RESUMO
Angiogenesis is crucial for the growth and metastasis of solid tumors with sizes larger than a few cubic millimeter Canstatin, the non-collagenous 1 (NC1) domain of alpha2 chain of type IV collagen, was previously shown to inhibit proliferation of endothelial cells in vitro and suppress in vivo tumor growth. Our previous studies showed that canstatin-N, the N-terminal 1-89 amino acid fragment of canstatin, inhibited the neovascularization in vivo, potently induced apoptosis of endothelial cells in vitro, and suppressed in vivo tumor growth in BALB/c mice. In the present study, we demonstrated that canstatin-C, the C-terminal 157-227 amino acid fragment of canstatin, also specifically inhibited in vitro the proliferation of human umbilical vein endothelial cells and induced apoptosis, but the apoptosis-inducing activity, while close to that of the full-length canstatin, was much lower than that of canstatin-N. Canstatin-C also suppressed in vivo tumor growth in BALB/c mice at a dosage of 10mg/kg/day. These results suggest that canstatin-C is an anti-angiogenic domain of canstatin mainly associated with the specific inhibition of proliferation of endothelial cells, whereas canstatin-N with the potential apoptosis-inducing activity on endothelial cells.
Assuntos
Colágeno Tipo IV/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/crescimento & desenvolvimento , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/tratamento farmacológico , Inibidores da Angiogênese/biossíntese , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Embrião de Galinha , Colágeno Tipo IV/biossíntese , Colágeno Tipo IV/química , Endotélio Vascular/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neovascularização Patológica/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Total RNA was extracted from placenta umbilical tissue. The canstatin cDNA was amplified from total RNA by net-RT-PCR technique and cloned into pSP72, and the resulted plasmid pSP72C was used as template to amplify its N-domain. The amplified 1-89 aa N-domain was then cloned into pET-3c. The resulted plasmid pET-CN was transformed into E. coli BL21(DE3). The N-domain was efficiently expressed after IPTG induction as a 10 kD band on SDS-PAGE. The expressed product accounted for approximately 35.3% of the total bacterial proteins, as estimated by densitometry and existed mainly as inclusion body. The inclusion bodies were washed, lysed and the reactivated proteins were purified by the Sephadex G-100 gel filtration to a purity of 92.6%. CAM assay showed that N-domain effectively inhibited the angiogenesis of chicken embryo microcapillary vessel.
