Evaluation of the Toxicological Efficacy of Protein Hydrolysates From Camel Milk β-Casein Against Different Types of Cancer Cell Lines in Vitro

Abstract

This study aimed to isolate and purify the β-CN protein from camel milk using ion exchange chromatography and gel filtration techniques and to verify its purity using polyacrylamide gel electrophoresis (PAGE), Then prepare hydrolyzates of β-CN isolated from camel milk by enzymatic hydrolysis using the enzyme pepsin and trypsin and their mixture in a ratio (1:1). The cytotoxicity of β-CN protein degraders was studied by conducting an MTT test to determine the toxic effect of β-CN degraders and their ability to inhibit the growth of cancer cells represented by HepG2 liver cancer cells, PC3 prostate cancer cells, and MCF-7 breast cancer cells and comparing them with normal HdFn cells in vitro. The results showed the presence of two peaks for the β-CN protein on DEAE-Cellulose and one peak on the Sephadex G-100 column and the appearance of a clear and pure protein band on the polyacrylamide gel. the highest decomposition rate was due to the synergistic action of the enzyme pepsin and trypsin at a ratio of 1:1 and amounted to 52.32%. The results also showed that β-CN protein hydrolyzates have an effective inhibitory effect on cancer cell growth at a concentration of 400µg/mL, as the inhibition rate reached (45.9, 59.5 and 63.3%) for the cell types, respectively. Based on the results of the MTT test, the MCF-7 cell line was chosen as it is the most affected by the β-CN degraders under study. Cytotoxicity was evaluated by performing an HCS test to detect some changes that may occur to cells by measuring cellular indicators, including the Viable Cell Count (VCC), total Nuclear Intensity (TNI), Cell Membrane Permeability (CMP), Mitochondrial Membrane Potential (MMP), and Cytochrome C releasing (CC) level. The results indicated a decrease in the number of (VCC) for MCF-7 cells, especially at concentrations of 100 and 200 µg/mL, by 31.53 and 47.58%, respectively, and an increase in (TNI), significantly at a significance level (P≤0.05), by 39.10 and 51.91%, respectively, there was a clear effect on (MMP) by 20.47 and 27.39%, respectively, and a significant increase in the level of Cytochrome C release by 16.16 and 25.84%, respectively. It can be concluded from the above that β-CN hydrolyzates have high inhibitory activity against the cancer cells under study in vitro.