Development of novel therapeutic strategies for lung cancer: targeting the cholinergic system
Authors: Patrizia A. Russo, Fausto Sessa, Alessia Catassi, Silvia Viaggi, Michele Cilli, Emanuela Ognio, Guido Doria-Miglietta, Massimo Paolucci, Andrea Calcaterra, Andrea Pericelli, Massimo Fini, Alfredo Cesario, Pierluigi Granone, Denis Servent, Lorenzo Dominioni
Journal: Cancer Research (2006)

4618   The concept of non-neuronal Acethlycholine synthesis and non-neuronal cholinergic system activity is longstanding. It was reported that nicotine in human NSCLC A549 cell line,  via  linking to alpha7-nicotinic receptors, potently induces Bad phosphorylation that is associated with reduced apoptotic cell death ( Jen et al. J Biol Chem. 2004; 279:23837 ). We have reported that mesothelioma cell growth is modulated by the cholinergic system in which agonists (i.e. nicotine) has a proliferative and anti-apoptotic effect and antagonists (i.e. curare) has an inhibitory and pro-apoptotic effect ( Trombino et al. Cancer Res. 2004; 64:135 ). Our objective, in this study, is to inhibit NSCLC & mesothelioma (A549, IST-MES2 cell lines & primary cell cultures obtained by human biopsies) cell growth in culture and in i.p.or ortotopically xenotransplanted SCID mice using a potent and specific antagonist of alpha-7-nicotinic receptor namely: alpha-Cobratoxin (alpha-CbT). Alpha-CbT, a member of the long alpha-neurotoxin family, is obtained from the venom of  Naja naja kaouthia  and 95% purified. We confirmed the presence of nicotinic receptors in all cells studied. Alpha-CbT is cytotoxic and induced apoptosis both in NSCLC and in mesothelioma cells.10 x IC 50  [10 nM] alpha-CbT, on A549 cells, induced reduction of mitochondrial membrane potential, release of cytochrome  c  and SMAC proteins, translocation of Bad to mitochondria, inhibition of BAD phosphorilation in Ser 112 and 136, dissociation of the complex BAD-14-3-3 and consequent formation of the complex BAD-BCL-XL and finally cleavage of caspase 9 and 3. This concentration induced mitotic catastrophe and destroyed tubulin arrangement. In SCID mice model the concentrations of 3.0 nM (1/ 3 in vitro  IC 50 ), given three times a week for two months (i.p. or i.v.), did not induce lethality, net body weight loss or any signs of stress (i.e. lethargy, ruffled coat, ataxia). Furthermore 3.0 nM alpha-CbT did not change hematological parameters & levels of serum liver enzymes. Histology of several organs (i.e. brain, liver, heart, kidney, spleen, testis) appeared normal. 3.0 nM, as well as 1.0 nM (1/10  in vitro  IC 50 ), alpha-CbT reduced, significantly, the tumor mass, induced “in vivo” apoptosis and decreased, significantly, the number of liver metastases. Cells, obtained after enzymatic digestion of tumors obtained from treated mice, showed high degree of apoptosis, low number of mitotic cells, are unable to clone in soft agar and to develop tumor when re-injected in SCID mice. Modified alpha-CbT (substitution in 33 R/E, not specific to nicotinic receptor) is ineffective. Interestingly, human proliferating T lymphocytes were not affected by alpha-CbT, also when treated at very high concentrations (1 microM). Cells without alpha-7-nicotinic receptors (Jurkat) are also not affected. In conclusion alpha-CbT shows clear and specific antitumor activity both in NSCLC and mesothelioma cells.