P2Y2 receptor-mediated modulation of estrogen-induced proliferation of breast cancer cells

Molecular and Cellular Endocrinology 338 (2011) 28–37

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P2Y2 receptor-mediated modulation of estrogen-induced proliferation of breast cancer cells Han-jun Li a,1 , Li-ya Wang b,1 , Hai-na Qu b , Li-hua Yu a , Geoffrey Burnstock c , Xin Ni a , Mingjuan Xu b,∗ , Bei Ma a,∗∗ a b c

Department of Physiology and The Key Laboratory of Molecular Neurobiology of Ministry of Education, Second Military Medical University, Shanghai 200433, PR China Department of Gynaecology and Obstetrics, Changhai Hospital, Shanghai Second Military Medical University, Shanghai 200433, PR China Autonomic Neuroscience Centre, University College Medical School, Royal Free Campus, Rowland Hill Street, London, NW3 2PF, UK

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Article history: Received 16 September 2010 Received in revised form 28 January 2011 Accepted 18 February 2011 Keywords: Estrogen Estrogen receptors P2Y2 receptor UTP Breast cancer

a b s t r a c t It is known that estrogen promotes the proliferation of breast cancer cells. Agonists to P2Y2 receptors promote or suppress proliferation in different cancers. In the present study, the methods of methylthiazoltetrazolium (MTT) assay, real-time RT-PCR, Western blot and fluorescent calcium imaging analysis were used to investigate whether P2Y2 receptors play a role in the effects of estrogen on the breast cancer cell lines, MCF-7 and MDA-MB-231. We found that P2Y2 receptors were expressed in both the estrogen receptor alpha (ER␣ )-positive breast cancer cell line MCF-7 and the ER␣ -negative breast cancer cell line MDA-MB-231. 17␤-Estradiol (17␤-E2 ) (1 pM to 1000 nM) promoted proliferation of MCF-7 cells, which was blocked by the ER antagonist ICI 182,780 (1 ␮M) and the ER␣ antagonist methyl-piperidino-pyrazole (MPP, 50 ␮M), but not by the ER␤ antagonist 4-[2-phenyl-5,7bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP, 50 ␮M) or ER␤ small interfering RNA. The P2Y2 and P2Y4 receptor agonist UTP (10–100 ␮M) suppressed the viability of breast cancer cells in both MCF-7 and MDA-MB-231 cells. The effect was blocked by suramin (10–100 ␮M), known to be an effective antagonist against P2Y2 , but not P2Y4 , receptor-mediated responses. 17␤-E2 played a more positive role in promoting proliferation in MCF-7 cells when suramin blocked the functional P2Y2 receptors. 17␤-E2 (0.1–1000 nM) downregulated the expression of P2Y2 receptors in terms of both mRNA and protein levels in MCF-7 cells. The effect was blocked by ICI 182,780 and MPP, but not PHTPP or ER␤ small interfering RNA. 17␤-E2 did not affect the expression of P2Y2 receptors in MDA-MB-231. UTP (10–100 ␮M) led to a sharp increase in intracellular Ca2+ in MCF-7 cells. Pre-incubation with 17␤-E2 (0.1 ␮M) attenuated UTP-induced [Ca2+ ]i , which was blocked by ICI182,780 and MPP, but not PHTPP. It is suggested that estrogen, via ER␣ receptors, promotes proliferation of breast cancer cells by downregulating P2Y2 receptor expression and attenuating P2Y2 -induced increase of [Ca2+ ]i . © 2011 Elsevier Ireland Ltd. All rights reserved.

Evidence has shown that 17␤-estradiol induces neoplastic transformation in human breast cancer cells (Russo and Russo, 2006), promotes the growth of breast cancer cells in vivo and in vitro (Dees et al., 1997), and significantly increases the risk of breast cancer (Berrino et al., 1996; Rock et al., 2008). Epidemiologically, the incidence of breast cancer is significantly higher in women who receive estrogen replacement therapy (ERT) or hormone replacement therapy (HRT), especially in post-menopausal women with long-term of hormone treatments (Willey and Cocilovo, 2007; Mourits and De Bock, 2006; Anonymous, 2001; Fernandez et al.,

∗ Corresponding author. ∗∗ Corresponding author. Fax: +86 21 81870979. E-mail addresses: [email protected] (H.-j. Li), [email protected] (L.-y. Wang), [email protected] (M. Xu), [email protected] (B. Ma). 1 These authors contributed equally to this work. 0303-7207/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2011.02.014

2003). Breast tumors express a high level of aromatase to biosynthesize estrogen locally (Yager and Davidson, 2006; Russo and Russo, 2006). The inhibition of aromatase by its chemical inhibitors or siRNA has been shown to suppress the proliferation of breast cancer cells in culture and reduce the growth of breast tumor in animal experiments as well as patients with breast cancer (Chen et al., 2008). The carcinogenic effect of estrogen has been shown to include genomic or/and non-genomic pathways, cell membrane-related, but ER-independent, phosphorylation of target molecules or direct genotoxic effects of estrogen metabolites on DNA damage, mutation and cell transformation (Chen et al., 2008). The relationship and the balance between ER␣ and ER␤ may be important. Functional experiments have demonstrated that ER␣ and ER␤ have completely different roles in breast cancers, in which ER␣ functions as a tumor promoter (Au et al., 2007; Taylor et al., 2002; Boggess et al., 2006; Zhang et al., 2006; Lin et al., 2007a,b) whereas ER␤ functions as a

H.-j. Li et al. / Molecular and Cellular Endocrinology 338 (2011) 28–37

tumor suppressor (Lin et al., 2007a,b; Behrens et al., 2007; Treeck et al., 2007a,b; Lazennec, 2006; Paruthiyil et al., 2004; Strom et al., 2004). Experiments on ER-knockout mice have also suggested a stimulatory role of ER␣ and an inhibitory effect of ER␤ in the proliferation of different estrogen-target tissues (Fuqua and Wolf, 1995; Dupont et al., 2000; Fuqua et al., 2003). Further, recent studies have indicated that estrogen can affect tumor cell proliferation and metastasis by interacting with G protein-coupled receptors (Chen et al., 2008). The cross-talk between a Gs-coupled A2A adenosine receptor and ER␣ is involved in the effects of ethanol on breast cancer cells (Etique et al., 2009). Another G protein-coupled receptor, the chemokine receptor CXCR4, strongly correlated with the aggressive and metastatic potential of breast cancer cells (Müller et al., 2001), which could be post-transcriptionally regulated by estrogens through ER via kinase pathways (Sengupta et al., 2009). These findings suggest that estrogen effects on G protein-coupled receptors may play a critical role in the onset and development of breast cancer. P2Y2 is a G protein-coupled receptor, which can be activated by ATP/UTP (Ralevic and Burnstock, 1998; Burnstock, 2007). P2Y2 receptors mediate proliferation in some tumours, e.g. melanoma (White et al., 2005), lung (Schafer et al., 2003), bladder (Shabbir et al., 2008a) and prostate cancer (Pines et al., 2005; Shabbir et al., 2008b,c) and antiproliferation in others, e.g. oesophageal (Janssens and Boeynaems, 2001), colorectal (Katzur et al., 1999), and ovarian cancer (Höpfner et al., 1998). These results indicate that P2Y2 receptors may qualify as promising targets for innovative treatment strategies of these cancers. In breast cancer there are reports that P2Y2 receptors are expressed in MCF-7 breast cancer tumor cells (Dixon et al., 1997; Wagstaff et al., 2000; Bilbao et al., 2010a,b) and play roles in cell proliferation (Dixon et al., 1997; Wagstaff et al., 2000). Therefore, it is speculated that G-protein-coupled P2Y2 receptors might be involved in the carcinogenic effect of estrogen in breast tumor. In this report, we have investigated the function of the P2Y2 receptor, and its possible involvement in the excitatory effect of estradiol in the breast cancer cell lines, MCF-7 and MDA-MB-231, which might open new perspectives for the therapy of estrogen-dependent breast cancer. 1. Materials and methods 1.1. Cell culture The experiments were conducted on two breast cancer cell lines. MCF-7 cells were obtained from the laboratory of the Cancer Research Institute of Second Military Medical University. MDA-MB-231 cells were obtained from the Department of Pathophysiology, Second Military Medical University. Both cell lines were grown in DMEM medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Si Ji Qing, HangZhou, China), Cells were maintained at 37 ◦ C in a humidified atmosphere of 95% air and 5% CO2 . In experiments, cells were incubated with steroid-free medium (SFM) for 1 days before applying drugs. This medium was composed of phenol red-deficient DMEM-Ham’s F-12 (GIBCO) containing 8% heat-inactivated FBS that was previously treated with charcoal to remove steroids. Preparation of charcoal-treated serum was as reported previously (Gorodeski, 1998). 1.2. MTT assay The MTT (methylthiazoltetrazolium) assays were conducted to assess cell viability. After incubation with drugs, cells seeded in 96-well plates were harvested and pre-incubated with MTT (0.5 mg/mL) for 3.5 h at 37 ◦ C. The culture media was discarded and DMSO (150 ␮L) was added; the cells were agitated on an orbital shaker for 15 min. The absorbance was then read at 490 nm.

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(sense), 5 -GCATCTCGGGCAAAGCGTA-3 (antisense). Primers of ␤-actin were used as previously described (Gao et al., 2008). The PCR solution consisted of 1.2 ␮L diluted cDNA, 0.5 ␮mol of each paired primers, 1.6 mmol Mg2+ , 200 ␮mol dNTPs, 2 U Taq DNA polymerase(TIANGEN, Beijing, China), and 1× PCR buffer. 10 ␮L of the reaction mixture was electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide, applying 100-bp DNA ladder (TIANGEN) to estimate the band sizes. Quantitative real-time PCR was carried out with Rotor-Gene 3000 (Corbett Research, Sydney, Australia) in a total volume of 25 ␮L reaction mixture. 2× Taq PCR master mix (TIANGEN) and 0.2 ␮M of each primer were used. Quantitative real-time PCR conditions were optimized according to preliminary experiments to achieve linear relationships between initial RNA concentration and PCR product. The specificity of the primers was verified by examining the melting curve as well as subsequent sequencing of the real-time RT-PCR products. Distilled water was used in place of cDNA as a negative control. Each sample was normalized on the basis of its ␤-actin mRNA content. The relative expression of the genes of interest was determined by using comparative threshold cycle (Ct) method (Livak and Schmittgen, 2001). Briefly, Ct in each group was yielded by subtracting the Ct of the housekeeping gene from the Ct of the target gene yields the Ct in each group (control and experimental groups). Then subtracting Ct of control group from the experimental group obtains the Ct, which was entered into the equation 2−Ct and calculated for the exponential amplification of PCR.

1.4. Western blotting analysis Cells were harvested and homogenized in cold lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% deoxycholic acid sodium salt, 0.1% SDS, and a protease inhibitor mixture) using a homogenizer. Total protein concentration was determined by the Bradford method using bovine serum albumin as a standard. Proteins were separated using SDS–PAGE on 10% Tris–HCl gels and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked in blocking buffer consisting of 20 mM Tris–HCl, pH 7.4, 137 mM NaCl, 0.1% Tween 20, and 5% nonfat milk at room temperature for 2 h and then incubated with the rabbit anti-human P2Y2 primary antibody (1:800, Santa Cruz), or ␤-actin (1:800, Santa Cruz) overnight at 4 ◦ C. The blots were washed, incubated with HRP-conjugated secondary antibody (1:1000) for 2 h at room temperature, and finally visualized in ECL solution. For 1 min and exposed onto Kodak film for 1–30 min. For control of correct gel loading, ␤-actin quantification was used. To quantify Western blot signals, band density was measured using UMAX PowerLook III and normalized with respect to the control.

1.5. Small interfering RNA (siRNA) of ERˇ Small interfering RNAs were synthesized by Genepharma Company (Shanghai, China). The target sequences for ER␤ siRNA were: 5 -GCCCUGCUGUGAUGAAUUATT (sense) and 5 -UAAUUCAUCACAGCAGGGCTT-3 (antisense). Scramble 3 siRNA duplexes were designed (5 -UUCUCCGAACGUGUCACGUTT-3 and 5 ACGUGACACGUUCGGAGAATT-3 ) as a negative control (NC). Cells were transfected with 25 nM target siRNAs or control siRNA using siPORTTM NeoFXTM Transfection Agent (Ambion) according to the manufacturer’s instructions, and transfection was carried out for 24 h. Subsequently, the culture medium was replaced with phenol red-deficient DMEM-Ham’s F-12 medium supplemented with 8% heat-inactivated FBS containing 17␤-E2 (0.1 ␮mol/L). After 36 h, MTT assays or Western blot analyses were conducted to assess cell viability or P2Y2 protein expression. 1.6. Measurement of [Ca2+ ]i in single cells MCF-7 cells were grown as a monolayer on rectangular glass coverslips (22 mm × 40 mm). Cell growth medium was replaced with 1 mL serum-free medium prior to loading with the calcium indicator Fluo-3 AM (Invitrogen) at 5 ␮M for 1 h at 37 ◦ C. Live confocal imaging was performed using a Leica CTS SP2 confocal spectral microscope (Olympus) equipped with a 20× dry objective (numerical aperture = 0.75). The calcium indicator was excited with an argon laser line (488 nm) and emissions recorded in the green channel (510–580 nm) for Fluo-3 after addition of vehicle control (PSS) or different drugs. PSS was prepared as previously described (Dai et al., 2002). Experiments were repeated in at least 3 different passages of cell line. Relative changes in [Ca2+ ]i were determined by calculations of F/F, where F/F = (Ft − F0 )/F0 . In this equation, Ft equals the fluorescence reading at each time point and F0 represents initial fluorescence (Laskey et al., 1998).

1.3. RT-PCR and quantitative real-time PCR

1.7. Drugs and reagents

Total RNA of cells was extracted from individual samples using the TRIzol reagent (Invitrogen) according to the manufacturer’s guidelines. Quantification of total RNA was performed by measuring absorbance at OD260. 2 ␮g RNA were reverse transcribed in a final volume of 25 ␮L using the Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) and then stored at −20 ◦ C. Primers of P2Y2 receptor used for PCR amplification were, 5 -TGGCTCGGCGACTGCTAAA-3

17␤-E2 (water soluble), UTP, suramin and charcoal were purchased from Sigma, Aldrich. ICI 182,780, MPP and PHTPP, were purchased from Tocris. DMEM/F-12 medium was from Gibco Co. Solutions of 17␤-E2 and other drugs were prepared using deionized water and stored frozen, except for ICI 182,780, MPP and PHTPP, which were dissolved in dimethylsulphoxide to 1 mM, and then diluted in the medium to the final concentration.

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1.8. Statistical analysis The results are presented as mean ± SEM. Data were analyzed using SPSS software version 11.5. Statistical significance of differences was determined by one-way ANOVA. Differences were considered significant at p < 0.05.

2. Results 2.1. Estrogen promoted the proliferation of MCF-7 cells through ER˛ To elucidate the effect of estrogen on proliferation of breast cancer cells, we carried out an MTT assay to measure cell viability. The results showed that the viability of MCF-7 cells were more than 130% of control after 48 h in different concentrations of 17␤E2 (0.01 nmol/L to 1 ␮mol/L, respectively) (Fig. 1A, n = 4, p < 0.05 in each concentration). The effect of 17␤-E2 was time-dependent from 24 to 96 h, with a peak of 486.3 ± 7.88% of control at 96 h application. The effect of 17␤-E2 was blocked by the ER antagonist ICI 182,780 (1 ␮mol/L, Fig. 1B, n = 4, p < 0.05).

To further confirm that the effect was mediated by ER, we treated MCF-7 cells with the ER␣ antagonist MPP (50 ␮mol/L) or the ER␤ antagonist PHTPP (50 ␮mol/L), which were applied 30 min before the application of 17␤-E2 (0.1 ␮mol/L). The proliferation of MCF-7 cells was 163.73 ± 8.41% of control 96 h after application of 17␤-E2 (Fig. 1C, n = 5, p < 0.01 vs control). The estrogen effect was blocked by MPP, where the cell viability was reversed to 105.45 ± 1.47% of control (n = 3, p < 0.05). PHTPP did not block the estrogen effect, with a cell viability of 151.84 ± 15.01% of control (Fig. 1D, n = 3, p > 0.05). These results suggest that estrogen could induce cell proliferation by activating ER␣ . We then used another type of breast cancer cell, the MDAMB-231 cell line, to investigate the estrogen effect. After 17␤-E2 (0.1 ␮mol/L) application, the cell viability was 104.93 ± 6.67% of control at 96 h, which was not significantly different with that of control cells at the same time point (Fig. 1E, n = 6, p > 0.05 vs control). 17␤-E2 in different concentrations (0.1 nmol/L to 1 ␮mol/L) had no effect on proliferation of MDA-MB-231 cells (Fig. 1E, n = 3, p > 0.05 vs control in each concentration). ICI 182,780 (1 ␮mol/L) did not affect

H.-j. Li et al. / Molecular and Cellular Endocrinology 338 (2011) 28–37 MCF-7

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UTP (μM) Fig. 2. MTT results showing facilitation of P2Y antagonist suramin at 10 ␮mol/L (A) and 100 ␮mol/L (B) in MCF-7 cells after 4-day application (n = 5 in each concentration). (C) Diagram showing P2Y2 agonist UTP promotes cell proliferation of MDA-MB-230 in a dose-dependant manner (n = 3 in each concentration). (D) Diagram showing P2Y antagonist suramin (10 ␮mol/L) could block UTP effect (n = 3). *p < 0.05, **p < 0.01 vs control.  p < 0.05 vs UTP 10 ␮M. ## p < 0.01 vs UTP 100 ␮M.

the viability of MDA-MB-231 cells after 96 h in 17␤-E2 (0.1 ␮mol/L) (Fig. 1F, n = 4, p > 0.05 vs control). 2.2. UTP suppressed the proliferation of MCF-7 cells via P2Y2 receptors The MTT results showed that, after 4 days application of the P2Y2 receptor agonist UTP (10 ␮mol/L, 100 ␮mol/L), the proliferation of MCF-7 cells decreased to 76.75 ± 7.28% and 71.86 ± 11.99% of the control, respectively, which were significantly lower than those in control groups (Fig. 2A and B, n = 5, p < 0.01, in each con-

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centration). After co-application of UTP (10 ␮mol/L or 100 ␮mol/L) with the selective P2Y2 receptor antagonist suramin (10 ␮mol/L), cell viability was reversed to 103.12 ± 14.04% and 95.76 ± 18.86% of control, respectively (Fig. 2A, n = 5, p < 0.05, p < 0.05), indicating that suramin could block the inhibitory effect of UTP. Suramin at a concentration of 100 ␮mol/L could also block the effect of UTP (Fig. 2B, n = 5, p < 0.05, p < 0.05). Parallel with MCF-7 cells, UTP (0.1–100 ␮mol/L) decreased the viability of MDA-MB-231 cells in a dose-dependent manner. The viability of MDA-MB-231 cells was 84.35 ± 2.29% and 81.20 ± 5.13% of the control after 4 days application of UTP (10 ␮mol/L, 100 ␮mol/L), respectively, which were

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Fig. 3. (A) MTT results showing that the viability of MCF-7 cells was 187.94 ± 22.71% (n = 4) and 182.35 ± 22.64% (n = 4) of control after 4-day co-application of 17␤-E2 (0.1 ␮mol/L) and suramin (10 ␮mol/L, 100 ␮mol/L), respectively, which were increased significantly compared with those in the control group or with that in the estrogen group. (B) MTT results showing 17␤-E2 have no effect on viability of MDA-MB-231 cells either in the absence or presence of suramin (10 ␮mol/L, n = 4). *p < 0.05, **p < 0.01 vs control. # p < 0.05 vs 17␤-E2 group.

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17β − E2 (nM) Fig. 4. (A) The inhibitory effect of different concentration of 17␤-E2 (0.1–1000 nmol/L) on the expression of P2Y2 mRNA in MCF-7 cells (n = 5 in each concentration). (B) The block effect of ER antagonist ICI 182,780 (1 ␮mol/L) on estrogen (0.1 ␮mol/L) inhibition on P2Y2 mRNA expression in MCF-7 cells (n = 3). (C) 17␤-E2 (0.1–1000 nmol/L) have no effect on the expression of P2Y2 mRNA in MDA-MB-230 cells (n = 3 in each concentration). (D) ER antagonist ICI 182,780 (1 ␮mol/L) has no effect on estrogen (0.1 ␮mol/L) inhibition on P2Y2 mRNA expression in MDA-MB-230 cells (n = 3). *p < 0.05, **p < 0.01 vs control.

significantly lower than those in the control group (Fig. 2C, p < 0.01, p < 0.01, n = 3 in each concentration). After suramin and UTP coapplication, the viability of MDA-MB-231 cells was reversed to 106.75 ± 7.79% of control (Fig. 2D, n = 3, p > 0.05 vs control). 2.3. P2Y2 receptors are involved in the process of estrogen promoting proliferation of breast cancer cells In order to investigate whether P2Y2 receptors were involved in the 17␤-E2 effect on cell proliferation, we applied suramin (10 ␮mol/L, 100 ␮mol/L) to block the function of P2Y2 receptors, and then detected the function of 17␤-E2 on the proliferation of MCF-7 cells. The MTT results showed that the viability of MCF-7 cells was 187.94 ± 22.71% and 182.35 ± 22.64% of control after 4 days co-application of 17␤-E2 (0.1 ␮mol/L) and suramin (10 ␮mol/L, 100 ␮mol/L, respectively), which were increased significantly compared with those in the control group (Fig. 3A, n = 4, p < 0.01, p < 0.01) or with that in the estrogen group (Fig. 3A, n = 4, p < 0.05). However, suramin did not affect the estrogen effect on the MDA-MB-231 cell line (Fig. 3B, n = 4, p > 0.05). 2.4. Estrogen inhibited P2Y2 receptor expression in MCF-7 cells Real-time RT-PCR and Western blot were used to measure the expression of P2Y2 receptors in two breast cancer cell lines. In ER␣ positive MCF-7 cells, after 24 h incubation with 17␤-E2 at a concentration of 1 nmol/L or 1 ␮mol/L, P2Y2 mRNA levels were significantly decreased to 81.92 ± 7.79% and 53.68 ± 5.9% of control, respectively (Fig. 4A, p < 0.05 and p < 0.01, n = 5 in each concentration), and P2Y2 protein levels were decreased to 53.59 ± 13.60% and 48.94 ± 10.31% of control, respectively (Fig. 5C, p < 0.01 and p < 0.01, n = 4 in each concentration). The inhibitory effect of 17␤-E2 on P2Y2 expression was blocked by the ER antagonist ICI 182,780.

17␤-E2 treatment at a concentration of 0.1 ␮mol/L decreased the P2Y2 mRNA levels to 56.5 ± 6.01% (Fig. 4B, n = 4, p < 0.01) and P2Y2 protein levels to 52.97 ± 8.36% of control (Fig. 5C, n = 4, p < 0.01). After 17␤-E2 and ICI 182,780 co-application, the expression of P2Y2 mRNA was reversed to 114 ± 3.89% of control (Fig. 4B, n = 3, p > 0.05) and P2Y2 protein was reversed to 96.68 ± 8.71% of control (Fig. 5C, n = 3, p > 0.05). In the ER␣ -negative cell line MDA-MB-231, 17␤-E2 (0.1 nmol/L to 1 ␮mol/L) did not affect the expression of P2Y2 receptors (Fig. 4C, n = 3, p > 0.05 vs control in each concentration). ICI 182,780 (1 ␮mol/L) and 17␤-E2 (0.1 ␮mol/L) co-application did not affect the expression of P2Y2 receptors (Fig. 4D, p > 0.05 vs control, n = 3). Further research was conducted with the ER␣ antagonist MPP and the ER␤ antagonist PHTPP in MCF-7 cells after 24 h treatment. MPP blocked the inhibitory effect of 17␤-E2 on the expression of P2Y2 receptor mRNA (Fig. 5A, n = 5) and protein (Fig. 5D, n = 3). MPP (50 ␮mol/L) alone did not affect P2Y2 mRNA or protein levels (p > 0.05, p > 0.05 vs control). When MPP and 17␤-E2 were co-applied, the expression of P2Y2 mRNA was to 84.6 ± 10.73% and P2Y2 protein was to 93.66 ± 18.83% of control, both of which were significantly different to that after 17␤-E2 treatment (p < 0.01, p < 0.01). PHTPP did not block the inhibitory effect of 17␤-E2 on the expression of P2Y2 receptors. After pre-incubation with PHTPP and 17␤-E2 , the expression of both P2Y2 mRNA (Fig. 5B, n = 6) and P2Y2 protein (Fig. 5D, n = 3) was not significantly different compared with those after 17␤-E2 treatment (p > 0.05, p > 0.05). 2.5. Estrogen effect was not changed after using siRNA for ERˇ in MCF-7 cells To confirm the role of ER␤ in estrogen-induced proliferation, we used the siRNA strategy to deplete ER␤ (Fig. 6A). MTT assays

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Fig. 5. (A) The block effect of MPP (50 ␮mol/L) on 17␤-E2 (0.1 ␮mol/L) inhibition on P2Y2 mRNA expression (n = 5). (B) PHTPP (50 ␮mol/L) cannot block the inhibitory effect of estrogen (0.1 ␮mol/L) on P2Y2 mRNA expression (n = 6). (C) Diagram showing the inhibitory effects of different concentration of 17␤-E2 (0.1–1000 nmol/L) on the expression of P2Y2 protein and the block effect of ER antagonist ICI 182,780 (1 ␮mol/L) on estrogen effect in MCF-7 cells (n = 3 in each concentration). (D) Effects of ER␣ antagonist MPP (50 ␮mol/L, n = 3) and ER␤ antagonist PHTPP (50 ␮mol/L, n = 3) on estrogen modulation of P2Y2 protein expression in MCF-7 cells. *p < 0.05, **p < 0.01 vs control.

Fig. 6. (A) Effects of ER␤ small interfering RNA (siRNA) on ER␤ protein expression in MCF-7 cells (n = 3). (B) MTT results showing ER␤ siRNA did not block the effect of estrogen (0.1 ␮mol/L, n = 3) on MCF-7 cells after 36 h application. (C) Effects of ER␤ depletion on estrogen modulation (0.1 ␮mol/L, n = 4) of P2Y2 protein expression in MCF-7 cells. *p < 0.05, **p < 0.01 vs those in the absence of estrogen.

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5

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Fig. 7. (A) UTP caused a sharply increase in [Ca ]i in 10 s after UTP treatment, followed by a slow return. [Ca2+ ]i increased by 100 ␮mol/L UTP (n = 4 slices, 37cells) was more significant than that by 10 ␮mol/L UTP (n = 4 slices, 36 cells). The averaged data obtained from multiple cells. Extracellular UTP at concentrations of 10 ␮mol/L and 100 ␮mol/L evoked peak increases of F/F to 3.76 ± 1.09 and 4.77 ± 0.79, respectively. The [Ca2+ ]i stimulated by 100 ␮mol/L UTP were significantly higher than by 10 ␮mol/L UTP. **p < 0.01 vs control. 2+

demonstrated that after 17␤-E2 (0.1 ␮mol/L) application, the cell viability was 131.97 ± 7.17% of control at 36 h, which was significantly different from that in the absence of estrogen (Fig. 6B, n = 3, p < 0.01). There was no difference between scramble siRNA and ER␤ siRNA after 17␤-E2 application (Fig. 6B, p > 0.05). Depletion of ER␤ concomitant with 17␤-E2 treatment led to a significant decrease in P2Y2 protein expression compared with that in the absence of estrogen (Fig. 6C, n = 4, p < 0.05), while there was no significant difference in the expression of P2Y2 protein compared with that in the absence of siRNA (Fig. 6C, n = 4, p > 0.05).

2.6. Estrogen inhibited P2Y2 receptor-induced intracellular calcium increase in MCF-7 cells To investigate the effect of UTP (10 ␮mol/L, 100 ␮mol/L) on intracellular free Ca2+ concentrations, MCF-7 cells were loaded with the Ca2+ -sensitive fluorescent probe, fura-3 AM. The intracellular calcium concentration was monitored in single cells that were exposed for 450 s to UTP at concentrations of 10 ␮mol/L or 100 ␮mol/L. As illustrated in Fig. 7A, UTP caused a sharp increase in [Ca2+ ]i within 10 seconds after UTP treatment, followed by a slow return. The [Ca2+ ]i increase by 100 ␮mol/L UTP (n = 4 slices, 37cells) was greater than that by 10 ␮mol/L UTP (n = 4 slices, 36 cells). Fig. 7B illustrates the averaged data obtained from multiple cells. Extracellular UTP at concentrations of 10 ␮mol/L and 100 ␮mol/L evoked peak increases of F/F to 3.76 ± 1.09 and 4.77 ± 0.79, respectively. The [Ca2+ ]i stimulated by 100 ␮mol/L UTP were significantly higher than by 10 ␮mol/L UTP (p < 0.01). To investigate the effect 17␤-E2 on the UTP-induced [Ca2+ ]i increase, MCF-7 cells in normal Ca2+ PSS were pre-incubated with 0.1 ␮mol/L 17␤-E2 for 1 day, followed by a 450-s stimulation with 100 ␮mol/L UTP in the presence of 0.1 ␮mol/L 17␤-E2 . Fig. 8A is a representative trace of F/F, depicting [Ca2+ ]i responses to either 100 ␮mol/L UTP alone, or 100 ␮mol/L UTP preceded by 0.1 ␮mol/L 17␤-E2 treatment or 17␤-E2 and ICI 182,780 co-treatment, or 17␤E2 and MPP co-treatment or 17␤-E2 and PHTPP co-treatment.

Fig. 8B illustrates the average F/F obtained from four independent experiments. While 100 ␮mol/L UTP alone evoked a peak increase of 4.77 ± 0.79 (n = 4 slices, 37 cells). 17␤-E2 pretreatment significantly decreased 100 ␮mol/L UTP-induced [Ca2+ ]i , with the peak of F/F being 3.67 ± 0.70 (n = 4 slices, 62 cells, p < 0.01 vs control). The effect of 17␤-E2 on UTP-induced [Ca2+ ]i increase was blocked by the ER antagonist ICI 182,780, which restored the F/F to a peak of 4.80 ± 0.88 (n = 3 slices, 35 cells, p > 0.05 vs control). Further, we found that the effect of 17␤-E2 on UTP-induced [Ca2+ ]i increases were also blocked by the ER␣ antagonist MPP, with the peak of F/F being 4.72 ± 1.13 (n = 3 slices, 25 cells, p > 0.05 vs control). However, the estrogen effect was not blocked by the ER␤ antagonist PHTPP. 17␤-E2 and PHTPP co-application decreased 100 ␮mol/L UTP-induced F/F to a peak of 4.00 ± 0.83 (n = 3 slices, 34 cells, p < 0.01 vs control), which was not significantly different from that in the 17␤-E2 group.

3. Discussion In the present study, we showed that UTP inhibited the proliferation of breast cancer cells via P2Y2 receptors in both MCF-7 and MDA-MB-231 cell lines, although this is in contrast to early findings of UTP-mediated promotion of proliferation in breast cancer cells via P2Y2 receptors (Dixon et al., 1997; Bilbao et al., 2010b). Estrogen, however, promoted proliferation of MCF-7 cells via activating ER␣ . 17␤-E2 promoted significantly greater proliferation of MCF-7 cells after co-application of the P2Y receptor antagonist, suramin. P2Y2 mRNA and protein expressions and P2Y2 -induced intracellular calcium increases in MCF-7 cells were reduced after 17␤-E2 treatment, which was blocked by the ER antagonist ICI 182,780, the ER␣ antagonist MPP, but not the ER␤ antagonist PHTPP. ER␤ siRNA did not block the effect of estrogen on MCF-7 cell proliferation or estrogen modulation of P2Y2 protein expression in MCF-7 cells. These results suggest that inhibition of P2Y2 receptors via ER␣ is probably involved in the regulatory role of estrogen at the onset and development of breast cancer.

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Fig. 8. (A) A representative trace of F/F which depicting [Ca2+ ]i responses to either 100 ␮mol/L UTP alone, or 100 ␮mol/L UTP preceded by 0.1 ␮mol/L 17␤-E2 treatment or 17␤-E2 and ICI 182,780 co-treatment, or 17␤-E2 and MPP co-treatment or 17␤-E2 and PHTPP co-treatment. The average F/F obtained from four independent experiments. While 100 ␮mol/L UTP alone evoked a peak increase of 4.77 ± 0.79 (n = 4 slices, 37 cells). 17␤-E2 pretreatment significantly decreased 100 ␮mol/L UTP-induced [Ca2+ ]i , with the peak of F/F as 3.67 ± 0.70 (n = 4 slices, 62 cells), **p < 0.01 vs control.

ATP was originally shown to cause a decrease in breast tumor cell growth in MCF-7 and MDA-MB 231 cell lines, probably via P2X7 receptors (Vandewalle et al., 1994) and later was reported to be involved in the inhibition of cell growth in various malignancies including oesophageal (Maaser et al., 2002), colon (Höpfner et al., 2001), ovarian (Schultze-Mosgau et al., 2000) and hormonerefractory prostate cancers (Calvert et al., 2004). This response has been found to be mediated via P2 receptor subtypes. In the breast cancer cell line MCF-7, P2Y2 and P2Y4 receptors were reported to be expressed and mediating cell proliferation (Dixon et al., 1997; Wagstaff et al., 2000; Bilbao et al., 2010a). Since ATP can activate both P2X and P2Y receptors, UTP was then chosen to be a specific agonist of P2Y2 and P2Y4 receptors in breast cancer cells. Our results showed that exogenous UTP suppressed cell proliferation in both MCF-7 and MDA-MB-231 cell lines, which was blocked by suramin. The ability of suramin to inhibit the antineoplastic action of UTP confirms the activation of P2Y2 purinergic receptors, and makes activation of P2Y4 unlikely (Bogdanov et al., 1998). This result is supported by the evidence that the cell cycle arresting and apoptosis inducing actions of ATP involved the activation of P2Y2 receptors in oesophageal cancer cells (Maaser et al., 2002), human colorectal HT29 cells (Höpfner et al., 1998), human colorectal carcinoma cells (Höpfner et al., 2001), and human endometrial cancer HEC-1A and Ishikawa cells (Katzur et al., 1999).

In contrast with the observations in this study, extracellular ATP was found to stimulate proliferation of breast cancer cells in vitro through P2Y2 receptors, whose activation led to release of intracellular Ca2+ (Dixon et al., 1997; Wagstaff et al., 2000). However, it is not clear why the activation of the same receptors in the same cell line leads to different effects on cell growth in the hands of different research groups. P2Y2 receptors are G protein-coupled receptors that usually activate the PLC pathway, which leads to the formation of inositol Ins(1,4,5)P3 and the mobilization of Ca2+ from intracellular stores (White and Burnstock, 2006). However, some other downstream intracellular signaling pathways are also reported. In human breast cancer cell line MCF-7, activation of P2Y2 receptors led to elevated calcium levels and increased proliferation (Dixon et al., 1997; Wagstaff et al., 2000). This effect might be induced by transcription of the immediate early gene c-fos, activation of extracellular signal-regulated kinase (ERK) and phosphorylation of the transcription factors CREB and Elk-1 (Wagstaff et al., 2000). In human ovarian EFO-21 and EFO-27 carcinoma cells, ATP attenuated cell proliferation through P2Y2 receptors, which activated both phospholipase C and the phospholipase D pathways, and then generated the production of several intracellular messengers (Schultze-Mosgau et al., 2000). Recently, it has been reported that activation of P2Y2/4 receptors in the MCF-7 cell line causes an increase in cell number, which was mediated via activation of the PI3 K/Akt signaling pathway through PLC/IP3 /Ca2+ , PKC and Src

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(Bilbao et al., 2010b). Although the mechanism underlying these differences is not fully understood, the simultaneous activation of G proteins-related different intracellular signaling pathways might play opposite roles in the P2Y2 effect on cell growth. In addition, the tissue of origin of the cancer and the anaplastic nature of cancers needs to be considered. It has been widely accepted that breast cancer risk is associated with relatively high concentrations of endogenous estradiol in postmenopausal women (Key et al., 2002; Folkerd et al., 2006; Hankinson, 2005; Schairer et al., 2000). Moreover, reports indicate that in post-menopausal women, estrogen levels in breast tissue are 10–50 times higher than the level in blood and concentrations of estrogen are higher in malignant tissues than in non-malignant tissues (Thomas et al., 1997; Yager and Davidson, 2006). Previous studies indicate that two nuclear estrogen receptors ER␣ and ER␤ respond differently to estrogen and selective estrogen receptor modulators (SERMs) at AP1 sites, and thus play different roles in the process of proliferation (Paech et al., 1997; Webb et al., 1995). ER␣ appears to promote the proliferation while ER␤ mainly suppresses the proliferation of tumor cells (Yager and Davidson, 2006; Pearce and Jordan, 2004; Lin et al., 2007a,b; Behrens et al., 2007; Treeck et al., 2007b; Au et al., 2007; Chan et al., 2008). Our result showed that estradiol promoted the proliferation of cells in the ER␣ -positive breast cancer cell line MCF-7, which was blocked by the ER␣ antagonist MPP but not the ER␤ antagonist PHTPP or ER␤ siRNA. In the ER␣ -negative breast cancer cell line MDA-MB-231, estrogen had no effect on cell growth. These results are consistent with previous reports (Chen et al., 2008). Recently, ER␤ -selective agonists or ER␣ antagonists have been shown to inhibit cell proliferation or to enhance apoptosis (Davis et al., 2006; Pravettoni et al., 2007). These findings further support that ER␣ and ER␤ play different roles in the onset and development of cancers, which may be due to different recruitment and expression of regulators, different ratios of ER␣ and ER␤ , and phosphorylation level of ERs (Barnard et al., 1994). In our research, when P2Y2 receptor function was blocked by suramin in the ER␣ -positive MCF-7 cell line, estrogen played a more significant role in promoting cell proliferation. However, in the ER␣ -negative MDA-MB-231 cell line, we did not see a similar phenomenon. These findings suggest that P2Y2 receptors might be involved in the estrogen effect on MCF-7 cells. Further research showed that estrogen inhibited expression of P2Y2 receptors at both the mRNA and protein levels in MCF-7 cells, in a concentration-dependent manner. The ER antagonist ICI 182,780 and the ER␣ antagonist MPP blocked the estrogen effect, while the ER␤ antagonist PHTPP did not. A similar phenomenon was not observed in MDA-MB-230 cells. ER␤ siRNA did not block estrogen modulation of P2Y2 protein expression in MCF-7 cells. Combined with the stimulatory effect of estrogen on MCF-7 cell growth and the inhibitory effect on UTP-induced [Ca2+ ]i , we speculate that estrogen might increase the proliferation of breast cancer cells by down-regulating the expression of P2Y2 receptors via an ER␣ related genomic pathway. In the genomic pathway, estrogen exerts its function via ER␣ and ER␤ . The genomic pathway typically occurs in a couple of hours. In the classical genomic pathway, activated ERs formats nuclear ER homo- or heterodimers, and subsequently binding of this nuclear estrogen-ER complex binds to estrogen response element (ERE) sequences in the promoter region of estrogenresponsive genes, leading to the recruitment of co-regulators to the promoter, which leads to an increase or decrease in mRNA levels. Another genomic pathway which is usually described as the non-classical genomic pathway, ERs can regulate transcription of about one third of estrogen-responsive genes independent of ERE by interacting with other DNA-bound transcription factors (Pearce and Jordan, 2004; Ascenzi et al., 2006; Pietras and Marquez-Garban,

2007). In the present study, the mechanisms underlying the altered P2Y2 receptor expression after exposure to estrogen in MCF-7 cells are explored. Further studies are required to clarify the mechanisms of the regulatory role of estrogen on P2Y2 receptors and its role in the metastatic potential of breast cancer cells. Acknowledgements This study was supported by the Natural Science Foundation of Shanghai Science and Technology Committee (06ZR14109) and The Key Laboratory of Molecular Neurobiology of Ministry of Education. We thank Dr. Jianxin Dai in the Cancer Research Institute of Second Military Medical University for providing us with the MCF-7 cell line, Dr. Jian Lu in the Department of Pathophysiology SMMU for providing us with the MDA-MB-231 cell line, and Dr. Yongji Yang for his help in [Ca2+ ]i measurements. We are deeply grateful to Dr. Gill Knight (from the Autonomic Neuroscience Centre, Royal Free and University College Medical School, UK) for her kind assistance in English writing. References Anonymous, 2001. Hormone replacement therapy and cancer. Gynecol. Endocrinol. 15, 453–465. Ascenzi, P., Bocedi, A., Marino, M., 2006. Structure–function relationship of estrogen receptor alpha and beta: impact on human health. Mol. Aspects Med. 27 (4), 299–402. Au, W.W., Abdou-Salama, S., Al-Hendy, A., 2007. Inhibition of growth of cervical cancer cells using a dominant negative estrogen receptor gene. Gynecol. Oncol. 104, 276–280. Bogdanov, Y.D., Wildman, S.S., Clements, M.P., King, B.F., Burnstock, G., 1998. Molecular cloning and characterization of rat P2Y4 nucleotide receptor. Br. J. Pharmacol. 124 (3), 428–430. Barnard, E.A., Burnstock, G., Webb, T.E., 1994. G protein-coupled receptors for ATP and other nucleotides: a new receptor family. Trends Pharmacol. Sci. 15 (3), 67–70. Behrens, D., Gill, J.H., Fichtner, I., 2007. Loss of tumourigenicity of stably ERbetatransfected MCF-7 breast cancer cells. Mol. Cell. Endocrinol. 274, 19–29. Berrino, F., Muti, P., Micheli, A., Bolelli, G., Krough, V., Sciajno, R., Pisani, P., Panico, S., Secreto, G., 1996. Serum sex hormone levels after menopause and subsequent breast cancer. J. Natl. Cancer Inst. 88, 291–296. Bilbao, P.S., Boland, R., Santillán, G., 2010a. ATP modulates transcription factors through P2Y2 and P2Y4 receptors via PKC/MAPKs and PKC/Src pathways in MCF-7 cells. Arch. Biochem. Biophys. 494 (1), 7–14. Bilbao, P.S., Santillán, G., Boland, R., 2010b. ATP stimulates the proliferation of MCF7 cells through the PI3K/Akt signaling pathway. Arch. Biochem. Biophys. 499 (1–2), 40–48. Boggess, J.F., Zhou, C., Bae-Jump, V.L., Gehrig, P.A., Whang, Y.E., 2006. Estrogenreceptor-dependent regulation of telomerase activity in human endometrial cancer cell lines. Gynecol. Oncol. 103, 417–424. Burnstock, G., 2007. Purine and pyrimidine receptors. Cell. Mol. Life Sci. 64 (12), 1471–1483. Calvert, R.C., Shabbir, M., Thompson, C.S., Mikhailidis, D.P., Morgan, R., Burnstock, G., 2004. Immunocytochemical and pharmacological characterisation of P2purinoceptor-mediated cell growth and death in PC-3 hormone refractory prostate cancer cells. Anticancer Res. 24, 2853–2859. Chan, K.K., Wei, N., Liu, S.S., Xiao-Yun, L., Cheung, A.N., Ngan, H.Y., 2008. Estrogen receptor subtypes in ovarian cancer: a clinical correlation. Obstet. Gynecol. 111 (1), 144–151. Chen, G.G., Zeng, Q., Tse, G.M., 2008. Estrogen and its receptors in cancer. Med. Res. Rev. 28 (6), 954–974. Dai, J., Inscho, E.W., Yuan, L., Hill, S.M., 2002. Modulation of intracellular calcium and calmodulin by melatonin in MCF-7 human breast cancer cells. J. Pineal Res. 32 (2), 112–119. Davis, A.M., Ellersieck, M.R., Grimm, K.M., Rosenfeld, C.S., 2006. The effects of the selective estrogen receptor modulators, methyl-piperidino-pyrazole (MPP), and raloxifene in normal and cancerous endometrial cell lines and in the murine uterus. Mol. Reprod. Dev. 73, 1034–1044. Dees, C., Askari, M., Foster, J.S., Ahamed, S., Wimalasena, J., 1997. DDT mimics oestrogen stimulation of breast cancer cells. Mol. Carcinog. 18, 107–114. Dixon, C.J., Bowler, W.B., Fleetwood, P., Ginty, A.F., Gallagher, J.A., Carron, J.A., 1997. Extracellular nucleotides stimulate proliferation in MCF-7 breast cancer cells via P2-purinoceptors. Br. J. Cancer 75 (1), 34–39. Dupont, S., Krust, A., Gansmuller, A., Dierich, A., Chambon, P., Mark, M., 2000. Effect of single and compound knockouts of estrogen receptors a and b on mouse reproductive phenotypes. Development 127, 4277–4291. Etique, N., Grillier-Vuissoz, I., Lecomte, J., Flament, S., 2009. Crosstalk between adenosine receptor (A2A isoform) and ERalpha mediates ethanol action in MCF7 breast cancer cells. Oncol. Rep. 21 (4), 977–981.

H.-j. Li et al. / Molecular and Cellular Endocrinology 338 (2011) 28–37 Fernandez, E., Gallus, S., Bosetti, C., Franceschi, S., Negri, E., LaVecchia, C., 2003. Hormone replacement therapy and cancer risk: a systematic analysis from a network of case–control studies. Int. J. Cancer 105, 408–412. Folkerd, E.J., Martin, L.A., Kendall, A., Dowsett, M., 2006. The relationship between factors affecting endogenous oestradiol levels in postmenopausal women and breast cancer. J. Steroid Biochem. Mol. Biol. 102 (1–5), 250–255. Fuqua, S.A., Wolf, D.M., 1995. Molecular aspects of estrogen receptor variants in breast cancer. Breast Cancer Res. Treat. 35, 233–241. Fuqua, S.A., Schiff, R., Parra, I., Moore, J.T., Mohsin, S.K., Osborne, C.K., Clark, G.M., Allred, D.C., 2003. Estrogen receptor beta protein in human breast cancer: correlation with clinical tumor parameters. Cancer Res. 63, 2434–2439. Gao, L., Lu, C., Xu, C., Tao, Y., Cong, B., Ni, X., 2008. Differential regulation of prostaglandin production mediated by corticotropin-releasing hormone receptor type 1 and type 2 in cultured human placental trophoblasts. Endocrinology 149 (6), 2866–2876. Gorodeski, G.I., 1998. Estrogen increases the permeability of the cultured human cervical epithelium by modulating cell deformability. Am. J. Physiol. 275 (3 Pt 1), C888–C899. Hankinson, S.E., 2005. Endogenous hormones and risk of breast cancer in postmenopausal women. Breast Dis. 24, 3–15. Höpfner, M., Lemmer, K., Jansen, A., Hanski, C., Riecken, E.O., Gavish, M., Mann, B., Buhr, H., Glassmeier, G., Scherübl, H., 1998. Expression of functional P2purinergic receptors in primary cultures of human colorectal carcinoma cells. Biochem. Biophys. Res. Commun. 251 (3), 811–817. Höpfner, M., Maaser, K., Barthel, B., von Lampe, B., Hanski, C., Riecken, E.O., Zeitz, M., Scherübl, H., 2001. Growth inhibition and apoptosis induced by P2Y2 receptors in human colorectal carcinoma cells: involvement of intracellular calcium and cyclic adenosine monophosphate. Int. J. Colorectal Dis. 16 (3), 154–166. Janssens, R., Boeynaems, J.M., 2001. Effects of extracellular nucleotides and nucleosides on prostate carcinoma cells. Br. J. Pharmacol. 132 (2), 536–546. ´ M., Schultze-Mosgau, A., Ortmann, O., Stojilkovic, Katzur, A.C., Koshimizu, T., Tomic, S.S., 1999. Expression and responsiveness of P2Y2 receptors in human endometrial cancer cell lines. J. Clin. Endocrinol. Metab. 84 (11), 4085–4091. Key, T., Appleby, P., Barnes, I., Reeves, G., Endogenous Hormones and Breast Cancer Collaborative Group, 2002. Endogenous sex hormones and breast cancer in postmenopausal women: reanalysis of nine prospective studies. J. Natl. Cancer Inst. 94 (8), 606–616. Laskey, A.D., Roth, B.J., Simpson, P.B., Russell, J.T., 1998. Images of Ca2+ flux in astrocytes: evidence for spatially distinct sites of Ca2+ release and uptake. Cell Calcium 23 (6), 423–432. Lazennec, G., 2006. Estrogen receptor beta, a possible tumor suppressor involved in ovarian carcinogenesis. Cancer Lett. 231, 151–157. Lin, C.Y., Strom, A., Li Kong, S., Kietz, S., Thomsen, J.S., Tee, J.B., Vega, V.B., Miller, L.D., Smeds, J., Bergh, J., Gustafsson, J.A., Liu, E.T., 2007a. Inhibitory effects of estrogen receptor beta on specific hormone-responsive gene expression and association with disease outcome in primary breast cancer. Breast Cancer Res. 9 (2), R25. Lin, Z., Reierstad, S., Huang, C.C., Bulun, S.E., 2007b. Novel estrogen receptor-alpha binding sites and estradiol target genes identified by chromatin immunoprecipitation cloning in breast cancer. Cancer Res. 67, 5017–5024. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25 (4), 402–408. Maaser, K., Höpfner, M., Kap, H., Sutter, A.P., Barthel, B., von Lampe, B., Zeitz, M., Scherübl, H., 2002. Extracellular nucleotides inhibit growth of human oesophageal cancer cells via P2Y(2)-receptors. Br. J. Cancer 86 (4), 636–644. Mourits, M.J., De Bock, G.H., 2006. Exogenous steroids for menopausal symptoms and breast/endometrial cancer risk. Int. J. Gynecol. Cancer 16 (Suppl. 2), 494–496. Müller, A., Homey, B., Soto, H., Ge, N., Catron, D., Buchanan, M.E., McClanahan, T., Murphy, E., Yuan, W., Wagner, S.N., Barrera, J.L., Mohar, A., Verástegui, E., Zlotnik, A., 2001. Involvement of chemokine receptors in breast cancer metastasis. Nature 410, 50–56. Paech, K., Webb, P., Kuiper, G.G., Nilsson, S., Gustafsson, J., Kushner, P.J., Scanlan, T.S., 1997. Differential ligand activation of estrogen receptors ERalpha and ERbeta at AP1 sites. Science 277 (5331), 1508–1510. Paruthiyil, S., Parmar, H., Kerekatte, V., Cunha, G.R., Firestone, G.L., Leitman, D.C., 2004. Estrogen receptor beta inhibits human breast cancer cell proliferation and tumor formation by causing a G2 cell cycle arrest. Cancer Res. 64, 423–428. Pearce, S.T., Jordan, V.C., 2004. The biological role of estrogen receptors alpha and beta in cancer. Crit. Rev. Oncol. Hematol. 50 (1), 3–22. Pietras, R.J., Marquez-Garban, D.C., 2007. Membrane-associated estrogen receptor signaling pathways in human cancers. Clin. Cancer Res. 13 (16), 4672–4676. Pines, A., Perrone, L., Bivi, N., Romanello, M., Damante, G., Gulisano, M., Kelley, M.R., Quadrifoglio, F., Tell, G., 2005. Activation of APE1/Ref-1 is dependent on reactive

37

oxygen species generated after purinergic receptor stimulation by ATP. Nucleic Acids Res. 33 (14), 4379–4394. Pravettoni, A., Mornati, O., Martini, P.G., Marino, M., Colciago, A., Celotti, F., Motta, M., Negri-Cesi, P., 2007. Estrogen receptor beta (ERbeta) and inhibition of prostate cancer cell proliferation: studies on the possible mechanism of action in DU145 cells. Mol. Cell. Endocrinol. 263, 46–54. Ralevic, V., Burnstock, G., 1998. Receptors for purines and pyrimidines. Pharmacol. Rev. 50 (3), 413–492. Rock, C.L., Flatt, S.W., Laughlin, G.A., Gold, E.B., Thomson, C.A., Natarajan, L., Jones, L.A., Caan, B.J., Stefanick, M.L., Hajek, R.A., Al-Delaimy, W.K., Stanczyk, F.Z., Pierce, J.P., Women’s Healthy Eating and Living Study Group, 2008. Reproductive steroid hormones and recurrence-free survival in women with a history of breast cancer. Cancer Epidemiol. Biomarkers Prev. 17, 204–211. Russo, J., Russo, I.H., 2006. The role of estrogen in the initiation of breast cancer. J. Steroid Biochem. Mol. Biol. 102, 89–96. Shabbir, M., Ryten, M., Thompson, C., Mikhailidis, D., Burnstock, G., 2008a. Purinergic receptor-mediated effects of ATP in high-grade bladder cancer. BJU Int. 101 (1), 106–112. Shabbir, M., Ryten, M., Thompson, C., Mikhailidis, D., Burnstock, G., 2008b. Characterization of calcium-independent purinergic receptor-mediated apoptosis in hormone-refractory prostate cancer. BJU Int. 101 (3), 352–359. Shabbir, M., Thompson, C., Jarmulowiczc, M., Mikhailidis, D., Burnstock, G., 2008c. Effect of extracellular ATP on the growth of hormone-refractory prostate cancer in vivo. BJU Int. 102 (1), 108–112. Schafer, R., Sedehizade, F., Welte, T., Reiser, G., 2003. ATP- and UTP-activated P2Y receptors differently regulate proliferation of human lung epithelial tumor cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 285 (2), L376–L385. Schairer, C., Lubin, J., Troisi, R., Sturgeon, S., Brinton, L., Hoover, R., 2000. Menopausal estrogen and estrogen-progestin replacement therapy and breast cancer risk. JAMA 283 (4), 485–491. Schultze-Mosgau, A., Katzur, A.C., Arora, K.K., Stojilkovic, S.S., Diedrich, K., Ortmann, O., 2000. Characterization of calcium mobilizing, purinergic P2Y2 receptors in human ovarian cancer cells. Mol. Hum. Reprod. 6, 435–442. Sengupta, S., Schiff, R., Katzenellenbogen, B.S., 2009. Post-transcriptional regulation of chemokine receptor CXCR4 by estrogen in HER2 overexpressing, estrogen receptor-positive breast cancer cells. Breast Cancer Res. Treat. 117, 243–251. Strom, A., Hartman, J., Foster, J.S., Kietz, S., Wimalasena, J., Gustafsson, J.A., 2004. Estrogen receptor beta inhibits 17beta-estradiol-stimulated proliferation of the breast cancer cell line T47D. Proc. Natl. Acad. Sci. U.S.A. 101, 1566–1571. Taylor, A.H., al-Azzawi, F., Pringle, J.H., Bell, S.C., 2002. Inhibition of endometrial carcinoma cell growth using antisense estrogen receptor oligodeoxyribonucleotides. Anticancer Res. 22, 3993–4003. Thomas, H.V., Reeves, G.K., Key, T.J., 1997. Endogenous estrogen and postmenopausal breast cancer: a quantitative review. Cancer Causes Control 8 (6), 922–928. Treeck, O., Pfeiler, G., Mitter, D., Lattrich, C., Piendl, G., Ortmann, O., 2007a. Estrogen receptor 1 exerts antitumoral effects on SK-OV-3 ovarian cancer cells. J. Endocrinol. 193 (3), 421–433. Treeck, O., Pfeiler, G., Horn, F., Federhofer, B., Houlihan, H., Vollmer, A., Ortmann, O., 2007b. Novel estrogen receptor beta transcript variants identified in human breast cancer cells affect cell growth and apoptosis of COS-1 cells. Mol. Cell. Endocrinol. 264, 50–60. Vandewalle, B., Hornez, L., Revillion, F., Lefebvre, J., 1994. Effect of extracellular ATP on breast tumor cell growth, implication of intracellular calcium. Cancer Lett. 85, 47–54. Wagstaff, S.C., Bowler, W.B., Gallagher, J.A., Hipskind, R.A., 2000. Extracellular ATP activates multiple signalling pathways and potentiates growth factor-induced c-fos gene expression in MCF-7 breast cancer cells. Carcinogenesis 21 (12), 2175–2181. Webb, P., Lopez, G.N., Uht, R.M., Kushner, P.J., 1995. Tamoxifen activation of the estrogen receptor/AP-1 pathway: potential origin for the cell-specific estrogenlike effects of antiestrogens. Mol. Endocrinol. 9 (4), 443–456. White, N., Ryten, M., Clayton, E., Butler, P., Burnstock, G., 2005. P2Y purinergic receptors regulate the growth of human melanomas. Cancer Lett. 224 (1), 81–91. White, N., Burnstock, G., 2006. P2 receptors and cancer. Trends Pharmacol. Sci. 27 (4), 211–217. Willey, S.C., Cocilovo, C., 2007. Screening and follow-up of the patient at high risk for breast cancer. Obstet. Gynecol. 110, 1404–1416. Yager, J.D., Davidson, N.E., 2006. Estrogen carcinogenesis in breast cancer. N. Engl. J. Med. 354 (3), 270–282. Zhang, Y., Liao, Q., Chen, C., Yu, L., Zhao, J., 2006. Function of estrogen receptor isoforms alpha and beta in endometrial carcinoma cells. Int. J. Gynecol. Cancer 16, 1656–1660.