Identification of the anti-angiogenic site within vascular basement membrane-derived tumstatin.
Components of vascular basement membrane are involved in regulating angiogenesis. Recently, tumstatin (the NC1 domain of alpha3 chain of type IV collagen) was identified as possessing anti-angiogenic activity. In the present study, the anti-angiogenic activity of tumstatin was localized to the putative 54-132-amino acid Tum-5 domain, and the activity mediated by alpha(v)beta(3) integrin interaction in an RGD-independent manner. The recombinant Tum-5 produced in Escherichia coli and Pichia Pastoris specifically inhibited proliferation and caused apoptosis of endothelial cells with no significant effect on nonendothelial cells. Tum-5 also inhibited tube formation of endothelial cells on Matrigel and induced G1 endothelial cell cycle arrest. Moreover, anti-angiogenic effect of Tum-5 was also examined in vivo using both a Matrigel plug assay in C57BL/6 mice and human prostate cancer (PC-3) xenografts in nude mice. The in vivo results demonstrate that Tum-5 at 1 mg/kg significantly inhibited growth of PC-3 tumors in association with a decrease in CD31 positive vasculature. These in vivo studies also show that, at molar equivalents, human Tum-5 is at least 10-fold more active than human endostatin. In addition, these studies for the first time suggest that through the action of endogenous inhibitors, alpha(v)beta(3) integrin may also function as a negative regulator of angiogenesis. Taken together, these findings demonstrate that Tum-5, a domain derived from tumstatin, is an effective inhibitor of tumor-associated angiogenesis and a promising candidate for the treatment of cancer.
Department of Medicine and the Cancer Center, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.
Y
Y
Maeshima
M
M
Manfredi
C
C
Reimer
K A
KA
Holthaus
H
H
Hopfer
B R
BR
Chandamuri
S
S
Kharbanda
R
R
Kalluri
D020533:Angiogenesis Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D000818:Animals; D001324:Autoantigens / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001485:Basement Membrane / Q000737:chemistry; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D002455:Cell Division; D002460:Cell Line; D003094:Collagen / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D024141:Collagen Type IV; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D006801:Humans; D051379:Mice; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D020533:Angiogenesis Inhibitors; D001324:Autoantigens; D024141:Collagen Type IV; D011994:Recombinant Proteins; C057112:type IV collagen alpha3 chain; D003094:Collagen; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases