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Published on: March 18, 2014
Non-DHFR-mediated effects of methotrexate in osteosarcoma cell lines: epigenetic alterations and enhanced cell
Martin Sramek1, Jakub Neradil1, Jaroslav Sterba2
1Laboratory 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.
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.
Insights
Methotrexate exhibits non-DHFR-mediated epigenetic effects in osteosarcoma, influencing cell differentiation and gene expression. Combining methotrexate with all-trans retinoic acid shows promise for osteosarcoma differentiation therapy.
Area of Science:
- Oncology
- Epigenetics
- Pharmacology
Background:
- Methotrexate (MTX) is a chemotherapy drug primarily acting as a dihydrofolate reductase (DHFR) inhibitor.
- Emerging evidence suggests non-DHFR-mediated effects of MTX may offer new therapeutic strategies for pediatric cancers.
- This study investigated MTX's impact on osteosarcoma cell lines, focusing on non-DHFR-mediated actions.
Purpose of the Study:
- To analyze the effects of clinically relevant methotrexate concentrations on osteosarcoma cell proliferation.
- To investigate methotrexate's influence on epigenetic regulation, including DNA methylation and histone acetylation.
- To evaluate methotrexate's role in inducing cell differentiation and modulating related gene expression in osteosarcoma.
Main Methods:
- Utilized Saos-2 and five patient-derived osteosarcoma cell lines.
- Assessed cell proliferation via MTT assay.
- Detected DNA methylation and histone acetylation using ELISA and Western blotting.
- Quantified gene expression with RT-qPCR and evaluated differentiation through Alizarin Red S staining.
Main Results:
- Methotrexate reduced proliferation only in the Saos-2 cell line, indicating DHFR-mediated sensitivity.
- Non-DHFR-mediated effects were observed in patient-derived lines, including DNA demethylation (similar to 5-aza-2'-deoxycytidine).
- Methotrexate increased histone H3 acetylation in one cell line and enhanced all-trans retinoic acid-induced differentiation in three lines, with associated gene expression changes.
Conclusions:
- Non-DHFR-mediated effects of methotrexate were evident in patient-derived osteosarcoma cells.
- Methotrexate functions as an epigenetic modifier, impacting osteosarcoma cell differentiation and gene expression.
- Combination therapy with methotrexate and all-trans retinoic acid presents a potential differentiation strategy for osteosarcoma.
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