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

Abstract

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.

Fig. 1.

Effects of nicotine and GABA on Panc-1 xenografts. Athymic nude mice were subcutaneously injected with human PDAC cells Panc-1 and observed for 30 days. Controls received no pharmacological treatment, one group was given nicotine in the drinking water for 30 days, one group received nicotine in the drinking water and additional intraperitoneal injections with GABA (five times per week) and one group was treated by intraperitoneal injections with GABA alone. At the end of the observation period, nicotine had significantly (P < 0.001) increased xenograft size, a response significantly (P < 0.001) inhibited by GABA. Data were mean values and standard errors (expressed as fold increase over untreated animals) of the eight tumor-bearing mice in the untreated and nicotine-treated groups and of the two mice that developed small xenografts in the group treated with nicotine plus GABA. None of the animals given GABA alone developed xenografts. * significantly (P < 0.001) different from untreated group; ** significantly (P < 0.001) different from group treated with nicotine alone.

Fig. 2.

A) Modulation of serum levels of adrenaline and noradrenaline by nicotine and GABA. Immunoassay revealed a 4.2-fold increase of noradrenaline (P < 0.001) and a 2-fold increase of adrenaline (P < 0.001) in the mice treated with nicotine. GABA completely abrogated these responses to nicotine. Data were mean values and standard errors of triplicate samples expressed as fold increase over the levels in untreated mice. * significantly (P < 0.001) different from untreated group; ** significantly (P < 0.001) different from group treated with nicotine alone. (B) Results of immunoassay for the determination of intracellular cAMP in the cellular fraction of blood samples in tumor tissue and in the skin of mice without tumors after treatment with GABA alone. Treatment with nicotine increased intracellular cAMP 1.86-fold in blood cells and 2.6-fold in xenograft tissue, a response completely blocked by GABA. Treatment with GABA alone significantly (P < 0.001) reduced cAMP levels below the base levels in untreated animals. Data were mean values and standard errors of triplicate samples per treatment group expressed as fold increase over the levels in untreated animals. * significantly (P < 0.001) different from untreated group; ** significantly (P < 0.001) different from group treated with nicotine alone.

Fig. 3.

Western blots showing protein expressions of the GABA-synthesizing enzymes GAD67, GAD65 and the inhibitory neurotransmitter GABA in xenografts of untreated mice (controls), animals treated with nicotine or with nicotine plus GABA and in skin samples from the group treated with GABA alone. Nicotine significantly (P < 0.001) suppressed GAD65 and GABA, an effect inhibited (P < 0.001) by treatment with GABA. The columns in the graph are mean values and standard errors of five densitometric readings per band calculated as ratio of GAD or GABA protein over actin and expressed as fold increase over controls. * significantly (P < 0.001) different from untreated group; ** significantly (P < 0.001) different from group treated with nicotine alone.

Fig. 4.

Western blots showing protein expression of phosphorylated and unphosphorylated CREB as well as phosphorylated and unphosphorylated ERK1/2 in xenografts of untreated mice (controls), animals treated with nicotine or with nicotine plus GABA and in skin samples from the group treated with GABA alone. Nicotine significantly (P < 0.001) induced the expression levels of p-CREB and p-ERK1/2. These effects were inhibited (P < 0.001) by GABA. The columns in the graph are mean values and standard errors of five densitometric readings per band calculated as ratio of GAD or GABA protein over actin and expressed as fold increase over controls. * significantly (P < 0.001) different from untreated group; ** significantly (P < 0.001) different from group treated with nicotine alone.