Behind anticancer molecules: lactoferricin dimeric peptides with fast, selective, persistent and broad-spectrum cytotoxic effect

Abstract

Aim: To identify peptides derived from bovine lactoferricin (LfcinB) as potential therapeutics for colon cancer treatment. We systematically modified dimeric peptides to enhance their selectivity against colon cancer cells and reduce toxicity. We examined the effects of specific changes, such as substituting L-arginine (Arg) with L-ornithine (Orn) and/or D-Arg, on cytotoxic activity in colon cancer cells, as well as activity in prostate and cervical cancer cell lines. Additionally, we assessed the type of cell death induced and the in vivo toxicity of the dimeric peptides.

Methods: The peptides were synthesized by manual solid-phase peptide synthesis, purified by reverse phase-solid phase extraction (RP-SPE), and characterized by RP-high performance liquid chromatography (RP-HPLC), and mass spectrometry (MS). Their cytotoxic effect on cancer and non-cancerous cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The most promising dimeric peptide underwent scale-up synthesis to yield approximately 1 g. The type of induced cell death was analyzed through cytometry assays, while preliminarily toxicity studies were conducted inGalleria mellonella, zebrafish, and CD1 mice.

Results: Our findings demonstrated that dimeric peptides containing L-Orn or D-Arg residues exhibited potent and selective cytotoxic effects against colon cancer cells (Caco-2 and HT-29), prostate cancer cells (DU-145), and cervical adenocarcinoma (HeLa). Notably, these modified peptides showed minimal toxicity in human erythrocytes, HEK 293 cells or fibroblasts, and Galleria mellonella larvae. Peptide 3: (R-Orn-WQWRFKKLG)₂-K-Ahx, emerged as particularly promising, preserving its integrity and anticancer activity during scaled-up synthesis. Furthermore, peptide 3 induced behavioral changes and sedation in CD1 mice and showed significantly lower toxicity in zebrafish.

Conclusions: The results suggested that specific modifications of Arg/Orn residues in dimeric peptides enhance their cytotoxicity against colon cancer cells and reduce in vivo toxicity. These modified peptides hold promise as safe and effective therapeutic candidates, potentially expanding the treatment options available for cancer.

Keywords

Dimeric peptides / bovine lactoferricin / cancer / toxicity / ornithine / Galleria mellonella / zebrafish

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