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Basic and Translational Investigations |
Childhood Cancer Research Unit, Department of Woman and Child Health (N.B., B.S., S.E., L.S., S.H., B.G., P.K., J.I.J.), and Department of Oncology and Pathology (A.O., C.O.Ö., B.K.), Karolinska Institutet, Stockholm, Sweden; Department of Cell Biology and Histology, University of Tromsö, Tromsö, Norway (B.S.)
Address correspondence to Per Kogner, Childhood Cancer Research Unit, Q6:05, Dept. of Woman and Child Health, Karolinska Institutet, S-171-76, Stockholm, Sweden (per.kogner{at}ki.se).
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Abstract |
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Key Words: angiogenesis • apoptosis • cyclooxygenase-2 • in vivo treatment • medulloblastoma • microsomal prostaglandin E synthase-1 • primitive neuroectodermal tumors • proliferation • prostaglandin E2 • prostanoid receptors
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Introduction |
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Increased levels of prostaglandin E2 (PGE2) have been detected in a variety of malignancies, including brain tumors.4–7 PGE2 is synthesized from arachidonic acid by the sequential action of cyclooxygenases (COX) and prostaglandin E synthases (PGES).6 COX-2, the inducible form of COX, has frequently been detected in both premalignant and malignant tissues.8 COX-2 is functionally coupled to microsomal prostaglandin E synthase-1 (mPGES-1), which converts prostaglandin H2 to PGE2.9 Overexpression of mPGES-1 in cancer tissues has been reported, further supporting an important function of prostanoids in tumorigenesis.10,11 PGE2 exerts its physiological effects by interacting with a subfamily of four distinct G-protein–coupled receptors designated EP1, EP2, EP3, and EP4. PGE2 promotes tumor growth by stimulating EP receptor signaling with subsequent enhancement of cellular proliferation, promotion of angiogenesis, inhibition of apoptosis, stimulation of invasion, and suppression of immune responses.12,13
In this study, we investigated the expression of key enzymes involved in the production of PGE2 as well as its prostanoid EP receptors in MB primary tumors and cell lines. We also studied the effects of PGE2, the EP2 agonist butaprost, and four EP antagonists, ONO-8713, ONO-AE3-240, ONO-AE3-208, and AH 23848, that specifically block the function of EP1, EP3, and EP4, respectively, on MB cell growth. Furthermore, we examined the effect of inhibiting COX-2 using RNA silencing by small interfering RNA or nonsteroidal anti-inflammatory drugs (NSAIDs) on MB cell proliferation, apoptosis, and PGE2 secretion in vitro and in nude mice carrying established MB xenografts.
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Materials and Methods |
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Ethical approval was obtained by the Karolinska University Hospital Research Ethics Committee (approval no. 03-708).
Immunohistochemistry
For detection of COX-2 and mPGES-1 in primary tumors, tissue sections were incubated with either a monoclonal mouse anti-COX-2 antibody (Zymed Laboratories Inc., Carlsbad, CA, USA) or a monoclonal mouse anti-mPGES-1 antibody (Cayman Chemicals, Ann Arbor, MI, USA) overnight at 4°C. Prostanoid receptors were detected by incubating tumor sections with rabbit anti-EP1, anti-EP2, anti-EP3, and anti-EP4 (Cayman Chemicals), respectively, overnight at 4°C. The SuperPicture polymer detection kit with appropriate secondary antibodies was used together with a diaminobenzidine (DAB) substrate chromogen system to visualize immunopositivity (Zymed).
Active caspase-3 expression in human MB xenografts isolated from NMRI nu/nu mice was investigated by incubating sections overnight at 4°C with a monoclonal rabbit antibody specifically detecting active caspase-3 (R&D Systems, Abingdon, UK). Proliferation was detected using a specific Ki-67 (SP6) antibody (Neomarkers, Fremont, CA, USA). Immunopositivity was visualized as described above. As a control for nonspecific background staining, corresponding sections were incubated with antimouse immunoglobulin G (IgG) isotype or antirabbit IgG isotype controls (Zymed). Caspase-3 activation in tumor sections was quanitified by counting the number of tumor cells staining positive for the active caspase-3 antibody in six representative regions of the tumor section. Proliferation was assessed by counting the number of Ki-67 positively staining nuclei and total number of cancer cells at x200 magnification, in six representative regions. For caspase-3 and Ki-67 staining, the results are expressed as the proportion of positively staining cells over the total number of cells.
Biotinylated Bandeiraea simplicifolia-1 (BS-1) lectin (Sigma-Aldrich, Solna, Sweden) was used to visualize endothelial cells. BS-1 was diluted 1:50 and incubated overnight at 4°C. Cells were detected with ABComplex conjugated to horseradish peroxidase (Dako A/S, Glostrup, Denmark). Sections were developed using DAB (SK-4100, Vector Laboratories Inc., Burlingame, CA, USA). Four tumor slides per treatment group and four fields per slide were quantified for microvessel density at x200 magnification. The results are expressed as an average number of microvessels per field.
Chemicals
Diclofenac (Cayman Chemicals) was dissolved in OptiMEM (Gibco BRL, Sundbyberg, Sweden) to achieve the concentrations desired. Celecoxib (Pfizer, Täby, Sweden); ONO-8713, ONO-AE3-240, and ONO-AE3-208 (a gift from ONO Pharmaceuticals Co., Ltd., Osaka, Japan); AH 23848 and PGE2 (Sigma-Aldrich); and butaprost (Cayman Chemicals) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and further diluted in OptiMEM or RPMI medium (Gibco BRL) to its final concentration (final DMSO concentration, 0.1%–0.7%).
Cell Lines
Cell lines used were kindly provided by Dr. T. Pietsch (University of Bonn Medical Center, Bonn, Germany), Dr. C. Redfern (Northern Institute for Cancer Research, Newcastle University, Newcastle, UK), and Dr. M. Nister (Karolinska Institutet, Stockholm, Sweden). In total, nine human MB/PNET cell lines were used, although DAOY and D324 MED, originally the same cell line, were obtained from different sources and cultured under slightly different conditions.14 The cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; DAOY and MEB-MED-8A cells), modified essential medium (MEM; D283 MED and D324 MED cells), Richter's improved MEM with zinc (IMEMZO/DMEM; (D425 MED and D458 MED cells), DMEM/F12 (UW228-3 cells), IMEMZO/N-2 growth factor (D384 MED cells), or RPMI (PFSK-1 cells). Medium was supplemented with 10% (D283 MED, D324 MED, and PFSK-1) or 15% (DAOY, MEB-MED-8A, D425 MED, D458 MED, D384 MED, and UW228-3) heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/ml penicillin G, and 100 µg/ml streptomycin (Life Technologies Inc., Stockholm, Sweden) at 37°C in a humidified 5% CO2 atmosphere. All media were purchased from Gibco BRL.
PGE2 Measurement and COX Activity Assay
The MB cell lines D324 MED and PFSK-1 were seeded in 96-well plates and cultivated in OptiMEM containing 80 µM arachidonic acid (Sigma-Aldrich). Cells were treated with increasing concentrations of either diclofenac (0.78–100 µM) or celecoxib (0.001–30 µM) for 24 h, respectively. Cell supernatants were harvested, and PGE2 levels were measured using a PGE2 ELISA (enzyme-linked immunosorbent assay) kit (Cayman Chemicals) according to the manu facturer's instructions. For measurements of COX enzymatic activity in MB cells, cell extracts from 1 x 108 D324 MED or PFSK-1 cells were preincubated with 0.001–10 µM celecoxib or 0.001–30 µM diclofenac for 5 min before addition of arachidonic acid. COX enzymatic activity was measured using a COX activity assay (Cayman Chemicals).
Proliferation and Clonogenic Assay
The effects of NSAIDs (diclofenac and celecoxib) and the EP receptor antagonists (ONO-8713, ONO-AE3-240, ONO-AE3-208, and AH 23848) on MB cell growth were determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay (Sigma-Aldrich) as previously described.15 Eight parallels of each treatment were performed in each experiment. The concentration that inhibited 50% of cell viability (EC50) was calculated.
For assessment of PGE2 and butaprost effects on cell proliferation, 1.0 x 104 cells were seeded in 96-well plates and incubated overnight in RPMI medium containing 10% FBS. Cells were then serum starved for 24 h before treatment with 5 nM to 1 µM PGE2 supplemented with 0.5% bovine serum albumin (Sigma-Aldrich) or 1–10 µM butaprost for 72 h. Proliferation was measured using MTT assay as described above.
To determine colony formation, D283 MED, D324 MED, PFSK-1, DAOY, and UW228-3 cells were seeded in 50 mm2 Cell+ Petri dishes (Sarstedt, Solna, Sweden) at a concentration of 150 cells/dish in triplicate. Cells were allowed to attach to the surface for 5 h before treatment with diclofenac (50 and 100 µM) or celecoxib (10 and 30 µM) for 48 h. After 12 days of incubation in drug-free medium, cell cultures were rinsed with phosphate-buffered saline, fixed in formaldehyde, and stained with Giemsa (Gibco BRL). Colonies (>75 cells) with 50% plate efficiency (PE) were counted manually using a colony counter. For each treatment combination, the surviving fraction was calculated as the ratio of the mean PE of treated cells over the PE of untreated control cells.
Plasmids and Lentivirus Infection
Plasmids containing COX-2 short hairpin RNA (shRNA; pLKO.1 COX-2 shRNA clone 0000045535, Open Biosystems, Huntsville, AL, USA), scrambled shRNA (Addgene pLKO.1, clone 1864, Addgene Inc., Cambridge, MA, USA), or enhanced green fluorescent protein plasmid (peGFP; clone 12257, Addgene Inc.) were cotransfected together with psPAX2 and pDM2.G (Addgene Inc.) into actively growing HEK-293T cells (kindly provided by J. Löfling, Karolinska Institutet) using polyethyleneimine (Polyscience Europe, Eppelheim, Germany); peGFP served as a control for transfection efficiency. MB cells were infected with approximately 1 x 106 lentivirus particles/ml. Western blot analysis on protein extracts and trypan blue dye exclusion to evaluate cell proliferation were carried out 5 days after infection.
Fluorescence-Activated Cell-Sorting Analysis
DNA content was assessed essentially as previously described.16 Briefly, D283 MED, D324 MED, and PFSK-1 cells were treated with the indicated concentrations of diclofenac and celecoxib for 48 h. Cells were harvested, stained with 4',6-diamidino-2-phenylindole (DAPI), and subjected to cell cycle analysis using single-parameter DNA flow cytometry. The multicycle program for cell cycle analysis (Phoenix Flow Systems, San Diego, CA, USA) was used for histogram analysis.
Western Blotting
Protein was extracted from cells in RIPA buffer (25 mM Tris [pH 7.8], 2 mM EDTA, 20% glycerol, 0.1% Nonidet P-40 [NP-40], 1 mM dithiothreitol, and protease inhibitors [Roche Diagnostic, Mannheim, Germany]). Protein concentration was measured using Bradford reagent (Bio-Rad, Sundbyberg, Sweden). Equal quantities were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nylon membranes (Millipore Inc., Sundbyberg, Sweden), and probed with antibodies against COX-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); polyclonal anti-mPGES-1 (Cayman Chemicals); EP1 receptor, EP2 receptor, EP3 receptor, and EP4 receptor (Cayman Chemicals); cleaved caspase-9, cleaved caspase-3, poly(ADP-ribose) (PARP), and the BH3 interacting domain death agonist (BID; Cell Signaling Technology, Beverly, MA, USA); and β-actin (Sigma-Aldrich). Antimouse IgG or antirabbit IgG, conjugated with horseradish peroxidase (Pharmacia Biosciences, Uppsala, Sweden), was used as secondary antibody. Pierce Super Signal (Pierce, Rockford, IL, USA) was used for chemiluminescent detection.
Xenografts and In Vivo Administration of NSAIDs
Female NMRI nu/nu mice (Taconic Laboratories, Ejby, Denmark) 4–8 weeks old were maintained at five of each per cage and were given sterile water and food ad libitum. Each NMRI nu/nu mouse was subcutaneously injected with 20 x 106 D283 MED MB cells. Treatment was started on the appearance of palpable tumors reaching the volume of 0.20 ml. The mean tumor volume at the start of the treatment was 0.24 ml. Tumors were measured every day, and tumor volume was calculated as (width)2 x length x 0.44. Tumor volume index was calculated using the measured volume divided by the volume measured at start of treatment. Two independent experiments were carried out. In the first experiment mice were randomly assigned to receive 250 mg/liter diclofenac (n = 8) in drinking water or no treatment (n = 9). In the second experiment, mice were randomized to receive 2 mg celecoxib (n = 7) once daily through a gastric feeding tube or no treatment (n = 10). Tumor weight was recorded at autopsy, after which tumors were fixed in formaldehyde for subsequent immunohistochemical analysis. All animal experiments were approved by the regional ethics committee for animal research (approval N234/05) in accordance with the Animal Protection Law (SFS 1988:534), the Animal Protection Regulation (SFS 1988:539), and the Regulation for the Swedish National Board for Laboratory Animals (SFS 1988:541)
Statistical Analysis
The EC50 values were evaluated from a plot of survival versus the logarithm of the molar drug concentration, using a standard dose–response curve defined by four parameters: the baseline response (Bottom), the maximum response (Top), the slope (Hill slope), and the drug concentration:
 | (1) |
Two independent populations were analyzed for statistically significant differences by the Mann-Whitney U-test; several independent populations were analyzed by the Kruskal-Wallis test (nonparametric analysis of variance) followed by Dunn's multiple comparison test.
Calculation of median values and their nonparametric approximate 95% confidence intervals (95% CIs) were based on the Wilcoxon sign-rank test as outlined by Tukey.19 The one-sample t-test was used to test whether the mean of a single sample differed significantly from control.
All statistical tests were two-sided.
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Results |
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PGE2 Induces Proliferation of MB Cells
Because most of the MB cell lines investigated expressed high levels of COX-2 as well as mPGES-1, we measured the levels of PGE2 in the supernatant from the MB cell lines D324 MED and PFSK-1. Both cell lines secreted PGE2, levels of which could be significantly reduced in a dose-dependent manner by treatment with the COX inhibitors diclofenac and celecoxib (p < 0.01; Fig. 2A). The concentrations that inhibited 50% of PGE2 secretion (IC50) from D324 MED and PFSK-1 cells were 40 µM diclofenac and 10 µM celecoxib, and 35 µM diclofenac and 12 µM celecoxib, respectively (Fig. 2A). Half-maximal inhibition (IC50) of COX enzymatic activity as measured by the conversion of arachidonic acid to prostaglandin H2 was achieved with 0.1 µM diclofenac or 0.005 µM celecoxib in cell extracts isolated from D324 MED or PFSK-1 cells (data not shown).
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Inhibition of COX-2 or EP Receptor Activity Suppresses Both Growth and Clonogenic Capacity of MB Cells
We first investigated the effect of the dual COX-1/COX-2 inhibitor diclofenac and the COX-2–specific inhibitor celecoxib on the growth of nine MB cell lines. The cell lines were treated with increasing concentrations of diclofenac or celecoxib for 48 h, and cell viability was determined by MTT at the end of the treatment period. All MB cell lines investigated demonstrated a concentration-dependent decrease in cell viability after 48 h of incubation (Table 2). The biological EC50 concentrations for the MB cell lines ranged from 27.5 µM to 79.1 µM (median, 47.3 µM) for diclofenac and from 7.8 µM to 13.5 µM (median, 9.3 µM) for celecoxib (Table 2). We also investigated the time-dependent effect of diclofenac and celecoxib on the MB cell lines D283 MED, D324 MED, and PFSK-1 (Fig. 3A). A significant reduction of cell viability was observed in MB cells treated with diclofenac or celecoxib with increasing incubation times (p < 0.001; Fig. 3A). Comparison of the EC50 values for the different MB cell lines incubated for 24–96 h with diclofenac or celecoxib demonstrated that the effect of the dual COX-1/COX-2 inhibitor diclofenac had a more definite time dependency and was more effective upon longer incubation periods compared with celecoxib (Fig. 3A).
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Clonogenic assay was performed on five of the MB cell lines to further determine the antitumor effects of diclofenac and celecoxib. Treatment showed a significant dose-dependent inhibition of colony formation (p < 0.001; Fig. 3B).
To investigate whether the observed treatment effect of NSAIDs was caused by a direct inhibition of COX-2 or by COX-2–independent mechanisms, we suppressed COX-2 expression in D324 MED and PFSK-1 MB cells using COX-2 small interfering RNA (siRNA). A marked reduction of COX-2 protein, as analyzed by Western blotting, was observed in both MB cell lines infected with lentiviral COX-2 siRNA compared with cells infected with the scrambled siRNA construct (Fig. 3C). Both MB cell lines infected with the COX-2–specific siRNA demonstrated significantly reduced cell proliferation (D324 MED, p < 0.0022; PFSK-1, p < 0.0152; Fig. 3D) after 5 days of incubation, compared with cells infected with the scrambled siRNA construct.
As shown in Fig. 1, A and B, all MB/sPNET primary tumors and cell lines investigated expressed the four PGE2 receptors EP1 through EP4. We therefore examined the effect of EP receptor antagonists on MB cell proliferation using the EP1-specific receptor antagonist ONO-8713, the EP3-specific antagonist ONO-AE3-240, and the EP4-specific antagonists ONO-AE3-208 and AH 23848. The biological EC50 values of the different receptor antagonists ranged from 14 µM to 30 µM for ONO-8713 and from 9 µM to 42 µM for ONO-AE3-240; both EP4 receptor antagonists, ONO-AE3-208 and AH 23848, were less efficient in inhibiting MB proliferation with EC50 concentrations varying from 55 µM to 175 µM for ONO-AE3-208 and from 70 µM to 270 µM for AH 23848 (Fig. 3E).
Inhibition of COX-2 Activity Induces Apoptosis of MB Cells
To further study the mechanisms of NSAID-mediated inhibition of MB cell growth, we evaluated the effects of diclofenac and celecoxib on the induction of apoptosis. Cell cycle analyses of D283 MED, D324 MED, and PFSK-1 cells treated with diclofenac or celecoxib for 48 h showed an accumulation of cells in the sub-G1 phase of the cell cycle (Fig. 4A). A pronounced G2 arrest was also observed in MB cells treated with diclofenac (Fig. 4A). Western blot performed on protein extract isolated from MB cells treated with diclofenac or celecoxib for 24 h and 48 h revealed activation of caspase-9 and caspase-3 followed by cleavage of PARP, a downstream substrate of caspase-3 (Fig. 4B). No cleavage of BID (BH3 interacting domain death agonist) was detected in MB cells treated with NSAIDs (Fig. 4B).
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Discussion |
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MB is considered to originate from primitive pluripotent neuroepithelial stem cells, located in the external germinal layer (EGL) of the cerebellum.20 Nontumorigenic cells located in the EGL do not express COX-2, whereas Purkinje cells, which are unlikely to give rise to MB tumors, have been shown to express COX-2.20–25 Using immunohistochemistry, we demonstrated that MB cells and Purkinje cells express COX-2, whereas in stroma and surrounding nonmalignant brain tissue, COX-2 was not detected (Fig. 1A). Hence, MB cells might acquire COX-2 expression at some stage during the development from pluripotent neuroepithelial progenitor cells. High expression of COX-2 has also been reported in other tumors of CNS origin, including glioma and ependymoma, as well as in meningioma.24,26–28 We also detected constitutive expression of mPGES-1 in both MB primary tumors and cell lines (Fig. 1A,B). Although information on mPGES-1 expression in brain tumors is limited, this finding is consistent with other reports describing increased expression of mPGES-1 in cancers.10,11
Compared to nonmalignant nervous tissue, MB and other tumors of CNS origin contain increased levels of arachidonic acid.4,29 The presence of high levels of COX-2 and mPGES-1 in MB cells may therefore generate a favorable environment for the efficient production of prostanoids.
Measurement of PGE2 using ELISA revealed that MB cells secrete PGE2 that could be reduced by inhibiting COX (Fig. 2A). Furthermore, exogenous addition of PGE2 to serum-starved MB cells resulted in increased proliferation (Fig. 2B). Other reports have shown that patients with malignant brain tumors have significantly higher concentrations of PGE2 in their tumors and plasma compared with patients with benign brain tumors or with nontumoral control patients.6,30 Moreover, the concentration of PGE2 in plasma decreases following neurosurgical resection of both malignant and benign brain tumors.5
Having shown that PGE2 stimulates MB cell growth, we looked for the expression of prostanoid receptors in both clinical MB samples and cell lines and found that all four EP receptors (EP1 through EP4) were abundantly expressed in MB (Fig. 1A,B). Although the precise role of each EP receptor in cancer development has not been fully characterized, the generation of specific EP receptor knockout mice has shown that all four prostanoid receptors may contribute to carcinogenesis, malignant aggressiveness, and tumor progression in a number of cancers.31 Despite structural and sequence similarities among the EP receptors they are coupled to different intracellular signaling pathways, and the receptors exhibit different functions depending on tumor type.12 Of interest, the stimulation of EP receptors by PGE2 has been shown to activate intracellular signaling pathways that are aberrantly regulated in MB. For instance, stimulation of EP2 receptor by PGE2 activates the kinase activity of the phosphatidylinositol-3-kinase (PI3K) oncogene and stimulates the transcription-factor activity of β-catenin, whereas PGE2-mediated activation of EP1 or EP4 induces phosphorylation of the epidermal growth factor receptor resulting in the activation of the Erk signaling pathway.32–34 Hence, activation of EP receptors by PGE2 may contribute to the enhancement of signal transduction by these pathways in MB. In our study, treatment with the EP1 antagonist ONO-8713 or EP3 antagonist ONO-AE3-240 reduced MB cell growth, whereas antagonists targeting the EP4 receptor were less effective in inhibiting MB cell growth. We also found that the EP2 agonist butaprost stimulated MB cell growth. To our knowledge, antagonists targeting the EP2 receptor are not yet commercially available for experimental research. Although more studies need to be performed in order to exactly demonstrate which EP receptors are most important in promoting MB cell proliferation, our data indicate that the prostanoid receptors EP1, EP2, and EP3 are important for PGE2-mediated stimulation of MB cell growth.
Because we showed that MB expressed high levels of both COX-2 and mPGES-1, we decided to investigate the effect of inhibiting PGE2 secretion by targeting COX-2. As shown in Fig. 3A and Table 2, both the dual COX-1/COX-2 NSAID diclofenac and the COX-2–specific NSAID celecoxib, inhibited MB cell growth and clonogenic capacity in all cell lines tested. The biological EC50 values ranged from 27.5 µM to 79.1 µM for diclofenac and from 7.8 µM to 13.5 µM for celecoxib (Table 2) after 48 h of drug incubation, which is comparable to EC50 values found in other cancer cell lines.15,35–38 Although the EC50 doses of diclofenac and celecoxib are higher than the concentrations of NSAIDs required to inhibit the enzymatic activity of COX in MB cells, they correspond to the concentrations required to inhibit 50% of PGE2 secretion from MB cells. These data, together with the demonstration that PGE2 stimulates MB cell proliferation (Fig. 2B), suggest that PGE2 is important for MB growth. The observed growth inhibitory effect of NSAIDs was associated with the induction of caspase-dependent apoptosis (Fig. 4B). To further test the importance of COX-2 expression in MB, we suppressed COX-2 expression by COX-2 siRNA (Fig. 3C). This suppression resulted in inhibition of MB cell proliferation (Fig. 3D). Although a number of other reports have shown that NSAIDs induce cytotoxicity in tumor cells by COX-2 inhibition followed by induction of apoptosis, it cannot be excluded that NSAID targets other than COX-2 are affected in MB cells.39,40 For instance, the COX-2–specific inhibitors, celecoxib and rofecoxib, have been shown to decrease cell survival in colon cancer and prostate cancer cell lines, independent of their COX-2 expression.41,42 In addition to specific COX-2 inhibition, celecoxib has also been shown to direct target protein-dependent kinase-1, the endoplasmic reticulum Ca2+ ATPase complex, cyclin-dependent kinases, and various carbonic anhydrases. Inhibition of these proteins leads to apoptosis or inhibition of cell cycle progression, angiogenesis, and metastasis.40,42–45 The concentration of NSAID required to induce these effects is higher than that achieved in the plasma of patients in clinical studies to obtain anticarcinogenic effect.40
We found that treatment with NSAIDs was associated with the suppression of growth of established human MB xenografts in nude mice (Fig. 5A–D). The doses of NSAID used for the in vivo study were based on our previously obtained results in experimental neuroblastoma, and the dose for celecoxib is significantly lower than the oncological dose of 250 mg/m2 orally twice daily established for children.36,46,47 All mice gained in weight during treatment and showed no signs of toxicity. To better understand the growth inhibitory effects of diclofenac and celecoxib, measurements of apoptosis, microvessel density, and cell proliferation were performed ex vivo (Fig. 5E,F). Immunohistochemistry of tumors from diclofenac- and celecoxib-treated animals demonstrated elevated expression of active caspase-3 compared with untreated tumors, indicating that both NSAIDs induce apoptosis of MB in vivo. We also detected significant reduction in the expression of the cell proliferation marker Ki-67 in tumors from animals treated with NSAIDs. The increased number of caspase-3–positive cells in tumors from animals treated with NSAIDs might be due to a direct effect of COX-2 inhibition. However, we also observed a significant reduction in microvessel density in the treated tumors compared with nontreated tumors (Fig. 5E,F). COX-2 is expressed within neovasculature in tumors, and a number of reports have shown that certain NSAIDs, including celecoxib and diclofenac, suppress angiogenesis.48–51 Thus, both diclofenac and celecoxib may inhibit MB growth by a combination of apoptosis of tumor cells and inhibition of angiogenesis.
The current treatment approach for patients with childhood MB is surgery followed by irradiation and chemotherapy. Inhibition of COX-2 activity has been shown to potentiate the effect of both irradiation and cytotoxic drugs.52–54 Hence, agents that target components in the eicosanoid cascade may prove beneficial, especially as an adjuvant therapy because their advantageous effect may be the synergism with irradiation or conventional cytotoxic treatment or novel agents targeted against other pro-carcinogenic pathways. Particularly the youngest children are at risk for irreversible adverse side effects from irradiation. Since ionizing radiation induces COX-2 expression the concomitant use of agents targeting the production of prostanoids may be beneficial for patients through both improved therapeutic efficacy and the putative limitation of adverse long-term neurological side effects.53
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Acknowledgments |
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Received for publication October 9, 2007. Accepted for publication February 8, 2008.
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