Preparation and identification of anti-breast cancer cells peptides released from yak milk casein

Abstract

Yak milk casein (YMC) is the main protein in the yak milk. Peptides released from Yak milk casein (YMC) have multiple bioactivities, including anti-inflammation and immune-regulation, suggesting that these peptides might be able to inhibit cancer theoretically. However, the anti-cancer peptides from YMC have only been sparsely studied. Breast carcinoma is the most common carcinoma in women worldwide. Thus, the paper herein was to identify yak milk casein (YMC)-derived anti-breast cancer peptides via gel filtration, reversed phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) for the first time. The inhibitory effects of the hydrolysates on the cell viabilities, cell cycles and apoptosis of breast cancer cells were evaluated with a cck8 kit and a flow cytometry. The result showed that YMC hydrolysates (YMCH) obtained by united hydrolyzation with trypsin (3 h) and alkaline protease (3 h) displayed the highest cell viability inhibition rate for MCF7 (20.74 ± 1.39%) and MDA-MB-231 (26.73 ± 2.87%) cells. Three peptides were identified in the RP-HPLC subfraction F3-4, and a nonapeptide (TPVVVPPFL) showed the most potent inhibitory effects on both cancer cells and displayed good gastrointestinal stability. TPVVVPPFL could induce G2-M cell cycle arrest in MCF7 cells and S cell arrest in MDA-MB-231 cells and induce apoptosis in both cancer cells. Moreover, in silico analysis indicated that the peptide had non-toxic and no inhibitory roles on P4502D6-enzyme. Together, this study shows that YMC is a good source of anti-breast cancer cells peptides.

Keywords: breast cancer cells; hydrolysate; identification; peptides; yak milk casein.

FIGURE 1

Anti-breast cancer cells activity and degree of hydrolysis of yak milk casein hydrolysates (YMCH). Yak milk casein (YMC) was united hydrolyzed by trypsin (3 h)+alkaline protease (0–3 h). MCF7 and MDA-MB-231 cells were incubated with the hydrolysates (final assay dose, 0.5 mg/mL) for 48 h, and the inhibitory effects of hydrolysates on the two breast cancer cells were evaluated by CCK8.

FIGURE 2

Elution profile (A) and anti-breast cancer cells activity (B,C) of yak milk casein hydrolysates (YMCH) fractions separated using a Sephadex G-25 gel column. The final dose of various YMCH was 0.1 mg/mL, the treatment time was 48 h.

FIGURE 3

Reversed phase high-performance liquid chromatography chromatogram of F2 fraction (A) and the cell viability inhibition rate of subfractions against MCF7 cells (B) and MDA-MB-231 cells (C). Both cancer cells were treated with 0.2 mg/mL of various subfractions (F3-1∼F3-4) for 48 h. The inhibitory effects of subfractions on the two breast cancer cells were evaluated by CCK8.

FIGURE 4

Identification of anti-breast cancer cells peptides. (A) LC-ESI MS/MS spectrum of the double-charged ion with m/z 510.2713. The spectrum fitted to TPVVVPPFL. (B) The primary structure of yak β-casein (UniProt KB number H2DZG3) and potential anti-cancer peptides identified in the yak casein hydrolysate fractions are colored yellow.

FIGURE 5

Synthesized peptides inhibited the cell viabilities of MCF7 and MDA-MB-231 cells. (A) The effect of synthesized peptides on the cell viabilities of the two cells. Both malignant cells were treated with synthesized peptides (TPVVVPPFL, NQFLPYPY, and VAPFPEVFGK) for 48 h at the concentration of 0, 62.5, 125, 250, 500, and 1,000°μg/mL. The cell viabilities were evaluated by CCK8. (B) The cell viability inhibition rate of TPVVVPPFL against MCF7 and MDA-MB-231 cells. The two malignant cells were incubated with TPVVVPPFL for 24–72 h at the concentration of 0, 62.5, 125, 250, 500, 1,000°μg/mL, respectively. The inhibitory effects of TPVVVPPFL on both breast cancer cells were evaluated by CCK8.

FIGURE 6

TPVVVPPFL elicited cell arrest of mammary carcinoma cells. MCF7 cells were incubated with TPVVVPPFL for 48 h at the concentration of 250°μg/mL. MDA-MB-231 cells were incubated with TPVVVPPFL for 48 h at the concentration of 500°μg/mL. The cell cycle distributions were analyzed using flow cytometry after treatment with PI.

FIGURE 7

TPVVVPPFL induced morphology alteration (A) and apoptosis of mammary carcinoma cells (B). MCF7 and MDA-MB-231 cells were incubated with TPVVVPPFL for 48 h at the concentration of 250 and 500°μg/mL, respectively. The image of the two cells was acquired by the inverted microscope, and the apoptotic cells were measured using a flow cytometry after treatment with Annexin-V and PI.