The Mechanism Of Tannic Acid Interferes The Proliferation Of Colon Cancer Cells Through Targeted Inhibition Of PKM2 Activity

Tannic acid(TA),a naturally occurring polyphenolic acid that is primarily found in pomegranate,gallnut,grape and mango.It has attracted much attention due to its potent antioxidant,anti-inflammatory,hypoglycemic,lipidlowering and anticarcinogenic characteristics.However,the underlying molecular mechanisms and targets of Tannic acid,which are responsible for cancer prevention,remain elusive.In the present study,we used tannic acidfunctionalized magnetite nanoparticles to identify pyruvate kinase isoenzyme M2(PKM2)as the direct target of tannic acid.We further studied how tannic acid can inhibit the proliferation of colon cancer cells through its target protein in vitro and investigated the site and interaction mode between tannic acid and target protein.The main contents of this study are as follows in three parts:1.We produced magnetic nanoparticles by synthesis.Then magnetic nanoparticles were used as carriers to combine with tannic acid in ethanol to synthesis tannic acid-functionalized magnetite nanoparticles.It was analyzed and confirmed by transmission electron microscopy and Fourier transform infrared spectrometer.In the solid-phase experiment,the binding proteins of tannic acid in colon cancer cells were separated by pull down assay.The bound proteins were analyzed by SDS-PAGE silver nitrate stained analysis.We identified that PKM2 is a target of tannic acid by mass spectrometry.2.The results of MTT and clonogenic assay demonstrated that tannic acid significantly suppressed DLD1 and HCT-116 cell growth.In contrary,tannic acid exerted almost no effects on the growth of normal colonic epithelial cells.The results of western blot and qPCR showed that tannic acid did not affect the endogenous expression profile of PKM2.Thus,we investigated that kinase activity of PKM2 were responsible for cancer prevention.Tannic acid treatment did not alter Akt phosphorylation levels induced by recombinant PKM2.These results demonstrated that the protein kinase activity of PKM2 was not responsible for the inhibitory effects of tannic acid on the proliferation of CRC cells.We examined cell viability rates in tannic acid-treated colon cancer cells with overexpressing the PKM1 protein to further assess the role of the metabolic activity of PKM2 in tannic acid-mediated antitumor activities and found that the inhibitory effects of tannic acid on DLD1-PKM1 and HCT-116-PKM1 cells were much lower than the effects on control cells.It suggested that the pyruvate kinase activity of PKM2 was critical for tannic acid-mediated CRC cell proliferation.Tannic acid slightly altered PKM1 activity,and tannic acid was a powerful selective inhibitor toward PKM2.3.Fluorescence spectroscopy was used to examine the binding of tannic acid to PKM2.Enzymatic kinetics results showed that inhibitory mode was noncompetitive.Purified recombinant PKM2 protein significantly bound TAfunctionalized magnetite nanoparticles.However,the combination of tannic acid and PKM1 protein was not detected.A glutaraldehyde crosslinking assay demonstrated that tannic acid treatment decreased the amount of PKM2 tetramers.PKM2 is allosteric regulated by FBP,while PKM1 is not affected by allosteric regulation.Tannic acid caused a dramatic reduction in PKM2 activity,which was only slightly and gradually improved by the addition of increasing concentrations of FBP.Therefore,the tannic acid binding sites on PKM2 may also be involved in the FBP binding sites.There are 6 important amino acid regulatory sites on PKM2 protein.The pyruvate kinase activity of PKM2 is only affected by R399 and K433.Pull down assays further demonstrated that tannic acid-functionalized magnetite nanoparticles bound R399 E but not K433 E.Tannic acid significantly impaired WT PKM2 activity,which was dramatically restrained in K433 E mutants.The results further support the obvious tannic acid suppression of cell proliferation in DLD1-GFP cells,but tannic acid exerted only a weak inhibitory effect on DLD1-K433 E cells.Collectively,these results indicate that lysine 433 is the key site for tannic acid binding to PKM2.In summary,the results of this study demonstrates that tannic acid impairs CRC cell proliferation by directly bind to K433 residue to impede CRC cell proliferation.