inhibition of nuclear factor- b dna binding by organoselenocyanates through covalent modification...

2007;67:10475-10483. Cancer Res Kun-Ming Chen, Thomas E. Spratt, Bruce A. Stanley, et al. p50 SubunitOrganoselenocyanates through Covalent Modification of the

B DNA Binding byκInhibition of Nuclear Factor-

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Inhibition of Nuclear Factor-KB DNA Binding by

Organoselenocyanates through Covalent

Modification of the p50 Subunit

Kun-Ming Chen,1Thomas E. Spratt,

1Bruce A. Stanley,

2Dan A. De Cotiis,

1

Maria C. Bewley,1John M. Flanagan,

1Dhimant Desai,

3Arunangshu Das,

1

Emerich S. Fiala,4Shantu Amin,

3and Karam El-Bayoumy

1

1Department of Biochemistry and Molecular Biology, 2Proteomics/Mass Spectrometry Core Facility of the Section of Research Resources,and 3Department of Pharmacology, Penn State College of Medicine, Hershey, Pennsylvania and 4New York University School ofMedicine, Tuxedo, New York

Abstract

Most known chemopreventive agents including certain sele-nium compounds suppress the activation of the nuclear factorKB (NF-KB), but the mechanisms remain largely elusive.Toward this end, we initially showed that the inhibition ofNF-KB DNA binding by benzyl selenocyanate (BSC) and1,4-phenylenebis(methylene)selenocyanate (p-XSC) was re-versed by the addition of DTT; this suggests the formation ofDTT-reducible selenium-sulfur bonds between selenocyanatemoieties and cysteine residues in NF-KB (p50) protein.Furthermore, the inhibitory effect of selenocyanates onNF-KB was not altered in the presence of physiologic level ofreduced glutathione (1 mmol/L), suggesting that selenocya-nates can also inhibit NF-KB in vivo . Using both matrix-assisted laser desorption/ionization-time of flight and tandemmass spectrometry fragmentation, we showed for the firsttime that the Cys62 residue in the active site of NF-KB (p50)protein was modified by BSC through the formation of aselenium-sulfur bond. In addition, p-XSC–bound NF-KB (p50)protein was also detected by a radiotracer method. To providefurther support, molecular models of both BSC and p-XSCpositioned in the DNA binding pocket of the p50 wereconstructed through the covalent modification of Cys62; themodels reveal that DNA substrate could be hindered to enterits DNA binding region. This study shows for the first timethat BSC and p-XSC may exert their chemopreventive activity,at least in part, by inhibiting NF-KB through covalent modi-fication of Cys62 of the p50 subunit of NF-KB. [Cancer Res2007;67(21):10475–83]

Introduction

Collectively, results from epidemiologic studies, laboratorybioassays, and human clinical intervention trials clearly supportthe protective role of selenium against cancer development (1–3).Although many different mechanisms, including protection againstoxidative damage, altered carcinogen metabolism, enhancedimmune surveillance, antiangiogenesis, and induction of apoptosishave been proposed to account for the anticarcinogenic effects ofselenium, the inhibition of nuclear factor-nB (NF-nB) by selenium-

containing compounds has been reported (4–6). The inhibition ofNF-nB by selenium, in particular, is appealing because it couldresult in apoptosis of tumor cells and thus may, in part, account forthe chemopreventive activity of selenium compounds (7–10).NF-nB, identified by Sen et al. in 1986, is one of the major

antiapoptotic transcription factors (11). It regulates more than150 genes including genes involved in tumor progression, such asinducible nitric oxide synthase, cyclooxygenase-2, and proliferativeand antiapoptotic cytokines (12). The activity of NF-nB has beenfound constitutively elevated in human tumors from eitherhematologic or solid origin, such as melanomas, breast, prostate,ovarian, pancreatic, colon, and thyroid carcinomas, and itcontributes to malignant progression and resistance to therapy inmost of the major forms of human cancer (13, 14). Five distinctNF-nB subunits have been identified and cloned in mammaliancells including NF-nB1 (p50/p105), NF-nB2 (p52/p100), RelA (p65),RelB, and c-Rel (12). NF-nB1 and NF-nB2 can undergo proteolysisto liberate p50 and p52, respectively. In the cytoplasm of restingcells, the NF-nB subunits can associate to form either homodimersor heterodimers and remain inactive via the association withinhibitory InB molecules (Fig. 1; ref. 15). On activation byextracellular stimuli such as growth factors, inflammatory cyto-kines, bacterial products, chemotherapeutic agents, or oxidativestress, NF-nB complexes can undergo phosphorylation of InB byspecific InB kinases and proteolytic degradation to yield freeNF-nB. This, in turn, enters the nucleus and binds to the nB sitesof gene promoters to regulate the specific genes typically involvedin immune and inflammatory responses and in cell growth control(16–18). Previous studies have shown that the inhibition ofconstitutive NF-nB activity blocks the oncogenic potential ofneoplastic cells in different ways: by sensitizing tumor cells tochemotherapeutic drug-induced apoptosis, by decreasing thehighly proliferative rate which characterizes transformed cells,by inhibiting tissue invasiveness and metastatic potential of highlymalignant cells, and by suppression of expression of genes involvedin carcinogenesis (19, 20). In addition, specific NF-nB (p50) decoyoligonucleotides that can prevent the binding of NF-nB (p50) to thepromoter region of target genes can efficiently promote cell deathin cancer cells (21). Therefore, NF-nB is an attractive moleculartarget for cancer therapy and cancer chemoprevention (22); in fact,most known chemopreventive agents suppress the activation ofNF-nB (20).Synthetic organoselenium compounds such as benzyl selenocya-

nate (BSC) and 1,4-phenylenebis(methylene)selenocyanate (p-XSC)have been developed in our laboratory (structures are shown inFig. 1), and these compounds are superior cancer-chemopreventive

Requests for reprints: Karam El-Bayoumy, Department of Biochemistry andMolecular Biology, Penn State College of Medicine, H171, 500 University Drive,Hershey, PA 17033. Phone: 717-531-1005; Fax: 717-531-0002; E-mail: [email protected].

I2007 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-07-2510

www.aacrjournals.org 10475 Cancer Res 2007; 67: (21). November 1, 2007

Research Article



agents than other known selenium compounds, as judged bynumerous animal models and cell culture systems (4). p-XSC wasrecently found to reduce the expression of NF-nB and several targetgenes regulated by NF-nB, such as cyclooxygenase-2, phospholipaseA2, and cyclin D1, in non–small cell lung cancer cells (7). In thehuman colorectal adenocarcinoma HCT-116 cells, p-XSC reducesthe binding activity of NF-nB in an electrophoretic mobility shiftassay; the inhibition was reversed in the presence of 1 mmol/LDTT, suggesting that the formation of -Se-S- bonds between p-XSCand essential sulfhydryl groups of NF-nB may account for theobserved inhibition (5). NF-nB (p50) monomer contains sevencysteine residues. The binding of NF-nB (p50) to its cognateregulatory element has been shown to depend on the integrity ofCys62, which primarily remains in its reduced state (23). Thisresidue is enveloped in a cationic environment that renders thethiol group highly reactive and particularly susceptible tooxidation. The present investigation was conducted to test ournovel hypothesis that the chemopreventive selenocyanate-contain-ing compounds BSC and p-XSC may inhibit tumorigenesis throughthe inhibition of NF-nB via the formation of -Se-S- bonds withcysteine residues in p50 protein, thereby down-regulating its targetgenes and leading to the inhibition of cell proliferation andinduction of apoptosis.

Materials and Methods

Cell culture and treatment. Squamous carcinoma SCC 1483 cells

(donated by Dr. Peter G. Sacks, New York University, New York, NY) were

maintained in a 1:1 mixture of DMEM/F12 supplemented with 10% fetalbovine serum (FBS) and penicillin/streptomycin (50 Ag/mL; ref. 24). Humannon–small cell lung cancer cells (NCI-H460) obtained from the American

Type Culture Collection were maintained in RPMI 1640 containing 10%

FBS and penicillin/streptomycin (50 Ag/mL). Both SCC 1483 and NCI-H460cells were kept in a humidified environment at 37jC with 5% CO2, grown

to 70% to 80% confluency, trypsinized with 0.1% trypsin-2 mmol/L EDTA,

and plated for experiment use as described below. BSC and p-XSC

were synthesized according to published methods (25, 26). The cells weretreated with various concentrations of BSC (6.25–200 Amol/L) or p-XSC

(0.625–20 Amol/L) dissolved in DMSO in the cell culture medium for 1 h,

followed by stimulation with tumor necrosis factor (TNF)-a (2 ng/mL)

for 30 min. Control cultures were treated with DMSO alone (not exceeding0.1% of volume) and were processed similarly.

Preparation of nuclear extracts from cells. Nuclear proteins wereprepared with the TransAM nuclear extract kit according to the manu-

facturer’s protocol (Active Motif North America). In brief, cells were scrapedinto PBS containing phosphatase inhibitor, centrifuged to remove the

supernatant, and then resuspended with 1� hypotonic buffer and kept on

ice for 15 min. After the addition of detergent, lysates were centrifuged at14,000 � g for 30 s at 4jC. The pellets were resuspended in complete lysisbuffer and vortexed vigorously. After incubation on ice for 30 min and

centrifugation at 14,000 � g for 10 min at 4jC, supernatants were collectedand protein concentration was determined by DC Protein assay (Bio-Rad).Assay of NF-KB (p50) DNA binding activity. The activity of the p50

subunit of NF-nB was analyzed by a 96-well colorimetric ELISA-based

procedure using a trans-AM NF-nB (p50) Transcription Factor Assay Kit(Active Motif North America) according to the manufacturer’s instructions.In brief, pure p50 protein (3 nmol/L, Active Motif North America) was

incubated with various concentrations of organoselenium compounds at

ambient temperature for 30 min. The incubation mixtures or nuclearextracts from cells were then added into a 96-well plate that had been

coated with immobilized oligonucleotide containing a consensus-binding

site for NF-nB. After 1 h of incubation with agitation, wells were washedthrice with washing buffer [100 mmol/L PBS (pH 7.5), 500 mmol/L NaCl,and 1% Tween 20] and incubated with p50 antibody (dilution 1:1,000 in

antibody binding buffer) for 1 h at room temperature. The wells were

washed thrice more with washing buffer before incubation with diluted

Figure 1. Schematic representation of our working hypothesis.

Cancer Research

Cancer Res 2007; 67: (21). November 1, 2007 10476 www.aacrjournals.org



horseradish peroxidase–conjugated antibody (dilution 1:1,000 in antibody

binding buffer) for 1 h. Wells were further washed thrice before the addition

of 100 AL of developing solution. After 5 min of incubation, the reaction wasstopped by the addition of 100-AL stop solution and quantified by

measuring the absorbance at 450 nm using a microplate spectrophotometer

(SPECTRAmax PLUS384, Molecular Devices).The absorbance is proportional to the amount of NF-nB (p50) bound to

the DNA. In each set of experiments, the total binding that occurred

without added BSC or p-XSC was normalized to 100%. Inhibition of NF-nB(p50) binding by BSC and p-XSC was analyzed by plotting the percentage ofDNA binding against the concentration of organoselenium compound using

Sigmaplot for Windows version 10.0. The inhibition was quantified using

the four-parameter Hill equation in which B is the amount of DNA binding;

Bmax, the DNA binding without inhibitor; Bmin, the amount of DNA bindingat infinite inhibitor concentration; C , the concentration of inhibitor; IC50,

the concentration required to achieve a 50% inhibition of DNA binding;

and n, the Hill slope.

B ¼ Bmin þ ðBmax �BminÞ1þ ðIC50=CÞn

Detection of BSC-modified p50 by mass spectrometry. Therecombinant NF-nB (p50) protein (2 Amol/L) was incubated with BSC

(1.67 mmol/L in DMSO) for 30 min at 37jC. Immediately, the reactionmixture was treated with urea (4 mmol/L) and N-ethylmaleimide (0.13

mmol/L) to avoid the rearrangement reaction with the other cysteine

residues that had not been modified, and then incubated for an additional

30 min at 37jC. The resulting solution was dialyzed for 1 h against

20 mmol/L Tris-HCl (pH 7.5) in a Slide-a-Lyzer dialysis cassette (7 kDamolecular mass cutoff); this was followed by digestion with Trypsin Gold

(Promega) overnight at 37jC with a protein/trypsin ratio of 10:1. The

mixture was separated by gradient elution through a Michrom Magic C185-Am 100-A, 0.1 � 150-mm column, with a flow rate of 900 nL/min,

provided by an Eksigent Nanoflow LC system. Separation of peptides was

achieved by a 30-min gradient of increasing acetonitrile in 0.1% TFA (buffer

A: 1% acetonitrile, 0.1% TFA; buffer B: 90% acetonitrile, 0.1% TFA; 0–1 min,0% B; 1–2 min, 0% ! 5% buffer B; 2–5 min, 5% buffer B; 5–30 min, 5% !55% buffer B; 30–30.5 min, 50% ! 0% buffer B). The nanoflow-LC eluent

was mixed with an equal volume of matrix-assisted laser desorption/

ionization (MALDI) matrix [2 mg/mL a-cyano-4-hydroxycinnamic acid,2 mg/mL (NH4)H2PO4, 0.1% TFA, 80% acetonitrile] and spotted every 20 s

(300 nL eluant + 300 nL matrix solution) directly onto the MALDI target

plate using a Probot spotting robot (LC-Packings/Dionex).

After drying, the samples were then analyzed using an AppliedBiosystems 4700 Proteomics Analyzer with TOF/TOF ion optics. Both

mass spectrometry (MS) and MS/MS data were acquired with a Nd:YAG

laser with 200 Hz repetition rate, accumulating 1,000 laser shots for eachMS spectrum in positive ion reflector mode at a laser attenuation setting of

2,600, and between 2,000 and 5,000 laser shots at a laser attenuation setting

of 3,650 for MS/MS spectra (accumulation continued until the signal-to-

noise ratio for at least six fragment peaks exceeded S/N 60 or 5,000 lasershots had been fired). Both MS and MS/MS data were acquired using a

Figure 2. Concentration-dependent effects on NF-nB (p50) DNA binding by synthetic organoselenium compounds: SCC 1483 cells + p-XSC (A), H460 cells + p -XSC(B), SCC 1483 cells + BSC (C ), and H460 cells + BSC (D ). Points, average of three experiments; bars, SD.

Modification of p50 by Organoselenium Compounds




recently updated instrument default calibration. Peak lists were submittedto Mascot version 2.0 using GPS Explorer software (ABI) as the front end

for data submission, and then manually interpreted using in silico

generated theoretical fragmentation data produced with the ABI Ion

Fragmentation Calculator software, which is part of the 4000 SeriesExplorer program suite.

Detection of p-XSC–bound p50 by radiotracer experiment. NF-nB(p50; 400 nmol/L) was incubated with 12 Amol/L p-[14C]XSC (0.15 ACi,100 Ci/mol) in 20 mmol/L Tris-HCl (pH 7.0), 0.2 mol/L NaCl, 2 mmol/LMgCl2, and 10% glycerol for 20 min at room temperature. The reaction

mixture was applied to a Superdex 75 HR 10/30 column (GE Healthcare)

and was eluted with 20 mmol/L sodium phosphate (pH 7.5), 150 mmol/L

NaCl at 0.5 mL/min. The 1-mL fractions were mixed with 10-mL Picofluor40 (Perkin-Elmer) and the radioactivity was measured with a liquid

scintillation counter.

Molecular modeling of the modification of p50 by BSC or p-XSC.Three-dimensional model structures of either BSC or p-XSC adducts were

positioned in the DNA binding region of the p50 subunit (residues 59–71).

In the case of both adducts, a novel residue consisting of the target cysteine

derivatized with the ligand (BSC or p-XSC) of interest by a selenium-sulfurlinkage was introduced into the parent structure (PDB code 1NFK; ref. 27).

CHARMM parameterization of the novel residue was done by semiempirical

quantum mechanics calculations using Parametric Model Number 3 (PM3)

as implemented by the program GAMESS (28). The composite derivatizedstructure was then subjected to 10,000 rounds of energy minimization in the

NPT ensemble using the program NAMD (29). Model manipulation andimage rendering were done by means of Chimera molecular modeling

software (30).

Results

Inhibition of NF-KB (p50) DNA binding activity by p-XSCand BSC. Although p-XSC was reported to reduce the bindingactivity of NF-nB in colon cancer cells (HCT-116) using electro-phoretic mobility shift assay (5), it is not known whetherselenocyanate-containing compounds including p-XSC and BSCcould inhibit NF-nB DNA binding in other cancer cells. Therefore,both TNF-a–activated oral squamous cell carcinoma SCC 1483 andnon–small cell lung cancer NCI-H460 cells were used to evaluatethe inhibitory effects of p-XSC or BSC on NF-nB (p50) protein byELISA. p-XSC clearly inhibited the NF-nB (p50) DNA binding inboth cell lines in a dose-dependent manner (Fig. 2A and B). Incontrast, the binding of NF-nB (p50) protein was enhanced by BSCat lower dose (V25 Amol/L), but at higher doses (z50 Amol/L) adecrease in binding was observed (Fig. 2C and D).Using a recombinant p50 protein, we determined whether the

inhibition could be due to direct binding of NF-nB (p50) protein toselenocyanates (Fig. 3A and B). p-XSC and BSC were found to

Figure 3. Inhibition of recombinant NF-nB (p50) DNA binding by the synthetic organoselenium compounds p-XSC (A and C ) and BSC (B and D ). Reactions werecarried out without reducing agents (.) or with 5 mmol/L DTT (n). The DTT was added during (A and B) or after (C and D) the incubation of the organoseleniumcompounds with NF-nB (p50). Points, average of two experiments (C and D ) or three experiments (A and B ; conducted in the absence of DTT); bars, SD.

Cancer Research




inhibit p50 DNA binding in a dose-dependent manner in theabsence of a reducing agent (DTT), with IC50 of 0.50 F 0.03 and2.20 F 0.12 Amol/L, respectively. The inhibitory effects were timedependent and reached a maximum between 20 and 40 min byboth p-XSC (1 Amol/L) and BSC (20 Amol/L; data not shown). Thisdose-dependent inhibition was eliminated when DTT (5 mmol/L)was present in the reaction mixture. The addition of DTT 30 minafter the incubation of selenocyanates and p50 protein and thenincubation for another 20 min attenuated the inhibition (Fig. 3Cand D). In addition, the inhibitory effects of p-XSC (1 Amol/L) andBSC (20 Amol/L) were partially reversed in a dose-dependentmanner by post-addition of DTT (Fig. 4A and B). Collectively, theseresults suggest that the formation of -Se-S- bonds is responsible forthe inhibition of the DNA binding activities of NF-nB (p50) protein.The inhibitory effects by either p-XSC or BSC were unchanged inthe presence or absence of physiologic levels of reducedglutathione (GSH; 1 mmol/L; Fig. 4C and D).Detection of BSC-induced modification of p50 by MS. The

fact that the addition of DTT can restore the DNA binding activityof NF-nB (p50) protein after treatment with BSC or p-XSC suggeststhat covalent interactions with thiol groups of cysteine residues inp50 were involved in the interaction of p50 with selenocyanates.Therefore, we attempted to characterize which residue of p50 was

chemically modified by organoselenocyanates. Thus, we selected,for practical purpose of obtaining interpretable mass spectrainformation, the monofunctional selenocyanate BSC. Figure 5Ashows the MALDI-time of flight (TOF) mass spectra of a high-performance liquid chromatography fraction obtained by thetryptic digestion of BSC-modified p50.A peptide from tryptic digestion of BSC treated p50 mixture with

an average mass of 1,944.6 was found to be consistent with thetheoretical mass of the YVCEGPSHGGLPGASSEK (monoisotopicmass of 1,774.8) peptide bearing a benzylselenyl (Se80) moiety; inaddition, another peptide with an average mass of 1,942.6 thatcorresponded to the same peptide bearing a benzylselenyl (Se78)moiety is also present (Fig. 5A). Neither m/z values were observedin the trypsin-digested p50 mixture without BSC treatment. Theobserved mass spectrum isotope cluster pattern is consistent withthe isotope distribution pattern of the presence of Se-containingpeptides. This peptide (m/z = 1,944.6) was subjected to MS/MSfragmentation to precisely map the cysteine residue that wascovalently bound to a benzyl selenyl moiety. The results of tandemmass spectrum support the formation of a selenium-sulfur bondbetween a benzyl selenyl moiety and Cys62 of the peptide (Fig. 5B).The fragment with a mass of 1,854.0 is consistent with the peptidewithout a benzyl moiety, and the fragment with a mass of 1,740.7 is

Figure 4. A and B, effect of DTT on the inhibition of NF-nB (p50) DNA binding by p-XSC or BSC, respectively. C and D, effect of GSH (1 mmol/L) on the inhibitoryeffects of p -XSC and BSC on NF-nB (p50) DNA binding, respectively. n, with 1 mmol/L GSH; ., without GSH. Points, average of two independent experiments.





Figure 5. Detection of BSC-modified p50 proteinby MALDI-TOF and MS/MS fragmentation.A, MALDI-TOF mass spectrum of BSC-boundpeptide corresponding to the peptideYVCEGPSHGGLPGASSEK. B, MS/MS spectrum ofthe peptide (1,944.6 Da) with BSC-modified Cys62.C, MALDI-TOF mass spectrum of BSC-bound peptidecorresponding to the peptide MTEACIR.

Cancer Research




consistent with the peptide without a selenium-sulfur–containing(C7H8SeS-) moiety. Fragments in the y ion series (y15, y14, y13, y11,y10, and y7) and b11 and a12 are also in agreement with theformation of a selenium-sulfur bond between a benzyl moiety andCys62 in the peptide. The tandem mass spectrum is entirelyconsistent not only with the expected peptide sequence but alsowith the modification of thiol group of Cys62 by BSC, and providesfor the first time a chemical evidence that Cys62 of NF-nB (p50)protein was modified by BSC through the formation of a selenium-sulfur linkage.Another peptide, present with a mass of 993.3, is also found to be

consistent with the theoretical mass of MTEACIR peptide bearinga benzylselenyl (Se80) adduct; in addition, the whole mass clusteris also consistent with the isotope effects of the presence ofSe-containing peptides; however, we were unable to obtain satis-factory tandem mass spectrum for unequivocal interpretation.Detection of p-XSC–bound p50 by radiotracer experiment.

The potential covalent bond between p50 and p-XSC was probedby incubation of the protein with p-[14C]XSC for 20 min. When thereaction mixture was fractionated by gel filtration, a radioactivepeak eluted at the elution volume expected for p50. In two separateexperiments, the amount of radioactivity that coeluted with theprotein indicated that there are 1.6 p-XSC molecules bound toeach p50.Molecular modeling of the modification of p50 by BSC or

p-XSC. The thiol group of Cys62 is important in DNA recognitionby p50; indeed, single-site substitution of this residue with a serine(Cys62Ser mutant) inhibits DNA binding (31). In the crystalstructure of p50, the DNA is in contact with both domains of p50and 12 of the 18 phosphates are involved in hydrogen bondinginteractions with the protein, with the six free phosphates residingat the ends of the DNA. Tyr60 and Cys62 interact with the sugar andphosphate backbones, respectively, consistent with the photo-chemical cross-linking experiments (32) and the observed sensi-tivity of DNA binding to the oxidation of Cys62 (Fig. 6A ; ref. 33).To provide further structural evidence that the organoselenium

compounds can react with Cys62 and inhibit DNA binding,molecular models of both ligands (BSC and p-XSC) positioned inthe DNA binding pocket of the p50 were constructed through theintroduction of a novel derivatized residue. Following a number ofrounds of whole molecule energy minimization, both adducts seemto be accommodated within the area of interest with limitedperturbation to the surrounding protein structure (Fig. 6B and C).Along with the proposed covalent linkage, both ligands could bestabilized by aromatic moiety (k-cloud)–induced van der Waalsforces and electrostatic interactions contributed by other aminoacid residues in the DNA binding pocket. Available k-stackinginteractions between the aromatic components of each ligand andTyr60 in the binding pocket can provide either T-shaped or off-center parallel displaced stabilized conformations (34). The DNAbinding pocket contains a number of charged amino acids, Glu63,Arg57, and Lys244, all of which may contribute to intermolecularelectrostatic interactions. This is particularly the case with Lys244

and selenium substituents of the para-substituted ligand (p-XSC),both of which contribute significant areas of charge to the overallelectrostatic potential map.

Discussions

In this report, we provide data supporting our novel hypothesisand show for the first time that the chemopreventive selenocya-

nates, BSC and p-XSC, can inhibit the binding of NF-nB (p50)protein to its oligonucleotide substrate through the formation of-Se-S- bonds between BSC or p-XSC and Cys62 in the DNA bindingdomain of p50 protein. However, the observation that DTT onlypartially reversed the inhibition suggests that the -Se-S- bonds aresomewhat stable. It is highly significant that physiologic level ofGSH (1 mmol/L) did not alter the inhibitory effects of BSC or

Figure 6. Computer-based modeling of the p50 protein monomer with or withoutmodification by organoselenocyanates. A, a drawing of DNA binding region ofp50. B, a drawing of BSC bound to Cys62 of p50 through a Se-S bond. C, adrawing of p-XSC bound to Cys62 of p50 through a Se-S bond. Both ligandresulted moieties are ball-and-stick representations with carbon atoms in gray,selenium atom in aqua, oxygen atom in red, nitrogen atom in purple, and sulfuratom in yellow.





p-XSC on p50, suggesting that both BSC and p-XSC might alsoinhibit p50 in vivo , and this needs to be explored in future studies.Both BSC and p-XSC inhibit DNA binding of NF-nB (p50) protein inTNF-a–stimulated oral cancer SCC 1483 and lung cancer non–small cell lung cancer NCI-H460 cells. Numerous previous studiesalso showed the inhibition of NF-nB in other cancer cells byselenium-containing compounds, such as p -XSC in humancolorectal adenocarcinoma HCT-116 cells (5), methylseleninic acidin fibrosarcoma HT1080 cells (35), and selenite in nuclear extractsfrom human Jurkat T cells and human lung adenocarcinoma NCI-H441 cells (36).Previous studies that examined numerous inhibitors of NF-nB

showed that inhibition of NF-nB activation can occur at severallevels: (a) blockage of the extracellular stimulations; (b) interfer-ence with a cytoplasmic step in the NF-nB activation pathway byblockage of a specific component of the cascade; and (c) blockageof the nuclear activity of NF-nB by inhibition of translocation to thenucleus or its binding to DNA (37). Among those, the directinhibition of NF-nB DNA binding by interfering with the DNAbinding region in the NF-nB was suggested to be a more effectiveapproach to design specific inhibitors (38). We showed that BSCand p-XSC inhibited the DNA binding of p50 protein throughmodifications in the DNA binding region in the NF-nB (p50)in vitro , and this may, in part, account for the mechanisms bywhich these compounds inhibit DNA binding in cell cultures. Atlower doses (V25 Amol/L), BSC unexpectedly increased the DNAbinding of NF-nB (p50) protein in both cell lines. However,enhancement of DNA binding does not necessarily indicate thatthe DNA binding step is specifically altered (37), but it couldinstead arise from enhancement of extracellular signals byselenium-containing compounds, differences between cell lines,or interaction with other components involved in the NF-nBactivation pathway (39, 40). In fact, we did not rule out thepossibility that low level of p-XSC (V1 Amol/L) may also increasethe DNA binding of NF-nB (as seen in Fig. 2A ) throughselenoproteins to control cellular redox potential, which has adifferent effect on the NF-nB activity than at higher level where thecovalent modification mechanisms take place.It is important to determine in future studies whether the

covalent modification of p50 by selenocyanates is responsible forthe inhibition observed in lung and oral cancer cell lines used here.To precisely map the particular cysteine residue that was

modified by selenocyanates, we incubated NF-nB (p50) proteinwith the monofunctional selenocyanate BSC, digested the mixturewith trypsin, and analyzed it by both MALDI-TOF and MS/MSfragmentation. We show for the first time that the Cys62 residue inthe active site of NF-nB (p50) protein was modified by BSC through

the formation of a -Se-S- bond. The thiol group of Cys62 is critical inthe DNA recognition by p50 subunit (31, 41–44) and is the onlycysteine, among the seven cysteines present in p50, located in theDNA recognition region (44). The formation of -Se-S- bond may bedue to an SN2 reaction by a nucleophilic attack of the thiol group ofCys62 at the electrophilic selenium moiety of the selenocyanatewith displacement of the cyano group; however, an SN1 reactionmechanism cannot be excluded.Using a radiotracer method, we showed that p-XSC also binds to

p50 in vitro . To further provide additional support of themodification of p50 protein by selenocyanates, we used a computermodeling approach to examine the potential effect of the bindingof both BSC and p-XSC at Cys62 on DNA binding activity. Thisapproach revealed that the modification of p50 protein by BSC orp-XSC could hinder its DNA substrate to enter its DNA bindingregion. Our results provide structural evidence showing that BSCand p-XSC exert chemopreventive or therapeutic activity, at least inpart, through inhibition of the DNA binding of NF-nB (p50) proteinby covalent modification of Cys62. This computer-based modelmay be used as a tool in the design of more effective cancer-chemopreventive selenium compounds.The rational strategy for the development of chemopreventive or

therapeutic agents to inhibit DNA binding of p50 protein has beendescribed (45, 46). In oral squamous cell carcinoma andnasopharyngeal carcinoma, the dominant NF-nB dimer is thep50/p50 homodimer, which was found to be highly expressed in allmalignant oral squamous cell carcinoma tissues but only at amoderate level in precancerous lesions. Because the composition ofNF-nB regulates transcriptional specificity, a p50/p50 homodimeris likely to mediate NF-nB activity to provide a better survival andproliferative advantage to the tumor cells than the p50/p65 form inoral carcinogenesis. In addition, deletion of the NF-nB p50 subunitresults in the inhibition of tumorigenesis in mice given theperoxisome proliferators ciprofibrate and Wy-14,643 (47). There-fore, inhibition of NF-nB (p50) DNA binding may represent aninnovative chemopreventive strategy (48, 49). Our results provide aplausible approach to develop more effective chemopreventiveagents based on their binding efficiency to NF-nB.

Acknowledgments

Received 7/5/2007; revised 7/26/2007; accepted 8/3/2007.Grant support: NCI grants R01-CA100924 and P01-CA70972.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 accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.We thank Anne Stanley of the Proteomics/Mass Spectrometry Core Facility, and the

Organic Synthesis Facility of the Section of Research Resources, Penn State College ofMedicine. We also thank Dr. Peter G. Sacks for providing us SCC 1483 cells.

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