title: Mechanism of action of (-)-epigallocatechin-3-gallate: auto-oxidation-dependent activation of extracellular signal-regulated kinase 1/2 in Jurkat cells.
abstract: OBJECTIVE
  (-)-Epigallocatechin-3-gallate (EGCG), a major compound of tea polyphenols, exhibited antitumor activity in previous studies. In these studies, EGCG usually inhibits EGFR, and impairs the ERK1/2 phosphorylation in tumor cells. The aim was to clarify the mechanism of ERK1/2 activation induced by EGCG.
  
  
  METHODS
  Jurkat and 293T cells were treated with EGCG in different culture conditions. Western Blotting (WB) was employed to analyze ERK1/2 and MEK phosphorylation. Cetuximab and FR180204 were used to inhibit cell signaling. The stability of EGCG was assessed by HPLC. The concentration of hydrogen peroxide generated by the auto-oxidation of EGCG was determined by photocolorimetric analysis.
  
  
  RESULTS
  Activation of ERK1/2 was observed to be both time-and dose-dependent. Stimulation of cell signaling was dependent on MEK activity, but independent of EGFR activity. Unexpectedly, EGCG was depleted within one hour of incubation under traditional culture conditions. Auto-oxidation of EGCG generated a high level of hydrogen peroxide in the medium. Addition of catalase and SOD to the acidic medium inhibited the oxidation of EGCG. However, this particular condition also prevented the phosphorylation of ERK1/2. The generation of ROS by hydrogen peroxide may also induce ERK1/2 activation in Jurkat cells.
  
  
  CONCLUSIONS
  ERK1/2 phosphorylation was caused by auto-oxidation of EGCG. Traditional culture conditions were determined to be inappropriate for EGCG research.
authors.affiliation: College of Life Sciences, Jilin University, Changchun, 130012, China; Key Laboratory of Puer Tea Science, Ministry of Education, Kunming, Yunnan Agriculture University, 650201, China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Song
authors.affiliation: College of Life Sciences, Jilin University, Changchun, 130012, China; Key Laboratory of Puer Tea Science, Ministry of Education, Kunming, Yunnan Agriculture University, 650201, China.
authors.forename: Ye-Wei
authors.initials: YW
authors.lastname: Huang
authors.affiliation: College of Life Sciences, Jilin University, Changchun, 130012, China; Key Laboratory of Puer Tea Science, Ministry of Education, Kunming, Yunnan Agriculture University, 650201, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Tian
authors.affiliation: Key Laboratory of Puer Tea Science, Ministry of Education, Kunming, Yunnan Agriculture University, 650201, China. Electronic address: wangxuanjun@gmail.com.
authors.forename: Xuan-Jun
authors.initials: XJ
authors.lastname: Wang
authors.affiliation: College of Life Sciences, Jilin University, Changchun, 130012, China; Key Laboratory of Puer Tea Science, Ministry of Education, Kunming, Yunnan Agriculture University, 650201, China. Electronic address: shengj@ynau.edu.cn.
authors.forename: Jun
authors.initials: J
authors.lastname: Sheng
mesh_terms: D028241:Camellia sinensis / Q000737:chemistry*; D002374:Catalase / Q000378:metabolism; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D019169:Jurkat Cells; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D048053:Mitogen-Activated Protein Kinase 6 / Q000378:metabolism*; D010084:Oxidation-Reduction; D010766:Phosphorylation; D010936:Plant Extracts / Q000494:pharmacology*; D059808:Polyphenols / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000378:metabolism
chemical_list: D010936:Plant Extracts; D059808:Polyphenols; D002392:Catechin; D006861:Hydrogen Peroxide; C045651:epigallocatechin gallate; D002374:Catalase; D013482:Superoxide Dismutase; D048052:Mitogen-Activated Protein Kinase 3; D048053:Mitogen-Activated Protein Kinase 6
keywords: (–)-Epigallocatechin-3-gallate (EGCG); Auto-oxidation; ERK1/2; Hydrogen peroxide; Phosphorylation