Nitrosamines as nicotinic receptor ligands

Nitrosamines are carcinogens formed in the mammalian organism from amine precursors contained in food, beverages, cosmetics and drugs. The potent carcinogen, NNK, and the weaker carcinogen, NNN, are nitrosamines formed from nicotine. Metabolites of the nitrosamines react with DNA to form adducts responsible for genotoxic effects. We have identified NNK as a high affinity agonist for the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) whereas NNN bound with high affinity to epibatidine-sensitive nAChRs. Diethylnitrosamine (DEN) bound to both receptors but with lower affinity. High levels of the alpha7nAChR were expressed in human small cell lung cancer (SCLC) cell lines and in hamster pulmonary neuroendocrine cells (PNECs), which serve as a model for the cell of origin of human SCLC. Exposure of SCLC or PNECs to NNK or nicotine increased expression of the alpha7nAChR and caused influx of Ca(2+), activation of PKC, Raf-1, ERK1/2, and c-myc, resulting in the stimulation of cell proliferation. Signaling via the alpha7nAChR was enhanced when cells were maintained in an environment of 10-15% CO(2) similar to that in the diseased lung. Hamsters with hyperoxia-induced pulmonary fibrosis developed neuroendocrine lung carcinomas similar to human SCLC when treated with NNK, DEN, or nicotine. The development of the NNK-induced tumors was prevented by green tea or theophylline. The beta-adrenergic receptor agonist, isoproterenol or theophylline blocked NNK-induced cell proliferation in vitro. NNK and nicotine-induced hyperactivity of the alpha7nAChR/RAF/ERK1/2 pathway thus appears to play a crucial role in the development of SCLC in smokers and could be targeted for cancer prevention.     

2‐Deoxy‐d‐Glucose Sensitizes Human Ovarian Cancer Cells to Cisplatin by Increasing ER Stress and Decreasing ATP Stores in Acidic Vesicles

Cisplatin is a commonly used chemotherapeutic agent; however, the development of acquired resistance limits its application. Here, we demonstrate that 2‐deoxy‐d ‐glucose (2‐DG) enhanced the antitumor effects of cisplatin in SKOV3 cells, which include inhibition of proliferation and promotion of apoptosis. Additionally, either cisplatin or 2‐DG alone could upregulate the endoplasmic reticulum (ER) stress‐associated protein glucose‐regulated protein‐78 (GRP78). Moreover, exposure to 2‐DG increased the expression of GRP78 induced by cisplatin. Cisplatin also upregulated ER stress‐associated apoptotic protein 153/C/EBP homology protein (CHOP) in SKOV3 cells. While treatment with 2‐DG alone could not upregulate the CHOP expression, a combination of both 2‐DG and cisplatin increased the protein levels of CHOP above those induced by Cisplatin alone. Finally, cisplatin mediated an increase in ATP stores within acidic vesicles, whereas 2‐DG decreased this effect. These data demonstrate that 2‐DG sensitizes SKOV3 cells to cisplatin by increasing ER stress and decreasing ATP stores in acidic vesicles.     

Thirdhand smoke: a new dimension to the effects of cigarette smoke on the developing lung

The underlying mechanisms and effector molecules involved in mediating in utero smoke exposure-induced effects on the developing lung are only beginning to be understood. However, the effects of a newly discovered category of smoke, i.e., thirdhand smoke (THS), on the developing lung are completely unknown. We hypothesized that, in addition to nicotine, other components of THS would also affect lung development adversely. Fetal rat lung explants were exposed to nicotine, 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA), or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the two main tobacco-specific N-nitrosamine constituents of THS, for 24 h. We then determined key markers for alveolar paracrine signaling [epithelial differentiation markers surfactant phospholipid and protein synthesis; mesenchymal differentiation markers peroxisome proliferator-activated receptor γ (PPAR-γ), fibronectin and calponin], the BCL-2-to-Bax ratio (BCL-2/Bax), a marker of apoptosis and the involvement of nicotinic acetylcholine receptors (nAChR)-α3 and -α7 in mediating NNA's and NNK's effects on the developing lung. Similar to the effects of nicotine, exposure of the developing lung to either NNK or NNA resulted in disrupted homeostatic signaling, indicated by the downregulation of PPAR-γ, upregulation of fibronectin and calponin protein levels, decreased BCL-2/Bax, and the accompanying compensatory stimulation of surfactant phospholipid and protein synthesis. Furthermore, nAChR-α3 and -α7 had differential complex roles in mediating these effects. NNK and NNA exposure resulted in breakdown of alveolar epithelial-mesenchymal cross-talk, reflecting lipofibroblast-to-myofibroblast transdifferentiation, suggesting THS constituents as possible novel contributors to in utero smoke exposure-induced pulmonary damage. These data are particularly relevant for designing specific therapeutic strategies, and for formulating public health policies to minimize THS exposure.     

Calcium rises induced by AMPA and nicotine receptors in the ventral tegmental area show differences in mouse brain slices prenatally exposed to nicotine

Nicotine exposure during gestation is associated with a higher risk of adverse behavioral outcomes including a heightened liability for dependency to drugs of abuse, which can exhibit drug‐specificity influenced by gender. This enhanced liability suggests that nicotine use during pregnancy alters neural development in circuits involved in motivation and reward‐based learning. The ventral tegmental area (VTA) is critical in motivated behaviors and we hypothesized that gestational exposure to nicotine alters the development of excitatory circuits in this nucleus. Accordingly, in VTA brain slices from male and female mice exposed to nicotine during the prenatal period (PNE) and controls, we compared cellular rises in calcium induced by AMPA receptor and nicotinic acetylcholine receptor (nAChR) stimulation by use of the ratiometric calcium binding dye, Fura‐2AM. We found that AMPA induced smaller amplitude calcium rises in the PNE VTA, which was an effect only detected in males. Further, while the amplitude did not vary between treatment and control in females, a greater number of cells responded with rises in calcium in the PNE. Conversely, the proportions of cells responding with calcium rises induced by nAChR stimulation did not change in either gender according to treatment. However, larger rises in calcium in PNE females were detected. When taken together our data show that excitatory signaling in the VTA is altered in a gender‐specific manner by PNE and suggest that alterations in signaling could play a role in drug‐specific differences in maladaptive, motivated behaviors exhibited by males and females born to mothers exposed to nicotine during pregnancy. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 2018     

Long‐term nicotine treatment down‐regulates α6β2* nicotinic receptor expression and function in nucleus accumbens

Long‐term nicotine exposure induces alterations in dopamine transmission in nucleus accumbens that sustain the reinforcing effects of smoking. One approach to understand the adaptive changes that arise involves measurement of endogenous dopamine release using voltammetry. We therefore treated rats for 2–3 months with nicotine and examined alterations in nAChR subtype expression and electrically evoked dopamine release in rat nucleus accumbens shell, a region key in addiction. Long‐term nicotine treatment selectively decreased stimulated α6β2* nAChR‐mediated dopamine release compared with vehicle‐treated rats. It also reduced α6β2* nAChRs, suggesting the receptor decline may contribute to the functional loss. This decreased response in release after chronic nicotine treatment was still partially sensitive to the agonist nicotine. Studies with an acetylcholinesterase inhibitor demonstrated that the response was also sensitive to increased endogenous acetylcholine. However, unlike the agonists, nAChR antagonists decreased dopamine release only in vehicle‐ but not nicotine‐treated rats. As antagonists function by blocking the action of acetylcholine, their ineffectiveness suggests that reduced acetylcholine levels partly underlie the dampened α6β2* nAChR‐mediated function in nicotine‐treated rats. As long‐term nicotine modifies dopamine release by decreasing α6β2* nAChRs and their function, these data suggest that interventions that target this subtype may be useful for treating nicotine dependence.

     

α6β2*‐subtype nicotinic acetylcholine receptors are more sensitive than α4β2*‐subtype receptors to regulation by chronic nicotine administration

Nicotinic acetylcholine receptors (nAChR) of the α6β2* subtype (where *indicates the possible presence of additional subunits) are prominently expressed on dopaminergic neurons. Because of this, their role in tobacco use and nicotine dependence has received much attention. Previous studies have demonstrated that α6β2*‐nAChR are down‐regulated following chronic nicotine exposure (unlike other subtypes that have been investigated – most prominently α4β2* nAChR). This study examines, for the first time, effects across a comprehensive chronic nicotine dose range. Chronic nicotine dose–responses and quantitative ligand‐binding autoradiography were used to define nicotine sensitivity of changes in α4β2*‐nAChR and α6β2*‐nAChR expression. α6β2*‐nAChR down‐regulation by chronic nicotine exposure in dopaminergic and optic‐tract nuclei was ≈three‐fold more sensitive than up‐regulation of α4β2*‐nAChR. In contrast, nAChR‐mediated [³H]‐dopamine release from dopamine‐terminal region synaptosomal preparations changed only in response to chronic treatment with high nicotine doses, whereas dopaminergic parameters (transporter expression and activity, dopamine receptor expression) were largely unchanged. Functional measures in olfactory tubercle preparations were made for the first time; both nAChR expression levels and nAChR‐mediated functional measures changed differently between striatum and olfactory tubercles. These results show that functional changes measured using synaptosomal [³H]‐DA release are primarily owing to changes in nAChR, rather than in dopaminergic, function.

     

The nicotine‐mediated decline in l‐dopa‐induced dyskinesias is associated with a decrease in striatal dopamine release

l ‐dopa‐induced dyskinesias (LIDs) are a side effect of Parkinson's disease therapy that is thought to arise, at least in part, because of excessive dopaminergic activity. Thus, drugs that regulate dopaminergic tone may provide an approach to manage LIDs. Our previous studies showed that nicotine treatment reduced LIDs in Parkinsonian animal models. This study investigates whether nicotine may exert its beneficial effects by modulating pre‐synaptic dopaminergic function. Rats were unilaterally lesioned by injection of 6‐hydroxydopamine (6‐OHDA) (2 × 3 ug per site) into the medial forebrain bundle to yield moderate Parkinsonism. They were then implanted with minipumps containing vehicle or nicotine (2.0 mg/kg/d) and rendered dyskinetic with l ‐dopa (8 mg/kg plus 15 mg/kg benserazide). Lesioning alone decreased the striatal dopamine transporter, nicotinic receptor (nAChR) levels, and nAChR‐mediated ³H‐dopamine release, consistent with previous results. Nicotine administration reduced l ‐dopa‐induced abnormal involuntary movements throughout the course of the study (4 months). Nicotine treatment led to declines in the striatal dopamine transporter, α6β2* nAChRs and various components of α6β2* and α4β2* nAChR‐mediated release. l ‐dopa treatment had no effect. These data suggest that nicotine may improve LIDs in Parkinsonian animal models by dampening striatal dopaminergic activity.     

Nicotine, acetylcholine and bombesin are trophic growth factors in neuroendocrine cell lines derived from experimental hamster lung tumors.

Neuroendocrine hamster lung tumors, induced by exposure to 60% hyperoxia and subcutaneous administration of the tobacco-specific nitrosamine 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK) for 12 weeks, were placed in cell culture. By subsequent selective transfer of epithelial cells and maintenance in an atmosphere of 8% CO2, cell lines with characteristics of neuroendocrine cells were established. The neuroendocrine markers expressed by these cell lines included electron dense neuroendocrine secretion granules as well as secretion of calcitonin and mammalian bombesin. In keeping with data previously reported for a human neuroendocrine lung tumor cell line, nicotine, acetylcholine, and mammalian bombesin (MB) acted as strong growth factors in these neuroendocrine hamster tumor lines. The mitogenic effect of nicotine and acetylcholine was abolished by nicotinic receptor inhibition while the effects of mammalian bombesin were inhibited by an antagonist of MB receptors. Our data suggest that a receptor-mediated mitogenic effect of nicotine on neuroendocrine lung cells may be instrumental in the induction of smoking-associated small cell lung cancer.     

Multiple roles of nicotine on cell proliferation and inhibition of apoptosis: implications on lung carcinogenesis

The genotoxic effects of tobacco carcinogens have long been recognized, the contribution of tobacco components to cancerogenesis by cell surface receptor signaling is relatively unexplored. Nicotine, the principal tobacco alkaloid, acts through nicotinic acetylcholine receptor (nAChR). nAChR are functionally present on human lung airway epithelial cells, on lung carcinoma [SCLC and NSCLC] and on mesothelioma and build a part of an autocrine-proliferative network that facilitates the growth of neoplastic cells. Different nAChR subunit gene expression patterns are expressed between NSCLC from smokers and non-smokers. Although there is no evidence that nicotine itself could induce cancer, different studies established that nicotine promotes in vivo the growth of cancer cells and the proliferation of endothelial cells suggesting that nicotine might contribute to the progression of tumors already initiated. These observations led to the hypothesis that nicotine might be playing a direct role in the promotion and progression of human lung cancers. Here, we briefly overview the role and the effects of nicotine on pulmonary cell growth and physiology and its feasible implications in lung carcinogenesis.     

Maternal nicotine exposure increases oxidative stress in the offspring

Fetal and neonatal nicotine exposure causes β-cell apoptosis and loss of β-cell mass, but the underlying mechanisms are unknown. The goal of this study was to determine whether maternally derived nicotine can act via the pancreatic nicotinic acetylcholine receptor (nAChR) during fetal and neonatal development to induce oxidative stress in the pancreas. Female Wistar rats were given saline or nicotine (1 mg/kg/day) via subcutaneous injection for 2 weeks prior to mating until weaning (postnatal day 21). In male offspring, nAChR subunit mRNA expression was characterized in the developing pancreas and various oxidative stress markers were measured at weaning following saline and nicotine exposure. The nAChR subunits 2-4, 6, 7, and β2–β4 were present in the pancreas during development. Fetal and neonatal exposure to nicotine significantly increased pancreatic GPx-1 and MnSOD protein expression, as well as islet ROS production. Furthermore, protein carbonyl formation was higher in nicotine-exposed offspring relative to controls, particularly within the mitochondrial fraction. There was also a nonsignificant trend toward higher serum 8-isoPG levels. These data suggest that β-cell apoptosis in the fetal and neonatal pancreas may be the result of a direct effect of nicotine via its receptor and that this effect may be mediated through increased oxidative stress.     

Nicotine enhances intracellular nicotinic receptor maturation: a novel mechanism of neural plasticity?

Nicotine addiction, the primary cause of tobacco consumption, is mediated through nicotine binding to brain nicotinic acetylcholine receptor (nAChRs). Upon chronic exposure, nicotine elicits a cascade of events, starting with nAChR activation and desensitization, followed by a long term up-regulation that corresponds to an increase in the number of the high affinity nAChRs, a paradoxical process that occurs in the brain of smokers. Recent investigation of the maturation and trafficking of the major brain alpha4beta2 nAChR demonstrates that up-regulation is initiated in the endoplasmic reticulum soon after protein translation. The data thus far accumulated provide evidence that nicotine elicits up-regulation by promoting maturation of nAChR precursors that would otherwise be degraded. This "maturational enhancer" action of nicotine probably contributes to the long term effect of chronic nicotine, and suggests a novel mechanism of neuronal plasticity through an yet unknown endogenous substance which would modulate the receptor expression under physiological conditions.     

α7 Nicotinic receptor activation reduces β-amyloid-induced apoptosis by inhibiting caspase-independent death through phosphatidylinositol 3-kinase signaling

The neurotoxicity of amyloid-β (Aβ) involves caspase-dependent and -independent programmed cell death. The latter is mediated by the nuclear translocation of the mitochondrial flavoprotein apoptosis inducing factor (AIF). Nicotine has been shown to decrease Aβ neurotoxicity via inhibition of caspase-dependent apoptosis, but it is unknown if its neuroprotection is mediated through caspase-independent pathways. In the present study, pre-treatment with nicotine in rat cortical neuronal culture markedly reduced Aβ(1-42) induced neuronal death. This effect was accompanied by a significant reduction of mitochondrial AIF release and its subsequent nuclear translocation as well as significant inhibition of cytochrome c release and caspase 3 activation. Pre-treatment with selective α7nicotinic acetylcholine receptor(nAChR) antagonist (methyllycaconitine), but not the α4 nAChR antagonist (dihydro-β-erythroidine), could prevent the neuroprotective effect of nicotine on AIF release/translocation, suggesting that nicotine inhibits the caspase-independent death pathway in a α7 nAChR-dependent fashion. Furthermore, the neuroprotective action of nicotine on AIF release/translocation was suppressed by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Pre-treatment with nicotine significantly restored Akt phosphorylation, an effector of PI3K, in Aβ(1-42) -treated neurons. These findings indicate that the α7 nAChR activation and PI3K/Akt transduction signaling contribute to the neuroprotective effects of nicotine against Aβ-induced cell death by modulating caspase-independent death pathways.     

Nicotine promotes atherosclerosis development in apolipoprotein E-deficient mice through α1-nAChR

α1 Nicotinic acetylcholine receptor (α1nAChR) is an important nicotine receptor that is widely distributed in vascular smooth muscle cells, macrophages, and endothelial cells. However, the role of α1nAChR in nicotine-mediated atherosclerosis remains unclear. The administration of nicotine for 12 weeks increased the area of the atherosclerotic lesion, the number of macrophages infiltrating the plaques, and the circulating levels of inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-α, in apolipoprotein E-deficient (ApoE^−/−) mice fed a high-fat diet. Nicotine also increased α1nAChR, calpain-1, matrix metalloproteinase-2 (MMP-2), and MMP-9 expression in the aortic tissue. Silencing of α1nAChR with an adenoassociated virus decreased the atherosclerotic size, lesion macrophage content, and circulating levels of inflammatory cytokines and suppressed α1nAChR, calpain-1, MMP-2, and MMP-9 expression in the nicotine group. In vitro, nicotine-induced α1nAChR, calpain-1, MMP-2, and MMP-9 expression in mouse vascular smooth muscle cells (MOVAS) and macrophages (RAW264.7), and enhanced the migration and proliferation of these cells. The silencing of α1nAChR inhibited these effects of nicotine MOVAS and RAW264.7 cells. Thus, we concluded that nicotine promoted the development of atherosclerosis partially by inducing the migration and proliferation of vascular smooth muscle cells and macrophages and inducing an inflammatory reaction. The effect of nicotine on atherogenesis may be mediated by α1nAChR-induced activation of the calpain-1/MMP-2/MMP-9 signaling pathway.     

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a nicotine derivative, induces apoptosis of endothelial cells

Smoking causes endothelial cell (EC) injury; however, neither the components of cigarette smoke nor the mechanisms responsible for this injury are understood. The nitrosated derivative of nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has been implicated in the carcinogenic effects of tobacco; however, the effects of NNK on the cardiovascular system are largely unknown. NNK binds to beta1- and beta2-adrenergic receptors. Because beta-adrenergic receptor activation causes arachidonic acid (AA) release and cellular injury, we postulated that NNK causes EC injury by a mechanism that involves beta-adrenergic-mediated release of AA. NNK stimulated [3H]AA release from ECs, and this effect was mediated by both beta1- and beta2-adrenergic receptors because pretreatment with atenolol or ICI 118,551 inhibited the response. NNK also induced EC apoptosis, as measured by terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling and annexin V staining. NNK-mediated apoptosis was attenuated by pretreatment with atenolol or ICI 118,551. Furthermore, depletion of cellular AA by incubation with eicosapentaenoic acid abolished the apoptotic effect of NNK. These data suggest that NNK causes EC apoptosis by a mechanism that involves beta1- and beta2-adrenergic receptor-mediated release of AA.     

Chronic Exposure to Combined Carcinogens Enhances Breast Cell Carcinogenesis with Mesenchymal and Stem-Like Cell Properties

Breast cancer is the most common type of cancer affecting women in North America and Europe. More than 85% of breast cancers are sporadic and attributable to long-term exposure to small quantities of multiple carcinogens. To understand how multiple carcinogens act together to induce cellular carcinogenesis, we studied the activity of environmental carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P), and dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) using our breast cell carcinogenesis model. Our study revealed, for the first time, that combined NNK and B[a]P enhanced breast cell carcinogenesis chronically induced by PhIP in both non-cancerous and cancerous breast cells. Co-exposure was more potent than sequential exposure to combined NNK and B[a]P followed by PhIP in inducing carcinogenesis. Initiation of carcinogenesis was measured by transient endpoints induced in a single exposure, while progression of carcinogenesis was measured by acquisition of constitutive endpoints in cumulative exposures. Transient endpoints included DNA damage, Ras-Erk-Nox pathway activation, reactive oxygen species elevation, and increased cellular proliferation. Constitutive endpoints included various cancer-associated properties and signaling modulators, as well as enrichment of cancer stem-like cell population and activation of the epithelial-to-mesenchymal transition program. Using transient and constitutive endpoints as targets, we detected that a combination of the green tea catechins ECG and EGCG, at non-cytotoxic levels, was more effective than individual agents in intervention of cellular carcinogenesis induced by combined NNK, B[a]P, and PhIP. Thus, use of combined ECG and EGCG should be seriously considered for early intervention of breast cell carcinogenesis associated with long-term exposure to environmental and dietary carcinogens.     

Type I and II positive allosteric modulators differentially modulate agonist-induced up-regulation of α7 nicotinic acetylcholine receptors

Long‐term treatment with nicotine or selective α7 nicotinic acetylcholine receptor (nAChR) agonists increases the number of α7 nAChRs and this up‐regulation may be involved in the mechanism underlying the sustained procognitive effect of these compounds. Here, we investigate the influence of type I and II α7 nAChR positive allosteric modulators (PAMs) on agonist‐induced α7 nAChR up‐regulation. We show that the type II PAMs, PNU‐120596 (10 μM) or TQS (1 and 10 μM), inhibit up‐regulation, as measured by protein levels, induced by the α7 nAChR agonist A‐582941 (10 nM or 10 μM), in SH‐EP1 cells stably expressing human α7 nAChR, whereas the type I PAMs AVL‐3288 or NS1738 do not. Contrarily, neither type I nor II PAMs affect 10 μM nicotine‐induced receptor up‐regulation, suggesting that nicotine and A‐582941 induce up‐regulation through different mechanisms. We further show in vivo that 3 mg/kg PNU‐120596 inhibits up‐regulation of the α7 nAChR induced by 10 mg/kg A‐582941, as measured by [¹²⁵I]‐bungarotoxin autoradiography, whereas 1 mg/kg AVL‐3288 does not. Given that type II PAMs decrease desensitization of the receptor, whereas type I PAMs do not, these results suggest that receptor desensitization is involved in A‐582941‐induced up‐regulation. Our results are the first to show an in vivo difference between type I and II α7 nAChR PAMs, and demonstrate an agonist‐dependent effect of type II PAMs occurring on a much longer time scale than previously appreciated. Furthermore, our data suggest that nicotine and A‐582941 induce up‐regulation through different mechanisms, and that this confers differential sensitivity to the effects of α7 nAChR PAMs. These results may have implications for the clinical development of α7 nAChR PAMs.     

α5‐nAChR contributes to epithelial‐mesenchymal transition and metastasis by regulating Jab1/Csn5 signalling in lung cancer

Recent studies have showed that α5 nicotinic acetylcholine receptor (α5‐nAChR) is closely associated with nicotine‐related lung cancer. Our previous studies also demonstrated that α5‐nAChR mediates nicotine‐induced lung carcinogenesis. However, the mechanism by which α5‐nAChR functions in lung carcinogenesis remains to be elucidated. Jab1/Csn5 is a key regulatory factor in smoking‐induced lung cancer. In this study, we explored the underlying mechanisms linking the α5‐nAChR‐Jab1/Csn5 axis with lung cancer epithelial‐mesenchymal transition (EMT) and metastasis, which may provide potential therapeutic targets for future lung cancer treatments. Our results demonstrated that the expression of α5‐nAChR was correlated with the expression of Jab1/Csn5 in lung cancer tissues and lung cancer cells. α5‐nAChR expression is associated with Jab1/Csn5 expression in lung tumour xenografts in mice. In vitro, the expression of α5‐nAChR mediated Stat3 and Jab1/Csn5 expression, significantly regulating the expression of the EMT markers, N‐cadherin and Vimentin. In addition, the down‐regulation of α5‐nAChR or/and Stat3 reduced Jab1/Csn5 expression, while the silencing of α5‐nAChR or Jab1/Csn5 inhibited the migration and invasion of NSCLC cells. Mechanistically, α5‐nAChR contributes to EMT and metastasis by regulating Stat3‐Jab1/Csn5 signalling in NSCLC, suggesting that α5‐nAChR may be a potential target in NSCLC diagnosis and immunotherapy.     

Developmental nicotine exposure alters cholinergic control of respiratory frequency in neonatal rats

Prenatal nicotine exposure with continued exposure through breast milk over the first week of life (developmental nicotine exposure, DNE) alters the development of brainstem circuits that control breathing. Here, we test the hypothesis that DNE alters the respiratory motor response to endogenous and exogenous acetylcholine (ACh) in neonatal rats. We used the brainstem‐spinal cord preparation in the split‐bath configuration, and applied drugs to the brainstem compartment while measuring the burst frequency and amplitude of the fourth cervical ventral nerve roots (C4VR), which contain the axons of phrenic motoneurons. We applied ACh alone; the nicotinic acetylcholine receptor (nAChR) antagonist curare, either alone or in the presence of ACh; and the muscarinic acetylcholine receptor (mAChR) antagonist atropine, either alone or in the presence of ACh. The main findings include: (1) atropine reduced frequency similarly in controls and DNE animals, while curare caused modest slowing in controls but no consistent change in DNE animals; (2) DNE greatly attenuated the increase in C4VR frequency mediated by exogenous ACh; (3) stimulation of nAChRs with ACh in the presence of atropine increased frequency markedly in controls, but not DNE animals; (4) stimulation of mAChRs with ACh in the presence of curare caused a modest increase in frequency, with no treatment group differences. DNE blunts the response of the respiratory central pattern generator to exogenous ACh, consistent with reduced availability of functionally competent nAChRs; DNE did not alter the muscarinic control of respiratory motor output. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1138–1149, 2016     

Arctigenin Enhances Chemosensitivity to Cisplatin in Human Nonsmall Lung Cancer H460 Cells through Downregulation of Survivin Expression

Arctigenin, a dibenzylbutyrolactone lignan, enhances cisplatin‐mediated cell apoptosis in cancer cells. Here, we sought to investigate the effects of arctigenin on cisplatin‐treated non‐small‐cell lung cancer (NSCLC) H460 cells. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and annexin‐V/propidium iodide staining were performed to analyze the proliferation and apoptosis of H460 cells. Arctigenin dose‐dependently suppressed cell proliferation and potentiated cell apoptosis, coupled with increased cleavage of caspase‐3 and poly(ADP‐ribose) polymerase. Moreover, arctigenin sensitized H460 cells to cisplatin‐induced proliferation inhibition and apoptosis. Arctigenin alone or in combination with cisplatin had a significantly lower amount of survivin. Ectopic expression of survivin decreased cell apoptosis induced by arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01). Moreover, arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01) induced G1/G0 cell‐cycle arrest. Our data provide evidence that arctigenin has a therapeutic potential in combina‐tion with chemotherapeutic agents for NSLC.