The tea catechin epigallocatechin gallate inhibits NF-κB-mediated transcriptional activation by covalent modification

Abstract

Potential health benefits of consuming tea are thought to include anti-inflammatory actions of its constituent flavonoids including catechins, which are well-recognized antioxidants. We analyzed and discovered a novel mechanism by which epigallocatechin gallate (EGCG), the most abundant polyphenol in tea and a putative health-promoting constituent, inhibits activation of the nuclear transcription factor NF-κB, which mediates inflammatory responses to cytokines and other agents. We found that EGCG inhibits NF-κB-p65 transcriptional activity, by preventing NF-κB-p65 binding to κBs in normal human bronchial epithelial cells. We also analyzed the chemical mechanism by which EGCG binds directly to NF-κB-p65, and found that it involves covalent reaction via enones within EGCG ring structures, as the oxidizer diamide, which prevents 1, 4-addition reactions, blocked adduct-forming reaction of biotinylated EGCG with NF-κB-p65. Such blockade was inhibited by competing unlabeled EGCG. Furthermore, such covalent binding reflected irreversible reaction of EGCG with sulfhydryls of NF-κB-p65, as it was inhibited by glutathione but not reversible by it. We identified the reactive sulfhydryl moiety as that of cysteine, as S-carboxymethylation to block cysteine sulfhydryls prevented NF-κB-p65-Cys-alkylation reaction with EGCG. We also tested if EGCG can inhibit NF-κB-p65 binding to DNA within the nucleus, after its phosphorylation and translocation (activation). EGCG did not alter intranuclear phosphorylation levels of NF-κB-p65, but strongly repressed DNA-binding ability of activated NF-κB-p65, indicating that EGCG inhibits NF-κB-p65 DNA binding activity even without altering NF-κB-p65 phosphorylation or expression. These findings thus reveal a novel mechanism by which EGCG inhibits transcriptional activity of NF-κB-p65, that may potentially contribute to anti-inflammatory and health-promoting effects of EGCG and consumption of tea.

Keywords: Covalent inhibitor; Cys-alkylation; Epigallocatechin gallate; Human bronchial epithelial cells; NF-κB.