title: Non-DHFR-mediated effects of methotrexate in osteosarcoma cell lines: epigenetic alterations and enhanced cell differentiation.
abstract: BACKGROUND
  Methotrexate is an important chemotherapeutic drug widely known as an inhibitor of dihydrofolate reductase (DHFR) which inhibits the reduction of folic acid. DHFR-mediated effects are apparently responsible for its primary antineoplastic action. However, other non-DHFR-mediated effects of methotrexate have been recently discovered, which might be very useful in the development of new strategies for the treatment of pediatric malignancies. The principal goal of this study was to analyze the possible impact of clinically achievable methotrexate levels on cell proliferation, mechanisms of epigenetic regulation (DNA methylation and histone acetylation), induced differentiation and the expression of differentiation-related genes in six osteosarcoma cell lines.
  
  
  METHODS
  The Saos-2 reference cell line and five other patient-derived osteosarcoma cell lines were chosen for this study. The MTT assay was used to assess cell proliferation, DNA methylation and histone acetylation were detected using ELISA, and western blotting was used for a detailed analysis of histone acetylation. The expression of differentiation-related genes was quantified using RT-qPCR and the course of cell differentiation was evaluated using Alizarin Red S staining, which detects the level of extracellular matrix mineralization.
  
  
  RESULTS
  Methotrexate significantly decreased the proliferation of Saos-2 cells exclusively, suggesting that this reference cell line was sensitive to the DHFR-mediated effects of methotrexate. In contrast, other results indicated non-DHFR-mediated effects in patient-derived cell lines. Methotrexate-induced DNA demethylation was detected in almost all of them; methotrexate was able to lower the level of 5-methylcytosine in treated cells, and this effect was similar to the effect of 5-aza-2'-deoxycytidine. Furthermore, methotrexate increased the level of acetylated histone H3 in the OSA-06 cell line. Methotrexate also enhanced all-trans retinoic acid-induced cell differentiation in three patient-derived osteosarcoma cell lines, and the modulation of expression of the differentiation-related genes was also shown.
  
  
  CONCLUSIONS
  Overall non-DHFR-mediated effects of methotrexate were detected in the patient-derived osteosarcoma cell lines. Methotrexate acts as an epigenetic modifier and has a potential impact on cell differentiation and the expression of related genes. Furthermore, the combination of methotrexate and all-trans retinoic acid can be effective as a differentiation therapy for osteosarcoma.
authors.affiliation: Laboratory of Tumor Biology, Department of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic ; Department of Pediatric Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Cernopolni 9, 613 00 Brno, Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Sramek
authors.affiliation: Laboratory of Tumor Biology, Department of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic ; Department of Pediatric Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Cernopolni 9, 613 00 Brno, Czech Republic.
authors.forename: Jakub
authors.initials: J
authors.lastname: Neradil
authors.affiliation: Department of Pediatric Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Cernopolni 9, 613 00 Brno, Czech Republic.
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Sterba
authors.affiliation: Laboratory of Tumor Biology, Department of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic ; Department of Pediatric Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Cernopolni 9, 613 00 Brno, Czech Republic.
authors.forename: Renata
authors.initials: R
authors.lastname: Veselska
keywords: All-trans retinoic acid; DNA methylation; Epigenetic regulation; Histone acetylation; Methotrexate; Osteogenic differentiation; Osteosarcoma