Stimulation of beta-adrenergic receptors inhibits the proliferation and increases the adhesion of MCF-10A human breast cells: evidence for cAMP-dependant effects via distinct signalling pathways

BRUZZONE A; LUTHY IA; PARIS H

Marsella

Congreso; 13vo Congreso de la Société Française de Pharmacologie et de Thérapeutique; 2009

Société Française de Pharmacologie et de Thérapeutique

The stimulation of Beta-adrenergic receptors has been previously demonstrated to inhibit the growth of several human breast cancer cells but has never been examined in non-tumoral cells. The effects Beta-adrenergic receptors are generally due to enhanced cAMP production and subsequent activation of protein kinase A (PKA), however, another pathway involving the exchange protein directly activated by cAMP (Epac) has been described. The objective of the present work was to study the effect of Beta-agonist treatment on the proliferation and the adhesion of non-tumoral human breast cells, MCF-10A, and to investigate the signalling pathways involved in these actions. Methods MCF-10A (human epithelial breast) were routinely cultured in Hepes-buffered DMEM/F12 medium containing 10 % fetal calf serum, 2 ug/ml insulin, 20 ng/ml EGF and 50 nM hydrocortisone. Proliferation was measured by cell-counting and Erk 1/2 phosphorylation was examined by Western-blotting. Effect on adhesion was investigated by counting attached cells after cell-layer treatment with trypsin-EDTA under mechanical agitation and by examining subcellular distribution of integrin beta1, E-cadherin and beta-catenin in cell-layer placed in Ca++/Mg++-free medium. Results Results The treatment of MCF-10A with 0.2 ìM isoproterenol caused a significant diminution of cell proliferation (37 ± 3 % reduction after 48 hours of treatment), that correlated with a large decrease of the level of Erk 1/2 phosphorylation. This effect was blocked by H-89 (PKA inhibitor) and mimicked by forskolin (direct activator of adenylyl cyclase) or 8-Br-cAMP (activator of PKA and Epac) or 6-Bnz-cAMP (specific activator of PKA) but not by 8-CPT-2´-O-Me-cAMP (specific activator of Epac), indicating involvement of PKA. The exposure of MCF-10A cells to 0.2 ìM isoproterenol for 4 hours caused a significant enhancement of cell adhesion (74 ± 2 % vs 1.2 ± 0.1 % of adherent cells after trypsin-EDTA treatment). Isoproterenol-induced cell adhesion was not affected by H-89. On the other hand cells treated with 8-CPT-2´-O-Me-cAMP, but not 6-Bnz-cAMP, exhibited enhanced adhesion, demonstrating involvement of Epac. As assessed by immuno-cytology with specific antibodies the effect of isoproterenol was related with a redistribution of integrin beta1 and with reinforced cell-cell contacts. vs 1.2 ± 0.1 % of adherent cells after trypsin-EDTA treatment). Isoproterenol-induced cell adhesion was not affected by H-89. On the other hand cells treated with 8-CPT-2´-O-Me-cAMP, but not 6-Bnz-cAMP, exhibited enhanced adhesion, demonstrating involvement of Epac. As assessed by immuno-cytology with specific antibodies the effect of isoproterenol was related with a redistribution of integrin beta1 and with reinforced cell-cell contacts. vs 1.2 ± 0.1 % of adherent cells after trypsin-EDTA treatment). Isoproterenol-induced cell adhesion was not affected by H-89. On the other hand cells treated with 8-CPT-2´-O-Me-cAMP, but not 6-Bnz-cAMP, exhibited enhanced adhesion, demonstrating involvement of Epac. As assessed by immuno-cytology with specific antibodies the effect of isoproterenol was related with a redistribution of integrin beta1 and with reinforced cell-cell contacts. Conclusion The present study demonstrates that beta-adrenergic stimulation of MCF-10A cells attenuates cell proliferation and enhances cell adhesion. Both effects are cAMP-dependant, however whereas growth inhibition is mediated via the PKA-Erk 1/2 pathway, enhancement of adhesion is due to Epac activation and subsequent redistribution of integrin beta1.