Antimicrobial peptides have gained much attention in clinical research due to their extensive possibilities of application beyond antimicrobial use. The modification of antimicrobial peptides enables the peptides to target particular cancer cells, improving the specificity and efficiency of the treatment. In this study, TP2-D-Tox, a derivative of TP-D-Tox, demonstrated a superior anti-tumor activity towards renal carcinoma, Caki-2, and breast carcinoma, SK-BR-3. TP-Tox was previously reported to inhibit tumor growth in a mouse model, increasing the overall survival. TP- and TP2-D-Tox were shown to penetrate the cells via clathrin-mediated endocytosis, triggered by binding to the subunits of non-muscle myosin IIa and S100A9. HSPB1 was observed to have a protective effect towards TP2-D-Tox against the immediate proteolytic inactivation. The intracellular presence of the peptides evoked mitochondrial permeability transition, generation of reactive oxygen species, and formation of MLKL oligomers in the plasma membrane. Our investigation revealed that TP- and TP2-D-Tox induced a similar but distinctly regulated cell death in Caki-2 and SK-BR-3 cells. Both peptides established toxicity without activating any caspases, suggesting the possibility of TP- and TP2-D-Tox as a promising approach to bypass the caspase-dependent apoptosis-resistance issue impairing therapeutic responses of many cancer treatments.

Keywords: MLKL; NMIIA; bifunctional peptide; cancer; clathrin‐mediated endocytosis.

© 2025 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.