Bisphenol A impairs estradiol-induced protective effects against DLD-1 colon cancer cell growth

Bisphenol A impairs estradiol-induced protective effects against DLD-1 colon cancer cell growth
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  Research Communication  Bisphenol A Impairs Estradiol-induced Protective EffectsAgainst DLD-1 Colon Cancer Cell Growth Alessandro Bolli 1,2 , Pamela Bulzomi 1 , Paola Galluzzo 1 , Filippo Acconcia 1 and Maria Marino 1,2 * 1  Department of Biology, University Roma Tre, Roma, Italy 2  National Institute of Biostructure and Biosystems, Roma, Italy Summary Bisphenol A (BPA), a prototype of endocrine disruptors,mimics 17 b -estradiol (E2)-induced proliferation in several can-cer cells by binding to estrogen receptor  a  (ER a ). However,scarce and conflicting data are available concerning the effectof BPA on estrogen receptor  b  (ER b )-mediated functions. Here,the detailed analysis of the effect of BPA, alone or in combina-tion with E2, on ER b -mediated cellular functions is reported inER b -expressing colon cancer cell line. BPA binds to ER b  with-out activating any receptor activities. On the other hand, BPAinhibits E2-induced genomic activity of ER b  as well as ER b extra-nuclear activities ( i.e. , ER b :p38 association and p38 acti-vation). As a consequence, BPA impairs the E2-induced activa-tion of the apoptotic cascade which is at the root of the protec-tive role played by the hormone against colon cancer growth.Thus, women may be considered a highly susceptible populationwith an increased risk of colon cancers after BPAexposures.    2010 IUBMBIUBMB  Life ,  62(9): 684–687, 2010Keywords  apoptosis; bisphenol A; endocrine disruptor; 17 b -estra-diol; estrogen receptor   b ; colon cancer cell line. INTRODUCTION Colorectal cancer (CRC), the second most common malig-nancy in industrialized countries, is more frequent in men thanwomen ( 1, 2 ). When patients are grouped by onset age, further stratification is observed. The incidence of colon cancer inyoung women is far lower than that in men. On the contrary,postmenopausal women display an increased risk in colon can-cer. This difference becomes more pronounced as age increases(  3, 4 ). This has been linked to the ability of the female sex ste-roid hormone, 17 b -estradiol (E2), to confer protection againstCRC [see (  5 ) and literature cited therein]. The mechanismsunderlying this E2-induced effect involves estrogen receptor   b (ER b ) isoform which represents the predominant estrogen re-ceptor (ER) subtype in the human colon. Actually, low ER b levels are associated with colonic tumsrcenesis and loss of ma-lignant colon cell differentiation ( 6–9 ). In particular, in ER b -containing DLD-1 colon cancer cell line, E2 binding to ER b both increases receptor association to caveolin-1, a membranescaffolding protein, and to p38, a member of the mitogen-acti-vated protein kinase family ( 10 ). The resulting signal transduc-tion pathways are required for the downstream activation of thecaspase-3-dependent pro-apoptotic cascade ( 10, 11 ).Besides E2, the binding site of ERs ( i.e. , both ER a  andER b ) can accommodate many ligands which are planar, aro-matic, and with 2 oxygen atoms spaced 11.5 A˚apart ( 12 ). Sev-eral natural and synthetic compounds can fit this need and bindto ER acting as E2 agonist or antagonist ( 13 ). Among ERligands, bisphenol A [BPA, 2,2-bis (4-hydroxyphenyl) propane]deserves particular attention. In fact, BPA is a monomer of pol-ycarbonate plastics and BPA-based resins are used in manyproducts including food cans, dental composites, baby bottles,mineral water bottles, and toys ( 14, 15 ). BPA is known to har-bor estrogenic activity: this compound is a weak ligand of bothER a  and ER b  ( 13 ) and moderately stimulates E2-independentproliferation in ER a -containing breast cancer cells and in ER a -overexpressed HeLa cells, a cervix carcinoma cell line ( 16–19 ).To our knowledge, scarce and conflicting investigations havebeen aimed at dissecting the role played by ER b  in the estro-genic activity of BPA ( 14, 20 ).Here, we report the modulating effect of BPA, alone and incombination with E2, on ER b  activities relevant for the E2-induced protection against colon cancer growth. Address correspondence to: Prof. Maria Marino, Department of Biol-ogy, University Roma Tre, Viale Guglielmo Marconi, 446, I-00146Roma, Italy. Tel:  1 390657336345. Fax:  1 390657336321.E-mail: m.marino@uniroma3.itAdditional Supporting Information may be found in the online ver-sion of this article.Received 13 July 2010; accepted 21 July 2010 ISSN 1521-6543 print/ISSN 1521-6551 onlineDOI: 10.1002/iub.370 IUBMB  Life ,  62(9): 684–687, September 2010  MATERIAL AND METHODS All materials were purchased as previously indicated ( 10, 11,18, 19 ).The affinity of BPA for ER b  was determined by competitiveradiometric binding assays by using [ 3 H]-E2 as tracer, as previ-ously reported ( 19 ). Values of the apparent ( Y  app ) and intrinsic ( Y  )molar fraction of E2- and BPA-bound ER b , as well as of theintrinsic equilibrium dissociation constant (  K  d ) for E2 and BPAbinding to ER b , were obtained at pH 7.4 and 25  8 C ( 19 ). Datawere analyzed according to a nonlinear four-parameter model (  21 ).The ER b  containing human colon adenocarcinoma cell line(DLD-1) were grown as previously described ( 10 ). DLD-1 cellswere plated in 96-well culture plates and stimulated with either vehicle (DMSO:PBS, 1:1) or E2 (1.0  3  10 2 8 M) in the pres-ence or absence of different BPA concentrations (from 1.0  3 10 2 6 M to 1.0  3  10 2 3 M). After 30 H, cell vitality wasassessed by using the XTT reaction solution according to themanufacturer’s instructions.The reporter plasmid containing the promoter of complementcomponent 3 gene (pC3), retaining a natural estrogen responsiveelement (ERE), has been previously described ( 19 ). Six Hoursafter DLD-1 transfection with lipofectamine, the medium waschanged and after 24 H cells were stimulated with differentBPA concentrations (from 1.0  3  10 2 6 M to 1.0  3  10 2 3 M) or with 1.0  3  10 2 5 M BPA in the presence of 1.0  3  10 2 8 M E2for 6 H. The measurement of promoter activity was performedas previously described ( 19 ).Cell lysates were immunoprecipitated with TrueBlot (eBio-science, San Diego, CA, USA). Equal amounts of soluble cellextracts were incubated with either 2.0  l g of anticaveolin-1 or anti-ER b  antibodies. Cell lysis, immunoprecipitation, and SDS-PAGE were performed as previously described ( 10 ). Sampleswere probed with the indicated antibodies ( 10 ), and the reactionwas visualized with indirect chemiluminescence. Densitometryof Western blot was performed with ImageJ software for Win-dows (data not shown).The statistical analysis was performed by utilizing Student’s t  -test with the INSTAT software system for Windows. Probabil-ity (  P ) values \ 0.05 were considered significant. RESULTS As shown in Fig. 1, BPA binds to human recombinant ER b .The value of   K  d  for BPA binding to ER b  ( 5  (4.8  6  0.6)  3 10 2 7 M) is higher by about three orders of magnitude than thatfor E2 association ( 5  (3.5  6  0.5)  3  10 2 10 M). Notably, the  K  d value for BPA binding to ER b  is about three fold lower thanthat for BPA association to ER a  ( 13, 19 ). Because the full re-ceptor occupancy is achieved for ligand concentrations higher than 10  3  K  d , the concentrations of E2 and BPA used in thisstudy were 1.0  3 10 2 8 M and 1.0  3 10 2 5 M, respectively.BPA stimulation does not affect DLD-1 cell vitality at anyof tested concentration (Fig. 2A). On the other hand, asexpected ( 10 ), 30 H of 1.0  3  10 2 8 M E2 stimulation reducesDLD-1 cell vitality of about 50% with respect to unstimulatedcells (Fig. 2B). Notably, the effect of E2 on cell vitality is com-pletely prevented when cells are stimulated with BPA in thepresence of E2 background (1.0  3  10 2 8 M). This effect is pres-ent even at 1.0  3  10 2 6 M BPA concentrations. On the other hand, higher concentrations (1.0  3  10 2 3 M) resulted cytotoxic,further decreasing the cell vitality (Fig. 2B). This result, whichindicates an antagonism of BPA toward E2 in colon cancer cellline, has been confirmed by evaluating the effects of BPA oncaspase-3 activation. Indeed, E2 activates caspase-3 increasingthe level of both active caspase ( i.e. , 17 kDa band) and thecleavage of its substrate PARP. BPA alone does not affect cas-pase-3 activation and PARP cleavage but prevents E2 effects onpro-apoptotic protein activation when cells are co-stimulatedwith BPA and E2 (Fig. 2C and 2D).Afterwards, the effects of BPA on ER b  genomic and extra-nuclear activities have been evaluated. Figure 3A shows theeffect of different BPA concentrations on the ERE-containingpC3 promoter activity in the absence or in the presence of 1.0 3  10 2 8 M E2. BPA does not exert any effect, whereas the E2treatment induces a two-fold increase of the pC3 promoter activity (Fig. 2A). When added with E2, BPA prevents the E2-induced ER b  transcriptional activity (Fig. 3A). Accordingly, theBPA treatment also impairs the E2-induced extra-nuclear activ-ities of ER b . In fact, the BPA and E2 co-treatment inhibits theE2-induced increase of p38 phosphorylation (Fig. 3B). Previ-ously, we reported that both genomic and extra-nuclear ER b activities are required for the E2-induced increased expressionof ER b , which represents an important step for the E2-mediatedprotection against colon cancer cell growth ( 11 ). This promptedus to evaluate the impact of BPA on the E2-induced increase of ER b  level. Figure 3C shows that BPA specifically impairs theE2-induced increase of ER b  level further confirming the antago-nistic behavior of this compound on E2-induced effects.Besides lipid modification, association of ERs to membraneand/or signalling proteins allows the activation of down-stream Figure 1.  Dependence of the molar fraction of the ligand-boundER b  ( Y  ) on the free E2 (circles) and BPA (squares) concentra-tion. 685BISPHENOL A IN COLON CANCER CELLS  cascade signals important for E2 effects ( 12 ). In particular, wepreviously reported that the physical association between ER b ,caveolin-1, and the un-phosphorylated form of p38 is present inDLD-1 cell line even in the absence of E2 ( 10 ). This promptedus to evaluate the effect of BPA on ER b  molecular interactionswith caveolin-1 and p38. As expected, in the resting state ( i.e. ,vehicle treated), ER b  results associated to caveolin-1 and theun-phosphorylated p38, whereas the association ER b :phosphory-lated p38 is barely detectable (Fig. 3D). Intriguingly, both inthe absence and in the presence of E2, BPA stimulation specifi-cally prevents the ER b :p38 association without affecting theER b :caveolin-1 complex formation (Fig. 3E). DISCUSSION Here we demonstrate that in an endogenous ER b  expressingcolon cancer cell line BPA behaves as a subtype-specific E2 an-tagonist by blocking the E2 ability to activate a pro-apoptoticcascade (Figs. 2). Notably, only higher BPA concentrations ( ‡ 1.0  3  10 2 3 M) result cytotoxic in DLD-1 cancer cell line, aspreviously reported in HeLa cells ( 19 ). Figure 2.  Panel A and Panel B, XTT assay of DLD-1 cell linetreated with different BPA concentration or with E2 in the ab-sence or presence of different concentrations of BPA. Absorb-ance was determined at 490 nm. Data are the mean of six dif-ferent experiments  6  SD. (*)  P \ 0.001 with respect to the ve-hicle. Western blot analyses, representative of three differentexperiments, of caspase-3 activation (Panel C) and PARP cleav-age (Panel D) was performed on DLD-1 cell line treated 30 Hwith E2, BPA or E2 plus BPA. The amount of protein levelswere normalized by comparison with  b -tubulin. Figure 3.  Luciferase assay detection in DLD-1 cell line stimu-lated 24 H with vehicle, E2, or different BPA concentrations, or E2 plus BPA (Panel A). Data are the mean of six differentexperiments  6  SD.  P \ 0.001 with respect to samples treatedwith vehicle (*) or E2 ( 8 ). Representative Western blot analysisof p38 phosphorylation (Panel B) and ER b  level (Panel C) inDLD-1 cell line treated 1 H with vehicle, E2 or BPA, or E2plus BPA.  b -tubulin expression was used for protein level nor-malization. After the same stimulation described above DLD-1cell line were lysated and subjected to caveolin-1 immunopreci-pitation (Panel D) or ER b  immunoprecipitation (Panel E) fol-lowed by Western blot with anticaveolin-1, anti-ER b , antiphos-phorylated-p38, or anti-p38 antibodies. 686 BOLLI ET AL.  The ER-dependent molecular mechanisms underlying BPAinterference with E2 protective effects involve both genomicand extra-nuclear ER b  activities. Most notably, we found thatBPA prevents the E2-induced transcriptional activity of ER b  to-ward an ERE containing reporter gene in colon cancer cell line.In transiently transfected HeLa cells where ER b , co-activators,and reporter genes were all over-expressed ( 14, 17  ) a generalagonistic behavior of the ER b :BPA complex on E2-inducedER b  transcriptional activity has been reported without any in-formation on the BPA effect on cell proliferation. In contrast, inprostate cancer cell line, where the loss of ER b  signaling resultsin increased cell proliferation, BPA reduced ER b  levels (  20 ) asan E2 antagonist. Present data showing that BPA prevents theE2 ability to increase ER b  levels, which requires both thegenomic and the extra-nuclear ER b  activities ( 11 ), further sustainthe antagonist character of the BPA:ER b  complex observed. More-over, BPA was able to completely silence also the E2-inducedER b  extra-nuclear activities further supporting its role as an ER b -specific E2 antagonist in colon cancer cell line. In particular, uponBPA binding ER b  still interacts with the plasma-membrane proteincaveolin-1, however, ER b  association with the signaling proteinp38 results impaired, thus BPA decouples ER b  from the down-stream signals important for the E2-induced pro-apoptotic cascade.The BPA-induced modulation of the ER b :p38 interaction indicatesthat different ligands, by inducing distinct conformational changesin ER b , could also modulate ER extra-nuclear signals changing,thus, the final cellular outcomes.As a whole, we describe an ER b -mediated mechanism of BPA action, which was never reported before. This mechanism,along with the well-known BPA ability to induce and/or pro-mote cancer cell proliferation in an ER a -dependent manner ( 19 ), allow us to depict BPA as a double-sided endocrine dis-ruptor, which promotes tumor incidence in breasts and other tar-get organs that predominantly express ER a , but the same mole-cule inhibits the E2 protective effects in the ER b -expressing co-lon. These two divergent aspects could act synergistically byincreasing the E2-disrupting potential of this widespread envi-ronmental polluter. In light with these results, women may beconsidered a highly susceptible population with an increasedrisk of colon cancers after BPA exposures. ACKNOWLEDGEMENTS The authors thank Prof. Paolo Ascenzi for helpful discussions.Dr. Alessandro Bolli is supported by a grant from the NationalInstitute of Biostructures and Biosystems (INBB) of Italy.This work was financially supported by the Italian HealthMinistry (Strategico, 2008). REFERENCES 1. Slattery, M. 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