Abstract
BackgroundPancreatic cancer is frequently resistant to cancer therapeutics. Smoking and alcoholism are risk factors and pancreatic cancer patients often undergo nicotine replacement therapy (NRT) and treatment for alcohol dependence. Based on our report that low dose nicotine within the range of NRT causes gemcitabine resistance in pancreatic cancer, our current study has tested the hypothesis that GABA or the selective GABA-B-R agonist baclofen used to treat alcohol dependence reverse nicotine-induced gemcitabine resistance in pancreatic cancer.MethodsUsing mouse xenografts from the gemcitabine--sensitive pancreatic cancer cell line BXPC-3, we tested the effects of GABA and baclofen on nicotine-induced gemcitabine resistance. The levels of cAMP, p-SRC, p-ERK, p-AKT, p-CREB and cleaved caspase-3 in xenograft tissues were determined by ELISA assays. Expression of the two GABA-B receptors, metalloproteinase-2 and 9 and EGR-1 in xenograft tissues was monitored by Western blotting. Mechanistic studies were conducted in vitro, using cell lines BXPC-3 and PANC-1 and included analyses of cAMP production by ELISA assay and Western blots to determine protein expression of GABA-B receptors, metalloproteinase-2 and 9 and EGR-1.ResultsOur data show that GABA was as effective as gemcitabine and significantly reversed gemcitabine resistance induced by low dose nicotine in xenografts whereas baclofen did not. These effects of GABA were accompanied by decreases in cAMP, p-CREB, p-AKT, p-Src, p-ERK metalloproteinases-9 and -2 and EGR-1 and increases in cleaved caspase-3 in xenografts whereas baclofen had the opposite effects. In vitro exposure of cells to single doses or seven days of nicotine induced the protein expression of MMP-2, MMP-9 and EGR-1 and these responses were blocked by GABA. Baclofen downregulated the protein expression of GABA-B-Rs in xenograft tissues and in cells exposed to baclofen for seven days in vitro. This response was accompanied by inversed baclofen effects from inhibition of cAMP formation after single dose exposures to stimulation of cAMP formation in cells pretreated for seven days.ConclusionsThese findings suggest GABA as a promising single agent for the therapy of pancreatic cancer and to overcome nicotine-induced gemcitabine resistance whereas treatment with baclofen may increase gemcitabine resistance.
Highlights
Pancreatic cancer is frequently resistant to cancer therapeutics
Having shown that GABA and baclofen each inhibit p-extra cellular signal regulated kinase (ERK) when administered as a single dose to pancreatic cancer cells in vitro by reducing the formation of cyclic adenosine monophosphate (cAMP) [9], our current experiments have tested the hypothesis that both agents reverse gemcitabine resistance induced by low dose nicotine in pancreatic cancer
The gemcitabine-induced reduction in xenograft volumes was significantly diminished by baclofen in weeks 2 and 4 (Figure 1B), suggesting an increase in gemcitabine resistance
Summary
Pancreatic cancer is frequently resistant to cancer therapeutics. Smoking and alcoholism are risk factors and pancreatic cancer patients often undergo nicotine replacement therapy (NRT) and treatment for alcohol dependence. We have previously reported that high doses of nicotine comparable to the blood nicotine levels in heavy smokers accelerated the progression of pancreatic cancer xenografts by increasing cell proliferation and that treatment of the mice with GABA in the drinking water blocked this effect via GABA-B-receptor (GABA-B-R)-mediated inhibition of cAMP-dependent pathways [7,8] In accord with these findings, single dose exposures of pancreatic cancer cells in vitro to GABA or baclofen inhibited via this mechanism cell proliferation and migration induced by the beta-adrenergic receptor agonist isoproterenol [9]. Having shown that GABA and baclofen each inhibit p-ERK when administered as a single dose to pancreatic cancer cells in vitro by reducing the formation of cAMP [9], our current experiments have tested the hypothesis that both agents reverse gemcitabine resistance induced by low dose nicotine in pancreatic cancer
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