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Angiogenesis, Metastasis, and the Cellular Microenvironment
CADPE Inhibits PMA-Stimulated Gastric Carcinoma Cell
Invasion and Matrix Metalloproteinase-9 Expression by
FAK/MEK/ERK–Mediated AP-1 Activation
Honghui Han, Bing Du, Xinhua Pan, Junchen Liu, Qufei Zhao, Xiaoyuan Lian, Min Qian, and Mingyao Liu
Abstract
Metastasis is one of the main causes of death for patients with malignant tumors. Aberrant expression of
matrix metalloproteinase-9 (MMP-9) has been implicated in the invasion and metastasis of various cancer cells.
Here, we found that caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE) could inhibit the migration and in-
vasion of human gastric carcinoma cells in Transwell migration assays. To understand the underlying mecha-
nism, we showed that CADPE significantly inhibited phorbol 12-myristate 13-acetate (PMA)–induced increases
in MMP-9 expression and activity in a dose-dependent manner. The inhibitory effect of CADPE on MMP-9
expression correlated well with the suppression of MMP-9 promoter activity and the reduction of MMP-9
mRNA. Reporter gene assay and electrophoretic mobility shift assay showed that CADPE inhibited
MMP-9 expression by suppressing the activation of the nuclear transcription factor activator protein-1
(AP-1) and c-Fos, but not NF-κB. Moreover, CADPE inhibited PMA-induced phosphorylation of protein
kinases involved in AP-1 activation, such as focal adhesion kinase (FAK), mitogen-activated protein kinase/
extracellular signal–regulated kinase (ERK) kinase (MEK), and ERK1/2, whereas CADPE had little effect on
the phosphorylation of p38 and c-jun NH
2
-terminal kinase. Taken together, our findings indicate that CADPE
could be a unique antitumor agent that specifically inhibits MMP-9 activity by targeting the activation of FAK/
MEK/ERK protein kinases and AP-1 transcription factor. Mol Cancer Res; 8(11); 1477–88. ©2010 AACR.
Introduction
Gastric carcinoma is the second most common cause of
death and the third most common cancer worldwide (1).
The current overall 5-year survival figures for gastric cancers
in western patients are in the range of 5% to 17%. The inci-
dence is also high in Europe, South America, and Eastern Asia
(2-4). Because of its limited treatment efficiency and poor
prognosis, the therapeutics of gastric cancer remains a major
clinical challenge (5). Loss of control of tumor cell invasion
and metastasis is the main cause of death in gastric cancer
patients. The formation of metastatic nodules to gastric
carcinoma is a multistep and complex process that includes
cell proliferation, digestion of the extracellular matrix
(ECM), cell migration to circulation system or lymph nodes,
and remigration and growth of tumors at metastatic sites. It
is widely believed that the aberrant expression of matrix
metalloproteinases (MMP) is involved in these processes (6).
MMPs are well-known ECM-degrading enzymes, which
comprise a family of 24 members. Based on their sub-
strates, MMPs are divided into four subclasses: collagenase,
gelatinase, stromelysin, and membrane-associated MMPs
(7). As a main ECM-degrading enzyme family, MMPs have
essential roles in physiologic processes such as tissue devel-
opment, remodeling, and wound healing (8). However,
they are also involved in some tissue destructive diseases,
such as atherosclerosis; inflammation; rheumatoid arthritis;
and tumor invasion, metastasis, and neoangiogenesis
(9, 10). Recent studies showed that MMPs were important
regulators of the tumor microenvironment, including
tumor progression, metastatic niche formation, and inflam-
mation in cancer (11). Among human MMPs, MMP-2
(gelatinase-A) and MMP-9 (gelatinase-B) are key enzymes
in the degradation of type IV collagen, which is an impor-
tant component of ECM. These two members are mostly
associated with tumor migration, invasion, and metastasis
for various cancers (12). An enhanced expression of
MMP-9 has been shown to be associated with the progres-
sion and invasion of tumors, whereas MMP-2 is usually
expressed constitutively (13, 14).
Authors' Affiliation: The Institute of Biomedical Sciences and School of
Life Sciences, East China Normal University, Shanghai, China
Note: Supplementary data for this article are available at Molecular Can-
cer Research Online (http://mcr.aacrjournals.org/).
H. Han and B. Du contributed equally to this work.
Corresponding Authors: Mingyao Liu, The Institute of Biomedical
Sciences, East China Normal University, 500 Dongchuan Road, Shanghai
200241, China. Phone: 86-21-5434-5124; Fax: 86-21-5434-4922.
E-mail: myliu@bio.ecnu.edu.cn or Min Qian, East China Normal University
School of Life Science, 3663 North Zhongshan, Shanghai 2000 62, PR
China. Phone: 86-021-62233569; Fax: 011-86-021-62233754. E-mail:
mqian@bio.ecnu.edu.cn
doi: 10.1158/1541-7786.MCR-10-0114
©2010 American Association for Cancer Research.
Molecular
Cancer
Research
www.aacrjournals.org 1477
The expression of MMP-9 can be stimulated by various
agents, such as inflammatory cytokine, growth factor, and
phorbol myristate acetate (PMA). PMA is a well-known
inflammatory stimulator and tumor promoter that acti-
vates almost all protein kinase C (PKC) isozymes and in-
creases the invasiveness of various types of cancer cells by
activating MMP-9 (15). Those stimulators can upregulate
the expression of MMP-9 by modulating the activation of
transcription factors such as activator protein-1 (AP-1) and
NF-κB through the Ras/Raf/extracellular signal-regulated
kinase (ERK), c-jun NH
2
-terminal kinase (JNK), and
phosphoinositide 3-kinase/Akt signaling pathways
(16-19). AP-1 has been shown to regulate the expression
of a number of genes, some of which are involved in tu-
morigenesis (20, 21). Thus, it will be an effective way to
find tumorigenesis and metastasis inhibitors from the
agents that can suppress the activities of AP-1 and MMP-9.
Caffeic acid 3,4-dihydroxyphenethyl ester (CADPE) was
originally isolated from Teucrium pilosum as a substance
named teucrol in 2000 (22) and can be synthesized by a
chemical process (23). Caffeic acid (CA) is a phenolic com-
pound and is largely found in food plants. CA has been
reported to posses a wide spectrum of biological effects
(e.g., antioxidant, anti-inflammatory, antitumor angiogen-
esis and antitumor invasion, and metastasis properties; ref.
24-26). Recent studies showed that both CA and CADPE
could inhibit tumor angiogenesis in human renal carcino-
ma cells by suppressing hypoxia-induced signal transducer
and activator of transcription-3 phosphorylation, signal
transducer and activator of transcription-3 nuclear translo-
cation, hypoxia-inducible factor-1αinduction, and vascu-
lar endothelial growth factor expression (24). However, the
effect and related molecular mechanisms of CADPE in the
regulation of MMPs in cancer cells have not been reported.
In this study, human gastric carcinoma cell line (MGC-
803) was used to investigate the effect of CADPE on
PMA-induced MMPs expression and the underlying mo-
lecular mechanism. We show that CADPE inhibits the
migration and invasion of gastric cancer cells by suppres-
sing MMP-9 expression and blocking the activation of
focal adhesion kinase (FAK)/mitogen-activated protein ki-
nase (MAPK)/ERK kinase (MEK)/ERK1/2 protein kinases
and AP-1 transcription factor.
Materials and Methods
Materials and cells
CADPE was synthesized by Dr. Xiaoyuan Lian (The
Institute of Biomedical Sciences, East China Normal
University, Shanghai, China; ref. 23). A 100 mmol/L
stock solution of CADPE was prepared in DMSO.
CA and PMA were purchased from Sigma-Aldrich.
Matrigel was purchased from BD Biosciences. Kinase
inhibitors PD98059, SB203580, SP600125, and
U0126 were purchased from Calbiochem. Antibodies
against MMP-9, MMP-2, c-Fos, c-Jun, p65, total and
phosphorylated FAK, MEK, ERK1/2, stress-activated
protein kinase/JNK, and p38 MAPK were from Cell
Signaling Technology; phospho-PKC antibody sample
kit (#9921) was obtained from Cell Signaling Tech-
nology; and antibody against β-actin was purchased from
Sigma-Aldrich.
Human gastric carcinoma cell lines MGC-803, HGC-27,
and AGS and human breast carcinoma cell line MDA-MD-
231 were obtained from the China Type Culture Collection
(Shanghai, China). MGC-803 and HGC-27 were cultured
in RPMI 1640 containing 10% fetal bovine serum. AGS
and MDA-MD-231 were cultured in DMEM and L-15
medium containing 10% fetal bovine serum, respectively.
Cell viability assay
For the cell viability assay, 2 × 10
4
MGC-803 cells per
well were treated with different concentrations of CADPE
for 24 hours. Cell viability was determined by the 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) method,
following the manual of CellTiter 96 Aqueous One Solu-
tion Cell Proliferation assay (Promega). Absorbance was
measured with a VERSAmax microplate reader.
Transwell migration assay
The Transwell migration assay was done according to a
previously reported method with some modifications (27).
Briefly, Transwell membrane (8-μm pore size, 6.5-mm
diameter; Corning Costar Corporation) was used. The bot-
tom chambers of the Transwell were filled with migration-
inducing medium (with 10% fetal bovine serum). The top
chambers were seeded with 2 × 10
4
MGC-803 cells per
well with different concentrations of CADPE (0, 5, 10,
and 20 μmol/L). After 8 to 10 hours, the filters were fixed
with 4% paraformaldehyde for 30 minutes at room tem-
perature; subsequently, the cells on the upper side of the
membrane were scraped with a cotton swab. Filters were
stained with hematoxylin for light microscopy. Images
were taken using an Olympus inverted microscope and
migratory cells were evaluated by manual counting. Per-
centage inhibition of migratory cells was quantified and
expressed based on untreated control wells.
Matrigel-based Transwell invasion assay
Cell invasion assays were carried out as previously re-
ported (27) with slight modifications. Transwell membrane
coated with Matrigel (100 μg/mL, 100 μL per well) was
used for invasion assay. Cells (5 × 10
4
) were seeded onto
the upper wells in the presence of different concentrations
ofCADPEorMMP-9inhibitor(28)withorwithout
PMA. The bottom chambers of the Transwell were filled
with condition medium. The inserts were incubated at
37°C for 24 hours. Cells that had invaded the lower surface
of the membrane were fixed, stained, and counted under a
light microscope. Percentage inhibition of invasive cells was
quantified and expressed based on untreated control wells.
Gelatin substrate gel zymography
Gelatin zymography was carried out as previous reported
(18). The MGC-803 cells were plated onto six-well plates at
Han et al.
Mol Cancer Res; 8(11) November 2010 Molecular Cancer Research1478
a density of 2 × 10
5
cells per well and incubated until they
reached 80% confluence; the medium then was changed to
fresh serum-free medium with or without CADPE or specif-
ic inhibitors of MAPKs (PD98059, U0126, SP600125, and
SB203580). After 2 hours of pretreatment, 100 nmol/L
PMA was added to the medium for 24 hours, and the super-
natant was collected and concentrated. The resultant super-
natant was subjected to SDS-PAGE in 8% polyacrylamide
gels that were copolymerized with 1 mg/mL gelatin. After
the electrophoresis runs, the gels were washed several times
with 2.5% Triton X-100 for 1 hour at room temperature to
remove the SDS and incubated for 12 hours at 37°C in a
buffer containing 5 mmol/L CaCl
2
and 1 μmol/L ZnCl
2
.
The gels were stained with Coomassie brilliant blue R250
(0.25%; Bio-Rad) for 1 hour and then destained for 1 hour
in a solution of acetic acid and methanol. Proteolytic activity
was evidenced as clear bands against the blue background of
the stained gelatin.
Western blot assay
For the Western blot assay, MGC-803 cells were treated
with different concentrations of CADPE in the presence of
100 nmol/L PMA for 24 hours. Cell lysates were prepared
in radioimmunoprecipitation assay buffer (20 mmol/L Tris,
2.5 mmol/L EDTA, 1% Triton X-100, 1% deoxycholate,
0.1% SDS, 40 mmol/L NaF, 10 mmol/L Na
4
P
2
O
7
,and
1 mmol/L phenylmethylsulfonyl fluoride). Aliquots of
cellular protein (40 μg/lane) were electrophoresed on
10% to 12% SDS-PAGE and transferred onto a polyviny-
lidene difluoride membrane (Millipore Corp.). The mem-
brane was blocked with 5% skim milk in PBS containing
0.1% Tween 20 and then reacted with specific antibodies.
Detection of specific proteins was carried out with an en-
hanced chemiluminescence Western blotting kit following
the manufacturer's instructions (Amersham-Pharmacia).
To examine the activation of transcription factors, nuclear
fractions were obtained from cells treated with PMA for
1 hour after 4 hours of pretreatment with CADPE. To
assess the changes in signaling pathway, starved MGC-803
cells were treated with CADPE for 2 hours and then stimu-
lated with 100 nmol/L PMA.
Reverse transcription-PCR
In the reverse transcription-PCR (RT-PCR) analysis, to-
tal RNA was extracted from the treated cells. For reverse
transcription reaction, cDNA was synthesized from 1 μg
of total RNA using Moloney murine leukemia virus reverse
transcriptase (Promega). The PCR primers used are as follows:
MMP-9 sense, 5′-TCCCTGGAGACCTGAGAACC-3′;
MMP-9 antisense, 5′-GGCAAGTCTTCCGAGTAGTTT-
3′;MMP-2 sense, 5′-GGATGATGCCTTTGCTCG-3′;
MMP-2 antisense, 5′-ATCGGCGTTCCCATACTT-3′;
MMP-7 sense, 5′-CTTCCTGTATGCTGCAACTC-3′,
MMP-7 antisense, 5′-GTGGAGGAACAGTGCTTATC-3′.
TIMP-1 sense, 5′-GGGGACACCAGAAGTCAACCAGA-
3′;TIMP-1 antisense, 5′-CTTTTCAGAGCCTTGGAG-
GAGCT-3′;TIMP-2 sense, 5′-TGCAGCTGCTCCCC-
GGTGCAC-3′;TIMP-1 antisense, 5′-TTATGGGTCCT‐
CGATGTCGAG-3′;β-actin sense, 5′-GCCATCGTCAC-
CAACTGGGAC-3′;andβ-actin antisense, 5′-CGATTT‐
CCCGCTCGGCCGTGG-3′. PCR products were analyzed
by agarose gel electrophoresis and visualized by treatment
with ethidium bromide.
Construction of human MMP-9 promoter
A 700-bp fragment at the 5′-flanking region of the hu-
man MMP-9 gene was amplified by PCR from human ge-
nomic DNA. Specific primers were designed to contain the
appropriate restriction enzyme site: sense 5′-CGG
GGTACCTGCTACTGTCCCCTTTACTG-3′(KpnI)
and antisense 5′-CCCAGATCTGTGAGGGCA-
GAGGTGTCT-3′(BglII). The amplified promoter DNA
was digested with KpnIandBglIIandthenclonedup-
stream of the luciferase gene in pGL3 plasmid. The
DNA sequence of the MMP-9 promoter was confirmed,
and the resultant reporter plasmid was named pGL3-
MMP-9-WT. The AP-1-1, AP-1-2, NF-κB, and SP-1 mu-
tants from pGL3-MMP-9-WT were generated using the
QuickChange Site-Directed Mutagenesis Kit (Stratagene);
all the mutants were confirmed by DNA sequencing.
Transient transfection and luciferase reporter
gene assays
MMP-9 wild-type (pGL3-MMP-9-WT), AP-1 site-
mutated (pGL3-MMP-9-Mut-AP-1-2), NF-κB site-mutated
(pGL3-MMP-9-Mut-NF-κB), and SP-1 site-mutated
MMP-9 luciferase promoter constructs (pGL3-MMP-
9-Mut-SP-1) were used in transient transfection assays.
MGC-803 cells were plated onto six-well plates at a density
of 2 × 10
5
cells per well and grown overnight. Cells were
cotransfected with 1 μg of MMP-9 promoter-luciferase
reporter constructs and 0.2 μg of the Renilla reporter plas-
mid for 6 hours using Lipofectamine reagent (Invitrogen)
according to the manufacturer's protocol. After transfec-
tion, the cells were cultured in condition medium with
PMA and incubated with different concentrations of
CADPE for 24 hours. Luciferase and Renilla activities
were determined by following the manufacturer's protocol
(Dual-Luciferase Reporter Assay System, Promega). Lucif-
erase activity was normalized with the Renilla activity in the
cell lysate and expressed as an average of three independent
experiments.
Electophoretic mobility shift assay
Cultured cells were collected by centrifugation, washed,
and suspended in buffer A [10 mmol/L HEPES (pH 7.9),
10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA,
1 mmol/L DTT, and 0.5 mmol/L phenylmethylsulfonyl
fluoride]. After 15 minutes on ice, the cells were vortexed
in the presence of 0.5% NP40. The nuclear pellet was then
collected by centrifugation and extracted with buffer B
[20 mmol/L HEPES (pH 7.9), 0.4 mol/L NaCl, 1 mmol/L
EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, and 1 mmol/L
PMSF] for 15 minutes at 4°C. Double-stranded oligonu-
cleotides containing the consensus sequences for AP-1 up-
stream (5′-GAGGAAGCTGAGTCAAAGAAGGC-3′),
CADPE Inhibits Gastric Carcinoma Invasion
Mol Cancer Res; 8(11) November 2010www.aacrjournals.org 1479
AP-1 proximal (5′-CTGACCCCTGAGTCAGCACTT-
GC-3′), and NF-κB(5′-CCCCAGTGGAATTCCCCA‐
GCCTTG-3′) were end- labeled with [γ-
32
P]ATP using
T4 polynucleotide kinase and used as probes for electro-
phoretic mobility shift assay (18). The nuclear extracts
(5 μg) were incubated at 4°C for 30 minutes in 25
mmol/L HEPES buffer (pH 7.9), 0.5 mmol/L EDTA,
0.5 mmol/L DTT, 0.05 mol/L NaCl, and 2.5% glycerol
with 1 μg of poly(deoxyinosinic-deoxycytidylic acid) and
0.5 pmol of labeled probe. The DNA-protein complex
FIGURE 1. Chemical structures of CADPE and CA and the effects of CADPE on the viability, migration, and invasion of MGC-803 cells. A, chemical
structures of CA and CADPE. B, effect of CADPE on cell viability. MGC-803 cells were treated with 5 to 20 μmol/L CADPE in serum or serum-free medium
for 24 h. Cell viability was determined by MTS assay. The percentage of cell viability was calculated as the ratio (A
570
) of treated cells to control cells.
Points, mean of three independent experiments; bars, SE. C, CADPE inhibits tumor cell migration. The Transwell migration assay was carried out in
MGC-803 cells. After 8 h of incubation with or without the indicated concentration of CADPE, cells that migrated to the low chamber were fixed, stained, and
counted by light microscopy as described in Materials and Methods (left). Random fields were scanned (four fields per filter of the well) for the
presence of cells on the lower side of the membrane (right image). Columns, mean number of migrated cells in three wells from three independent
experiments; bars, SE. *, P< 0.05. D, the effect of CADPE on cell invasion was determined using the Matrigel invasion assay system. For invasion assay, the
upper chamber was coated with Matrigel, and MGC-803 cells with various concentrations of CADPE in the presence of 100 nmol/L PMA were added.
After 24 h, cells on the bottom side of filter were fixed, stained, and counted under a light microscope (D, left images). The inhibitory effects of CADPEon
two additional gastric cancer cell lines, HGC-27 and AGS, were also examined (D, right columns). Columns, mean number of migrated cells in
three wells from three independent experiments; bars, SE. *, P< 0.05.
Han et al.
Mol Cancer Res; 8(11) November 2010 Molecular Cancer Research1480
was separated by electrophoresis at 4°C in 6% polyacryl-
amide gels in 0.5× Tris-borate EDTA buffer. For compe-
tition assay to confirm the binding specificity, nuclear
extracts were preincubated at 4°C for 30 minutes with
a 100-fold excess of an unlabeled oligonucleotide. Gels
were dried and imaged using the Personal Molecular Im-
ager system (Bio-Rad).
Statistical analysis
The results are presented as mean ± SE, and statistical
comparisons between groups were done using one-way
ANOVA followed by Student's ttest. P≤0.05 was consid-
ered statistically significant.
Results
Effects of CADPE on the viability and invasion of
gastric carcinoma cells
As a synthetic derivative of CA, CADPE preserved
the key structure of CA (Fig. 1A). To evaluate the
effect of CADPE on cell proliferation, we treated
MGC-803 cells with different concentrations of
CADPE or CA in serum-free and serum-containing me-
dium, respectively, for MTS assays. Only 9% and 15%
decreases in cell viability were found in cells treated
with 25 μmol/L CADPE in serum-containing and
serum-free medium (Fig. 1B). No significant cytotoxi-
city was found for CA at the concentration of
100 μmol/L (data not shown). To investigate the effects
of CADPE on tumor cell migration and invasion,
the Transwell migration assay and the Matrigel-based
Transwell invasion assay were done using MGC-803
cells at different concentrations of CADPE, ranging
from 5 to 20 μmol/L. As shown in Fig. 1C, CADPE
significantly inhibited MGC-803 cancer cell migration
(Fig. 1C). Moreover, CADPE dramatically inhibited
PMA-induced cell invasion in a dose-dependent
manner (Fig. 1D), suggesting that CADPE is an effec-
tive inhibitor of cancer cell migration, invasion,
and metastasis.
FIGURE 2. Inhibition of MMP-9 activity by
CADPE in tumor cells. A, CADPE inhibits
PMA-induced MMP-9 activity. MGC-803,
HGC-27, AGS, and MDA-MB-231 cells were
preincubated with varying concentrations of
CADPE for 2 h, followed by PMA stimulation for
24 h. Conditional media were collected and
MMP activity was analyzed by gelatin
zymography. B, CADPE inhibits EGF-induced
MMP-9 activity. MGC-803 cells were incubated
with varying concentrations of CADPE in the
presence of EGF for 24 h and gelatin
zymography was done. C, CA inhibits
PMA-induced MMP-9 activity. MGC-803 cells
were incubated with 25 to 100 μmol/L CA in the
presence of PMA, and MMP-9 enzyme activity
in the conditioned medium was analyzed by
zymography. D, MMP-9 and MMP-2 derived
from PMA-treated conditioned medium were
incubated with CADPE (5-20 μmol/L) for 30 min
and then subjected to gelatin zymography.
CADPE Inhibits Gastric Carcinoma Invasion
Mol Cancer Res; 8(11) November 2010www.aacrjournals.org 1481
CADPE inhibits MMP-9 expression and activity
MMP-9 and MMP-2 are important ECM-degrading
enzymes. It has been reported that both enzymes were in-
volved in cancer cell invasion and metastasis (14, 29). The
fact that CADPE inhibited cancer cell invasion prompted
us to examine the effect of CADPE on MMPs activity
using gelatin zymography. The secretion of MMP-9 in
the conditioned medium of MGC-803 was dramatically
induced by PMA (10-100 nmol/L) in a dose-dependent
manner, whereas no detectable change of MMP-2 (data
not shown) was found in the same condition. To examine
the effects of CADPE on MMP-9 expression in cancer
cells, we chose 100 nmol/L PMA to induce the activation
of MMP-9 in different cancer cells. As shown in Fig. 2A,
treatment of MGC-803 cells with CADPE (5-20 μmol/L)
suppressed PMA-induced MMP-9 activity in a dose-
dependent manner, whereas the activity of MMP-2 did
not significantly decrease. Similar results were obtained
in three other cancer cell lines, including two gastric can-
cer cell lines (AGS and HGC-27) and one breast cancer
cell line (MDA-MB-231; Fig. 2A), suggesting that
CADPE significantly inhibits the secretion of MMP-9 in
FIGURE 3. Inhibition of PMA-induced MMP-9
expression by CADPE. A, CADPE inhibits
the protein expression of MMP-9, but not
MMP-2. The expression levels of MMP-9 and
MMP-2 in MGC-803 cells treated with
CADPE in the presence of PMA for 24 h were
evaluated by Western blot analysis with MMP-9
and MMP-2 antibodies. Expression of β-actin
in cell lysates was used as a control. B, CADPE
inhibits MMP-9, but not MMP-2, mRNA level.
MGC-803 cells were treated with or without
CADPE (20 μmol/L) in the presence of PMA for
0, 3, 6, 12, 18, and 24 h, and mRNA levels of
MMP-9 and MMP-2 were examined. C, effects
of CADPE on the mRNA levels of MMPs and
their regulators. MGC-803 cells were incubated
with CADPE for 24 h. The mRNA expression
levels of MMP-9, MMP-2, MMP-3, MMP-7,
TIMP-1, and TIMP-2 were analyzed by RT-PCR;
β-actin expression was included as an internal
control. The expression levels of MMP-9 in
CADPE-treated or untreated MGC-803 cells
were determined by real-time PCR analysis.
D, MMP-9 inhibitor can block PMA-induced
MGC-803 cell invasion in the Matrigel
invasion assay.
Han et al.
Mol Cancer Res; 8(11) November 2010 Molecular Cancer Research1482
invasive cancer cell lines. Compared with CADPE, CA
also decreased MMP-9 activity in MGC-803 cells, but
at a much higher concentration (25-100 μmol/L). CA
had little effect on MMP-2 activity (Fig. 2C). Further-
more, we show that CADPE inhibited epidermal growth
factor (EGF)–induced MMP-9 expression and activity in
MGC-803 cells (Fig. 2B). To investigate whether CADPE
directly affects MMP-9 enzyme activity, conditioned me-
dium derived from PMA-treated MGC-803 was incubated
with different concentrations of CADPE in the gelatin zy-
mography assays. As shown in Fig. 2D, there was no vis-
ible difference between the CADPE- treated and untreated
groups (Fig. 2D), suggesting that CADPE has no direct
influence on MMP-9 activity. Taken together, our data
suggest that CADPE inhibits PMA- and EGF-induced
MMP-9 activation in different cancer cell lines.
CADPE suppresses MMP-9 transcription in a
dose-dependent manner
As shown in Fig. 3A, the expression levels of MMP-9
gradually decreased in a dose-dependent manner in
Western blot assay, indicating that CADPE inhibits
MMP-9 enzyme activity by reducing the protein level
of MMP-9 (Fig. 3A). Similar to prior observations,
FIGURE 4. Analysis of CADPE on MMP-9
promoter activity through AP-1 and NF-κB
binding sites. A, CADPE inhibits PMA-induced
MMP-9 luciferase activity. B and C, mutations
at NF-κB and SP-1 binding sites have little
influence on the inhibitory effects of CADPE in
MGC-803 cells. D, mutations at the two AP-1
binding sites of the MMP-9 promoter diminish
the inhibitory effects of CADPE, suggesting that
CADPE suppresses the expression of MMP-9
through AP-1 binding sites. E and F, CADPE
inhibits the AP-1-luciferase, but not the
NF-κB-luciferase, activity. MGC-803 cells were
transfected with reporter vectors containing
binding sites for AP-1 and NF-κB. The cells
were cultured in the presence of CADPE
(5-20 μmol/L) for 24 h, and luciferase activity
was measured. Columns, mean of at least three
independent experiments; bars, SE.
*, P< 0.05.
CADPE Inhibits Gastric Carcinoma Invasion
Mol Cancer Res; 8(11) November 2010www.aacrjournals.org 1483
CADPE had little effect on the protein expression of
MMP-2 (Fig. 3A). Furthermore, we performed RT-
PCR to determine the CADPE regulation of MMPs at
the mRNA level. In MGC-803 cells, the mRNA level of
MMP-9 was induced by PMA after 3 hours, peaked in
12 hours, and persisted for least 24 hours (Fig. 3B).
CADPE significantly inhibited PMA-induced MMP-9
mRNA expression in a time- and dose-dependent man-
ner (Fig. 3B and C). CADPE had no effect on the
mRNA level of MMP-2 (Fig. 3B and C). The inhibitory
effect of CADPE on MMP-9 mRNA expression was fur-
ther confirmed by real-time quantitative PCR (Fig. 3C).
Because the activity of MMP-9 is tightly regulated by
endogenous inhibitors, tissue inhibitors of metalloprotei-
nases (TIMP), we examined the expression levels of
TIMP-1 and TIMP-2 by RT-PCR. As shown in Fig. 3C,
TIMP-1, but not TIMP-2, can be slightly stimulated by
PMA. However, CADPE had no effect on the mRNA
levels of both TIMP-1 and TIMP-2 (Fig. 3C). Further-
more, we examined the expression level of MMP-7 in
CADPE-treated MGC-803 cells and showed that
MMP-7 remained essentially unchanged (Fig. 3C). These
results indicate that CADPE selectively suppressed MMP-
9 expression both at the protein and mRNA levels in a
time- and dose-dependent manner.
To understand the relationship of cell invasion and
MMP-9 in gastric cancer, we performed a Matrigel inva-
sion assay with MMP-9 inhibitor. Results showed that
MMP-9 inhibitor blocked MGC-803 cell invasion in a
dose-dependent manner, suggesting that MMP-9 was
largely responsible for the invasion of MGC-803 cells
(Fig. 3D). The inhibitory effect of MMP-9 inhibitor on
MGC-803 cell invasion was almost same as that of
CADPE (Supplementary Fig. S2B). The specificity of
MMP-9 inhibitor was tested by gelatin zymography assay
in different gastric carcinoma cell lines (Supplementary
Fig. S1). We also detected the effects of MMP-9 inhibitor
on the migration of gastric cancer cells. Results showed
that MMP-9 inhibitor had less inhibitory effects when
compared with CADPE (Supplementary Fig. S2A).
CADPE inhibits MMP-9 expression by suppressing
AP-1 binding and AP-1–dependent
transcription activity
To understand the molecular mechanism underlying
the inhibitory effects of CADPE on MMP-9 expression,
we find that there are two AP-1 binding sites (located at
−79 bp and −533 bp) and an NF-κB binding site (loca-
ted at −600 bp) in the MMP-9 promoter. It has been
shown that NF-κB and AP-1 play an important role in
controlling basal and cytokine-induced MMP-9 expres-
sion in various cancer cell lines (18). To determine the
effect of CADPE on the promoter activity of MMP-9,
luciferase-report gene that contains the MMP-9 promoter
region was transiently transfected into MGC-803 cells. As
shown in Fig. 4A, MMP-9-luciferase activity was activat-
ed up to ∼10-fold in cells treated with PMA. CADPE
inhibited the PMA-induced MMP-9-luciferase activity
in a dose-dependent manner (Fig. 4A), suggesting that
CADPE could inhibit MMP-9 expression at the tran-
scriptional level.
To determine which of these transcription factors may
participate in the regulation of MMP-9 transcription in
MGC-803 cells, we mutated the potential binding sites
for different transcription factors found in the MMP-9
promoter, including NF-κB, SP-1, and two AP-1 sites
(Fig. 4B-D). MGC-803 cells were transiently transfected
with MMP-9 reporter genes with mutations in different
transcription binding sites. Mutations at the NF-κB and
SP-1 binding sites have little effect on the inhibitory
effects of CADPE on PMA-induced MMP-9 activity
(Fig. 4B and C). However, mutations at the two AP-1
binding sites completely abolished the inhibitory effects
of CADPE on PMA-induced MMP-9 promoter activity
(Fig. 4D), suggesting that the regulation of CADPE on
the MMP-9 promoter region was facilitated by AP-1
transcription factor. To further confirm this observation,
the luciferase report vectors that contain tandem repeats
of the AP-1 or NF-κB binding sites were used to examine
the effects of CADPE in the luciferase assays. As shown
in Fig. 4E, luciferase activity in the cells transfected with
the AP-1 reporter was significantly reduced by treatment
with CADPE in the range of 5 to 20 μmol/L, whereas no
statistically significant changes were found in the cells
transfected with the NF-κB reporters in the presence of
CADPE (Fig. 4F). These results suggest that AP-1 tran-
scription factor and AP-1 binding sites in the MMP-9
promoter region contribute to the inhibition of PMA-
dependent MMP-9 activation by CADPE.
CADPE decreases transcription factor binding to AP-1
motifs in the MMP-9 promoter region
To determine whether CADPE inhibited the transcrip-
tional binding activity of AP-1 to its DNA motifs, we per-
formed electrophoretic gel mobility shift assay using the
consensus sequences of AP-1 or NF-κBasprobesin
CADPE-treated cells. MGC-803 cells were pretreated with
different concentrations of CADPE for 4 hours, followed
by treatment with 100 nmol/L PMA for 1 hour. Then, nu-
clear extracts were prepared and analyzed for AP-1 and
NF-κB DNA binding activities, respectively. As shown in
Fig. 5A, CADPE significantly decreased PMA-induced
AP-1 DNA binding ability, whereas CADPE has no effect
on PMA-induced NF-κB binding activity (Fig. 5B). These
data were consistent with the reporter gene analysis, sug-
gesting that CADPE blocks MMP-9 expression, at least
in part, by inhibiting the expression or DNA binding ac-
tivity of AP-1 transcription factor. To determine which
subunit of AP-1 transcritpion factor is regulated by
CADPE, we examined the expression levels of c-Fos and
c-Jun with CADPE treatment. Our data showed that
CADPE significantly reduced PMA-induced c-Fos expres-
sion but had little effect on the expression of c-Jun or p65
in Western blot assays (Fig. 5C), suggesting that CADPE
inhibits AP-1 transcription activity by suppressing the
expression of c-Fos in the gastric cancer cells.
Han et al.
Mol Cancer Res; 8(11) November 2010 Molecular Cancer Research1484
CADPE blocks PMA-induced activation of FAK, MEK,
and ERK1/2 in gastric cancer cells
Activation of one or more mitogen-activated protein
kinase (MAPK) pathways is important for the MMP-9 in-
duction by PMA in various cell types (30). As shown in
Fig. 6A, the phosphorylation of ERK, JNK, and p38 was
increased by the stimulation with PMA. Addition of
CADPE decreased only ERK phosphorylation in MGC-
803 cells (Fig. 6B). On the other hand, the levels of phos-
phorylated JNK and p38 had little change after CADPE
treatment (Fig. 6B). We further examined the phosphory-
lation of upstream regulators in the ERK signaling pathway
by Western blot with specific phosphorylation antibodies.
CADPE significantly inhibited MEK and FAK phosphor-
ylation induced by PMA treatment (Fig. 6B). As PKC is
also involved in MMP-9 expression, we examined the effects
of CADPE on PKC activation using specific phosphor-
antibodies for different PKC isoforms as described in
Materials and Methods. Our results showed that CADPE
has little effect on PKC activation (Fig. 6B), suggesting
that the effects of CADPE are mediated by pathways other
than PKC. To further confirm our conclusion, we measured
MMP-9 activity using specific protein kinase inhibitor as-
says. Overnight starved MGC-803 cells were pretreated
with PD98059, U0126, SP600125, and SB203580 (inhi-
bitors of MEK, ERK, JNK, and p38, respectively) for
2hoursandthenstimulatedwithPMAfor24hours;
conditioned media were collected for gelatin zymography
assay of MMP-9 activity. As shown in Fig. 6C, MEK
and ERK inhibitors can significantly suppress MMP-9
expression and activity, whereas JNK and p38 inhibitors
have little inhibitory activity on MMP-9. These results
suggest that CADPE inhibits the PMA-induced activation
of AP-1 by blocking FAK and MEK/ERK1/2 activation in
MGC-803 cells.
Discussion
Metastasis and invasion are major properties of various
malignant tumors that are associated with a poor progno-
sis. It was thought that MMP-9 participated in promoting
these processes. Recent studies show that MMP-9 has
statistically significantly different expression patterns
between well-differentiated and poorly differentiated tissue
samples and may play key roles during the development of
gastric cancer (31). In addition, MMP-9 also correlated
with the invasion, metastasis, and angiogenesis of gastric
cancer cells (32, 33). Therefore, development of various
compounds that can inhibit MMP-9 would be useful in
the treatment of gastric carcinoma.
FIGURE 5. Effects of CADPE on
the DNA binding activities and
expression of AP-1 and NF-κB.
A and B, CADPE suppresses
PMA-induced AP-1 DNA binding
activity but has little effect on
NF-κB activity. MGC-803 cells
were pretreated with CADPE for
4 h and then stimulated with PMA
for 1 h. Nuclear extract (5 μg)
prepared from these treated cells
was mixed with radioactive
oligonucleotides containing the
AP-1 (A) or NF-κB (B) motif of the
MMP-9 promoter. Bound
complexes were analyzed by
electrophoresis. C, effects of
CADPE on the expression levels of
AP-1 and NF-κB subunits.
MGC-803 cells were pretreated
with CADPE for 4 h followed by
PMA stimulation for 1 h. Western
blot analysis was done to
determine the nuclear levels of
AP-1 (c-Fos and c-Jun) and NF-κB
(p65) subunits using specific
antibodies for the proteins; β-actin
was used as an internal control.
CADPE Inhibits Gastric Carcinoma Invasion
Mol Cancer Res; 8(11) November 2010www.aacrjournals.org 1485
As a structure analogue of CADPE, CA has a wide spec-
trum of biological effects, including antitumor invasion by
targeting MMP-9 through the AP-1 and NF-κB signaling
pathways (18). The ability to inhibit tumor cell invasion
was also extended to other caffeic acid derivatives, such
as caffeic acid phenethyl ester, which was originally isolated
from honeybee propolis. However, the inhibitory role of
CADPE against MMP expression and the invasiveness of
gastric carcinoma has not been reported. In this study, we
show that CADPE is a potential anticancer agent due to its
ability to inhibit PMA-induced phosphorylation of protein
kinases (FAK, MEK, and ERK1/2), AP-1 nuclear translo-
cation, and MMP-9 expression in invasive tumor cells.
Gelatin zymography is a classic method to detect the ac-
tivity and expression of gelatinases A and B, and thus we
used this method to detect the secretion of MMP-9 and
MMP-2 in the condition medium. In this study, treatment
of MGC-803 cells with CADPE selectively suppressed the
PMA-induced activity of MMP-9, whereas the activity of
MMP-2 was not affected. A similar result was also ob-
served in two more gastric carcinoma cell lines, AGS and
HGC-27, and a breast cancer cell line, MDA-MB-231.
When compared with its precursor CA, CADPE showed
a better inhibitory effect on MMP-9 at a relatively lower
concentration, suggesting that CADPE inhibits tumor in-
vasion through MMP-9 in these invasive tumor cell lines.
The activity of MMPs is precisely regulated at three
levels: gene transcription, posttranscriptional activation
of zymogens, and endogenous expression of tissue inhi-
bitors of metalloproteinases (34). It was thought that the
key step in the regulation of MMPs was at the transcrip-
tion level. To determine which step was affected by
CADPE, gelatin zymography, RT-PCR, and Western blot
analysis were performed to show that CADPE inhibits
the expression of MMP-9 at both the mRNA and pro-
tein levels but has little effect on the enzymatic activity.
FIGURE 6. CADPE inhibits
the activation of FAK, MEK, and
ERKs. A, time-dependent
phosphorylation of ERK, JNK, and
p38 induced by PMA in MGC-803
cells. B, effects of CADPE on
the phosphorylation of ERK, JNK,
p38, MEK, FAK, and PKC.
MGC-803 cells were pretreated
with CADPE for 4 h and stimulated
with PMA for 15 min; the levels of
phosphorylated protein kinases,
including pERK, p-JNK, p-p38,
p-MEK, p-FAK, and p-PKC, were
determined by Western blotting
with phospho-specific antibodies.
C, inhibition of PMA-induced
MMP-9 activity by protein kinase
inhibitors for MEK and ERK.
MGC-803 cells were pretreated
with U0126 (MEK inhibitor; 10
μmol/L), SP600125 (JNK inhibitor;
10 μmol/L), PD98059 (ERK
inhibitor; 20 μmol/L), and
SB203580 (p38 inhibitor;
10 μmol/L) for 2 h and then treated
with PMA for 24 h; MMP-9
levels were tested by gelatin
zymography. D, possible molecular
mechanisms underlying the
antitumor activities of CADPE in
PMA-treated MGC-803 cells.
Han et al.
Mol Cancer Res; 8(11) November 2010 Molecular Cancer Research1486
MMPs activity is also regulated by tissue-specific inhibi-
tors (TIMPs), of which there are four identified members
(TIMP-1 to TIMP-4; ref. 8). These proteins bind MMPs
in a 1:1 stoichiometry and directly affect the level of
MMP activity. Because TIMP-1 is a major inhibitor of
MMP-9, TIMP-1 and TIMP-2 are differentially regulated
in vivo as well as in cultured cells (35, 36). We tested the
mRNA levels of these two proteins and ruled out the ef-
fects of CADPE on TIMP-1 and TIMP-2. Therefore, the
inhibitory effect of CADPE on MMP-9 activity was
mainly due to the transcriptional regulation and protein
expression of MMP-9, which was further confirmed by
MMP-9-luciferase report gene assay.
AP-1 and NF-κB are two key transcription factors in-
volved in the regulation of MMP-9 gene expression (19).
Activation of these transcription factors is centrally in-
volved in the process of tumor invasion and metastasis
by various agents including PMA, growth factors (e.g.,
EGF, vascular endothelial growth factor, platelet-derived
growth factor, and transforming growth factor-β;refs.
37-39), and inflammatory cytokines (e.g., CXCL12 and
tumor necrosis factor-α; refs. 40, 41). Thus, the regulation
of AP-1 and NF-κB downstream of the FAK, phosphoino-
sitide 3-kinase/Akt, and MAPK pathways might be in-
volved in PMA-induced MMP-9 expression and invasion
in MGC-803 cells. Luciferase reporter gene assay and mu-
tation analysis of the promoter revealed that the major tar-
get of CADPE in the MMP-9 promoter was AP-1, whereas
NF-κB had little effect on the transcriptional regulation of
MMP-9. AP-1 is composed of members of the c-Jun and
c-Fos families (21, 42, 43). They associate to form a variety
of homodimers and heterodimers to regulate gene expres-
sion and localize to the nucleus when AP-1 is activated.
Our results showed that CADPE suppressed nuclear
c-Fos in a dose-dependent manner, whereas c-Jun or p65
was not affected, suggesting that the suppression of AP-1 is
responsible for CADPE-induced inhibition of MMP-9 in-
duction and cell invasion. AP-1 activity, including tran-
scriptional activation, protein stability, and intracellular
localization, was modulated by protein kinases such as
MAPKs. It was reported that the intracellular localization,
protein stability, and chromatin association of c-Fos family
proteins were mainly regulated by ERK phosphorylation
(44, 45). In our experiments, CADPE specifically sup-
pressed PMA-mediated MEK and ERK phosphorylation
without affecting pathways involving p38 and JNK. Acti-
vation of PKC was also little affected by CADPE, suggest-
ing that the effects of CADPE are mediated by pathways
other than PKC. Furthermore, CADPE also inhibits
PMA-induced phosphorylation of FAK. FAK plays a
critical role in contact formation between ECM and
cytoskeleton and has been linked with cancer cell migra-
tion, invasion, proliferation, and survival (46). Therefore,
suppression of FAK may partially explain the inhibition of
cell migration in our assays.
In summary, our studies show that CADPE inhibits tu-
mor cell migration and invasion by suppressing MMP-9
expression by blocking the phosphorylation of protein
kinases (FAK, MEK, and ERK) and the activation of
AP-1 transcriptional factor. Therefore, CADPE is a poten-
tial agent for clinical use in preventing the invasion and
metastasis of human malignant tumors, such as gastric cancer.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant Support
Research Platform for Cell Signaling Networks (06DZ22923), Pujiang Program
(09PJ1403900), Key Science and Technology Projects (074319104 and
09JC1405200) from the Science and Technology Commission of Shanghai Muni-
cipality, “Chen Guang”Project (2008CG27) supported by Shanghai Municipal
Education Commission and Shanghai Education Development Foundation.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 03/22/2010; revised 09/03/2010; accepted 09/05/2010; published
OnlineFirst 10/06/2010.
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