Neuropeptide Y inhibits cholangiocarcinoma cell growth and invasion

Sharon DeMorrow,2,4* Paolo Onori,7* Julie Venter,4 Pietro Invernizzi,6 Gabriel Frampton,4

Mellanie White,4 Antonio Franchitto,5 Shelley Kopriva,4 Francesca Bernuzzi,6 Heather Francis,3

Monique Coufal,3 Shannon Glaser,2,4 Giammarco Fava,8 Fanyin Meng,3 Domenico Alvaro,9

Guido Carpino,10 Eugenio Gaudio,5 and Gianfranco Alpini1,2,4

1Research, Central Texas Veterans Health Care System; 2Scott & White Digestive Disease Research Center, Scott & White,3Division of Research and Education, Scott & White; 4Department of Medicine, Division Gastroenterology, Texas A&MHealth Science Center, College of Medicine, Temple, Texas; 5Department of Human Anatomy, University of Rome “LaSapienza,” Rome, Italy; 6Center for Autoimmune Liver Diseases, Division of Internal Medicine, Istituto di Ricovero eCura a Carattere Scientifico (IRCCS), Istituto Clinico Humanitas, Rozzano, Milan, Italy; 7Experimental Medicine,University of L’Aquila, L’Aquila, Italy; 8Division of Gastroenterology, “Ospedali Riuniti,” Ancona, Italy;9Gastroenterology, Sapienza University of Rome, Italy; and 10Department of Health Science, University of Rome “ForoItalico,” Rome, Italy

Submitted 2 September 2010; accepted in final form 22 January 2011

DeMorrow S, Onori P, Venter J, Invernizzi P, Frampton G, WhiteM, Franchitto A, Kopriva S, Bernuzzi F, Francis H, Coufal M, Glaser S,Fava G, Meng F, Alvaro D, Carpino G, Gaudio E, Alpini G. Neuropep-tide Y inhibits cholangiocarcinoma cell growth and invasion. Am J PhysiolCell Physiol 300: C1078–C1089, 2011. First published January 26, 2011;doi:10.1152/ajpcell.00358.2010.—No information exists on the role ofneuropeptide Y (NPY) in cholangiocarcinoma growth. Therefore, weevaluated the expression and secretion of NPY and its subsequenteffects on cholangiocarcinoma growth and invasion. Cholangiocarci-noma cell lines and nonmalignant cholangiocytes were used to assessNPY mRNA expression and protein secretion. NPY expression wasassessed by immunohistochemistry in human liver biopsies. Cellproliferation and migration were evaluated in vitro by MTS assaysand matrigel invasion chambers, respectively, after treatment withNPY or a neutralizing NPY antibody. The effect of NPY or NPYdepletion on tumor growth was assessed in vivo after treatment withNPY or the neutralizing NPY antibody in a xenograft model ofcholangiocarcinoma. NPY secretion was upregulated in cholangiocar-cinoma compared with normal cholangiocytes. Administration ofexogenous NPY decreased proliferation and cell invasion in allcholangiocarcinoma cell lines studied and reduced tumor cell growthin vivo. In vitro, the effects of NPY on proliferation were blocked byspecific inhibitors for NPY receptor Y2, but not Y1 or Y5, and wereassociated with an increase in intracellular D-myo-inositol 1,4,5-trisphosphate and PKC� activation. Blocking of NPY activity using aneutralizing antibody promoted cholangiocarcinoma growth in vitroand in vivo and increased the invasiveness of cholangiocarcinoma invitro. Increased NPY immunoreactivity in human tumor tissue oc-curred predominantly in the center of the tumor, with less expressiontoward the invasion front of the tumor. We demonstrated that NPYexpression is upregulated in cholangiocarcinoma, which exerts localcontrol on tumor cell proliferation and invasion. Modulation of NPYsecretion may be important for the management of cholangiocarci-noma.

migration; microenvironment; cholangiocyte; protein kinase C

CHOLANGIOCARCINOMA IS A DEVASTATING tumor that is relativelyresistant to treatment with chemotherapy or radiation therapy

(6, 57). Cholangiocarcinoma arises from the neoplastic trans-formation of the epithelial cells (i.e., cholangiocytes) that linethe intra- and extrahepatic bile ducts (6, 57). Typically, cholan-giocarcinomas are adenocarcinomas and have a poor prognosisand limited treatment options. This is due, at least in part, to thelate presentation of symptoms and the relative resistance tocurrent treatment options (57, 60). The incidence of this canceris increasing in Western societies, and the mortality rates of theintrahepatic cancer are increasing (57). Therefore, research intothe mechanisms by which cholangiocarcinoma growth anddisease progression are regulated is imperative in an attempt todesign more effective treatment options for this cancer.

We have previously demonstrated that cholangiocarcinomasecretes factors capable of regulating its own proliferation (2,8). For example, cholangiocarcinoma synthesizes and secreteshigher amounts of serotonin (2) and dopamine (8), which canbe detected in bile (but not serum) of patients with cholangio-carcinoma (2, 8). Both of these neuroendocrine modulatorsexert growth-promoting effects on cholangiocarcinoma (2, 8),and blocking their synthesis slowed the rate of cholangiocar-cinoma tumor growth in vitro and in an in vivo xenograftmodel of cholangiocarcinoma.

Neuropeptide Y (NPY) is a neurotransmitter, mainly foundin the brain but also present in neurons throughout the gastro-intestinal tract, around the walls (tunica adventitia and tunicamedia) of hepatic vessels, and in high concentrations in thebiliary tree (25, 32). Other studies have shown that immuno-histochemical NPY reactivity is present not only in intrahepaticnerve fibers and ganglion cells but also in cholangiocytes (15).Limited information exists regarding the role of NPY in theregulation of biliary function. NPY has been shown to play arole in the neural control of biliary motility and secretion (14,53). NPY has inhibitory effects on cancer cell lines of variousorigins (51), but nothing is known about the effects of NPY oncholangiocarcinoma growth.

NPY exerts its many functions through six main receptorsubtypes (Y1 through Y6) (7). These receptors are a class of Gprotein-coupled receptors that can either inhibit 3=-5=-cyclicadenosine monophosphate (cAMP) synthesis (22, 35) or in-crease D-myo-inositol 1,4,5-trisphosphate (IP3)/Ca2�-signaling(1, 39). We have previously shown that both cAMP- andIP3/Ca2�-signaling modulate cholangiocarcinoma growth (3,

* S. DeMorrow and P. Onori contributed equally to this work.Address for reprint requests and other correspondence: G. Alpini, Texas

A&M Health Science Center College of Medicine, 702 SW H. K. DodgenLoop, Temple, TX, 76504 (e-mail: galpini@tamu.edu or galpini@medicine.tamhsc.edu).

Am J Physiol Cell Physiol 300: C1078–C1089, 2011.First published January 26, 2011; doi:10.1152/ajpcell.00358.2010.

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27). For example, while both the �2-adrenergic receptor ago-nist, UK14,304, and secretin inhibit cholangiocarcinomagrowth by cAMP-dependent mechanism (28, 44), other mole-cules such as tauroursodeoxycholate, the H3 histamine recep-tor agonist, RAMH, and gastrin inhibit cholangiocarcinomaproliferation by activation of the IP3/Ca2�-dependent PKC�isoform (3, 19, 27). Furthermore, the neurotransmitter, �-ami-nobutyric acid, inhibits the growth of biliary cancer by activa-tion of both cAMP- and IP3/Ca2�-dependent signaling (17).

Thus, the aims of our studies were to 1) assess the expres-sion of NPY and the NPY receptors in normal liver andcholangiocarcinoma tissue samples and nonmalignant andcholangiocarcinoma cell lines; 2) determine the effects of NPYtreatment on cholangiocarcinoma cell growth and elucidate theintracellular mechanism by which this occurs; and 3) evaluatethe effects of NPY depletion on cholangiocarcinoma cellgrowth in vitro.

MATERIALS AND METHODS

Materials

Reagents were purchased from Sigma Chemical (St. Louis, MO)unless otherwise indicated. The nuclear dye 4,6-diamidino-2-phe-nylindole (DAPI) was obtained from Molecular Probes (Eugene, OR).The antibody against proliferating cellular nuclear antigen (PCNA)was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Theantibodies recognizing the different subtypes of NPY receptors (Y1 toY6) were purchased from Santa Cruz Biotechnology. The affinity-purified goat polyclonal antibody (A-17, against Y1) was raisedagainst a peptide mapping near the NH2 terminus of Y1 of humanorigin. The affinity-purified goat polyclonal antibody (L-17, againstY2) was raised against a peptide mapping near the COOH terminus ofY2 of human origin. The synthetic peptide, corresponding to NH2-

terminal amino acids 1-14 of Y3, was purchased from Tocris Biosci-ences (Ellisville, MO). The affinity-purified goat polyclonal antibody(C-20, against Y4) was raised against a peptide mapping at the COOHterminus of Y4 of human origin. The affinity-purified goat polyclonalantibody (N-20, against Y5) was raised against a peptide mappingwithin an extracellular domain of Y5 of human origin. The affinity-purified goat polyclonal antibody (P-20, against Y6) was raisedagainst a peptide mapping within an internal region of Y6 of mouseorigin. All the purchased antibodies are recommended for the detec-tion of all NPY receptor subtypes of mouse, rat, and human origin byimmunoblots and immunofluorescence. The anti-NPY antibody wasdeveloped in rabbits using synthetic NPY (porcine) conjugated tokeyhole limpet hemocyanin as the immunogen (Sigma Chemical).The highly selective and potent antagonist of Y1, BVD 10 (4), waspurchased from Tocris Biosciences. The potent, selective, and com-petitive nonpeptide antagonist for Y2, BIIE 0246 (12), was purchasedfrom Tocris Biosciences. The selective, nonpeptide Y5 antagonist,CGP 71683 hydrochloride (13), was purchased from Tocris Biosci-ences. Specific inhibitors for Y3, Y4, and Y6 are not commerciallyavailable. The radioimmunoassay (RIA) kits for the measurement ofintracellular cAMP ([125I] Biotrak Assay System, RPA509) and IP3

([3H] Biotrak Assay System, TRK1000) levels were purchased fromGE Healthcare (Piscataway, NJ).

Cell Lines

The in vitro experiments were performed in six human cholangio-carcinoma cell lines (Mz-ChA-1, HuH28, HuCC-T1, CCLP1, SG231,and TFK-1) with different origins. Mz-ChA-1 cells, from humangallbladder (29), were a gift from Dr. G. Fitz (University of TexasSouthwestern Medical Center, Dallas, TX). HuH28 cells, from humanintrahepatic bile duct (31), and TFK-1 cells, from extrahepatic chol-

angiocarcinoma (52), were acquired from Cancer Cell Repository,Tohoku University (Tohoku, Japan). These cells were maintained atstandard conditions as previously described (10, 27). CCLP-1 (56),HuCC-T1 (40), and SG231 (58) (from intrahepatic bile ducts) were akind gift from Dr. A. J. Demetris (University of Pittsburgh, Pittsburgh,PA) and were cultured as previously described (40, 56, 58). Thehuman immortalized, nonmalignant cholangiocyte cell line, H69(from Dr. G. J. Gores, Mayo Clinic, Rochester, MN), was cultured aspreviously described (21). Human intrahepatic biliary epithelial cells(HIBECs) were purchased from Sciencell (Carlsbad, CA) and culturedas previously described (8).

Real-Time PCR for NPY and Y Receptors

The mRNA expression of NPY and its receptors was assessed inthe selected cell lines by real-time PCR (2, 10) using commerciallyavailable primers against NPY (accession no. NM_000905; referenceposition �451; expected product size 93 bp), Y1 (accession no.NM_000909; reference position �1,016; expected product size 172bp), Y2 (accession no. NM_000910; reference position �533; ex-pected product size 164 bp), Y3 (accession no. NM_003467; refer-ence position �192; expected product size 150 bp), Y4 (accession no.NM_005972; reference position �440; expected product size 161 bp),Y5 (accession no. NM_006174; reference position �312; expectedproduct size 116 bp), and Y6 (accession no. NR_002713; referenceposition �480; expected product size 174 bp; SA Bioscience, Fred-erick, MD). A ��CT analysis was performed (34) using normalcholangiocytes as the control sample and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) as the housekeeping gene. Data are ex-pressed as relative mRNA levels � SE of gene expression-to-GAPDHratio (n � 3).

NPY Secretion

All cell lines (H69, HIBEC, Mz-ChA-1, HuH28, HuCCT-1, SG231,TFK-1, and CCLP-1) were trypsinized, and the resulting cell pellet wasresuspended in 1� Hanks’ buffered saline buffer (1 � 107 cells/ml).Cells were incubated for 6 h at 37°C, and the amount of NPY releasedinto the media was assayed using a commercially available NPY enzyme-linked immunoassay (EIA) kit (Bachem Americas; Torrance, CA) ac-cording to the manufacturer’s instructions. NPY secretion from each cellline was assayed in triplicate, and each data point from nonmalignantcells or cholangiocarcinoma cells was collated and plotted on a scatterplot.

In parallel, NPY secretion was assessed in serum and bile samplesobtained from cholangiocarcinoma patients and age-matched controlsas described previously (2, 8) by an NPY EIA kit (Bachem Americas).The human sera and bile samples were obtained from an unidentifiedtissue bank from the laboratory of P. Invernizzi (coauthor of thisarticle). The de-identified samples were analyzed in a coded fashion inthe laboratory of Dr. Invernizzi. The human liver samples wereobtained from a tissue bank from the laboratory of E. Gaudio (coau-thor of this article). The samples were analyzed in a coded fashion inthe laboratory of Dr. Gaudio by three board-certified pathologists in ablinded fashion. Written informed consent was obtained from allpatients included in the study.

Cholangiocarcinoma Tissue Array Analysis

NPY immunoreactivity was assessed in commercially availableAccumax tissue arrays (Isu Abxis, Seoul, Korea) by immunohisto-chemistry as previously described (2, 8). The tissue arrays contain 48well-characterized cholangiocarcinoma biopsy samples from a varietyof tumor differentiation grades as well as four control liver biopsysamples. Semiquantitative analysis was performed by three indepen-dent board-certified pathologists, in a blind fashion, using the follow-ing parameters. Staining intensity was assessed on a scale from 1 to 4(1, no staining; 4, intense staining), and the abundance of positively

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stained cells was given a score from 1 to 5 (1, no cells stained; 5,100% stained). The staining index was then calculated by the stainingintensity multiplied by the staining abundance that gave a range from1 to 20.

Immunoblots for NPY Receptors

The expression of NPY receptor subtypes was evaluated by immu-noblotting in protein (10 �g) from whole cell lysate from the selectednormal and cholangiocarcinoma cell lines as previously described (2)using the aforementioned specific antibodies for each receptor sub-type.

Immunofluorescence

The expression of NPY receptors was assessed in the normalhuman cell lines, H69 and HIBEC, and the cholangiocarcinoma cellline, Mz-ChA-1, by immunofluorescence as previously described byus (10, 18) using the aforementioned antibodies to all the NPYreceptor subtypes (Y1 to Y6). Preimmune sera were substituted for theprimary antibodies as a negative control. Coverslips were visualizedusing an Olympus IX-71 inverted confocal microscope (Tokyo, Ja-pan).

MTS Cell Proliferation Assays

To assess the effects of NPY on cell proliferation, all cell lines werestimulated with various concentrations of recombinant NPY (106 to1010 M), and cell proliferation was assessed by CellTiter 96 CellProliferation Assay (Promega, Madison, WI) as described previously(10, 18). Absorbance was measured at 490 nm on a microplatespectrophotometer (Molecular Devices, Sunnyvale, CA). Data areexpressed as the fold change of treated cells compared with BSA-treated cells. In separate sets of experiments, Mz-ChA-1 cells weretreated at 37°C for 48 h with saline or NPY (107 M) for 48 h in theabsence/presence of preincubation with 1) BVD 10 (107 M, aselective and competitive nonpeptide antagonist for Y1) (4); 2) BIIE0246 [107 M, a selective and competitive nonpeptide antagonist forY2 (12)]; or 3) CGP 71683 hydrochloride (107 M, a selective,nonpeptide Y5 antagonist) (13) before evaluation of proliferation byCellTiter 96 Cell Proliferation Assay (Promega). Conversely, theeffects of NPY depletion on cell proliferation were also assessed.Specifically, Mz-ChA-1 cells were treated with a 1:500 or 1:1,000dilution of a neutralizing NPY antibody (26) for 48 h and cellproliferation was assessed by CellTiter 96 Cell Proliferation Assay(10, 18).

Evaluation of the Intracellular Mechanisms by Which NPYRegulates Cholangiocarcinoma Growth In Vitro

Effect of NPY on intracellular cAMP and IP3 levels. Aftertrypsinization, Mz-ChA-1 cells were incubated at 37°C for 1 h toregenerate membrane proteins damaged by trypsin (17). Mz-ChA-1cells (1 � 105 for determination of cAMP levels, and 1 � 106 cells forthe measurement of IP3 levels) (17) were incubated at room temper-ature with 0.2% BSA (basal) or NPY (107 M) for 5 min (for cAMPevaluations; 17) or 10 min (for IP3 measurements; 17). IntracellularcAMP and IP3 levels were measured by RIA as previously described(17, 19, 20).

Effect of NPY on PKC� phosphorylation and translocation. Tobegin to assess the involvement of PKC� activation on the antipro-liferative effects of NPY, Mz-ChA-1 cells were pretreated with aspecific inhibitor for PKC� (Gö6976, 106 M) (38) for 1 h before theaddition of NPY (107 M). Cells were incubated for a further 48 h,after which time the protein lysates were made as previously de-scribed (2). PCNA expression was used as an indicator of theproliferative capacity of the cells and was assessed by immunoblots(2). The amount of protein loaded (10 �g) was normalized byimmunoblots for -actin (19). Band intensity was determined by

scanning video densitometry using the phospho-imager Storm 860(GE Healthcare) and the Image-Quant TL software (version 2003.02,GE Healthcare). Furthermore, to assess the phosphorylation of PKC�,Mz-ChA-1 cells were treated with NPY (107 M) for 2 h at 37°C.Following stimulation, we assessed the expression of the phosphory-lated form for PKC� by immunoblotting using a phosphospecificPKC� antibody (Santa Cruz Biotechnology) and expressed the data asa ratio to total PKC� expression (19).

To detect membrane translocation of PKC�, Mz-ChA-1 cells wereplated, stimulated with NPY (107 M) for 2 h, and processed forimmunofluorescence (10, 18) as described above using the antibodyfor total PKC�. Negative controls were done with the use of preim-mune serum instead of the respective primary antibody. Slides werevisualized using an Olympus IX-71 inverted confocal microscope.

Invasion Assays

The effects of NPY on the invasive properties of cholangiocarci-noma were performed using growth factor-reduced matrigel invasionchambers (BD Biosciences, San Jose, CA) according to the manufac-turer’s instructions. Specifically, Mz-ChA-1 cells were plated in theupper chamber and allowed to adhere overnight. Cells were treatedwith either NPY (107 M) or anti-NPY neutralizing antibody (1:500dilution) for 24 h. The number of cells that had migrated to theunderside of the upper chamber were then counted in 10 fields, anddata are expressed as the average (� SE) number of cells as anindication of the invasion index.

Nude Mice Treatment

The effects of NPY or anti-NPY antibody on cholangiocarcinomagrowth were assessed in vivo using a xenograft model of cholangio-carcinoma as previously described by us (9) with prior approval fromthe Scott & White and Texas A&M Health Science Center Institu-tional Animal Care and Use Committees. Briefly, Mz-ChA-1 cells(5 � 106) were suspended in 0.25 ml of extracellular matrix gel andinjected subcutaneously in the flanks of these animals. After theestablishment of the tumors, mice received 0.5 �g per tumor per dayof NPY, or 50 �l per tumor per day of a 1:500 dilution anti-NPYantibody injected three times per week, and tumor dimensions weremeasured using electronic callipers (8, 9). After 60 days, mice wereanesthetized with pentobarbital sodium (50 mg/kg body wt ip) andeuthanized according to institutional guidelines. Serum was collected,and aspartate aminotransferase (AST) and alanine aminotransferase(ALT) levels were measured using a Dimension RxL Max IntegratedChemistry system (Dade Behring, Deerfield, IL) by the Scott & WhiteHospital Chemistry Department.

Tumor tissues were dissected from the flank of these mice. Neo-plastic tissues were fixed in formalin, embedded in paraffin, processedfor histopathology, and stained for hematoxylin and eosin for routineexamination and Masson’s trichrome for collagen. For immunohisto-chemistry, glass slides were deparaffinated, and endogenous peroxi-dase activity was blocked by a 30-min incubation in methanolichydrogen peroxide (2.5%). The endogenous biotin was blocked by abiotin blocking system (code X0590; DAKO, Copenhagen, Denmark)according to the instructions supplied by the vendor. Sections werethen hydrated in graded alcohol and rinsed in 1� phosphate-bufferedsaline (PBS, pH 7.4) before application of the primary antibody.Sections were incubated overnight at 4°C with polyclonal antibodiesfor NPY (E-17, 1:50 dilution; Santa Cruz Biotechnology), and PCNA(PC10, 1:100 dilution; DAKO). Samples were then rinsed with 1�PBS for 5 min, incubated for 10 min at room temperature withsecondary biotinylated antibody (LSAB Plus System, DAKO, Milan,Italy), then with DAKO ABC (LSAB Plus system), and finallydeveloped with 3–3= diaminobenzidine.

To demonstrate the specificity of the immunoreaction, negativecontrols [the primary antibody was replaced (same dilution) withnormal serum from the same species] were performed for all immu-

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noreactions. Apoptosis was measured by quantitative terminal deoxy-nucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) kit(Apoptag; Chemicon International). Sections were analyzed in acoded manner using BX-51 light microscopy (Olympus, Tokyo,Japan) with a video cam (Spot Insight; Diagnostic Instrument, SterlingHeights, MI) and processed with an Image Analysis System (DeltaSistemi, Rome, Italy).

NPY Immunohistochemistry in Human Tumor Samples

NPY immunoreactivity was assessed in excised tumor samplesfrom cholangiocarcinoma patients, where the invasion front of thecancer was clearly visible. Immunohistochemical staining for NPYwas performed as outlined above.

Statistical Analysis

All data are expressed as means � SE. Differences between groupswere analyzed by Student’s unpaired t-test when two groups wereanalyzed and by ANOVA when more than two groups were analyzed,followed by an appropriate post hoc test. P � 0.05 was used toindicate statistical significance.

RESULTS

Cholangiocarcinomas Express and Synthesize More NPYThan Normal Cholangiocytes

NPY mRNA expression was assessed in six cholangiocar-cinoma cell lines and in two nonmalignant cholangiocyte celllines and was found to be elevated in all cholangiocarcinomacell lines studied (Fig. 1A). The increase in NPY expressionwas also reflected by increased NPY secretion from all chol-angiocarcinoma cell lines studied compared with nonmalignantcholangiocytes (Fig. 1B). In addition, immunohistochemicalanalysis of human liver biopsy samples indicated that there isalso increased NPY immunoreactivity in cholangiocarcinomasamples compared with controls as assessed in a coded fashionby three independent board-certified pathologists (Fig. 1C).Analysis of serum and bile samples from cholangiocarcinomapatients vs. age-matched normal controls revealed no signifi-cant difference in NPY levels (data not shown), suggesting that

Fig. 1. Cholangiocarcinoma cells express and secrete increased amounts of neuropeptide Y (NPY) compared with normal cholangiocytes. A: NPY mRNAexpression was assessed in six cholangiocarcinoma cell lines as well as the nonmalignant cholangiocyte cell lines H69 and human intrahepatic biliary epithelialcells (HIBEC) by real-time PCR. Data are expressed as averages � SE (n � 3). *Significance (P � 0.05) compared with NPY expression in H69 cells. B: NPYsecretion from the cell lines was assessed using an enzyme-linked immunoassay (EIA) kit. Secretion from each cell line was determined by EIA after 6 h, andassays were performed in triplicate. Each data point from nonmalignant cells or cholangiocarcinoma cells (CCA) was collated and plotted on a scatter plot.*Significance (P � 0.05) compared with NPY secretion from cholangiocyte cell lines. C: NPY levels were also assessed in biopsy samples from 48cholangiocarcinoma patients and healthy controls by immunohistochemistry. Representative photomicrographs of NPY immunoreactivity are shown (magnifi-cation �40). Staining intensity was assessed as described in MATERIALS AND METHODS, and values are expressed as averages � SE of all cholangiocarcinomapatients compared with control samples. *Significance (P � 0.05) compared with NPY immunoreactivity in control biopsy samples.

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the expression and secretion of NPY from cholangiocarcinomacells is a local event.

Increased Local Release of NPY DecreasesCholangiocarcinoma Growth and Invasion

We first determined the presence of the NPY receptors inmalignant and normal cholangiocyte cell lines. By real-timePCR, all NPY receptor subtypes were expressed by nonmalig-nant (H69 and HIBEC) and Mz-ChA-1 cells (Fig. 2A) inaddition to all the other cholangiocarcinoma cell lines used(data not shown). Similarly, by immunoblots, NPY receptors

were expressed by Mz-ChA-1 and H69 cells (Fig. 2B) andHIBECs (not shown). By immunofluorescence, immunoreac-tivity for all NPY receptors was predominantly located in themembrane of both H69 cells (nonmalignant cholangiocytes)and Mz-ChA-1 cells (cholangiocarcinoma cells; Fig. 3) as wellas in the other malignant and nonmalignant cholangiocyte celllines studied (data not shown).

Treatment of the nonmalignant human cholangiocyte celllines, H69 and HIBEC, with various concentrations of NPYhad no significant impact on cell proliferation (data notshown), whereas treatment of all of the selected cholangiocar-

Fig. 2. Evaluation of NPY receptors in nonmalignant cholangiocytes and Mz-ChA-1 cells by real-time PCR (A) and immunoblotting (B). A: by real-time PCR,all NPY receptor subtypes were expressed by H69 and HIBEC and Mz-ChA-1 cells. Data are expressed as averages � SE (n � 3). B: by immunoblot analysis,NPY receptors were expressed by Mz-ChA-1 and H69 cells. The comparability of the protein used was evaluated by immunoblotting for -actin, thehousekeeping gene.

Fig. 3. NPY receptor localization was assessed by immunofluorescence in H69 and Mz-ChA-1 cells. Specific receptor immunoreactivity is shown in red. Nucleiwere counterstained with DAPI (blue). Negative controls were performed by substitution of the primary antibodies with preimmune serum. Scale bar, 20 �m.

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cinoma cell lines with NPY (given as a single dose of 106 to1010 M) caused a significant (P � 0.05) decrease in cellproliferation after 48 h as demonstrated by cell proliferationassay (Fig. 4A). Repeated daily administration of NPY had noadditional antiproliferative effects beyond that after a singledose, suggesting that the recombinant NPY is stable in theseculture conditions (data not shown). In separate sets of exper-iments, we demonstrated that 1) NPY decreased the growth ofMz-ChA-1 cells (Fig. 4B) and 2) the antiproliferative effects ofNPY (107 M) on cholangiocarcinoma growth was preventedby the specific inhibitor of Y2, BIIE 0246 (12), but notinhibitors for Y1 and Y5 (Fig. 4B), suggesting that Y2 areinvolved in the inhibitory effects of NPY on cholangiocarci-noma growth. Alone, inhibitors of Y1 and Y5 did not affect the

growth of Mz-ChA-1 cell growth (not shown). We did notevaluate the effects of Y3, Y4, and Y6 on NPY inhibition ofcholangiocarcinoma growth since these receptor inhibitors arenot available.

NPY receptor activation can result in changes to eithercAMP levels or to intracellular Ca2�/IP3 levels in other celltypes (1, 22, 35, 39). Therefore, we assessed the effects of NPYon intracellular IP3 and cAMP. NPY (107 M) significantlyincreased intracellular IP3 levels in Mz-ChA-1 cells comparedwith the basal levels (Fig. 5A) and had no effect on intracellularcAMP (data not shown), suggesting that NPY is acting throughan IP3/Ca2�-dependent signaling pathway.

We have previously demonstrated that one of the majordownstream effectors of Ca2� signaling in the regulation of

Fig. 4. A: NPY decreases cholangiocarcinoma cell proliferation in vitro. Cholangiocarcinoma cells were treated with various concentrations of NPY (1010 Mto 106 M) for 48 h. Cell proliferation was assessed using MTS cell proliferation assays. Data are expressed as fold change in proliferation (averages � SE,n � 7). *P � 0.05 compared with basal treatment within each cell line. B: inhibitory effects of NPY could be prevented by a specific inhibitor of Y2, but notY1 and Y5. Mz-ChA-1 cells were pretreated with BVD 10 (107 M; Y1 inhibitor), BIIE 0246 (107 M; Y2 inhibitor), or CPG 71683 hydrochloride (107 M;Y5 inhibitor) for 1 h before the addition of NPY (107 M) for 48 h. Cell proliferation was assessed using MTS cell proliferation assays. Data are expressed asfold change in proliferation (averages � SE, n � 7). *P � 0.05 compared with basal treatment.

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cholangiocarcinoma cell growth is PKC� (3, 19, 20, 27, 28).Pretreatment of Mz-ChA-1 cells with the specific inhibitor ofPKC� before the addition of NPY (107 M) prevented theNPY-induced decrease in PCNA protein expression (as amarker of proliferative capacity; Fig. 5B), suggesting thatPCK� is involved in the effects of NPY on cholangiocarci-noma growth. Indeed, treatment of Mz-ChA-1 cells with NPY(107 M) increased the phosphorylation of PKC� (Fig. 5C)compared with the basal levels. Furthermore, by immunofluo-rescence, there was a distinct positive stain for PKC� underbasal conditions localized in the cytoplasm (Fig. 5D), where-as after NPY stimulation, there was translocation of PKC�from the cytosolic region to the membrane domain of the cells(Fig. 5D).

In addition, the effects of NPY on cell invasion wereassessed using Mz-ChA-1 cells plated in the matrigel invasionupper chamber. The average number of cells that invaded the

lower chamber in 24 h under basal conditions was 123.25 �1.29, whereas the number of cells that had invaded the lowerchamber after 24 h of NPY treatment decreased to 100.95 �1.64 (P � 0.05 vs. corresponding basal value).

In support of the in vitro data, treating an in vivo xenograftmodel of cholangiocarcinoma tumors with NPY significantlysuppressed tumor growth (Fig. 6A). In addition, the latency oftumor growth (i.e., time taken for tumor volume to increase to150% of the original size) was increased after NPY treatmentcompared with vehicle treatment (Fig. 6A). Analysis of liverenzymes in the serum revealed that there was no significantdifference in AST levels (vehicle, 80.67 � 8.25 U/l vs. NPY,108.67 � 21.38 U/l) and ALT levels (vehicle, 28.33 � 3.08 U/lvs. NPY, 35.0 � 2.54 U/l) between NPY-treated and vehicle-treated animals, both of which fell within normal range, sug-gesting that the NPY treatment was well tolerated and did notcause any liver damage. Histological analysis of liver, heart,

Fig. 5. In vitro effect of NPY on D-myo-inositol 1,4,5-trisphosphate (IP3) levels (A), PCNA (B), and phosphorylation (C) and translocation (D) of PKC� inMz-ChA-1 cells. A: Mz-ChA-1 cells were treated with 0.2% BSA (basal) or NPY (107 M) for 10 min at room temperature before evaluation of IP3 levels byradioimmunoassay. NPY increased IP3 levels of Mz-ChA-1 cells compared with Mz-ChA-1 cells treated with 0.2% BSA. *P � 0.05 vs. its corresponding basalvalue (n � 6). B: Mz-ChA-1 cells were pretreated with Gö6976 (a specific PKC� inhibitor) for 1 h before the addition of NPY (107 M). Pretreatment ofMz-ChA-1 cells with Gö6976 before the addition of NPY prevented the NPY-induced decrease in PCNA protein expression. *P � 0.05 vs. its correspondingbasal value (4 immunoblots from cumulative preparations of cholangiocytes). C and D: following treatment of Mz-ChA-1 cells with 0.2% BSA (basal) or NPY(107 M), we evaluated the phosphorylation (by immunoblots; C) and translocation (by immunofluorescence in cell smears; D) of PKC�. In C, treatment ofMz-ChA-1 cells with NPY (107 M) increased the phosphorylation of PKC� (pPKC�) compared with the basal levels. tPKC�, total PKC�. *P � 0.05 vs. itscorresponding basal value (4 immunoblots from cumulative preparations of cholangiocytes). In D, by immunofluorescence, there was a distinct positive stainfor PKC� under basal conditions localized in the cytoplasm, whereas after NPY stimulation, there was translocation of PKC� from the cytosolic region to themembrane domain of the cells. Scale bar, 50 �m.

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and kidney also indicated no significant organ damage causedby the chronic NPY treatment (data not shown).

Histological analysis of the excised tumors revealed that alltumor cells within tumors from NPY-treated and vehicle-treated animals were positive for cytokeratin-19, a specificmarker for cholangiocytes (2, 8), indicating biliary phenotypes(data not shown). Using PCNA immunoreactivity as a markerof proliferative capacity, NPY treatment decreased the numberof PCNA-positive nuclei per field compared with vehicletreatment (Fig. 6B). In parallel, using TUNEL staining as amarker of apoptosis, NPY treatment significantly increased thepercentage of TUNEL-positive nuclei per field compared withvehicle treatment (Fig. 6B).

Inhibition of NPY Function Increases CholangiocarcinomaCell Growth and Invasion In Vitro

Because NPY expression and secretion increased in cholan-giocarcinoma, but then had an apparently contradictive, anti-proliferative effect on cell growth, we designed experimentsaimed to demonstrate that blocking of NPY activity had growth-

promoting effects on cholangiocarcinoma. Indeed, treating Mz-ChA-1 cells with an NPY-specific antibody that has neutralizingactivity (26) increased cell proliferation as shown by MTS assays(Fig. 7A). Furthermore, Mz-ChA-1 cells treated with anti-NPYantibody for 24 h significantly (P � 0.05) increased the rate of cellinvasion compared with that seen under basal conditions (basal123.35 � 1.29, anti-NPY antibody 162.0 � 3.25).

In addition, treatment of an in vivo xenograft model ofcholangiocarcinoma tumors with anti-NPY antibody signifi-cantly increased the rate of tumor growth (Fig. 7B) and de-creased the latency of tumor growth (Fig. 7B). Analysis of liverenzymes in the serum revealed that there was no significantdifference in AST (vehicle, 69.66 � 8.95 U/l vs. anti-NPY,53.67 � 13.79 U/l) and ALT levels (vehicle, 38.67 � 13.68 U/lvs. anti-NPY, 30.00 � 8.15 U/l) between anti-NPY antibody-treated and vehicle-treated animals, both of which fell withinnormal range, suggesting that the inhibition of NPY did notcause any liver damage. Histological analysis of liver, heart,and kidney also indicated no significant organ damage causedby the chronic inhibition of NPY (data not shown).

Fig. 6. NPY treatment decreases tumor growth in an in vivo xenograft model of cholangiocarcinoma. A: Mz-ChA-1 cells were injected into the flank of athymicmice. After tumors were established, mice were treated with 0.5 �g NPY per tumor per day (intratumoral), 3 days per week for 60 days, and tumor volume wasassessed (left). Tumor latency was assessed as the time taken for the tumor to grow to 150% of the original size (right). Data are expressed as average latency(days � SE). *Significance (P � 0.05) compared with vehicle-treated tumors. B: immunohistochemistry on tumors from vehicle- and NPY-treated mice wasperformed using a specific antibody against PCNA. Apoptosis was detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL)staining. Representative photomicrographs of the immunoreactivity are shown (original magnification �40). Semiquantitative analyses of PCNA immunore-activity and TUNEL-positive nuclei were performed, and data are expressed as averages � SE of positive nuclei per field. *Significance (P � 0.05) comparedwith vehicle-treated tumors.

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Immunohistochemical Analysis of Tumor Tissue Reveals aGradient of NPY Expression

The concept that cholangiocarcinoma cells are overproducingNPY, which in turn slows the growth rate of the tumor, appearssomewhat counterintuitive. Therefore, we wished to determinewhether the increased expression of NPY occurs uniformlythroughout the tumor. Interestingly, the increased expression ofNPY occurred predominantly in the center of the tumor (Fig. 8, Aand B), particularly near the necrotic areas, with considerably lessexpression toward the periphery of the tumor (Fig. 8C). Further-more, the nonmalignant hepatocytes in the normal liver tissuesurrounding the tumor appear to also have an increased expressionof the antiproliferative NPY (Fig. 8A).

DISCUSSION

The major findings of the study relate to the local regulationof cholangiocarcinoma cell growth and migration by NPY. Wedemonstrated that cholangiocarcinoma cells express and pro-duce more NPY than normal cholangiocytes and that there is agradient of NPY expression within the tumor, with the maxi-mum expression occurring toward the center of the tumor, nearthe necrotic areas. Furthermore, treatment of cholangiocarci-noma cells with NPY in vitro and in vivo decreases bothproliferation and migration. In vitro, NPY inhibition of chol-

angiocarcinoma growth was prevented by the specific inhibitorof Y2, BIIE 0246 (12), but not inhibitors for Y1 and Y5 (Fig.4B), suggesting that Y2 are involved in the inhibitory effects ofNPY on cholangiocarcinoma growth; inhibitors for Y3, Y4,and Y6 are not available. In addition, we have shown that1) NPY-induced inhibition of cholangiocarcinoma cell growthwas associated with an increase in intracellular IP3 and acti-vation of PKC� and 2) the antiproliferative effects of NPY areblocked by a specific PKC� inhibitor. Taken together thesedata suggest that the gradient expression of NPY in cholangio-carcinoma may be a key regulatory feature of the local regu-lation of cholangiocarcinoma growth and progression.

A physiological function of NPY is to regulate food intakeand increase fat storage (23). Furthermore, NPY has beenshown to be upregulated in obesity (5), which is a risk factorfor cholangiocarcinoma (59). Therefore, because of the linkbetween obesity and cholangiocarcinoma, it is conceivable thatmolecules that are upregulated during obesity, such as NPY,may also play a role in cholangiocarcinoma initiation, growth,and progression. Indeed, recently, we demonstrated that an-other obesity-related molecule, leptin, was involved in thedevelopment and growth of cholangiocarcinoma in a thioacet-amide rat model of cholangiocarcinoma (16).

In support of our observation that cholangiocarcinoma cellssecrete increased levels of NPY, this neuropeptide is secreted

Fig. 7. Blocking of NPY activity promotes cholangiocarcinoma cell growth in vitro and in vivo. A: Mz-ChA-1 cells were treated with various concentrations ofanti-NPY antibody (1:500 and 1:1,000 dilution) for 48 h. Cell proliferation was assessed using an MTS cell proliferation assay. Data are expressed as fold changein proliferation (averages � SE, n � 7). *P � 0.05 compared with basal treatment within each cell line. B: Mz-ChA-1 cells were injected into the flank of athymicmice. After tumors were established, mice were treated with 50 �l of a 1:500 dilution anti-NPY antibody (intratumoral), 3 days per week for 60 days, and tumorvolume was assessed (left). Tumor latency was assessed as the time taken for the tumor to grow to 150% of the original size (right). Data are expressed as averagelatency (days � SE). *Significance (P � 0.05) compared with vehicle-treated tumors.

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in a number of other tumors. For example, NPY immunoreac-tivity has been shown in �75% of prostate cancer tumorspecimens studied (36). Furthermore, NPY treatment of threedifferent prostate cancer cell lines stimulated proliferation inone cell line and decreased proliferation in the other two (49).Both the growth-enhancing and antiproliferative actions ofNPY were through Y1, and the difference between the prolif-erative and antiproliferative effects appears to be via themechanism by which extracellular signal-regulated kinase 1/2is activated (49). NPY has also been shown to be upregulatedin a number of neuroendocrine tumors, such as pheochromo-cytomas (11, 24), and neuroblastomas (30, 41), and are thoughtto be of diagnostic importance; however, its contribution totumor progression still remains to be clarified. We have dem-onstrated that cholangiocarcinomas display typical markers ofa neuroendocrine phenotype such as the expression of chro-mogranin A and neuron-specific enolase (2); therefore it is notsurprising that cholangiocarcinomas also expressed NPY. Inthe present study, the diffuse positivity of the neuroendocrinemarker, NPY, observed in human ductular cholangiocarcinomasamples is supported by a number of studies. Indeed, Liu et al.(33) have demonstrated higher expression of NPY in nonag-gressive prostate epithelial tumors. Moreover, these observa-tions about the nonconventional immunolocalization of neu-roendocrine proteins are previously discussed in cholestasisand cholangiocarcinoma where the important role of GABA,chromogranin A, glycolipid A2-B4, S-100 protein, and neuralcell adhesion molecule are demonstrated by us and other

groups (17, 37, 46–48), suggesting that the biliary epitheliumis a significant target for the neuroendocrine system. A directoutgrowth of our findings will be to study the expression ofNPY in hepatocytes and cholangiocytes in cholangiocarcinomaperitumoral tissue.

While the majority of studies into the effects of NPY oncancer growth and invasion demonstrate a growth-promotingand invasive effect (45, 50, 55), there are a number of studiesthat have demonstrated antiproliferative effects of NPY. Asmentioned above, NPY administration to a number of prostatecancer cell lines inhibited the proliferation through a Y1-dependent mechanism (49). In addition, NPY administration tocolon carcinoma cells in vitro reduced the invasion potential ofthese tumor cells in a concentration-dependent manner (42).The data that we present here support an antiproliferativeaction of NPY on cholangiocarcinoma cell growth in vitro andin vivo and also an inhibitory effect on cholangiocarcinomamigration and invasion. It is conceivable that the differenteffects of NPY on cell growth may be due to the number ofspecific NPY receptors through which NPY may exert itseffects. We demonstrated that all receptors are present incholangiocarcinoma, but because of the lack of reliable specificantagonists for Y3, Y4, and Y6, we were unable to pinpoint thespecific NPY receptor that is responsible for the actions ofNPY on cholangiocarcinoma, and this is a limitation of thepresent study. However, since a specific inhibitor of Y2, BIIE0246 (12), blocks the in vitro inhibitory effect of NPY oncholangiocarcinoma growth, likely Y2 play an important role

Fig. 8. Immunohistochemical analysis of NPY expression across human cholangiocarcinoma tumors. Representative photomicrographs of the immunoreactivityare shown (original magnification �10, particular �40). The NPY expression, present in all tumor tissues (A and B), appeared higher near the neoplastic necroticareas (C). Furthermore, the nonmalignant hepatocytes in the normal liver tissue surrounding the tumor appear to also have increased expression of theantiproliferative NPY (A).

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in the inhibitory effect of NPY on cholangiocarcinoma growth.Further experiments (gene silencing of NPY receptors withsmall interfering RNAs) are necessary to pinpoint the NPYreceptors involved in NPY effects on cholangiocarcinoma. Asmentioned previously, NPY receptor activation can elicitcAMP- or Ca2�-mediated signal transduction pathways (1, 22,35, 39). The data presented here support a role for IP3/Ca2�-mediated PKC� activation in the antiproliferative action ofNPY since NPY increased IP3 (but not cAMP) levels andinduced the activation of PKC�. These findings support theconcept that Ca2�-dependent PKC� is a key regulator of thehyperplastic and neoplastic growth of cholangiocytes. Indeed,we have previously shown that the inhibition of cholangiocar-cinoma growth (e.g., by gastrin, H3 histamine receptor ago-nists, �2-adrenergic receptor agonists, and the bile salt, taur-oursodeoxycholate) is associated with enhanced phosphoryla-tion of PKC�.

In the present study, we demonstrated a gradient of NPYexpression in the cholangiocarcinoma tumor, with the highestexpression found in the center of the tumor near the necroticareas. The concept of a gradient of expression to accommodatethe different microenvironments within the tumor is not new.Recently, a large-scale expression analysis of melanoma me-tastases was performed to identify genes that exhibit differen-tial expression between the invasion front and the central tumorareas (54). The authors identified 248 genes that were differ-entially expressed within the tumor, 97 of which had higherexpression within the center of the tumor compared with theinvasion front (54). Some of the genes that were expressedhigher at the invasion front, generally, had known functions incell invasion (54); however, the function, with respect tocancer progression, of the genes predominantly expressed inthe central areas of the tumor was largely unknown (54). Inaddition, Ohira et al. (43) recently demonstrated that a gradientof transforming growth factor- expression is evident in chol-angiocarcinoma with little to no expression in the invasionfront and strong expression in tumors growing in the bile ductlumen (43). Here we speculate that NPY may be expressedhigher in the center of the tumor where it exerts growth-suppressive and antiinvasive effects to allow for the recruit-ment of adequate stromal support.

In conclusion, the data presented here demonstrate a role ofNPY in the local control of cholangiocarcinoma cell growth.Specifically, NPY appears to be expressed to a greater extent inthe center of the tumor, where we speculate that it exerts a localantiproliferative and antimigratory effect on cholangiocarci-noma cells. Modulation of NPY signaling may be a usefultarget for the design of therapeutic tools for the treatment ofthis devastating cancer.

GRANTS

This work was supported partly by the Dr. Nicholas C. Hightower Centen-nial Chair of Gastroenterology from Scott & White, the VA Research ScholarAward, a VA Merit Award, and National Institutes of Health (NIH) GrantsDK58411, DK062975, and DK09768 to G. Alpini, an NIH K01 grant award(DK078532) to S. DeMorrow, an NIH grant (DK081442) to S. Glaser, and byUniversity funds to P. Onori and PRIN 2007 and Federate Athenaeum fundsfrom University of Rome “La Sapienza” to E. Gaudio.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

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