Inhibition Of Proliferation Induced By Papain Protease Hydrolysate Of Walnut Protein On MCF-7 Cells

Protein was prepared from walnut kernel,and walnut protein hydrolysates were prepared by alkaline protease,trypsin,pepsin,papain and neutral protease.The effect of walnut protein and the hydrolysates on proliferation of human breast cancer MCF-7 cell was investigated by MTT assay.The mechanism underlying the activity was studied using flow cytometry,immunofluorescence,westernblotting and other methods.The main results were as follows:1)Walnut protein hydrolyzates were prepared with alkaline protease,trypsin,pepsin,papain and neutral protease.The effect of walnut protein and hydrolyzates on proliferation of human breast cancer cell MCF-7 was studied by MTT assay.The results showed that the walnut protein hydrolyzed by papain protease inhibited significantly proliferation of the cells(IC50 value was 1.62 mg/mL),while walnut protein and the protein hydrolyzate by other four proteases exhibited no obvious inhibitory effect2)The papain protease hydrolysate of walnut protein also inhibited significantly proliferation of human colon carcinoma cell(Caco-2,IC50 value was 2.04 mg/mL),and human cervical carcinoma cell(Hela,IC50 value was 2.21 mg/mL).However,it did not show inhibitory effect on the growth of rat small intestinal crypt epithelial cell(IEC-6).3)Cell cycle distribution was analyzed by flow cytometry.MCF-7 cells were treated with papain protease hydrolysate of walnut protein(2 or 4 mg/mL),which significantly increased the number of cells in sub-G1 phase to 7.67%or 1 8.24%compared with control(3.79%)at the concentration of 2 or 4 mg/mL.The cell proliferation index at o,2 and 4 mg/mL were 0.4561,0.4321,0.3796 respectively.This results suggested that the walnut protein hydrolysate inhibited growth of cells in G0/G1 phase.4)Annexin V-FITC/PI and TUNEL assay showed that the walnut protein hydrolysate induced apoptosis in a does-dependent manner,and the apoptosis rate were 8.17%and 18.11%at concentration 2 mg/mL and 4 mg/mL.5)LC3-? is known to correlate with the extent of autophagy,which was determined by western blotting analysis.The walnut protein hydrolysate significantly increased the level of expression LC3-?in does dependent manner.The result of laser confocal immunofluorescence experiment indicated that LC3-? was in the cytoplasm and the walnut protein hydrolysate induced autophagy.These results suggest that bioactive peptides with inhibitory effect on proliferation of cancer cells were contained in walnut protein,and the biological activity was exhibited when the walnut protein was hydrolyzed by papain.The inhibition was induced by apoptosis and autophagy.