Molecular bases of thioredoxin and thioredoxin reductase-mediated prooxidant actions of (-)-epigallocatechin-3-gallate.
Thioredoxin (Trx) and thioredoxin reductase (TrxR) function as antioxidant and anti-apoptotic proteins, which are often up-regulated in drug-resistant cancer cells. (-)-epigallocatechin-3-gallate (EGCG) is a naturally occurring antioxidant in green tea, but also exhibits prooxidant and apoptosis-inducing properties. We have previously showed a linkage between EGCG-induced inactivation of TrxR and decreased cell survival, revealing TrxR as a new target of EGCG. However, the molecular events underlying the importance of Trx/TrxR in EGCG-induced cytotoxicity remain unclear. Here, we show that the crosstalk between EGCG and Trx/TrxR occurred in a redox-dependent manner, and EGCG induced inactivation of Trx/TrxR in parallel with increased ROS levels in HeLa cells. Moreover, EGCG displayed great reactivity with Cys/Sec residues that have low pK(a) values. The structure of EGCG suggests that its quinone form would readily react with thiolate and selenolate nucleophiles. Using mass spectrometry, we have demonstrated the formation of EGCG-Trx1 (Cys(32)) and EGCG-TrxR (Cys/Sec) conjugates, confirming that EGCG quinone specifically conjugates with active-site Cys(32) in Trx or C-terminal Cys/Selenocysteine (Sec) couple in TrxR under conditions where Trx/TrxR are reduced. Non-reduced form of Trx/TrxR could escape from EGCG inhibition. These data reveal a potential mechanism for enhancing EGCG-induced cancer cell death by the NADPH-dependent reduction of Trx/TrxR.
Zhang
Huihui
H
College of Life Sciences, Graduate University of Chinese Academy of Sciences, Beijing, China.
Cao
Dong
D
Cui
Wei
W
Ji
Mingjuan
M
Qian
Xiaohong
X
Zhong
Liangwei
L
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002417:Cattle; D003545:Cysteine / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010084:Oxidation-Reduction; D011485:Protein Binding; D051381:Rats; D017382:Reactive Oxygen Species / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism*
D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D013879:Thioredoxins; D002392:Catechin; C045651:epigallocatechin gallate; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine