Determination Of The Major Factors In EGCG Stability For Cell Culture And The Insulin Receptor Phosphorylation Induced By EGCG

Tea is the most widely consumed beverage in the world, it has been demonstratedtea is of great benefit to human health, for example, its effects including antioxidation,anti aging, antibacterial, antiviral antitumor and other functions.A tea polyphone play fundamental role in metabolism of body, and it was namedas the eighth nutrient element. Among them EGCG was a key component of green tea.Recent researches have shown that EGCG possess anti-tumor properties.ERK1/2phosphorylation induced by EGCG was discovered in our study.An EGCG concentration of50μg/ml induced significant phosphorylation of ERK1/2as early as the2min time-point, and this effect persisted through the30mintime-point.The critical concentration of EGCG was determined to be12.5μg/ml, andincreasing EGCG concentrations produced increasing levels of ERK1/2phosphorylation. However,293T cells exhibited a resistance to EGCG-inducedphosphorylation of ERK1/2, even at high concentrations of EGCG.A similar phosphorylation of MEK was also observed. The results suggest thatsignaling transduction form MEK to ERK.Although cetuximab inhibited EGFR signaling, it did not affect the EGCG-induced activity of ERK1/2. Cetuximab was unable to block the stimulation ofERK1/2by EGCG.The results suggest that50μg/ml EGCG was sufficient to activate ERK; bycontrast, even high concentrations of EGF were insufficient for activation. Nocombination of the two molecules produced an additive effect. These resultssuggested that ERK1/2phosphorylation induced by EGCG independent on EGFRsignaling activation.However, EGCG has some effects on tumor therapy were not accept widely bytea-researcher. The reason of this argument may attribute to the oxidation of EGCG.In our study, the half-life of EGCG was short than30min in the surem absentculture medium. After a30min incubation, an initial EGCG concentration of50μg/ml produced a significant level of H2O2(100μM).In order to protect EGCG from oxidation catalase SOD,acidic medium and cellco-incubation were employed. However, EGCG-induced ERK1/2phosphorylationwas minimal in this novel condition. Phosphorylation was suppressed by the acidity ofthe medium or the presence of catalase and SOD. At pH6.0, EGCG was unable topromote ERK1/2phosphorylation. EGCG-induced phosphorylation was also inhibitedby catalase and SOD, even at pH7.4.These measures employed to prevent EGCG oxidation also inhibited ERK1/2phosphorylation. Based on these observations and the results of previous studies, wepropose that stimulation of ERK1/2phosphorylation may be dependent on amechanism involving the EGCG auto-oxidation. To test this hypothesis, H2O2and EGCG polymers were used. The results revealthat H2O2is the primary species responsible for ERK1/2activation. EGCG oxidationproducts also stimulated a slight phosphorylation of ERK1/2. These results enough toprove that ERK1/2induced by EGCG oxidation independent on EGFR signalingactivation.Our results revealed that the auto-oxidative susceptibility of EGCG hadpreviously masked its true bio-activity. We found that apoptosis of P12cell andERK1/2phosphorylation in Jurkat cell were induced by EGCG oxidation, which wasabolished by addition of catalase and superoxide dismutase.No evidence provided for oxidation of EGCG induced effects on cell occur invivo, whether effects on cell induced by oxidation of EGCG occur in vivo have beenunclear. Because EGCG is consumed quickly, it cannot persist as an anti-tumor drugin this short time of period.The results suggest that the research was misleaded by the mechanism acquiredfrom in vitro in typical culture condition. Thus, auto-oxidation of EGCG remains amajor problem in EGCG research.To address this problem, different culture conditions were tested for the ability toimprove EGCG stability (Table1). Several factors were identified that increased thestability of EGCG. In the improved culture system, the half-life of EGCG wasextended to greater than24h.Under stable condition the research of EGCG start in a new direction. Theinvestigation for target of EGCG focuses on receptor on cell surface. Phosphorylation of insulin receptor was induced by EGCG in stable culture condition. EGCG binds toInsulin receptor was demonstrated by biolayer interferometry（BLI）. Those resultssuggest that EGCG binding to insulin receptor(KD=4.72μM), and that binding wasthe reason for the phosphorylation of insulin receptor. The result in this study willbecome the basestone for EGCG regulate the insulin signaling, explain the benefit ofEGCG on diabetes mellitus treatment in molecular mechanism.Stable condition provide a probability for discover of EGCG for other function.