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.
Krol
Janna
J
Klinische Forschergruppe der Frauenklinik der Technischen Universität München, Klinikum rechts der Isar, D-81675 München, Germany.
Sato
Sumito
S
Rettenberger
Peter
P
Assfalg-Machleidt
Irmgard
I
Schmitt
Manfred
M
Magdolen
Viktor
V
Magdolen
Ulla
U
D045744:Cell Line, Tumor; D015853:Cysteine Proteinase Inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D005260:Female; D006801:Humans; D061965:Matrix Metalloproteinase Inhibitors; D009361:Neoplasm Invasiveness / Q000517:prevention & control; D010051:Ovarian Neoplasms / Q000201:enzymology / Q000378:metabolism; D010206:Papain / Q000037:antagonists & inhibitors; D010957:Plasmids; D011956:Receptors, Cell Surface / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000096:biosynthesis / Q000235:genetics; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D015853:Cysteine Proteinase Inhibitors; D061965:Matrix Metalloproteinase Inhibitors; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D019717:Tissue Inhibitor of Metalloproteinase-3; D010450:Endopeptidases; D010206:Papain