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The FASEB Journal Research Communication

Thioredoxin reductase inhibition by antitumor quinols:a quinol pharmacophore effect correlating toantiproliferative activity

Eng-Hui Chew,* Jun Lu,* Tracey D. Bradshaw, and Arne Holmgren*,1

*Department of Medical Biochemistry and Biophysics, Medical Nobel Institute for Biochemistry,Karolinska Institutet, Stockholm, Sweden; and School of Pharmacy, Centre for BiomolecularSciences, University of Nottingham, Nottingham, UK

ABSTRACT Novel heteroaromatic-substituted 4-hy-droxycyclohexa-2,5-dienones (quinols) demonstrate po-tentin vitroantiproliferative activity andin vivoantitu-mor activity in tumor xenografts. The mechanism ofaction of these promising novel anticancer agents,however, remains to be fully elucidated. The thiore-

doxin (Trx) system comprising Trx, thioredoxin reduc-tase (TrxR), and NADPH participates in a broad rangeof cellular functions involved in cell survival and pro-liferation. Accumulating evidence has indicated that theselenocysteine-containing mammalian TrxR is a validmolecular target for development of novel cancertherapeutics. In this study, we demonstrate that struc-tural analogs containing a quinol pharmacophore inhib-ited TrxR with potencies correlated with their antipro-liferative and cytotoxic efficacies. Benzenesulfonyl-6F-indole-substituted quinol (compound 6) irreversiblyinhibited TrxR most strongly with a half-maximal inhib-itory concentration of 2.7 M after 1 h of incubation

with recombinant rat TrxR. The inhibition was shownto be concentration-, time-, and NADPH-dependentand mediated through a direct quinol attack on thepenultimate C-terminal selenocysteine residue. More-over, TrxR activity in lysates of HCT 116 cells treatedwith apoptosis-inducing doses of quinols was signifi-cantly reduced. From the results obtained, we proposethat TrxR inhibition is a critical cellular event thatcontributes to the proapoptotic effects of quinols.Chew, E. H., Lu, J., Bradshaw, T. D., Holmgren, A.Thioredoxin reductase inhibition by antitumor quinols:a quinol pharmacophore effect correlating to antipro-liferative activity.FASEB J. 22, 20722083 (2008)

Key Words: Michael acceptors anticancer agents antitumormechanism of action selenocysteine

Cancer remains one of the leading causes of deathworldwide. In addition, drug resistance is causing manycancers to become resistant to conventional chemo-therapeutic regimens. Persistent efforts to uncovernovel molecular targets and new anticancer agents aretherefore of utmost importance.

The thioredoxin system, comprising thioredoxin

(Trx), thioredoxin reductase (TrxR), and NADPH, is amajor thiol redox system that plays key roles in numer-ous cellular signaling pathways involved in cell survivaland proliferation (1, 2). TrxR is the only knownenzyme to catalyze the NADPH-dependent reduction ofthe active site disulfide (Cys32 and Cys35) of oxidized

Trx; reduced Trx performs biological functions such asthe following:1) protection of cellular proteins againstoxidative insult by reducing protein disulfides; 2) anantioxidant acting as an electron donor to peroxide-scavengers peroxiredoxins; 3) redox control of tran-scription factors such as nuclear factor-B (NF-B),activator protein-1 (AP-1), and hypoxia-inducible fac-tor-1 (HIF-1); and 4) as an electron donor for theenzyme ribonucleotide reductase (RNR) involved inDNA synthesis (14). Essentially, these genes are notfunctionally redundant, as demonstrated in studies(58) where deletion of cytosolic and mitochondrialisoforms ofTrxandTrxRleads to embryonic lethality in

homozygous mice.On the other hand, in cancer, the properties andbiological effects of the Trx system contribute to tumorgrowth and progression (9). Accumulating evidenceindicates that the Trx system serves as a valid therapeu-tic target. Overexpression of the Trx system is reportedin several human cancers (1012); it is reported thatincreased Trx expression is associated with resistance todocetaxel in primary breast cancer (13) and decreasedsurvival in colorectal cancer patients (14). In recentyears, the essential role of TrxR in tumorigenesis hasbeen further assessed: mouse lung carcinoma (LLC1)cells stably transfected with a TrxR small interfering

RNA construct achieve a reversal of their tumor phe-notype to those of normal cells, and when injected intoa mouse model, tumor growth and metastasis werelargely reduced (15). In the field of anticancer re-search, novel strategies based on targeting Trx andTrxR are thus pertinent with regards to the goal of

1 Correspondence: Medical Nobel Institute for Biochemis-try, Department of Medical Biochemistry and Biophysics,Karolinska Institute, SE-17177 Stockholm, Sweden. E-mail:[email protected]

doi: 10.1096/fj.07-101477

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achieving improved clinical outcome among patientswith intractable malignancies.

As part of the drug discovery program, generation ofnovel and structurally diverse chemical oxidation prod-ucts of bioactive phenols with potential biological prop-erties has been actively pursued. This has led to thesyntheses of heteroaromatic-substituted 4-hydroxycyclo-hexa-2,5-dienones (quinols) possessing selective andpotent antiproliferative activity against colon, renal,and certain breast carcinoma cell lines [median growthinhibition (GI50)0.5 M; refs. 1619]. Previous workhas provided in vitro evidence of quinol binding tocysteine residues in cytosolic thioredoxin-1 (Trx1) pro-tein and of quinol-mediated dose-dependent inhibitionof TrxR/Trx signal transduction [half-maximal inhibi-tory concentration (IC50)6 M], suggesting that Trxis a molecular target of quinols (20). Owing to a highlyaccessible C-terminal -Gly-Cys-Sec-Gly- active site andthe low pKa of the free selenol group (-SeH) in theselenocysteine residue (Sec) that ensures full ionizationto selenolate (Se) at physiological pH, mammalianTrxR is known to be targeted by electrophilic com-

pounds for inhibition (4, 21). Given that quinols areMichael acceptors with electrophilic propensity, wewanted to find out whether they could target TrxR. Weaddressed this hypothesis by testing a series of quinolanalogs to analyze possible inhibition of mammalianTrxR and found that analogs containing a quinolpharmacophore inhibited mammalian TrxR with or-ders of potencies correlating to their cell growth inhib-itory and cytotoxicity potencies. We found that activequinols exhibited a concentration-, time-, and NADPH-dependent selective inhibition of mammalian TrxRand that the mechanism would involve direct irrevers-ible modification of the C-terminal Sec residue. Consis-

tent with the previous finding that glutathione (GSH)protects cells from the cytotoxic effects of quinols (22),we show here that GSH attenuated quinol-mediatedTrxR inhibition to a similar magnitude. Based on theevidence obtained from cell-free and cell-based assaysundertaken in this study, we propose that TrxR is amechanistic target of antitumor quinols.

MATERIALS AND METHODS

Materials and cell culture

Recombinant rat TrxR was prepared as described previously(23). The enzyme exhibited 50% specific activity of wild-typeTrxR as determined by 5,5-dithiobis-(2-nitrobenzoic acid;DTNB) assay. Preparation of recombinantEscherichia coliTrxand TrxR and double mutant human C62S/C73S Trx was asdescribed previously (24, 25). Yeast glutathione reductase(GR), insulin, and glutathione disulfide (GSSG) were pur-chased from Sigma (St. Louis, MO, USA). Biotin-conjugatediodoacetamide (BIAM) was from Molecular Probes (Eugene,OR, USA). Quinol compounds 1 to 10 were kindly obtainedfrom Malcolm Stevens (University of Nottingham, Notting-ham, UK), of which the chemical syntheses of compounds 1to 9 have been described previously (1619). Quinol stocks

(10 mM) were prepared in dimethyl sulfoxide (DMSO),stored, and protected from light at 4C.

Colon carcinoma HCT 116 cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum,100 U/ml penicillin, and 100 g/ml streptomycin and incu-bated at 37C in an humidified atmosphere of 95% air-5%CO2.

TrxR activity by DTNB reduction assay

TrxR activity was determined using previously describedmethods (25, 26). Quinols of different concentrations wereincubated with 110 nM recombinant rat TrxR and 200 MNADPH in a volume of 100 l of 50 mM Tris-HCl and 1 mMEDTA, pH 7.5 (TE buffer), for different time points in 96-wellplates at room temperature. Then, 100 l of TE buffercontaining DTNB and NADPH was added (final concentra-tion: 5 mM and 200 M, respectively), and the linear increasein absorbance at 412 nm during the initial 2 min wasmeasured with a VERSA microplate reader (Molecular De-

vices, Sunnyvale, CA, USA). TrxR activity was calculated as apercentage of enzyme activity of that of DMSO vehicle treatedsample.

TrxR activity by spectrophotometric insulin reduction assay

Assays were carried out in 96-well plates using publishedmethods with modifications (25, 26). In a volume of 100 l of50 mM Tris-HCl, 1 mM EDTA, pH 7.5 (TE buffer), and 200M NADPH, 50 nM recombinant rat TrxR and 2 M humanTrx1 were incubated with quinols of concentrations 150 Mfor 30 min. An aliquot of 100 l of TE buffer containinginsulin and NADPH was then added (final concentration forboth: 200 M), and the absorbance at 340 nm was followed.In a separate experiment, human Trx1 (2 M) was excludedfrom the 30 min incubation and then added together withinsulin before the reaction was followed at 340 nm. TrxRactivity was calculated as the linear change in absorbance at

340 nm per min of the initial 10 min. Experiments wereperformed with similar procedures to assess inhibition of theE. coli Trx system, where quinols of various concentrationswere incubated with 25 nM E. coliTrxR, 2 ME. coliTrx1, and200 M NADPH.

Preparation of reduced TrxR

For experiments to isolate NADPH- or dithiothreitol (DTT)-reduced TrxR free of excess reductant, 110 nM TrxR waspreincubated in TE buffer containing 200 M NADPH or 1mM DTT and then desalted by passing through PD-10desalting columns (Amersham Biosciences, Uppsala, Swe-den) equilibrated with TE buffer. The eluted enzyme frac-

tions were used immediately for quinol inhibition assays, andTrxR enzyme activity was determined using the DTNB andspectrophotometric insulin reduction assays, respectively.

Cell viability assay

HCT116 cells were seeded into 96-well plates at a density of3 103 per well and allowed to attach for 24 h before drugs at

various concentrations prepared as serial dilutions in mediumfrom DMSO stocks were introduced. After 72 h of drugtreatment, cell viability was determined by reduction of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideas described previously (20).

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Preparation of cell lysate

HCT116 cells (3106) were plated onto 100 mm plates andincubated for 24 h to allow exponential growth beforetreatment with quinols for 16 h. Whole cell lysates of DMSOcontrol or quinol-treated cells were prepared by lysing cellpellets (comprised of attached and floating cells) in reducingagent-free lysis buffer comprised of 25 mM Tris-HCl (pH 7.5),100 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1mM Na3VO4, 20 mM sodium -glycerophosphate, 10 mM

sodium pyrophosphate, and 0.5% Triton X-100 containingfreshly added protease inhibitor cocktail (Roche Diagnostics,Mannheim, Germany). Lysates were precleared by centrifu-gation before use, and protein concentrations were deter-mined using a modified Bradford assay (Bio-Rad Laborato-ries, Hercules, CA, USA) as described in the manufacturersmanual.

Immunoblotting

For Western blot analysis, equal amounts of protein (50 g) ineach lysate sample were separated by SDS-PAGE. The followingantibodies were used: goat anti-human TrxR (Santa Cruz Bio-technology, Santa Cruz, CA, USA), goat anti-human Trx (IMCOCorporation, Stockholm, Sweden; www.imcocorp.se), and goatanti--actin (Santa Cruz). All Western blots shown representthree independent experiments.

Determination of TrxR and Trx activity in cell lysates

TrxR and Trx activity in cell lysates was measured in 96-wellplates using an end point insulin assay as described previously(27). For TrxR activity measurement, 25 g of cell lysate wasincubated in a final reaction volume of 50 l containing 85mM HEPES (pH 7.6), 0.3 mM insulin, 660 M NADPH, 2.5mM EDTA, and 5 M human C62S/C73S Trx (24) for 20 minat room temperature. Controls were set up alongside with

incubation of cell lysates in reaction solutions without thepresence of human Trx. The reaction was quenched byadding 250 l of 1 mM DTNB and 240 M NADPH in 6 Mguanidine hydrochloride and 200 mM Tris-HCl, pH 8.0solution, where the amount of free thiols generated frominsulin reduction was determined by DTNB reduction mea-sured at 412 nm (extinction coefficient for 2-nitro-5-thioben-zoic acid 13.6 mM1cm1). TrxR activity was represented asthe absorbance at 412 nm subtracted from that of control. ForTrx activity measurement, procedures were the same as forthe TrxR activity assay, where in control wells, cell lysates (25g) were incubated in reaction solutions without 600 nMrecombinant rat TrxR. Trx activity for each lysate sample wasobtained as the absorbance at 412 nm subtracted from that ofthe corresponding control.

GR assay

Quinols of concentrations 150M were incubated with 10nM yeast GR and 200 M NADPH in a volume of 100 l TEbuffer for 30 min in 96-well plates at room temperature.

When substrate GSSG and fresh NADPH (final concentra-tion: 1 mM and 200 M, respectively) were added, GR activity

was determined by measuring the decrease in absorbance at340 nM during the initial 3 min and expressed as a percent-age of enzyme activity of that of the DMSO vehicle treatedsample.

BIAM-labeling assay

The biotin labeling of free Sec in mammalian TrxR was usedto determine whether the Sec residue is susceptible to quinoltreatment. Briefly, a 100 M BIAM stock solution in 100 mMTris-HCl and 1 mM EDTA, pH 6.5, was freshly prepared foreach experiment. In a tube, NADPH-reduced recombinantrat TrxR (0.9 M) was incubated with compound 1 or 6 (100M) or DMSO vehicle in 20 mM Tris-HCl, 1 mM EDTA, pH7.5, and 200 M NADPH at room temperature. At different

time points, a 3 l aliquot was withdrawn and mixed with 20M BIAM in 100 mM Tris-HCl and 1 mM EDTA, pH 6.5 (finalvolume30 l), for 15 min at 37C. Alkylation of free selenolwas quenched by adding freshly made iodoacetamide solu-tion (final concentration50 mM). The protein samples werethen denatured in SDS sample buffer and subjected toSDS-PAGE and Western blotting. The biotin was detected byhorseradish perioxidase (HRP) -conjugated streptavidin us-ing the enhanced chemiluminescence system (Perkin-ElmerLife Sciences). Membranes were stripped with strippingbuffer (0.15 M glycine, pH 2.5, 0.4% SDS) and reprobed withpolyclonal antirat TrxR1 antibody purified from rabbit anti-serum against rat liver TrxR1 (28); protein levels were deter-mined using the same detection system. At each time point,40 l of TrxR protein mixture was also removed for TrxR

activity determination using the DTNB assay.

RESULTS

Quinols with an active pharmacophore inhibit TrxRin a dose- and time-dependent manner

A novel series of quinols with heteroaromatic or (aryl-sulfonyl)indole substitutions at the 4 position has beenreported to exhibit potent in vitroantitumor activity

(1619). To study the structure-activity relationship ofthese quinol compounds on mammalian TrxR inhibi-tion, eight compounds (1 to 8) containing a quinolpharmacophore with various heterocyclic substituentsand two structural derivatives of compound 1 lackinga quinol pharmacophore (9 and 10 contain a phenoland 4-hydroxy-cyclohexane moiety, respectively) wereselected for evaluation (Fig. 1; Table 1). The com-pounds were incubated at different concentrationswith NADPH-reduced recombinant rat TrxR (110 nM)for various time points at room temperature, followedby TrxR activity determination using DTNB reductionassay. Quinol compounds 1 to 8 caused a dose-depen-

dent (Fig. 2A) and time-dependent (Fig. 2B) decreasein TrxR activity, while compounds 9 and 10 showed noenzyme inhibition even at a high dose of 150 M. Asillustrated in Table 1, compounds 5 and 6 with abenzenesulfonyl indole substitution that possess themost potentin vitroantiproliferative activity also exhib-ited the strongest TrxR inhibitory activity with a respec-tive IC50value of 2.9 and 2.7 M at 1 h of incubation.Their respective more polar propionic acid derivatives,compounds 7 and 8, display an overall decrease in invitro activity against TrxR (IC508.4 and 9.8 M, re-spectively, at 1 h incubation) and cell growth. Benzo-

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thiazole-substituted quinol compounds 1 and 2 showed10-fold enhancement in TrxR inhibitory activity(IC506.5 and 6.3 M, respectively, at 1 h incubation)compared to thiophen- and benzoxazole-substitutedquinols (compounds 3 and 4; IC50104.3 and 76.1 M,respectively, at 1 h of incubation; Table 1). Among thecompounds tested, it was observed that the trend of

TrxR activity inhibition, a property attributed to thepresence of a quinol pharmacophore, was positivelycorrelated to antitumor potency.

TrxR inhibition by quinols in cells

The effect of four of the more potent TrxR inhibitoryquinols (compounds 1, 2, 5, and 6) on the Trx systemin intact cells was assessed. As previously reported,quinols induce apoptosis within 12 to 24 h in quinol-sensitive cell lines, and the effect is most profound incolon carcinoma HCT116 cells (22). Here, we exposedHCT116 cells to a similar range of drug concentrationsfor 16 h, after which collected cell lysates were assayedfor TrxR and Trx activity and protein expression. Asillustrated in Fig. 3A, quinols at apoptosis-inducingdoses of 3 and 5 M brought about a marked decreasein TrxR activity to 60% that of DMSO control cells,whereas at sublethal concentrations (0.5 and 1 M), anelevation in TrxR activity to as high as 130% wasobserved. These low quinol doses resulted in a similarmarginal rise in cellular Trx activity (a possible conse-

quence of the increase in cellular TrxR activity), but thehigher doses (3 and 5 M) did not have a significanteffect on the activity of Trx (Fig. 3B). Western blotresults showed that TrxR expression was induced incells treated with quinols at 0.5 and 1 M and was atcomparable levels as in DMSO control cells whenexposed to concentrations 3 M (Fig. 3C). Trx pro-tein levels remained unchanged for all drug concentra-tions tested (data not shown). Taken together, theseresults indicated that the quinol-mediated decrease incellular TrxR activity was due to an inhibition ofenzyme function and not a result of a decrease inprotein synthesis or an increase in protein degradation.

Figure 1.Chemical structures of quinols with heteroaromaticor (arylsulfonyl)indole substitution at the 4 position.

TABLE 1. Mammalian TrxR inhibitory and antiproliferative activities of quinol analogs

Compound Heterocyclic substituent

TrxR inhibitory activitya Antiproliferative activityagainst NCI 60-cell panel

b

GI50 (M) onHCT116 cellsc

MeanIC50 (30 min)

(M)

MeanIC50 (60 min)

(M)Mean

GI50 (M)Mean

LC50 (M)

1 Benzothiazole 16.5 7.1 6.5 2.3 0.23d,e 3.39d,e 0.11f

2 5F-benzothiazole 11.7 3.3 6.3 2.8 NA NA 0.08f

3 Thiophen ND 104.3 16.6 1.78d 30.9d 0.66d

4 Benzoxazole 124.5 7.1 76.1 0.3 0.46d 7.08d 0.18d

5 Benzenesulfonyl indole 5.2 1.9 2.9 0.4 0.039e 2.95e 0.083 0.009g

6 Benzenesulfonyl 6F-indole 4.3 0.7 2.7 0.1 0.016

e

2.24

e

0.071 0.018

g

7Indolyl-sulfonyl-phenyl propanoic

acid 14.6 5.7 8.4 0.1 2.34h 23.4h NA

86F-indolyl-sulfonyl-phenyl propanoic

acid 14.8 0.4 9.8 2 2.5h 29.5h NA9i Benzothiazole ND ND 33.9d 100d 100d

10i Benzothiazole ND ND NA NA 100g

Values are presented as means or means sd. GI50, growth inhibition median; IC50, inhibitory concentration median; LC50, lethalconcentration median; NA, not available; ND, TrxR inhibitory activity not detected using concentrations up to 150 M. aIC50values determinedafter 30 and 60 min incubation of recombinant rat TrxR (110 nM) with quinols using DTNB assay. bThrough the National Cancer Institute(NCI) Developmental Therapeutics Screening Program (ref. 29), quinols have been screened across 60 human cancer cell lines and growthinhibitory profiles are determined by sulforhodamine B colorimetry after 48 h drug exposure. The mean GI50 and LC50 values obtained (seeref. 29 for definitions) are presented. cGrowth inhibitory profiles were determined by MTT assay (see Materials and Methods) after 72 h drugexposure. dSee ref. 16. eSee ref. 17. fSee ref. 18. gAs determined in our laboratory. hSee ref. 19. iLacks quinol pharmacophore.


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Quinols irreversibly inhibit TrxR in a

NADPH-dependent manner

Experiments were carried out to determine the influ-ence of NADPH on TrxR inhibition by selected quinolcompounds 1, 2, 5, and 6. As compounds 1 and 2, aswell as compounds 5 and 6, share similar activityinhibition profiles, only results of assays performed oncompounds 1 and 6 are shown in Fig. 4. MammalianTrxR was preincubated with quinols in the presence orabsence of NADPH for 30 min, before enzyme activitywas determined by the DTNB reduction assay. Theoxidized enzyme remained unaffected, while the re-duced form was inhibited. A sample of NADPH-re-

duced TrxR removed of excess NADPH by desaltingover a PD-10 column was inhibited to a similar extentwith comparable IC50 values (Fig. 4A, B), suggestingthat NADPH is not directly involved in the quinol-mediated enzyme inhibition. The effect of reductantDTT on quinol inhibition of mammalian TrxR was alsoinvestigated. Interestingly, DTT-reduced TrxR with ei-ther excess DTT removed over a PD-10 desalting col-umn (Fig. 4A, B) or not removed (results not shown)did not show susceptibility to quinol inhibition. It maybe appreciated that the enzyme activity herein wasdetermined using the insulin reduction assay to follow

the reaction rate of NADPH consumption at 340 nm

over a 10 min period. Thus enzyme inhibition pro-duced at higher quinol concentrations (compound 1:50 M, Fig. 4A; compound 6: 25 and 50 M, Fig. 4B)was not negligible. Taken together, these results high-light the NADPH dependence of TrxR inhibition byquinols.

To investigate whether the inhibition was reversibleor irreversible, NADPH-reduced mammalian TrxR (0.9M) was incubated with selected quinol compounds 1and 6 (100 M) and when complete inhibition wasachieved (TrxR activity of a withdrawn aliquot deter-mined by DTNB assay), quinols were removed bypassing the enzyme sample through a PD-10 column. At

various time points over a 24 h period, aliquots of theeluted enzyme were assayed for activity in the presenceof NADPH. No TrxR activity was recovered (data notshown), indicating that mammalian TrxR was irrevers-ibly inhibited by quinols.

GSH modulation of quinol-mediated inhibition ofmammalian TrxR

The modulatory role of GSH in quinol-induced cyto-toxicity has been investigated in a previous study (22)

Figure 2. Inhibitory activity of quinols on mammalian TrxR in vitro. A) Quinol compounds 1 8, containing a quinolpharmacophore, display a concentration-dependent inhibition of TrxR activity. TrxR activity was evaluated by DTNB reductionassay (see Materials and Methods) after 1 h incubation of compounds with 110 nM recombinant rat TrxR. Compounds 9 and10, lacking a quinol pharmacophore, showed no enzyme inhibition at all concentrations indicated. B) Illustration oftime-dependence inhibition of quinols 1, 2, and 58 (15 M) and compunds 3 and 4 (100 M) on mammalian TrxR in contrastto compounds 9 and 10 (100 M), which failed to inhibit TrxR at all time points indicated. All data points are means sdof2 independent experiments.


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that reports a 6- to 10-fold increased sensitivity ofGSH-depleted cells to quinols, whereas GSH monoethylester supplementation reduces quinol potencies by23. We were interested to investigate the effect ofGSH on quinol inhibition of mammalian TrxR. Recom-binant rat TrxR was preincubated with NADPH, differ-ent concentrations of quinol compounds 1, 2, 5, and 6

and with or without the presence of GSH for 30 min.Again, compounds 2 and 5 gave activity profiles similarto compounds 1 and 6, respectively; only assay results ofcompounds 1 and 6 are presented inFig. 5. Addition ofGSH at physiological concentrations 0.5 and 1 mMimpeded inactivation of mammalian TrxR with result-ing IC50s raised by 2- to 5-fold (Fig. 5A,B). These resultsconcurred with the magnitude of influence GSH hadon quinol-induced cytotoxicity, which, taken together,

underpin the protective role the GSH system plays incell survival against quinol-mediated TrxR inactivation.

Figure 3.Effects of quinols on TrxR activity and protein levelsin HCT116 cells. Cells were exposed to indicated quinolcompounds (Cpd) for 16 h at concentrations as indicated,and cell lysates were collected for enzyme activity and proteinexpression determination. TrxR (A) and Trx activity (B) wasexpressed as a percentage of DMSO control. *Statisticallysignificant difference (P0.05) in enzyme activity from thecontrol. All data points are means se of 4 independentexperiments. C) Aliquots of cell lysates with equal proteincontent were analyzed by Western blotting with the indicatedantibodies.

Figure 4. NADPH dependence of the inhibition of mamma-lian TrxR by quinols. A 110 nM concentration of oxidizedrecombinant rat TrxR was either not reduced with NADPH,reduced with NADPH (200 M), or reduced with NADPH(200 M) and stripped of excess NADPH by desalting over aPD-10 column; and 50 nM of oxidized recombinant rat TrxR

was reduced with DTT (1 mM) and stripped of excess DTT bydesalting over a PD-10 column. The enzyme was next exposedto quinol compounds 1 (A) and 6 (B) at concentrations asindicated for 30 min. For reactions involving incubation ofenzyme with or without NADPH, TrxR activity was thenmeasured by DTNB reduction assay (see Materials and Meth-ods). For reactions involving enzyme reduction by DTT, TrxRactivity was measured by insulin reduction assay, in which theabsorbance at 340 nm was followed after adding insulin,human Trx1, and NADPH (see Materials and Methods). Alldata points are means of 2 independent experiments. Assays

were also carried out on compounds 2 and 5, and similaractivity inhibition profiles were observed as for compounds 1and 6, respectively (results not shown).


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Quinols are selective for mammalian TrxR inhibitionand target the penultimate Sec residue

In the TrxR inhibition reaction, quinols did not induceincreased NADPH oxidase activity (data not shown),

suggesting that quinols are not likely substrates of theflavoenzyme. Moreover, presence of superoxide dis-mutase (25 g/ml) and catalase (30 g/ml) failed toprotect TrxR from quinol-mediated inactivation (datanot shown).

To test the specificity of mammalian TrxR inhibitionby quinol compounds, we determined the effect ofcompounds 1, 2, 5, and 6 on bacterial TrxR and yeastGR. Mammalian TrxR differs structurally from bacterialTrxR: the former possesses an additional redox activesite at the C terminus, the penultimate Sec residue ofwhich is indispensable for its Trx reduction activity

(3032). The absence of this C-terminal -Cys-Sec- activesite is also apparent in GR, to which mammalian TrxRshares closer similarity (compared to E. coliTrxR) onthe basis of sequence homology, domain structurearchitecture (33, 34), and reaction mechanism (35).Similarities in inhibitory profiles were obtained forcompounds 1 and 2 and compounds 5 and 6, and onlyresults obtained for compounds 1 and 6 are presentedin Fig. 6. Compounds 5 and 6, the most potent mam-malian TrxR inhibitors, exhibited no inhibitory effecton bacterial TrxR and GR (Fig. 6B). Compounds 1 and2, next in line on the TrxR inhibition potency tally(Table 1), did not inhibit GR and, interestingly, at

Figure 5. GSH modulation of quinol-mediated inhibition ofmammalian TrxR. A 50 nM concentration of recombinant ratTrxR in the presence or absence of reduced GSH (0.5 and 1mM) was incubated with 200 M NADPH and quinol com-pounds 1 (A) and 6 (B) at concentrations as indicated for 30

min. TrxR activity was determined by insulin reduction assay,in which absorbance at 340 nm was followed after addinginsulin, human Trx1, and NADPH (see Materials and Meth-ods). All data points are means of 4 independent experi-ments. Assays were also carried out on compounds 2 and 5,and similar activity inhibition profiles were observed as forcompounds 1 and 6, respectively (results not shown).

Figure 6. Effect of quinols on TrxR and GR activity. Recom-binant rat (r) TrxR (50 nM) alone or in the presence of 2 M

human (h) Trx1 or E. coli TrxR (25 nM) alone or in thepresence of 2 M E. coliTrx was preincubated with 200 MNADPH and quinol compounds 1 (A) and 6 (B) at concen-trations as indicated for 30 min. TrxR activity was determinedby insulin reduction assay (see Materials and Methods). YeastGR (10 nM) was preincubated with 200 M NADPH andquinol compounds 1 (A) a n d 6 (B) at concentrations asindicated for 30 min. GR activity was determined by glutathi-one reduction assay, in which absorbance at 340 nm wasfollowed after adding GSSG and NADPH (see Materials andMethods). All data points are means of 2 independentexperiments. Assays were also carried out on compounds 2and 5, and similar activity inhibition profiles were observed as

with compounds 1 and 6, respectively (results not shown).


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higher concentrations of 25 and 50 M caused arespective 20 and 30% inactivation ofE. coliTrxR (Fig.6A). Although relatively insignificant in comparison tothe degree of inhibition observed with mammalianTrxR, the difference in the behavior between benzo-thiazole-substituted (compounds 1 and 2) and (arylsul-fonyl)indole-substituted (compounds 5 and 6) quinolssuggests the possibility to rationally design and developstructural derivatives of the former class of quinols asinhibitors of bacterial TrxR with minimal inhibition ofits mammalian counterpart. Furthermore, the inhibi-tory effect of quinols on the whole Trx system wascompared with that on TrxR only (independent of Trx)using the insulin reduction assay. The compounds werefound to inhibit the catalyzed reduction of insulindisulfide to the same extent with comparable IC50s (Fig.6A, B), suggesting that the presence of Trx did notaffect the inhibitory efficiency and that TrxR was in-deed the specific target of quinols.

Results obtained so far have revealed the susceptibil-ity of mammalian TrxR to quinol inactivation, whilebacterial TrxR and GR activity remained unaltered.

Given that the selenol in the penultimate C-terminalSec residue has a low pKa of 5.2 (36) that results inreduced mammalian TrxR existing as a deprotonatedselenol (Se) species at physiological pH, we hypothe-sized that the Sec residue was targeted by quinols. Toconfirm this hypothesis, we made use of the BIAM-labeling reaction at low pH to monitor the extent offree Sec modified by the presence of quinols. Previousstudies (3739) have reported the selective alkylation ofthe Sec at pH 6.5 due to its low pKa that renders thedeprotonated species more susceptible to alkylation; aSec that is oxidized or chemically modified would beunavailable for BIAM labeling, which could then be

monitored by streptavidin blot analysis. As shown in thebottom panel of Fig. 7, the HRP-streptavidin-blottedbands of the 55 kDa TrxR monomer depicting freeselenol decreased in intensity along increasing incuba-tion time with compounds 1 and 6. This observeddecrease in free selenolate available for BIAM labelingwas consistent with the decline in enzyme activity overtime (Fig. 7, top), strongly suggesting that quinols

target the Sec residue to bring about mammalian TrxRinhibition.

DISCUSSION

TrxR is a homodimeric enzyme belonging to the fla-voprotein family of pyridine nucleotide-disulfide oxi-doreductases that also includes GR (40). Two distinct

types of TrxR exist: the low molecular mass (35 kDasubunit) TrxR is present in bacteria, archaea, plants,and yeast and uses a -Cys-Ala-Thr-Cys- catalytic motif fora narrow substrate spectrum; a large subunit (55 kDa)TrxR is present in higher eukaryotes and has both anN-terminal redox active dithiol/disulfide in commonwith GR and a second C-terminal active site that ac-counts for its broad substrate specificity, including Trx,DTNB, protein disulfide isomerase, NK-lysin, perox-ides, GSH peroxidase, selenite, and others (2, 4, 41).Generally, mammalian TrxR1 localized in the cytosol(31, 32, 34, 42), TrxR2 in the mitochondria (38, 43),and thioredoxin/glutathione reductase (TGR) in the

testis (44) have a common domain structure comprisedof a flavin adenine dinucleotide (FAD) and NADPH-binding domain and an interface domain that is similarto GR; also conserved in mammalian TrxRs are theN-terminal -Cys59-Val-Asn-Val-Gly-Cys64- dithiol/disul-fide active site contained in the FAD-binding domainand the second -Gly-Cys497-Sec498-Gly- active site thatresides in the 16-residue extension in the C terminus.The essential role of the penultimate C-terminal Secresidue for TrxR catalytic activity has been well studied(32, 3739, 45, 46). The catalytic mechanism of mam-malian TrxR, as supported by biochemical and X-raycrystallographic studies, involves the transfer of reduc-

ing equivalents from NADPH to bound FAD and thento the N-terminal active site disulfide; the resultantdithiol subsequently reduces the C-terminal selenenyl-sulfide of the other subunit of the homodimer, and theselenolthiol motif finally reduces a substrate of theenzyme, for example, the active site disulfide withinoxidized Trx (34, 35, 42, 43, 47). On NADPH reduc-tion, the flexible C-terminal tail moves such that the

Figure 7.Quinols target the Sec residue of mammalian TrxR. A 0.9 M concentration of recombinant rat TrxR was incubatedwith 200 M NADPH and 100 M quinol compounds 1 and 6. At different time points, an aliquot of enzyme mixture waswithdrawn for TrxR activity measurment and BIAM labeling. Top: time course of TrxR activity of the mixture. Bottom:HRP-conjugated streptavadin detection of BIAM labeling of free selenol in the mixture at different incubation times. Resultsshown are representative of 3 independent experiments.


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selenothiol motif becomes surface exposed (34, 42).This unique feature of reduced mammalian TrxR, thehighly accessible Sec and high reactivitiy of the nucleo-philic selenolate moiety, has made the enzyme a selec-tive target of many electrophilic compounds (4, 21).

Novel heteroaromatic-substituted quinols exhibit invitro antiproliferative activity against tumor cell linesand in vivo antitumor activity in xenograft models(1619). However, the precise mechanisms of antitu-

mor action of these promising novel anticancer agentsremain elusive. A previous study (20) using mass spec-trometry has shown in vitrobinding of quinol to cys-teine residues in Trx1 protein, suggesting that proteinthiol inactivation contributes at least in part to theproapoptotic and antiproliferative effect of quinols.Quinols are Michael acceptors, which are chemicalcompounds containing an ,-unsaturated carbonylmoiety where the electrophilic carbon atom mayinteract with nucleophiles such as sulfhydryl groups.Michael acceptors are known to have sulfhydryl reactiv-ities (48). Compared to cysteine, Sec is a strongernucleophile due to a larger atomic radius and a lower

pKa to ensure full ionization under physiological con-ditions (36). Given that compounds reported to inhibitmammalian TrxR such as quinones (49), curcumin(50), and flavonoids quercetin and myricetin (51) areknown Michael acceptors, we sought to investigatewhether quinols could inhibit mammalian TrxR. Wescreened a number of quinol analogs with differentheteroaromatic or (arylsulfonyl)indole substitutions atthe 4 position (Fig. 1) for their effects on mammalianTrxR activity. A structure-activity relationship was estab-lished, which positively correlated with the cell-growthinhibition and cytotoxicity profile of each analog. Thepresence of a quinol pharmacophore was accountablefor TrxR inactivation. A benzenesulfonyl indole substi-tution resulted in the most potent analogs (compounds5 and 6), but coupling a propionate group to thissubstituent (compounds 7 and 8) led to a detrimentaleffect on potency; benzothiazole substitutions gave riseto more potent analogs (compounds 1 and 2) thanthiophen (compound 3) or benzoxazole (compound4) substitutions.

Here, we have shown that quinols irreversibly inhib-ited mammalian TrxR in a concentration-, time-, andNADPH-dependent manner. Earlier reports (35, 5053) have shown that most inhibitors fail to inactivate

oxidized TrxR in the absence of NADPH. Similarly,oxidized mammalian TrxR, which contains a nonreac-tive C-terminal Cys497-Sec498 selenenylsulfide bridge(47), was resistant to inactivation by quinols. Across arange of cytotoxic doses, potent quinols, for instance,compound 6, selectively inhibited mammalian TrxRwith an IC50of 4.3 M (at 30 min incubation; Table 1)but had no effect on GR and bacterial TrxR. Indeed, atime-related decrease in BIAM labeling of free selenol(-SeH) corresponding to a decline in catalytic activity(Fig. 7) indicated that quinols target the Sec in theenzyme. Collectively, we demonstrated that mammalian

TrxR is a mechanistic target for quinols based onseveral lines of evidence: 1) a correlating trend ofquinol cytotoxicity with the in vitro TrxR inhibitionpotency, 2) cell lysates of HCT116 cells treated withquinol concentrations and for a time point that waspreviously found to induce apoptosis (22) showed areduction in TrxR activity, and 3) the results of our invitroexperiment where the presence of physiologicalconcentrations of GSH impeded quinol-mediated TrxR

inactivation, which is in agreement with the findingthat depletion or supplementation of intracellular GSHhas modulated quinol-mediated cytotoxicity (previouslyreported in ref. 22).

Quinol-modified TrxR did not show induction ofNADPH oxidase activity, suggesting that quinols are notsubstrates of TrxR. Based on the obtained results, aproposed mechanism of mammalian TrxR inactivationby quinols is outlined in Fig. 8: the quinol electrophilic carbon atom directly attacks the selenolate moiety inNADPH-reduced TrxR, and the resultant enzyme ischemically modified to lose catalytic activity.

With the use of in vitro experimental approach, aprevious study (20) suggested Trx as a molecular target

Figure 8. Proposed mechanism of inhibition of mammalianTrxR by quinols. The electrophilic carbon atom in activequinols, for example, compound 6, directly attacks the Secresidue in NADPH-reduced TrxR. The C-terminal-modifiedenzyme is unable to reduce oxidized Trx.


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of quinols; it remains to be determined whether quin-ols inhibit Trx directly in intact cells. In this presentstudy, although a decrease in TrxR activity was detectedin lysates of cells exposed to apoptosis-inducing dosesof quinols, Trx activity remained unaffected (Fig. 3A,B). We also performed the insulin reduction assaywhere the whole Trx system was preincubated withquinols, and the results obtained were consistent withobservations in the work of Bradshaw et al. (20) thatshow quinol inhibition of TrxR/Trx protein disulfidereduction. We thus investigated whether quinols wouldinhibit TrxR independent of Trx by performing theassays where TrxR alone was preincubated with quinols;the results indeed showed that quinols caused the samedegree of TrxR/Trx disulfide reduction. As supportedbyin vitroevidence of quinol binding to cysteines in Trx(20), quinols have a propensity toward nucleophilessuch as reactive cysteines in cellular proteins. However,in intact cells, within which numerous reactive cellularproteins are contained, we would surmise from theevidence presented here that quinols at therapeuticdoses preferentially target TrxR instead of Trx.

The Trx and GSH systems exert redox control overmany cellular processes involved in cell proliferationand apoptosis (54). Accumulating evidence has shownthat components of the Trx system are overexpressedin tumors and are involved in the carcinogenic process(1012, 15). Numerous chemotherapeutic agents suchas nitrosoureas (35, 55), cisplatin (53), cyclophospha-mide (56), motexafin gadolinium (57), and arsenictrioxide (58) have been reported to inhibit TrxR,suggesting that mammalian TrxR is an important mo-lecular target for anticancer drug development. Trx isinvolved in DNA synthesis/repair and redox balancethrough respective reduction of RNR and peroxiredox-

ins, as well as in the modulation of gene expression ofcellular proteins through redox regulation of transcrip-tional activity of NF-B, AP-1, p53, and HIF-1 (14).TrxR inhibition leads to Trx oxidation and culminatesin disruption or deregulation of Trx-regulated cellularactivities and responses, such as DNA synthesis andrepair. Reduced Trx is known to inhibit apoptosis bybinding to apoptosis signal regulating kinase 1 (ASK1);oxidized Trx dissociates from the complex, leading toASK1 activation and ultimately to apoptosis induction(59). Moreover, studies have shown that TrxR modifiedat the Sec residue by alkylating agents (60) or endoge-nous lipid electrophiles (61) is the species (not the

full-length Sec-containing enzyme) that is necessary forinducing apoptosis. We therefore suggest that cytotox-icity due to quinol inhibition of TrxR is the result of thecatastrophic events after Trx oxidation and quinol-modified TrxR directly inducing apoptosis.

In summary, we have demonstrated that quinolsirreversibly inhibit mammalian TrxR by targeting thepenultimate C-terminal Sec residue. Particularly, wehave identified compound 6, a quinol with a benzene-sulfonyl-6-fluoroindolyl substitution, to exhibit TrxRinhibition with remarkable potency that correlates wellwith its superior antitumor potency against sensitive

cancer cell lines. In the context of anticancer researchfocused on developing novel cancer therapeuticsagainst novel molecular targets, quinols as TrxR inhib-itors certainly make themselves promising clinicalcandidates.

We are grateful to Malcolm Stevens, School of Pharmacy,University of Nottingham, for kindly providing the quinolcompounds. We thank Elias Arner, Qing Cheng, and OlleRengby (Medical Nobel Institute for Biochemistry, Karolinska

Institutet) for kind provision of recombinant rat TrxR and R.Eliasson for expert technical assistance in the affinity purifi-cation of polyclonal antirat TrxR1 antibodies. This work wassupported by Swedish Cancer Society Grant 961, SwedishResearch Council Medicine Grant 13x-3529, the K.A. Wallen-berg Foundation, and the Karolinska Institutet.

REFERENCES

1. Arner, E. S., and Holmgren, A. (2000) Physiological functions ofthioredoxin and thioredoxin reductase. Eur. J. Biochem. 267,61026109

2. Gromer, S., Urig, S., and Becker, K. (2004) The thioredoxinsystemfrom science to clinic. Med. Res. Rev. 24, 4089

3. Lillig, C. H., and Holmgren, A. (2007) Thioredoxin and relatedmoleculesfrom biology to health and disease. Antioxid. RedoxSignal. 9, 2547

4. Papp, L. V., Lu, J., Holmgren, A., and Khanna, K. K. (2007)From selenium to selenoproteins: synthesis, identity, and theirrole in human health. Antioxid. Redox Signal. 9, 775806

5. Matsui, M., Oshima, M., Oshima, H., Takaku, K., Maruyama, T.,Yodoi, J., and Taketo, M. M. (1996) Early embryonic lethalitycaused by targeted disruption of the mouse thioredoxin gene.

Dev. Biol.178, 1791856. Nonn, L., Williams, R. R., Erickson, R. P., and Powis, G. (2003)

The absence of mitochondrial thioredoxin 2 causes massiveapoptosis, exencephaly, and early embryonic lethality in ho-mozygous mice. Mol. Cell. Biol. 23, 916922

7. Jakupoglu, C., Przemeck, G. K., Schneider, M., Moreno, S. G.,Mayr, N., Hatzopoulos, A. K., de Angelis, M. H., Wurst, W.,

Bornkamm, G. W., Brielmeier, M., and Conrad, M. (2005)Cytoplasmic thioredoxin reductase is essential for embryogene-sis but dispensable for cardiac development. Mol. Cell. Biol. 25,19801988

8. Conrad, M., Jakupoglu, C., Moreno, S. G., Lippl, S., Banjac, A.,Schneider, M., Beck, H., Hatzopoulos, A. K., Just, U., Sinowatz,F., Schmahl, W., Chien, K. R., Wurst, W., Bornkamm, G. W., andBrielmeier, M. (2004) Essential role for mitochondrial thiore-doxin reductase in hematopoiesis, heart development, andheart function. Mol. Cell. Biol. 24, 94149423

9. Arner, E. S., and Holmgren, A. (2006) The thioredoxin systemin cancer. Semin. Cancer Biol. 16, 420426

10. Berggren, M., Gallegos, A., Gasdaska, J. R., Gasdaska, P. Y.,Warneke, J., and Powis, G. (1996) Thioredoxin and thioredoxinreductase gene expression in human tumors and cell lines, andthe effects of serum stimulation and hypoxia. Anticancer Res. 16,34593466

11. Soini, Y., Kahlos, K., Napankangas, U., Kaarteenaho-Wiik, R.,Saily, M., Koistinen, P., Paaakko, P., Holmgren, A., and Kinnula,V. L. (2001) Widespread expression of thioredoxin and thiore-doxin reductase in non-small cell lung carcinoma. Clin. CancerRes.7, 17501757

12. Kahlos, K., Soini, Y., Saily, M., Koistinen, P., Kakko, S., Paakko,P., Holmgren, A., and Kinnula, V. L. (2001) Up-regulation ofthioredoxin and thioredoxin reductase in human malignantpleural mesothelioma.Int. J. Cancer 95,198204

13. Kim, S. J., Miyoshi, Y., Taguchi, T., Tamaki, Y., Nakamura, H.,Yodoi, J., Kato, K., and Noguchi, S. (2005) High thioredoxinexpression is associated with resistance to docetaxel in primarybreast cancerClin. Cancer Res. 11, 84258430

14. Raffel, J., Bhattacharyya, A. K., Gallegos, A., Cui, H., Einspahr,J. G., Alberts, D. S., and Powis, G. (2003) Increased expression


8/13/2019 quinol

11/12

of thioredoxin-1 in human colorectal cancer is associated withdecreased patient survival. J. Lab. Clin. Med. 142, 4651

15. Yoo, M. H., Xu, X. M., Carlson, B. A., Gladyshev, V. N., andHatfield, D. L. (2006) Thioredoxin reductase 1 deficiencyreverses tumor phenotype and tumorigenicity of lung carci-noma cells. J. Biol. Chem. 281, 1300513008

16. Wells, G., Berry, J. M., Bradshaw, T. D., Burger, A. M., Seaton,A., Wang, B., Westwell, A. D., and Stevens, M. F. (2003)4-Substituted 4-hydroxycyclohexa-2,5-dien-1-ones with selectiveactivities against colon and renal cancer cell lines. J. Med. Chem.46,532541

17. Berry, J. M., Bradshaw, T. D., Fichtner, I., Ren, R., Schwalbe,

C. H., Wells, G., Chew, E.-H., Stevens, M. F., and Westwell, A. D.(2005) Quinols as novel therapeutic agents. 2.(1) 4-(1-Arylsul-fonylindol-2-yl)-4-hydroxycyclohexa-2,5-dien-1-ones and relatedagents as potent and selective antitumor agents. J. Med. Chem.48,639644

18. Lion, C. J., Matthews, C. S., Wells, G., Bradshaw, T. D., Stevens,M. F., and Westwell, A. D. (2006) Antitumour properties offluorinated benzothiazole-substituted hydroxycyclohexa-2,5-di-enones (quinols). Bioorg. Med. Chem. Lett. 16, 50055008

19. McCarroll, A. J., Bradshaw, T. D., Westwell, A. D., Matthews,C. S., and Stevens, M. F. (2007) Quinols as novel therapeuticagents. 7.1 Synthesis of antitumor 4-]1-(arylsulfonyl-1H-indol-2-yl)]-4-hydroxycyclohexa-2,5-dien-1-ones by Sonogashira reac-tions.J. Med. Chem. 50, 17071710

20. Bradshaw, T. D., Matthews, C. S., Cookson, J., Chew, E.-H.,Shah, M., Bailey, K., Monks, A., Harris, E., Westwell, A. D., Wells,G., Laughton, C. A., and Stevens, M. F. (2005) Elucidation of

thioredoxin as a molecular target for antitumor quinols.CancerRes.65, 39113919

21. Urig, S., and Becker, K. (2006) On the potential of thioredoxinreductase inhibitors for cancer therapy. Semin. Cancer Biol. 16,452465

22. Chew, E.-H., Matthews, C. S., Zhang, J., McCarroll, A. J., Hagen,T., Stevens, M. F., Westwell, A. D., and Bradshaw, T. D. (2006)Antitumor quinols: role of glutathione in modulating quinol-induced apoptosis and identification of putative cellular proteintargets. Biochem. Biophys. Res. Commun. 346, 242251

23. Arner, E. S., Sarioglu, H., Lottspeich, F., Holmgren, A., andBock, A. (1999) High-level expression in Escherichia coli ofselenocysteine-containing rat thioredoxin reductase utilizinggene fusions with engineered bacterial-type SECIS elements andco-expression with the selA, selB and selC genes. J. Mol. Biol.292,10031016

24. Ren, X., Bjornstedt, M., Shen, B., Ericson, M. L., and Holmgren,A. (1993) Mutagenesis of structural half-cystine residues inhuman thioredoxin and effects on the regulation of activity byselenodiglutathione. Biochemistry32, 97019708

25. Holmgren, A., and Bjornstedt, M. (1995) Thioredoxin andthioredoxin reductase.Methods Enzymol. 252, 199208

26. Luthman, M., and Holmgren, A. (1982) Rat liver thioredoxinand thioredoxin reductase: purification and characterization.Biochemistry21, 66286633

27. Arner, E. S., and Holmgren, A. (2000) Measurement of thiore-doxin and thioredoxin reductase. In Current Protocols in Toxicol-ogy, Supplement 5 (Maines, M.D., Costa, L.G., Reed, D.J., Sassa,S., and Sipes, I.G., eds) pp. 7.4.17.4.14, John Wiley & Sons, Inc.,New York

28. Rozell, B., Hansson, H. A., Luthman, M., and Holmgren, A.(1985) Immunohistochemical localization of thioredoxin andthioredoxin reductase in adult rats. Eur. J. Cell Biol. 38, 7986

29. Boyd, M. R., and Paull, K. D. (1995) Some practical consider-ations and applications of the National Cancer Institute in vitroanticancer drug discovery screen. Drug Dev. Res. 34, 91104

30. Tamura, T., and Stadtman, T. C. (1996) A new selenoproteinfrom human lung adenocarcinoma cells: purification, proper-ties, and thioredoxin reductase activity. Proc. Natl. Acad. Sci.U. S. A. 93, 10061011

31. Gladyshev, V. N., Jeang, K. T., and Stadtman, T. C. (1996)Selenocysteine, identified as the penultimate C-terminal residuein human T-cell thioredoxin reductase, corresponds to TGA inthe human placental gene. Proc. Natl. Acad. Sci. U. S. A. 93,61466151

32. Zhong, L., Arner, E. S., Ljung, J., Aslund, F., and Holmgren, A.(1998) Rat and calf thioredoxin reductase are homologous toglutathione reductase with a carboxyl-terminal elongation con-

taining a conserved catalytically active penultimate selenocys-teine residue. J. Biol. Chem. 273, 85818591

33. Gasdaska, P. Y., Gasdaska, J. R., Cochran, S., and Powis, G.(1995) Cloning and sequencing of a human thioredoxin reduc-tase. FEBS Lett. 373, 59

34. Sandalova, T., Zhong, L., Lindqvist, Y., Holmgren, A., andSchneider, G. (2001) Three-dimensional structure of a mamma-lian thioredoxin reductase: implications for mechanism andevolution of a selenocysteine-dependent enzyme. Proc. Natl.Acad. Sci. U. S. A. 98, 95339538

35. Arscott, L. D., Gromer, S., Schirmer, R. H., Becker, K., andWilliams, C.H. Jr. (1997) The mechanism of thioredoxin reduc-

tase from human placenta is similar to the mechanisms oflipoamide dehydrogenase and glutathione reductase and isdistinct from the mechanism of thioredoxin reductase fromEscherichia coli. Proc. Natl. Acad. Sci. U. S. A. 94, 36213626

36. Stadtman, T. C. (1996) Selenocysteine. Annu. Rev. Biochem. 65,83100

37. Gorlatov, S. N., and Stadtman, T. C. (1998) Human thioredoxinreductase from HeLa cells: selective alkylation of selenocysteinein the protein inhibits enzyme activity and reduction withNADPH influences affinity to heparin. Proc. Natl. Acad. Sci.U. S. A. 95, 85208525

38. Lee, S. R., Kim, J. R., Kwon, K. S., Yoon, H. W., Levine, R. L.,Ginsburg, A., and Rhee, S. G. (1999) Molecular cloning andcharacterization of a mitochondrial selenocysteine-containingthioredoxin reductase from rat liver. J. Biol. Chem. 274, 47224734

39. Lee, S. R., Bar-Noy, S., Kwon, J., Levine, R. L., Stadtman, T. C.,

and Rhee, S. G. (2000) Mammalian thioredoxin reductase:oxidation of the C-terminal cysteine/selenocysteine active siteforms a thioselenide, and replacement of selenium with sulfurmarkedly reduces catalytic activity. Proc. Natl. Acad. Sci. U. S. A.97,25212526

40. Williams, C. H., Jr. (1992) Lipoamide dehydrogenase, glutathi-one reductase, thioredoxin reductase, and mercuric ion reduc-taseA family of flavoenzyme transhydrogenases. In Chemistryand Biochemistry of Flavoenzymes, Vol. 3 (Muller, F., ed) pp.121211, CRC Press, Boca Raton, FL, USA

41. Novoselov, S. V., and Gladyshev, V. N. (2003) Non-animal originof animal thioredoxin reductases: implications for selenocys-teine evolution and evolution of protein function throughcarboxy-terminal extensions.Protein Sci. 12, 372378

42. Fritz-Wolf, K., Urig, S., and Becker, K. (2007) The structure ofhuman thioredoxin reductase 1 provides insights into C-termi-

nal rearrangements during catalysis. J. Mol. Biol. 370, 11612743. Biterova, E. I., Turanov, A. A., Gladyshev, V. N., and Barycki, J. J.(2005) Crystal structures of oxidized and reduced mitochon-drial thioredoxin reductase provide molecular details of thereaction mechanism. Proc. Natl. Acad. Sci. U. S. A. 102, 1501815023

44. Sun, Q. A., Kirnarsky, L., Sherman, S., and Gladyshev, V. N.(2001) Selenoprotein oxidoreductase with specificity for thiore-doxin and glutathione systems. Proc. Natl. Acad. Sci. U. S. A. 98,36733678

45. Gromer, S., Wissing, J., Behne, D., Ashman, K., Schirmer, R. H.,Flohe, L., and Becker, K. (1998) A hypothesis on the catalyticmechanism of the selenoenzyme thioredoxin reductase. Bio-chem. J. 332, 591592

46. Zhong, L., and Holmgren, A. (2000) Essential role of seleniumin the catalytic activities of mammalian thioredoxin reductaserevealed by characterization of recombinant enzymes with sel-enocysteine mutations. J. Biol. Chem. 275, 1812118128

47. Zhong, L., Arner, E. S., and Holmgren, A. (2000) Structure andmechanism of mammalian thioredoxin reductase: the active siteis a redox-active selenolthiol/selenenylsulfide formed from theconserved cysteine-selenocysteine sequence.Proc. Natl. Acad. Sci.U. S. A. 97, 58545859

48. Dinkova-Kostova, A. T., Massiah, M. A., Bozak, R. E., Hicks, R. J.,and Talalay, P. (2001) Potency of Michael reaction acceptors asinducers of enzymes that protect against carcinogenesis de-pends on their reactivity with sulfhydryl groups. Proc. Natl. Acad.Sci. U. S. A. 98, 34043409

49. Cenas, N., Nivinskas, H., Anusevicius, Z., Sarlauskas, J., Lederer,F., and Arner, E. S. (2004) Interactions of quinones withthioredoxin reductase: a challenge to the antioxidant role of themammalian selenoprotein.J. Biol. Chem. 279, 25832592


8/13/2019 quinol

12/12

50. Fang, J., Lu, J., and Holmgren, A. (2005) Thioredoxin reductaseis irreversibly modified by curcumin: a novel molecular mecha-nism for its anticancer activity. J. Biol. Chem. 280, 2528425290

51. Lu, J., Papp, L. V., Fang, J., Rodriguez-Nieto, S., Zhivotovsky,B., and Holmgren, A. (2006) Inhibition of mammalianthioredoxin reductase by some flavonoids: implications formyricetin and quercetin anticancer activity. Cancer Res. 66,44104418

52. Gromer, S., Arscott, L. D., Williams, C. H. Jr., Schirmer, R. H.,and Becker, K. (1998) Human placenta thioredoxin reductase.Isolation of the selenoenzyme, steady state kinetics, and inhibi-tion by therapeutic gold compounds. J. Biol. Chem. 273,20096

2010153. Becker, K., Herold-Mende, C., Park, J. J., Lowe, G., andSchirmer, R. H. (2001) Human thioredoxin reductase is effi-ciently inhibited by (2,2:6,2-terpyridine)platinum(II) com-plexes. Possible implications for a novel antitumor strategy.

J. Med. Chem.44, 2784 279254. Davis, W. Jr., Ronai, Z., and Tew, K. D. (2001) Cellular thiols and

reactive oxygen species in drug-induced apoptosis. J. Pharmacol.Exp. Ther. 296, 1 6

55. Witte, A. B., Anestal, K., Jerremalm, E., Ehrsson, H., and Arner,E. S. (2005) Inhibition of thioredoxin reductase but not ofglutathione reductase by the major classes of alkylating andplatinum-containing anticancer compounds. Free Radic. Biol.Med.39, 696703

56. Wang, X., Zhang, J., and Xu, T. (2007) Cyclophosphamide as apotent inhibitor of tumor thioredoxin reductase in vivo. Toxicol.Appl. Pharmacol. 218, 8895

57. Hashemy, S. I., Ungerstedt, J. S., Zahedi Avval, F., andHolmgren, A. (2006) Motexafin gadolinium, a tumor-selectivedrug targeting thioredoxin reductase and ribonucleotide reduc-tase. J. Biol. Chem. 281, 1069110697

58. Lu, J., Chew, E.-H., Holmgren, A. (2007) Targeting thioredoxinreductase is a basis for cancer therapy by arsenic trioxide. Proc.Natl. Acad. Sci. U. S. A. 104, 1228812293

59. Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K.,Sawada, Y., Kawabata, M., Miyazono, K., and Ichijo, H. (1998)

Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO J. 17, 2596 260660. Anestal, K., and Arner, E. S. (2003) Rapid induction of cell

death by selenium-compromised thioredoxin reductase 1 butnot by the fully active enzyme containing selenocysteine. J. Biol.Chem.278, 1596615972

61. Cassidy, P. B., Edes, K., Nelson, C. C., Parsawar, K., Fitzpatrick,F. A., and Moos, P. J. (2006) Thioredoxin reductase is requiredfor the inactivation of tumor suppressor p53 and for apoptosisinduced by endogenous electrophiles. Carcinogenesis27, 25382549

Received for publication October 29, 2007.Accepted for publication November 29, 2007.


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