Mechanisms of In Vitro Cytotoxicity of Honeybee Venom Components and Melittin-Functionalized Fe3O4 Nanoparticles on HaCaT Keratinocytes and A375 Melanoma Cells

Melittin (Mel), the principal cytolytic peptide in honeybee venom (BV), has anticancer activity but limited selectivity. This study examined whether adsorption of Mel to Fe3O4 magnetic nanoparticles (MNPs and MNPs-Mel, respectively), alone or combined with magnetic hyperthermia (MH), modifies cytotoxicity and cell-death phenotype in A375 melanoma cells relative to HaCaT keratinocytes. BV, free Mel, and phospholipase A2 (PLA) were first screened by Alamar Blue assay to select dose-matched conditions; MNPs-Mel (50 or 100 µg/mL MNPs, equivalent to approximately 3 or 6 µg/mL Mel) was then compared with dose-matched free Mel and unfunctionalized MNPs, and the MNP-containing groups were additionally evaluated with MH. Outcomes included metabolic activity, Annexin V/propidium iodide flow cytometry, transmission electron microscopy (TEM), and clonogenic potential. At the lower dose, MNPs-Mel produced a larger Annexin-positive fraction in A375 than in HaCaT cells (28.30% vs. 11.80%) and a lower viable-cell fraction (71.64% vs. 87.55%); relative to dose-matched free Mel, the A375 response shifted toward early apoptosis. After MNPs-Mel plus MH, too few A375 cells remained for reliable cytometric acquisition, whereas HaCaT populations remained quantifiable and showed 25.56–29.45% apoptosis, predominantly late apoptosis. Free PLA produced smaller changes in metabolic activity than BV or Mel at composition-matched concentrations. TEM supported nanoparticle internalization and treatment-associated mitochondrial and lysosomal alterations. These findings suggest that MNP association changes the cellular presentation of low-dose Mel, while MH increases overall treatment intensity but may narrow the separation between malignant and nonmalignant cells.