RESUMEN
The solid-phase synthesis, structural characterization, and biological evaluation of a small library of cancer-targeting peptides have been determined in HepG2 hepatoblastoma cells. These peptides are based on the highly specific Pep42 motif, which has been shown to target the glucose-regulated protein 78 receptors overexpressed and exclusively localized on the cell surface of tumors. In this study, Pep42 was designed to contain varying lengths (3-12) of poly(arginine) sequences to assess their influence on peptide structure and biology. Peptides were effectively synthesized by 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis, in which the use of a poly(ethylene glycol) resin provided good yields (14-46%) and crude purities >95% as analyzed by liquid chromatography-mass spectrometry. Peptide structure and biophysical properties were investigated using circular dichroism spectroscopy. Interestingly, peptides displayed secondary structures that were contingent on solvent and length of the poly(arginine) sequences. Peptides exhibited helical and turn conformations, while retaining significant thermal stability. Structure-activity relationship studies conducted by flow cytometry and confocal microscopy revealed that the poly(arginine) derived Pep42 sequences maintained glucose-regulated protein 78 binding on HepG2 cells while exhibiting cell translocation activity that was contingent on the length of the poly(arginine) strand. In single dose (0.15 mM) and dose-response (0-1.5 mM) cell viability assays, peptides were found to be nontoxic in human HepG2 liver cancer cells, illustrating their potential as safe cancer-targeting delivery agents.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Péptidos de Penetración Celular , Sistemas de Liberación de Medicamentos/métodos , Neoplasias Hepáticas/tratamiento farmacológico , Péptidos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Péptidos de Penetración Celular/síntesis química , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacología , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteínas de Neoplasias/metabolismo , Péptidos/síntesis química , Péptidos/química , Péptidos/farmacología , Unión Proteica , Relación Estructura-ActividadRESUMEN
Linear, branch and hyperbranch siRNAs were effectively prepared for down-regulating GRP78 expression and inducing cell death in HepG2 liver cancer cells. Branch and hyperbranch GRP78 siRNAs were synthesized by automated solid-phase synthesis in good yields (44-78%) and isolated in excellent purities (>99%) following HPLC purification. Moreover, siRNAs adopted stable intramolecular hybrids as discerned by native PAGE and thermal denaturation studies. These sequences also exhibited the pre-requisite A-type helical trajectory for triggering RNAi activity as determined by CD spectroscopy. Biological studies confirmed potent suppression of GRP78 expression (50-60%) while compromising cancer cell viability by ~20%. Thus, branch and hyperbranch siRNAs may serve as potent siRNA candidates in cancer gene therapy applications.