Prevention of pancreatic cancer by the beta-blocker propranolol

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is among the leading causes of cancer deaths and is unresponsive to existing therapy. Smoking and alcohol-induced pancreatitis are among the risk factors for PDAC. We have previously reported that beta-adrenergic receptors (beta-ARs) stimulate the proliferation and migration of human PDAC cells in vitro by cAMP-dependent signaling and that the nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) activates this pathway directly in vitro while additionally stimulating the release of noradrenaline/adrenaline by binding to alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) in hamsters. In this study, we have tested the hypothesis that the beta-AR antagonist propranolol prevents the development of PDAC induced in hamsters with ethanol-induced pancreatitis by NNK. We found that propranolol had strong cancer preventive effects in this animal model. Western blots of pancreatic duct cells and PDAC cells harvested by laser capture microscopy showed significant upregulation of the alpha7 nAChR associated with significant inductions of p-CREB, p-ERK1/2, and increases in epidermal growth factor and vascular endothelial growth factor in PDAC cells of hamsters not treated with propranolol. These effects were reversed by treatment with propranolol. Our data suggest that propranolol may prevent the development of PDAC by blocking cAMP-dependent intracellular signaling, cAMP-dependent release of epidermal growth factor, and PKA-dependent release of vascular endothelial growth factor while additionally downregulating the alpha7 nAChR by inhibiting cAMP-mediated subunit assembly. We conclude that increased cAMP signaling is an important factor that drives the development and progression of PDAC and that the inhibition of cAMP formation is a promising new target for the prevention and adjuvant therapy of PDAC.

Figure 1

Survival curves of hamsters expressed as percent surviving animals per treatment group (n=12 in each group) over time. Cancer preventive treatment with propranolol significantly (p<0.001 by Logrank test) increased the survival time of hamsters induced for the development of pancreatic cancer by prenatal treatment with ethanol and NNK.

Figure 2

Western blot showing upregulation (1.9-fold; p<0.001) of the α7nAChR in cells harvested from ETOH+NNK-induced PDACs and dowregulation of this receptor below the levels of the ETOH controls by propranolol (p<0.001). Columns in the graph represent mean values and standard errors of five densitometric readings per band expressed as ratio of α7nAChR over actin. This Western was conducted three times with lysates from three separate samples per treatment group and yielded similar data.

Figure 3

Western blots showing induction of p-CREB (2.9-fold, p<0.001) and p-ERK1/2 (2.2-fold, p<0.001) in cells harvested from ETOH/NNK-induced PDACs and inhibition of these responses (p<0.001) by treatment with propranolol. Columns in the graph represent mean values and standard errors of five densitometric readings per band expressed as ratio of p-CREB over CREB or p-ERK1/2 over ERK1/2. Each Western was conducted three times with lysates from three separate samples per treatment group and yielded similar data.

Figure 4

Western blots exemplifying the induction of VEGF (2.9-fold, p<0.001) and EGF (1.8-fold, p<0.001) in cells from ETOH/NNK-induced PDACs and inhibition of these responses (p<0.001) by propranolol. Columns in the graph represent mean values and standard errors of five densitometric readings per band expressed as ratio of VEGF or EGF over actin. Each Western was conducted three times with lysates from three separate samples per treatment group and yielded similar data.

Figure 5

Working model of the proposed mechanisms how propranolol prevented the development of PDAC induced in hamsters by ethanol+NNK. NNK-induced upregulation of the α7nAChR increased the production and release of the stress neurotransmitter noradrenaline from which adrenaline is formed enzymatically. Both neurotransmitters bind as agonists to β-ARs, activating its effector adenylyl cyclase, the rate-limiting step for the formation of intracellular cAMP. In turn, cAMP causes the release of EGF [34] and activates PKA that phosphorylates the transcription factor CREB while additionally stimulating the release of VEGF [32] and transactivating the EGFR. This signaling cascade is further intensified by direct agonist binding of NNK to β-ARs and activation of adenylyl cyclase by ethanol [36]. Propranolol blocks all signaling events downstream of β-ARs by binding as an antagonist to these receptors. In addition, propranolol downregulates the α7nAChR by inhibiting cAMP-mediated assembly of its subunits [28], thus indirectly reducing the synthesis and release of noradrenaline and adrenaline. Collectively, these multiple actions of propranolol have strong inhibiting effects on the proliferation, migration and angiogenesis of PDAC.