Physicochemical Characteristics of Amphipathic Peptides and Their Cytotoxic Effects on Cancer and Normal Cell Lines

The aim of this study was to investigate which physicochemical and structural properties of cationic peptides P1-P6 may determine their selective anticancer activity against melanoma cells and their interactions with tumor cell membranes. An integrated approach was applied, including characterization in solution (osmotic pressure, NaCl stability, surface tension); cytotoxicity evaluation against Me45, B16F10, and HaCaT cells; analysis of interactions with phosphatidylglycerol (POPG) model membranes using isothermal titration calorimetry and steady-state fluorescence spectroscopy; membrane permeability assays; and F-actin staining. Anticancer activity depended on positively charged residues, hydrophobic amino acids, and sequence arrangement. Tryptophan-rich peptides P2 and P5 exhibited strong membrane interactions and high efficacy after 72 h. Highly hydrophobic P4, containing long C₁₂ chains with a relatively low net charge, caused nonselective lysis. P3 showed reduced activity due to insufficient amphipathicity, whereas P6, with excessive WWW and KKKK motifs, exhibited weak or nonselective effects. Thermodynamic and fluorescence analyses indicated that P2 and P5 initially bind POPG membranes via entropy-driven electrostatic interactions, followed by hydrophobic insertion of tryptophan residues, evidenced by increased fluorescence intensity and a blue shift of the emission maximum. P2, P4, and P5 induced actin cytoskeleton reorganization and increased membrane permeability, emphasizing the role of balanced amphipathicity and charge-hydrophobicity in designing selective anticancer peptides.