[Chidamide Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stromal Cells from Patients with Myelodysplastic Syndromes].

OBJECTIVE: To explore the effects and mechanisms of chidamide on the osteogenic differentiation of bone marrow mesenchymal stromal cells (MSC) from myelodysplastic syndromes (MDS). METHODS: MSC were isolated and cultured from bone marrow of MDS patients and healthy donors. CCK-8 assay was used to detect the effects of chidamide on the proliferation of MSC. The effects of chidamide on the activity of histone deacetylase (HDAC) in MSC was measured by a fluorescence assay kit and Western blot. Alkaline phosphatase (ALP) activity was detected on day 3 and calcium nodule formation was observed by Alizarin Red staining on day 21 after osteogenic differentiation. The expression of early and late osteogenic genes was detected on day 7 and day 21, respectively. RT-PCR and Western blot were used to detect the effects of chidamide on mRNA and protein expression of RUNX2 which is the key transcription factor during osteogenesis. RESULTS: As the concentration of chidamide increased, the proliferation of MSC was inhibited. However, at a low concentration (1 µmol/L), chidamide had no significant inhibitory effect on MSC proliferation but significantly inhibited HDAC activity. In MSC from both MDS patients and healthy donors, chidamide (1 µmol/L) significantly increased ALP activity, calcium nodule formation, thereby mRNA expression of osteogenic genes, and restored the reduced osteogenic differentiation ability of MDS-MSC compared to normal MSC. Mechanistic studies showed that the osteogenic-promoting effect of chidamide may be related to the upregulation of RUNX2 . CONCLUSION: Chidamide can inhibit HDAC activity in MSC, upregulate the expression of the osteogenic transcription factor RUNX2, and promote the osteogenic differentiation of MDS-MSC.

ROBO1 deficiency impairs HSPC homeostasis and erythropoiesis via CDC42 and predicts poor prognosis in MDS.

Myelodysplastic syndrome (MDS) is a group of clonal hematopoietic neoplasms originating from hematopoietic stem progenitor cells (HSPCs). We previously identified frequent roundabout guidance receptor 1 (ROBO1) mutations in patients with MDS, while the exact role of ROBO1 in hematopoiesis remains poorly delineated. Here, we report that ROBO1 deficiency confers MDS-like disease with anemia and multilineage dysplasia in mice and predicts poor prognosis in patients with MDS. More specifically, Robo1 deficiency impairs HSPC homeostasis and disrupts HSPC pool, especially the reduction of megakaryocyte erythroid progenitors, which causes a blockage in the early stages of erythropoiesis in mice. Mechanistically, transcriptional profiling indicates that Cdc42, a member of the Rho-guanosine triphosphatase family, acts as a downstream target gene for Robo1 in HSPCs. Overexpression of Cdc42 partially restores the self-renewal and erythropoiesis of HSPCs in Robo1-deficient mice. Collectively, our result implicates the essential role of ROBO1 in maintaining HSPC homeostasis and erythropoiesis via CDC42.

The bcl6 corepressor mutation regulates the progression and transformation of myelodysplastic syndromes by repressing the autophagy flux.

The occurrence of autophagy dysregulation is vital in the development of myelodysplastic syndrome and its transformation to acute myeloid leukemia. However, the mechanisms are largely unknown. Here, we have investigated the mechanism of the bcl6 corepressor mutation in myelodysplastic syndrome development and its transformation to acute myeloid leukemia. We identified a novel pathway involving histone deacetylase 6 and forkhead box protein O1, which leads to autophagy defects following the bcl6 corepressor mutation. And this further causes apoptosis and cell cycle arrest. The bcl6 corepressor-mutation-repressed autophagy resulted in the accumulation of damaged mitochondria, DNA, and reactive oxygen species in myelodysplastic syndrome cells, which could then lead to genomic instability and spontaneous mutation. Our results suggest that the bcl6 corepressor inactivating mutations exert pro-carcinogenic effects through survival strike, which is only an intermediate process. These findings provide mechanistic insights into the role of the bcl6 corepressor gene in myelodysplastic syndrome.

The non-cell-autonomous function of ID1 promotes AML progression via ANGPTL7 from the microenvironment.

The bone marrow microenvironment (BMM) can regulate leukemia stem cells (LSCs) via secreted factors. Increasing evidence suggests that dissecting the mechanisms by which the BMM maintains LSCs may lead to the development of effective therapies for the eradication of leukemia. Inhibitor of DNA binding 1 (ID1), a key transcriptional regulator in LSCs, previously identified by us, controls cytokine production in the BMM, but the role of ID1 in acute myeloid leukemia (AML) BMM remains obscure. Here, we report that ID1 is highly expressed in the BMM of patients with AML, especially in BM mesenchymal stem cells, and that the high expression of ID1 in the AML BMM is induced by BMP6, secreted from AML cells. Knocking out ID1 in mesenchymal cells significantly suppresses the proliferation of cocultured AML cells. Loss of Id1 in the BMM results in impaired AML progression in AML mouse models. Mechanistically, we found that Id1 deficiency significantly reduces SP1 protein levels in mesenchymal cells cocultured with AML cells. Using ID1-interactome analysis, we found that ID1 interacts with RNF4, an E3 ubiquitin ligase, and causes a decrease in SP1 ubiquitination. Disrupting the ID1-RNF4 interaction via truncation in mesenchymal cells significantly reduces SP1 protein levels and delays AML cell proliferation. We identify that the target of Sp1, Angptl7, is the primary differentially expression protein factor in Id1-deficient BM supernatant fluid to regulate AML progression in mice. Our study highlights the critical role of ID1 in the AML BMM and aids the development of therapeutic strategies for AML.

The use of carbon monoxide breath test to detect the effect of iron overload on erythrocyte lifespan in MDS.

Objective: To investigate the effect of iron overload (IO) on red blood cell (RBC) lifespan in MDS patients with the use of carbon monoxide breath test. Methods: The red blood cell lifespan of 93 patients with myelodysplastic syndrome (MDS) and 22 healthy volunteers in the control group were measured by alveolar gas carbon monoxide (CO) assay, with the detection of liver iron concentration, iron metabolism index, erythropoietin (EPO) concentration, peripheral blood inflammatory cytokines, etc. The MDS patients were divided into the severe IO group, mild IO group and non IO group according to liver iron concentration. The effect of IO on RBC lifespan was analyzed in MDS patients. Results: The RBC lifespan of MDS patients in the severe IO group was significantly lower than that in the mild IO group (p<0.05), while the RBC life span in the mild IO group was significantly lower than that in the non IO group (p<0.05). The expression of inflammatory cytokines in the severe IO group was significantly higher than that of the mild and non IO groups. After receiving iron removal treatment(ICT), the expression of inflammatory cytokines was decreased significantly, and the RBC lifespan was significantly prolonged (p<0.05).Besides, liver iron concentration was significantly positively correlated with EPO concentration, while EPO concentration was significantly negatively correlated with RBC lifespan, especially in the MDS-RS subgroup. The RBC lifespan in the EPO>1000 group was significantly lower than that in the EPO<1000 group. Conclusion: IO can shorten RBC lifespan in MDS patients, which may be result from the increase of endogenous EPO and the over-expression of inflammatory cytokines. After ICT, the ineffective hematopoiesis caused by increased EPO may reduced and the decrease of inflammatory cytokine may significantly prolong the RBC lifespan in MDS patients.

Targeting UHRF1-SAP30-MXD4 axis for leukemia initiating cell eradication in myeloid leukemia.

Aberrant self-renewal of leukemia initiation cells (LICs) drives aggressive acute myeloid leukemia (AML). Here, we report that UHRF1, an epigenetic regulator that recruits DNMT1 to methylate DNA, is highly expressed in AML and predicts poor prognosis. UHRF1 is required for myeloid leukemogenesis by maintaining self-renewal of LICs. Mechanistically, UHRF1 directly interacts with Sin3A-associated protein 30 (SAP30) through two critical amino acids, G572 and F573 in its SRA domain, to repress gene expression. Depletion of UHRF1 or SAP30 derepresses an important target gene, MXD4, which encodes a MYC antagonist, and leads to suppression of leukemogenesis. Further knockdown of MXD4 can rescue the leukemogenesis by activating the MYC pathway. Lastly, we identified a UHRF1 inhibitor, UF146, and demonstrated its significant therapeutic efficacy in the myeloid leukemia PDX model. Taken together, our study reveals the mechanisms for altered epigenetic programs in AML and provides a promising targeted therapeutic strategy against AML.

[Research Progress in the Role of Tim3/galectin-9 in Hematological Malignancy--Review].

The incidence of hematological malignant tumor is increasing year by year, and seriously affecting the human health. In addition to the traditional radiation and chemotherapy, immunotherapy has achieved a certain effect in the treatment of blood tumor, but it is limited by exhaustion of CD8+ T cell. The exhaustion of CD8+ T cells is mainly related to the activation of some immune checkpoint inhibitors, such as (Tim3), programmed death 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), etc. Among them, Tim3 is getting more and more attention. The combination of Tim3 and its ligand galectin-9 produced a strong cellular immunosuppressive effect. Tim3 has been proved to be associated with CD8+ T cell exhaustion in many tumors. In this review, the application of Tim3/galectin-9 in hematologic tumors is briefly summarized so as to provide theoretical basis for clinical diagnosis and treatment of these diseases.

Dynamics of epigenetic regulator gene BCOR mutation and response predictive value for hypomethylating agents in patients with myelodysplastic syndrome.

BACKGROUND: BCOR (BCL6 corepressor) is an epigenetic regulator gene involved in the specification of cell differentiation and body structure development. Recurrent somatic BCOR mutations have been identified in myelodysplastic syndrome (MDS). However, the clinical impact of BCOR mutations on MDS prognosis is controversial and the response of hypomethylating agents in MDS with BCOR mutations (BCORMUT) remains unknown. RESULTS: Among 676 MDS patients, 43 patients (6.4%) harbored BCOR mutations. A higher frequency of BCOR mutations (8.7%) was investigated in patients with normal chromosome, compared to 4.2% in patients with abnormal karyotype (p = 0.040). Compared to the BCORWT patients, the BCORMUT patients showed a higher ratio of refractory anemia with excess blasts subset (p = 0.008). The most common comutations with BCOR genes were ASXL1 (p = 0.002), DNMT3A (p = 0.114) and TET2 (p = 0.148). When the hierarchy of somatic mutations was analyzed, BCOR mutations were below the known initial mutations (ASXL1 or TET2) but were above U2AF1 mutations. Transformation-free survival was significantly shorter in BCORMUT patients than that in BCORWT patients (16 vs. 35 months; p = 0.035). RNA-sequencing was performed in bone marrow mononuclear cells from BCORMUT and BCORWT patients and revealed 2030 upregulated and 772 downregulated genes. Importantly, HOXA6, HOXB7, and HOXB9 were significantly over-expressed in BCORMUT patients, compared to BCORWT patients. Eight of 14 BCORMUT patients (57.1%) achieved complete remission (CR) with decitabine treatment, which was much higher than that in BCORWT patients (28.7%, p = 0.036). Paired sequencing results (before and after decitabine) showed three of 6 CR patients lost the mutated BCOR. The median survival of CR patients with a BCORMUT was 40 months, which was significantly longer than that in patients with BCORWT (20 months, p = 0.036). Notably, prolonged survival was observed in three BCORMUT CR patients even without any subsequent therapies. CONCLUSIONS: BCOR mutations occur more frequently in CN MDS patients, predicting higher risk of leukemia transformation. BCORMUT patients showed a better response to decitabine and achieved longer post-CR survival.

Comparative Study on Iron Content Detection by Energy Spectral CT and MRI in MDS Patients.

Objective: The purpose of this study was to identify the difference between dual energy spectral computed tomography (DECT) and magnetic resonance imaging (MRI) used to detect liver/cardiac iron content in Myelodysplastic syndrome (MDS) patients with differently adjusted serum ferritin (ASF) levels. Method: Liver and cardiac iron content were detected by DECT and MRI. Patients were divided into different subgroups according to the level of ASF. The receiver operating characteristic curve (ROC) analysis was applied in each subgroup. The correlation between iron content detected by DECT/MRI and ASF was analyzed in each subgroup. Result: ROC curves showed that liver virtual iron content (LVIC) Az was significantly less than liver iron concentration (LIC) Az in the subgroup with ASF < 1,000 ng/ml. There was no significant difference between LVIC Az and LIC Az in the subgroup with 1,000 ≤ ASF < 2,500 ng/ml and 2,500 ≤ ASF < 5,000 ng/ml. LVIC Az was significantly higher than LIC Az in the subgroup with ASF <5,000 and 5,000 ≤ ASF ng/ml. In patients undergoing DECT and MRI examination on the same day, ASF was significantly correlated with LVIC, whereas no significant correlation was observed between ASF and LIC. After removing the data of ASF > 5,000 mg/L in LIC, LIC became correlated with ASF. There was no significant difference between the subgroup with 2,500 ≤ ASF < 5,000 ng/ml and 5,000 ng/ml ≤ ASF in LIC expression. Furthermore, both LIC and liver VIC had significant correlations with ASF in patients with ASF < 2,500 ng/ml, while LVIC was still correlated with ASF, LIC was not correlated with ASF in patients with 2,500 ng/ml ≤ ASF. Moreover, neither cardiac VIC nor myocardial iron content (MIC) were correlated with ASF in these subgroups. Conclusion: MRI and DECT were complementary to each other in liver iron detection. In MDS patients with high iron content, such as ASF ≥ 5,000 ng/ml, DECT was more reliable than the MRI in the assessment of iron content. But in patients with low iron content, such as ASF < 1,000 ng/ml, MRI is more reliable than DECT. Therefore, for the sake of more accurately evaluating the iron content, the appropriate detection method can be selected according to ASF.

Analysis of the influencing factors related to liver and cardiac iron overload in MDS patients detected by MRI in the real world.

OBJECTIVES: We aim to explore and analyze the related influencing factors of liver and cardiac iron overload in MDS patients detected by magnetic resonance imaging (MRI). METHODS: We have detected cardiac T2* and liver T2* by MRI in 105 MDS patients. Among them, 20 patients accepted MRI examination before and after iron chelation therapy (ICT). Results: We found that adjusted ferritin (ASF) was significantly correlated with liver T2* and cardiac T2*. RBC transfusion volume, brain natriuretic peptide (BNP) and age were the related factors of cardiac T2*, while RBC transfusion volume and erythropoietin (EPO) were related factors of liver T2*. After ICT, the changes of ASF and liver T2* were earlier than cardiac T2*. Chronic hepatitis but virus copy normal's has no significant effect on liver iron deposition. CONCLUSION: These results showed special attention should be paid to these related influencing factors of liver and cardiac T2* expression when we evaluated iron overload and detected the efficacy of ICT in MDS patients.

[Iron Overload and Immune Inflammatin Are Risk Factors for the Progression of Myelodysplastic Syndrome--Review].

Abstract　　Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal disorders derived from haematopoietic stem and progenitor cells, also is a common malignant hematological diseases. It is characterized by ineffective bone marrow (BM) haematopoiesis, peripheral blood cytopaenias and a risk of progression to acute myeloid leukaemia. Iron overload is caused from transfusion dependence and ineffective hematopoiesis, which seriously affects the survival and prognosis of MDS patients. The role of immune inflammation in the development of MDS has been widely concerned. Oxidative stress and metabolic disorder caused from iron overload enhance the immune inflammatory response and accelerate the disease progression. Iron overload and immune inflammation are risk factors for the progression of MDS.

A genetic development route analysis on MDS subset carrying initial epigenetic gene mutations.

MDS development is a dynamic process during which the accumulation of somatic mutations leads to specific malignant evolution. To elucidate the differential roles of gene mutations in typical MDS, we used targeted sequencing to investigate clonal patterns from 563 patients and focused on cases (199/563 cases) with initial mutations (ASXL1, DNMT3A and TET2) at MDS diagnosis. The consistency of frequency and distribution in patients with or without aberrant chromosomes suggested early events of these initial mutations. Some additional driver mutations (SF3B1, U2AF1 or RUNX1) played roles to keep the basic disease features, or give rise to different phenotypes (BCOR, EZH2 or TP53) in individual patients. Notably, analysis in paired samples before and after MDS progression showed that the mutations identified as last events (involving active signaling, myeloid transcription or tumor suppressor) seemed necessary for MDS development to be AML. Last mutations can exist at MDS diagnosis, or emerge at AML transformation, and involve a small group of genes. Single-allele CEBPA mutations and diverse TP53 mutations were checked as the most common last event mutations. Considering the necessity of last event mutations and limited gene involvement in AML transformations, it is possible to validate a small group of last events involved mutations to develop some new strategies to block MDS progression.

Integration Analysis of JAK2 or RUNX1 Mutation With Bone Marrow Blast Can Improve Risk Stratification in the Patients With Lower Risk Myelodysplastic Syndrome.

Despite the improvements in prognostication of the revised International Prognostic Scoring System (IPSS-R) in myelodysplastic syndrome (MDS), there remain a portion of patients with lower risk (low/intermediate risk, LR) but poor prognostics. This study aimed to evaluate the relative contribution of mutational status when added to the IPSS-R, for estimating overall survival (OS) and progression-free survival (PFS) in patients with LR-MDS. We retrospectively analyzed clinical and laboratory variables of 328 patients diagnosed with MDS according to the FAB criteria. Twenty-nine-gene NGS assay was applied to bone marrow samples obtained at diagnosis. 233 (71.04%) patients were classified as LR-MDS. Univariate analysis showed association between inferior outcome (OS and PFS) and presence of JAK2 (p = 0.0177, p = 0.0002), RUNX1 (p = 0.0250, p = 0.0387), and U2AF1 (p = 0.0227, p = 0.7995) mutations. Multivariable survival analysis revealed JAK2 (p < 0.0001) and RUNX1 (p = 0.0215) mutations were independently prognostic for PFS in LR-MDS. Interestingly, bone marrow blast >1.5% could further predict disease progression of patients with LR-MDS (HR 8.06, 95%CI 2.95-22.04, p < 0.0001). Incorporation of JAK2, RUNX1 mutation and bone marrow blast in the IPSS-R can improve risk stratification in patients with LR-MDS. In summary, our result provided new risk factors for LR-MDS prognostics to identify candidates for early therapeutic intervention.

Decitabine Induces Change of Biological Traits in Myelodysplastic Syndromes via FOXO1 Activation.

Decitabine (DAC) is considered to be a profound global DNA demethylation, which can induce the re-expression of silenced tumor suppressor genes. Little is known about the function of tumor suppressor gene FOXO1 in myelodysplastic syndromes (MDS). To address this issue, the study firstly investigated differentially expressed genes (DEGs) for DAC treatment in MDS cell lines, then explored the role of FOXO1 through silencing its expression before DAC treatment in MDS. The results showed that FOXO1 exists in a hyperphosphorylated, inactive form in MDS-L cells. DAC treatment both induces FOXO1 expression and reactivates the protein in its low phosphorylation level. Additionally, the results also demonstrated that this FOXO1 activation is responsible for the DAC-induced apoptosis, cell cycle arrest, antigen differentiation, and immunoregulation in MDS-L cells. We also demonstrated DAC-induced FOXO1 activation upregulates anti-tumor immune response in higher-risk MDS specimens. Collectively, these results suggest that DAC induces FOXO1 activation, which plays an important role in anti-MDS tumors.

[Research Advances on Role of Extracellular Vesicles in the Bone Marrow Microenvironment of Patients with Hematological Malignancies--Review].

Bone marrow (BM) microenvironment appears to play an important role in the pathogenesis of hematological malignancies. Apart from soluble factors and direct cell-cell contact, the extracellular vesicles (EVs) were identified as a third mediator for cell communication within BM microenvironment. Recently, more and more evidences have demonstrated that EVs are also involved in the dysregulation of the BM microenvironment in patients with hematological malignancies. Therefore this review focuses on the biological characteristics of EVs, the clinical value of EVs as biomarkers, the BM microenvironment reprogramming in hematological malignancies by EVs, and the potential role of EVs in drug resistance and therapy of hematological malignancies.

[Effects of Abnormal Iron Metabolism on EPO-STAT5 Signaling Pathway in Anemia Patients].

OBJECTIVE: To study the effects of iron metabolism abnormality on EPO-STAT5 signaling pathway in anemia patients. METHODS: According to diseases, the patients were divided into 3 groups: lower risk myelodysplastic syndrome (MDS) group (30 cases) including 14 cases of non-iron over load and 16 cases of iron over load, 12 cases of them were treated by iron chelation therapy; anemia of chronic disease (ACD) group (12 cases) and iron deficiency anemia (IDA) group (12 cases). In addition, the healthy control group was selected. The iron metaloslism index (SF, SI, TIBC), serum level of EPO, plasma level of P-STAT5 and STAT-5 mRNA expression in peripheral blood cells were detected and compared in different groups. Moreover, the effects of iron metabolism abnormality on the expression of EPO and STAT5 in anemia patients were analyzed. RESULTS: compared with non-iron over load group, the EPO level in iron over load group significantly increased (P<0.05), the expression of STAT5 mRNA and P-STAT5 significantly decreased (P<0.05). After iron chelation therapy, the EPO level in serum significantly decreased (P<0.05), the expression of STAT5 mRNA and P-STAT5 was up-regulated significantly (P<0.05). Compared with healthy control group, the expression of EPO in ACD group was down-regulated significantly, while the expression of STAT5 mRNA was not different, but the P-STAT5 expression was down-regulated significantly (P<0.05). Compared with the healtly control group, the EPO expression in IDA group was enhanced significantly (P<0.05), the expression of STAT5 mRNA and P-STAT5 were also significantly enhanced (P<0.05). CONCLUSION: The excessive iron load or chronic inflammation may inhibit the activation of EPO-STAT5 signaling pathway and aggravate the anemia.

Clinical significance of hyaluronan levels and its pro-osteogenic effect on mesenchymal stromal cells in myelodysplastic syndromes.

BACKGROUND: Hyaluronan (HA), a major component of the extracellular matrix, has been proven to play a crucial role in tumor progression. However, it remains unknown whether HA exerts any effects in myelodysplastic syndromes (MDS). METHODS: A total of 82 patients with MDS and 28 healthy donors were investigated in this study. We firstly examined the bone marrow (BM) serum levels of HA in MDS by radioimmunoassay. Then we determined HA production and hyaluronan synthase (HAS) gene expression in BM mesenchymal stromal cells (MSC) and mononuclear cells derived from MDS patients. Finally, we investigated the effects of HA on osteogenic differentiation of MSC. RESULTS: The BM serum levels of HA was increased in higher-risk MDS patients compared to normal controls. Meanwhile, patients with high BM serum HA levels had significantly shorter median survival than those with low HA levels. Moreover, the HA levels secreted by MSC was elevated in MDS, especially in higher-risk MDS. In addition, HAS-2 mRNA expression was also up-regulated in higher-risk MDS-MSC. Furthermore, we found that MSC derived from MDS patients with high BM serum HA levels had better osteogenic differentiation potential. Moreover, MSC cultured in HA-coated surface presented enhanced osteogenic differentiation ability. CONCLUSIONS: Our results show that elevated levels of BM serum HA are related to adverse clinical outcome in MDS. Better osteogenic differentiation of MSC induced by HA may be implicated in the pathogenesis of MDS.

[Expression of Insulin-like Growth Factor Recepter Type I in CD34+ Cells of Patients with Myelodysplastic Syndromes].

OBJECTIVE: To explore the expression level of insulin-like growth facter (IGF-IR) in CD34+ cells of patients with myelodysplastic syndromes(MDS). METHODS: Flow cytometry was used to detect the expression of IGF-IR in the CD34+ cells of 100 MDS patients and 18 normal controls. RESULTS: The average IGF-IR expression level in the CD34+ cells of 100 MDS patients (41.0±28.1)% was statistically and significantly elevated in comparison with the corresponding level in normal controls(4.3±1.8)%,(P<0.0001). The average expression level of 22 cases in high-risk groups was very significantly increased, compared with that in 78 cases of low-risk groups[(66.5±27.8)% vs (34.5%±24.9)%](P<0.0001), and the average expression level in 23 patients with chromosome abnormality was very significantly increased in comparison with that in rest 77 patients [(56.0±30.9)% vs (36.9%±26.2)%](P<0.01). CONCLUSION: The over-expression of IGF-IR in CD34+ cells of MDS patients suggests that the IGF-IR may involve in the origin, occurrence and progress. The average IGF-IR expression level is markedly elevated in high-risk groups and the patients who showed chromosome abnormality, this trend revealed that IGF-IR correlates with malignant clonal proliferation in MDS patients, thus providing a basis for their prognosis and outcome evaluation.

[Anti-Tumor Effect of Rigosertib on HEL and K562 Cells and Its Related Mechanism].

OBJECTIVE: To investigate the effects of rigosertib on the apoptosis, proliferation and cell cycle of HEL and K562 cells. METHODS: The HEL and K562 cells were treated with different concentration of rigosertib at different time points, the cell apoptosis, proliferation and cycle were determined by using flow cytometry with Annexin V/PI double staining, WST-1 method and 7-AAD assay, respectively. Intracellular signaling proteins were detected by flow cytometry (FCM). RESULTS: Rigosertib induced obvious apoptosis in HEL and K562 cells, and the apoptotic effect was both time-dependent and dose-dependent manner (P<0.05). The low dose of rigosertib inhibited obviously the proliferation of HEL and K562 cells after treatment from 6 to 54 h, Rigosertib arrested HEL and K562 cells into G2/M phase. In addition, Rigosertib obviously increased the expression of apoptosis-related proteins such as cleaved caspase 3 and PARP, and reduced the proliferation-related proteins such as BCL-2 and Cyclin D1. Rigosetib inhibited the activation of AKT-GSK signaling through decreasing the expression of AKT, pAKT(Ser473) and GSK-3α/ß (S21/9). CONCLUSION: Rigosertib inhibites proliferation, induces apoptosis and cell cycle arrest in G2/M phase of HEL and K562 cells. This agent may have potential application prospect in leukemia therapy.