Addition of an aminopeptidase N-binding sequence to human endostatin improves inhibition of ovarian carcinoma growth.
BACKGROUND
Blood vessels in tumors express higher level of aminopeptidase N (APN) compared with normal tissues. It has been reported that peptides that contain asparagine-glycine-arginine (NGR) sequence home to APN in tumor vasculature. Increased expression of APN in tumor vascular endothelium, therefore, offers an opportunity to target NGR peptide-linked therapeutic reagents to tumors.


METHODS
To determine whether an additional NGR sequence could improve endothelial homing and biologic activity, human endostatin was modified genetically to introduce an NGR motif (NGR-endostatin) and was expressed in yeast. In vitro biologic activity of NGR-endostatin was compared with the native protein in endothelial cell proliferation and migration. NGR-modified endostatin was used in tumor localization studies. Finally, the effects of endostatin and NGR-endostatin on tumor growth were determined in two model systems.


RESULTS
Human endostatin has an internal NGR sequence, which is not accessible to bind APN. However, the addition of an NGR-sequence at the amino terminus resulted in strong binding and inhibition of endothelial cell APN. NGR-endostatin showed increased binding to endothelial cells compared with the native protein. Increased binding of endostatin also coincided with improved antiangiogenic properties of endostatin. NGR modification improved tumor localization and, as a consequence, effectively inhibited ovarian carcinoma growth in athymic nude mice.


CONCLUSIONS
These studies demonstrated that human endostatin can be modified genetically to improve its ability to inhibit tumor growth.
Yokoyama
Yumi
Y
Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Ramakrishnan
Sundaram
S
D020533:Angiogenesis Inhibitors / Q000627:therapeutic use*; D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use; D001120:Arginine; D001216:Asparagine; D018826:CD13 Antigens / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D043169:Endostatins / Q000235:genetics / Q000627:therapeutic use*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005998:Glycine; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic / Q000188:drug therapy; D009842:Oligopeptides / Q000627:therapeutic use*; D010051:Ovarian Neoplasms / Q000188:drug therapy*; D011993:Recombinant Fusion Proteins / Q000627:therapeutic use*
D020533:Angiogenesis Inhibitors; D000970:Antineoplastic Agents; D043169:Endostatins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D001216:Asparagine; D001120:Arginine; D018826:CD13 Antigens; D005998:Glycine