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Among matrix metalloproteinases (MMPs), gelatinases MMP-2 (gelatinase A) and MMP-9 (gelatinase B) play a key role in a number of physiological processes such as tissue repair and fibrosis. Many evidences point out their involvement in a series of pathological events, such as arthritis, multiple sclerosis, cardiovascular diseases, inflammatory processes and tumor progression by degradation of the extracellular matrix. To date, the identification of non-specific MMP inhibitors has made difficult the selective targeting of gelatinases. In this work we report the identification, design and synthesis of new gelatinase inhibitors with appropriate drug-like properties and good profile in terms of affinity and selectivity. By a detailed in silico protocol and innovative and versatile solid phase approaches, a series of 4-thiazolydinyl-N-hydroxycarboxyamide derivatives were identified. In particular, compounds 9a and 10a showed a potent inhibitory activity against gelatinase B and good selectivity over the other MMP considered in this study. The identified compounds could represent novel potential candidates as therapeutic agents., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The tumor microenvironment has emerged as a novel chemotherapeutic strategy in the treatment of cancer. This is most clearly exemplified by the antiangiogenesis class of compounds. Therapeutic strategies that target fibroblasts within the tumor stroma offer another treatment option. However, despite promising data obtained in preclinical models, such strategies have not been widely used in the clinical setting, largely due to a lack of effective treatments that specifically target this population of cells. The identification of fibroblast activation protein α (FAP) as a target selectively expressed on fibroblasts within the tumor stroma or on carcinoma-associated fibroblasts led to intensive efforts to exploit this novel cellular target for clinical benefit. FAP is a membrane-bound serine protease of the prolyl oligopeptidase family with unique post-prolyl endopeptidase activity. Until recently, the majority of FAP-based therapeutic approaches focused on the development of small-molecule inhibitors of enzymatic activity. Evidence suggests, however, that FAP's pathophysiological role in carcinogenesis may be highly contextual, depending on both the exact nature of the tumor microenvironment present and the cancer type in question to determine its tumor-promoting or tumor-suppressing phenotype. As an alternative strategy, we are taking advantage of FAP's restricted expression and unique substrate preferences to develop a FAP-activated prodrug to target the activation of a cytotoxic compound within the tumor stroma. Of note, this strategy would be effective independently of FAP's role in tumor progression because its therapeutic benefit would rely on FAP's localization and activity within the tumor microenvironment rather than strictly on inhibition of its function.

The concept of targeting antigens selectively expressed on the surface of tumor capillary endothelial cells or in tumor stroma has emerged as a promising strategy for cancer therapeutics. Identification of stromal targets for anticancer therapy and development of selective inhibitors of these targets are of great clinical interest. Fibroblast activation protein (FAP), a member of the serine protease family, selectively expressed in the stromal fibroblasts associated with epithelial cancers, whereas with low or undetectable expression in the resting fibroblasts of normal adult tissues. The proteolytic activity of FAP has been shown to support tumor growth and proliferation, making it a potential target for novel anticancer therapies, such as those by immune-based approaches.

Fibroblast activation protein-α (FAP) is a serine protease that can provide target specificity to therapeutic agents because in adults its expression is restricted to pathologic sites, including cancer, fibrosis, arthritis, wounding, or inflammation. It is not expressed in most normal tissues. The majority of FAP is expressed by activated fibroblasts responding to the pathologic situations. FAP is typically found as a type II transmembrane protein physically attached to cells and with the bulk of the protein, including the catalytic domain, exposed to the extracellular space and accessible to small molecules. In this chapter, we review the structure, substrate specificities, signaling functions, and current design of FAP inhibitors. Evidence indicating the presence of FAP in multiple cancers, arthritis, fibrosis, keloids, and other pathologies is described and indicates possible roles for FAP in facilitating cell invasion and growth. Separate sections are devoted to the role of FAP in coordinating the stromal response to cancer, including a role in angiogenesis and a potential role in modulation of the antitumor immune response. Finally studies attempting to demonstrate the clinical potential of FAP are discussed, as well as some novel applications employing FAP in therapy or diagnosis. Throughout this review, effort is made to highlight areas where information is lacking and to highlight important questions that require further investigation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The interest in gelatinases is increased because of their association in diverse human diseases, though the relationship between MMP expression and disease progression is very complex and varies in cell to cell. Targeting gelatinases in disease treatment is complicated by the fact that gelatinases are indispensable for normal development and physiology due to their multifunctionality, possible functional redundancy, context-dependent expression, and activity. They are secreted as inactive zymogens which are processed to become active by removal of N-terminal propeptide. The folded conformation of zymogen is required to keep the gelatinases in its latency. Acting on a broad spectrum of extracellular substrates, the gelatinases (both MMP-2 and MMP-9) are critical to the biological processes. Three-dimensional structures of gelatinase-inhibitor complexes and inhibition profiles of compounds screened on them provide an invaluable source to gain insight into the structural determinants as well as functional selectivity. The quest for selective MMP inhibitors (MMPIs) still remains a challenge in search of successful clinical candidates. An increased understanding of the structure, regulation, and function of the individual MMPs will likely lead to more effective strategies in the development of highly selective inhibitors for any given MMP that can then be exploited to achieve the desired drugs.

1. The potential of the matrix metalloproteinase (MMP) inhibitor ABT-518 to affect pre-adipocyte differentiation in vitro and adipose tissue development in vivo was investigated using mouse models of adipogenesis and obesity. 2. Differentiation of 3T3-F442A pre-adipocytes into mature adipocytes was enhanced in a dose-dependent manner by the addition of ABT-518 (0-100 μmol/L). This was associated with increased expression of the adipogenic markers adipocyte fatty acid-binding protein 2 (AP2), peroxisome proliferator-activated receptor γ and adiponectin. 3. Feeding 5-week-old male wild-type mice with a high-fat diet, with or without ABT-518 (to achieve a dose of 100 mg/kg per day), for 16 weeks resulted in a significant reduction in bodyweight throughout the experimental period. Magnetic resonance spectroscopy revealed that the lipid : water ratio was significantly lower in ABT-518-treated mice. The total weight of isolated subcutaneous or gonadal fat depots did not differ significantly following ABT-518 treatment, but adipocyte and blood vessel size were significantly reduced in the gonadal fat. 4. Administration of ABT-518-2 (100 mg/kg per day for 10 weeks) to 5-week-old male wild-type mice with established obesity maintained on a high-fat diet had no effect on total bodyweight at the end of the experiment, but was associated with reduced blood vessel size in the fat depots. 5. Thus, the MMP inhibitor ABT-518 stimulates differentiation of 3T3-F442A pre-adipocytes in vitro. It mildly reduces bodyweight gain in a murine model of diet-induced obesity, but does not affect established obesity., (© 2011 The Authors. Clinical and Experimental Pharmacology and Physiology © 2011 Blackwell Publishing Asia Pty Ltd.)

Aims: Collagen degradation in atherosclerotic plaques with thin fibrous caps renders them more prone to rupture. Fibroblast activation protein (FAP) plays a role in arthritis and tumour formation through its collagenase activity. However, the significance of FAP in thin-cap human fibroatheromata remains unknown., Methods and Results: We detected enhanced FAP expression in type IV-V human aortic atheromata (n = 12), compared with type II-III lesions (n = 9; P < 0.01) and healthy aortae (n = 8; P < 0.01) by immunostaining and western blot analyses. Fibroblast activation protein was also increased in thin-cap (<65 µm) vs. thick-cap (≥ 65 µm) human coronary fibroatheromata (n = 12; P < 0.01). Fibroblast activation protein was expressed by human aortic smooth muscle cells (HASMC) as shown by colocalization on immunofluorescent aortic plaque stainings (n = 10; P < 0.01) and by flow cytometry in cell culture. Although macrophages did not express FAP, macrophage burden in human aortic plaques correlated with FAP expression (n = 12; R(2)= 0.763; P < 0.05). Enzyme-linked immunosorbent assays showed a time- and dose-dependent up-regulation of FAP in response to human tumour necrosis factor α (TNFα) in HASMC (n = 6; P < 0.01). Moreover, supernatants from peripheral blood-derived macrophages induced FAP expression in cultured HASMC (n = 6; P < 0.01), an effect abolished by blocking TNFα (n = 6; P < 0.01). Fibroblast activation protein associated with collagen-poor regions in human coronary fibrous caps and digested type I collagen and gelatin in vitro (n = 6; P < 0.01). Zymography revealed that FAP-mediated collagenase activity was neutralized by an antibody directed against the FAP catalytic domain both in HASMC (n = 6; P < 0.01) and in fibrous caps of atherosclerotic plaques (n = 10; P < 0.01)., Conclusion: Fibroblast activation protein expression in HASMC is induced by macrophage-derived TNFα. Fibroblast activation protein associates with thin-cap human coronary fibroatheromata and contributes to type I collagen breakdown in fibrous caps.

Background: Matrix metalloproteinases (MMP) are strongly associated with cancer progession. Broad-spectrum MMP inhibition is rarely beneficial clinically due to adverse effects. Of all MMPs, the gelatinases are associated with the spread of several types of cancer, including oral carcinoma. We have developed gelatinase-specific peptides, as well as their fusion with green fluorescent protein (GFP), capable of effectively targeting carcinomas., Materials and Methods: Effects on tumor growth and lymphatic micrometastatic spread in vivo was studied by use of HSC-3-cell xenografted athymic nude mice. Antigelatinolytic mono- vs. polytherapies, as well as biological activity of peptide-GFP fusion, were also analyzed in vivo., Results: Antigelatinolytic therapy effectively inhibited growth of xenografted tumors in mice but the proportion of enlarged lymph nodes remained the same; antigelatinolytic polytherapy seemed not to potentiate the antitumor effects. The peptide-GFP chimera sustained its activity in vivo and effectively homed to the primary tumors., Conclusion: Peptide gelatinase inhibitors are effective in inhibiting primary tumor growth but alone do not prevent the spread of carcinoma cells; however, their bioactive GFP fusion is a candidate for tumor characterization and imaging.

SB-3CT (1), a selective and potent thiirane-based gelatinase inhibitor, is effective in animal models of cancer metastasis and stroke; however, it is limited by poor aqueous solubility and extensive metabolism. We addressed these issues by blocking the primary site of metabolism and capitalizing on a prodrug strategy to achieve >5000-fold increased solubility. The amide prodrugs were quantitatively hydrolyzed in human blood to a potent gelatinase inhibitor, ND-322 (3). The arginyl amide prodrug (ND-478, 5d) was metabolically stable in mouse, rat, and human liver microsomes. Both 5d and 3 were nonmutagenic in the Ames II mutagenicity assay. The prodrug 5d showed moderate clearance of 0.0582 L/min/kg, remained mostly in the extracellular fluid compartment (Vd = 0.0978 L/kg), and had a terminal half-life of >4 h. The prodrug 5d had superior pharmacokinetic properties than those of 3, making the thiirane class of selective gelatinase inhibitors suitable for intravenous administration in the treatment of acute gelatinase-dependent diseases.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Previously we found that exposure to electromagnetic pulse (EMP) induced an increase in blood-brain-barrier (BBB) permeability and the degradation of tight junction protein ZO-1 in rats. Matrix metalloproteinases (MMPs), in particular gelatinases (MMP-2 and MMP-9), play a key role in degradation of tight junction proteins, are known mediators of BBB compromise. We hypothesized that the degradation of ZO-1 by gelatinases contributed to EMP-induced BBB opening. To test this hypothesis, the mRNA level of ZO-1, protein levels of MMP-2, MMP-9 and tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2) were detected in rat cerebral cortex after exposing rats to EMP at 200 kV/m for 200 pulses. It was found that the mRNA level of ZO-1 was unaltered at different time points after EMP exposure. The protein levels of MMP-2 and MMP-9 significantly increased at 3 h and 0.5 h, respectively. However, TIMP-1 (inhibitor of MMP-9) and TIMP-2 (inhibitor of MMP-2) only moderately increased after EMP exposure. In addition, in situ zymography results showed that the gelatinase activity increased in cerebral microvessels at 3 h after EMP exposure. When rats were treated with gelatinases inhibitor (SB-3CT) before EMP exposure, the EMP-induced BBB opening was attenuated and the ZO-1 degradation was reversed. Our results suggested that EMP-induced BBB opening was related to gelatinase mediated ZO-1 degradation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Administration of Tolylsam, a MMP inhibitor with relative specificity for gelatinases, at a dose of 100 mg/kg/day to leptin-deficient (ob/ob) mice kept on high fat diet for 15 weeks, was associated with significantly reduced weight gain as compared to controls (p < 0.0005), resulting in lower body weight (p < 0.0005) at the end of the experiments. Food intake, physical activity and body temperature were not affected. Subcutaneous (SC) (2.9 ± 0.1g vs. 3.4 ± 0.2g in controls; p < 0.05) and gonadal (GON) (3.4 ± 0.1g vs. 3.7 ± 0.1g in controls; p = NS) fat mass were reduced by Tolylsam treatment. Reduced MMP-2 (gelatinase A) activity in adipose tissue extracts was confirmed by zymography. Mild adipocyte hypotrophy was observed in treated SC and GON adipose tissues. Blood vessel density was significantly reduced in Tolylsam treated SC (p < 0.05) and GON (p < 0.005) adipose tissues. Sirius red staining revealed comparable collagen content in both SC and GON fat of treated mice, whereas collagen disorganization (ratio thick/thin fibers) was also similar. Thus, gelatinase inhibition in mice with leptin deficiency resulted in lower body and fat pad weights, associated with mild adipocyte hypotrophy. This indicates that MMP inhibition may impair adipose tissue development independently of leptin.

The mortality rate due to snakebite is reduced markedly by the use of anti-venoms, which are the only medically approved remedial agents available. The anti-venoms effectively neutralize the systemic toxicity but offer no protection towards local tissue degradation. In viperid snake envenomations, SVMPs and SVHYs are the major agents responsible for brutal local tissue damage as they degrade ECM and basement membrane surrounding the blood vessels. Thus, the usage of inhibitor(s) against ECM degrading enzymes in the treatment of viper bites is an affirmative therapeutic choice. The present study assessed the efficacy of N-acetyl cysteine (NAC) to inhibit gelatinase, hyaluronidase, hemorrhagic and defibrinogenating activities of Vipera russelli and Echis carinatus venoms. NAC inhibited these activities dosedependently, but it did not inhibit the PLA2, 5' nucleotidase, procoagulant and edema inducing activities of both the venoms. NAC showed complete inhibition of hemorrhagic activity when incubated with venom prior to testing. Whereas little inhibition was observed when venom and NAC were injected independently. Inhibition of the basement membrane degradation and accumulation of inflammatory leukocytes at the site of venom injection in histological sections further corroborate the inhibitory property of NAC. The observed inhibition of hemorrhage was likely due to zinc chelation as supported by spectral studies. Further, docking predictions suggested the role of -SH and -NH-CO-CH3 groups of NAC in the inhibition of SVMPs and SVHYs. Future studies related to the protective role of NAC against the venom induced systemic hemorrhage and secondary complications are highly exciting.

Dipeptidyl peptidase-IV (DPP-IV), a serine protease that specifically cleaves the N-terminal dipeptide with a preference for L-proline or L-alanine at the penultimate position, is involved in the degradation of incretin hormones, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). GLP-1 regulates glucose homeostasis by stimulating insulin secretion, inhibiting glucagon release, and delaying gastric emptying. Intravenous GLP-1 has been shown to increase insulin secretion in response to elevated glucose levels and offers therapeutic benefit for patients with type 2 diabetes. However, the therapeutic application of GLP-1 is severely compromised by its lack of oral activity and its rapid degradation by plasma DPP-IV. Consequently, small-molecule DPP-IV inhibitors that could extend the duration of action of GLP-1 and prolong its beneficial effects have been investigated as potential therapeutics for type 2 diabetes. This review summarizes important structural classes of DPP-IV inhibitors, focusing mainly on their inhibitory potency and selectivity for DPP-IV over other related peptidases such as DPP-II, DPP8, DPP9, and FAP. Because inhibition of DPP8 and/or DPP9 has been shown to cause severe toxicity in preclinical species, high selectivity is an important criterion in selecting DPP-IV inhibitors for clinical development. As of today, several DPP-IV inhibitors have completed phase III clinical studies for the treatment of type 2 diabetes. A brief overview of clinical efficacy data on these inhibitor drugs is provided here. In addition, biological activities of other related dipeptidyl peptidases (DPP-II, DPP8, DPP9, and FAP) will be summarized. Selective inhibitors for these peptidases and their therapeutic potential will be discussed.

Investigation of collagenase and gelatinase inhibitory natural components afforded two isoflavonoids. Two isoflavonoids, tectorigenin-7-O-β-D-glucoside (1) and luteolin-7-O-β-D-glucuronopyranoside (2), were isolated from ethyl acetate fraction of Viola patrinii fermentation extracts (VPFE). Of these, compounds 1 and 2 exhibited collagenase inhibitory activity (IC(50)) at a concentration of less than 1.5 μM, and compound 2 showed gelatinases A and B inhibitory activity (IC(50)) at 0.3 μM and 0.8 μM, respectively.

Fibroblast activation protein (FAP) belongs to the prolyl peptidase family. FAP inhibition is expected to become a new antitumor target. Most known FAP inhibitors often resemble the dipeptide cleavage products, with a boroproline at the P1 site; however, these inhibitors also inhibit DPP-IV, DPP-II, DPP8, and DPP9. Potent and selective FAP inhibitor is needed in evaluating that FAP as a therapeutic target. Therefore, it is important to develop selective FAP inhibitors for the use of target validation. To achieve this, optimization of the nonselective DPP-IV inhibitor 8 led to the discovery of a new class of substituted 4-carboxymethylpyroglutamic acid diamides as FAP inhibitors. SAR studies resulted in a number of FAP inhibitors having IC(50) of <100 nM with excellent selectivity over DPP-IV, DPP-II, DPP8, and DPP9 (IC(50) > 100 μM). Compounds 18a, 18b, and 19 are the only known potent and selective FAP inhibitors, which prompts us to further study the physiological role of FAP.

Hemiscorpius lepturus envenomation exhibits various pathological changes in the affected tissues, including skin, blood cells, cardiovascular and central nervous systems. The enzymatic activity and protein component of the venom have not been described previously. In the present study, the electrophoretic profile of H. lepturus venom was determined by SDS-PAGE (12 and 15%), resulting in major protein bands at 3.5-5, 30-35 and 50-60 kDa. The enzymatic activities of the venom was, for the first time, investigated using various zymography techniques, which showed the gelatinolytic, caseinolytic, and hyaluronidase activities mainly at around 50-60 kDa, 30-40 kDa, and 40-50 kDa, respectively. Among these, the proteolytic activities was almost completely disappeared in the presence of a matrix metalloproteinase inhibitor, 1, 10-phenanthroline. Antigen-antibody interactions between the venom and its Iranian antivenin was observed by Western blotting, and it showed several antigenic proteins in the range of 30-160 kDa. This strong antigen-antibody reaction was also demonstrated through an enzyme-linked immunosorbent assay (ELISA). The gelatinase activity of the venom was suppressed by Razi institute polyvalent antivenin, suggesting the inhibitory effect of the antivenin against H. lepturus venom protease activities. Prudently, more extensive clinical studies are necessary for validation of its use in envenomed patients., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The extent to which a drug inhibits a target responsible for a therapeutic effect is a more rational primary endpoint for dose-finding studies of more selective anticancer drugs than the conventional endpoint of dose-limiting toxicity (DLT) used for cytotoxic agents. An adaptive phase I trial design incorporating maximum target inhibition as the primary endpoint was developed to define the optimal dose of talabostat, a dipeptidyl peptidase (DPP) inhibitor, in children with relapsed or refractory solid tumors. The relationship between dose and effect (percent inhibition of serum DPP-4) was assessed using a maximum effect model. Maximum target inhibition was defined as greater than 90% DPP-4 inhibition in five or more of six patients 24 hours post-dose. If DLT was to occur, the trial would adapt to a traditional phase I design with a more conservative dose escalation. At the 600 microg/m(2) dose level, serum DPP-4 inhibition at 24 hours was 85%. No talabostat-related DLT occurred. The maximum effect model predicted that 1200 microg/m(2) of talabostat would maximally inhibit DPP-4. This adaptive trial design appears to be feasible, safe, and efficient and warrants further evaluation for development of molecularly targeted agents.

Objective: Since fibroblasts in the synovium of patients with rheumatoid arthritis (RA) express the serine proteases fibroblast activation protein (FAP) and dipeptidylpeptidase 4 (DPP-4)/CD26, we undertook the current study to determine the functional role of both enzymes in the invasion of RA synovial fibroblasts (RASFs) into articular cartilage., Methods: Expression of FAP and DPP-4/CD26 by RASFs was analyzed using fluorescence-activated cell sorting and immunocytochemistry. Serine protease activity was measured by cleavage of fluorogenic substrates and inhibited upon treatment with L-glutamyl L-boroproline. The induction and expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in RASFs were detected using real-time polymerase chain reaction. Densitometric measurements of MMPs using immunoblotting confirmed our findings on the messenger RNA level. Stromal cell-derived factor 1 (SDF-1 [CXCL12]), MMP-1, and MMP-3 protein levels were measured using enzyme-linked immunosorbent assay. The impact of FAP and DPP-4/CD26 inhibition on the invasiveness of RASFs was analyzed in the SCID mouse coimplantation model of RA using immunohistochemistry., Results: Inhibition of serine protease activity of FAP and DPP-4/CD26 in vitro led to increased levels of SDF-1 in concert with MMP-1 and MMP-3, which are downstream effectors of SDF-1 signaling. Using the SCID mouse coimplantation model, inhibition of enzymatic activity in vivo significantly promoted invasion of xenotransplanted RASFs into cotransplanted human cartilage. Zones of cartilage resorption were infiltrated by FAP-expressing RASFs and marked by a significantly higher accumulation of MMP-1 and MMP-3, when compared with controls., Conclusion: Our results indicate a central role for the serine protease activity of FAP and DPP-4/CD26 in protecting articular cartilage against invasion by synovial fibroblasts in RA.

Four oligomeric procyanidins were isolated from the MeOH extracts of the leaves of Crataegus pinnatifida (Rosaceae). Investigation of collagenase and gelatinase inhibitory natural components afforded four oligomeric procyanidins. Of these, 2 and 3 exhibited collagenase inhibitory activity (IC(50)) at a concentration of less than 1 microM, and 3 showed gelatinases A and B inhibitory activity (IC(50)) at 0.4 and 2.3 microM, respectively., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Aims: To determine the ability of a novel Bacillus subtilis AMR isolated from poultry waste to hydrolyse human hair producing peptidases including keratinases and hair keratin peptides., Methods and Results: The Bacillus subtilis AMR was identified using biochemical tests and by analysis of 16S rDNA sequence. The isolate was grown in medium containing human hair as the sole source of carbon and nitrogen. The supplementation of hair medium (HM) with 0.01% yeast extract increased the keratinolytic activity 4.2-fold. B. subtilis AMR presented high keratinase production on the 8th day of fermentation in hair medium (HM) supplemented with 0.01% yeast extract (HMY) at pH 8.0. Keratinase yield was not correlated with increase in biomass. Zymography showed keratin-degrading peptidases migrating at c. 54, 80 and 100 kDa and gelatin-degrading bands at c. 80, 70 63, 54 32 and 15 kDa. Keratinases were optimally active at 50 degrees C and pH 9.0 and was fully inhibited by the serine proteinase inhibitor (PMSF). Scanning electron microscopy showed complete degradation of the hair cuticle after exposure to B. subtilis AMR grown in HMY. MALDI-TOF analysis of culture supernatant containing peptides produced during enzymatic hydrolysis of hair by B. subtilis AMR revealed fragments in a range of 800-2600 Da., Conclusions: This study showed that B. subtilis AMR was able to hydrolyse human hair producing serine peptidases with keratinase and gelatinase activity as well as hair keratin peptides., Significance and Impact of the Study: This is the first report describing the production and partial characterization of keratinases by a B. subtilis strain grown in a medium containing human hair. These data suggest that peptides obtained from enzymatic hair hydrolysis may be useful for future applications on pharmaceutical and cosmetic formulations.

Matrix metalloproteinases (MMPs) have been implicated in the degradation of the extracellular matrix in normal and pathological tissue remodelling. Among the MMPs, MMP-2 is the most commonly studied protease that has been involved in cancer, inflammation, infective diseases, degenerative diseases of the brain and vascular diseases. In this study, monoclonal antibodies (MAbs) were generated against human MMP-2, purified, characterized and tested for their ability to inhibit the enzymatic activity of MMP-2. Out of 12 positive clones generated against MMP-2, 2 clones (F2-1-11 and G8-25-5) were selected for further characterization. The selected clones react specifically with human pro and active form of MMP-2 in enzyme linked immunosorbant assay (ELISA), dot immunobinding assay (DIA) and Western blot and do not cross react with other human metalloproteinases or MMP-2 from other species. Additionally, these MAbs (F2-1-11 and G8-25-5) selectively inhibit collagenolytic and gelatinolytic activity of APMA ((p-aminophenylmercuric acetate)-activated-pro-MMP-2 and MMP-2, respectively.

Gelatinases (MMP-2 and MMP-9) have been implicated in a number of pathological conditions, including cancer and cardiovascular disease. Hence, small molecule inhibitors of these enzymes are highly sought for use as potential therapeutic agents. 2-(4-Phenoxyphenylsulfonylmethyl)thiirane (SB-3CT) has previously been demonstrated to be a potent and selective inhibitor of gelatinases, however, it is rapidly metabolized because of oxidation at the para position of the phenoxy ring and at the alpha-position to the sulfonyl group. alpha-Methyl variants of SB-3CT were conceived to improve metabolic stability and as mechanistic probes. We describe herein the synthesis and evaluation of these structural variants as potent inhibitors of gelatinases. Two (compounds 5b and 5d) among the four synthetic stereoisomers were found to exhibit slow-binding inhibition of gelatinases and MMP-14 (MT1-MMP), which is a hallmark of the mechanism of this class of inhibitors. The ability of these compounds to inhibit MMP-2, MMP-9, and MMP-14 could target cancer tissues more effectively. Metabolism of the newly synthesized inhibitors showed that both oxidation at the alpha-position to the sulfonyl group and oxidation at the para position of the terminal phenyl ring were prevented. Instead oxidation on the thiirane sulfur is the only biotransformation pathway observed for these gelatinase inhibitors.

Membrane-bound proteases have recently emerged as critical mediators of tumorigenesis, angiogenesis, and metastasis. However, the mechanisms by which they regulate these processes remain unknown. As the cell surface serine protease fibroblast activation protein (FAP) is selectively expressed on tumor-associated fibroblasts and pericytes in epithelial tumors, we set out to investigate the role of FAP in mouse models of epithelial-derived solid tumors. In this study, we demonstrate that genetic deletion and pharmacologic inhibition of FAP inhibited tumor growth in both an endogenous mouse model of lung cancer driven by the K-rasG12D mutant and a mouse model of colon cancer, in which CT26 mouse colon cancer cells were transplanted into immune competent syngeneic mice. Interestingly, growth of only the K-rasG12D-driven lung tumors was also attenuated by inhibition of the closely related protease dipeptidyl peptidase IV (DPPIV). Our results indicate that FAP depletion inhibits tumor cell proliferation indirectly, increases accumulation of collagen, decreases myofibroblast content, and decreases blood vessel density in tumors. These data provide proof of principle that targeting stromal cell-mediated modifications of the tumor microenvironment may be an effective approach to treating epithelial-derived solid tumors.

Great inroads have been made in defining the oncogenic pathways intrinsic to neoplastic cells and the mechanisms by which they are activated in tumors. Knowledge of these pathways provides numerous opportunities that are actively being pursued to develop targeted therapies for cancer. Complementary studies, focused on the non-transformed components of the tumor microenvironment (TME), have revealed that the extrinsic cues provided by the TME are also essential for tumor cells to manifest a fully transformed phenotype, angiogenesis and metastasis. Delineation of these cues and their underlying cellular and molecular pathways will thus lead to a new era of integrative cancer therapy based on combinatorial drug regimens that act synergistically to destroy the neoplastic cells by targeting both the intrinsic and extrinsic pro-oncogenic pathways. Tumor-associated fibroblasts (TAFs) and proteases are two of the key regulators of epithelial-derived tumors that represent potential targets of such integrative therapies. Herein, we consider the potential therapeutic benefit of inhibiting the function of fibroblast activation protein (FAP), a cell surface serine protease with dipeptidyl peptidase and endopeptidase activity that is expressed on TAFs and pericytes, in an integrative approach to treating cancer.

Titanocene dichloride, the most studied metallocene, exhibits antiproliferative activity in a wide spectrum of murine and human tumours. In this article it is demonstrated that titanocene dichloride inhibits tumour gelatinases in a dose-dependent manner. Substrate saturation experiments and the fact that the IC(50) values were increased in correlation with collagen substrate concentrations indicate that the titanocene dichloride induced inhibition is of a competitive type. Titanocene dichloride also specifically inhibits clostridium collagenase and trypsin, particularly when collagens are used as substrates. Binding experiments demonstrate that cyclopentadiene-Ti(IV) moieties, resulting from titanocene dichloride at physiological pH, are bound mainly to different types of collagens and to a lesser extent to casein or bovine serum albumin, forming soluble and stable adducts. These results indicate that titanocene dichloride behaves as a competitive inhibitor against various proteolytic enzymes by binding to the substrate rather than to the enzyme active site. This property may be responsible for the antiangiogenic effect of titanocene dichloride and additionally contributes to its anticancer action.

Circulating antiplasmin-cleaving enzyme (APCE), a prolyl-specific serine proteinase, is essentially identical to membrane-inserted fibroblast activation protein (FAP) that is transiently expressed during epithelial-derived cancer growth. Human precursive alpha(2)-antiplasmin (Met-alpha(2)AP), the only known physiologic substrate for APCE, is cleaved N-terminally to Asn-alpha(2)AP that is rapidly cross-linked to fibrin and protects it from digestion by plasmin. Identifying a specific inhibitor of APCE/FAP continues to be intensely pursued. Recombinant FAP cleavage of peptide libraries of short amino acid sequences surrounding the scissile bond, -Pro(12)-Asn(13)-, indicated that P2 Gly and P1 Pro are required, just as we found for APCE. We examined cleavage of P4-P4' peptides, using 19 amino acid substitutions at each position and selected ones in P8-P5. K(m) values determined for peptide substrates showed that P7 Arg has the highest affinity for APCE. Peptide cleavage rate increased with Arg in P6 rather than P5 or native P7. Placing Arg in P4 or P8 reduced cleavage rates dramatically. Cleavage of substrates with extended peptide sequences before or after the scissile bond showed endopeptidase to be superior to dipeptidase activity for APCE. A substrate analogue inhibitor, Phe-Arg-(8-amino-3,6-dioxaoctanoic acid)-Gly-[r]-fluoropyrrolidide, inhibited APCE with a K(i) of 54 microM but not dipeptidyl peptidase IV even at 2 mM. The inhibitor also blocked cleavage of Met-alpha(2)AP with an IC(50) of 91 microM. Replacing Arg with Gly at the same distance from fluoropyrrolidide as P7 Arg is from P1 Pro reduced its inhibition of APCE approximately 10-fold. Results indicate that Arg at P5, P6, or P7 distances from P1 enhances affinity and efficiency of substrates or inhibitors toward APCE or FAP.

A number of recent researches have demonstrated the therapeutic effects of glucosamine (Glc) in a range of human diseases including arthritis. For the first time, we identified that a novel Glc derivative having quaternized amino functionality (QAGlc) suppresses MMP-9 and MMP-2, gelatinases in HT1080, human fibrosarcoma cells at 40microg/ml, following stimulation with PMA. Reporter gene assay results revealed that, the mechanism of suppression involves decreased transcriptional activation of MMP-9 and MMP-2 via transcription factors NF-kappaB and AP-1. However based on western blot results, QAGlc did not attenuate the nuclear translocation of both NF-kappaB and AP-1. Apparently, phorbol myristate acetate (PMA) stimulated expressions of ERK, JNK and p38 were altered in the presence of potent tumour inducer, phorbol myristate acetate QAGlc, suggesting their suppression also contributes to QAGlc-mediated inhibition of MMP-9 and MMP-2. Moreover, the ability of QAGlc to inhibit gelatinases was confirmed by its ability to act against invasiveness of HT1080 cells through extracellular matrix components.

Mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury in peripubertal rodents results in the stimulation of germ cell apoptosis through an interaction of FAS/FASL between these two cell types. During this peripubertal period, an early spike in the incidence of germ cell apoptosis occurs during the first wave of spermatogenesis and is essential for the development of functional spermatogenesis in adults. Our previous observations revealed that soluble tumor necrosis factor alpha (sTNFA) released by germ cells after MEHP exposure consequently resulted in a robust induction of FASL by Sertoli cells. Metalloproteinases (MPs) are essential for processing the TNFA precursor to its soluble form and its ability to bind to TNFRSF1A. The activity of MPs is regulated by the tissue inhibitors of MPs (TIMPs) family. Herein we report that TIMP2 is predominately expressed in Sertoli cells and that protein levels decrease in a time-dependent manner after MEHP exposure. The secretion of matrix MP 2 (MMP2) in primary rat Sertoli cell-germ cell cocultures is induced after MEHP exposure, and its activity increases in a time-dependent manner. The addition of SB-3CT, a specific gelatinase inhibitor, decreases the activity of MMP2 and significantly reduces MEHP-enhanced sTNFA production in primary cocultures. In vivo challenges with SB-3CT decrease sTNFA and reduce MEHP-induced testicular germ cell apoptosis. In primary cocultures, MEHP exposure causes a 9.46-fold increase in sTNFA, while the addition of recombinant MMP2 protein results in a 5.4-fold increase in sTNFA, suggesting that MEHP-induced MMP2 is in part responsible for the activation of TNFA in the testis. Taken together, these observations indicate the distinct role of specific MPs in response to toxicant-induced Sertoli cell injury, providing further insights into the mechanism by which Sertoli cells control the sensitivity of germ cells to undergo apoptosis.

Metastatic tumors lead to more than 90% fatality. Despite the importance of invasiveness of tumors to poor disease outcome, no anti-invasive compounds have been commercialized. We describe herein the synthesis and evaluation of 4-(4-(thiiranylmethylsulfonyl)phenoxy)-phenyl methanesulfonate (compound 2) as a potent and selective inhibitor of gelatinases (matrix metalloproteinases-2 and -9), two enzymes implicated in invasiveness of tumors. It was demonstrated that compound 2 significantly attenuated the invasiveness of human fibrosarcoma cells (HT1080). The metabolism of compound 2 involved hydroxylation at the alpha-methylene, which generates sulfinic acid, thiirane ring-opening, followed by methylation and oxidation, and cysteine conjugation of both the thiirane and phenyl rings.

Matrix metalloproteinase (MMP)-2 and MMP-9 have been associated with the ability of tumor cells to metastasize because of their capacity to degrade type IV collagen, the main component of basement membrane, and to their elevated expression in malignant tumors. (S)-methyl 6-(benzyloxycarbonylamino)-2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenzamido) piperidin-1-yl) acetamido) hexanoate (CH1104I) is a galloyl cyclic-imide derivative designed to fit and extend into the S1' active pocket of MMP-2 and MMP-9. We aimed to evaluate the efficacy of CH1104I as a candidate compound for antiinvasion and antimetastasis of tumor cells. CH1104I significantly blocked gelatinase activity as evidenced by a decrease in the degradation of succinylated gelatin. Gelatin zymography analysis showed that the compound (7-210 micromol/l) inhibited the activity of MMP-2 and MMP-9 produced by human ovarian carcinoma SKOV3 cells. Inhibition of MMP-2 and MMP-9 expression was also observed using the assays of immunocytochemical staining and western blot analysis. The results showed that CH1104I suppressed the expression of zymogens and active MMP-2 and MMP-9. The effects of CH1104I on the invasion and migration of SKOV3 cells were then measured. Both the trans-well motility assay and wound scratch assay indicated that CH1104I was very effective for the antiinvasion and antimigration of SKOV3 cells. Furthermore, the Lewis lung carcinoma model was used to evaluate the efficacy of CH1104I in vivo. A significant inhibition of pulmonary metastasis of carcinoma cells was observed in CH1104I-administrated mice (25-100 mg/kg). These results suggest that CH1104I is a potential MMP-2 and MMP-9 inhibitor that may effectively suppress tumor invasion and metastasis.

Aim: To investigate the effects of substance P (SP) on gelatinolytic activity of matrix metalloproteinases (MMPs) in human pulp cells., Methodology: Human dental pulp cells were isolated and cultured. Subconfluent cells, between the third and sixth passages, were maintained under serum deprivation for 18 h followed by the treatment of varying doses of SP (1 pmol L(-1), 100 pmol L(-1), 10 nmol L(-1), 1 micromol L(-1) and 100 micromol L(-1)). Conditioned media were then subjected to gelatin zymography using 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis minigels containing 1.5 g L(-1) gelatin. The effect of SP on intracellular reactive oxygen species (ROS) was also examined by confocal microscopy. ROS scavenger N-Acetyl-L-cysteine (NAC, 5 mmol L(-1)) was utilized to evaluate the roles of ROS pathway in mediating the impact of SP on cellular gelatinolytic activity. Data were analysed using analysis of variance with Bonferroni correction for multiple comparisons or an unpaired Student's t-test., Results: Substance P, at levels above 1 micromol L(-1), remarkably enhanced MMP-2 activity reflected by the band migrating at 66 kDa (P < 0.05). A gelatinolytic band at approximately 44 kDa appeared to be intensified in a SP dose-dependent manner. In addition, it was demonstrated that SP could induce ROS production in pulp cells and ROS scavenger NAC was further found to significantly reduce MMP-2 activity (P < 0.05), as well as other bands of gelatinolytic proteinases., Conclusion: Substance P can influence gelatinolytic activity in human pulp cells via ROS pathway.

Altered activity of matrix metalloproteinases (MMPs) is implicated in the vascular remodeling of hypertension. We examined whether increased MMP-2 expression/activity plays a role in the vascular remodeling and dysfunction found in the two-kidney, one-clip (2K-1C) hypertension. Sham operated or 2K-1C hypertension rats were treated with doxycycline 30mg/(kgday) (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes, collagen, and elastin contents in the aortic wall were studied in hematoxylin/eosin, Sirius Red, and Orceine stained aortic sections, respectively. Aortic MMP-2 levels were determined by gelatin zymography and aortic MMP-2 proteolytic activity was measured using DQ gelatin as the substrate after MMP-2 was captured by a specific antibody and immobilized on a microplate. Aortic MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by real time RT-PCR. Doxycycline attenuated 2K-1C hypertension (215+/-8mmHg versus 167+/-13mmHg in 2K-1C rats and 2K-1C+doxy rats, respectively; P<0.01) and prevented the 35% reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Doxycycline prevented the increases in media thickness, and was associated with lower media/lumen and cross-sectional areas (all P<0.01). Doxycycline also prevented excessive collagen and elastin deposition in the vascular wall. Increased MMP-2 and Pro-MMP-2 levels and MMP-2 activity were found in the aortas of 2K-1C rats (all P<0.05). A 21-fold increase (P<0.001) in the ratio of MMP-2/TIMP-2 mRNA expression was found in the 2K-1C group, whereas this ratio remained unaltered in 2K-1C+doxy rats. Our results suggest that MMP-2 plays a role in 2K-1C hypertension and its structural and functional vascular changes, which were attenuated by doxycycline.

SB-3CT is a thiirane-containing inhibitor of the gelatinase class of matrix metalloprotease enzymes. In support of the mechanistic study of this inhibition, the conformational analyses of SB-3CT (and of two methyl-substituted derivatives) were undertaken using X-ray crystallography and molecular dynamics simulation.

(4-Phenoxyphenylsulfonyl)methylthiirane (compound 1) is a highly selective and potent inhibitor of gelatinases that shows considerable promise in animal models for cancer and stroke. The metabolism of compound 1 was investigated in mice, following intraperitoneal administration at 100 mg/kg. Eight metabolites were identified in plasma and urine. The primary routes of metabolism of 1 were hydroxylation at the para-position of the terminal phenyl ring, hydroxylation at the alpha-methylene to the sulfonyl, which lead to the generation of a sulfinic acid, and cysteine conjugation of the thiirane ring. The cysteine adducts arose through addition of glutathione to the thiirane ring. The molecule is extensively metabolized and

Recent studies have shown significantly increased expression of matrix metalloproteinases (MMP) and disintegrin-type metalloproteinases (ADAM) during allograft rejection. In this regard, our previous studies have demonstrated contrasting roles for MMP-2 and MMP-9 during allograft rejection: MMP-2-deficiency enhanced allograft survival while MMP-9-deficiency decreased allograft survival. The aim of this study was to determine the effect of broad-spectrum MMP/ADAM inhibition on the pathogenesis of allograft rejection. Toward this, heterotopic BALB/c cardiac allografts were transplanted into C57BL/6 recipients treated with MMP/ADAM inhibitors, GM6001 or doxycycline. Systemic MMP/ADAM inhibition significantly enhanced allograft survival. Functioning allografts recovered from MMP/ADAM inhibitor-treated recipients showed lower cellular infiltration and tissue remodeling than rejected allografts recovered from control recipients. In addition, decreased chemotaxis of CD4+ and CD8+ T cells, B cells and macrophages was observed in vitro in the presence of MMP/ADAM inhibitors. Enhanced T-cell alloreactivity was also observed ex vivo in MMP/ADAM inhibitor-treated recipients and in vitro in the presence of MMP/ADAM inhibitors. These observations were associated with enhanced cytokine, chemokine and growth factor production. These results indicate that MMPs and ADAMs play a critical role in the pathogenesis of allograft rejection and may represent novel therapeutic targets for the treatment and/or prevention of this disease.

The introduction and the optimization of an alpha-amino substituent based on a series of alpha-hydroxy-beta-N-biaryl ether sulfonamide hydroxamates is described. The modification leads to a new series of MMP-2/MMP-9 inhibitors with enhanced inhibitory activities and improved ADME properties. An efficacy study on reducing the ischemia-induced brain edema in the rat transient middle cerebral artery occlusion (tMCAo) model is also demonstrated.

A new series of beta-N-biaryl ether sulfonamide hydroxamates as novel gelatinase inhibitors is described. These compounds exhibit good potency for MMP-2 and MMP-9 without inhibiting MMP-1. The structure-activity relationships (SAR) reveal the biaryl ether type P1' moiety together with methanesulfonamide is the optimal combination that provides inhibitory activity of MMP-9 in the single-digit nanomolar range.

N-Acetylglucosaminyltransferase-V (GnT-V) has been reported to be up-regulated in invasive/metastatic cancer cells, but a comprehensive understanding of how the transferase correlates with the invasive/metastatic potential is not currently available. Through a glycomics approach, we identified 30 proteins, including tissue inhibitor of metalloproteinase-1 (TIMP-1), as a target protein for GnT-V in human colon cancer cell WiDr. TIMP-1 was aberrantly glycosylated as characterized by the addition of beta1,6-N-acetylglucosamine, polylactosaminylation, and sialylation in GnT-V-overexpressing WiDr cells. Compared with normal TIMP-1, the aberrantly glycosylated TIMP-1 showed the weaker inhibition on both matrix metalloproteinase (MMP)-2 and MMP-9, and this aberrancy was closely associated with cancer cell invasion and metastasis in vivo as well as in vitro. Integrated data, both of TIMP-1 expression level and aberrant glycosylation, could provide important information to aid to improve the clinical outcome of colon cancer patients.

Background: Osteoarthrosis is characterized by cartilage erosion, proteolysis of aggrecan and collagen, and disturbed rates of synthesis of aggrecan and hyaluronan by chondrocytes, with hyaluronan over-production being an early reaction. We considered that inhibition of hyaluronan export might prevent subsequent proteoglycan loss and collagen degradation., Methods: To test this hypothesis, we studied a tissue culture model using bovine cartilages explants activated with IL-1alpha to induce osteoarthritic reactions using the phosphodiesterase-5 inhibitors tadalafil, zaprinast and vardenafil., Results: These drugs inhibited hyaluronan export, but they did not inhibit hyaluronan synthase activity. Simultaneously, they inhibited proteoglycan loss and collagen degradation, but not their synthesis. They also reduced the release of gelatinases into the culture media and diffusion of the indicator protein horseradish peroxidase through the cartilage explants. The mechanism of action of these compounds may be through inhibition of hyaluronan exporter multidrug resistance-associated protein 5 (MRP5), because the effective drug concentrations were much higher than required for phosphodiesterase-5 inhibition and intracellular cGMP accumulation., Conclusion: Inhibition of hyaluronan over-production may be an appropriate target to attenuate IL-1-induced reactions in osteoarthritic cartilage.

(4-Phenoxyphenylsulfonyl)methylthiirane (inhibitor 1) is a highly selective inhibitor of gelatinases (matrix metalloproteinases 2 and 9), which is showing considerable promise in animal models for cancer and stroke. Despite demonstrated potent, selective, and effective inhibition of gelatinases both in vitro and in vivo, the compound is rapidly metabolized, implying that the likely activity in vivo is due to a metabolite rather than the compound itself. To this end, metabolism of inhibitor 1 was investigated in in vitro systems. Four metabolites were identified by LC/MS-MS and the structures of three of them were further validated by comparison with authentic synthetic samples. One metabolite, 4-(4-thiiranylmethanesulfonylphenoxy)phenol (compound 21), was generated by hydroxylation of the terminal phenyl group of 1. This compound was investigated in kinetics of inhibition of several matrix metalloproteinases. This metabolite was a more potent slow-binding inhibitor of gelatinases (matrix metalloproteinase-2 and matrix metalloproteinase-9) than the parent compound 1, but it also served as a slow-binding inhibitor of matrix metalloproteinase-14, the upstream activator of matrix metalloproteinase-2.

Matrix metalloproteinases (MMPs) play important roles in solid tumor invasion and migration. In this study, we showed that 1-furan-2-yl-3-pyridin-2-yl-propenone (FPP-3) dose-dependently inhibited HT1080 cell invasion and migration, and decreased MMP-2 and MMP-9 activities. Furthermore, FPP-3 reduced MMP-2 expression at protein and mRNA levels, and suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-enhanced expression of MT1-MMP without changing tissue inhibitors of metalloproteinase (TIMP)-2 level. FPP-3 also suppressed TPA-induced increases in MMP-9 protein and mRNA levels, but did not alter TIMP-1 level. Our results suggest that FFP-3 may be a valuable anti-invasive drug candidate for cancer therapy by suppressing MMP-2, MMP-9, and MT1-MMP.

Liposarcoma, a malignancy of fat cells, is the most common soft tissue sarcoma. Though rare, poorly differentiated liposarcomas commonly metastasize to lungs and liver, leading to poor prognosis. Prevention of Extracellular matrix (ECM) degradation by inhibition of matrix metalloproteinases (MMPs) activity has been shown to be a promising therapeutic approach to inhibition of cancer progression. A nutrient mixture (NM) containing lysine, proline, ascorbic acid, and green tea extract has shown significant anticancer activity against a number of cancer cell lines. We investigated the effect of NM on liposarcoma cell line SW-872 proliferation (MTT assay), MMP secretion (gelatinase zymography), invasion through Matrigel, and apoptosis and morphology (live green caspase kit and H&E). Liposarcoma cell growth was inhibited by 36 and 61% at 500 and 1,000 microg/ml NM. Zymography demonstrated both MMP-2 and MMP-9 secretion, with PMA-enhanced MMP-9 activity. NM inhibited both MMPs with virtual total inhibition at 500 microg/ml NM. Invasion through Matrigel was inhibited at 100, 500, and 1,000 microg/ml by 44, 75, and 100%, respectively. Dose-dependent apoptosis of liposarcoma cells was evident with NM challenge, with virtually all cells exposed to 1,000 microg/ml NM in late apoptosis. H&E staining did not demonstrate any changes in morphology at lower concentrations. However, some apoptotic changes were evident at higher concentrations. In conclusion, NM significantly inhibited liposarcoma cell growth, MMP activity, and invasion and induced apoptosis in vitro-important parameters for cancer development, suggesting NM as a potential treatment strategy for liposarcoma.

Available treatments for multiple sclerosis (MS) require frequent injections and have significant side effects. Proteases generated during inflammation are involved in the induction of tissue damage during inflammatory demyelination in the central nervous system (CNS). The Bowman-Birk Inhibitor (BBI), a soy-derived protease inhibitor with anti-carcinogenic and anti-inflammatory properties, has been shown to be well tolerated in clinical trials for pre-cancerous conditions, such as oral leukoplakia and the inflammatory disease, ulcerative colitis. We hypothesized that BBI may modulate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The BBI concentrate (BBIC), a soybean extract enriched in BBI, was administered to myelin basic protein (MBP)-immunized Lewis rats by gastric gavage in different treatment regimens, during the induction or the effector phase of disease. BBIC significantly delayed disease onset and suppressed disease severity, clinically and pathologically, in all treatment protocols. Both in vitro and ex vivo, BBIC inhibited MBP-specific proliferation of lymph node cells. BBIC reduced the activity of matrix metalloproteinase (MMP)-2 and -9 in spleen cell supernatants and was detected in the CNS of treated rats. BBIC suppresses EAE, it can be administered orally, and it is safe and relatively inexpensive. It may have a therapeutic role in patients with MS.

Background: Glucosamine sulfate may have an ex vivo inhibitory effect on the plasminogen activator (PA)/plasmin system and gelatinases expression during the early development of osteoarthritis (OA)., Methods: We compared the levels of urokinase-type PA (u-PA), PA inhibitor-1 (PAI-1) and gelatinases (matrix metalloproteinase-2 and -9 [MMP-2 and -9]) in a series of chondral, meniscal, and synovial cultures of early OA after treatment with or without glucosamine sulfate., Results: Gelatin zymography revealed that glucosamine sulfate could suppress MMP-2 secretion in chondral, meniscal and synovial cultures and also decrease MMP-9 production in synovial and meniscal cultures. ELISA data also showed the suppressive effects of glucosamine sulfate on u-PA and PAI-1 production in synovial cultures at 48 h., Conclusions: Our data suggest that one of the therapeutic effects of glucosamine sulfate is to down-regulate the expressions of u-PA, PAI-1, MMP-2 and MMP-9 that underlie the destruction of articular cartilage in the early stage of OA, and therefore to delay the joint failure.

Tumor metastasis is usually a serious problem in tumor patients because of the lack of therapeutic approaches. A new compound, N-all-trans-retinoyl-L-proline (ATRP), has been developed and its metastasis inhibition activity has been studied. Low concentrations of ATRP have already been found to inhibit hepatocellular carcinoma cells (HCC) in a dose- and time-dependent manner by inducing the expression of p27(kip). We found that ATRP inhibited metastasis-associated behaviors in Hep3B cells, such as cell migration, invasion, collagen adhesion and gelatinase expression, more significantly than retinoic acid. Further, such inhibitory activities were observed in the regulation of cellular surface fucosylated epitope functions, such as binding of ulex europaeus lectin, expression of Lewis x, y and b, and activity of alpha1,3 fucosyltransferase. Hep3B cells pretreated with ATRP showed a significantly reduced incidence of experimental intrahepatic metastasis in nude mice. We conclude that ATRP is an alternative inhibitor and potential therapeutic agent for HCC metastasis with a different mechanism of action from ATRP.

Metastasis to the bone is a major clinical complication in patients with prostate cancer (PC). However, therapeutic options for treatment of PC bone metastasis are limited. Gelatinases are members of the matrix metalloproteinase (MMP) family and have been shown to play a key role in PC metastasis. Herein, we investigated the effect of SB-3CT, a covalent mechanism-based MMP inhibitor with high selectivity for gelatinases, in an experimental model of PC bone metastases. Intraperitoneal (i.p.) treatment with SB-3CT (50 mg/kg) inhibited intraosseous growth of human PC3 cells within the marrow of human fetal femur fragments previously implanted in SCID mice, as demonstrated by histomorphometry and Ki-67 immunohistochemistry. The anti-osteolytic effect of SB-3CT was confirmed by radiographic images. Treatment with SB-3CT also reduced intratumoral vascular density and bone degradation in the PC3 bone tumors. A direct inhibition of bone marrow endothelial cell invasion and tubule formation in Matrigel by SB-3CT in vitro was also demonstrated. The use of the highly selective gelatinase inhibitors holds the promise of effective intervention of metastases of PC to the bone.

Matrix metalloproteinases (MMPs), zinc-dependent endopeptidases, are implicated in tumor progression. We describe herein the development of a resin-immobilized, broad-spectrum synthetic MMP inhibitor for selective binding of the active forms of MMPs from different experimental samples. We confirmed the activity-based binding of MMPs to the inhibitor-tethered resin with purified human recombinant MMP-2, -9, and -14, samples of cultured cells, and tissue extracts. Our results show that only the free active MMPs, and not the zymogens or MMP/TIMP (enzyme-protein inhibitor) complexes, bound specifically to the resin. In our comparison of benign and carcinoma tissue extracts, we detected active MMP-2 and MMP-14 forms only in the cancerous tissue samples, indicating that a pool of the tumor MMPs is free of endogenous inhibitors (TIMPs), and is thus likely to contribute to proteolytic events that precipitate tumor metastasis.

A series of novel galloyl pyrrolidine derivatives were synthesized as potential anti-tumor agents. Their inhibiting activities on gelatinase (MMP-2 and -9) were tested with succinylated gelatin as the substrate. Structure-activity analyses demonstrate that introduction of longer and more flexible side chains at the C(4) position of the pyrrolidine ring brings higher activity against gelatinase. Free phenol hydroxyl group is more favorable than the methylated one, which confirms the important role of the phenol hydroxyl group when inhibitors interact with gelatinase. In particular, (2S,4S)-4-(3-(3,4-dimethoxyphenyl)acrylamido)-N-hydroxy-1-(3,4,5- trimethoxybenzoyl)pyrrolidine-2-carboxamide (18) stood out as the most attractive compound (IC(50) = 0.9 nM). The anti-metastasis model of mice bearing H(22) tumor cells was used to evaluate their anti-tumor activities in vivo. The assay in vivo revealed that most of these inhibitors displayed favorable inhibitory activities (inhibitory rate >35%) and no significant toxic effects were observed. The inhibition for 62.37% of 19 indicates the strategy used to design MMP inhibitors (MMPIs) of galloyl pyrrolidine derivatives as potential anti-tumor agents is promising.