Osteopontin Induces Nuclear Factor kappa B-mediated Promatrix Metalloproteinase-2 Activation through Ikappa Balpha /IKK Signaling Pathways, and Curcumin (Diferulolylmethane) Down-regulates These Pathways*

Subha Philip and Gopal C. KunduDagger

From the National Center for Cell Science (NCCS), NCCS Complex, Pune-411 007, India

Received for publication, July 20, 2002, and in revised form, December 1, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have recently reported that osteopontin (OPN) stimulates tumor growth and activation of promatrix metalloproteinase-2 (pro-MMP-2) through nuclear factor kappa B (NFkappa B)-mediated induction of membrane type 1 matrix metalloproteinase (MT1-MMP) in murine melanoma cells (Philip, S., Bulbule, A., and Kundu, G. C. (2001) J. Biol. Chem. 276, 44926-44935). However, the molecular mechanism by which OPN activates NFkappa B and regulates pro-MMP-2 activation in murine melanoma (B16F10) cells is not well defined. We also investigated the mechanism of action of curcumin (diferulolylmethane) on OPN-induced NFkappa B-mediated activation of pro-MMP-2 in B16F10 cells. Here we report that OPN induces phosphorylation and degradation of the inhibitor of nuclear factor kappa B (Ikappa Balpha ) by inducing the activity of Ikappa B kinase (IKK) in these cells. OPN also induces the nuclear accumulation of NFkappa B p65, NFkappa B-DNA binding, and transactivation. However, curcumin a known anti-inflammatory and anticarcinogenic agent suppressed OPN-induced Ikappa Balpha phosphorylation and degradation by inhibiting the IKK activity. Moreover, our data revealed that curcumin inhibited the OPN-induced translocation of p65, NFkappa B-DNA binding, and NFkappa B transcriptional activity. The OPN-induced pro-MMP-2 activation and MT1-MMP expression were also drastically reduced by curcumin. Curcumin also inhibited OPN-induced cell proliferation, cell migration, extracellular matrix invasion, and synergistically induced apoptotic morphology with OPN in these cells. Most importantly, curcumin suppressed the OPN-induced tumor growth in nude mice, and the levels of pro-MMP-2 expression and activation in OPN-induced tumor were inhibited by curcumin. To our knowledge, this is the first report that OPN induces NFkappa B activity through phosphorylation and degradation of Ikappa Balpha by activating IKK that ultimately triggers the activation of pro-MMP-2 and further demonstrates that curcumin potently suppresses OPN-induced cell migration, tumor growth, and NFkappa B-mediated pro-MMP-2 activation by blocking the IKK/Ikappa Balpha signaling pathways.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nuclear factor kappa B (NFkappa 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 kappa B sequence (3). It promotes transcription of varieties of cytokines such as IL-1, IL-2, IL-6, IL-8, TNF-alpha and cell adhesion molecules such as E-selectin, ICAM-1, and VCAM-1 (4-6). NFkappa 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, NFkappa B is present as a latent inactive, Ikappa B bound complex in the cytoplasm, but upon activation by extracellular stimuli or by other factors, NFkappa 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 NFkappa B is not well understood. However, most signals induce the activity of a large multi subunit protein kinase, called Ikappa B kinase (IKK). Active IKK phosphorylates Ikappa Balpha (12, 13), which targets Ikappa Balpha for ubiquitination and degradation by proteases (14, 15). The free NFkappa B can then translocate into the nucleus. Aberrant NFkappa B activity has been reported in several cancers including breast, colon, prostate, and lymphoid cancers (16-19). NFkappa B-inducible genes play important role in various disorders especially in cancers. NFkappa 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 NFkappa 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 beta -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 alpha vbeta 3 integrin in various cell types (31). OPN induces pro-MMP-2 activation and NFkappa B-mediated signaling pathways by binding to its receptor alpha vbeta 3 integrin (21, 32). All these above effects contribute to the tumor growth and progression. However, the molecular mechanism by which OPN activates the NFkappa 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 NFkappa 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-alpha and phorbol myristate acetate (PMA) (44) which require NFkappa B activation. There have been reports that curcumin can inhibit NFkappa 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 Ikappa Balpha phosphorylation and degradation by activating IKK in B16F10 cells. The translocation of p65 subunit of NFkappa 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 NFkappa B transcriptional activity and EMSA data showed that OPN enhances NFkappa B-DNA binding activity. Supershift assay using p65 or p50 antibody showed the shift of the NFkappa B-specific band toward higher molecular weight. Curcumin suppressed the OPN-induced IKK kinase activity, Ikappa Balpha phosphorylation, p65 nuclear translocation, NFkappa 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 NFkappa B-mediated pro-MMP-2 activation through IKK-regulated phosphorylation of Ikappa Balpha and further demonstrate that curcumin inhibits OPN-induced cell migration, tumor growth, and NFkappa B-mediated MMP-2 activation by inhibiting signal leading to IKK activity.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Materials-- Rabbit polyclonal anti-NFkappa B p65, anti-p50, anti-NFkappa B X TransCruz, anti-IKKalpha /beta , anti-Ikappa Balpha , mouse monoclonal anti-Ikappa Bbeta and goat polyclonal anti-actin antibodies, NFkappa B consensus oligonucleotide, and Ikappa Balpha recombinant protein were purchased from Santa Cruz Biotechnology. The rabbit polyclonal phospho-specific anti-Ikappa Balpha , 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 [gamma -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-kappa 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 NFkappa B oligonucleotide for 15 min prior to the addition of labeled probe and the samples were further analyzed.

NFkappa B Luciferase Reporter Gene Assay-- The semiconfluent cells grown in 24-well plates were transiently transfected with a luciferase reporter construct (pNFkappa B-Luc) containing five tandem repeats of the NFkappa 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 beta -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-IKKalpha /beta antibody in immunoprecipitation buffer (40 mM Tris-HCl (pH 8.0), 500 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM beta -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 Ikappa Balpha (4 µg) in kinase buffer (20 mM Hepes, (pH 7.7), 2 mM MgCl2, 10 µM ATP, 3 µCi of [gamma -32p]ATP, 10 mM beta -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-IKKalpha /beta 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-Ikappa Balpha antibody.

Western Blot Analysis-- For Ikappa Balpha 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-Ikappa Balpha 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-Ikappa Balpha (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-Ikappa Bbeta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

OPN Induces Translocation of p65 subunit of NFkappa 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 NFkappa 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 NFkappa B-DNA Binding and NFkappa 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 NFkappa B oligonucleotides. The results in Fig. 2A showed that OPN induced NFkappa B-DNA binding (lane 2) compared with untreated cells (lane 1). Curcumin (50 and 100 µM) suppressed the OPN-induced NFkappa B-DNA-binding in a dose-dependent manner (lanes 3 and 4, respectively). The NFkappa 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 NFkappa B-DNA binding (A and B) and NFkappa 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 NFkappa B-specific band. OPN treatment (lane 2) induces NFkappa B binding compared with the untreated cells (lane 1). Curcumin inhibits OPN-induced NFkappa 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 (pNFkappa 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 NFkappa 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 NFkappa 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 NFkappa 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 NFkappa B-specific band (lane 7). As expected, no band was obtained when EMSA was performed without nuclear extract (lane 1).

The induction of NFkappa B transcriptional activity by OPN was also monitored by luciferase reporter gene assay. The cells were transiently transfected with NFkappa B luciferase reporter construct (pNFkappa 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 NFkappa B transcriptional activity, and curcumin inhibits the OPN-induced NFkappa B activity in a dose-dependent manner (Fig. 2C). PMA, as a positive control, induces the NFkappa 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 Ikappa Balpha Phosphorylation by Inducing IKK Activity, and Curcumin Inhibits OPN-induced Ikappa Balpha Phosphorylation and IKK Activity-- Since we have shown earlier that OPN-induced NFkappa B activity is suppressed by the super-repressor form of Ikappa Balpha (21), we sought to determine whether OPN-induced NFkappa B activation is occurred through phosphorylation/degradation of Ikappa Balpha . 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 Ikappa Balpha was detected by Western blot analysis using anti-phospho-Ikappa Balpha antibody. The data demonstrated that OPN induces Ikappa Balpha phosphorylation in 10 min, and the levels of phospho-Ikappa Balpha disappeared in 60 min and reappeared in 120 min (Fig. 3A, upper panel a). The blot was reprobed with anti-Ikappa Balpha antibody, and the data indicated that the maximum OPN-induced degradation was observed in 60 min (middle panel a). After that, Ikappa Balpha synthesis was reactivated possibly by NFkappa B in 180 min (middle panel a). The lack of phosphorylated Ikappa Balpha at 60 min indicates that the rate of degradation exceeded the rate of Ikappa Balpha phosphorylation at this time point (upper panel a).


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Fig. 3.   OPN stimulates Ikappa Balpha phosphorylation (A) and IKK activity (B) and curcumin blocks the OPN-induced Ikappa Balpha 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-Ikappa Balpha antibody (upper panels a and b). The blots were reprobed with anti-Ikappa Balpha (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-Ikappa Balpha or Ikappa Balpha in terms of -fold changes are indicated. Note that the maximum phosphorylation of Ikappa Balpha 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-IKKalpha /beta antibody and used for kinase assay using recombinant Ikappa Balpha as substrate (upper panel). The immunoprecipitated samples were analyzed by Western blot analysis using anti-IKKalpha /beta antibody (middle panel). An equal volume of samples from kinase assay was analyzed by Western blot analysis using anti-Ikappa Balpha 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 IKKalpha /beta - and Ikappa Balpha -specific bands are obtained in the autoradiographs, indicating that an identical amount of IKK was expressed in the cells, and an equal amount of Ikappa Balpha was used. The results shown here represent three experiments exhibiting similar effects.

In other experiments, the OPN-treated cell lysates were immunoprecipitated with anti-Ikappa Bbeta antibody, separated by SDS-PAGE, and detected by phosphoserine detection system. The data indicated that there was no phosphorylated Ikappa Bbeta -specific band in the OPN-treated sample, suggesting that OPN phosphorylates Ikappa Balpha but not Ikappa Bbeta 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 Ikappa Balpha or anti-Ikappa Balpha antibody as described above. The results indicated that curcumin inhibited OPN-induced Ikappa Balpha 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 Ikappa Balpha at serine residues 32 and 36 (14, 15). Therefore we sought to determine whether OPN controls the Ikappa Balpha 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-IKKalpha /beta antibody. Half of the immunoprecipitated samples were used for kinase assay using recombinant Ikappa Balpha as substrate. The radiolabeled, phosphorylated Ikappa Balpha -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-IKKalpha /beta 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 Ikappa Balpha was detected when the equal volume of kinase reaction mixture was loaded into SDS-PAGE and analyzed by Western blot using anti-Ikappa Balpha antibody (lower panel). These data further suggested that curcumin suppressed the OPN-induced NFkappa B activation at a step prior to the Ikappa Balpha 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).

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).

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 NFkappa 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.

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.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In a recent study (21), we have shown that OPN stimulated activation of pro-MMP-2 through NFkappa 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 NFkappa B activation. We found that OPN induces the Ikappa Balpha phosphorylation by enhancing the activity of IKK in B16F10 cells. This ultimately causes the degradation of Ikappa Balpha and translocation of NFkappa B into the nucleus and transcriptional activation.

The number of functions attributed to being regulated by the transcription factor NFkappa 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, NFkappa B is activated in several pathological conditions like arthritis, inflammation, asthma, neurodegenerative diseases, heart diseases, and cancers. Inappropriate NFkappa B activity has been reported in several cancers (15-18). Activation of NFkappa B is known to confer resistance to apoptotic signals (5, 49). The involvement of most of NFkappa 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 NFkappa B by TNF-alpha , phorbol esters, and hydrogen peroxide (45). Curcumin also blocks cytokine-mediated activation of NFkappa B in various cell types (50).

Previous reports have indicated that OPN ligation to its alpha vbeta 3 integrin receptor protected the endothelial cells from apoptosis through a NFkappa B-mediated pathway (32). Earlier results have also shown that NFkappa 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 NFkappa 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 NFkappa B activation with concomitant down-regulation of MT1-MMP expression and pro-MMP-2 activation. Inhibition of OPN-induced NFkappa 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 NFkappa B activation. NFkappa B activity is regulated by an endogenous inhibitor Ikappa Balpha ; interaction of NFkappa B with Ikappa Balpha blocks the nuclear transport signal and keeps it sequestered in the cytoplasm. Following any kind of stimulation, Ikappa Balpha is phosphorylated at serine residues 32 and 36, which leads to its ubiquitination and degradation. The free NFkappa B then translocates to nucleus and activates the transcription of target genes (51). Inducible phosphorylation of Ikappa Balpha is mediated by a multisubunit complex of kinases, IKKalpha /beta (12, 16). Our results indicate that inhibition of OPN-induced NFkappa B activity by curcumin involved suppression of OPN-induced Ikappa Balpha phosphorylation and inhibition of IKK activity.

The signaling pathways that involve the phosphorylation and degradation of Ikappa Balpha by inducing the IKK activity and subsequent activation of NFkappa B in presence of various stimuli are not well defined. A number of upstream kinases such as NFkappa 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 NFkappa 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 NFkappa 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 NFkappa B activity through phosphorylation and degradation of Ikappa Balpha 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 Ikappa Balpha phosphorylation/degradation and NFkappa B activation. Curcumin could thus be a potential therapeutic candidate for cancers such as melanoma and other inflammatory disorders that involve NFkappa B-mediated MMP-2 activation.


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Fig. 8.   Molecular mechanism of OPN-induced NFkappa 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 NFkappa B signaling pathway by inducing the activity of IKKalpha /beta followed by phosphorylation and breakdown of Ikappa Balpha . NFkappa 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/Ikappa Balpha signaling pathways.


    ACKNOWLEDGEMENT

We thank Dr. Rainer de Martin for providing the luciferase reporter construct (pNFkappa B-Luc) containing five tandem repeats of NFkappa B binding site.

    FOOTNOTES

* This work was supported by funds from the Department of Biotechnology (to the National Center for Cell Science) and by an extramural fund from the Department of Science and Technology (to G. C. K.) of the Government of India.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: National Center for Cell Science, NCCS Complex, Pune-411 007. Tel.: 91-20-5690931 (ext. 203); Fax: 91-20-5692259; E-mail: gopalkundu@hotmail.com.

Published, JBC Papers in Press, December 7, 2002, DOI 10.1074/jbc.M207309200

    ABBREVIATIONS

The abbreviations used are: NFkappa B, nuclear factor kappa B; OPN, osteopontin; MMP, matrix metalloproteinase; Ikappa Balpha , inhibitor of nuclear factor kappa B; IKK, Ikappa B kinase; EMSA, electrophoretic mobility shift assay; PMA, phorbol myristate acetate; MT1, membrane type 1; ECM, extracellular matrix; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IL, interleukin; TNF, tumor necrosis factor; TIMP, tissue inhibitor of matrix metalloproteinase; FITC, fluorescein isothiocyanate; DTT, dithiothreitol; pNPP, p-nitrophenyl phosphate; PI, propidium iodide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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