Gao-Song Wu, Sheng-Quan Zou, Zheng-Ren Liu, Da-Yu Wang, Department of General Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei Province, China
Correspondence to: Dr Gao-Song Wu, Department of General Surgery of Tongji Hospital, 1095 Jiefang Road, Wuhan, 430030, Hubei Province, China.  wugaosong9172@sina.com
Telephone: +86-27-83662851    Fax: +86-27-83662851
Received: 2002-08-24    Accepted: 2002-10-12

Abstract
AIM: To explore the effects of COX-2 gene in the proliferative activity induced by bile from anomalous pancreaticobiliary ductal union (APBDU) on human cholangiocarcinoma cell line.

METHODS: Bile sample from APBDU and normal bile sample were used for this study. The proliferative effect of bile was measured by methabenzthiazuron (MTT) assay; COX-2 mRNA was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Cell cycle was analyzed by flow cytometry (FCM), and the PGE2 levels in the supernatant of cultured cholangiocarcinoma cells were quantitated by enzyme-linked immunoabsordent assay (ELISA).

RESULTS: Bile from APBDU can significantly promote the proliferation of human cholangiocarcinoma QBC939 cells compared with normal bile (P=0.005) and up-regulated remarkably their COX-2 mRNA expression (P=0.004). The proliferative activity of APBDU bile can be abolished by addition of cyclooxygenase-2 specific inhibitor celecoxib.

CONCLUSION: Bile from APBDU can promote the proliferation of human cholangiocarcinoma QBC939 cells via COX-2 pathway.

Wu GS, Zou SQ, Liu ZR, Wang DY. Bile from a patient with anomalous pancreaticobiliary ductal union promotes the proliferation of human cholangiocarcinoma cells via COX-2 pathway. World J Gastroenterol  2003; 9(5): 1094-1097
http://www.wjgnet.com/1007-9327/9/1094.asp

INTRODUCTION
It is well known that APBDU is associated with choledochal cyst^[1-9]. Recently, a frequent association of biliary tract carcinoma and APBDU without choledochal cyst is well recognized, but its underlying mechanism is unclarified. For the purpose of resolving these mechanism we used the APBDU bile to act directly on the QBC939 cells to determine the effects of bile from APBDU on the cholangiocarcinoma cells growth.

MATERIALS AND METHODS
Materials
Bile samples collection and treatment: APBDU bile was obtained from the common bile duct of a patient (male, 39) with polypoid lesion of the gallbladder who underwent cholecystectomy in the Department of Surgery, Tongji hospital, Wuhan, China. Preoperative MRCP revealed the length of the pancreaticobiliary common channel was 19 mm with absence of dilation of the common bile duct, the pancreatic duct merged with the bile duct (P-B type) and the pancreaticobiliary ductal union was located proximal to the narrow distal segment, which represented the sphincter of Oddi, APBDU was diagnosed by intraoperative cholangiography concordant with the MRCP diagnosis. Normal control bile was obtained from the common bile duct of another patient (male, 41) with gastric cancer and a normal hepatobiliary tract. Both patients had not taken any nonsteroidal anti-inflammatory drugs, antibiotics or anti-tumor drugs before the operation. Bile samples were sterile and filtered (0.22 祄, Millipore) twice immediately and stored at -80 ℃. PBS (pH7.2) was used as negative control. Human extrahepatic cholangiocarcinoma cell line QBC939 was gifted by professor Wang (Third Military Medical University,China)^[10], the cells were maintained as monolayers in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS, Gibco. USA.), 100 units/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere of 95 % air and 5 % CO2 at 37 ℃. PGE2 ELISA detection kit was purchased from Jingmei Biotech Co., Wuhan, China. Celecoxib was provided by Dr. Mei (Wuhan University, China)^[11]. Stock solution was prepared in dimethylsulfoxide (DMSO) and stored at -20 ℃. In all experiments the final concentration of DMSO in the medium was ≤0.1 %.

Methods
Cytotoxicity pretesting  Cytotoxicity pretesting was taken with each of the gradient in diluted bile sample to determine the concentration of experimental bile samples. Our results showed that 1 % bile (10 ml bile/mL medium) was not cytotoxic to QBC939 cells.
MTT assay  The proliferating status of human cholangiocarcinoma cells QBC939 was determined by MTT assay. Cholangiocarcinoma cells were seeded at a density of 1×10⁴ cells per well in flat-bottomed 96-well microplates. 12 h after incubation, the cells were treated with 1 % bile with or without 20 mol/L celecoxib. After 72 h incubation, 20 ml MTT (5 g/L) was added to each well and cultured for 4 h. Upon removal of the supernatant, added DMSO 150 ml and shook for 5 min untill the crystals were dissolved. OD490nm value was measured by enzyme-linked immunoabsorbent assay. The negative control well was used as zero point of absorbance. Each assay was performed three times in triplicate.
ELISA  The PGE₂ levels in the supernatant of the cultured human cholangiocarcinoma cells QBC939 were quantitated by ELISA. Cells were seeded into 24-well microplates (4.0×10⁵/well) and allowed to adhere overnight. The cells were then incubated in presence 1 % bile with or without 20 mol/L celecoxib for 48 h. The supernatants were aspirated and centrifuged to prepare for the detection of PGE₂. 0.5 mL supernatant was added into 1 N HCl 0.1 mL and centrifuged for 10 min at room temperature; then 1.2 N NaOH 0.1 mL was used to neutralize the acidified samples. Standard solution (200 ml/well) or activated samples were added into the microplates. Then the steps were proceeded as instructed. The value of OD of each well was determined at 450nm. The supernatants were harvested in triplicate and the experiment was performed three times.
Flow cytometric analysis  Human cholangiocarcinoma cells QBC939 were trypsinized and plated in 6-well culture dishes in presence of 1 % APBDU bile or 1 % normal bile. After 24 h, the cells were harvested, centrifuged at low speed and fixed in 70 % ethanol. After overnight incubation at 4 ℃, the cells were stained with 50 ml/ml propidium iodide in presence of RNAsin A (10 ml/ml) and 0.1 % Triton X-100 and determined by flow cytometer.
RT-PCR  QBC939 cells were cultured in presence of 1 % APBDU bile, 1 % normal bile or 1 % PBS for 3 d. The total RNA was prepared from subconfluent cultures with RNA-SOLV reagent (Omega) according to the manufacture instruction. The primers were designed to amplify a fragment of COX-2 cDNA based on the reported sequence for human COX-2. To normalize the amount of input RNA, RT-PCR was performed with primers for the constitutively expressed ?-actin gene. The COX-2 primers were 5' -ACAATGCTGACTATGGCTAC-3' (sense) and 5 -AACTGATGCGTGAAGTGCTG-3 (antisense), giving rise to a 238 base pair polymerase chain reaction production. The b-actin primers were 5' -GTGCGTGACATTAAGGAG- 3' (sense) and 5' -CTAAGTCATAGTCCGCCT- 3' (antisense), giving rise to a 520 base pair polymerase chain reaction production. The first strand cDNA synthesis and the subsequent PCR were performed with RNA PCR kit (AMV) using a programmed temperature control system set for 30 cycles of denaturation at 94 ℃ for 45 s, annealing at 58 ℃ for 30 s, and extension at 72 ℃ for 60 s. 10 ml reaction mixture was electrophoresed on a 1.5 % agarose gel, and the PCR products were visualized by ethidium bromide staining and quantified by an ImageQuant software. COX-2 mRNA expression level was determined by COX-2/b-actin protein.

Statistical analysis
The data were expressed as x±s. Student's t-test was used for statistical analysis. P<0.05 indicated significant difference.

RESULTS
Assay of COX-2 activity
PGE2 levels in the supernatant released by the cultured human cholangiocarcinoma cells QBC939 determined the COX-2 activity. The concentrations of PGE₂ in culture medium of QBC939 cells treated with 1 % APBDU bile or 1 % normal bile with or without 20 mol/L celecoxib for 48 h were quantitated by ELISA. APBDU bile could induce the release of PGE₂ in QBC939 cells: the PGE₂ level was higher significantly (P=0.004) in APBDU bile group (187.1±14.0 ng/well) as compared with that in normal bile group (139.4±15.3 ng/well). Celecoxib could suppress PGE₂ production of the QBC939 cells, the PGE₂ concentration was (65.2±10.6) ng/well and (57.0±9.8) ng/well in APBDU bile group and normal bile group respectively when pre-treated with 20 mol/L celecoxib, there was no statistical difference between the two group (P=0.09).

Effects of bile on the proliferation activity of QBC939
QBC939 cells were incubated in 1 % bile with or without 20 mol/L celecoxib, and the cells density was measured by MTT assay. APBDU bile could significantly promote the proliferation of QBC939 cells as compared with normal bile (P=0.005), and the proliferative effect of APBDU bile could be abolished by addition of 20 mol/L celecoxib (P=0.103, Table 1).

Table 1  Effects of bile on the growth of QBC939 with or without celecoxib

QBC939 cells were incubated in 1 % APBDU bile (A), 1 % normal bile (B) or 1 % PBS (C) with or without 20 mol/L celecoxib (+CE), and the cells density (OD490nm) was measured by using MTT assay. Data were expressed as x±s, b vs C, d vs B.

Flow cytometric analysis of proliferative index of QBC939 cells
The QBC939 cells proliferative index (PI) increased significantly (P=0.003) after treatment with 1 % APBDU bile (60.59±4.06) as
compared with that of the normal bile(28.69±1.79, Figure 2), PI =S+G2/M)100 %.

Figure 1(PDF) Expression of COX-2 mRNA, b-actin served as control. M: DL2,000 marker; 1: normal bile; 2 and 4: APBDU; 3: PBS.
Figure 2(PDF) Representative data of cell cycle from QBC939 cells in the presence of 1 % APBDU bile (S+G2/M=65.12 %) or 1 % normal bile (S+G2/M=30.47 %) for 24 h was analyzed by flow cytometry.

Expression level of COX-2 mRNA
APBDU bile could markedly (P=0.004) up-regulate the COX-2 mRNA expression of QBC939 cells (Figure 1, Ttable 2).

Table 2  Expression level of COX-2 mRNA

COX-2 mRNA expression level was determined by COX-2/b-actin protein. Data were expressed as x±s, b vs C (PBS), d vs B (normal bile), A: APBDU.

DISCUSSION
A frequent association of biliary tract carcinoma and APBDU is well recognized^[12-19], especially in the undilated type APBDU^{[20, 21]}. Mori[7] had reported that among 698 patients subjected to endoscopic retrograde cholangiopancreatography, APBDU was found in 6 patients (0.9 %). 4 of these 6 patients had no associated congenital choledochal cyst, and two patients had advanced gallbladder cancer. The remaining 2 patients had no associated carcinoma of the biliary tract. They further studied 28 such APBDU without choledochal cyst cases. The clinicopathological data showed that the thickness of the gallbladder wall was visualized in 26/28 (92.9 %) patients. Some researchers^[20-22] had reported that patients with adenomyomatosis (a presumed premalignant lesion of the gallbladder) were frequently associated with the undilated type APBDU. Tanno^[23] reported 15/24 (63 %) of APBDU patients had epithelial hyperplasia of the gallbladder, the incidence of which was significantly higher in the gallbladders of undilated type APBDU patients (91 %) than that in dilated type patients (38 %). Ki-67 labeling index was significantly higher in hyperplastic mucosa than that in the control gallbladder mucosa. 2/9 (22 %) high grade hyperplasia cases had K-ras mutations. Their results suggested that hyperplasia of the gallbladder mucosa in APBDU patients was an early change. Cell kinetic studies of gallbladder epithelial cells by Yang^[24] had shown the Ki-67 labeling index, PCNA labeling index and BrdU labeling index of the noncancerous mucosa in patients with APBDU and/or gallbladder carcinoma were significantly higher than those in patients without APBDU and gallbladder carcinoma.
      Increase of the secondary and free bile acid concentrations is considered a risk factor for biliary carcinogenesis in APBDU patients. Sugiyama^[25] had suggested that elevation of the lysolecithin (LL) in the bile was one of the factors for development of biliary tract carcinoma in patients with APBDU: the LL in the phospholipid produced from lecithin by activated phospholipase A2 in the refluxed pancreatic juice, was significantly elevated in the APBDU group. Yoon^[26] also indicated that bile acids induced both EGFR phosphorylation and enhanced COX-2 protein expression. EGFR was activated by bile acids to induce COX-2 expression by a MAPK cascade. The induction of COX-2 might participate in the genesis and progression of cholangiocarcinoma.
     In an effort to delineate the underlying mechanism of the carcinogenesis in APBDU and the effects of COX-2 gene in the proliferative activity induced by APBDU bile, we used the bile from APBDU to see the direct effect on the human cholangiocarcinoma QBC939 cells in vitro to determine the effect of APBDU bile on the growth of human cholangiocarcinoma cells. Our data show that APBDU bile could significantly promote the proliferation of human cholangiocarcinoma QBC939 cells and up-regulated remarkably their COX-2 mRNA expression, and the proliferative activity of APBDU bile could be abolished by adding cyclooxygenase-2 specific inhibitor celecoxib. Our study indicated that APBDU bile promoted the proliferation of human cholangiocarcinoma QBC939 cells via COX-2 pathway.
      Substantial evidences have shown that COX-2 is important in carcinogenesis^[27-33]. Celecoxib as a new COX-2 selective inhibitor has shown its safety and efficiency in human and animals. Several studies have demonstrated that celecoxib has significant efficacy in animal models: Celecoxib inhibited intestinal tumor multiplicity up to 71 % as compared with controls in the Min mouse model, and inhibited colorectal tumor burden in the rat azoxymethane (AOM) model^[34-36]. Recently celecoxib has been approved by the FDA to reduce the number of adenomatous colorectal polyps in patients with familial adenomatous polyposis (FAP). Our data suggested that celecoxib as a chemopreventive and chemotherapeutic agent might be effective in cholangiocarcinoma and could be used as a chemopreventive strategy in the people of high-risk conditions for the development of cholangiocarcinoma such as APBDU. Our study demonstrated that the QBC939 cells proliferative index increased significantly after treated with APBDU bile for 24 h. These data suggested that APBDU bile could affect the QBC939 cell proliferation cycle.
     In conclusion, APBDU bile can promote the proliferative activity of human cholangiocarcinoma QBC939 cells and the effect is via COX-2 pathway.

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Edited by  Wu XN 

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