Novel bi- and trifunctional inhibitors of tumor-associated proteolytic systems.

Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCys-uPA19-31) directed against cysteine proteases and the urokinase-type plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)-binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the N-terminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCys-uPA19-31 or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OV-MZ-6#8). Additionally, OV-MZ-6#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitor-producing cells was significantly reduced compared to vector-transfected control cells. Thus, these novel, compact, and small-size inhibitors directed against up to three different tumor-associated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis.

Inhibition of the tumor-associated urokinase-type plasminogen activation system: effects of high-level synthesis of soluble urokinase receptor in ovarian and breast cancer cells in vitro and in vivo.

Tumor cell invasion and metastasis depend on the coordinated and temporal expression of proteolytic enzymes to degrade the surrounding extracellular matrix and of adhesion molecules to remodel cell-cell and/or cell-matrix attachments. The tumor cell-associated urokinase-type plasminogen activator system, consisting of the serine protease uPA, its substrate plasminogen, its membrane-bound receptor uPAR, as well as its inhibitors PAI-1 and PAI-2, plays an important role in these pericellular processes. Especially, association of the proteolytic activity of uPA with the cell surface via interaction with uPAR significantly increases the invasive capacity of tumor cells. Consequently, various approaches have been pursued to interfere with the expression or activity of uPA and/or uPAR, including antisense strategies and the development of active-site inhibitors of uPA or inhibitors of uPA/uPAR interaction. In this review, we focus on the results obtained in vitro and in vivo with tumor cells producing high levels of a recombinant soluble form of uPAR, which efficiently inhibits uPA binding to cell surface-associated uPAR and, by this, acts as a scavenger for uPA.