Probing the cytotoxicity and the dynamic interaction of IIKK cationic peptides with human melanoma cells.

Cationic peptides have emerged as promising candidates in anticancer therapy due to their ability to directly target the plasma membrane of cancer cells, a mechanism that could potentially bypass traditional drug resistance pathways. In this study, we evaluated the cytotoxic activity and cell-membrane binding properties of three amphiphilic cationic peptides from the G(IIKK)ₙI-NH₂ family (n = 2-4) against human melanoma cells (SK-MEL-28). By performing MTT assays and tracking the propidium iodide (PI) uptake throughout peptide-cell interaction, we evaluated peptides' cytotoxicity. Assessment of the interaction dynamics was conducted by fluorescence spectroscopy assays with FPE, a surface potential sensitive probe. This evaluation indicated that an increase in net positive charge was relatable to a lower dissociation constant (Kd). Notably, G(IIKK)₄I-NH₂ showed the highest cytotoxicity, significant morphological alterations, rapid membrane permeabilization, and the lowest Kd, indicating a stronger membrane affinity when compared to the other peptides. G(IIKK)3I-NH₂, in the same manner as G(IIKK)₄I-NH₂, revealed a cooperative binding behavior, evidenced by a Hill coefficient > 1. An inverse correlation between peptide-cell membrane dissociation constants and cytotoxicity was established, supporting the notion that membrane interaction is a critical determinant of anticancer activity. In addition, we used a cell surface membrane potential probe to possibly anticipate the in vitro activity of cationic peptides. Altogether, these findings provide mechanistic insights into peptide-cell membrane interactions and may offer a basis for the rational design of novel anticancer peptides targeting melanoma.