Beta adrenergic action on human breast cancer cell lines

PEREZ PIñERO C; CASTILLO LF; BRUZZONE A; LUTHY IA

Orlando

Congreso; Amercian Association for Cancer Research; 2011

Amercian Association for Cancer Research

Breast cancer, the most frequent can­­cer among women in the majority of countries, is also a stressful disease. The principal effectors of the stress system include the epinephrine (EPI) and norepinephrine which bind to a1-, a2- and b-adrenoceptors (AR). We have described á2-AR in human tumor and non-tumor breast cell lines, associated with increased cell proliferation and tumor growth. We have also studied the effect of â-AR agonists and antagonists in different breast cancer experi­men­tal models. When animals were treated with the agonist isoproterenol (ISO) and/or salbutamol (SALB), tumor growth was significantly reduced correlating with Erk 1/2 phosphorylation status in whole protein tumor samples by Western blotting. The aims of the present work were: to assess the expression of â2-AR in human breast cancer cell lines (IBH-4, IBH-6, MDA-MB-231, MCF-7, among others) by RT-PCR and immunofluorescence (IF) techniques; to study the biological effect of b-AR agonists by means of proliferation assays and compare these results with the effect of the natural agonist (EPI); to find out the signaling pathways involved in reduced Erk phosphorylation induced by b-AR stimulation.   b2-AR expression was detected by RT-PCR, using GAPDH as a control. A positive staining was seen in all cells by IF. After the incubation of the cells with increasing doses (from 10-13M to 10-6M) of the â-AR agonist (ISO or SALB), a significant reduction in [3H]-thymidine incorporation to the cells nuclei was observed as compared to control values (expressed as percentage of control incorporation). IBH-4: ISO 0.1ìM, 75.87±8.49% vs control 100±7.18% p<0.05; SALB 0.1nM, 63.86±6.42% vs 100±7.79% p<0.05. IBH-6: ISO 0.1ìM, 67.02±6.11% vs control 100±6.64% p<0.05; SALB 0.1nM, 59.88±5.62% vs control 100±7.34% p<0.05. A similar inhibition was detected in MDA-MB-231, MCF-7 and HS-578T cell lines. In all the cell lines studied, the adrenergic agonist EPI exerted a stimulating cell proliferation effect. Erk1/2 phosphorylation was significantly inhibited by SALB or ISO studied by Western blotting. Erk1/2 phosphorylation decreased when the cells were treated with 8-Br-cAMP, or the PKA specific analog (6-Bnz) and this effect was mimic with forskolin. The effect on Erk1/2 phosphorylation was reversed by H-89 (PKA inhibitor) in the presence and absence of â-agonists. We conclude that both ISO and SALB, acting through b-AR, inhibit cell proliferation.  On the other hand, EPI increases cell proliferation acting through a2-AR. We propose that in the cell lines studied, the adrenergic compounds act via classic Gs pathways, augmenting cAMP and activating PKA and not EPAC. The results are promising and in agreement with the in vivo experiments presented before. These compounds, devoid of serious side-effects, could eventually be employed to inhibit tumor growth. In the future, it might be useful to analyse the metastatic potential of the cells.