Myelodysplastic Syndrome Cell Line SKM-1 Research Articles

Whole-Genome DNA Methylation Sequencing Reveals Epigenetic Changes in Myelodysplastic Syndromes.

Epigenetic dysregulation of cancer-associated genes has been identified to contribute to the pathogenesis of myelodysplastic syndromes (MDS). However, few studies have elucidated the whole-genome DNA methylation in the initiation pathogenesis of MDS. Reduced representation bisulfite sequencing was performed in five de novo MDS patients and four controls to investigate epigenetic alterations in MDS pathogenesis. The mean global methylation in five MDS patients showed no significant difference compared with the four controls. In depth, a total of 1,459 differentially methylated fragments, including 759 hypermethylated and 700 hypomethylated fragments, were identified between MDS patients and controls. Targeted bisulfite sequencing further identified that hypermethylation of DLEU7, FOXR1, LEP, and PANX2 were frequent events in an additional cohort of MDS patients. Subsequently, LEP hypermethylation was confirmed by real-time quantitative methylation-specific PCR in an expanded cohort of larger MDS patients. In clinics, LEP hypermethylation tended to be associated with lower bone marrow blasts and was significantly correlated with U2AF1 mutation. Survival analysis indicated that LEP hypermethylation was associated with a markedly longer survival time but was not an independent prognostic biomarker in MDS patients. Functional studies revealed pro-proliferative and anti-apoptotic effects of leptin in the MDS cell line SKM-1, and it was significantly associated with cell growth and death as well as the Toll-like receptor and NF-kappa B signaling pathways. Collectively, our findings demonstrated that whole-genome DNA methylation analysis identified novel epigenetic alterations such as DLEU7, FOXR1, LEP, and PANX2 methylations as frequent events in MDS. Moreover, LEP might play a role in MDS pathogenesis, and LEP hypermethylation was associated with longer survival but not as an independent prognostic biomarker in MDS.

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

BackgroundPrevious 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 MethodsTen 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.ResultsFive 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.ConclusionThese data show a potential clinical application of As2S2 as an innovative hypermethylation agent in MDS.

High expression of PIM2 induces HSC proliferation in myelodysplastic syndromes via the IDH1/HIF1-α signaling pathway.

PIM2 proto-oncogene, serine/threonine kinase (PIM2) is a serine/threonine protein kinase that is upregulated in different types of cancer and serves essential roles in the regulation of signal transduction cascades, which promote cell survival and cell proliferation. The present study demonstrated that PIM2 was highly expressed in CD34+ cells derived from the bone marrow of patients with myelodysplastic syndromes (MDS)/acute myeloid leukemia. The mRNA expression level of PIM2 was quantified in MDS cell lines and mRNA expression was significantly decreased compared with that in KG-1 cells. In vitro, downregulation of PIM2 by short interfering RNA (siRNA) inhibited cell proliferation and delayed G0/G1 cell cycle progression in the MDS cell line SKM-1. Western blotting revealed that cyclin dependent kinase 2 was markedly downregulated and cyclin dependent kinase inhibitor 1A was markedly upregulated following transfection with PIM2 siRNA. Cell Counting Kit-8 analysis demonstrated that cell proliferation of si-PIM2-transfected cells was significantly decreased compared with control cells. Reverse-transcription quantitative polymerase chain reaction and western blotting revealed that PIM2 expression was negatively correlated with isocitrate dehydrogenase [NADP(+)]1 cytosolic (IDH1) and positively correlated with hypoxia inducible factor 1 subunit α (HIF1A) in CD34+ MDS cells. Collectively, these results suggested that the expression of PIM2 induced increased expression of HIF1A by decreasing the expression of IDH1, resulting in increased CD34+ cell proliferation. Therefore, PIM2 may be a potential biomarker for the diagnosis of MDS and AML or a target for novel therapeutic agents.

A novel all-trans retinoic acid derivative inhibits proliferation and induces apoptosis of myelodysplastic syndromes cell line SKM-1 cells via up-regulating p53

Myelodysplastic syndromes (MDS) are a varied set of hematologic neoplasms and a high risk of progression to acute myeloid leukemia (AML). 4-Amino-2-trifluoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative, play an important role in various types of cancer cells as a tumor inhibitor. However, little is known concerning its antitumor effect on MDS. The cell viability and the percentage of apoptotic cells were used to measure MTT, Flow Cytometry and Hoechst 33342/PI staining. In addition, real-time quantitative RT-PCR (qRT-PCR) and western blotting were used to analyzed the expression of p53, as well as the levels of BNIP3, apoptosis proteins of Caspase-3, BAX and BCL-2. After SKM-1 cells were incubated with DAC, ATRA and ATPR, the viability of the SKM-1 cells was inhibited in a dose- and time-dependent manner. Both Hoechst staining and flow cytometry showed the apoptosis of SKM-1 cells was increased. Moreover, SKM-1 cells treated with ATPR unveiled elevated mRNA and protein levels of p53, BNIP3, BAX and Caspase-3 expression and decreased BCL-2 expression. However, silencing p53 suppressed the pro-apoptosis function of ATPR. Consequently, these data provide the first evidence for ATPR increased apoptosis in SKM-1 cells by p53 that is mutually dependent on and obligatorily linked to BNIP3 gene activation.

Pyrosequencing quantified methylation level of miR-124 predicts shorter survival for patients with myelodysplastic syndrome

BackgroundAberrant CpG island methylation has been increasingly recognized as a common event in myelodysplastic syndrome (MDS). To date, most of the previous studies of miR-124 in MDS have focused on epigenetic changes and little is known about the underlying mechanism through which miR-124 regulates CDK6 expression.ResultsIn the present study, we employed pyrosequencing analysis to quantify the methylation levels of upstream regions of the miR-124 genes (miR-124-1, miR-124-2 and miR-124-3) in 56 primary MDS patients. We found the three miR-124 genes were methylated in MDS patients. Univariate analysis revealed that the World Health Organization (WHO) classification, marrow blast count, karyotype, International Prognostic Scoring System (IPSS), mean corpuscular volume, as well as high methylation of miR-124-1, miR-124-2 and miR-124-3 were significantly related to overall survival. In leukaemia-free survival, patients who were older and had an advanced WHO classification, high marrow blast counts, high IPSS risk and high methylation of miR-124-1 and miR-124-2 progressed rapidly to acute myeloid leukaemia. Multivariate analysis demonstrated that high methylation of miR-124-3 was an independent factor of overall survival. Median survival of patients with high miR-124-3 methylation was significantly shorter (7.6 months) than patients with low methylation (32.7 months; P = 0.010). A functional study revealed that silencing of miR-124 resulted in upregulation of its target gene, cyclin dependent kinase CDK6, which in turn promoted cell proliferation in the MDS cell line SKM-1.ConclusionsHigh methylation of miR-124-3 predicts shorter survival for patients with MDS, which may be a useful prognostic marker in MDS.

Decitabine-Induced Changes in Human Myelodysplastic Syndrome Cell Line SKM-1 Are Mediated by FOXO3A Activation.

The epigenetic silencing of tumor suppressor genes in myelodysplastic syndromes (MDS) can potentially confer a growth advantage to individual cellular clones. Currently, the recommended treatment for patients with high-risk MDS is the methylation agent decitabine (DAC), a drug that can induce the reexpression of silenced tumor suppressor genes. We investigated the effects of DAC treatment on the myeloid MDS cell line SKM-1 and investigated the role of FOXO3A, a potentially tumor-suppressive transcription factor, by silencing its expression prior to DAC treatment. We found that FOXO3A exists in an inactive, hyperphosphorylated form in SKM-1 cells, but that DAC both induces FOXO3A expression and reactivates the protein by reducing its phosphorylation level. Furthermore, we show that this FOXO3A activation is responsible for the DAC-induced differentiation of SKM-1 cells into monocytes, as well as for SKM-1 cell cycle arrest, apoptosis, and autophagy. Collectively, these results suggest that FOXO3A reactivation may contribute to the therapeutic effects of DAC in MDS.

Dysregulation of ANKRD11 Influenced Hematopoisis By Histone Acetylation-Mediated Gene Expression in Myelodysplastic Syndrome

Background and Object In addition to histone deacetylation, the importance of histone over-acetylation induced oncogene transcription in initiation and progression of myelodysplastic syndrome (MDS) has been proposed recently. Our previous whole-exome sequencing identified a new somatic mutation, ANKRD11, an important factor in histone acetylation regulation. Its roles in MDS pathophysiology need to be clarified.Methods The next generation target sequencing (Including ANKRD11) was carried out in 320 patients with MDS using the MiSeq Benchtop Sequencer. ANKRD11 mRNA expression in bone marrow of MDS was measured by real-time PCR. Loss and gain of function assay were carried out in myeloid cell lines K562, MEG-01£¬or SKM-1 to observe the influence on cell proliferation and differentiation . The levels of histone acetylation at H3 and H4 were detected by Western blot.Results Target sequencing in a cohort of 320 MDS patients identified 14 of ANKRD11 mutations (4.38%, Fig.1), which were confirmed by Sanger sequencing. Meanwhile, no ANKRD11 mutations in 100 normal controls were defined. ANKRD11 mutations occurred frequently in exons 10 and 9. The mRNA expression levels of ANKRD11 were significantly decreased in MDS patients, especially in ANKRD11mutant patients (Fig.2). ANKRD11 knockdown in K562 and MEG-1 resulted in growth inhibition, cell cycle arrest and erythroid/megakaryocytic differentiation retardant. In MDS cell line SKM-1, the arrested differentiation was rescued by over-expression of ANKRD11. Consistent with a role for ANKRD11 in histone acetylation, ANKRD11 KD increased acetylation of histones H3 and H4 at H3K14 and H4K5 and resulted in the upregulation of genes involved in differentiation inhibilation (SOX6, P21, et al). Finally, the ANKRD11 KD-mediated influence on cell proliferation and differentiation were reversed by inhibiting histone acetyltransferase activity.Conclusion Our assay defined that ANKRD11 was a crucial chromatin regulator that suppress histone acetylation and then decrease gene expression during myeloid differentiation, providing a likely explanation for its role in MDS pathogenesis. This study further support histone acetylase inhibitor as a potential treatment in MDS. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Synergistic Effects of Decitabine Combined with Arsenic Trioxide (ATO) in the Human Myelodysplastic Syndrome Cell Line SKM-1.

Despite recent advances in the treatment of myelodysplastic syndrome (MDS), single-agent clinical effects remain unsatisfactory, and decitabine monotherapy is also associated with a relatively low rate of complete remission. To study the combined effects and mechanism of decitabine (DAC) and arsenic trioxide (ATO) on the human myelodysplastic cell line SKM-1,we used the MTS assay and CalcuSyn software to determine the cytotoxicity and potential synergistic effects, respectively. Furthermore, we determined apoptosis and measured the mRNA expression level of two genes that are considered main regulators of the apoptosis process. The results showed that DAC and/or ATO can inhibit the proliferation of SKM-1 cells and demonstrated significant synergy between the two agents (CI<1). Additionally, combination of 2.5μmol/L DAC and 5μmol/L ATO led to a significantly higher apoptosis rate and more significantly decreased the Bcl2/Bax ratio than either compound alone (P<0.001). Based on the observations of this study, we suggest that combined administration of these two drugs might be considered a novel therapeutic regimen for treating MDS.

Chidamide, a Novel Histone Deacetylase Inhibitor, Displays Potent Antitumor Activity Against MDS Cells Mainly through JAK2/STAT3 Signaling Inhibition

Background:Many studies have indicated that histone deacetylases (HDAC) activity is always increased in a lot of human tumors, and inhibition of HDAC activity are promising new strategies in the treatment of cancers. Chidamide (CS055/HBI-8000), a novel histone deacetylases inhibitor (HDACi) of the benzamide class, is currently under clinical trials. Despite emerging information on the effect of chidamide in multiple cancers, little is yet known about mechanism of action, and there were few reports about this drug's effects on myelodysplastic syndromes (MDS). In this study, we aimed to investigate the antitumor activities of chidamide on MDS cell line SKM-1 and to explore the possible mechanism.Methods:We treated MDS cells with different concentrations of chidamide. The effect of chidamide on HDAC activity of MDS cells was studied by using a HDAC Fluorometric Activity Assay Kit. The induction of histone acetylation was confirmed by detecting acetylated histone H3 and H4 using Western blot analysis. The effect of chidamide on the proliferation of MDS cells was analyzed by Cell Counting Kit (CCK-8) assay. Apoptosis were detected by Annexin V/propidium iodide (PI) double-labeled cytometry. Cell cycle was analyzed by a PI method. The acetylation levels of genes promoterassociated histone H3 and H4 were examined by chromatin immunoprecipitation (ChIP) and quantitative real-time PCR. Quantitativereal-time PCR and Western blot were used to detect the expression of signaling pathway factors (SOCS3, JAK2, STAT3,Bcl-XL,Bcl-2 and Mcl-1).Results:Our results demonstrated chidamide suppressed MDS cells growth in a time- and dose-dependent manner, and induced cell apoptosis and cell cycle arrest at G0/G1phase. Chidamide was able to significantly inhibit the HDAC activity and increase the acetylated histoneH3 and H4 of MDS cells. ChIP analysis further indicated that chidamide induced acetylated histones accumulation in the promoter of SOCS3.Moreover, chidamide upregulated the mRNA and protein expression ofSOCS3, and also significantly downregulated JAK2/STAT3 signaling. Further, we identified reduced expression of STAT3 downstream targets with treatment of chidamide, including Bcl-XL, Bcl-2 and Mcl-1, which may explain the cytotoxic effects of chidamide on MDS cells.Conclusion:These results demonstrate that chidamide possesses significant cytotoxic effects on MDS cells mainly through histone modifications of SOCS3 and consequently JAK2/STAT3 signaling inhibition. Therefore, Our data provide rationale for clinical investigation of chidamide in MDS. DisclosuresNo relevant conflicts of interest to declare.

Synergistic Effect and Molecular Mechanism of Homoharringtonine and Bortezomib on SKM-1 Cell Apoptosis.

BackgroundMyelodysplastic syndromes (MDS) are clonal marrow stem-cell disorders with a high risk of progression to acute myeloid leukemia (AML). Treatment options are limited and targeted therapies are not available for MDS. In the present study, we investigated the cytotoxicity and the molecular mechanism of Homoharringtonine (HHT) and Bortezomib towards high-risk MDS cell line SKM-1 in vitro and the role of miR-3151 was first evaluated in SKM-1 cells.MethodsSKM-1 cells were treated with different concentrations of HHT or Bortezomib, and cell viability was analyzed with CCK-8 assay. The influence on cell proliferation, cell cycle distribution and the percentage of apoptosis cells were analyzed by flow cytometry. Calcusyn software was used to calculate combination index (CI) values. Western blot was used to analysis phosphorylation of Akt and nuclear NF-κB protein expression in SKM-1 cells. Mature miR-3151 level and p53 protein level were detected after HHT or Bortezomib treatment. The cell proliferation and p53 protein level were reassessed in SKM-1 cells infected with lentivirus to overexpress miR-3151.ResultsSimultaneous exposure to HHT and Bortezomib (10.4:1) resulted in a significant reduction of cell proliferation in SKM-1 cells (P < 0.05). Cell cycle arrest at G0/G1 and G2/M phase was observed (P < 0.05). HHT and Bortezomib synergistically induced cell apoptosis by regulating members of caspase 9, caspase 3 and Bcl-2 family (P < 0.01). The mechanisms of the synergy involved Akt and NF-κB signaling pathway inhibition, downregulation of mature miR-3151 and increment of downstream p53 protein level. Overexpression of miR-3151 promoted cell proliferation and inhibited p53 protein expression in SKM-1 (P < 0.01).ConclusionsHHT and Bortezomib synergistically inhibit SKM-1 cell proliferation and induce apoptosis in vitro. Inhibition of Akt and NF-κB pathway signaling contribute to molecular mechanism of HHT and Bortezomib. miR-3151 abundance is implicated in SKM-1 cell viability, cell proliferation and p53 protein expression.

Downregulation of HO-1 promoted apoptosis induced by decitabine via increasing p15INK4B promoter demethylation in myelodysplastic syndrome.

Decitabine, which reverses hypermethylation of the p15(INK4B) gene in vitro, has been used to relieve cytopenias and blast excess in over 50% of patients with high-risk myelodysplastic syndrome (MDS). In this study, heme oxygenase-1 (HO-1) was overexpressed in MDS cell line SKM-1, which was closely related to resistance to decitabine-induced apoptosis. We aimed to further investigate the role of HO-1 in apoptosis induced by low-dose decitabine in SKM-1 cells. Upregulation of HO-1 by transfecting it into SKM-1 cells with lentivirus vector promoted cell proliferation and protected them against apoptosis. In contrast, downregulation of HO-1 enhanced decitabine-induced apoptosis but reduced accumulation of the S phase in cell cycle. To explore the mechanism, the expressions of cell cycle-related proteins were detected after the cells were treated by decitabine in each group. p15(INK4B) and CDK4 were overexpressed in SKM-1 cells in which HO-1 was inhibited, and the expression-depending apoptosis was related to the caspase-3 pathway. Even though HO-1 was silenced, the apoptotic rate never increased as the caspase-3 pathway was blocked. It is well known that p15(INK4B) dominantly regulates the S phase of the cell cycle. p15(INK4B) was herein demethylated more evidently in the group of SKM-1 cells in which HO-1 was downregulated, as well as in the mononuclear cells of patients suffering from MDS. In the case of poor prognosis, the mRNA level of HO-1 was raised. In conclusion, overexpression of HO-1 indicated resistance to demethylation of p15(INK4B) induced by decitabine.

Reversal of Bortezomib Resistance in Myelodysplastic Syndrome Cells by MAPK Inhibitors

The myelodysplastic syndromes (MDS) comprise a heterogeneous group of malignant neoplasms with distinctive clinicopathological features. Currently, there is no specific approach for the treatment of MDS. Here, we report that bortezomib (BTZ), a proteasome inhibitor that has been used to treat plasma cell myeloma, induced G2/M phase cycle arrest in the MDS cell line SKM-1 through upregulation of Wee1, a negative regulator of G2/M phase transition. Treatment by BTZ led to reduced SKM-1 cell viability as well as increased apoptosis and autophagy. The BTZ-induced cell death was associated with reduced expression of p-ERK. To elucidate the implications of downregulation of p-ERK, we established the BTZ resistant cell line SKM-1R. Our data show that resistance to BTZ-induced apoptosis could be reversed by the MEK inhibitors U0126 or PD98059. Our results suggest that MAPK pathway may play an important role in mediating BTZ resistance.

Methylation of Wnt antagonist genes: a useful prognostic marker for myelodysplastic syndrome

Activation of the Wnt signaling pathway has been implicated in the pathogenesis of many tumors as well as in leukemia. However, its role in myelodysplastic syndrome (MDS) is unknown. In this study, we employed methylation-specific PCR to examine the methylation status of six Wnt antagonist genes in 144 MDS patients and in the MDS cell line SKM-1. We also used real-time PCR to examine the expression of Wnt antagonist genes and Wnt pathway genes in the SKM-1 cell line after treatment with 5-aza-2'-deoxycytidine. We found that methylation of the gene promoters of each of the six genes were observed in MDS patients at the following methylation frequencies: 41% for sFRP1, 89.6% for sFRP2, 43.1% for sFRP4, 50.7% for sFRP5, 44.4% for DKK-1, and 69.4% for DKK-3. In the SKM-1 cell line, the gene promoters sFRP1, sFRP2, sFRP5, DKK-1, and DKK-3 were methylated, while sFRP4 was not methylated. Treatment of the SKM-1 cell line with 5-aza-2'-deoxycytidine induced re-expression of methylated Wnt antagonists and inactivation of the Wnt pathway. Survival analysis showed that methylation status of sFRP1, sFRP4, and sFRP5 was associated with worse survival in MDS and sFRP5 methylation also predicted a high risk of leukemia evolution (P = 0.018). Our results indicate that epigenetic regulation of the Wnt pathway in MDS cell line, and the methylation status of Wnt antagonists predicts prognoses of MDS patients.