RESUMEN
Angiogenesis is associated with several pathological disorders as well as with normal physiological maintenance. Components of vascular basement membrane are speculated to regulate angiogenesis in both positive and negative manner. Recently, we reported that tumstatin (the NC1 domain of alpha 3 chain of type IV collagen) and its deletion mutant tum-5 possess anti-angiogenic activity. In the present study, we confirm that the anti-angiogenic activity of tumstatin and tum-5 is independent of disulfide bond requirement. This property of tum-5 allowed us to use overlapping synthetic peptide strategy to identify peptide sequence(s) which possess anti-angiogenic activity. Among these peptides, only the T3 peptide (69-88 amino acids) and T7 peptide (74-98 amino acids) inhibited proliferation and induced apoptosis specifically in endothelial cells. The peptides, similar to tumstatin and the tum-5 domain, bind and function via alpha(v)beta(3) in an RGD-independent manner. Restoration of a disulfide bond between two cysteines within the peptide did not alter the anti-angiogenic activity. Additionally, these studies show that tumstatin peptides can inhibit proliferation of endothelial cells in the presence of vitronectin, fibronectin, and collagen I. Anti-angiogenic effect of the peptides was further confirmed in vivo using a Matrigel plug assay in C57BL/6 mice. Collectively, these experiments suggest that the anti-angiogenic activity of tumstatin is localized to a 25-amino acid region of tumstatin and it is independent of disulfide bond linkage. Structural features and potency of the tumstatin peptide make it highly feasible as a potential anti-cancer drug.
Asunto(s)
Autoantígenos/metabolismo , Colágeno Tipo IV , Colágeno/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Neovascularización Patológica , Neovascularización Fisiológica , Fragmentos de Péptidos , Receptores de Vitronectina/metabolismo , Alquilación , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Autoantígenos/química , Autoantígenos/farmacología , Caspasa 3 , Caspasas/metabolismo , Bovinos , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Colágeno/química , Colágeno/farmacología , Disulfuros/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Activación Enzimática , Proteínas de la Matriz Extracelular/química , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Oxidación-Reducción , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Células Tumorales Cultivadas , Vitronectina/metabolismoRESUMEN
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.
Asunto(s)
Inhibidores de la Angiogénesis/química , Autoantígenos/química , Colágeno Tipo IV , Colágeno/química , Endotelio Vascular/química , Inhibidores de la Angiogénesis/aislamiento & purificación , Animales , Autoantígenos/genética , Autoantígenos/aislamiento & purificación , Membrana Basal/química , Caspasa 3 , Caspasas/metabolismo , Bovinos , División Celular , Línea Celular , Colágeno/genética , Colágeno/aislamiento & purificación , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Ratones , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Vascular basement membrane is an important structural component of blood vessels. During angiogenesis this membrane undergoes many alterations and these changes are speculated to influence the formation of new capillaries. Type IV collagen is a major component of vascular basement membrane, and recently we identified a fragment of type IV collagen alpha2 chain with specific anti-angiogenic properties (Kamphaus, G. D., Colorado, P. C., Panka, D. J., Hopfer, H., Ramchandran, R., Torre, A., Maeshima, Y., Mier, J. W., Sukhatme, V. P., and Kalluri, R. (2000) J. Biol. Chem. 275, 1209-1215). In the present study we characterize two different antitumor activities associated with the noncollagenous 1 (NC1) domain of the alpha3 chain of type IV collagen. This domain was previously discovered to possess a C-terminal peptide sequence (amino acids 185-203) that inhibits melanoma cell proliferation (Han, J., Ohno, N., Pasco, S., Monboisse, J. C., Borel, J. P., and Kefalides, N. A. (1997) J. Biol. Chem. 272, 20395-20401). In the present study, we identify the anti-angiogenic capacity of this domain using several in vitro and in vivo assays. The alpha3(IV)NC1 inhibited in vivo neovascularization in matrigel plug assays and suppressed tumor growth of human renal cell carcinoma (786-O) and prostate carcinoma (PC-3) in mouse xenograft models associated with in vivo endothelial cell-specific apoptosis. The anti-angiogenic activity was localized to amino acids 54-132 using deletion mutagenesis. This anti-angiogenic region is separate from the 185-203 amino acid region responsible for the antitumor cell activity. Additionally, our experiments indicate that the antitumor cell activity is not realized until the peptide region is exposed by truncation of the alpha3(IV)NC1 domain, a requirement not essential for the anti-angiogenic activity of this domain. Collectively, these results effectively highlight the distinct and unique antitumor properties of the alpha3(IV)NC1 domain and the potential use of this molecule for inhibition of tumor growth.
Asunto(s)
Inhibidores de la Angiogénesis/toxicidad , Antineoplásicos/toxicidad , Autoantígenos/toxicidad , Membrana Basal/fisiología , Carcinoma de Células Renales/patología , Colágeno Tipo IV , Colágeno/toxicidad , Endotelio Vascular/efectos de los fármacos , Neoplasias Renales/patología , Fragmentos de Péptidos/toxicidad , Neoplasias de la Próstata/patología , Animales , Apoptosis , Membrana Basal/química , Carcinoma de Células Renales/irrigación sanguínea , Carcinoma de Células Renales/tratamiento farmacológico , División Celular/efectos de los fármacos , Línea Celular , Colágeno/química , Combinación de Medicamentos , Endotelio Vascular/citología , Humanos , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/tratamiento farmacológico , Laminina , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/tratamiento farmacológico , Proteoglicanos , Proteínas Recombinantes/toxicidad , Transfección , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Ganglioside sialic acid content was examined in the U87-MG human glioma grown as cultured cells and as a xenograft in severe combined immunodeficiency (SCID) mice. The cultured cells and the xenograft possessed N-glycolylneuraminic acid (NeuGc)-containing gangliosides, despite the inability of human cells to synthesize NeuGc. Human cells express only N-acetylneuraminic acid (NeuAc)-containing gangliosides, whereas mouse cells express both NeuAc- and NeuGc-containing gangliosides. Small amounts of NeuGc ganglioside sialic acid (2-3% of total ganglioside sialic acid) were detected in the cultured cells, whereas large amounts (66% of total ganglioside sialic acid) were detected in the xenograft. The NeuGc in gangliosides of the cultured cells was derived from gangliosides in the fetal bovine serum of the culture medium, whereas that in the U87-MG xenograft was derived from gangliosides of the SCID host. The chromatographic distribution of U87-MG gangliosides differed markedly between the in vitro and in vivo growth environments. The neutral glycosphingolipids in the U87-MG cells consisted largely of glucosylceramide, galactosylceramide, and lactosylceramide, and their distribution also differed in the two growth environments. Asialo-GM1 (Gg4Cer) was not present in the cultured tumor cells but was expressed in the xenograft, suggesting an origin from infiltrating cells (macrophages) from the SCID host. The infiltration of mouse host cells and the expression of mouse sialic acid on human tumor cell glycoconjugates may alter the biochemical and immunogenic properties of xenografts.