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

Abstract

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 α4β2nAChR 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.

Fig. 2.

Total (secreted plus intracellular) GABA levels (a–c) and total (secreted plus intracellular) adrenaline levels (d–f) in HPDE6-C7, BxPC-3 and Panc-1 cells treated with single doses of nicotine from 10 pM through 10 µM for 30min or pretreated with 1 µM nicotine for 7 days and then exposed to identical concentrations of nicotine. Data points are mean and ±SD from five samples per treatment group.

Fig. 1.

Secreted (a) and intracellular (b) GABA levels in HPDE6-C7, BxPC-3 and Panc-1 cells treated with 1 µM nicotine from 1 to 30min. (c) ELISAs showing intracellular GABA levels in cells in the presence and absence of gene knockdown of the α4-nAChR in the presence and absence of 1 µM nicotine for 30min. Data points are mean and ±SD from five samples per treatment group. (d) Western blots showing the effects of gene knockdown on protein expression of the β4-nAChR in the presence and absence of nicotine (1 µM for 30min). The house keeping protein β-actin was used as a control to ensure equal loading of proteins. The columns in the graph represent means and ±SD of two mean density readings per band from three independent western blots (n = 6) expressed as fold changes in expression of β4-nAChR.

Fig. 3.

Total (secreted plus intracellular) noradrenaline levels (a–c) in HPDE6-C7, BxPC-3 and Panc-1 cells treated with single doses of nicotine from 10 pM through 10 µM for 30min or pretreated with 1 µM nicotine for 7 days and then exposed to identical concentrations of nicotine. Levels of dopamine beta-hydroxylase (d) in HPDE6-C7, BxPC-3 and Panc-1 cells treated with 1 µM nicotine for 30min, pretreated with 1 µM nicotine for 7 days, or pretreated with 1 µM nicotine for 7 days followed by 30-min nicotine treatment prior to harvesting. Data points are mean and ±SD from five samples per treatment group.

Fig. 4.

Western blots assessing protein expression of nAChR subunits α3, α4, α5 and α7 in HPDE6-C7, BxPC-3 and Panc-1 cells treated with 1 µM nicotine, 30 µM GABA or GABA + nicotine for 7 days. The house keeping protein β-actin was used as a control to ensure equal loading of proteins (a). The columns in graph (b) represent means and ±SD of two mean density readings per band from three independent western blots (n = 6) expressed as fold changes in expression of α3, α4, α5 and α7-nAChRs.

Fig. 5.

Western blots assessing phosphorylation of AKT, Src, GAD65 and GAD67 in HPDE6-C7, BxPC-3 and Panc-1 cells treated with 1 µM nicotine, 30 µM GABA and GABA + nicotine for 7 days (a). The columns in graph (b) and (c) represent means and ±SD of two mean density readings per band from three independent western blots (n = 6) expressed as fold changes in protein phosphorylation. The unphosphorylated proteins AKT, Src and β-actin were used as controls to ensure equal loading of proteins.

Fig. 6.

Phosphorylation of CREB and ERK (a) assessed by ELISA in HPDE6-C7, BxPC-3 and Panc-1 cells showing inhibition of nicotine-induced phosphorylation, cell proliferation and migration by GABA. Cell proliferation (b) assessed by MTT assays and metastatic potential (c) assessed by cell migration assays: data from all three cell lines were significantly (P < 0.0001) different from controls (*), from nicotine 30’ (**) and from nicotine 7 days + 30’ nicotine (***). The columns represent means and ±SD of five samples per treatment group.