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Originally published In Press as doi:10.1074/jbc.M414645200 on May 6, 2005

J. Biol. Chem., Vol. 280, Issue 26, 25284-25290, July 1, 2005
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Thioredoxin Reductase Is Irreversibly Modified by Curcumin

A NOVEL MOLECULAR MECHANISM FOR ITS ANTICANCER ACTIVITY*

Jianguo Fang, Jun Lu, and Arne Holmgren{ddagger}

From the Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, SE-17177 Stockholm, Sweden

Received for publication, December 29, 2004 , and in revised form, April 25, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (1-6). Several studies in recent years have shown that curcumin is a potent inhibitor of tumor initiation in vivo (7-10) and possesses antiproliferative activities against tumor cells in vitro (11-14). Curcumin is also a potent chemopreventive agent inhibiting tumor promotion against skin, oral, intestinal, and colon carcinogenesis (15, 16).

Thioredoxin reductase (TrxR)1 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 (17-19). 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 (20). Thioredoxin is also a key enzyme for DNA synthesis by directly serving as an electron donor to ribonucleotide reductase (19). 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 (21-23). 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 potential target for anticancer drugs (24).

Much work has been done on the cancer preventative activity of curcumin, and also many mechanistic explanations on this property have been proposed (Refs. 1 and 2 and references cited therein). TrxR can serve as a potential target for anticancer drugs, and curcumin has been reported to be a potent anticancer agent. The combination of these two factors motivated us to investigate whether TrxR can be inhibited by curcumin. In this study we found that TrxR can be irreversibly inhibited by curcumin to form a 1:2 covalent adduct. However, the modified enzyme, which lacked Trx reducing activity, had a greatly increased NADPH oxidase activity producing reactive oxygen species (ROS). We propose that modification of TrxR by curcumin may be a novel pathway to explain the molecular mechanism of cancer preventative activity of curcumin.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Enzyme—Recombinant rat TrxR1 was essentially prepared as described previously (25). The enzyme was pure as judged by Coomassie-stained SDS-PAGE and had a specific activity of 50% of wild thioredoxin reductase with DTNB assay (26). NADPH, curcumin, insulin, 1-chloro-2,4-dinitrobenzene (DNCB), and DTNB were from Sigma. Trypsin was from Promega. Cytochrome c from horse heart was a product of Roche Applied Science. Dimethyl sulfoxide (Me2SO) was from Merck. Biotin-conjugated iodoacetamide (BIAM) was from Molecular Probes. BIAM was dissolved in 0.2 M Tris-Cl, 1 mM EDTA, pH 6.5 and 8.5, respectively. Curcumin was dissolved in fresh Me2SO before addition into the aqueous solvents. Concentrations of Me2SO were no more than 5% of the solvent buffer, were effective in dissolving the drug, and had no influence on the enzyme activity. All other reagents were of analytical grade.

Enzyme Activity Assays—The activity of enzyme was determined at room temperature using a thermostatic Ultrospec UV/visible spectrophotometer (Shimadzu, CPS-260) in a buffer containing 50 mM Tris-Cl, 1 mM EDTA, pH 7.5 (TE buffer). TrxR was first reduced by incubation with excess NADPH at room temperature for 5 min. Then appropriate amounts of curcumin were added to the preincubation buffer followed by incubating at room temperature for the appropriate time. The same amounts of Me2SO were added to the control experiments. The enzyme activities were measured by DTNB reducing assay, which provides a direct measurement of TrxR activity (21, 22, 25, 26), and insulin disulfide reduction assays using Trx according to the available protocols (26).

Identification of Curcumin-modified Peptides—Reduced TrxR (10 µM) and curcumin (250 µM) were incubated at room temperature for 2 h in TE buffer. The final concentration of Me2SO was 5%. After incubation, unreacted curcumin was removed by centrifugation using a Microspin G-25 column (Amersham Biosciences). Twenty microliters of the filtered sample were taken out, added to a tube containing 80 µl of 8 M guanidine hydrochloride, and incubated at 60 °C for 1 h. Then 900 µl of TE buffer were added, and 10 µl of 200 µg/ml trypsin were added to digest the protein. This digestion process was incubated at 37 °C for 1 h. The molecular weight of the trypsin-digested peptides were recorded with matrix-assisted laser desorption ionization mass spectrometry (Applied Biosystems).

Detection of the Curcumin-modified Sites—NADPH-reduced TrxR (0.9 µM) and different concentrations of curcumin were incubated at room temperature for 2 h. The same amounts of Me2SO were added to the control experiments. After incubation, 1 µl of the reaction mixture was taken out and added to new tubes containing 19 µl of 100 µM BIAM (pH 6.5 and 8.5, respectively) following incubation at 37 °C for another 30 min to alkylate the remaining free -SeH and -SH groups in the enzyme (27, 28). Twenty microliters of BIAM-modified enzyme were mixed with 20 µl of loading buffer, 20 µl of the samples were subjected to SDS-PAGE on a 7.5% gel, and the separated proteins were transferred to nitrocellulose membrane. Proteins labeled with BIAM were detected with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection.

Induction of NADPH Oxidase Activity and Production of Superoxide Anion by Curcumin-modified Enzyme—Prereduced TrxR (0.2 µM) was incubated with either 50 µM curcumin or 50 µM DNCB at room temperature for 1 h in TE buffer. The remaining enzyme activity was 12% for curcumin and 2% for DNCB, respectively, using the DTNB activity assay. To determine the NADPH oxidase activity, 200 µl of modified enzyme were added to 400 µl of TE buffer containing 200 µM NADPH. Oxidation of NADPH was followed at 340 nm using a molar extinction coefficient of 6200 M-1 cm-1 in calculations. The production of superoxide anion was determined by addition of 30 µl of 1.5 mM cytochrome c to the reaction mixture, and the amount of superoxide anion production was calculated using a molar extinction of 21,000 M-1 cm-1 at 550 nm due to the reduction of cytochrome c.

Inhibition of TrxR in Vivo—HeLa cells were cultivated in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (PAA Laboratories, Pasching, Austria), 2 mM glutamine (PAA Laboratories), and 100 units/ml penicillin/streptomycin (PAA Laboratories). The cells were cultured at 37 °C in an incubator with 90% humidified atmosphere containing 5% CO2. HeLa cells were incubated with different concentrations (5, 10, 25, and 50 µM) of curcumin for 6 h in the incubator. The control group contained the same amount of Me2SO (1%, v/v). Before harvesting, cells were washed with phosphate-buffered saline and then cells were lysed with cell lysis buffer (0.5% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 150 mM NaCl, and 1 mM EDTA in TE buffer) in the presence of protease inhibitors (Complete, EDTA-free, catalog number 1873580, Roche Applied Science). The activity of TrxR in the cell extracts was determined as described elsewhere (26). Protein concentration was quantified using the Bio-Rad procedure. A volume corresponding to 50 µg of protein from each cell extract was incubated with TE buffer containing 300 µM NADPH, 1.5 mg/ml insulin, and 17 µM Escherichia coli Trx at room temperature for 30 min in a final volume of 40 µl. By the addition of 500 µl of 1 mM DTNB in 6 M guanidine hydrochloride (pH 8.0) the reaction was terminated. A blank sample, containing everything except Trx, was treated in the same manner. The absorbance at 412 nm was measured, and the blank value was subtracted from the corresponding absorbance value of the sample. The activity of the enzyme was expressed as the percentage of the control.



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  		FIG. 1.
Dose-dependent inhibition of TrxR by curcumin. A, DTNB assay. A mixture of 0.5 µM TrxR and 100 µM NADPH in TE buffer was preincubated at room temperature for 5 min. Then different concentrations of curcumin were added, and incubation continued at room temperature for 2 h (the final volume of the mixture was 80 µl). The final concentration of Me2SO was 1% (v/v), and the control had the same amount of Me2SO. To assay the enzyme activity, 60 µl of the incubation mixture were taken out and added to a cuvette containing 415 µl of 2 mM DTNB and 25 µl of 4 mM NADPH. The increase of absorbance at 412 nm (A412) was recorded immediately at the initial 1 min with a blank reference. The activity was expressed as the percentage of the control. B, Trx-mediated insulin reduction assay. A mixture of 0.9 µM TrxR and 100 µM NADPH in TE buffer was preincubated at room temperature for 5 min. Then different concentrations of curcumin were added, and incubation continued at room temperature for 2 h (the final volume of the mixture was 51 µl). The final concentration of Me2SO was 2% (v/v), and the control had the same amount of Me2SO. To assay the enzyme activity, 45 µl of the incubation mixture were taken out and added to a cuvette containing 315 µl of TE, 50 µl of 10 mg/ml insulin, 25 µl of 4 mM NADPH, and 65 µl of 80 µM E. coli Trx. The decrease of absorbance (A340) was recorded immediately at the initial 1 min with a blank reference. The activity was expressed as the percentage of the control based on the rate of NADPH oxidation.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Curcumin Can Irreversibly Inhibit TrxR in a Dose- and Time-dependent Manner—When NADPH-reduced TrxR was incubated with curcumin, its DTNB reduction and Trx-dependent NADPH oxidation activity due to insulin disulfide reduction were lost (Fig. 1). The IC50 value for curcumin was determined to be 3.6 µM by DTNB assay as shown in Fig. 1A. The slightly higher value for Trx-mediated insulin reduction assay (around 10 µM) can be explained as follows. First, the enzyme concentration used in the incubation with curcumin is higher than that in the DTNB assay, and this would cause a higher concentration to modify the enzyme. Second, this assay was based on the oxidation of NADPH. The modified enzyme-induced NADPH oxidase activity (see below) should also contribute partly to the obtained rate of NADPH oxidation. The curcumin-induced inactivation of TrxR was time-dependent. With longer incubation time, less activity of the enzyme remained (Fig. 2). After removing the curcumin from the incubation buffer by filtering through an Ultrafree-MC Millipore 30,000 cutoff filter, the enzyme activity could not be recovered (Fig. 3), demonstrating that TrxR was irreversibly inhibited by curcumin. By preincubation of oxidized TrxR with curcumin and then removal of the compound from the solution, the enzyme activity was not influenced (data not shown).



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  		FIG. 2.
Time-dependent inhibition of TrxR by curcumin. A mixture of 0.9 µM TrxR and 100 µM NADPH in TE buffer was preincubated at room temperature for 5 min. Then 50 µM curcumin was added, and incubation continued at room temperature. The final concentration of Me2SO was 1% (v/v), and the control had the same amount of Me2SO. To assay the enzyme activity, 30 µl of the incubation mixture were taken out at the appropriate time interval (0.5, 1, and 2 h) and added to a cuvette containing 440 µl of 2 mM DTNB and 30 µl of 4 mM NADPH. The increase of A412 was recorded immediately at the initial 1 min with a blank reference. The activity was expressed as the percentage of the control. There was no influence of the enzyme activity after it was incubated with only Me2SO at room temperature for 2 h.

 
Identification of the Covalent Adduct between Curcumin and TrxR—Because curcumin irreversibly inhibited NADPH-prereduced TrxR but had no effect on the oxidized enzyme, this suggested that curcumin might modify the reduced enzyme by forming a covalent adduct with the nascent selenol/thiol of the active site (29). To confirm this, we preincubated the reduced enzyme with a large excess of curcumin (25-fold) and then digested the protein with trypsin. By analyzing the mass of the peptides, we could identify the modified residue. The trypsin-digested C-terminal peptide sequence is -SGGDILQSGCUG, which has molecular weight of 1143.2801 (M + 1). After incubation with curcumin, we got a new fragment with molecular weight of 1879.9850 (M + 1). The mass difference between the two peptides is 736.7049, which just equals twice the molecular weight of curcumin (368.39 x 2 = 736.78), demonstrating that one unit of the enzyme can react with two molecules of curcumin to form a 1:2 adduct. The mass spectrum of the adduct is illustrated in Fig. 4.

The Redox-active Sites Cys496 and Sec497 Were Targets for Curcumin Modification—By comparing the peptide mass difference between the treated and untreated enzymes, we proved that curcumin can modify the C-terminal residue. Are the redox-active sites Cys496 and Sec497 targets for curcumin modification? When TrxR was incubated with curcumin prior to BIAM, which can alkylate free -SH and -SeH groups of Cys496 and Sec497, the blotting band intensity was weaker than the control (Fig. 5). This result proved that TrxR was first alkylated by curcumin. With the higher concentration of curcumin, the weaker band intensity could be observed. It is reported that BIAM can selectively alkylate TrxR by adjusting the pH value (27). At high pH value (pH 8.5), both -SH group and -SeH groups were alkylated (Fig. 5, lane 3), but at low pH value (pH 6.5), only the -SeH group was alkylated (lane 4). This blotting result confirmed that the redox-active sites Cys496 and Sec497 in the enzyme were targets for curcumin covalent modification.



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  		FIG. 3.
Curcumin can irreversibly inhibit TrxR. A mixture of 0.9 µM TrxR and 100 µM NADPH in TE buffer was preincubated at room temperature for 5 min. Then different concentrations of curcumin were added, and incubation continued at room temperature for 2 h (the final volume of the mixture was 51 µl). The final concentration of Me2SO was 2% (v/v), and the control had the same amount of Me2SO. Curcumin in the incubation mixture was removed by filtering through an Ultrafree-MC Millipore 30,000 cutoff filter. The remaining enzyme was washed again with 50 µl of TE buffer and then redissolved in 60 µl of TE buffer. To assay the enzyme activity, 50 µl of the sample were taken out and added to a cuvette containing 420 µl of 2 mM DTNB and 30 µl of 4 mM NADPH. The increase of A412 was recorded immediately at the initial 1 min with a blank reference. The activity was expressed as the percentage of the control.

 



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  		FIG. 4.
Mass spectrum of the adduct between TrxR and curcumin. The obtained mass of the adduct is 1879.9850, which equals the addition of two curcumin molecules.

 



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  		FIG. 5.
The redox-active sites Cys496 and Sec497 were targets for curcumin modification. Different concentrations of curcumin were added to reduced TrxR (0.9 µM) and incubated at room temperature for 2 h. The same amounts of Me2SO were added to the control experiments. Lanes 1-3, alkylation of TrxR by BIAM at pH 8.5; lanes 4-6, alkylation of TrxR by BIAM at pH 6.5. Lane 1, 10 µM curcumin; lane 2, 50 µM curcumin; lanes 3 and 4, control; lane 5, 10 µM curcumin; lane 6, 50 µM curcumin.

 
Curcumin-modified Enzyme Showed Increased NADPH Oxidase Activity and Produced Superoxide Anion—As shown in Fig. 6A, curcumin-modified enzyme displayed a much higher NADPH oxidase activity and was comparable to that of DNCB-modified enzyme, which was used as a positive control in our experiment (29). By addition of cytochrome c to the reaction mixture, a peak of the spectrum at 550 nm was observed (Fig. 6B) that corresponds to the reduction of cytochrome c by superoxide anion. Taking into account that oxidation of one molecule of NADPH would produce two molecules of superoxide anion, the rate of NADPH oxidation, calculated to be 0.15 µM min-1, correlated well with that of superoxide anion production, 0.32 µM min-1.



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  		FIG. 6.
Induction of NADPH oxidase activity and production of superoxide anion. The modified enzymes were incubated with either NADPH or NADPH with cytochrome c at room temperature. A, the decrease of A340 was monitored for 30 min for determination of NADPH oxidase activity. B, after monitoring the NADPH oxidase activity, cytochrome c was added to the above reaction mixture, and the spectrum was scanned from 500 to 700 nm every 5 min at room temperature. The inset in B was the change of A550 after addition of cytochrome c. Curc, curcumin.

 
Curcumin Can Inhibit TrxR Dose Dependently in Vivo—Because of its lipid solubility, curcumin can permeate the cell membrane easily. Exposure of HeLa cells to 5-50 µM curcumin significantly attenuated TrxR activity as shown in Fig. 7. From these results we estimated that the IC50 value of curcumin is around 15 µM. When the concentration of curcumin reached 50 µM, most TrxR activity in the HeLa cell extract was inhibited.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Curcumin, the well known yellow dietary pigment from curry, is a promising anticancer drug (1-3), but its in vivo target molecules remain to be clarified. Curcumin inhibits cell proliferation, interrupts the cell cycle, and induces apoptosis in cancer cells (30). Curcumin inhibits proliferation of normal as well as malignant cells, but it is induces apoptosis mainly in malignant cells. However, the molecular mechanisms underlying the different effects of curcumin on normal and malignant cells have been unclear. The results of this work show that curcumin can irreversibly inhibit TrxR activity by forming covalent adducts and that the inhibition is NADPH-dependent. The modified residues of the enzyme are in the active site, i.e. Cys496 and Sec497, which form a selenol/thiol after NADPH reduction (21-23). Obviously inhibition of TrxR, which will directly effect the many redox functions of Trx, should be an important mechanism to explain the antitumor effects of curcumin. However, even more important is our discovery that the curcumin-modified enzyme had a strongly induced NADPH oxidase activity and produced ROS in the presence of oxygen. We believe that our results explain the mechanism of the cancer preventive property of curcumin and will discuss these in relation to the known cellular effects described for this dietary polyphenol.



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  		FIG. 7.
Inhibition of TrxR by curcumin in HeLa cells. Cells were cultured in the presence of 5, 10, 25, and 50 µM curcumin for 6 h, and the enzyme activity was measured and expressed as the percentage of the control, which contains the same amount of Me2SO (1%).

 
The thioredoxin system, composed of TrxR, Trx, and NADPH, is the major disulfide reducing enzyme system in all cells responsible for maintaining the intracellular redox milieu with a high content of free protein thiols and rare disulfides (17-19). Separate TrxR and Trx enzymes operate in the cytosol and the mitochondria, and both Trx and TrxR have been described to move into the nucleus in cells consistent with their role in regulation of binding of transcription factors to DNA directly or via the redox activity of ApeI/Ref-1 (31). Trx and TrxR, the major protein disulfide reductase of the cell, have a large range of functions in enzymatic reductions and play multiple roles in intracellular signaling and resistance against oxidative stress. In particular, TrxR levels in tumor cell lines are often 10 times higher than those in normal tissues (32, 33) perhaps because tumor cell proliferation is dependent on a constant supply of deoxyribonucleotides and initiation of protein synthesis, both of which require an active thioredoxin system (17, 18, 34, 35). Another role of the thioredoxin system is to protect against apoptosis, which may be important particularly in tumor cells. Extracellular thioredoxin acts as a growth factor, protecting the tumor cell from natural killerlysin and tumor necrosis factor-{alpha} and from respiratory burst of immune cells (36, 37). Considering the tumor-promoting effect of TrxR described above and the much higher levels of the enzyme in tumor cells, with up to 0.5% of total soluble proteins in mammary adenocarcinoma cell lines (32, 33), it is clear that this selenoenzyme has major roles in transformed cells (Scheme 1).

A specific function of reduced thioredoxin in prevention of apoptosis is via binding with ASK-1. This mitogen-activated protein kinase kinase kinase plays an essential role in apoptosis and is activated by many stress- and cytokine-related stimuli. Saitoh et al. (20) found that reduced Trx, but not oxidized Trx, bound directly to the N terminus of ASK-1 and inhibited ASK-1 kinase activity as well as the ASK-1-dependent apoptosis. Curcumin will inactivate TrxR and make it unable to reduce oxidized Trx with loss of the activity to bind ASK-1, causing the signaling cascades ultimately inducing apoptosis. Reduced thioredoxin is a direct electron donor to peroxiredoxins or thioredoxin peroxidases, which are major hydrogen peroxide-scavenging enzymes that normally keep the level of reactive oxygen species in the cell under control. Methionine sulfoxide reductases utilize reduced thioredoxin as an electron donor, and these enzymes serve as specific scavengers of ROS via reversible oxidation of methionine residues (34, 35). The curcumin inhibition of TrxR will lead to an oxidized intracellular environment, and this oxidative stress should stop cell proliferation in normal cells, which do not overproduce cyclins.



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  		SCHEME 1.
Cascade effects after modification of TrxR by curcumin. AP-1, activator protein 1.

 
An outstanding electron acceptor for reduced thioredoxin is ribonucleotide reductase, the S phase enzyme essential for synthesis of deoxyribonucleotides for DNA synthesis. Dys-regulation of ribonucleotide reductase by lack of electrons from Trx/TrxR may lead to catastrophic events in S phase cells of tumor origin, which have no chance to complete their DNA replication and will go into apoptosis. In the nucleus, the role of ribonucleotide reductase subunit R2 induced by p53 (38) is essential for DNA repair. It is most likely that thioredoxin and thioredoxin reductase act as electron donors to this form of ribonucleotide reductase, which operates in the nucleus. Inhibition of thioredoxin reductase will stop this activity. Curcumin-induced conversion into an NADPH oxidase with a high ROS production, by a mechanism similar to that of the well known immunomodulatory agent DNCB (29), should be catastrophic via direct damage to DNA by TrxR located in the nucleus. Targets of Trx-dependent transcription factor up-regulation include ApeI/Ref-1, which is the most important DNA repair enzyme and has dual functions (39). It is both the apurinic/apyrimidinic endonuclease responsible for base excision repair and a transcription factor regulator together with Trx (40). There is evidence that only the reduced form of ApeI/Ref-1 is required for its DNA repair activity. Nonrepaired abasic sites will stall the replication forks and lead to loss of coding information.

The most dramatic outcome of the curcumin-modified TrxR was its strongly induced NADPH oxidase activity producing ROS (Scheme 1). Thus, the enzyme is converted into a prooxidant rather than an antioxidant. Now the higher levels of TrxR in tumor cells will not act as a protection but on the contrary will produce a lot more ROS. Low concentrations of ROS are involved in proliferation, differentiation, and regulation of transcription factors such as NF-{kappa}B (41). ROS activate transcription of NF-{kappa}B to the nucleus where ApeI/Ref-1 is involved together with reduced Trx in binding to DNA. Excessive ROS production by modified TrxR will obviously destroy the NF-{kappa}B survival mechanism. Perhaps a defect in up-regulation of Trx and TrxR in malignant cells makes them more susceptive to the oxidative stress following inactivation of TrxR (42). Recent reports by Anestal and Arnér (43) also show that TrxR modified by alkylating agents at the Sec residue can directly induce apoptosis, pointing to the importance of this selenoenzyme as a target for cancer therapy.



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  		SCHEME 2.
Proposed mechanism of inhibition of TrxR by curcumin.

 
Why does curcumin only inhibit the reduced form of TrxR? In the reduced form generated by NADPH, the active site residues Cys496 and Sec497 are present in the form of free -SH/-SeH groups and exposed at the surface of the enzyme (23), making them easily attacked by alkylating agents (29). In the oxidized enzyme there is a nonreactive Cys496-Sec497 selenenylsulfide bridge (21). Both mass analysis of peptides and the blotting results suggested that both the active site Cys496 and Sec497 residues were modified when the enzyme was incubated with curcumin. The {alpha},{beta}-unsaturated ketone structure in curcumin makes it act as a potential alkylator. There is an equilibrium between {alpha},{beta}-unsaturated ketone and its enol form, and the latter is more sensitively attacked by nucleophilic agents. The pKa value for selenocysteine is 5.1 (44), so it is clear that the selenocysteine in the enzyme is present in the predominant form of selenide (-Se-) in our experimental conditions (pH 7.5). The selenide, with high nucleophilicity and exposed to the surface of the enzyme, can attack the carbon cation in the enol form of curcumin effectively and produce the covalent adduct. The same mechanism will make the -SH group react with curcumin. A proposed mechanism of curcumin inactivation by TrxR is shown in Scheme 2. The presence of a large excess of curcumin (250 µM) may explain why there was no monoadduct found in our experiment. Liu et al. (45) reported recently that N-ethylmaleimide, acrolein, and some other {alpha},{beta}-unsaturated aldehydes can inactivate the reduced form of protein-disulfide isomerase but have no effect on its oxidized form. This can also be explained by the mechanism proposed here because protein-disulfide isomerase has two thioredoxin domain -CGHC- active sites with a relatively low pKa value.

One major physiological function of GSH is to detoxify many alkylating reagents by competing with other potential targets to form covalent adducts in vivo. Can GSH protect TrxR from alkylation by curcumin? Awasthi et al. (46) studied the interaction between curcumin and GSH and found that curcumin had very low reactivity toward GSH. In a co-incubation of GSH (1 mM) and reduced TrxR (0.9 µM) with curcumin in our experiments, GSH had little protective effect on the inactivation of TrxR (data not shown). That curcumin inhibited TrxR in vivo was also proved by our experiments with HeLa cells.

Several effects of curcumin on regulation of signaling pathways can be directly explained by inhibition of TrxR and excessive generation of ROS. Curcumin can impair p53 function in colon cancer cells and B cells as reported by Moos et al. (47) and Han et al. (48), respectively. Also the activity of activator protein 1 (AP-1) was suppressed by curcumin in cultured human promyelocytic leukemia cells (49). Four of the nine cysteines present in p53 DNA-binding domain are essential for the activity of this factor (50). This site-specific DNA binding of p53 and transcriptional activity are controlled by thiol redox state, which is regulated by the thioredoxin system. The DNA binding activity of activator protein 1 is facilitated by a nuclear redox protein, ApeI/Ref-1 (51). As discussed above, Trx associates directly with ApeI/Ref-1 in the nucleus and modulates Ref-1 activity. This protein-protein interaction requires the active site cysteine residues in the reduced Trx active site (52). The thioredoxin system, besides being the main electron donor to peroxiredoxins and directly acting as a ROS scavenger (53), can regenerate vitamin C from ascorbate free radical (54) and dehydroascorbate (55), lipoic acid (56), and ubiquinone (57) to maintain the low molecular weight antioxidant levels in cells. Inhibition of TrxR will impair the antioxidant defense against oxidative stress, ROS will accumulate from mitochondrial sources, and most importantly TrxR modified by curcumin will generate ROS. Is there any evidence that this happens in cells? Recent reports by Kang et al. (58) showed that high concentrations (higher than 25 µM) of curcumin promoted ROS generation, whereas low concentrations (less than 10 µM) usually diminished ROS in Hep3B cells. The effect was not inhibitable by cycloheximide, consistent with modification of preexisting TrxR. Some other reports that curcumin can induce cell apoptosis through production of ROS (59, 60) can also be explained by our results.

How does TrxR compare with other targets? It was described previously that curcumin can interact with several enzymes, but this requires relatively high concentrations of curcumin to show effects (58, 61-65). We also determined the effects of curcumin on Trx, glutathione reductase, and glutaredoxin, all of which contain free thiol groups in their catalytic sites, in vitro. The result was very weak inhibition observed in the presence of 50 µM curcumin. In contrast, curcumin inhibited TrxR both in vitro and in vivo with IC50 values of 3.6 and 15 µM, respectively. The common dosage for curcumin in cell experiments is between 10 and 100 µM; this will inhibit and modify most activity of TrxR. The requirement of a low concentration and its irreversibility modifying TrxR makes this enzyme a most likely target of curcumin. Curcumin is used world wide as a coloring and flavoring additive in many foods, and the consumption in a normal diet is at the rate of up to 100 mg/day by people in certain countries for centuries (64). Human studies indicate that curcumin is tolerated in large oral doses, as high as to 8,000 mg/day, without apparent toxicity (66). The in vivo IC50 for TrxR by curcumin is in the low range of values, 10-100 µM, which is required for the preventive effect and can be achieved by sufficient dietary doses (67).

In conclusion, herein we have characterized an irreversible inhibition mechanism of TrxR by curcumin. The inhibition was caused by covalent modification of active site Cys496 and Sec497 residues to destroy the Trx reduction activity. In addition and more importantly, the curcumin-modified enzyme was converted into an NADPH oxidase with production of ROS to which cancer cells appear more sensitive (68, 69). This shift in activity means that TrxR as part of the antioxidant thioredoxin system is converted into a prooxidant. TrxR is present in cytosol and mitochondria, and both TrxRs have the same active site and are targets for curcumin. Because curcumin has obvious potential in the prevention and therapy of cancer, this modification of TrxR, i.e. inhibition of Trx reduction but induction of NADPH oxidase activity producing ROS, provides a likely mechanism for how curcumin works.


   FOOTNOTES
 
* This work was supported by Swedish Cancer Society Grant 961, Swedish Research Council Medicine Grant 13x 3529, The K. A. Wallenberg Foundation, and the Karolinska Institutet. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 46-8-52487686; Fax: 46-8-7284716; E-mail: Arne.Holmgren{at}mbb.ki.se.

1 The 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. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Elias Arnér and Olle Rengby for kindly providing recombinant TrxR.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Z.-H. Chen, Y. Saito, Y. Yoshida, A. Sekine, N. Noguchi, and E. Niki
4-Hydroxynonenal Induces Adaptive Response and Enhances PC12 Cell Tolerance Primarily through Induction of Thioredoxin Reductase 1 via Activation of Nrf2
J. Biol. Chem., December 23, 2005; 280(51): 41921 - 41927.
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