RESUMEN
The expression level of folate receptor alpha (FRα) is located highly rate in ovarian cancer though it is remained absent in normal tissues. This highly tumor restricted expression profile makes FRα a promising target for tumor therapy and diagnosis. In this research we report a FRα binding peptide C7(Met-His-Thr-Ala-Pro-Gly-Trp-Gly-Tyr-Arg-Leu-Ser) discovered by phage display and this peptide showed specific binding to FRα expressing cells by cell ELISA and flow cytometry. Tumor targeting ability of C7 was proved in vivo by both phage homing experiment and fluorescence imaging. C7 can be internalized by SKOV3 cells and its affinity to FRα was determined by MST. The molecular recognition was revealed by structure modeling, suggesting its binding mode with FRα.
Asunto(s)
Técnicas de Visualización de Superficie Celular , Receptor 1 de Folato/metabolismo , Terapia Molecular Dirigida , Neoplasias Ováricas/patología , Péptido Hidrolasas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Femenino , Receptor 1 de Folato/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Simulación del Acoplamiento Molecular , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Estructura Secundaria de Proteína , Transporte de Proteínas/efectos de los fármacosRESUMEN
The present research has been carried out to elicit the mechanism of antiangiogenic activity of a fusion peptide P2. Peptide P2 was designed by the connection of a heptapeptide MMP inhibitor to ES-2, a fragment of Endostatin. In a previous study, P2 demonstrated strong antiangiogenic and antitumor effect, and the current work explains the antiangiogenic mechanism of P2 through endothelial cell proliferation and apoptosis. In our study, it was shown that P2 inhibited HUVECs proliferation at a low serum concentration and this effect might be achieved through arresting cell cycle by decreasing the expression level of Cyclin D1. In addition, P2 was found to induce apoptosis of HUVECs. Using Western blot, it was indicated that P2 induced the cleavage of Caspase-3, the hallmark protease of apoptosis. The activation and expression of the upstream regulator Caspase-9 can also be affected by P2 treatment. Furthermore, P2 reduced the protein level of antiangiogenic factors Bcl-xL and Bcl-2. These results revealed that P2 regulates endothelial cell apoptosis through intrinsic apoptotic pathway.
Asunto(s)
Antineoplásicos/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Péptidos/farmacología , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Caspasa 3/efectos de los fármacos , Caspasa 3/metabolismo , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ciclina D1/biosíntesis , Ciclina D1/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Péptidos/químicaRESUMEN
Phage peptide display is a powerful technique for discovery of various target-specific ligands. However, target-unrelated peptides can often be obtained and cause ambiguous results. Peptide PB-TUP has been isolated repeatedly in our laboratory on different targets and we conducted a research on PB-TUP phage to investigate their binding properties and rate of propagation. ELISA and phage recovery assay demonstrated that PB-TUP phage had a significant superior affinity to polystyrene solid surface compared with control phage clones. In this study, some incidental bindings are excluded like blocking agents and non-specific binding of secondary antibodies. Propagation rate assays of the selected phage clones showed that the growth rate of PB-TUP phage was not superior to the control phages. Furthermore, the binding of PB-TUB to polystyrene was concentration dependent and varied with solution pH. Molecular modeling revealed that stable structures of α-helix and ß-turn may contribute to the binding of PB-TUP to polystyrene plate. The PB-TUP sequence was fused to the N-terminus of peptide P2 and the fusion peptide significantly increased the binding affinity to polystyrene. The fusion peptide also enhanced the cell adhesion ability of peptide P2 with human umbilical vein endothelial cell (HUVEC). The addition of the polystyrene binding peptide provided a convenient method for peptide immobilization.