Karyotype and DNA-Methylation Responses in Myelodysplastic Syndromes following Treatment with Traditional Chinese Formula Containing Arsenic

We have previously shown that arsenic-containing Chinese herbal formula, Qing-Huang powder capsule (containing tetraarsenic tetrasulfide, As4S4), is effective in the treatment of myelodysplastic syndrome (MDS); yet the underlined mechanisms remain unclear. In this study, using standard cytogenetic analysis (G-banded) and global DNA methylation method (ChIP-on-chip assays), we aimed to determine the effect of arsenic-containing Chinese herbal formula on karyotype status and the genomic methylation level in primarily diagnosed MDS patients. Correlation of aberrant DNA methylation and chromosome aberrations in MDS was also investigated. We found that the number of genes with aberrant DNA methylation was highest in MDS patients with normal karyotype, followed by trisomy 8 karyotype, and relatively low in patients with cytogenetic abnormalities other than trisomy 8. Treatment with arsenic-containing Chinese herbal formula had no effects on karyotype status, but resulted in a significant genome-wide demethylation. Our research uncovered a DNA demethylating activity of arsenic-containing Chinese herbal formula in the treatment of MDS.


Introduction
Myelodysplastic syndromes (MDS) are characterized by functional abnormalities of hematopoietic lineages.Due to ineffective hematopoiesis, patients with MDS present with cytopenia(s) associated with morphological dysplasia and/or increase in number of blasts, and can progress to acute myeloid leukemia.The laboratory diagnostic strategy in MDS has evolved significantly over the years.Currently the integrated cytogenetic/genetic approach is critical for diagnosis and prognosis of MDS.DNA methylation is a biochemical process that is important for normal development in higher organisms.It involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring carried out by DNA methyltransferases.It has been shown that MDS patients, especially high-risk MDS, have abnormal hypermethylation of tumor suppressor genes [1].
Our previous works indicated that Chinese herbal formula Qing-Huang powder (containing Realgar As 4 S 4 ) is effective in the treatment of myelodysplastic syndrome (MDS) [2][3][4].In the year 2011, we reported that of 124 cases of MDS patients treated with arsenic-containing herbal formula, complete remission and partial remission rate was achieved in 29 (23.4%)patients; hematologic improvement was achieved in 61 (49.2%) patients.The total efficacy rate was 80.6%.The clinical outcome after treatment was closely correlated with WHO Classification of myelodysplastic syndromes, MDS-risk scores, "International Prognostic Scoring System" (IPSS), and cyogenetic abnormality.However the underlined mechanisms remain unclear.To address the value of traditional medicine, efforts are required to delineate how the clinically effective formulae work at the molecular, cellular, and organism levels.
In this paper, using standard cytogenetic analysis (Gbanded) and global DNA methylation method (ChIP-onchip assays), we aimed to determine the effect of arseniccontaining Chinese herbal formula on karyotype status, the genomic methylation level as well as the correlation between aberrant DNA methylation and chromosome aberrations.

Patients and Treatment
Regimen.Twenty-five patients enrolled in this study were from outpatient clinics between 2009 and 2011 after giving informed consent.All patients were diagnosed according to WHO classification system (2008) and international prognostic scoring system (IPSS) [5].The inclusion criteria: measurements of clinical response were defined according to standardized response criteria for myelodysplastic syndromes established by the International Working Group [6,7]; patient had to be 18 to 75 years old; patients must not have received another investigational or approved therapy for MDS within 4 weeks of study enrollment; patients had no history of severe dysfunction of heart, liver, or kidney; all patients provided informed consent before this study.The exclusion criteria: patients had severe adverse effects during treatment; patients were allergic to tested drugs; patients were pregnant, breast-feeding, or had a mental disorder during treatment.

Detection of Blood Arsenic Concentration.
After treatment with Qing-Huang powder capsule, arsenic level in peripheral blood was detected by the coupling of high performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometry (ICP-MS) with the lowest detection limitation of 0.03 ug/L.

Cytogenetic Analysis.
Cytogenetic analysis was performed on marrow aspirates according to the standard methods.Chromosome preparations were G-banded using trypsin and Giemsa, and karyotypes were described according to International System for Human Cytogenetic Nomeclature (ICSN) [8].Major aberrant clone was confirmed by fluorescence in situ hybridization (FISH), probes including CEP8, D5S23-5q31, D7S522-CEP7, and D20S108.

DNA Methylation and Data Analysis.
Methylation detection was performed using Affymetrix USA GeneChip Human Promoter 1.0 Array according to the manufacturer's protocol (detailed protocol can be found at http://www .affymetrix.com/).Briefly, bone marrow mononuclear cells from MDS patients were collected before and after treatment.Methylated fraction of DNA was obtained using EpiQuik Methylated DNA Immunoprecipitation Kit (Promega).DNA target was PCR-amplified, fragmented, and terminally labeled.After hybridization, washing, and staining, the chip was scanned following the instructions.Conditions of chips before and after standardization were shown in supplemental Figures 1 and 2 (see Supplementary Material available online at doi:10.1155/2012/969476).
DNA methylation detection was achieved in all 25 patients before treatment, in 14 patients after treatment.DNA methylation information from 5 normal cases traced from NCBI public data Bank served as control <http:// www.ncbi.nlm.nih.gov/>.Patient characteristics are listed in Table 1.

Data Analysis and Statistics.
All data were analyzed using two tools-Gene Ontology (GO) analysis and Pathway analysis-from Gene Ontology database and the KEGG database, respectively.
Gene Ontology online database was established by the Gene Ontology Consortium in 2000.The Gene Ontology project is a major bioinformatics initiative with the aim of standardizing the representation of gene and gene product attributes across species and databases.The database can be used to search or predict disease-related aberrantly expressed gene/function [9,10].The KEGG database has been in development by Kanehisa Laboratories since 1995.KEGG is a collection of online databases dealing with genomes, enzymatic pathways, and biological chemicals.KEGG connects known information on molecular interaction networks, such as pathways and complexes (this is the Pathway Database), information about genes and proteins generated by genome projects (including the gene database), and information about biochemical compounds and reactions (including compound and reaction databases).The higher order functional information is stored in the Pathway Database, which contains graphical representations of cellular processes, such as metabolism, membrane transport, signal transduction, and cell cycle [11][12][13][14][15].
GO analysis and Pathway analysis are two major tools based on the above two bioinformatics databases and are frequently used to analyze gene and gene function via gene Chip assay.GO analysis is more specific about single gene function, while Pathway analysis is more about networks of gene interactions in the cells, and integrating the two tools will yield better results on gene function analysis.
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) < 0.05 as filtering cut-off point.

Karyotype and Gene
Methylation.Among 25 MDS patients, normal karyotypes were observed in 12 cases, trisomy +8 was observed in 6 cases.The remaining 7 patients were cases of other type of aberrant karyotype, including 2 cases of −7, one case of 4p-, +9, dup(1q), −X/Xq-, and complex karyotype, respectively (Table 1).Compared to healthy control, as analyzed by GO analysis, the number of aberrant DNA methylation is higher in patients with normal karyotype (1656 counts) and trisomy 8 karyotype (880 counts).DNA methylation is relative low in patients with cytogenetic abnormalities other than trisomy 8 (39 counts) (Figure 1(a)).The same DNA hypermethylation pattern is observed analyzed by Pathway analysis (Figure 1(b)).

Karyotype Status after
Treatment.Certain amount of blood arsenic was observed in most patients (7.6 ug/L to 35.4 ug/L) after treatment.Hematologic responses were acquired after treatment with arsenic-containing Chinese herbal formula in 14 patients that have completed treatment for 6 months, including 2 CR and 12 hematologic improvements.Of these 14 patients, no karyotype change was observed after treatment in most cases, with the exception of one case, whose karyotype changed from 46, XX [20], trisomy +8 signal < 1.0% (by FISH) before treatment, to 47, XX, +8 [6], -X[2]/46, XX [23], trisomy +8 signal > 40% (by FISH) after treatment.We concluded that arsenic-containing Chinese herbal formula had no obvious effects on karyotype status.

Demethylation Effects of Arsenic-Containing Chinese
Herbal Formula.Six months of treatment with arseniccontaining Chinese herbal formula significantly decreased number of methylated genes and their functional signaling pathways in MDS patients.
According to GO analysis tool, the number of hypermethylated gene decreased from 1063 to 75 after treatment.The functional pathways that involved gene hypermethylated decreased from 156 to 18 after treatment (Table 4).Further analysis indicated that the methylated genes involved in multicellular organismal development, signal transduction and apoptosis were demethylated after treatment.
Analyzed by Pathway analysis tool, the number of hypermethylated gene decreased from 318 to 21 after treatment.The involved functional pathways of the hypermethylated genes decreased from 47 to 8 (Table 5).The methylated genes involved in cancer signals, chemokine signaling pathway, MAPK signaling pathway, calcium signaling, and so forth, were demethylated after treatment.
Overall, our results indicated that after treatment with arsenic-containing Chinese herbal formula, the numbers of hypermethylated genes and the involved functional pathways were significantly decreased.The methylated genes involved

Discussion
Aberrant DNA methylation is frequently observed in the myeloid malignancies, including myelodysplastic syndrome (MDS).With most researches focusing on single gene methylation [17][18][19][20][21][22][23], the limitations of these researches are obvious regarding global DNA methylation in MDS.In this study, using global DNA methylation method (ChIP-on-chip assays), we investigated genomic wide DNA methylation level in MDS thus reveal overall methylation profiling in MDS.
After analyzing the correlation of DNA methylation status and karyotypes, we found that the number of genes with aberrant methylation is the highest in MDS patients with normal karyotype, higher in trisomy 8 karyotype, and relative low in patients with cytogenetic abnormalities other than trisomy 8 karyotype.Interestingly, we found that after treatment with arsenic-containing Chinese herbal formula, clinical outcome of MDS patients with normal karyotype or trisomy 8 is better than that of the patients with other cytogenetic abnormalities [4].However arsenic-containing Chinese herbal formula had no effects on karyotype status of the patients.
Furthermore, after treatment with arsenic-containing Chinese herbal formula, the number of hypermethylated genes decreased from 1063 to 75; the functional pathways affected by these hypermethylated genes decreased from 156 to 18.The methylated genes involved in multicellular organismal development, signal transduction, and apoptosis were demethylated after treatment.We identified 13 genes, whose function involving in multicellular organismal development, transcription control, signal transduction, apoptosis, cell development, and adhesion, as well as pathways in cancer, had been completely demethylated after treatment.The identified 13 demethylated genes including WNT8B, DCC, DACT1, E2F3, NCOA4, HDAC1, PIAS3, ATF3, ZBTB33, LHX9, FASLG, TRAF3, and PIK3R2), are critical for pathogenesis of MDS.Our research uncovered a novel DNA demethylating activity of Realgar-containing Chinese herbal formula in the treatment of MDS.
Currently DNA hypomethylating agents like the cytidine nucleoside analogs azacitidine and decitabine are widely used for the treatment of patients with myelodysplastic syndromes.Both drugs effected DNA methyltransferase-1 depletion and DNA hypomethylation [24][25][26][27].However based on detoxification process of arsenic, we reason that arsenic hypomethylates DNA in a different pathway from azacitidine.Methylation of arsenic had been recognized as a detoxification process through methyltransferase enzymes using S-adenosylmethionine as methyl donor.Therefore the observed hypomethylating effects of arsenic in this study might be due to its competitive exhaustion of methyl during detoxification process of arsenic.Details regarding this mechanism are currently under investigation in the lab.
In summary, in this study we found that aberrant DNA methylation correlates with MDS pathogenesis; the number of aberrant DNA methylation is highest in patients with normal karyotype, followed by trisomy 8 karyotype and relative low in patients with other cytogenetic abnormalities; treatment with arsenic-containing Chinese herbal formula resulted in a significant genome-wide demethylation, with no effects on karyotype status; the function of the demethylated genes is largely involved in transcription control, signal transduction, cell apoptosis, and multicellular organismal development.Our research uncovered a novel DNA demethylating activity of arsenic-containing Chinese herbal formula in the treatment of MDS.

4 Evidence-
Based Complementary and Alternative Medicine

Table 2 :
Functions of genes with hypermethylation.
This table shows parts of 156 category functions of genes with hypermethylation in 25 MDS patients.go id: gene ID number in Gene Ontology database; go name: category of function of gene; diffgene count: number of gene methylated; P value: P < 0.01 indicating methylation is significant; FDR: false discovery rate, P < 0.05 as filter cutoff rate.

Table 3 :
Pathways of hypermethylated genes.This table shows parts of 46 pathways of genes with hypermethylation in 25 MDS patients.Path id: gene ID number in KEGG database; Path name: pathway of gene; diffgene count: number of gene methylated in specific pathway; P value: P < 0.01 indicating methylation is significant; FDR: false discovery rate, P < 0.05 as filter cutoff rate.

Table 4 :
Functions of genes with hypermethylation after treatment.This table shows functions of genes with hypermethylation after treatment.go id: gene ID number in Gene Ontology database; go name: category of function of gene; diffgene count: number of gene methylated; P value: P < 0.01 indicating methylation is significant; FDR: false discovery rate, P < 0.05 as filter cutoff rate.

Table 5 :
Pathways of hypermethylated genes after treatment.This table shows pathways of hypermethylated genes after treatment.Path id: gene ID number in KEGG database; Path name: pathway of gene; diffgene count: number of gene methylated in specific pathway; P value: P < 0.01 indicating methylation is significant; FDR: false discovery rate, P < 0.05 as filter cutoff rate.