Effect of methotrexate/vitamin B12 on DNA methylation as a potential factor in leukemia treatment-related neurotoxicity

Abstract

Methotrexate (MTX) is administered to treat childhood acute lymphoblastic leukemia (ALL). It acts by inhibiting dihydrofolate reductase which reduces methyltetrahydrofolate, a key component in one carbon metabolism, thus reducing cell proliferation. Further perturbations to one carbon metabolism, such as reduced vitamin B₁₂ levels via the use of nitrous oxide for sedation during childhood ALL treatment, may increase neurotoxicity risk. With B₁₂ as an enzymatic cofactor, methyltetrahydrofolate is essential to produce methionine, which is critical for DNA methylation. We investigated global and gene specific DNA methylation in neuronal cell lines in response to MTX treatment and vitamin B₁₂ concentration individually, and in combination.

Results: MTX treatment alone significantly increased LINE-1 methylation in SH-SY5Y (p = 0.040) and DAOY (p < 0.001), and increased FKBP5 methylation in MO3.13 cells (p = 0.009).

Conclusion: We conclude that altered DNA methylation of brain/central nervous system cells could be one mechanism involved in MTX treatment-related neurotoxicities and neurocognitive late effects in ALL survivors.

Keywords: childhood acute lymphoblastic leukemia; epigenetic; late effects; methotrexate; neurocognition; neurotoxicity; one carbon metabolism; vitamin B12.

Figure 1.. Summary diagram of the main pathways involved in cellular one-carbon metabolism including the production of S-adenosylmethionine for methylation of DNA.

Dietary factors able to influence this pathway are highlighted in red, enzymes driving biological reactions are highlighted in blue/bold. The influence of methotrexate on this pathway through the inhibition of dihydrofolate reductase enzyme is highlighted in red/italics. Additional abbreviations: DHF, THF, SHMT, MTRR, MTHFR, BHMT, CBS, COMT, DNMT, SAH. BHMT: Betaine-homocysteine S-methyltransferase; CBS: Cystathione beta synthase; COMT: cystathionine beta synthase; DHF: Dihydrofolate; DHFR: Dihydrofolate reductase; DNMT: DNA methyltransferase; MTHFR: Methylenetetrahydrofolate reductase; MTRR: Methionine synthase reductase; MTX: Methotrexate; SAH: S-adenosylhomocysteine; SAM: S-adenosylmethionine; SHMT: Serine hydroxymethyltransferase; THF: Tetrahydrofolate. Modified with permission from [1] (2011).

Figure 2.. Schema depicting 3 × 3 factorial study design.

The experiment was carried out in triplicate for each cell line, resulting in an n = 9 per group to investigate the main effects of MTX and vitamin B₁₂ treatments alone, and n = 3 to investigate the interaction between treatments. MTX: Methotrexate.

Figure 3.. Effect of (A) MTX treatment (B) vitamin B₁₂ concentration and (C) interaction between treatments on proliferation of DAOY, SH-SY5Y and MO3.13 cells 4 days after seeding.

Cells were seeded in T25 cm² flasks at the following starting densities (total cell number): MO3.13 and DAOY; 1 × 10⁵ - SH-SY5Y - 2 × 10⁵. Data were analyzed using univariate ANOVA, n = 9 per treatment group for main effects (i.e., MTX and vitamin B₁₂ treatments) and n = 3 for interaction, error bars represent 95% CIs. P-values relate to effect of treatment on methylation; where groups do not share the same letter post hoc tests revealed significant differences (p > 0.05). MTX: Methotrexate.

Figure 4.

Effect of MTX treatment on (A) LINE1 (B) HOXA4 and (C) FKBP5 DNA methylation in DAOY, SH-SY5Y and MO3.13 cells. Data were analyzed using univariate ANOVA, n = 9 per treatment group*, error bars represent 95% CIs. P-values relate to effect of treatment on methylation; where groups do not share the same letter post hoc tests revealed significant differences (p > 0.05). (*with the exception of HOXA4 methylation in MO3.13 cells where n = 7, 9 and 8 respectively for 0, 1 and 5 μM MTX groups). MTX: Methotrexate.

Figure 5.. Mean LINE1 methylation of DAOY treated with various concentrations of methotrexate and vitamin B₁₂.

n = 3 per treatment group. Data were analyzed using univariate ANOVA, p = 0.038 for interaction between MTX and vitamin B₁₂ treatments. Error bars represent 95% CIs. MTX: Methotrexate.