Cross-talk between β-adrenoreceptor and α7-nicotinic acetylcholine receptor (α7-nAChR) signaling in gastric carcinogenesis

2238 Cigarette smoking is associated with various cancers, but it is unclear which component(s) imposed the carcinogenic effect in stomach. Nicotine, the major component in cigarette smoke, has been shown to exert mitogenic effect on tumor growth and angiogenesis. Moreover, nicotine-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is carcinogenic in lung cancer. However, there are few studies that examined the effects of these two components in gastric carcinogenesis. The aim of this study is to determine if nicotine and/or NNK would promote gastric tumor growth and to delineate the molecular mechanism(s) involved, which may shed light on new gastric cancer drug discovery. Human gastric carcinoma cells (AGS) were incubated with nicotine and NNK at various concentrations for 5 hours. High dose of nicotine (200 μg/ml) and NNK (100 nM) significantly increased cell proliferation by 40% and 96%, respectively. The increased cancer growth is due to the modulation of several G1 regulatory proteins, leading to the cell cycle transitions. Cyclin D1 expression and its association with cyclin-dependent kinases (CDK4/6) was analyzed by immunoprecipitation. The complexes immunoprecipitated with anti-cyclin D1 antibody exhibited higher amounts of CDK4 in NNK than nicotine treatment. Furthermore, phosphorylation of retinoblastoma protein (Rb) was activated which released the transcription factor E2F and DP-1 to form heterodimers and facilitate cell cycle progression. Pretreatment with a selective COX-2 inhibitor (SC-236) prevented the upregulation of G1 regulatory proteins and caused cell cycle arrest at G1 phase. Furthermore, nicotine- and NNK-induced COX-2 activity and protein expressions were abrogated by SC-236. The increased production of PGE 2 was blocked by SC-236. Pretreatment of cells with propranolol (non-specific β-adrenoreceptor antagonist), but not α-bungarotoxin (α-BTX, α7nAChR antagonist), abolished the nicotine-induced cell proliferation. On the other hand, α-BTX suppressed COX-2 and PGE 2 production in NNK-induced cell proliferation, and was partially abrogated by propranolol. These results pointed to the importance of adrenergic and nicotinic receptors in gastric tumor growth promoted by cigarette smoke. These data revealed that nicotine and NNK are two major components in cigarette smoke that are responsible for the tumor promoting effects on gastric carcinogenesis through the cooperation of different receptors to regulate the progression of cell cycle via COX-2 dependent pathway.