Cancer Proliferation Inhibiton And Cytotoxicity Effects Of EGCG Auto-oxidation Products(EAOPs) And The Mechanisms Involved

EGCG,a kind of catechins mainly from tea leaves,is the most abundant bioactive content in tea.EGCG was reported to exhibit both cancer proliferation inhibiting activity and cytotoxicity.The present study showed that EGCG was certainly effective to inhibit human squamous carcinoma cell line Tca8113 and murine colon carcinoma cell line CT26 proliferation.In this study,we found that EGCG(30 ?g/mL)treatment inhibited Tca8113 90% proliferation,and EGCG(20 ?g/mL)treatment killed more than 70% CT26 cancer cell.Moreover,EGCG(50?g/mL)treatment killed 80% Tca8113 cancer cells and EGCG(100 ?g/mL)treatment killed more than 60% CT26 cancer cells.The inhibition and cytotoxicity effects of EGCG on carcinoma were partly attributed to its function on cell redox state.Firstly,EGCG undering autoxidation produce a large number of reactive oxygen species(ROS),which plays a vital role in oxidative stress state.EGCG-mediated ROS production at least included superoxide anion and hydrogen peroxide,which action was abolished by the presence of superoxide dismutase(SOD)or catalase(CAT).In addition,the pivotal antioxidant enzyme thioredoxin reductase(TrxR)and the essential antioxidant glutathione(GSH)as well as cysteine(Cys)were all targets for EGCG.Polyphenol oxidase and peroxidase existing in tea leaves play important roles in EGCG oxidation during black tea fermentation process with the formation of polymers,such as theaflavins and thearubigin.EGCG oxidation in vitro can be realized by enzyme catalysis or other conditions.In present study,we obtained a series of 5 mg/mL EGCG auto-oxidation products(EAOPs)resolved in 200 mM phosphate buffer solution(pH8.0)in a 37 water bath.The auto?-oxidation time was setted at 2h,4h,8h,16 h and 32 h,respectively.During the autoxidation,color of the solution gradually changed to yellow,next to red and then to dark red ultimately.The visible-ultraviolet spectrum scan showed that the solution absorption at 510 nm emerged and increased gradually,and regular change was appeared around EGCG maximum absorption wavelength 280 nm.HPLC analysis indicated that the speed of EGCG oxidation under mentioned condition seems quite rapid,which can be confirmed that only 20% EGCG retention after 2h oxidation.The most newly appeard peaks at 278 nm from EAOP-2h were near the EGCG peak,which were far from the theaflavins.With respect to the light brown color of EAOP-2h,it's reasonable to speculate that the oxidation degree of EAOP-2h has outstripped theaflavins.After oxidation of 4h,EGCG can not be dectected in the solution.The peak of EGCG and the peaks around EGCG peak were all disappeared gradually,at the same time,the peak area of gallic acid(GA)increased gradually which is high related to the EGCG degradation.The present study found that EAOP-2h~16h remain almost identical bioactivity on Tca8113 and CT26 proliferation inhibition and cytotoxicity compared with EGCG control,while these effects of EAOP-32 h was invalid.The anti-cancer mechanisms of EGCG were high related with its function of ROS and H2O2 production,TrxR activity inhibition and GSH/Cys depletion.Compared with EGCG,hydrogen peroxide production of the EAOPs gradually decreased,in contrast,glutathione and cysteine depletion capacity of the EAOPs substantially increased.On the other hand,EGCG and the EAOPs had same capacity in inhibiting activity of purified thioredoxin reductase 1 that is highly expressed in cancer cells and a putative target for cancer prevention and treatment.