The Multi-targets Inhibition Mechanism Of Apple Polyphenols On Tumor Cells Based On The Caco-2 Cell Model

Phenolic compounds are one of the main biologically active substances in apples,which can reduce the incidence of colon cancer.At present,there have been many studies on the anti-proliferative activity of apple polyphenols on human colon cancer(Caco-2)cells,but the relevant mechanism of action is still unclear.In this paper,targeted screening was used to obtain the potentially highly active apple polyphenol monomer,cyanidin-3-O-arabinoside,and verified its anti-tumor activity using cell experiments.Metabolomics technology is used to explore its effect on Caco-2 cell metabolites and clarify its anti-tumor mechanism.The main research contents are as follows:1.Aminopeptidase N(APN),janus kinase(JAK)and enhancer of zeste homolog 2(EZH2)were as targets using molecular docking technology.Cyanidin-3-O-arabinoside with potentially high activity and non-toxicity which can bind tightly to three target proteins was screened form88 apple polyphenols and was analyzed their interaction mechanism;2.The Caco-2 cell model was used to evaluate the anti-proliferative effect of cyanidin-3-O-arabinoside and anti-tumor activity.The CCK-8 experiment results showed that at the same concentration of cyanidin-3-O-arabinoside,the inhibition rate of Caco-2 cell proliferation increased with the increase of incubation time.At the same incubation time,the inhibition rate increased as the concentration increased.Compared with the positive control group,as the incubation time increased,the anti-proliferation effect of cyanidin-3-O-arabinoside was more significant.After 72 h of simultaneous incubation,the inhibitory rate of 120?g m L-1cyanidin-3-O-arabinoside was about 4 times that of the positive control group.The EC₅₀ value of72 h is the lowest than that of 24 h and 48 h,which is 112.40±4.36?g m L^-1 and has the most significant inhibitory effect on the proliferation of Caco-2 cells.That proved that cyanidin-3-O-arabinoside can inhibit the proliferation of Caco-2 cells.The flow cytometry was used to detect the effect of cyanidin-3-O-arabinoside on the apoptosis and cell cycle of Caco-2cells,and the results showed that apoptosis can be induced by blocking cells of the S phase.Western Blot experiment results showed that the pro-apoptotic protein Bax protein level was up-regulated,and the anti-apoptotic protein Bcl-2 protein level was down-regulated in the experimental group;3.The metabonomic analysis based on LC-Q-TOF-MS identified the effects of cyanidin-3-O-arabinosides(120?g m L^-1)on the metabolites of Caco-2 cells after incubation for72 h.Thirty-eight significant differential metabolites were identified in the positive and negative ion mode.And five potential metabolic markers closely related to the enrichment pathway of the KEGG pathway were screened,including cytidine,sphingosine,phosphoethanolamine,uracil and UDP-glucose.The metabolic pathways related to these metabolites included apoptosis,pyrimidine metabolism,?-alanine metabolism,sphingolipid metabolism and sphingolipid signaling pathway.In summary,cyanidin-3-O-arabinoside with anti-tumor effect in apple polyphenol was obtained using targeted screening technology,and the anti-tumor mechanism of cyanidin-3-O-arabinoside on Caco-2 cells was studied using non-targeted metabonomics technology,and the potential metabolic markers were screened.This study provided a theoretical basis for elucidating the anti-tumor mechanism of apple polyphenols.