Curcumin inhibits TGF-??1-induced MMP-9 and invasion through ERK and Smad signaling in breast cancer MDA- MB-231 cells

Abstract

Objective:
To evaluate the effects of curcumin on matrixmetalloproteinase-9 (MMP-9) and invasion ability induced by transforming growth factor-β1 (TGF-β1) in MDA-MB-231 cells and potential mechanisms.

Methods:
Human breast cancer MDA- MB-231 cells were used with the CCK-8 assay to measure the cytotoxicity of curcumin. After treatment with 10 ng/ml TGF-β1, with or without curcumin (≤10 μM), cell invasion was checked by transwell chamber. The effects of curcumin on TGF-β1-stimulated MMP-9 and phosphorylation of Smad2, extracellular-regulated kinase (ERK), and p38 mitogen activated protein kinases (p38MAPK) were examined by Western blotting. Supernatant liquid were collected to analyze the activity of MMP-9 via zymography. Following treatment with PD98059, a specific inhibitor of ERK, and SB203580, a specific inhibitor of p38MAPK, Western blotting and zymography were employed to examine MMP-9 expression and activity, respectively.

Results:
Low dose curcumin (≤10 μM) did not show any obvious toxicity to the cells, while 0~10 μmol/L caused a concentration-dependent reduction in cell invasion provoked by TGF-β1. Curcumin also markedly inhibited TGF-β1-regulated MMP-9 and activation of Smad2, ERK1/2 and p38 in a dose- and time-dependent manner. Additionally, PD98059, but not SB203580, showed a similar pattern of inhibition of MMP-9 expression.

Conclusion:
Curcumin inhibited TGF-β1-stimulated MMP-9 and the invasive phenotype in MDA-MB-231 cells, possibly associated with TGF-β/Smad and TGF-β/ERK signaling.

Full text

Asian Pacic Journal of Cancer Prevention, Vol 13, 2012 5709
DOI:http://dx.doi.org/10.7314/APJCP.2012.13.11.5709
Curcumin Inhibits TGF-β1-Induced MMP -9 and Invasion MDA-MB-231 Cells through ERK, Smad Signaling
Asian Pacic J Cancer Prev, 13 (11), 5709-5714
Introduction
Breast cancer is one of most common malignant tumor
in global female. And the leading cause of morbidity and
mortality in breast cancer patients is cancer metastases, but
not the primary tumor (Porter, 2009; Sanchez-Zamorano
et al., 2011). It has been demonstrated that tumor cell
invasion is the key event in the metastatic steps (Geiger
et al., 2009). However, recent studies show that cancer
progression toward metastasis is not entirely dependent on
tumor cells themselves, but the result of the interactions
of tumor cells and the primary tumor microenvironment
(Allen et al., 2011). Cancer cells, specially those at the
edges of the tumor, can attract and stimulate host cells
to secret many kinds of factors (TGF-β1, IL-1, TNF-α,
etc.) in the tumor microenvironment. Those factors can
generate a series of invasion-related biological effects
through the tumor cell signal transduction pathways, and
then cause the cells to separate from the primary lesion
focus and result in local invasion and distant mestastasis
nally (Allen et al., 2011). So far, novel ndings indicates
that TGF-β1 is one of the factors which could be highly
1Department of Pathology, Chongqing Medical University, Chongqing 2Department of Anesthesiology, Peking University Third
Hospital (PUTH), Beijing, China *For correspondence: cydcyj@163.com
Abstract
Objective: To evaluate the effects of curcumin on matrixmetalloproteinase-9 (MMP-9) and invasion ability
induced by transforming growth factor-β1 (TGF-β1) in MDA-MB-231 cells and potential mechanisms. Methods:
Human breast cancer MDA- MB-231 cells were used with the CCK-8 assay to measure the cytotoxicity of
curcumin. After treatment with 10 ng/ml TGF-β1, with or without curcumin (≤10 μM), cell invasion was checked
by transwell chamber. The effects of curcumin on TGF-β1-stimulated MMP-9 and phosphorylation of Smad2,
extracellular-regulated kinase (ERK), and p38 mitogen activated protein kinases (p38MAPK) were examined by
Western blotting. Supernatant liquid were collected to analyze the activity of MMP-9 via zymography. Following
treatment with PD98059, a specic inhibitor of ERK, and SB203580, a specic inhibitor of p38MAPK, Western
blotting and zymography were employed to examine MMP-9 expression and activity, respectively. Results: Low
dose curcumin (≤10 μM) did not show any obvious toxicity to the cells, while 0~10 μmol/L caused a concentration–
dependent reduction in cell invasion provoked by TGF-β1. Curcumin also markedly inhibited TGF-β1-regulated
MMP-9 and activation of Smad2, ERK1/2 and p38 in a dose- and time-dependent manner. Additionally, PD98059,
but not SB203580, showed a similar pattern of inhibition of MMP-9 expression. Conclusion: Curcumin inhibited
TGF-β1-stimulated MMP-9 and the invasive phenotype in MDA-MB-231 cells, possibly associated with TGF-β/
Smad and TGF-β /ERK signaling.
Keywords: Curcumin - breast cancer - TGF-β1 - MMP-9 - MAPKs - invasion
RESEARCH ARTICLE
Curcumin Inhibits TGF-β1-Induced MMP-9 and Invasion
through ERK and Smad Signaling in Breast Cancer MDA-
MB-231 Cells
Na Mo1, Zheng-Qian Li2, Jing Li1, You-De Cao1*
relevant to breast cancer invasion and metastasis (Perera
et al., 2010).
TGF-β1 is known to be one of the TGF-β superfamily
members that plays a dual role in breast cancer generation
and progression. In the early stages, TGF-β1 acts as
a tumor suppressor, nevertheless, it promotes cancer
invasion and metastasis in later stages (Biswas et al.,
2007). After binding to TGF-β1, the type II TGF-β1
receptor recruits and phosphorylates the type I receptor.
The activated type I receptor subsequently initiates the
activation of Receptor-associated Smads (R-Smads), then
the latter forms heteromeric complexes with common
mediator Smad4 (co-Smad4) and translocate into the
nucleus. The Smad complexes regulate specic target
genes by directly or indirectly combining and interacting
with transcription factors, co-repressors, and co-activators
(Smith et al., 2012). In addition to the classical TGF-β/
Smad signaling pathway, TGF-β1 can also directly
activate non-Smad signaling pathways (Zhang, 2009).
Previous reports have shown the direct function of MAPK
pathways, including c-Jun N-terminal kinase (JNK),
extracellular- regulated kinase ERK, and p38 MAPK, in

Na Mo et al
Asian Pacic Journal of Cancer Prevention, Vol 13, 2012
5710
signal transduction of TGF-β1-regulated cell migration
and invasion (Sana et al., 2007).
Researchers have demonstrated that the serum TGF-β1
level in early-stage breast cancer patients is uplifted
and the high level of TGF-β1 has positive correlation
with the effects of anti-tumor. However, it accelerates
cancer invasion and formation of metastases in late-stage
breast cancer (Cheung, 2007). Serra R et al. have found
that radiation and chemotherapy can quicken tumor
cells diffusion in mouse models of breast cancer. The
experimental datas indicate that the levels of TGF-β1
in those mouse models have been rised remarkably
. In contrast, there is no far metastasis in the mouse
models with low level of TGF-β1 (Serra et al., 2005).
Therefore, antagonism of TGF-β1 signaling may provide
a therapeutic target for late-stage breast cancer, blocking
metastasis without detrimental side effects.
Curcumin is a natural phenolic pigment extracted from
the roots of turmeric. Numerous studies have reported that
it has positive pharmacological effects, such as anti-tumor,
anti-oxidation, anti-inammation, anti-rheumatism and so
on (Yan et al., 2012). The U.S. National Cancer Institute
has listed it as the third-generation anti-cancer drug to
study, so that curcumin will likely to be a promising
clinical anti-cancer drug (Park et al., 2008). Nevertheless,
the precise molecular mechanisms underlying its anti-
tumor invasion and metastasis are not entirely clear (Park
et al., 2008). Most of the reports pay more attention to the
anti-brosis activity of curcumin on TGF-β1-stimulated
organs (Tubulointerstitial, corneal, liver, etc.) brosis via
blocking TGF-β signing pathway (Smith et al., 2010; Yao
et al., 2012; Zhang et al., 2012). Kim et al. have conrmed
that curcumin inhibites TGF-β1-induced MMPs in mouse
keratinocytes (Santibáñez et al., 2000; Santibáñez et al.,
2002), but the effects on breast cancer MDA-MB -231
have not been reported.
Therefore, the present study investigated the effect
of curcumin on the exogenous TGF-β1–stimulated
expression and activity of MMP-9 in human breast
cancer MDA-MB-231 cells. Furthermore, the underlying
mechanisms were also probed.
Materials and Methods
Cell culture
MDA-MB-231 cells were incubated with RPMI-1640
medium (Gibco, California, USA) containing 10% fetal
calf serum at 37°C in 5% CO2 incubator. Cells were
cultured in serum-free media for 24h to synchronize
cell growth before the experiments, then the media
were exchanged for fresh serum-free medium, treated
with various agents at the concentrations specied, and
cells were harvested at different time points for various
analyses.
Cytotoxicity assay
MDA-MB-231 cells were trypsinized and seeded in
96-well plates at 5 × 103 cells/well. After 24 h, escalating
doses of curcumin (Sigma, USA) were added, and
incubated for another 24 h, 48h, and 72h respectively.
Cells without any treatment were used as control. Then,
10 μL CCK-8 (Dojindo, Tokyo, Japan) solution in culture
medium was added to each well. Plates were incubated
for an additional 2 h. The optical density of each well was
measured using microplate absorbance reader at a 450
nm wavelength. Cell viability was calculated as follows.
The cell survival rate (%) = [(A Treatment group- A blank
wells) / (A negative control group-A blank wells)] × 100%.
Invasion assay
The MDA-MB-231 cells invasion behavior with
or without indicated treatment was tested by Matrigel
transwell system as described previously (Ye et al., 2012) .
After cultured in 6 well plates for 48h, the cells in different
experimental groups were trypsinized, centrifuged, and
resuspended at 1×106 cells/mL in serum-free medium
respectively. 100μl cell suspension per well were seeded
onto the upper wells of transwells (8-µm-diameter pores;
Millipore), which precoated with Matrigel (0.5 mg/Ml,
BD Biosciences Discovery Labware). Lower chamber of
the transwells contained the medium containing 10% FBS
as chemoattractant. After 8 h of incubation, the cells on
the upper chamber were carefully wiped with the cotton
swab. The wells were washed 3 times with PBS, then
xed with 4% paraformaldehyde, and stained with crystal
violet solution (Sigma Chemical, USA). The cells on the
lower surface of the membrane were counted under a light
microscope (magnication, ×100). The experiments were
performed three times, each time in triplicate.
Gelatin zymography
Cells in the logarithmic phase were seeded in 6-well
plate at the desity of 3 × 105 cells per well. After incubated
in serum-free medium with or without curcumin (2.5, 7.5
and 10 μM) and 10 μM TGF-β1 treatment for 48h. The
supernatants were collected, and gelatin zymography
assay was performed as described formerly (Zayani et al.
2012). After electrophoresis, the gels were washed three
times with renaturing buffer containing 50 mM Tris–HCl,
5 mM CaCl2 and 2.5% Triton X-100 (v/v), pH 7.5 for
30 min, followed by a brief rinsing with washing buffer
(50 mM Tris–HCl , 5 mM CaCl2), pH 7.5. Then the gels
were incubated at 37°C for 42 h in developing buffer
containing 50 mM Tris–HCl, 5 mM CaCl2, 0.2 M NaCl,
and 0.02% Brij 35, pH 7.5. The gels were subsequently
stained with 0.25% Coomassie Brilliant Blue (G250)
followed by destaining with a solution containing 10%
acetic acid and 20% methanol. Enzyme-digested regions
were visualized as light bands against a dark background.
Zones of enzymatic activity were regarded as negatively
stained bands.
Western blotting
Cells in experimental groups were collected and
lysed in RIPA buffer respectively. Supernatants of the
cell lysates were used in the western blot analysis for
MMP-9 (Bioss, Beijing, China), β-actin (Beyotime,
Beijing, China), and phosphorylation levels of ERK1/2,
Smad2, p38MAPK (Cell signing technology, USA). After
electrophoresis and trarsmembrane, the PVDF membranes
(Millipore, USA)containing the proteins were blocked
with 5% bovine serum albumin in TBST buffer (0.01%

Asian Pacic Journal of Cancer Prevention, Vol 13, 2012 5711
DOI:http://dx.doi.org/10.7314/APJCP.2012.13.11.5709
Curcumin Inhibits TGF-β1-Induced MMP -9 and Invasion MDA-MB-231 Cells through ERK, Smad Signaling
Figure 1. The Cell Toxicity of MDA-MB-231 Cells
Treated by Curcumin. * P<0.05 vs. 0 μM Cur (24h), ﹟P<0.05
vs. 0 μM Cur (48h), ﹠P<0.05 vs. 0 μM Cur (72h)
Figure 2. Effect of Curcumin on TGF-β1-induced
Invasion Ability of MDA-MB-231 Cells in vitro (crystal
violet ×100) (a) MDA-MB-231 cells were incubated with
different doses of Cur ,with or without TGF-β (10 ng/ml)
for 48h, then checked invasion ability by Transwell assay. (b)
Quantitation of the cells which invasive matrigel to lower surface
of the membrane by cell counting under microscope (×100)
(mean±SEM from 3 independent tests).*P<0.05 vs. control,
**P<0.05 vs. TGF-β1 alone
Figure 3. Effect of Curcumin on MMP-9 Protein
Expression Activated by TGF-β1 (a, b) Cell were incubated
with different doses of curcumin and with or without TGF-β1
for 48h , and the MMP-9 Protein expression was checked by
Western blotting . (mean ± SEM from 3 separate tests) *P<0.05
vs. control, **P<0.05 vs. TGF-β1 alone. (c, d) MDA-MB-231
cells were treated with 10μM Cur, followed by TGF-β1 for
different times, and the MMP-9 Protein expression was analyzed
by Western blotting (mean ± SEM from 3 separate tests) *P<0.05
vs. control, **P<0.05 vs. TGF-β1 alone
a b
c d
Tween 20 in TBS) at room temperature(RT) for about 2h,
following by incubated with the primary antibody (i.e.,
specic antibodies against the target proteins described
above) in TBST at 4℃ overnight. The membranes were
washed three times in TBST and subsequently covered
with anti-rabbit HRP-conjugated goat anti-rabbit IgG
(Bioss, Beijing, China) or goat anti-mouse IgG (Beyotime,
Beijing, China) at RT for 2h in TBST buffer. After a
2h incubation, all blots were washed three times and
ECL reagents (Beyotime, Beijing, China) were used for
development.
Statistical analysis
Data are presented as means ± SEM and analyzed for
statistical signicance using independent samples T-test.
All Statistical analysis was performed with SPSS for
Windows (version 14.0; SPSS, Chicago, IL). Statistical
signicance was considered as P <0.05.
Results
The cytotoxicity effect of curcumin on the growth of MDA-
MB-231 cells
To evaluate cytotoxicity of curcumin on the growth
of breast cancer cells, MDA- MB-231 cells were treated
with 5, 10, 15, 20, 30 and 50 μM curcumin for 24h, 48h,
72h respectively and then cell viability was detected by
CCK-8 assay. As shown in Figure1, low-dose curcumin
(≤10 μM) did not affect the viability of MDA-MB-231
cells, and survival rates of all the low-dose groups had
exceeded 90% (except 10 μM curcumin for 72 h). But
when the concentrations was above 10μM, curcumin
time- and dose- dependently inhibited the growth of
MDA-MB-231 cells. Therefore, the cells were treated
with selected doses (≤ 10μM) for no more than 48 hours
in subsequent experiments.
Effects of curcumin on TGF-β1-induced invasiveness of
MDA -MB -231 cells
We next examined the effect of curcumin on TGF-β1-
induced cell invasion in MDA-MB-231 breast cancer cells
using the transwell chamber assay. Our results showed that
the invasiveness of MDA-MB-231 cells was increased by
TGF-β1 treatment (Figure 2). On the other hand, TGF-
β1-stimulated invasiveness of cells was decreased by
curcumin in a dose-dependent manner (Figure 2). These
results were inconsistent with the results of the wound
healing assay (data did not shown). This suggests that
curcumin can prevent the TGF-β1-induced invasion in
MDA-MB-231 cells.
Effects of curcumin on TGF-β1-Mediated MMP-9 protein
expression and activity in MDA -MB -231 cells
We examined whether curcumin involved with the
TGF-β1-induced MMP-9 protein expression. After
pretreatment with different concentrations of curcumin
for 30 min, the cells were cultured with TGF-β1
and various doses of curcumin for 48h. Western blot
analyses revealed that TGF-β1-induced MMP-9 protein
expression was signicantly decreased by curcumin in
a dose-dependent manner. The level of MMP-9 protein
expressions was increased to 1.93-fold of the control
level by 10 nM TGF-β1 treatment, while the TGF-β1-
induced MMP-9 protein expressions was decreased to
81.5%, 70.4%, 55.0% of the 10 nM TGF-β1 group level
by 5, 7.5 and 10μM curcumin treatment, respectively
(Figure 3A, 3B). So, 10 μM curcumin has the maximal
inhibitory effect. After pretreatment with 10μM curcumin

Na Mo et al
Asian Pacic Journal of Cancer Prevention, Vol 13, 2012
5712
for 30 min, the cells were treated with 10 nM TGF-β1
and 10μM curcumin for 12h, 24h, 48h, respectively. Our
results showed that the TGF-β1-induced MMP-9 protein
expressions were evidently suppressed by curcumin in
a time-dependent way. The TGF-β1-induced MMP-9
protein expressions was decreased by 77.5%, 60.4%,
58.4% of the 10 nM TGF-β1 group level in 12h, 24h,
48h respectively. Therefore, curcumin had the best
inhibitory effect at 48h. As exhibited in Figure 3 C and
D, treatment with 10 nM TGF-β1 for 24h led to increase
enzymatic activity of MMP-9. In contrast, curcumin dose-
dependently inhibited this effect, with 10 μM curcumin
showing optimum inhibitory effect.
Effect of TGF-β1 on phosphorlation of Smad2, ERK and
p38MAPK in breast cancer MDA-MB-231 cells
As shown in Figure 4, 10μM TGF-β1 stimulated
phosphorylation of Smad2, ERK and p38MAPK as early
as 15 minutes while p-Smad2 peaked at 30 minutes. And
the p-ERK and p-p38MAPK reached maximum at 60 min.
The levels of total Smad2, ERK and p38 did not altered.
Effect of curcumin on activation of Smad and MAPK
Pathway by TGF-β1
Cells were treated with 10μM curcumin for 0, 6,
12, and 24 h, with or without inducing of TGF-β1 for
an additional 15 min. Western blot analyses indicated
that TGF-β1 markedly increased activation of Smad2,
ERK1/2, and p38MAPK at 12h and 24h, but did not
increase levels of total Smad2, ERK1/2, p38MAPK.
Curcumin at 10μM exhibited signicantly inhibitive effect
on TGF-β1-induced phosphorylation of Smad2, ERK1/2,
p38MAPK at different intervals, and the 12 h treated group
was more effective. Compared with the same time point
of TGF-β1 alone treatment group, the difference was
signicant (P <0.001) as shown in Figure 5 A and B.
Next, cells were incubated with different concentrations
(0, 5, 7.5, 10 μM) of curcumin for 12 h, followed by
treatment of TGF-β1 for an additional 15 min. The results
revealed that TGF-β1 at 10μM dependently induced
p-Smad2, p-ERK1/2 and p-p38MAPK when compared
with constant levels of total Smad2, ERK1/2, p38 MAPK.
Curcumin significantly inhibited TGF-β1-induced
phosphorlation of Smad2, ERK1/2 and P38MAPK in
MDA-MB-231 cells at protein levels,and the inhibition
effect was characterized by a concentration-dependent
way, and the dose of 10 μM was regarded as the best
inhibition effect (Figure 5 C, D)
Effect of PD98059, SB202580, curcumin on MMP-9
enzymatic activity and expression by TGF-β1
Cells were treated with curcumin (10μM) , PD98059
(30μM) , SB202580 (20μM) for 48 h, combination with
or without treatment of TGF-β1 (10 ng/ml). The inhibitory
effect of PD98059 on MMP-9 was similar to curcumin,
whereas SB202580 group shown no signicant change
when compared with the TGF-β1 alone group (Figure 6).
Discussion
About one-third of women with breast cancer develop
distant metastasis and ultimately die worldwide each year
(Allen et al., 2011; Nasser et al., 2012). Thus, metastatic
breast cancer has been thought to be the principal
challenge for the effective treatment and prevention
Figure 4. Effects of TGF-β1 on p-smad2, p-ERK1/2,
p-p38 Expression in Different Time Periods. MDA-
MB-231 cells were treated with TGF-β1 (10 ng/ml) for the
indicated times, and the phosphorylation of Smad2< ERK1/2 and
p38 was analyzed by Western blotting. The same membrane was
retested with anti-Smad2<ERK1/2 and p38 antibody
Figure 5. Effects of Curcumin on p-smad2, p-ERK1/2,
p-p38 Expression Activated by TGF-β1. (a) (b) MDA-
MB-231 cells were treated with 10μM Cur for different times,
with or without TGF-β1 for an additional 15min, and the
phosphorylation of Smad2, ERK1/2, p-p38 was analyzed by
Western blotting (mean ± SEM from 3 separate tests) *P<0.05 vs.
TGF-β1 alone. (c) (d) Cells were incubated with different doses
of Cur for 12h,and with or without TGF-β1 for an additional
15min , and the phosphorylation of Smad2, ERK1/2, p-p38 was
checked by Western blotting . (mean ± SEM from 3 independent
tests) *P<0.05 vs. control, **P<0.05 vs. TGF-β1 alone
a b
c d
Figure 6. Effects of PD98059, SB202580, and Curcumin
on TGF-β1-induced MMP-9 in MDA-MB-231 Cells.
The MMP-9 expression and enzymatic activity were analyzed by
Western blot and zymography, respectively (*P<0.05 vs. control
﹟P<0.05 vs. TGF-β1 alone)

Asian Pacic Journal of Cancer Prevention, Vol 13, 2012 5713
DOI:http://dx.doi.org/10.7314/APJCP.2012.13.11.5709
Curcumin Inhibits TGF-β1-Induced MMP -9 and Invasion MDA-MB-231 Cells through ERK, Smad Signaling
(Hortobagyi et al., 2002). Extensive evidence now
indicates that degradation of extracelluar matrix assisting
cancer cell to invade neighbouring tissue, blood vessels
and spread to other sites is the essential process of distant
metastases in invasive breast cancer (Hassan et al., 2012).
Reportedly, MMP-9 plays a signicant role in breast
cancer invasion and metastasis via degrading type IV
collagen-rich extracelluar matrix (Chen et al., 2011) , and
also be regulated by TGF-β1, epidermal growth factor
(EGF), broblast growth factor (FGF), Nerve growth
factor (NGF), vascular endothelial growth factor (VEGF),
etc., which secreted by cancer cells and/or host cells in
the tumor microenvironment (Chou et al., 2006; Belotti
et al., 2008; Blair et al., 2011). Among those factors,
TGF-β1 generated by autocrine and paracrine is high
related to malignant tumor (Na et al., 2010). And TGF-β
signing pathway is of great importance of breast cancer
invasivness and metastasis (Imamura et al., 2012). Based
on these reports, compounds that can suppress TGF-β1-
induced MMP-9 are applied for the treatment of metastatic
breast cancer. Many researchers focus mostly on the anti-
brotic effects of curcumin via inhibiting TGF-β signing
pathway (Smith et al., 2010; Yao et al., 2012; Zhang et al.,
2012). However, whether curcumin can suppress TGF-β1-
stimulated MMP-9 expression was not clear.
In accordance with previous reports (Yodkeeree et al.,
2010), invasion assay in our study shown that curcumin at
a nontoxic concentration makedly decreased invasiveness
in response to TGF-β1 in a dose-dependent manner. Our
results demonstrated that the levels of MMP-9 protein
expression and enzymatic activity were signicantly
increased stimulated by TGF-β1 in the MDA-MB-231
breast cancer cells, and this increase can be inhibited
obviously by curcumin. So, we came to the conclusion
that TGF-β1-stimulated MMP-9 expression and activity
might mediate cell invasion during wound healing and
curcumin inhibited this process in MDA-MB-231 cells.
In classical TGF-β/Smad pathway, TGF-β1 signal
through TGF-β/receptor serine- threonine kinases. The
activated receptor complex phosphorylated receptor-
regulated Smads (R-Smads), the latter forms heteromeric
complexes with Smad4 that translocate into the nucleus
and regulate transcription of specic target genes (Zhang,
2009). Besides TGF-β/Smad pathway, TGF-β1 can also
directly activate non-Smad signaling pathways, including
the MAPKs (JNK, ERK, p38) (Smith et al., 2012). In
other words, different transcriptional responses to TGF-β1
depend on activation of either or both Smad and Smad-
independent pathways. So far, several study revealed
that TGF-β1 might regulate MMP-9 expression through
rapidly activating intracellular Smads and MAPKs signing
pathway (Chou et al., 2006). However, the previous
study have shown that TGF-β/MAPKs pathway which
participate in regulating expression and activity of
MMP-9 has specicity in defferent types of cells (Kim et
al., 2005; Dziembowska et al., 2007; Szuster-Ciesielska
et al., 2011). For example, Hsieh et al. have found that
TGF-β1 induced MMP-9 expression is associated with
the ERK signing pathway in gastric cancer cells (Hsieh
et al., 2010). Nevertheless, this process is inconclusive
in breast cancer MDA-MB-231 cells. Some researchers
have also used kinase inhibitors to implicate both p38 and
ERK signing pathways in TGF-β1-mediated regulation
of MMP-9 (Iiunga et al., 2004). However, other results
have revealed that p38 inhibitor in minimal effective
dose has no effects on TGF-β1-induced MMP-9, while
excess p38 inhibitors do. The subsequent research have
found that excess p38 inhibitors not only decreased
phosphorylation of p38, but also can effectively block the
activation of TGF-β receptors kinases (Sana et al., 2007).
Our results indicated that non-toxic dose of curcumin
(≤10μM) markedly inhibited phosphorylation of Smad2,
ERK1/2, p38 mediated by TGF-β1 in a dose- and time-
dependent way. Furthermore, PD98059 and curcumin had
the similar inhibitory effects on TGF-β1-induced MMP-9.
Nevertheless, the lowest effect concentration of SB203580
did not affect regulation of MMP-9. These results were
consistent with the results have reported by Sana A
et al.. Accordingly, our ndings argued that curcumin
perhaps down-regulated TGF-β-induced MMP-9 via a
mechanism involving ERK, Smad2 but not p38MAPK
in MDA-MB-231 cells.
Moreover, previous studys have revealed that the
p38 pathway might enhance cell metastasis ability by
regulating actin remodeling factor HSP27 (Hedges et
al., 1999) and affecting actin polymerization and cell
contractility (Srinivasan et al. 2008). ERK may regulate
cell motility by preventing formation of extensive actin
stress bers via suppression of tropomyosin induction
by TGF-β1 or through inhibition of RhoA-Rho kinase
pathway (Bakin et al., 2004; Helfman et al., 2005). So,
perhaps activation of ERK, Smads and p38 MAPK signing
pathways is all required for the suppression of curcumin
on TGF-β1-mediated cell migration. This speculative
conclusion will also be our future research directions.
Our data revealed that the change of ERK1/2 and
Smad2 phosphorylation was the key point of the anti-
invasion effect of curcumin. Although our results also
found that curcumin was able to reduce TGF-β RII
expression in MDA-MB-231 cells (data does not show).
However, as the lack of specic protein phosphatase
inhibitors, we cannot exclude the possibility that
curcumin inhibits TGF-β1-stimulated MMP-9 via directly
suppressing activiation of the TGF-β receptors which acts
as an upstream regulator of ERK and Smad pathway .
In conclusion: In the present study, we explored
the inhibition action of curcumin on TGF-β1–induced
invasion in human breast cancer MDA-MB-231 cells and
disclosed the potential mechanisms of the anti–invasion
and metastasis effect. We demonstrated that (1) Curcumin
dose-dependently inhibited the invasion ablity induced by
TGF-β1 in MDA-MB-231 cells. (2) Curcumin inhibited
TGF-β1-induced MMP-9 protein expression and activity
in MDA-MB-231 cells in a time- and concentration- way.
(3) Curcumin time- and dose-dependently inhibited
Smad2, ERK1/2, p38MAPK phosphorylation induced
by TGF-β1. (4) PD98059 and curcumin had the similar
suppression effect on TGF-β1-induced MMP-9 protein
expression and activity. All together, these findings
highlight the protable effect of curcumin, and it serves
as an anti-MMP-9 factor through inhibition of the TGF-β/
Smad and TGF-β/Erk signaling pathway.

Na Mo et al
Asian Pacic Journal of Cancer Prevention, Vol 13, 2012
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