Thioredoxin Reductase Is Irreversibly Modified by Curcumin
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 IC50 value for the enzyme was 3.6 μm 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 (Cys496/Sec497) 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 IC50 of around 15 μm. Modification of TrxR by curcumin provides a possible mechanistic explanation for its cancer preventive activity, shifting the enzyme from an antioxidant to a prooxidant. 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 IC50 value for the enzyme was 3.6 μm 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 (Cys496/Sec497) 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 IC50 of around 15 μm. Modification of TrxR by curcumin provides a possible mechanistic explanation for its cancer preventive activity, shifting the enzyme from an antioxidant to a prooxidant. Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione; diferuloylmethane), a natural lipid-soluble yellow compound from the plant Curcuma longa, is used as a spice to give a specific flavor and yellow color to curry and is consumed daily by millions of people in the world. It was originally isolated from turmeric, belongs to the group of diarylheptanoids found in various natural products, and has been used for centuries in indigenous medicine for the treatment of a variety of diseases (1Aggarwal B.B. Kumar A. Bharti A.C. Anticancer Res. 2003; 23: 363-398PubMed Google Scholar, 2Joe B. Vijaykumar M. Lokesh B.R. Crit. Rev. Food Sci. Nutr. 2004; 44: 97-111Crossref PubMed Scopus (588) Google Scholar, 3Egan M.E. Pearson M. Weiner S.A. Rajendran V. Rubin D. Glockner-Pagel J. Canny S. Du K. Lukacs G.L. Caplan M.J. Science. 2004; 304: 600-602Crossref PubMed Scopus (520) Google Scholar, 4Naidu K.A. Thippeswamy N.B. Mol. Cell. Biochem. 2002; 229: 19-23Crossref PubMed Scopus (109) Google Scholar, 5Li C.J. Zhang L.J. Dezube B.J. Crumpacker C.S. Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1839-1842Crossref PubMed Scopus (221) Google Scholar, 6Chen H.W. Huang H.C. Br. J. Pharmacol. 1998; 124: 1029-1040Crossref PubMed Scopus (208) Google Scholar). Several studies in recent years have shown that curcumin is a potent inhibitor of tumor initiation in vivo (7Lin J.K. Pan M.H. Lin-Shiau S.Y. Biofactors. 2000; 13: 153-158Crossref PubMed Scopus (165) Google Scholar, 8Sharma R.A. Mclelland H.R. Hill K.A. Ireson C.R. Euden S.A. Manson M.M. Pirmohamed M. Marnett L.J. Gescher A.J. Steward W.P. Clin. Cancer Res. 2001; 7: 1894-1900PubMed Google Scholar, 9Ireson C.R. Jones D.J. Orr S. Coughtrie M.W. Boocock D.J. Williams M.L. Fanner P.B. Steward W.P. Gescher A.J. Cancer Epidemiol. Biomarkers Prev. 2002; 11: 105-111PubMed Google Scholar, 10Huang M.T. Smart R.C. Wong C.Q. Conney A.H. Cancer Res. 1988; 48: 5941-5946PubMed Google Scholar) and possesses antiproliferative activities against tumor cells in vitro (11Elattar T.M. Virji A.S. Anticancer Res. 2000; 20: 1733-1738PubMed Google Scholar, 12Choudhuri T. Pal S. Agwarwal M.L. Das T. Sa G. FEBS Lett. 2002; 512: 334-340Crossref PubMed Scopus (349) Google Scholar, 13Somasudaram S. Edmund N.A. 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Thioredoxin reductase (TrxR) 1The abbreviations used are: TrxR, thioredoxin reductase; ApeI/Ref-1, apurinic/apyrimidinic endonuclease/redox factor-1; ASK-1, apoptosis signaling kinase-1; BIAM, biotin-conjugated iodoacetamide; DNCB, 1-chloro-2,4-dinitrobenzene; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); ROS, reactive oxygen species; Trx, thioredoxin; Sec, selenocysteine. 1The abbreviations used are: TrxR, thioredoxin reductase; ApeI/Ref-1, apurinic/apyrimidinic endonuclease/redox factor-1; ASK-1, apoptosis signaling kinase-1; BIAM, biotin-conjugated iodoacetamide; DNCB, 1-chloro-2,4-dinitrobenzene; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); ROS, reactive oxygen species; Trx, thioredoxin; Sec, selenocysteine. catalyzes NADPH-dependent reduction of the redox-active disulfide in thioredoxin (Trx), which serves a wide range of functions in cellular proliferation and redox control (17Gromer S. Urig S. Becker K. Med. Res. Rev. 2004; 24: 40-89Crossref PubMed Scopus (398) Google Scholar, 18Arnér E.S. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (1976) Google Scholar, 19Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). The thioredoxin system can regulate the apoptosis signaling transfer pathway by the redox state of Trx. For example, reduced Trx can inhibit apoptosis by binding to apoptosis signaling kinase-1 (ASK-1), whereas oxidized Trx cannot (20Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2065) Google Scholar). Thioredoxin is also a key enzyme for DNA synthesis by directly serving as an electron donor to ribonucleotide reductase (19Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). Mammalian TrxRs have a remarkably wide substrate specificity explained by their easily accessible C-terminal active site redox center, which contains an essential selenocysteine residue (21Zhong L. Arnér E.S. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5854-5859Crossref PubMed Scopus (402) Google Scholar, 22Zhong L. Holmgren A. J. Biol. Chem. 2000; 275: 18121-18128Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 23Sandalova T. Zhong L. Lindqvist Y. Holmgren A. Schneider G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9533-9538Crossref PubMed Scopus (295) Google Scholar). TrxR is a ubiquitous enzyme present in all living cells; however, the level of TrxR in tumor cells is often 10-fold or even greater than in normal tissues, and tumor proliferation seems to be crucially dependent on an active thioredoxin system, making it a for anticancer K. S. S. Eur. J. Biochem. 2000; 267: PubMed Scopus Google Scholar). has been on the cancer activity of and also mechanistic on this have been B.B. Kumar A. Bharti A.C. Anticancer Res. 2003; 23: 363-398PubMed Google and 2Joe B. Vijaykumar M. Lokesh B.R. Crit. Rev. Food Sci. Nutr. 2004; 44: 97-111Crossref PubMed Scopus (588) Google and TrxR can as a for anticancer and curcumin has been to be a potent anticancer The of to TrxR can be inhibited by curcumin. In this study we found that TrxR can be inhibited by curcumin to a However, the which Trx activity, a NADPH oxidase activity reactive oxygen that of TrxR by curcumin be a pathway to the of cancer activity of curcumin. and rat TrxR1 was as E.S. H. Holmgren A. A. J. Mol. Biol. PubMed Scopus Google Scholar). The enzyme was as by and a specific activity of of thioredoxin reductase with E.S. Holmgren A. M. L. D. S. in Scholar). and from was from from was a of Science. was from was from was in and Curcumin was in the of than of the in the and on the enzyme of activity of enzyme was at room temperature using a in a TrxR was reduced by incubation with NADPH at room temperature for of curcumin added to the by at room temperature for the The of added to the control The enzyme activities by which provides a of TrxR activity (21Zhong L. Arnér E.S. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5854-5859Crossref PubMed Scopus (402) Google Scholar, 22Zhong L. Holmgren A. J. Biol. Chem. 2000; 275: 18121-18128Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, E.S. H. Holmgren A. A. J. Mol. Biol. PubMed Scopus Google Scholar, E.S. Holmgren A. M. L. D. S. in and disulfide reduction using Trx to the E.S. Holmgren A. M. L. D. S. in Scholar). of TrxR and curcumin at room temperature for 2 h in The of was curcumin was by using a of the added to a of and at for of and of added to the This was at for The of the with mass spectrometry of the TrxR and of curcumin at room temperature for 2 The of added to the control of the was and added to of μm and incubation at for to the and in the enzyme H.W. Biochem. 2000; PubMed Scopus Google Scholar, J. FEBS Lett. 2004; PubMed Scopus Google Scholar). of enzyme with of of the to on a and the to with with and of NADPH and of by TrxR was with μm curcumin or μm at room temperature for h in The enzyme activity was for curcumin and for DNCB, using the activity the NADPH oxidase activity, of enzyme added to of μm of NADPH was at using a of in The of was by of of to the and the of was using a of at to the reduction of Inhibition of TrxR in cells in with 2 and The cells cultured at in an with HeLa cells with and of curcumin for h in the The control group the of cells with and cells with and in in the presence of number The activity of TrxR in the was as E.S. Holmgren A. M. L. D. S. in Scholar). was using the to of from was with μm and μm Trx at room temperature for in a of By the of of in the was Trx, was in the The at was and the value was from the value of the The activity of the enzyme was as the of the control. Curcumin TrxR in a and TrxR was with its reduction and Trx-dependent NADPH activity to disulfide reduction The IC50 value for curcumin was to be 3.6 μm by as shown in The value for reduction can be explained as the enzyme used in the incubation with curcumin is than that in the and this a to the enzyme. this was on the of The NADPH oxidase activity also to the of NADPH The of TrxR was incubation activity of the enzyme the curcumin from the incubation by an the enzyme activity be that TrxR was inhibited by curcumin. By of oxidized TrxR with curcumin and removal of the compound from the the enzyme activity was inhibition of TrxR by curcumin. of μm TrxR and μm NADPH in was at room temperature for μm curcumin was and incubation at room The of was and the control the of the enzyme activity, of the incubation at the and 2 and added to a of 2 and of The of was at the with a The activity was as the of the control. was of the enzyme activity after it was with only at room temperature for 2 can inhibit of μm TrxR and μm NADPH in was at room temperature for of curcumin and incubation at room temperature for 2 h of the was The of was and the control the of Curcumin in the incubation was by an The enzyme was with of and in of the enzyme activity, of the and added to a of 2 and of The of was at the with a The activity was as the of the of the Curcumin and curcumin inhibited TrxR on the oxidized this that curcumin the reduced enzyme by a with the of the active site J. Zhong L. Holmgren A. Arnér E.S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). we the reduced enzyme with a of curcumin and the with By the mass of the we the The C-terminal is which has of incubation with we a with of The mass the is which the of curcumin 2 that of the enzyme can with of curcumin to a The mass of the is in The and for Curcumin the mass the and we proved that curcumin can the C-terminal the redox-active and for curcumin TrxR was with curcumin to BIAM, which can and of and the blotting was than the control This proved that TrxR was by curcumin. the of the be It is that can TrxR by the value H.W. Biochem. 2000; PubMed Scopus Google Scholar). value both group and at value only the group was This blotting that the redox-active and in the enzyme for curcumin NADPH and shown in curcumin-modified enzyme a NADPH oxidase activity and was to that of which was used as a control in J. Zhong L. Holmgren A. Arnér E.S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). By of to the a of the at was observed that to the reduction of by that of of NADPH produce of the of NADPH to be μm well with that of μm Curcumin TrxR in of its curcumin can the of HeLa cells to μm curcumin TrxR activity as shown in we that the IC50 value of curcumin is around 15 μm. the of curcumin TrxR activity in the HeLa was Curcumin, the well known yellow from is a anticancer (1Aggarwal B.B. Kumar A. Bharti A.C. Anticancer Res. 2003; 23: 363-398PubMed Google Scholar, 2Joe B. Vijaykumar M. Lokesh B.R. Crit. Rev. Food Sci. Nutr. 2004; 44: 97-111Crossref PubMed Scopus (588) Google Scholar, 3Egan M.E. Pearson M. Weiner S.A. Rajendran V. Rubin D. Glockner-Pagel J. Canny S. Du K. Lukacs G.L. Caplan M.J. Science. 2004; 304: 600-602Crossref PubMed Scopus (520) Google its in vivo to be Curcumin the and apoptosis in cancer cells T. Pal S. Das T. Sa G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Curcumin proliferation of normal as well as it is apoptosis in However, the the of curcumin on normal and cells have been The of this that curcumin can inhibit TrxR activity by and that the inhibition is The residues of the enzyme are in the active and which a after NADPH reduction (21Zhong L. Arnér E.S. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5854-5859Crossref PubMed Scopus (402) Google Scholar, 22Zhong L. Holmgren A. J. Biol. Chem. 2000; 275: 18121-18128Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 23Sandalova T. Zhong L. Lindqvist Y. Holmgren A. Schneider G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9533-9538Crossref PubMed Scopus (295) Google Scholar). inhibition of TrxR, which directly the redox functions of Trx, be an to the of curcumin. However, even is that the curcumin-modified enzyme a strongly induced NADPH oxidase activity and in the presence of that the of the cancer preventive of curcumin and in to the known cellular for this The thioredoxin system, of TrxR, Trx, and is the disulfide enzyme system in all cells for the redox with a of and (17Gromer S. Urig S. Becker K. Med. Res. Rev. 2004; 24: 40-89Crossref PubMed Scopus (398) Google Scholar, 18Arnér E.S. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (1976) Google Scholar, 19Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). TrxR and Trx enzymes in the cytosol and the mitochondria, and both Trx and TrxR have been to the nucleus in cells with their in regulation of binding of to DNA directly or the redox activity of M. C. S. A. Schneider M. H. U. W. K.R. W. G.W. M. Mol. Cell. Biol. 2004; 24: PubMed Scopus Google Scholar). 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Google Scholar). the of TrxR and the of the enzyme in tumor with to of in T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google it is that this has roles in cells specific of reduced thioredoxin in of apoptosis is binding with This an essential in apoptosis and is by and (20Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2065) Google Scholar) found that reduced Trx, oxidized Trx, directly to the of and inhibited activity as well as the Curcumin TrxR and it to oxidized Trx with of the activity to ASK-1, the signaling thioredoxin is a electron donor to or thioredoxin which are enzymes that the level of reactive oxygen in the control. reduced thioredoxin as an electron and enzymes as specific of of residues J. Arnér E.S. Biol. Med. 2001; PubMed Scopus Google Scholar, A. 2000; PubMed Scopus Google Scholar). 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DNA PubMed Scopus Google Scholar). is that only the reduced of is for its DNA the and to of The of the curcumin-modified TrxR was its strongly induced NADPH oxidase activity the enzyme is a than an the of TrxR in tumor cells as a on the produce a of are in and regulation of as M. Y. Rev. Cancer. 2002; PubMed Scopus Google Scholar). of to the nucleus is with reduced Trx in binding to by TrxR the a in of Trx and TrxR in cells to the oxidative of TrxR Y. Y. M. G. L. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). by and Arnér K. Arnér E.S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) also that TrxR by at the residue can directly to the of this as a for cancer curcumin only inhibit the reduced of In the reduced by the active site residues and are present in the of and at the of the enzyme T. Zhong L. Lindqvist Y. Holmgren A. Schneider G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9533-9538Crossref PubMed Scopus (295) Google making easily by J. Zhong L. Holmgren A. Arnér E.S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In the oxidized enzyme is a (21Zhong L. Arnér E.S. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5854-5859Crossref PubMed Scopus (402) Google Scholar). mass of and the blotting that both the active site and residues the enzyme was with curcumin. The in curcumin it as a is an and its and the is by The value for selenocysteine is Annu. Rev. Biochem. PubMed Scopus Google it is that the selenocysteine in the enzyme is present in the of in The with and to the of the can the in the of curcumin and produce the The the group with curcumin. of curcumin by TrxR is shown in The presence of a of curcumin was found in Biol. 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