Abstract
Rhabdomyosarcomas (RMS) are the most prevalent soft tissue sarcomas affecting children and adolescents. Despite intensive treatment consisting of multimodal chemotherapy and surgery RMS patients diagnosed with metastatic disease expect long term survival rates of only 20%. Often multidrug resistance arises upon initial response emphasizing the need for new therapeutic drugs to improve treatment efficiency. Previously, we demonstrated the efficacy of the FDA approved drug arsenic trioxide (ATO) specifically inhibiting viability and clonal growth as well as inducing cell death in human RMS cell lines of different subtypes. In this study, we combined low dose ATO with lithium chloride (LiCl), which is approved as mood stabilizer for the treatment of bipolar disorder, but also inhibits growth and survival of different cancer cell types in pre-clinical research. Indeed, we could show additive effects of LiCl and ATO on viability reduction, decrease of colony formation as well as cell death induction. In the course of this, LiCl induced inhibitory glycogen synthase kinase-3β (GSK-3β) serine 9 phosphorylation, whereas glioma associated oncogene family 1 (GLI1) protein expression was particularly reduced by combined ATO and LiCl treatment in RD and RH-30 cell lines, showing high rates of apoptotic cell death. These results imply that combination of ATO with LiCl or another drug targeting GSK-3 is a promising strategy to enforce the treatment efficiency in resistant and recurrent RMS.
Highlights
The IC50 values obtained for the RMS cell lines ranged from 20.26 mM in RH-30 to 32.14 mM in SRH, whereas skeletal muscle cells from adult donors (SKMC) were less sensitive (50.12 mM)
Combination of 1 μM arsenic trioxide (ATO) and 25 mM lithium chloride (LiCl) significantly enhanced viability reduction compared to single treatment in SRH, RD and RH-30 cells (Fig 1)
We previously demonstrated that ATO mediated inhibition of viability and colony formation as well as cell death induction in human ERMS and ARMS cell lines is associated with the reduction of glioma associated oncogene family 1 (GLI1) protein abundance [9]
Summary
ATO (Trisenox, Pharmacy of University Hospital Tuebingen) and LiCl (Sigma-Aldrich, Taufkirchen, Germany) were dissolved in deionised water. For cell culture treatment stock solutions were further diluted in culture medium. SRH (sclerosing spindle cell RMS) were established and characterized at the University Children’s Hospital Tuebingen. The patient has given her written informed consent to the scientific analysis and cell line establishment from tissue samples, the study of which was approved by the ethics committee of the University of Tuebingen (008/2014BO2). Normal skeletal muscle cells from adult donors (SKMC) were purchased from PromoCell (Heidelberg, Germany). All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAX, 4.5 g/l D-glucose (Gibco, Life Technologies, Darmstadt, Germany) supplemented with 10% FCS (Biochrom, Berlin, Germany) at 37 ̊C in humidified atmosphere containing 5% CO2
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