Role of matrix metalloproteinase-7 (matrilysin) in human cancer invasion, apoptosis, growth, and angiogenesis

Matrix metalloproteinase (MMP)-7, also known as matrilysin, is a "minimal domain MMP" that exhibits proteolytic activity against components of the extracellular matrix (ECM). Matrilysin is frequently overexpressed in human cancer tissues and is associated with cancer progression. Tumorigenesis is a multistep process involving cell growth, invasion, metastasis, and angiogenesis. Matrilysin has been shown to play important roles not only in degradation of ECM proteins, but also in the regulation of several biochemical processes such as activation, degradation, and shedding of non-ECM proteins. This minire-view provides a summary of the current literature on the roles of matrilysin in tumorigenesis with a focus on the roles of modifications of non-ECM proteins by matrilysin and other related MMPs in tumorigenesis. Proteolysis of insulin-like growth factor binding protein by matrilysin results in increased bioavailability of insulin-like growth factors and enhanced cellular proliferation. Matrilysin has also been implicated in the ectodomain shedding of several cell surface molecules. Heparin-binding epidermal growth factor precursor (proHB-EGF) is cleaved by matrilysin into mature HB-EGF, which promotes cellular proliferation. Membrane-bound Fas ligand (FasL) is cleaved into soluble FasL, which increases apoptosis of cells adjacent to tumor cells. E-cadherin is converted to soluble E-cadherin to promote invasion. Tumor necrosis factor (TNF)-alpha precursor is cleaved to release soluble TNF-alpha to increase apoptosis. We propose that these matrilysin-mediated pathways provide the necessary and logical mechanisms to promote cancer progression.     

Osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin)

Osteopontin (OPN) is a secreted phosphoprotein shown to function in wound healing, inflammation, and tumor progression. Expression of OPN is often co-localized with members of the matrix metalloproteinase (MMP) family. We report that OPN is a novel substrate for two MMPs, MMP-3 (stromelysin-1) and MMP-7 (matrilysin). Three cleavage sites were identified for MMP-3 in human OPN, and two of those sites were also cleaved by MMP-7. These include hydrolysis of the human Gly166-Leu167, Ala201-Tyr202 (MMP-3 only), and Asp210-Leu211 peptide bonds. Only the N-terminal Gly-Leu cleavage site is conserved in rat OPN (Gly151-Leu152). These sites are distinct from previously reported cleavage sites in OPN for the proteases thrombin or enterokinase. We found evidence for the predicted MMP cleavage fragments of OPN in vitro in tumor cell lines, and in vivo in remodeling tissues such as the postpartum uterus, where OPN and MMPs are co-expressed. Furthermore, cleavage of OPN by MMP-3 or MMP-7 potentiated the function of OPN as an adhesive and migratory stimulus in vitro through cell surface integrins. We predict that interaction of MMPs with OPN at tumor and wound healing sites in vivo may be a mechanism of regulation of OPN bioactivity.     

Identification of novel matrix metalloproteinase-7 (matrilysin) cleavage sites in murine and human Fas ligand

Soluble Fas ligand (sFasL) is released from the cell surface by matrix metalloproteinases (MMPs), one of which is MMP-7. We have reported that MMP-7-generated sFasL is pro-apoptotic in both in vitro and in vivo systems. However, there are contradictory reports that the soluble form of FasL is inactive or anti-apoptotic, resulting in significant controversy in the literature. One potential explanation for these discrepancies is that forms of sFasL with different amino-terminal sequences have been demonstrated to have varying activities. Here we report that MMP-7 cleaves murine and human FasL at sites that are distinct from previously reported cleavage sites resulting in production of novel forms of sFasL. Cleavage of FasL by MMP-7 occurs at the leucine residues in the sequence "ELAELR" within the region between the transmembrane and trimerization domains. When this site is unavailable, a more c-terminal site, "SL," is cleaved. MMP-7 differentially processes murine and human FasL since it cleaves human FasL not only at the "ELAELR" site but also at a previously identified site. Additionally, MMP-3, but not MMP-2, was found to have the same cleavage specificity for murine FasL as MMP-7. We conclude that the controversy regarding the biological activity of sFasL may be explained, in part, by the generation of distinct forms of sFasL as a result of cleavage at specific sequences.     

Production of metalloproteinase-7 (matrilysin) by human myeloma cells and its potential involvement in metalloproteinase-2 activation.

Matrix metalloproteinases (MMPs) play a critical role in bone remodeling and tumor spreading. Multiple myeloma (MM) is a plasma cell malignancy primarily localized within the bone marrow and characterized by its capacity to destroy bone matrix and to disseminate. We have reported recently that human myeloma cells were able to induce the conversion of pro-MMP-2 produced by the tumoral environment in its activated form. In the current study, we have investigated the mechanism involved in this process. We demonstrate that a soluble MMP constitutively produced by myeloma cells was responsible for pro-MMP-2 activation. Furthermore, we show that the soluble MMP, MMP-7, also known as matrilysin, was able to activate the MMP-2 produced in its latent form by bone marrow stromal cells. Finally, we demonstrate that myeloma cells constitutively produce MMP-7 with expected proteolytic activity. Our results suggest that MMP-7 produced by myeloma cells could participate in bone destruction and tumor spreading in MM, on one hand by its own proteolytic activity and on the other hand by its capacity to activate pro-MMP-2. These findings strengthen the idea that inhibition of MMP activity could represent an interesting therapeutic approach in MM.     

Morusin induces apoptosis and suppresses NF-kappaB activity in human colorectal cancer HT-29 cells

Morusin is a pure compound isolated from root bark of Morusaustralis (Moraceae). In this study, we demonstrated that morusin significantly inhibited the growth and clonogenicity of human colorectal cancer HT-29 cells. Apoptosis induced by morusin was characterized by accumulation of cells at the sub-G₁ phase, fragmentation of DNA, and condensation of chromatin. Morusin also inhibited the phosphorylation of IKK-α, IKK-β and IκB-α, increased expression of IκB-α, and suppressed nuclear translocation of NF-κB and its DNA binding activity. Dephosphorylation of NF-κB upstream regulators PI3K, Akt and PDK1 was also displayed. In addition, activation of caspase-8, change of mitochondrial membrane potential, release of cytochrome c and Smac/DIABLO, and activation of caspase-9 and -3 were observed at the early time point. Downregulation in the expression of Ku70 and XIAP was exhibited afterward. Caspase-8 or wide-ranging caspase inhibitor suppressed morusin-induced apoptosis. Therefore, the antitumor mechanism of morusin in HT-29 cells may be via activation of caspases and inhibition of NF-κB.     

Rosiglitazone enhances the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells

Combined-modality treatment has improved the outcome in cases of various solid tumors, and radiosensitizers are used to enhance the radiotherapeutic efficiency. Rosiglitazone, a synthetic ligand of peroxisome proliferator-activated receptors γ used in the treatment of type-2 diabetes, has been shown to reduce tumor growth and metastasis in human cancer cells, and may have the potential to be used as a radiosensitizer in radiotherapy for human colorectal cancer cells. In this study, rosiglitazone treatment significantly reduced the cell viability of p53-wild type HCT116 cells but not p53-mutant HT-29 cells. Interestingly, rosiglitazone pretreatment enhanced radiosensitivity in p53-mutant HT-29 cells but not HCT116 cells, and prolonged radiation-induced G₂/M arrest and enhanced radiation-induced cell growth inhibition in HT-29 cells. Pretreatment with rosiglitazone also suppressed radiation-induced H2AX phosphorylation in response to DNA damage and AKT activation for cell survival; on the contrary, rosiglitazone pretreatment enhanced radiation-induced caspase-8, -9, and -3 activation and PARP cleavage in HT-29 cells. In addition, pretreatment with a pan-caspase inhibitor, zVAD-fmk, attenuated the levels of caspase-3 activation and PARP cleavage in radiation-exposed cancer cells in combination with rosiglitazone pretreatment. Our results provide proof for the first time that rosiglitazone suppresses radiation-induced survival signals and DNA damage response, and enhances the radiation-induced apoptosis signaling cascade. These findings can assist in the development of rosiglitazone as a novel radiosensitizer.     

Endothelium specific matrilysin (MMP-7) expression in human cancers.

Over-expression of matrilysin (MMP-7) is predominantly associated with epithelial (pre)malignant cells. In the present study MMP-7 expression is also found in endothelial cells in various human cancer types. Endothelial MMP-7 was associated with CD34 and/or CD105 expression. These immunohistochemical data were confirmed by RT-PCR on VEGF-stimulated endothelial cells. In addition, MMP-7 was also identified in sprouting endothelial cells in vitro. The potential clinical relevance of endothelial MMP-7 was assessed for cervical cancer patients by evaluating the association with overall survival. In contrast to MMP-7 in malignant epithelial cells, MMP-7 expression in endothelial cells showed a significant association with poor survival (LR 5.12, P=0.02, n=30). Our data suggest that MMP-7 is involved in tumor angiogenesis, thereby contributing to malignant growth and hence associated with decreased survival.     

Lack of cyclooxygenase-2 activity in HT-29 human colorectal carcinoma cells

Induction of cyclooxygenase-2 (COX-2) is an early event in the sequence of polyp formation to colon carcinogenesis. COX-2 is at elevated levels in human colorectal cancers and in tumors and polyps of mouse models of colorectal cancer. Mutation of the adenomatous polyposis coli (APC) gene is the initial event leading to colorectal cancer. Colorectal cells in culture which express mutant APC are often used to examine the association of COX-2 expression and apoptosis. The expression of full-length APC in HT-29 cells, a human colorectal carcinoma cell line which normally expresses truncated APC and highly expresses COX-2, inhibits cell growth through increased apoptosis and results in a down-regulation of COX-2 protein. In this report, we examine whether down-regulation of COX-2 is directly linked to the increase in apoptosis observed in these HT-29-APC cells. We present evidence that COX-2 and apoptosis are not linked since COX-2, although expressed, is catalytically inactive. Interestingly, the COX-2 cloned from HT-29 cells is catalytically active when transfected into HCT-116 cells, a colorectal cell line which normally does not express COX-2, but is not active in the HT-29 cell line itself.     

Syndecan-2 expression in colorectal cancer-derived HT-29 M6 epithelial cells induces a migratory phenotype

Members of the heparan sulfate proteoglycan family, the syndecans have emerged as integrators of extracellular signals, such as ECM components or growth factors, that activate cytoplasmic signaling cascades and regulate cytoskeletal functions. Specifically, syndecan-2 has been implicated in various cellular processes, from differentiation to migration, including its participation in cell-cell and cell-matrix adhesion. Here, we focused on the involvement of syndecan-2 in epithelial versus mesenchymal differentiation. Colorectal cancer-derived HT-29 M6 epithelial cells were stably transfected with full-length syndecan-2 cDNA, and the effect on cell morphology, adhesion, and mobility was evaluated. Characteristic features of migratory cells such as loss of intercellular contacts, flatter shape and multiple membrane projections were observed in syndecan-2 transfectants. Western blot analysis of the major component of epithelial adherens junctions, E-cadherin, revealed decreased expression levels. Furthermore, syndecan-2 induced stronger adhesion to collagen type I, specifically inhibited by heparin. This was correlated with an increased ability for migration, as demonstrated by wound healing experiments and transwell assays, without affecting their growth rate. These results indicate that syndecan-2 expression in mucus-secreting HT-29 M6 cells induces differentiation toward a migratory mesenchymal-like phenotype.     

Effect of Carica papaya on beta catenin and Wnt mRNA expression in human colon cancer (HT-29) cells in vitro

Colon cancer is the third most frequent cancer in humans. Carica papaya leaves are vegetable foods consumed by most people around the world; it has potential as an anticancer. Therefore it is of interest to investigate the effect of Carica papaya on beta catenin and Wnt mRNA expression in human colon cancer (HT-29) cells in vitro. Human Colon cancer cell line (HT-29) was purchased from the National Centre for Cell Sciences, Pune, India. Cell viability test was done by MTT assay. Gene expression analysis was done by Real Time-PCR. The obtained data were analyzed statistically by one-way analysis of variance and Duncan''s multiple range test with Graph Pad Prism version 5 to analyze the significance of individual variations among the control and experimental groups. The significance was considered at p<0.05 level in Duncan''s test. Carica papaya caused a marked increase in cell death in a dose dependent manner. At the end of 48 hours, maximum inhibition was at 300 and 400 µg/ml. Carica papaya has significantly reduced the mRNA expression of Wnt and beta catenin (p<0.05). Data showed that Carica papaya leaf extract has anticancer activity on Colon cancer cell lines (HT-29).     

Inhibitory effects of alcohols on the activity of human matrix metalloproteinase 7 (matrilysin)

Aliphatic alcohols inhibited the activity of human matrix metalloproteinase 7 (matrilysin) competitively with K(i) of 6.1-19.4% (v/v) or 0.66-4.80 M. From the relationship between the structures of alcohols and their K(i) values, alcohols are considered to bind the hydrophobic S1' subsite most plausibly, and the size of the pocket was estimated to be large enough to accommodate the length of 1-butanol (4-carbon chain) and the bulk of tertiary alcohols. Alcohols might be suitable probes for exploring the active-site geometry of enzymes.     

Interactions of human matrix metalloproteinase 7 (matrilysin) with the inhibitors thiorphan and R-94138

The effects of the metalloproteinase inhibitors thiorphan and R-94138 on the matrilysin-catalyzed hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2) [MOCAc-PLGL(Dpa)AR] were examined. The inhibitor constants (K(i)) of thiorphan and R-94138 for matrilysin at pH 7.5, 25 degrees C were determined to be 11.2 and 7.65 microM, respectively. From the temperature dependence of the K(i) values at pH 7.5, the standard enthalpy change (Delta H degrees ') values for the binding of matrilysin with thiorphan and R-94138 were determined to be -(18.2 +/- 0.9) and (1.65 +/- 1.07) kJ x mol(-1), respectively. The binding of matrilysin to thiorphan is exothermic and the free energy change in the complex formation depends mainly on the change in enthalpy, while the binding to R-94138 is endothermic and typically entropy-driven. Hydrophobic interactions are suggested to contribute significantly to the binding of matrilysin to R-94138 as well as to the substrate. The pH dependence of the K(i) value suggests that at least two ionizing groups with pK(a) values of 4.5 and 9.1--9.3 are involved in the binding. The matrilysin activity is regulated by ionizing groups with pK(a) values of 4.3 and 9.6. Both inhibition and hydrolysis are suggested to be controlled by the same residues in matrilysin, most likely Glu 198 and Tyr 219, respectively.     

UDN glycoprotein inhibits activator protein-1 and matrix metalloproteinase-9 via blocking of oxygen radicals in HT-29 cells

The present study was performed to investigate the anti-inflammatory potential of a 116-kDa glycoprotein isolated from Ulmus davidiana Nakai (UDN glycoprotein) in lipopolysaccaride (LPS)-treated cancerous human colon epithelial cells (HT-29 cells). UDN glycoprotein inhibited the production of intracellular superoxide anion (O₂^·−), hydrogen peroxides (H₂O₂), and nitric oxide (NO), whereas normalized the activity of anti-oxidant enzymes [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX)], accompanying the inhibition of manganese-superoxide dismutases (Mn-SOD) activity in LPS-treated HT-29 cells. In addition, UDN glycoprotein blocked the DNA binding activity of activator protein-1 (AP-1) through suppression of c-Jun and c-Fos activities, respectively. We also evaluated the anti-inflammatory potential of UDN glycoprotein based on the activity of the pro-inflammatory signal mediators [inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and matrix metalloproteinases-9 (MMP-9)]. The results showed that UDN glycoprotein (200 μg/ml) has an inhibitory effect on the activation of iNOS, COX-2, and MMP-9 proteins in the LPS-treated HT-29 cells. From these results, we suggest that UDN glycoprotein is one of the potential anti-inflammatory agents that blocks LPS-mediated inflammatory signal pathway in HT-29 cells. Here, we speculate that UDN glycoprotein could be used as an antioxidative agent for inflammatory gastrointestinal cancers.     

Function of CSE1L/CAS in the secretion of HT-29 human colorectal cells and its expression in human colon

The secretion of colorectal epithelium is important for maintaining the physiological function of colorectal organ. Herein, we report that cellular apoptosis susceptibility (CAS) (or CSE1L) protein regulates the secretion of HT-29 human colorectal cells. Polarity is essential for directed secretion of substances produced by epithelial cells to the external (luminal) compartment; CAS overexpression induced polarization of HT-29 cells. CAS was punctate stained in the cytoplasm of HT-29 cells, and CAS overexpression increased the translocation of CAS-stained vesicles to the cytoplasm near cell membrane and cell protrusions. CAS overexpression increased the secretion of carcinoembryonic antigen (CEA) and cathepsin D. Immunohistochemistry showed CAS was positively stained in the goblet cells of colon mucosa and cells in the crypts of Lieberkühn of human colon as well as the glands in metastatic colorectal cancer tissue. Our results suggest that CAS regulates the secretion of colorectal cells and may regulate the metastasis of colorectal cancer.     

Nobiletin, a citrus flavonoid, suppresses invasion and migration involving FAK/PI3K/Akt and small GTPase signals in human gastric adenocarcinoma AGS cells

Nobiletin, a compound isolated from citrus fruits, is a polymethoxylated flavone derivative shown to have anti-inflammatory, antitumor, and neuroprotective properties. This study has investigated that nobiletin exerted inhibitory effects on the cell adhesion, invasion, and migration abilities of a highly metastatic AGS cells under non-cytotoxic concentrations. Data also showed nobiletin could inhibit the activation of focal adhesion kinase (FAK) and phosphoinositide-3-kinase/Akt (PI3K/Akt) involved in the downregulation of the enzyme activities, protein expressions, messenger RNA levels of matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-2 (MMP-9). Also, our data revealed that nobiletin inhibited FAK/PI3K/Akt with concurrent reduction in the protein expressions of Ras, c-Raf, Rac-1, Cdc42, and RhoA by western blotting, whereas the protein level of RhoB increased progressively. Otherwise, nobiletin-treated AGS cells showed tremendously decreased in the phosphorylation and degradation of inhibitor of kappaBα (IκBα), the nuclear level of NF-κB, and the binding ability of NF-κB to NF-κB response element. Furthermore, nobiletin significantly decreased the levels of phospho-Akt and MMP-2/9 in Akt1-cDNA-transfected cells concomitantly with a marked reduction in cell invasion and migration. These results suggest that nobiletin can reduce invasion and migration of AGS cells, and such a characteristic may be of great value in the development of a potential cancer therapy.     

The phosphoinositide 3-kinase inhibitor LY294002, decreases aminoacyl-tRNA synthetases, chaperones and glycolytic enzymes in human HT-29 colorectal cancer cells

The proposed anticancer drug LY294002, inhibits phosphoinositide-3 kinase (PI3K) that initiates a signalling pathway often activated in colorectal cancer (CRC). The effects of LY294002 (10 μM, 48 h) on the cytosolic, mitochondrial and nuclear proteomes of human HT-29 CRC cells have been determined using iTRAQ (isobaric tag for relative and absolute quantitation) and tandem mass spectrometry (MS/MS). Analysis of cells treated with LY294002 identified 26 differentially abundant proteins that indicate several mechanisms of action. The majority of protein changes were directly or indirectly associated with Myc and TNF-α, previously implicated in CRC progression. LY294002 decreased the levels of 6 aminoacyl-tRNA synthetases (average 0.39-fold) required for protein translation, 5 glycolytic enzymes (average 0.37-fold) required for ATP synthesis, and 3 chaperones required for protein folding. There was a 3.2-fold increase in lysozyme C involved in protein-glycoside hydrolysis. LY294002 increased cytosolic p53 with a concomitant decrease in nuclear p53, suggesting transfer of p53 to the cytosol where apoptosis might be initiated via the intrinsic mitochondrial pathway. Protein changes described here suggest that the anti-angiogenic effects of LY294002 may be related to p53; the mutational status of p53 in CRC may be an important determinant of the efficacy of PI3K inhibitors for treatment.