The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin
Angiostatin, a potent naturally occurring inhibitor of angiogenesis and growth of tumor métastases, is generated by cancer-mediated proteolysis of plasminogen. Human prostate carcinoma cells (PC-3) release enzymatic activity that converts plasminogen to angiostatin (Gately et al., Can. Res. 56:4887, 1996). In the current study, we have identified two components released by PC-3 cells, urinary-type plasminogen activator (uPA) and free sulfhydryl donors, that are sufficient for angiostatin generation. In a defined cell-free system plasminogen activators (uPA, tissue-type PA, or streptokinase) in combination with one of a series of free sulfhydryl donors (N-acetyl-L-cysteine, D-penicillamine, captopril, L-cysteine, or reduced glutathione) generate angiostatin from plasminogen. An essential requirement of plasmin catalytic activity for angiostatin generation was identified using recombinant mutant plasminogens (D646E active site mutant and R561A activation site mutant). Wild-type recombinant plasminogen was converted to angiostatin in the presence of uPA and a free sulfhydryl donor, however both the active site and activation site mutants failed to generate angiostatin. Angiostatin generated in the cell-free system was biologically active and inhibited bFGF-induced endothelial cell migration and proliferation as well as the growth of Lewis lung carcinoma métastases. These findings define a direct mechanism for cancer cellmediated angiostatin generation, and permit large scale production of bioactive angiostatin for investigation and potential therapeutic application.
Gately, S; Twardowski, P; Sharon Stack, M; Cundiff, DL; Grella, D; Castellino, FJ; Enghild, J; Kwaan, HC; Lee, F; Kramer, RA; Soff, GA
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