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 in Galleria 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.

Synthetic peptides are commonly obtained by means of solid-phase peptide synthesis (SPPS), in which separation of the peptide from the solid support requires treatment with 92.5% v/v trifluoroacetic acid (TFA); therefore, peptides are obtained as trifluoroacetate salts. For promising anticancer/antibacterial peptides it is essential to exchange the counterion from trifluoroacetate to hydrochloride or acetate, since the former are more widely studied in biological activity assays. In this research, RP-SPE-based methodologies were designed, developed, and implemented for simultaneous counterion exchange and peptide purification. Critical process steps were identified and parameters such as mobile phase composition, elution, and program were optimized. Analysis of the counterion exchange reaction and characterization of the final products was performed using high-performance liquid chromatography, attenuated total reflectance, nuclear magnetic resonance, and mass spectrometry. Peptides with purities between 82–97% and a trifluoroacetate ion content less than 0.36% were obtained. This novel counterion exchange proved efficient for peptides with different characteristics such as length, polarity, polyvalency, and presence of non-natural amino acids or non-protein molecules, therefore showing a wide range of applications in the field of therapeutic peptides. The methods developed are fast, efficient, low-cost, and do not require robust instrumentation and can be routinely implemented in SPPS.

This study focuses on the chimeric peptide KKWQWK-Ahx-RLLRRLLR and its interaction with cancer cells, specifically HPV18-positive cervical cancer HeLa and Ca Ski cells. The main objective of this study was to understand the mode of action of this chimera related to its cytotoxic activity, as well as the internalization processes and the type of cell death induced. For this purpose, several in vitro and in vivo assays were performed, showing that the uptake of the chimera is not energy-dependent and could involve a passive transport process. The results suggested that chimera internalization can be mediated by a specific interaction of the peptide with molecules on the cell membrane. The cytotoxic effect of the chimera in cervical cancer cells causes severe morphological changes, including rounding, shrinking, and vacuole formation. It was also determined that the chimera primarily induces early and late apoptosis in HeLa cells, without causing necrosis, and activates caspases 3 and 7. The chimera was localized in both the cytoplasm and the nucleus of the cancer cells, suggesting that the peptide could interact with intracellular targets. In conclusion, this study provides a broader understanding of the mechanism of action of the KKWQWK-Ahx-RLLRRLLR chimera on cancer cells, highlighting its ability to induce a fast, selective, and significantly cytotoxic effect in cervical cancer cells, which involves cell death through the apoptotic pathway. The toxicity assays in Galleria mellonella and zebra fish showed that the chimera is safe and can be considered for preclinical studies. This study demonstrated that the chemical binding of two sequences with low activity produces a chimeric entity with enhanced cytotoxic activity capable of cellular internalization and inducement of apoptosis.