Antitumor Effect of Cationic INKKI Peptide from Bovine β-Casein on Melanoma B16F10

Cationic peptide with the sequence INKKI 41-45 was isolated from bovine β-casein after tryptic hydrolysis and synthetized. The aim of this work was to evaluate the antiproliferative activity in vitro and antitumor effect in animal model. The in vitro cytotoxicity was evaluated on B16F10 melanoma cells by MTT assay. Detection of apoptosis was measured using the annexin V/PI double staining and cell cycle analysis performed flow cytometry. Caspase-3 activity was analyzed with substrate specific fluorogenic DEVD-MCA. In vivo, antitumor activity was evaluated in B16F10 melanoma tumor-bearing C57BL/6J mice. The animals were treated with 55 mg/kg INKKI administered into peritumoral region, while control group received saline solution. The following antitumor parameters were examined: tumor volume, number of metastases, tumor delayed time, tumor doubling time. Histological analyses were performed with H & E staining. The results showed that INKKI induced dose-response cytotoxicity selective for B16F10 melanoma cells (IC50 1.7 μM) and did not present cytotoxic effects for FN1 fibroblast cells. INKKI-induced apoptosis detected trough of annexin V/PI assay and it was accompanied with an increase of sub-G1 apoptotic fractions and significant increase of caspase-3 cleavage. The tumor-bearing mice treated with INKKI showed a significant reduction in tumor volume of 72.62% and decreased of metastasis number loci. In addition, INKKI caused a significant delay in tumor growth and prolonged the tumor doubling time. Histological analysis revealed an increased of necrosis areas and reduction of tumor cells in tumor treated with INKKI, it was a many hallmark of its antitumor effects observed from in vivo experiments. In conclusion, we show that INKKI is a peptide that could be considered a new putative candidate development to anticancer therapy drug.