[CANCER RESEARCH 64, 7210 –7215, October 15, 2004] Advances in Brief Cyclooxygenase-2 Is Expressed in Neuroblastoma, and Nonsteroidal Anti- Inflammatory Drugs Induce Apoptosis and Inhibit Tumor Growth In vivo John I. Johnsen, 1 Magnus Lindskog, 1 Frida Ponthan, 1 Ingvild Pettersen, 2 Lotta Elfman, 1 Abiel Orrego, 3 Baldur Sveinbjo ¨rnsson, 2 and Per Kogner 1 1 Childhood Cancer Research Unit, Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden; 2 Department of Experimental Pathology, Faculty of Medicine, University of Tromso ¨, Tromso ¨, Norway; and 3 Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden Abstract Neuroblastoma is the single most common and deadly tumor of child- hood and is often associated with therapy resistance. Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to prostaglandins. COX-2 is up-regulated in several adult epithelial cancers and is linked to proliferation and resistance to apoptosis. We detected COX-2 expression in neuroblastoma primary tumors and cell lines but not in normal adrenal medullas from children. Treatment of neuroblastoma cells with nonste- roidal anti-inflammatory drugs, inhibitors of COX, induced caspase- dependent apoptosis via the intrinsic mitochondrial pathway. Treatment of established neuroblastoma xenografts in nude rats with the dual COX- 1/COX-2 inhibitor diclofenac or the COX-2–specific inhibitor celecoxib significantly inhibited tumor growth in vivo (P < 0.001). In vitro, arachi- donic acid and diclofenac synergistically induced neuroblastoma cell death. This effect was further pronounced when lipooxygenases were simultaneously inhibited. Proton magnetic resonance spectroscopy ( 1 H MRS) of neuroblastoma cells treated with COX inhibitors demon- strated accumulation of polyunsaturated fatty acids and depletion of choline compounds. Thus, 1 H MRS, which can be performed with clinical magnetic resonance scanners, is likely to provide pharmacodynamic markers of neuroblastoma response to COX inhibition. Taken together, these data suggest the use of nonsteroidal anti-inflammatory drugs as a novel adjuvant therapy for children with neuroblastoma. Introduction Neuroblastoma is the most common neoplasia during infancy. The majority of these embryonal sympathetic nervous system tumors arise from primitive cells in the adrenal medulla. Patients  1 year of age with metastatic disease and those with MYCN-amplified tumors have poor prognosis and often develop resistance to conventional therapy (1). Alternative treatments for these patients are therefore needed. Arachidonic acid (AA) is released from cellular phospholipids by phospholipase A 2 and converted to prostaglandins by two cyclooxy- genase (COX) enzymes, COX-1 and COX-2 (2). COX-1 is constitu- tively expressed in most tissues, whereas inflammatory stimuli, hor- mones and mitogens may induce COX-2 expression (2). COX-2 is overexpressed in a variety of adult cancers and has been implicated in resistance to apoptosis as well as induction of metastases and angio- genesis (3). We thus investigated the expression of COX-2 in neuro- blastoma tumors and the therapeutic effect of nonsteroidal anti-in- flammatory drugs (NSAIDs) against neuroblastoma cell lines in vitro and xenografts in vivo. Materials and Methods Human Tissue Samples. Twenty-eight neuroblastomas derived from chil- dren of different ages and all clinical stages, including different biological subsets (MYCN amplification, 7 of 28; 1p deletion, 9 of 26; Table 1) were analyzed. Three childhood ganglioneuromas and three samples of nonmalig- nant adrenal medulla (and cortex) from children were also included. All tissue samples were frozen at surgery and kept at -80°C until fixation. Immunohistochemistry. Paraffin-embedded sections were incubated with a monoclonal mouse anti–COX-2 antibody (Zymed Laboratories Inc., South San Francisco, CA) overnight at 4°C. As a control for nonspecific background staining, sections were incubated with mouse IgG isotype control. Biotinylated antimouse IgG and streptavidin-horseradish peroxidase (HRP) complex were used as secondary antibodies (Zymed Laboratories Inc.). Reaction products were visualized with 3,3'-diaminobenzidine substrate chromogen system (DakoCytomation, Carpinteria, CA). For identification of caspase-3 activity, sections of neuroblastoma xenografts were incubated overnight at 4°C with a polyclonal antibody specifically detecting cleaved caspase-3 (R&D Systems, Abingdon, United Kingdom). Sections were subsequently washed and incubated with secondary biotinylated antibody and streptavidin-HRP complex (Zymed Laboratories Inc.). Chemicals. Diclofenac (Cayman Chemicals, Ann Arbor, MI) was dis- solved in culture medium to achieve the concentrations desired. Celecoxib (Pharmacia, La Jolla, CA) was dissolved in dimethyl sulfoxide and further diluted in medium (final dimethyl sulfoxide concentration, 0.1– 0.7‰). Nor- dihydro-guaiaretic acid [NDGA (10 mol/L)], reduced glutathione (10 mmol/ L), N-acetylcysteine (100 mol/L), L-cycloserine (5 mmol/L), and -tocoph- erol (100 mol/L) were all from Sigma (Stockholm, Sweden). Cell Lines. Neuroblastoma cell lines were grown in Eagle Minimal Essen- tial Medium (SH-SY5Y) or RPMI 1640 [SK-N-BE(2), SK-N-SH, SK-N-AS, SK-N-FI, SK-N-DZ and IMR-32] medium supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 IU/ml penicillin G, and 100 g/mL streptomycin (Life Technologies, Inc., Stockholm, Sweden) at 37°C in a humidified 5% CO 2 atmosphere. Cytotoxcity Assay and Fluorescence-Activated Cell-Sorting Analysis. Cells were incubated with the indicated concentrations of drugs for 48 hours. Cell viability was assessed using a colorimetric 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide assay (Sigma). The mitochondrial transmem- brane potential was determined using tetramethylrhodamine ethyl ester (TMRE; Molecular Probes, Eugene, OR). After labeling (25 nmol/L TMRE, 30 minutes), cells were harvested, rinsed, resuspended in PBS, and analyzed on the FL2 channel on a FACSCalibur flow cytometer, using Cell Quest Software (Becton Dickinson, San Jose, CA). Quantification of apoptosis was performed by counting 4',6-diamidino-2-phenylindole–stained nuclei using a fluores- cence microscope. DNA content was assessed by fluorescence-activated cell- sorting analysis as described previously (4). Western Blotting. Protein was extracted from cells in a buffer containing 25 mmol/L Tris (pH 7.8), 2 mmol/L EDTA, 20% glycerol, 0.1% Nonidet P-40, 1 mmol/L dithiothreitol, and protease inhibitors (Roche Diagnostic, Mannheim, Germany). Protein content was measured using Bradford reagent (Bio-Rad, Sund- byberg, Sweden). Equal quantities were separated by SDS-PAGE, transferred to Received 5/21/04; revised 8/7/04; accepted 8/18/04. Grant support: The Swedish Children’s Cancer Foundation, Swedish Cancer Society, Mary Beve’s Foundation, and The Cancer Society of Stockholm. This work was presented in part at the AACR 95th Annual Meeting in Orlando, Florida, March 27–31, 2004 and at the 11th Symposium of Advances in Neuroblastoma Research in Genoa, Italy, June 16 –19, 2004, where it was awarded the Audrey E. Evans Prize. 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: Per Kogner, Childhood Cancer Research Unit, Q6:05, Depart- ment of Woman and Child Health, Karolinska Institutet, Karolinska Hospital, S-171 76, Stockholm, Sweden. Phone: 46-851-773-534; Fax: 46-851-773-475; e-mail: Per.Kogner@ kbh.ki.se. ©2004 American Association for Cancer Research. 7210 Research. on July 1, 2015. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from