The tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone stimulates proliferation of immortalized human pancreatic duct epithelia through beta-adrenergic transactivation of EGF receptors

Abstract

Purpose: Pancreatic ductal adenocarcinoma is an aggressive smoking-associated human cancer in both men and women. The nicotine-derived 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is thought to contribute to the development of these neoplasms in smokers through genotoxic effects. However, NNK has been recently identified as an agonist for both beta(1)- and beta(2)-adrenergic receptors. Binding of NNK to these receptors stimulates proliferation of pulmonary and pancreatic adenocarcinomas cells in vitro and in hamster models. The goal of this study was to elucidate the NNK effects on the signal transduction pathways downstream of both beta(1)- and beta(2)-adrenergic receptors in immortalized human pancreatic HPDE6-c7 cells.

Methods: The HPDE6-c7 cells are developed from normal pancreatic duct epithelial cells which are the putative cells of origin of pancreatic ductal adenocarcinoma. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) cell proliferation assays, Western blot and cyclic AMP assays were employed to demonstrate the effects of NNK and other beta(1)- and beta(2)-adrenergic agonists and antagonist treatments on these cells.

Results: MTT cell proliferation assays demonstrated that NNK and the classic beta-adrenergic agonist, isoproterenol, increased cell proliferation in HPDE6-c7 cells. Western blot and cyclic AMP assays demonstrated that NNK treatments also resulted in: (1) transactivation of the epidermal growth factor receptor, EGFR, (2) an increase in intracellular cyclic AMP accumulation, and (3) phosphorylation of mitogen-activated protein kinase, Erk1/2. The proliferative response to NNK and isoproterenol were inhibited by the use of beta-blockers (propranolol), and the inhibitors of adenylyl cyclase (SQ 22536), EGFR-specific tyrosine kinase (AG 1478) and Erk (PD 98059).

Conclusion: These findings suggest that the NNK -mediated beta-adrenergic receptor transactivation of the EGFR and phosphorylation of Erk1/2 in immortalized human pancreatic duct epithelial cells as a novel mechanism might contribute to the development of tobacco-associated pancreatic carcinogenesis.

Fig. 1

Expression of mRNA for β₁- and β₂-adrenergic receptors in the human pancreatic cell line HPDE6-c7. Total RNA was isolated and subjected to RT-PCR. a Lane 2: HPDE6-c7, β₁ primers; lane 4, transfected CHO cell line Rex 50 with β₁ primers (positive control); lanes 1 and 3 are negative controls without MMLV reverse transcriptase. b Lane 2 HPDE6-c7, β₂ primers; lane 4, transfected CHO cell line NBR29 with β₂ primers (positive control).β₂ and cyclophylin primers; lane 4, cyclophylin primer; lanes 1 and 3 are negative controls without MMLV reverse transcriptase

Fig. 2

Ten minutes treatments of HPDE6-c7 cells with NNK increased the intracellular levels of cAMP in a concentration-dependent manner. Representative data points are mean ± SD from triplicate measurements in a single experiment

Fig. 3

Accumulation of cyclic AMP in response to β-adrenergic receptor agonists and antagonist. HPDE6-c7 cells were treated with the indicated agents and processed for cyclic AMP activity. Data represent means ± SD of three different experiments

Fig. 4

Phosphorylation of EGFR in response to NNK exposure. Western blot analysis of HPDE6c-7 cells stimulated with NNK (1 nM, 10 min), propranolol, (1 μM, 30 min), or propranolol followed by NNK treatment are presented. Blots were detected using monoclonal antibody against a EGFR and also phosphorylated EGF receptors at b Tyr 992, c Tyr 1068 and d Tyr 1173. These panels show phosphorylation of the EGFR at all three sites upon NNK treatment. Data represent means ± SD of three different experiments

Fig. 5

Activation of ERK1/2 by NNK treatment. Lysates from untreated cells or cells treated with NNK (1 nM) or propranolol (1 uM) or propranolol and NNK were subjected to immunoblotting with rabbit anti ERK1/2 antibody, or rabbit anti phosphorylated ERK1/2 antibody. The house keeping protein β-actin was used as a control to ensure equal loading of the proteins. Data represent means ± SD of three different experiments

Fig. 6

Isoproterenol and MEK inhibitor effects on phosphorylation ERK1/2. Analysis of HPDE6c-7 cells in response to NNK (1 nM, 10 min) and isoproterenol, (10 nM, 10 min) alone or subsequent to PD98059 (MEK inhibitor) treatments and AG1478 (EGFR tyrosine kinase inhibitor). Blots were detected using monoclonal antibody against (a) ERK1/2 and b phosphorylated ERK1/2. Data represent means ± SD of three different experiments

Fig. 7

Effects of NNK on AA release in HPDE6-c7 cells. a Treatment of the cells with NNK (1 nM) or isoproteronl did not induce an increase in AA release. Methylanthracene (30 μM) was used as positive control

Fig. 8

NNK-dependent cell proliferation. HPDE6-c7 cells were grown in 96-well microplates, treated and cell numbers were analyzed using MTT assays. NNK and isoproterenol enhanced cell proliferation, whereas, propranolol, SQ 22536, PD98059 andAG1478 treatments did not stimulate increase in cell number. All data points are mean ± SD from quadruplicate determinations from three separate experiments