PHARMACOLOGICAL AND FUNCTIONAL CHARACTERISATION OF NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS IN LUNG CANCER CELL LINES: A CHALLENGE FOR NEW THERAPEUTICAL STRATEGIES?

Lung cancer is the leading cause of cancer-related deaths worldwide and cigarette smoking is related to 90% of all deaths due to lung cancer. Tobacco smoke contains many classes of carcinogens and, although nicotine, the addictive and most active component of tobacco smoke, is unable to initiate tumourigenesis in humans and rodents, it promotes tumour growth and metastasis by inducing cell-cycle progression, cell migration, angiogenesis and the evasion of apoptosis in a variety of systems. Nicotine and its metabolites are highly lipophilic compounds that bind and activate a family of ligand-gated cation channels (the neuronal nicotinic acetylcholine receptors, nAChRs) that are widely expressed in the central and peripheral nervous systems. Over the last few decades, the extra-neuronal localisation of nAChRs has been demonstrated in a large number of cell types including endothelial cells, glia, immune cells, lung tissue and cancer cells, indicating that they might have functions well beyond simple neurotransmission. Recent studies have shown that most of the tumour-promoting effects of nicotine are primarily due to the binding and the activation of nAChRs, which lead to downstream intracellular signalling cascades. However, identifying the receptor subtypes expressed in lung tumour cells and their signalling pathways is still in its early stage. For these reasons, we investigated the pathophysiological role of nAChRs in lung cancer cells. We found that different non-small cell lung cancer (NSCLC) cells express distinctive nicotinic receptor subtypes and that this variety affects nicotine-induced proliferation and migration. In the A549 adenocarcinoma cell line, nAChRs containing the \u3b17, \u3b19 and \u3b15 subunits regulate not only the nicotine-induced cell proliferation and migration but also the activation of anti-apoptotic and proliferative pathways. Blocking nAChRs containing the \u3b17 or \u3b19 or \u3b15 subunits with specific toxins or silencing their expression by means of subunit-specific siRNAs abolishes the nicotine-induced proliferation, migration and signalling. Prompted by these results, we also studied oxystylbene compounds previously characterised by our group and started to syntehesise some new oxystylbene/resveratrol derivatives with specific modifications. We found that these 4-oxystilbene derivates act on both \u3b17 and \u3b19-containing receptors and block NSCLC cell proliferation and viability in a dose-dependent manner. These results highlight the pathophysiological role of specific nAChR subtypes in promoting NSCLC cell growth and migration and raise the possibility of targeting them in order to treat tobacco related cancer

Nicotinic Receptors Underlying Nicotine Dependence: Evidence from Transgenic Mouse Models.

Nicotine underlies the reinforcing properties of tobacco cigarettes and e-cigarettes. After inhalation and absorption, nicotine binds to various nicotinic acetylcholine receptor (nAChR) subtypes localized on the pre- and postsynaptic membranes of cells, which subsequently leads to the modulation of cellular function and neurotransmitter signaling. In this chapter, we begin by briefly reviewing the current understanding of nicotine's actions on nAChRs and highlight considerations regarding nAChR subtype localization and pharmacodynamics. Thereafter, we discuss the seminal discoveries derived from genetically modified mouse models, which have greatly contributed to our understanding of nicotine's effects on the reward-related mesolimbic pathway and the aversion-related habenulo-interpeduncular pathway. Thereafter, emerging areas of research focusing on modulation of nAChR expression and/or function are considered. Taken together, these discoveries have provided a foundational understanding of various genetic, neurobiological, and behavioral factors underlying the motivation to use nicotine and related dependence processes, which are thereby advancing drug discovery efforts to promote long-term abstinence

Different physiological and behavioural effects of e-cigarette vapour and cigarette smoke in mice

Nicotine is the primary addictive substance in tobacco smoke and electronic cigarette (e-cig) vapour. Methodological limitations have made it difficult to compare the role of the nicotine and non-nicotine constituents of tobacco smoke. The aim of this study was to compare the effects of traditional cigarette smoke and e-cig vapour containing the same amount of nicotine in male BALB/c mice exposed to the smoke of 21 cigarettes or e-cig vapour containing 16.8mg of nicotine delivered by means of a mechanical ventilator for three 30-min sessions/day for seven weeks. One hour after the last session, half of the animals were sacrificed for neurochemical analysis, and the others underwent mecamylamine-precipitated or spontaneous withdrawal for the purposes of behavioural analysis. Chronic intermittent non-contingent, second-hand exposure to cigarette smoke or e-cig vapour led to similar brain cotinine and nicotine levels, similar urine cotinine levels and the similar up-regulation of α4β2 nicotinic acetylcholine receptors in different brain areas, but had different effects on body weight, food intake, and the signs of mecamylamine-precipitated and spontaneous withdrawal episodic memory and emotional responses. The findings of this study demonstrate for the first time that e-cig vapour induces addiction-related neurochemical, physiological and behavioural alterations. The fact that inhaled cigarette smoke and e-cig vapour have partially different dependence-related effects indicates that compounds other than nicotine contribute to tobacco dependence

alpha9- and alpha7-containing receptors mediate the pro-proliferative effects of nicotine in the A549 adenocarcinoma cell line

Background and Purpose: Tobacco smoke contains many classes of carcinogens and although nicotine is unable to initiate tumourigenesis in humans and rodents, it promotes tumour growth and metastasis in lung tumours by acting on neuronal nicotinic ACh receptors (nAChRs). The aim of this study was to identify molecularly, biochemically and pharmacologically which nAChR subtypes are expressed and functionally activated by nicotine in lung cancer cell lines. Experimental Approach: We used A549 and H1975 adenocarcinoma cell lines derived from lung tumours to test the in vitro effects of nicotine, and nAChR subtype-specific peptides and compounds. Key Results: The two adenocarcinoma cell lines express distinctive nAChR subtypes, and this affects their nicotine-induced proliferation. In A549 cells, nAChRs containing the alpha7 or alpha9 subunits not only regulate nicotine-induced cell proliferation but also the activation of the Akt and ERK pathways. Blocking these nAChRs by means of subtype-specific peptides, or silencing their expression by means of subunit-specific siRNAs, abolishes nicotine-induced proliferation and signalling. Moreover, we found that the alpha antagonist MG624 also acts on alpha9-alpha10 nAChRs, blocks the effects of nicotine on A549 cells and has dose-dependent cytotoxic activity. Conclusions and Implications: These results highlight the pathophysiological role of alpha7- and alpha9-containing receptors in promoting non-small cell lung carcinoma cell growth and intracellular signalling and provide a framework for the development of new drugs that specifically target the receptors expressed in lung tumours

Potent Antiglioblastoma Agents by Hybridizing the Onium-Alkyloxy-Stilbene Based Structures of an α7-nAChR, α9-nAChR Antagonist and of a Pro-Oxidant Mitocan

Adenocarcinoma and glioblastoma cell lines express \u3b17- and \u3b19\u3b110-containing nicotinic acetylcholine receptors (nAChRs), whose activation promotes tumor cell growth. On these cells, the triethylammoniumethyl ether of 4-stilbenol MG624, a known selective antagonist of \u3b17 and \u3b19\u3b110 nAChRs, has antiproliferative activity. The structural analogy of MG624 with the mitocan RDM-4'BTPI, triphenylphosphoniumbutyl ether of pterostilbene, suggested us that molecular hybridization among their three substructures (stilbenoxy residue, alkylene linker, and terminal onium) and elongation of the alkylene linker might result in novel antitumor agents with higher potency and selectivity. We found that lengthening the ethylene bridge in the triethylammonium derivatives results in more potent and selective toxicity toward adenocarcinoma and glioblastoma cells, which was paralleled by increased \u3b17 and \u3b19\u3b110 nAChR antagonism and improved ability of reducing mitochondrial ATP production. Elongation of the alkylene linker was advantageous also for the triphenylphosphonium derivatives resulting in a generalized enhancement of antitumor activity, associated with increased mitotoxicity