Copper(II) binding properties and anticancer activity of peptide fragments from a β-strand region of angiogenin protein

Angiogenin is a potent angiogenic factor overexpressed in many cancers. In this study, we report the characterization of copper(II) complexes of two peptides from the 40–50 angiogenin domain by means of analytical techniques such as potentiometry, UV–visible, circular dichroism and by DFT calculations. Potentiometry and spectroscopic analysis revealed that at physiological pH, the predominant species formed by the peptide 40–48 is [CuLH−2] in which the metal ion is coordinated by one imidazole nitrogen and three deprotonated amide nitrogen atoms. Differently, for the longer peptide Ang40-50 the main species is [CuL], with metal ion bound to one imidazole and two deprotonated amides. DFT calculations highlight the different distortion of tetragonal geometry in complex species, which increases with the involvement of more deprotonated amides. It is to note that Cu2+ center holds similar electronic features even though the number of deprotonated amides coordinated to the metal ion differs. Cytotoxic effects of peptides and their complexes were evaluated against normal human osteoblasts hFob and U2OS bone osteosarcoma cell lines using MTT assay. The free peptides show no cytotoxicity on both cell lines, whereas copper complexes are active only towards cancer cells with the most pronounced effect observed for the Cu(II)-Ang40–50 system. The effect of two peptides on mitochondrial membrane potential (ψm) was also assessed. Peptides and their complexes do not affect ψm of normal hFob cells. However, while the peptides do not affect mitochondrial potential in cancer U2OS cells, their complexes decreased the level of ψm.These findings show that copper complexes exhibit selective cytotoxicity towards cancer cells by perturbing mitochondrial membrane potential.