br DNA and protein demethylation Regarding SAM metabolism

DNA and protein demethylation Regarding SAM metabolism, both DHFR- and non-DHFR-mediated effects of MTX can be involved. On one hand, inhibition of DHFR decreases THF levels. This can affect SAM metabolism since the reduced derivative of 5,10-methylene THF, 5-methyl THF, provides the methyl group to regenerate methionine from homocysteine. On the other hand, MTXPGs and DHF polyglutamates have an inhibitory effect on methylenetetrahydrofolate reductase (MTHFR), which catalyzes the conversion of 5,10-methylene THF to 5-methyl THF [5]. Moreover, MTX inhibits methionine adenosyltransferase (MAT) expression and MAT enzyme activity. MAT is a key enzyme for SAM metabolism, as it catalyzes the synthesis of SAM from methionine and ATP [50]. Several studies showed that MTX also affects DNA methylation. Folate deficiency is associated with hypomethylated DNA in rat liver A-803467 [3]. In mice, the total level of DNA methylation showed a significant reduction compared to the control group when different doses of MTX were used [12]. In zebrafish embryos, treatment with MTX reduced overall methylation levels and altered gene-specific methylation patterns [26]. In humans, administration of MTX is unable to induce genomic DNA hypomethylation in patients with inflammatory arthritis, but this disease is associated with a significant degree of systemic DNA hypomethylation [18]. In cutaneous T-cell lymphoma cells, MTX treatment significantly reduces SAM levels, and this effect is accompanied by reduction in promoter methylation at CpG islands and by an increase in the expression of Fas protein [54]. Treatment with MTX also decreases global DNA methylation in A549 human lung carcinoma cells [19] and reduces the FAS promoter methylation in melanoma cell lines, which is associated with increased levels of Fas protein [31]. In patients suffering with rheumatoid arthritis, treatment with MTX restores defective Treg cell function through demethylation of the FOXP3 locus, leading to the subsequent increase in FoxP3 expression [6]. Very recently, MTX was shown to decrease global DNA methylation in several osteosarcoma cell lines [44]. Methylation of DNA is not the only situation in which MTX can change the methylation status of biomolecules. For example, MTX inhibits Ras carboxyl methylation. Ras is a prototypic GTPase that functions as a molecular switch to control cell growth and differentiation. After MTX treatment of DKOB8 cells, Ras methylation is decreased by almost 90%. This hypomethylation is accompanied by mislocalization of Ras to the cytoplasm and a substantial decrease in the activation of p44 mitogen-activated protein kinase and Akt [53]. Another example is the methylation of phosphatase-2A (PP2A). MTX reduces the levels of methylated PP2A in primary rat neurons. This effect is accompanied by an increase in phosphorylation of Tau-protein because methylated PP2A is the active form of PP2A [56]. There is also evidence that MTX is able to promote demethylation of the transcription factor E2F1 [36].