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.
Fortier
A H
AH
EntreMed Inc., 9640 Medical Center Drive, Rockville, MD 20850, USA. annef@entremed.com
Holaday
J W
JW
Liang
H
H
Dey
C
C
Grella
D K
DK
Holland-Linn
J
J
Vu
H
H
Plum
S M
SM
Nelson
B J
BJ
D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D016228:Endothelial Growth Factors / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D008222:Lymphokines / Q000494:pharmacology; D008297:Male; D009389:Neovascularization, Pathologic; D010843:Pichia / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017430:Prostate-Specific Antigen / Q000494:pharmacology*; D011994:Recombinant Proteins; D012697:Serine Endopeptidases / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D020533:Angiogenesis Inhibitors; D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D012697:Serine Endopeptidases; D017430:Prostate-Specific Antigen