effect of bisphenol-a on the expression of selected genes involved in cell cycle and apoptosis in...

6
Toxicology Letters 202 (2011) 30–35 Contents lists available at ScienceDirect Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet Effect of bisphenol-A on the expression of selected genes involved in cell cycle and apoptosis in the OVCAR-3 cell line Anna Ptak , Anna Wróbel, Ewa L. Gregoraszczuk Department of Physiology and Toxicology of Reproduction, Chair of Animal Physiology, Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060, Krakow, Poland article info Article history: Received 18 November 2010 Received in revised form 14 January 2011 Accepted 19 January 2011 Available online 26 January 2011 Keywords: BPA Apoptosis Cell cycle OVCAR-3 abstract To support the argument that bisphenol-A (BPA) poses a risk for ovarian cancer, OVCAR-3 cell line was exposed to environmentally relevant concentration of BPA. Expression of selected genes involved in cell cycle and apoptosis were evaluated by real-time PCR. In a dose-dependent manner, BPA increased OVCAR- 3 cell proliferation and decreased caspase-3 activity, but it had no effect on DNA fragmentation. We noted 1.2–1.5-fold induction of genes responsible for inducing cell proliferation and 1.2–46-fold suppression of genes responsible for inhibition of proliferation. Moreover, 1.6–8-fold suppression of genes involved in the extrinsic apoptotic pathway was observed. In parallel, 1.3–2.5-fold suppression pro-apoptotic genes and 1.6–51-fold induction of pro-survival genes involved in the intrinsic apoptotic pathway were observed. Additionally, 1.7-fold induction of p53 and 5-fold induction of endonuclease G genes involved in CAD-independent DNA fragmentation were noted under the influence of BPA. In conclusion, we hypothesize that induction of p53 and suppression of caspase-3 and 7 gene expression observed in this study activate the DNA repair process. Therefore, despite the observed induction of endo G gene expression, the action of BPA on DNA fragmentation was not observed. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Bisphenol A (BPA) is a small monomer (228 Da) commonly used as plasticizer in the manufacture of polycarbonate plastics and epoxy resins. It is present in a multitude of products, includ- ing the interior coatings of food cans, milk containers, and baby formula bottles, as well as in dental sealants (Welshons et al., 2006). BPA was found in the serum, milk, saliva, and urine of humans at nanomolar concentrations. Many studies in the United States, Europe, and Japan have documented BPA levels ranging from 0.2 to 10 ng/mL (0.5–40 nM) in adult and fetal human serum (Calafat et al., 2005; Sun et al., 2004; Vandenberg et al., 2007; Welshons et al., 2006). Epidemiological studies have highlighted the correlation between the increase of BPA level in the environment and the incidence of cancer in humans (Keri et al., 2007). As a mitogen, BPA has moreover been suggested to induce susceptibility to neo- plastic transformation (Durando et al., 2007; Keri et al., 2007). Studies with human breast cancer cells have yielded inconsistent data with respect to the mitogenic, apoptotic, and transcrip- tional properties of BPA (Dairkee et al., 2008; Diel et al., 2002; Corresponding author. Tel.: +48 126632615; fax: +48 126343716. E-mail address: [email protected] (A. Ptak). Singleton et al., 2006). Betabcourt et al. (2010) demonstrated changes in mammary gland protein expression of vimentin, SPARC, 14–3–3, phospho-AKT c-Raf, phospho-ERKs, and TGF- are con- sistent with increased susceptibility for cancer development in rat prenataly exposure to BPA. There is only one data of Park et al. (2009), who using thymidine incorporation assay, showed that BPA increased BG-1 human ovarian cancer cell prolifera- tion by a genomic effect through ERs. To our knowledge there are lack of data concerning action of BPA on gene expression involved in cell proliferation and apoptosis in ovarian cancer cell. Ovarian cancer that originates from the ovarian surface epithe- lium constitutes 90% of all cancers in the ovaries (Auersperg et al., 2001). In the present study we used human ovarian epithe- lial carcinoma cell line, OVCAR-3 to examine the effects of BPA on expression of selected genes involved in cell cycle and apo- ptosis. 2. Materials and methods 2.1. Reagents RPMI 1640 medium without phenol red, fetal bovine serum (FBS, heat inactivated), penicillin, streptomycin, and trypsin EDTA were obtained from PAA Laboratories GmbH (Colbe, Germany). Trypan blue was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Bisphenol A (AccuStandard Inc., New Haven, CT, USA) was dissolved in absolute ethanol. The final concen- tration of ethanol in the medium in each case was 0.1%. Ethanol at this concentration has no effect on cell proliferation and apoptosis (data not shown). 0378-4274/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2011.01.015

Upload: anna-ptak

Post on 29-Oct-2016

219 views

Category:

Documents


1 download

TRANSCRIPT

Page 1: Effect of bisphenol-A on the expression of selected genes involved in cell cycle and apoptosis in the OVCAR-3 cell line

Ea

AD

a

ARRAA

KBACO

1

uaif2

aEtee

biBpSdt

0d

Toxicology Letters 202 (2011) 30–35

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

ffect of bisphenol-A on the expression of selected genes involved in cell cyclend apoptosis in the OVCAR-3 cell line

nna Ptak ∗, Anna Wróbel, Ewa L. Gregoraszczukepartment of Physiology and Toxicology of Reproduction, Chair of Animal Physiology, Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060, Krakow, Poland

r t i c l e i n f o

rticle history:eceived 18 November 2010eceived in revised form 14 January 2011ccepted 19 January 2011vailable online 26 January 2011

eywords:

a b s t r a c t

To support the argument that bisphenol-A (BPA) poses a risk for ovarian cancer, OVCAR-3 cell line wasexposed to environmentally relevant concentration of BPA. Expression of selected genes involved in cellcycle and apoptosis were evaluated by real-time PCR. In a dose-dependent manner, BPA increased OVCAR-3 cell proliferation and decreased caspase-3 activity, but it had no effect on DNA fragmentation. We noted1.2–1.5-fold induction of genes responsible for inducing cell proliferation and 1.2–46-fold suppressionof genes responsible for inhibition of proliferation. Moreover, 1.6–8-fold suppression of genes involved

PApoptosisell cycleVCAR-3

in the extrinsic apoptotic pathway was observed. In parallel, 1.3–2.5-fold suppression pro-apoptoticgenes and 1.6–51-fold induction of pro-survival genes involved in the intrinsic apoptotic pathway wereobserved. Additionally, 1.7-fold induction of p53 and 5-fold induction of endonuclease G genes involvedin CAD-independent DNA fragmentation were noted under the influence of BPA.

In conclusion, we hypothesize that induction of p53 and suppression of caspase-3 and 7 gene expressionobserved in this study activate the DNA repair process. Therefore, despite the observed induction of endo

tion o

G gene expression, the ac

. Introduction

Bisphenol A (BPA) is a small monomer (228 Da) commonlysed as plasticizer in the manufacture of polycarbonate plasticsnd epoxy resins. It is present in a multitude of products, includ-ng the interior coatings of food cans, milk containers, and babyormula bottles, as well as in dental sealants (Welshons et al.,006).

BPA was found in the serum, milk, saliva, and urine of humanst nanomolar concentrations. Many studies in the United States,urope, and Japan have documented BPA levels ranging from 0.2o 10 ng/mL (0.5–40 nM) in adult and fetal human serum (Calafatt al., 2005; Sun et al., 2004; Vandenberg et al., 2007; Welshonst al., 2006).

Epidemiological studies have highlighted the correlationetween the increase of BPA level in the environment and the

ncidence of cancer in humans (Keri et al., 2007). As a mitogen,PA has moreover been suggested to induce susceptibility to neo-

lastic transformation (Durando et al., 2007; Keri et al., 2007).tudies with human breast cancer cells have yielded inconsistentata with respect to the mitogenic, apoptotic, and transcrip-ional properties of BPA (Dairkee et al., 2008; Diel et al., 2002;

∗ Corresponding author. Tel.: +48 126632615; fax: +48 126343716.E-mail address: [email protected] (A. Ptak).

378-4274/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2011.01.015

f BPA on DNA fragmentation was not observed.© 2011 Elsevier Ireland Ltd. All rights reserved.

Singleton et al., 2006). Betabcourt et al. (2010) demonstratedchanges in mammary gland protein expression of vimentin, SPARC,14–3–3, phospho-AKT c-Raf, phospho-ERKs, and TGF-� are con-sistent with increased susceptibility for cancer development inrat prenataly exposure to BPA. There is only one data of Parket al. (2009), who using thymidine incorporation assay, showedthat BPA increased BG-1 human ovarian cancer cell prolifera-tion by a genomic effect through ERs. To our knowledge thereare lack of data concerning action of BPA on gene expressioninvolved in cell proliferation and apoptosis in ovarian cancer cell.Ovarian cancer that originates from the ovarian surface epithe-lium constitutes 90% of all cancers in the ovaries (Auersperget al., 2001). In the present study we used human ovarian epithe-lial carcinoma cell line, OVCAR-3 to examine the effects of BPAon expression of selected genes involved in cell cycle and apo-ptosis.

2. Materials and methods

2.1. Reagents

RPMI 1640 medium without phenol red, fetal bovine serum (FBS, heatinactivated), penicillin, streptomycin, and trypsin EDTA were obtained from

PAA Laboratories GmbH (Colbe, Germany). Trypan blue was obtained fromSigma Chemical Co. (St. Louis, MO, USA). Bisphenol A (AccuStandard Inc.,New Haven, CT, USA) was dissolved in absolute ethanol. The final concen-tration of ethanol in the medium in each case was 0.1%. Ethanol at thisconcentration has no effect on cell proliferation and apoptosis (data notshown).
Page 2: Effect of bisphenol-A on the expression of selected genes involved in cell cycle and apoptosis in the OVCAR-3 cell line

gy Letters 202 (2011) 30–35 31

2

Aic(3rs

2

pBawo2auVby

2

2af−e1cPp(mIocMVei

2

EtBstDpuaatVe

2

iftoaSmtLARP

0.8

1

1.2

1.4

1.6

** **

******

0

0.2

0.4

0.6

BPA 1nMC BPA 10nM BPA 40nM BPA 100nM

BrdU

Inco

rpor

a�on

(Rel

a�ve

Abs

orba

nce

A. Ptak et al. / Toxicolo

.2. Cell culture and treatments

The human ovarian epithelial carcinoma cell line OVCAR-3 was obtained frommerican Type Culture Collection (Manassas, VA, USA). Cells were routinely cultured

n RPMI 1640 medium without phenol red supplemented with 50 units/mL peni-illin, 50 �g/mL streptomycin, and 10% heat inactivated fetal bovine serum (FBS)PAA Laboratories GmbH, Colbe, Germany) in a humidified incubator with 5% CO2 at7 ◦C. For individual experiments, cells were cultured in RPMI 1640 without phenoled supplemented with 5% heat inactivated FBS or RPMI 1640 without phenol redupplemented with 0.1% BSA.

.3. Cell proliferation

DNA synthesis in proliferating cells was determined by measuring BrdU incor-oration with the commercial Cell Proliferation ELISA System (Roche Moleculariochemicals, Mannheim, Germany). The cells were seeded in 96-well culture platest a density of 1.5 × 104 cells/well and then incubated in RPMI 1640 supplementedith 5% FBS as a control medium or with four different doses (1, 10, 40 and 100 nM)

f BPA for 72 h. Afterward, medium was removed and the cells were incubated forh with a BrdU labeling solution (provided by the kit) containing 10 �M BrdU. Thessay was performed according to the manufacturer’s instructions. Absorbance val-es were measured at 450 nm using an ELISA reader (ELx808 BIO-TEK Instruments,inooski, VT, USA). Culture medium alone was used as a control for nonspecificinding. Data from three independent experiments were pooled for statistical anal-sis.

.4. Measurement of caspase-3 activity

OVCAR-3 cells were seeded in 96-well culture plates at a density of× 104 cells/well and then incubated in RPMI 1640 supplemented with 0.1% BSAs a control medium or with four different doses (1, 10, 40 and 100 nM) of BPAor 24 h. After exposure, the medium was removed, and the plates were stored at70 ◦C. A fluorometric assay for caspase-3 was performed as described by Nicholsont al. (1995). Cells were lysed in Caspase Assay Buffer (50 mM HEPES, pH 7.4,00 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol and 10 mM DTT). Proteinoncentrations in the lysates were determined by the Bradford method (Bio-Radrotein). Equal amounts of cytosolic extract (50 �g of protein) from each sam-le were analyzed. The assay was carried out by adding 50 �M of Ac DEVD-AMCSigma Chemical Co., MO, USA) at 37 ◦C. The amount of fluorescent product was

onitored continuously for 120 min with a spectrofluorometer (FLx800 BIO-TEKnstruments, USA) at excitation wavelength of 355 nm and emission wavelengthf 460 nm. Additionally, to confirm the correlation between signal detection andaspase-3 activity, we used 100 �M Ac-DEVD-CHO inhibitor (Sigma Chemical Co.,O, USA). Data were analyzed with KC JUNIOR software (BIO-TEK Instruments,

inooski, VT, USA), normalized to the fluorescence in vehicle-treated cells andxpressed as fluorescence. The results presented are the average value of threendependent experiments.

.5. Measurement of DNA fragmentation

DNA fragmentation was determined using the Cellular DNA FragmentationLISA kit (Roche Applied Science, Mannheim, Germany). This assay is based onhe quantitative detection of bromodeoxyuridine (BrdU)-labeled DNA fragments.riefly, after exposure to BrdU for 18 h, cells were reseeded into a RPMI 1640upplemented with 0.1% BSA in a 96-well microplate (at 2 × 104 cells/well) andreated for 24 h with the test compounds (BPA at 1, 10, 40 and 100 nM) or Ac-EVD-CHO (100 �M) as a negative control. After 24 h, the cells were washed withhosphate buffered saline (PBS) and lysed with the kit buffer according to the man-facturer’s instructions. The cytoplasmic fraction was transferred separately inton anti-DNA precoated microtiter plate and analyzed using the ELISA procedures recommended by the manufacturer. DNA fragmentation was measured spec-rophotometrically at 450 nm using an ELISA reader (ELx808 BIO-TEK Instruments,inooski, VT, USA). The results presented are the average value of three independentxperiments.

.6. Measurement of gene expression by real-time PCR analysis

The cells were seeded in 6-well culture plates at a density of 1 × 106 cells/welln RPMI1640 supplemented with 0.1% BSA as a control medium or with 40 nM BPAor 24 h. Total RNA from OVCAR-3 cells was isolated using the High Pure RNA Isola-ion Kit (Roche Applied Science, Mannheim, Germany). The purity and the quantityf the RNA were determined by spectrophotometry according to optical densitiest 260 and 280 nm. cDNA synthesis was performed using the Transcriptor Firsttrand cDNA Synthesis Kit (Roche Applied Science, Mannheim, Germany) with a

ixture of anchored-oligo(dT)18 primer and random hexamer primers according to

he manufacturer’s protocol. Amplifications were performed in duplicate using theightCycler 480 System (Roche Applied Science) and the Real Time Ready Humanpoptosis Panel (Cat. No. 05 392 063 001) and Real Time Ready Human Cell Cycleegulation Panel (Cat. No. 05 339 359 001) in combination with LightCycler 480robes Master (Roche Applied Science) according to the manufacturer’s instruc-

Fig. 1. Effects of BPA on the proliferation of OVCAR-3 cells. Cells were treated withvarious concentration of BPA for 72 h, and then analyzed by BrdU incorporationassay. Values are mean ± SEM. All means marked with **p < 0.01, ***p < 0.001 aresignificantly different from the control.

tions. PCR was performed in a final volume of 20 �l including 50 ng/reaction cDNA.The PCR conditions were as follows: pre-incubation for 10 min at 95 ◦C, amplifica-tion for 45 cycles (10 s at 95 ◦C, 30 s at 60 ◦C, and 1 s at 72 ◦C). A positive control(checks for degradation of RNA) and negative control (detects residual genomicDNA) were run simultaneously with every assay according to the manufacturer’sprotocol. The relative expression of genes analyzed was normalized to seven ref-erence genes in the panel gene using the E-Method from Roche Applied Science.The E-Method analyses the amplification efficiency of target and reference genes byusing the so-called relative standards. These standards are serial dilutions of a singlesample (for example, undiluted, 1:10 and 1:100), where concentration is expressedin relative units (for example, 1, 0.1, and 0.01). By using such dilutions to generatea standard curve, the E-Method avoids the time-consuming preparation of artifi-cial or cloned standards and the determination of their absolute values (Tellmann,2006).

2.7. Statistical analysis

Each experiment was repeated three times (n = 3), and each The aver-age of the four values was used for statistical calculations. Statistical analyseswere performed using GraphPad Prism 5. Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s honestly significantdifferences (HSD) multiple range test. Groups that are significantly differ-ent from control are indicated in the figures as *p < 0.05, **p < 0.01, and***p < 0.001.

3. Results

3.1. Cell proliferation

As shown in Fig. 1, BPA increased BrdU incorporation in a dose-dependent manner. We noted 1.2-fold increase after treatmentwith 1 and 10 nM of BPA (p < 0.01) and 1.5-fold increase after treat-ment with 40 and 100 nM of BPA (p < 0.001).

3.2. Measurement of cell cycle gene expression by real-time PCRanalysis

We noted 1.2–1.5-fold induction of G1/S-specific cyclin-D1,G2/mitotic-specific cyclin-F and -A, cell division protein kinase 4,transcription factors E2F1 and E2F3 and proliferating cell nuclearantigen (PCNA) in cells exposed to BPA. All of these genes areresponsible for inducing cell proliferation. In parallel 1.2-foldsuppression of cyclin-dependent kinase inhibitor 1 (p21), 5-foldsuppression of growth arrest and DNA-damage-inducible pro-tein GADD45 alpha, and 46-fold suppression of Wee1-like proteinkinase were noted. All of these genes are responsible for inhibition

of proliferation.

No change was found in levels of the S-phase regulatory proteincyclin-E and its partner protein CDK2. Also, the abundance of pRb,which is a key regulator of G1 progression, did not change (Table 1).

Page 3: Effect of bisphenol-A on the expression of selected genes involved in cell cycle and apoptosis in the OVCAR-3 cell line

32 A. Ptak et al. / Toxicology Letters 202 (2011) 30–35

Table 1Selected cell cycle gene expression changes in OVCAR-3 cells exposed to 40 nM BPA for 24 h.

Assay ID Transcript Description Gen symbol(Alias)

Average foldchange

G1/S phase100844 ENST00000227507 G1/S-specific cyclin-D1 (PRAD1 oncogene) CCND1 1.5**

102813 ENST00000372991 G1/S-specific cyclin-D3 CCND3 1.0101418 ENST00000257904 Cell division protein kinase 4 CDK4 1.2*

101427 ENST00000265734 Cell division protein kinase 6 CDK6 1.0100848 ENST00000262643 G1/S-specific cyclin-E1 CCNE1 1.0100850 ENST00000308108 G1/S-specific cyclin-E2 CCNE2 1.0101416 ENST00000266970 Cell division protein kinase 2 CDK2 1.0101538 ENST00000267163 Retinoblastoma-associated protein (pRb) RB1 (pRb) 1.0102827 ENST00000343380 Transcription factor E2F1 (E2F-1) E2F1 1.5**

102834 ENST00000346618 Transcription factor E2F3 (E2F-3) E2F3 1.3*

101524 ENST00000379160 Proliferating cell nuclear antigen (PCNA) PCNA 1.4**

G2/M phase102811 ENST00000274026 Cyclin-A2 (Cyclin-A) CCNA2 1.3*

101373 ENST00000256442 G2/mitotic-specific cyclin-B1 CCNB1 1.0101376 ENST00000288207 G2/mitotic-specific cyclin-B2 CCNB2 1.0101387 ENST00000293968 G2/mitotic-specific cyclin-F CCNF 1.5**

101406 ENST00000293968 Cell division control protein 2 homolog CDC2 (CDK1) 1.0102820 ENST00000302506 M-phase inducer phosphatase 1 CDC25A 1.0102823 ENST00000245960 M-phase inducer phosphatase 2 CDC25B 1.0102826 ENST00000323760 M-phase inducer phosphatase 3 CDC25C 1.0102849 ENST00000299613 Wee1-like protein kinase WEE1 −46.0***

Cell cycle progression inhibitors102909 ENST00000244741 Cyclin-dependent kinase inhibitor 1 (p21) CDKN1A

(p21CIP1)−1.2*

100855 ENST00000228872 Cyclin-dependent kinase inhibitor 1B(Cyclin-dependent kinase inhibitor p27)

CDKN1B(P27KIP1)

1.0

101471 ENST00000370986 Growth arrest and DNA-damage-inducibleprotein GADD45 alpha (DNA-damage-inducible transcript 1)

GADD45A −5.0***

A

3

dA5eF

3

m

Fwnw

ll means marked with* p < 0.5 is significantly different from the control.

** p < 0.01 is significantly different from the control.*** p < 0.001 is significantly different from the control.

.3. Caspase-3 activity

Ac-DEVD-CHO (a caspase-3 inhibitor) at a dose of 100 �Mecreased basal caspase-3 activity to 25% that of control cells.statistically significant inhibition of caspase-3 activity to 56%,

8%, 47% and 39% of control values was observed under the influ-nce of BPA at 1, 10, 40 and 100 nM, respectively (p < 0.001;ig. 2).

.4. DNA fragmentation

BPA at all doses used had no effect on DNA fragmentation aseasured by the ELISA assay (Fig. 3).

16000

4000

6000

8000

10000

12000

14000

ase-

3ac�

vity

o r

esce

nc U

nit /

50μg

pr

otei

n ***

******

******

0

2000

4000

Casp

aRe

la�v

e F

luo

ig. 2. Effects of BPA on the caspase-3 activity of OVCAR-3 cells. Cells were treatedith various concentration of BPA for 24 h. Ac-DEVD-CHO (100 �M) was used as aegative control for caspase-3 activation. Values are mean ± SEM. All means markedith ***p < 0.001 are significantly different from the control.

3.5. Measurement of apoptosis gene expression by real-timeanalysis

To understand the impact of BPA on ovarian cancer, we con-ducted expression studies to characterize gene expression changesin the OVCAR-3 cell line that result from BPA exposure.

As shown in Table 2, BPA had no effect on TNFR1 mRNA lev-els, whereas 8-fold suppression of Fas was observed, suggesting

lack of activation of the TNFR apoptotic pathway and inhibition theFas-dependent apoptotic pathways. Also, 1.6–2-fold suppressionof FADD, RAIDD, caspase-8 and caspase-10 genes was observed.All of these genes are involved in the extrinsic apoptotic path-way. In parallel, 1.2–2.5-fold suppression of Bax, Bak and Bok

3

1.5

2

2.5

3

men

ta�o

n A

bosr

banc

e U

nits

)

0

0.5

1

C 1nMBPA 10nMBPA 40nMBPA 100nM

DN

Afr

agm

(Rel

a�ve

A

Fig. 3. Effects of BPA on the DNA fragmentation of OVCAR-3 cells. Cells were treatedwith various concentration of BPA for 24 h, and then analyzed by Cellular DNA Frag-mentation ELISA kit. Values are mean ± SEM. All means marked with ***p < 0.001 aresignificantly different from the control.

Page 4: Effect of bisphenol-A on the expression of selected genes involved in cell cycle and apoptosis in the OVCAR-3 cell line

A. Ptak et al. / Toxicology Letters 202 (2011) 30–35 33

Table 2Selected apoptosis gene expression changes induced in OVCAR-3 cells by exposure to 40 nM BPA for 24 h.

Assay ID Transcript Description Gen symbol(Alias)

Average foldchange

Extrinsic pathway102679 ENST00000162749 Tumor necrosis factor receptor superfamily

member 1A precursor (p60)TNFRSF1A(TNFR1)

1.0

100426 ENST00000371875 Tumor necrosis factor receptor superfamilymember 6 precursor (FASLG receptor)

FAS −8.0***

100417 ENST00000301838 Protein FADD (FAS-associated death domainprotein)

FADD −1.6*

100289 ENST00000332896 Death domain-containing protein CRADD (Caspaseand RIP adapter with death domain)

CRADD(RAIDD)

−1.7**

100227 ENST00000358485 Caspase-8 precursor CASP8 −2.0***

100204 ENST00000272879 Caspase-10 precursor CASP10 −1.9***

Intrinsic pathwayPro-survival100083 ENST00000398117 Apoptosis regulator Bcl-2 BCL2 −1.4**

104039 ENST00000250405 Apoptosis regulator Bcl-W (Bcl-2-like 2 protein) BCL2L2(BCL-W)

−1.2*

100088 ENST00000307677 Apoptosis regulator Bcl-X (Bcl-2-like 1 protein) BCL2L1 (Bcl-X) 1.6**

102930 ENST00000369026 Induced myeloid leukemia cell differentiationprotein Mcl-1 (Bcl-2- related protein EAT/mcl1)

MCL1 (Mcl-1) 51.0***

Pro-apoptotic104034 ENST00000394532 Bcl2 antagonist of cell death (BAD) BAD −1.45**

100068 ENST00000374467 Bcl-2 homologous antagonist/killer (Apoptosisregulator BAK)

BAK1 −2.5***

102861 ENST00000293288 Apoptosis regulator BAX BAX −1.25*

100122 ENST00000342111 BH3-interacting domain death agonist (BID) BID 1.1100126 ENST00000216115 Bcl-2-interacting killer (Apoptosis inducer NBK) BIK 1.0100165 ENST00000318407 Bcl-2-related ovarian killer protein (Hbok) BOK −1.5**

100233 ENST00000333868 Caspase-9 precursor CASP9 6.5***

102892 ENST00000333991 Apoptotic protease-activating factor 1 (Apaf-1) APAF1 −1.6**

100213 ENST00000308394 Caspase-3 precursor CASP3 −2.3***

100222 ENST00000265164 Caspase-6 precursor CASP6 −1.4**

100224 ENST00000369331 Caspase-7 precursor CASP7 −1.5**

102620 ENST00000264705 CAD protein CAD −1.8***

102630 ENST00000377038 DNA fragmentation factor subunit alpha (DNAfragmentation factor 45 kDa subunit)

DFFA (ICAD) −1.3*

Caspase-independent pathway102632 ENST00000372642 Endonuclease G, mitochondrial precursor endoG 5.1***

101277 ENST00000396473 Cellular tumor antigen p53 (Tumor suppressorp53)

TP53 (p53) 1.7**

All means marked with

(w5nsfkritff(

4

lOowb

* p < 0.5 is significantly different from the control.** p < 0.01 is significantly different from the control.

*** p < 0.001 is significantly different from the control.

proapoptotic genes) and Bcl-2 and Bcl-w (antiapoptotic genes)as observed. We also noted 1.6-fold induction of Bcl-xL and

1-fold induction of Mcl-1 (pro-survival gene) expression. Weoted 6.5-fold induction of caspase-9 gene expression but 1.6-folduppression of its partner protein, Apaf-1. We observed 1.3–2.3-old suppression of caspase 3, 6 and 7 genes, which are wellnown effector caspases, and CAD and ICAD genes, which areesponsible for DNA fragmentation. All of these genes are involvedn the intrinsic apoptotic pathway. Additionally, 5.1-fold induc-ion of endonuclease G genes involved in CAD-independent DNAragmentation was noted under the influence of BPA. Also, 1.7-old induction of the tumor suppressor gene p53 was observedTable 2).

. Discussion

In this study, we evaluated the effects of bisphenol A on pro-

iferation and apoptosis in the human ovarian cancer cell lineVCAR-3. Because we found dose-dependent stimulatory actionn BrdU incorporation and inhibitory action on caspase-3 activity,e expected that gene products involved in these processes would

e affected.

4.1. Action on cell proliferation

There is no data available in the literature concerning action ofBPA on cell cycle regulation in ovarian cancer cells. However, recentdata of Park et al. (2009) showed that BPA increased BG-1 humanovarian cancer cell proliferation in a dose-dependent manner andconfirms results of our experiments. In this study we demonstratethat the low doses of BPA stimulate human ovarian cancer cell lineOVCAR-3 proliferation.

It has also been reported that BPA can promote proliferation ofMCF-7 breast cancer cells (Olsen et al., 2003; Ricupito et al., 2009),HeLa cells (Bolli et al., 2008) and JKT-1 cells (Bouskine et al., 2009).

Using real-time PCR, we found that mRNA levels of gene prod-ucts involved in biological processes, including the cell cycle wereaffected, confirming the results from the proliferation assay.

In the present investigation, the most up-regulated mRNAsinvolved in cell cycle were cyclin D1, CDK4, cyclin A, PCNA, E2F1,and E2F3, all of which are genes responsible for inducing cell pro-

liferation. The most down-regulated mRNAs were p21, Weel-1 andGADD45 alpha, genes responsible for inhibition of proliferation.

Basal and mitogen-induced cell growth is regulated by multipleproteins that control cell cycle progression from G0/G1 to M phase,including cyclins, cyclin-dependent kinases, and their inhibitors.

Page 5: Effect of bisphenol-A on the expression of selected genes involved in cell cycle and apoptosis in the OVCAR-3 cell line

3 gy Lett

Capaimemouaem2inoc(otoir

4

ewfieacfluoclBdi1t

TctsagfoTirc

ft(2re

4 A. Ptak et al. / Toxicolo

entral to these cell cycle control mechanisms are cyclin D1-Cdk4nd cyclin E-Cdk2, which phosphorylate substrates, including theroduct of the retinoblastoma susceptibility gene pRB, and therebyllow the initiation of DNA synthesis (Weinberg, 1995). Studiesn human neuroblastoma cells have shown that BPA can pro-

ote growth by increasing expression of Cdk4 and cyclin D1 (Zhut al., 2009). We also observed that BPA up-regulated cyclin ARNA. Watanabe et al. (2007) showed that the stimulatory effect

f estrogen on Ishikawa cell proliferation was mediated by thep-regulation of cyclin D1 and cyclin A. In the present study, welso observed suppression of p21/WAF mRNA under the influ-nce of BPA. Low levels of p21/WAF1 have been shown to be aarker of poor overall survival in ovarian cancer patients (Bali et al.,

004). Furthermore, down-regulation or inactivation of the CDKnhibitors, including p21Waf1/Cip1, p27Kip1, and p16Ink4a, whichormally cause G1 arrest by binding to cyclin-CDK complexes, areften observed in diverse human tumors, further rendering theell susceptible to uncontrolled extracellular proliferation signalsSherr and Roberts, 1999). It must be pointed out that inductionf apoptosis appears to activate multiple cell cycle-associated fac-ors through cleavage and inactivation of their inhibitors. A numberf cell cycle inhibitory factors are cleaved during apoptosis. Thosenclude Cdc27, Wee1, and two Cdk inhibitors: p21 and p27 (seeeview Jin and El-Deiry, 2005).

.2. Action on cell apoptosis

Cells undergo apoptosis through two major pathways, thextrinsic pathway (death receptor pathway) or the intrinsic path-ay (the mitochondrial pathway). Increasing attention is being

ocused on alternative signaling pathways leading to cell deathncluding necrosis, autophagy, and mitotic catastrophe (Brokert al., 2005). However, no information is available in the literaturebout the effect of BPA on regulation of apoptosis in ovarian cancerells. Studies on the effects of BPA on apoptosis have generated con-icting results, largely due to the micromolar concentrations of BPAtilized by most studies. Diel et al. (2002) showed that BPA in dosesf 10, 1 and 0.1 mM was a potent inhibitor of apoptosis in MCF-7ells. Xu et al. (2002) observed that BPA decreased mice granu-osa cell viability in a dose- and time-dependent manner. Moreover,PA at 100 �M increased the expression of Bax and concomitantlyecreased the expression of Bcl2 at both protein and mRNA levels

n granulosa cells. Also, Bredhult et al. (2007) observed that BPA at00 �M decreased cell viability and increased necrosis compared tohe DMSO control in human endometrial endothelial cells (HEECs).

A hallmark of apoptosis is the fragmentation of nuclear DNA.his process involves the caspase-3 dependent DNAse CAD oraspase-independent DNA fragmentation. In this study, we showedhat BPA acts by suppressing the expression of pro-apoptotic genes,uch as FAS, FADD, RAIDD, caspase-8, -10, -3, -6, -7, CAD, Bax, Baknd Bok, and Apaf-1, and inducing the expression of pro-survivalenes, such as Bcl-xL and Mcl-1. On the other hand, a strong (5-old) induction of endonuclease G gene expression points to actionf BPA by activation of caspase-independent DNA fragmentation.herefore, observed by us inhibition of caspase-3 activity (Fig. 2)s probably related to the reduction in protein expression, as aesult of suppressing the expression of gene, not the reduction ofaspase-3 activity.

In response to apoptotic stimuli, endonuclease G is also releasedrom the mitochondria into the cytosol, where it translocates to

he nucleus and generates oligonucleosomal DNA fragmentationLi et al., 2001). Donovan and Cotter (2004) showed that Bcl-

prevents mitochondrial membrane permeabilization and, as aesult, prevents release of potent pro-apoptotic molecules such asndonuclease G.

ers 202 (2011) 30–35

It is difficult to explain the lack of DNA fragmentation as mea-sured by the BrdU incorporation test. It is likely that the observedinduction of p53 (a nuclear transcription factor that accumulatesin response to cellular stress, including DNA damage and oncogeneactivation) and suppression of caspase-3 and 7 activates the DNArepair process. Therefore, despite the observed induction of endoG gene expression, an action of BPA on DNA fragmentation was notobserved. One of the essential substrates cleaved by both caspase-3and -7 is poly-ADP-ribose polymerase (PARP), an abundant DNA-binding enzyme that detects and signals DNA strand breaks (Deckerand Muller, 2002). The presence of cleaved PARP-1 is one of themost used diagnostic tools for the detection of apoptosis in manycell types. The cleavage of PARP-1 into two fragments of 89 and24 kDa has been considered indicative of functional caspase activa-tion (Koh et al., 2005).

It is evident from our study that suppression of the caspase-dependent apoptotic pathway could indirectly explain theobserved antagonizing effects of BPA on multiple anticancer drugs(LaPensee et al., 2009, 2010). It has been showed that tumors canacquire drug resistance by overexpressing anti-apoptotic proteins(e.g., Bcl-2 and Bcl-xL) or down-regulating pro-apoptotic proteins(e.g., Bax). Breast cancer cells that overexpress Bcl-xL are less sen-sitive to paclitaxel, which correlates with failure of the drug toactivate caspase 9 (Wang et al., 2005). In addition, Bax overexpres-sion in MCF7 cells restores their sensitivity to various apoptoticagents. Higher Bax expression levels are detected in normal breastepithelium than in adjacent tumors (Bargou et al., 1996).

In conclusion, this is the first report to show action of BPAon expression of proliferation and apoptosis genes in ovariancancer cells. We showed that (1) BPA in an environmentally rele-vant concentration enhances proliferation by up-regulating genesresponsible for inducing cell proliferation and down-regulatinggenes involved in inhibition of cell proliferation, and (2) BPA hasa dual action on ovarian cancer cell apoptosis by up-regulatinganti-apoptotic and down-regulating pro-apoptotic factors in thecaspase-dependent pathway and up-regulating endo G geneexpression in the caspase-independent pathway. However, it is dif-ficult to explain the lack of DNA fragmentation as measured by theBrdU incorporation test. We hypothesize that the observed induc-tion of p53 and suppression of caspase-3 and 7 gene expressionactivate the DNA repair process. Therefore, despite the observedinduction of endo G gene expression, action of BPA on DNA frag-mentation was not observed. This hypothesis requires clarification.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by the Polish Committee for ScientificResearch from 2010 to 2013 as a project 0050/B/PO1/2010/38.

References

Auersperg, N., Wong, A.S., Choi, K.C., Kang, S.K., Leung, P.C., 2001. Ovarian surfaceepithelium: biology, endocrinology, and pathology. Endocr. Rev. 22, 255–288.

Bali, A., O’Brien, P.M., Edwards, L.S., Sutherland, R.L., Hacker, N.F., Henshall, S.M.,2004. Cyclin D1, p53, and p21Waf1/Cip1 expression is predictive of poor clinicaloutcome in serous epithelial ovarian cancer. Clin. Cancer Res. 10, 5167–5177.

Bargou, R.C., Wagener, C., Bommert, K., Mapara, M.Y., Daniel, P.T., Arnold, W.,Dietel, M., Guski, H., Feller, A., Royer, H.D., Dorken, B., 1996. Overexpression

of the death-promoting gene bax-alpha which is downregulated in breast can-cer restores sensitivity to different apoptotic stimuli and reduces tumor growthin SCID mice. J. Clin. Invest. 97, 2569–2651.

Betabcourt, A.M., Mobley, J.A., Russo, J., Lamartiniere, C.A., 2010. Proteomic anal-ysis in mammary glands of rat offspring exposed in utero to bisphenol A. J.Proteomics 73, 1241–1253.

Page 6: Effect of bisphenol-A on the expression of selected genes involved in cell cycle and apoptosis in the OVCAR-3 cell line

gy Let

B

B

B

B

C

D

D

D

D

D

J

K

K

L

L

L

A. Ptak et al. / Toxicolo

olli, A., Galluzzo, P., Ascenzi, P., Del Pozzo, G., Manco, I., Vietri, M.T., Mita, L., Altucci,L., Mita, D.G., Marino, M., 2008. Laccase treatment impairs bisphenol A-inducedcancer cell proliferation affecting estrogen receptor alpha-dependent rapid sig-nals. IUBMB Life. 60, 843–852.

ouskine, A., Nebout, M., Brucker-Davis, F., Benahmed, M., Fenichel, P., 2009. Lowdoses of bisphenol A promote human seminoma cell proliferation by activatingPKA and PKG via a membrane G-protein-coupled estrogen receptor. EnvironHealth Perspect. 117, 1053–1058.

redhult, C., Backlin, B.M., Olovsson, M., 2007. Effects of some endocrine disruptorson the proliferation and viability of human endometrial endothelial cells in vitro.Reprod. Toxicol. 23, 550–559.

roker, L.E., Kruyt, F.A., Giaccone, G., 2005. Cell death independent of caspases: areview. Clin. Cancer Res. 11, 3155–3162.

alafat, A.M., Kuklenyik, Z., Reidy, J.A., Caudill, S.P., Ekong, J., Needham, L.L., 2005.Urinary concentrations of bisphenol A and 4-nonylphenol in a human referencepopulation. Environ Health Perspect. 113, 391–395.

airkee, S.H., Seok, J., Champion, S., Sayeed, A., Mindrinos, M., Xiao, W., Davis, R.W.,Goodson, W.H., 2008. Bisphenol A induces a profile of tumor aggressiveness inhigh-risk cells from breast cancer patients. Cancer Res. 68, 2076–2080.

ecker, P., Muller, S., 2002. Modulating poly (ADP-ribose) polymerase activity:potential for the prevention and therapy of pathogenic situations involving DNAdamage and oxidative stress. Curr. Pharm. Biotechnol. 3, 275–283.

iel, P., Olff, S., Schmidt, S., Michna, H., 2002. Effects of the environmental estro-gens bisphenol A, o,p′-DDT, p-tert-octylphenol and coumestrol on apoptosisinduction, cell proliferation and the expression of estrogen sensitive molecularparameters in the human breast cancer cell line MCF-7. J. Steroid. Biochem. Mol.Biol. 80, 61–70.

onovan, M., Cotter, T.G., 2004. Control of mitochondrial integrity by Bcl-2 fam-ily members and caspase-independent cell death. Biochim. Biophys. Acta 1644,133–147.

urando, 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.

in, Z., El-Deiry, W.S., 2005. Overview of cell death signaling pathways. Cancer Biol.Therapy 4, 139–163.

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.

oh, D.W., Dawson, T.M., Dawson, V.L., 2005. Mediation of cell death by poly(ADP-ribose) polymerase-1. Pharmacol. Res. 52, 5–14.

aPensee, E.W., Tuttle, T.R., Fox, S.R., Ben-Jonathan, N., 2009. Bisphenol A at lownanomolar doses confers chemoresistance in estrogen receptor-alpha-positive

and -negative breast cancer cells. Environ. Health Perspect. 117, 175–180.

aPensee, E.W., LaPensee, C.R., Fox, S., Schwemberger, S., Afton, S., Ben-Jonathan,N., 2010. Bisphenol A and estradiol are equipotent in antagonizing cisplatin-induced cytotoxicity in breast cancer cells. Cancer Lett. 290, 167–173.

i, L.Y., Luo, X., Wang, X., 2001. Endonuclease G is an apoptotic DNase when releasedfrom mitochondria. Nature. 412, 95–99.

ters 202 (2011) 30–35 35

Nicholson, D.W., Ali, A., Thornberry, N.A., Vaillancourt, J.P., Ding, C.K., Gallant, M.,Gareau, Y., Griffin, P.R., Labelle, M., Lazebnik, Y.A., et al., 1995. Identification andinhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature6, 37–43.

Olsen, C.M., Meussen-Elholm, E.T., Samuelsen, M., Holme, J.A, Hongslo, J.K., 2003.Effects of the environmental oestrogens bisphenol A, tetrachlorobisphenolA, tetrabromobisphenol A, 4-hydroxybiphenyl and 4,4′-dihydroxybiphenyl onoestrogen receptor binding, cell proliferation and regulation of oestrogen sen-sitive proteins in the human breast cancer cell line MCF-7. Pharmacol. Toxicol.92, 180–188.

Park, 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 anestrogen-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.

Ricupito, A., Del Pozzo, G., Diano, N., Grano, V., Portaccio, M., Marino, M., Bolli, A.,Gzlluzzo, P., Bontempo, P., Mita, L., Altucci, L., Mita, D.G., 2009. Effect of bisphenolA with or without enzyme treatment on the proliferation and viability of MCF-7cells. Environ. Int. 35, 21–26.

Sherr, C.J., Roberts, J.M., 1999. CDK inhibitors: positive and negative regulators ofG1-phase progression. Genes Dev. 13, 1501–1512.

Singleton, D.W., Feng, Y., Yang, J., Puga, A., Lee, A.V., Khan, S.A., 2006. Gene expressionprofiling reveals novel regulation by bisphenol-A in estrogen receptor-alpha-positive human cells. Environ. Res. 100, 86–92.

Sun, Y., Irie, M., Kishikawa, N., Wada, M., Kuroda, N., Nakashima, K., 2004. Deter-mination of bisphenol A in human breast milk by HPLC with column-switchingand fluorescence detection. Biomed. Chromatogr. 18, 501–507.

Tellmann, G., 2006. The E-Method: a highly accurate technique for gene-expressionanalysis. Nat. Methods 3, doi:10.1038/nmeth894.

Wang, Z., Goulet, R., Stanton, K.J., Sadaria, M., Nakshatri, H., 2005. Differential effectof anti-apoptotic genes Bcl-xL and c-FLIP on sensitivity of MCF-7 breast cancercells to paclitaxel and docetaxel. Anticancer Res. 25, 2367–2379.

Watanabe, J., Kamata, Y., Seo, N., Okayasu, I., Kyramoto, H., 2007. Stimulatory effectof estrogen on the growth of endometrial cancer cells is regulated by cell-cycleregulators. J. Steroid Biochem. Mol. Biol. 107, 163–171.

Weinberg, R.A., 1995. The retinoblastoma protein and cell cycle control. Cell 81,323–330.

Welshons, W.V., Nagel, S.C., vom Saal, F.S., 2006. Large effects from small exposuresIII. Endocrine mechanisms mediating effects of bisphenol A at levels of humanexposure. Endocrinology 147, S56–S69.

Vandenberg, L.N., Hauser, R., Marcus, M., Olea, N., Welshons, W.V., 2007. Humanexposure to bisphenol A (BPA). Reprod. Toxicol. 24, 139–177.

Xu, J., Osuga, Y., Yano, T., Morita, Y., Tang, X., Fujiwara, T., Takai, Y., Matsumi, H.,Koga, K., Taketani, Y., Tsutsumi, O., 2002. Bisphenol A induces apoptosis and G2-to-M arrest of ovarian granulosa cells. Biochem. Biophys. Res. Commun. 292,456–462.

Zhu, H., Xiao, X., Zheng, J., Zheng, S., Dong, K., Yu, Y., 2009. Growth-promoting effect ofbisphenol A on neuroblastoma in vitro and in vivo. J. Pediatr. Surg. 44, 672–680.