Bile acids induce cyclooxygenase-2 expression in human pancreatic cancer cell lines
Olga N. Tucker1,4,
Andrew J. Dannenberg2,3,
Eun K. Yang2,3 and
Thomas J. Fahey, III1,3
1 Departments of Surgery and 2 Medicine, New York Presbyterian Hospital and Weill Medical College of Cornell University, New York, NY 10021, USA and 3 Strang Cancer Prevention Center, New York, NY 10021, USA
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Abstract
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To investigate a possible link between bile acids and the pathogenesis of pancreatic cancer, we determined whether conjugated or unconjugated bile acids induced cyclooxygenase-2 (COX-2) in two human pancreatic cancer cell lines, BxPC-3 and SU 86.86. Bile acids are known promoters of gastric and colon cancer. We demonstrated previously that COX-2, an enzyme that catalyzes the synthesis of prostaglandins, is over-expressed in human pancreatic adenocarcinoma. Both human pancreatic cell lines were treated with conjugated and unconjugated bile acids. COX-2 mRNA and protein were determined. In addition, prostaglandin E2 (PGE2) synthesis was measured. Treatment with conjugated or unconjugated bile acids for 3 h up-regulated COX-2 mRNA. Chenodeoxycholate (CD) or deoxycholate at concentrations ranging from 12.5 to 100 µM caused a dose-dependent induction of COX-2 protein with a maximal effect at 100 µM. Induction of COX-2 protein by CD and deoxycholate was detected after treatment for 6 h with maximal induction at 12 h. Taurochenodeoxycholate, a conjugated bile acid, also caused dose-dependent induction of COX-2 but higher concentrations of bile acid (2001200 µM) were required. Levels of cyclooxygenase-1 were unaffected by bile acid treatment. Unconjugated and conjugated bile acids caused 7- and 4-fold increases in PGE2 production, respectively. Taken together, these findings suggest a possible role for bile acids in the pathogenesis of pancreatic cancer.
Abbreviations: CD, chenodeoxycholic acid; COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; cPLA2, cytoplasmic phospholipase A2; DC, deoxycholic acid; FCS, fetal calf serum; GCDC, glycochenodeoxycholic acid; PGE2, prostaglandin E2; TCDC, taurochenodeoxycholic acid
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Introduction
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Pancreatic cancer is one of the most lethal neoplasms. The 5-year survival rate is <1.3% in the US, with a median survival of 4.1 months (1). Pancreaticoduodenectomy is the only potentially curable treatment. However, by the time most patients seek medical treatment for symptoms the disease is advanced, which precludes curative therapy. Future advances to successfully prevent and treat pancreatic cancer require a better understanding of the pathogenesis of this disease. This requires identification of specific etiological factors associated with an increased risk of pancreatic cancer and the elimination or reduction of exposure to these factors.
There appears to be an etiological relationship between the pathogenesis of pancreatic cancer and dietary factors, but direct evidence linking specific dietary carcinogens to pancreatic cancer has been difficult to establish. Epidemiological studies have demonstrated a positive correlation between ingestion of a western-style high fat diet and the incidence of pancreatic cancer (24). Diets with a high fat content stimulate bile secretion, which increases the availability of cholesterol, bile acids and their metabolites. There is considerable experimental evidence that bile acids play a role in the development of gastrointestinal tumors. Bile acids have been shown to act as tumor promoters both in vivo (57) and in vitro (8). Recent evidence suggests that bile acids may promote carcinogenesis by up-regulating cyclooxygenase-2 (COX-2) and prostaglandin production (911).
Cyclooxygenases catalyze the formation of prostaglandins from arachidonic acid. Two distinct COX enzymes have been described, a constitutive enzyme (COX-1) and an inducible form (COX-2). COX-1 is a housekeeping gene that is detected in most tissues under basal conditions, and is involved in homeostasis. COX-2 is an early-response gene that is induced rapidly by growth factors, tumor promoters, oncogenes and carcinogens (12). A large body of experimental data from animal and human studies suggests that COX-2 is important for tumor formation. COX-2 is up-regulated in transformed cells (13) and in a variety of cancers (1417), whereas levels of COX-1 are relatively constant. Moreover, a null mutation for COX-2 caused a marked reduction in the number and size of intestinal polyps in APCD716 mice, a murine model of familial adenomatous polyposis (18). In addition to the genetic evidence implicating COX-2 in carcinogenesis, newly developed selective inhibitors of COX-2 protect against gastrointestinal tumor formation (1921).
We and others have reported increased amounts of COX-2 in human pancreatic adenocarcinoma; COX-2 expression was localized to tumor cells (17,20,21). Most adenocarcinomas of the pancreas occur in the head of the gland, which is in close proximity to bile. In the current study, we examined the effect of bile acids on the expression of COX and synthesis of prostaglandins in cultured human pancreatic cancer cells. Evidence is presented that COX-2 is up-regulated by both unconjugated and conjugated bile acids.
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Materials and methods
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Materials
RPMI 1640, fetal calf serum (FCS), penicillin, streptomycin, COX-1, COX-2, cytoplasmic phospholipase A2 (cPLA2) and ß2-microglobulin primers were from Life Technologies (Grand Island, NY). Lowry protein assay kits, bile acids, phorbol 12-myristate 13-acetate, and secondary antibody to IgG conjugated to horseradish peroxidase were from Sigma Chemical (St Louis, MO). Goat polyclonal anti-human COX-1 and COX-2 antiserum were from Santa Cruz Biotechnology (Santa Cruz, CA). RNA isolation kit was from Qiagen (Chatsworth, CA). GeneAmp RNA PCR kits were from Perkin Elmer (Norwalk, CT). GenEluteTM Agarose Spin Columns were from Supelco (Bellefonte, PA). Enzyme immunoassay reagents for prostaglandin E2 (PGE2) assays, and COX-1 and COX-2 standards for immunoblotting were from Cayman Chemical Co. (Ann Arbor, MI). Nitrocellulose membranes were from Schleicher & Schuell (Keene, NH). Western blotting detection reagents (ECL) were from Amersham Pharmacia Biotech (Pistacaway, NJ).
Tissue culture
Two human pancreatic adenocarcinoma cell lines, SU 86.86 and BxPC-3, were obtained from American Type Culture Collection (Manassas, VA). The cell lines were maintained in RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were plated for experimental use in complete media and allowed to attach and grow for 48 h in a 5% CO2/water saturated incubator at 37°C. The media was then replaced with serum-free media. Twenty-four hours later, cells were treated with vehicle or bile acid under serum-free conditions. Cellular cytotoxicity was assessed by measurements of cell number and trypan blue exclusion as described previously (9).
RNA isolation and reverse transcription
Total RNA was isolated from cell monolayers using an RNeasy Mini Kit from Qiagen. One microgram of total RNA was reverse transcribed using the GeneAmp RNA PCR kit according to the manufacturer's protocol.
Construction of a COX-2 competitor template containing a nucleotide deletion
A competitive RTPCR deletion construct (mimic) for COX-2 was synthesized using a mutant sense primer (nucleotides 932955 attached to nucleotides 11111130; 5'-GGTCTGGTGCCTGGTCTGATGATGGAGTGGCTATCACTTCAAAC-3') and an antisense primer (nucleotides 16341655; 5'-GTCCTTTCAAGGAGAATGGTGC-3'), producing a 569 bp PCR product. The mutant sense primer contains the primer-binding sequence of endogenous target (from nucleotides 932 to 955) attached to the end of an intervening DNA sequence (a 156 bp deletion from nucleotides 956 to 1110). Thus, the mimic DNA has primer-binding sequences identical to the target cDNA. The 569 bp mimic was further amplified using the sense primer (5'-GGTCTGGTGC CTGGTCTGATGATG-3') and the antisense primer (5'-GTCCTTTCAAGGAGAATGGTGC-3') in a reaction mixture containing 10 mM TrisHCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM deoxynucleotide triphosphate, 2.5 U AmpiTaq DNA polymerase and 400 nM primers for 35 cycles consisting of denaturation at 94°C for 20 s, annealing at 60°C for 20 s, and extension at 72°C for 30 s in a Perkin Elmer 2400 thermal cycler. The PCR products were electrophoresed on 1% agarose gels and gel-purified using GenEluteTM Agarose Spin Columns according to the manufacturer's protocol.
Quantitative PCR for COX-2 in human pancreatic cancer cell lines
Each PCR was carried out in 25 µl of a reaction mix, containing 10 mM TrisHCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM deoxynucleotide triphosphate, 2.5 U Amplitaq DNA polymerase and 400 nM primers (the sense primer: 5'-TGCCCAGCTCCTGGCCCGCCGCTT-3' and the antisense primer: 5'-GTGCATCAACACAGGCGCCTCTTC-3'). Three to five microliter aliquots of the reverse-transcribed cDNA samples and various known amounts of COX-2 mimic (between 0.0001 and 0.05 pg) adjusted to the abundance of the target cDNA, were added to the reaction mix and co-amplified for 35 cycles of denaturation at 94°C for 20 s, annealing at 65°C for 20 s, extension at 72°C for 90 s, and final extension at 72°C for 10 min. Ten microliters of PCR products, 724 bp fragments from endogenous target cDNA and 569 bp fragments from mimic COX-2, were then separated by electrophoresis on 1% agarose gels and visualized by ethidium bromide staining.
Semi-quantitative PCR for COX-1, cPLA2 and ß2-microglobulin in human pancreatic cancer cell lines
The semi-quantitative analysis for COX-1 was performed in 25 µl of a reaction mix containing 5 µl aliquots of reverse transcribed cDNA samples, 10 mM TrisHCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM deoxynucleotide triphosphate, 2.5 U AmpliTaq DNA polymerase and 400 nM primers (the sense primer: 5'-TGCCCAGCTCCTGGCCCGCCGCTT-3' and the antisense primer: 5'-GTGCATCAACACAGGCGCCTCTTC-3') for 35 cycles consisting of denaturation at 94°C for 20 s, annealing at 68°C for 20 s, extension at 72°C for 30 s and final extension at 72°C for 10 min, generating a 217 bp PCR product. The semi-quantitative analysis for cPLA2 was performed in 25 µl of a reaction mix containing 5 µl aliquots of reverse transcribed cDNA samples, 10 mM TrisHCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM deoxynucleotide triphosphate, 2.5 U AmpliTaq DNA polymerase and 400 nM primers (the sense primer: 5'-GAGCTGATGTTTGCAGATTGGGTTG-3' and the antisense primer: 5'-GTCACTCAAAGGAGACAGTGGATAAGA-3') for 35 cycles consisting of denaturation at 94°C for 20 s, annealing at 65°C for 20 s, extension at 72°C for 25 s and final extension at 72°C for 3 min, generating a 509 bp PCR product. A constitutively expressed gene, ß2-microglobulin was used as an internal control, generating a 266 bp PCR product. The primers for ß2-microglobulin (from nucleotides 75 to 340) were 5'-AGCAGAGAATGGAAAGTCAAA-3' for sense and 5'-ATGCTGCTTACATGTCTCGAT-3' for antisense. The PCR conditions for ß2-microglobulin were identical to that for COX-2 except for annealing at 55°C for 20 s. Ten microliters of PCR products, 217, 509 and 266 bp fragments for COX-1, cPLA2 and ß2-microglobulin, respectively, were separated by electrophoresis on 1% agarose gels and visualized by ethidium bromide staining.
Western blotting
Cellular lysates were prepared by treating cells with an ice-cold homogenization buffer containing 150 mM NaCl, 100 mM Tris-buffered saline (pH 8), 1% Tween-20, 50 mM diethyldithiocarbamate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml trypsin chymotrypsin inhibitor and 10 µg/ml pepstatin. Lysates were sonicated for 20 s on ice and centrifuged at 11 750 g for 10 min to sediment the particulate material. The protein concentration of the supernatant was measured using the Lowry protein assay kit. Immunoblot analysis for COX-1 and COX-2 was performed as described in previous studies (17).
PGE2 production by cells
Cells (1 x 104/well) were plated in 24-well dishes and grown to 70% confluence in RPMI containing 10% FCS. The medium was replaced with serum-free RPMI, and 24 h later cells were treated with vehicle or bile acid under serum-free conditions for 24 h. At the end of the treatment period, the culture medium was collected to determine spontaneous production of PGE2 by these cells. The levels of PGE2 were measured by enzyme immunoassay (9). The amount of PGE2 produced was normalized to protein concentrations.
Statistical analysis
Comparisons between groups were made by the Student's t test. A difference between groups of P < 0.05 was considered significant.
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Results
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Bile acids induce COX-2 in human pancreatic cancer cell lines
To quantify the expression of COX-2, we utilized a sensitive competitive RTPCR assay. This method relies on the co-amplification in the same tube of known amounts of competitor COX-2 cDNA with endogenous target obtained after reverse transcription from total cellular RNA (Figure 1A). The competitor and target use the same PCR primers but yield amplicons with a different size (Figure 1A), allowing their separation on an agarose gel at the end of the reaction. Increased amounts of COX-2 mRNA were detected in bile acid-treated cells compared with control cells (Figure 1B). Treatment with the unconjugated bile acids chenodeoxycholate (CD) and deoxycholate (DC) resulted in greater induction of COX-2 mRNA (mean 41 667 and 28 136 fg/mg total RNA, respectively) than treatment with the conjugated bile acids, taurochenodeoxycholate (TCDC) and glycochenodeoxycholate (GCDC) (mean 8333 and 7500 fg/mg total RNA, respectively). Overall, amounts of COX-2 mRNA were increased by >15-fold by CD, 10-fold by DC and 3-fold by TCDC and GCDC. There was no evidence of cytotoxicity under these conditions (data not shown).

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Fig. 1. Increased levels of COX-2 in human pancreatic cancer cell lines following bile acid treatment. (A) Representative quantitative RTPCR for COX-2 in BxPC-3 cells following treatment with CD under serum-free conditions. Three microliters of endogenous cDNA and known concentrations (3.57.5 pg) of hCOX-2 mimic are competing for a fixed amount of hCOX-2 primer in each reaction lane, giving rise to relative proportions of 569 bp mimic product and 724 bp target cDNA product. At 4.5 pg of mimic, there is more 724 bp target cDNA product relative to mimic product. At 5.5 pg, there is more mimic product relative to target cDNA product. Using densitometry, there are equal amounts of both products formed at the mimic concentration of 5 pg, indicating that 3 µl of target cDNA are equivalent to 5 pg of COX-2 mimic. This is calculated to be 41 667 fg of COX-2 mRNA/µg of total RNA. MWM, molecular weight marker. (B) Quantitative RTPCR was used to determine amounts of COX-2 mRNA in BxPC-3 cells following treatment with vehicle (C), unconjugated bile acid (100 µM CD, 100 µM DC) or conjugated bile acid (600 µM TCDC, 600 µM GCDC) for 3 h. There was a 15-fold induction of COX-2 mRNA with CD (mean 41 667 fg/mg total RNA) and a 10-fold induction with DC (mean 28 136 fg/mg total RNA). In contrast, there was a 3-fold induction in COX-2 mRNA following treatment with TCDC or GCDC (mean 8333 and 7500 fg/mg total RNA, respectively). Data are represented are mean ± SD (n = 3); *P < 0.05.
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To determine if the differences in COX-2 mRNA resulted in altered production of COX-2 protein, we performed western blot analysis. COX-2 protein was detectable under basal conditions in both the SU 86.86 and BxPC-3 cell lines (Figure 2). Treatment of both cell lines with unconjugated bile acids (CD, DC) (Figure 2) and conjugated bile acids (TCDC, GCDC) (Figure 3) caused dose- and time-dependent induction of COX-2 protein. Maximal induction of COX-2 was observed following treatment with 100 µM CD and DC (Figure 2A). In contrast, 6001000 µM TCDC and GCDC (data not shown) was required to maximally induce COX-2 (Figure 3A). 100 µM CD induced COX-2 in a time-dependent manner over 24 h, with increased expression as early as 6 h and maximal induction by 12 h (Figure 2B). A similar result was observed with 100 µM DC (data not shown). Treatment with 600 µM TCDC resulted in maximal induction of COX-2 protein from 1224 h (Figure 3B). Bile acid-mediated induction of COX-2 was still apparent after 24 h with all treatments.

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Fig. 2. COX-2 is induced by treatment with unconjugated bile acids in human pancreatic cancer cell lines. (A) CD and DC cause a dose-dependent increase in COX-2. SU 86.86 cells were treated with CD (lanes 26) or DC (lanes 711) in the dose range 12.5100 µM or vehicle (C) for 12 h. (B) CD induces COX-2 in a time-dependent manner with maximal induction at 12 h. SU 86.86 cells were treated with CD 100 mM or vehicle (C) for 624 h. In (A) and (B), lysate protein (30 mg/lane) was loaded onto a 10% sodium dodecyl sulphate gel, electrophoresed and transferred to a nitrocellulose membrane. The immunoblot was probed with antibody specific for COX-2. Ovine COX-2 was used as a standard.
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Fig. 3. COX-2 is induced by treatment with conjugated bile acids in human pancreatic cancer cell lines. (A) TCDC causes a dose-dependent increase in COX-2, with maximal induction in the range 6001000 µM. BxPC-3 cells were treated with TCDC in a dose range 2001000 µM or vehicle (C) for 12 h. (B) TCDC induces COX-2 in a time-dependent manner. BxPC cells were treated with TCDC 600 µM or vehicle (C) for 624 h. In both (A) and (B) lysate protein (30 µg/lane) was loaded onto a 10% sodium dodecyl sulphate gel, electrophoresed and transferred to a nitrocellulose membrane. The immunoblot was probed with antibody specific for COX-2. Ovine COX-2 was used as a standard.
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Bile acids do not induce COX-1 or cPLA2
Because COX-1 and cPLA2 are also important in eicosanoid synthesis, we measured levels of these enzymes following bile acid treatment. Semi-quantitative RTPCR for COX-1 and cPLA2 mRNAs was performed following treatment of the BxPC-3 cells with unconjugated and conjugated bile acids (Figure 4). In contrast to COX-2, COX-1 (Figure 4A) and cPLA2 mRNAs (Figure 4B) were unaffected by bile acids. COX-1 protein was not induced by bile acids (data not shown). Similarly, neither COX-1 nor cPLA2 were induced in SU 86.86 cells with bile acid treatment.

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Fig. 4. Cyclooxygenase-1 and cPLA2 are unaffected by bile acids. BxPC-3 cells were treated with vehicle (C), unconjugated (100 µM CD, 100 µM DC) or conjugated (600 µM TCDC, 600 µM GCDC) bile acids. Total RNA was isolated and 1 mg of total RNA was reverse transcribed. Semi-quantitative RTPCR for COX-1 and cPLA2 was performed. (A) COX-1 mRNA expression is unchanged with bile acid treatment. (B) cPLA2 expression is unaffected with bile acid treatment. (C) ß2-Microglobulin, a constitutively expressed gene was used as an internal control generating a 266 bp PCR product.
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Bile acids increase PGE2 production in human pancreatic cancer cell lines
SU 86.86 cells were treated with bile acids or vehicle for 24 h. Figure 5 shows the effects of unconjugated and conjugated bile acids on the production of PGE2. PGE2 production was increased by treatment with both unconjugated (CD, DC) and conjugated bile acids (TCDC, GCDC). At 24 h, there was a 7-fold induction of PGE2 following treatment with either CD or DC (Figure 5A). Treatment with either TCDC or GCDC resulted in a 4-fold induction of PGE2 (Figure 5B).

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Fig. 5. Bile acids enhance the production of PGE2 in human pancreatic cancer cell lines. SU 86.86 cells were treated with vehicle (C) or bile acid (CD 100 µM, DC 100 µM, TCDC 600 µM, GCDC 600 µM) for 24 h. The post-culture medium was collected to determine spontaneous release of PGE2. Production of PGE2 was determined by enzyme immunoassay as described under Materials and methods. The amounts of PGE2 produced were normalized to protein concentrations. Data are represented as mean ± SD (n = 4); *P < 0.05.
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Discussion
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Despite recent improvements in diagnosis and staging of pancreatic cancer, most patients present with advanced local or distant disease. A lack of understanding of the pathogenesis of pancreatic cancer contributes significantly to the poor prognosis of this disease. We have reported previously over-expression of COX-2 in human pancreatic adenocarcinoma (17). In the current study, we demonstrate that bile acids induce COX-2 and increase prostaglandin synthesis in cultured human pancreatic cancer cells. This result suggests a possible mechanism by which bile acids could promote pancreatic cancer.
Epidemiological studies have suggested that consumption of a western-style diet high in animal fat is associated with an increased risk of pancreatic cancer (24). Experimental animal studies support this observation. Dietary fat has been shown to enhance the incidence of nitrosamine-induced pancreatic adenocarcinomas in hamsters (22). Biliary excretion of cholic and deoxycolic acid is higher in rats fed a high fat diet containing 20% corn oil or lard than in rats fed diets containing 5% corn oil or lard (5). A large body of evidence suggests that bile acids play a role in the development of gastrointestinal tumors. CD and DC increase cellular proliferation and the number of mitotic events in colonic mucosa (23). Enhanced DNA synthesis has been demonstrated in the epithelium of the large intestine of rats treated with bile acids (24,25). Reduced susceptibility to apoptosis occurs in animal and human models of colon cancer following bile acid treatment (26,27). Administration of CD resulted in a 9-fold increase in duodenal tumor formation in a murine model of familial adenomatous polyposis (28). Taken together, the data suggest that bile acids are important mediators of carcinogenesis.
The mechanisms by which bile acids promote carcinogenesis are areas of current active research. Bile acids induce AP-mediated gene transcription (29) and enhance the activity of protein kinase C (30,31). Recent evidence has linked bile acid-induced tumorigenesis with increased activity of COX-2. Induction of COX-2 and increased PGE2 production has been demonstrated in human esophageal adenocarcinoma cell lines after treatment with CD and DC (32). Ex vivo organ culture of tissue from Barrett's esophagus and esophageal adenocarcinoma treated with bile acids demonstrated increase COX-2 expression, which was blocked with a COX-2 inhibitor (10). Similarly, DC and CD induced COX-2 promoter activity in a colorectal carcinoma cell line (11). Finally, the p44/p42 and p38 mitogen-activated protein kinase pathways have been implicated in post-transcriptional regulation of COX-2 expression by bile acids (33).
In the present study, we found that both conjugated and unconjugated bile acids were capable of stimulating COX-2 and PGE2 production in two different human pancreatic cancer cell lines. The dihydroxy unconjugated bile acids DC and CD induced COX-2 at lower concentrations than the conjugated bile acids TCDC and GCDC, which is likely to reflect their difference in hydrophobicity. Importantly, concentrations of bile acids used in these studies are below physiologic concentrations present in normal bile (34).
Whether pancreatic tissue is exposed to bile and bile acids through biliopancreatic reflux is controversial. In one study, the kinetics of biliopancreatic reflux was examined in patients 1015 days after cholecystectomy for chronic calculous cholecystitis (35). Pancreatic reflux was demonstrated in six out of the seven patients, which was extensive at times reaching the pancreatic tail. No structural anatomic anomaly was found in these patients. This observation lends support to the theory that pancreatic ductal cells can be exposed to bile acids in vivo.
Multiple lines of evidence suggest that COX-2 is important in gastrointestinal carcinogenesis. We have now demonstrated that both conjugated and unconjugated bile acids induce COX-2 in human pancreatic cancer cell lines. Based on the results of this study, and our previous demonstration of up-regulation of COX-2 in human pancreatic cancer, these findings suggest a possible role for bile acids in the pathogenesis of pancreatic cancer.
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Notes
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4 To whom correspondence should be addressed at: The Department of Surgery, The Mater Misericordiae Hospital, Eccles Street, Dublin 7, Republic of Ireland Email: olga_tucker{at}hotmail.com 
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Acknowledgments
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This work was supported by the Frederick and Sandra P.Rose Foundation.
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Received April 2, 2003;
revised September 30, 2003;
accepted October 27, 2003.