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Mol Cancer Ther. 2004;3:299-307
© 2004 American Association for Cancer Research

The cyclooxygenase-2 inhibitor celecoxib blocks phosphorylation of Akt and induces apoptosis in human cholangiocarcinoma cells

Tong Wu1, Jing Leng1,2, Chang Han1 and Anthony Jake Demetris1

1 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA and 2 Department of Pathology, Nanjing Medical University, Nanjing, China

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The expression of cyclooxygenase (COX)-2 is increased in human cancers including cholangiocarcinoma. This study was designed to evaluate the effect and mechanisms of the selective COX-2 inhibitor celecoxib in the growth control of human cholangiocarcinoma cells. Immunohistochemical analysis using human cholangiocarcinoma tissues showed increased levels of COX-2 as well as phospho-Akt (Thr 308), a protein kinase activated by COX-2-mediated prostaglandins, in human cholangiocarcinoma cells. Treatment of cultured human cholangiocarcinoma cells (HuCCT1, SG231, and CCLP1) with celecoxib resulted in a dose- and time-dependent reduction of cell viability. Fluorescence microscopy, Western blot, and caspase activity assays demonstrated that celecoxib induced morphological features of apoptosis, activation of caspase-9 and caspase-3, and release of cytochrome c. The celecoxib-induced cell death was significantly blocked by N-benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone, a wide-spectrum caspase inhibitor. Furthermore, cholangiocarcinoma cells treated with celecoxib showed significant reduction of Akt phosphorylation, whereas the levels of Bcl-2 and Bax were not altered. Inhibition of Akt activation by LY294002 significantly decreased the viability of human cholangiocarcinoma cells. These findings suggest that celecoxib inhibits cholangiocarcinoma growth partly through induction of apoptosis and inhibition of Akt phosphorylation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cholangiocarcinoma is the second most common primary hepatobiliary malignancy in adults with high mortality. It often arises from 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. Although the molecular mechanisms underlying cholangiocarcinogenesis have yet to be determined, recent evidences indicate that mediators of inflammation, such as prostaglandins (PGs), may play an important role in the growth regulation of human cholangiocarcinoma ( 1 –8). This is highlighted by the observations that the expression of cyclooxygenase (COX)-2 is increased in human cholangiocarcinoma cells ( 1, 3, 6) and that PGs promote cholangiocarcinoma cell growth ( 4, 5, 7, 8).

The synthesis of PGs is controlled by two COX enzymes—COX-1 and COX-2 isoforms. Whereas COX-1 is constitutively expressed in human cells, COX-2 is an inducible enzyme that is up-regulated during pathological conditions such as inflammation and cancer. Recent evidence shows that COX-2 overexpression may increase tumorigenic potential via prevention of cell apoptosis ( 8, 9). Consistent with this, null mutation of COX-2 in Apc716 knockout mice, a murine model of familial adenomatous polyposis, restored apoptosis and reduced the size and the number of colorectal adenomas ( 10). These observations suggest that modulation of apoptotic pathway may represent a critical intracellular mechanism by which COX-2-controlled PGs regulate cancer cell growth.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are COX inhibitors that have been shown to inhibit cancer cell growth in various in vitro and in vivo experimental studies ( 11 –22). The clinical potential of COX inhibitors was suggested by epidemiological studies, which showed a lower incidence of cancers in patients with NSAID use ( 15, 23). The clinical efficacy of COX inhibitors in cancer chemoprevention was demonstrated by randomized studies using COX inhibitors in patients with a precancerous condition ( i.e., familial adenomatous polyposis; 24 –27). Consistent with the antiapoptotic effect of COX-2, induction of cancer cell apoptosis is the principle mechanism by which NSAIDs inhibit cancer cell growth ( 11, 19, 20, 28 –30). Celecoxib (SC-58635) 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 progression of colon tumor in human and animal models and inhibits the in vitro growth of several other tumor cell types ( 5, 27, 31 –42). These findings suggest that celecoxib may provide effective chemoprevention and treatment for human cancer with fewer side effects.

In spite of the established role of COX-2 and NSAIDs in human cancer, little is known about the effect and mechanism of COX-2-selective NSAIDs in the growth control of cholangiocarcinoma cells. In this study, we show that the COX-2 inhibitor celecoxib blocks Akt phosphorylation and induces apoptosis in human cholangiocarcinoma cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
MEM, fetal bovine serum, glutamine, trypsin, and antibiotics were purchased from Life Technologies, Inc. (Rockville, MD). PGE 2, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], and caspase inhibitor N-benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) were purchased from Calbiochem (San Diego, CA). The cell growth assay reagent WST-1 was purchased from Roche Molecular Biochemicals (Indianapolis, IN). The COX-2 inhibitor celecoxib (SC-58635) was kindly provided by Pharmacia (Chicago, IL). The antibody for human COX-2 was purchased from Cayman Chemical Co. (Ann Arbor, MI). The polyclonal rabbit anti-human caspase-3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-human ß-actin monoclonal antibody was purchased from Sigma Chemical Co. (St. Louis, MO). The horseradish peroxidase-linked streptavidin and chemiluminescence detection reagents were from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ). The PGE 2 enzyme immunoassay system was also from Amersham Pharmacia Biotech. The rabbit polyclonal antibodies against human Akt, phospho-Akt (Thr 308), phospho-PTEN (phosphatase and tensin homologue), caspase-9, and Bax were obtained from Cell Signaling Technology (Beverly, MA). The mouse antibodies against human Bcl-2 and Bax were obtained from Stressgen Biotechnologies (Victoria, BC, Canada). The mouse anti-human cytochrome c was purchased from BD Biosciences (San Diego, CA). The Bio-Rad protein assay system was obtained from Bio-Rad Laboratories (Hercules, CA). The Tris-glycine gels were obtained from Invitrogen Life Technologies (Carlsbad, CA). The anti-phospho-Akt (Thr 308) antibody for immunohistochemical staining was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). The indirect avidin-biotin complex immunohistochemistry assay detection kit was from Vector Laboratories, Inc. (Burlingame, CA). The caspase-3 and caspase-9 colorimetric protease assay kit was purchased from BioSource International, Inc. (Camarillo, CA).

Human Cholangiocarcinoma Tissues
Archival formalin-fixed, paraffin-embedded specimens of human cholangiocarcinoma and nonneoplastic liver tissues were obtained from the University of Pittsburgh Medical Center. The tissue specimens were used for immunohistochemical analysis for COX-2 and phospho-Akt (Thr 308) according to the protocol approved by the University of Pittsburgh Institutional Review Board (020134). None of the cases used in this study had patient identifiers and strict confidentiality was maintained in accordance with the approval granted by the Institutional Review Board.

Cell Culture and Experimental Design
Three human intrahepatic cholangiocarcinoma cell lines including HuCCT1 (obtained from Japanese Cancer Research Resources Bank; 43), SG231 ( 44), and CCLP1 ( 45) were used in this study. The cells were cultured according to our previously described conditions ( 4, 46). The experiments were performed when cells reached 80% confluence and conducted in serum-free medium with serum deprivation for 12 h prior to experiments.

For experiments with COX-2 inhibitor celecoxib, the HuCCT1, SG231, and CCLP1 cells were treated with various concentrations of celecoxib (0.1–100 µ M) in serum-free medium for 4–72 h (100 m M stock solution was prepared in DMSO and the control cells were treated with DMSO vehicle). For experiments evaluating the effect of PGE 2 on cell viability, the cells were treated with increasing concentrations of PGE 2 (5–100 µ M) or vehicle (ethanol, <0.1%) in serum-free medium for 24–48 h and the cell viability was measured by WST-1 assay. At the end of treatment, the cell lysates were obtained for Western blot analysis for phospho-Akt and apoptosis-associated proteins and the cell viability was determined. In some experiments, the cells were subjected to light and fluorescence microscopy to assess morphological features of cell apoptosis. For experiments with PI3K inhibitor LY294002, the cells were treated with PI3K inhibitor LY294002 (0.1–50 µ M) for 24 h in serum-free medium and the cell viability was measured by WST-1 assay.

PGE 2 Immunoassay
Human cholangiocarcinoma cells with or without celecoxib treatment and with or without overexpression or antisense inhibition of COX-2 were cultured in 6- or 12-well plates in 0.5 or 1.0 ml of serum-free medium. The 8-h supernatants were then collected and assayed for PGE 2 production by enzyme immunoassay. The centrifuged supernatant (100 µl) was analyzed for each sample.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increased COX-2 and phospho-Akt in Human Cholangiocarcinoma Cells
Immunohistochemical stains for COX-2 and phospho-Akt (Thr 308) were performed on 12 human cholangiocarcinoma tissue specimens and nonneoplastic liver tissues. Significantly increased cytoplasmic staining of COX-2 and phospho-Akt was observed in cholangiocarcinoma cells when compared with the nonneoplastic bile duct epithelium. The average staining intensity for COX-2 expression in cholangiocarcinoma is 2.14, which is significantly higher than that in nontumor liver tissue (0.95; P < 0.01). The level of phospho-Akt in human cholangiocarcinoma tissue is also significantly higher than that in the nonneoplastic liver tissue, with the average staining intensity for phospho-Akt being 1.94 in cholangiocarcinoma versus 0.71 in nontumor liver tissue ( P < 0.01). No phospho-Akt and COX-2 are detected in the lobular hepatocytes, interlobular bile ducts, or nonparenchymal cells such as Kupffer cells and endothelial cells. The expression of COX-2 and phospho-Akt in human cholangiocarcinoma tissue is depicted in Fig. 1 . Immunocytochemical staining showed cytoplasmic expression of COX-2 and phospho-Akt in cultured human cholangiocarcinoma cells ( Fig. 2A ). The expression of COX-2 and phospho-Akt in these cells was also confirmed by Western blot analysis ( Fig. 2B).



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  		Figure 1. Increased levels of COX-2 and phospho-Akt in human cholangiocarcinoma tissue. Human cholangiocarcinoma tissue sections were stained by avidin-biotin complex immunohistochemistry assay using antibodies against human COX-2 and phospho-Akt (Thr 308) according to described methods ( 55). After immunostaining, the slides were counterstained by hematoxylin. Images show the expression of COX-2 and phospho-Akt in human cholangiocarcinoma cells. A, COX-2 expression [3-amino-9-ethylcarbazole (AEC), x200; inset, x400]. B, negative control for COX-2 (AEC, x200). C, phospho-Akt level (3,3'-diaminobenzidine, x200; inset, x400). D, negative control for phospho-Akt (3,3'-diaminobenzidine, x200). No signal staining was observed in the negative controls (the primary antibodies were substituted with corresponding nonimmunized IgG).

 


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  		Figure 2. Expression of COX-2 and phospho-Akt in human cholangiocarcinoma cell lines. A, immunocytochemical analysis showing cytoplasmic staining of COX-2 and phospho-Akt (Thr 308) in HuCCT1 cells. The stains were performed according to described methods ( 55). a, immunostaining for COX-2 (AEC, x400); b, negative control for COX-2 (hematoxylin counterstain); c, immunostaining for phospho-Akt (Thr 308; AEC, x400); d, negative control for phospho-Akt (hematoxylin counterstain). B, Western blot analysis showing the levels of COX-2, phospho-Akt (Thr 308), and total Akt in human cholangiocarcinoma cells, with cell lysate from a human hepatocellular carcinoma cell line ( Hep3B) as positive control.

 
Celecoxib Inhibits Akt Phosphorylation and Prevents Cholangiocarcinoma Cell Growth
The expression of both COX-2 and phospho-Akt in human cholangiocarcinoma cells suggests a potential link between these two growth-promoting pathways for cholangiocarcinoma growth. To further investigate this, we examined the effect of COX-2 inhibitor celecoxib on Akt phosphorylation in human cholangiocarcinoma cells. As shown in Fig. 3A , celecoxib significantly inhibits the phosphorylation of Akt, which is reflected by the marked reduction of phospho-Akt and concomitant increase of non-phospho-Akt. Because recent studies in prostate and colon cancers have associated Bcl-2 regulation with COX-2 inhibition ( 47, 48), the potential effect of celecoxib on Bcl-2 expression was also evaluated. Figure 3B shows that the Bcl-2 level was not altered after celecoxib treatment. Furthermore, protein concentrations of Bax, another proapoptotic Bcl-2 family member, were also unchanged in cells treated with celecoxib. The above results indicate that inhibition of Akt phosphorylation is an important signaling event induced by celecoxib in human cholangiocarcinoma cells.



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  		Figure 3. Celecoxib inhibits the phosphorylation of Akt in human cholangiocarcinoma cells. HuCCT1 cells were treated with celecoxib (50 µ M) for 4–24 h and the cell lysates were obtained for Western blot analysis using antibodies against phospho-Akt (Thr 308) and Bcl-2. Cellular protein (20 µg) was used for each sample and the immunoblot for ß-actin was used as loading control. A, celecoxib treatment inhibited Akt phosphorylation as reflected by the decrease of phospho-Akt with concomitant increase of nonphosphorylated Akt. B, the level of Bcl-2 was not altered after celecoxib treatment. Similar effects were observed in SG231 and CCLP1 cells.

 
The phosphorylation status of Akt is controlled by the PI3K/3-phosphoinositide-dependent protein kinase-1 (PDK1) pathway. Phosphorylation of PDK1 by PI3K increases its enzyme activity that subsequently activates Akt via phosphorylation at Thr 308. This process is inhibited by PTEN, a phosphatase that converts phosphatidylinositol 3,4,5-triphosphate back to phosphatidylinositol 4,5-bisphosphate, thus inhibiting PI3K/PDK1 activation (phosphorylation of PTEN down-regulates its phosphatase activity leading to increased PI3K/PDK1 activity). To further document the mechanism by which celecoxib inhibits Akt phosphorylation in cholangiocarcinoma cells, the effect of celecoxib on the phosphorylation of PDK1 and PTEN was also examined. As shown in Fig. 4 , the cells treated with celecoxib (50 µ M) showed significantly decreased phosphorylation of PDK1 and PTEN. This effect is unlikely due to nonspecific inhibition of serine/threonine phosphorylation, because celecoxib treatment did not alter the level of other serine/threonine phosphoproteins such as phospho-p21 Thr 145 and phospho-p21 Ser 146. These findings indicate that celecoxib may inhibit Akt phosphorylation through mechanisms involving inhibition of PDK1 and PTEN phosphorylation, although the kinases responsible for celecoxib-induced inhibition of PDK1 and PTEN phosphorylation remain to be further defined.



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  		Figure 4. Celecoxib inhibits phosphorylation of PDK1 and PTEN in human cholangiocarcinoma cells. HuCCT1 cells were treated with celecoxib (50 µ M) for 8 h and the cell lysates were obtained for Western blot analysis for p-PDK1 (Ser 241) and p-PTEN (Ser 380). Cellular protein (20 µg) was used for each sample and the immunoblot for ß-actin was used as loading control. Results are representative of three individual experiments.

 
Consistent with the inhibition of Akt phosphorylation, celecoxib induces a dose- and time-dependent reduction of cholangiocarcinoma cell viability. As shown in Fig. 5 , treatment of cells with 75 and 50 µ M celecoxib for 24 h caused a loss of viability up to 80% and 50%, respectively.



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  		Figure 5. Celecoxib decreases cholangiocarcinoma cell viability. Human cholangiocarcinoma cells were treated with increasing concentrations of celecoxib (25–100 µ M) or DMSO vehicle (0.1%) for 24 h and the cell viability was measured by WST-1 assay. The cells treated with celecoxib showed a dose-dependent reduction of cell viability ( P < 0.01; n = 6).

 
Celecoxib-Induced Inhibition of Cholangiocarcinoma Cell Growth Is Mediated in Part by Apoptosis
Because Akt has been shown to promote cell survival through mechanisms including inhibition of apoptosis ( 49 –51), we then examined if cholangiocarcinoma cells treated with celecoxib undergo apoptosis. Figure 6A shows that treatment with celecoxib (25 and 50 µ M) for 24 h resulted in morphological features of cell death characterized by cell shrinkage and detachment. Fluorescence microscopy following propidium iodine staining revealed nuclear features of apoptosis characterized by marked chromatin condensation and fragmentation. Western blot analysis revealed that celecoxib induced activation of caspase-9 and caspase-3, which was detected 4–24 h after celecoxib (50 µ M) treatment ( Fig. 6B). These observations were also confirmed by the assays for caspase activity, which revealed ~12.8–13.4-fold increase of caspase-3 activity ( P < 0.01) and 2.3–3.2-fold increase of caspase-9 activity ( P < 0.01) 4–8 h after celecoxib treatment. Consistent with these findings, the release of cytochrome c was also increased after celecoxib treatment ( Fig. 6C). To further determine whether the cytotoxic effect of celecoxib was due to apoptosis, the cells were treated with celecoxib in the presence of the wide-spectrum caspase inhibitor Z-VAD-fmk. In the presence of Z-VAD-fmk, a reduction in celecoxib-induced cell death was observed ( Fig. 6D). These findings demonstrated that celecoxib reduced cholangiocarcinoma cell viability at least in part through induction of apoptosis.



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  		Figure 6. Celecoxib inhibits cholangiocarcinoma cell growth through induction of apoptosis. A, phase light microscopy (x100) showing the morphological changes of cholangiocarcinoma cells after celecoxib treatment for 24 h; 25 µ M celecoxib decreased cell number and 50 µ M celecoxib resulted in cell floating and cell death. B, Western blot analysis showing activation of caspase-3 and caspase-9 after celecoxib treatment. HuCCT1 cells were treated with celecoxib (50 µ M) for 4–24 h and the cell lysates were obtained for Western blot analysis. Activation of caspase-3 (reflected by the presence of 20-kDa proteolytic cleaved band) and caspase-9 (reflected by the appearance of 35- and 37-kDa cleaved bands) was observed 4, 8, 12, and 24 h after celecoxib treatment. C, increased cytochrome c release after celecoxib treatment. The HuCCT1 cells were treated with celecoxib and the cytosolic protein was obtained for Western blot analysis for cytochrome c according to the described method ( 55). D, caspase-3 inhibitor Z-VAD-fmk blocks the celecoxib-induced inhibition of growth. Human cholangiocarcinoma cells were treated with celecoxib (50–100 µ M) in the presence or absence of Z-VAD-fmk (100 µ M) in serum-free medium for 24 h (Z-VAD-fmk was added to the cells 1 h prior to celecoxib treatment; DMSO vehicle concentration, 0.1%). The cell viability was measured by WST-1 assay. Celecoxib-induced reduction of cell viability was partially prevented by cotreatment with Z-VAD-fmk ( P < 0.01). a, HuCCT1 cells; b, SG231 cells; c, CCLP1 cells. Results were obtained from three individual experiments.

 
Inhibition of Akt by LY294002 Blocks Human Cholangiocarcinoma Cell Growth
The effect of LY294002 (inhibitor of Akt activation) on cholangiocarcinoma cell growth was examined. Figure 7 shows that treatment of cells with LY294002 (0.1–50 µ M) caused a dose-dependent inhibition of cell growth. These results support the involvement of Akt in human cholangiocarcinoma cell growth, although the IC 50 of LY294002 is higher than the reported IC 50 of 1.4 µ M ( 52). Our data showed that the celecoxib-induced reduction of Akt phosphorylation occurred earlier than the morphological evidence of cell apoptosis in human cholangiocarcinoma cells. Whereas celecoxib (50 µ M)-induced inhibition of Akt phosphorylation was observed at 4 h, morphological evidence of cell death was detected at later incubation periods (18–24 h), as assessed by phase light microscopy, fluorescence microscopy, and WST-1 assay. These findings suggest that Akt inhibition is likely the cause of celecoxib-induced apoptosis.



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  		Figure 7. Inhibition of Akt by LY294002 blocks human cholangiocarcinoma cell growth. HuCCT1 cells were treated with increasing concentrations of LY294002 in serum-free medium (DMSO vehicle concentration, 0.1%) for 24 h and the viable cells were measured by WST-1 assay. LY294002 induced a dose-dependent inhibition of cell growth ( P < 0.01). Columns, percentage of control from three individual experiments; bars, SD. A similar effect was also observed in SG231 and CCLP1 cells.

 
Celecoxib-Induced Cholangiocarcinoma Cell Death Is Partially Reversed by PGE 2
Further experiments were performed to examine if PGE 2 could protect cholangiocarcinoma cells from celecoxib-induced cell death. As shown in Fig. 8 , cells treated with celecoxib showed decreased PGE 2 production, indicating an inhibition of COX-2 by celecoxib in these cells. Addition of PGE 2 partially prevented the celecoxib-induced cell death ( Fig. 9 ). These findings suggest that celecoxib-induced apoptosis may involve inhibition of COX-2 in human cholangiocarcinoma cells. However, the high concentration of celecoxib required for apoptosis (50 µ M) and the incomplete prevention of celecoxib-induced cell death by PGE 2 also suggest the existence of COX-2-independent mechanism in celecoxib-mediated cholangiocarcinoma cell apoptosis.



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  		Figure 8. Celecoxib inhibits PGE 2 production in human cholangiocarcinoma cells. HuCCT1 cells cultured in 12-well plate were treated with celecoxib (10 µ M) in serum-free RPMI 1640 (500 µl) for 8 h. The supernatant (100 µl) was collected from each well for PGE 2 measurement and the result was compared with that from control cells. Columns, percentage of inhibition of PGE 2 production from three separate experiments; bars, SD ( P < 0.01).

 


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  		Figure 9. PGE 2 partially prevents the celecoxib-induced inhibition of cholangiocarcinoma cell growth. HuCCT1 cells were treated with celecoxib (50 µ M) for 24 h in the presence of increasing concentration of PGE 2 and the cell viability was measured by WST-1 assay. Addition of PGE 2 partially reversed the celecoxib-induced growth inhibition ( P < 0.01). Results represent the average data from three experiments for each individual time point.

 
Effect of Antisense Depletion of COX-2 on Celecoxib-Induced Cell Death
HuCCT1 cells with antisense depletion of COX-2 were selected to further determine the role of COX-2 in celecoxib-induced cell death. The 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) and the stably transfected cells were selected using G418. The expression of antisense COX-2 RNA was induced by treatment with doxycycline (2 µg/ml) for 4 and 5 days. As shown in Fig. 10A , ~80% reduction of COX-2 protein was achieved in cells expressing antisense COX-2. These cells were then treated with celecoxib to determine cell viability. Figure 10B shows that celecoxib induced more prominent reduction of cell viability in HuCCT1 cells with antisense depletion of COX-2 when compared with the cells transfected with the COX-2 antisense plasmid but without doxycycline induction or cells transfected with the empty control vector plus doxycycline treatment. However, the COX-2 antisense cells and control cells show a comparable degree of PGE 2 reduction after celecoxib treatment and the level of Akt phosphorylation was not significantly altered by antisense depletion of COX-2. These findings suggest that the celecoxib-induced cell death and inhibition of Akt phosphorylation are mediated at least in part through mechanisms independent of COX-2 inhibition.



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  		Figure 10. Effect of celecoxib on the viability of cholangiocarcinoma cells with antisense depletion of COX-2. 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. The cells with stable transfection of 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 and 5 days. A, Western blot analysis showing the level of COX-2 protein in antisense COX-2 cells. B, cell growth in cells with or without antisense depletion of COX-2. Celecoxib induced a more prominent reduction of viability in cells with antisense depletion of COX-2 ( P < 0.01; n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study reveals that celecoxib inhibits human cholangiocarcinoma cell growth via induction of apoptosis. Celecoxib treatment induces marked morphological characteristics and biochemical changes of apoptosis in these cells. In contrast to the previous reports that correlated the apoptotic effect of COX-2 inhibitors with Bcl-2 down-regulation in intestine epithelial and colon carcinoma cells ( 47, 48), our data indicate that the induction of apoptosis by celecoxib is independent of Bcl-2 in human cholangiocarcinoma cells. We show that celecoxib induces apoptosis at least in part by blocking the activation of Akt. Moreover, celecoxib inhibited PDK1 and PTEN phosphorylation in cholangiocarcinoma cells, suggesting their involvement in celecoxib-mediated inhibition of Akt activation. The latter findings are consistent with the documented role of PDK1 and PTEN in the regulation of Akt phosphorylation in other cell types ( 49 –51).

Akt plays a key role in tumorigenesis and cancer progression by stimulating cell proliferation and inhibiting apoptosis ( 49 –51); phosphorylation of Akt has recently been implicated in the COX-2-mediated lung cancer and hepatocellular carcinoma cell survival ( 54, 55). The Akt is composed of a NH 2-terminal pleckstrin homology domain and a COOH-terminal kinase catalytic domain. It is activated by a dual regulatory mechanism that requires both translocation to the plasma membrane and phosphorylation. The generation of phosphatidylinositol 3,4,5-triphosphate on the inner layer of the plasma membrane, following PI3K activation, recruits Akt by direct interaction with its pleckstrin homology domain. At the membrane, Akt is phosphorylated on Thr 308 by PDK1. The phospho-Akt dissociates from the membrane and enters the cytoplasm and nucleus, where it phosphorylates several key proteins resulting in promotion of cell cycle and inhibition of apoptosis. In this study, we provide novel evidence for increased level of phospho-Akt in human cholangiocarcinoma tissue. The importance of Akt in cholangiocarcinoma cell survival is also supported by the observation that inhibition of Akt activation by LY294002 significantly decreased cholangiocarcinoma cell viability. Furthermore, the results in this study also reveal that Akt is an important target that is inhibited by celecoxib in human cholangiocarcinoma cells.

Our findings provide evidence for the involvement of COX-2-independent mechanism in celecoxib-mediated cholangiocarcinoma cell apoptosis. The observations that celecoxib inhibits the production of PGE 2 and that PGE 2 partially protects the cells from celecoxib-induced cell death suggest the inhibition of COX-2 by celecoxib in these cells. However, the relatively high concentrations of celecoxib required for apoptosis, the incomplete protection of celecoxib-induced cell death by PGE 2, and that antisense depletion of COX-2 failed to alter the level of phospho-Akt indicate the existence of COX-2-independent effect. Thus, although celecoxib inhibits human cholangiocarcinoma cell growth, its antitumor effect is mediated at least in part through mechanisms independent of COX-2 inhibition. This assertion is also supported by the recent studies from other investigators showing that celecoxib induces apoptosis via COX-2-independent mechanism in other human cancer cells ( 32, 33, 36, 37).

One limitation of this study is the high concentration of celecoxib required for induction of cholangiocarcinoma cell apoptosis in our in vitro system (50 µ M). This concentration is ~10–25 times the plasma concentration in patients receiving celecoxib treatment (2–5 µ M; 56) and will unlikely be achieved in human or animal tissues. Therefore, nonspecific actions may also take place in these in vitro experiments. Further studies are needed to determine whether celecoxib at therapeutic doses will inhibit cholangiocarcinoma growth and Akt phosphorylation in vivo.


   Acknowledgments
 
We thank Dr. J. Morrow (Vanderbilt University) for providing the Tet-On antisense COX-2 construct and Pharmacia Corp. (St. Louis, MO) for providing celecoxib.


   Footnotes
 
Grant support: American Liver Foundation (T. Wu), Cancer Research Foundation of America (T. Wu), Wendy Will Case Cancer Fund (T. Wu), NIH grant DK49615 (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.

Received 7/ 8/03; revised 12/ 4/03; accepted 12/15/03.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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