Non-DHFR-mediated effects of methotrexate in osteosarcoma cell lines: epigenetic alterations and enhanced cell differentiation.

Martin Sramek,Jaroslav Sterba,Jakub Neradil,Renata Veselska

doi:10.1186/s12935-016-0289-2

Martin Sramek, Jaroslav Sterba + Show 2 more

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https://doi.org/10.1186/s12935-016-0289-2

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Abstract

BackgroundMethotrexate 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.MethodsThe 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.ResultsMethotrexate 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.ConclusionsOverall 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.

Highlights

Methotrexate is an important chemotherapeutic drug widely known as an inhibitor of dihydrofolate reductase (DHFR) which inhibits the reduction of folic acid
Our results showed that the non-DHFR mediated effects of MTX were detectable, especially in patient-derived cell lines and that MTX act as an epigenetic modifier with an impact both on DNA demethylation and the accumulation of acetylated histones
We showed that MTX treatment significantly decreased the proliferation activity in the Saos-2 reference cell line, but all five patient-derived OSA cell lines were much less sensitive to MTX action

Summary

Introduction

Methotrexate is an important chemotherapeutic drug widely known as an inhibitor of dihydrofolate reductase (DHFR) which inhibits the reduction of folic acid. Methotrexate (MTX; amethopterin; 4-amino-10-methylfolic acid), a structural analogue of folic acid, is a chemotherapeutic drug which is still very frequently used as a treatment of osteosarcomas—the most common primary malignant bone tumors affecting both children and adults [1]. Polyglutamate MTX reversibly inhibits dihydrofolate reductase (DHFR) and inhibits other enzymes, for example, phosphoribosylaminoimidazolecarboxamide formyltransferase (AICAR transformylase) or thymidylate synthase (TS). Inhibition of DHFR affects the reduction of folic acid and leads to a lack of 5,10-methylenetetrahydrofolate, which is used as a coenzyme in the biosynthesis of thymidine. Purine precursor biosynthesis is affected by the deficiency of another folate co-factor, 10-formyltetrahydrofolate and by MTX inhibition of AICAR transformylase. The inhibition of dTMP and purine synthesis causes MTX-induced cell death [7]

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