p38-Dependent Sensitivity of Ewing’s Sarcoma Family of Tumors to Fenretinide-Induced Cell Death

Purpose:There is an urgent need for new therapeutic strategies in Ewing’s sarcoma family of tumors (ESFT). In this study, we have evaluated the effect of fenretinide [N-(4-hydroxyphenyl)retinamide] in ESFT models. Experimental Design:The effect of fenretinide on viable cell number and apoptosis of ESFT cell lines and spheroids and growth of s.c. ESFT in nu/nu mice was investigated. The role of the stress-activated kinases p38 and c-Jun NH2-terminal kinase in fenretinide-induced death was investigated by Western blot and inhibitor experiments. Accumulation of reactive oxygen species (ROS) and changes in mitochondrial transmembrane potential were investigated by flow cytometry. Results: Fenretinide induced cell death in all ESFT cell lines examined in a doseand timedependent manner. ESFT cells were more sensitive to fenretinide than the neuroblastoma cell lines examined. Furthermore, fenretinide induced cell death in ESFT spheroids and delayed s.c. ESFT growth in mice. p38 was activated within 15 minutes of fenretinide treatment and was dependent on ROS accumulation. Inhibition of p38 activity partially rescued fenretinide-mediated cell death in ESFT but not in SH-SY5Y neuroblastoma cells. c-Jun NH2-terminal kinase was activated after 4 hours and was dependent on ROS accumulation but not on activation of p38. After 8 hours, fenretinide induced mitochondrial depolarization (#wm) and release of cytochrome c into the cytoplasm in a ROSand p38-dependent manner. Conclusions:These data show that the high sensitivity of ESFTcells to fenretinide is dependent in part on the rapid and sustained activation of p38.The efficacy of fenretinide in preclinical models demands the evaluation of fenretinide as a potential therapeutic agent in ESFT. The Ewing’s sarcoma family of tumors (ESFT) are small round cell tumors with limited neural differentiation, characterized by the fusion of the 5V portion of the EWS gene with the 3V portion of a gene of the ETS family of transcription factors (1). ESFT constitutes 3% of all pediatric malignancies (2) and are most frequently diagnosed in adolescents and young adults between the ages 10 and 25 years. Treatment intensification has improved prognosis for some patients (2, 3), but 30% to 40% of those with localized and 80% with metastatic disease still die due to disease progression. Recurrence and metastases continue to pose the most difficult challenge for management and treatment of these solid cancers, emphasizing the urgent need for new treatment strategies in ESFT. Retinoic acid has well-described antiproliferative and differentiation-inducing activities in several malignant cell types and is clinically effective in the treatment of some cancers (e.g., alltrans retinoic acid in acute promyelocytic leukemia; refs. 4, 5) and 13-cis retinoic acid in disseminating neuroblastoma (6). Unfortunately, the clinical use of retinoic acid in many solid tumors has been restricted by dose-limiting side effects and low therapeutic efficacy; in ESFT, retinoic acid has no effect on cell growth or survival (7, 8). However, the identification of selective retinoic acid derivatives, which are capable of inducing apoptosis and display synergy with other anticancer therapies, promises more effective and less toxic strategies for treatment. Fenretinide [N-(4-hydroxyphenyl)retinamide] is a synthetic vitamin A analogue with recognized chemopreventive (9) and antitumor activity (10–12). Phase I studies have shown that it is well tolerated in both adults (13, 14) and children (15) and may have efficacy in tumors that do not respond to other retinamides or retinoic acid derivatives. Furthermore, fenretinide acts to additively or synergistically increase the apoptotic response of some cancer cells to chemotherapeutic drugs (16, 17). These important studies suggest that fenretinide may be useful in different clinical situations: increasing response rates to initial therapy by pretreating patients with fenretinide before conventional therapy, targeting disease that develops chemoresistance, treating minimal residual disease, potentially sustaining remission, and preventing recurrence and relapse as a maintenance therapy. www.aacrjournals.org Clin Cancer Res 2005;11(8) April 15, 2005 3136 Authors’Affiliations: Candlelighter’s Children’s Cancer Research Laboratory, Cancer Research UK Clinical Centre, Leeds, United Kingdom and Northern Institute for Cancer Research, University of Newcastle uponTyne, Newcastle upon Tyne, United Kingdom Received10/8/04; revised12/17/04; accepted1/25/05. Grant support: MJP Cancer Care Trust Fund and Candlelighter’s Trust, United Kingdom (S.A. Burchill) and University of Leeds research scholarship (S.S. Myatt). 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 with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Susan A. Burchill, Candlelighter’s Children’s Cancer Research Laboratory, Cancer Research UK Clinical Centre, St. James’s University Hospital, Beckett Street, Leeds LS9 7TF, United Kingdom. Phone: 44-1132065873; Fax: 44-113-2429886; E-mail: [email protected]. F2005 American Association for Cancer Research. Cancer Therapy: Preclinical Research. on July 16, 2017. © 2005 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Although the in vivo efficacy of fenretinide has been widely documented, the mechanism by which fenretinide induces cell death is not fully understood. The mode of action of fenretinide and the mode of action of classic retinoids are distinct. Unlike cell death mediated by the classic retinoids, fenretinidemediated cell death is largely independent of retinoic acid receptors (18, 19), although accumulation of reactive oxygen species (ROS) has been described in many different tumor cell types following exposure to fenretinide (12, 20). Although the mechanisms of fenretinide-mediated ROS production are not well characterized, the mitochondrial electron transport chain (21, 22) and lipoxygenase enzymes (23, 24) have both been implicated. The ability of antioxidants to prevent fenretinide induced cell death suggests a critical role for ROS in the apoptotic signal transduction (21). Interestingly, ROS activate members of the mitogen-activated protein kinase (MAPK) cascade, including c-Jun NH2-terminal kinase (JNK), p38 , and Big MAPK-1 (25). Furthermore, JNK-dependent, fenretinide-induced cell death has been observed in prostate carcinoma cell lines (26). Given the efficacy of fenretinide-induced cell death in neuroblastoma and other cancers, the aim of this study was to investigate the effect of fenretinide on ESFT cells in vitro and on s.c. growing human ESFT in nude mice. Because p38 plays a key role in cell death of ESFT cells (27), we have also asked whether this stress-activated kinase mediates the effects of fenretinide on ESFT cells. Materials andMethods Fenretinide (Janssen-Cilag, Basserdorf, Switzerland) was stored protected from light as a 10 mmol/L stock in ethanol (BDH, United Kingdom) at 20jC. Basic fibroblast growth factor (Sigma, Dorset, United Kingdom) was dissolved in filter-sterilized 1% bovine serum albumin (Sigma) and aliquoted before storing as a stock solution (25 Ag/mL) at 20jC. 4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4pyridyl)-imidazole (SB202190, Calbiochem, San Diego) was protected from light and stored at 20jC as a stock solution of 5 mg/mL in DMSO (Sigma) and used at a final concentration of 20 Amol/L. Vitamin C (L-ascorbic acid, Sigma) was stored at room temperature and prepared as a 1 mol/L stock in filter-sterilized double-distilled H2O immediately before use. Selective inhibitors of ROS generation pathways were used and stored in ethanol and were purchased from Calbiochem unless otherwise stated. Ketoconazole, a cytochrome P450 inhibitor (Sigma), was stored as a stock solution in DMSO and used at a final concentration of 5 Amol/L. The nitric oxide synthase inhibitor nitro-L-arginine methyl ester (Sigma) was used at 400 Amol/L. AACOCF3, a phospholipase A2 inhibitor, was used at a final concentration of 10 Amol/L. The NADPH oxidase inhibitor diaphenylene iodonium was stored in DMSO and used at a final concentration of 10 Amol/L. Nordihydroguairetic acid, a pan-lipoxygenase inhibitor, was used at a final concentration of 50 Amol/L. The selective 5lipoxygenase inhibitor MK886 was used at a final concentration of 1 Amol/L. Caffeic acid, a selective 5and 15-lipoxygenase inhibitor, was used at a final concentration of 10 Amol/L. 15-Lipoxygenase was selectively inhibited using PD146176 at 0.3 Amol/L and was a gift from Parke-Davis Pharmaceutical Research, Ann Arbor, MI. The 12lipoxygenase-selective inhibitor baicalein was used a final concentration of 1 Amol/L. N-vanillylnononamide, a NADPH oxidase inhibitor, was used at 10 Amol/L (vehicle methanol) and was purchased from Sigma. The mitochondrial respiratory chain inhibitor AEBSF (Sigma) was used at final concentration of 100 Amol/L (in H2O). The ROSresponsive dye CM2-DCFDA (Molecular Probes, Eugene, OR) was prepared in DMSO as a 5 Amol/L stock solution, placed on ice, and used immediately. H2O2 (Sigma) was prepared as a 100 Amol/L stock solution in double-distilled H2O and used immediately. 3,3VDihexyloxacarbocyanine (Sigma) was stored as a stock solution in DMSO (5 mmol/L) and used at a final concentration of 40 nmol/L. Anti-human poly(ADP-ribose) polymerase (PARP) antibody (BD PharMingen/Becton Dickinson, Oxford, United Kingdom) was used at a final concentration of 1:500. Anti-human tubulin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used at 1:5,000. Anti-human total p38 (rabbit polyclonal), phospho-p38, total JNK (rabbit polyclonal), phospho-JNK, and cytochrome c (rabbit polyclonal) antibodies were purchased from Cell Signaling (Hertfordshire, United Kingdom) and used at 1:1,000. All antibodies were mouse monoclonal unless otherwise stated. Alexa Flour 680 IgG rabbit antimouse and goat anti-rabbit secondary antibodies (Molecular Probes) were used at 1:5,000. Antibodies purchased from Cell Signaling were stored at 20jC; all other antibodies were stored at 4jC. Cell culture. The substrate adherent ESFT cell lines TC-32, RD-ES, and TTC-466 were grown in RPMI 1640 (Sigma) containing 10% FCS (SeraLab, Sussex, United Kingdom); medium for TTC-466 cells was supplemented with 10% tumor cell conditioned medium. A673 cells were grown in DMEM (Sigma) and 10% FCS, SK-ES1 cells in McCoy’s medium (Sigma) plus 15% FCS, and SK-N-MC and SH-SY5Y cells in DMEM/F-12 plus 10% FCS. SH-EP1 and SK-N-SH cells were grown in a 1:1 mix of DMEM and MEM and 10% FCS. All cell lines were purchased from the American Type Culture Collection, Manassas, VA, except for the following cells that were kind gifts: TC-32 cells from Dr. J. Toretsky (Division of Pediatrics, University of Maryland, Baltimore, MD), the TTC-466 cells from Dr. P. Sorenson (British Columbia Children’s Hospital, Vancouver, British Columbia, Canada), and the SH-SY5Y cells from Dr. R.A. Ross (Fordham University, Bronx, NY). Spheroid cultures were initiated by seeding cells into flasks coated with 1.4% agar (Sigma); after 3 to 4 days, cell aggregates were transferred to Integra Bioscience, Switzerland spinner flasks (100 or 250 mL); spheroid formation was promoted and preserved by incubation in spinner flasks with continuous stirring (15 g). Medium was replenished every 3 days by allowing the spheroids to settle in the bottom of the flask, aspirating 80% of the used medium and replacing with fresh medium. All incubations and treatments of cells, unless otherwise stated, were carried out in a humidified atmosphere of 5% CO2, 95% air at 37jC (Sanyo Gallenkamp, Loughborough, United Kingdom). Viable cell counts. Cells were seeded into Primeria six-well plates (Fahrenheit, Leeds, United Kingdom), maintained for 24 hours, and treated with fenretinide (0-10 Amol/L) for 0 to 24 hours. Control cultures were treated with ethanol vehicle. Substrate adherent and nonadherent cells were harvested by trypsinization and centrifugation and resuspended in 1 mL normal growth medium, and viable cell number was counted using the Vi-cell automated trypan blue exclusion assay (Becton Dickinson). To assess spheroid viability and growth characteristics after treatment with fenretinide (3-10 Amol/L, 24-48 hours), spheroids were incubated in 100 mL medium, spheroid slurry was allowed to settle for 5 minutes, and spheroid-free medium (90 mL) was removed and discarded. Three aliquots (2 mL) of spheroid slurry were collected while gently agitating the spheroid suspension to ensure even sampling. Spheroids were either fixed in 4% formal saline for 30 minutes, suspended in 1.4% agar and paraffin embedded for the preparation of sections for staining (see Immunohistochemistry), or disaggregated using EDTA and trypsin to generate a single cell suspension that was analyzed using the Vi-cell automated trypan blue exclusion assay for total and viable cell number. In vivo methodology. The effect of fenretinide (100 mg/kg/d s.c. or 5 days treatment, 2 days rest by oral gavage) on s.c. ESFT growth was examined in nu/nu mice. Mice (n = 18) were injected in one (n = 10) or both (n = 8) flanks s.c. with a single cell suspension of RD-ES cells (2.5 10 in 0.2 mL medium). On day 15, after the development of a palpable tumor, mice were (a) injected with either vehicle alone or fenretinide or (b) treated by oral gavage with fenretinide or vehicle. A stock solution of fenretinide (250 mg/mL) was prepared in ethanol and diluted in 0.9% saline for s.c. injection into mice or in corn oil and www.aacrjournals.org Clin Cancer Res 2005;11(8) April 15, 2005 3137 p38-Dependent Fenretinide-Induced Death in ESFT Research. on July 16, 2017. © 2005 American Association for Cancer clincancerres.aacrjournals.org Downloaded from cremaphor for oral gavage (28). Tumor size was measured twice weekly and mice were sacrificed when s.c. tumors reached a size of 1.4 cm, at the end of the experiment, or if the mouse showed signs of distress. Administration of fenretinide to the test group was stopped when all mice in the control group had been sacrificed. Tumors were excised, fixed in 10% phosphate-buffered formalin, and paraffin embedded for the preparation of tissue sections. Immunohistochemistry. Sections (3 Am) of paraffin-embedded spheroids or RD-ES tumors were heat sealed (1 hour) onto glass slides. Sections were stained with H&E and morphology was observed by light microscopy (Zeiss Axioplan microscope, United Kingdom). Apoptosis was detected using the Apoptag Apoptosis Detection kit (Chemicon, Temecula, CA) according to the manufacturer’s instructions. Proliferation was detected by immunohistochemistry for Ki-67 after dewaxing and rehydration of sections. Endogenous peroxidases were blocked by incubating in hydrogen peroxide (H2O2 3%) for 10 minutes. After antigen retrieval (microwaving for 12 minutes in citric acid), sections were incubated in avidin followed by biotin and finally in normal rabbit serum (1:10) for 10-minute periods. Sections were then incubated with Ki-67 antibody (1:50, DAKO, Carpintaria, CA) for 1 hour at room temperature and finally with the rabbit anti-mouse secondary antibody (1:50; DAKO). Specific binding was detected using streptavidin AB and 3,3V-diaminobenzidine (DAKO), and sections were counterstained with H&E and mounted in DePex. Assessment of apoptosis using Annexin V and propidium iodide. Cells were seeded at 2 10 cells per well into six-well plates (Primeria) and left overnight to adhere. Fenretinide (1.5 Amol/L) was added at 0 to 24 hours before harvest by trypsinization and resuspension in RPMI 1640. Labeling of cells with Annexin V and propidium iodide (PI) was determined using the BD PharMingen Annexin V-FITC apoptosis detection kit. Fluorescence-activated cell sorting was conducted using the FACSCalibur (Becton Dickinson) and analyzed using the flow cytometry software CellQuest. Determination of reactive oxygen species. ROS production was detected using CM2-DCFDA. Cells (2 10 per well) were seeded into Primeria six-well plates and left to adhere overnight. Cells were then treated with fenretinide (1.5 Amol/L, 0-10 hours, 37jC) and harvested by trypsinization. Cells were pelleted (403 g , 5 minutes) and resuspended in CM2-DCFDA (5 Amol/L in PBS). Cells were incubated at 37jC in the dark for 30 minutes and then washed twice in PBS (403 g , 5 minutes). To unstained samples, DMSO was added as a vehicle control. Treatment with H2O2 (100 Amol/L, 15 minutes) served as a positive control. FACSCalibur flow cytometry and CellQuest Pro were used to assess fluorescence; 10,000 events were counted for each sample. To assess the role of ROS in cell death, the antioxidant vitamin C was used. Cells were incubated for 1 hour at 37jC with vitamin C (100 Amol/L in doubledistilled H2O) before the addition of fenretinide (1.5 Amol/L). Cells were pretreated with specific ROS-generating inhibitors for 2 hours before incubation with fenretinide (1.5 Amol/L). At 24 hours, cells were collected and viable cell number was determined by the trypan blue exclusion assay. ESFT cells were exposed to the inhibitors alone at previously published doses (23, 29) to determine whether the inhibitor had any toxic effects; for PD146176, MK886, baicalein, AACOCF3, and nordihydroguairetic acid, the highest dose tolerated by ESFT was also evaluated. The effects of diaphenylene iodonium and AEBSF on fenretinide-induced cell death were not assessed, as these agents alone were toxic to ESFT cells at doses required to inhibit ROS accumulation. Determination of protein phosphorylation and poly(ADP-ribose) polymerase cleavage. Cells (1 10) were seeded in 10 cm Primeria dishes, allowed to adhere overnight, and either untreated or treated with fenretinide (1.5 Amol/L, 0-24 hours). Cells were collected by brief centrifugation (11,600 g , 10 minutes, 4jC) and resuspended in lysis buffer [50 mmol/L HEPES (pH 7.5), 100 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L DTT, 10 mmol/L MgCl2, 1% Triton X-100, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 1 mmol/L sodium orthovanadate, 25 mmol/L NaF]. Cells were then incubated for 30 minutes on ice, vortexed at 10-minute intervals, and centrifuged (11,600 g , 4jC) for 10 minutes to pellet the insoluble material. Lysate was stored as aliquots in an equal volume of 2 SDS nonreducing loading buffer [100 mmol/L Tris-HCl (pH 6.8,), 20% (w/v) glycerol, 4% (w/v) SDS, 0.2% (w/v) bromophenol blue] at 20jC. Before storage, samples were heated to 99.9jC for 5 minutes (Hybond, Omnigene, Cambridge, MA) and placed quickly on ice. Protein concentration was determined using the Bio-Rad DC Protein Assay kit (Bio-Rad, Herts, United Kingdom). To detect protein phosphorylation and PARP cleavage, Western blotting was carried out using the Li-cor Odyssey IR imaging system (Lincoln, NE). Inhibition of p38 using SB202190. The cell-permeable p38a and p38h selective inhibitor SB202190 was used to assess the role of p38 in fenretinide (1.5 Amol/L)– induced cell death. Cells were incubated with SB202190 (20 Amol/L) for 1 hour at 37jC before addition of fenretinide. Control cells were treated with