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[Cancer Research 64, 1369-1376, February 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Cyclooxygenase-2 Promotes Human Cholangiocarcinoma Growth

Evidence for Cyclooxygenase-2-Independent Mechanism in Celecoxib-Mediated Induction of p21waf1/cip1 and p27kip1 and Cell Cycle Arrest

Chang Han1, Jing Leng1,2, A. Jake Demetris1 and Tong Wu1

1 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, and2 Department of Pathology, Nanjing Medical University, Nanjing, China


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The expression of cyclooxygenase-2 (COX-2) is increased in human cholangiocarcinoma. However, the biologic function and molecular mechanisms of COX-2 in the control of cholangiocarcinoma cell growth have not been well established. This study was designed to examine the direct effect of COX-2 and its inhibitor celecoxib on the growth of human intrahepatic cholangiocarcinoma cells. Overexpression of COX-2 or treatment with prostaglandin E2 (PGE2) enhanced human cholangiocarcinoma cell growth, whereas antisense depletion of COX-2 in these cells decreased PGE2 production and inhibited growth. These findings demonstrate a direct role of COX-2-mediated PGE2 in the growth regulation of human cholangiocarcinoma cells. Furthermore, the COX-2 inhibitor celecoxib induced a dose-dependent inhibition of cell growth, cell cycle arrest at the G1-S checkpoint, and induction of cyclin-dependent kinase inhibitors p21waf1/cip1 and p27kip1. However, the high concentration of celecoxib (50 µM) required for inhibition of growth, the incomplete protection of celecoxib-induced inhibition of cell growth by PGE2 or COX-2 overexpression, and the fact that overexpression or antisense depletion of COX-2 failed to alter the level of p21waf1/cip1 and p27kip1 indicate the existence of a COX-2-independent mechanism in celecoxib-induced inhibition of cholangiocarcinoma cell growth.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cholangiocarcinoma is a malignant neoplasm originating from epithelium of the biliary tree with high mortality (1) . It often occurs in patients with background conditions that cause long-standing inflammation, injury, and reparative biliary epithelial cell proliferation, such as primary sclerosing cholangitis, clonorchiasis, hepatolithiasis, or complicated fibropolycystic diseases. This association strongly indicates that chronic inflammatory processes involving bile duct epithelium predispose the development of cholangiocarcinoma. However, the exact molecular mechanisms by which inflammatory cascades promote malignant transformation of biliary epithelium are not well understood. Recent evidence reveals that cyclooxygenase-2 (COX-2), an inducible enzyme controlling the synthesis of lipid inflammatory mediator prostaglandins (PGs), is increased in cholangiocarcinoma cells (2, 3, 4, 5) , which suggests a potential role of COX-2-mediated PG pathway in biliary carcinogenesis.

The synthesis of PGs is controlled by coordinated activation of eicosanoid-forming enzymes, including COXs (6) . There are two isoforms of COXs, COX-1 and COX-2, which catalyze the same enzymatic reaction but are regulated differently. Whereas COX-1 is expressed constitutively in most tissues, COX-2 is an immediate early gene that is induced by a variety of stimuli, such as cytokines, hormones, mitogens, and growth factors (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) . Recent evidence shows that the expression of COX-2 is increased in human cancers and that the COX-2-generated PGs promote tumor cell proliferation, survival, and angiogenesis (6, 7, 8, 9, 10, 11, 12 , 21, 22, 23, 24, 25) . Targeted expression of COX-2 in mammary gland (26) and skin (27) induces tumorigenesis in mice; knockout of the COX-2 gene suppresses intestinal tumorigenesis (28) . These findings highlight the important role of COX-2-controlled PG metabolism in cancer development and progression.

The contribution of PG pathway in cancer growth is also supported by the substantial epidemiologic, experimental, and clinical studies using the COX inhibitor nonsteroidal anti-inflammatory drugs (NSAIDs). Large epidemiologic studies reveal that aspirin and other NSAIDs reduce the risk of colorectal cancer (7 , 9 , 13 , 14 , 16 , 19) and several other cancers (15 , 17 , 18 , 29, 30, 31) . Randomized clinical studies show that NSAIDs decrease colon polyps in patients with familial adenomatous polyposis (32, 33, 34, 35) . Experimental studies using various in vitro and in vivo models demonstrate that NSAIDs decrease the growth of human cancer cells, including cholangiocarcinoma (3 , 5 , 25 , 36) . The proposed mechanisms by which NSAIDs prevent cancer growth include inhibition of cell proliferation, potentiation of immune response, inhibition of angiogenesis, and induction of apoptosis (7 , 9 , 19 , 20 , 37 , 38) . However, interpretation of the NSAID-mediated antitumor effect is complicated by the presence of COX-independent effect (7 , 19 , 21 , 39, 40, 41, 42, 43) . Celecoxib belongs to the new generation of NSAIDs that selectively inhibits COX-2 activity without inhibition of COX-1 and thus lacks the side effects associated with the traditional NSAIDs. Recent studies have shown that celecoxib inhibits the growth of several tumor cell types (35 , 44, 45, 46, 47, 48, 49, 50, 51, 52) , although its effect on cholangiocarcinoma growth remains to be further defined.

Despite increased COX-2 expression in human cholangiocarcinoma, the direct biologic role of COX-2-mediated prostanoids in the control of cholangiocarcinoma cancer growth has not been established with molecular approach. In this study, we performed in vitro studies to address the direct role of COX-2 in cholangiocarcinoma cell growth by overexpression and antisense depletion of COX-2 and to evaluate the efficacy and mechanisms of celecoxib in the growth of human intrahepatic cholangiocarcinoma cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
{alpha}-MEM, DMEM, RPMI 1640, fetal bovine serum (FBS), glutamine, antibiotics, and Lipofectamine plus reagent were purchased from Life Technologies, Inc. (Rockville, MD). PGE2 was purchased from Calbiochem (San Diego, CA). The cell proliferation assay reagent WST-1 was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Pharmacia (Chicago, IL) provided the COX-2 inhibitor celecoxib (SC58635). The antibody for human COX-2 was purchased from Cayman Chemical Company (Ann Arbor, MI). The antibodies against human p27, p21, cyclin E, and cyclin-dependent kinase 2 (cdk2) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antihuman ß-actin monoclonal antibody was purchased from Sigma (St. Louis, MO). The horseradish peroxidase-linked streptavidin and chemiluminescence detection reagents were from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ). The PGE2 enzyme immunoassay system also was from Amersham Pharmacia Biotech, Inc. The Bio-Rad protein assay system was obtained from Bio-Rad Laboratories (Hercules, CA). The Tris-glycine gels were obtained from Invitrogen Life Technologies, Inc. (Carlsbad, CA). The Tet-On antisense COX-2 construct (containing a 1.93-kb human COX-2 cDNA in antisense orientation that is controlled by the tetracycline response element) was provided by Dr. J. Morrow at Vanderbilt University (53) . Dr. T. Hla at the University of Connecticut Health Center provided the COX-2 expression plasmid (containing full length of human COX-2 cDNA in sense orientation cloned in mammalian expression vector PCDNA3).

Cell Culture.
Three human intrahepatic cholangiocarcinoma cell lines—HuCCT1 (Ref. 54 ; obtained from Japanese Cancer Research Resources Bank), SG231 (55) , and CCLP1 (56) —were used in this study. HuCCT1 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 50 µg/ml gentamicin. SG231 cells were cultured in {alpha}-MEM with 2 mM L-glutamine, 50 µg/ml gentamicin, 10 mM HEPES, and 10% FBS. CCLP1 cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 50 µg/ml gentamicin. The cells were cultured at 37°C in a humidified CO2 incubator. The experiments were performed when cells reached ~80% confluence and conducted in serum-free medium with serum deprivation for 24 h before experiments.

Cell Growth Assay.
Cell growth was determined using the cell proliferation reagent WST-1, a tetrazolium salt that is cleaved by mitochondrial dehydrogenases in viable cells. Briefly, 100 µl of cell suspension (containing 0.5–2 x 104 cells) were plated in each well of 96-well plates. After 24-h culture to allow reattachment, the cells then were treated with specific reagents such as PGE2 or celecoxib for indicated time points. At the end of each experiment, the cell proliferation reagent WST-1 (10 µl) was added to each well, and the cells were incubated at 37°C for 0.5–5 h. A450 nm was measured using an automatic ELISA plate reader.

Cell Cycle Analysis.
The cell cycle of human cholangiocarcinoma cells was analyzed by flow cytometry. Cells were fixed in ice-cold 70% ethanol, washed with PBS, treated with RNase (Sigma; 0.2 mg/ml) at room temperature for 30 min, and stained with propidium iodide (Sigma; 0.05 mg/ml). Cell cycle analysis was performed using EPICS XL flow cytometry (Beckman Coulter, Fullerton, CA). Fifteen thousand cells were analyzed for each point, and quantitation of cell cycle distribution was performed using the EXP032 software program provided by the manufacturer.

Immunoprecipitations.
Equal amount of cellular protein from the treated cells was incubated with 10 µl of mouse antihuman cdk2 monoclonal antibody at 4°C for 1 h, followed by addition of 20 µl Protein A/G PLUS agarose (Santa Cruz Biotechnology). The mixture was incubated overnight and then washed three times with the cell lysis buffer [50 mM HEPES (pH 7.55), 1 mM EDTA, 1 mM DTT, and protease inhibitor cocktail tablets from Roche Diagnostics (Basel, Switzerland)]. The final pellets were dissolved in 20 µl 1x protein loading buffer, and the samples were subjected to SDS-PAGE and Western blot analysis using 1:1000 dilution rabbit antihuman p27 or p21 polyclonal antibodies and enhanced chemiluminescence (Amersham Pharmacia Biotech, Inc.) Western blot detection system.

cdk2 Kinase Activity Assays.
The cdk2 kinase was immunoprecipitated from 100 µg of cellular extracts with polyclonal antibodies against cdk2 (Upstate Biotechnology, Inc., Lake Placid, NY) for 2 h at 4°C. The obtained immunoprecipitates then were used to measure cdk2 kinase activity with histone H1 as the substrate using the kinase assay kit (Upstate Biotechnology, Inc.). The reaction mixture consisted of 20 mM 4-morpholinepropanesulfonic acid (MOPS; pH 7.2), 15 mM MgCl2, 50 mM ATP, 25 mM ß-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, and 10 µCi of [{gamma}-32P] ATP in a 50-µl volume in the presence of 4 µM protein kinase C inhibitor peptide, 0.5 µM protein kinase A inhibitor peptide, and 0.5 µM compound R24517. The reaction was started by addition of the enzyme source from immunoprecipitates and was allowed to proceed for 20 min at 30°C and stopped by taking 25-µl aliquots onto p81 phosphocellulose squares and then washing three times with 0.75% phosphoric acid. After acetone rinse, the radioactivity was measured by scintillation counting.

Transfection of COX-2 Expression Plasmid in Human Cholangiocarcinoma Cells.
CCLP1 cells were exposed to the mixture of Lipofectamine plus reagents and COX-2 expression plasmid (full-length human COX-2 cDNA cloned in pcDNA3 vector) or pcDNA3 control vector for 4 h. Following removal of the transfection mixtures, fresh medium with 10% FBS was added. On day 2, the medium was replaced with fresh medium containing 10% FBS and 800 µg/ml G418 sulfate (Calbiochem). Colonies of resistant cells appeared after ~14 days and were subcultured at ~18 days. Subsequent cultures of selected cells were routinely grown in the presence of the same selective pressure. Western blot analysis for COX-2 then was performed in the selected cells transfected permanently with the COX-2 expression plasmid or control plasmid. The selected cells with successful overexpression of COX-2 protein were used for subsequent experiments.

Antisense Inhibition of COX-2 in HuCCT1 Cells.
The HuCCT1 cells (containing high basal level of COX-2 protein) were used for antisense inhibition of COX-2. The cells were transfected with the Tet-On antisense COX-2 plasmid (containing a 1.93-kb human COX-2 cDNA in antisense orientation that is controlled by the tetracycline response element) according to the previously described procedure with modification (53) . Briefly, the cells were exposed to the mixture of Lipofectamine plus reagents and the antisense COX-2 construct in serum-free medium for 4 h. Following removal of the transfection mixtures, the cells were cultured in RPMI 1640 medium containing 10% FBS. On day 2, fresh medium containing 10% FBS and 800 µg/ml G418 sulfate was added. Colonies of resistant cells appeared after ~14 days and were subcultured at ~18 days. Subsequent cultures of selected HuCCT1 cells were routinely grown in the presence of the same selective pressure. The level of COX-2 expression was determined 96–120 h after cells were exposed to doxycycline (2 µg/ml) by Western blot analysis. The selected cells with optimal depletion of COX-2 were used for subsequent experiments.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Antisense Depletion of COX-2 Inhibits Cholangiocarcinoma Cell Growth.
The direct effect of COX-2 on cholangiocarcinoma cell growth was evaluated using antisense inhibition of COX-2. The HuCCT1 cells were selected for this approach because these cells contain high basal level of COX-2 protein. The HuCCT1 cells were transfected with the COX-2 antisense plasmid containing a 1.93-kb human COX-2 cDNA in antisense orientation that is controlled by the tetracycline response element (53) . Fig. 1Citation shows that ~80% reduction of COX-2 protein was achieved in cells with antisense inhibition of COX-2. The cells with antisense depletion of COX-2 exhibited significant reduction of PGE2 and decrease of cell growth. Furthermore, addition of PGE2 partially reverses the growth inhibition caused by antisense depletion of COX-2 (Fig. 1)Citation . These findings demonstrate a direct effect of COX-2-controlled PGE2 in human cholangiocarcinoma cell growth.



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  		Fig. 1. Antisense depletion of cyclooxygenase-2 (COX-2) inhibits cholangiocarcinoma cell growth. The HuCCT1 cells were transfected with the COX-2 antisense plasmid (pUHD.2 neo) containing a 1.9- kb human COX-2 cDNA in antisense orientation that is controlled by the tetracycline response element (Ref. 53 ; provided by J. Morrow at Vanderbilt University). The HuCCT1 cells stably transfected with the COX-2 antisense plasmid were selected using G418, and the expression of antisense COX-2 RNA was induced by treatment with doxycycline (2 µg/ml for 4–5 days). The cells transfected with the empty control vector were selected as control. A, decreased growth of HuCCT1 cells with antisense inhibition of COX-2. Equal numbers of cells (1 x 105/ml) were seeded onto the 96-well plates and cultured in serum-free medium for 48 h either with induction by 2 µg/ml doxycycline or without induction. The cell growth was determined by using WST-1 assay, and the data are presented as percentage of control ± SD from six experiments (*P < 0.01 compared with antisense COX-2 cells without doxycycline treatment or control vector cells with doxycycline treatment). B, prostaglandin (PG) E2 production in HuCCT1 cells with or without antisense inhibition of COX-2. Equal numbers of cells (3 x 105/ml) were cultured on 12-well plates, and the 8-h spent media were collected. One hundred µl of centrifuged supernatant from each sample were analyzed for PGE2 production using the PGE2 enzyme immunoassay system. The production of PGE2 in cells with antisense inhibition of COX-2 is decreased significantly when compared with controls (antisense COX-2 cells without doxycycline treatment or control vector cells with doxycycline treatment; *P < 0.05). The results were obtained from four individual experiments and are presented as mean ± SD. C, Western blot analysis showing successful reduction of COX-2 protein level in cells with antisense inhibition of COX-2. Equal amount of cellular protein (20 µg) was subjected to SDS-PAGE, followed by Western blot analysis for COX-2. The same blot then was stripped and reprobed for ß-actin as loading control. D, addition of PGE2 partially reconstitutes the growth inhibition caused by antisense depletion of COX-2. HuCCT1 cells expressing antisense COX-2 were incubated with or without 10 µM PGE2 in serum-free medium for 48 h, and the cell growth was determined using WST-1 assay. The data are presented as percentage of control ± SD from six experiments (*P < 0.05 when compared with doxycycline-treated antisense COX-2 cells).

 
Overexpression of COX-2 Promotes Cholangiocarcinoma Cell Growth.
The direct contribution of COX-2 to cholangiocarcinoma cell growth was examined further by stable transfection of a COX-2 expression plasmid. The CCLP1 cells were selected for this approach because these cells contain low basal level of COX-2 protein. The cells were transfected with the human COX-2 constitutive expression plasmid pcDNA3-COX-2 and the control vector pcDNA3. After transfection, the cells were cultured in media containing 800 ng/ml G418 to select the cells with stably transfected cells. The selected cells transfected with COX-2 expression plasmid then were analyzed for their growth in vitro under spontaneous culture conditions and showed significantly increased cell growth over the control vector cells (Fig. 2)Citation . The efficiency of COX-2 expression was confirmed by Western blot analysis showing that the COX-2 overexpressed cells exhibit ~12-fold increase of COX-2 protein when compared with the vector control cells as evaluated by densitometry (see below; Fig. 7Citation ). These observations further demonstrate an important role of COX-2 in human cholangiocarcinoma cell growth.



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  		Fig. 2. Overexpression of cyclooxygenase-2 (COX-2) promotes human cholangiocarcinoma cell growth. CCLP1 cells were transfected with the COX-2 expression vector or control vector (pcDNA3), and the stably transfected cells were selected using G418 as described in "Materials and Methods." A, effect of COX-2 overexpression on cell growth. Equal numbers of cells were seeded onto the 96-well plates, and the cell growth was determined using WST-1 assay. The data are presented as percentage of control ± SD from six experiments. Cells transfected with COX-2 expression plasmid show ~26% increase in cell growth when compared with cells transfected with pcDNA3 vector (*P < 0.05). In addition, the cells stably transfected with the COX-2 expression plasmid or pcDNA3 control vector were also treated with the COX-2 inhibitor celecoxib (50 µM) in serum-free medium for 24 h to determine cell growth. COX-2 overexpression partially prevents the celecoxib-induced inhibition of cell growth (**P < 0.01 compared with pcDNA3 cells treated with celecoxib). B, prostaglandin (PG) E2 production in cells with or without COX-2 overexpression. Equal numbers of cells (3 x 105/ml) were cultured on 12-well plates; the 8-h supernatants were collected for PGE2 measurement; and the result is expressed as mean ± SD from four experiments. The production of PGE2 in cells with overexpression of COX-2 is significantly higher than in cells transfected with control vector under spontaneous culture conditions (*P < 0.05; n = 4). Increased PGE2 production is also observed in COX-2 overexpressed cells in the presence of celecoxib (50 µM; **P < 0.01 compared with pcDNA3 cells treated with celecoxib; n = 4).

 


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  		Fig. 7. The level of p27 and p21 in human cholangiocarcinoma cells with overexpression of cyclooxygenase-2 (COX-2). CCLP1 cells transfected with COX-2 expression plasmid or control vector pcDNA3 were treated with 50 µM celecoxib or DMSO vehicle for 24 h. Equal amount of cellular protein (20 µg) was subjected to SDS-PAGE, followed by Western blot analysis for COX-2, p27, and p21. Western blot analysis for ß-actin was used as the loading control. Overexpression of COX-2 did not alter the basal level or celecoxib-induced expression of p21 and p27.

 
Celecoxib Inhibits Growth of Human Cholangiocarcinoma Cells.
We then examined whether the selective COX-2 inhibitor celecoxib would prevent cholangiocarcinoma cell growth. Fig. 3ACitation shows that celecoxib treatment induced a dose-dependent inhibition of cell growth in SG231, HuCCT1, and CCLP1 cells. Despite the higher basal level of COX-2 expression in HuCCT1 cells, a comparable degree of inhibition of growth was observed in all of the three human cholangiocarcinoma cell lines.



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  		Fig. 3. The cyclooxygenase-2 (COX-2) inhibitor celecoxib inhibits human cholangiocarcinoma cell growth, which is partially prevented by prostaglandin (PG) E2. A, SG231, HuCCT1, and CCLP1 cells were treated with increasing concentrations of celecoxib (25–100 µM) or vehicle DMSO (0.1%) for 24 h, and the cell growth was measured by WST-1 assay. The cells treated with celecoxib showed a dose-dependent inhibition of cell growth (P < 0.01). The results were obtained from at least six individual experiments for each time point and are expressed as percentage of control ± SD. B, addition of PGE2 partially prevents the celecoxib-mediated inhibition of growth. HuCCT1 cells were treated with 50 µM celecoxib for 24 h in the presence or absence of 10 µM PGE2. The data are presented as percentage of control ± SD from six experiments (**P < 0.01 compared with control; *P < 0.05 compared with celecoxib-treated cells).

 
Celecoxib-Induced Inhibition of Cholangiocarcinoma Cell Growth Is Partially Reversed by COX-2 and PGE2.
Additional experiments were performed to examine whether COX-2 could protect cholangiocarcinoma cells from celecoxib-induced inhibition of growth. As shown in Fig. 2Citation , overexpression of COX-2 in CCLP1 cells partially prevented the celecoxib-mediated inhibition of growth; increased PGE2 production was observed in COX-2 overexpressed cells in the presence of celecoxib. In addition, cells treated with celecoxib showed decreased PGE2 production (Fig. 2)Citation , and exogenous PGE2 partially prevented the celecoxib-induced inhibition of growth (Fig. 3B)Citation . Consistent with these findings, celecoxib induced more prominent inhibition of growth in HuCCT1 cells with antisense depletion of COX-2 (see below; Fig. 6Citation ). These observations suggest that COX-2 and PGE2 can partially prevent the celecoxib-mediated inhibition of cholangiocarcinoma cell growth.



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  		Fig. 6. Effect of celecoxib on prostaglandin (PG) E2 production, p21/p27 expression, and cell growth in cholangiocarcinoma cells with or without antisense depletion of cyclooxygenase-2 (COX-2). A, effect of celecoxib on cell growth. HuCCT1 cells stably transfected with COX-2 antisense plasmid or control vector were cultured with or without doxycycline (2 µg/ml; 5 days). Equal numbers of cells with or without COX-2 antisense depletion (1 x 105/ml) were seeded onto the 96-well plates and treated with celecoxib or DMSO vehicle for 48 h. The cell growth was determined using WST-1 assay. Although celecoxib inhibits growth in cells without COX-2 depletion, this inhibitory effect is more prominent in cells with COX-2 depletion (*P < 0.05 compared with cells transfected with COX-2 antisense vector without doxycycline induction or cells transfected with control vector plus doxycycline treatment). The data represent percentage of control ± SD from six individual experiments. B, effect of celecoxib on PGE2 production. Equal numbers of cells (3 x 105/ml) were cultured on 12-well plates and treated with celecoxib or DSMO vehicle for 8 h. The supernatants were collected for PGE2 measurement. Celecoxib treatment resulted in a similar reduction of PGE2 release in cells with or without antisense depletion of COX-2. The data are presented as mean ± SD from four experiments. C, representative Western blot analysis showing the levels of COX-2, p21, and p27 in cells with antisense COX-2 depletion and celecoxib treatment. HuCCT1 cells transfected with COX-2 antisense plasmid or control vector were cultured in the presence or absence of doxycycline and treated with or without 50 µM celecoxib for 24 h. Equal amount of cellular protein (20 µg) was subjected to SDS-PAGE, followed by Western blot analysis for COX-2, p21, and p27. The same blot then was stripped and reprobed for ß-actin as the loading control.

 
Celecoxib-Induced Inhibition of Cell Growth Is Mediated in Part through G1-S Arrest.
To document further the mechanisms by which celecoxib inhibits cholangiocarcinoma cell growth, flow cytometry was performed to determine the cell cycle progression in cells with or without celecoxib treatment. As shown in Table 1Citation , celecoxib induced a significant inhibition of cell cycle progression at the G1-S checkpoint, with no effect on G2-M transit. This result suggests that the celecoxib-induced inhibition of cholangiocarcinoma cell growth is mediated, at least in part, through cell cycle inhibition at the G1-S checkpoint.


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  		Table 1 Cell cycle analysis of celecoxib-treated cholangiocarcinoma cells

Data are presented as mean (%) ± SD (n = 5). Cholangiocarcinoma cells were subjected to celecoxib treatment (50 µM) for 48 h. Cells then were harvested, and the cell cycle profile was analyzed using flow cytometry.

 
Celecoxib Increases Expression of cdk Inhibitors p21waf1/cip1 and p27kip1.
Because p21 and p27 play an important role in human cholangiocarcinoma growth (57, 58, 59, 60) , additional experiments were performed to examine whether celecoxib treatment might alter their expression. Fig. 4Citation shows that celecoxib treatment induced a significant increase of p21 and p27 in human cholangiocarcinoma cells. Basal level of p27 is detected in all of the three human cholangiocarcinoma cells lines (HuCCT1, SG231, and CCLP1); its expression is elevated significantly after treatment with 50 µM celecoxib. In HuCCT1 and CCLP1 cells, the induction of p27 appeared at 6 h (with ~2-fold increase) and progressed with longer time periods (~4.5-fold increase is observed at 48 h). In SG231 cells, the induction of p27 occurred at 12 h and peaked at 48 h. Whereas HuCCT1 cells express little or no p21 at baseline culture conditions, ~2.5-fold increase of p21 was observed 6 h after celecoxib treatment, and this effect peaked at 12 h. Celecoxib treatment also increased the expression of p21 in SG231 and CCLP1 cells. In contrast, the levels of p18INK4c (another G1 cdk inhibitor) and GADD45 (cdc2 inhibitor) were not altered after celecoxib treatment (data not shown). These results suggest the participation of p21waf1/cip1 and p27kip1 in celecoxib-mediated G1-S arrest in human cholangiocarcinoma cells.



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  		Fig. 4. Celecoxib induces the expression of p27 and p21 in human cholangiocarcinoma cells. HuCCT1, SG231, and CCLP1 cells were treated with 50 µM celecoxib or vehicle DMSO (0.1%) for 6–48 h, and the cell lysates were obtained for Western blot analysis. Equal amount of cellular protein (20 µg) was subjected to SDS-PAGE, followed by Western blot analysis for p27 and p21. Western blot for ß-actin was used as loading control.

 
We then measured the G1-S cdk2 activity in cells with or without celecoxib treatment. Fig. 5ACitation shows that celecoxib significantly inhibited the cdk2 activity in all of the three human cholangiocarcinoma cell lines (P < 0.05). The cells treated with 50 µM celecoxib for 24 h exhibited ~40–60% reduction of cdk2 activity. Western blot analysis showed that celecoxib did not alter the protein levels of cdk2 and cyclin E, indicating that the celecoxib-induced reduction of cdk2 activity is not mediated through cyclin E degradation or alteration of cdk2 protein level (Fig. 5B)Citation . Additional experiments were performed to evaluate whether celecoxib would alter the in vitro binding between cdk2 and p27 or p21. Human cholangiocarcinoma cells were treated with 50 µM celecoxib or DMSO vehicle for 24 h, and the cell lysates were obtained for immunoprecipitation using anti-cdk2 antibody, followed by Western blot for p27 and p21. Fig. 5CCitation shows that treatment with 50 µM celecoxib increases the binding of p27 to cdk2 kinase complex in all three of the human cholangiocarcinoma cell lines. Celecoxib treatment also increased the association between cdk2 complex and p21 in HuCCT1 and SG231 cells, although this effect is weak in CCLP1 cells. These results further demonstrate that celecoxib induces G1-S cell cycle arrest through induction of p27 and p21 in human cholangiocarcinoma cells.



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  		Fig. 5. Effect of celecoxib on cyclin-dependent kinase 2 (cdk2) kinase activity and cdk2/cyclin E protein level in cholangiocarcinoma cells. A, celecoxib inhibited cdk2 kinase activity. Human cholangiocarcinoma cells (HuCCT1, SG231, and CCLP) were treated with 50 µM celecoxib or 0.1% DMSO as control in serum-free medium for 24 h. The cell lysates were then obtained, and the cdk2 kinase was immunoprecipitated from 100 µg of cellular protein with antibody against cdk2 for 2 h at 4°C. The kinase assay was performed in 50 µl reaction buffer containing 20 µg histone H1 substrate and 10 µCi of [{gamma}-32P] ATP as described in "Materials and Methods." Celecoxib significantly inhibited the cdk2 kinase activity in all of the three cholangiocarcinoma cell lines (*P < 0.01 compared with DMSO control). The data are presented as percentage of control ± SD from four different experiments. B, celecoxib did not alter the protein levels of cdk2 and cyclin E. The cells were treated with 50 µM celecoxib or 0.1% DMSO in serum-free medium for 24 h. Equal amount of cell lysate was obtained for Western blot analysis using antibodies against cdk2 and cyclin E with ß-actin as loading control. C, celecoxib increased binding of p27 and p21 to cdk2 kinase complex. Cholangiocarcinoma cells were treated with 50 µM celecoxib for 24 h, and the cell lysates were obtained for immunoprecipitation using monoclonal anti-cdk2 antibody, followed by Western blot analysis for p27 and p21. Cell lysate incubated with mouse IgG was used as the negative control for immunoprecipitation. The cells treated with celecoxib showed increased association between cdk2 kinase complex and p27 or p21.

 
Effect of Altered COX-2 Expression on Celecoxib-Induced Cell Growth, PGE2 Production, and Expression of p21waf1/cip1 and p27kip1.
The direct effect of COX-2 on the expression of p21 and p27 in human cholangiocarcinoma cells was examined further by antisense inhibition and overexpression of COX-2. For antisense depletion of COX-2, HuCCT1 cells stably transfected with COX-2 antisense plasmid or control vector were cultured with or without doxycycline, and the cells were treated with celecoxib. Fig. 6ACitation shows that celecoxib induced more prominent inhibition of growth in HuCCT1 cells with antisense depletion of COX-2 when compared with the control cells (cells transfected with the COX-2 antisense plasmid but without doxycycline induction or cells transfected with the empty vector plus doxycycline treatment). However, the COX-2 antisense cells and control cells show a comparable degree of PGE2 reduction after celecoxib treatment (Fig. 6B)Citation . Furthermore, the celecoxib-mediated induction of p21 and p27 was not altered by antisense depletion of COX-2. As shown in Fig. 6CCitation , although ~80% reduction of COX-2 protein was achieved in cells expressing antisense COX-2, the level of p27 and p21 was not significantly altered either under spontaneous culture conditions or after celecoxib treatment. These findings suggest that celecoxib-induced growth inhibition is mediated, at least in part, through mechanisms independent of COX-2 inhibition.

We then performed experiments to examine whether overexpression of COX-2 would alter celecoxib-induced p21 and p27 expression. CCLP1 cells were transfected with the human COX-2 expression plasmid or control vector, and the cell lysates were obtained to determine the expression of p27 and p21. Fig. 7Citation shows that although an ~12-fold increase of COX-2 protein was achieved after transfection with the COX-2 expression plasmid, the level of p27 and p21 in these cells was not significantly altered when compared with the vector control cells either under basal culture conditions or after celecoxib treatment. Taken together, these findings suggest that the celecoxib-mediated induction of p21waf1/cip1 and p27kip1 in cholangiocarcinoma cells is mediated predominantly through mechanism(s) independent of COX-2 inhibition.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results in this study establish a direct role of COX-2 in the growth control of intrahepatic human cholangiocarcinoma cells. This conclusion is based on observations that overexpression of COX-2 increases PGE2 production in cholangiocarcinoma cells and enhances cell viability, that antisense depletion of COX-2 inhibits PGE2 production in these cells and decreases cell viability, and that PGE2 increases cholangiocarcinoma cell growth. Moreover, our findings demonstrate that the COX-2 inhibitor celecoxib potently inhibits cholangiocarcinoma cell growth, and this effect is mediated, in part, through inhibition of cell cycle progression. Because this study uses only intrahepatic cholangiocarcinoma cells, the potential role of COX-2 and celecoxib in the growth of extrahepatic cholangiocarcinoma cells remains to be further defined.

In eukaryotes, the cell cycle is regulated tightly at G1-S and G2-M checkpoints by several protein kinases composed of a cdk subunit(s) and corresponding regulatory cyclin subunit(s), and cdk inhibitors (61 , 62) . The G1-S progression is mediated by the cdks (cdk2, cdk4, and cdk6) in complex with the G1 cyclins (cyclin D1 and cyclin E), which phosphorylate Rb, resulting in cell cycle transit. The G1-S checkpoint is controlled by the CIP/KIP and INK4 families of cdk inhibitors, including p21waf1/cip1, p27kip1, and p18ink4c, which bind to G1 cyclin/cdk complexes and inhibit their catalytic activity, thus preventing the transition of cells from G1 to S phase (63 , 64) . The transition from G2 to M is regulated mainly by the G2-specific kinases consisting of cdc2 and cyclin B1 (65 , 66) . The G2-M checkpoint is controlled by regulatory molecules, including GADD45 (65 , 66) , which interact with the cdc2/cyclin B1 complex and inhibit the cdc2 activity, thus preventing the transition of cells from G2 to M phase. The expression of p27 is increased in lung cancer cells after treatment with the COX-2 inhibitor NS-398, suggesting that cdk inhibitors may be potential targets for COX-2 inhibitor-mediated inhibition of tumor growth (67) . The cdk inhibitors p21waf1/cip1, p27kip1, and GADD45 have been shown recently to play a critical role in the regulation of human intrahepatic cholangiocarcinoma growth (57, 58, 59, 60 , 68) . For example, decreased expression of p21waf1/cip1 and p27kip1 in human cholangiocarcinoma tissue is associated with poor patient survival (57, 58, 59, 60) ; induction of GADD45 is an important mechanism for agonist-induced G2-M phase arrest in human cholangiocarcinoma cells (68) . In the present study, we found that celecoxib treatment significantly increased the p21waf1/cip1 and p27kip1 protein level in a dose- and time-dependent fashion in human cholangiocarcinoma cells, whereas the protein levels of p18ink4c and GADD45 were not altered. The cells treated with celecoxib show increased binding of p21 and p27 to cdk2 kinase complex and decreased cdk2 kinase activity but no change in cdk2 and cyclin E protein levels. Consistent with these findings, flow cytometric analysis showed that celecoxib induced G1-S arrest with no significant effect on G2-M transition. These results provide a novel link between p21/p27 and celecoxib-mediated inhibition of intrahepatic cholangiocarcinoma cell growth.

Our findings provide evidence for the involvement of COX-2-independent mechanism in celecoxib-mediated inhibition of human intrahepatic cholangiocarcinoma cell growth. The observations that celecoxib inhibits the production of PGE2 and that overexpression of COX-2 or addition of exogenous PGE2 partially protects the cells from celecoxib-induced inhibition of growth suggest the involvement of COX-2 inhibition. However, the high concentration of celecoxib (50 µM) required for inhibition of growth, the incomplete protection of celecoxib-induced inhibition of cell growth by PGE2 or COX-2 overexpression, and the fact that overexpression or antisense depletion of COX-2 failed to alter the level of p21waf1/cip1 and p27kip1 indicate the existence of COX-2-independent effect. Thus, although celecoxib potently inhibits human cholangiocarcinoma cell growth, its antitumor effect is mediated, at least in part, through mechanisms independent of COX-2 inhibition.

The exact mechanism by which COX-2 promotes cholangiocarcinoma cell growth remains to be defined further. Because the level of p21 and p27 was not altered significantly by COX-2 overexpression and antisense depletion or by PGE2 treatment, it is likely that COX-2 and PGs may promote cholangiocarcinoma cell growth through other intracellular targets. In light of the limited survival benefit from current chemotherapy and the lack of effective chemoprevention for cholangiocarcinomas (69) , additional studies delineating the molecular mechanisms by which COX-2 and its inhibitors modulate cholangiocarcinogenesis will likely provide important future therapeutic implications.


   ACKNOWLEDGMENTS
 
We thank Dr. Timothy Hla at the University of Connecticut Health Center for providing the human COX-2 expression plasmid, Dr. J. Morrow at Vanderbilt University for providing the Tet-On antisense COX-2 construct, and Pharmacia Corp. (St. Louis, MO) for providing celecoxib.


   FOOTNOTES
 
Grant support: Grants from the American Liver Foundation (to T. Wu), Cancer Research Foundation of America (to T. Wu), Wendy Will Case Cancer Fund (to T. Wu), National Institutes of Health (DK49615 to A. J. Demetris), and Department of Pathology, University of Pittsburgh Medical Center.

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Tong Wu, Department of Pathology, University of Pittsburgh School of Medicine, Presbyterian University Hospital C902, 200 Lothrop Street, Pittsburgh, PA 15213. Phone: 412-647-9504; Fax: 412-647-5237; E-mail: wut{at}msx.upmc.edu

Received 4/21/03. Revised 12/ 4/03. Accepted 12/11/03.


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