Nicotine induces self-renewal of pancreatic cancer stem cells via neurotransmitter-driven activation of sonic hedgehog signalling.

A small subpopulation of pancreatic cancer cells with characteristics of stem cells drive tumour initiation, progression and metastasis. A better understanding of the regulation of cancer stem cells may lead to more effective cancer prevention and therapy. We have shown that the proliferation and migration of pancreatic cancer cell lines is activated by the nicotinic receptor-mediated release of stress neurotransmitters, responses reversed by γ-aminobutyric acid (GABA). However, the observed cancer inhibiting effects of GABA will only succeed clinically if GABA inhibits pancreatic cancer stem cells (PCSCs) in addition to the more differentiated cancer cells that comprise the majority of cancer tissues and cell lines. Using PCSCs isolated from two pancreatic cancer patients by cell sorting and by spheroid formation assay from pancreatic cancer cell line Panc-1, we tested the hypothesis that nicotine induces the self-renewal of PCSCs. Nicotinic acetylcholine receptors (nAChRs) α3, α4, α5 and α7 were expressed and chronic exposure to nicotine increased the protein expression of these receptors. Immunoassays showed that PCSCs produced the stress neurotransmitters epinephrine and norepinephrine and the inhibitory neurotransmitter GABA. Chronic nicotine significantly increased the production of stress neurotransmitters and sonic hedgehog (SHH) while inducing Gli1 protein and decreasing GABA. GABA treatment inhibited the induction of SHH and Gli1. Spheroid formation and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assays showed significant nicotine-induced increases in self renewal and cell proliferation, responses blocked by GABA. Our data suggest that nicotine increases the SHH-mediated malignant potential of PCSCs and that GABA prevents these effects.

Chronic nicotine inhibits the therapeutic effects of gemcitabine on pancreatic cancer in vitro and in mouse xenografts.

AIM OF STUDY: Smoking is an established risk factor for pancreatic cancer and nicotine replacement therapy (NRT) often accompanies chemotherapy. The current study has tested the hypothesis that chronic exposure to low dose nicotine reduces the responsiveness of pancreatic cancer to the leading therapeutic for this cancer, gemcitabine. METHODS: The effects of chronic nicotine (1 µm/L) on two pancreatic cancer cell lines in vitro and in a xenograft model were assessed by immunoassays, Western blots and cell proliferation assays. RESULTS: Exposure in vitro to nicotine for 7 days inhibited the gemcitabine-induced reduction in viable cells, gemcitabine-induced apoptosis as indicated by reduced expression of cleaved caspase-3 while inducing the phosphorylation of signalling proteins extracellular signal-regulated kinase (ERK), v-akt thymoma viral oncogene homolog (protein kinase B, AKT) and Src. Nicotine (1 µm/L) in the drinking water for 4 weeks significantly reduced the therapeutic response of mouse xenografts to gemcitabine while reducing the induction of cleaved caspase-3 and the inhibition of phosphorylated forms of multiple signalling proteins by gemcitabine in xenograft tissues. CONCLUSIONS: Our experimental data suggest that continued moderate smoking and NRT may negatively impact therapeutic outcomes of gemcitabine on pancreatic cancer and that clinical studies in cancer patients are now warranted.

Gamma-amino butyric acid (GABA) prevents the induction of nicotinic receptor-regulated signaling by chronic ethanol in pancreatic cancer cells and normal duct epithelia.

Pancreatic cancer has a high mortality rate and alcoholism is a risk factor independent of smoking. We have shown that nicotinic acetylcholine receptors (nAChR) regulate pancreatic ductal epithelia and pancreatic ductal adenocarcinoma (PDAC) cells in an autocrine fashion by stimulating their production of the stress neurotransmitters noradrenaline and adrenaline that signal through ß-adrenergic receptors (ß-AR). Our current study has investigated the modulation of this autocrine regulatory loop by chronic ethanol and explored the potential prevention of these effects by γ-amino butyric acid (GABA). Using MTT assays, cell migration assays, Western blotting, immunoassays, and gene knockdown of individual nAChRs in two PDAC cell lines and in immortalized human pancreatic duct epithelial cells, our data show that treatment for seven days with ethanol induced the protein expression and sensitivity of nAChRs α3, α5, and α7 resulting in increased production of noradrenaline and adrenaline, which drive proliferation and migration via cyclic AMP (cAMP)-dependent signaling downstream of ß-ARs. Treatment with GABA prevented all of these responses to chronic ethanol, reducing cell proliferation and migration below base levels in untreated cells. Our findings suggest that alcoholism induces multiple cAMP-dependent PDAC stimulating signaling pathways by upregulating the protein expression and sensitivity of nAChRs that regulate stress neurotransmitter production. Moreover, our data identify GABA as a promising agent for the prevention of PDAC in individuals at risk due to chronic alcohol consumption.

Celecoxib and GABA cooperatively prevent the progression of pancreatic cancer in vitro and in xenograft models of stress-free and stress-exposed mice.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and is associated with high levels of psychological distress. We have shown that beta-adrenergic receptors (ß-ARs), which are activated by stress neurotransmitters, regulate PDAC cells via cyclic AMP (cAMP)-dependent signaling in vitro, that social stress promotes PDAC progression in mouse xenografts and that γ-aminobutyric acid (GABA) inhibits these responses in vitro and in vivo. The targeted inhibition of stress-induced regulatory pathways may abolish the potentially negative impact of psychological stress on clinical outcomes. Our current data show that chronic exposure of PDAC cell lines Panc-1 (activating point mutations in K-ras) and BXPC-3 (no mutations in K-ras) in vitro to the stress neurotransmitter epinephrine at the concentration (15 nM) previously measured in the serum of mice exposed to social stress significantly increased proliferation and migration. These responses were inhibited in a concentration-dependent manner by celecoxib. The effects of celecoxib alone and in combination with γ-aminobutyric acid (GABA) on the progression of subcutaneous mouse xenografts from the cell line (BXPC-3) most responsive to epinephrine were then investigated in the presence and absence of social stress. Cancer-stimulating factors (VEGF & prostaglandin E(2) [PGE(2)]) and levels of cAMP were measured by immunoassays in blood and xenograft tissue. Phosphorylation of the signaling proteins ERK, CREB, Src, and AKT was assessed by ELISA assays and Western blotting. Expression of COX-2, 5-lipoxygenase, and p-5-LOX were determined by semi-quantitative Western blotting. Celecoxib alone significantly inhibited xenograft progression and decreased systemic and tumor VEGF, PGE2, and cAMP as well as phosphorylated signaling proteins in stress-exposed and stress-free mice. These responses were significantly enhanced by co-treatment with GABA. The celecoxib-induced downregulation of COX-2 protein and p-5-LOX was also significantly enhanced by GABA under both experimental conditions. Our findings identify the targeted inhibition of stress-induced pathways as a promising area for more effective cancer intervention in pancreatic cancer.

Effects of chronic nicotine on the autocrine regulation of pancreatic cancer cells and pancreatic duct epithelial cells by stimulatory and inhibitory neurotransmitters.

Pancreatic ductal adenocarcinoma (PDAC) has a mortality rate near 100%. Smoking is a documented risk factor. However, the mechanisms of smoking-associated pancreatic carcinogenesis are poorly understood. We have shown that binding of nicotine to nicotinic acetylcholine receptors (nAChRs) expressing subunits α7, α3 and α5 in PDAC and pancreatic duct epithelial cells in vitro triggered the production of the neurotransmitters noradrenaline and adrenaline by these cells. In turn, this autocrine catecholamine loop significantly stimulated cell proliferation via cyclic adenosine 3',5'-monophosphate-dependent signaling downstream of beta-adrenergic receptors. However, the observed responses only represent acute cellular reactions to single doses of nicotine whereas nicotine exposure in smokers is chronic. Using the PDAC cell lines BxPC-3 and Panc-1 and immortalized pancreatic duct epithelial cell line HPDE6-C7, our current experiments reveal a significant sensitization of the nAChR-driven autocrine catecholamine regulatory loop in cells pre-exposed to nicotine for 7 days. The resulting increase in catecholamine production was associated with significant inductions in the phosphorylation of signaling proteins ERK, CREB, Src and AKT, upregulated protein expression of nAChR subunits α3, α4, α5 and α7 and increased responsiveness to nicotine in 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide and cell migration assays. All three cell lines produced the inhibitory neurotransmitter γ-aminobutyric acid, an activity inhibited by gene knockdown of the α4ß2nAChR and suppressed by chronic nicotine via receptor desensitization. All of the observed adverse effects of chronic nicotine were reversed by treatment of the cells with γ-aminobutyric acid, suggesting the potential usefulness of this agent for the improvement of PDAC intervention strategies in smokers.

Intercepting neoplastic progression in lung malignancies via the beta adrenergic (ß-AR) pathway: implications for anti-cancer drug targets.

The understanding of signaling cascades involved in the induction, promotion, and progression of cancer, although advanced in recent years, is still incomplete. Tracing the imbalance of the impaired, physiologically-essential cellular signaling that drives the neoplastic process is a complex issue. This review discusses the role of the regulator of the fight or flight response, the beta-adrenergic signaling cascade, as a mediator of cancer growth and progression in in vitro and in vivo cancer models. We review a series of experiments from our own laboratory and those of others examining the contribution of this signaling network to lung and other human malignancies and thereby identifying potential targets for chemotherapeutic interventions. The stimulation of the ß-adrenergic receptor by lifestyle and environmental factors, as well as a preexisting risk for neoplasm, activates downstream effector molecules (adenylyl cyclase/cAMP/PKA/CREB) concomitant to the transactivation of related pathways (EGFR) that lead to pro-oncogenic signaling; this ß-adrenergic pathway thereby encourages cancer growth by evasion of apoptosis, invasion, angiogenesis, and metastasis. GABAergic signaling acts as an antagonist to the ß-adrenergic cascade by intercepting adenylyl cyclase activation, and thereby neutralizing the pro-oncogenic effects of ß-adrenergic stimulation. The regulation of cancer cell growth by neurobiological signals expands the possibilities for pharmacological interventions in cancer therapy.

Cooperative regulation of non-small cell lung carcinoma by nicotinic and beta-adrenergic receptors: a novel target for intervention.

Lung cancer is the leading cause of cancer death; 80-85% of lung cancer cases are non-small cell lung cancer (NSCLC). Smoking is a documented risk factor for the development of this cancer. Although nicotine does not have the ability to initiate carcinogenic events, recent studies have implicated nicotine in growth stimulation of NSCLC. Using three NSCLC cell lines (NCI-H322, NCI-H441 and NCI-H1299), we identified the cooperation of nicotinic acetylcholine receptors (nAChRs) and ß-adrenergic receptors (ß-ARs) as principal regulators of these effects. Proliferation was measured by thymidine incorporation and MTT assays, and Western blots were used to monitor the upregulation of the nAChRs and activation of signaling molecules. Noradrenaline and GABA were measured by immunoassays. Nicotine-treated NSCLC cells showed significant induction of the α7nAChR and α4nAChR, along with significant inductions of p-CREB and p-ERK1/2 accompanied by increases in the stress neurotransmitter noradrenaline, which in turn led to the observed increase in DNA synthesis and cell proliferation. Effects on cell proliferation and signaling proteins were reversed by the α7nAChR antagonist α-BTX or the ß-blocker propranolol. Nicotine treatment also down-regulated expression of the GABA synthesizing enzyme GAD 65 and the level of endogenous GABA, while treatment of NSCLC cells with GABA inhibited cell proliferation. Interestingly, GABA acts by reducing ß-adrenergic activated cAMP signaling. Our findings suggest that nicotine-induced activation of this autocrine noradrenaline-initiated signaling cascade and concomitant deficiency in inhibitory GABA, similar to modulation of these neurotransmitters in the nicotine-addicted brain, may contribute to the development of NSCLC in smokers. Our data suggest that exposure to nicotine either by tobacco smoke or nicotine supplements facilitates growth and progression of NSCLC and that pharmacological intervention by ß blocker may lower the risk for NSCLC development among smokers and could be used to enhance the clinical outcome of standard cancer therapy.

Social stress promotes and γ-aminobutyric acid inhibits tumor growth in mouse models of non-small cell lung cancer.

Psychologic distress is associated with increased lung cancer incidence and mortality. We have shown that non-small cell lung cancer (NSCLC) cells in vitro are stimulated by the cyclic AMP (cAMP)-dependent activation of cAMP-responsive element binding protein (CREB) and extracellular signal-regulated kinase (ERK) downstream of ß-adrenergic receptors and that this pathway is inhibited by the neurotransmitter γ-aminobutyric acid (GABA). Because the stress neurotransmitters noradrenalin and adrenalin are ß-adrenergic agonists, the current study has tested the hypothesis that social stress stimulates NSCLC growth in vivo and that GABA inhibits this effect. Social stress was induced in mice carrying xenografts from two NSCLC cell lines in the presence and absence of treatment with GABA. Xenograft sizes were measured after 30 days. Noradrenalin, adrenalin, cortisol, GABA, and cAMP were measured in blood and tumor tissues by immunoassays. Expression of nicotinic receptors in the xenografts was assessed by real-time PCR and Western blotting. Protein expression of phospho (p)-CREB, CREB, phospho (p)-ERK, ERK, and glutamate decarboxylase (GAD) 65 and 67 were determined by Western blotting. Xenograft sizes in stress-exposed mice were significantly increased. Nicotinic acetylcholine receptor (nAChR) subunits α3, α4, α5, and α7 in xenograft tissues showed posttranscriptional induction. Noradrenalin, adrenalin, and cortisol were elevated in serum and xenograft tissue whereas GABA was suppressed. Levels of cAMP, p-CREB, and p-ERK were increased whereas GAD65 and GAD67 were suppressed in tumor tissue. Treatment with GABA reversed the effects of stress. Our findings suggest that social stress stimulates NSCLC by increasing nAChR-mediated stress neurotransmitter signaling and that GABA is a promising novel agent for NSCLC intervention.

Pancreatic cancer cells and normal pancreatic duct epithelial cells express an autocrine catecholamine loop that is activated by nicotinic acetylcholine receptors α3, α5, and α7.

Pancreatic cancer is the fourth leading cause of cancer deaths in developed countries. Smoking is an established risk factor for this malignancy but the underlying mechanisms are poorly understood. Previous reports have provided evidence that nicotinic acetylcholine receptors (nAChR) and beta adrenergic receptors (ß-AR) stimulate the growth and migration of pancreatic cancer cells. However, a potential cooperation of these two receptor families in the regulation of pancreatic cancer has not been studied to date. Using two pancreatic cancer cell lines and immortalized pancreatic duct epithelia in vitro, our current data show that all three cell lines synthesized and released the catecholamine neurotransmitters noradrenaline and adrenaline upon exposure to nicotine and that this activity was regulated by α3, α5, and α7-nAChRs. In accordance with the established function of these catecholamines as ß-AR agonists, nicotine-induced cell proliferation was blocked by the ß-AR antagonist propranolol. Nicotine-induced proliferation was also abolished by the α7-nAChR antagonist α-bungarotoxin, whereas catecholamine production in response to nicotine was blocked by gene knockdown of the α3, α5, and α7-nAChRs. The nicotinic agonists acetylcholine, nicotine, and its nitrosated carcinogenic derivative NNK induced the phosphorylation of CREB, ERK, Src, and AKT and these responses were inhibited by propranolol. Our findings identify this hitherto unknown autocrine catecholamine loop as an important regulatory cascade in pancreatic cancer that may prove a promising new target for cancer intervention.

Beta-adrenergic signaling in the development and progression of pulmonary and pancreatic adenocarcinoma.

Small airway epithelial cells from, which most pulmonary adenocarcinomas (PACs) derive, and pancreatic duct epithelia, from which pancreatic ductal adenocarcinomas (PDACs) originate, share the ability to synthesize and release bicarbonate. This activity is stimulated in both cell types by the α7nicotinic acetylcholine receptor (α7nAChR)-mediated release of noradrenaline and adrenaline, which in turn activate ß-adrenergic receptor (ß-AR) signaling, leading to the cAMP-dependent release of bicarbonate. The same signaling pathway also stimulates a complex network of intracellular signaling cascades which regulate the proliferation, migration, angiogenesis and apoptosis of PAC and PDAC cells. The amino acid neurotransmitter γ-aminobutyric acid (GABA) serves as the physiological inhibitor of this cancer stimulating network by blocking the activation of adenylyl cyclase. This review summarizes experimental, epidemiological and clinical data that have identified risk factors for PAC and PDAC such as smoking, alcoholism, chronic non neoplastic diseases and their treatments as well as psychological stress and analyzes how these factors increase the cancer-stimulating effects of this regulatory cascade in PAC and PDAC. This analysis identifies the careful maintenance of balanced levels in stimulatory stress neurotransmitters and inhibitory GABA as a key factor for the prevention of PDAC and suggests the marker-guided use of beta-blockers, GABA or GABA-B receptor agonists as well as psychotherapeutic or pharmacological stress reduction as important tools that may render currently ineffective cancer intervention of PAC and PDAC more successful.

Regulation of pancreatic cancer by neuropsychological stress responses: a novel target for intervention.

Pancreatic cancer has a poor prognosis and is associated with high levels of psychological stress that may adversely affect clinical outcomes. However, the potential influence of neuropsychological factors on pancreatic cancer has not been investigated to date. Using a mouse model of social stress, we have tested the hypothesis that psychological stress promotes the progression of pancreatic cancer xenografts via neurotransmitter-induced activation of multiple pathways and that the inhibitory neurotransmitter γ-aminobutiric acid (GABA) inhibits these responses. Sytemic and xenograft levels of noradrenalin, adrenalin, GABA, cortisol, vascular endothelial growth factor (VEGF) and cyclic adenosine 3', 5'-monophosphate (cAMP) were measured by immunoassays. Xenograft expression of nicotinic acetylcholine receptors (nAChRs) α3, α4, α5, α6 and α7 and ß-adrenergic receptors 1 and 2 were assessed by real-time PCR and western blots. Expression of glutamate decarboxylases GAD65 and GAD67 and phosphorylated and unphosphorylated signaling proteins of relevance to pancreatic cancer were determined in tumor tissue by western blots. Psychological stress significantly promoted xenograft growth and increased systemic and tumor levels of noradrenalin, adrenalin, cortisol, VEGF and cAMP while GABA and GAD were suppressed. Stress upregulated nAChR proteins but not RNAs and induced phosphorylated ERK, CREB, Src and AKT in xenografts. Reduction of cAMP by treatment with GABA prevented tumor progression and activation of signaling proteins. Our findings suggest that neurotransmitter responses to psychological stress negatively impact clinical outcomes of pancreatic cancer via the activation of multiple pathways and that replacement of the suppressed inhibitory neurotransmitter GABA prevents these effects.

GABA (γ-aminobutyric acid), a non-protein amino acid counters the ß-adrenergic cascade-activated oncogenic signaling in pancreatic cancer: a review of experimental evidence.

GABA is a bioactive constituent of fruits, vegetables, cereals and is believed to play a role in defense against stress in plants. In animals, it acts as an inhibitory neurotransmitter in brain while also expressed in non-neuronal cells. Studies have implicated the regulator of fight or flight stress responses, ß-AR signaling cascade, as mediators of cancer growth and progression in in vitro and in vivo models of pancreatic malignancies. Pancreatic cancer is the fourth leading cause of cancer mortality in western countries. This malignancy is generally unresponsive to conventional radio- and chemotherapy, resulting in mortality rate near 100% within 6 months of diagnosis. We review a series of experiments from our laboratory and those of others examining the contribution of this signaling network to pancreatic and other human malignancies. Stimulation of the ß-adrenergic receptor by lifestyle and environmental factors, as well as a pre-existing risk of neoplasm, activates downstream effector molecules that lead to pro-oncogenic signaling and thereby aid cancer growth. GABAergic signaling mediated by the serpentine receptor GABA(B) acts as an antagonist to ß-adrenergic cascade by intercepting adenylyl cyclase. These evidences enhance the pharmacological value of human diets rich in GABA for use as an adjuvant to standard therapies.

Chronic exposure to estrogen and the tobacco carcinogen NNK cooperatively modulates nicotinic receptors in small airway epithelial cells.

Small airway epithelial cell-derived adenocarcinoma is the most common human lung cancer and is particularly prevalent in women. We have previously reported that the proliferation of immortalized human small airway epithelial cells HPL1D is stimulated by a single dose of the tobacco carcinogen NNK via cAMP signaling downstream of the beta-1-adrenergic receptor (beta1-AR) and that estrogen enhances this response. In the current study we show that gamma-aminobutyric acid (GABA) blocks this cooperative signaling of NNK and estrogen in HPL1D cells. NNK additionally stimulated the production of noradrenaline, an effect mediated by the alpha7 nicotinic acetylcholine receptor (alpha7nAChR), while reducing GABA production via desensitization of the alpha4nAChR. Chronic exposure to NNK, estrogen or the combination of both upregulated and sensitized the alpha7nAChR, resulting in an enhanced noradrenergic response to agonist. At the same time, chronic NNK and estrogen suppressed the production of GABA by desensitizing its regulatory alpha4beta2nAChR. This selective imbalance in stimulatory and inhibitory signaling may contribute to the development and progression of small airway-derived adenocarcinoma in women who smoke.

beta-Carotene promotes the development of NNK-induced small airway-derived lung adenocarcinoma.

AIM: beta-Carotene has shown cancer-preventive effects in preclinical studies while increasing lung cancer mortality in clinical trials. We have shown that beta-carotene stimulates cAMP signalling in vitro. Here, we have tested the hypothesis that beta-carotene promotes the development of pulmonary adenocarcinoma (PAC) in vivo via cAMP signalling. METHODS: PAC was induced in hamsters with the carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), followed by beta-carotene for 1.5 years. Incidence, multiplicity and size of lung tumours were recorded, and phosphorylated CREB and ERK1/2 in tumour cells were determined by Western blots. Cyclic AMP in blood cells was analysed by immunoassays, retinoids in serum and lungs by HPLC. RESULTS: beta-Carotene increased lung tumour multiplicity, lung tumour size, blood cell cAMP, serum and lung levels of retinoids and induced p-CREB and p-ERK1/2 in lung tumours. CONCLUSIONS: Our data suggest that beta-carotene promotes the development of PAC via increased cAMP signalling.

Non-genomic inhibitory signaling of beta-carotene in squamous cell carcinoma of the lungs.

Studies have suggested that retinoids prevent lung cancer by interacting with nuclear retinoid receptors. However, clinical trials with beta-carotene increased lung cancer mortality. We recently showed that beta-carotene stimulates the proliferation of small airway-derived adenocarcinoma by increasing cAMP signaling. Here, we have tested the hypothesis that beta-carotene may stimulate squamous cell carcinoma cells via similar mechanisms. We determined the effects of beta-carotene in cell lines from squamous cell carcinomas and large airway epithelia on proliferation by MTT assays in the presence and absence of inhibitors. Signaling via cAMP/PKA was measured by immunoassays and PKA activation assay. Phosphorylated ERK1/2 was determined by Western blotting. beta-carotene significantly inhibited proliferation and phosphorylation of ERK1/2 by Galphas-mediated signaling involving adenylyl cyclase, cAMP, PKA and ERK1/2. These findings introduce a non-genomic inhibitory mechanism of beta-carotene and emphasize the need for the development of marker-guided lung cancer prevention.

Nicotinic receptor-associated modulation of stimulatory and inhibitory neurotransmitters in NNK-induced adenocarcinoma of the lungs and pancreas.

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 signalling stimulated by the stress neurotransmitters adrenaline and noradrenaline as an important stimulator of 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 alpha(7)- and alpha(4)beta(2)nAChRs, causing an increase in stress neurotransmitters and a decrease in the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). 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 on microdissected control small airway epithelia, alveolar epithelia, pancreatic islet and pancreatic duct epithelia, and from NNK-induced PACs and PDACs showed that the GABA-synthesizing enzyme glutamate decarboxylase 65 (GAD65) and GABA were suppressed in NNK-induced PACs and PDACs. In contrast, protein expression of the alpha(7)nAChR, alpha(4)nAChR as well as p-CREB and p-ERK1/2 were up-regulated. These findings suggest 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.

Nicotine stimulates pancreatic cancer xenografts by systemic increase in stress neurotransmitters and suppression of the inhibitory neurotransmitter gamma-aminobutyric acid.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality in Western countries. We have shown previously that four representative human PDAC cell lines were regulated by beta-adrenoreceptors via cyclic adenosine 3',5'-monophosphate (cAMP)-dependent signaling. In the current study, we have tested the hypothesis that nicotine stimulates the growth of PDAC xenografts in nude mice by increasing the systemic levels of the stress neurotransmitters adrenaline and noradrenaline, which are the physiological agonists for beta-adrenoreceptors and that inhibition by gamma-aminobutyric acid (GABA) of the adenylyl cyclase-dependent pathway downstream of adrenoreceptors blocks this effect. The size of xenografts from PDAC cell line Panc-1 was determined 30 days after inoculation of the cancer cells. Stress neurotransmitters in serum as well as cAMP in the cellular fraction of blood and in tumor tissue were assessed by immunoassays. Levels of GABA, its synthesizing enzymes GAD65 and GAD67 and beta-adrenergic signaling proteins in the tumor tissue were determined by western blotting. Nicotine significantly increased the systemic levels of adrenaline, noradrenaline and cAMP while increasing xenograft size and protein levels of cAMP, cyclic AMP response element-binding protein and p-extracellular signal-regulated kinase 1/2 in the tumor tissue. Nicotine additionally reduced the protein levels of both GAD isozymes and GABA in tumor tissue. Treatment with GABA abolished these responses to nicotine and blocked the development of xenografts in mice not exposed to nicotine. These findings suggest that the development and progression of PDAC is subject to significant modulation by stimulatory stress neurotransmitters and inhibitory GABA and that treatment with GABA may be useful for marker-guided cancer intervention of PDAC.

Gamma-aminobutyric acid, a potential tumor suppressor for small airway-derived lung adenocarcinoma.

Pulmonary adenocarcinoma (PAC) is the leading type of lung cancer in smokers and non-smokers that arises in most cases from small airway epithelial cells. PAC has a high mortality due to its aggressive behavior and resistance to cancer therapeutics. We have shown previously that the proliferation of human PAC cells NCI-H322 and immortalized human small airway epithelial cells HPL1D is stimulated by cyclic adenosine monophosphate (cAMP)/protein kinase A-dependent phosphorylation of cyclic adenosine monophosphate response element-binding (CREB) protein and transactivation of the epidermal growth factor receptor and that this pathway is activated by beta-1-adrenoreceptors (beta(1)-ARs) and the non-genomic estrogen receptor beta. Our current in vitro studies with HPL1D and NCI-H322 cells showed that signaling via the gamma-amino butyric acid receptor (GABA(B)R) strongly inhibited base level and isoproterenol-induced cAMP, p-CREB, cyclic adenosine monophosphate response element-luciferase activity and p-extracellular regulated kinase-1 (ERK1)/2 and effectively blocked DNA synthesis and cell migration. The inhibitory effects of gamma-amino butyric acid (GABA) were disinhibited by the GABA(B)R antagonist CGP-35348 or GABA(B)R knockdown. Immunohistochemical investigation of hamster lungs showed significant underexpression of GABA in animals with small airway-derived PACs induced by the nicotine-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These findings suggest that GABA may have tumor suppressor function in small airway epithelia and the PACs derived from them and that downregulation of GABA by NNK may contribute to the development of this cancer in smokers. Our findings suggest that marker-guided treatment with GABA or a GABA(B)R agonist of individuals with downregulated pulmonary GABA may provide a novel targeted approach for the prevention of PAC in smokers.

GABA B receptor is a novel drug target for pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer death. Smoking, diabetes, and pancreatitis are risk factors. It has been shown that the growth of PDAC and pancreatic duct epithelial cells is regulated by beta-adrenoreceptors (beta-ARs). The activity of beta-ARs in the central nervous system is counteracted by gamma-aminobutyric acid (GABA) via GABA B receptor-mediated inhibition of adenylyl cyclase. The aim of the study was to investigate if GABA B R inhibits beta-AR signaling in PDAC and pancreatic duct epithelial cells, thus blocking driving forces of cancer progression, such as cell proliferation and cell migration. METHODS: Intracellular cAMP was measured by immunoassays, DNA synthesis by BrdU incorporation assays, activation of ERK1/2 by ERK activation assays, and Western blots and metastatic potential by cell migration assays in the human PDAC cell lines PANC-1 and BXPC-3 and immortalized human pancreatic duct epithelial cells HPDE6-C7. The expression of norepinephrine, PKAR IIalpha, and GABA in PDAC microarrays was assessed by immunohistochemistry. RESULTS.: Stimulation of the GABA B R by GABA or baclofen inhibited isoproterenol-induced cAMP signaling below base levels. ERK1/2 activity in response to isoproterenol was blocked by GABA, an effect enhanced by transient overexpression of the GABA B R and abolished by GABA B R knockdown. DNA synthesis and cell migration were stimulated by isoproterenol, responses blocked by GABA and baclofen. Norepinephrine and PKAR IIalpha were overexpressed while GABA was underexpressed in human PDAC tissue arrays. CONCLUSIONS: The data suggest the stimulation of GABA B R signaling as a novel target for the treatment and prevention of pancreatic cancer.

Low concentrations of beta-carotene stimulate the proliferation of human pancreatic duct epithelial cells in a PKA-dependent manner.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is among the most common causes of cancer death. Preclinical and clinical studies on the preventive effects of beta-carotene or other retinoids have used dietary supplements that yielded high systemic concentrations (1-50 microM). While some of the preclinical data suggested cancer preventive effects of these agents, they have disappointed in clinical investigations. MATERIALS AND METHODS: The effects of low concentrations (10 fM-200 nM)of beta-carotene on the proliferation, intracellular cAMP levels, PKA activation status and phosphorylation of EGFR-specific tyrosine kinases and ERK1/2 in immortalized human pancreatic duct epithelial cells was investigated. RESULTS: Our data show significant concentration-dependent and PKA-dependent stimulation of all measured endpoints. Similar responses were achieved with forskolin. Our data indicate that low concentrations of beta-carotene stimulate the proliferation of the putative origin of PDAC, pancreatic duct epithelial cells via cAMP and PKA-dependent transactivation of the EGFR pathway. This could potentially have promoting effects on the development of PDAC.