Dna-Methylation Responses In Myelodysplastic Syndromes Following Treatment With Traditional Chinese Formula Containing Arsenic

Part1DNA-Methylation Responses in Myelodysplastic Syndromes following Treatment with Traditional Chinese Formula Containing ArsenicObjectives:In order to understand the importance of aberrant methylation in the pathology of MDS, we check the methylation status in MDS patients; Using global DNAmethylation method (ChIP-on-chip assays), we aimed to determine the effect of arsenic-containing Chinese herbal formula on the genomic methylation changes in primarily diagnosed MDS patients.Methods:Methylation detection was performed using Affymetrix USA GeneChip Human Promoter1.0Array according to the manufacturer’s protocol. DNA methylation detection was achieved in all28healthy patients and15MDS patients before and after treatment. DNA methylation information from28healthy patients served as control. All data were analyzed using two tools桮ene Ontology (GO) analysis and Pathway analysis-from Gene Ontology database and the KEGG database, respectively. In this study, we used both GO analysis (based on Gene Ontology database) and Pathway analysis (based on KEGG database) to map the corresponding genomic position and function. Statistical significance of the difference between values of methylation levels for different samples/groups were assessed using Fisher and multiple comparison test with P<0.05, false discovery rate (FDR)<0.05as filtering cut-off point.Results:1The aberrant hypomethylation and hypermethylation in MDS patients7724hypermethylated genes and32hypomethylated genes were identified in15MDS patients compared with normal control. Gene methylation differences above cut-off of a1.4-fold change were analyed by Go analysis and Pathway analysis. Based on Go analysis,554hypermethylated genes involve237functional pathways, including apoptosis, cell cycle, Wnt signaling pathway, toll-like receptor signaling pathway, BMP signaling pathway and so forth.25hypomethylated genes were observed and these genes involve75functional pathways, including positive regulation of cell proliferation, Wnt receptor signaling pathway, positive regulation of B cell differentiation and so forth. Using Pathway analysis, we observed that185hypermethylated genes were identified in15MDS patients compared with control. The functions of these genes involve35different pathways, including pathways in cancer, Wnt signaling pathway, MAPK signaling pathway, PI3K-Akt signaling pathway and so forth. There were two hypomethylated genes which take part in one functional pathway.2. Arsenic-Containing Chinese Herbal Formula induce double effect on DNA methylation in MDS patients2.1the methylation changes of15MDS patients after the treatment Compared with the MDS patients before treatment, Six months of treatment with arsenic containing Chinese herbal formula significantly decreased the methylation level of11311genes and increased methylation level of57genes. Gene methylation differences above cut-off of a1.5-fold change were analyed by Go analysis and Pathway analysis. Based on Go analysis,1895hypomethylated genes involve640functional pathways, including apoptosis, cell cycle, Wnt signaling pathway, toll-like receptor signaling pathway, Notch signaling pathway and so forth.13hypermethylated genes were observed and these genes involve49functional pathways, including regulation of small GTPase mediated signal transduction, UTP biosynthetic process, CTP biosynthetic process and so on. Using Pathway analysis, we observed that861hypomethylated genes take part in125functional pathways that involved pathways in cancer, Wnt signaling pathway, MAPK signaling pathway, NF-KB signaling pathway, PI3K-Akt signaling pathway and so forth. There were2hypermethylated genes which take part in2functional pathway:Pyrimidine metabolism and Purine metabolism.2.2the double effect of arsenic containing Chinese herbal formula on methylation in MDS patients compared with the control After treatment with arsenic-containing Chinese herbal formula, the number of hypermethylated genes decreased from7724to3467and hypomethylated genes increased from32to107. Gene methylation differences above cut-off of a1.4-fold change were analyed by Go analysis and Pathway analysis. Based on Go analysis, hypermethylated genes decreased from579to117and the hypomethylated genes disappeared. The disappeared genes involve apoptosis, cell proliferation, Wnt signaling pathway, toll-like receptor signaling pathway, BMP signaling pathway and so forth. Through Pathway analysis, the number of hypermethylated genes decreased from187to30and2hypomethylated genes vanished. The disappeared genes involve pathways in cancer, Wnt signaling pathway, MAPK signaling pathway, PI3K-Akt signaling pathway and so forth.Conclusions:In summary, in this study we found that aberrant DNA methylation correlates with MDS pathogenesis and these aberrant methylation genes include many hypermethylated genes and some hypomethylated genes. Based on Go and Pathway analysis, the functions of these aberrant hypermethylated genes or hypomethylated genes involve many cancer pathways. Treatment with arsenic-containing Chinese herbal formula induced a double effect on DNA methylation:hypermethylation and hypomethylation, which may be the main action mechanism in treatment of MDS. Our research uncovered a novel DNA methylation activity of arsenic-containing Chinese herbal formula in the treatment of MDS. Part2Arsenic sulfide (As2S2) induces DNA hypomethylation and hypermethylation in MDS derived cell line F-36P:implications for arsenic sulfide as DNA methylation-targeted therapyObjectives:To investigate the effects of Arsenical sulfide (AS2S2) on DNA methylation using F-36P and explore the change of DNA methylation after the treatment treatment of realgar; Dectect the change of DNA Methyltransferase (Tl,3a,3b) expression changes after the treatment of realgar and discusse the mechanism of realgar on the effect of methylation. Methods:Global DNA methylation was detected with the Methylamp Global DNA Methylation Quantification Ultra Kit (Epigentek, NY) according to the manufacturer’s instructions. The450K DNA methylation array by Illumina is an established, highly reproducible method for DNA methylation detection and has been validated in two independent laboratories. Three DNA samples from F-36P cell line treated by different concentrations of As2S2were analyzed with the Illumina Human Methylation450K array after the integrity of DNA was analyzed in a1.0%agarose gel according to the manufacturer’s protocol. To further analyze the effects of As2S2on DNMTs and whether methylation status influences the gene expression of specific genes,5genes were selected for RQ-PCR.Results:1Treatment with As2S2decreased the levels of global methylation in F-36P, HL-60After72hours of exposure to As2S2at the concentrations of1umol and2umol, the degree of global methylation in F-36P declined. For HL-60, the decline of the whole methylation was observed after the treatment of As2S2at2umol and10umol.2As2S2induced a double effect on DNA methylation in F-36P cell line Of the475,376CpGs studied, treatment with As2S2at2umol induced significant DNA methylation differences comparing with Control group. The methylation levels of19,832CpGs(4.17%of the CpGs) changed significantly after the treatment of2umol As2S2. Among the19,832CpGs, there was37.37%(7,412CpGs) of the observed DNA methylation changes which corresponded to a CpG hypermethylation event and62.63%(12,420CpGs) of the DNA methylation changes corresponded to a CpG hypomethylation event in F-36P cell line treated with2umol As2S2. Treatment with As2S2at4umol caused smilar situation in F-36P cell line (8,704hypermethylated CpG sites and13,033hypomethylated CpG sites). No special association was found between the DNA methylation events and the type of related transcript:both hyper-and hypo-methylation changes occured in mRNA, ncRNA and non-RNA-associated CpG sites. The changes of gene-specific methylation at the promoter region were examined. Gene-specific methylation levels at promoter region increased or decreased in F-36P cells after incubation with As2S2. The event also exsited in CpG islands. 3The influence of AS2S2on the mRNA expression of hypomethylated and hypermethylated genes in F-36P cell line BAK1(hypomethylated gene on the promoter region) and FOLH1(hypermethylated gene on the promoter region) were chosen for RT-PCR according to the results from Humanmethylation450K. Treatment with4umol AS2S2for72hours resulted in a significant increase in BAK1mRNA expression compared with control group, but2umol AS2S2treatment for72hours did not increase the expression; For the hypermethylated gene FOLHl,2umol AS2S2signicantly reduced the expression and the decrease of FOLH1expression in F-36P exposed to AS2S2at4umol was also observed but was not statistically significant.4Treatment with As2S2generated differential mRNA expression of DNA Methyltransferases(DNMTs)1,3a and3b in F-36P, HL-60and K562cell lines no statistically siganificant changes of DNMTl mRNA expression were found in F-36P, HL-60and K562cell lines after the treatment of AS2S2; For DNMT3a, the mRNA expression siganificantly increased in F-36P, HL-60and K562cell lines after exposure to AS2S2for72hours; Treatment with AS2S2had no effect on mRNA expression of DNMT3b in F-36P and K562cell lines, however,10umol AS2S2treatment increased level of DNMT3b mRNA expression in HL-60cell line.Conclusions:AS2S2has double effects on DNA methylation in F-36P cell lines mainly through increasing the expression of DNMT3a. Further more, AS2S2treatment can demethylate the tumor suppressor genes and methylate the oncogenes, which may be the main action mechanism in treatment of malignant diseases.