Mechanism Of EGCG Inhibiting STAT3Signaling Pathway

BackgroundTea is one of the most popular beverages in the world and has been well known to promote goodhealth in multiple ways over two thousand years. The daily consumption of tea might reduce cholesteroland chance of heart disease, boost immunity, and benefit human skin et al. Particularly, tea may lower therisk of various types of cancers, including gastric, pancreatic and colorectal, in the human population.EGCG, which contributes to more than40%of the total polyphenol mixture in tea, plays a very importantrole in the chemotherapeutic and chemo-preventive effects of tea. In fact, the anti-oxidative activity andmetal chelating functions from EGCG may contribute to the tea’s inhibitory activity against carcinogenesis.Additionally, there is considerable evidence that EGCG has anticancer nature by modulating theintracellular signaling network.Stat3is one of the seven members of the Stat protein family that mediates the actions of manycytokines and growth factors. Stat3shows constitutive activity in many different types of cancers, includingbreast, prostate, head and neck, lung, colon, liver and pancreas cancers, large granular lymphocyticleukemia, and multiple myeloma et al. In addition, human tumor xenograft studies in mice have repeatedlydemonstrated that inhibiting STAT3signaling results in decreased tumor growth and improved animalsurvival by inducing apoptosis in tumor cells, inhibiting angiogenesis, and enhancing anti-tumorimmune-mediated cytotoxicity. Thus, STAT3has been identified as a potential high-yield target forpharmaceutical prevention in treating many types of cancers.PurposesTo explore the possible molecular mechanisms of EGCG through inhibiting JAK-STAT3signaling pathway, which regulates the proliferation of cancer cells.MethodsThis research method is divided into two parts.The first part: In this study, We explored themechanism of epigallocatechin-3-gallate (EGCG) inhibiting STAT3signaling by Surface PlasmonResonance (SPR) binding assays and docking in silico. And the second part Cancer cells were traditionallycultured in vitro and intervened by EGCG,①MTT assay was employed to evaluate the effect on the proliferative inhibition of cancer cells, after which time the relationship between drug concentration andproliferation inhibition rate was analyzed;②The percentage of apoptotic cells was examined by flowcytometry to analyze the effect of EGCG on cell apoptosis;③After the intervention of EGCG, cancer cellnuclear total-Stat3and phosphorylated Stat3protein content were assayed by Western blot and cell-basedELISA;④The mRNA levels of Bcl-xl, C-myc, VEGF and cyclin D1in QGY-7704cells treated withEGCG were detected by Semi-quantitative RT-PCR.ResultsBased on our study on BIAcore binding assay in micromole, EGCG blocked Stat3binding to itsphosphopeptide ligand, according to SPR testing. Furthermore, the EGCG molecule had major interactionswith two key residues–R-609and K-591–localized in Stat3SH2domain, as found through dockingsimulation analysis. The study of the mechanism of inhibiting the proliferation and apoptosis ofhepatocellular carcinoma by EGCG through JAK-STAT3signaling pathway revealed the following:①cellproliferation inhibition rates in HCC cell lines Bel7402and QGY7703had an upward trend after a48-hourintervention with different concentrations of EGCG, according to cell proliferation assay. As theconcentration increases, the inhibitory rate also increases in a dose-dependent pattern; P<0.05is consideredsignificant.②After the48-hour treatment of EGCG in Bel-7402and QGY-7703cells, the concentration ofEGCG increases, causing a corresponding increase in the apoptosis rate of the QGY-7703cell line in adose-dependent manner (P<0.05), by flow cytometry.③As the concentration of EGCG increased, theintracellular core protein expression of t-Stat3levels remained the same, while the p-Stat3protein in theexperimental group cells decreased significantly in comparison to the negative control group（P<0.05）,according to Western blot experiment. In another words, the higher the concentration, the lower theexpression. At the same time, the relative intensity of p-Stat3is the same for AG490at50μM as for EGCGat40μM.④mRNA expressions in BCL-XL, c-myc, VEGF and cyclinD1were significantly lower than thatin the negative control group （P<0.05）, as shown in a dose-dependent pattern according toSemi-quantitative RT-PCR.ConclusionsOur research data supports that green tea’s anti-cancer function may have been resulted fromEGCG inhibition of STAT3signaling pathway. EGCG is a STAT3signaling inhibitor that competitively binds to Stat3SH2domain, contributing to the regulation of cellular signaling network that helps to explaintea’s anticancer effects.