A Novel Gelatinase from Marine Flocculibacter collagenilyticus SM1988: Characterization and Potential Application in Collagen Oligopeptide-Rich Hydrolysate Preparation.

Although the S8 family in the MEROPS database contains many peptidases, only a few S8 peptidases have been applied in the preparation of bioactive oligopeptides. Bovine bone collagen is a good source for preparing collagen oligopeptides, but has been so far rarely applied in collagen peptide preparation. Here, we characterized a novel S8 gelatinase, Aa2_1884, from marine bacterium Flocculibacter collagenilyticus SM1988T, and evaluated its potential application in the preparation of collagen oligopeptides from bovine bone collagen. Aa2_1884 is a multimodular S8 peptidase with a distinct domain architecture from other reported peptidases. The recombinant Aa2_1884 over-expressed in Escherichia coli showed high activity toward gelatin and denatured collagens, but no activity toward natural collagens, indicating that Aa2_1884 is a gelatinase. To evaluate the potential of Aa2_1884 in the preparation of collagen oligopeptides from bovine bone collagen, three enzymatic hydrolysis parameters, hydrolysis temperature, hydrolysis time and enzyme-substrate ratio (E/S), were optimized by single factor experiments, and the optimal hydrolysis conditions were determined to be reaction at 60 ℃ for 3 h with an E/S of 400 U/g. Under these conditions, the hydrolysis efficiency of bovine bone collagen by Aa2_1884 reached 95.3%. The resultant hydrolysate contained 97.8% peptides, in which peptides with a molecular weight lower than 1000 Da and 500 Da accounted for 55.1% and 39.5%, respectively, indicating that the hydrolysate was rich in oligopeptides. These results indicate that Aa2_1884 likely has a promising potential application in the preparation of collagen oligopeptide-rich hydrolysate from bovine bone collagen, which may provide a feasible way for the high-value utilization of bovine bone collagen.

The role of fibroblast activation protein in health and malignancy.

Fibroblast activation protein-α (FAP) is a type-II transmembrane serine protease expressed almost exclusively to pathological conditions including fibrosis, arthritis, and cancer. Across most cancer types, elevated FAP is associated with worse clinical outcomes. Despite the clear association between FAP and disease severity, the biological reasons underlying these clinical observations remain unclear. Here we review basic FAP biology and FAP's role in non-oncologic and oncologic disease. We further explore how FAP may worsen clinical outcomes via its effects on extracellular matrix remodeling, intracellular signaling regulation, angiogenesis, epithelial-to-mesenchymal transition, and immunosuppression. Lastly, we discuss the potential to exploit FAP biology to improve clinical outcomes.

The gelatinase MMP-9like is involved in regulation of LPS inflammatory response in Ciona robusta.

Matrix metalloproteinases (MMPs) are a family of endopeptidases collectively able to degrade the components of the extracellular matrix (ECM), with important roles in many biological processes, such as embryogenesis, normal tissue remodelling, angiogenesis and wound healing. New views on the function of MMPs reveal that they regulate inflammatory response and therefore might represent an early step in the evolution of the immune system. MMPs can affect the activity of cytokines involved in inflammation including TGF-ß and TNF-α. MMPs are widely distributed in all kingdoms of life and have likely evolved from a single-domain protein which underwent successive rounds of duplications. In this study, we focused on the Ciona robusta (formerly known as Ciona intestinalis) MMP gelatinase homologue. Gene organization, phylogenetic analysis and 3D modeling supported the closest correlation of C. robusta gelatinase with the human MMP-9. Real-time PCR analysis and zymographic assay showed a prompt expression induced by LPS inoculation and an upregulation of enzymatic activity. Furthermore, we showed that before of the well-known increase of TGF-ß and TNF-α levels, a MMP-9like boost occurred, suggesting a possible involvement of MMP-9like in regulating inflammatory response in C. robusta.

Characterization of gelatinase produced by Antarctic Mrakia sp.

In the present study, 20 psychrotolerant yeast species isolated from the soils of King George Island in the sub-Antarctic region were evaluated for the production of extracellular gelatinase, an enzyme with high potential for applications in diverse areas, such as food and medicine. The production of extracellular gelatinase was confirmed in the yeasts Metschnikowia sp., Leucosporidium fragarium, and Mrakia sp., the last one being the yeast in which the highest gelatinase activity was detected. The enzyme was purified from cultures of Mrakia sp., and the effect of different physical-chemical factors on its activity was determined. The gelatinase produced by Mrakia sp. would correspond to a protein of relative molecular weight (rMW) 37,000, which displayed the highest activity at 36°C, pH 7.0, 10 mM CaCl 2 , and 5 mM ZnSO 4 .

In†Situ Self-Assembled Nanofibers Precisely Target Cancer-Associated Fibroblasts for Improved Tumor Imaging.

Tumor complexity makes the development of highly sensitive tumor imaging probes an arduous task. Here, we construct a peptide-based near-infrared probe that is responsive to fibroblast activation protein-α (FAP-α), and specifically forms nanofibers on the surface of cancer-associated fibroblasts (CAFs) in situ. The assembly/aggregation-induced retention (AIR) effect results in enhanced accumulation and retention of the probe around the tumor, resulting in a 5.5-fold signal enhancement in the tumor 48 h after administration compared to that of a control molecule that does not aggregate. The probe provides a prolonged detectable window of 48 h for tumor diagnosis. The selective assembly of the probe results in a signal intensity over four- and fivefold higher in tumor than in the liver and kidney, respectively. With enhanced tumor imaging capability, this probe can visualize small tumors around 2 mm in diameter.

A specific, transmembrane interface regulates fibroblast activation protein (FAP) homodimerization, trafficking and exopeptidase activity.

Fibroblast activation protein (FAP) is a cell-surface serine protease which promotes invasiveness of certain epithelial cancers and is therefore a potential target for cancer drug development and delivery. Unlike dipeptidyl peptidase IV (DPPIV), FAP exhibits prolyl endopeptidase activity and is active as a homodimer with specificity for type I collagen. The mechanism that regulates FAP homodimerization and its relation to prolyl endopeptidase activity is not completely understood. Here, we investigate key residues in the FAP TM domain that may be significant for FAP homodimerization. Mutations to predicted TM interfacial residues (G10L, S14L, and A18L) comprising a small-X3-small motif reduced FAP TM-CYTO dimerization relative to wild type as measured using the AraTM assay, whereas predicted off-interface residues showed no significant change from wild type. The results implied that the predicted small-X3-small dimer interface affect stabilization of FAP TM-CYTO homodimerization. Compared with FAPwild-type, the interfacial TM residue G10L significantly decreased FAP endopeptidase activity more than 25%, and also reduced cell-surface versus intracellular expression relative to other interfacial residues S14L and A18L. Thus, our results suggest FAP dimerization is important for both trafficking and protease activity, and is dependent on a specific TM interface.

Inhibition of pro-/active MMP-2 by green tea catechins and prediction of their interaction by molecular docking studies.

Matrix metalloproteinases (MMPs) play a crucial role in developing different types of lung diseases, e.g., pulmonary arterial hypertension (PAH). Green tea polyphenolic catechins such as EGCG and ECG have been shown to ameliorate various types of diseases including PAH. Our present study revealed that among the four green tea catechins (EGCG, ECG, EC, and EGC), EGCG and ECG inhibit pro-/active MMP-2 activities in pulmonary artery smooth muscle cell (PASMC) culture supernatant. Based on the above, we investigated the interactions of pro-/active MMP-2 with the green tea catechins by computational methods. In silico analysis revealed a strong interaction of pro-/active MMP-2 with EGCG/ECG, and galloyl group has been observed to be responsible for this interaction. The in silico analysis corroborated our experimental observation that EGCG and ECG are active in preventing both the proMMP-2 and MMP-2 activities. Importantly, these two catechins appeared to be better inhibitors for proMMP-2 in comparison to MMP-2 as revealed by gelatin zymogram and also by molecular docking studies. In many type of cells, activation of proMMP-2 occurs via an increase in the level of MT1-MMP (MMP-14). We, therefore, determined the interactions of MT1-MMP with the green tea catechins by molecular docking analysis. The study revealed a strong interaction of MT1-MMP with EGCG/ECG, and galloyl group has been observed to be responsible for the interaction.

Preventive Effects of Rhodiola rosea L. on Bleomycin-Induced Pulmonary Fibrosis in Rats.

Rhodiola rosea L. (RRL) possesses a wide range of pharmacological properties, including lung-protective activity, and has been utilized in folk medicine for several 100 years. However, the lung-protective mechanism remains unclear. This study investigated the possible lung-protective activity mechanism of RRL in a pulmonary fibrosis (PF) rat model. Lung fibrotic injury was induced in Sprague-Dawley rats by single intratracheal instillation of saline containing bleomycin (BLM; 5 mg/kg). The rats were administered 125, 250, or 500 mg/kg of a 95% ethanol extract of RRL for 28 days. The animals were killed to detect changes in body weight, serum levels of glutathione (GSH) and total superoxide dismutase (T-SOD), as well as lung tissue hydroxyproline (HYP) content. Tumor necrosis factor-α (TNF-α), transforming growth factor-ß1 (TGF-ß1), and interleukin 6 (IL-6) levels were measured in bronchoalveolar lavage fluid (BALF) by enzyme-linked immunosorbent assay. Hematoxylin and eosin, Masson's trichrome, and immunohistochemical staining were performed to observe the histopathological changes in lung tissues. Additionally, target-related proteins were measured by Western blotting. RRL alleviated the loss of body weight induced by instilling BLM in PF rats, particularly at the 500 mg/kg per day dose. RRL reduced HYP (p < 0.01) and increased GSH and T-SOD contents. BALF levels of TNF-α, TGF-ß1, and IL-6 decreased significantly in the RRL-treated groups. Expression levels of matrix metalloproteinase-9 (MMP-9) and α-smooth muscle actin decreased significantly in a dose-dependent manner in response to RRL. Moreover, the levels of TGF-ß1 and tissue inhibitor of metalloproteinase-1 in lung tissues also decreased in the RRL-treated groups. RRL alleviated BLM-induced PF in rats. Our results reveal that the protective effects of RRL against fibrotic lung injury in rats are correlated with its anti-inflammatory, antioxidative, and anti-fibrotic properties. MMP-9 may play important roles in BLM-induced PF.

Design and synthesis of potent hydroxamate inhibitors with increased selectivity within the gelatinase family.

MMP-2 is a validated target for the development of anticancer agents. Herein we describe the synthesis of a new series of potent phenylalanine derived hydroxamates, with increased MMP-2/MMP-9 selectivity compared to analogous hydroxamates described previously. Docking and molecular dynamics experiments have been used to account for this selectivity, and to clarify the role of the triazole ring in the binding process.

A new pepstatin-insensitive thermopsin-like protease overproduced in peptide-rich cultures of Sulfolobus solfataricus.

In this study, we gain insight into the extracellular proteolytic system of Sulfolobus solfataricus grown on proteinaceous substrates, providing further evidence that acidic proteases were specifically produced in response to peptide-rich media. The main proteolytic component was the previously isolated SsMTP (Sulfolobus solfataricus multi-domain thermopsin-like protease), while the less abundant (named SsMTP-1) one was purified, characterized and identified as the sso1175 gene-product. The protein revealed a multi-domain organization shared with the cognate SsMTP with a catalytic domain followed by several tandemly-repeated motifs. Moreover, both enzymes were found spread across the Crenarchaeota phylum and belonging to the thermopsin family, although segregated into diverse phylogenetic clusters. SsMTP-1 showed a 75-kDa molecular mass and was stable in the temperature range 50-90 °C, with optimal activity at 70 °C and pH 2.0. Serine, metallo and aspartic protease inhibitors did not affect the enzyme activity, designating SsMTP-1 as a new member of the pepstatin-insensitive aspartic protease family. The peptide-bond-specificity of SsMTP-1 in the cleavage of the oxidized insulin B chain was uncommon amongst thermopsins, suggesting that it could play a distinct, but cooperative role in the protein degradation machinery. Interestingly, predictions of the transmembrane protein topology of SsMTP and SsMTP-1 strongly suggest a possible contribution in signal-transduction pathways.

Proteolytic action of kallikrein-related peptidase 7 produces unique active matrix metalloproteinase-9 lacking the C-terminal hemopexin domains.

The gelatinases, matrix metalloproteinase (MMP)-9 and -2, are produced as latent, inactive enzymes that can be proteolytically activated by a number of proteases. In many normal and pathological conditions, where the expression of MMPs is deregulated, changes in the expression of other proteases have also been reported. Human kallikrein-related peptidase 7 (KLK7), a chymotryptic-like serine protease, is overexpressed in many different types of neoplastic conditions, which have also been shown to express high levels of both MMP-9 and -2. Since the activation of MMPs by KLK7 has never been examined, we sought to determine whether KLK7 can activate these MMPs. To test this hypothesis KLK7 was incubated with the recombinant MMPs and the products of the reaction were analyzed for their activity. Incubation of proMMP-9 with KLK7 resulted in the production of a novel truncated, active MMP-9 lacking the C-terminal hemopexin domains. In contrast, KLK7 degraded, but did not activate, proMMP-2. The novel activation of proMMP-9 by KLK7 was further confirmed using conditioned medium prepared from an MMP-9-expressing cell line, MDA-MMP-9. Our results clearly establish that KLK7 activates proMMP-9 to produce a novel truncated, active MMP-9 product not generated by other proteases. These findings suggest that KLK7 may play an important role in the activation of MMP-9 in tumors that express high levels of both these proteases and the resulting truncated MMP may possess altered substrate specificities compared with full-length MMP-9 activated by other proteases.

Activatable near-infrared fluorescent probe for in vivo imaging of fibroblast activation protein-alpha.

Fibroblast activation protein-alpha (FAPα) is a cell surface glycoprotein which is selectively expressed by tumor-associated fibroblasts in malignant tumors but rarely on normal tissues. FAPα has also been reported to promote tumor growth and invasion and therefore has been of increasing interest as a promising target for designing tumor-targeted drugs and imaging agents. Although medicinal study on FAPα inhibitors has led to the discovery of many FAPα-targeting inhibitors including a drug candidate in a phase II clinical trial, the development of imaging probes to monitor the expression and activity of FAPα in vivo has largely lagged behind. Herein, we report an activatable near-infrared (NIR) fluorescent probe (ANP(FAP)) for in vivo optical imaging of FAPα. The ANP(FAP) consists of a NIR dye (Cy5.5) and a quencher dye (QSY21) which are linked together by a short peptide sequence (KGPGPNQC) specific for FAPα cleavage. Because of the efficient fluorescence resonance energy transfer (FRET) between Cy5.5 and QSY21 in ANP(FAP), high contrast on the NIR fluorescence signal can be achieved after the cleavage of the peptide sequence by FAPα both in vitro and in vivo. In vitro assay on ANP(FAP) indicated the specificity of the probe to FAPα. The in vivo optical imaging using ANP(FAP) showed fast tumor uptake as well as high tumor to background contrast on U87MG tumor models with FAPα expression, while much lower signal and tumor contrast were observed in the C6 tumor without FAPα expression, demonstrating the in vivo targeting specificity of the ANP(FAP). Ex vivo imaging also demonstrated ANP(FAP) had high tumor uptake at 4 h post injection. Collectively, these results indicated that ANP(FAP) could serve as a useful NIR optical probe for early detection of FAPα expressing tumors.

The potential mechanism for the different expressions of gelatinases induced by all-trans retinoic acid in different cells.

Gelatinases include matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). The increased expressions of gelatinases are implicated in the pathogenesis of cell injury and cell death. All-trans retinoic acid (ATRA) is an import biological agent which can regulate the expressions of gelatinases and take part in cell injury and cell death. ATRA exerts its biological effect by the high-affinity binding to retinoic acid receptors (RARs). The RARs consist of three isoforms: RAR-α, RAR-ß and RAR-γ. However, it is interesting that the effect of ATRA on the expressions of gelatinases is different in different cells. There is no report to explore the possible mechanism for it at present. In this context, we review the published reports and draw a hypothesis that: (i) The distributions of RARs isoforms are different in different cells; (ii) ATRA activates the different RARs isoforms in different cells; (iii) The roles of different RARs isoforms for regulating the expression of MMP-2 or MMP-9 are different in different cells. So, ATRA takes a different function on the expressions of MMP-2 and MMP-9 in different cells. Once the potential strategy can be successfully confirmed, it would be prone to comprehend why the ATRA regulates the different expressions of gelatinases in different cells.

Ultrasensitive Fibroblast Activation Protein-α-Activated Fluorogenic Probe Enables Selective Imaging and Killing of Melanoma In Vivo.

Melanoma is a malignant cancer with a high risk of metastasis and continued increase in death rates over the past decades, and its prognosis is highly related to the disease's stage, while early detection and treatment of melanoma are significant to the improvement of its therapy outcome. Different from the traditional methods for disease diagnosis, enzyme-activated fluorescent probes were developed rapidly due to their high sensitivity and temporal-spatial ratio and have been widely applied in tumor detection, surgical navigation, and cancer-related research. Fibroblast activation protein-α (FAPα), a serine-type cell surface protease that plays important roles in cell invasion and extracellular matrix degradation, is widely involved in tumor progression such as malignant melanoma, so developing a FAPα activity-based molecular tool would be of great potential for the early diagnosis and therapy of melanoma. However, few fluorescent probes targeting FAPα have been applied in melanoma-related studies, and thus, the construction of FAPα activity-based fluorescent probes for melanoma detection is in urgent need. By incorporating the selective recognition unit with a red-emission fluorophore, cresyl violet, we herein report an ultrasensitive (limit of detection = 5.3 ng/mL) fluorogenic probe for FAPα activity sensing, named CV-FAP; the acquired probe showed a significantly higher binding affinity (15.7-fold) and overall catalytic efficiency (2.6-fold) when compared with those of the best reported FAPα probes. The good performance of CV-FAP made it possible to discriminate malignant melanoma cells and tumor-bearing mice from normal cells and mice with high contrast. More importantly, CV-FAP showed significant antitumor activity toward melanoma in cultured cells and tumor-bearing nude mice (over 95% inhibited tumor growth) with good safety, which made it an ideal theranostic agent for melanoma.

N-substituted homopiperazine barbiturates as gelatinase inhibitors.

Matrix metalloproteinases are implicated in a wide range of pathophysiological processes and potent selective inhibitors for these enzymes continue to be eagerly sought. 5,5-Disubstituted barbiturates hold promise as inhibitor types being stable in vivo and relatively selective for the gelatinases (MMP-2 and MMP-9). In this paper we describe the synthesis of 5-piperazine and -homopiperazine substituted barbiturates. The activity of these compounds as gelatinase inhibitors was evaluated using supernatants from 12-O-tetradecanoylphorbol-13-acetate (PMA)-stimulated HT-1080 cells as well as using recombinant human MMPs. N-Acyl homopiperazine compounds were found to be potent inhibitors of the gelatinases (range in nM) and generally more potent than the corresponding piperazine analogues. The panel of N-acyl homopiperazines was enlarged in order to exploit differences between the gelatinases at the S2' site in order to design MMP-2- or MMP-9-selective inhibitors. Compounds in this group exhibited single digit nano-molar potency and some selectivity between the two enzymes. Representative potent compounds were effective inhibitors of cancer cell migration.

Presence of 46 kDa gelatinase on the outer membrane of Leptospira.

Leptospira infection involves the adhesion of the bacteria followed by invasion of the host crossing the extracellular matrix barrier. In an effort to understand the molecular mechanism of this process, the possibility of occurrence of matrix degrading enzymes from Leptospira was investigated. Zymographic analysis showed that the outer membrane of Leptospires contains a gelatinase of average molecular size of 46 kDa. The gelatinase exhibited maximum activity at neutral pH and was inhibited by metal chelators such as EGTA, EDTA, and Orthophenanthroline and was activated by calcium, magnesium, zinc, and copper, suggesting that it is a membrane-associated neutral matrix metalloproteinase. Analysis of the production of the enzyme by various serovars showed that the pathogenic serovars expressed significant amount of this enzyme while nonpathogenic forms either did not express or showed only very low activity, suggesting that this enzyme may be associated with pathogenesis of leptospirosis.

Gene expression of microbial gelatinase activity for porcine gelatine identification.

In order to invent a porcine gelatine detection device using microbial resources, bacterial enzymes with a preference towards porcine gelatine and their candidate genes were evaluated. Five (n = 5) bacterial strains isolated from hot spring water and wet clay, Malaysia were screened for their gelatinase activity. The gelatinase enzyme was extracted and purified using ammonium sulphate precipitation prior to performing gelatinase assay on porcine, bovine and fish gelatine medium substrates. The G2 strain or Enterobacter aerogenes (Strain EA1) was selected for whole genome sequenced after showing a consistent trend of preference towards porcine gelatine. The gelatinase candidate gene gelEA1_9 was cloned and expressed. Based on one-way analysis of variance (ANOVA) with POST-HOC Duncan test (α = 0.05), the final product of gelEA1_9 was identified as a novel gelatinase. This gelatinase presented no significant difference in activity towards porcine gelatine. Hence, the present study demonstrated an enzyme-substrate interaction for porcine gelatine identification.

Hydrophilic residues surrounding the S1 and S2 pockets contribute to dimerisation and catalysis in human dipeptidyl peptidase 8 (DP8).

Dipeptidyl peptidase (DP) 8 belongs to the dipeptidyl peptidase IV gene family. DP8 has been implicated in immune function and asthma, although its biological function is yet unknown. Structures of the homologs, fibroblast activation protein (FAP) and DPIV, are known but the DP8 structure is yet to be resolved. To help characterise the DP8 substrate pocket, mutants of residues lining the pocket were produced at DP8(D772), DP8(Y315), DP8(H434) and DP8(D435) and assessed by substrate kinetics and size-exclusion chromatography. Mutations of DP8(D772A/E/S/V) affected catalysis but did not confer endopeptidase activity. Mutations of DP8(H434F), DP8(D435F) and DP8(Y315F) reduced catalytic activity. Furthermore, mutations to DP8(D772A/E/S/V), DP8(H434F), DP8(D435F) and DP8(Y315F) affected dimer stabilisation. Homology modelling of DP8 using DPIV and FAP crystal structures suggested that DP8(D772), DP8(H434) and DP8(D435) were located at the edge of the S2 catalytic pocket, contributing to the junction between the alpha-beta hydrolase and beta-propeller domains. This study provides insights into how the DP8 substrate pocket and dimer interface differ from DPIV and FAP which could be utilised for designing more selective DP8 inhibitors.

Co-purified gelatinases alter the stability and biological activities of human plasma fibronectin preparations.

BACKGROUND AND OBJECTIVE: Fibronectin (FN) is an important cell adhesion molecule that is used widely to characterize cell behavior. Preparations of FN purified from human plasma by gelatin-Sepharose affinity chromatography typically also contain gelatin-binding gelatinases that may cleave FN, reduce its stability and alter its biological activities. Available methods for separating gelatinases from FN are resource demanding. Therefore, our objective was to devise a time- and cost-efficient protocol for purification of gelatinase-free FN. MATERIAL AND METHODS: Experiments tested the elution profiles for FN and gelatinases from gelatin-Sepharose using a concentration range (1-7%) of dimethyl sulfoxide (DMSO) and 4 m urea as eluants. Subsequently, we explored the sequential application of those eluants for differential elution of gelatinases and FN using a single affinity column. Finally, experiments characterized the stability of purified FN with or without contaminating gelatinases, as well as the effects of FN degradation on cell attachment and migration. RESULTS: Assay optimization demonstrated that pre-elution with 3% DMSO efficiently eliminated gelatinases but not FN from gelatin-Sepharose, whereas subsequent elution with 4 m urea released FN. Sequential elutions with DMSO and urea produced gelatinase-free FN, which was more stable than FN eluted by urea only. Fibronectin degradation did not affect human gingival fibroblast attachment, but increased cell migration significantly. CONCLUSION: The present experiments devised a time- and cost-efficient protocol for eliminating gelatinases during purification of human plasma FN. Gelatinase-free FN preparations had greater stability, which may be essential for experiments because FN fragments have altered biological activities compared with intact FN.

[Methods for determining matrix metalloproteinase activity].

The paper describes methods for determining the activity of matrix metalloproteinases (MMP), primarily collagenases and gelatinases, by applying natural protein substrates. Reconstructed fluorescein-labeled type I collagen fibrils were used to determine collagenases as a substrate. The zymographic technique in polyacrylamide gel with copolymerized gelatin was employed for the identification and assay of gellatinases. These methods are rather sensitive, reproducible and may be used for screening. Collagenases specifically trigger the hydrolysis of fibrillar collagens; gelatinases are responsible for the hydrolysis of type IV collagen, the basis of basement membranes. MMP activity characterizes the development of a destructive or invasive process in the connective tissue matrix; this indicator is borne in mind when choosing therapeutic agents, has a prognostic value, determines targets for the development of pharmacological agents, and is required to understand the mechanism responsible for the destruction of matrix and for the development of invasion processes.