL, Liang WH, Lee YJ, Chuang SK, Tseng TH. Antitumor progression
Biol Interact 2010;188(3):607-15.
Gencer S, Cebeci A, Irmak-Yazicioglu MB. Matrix metalloproteinase gene
d
26.
M
potential of caffeic acid phenethyl ester involving p75(NTR) in C6 glioma cells. Chem
te
expressions might be oxidative stress targets in gastric cancer cell lines. Chin J
Ac ce p
Cancer Res 2013;25(3):322-33.
Page 17 of 30
Table 1- Primer sequences used for quantitative real-time PCR. Gene
Primer sequence
Size (bp)
Accession number
HPRT1
F 5’TGCTCGAGATGTGATGAAGG3’
192
NM_000194.2
183
HF583680.1
106
NM_004994.2
206
NM_003254.2
R 5’TCCCCTGTTGACTGGTCATT3’
MMP-1
F 5’TGGACCTGGAGGAAATCTTGC3’
MMP-9
F 5’GAGGTGGACCGGATGTTCC3’
TIMP-1
F5’ACTGCAGGATGGACTCTTGCA3’ R 5’TTTCAGAGCCTTGGAGGAGCT3’
HPRT1,
hypoxanthine
phosphoribosyltransferase
1;
cr
R 5’AACTCACGCGCCAGTAGAAG3’
ip t
R 5’AGAGTCCAAGAGAATGGCCGA3’
MMP-1,
matrix
us
metalloproteinase-1; MMP-9, matrix metalloproteinase-9; TIMP-1, tissue inhibitor of
Ac ce p
te
d
M
an
metalloproteinase-1; F, forward; R, reverse.
Page 18 of 30
Figure legends
Fig. 1 - Evaluation of the viability of THP-1 cells by the MTS assay. Absorbance after
ip t
treatment with different concentrations of CAPE (10, 60, 100 and 200 μM), expressed as percentage in relation to the group showing 100% viability (0 μM CAPE). * Only
cr
the concentration of 200 μM CAPE was significantly cytotoxic to the cells. ANOVA
us
and Tukey’s test (p ≤ 0.05).
an
Fig. 2 – Real-time PCR analysis for MMP-1. Human monocytes (THP-1) were treated with CAPE (10 and 60 μM) combined with LPS (1 μg/mL) for 24 h. The level of RNA
M
expression in the treated groups was compared to the control group (A = exposed to 1 μg/mL of LPS) after normalization for HPRT1 gene expression. The graph
d
represents the median and range of two independent experiments. Kruskal-Wallis
te
and Dunn’s test (p ≤ 0.05). * p < 0.05 versus the control group (A).
Ac ce p
Fig. 3 – Real-time PCR analysis for MMP-9. Human monocytes (THP-1) were treated with CAPE (10 and 60 μM) combined with LPS (1 μg/mL) for 24 h. The level of RNA expression in the treated groups was compared to the control group (A = exposed to 1 μg/mL of LPS) after normalization for HPRT1 gene expression. The graph represents the median and range of two independent experiments. Kruskal-Wallis and Dunn’s test (p ≤ 0.05). * p < 0.05 versus the B group.
Fig. 4 – Real-time PCR analysis for TIMP-1. Human monocytes (THP-1) were treated with CAPE (10 and 60 μM) combined with LPS (1 μg/mL) for 24 h. The level of RNA expression in the treated groups was compared to the control group (A = exposed to
Page 19 of 30
1 μg/mL of LPS) after normalization for HPRT1 gene expression. The graph represents the median and range of two independent experiments. Kruskal-Wallis
ip t
and Dunn’s test (p ≤ 0.05). * p < 0.05 versus the untreated group (A).
Fig. 5 – Analysis of MMP-1 production by ELISA. Human monocytes (THP-1) were
cr
incubated with CAPE (10 and 60 μM) combined with LPS (1 μg/mL). The cell supernatant was collected after 24 h and the level of MMP-1 (pg/mL) was quantified.
us
The graph represents the mean and standard deviation of the results of two
an
independent experiments. ANOVA and Tukey’s test (p ≤ 0.05). * p < 0.05 versus the
M
control group (A); ** p < 0.05 the B group versus the C group.
Fig. 6 – Analysis of MMP-9 production by ELISA. Human monocytes (THP-1) were
d
incubated with CAPE (10 and 60 μM) combined with LPS (1 μg/mL). The cell
te
supernatant was collected after 24 h and the level of MMP-9 (pg/mL) was quantified. The graph represents the mean and standard deviation of the results of two
Ac ce p
independent experiments. ANOVA and Tukey’s test (p ≤ 0.05). * p < 0.05 versus the control group (A); ** p < 0.05 the B group versus the C group.
Fig. 7 – Zymography analysis for MMP-9. The intensity of bands corresponding to gelatinolytic activity of MMP-9. MMP-9 standard; A) CAPE [0 μM] + LPS [1 μg/mL]; B) CAPE [10 μM] + LPS [1 μg/mL]; C) CAPE [60 μM] + LPS [1 μg/mL]. 92 kDa: proMMP-9; 83 kDa: MMP-9.
Page 20 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 21 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 22 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 23 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 24 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 25 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 26 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 27 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 28 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 29 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 30 of 30
S0003-9969(15)00098-9 http://dx.doi.org/doi:10.1016/j.archoralbio.2015.04.009 AOB 3381
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
3-7-2014 24-3-2015 16-4-2015
Please cite this article as: Vilela PGF, Oliveira JR, Barros PP, Le˜ao MVP, Oliveira LD, Jorge AOC, In vitro effect of caffeic acid phenethyl ester on matrix metalloproteinases (MMP-1 and MMP-9) and their inhibitor (TIMP-1) in lipopolysaccharide-activated human monocytes, Archives of Oral Biology (2015), http://dx.doi.org/10.1016/j.archoralbio.2015.04.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
ip t
CAPE inhibited the expression, production and activity of MMP in human
cr
monocytes.
us
CAPE showed an important role in the balance between MMP and TIMP.
an
This study provides an additional evidence for a therapeutic potential of
Ac ce p
te
d
M
CAPE.
Page 1 of 30
Title In vitro effect of caffeic acid phenethyl ester on matrix metalloproteinases (MMP-1 and MMP-9) and their inhibitor (TIMP-1) in lipopolysaccharide-activated human
ip t
monocytes
Running Title
cr
The effect of CAPE in MMP expression
us
Authors
M
an
Polyana das Graças Figueiredo Vilela* Laboratory of Microbiology and Immunology. Department of Biosciences and Oral Diagnosis. Institute of Science and Technology - Univ. Estadual Paulista- UNESP. Address: Engenheiro Francisco José Longo, 777, Jardim São Dimas, CEP 12245-000, São José dos Campos, SP, Brazil. *Corresponding author. +1 201 9138230 [email protected]
te
d
Jonatas Rafael de Oliveira Laboratory of Microbiology and Immunology. Department of Biosciences and Oral Diagnosis. Institute of Science and Technology - Univ. Estadual Paulista- UNESP São José dos Campos, SP, Brazil. [email protected]
Ac ce p
Patrícia Pimentel de Barros Laboratory of Microbiology and Immunology. Department of Biosciences and Oral Diagnosis. Institute of Science and Technology - Univ. Estadual Paulista- UNESP. São José dos Campos, SP, Brazil. [email protected] Mariella Vieira Pereira Leão Bioscience Basic Institute, University of Taubaté. Taubaté, SP, Brazil. [email protected] Luciane Dias de Oliveira Laboratory of Biochemistry and Pharmacology. Department of Biosciences and Oral Diagnosis. Institute of Science and Technology - Univ. Estadual Paulista- UNESP São José dos Campos, SP, Brazil. [email protected] Antonio Olavo Cardoso Jorge Laboratory of Microbiology and Immunology. Department of Biosciences and Oral Diagnosis. Institute of Science and Technology - Univ. Estadual Paulista- UNESP. São José dos Campos, SP, Brazil. [email protected]
Page 2 of 30
Abstract
Objective: The role of matrix metalloproteinases (MMPs) in tissue degradation has
ip t
become evident in many diseases and great interest therefore exists in the pharmacological control of the activity of these enzymes. This study evaluated the
cr
effect of caffeic acid phenethyl ester (CAPE) on the production of MMPs and their inhibitor (TIMP) in monocytes activated by lipopolysaccharide (LPS). Design: The
us
human monocytic cell line (THP-1) was treated with non-cytotoxic concentrations of
an
CAPE (10 and 60 μM) combined with 1 μg/mL of LPS. The gene expression of MMP1, MMP-9 and TIMP-1 was evaluated by quantitative real-time polymerase chain
M
reaction. The protein secretion into the culture medium was assessed via enzymelinked immunosorbent assay and the gelatinolytic activity of MMP-9 by zymography.
d
Results: CAPE, especially at the highest concentration, down-regulated MMP-1 and
te
MMP-9 gene expression but up-regulated the gene expression of TIMP-1. Furthermore, CAPE reduced the secreted protein level of MMP-1 and MMP-9 as well
Ac ce p
as the gelatinolytic activity of MMP-9. Conclusion: CAPE was able to inhibit the gene expression, production and the activity of MMPs induced by LPS and also increased the gene expression of TIMP-1. The present observations suggest that CAPE exerted a positive effect on the regulatory mechanism between MMPs and TIMP, which is important for the control of different diseases.
Keywords: matrix metalloproteinases; lipopolysaccharide; monocytes; CAPE.
Page 3 of 30
Introduction
Studies have shown the cytotoxic effect and the activity of caffeic acid
its
ability
to
suppress
the
enzymatic
activity
or
expression
of
matrix
cr
metalloproteinases 9 (MMP-9).3,4
ip t
phenethyl ester (CAPE), an active component of propolis, against tumor cells,1,2 and
MMPs are involved in a variety of physiological processes associated with the
us
modulation and regulation of extracellular matrix (ECM) turnover.5-7 However, in
an
recent years it has become evident that these enzymes also play a role in tissue degradation in different diseases such as cancer and chronic inflammatory
M
processes.6-13 The MMPs have been also associated with periodontitis and apical periodontitis in which bacteria and their components induce an inflammatory response that leads to the excessive production and the activation of these enzymes
te
d
which, in turn, cause tissue destruction.13-16 In addition, it is also known that an imbalance between MMP and tissue
Ac ce p
inhibitors of metalloproteinase (TIMP) is responsible for tissue degradation.13 TIMP is the main endogenous inhibitors of MMP and comprise a group of four different proteins, TIMP-1, TIMP-2, TIMP-3 and TIMP-4.7 Knowledge of the factors that regulate the synthesis and secretion of MMPs
and TIMPs is important for the understanding of the pathogenesis of oral disease.13,17 Therefore, growing interest exists in the pharmacological control of the activity of these enzymes6 once the MMP inhibitors may be useful therapeutic agents for the prevention and treatment of different oral diseases.14 Since most of the studies have demonstrated the effects of CAPE in cancer cells and the main sources of MMPs in pathogenic processes are the cells of
Page 4 of 30
inflammatory line, the objective of the present study was to evaluate the effect of CAPE on the activity and production of MMPs and on the gene expression of MMP-1, MMP-9 and TIMP-1 in human monocytes (THP-1) activated by lipopolysaccharide
ip t
(LPS).
cr
Material and Methods
an
us
Cell culture
Cells of a human monocytic cell line (THP-1) were obtained from the cell bank
M
of the Paul Ehrlich Technical-Scientific Association (Associação Técnico Científica Paul Ehrlich - APABCAM, Rio de Janeiro, Brazil). The cells were cultured in RPMI-
d
1640 medium (Gibco, Life Technologies Corporation, Grand Island, NY, USA),
te
supplemented with 10% fetal bovine serum, 1% MEM vitamin solution, 1% sodium pyruvate, 1% MEM non-essential amino acid solution, 0.01% beta-mercaptoethanol,
Ac ce p
and 1% penicillin-streptomycin (all from Gibco, Life Technologies Corporation). The cells were maintained in an incubator at 37ºC with 5% CO2 and the culture medium was changed at 2-day intervals.
Determination of cell viability by the MTS assay
The cytotoxicity of CAPE (Sigma- Aldrich, St. Louis, MO, USA) was evaluated by the MTS assay. After exposure to different concentrations of CAPE (0, 10, 60, 100 and 200 μM), 5 x 104 cells were incubated for 2 h in CellTiter 96® AQueous solution (Promega, Madison, WI, USA). The absorbance was read in a microplate reader
Page 5 of 30
(Biotek®, Winooski, VT, USA) at 490 nm and converted into percentage of cell viability.
ip t
Experimental assays
cr
THP-1 cells (0.5 x 106 cells per well) were transferred to 6-well plates (TPP, Trasadingen, Switzerland) in RPMI medium not containing fetal bovine serum or
us
additives. The cells were treated with non-cytotoxic concentrations of CAPE
an
combined with 1 μg/mL of LPS (Escherichia coli 0055:B5; Sigma- Aldrich). The control group consisted of cells exposed only to 1 μg/mL of LPS (n=6 for each group,
M
2 independent experiments). The plates were maintained in an incubator at 37ºC with 5% CO2 for 24 h.
d
After incubation, the cell culture was collected and centrifuged (2400 x g for 5
te
min). The cell supernatant was collected and frozen at -20ºC for zymography and enzyme-linked immunosorbent assay (ELISA). The cell pellet was resuspended in
Ac ce p
Trizol® solution (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA), transferred to new tubes and frozen at -80ºC for subsequent RNA extraction and quantitative real-time polymerase chain reaction analysis (PCR).
RNA extraction, synthesis of complementary DNA (cDNA), and Quantitative Realtime PCR
The total RNA was extracted with Trizol® reagent according to manufacturer instructions. RNA (1 µg) was treated with DNAse (Deoxyribonuclease I kit, Amplification Grade; Invitrogen, Life Technologies Corporation) and then used for
Page 6 of 30
cDNA synthesis (SuperScript III, First-Strand Synthesis SuperMix, Invitrogen, Life Technologies Corporation). The resulting cDNA was amplified by real-time PCR (Platinum®SYBR Green, Invitrogen, Life Technologies Corporation) and quantified
ip t
using the Line-Gene K system (Bior Technology Hitech, Binjiang, Hangzhou, China). The sequences of the primers used were checked at Primer-Blast software from the
cr
National Center for Biotechnology Information nucleotide sequence database and is shown in Table 1.
us
The reactions were performed in triplicate and previously standardized based
an
on efficiency curves. Hypoxanthine phosphoribosyl transferase 1 (HPRT1) was used as housekeeping gene to normalize the PCR array data.
M
Relative RNA levels were presented as ratio relative to control cells after normalization for HPRT1 expression. The results were obtained as threshold cycle
d
(Ct) values. Delta delta Ct (∆∆Ct) method was performed to analyze RNA expression
te
levels by using the Line Gene K software.
Ac ce p
Enzyme-linked immunosorbent assay (ELISA)
The production of MMP-9 and MMP-1 was evaluated by ELISA according to
the recommendations of the manufacturers of the DuoSet® ELISA kits for human MMP-9 (R&D Systems, Minneapolis, MN, USA) and human MMP-1 (Sigma Chemical Co., St. Louis, MO, USA). Briefly, the plates were coated with anti-MMP capture antibody and kept overnight. The next day, the samples and standard curve were added and after incubation and the washed, the wells are incubated with biotinylated anti-MMP detection antibody. The reaction was developed with a chromogen solution. The absorbance was read in a microplate reader at 450 nm and the results
Page 7 of 30
were converted into concentrations (pg/mL) of MMP using the GraphPad Prism® 5 Program.
ip t
Zymography
cr
The gelatinolytic activity of MMP-9 was determined by zymography. First, total protein concentration in the cell supernatant was quantified by the method of Lowry
us
(Sigma Chemical Co.). Next, a known concentration of total protein in the samples
an
and of the MMP-9 standard (R&D Systems) was mixed with non-reducing sample buffer (62.5 mM Tris-HCl, 3% SDS, 10% glycerol, 0.25% bromophenol blue) and the
M
mixture was loaded onto 10% polyacrylamide gel containing 0.2% gelatin (Sigma Chemical Co.). After electrophoresis, the gel was incubated in a solution of 10 mM
d
Tris (pH 8.0) and 2.5% Triton X-100 (Sigma Chemical Co.) for 1 h at 37ºC. Next, the
te
gel was incubated with developing solution containing 50 mM Tris buffer (pH 8.8) and 5 mM CaCl2 for 20 h at 37ºC. The gel was stained with Coomassie blue R-250
Ac ce p
(Sigma Chemical Co.), followed by decolorization with a solution containing 40% methanol, 20% acetic acid and distilled water, which resulted in the formation of clear bands of lysis against a blue background, corresponding to enzymes with gelatinolytic activity. For an objective quantification, the optical intensities of the bands were measured with the Image J software.
Statistical analysis
The cell viability and ELISA results were analyzed statistically by analysis of variance (ANOVA) and Tukey’s test, and the quantitative real-time PCR results were
Page 8 of 30
performed using Kruskal-Wallis test followed by Dunn’s test. p values of 0.05 or less were considered statistically significant.
cr
Evaluation of the viability of THP-1 cells by the MTS assay
ip t
Results
us
Cell viability, expressed as the percentage in relation to the group showing
an
100% viability (0 μM CAPE), was 98.79% for cells treated with 10 μM CAPE, 74.83% for cells treated with 60 μM CAPE, and 71.47% for cells treated with 100 μM CAPE.
M
Compared with the other concentrations, only the concentration of 200 μM CAPE
d
was significantly cytotoxic to the cells (p < 0.05) (Fig. 1).
te
Effect of CAPE on the gene expression of MMP-1, MMP-9 and TIMP-1
Ac ce p
When compared to the control group, all concentrations of CAPE were able to
significantly inhibit (p < 0.05) the gene expression of MMP-1 induced by LPS (Fig. 2). With respect to MMP-9, only the concentration of 60 μM CAPE inhibited the expression of this enzyme (Fig. 3). Although all CAPE concentrations induced an increase in the expression of TIMP-1, this increase was only significant for the concentration of 60 μM (p < 0.05) (Fig. 4).
Effect of CAPE on the MMP-1 and MMP-9 production
Page 9 of 30
Analysis of MMP production in the cell supernatant by ELISA revealed a significant reduction (p < 0.05) in the production of MMP-1 after treatment with the two CAPE concentrations (10 and 60 μM) compared to the control group (Fig. 5).
ip t
There was also a reduction in the production of MMP-9; however, this reduction was only significant (p < 0.05) when a CAPE concentration of 60 μM was used (Fig. 6).
us
were compared (the B and the C groups) (Fig. 5 and 6)
cr
Statistically significant difference (p < 0.05) was observed when the treated groups
an
Effect of CAPE on MMP-9 activity
M
In the control group (A), the strong lysis bands are clearly visible corresponding to active MMP-9. After the treatment with CAPE the intensity of the
d
bands was changed. Mainly in the group treated with 60 μM of CAPE, weak lysis
te
bands are visible indicating that CAPE was able to reduce the MMP-9 activity in comparison with the control group (Fig. 7). To confirmed this analysis we have
Ac ce p
measured the intensities of the bands and observed a reduction of 64.17% of the band density in the group treated with 60 μM of CAPE and 30.22% in the group treated with 10 μM of CAPE, in relation to the density of the control group (A).
Discussion
The biological and pharmacological properties of propolis have gained attention in the scientific community,4 and its main active compound, CAPE, has been the subject of several studies.4, 18-20 The different properties of CAPE include its anticarcinogenic activity through the inhibition of the activity of gelatinases such as
Page 10 of 30
MMP-9. This enzyme plays an important role in extracellular matrix degradation during tumor growth and invasion3 and in pathological events that occur in the oral environment.13-16
ip t
MMPs are involved in tissue remodeling during both physiological and pathological processes. An increase in the expression of the MMPs is observed
cr
under different pathological condition such as inflammatory processes and tumor invasion.14 To gain knowledge of the mechanisms whereby CAPE alters the
us
production of MMPs in phagocytic cells involved in inflammatory processes,21 the
an
present study evaluated the in vitro effect of CAPE on the expression of MMP-1, MMP-9 and TIMP-1 in LPS-activated human monocytes (THP-1).
M
With respect to cell viability after exposure to CAPE, the results reported in the literature regarding the cytotoxic concentrations of this substance are divergent. Jin
d
et al. (2005)4 observed cytotoxicity in hepatocellular cell cultures when a CAPE
te
concentration higher than 40 μg/mL was used. However, the authors also found that a concentration 200 μg/mL of CAPE was not cytotoxic to primary mouse
Ac ce p
hepatocytes. In the present study, the CAPE concentrations used (10 and 60 μM) resulted in cell viability higher than 74%. These findings agree with Peng et al. (2012),20 who observed cell viability higher than 80% for all CAPE concentrations tested (0 to 40 μM) in both a human carcinoma cell line (SCC-9) and normal gingival fibroblasts.
The present study showed that CAPE was able to inhibit the gene expression,
production and the activity of MMP-9 in LPS-activated human monocytes. These results agree with Chung et al. (2004)22 who observed a significant reduction in the expression and activity of MMP-9 mediated by CAPE. The authors demonstrated that
Page 11 of 30
this reduction occurred through inhibition of the NF-kB pathway and blockade of the catalytic site of this MMP, respectively. The inhibition observed in the present study was only significant when CAPE
ip t
was used at the highest non-cytotoxic concentration (60 μM). This result corroborates the findings of Peng et al. (2012)20 who studied different CAPE concentrations (0, 5,
cr
10, 20 and 40 μM) and observed a significant reduction in the expression of gelatinase MMP-2 at the highest concentrations tested. Furthermore, Hwang et al.
an
cells (HT1080) in a dose-dependent manner.
us
(2006)3 showed that CAPE inhibited the expression of MMP-9 in human fibrosarcoma
In addition, the highest concentration of CAPE was able to enhance
M
significantly the gene expression of TIMP-1. Similarly, Peng et al. (2012)20 demonstrated an increase in the gene expression of the MMP inhibitor TIMP-2 and this increase was more expressive when a higher CAPE concentration (40 μM) was
te
d
used. On the other hand, another study3 observed the suppression of TIMP-2 after 24 h of treatment by CAPE. Although TIMP-2 is also an inhibitor of MMPs, the
Ac ce p
expression of this protein is regulated differently in vivo and in cell culture.23 TIMP-1 is considered to be the most important inhibitor of the mechanism of extracellular matrix degradation because it suppresses the activity of all MMPs, except for MTMMP.24
In agreement with other studies,3, 4, 18, 20, 22, 25 the present results showed that
CAPE was able to inhibit the activity of MMP-9. This inhibition was probably the result of reduced gene expression and consequent decreased production of this enzyme and because of the action of TIMP-1 which inhibited its activity. Studies4, 22 have shown that the addition of TIMP-1 significantly reduced the gelatinolytic activity of MMP-9. According to Hwang et al. (2006),3 TIMP-1 binds to the active site of
Page 12 of 30
MMP-9 and blocks its binding to the substrate, reducing the gelatinolytic activity of this MMP. With respect to MMP-1 that serves as an initiator of ECM breakdown13 and
ip t
has been detected in high level in teeth with apical periodontitis,16 the present study showed that CAPE was capable to inhibit the gene expression and the production of
cr
this enzyme in the human monocytic cell line THP-1. These results agree with Gencer et al. (2013)26 who also observed a significant reduction in the gene
us
expression of MMP-1 after treatment of human adenocarcinoma cells (MKN-45) with
an
CAPE. Since few studies have evaluated the effect of CAPE on the expression of MMP-1 and TIMP-1, the present results are important and contribute to the
M
discussion of future studies in this area.
In the present study especially the highest concentration of CAPE was able to
d
inhibit the gene expression, production and the activity of MMPs induced by LPS and
te
also increased the gene expression of TIMP-1. The results suggested that CAPE determined a positive correlation in the regulation between MMPs and TIMPs. This
Ac ce p
finding has important implications for the therapeutic potential of CAPE. The understanding of the mechanisms whereby CAPE interferes with the expression and activity of MMP and TIMP is fundamental for the development of in vivo studies in order to determine the ideal conditions (concentration, time, and indications) for the prevention and treatment of different diseases.
Acknowledgements
This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) through a PhD fellowship in Brazil with overseas training
Page 13 of 30
(PDEE/Grant 6608/10-8) and by Fundação de Amparo à Pesquisa de São Paulo (FAPESP) through a PhD fellowship (Grant 2010/18093-7).
He YJ, Liu BH, Xiang DB, Qiao ZY, Fu T, He YH. Inhibitory effect of caffeic
cr
1.
ip t
References
acid phenethyl ester on the growth of SW480 colorectal tumor cells involves beta-
us
catenin associated signaling pathway down-regulation. World J Gastroenterol
2.
an
2006;12(31):4981-5.
Wang D, Xiang DB, He YJ, Li ZP, Wu XH, Mou JH, et al. Effect of caffeic acid
M
phenethyl ester on proliferation and apoptosis of colorectal cancer cells in vitro. World J Gastroenterol 2005;11(26):4008-12.
Hwang HJ, Park HJ, Chung HJ, Min HY, Park EJ, Hong JY, et al. Inhibitory
d
3.
down-regulation of
te
effects of caffeic acid phenethyl ester on cancer cell metastasis mediated by the matrix
metalloproteinase
expression
in
human
HT1080
4.
Ac ce p
fibrosarcoma cells. J Nutr Biochem 2006;17(5):356-62. Jin UH, Chung TW, Kang SK, Suh SJ, Kim JK, Chung KH, et al. Caffeic acid
phenyl ester in propolis is a strong inhibitor of matrix metalloproteinase-9 and invasion inhibitor: isolation and identification. Clin Chim Acta 2005;362(1-2):57-64. 5.
Llano E, Pendás AM, Knäuper V, Sorsa T, Salo T, Salido E, et al. Identification
and structural and functional characterization of human enamelysin (MMP-20). Biochemistry 1997;36(49):15101-8. 6.
Sorsa T, Tjäderhane L, Salo T. Matrix metalloproteinases (MMPs) in oral
diseases. Oral Dis 2004;10(6):311-18.
Page 14 of 30
7.
Klein
T,
Bischoff
R.
Physiology
and
pathophysiology
of
matrix
metalloproteases. Amino Acids 2011;41(2):271-90. 8.
Huang JL, Wu SY, Xie XJ, Wang MX, Zhu S, Gu JR. Inhibiting effects of
differentiated THP-1 cells. Eur J Pharmacol 2011;670(1):304-10.
Chou YC, Sheu JR, Chung CL, Chen CY, Lin FL, Hsu MJ, et al. Nuclear-
cr
9.
ip t
Leflunomide metabolite on overexpression of CD147, MMP-2 and MMP-9 in PMA
targeted inhibition of NF-kappaB on MMP-9 production by N-2-(4-bromophenyl) ethyl
Chuang SY, Yang SH, Chen TY, Pang JH. Cilostazol inhibits matrix invasion
an
10.
us
caffeamide in human monocytic cells. Chem Biol Interact 2010;184(3):403-12.
and modulates the gene expressions of MMP-9 and TIMP-1 in PMA-differentiated
11.
M
THP-1 cells. Eur J Pharmacol 2011;670(2-3):419-26.
Lee WR, Chung CL, Hsiao CJ, Chou YC, Hsueh PJ, Yang PC, et al.
d
Suppression of matrix metalloproteinase-9 expression by andrographolide in human
4):270-7.
Desai A, Darland G, Bland JS, Tripp ML, Konda VR. META060 attenuates
Ac ce p
12.
te
monocytic THP-1 cells via inhibition of NF-κB activation. Phytomedicine 2012;19(3-
TNF-α-activated
inflammation,
endothelial-monocyte
interactions,
and
matrix
metalloproteinase-9 expression, and inhibits NF-κB and AP-1 in THP-1 monocytes. Atherosclerosis 2012;223(1):130-6. 13.
Wu Y, Zhu L, Wei H, Peng B. Regulation of matrix metalloproteinases, tissue
inhibitor of matrix metalloproteinase-1, and extracellular metalloproteinase inducer by interleukin-17 in human periodontal ligament fibroblasts. J Endod 2013;39(1):62-7. 14.
Hannas AR, Pereira JC, Granjeiro JM, Tjäderhane L. The role of matrix
metalloproteinases in the oral environment. Acta Odontol Scand 2007;65(1):1-13.
Page 15 of 30
15.
Itoh T, Nakamura H, Kishi J, Hayakawa T. The activation of matrix
metalloproteinases by a whole-cell extract from Prevotella nigrescens. J Endod 2009;35(1):55-9. Paula-Silva FW, da Silva LA, Kapila YL. Matrix metalloproteinase expression
ip t
16.
in teeth with apical periodontitis is differentially modulated by the modality of root
cr
canal treatment. J Endod 2010;36(2):231-7. 17.
Wisithphrom K, Windsor LJ. The effects of tumor necrosis factor-alpha,
us
interleukin-1beta, interleukin-6, and transforming growth factor-beta1 on pulp
an
fibroblast mediated collagen degradation. J Endod 2006;32(9):853-61. 18.
Lee KW, Kang NJ, Kim JH, Lee KM, Lee DE, Hur HJ, et al. Caffeic acid
M
phenethyl ester inhibits invasion and expression of matrix metalloproteinase in SKHep1 human hepatocellular carcinoma cells by targeting nuclear factor kappa B.
d
Genes Nutr 2008;2(4):319-22.
Jin UH, Song KH, Motomura M, Suzuki I, Gu YH, Kang YJ, et al. Caffeic acid
Genes
Nutr
te
19.
2008;2(4):319-22.phenethyl
ester
induces mitochondria-mediated
Ac ce p
apoptosis in human myeloid leukemia U937 cells. Mol Cell Biochem 2008;310(12):43-8. 20.
Peng CY, Yang HW, Chu YH, Chang YC, Hsieh MJ, Chou MY, et al. Caffeic
Acid phenethyl ester inhibits oral cancer cell metastasis by regulating matrix metalloproteinase-2 and the mitogen-activated protein kinase pathway. Evid Based Complement Alternat Med 2012;2012:732578. 21.
Melgarejo E, Medina MA, Sánchez-Jiménez F, Urdiales JL. Epigallocatechin
gallate reduces human monocyte mobility and adhesion in vitro. Br J Pharmacol 2009;158(7):1705-12.
Page 16 of 30
22.
Chung TW, Moon SK, Chang YC, Ko JH, Lee YC, Cho G, et al. Novel and
therapeutic effect of caffeic acid and caffeic acid phenyl ester on hepatocarcinoma cells: complete regression of hepatoma growth and metastasis by dual mechanism.
23.
ip t
FASEB J 2004;18(14):1670-81. Stetler-Stevenson WG, Brown PD, Onisto M, Levy AT, Liotta LA. Tissue
human tumor tissues. J Biol Chem 1990;265(23):13933-8.
Lee JH, Chung JH, Cho KH. The effects of epigallocatechin-3-gallate on
us
24.
cr
inhibitor of metalloproteinases-2 (TIMP-2) mRNA expression in tumor cell lines and
25.
an
extracellular matrix metabolism. J Dermatol Sci 2005;40(3):195-204.
Lin WL, Liang WH, Lee YJ, Chuang SK, Tseng TH. Antitumor progression
Biol Interact 2010;188(3):607-15.
Gencer S, Cebeci A, Irmak-Yazicioglu MB. Matrix metalloproteinase gene
d
26.
M
potential of caffeic acid phenethyl ester involving p75(NTR) in C6 glioma cells. Chem
te
expressions might be oxidative stress targets in gastric cancer cell lines. Chin J
Ac ce p
Cancer Res 2013;25(3):322-33.
Page 17 of 30
Table 1- Primer sequences used for quantitative real-time PCR. Gene
Primer sequence
Size (bp)
Accession number
HPRT1
F 5’TGCTCGAGATGTGATGAAGG3’
192
NM_000194.2
183
HF583680.1
106
NM_004994.2
206
NM_003254.2
R 5’TCCCCTGTTGACTGGTCATT3’
MMP-1
F 5’TGGACCTGGAGGAAATCTTGC3’
MMP-9
F 5’GAGGTGGACCGGATGTTCC3’
TIMP-1
F5’ACTGCAGGATGGACTCTTGCA3’ R 5’TTTCAGAGCCTTGGAGGAGCT3’
HPRT1,
hypoxanthine
phosphoribosyltransferase
1;
cr
R 5’AACTCACGCGCCAGTAGAAG3’
ip t
R 5’AGAGTCCAAGAGAATGGCCGA3’
MMP-1,
matrix
us
metalloproteinase-1; MMP-9, matrix metalloproteinase-9; TIMP-1, tissue inhibitor of
Ac ce p
te
d
M
an
metalloproteinase-1; F, forward; R, reverse.
Page 18 of 30
Figure legends
Fig. 1 - Evaluation of the viability of THP-1 cells by the MTS assay. Absorbance after
ip t
treatment with different concentrations of CAPE (10, 60, 100 and 200 μM), expressed as percentage in relation to the group showing 100% viability (0 μM CAPE). * Only
cr
the concentration of 200 μM CAPE was significantly cytotoxic to the cells. ANOVA
us
and Tukey’s test (p ≤ 0.05).
an
Fig. 2 – Real-time PCR analysis for MMP-1. Human monocytes (THP-1) were treated with CAPE (10 and 60 μM) combined with LPS (1 μg/mL) for 24 h. The level of RNA
M
expression in the treated groups was compared to the control group (A = exposed to 1 μg/mL of LPS) after normalization for HPRT1 gene expression. The graph
d
represents the median and range of two independent experiments. Kruskal-Wallis
te
and Dunn’s test (p ≤ 0.05). * p < 0.05 versus the control group (A).
Ac ce p
Fig. 3 – Real-time PCR analysis for MMP-9. Human monocytes (THP-1) were treated with CAPE (10 and 60 μM) combined with LPS (1 μg/mL) for 24 h. The level of RNA expression in the treated groups was compared to the control group (A = exposed to 1 μg/mL of LPS) after normalization for HPRT1 gene expression. The graph represents the median and range of two independent experiments. Kruskal-Wallis and Dunn’s test (p ≤ 0.05). * p < 0.05 versus the B group.
Fig. 4 – Real-time PCR analysis for TIMP-1. Human monocytes (THP-1) were treated with CAPE (10 and 60 μM) combined with LPS (1 μg/mL) for 24 h. The level of RNA expression in the treated groups was compared to the control group (A = exposed to
Page 19 of 30
1 μg/mL of LPS) after normalization for HPRT1 gene expression. The graph represents the median and range of two independent experiments. Kruskal-Wallis
ip t
and Dunn’s test (p ≤ 0.05). * p < 0.05 versus the untreated group (A).
Fig. 5 – Analysis of MMP-1 production by ELISA. Human monocytes (THP-1) were
cr
incubated with CAPE (10 and 60 μM) combined with LPS (1 μg/mL). The cell supernatant was collected after 24 h and the level of MMP-1 (pg/mL) was quantified.
us
The graph represents the mean and standard deviation of the results of two
an
independent experiments. ANOVA and Tukey’s test (p ≤ 0.05). * p < 0.05 versus the
M
control group (A); ** p < 0.05 the B group versus the C group.
Fig. 6 – Analysis of MMP-9 production by ELISA. Human monocytes (THP-1) were
d
incubated with CAPE (10 and 60 μM) combined with LPS (1 μg/mL). The cell
te
supernatant was collected after 24 h and the level of MMP-9 (pg/mL) was quantified. The graph represents the mean and standard deviation of the results of two
Ac ce p
independent experiments. ANOVA and Tukey’s test (p ≤ 0.05). * p < 0.05 versus the control group (A); ** p < 0.05 the B group versus the C group.
Fig. 7 – Zymography analysis for MMP-9. The intensity of bands corresponding to gelatinolytic activity of MMP-9. MMP-9 standard; A) CAPE [0 μM] + LPS [1 μg/mL]; B) CAPE [10 μM] + LPS [1 μg/mL]; C) CAPE [60 μM] + LPS [1 μg/mL]. 92 kDa: proMMP-9; 83 kDa: MMP-9.
Page 20 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 21 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 22 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 23 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 24 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 25 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 26 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 27 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 28 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 29 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 30 of 30
