Activation of non neuronal cholinergic system induces proliferation and angiogenesis in mcf-7 human mammary adenocarcinoma cells

FISZMAN GABRIEL; MIDDONNO MARIA C,; DE LA TORRE EULALIA; FARINA MARIANA; ESPAÑOL ALEJANDRO; SALES MARIA ELENA

Los Angeles CA., USA

Congreso; Annual Meeting of the American Association for Cancer Research (AACR); 2007

Muscarinic acetylcholine receptors (mAChR) are members of the G-protein coupled receptor family. These membrane receptors play key physiological roles and changes in their expression and/or function can be involved in several diseases. We had previously demonstrated that mAChR expression is up-regulated in LM2, LM3 and LMM3 cells, three different cell lines derived from distinct murine mammary adenocarcinomas that spontaneously arose in BALB/c female mice, in comparison with normal murine mammary cells. Stimulation of mAChR with the synthetic muscarinic agonist carbachol (CARB) potentiated different steps of tumor progression. Less knowledge is available about the response of different mAChR subtypes to muscarinic agonists in human tumor mammary cells. Here we investigate the actions of CARB on human adenocarcinoma MCF-7 cells proliferation and in vivo neovascular response. We observe that CARB stimulates human breast tumor cells proliferation measured using the conversion of MTS tetrazolium salt to formazan. The maximal proliferation effect was observed with 10-9 M CARB. This action was due to M3 and M1 receptors activation because 4-diphenylacetoxi-N-methylpiperidine (4-DAMP) or pirenzepine, respectively, reduced CARB action. We also demonstrated the participation of phospholipase C, protein kinase C and nitric oxide synthase (NOS) signaling pathway in CARB-induced MCF-7 cells proliferation. The agonist action was reverted by preincubating cells with the correspondent enzymatic inhibitors: 10-6M carboxyphenyl-N,N diphenylcarbamate,  10-7M H-7 or 10-4M NGmonomethyl-L-arginine. Particularly  we confirmed the expression of NOS1 and NOS3 isoforms by Western blot. Nitric oxide production derived from calcium dependent NOS was tested by measuring nitrite accumulation in culture supernatants by Griess reagent. In addition 10-9M CARB that was the most potent concentration to produce nitrite accumulation, stimulates blood vessels formation induced in vivo by tumor cells. This response was evaluated in oestrogenized nude mice inoculated intradermically in both flanks with breast tumor cells. We can conclude that non neuronal cholinergic system activation stimulates MCF-7 tumor cells growth and neovascular response promoting tumor progression.