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Nutrition and Cancer, 61(3), 322–331Copyright © 2009, Taylor & Francis Group, LLCISSN: 0163-5581 print / 1532-7914 onlineDOI: 10.1080/01635580802521338

Sodium Selenite Increases the Activity of the TumorSuppressor Protein, PTEN, in DU-145 Prostate Cancer Cells

Margareta BerggrenDepartment of Nutritional Sciences, College of Agriculture and Life Sciences, The Universityof Arizona, Tucson and Arizona Cancer Center, Tucson, Arizona, USA

Sivanandane SittadjodyDepartment of Nutritional Sciences, College of Agriculture and Life Sciences, The Universityof Arizona, Tucson, Arizona, USA

Zuohe Song and Jean-Louis SamiraDepartment of Nutritional Sciences, College of Agriculture and Life Sciences, The Universityof Arizona, Tucson and Arizona Cancer Center, Tucson, Arizona, USA

Randy BurdDepartment of Nutritional Sciences, College of Agriculture and Life Sciences, The Universityof Arizona, Tucson, Arizona, USA

Emmanuelle J. MeuilletDepartment of Nutritional Sciences, College of Agriculture and Life Sciences, and the Departmentof Molecular and Cellular Biology, The University of Arizona, Tucson; Arizona Cancer Center, Tucson,Arizona, USA

Epidemiological and clinical data suggest that selenium mayprevent prostate cancer; however, the cellular effects of seleniumin malignant prostate cells are not well understood. We previouslyreported that the activity of the tumor suppressor PTEN is modu-lated by thioredoxin (Trx) in a RedOx-dependent manner. In thisstudy, we demonstrated that the activity of Trx reductase (TR)is increased by sevenfold in the human prostate cancer cell line,DU-145, after 5 days of sodium selenite (Se) treatment. The treat-ment of DU-145 cells with increasing concentrations of Se inducedan increase in PTEN lipid phosphatase activity by twofold, whichcorrelated with a decrease in phospho-ser473-Akt, and an increasein phospho-Ser370-PTEN levels. Se also increased casein kinase-2(CK2) activity; and the use of apigenin, an inhibitor of CK2, re-vealed that the regulation of the tumor suppressor PTEN by Se maybe achieved via both the Trx-TR system and the RedOx control ofthe kinase involved in the regulation of PTEN activity.

Submitted 25 January 2008; accepted in final form 3 September2008.

Address correspondence to Emmanuelle J. Meuillet, The Universityof Arizona, Department of Nutritional Sciences, College of Agricultureand Life Sciences, 1177 East 4th Street, P.O. Box 210038, Tucson,AZ 85721-0038. Phone: 520-626-5794. Fax: (520) 626-4455. E-mail:[email protected]

INTRODUCTIONSelenium (Se) is an essential dietary nutrient for all mam-

malian species including humans (1). The mechanism of actionof Se compounds, either via a prooxidant pathway (as seenin cytotoxicity and apoptosis) or an antioxidant pathway (asproposed in cancer chemoprevention), is still unclear but isnonetheless intriguing. Several studies have shown an effectof seleno-compounds on phospho-Akt in human prostate can-cer cells (2–5). However, the mechanisms for such effects onthe phosphatidylinositol-3 kinase (PtdIns-3 K)/Akt cell survivalpathway remain unclear.

Thioredoxin (Trx) is a 12-kDa protein thiol-disulfide oxi-doreductase that, in mammalian cells, has a variety of biologicalfunctions related to cell proliferation and apoptosis (6). The ox-idized form (Trx-S2) contains a disulfide bridge in the active sitethat is bioreduced to a dithiol by NADPH and the flavoproteinThioredoxin reductase (TR) (7). Sodium selenite increases theactivity of TR and stabilizes TR mRNA, leading to an increasein TR expression in several cancer cell lines including MCF-7breast, HT-29 colon, and A549 lung cancer cells (8). Trx hasbeen implicated in many cellular functions such as DNA syn-thesis as electron donors (9), RedOx regulation of transcriptionfactors such as NF-κB (10) and AP1 (11) as well as the mod-ulating the activity of several proteins including PK C (12),ASK1 (13) and PTEN (14,15). In fact, we recently reported that

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SELENIUM INCREASES PTEN ACTIVITY 323

Trx is a physiological inhibitor of the tumor suppressor pro-tein deleted on chromosome 10/mutated in multiple advancedcancer (PTEN/MMAC1phosphatase and tensin homolog), thephosphatidylinositol (PtdIns)-3-phosphate phosphatase that reg-ulates Akt activation in normal cells (14). In PTEN-deficientmouse embryo fibroblasts, constitutively elevated activity ofAkt together with decreased sensitivity to apoptosis suggests arole for PTEN as a negative regulator of cell survival.

PTEN is a 54 kDa protein, which exhibits a dual specificitytyrosine/serine/threonine and lipid phosphatase, but the mainsubstrate of PTEN, both in vitro and in living cells, is the lipidPtdIns-(3,4,5)-trisphosphate. PTEN is also a phosphoprotein,and this posttranscriptional modification of the protein plays animportant role in its stability (16). Specifically, the phosphory-lation of Ser380 and Thr382 has been shown to affect the half-lifeof PTEN in T lymphocytes (17). Thus, PTEN is phosphorylatedat several serine and Threonine residues in the C-terminal of theprotein, particularly in the PDZ (PSD95, Dlg and ZO1)-bindingmotif. Interestingly, the phosphorylation of the PTEN tail resultsin an inhibition of PTEN activity, at least in part, by prevent-ing its interaction with PDZ domain-containing proteins such asMAGI-2. Moreover, the phosphorylation on Thr366 by glycogensynthase kinase 3 (GSK3) was recently shown to destabilizePTEN (18). Taken together, PTEN activity may be regulated viaits phosphorylation on the C-terminus and its association withPDZ binding partners.

The activation and the regulation of the kinases involved inPTEN phosphorylation and their effects on PTEN activity inprostate cancer cells remain an important topic to be investi-gated. Our laboratory is interested in understanding the possiblemechanisms for PTEN regulation in normal and cancer cells. Wehave shown that the small RedOx protein, Trx, inhibits PTENactivity in MCF-7 breast cancer cells (14,15). Trx, by bindingdirectly to the C-terminal part of PTEN, inhibits PTEN lipidphosphatase activity in vitro and in cells. The interaction be-tween Trx and PTEN is dependent on the RedOx status of Trxin that the interaction is observed only under reducing conditions(14). Our observations strongly suggest that Trx regulates PTENactivity in the cells and that this interaction may partially clarifythe antiapoptotic functions of Trx in the cells (14). Therefore,in this study, we have investigated the effects of the endogenousmodulation of the Trx-TR RedOx system using sodium seleniteand tested our hypothesis that PTEN activity can be modulatedvia the Trx system. We show that sodium selenite increases theactivity of the tumor suppressor PTEN in prostate cancer cells.Interestingly, at a similar concentration, Se increases the activ-ity of casein kinase-2 (CK2) involved in PTEN phosphorylation,thereby also regulating the tumor suppressor’s activity.

MATERIALS AND METHODS

MaterialsRabbit anti-PTEN, rabbit anti-phospho-(Ser380-Thr382/383)

PTEN, rabbit antiphospho-Ser473-Akt, rabbit total Akt, andrabbit PARP antibodies were purchased from Cell Signaling-

Technology (Danvers, MA). Rabbit anti-phospho-Ser370-PTENand rabbit antiphospho-Ser209-CK2 were obtained from NovusBiochemicals (Littletown, CO). Mouse anti-CK2 (clone mAb6D5) antibody was purchased from Calbiochem (La Jolla, CA).Pleurotin (i.e., NSC131233) was recently identified as an irre-versible inhibitor of TR with a Ki of 0.28 µM (19). Pleurotinand rabbit polyclonal antibodies against Trx were obtained as agift from Dr Garth Powis (MD Anderson Cancer Center, Hous-ton, TX). TR polyclonal antibodies were obtained from Upstate(Lake Placid, NY). Apigenin was obtained from Tocris Bio-science (Ellisville, MO). All other biochemicals were purchasedfrom Sigma (St. Louis, MO).

Cell Culture, Sodium Selenite, and Drug TreatmentHuman DU-145, LNCaP and PC-3 prostate cancer cell lines

were obtained from the American Type Culture Collection(Rockville, MD). The cells were maintained in bulk culturein Dulbecco’s modified eagle medium (DMEM) supplementedwith 10% fetal bovine serum (FBS). Cells were passaged us-ing 0.25% trypsin and 0.02% EDTA. The cells were confirmedto be mycoplasma free using an ELISA kit (Roche-BoehringerMannheim, Indianapolis, IN). Sodium selenite was dissolved indistilled water at the stock concentrations of 10 mM and addedevery other day at the indicated concentrations to the cell culturemedia for 5 days. For experiments using pleurotin or apigenin,cells were preincubated for 5 days with sodium selenite followedby 1 h of drug at the concentrations indicated.

PTEN Lipid Phosphatase ActivityLipid phosphatase activity of PTEN was measured as de-

scribed by Georgescu et al. (20). Briefly, PTEN was immuno-precipitated from DU-145 cells using anti-PTEN antibody, andthe lipid phosphatase activity was measured by detecting the re-lease of phosphate from the water soluble substrate diC8-PdtIns-(3,4,5)-trisphosphate (Echelon, Salt Lake City, UT). Briefly, af-ter immunoprecipitation, the protein A/G-agarose beads (SantaCruz Laboratories, Santa Cruz, CA) were washed twice: oncein a low-stringency buffer containing 20 mM HEPES (pH 7.7),50 mM NaCl, 0.1 mM EDTA, and 2.5 mM MgCl2, and oncein phosphatase assay buffer lacking the substrate. The reac-tion was incubated for 40 min at 37◦C and transferred to a96-well plate. The release of phosphate from the substrate wasmeasured in a colorimetric assay by using the Biomol Green

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Reagent (Biomol, Plymouth Meeting, PA) in accordance withthe instructions of the manufacturer. The absorbance at 650 nmwas recorded in an ELISA plate reader. A standard curve wasperformed in each assay, and the amount of free phosphate wascalculated from the standard curve line-fit data. Each assay wasperformed in triplicate, and the data are reported as nmoles ofphosphate liberated/mg of total protein.

Akt and CK2 Activity and Expression in Controland Selenite-Treated DU-145 Cancer Cells

Cells were grown in 6-well plates to 75% confluency inDMEM/10% heat-inactivated FBS, 4.5 g/l glucose, 100 U/ml

324 M. BERGGREN ET AL.

penicillin, and 100 µg/ml streptomycin in a 5% CO2 atmo-sphere. Following drug and/or selenite treatment, the culturemedia was aspirated and cells were lysed in RIPA lysis buffermade up of 0.05 M Tris, pH 7.4, 0.15 M NaCl, 1% Triton-X100, 0.1% sodium dodecyl sulfate (SDS), and 1% sodium deoxy-cholate supplemented with protease and phosphatase inhibitors.Thirty micrograms of the lysates were diluted into a ×4 reducingsample buffer and boiled for 5 min. Samples were then loadedon a 7.5% SDS-PAGE. Proteins were transferred to PVDF mem-branes, pre-incubated in blocking buffer [137 mM NaCl, 2.7 mMKCl, 897 µM CaCl2, 491 µM MgCl2, 3.44 mM Na2HPO4, 593mM KH2PO4 and 5% bovine serum albumin (BSA)], and in-cubated with the antibodies of interest. Immunoreactive bandswere detected using an anti-rabbit or anti-mouse coupled toperoxidase (Amersham, Arlington Heights, IL) and the Renais-sance chemilluminescence kit (NEN, Life Science Products,Inc., Boston, MA). Bands corresponding to phospho-CK2 werequantified using Eagle Eye software (BioRad, Richmond, CA)and Kodak X-Omat Blue XB (NEN, Life Science Products).Actin or CK2 were used as respective loading controls.

TR Activity and Expression in Controland Selenite-Treated DU-145 Cells

TR activity was measured as previously described (21). Celllysates were briefly incubated with adenosine 2′5′ di-phosphatebeads that were preswollen in 15 ml of water and rotated foran hour at 4◦C. The lysates mixed with the beads were rotatedfor 1 h at 4◦C. Lysates were washed twice in 1 ml 0.1M NaCl,50 mM HEPES, 5mM EDTA pH 7.6, and 50 µl of 1M NaCl.50mM HEPES and 5mM EDTA pH 7.6 was added to the beadsand rotated for 1 h at 4◦C. The supernatants obtained aftercentrifugation at 10,000 g for 5 min were used to measure TRactivity. TR activity was determined spectrophotometrically atroom temperature by the oxidation of NADPH at 339 nm in thepresence of 15 µM recombinant human Trx and 1 mg/ml bovineinsulin.

Measurement of Trx and Glutathione (GSH)Bioreduction Capacity in DU-145 Cells

To measure Trx and GSH bioreduction capacity in live cells,a modified method described by Biaglow et al. (22) was used.Briefly, DU-145 cells were treated with various concentrationsof sodium selenite for 5 days. In some experiments, prior tothe bioreduction assay cells were treated with 1 mM buthioninesulphoximine (BSO) for 2 h to deplete GSH. To perform theassay, cells were washed twice with incubation buffer [×1 PBScontaining 5 mM CaCl2, 10 mM KCl, and 5mM MgCl2]. Cellswere then incubated at 37◦C in the incubation buffer supple-mented with 5 mM glucose plus vehicle, 5 mM oxidized lipoate,or 5 mM hydroxyethyl disulfide (HEDS). Lipoate or HEDSfreely diffuses into the cells and are bioreduced primarily bythe Trx or glutathione systems, respectively (23–26). Followingbioreduction, the compounds diffuse back into the supernatant.

Samples of the supernatant were taken every 10 min for 1 hand added to 5 mM of 5,5′-dithiobisnitrobenzoic acid (DTNB),which reacts rapidly with the reduced compounds. Disappear-ance of DTNB was read on a spectrophotometer at 412 nm.This technique is able to measure the following reactions asillustrated below as described in Ref. 22:

1. Trx-(SH)2+ lipoate-(S-S) → Trx-(S-S) + lipoate-(SH)2

2. TR-(SH)2+ Trx-(S-S) → Trx-(SH)2+ TR-(S-S)3. TR-(S-S) + NADPH + H+ → TR-(SH)2+ NADP+

Apoptosis MeasurementApoptosis was ascertained microscopically using ethidium

bromide and acridine orange (27). Briefly, cells were seeded ina 30 mm plates at 1 × 105 cells for 24 h until they reachedconfluency. They were then treated with various concentrationsof sodium selenite for 5 days. Both the floating and adherent cellswere collected and centrifuged. They were then washed in PBS,pH 7.0, and resuspended in 1 ml of DMEM media. To detectapoptosis, 10 µl of cells were mixed with ethidium bromideand acridine orange solution (100 µg/ml each in DMEM) andvisualized for morphological changes. Acridine orange stainsthe nuclei green in viable cells, and the ethidium bromide isexcluded. However, in apoptotic or necrotic cells, the nuclei arestained red by the ethidium bromide, and the morphology ofthe nucleus easily determines whether the cell is apoptotic ornecrotic. A minimum count of 200 cells was counted and thepercentage of apoptotic cells determined. All experiments wereperformed in triplicate.

Statistical AnalysisData are presented as means ± SE. Significance was achieved

with P values referred as * for P < 0.01, ** for P < 0.005,and *** for P < 0.001.

RESULTS

Sodium Selenite Increases TR Activity in ProstateCancer Cells

TR is a known selenoprotein, and its activity can be regu-lated by seleno-compounds (8). We have measured TR activityin control and sodium selenite-treated prostate cancer cells. DU-145, LNCaP, and PC-3 cells lines were grown in the presenceof the indicated concentration of sodium selenite (Se) for 5days. Following this treatment, TR activity was measured asdescribed previously (8). TR activity was increased in a dose-dependent manner in the presence of increasing concentrationsof sodium selenite in DU-145 cells (Fig. 1A). In the presence of10 µM selenite, TR was inhibited (data not shown), potentiallysuggesting some level of toxicity. TR was not significantly mod-ulated in the presence of increasing concentrations of selenite inLNCaP and PC-3 cells. In fact, the level of enzyme activity waslow in LNCaP as already observed by Lim and collaborators


FIG. 1. Thioredoxin reductase activity is increased by sodium selenite in DU-145 prostate cancer cells. Prostate cancer cells [DU-145, PC-3, and LNCaP)]were treated for 5 days with 0.1 or 1.0 µM sodium selenite. TR activity wasmeasured as described in Materials and Methods (Panel A). TR was increasedsignificantly in a dose-dependent manner by sodium selenite in DU-145 cells.Bars represent means ± SE from two independent experiments performed intriplicate (n = 6). Significance was achieved: ***P < 0.001. Panel B repre-sents the expression of TR in DU-145, PC-3, and LNCaP prostate cancer cellsfollowing 0.1 and 1.0 µM sodium selenite treatment. Note the large increase inTR protein expression in DU-145 cells as compared to LNCaP and PC-3 on 1.0µM sodium selenite treatment.

(28) who reported a TR activity of 3 times lower than in PC-3cells. TR activity was also constitutively high in PC-3 (Fig. 1A).Figure 1B is a representative Western blot of TR protein expres-sion in DU-145, PC-3, and LNCaP cells after sodium selenitetreatment. TR protein expression was considerably increased inthe presence of increasing concentration of selenite in DU-145cells. However, LNCaP cells did not express detectable levels ofTR, and sodium selenite treatment had no significant effect onTR expression and activity (Figs. 1A and 1B). In PC-3 cells, al-though a marked increase in the expression of TR was observedfollowing 1 µM treatment, the measure of TR activity did notcorrelate with this increase. These results may reflect other Re-dOx systems being measured using this technique. Nonetheless,we show TR expression and activity was significantly increasedby sodium selenite in DU-145 cells. The increase in TR activ-ity has been shown to occur over a range of Se concentrationsfrom <0.01 to 10 µM (29), which encompasses the range of Seconcentrations found in human serum (between 1 and 5 µM)(30).

FIG. 2. Effect of sodium selenite on glutathione peroxidase activity. DU-145,PC-3, and LNCaP prostate cancer cells were treated with various concentra-tions of sodium selenite for 5 days at the indicated concentrations. The graphrepresents the activity of glutathione peroxidase as measured by the amount ofreduced NADPH produced/min/mg of total protein/sample. Note that the levelof glutathione peroxidase activity remains low in DU-145 and LNCaP cells ascompared to the level in PC-3 cells.

We next measured GSH peroxidase activity in these samecells. GSH peroxidase activity was increased by a factor of 8in PC-3 on increasing concentrations of sodium selenite (Fig.2). This observation could explain, at least in part, the highTR activity measured in Fig. 1A. In DU-145 and LNCaP cells,GSH peroxidase was slightly increased by sodium selenite in adose-dependent manner. However, this increase was consideredas negligible when compared to the increase observed in PC-3 cells. In all the 3 cell lines tested, higher concentrations ofsodium selenite (10 µM) were shown also to become toxic(data not shown). In conclusion, we showed that sodium seleniteincreases TR in DU-145 cells, and that increase is not due toGST peroxidase activity.

Sodium Selenite Increases the RedOx Rate of Trx inDU-145 Prostate Cancer Cells Independently of GSH

We have shown previously that the covalent interaction be-tween Trx and PTEN occurs via disulfur bridges between Cys-32of Trx and Cys-212 of the C2 domain of PTEN (14). BecauseTR activity was increased by sodium selenite, the bioreductioncapacity of the Trx system was determined using lipoate as asubstrate in DU-145 cells (Fig. 3A). The rate of lipoate biore-duction is known to be primarily coupled to Trx as describedpreviously (23–26). As observed with TR activity, increasingconcentrations of sodium selenite increases the bioreductionrate of lipoate in a dose-dependent manner in DU-145 cells (Fig.3A). Incubation of DU-145 cells with 0.1 or 1.0 µM sodiumselenite increased the rate of lipoate bioreduction by 36% and


FIG. 3. Effect of sodium selenite on Trx and GSH bioreduction capacity.DU-145 cells were treated with various concentrations of sodium selenite for 5days at the indicated concentrations. Panel A: DU-145 cells were treated withsodium selenite for 5 days and BSO at 1 mM for 1 h prior to the bioreductionassay. Cells were incubated with buffer containing 5 mM glucose plus vehicleor 5 mM oxidized lipoate. The rate of Trx bioreduction was determined using 6measurements over a 1-h period in duplicate treatment samples. Note that Trx’sbioreduction potential increased significantly on sodium selenite treatment. Barsrepresent means ± SE from three independent experiments performed in trip-licate. Significance was achieved: ***P < 0.001 when compared to respectivevehicles; #P < 0.01 when compared to lipoate reduction in control. Panel B:DU-145 cells were treated with sodium selenite for 5 days, and then cells wereincubated with buffer containing 5 mM glucose plus vehicle or 5mM HEDS.Note that there was no effect of selenite on GSH bioreduction. Bars representmeans ± SE from 3 independent experiments performed in duplicate. BSO,buthionine sulphoximine.

75%, respectively, as compared to control. Depletion of GSH byBSO did not affect the rate of lipoate bioreduction, confirmingthat GSH was not contributing to the reaction and that selenitespecifically acts on the Trx system. These data strongly suggestthat the RedOx rate of Trx is increased and that Trx may not be

available to bind PTEN. The specificity of the selenite was fur-ther confirmed using the HEDS in the bioreduction assay (Fig.3B). The bioreduction of HEDS, which is primarily mediated byGSH, was unaffected by selenite (Fig. 3B) and further indicatesthat sodium selenite specifically increased TR expression and itsactivity in DU-145 prostate cancer cells. Taken together, selen-ite increases TR expression and activity leading to an increasein the RedOx rate of Trx.

Sodium Selenite Increases PTEN Lipid Activityin DU-145 Cells

We have recently reported that Trx binds to PTEN in aRedOx-dependent manner and inhibits its lipid phosphatase ac-tivity (14,15). Moreover, TR is a selenium-dependent enzyme,and increasing concentrations of sodium selenite increase TRexpression and activity (Fig. 1 and Ref. 8). Consequently, we hy-pothesized that by increasing TR activity via increasing amountsof sodium selenite, the RedOx rate of Trx increases in the cells.Trx would then not be able to interact with PTEN and thuswould allow PTEN to be active. To test this hypothesis, DU-145 cells were treated with increasing amounts of sodium se-lenite, and PTEN lipid phosphatase activity was measured aspreviously described (20). Figure 4A summarizes the effects ofsodium selenite in DU-145 cells on PTEN lipid phosphatase ac-tivity using phosphatidyl-myo-inositol(3,4,5)-trisphosphate assubstrate. PTEN activity from these cells was significantly in-creased by sodium selenite in a dose-dependent manner. Therewas no change in PTEN protein expression in DU-145 cells inthe presence of selenite as compared to control cells (Fig. 4A,insert).

To test whether this interaction is also dependent of TR ac-tivity, we used pleurotin (i.e., NSC131233; Ref. 19), a knowninhibitor for TR, and measured PTEN lipid phosphatase ac-tivity in DU-145 cells (Fig. 4B). Cells were pretreated withsodium selenite (1 µM), pleurotin (5 µM), or the combination,and PTEN lipid phosphatase was measured in DU-145 cells.As shown in Fig. 4A, sodium selenite increased PTEN activity.Pleurotin decreased PTEN activity, and the combination wasable to restore PTEN activity to its basal activity as comparedto control nontreated cells. Therefore, sodium selenite regulatesPTEN activity, which may be in part via the activation of theTrx-TR system in DU-145 prostate cancer cells.

Sodium Selenite Inhibits Akt Phosphorylation and InducesApoptosis in Prostate Cancer Cells

We have shown that PTEN lipid phosphatase is activatedfollowing sodium selenite treatment in DU-145 prostate cancercells. Activation of PTEN has been correlated with a decreasein Akt activation in cells and an increase in apoptosis (31). Tocorrelate the activity of PTEN with that of Akt, we have treatedDU-145 cells with sodium selenite for 5 days and measuredsodium-selenite-induced apoptosis as well as the phosphory-lation status of Akt. The results are summarized in Fig. 5. A


FIG. 4. PTEN lipid phosphatase is increased by sodium selenite via the Trx-TR system. Panel A represents PTEN lipid phosphatase activity as measuredby the release of nmoles of phosphate from the substrate PtdIns-3,4,5-P3 asdescribed in Materials and Methods section. Note that increasing concentra-tions of sodium selenite stimulates PTEN lipid phosphatase activity in DU-145cells. Inset shows the total PTEN protein expression in the corresponding sam-ples as compared to internal control, β-actin. Panel B represents PTEN activityfollowing 1 µM sodium selenite, 5 µM pleurotin (an inhibitor of TR), or thecombination treatment. The combination treatment restores PTEN activity backto basal activity as compared to that in control cells. Bars represent means ± SEfrom three independent experiments performed in triplicate. Significance wasachieved: ***P < 0.001.

statistically significant increase in apoptosis was observed fol-lowing 1 µM and 5 µM sodium selenite treatments in DU-145cells (Fig. 5, Panel A). As already shown by Menter et al. (32),apoptosis was also confirmed by monitoring PARP cleavagefollowing 5 µM sodium selenite treatment in these cells (Fig.5, Panel B). A dose-dependent decrease in Akt phosphorylationon Ser473 was also observed in sodium-selenite-treated DU-145 cells (Fig. 5, insert, Panel A). These observations are inagreement with the increase in PTEN activity, correlating witha decrease in Akt phosphorylation, leading to the induction of

FIG. 5. Sodium selenite inhibits Akt activation and induces apoptosis andPARP cleavage in DU-145 prostate cancer cells. The insert of Panel A representsa typical Western blot for the inhibition of Akt phosphorylation by increasingconcentrations of sodium selenite (Se) in DU-145 cells. Briefly, cells werelysed, and phosphorylated Akt was detected using specific antiphospho-Ser473-Akt. The numbers at the bottom of the Western blot represent the ratio ofpAkt/β-actin. Akt phosphorylation was decreased in the presence of increasingconcentration of sodium selenite in DU-145 cells, correlating with the increase inPTEN lipid phosphatase activity. The graph in Panel A represents the inductionof apoptosis in DU-145 cells by selenite as measured by the morphological assaydescribed in Materials and Methods. Bars represent means ± SE from twoindependent experiments performed in triplicate. Significance was achieved:**P < 0.01. Panel B represents a typical Western blot for the cleavage of PARPby increasing concentrations of sodium selenite in DU-145 cells. Actin wasused a loading control. Note the lower band appearing in cells treated with 5µM selenite, which corresponds to cleaved PARP and induction of apoptosis(Panel A).

apoptosis in DU145 cells. In conclusion, we showed that sodiumselenite treatment caused a decrease in Akt phosphorylation anda significant increase in apoptosis in DU-145 cells.

Sodium Selenite Modulates PTEN Activity Also Via CaseinKinase 2 in DU-145 Prostate Cancer Cells

PTEN activity has been recently shown to be strongly reg-ulated by its phosphorylation status (16,17). Because sodiumselenite is also known to affect the expression as well as theactivity of several enzymes, we have also tested the effects of


FIG. 6. Sodium selenite modulated PTEN phosphorylation in DU-145 cells.DU-145 cells were treated with increasing concentrations of sodium selenitefor 5 days. Cells were lysed, and samples were probed for PTEN phosphory-lation using specific phospho-antibodies. Panel A represents a typical Westernblot for PTEN phosphorylation on residues Ser380/Thr382/383, PTEN phospho-rylation on Ser380, and on Ser370 as compared to loading control with β-actin.Note the decrease in PTEN phosphorylation with 5 µM of sodium seleniteon Ser380/Thr382/383 and a slight increase in PTEN phosphorylation on Ser370.Panel B represents the expression and the phosphorylation status of CK2 in-volved in the phosphorylation of PTEN following increasing concentrations ofsodium selenite in DU-145, LNCaP and PC-3 prostate cancer cells. Cells weretreated with 1 µM of Se. Note the high levels of CK2 and phospho-(Ser209)-CK2in DU-145 cells as compared to the other cell lines.

sodium selenite on PTEN phosphorylation status in DU-145cells using specific phosphoantibodies as well as the expres-sion of the kinases involved in the phosphorylation of PTEN.Phosphorylation on Ser380/Thr382/383 was slightly decreased ina dose-dependent manner, but phospho-Ser380-PTEN remainedunchanged in the presence of increasing concentrations of se-lenite (Fig. 6A). Phospho-Ser370-PTEN appeared to increaseslightly on selenium treatment. Therefore, we hypothesized thatthe activity of the kinases involved in the phosphorylation andregulation of PTEN activity may be also affected by sodiumselenite.

More specifically, we investigated the expression levels aswell as the activity of CK2, a kinase known to phosphory-late PTEN (33). DU-145 cells expressed high levels of CK2 ascompared to LNCaP and PC-3 cells (Fig. 6B). Moreover, theaddition of 1 µM of Se increased pSer209-CK2 levels only inDU-145 cells and reduced it in the other cell lines. The activ-ity of CK2, as measured by the ratio of phospho-Ser209-CK2over total CK2, was significantly increased in DU-145 follow-ing 1 µM of selenite treatment (Fig. 7A). Apigenin, a knowninhibitor of CK2 (34), was also shown to reduce phospho-Ser209-CK2 and confirmed its inhibitory effects on the kinase (Fig.7B). Apigenin-induced inhibition could partially be rescued by

FIG. 7. Sodium selenite increases the activity of CK2 in DU-145 cells. DU-145 cells were treated with different concentrations of sodium selenite for5 days. Cells were lysed, and samples were probed for phospho-Ser209CK2and total CK2 using specific phospho- and total antibodies. Panel A repre-sents the quantification of five separate experiments. Note the significant in-crease in the ratio phospho-Ser209CK2/Total CK2 following Se treatment inDU-145 cells. Bars represent means ± SE from five independent experimentsperformed in triplicate. Significance was achieved: ***P < 0.001. The insetin Panel B represents a typical Western blot in which phospho-Ser209-CK2,total CK2, and β-actin were probed following treatment of the cells with Sewith or without 60 µM of apigenin. The graph represents the quantificationof the typical Western blot shown in the inset. Note that apigenin counter-acts the effects of Se on CK2 phosphorylation. Panel C represents a typicalWestern blot from DU-145 cells that were pretreated with increasing con-centrations of Se and then incubated with apigenin or vehicle control. Spe-cific antiphospho-Ser209CK2, phospho-Ser473Akt, phospho-Ser370PTEN andphospho-Ser380/Thr382/383PTEN antibodies were used to probe the membrane.Note that Se induces CK2 and PTEN phosphorylation (Ser380/Thr382/383) anddecreases Akt phosphorylation. Se can also partially rescue the effects of api-genin on CK2.

1 µM of selenite. CK2 is one of the kinases that phosphory-lates PTEN and stabilizes the protein in an active form (33).Thus, to test the hypothesis that by increasing the activity of


CK2 sodium selenite could also modulate the activity of PTEN,DU-145 cells were pretreated with Se and then incubated withapigenin or vehicle control. Figure 7, Panel C, represents a rep-resentative Western blot correlating the phosphorylation statusof PTEN, AKT, and CK2. We show that phospho-Ser209-CK2remains high in presence of 1 µM selenite and that conse-quently, phospho-Ser370-PTEN is also high correlating with alow phosphor-Ser473-AKT. Apigenin decreased CK2 phospho-rylation, which in turn also decreased Ser380/Thr382/383-PTENphosphorylation as well as Ser473-Akt. Although these phospho-rylation sites on PTEN have been suggested to act to facilitatePTEN phosphorylation on other residues but do not affect thelipid phosphatase activity, they may play a role in selenite-mediated effects on the PTEN/Akt pathway in prostate cancercells.

DISCUSSIONEpidemiological studies have shown that there is a consis-

tent trend for populations residing in geographic areas that havelow sodium selenite levels in the soil to have a high mortalityfrom cancer. Other studies have show a lower risk for prostatecancer in individuals with selenium supplementation (35), andhigher serum selenium concentrations correlates with a mod-erately reduced risk of prostate cancer (36). These cancers in-clude cancers of the bladder, pancreas, thyroid, stomach, lung,esophagus, melanoma, head and neck tumors, and brain tumors.More specifically, several studies have suggested a protectiverole of selenium compounds against several cancers includingprostate cancer (reviewed in Ref. 37). In an attempt to bet-ter understand the protective effects of Se on prostate cancercells, we have studied the effects of sodium selenite on theregulation of the cell survival pathway, phosphatidylinositol-3kinase/Akt/PTEN. Previously, we have shown in cell culturesystems that human Trx binds in a RedOx-dependent manner toPTEN and inhibits its PtdIns-3-phosphatase activity, resultingin increased Akt activation (14). Molecular docking and site-specific mutation studies have shown that the binding of hTrx toPTEN occurs through a disulfide bond between the active siteCys32 of hTrx and Cys218 of the C2 domain of PTEN, leadingto steric interference by bound Trx of the catalytic site of PTENand of the C2 lipid membrane-binding domain (14). Se is anessential element in RedOx pathways and acts as an effectiveoxidant of bioreduced Trx and bioreduced TR, which dimin-ishes the Trx-TR pool. This important role of sodium selenitemay explain its inhibitory influence on cell growth and can-cer progression (32). Consequently, we have hypothesized thatby modulating the Trx-TR RedOx system endogenously withsodium selenite, PTEN activity can be regulated in prostatecancer cells. We show that sodium selenite decreases Akt phos-phorylation by increasing PTEN lipid phosphatase activity inDU-145 cells. Consistent with others, it has been shown thatsodium selenite induces a dose-dependent growth inhibition ofprostate cancer cells such as DU-145, PC-3, and LNCaP (32)

and an induction of apoptosis in selenite-treated prostate can-cer cells (32). Our observations are in agreement with thesestudies and confirm the inhibitory effects of sodium seleniteon Akt phosphorylation, leading to the increase in apoptosis.However, we show here that it is by releasing PTEN fromthe inhibitory binding of Trx that Se treatment leads to a de-crease in Akt phosphorylation and consequently to apoptosis inDU-145 cells.

In this study, we also showed that selenite increased the ac-tivity of a kinase involved in PTEN phosphorylation and therebyregulated the tumor suppressor’s activity in these cells. In theDU-145 prostate cancer cells, which express an active PTEN(38), we expected a bigger effect on Akt; however, Akt phospho-rylation was only slightly decreased in the presence of sodiumselenite. The reason for these observations may reflect the dualrole of CK2. CK2 is a ubiquitous serine/threonine kinase forwhich regulation and function is not well understood yet (39).Originally, CK2 was defined as a constitutively active protein ki-nase, although recently several reports have suggested that CK2acts as a regulated kinase participating in signal transduction.Indeed, CK2 was recently identified as the kinase that phospho-rylates the C-terminus of PTEN on residues Ser385 and Ser370

(33,40,41). CK2 has been demonstrated to also phosphorylatePTEN on Ser380 and thereby affects its stability (33). The phos-phorylation by CK2 hinders the cleavage of PTEN by caspases.On the other hand, CK2 also phosphorylates at Ser129 of Aktand upregulates Akt activity in Jurkat cells (42). In this study,we showed that CK2 activity is increased upon selenite treat-ment. The increase in phosphorylation of CK2 may translateitself in an increase in Ser129 phosphorylation of Akt, leadingto an increase in Akt activity and thereby counterbalancing theeffects of selenite on PTEN activity via the Trx-TR system. In-terestingly, using apigenin, an inhibitor for CK2, we showedthat the decrease in Akt phosphorylation due to the increase inPTEN activity can be enhanced. This suggests that upregulationof CK2 activity with selenite may contribute to the observed de-crease in the effects of selenite on Akt. Therefore, although weobserved an increase in phosphorylation status of CK2, overallthe effects of sodium selenite are counteracted by the fact thatCK2 directly phosphorylates Akt to increase its activity and itsphosphorylation status at Ser473.

Sodium selenite may modulate the activity of protein ki-nases and other enzymes through phosphorylation-independentmechanisms such as RedOx modification. In this study, we haveshown that sodium selenite increased CK2 activity in prostatecancer cells. The regulation of this enzyme is then affectingPTEN activity because it has been recently found to phospho-rylate PTEN and influence the stability and the activity of thetumor suppressor. Interestingly, recent studies that have usedapigenin have shown that the downregulation of CK2 in prostatecancer cells promotes apoptosis (43). These observations cor-relate well with our study presented in this article. Finally, al-though apigenin’s mechanism of action remains to be clearlyestablished and probably extends beyond the inhibition of CK2,


the novel finding that Se increases CK2 activity, which in turncan affect PTEN activity, is of interest for the treatment ofprostate cancer.

In conclusion, we have demonstrated that the regulation ofPTEN activity may be achieved at least in part via the modulationof the Trx-TR system by selenite as well as the RedOx propertiesof the kinases involved in PTEN phosphorylation. These resultsmay explain, at least in part, and contribute to understandingthe chemopreventive effects of sodium selenite against prostatecancers. However, they also suggest the importance of PTENstatus (expression as well as phosphorylation) in predicting thepossible therapeutic effects of sodium selenite.

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