Figure 3

Effect of GPCR antagonism on 17β-oestradiol and EGF induced cell proliferation, Raf phosphorylation and cAMP production in breast cancer cells. (a) SKBR3 and MCF-7 breast cancer cells pretreated with or without the GPCR antagonist PTX (50 ng/ml) 1 hour before 24 hours of incubation EGF (10 ng/ml) and 17β-oestradiol (10^-8 mol/l) alone and in combination. Cell proliferation assays were carried out using MTT thiozolyl blue. Proliferative index of the control group is standardized to 1. The results shown are expressed as mean ± standard error (n = 9). Statistical analysis was performed using Mann Whitney U test (*P < 0.02 versus without PTX). (b) Western blot analysis of phospho-Raf and total Raf in SKBR3 and MCF-7 cells pretreated with or without the GPCR antagonist pertussis toxin (50 ng/ml) for 1 hour before 10 minutes of incubation with EGF (10 ng/ml) and 17β-oestradiol (10^-8 mol/l) alone and in combination. Results are representative of those obtained in three separate experiments. (c) Intracellular cAMP levels in SKBR3 cells treated with EGF (10 ng/ml) and 17β-oestradiol (10^-8 mol/l) alone and in combination. Results are expressed as mean ± standard error (n = 9). Statistical analysis was performed using Mann-Whitney U test (*P < 0.02 versus control). E2, 17β-oestradiol; EGFR, epidermal growth factor receptor; GPCR, G-protein-coupled receptor; PI, proliferative index.