The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin

Abstract

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...

The possible role of matrix metalloproteinase (MMP)-2 and MMP-9 in cancer, e.g. acute leukemia

“…angiostatin) or fragments of MMPs (e.g. PEX) are associated with inhibition of tumor angiogenesis [115][116][117][118][119][120][121][122][123][124]. Some mechanisms of action of tumstatin and endostatin have recently been elucidated.…”

Role of plasminogen activator-plasmin system in tumor angiogenesis

“…Much attention has been focused on angiostatin and endostatin, which are antiangiogenic molecules derived from plasminogen and collagen XVIII, respectively [77,78]. Recent studies suggest that angiostatin can be generated by limited proteolysis of plasminogen by plasmin, uPA, tPA, or MMPs [79,80]. Other negative regulators are fragments of collagen type XV/restin [81] or collagen type IV, such as arresten, from the α1 chain [82], canstatin, from the α2 chain [83], and tumstatin, from the α3 chain [84][85][86].…”

Synergistic antitumor effect of antiangiogenic factor genes on colon 26 produced by low-voltage electroporation

“…[1][2][3] However, long-term systemic delivery of these proteins poses a number of difficult logistic and pharmacological problems in preclinical models due to the stability and solubility of these agents. 4,5 In order to facilitate the delivery of such recombinant proteins, transfer of their genes into neoplastic tissues in vivo using a variety of viral and nonviral carriers has been proposed, and several gene therapy studies have demonstrated tumor shrinkage. [6][7][8][9][10][11] Electroporation-mediated gene transfer relies upon direct delivery of plasmids into cells permeabilized by electric fields, stimulating increased cell permeability by a variety of genes entering the cell through the membrane micropores.…”