Cytotoxic effects of branched cationic peptides derived from bovine lactoferricin on oral squamous cell carcinoma

Aim: Squamous cell carcinoma (SCC) is one of the most common types of cancer affecting the oral cavity and the upper aerodigestive tract. In many cases, misdiagnosis leads to the progression of the disease to life-threatening stages, highlighting the importance of advancing anti-oral cancer therapeutics. In this study, we evaluated the cytotoxic activity against SCC of a series of synthetic peptides derived from bovine lactoferricin (bLFcin). To enhance the cationic and amphiphilic properties of lactoferricin-derived peptides, we designed branched and palindromic sequences from bLFcin(20-25) (i.e., 20RRWQWR25). Methods: A total of 14 peptides were synthesized using solid support synthesis. More than 90% peptide purity was obtained by solid-phase extraction, and the final products were characterized using reversed-phase liquid chromatography and mass spectrometry. The in vitro cytotoxic activity of the peptides was assessed in cancer cell lines SCC9 and FaDu; HEK001 keratinocytes were used as a non-cancerous control cell line. Additionally, scanning electron microscopy and flow cytometry provided further insights into the peptide-cell interaction. Finally, the dimeric peptide bLFcin(20-25)2 was evaluated in a chemically-induced carcinoma in vivo model. Results: Tetrameric and dimeric peptides with higher cationic charge and improved amphiphilic properties were cytotoxic to the oral carcinoma cell line FaDu. Scanning electron microscopy revealed membrane disruption and pore formation. In addition, flow cytometric analyses of cells treated with fluorescently tagged bLFcin(20-25)2 confirmed the interaction of the cationic peptide with the anionic cancer cells. Furthermore, the dimeric peptide bLFcin(20-25)2 inhibited tumor growth of chemically induced oral carcinoma in hamsters, demonstrating its in vivo efficacy. Conclusions: Branched peptides with a higher cationic charge were cytotoxic to the cancer cell line FaDu; they induced cell membrane lysis, as evidenced by electron microscopy, and inhibited tumor progression in hamsters. These preliminary results support further design and mechanistic evaluation of branched cationic peptides as potential oral cancer therapeutics.