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Aminopeptidase N (APN)/CD13 (EC 3.4.11.2) is a transmembrane protease present in a wide variety of human tissues and cell types (endothelial, epithelial, fibroblast, leukocyte). APN/CD13 expression is dysregulated in inflammatory diseases and in cancers (solid and hematologic tumors). APN/CD13 serves as a receptor for coronaviruses. Natural and synthetic inhibitors of APN activity have been characterized. These inhibitors have revealed that APN is able to modulate bioactive peptide responses (pain management, vasopressin release) and to influence immune functions and major biological events (cell proliferation, secretion, invasion, angiogenesis). Therefore, inhibition of APN/CD13 may lead to the development of anti-cancer and anti-inflammatory drugs. This review provides an update on the biological and pharmacological profiles of known natural and synthetic APN inhibitors. Current status on their potential use as therapeutic agents is discussed with regard to toxicity and specificity.

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Enhanced expression of (pro)matrix metalloproteinase-9 (MMP-9) is associated with human tumor invasion and/or metastasis. COH cells derived from a highly invasive and metastatic Ewing's sarcoma constitutively express proMMP-9. Transfection of a double stranded RNA that targets the MMP-9 mRNA into COH cells depleted the corresponding mRNA and protein as demonstrated by reverse transcriptase-PCR, enzymelinked immunosorbent assay, and gelatin zymography. proMMP-9 extinction resulted in the following: (i) decreased spreading on extracellular matrix (fibronectin, laminin, collagen IV)-coated surfaces, (ii) inhibition of migration toward fibronectin, and (iii) induced aggregation, which was specifically disrupted by a functionblocking E-cadherin antibody. MMP-9 knockdown concomitantly resulted in increased levels of surface E-cadherin, redistribution at the plasma membrane of ␤-catenin, and its physical association with E-cadherin. Moreover, induction of E-cadherin-mediated adhesion was associated with RhoA activation and changes in paxillin cytoskeleton. Finally, an inhibitor of gelatinolytic activity of pro-MMP9 did not reduce COH cell migration confirming that the enzymatic property of COH MMP-9 was not required for migration toward fibronectin. Overall, our observations define a novel critical role for proMMP-9 in providing a cellular switch between stationary and migratory cell phases.Invasion and metastasis of tumor cells is a multiple process that depends on uncontrolled interactions between adjacent cells and/or cells and their extracellular environment (1, 2). These interactions are mediated directly by specific adhesion receptors and indirectly by extracellular proteinases that mediate degradation of the extracellular matrix (ECM).

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Invasion and metastasis of tumor cells is a multiple process, in which cell motility is accompanied by uncontrolled degradation of basement membrane and components of the extracellular matrix (1, 2). Matrix metalloproteinases (MMPs) 1 are a family of related zinc-containing proteinases that have the ability to degrade most extracellular matrix (3, 4). One member of the MMP family, MMP-9 (gelatinase B, 92-kDa), contains fibronectin-like domains for collagen binding and is capable of degrading type I, IV, V, VII, and XI collagens and laminin (1, 3-5). Such proteolytic ability suggests that MMP-9 ultimately regulates cell migration, tumor growth, and angiogenesis (1-4). MMP-9 is overexpressed in many human malignancies including solid tumors and hematological neoplasms (3, 4, 6 -8). MMP-9 promoter activity is induced coincidentally with invasion during tumor progression (9). In vitro overexpression of MMP-9 confers metastatic phenotype (10 -12).The promoter of MMP-9 possesses several functional enhancer element-binding sites including three AP-1 sites, a nonconsensus NF-B site, an Ets site, an Sp-1 site, and a retinoblastoma element (5,(13)(14)(15)(16)(17). A functional AP-2 element downstream to the previous AP-1-responsive element in the MMP-9 promoter was recently identified, and both of these factors appear important for MMP-9 transcription in keratinocytes (18).

A dipeptidyl aminopeptidase (DPP) was detected in plasma membranes from normal (3T3) and transformed (3T12) mouse fibroblasts. This enzyme was active in cleaving the prolyl bond in the synthetic dipeptide nitroanilide Gly-Pro-NH-Np, which is a specific substrate for DPP IV (Km 0.63 mM and Vmax 6.1 nmol/min per mg at pH 6.0 and 37 degrees C). However, it did not degrade Pro-NH-Np or other dipeptide nitroanilides such as Gly-Arg-NH-Np or Val-Ala-NH-Np. The enzyme was totally inhibited by di-isopropyl phosphorofluoridate (Pri2-P-F) and by phenylmethanesulphonyl fluoride, indicating a serine catalytic site for the proteinase. DPP IV is a glycoprotein that specifically recognized immobilized gelatin and type I collagen. Upon molecular exclusion chromatography, the proteinase exhibited an apparent Mr of 100,000. SDS/polyacrylamide-gel electrophoresis under non-reducing and reducing conditions revealed that the [3H]Pri2-P-protein was exclusively represented by a polypeptide of Mr 55,000. This suggested that DPP IV consists of two non-covalently linked 55,000-Mr subunits. Fibroblast adhesion to native or denatured collagen was significantly inhibited by the two dipeptide inhibitors of DPP IV, Gly-Pro-Ala and Ala-Pro-Gly, but not by the peptides Gly-Pro and Gly-Gly-Gly, which are not inhibitors of the proteinase. Moreover, preliminary fractionation of DPP IV by molecular exclusion chromatography and affinity chromatography indicated that this material was active in disrupting cell adhesion to collagens. Taken together, the above data suggest that a fibroblast membrane-associated collagen-binding glycoprotein, DPP IV, may play a role in cell attachment to collagen.