RESUMO
Kringle 1-3 of human plasminogen is a potent inhibitor of endothelial cell proliferation. To understand a possible role for the unique cystine bridge between kringle 2 and kringle 3, we disrupted the interkringle disulfide bond by mutating Cys(169) and Cys(297) to serine residues. The yield of the mutant during the refolding process was decreased significantly. Anti-endothelial cell proliferative activity of the mutant was similar to that of the wild type. There was no significant difference in in vivo antiangiogenic activity between the wild type and the mutant in chorioallantoic membrane assay. However, in the mutant, the weak lysine binding capability of kringle 2 was not detected and its mobility in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis is different from that of the wild type. These results support the notion that the overall antiangiogenic function of angiostatin is mediated by individual kringles, and suggest that the lysine binding capability of kringle 2 is likely not important for the antiangiogenic activity of kringle 1-3.
Assuntos
Dissulfetos/metabolismo , Kringles/fisiologia , Lisina/metabolismo , Neovascularização Fisiológica , Plasminogênio/química , Plasminogênio/metabolismo , Substituição de Aminoácidos/genética , Inibidores da Angiogênese/química , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Bovinos , Divisão Celular/efeitos dos fármacos , Embrião de Galinha , Córion/citologia , Córion/efeitos dos fármacos , Córion/fisiologia , Cisteína/genética , Cisteína/metabolismo , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Humanos , Kringles/genética , Ligantes , Mutação/genética , Neovascularização Fisiológica/efeitos dos fármacos , Plasminogênio/genética , Plasminogênio/farmacologia , Ligação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , TermodinâmicaRESUMO
Human malignant gliomas are highly vascularized and aggressive tumors. Angiogenesis inhibitors have been shown to induce regression of a variety of primary and metastatic tumors in vivo. However, their usefulness in treating brain tumors is not well understood. Angiostatin, a multiple kringle (1-4 of 5)-containing fragment of plasminogen, is one of the highly effective natural cryptic angiogenesis inhibitors. In our study, the therapeutic efficacy of non-glycosylated and small molecular size recombinant kringles 1-3 (rPK1-3) was examined in the treatment of brain tumors generated by stereotactic intracerebral implantation of U-87 human glioma cells in nude mice. Mice bearing tumors 7 days post-implant were treated daily with rPK1-3 (100 mg/kg) s.c. for 21 days. Treated animals showed suppressed brain tumor growth by greater than 71.2% along with a 3-fold increase of apoptotic index and suppressed vascularization by 78.9%, without any observable signs of toxicity. Analysis of bFGF and VEGF expression in the tumors of treated animals using immuno-histochemical methods showed near complete absence of growth factors. Our results indicate that the non-glycosylated, small molecular size rPK1-3 is an efficient tumoristatic agent for the treatment of intracranial human glioma xenografts in mice and might provide new strategies for the treatment of brain tumors.
Assuntos
Glioma/patologia , Kringles/genética , Neoplasias Experimentais/prevenção & controle , Plasminogênio/uso terapêutico , Animais , Apoptose , Neoplasias Encefálicas/patologia , Divisão Celular/efeitos dos fármacos , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento de Fibroblastos/biossíntese , Humanos , Linfocinas/biossíntese , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neovascularização Patológica , Fragmentos de Peptídeos/uso terapêutico , Plasminogênio/genética , Proteínas Recombinantes/uso terapêutico , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Endostatin, a carboxyl-terminal fragment of collagen XVIII is known as an anti-angiogenic agent, that specifically inhibits the proliferation of endothelial cell and the growth of several primary tumor. We report here the purification and characterization of the recombinant murine endostatin (rmEndostatin) which was expressed in a prokaryotic expression system. This rmEndostatin has similar physiochemical properties of yeast-produced recombinant endostatin, and it also specifically inhibits the proliferation and migration of bovine capillary endothelial cells stimulated by basic fibroblast growth factor. The biological activity of rmEndostatin was also shown by its anti-angiogenic ability on the chorioallantoic membrane of chick embryo in vivo. In this article, we demonstrate the refolding and purification of rmEndostatin, expressed using E. coli system, to a biologically active and soluble form. In addition, these results confirm the activity of endostatin as a potent anti-angiogenic agent.