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ENDOGENOUS THIOREDOXIN IS REQUIRED FOR REDOX-CYCLING OFANTHRACYCLINES AND P53-DEPENDENT APOPTOSIS IN CANCER CELLS

Dashnamoorthy Ravi, Harish Muniyappa and Kumuda C. Das*From the Department of Pathology and Arkansas Cancer Research Center, University of Arkansas

for Medical Sciences, 4301 West Markham, Slot # 845, Little Rock, AR 72205.Running Title: Thioredoxin enhances apoptosis

Address for Correspondence to: Kumuda C. Das, Department of Pathology, 4301 West Markham Slot#845, Little Rock, AR 72205, Tel. 501-526-4597; Fax. 501-526-4601; Email: [email protected]

Apoptosis is a major mechanism of cancer celldestruct ion by chemotherapy andradiotherapy. Anthracycline class of antitumordrugs undergo redox-cycling in living cellsproducing increased amounts of reactiveoxygen species and semiquinone radical, bothof which can cause DNA damage, andconsequently trigger apoptotic death of cancercells. We show here that MCF-7 cellsoverexpressing thioredoxin (Trx) were moreapoptotic in response to daunomycin. Trxoverexpression in MCF-7 cells increased thegeneration of superoxide anion (O2

-.) inanthracycline-treated cell extracts. Enhancedgeneration of O2

-. in response to daunomycin inTrx-overexpressing MCF-7 cells was inhibitedby DPIC, a general NADPH reductaseinhibitor, demonstrating that Trx providesreducing equivalents to a bioreductive enzymefor redox-cycling of daunomycin. Additionally,Trx increased p53 DNA binding and expressionin response to anthracyclines. MCF-7 cellsexpressing mutant redox-inactive Trx showeddecreased superoxide generation, apoptosis andp53 protein and DNA binding. In addition,down regulation of endogenous Trx expressionby siRNA resulted in decreased expression ofcaspase-7 and cleaved PARP expression inresponse to daunomycin. These results suggestthat endogenous Trx is required foranthracycline-mediated apoptosis of breastcancer cells. Taken together, our datademonstrate a novel pro-oxidant and pro-apoptotic role of Trx in anthracycline-mediatedapoptosis in anthracycline chemotherapy.

Thioredoxin (Trx) is a low molecular weightprotein (12 kD) that is widely distributed; Trx isfound within the cytoplasmic, membrane, extracellular and mitochondrial cellular fractions (1,2).

The Trx system includes Trx, and Trx reductase(TR) and peroxiredoxins. TR is an efficientprotein disulfide reductase that uses NADPH as asource of reducing equivalents. Besides being anantioxidant itself (3,4), Trx also plays an importantrole in regulating the expression of otherantioxidant gene such as manganese superoxidedismutase (5). Trx overexpression also enhancesthe expression of peroxiredoxin that could reduceperoxides to molecular oxygen and H2O (6). Trxhas been shown to regenerate oxidativelyinactivated proteins (7,8). In addition to its role asan antioxidant protein, Trx has been shown tohave growth promoting properties (9). In contrast,a recent study has demonstrated thatoverexpression of redox-active Trx could promotecell death via activation of caspase-8 (10).Additional studies have shown that TR is criticalfor cell death, and a Trx-dependent mechanism hasbeen suggested (11). Recent studies also indicatethat caspases, the executioner of cell death byapoptosis could be activated by Trx due to itsdisulfide reducing properties (12). Caspases arerich in cysteine motif that is required for itscatalytic activity. Therefore, oxidation couldinhibit caspase activity, which could be restoredby Trx system. (13). Furthermore, Trx has alsobeen shown to promote p53 DNA binding due toits reducing actions on DNA binding cysteinemotifs on p53 (14). Taken together, accumulatingevidence suggest that Trx is a multi-functionalprotein, which can participate in proliferation, aswell as cell death process. The antioxidative actionof Trx could be due to its MnSOD inducingproperties (5,15), as well as direct scavenging ofhydroxyl radicals or singlet oxygen.

Anthracycline class of anticancer drugs such asdoxorubicin or daunomycin has been shown toinduce p53-dependent apoptosis in cancer cells

JBC Papers in Press. Published on September 13, 2005 as Manuscript M507192200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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(16,17). Additionally, anthracyclines have alsobeen shown to cause DNA damage, whichincreases p53 expression (18,19). p53 is asequence-specific transcription factor, which caninduce pro-apoptotic or suppress anti-apoptoticgenes in response to DNA damage or irreparablecell cycle arrest (20). Phosphorylation of p53 onser-15 residue dissociates MDM2 and activatesp53 as a transcription factor, which binds tovarious p53-dependent genes resulting theiractivation or repression (20). While evaluating theprotective effect of Trx in daunomycin-inducedcytotoxicity we observed increased death of MCF-7 cells overexpressing Trx. Since Trx has beenshown to protect against oxidative stress anddaunomycin-mediated cytotoxicity has beenshown to be mediated in part by ROS, ourobservation was rather surprising and novel.Anthracyclines contain quinone moieties in theirstructure, which can undergo biochemicalreduction by one or two electrons catalyzed byflavoenzymes in the cell using NADPH as anelectron donor (21-23). This bio-reductive processgenerates semiquinone radical with concomitantproduction of superoxide anion (O2

.-). Thesemiquinone radical intercalates with the DNAresulting in DNA damage. The formation of O2

.-

is the beginning of a cascade that generateshydrogen peroxide and hydroxyl radicals,generally referred to as reactive oxygen species(24). In addition to various bioreductive enzymes,low molecular weight protein or non-protein thiolsmay also take part in the redox cycling process(25).

In the present investigation we report thatendogenous Trx is required for daunomycin-induced apoptosis of cancer cells. In addition, wealso demonstrate that Trx enhances the apoptoticdeath of cancer cells in response to daunomycindue to enhanced redox-cycling of anthracyclines.In contrast, cells that express redox-inactive Trx ortransfected with Trx siRNA show resistance toapoptosis.

Experimental ProcedureReagentsDaunomycin was purchased from Sigma(St.Louis, MO) and 5-iminodaunomycin wasobtained from National Cancer Institute (NCI).Anti-p53 (full length), anti-caspase 7, and anti-

caspase-1 antibodies were purchased from SantaCruz Biotech (Santa Cruz, CA); anti-p53 phosphoser-15, anti-caspase-6, anti-caspase-8 (recognizescleaved fragment) and anti-Poly ADP RibosePolymerase (PARP) antibodies were purchasedfrom Cell Signaling Technology (Beverly, MA).Anti-thioredoxin antibody was purchased fromResearch Diagnostics (Flanders, NJ).

Cell culture and adenovirus productionMCF7 cells were cultured in DMEM with 10%fetal bovine serum and 100 units ofpenicillin/streptomycin. MCF7 clones expressingTrx (Trx9), dominant negative redox inactive Trx(Serb4) and only vector (Vector) were generouscontribution of Dr. Garth Powis (Arizona CancerCenter, Tucson, Arizona) and have been described(26,27). MCF-7 clones were cultured in DMEMcontaining G418 (300 µg/ml). A549 cells wereobtained from ATCC and propagated in F12Kmedium. AdenoX system was obtained fromStratagene Corporation (La Jolla, California) andTrx or mutant Trx ORF (26) was cloned intopAdenoX vector. Recombinant virus was allowedto infect HEK293 cells for generation of viralparticles. For transfection, MCF-7 cells wereinfected with approximately 1X108 infectious units(per million cells) and after 48 hours proteinexpression was determined using ELISA.

RNA interference:p53, Trx and scrambled non-targeting siRNA werepurchased from Dharmacon RNA technologies(Lafayette, CO). For transfection, MCF-7 or A549cells were seeded in 35 mm dishes to obtain 20%confluency at the time of transfection. XtremesiRNA transfection reagent (Roche MolecularDiagnostics, Indianapolis, IN) was used totransfect siRNA to a final concentration of 100nM. Inhibition of gene expression by siRNA wasdetermined after 72 hours by western analysis.

Thioredoxin Activity Assay:Thioredoxin activity assay was performed asdescribed by Holmgren et al. (1). Briefly, thereaction mixture contained NADPH (200 µM),porcine insulin (80 µM; Sigma), and bovine TR(0.1 µM) in 0.05 M potassium phosphate buffer(pH 7.0) containing EDTA (1 mM) in a totalvolume of 0.5 ml. The reaction was started byaddition of bovine TR (0.1 µM). TRX activity


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was calculated as µmoles of NADPH oxidized perminute per mg protein at 25° C (Beckman DU800spectrophotometer).

TUNEL assay:Apoptotic cells were detected using In Situ CellDeath detection, POD kit (Roche MolecularBiochemicals, Indianapolis, Indiana). ApoptoticDNA strand breaks were identified by labeling 3’-OH termini with fluorescein-dUTP using Terminaldeoxy Transferase as per manufacturer’s protocol.Cells were allowed to adhere overnight inchambered glass slides (Nunc) to a final density of25,000 cells per well. Following treatment withappropriate concentration of drugs media wasremoved, and cells were washed twice with PBScontaining 1% BSA and fixed in 4%paraformaldehyde for 30 mins. Cells were thenpermeabilized with 0.1% Triton X-100 in 0.1%sodium citrate for 2 mins on ice, and washed twicewith PBS containing 1% BSA. The labelingreaction was performed using FITC labeled dUTPalong with other nucleotides by terminaldeoxynucleotidyl transferase for 60 mins in dark at37OC in humidified chamber. Further, cells werewashed with PBS-1% BSA, mounted and theincorporated fluorescein-dUTP was analyzedusing fluorescent microscope (Carl Zeiss,Axiovert M200).

Flow cytometry:Cells were treated with drugs for 48 hours.Floating cells were collected, and adherent cellswere washed with PBS and trypsinized. Floatingand adherent cells were pooled and centrifuged at500 X g for 3 mins. Cells were washed again withPBS containing 1% FBS and resuspended in 500ml of PBS followed by fixing in 7.5 ml of ice-coldethanol (70%), added drop wise while vortexingand stored in -200C overnight. After two washeswith PBS containing 1% FBS, cells wereresuspended in the same buffer and stained withPropidium Iodide 10 mg/ml (Sigma, St.Louis,USA) in the presence of RNase 250 mg/ml at 370Cfor 30 minutes in dark. Stained cells wereanalyzed using Epics Elite ESP Coulter, usingargon laser at 488 nm wavelength. Flowcytometric results were analyzed and apoptosiswas defined as ‘sub G1’ peak (6) using Multicyclesoftware.

Western blotting:Protein lystates were prepared using radioimmunoprecipitation assay (RIPA) buffercontaining 5% sodium deoxycholate, 1% SDS, 1%Igepal in PBS with protease inhibitors and proteinconcentration was determined using Bioradprotein assay reagent (Biorad). Equal amounts ofprotein was resolved on 10% SDS-polyacrylamide gel electrophoresis, andtransferred onto nitrocellulose membrane(Hybond-ECL, Amersham Pharmacia Biotech).The blot was treated with appropriate dilutions ofprimary antibody and visualized using eitherLumiglo (Cell Signaling Technology, Beverly,MA) or ECL plus system (Amersham PharmaciaBiotech, Piscataway, New Jersey) with appropriateHRP conjugated secondary antibody.

Determination of O2.- production by reduction of

ferricytochrome c:Superoxide production was measured assuperoxide dismutase (SOD) inhibitable reductionof ferricytochrome c (28). Cells were sonicated inpotassium phosphate buffer (0.05M, pH 7.8 plus 1mM EDTA), centrifuged and the supernatant wasused for the assay. To determine the O2

.-

generation in the cell lysate, the supernatant wasincubated with 10 µM drug and 10 µMcytochrome c, with or without 1unit of SOD todetermine SOD inhibitable rate. All reactions wereperformed in triplicate. The reduction offerricytochrome c was measured both in kineticand end point mode with path check on for 1hourduration at a wavelength 550 nM usingSpectramax 190 plate reader (Molecular Devices).Total protein was quantified using Bradfordprotein assay (Biorad).

In situ detection of O2.- by fluorescent probe

DiCarboxyFluorescein-DiAcetate (DCF-DA).Cells were grown in chambered glass slides(Nunc) to a final density of 25, 000 cells per well.Cells were pre-incubated with 20 µM DCF-DA(Sigma, St. Louis, Missouri) in 20 mM HEPES inPBS containing BSA, (5mg/ml) at 37O C for 30minutes followed by washing with PBS buffer,and the drug was added and observed for 300seconds in a Nikon laser confocal microscopeusing laser beam wavelength 488 nm analyzed byUltraview software (Perkin-Elmer).


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Clonogenic assayCells were trypsinzed and seeded to a final densityof 1 X 106 viable cells per100 mm dishes andallowed to attach overnight. Cells were thentreated with appropriate concentration of drugs for24 hours, trypsinized and seeded to a final densityof 500,000 viable cells per 100 mm dish. Viablecells were determined using Vicell counter(Beckman Coulter). After 14 days, the survivingcolonies were washed in PBS, fixed in 70%ethanol and stained using 0.1% crystal violet in90% ethanol. Colony containing a minimum of 30cells were counted using colony counting featurepresent in the quantity one software from Biorad.The assays were performed in triplicate and thedata was statistically analyzed using instat 2.01software.

Nuclear Extract preparationNuclear extract was prepared as describedpreviously (29). Briefly, cells were washed in icecold PBS and harvested in 2 ml of ice cold PBS bycentrifugation. Cell pellets were resuspended in400 µl of Buffer A (10 mM HEPES, pH 7.8, 10mM KCl, 0.1mM EDTA, 1 mM Dithiothreitol,1mM Phenylmethylsulfonyl fluoride (PMSF) and50 µg/ml of leupeptin and antipain by gentlepipetting. Cells were allowed to swell on ice for 15mins followed by addition of 25 ml 10% Nonidet-P40 and vortexed at full speed for 10 seconds. Thehomogenate was centrifuged for 30s at 14,000rpm. The nuclear pellet was resuspended in bufferC (20 mM HEPES, pH 7.8, 0.42M NaCl, 5 mMEDTA, 1mM Dithiothreitol, 1mM PMSF in 10%v/v glycerol) and tubes were rocked gently at 4OCfor 30 mins on a shaking platform. The extractswere then centrifuged at 14,000 rpm for 25 mins,and the supernatant was saved as nuclear extract in-700C for further experiments. Protein wasquantified using Bradford protein assay (Biorad).

Electrophoretic Mobility shift assay (EMSA)For the EMSA, the p53 consensus oligonucleotidew a s o b t a i n e d f r o m Genosys (5’-GGCATGTCCGGGCATGTCC-3’), and was endlabeled using T4 Poly nucleotide kinase (NewEngland Biolabs, Beverly, MA) and [γ-32P] ATP(Perkin Elmer, Boston, MA) in 10X kinase buffersupplied with the enzyme. Ten microgram of

nuclear protein was pre-incubated in 5µl of 5Xbinding buffer (20% glycerol, 5 mM MgCl2, 5 mMEDTA, 5 mM DTT, 500 mM NaCl, 50 mM TrisHCL, 0.4 mg/ml calf thymus DNA), 200 ng anti-p53 pAb 421 and 2 µg of poly dIdC for 15 minsfollowed by binding with labeled oligonucleotidefor 30 mins. The nuclear protein was separated byelectrophoresis using 4% native polyacrylamidegel and 0.25X of TBE (Tris-Borate- EDTA) asrunning buffer. Gels were dried and exposed toKodax Biomax X-ray film overnight.

Thioredoxin ELISACells were homogenized in 50 mM Tris.HCl (pH7.5) containing EDTA (1mM), PMSF (1 mM),leupeptin (20 µg/ml), and antipain (20 µg/ml).Lysates were microcentrifuged for 10 min at14000 rpm. The protein concentration in thesupernatant was measured using the Bradfordmethod (Biorad) with bovine serum albumin asstandard. ELISA was performed as previouslydescribed (30).

Statistical Analysis.All statistical analysis was performed using In Statsoftware program (V2.01, and 3.0). Allexperiments were repeated at least twice.

ResultsIncreased expression of redox-active Trxenhances apoptosis in response to daunomycin.To test whether Trx overexpression protects MCF-7 cells against daunomycin mediated apoptosis,we treated vector, Trx9 or serb4 cells (clones ofMCF-7 cells) with daunomycin and determinedapoptosis as described in ‘ExperimentalProcedures’. First we determined apoptosis usingTUNEL assay, which detects nicks in the DNAthat are generated during DNA damage andapoptosis. We expected to find more TUNEL-positive nuclei in Serb4 cells compared to Trx9cells in response to daunomycin, since previousstudies show that Trx could protect against thecytotoxic actions of other anticancer drugs such ascisplatin and bleomycin (31-33). However, to oursurprise we observed a higher number of TUNELpositive Trx9 cells (Fig 1A & B), in response todaunomycin. TUNEL assay detects nicksgenerated by drug-induced DNA damage andendonuclease activation during apoptosis. MCF-7


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cells lack caspase 3 and do not undergo classicalDNA laddering during apoptosis. Therefore, wefurther determined apoptotic cells as ‘Sub-G1’population by flow cytometry by propidiumiodide staining. Treatment of vector, Trx9 or serb4clones with daunomycin resulted in the appearanceof apoptotic cells (Fig. 1 C & D). Trx9 cellsshowed a higher percentage of apoptotic cells(23%) compared to vector or Serb4 cells (8%).These data also agree with our TUNEL data thatshow increased apoptosis of Trx9 cells in responseto daunomycin. Trx activity was assayed in vector,serb4 and Trx9 cells using insulin reduction assayas described in experimental procedure (Fig 1E).

Decrease clonogenic survival of MCF-7 cellsoverexpressing Trx.After cytotoxic treatment, cells can survive DNAdamage through various repair processes and maycontinue to propagate into colonies (34,35).Therefore we compared the clonogenic survivalof Trx9, serb4 or vector cells treated withdaunomycin at concentrations as low as 0.025µM. A clonogenic assay is a more stringentassessment of chemosensitivity than TUNEL orSub-G1 peak measurements (34,35). Trx9 cellstreated with daunomycin formed significantlyless colonies at the end of 14 days, whereasvector cells treated with daunomycin formedseveral colonies (Fig. 2A). Thus MCF-7 cellsoverexpressing Trx exhibited increased apoptosisand decreased clonogenic survival in the presenceof daunomycin, as compared with vector-transfected cells.

In addition to MCF-7 clones, we also generatedTrx or dnTrx expressing clones in A549 cells totest whether the observations with MCF-7 cellscould be reproduced in other cell types. Asdemonstrated in Fig 2B, A549-vector only clonesor A549-dnTrx clones showed several colonies atthe end of 14 days, whereas A549-Trx clones didnot show significant number of colonies. Thesedata demonstrate that either A549 cells or MCF-7cells expressing higher level of Trx undergoincreased apoptosis and decreased clonogenicsurvival in response to daunomycin. Next, todetermine whether endogenous Trx contributes toapoptosis induced by anthracyclines, we usedRNA interference to down regulate Trxexpression.

Silencing Trx expression by siRNA decreasesapoptosis in MCF-7 cells, as well as in A549 cellsin response to daunomycin.The apoptosis experiments described above weredone using Trx or dnTrx overexpressing clones ofMCF-7 cells. Therefore, there is reason to believethat the data obtained could be specificallyapplicable to a specific clone of Trx or dnTrx ofMCF-7 cells. In addition, it is not clear whethereffects that were observed is only related to theoverexpression of Trx. Therefore, to understandthe role of endogenous Trx in apoptosis inducedby daunomycin, we down regulated the level ofTrx in MCF-7 cells using siRNA approach. Asshown in Fig 3A, treatment of MCF-7 cells withTrx siRNA down regulated Trx protein level. Thedecrease was about 95% compared to cellstransfected with non-targeting siRNA. FollowingsiRNA transfection, cells were treated withdaunomycin and the level of apoptotic markerswere evaluated by western analysis. As shown inFig 3B (top panel) and Fig 3C, MCF-7 cellstransfected with non-targeting siRNA and treatedwith daunomycin demonstrated a significantincrease in p53 protein level. In contrast, cellstreated with Trx siRNA demonstrated asignificantly (p<0.001) lower level of p53 proteinin response to daunomycin treatment (Fig 3C).Additionally, the level of active caspase-7 (Fig 3B,2nd panel and Fig 3D), and the level of cleavedPARP (Fig 3B 3rd panel; Fig 3E) were alsosignificantly (p<0.001) decreased in Trx siRNAtransfected cells compared to non-targeting siRNAtransfected cells. These data demonstrate a crucialrole of endogenous Trx in daunomycin-inducedapoptosis in MCF-7 cells.

Recent studies have demonstrated that caspases-1expression is up regulated in response todoxorubicin in a p53-dependent manner in MCF-7cells (36,37). Since caspases-1 expression is pro-apoptotic, and its expression is dependent on p53,we examined the level of caspases-1 in response todaunomycin treatment and the effect of silencingTrx on the expression of caspases-1. In addition,we also examined the expression of caspases-6and cleaved caspases-8 expression in response toTrx silencing in daunomycin treated MCF-7 cells.As demonstrated in Fig 3F and Fig 3G, caspases-1expression was significantly up regulated in


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response to daunomycin in MCF-7 cellstransfected with non-targeting siRNA. In contrast,cells transfected with Trx siRNA demonstratedsignificant inhibition of caspases-1 up regulationin response to daunomycin. However, the level ofcaspases-6 remained unchanged in response todaunomycin (Fig 3F, middle panel). Treatment ofcells with daunomycin resulted in the appearanceof cleaved caspases-8 product (Fig 3F, lowerpanel), but the level of cleaved caspases-8 levelremain unchanged in Trx siRNA treated cellscompared to non-targeting siRNA treated cells.Taken together, these data suggest that caspases-1and caspases-7 levels are modulated bydaunomycin treatment, and Trx plays an importantrole in regulation of expression of these caspases.

In order to determine whether the role played byTrx in MCF-7 cells could be reproduced in othercell lines, we transfected lung adenocarcinomacells A549 with siRNA for Trx or a non-targetingsiRNA sequence, and treated these cells withdaunomycin. As demonstrated in Fig 4A,transfection of Trx siRNA significantly downregulated Trx level in A549 cells. These cells weretreated with daunomycin and the level of p53 wasdetermined by quantitative immunoblotting. Asshown in Fig 4B and C, down regulation of Trxprotein levels by siRNA significantly lowered p53protein level similar to that observed with MCF-7cells. Taken together, these data demonstrate thatendogenous Trx is required for daunomycin-induced apoptosis of cancer cells. Next, wedetermined how p53 expression is modulated inthe presence of higher level of redox-active Trx orin the absence of redox-active Trx.

MCF-7 cells expressing higher Trx activity showhigher p53 protein levels in response todaunomycinAnticancer agents that induce DNA damage alsoinduce p53-mediated apoptosis (38). In the eventof DNA damage, p53 is activated byphosphorylation and binds to the consensussequence of the DNA, resulting in the regulationof gene expression required for apoptosis or cellcycle arrest (39). Phosphorylation of p53 blocksMdm-2 binding and thereby prevents degradationof p53 protein resulting in accumulation of p53(40). To determine the role of Trx in daunomycin-induced p53 protein expression, we examined the

level and the extent of phosphorylation of p53 inTrx9 cells. Treatment of cells with 1 µMdaunomycin increased p53 protein in all clones(Fig. 5A, upper panel); however, in Trx 9 cellsthere was 2-3 fold increase in p53 protein levelthan in vector or Serb4 cells. Phosphorylation ofp53 at ser15 has been shown to be crucial inactivating p53 as a transcription factor (17).Conformational changes due to phosphorylation isrequired for its DNA binding activity (41).Therefore, we next, evaluated the phosphorylationstate of p53 on ser15 in daunomycin-treated cellsusing phospho-specific antibodies (Ser-15). Trx9cells exhibited higher phospho-p53 (Ser-15) levelin response to daunomycin compared to vector orSerb4 cells, suggesting that higher level of Trxcould be potentiating the apoptotic potential ofdaunomycin (Fig. 5A, lower panel).

To determine whether the results obtained with aspecific clone overexpressing Trx could bereproduced in other clones of MCF-7 cellsoverexpressing Trx, we generated several clonesof Trx and dnTrx as described in the experimentalprocedure section in MCF-7 cells as shown in Fig5B. These clones were treated with 1 µMdaunomycin for 24 h followed by detection of p53expression using western analysis. As shown inFig 5C MCF-7 clones Trx25 and Trx26 showedhigher p53 expression in response to daunomycin.In contrast, dnTrx clones 1 & 4 did not showhigher p53 expression compared to vector onlyclones or Trx clones. These studies show that theresults observed in vector, Trx9 or Serb4 cellscould be reproduced in other MCF-7 clones.Therefore, using different clones of MCF-7 cellsor other cells (A549) we observed increased p53expression in response to daunomycin in Trxoverexpressed cells.

Next, we examined whether transientoverexpression of Trx in MCF-7 cells couldproduce similar results obtained using stableclones, because there is reason to believe thatstable expression of a protein could be clone-specific. Additionally, the physiological responseof stable clones could be different than that oftransient overexpression. We used adenovirusmediated gene delivery, as well as usedlipofectamine-mediated transfection tooverexpress Trx or dnTrx in MCF-7 cells and


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studied its effect on p53 expression in response todaunomycin. As demonstrated in Fig 5D,adenovirus infection increased Trx or dnTrx levelin MCF-7 cells. When these cells were treatedwith daunomycin increased p53 expression wasobserved in MCF-7 cells overexpressing redox-active Trx (Fig 5E). In contrast, dnTrx-overexpressing cells demonstrated almost noinduction of p53 (Fig 5E). Additionally, as shownin Fig 5F, transient transfection also increased Trxor dnTrx levels in MCF-7 cells (data not shown).When these cells were treated with daunomycinwe observed similar pattern of p53 expression (Fig5F). Taken together, these data indicate thatoverexpression of Trx enhances p53 expression inresponse to daunomycin, whereas expressionredox-inactive Trx inhibits the expression of p53in response to daunomycin.

p53 DNA binding is enhanced in Trx9 cells inresponse to daunomycin:Since phosphorylation of p53 activates its DNAbinding that results in the regulation of theexpression of many different genes involved inapoptosis or DNA repair, we used electrophoreticmobility shift assays (EMSA) to measure p53binding to the DNA in nuclear extracts of cellstreated with or without daunomycin. Asdemonstrated in Fig 6A, daunomycin-induced p53DNA binding was several folds higher in Trx9cells than in vector or serb4 cells. Increased DNAbinding implies increased transcription of p53-dependent genes, which may lead to increasedapoptosis. We also determined the p53 DNAbinding in MCF-7 cells transiently overexpressingTrx or its mutant form. As shown in Fig. 6B,higher levels of p53 DNA binding was observed incells expressing higher levels of Trx. Thesestudies indicate that higher Trx level couldenhance the transcription of p53-dependent genesthat could enhance apoptosis.

Silencing p53 decreases expression of apoptoticproteins such as active caspase-7 or cleavedPARP.Since daunomycin treatment in Trx-siRNAtransfected cells resulted in decreased p53accumulation and apoptosis, we sought todetermine whether the observed decrease inapoptosis in daunomycin is dependent on the level

of p53 accumulation. We used p53 siRNA toinhibit p53 expression in MCF-7 cells (Fig 7,upper panel), and treated these cells withdaunomycin. Transfection of MCF-7 cells withp53 siRNA resulted in about 90% decrease in theexpression of p53 (Fig 7A, upper panel and Fig7B). We also observed a decrease in the level ofcaspase 7 (Fig 7A middle panel and Fig 7C) and adecrease PARP cleavage (82 kDa cleaved PARP;Fig 6A, third panel, and Fig 7D) in p53 siRNAtransfected cells compared to non-targeting siRNAtransfected cells. However cleavage of PARP wasnot completely inhibited in the presence of p53siRNA suggesting that apoptotic degradation ofPARP is partially dependant on p53. These dataindicate that p53 is required for daunomycin-induced apoptosis in MCF-7 cells. Therefore, thedata generated using Trx overexpression suggestthat apoptotic potential of MCF-7 could beincreased in the presence of higher levels of redox-active Trx.

Since daunomycin is a quinone containinganthracycline, and undergoes redox-cycling thatgenerates reactive oxygen species (ROS) and thesemiquinone radical, we tested whether Trx couldcontribute to enhanced redox cycling in vivo thatcould increase the expression of p53-mediatedapoptosis.

Daunomycin treatment increases the generationof ROS in Trx9 cells:We estimated the total load of oxidative stress byusing the ROS-sensit ive probe 2',7'-dichloroflourescin diacetate (DCF-DA) to measurethe total cellular peroxide levels in vector, Trx9 orSerb4 cells in response to daunomycin. Pre-incubation of cells with non-fluorescent DCF-DAdye followed by treatment with daunomycinresulted in enhanced fluorescence due to increasedoxidation of DCF-DA in Trx9 cells compared tovector or serb4 cells (Fig 8 A & B). We also used5-iminodaunomycin, a non-redox cycling form ofdaunomycin as a negative control (Fig 8A&C). 5-imminodaunomycin does not contain the quinonemoiety of anthracyclines, and therefore does notgenerate superoxide anions (42). Treatment with5-iminodaunomycin did not induce any detectableoxidation of DCF-DA in any of these Trx clones(Fig. 8 A & C). These data demonstrate that


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daunomycin could undergo a higher rate of redoxcycling in the presence of higher level of Trx

Redox cycling of daunomycin generatessemiquinone radical and O2

.-, both of which couldinduce DNA damage and cell death. Since Trx9cells showed enhanced DNA damage andapoptosis, we sought to determine the rate ofredox cycling of daunomycin in Trx9 cells. Wemeasured the production of O2

.- in the presence ofdaunomycin in Trx clones as an indicator of redoxcycling (43). As shown in Fig. 8D, treatment ofcells with daunomycin produced significantlyhigher level of O2

.- in Trx9 cells compared tovector or Serb4 cells. We have previously shownthat Trx being an antioxidant can scavengehydroxyl radicals; however, it does not scavengeO2

.- (4). In addition, we have also shown thatE.coli Trx could participate in the redox cycling ofanthracyclines generating O2

.- (44). Therefore, wefurther investigated whether human Trx could alsoredox cycling daunomycin using additionalcontrols. As demonstrated in Fig 8D, treatment ofMCF-7 clones with 5-Imminodaunomycin did notincrease the generation of superoxide anions inTrx9 or Serb4 cells. These data demonstrate thatquinone redox cycling is enhanced in the presenceof increased level of Trx. Next, we evaluated theeffect of transient overexpression of Trx ondaunomycin redox cycling. As demonstrated inFig 8E, MCF-7 cells expressing higher level ofTrx in adenovirus-mediated overexpressiongenerated higher level of superoxide anionscompared to vector only or dnTrx transfectedcells. In additional experiments (Fig 8F) we downregulated Trx expression using siRNA approach,and evaluated the response of these cells togenerate superoxide anion in presence ofdaunomycin. MCF-7 cells treated with siRNAdemonstrated lower level of superoxide aniongeneration in response to daunomycin. These datademonstrate that endogenous Trx is required forthe redox cycling of daunomycin, andoverexpression of Trx could enhance the level ofredox cycling in these cells.

In order to determine whether the responseobtained in MCF-7 cells with respect to generationof superoxide anions could be reproduced in othercell types, we down regulated Trx expression inlung adenocarcinoma cell line A549 using siRNA

approach, and evaluated the response of these cellsto daunomycin-mediated superoxide aniongeneration. As demonstrated in Fig 8G, there wassignificant inhibition of superoxide aniongeneration in cell with reduced level of Trx. Thesedata indicate that endogenous Trx is required incancer cells for redox-cycling of anthracyclines.

Effect of bioreductive enzyme inhibitor on O2.-

anion generation in Trx9 cells:

We observed that Trx9 cells generate more O2.- in

the presence of anthracyclines. Redox-cycling ofanthracyclines has been shown to be mediated byone electron reduction by NADPH-cytochrome P-450 reductase (45), mitochondrial NADHdehydrogenase (46) and nitrate reductase (47)from Neurospora. Therefore, to determine theinvolvement of bioreductive enzyme(s) in theredox-cycling of daunomycin in the presence ofincreased thioredoxin we treated cells withdipheyleneiodonium chloride (DPIC) a non-specific inhibitor of NADPH-dependent reductases(48) As demonstrated in Fig 9, treatment of cellswith DPIC inhibited generation of O2

.- suggestingthat Trx-mediated O2

.- generation is dependant onbioreductive enzymes for anthracyclines redox-cycling. These results indicate that reductasescould be involved in the redox-cycling ofanthracyclines using reducing equivalents fromreduced Trx.

Redox cycling contributes to increased p53 DNAbinding in the presence of higher level of redox-active Trx.Next, we determined whether the increase in p53-DNA binding observed in Trx9 cells depends onthe redox cycling of daunomycin. By treating Trxclones with 5-iminodaunomycin, which cannotundergo redox cycling but intercalates with DNAwe show that each of these clones vector, Trx9 andSerb4 exhibited enhanced p53-DNA binding (Fig10A). However, there were minor differences inthe intensity of DNA binding in these cells. Todetermine whether ROS mediate p53 DNAbinding due to daunomycin redox-cycling we usedH2O2 as a positive control for ROS, and Taxol as anegative control for ROS. Additionally, we usedmenadione as a positive control for quinonecontaining compound. As shown in Fig 10B,H2O2, Menadione and taxol treatment did not


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induce p53–DNA binding in Trx clones, exceptthat 500 µM H2O2 showed very weak p53–DNAbinding (Fig 10B). These data indicate that eitherH2O2 is scavenged by peroxiredoxins that has beenshown to be up regulated in Trx9 cells (6), or H2O2is not an effective inducer of p53 response.However, menadione that contain a quinonemoiety also did not show p53 DNA binding in ourprotocol, suggesting that daunomycin ordoxorubicin could specifically interact with Trx.To further evaluate the role of ROS in p53 DNAbinding, we treated Trx9 cells with other ROSgenerating compounds such as pyocyanine orphenazinemethosulfate (PMS, (49,50). We did notobserve any detectable increase in p53-DNAbinding (Fig 10C). These studies indicate thatROS may not contribute substantially to p53activation in MCF-7 cells.

To understand the relative contribution of ROS orintercalation of drug due to redox-cycling on p53activation, we used MnTBAP to scavenge O2

-. orH2O2 (51) in daunomycin treated Trx9 cells. Asdemonstrated in Figure 10C (upper panel),MnTBAP treatment resulted in about 30%(densitometric analysis) reduction in the p53binding activity. This data indicate that activationof p53 is not fully dependent on ROS. Since ser15phosphorylation on p53 enables it as a DNAbinding transcription factor we also determinedser-15 phosphorylation in response to MnTBAPtreatment. As demonstrated in Fig. 10C (lowerpanel, lanes 3-4), there was no appreciabledecrease in ser-15 phosphorylation in response toMnTBAP in daunomycin treated cells. These datafurther support the notion that ROS is not a majorinducer of p53 activation due to redox-cycling ofdaunomycin, but due to DNA damage by theinteraction of the drug to DNA. Next, wedetermined the role of reductases that redox-cycleanthracyclines. Treatment of cells with a generalreductase inhibitor DPIC abolished p53 DNAbinding as well as p53 (ser-15) phosphorylation(Fig. 10C, lanes 5-6). These data indicate thatgeneration of semiquinone by redox-cycling maycontribute to p53 activation as a result of DNAdamage in Trx9 cells, and ROS plays a minor rolein the activation of p53.

Discussion

In the present study we have demonstrated thatincreased expression of Trx enhances apoptosis inMCF-7 cells in response to daunomycin. Incontrast, silencing endogenous Trx expressionresulted in significant decrease in p53 expressionand apoptosis in MCF-7 cells, as well as othercells in response to daunomycin. The expressionand DNA binding of p53 protein was alsoincreased in response to daunomycin in Trx9 cellscompared to vector or Serb4 cells. Additionally,there was increased production of O2

-. in extractsof Trx9 cells in response to daunomycin. DPIC, ageneral reductase inhibitor decreased thegeneration of O2

- as well as p53-DNA binding inTrx9 cells in response to daunomycin. Takentogether, our studies demonstrate that Trxincreases the redox-cycling of daunomycin andenhances the apoptotic potential of daunomycin.Our data also indicate that endogenous Trx isessential for anthracycline-dependant p53expression and the activation of apoptotic cascade.These are novel pro-oxidant and pro-apoptotic roleof Trx in response to anthracycline drugs.Additionally, the present study also indicate acrucial role of Trx in daunomycin-inducedcaspases-1 expression.

Thioredoxin is widely considered to be anantioxidant protecting cells from a variety ofoxidative stress conditions (1,2). Our data indicatethat Trx could provide reducing equivalents tobioreductive enzymes that can redox-cycleanthracyclines, and this process increases theapoptotic potential of anthracyclines.Interestingly, Trx has been shown to conferresistance against ROS-generating anticancerdrugs due to its antioxidant properties (31,33).Conversely, recent studies have also shown thatTrx does not confer resistance against doxorubicin(52). Furthermore, thioredoxin failed to protectMCF-7 cells from apoptosis induced by ROSgenerating drugs. The study published by Wang etal (53) is a correlative study that demonstrated acorrelation between increased Trx expression invarious leukemia cell lines and the drug resistanceto adriamycin. Therefore, there was nomechanistic evaluation of the role of Trx in drugresistance or apoptosis. The study also comparedseveral cell lines in terms of Trx expression anddrug resistance. Our present investigation hasshown, using different expression systems and


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enhancing endogenous Trx in multiple cells thatincreased Trx expression could enhance theapoptotic potential of anthracyclines. Additionally,the role of endogenous Trx in apoptosis of cancercells in response to daunomycin was clearlyelucidated using siRNA approach. In contrast tothe study of Wang et al, studies by Berggren et al(6) has clearly demonstrated that although Trxprotect cells against H2O2-mediated apoptosis, itdoes not protect against adriamycin-inducedapoptosis. They also demonstrated that protectiveeffect of Trx against H2O2 is due to enhancedperoxiredoxin expression (6). This study clearlydemonstrated that Trx could remove H2O2 due tohigher Prx activity, but it can not protect againstdoxorubicin-induced toxicity (6). These resultssupport our data that H2O2 does not play a majorrole in apoptosis of daunomycin treated Trxoverexpressed cells. Therefore, if H2O2 were theonly cytotoxic compound that mediatesdoxorubicin-induced apoptosis then probably Trxoverexpression would protect against doxorubicin-induced apoptosis. However, the toxicity ofanthracyclines predominantly comes from thesemiquinone intercalation with the DNA resultingin DNA damage and apoptosis. In our studies weobserved that Trx9 cells not only failed to protectagainst daunomycin-mediated apoptosis, but alsoenhanced apoptosis in these cells. Thus, dataobtained using multiple systems using differentapproaches confirm a crucial role of Trx inapoptotic response of cancer cells in treatmentwith daunomycin. Taken together, these dataindicate that protective role of Trx could beimportant in ROS-mediated cytotoxicity. Asdemonstrated in our data, removal of superoxideanion or H2O2 by using MnTBAP decreased p53DNA binding by about 25-30 percent.Additionally, treating cells with H2O2 or otherROS-generating systems did not induce significantp53 DNA binding. This fact demonstrates thatROS are generated due to redox-cycling, but theydo not significantly contribute to p53-dependentapoptotic process. Therefore, Trx could inhibit theROS-mediated cytotoxicity such as that of H2O2 ashas been shown, but it does not inhibit the redox-cycling process that facilitate the intercalation ofthe drug with the DNA.

We have earlier shown that Trx induces MnSOD(5). Additionally, Trx does not scavenge O2

.- (4).

However, it can scavenge hydroxyl radicals orsinglet oxygen (4). O2

.- is the first reductionproduct of redox-cycling that could produce H2O2,which could be scavenged by Prx or otherperoxidases. Therefore, in this circumstanceredox-cycling enhancing action of Trx by itselectron donating function will remain unaffected.Additionally, our data indicated that NADPH-dependent reductase inhibitor inhibited O2

.-

generation in Trx9 cells, which suggests thatredox-cycling produces enhanced DNA damage inTrx9 cells resulting in p53 activation. Thesefindings are further verified by the fact thatdominant-negative expression of redox inactivemutant Trx or silencing of Trx using Trx siRNAfailed to induce p53 protein in response todaunomycin suggesting that Trx is required forbioreductive activation of daunomycin.

Our data presented here indicate a fundamentalrole of endogenous Trx in redox-cycling ofanthracyclines, which has not been recognizedpreviously. We have shown previously that E.coliTrx could enhance redox cycling of anthracyclinesand induce DNA damage both, in vivo and in vitro(44). We have also shown that E.coli Trx providesreducing equivalents to TR and mammaliancytochrome P450 reductase that enhanced theredox cycling of anthracyclines (44). It is thereforeconceivable that Trx may indirectly enhance redoxcycling of daunomycin involving NADPHdependant reductase. We also demonstrated thatDPIC abolished daunomycin-induced p53 DNAbinding, suggesting that redox-cycling ofdaunomycin depends on NADPH-dependantbioreductive enzymes.

Although daunomycin potently induced p53-DNAbinding, other ROS generating agents includingmenadione did not induce p53-DNA binding (Fig10B) at concentrations that was used in our study.A previous study has also observed failure ofmenadione to induce p53–DNA binding in MCF-7cells (18). However, p53 expression did increaseseveral hours after menadione was removed,indicating that p53 was activated in the DNAdamage repair phase (18). Thus, it is unclear whymenadione did not activate p53, whereasdaunomycin did induce p53 activation. H2O2 didnot induce a strong p53 response at 500 µMconcentration. Increased Trx expression could


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have increased H2O2 scavenging byperoxiredoxins, which obtain reducing equivalentsfrom Trx. A lower level of p53-DNA binding wasobserved in Trx mutant Serb4 cells that expressesredox-inactive form of Trx, which does not showhigher peroxiredoxin expression (6). This factsuggest that removal of H2O2 by overexpression ofredox-active Trx in Trx9 cells could not haveaccounted for less p53 DNA binding in responseto H2O2. Further, DNA intercalation appears to beessential for induction of p53-DNA binding since5-iminodaunomycin did induce p53-DNA binding.This fact was further strengthened since there wasno involvement of redox cycling in 5-iminodaunomycin, and p53-DNA binding wasunaffected in both Trx9 and Serb4 cells.

We also demonstrated decreased O2.- production,

p53 expression and apoptosis in daunomycintreated MCF-7 cells deficient in Trx using Trx-siRNA. Accumulating evidence from variousstudies indicate that p53 is required to enhancechemosensitivity in cancer cells (38,39). In thepresent study we used MCF-7 cells which aredefective in caspase 3, and does not undergoclassical 180 bp DNA fragmentation duringapoptosis (54), and therefore is dependant onalternate or compensatory mechanisms to induceapoptosis. Further, in p53 defective cells there arealternate regulatory proteins such as Rb or E2F,which could trigger apoptosis. In the presentcontext it becomes more important for cells suchas caspase 3 deficient MCF-7 cell lines with wildtype p53 to utilize p53 mediated response toinduce apoptosis. In summary, our investigationdemonstrates a novel pro-oxidant and pro-apoptotic role of Trx in response to anthracyclineclass of antitumor agents.

Acknowledgements.This study was supported in part by a researchproject grant from the American Cancer Society(KCD), and National Institutes of Health 1R01HL071558.

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Figure legends

Figure 1. Increased apoptosis of Trx9 cells inresponse to daunomycin: (A) Trx clones weretreated with daunomycin for 24 hours followed bydetection of apoptosis using TUNEL assay kit(Roche Molecular Biochemicals, Indianapolis,Indiana) as described in the methods. Left panel,untreated control cells; right panel, Trx clonestreated with daunomycin (1µM, 24h). FITC panelshows incorporation of Fluorescein dUTP by nickgenerated in DNA and PI panel shows counterstain with propidium iodide. (B) Bar graphrepresenting percentage of TUNEL positive nucleiin daunomycin treated Trx clones at 24 or 48h. (C)Trx clones were treated with daunomycin (1 µM,16h) and cell cycle analysis was performed asdescribed in the methods. Left panel, histogram ofcell cycle distribution of untreated control cells;right panel, histogram of cell cycle distribution ofTrx clones treated with daunomycin. Arrow inright panel indicates “Sub-G1” peak. (D) Data insub-G1 peak in Fig. 1D is represented as percentapoptotic cells in a bar graph (average of twoexperiments). (E) Trx activity in MCF-7 clones.

Unit of activity is expressed as µmoles of NADPHoxidized/min/mg protein.

Figure 2. Enhanced clonogenic death of MCF-7cells and A549 cells overexpressing redox-activeTrx, but not redox-inactive Trx. (A). Vector,serb4 or Trx9 cells were trypsinzed and seeded toa final density of 1 X 106 viable cells per100 mm2

dishes and allowed to attach overnight. Cells werethen treated with appropriate concentration ofdrugs for 24 hours, trypsinized and seeded to afinal density of 500,000 viable cells per 100 mm2

dish. Viable cells were determined using Vicellcounter (Beckman Coulter). After 14 days, thesurviving colonies were washed in PBS, fixed in70% ethanol and stained using 0.1% crystal violetin 90% ethanol. (B). Stable clones of A549 cellsexpressing Trx (Trx) or redox-inactive Trx(dnTrx) were processed as described for Fig 2A.

Figure 3. Silencing Trx expression by RNAidecreases apoptosis in MCF-7 cells in responseto anthracyclines. (A) Transfection of Trx-specific RNAi inhibits Trx expression in MCF-7cells. MCF-7 cells were transfected with indicatedconcentrations of non-targeting or Trx siRNA asdescribed in methods. Expression of Trx wasdetermined by western blot as described inmethods. β-actin is shown as loading control. (B)Trx siRNA down regulates expression of p53,active caspase-7 and cleaved PARP expressionin response to daunomycin. Non-targetingsiRNA or Trx siRNA transfected MCF-7 cellswere treated with 1 µM daunomycin for 16 hours.Western analysis was performed for the detectionof p53, caspase 7 and cleaved PARP as describedin the method. Lanes 1-3, Control cells transfectedwith non targeting siRNA in triplicates; lanes 4-6,untreated Trx siRNA transfected cells intriplicates; Lanes, 7-9, cells transfected with non-targeting siRNA and treated with 1 µMdaunomycin (16h); lanes 10-12, cells transfectedwith Trx siRNA and treated with 1 µMdaunomycin (1 µM). β-actin is shown as loadingcontrol. (C) Ratio of p53 level (upper panel of Fig3B) to β -actin level. ** Significantly lesscompared to p53 level in NT transfected anddaunomycin-treated cells in Fig 3B. (D). Ratio ofcaspase-7 level (middle panel of Fig 3B) to β-actinlevel. ** Significantly less compared to caspase-7


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level in NT (Non-targeting) transfected anddaunomycin-treated cells in Fig 3B. (E). Ratio ofcleaved PARP level (lower panel of Fig 3B) to β-actin level. ** Significantly less compared to p53level in NT transfected and daunomycin-treatedcells in Fig 3B. Fig 3F. Effect of Trx silencing oncaspases-1. –6, and –8 expression in response todaunomycin. MCF-7 cells were treated with TrxsiRNA or NT siRNA, and treated withdaunomycin (1 µM) for 16 hours. Immunoblottingwas performed for caspases-1, caspases-6 andcleaved caspases-8 using respective specificantibodies. Upper panel, caspases-1; middle panel,caspases-6; lower panel, caspases-8; lowest panel,β-actin. Lanes, 1-3 cells transfected with NTsiRNA; lanes 4-6, cells transfected with TrxsiRNA; lanes 7-9, cells treated with NT siRNAand treated with daunomycin; lanes 10-12, cellstreated with Trx siRNA and treated withdaunomycin. Fig 3G. Densitometry of caspases-1western blotting.

Figure 4. Silencing Trx expression by RNAidecreases expression of p53 in response toanthracyclines in A549 cells. (A). Transfectionof Trx-specific RNAi inhibits Trx expression inA549 cells. A549 cells were transfected withindicated concentrations of non-targeting or TrxsiRNA as described in methods. Expression of Trxwas determined by western blot as described inmethods. β-actin is shown as loading control. (B)Trx siRNA down regulates expression of p53 inresponse to daunomycin. Non-targeting siRNAor Trx siRNA transfected A549 cells were treatedwith 1 µM daunomycin for 16 hours. Westernanalysis was performed for the detection of p53 asdescribed in the method. Lanes 1-3, Control cellstransfected with non targeting siRNA intriplicates; lanes 4-6, untreated Trx siRNAtransfected cells in triplicates; Lanes, 7-9, cellstransfected with non-targeting siRNA and treatedwith I µM daunomycin (16h); lanes 10-12, cellstransfected with Trx siRNA and treated with 1 µMdaunomycin (1 µM). β-actin is shown as loadingcontrol. (C) Ratio of p53 level (upper panel of Fig4A) to β-actin level** Significantly less comparedto p53 level in NT transfected and daunomycin-treated cells in Fig 4A.

Figure 5. Effect of Trx overexpression ondaunomycin-induced p53 expression: (A) Trxclones were treated with 1 µM daunomycin for 16hours followed by lysate preparation as describedin the method. p53 or phospho-p53 (ser-15) wasdetected as described in the method. Lanes 1-3,untreated control cells; lanes 4-6, Trx clonestreated with daunomycin. Upper panel, p53;middle panel, p53 (ser-15); lower panel, loadingcontrol (β-actin). (B) Stable clones of MCF-7expressing redox-active Trx or redox-inactive Trx(dnTrx) were generated by transfecting cells withpCMV-Trx or pCMV-dnTrx constructs andselecting clones using G418, 800ug/ml.Expression of Trx was determined by westernanalysis as described previously. (C). MCF-7clones were treated with 1 µM daunomycin for 16hours, followed by p53 western analysis asdescribed previously. Lower panel, β-actin ofsame blot. (D) Overexpression of Trx or dnTrx inMCF-7 cells using adenox infection. MCF7 cellswere infected with AdenoX LacZ, Adenox-Trx orAdenox-dnTrx, and Trx was determined using anELISA assay as described (30). The amount of Trxwas expressed as picograms of Trx per mg protein.(E). MCF-7 cells were infected with adenovirus(AdenoX LacZ, AdenoX Trx or Adx dnTrx) asdescribed in the method. After 48 hours infectedcells were treated with 1 µM daunomycin for 16hours. Western analysis for p53 was performed asdescribed in the method. Lane 1, cells infectedwith AdenoX LacZ; lane 2, cells treated withAdenoX Trx; lane 3, cells treated with AdenoXdnTrx; lanes 4-6, cells were infected with AdenoXLacZ, pAdenoX Trx or Adenox dnTrx and treatedwith 1 µM daunomycin for 16 hours. β-actin isshown as loading control. (F) MCF-7 cells weretransfected with Trx or dnTrx vectors as describedin the methods section. Cells were treated with 1µM daunomycin after 48h, and following 16 hoursof incubation with the drug western analysis wasperformed for p53 and β-actin as described in themethods. (G)

Figure 6. (A) Effect of Trx overexpression onp53 DNA binding in response to daunomycin.Trx clones were treated with 1 µM daunomycin(4h) and nuclear extract was prepared and gel-shiftassay was performed as described in the method.Lanes 1-3, untreated control cells; lanes 4-6, Trx


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clones cells treated with daunomycin. (B) Effectof adenovirus-mediated overexpression ofthioredoxin in daunomycin induced p53-DNAbinding: MCF-7 cells were infected withadenovirus (AdenoX LacZ, AdenoX Trx orAdenoX dnTrx) as described in the method. After48 hours infected cells were treated with 1 µMdaunomycin (4h) and nuclear extract was preparedand gel-shift assay was performed as described inthe method. Lanes 1-3, untreated control cells;lanes 4-6, daunomycin treated cells.

Figure 7. Effect of p53 down regulation usingRNA interference on apoptosis in MCF-7 cells.Non-targeting siRNA or p53siRNA transfectedMCF-7 cells were treated with 1 µM daunomycinfor 16 hours. Western analysis was performed forthe detection of p53, caspase 7 and PARP asdescribed in the method. Lanes 1-6, Control cellstransfected with non-targeting or Trx-siRNA intriplicates, lanes 7-12, Non-targeting siRNA orTrx-siRNA transfected MCF-7 cells were treatedwith 1 µM daunomycin in triplicates. b-actin isshown as loading control. (B) Ratio of p53 level(upper panel of Fig 7A) to β -actin level. **Significantly less compared to p53 level in NTtransfected and daunomycin-treated cells in Fig7A. (C) Ratio of caspase-7 level (2nd panel of Fig7A) to β -actin level. **Significantly lesscompared to caspase-7 level in NT transfected anddaunomycin-treated cells in Fig 7A. (D) Ratio ofcleaved PARP level (upper panel of Fig 7A) to b-actin level. ** Significantly less compared tocleaved PARP level in NT transfected anddaunomycin-treated cells in Fig 7A.

Figure 8. Generation of ROS in Trx clones inresponse to daunomycin. (A) Trx clones weretreated with daunomycin (1µM) or 5-iminodaunomycin (1 µM) and processed for DCF-DA assay as described in the method. Left panel,untreated control cells; middle panel, cells treatedwith daunomycin; right panel, cells treated with 5-iminodaunomycin; Inset, mid section of confocalmicroscopy scan showing most of the fluorescencelocalized in the cytosol. (B) Graph showingchange in intensity of DFC-DA fluorescence overtime indicated in seconds. (C) Control and 5-iminodaunomycin treated Trx clones. ( D )Generation superoxide anion in Trx clones inresponse to daunomycin. Trx clones were treated

with daunomycin and the generation of O2-. was

determined by reduction of SOD-inhibitablecytochrome c as described in the method. Datawas presented as nmoles of O2

-. produced per mgtotal cell protein. (E) Effect of adenovirus-mediated overexpression of thioredoxin onredox cycling of daunomycin. Adenovirus-mediated Trx transfected MCF-7 cells were treatedwith daunomycin and the generation of O2

-. wasdetermined by reduction of SOD-inhibitablecytochrome c as described in the method. Datawas presented as nmoles of O2

-. produced per mgtotal cell protein. (F) Trx siRNA decreases redoxcycling of daunomycin. Non-targeting or TrxsiRNA transfected MCF-7 cells were treated withdaunomycin and the generation of O2

-. wasdetermined by reduction of SOD-inhibitablecytochrome c as described in the method. Datawas presented as nmoles of O2

-. produced per mgtotal cell protein. (G) Effect of silencing Trx ondaunomycin redox cycling in A549 cells. A549cells were transfected with NT or Trx siRNA, andO2

.- generation was determined as described forFig 8F.

Figure 9. Effect of reductase inhibitor DPIC onO2

-. generation: Trx9 cells were treated withDPIC as indicated in the Figure, and O2

-.

generation was assayed as described in themethod. Lane 1, Trx9 cells treated with 10 µMdaunomycin. The generation of O2

-. in thistreatment was considered 100%. Lane 2, Trx9cells pre-incubated with 25 µM DPIC followedwith 10 µM daunomycin treatment; lane 3, Trx9cells pre-incubated with 50 µM DPIC followedwith 10 µM daunomycin treatment.

Figure 10. (A) Effect of 5-Iminodaunomycinand ROS generating agents on p53-DNAbinding. Trx clones were treated with indicatedconcentration of superoxide-generating agents for4-hr. Following incubation nuclear extract wasprepared and p53 EMSA was performed asdescribed. (B) Trx9 cells were treated withindicated concentration of superoxide-generatingagents for 4-hr. Following incubation nuclearextract was prepared and p53 EMSA wasperformed as described in the methods. (C) Effectof MnTBAP and DPIC on daunomycin-inducedp53 DNA binding in Trx9 cells: Trx9 cells weretreated with daunomycin either in presence of


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MnTBAP (20 µM) or DPIC (50 µM) for 4 hoursand gel-shift assay was performed as described inthe methods. Lane 1, untreated Trx9 cells; lane 2,Trx9 cells treated with daunomycin (1 µM); lane3, Trx 9 cells treated with 100 µM MnTBAP; lane4, Trx9 cells treated with daunomycin +MnTBAP; lane 5, Trx9 cells treated with DPIC;

lane 6, Trx9 cells treated with daunomycin +DPIC. Lower panel; expression of phospho-p53-Ser15 in response to daunomycin. Trx9 cells wereexposed to various treatments as described for theupper panel. The level of phospho-p53-Ser15 wasdetermined by western analysis as described in themethods section.


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Figure 1A

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Figure 1C


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Figure 1D

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Figure 2A

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Figure 3A

Figure 3B

Thioredoxin

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Trx siRNA (nM)

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Figure 3C

Control Daunomycin

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Figure 3D

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Figure 3E

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Figure 3F

Figure 3G

0

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Control Daunomycin

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caspase-8

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Figure 4A

Figure 4B

Figure 4C

A549 40nM 50nM 60nMTrx siRNA

Trx

β-actin

A549 Trx siRNA A549 Trx siRNAControl Daunomycin

p53

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Figure 5A

Vector Serb4 Trx9 vector Serb4 Trx9

Daunomycin

Phospho-p53

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Figure 5B

Figure 5C

Control Daunomycin, 1 µM


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Figure 5D

Figure 5E

p53

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Adx Adx Adx Adx Adx AdxLacZ Trx dnTrx LacZ Trx dnTrxC

ontro

lDaunomycin

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Figure 5F

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pCMVpCMV-Trx

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+ - -- + -

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+ - -- + -

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Figure 6A

Figure 6B

p53-DNAcomplex

Adx Adx Adx Adx Adx AdxLacZ Trx dnTrx LacZ Trx dnTrx

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Daunomycin

Vector Serb4 Trx9 vector Serb4 Trx9

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Figure 7A

Figure 7B

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Figure 7C

Figure 7D

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Figure 8A


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Figure 8B

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Figure 8C

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Figure 8D

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Figure 8E


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Figure 8F

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Figure 8G

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Figure 9

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Figure 10A

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Figure 10C

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Dashnamoorthy Ravi, Harish Muniyappa and Kumuda C. Dasp53-dependent apoptosis in cancer cells

Endogenous thioredoxin is required for redox-cycling of anthracyclines and

published online September 13, 2005J. Biol. Chem.

10.1074/jbc.M507192200Access the most updated version of this article at doi:

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When a correction for this article is posted•

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endogenous thioredoxin is required for … · xtreme sirna transfection reagent (roche molecular...

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