title: The nicotinic receptor antagonists abolish pathobiologic effects of tobacco-derived nitrosamines on BEP2D cells.
abstract: Identification of the mechanisms leading to malignant transformation of respiratory cells may prove useful in the prevention and treatment of tobacco-related lung cancer. Nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) can induce tumors both locally and systemically. In addition to the genotoxic effect, they have been shown to affect lung cells due to ligating the nicotinic acetylcholine receptors (nAChRs) expressed on the plasma membrane. In this study, we sought to establish the role for nAChRs in malignant transformation caused by NNK and NNN. We used the BEP2D cells that represent a suitable model for studying the various stages of human bronchial carcinogenesis. We found that these cells express alpha1, alpha3, alpha5, alpha7, alpha9, alpha10, beta1, beta2, and beta4 nAChR subunits that can form high-affinity binding sites for NNK and NNN. Exposure of BEP2D cells to either NNK or NNN in both cases increased their proliferative potential which could be abolished in the presence of nAChR antagonists alpha-bungarotoxin, which worked most effectively against NNK, or mecamylamine, which was most efficient against NNN. The BEP2D cells stimulated with the nitrosamines showed multifold increases of the transcription of the PCNA and Bcl-2 genes by both real-time polymerase chain reaction and in-cell western assays. To gain a mechanistic insight into NNK- and NNN-initiated signaling, we investigated the expression of genes encoding the signal transduction effectors GATA-3, nuclear factor-kappaB, and STAT-1. Experimental results indicated that stimulation of nAChRs with NNK led to activation of all three signal transduction effectors under consideration, whereas NNN predominantly activated GATA-3 and STAT-1. The GATA-3 protein-binding activity induced by NNK and NNN correlated with elevated gene expression. The obtained results support the novel concept of receptor-mediated action of NNK and NNN placing cellular nAChRs in the center of the pathophysiologic loop, and suggest that an nAChR antagonist may serve as a chemopreventive agent.
authors.affiliation: Department of Dermatology, University of California, Davis, 3301 C Street, Suite #1400, Sacramento, CA 95816, USA.
authors.forename: Juan
authors.initials: J
authors.lastname: Arredondo
authors.forename: Alex I
authors.initials: AI
authors.lastname: Chernyavsky
authors.forename: Sergei A
authors.initials: SA
authors.lastname: Grando
mesh_terms: D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D001665:Binding Sites; D002038:Bungarotoxins / Q000037:antagonists & inhibitors; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D050990:GATA3 Transcription Factor / Q000235:genetics; D006801:Humans; D008168:Lung / Q000166:cytology / Q000187:drug effects*; D016328:NF-kappa B / Q000235:genetics; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D009602:Nitrosamines / Q000037:antagonists & inhibitors* / Q000633:toxicity; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics; D021122:Protein Subunits / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D011978:Receptors, Nicotinic / Q000378:metabolism; D050794:STAT1 Transcription Factor / Q000235:genetics; D014026:Nicotiana / Q000737:chemistry; D014158:Transcription, Genetic; D015854:Up-Regulation
chemical_list: D016588:Anticarcinogenic Agents; D002038:Bungarotoxins; D050990:GATA3 Transcription Factor; D016328:NF-kappa B; D018733:Nicotinic Antagonists; D009602:Nitrosamines; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; D019253:Proto-Oncogene Proteins c-bcl-2; D011978:Receptors, Nicotinic; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; C008655:N'-nitrosonornicotine