As expected, done E2, G1 or Tam stimulates phosphorylation of Erk1 2 in MCF 7 cells. Interestingly, a stronger and earlier phosphorylated Inhibitors,Modulators,Libraries Erk1 2 was observed in TAM R cells during E2, G1 and Tam treatment, respectively, although there was no significant difference in basal levels of Erk1 2 between MCF 7 and TAM R cells. Moreover, these increased activations of Erk1 2 were coincident with EGFR phosphorylation in TAM R cells. The GPR30 specific antagonist G15 could significantly inhibit phosphorylation of Erk1 2 and EGFR as did the EGFR inhibitor AG1478. We noted that GPR30 activation increased ligand dependent EGFR activity, lead ing to an Erk1 2 mediated transcriptional response, thus contributing to the development of tamoxifen resistance in breast cancer cells.

As these observations indicate, GPR30 interaction with the EGFR signaling pathway could be an important mechanism in the development Inhibitors,Modulators,Libraries of tamoxifen resistance in MCF 7 cells. In human breast cancer MTs, endocrine treatment increases expression of GPR30 compared to corresponding PTs. Further experiments Inhibitors,Modulators,Libraries showed that in creased GPR30 expression mainly occurred in mem branes of TAM R cells, whereas the total GPR30 expression did not change. GPR30 seemed to enhance interaction with Inhibitors,Modulators,Libraries the EGFR signaling pathway through its translocation to the cell membrane. Redistribution of ER has been proposed as the mech anism of acquired tamoxifen resistance in breast cancer, but, this hypothesis is disputed. ER protein has no hydrophobic transmembrane domains or membrane localizing sequences, and any potential role of cytoplasmic ER interaction in the EGFR pathway in de veloping tamoxifen resistance is unclear.

ER and EGFR expression in human breast cancer tissue are also in versely correlated, ER seems to repress EGFR in breast cancer cells. On the other hand, the Gs subunit of GPR30 has been suggested to be responsible Inhibitors,Modulators,Libraries for E2 stimulation of adenylate cyclase and the ensuing increase in cAMP generation in breast cancer cells. Production of cAMP triggered by GPR30 can attenuate Erk1 2 activity by suppressing protein kinase A on RAF1. It is likely that there is an exact balance between inhibition and stimulation of the Erk1 2 pathway in MCF 7 cells. In our study, the basal cAMP level of MCF 7 cells was similar to that of TAM R cells, but E2 induced, G1 induced or Tam induced cAMP generation in TAM R cells was significantly lower than in MCF 7 cells.

These reductions of cAMP production which receded as a re sult of PKA inhibition led to increased activation of Erk1 2 in TAM R cells. All these results, showing that GPR30 destroyed the exact balance mentioned above, would promote the development of tamoxifen resistance in MCF 7 cells Imatinib Mesylate during endocrine treatment, but the pre cise molecular mechanism to explain how GPR30 causes an imbalance between inhibition and stimulation of the Erk1 2 pathway induced by cAMP is unclear at the present time. Further studies are needed to investigate this process.