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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Recently, we demonstrated that the cyclooxygenase-2 (COX-2) inhibitor celecoxib acts to significantly suppress the growth of rat C611B cholangiocarcinoma (ChC) cells in vitro. To establish a molecular mechanism for this growth suppression, we investigated the effects of celecoxib on apoptotic signaling pathways in cultured rat C611B ChC cells. Celecoxib and another COX-2 inhibitor, rofecoxib, at 5 μM were almost equally effective in inhibiting prostaglandin E2 (PGE2) production by these cells, but at this low concentration, neither inhibitor suppressed growth or induced apoptosis. Celecoxib at 50 μM induced prominent apoptosis in these cells, whereas rofecoxib at 50 μM was without effect in either suppressing growth or inducing apoptosis. Celecoxib (50 μM) did not alter Bcl-2, Bcl-xL, or COX-2 protein levels, nor did it inhibit p42/44 mitogen-activated protein kinase (MAPK) phosphorylation; however, it significantly suppressed serine/threonine kinase Akt/PKB (Akt) phosphorylation and kinase activity in cultured C611B cells. This effect, in turn, directly correlated with Bax translocation to mitochondria, cytochrome c release into cytosol, activation of caspase-9 and caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP). Addition of 25 μM PGE2 to C611B cell cultures blocked the apoptotic actions of celecoxib. Rofecoxib (50 μM) was without effect in suppressing Akt phosphorylation and caspase-3 activation. In vivo, celecoxib partially suppressed tumorigenic growth of C611B ChC cells. In conclusion, our results indicate that celecoxib preferentially acts in vitro to induce apoptosis in ChC cells through a mechanism involving Akt inactivation, Bax translocation, and cytochrome c release. Our in vivo results further suggest celecoxib might have potential therapeutic or chemopreventive value against ChC. (HEPATOLOGY 2004;39:1028–1037.)

Accumulating evidence suggests that cyclooxygenase-2 (COX-2), the inducible form of prostaglandin endoperoxidase, may play an important role in cholangiocarcinogenesis in both the human and in the furan rat model.1–3 COX-2 has been demonstrated to be significantly up-regulated in the neoplastic epithelium of a large percentage of analyzed cases of human intra- and extrahepatic cholangiocarcinoma (ChC),4–6 being more prominent in its immunochemical staining intensities in well-differentiated tumors than in moderately and poorly differentiated tumors.4, 6 In addition, using reverse-transcription polymerase chain reaction, we could detect COX-2 messenger RNA (mRNA) in neoplastic glands obtained by laser capture microdissection from furan-induced transplantable rat ChC, but not in hyperplastic bile ducts microdissected from the liver of bile duct-ligated rat.2 Recently, COX-2 mRNA has also been found to be constitutively expressed in a number of human ChC cell lines.5 Moreover, COX-2 mRNA and protein are abundantly expressed in a novel rat ChC cell line, designated C611B, established in our laboratory from a transplantable ChC derived from the furan rat cholangiocarcinogenesis model.2, 7, 8

Recently, Hayashi et al.5 demonstrated that the select COX-2 inhibitors JTE-522 and NS-398 (at concentrations of 100 μM and 200 μM, respectively) maximally inhibited the in vitro growth of 5 separate human COX-2-expressing ChC cell lines by greater than or equal to 90%. NS-398 has also recently been found by Wu et al.9 to significantly inhibit hepatocyte growth factor and IL-6-induced release of arachidonic acid, prostaglandin synthesis, and cell growth in 3 separate cultured human ChC cell lines. Furthermore, we recently showed that the clinically relevant COX-2 inhibitor celecoxib produces a significant dose-dependent inhibition of cell growth of rat C611B ChC cells in vitro, which could be circumvented in large part by the addition of prostaglandin E2 (PGE2) ethanolamide to the culture medium.2 While these data suggest COX-2 inhibitors might have therapeutic potential in the management of and/or chemoprevention of ChC, evidence establishing specific molecular mechanisms underlying their action to suppress the growth of cultured human and rat ChC cell types in vitro is lacking. There also have been no reported studies aimed at ascertaining the effect of COX-2 inhibitors on suppressing the growth of COX-2-expressing ChC cells in vivo. We now report that the COX-2 inhibitor celecoxib acts to suppress the growth of rat C611B ChC cells in vitro by a mechanism involving Akt inactivation, leading to translocation of Bax to mitochondria, cytochrome c release to the cytosol, and associated activation of caspase-9 and caspase-3, culminating in prominent apoptosis. We further show that celecoxib and another clinically important selective COX-2 inhibitor, rofecoxib, differ significantly in their ability to suppress growth and induce apoptosis in C611B ChC cells in vitro. Moreover, we demonstrate the ability of celecoxib to partially suppress tumorous growth of ChC cells following their subcutaneous transplantation into syngeneic recipient rats.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Drugs and Reagents.

Celecoxib was kindly provided by Pharmacia Corp. (St. Louis, MO) and rofecoxib was obtained as a generous gift from Merck & Co., Inc. (Whitehouse Station, NJ). PGE2 was purchased from Cayman Chemical, Ann Arbor, MI. Dimethylsulfoxide (DMSO) together with 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich Co., St. Louis, MO. The phosphatidylinositol 3-kinase (PI3)-kinase) inhibitor LY 294002 was purchased from Cell Signaling Technology, Beverly, MA.

C611B ChC Cell Line and In Vitro COX-2 Inhibitor Treatments.

The tumorigenic rat C611B ChC cell line used in this study was established in our laboratory,7 and previously characterized in terms of its significant up-regulation of COX-2.1, 2 Our standard conditions for culturing C611B ChC cells on plastic substratum were also previously published.7, 10 Celecoxib or rofecoxib were administered separately to individual cultures at different concentrations in DMSO at a final solvent concentration of 0.1%, essentially according to previously described treatment conditions.2, 8

Western Blotting and Primary Antibodies.

Western blot analysis was performed as previously described,8, 11 with each of the following primary antibodies: (1) cleaved caspase-9 (Asp353) rat-specific antibody (#9507), an affinity-purified rabbit polyclonal antibody detecting only the large active subunit (35 kd/17 kd) of caspase-9 when cleaved at Asp 353; (2) caspase-3 antibody (#9662), an affinity-purified rabbit polyclonal antibody detecting the full length (32 kd) and cleaved large fragment (17 kd) of caspase-3; (3), cleaved poly (ADP-ribose) polymerase (PARP; Asp214) rat specific antibody (#9545), an affinity-purified rabbit polyclonal antibody detecting cleaved large fragment (89 kd) of rat nuclear PARP; (4) Bcl-2 (C-2)—sc-7382, mouse monoclonal IgG1 raised against amino acids 1–205 of human Bcl-2; (5) Bcl-xL (H-62)—sc-7195, rabbit polyclonal IgG raised against an epitope corresponding to amino acids 126–188, mapping at the carboxy (COOH) terminus of human Bcl-xSL, but not present in Bcl-xS; (6) actin (C-11)—sc-1615, affinity-purified goat polyclonal antibody against a peptide mapping at the COOH terminus of human actin, and identical to the corresponding rat sequence; (7) affinity-purified rabbit polyclonal p44/42 mitogen-activated protein kinase (MAPK) antibody (#9102), raised against a synthetic peptide derived from the sequence of rat p42 MAPK; (8) affinity-purified rabbit polyclonal phospho-p44/42 MAPK (Thr202/Tyr204) antibody (#9101S); (9) COX-2 (M-19)—sc-1747, affinity-purified goat polyclonal antibody raised against a peptide mapping at the COOH terminus of rat COX-2 and reactive with rat C611B ChC8; (10) affinity-purified rabbit polyclonal Akt antibody (#9272) raised against a synthetic peptide corresponding to residues 466–479 of mouse Akt; (11) affinity-purified rabbit polyclonal phospho-Akt (Ser 473) antibody (#9271); (12) affinity-purified Bax antibody (#2772) raised against a synthetic peptide corresponding to the N-terminal residues of human Bax; (13) cytochrome c oxidase subunit IV (COX4) antibody raised in mouse; and (14) cytochrome c antibody raised in rabbit. Antibodies 1–3, 7, 8, and 10–12 were purchased from Cell Signaling Technology; 4–6 and 9 were obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA; and 13 and 14 were included as part of the ApoAlert Cell Fractionation Kit purchased from Clontech, Palo Alto, CA. This kit was used to separate a highly enriched mitochondrial fraction from the cytosolic fraction of apoptotic and non apoptotic C611B ChC cultured cells.

Assays.

Cell density determinations were made using the CellTiter 96 Aqueous Nonradioactive Cell Proliferation Assay from Promega (Madison, WI), as previously described.8 Activated caspase-9 activity was determined using the Caspase-9 Colorimetric Assay Kit (catalogue no. 218824), purchased from Calbiochem-Novabiochem Corp., San Diego, CA.Activated caspase-3 activity was measured using the ApoAlert Caspase-3 Colorimetric Assay Kit from Clontech. Apoptosis was assessed by standard DNA laddering and by DAPI fluorescence staining. Akt kinase activity was assayed using the Akt Kinase Assay Kit (catalogue no. 9840) from Cell Signaling Technology, as previously described.8 PGE2 concentration in culture medium was assayed using the Prostaglandin E2 EIA Kit-Monoclonal (catalogue no. 514010), purchased from Cayman Chemical. Total cell protein per culture dish was measured using the DC Protein Assay Kit (catalogue no. 500-0120), purchased from Bio-Rad Laboratories, Bio-Rad, CA.

In Vivo Studies.

Two separate animal studies were conducted to evaluate the ability of celecoxib to suppress tumorous growth of rat C611B ChC cells using a protocol approved by the Institutional Animal and Care Committee at Virginia Commonwealth University. Briefly, young adult male Fischer 344 rats (Harlan, Indianapolis, IN) were each inoculated subcutaneously in their right inguinal region with 2 × 106 C611B cells (≈98% viable) in Hanks' Balanced Salt Solution, pH 7.2-7.4 (Sigma-Aldrich). The rats were then randomized into celecoxib treatment and vehicle control groups and housed 2 per cage in plastic cages in a controlled animal facility (12-hour light/dark cycle, 70°F ± 5°F room temperature, and 50% ± 10% relative humidity. Drug and control treatments were initiated 2 weeks after tumor-cell inoculation, when all rats in each group exhibited evidence of a palpable tumor (≈1-3 mm) at the cell transplantation site. Celecoxib in a vehicle composed of 0.5% hydroxypropyl methyl cellulose (Methocel * K15M, product code 53984; Dow Chemical Co., Midland, MI) and 0.1% polyoxyethylenesorbitan monooleate (Tween 80; Sigma-Aldrich) was administered by gavage at a dose of 30 mg/kg body weight in a 0.1% body weight volume and given twice daily, first at between 9:30 and 10:00 AM and then again between 5:30 and 6:00 PM. Control rats received vehicle only,according to the same schedule. In experiment 1, the treatments were carried out 5 days per week over a 33-day period. In experiment 2, treatments were carried out 7 days per week for 40 days. Body weights were recorded daily. At the end of each treatment period, the rats were euthanized and tumors immediately resected. Tumor wet weights were measured directly and tumor volume was determined using the formula

  • equation image

Statistical Analysis.

Mean values ± SD were calculated from pooled data from repeat experiments where n = 3 or 4, with a minimum of 3 and a maximum of 8 separate determinations being made per individual experiment. Student's 2-tailed t test was used to determine P, with a P value of less than or equal to .05 considered to be significant. Specific P and n values are given in individual figure legends.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Celecoxib produced significant dose-dependent growth inhibition of C611B ChC cells cultured on plastic, with the most prominent responses being elicited by drug at concentrations greater than or equal to 50 μM. (Fig. 1A). This in vitro growth-inhibitory effect could be partially circumvented, in a dose-dependent manner, by exogenous addition of 5 –to 25 μM PGE2 to the medium (Fig. 1B). However, as demonstrated in Fig. 2, celecoxib at 50 μM, and in the absence of added PGE2, induced extensive apoptosis in cultured C611B cells, which correlated with significant activation of caspase-9 and caspase-3, respectively (Figs. 1C and D, Fig. 3), together with cleavage of PARP (Fig. 3).

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Figure 1. (A) Celecoxib-induced dose-dependent growth suppression of cultured rat C611B ChC cells. This growth-inhibitory effect was circumvented (B) by the addition of PGE2 to the medium and correlated with dose-dependent activation of (C) caspase-9 and (D) caspase-3 activity in the celecoxib-treated cultures. *P values significant at less than or equal to .001.

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Figure 2. Celecoxib-induced apoptosis in cultured C611B ChC cells demonstrated by (A and B) DAPI fluorescent staining and DNA laddering. (C) Arrows point to representative apoptotic cells (all cells in this field have undergone apoptosis).

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Figure 3. Western blot demonstrating dose-dependent cleavage (activation) of caspase-9 and caspase-3, and cleavage of PARP in C611B ChC cells following in vitro treatment with celecoxib. LYB refers to the lysis buffer (noncellular) control.

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As shown in Fig. 4, celecoxib over a concentration range of 5 –to 50 μM did not affect levels of either Bcl-2, Bcl-xL, or COX-2 protein expressed in cultured C611B ChC cells, nor, at these concentrations, did celecoxib alter levels of p42/44 MAPK protein or phosphorylation constitutively expressed by these malignant cells. In contrast, celecoxib at the 50 μM concentration markedly suppressed Akt phosphorylation (Fig. 5A) and kinase activity (Fig. 5B) of the C611B ChC cells but did not affect their level of expressed Akt protein relative to that of the DMSO control cultures (Fig. 5A). In this context, and with respect to molecular mechanism, it is particularly noteworthy that celecoxib-induced suppression of Akt phosphorylation and kinase activity correlated with translocation of cytosolic Bax to mitochondria and release of cytochrome c from mitochondria to cytosol (Figs. 6 and 7). It is of further note that inclusion of 25 μM PGE2 in the culture medium of C611B ChC cells exposed to the 50 μM, apoptosis-inducing concentration of celecoxib largely blocked the effect of celecoxib to induce suppression of Akt phosphorylation (Fig. 5C), leading to a concomitant reduction in Bax translocation and cytochrome c release in these cells and to distinct decreases in the levels of the cleaved (activated) forms of caspase-9 and caspase-3 (Fig. 7). PGE2 alone was in itself nonapoptotic, but we found it to stimulate modest growth in C611B ChC cultures.2 Moreover, the ability of PGE2 to partially circumvent the growth-inhibitory effect of the 50 μM concentration of celecoxib on C611B ChC cells (Fig. 1B) closely paralleled its effect on partially blocking various key steps in the apoptosis-signaling pathway. It is also interesting that the select PI 3-kinase inhibitor LY294002 acted in a comparable manner to celecoxib also by suppressing Akt phosphorylation and kinase activity (Figs. 5A and B), inducing Bax translocation and cytochrome c release (Figs. 6 and 8), and stimulating activation of caspase-9 and caspase-3 and cleavage of PARP (Fig. 3) in cultured C611B ChC cells.

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Figure 4. Western blot demonstrating lack of effect of celecoxib treatment on levels of Bcl-2, Bcl-xL, COX-2, and p44/42 MAPK protein, and on p44/42 MAPK phosphorylation expressed in cultured C611B ChC cells.

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Figure 5. Western blots demonstrating (A) dose-dependent dephosphorylation of Akt by celecoxib treatment without affecting Akt protein expression and (B) celecoxib-induced inhibition of Akt kinase activity in cultured C611B cells, using glycogen synthase kinase-3 (GSK-3α and GSK-3β) as substrate. Positive control cultures treated with the PI 3-kinase/Akt-specific inhibitor LY294002. (C) Suppression of Akt phosphorylation by celecoxib prevented by PGE2 added to the medium. RIPA and LYB indicate buffer controls without cells.

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Figure 6. Western blot demonstrating translocation of Bax to mitochondria and release of cytochrome c into cytosol in C611B ChC cells treated in vitro with either 50 μM celecoxib or 75 μM LY294002. COX-4-mitochondrial protein marker; actin-cytosol protein marker.

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Figure 7. Western blot demonstrating that addition of PGE2 to the culture medium of celecoxib-treated C611B ChC cells partially prevented Bax translocation, cytochrome c release, and cleavage of caspase-9 and caspase-3 to activated forms in these cells.

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Figure 8. Western blot demonstrating dose-dependent action of the PI 3-kinase/Akt inhibitor LY294002 on promoting Bax translocation to mitochondria and cytochrome c release into cytosol in cultured C611B ChC cells.

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Not surprisingly, the COX-2 inhibitor rofecoxib, like celecoxib, significantly inhibited COX-2 activity in cultures of C611B ChC cells, as reflected by a dose-dependent decrease of PGE2 production into medium from exogeneously added arachidonic acid (Fig. 9A and B). It should be emphasized that, while celecoxib at 1 to 5 μM was quite effective in inhibiting COX-2 activity in C611B ChC cells, these lower concentrations of drug were without effect on suppressing in vitro growth or inducing detectable levels of apoptosis in these cells. Rofecoxib at 1 to 5 μM also significantly inhibited PGE2 production by cultured C611B ChC cells. However, in contrast to celecoxib, rofecoxib at greater than or equal to 50 μM was without effect in suppressing C611B ChC in vitro cell growth (Fig. 9C). This, in turn, directly related to the ineffectiveness of rofecoxib, but not celecoxib, at 50 μM to block Akt phosphorylation (Fig. 9D), promote Bax translocation and cytochrome c release (Fig. 9E), and induce cleavage of caspase-3 proform to its activated form (Fig. 9F) in C611B ChC cultured cells.

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Figure 9. (A and B) Low concentrations of rofecoxib, like celecoxib, significantly inhibited PGE2 production by cultured C611B ChC cells. Rofecoxib at higher concentrations, and in contrast to the effects observed for celecoxib, was without effect in (C) suppressing in vitro growth, (D) inhibiting Akt phosphorylation, (E) inducing Bax translocation to mitochondria and cytochrome c release into cytosol, and (F) activating the proform of caspase-3 to its cleaved active form. *P values significant at less than or equal to .001.

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In an initial effort to determine if celecoxib might be effective in suppressing ChC growth in vivo, we tested celecoxib in 2 separate experiments for its ability to suppress the tumorous growth of C611B ChC cells transplanted subcutaneously into the inguinal region of syngeneic young adult male rats. The results of these experiments are collectively shown in Table 1. As demonstrated, celecoxib treatment resulted in a partial, but significant suppression of tumorous growth of ChC xenografts, without notable toxicity to the host animals.

Table 1. Effect of Celecoxib Treatment on Tumorous Growth of Rat C611B ChC Cells Implanted Subcutaneously Into Syngeneic Fischer 344 Rats
 TreatmentNumber of Rats per GroupInitial Body Weight (g)Final Body Weight (g)Tumor Volume (cm3)Tumor Wet Weight (g)
  • NOTE. Results are expressed as mean ± SD. Numbers in parentheses represent % tumor inhibition relative to vehicle control.

  • *

    Not significant relative to vehicle control values.

  • P ≤ .02 compared with vehicle control.

  • P ≤ .04 compared with vehicle control.

  • §

    P ≤ .01 compared with vehicle control.

  • P ≤ .0001 compared with vehicle control.

EXPT 1Celecoxib7205 ± 9.4*268 ± 9.6*0.69 ± 0.421.02 ± 0.27
    (54%)(37%)
 Vehicle control7205 ± 6.5265 ± 10.61.51 ± 0.721.63 ± 0.65
EXPT 2Celecoxib8220 ± 12.8*289 ± 17.5*0.96 ± 0.25§1.64 ± 0.26
    (47%)(30%)
 Vehicle control8222 ± 11.5292 ± 20.61.82 ± 0.622.35 ± 0.28

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Celecoxib, at a concentration approximately 10 times higher than that needed to markedly inhibit COX-2 activity in rat C611B ChC cells in vitro, induced significant apoptosis in these cultured biliary cancer cells by a mechanism compatible with that shown schematically in Fig. 10. As depicted, our data indicate apoptosis induced by celecoxib in cultured C611B ChC cells appears to occur through a mechanism in which suppression of Akt phosphorylation plays a key role. This, in turn, promotes translocation of Bax to mitochondria, subsequent release of cytochrome c from mitochondria to cytosol, associated activation of caspase-9 and then caspase-3, followed by cleavage of PARP, and subsequent nuclear fragmentation. As with celecoxib, we found that the COX-2 inhibitor NS-398 also suppresses growth1 and induces apoptosis (Z.Z. and A.E.S., unpublished data, 2003) in cultured C611B ChC cells, but at in vitro concentrations (150-200 μM) that were approximately 3 to 4 times higher than those observed for celecoxib to produce a comparable effect. As already noted, NS-398 at 200 μM was previously demonstrated by Hayashi et al.5 to maximally suppress in vitro growth of a number of different human ChC cell lines. Wu et al.13 have also just recently reported that celecoxib at 20 –to 40 μM suppresses growth and induces apoptosis in vitro in the human QBC939 ChC cell line. However, as observed in the present study, rofecoxib, at concentrations 10 –to 20 times higher than needed for this agent to effectively inhibit COX-2 activity, was without effect in either suppressing growth or inducing apoptosis in cultured C611B ChC cells.

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Figure 10. Diagram schematically showing mechanism of high-dose celecoxib-induced apoptosis in C611B ChC cells in vitro that is consistent with our experimental findings.

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The differential ability of celecoxib to induce apoptosis in C611B ChC cells, in comparison with rofecoxib, was likely not due to solubility differences between the 2 agents. This is because we did not detect any evidence of precipitated drug in the culture medium either (1) by direct microscopic observation under phase contrast or (2) following centrifugation (at 15,000g for 30 minutes) of medium collected at both 1 and 24 hours after addition of either 50 or 100 μM rofecoxib or 50 μM celecoxib, respectively, in a manner comparable to that of our experimental culture conditions. Moreover, our findings are in agreement with those of a number of recent independent reports demonstrating celecoxib to be considerably more potent than rofecoxib in suppressing in vitro cell growth by inducing apoptosis in several different human malignant cell types, including HT-29 colon adenocarcinoma cells,14 A549 non–small-cell lung carcinoma cells,15 PC-3 prostate carcinoma cells,16 and DAOY and PFSK primitive neuroectoderm tumor cell lines.17 Structure-function studies by Zhu et al.16 further suggest that differences in the apoptotic-inducing activity of celecoxib versus rofecoxib are related to important differences in surface electrostatic potential surrounding the heterocyclic system of these 2 compounds.

Specific steps in the scheme shown in Figure 10 for the apoptotic signaling pathway activated by celecoxib in C611B ChC cells in vitro have also been supported by recent data from other groups for a number of different human cancer cell lines. Celecoxib, at apoptosis-inducing concentrations in vitro, was shown to block Akt phosphorylation in cultured LNCaP and PC-3 prostate carcinoma cells,16, 18 in HT-29 cells,14 in DAOY and PFSK cells,17 and in cultured human hepatocellular carcinoma cell lines transfected with COX-2 expression vector.19 NS-398-induced apoptosis in cultured human colon cancer cell lines was demonstrated to be mediated by release of cyrochrome c from mitochondria, and, consequently, with caspase-9 and caspase-3 activation20, 21 and cleavage of PARP.20 In addition, our results demonstrate, as an important part of this scheme, that celecoxib-mediated inhibition of Akt phosphorylation in C611B ChC cells correlates with the translocation of Bax to mitochondria, which is a key step leading to altered mitochondrial membrane potential and cytochrome c release.22, 23

The induction of apoptosis by 50 μM celecoxib in cultured C611B ChC cells was independent of Bcl-2 and Bcl-xL protein expression. Similarly, celecoxib at 50 μM was previously shown not to alter cellular Bcl-2 levels in human LNCaP and PC-3 human prostate cell lines18 or in human Huh7 liver tumor cells.24 However, Kern et al.24 reported that apoptosis induced after COX-2 inhibition with 50 μM celecoxib did not correlate with the phosphoryation status of Akt for cultured Huh7 cells. This is in sharp contrast to our findings for C611B ChC cells and the findings of others for other tumor cell lines.16–18 The basis for such a difference is presently unclear, but it is important to point out that we were able to obtain results comparable to those produced by celecoxib in cultures of C611B ChC cells treated with the PI3-kinase/Akt inhibitor LY 294002. It is also noteworthy that inclusion of 25 μM PGE2 in the medium of celecoxib-treated C611B ChC cells prevented Akt dephosphorylation, which, in turn, correlated with a protective effect against apoptosis. Moreover, rofecoxib, which did not inhibit Akt phosphorylation in C611B ChC cells, was also without effect in suppressing growth or in stimulating Bax translocation to mitochondria, cytochrome c release, and caspase-3 activation in these cells.

Apoptosis induced by celecoxib in cultured tumor cell lines has also been reported to involve concomitant dephosphorylation of P42/44 MAPK dephosphorylation.16 In the case of C611B ChC cells, however, we did not observe any differences between our control and celecoxib-treated cultures with respect to their constitutive levels of p42/44 MAPK protein expression and phosphorylation, suggesting that signaling through the PI 3-kinase/Akt pathway may play a more prominent role than p42/44 MAPK in regulating survival of these malignant biliary cells.

Our data, as well as those of Wu et al.,13 demonstrate the ability of PGE2 to abolish the growth-suppressive effect of celecoxib on cultured ChC cells. Wu et al.13 concluded from their dose-response data that COX-2 might play a central role in the production of PGE2 and that the inhibition of COX-2 by celecoxib suppresses in vitro growth and induces apoptosis of human QBC939 ChC cells via suppression of PGE2 production, thus appearing to function through a COX-2-dependent mechanism. However, in addition to the marked disparity we observed between μM concentration levels required for celecoxib to significantly inhibit PGE2 production by cultured C611B ChC cells (Fig. 9B), and those needed for this agent to definitively suppress in vitro cell growth (Figs. 1A and 9C) and activate caspase-9 and caspase-3 (Figs. 1C and D), our results with rofecoxib also appear to support involvement of a COX-2-independent mechanism underlying, in some part, celecoxib's actions on suppressing growth and inducing apoptosis in C611B ChC cells in vitro. In this context, it is interesting that COX-2 inhibitors have recently been demonstrated to sensitize tumor cells specifically to death receptor-induced apoptosis independently of COX-2 inhibition.25 Furthermore, our results strongly suggest that exogenous addition of PGE2 acts to circumvent the growth-inhibitory/apoptotic effects of celecoxib through a mechanism involving maintenance of Akt in its activated phosphorylated state (Fig. 5C). In this regard, it is of further interest that PGE2 stimulation of EP4 prostanoid receptors has recently been shown to induce phosphorylation of both Akt kinase and glycogen synthase kinase-3 (GSK-3) substrate.26, 27 PGE2 has also been shown by Nzeako et al.28 to reduce Fas-mediated apoptosis and mitochondrial depolarization in cultured human KMBC extrahepatic biliary carcinoma cells, likely by up-regulating Mcl-1, an antiapoptotic member of the Bcl-2 family. In preliminary Western blot experiments, we were not able to detect Mcl-1 protein expression in our cultured C611B ChC cells (Z.Z. and A.E.S, unpublished data, 2003).

Although we have not yet established whether our in vitro findings have any direct bearing on our in vivo results, it is important to note that, in 2 separate animal studies, we have shown the ability of celecoxib to significantly, albeit partially, suppress the tumorous growth of ChC cells in vivo. Clearly, further studies are now needed to define the mechanism(s) for celecoxib effect on suppressing ChC tumor growth in vivo. We are presently pursuing such studies, with a focus on determining if Akt inactivation represents a key step in the mechanism by which celecoxib suppressed ChC tumor growth in vivo, either through a direct action on the cancerous cells themselves or by indirectly inhibiting tumor angiogenesis. Our recently published findings demonstrating that celecoxib acts in synergy with the ERBB-2 tyrosine kinase inhibitor emodin to suppress rat ChC cell growth in vitro through a mechanism involving enhanced suppression of Akt activation8 further supports combinatorial upstream targeting of Akt as a potential therapeutic strategy for the adjuvant treatment of ChC. This strategy is also now being tested by us in vivo.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Dr. Timothy J. Mazaisz of Pharmacia Corp., Skokie, IL and Dr. Ian W. Rodger of Merck & Co., Inc., Whitehouse Station, NJ for their helpful comments and careful review of this work.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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