Cancer Investigation, 31:374–384, 2013ISSN: 0735-7907 print / 1532-4192 onlineCopyright C© 2013 Informa Healthcare USA, Inc.DOI: 10.3109/07357907.2013.802798

CELLULAR AND MOLECULAR BIOLOGY

Peroxiredoxin Overexpression in MCF-7 Breast Cancer Cells andRegulation by Cell Proliferation and Oxidative Stress

Lauren Tehan, Kekoa Taparra, and Shelley Phelan

Department of Biology, Fairfield University, Fairfield, Connecticut, USA

Peroxiredoxins are thiol-specific antioxidant proteins thatprotect cells from ROS-induced cell death and are elevated inseveral cancers. We found that five of the six mammalianperoxiredoxins are overexpressed in MCF-7 breast cancer cellsat the mRNA and protein levels, compared to noncancerousMCF-10A cells. Inhibition of MCF-7 proliferation reduced thelevels of several peroxiredoxins. In contrast, all six proteinswere strongly and transiently induced in MCF-7 cells by H2O2.These data suggest that coordinate overexpression ofperoxiredoxins may be an important cancer cell adaptation,and that these proteins can be regulated by cell proliferationand oxidative stress.

Keywords: Breast cancers, Signal transduction, Gene expression

INTRODUCTION

It has long been recognized that cancer cells possess an en-vironment of increased oxidative stress, as a result of aber-rant cell signaling and altered cellular metabolism (1, 2).Also well established is the dysregulation of antioxidant pro-teins in cancer cells, which are often elevated to counter-balance the higher level of reactive oxygen species (ROS)(2, 3). In the last decade, the peroxiredoxin (Prdx) familyof proteins has emerged as an important and unique groupof antioxidants. Peroxiredoxins are evolutionarily conservedthiol-specific antioxidant proteins that serve to reduce ROSwithin the cell and protect cells from oxidative stress-induceddamage and death (4–8). There are six mammalian Prdxs,which can be classified into three subgroups: the 2-Cys Prdxs(Prdx1–4), the atypical 2-Cys Prdx (Prdx5), and the 1-CysPrdx (Prdx6). Unlike alternative cellular antioxidants suchas catalase and superoxide dismutase, Prdxs utilize internalcysteine residues, rather than cofactors, for activation (4–8).Over the last several years, many studies have reported Prdxoverexpression in various cancers (9–18), and there are sig-nificant data supporting a protective role for these proteinsin cancer cell survival (19–28).

Correspondence to: Shelley Phelan, PhD, Department of Biology, Fairfield University, North Benson Road, Fairfield, CT 06824, USA.email: sphelan@fairfield.eduReceived 26 October 2012; accepted 2 May 2013.

With the vast data demonstrating peroxiredoxin overex-pression in cancer cells, much attention has been given tounderstanding the structural and functional modificationsof Prdx proteins in these states. In contrast, the mechanismby which cancer cells upregulate Prdx expression has hardlybeen investigated. The Prdxs have been shown to be inducibleby oxidative stress in several systems (19, 24, 28–32), andROS-induced modifications include regulation at both thetranscriptional and posttranscriptional levels. Cancer cellsalso exhibit aberrant signaling of many redox-sensitive path-ways, most notably the MAP kinases (3), but the role ofROS and redox-regulated kinases in Prdx regulation is notunderstood.

Several studies have shown that breast cancer cells haveelevated levels of peroxiredoxins (10, 12, 16). While expres-sion studies in clinical samples are important, a comparisonof relevant cell lines can be a valuable tool for the dissectionof this type of question. Toward this end, a previous studyfrom our laboratory compared Prdx1 and Prdx6 expressionbetween the MCF-7 adenocarcinoma cell line and the non-cancerous MCF10A cell line and found significant overex-pression of Prdx1 (31). A separate study from Bae et al. alsodemonstrated overexpression of Prdx1 and Prdx2 (24), al-though other Prdxs were not examined. Since few studieshave investigated the entire set of Prdxs in these cells, andlittle is known about the regulators of these proteins in breastcancer, we sought to use these cell lines to analyze expres-sion of the whole Prdx family and investigate possible mech-anisms of regulation.

MATERIAL AND METHODS

Cell cultureThe MCF-7 mammary adenocarcinoma cell line (ATCC)was cultured in ATCC-formulated Eagle’s minimum essen-tial medium, supplemented with 0.01 mg/mL bovine insulinand 10% fetal bovine serum. The MCF-10A cell line wascultured in MEBM medium containing 0.5 mg/mL hydro-cortisone, 5 mg/mL insulin, 13 mg/mL BPE, and 10 μg/mL

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hEGF. Both cell lines were maintained at 37 ◦C in a 5% CO2atmosphere.

Cell treatmentsMCF-7 and MCF-10A cells were cultured under their nor-mal growth conditions for measurement of basal peroxire-doxin levels. For serum deprivation experiments, MCF-7cells seeded the previous day were rinsed and cultured inthe absence of serum and insulin for 48 hr. For kinase in-hibition experiments, MCF-7 cells were seeded in a 6-wellplate at a density of approximately 50% confluency. Twenty-four hours after seeding, cells were treated with either theMEK inhibitor PD 98059 (Calbiochem) or the JNK inhibitorSP 600125 (ENZO) at a concentration of 25 or 10 μM, re-spectively. Cells were treated with inhibitors for either 8 or24 hr. Because the inhibitor solutions are soluble in DMSO,corresponding DMSO vehicle controls were also prepared ineach experiment. For oxidative stress experiments, cells weretreated with either 0.5 mM NAC or 0.5 mM hydrogen perox-ide for various periods of time.

Real-time PCRTo measure mRNA levels, cells seeded in 96-well plateswere washed in cold PBS and lysed in the wells using theTaqMan R© Gene Expression Cells-to-CT

TM Kit (AppliedBiosystems), according to manufacturer’s recommendations.Lysates were immediately used for reverse transcriptionreactions using the TaqMan R© Gene Expression Cells-to-CT

TM Kit. Twenty-microliter real-time PCR reactionswere performed in triplicate for each reverse transcriptionreaction with each target gene using the TaqMan R© GeneExpression Cells-to-CT

TM Kit reagents and protocol. Re-actions included 1 μL of the appropriate human TaqManassay: Prdx1 (Hs00602020 mH), Prdx2 (Hs03044802 g1),Prdx3 (Hs00428953 g1), Prdx4 (Hs00197394 A1), Prdx5(Hs00738905 g1), Prdx6 (Hs00705355 s1), or β-actin(ACTHs00242273 ml). To ensure that PCR amplificationwas not due to genomic DNA contamination, mock-reversetranscription reactions containing no reverse transcriptasewere conducted for each experiment and used in parallelPCR reactions with each target primer set. In all cases,reactions lacking reverse transcriptase amplified no prod-uct, or resulted in cycle threshold (Ct) values beyond 35and at least seven Cts higher than the positive reactions,representing negligible genomic DNA contamination.Relative expression was calculated using the ��CT method(33), with each target gene normalized against β-actinlevels for the same sample. For each experiment, an ap-propriate sample was selected as calibrator for relativequantification.

Western blottingMCF-7 and MCF10A cells seeded in 6-well plates were usedfor protein analysis. Cells were rinsed with 1 mL 1X PBSand then lysed in 100 μL Mammalian Protein ExtractionReagent (MPER) (Thermo Scientific) according to productspecifications. Total protein was quantified using CoomassieBlue Protein Assay Reagent (BioRad). Equal amounts of pro-

tein (between 20 and 50 μg) were separated on a 10–12%Tris-HCL gel (BioRad) and electrophoretically transferredto a PVDF membrane (BioRad). The blots were blockedin 4% milk in TBST, incubated with primary antibodies,and subsequently processed with the appropriate alkalinephosphatase-conjugated secondary antibody. The chemilu-minescent CDP-Star reagent (GE) was used as a substrate,and blots were imaged with X-OMAT film (Kodak). Anti-bodies used for western blotting include human antibodies toPrdx1, Prdx2, Prdx4, Prdx5, and Prdx6 (Abcam), Prdx3 (Ab-nova), ERK1/2 (Abcam), phospho-ERK1/2 (Abcam), JNK1/2(Abcam), phospho-c-Jun (Cell Signaling), and LC3 (Sigma).An antibody for Gapdh (Sigma) was used as a loading con-trol for all western blots. Band intensity was quantified usingImage J software, and relative intensity differences were nor-malized to Gapdh levels.

ELISA assay (eBioscience)The phospho-ERK1/2 and phospho-c-Jun InstantOne ELISAassays (eBioscience) were used to measure ERK and JNKactivity, respectively. For cell-line comparison, quantifiedlysates were diluted in 1X lysis buffer to 0.2 μg/μL. For ki-nase inhibition experiments, lysates were used directly, andthe final activity was normalized against protein concentra-tion. Lysates were incubated with the appropriate antibodysolutions, and rinsed following product suggestions. After in-cubation with the detection reagent and stop solutions, theabsorbance values were measured at 450 nm, were normal-ized for protein amount, and were compared against the con-trol. Each treatment was conducted in triplicate or quadrupli-cate wells, and each lysate was assayed by ELISA in duplicateor triplicate wells.

MTT assayFor cell proliferation measurements, an MTT assay was used.Briefly, the cell medium was removed and cells were rinsedwith PBS to eliminate traces of phenol red. Cells were incu-bated with 0.5 mg/mL MTT (diluted in phenol red-free me-dia) for 2 hr at 37◦C. This solution was removed from wells,and crystallized MTT was solubilized with acidic isopropanolfor 30 min. The absorbance of solubilized MTT in each wellwas measured at 410 nm. Averages from 4–6 wells were cal-culated for each treatment.

Vybrant assay for cell deathThe Vybrant assay (InVitrogen) was used to measure celldeath in MCF-7 cells grown with or without serum. After 48hr of culture in the appropriate medium, cells were rinsed andstained with propidium iodide and Annexin V according tothe manufacturer’s suggestions, and cells were photographedusing phase contrast microscopy and fluorescence. Sev-eral fields (including hundreds of cells) were quantifiedfor the percentage of cells staining positive for necrosis orapoptosis.

ENZO assayFor measurements of intracellular ROS levels, the to-tal ROS detection assay (ENZO) was used, according to

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Figure 1. mRNA and protein expression of peroxiredoxins in MCF-7 and MCF-10A cells. (A) Prdx1, Prdx2, Prdx3, Prdx4, Prdx5, and Prdx6 mRNAwas measured in both lines by real time PCR in separate triplicate reactions and was normalized to β-actin levels for relative quantification (RQ)between lines. Average RQ of each mRNA (± stdev) in MCF-10A cells, compared to levels in MCF-7 cells, is shown (#p < .0005; ∗p < .05). (B)Prdx protein expression was assayed by western blotting and bands quantified on replicate blots. Corresponding numbers refer to average relativeexpression in MCF-7 cells versus MCF-10A (∗ denotes a statistically significant increase in MCF-7 expression).

manufacturer’s suggestions. Briefly, cells were plated in a 48-well plate and allowed to reattach overnight in normal growthmedia. Cells were treated with nothing, 0.5 mM NAC for30 min, or NAC for 30 min followed by 0.5 mM hydrogenperoxide for 15 min. For ROS detection, the medium was

replaced with the phenol red-free medium containing theROS detection reagent. Cells were incubated at 37◦C for 1hr, washed with wash buffer, and visualized in wash bufferby phase contrast and fluorescence microscopy at 100×. Forquantification of fluorescence, micrographs were analyzed

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Figure 2. Elevated kinase activity and ROS in MCF-7 cells. (A) Total levels of ERK and JNK were measured by western blotting. Correspondingnumbers refer to relative expression in MCF-7 cells versus MCF-10A (+ means present only in MCF-7 cells). (B) Activity of ERK and JNK wasmeasured in each cell line by ELISA. Average activity from triplicate wells is shown (± stdev) for each line (∗∗p < .01). (C–F) Cell lines were culturedunder normal conditions and cells were stained for ROS with the ENZO total ROS detection assay and photographed at 100× by phase contrastmicroscopy (C, E) or fluorescence microscopy (D, F). MCF-7 cells are shown in the left panels, while MCF-10 cells are shown in the right panels.(G) The fluorescence intensity was measured in 9–10 individual cells from micrographs of each line using Image J, and the mean is shown (± stdev)(#p < .0005).

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Figure 3. Effect of serum deprivation on peroxiredoxin expression in MCF-7 cells. MCF-7 cells were cultured in the presence of normal growthmedium (serum) or base medium (serum-free) for 48 hr. Cells grown in the presence of serum (A) or absence of serum (B) were photographedat 100× using phase contrast microscopy. (C) Cell proliferation was measured using an MTT assay. Samples were treated in replicates of fourto six, and average absorbance is shown for each group (± stdev) (∗p < .05). (D) Cell death was measured using the Vybrant assay. The averagepercentage of cells staining positive for necrosis is shown for each group (± stdev). (E) Peroxiredoxin protein levels were measured by westernblotting. Corresponding numbers refer to relative expression in growing cells versus serum-deprived cells (“>” means higher in MCF-7 cells, butnot able to quantify due to nearly undetectable levels in MCF-10A cells).

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Figure 4. Effect of ERK inhibition on peroxiredoxin expression in MCF-7 cells. (A) MCF-7 cells were treated with 25 μM PD or DMSO vehiclecontrol for 24 hr. Western blotting was used to demonstrate pERK levels. Corresponding numbers refer to relative expression in treated cells versuscorresponding control cells. (B) ERK activity was measured in each treatment by ELISA. Average activity from triplicate wells is shown (± stdev)(∗∗p < .01). (C) MCF-7 cells were treated with 25 μM PD or DMSO vehicle control for 8 or 24 hr. Peroxiredoxin expression was measured bywestern blotting. Corresponding numbers refer to relative expression in each treatment versus corresponding control cells (“nq” means unable toquantify due to nearly undetectable levels).

using Image J software. Individual cells, or groups of cells,were measured for mean fluorescence.

Statistical analysisAll quantitative data shown in graphs were analyzed statis-tically using Student’s t-tests. The p value corresponding toeach comparison is indicated on the graph, as noted in thelegend.

RESULTS

Peroxiredoxin expression in MCF-7 and MCF-10A cellsWe first compared mRNA expression of all six peroxiredoxingenes in the MCF-7 breast cancer line and the noncancerousMCF-10A cell line. This data is shown in Figure 1(A). Thecancer line exhibited significantly elevated mRNA expressionof five of the six Prdx genes (Prdx1, Prdx2, Prdx3, Prdx4, andPrdx5). In contrast, there was only a small (20%) increase inPrdx6 in MCF-7 cells. The overexpression of Prdx1–5 in thecancer line was even more dramatic at the protein level, asshown in Figure 1(B), while Prdx6 protein levels were simi-lar between the two cell types. These data show that produc-tion of five of the six Prdx proteins is markedly elevated inMCF-7 cells, and that this upregulation occurs at the mRNAlevel.

Upregulation of MAP kinases and ROS levels in MCF-7 breastcancer cellsWe sought to compare the cancerous and noncancerous celllines for MAP kinase and ROS levels, since both have beendemonstrated to be elevated in breast cancer. Figure 2 showsthe expression of total and active ERK1/2 and JNK1/2 inMCF-7 and MCF-10A cells. As shown in Figure 2(A), MCF-7 cells express both ERK1 and ERK2, while MCF-10A cellsonly express ERK2. The expression of JNK is elevated in thecancer line (Figure 2(A)). In contrast, the activity of bothERK and JNK was significantly elevated in MCF-7 cells, withMCF-10A cells expressing only approximately 60% of theactivity of the cancer line (Figure 2(B)). We also examinedthe intracellular ROS levels in both cell types. MCF-7 cellsgrown under normal growth conditions show higher levelsof ROS than MCF-10A cells under normal growth conditions(Figures 2(C)–(F)). Quantification of mean cellular fluores-cence in a representative experiment is shown in Figure 2(G),demonstrating a significant increase in ROS levels in the can-cer line. Together, these data demonstrate that MCF-7 cellsexhibit elevated MAP Kinase activity and ROS levels, as com-pared with MCF-10A cells.

Effect of growth inhibition on peroxiredoxin expressionin MCF-7 cellsTo begin to sort out possible mechanisms of Prdx upreg-ulation in MCF-7 cells, we examined the effect of serumdeprivation on peroxiredoxin expression. As shown in

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Figures 3(A) and (B), 48-hr serum deprivation of MCF-7cells led to a marked change in cell morphology, includingtransition to a less rounded and more elongated attachedstate. This change was associated with a significant inhibi-tion of cell proliferation, as shown in Figure 3(C), but no sta-tistically significant increase in cell death (Figure 3(D)). Ex-amination of peroxiredoxin expression showed that growthinhibition led to a marked reduction in several peroxire-doxin proteins, namely, Prdx1, Prdx2, Prdx3, and Prdx4(Figure 3(E)). To rule out the possibility of autophagy-induced proteolysis, lysates from serum-deprived cells wereanalyzed for LC3 expression, a marker of autophagy, but noLC3 expression was detected (data not shown).

Effect of MAP kinase inhibition on peroxiredoxin expressionin MCF-7 cellsGiven the upregulation of ERK and JNK activity in MCF-7cells, we sought to determine if selective inhibitors of thesekinases would lead to a downregulation of Prdx expression.As shown in Figure 4(A), treatment of MCF-7 cells withthe MEK inhibitor PD 98069 for 18 hr led to an approxi-mately 80% decrease in p-ERK1/p-ERK2 levels, as measuredby western blotting. Measurement of p-ERK levels by ELISAalso showed a significant reduction in treated cells, confirm-ing that the inhibitor was effective (Figure 4(B)). However,there was no major difference in Prdx expression after 8or 18 hr of inhibitor treatment (Figure 4(C)). To examinethe role of JNK in peroxiredoxin expression, we used theselective inhibitor SP600125. Examination of p-cJUN lev-els by ELISA demonstrated an approximately 50% decreasein inhibitor-treated cells, confirming effectiveness of the in-hibitor (Figure 5(A)). Subsequent analysis of Prdx expressionafter 8 and 24 hr of treatment revealed a slight increase inexpression of several peroxiredoxins (Figure 5(B)). Together,these data suggest no significant effect of ERK inhibition,but a mild Prdx induction upon JNK inhibition, and suggestthat neither pathway is mediating Prdx upregulation in thesecells.

Effect of ROS modulation on peroxiredoxin expressionin MCF-7 cellsTo examine a potential role of ROS in the regulation of Prdxexpression in these cells, we first measured the effect of ex-ternal ROS modulators in these cells. As shown in Figure 6,MCF-7 cells treated with NAC for 30 min led to a notice-able reduction in ROS fluorescence (Figure 6(B) vs Figure6(D)). Subsequent treatment with H2O2 for 15 min caused amarked increase in ROS-fluorescence (Figure 6(D) vs Figure6(F)). These data demonstrate that NAC and H2O2 are ca-pable of modulating intracellular ROS levels in MCF-7 cells.Analysis of Prdx expression in NAC-treated cells revealed nosignificant change in any of the Prdx proteins (Figure 7(A)).In contrast, H2O2 treatment led to a significant increase inall six Prdx proteins after 4 hr, to varying levels, returningto near-normal levels by 18 hr (Figure 7(B)). Together, thesedata confirm that MCF-7 cells upregulate Prdx expression inresponse to hydrogen peroxide.

Figure 5. Effect of JNK inhibition on peroxiredoxin expression inMCF-7 cells. (A) MCF-7 cells were treated with 10 μM SP or DMSOvehicle control for 24 hr. JNK activity was measured in each treatmentby ELISA. Average activity from triplicate wells is shown (± stdev)(∗∗∗p < .001). (B) MCF-7 cells were treated with 10 μM SP or DMSOvehicle control for 8 or 24 hr. Peroxiredoxin expression was measuredby western blotting. Corresponding numbers refer to relative expres-sion in each treatment versus corresponding control cells (“nq” meansunable to quantify due to nearly undetectable levels).

DISCUSSION

In this study, we investigated the expression and regulationof all six mammalian peroxiredoxin proteins in the MCF-7 breast cancer cell line, compared to the noncancerousMCF-10A breast epithelial line. We showed elevated expres-sion of five of the six Prdx genes at the mRNA and proteinlevels. We demonstrated that inhibition of MCF-7 prolifer-ation led to a decrease in several Prdx proteins. We showedthat inhibition of MEK or JNK had no significant effect onPrdx expression. We further demonstrated that while NAC-stimulated ROS reduction in MCF-7 cells had no effect onPrdx expression, H2O2-stimulated ROS induction led to asignificant and transient increase in expression of all six Prdxproteins.

Thus far, no human Prdx mutations have been foundto be associated with cancers, suggesting that the dysreg-ulation of these genes in cancer is likely an epigeneticadaptation to the cancerous state. Our findings are the firstto analyze expression of the entire peroxiredoxin family ina well-established breast cancer cell line. Our observation

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Figure 6. ROS levels in MCF-7 cells. Cells were treated with nothing (A, B), 0.5 mM NAC (C, D), or 0.5 mM NAC followed by 15 min with 0.5 mMhydrogen peroxide (E, F). Cells were stained for ROS with the ENZO total ROS detection assay and photographed at 100× by phase contrastmicroscopy (A, C, E) or fluorescence (B, D, F). (G) The mean fluorescence intensity was measured in three confluent equal-sized micrograph areasusing Image J, and the mean is shown (± stdev). Statistical comparisons were made between control versus NAC, and NAC versus NAC + H2O2(∗p < .05).

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Figure 7. Effect of ROS modulation on peroxiredoxin expression in MCF-7 cells. (A) MCF-7 cells were treated with 0.5 mM NAC for 4 or 18 hr andperoxiredoxin expression was measured by western blotting. Corresponding numbers refer to relative expression in each treatment versus controlcells (“nq” means unable to quantify due to nearly undetectable levels). (B) MCF-7 cells were treated with 0.5 mM hydrogen peroxide for 4 or 18hr, and peroxiredoxin expression was measured by western blotting. Corresponding numbers refer to relative expression in each treatment versuscontrol cells.

that five of the six family members are overexpressed at thetranscriptional level not only supports the notion that Prdxelevation may be a mechanism to evade ROS-induced celldeath but also suggests possible coordinate regulation of thefamily by some transcriptional mechanism. Noh et al. foundelevated expression of Prdx1, Prdx2, and Prdx3 in a large ma-jority of breast cancer tissues analyzed, but the study did notinvestigate other Prdxs (10). Karihtala et al. analyzed expres-sion of all six Prdxs in a large number of normal and breastcancer tissues and found significant overexpression of Prdx3,Prdx4, and Prdx5 (12). Together, these data suggest that Prdxoverexpression in MCF-7 cells is consistent with data frompatient samples, and can serve as a useful model system forinvestigation of potential mechanisms.

It is of interest that Prdx6 is the only family member that isnot elevated in MCF-7 cells. Indeed, Prdx6 is a unique mem-ber of the family, in that it is the only peroxiredoxin withphospholipase A(2) activity. Previous studies reported thatPrdx6 expression was elevated in a highly metastatic mam-mary adenocarcinoma cell line (compared to its parentalcounterpart) and that expression correlated with metastaticpotential (16). Furthermore, Prdx6 was also recently impli-cated in invasion and metastasis of lung cancer cells, andthe invasiveness was attributable to its phospholipase activity(34). Therefore, it is possible that this protein has a uniquerole in carcinogenesis.

Our serum deprivation experiments demonstrate thatPrdx1–4 overexpression is regulated by the proliferative stateof the cell. Our data also show that this regulation is notdue to increased cell death. Therefore, this downregulationmay be a consequence of the growth-suppressed state of thecells, or may indicate a functional role for Prdxs in MCF-7 cell proliferation. There is growing evidence supporting arole for peroxiredoxins in regulating cancer cell prolifera-

tion. Cha et al. found that Prdx1 expression in breast can-cer tissues correlated with tumor grade (35); another recentstudy demonstrated that Prdx3 expression correlated withPCNA immunostaining, a proliferative marker, in breast can-cer patient samples (36). This study also found that Prdx3suppression in MDA-MB-231 cells induced cell cycle arrest,suggesting a functional role in mediating proliferation sig-nals (35). Furthermore, Prdx4 was shown to mediate theextra 16α-hydroxyestrone-induced proliferation of MCF-7 cells (37). Together, these observations suggest thatbreast cancer cell proliferation may be modulated by Prdxexpression.

While much attention has been paid to the biochemicalproperties and enzymatic activities of peroxiredoxins in can-cer cells, relatively little research has focused on understand-ing the molecular basis of their upregulation. We decided tofocus on ERK and JNK as potential candidates for such up-regulation in MCF-7 cells given their known roles as stress-activated protein kinases. While we showed no statisticallysignificant effect of ERK or JNK inhibition on Prdx regu-lation, the contribution of these kinases might have beenmasked by the extent of our kinase suppression or period an-alyzed. However, it is also possible that these kinases do notplay a role in the basal Prdx upregulation in MCF-7 cells, butmay play an important role in response to external oxidativestresses. Further analysis of these and other redox-sensitivekinase pathways is necessary.

Despite the increased oxidative stress environment ofMCF-7 cells, as compared with MCF-10A cells, we found noeffect of ROS suppression on Prdx expression. These datamay suggest that intracellular ROS levels are not directly in-volved in Prdx upregulation in these cells, or alternatively,that the magnitude and extent of ROS suppression in ourstudy was not sufficient to reveal such regulation. Although

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suppression of ROS by NAC was enough to reduce intracel-lular ROS levels, it is likely that many pathways dysregulatedin these cells by their oxidative state or oncogenic activationwould remain aberrantly regulated. Since Prdxs may be indi-rectly regulated by such pathways, it is difficult to interpretthe significance of these findings and the role of ROS in Prdxupregulation.

However, elevation of ROS by hydrogen peroxide was suf-ficient to transiently and markedly upregulate expression ofall of the Prdx proteins in MCF-7 cells, suggesting that theseproteins can, in fact, be regulated by oxidative stress in thesecells. Bae et al. showed that exposure of these cells to hydro-gen peroxide for 24 hr led to a reduction in expression, butthese experiments did not examine earlier time points, andthe reduction was only observed at higher peroxide concen-trations (24). Further examination of Prdx regulation by ROSmodulation in breast cancer cells versus normal cells will helpto elucidate whether differential mechanisms exist in the can-cerous state.

Together, these data suggest that MCF-7 cells engage sometranscriptional mechanism to increase the expression of mostof the peroxiredoxin family. These data also suggest involve-ment of peroxiredoxin upregulation in cell proliferation, andthat ROS and stress-activated kinases are likely to play an im-portant role in maintaining proper Prdx levels to promotebreast cancer cell survival.

DECLARATION OF INTEREST

The authors report no conflicts of interest. The authors aloneare responsible for the content and writing of the paper.

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