Recombinant prostate specific antigen inhibits angiogenesis in vitro and in vivo.

BACKGROUND: Prostate specific antigen (PSA) is a kallikrein family member with serine protease activity commonly used as a diagnostic marker for prostate cancer. We recently described anti-angiogenic properties of PSA [Fortier et al.: JNCI 91:1635-1640]. METHODS: Two forms of PSA were cloned and expressed in Pichia pastoris: one, an intact PSA with an N-terminus of IVGGVS em leader; the second, an N-1 PSA variant. The recombinant proteins were tested for serine protease activity and for anti-angiogenic activity in vitro and in vivo. RESULTS: The rate of substrate hydrolysis by the intact recombinant PSA was similar to that of PSA isolated and purified from human seminal plasma. In contrast, the N-1 PSA variant lacked serine protease activity. In an endothelial cell migration assay, the concentration that resulted in 50% inhibition (IC(50)) was: 0.5 microM for native PSA, 0.5 microM for intact recombinant protein, and 0.1 microM for the N-1 variant PSA. Both the intact recombinant and the N-1 recombinant PSA inhibited angiogenesis in vivo. CONCLUSIONS: Purified recombinant PSA inhibits angiogenesis, proving the concept that PSA is an anti-angiogenic, and serine protease activity, as determined by synthetic substrate hydrolysis, is distinct from the anti-angiogenic properties of PSA.

Endostatin binds tropomyosin. A potential modulator of the antitumor activity of endostatin.

The mechanism of action of Endostatin, an endogenous inhibitor of angiogenesis and tumor growth, remains unknown. We utilized phage-display technology to identify polypeptides that mimic the binding domains of proteins with which Endostatin interacts. A conformed peptide (E37) was identified that shares an epitope with human tropomyosin implicating tropomyosin as an Endostatin-binding protein. We show that recombinant human Endostatin binds tropomyosin in vitro and to tropomyosin-associated microfilaments in a variety of endothelial cell types. The most compelling evidence that tropomyosin modulates the activity of Endostatin was demonstrated when E37 blocked greater than 84% of the tumor-growth inhibitory activity of Endostatin in the B16-BL6 metastatic melanoma model. We conclude that the E37 peptide mimics the Endostatin-binding epitope of tropomyosin and blocks the antitumor activity of Endostatin by competing for Endostatin binding. We postulate that the Endostatin interaction with tropomyosin results in disruption of microfilament integrity leading to inhibition of cell motility, induction of apoptosis, and ultimately inhibition of tumor growth.