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Toxicology Letters 210 (2012) 332– 337

Contents lists available at SciVerse ScienceDirect

Toxicology Letters

jou rn al h om epa ge: www.elsev ier .com/ locate / tox le t

isphenol A induces leptin receptor expression, creating more binding sites foreptin, and activates the JAK/Stat, MAPK/ERK and PI3K/Akt signalling pathways inuman ovarian cancer cell

nna Ptak ∗, Ewa L. Gregoraszczukepartment of Physiology and Toxicology of Reproduction, Chair of Animal Physiology, Institute of Zoology, Jagiellonian University, Krakow, Poland

r t i c l e i n f o

rticle history:eceived 16 December 2011eceived in revised form 27 January 2012ccepted 3 February 2012vailable online 10 February 2012

eywords:PA

a b s t r a c t

We previously demonstrated that bisphenol A (BPA) promotes proliferation in OVCAR-3 human ovariancancer cells. This study was designed to investigate the effects of BPA on leptin expression and activityin ovarian cancer. Real-time PCR, Western blot analysis and ELISA assays were used to quantify leptinreceptor expression and leptin gene and protein expression after treatment with BPA at doses of 0.2,2, 8 and 20 ng/ml. Our data reveal leptin receptor expression but an absence of leptin gene and proteinexpression in OVCAR-3 cells. At doses of 8 and 20 ng/ml, BPA had stimulatory effects on leptin receptorgene and protein expression. Leptin and BPA alone stimulated cell proliferation but BPA did not potentiate

eptineptin receptorroliferationVCAR-3

leptin activity. Similarly to leptin, but with different kinetics and duration, BPA induced phosphorylationof Stat3, ERK1/2 and Akt. In co-treatment experiments, the timing of protein phosphorylation representedan additive effect of BPA and leptin treatment.

In conclusion, taking into consideration limitation of in vitro study, whether BPA by creating morebinding sites for leptin and extending the time of leptin-induced Stat3, ERK1/2 and Akt phosphorylation,can potentiated leptin action in cancer cells, require confirmation by in vivo study.

. Introduction

A number of studies have identified an association betweenbesity and hormone-dependent cancers, including endometrialCalle and Thun, 2004), breast (Rapp et al., 2005) and ovarian can-er (Olsen et al., 2007). A recent epidemiological study has foundhat among women who have never used menopausal hormoneherapy, the association between obese women and ovarian cancers increased compared with women of normal weight (Leitzmannt al., 2009). Although a hormonal mechanism has been suggesteds a link between ovarian cancer and obesity, at present, a cleariological explanation for risk association between obesity andvarian cancer is not fully known. Several studies have addressedhe possible role of leptin, a hormone produced by adipose tissue,n cancer development and progression. Serum leptin levels areositively associated with the occurrence of endometrial (Petridou

t al., 2002; Yuan et al., 2004) and breast cancer (Cleary et al., 2003;ieudonne et al., 2002).

∗ Corresponding author at: Department of Physiology and Toxicology of Repro-uction, Institute of Zoology, Jagiellonian University, Gronostajowa 9, 30-387rakow, Poland. Tel.: +48 12 6645101; fax: +48 12 6445004.

E-mail address: anna.ptak@uj.edu.pl (A. Ptak).

378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2012.02.003

© 2012 Elsevier Ireland Ltd. All rights reserved.

Leptin exerts its biological activity through the leptin receptorOb-R, which belongs to the cytokine receptor family. The bind-ing of leptin to its receptor activates different signalling pathways,including the Janus-activated kinase/signal transducers and activa-tors of transcription (JAK/Stat), as well as mitogen-activated proteinkinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3K/Akt)pathways (Tartaglia et al., 1995; Frühbeck, 2006). Leptin and Ob-Rare over-expressed in invasive breast carcinomas (Garofalo et al.,2006; Ishikawa et al., 2004) and Ob-R is over-expressed in 59.2%of epithelial ovarian cancers, which is significantly associated withpoor progression-free survival (Uddin et al., 2009).

Bisphenol A, one of the most ubiquitous endocrine disruptors,has been chosen as a chemical model for xenoestrogen action(Welshons et al., 2006). BPA has been detected in the serum,milk, saliva, urine, follicular fluid and adipose tissue of humansat nanomolar concentrations. Many studies in the United States,Europe, and Japan have documented BPA levels ranging from0.2 to 18.9 ng/ml in adult and fetal plasma human samples (seereview Dekant and Völkel, 2008). Higher concentrations of BPAhave been detected in obese women as compared to non-obesewomen (Takeuchi et al., 2004). As a mitogen, BPA induces suscepti-

bility to neoplastic transformation (Durando et al., 2007; Keri et al.,2007). Recently, an association of environmental chemicals withthe development of obesity has been proposed (Baillie-Hamilton,2002; Heindel, 2003, Newbold et al., 2008, 2009). Phrakonkham

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t al. (2008) reported that BPA induced mRNA expression of leptinn the 3T3-L1 preadipocyte cell line.

In our previous study, we showed that BPA promoted pro-iferation in OVCAR-3 human epithelial ovarian cancer cells byp-regulating genes associated with the cell cycle (Ptak et al., 2011).he present study investigates the effect of BPA on leptin and Ob-Rxpression in epithelial ovarian cancer cells (OVCAR-3) and analy-es the effect of leptin, BPA or both on cell proliferation by assessinghe activation statuses of the JAK/Stat, MAPK/ERK1/2 and PI3K/Aktignalling pathways.

. Materials and methods

.1. Cell culture and chemicals

The human ovarian epithelial carcinoma cell line OVCAR-3 was obtained fromhe American type culture collection (Manassas, VA, USA). Cells were routinely cul-ured in RPMI 1640 medium without phenol red and supplemented with 50 U/mlenicillin, 50 �g/ml streptomycin, and 10% heat inactivated fetal bovine serum (FBS)PAA Laboratories GmbH, Colbe, Germany) in a humidified incubator with 5% CO2

t 37 ◦C. Cells were switched to medium without serum 24 h before each experi-ent. Human leptin and trypan blue were obtained from Sigma Chemical Co. (St.

ouis, MO, USA). Bisphenol A (AccuStandard Inc., New Haven, CT, USA) was dis-olved in absolute ethanol. The final concentration of ethanol in the medium was.01% ethanol. At this concentration, ethanol had no effect on cell proliferation (dataot shown). Control cells were treated with an equivalent volume of solvent.

.2. Real-time PCR analysis of leptin receptor gene expression

The cells were seeded in 96-well culture plates at a density of 2 × 104 cells/welln RPMI 1640 alone or with four different doses of BPA (0.2, 2, 8 and 20 ng/ml)or 24 h. Total RNA isolation and cDNA synthesis were performed using the Taq-

an Gene Expression Cells-to-CT kit (Applied Biosystems, Foster City, CA, USA)ccording to the manufacturer’s protocol. The lysis solution contained DNase I toemove genomic DNA during cell lysis. The resulting pre-amplified cDNA prepa-ations were analysed by real-time PCR in a StepOnePlus Real-time PCR SystemApplied Biosystems) using the TaqMan Gene Expression Assay in combinationith the TaqMan Gene Expression Master Mix containing ROX (Applied Biosys-

ems) according to the manufacturer’s instructions. The PCR conditions were asollows: incubation for 2 min at 50 ◦C, followed by incubation for 10 min at 95 ◦C,nd finally 40 cycles (denaturation step: 15 s at 95 ◦C; annealing/elongation step:0 s at 60 ◦C). Duplicate negative controls without cDNA showed no DNA contamina-ion. The relative expression levels of leptin receptors (Hs00174497 m1) and leptinHs00174877 m1) were normalised to 18S rRNA (Hs99999901 s1) (�Ct) to com-ensate for differences in the amount of cDNA and were calculated using the 2−��Ct

ethod (Livak and Schmittgen, 2001). We used the TaqMan Gene Expression AssayHs00174877 m1), which coded the leptin (NCBI Reference Sequence: NM 000230),nd the TaqMan Gene Expression Assay (Hs00174497 m1), which coded the longesttranscript variant 1; NM 002303) and two of the shortest (transcript variant 2 and; NM 001003680; NM 001003679) splice variants of the leptin receptor.

.3. Western blot analysis

.3.1. Leptin receptor protein expressionFor leptin and leptin receptor protein analysis the cells were seeded in 60 mm

ishes at a density of 2 × 106 cells/dish in RPMI 1640 alone or with RPMI 1640 plusour different doses of BPA (0.2, 2, 8 and 20 ng/ml) for 24 h. The cells were washedith ice-cold PBS and lysed in ice-cold buffer. The lysate protein concentrationsere determined using the Bradford assay. Sixty micrograms of protein from each

reatment group were separated by 7% and 15% SDS-PAGE for the leptin receptornd leptin, respectively, and transferred to PVDF membranes using a Bio-Rad Mini-rotean 3 apparatus (Bio-Rad Laboratories, Inc., USA). The blots were blocked for

h with 5% BSA and 0.1% Tween 20 in 0.02 M TBS buffer. The blots were incubatedvernight at 4 ◦C with antibodies specific for the human short and long forms ofb-R (sc-8391) (Santa Cruz Biotechnology Inc, CA, USA), human leptin (L 3160) and-actin (A5316) (Sigma Chemical Co., MO, USA). After incubation with the primaryntibody, the membranes were washed three times and incubated for 1 h with aorseradish peroxidase-conjugated secondary antibody (P0447) (DakoCytomation,enmark). Immunopositive bands were visualised using Western Blotting Luminoleagents (sc-2048) (Santa Cruz Biotechnology Inc, CA, USA) and quantified usingensitometry analysis (EasyDens, Cortex Nowa, Poland).

.3.2. Stat3, ERK1/2 and Akt protein expression

To study leptin receptor signalling, the cells were treated with 40 ng/ml of lep-

in or 8 ng/ml of BPA alone or in combination for 5, 15 and 30 min, and 1, 2, 4, and 24 h. Thirty micrograms of protein from each treatment group were sep-rated by 10% SDS-PAGE. Blots were incubated overnight at 4 ◦C with antibodiespecific for phospho-Stat3 (Tyr705) (#9131), Stat3 (#9132), phospho-p44/42 MAPK

y Letters 210 (2012) 332– 337 333

(#9101), p44/42 MAPK (#9102), phospho-Akt (Ser473) (#9271) and Akt (#9272)(Cell Signaling Technology, Danvers, MA, USA). After incubation with the primaryantibody, the membranes were washed three times and incubated for 1 h witha horseradish peroxidase-conjugated secondary antibody (#7074) (Cell SignalingTechnology). Immunopositive bands were visualised using Western Blotting Lumi-nol Reagents (sc-2048) (Santa Cruz Biotechnology Inc., CA, USA) and quantified usingdensitometry analysis (EasyDens, Cortex Nowa, Poland).

2.4. Leptin assay

The cells were seeded in 12-well culture plates at a density of 5 × 105 cells/wellin RPMI 1640 alone or in RPMI 1640 plus four different doses of BPA (0.2, 2, 8and 20 ng/ml) for 24 h. After 24 h, the medium was collected and frozen at −70 ◦C.Cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed inice-cold lysis buffer (PBS pH 7.2, 0.5% Tween-20, 1 nM EDTA and 1 nM phenyl-methylsulfonyl fluoride (PMSF)). Leptin concentrations were quantified usingthe enzyme immunoassay Human Leptin ELISA (BioVendor GmbH, Heidelberg,Germany) according to the manufacturer’s instructions. The sensitivity of the assaywas 0.2 ng/ml, and the linear measuring range was 0–50 ng/ml. The absorbance val-ues were measured at 450 nm using an ELISA reader (ELx808 BIO-TEK Instruments,USA).

2.5. Cell proliferation

Cell proliferation was measured by alamarBlue Cell Viability Reagent (Invitro-gen, Paisley, UK) according to the manufacturer’s instructions. The cells were seededin 96-well culture plates at a density of 1.5 × 104 cells/well in RPMI 1640 and thenincubated with five different doses (10, 20, 50, 100 and 200 �M) of AG490, PD098059(Sigma Chemical Co.) and LY294002 (Cell Signaling Technology). For combinationtreatments, (1) cells were treated with two different doses (2 and 40 ng/ml) of leptinalone or two different doses (0.2 and 8 ng/ml) of BPA alone, or both compounds incombination for 72 h and (2) pretreated with 50 �M AG490, 10 �M LY294002, or100 �M PD098059 for 1 h followed by the addition of 40 ng/ml of leptin, 8 ng/mlof BPA or both for 72 h. Afterwards, alamarBlue was aseptically added to the wellsin an amount equal to 10% of the incubation volume. After incubating for 4 h, thefluorescence of the medium was measured at 540 and 590 nm wavelengths using aBioTek FLx800 plate reader (BIO-TEK Instruments, USA). Cell-free RPMI 1640 with10% alamarBlue was used as a blank for the fluorometric measurements.

2.6. Statistical analysis

ELISA and real-time PCR experiments were repeated three times (n = 3) and eachsample was run in quadruplicate. Data were plotted as the mean ± S.E.M. Westernblot experiments were repeated three times. Densitometry data were represented asthe mean ± S.D. Statistical analyses were performed using GraphPad Prism 5. Datawere analysed using the one-way analysis of variance (ANOVA) test followed byTukey’s honestly significant difference (HSD) test. A p-value of <0.05 was consideredstatistically significant.

3. Results

3.1. The effect of BPA on leptin receptor gene and proteinexpression

The expression of Ob-R mRNA was quantified using primers thatamplify the long variant and two short splice variants of the leptinreceptor. No change in Ob-R mRNA expression was observed aftertreatment with 0.2 and 2 ng/ml of BPA, but a 2-fold increase wasnoted in cells exposed to 8 and 20 ng/ml of BPA (Fig. 1, p < 0.001).

The expression of Ob-R protein was examined using the anti-body recognising the short (Ob-Rt) and long (Ob-Rb) forms ofOb-R. Both short and long isoforms of Ob-R protein are present inOVCAR-3 cells. BPA had no effect on the protein expression of theshort isoform of ObR; however, a statistically significant increaseof the long isoform was observed following treatment with 8 and20 ng/ml of BPA (Fig. 2, p < 0.001).

3.2. The effect of BPA on leptin gene and protein expression andleptin protein secretion

Real-time PCR, Western blot and ELISA analyses showed a lackof basal leptin gene expression, protein expression and proteinsecretion in OVCAR-3 cells (data not shown).

334 A. Ptak, E.L. Gregoraszczuk / Toxicology Letters 210 (2012) 332– 337

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Fig. 1. The effect of BPA (0.2, 2, 8 and 20 ng/ml) on leptin receptor (Ob-R) mRNAexpression. Ob-R mRNA levels were determined by real-time PCR and expressed asrelative values compared to those of cells under basal conditions. The expressionosm

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Based on the above dose response experiments, inhibitors wereadded at doses of 50 �M for AG490, 100 �M for PD098059 and10 �M for LY294002. As shown in Fig. 4A, leptin and BPA alone,

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.3. Involvement of Stat3, ERK1/2 and Akt in leptin- andPA-induced cell proliferation

To choose the appropriate dose of JAK/Stat, Ras/ERK1/2 andI3K/Akt pathway inhibitors, cells were incubated for 72 h withncreasing concentrations (10–200 �M) of AG490 (JAK2 inhibitor),

D098059 (ERK1/2 inhibitor) or LY294002 (PI3K inhibitor). Ashown in Fig. 3, all inhibitors decreased basal cell proliferation in aose-dependent manner (AG490- 66, 62, 58, 36 and 28% of control;

ig. 2. The effect of BPA (0.2, 2, 8 and 20 ng/ml) on leptin receptor protein expression.he expression of Ob-R protein was examined using the antibody that recogniseshe short and long forms of Ob-R. ˇ-Actin was used as a loading control for Westernlot analysis. The blot is representative of three experiments. Ob-R densitometryesults were normalised to ˇ-actin loading controls to obtain an Ob-R:ˇ-actin ratio.alues are the mean ± S.D. All means marked with (***) are significantly different

rom the control (p < 0.001).

OVCAR-3 cells. Cells were treated with different doses of inhibitors (10, 20, 50, 100and 200 �M). Values are the mean ± S.E.M. All means marked with (*p < 0.05), and(***p < 0.001) are significantly different from the control.

PD098059- 86, 71, 70, 59 and 50% of control; LY294002- 59, 50, 36,22 and 7% of control, respectively, after 10, 20, 50, 100 and 200 �M(Fig. 3, p < 0.05, p < 0.001).

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Fig. 4. The effects of leptin and BPA alone and co-treatment on cell proliferation.(A) OVCAR-3 cells were treated with BPA (0.2 and 8 ng/ml), leptin (2 and 40 ng/mL),or both compounds in combination for 72 h (B) OVCAR-3 cells were pretreated withAG490 (50 �M), PD098059 (100 �M) or LY294002 (10 �M) for 1 h, then the cellswere treated with leptin (40 ng/ml) or BPA (8 ng/ml) alone or both and an alamar-Blue assay was performed. Values are the mean ± S.E.M. All means marked with(*p < 0.05), and (***p < 0.001) are significantly different from the control.

A. Ptak, E.L. Gregoraszczuk / Toxicology Letters 210 (2012) 332– 337 335

Fig. 5. The effects of leptin, BPA or both on the activation of Stat-3, ERK1/2 and Akt in OVCAR-3 cells. (A) Western blot analysis was performed following treatment withleptin, BPA or both in a time-dependent manner (5, 15, and 30 min and 1, 2, 4, 6 and 24 h). Total Stat-3, ERK1/2 and Akt were used to normalise the level of phosphorylatedS s. (B)

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tat-3, ERK1/2 and Akt, respectively. The blot is representative of three experimentn a three independent experiments with similar results. The intensities of signal**p < 0.01), and (***p < 0.001) are significantly different from the control.

ncreased OVCAR-3 cell proliferation (120 ± 15%, 124 ± 8%; p < 0.05nd 164 ± 18%, 162 ± 25%; p < 0.001, respectively after low and highoses). In co-treatment experiments no additional effect was noted.ll inhibitors that were tested decreased cell proliferation to 60%f control cells and 50% of BPA, leptin and both stimulated cellsFig. 4B, p < 0.001).

Stat3, ERK1/2 and Akt protein expression and their activationtatuses in OVCAR-3 cells were determined by Western blot. Ashowed in Fig. 5, the addition of leptin increased the phosphoryla-ion of Stat3 from 15 min to 4 h after treatment, while BPA increasedt from 5 min to 2 h and co-treatment increased it from 5 min to 4 h.RK1/2 phosphorylation was also stimulated by leptin (from 5 mino 30 min), BPA (from 5 min to 2 h) and co-treatment with bothfrom 5 min to 2 h). Finally, Akt phosphorylation was also increasedfter leptin treatment (from 30 min to 4 h), BPA (from 5 min to

0 min) and after co-treatment (from 5 min to 4 h). Immunoblotsere reblotted with antibodies against Stat3, Akt and ERK1/2 to

nsure that the increase in phosphorylation was not due to thencreased total protein expression.

Densitometry of pStat-3/Stat3, p-ERK/ERK, p-Akt/Akt bands ratio are shown basede expressed as arbitrary units. Values are the mean ± S.D. All means marked with

4. Discussion

The data presented in this study confirmed the presence of leptinreceptor gene expression in OVCAR-3 cells (Choi et al., 2005) and,for the first time, demonstrated leptin receptor protein expressionin OVCAR-3 cells. Additionally, results from real-time PCR, West-ern blot and ELISA studies suggest that leptin is not expressedin OVCAR-3 cells. The results of our research differ from thoseof a recent study by Uddin et al. (2009), who detected leptinimmunohistochemically in 89.5% of tested explants of epithelialovarian cancer. The differences can likely be attributed to our useof an ovarian cancer cell line versus the primary tissue samplesused for the previous study. The discrepancy in the results mayalso be due to the different methods used in these two stud-ies. Real-time PCR provides information on the synthesis of leptin

mRNA in a single cell population when using a cell line, whilein a heterogeneous tissue explants, one cannot exclude that lep-tin, a secretory protein, was transported via the plasma. However,our research and the studies by Uddin et al. (2009) do not differ

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36 A. Ptak, E.L. Gregoraszczuk / Tox

n regards to the expression of leptin receptors, a non-secretoryrotein.

In the second part of our experiment, we showed that BPAn concentrations of 0.2 and 2 ng/ml, a level noted in the ovar-an follicular fluid of healthy women (Ikezuki et al., 2002), doesot alter leptin receptor mRNA and protein expression, whereast concentrations of 8 and 20 ng/ml, the level found in womenPadmanabhan et al., 2008), increased leptin receptor mRNA androtein expression. We showed the stimulatory effects of BPA (Ptakt al., 2011) or leptin alone (unpublished data) on the prolifera-ion of OVCAR-3 cells. The fact that BPA increases leptin receptorene and protein expression, as observed in the present study, sug-ests a possible intensification of leptin activity in ovarian cancerells. To test this hypothesis, we focused on a known mechanismf leptin for inducing the proliferation of target cells. The biologi-al activities of leptin in target tissues are carried out through itsnteraction with its specific receptors, leading to the secondaryctivation of the JAK/Stat3, MAPK/ERK, and PI3K/Akt pathways.resented data showed that while BPA alone and leptin alonencreased cell proliferation, in co-treatment experiments no addi-ional effect was noted. Using chemical inhibitors of the JAK/Stat3,

APK/ERK and PI3K/Akt pathways, we have shown the inhibitionf the proliferative effects of BPA, leptin and both. Activation ofhese second messenger systems under the influence of BPA andeptin were confirmed by Western blot analysis. Our results indi-ate a difference in the kinetics and duration of Stat3, ERK1/2 andkt phosphorylation under the influence of BPA or leptin. Thisesult is in agreement with Choi et al., who used BG-1 humanvarian cancer cells to demonstrate a dose-dependent effect of lep-in on cell proliferation by activation of the ERK pathway (2005),s well as increased Stat3 and Akt signalling Choi et al. (2011).he activation of multiple pathways by leptin was also observedn endometrial (Gao et al., 2009) and liver cancer (Saxena et al.,007). There are also data showing that BPA triggers a rapid bio-

ogical response through the phosphorylation of Stat3, ERK1/2 andkt. Canesi et al. (2005) observed that BPA led to an increase intat3 and ERK1/2 phosphorylation in mussel hemocytes. Masunot al. (2005) demonstrated that treatment with BPA increased theevel of phospho-Akt, and LY294002 blocked this effect in 3T3-L1ells. Recently, Dong et al. (2011) demonstrated that BPA induces aapid activation of ERK1/2 in both ER˛/ˇ-positive and -negativereast cancer cells and that this effect is not blocked with anR antagonist, suggesting the involvement of an ER-independentathway. Also, Park et al. (2009) observed that similar concentra-ions of BPA stimulated cell proliferation and phosphorylation ofRK1/2 in BG-1 ovarian epithelial cancer cells. Results presentedn this paper indicated a similar mechanism of action of BPA inVCAR-3 ovarian epithelial cancer cells.

The most interesting and novel finding of the present study washe observation in co-treatment experiments that the activationimes of secondary messengers spanned the individual times of BPAnd leptin. The results suggested that leptin and BPA activate theame intracellular signalling pathways to induce cell proliferation.he lack of an additive, potentiating or synergistic effect of BPAnd leptin co-treatment on proliferation can be explained by anbsence of change in total target protein expression. To our knowl-dge, there are no data showing that two different compounds thathare the same intracellular pathway targets can activate cell prolif-ration or apoptosis in ovarian cancer cells. An indirect explanationf our suggestion can be drawn from the data of Chen et al. (2006)ho showed a synergistic effect of leptin with 17ˇ-estradiol on theroliferation of ZR-75-1 human breast cancer cells. These authors

uggested that leptin and 17ˇ-estradiol did not completely sharehe same intracellular signalling pathways and their cooperationtimulated further cell proliferation. Therefore, it is reasonable toypothesise that BPA, which is an estrogenic endocrine disruptor

y Letters 210 (2012) 332– 337

that influences various physiological functions at very low doses,and 17ˇ-estradiol may interact with leptin differently and inducedifferent signalling pathways. One possible explanation stems fromdata which showed that BPA binds to estrogen-related receptor-gamma (ERR-�), which is a member of the human estrogen relatedreceptor (ERR) subfamily (Okada et al., 2008; Liu et al., 2010), while17ˇ-estradiol does not bind to any of the ERR family members(Horard and Vanacker, 2003).

In summary, our presented results clearly showed that BPAincreased leptin receptor expression and induced proliferation,similarly to leptin, by activation of the Stat3, ERK1/2 and Akt sig-nalling pathways. Lack of synergistic effect seen with co-treatmentis probably due to activation of the same intracellular signallingpathways by both BPA and leptin. Taking into consideration limita-tion of in vitro study, whether BPA by creating more binding sitesfor leptin and extending the time of leptin -induced Stat3, ERK1/2and Akt phosphorylation, potentiated leptin action in cancer cells,require confirmation by in vivo study.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgment

This work was supported by the Polish Committee for Scien-tific Research from 2010 to 2013 as a project 0050/B/PO1/2010/38(Poland).

References

Baillie-Hamilton, P.F., 2002. Chemical toxins: a hypothesis to explain the globalobesity epidemic. J. Altern. Complement. Med. 8, 185–192.

Calle, E.E., Thun, M.J., 2004. Obesity and cancer. Oncogene 23, 6365–6378.Canesi, L., Betti, M., Lorusso, L.C., Ciacci, C., Gallo, G., 2005. ‘In vivo’ effects of Bisphenol

A in Mytilus hemocytes: modulation of kinase-mediated signaling pathways.Aquat. Toxicol. 71, 73–84.

Chen, C., Chang, Y.C., Liu, C.L., Chang, K.J., Guo, I.C., 2006. Leptin-induced growth ofhuman ZR-75-1 breast cancer cells is associated with up-regulation of cyclin D1and c-Myc and downregulation of tumor suppressor p53 and p21WAF1/CIP1.Breast Cancer Res. Treat. 98, 121–132.

Choi, J.H., Park, S.H., Leung, P.C., Choi, K.C., 2005. Expression of leptin receptors andpotential effects of leptin on the cell growth and activation of mitogen-activatedprotein kinases in ovarian cancer cells. J. Clin. Endocrinol. Metab. 90, 207–210.

Choi, J.H., Lee, K.T., Leung, P.C., 2011. Estrogen receptor alpha pathway is involvedin leptin-induced ovarian cancer cell growth. Carcinogenesis 32, 589–596.

Cleary, M.P., Phillips, F.C., Getzin, S.C., Jacobson, T.L., Jacobson, M.K., Christensen,T.A., Juneja, S.C., Grande, J.P., Maihle, N.J., 2003. Genetically obese MMTV-TGF-alpha/Lep(ob)Lep(ob) female mice do not develop mammary tumors. BreastCancer Res. Treat. 77, 205–215.

Dekant, W., Völkel, W., 2008. Human exposure to bisphenol A by biomonitoring:methods, results and assessment of environmental exposures. Toxicol. Appl.Pharmacol. 228, 114–134.

Dieudonne, M.N., Machinal-Quelin, F., Serazin-Leroy, V., Leneveu, M.C., Pecquery,R., Giudicelli, Y., 2002. Leptin mediates a proliferative response in human MCF7breast cancer cells. Biochem. Biophys. Res. Commun. 293, 622–668.

Dong, S., Terasaka, S., Kiyama, R., 2011. Bisphenol A induces a rapid activationof Erk1/2 through GPR30 in human breast cancer cells. Environ. Pollut. 159,212–228.

Durando, M., Kass, L., Piva, J., Sonnenschein, C., Soto, A.M., Luque, E.H., Munoz-de-Toro, M., 2007. Prenatal bisphenol A exposure induces preneoplastic lesions inthe mammary gland in Wistar rats. Environ. Health Perspect. 115, 80–86.

Frühbeck, G., 2006. Intracellular signalling pathways activated by leptin. Biochem.J. 393, 7–20.

Gao, J., Tian, J., Lv, Y., Shi, F., Kong, F., Shi, H., Zhao, L., 2009. Leptin induces functionalactivation of cyclooxygenase-2 through JAK2/STAT3, MAPK/ERK, and PI3K/AKTpathways in human endometrial cancer cells. Cancer Sci. 100, 389–395.

Garofalo, C., Koda, M., Cascio, S., Sulkowska, M., Kanczuga-Koda, L., Golaszewska, J.,Russo, A., Sulkowski, S., Surmacz, E., 2006. Increased expression of leptin andthe leptin receptor as a marker of breast cancer progression: possible role of

obesity-related stimuli. Clin. Cancer Res. 12, 1447–1453.

Heindel, J.J., 2003. Endocrine disruptors and the obesity epidemic. Toxicol. Sci. 76,247–249.

Horard, B., Vanacker, J.M., 2003. Estrogen receptor-related receptors: orphan recep-tors desperately seeking a ligand. J. Mol. Endocrinol. 31, 349–357.

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kezuki, Y., Tsutsumi, O., Takai, Y., Kamei, Y., Taketani, Y., 2002. Determination ofbisphenol A concentrations in human biological fluids reveals significant earlyprenatal exposure. Hum. Reprod. 17, 2839–2841.

shikawa, M., Kitayama, J., Nagawa, H., 2004. Enhanced expression of leptin andleptin receptor (Ob-R) in human breast cancer. Clin. Cancer Res. 10, 4325–4331.

eri, R.A., Ho, S.M., Hunt, P.A., Knudsen, K.E., Soto, A.M., Prins, G.S., 2007. An evalu-ation of evidence for the carcinogenic activity of bisphenol A. Reprod. Toxicol.24, 240–252.

eitzmann, M.F., Koebnick, C., Danforth, K.N., Brinton, L.A., Moore, S.C., Hollenbeck,A.R., Schatzkin, A., Lacey Jr., J.V., 2009. Body mass index and risk of ovarian cancer.Cancer 115, 812–822.

iu, X., Matsushima, A., Okada, H., Shimohigashi, Y., 2010. Distinction of the bind-ing modes for human nuclear receptor ERRgamma between bisphenol A and4-hydroxytamoxifen. J. Biochem. 148, 247–254.

ivak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data usingreal-time quantitative PCR and the 2(-Delta delta C(T)) method. Methods 25,402–408.

asuno, H., Iwanami, J., Kidani, T., Sakayama, K., Honda, K., 2005. Bisphenol aaccelerates terminal differentiation of 3T3-L1 cells into adipocytes through thephosphatidylinositol 3-kinase pathway. Toxicol. Sci. 84, 319–327.

ewbold, R.R., Padilla-Banks, E., Jefferson, W.N., Heindel, J.J., 2008. Effects ofendocrine disruptors on obesity. Int. J. Androl. 31, 201–208.

ewbold, R.R., Padilla-Banks, E., Jefferson, W.N., 2009. Environmental estrogens andobesity. Mol. Cell. Endocrinol. 304, 84–89.

kada, H., Tokunaga, T., Liu, X., Takayanagi, S., Matsushima, A., Shimohigashi, Y.,2008. Direct evidence revealing structural elements essential for the high bind-ing ability of bisphenol A to human estrogen-related receptor-gamma. Environ.Health Perspect. 116, 32–38.

lsen, C.M., Green, A.C., Whiteman, D.C., Sadeghi, S., Kolahdooz, F., Webb, P.M., 2007.Obesity and the risk of epithelial ovarian cancer: a systematic review and meta-analysis. Eur. J. Cancer 43, 690–709.

admanabhan, V., Siefert, K., Ransom, S., Johnson, T., Pinkerton, J., Anderson, L., Tao,L., Kannan, K., 2008. Maternal bisphenol A levels at delivery: a looming problem?J. Perinatol. 28, 258–263.

ark, S.H., Kim, K.Y., An, B.S., Choi, J.H., Jeung, E.B., Leung, P.C., Choi, K.C., 2009.Cell growth of ovarian cancer cells is stimulated by xenoestrogens through an

y Letters 210 (2012) 332– 337 337

estrogen-dependent pathway, but their stimulation of cell growth appears notto be involved in the activation of the mitogen-activated protein kinases ERK-1and p38. J. Reprod. Dev. 55, 23–29.

Petridou, E., Belechri, M., Dessypris, N., Koukoulomatis, P., Diakomanolis, E., Spanos,E., Trichopoulos, D., 2002. Leptin and body mass index in relation to endometrialcancer risk. Ann. Nutr. Metab. 46, 147–151.

Phrakonkham, P., Viengchareun, S., Belloir, C., Lombès, M., Artur, Y., Canivenc-Lavier,M.C., 2008. Dietary xenoestrogens differentially impair 3T3-L1 preadipocyte dif-ferentiation and persistently affect leptin synthesis. J. Steroid Biochem. Mol. Biol.110, 95–103.

Ptak, A., Wróbel, A., Gregoraszczuk, E.L., 2011. Effect of bisphenol-A on the expres-sion of selected genes involved in cell cycle and apoptosis in the OVCAR-3 cellline. Toxicol. Lett. 202, 30–35.

Rapp, K., Schroeder, J., Klenk, J., Stoehr, S., Ulmer, H., Concin, H., Diem, G., Oberaigner,W., Weiland, S.K., 2005. Obesity and incidence of cancer: a large cohort study ofover 145,000 adults in Austria. Br. J. Cancer 93, 1062–1067.

Saxena, N.K., Sharma, D., Ding, X., Lin, S., Marra, F., Merlin, D., Anania, F.A., 2007. Con-comitant activation of the JAK/STAT, PI3K/AKT, and ERK signaling is involved inleptin-mediated promotion of invasion and migration of hepatocellular carci-noma cells. Cancer Res. 67, 2497–2507.

Takeuchi, T., Tsutsumi, O., Ikezuki, Y., Takai, Y., Taketani, Y., 2004. Positive relation-ship between androgen and the endocrine disruptor, bisphenol A, in normalwomen and women with ovarian dysfunction. Endocr. J. 51, 165–169.

Tartaglia, L.A., Dembski, M., Weng, X., Deng, N., Culpepper, J., Devos, R., Richards, G.J.,Campfield, L.A., Clark, F.T., Deeds, J., Muir, C., Sanker, S., Moriarty, A., Moore, K.J.,Smutko, J.S., Mays, G.G., Wool, E.A., Monroe, C.A., Tepper, R.I., 1995. Identificationand expression cloning of a leptin receptor, Ob-R. Cell 83, 1263–1271.

Uddin, S., Bu, R., Ahmed, M., Abubaker, J., Al-Dayel, F., Bavi, P., Al-Kuraya, K.S., 2009.Overexpression of leptin receptor predicts an unfavorable outcome in middleeastern ovarian cancer. Mol. Cancer 8, 74.

Welshons, W.V., Nagel, S.C., vom Saal, F.S., 2006. Large effects from small exposures.

III. Endocrine mechanisms mediating effects of bisphenol A at levels of humanexposure. Endocrinology 147, 56–69.

Yuan, S.S., Tsai, K.B., Chung, Y.F., Chan, T.F., Yeh, Y.T., Tsai, L.Y., Su, J.H., 2004. Aber-rant expression and possible involvement of the leptin receptor in endometrialcancer. Gynecol. Oncol. 92, 769–775.