Jianmin Luo

Artesunate induces apoptosis through caspase-dependent and -independent mitochondrial pathways in human myelodysplastic syndrome SKM-1 cells

Artesunate (ART) is a semi-synthetic derivative of artemisinin extracted from Artemisia annua (sweet wormwood) that is conventionally used in anti-malarial drugs and more recently in medications that induce tumor cell apoptosis. Here, we investigated the effects and mechanistic pathways of ART in human myelodysplastic syndrome (MDS), a condition that commonly progresses to acute myeloid leukemia (AML). Human MDS SKM-1 cells, primary bone marrow (PBM) mononuclear cells from patients with refractory anemia with excess blasts (RAEB) or MDS-AML (MDS cell group), and PBM stromal cells from three patients without hematological diseases (non-MDS cell group) were cultured for 24, 48, or 72 h with or without various ART concentrations. CCK-8, western blot, JC-1 fluorescence, and Annexin-V/Propidium iodide (PI) labeling were used to assess cell proliferation, protein levels, mitochondrial membrane potentials (MMPs) and apoptosis, respectively. ART administration dose- and time-dependently inhibited SKM-1 proliferation. At 24, 48, and 72 h, ART IC₅₀ values were 89.92, 4.24, and 1.28 μmol/L, respectively. ART only significantly inhibited proliferation in the MDS cell group, but it has little impact on proliferation of non-MDS cells. ART decreased MMPs, and dose-dependently induced SKM-1 cell apoptosis, peaking at 82.9% when treated with 200 μmol/L ART for 24h. Caspase-3 and -9 activation, poly(ADP-ribose) polymerase cleavage, decreased Bcl-2/Bax ratio and apoptosis inducing factor nuclear localization were implicated in apoptosis. Our results indicate that ART effectively induces apoptosis in SKM-1 cells through both caspase-dependent and -independent mitochondrial pathways.

A novel recombinant human thrombopoietin therapy for the management of immune thrombocytopenia in pregnancy

The aim of this study was to determine the safety and efficacy of recombinant human thrombopoietin (rhTPO) for the management of immune thrombocytopenia (ITP) during pregnancy. Pregnant patients with ITP were enrolled in the study if they had a platelet count less than 30 × 109/L, were experiencing bleeding manifestations, had failed to respond to corticosteroids and/or intravenous immunoglobulin (IVIG), and had developed refractoriness to platelet transfusion. Thirty-one patients received rhTPO at an initial dose of 300 U/kg once daily for 14 days. Twenty-three patients responded (74.2%), including 10 complete responders (>100 × 109/L) and 13 responders (30-100 × 109/L). It appears that rhTPO ameliorated the bleeding symptoms remarkably, even in the nonresponders. rhTPO was well tolerated. Dizziness, fatigue, and pain at an injection site were reported in 1 patient each. No congenital disease or developmental delays were observed in the infants in a median follow-up of 53 (range, 39-68) weeks. In conclusion, rhTPO is a potentially safe and effective treatment choice for patients with ITP during pregnancy. Our work has paved the way for further study on the clinical application of rhTPO and other thrombopoietic agents for the management of ITP during pregnancy. This study is registered at www.clinicaltrials.gov as NCT02391272.

Long noncoding RNA MEG3 inhibits proliferation of chronic myeloid leukemia cells by sponging microRNA21

The long noncoding RNA (lnc) maternally expressed 3 (MEG3) is downregulated in many types of cancers. However, the relationship between lncRNA MEG3, microRNA-21 (miR-21) and chronic myeloid leukemia (CML) blast crisis is unknown. This study examined bone marrow samples from 40 CML patients and 10 healthy donors. Proliferation and apoptosis assays, real-time polymerase chain reaction (PCR), bisulfite sequencing PCR, Western blotting, luciferase assay, RNA pull-down, RNA immunoprecipitation (RIP), co-immunoprecipitation (CoIP) and Chromatin immunoprecipitation (ChIP) were performed. We found that MEG3 and PTEN expression were down-regulated, whereas, MDM2, DNMT1 and miR-21 were up-regulated in the accelerated and blast phases of CML. Treated with 5-azacytidine decreased the level of MDM2, DNMT1 and miR21, but increased the level of MEG3 and PTEN. Overexpression of MEG3 and silencing the expression of miR-21 inhibited proliferation and induced apoptosis. MEG3 overexpression and silencing the expression of miR21 influence the levels of MMP-2, MMP-9, bcl-2 and Bax. MEG3 was able to interact with MDM2 and EZH2. MDM2 could interact with DNMT1 and PTEN. MYC and AKT can interact with EZH2. ChIP-seq showed that the promoter of KLF4 and SFRP2 interacts with DNMT1. In conclusion, lncRNA MEG3 and its target miR21 may serve as novel therapeutic targets for CML blast crisis; and demethylation drugs might also have potential clinical application in treating CML blast crisis.

Notable roles of EZH2 and DNMT1 in epigenetic dormancy of the SHP1 gene during the progression of chronic myeloid leukaemia

Tumor development is associated with the methylation of cytosine-guanine (CpG) islands. The occurrence of methylation requires several factors, such as DNA methylation systems and polycomb group (PcG) proteins. At present, novel drugs are needed for the treatment of chronic myeloid leukaemia (CML), particularly considering the current prognosis of CML. The methylation status of the Src homology 2 domain-containing tyrosine phosphatase 1 (SHP1) gene, a negative regulator of signal transduction, has been identified as being altered in numerous haematological malignancies. DNA methyltransferase 1 (DNMT1) and the PcG protein complex member enhancer of zeste homolog 2 (EZH2) participate in a number of gene methylation processes. The present study investigated the methylation status of the SHP1 gene in CML, and examined the association between DNMT1 and EZH2 activity and the SHP1 gene methylation status to develop novel strategies for the treatment of CML. The results revealed that SHP1 gene methylation status was altered during the progression of CML. These data indicated that SHP1 gene methylation is associated with the progression of this disease. The associations of DNMT1 and EZH2 activities with the methylation status of the SHP1 gene were additionally investigated via chromatin immunoprecipitation. DNMT1 and EZH2 were revealed to be bound to the promoter region of the SHP1 gene, and were involved in the process of SHP1 methylation. Furthermore, DNMT1 and EZH2 were associated with disease progression. Thus, the findings of the present study suggest a new target for the treatment of CML, particularly for future drug development.

Research on the epigenetic regulation mechanism of the PTPN6 gene in advanced chronic myeloid leukaemia

PTPN6, a tyrosine phosphatase protein, plays a negative role in cell signal transduction and is negatively correlated with tumour formation and growth. However, epigenetic regulation mechanism of the PTPN6 gene in advanced chronic myeloid leukaemia (CML) remains unclear. This study investigated bone marrow or blood samples from 44 CML patients and 10 healthy volunteers. KCL22 and K562 cells were cultured and treated with demethylation drugs and histone deacetylase inhibitors. Real time quantitative polymerase chain reaction (qPCR), methylation‐specific PCR, bisulfite sequencing PCR, Western blotting, co‐immunoprecipitation and chromatin immunoprecipitation (ChIP) was performed. PTPN6 was down‐regulated in cell lines and patients with advanced phase CML, whereas DNMT1, DNMT3A, MECP2, MBD2 and HDAC1 were up‐regulated. Treatment with 5‐azacytidine, decitabine, sodium valproate and LBH589 increased PTPN6 expression, but decreased that of DNMT1, DNMT3A, MECP2, MBD2 and HDAC1. Immunoprecipitation and mass spectrometry showed that HDAC1 combined directly with PTPN6. ChIP‐seq showed that HDAC1 did not combine with the promoter region of PTPN6, while MAPK, AKT, STAT5, JAK2 and MYC promoter regions all combined with HDAC1. PTPN6 is associated with progression of CML. Low expression level of PTPN6 was associated with DNA methylation and regulated by histone acetylation. HDAC1 participates in the regulation of PTPN6.

The Long Noncoding RNA MEG3 and its Target miR-147 Regulate JAK/STAT Pathway in Advanced Chronic Myeloid Leukemia

Background Long non-coding (lnc) RNAs plays an important role in chronic myeloid leukemia (CML). In this study, we aimed to uncover the mechanism of the lncRNA maternally expressed 3 (MEG3) and its target microRNA-147 (miR-147) in CML. Methods Sixty CML patients and 10 healthy donors were included in the study. The methylation of MEG3 and miR-147 promoter was determined by methylation-specific PCR. The relationship of MEG3 and miR-147 was explored by luciferase assay. The interactions of proteins were studied by RNA pull-down assay, RNA immunoprecipitation and co-immunoprecipitation. Findings Patients in accelerated phase CML (CML-AP) and blast phase CML (CML-BP) showed lower expressions of MEG3 and miR-147 and higher expressions of DNMT1, DNMT3B, MBD2, MECP2 and HDAC1 compared to the controls. These patients also showed a higher degree of methylation of MEG3 and miR-147 while there was a reduction after chidamide treatment. Furthermore, the overexpression of MEG3 and miR-147 inhibited cell proliferation both in vivo and in vitro, promoted apoptosis and decreased the expressions of DNMT1, DNMT3A, DNMT3B, MBD2, HDAC1 and MECP2. We also found MEG3 interacted with DNMT1, JAK2, STAT3, HDAC1, and TYK2, and JAK2 was bound to STAT3, STAT5 and MYC. More interestingly, JAK2 was bound to TYK2 by the bridge of MEG3. Interpretation LncRNA MEG3 and its target miR-147 may serve as a novel therapeutic target for CML blast crisis, and chidamide might have a potential clinical application in treating CML blast crisis.

THE CLINICAL SIGNIFICANCE OF MULTIDRUG RESISTANCE GENE (mdrl) EXPRESSION IN ACUTE LEUKEMIA

Objective: To study the clinical significance of multidrug resistance gene expression in acute leukemia. Methods: The relationships between drug resistance of leukemia cells and prognosis, multidrug resistance gene (mdr1) were examined in 85 patients with acute leukemia and 20 normal controls by reverse transcriptase poly-merase chain reaction (RT-PCR). Results: The mdrl positive rate in untreated group was 44.7%. The complete remission (CR) rate of mdr1 positive patients (23.9%) was significantly lower than that of mdr1 negative patients (88.5%) (P<0.005). The mdr1 expression level in relapsed-refractory group was higher than that of CR group. A gradually increased mdr1 mRNA level in CR patients indicated early relapse. Conclusion: The mdr1 positive rate in normal control and long-term survival patients was very low. The mdr1 expression was correlated with French-American-British Cooperative Group (FAB) classification. The mdr1 expression level was correlated with chemotherapeutic effect and prognosis. It is an unfavorable prognostic factor for patients with acute leukemia.