Camel whey protein hydrolysates induced G2/M cellcycle arrest in human colorectal carcinoma

Abstract

Camel milk has been gaining immmense importance due to high nutritious value and medicinal properties. Peptides from milk proteins is gaining popularity in various therapeutics including human cancer. The study was aimed to investigate the anti-cancerous and anti-inflammatory properties of camel whey protein hydrolysates (CWPHs). CWPHs were generated at three temperatures (30 ℃, 37 ℃, and 45 ℃), two hydrolysis timepoints (120 and 360 min) and with three different enzyme concentrations (0.5, 1 and 2 %). CWPHs demonstrated an increase in anti-inflammatory effect between 732.50 (P-6.1) and 3779.16 (P-2.1) µg Dicolfenac Sodium Equivalent (DSE)/mg protein. CWPHs (P-4.3 & 5.2) inhibited growth of human colon carcinoma cells (HCT116) with an IC₅₀ value of 231 and 221 μg/ml, respectively. P-4.3 induced G2/M cell cycle arrest and modulated the expression of Cdk1, p-Cdk1, Cyclin B1, p-histone H3, p21 and p53. Docking of two peptides (AHLEQVLLR and ALPNIDPPTVER) from CWPHs (P-4.3) identified Polo like kinase 1 as a potential target, which strongly supports our in vitro data and provides an encouraging insight into developing a novel peptide-based anticancer formulation. These results suggest that the active component, CWPHs (P-4.3), can be further studied and modeled to form a small molecule anti-cancerous therapy.

Figure 1

Peptide profiling and anti-proliferative screening of camel whey protein hydrolysates (P-4.3 and P-5.2). (a) RP-UPLC peptide profile of CWP and their pepsin generated hydrolysates P-5.2 and P-4.3. (b) Camel whey protein hydrolysates P-4.3 and P-5.2 inhibit growth of HCT116 cells. Viability of HCT116 cells after treatment with increasing concentrations of the CWPHs P-4.3 and P-5.2 for a period of 48 h. 231 μg/ml is the IC₅₀ for CWPH P-4.3, 221 μg/ml is the IC₅₀ for CWPH P-5.2. (c) Quantitative distribution of HCT116 cells in different phases of the cell cycle after treatment with camel CWPH P-4.3 (231 μg/ml) over a period of 24 h, 48 h and 72 h. Statistical analysis was carried out by student’s t-test using GraphPad Prism software and p < 0.05 was considered as statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 2

Inhibitory effect of CWPH P-4.3 on cell cycle progressive markers in HCT116 cells. (a, c) Western blot analysis of cell cycle regulatory proteins from HCT116 treated with CWPH P-4.3 (231 μg/ml) over a period of 24 h, 48 h and 72 h. (b, d) Each band intensity was quantified to analyze the protein expression using ImageJ, normalized relative to their respective loading control bands. Values are expressed as ratio of untreated control in log fold. Statistical analysis was carried out by student’s t-test using GraphPad Prism software and p < 0.05 was considered as statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 3

Investigated peptides binding modes within the active site of PLK-1 Polo box domain (PDB-ID: 3RQ7). (a) Binding mode of peptide-1 (AHLEQVLLR) from CWPHs P-4.3 within the active site of PLK-1, (b) Binding mode of peptide-2 (ALPNIDPPTVER) from CWPHs P-4.3 within the active site of PLK-1. Left panels illustrated the overall binding site, while the right panels illustrated hydrogen bond interactions with the residues of the active site. Ligands and important amino acids residues are presented in stick rendering, while H-bond interactions are shown as green dashed lines with their corresponding distances in angstrom.

Figure 4

Camel whey protein hydrolysates induce G2/M cellcycle arrest in HCT116 cells. This graphical abstract shows the molecular mechanism employed by the camel whey protein hydrolysates in inducing anti-proliferative effect on the human colorectal cancer cells implicating PLK1 as a potential target.