Recombinant prostate specific antigen inhibits angiogenesis in vitro and in vivo
- Prostate. 2003 Aug 1;56(3):212-9. doi: 10.1002/pros.10256.
- 1. EntreMed Inc., 9640 Medical Center Drive, Rockville, MD 20850, USA. [email protected]
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.