Int J Oncol. 2016 Feb;48(2):801-12. doi: 10.3892/ijo.2015.3293.

Targeting hedgehog signalling by arsenic trioxide reduces cell growth and induces apoptosis in rhabdomyosarcoma.
 

Boehme KA1, Zaborski JJ1, Riester R1, Schweiss SK1, Hopp U1, Traub F2, Kluba T2, Handgretinger R3, Schleicher SB3.
  • 1Laboratory of Cell Biology, Department of Orthopaedic Surgery, Eberhard Karls University Tuebingen, Tuebingen, Germany.
  • 2Department of Orthopaedic Surgery, Eberhard Karls University, Tuebingen, Germany.
  • 3Department of Haematology and Oncology, Children’s Hospital, Eberhard Karls University Tuebingen, Tuebingen, Germany.

 

Abstract

Rhabdomyosarcomas (RMS) are soft tissue tumours treated with a combination of surgery and chemotherapy. However, mortality rates remain high in case of recurrences and metastatic disease due to drug resistance and failure to undergo apoptosis. Therefore, innovative approaches targeting specific signalling pathways are urgently needed. We analysed the impact of different hedgehog (Hh) pathway inhibitors on growth and survival of six RMS cell lines using MTS assay, colony formation assay, 3D spheroid cultures, flow cytometry and western blotting. Especially the glioma-associated oncogene family (GLI) inhibitor arsenic trioxide (ATO) effectively reduced viability as well as clonal growth and induced cell death in RMS cell lines of embryonal, alveolar and sclerosing, spindle cell subtype, whereas normal skeletal muscle cells were hardly compromised by ATO. Combination of ATO with itraconazole potentiated the reduction of colony formation and spheroid size. These results show that ATO is a promising substance for treatment of relapsed and refractory RMS by directly targeting GLI transcription factors. The combination with itraconazole or other chemotherapeutic drugs has the opportunity to enforce the treatment efficiency of resistant and recurrent RMS.

PMID: 26676886

 

 

Supplement

The standard chemotherapeutic treatment of rhabdomyosarcomas (RMS) includes vincristine, actinomycin D and cyclophosphamide (VAC). Besides, in some protocols doxorubicin is administered. Etoposide in combination with ifosfamide (IE) is added for treatment of patients with metastatic RMS [1]. However, multidrug resistance of sarcomas often arises upon initial response [2, 3]. Mechanisms responsible for emergence of multidrug resistance include exclusion of drugs from the cell, failure to activate prodrugs, increased detoxification, alterations of the drug target, enhanced repair of damage or failure to undergo cell cycle arrest and apoptosis [4]. Interestingly, in addition to multidrug resistance associated genes, RMS cells exposed to cytotoxic agents increase the expression of marker genes associated with myogenic differentiation [5]. Indeed, RMS express the ABC transporter Multidrug Resistance Protein 1 (MDR1) and Multidrug Resistance Associated Protein 1 (MRP1) [4, 6] and transcriptional regulation of both involves GLI [7].

 

 

Figure 1 SKMC RD MTS Kombi ATO etoposide finalFigure 1: ATO in combination with etoposide enhances viability reduction in the human RMS cell line RD. MTS assays were performed four days after single or combined treatment with ATO and etoposide in the RMS cell line RD and primary skeletal muscle cells (SKMC) in quadruplicate. Mock treated control was set to 100% viability. Error bars indicate the standard deviations (*** p ≤ 0.001 relative to single treatment).

 

Since we could prove the efficiency of ATO in reducing viability and inducing apoptotic cell death in different RMS cell lines [8], we analysed its potency in combination with etoposide. Actually, the combination of 2 µM ATO and 10 µM etoposide potentiated viability reduction (figure 1) and cell death induction (figure 2) in the human ERMS cell line RD, whereas primary skeletal muscle cells (SKMC) were only marginally affected.

One potential mechanism implicated in the additive effect of ATO and etoposide is accumulation of the MDR1 and MRP1 substrate etoposide in the cells upon ATO dependent downregulation of ABC transporter expression. However, it is reported that MDR1 and MRP1 expression remains essentially unchanged upon chemotherapy of RMS, whereas Lung Resistance-Related Protein (LRP) expression is increased, indicating this transporter to be more important for acquired multidrug resistance [4, 9].

Another conceivable mechanism involves topoisomerases, which relax DNA supercoiling by forming transient covalent DNA cleavage complexes. Topoisomerase II is a target of all chemotherapeutic drugs that are DNA intercalating and its altered activity is implicated in drug resistance [4]. Trapping of topoisomerase II by etoposide initiates DNA double-strand break induced apoptosis and subsequently promotes ROS and oxidative DNA damage dependent formation of apoptotic topoisomerase I-DNA complexes [10]. Since also ATO induces ROS generation and trapped topoisomerase I-DNA complexes [11] both drugs might cooperate in DNA damage induced cell cycle arrest and apoptosis induction.

Although the exact mechanism of action still has to be determined, combination of ATO and etoposide turns out to be a promising strategy for viability reduction und cell death induction in the ERMS cells line RD. Whether this is a general feature of RMS and may also be extended to combination of other chemotherapeutic drugs with ATO has to be investigated.

 

 

Figure 2 SKMC RD FACS Kombi ATO etoposide finalFigure 2: ATO in combination with etoposide enhances cell death induction in the human RMS cell line RD. Incorporation of the fixable viability dye eFluor® 450 was analysed by flow cytometry three days after single or combined treatment with ATO and etoposide in the RMS cell line RD and SKMC in triplicate. Mock treated control was set to zero % cell death. Error bars indicate the standard deviations (** p ≤ 0.01 relative to single treatment).

 

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  8. Boehme, K.A., et al., Targeting hedgehog signalling by arsenic trioxide reduces cell growth and induces apoptosis in rhabdomyosarcoma. Int J Oncol, 2016. 48(2): p. 801-12.
  9. Marchal, J.A., et al., GR-891: a novel 5-fluorouracil acyclonucleoside prodrug for differentiation therapy in rhabdomyosarcoma cells. Br J Cancer, 1999. 79(5-6): p. 807-13.
  10. Sordet, O., A. Goldman, and Y. Pommier, Topoisomerase II and tubulin inhibitors both induce the formation of apoptotic topoisomerase I cleavage complexes. Mol Cancer Ther, 2006. 5(12): p. 3139-44.
  11. Sordet, O., et al., Topoisomerase I-DNA complexes contribute to arsenic trioxide-induced apoptosis. J Biol Chem, 2004. 279(32): p. 33968-75.

 

 

 

 

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