Prevention of pancreatic cancer in a hamster model by cAMP decrease

Abstract

Smoking and alcoholism are risk factors for the development of pancreatitis-associated pancreatic ductal adenocarcinoma (PDAC). We have previously shown that these cancers overexpressed stress neurotransmitters and cyclic adenosine monophosphate (cAMP) while the inhibitory neurotransmitter γ-aminobutyric acid (GABA) was suppressed. Using a hamster model, the current study has tested the hypothesis that cAMP decrease by GABA supplementation in the drinking water prevents the development of pancreatitis-associated PDAC. Our data reveal strong preventive effects of GABA supplementation on the development of PDAC and pancreatic intraductal neoplasia (PanIN). ELISA assays and immunohistochemistry revealed significant decreases in the levels of cAMP and interleukin 6 accompanied by reductions in the expression of several cancer stem cell markers and phosphorylated signaling proteins, which stimulate cell proliferation, and migration in pancreatic exocrine cells of GABA treated animals. We conclude that cAMP decrease by GABA supplementation inhibits multiple cancer stimulating pathways in cancer stem cells, differentiated cancer cells and the immune system, identifying this approach as promising novel tool for the prevention of PDAC in individuals with a history of smoking and alcoholism.

Keywords: cancer stem cell signaling; cyclic adenosine monophosphate; pancreatic cancer; prevention; γ-aminobutyric acid.

Figure 1. Effects of GABA supplementation on the incidence (%) of PDACs and PanINs in controls, hamsters treated prenatally with ETOH and NNK and in hamsters treated prenatally with ETOH and NNK followed by GABA supplementation in the drinking water at 4 weeks of age

GABA had strong preventive effects on PDAC and PanIN. Significantly (p < 0.0001) different from controls (one asterix); significantly (p < 0.0001) different from group treated with ETOH and NNK alone (double asterix).

Figure 2. Levels of the transcription factor SOX9 that serves as a marker for pancreatic ductal lineage as assessed by ELISA assay in cells harvested by laser capture micro dissection from pancreatic ducts of control and GABA treated hamsters versus cells harvested from ETOH/NNK-induced PDACs

SOX9 was strongly expressed in the tumor cells (control levels: 4.07 ± 0.41 ng/ml). Significantly (p < 0001) different from controls (asterix).

Figure 3. Modulation of intracellular cAMP and the pro-inflammatory cytokine IL-6 in exocrine pancreatic cells and tumor cells as assessed by ELISA assays

PDAC cells (Tumor) in animals treated prenatally with ETOH plus NNK alone showed significant increases of cAMP (control levels: 5.1 ± 0.29 pmol/ml) and IL-6 (control levels: 10.81 ± 0.62 pg/ml) over the levels measured in exocrine pancreatic cells of controls. GABA supplementation completely reversed these responses, reducing cAMP and IL-6 in pancreatic exocrine cells below control levels. Significantly (p < 0001) different from controls (one asterix); significantly (p < 0.0001) different from tumor cells (double asterix).

Figure 4. Results of ELISA assays for the determination of the phosphorylated forms of signaling proteins ERK and Src in micro-dissected PDAC cells induced by ETOH plus NNK and in exocrine pancreatic cells of controls and hamsters treated prenatally with ETOH plus NNK followed by GABA supplementation starting at 4 weeks of age

The tumor cells showed elevated levels of p-ERK (control levels: 0.72 ± 0.5 ng/ml) and p-Src (control levels: 0.71 ± 0.07 ng/ml), responses reduced below control levels in exocrine pancreatic cells after GABA supplementation. Significantly (p < 0.0001) different from controls (on asterix); significantly (p < 0.0001) different from PDACs induced by ethanol and NNK (double asterix).

Figure 5. Results of ELISA assays for the determination of the cancer stem cell markers ALDH-1 and SHH in exocrine pancreatic cells and PDAC cells harvested by laser capture micro-dissection

Both markers were significantly (p < 0.001) increased in PDACs induced by treatment with ETOH and NNK alone while GABA supplementation significantly (p < 0.0001) reduced both markers in exocrine pancreatic cells (Control SHH levels: 102.9 ± 6.1 pg/ml; control ALDH-1 levels: 2.03 ± 0.10 ng/ml). Significantly (p < 0.001) different from controls (one asterix); significantly (p < 0.0001) different fro group treated with ETOH + NNK alone (double asterix).

Figure 6. Photomicrographs exemplifying the over-expression of the cancer stem cell markers CD133 (brown stain) and Gli-1 (red stain)

Photos A and B show a preneoplastic lesion PanIN-3 indicated by arrow in the pancreas of a hamster treated prenatally with ETOH and NNK while photos C and D show tumor tissue. Immunohistochemical stain was done with the Vectastain Elite ABC kit using incubation with exposure to primary anti-CD133 antibody (1:200) or Gli-1 (1:500) for 1 hour, DAB or AEC as substrate and hematoxylin as counterstain.

Figure 7. Photomicrographs showing faint immunoreactivity to the cancer stem cell marker CD133

A. In pancreatic tissue sections from a control (A) and from an animal that received cancer preventive treatment with GABA B. after prenatal treatment with ETOH and NNK and faint immunoreactivity for Gli-1 in a control C. and GABA treated D. animal. Pancreatic ducts are indicated by arrows.

Figure 8. Results of densitometry for the quantitative assessment of CD133 immunoreactivity

Tumor tissues and PanINs expressed significantly (p < 0.0001; asterix)) higher levels of CD133 immunoreactivity than duct epithelia in controls and GABA treated hamsters. Data are expressed as mean values and standard deviations at the 95% confidence interval from 50 mean density measurements per column.

Figure 9. Results of densitometry for the quantitative determination of Gli-1 immunoreactivity

Tumor tissues and PanINs expressed significantly (p < 0.0001; asterix)) higher levels of CD133 immunoreactivity than duct epithelia in controls and GABA treated hamsters. Data are expressed as mean values and standard deviations at the 95% confidence interval from 50 mean density measurements per column.

Figure 10

Cartoon illustrating the mechanisms of pancreatic cancer induction and progression induced by ethanol and NNK A. and the cancer preventive mechanisms of cAMP control by GABA supplementation B.