PEPAD: A Promising Therapeutic Approach for the Treatment of Murine Melanoma (B16F10-Nex2)

<b>Background/Objectives</b>: Cancer is one of the leading causes of death worldwide, and skin cancer is especially prevalent and lethal in Brazil. Despite advancements in treatment, there is still a need for new anticancer agents that are effective, selective, and less toxic. This study aimed to evaluate the cytotoxic and therapeutic potential of the peptide PEPAD. <b>Methods:</b> The cytotoxicity of PEPAD was assessed by MTT assay in murine melanoma (B16F10-Nex2), human melanoma (SK-MEL-28), breast (MCF-7), and cervical (HeLa) cancer cell lines. Selectivity was evaluated in healthy cells (RAW 264.7 and FN1). Morphological changes were analyzed by microscopy. Cell migration was assessed using scratch assays. Apoptotic features were evaluated using MitoTracker Deep Red, NucBlue, CaspACETM labeling, and flow cytometry. Immunogenic cell death was investigated by calreticulin and HMGB1 release. Molecular dynamics simulations explored peptide structure and interaction with lipid membranes. <b>Results:</b> PEPAD showed IC₅₀ values of 7.4 µM and 18 µM in B16F10-Nex2 and SK-MEL-28 cells, respectively, and >60 µM in MCF-7 and HeLa cells. Low toxicity was observed in healthy cells (IC₅₀ > 56 µM), indicating high selectivity. Apoptotic morphology and reduced cell migration were observed. Flow cytometry and fluorescence probes confirmed apoptosis and mitochondrial swelling. Calreticulin and HMGB1 release indicated immunogenic cell death. Simulations showed that PEPAD maintains a stable α-helical conformation and interacts with membranes. <b>Conclusions:</b> These findings highlight PEPAD's selective cytotoxicity and its potential as an anticancer agent with apoptotic and immunogenic properties, making it a promising candidate for therapeutic development.