Thioredoxin reductase is irreversibly modified by curcumin: a novel molecular mechanism for its anticancer activity.
The thioredoxin reductase (TrxR) isoenzymes, TrxR1 in cytosol or nucleus and TrxR2 in mitochondria, are essential mammalian selenocysteine (Sec)-containing flavoenzymes with a -Gly-Cys-Sec-Gly active site. TrxRs are the only enzymes catalyzing the NADPH-dependent reduction of the active site disulfide in thioredoxins (Trxs), which play essential roles in substrate reductions, defense against oxidative stress, and redox regulation by thiol redox control. TrxRs have been found to be overexpressed by a number of human tumors. Curcumin, which is consumed daily by millions of people, is a polyphenol derived from the plant Curcuma longa. This phytochemical has well known anticancer and antiangiogenic properties. In this study we report that rat TrxR1 activity in Trx-dependent disulfide reduction was inhibited by curcumin. The IC(50) value for the enzyme was 3.6 microM after incubation at room temperature for 2 h in vitro. The inhibition occurred with enzyme only in the presence of NADPH and persisted after removal of curcumin. By using mass spectrometry and blotting analysis, we proved that this irreversible inhibition by curcumin was caused by alkylation of both residues in the catalytically active site (Cys(496)/Sec(497)) of the enzyme. However, the curcumin-modified enzyme showed a strongly induced NADPH oxidase activity to produce reactive oxygen species. Inhibition of TrxR by curcumin added to cultured HeLa cells was also observed with an IC(50) of around 15 microM. Modification of TrxR by curcumin provides a possible mechanistic explanation for its cancer preventive activity, shifting the enzyme from an antioxidant to a prooxidant.
Fang
Jianguo
J
Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, SE-17177 Stockholm, Sweden.
Lu
Jun
J
Holmgren
Arne
A
D000818:Animals; D000838:Anions; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D030024:Curcuma; D003474:Curcumin / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D013058:Mass Spectrometry; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D008956:Models, Chemical; D009249:NADP; D019255:NADPH Oxidases / Q000737:chemistry / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D010936:Plant Extracts / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017279:Selenocysteine / Q000737:chemistry; D013481:Superoxides; D013696:Temperature; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
D000838:Anions; D000970:Antineoplastic Agents; D016877:Oxidants; D010455:Peptides; D010936:Plant Extracts; D017279:Selenocysteine; D013481:Superoxides; D009249:NADP; D019255:NADPH Oxidases; D013880:Thioredoxin-Disulfide Reductase; D003474:Curcumin; D003545:Cysteine