Research Article

Gamma-aminoButyric Acid (GABA) Prevents the Induction ofNicotinic Receptor–Regulated Signaling by Chronic Ethanolin Pancreatic Cancer Cells and Normal Duct Epithelia

Mohammed H. Al-Wadei1, Hussein A.N. Al-Wadei1,2, and Hildegard M. Schuller1

AbstractPancreatic cancer has a high mortality rate and alcoholism is a risk factor independent of smoking. We

have shown that nicotinic acetylcholine receptors (nAChR) regulate pancreatic ductal epithelia and

pancreatic ductal adenocarcinoma (PDAC) cells in an autocrine fashion by stimulating their production

of the stress neurotransmitters noradrenaline and adrenaline that signal through b-adrenergic receptors

(b-AR). Our current study has investigated the modulation of this autocrine regulatory loop by chronic

ethanol and explored the potential prevention of these effects by g-amino butyric acid (GABA).

Using MTT assays, cell migration assays, Western blotting, immunoassays, and gene knockdown of

individual nAChRs in two PDAC cell lines and in immortalized human pancreatic duct epithelial cells, our

data show that treatment for seven days with ethanol induced the protein expression and sensitivity of

nAChRs a3, a5, and a7 resulting in increased production of noradrenaline and adrenaline, which drive

proliferation and migration via cyclic AMP (cAMP)-dependent signaling downstream of b-ARs. Treatment

with GABA prevented all of these responses to chronic ethanol, reducing cell proliferation and migration

below base levels in untreated cells.

Our findings suggest that alcoholism induces multiple cAMP-dependent PDAC stimulating signaling

pathways by upregulating the protein expression and sensitivity of nAChRs that regulate stress neurotrans-

mitter production. Moreover, our data identify GABA as a promising agent for the prevention of PDAC in

individuals at risk due to chronic alcohol consumption. Cancer Prev Res; 6(2); 139–48. �2012 AACR.

IntroductionPancreatic ductal adenocarcinoma (PDAC), which has

phenotypic and functional features of pancreatic duct epi-thelial cells, is one of themost aggressive neoplastic diseaseswith amortality rate near 100%withinone year of diagnosis(1). Smoking, chronic alcohol consumption, and pancre-atitis of any etiology, including alcoholism, are documen-ted risk factors for PDAC (2, 3). Smoking and drinking areoften correlated. However, a recent study has identified asignificant association of chronic alcohol intake with pan-creatic cancer mortality in never smokers, thus identifyingchronic alcohol consumption as a PDAC risk factor inde-pendent of smoking (4).

Numerous publications have focused on themechanismsof pancreatic cancer development and progression asso-ciated with exposure to nicotine, its nitrosated carcinogenicderivative 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone(NNK), and other tobacco-related carcinogens (5). Nicotine(6) and NNK (7, 8) are both high-affinity agonists fornicotinic acetylcholine receptors (nAChR), and this receptorfamily has been identified as an important regulator of cellproliferation, apoptosis, migration, and angiogenesis in themost common epithelial human cancers (9), includingcancer of the lung (10–13), colon (14), breast (15), oralcavity (8), stomach (16), and pancreas (15, 17–19). It wasinitially thought that cellular responses after treatment ofcancer cellswithnAChRagonistswere causedby intracellularsignaling pathways activated immediately downstream ofnAChRs (9). However, emerging research suggests that mostof these reported signaling events are in fact indirectresponses caused by the nAChR-mediated synthesis andrelease of neurotransmitters by cancer cells and the epitheliafrom which they arise (9). In accord with these findings,we have recently shown that PDAC and pancreatic ductepithelial cells also synthesize and release the stress neuro-transmitters adrenaline and noradrenaline in response toactivation of nAChRs a3, a5, and a7 resulting in increasedproliferation and migration via the activation of multiple

Authors' Affiliations: 1Experimental Oncology Laboratory, Department ofBiomedical and Diagnostic Sciences, College of Veterinary Medicine,University of Tennessee, Knoxville, Tennessee; and 2Sana'a University,Sana'a, Yemen

Corresponding Author: Hildegard M. Schuller, Experimental Oncol-ogy Laboratory, Department of Biomedical and Diagnostic Sciences,College of Veterinary Medicine, University of Tennessee, 2407 RiverDrive, Knoxville, TN 37996. Phone: 865-974-8217; Fax: 865-974-5616;E-mail: hmsch@utk.edu

doi: 10.1158/1940-6207.CAPR-12-0388

�2012 American Association for Cancer Research.

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cAMP-dependent signaling pathways downstream of b-ARs(18, 19).

It has been reported that exposure of immortalized pan-creatic duct epithelial cells to ethanol in vitro enhancedNNK-induced activation of ERK1/2 and cell proliferationin a cAMP-dependent manner (20), whereas investigationsin brain cells and recombinant nAChRs in oocytes haveshown that chronic in vitro exposure to ethanol modulatedthe number and sensitivity of nAChRs (21, 22). Thesefindings suggest that chronic exposure to ethanol mayenhance pancreatic cancer–promoting b-adrenergic path-ways by modulating the expression and sensitivity ofnAChRs. In the current experiments, we have thereforeinvestigated the effects of chronic ethanol on the proteinexpression and sensitivity of nAChRs a3, a5, and a7 inimmortalized pancreatic duct epithelial cells and 2 PDACcell lines. Moreover, we have tested the potential inhibitionof these responses by g-amino butyric acid (GABA).

Materials and MethodsChemicals, primers, antibodies, and assay kits

The MTT colorimetric assay kit was purchased fromSigma-Aldrich. The CytoSelect Cell Migration Assay kit waspurchased from Cell BioLabs, Inc. The 2-Cat ELISA Kit waspurchased from Rocky Mountain Diagnostics Incorpo-ration. The cAMP and protein kinase A (PKA) activity ELISAassays were purchased from Enzo Life Sciences.

ELISA kits for Akt, ERK1/2, and CREB (Invitrogen Cor-poration) are designed to detect pAKT at serine 473, pERK1/2 at threonine 202 and tyrosine 204 for ERK1, and threo-nine 185 and tyrosine 187 for ERK2, and pCREB at serine133, respectively. The CycLex c-Src Kinase assay (MBLInternational) is designed to detect p-Src at tyrosinekinase-substrate-1. All 4 assays use the chromogenic tetra-methylbenzidine substrate for signal detection.

Lipofectamine 2000 Reagent, stealth-1079 for theCHRNA3 gene, stealth-873 for the CHRNA5 gene,stealth-183 for the CHRNA7 gene, stealth RNAi NegativeControl Low GC Duplex, and Opti-MEM I-reduced serummedium 1� were purchased from Invitrogen Corporation.The primer used to interfere with thea3 subunitmRNAwassense, GCU CUU CCA UGA ACC UCA AGG ACU A andantisense, UAG UCC UUG AGG UUC AUG GAA GAG C.The primer used to interfere with thea5 subunitmRNAwassense, GGG AGC AAA GGA AAC AGA ACC GAC A andantisense, UGU CGG UUC UGU UUC CUU UGC UCC C.The primer used to interfere with thea7 subunitmRNAwassense, GGA AGC UUU ACA AGG AGC UGG UCA A andantisense, UUG ACC AGC UCC UUG UAA AGC UUC C.

The antibodies anti-rabbit and anti-mouse were pur-chased from Cell Signaling. The nAChR subunits a7 (56kDa), a3 (57 kDa), and a5 (53 kDa) antibodies as well asb-actin (42 kDa) were all purchased from Abcam. Nicotine[(�)Nicotine hydrogen tartrate salt, minimum 98% TLC)],GABA, and isobutylmethylxanthine (IBMX) were pur-chased from Sigma-Aldrich. Ethanol was purchased fromFisher Scientific International. The TE Buffer 1� was pur-chased from Promega Corporation. The lysis buffer used to

extract proteins along with Pierce ECL Western blottingsubstrate was purchased from Thermo Scientific.

Maintenance and chronic treatments of cultured cellsThe immortalized human pancreatic duct epithelial

cell line, HPDE6-C7, was kindly provided to us in theyear 2005 by Dr. Tsao (Division of Cellular and Molecu-lar Biology, Department of Pathology, Ontario CancerInstitute/Princess Margaret Hospital, University ofToronto, Toronto, Ontario, Canada). This cell line wasclonally established after transduction of the HPV16-E6E7 genes into primary cultures of pancreatic ductepithelial cells. The human PDAC cell lines Panc-1 (withactivating point mutation in K-ras) and BxPC-3 (withoutras mutation) were purchased in the year 2001 from theAmerican Type Culture Collection. We chose these par-ticular PDAC cell lines for the current study to ensureinclusion of cells with and without ras mutations andbecause our laboratory has generated substantial infor-mation regarding nAChR-mediated regulation of thesecell lines in vitro and in vivo (23–26). All 3 cell lineswere last authenticated at the beginning of this projectin the year 2010 by Research Animal Diagnostic Labo-ratory (RADIL, Columbia, MO) by species-specific PCRevaluation.

Cell lines Panc-1 and BxPC-3 were maintained inDulbecco’s modified Eagle’s medium and RPMI media,respectively, and were both supplemented with 10% FBS.HPDE6-C7 cells were maintained in keratinocyte serum-free medium (KSFM) supplemented with 25 mg/500 mLbovine pituitary extract (BPE) and 2.5 mg/500 mL EGF;GIBCO Invitrogen Corporation). All cell lines were grownwithout any antibiotics in an atmosphere of 5% CO2, 99%relative humidity, and 37�C.

Chronic treatment of cells in complete medium withethanol (17 mmol/L) and GABA (30 mmol/L) was for 7days. Fresh medium containing each drug was replacedevery 24 hours. In the untreated control cells, the mediumwas replaced every 24 hours. Treatment groups were asfollows:

(i) Control: cells maintained for 7 days in completemedium without drugs;

(ii) Ethanol: cells treated with ethanol for 7 days;(iii) Ethanol þ GABA: cells treated simultaneously for 7

days with ethanol and GABA;(iv) GABA: cells treated with GABA for 7 days.

The concentration (17 mmol/L) of ethanol used is theequivalent of the legal blood alcohol limit of 0.08% in theUnited States. The concentration of GABA (30 mmol/L) isthe equivalent of 21.7 mg/person/d and is within therecommended range of GABA as a nutritional supplement.

Protein analyses of nAChR subunits by Westernblotting

Theprotein expression of nAChR subunitsa3,a5, anda7was determined in cells chronically treated with ethanol,

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GABA, or the combination of both. Protein samples wereprepared using lysis buffer (RIPA Buffer 1�; Halt ProteaseInhibitor Single-Use Cocktail, EDTA-Free (100�; ThermoScientific); 10 mL of 100 mmol/L phenylmethysulfonyl-fluoride/mL RIPA; 10 mL of 100mmol/L Na3VO4/mL RIPA;10 mL of 1 mol/L NaF/mL RIPA). After heat denaturation,protein samples were electrophoresed using 12% SDS gels(Invitrogen) and were then blotted onto membranes. Themembranes were then blocked (5% nonfat dry milk solu-tion in 1� TBST) for 1 hour at room temperature. Mem-branes were then incubated overnight at 4�C with thefollowing primary antibodies: nAChR subunits a3, a5, anda7. The primary antibody b-actin was used as a loadingcontrol to ensure equal loading of proteins. All membraneswere then washed (0.5% Tween 20/TBS) and incubatedwith their respective fluorescent secondary antibodies for 2hours. Protein bands were then visualized with enhancedchemiluminescence reagent (Pierce ECL Western BlottingDetection Substrate). Following background subtraction,mean densities of 2 rectangular areas of standard size perband from 3 independent Western blots were determinedandmean values and SD (n¼ 6) of protein expression werecalculated.

Assessment of responsiveness to ethanol byimmunoassays for the detection of neurotransmittersTo test the hypothesis that chronic exposure to ethanol

modulates the responsiveness of nAChR-mediated neuro-transmitter production to ethanol itself, dose–responsecurves for ethanol were established in both unpretreatedand chronically pretreated cells. Unpretreated cells from all3 cell lines and cells pretreated for 7 days with 17 mmol/Lethanol were exposed to a range of ethanol concentrations(1 mmol/L, 50 mmol/L, 500 mmol/L, 1mmol/L, 17mmol/L,and 35 mmol/L) for 30 minutes before harvesting. EC50

values for ethanolwere then compared in unpretreatedwithethanol-pretreated cells. The culture media, containing thesecreted stress neurotransmitters noradrenaline and adren-alinewere then collected in 15mL test tubes. The cellswhichcontained synthesized intracellular catecholamines werelysed and harvested into 1.5 mL eppendorf tubes after aone time wash with warm 1� PBS. Total (secreted plusintracellular) stress neurotransmitters of 5 samples pertreatment group were analyzed using 2-Cat ELISA kitsfollowing the vendors’ recommendations. Absorbance ofsamples was read using an uQuant Bio-Tek InstrumentELISA reader at 450 nm primary wavelength with a 630nm reference wavelength.

Quantitative assessment of accumulated intracellularcAMP levelsChronic treatment groups for this assay were identical

to those of the above Western blotting experiment.On the sixth day of treatment, cells were washed twicewith warm 1� PBS then incubated for 30 minutes with1 mmol/L of the phosphodiesterase inhibitor IBMXto prevent enzymatic breakdown of the cAMP formed.Once the 30 minutes incubation was over, cells were

washed with 1� PBS twice then treated with 17 mmol/Lethanol for 24 hours. The cells were then lysed andharvested into 1.5 mL Eppendorf tubes following thecAMP ELISA kit’s instructions. Quantitative analyses ofcAMP levels from 5 samples per treatment group wereconducted using the cAMP ELISA kit following thevendor’s recommendations. Absorbance of samples wasread using an uQuant Bio-Tek Instrument ELISA reader at405 nm primary wavelength with a 550 nm referencewavelength.

Quantitative assessment of PKA activation andphosphorylation levels of signaling proteins byimmunoassays

Following identical chronic treatments, the cells werelysed and harvested into 1.5 mL Eppendorf tubes followingELISA kit’s instructions. Quantitative analyses of PKA activ-ity, Akt, CREB, Src, and ERK1/2 phosphorylation from5 samples per treatment group were conducted usingPKA activity, Akt, CREB, c-Src, and ERK1/2 ELISA kits,respectively, following the vendors’ recommendations.Absorbance of samples was read using an uQuant Bio-TekInstrument ELISA reader at 450 nm primary wavelengthwith a 630 nm reference wavelength.

Gene knockdown of the a3, a5, and a7-nAChRsAll 3 cell lines were grown for 24 hours in their respective

complete media. After which they were switched to Opti-MEM Imedia and were divided into several groups. Groups1 and2 fromeach cell linewere left untreated inOpti-MEMImedia for 24 hours. Group 3 was transfected for 24 hourswith stealth RNAi Negative Control Low GC Duplex.Groups 4 and 5 were transfected with either stealth-183,1079, or 873 for the CHRNA7, 3, and 5 genes, respectively,for 24 hours in Opti-MEM I media. Once the 24-hourtransfection was complete, all cells were switched into theirrespective complete media. Groups 1, 3, and 4 were leftuntreated for 7 days in complete media, whereas groups 2and 5 were treated with 17 mmol/L ethanol for 7 daysin complete media. All transfections were transient andwere done using Lipofectamine 2000 reagent followingthe instructions of the manufacturer. Quantification of theexpression of a3, a5, and a7 nAChRs for all groups wereassessed via Western blot analyses at the end of the 7 dayschronic exposure to ethanol for groups 2 and 5. Similartreatment groups, to the one in this experiment, were usedto assess cell proliferation and migration of all 3 cell linesusing MTT and cell migration assays following the proce-dure outlined below.

Assessment of cell proliferation by MTT assayAll 3 cell lineswere seeded into6-well plates at adensity of

20,000 cells per well (n ¼ 5) in their respective completemedia. Cells were then left untreated or treated with etha-nol, GABA, or both for 7 days. The MTT colorimetric assaywas then used for the assessment of cell proliferationfollowing instructions provided by the vendor. Absorbanceof samples was read using an uQuant Bio-Tek Instrument

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ELISA reader at a primary and reference wavelengths of 570nm and 650 nm, respectively.

Assessment of cell migration by CytoSelect cellmigration assay

The 3 cell lines were seeded onto the top chamber ofpolycarbonate membrane filter inserts (8 mmol/L pore size)in 6-well plates at a density of 20,000 cells per well in theirrespective complete media. Using identical treatmentgroups as for the MTT assay (above), a cell migration assaykit was then used to assess themigratory ability of these cellsfollowing the vendor’s instructions. Optical density of cellsamples was read at 560 nm using an uQuant Bio-TekInstrument ELISA reader.

Statistical analysisUsing GraphPad Instat software (GraphPad), all data

presented in the figures as bar graphs were analyzed forstatistically significant differences among treatment groupsby one-way ANOVA and Tukey–Kramer multiple compar-ison tests when the data followed a normal distribution.Data that did not pass normality tests were tested forsignificant differences by nonparametric Kruskal–WallisANOVA followed by Dunn multiple comparison test.GraphPad Prism 5 software (GraphPad) was used to estab-lish sigmoidal dose–response curves and to calculate EC50

values by nonlinear regression analysis. Data of the immu-noassays are expressed as mean � SD of 5 samples pertreatment group. Using NIH ImageJ, 2 densitometric deter-minations per band were assessed from 3 independentWestern blots (n ¼ 6) per protein and expressed as meanvalues and SDs.

ResultsEffects of chronic ethanol and GABA on the proteinexpression of nAChRs

We have previously shown that nAChRs expressing thesubunits a3, a5, and a7 regulate the proliferation ofPDAC cell lines Panc-1 and BxPC-3 as well as immortal-ized pancreatic duct epithelial cells HPDE6-C7 by acti-vating an autocrine stress neurotransmitter loop (18). Toinvestigate a potential modulation of this regulatory loopby chronic ethanol, we tested the effects of chronicethanol and GABA on the protein expression of thesenAChRs. Our data show that chronic exposure to ethanolalone significantly induced (P < 0.0001) protein expres-sion of nAChR subunits a3 (4.5-fold), a5 (5-fold), anda7 (5-fold). Simultaneous treatment of cells with GABAsignificantly (P < 0.0001) reduced these effects of ethanol(Figure 1).

Concentration-dependent effects of chronic ethanolon total adrenaline and noradrenaline

To assess the functional significance of the observedchanges in chronic ethanol-induced protein expression ofnAChRs on their effectors, we measured total (intracellularplus secreted) levels of the stress neurotransmitters nor-adrenaline and adrenaline in 7-day ethanol pretreated

versus nonpretreated cells exposed to a range of ethanolconcentrations (1–35 mmol/L). As shown in Fig. 2, theconcentration-dependent increases in total levels ofthe stress neurotransmitters noradrenaline and adrenalinewere significantly (P < 0.0001) enhanced by a 7-day pre-exposure to ethanol in all 3 cell lines and EC50 values ofethanol for these increases were significantly (P < 0.0001)reduced (Fig. 2).

Effects of chronic ethanol and GABA on intracellularaccumulation of cAMP and PKA activation

We have previously shown that nAChRs expressing sub-units a3, a5, and a7 jointly regulate the synthesis andrelease of the stress neurotransmitters noradrenaline andadrenaline in PDAC cells andpancreatic duct epithelial cells(18). In turn, noradrenaline and adrenaline are b-AR ago-nists (27, 28) that increase intracellular cAMP levels viaactivation of the stimulatory G-protein Gas of these recep-tors, resulting in the activation of PKA. (18). We thereforemeasured intracellular cAMP levels and activated PKA in thecurrent experiments to assess the effects of chronic ethanolon these b-AR effectors. As Fig. 3 shows, chronic treatmentof cells with ethanol significantly (P < 0.0001) increasedintracellular cAMP levels (3, 4-fold) in the 3 investigated celllines while also significantly (p < 0.0001) increasing thelevels of activated PKA (2.8–3.4 fold). Simultaneous expo-sure of these cells to GABA during the 7-day ethanoltreatment period significantly (P < 0.0001) reduced bothof these responses to ethanol (Fig. 3). In addition, chronicexposure of cells to GABA alone significantly (P < 0.0001)suppressed base levels of cAMP and activated PKA in cellsnot pretreated with ethanol (Fig. 3).

Effects of chronic ethanol in the presence and absenceof GABA on the phosphorylation of multiple signalingproteins

We have previously shown that activation of cAMP-dependent PKA results in the phosphorylation of multiplesignaling proteins involved in the regulation of prolifera-tion, angiogenesis, metastatic potential, and apoptosis inPDAC cells (18, 24–26).We therefore assessed the potentialmodulation in phosphorylation levels of these signalingproteins by chronic ethanol in the presence and absence ofsimultaneous treatment with GABA. Our data show thatchronic exposure of normal pancreatic duct epithelia andboth PDAC cell lines to ethanol significantly induced (P <0.0001) phosphorylation of ERK, CREB, Akt, and Src (Fig.4). In analogy to our findings with cAMP and PKA, simul-taneous exposure of the cells to GABA during the ethanoltreatment period significantly (P < 0.0001) reduced each ofthese responses (Fig. 4).

Effects of nAChR gene knockdown on ethanol-mediated regulationof cell proliferationandmigration

To determine whether the protein induction of nAChRsa3, a5, and a7 by chronic ethanol shown in Fig. 1 wereassociated with stimulation of proliferation and migration,we investigated the effects of gene knockdown of each of

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these nAChRs in the presence and absence of chronicethanol. At the end of the 7-day ethanol pretreatment, thetransient gene knockdown was still detectable by Westernblot analyses (Fig. 5). Gene knockdown of each of the 3receptor subunits caused a significant reduction (P < 0.001)in base level cell proliferation (Fig. 6A) and migration (Fig.6B) in all 3 cell lines. Chronic exposure to ethanol signif-icantly (P < 0.001) increased cell proliferation (2.7-fold)and migration (3-fold) in each of the investigated cell lines(Fig. 6A and B). Both of these responses to chronic ethanolwere significantly (P < 0.001) reduced by gene knockdownof nAChRs a3, a5, or a7 in all 3 cell lines (Fig. 6A and B).

Effects of chronic ethanol in the presence and absenceof GABA on cell proliferation and migrationUsing an ethanol concentration (17 mmol/L) equivalent

to the legal blood alcohol limit of 0.08% in the UnitedStates, we found that all 3 investigated cell lines respondedwithmore than 2-fold increases (P < 0.0001) in the numberof viable cells when exposed for 7 days to ethanol (Fig. 6C).This response was significantly (P < 0.001) inhibited bysimultaneous exposure of these cells to GABA (Fig. 6C) at aconcentration (30 mmol/L) within the recommendeddaily dose of GABA as a nutritional supplement. In addi-

tion, 7-day exposure to GABA significantly (P < 0.0001)decreased the number of viable cells in the 3 cell lines notpretreated with ethanol (Fig. 6C). These findings weremirror-imaged by the results of cell migration assays thatused identical treatment groups (Fig. 6D), with more than2.5-fold increase inmigrated cells after 7-day treatmentwithethanol (P<0.0001) and significant (P<0.0001) inhibitionof this effect by GABA.

DiscussionSmoking and chronic alcohol consumption are often

correlated and represent established risk factors for pancre-atic cancer (1–3). Heavy alcohol consumption is thought tofacilitate pancreatic cancer development in smokers byincreasing the risk for diabetes and chronic pancreatitis,which are independent risk factors for this malignancy (3).However, a recent study has identified chronic consump-tion ofmoderate amounts of alcohol in never smokers as anindependent risk factor for pancreatic cancer (4). Themechanisms responsible for this effect of ethanol on pan-creatic cancer are far from understood.

It has been shown that nicotine addiction and alcoholdependence are highly correlated (22, 29) and that chronicnicotine-induced upregulation of nAChR proteins in brain

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Figure 1. Western blots assessing protein expression of nAChR subunits in cells treated with 17 mmol/L ethanol, 30 mmol/L GABA, or the combination of thetwo for 7 days. Protein b-actin was used as a loading control (A). The columns in (B) represent mean and � SD of 2 mean density readings per band from3 independent Western blots (n ¼ 6).

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cells is significantly enhanced by simultaneous exposure tochronic ethanol (22). While nAChRs were initially thoughtto be uniquely expressed in the nervous system,more recentstudies have identified these receptors in a majority ofmammalian cells, including epithelia (30). In accord with

these reports, we have recently shown that the stress neuro-transmitters noradrenaline and adrenaline are synthesizedand released by normal pancreatic duct epithelia and PDACcells in vitro when nicotine binds to nAChRs expressing thesubunits a3,a5, or a7 in these cells (18). In accord with theestablished function of noradrenaline and adrenaline asb-adrenergic receptor agonists (27, 28), a single dose ofnicotine thus increased cAMP and activated PKA, causingthe phosphorylation of multiple signaling proteins (p-ERK,p-CREB, p-Akt, p-Src) frequently overexpressed in pancre-atic cancer while also stimulating cell proliferation (18).Wehave also shown that exposure of immortalized pancreaticduct epithelial cells to a single dose of ethanol at a concen-tration (17 mmol/L), equivalent to the legal blood alcohollimit (0.08%) in the United States, stimulates cell prolifer-ation by increasing intracellular cAMP levels, resulting in asignificant induction of activated PKA and PKA-dependentphosphorylation of CREB and ERK1/2 (20). Our currentstudy shows, for the first time, that chronic exposure to thismoderate ethanol concentration significantly induces theprotein expression of nAChRs a3, a5, and a7 in pancreaticduct epithelial and PDAC cells. The observed significantincreases in total noradrenaline and adrenaline after chron-ic ethanol and associated significant decrease in EC50 valuesof ethanol required to stimulate the production of theseneurotransmitters indicate functional sensitization ofnAChRsa3,a5, anda7. The resulting significant inductionsof activated PKA, CREB, ERK, Akt and Src after chronicethanol alone additionally identifies these signalingproteins as the driving forces of chronic ethanol-inducedcell proliferation and migration in the 3 investigated celllines.

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Figure 2. Total (secreted and intracellular) adrenaline (A–C) and noradrenaline (D–F) levels in cells treated with single doses of ethanol from 1 mmol/L to35mmol/L for 30minutes or pretreatedwith 17mmol/L ethanol for 7 days and then exposed to identical concentrations of ethanol. Data points aremean�SDfrom 5 samples per treatment group.

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Figure 3. ELISA assays showing cAMP levels (A) and PKA activity (B) incells treated with 17 mmol/L ethanol, 30 mmol/L GABA, or thecombination of the two for 7 days. Data points are mean � SD from 5samples per treatment group.

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The mechanisms of how chronic ethanol increased theprotein expression and sensitivity of nAChRs in the currentexperiment remain to be elucidated. It is widely acceptedthat changes in nAChR number and sensitivity in response

to chronic nAChR agonists are mediated by rapid posttran-scriptional mechanisms without concomitant changes inmRNA levels (31). Ethanol is not a ligand for nAChRs butchronic exposure to ethanol modulates the responsiveness

Figure 5. Western blots assessingthe effects of gene knockdown onprotein expression of nAChRsubunits in the presence andabsence of 7 days ethanol. Proteinb-actin was used as a loading control(A). The columns in figure (B)represent mean � SD of 2 meandensity readings per band from 3independent Western blot analyses(n ¼ 6).

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Figure 4. ELISA assays showingphosphorylation of CREB and ERK(A) and of Src and Akt (B) in cellstreated with 17 mmol/L ethanol, 30mmol/L GABA, or the combination ofthe two for 7 days. Data points aremean � SD from 5 samples pertreatment group.

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GABA Prevents Ethanol Promotion of Pancreatic Cancer

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of these receptors to agonist by increasing the number ofagonist-binding sites (22). Epithelial cells produce thephysiologic nAChR agonist acetylcholine (30). According-ly, chronic ethanol increased responsiveness of nAChRsa3,a5, anda7 to this physiologic agonist in the current study. Ithas been suggested that ethanol-induced activation of PKCis involved in the increase in nAChR-binding sites observedin PC12 cells after chronic exposure to ethanol (22). Alter-natively, it has been shown that chronic ethanol increasedthe gene expression for enzymes that synthesize noradren-aline and adrenaline (32), a mechanism that may addition-ally have contributed to the ethanol-induced increase instress neurotransmitter production in the current experi-ments. In turn, this may have triggered an increase in theposttranscriptional assembly andmaturation of nAChRs, asthese events are induced by increases in intracellular cAMP(31). Further studies are needed to obtain a more completeunderstanding of the complex mechanisms involved inethanol-induced modulation of nAChRs in pancreatic can-cer cells.

The importance of the observed in vitro effects ofchronic ethanol on the b-adrenergic signaling cascade forPDAC development is supported by findings that chronictreatment with ethanol and NNK induced the develop-ment of PDAC in hamsters (33). These tumors overex-pressed nAChR proteins as well as p-CREB and p-ERK and

were prevented by treatment with the b-blocker propran-olol (34). While our findings are in accord with thereports from other laboratories that alcohol activatessignaling proteins such as ERK, JNK, PKC, and Stat3 inliver cells (35–39), they suggest that these proteins aredownstream effectors of noradrenaline and adrenaline-induced b-adrenergic signaling that is regulated bynAChRs.

Our findings suggest that changes in nAChR expressionand function in the brain that are associated with alcoholdependence are also induced by chronic ethanol in PDACcells and pancreatic duct epithelial cells where they coop-erate to stimulate proliferation and migration, thus facil-itating the development and progression of PDAC. More-over, the strong inhibitory effects of GABA on theobserved cellular responses to chronic ethanol identifythis neurotransmitter, which is widely used as a nutri-tional supplement, as a promising agent for the preven-tion of PDAC in individuals at increased risk due tochronic consumption of alcohol. In light of the fact thatthe GABA-B receptor agonist baclofen has been identifiedas an effective therapeutic for alcohol dependence(40, 41), nutritional supplementation with GABA maynot only have protective effects against PDAC develop-ment, but simultaneously reduce the craving of alcoholwithdrawal in dependent patients.

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Figure 6. Effects of gene knockdown of nAChR subunits on cell proliferation (A) and migration (B) in the presence and absence of chronic ethanol. Cellproliferation (C) and migration (D) assays showing stimulation of both proliferation and migration upon ethanol exposure and a reversal of theseeffects upon exposure to GABA. The columns represent mean � SD of 5 samples per treatment group.

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b-adrenergic signaling has emerged as an importantregulatory cascade in some of the most common humanmalignancies, including cancer of the breast (42–44),colon (14, 45), prostate (46), stomach (16), ovary(47), pancreas (18, 24, 34, 48), and non–small cell lungcancer (49). In fact, several recent studies have shownimproved clinical outcomes in patients with breastcancer who received b-blocker treatment for incidentalcardiovascular disease while undergoing cancer therapy(42, 43). Moreover, we have reported that the b-blockerpropranolol prevents experimentally induced PDAC andnon–small cell lung carcinoma in hamsters (34, 50). Thesignificant activation of b-adrenergic signaling by chronicethanol via sensitization of nAChRs upstream of stressneurotransmitter production observed in the currentexperiments suggests that chronic consumption of alco-hol may also promote the development and progressionof non-PDAC cancers that are under b-adrenergic controland that GABA may have preventive effects on thesecancers as well.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: H.M. SchullerAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): M.H. Al-WadeiAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): M.H. Al-WadeiWriting, review, and/or revision of themanuscript:M.H. Al-Wadei, H.A.N. Al-Wadei, H.M. SchullerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, H.A.N. Al-Wadei constructing databases): M.H. Al-WadeiStudy supervision: H.A.N. Al-Wadei, H.M. Schuller

Grant SupportThis study was supported by the National Cancer Institute grants

R01CA042829 and RO1CA130888.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 12, 2012; revised November 12, 2012; acceptedNovember 24, 2012; published OnlineFirst December 4, 2012.

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2013;6:139-148. Published OnlineFirst December 4, 2012.Cancer Prev Res   Mohammed H. Al-Wadei, Hussein A.N. Al-Wadei and Hildegard M. Schuller  Pancreatic Cancer Cells and Normal Duct Epithelia

Regulated Signaling by Chronic Ethanol in−Nicotinic Receptor Gamma-amino Butyric Acid (GABA) Prevents the Induction of

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