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Small airway-derived pulmonary adenocarcinoma (PAC) and pancreatic ductal adenocarcinoma (PDAC) are among the most common human cancers and smoking is a risk factor for both. Emerging research has identified cAMP signaling stimulated by the stress neurotransmitters adrenaline and noradrenaline as important stimulators of several adenocarcinomas, including PAC and PDAC. The nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent mutagen and the most powerful tobacco carcinogen. NNK is also an agonist for nicotinic acetylcholine receptors (nAChRs). Using hamster models of NNK-induced PAC and PDAC, we have tested the hypothesis that in analogy to chronic effects of nicotine in the brain, NNK may modulate the α7- and α4β2nAChRs, causing an increase in stress neurotransmitters and decrease in the inhibitory neurotransmitter γ-aminobutyric acid (GABA). In support of our hypothesis, immunoassays showed a significant increase in serum adrenaline/noradrenaline and increased intracellular cAMP in the cellular fraction of blood of NNK treated hamsters. Western blots were done with cells harvested by laser capture microcopy from control small airway epithelia, alveolar epithelia, pancreatic islet and pancreatic duct epithelia and from NNK-induced PACs and PDACs. The GABA synthesizing enzyme glutamate decarboxylase 65 (GAD65) and GABA were suppressed in NNK-induced PACs and PDACs whereas protein expression of the α7nAChR, α4nAChR as well as p-CREB and p-ERK1/2 were upregulated. These findings suggest, for the first time, that NNK-induced alterations in regulatory nAChRs may contribute to the development of smoking-associated PAC and PDAC by disturbing the balance between cancer stimulating and inhibiting neurotransmitters.
Small airway-derived pulmonary adenocarcinoma (PAC) and pancreatic ductal adenocarcinoma (PDAC) are among the most common and most deadly human cancers and smoking is a risk factor for both [1, 2]. The carcinogenic nitrosamine 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK) is formed from nicotine by nitrosation during the processing of tobacco and in the mammalian organism . Metabolites formed from NNK are strong mutagens that induce activating point mutations in k-Ras and inactivating mutations in p53 , both of which are common in human PAC and PDAC [5–8]. In addition to the mutational activities of its metabolites, the parent NNK itself is an agonist for nicotinic acetylcholine receptors (nAChRs) [9, 10]. This receptor family has been extensively studied in the nervous system where it regulates the synthesis and release of neurotransmitters [11, 12]. The nAChRs also regulate the synthesis and release of the stress neurotranmitters adrenaline and noradrenaline in the adrenal medulla and in sympathetic nerve endings [13–15]. Emerging research suggests that nAChRs are not restricted to the nervous system but are ubiquitously expressed in non-neuronal mammalian cells where they regulate diverse cellular functions, including differentiation, proliferation, apoptosis and angiogenesis . The discoveries that nAChRs regulate the proliferation  and apoptosis  of small cell lung cancer cells and that NNK is an agonist for nAChRs  marked the beginning of intense studies on the role of nAChRs in the regulation of cancer. It has thus been shown that nAChRs stimulate cancer cells via direct intracellular signaling and indirectly, by triggering the release of growth factors and angiogenic factors . On the other hand, recent studies have shown that the stress neurotransmitters adrenaline and noradrenaline stimulate via beta-adrenoreceptor signaling the growth and metastasis of some of the most common human cancers, including PAC [20–22], PDAC [23, 24], gastric cancer , colon cancer [26, 27], prostate cancer  and ovarian cancer [29, 30].
In the nervous system (Figure 1), the effects of the excitatory neurotransmitters glutamate, noradrenaline, serotonin and dopamine are counteracted by the inhibitory neurotransmitter γ-aminobutyric acid (GABA), which inhibits the formation of adenylyl cyclase-dependent cAMP [31, 32]. The release of the excitatory neurotransmitters from neurons is regulated by the homomeric α7nAChR  whereas the heteromeric α4β2nAChR regulates the release of GABA . Chronic exposure to nicotine upregulates both receptors, resulting in an increased number of nAChRs [11, 34]. However, the α4β2nAChR is additionally desensitized by chronic nicotine, resulting in decreased GABA release associated with nicotine addiction [13, 33]. By contrast, the sensitivity of the upregulated α7nAChR to agonists remains undiminished, resulting in increased synthesis and release of excitatory neurotransmitters triggered by the increased number of this receptor . In vitro studies have shown that GABA inhibits the beta-adrenoreceptor-mediated proliferation and migration of colon cancer , PDAC  and PAC  and is under expressed in the majority of human PDACs . These findings suggest that GABA may have tumor suppressor function in beta-adrenoreceptor-regulated cancers. Using well characterized hamster models of PAC [38, 39] and PDAC [40, 41], we have tested the hypothesis that in analogy to the chronic effects of nicotine in the nervous system, NNK may modulate the α7 -and α4β2nAChR in PAC and PDAC, resulting in enhanced stimulatory cAMP signaling and reduced inhibitory GABA.
The animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee. Outbred six week old Syrian golden hamsters were purchased from Charles River (Wilmington, DL) and housed one animal per cage in plastic shoebox-type cages under standard laboratory conditions. The animals had free access to food (Purina Rodent Chow, Agri Feed, Knoxville, TN) and tap water. Ten male hamsters were treated by subcutaneous injections with NNK (2.5mg/100g bodyweight three times/week for 20 weeks). This treatment reproducibly induces the development of small airway-derived PAC in 80–100% of the animals [38, 42]. All of these animals developed multiple PACs. For the induction of pancreatic ductal adenocarcinoma (PDAC) in their offspring, pregnant hamsters were given ethanol (10%) in their drinking water from day 5 through the end of their pregnancy and one subcutaneous injection of NNK (5mg/100g) on day 15 of gestation and the offspring were observed until eight moths old. This treatment reproducibly induces pancreatitis-associated PDAC in 50–70% of the animals [40, 41]. Among the 15 surviving offspring of this treatment group, eight developed PDACs. Ten untreated hamsters served as controls. The animals were killed by CO2 asphyxiation. Blood samples were collected from each hamster and pooled for each treatment group for the analysis of three replicate samples per group of adrenaline, noradrenaline, and cAMP. Lungs and pancreas were fixed in 70% ethanol, embedded in paraffin and used for the harvesting of lung and pancreatic cells by laser capture microscopy.
It is well established that binding of nicotine to nAChRs stimulates the release of the stress neurotranmitters adrenaline and noradrenaline, thereby increasing their systemic levels [13, 14]. Since NNK is an agonist for nAChRs [10, 14], we assessed the levels of both neurotransmitters in serum from control hamsters and from hamsters treated for 20 weeks with NNK using an enzyme immunoassay kit according to the manufacture instruction(2-CAT ELISA, Rocky Mountain Diagnostic, Inc., Colorado Spring, CO, USA). Absorbance was read with an ELISA reader at 450 nm. The quantification of the serum samples was achieved by comparing their absorbance with a reference curve prepared with known standard concentrations of adrenaline and noradrenaline. Mean values and standard errors from triplicate samples per treatment group were expressed as fold increase over controls. Statistical significance of data was assessed by one-way ANOVA, Tukey-Kramer multiple comparisons test and two-tailed unpaired t-test.
It has been shown that adrenaline and noradrenaline stimulate the proliferation and migration of numerous human cancers, including PAC and PDAC via cAMP signaling downstream of beta-adrenoreceptors [20–30]. We therefore determined the systemic levels of cAMP in control hamsters and in animals treated for 20 weeks with NNK. The cellular fraction of blood samples comprised of erythrocytes, lymphocytes, granulocytes and thrombocytes was used for this analysis. Each of these blood cells expresses beta-adrenoreceptors, and lymphocytes have previously been used in human patients for the assessment of systemic cAMP-dependent signaling activity . After 3 washes with water, cells were treated with 0.1 M HCL for 20 min, then, lysed by sonication. After centrifugation, samples were analyzed for cAMP levels using a direct cyclic AMP enzyme immunoassay kit according to the manufacturer’s instructions (Assay Designs Inc, Ann Arbor, MI, USA). Reactions were stopped and color intensity was measured immediately at 405 nm. Data are expressed as fold increase over controls of mean values and standard errors from triplicate samples per treatment group. Statistical analysis of data was by one-way ANOVA, Tukey-Kramer multiple comparisons test and two-tailed unpaired t-test.
This technique was used to harvest cells from lung and pancreatic tissue sections, using a PixCell IIe Laser Capture Microdissection system (Arcturus, Mountain View, CA. The simple respiratory epithelium (termed “small airway epithelial cells SAECs in this manuscript) comprised of Clara cells and ciliated cells from subsegmental bronchi and bronchioles, alveolar epithelial cells, and cells from PACs were harvested from the lung slides. Cells form pancreatic islets, pancreatic duct epithelia, and cells from PDACs were harvested from the pancreatic slides. The tissue sections were dewaxed by placing them in two baths of xylene for 5 min each, passed through decreasing concentrations of ethanol to rehydrate (100%, 95%, 70%), and water for 30 s each. The tissue was stained according to the procedure outlined in the Histogene Kit (Arcturus). Cells were harvested from histological sections of all hamsters per treatment group to obtain a total of 30,000 cells of each investigated cell type. These cells were allocated to three equal aliquots per treatment group for analysis. The proteins were extracted from the CapSure caps using protein extract buffer (Pierce) within 30 minutes after cell harvesting.
It has been shown in the nervous system and in colon cancer cells that the α7nAChR regulates the synthesis and release of adrenaline and noradrenaline which activate cAMP signaling downstream of beta-adrenoreceptors [13, 14, 27]. In turn, we have shown that the activation of the beta-adrenergic signaling pathway in human PAC and PDAC cells results in the PKA-dependent activation of the transcription factor CREB and transactivation of the EGFR, leading to the phosphorylation of its downstream effector ERK1/2 [24, 44]. The α7nAChR additionally regulates the release of glutamate  from which the formation of GABA is catalyzed by the two isoforms of the enzyme glutamate decarboxylase, GAD65 and GAD67 . On the other hand, the synthesis and release of GABA are regulated by the α4β2nAChR . We therefore analyzed the protein expression of nAChRs expressing the α7 or α4 subunits, GABA and its synthesizing enzymes as well as p-CREB and p-ERK1/2 in NNK-induced PAC and PDAC cells, small airway epithelial cells, alveolar epithelial cells, as well as cells from pancreatic ducts and pancreatic islets of control hamsters. Following lysis of the thawed tissues, protein was determined using the BCA Protein Assay (Pierce, Rockford, IL). Equal amounts of protein were treated with loading buffer at 100°C for five minutes and applied to each lane for electrophoresis in 12% polyacrylamide gel. The electrophoresed proteins were transferred onto nitrocellulose membranes in transfer buffer at 100 mV for one hour. After transfer, the membranes were treated with blocking buffer (5% nonfat dry milk in tris buffered saline with Tween 20, TBST) for one hour, and incubated with primary antibody overnight at 4°C. Primary antibodies against the following signaling proteins were used: total CREB (Upstate Biotechnology, Lake Placid, NY, USA), p-CREB, p-ERK1/2 and ERK1/2 (Cell Signaling, Danvers, MA, USA), α4- and α7nAChR, GAD65 and GAD67 and GABA (Millipore, Billerica, MA, USA and beta-actin (Sigma). After being washed with TBST, the membranes were incubated with horseradish peroxidase-labeled secondary antibody (goat anti-mouse, or goat anti-rabbit, Cell Signaling) for one hour. Immunoreactive bands were detected using a chemiluminescent reaction (ECL, Amersham Biosciences, Piscataway, NJ) via autoradiography on Kodak Bio-Max XAR film. Three separate Western blots were conducted for each antibody per sample and yielded similar data. Relative densities of the bands were determined by image analysis using NIH SCION image analysis software. Mean values and standard errors from five densitometric readings per band were analyzed by one way ANOVA and Tukey-Kramer multiple comparison test.
Analysis of serum samples by immunoassay showed that noradrenaline was increased 3.8-fold (p<0.001) and adrenaline 2.3-fold (p<0.001) in the NNK treated hamsters (Figure 2). These NNK-induced changes in neurotransmitter levels were accompanied by a significant (p<0.001) increase (2.2-fold) of cAMP levels in the cellular fraction of blood samples (Figure 2). These findings indicate, that similar to nicotine, NNK increases the systemic levels of stress neurotransmitters which in turn trigger a generalized increase in intracellular cAMP downstream of adrenoreceptors.
Western blots of protein samples from cells harvested by laser capture microscopy showed that nAChRs expressing the α4 subunit predominated over the α7nAChR in normal lung tissues (Figure 3). Small airway epithelial cells, from which NNK-induced PAC arises in this species , expressed significantly (p<0.001) higher protein levels of either receptor than alveolar epithelial cells. Both receptors were prominently upregulated (p<0.001) in NNK-induced PACs with a 3.2-fold increase over the levels observed in small airway epithelial cells in α4nAChR protein expression and a 3.4-fold increase in α7nAChR protein (Figure 3). These findings indicate that chronic exposure to NNK has similar upregulating effects on nAChRs in lung cells as the reported upregulation of this receptor family by chronic nicotine exposure in the brain .
Both GAD isozymes as well as GABA were expressed in all investigated lung cells, with the highest levels of all three proteins observed in small airway epithelial cells from control hamsters (Figure 4). GAD65 was the predominating isozyme in lung cells. The expression levels of GAD65 and GABA were significantly (p<0.001) suppressed below the levels of small airway epithelial cells in NNK-induced PAC (Figure 4; GAD65: 25% of small airway epithelial cells, GABA: 26% of small airway epithelial cells). Expression levels of GAD67 were less affected by NNK (reduction to 73% of small airway epithelial cell levels). In contrast to the observed suppression of GABA and its synthesizing enzymes, both, p-CREB (1.7-fold) and p-ERK1/2 (1.8-fold) were significantly (p<0.001) induced in the PAC cells from NNK-treated hamsters while expression of the unphosphorylated proteins remained unchanged (Figure 5).
In cells harvested from pancreatic tissues, Western blots identified the α7nAChR as the predominating nAChR in normal duct cells from control hamsters (Figure 6). Significantly (p<0.001) higher levels of both nAChRs were observed in duct epithelial cells than in islet cells. Similar to the observations in PAC cells, each of the two investigated nAChRs was significantly (p<0.001) upregulated in the NNK-induced PDACs (Figure 6; α4nAChR: 2.3-fold, α7nAChR: 1.8-fold). The upregulation of these receptors was contrasted sharply by prominent (p<0.001) suppression of GABA and GAD65 to barely detectable levels in PDAC cells (Figure 7). GAD65 predominated in control duct epithelial cells whereas GAD67 predominated in control islet cells (Figure 7). Similar to the observations in PAC cells, p-CREB (2.65-fold) and p-ERK1/2 (2.55-fold) were significantly (p<0.001) induced in the PDAC cells from NNK-treated hamsters (Figure 8) whereas the levels of the unphsophorylated proteins remained unchanged.
Collectively, NNK upregulated the investigated nAChRs and representative phosphorylated proteins of the cAMP (p-CREB) and EGFR (p-ERK1/2) pathways while suppressing GAD65 and GABA in both types of NNK-induced adenocarcinomas.
Our data show, for the first time, that the nicotine-derived carcinogenic nitrosamine NNK causes a systemic increase in the stress neurotransmitters adrenaline and noradrenaline associated with a generalized increase in intracellular cAMP in cells expressing β-adrenoreceptors. While in vitro studies have shown that NNK can stimulate PAC [21, 22, 44] and PDAC [23, 24] cells directly by binding as an agonist to their beta-adrenoreceptors (Figure 9), the observed systemic responses add a novel aspect to the mechanisms of action of this powerful tobacco carcinogen. These findings are in accord with reports on significant stimulating effects of these stress neurotransmitters on the proliferation and metastasis of adenocarcinoma of the colon [26, 27], stomach , prostate  and ovary [29, 30]. Accordingly, the NNK-induced systemic increase in these neurotransmitters may contribute to the development of these cited tumors as well as PAC and PDAC in smokers. In addition, smoking-induced increase in systemic adrenaline/noradreanline is an important factor in the etiology of cardiovascular disease that has been solely attributed to the nicotine contained in cigarette smoke . By contrast, our data suggest that NNK also contributes to these adverse cardiovascular effects observed in smokers.
The observed significant upregulation of the α7-and α4nAChRs in NNK-induced PAC and PDAC cells is in accord with the finding that this nitrosamine is an nAChR agonist that causes similar responses downstream of these receptors as nicotine [9, 10]. Chronic exposure to nicotine typically upregulates all nAChRs (Figure 1)  and our data show that NNK has similar effects (Figure 9). In analogy to the effects of chronic nicotine on brain cells  the simultaneous strong suppression of GAD65 and GABA in NNK-induced PAC and PDAC suggests that the upregulated α4nAChR is desensitized. This nAChR regulates the synthesis and release of GABA in brain cells and its desensitization has been associated with reduced GABA levels that contribute to nicotine addiction and withdrawal symptoms . It has been shown that GABA acts as an important tumor suppressor in beta-adrenoreceptor-regulated cancers such as PAC, PDAC [37, 47], as well as adenocarcinoma of the colon , and breast . Chronic agonist-induced desensitization of its upstream regulator, the α4β2nAChR, and the resulting GABA deficiency may thus contribute to the development of these cancers in smokers.
The observed significant increases in p-CREB and p-ERK1/2 in both investigated NNK-induced cancers is in accord with in vitro studies that have identified beta-adrenoreceptor-mediated signaling via cAMP/PKA/p-CREB and PKA-dependent transactivation of the EGFR as a key regulatory cascade in human PAC [21, 44, 49] and PDAC [23, 24] cells (Figuren9). Additional in vitro studies have shown that agents that increase intracellular cAMP enhance the responsiveness of this signaling cascade [49–52] whereas inhibitors of adenylyl cyclase, cAMP or PKA had blocking effects [24, 36, 44, 53]. These data identify GABA, which inhibits the adenylyl cyclase-dependent formation of camp by binding to the Gαi-coupled GABABR , as a key physiological defense against the hyperactive cAMP signaling that appears to drive the development and progression of PAC and PDAC (Figure 9). While the focus of prior investigations has been on beta-adrenoreceptors as the regulators of these signaling events, the current data suggest that the α7nAChR acts upstream of these receptors by regulating the systemic levels of adrenaline and noradrenaline. This interpretation is in accord with the documented regulatory role of this receptor for adrenaline/noradrenaline in the brain and sympathetic nerves [13, 14] and is supported by a recent report of α7nAChR-mediated synthesis of these neurotransmitters in colon cancer cells . It remains to be investigated if PAC and PDAC cells are also able to synthesize adrenaline and noradrenaline.
In conjunction with the cited literature, the current data suggest that due to their centralized regulatory functions, the upregulation of α7nAChRs and the upregulation and desensitizion of the α4β2nAChR associated with nicotine addiction [33, 34] may also play crucial roles in the development and progression of smoking-associated PAC and PDAC and possibly other cancer stimulated by the stress neurotransmitters and camp signaling.
Financial support: RO1CA042829 and RO1CA096128 with the National Cancer Institute and grant 003650 with the National Lung Cancer Partnership.
Conflict of interest: None to declare.