serotonin metabolism is dysregulated in cholangiocarcinoma, which has implications for tumor growth

Serotonin Metabolism Is Dysregulated in Cholangiocarcinoma,

which Has Implications for Tumor Growth

Gianfranco Alpini,1,2,3

Pietro Invernizzi,5Eugenio Gaudio,

6Julie Venter,

1Shelley Kopriva,

3

Francesca Bernuzzi,5Paolo Onori,

7Antonio Franchitto,

6Monique Coufal,

1Gabriel Frampton,

4

Domenico Alvaro,8Sum P. Lee,

9Marco Marzioni,

10Antonio Benedetti,

10and Sharon DeMorrow

1

1Department of Medicine, Texas A&M Health Science Center, College of Medicine, Scott & White Hospital; 2Systems Biology andTranslational Medicine, Texas A&M University System Health Science Center, College of Medicine; 3Division of Research, Central TexasVeterans Health Care System; 4Texas Biosciences Institute, Temple College, Temple, Texas; 5Department of Internal Medicine,Instituto Clinico Humanitas IRCCS, University of Milan, Milan, Italy; 6Division of Anatomy, University ‘‘La Sapienza,’’ Rome, Italy;7Department of Experimental Medicine, University of L’Aquila, L’Aquila, Italy; 8Department of Clinical Medicine,Division of Gastroenterology, University of Rome ‘‘La Sapienza’’ Polo Pontino, Latina, Italy; 9Department ofMedicine, University of Washington, Seattle, Washington; and 10Department of Gastroenterology,Universita Politecnica delle Marche, Ancona, Italy

Abstract

Cholangiocarcinoma is a devastating cancer of biliary originwith limited treatment options. Symptoms are usually evidentafter blockage of the bile duct by the tumor, and at this latestage, they are relatively resistant to chemotherapy andradiation therapy. Therefore, it is imperative that alternativetreatment options are explored. We present novel dataindicating that the metabolism of serotonin is dysregulatedin cholangiocarcinoma cell lines, compared with normalcholangiocytes, and tissue and bile from cholangiocarcinomapatients. Specifically, there was an increased expression oftryptophan hydroxylase 1 and a suppression of monoamineoxidase A expression (enzymes responsible for the synthesisand degradation of serotonin, respectively) in cholangiocarci-noma. This resulted in an increased secretion of serotoninfrom cholangiocarcinoma and increased serotonin in the bilefrom cholangiocarcinoma patients. Increased local serotoninrelease may have implications on cholangiocarcinoma cellgrowth. Serotonin administration increased cholangiocarci-noma cell growth in vitro , whereas inhibition of serotoninsynthesis decreases tumor cell growth both in vitro andin vivo . The data presented here represent the first evidencethat serotonin metabolism is dysregulated in cholangio-carcinoma and that modulation of serotonin synthesis mayrepresent an alternative target for the development oftherapeutic strategies. [Cancer Res 2008;68(22):9184–93]

Introduction

Cholangiocarcinomas are devastating cancers of intrahepaticand extrahepatic origin that are increasing in both their worldwideincidence and mortality rates (1, 2). The challenges posed by theseoften lethal biliary tract cancers are daunting, with conventionaltreatment options being limited and the only hope for long-termsurvival being that of complete surgical resection of the tumor(1, 2). Conventional chemotherapy and radiation therapies are not

effective in prolonging long-term survival (1); therefore, it isimportant to understand the intracellular mechanisms of chol-angiocarcinoma cell growth, with a view to develop novelchemopreventive strategies.Serotonin or 5-hydroxytryptamine is a neuromodulator, with

both neuroendocrine and neurotransmitter functions, which issynthesized in serotonergic neurons in the central nervous system(3) and enterochromaffin cells throughout the gastrointestinal tract(4). Serotonin is synthesized by the systematic hydroxylation anddecarboxylation of the amino acid tryptophan by the enzymestryptophan hydroxylase (TPH1) and amino acid decarboxylase,respectively (3). There are 16 serotonin receptor subtypes spreadover seven receptor families, through which serotonin exerts itsmultiple effects (5). With the exception of the serotonin 3 receptor,a ligand-gated ion channel, all other serotonin receptors are Gprotein–coupled, seven-transmembrane receptors that activateintracellular second messenger systems (5). Once serotonin hasactivated the receptor, it is cleared from the extracellular space byspecific reuptake transporters where it undergoes catabolism (6).Degradation of serotonin is carried out primarily by the enzymemonoamine oxidase (MAO), which occurs as two molecularsubtypes, called MAO A and MAO B, and have some differencesin their tissue and cellular distributions (7). MAO A is moreselective for serotonin oxidation by being able to metabolizeserotonin with a much lower Km value (and higher affinity for thesubstrate) than MAO B (8).Several opposing effects of serotonin on tumor growth have been

reported (9). On one hand, serotonin is known as a growth factorfor several types of nontumoral cells (10, 11), and it has beenproposed to take part in the autocrine loops of growth factorscontributing to cell proliferation in aggressive tumors, such assmall cell lung carcinoma (12), prostate cancer cells (13). humanbreast cancer cell lines (14), and bladder cancer (15). In contrast,several studies have also reported that serotonin can inhibit tumorgrowth, mainly via the specific vasoconstrictive effects of serotoninon the vessels irrigating the tumors (9, 16–18). In addition, thesynthesis and secretion of serotonin has previously been shownto be dysregulated in neuroendocrine tumors, with these cellspossessing a higher biogenic amine content than normal cells(19–21).Serotonin is involved in the pathogenesis of certain clinical

features of cholangiopathies, pruritus, and fatigue, in particular(22, 23). In animal models of chronic cholestasis, this may be due toan enhanced release of serotonin in the central nervous system and

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

Requests for reprints: Sharon DeMorrow, Department of Internal Medicine, Scottand White Hospital and Texas A&M Health Science Center, Medical Research Building,702 SW H.K. Dodgen Loop, Temple, TX 76504. Phone: 254-724-6240; Fax: 254-724-8070;E-mail: [email protected].

I2008 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-08-2133

Cancer Res 2008; 68: (22). November 15, 2008 9184 www.aacrjournals.org

Research Article

Research. on May 2, 2016. © 2008 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

its interactions with subtype 1 serotonin receptors (23). Chol-angiocytes synthesize and secrete serotonin, which is increased inproliferating rat cholangiocytes after bile duct ligation (24). Wepostulate that this autocrine loop is integral in limiting the growthof the biliary tree as a result of chronic cholestasis. However, to

date, nothing is known about the involvement of serotonin in theneoplastic transformation and growth of cholangiocarcinoma.In the present study, we show a dysregulation of the cellular

machinery responsible for the metabolism of serotonin incholangiocarcinoma cell lines and human samples, which results

Figure 1. TPH1 expression is increased in cholangiocarcinoma. TPH1 levels were assessed in six cholangiocarcinoma cell lines, as well as a nonmalignantcholangiocyte cell line H69, by real-time PCR (A) and immunoblotting (B ). Columns, average (n = 3); bars, SE. Asterisk denotes significance (P < 0.05)compared with TPH1 expression in H69 cells. TPH1 levels were also assessed in biopsy samples from 48 cholangiocarcinoma patients and healthy controls byimmunohistochemistry. C, representative photomicrographs of the TPH1 immunoreactivity. Magnification, 40�. D, staining intensity was assessed as described inMaterials and Methods. Columns, average of all cholangiocarcinoma patients compared with control samples, as well as a function of tumor grade (degree ofdifferentiation); bars, SE. Asterisk denotes significance (P < 0.05) compared with TPH1 immunoreactivity in control biopsy samples.

Serotonin and Cholangiocarcinoma

www.aacrjournals.org 9185 Cancer Res 2008; 68: (22). November 15, 2008



in an increased production and secretion of serotonin fromcholangiocarcinoma. Furthermore, we show that the increasedsecretion of serotonin has growth-promoting effects on cholangio-carcinoma cells and that inhibiting serotonin synthesis significant-ly blocks cholangiocarcinoma cell proliferation in vitro and in vivo .

Materials and Methods

Cell lines. We used six human cholangiocarcinoma cell lines (Mz-ChA-1,HuH-28, HuCC-T1, CCLP1, SG231, and TFK-1) with different origins. Mz-ChA-1 cells from human gallbladder (25) were a gift from Dr. G. Fitz

(University of Texas Southwestern Medical Center). HuH-28 cells from

Figure 2. MAO A expression is decreased in cholangiocarcinoma. MAO A levels were assessed in six cholangiocarcinoma cell lines, as well as a nonmalignantcholangiocyte cell line H69, by real-time PCR (A) and immunoblotting (B ). Columns, average (n = 3); bars, SE. Asterisk denotes significance (P < 0.05)compared with MAO A expression in H69 cells. MAO A levels were also assessed in biopsy samples from 48 cholangiocarcinoma patients and healthy controlsby immunohistochemistry. C, representative photomicrographs of the MAO A immunoreactivity. Magnification, 40�. D, staining intensity was assessed as described inMaterials and Methods. Columns, average of all cholangiocarcinoma patients compared with control samples, as well as a function of tumor grade (degree ofdifferentiation; bars, SE. Asterisk denotes significance (P < 0.05) compared with MAO A immunoreactivity in control biopsy samples.

Cancer Research




human intrahepatic bile duct (26) and TFK-1 cells from extrahepaticcholangiocarcinoma (27) were acquired from Cancer Cell Repository,

Tohoku University. These cells were maintained at standard conditions, as

described (28). In addition, CCLP-1 (29), HuCC-T1 (30), and SG231 (31), also

from intrahepatic bile ducts, were a kind gift from Dr. A.J. Demetris(University of Pittsburg) and were cultured as described (29–31). The

human immortalized, nonmalignant cholangiocyte cell line H69 ( from

Dr. G.J. Gores, Mayo Clinic) was cultured as described (32).

Real-time PCR. RNA was extracted from all cell lines using the RNeasyMini kit (Qiagen, Inc.), according to the instructions provided by the vendor,

and reverse transcribed using the Reaction Ready First Strand cDNA

Synthesis kit (SuperArray). These reactions were used as templates for the

PCR assays using a SYBR Green PCR Master Mix (SuperArray) in the real-time thermal cycler (ABI Prism 7900HT sequence detection system) using

commercially available primers designed against human TPH1, MAO A, andthe specific serotonin receptor subtypes (SuperArray). A DDCT analysis wasperformed using the normal cholangiocytes as the control sample. Data are

expressed as relative mRNA levels F SE (n = 3).

Immunoblotting. After trypsinization, all cell lines (1 � 106) wereresuspended in lysis buffer (33) and sonicated. Immunoblots to detect TPH,

MAO A, and h-actin were performed, as previously described (33), using

specific antibodies against each protein (Santa Cruz Biotechnology). Data

are expressed as fold change (mean F SE) of the relative expression afternormalization with h-actin.

Cholangiocarcinoma tissue array analysis. Immunoreactivity for

serotonin, TPH1, MAO A, the cholangiocyte marker CK-19, and the

neuroendocrine markers chromagranin A and neuron-specific enolase(NSE) were assessed in commercially available Accumax tissue arrays

Figure 3. Serotonin secretion in cholangiocarcinoma. A, serotonin levels in the supernatant of cell suspensions of cholangiocarcinoma cell lines and the nonmalignantcholangiocyte cell line H69 were determined by EIA after 6 h. Columns, average serotonin concentration (ng/mL; n = 3); bars, SE. Asterisk denotes significance(P < 0.05) compared with serotonin levels secreted from H69 cells. Serotonin immunoreactivity was assessed in cholangiocarcinoma biopsy samples byimmunohistochemistry. B, representative photomicrographs of the serotonin immunoreactivity. Magnification, 40�. C, staining intensity was assessed as described inMaterials and Methods. Columns, average of all cholangiocarcinoma patients compared with control samples; bars, SE. Asterisk denotes significance (P < 0.05)compared with serotonin immunoreactivity in control biopsy samples. D, serotonin levels in bile samples from cholangiocarcinoma patients and patients withintrahepatic cholelithiasis by EIA. Points, average serotonin concentration (ng/mL); bars, SE.





(Isu Abxis Co., Ltd.) by immunohistochemistry, as described (24), using

specific antibodies. These tissue arrays contain 48 well-characterized

cholangiocarcinoma biopsy samples from a variety of tumor differentiation

grades, as well as four control liver biopsy samples. Semiquantitative analysiswas performed by three independent observers, in a blind fashion, using the

following variables. Staining intensity was assessed on a scale from 1 to 4 (1,

no staining; 4, intense staining), and the abundance of positively stained cells

was given a score from 1 to 5 (1, no cells stained; 5, 100% stained). Thestaining index was then calculated by the staining intensity multiplied by the

staining abundance that gave a range from 1 to 20.

Serotonin secretion. All cell lines were trypsinized, and the resultingcell pellet was resuspended in HBS buffer (1 � 107 cells/mL). Cells were

then incubated for 6 h at 37jC, and the amount of serotonin released intothe medium was assayed using a commercially available serotonin EIA kit

(Invitrogen) according to the manufacturer’s instructions.Hepatic bile was collected aseptically from T-tube drainage during

postoperative days 1 to 3 from patients with intrahepatic stones (n = 12) or

gallstones in association with common bile duct stones (n = 11) and from

cholangiocarcinoma patients (n = 14). Bile samples were immediatelyfrozen at �80jC until analysis.

MTS cell proliferation assays. Cell lines were seeded into 96-well plates(10,000 per well) in a final volume of 200 AL of growth medium and allowed

to adhere to the plate overnight. Cells were serum-starved for 24 h beforestimulation with serotonin (10�8 to 10�6 mol/L) or p-chlorophenylalanine(CPA; a specific TPH1 inhibitor; 0.25–1 mmol/L, IC50 250 Amol/L; ref. 34)for 48 h. In parallel experiments, cells were pretreated with commerciallyavailable specific serotonin receptor antagonists, all at 10 Amol/L (5HTR 1Aantagonist, (S)-Way 100135 dihydrochloride; 5HTR 1B antagonist, SB216641

hydrochloride; 5HTR 1D antagonist, BRL 15572 hydrochloride; 5HTR 2A

antagonist, spiperone hydrochloride, 5HTR 2B, SB204741; 5HTR 2C, N-desmethylclozapine; pan 5HTR 3 antagonist, tropisetron hydrochloride;

5HTR 4 antagonist, GR125487 sulfamate; 5HTR 6 antagonist, SB258585

hydrochloride; and 5HTR 7 antagonist, SB269970 hydrochloride; all

purchased from Tocris Bioscience), for 1 h before the addition of serotonin

(10�7 mol/L). Cell proliferation was assessed using a colorimetric cell

proliferation assay (CellTiter 96Aqueous, Promega Corp.), and absorbance

measured at 490 nm by a microplate spectrophotometer (Versamax,Molecular Devices). In all cases, data were expressed as the fold change of

treated cells compared with vehicle-treated controls.

Bromodeoxyuridine incorporation assays. Bromodeoxyuridine

(BrdUrd) assays were performed, as described previously (28), using Mz-ChA-1 cells stimulated with serotonin (1 Amol/L) and CPA (0.25 mmol/L)

for 48 h. The number of BrdUrd-positive nuclei was counted and expressed

as a percentage of total cells in five random fields for each treatment group.Data are average F SE of five fields in three independent experiments.

Nude mice treatment. In vivo experiments were performed as described

previously (35). Male BALB/c 8-wk-old nude (nu/nu) mice were kept in a

temperature-controlled environment (20–22jC) with a 12-h light-dark cycleand with free access to drinking water and standard mouse chow. Mz-ChA-

1 cells (5 � 106) were suspended in 0.25 mL of extracellular matrix gel and

injected s.c. in the left back flank of these animals. After the establishment

of the tumors, mice received CPA (150 mg/kg/d i.p.) injected thrice perweek. Tumor variables were measured twice a week by an electronic caliper,

and volume was determined as follows: tumor volume (mm3) = 0.5 �[length (mm) � width (mm) � height (mm)]. After f2 mo, mice were

anaesthetized with sodium pentobarbital (50 mg/kg i.p.) and sacrificedaccording to institutional guidelines. Serum was collected, and aspartate

aminotransferase (AST) and alanine aminotransferase (ALT) levels were

measured using a Dimension RxL Max Integrated Chemistry System (DadeBehring, Inc.) by Scott & White Hospital, Chemistry Department. Heart,

liver, and kidneys were isolated, fixed in formalin, embedded in paraffin,

processed for histopathology, and stained with H&E for the detection of

tissue damage.Tumor tissues were also excised from the flank of these mice, fixed in

formalin, and embedded in paraffin. Histologic tests included H&E staining

for histopathology or Masson’s trichrome staining for collagen visualization.

Figure 4. Cholangiocarcinoma display features of aneuroendocrine tumor. Immunohistochemical analysisof biopsy samples from nonmalignant liver andcholangiocarcinoma tumors were performed usingantibodies against CK-19 (cholangiocyte marker),chromagranin A, and NSE (neuroendocrine markers).Representative photomicrographs of the immunoreactivityare shown. Magnification, 40�.

Cancer Research




Tumor protein, selectively expressed by cholangiocytes, was evaluated afterCK-7 immunohistochemical staining (36). Furthermore, the expression of

specific neuroendocrine markers chromagranin A and NSE was assessed

using immunohistochemistry (36). In each case, sections were counter-

stained with hematoxylin before analysis.Light microscopy and immunohistochemistry observation were taken by

BX-51 light microscopy (Olympus) with a videocam (Spot Insight,

Diagnostic Instrument, Inc.) and processed with an Image Analysis System

(Delta Sistemi). Three pathologists independently performed analysis in ablind manner. The degree of inflammation and fibrosis was evaluated in five

randomly nonoverlapping fields (magnification, 20�) for each slide using

light microscopy of Masson’s stained sections, as previously described (37);

the necrotic mass was evaluated by quantitative morphometry on LMimages, as previously described (38, 39), and expressed as area of necrosis /

total area of tumor � 100. For each sample, more than five nonoverlapping

fields (magnification, 20�) were studied.

Results

Expression of metabolic enzymes for serotonin is dysregu-lated in cholangiocarcinoma. The de novo synthesis of serotoninis predominantly performed by TPH1 in the gastrointestinal tract

(3, 34). The expression of TPH1 mRNA was significantly up-regulated ( from 2.5-fold to 50-fold) in five of six cholangiocarci-noma cell lines when compared with the nonmalignant H69 cells(Fig. 1A). This trend was confirmed by TPH1 protein expression, asshown by immunoblotting (Fig. 1B). In addition, immunohisto-chemical analysis of human liver biopsy samples indicated thatthere is also increased TPH1 immunoreactivity in cholangiocarci-noma samples compared with control, as assessed by threeindependent observers (Fig. 1C and D and data not shown).Analysis of the TPH1 immunoreactivity as a function of thedifferentiation grade of the tumor showed a correlation betweenstaining intensity and the degree of differentiation (Fig. 1D). Morespecifically, whereas there is an increased expression of TPH1 in allcholangiocarcinoma samples compared with normal liver samples,the increase is more evident in tumors with a differentiation gradeof 1 (well differentiated) compared with the less-differentiatedgrade 3 (Fig. 1D).In contrast to TPH1 expression, the MAO A mRNA levels were

significantly decreased in cholangiocarcinoma cell lines comparedwith H69 cells (Fig. 2A), which was paralleled by the protein

Figure 5. A, serotonin increasescholangiocarcinoma cell proliferationin vitro . Mz-ChA-1 cells and thenonmalignant cholangiocyte cell line H69were treated with various concentrations ofserotonin (10�8 to 10�6 mol/L) for 48 h. Cellproliferation was assessed using an MTScell proliferation assay. Data are expressedas fold change in proliferation. Columns,average (n = 7); bars, SE. Asteriskdenotes P < 0.05 compared with basaltreatment within each cell line. B, inhibitionof serotonin synthesis decreasescholangiocarcinoma cell proliferationin vitro . Mz-ChA-1 cells and thenonmalignant cholangiocyte cell line H69were treated with various concentrations ofCPA (0.25–1 mmol/L) for 48 h. Cellproliferation was assessed using an MTScell proliferation assay. Data are expressedas fold change in proliferation. Columns,average (n = 7); bars, SE. Asteriskdenotes P < 0.05 compared with basaltreatment within each cell line. C, BrdUrdlabeling of cholangiocarcinoma cellsindicates changes in cell cycle progressionafter serotonin and CPA. Mz-ChA-1 cellswere treated with serotonin (10�6 mol/L)and CPA (0.25 mmol/L) for 48 h, andBrdUrd uptake was determined. Thenumber of BrdUrd-positive cells wasexpressed as a percentage of total cells.Columns, average of five random fieldsfrom three independent experiments; bars,SE. *, significance (P < 0.05) whencompared with basal treatment.





expression, as shown by immunoblotting (Fig. 2B). Similarly,immunohistochemical analysis showed a suppression of MAO Aexpression in human biopsy samples, as assessed by threeindependent observers (Fig. 2C and D and data not shown).However, when MAO A immunoreactivity was expressed as afunction of tumor differentiation grade, there was no correlationbetween the degree of differentiation and the expression of MAO A(Fig. 2D).

Serotonin secretion is increased in cholangiocarcinoma.Taking the changes in the expression of serotonin synthesis anddegradation enzymes together, it would be reasonable to expect anoverall increase in serotonin production and secretion fromcholangiocarcinoma cells. Indeed, the secretion of serotonin wasincreased in five of six cholangiocarcinoma cell lines (Fig. 3A).Serotonin immunoreactivity was also increased in the humanbiopsy samples contained on the cholangiocarcinoma tissue array,as assessed by three independent observers (Fig. 3B and C and datanot shown). Analysis of serum samples from cholangiocarcinomapatients versus age-matched controls revealed no significantdifference in serotonin levels (data not shown), which is notsurprising, given that the normal tissue surrounding the tumorpresumably has normal degradation machinery (i.e., MAO Aexpression) and would possibly take up the excess serotonin andmetabolize it. We then performed a pilot study with a limited

number of bile samples taken from cholangiocarcinoma patients.As controls, we used bile samples collected from patients affectedby intrahepatic cholelithiasis (e.g., nonmalignant disease). Analysisof these treatment groups revealed an increase in serotonin levelsin cholangiocarcinoma patients compared with controls (Fig. 3D).

Expression of neuroendocrine markers in cholangiocarci-noma samples. Because serotonin production is dramaticallyincreased in a number of neuroendocrine tumors, we assessed theexpression of neuroendocrine markers and cholangiocyte markersin the tumor biopsy samples. CK-19 immunoreactivity wasobserved in a similar intensity in cholangiocarcinoma tissue andnonmalignant liver tissue (Fig. 4). There was no observableexpression of the neuroendocrine markers chromagranin A andNSE in nonmalignant livers but was expressed in a number of thecholangiocarcinoma biopsy samples studied (44% and 63% ofcholangiocarcinoma samples studied for chromagranin A and NSE,respectively), suggesting the emergence of a neuroendocrine or‘‘carcinoid-like’’ phenotype.

Increased local serotonin secretion has implications oncholangiocarcinoma cell growth in vitro . The potentialimplications of the increased local serotonin secretion wereexplored in vitro using the cholangiocarcinoma cell lines.Treatment of the SV40-transformed human cholangiocyte cell lineH69 with various concentrations of serotonin had no significant

Figure 6. Inhibition of serotonin synthesis decreasestumor growth in an in vivo xenograft model ofcholangiocarcinoma. Mz-ChA-1 cells were injected intothe flank of athymic mice. After tumors were established,mice were treated with 150 mg/kg/d (i.p.) CPA, 3 d/wk for62 d, and tumor volume was assessed (A). Tumors wereexcised and photographed before histologic analysis (B).Tumor latency was assessed as the time taken for thetumor to grow to 150% of the original size (C ). Dataare expressed as average latency (days F SE), andthe asterisk denotes significance (P < 0.05) fromvehicle-treated tumors.

Cancer Research




effect on cell proliferation (Fig. 5A), whereas treatment of all of thecholangiocarcinoma cell lines with serotonin (10�7 to 10�6 mol/L)caused a significant increase in cell proliferation after 48 hours asshown by MTS cell proliferation assays (Fig. 5A and SupplementaryFig. S1). Cholangiocarcinoma cell lines expressed all serotoninreceptor subtypes, with the vast majority being up-regulatedcompared with the H69 cholangiocyte cell line, except for 5HTR 1B,5HTR 1F, 5HTR 2B, 5HTR 3C, and 5HTR 7 (which are all down-regulated; Supplementary Table S1). Furthermore, specific inhibi-tion of the serotonin receptors 5HTR 1A, 5HTR 2A, 5HTR 2B, 5HTR4, and 5HTR 6 effectively prevented the growth-promoting effectsof serotonin (Supplementary Fig. S2).Conversely, blocking serotonin synthesis, CPA, a specific TPH1

inhibitor, markedly inhibited cholangiocarcinoma cell proliferation,particularly at 1 mmol/L (Fig. 5B and Supplementary Fig. S3). Onceagain, no effect was observed in H69 cells. Taken together, thisindicates that the increased serotonin secretion from cholangio-carcinoma cells may have a growth-promoting effect on tumorprogression.The effects of serotonin and CPA on cell cycle progression were

assessed by BrdUrd uptake assays in Mz-ChA-1 cells. Consistentwith the MTS assays described above, there was an increase in thenumber of BrdUrd-positive nuclei per field after serotonintreatment (Fig. 5C), whereas treatment with CPA decreased thenumber of BrdUrd-positive cells (Fig. 5C). This suggests thatincreased serotonin secretion from cholangiocarcinoma cells mayexert its effects by regulating cell cycle progression.

Inhibition of serotonin synthesis inhibits cholangiocarci-noma tumor growth in vivo . By treating an in vivo xenograftmodel of cholangiocarcinoma tumors with the TPH inhibitor, wesignificantly suppressed tumor growth (Fig. 6A and B). In addition,the latency of tumor growth (i.e., time taken for tumor volume toincrease to 150% of the original size) was increased after CPAtreatment compared with vehicle treatment (Fig. 6C). Analysis ofliver enzymes in the serum revealed that there was no significantdifference in AST (vehicle, 96.0 F 11.7 versus CPA, 75.6 F 13.5) andALT levels (vehicle, 42.3 F 13.1 versus CPA, 33.0 F 4.4) betweenCPA-treated and vehicle-treated animals, both of which fell withinthe reference range, suggesting that the CPA treatment was welltolerated and did not cause any liver damage. Histologic analysis ofliver, heart, and kidney also indicated no significant organ damagecaused by the chronic CPA treatment (data not shown).Histologic analysis of the excised tumors revealed that all cells

within tumors from CPA-treated and vehicle-treated animals wereCK-7–positive, indicating cholangiocyte phenotype (SupplementaryFig. S4). CPA treatment significantly decreases the area of tumornecrosis (Supplementary Fig. S4), and semiquantitative analysisfibrosis shows that CPA increased fibrosis within the tumor(Supplementary Fig. S4). Furthermore, the xenograft tumors alsoexpressed chromagranin A and NSE, in both vehicle-treated andCPA-treated animals (Supplementary Fig. S5).

Discussion

The major findings of this study relate to the dysregulation ofserotonin metabolism in cholangiocarcinoma. We showed that (a)expression of the enzyme responsible for serotonin synthesis in thegastrointestinal tract, TPH1, is up-regulated in cholangiocarci-noma; (b) the enzyme responsible for serotonin degradation, MAOA, is markedly decreased in cholangiocarcinoma samples; and (c)this results in an overall increase in serotonin secretion from

cholangiocarcinoma cells and in the bile from cholangiocarcinomapatients. This increase in serotonin production and secretionresulted in an increased cholangiocyte proliferation in vitro , andinhibition of serotonin synthesis by CPA decreased cell prolifera-tion in vitro and in vivo xenograft model of cholangiocarcinoma.These data suggest that the dysregulation of serotonin metabolismmay be a key feature associated with the progression ofcholangiocarcinoma, and the modulation of this metabolicpathway may result in the development of an effective adjuncttherapy to treat this deadly disease.Consistent with our findings that serotonin metabolism is

dysregulated in cholangiocarcinoma, increased serotonin secretionis the defining feature of a number of other neuroendocrine tumorsclassified as carcinoid tumors (19–21). Within these tumors,serotonin is thought to stimulate cell proliferation (40, 41) andcause symptoms of the carcinoid syndrome, including diarrhea,and damage to the valves of the heart (42). Here, we show thatcholangiocarcinoma, while not strictly classified as carcinoidtumor, not only secreted serotonin but displayed features of aneuroendocrine phenotype, such as chromagranin A and NSEexpression (43).The expression of TPH1 has been shown to be strongly expressed

in midgut carcinoid tissue (44). The authors postulate that theexpression of TPH and other specific proteins on the cell surfacecan be recognized by CD8+ T cells and constitute an immunerecognition of the tumors, which may be of great interest whenpursuing an immunotherapeutic treatment strategy (44). The datapresented here supports the idea that the expression of TPH1 maybe involved in the process of tumor progression and cellproliferation.In support of our findings that MAO A expression is suppressed

in cholangiocarcinoma, many other neuroendocrine tumors thathave a dysregulated serotonin synthesis and secretion often have aconcomitant decrease in the expression of MAO A (45). In a normalcell, these amine oxidases exert an antiproliferative effect morethan likely because the products of amine oxidation, aldehydes,hydrogen peroxide, and other reactive oxygen species aresomewhat cytotoxic (45–47). Because in certain tumor cells, theexpression of such amine oxidases (including MAO A) aresuppressed, in addition to the mitogenic effects of the accumulat-ing serotonin, the potential cytotoxic, antiproliferative effects ofamine oxidation are bypassed (45, 46). The mechanism of thisdecrease in MAO A expression is unclear. However, researchershave shown that the promoter region of MAO A can behypermethylated under certain conditions (48). Because hyper-methylation and subsequent silencing of genes, such as tumorsuppressor genes, are a common event in the malignanttransformation of cholangiocarcinoma (49), it is therefore conceiv-able that hypermethylation of the MAO A promoter contributes tothe suppression of expression. Indeed, treatment of cholangiocar-cinoma cell lines with a DNA methyltransferase inhibitor restoresthe expression of MAO A.11 This is a topic of ongoing research inour laboratory.As mentioned previously, the consequences of increased

serotonin production on tumor growth depend upon the tumortype in question and, perhaps, the predominant serotonin receptorsubtype present (9). On one hand, serotonin is known as a growth

11 S. DeMorrow et al., unpublished observation.





factor for several types of nontumoral cells (10, 11), and it has beenproposed to take part in the autocrine loops of growth factorscontributing to cell proliferation in aggressive tumors, such assmall cell lung carcinoma (12), choriocarcinoma (50), and bladdercancer (15). Depending on the tumor type, either serotonin type 2or serotonin type 1 receptor antagonists have been found to inhibitthe serotonin-induced increase in tumor growth (13–15, 50). Incontrast, several studies have also reported that serotonin and aserotonin type 2 receptor agonist can inhibit tumor growth (18).This effect may be related with the specific vasoconstrictive effectof serotonin and a serotonin type 2 receptor agonist on the vesselsirrigating the tumor (16–18). Here, we show a proliferative effect ofserotonin on cholangiocarcinoma growth, and the inhibition ofserotonin production effectively inhibits tumor growth. Further-more, we could show that inhibition of the serotonin receptors5HTR 1A, 5HTR 2A, 5HTR 2B, 5HTR 4, and 5HTR 6 effectivelyblocked the growth-promoting effects of serotonin. The effects ofincreased serotonin accumulation on other aspects of cholangio-carcinoma tumorogenesis, such as malignant transformation,metastatic activity, and angiogenesis, are also being studied inour laboratory.In conclusion, the data presented here indicate a dysregulated

serotonin metabolic pathway in cholangiocarcinoma comparedwith nonmalignant cholangiocytes. Specifically, there is an increasein the expression of TPH1, the enzyme responsible for serotonin

synthesis, and a decreased expression of MAO A, the enzymeresponsible for serotonin degradation. This leads to an increasedaccumulation and secretion of serotonin from cholangiocarcinomaand an increase in serotonin in the bile from cholangiocarcinomapatients. Specific inhibition of serotonin production leads to asuppression of tumor growth in a xenograft model of cholangio-carcinoma, which suggests that agents that modulate themetabolism of serotonin may be useful therapeutic tools for thetreatment of this devastating cancer.

Disclosure of Potential Conflicts of Interest

The authors of this article have no financial arrangements to disclose.

Acknowledgments

Received 6/4/2008; revised 8/6/2008; accepted 9/4/2008.Grant support: NIH K01 grant DK078532 (S. DeMorrow); Veterans Affairs Merit

Award, Veterans Affairs Research Scholar Award, and NIH grants DK062975 andDK58411 (G. Alpini); MIUR grant 2005067975_004 (M. Marzioni); MIUR grant2006068958_001 (Department of Gastroenterology, Universita Politecnica delleMarche, Ancona, Italy); MIUR grant PRIN 2005 and faculty funds (E. Gaudio); andMIUR grant 2005067975_002 (D. Alvaro).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Glen Cryer of Scott & White Hospital Grants Administration Office forhis assistance with proof reading and the Scott & White Hospital animal facility stafffor assistance with animal surgical models.

Cancer Research


References

1. Alpini G, McGill JM, LaRusso NF. The pathobiology ofbiliary epithelia. Hepatology 2002;35:1256–68.

2. Sirica AE. Cholangiocarcinoma: molecular targetingstrategies for chemoprevention and therapy. Hepatology2005;41:5–15.

3. Diksic M, Young SN. Study of the brain serotonergicsystem with labeled a-methyl-L-tryptophan. J Neuro-chem 2001;78:1185–200.

4. Ormsbee HS III, Fondacaro JD. Action of serotonin onthe gastrointestinal tract. Proc Soc Exp Biol Med 1985;178:333–8.

5. Kroeze WK, Kristiansen K, Roth BL. Molecular biologyof serotonin receptors structure and function at themolecular level. Curr Top Med Chem 2002;2:507–28.

6. Martel F. Recent advances on the importance of theserotonin transporter SERT in the rat intestine.Pharmacol Res 2006;54:73–6.

7. Shih JC. Cloning, after cloning, knock-out mice, andphysiological functions of MAO A and B. Neurotoxicol-ogy 2004;25:21–30.

8. Ekstedt B. Substrate specificity of monoamine oxidasein pig liver mitochondria. Med Biol 1979;57:220–3.

9. Vicaut E, Laemmel E, Stucker O. Impact of serotoninon tumour growth. Ann Med 2000;32:187–94.

10. Nocito A, Georgiev P, Dahm F, et al. Platelets andplatelet-derived serotonin promote tissue repair afternormothermic hepatic ischemia in mice. Hepatology2007;45:369–76.

11. Yang M, Li K, Ng PC, et al. Promoting effects ofserotonin on hematopoiesis: ex vivo expansion of cordblood CD34+ stem/progenitor cells, proliferation ofbone marrow stromal cells and anti-apoptosis. StemCells 2007;25:1800–6.

12. Vicentini LM, Cattaneo MG, Fesce R. Evidence forreceptor subtype cross-talk in the mitogenic action ofserotonin on human small-cell lung carcinoma cells. EurJ Pharmacol 1996;318:497–504.

13. Siddiqui EJ, Shabbir M, Mikhailidis DP, ThompsonCS, Mumtaz FH. The role of serotonin (5-hydroxytryp-tamine1A and 1B) receptors in prostate cancer cellproliferation. J Urol 2006;176:1648–53.

14. Sonier B, Arseneault M, Lavigne C, Ouellette RJ,Vaillancourt C. The 5-HT2A serotoninergic receptor is

expressed in the MCF-7 human breast cancer cell lineand reveals a mitogenic effect of serotonin. BiochemBiophys Res Commun 2006;343:1053–9.

15. Siddiqui EJ, Shabbir MA, Mikhailidis DP, Mumtaz FH,Thompson CS. The effect of serotonin and serotoninantagonists on bladder cancer cell proliferation. BJU Int2006;97:634–9.

16. Huhnt W, Lubbe AS. Venules and arterioles inxenotransplanted human colon adenocarcinoma criti-cally constrict with hyperthermia and serotonin. JCancer Res Clin Oncol 1995;121:267–74.

17. Huhnt W, Lubbe AS. Growth, microvessel densityand tumor cell invasion of human colon adenocar-cinoma under repeated treatment with hyperthermiaand serotonin. J Cancer Res Clin Oncol 1995;121:423–8.

18. Stucker O, Vicaut E, Teisseire B. Specific response to5-HT2 agonists by arterioles linked to Meth A tumors inmice. Am J Physiol 1992;262:H704–9.

19. di Sant’Agnese PA. Neuroendocrine cells of theprostate and neuroendocrine differentiation in prostaticcarcinoma: a review of morphologic aspects. Urology1998;51:121–4.

20. Lertprasertsuke N, Tsutsumi Y. Neuroendocrinecarcinoma of the urinary bladder: case report andreview of the literature. Jpn J Clin Oncol 1991;21:203–10.

21. Memon MA, Nelson H. Gastrointestinal carcinoidtumors: current management strategies. Dis ColonRectum 1997;40:1101–18.

22. Jones EA, Bergasa NV. The pathogenesis andtreatment of pruritus and fatigue in patients withPBC. Eur J Gastroenterol Hepatol 1999;11:623–31.

23. Swain MG, Maric M. Improvement in cholestasis-associated fatigue with a serotonin receptor agonistusing a novel rat model of fatigue assessment.Hepatology 1997;25:291–4.

24. Marzioni M, Glaser S, Francis H, et al. Autocrine/paracrine regulation of the growth of the biliary tree bythe neuroendocrine hormone serotonin. Gastroenterol-ogy 2005;128:121–37.

25. Knuth A, Gabbert H, Dippold W, et al. Biliaryadenocarcinoma. Characterisation of three new humantumor cell lines. J Hepatol 1985;1:579–96.

26. Kusaka Y, Muraoka A, Tokiwa T, Sato J. [Establish-

ment and characterization of a human cholangiocellularcarcinoma cell line]. Hum Cell 1988;1:92–4.

27. Saijyo S, Kudo T, Suzuki M, et al. Establishment of anew extrahepatic bile duct carcinoma cell line, TFK-1.Tohoku J Exp Med 1995;177:61–71.

28. DeMorrow S, Glaser S, Francis H, et al. Opposingactions of endocannabinoids on cholangiocarcinomagrowth: recruitment of fas and fas ligand to lipid rafts.J Biol Chem 2007;282:13098–113.

29. Shimizu Y, Demetris AJ, Gollin SM, et al. Two newhuman cholangiocarcinoma cell lines and their cytoge-netics and responses to growth factors, hormones,cytokines or immunologic effector cells. Int J Cancer1992;52:252–60.

30. MiyagiwaM, Ichida T, Tokiwa T, Sato J, Sasaki H. A newhuman cholangiocellular carcinoma cell line (HuCC-T1)producing carbohydrate antigen 19/9 in serum-freemedium. In vitro Cell Dev Biol 1989;25:503–10.

31. Storto PD, Saidman SL, Demetris AJ, Letessier E,Whiteside TL, Gollin SM. Chromosomal breakpoints incholangiocarcinoma cell lines. Genes ChromosomesCancer 1990;2:300–10.

32. Grubman SA, Perrone RD, Lee DW, et al. Regulationof intracellular pH by immortalized human intra-hepatic biliary epithelial cell lines. Am J Physiol 1994;266:G1060–70.

33. Kanno N, Glaser S, Chowdhury U, et al. Gastrininhibits cholangiocarcinoma growth through increasedapoptosis by activation of Ca2+-dependent proteinkinase C-a. J Hepatol 2001;34:284–91.

34. Liu Q, Yang Q, Sun W, et al. Discovery andcharacterization of novel tryptophan hydroxylase inhib-itors that selectively inhibit serotonin synthesis in thegastrointestinal tract. J Pharmacol Exp Ther 2008;325:47–55.

35. Fava G, Marucci L, Glaser S, et al. g-Aminobutyricacid inhibits cholangiocarcinoma growth by cyclicAMP-dependent regulation of the protein kinase A/extracellular signal-regulated kinase 1/2 pathway. Can-cer Res 2005;65:11437–46.

36. LeSage G, Glaser S, Ueno Y, et al. Regression ofcholangiocyte proliferation after cessation of ANITfeeding is coupled with increased apoptosis. Am JPhysiol Gastrointest Liver Physiol 2001;281:G182–90.





37. Carpino G, Morini S, Ginanni Corradini S, et al. a-SMA expression in hepatic stellate cells and quantitativeanalysis of hepatic fibrosis in cirrhosis and in recurrentchronic hepatitis after liver transplantation. Dig LiverDis 2005;37:349–56.

38. Alvaro D, Alpini G, Onori P, et al. Estrogens stimulateproliferation of intrahepatic biliary epithelium in rats.Gastroenterology 2000;119:1681–91.

39. Gaudio E, Onori P, Pannarale L, Alvaro D. Hepaticmicrocirculation and peribiliary plexus in experimentalbiliary cirrhosis: a morphological study. Gastroenterol-ogy 1996;111:1118–24.

40. Cattaneo MG, Palazzi E, Bondiolotti G, VicentiniLM. 5-HT1D receptor type is involved in stimula-tion of cell proliferation by serotonin in humansmall cell lung carcinoma. Eur J Pharmacol 1994;268:425–30.

41. Codignola A, Tarroni P, Cattaneo MG, Vicentini LM,Clementi F, Sher E. Serotonin release and cell prolifer-

ation are under the control of a-bungarotoxin-sensitivenicotinic receptors in small-cell lung carcinoma celllines. FEBS Lett 1994;342:286–90.

42. van der Horst-Schrivers AN, Wymenga AN, Links TP,Willemse PH, Kema IP, de Vries EG. Complications ofmidgut carcinoid tumors and carcinoid syndrome.Neuroendocrinology 2004;80 Suppl 1:28–32.

43. Kasprzak A, Zabel M, Biczysko W. Selected markers(chromogranin A, neuron-specific enolase, synaptophy-sin, protein gene product 9.5) in diagnosis andprognosis of neuroendocrine pulmonary tumours. PolJ Pathol 2007;58:23–33.

44. Vikman S, Giandomenico V, Sommaggio R, Oberg K,Essand M, Totterman TH. CD8(+) T cells againstmultiple tumor-associated antigens in peripheral bloodof midgut carcinoid patients. Cancer Immunol Immun-other 2008;57:399–409.

45. Pietrangeli P, Mondovi B. Amine oxidases andtumors. Neurotoxicology 2004;25:317–24.

46. Toninello A, Salvi M, Pietrangeli P, Mondovi B.Biogenic amines and apoptosis: minireview article.Amino Acids 2004;26:339–43.

47. Ou XM, Chen K, Shih JC. Monoamine oxidase A andrepressor R1 are involved in apoptotic signalingpathway. Proc Natl Acad Sci U S A 2006;103:10923–8.

48. Pinsonneault JK, Papp AC, Sadee W. Allelic mRNAexpression of X-linked monoamine oxidase A (MAO A)in human brain: dissection of epigenetic and geneticfactors. Hum Mol Genet 2006;15:2636–49.

49. Stutes M, Tran S, DeMorrow S. Genetic andepigenetic changes associated with cholangiocarci-noma: from DNA methylation to microRNAs. World JGastroenterol 2007;13:6465–9.

50. Sonier B, Lavigne C, Arseneault M, Ouellette R,Vaillancourt C. Expression of the 5-HT2A serotoninergicreceptor in human placenta and choriocarcinoma cells:mitogenic implications of serotonin. Placenta 2005;26:484–90.



2008;68:9184-9193. Cancer Res Gianfranco Alpini, Pietro Invernizzi, Eugenio Gaudio, et al. GrowthCholangiocarcinoma, which Has Implications for Tumor Serotonin Metabolism Is Dysregulated in

Updated version

http://cancerres.aacrjournals.org/content/68/22/9184

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2008/11/12/68.22.9184.DC1.html

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/68/22/9184.full.html#ref-list-1

This article cites 49 articles, 8 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/68/22/9184.full.html#related-urls

This article has been cited by 11 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

[email protected] at

To request permission to re-use all or part of this article, contact the AACR Publications


http://cancerres.aacrjournals.org/content/68/22/9184

http://cancerres.aacrjournals.org/content/suppl/2008/11/12/68.22.9184.DC1.html

http://cancerres.aacrjournals.org/content/68/22/9184.full.html#ref-list-1

http://cancerres.aacrjournals.org/content/68/22/9184.full.html#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

mailto:[email protected]


serotonin metabolism is dysregulated in cholangiocarcinoma, which has implications for tumor growth

Documents