Epidermal growth factor receptor-mediated regulation of matrix metalloproteinase-2 and matrix metalloproteinase-9 in MCF-7 breast cancer cells.

In breast cancer, increased epidermal growth factor receptor (EGFR) expression and phosphorylation have been correlated with increased invasive potential and poor prognosis. Interaction of EGFR with its ligand epidermal growth factor (EGF) activates cellular signalling cascades promoting tumour invasion. Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are upregulated in most cancers and play crucial roles in modulating invasion and metastasis. EGFR-mediated regulation of MMP-2 and MMP-9 in breast cancer was investigated using metastatic human breast ductal carcinoma cell line MCF-7. Culture of MCF-7 cells on 1 µg/ml EGF-coated culture dishes for 24 h led to appreciable increase in MMP-2 and MMP-9 expression and activity. Expression of membrane type-1 matrix metalloproteinase (MT1-MMP) and focal adhesion kinase (FAK), phosphorylation of EGFR and phosphatidylinositol 3' kinase (PI3K), and nuclear translocation of EGFR and cellular migration were also appreciably increased. Targeting EGFR-EGF interactions by treatment of MCF-7 cells with anti-EGFR monoclonal antibodies prior to culture on EGF prevented appreciable upregulation of MMP-2 and MMP-9 expression and activity. Increased expression of MT1-MMP and FAK, phosphorylation of EGFR and PI3K and enhanced cell migration were also inhibited. Treatment of cells with PI3K inhibitor LY294002 prevented upregulation of MMP-2 and MMP-9 indicating that EGFR-mediated signalling for MMP regulation occurs through PI3K. As increased EGFR activity has been observed in highly invasive breast cancers, targeting EGFR-EGF interactions might render such cancers less invasive by inhibiting EGFR-mediated upregulation of MMP-2 and MMP-9 and therefore could be of importance in their clinical management.

Fibronectin-integrin mediated signaling in human cervical cancer cells (SiHa).

Interaction between cell surface integrin receptors and extracellular matrix (ECM) components plays an important role in cell survival, proliferation, and migration, including tumor development and invasion of tumor cells. Matrix metalloproteinases (MMPs) are a family of metalloproteinases capable of digesting ECM components and are important molecules for cell migration. Binding of ECM to integrins initiates cascades of cell signaling events modulating expression and activity of different MMPs. The aim of this study is to investigate fibronectin-integrin-mediated signaling and modulation of MMPs. Our findings indicated that culture of human cervical cancer cell (SiHa) on fibronectin-coated surface perhaps sends signals via fibronectin-integrin-mediated signaling pathways recruiting focal adhesion kinase (FAK) extracellular signal regulated kinase (ERK), phosphatidyl inositol 3 kinase (PI-3K), integrin-linked kinase (ILK), nuclear factor-kappa B (NF-kappaB), and modulates expression and activation of mainly pro-MMP-9, and moderately pro-MMP-2 in serum-free culture medium.

Studies on Multifunctional Effect of All-Trans Retinoic Acid (ATRA) on Matrix Metalloproteinase-2 (MMP-2) and Its Regulatory Molecules in Human Breast Cancer Cells (MCF-7).

Background. Vitamin A derivative all-trans retinoic acid (ATRA) is considered as a potent chemotherapeutic drug for its capability of regulating cell growth and differentiation. We studied the effect of ATRA on MMP-2 in MCF-7, human breast cancer cells, and the probable signaling pathways which are affected by ATRA on regulating pro-MMP-2 activity and expression. Methods. Gelatin zymography, RT-PCR, ELISA, Western blot, Immunoprecipitation, and Cell adhesion assay are used. Results. Gelatin zymography showed that ATRA caused a dose-dependent inhibition of pro-MMP-2 activity. ATRA treatment downregulates the expression of MT1-MMP, EMMPRIN, FAK, NF-kB, and p-ERK. However, expression of E-cadherin, RAR, and CRABP increased upon ATRA treatment. Binding of cells to extra cellular matrix (ECM) protein fibronectin reduced significantly after ATRA treatment. Conclusions. The experimental findings clearly showed the inhibition of MMP-2 activity upon ATRA treatment. This inhibitory effect of ATRA on MMP-2 activity in human breast cancer cells (MCF-7) may result due to its inhibitory effect on MT1-MMP, EMMPRIN, and upregulation of TIMP-2. This study is focused on the effect of ATRA on MMP, MMP-integrin-E-cadherin interrelationship, and also the effect of the drug on different signaling molecules which may involve in the progression of malignant tumor development.

Multifunctional effect of epigallocatechin-3-gallate (EGCG) in downregulation of gelatinase-A (MMP-2) in human breast cancer cell line MCF-7.

AIMS: The tumor inhibiting property of green tea polyphenol epigallocatechin-3-gallate (EGCG) is well documented. Studies reveal that matrix-metalloproteinases (MMPs) play pivotal roles in tumor invasion through degradation of basement membranes and extracellular matrix (ECM). We studied the effect of EGCG on matrixmetalloproteinases-2 (MMP-2), the factors involved in activation, secretion and signaling molecules that might be involved in the regulation of MMP-2 in human breast cancer cell line, MCF-7. MAIN METHODS: MCF-7 was treated with EGCG (20 muM, 24 h), the effect of EGCG on MMP-2 expression, activity and its regulatory molecules were studied by gelatin zymography, Western blot, quantitative and semi-quantitative real time RT-PCR, immunoflourescence and cell adhesion assay. KEY FINDINGS: EGCG treatment reduced the activity, protein expression and mRNA expression level of MMP-2. EGCG treatment reduced the expression of focal adhesion kinase (FAK), membrane type-1-matrix metalloproteinase (MT1-MMP), nuclear factor-kappa B (NF-kB), vascular endothelial growth factor (VEGF) and reduced the adhesion of MCF-7 cells to ECM, fibronectin and vitronectin. Real time RT-PCR revealed a reduced expression of integrin receptors alpha5, beta1, alphav and beta3 due to EGCG treatment. SIGNIFICANCE: Down regulation of expression of MT1-MMP, NF-kB, VEGF and disruption of functional status of integrin receptors may indicate decreased MMP-2 activation; low levels of FAK expression might indicate disruption in FAK-induced MMP-2 secretion and decrease in activation of phosphatidyl-inositol-3-kinase (PI-3K), extracellular regulated kinase (ERK) indicates probable hindrance in MMP-2 regulation and induction. We propose EGCG as potential inhibitor of expression and activity of pro-MMP-2 by a process involving multiple regulatory molecules in MCF-7.

Culture of human A375 melanoma cells in the presence of fibronectin causes expression of MMP-9 and activation of MMP-2 in culture supernatants.

UNLABELLED: Interactions between tumor cell surface integrin receptors and extracellular matrix (ECM) ligands play an important role in tumor development, affecting cell survival, proliferation, and migration. Integrin-ECM ligand interaction leads to phosphorylation of focal adhesion kinase (FAK) and activation of mitogen-activated protein kinase (MAPK) pathways. It has been reported that integrins also regulate expression and function of matrix metalloproteinases (MMPs). In this present work, we cultured human A375 melanoma cells in the presence of fibronectin to study fibronectin-integrin mediated modulation of MMP activity. METHODS: A375 cells were cultured in serum-free culture medium (SFCM) in the presence of fibronectin (25 microg/0.75 ml), SFCM was collected and gelatin zymography was performed. Western blot and RT-PCR were performed with A375 cells cultured in the presence of fibronectin. RESULTS: Culture of A375 cells in the presence of fibronectin led to expression of MMP-9 and activation of MMP-2 within 2 h. When cells were treated with ERK inhibitor (PD98059) or PI-3K inhibitor (LY294002) and grown in the presence of fibronectin, MMP-9 expression and MMP-2 activation was inhibited. Tyrosine phosphorylation of FAK and ERK were increased in A375 cells grown in the presence of fibronectin. Increased MMP-9 mRNA expression and processing of MT1-MMP were also observed in A375 cells grown in the presence of fibronectin. CONCLUSIONS: Our findings indicate culture of A375 cells in SFCM in the presence of fibronectin perhaps generates a signaling cascade that leads to expression of MMP-9 and activation of MMP-2 in culture supernatants within 2 h. The signaling pathway activated is probably the FAK/ERK pathway.

Rapid expression and activation of MMP-2 and MMP-9 upon exposure of human breast cancer cells (MCF-7) to fibronectin in serum free medium.

Interactions between tumour cells and the extracellular matrix (ECM) strongly influence tumour development, affecting cell survival, proliferation and migration. Many of these interactions are mediated through a family of cell surface receptors named integrins. Fibronectin and its integrin receptors play important roles in tumour development. The alpha5beta 1 integrin interacts with the central cell adhesive region of fibronectin and requires both the RGD and synergy sites for maximal binding. Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases. They are capable of digesting the different components of the ECM and basement membrane. The ECM gives structural support to cells and plays a central role in cell adhesion, differentiation, proliferation and migration. Binding of ECM to integrins modulates expression and activity of the different MMPs. Our experimental findings demonstrate that cultivation of human breast cancer cells, MCF-7, in serum free medium in the presence of fibronectin upregulates the activity of MMP-2 and MMP-9. Blocking of alpha5beta 1 integrin with anti-alpha5 monoclonal antibody inhibits the fibronectin-induced MMP activation response appreciably. This strongly indicates alpha5beta 1 mediated signalling events in activation of MMP-2 and MMP-9. Phosphorylation of FAK and PI-3 kinase and the nuclear translocation of ERK and NF-kappaB upon fibronectin binding demonstrate possible participation of the FAK/PI-3K/ERK signalling pathways in the regulation of MMP-2 activity.

Culture of human cervical cancer cells, SiHa, in the presence of fibronectin activates MMP-2.

PURPOSE: Several studies indicate that integrin receptors are involved in the regulation of matrix metalloproteinase (MMP) expression. Integrin-ECM ligand interaction leads to phosphorylation of focal adhesion kinase (FAK) and activation of mitogen activated protein kinase pathways. In this present communication, we cultured human cervical cancer cells, SiHa, in the presence of fibronectin to study fibronectin-integrin mediated modulation of MMP activity. METHODS: SiHa cells were cultured in serum-free medium (SFCM) in the presence of fibronectin, SFCM was collected and gelatin zymography was performed. Western blot, RT-PCR and immunocytochemistry were performed with SiHa cells cultured in the presence of fibronectin. RESULTS: The culture of SiHa cells in the presence of 50 microg/1.5 ml fibronectin led to expression of pro-MMP-9 and activation of MMP-2 within 2 h. When cells were treated with ERK inhibitor (PD98059) and grown in the presence of fibronectin MMP-2 activation was partially inhibited, but when cells were treated with PI-3K inhibitor (LY294002) and grown in the presence of fibronectin MMP-2 activation was appreciably reduced. Tyrosine phosphorylation of FAK, PI-3K and ERK and nuclear trafficking of ERK were increased in SiHa cells grown in the presence of fibronectin. Increased MT1-MMP mRNA expression and processing of MT1-MMP were also observed in SiHa cells grown in the presence of fibronectin. CONCLUSIONS: Our findings indicate that the culture of SiHa cells in SFCM in the presence of fibronectin perhaps generates a signalling cascade which leads to the expression of pro-MMP-9 and the activation of MMP-2 within 2 h. The signalling pathways activated seem to be the FAK/ERK/PI-3K pathway.

Cell membrane-associated MT1-MMP dependent activation of MMP-2 in SiHa (human cervical cancer) cells.

UNLABELLED: Among the soluble MMPs, MMP-2 (gelatinase A) is particularly important in the invasive property of tumor cells. Cell membrane-associated MMP-2 activation is one of the challenging areas in tumor biology. In the present communication, we studied the membrane dependent activation of MMP-2 in SiHa cells. METHODS: Activation of pro-MMP-2 by membrane fraction, membrane extract, and live SiHa cells was studied by gelatin zymography. The role of MT1-MMP in MMP-2 activation was studied by incubating SiHa cells and cell membrane fractions with anti-MT1-MMP antibody. RESULTS: Activation of purified pro-MMP-2 by membrane fraction isolated from SiHa cells, by SiHa cell membrane extract and by SiHa cells, pro-MMP-2 from Con A treated HT-1080 conditioned medium by SiHa cells, and pro-MMP-2 from serum free culture medium of SiHa cells and cervical tissue homogenate by SiHa cell membrane fraction was shown by gelatin zymography. SiHa membrane fraction activated only pro-MMP-2 from purified MMP-9/MMP-2 mixture, indicating that the activation is specific for MMP-2. Inhibition of MMP-2 activation in the presence of anti-MT1-MMP antibody strongly indicated that the cell membrane mediated MMP-2 activation is MT1-MMP dependent. Immunocytochemistry of SiHa cells demonstrated expression of MT1-MMP at focal points. Invasion assay showed that invasiveness of anti-MT1-MMP antibody treated SiHa cells through Matrigel was drastically reduced compared to control SiHa cells. CONCLUSIONS: Our findings furnish an example of the cell membrane-associated MT1-MMP mediated MMP-2 activation in SiHa cells and suggest that this MT1-MMP mediated MMP-2 activation is of importance in tumor invasion and metastasis. This MT1-MMP mediated MMP-2 activation on tumor cell surface could be a realistic target for managing metastatic diseases.

Culture of human fibrosarcoma HT-1080 cells in presence of fibronectin activates MMP-2.

UNLABELLED: The importance of tumor cell surface integrin receptors in regulation of matrix metalloproteinase (MMP) expression and function has been reported. Integrin-ECM ligand interaction leads to phosphorylation of focal adhesion kinase (FAK) and activation of mitogen activated protein kinase (MAPK) pathways. In this present study, we cultured human fibrosarcoma cells, HT-1080, in presence of fibronectin to study fibronectin-integrin mediated modulation of MMP activity. METHODS: HT-1080 cells were cultured in serum free medium (SFCM) in presence of fibronectin, SFCM was collected, and gelatin zymography was performed. Western blot and immunocytochemistry were performed with HT-1080 cells cultured in presence of fibronectin. RESULTS: Culture of HT-1080 cells in presence of 50 microg/1.5 ml fibronectin led to expression of pro-MMP-9 and activation of MMP-2 within 1 hr. When HT-1080 cells were treated with PI-3K inhibitor (LY294002) and grown in presence of fibronectin, MMP-2 activation was partially inhibited, but when cells were treated with ERK inhibitor (PD98059) and grown in presence of fibronectin, MMP-2 activation was almost completely inhibited. Tyrosine phosphorylation of FAK and ERK were increased in HT-1080 cells grown in presence of fibronectin. Processing of MT1-MMP was also observed in HT-1080 cells grown in presence of fibronectin. The reorganization of actin filaments in fibronectin treated HT-1080 cells was also noticeable. CONCLUSIONS: Our findings indicate that culture of HT-1080 cells in SFCM in presence of fibronectin perhaps generates a signaling cascade that leads to expression of pro-MMP-9 and activation of MMP-2 within 1 hr. The signaling pathway activated seems to be the FAK/ERK pathway.

Curcumin, a potential inhibitor of MMP-2 in human laryngeal squamous carcinoma cells HEp2.

UNLABELLED: Curcumin (diferuloylmethane) has been widely studied for its tumor inhibiting and anticarcino-genic properties. In the present communication, we studied the effect of curcumin on matrix-metalloproteinase-2 (MMP-2), integrin receptors, and focal adhesion kinase (FAK) in human laryngeal cancer cells, HEp2. METHODS: HEp2 cells were treated with curcumin (5 microM) for 30 days and then grown without curcumin for 28 days. Effect of curcumin on MMP-2 expression and activity and on membrane type matrixmetalloproteinase-1 (MT1-MMP), FAK, and integrin receptors was studied by zymography, Western blot, ELISA, RT-PCR, and cell adhesion assay. RESULTS: Treatment of HEp2 cells with curcumin downregulated MMP-2 expression and activity and expression of integrin receptors, FAK, and MT1-MMP to almost background levels. MMP-2 (but not MMP-9) mRNA expression was abolished on curcumin treatment, indicating specific inhibition of MMP-2. Invasive potential of HEp2 cells was also significantly reduced. After drug withdrawal, expression of MMP-2, integrin receptors, MT1-MMP, and FAK returned to control levels. However, MMP-2 activity in serum free medium remained low. CONCLUSIONS: Downregulation of integrin receptors and low levels of FAK may hinder integrin-mediated signal transduction, preventing upregulation of MMP-2 activity. Reduction of MMP-2 activity and inhibition of HEp2 cell invasion by curcumin strongly indicate the potential of curcumin as an inhibitor of tumor cell invasion and metastasis.

Cell membrane-associated MT1-MMP-dependent activation of pro-MMP-2 in A375 melanoma cells.

UNLABELLED: Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, can degrade extracellular matrix components under physiological conditions and during cancer invasion and metastasis. Among the MMPs, the 72 kDa type IV collagenase MMP-2 (gelatinase A) is activated in a membrane-associated manner by an activation complex composed of membrane type 1 matrix metalloproteinase (MT1-MMP), tissue inhibitor of matrixmetalloproteinase-2 (TIMP-2), and pro-MMP-2 in the presence of alphavbeta3 integrin receptor. The activation of pro-MMP-2 correlates with increased occurrence of metastases. Increased MMP-2 activity has been demonstrated in many human tumors. In the present communication, we studied cell surface-associated activation of MMP-2 (72 kDa collagenase type IV) in the moderately metastatic human melanoma cell line A375. RESULTS: Activation of purified 72 kDa collagenase type IV, pro-MMP-2 from cervical cancer tissue homogenate and from serum-free culture medium of HT1080 cells grown in presence of concanavalin A, by A375 cells, was shown by gelatin zymography. A375 cells activated only pro-MMP-2 from purified MMP-9/MMP-2 mixture indicating that the activation is specific for MMP-2. Activation of MMP-2 and purified collagenase type IV by A375 membrane fraction and membrane extract was also demonstrated by gelatin zymography. When A375 cells were first incubated with anti-MT1-MMP polyclonal antibody, activation of collagenase type IV was significantly decreased, indicating that membrane-associated MMP-2 activation is MT1-MMP-mediated. Immunocytochemistry showed MT1-MMP localization at focal adhesion sites. The presence of the components of activation complex-MT1-MMP and integrin alphavbeta3-were confirmed by Western blot, cell adhesion assay, and integrin subunit assay. CONCLUSION: Our experimental findings furnish another example of the unique membrane-associated MMP-2 activation mechanism in A375 melanoma cells and clearly indicate the role of MT1-MMP in MMP-2 activation. The information could help in developing new therapies designed to interfere with MMP activation and management of cancer and metastases.

Binding of alpha2 monoclonal antibody to human cervical tumor cell (SiHa) surface alpha2beta1 integrin modulates MMP-2 activity.

OBJECTIVE(S): The purpose was to study the interrelationship between cell surface integrin receptor (alpha2beta1) and matrixmetalloproteinases. METHODS: Immunoprecipitation and cell adhesion assay were done to assay alpha2beta1 and alpha3beta1 on SiHa cell surface. Zymogram was developed to assay secreted MMP activity of cells grown in presence of alpha2 monoclonal antibody. Immunoblot was developed to assay expression of MMP-2, FAK, and p-FAK. Plasma membrane-dependent activation of MMP2 was performed by incubating pure MMP-2 with membrane-enriched fraction isolated from SiHa cells. RESULTS: Immunoprecipitation and cell adhesion assay results confirmed the presence of alpha2beta1 receptor on SiHa cells. Zymographic analysis of serum-free media collected at different time points from SiHa cells grown on alpha2 monoclonal antibody-coated culture dishes showed the expression and activation of MMP-2 within 2-4 h, confirmed by immunoblot. Western blot of cells grown on alpha2-coated dishes for 30 min-4 h showed increased phosphorylation of FAK. Membrane-enriched fraction isolated from SiHa cells was found to specifically activate proMMP-2 to its activated forms within 30 min. CONCLUSION(S): The experimental findings strongly indicate that SiHa cell surface alpha2beta1 regulates MMP-2 expression. Increased phosphorylation of focal adhesion kinase (FAK) strongly indicates the possible role of FAK in signaling cascade. Incubation of SiHa cell membrane fraction with pure MMP-2 strongly confirms the cell membrane-dependent activation of proMMP2.

Effect of curcumin on gelatinase A (MMP-2) activity in B16F10 melanoma cells.

Treatment of highly metastatic murine melanoma cells B16F10 with curcumin (15 microM) for 15 days significantly inhibited matrixmetalloproteinase-2 (MMP-2) activity. Expression of membrane type-1 matrix metalloproteinase (MT1-MMP) and focal adhesion kinase (FAK), an important component of the intracellular signalling pathway, were also reduced to almost background levels. MMP-2, MT1-MMP and FAK did not return to control levels even after 28 days of drug withdrawal. However, effect of curcumin on ligand binding property of integrin receptors was reversible. Downregulation of FAK (which would impair integrin mediated signal transduction cascade) and reduction of MMP-2 activity could be important reasons for anti-metastatic property of curcumin.