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INTRODUCTION |
Nuclear factor
B
(NF
B)1 is a family of
transcription factor that have been shown to be involved in gene
regulation of cellular processes like inflammation, immune response,
cell proliferation, and apoptosis (1, 2). This DNA-binding protein
binds to the
B sequence (3). It promotes transcription of varieties of cytokines such as IL-1, IL-2, IL-6, IL-8, TNF-
and cell
adhesion molecules such as E-selectin, ICAM-1, and VCAM-1 (4-6).
NF
B forms various homodimers and heterodimers mainly between p65
(Rel A) and p50 proteins. The N-terminal sequences of both p50 and p65
are homologous (7, 8). This family of proteins is particularly interesting due to its implication for therapies of diseases like cancer and AIDS.
In most cells, NF
B is present as a latent inactive, I
B bound
complex in the cytoplasm, but upon activation by extracellular stimuli
or by other factors, NF
B rapidly translocates to nucleus and
activates gene expression (9-11). The exact molecular mechanism by
which various extracellular stimuli lead to the activation of NF
B is
not well understood. However, most signals induce the activity of a
large multi subunit protein kinase, called I
B kinase (IKK). Active
IKK phosphorylates I
B
(12, 13), which targets I
B
for
ubiquitination and degradation by proteases (14, 15). The free NF
B
can then translocate into the nucleus. Aberrant NF
B activity has
been reported in several cancers including breast, colon, prostate, and
lymphoid cancers (16-19). NF
B-inducible genes play important role
in various disorders especially in cancers. NF
B induces
anti-apoptotic genes and protects cancer cells from apoptosis
contributing to tumor growth (19, 20). They also regulate expression
and activation of MMPs, which play significant role in ECM degradation
and facilitate cell motility, tumor growth and metastasis (21, 22).
Therefore, compounds that block NF
B activity can be used as a means
for inhibiting tumor growth or sensitizing cells to more conventional
therapies such as chemotherapy.
OPN is a member of the extracellular matrix protein. It is a
non-collagenous, sialic acid-rich, and glycosylated phosphoprotein (23,
24). It has an N-terminal signal sequence, a highly acidic region
consisting of nine consecutive aspartic acid residues, and a GRGDS cell
adhesion sequence predicted to be flanked by the
-sheet structure
(25). This protein has a functional thrombin cleavage site and is a
substrate for tissue transglutaminase (24). OPN binds with type I
collagen (26), fibronectin (27), and osteocalcin (28). Several highly
metastatic transformed cells synthesize higher level of OPN than the
nontumorigenic cells (29). It has been shown that OPN also interacts
with CD44 receptorglobulin (30). OPN causes cell adhesion, cell
migration, ECM invasion, and cell proliferation by interacting with its
receptor
v
3 integrin in various cell
types (31). OPN induces pro-MMP-2 activation and NF
B-mediated
signaling pathways by binding to its receptor
v
3 integrin (21, 32). All these above
effects contribute to the tumor growth and progression. However, the
molecular mechanism by which OPN activates the NF
B and regulates
MMP-2 activity in melanoma cells is not well understood. We have also
investigated whether we could reverse these above effects by blocking
the NF
B activation pathways.
MMPs are ECM degrading enzymes that play critical role in
embryogenesis, tissue remodeling, inflammation, and angiogenesis (33).
MMP-2 (also called type IV collagenase or gelatinase A) degrades
several ECM proteins such as fibronectin, laminin, type I collagen, and
proteoglycans (34). MMPs and tissue inhibitor of matrix
metalloproteinase (TIMP) play major role in regulation of cancer cell
migration, ECM invasion, and metastasis (35, 36). Earlier reports have
indicated that the increased levels of MMP-2 correlate with the
invasive properties of several tumor cells (34, 37). The TIMP-2 is the
specific inhibitor of MMP-2. TIMP-2 is a non-glycosylated protein (21 kDa) that forms a complex with both the inactive and active form of the
MMP-2 (38). Several inhibitors of MMPs (e.g. TIMPs) are
under clinical trial as therapy for cancer; however, there is an urgent
need to identify active compound from natural sources that can inhibit
pro-MMP-2 activation.
Malignant melanoma is the seventh leading cancer in the United States
and around the world. The epidemiological data suggested that dietary
modification might reduce this disease by as much as 85%. Curcumin
(diferuloylmethane) is a major component of turmeric (Curcuma
longa). This compound has been traditionally used to treat various
inflammatory disorders (39, 40). Several reports have indicated the
anti-inflammatory and anticarcinogenic properties of curcumin (41-43).
It has also been shown that curcumin inhibits type 1 human
immunodeficiency virus long terminal repeat-directed gene
expression and virus replication induced by TNF-
and phorbol myristate acetate (PMA) (44) which require NF
B activation. There
have been reports that curcumin can inhibit NF
B activation induced
by various agents (45). The molecular mechanisms by which curcumin
suppressed these effects are not well understood.
In this paper, we have shown that OPN induces I
B
phosphorylation
and degradation by activating IKK in B16F10 cells. The translocation of
p65 subunit of NF
B into the nucleus by OPN is shown by
immunofluorescence in a time-dependent manner and also by
Western blot analysis. Reporter gene assay indicated that OPN induces
NF
B transcriptional activity and EMSA data showed that OPN enhances
NF
B-DNA binding activity. Supershift assay using p65 or p50 antibody
showed the shift of the NF
B-specific band toward higher molecular
weight. Curcumin suppressed the OPN-induced IKK kinase activity,
I
B
phosphorylation, p65 nuclear translocation, NF
B-DNA
binding, and transactivation. OPN induced pro-MMP-2 activation; MT1-MMP
expression, cell migration, and ECM-invasion were also blocked by
curcumin. Curcumin also inhibited the OPN-induced tumor growth and
lowered the MMP-2 levels in the OPN-induced tumor of nude mice. Taken
together, these data demonstrate that OPN induces the NF
B-mediated
pro-MMP-2 activation through IKK-regulated phosphorylation of I
B
and further demonstrate that curcumin inhibits OPN-induced cell
migration, tumor growth, and NF
B-mediated MMP-2 activation by
inhibiting signal leading to IKK activity.
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EXPERIMENTAL PROCEDURES |
Materials--
Rabbit polyclonal anti-NF
B p65, anti-p50,
anti-NF
B X TransCruz, anti-IKK
/
, anti-I
B
, mouse
monoclonal anti-I
B
and goat polyclonal anti-actin antibodies,
NF
B consensus oligonucleotide, and I
B
recombinant protein were
purchased from Santa Cruz Biotechnology. The rabbit polyclonal
phospho-specific anti-I
B
, mouse monoclonal anti-MT1-MMP,
anti-MMP-2 antibodies, and normal rabbit IgG were from Oncogene
Research. The phosphoserine detection kit was purchased from
Calbiochem. The FITC-conjugated goat anti-rabbit IgG was obtained from
Pharmingen. The dual luciferase reporter assay system was purchased
from Promega. Curcumin (diferulolylmethane) was from Sigma. The
[
-32P]ATP was purchased from Board of Radiation and
Isotope Technology (Hyderabad, India). Boyden-type cell migration
chambers were obtained from Corning and BioCoat MatrigelTM
invasion chambers were from Collaborative Biomedical. The human OPN was
purified from human milk as described previously (21) and used
throughout these studies. The nude mice (NMRI, nu/nu) were obtained
from the National Institute of Virology (Pune, India). All other
chemicals were analytical grade.
Cell Culture--
The B16F10 cells were obtained from American
Type Culture Collection (Manassas, VA). These cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine in a humidified atmosphere of 5%
CO2 and 95% air at 37 °C.
Immunofluorescence Study--
The B16F10 cells were grown in
monolayer on glass slides and then treated with purified human OPN (5 µM) at 37 °C for a period of 0-6 h. In separate
experiments, the cells were pretreated with curcumin (50 µM) for 45 min and then treated with OPN (5 µM) for 3 h at 37 °C in serum-free Dulbecco's
modified Eagle's medium. The curcumin was dissolved in ethanol to a
stock solution of 20 mM and used for the treatment. The
cells were fixed in ice-cold methanol for 10 min, blocked with 5%
bovine serum albumin in phosphate-buffered saline (pH 7.4) for 30 min,
and washed with phosphate-buffered saline (pH 7.4). The fixed cells
were incubated with rabbit polyclonal anti-p65 antibody (1:100
dilution) for 1 h at room temperature. The cells were washed and
incubated with FITC-conjugated anti-rabbit IgG (1:100 dilution) for
1 h at room temperature. The cells were washed, mounted with cover
slips, and analyzed under confocal microscopy (Ziess).
Nuclear and Cytoplasmic Extracts and Western Blot--
The cells
were either treated with OPN (5 µM) for 3 h or with
curcumin (50 µM) for 45 min followed by OPN (5 µM) for 3 h at 37 °C. The nuclear extracts were
prepared as described (21). Briefly, the cells were scraped, washed
with phosphate-buffered saline (pH 7.4) and resuspended in hypotonic
buffer (10 mM Hepes (pH 7.9), 1.5 mM
MgCl2, 10 mM KCl, 0.2 mM
phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol),
and allowed to swell on ice for 10 min. Cells were homogenized in a
Dounce homogenizer. The nuclei were separated by spinning at 3300 × g for 5 min at 4 °C. The supernatant was used as
cytoplasmic extract. The nuclear pellet was extracted in nuclear
extraction buffer (20 mM Hepes (pH 7.9), 0.4 M
NaCl, 1.5 mM MgCl2, 0.2 mM EDTA,
25% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and
0.5 mM DTT) for 30 min on ice and centrifuged at
12,000 × g for 30 min. The supernatant was used as
nuclear extract. The protein concentrations in the supernatants of both
nuclear, and cytoplasmic extracts were measured by the Bio-Rad protein
assay. The nuclear and cytoplasmic extracts (30 µg) were resolved by
SDS-PAGE and then electrotransferred to the nitrocellulose membrane.
The membranes were incubated with anti-p65 antibody, washed, incubated
further with horseradish peroxidase-conjugated anti-rabbit IgG (1:2000
dilution), and detected using an ECL detection system (Amersham
Biosciences) as described previously (21).
EMSA--
EMSA was performed as described previously (45). The
nuclear extracts were prepared either by treating the cells with 5 µM OPN alone for 3 h or with curcumin (0-100
µM) for 45 min and then with 5 µM OPN for
3 h. The nuclear extracts (10 µg) were incubated with 16 fmol of
32P-labeled double-stranded NF-
B oligonucleotide (5'-AGT
TGA GGG GAC TTT CCC AGG C-3') in binding buffer (25 mM
Hepes (pH 7.9), 0.5 mM EDTA, 0.5 mM DTT, 1%
Nonidet P-40, 5% glycerol, and 50 mM NaCl) containing 2 µg of polydeoxyinosinic deoxycytidylic acid (poly(dI-dC)). The
DNA-protein complex was resolved on a native polyacrylamide gel and
analyzed by autoradiography. For supershift assay, the nuclear extracts
from OPN-treated cells were incubated with anti-p65 or anti-p50
antibody for 30 min at room temperature and analyzed by EMSA. As
controls, the nuclear extracts were also treated with normal rabbit
IgG. In separate experiments, the nuclear extracts were preincubated
with 100-fold excess of unlabeled NF
B oligonucleotide for 15 min
prior to the addition of labeled probe and the samples were further analyzed.
NF
B Luciferase Reporter Gene Assay--
The semiconfluent
cells grown in 24-well plates were transiently transfected with a
luciferase reporter construct (pNF
B-Luc) containing five tandem
repeats of the NF
B-binding site (a generous gift from Dr. Rainer de
Martin, University of Vienna, Vienna, Austria) using LipofectAMINE Plus
reagent (Invitrogen). The transfection efficiency was normalized by
cotransfecting the cells with pRL vector (Promega) containing a
full-length Renilla luciferase gene under the control of a
constitutive promoter. After 24 h of transfection, the cells were
treated with varying doses of OPN (0-10 µM) for 6 h
or with curcumin (0-100 µM) for 45 min and then with 5 µM OPN for 6 h. The cells were also treated with PMA
(50 ng/ml) at 37 °C for 6 h as control. Cells were harvested in
passive lysis buffer (Promega). The luciferase activities were measured
by luminometer (Lab Systems) using the dual luciferase assay system
according to the manufacturer's instructions (Promega). Changes in
luciferase activity with respect to the control were calculated.
IKK Assay--
The IKK activity was measured as described
previously (16). The semiconfluent cells were either treated with 5 µM OPN alone for 10 min or with curcumin (50 µM) for 45 min and then treated with 5 µM
OPN for 10 min at 37 °C. The cells were scraped, washed and lysed in
cold kinase assay lysis buffer (20 mM Tris-HCl (pH 8.0),
500 mM NaCl, 1 mM EDTA, 1 mM EGTA,
10 mM
-glycerophosphate, 10 mM NaF, 10 mM pNPP, 300 µM
Na3VO4, 1 mM benzamidine, 2 µM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM DTT, and
0.25% Nonidet P-40). The supernatant was obtained by centrifugation at
12,000 × g for 10 min at 4 °C. Protein
concentrations were measured using Bio-Rad protein assay. The cell
lysates (300 µg) were immunoprecipitated with anti-IKK
/
antibody in immunoprecipitation buffer (40 mM Tris-HCl (pH
8.0), 500 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM
-glycerophosphate, 10 mM NaF, 10 mM pNPP, 300 µM Na3VO4, 1 mM benzamidine, 2 µM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM DTT, and 0.1%
Nonidet P-40). Half of the immunoprecipitated samples were incubated
with recombinant I
B
(4 µg) in kinase buffer (20 mM
Hepes, (pH 7.7), 2 mM MgCl2, 10 µM ATP, 3 µCi of [
-32p]ATP, 10 mM
-glycerophosphate, 10 mM NaF, 10 mM pNPP, 300 µM Na3VO4, 1 mM benzamidine, 2 µM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM DTT) at
30 °C for 1 h. The kinase reaction was stopped by addition of
SDS-sample buffer. The sample was resolved by SDS-PAGE, dried, and
autoradiographed. The remaining half of the immunoprecipitated samples
were subjected to SDS-PAGE and analyzed by Western blot analysis using
anti-IKK
/
antibody. A fraction of equal volume of samples from
the kinase reaction mixture were resolved by SDS-PAGE and analyzed by
Western blot analysis using anti-I
B
antibody.
Western Blot Analysis--
For I
B
phosphorylation studies,
the cells were either treated with 5 µM OPN for 0-3 h or
with 50 µM curcumin for 45 min and then with 5 µM OPN for 0-2 h. The cells were lysed in lysis buffer
(50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1%
Nonidet P-40, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mM iodoacetamide, and 2 mM phenylmethylsulfonyl
fluoride), and the protein concentrations in cleared supernatants were
measured using Bio-Rad protein assay. The supernatant (lysates)
containing equal amount of total proteins (50 µg) were resolved by
SDS-PAGE and electrotransferred from gel to nitrocellulose membranes.
The membranes were incubated with rabbit anti-phospho-I
B
antibody
(1:500 dilution) and further incubated with horseradish
peroxidase-conjugated anti rabbit IgG and detected by ECL detection
system (Amersham Biosciences) according to the manufacturer's
instruction. The same blots were reprobed with rabbit
anti-nonphospho-I
B
(1:500 dilution) or anti-actin (1:1000
dilution) antibody and detected by ECL detection system as described
above. In other experiments, the OPN-treated cell lysates were
immunoprecipitated with anti-I
B
antibody, resolved by SDS-PAGE,
and analyzed by using phosphoserine detection system.
In separate experiments, the cells were either treated with 5 µM OPN for 12 h or curcumin (0-100
µM) for 45 min and then with 5 µM OPN for
12 h, and the cell lysates were analyzed by SDS-PAGE followed by
Western blot analysis using mouse monoclonal anti-MT1-MMP (1:1000
dilution) antibody. As control, the expression of actin was also
detected by reprobing the blot with anti-actin antibody.
Cell Migration and ECM Invasion Assay--
The migration assay
was conducted using transwell cell culture chamber according to the
standard procedure as described (21, 31). Briefly, the confluent
monolayer of B16F10 cells were harvested with trypsin-EDTA and
centrifuged at 800 × g for 10 min. The cell suspension
(5 × 105 cells/well) was treated in absence or
presence of varying concentrations of curcumin (0-100
µM) for 30 min at 37 °C and added to the upper chamber
of the prehydrated polycarbonate membrane filter. The lower chamber was
filled with fibroblast conditioned medium, which acted as
chemoattractant. Purified OPN (5 µM) was added to the upper chamber. The cells were incubated in a humidified incubator with
5% CO2 and 95% air at 37 °C for 16 h. The
non-migrating cells on the upper side of the filter were scraped and
washed. The migrating cells on the reverse side of the filter were
stained with Giemsa. The migrating cells on the filter were counted and
a photomicrograph was taken under an Olympus inverted microscope.
The ECM invasion assay was performed using
MatrigelTM-coated invasion chamber as described (21, 31).
The cell suspension (5 × 105 cells/well) were
pretreated in absence or presence of varying concentrations of curcumin
(0-100 µM) for 30 min, then treated with OPN (5 µM) and added to the upper chamber. The lower chamber was
filled with fibroblast-conditioned medium that acted as a chemoattractant. The cells were incubated at 37 °C for 16 h.
The non-migrating cells and MatrigelTM from the upper side
of the filter were scraped and removed using a moist cotton swab. The
invaded cells in the lower side of the filter were stained with Giemsa
and washed with phosphate-buffered saline (pH 7.6). The invaded cells
were then counted, and photomicrographs were taken under the inverted
microscope. In both these cases, the experiments were repeated in
triplicate. Preimmune IgG served as nonspecific control.
Zymography Experiments--
The gelatinolytic activity was
measured as described previously (21). To check the effect of curcumin
on OPN induced MMP-2 expression and activation, the cells were
pretreated with curcumin (0-100 µM) in serum-free medium
for 45 min and then incubated with OPN (5 µM) for 12 h at 37 °C. The conditioned medium was collected by centrifugation,
concentrated, and dialyzed. Protein concentrations were measured using
Bio-Rad protein assay. The samples containing equal amount of total
proteins were mixed with sample buffer in absence of reducing agent,
incubated at room temperature for 30 min, and loaded onto
zymography-SDS-PAGE containing gelatin (0.5 mg/ml) as described
previously (21, 46). The gels were washed and incubated in incubation
buffer (50 mM Tris-HCl (pH 7.5) containing 100 mM CaCl2, 1 µM ZnCl2,
1% (v/v) Triton-X100, and 0.02% (w/v) NaN3) for 16 h. The gels were stained with Coomassie Blue and destained. Negative
staining showed the zones of gelatinolytic activity.
Cell Viability Assay--
The effect of curcumin on OPN-induced
cell growth was assessed by MTT assay. Briefly, the cells (3 × 103) grown in 96-well plates were treated with varying
concentrations of curcumin (0-100 µM) for 45 min
followed by treatment with or without OPN (5 µM) for
12 h at 37 °C. The cells were further incubated with MTT (0.5 mg/ml) at 37 °C for 3 h followed by addition of 200 µl of
isopropanol. The color intensity was measured at 570 nm using an
enzyme-linked immunosorbent assay reader (Dynatech). The experiments
were performed in triplicate. The cell viability was plotted as percent
of control.
Propidium Iodide (PI) Staining--
The cells grown on sterile
glass coverslips were pretreated with 50 µM curcumin for
45 min and then treated in absence or presence of OPN (5 µM) at 37 °C for 6 h. The cells were washed with
phosphate-buffered saline (pH 7.4) and fixed in 1:1
acetone:methanol for 10 min. The cells on the cover slips were dried
and treated with PI (50 µg/ml) solution containing RNase A (20 µg/ml) for 20 min. The cells were washed again, mounted on slides,
and visualized under fluorescence confocal microscopy (Ziess).
In Vivo Tumorigenicity Experiments--
The tumorigenicity
experiments were performed as described previously (21, 47). The cells
were treated in absence or presence of purified OPN (10 µM) in serum-free medium at 37 °C for 16 h. After
that, the cells (1 × 106 cells/0.2 ml) were detached,
centrifuged, washed, and injected subcutaneously into the flanks of
male athymic NMRI (nu/nu) mice (6-8 weeks old). In separate
experiments, the cells were pretreated with various doses of curcumin
(0-100 µM) for 45 min and then treated with 10 µM OPN for 16 h and injected into the nude mice. Four mice were used in each set of experiments. The mice were kept
under specific pathogen-free conditions. OPN (10 µM)
alone or mixture of curcumin (0-100 µM) and OPN (10 µM) was again injected into the tumor sites twice a week
for up to 4 weeks. After 4 weeks, the mice were killed, and the tumor
weights were measured. The tumor tissues were homogenized; lysed in
lysis buffer composed of 50 mM Tris-HCl (pH 7.5) containing
150 mM NaCl, 1% Nonidet P-40, 15 µg/ml leupeptin, and
0.5 mM phenylmethylsulfonyl fluoride; and centrifuged at
12,000 × g for 10 min. The clear supernatants were
collected, and the levels of pro- and active MMP-2 were detected by
Western blot analysis. Briefly, the sample containing equal amount of
total proteins was resolved by SDS gel and analyzed by Western blot
analysis using anti-MMP-2 antibody. The levels of pro- and active MMP-2
in tumor samples were also detected by zymography as described
previously (21).
 |
RESULTS |
OPN Induces Translocation of p65 subunit of NF
B
into the Nucleus, and Curcumin Suppresses This
Translocation--
To check the effect of OPN on translocation of p65
into the nucleus in a time dependent manner, the cells were treated
with 5 µM OPN in basal medium for 0-6 h at
37 °C. The cells were fixed, incubated with rabbit anti-p65
antibody, incubated further with FITC-conjugated anti-rabbit IgG, and
analyzed under confocal microscopy. Fig.
1A showed that OPN induces
translocation of p65 into nucleus in a
time-dependent manner (panels a-f). In
OPN-treated cells, the majority of p65 staining resided in the
cytoplasm upto 10 min (panel b). At 30 min (panel
c) and 60 min (panel d) little nuclear translocation of
p65 were observed. However, the complete nuclear accumulation of p65
was noticed at 3 h (panel e) and continued upto 6 h (panel f). To examine whether curcumin inhibits the
OPN-induced p65 translocation, the cells were pretreated with curcumin
for 45 min and then treated with OPN for 3 h. The data indicated
that curcumin inhibited OPN-induced nuclear translocation, because most
of the p65 was localized in the cytoplasm (Fig. 1B,
panel c). In absence of OPN, majority of p65 resided in the
cytoplasm (panel a) and in presence of OPN, the p65 was
translocated into the nucleus (panel b). To further prove
whether OPN induces p65 translocation and curcumin suppresses
OPN-induced translocation at the protein level, both the nuclear and
cytoplasmic fractions were prepared from the untreated and treated
cells. The levels of p65 in these fractions were analyzed by Western
blot analysis using anti-p65 antibody (Fig. 1C). In the
OPN-untreated cells, the p65 was localized mostly in the cytoplasm
(lane 1) compared with the nucleus (lane 2),
whereas in the OPN-treated cells, it was translocated from the
cytoplasm (lane 3) to the nucleus (lane 4). The
p65 was localized in the cytoplasm (lane 5) compared with the nucleus (lane 6) when the cells were pretreated with
curcumin and then treated with OPN. The Western blot data were
quantified densitometrically (Kodak Digital Science) and analyzed
statistically (Fig. 1C, lower panel) using
Student's t test (p < 0.005). These data
are corroborated by the immunofluorescence data.

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Fig. 1.
Effects of OPN and curcumin on
cellular localization of p65 subunit of NF B by
immunofluorescence (A and B) and
Western blot analysis (C) in B16F10 cells.
A, cells grown on glass slides were treated with 5 µM OPN for 0-6 h. The cells were fixed, incubated with
anti-p65 antibody, followed by further incubation with FITC-conjugated
anti-rabbit IgG, and analyzed under confocal microscopy. p65 is
localized in the cytoplasm in OPN-treated cells at 0 min (panel
a) and at 10 min (panel b). Little nuclear accumulation
of p65 was observed at 30 min (panel c) and at 60 min
(panel d) in the OPN-treated cells. However, complete
nuclear accumulation of p65 was noticed at 3 h (panel
e) and continued up to 6 h (panel f).
B, cells were treated with 5 µM OPN for 3 h or pretreated with 50 µM curcumin for 45 min followed
by treatment with OPN for 3 h. The cells were
immune-stained as described above and analyzed under confocal
microscopy. In the untreated cells, the majority of p65 was detected in
the cytoplasm (panel a), but in the OPN-treated cells, p65
was translocated from the cytoplasm to the nucleus (panel
b). In contrast, upon treatment of cells with curcumin and then
with OPN, the majority of p65 staining was detected in the cytoplasm
(panel c). In Fig. 1, both A and B,
these experiments were performed in triplicates. C, nuclear
and cytoplasmic extracts from untreated and treated cells were
immunoblotted with rabbit polyclonal anti-p65 antibody. In the
untreated cells, p65 was detected in the cytoplasm (lane 1)
but not in the nucleus (lane 2). In the OPN-treated cells,
p65 translocated from cytoplasm (lane 3) into the nucleus
(lane 4). The p65 remained in the cytoplasm (lane
5) and did not translocate to nucleus (lane 6) when the
cells were pretreated with curcumin prior to the incubation with OPN.
The levels of p65 were quantified by densitometric analysis and
analyzed statistically using Student's t test (*,
p < 0.005). The data are represented in the form of a
bar graph (lower panel), and the mean value of
triplicate experiments is indicated.
|
|
OPN Induces NF
B-DNA Binding and NF
B Activation, and Curcumin
Blocks These Effects--
The cells were either treated with 5 µM OPN alone for 3 h or pretreated with different
concentrations of curcumin (0-100 µM) for 45 min followed by treatment with OPN (5 µM) for 3 h.
The nuclear extracts were prepared and used for EMSA using
32P-labeled NF
B oligonucleotides. The results in Fig.
2A showed that OPN induced
NF
B-DNA binding (lane 2) compared with untreated cells
(lane 1). Curcumin (50 and 100 µM) suppressed
the OPN-induced NF
B-DNA-binding in a dose-dependent manner
(lanes 3 and 4, respectively). The
NF
B-specific bands were quantified densitometrically, and the -fold
changes are calculated. The data indicated that there are a 4.5-fold
increase of DNA binding in OPN-treated cells compared with untreated
cells.

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Fig. 2.
OPN induces NF B-DNA
binding (A and B) and
NF B transactivation (C), and
these effects are suppressed by curcumin. A,
electrophoretic mobility shift assay. The cells were stimulated with 5 µM OPN for 3 h or pretreated with curcumin (0-100
µM) for 45 min and then stimulated with 5 µM OPN for 3 h. Nuclear extracts were prepared and
analyzed by EMSA as described under "Experimental Procedures." The
arrow indicates the NF B-specific band. OPN treatment
(lane 2) induces NF B binding compared with the untreated
cells (lane 1). Curcumin inhibits OPN-induced NF B-DNA
binding in a dose-dependent manner with 50 µM
(lane 3) and 100 µM (lane 4)
concentrations. The bands were analyzed densitometrically, and -fold
changes are indicated. B, supershift assay. The nuclear
extracts from OPN-treated cells were incubated with anti-p65 or
anti-p50 or both antibodies and analyzed by EMSA. Lane 1,
without nuclear extract; lane 2, with nuclear extract;
lane 3, with p65 antibody; lane 4, with p50
antibody; lane 5, with p65 and p50 antibodies; lane
6, with rabbit IgG; and lane 7, with excess unlabeled
probe. The upper arrow indicates the supershifted bands. The
results shown in Fig. 2, A and B, represent three
experiments exhibiting similar effects. C, luciferase
reporter gene assay. The cells were transiently transfected with
luciferase reporter construct (pNF B-Luc) with LipofectAMINE Plus.
Transfected cells were either stimulated with PMA (50 ng/ml) or
different doses of OPN (0-10 µM) for 6 h or with
various doses of curcumin (0-100 µM) for 45 min and then
treated with OPN (5 µM) for 6 h. The cell lysates
was used to measure the luciferase activity. The values were normalized
to Renilla luciferase activity. The -fold changes were
calculated, and mean ± S.E. of triplicate determinations are
plotted. The values were also analyzed by Student's t test
(*, p < 0.001).
|
|
To show that the band (Fig. 2A, lane 2) obtained
by EMSA in OPN-treated cells is indeed NF
B, the nuclear extracts
were incubated with anti-p65 or anti-p50 antibody or in combination and
then analyzed by EMSA. Fig. 2B showed the shift of NF
B
specific band to higher molecular weight when the nuclear extracts were
treated with anti-p65 (lane 3), anti-p50 (lane
4), or in both antibodies (lane 5), suggesting that the
OPN-activated complex consisted of p65 and p50 subunits. Normal rabbit
IgG, as control, had no effect on NF
B mobility (lane 6).
Specificity of binding was also confirmed by incubating the nuclear
extract with 100-fold excess of unlabeled oligonucleotide, and the data
showed that there is complete displacement of the NF
B-specific band
(lane 7). As expected, no band was obtained when EMSA was
performed without nuclear extract (lane 1).
The induction of NF
B transcriptional activity by OPN was also
monitored by luciferase reporter gene assay. The cells were transiently
transfected with NF
B luciferase reporter construct (pNF
B-Luc) in
presence of LipofectAMINE Plus. Transfected cells were either
stimulated with PMA (50 ng/ml), which served as a positive control, or
with increasing concentrations of OPN (0-10 µM). In
separate experiments, cells were pretreated with different doses of
curcumin (0-100 µM) and then treated with OPN (5 µM). The cell lysates were used to measure luciferase
activity. The data demonstrated that OPN stimulates the NF
B
transcriptional activity, and curcumin inhibits the OPN-induced NF
B
activity in a dose-dependent manner (Fig. 2C).
PMA, as a positive control, induces the NF
B activity in these cells.
The values were normalized to Renilla luciferase activity. The
-fold changes were calculated, and mean ± S.E. of triplicate
determinations are plotted. The values were also analyzed by Student's
t test (p < 0.001).
OPN Stimulates I
B
Phosphorylation by Inducing IKK Activity,
and Curcumin Inhibits OPN-induced I
B
Phosphorylation and IKK
Activity--
Since we have shown earlier that OPN-induced NF
B
activity is suppressed by the super-repressor form of I
B
(21), we
sought to determine whether OPN-induced NF
B activation is occurred
through phosphorylation/degradation of I
B
. Accordingly, cells
were treated with OPN (5 µM) for 0-180 min and lysed.
The lysates containing an equal amount of total proteins were resolved
by SDS-PAGE, and phosphorylated I
B
was detected by
Western blot analysis using anti-phospho-I
B
antibody. The data
demonstrated that OPN induces I
B
phosphorylation in 10 min, and
the levels of phospho-I
B
disappeared in 60 min and reappeared in
120 min (Fig. 3A, upper panel a). The blot was reprobed with anti-I
B
antibody, and
the data indicated that the maximum OPN-induced degradation was
observed in 60 min (middle panel a). After that, I
B
synthesis was reactivated possibly by NF
B in 180 min (middle
panel a). The lack of phosphorylated I
B
at 60 min indicates
that the rate of degradation exceeded the rate of I
B
phosphorylation at this time point (upper panel a).

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Fig. 3.
OPN stimulates
I B phosphorylation
(A) and IKK activity (B) and curcumin
blocks the OPN-induced I B
phosphorylation and IKK activity. A, cells were
either treated with 5 µM OPN alone for 0-180 min
(panel a) or with 50 µM curcumin for 45 min
and then with 5 µM OPN for 0-120 min (panel
b). The cells were lysed, and the lysates were used for Western
blot analysis using anti phospho-I B antibody (upper panels
a and b). The blots were reprobed with anti-I B
(middle panels a and b) or anti-actin
(lower panels a and b) antibody. The blots were
analyzed densitometrically, and the values were normalized to actin.
The relative values of phospho-I B or I B in terms of -fold
changes are indicated. Note that the maximum phosphorylation of
I B occurs at 10 min in OPN-treated cells in panel
"a," whereas in panel "b"
curcumin blocks the OPN-induced phosphorylation and degradation.
B, cells were stimulated with 5 µM OPN for 10 min or with 50 µM curcumin for 45 min followed by 5 µM OPN for 10 min. The cell lysates were
immunoprecipitated with anti-IKK / antibody and used for kinase
assay using recombinant I B as substrate (upper panel).
The immunoprecipitated samples were analyzed by Western blot analysis
using anti-IKK / antibody (middle panel). An equal
volume of samples from kinase assay was analyzed by Western blot
analysis using anti-I B antibody (lower panel).
Lane 1, without OPN; lane 2, with 5 µM OPN; and lane 3, with 50 µM
curcumin and 5 µM OPN. Note that equal intensities of
IKK / - and I B -specific bands are obtained in the
autoradiographs, indicating that an identical amount of IKK was
expressed in the cells, and an equal amount of I B was used. The
results shown here represent three experiments exhibiting similar
effects.
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In other experiments, the OPN-treated cell lysates were
immunoprecipitated with anti-I
B
antibody, separated by SDS-PAGE, and detected by phosphoserine detection system. The data indicated that
there was no phosphorylated I
B
-specific band in the OPN-treated sample, suggesting that OPN phosphorylates I
B
but not I
B
in these cells (data not shown).
In separate experiments, cells were pretreated with curcumin (50 µM) for 45 min and then stimulated with OPN (5 µM) for 0-120 min. The cells were lysed, and an equal
amount of total proteins from the lysates was resolved by SDS-PAGE and
analyzed by Western blot analysis using anti-phospho-specific I
B
or anti-I
B
antibody as described above. The results indicated
that curcumin inhibited OPN-induced I
B
phosphorylation and
degradation in these cells (upper and middle panels
b). As loading controls, both these blots were reprobed with
anti-actin antibody (lower panels a and b). These
bands were quantified by densitometry, and the values were normalized
with respect to actin expression. The -fold changes, as compared with
control, were calculated.
Previous reports have indicated that IKK play major role in the
cytokine-induced phosphorylation of I
B
at serine residues 32 and
36 (14, 15). Therefore we sought to determine whether OPN controls the
I
B
phosphorylation through modulating the activation of IKK and
whether curcumin has any effect on IKK activity. Accordingly, cells
were either treated with 5 µM OPN for 10 min or
pretreated with 50 µM curcumin for 45 min and then with 5 µM OPN for 10 min. Cells were lysed and
immunoprecipitated with anti-IKK
/
antibody. Half of the
immunoprecipitated samples were used for kinase assay using recombinant
I
B
as substrate. The radiolabeled, phosphorylated I
B
-specific band is detected in OPN-treated cells, demonstrating that OPN induces the IKK activity (Fig. 3B, upper
panel, lane 2). In contrast, this IKK activity was
undetectable in the untreated or curcumin treated cells (upper
panel, lanes 1 and 3, respectively), suggesting that OPN-induced IKK activity was blocked by curcumin. The
remaining half of the immunoprecipitated samples were analyzed by
Western blot analysis using anti-IKK
/
antibody. Fig.
3B showed the identical level of expression of IKK,
suggesting that IKK is expressed in these cells (middle
panel). The identical amount of I
B
was detected when the
equal volume of kinase reaction mixture was loaded into SDS-PAGE and
analyzed by Western blot using anti-I
B
antibody (lower
panel). These data further suggested that curcumin suppressed the
OPN-induced NF
B activation at a step prior to the I
B
phosphorylation.
Curcumin Suppresses OPN-induced in Vitro Cell Migration and ECM
Invasion--
We have shown previously that OPN enhances the cell
migration and ECM invasion in a dose dependent manner. In this study, we have assessed the effect of curcumin on OPN-induced cell migration and ECM invasion in these cells. The cells were pretreated with varying
concentrations of curcumin (0-100 µM) and added to the upper chamber. The purified OPN (5 µM) was used in the
upper chamber. Fig. 4A showed that
curcumin suppressed the OPN-induced cell migration (168-23%) in a
dose-dependent manner. The number of cells migrated in
absence of OPN were used as control (100% migration). Similarly, there
was dramatic reduction of OPN-induced ECM invasion (161-18%), when
the cells were pretreated with increasing concentrations of curcumin
followed by treatment with OPN (Fig. 4B). The number of
cells invaded in the absence of OPN was used as control (100% invasion). When preimmune IgG used in the upper chamber, no significant changes in migration or ECM invasion was observed, suggesting that the
migration or invasion of B16F10 cells are OPN and curcumin specific
(data not shown). The results are expressed as the mean of three
determinations ± S.E. The data are analyzed statistically using
Student's t test and were statistically significant
(p < 0.001).

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Fig. 4.
OPN-induced cell migration
(A) and ECM invasion (B) are
inhibited by curcumin. A, the migration assay was
performed either by using untreated cells (5 × 105
cells/well) or the cells pretreated with different doses of curcumin
(0-100 µM) for 30 min. The purified human OPN (5 µM) was added in the upper chamber. B, the ECM
invasion assay was done either by using untreated cells (5 × 105 cells/well) or cells treated with different doses of
curcumin (0-100 µM) for 30 min, and then OPN (5 µM) was added in the upper chamber. In both cases, the
results are expressed as the means + S.E. of three determinations. The
values were analyzed statistically by Student's t test (*,
p < 0.001).
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Curcumin Blocks OPN-induced MT1-MMP Expression and Pro-MMP-2
Activation--
Our earlier data indicated that OPN stimulates
pro-MMP-2 activation by inducing the expression of MT1-MMP. We sought
to determine whether curcumin had any effect on OPN-induced MT1-MMP
expression and pro-MMP-2 activation. Accordingly, we have treated the
cells with varying concentrations of curcumin (0-100 µM)
for 45 min. The cells were also treated with different doses of
curcumin (0-100 µM) and then treated with 5 µM OPN for 12 h. The cells were lysed, and the
lysates containing equal amount of total proteins were separated by
SDS-PAGE, and the level of MT1-MMP was detected by Western blot
analysis using anti-MT1-MMP antibody. There was a significant increase
of MT1-MMP expression in OPN-treated cells (Fig.
5A, upper panel,
lane 4) compared with untreated cells (lane 1),
and these data are consistent with our previous data. However, curcumin
dose-dependently suppressed the OPN-induced MT1-MMP
expression (lanes 5 and 6). The MT1-MMP
expression was also almost abolished when increasing concentrations of
curcumin alone were used (lanes 2 and 3). The
blots were reprobed with anti-actin antibody (lower panel).
The bands were quantified densitometrically and normalized with actin.
-Fold changes with respect to control were calculated. There were at
least a 1.4- and 3-fold decrease in MT1-MMP expression when the cells
were pretreated with 50 and 100 µM, curcumin,
respectively, prior to the OPN treatment compared with the cells
treated with OPN alone.

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Fig. 5.
Curcumin blocks OPN induced MT1-MMP
expression (A) and pro-MMP-2 activation
(B). A, the cells were pretreated with
different doses of curcumin (0-100 µM) and then treated
in the absence or presence of 5 µM OPN. The cell lysates
were used for the detection of MT1-MMP by Western blot analysis
(upper panel). The same blot was reprobed with anti-actin
antibody (lower panel). Lane 1, untreated cells;
lane 2, with 50 µM curcumin; lane
3, with 100 µM curcumin; lane 4, with 5 µM OPN; lane 5, with 50 µM
curcumin and 5 µM OPN; and lane 6, with 100 µM curcumin and 5 µM OPN. Note that
curcumin suppressed the MT1-MMP expression in both untreated and
OPN-treated cells in a dose-dependent manner. The
arrows indicate MT1-MMP and actin-specific bands. The bands
were analyzed by densitometry and normalized to actin expression. The
-fold changes were calculated. The results shown here represent three
experiments exhibiting similar effects. B, the cells were
either treated with 5 µM OPN alone or with varying doses
of curcumin (0-100 µM) and then with 5 µM
OPN. The conditioned medium was collected, and MMP-2 activity was
analyzed by gelatin zymography. Lane 1, untreated cells;
lane 2, with 5 µM OPN; lane 3, with
50 µM curcumin and 5 µM OPN; and lane
4, with 100 µM curcumin and 5 µM OPN.
The arrows indicate both 72-kDa pro-MMP2- and 66-kDa active
MMP-2-specific bands. The bands were analyzed by densitometry and are
represented in the form of a bar graph (lower
panel). The mean values of triplicate experiments are indicated.
The relative intensities were analyzed statistically using Student's
t test (*, p < 0.005).
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To check whether curcumin suppressed the OPN-induced pro-MMP-2
activation in these cells, the cells were treated with 5 µM OPN for 12 h or with curcumin (0-100
µM) for 45 min and then with 5 µM OPN for
12 h. The conditioned medium was collected, and the MMP-2 activity
was detected by zymography. The results showed that the levels of both
pro- and active MMP-2 were higher in OPN-treated cells (Fig.
5B, upper panel, lane 2) compared with
untreated cells (lane 1). However, the cells pretreated with
50 or 100 µM curcumin followed by treatment with OPN
showed drastic reduction of both pro-MMP-2 expression and activation
(lanes 3 and 4, respectively), indicating that
curcumin blocked the OPN-induced pro-MMP-2 activation. The
MMP-2-specific protein bands were quantified by densitometry and
analyzed by Student's t test (p < 0.005)
(lower panel).
Curcumin Suppresses OPN-induced Cell Proliferation and Induces
Apoptotic Morphology--
We have reported that OPN induced cell
proliferation in B16F10 cells; we therefore checked the effect of
curcumin on OPN-induced cell viability by MTT assay. The cells were
pretreated with various concentrations of curcumin (0-100
µM) for 45 min and then treated without or with OPN (5 µM) for 12 h. The cell viability in OPN-treated cells was higher than untreated cells (Fig.
6A). The cell viability was
reduced by curcumin in a dose-dependent manner in the
absence or presence of OPN (Fig. 6A). The data are
represented in the form of a bar graph and plotted using means + S.E.
of triplicate determinations. The values were analyzed by student's
t test (p < 0.005). To check whether OPN or
curcumin has any role in regulation of apoptotic morphology, these
cells were either treated with 5 µM OPN for 6 h or
with 50 µM curcumin for 45 min and then with 5 µM OPN for 6 h. The cells were fixed, nuclei were
stained with PI, and photographs were taken under confocal microscopy.
OPN alone did not induce apoptotic morphology in these cells (Fig. 6B, panel b). However, curcumin, in the presence
of OPN, synergistically induced apoptotic morphology within 6 h (panel c). Untreated cells, as expected, did not show any
apoptotic morphology (panel a). These data suggested that
curcumin can sensitize OPN-treated cells to apoptosis by selectively
inhibiting the signaling pathway for NF
B activation.

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Fig. 6.
Curcumin suppresses OPN-induced cell
proliferation (A) and induces apoptotic morphology in
presence of OPN (B). A,
the cells were pretreated with various concentrations of curcumin
(0-100 µM) for 45 min and then treated in absence or
presence of 5 µM OPN for 12 h. The cell viability
was determined by the MTT assay. The data are represented in the form
of a bar graph and plotted using the means ± S.E. of
triplicate determinations. The values were analyzed by Student's
t test (*, p < 0.005). B, the
cells were treated with 5 µM OPN for 6 h or with 50 µM curcumin for 45 min and then with 5 µM
OPN for 6 h. The cells were fixed and stained with PI. Panel
a, untreated cells; panel b, with 5 µM
OPN; and panel c, with 50 µM curcumin and 5 µM OPN. The typical photograph shown here represents
three experiments exhibiting similar effects.
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Curcumin Suppresses OPN-induced Tumor Growth and Inhibits
OPN-induced MMP-2 Activation in Tumor of Nude Mice--
The in
vitro results prompted us to examine whether curcumin has any role
on OPN-induced tumor growth and regulating the OPN-induced pro-MMP-2
activation in an in vivo system. Accordingly, B16F10 cells
were treated with OPN (10 µM) for 16 h and then
injected subcutaneously into the flanks of nude mice. In separate
experiments, cells were pretreated with curcumin, then treated with OPN
and injected into the nude mice. Fig.
7A (panels a-d)
show typical photographs of tumors grown in 4-week-old nude mice. After
4 weeks, the mice were killed, and tumor weights were measured. The
weights of the OPN-induced tumors were increased at least 3.1-fold
compared with the tumors of the non-OPN-injected mice (Table
I). These data are consistent with our
previous data. However, the weights of the OPN-induced tumors were
reduced drastically (6- and 30-fold) when two doses of curcumin (50 and
100 µM, respectively) were injected into the sites of the
tumors (Table I). Four mice were used in each set of experiments. The
changes in tumor weights were analyzed statistically by Student's
t test (p < 0.005).

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Fig. 7.
Curcumin inhibits OPN-induced tumor growth
and MMP-2 activation in OPN-induced tumors of nude mice.
A, typical photographs of tumors in nude mice. The
cells were treated with or without OPN (10 µM) and
injected subcutaneously into the flanks of nude mice. In separate
experiments, the cells were pretreated with curcumin followed by
treatment with OPN and injected into the nude mice. OPN (10 µM) alone or a mixture of curcumin (0-100
µM) and OPN (10 µM) was injected into the
tumor sites. Four mice were used in each set of experiments.
Panel a, cells with phosphate-buffered saline; panel
b, cells with OPN; panel c, cells with OPN and curcumin
(50 µM); and panel d, cells with OPN and
curcumin (100 µM). B, detection of MMP-2
expression in the tumors of nude mice by gelatin zymography. The tumor
samples from A were lysed in lysis buffer and analyzed by
gelatin zymography. Equal amounts of total proteins were used in each
lane. Lane 1, phosphate-buffered saline; lane 2,
with 10 µM OPN; lane 3, with 10 µM OPN and 50 µM curcumin; and lane
4, with 10 µM OPN and 100 µM curcumin.
C, detection of MMP-2 expression in the tumors of nude mice
by Western blotting. The tumor samples from A were lysed.
Equal amounts of total proteins were electrophoresed and analyzed by
Western blotting using mouse monoclonal anti-MMP-2 antibody. Lane
1, phosphate-buffered saline; lane 2, with 10 µM OPN; lane 3, with 10 µM OPN
and 50 µM curcumin; and lane 4, with 10 µM OPN and 100 µM curcumin. The
arrows indicate the 72- and 66-kDa MMP-2-specific bands. The
results show the representative of four mice used in each set
experiments.
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Table I
Effect of curcumin on OPN induced tumor growth in nude mice
B16F10 cells were treated with OPN (10 µM) for 16 h
and injected into nude mice (NMRI). In separate experiments, cells were
pretreated with curcumin (0-100 µM), then treated with
OPN (10 µM) and injected into the nude mice. After that,
OPN (10 µM) alone or mixture of curcumin (0-100
µM) and OPN (10 µM) were injected into the
tumor sites. The injection was performed twice a week for 4 weeks. The
mice were killed, and the tumor weights were measured and analyzed
statistically by Student's t test (p < 0.005). Mice injected with cells in PBS were used as controls.
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To detect the levels of pro-MMP-2 and active MMP-2 expressions in the
tumors, the samples were lysed, and MMP-2 expression was analyzed by
zymography (B). The levels of both the pro and active forms
of MMP-2 in the tumors produced by OPN (10 µM) were significantly higher (lane 2) compared with the levels of
MMP-2 in the tumors in non-OPN-injected mice (lane 1).
However, the levels of MMP-2 (especially the active form) were reduced
significantly in the two different doses of curcumin-injected mice
(lanes 3 and 4, respectively). MMP-2 expression
in tumors was further confirmed by Western blot analysis
(C). Both latent and active MMP-2 expressions were reduced
dramatically in curcumin-injected mice (lanes 3 and 4) compared with the levels of MMP-2 in the tumors produced
by OPN (lane 2) or in non-OPN-injected (lane 1)
mice, and these data are corroborated by the zymography data
(B). Taken together, these data strongly suggest that
curcumin suppressed OPN-induced MMP-2 expression and activation in a
dose-dependent manner, and these data correlate with tumor growth
(melanoma formation) in nude mice.
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DISCUSSION |
In a recent study (21), we have shown that OPN stimulated
activation of pro-MMP-2 through NF
B-mediated induction of MT1-MMP, and these data correlated with enhanced cell migration, ECM invasion, and tumor growth by OPN in melanoma (B16F10) cells. In this paper, we
have further investigated the mechanism of OPN-induced NF
B activation. We found that OPN induces the I
B
phosphorylation by
enhancing the activity of IKK in B16F10 cells. This ultimately causes
the degradation of I
B
and translocation of NF
B into the
nucleus and transcriptional activation.
The number of functions attributed to being regulated by the
transcription factor NF
B is rapidly increasing. It is involved in
the control of a large number of normal cellular processes such as
inflammatory and immune responses, developmental processes, cell
growth, and apoptosis. In addition, NF
B is activated in several
pathological conditions like arthritis, inflammation, asthma,
neurodegenerative diseases, heart diseases, and cancers. Inappropriate
NF
B activity has been reported in several cancers (15-18).
Activation of NF
B is known to confer resistance to apoptotic signals
(5, 49). The involvement of most of NF
B target genes in
several disease conditions makes its inhibitors attractive candidates
as therapeutic agents. Curcumin (diferulolylmethane), a constituent of
turmeric, is one such pharmacologically safe, non-toxic compound with
known anti-inflammatory and anticarcinogenic properties. Earlier
reports have indicated that curcumin blocks activation of NF
B by
TNF-
, phorbol esters, and hydrogen peroxide (45). Curcumin also
blocks cytokine-mediated activation of NF
B in various cell types
(50).
Previous reports have indicated that OPN ligation to its
v
3 integrin receptor protected the
endothelial cells from apoptosis through a NF
B-mediated pathway
(32). Earlier results have also shown that NF
B induces
anti-apoptotic genes (49). Enhanced proliferation rates and resistance
to apoptosis-inducing signals are important factors contributing to
tumor growth; therefore, anti-proliferative and apoptosis-inducing
properties of curcumin could prove to be potential candidates in the
control of various cancers.
In this study, we analyzed the effect of curcumin on OPN-induced NF
B
activation and its downstream effects such as MT1-MMP expression,
pro-MMP-2 activation, cell migration, and tumor growth. We found that
pretreatment of cells with curcumin resulted in inhibition of
OPN-induced NF
B activation with concomitant down-regulation of
MT1-MMP expression and pro-MMP-2 activation. Inhibition of OPN-induced
NF
B binding and transactivation by curcumin were accompanied by an
inhibition of p65 translocation into the nucleus.
To determine whether these in vitro results could be
extended to an in vivo nude mice model, tumorigenicity
experiments were carried out. OPN-induced tumor growth is suppressed by
curcumin. The expression and activation of pro-MMP-2 in OPN induced
tumors are also reduced drastically by curcumin in a
dose-dependent manner. OPN induces proliferation in these
cells. MTT assay indicated that curcumin suppresses OPN-induced cell
viability. Curcumin also induces apoptotic morphology in
OPN-treated cells. These results may also in part explain the reduced
tumor growth in curcumin injected mice.
We have delineated the molecular mechanism by which curcumin blocked
the OPN-induced NF
B activation. NF
B activity is regulated by an
endogenous inhibitor I
B
; interaction of NF
B with I
B
blocks the nuclear transport signal and keeps it sequestered in the
cytoplasm. Following any kind of stimulation, I
B
is
phosphorylated at serine residues 32 and 36, which leads to its
ubiquitination and degradation. The free NF
B then translocates to
nucleus and activates the transcription of target genes (51). Inducible phosphorylation of I
B
is mediated by a multisubunit complex of
kinases, IKK
/
(12, 16). Our results indicate that inhibition of
OPN-induced NF
B activity by curcumin involved suppression of
OPN-induced I
B
phosphorylation and inhibition of IKK activity.
The signaling pathways that involve the phosphorylation and degradation
of I
B
by inducing the IKK activity and subsequent activation of
NF
B in presence of various stimuli are not well defined. A number of
upstream kinases such as NF
B-inducing kinase (NIK),
phosphatidylinositol 3-kinase, and MEKK play
significant roles in regulation of activation of IKK. Therefore, it is
possible that OPN may induce the IKK activity directly or by inducing
the activation of upstream kinase that ultimately activates NF
B. Moreover, curcumin may block the upstream kinase activity. Further work
in this area is in progress in our laboratory.
Use of a natural product is emerging as an alternative to traditional
medicines in the treatment of cancer. Curcumin is a non-toxic natural
product that has been used as a food additive (52). The non-toxicity is
proved by its consumption by humans in several countries including the
country of its origin. Curcumin is also shown to be a non-mutagenic
compound (48). The ability of curcumin to inhibit NF
B
activity provides a major reason to further investigate the
effect of this compound in in vivo animal models.
In summary, we have demonstrated that OPN induces NF
B activity
through phosphorylation and degradation of I
B
by inducing the
activity of IKK. This ultimately leads to the activation of pro-MMP-2
and regulation of cell migration, ECM invasion, and tumor growth.
Curcumin could block OPN-induced MT1-MMP expression and pro-MMP-2
activation. Curcumin also suppresses OPN-induced cell proliferation,
cell migration, and ECM invasion. It induces apoptotic morphology in
OPN-treated cells. Both the tumor size and MMP-2 production and
activation in OPN-induced tumors are reduced by curcumin in nude mice.
We have also delineated the mechanism by which curcumin suppressed
these OPN-induced effects. Curcumin blocks MT1-MMP expression and
pro-MMP-2 activation by inhibiting signals that lead to IKK activation
(Fig. 8). This in turn inhibits I
B
phosphorylation/degradation and NF
B activation. Curcumin could thus
be a potential therapeutic candidate for cancers such as melanoma and
other inflammatory disorders that involve NF
B-mediated MMP-2
activation.

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Fig. 8.
Molecular mechanism of OPN-induced
NF B-mediated pro-MMP-2 activation and action
of curcumin on these pathways. Binding of OPN to its integrin
receptor on cell surface induces the NF B signaling pathway by
inducing the activity of IKK / followed by phosphorylation and
breakdown of I B . NF B then moves from cytoplasm into the
nucleus and elevates the levels of MT1-MMP mRNA. Increased MT1-MMP
on the cell surface facilitates the activation of pro-MMP-2. Curcumin
inhibits this activation process by blocking the IKK/I B signaling
pathways.
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