Arsenic Disulfide Promoted Hypomethylation by Increasing DNA Methyltransferases Expression in Myelodysplastic Syndrome

Abstract

Background: Previous studies have shown that DNA methylation plays a significant role in myelodysplastic syndrome (MDS). In addition to hypermethylation, aberrant hypomethylation can result in the transcriptional activation of oncogenes in cancer, including MDS. Therefore, drugs targeting DNA hypomethylation are needed for the treatment of MDS. This study aimed to investigate whether As₂S₂ promoted hypomethylation by increasing DNA methyltransferases (DNMTs) expression in MDS.

Patients and methods: Ten bone marrow samples from MDS patients and 3 healthy donors were obtained for the examination of the DNA methylation with a Human Methylation 850K BeadChip. The mRNA expressions for the DNMTs in the ten MDS patients and 3 controls were compared by Q-PCR. Then, the MDS cell line SKM-1 was treated with As₂S₂. After 2 days of treatment, Human Methylation 850K BeadChip was applied to analyze the changes of gene methylation status in the cells. Q-PCR and Western blot were taken to test the changes of mRNA and protein expressions for DNMTs in SKM-1 cells after treatment.

Results: Five hundred ninety-two abnormally hypomethylated genes were found in MDS patients compared to those in controls by Human Methylation 850K. The mRNA expressions of DNMTs (DNMT1, DNMT3a and DNMT3b) in MDS patients were significantly lower than those in healthy individuals. The IC50 value of As₂S₂ for SKM-1 cells was 4.97 μmol/L.Treatment with As₂S₂ at 2 μmoL/L resulted in significant alterations in the methylation levels at 1718 sites in SKM-1 cells compared to those in the controls. Hypermethylation was observed in 1625 sites (94.58%), corresponding to 975 genes, compared to those in the controls. Finally, the expression levels of DNMTs (DNMT1, DNMT3a, and DNMT3b) significantly increased in SKM-1 cells treated with As₂S₂ at 2 μmoL/L and 4 μmoL/L.

Conclusion: These data show a potential clinical application of As₂S₂ as an innovative hypermethylation agent in MDS.

Keywords: SKM-1 cell line; hypermethylation; myelodysplastic syndrome; arsenic disulfide.

Figure 1

Differential methylation study in ten MDS patients vs 3 healthy individuals from bone marrow samples. (A) Heatmap representing a supervised cluster centred on the median of the methylation levels at the 2421 CpG sites between ten MDS patients (A) vs 3 healthy individuals (N). Samples represented as A (Salmon orange) and N samples (purple). Hypermethylated CpG probes in MDS patients (orange) and hypomethylated probes (blue). (B) Volcano plot representation of methylation for significant CpG sites of genes. Hypomethylated probes are represented in green colour and hypermethylated probes are represented in red. Red lines delimit ±0.1 methylation differences between MDS patients vs healthy donors and the dotted line represents a p-value threshold of 0.05. (C) Significantly changed GOs of hypomethylated genes in MDS patients. The y axis shows category and the x axis, -LgP. The larger –LgP indicated a smaller P value.

Figure 2

The mRNA expressions for DNMTs in MDS patients were lower than those in controls. Bone marrow cells were extracted from ten untreated MDS patients and 3 healthy donors and then subjected to real-time PCR to measure the mRNA levels of DNMT1 (A), DNMT3a (B) and DNMT3b (C). The error bars indicate mean ± SEM. *, P<0.05, compared to those in healthy donors.

Figure 3

Effects of As₂S₂ on cell proliferation of SKM-1 cells. (A) Chemical structure of As₂S_2. (B) Dose–response curve for the proliferation of SKM-1 cell line after treatment with As₂S₂ for 48h. The error bars indicate mean ± SEM. Results from three independent experiments were shown.

Figure 4

Nine samples in 3 groups were checked by Human Methylation 850K. (A) Mean methylation level of cytosine in 3 groups by Human Methylation 850K: Control group contains A1, A2 and A3; B1, B2, B3 and C1, C2, C3 represents 1μmol/L -As₂S₂ treatment group and 2μmol/L -As₂S₂ treatment group, respectively. Distribution of differently methylated sites in chromosomes between 1μmol/L -As₂S₂ treatment group and control group (B) and 2μmol/L -As₂S₂ treatment group and control group (C): the red represents hypermethylated sites; the green represents hypomethylated sites.

Figure 5

As₂S₂ inhibited the mRNA expression of FGF1 and IGF1 in SKM-1 cells. SKM-1 cells were treated with A_S2S₂ (0, 1 and 2μmol/L) for 48 hours and then subjected to real-time PCR to measure the mRNA levels of FGF1 (A), and IGF1 (B). The error bars indicate mean ± SEM. Results from three independent experiments were shown. Each bar represents the mean ± SD of three independent experiments. *，P<0.05, compared with control group.

Figure 6

As₂S₂ increases the mRNA expression of DNMTs in SKM-1 cells. SKM-1 cells were treated with As₂S₂ (0, 1, 2 and 4μmol/L) for 48 hours, and then real-time PCR was used to check the mRNA levels of DNMT1 (A), DNMT3a (B) and DNMT3b (C). Results from three independent experiments were shown. Each bar represents the mean ± SD of three independent experiments. *, P<0.05, compared with control group.

Figure 7

As₂S₂ increased the protein expression of DNMTs in SKM-1 cells. (A) SKM-1 cells were treated with As₂S₂ (0, 1, 2 and 4μmol/L) for 48 hours, and Western blotting was used to check protein levels of DNMT1, DNMT3a and DNMT3b. Gray values of DNMT1 (B), DNMT3a (C) and DNMT3b (D) were showed. Results were from three independent experiments. Each bar represents the mean ± SD of three independent experiments. *, P<0.05, compared with control group.