Aminobutyric Acid Inhibits Cholangiocarcinoma Growth by Cyclic AMP–Dependent Regulation of the Protein Kinase A/Extracellular Signal-Regulated Kinase 1/2 Pathway

Research Article

;-Aminobutyric Acid Inhibits Cholangiocarcinoma Growth by Cyclic AMP–Dependent Regulation of the Protein Kinase A/Extracellular Signal-Regulated Kinase 1/2 Pathway

Giammarco Fava,3,5 Luca Marucci,5 Shannon Glaser,4 Heather Francis,4 Sharon De Morrow,4 Antonio Benedetti,5 Domenico Alvaro,6 Julie Venter,3 Cynthia Meininger,3 Tushar Patel,2 Silvia Taffetani,2,5 Marco Marzioni,5 Ryun Summers,3 Ramona Reichenbach,1 and Gianfranco Alpini1,2,3

1Central Texas Veterans Health Care System, Research Service; Departments of 2Medicine and 3Medical Physiology; 4Division R&E, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, Temple, Texas; 5Department of Gastroenterology, Polytechnic University of Marche, Ancona, Italy; and 6Division of Gastroenterology, La Sapienza University, Rome, Italy

Abstract and tissues (2–4). In addition to the CNS, the liver represents the We studied the effect of the inhibitory neurotransmitter, most important site of synthesis and metabolism of GABA (5). ;-aminobutyric acid (GABA), in the regulation of cholangio- Hepatocytes absorb GABA from the portal circulation (6), express GABA receptors (7), and contain enzymes responsible for GABA carcinoma growth. We determined the in vitro effect of GABA on the proliferation of the cholangiocarcinoma cell lines (Mz- synthesis (e.g., monoamine oxidase, diaminooxidase, and glutamic ChA-1, HuH-28, and TFK-1) and evaluated the intracellular acid decarboxylase; ref. 8) and catabolism (e.g., GABA-transaminase; pathways involved. The effect of GABA on migration of ref. 9). GABA action is mediated by two classes of receptors. The Mz-ChA-1 cells was also evaluated. In vivo, Mz-ChA-1 cells ionotropic GABAA and GABAC receptors are pentameric chloride were s.c. injected in athymic mice, and the effects of GABA on channels and are constituted, respectively, by combinations of a h g y q u tumor size, tumor cell proliferation, apoptosis, collagen several subunit types ( 1-6, 1-3, 1-3, , , , and k) and by only single quantity, and the expression of vascular endothelial growth or multiple subunits (U1-3). The metabotropic GABAB receptor is factor-A (VEGF-A) and VEGF-C (cancer growth regulators) associated with G proteins and exists as R1a, R1b, and R2 isoforms were measured after 82 days. GABA decreased in vitro (10). GABA has inhibitory effects on gastric cancer (11), colon carcinoma (12), and hepatocarcinoma (13); GABA content and GAD cholangiocarcinoma growth in a time-dependent and dose- dependent manner, by both cyclic AMP/protein kinase A– and activity are increased in neoplastic tissues, such as colon (14) and 2+ gastric cancer (15). The role of GABA in the regulation of D-myo-inositol-1,4,5-thriphosphate/Ca -dependent pathways, leading to down-regulation of extracellular signal-regulated cholangiocarcinoma growth is unknown. We posed these questions: kinase 1/2 phosphorylation. Blocking of GABA , GABA , and (a) Do malignant cholangiocytes express GABA receptors? (b) Does A B GABA regulate cholangiocarcinoma growth? (c) Do all three GABA GABAC receptors prevented GABA inhibition of cholangiocarcinoma proliferation. GABA inhibited Mz-ChA-1 cell migra- receptor subtypes play a role in mediating this GABA effect on tion and, in vivo, significantly decreased tumor volume, tumor malignant cholangiocytes? (d) Is GABA regulation of cholangio- cell proliferation, and VEGF-A/C expression whereas increas- carcinoma growth associated with increased D-myo-inositol-1,4,5- ing apoptosis compared with controls. An increase in collagen thriphosphate (IP3) and cyclic AMP (cAMP) synthesis, which leads was evident in GABA-treated tumors. GABA decreases biliary to changes in phosphorylation of protein kinase A (PKA)/ cancer proliferation and reduces the metastatic potential of extracellular signal-regulated kinase 1/2 (ERK1/2)? (e) Does GABA cholangiocarcinoma. GABA may represent a therapeutic agent reduce the migration of cholangiocarcinoma cells? (f ) Does GABA inhibit cholangiocarcinoma growth in an in vivo model of biliary for patients affected by malignancies of the biliary tract. (Cancer Res 2005; 65(24): 11437-46) cancer?

Introduction Materials and Methods Cholangiocarcinoma is characterized by the malignant prolifer- Materials ation of cholangiocytes, which line intrahepatic and extrahepatic Reagents were purchased from Sigma (St. Louis, MO) and antibodies for biliary ducts. Biliary tumors display high malignance and poor immunohistochemistry and immunoblotting from Santa Cruz Biotechnol- prognosis (1). No established treatments exist for this tumor. ogy (Santa Cruz, CA), unless differently indicated. The GABAA receptor g-Aminobutyric acid (GABA) is the most important inhibitory antagonist, bicuculline, was purchased from Tocris Cookson, Inc. (Ellisville, neurotransmitter in the central nervous system (CNS; ref. 2). GABA MO). The cleaved caspase-3 antibody was purchased from Cell Signaling is also present in the peripheral nervous system and several organs (Beverly, MA). RIA kits for the determination of intracellular cAMP and D-myo-inositol-1,4,5-thriphosphate (IP3) levels were purchased from Amer- sham (Arlington Heights, IL). Nitrocellulose membranes (0.2 Am) were purchased from Pierce Biotechnology (Rockford, IL). Slides were examined Requests for reprints: Gianfranco Alpini, The Texas A&M University System with a microscope Olympus BX 40, Olympus Optical Co. Ltd. (Tokyo, Japan). Health Science Center, College of Medicine, Medical Research Building, 702 Southwest H.K. Dodgen Loop, Temple, TX 76504. Phone: 254-742-7044; Fax: 254-724-5944; E-mail: [email protected]. Cell Culture I2005 American Association for Cancer Research. We used three human cholangiocarcinoma cell lines (Mz-ChA-1, HuH-28, doi:10.1158/0008-5472.CAN-05-1470 and TFK-1) with different origins. Mz-ChA-1 cells, from human gallbladder www.aacrjournals.org 11437 Cancer Res 2005; 65: (24). December 15, 2005

(16), were a gift from Dr. G. Fitz (University of Texas Southwestern Medical 24 hours of incubation, and fresh GABA at the specified concentrations, in Center, Dallas, TX). HuH-28 cells, from human intrahepatic bile duct (17), the absence or presence of the preincubating factors, was added. Mz-ChA-1, and TFK-1 cells, from human extrahepatic bile duct (18), were acquired HuH-28, and TFK-1 cells were also stimulated with GABA (100 Amol/L) up from Cancer Cell Repository, Tohoku University, Japan. Cells were to 72 hours. Cell proliferation was assessed using a colorimetric cell maintained at standard conditions as described (19). The human proliferation assay (CellTiter 96Aqueous, Promega Corp., Madison, WI), and immortalized, nonmalignant cholangiocyte cell line, H69 (from Dr. G.J. absorbance was measured at 490 nm by a microplate spectrophotometer Gores, Mayo Clinic, MN), was cultured as described (20). (Versamax, Molecular Devices, Sunnyvale, CA). Proliferation index was ; calculated as the ratio of the absorbance of cells incubated with GABA Expression of -Aminobutyric Acid Receptors compared with controls. h Immunofluorescence. We detected GABAA -3 and GABAB R1 receptor Proliferating cell nuclear antigen protein expression. Mz-ChA-1 cells subunits in H-69, Mz-ChA-1, HuH-28, and TFK-1 cells. We selected these (1.0 Â 106) were incubated in six-well plates until 70% confluence and h particular isoforms because GABAA -3 and GABAB R1 subunits were already subsequently stimulated for up to 48 hours with 0.2% BSA (basal), GABA detected in the liver, and no studies about their expression and function on (1 and 10 Amol/L), or GABA (100 Amol/L) in the presence or absence of 1 hour cholangiocytes were available (7, 21). There are no commercially available preincubation with Rp-cAMP116816 (100 Amol/L; ref. 26), BAPTA/AM antibodies against the GABAC receptor. Cells were seeded into six-well dishes (an intracellular Ca2+chelator, 5 Amol/L; ref. 19), bicuculline (100 Amol/L; containing a sterile coverslip on the bottom of each well. Cells were allowed ref. 27), phaclofen (100 Amol/L; ref. 27), or TPMPA (50 Amol/L; ref. 27). to adhere overnight, washed once in cold PBS, fixed to the coverslip with Medium was replaced every 24 hours, and fresh GABA at the specified 4% paraformaldehyde (in PBS) at room temperature for 5 minutes, concentrations, in the absence or presence of preincubating factors, was permeabilized in PBS containing 0.2% Triton X-100 (PBST), and blocked in added. 4% bovine serum albumin (BSA in PBST) for 1 hour. GABAA and GABAB Following two washes with ice-cold PBS, cells were resuspended in lysis receptor antibodies were diluted 1:100 in 1% BSA/PBST, added to the buffer and sonicated six times (30-second bursts). After electrophoresis, j Â coverslips, and incubated overnight at 4 C. Cells were washed 3 10 minutes protein samples were transferred to nitrocellulose membranes. Immuno- in PBST, and a 1:100 dilution (in 1% BSA/ PBST) of cy3-conjugated secondary blots were done as described (19, 22). The amount of protein loaded was antibodies (Jackson Immunochemicals, West Grove, PA) was added for evaluated by immunoblots for h-actin (30). The intensity of the bands was 2 hours at room temperature. Cells were washed again and mounted determined by scanning video densitometry using the phospho-imager, V into microscope slides with Antifade gold containing 4 ,6-diamidino-2- Storm 860 (Amersham Biosciences, Piscataway, NJ) using the ImageQuant phenylindole as a counterstain (Molecular Probes, Eugene, OR). Negative TLV 2003.02 (Little Chalfont, Buckinghamshire, England). controls were done with the omission of the respective primary antibodies. Images were taken on an Olympus IX71 fluorescence microscope with a DP70 Evaluation of the Intracellular Mechanism by which digital camera. For the merged pictures, images from each channel were ;-Aminobutyric Acid Regulates Cholangiocarcinoma Growth overlaid electronically using the Adobe Photoshop software. Effect of ;-aminobutyric acid on cyclic AMP and D-myo-inositol- Immunoblots. Following trypsinization, H-69, Mz-ChA-1, TFK-1, and 1,4,5-thriphosphate levels. After trypsinization, Mz-ChA-1 cells were HuH-28 cells (1 Â 106) were resuspended in lysis buffer as described (19, 22) incubated at 37jC for 1 hour (22) to regenerate membrane proteins damaged and sonicated six times (30-second bursts). We did immunoblots to detect by trypsin (22). Cells (1 Â 105) were incubated at room temperature for GABAA h-3 and GABAB R1 receptor subunits as described (19, 22). 10 minutes with 0.2% BSA (basal) or GABA (100 Amol/L) in the absence or Molecular analysis. We evaluated the expression of GABAC receptor by presence of a 10-minute preincubation with bicuculline (100 Amol/L; ref. 27), reverse transcription-PCR (RT-PCR) using total RNA from H-69, Mz-ChA-1, phaclofen (100 Amol/L; ref. 28), or TPMPA (50 Amol/L; ref. 27). cAMP levels HuH-28, and TFK-1 cholangiocarcinoma cells. cDNA synthesis was done as were measured by RIA. Mz-ChA-1 cells (5 Â 106) were stimulated for described (23). For PCR, we used primers specific for human GABAC (sense 10 minutes at 22jC with 0.2% BSA (control) or GABA (100 Amol/L). The 5V-CAGTGAGTCTGAGGCCAACA-3V, corresponding to nucleotides 1144- intracellular levels of IP3 were measured by the RIA assay system. 1164; antisense 5V-AATGGCATGGGTATTCTGGA-3V,correspondingto Is ;-aminobutyric acid modulation of cholangiocarcinoma growth nucleotides 1306-1326; refs. 23, 24). PCR was done at the same conditions associated with changes in the protein kinase A/mitogen-activated described (23). Ten microliters of each PCR product were separated on a protein kinase (extracellular signal-regulated kinase1/2, p38, and

1.5% agarose gel and stained with ethidium bromide. The intensity of the c-Jun NH2-terminal kinase) pathway? During incubation, medium was bands was determined by scanning video densitometry using the replaced every 24 hours, and fresh GABA at the specified concentrations ChemiImager low-light imaging system (Alpha Innotech Corp., San was added. After incubation of Mz-ChA-1 cells with 0.2% BSA (basal) or Leandro, CA). Sequence analysis indicates that the RT-PCR fragment for GABA (100 Amol/L) for 48 hours, culture plates were kept on ice for human GABAC is about 100% homologous to the published human GABAC 30 minutes, then cells were scraped and lysed. Proteins (2.5 Ag/lane) were receptor (refs. 23, 24; Genbank accession no. NM_002043). resolved, and immunoblots were done (19, 22). Membranes were ; subsequently stripped and reprobed to evaluate the expression of the Effect of -Aminobutyric Acid on Malignant Cholangiocyte corresponding total proteins. Growth 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul- Evaluation of the Effect of ;-Aminobutyric Acid on Mz-ChA-1 fophenyl)-2H-tetrazolium, inner salt proliferation assay. Cholangiocar- Cell Migration cinoma growth was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3- Mz-ChA-1 cells were plated on six-well culture dishes and grown using carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) the Connaught Medical Research Laboratories medium 1066 supplemented proliferation assay (an index of changes in cell number; ref. 25) in the with 10% fetal bovine serum. After reaching confluence, cell layers were Mz-ChA-1, HuH-28, and TFK-1 cell lines. After trypsinization, cells were disrupted by producing a linear wound with a sterile pipette tip (31). The seeded into 96-well plates (10,000 per well) in a final volume of 200 AL cells were then washed with PBS to remove debris and incubated in the medium. Cells were stimulated for 48 hours with GABA (1 Amol/L to absence or presence of GABA (100 Amol/L). Hydroxyurea (5 mmol/L) was 3 mmol/L) in the absence or presence of 1 hour preincubation with added to the system to arrest cell proliferation (32). At time 0 and after Rp-cAMP116816 (a PKA inhibitor, 100 Amol/L; ref. 26), bicuculline (a GABAA 24, 48, 72, and 96 hours, samples were examined by a phase-contrast receptor antagonist, 100 Amol/L; ref. 27), phaclofen (a GABAB receptor microscope (Olympus, CK2, Tokyo, Japan), and the wound size was antagonist, 100 Amol/L; ref. 28), (1,2,5,6-tetrahydropyridin-4-yl)methylphos- measured at five random sites perpendicular to the wound. At time 0 and phinic acid (TPMPA; 50 Amol/L, a GABAC receptor antagonist; ref. 27), or after 96 hours of incubation, MzChA-1 cells were resuspended in PBS, and sodium valproate (an inhibitor of GABA catabolism, 250-1,000 Amol/L; cell size was assessed by flow cytometry (FACSCalibur, Becton Dickinson, ref. 29). Because bicuculline was dissolved in 0.1% DMSO, we used Mz-ChA-1 San Jose, CA). The images were acquired by Cellquest software (10,000 cells treated with 0.1% DMSO as control. Medium was replaced every events evaluated).

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Effect of ;-Aminobutyric Acid on Cholangiocarcinoma in Mz-ChA-1 cells incubated for 48 hours with GABA at increasing Tumor Implanted in Nude Mice concentrations (1-100 Amol/L; Fig. 2C). The inhibitory effect of Treatment Schedule. Male 8-week-old BALB/c nude (nu/nu) mice were GABA (100 Amol/L) persisted up to 72 hours (Fig. 2D). Similar purchased from Taconic Farms (Germantown, NY). The mice were kept in a results were obtained using HuH-28 and TFK-1 cell lines (data not temperature-controlled environment (20-22jC) with a 12-hour light/dark shown). MTS assays and immunoblots for PCNA showed that cycle and with free access to drinking water and to standard mouse chow. Mz-ChA-1 cells (3 Â 106) were suspended in 0.5 mL of extracellular matrix GABA effects were significantly blocked by bicuculline, phaclofen, gel and injected s.c. in the left back flank of these animals. Mice were and TPMPA (Fig. 3A-C). As shown in other cell lines (37), 0.1% divided into two groups: (a) the first group (n = 4, control) received 0.9% DMSO had no inhibitory effect on Mz-ChA-1 cell proliferation NaCl injection (150 AL) into the implanted tumor; and (b) the second group (n = 4) received GABA injections (1000 mg/kg body weight dissolved in 150 AL of 0.9% NaCl; ref. 33) into the tumor. The same operator did the injections every other day starting from ‘‘day 0,’’ when the tumors were implanted, for 82 days. Tumor variables were measured twice a week by an electronic caliper, and volume was determined as tumor volume (mm3)= 0.5 Â [length (mm) Â width (mm) Â height (mm)]. The measurements started from the third week, day 23, when the tumor mass was well established. A third operator, in a coded and blinded fashion, evaluated morphometric variables. Latency represents the time for the tumor to increase to 150% of the initial volume. After 82 days, mice were anaesthetized with sodium pentobarbital (50 mg/kg IP) and sacrificed according to institutional guidelines. Before sacrifice, serum was obtained, and aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine were measured. 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. Morphologic Analysis of Tumor Tissues. A third operator dissected tumor tissues from mice. Neoplastic tissues were fixed in formalin, embedded in paraffin, processed for histopathology, stained with H&E for routine examination or with Sirius red for collagen visualization, and examined by light microscopy. Tumor protein selectively expressed by cholangiocytes was evaluated after CK-7 immunohistochemical staining; cell proliferation was assessed by immunohistochemistry for proliferating cell nuclear antigen (PCNA; ref. 34). Tumor sections (n = 4) from each group were stained for CK-7 and PCNA, as described (34, 35), and for

GABAA and GABAB receptors. Following staining, sections were counter- stained with hematoxylin and examined with a microscope. Tumor sections (n = 4) from each selected group of animals were stained by immunohistochemistry with antibodies specific for vascular endothelial growth factor-A (VEGF-A) or VEGF-C, diluted 1:400. Apoptosis was evaluated by staining for cleaved caspase-3 (36). Negative controls were obtained by incubating the tumor sections only with the secondary antibody. Statistical Analysis All data are expressed as mean F SE. Differences between groups were analyzed by the Student’s unpaired t test, when two groups were analyzed and ANOVA, when more than two groups were analyzed. P < 0.05 was used to indicate statistical significance.

Results

Expression of ;-aminobutyric acid receptors. GABAA h-3 and GABAB R1 receptor subunits were detected in H-69, Mz-ChA-1, HuH28, and TFK-1 cells by immunofluorescence (Fig. 1A). The four cell lines expressed two bands, migrating, respectively, at f59 and f70 kDa for the GABAA h-3 and GABAB R1 receptor subunits on immunoblots (Fig. 1B) and a band for the GABAC receptor by RT- PCR (Fig. 1C). Effect of ;-aminobutyric acid on cholangiocarcinoma growth. GABA (1-100 Amol/L) decreased the proliferation of Mz-ChA-1 cells at 24 and 48 hours (Fig. 2A). The significant Figure 1. Immunofluorescence (A) and immunoblots (B) for GABAA and GABAB receptors in H-69 cholangiocytes and Mz-ChA-1, HuH-28, and TFK-1 inhibitory effect of GABA (100 Amol/L) persisted up to 72 hours cholangiocarcinoma cells. All the cell lines express GABAA and GABAB (Fig. 2B). GABA inhibited HuH-28 and TFK-1 cell proliferation up receptors. Two bands, migrating at f59 and f70 kDa, corresponding, respectively, to GABA h-3 and GABA R1 receptor subunits. C, RT-PCR to 72 hours of incubation (P < 0.05 versus basal values; data not A B analysis for GABAC receptor using total RNA from H-69, Mz-ChA-1, HuH-28, and shown). Immunoblots showed decreased PCNA protein expression TFK-1 cells. GABAC receptor subunit is expressed by all these cell lines. www.aacrjournals.org 11439 Cancer Res 2005; 65: (24). December 15, 2005

Figure 2. Dose-dependent and time-dependent effect of GABA on Mz-ChA-1 cell proliferation. A-C, GABA at (1-100 Amol/L) inhibits DNA synthesis of MzChA-1 cells at 24 and 48 hours of incubation. The inhibitory effect was assessed by MTS assay (A) and immunoblots for PCNA protein expression (C). The decrease in proliferation was significant using GABA at 10 and 100 Amol/L. Furthermore, MTS assay (B) and PCNA protein expression (D) show that GABA (100 Amol/L) decreases Mz-ChA-1 cell proliferation in a time-dependent fashion. Columns, means; bars, SE. *, P < 0.05 versus corresponding basal value.

(data not shown) when compared with untreated cells. The noma (22), the increase of intracellular cAMP levels, the 2+ inhibitory effect on cholangiocarcinoma proliferation was more subsequent activation of PKA, and also changes of [Ca ]i levels evident with higher GABA concentrations (up to 3 mmol/L; (19) by GABA stimulation play a role in GABA inhibition of Fig. 3B), even if, as previously shown (19), lower dose of gastrin cholangiocarcinoma growth. (10À7 mol/L) inhibited in vitro cholangiocarcinoma growth at Intracellular pathways associated with the inhibitory effect higher extent (40% with respect to basal values). The inhibition of of ;-aminobutyric acid on cholangiocarcinoma growth. In GABA intracellular catabolism, thus favoring stable levels of Mz-ChA-1 cells, GABA (100 Amol/L) significantly increased cAMP neuropeptide, potentiates GABA inhibitory effect on cell prolifer- levels compared with controls (168.3 F 3.2 versus 141.71 F 3.3 ation (Fig. 3B). Rp-cAMP116816 and BAPTA/AM partially fmol/100,000 cells, P < 0.05). This increase was prevented by impaired GABA inhibitory effect on cholangiocarcinoma growth preincubation with the GABAA, GABAB, and GABAC receptor when added individually (Fig. 3C). However, when added together, antagonists (115.52 F 5.1, 115.1 F 4.0, and 123.72 F 0.8 fmol/105 Rp-cAMP116816 and BAPTA/AM completely blocked GABA’s cells, respectively; P < 0.05 versus GABA-treated cells). inhibitory effect on cholangiocarcinoma growth (Fig. 3C). These Enhanced IP3 levels were evident following incubation of results indicate that similar to other studies in cholangiocarci- Mz-ChA-1 cells with 100 Amol/L GABA (1.51 F 0.08 versus 2.33

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F 0.14 pmol/106 cells, P < 0.05). In Mz-ChA-1 cells, GABA (100 Amol/L) increased the protein expression of phosphorylated PKA (p-PKA) and decreased ERK1/2 phosphorylation, despite no change of total isoforms, with respect to controls (Fig. 4). GABA did not alter the phosphorylation of p38 and c-Jun NH2-terminal kinase (JNK; data not shown). Evaluation of the effect of ;-aminobutyric acid on the migration of Mz-ChA-1 cells. The addition of GABA (100 Amol/L) to Mz-ChA-1 cells in culture increases the time necessary for wound closure. After 72 and 96 hours of incubation, there was a significant difference in wound size between the two groups of stimulated cells (Fig. 5B). After 96 hours, incubation with GABA induced an incomplete wound closure (Fig. 5B), and no difference in cell volume was evident in GABA-treated cells with respect to controls (Fig. 5A). The data supports the hypothesis that GABA inhibits Mz-ChA-1 cell migration. Effect of ;-aminobutyric acid on the growth of tumors implanted in nude mice. At 82 days, a significant difference in tumor size was found in GABA-treated mice compared with mice injected with vehicle only (control; 517.48 F 46.05 versus 1,122.89 F 137.31 mm3; P < 0.02; Fig. 5C-D). No significant difference in body weight was detected between the two groups (Table 1). There was a significant decrease in the tumor latency (25) in control mice versus mice treated with GABA (Table 1). Normal serum values of AST, ALT, and creatinine were detected in the two groups (Table 1). Immunohistochemistry for PCNA shows a decrease of proliferation of the tumor cells from GABA-treated mice compared with tumor cells from controls (Fig. 6). A decrease of positivity for CK-7 and for VEGF-A and VEGF-C of GABA-treated versus control tumors is shown (Fig. 6). This represents the first evidence for the presence of VEGF-A and VEGF-C proteins in Mz-ChA-1 cells. Immunohistochemistry for cleaved caspase-3 (Fig. 6) shows an increase in apoptotic tumor cells in GABA-treated versus control tumors. In mice, the morphologic evaluation of xenografts (Fig. 6, H&E) showed encapsulated tumors with several acinar or tubular structures resembling those of other adenocarcinomas and fibrotic tissue that is more prominent in GABA-treated animals (Fig. 6, Sirius red). An amorphous matrix negative after staining with Sirius red, Gomory (collagen), and Congo red (amyloid) was often observed between groups of cells in all the tumors. Randomly distributed necrotic areas were common without difference between control and treated tumors, whereas apoptotic bodies, evaluated either after cleaved caspase-3 or H&E staining (Fig. 6), were significantly higher in GABA-treated tumors. Moreover, the expression of GABAA and GABAB receptors is maintained in GABA

Figure 3. A-C, effect of GABA on Mz-ChA-1 cell proliferation in the presence of the selected inhibitors. A, MTS assay shows that GABA-induced decrease of cholangiocarcinoma proliferation at 48 hours is blocked by preincubation with bicuculline (100 Amol/L), phaclofen (100 Amol/L), and TPMPA (50 Amol/L) and partially by RpcAMP116816 (100 Amol/L). *, P < 0.05 versus other values. Columns, means; bars, SE. B, inhibitory effect on cholangiocarcinoma cell proliferation was more evident by using higher GABA concentrations (up to 3 mmol/L) and by inhibiting GABA intracellular catabolism with sodium valproate (250-100 Amol/L). C, measurement of PCNA protein expression in Mz-ChA-1 cells incubated for 48 hours in the absence or presence of GABA (100 Amol/L) plus selected inhibitors. GABA-induced decrease of PCNA protein expression was prevented by preincubation with bicuculline (100 Amol/L), phaclofen (100 Amol/L), TPMPA (50 Amol/L), and Rp-cAMP116816 (100 Amol/L) plus BAPTA/ AM (5 Amol/L) and attenuated with Rp-cAMP116816 and BAPTA/AM. *, P < 0.05 versus corresponding basal value. Columns, means; bars, SE. www.aacrjournals.org 11441 Cancer Res 2005; 65: (24). December 15, 2005

Figure 4. Effect of GABA on the phosphorylation of PKA/ERK1/2 pathway evaluated by immunoblots. GABA inhibition of cholangiocarcinoma growth was associated with an increase of p-PKA (A) and a decrease of phosphorylated ERK1/2 (p-ERK1/2) protein expression (B) at 48 hours of incubation. Columns, means of four experiments; bars, SE. *, P < 0.05 versus basal. treated, with respect to the control tumors, at the end of the form a complex loop leading to the decrease of cholangiocarci- treatment (Fig. 6). No organ damage was found by immunohis- noma cell proliferation. The importance of the crosstalk between 2+ tochemical analysis in the two groups of animals (data not cAMP- and Ca /IP3-related pathways has been described in 2+ shown). cholangiocytes (40, 41). We propose that cAMP- and Ca /IP3- dependent pathways are both activated in GABA-treated cholangiocarcinoma cells and have a synergistic effect in the inhibition Discussion of cholangiocarcinoma proliferation. We found that H-69 cholangiocytes and Mz-ChA-1, TFK-1, and Substances, such as gastrin, inhibit in vitro cholangiocarcinoma HuH-28 cholangiocarcinoma cells express GABAA, GABAB, and growth to a greater extent than GABA. Nevertheless, the inhibitory GABAC receptors. We show that GABA inhibits Mz-ChA-1, HuH-28, effect of GABA on cholangiocarcinoma cell proliferation was and TFK-1 cholangiocarcinoma growth, and GABA inhibition of confirmed by the in vivo study in nude mice. Injections of GABA cholangiocarcinoma growth is associated with increased intracel- into cholangiocarcinoma tissue implanted s.c. in nu/nu athymic lular IP3 and cAMP levels, PKA phosphorylation, and dephosphor- mice induced a decrease in cholangiocarcinoma proliferation. This ylation of ERK1/2 but not of p38 and JNK1/2. GABA inhibition of effect was macroscopically evident by the significant reduction of cholangiocarcinoma growth was mediated by interaction with the tumor mass in the GABA-treated mice at 82 days. We speculate GABAA, GABAB, and GABAC receptors. In a nude mouse model, we that the inhibition of the tumor growth by GABA is also due to a showed that GABA inhibits cholangiocarcinoma growth in vivo. blocking of the production or the release of trophic factors (e.g., In several types of cancers, the increase in cAMP levels is VEGF-A and VEGF-C), which are determinant for the vasculariza- accompanied by a decrease in cell growth (22, 38, 39). Enhanced tion, the development, and the growth of neoplasias. At 27 days cAMP levels in human meningioma cells inhibit growth (38), and after tumor implantation, the inhibitory effect of GABA (40%) on up-regulation of the cAMP/PKA pathway causes apoptosis leading cholangiocarcinoma growth in vivo was similar to lanreotide, a to a decrease in proliferation of malignant gliomas (39). somatostatin analogue (42). Moreover, the immunohistochemical Stimulation of a2-adrenergic receptors by UK14,304 decreases analysis of the malignant specimens showed decreased protein cholangiocarcinoma growth by enhancing cAMP levels, which expression of PCNA, VEGF-A, and VEGF-C, confirming the inhibit mitogen-activated protein kinase activity (22). inhibitory effect of GABA on cholangiocarcinoma growth. The We showed that GABA inhibits cholangiocarcinoma growth by inhibition of cancer growth induced by GABA was accompanied by interacting with the three GABA receptors subtypes (sensitivity to increased apoptosis in malignant cells and abundant fibrosis. In GABAA, GABAB, and GABAC receptor antagonists). Maintaining addition, an amorphous matrix of unknown significance was often stable levels of GABA by blocking GABA intracellular catabolism, observed between groups of cells in all tumors. Previous studies cholangiocarcinoma growth was inhibited to a greater extent. The suggest that GABA stimulates collagen synthesis and proliferation inhibition of cholangiocarcinoma growth by GABA was partially of human fibroblasts (43). Furthermore, the fibrogenic pathways blocked by incubation with RpcAMP116816 and BAPTA/AM but could be activated in response to apoptotic and necrotic processes, was totally blocked by incubation with RpcAMP116816 and more evident in GABA-treated tumors (44) with respect to the BAPTA/AM added together to the medium. These findings support controls. Studies have shown the relationship between the the hypothesis that GABA, through GABAA, GABAB, and GABAC GABAergic system and tumors originating from different organs receptor stimulation, activates several intracellular pathways, (e.g., colon, breast, stomach, and ovary). High levels of diamine 2+ involving cAMP and IP3/Ca , which, interacting with each other, oxidase, an enzyme producing GABA, are described in at least nine

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Figure 5. GABA reduces in vitro wound healing. Mz-ChA-1 cells were grown to confluence, and the monolayers were disrupted by generating a straight scrape wound. The cells were incubated in the absence or presence of GABA (100 Amol/L) and observed at time 0 and after 24, 48, 72, and 96 hours. A, after 96 hours, there was no significant alteration in volume of the cells incubated with GABA with respect to controls. B, an increase in time necessary to wound closure was observed following GABA stimulation. Columns, means of at least three separate experiments; bars, SE. *, P < 0.01 versus control. C-D, malignant xenograft growth was evaluated in mice treated with periodic injections of GABA or NaCl (controls) into the tumors. C, at 82 days, a significant difference in tumor size was macroscopically evident in GABA-treated mice compared with controls. D, at 82 days, the cholangiocarcinoma xenograft volume of mice periodically injected with GABA (1,000 mg/kg) was 517.48 F 46.05 versus 1,122.89 33 F 137.31 mm3 for the control mice. Points, mean tumor size (mm3); bars, SE.

human malignancies (45, 46). Ovarian cancers are associated with Table 1. Effect of GABA on cholangiocarcinoma xeno- elevated GABA levels, which are reflected in high urinary grafts growth and nude mice variables after 82 days of neurotransmitter levels, supporting the hypothesis that the mea- treatment surement of GABA, combined with other cancer-associated markers, may be useful as a noninvasive indicator for cancer Variable Control solution GABA diagnosis or during follow-up to evaluate the tumor response to therapy (47). Body weight (g) 5.32 F 0.47 5.35 F 1.1 (NS) Tumor latency (d) 11.7 F 2.80 34.7 F 3.9* In malignant conditions, an increase of diamines, polyamines, c 3 F F and activity of diamine oxidase could enhance GABA production Tumor size (mm ) 1,122.89 137.31 517.48 46.05 A F F (46). We suppose that the increase in content, production, and ALT (n.v. 28-132 g/L) 32.3 6.9 52.7 11.1 AST (n.v. 59-247 Ag/L) 55.2 F 17.3 62.0 F 11.9 secretion of GABA could represent a cell response and an immune Creatinine (n.v. 0.2-0.8 mg/dL) 0.2 F 0.02 0.2 F 0.02 defense mechanism against tumor development. However, even if malignant cells produce high GABA concentrations, such levels are much less (46, 47) than the doses of neuropeptide required to NOTE: Data are mean F SE of four mice. inhibit cancer cells proliferation. Thus, we suppose that the Abbreviations: NS, not significant; n.v., normal value. increased production of GABA by tumoral cells is insufficient to *P < 0.05 vs the corresponding value of control solution-treated mice. c block tumor growth, because inadequate concentrations of the P < 0.02 vs the corresponding value of control solution-treated mice. neuropeptide are achieved. www.aacrjournals.org 11443 Cancer Res 2005; 65: (24). December 15, 2005

Figure 6. A, GABA decreases proliferation and increases apoptosis of cholangiocarcinoma cells in an in vivo mouse model. Columns, means of four experiments; bars, SE. *, P < 0.05 versus PCNA-positive malignant cholangiocytes from control tumors. *, P < 0.02 versus CK-7-positive tumor cells from control mice. VEGF-A and VEGF-C expression were reduced in malignant cells from GABA-treated versus control mice. Columns, mean of four experiments; bars, SE. *, P < 0.05 versus VEGF A/C positive tumor cells from control mice. Expression of cleaved caspase-3 is increased in GABA-treated tumors. Columns, means of four experiments; bars, SE. *, P < 0.02 versus cleaved caspase-positive tumor cells from control mice. Negative controls were done with the omission of the respective primary antibodies.

From this assumption, it can be speculated that malignant that are known to be tropic factors for malignant cells. These tumor growth can be modulated by using agonists of the neurotransmitters are thus potentially able to inhibit the growth of GABAergic system. GABA has been shown to be an inhibitory tumors expressing such hormone receptors. However, we did not regulator for the migration of SW480 colon carcinoma cells (12). In find any evidence related to any change of the hormonal status in our study, we also showed that GABA has an antimigratory action GABA-treated mice. on Mz-ChA-1 cells; thus, it might reduce the metastatic potential of Although studies showed that chronic exposure of cultured cholangiocarcinoma. cerebral cortical neurons to GABA decreases GABAA receptor Because GABA and its receptor agonists (e.g., baclofen) cross the subunit protein (49), other works showed that prolonged exposure h blood-brain barrier, this neurotransmitter may interact with the of HEK 293 cells stably expressing recombinant a1 2g2s GABAA CNS and perhaps inhibit cancer cell proliferation by nervous receptors for GABA and muscimol results in up-regulation of system regulation of malignant tissue growth. The inhibitory effect receptor number (50). We show that at the end of the treatment, of GABA could also be due to the systemic modulation of the the expression of GABAA and GABAB was maintained in GABA- complex hormonal system. GABA and its agonists, interacting with treated tumors. the hypothalamic-hypophyseal axis, regulate the secretion of Prolonged systemic effects of GABA administration are usually several hormones (e.g., thyroid hormone, prolactin, GnRH, associated with decreased body weight. Further evidence that adrenocorticorticotropic hormone, and growth hormone; ref. 48) in vivo GABA acts directly on Mz-ChA-1 cells and not through

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Figure 6 Continued. B, cholangiocarcinoma cells forming several acinar or tubular structures (H&E). An amorphous matrix (#) localized between groups of cancer cells is observed in all the tumors. A damaged area (arrows) characterized by several apoptotic bodies and a few necrotic cells is present in GABA-treated tumors. Sirius red staining shows fibrotic septa (red) surrounding cholangiocarcinoma nodules. In GABA-treated tumor, a wide area of fibrotic tissue is present. #, amorphous matrix. Columns, means of four experiments; bars, SE. *, P < 0.05 versus control solution-treated tumor. The expression of GABAA and GABAB receptors is maintained in GABA treated with respect to control tumors at the end of the treatment. Original magnification, Â50; Â100 (cleaved caspase-3) and Â75 (GABA receptors).

systemic interaction with nervous or hormonal systems is given Acknowledgments by the fact that we did not find any change in body weight in Received 4/29/2005; revised 8/26/2005; accepted 9/29/2005. mice treated with control solutions compared with GABA- Grant support: Scott & White Hospital (G. Alpini, S. Glaser, and H. Francis); The treated mice. The absence of systemic toxicity of GABA was Texas A&M University System Health Science Center, College of Medicine (G. Alpini, confirmed by normal values of serum AST, ALT, and creatinine S. Glaser, and H. Francis); VA Research Scholar Award; VA Merit Award; NIH grants DK58411 (G. Alpini), DK062975 (G. Alpini), and DK69370 (T. Patel); and Ministero and by no evidence of organ damage in the two groups of dell’Istruzione, Universita`e Ricerca grants 2004068113_001 (A. Benedetti) and COFIN treated animals. In conclusion, our findings raise the possibility 2003060498_002 (D. Alvaro). of a novel therapeutic approach for the treatment of tumors of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance the biliary tract based on the modulation of the GABAergic with 18 U.S.C. Section 1734 solely to indicate this fact. system. We thank Stefania Saccomanno for her technical assistance.

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