Fax: (865) 974-5616
GABAB receptor is a novel drug target for pancreatic cancer
Article first published online: 20 DEC 2007
Copyright © 2007 American Cancer Society
Volume 112, Issue 4, pages 767–778, 15 February 2008
How to Cite
Schuller, H. M., Al-Wadei, H. A. N. and Majidi, M. (2008), GABAB receptor is a novel drug target for pancreatic cancer. Cancer, 112: 767–778. doi: 10.1002/cncr.23231
- Issue published online: 1 FEB 2008
- Article first published online: 20 DEC 2007
- Manuscript Accepted: 6 SEP 2007
- Manuscript Received: 10 MAY 2007
- Manuscript Revised: 4 SEP 2006
- NIH. Grant Number: RO1 CA42829
- pancreatic cancer
Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer death. Smoking, diabetes, and pancreatitis are risk factors. It has been shown that the growth of PDAC and pancreatic duct epithelial cells is regulated by beta-adrenoreceptors (β-ARs). The activity of β-ARs in the central nervous system is counteracted by γ-aminobutyric acid (GABA) via GABAB receptor-mediated inhibition of adenylyl cyclase. The aim of the study was to investigate if GABABR inhibits β-AR signaling in PDAC and pancreatic duct epithelial cells, thus blocking driving forces of cancer progression, such as cell proliferation and cell migration.
Intracellular cAMP was measured by immunoassays, DNA synthesis by BrdU incorporation assays, activation of ERK1/2 by ERK activation assays, and Western blots and metastatic potential by cell migration assays in the human PDAC cell lines PANC-1 and BXPC-3 and immortalized human pancreatic duct epithelial cells HPDE6-C7. The expression of norepinephrine, PKARIIα, and GABA in PDAC microarrays was assessed by immunohistochemistry.
Stimulation of the GABABR by GABA or baclofen inhibited isoproterenol-induced cAMP signaling below base levels. ERK1/2 activity in response to isoproterenol was blocked by GABA, an effect enhanced by transient overexpression of the GABABR and abolished by GABABR knockdown. DNA synthesis and cell migration were stimulated by isoproterenol, responses blocked by GABA and baclofen. Norepinephrine and PKARIIα were overexpressed while GABA was underexpressed in human PDAC tissue arrays.
The data suggest the stimulation of GABABR signaling as a novel target for the treatment and prevention of pancreatic cancer. Cancer 2008. © 2007 American Cancer Society.
Pancreatic cancer is the fourth leading cause of cancer mortality in Western countries.1 About 95% of pancreatic cancers are ductal adenocarcinomas (PDAC). PDAC is among the most aggressive of human cancers and is generally unresponsive to conventional therapy, resulting in a mortality near 100% within 1 year of diagnosis.1
The regulation of PDAC and pancreatic duct epithelia is poorly understood. Most PDACs harbor activating point mutations in K-ras, while also overexpressing the epidermal growth factor receptor (EGFR), leading to the hypothesis that EGFR signaling via ras and the extracellular signal regulated protein kinases (ERK1/2) pathway regulate PDAC.2 Moreover, the arachidonic acid-metabolizing enzyme cyclooxygenase 2 (COX-2) is overexpressed in about 90% of PDACs.3 Studies in vitro and in human pancreatic cancer mouse xenografts have shown significant reductions in tumor growth by inhibitors of farnesyltransferase, EGFR tyrosine kinases, ERK1/2.1, 4 Likewise, inhibitors of COX-2 have yielded impressive antitumorigenic effects in vitro.4 Suppression of vascular endothelial cell growth factor (VEGF) has also shown promising responses in vitro and in an orthotopic mouse model of PDAC.5 However, clinical trials with inhibitors of tyrosine kinases, ras, COX-2, VEGF, or the combination of such agents have had disappointing results.6
Smoking, diabetes mellitus, and pancreatitis are risk factors for PDAC.7 However, the mechanisms by which these risk factors contribute to the development of PDAC are poorly understood.
We have shown that cell lines derived from human PDACs express β1- and β2-adrenoreceptors (ARs) and respond to their stimulation by agonists with the release of arachidonic acid and cell proliferation.8, 9 Our findings provided evidence, for the first time, that neurotransmitter receptors of the beta-adrenergic family participate in the growth regulation of PDAC.
Nicotine causes the release of the physiologic agonists for β-ARs, the catecholamine stress hormones epinephrine and norepinephrine, from the adrenal medulla.10 Identification of beta-adrenergic signaling as a growth regulator of pancreatic cancer cells8 suggests that the elevated systemic catecholamine levels in smokers may contribute to the development of pancreatic cancer. Our studies showed that the nicotine-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) mimicked the actions of the catecholamines by binding as an agonist to β1- and β2-ARs.8 We recently showed that the beta-adrenergic agonist isoproterenol and NNK stimulated the proliferation of human pancreatic duct epithelial cells by signaling via cAMP/PKA/p-CREB, while in addition transactivating the EGFR and ERK1/2 in a PKA-dependent manner.11 Beta-adrenergic activity of NNK resulting in mitogenic and/or antiapoptotic signaling has in addition been reported in cell lines from human small airway-derived adenocarcinomas,12, 13 human small airway epithelia,12 and in colon cancer cells.14 It has been shown that the migration and invasiveness of adenocarcinomas of the colon, prostate, and breast are also under beta-adrenergic control,15–17 and that ERK1/2-mediated proliferation of breast cancer cells is stimulated by beta-adreneregic agonists.18 Recent publications in addition demonstrated that the nicotine-induced increase in systemic catecholamines stimulated the growth of xenografts from human gastric carcinoma via activation of β-ARs19 and that epinephrine increased the invasiveness of ovarian cancer cells via stimulation of β-ARs.20 A novel concept is thus emerging of β-AR hyperstimulation as an important etiologic factor for some of the most common human cancers, including PDAC.
Beta-adrenoreceptors consist of β1, β2, and β3-ARs and are G-protein-coupled cell membrane receptors. Beta1 and β2-ARs are expressed in most mammalian cells. Binding of an agonist to β-ARs activates the adenylyl cyclase stimulating G-protein, Gαs, resulting in the formation of cAMP. In turn, cAMP activates PKA by binding to the PKA subunit complex, resulting in the release of inhibitory R subunits and activation of the transcription factor, cAMP response element binding protein (CREB).21 It has been shown that β1- and β2-ARs can in addition transactivate the EGFR pathway in a PKA-dependent manner22 and we have shown that such transactivation occurs in human pancreatic duct epithelial cells.11, 23
Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system and counteracts the stimulatory actions of the physiological agonists for β-ARs, epinephrine, and norepinephrine in the brain.24 Three types of GABA receptors have been identified: the ion channel receptor families GABAA and GABAC, and GABAB receptors which exist as 2 isoforms termed GABABR1 and GABABR2.25 Both GABABR isoforms are coupled to the adenylyl cyclase inhibiting G-protein, Gαi.25 GABAA and GABAC receptors mediate excitatory functions of GABA, whereas the GABAB receptors mediate the inhibitory actions of GABA.25 In the pancreas, GABA, its synthesizing enzyme, glutamic acid decarboxylase, and its metabolizing enzyme, GABA-transaminase, are expressed in the beta cells of pancreatic islets at high concentrations similar to those in the central nervous system, and GABA is secreted from beta-cells into the extracellular space.24 However, the function of GABA in the pancreas is poorly understood. GABA is decreased in the pancreas of individuals with diabetes mellitus24 due the reduction of functional islet beta cells. The number of functional pancreatic islets is also diminished in pancreatitis. The inhibitory role of GABA in the central nervous system and the finding that the GABABRs inhibits adenylyl cyclase via activation of Gαi prompted us to explore a potential inhibitory action of GABA on the cAMP-mediated beta-adrenergic stimulation of human PDAC and pancreatic duct epithelial cells. Our data show that stimulation of the GABAB receptor potently inhibited isoproterenol-induced cAMP signaling, cell proliferation, and cell migration. Moreover, human PDACs overexpressed norepinephrine and PKARIIα while underexpressing GABA. These data suggest that the reduction in pancreatic GABA may contribute to the development of PDAC. Therapeutic administration of GABA or a GABABR selective agonist may thus provide an effective novel tool for the treatment and prevention of pancreatic cancer.
MATERIALS AND METHODS
The human PDAC cell line, PANC-1, which carries an activating point mutation in K-ras and BXPC-3, without ras mutation (American Type Culture Collection, Rockville, Md) were cultured as suggested by the vendor: RPMI medium supplemented with 10% fetal bovine serum and L-glutamine (2 mM) for BXPC-3 and DMEM medium supplemented with 10% fetal bovine serum, L-glutamine (2 mM) for PANC-1. The immortalized human pancreatic duct epithelial cell line HPDE6-C726 (Dr. M.S. Tsao, Ontario Cancer Center, Ontario, Canada) was maintained in serum-free keratinocyte medium supplemented with bovine pituitary extract (25 mg/500 mL), EGF (2.5 mg/500 mL). Cells were washed to remove supplements before each assay, and all assays were conducted in basal media without supplements.
Analysis of Intracellular cAMP by Immunoassay
Cells were plated at 4 × 105 cells/well in 6-well plates in their growth medium until 65% to 70% confluence. The medium was replaced by basal medium without supplements after 3 washes with phosphate-buffered saline (PBS) and incubated for 24 hours. The cells were preincubated (30 minutes) with the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 1 mM) to prevent the enzymatic breakdown of cAMP formed in response to beta-adrenergic stimulation. The beta-adrenergic agonist isoproterenol (1 μM; Sigma, St. Louis, Mo) was added to the culture medium with or without a 4-hour preincubation with GABA (30 μM) or the selective GABAB receptor agonist baclofen (30 μM). After 3 washes with water, cells were treated with 0.1 M HCL for 20 minutes, then lysed by sonication. Intracellular cAMP was measured with an enzyme immunoassay kit (Assay Designs, Ann Arbor, Mich) as previously described.27 Assays were conducted in triplicate, each with triplicate samples. Statistical analysis of data was by 1-way analysis of variance (ANOVA), Tukey-Kramer multiple comparison test, and 2-tailed unpaired t-test.
BrdU Incorporation Assays
BrdU incorporation assays were conducted with a kit (Roche Applied Science, Nutley, NJ) as previously described.12 Cells were cultured in 96-well plates (1 × 104/well), deprived of serum and supplements for 24 hours, and then either treated with isoproterenol (10 nM) for 72 hours, pretreated for 4 hours with GABA (30 mM) or baclofen (30 μM), or they were exposed to each of the inhibitors alone for the duration of the assay. Cells were labeled with 10 mL/well BrdU and reincubated at 37°C for 4 hours. After removal of the labeling medium, cells were fixed and probed with anti-BrdU monoclonal antibody and its substrate, tetramethyl-benzidine, for 1 hour. After removal of the antibody conjugate the cells were rinsed 3 times with washing solution and substrate solution (100 μL/well) was added. After color development the absorbance of each sample was measured in an enzyme-linked immunosorbent assay (ELISA) reader at 370 nm. A blank was run in each experiment to provide information about BrdU and anti-BrdU nonspecific binding. The nonspecific binding was subtracted from all other values. Each experiment was conducted twice with 5 replicates per data point. Statistical analysis of data was by 1-way ANOVA and Tukey-Kramer multiple comparison test.
Cell Migration Assay
The metastasis of cancer cells is facilitated by their ability to migrate. Measurement of cell migration is therefore frequently used as a tool to assess the metastatic potential of cancer cells. We used a colorimetric cell migration assay kit (Cell Biolabs, San Diego, Calif) consisting of 24-well plates that contain polycarbonate membrane filter inserts (8 μM pore size). Cells (0.5 × 106 cells per mL of basal medium) were seeded onto the top chamber above the filter insert and pretreated for 4 hours with GABA (30 μM) or baclofen (30 μM). Isoproterenol (10 nM) was then added. After a 24-hour incubation period, nonmigratory cells were removed from the top of the filters by cotton swab. The filter with cells that had migrated to its bottom surface were incubated with staining solution for 10 minutes, washed 3 times with tapwater, air-dried, and photographed. Each filter was extracted and 100 μL per sample of the extract was transferred to a 96-well microtiter plate. Optical density at 560 nm was read with a plate reader. Each assay was conducted in triplicate. Statistical analysis of data was by ANOVA and Tukey-Kramer multiple comparison test.
Transient Transfection With Stealth Select RNAi for GABAB-R1 or With GABAB-R1 cDNA
Cells (>90% viable) were plated at 3 × 104 cells/well in 24-well plates in complete medium without antibiotics and allowed to reach 60% confluence. They were then transfected in triplicate for each treatment group with 100 mL of 100 nM GABAB-R1 Stealth RNAI (Invitrogen, La Jolla, Calif) or GABAB-R1 cDNA complexed with 2 mg/mL Lipofectamine (Invitrogen). After a 24-hour incubation in a humidified incubator (5% CO2, 37°C), transfection efficiency, transfection toxicity, and percent of transfected cells were determined (Block-iT Alexa Fluor Red Fluorescent Control, dead cell stain ethidium homodimer-1, Nuclear stain Hoechst 33,342, Invitrogen). The growth medium was then replaced by basal medium without additives and responses to isoproterenol and GABA assessed in untransfected cells versus cells transiently overexpressing the GABABR or cells with GABABR knockdown using the ERK1/2 activation assay described below. Negative Stealth RNAi provided by the vendor served as negative control.
ERK1/2 Activation Assays12
This assay takes advantage of the finding that ELK-1 is a downstream effector of activated ERK1/2. Equal amounts of protein were incubated overnight with 15 mL of agarose hydrazide beads immobilized p44 of 42 (Cell Signaling Technology, Beverly, MA). Immune precipitates were washed 3 times in 100 mM Tris (pH 7.5), 1% Nonidet P-40, 2 mM sodium orthovanadate; once in 100 mM Tris (pH 7.5), 0.5 M lithium chloride; and once in kinase buffer (12.5 mM MOPS, pH 7.5, 12.5 mM b-glycerophosphate, 7.5 mM MgCl2). Proteins were incubated for 20 minutes at 30°C in a 30-mL kinase reaction containing 2 mg ELK-1 fusion protein (GST-ELK-1 codons 307–428; Cell Signaling), and 10 mM ATP. Proteins were separated by electrophoresis on a 12% SDS-polyacrylamide gel, transferred to nitrocellulose membranes, and probed with anti-phospho ELK (Ser383) antibody. After incubation with the secondary antibody, bands were observed by enhanced chemiluminescence detection. The density of p-ELK-1 and ERK1/2 protein bands was quantified by densitometry (4 densitometric readings per band using NIH Scion Image analysis software). The ratios of p-ELK-1/ERK were statistically analyzed by 1-way ANOVA and Tukey-Kramer multiple comparison test.
To provide in vivo evidence for a potential role of cAMP-mediated and GABA signaling in the regulation of human pancreatic cancer, slides from human tissue arrays (US Biomax, Rockville, Md) were used. Microarray PA242 contained sections of 24 human PDACS and 4 normal unrelated pancreatic tissues. Microarray PA241 contained sections from 6 additional PDACs with matched normal tissues 1.5 cm away from tumor. The sections were deparaffinized in xylene, washed with PBS (pH 7.4), and incubated with 3% hydrogen peroxide in 50% methanol for 20 minutes at room temperature. Incubations with protein block solution, primary and biotinylated secondary antibodies, and diaminobenzidine substrate were conducted with reagents provided by the universal Vectastain ABC kit (Vector Laboratories, Burlingame, Calif) according to instructions with the kit. Incubations with primary polyclonal antibodies (Chemicon Millipore, Billerica, MA) to mammalian GABA (1:1000), human norepinephrine (1:1000), or human PKARIIα (1:1000; BD Biosciences, San Jose, Calif), which recognizes the regulatory RIIα subunit when it has been released from the PKA subunit complex upon binding of cAMP,28 were conducted at 4°C overnight in a humid chamber. Tissue slides processed without exposure to primary antibodies served as negative controls and showed no detectable immunoreactivity. Hematoxylin was used as counterstain.
In accordance with our earlier publications,8, 11, 23 the beta-adrenergic agonist isoproterenol caused a highly significant (P < .001) increase in intracellular cAMP in the pancreatic duct epithelial cells and in both PDAC cell lines (Fig. 1). This response was completely abrogated (P < .001) by preincubation with GABA, which is an agonist for all GABA receptors (Fig. 1). Preincubation of the cells with the selective agonist for GABABRs baclofen similarly blocked the responses of all 3 cell lines to isoproterenol (P < .001; Fig. 1), suggesting involvement of the GABABR in the observed inhibition. Exposure of unstimulated pancreatic duct or PDAC cells to GABA or baclofen significantly (P < .001) reduced base levels of cAMP below that of control cells (Fig. 1).
We have previously shown that β-AR-induced cAMP activates the ERK1/2 cascade via transactivation of EGFR in a PKA-dependent manner in human pancreatic duct epithelial cells and that the resulting activation of ERK1/2 serves as a major mitogenic stimulus.11 In the current study we therefore used an ERK1/2 kinase reaction and Western blot in anti-p-ERK1/2 immunoprecipitates of p-ELK-1 protein phosphorylated by activated ERK1/2 to assess the modulating effects of GABABR on ispoproterenol-evoked mitogenic signaling in these cells. Our data show that isoproterenol caused a significant (P < .001) increase in p-ELK-1 protein (Fig. 2, lane 2), a response significantly (P < .001) reduced by preincubation with GABA (Fig. 2, lane 3). Preincubation with GABA failed to reduce isoproterenol-induction of p-ELK-1 in cells with GABABR1 knockdown by transient transfection with GABABR1 stealth RNAi (P < .001; Fig. 2, lane 4). Conversely, overexpression of the GABABR1 by transient transfection of GABABR1 cDNA reduced (P < .001) isoproterenol-induced p-ELK-1 below control levels in cells preincubated with GABA (Fig. 2, lane 5). The inhibitory effects of GABA in cells overexpressing GABABR1 were significantly (P < .001) greater than in cells without GABABR1 overexpression (compare lanes 5 and 3 in Fig. 2). Collectively, these findings strongly suggest that activation of GABABR may inhibit mitogenic signaling in response to β-AR stimulation in pancreatic duct epithelial cells and the cancers derived from them. This interpretation is supported by the results generated by BrdU incorporation assays that monitored modulation of DNA synthesis in pancreatic duct epithelial cells HPDE6-C7 and the 2 PDAC cell lines PANC-1 and BXPC-3. As Figure 3 shows, isoproterenol strongly (P < .001) induced DNA synthesis in all 3 cell lines. Preincubation with GABA or baclofen each significantly (P < .001) reduced the response below base levels observed in the controls. In addition, both agents significantly (P < .001) reduced base level DNA synthesis in unstimulated cells from each of the 3 cell lines (Fig. 3).
The high mortality rate of pancreatic cancer is caused by the aggressive behavior of this malignancy, resulting in extensive invasiveness and metastasis. We therefore assessed the effects of β-AR stimulation on the migration of PDAC cells and the potential modulation of this response by GABA and baclofen. As Figures 4 and 5 show, both PDAC cell lines demonstrated a highly significant (P < .001) increase in cell migration when treated with isoproterenol. In both cell lines this response was suppressed below control levels (P < .001) by preincubation with GABA or baclofen. In addition, the migration of cells not stimulated by isoproterenol was significantly (P < .001) reduced by each of these GABA-ergic agents (Figs. 4, 5). These data suggest, for the first time, that beta-adrenergic signaling increases the invasiveness and metastatic potential of PDAC and that activation of the GABABR by agonists may serve as a valuable tool to suppress this aggressive behavior.
Our in vitro data suggest an important role of cAMP/PKA-mediated signaling in the aggressive behavior of PDAC while indicating that activation of GABA signaling could be useful for the prevention and treatment of this malignancy. However, there is no published evidence to date that identifies adrenergic agonists in the tumor environment of human PDAC or indicates hyperactivity of cAMP-mediated signaling in these tumor cells. Similarly, there are no reports on GABA levels in human PDAC. Our immunohistochemical data generated in human PDAC tissue arrays show low but detectable levels of the physiological agonist for β-ARs, norepinephrine, in all normal tumor adjacent (Fig. 6A) and unrelated tissues, whereas norepinephrine was overexpressed in all tumor tissues (Fig. 6B). In addition, we observed a strong overexpression of PKARIIα that is released from the PKA subunit complex upon binding of cAMP in all PDAC samples (Fig. 7B) as compared with normal tumor adjacent (Fig. 7A) and unrelated pancreatic tissues. This finding suggests hyperactivity of cAMP/PKA signaling in PDAC. By contrast, positive immunoreactivity to GABA was below detectable levels under the conditions used in 29 of the 30 investigated PDAC samples (Fig. 8B), whereas each of the adjacent tumors (Fig. 8A) and unrelated normal pancreatic tissues showed strong positive immunoreactivity to GABA. These findings are suggestive of a pronounced underexpression of GABA in most human PDACs.
This is the first report that implicates GABABR as a target for the prevention and therapy of PDAC. Our data show that this receptor, which inhibits adenylyl cyclase by activating Gαii,24 acts as a powerful control for cAMP-dependent signaling, including the transactivated EGFR-mediated ERK1/2 cascade, that drive the proliferation and migration of PDAC. Unlike therapeutics that block only individual downstream components of this signaling network, agonists for GABABR act as the “physiological brake” to counterbalance the entire cAMP-driven stimulatory signaling network activated by cell surface receptors coupled to the stimulatory G-protein Gαs. As our current and published8, 9, 11, 23 data suggest, among the family of Gαs-coupled receptors that are counterbalanced by the GABABR, β-ARs play a prominent role in pancreatic carcinogenesis. Recent studies by other laboratories have also implicated β-ARs as important mediators of cancer growth and/or invasiveness in adenocarcinoma of the lungs,12, 29, 30 prostate,16 colon,15 stomach,19 breast,17 and ovary.20 In addition, it has been shown that GABA and baclofen inhibit β-AR-mediated migration of breast cancer cells17 and colon cancer cells.31 Accordingly, GABABR may be a promising target for the prevention and treatment of all of these cancers.
Intracellular cAMP can be increased by the activation of numerous Gαs-coupled receptors, by activators of adenylyl cyclase or cAMP, as well as phosphodiesterase inhibitors. All of these factors could contribute to pancreatic carcinogenesis. The more pronounced overexpression of PKARII than norepinephrine in PDACs detected by us suggest that cAMP stimulators in addition to β-AR agonists may contribute to this response.
The 3 known risk factors for PDAC, smoking, diabetes, and pancreatitis, disturb the balance between stimulatory β-adrenergic signaling and inhibitory GABABR signaling in the pancreas. A classic biological effect of nicotine is the enhanced release of the stress hormones epinephrine and norepinephrine,10, 32 resulting in increased stimulation of β-ARs. β-ARs in smokers are in addition stimulated by the nicotine-derived carcinogenic nitrosamine NNK that acts as a high-affinity agonist for these receptors.8, 29 Conversely, diabetes and pancreatitis are associated with the loss of pancreatic islet beta cells, the major source of inhibitory GABA in the pancreas.24 It stands to reason that a patient diagnosed with PDAC, a malignancy with an extremely poor prognosis, will suffer extreme anxiety. In turn, anxiety has been shown to increase the sensitivity of β-ARs to agonists.33 In addition, any kind of stress, including anxiety, causes the release of the physiological agonists for β-ARs, norepinephrine and epinephrine, from the sympathetic nervous system and the adrenal medulla, increasing their systemic levels.34 Increased systemic levels of norepinephrine have also been reported as a response to gastrointestinal cancer surgery.35, 36 While these stress responses are not unique to pancreatic cancer, they have the potential to contribute to the poor prognosis of this malignancy. The overexpression of norepinephrine in the cancer cells suggest up-regulation of the β-ARs that mediate its uptake.
GABA was below detectable levels in 29 of 30 human PDAC samples investigated, whereas each of the adjacent tumors and unrelated normal pancreatic tissue samples demonstrated pronounced positive immunoreactivity to GABA. In accordance with its function in the regulation of secretion by pancreatic duct epithelia,37 acinar cells,38 and islet cells,39 norepinephrine was expressed in all normal and cancerous pancreatic tissues. While positive immunoreactivity to norepinephrine was higher in cancer cells than in normal cells, PKARIIα that is released from the PKA subunit complex upon activation of PKA by cAMP28 demonstrated a more pronounced overexpression in the cancer cells of each PDAC sample. These findings suggest that the balance between stimulatory and inhibitory signaling in the human pancreatic cancer samples was significantly shifted in favor of stimulatory cAMP/PKA signaling. In addition, the marked underexpression of GABA in the human PDACs in conjunction with our in vitro data suggest GABA as a novel type of tumor suppressor for PDAC.
GABA and baclofen are FDA-approved for the treatment of anxiety. In addition, GABA is widely used as a dietary supplement to ease anxiety and promote sleep. Our data suggest that the use of these GABA-ergic agents as adjuvants to standard cancer therapy may have significant beneficial effects on the outcome of pancreatic cancer by inhibiting the proliferation and migration of cancer cells. In addition, the regular intake of GABA as a dietary supplement may significantly reduce the risk for the development of PDAC in individuals with diabetes and pancreatitis.
It is intriguing that GABA is a natural product of plants that mediates communication between plants and other organisms40 and participates in the plants' defenses against stress, wounding, and attack by parasites and microbes.41 Significant levels of GABA have been reported in grape berries and the Merlot wine made from them,42 in tomatoes,40 and in soy.43 These natural products also have significant cancer preventive activities.44, 45 While research into the mechanisms of action of these and other plant-derived cancer preventive agents have focused on their antioxidant effects and inhibition of carcinogen metabolism,45 our data suggest that inhibition of Gαs-mediated signaling by GABA may be a novel and hitherto unexplored mechanism of action of some plants with cancer preventive activity.
- 11The 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. J Cancer Res Clin Oncol. 2005; 131: 639–648., , .
- 29The tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone is a beta-adrenergic agonist and stimulates DNA synthesis in lung adenocarcinoma via beta-adrenergic receptor-mediated release of arachidonic acid. Cancer Res. 1999; 59: 4510–4515., , , .
- 38Localization of cAMP-dependent protein kinase subunits along the secretory pathway in pancreatic and parotid acinar cells and accumulation of the catalytic subunit in parotid secretory granules following beta-adrenergic stimulation. Eur J Cell Biol. 1990; 51: 76–84., .