Mechanism of action of(-)-epigallocatechin-3-gallate:autooxidation-dependent activation of extracellular signal-regulated kinase 1/2 in Jurkat cells

AIM:(-)-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.CONCLUSION:ERK1/2 phosphorylation was caused by auto-oxidation of EGCG.Traditional culture conditions were determined to be inappropriate for EGCG research.