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

It is known that estrogen promotes the proliferation of breast cancer cells. Agonists to P2Y(2) 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 P2Y(2) receptors play a role in the effects of estrogen on the breast cancer cell lines, MCF-7 and MDA-MB-231. We found that P2Y(2) receptors were expressed in both the estrogen receptor alpha (ER alpha)-positive breast cancer cell line MCF-7 and the ER alpha-negative breast cancer cell line MDA-MB-231. 17 beta-Estradiol (17 beta-E-2) (1 pM to 1000 nM) promoted proliferation of MCF-7 cells, which was blocked by the ER antagonist ICI 182,780 (1 mu M) and the ER alpha antagonist methyl-piperidino-pyrazole (MPP, 50 mu M), but not by the ER beta antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP, 50 mu M) or ER beta small interfering RNA. The P2Y(2) and P2Y(4) receptor agonist UTP (10-100 mu 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 mu M), known to be an effective antagonist against P2Y(2), but not P2Y(4), receptor-mediated responses. 17 beta-E-2 played a more positive role in promoting proliferation in MCF-7 cells when suramin blocked the functional P2Y(2) receptors. 17 beta-E-2 (0.1-1000 nM) downregulated the expression of P2Y(2) 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 interfering RNA. 17 beta-E-2 did not affect the expression of P2Y(2) receptors in MDA-MB-231. UTP (10-100 mu M) led to a sharp increase in intracellular Ca2+. in MCF-7 cells. Pre-incubation with 17 beta-E-2 (0.1 mu M) attenuated UTP-induced [Ca2+](i), which was blocked by ICI182,780 and MPP, but not PHTPP. It is suggested that estrogen, via ER alpha receptors, promotes proliferation of breast cancer cells by downregulating P2Y(2) receptor expression and attenuating P2Y(2)-induced increase of [Ca2+](i) (C) 2011 Elsevier Ireland Ltd. All rights reserved.