Nicotinic receptors mediate tumorigenic action of tobacco-derived nitrosamines on immortalized oral epithelial cells.

Frequent users of smokeless tobacco (ST) have an increased risk for developing oral cancer. Nicotine and its derivatives may contribute to tumorigenesis through stimulation of nicotinic acetylcholine receptors (nAChRs) in target cells. Emerging evidence indicates that nAChRs can be stimulated by the nicotine-derived nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) that can induce oral cavity tumors in laboratory animals. This study was designed to elucidate the receptor-mediated mechanisms of the initiation and progression of NNK-, and NNN-induced oral cancers. We used Het-1A cells that were found to express alpha3, alpha5, alpha7, alpha9, beta2 and beta4 nAChR subunits. Both NNK and NNN competed with nicotinic radioligands for binding to Het-1A cells. NNK showed a higher than NNN affinity to the [3H]nicotine-labeled binding sites, and NNN-to the [3H]epibatidine-sensitive nAChRs. NNK and NNN increased proliferative potential of Het-1A cells and produced an anti-apoptotic effect, which was alleviated by antagonists. alpha-Bungarotoxin was most effective against NNK and mecamylamine against NNN. Treatment of Het-1A cells with either NNK or NNN led to acquisition of capability of anchorage independent growth and ability to produce tumors in nude mice, both of which can be by inhibited by antagonists. To elucidate the signaling mechanisms, we studied transcription of the genes encoding the cell cycle, apoptosis and signal transduction regulators at both the mRNA and protein levels. The Het-1A cells stimulated with nitrosamines showed multifold increases of the mRNA transcripts encoding PCNA and Bcl-2, and upregulated expression of the transcription factors GATA3, nuclear factor-kappaB, and STAT-1. The STAT-1 protein-binding activity induced by NNK and NNN correlated with elevated gene expression. The obtained results establish the role of specific nAChR subtypes in tobacco-related carcinogenesis and open a novel avenue for oral cancer chemoprevention.