INTERNATIONAL JOURNAL OF ONCOLOGY 49: 2135-2146, 2016 Arsenic trioxide potentiates the effectiveness of etoposide in Ewing sarcomas KAREN A. BOEHME1*, JULIANE NITSCH1*, ROSA RIESTER1, RUPert HANDGRETINGER2, SABINE B. SCHLEICHER2, TORSTEN KLUBA3,4 and FRANK TRAUB1,3 1Laboratory of Cell Biology, Department of Orthopaedic Surgery, Eberhard Karls University Tuebingen; 2Department of Haematology and Oncology, Children's Hospital, Eberhard Karls University Tuebingen; 3Department of Orthopaedic Surgery, Eberhard Karls University Tuebingen, Tuebingen; 4Department for Orthopaedic Surgery, Hospital Dresden-Friedrichstadt, Dresden, Germany Received June 12, 2016; Accepted July 28, 2016 DOI: 10.3892/ijo.2016.3700 Abstract. Ewing sarcomas (ES) are rare mesenchymal tumours, the drug concentrations used. With the exception of ATO in most commonly diagnosed in children and adolescents. Arsenic RD-ES cells, all drugs induced apoptosis in the ES cell lines, trioxide (ATO) has been shown to efficiently and selectively indicated by caspase-3 and PARP cleavage. Combination of target leukaemic blasts as well as solid tumour cells. Since the agents potentiated the reduction of viability as well as the multidrug resistance often occurs in recurrent and metastatic inhibitory effect on clonal growth. In addition, cell death induc- ES, we tested potential additive effects of ATO in combina- tion was obviously enhanced in RD-ES and SK-N-MC cells tion with the cytostatic drugs etoposide and doxorubicin. The by a combination of ATO and etoposide compared to single Ewing sarcoma cell lines A673, RD-ES and SK-N-MC as well application. Summarised, the combination of low dose, physi- as mesenchymal stem cells (MSC) for control were treated ologically easily tolerable ATO with commonly used etoposide with ATO, etoposide and doxorubicin in single and combined and doxorubicin concentrations efficiently and selectively application. Viability and proliferation (MTS assay, colony suppressed viability and colony formation in ES cell lines, formation, 3D spheroid culture) as well as cell death induction whereas a combination of ATO and etoposide was favourable (western blot analysis, flow cytometry) were analysed. In the for cell death induction. In addition to an increase of the effec- MTS viability assays ATO treatment significantly reduced tiveness of the cytostatic drugs and prevention of potential drug the metabolic activity of all three ES cell lines (A673, RD-ES resistance, this approach may also reduce toxicity effects, since and SK-N-MC) examined. Moreover, all ES cell lines were the individual doses can be reduced. sensitive to etoposide, whereas MSC remained unaffected by Introduction Ewing sarcomas (ES) are rare, aggressive mesenchymal tumours affecting bones and soft tissues with a peak inci- Correspondence to: Dr Karen A. Boehme, Laboratory of Cell dence in childhood and adolescence (1). In ~85% of patients a Biology, Department of Orthopaedic Surgery, Eberhard Karls University Tuebingen, Waldhoernlestrasse 22, D-72072 Tuebingen, t(11;22) (q24;q12) chromosomal translocation can be detected Germany leading to expression of a chimeric fusion protein composed E-mail: [email protected] of the Ewing sarcoma breakpoint region 1 (EWS) gene on chromosome 22 and the Fried leukaemia integration 1 (FLI1) Abbreviations: ATO, arsenic trioxide; EGFR, epidermal growth gene on chromosome 11. Moreover, other less common fusion factor receptor; ES, Ewing sarcoma; EWS, Ewing sarcoma breakpoint proteins have been described mostly comprising the EWS region 1; FLI1, Fried leukaemia integration 1; GLI, glioma associated transactivation domain (2). The aberrant transcription factor oncogene family; GSH, glutathion; Hh, hedgehog; IGF-1R, insulin- EWS-FLI1 activates several signalling pathways promoting like growth factor receptor 1; MAPK, mitogen activated protein proliferation and apoptosis resistance (3-5). kinase; MDR1, multidrug resistance protein 1; MRP1, multidrug The standard of care for ES is a neo-adjuvant chemo- resistance associated protein 1; MSC, mesenchymal stem cells; therapy including doxorubicin, vincristine, cyclophosphamide, mTOR, mammalian target of rapamycin; PARP, poly (ADP-ribose) polymerase 1; PDGFR, platelet-derived growth factor receptor; actinomycin D, ifosfamide and etoposide followed by surgery, PTCH1, patched 1; RMS, rhabdomyosarcoma; SKMC, primary conceivably radiation therapy and adjuvant chemotherapy skeletal muscle cells; SMO, smoothened; VEGFR, vascular growth (6,7). Five-year survival rates for localised disease are ~70%. factor receptor However, patients with metastases expect only 5-year survival rates of 20-30% (1). Key words: Ewing sarcoma, hedgehog signalling, arsenic trioxide, Multidrug resistance is a common problem in metastatic GANT61, etoposide, doxorubicin and recurrent ES and increases during prolonged therapy (8). Multidrug resistance associated protein 1 (MRP1) overexpres- 2136 BoeHme et al: COMBINED EFFECTS OF ARSENIC TRIOXIDE AND ETOPOSIDE IN EWING SARCOMAS sion is a common feature of ES, whereas multidrug resistance Faculty, project no. 401/2013 BO2), propagated as described protein 1 (MDR1) expression is less common (9). Substrate before (18) and confirmed to represent multi-lineage differ- specificity of both ABC transporters includes etoposide, doxo- entiation potential toward chondrocytes, adipocytes and rubicin and vincristine implicating their role in therapeutic osteocytes (data not shown). All cells were cultivated at 37˚C resistance of ES (10). in humidified atmosphere containing 5% CO2. Several clinical trials specifically targeting receptor tyrosine kinases such as insulin-like growth factor receptor 1 RNA isolation and qRT-PCR. RNA was isolated using the (IGF-1R), kit, platelet-derived growth factor receptor innuPREP RNA Mini kit (Analytik Jena AG, Jena, Germany). (PDGFR), epidermal growth factor receptor (EGFR), vascular RNA (1 µg) was reverse transcribed using the innuSCRIPT growth factor receptor (VEGFR) or other proteins including reverse transcriptase (Analytik Jena AG). cDNA (50 ng) Aurora kinase A, mammalian target of rapamycin (mTOR) was analysed in duplicate reactions by quantitative RT-PCR or poly (ADP-ribose) polymerase 1 (PARP1) in ES family (qRT‑PCR) using gene-specific primers and the SYBR tumours are currently in phase I and II. However, response Select Master mix for CFX (Life Technologies GmbH) in a rates in ES are usually <30% (1). Combination therapy using total volume of 10 µl. qRT-PCR was carried out in a CFX96 cytotoxic drugs and targeted therapeutics may be an opportu- real-time device (Bio-Rad, Munich, Germany) and was anal- nity to overcome drug resistance and improve survival. ysed using the CFX ManagerTM software (Bio-Rad). Relative The glioma associated oncogene family 1 (GLI1) transcrip- expression levels were calculated as fold change compared to tion factor is a direct transcriptional target of the EWS-FLI1 MSC using the ΔΔCt (2-ΔΔCt) method with TATA box binding fusion protein (11-13). In several types of cancer, including protein (TBP) as a reference gene. Hh pathway primers were ES and rhabdomyosarcoma (RMS), GLI1 has been associ- used according to Laurendeau et al (19). ated with proliferation and survival (12,14). Targeting GLI by arsenic trioxide (ATO) has been shown to reduce viability of Cytotoxicity assay. Cell Titer 96® AQueous One Solution Cell several ES cell lines with an IC50 of ~1 µM using a WST-1 Proliferation (MTS) assay (Promega, Mannheim, Germany) viability assay (15). Additionally, ATO inhibits migration and was used to measure cell viability via redox enzyme activity, invasion capacity of RD-ES and A673 cells (16). Moreover, according to the protocol provided by the manufacturer. A673, Matsumoto et al reported that GANT61, another GLI inhibitor, RD-ES, SK-N-MC and MSC (0.5-1x104 cells/well) were was capable of inducing caspase-3 and -7 independent cell grown in 96-well plates. Twenty-four hours after seeding, death in the ES cell line SK-N-LO (17). the cells were incubated in the presence of ATO, GANT61, In this study, we determined whether GLI inhibition using etoposide, doxorubicin or inhibitor combinations for another ATO or GANT61 specifically and efficiently compromises 96 h at 37˚C in a humidified atmosphere of 5% CO2 in air. three different ES cell lines compared to mesenchymal stem At the end of the incubation period, MTS reagent was added cells (MSC). Based on our results, we selected ATO for combi- to the wells, and the plate was incubated for 1.5 h protected nation experiments with etoposide and doxorubicin. Especially, from light. Absorbance was recorded at 490 nm with a refer- the combination of ATO and etoposide significantly exceeded ence wavelength of 630 nm using an EL 800 reader (BioTek, the effect of each single drug on viability reduction, clonal Winooski, VT, USA). growth and cell death induction in ES cell lines, whereas MSC were hardly compromised by the drug doses applied in the IC50 determination. IC50 values of ATO, GANT61, etoposide experiments. and doxorubicin were determined for the different cell lines by non-linear regression using GraphPad Prism V6.0 software. Materials and methods Colony formation assay. A673 cells were plated at a density Reagents. ATO (Trisenox, Pharmacy of University Hospital of 1x103 cells/well, RD-ES cells were plated at a density of Tuebingen) was dissolved in purified water, GANT61 (Abcam, 0.5x103 cells/well and SK-N-MC were plated at a density Cambridge, UK), etoposide and doxorubicin (Selleckchem,