Inhibitory effect of green tea extract and (-)-epigallocatechin-3-gallate on mammalian thioredoxin reductase and HeLa cell viability.
Mammalian cytosolic thioredoxin reductase (TrxR1) is an attractive target for developing cancer chemopreventive agents since its inhibition is associated with a reduced growth of cancer cells. However, the known inhibitors of this enzyme mostly have a toxic effect on human health. We report on a non-toxic inhibitor, green tea. TrxR1 was found to be inhibited by green tea extracts (Gte) with an IC50 value of 256 microg/ml. Catechins, the major components of Gte, showed various inhibitory effects, in which (-)-epigallocatechin-3-gallate (EGCG) exhibited a stronger inhibition than any other catechins tested. The inhibition of TrxR1 by EGCG was close to competitive (Ki = 64 microM) with substrate DTNB and was non-competitive (Ki = 92 microM) with co-enzyme NADPH. The preincubation of TrxR1 with EGCG led to irreversible enzyme inactivation in a time-dependent manner, which was highly effective in the presence of NADPH. The inactivation included an equilibrium step used to form a reversible TrxR1-EGCG complex (EI) (dissociation constant Ki* = 43 microM), and an isomerization step used to form an irreversible complex (E*I) (rate constant k3 = 4.8 x 10(-3) s(-1)). We have identified thiol/selenol groups in the active site as reactive sites that mediated TrxR1 inhibition by EGCG. When cultured HeLa cells were treated with Gte or EGCG for 22-24 h, TrxR1 activity in cell extracts was significantly inhibited, accompanied by a reduction of cell viability in a concentration-dependent manner (IC50 = 40 microg/ml for Gte and 107 microM for EGCG). The inactivation of TrxR1 by Gte/EGCG is most likely linked to a reduction of HeLa cell viability.
Wang
Yan
Y
College of Life Sciences, Graduate University of Chinese Academy of Sciences, 100049 Beijing, P.R. China.
Zhang
Huihui
H
Holmgren
Arne
A
Tian
Weixi
W
Zhong
Liangwei
L
D000818:Animals; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002417:Cattle; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008956:Models, Chemical; D009249:NADP / Q000737:chemistry; D013662:Tea / Q000378:metabolism*; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism
D013662:Tea; D013879:Thioredoxins; D009249:NADP; D002392:Catechin; C045651:epigallocatechin gallate; D013880:Thioredoxin-Disulfide Reductase