Angiostatin, a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, is generated by cancer-mediated proteolysis of plasminogen. Human prostate carcinoma cells (PC-3) release enzymatic activity that converts plasminogen to angiostatin. We have now identified two components released by PC-3 cells, urokinase (uPA) and free sulfhydryl donors (FSDs), that are sufficient for angiostatin generation. Furthermore, in a defined cell-free system, plasminogen activators [uPA, tissuetype plasminogen activator (tPA), or streptokinase], in combination with one of a series of FSDs (N-acetyl-L-cysteine, D-penicillamine, captopril, L-cysteine, or reduced glutathione] generate angiostatin from plasminogen. An essential role of plasmin catalytic activity for angiostatin generation was identified by using recombinant mutant plasminogens as substrates. The wild-type recombinant plasminogen was converted to angiostatin in the setting of uPA͞FSD; however, a plasminogen activation site mutant and a catalytically inactive mutant failed to generate angiostatin. Cell-free derived angiostatin inhibited angiogenesis in vitro and in vivo and suppressed the growth of Lewis lung carcinoma metastases. 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.Because tumor growth and metastases are dependent upon angiogenesis (1-3), the identification of agents that inhibit angiogenesis now represents a potential therapeutic approach for the control of cancer (4-7). Angiostatin, consisting of the first four of five kringle domains of plasminogen (8), is one of a number of angiogenesis inhibitors that are internal fragments of larger nonangiogenic precursor proteins (8-14); however, the mechanisms by which these fragments are generated in vivo remains unknown. Although the activity sufficient to cleave plasminogen to angiostatin is present in tumor-bearing animals and serum-free conditioned medium (SFCM) of human prostate carcinoma cells (9), the cancer-dependent mechanism of angiostatin generation has remained unknown. Recently, macrophage-derived metalloelastase was shown to produce angiostatin from plasminogen and may contribute to angiostatin generation in the murine Lewis lung carcinoma model (15). We now describe the enzymatic mechanism for the direct generation of human angiostatin from plasminogen by human prostate cancer cells and demonstrate the generation of bioactive angiostatin from human plasminogen in a defined cell-free system. MATERIALS AND METHODSAngiostatin Generation. Angiostatin was generated from PC-3 cell SFCM as described (9). To generate angiostatin in a cell-free system, human plasminogen (0.2 M) was incubated with 0.2 nM recombinant human urokinase (uPA; Abbott), 1.0 nM recombinant human two-chain tissue-type plasminogen activator (tPA; a gift from Henry Berger, Glaxo-Wellcome), or 8.0 nM streptokinase (Sigma) and with 100 M N-acetyl-L-cys...
To our knowledge, this is the first report that PSA may function in tumors as an endogenous antiangiogenic protein. This function may explain, in part, the naturally slow progression of prostate cancer. Our findings call into question various strategies to inhibit the expression of PSA in the treatment of prostate cancer.
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