Effect of CXCR4 on the Treatment Response and Prognosis of Carfilzomib in Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of CXCR4 on the treatment response and prognosis of Carfilzomib (CFZ) in multiple myeloma.@*METHODS@#Dataset GSE69078 based on microarray data from two CFZ-resistant MM cell lines and their corresponding parental cell lines (KMS11-KMS11/CFZ and KMS34-KMS34/CFZ) were downloaded from Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) were identified, and Protein-protein interaction (PPI) network was established to identify the key genes involved in CFZ resistance acquisition. Finally, the prognostic roles of the CFZ risistance key genes in MM using MMRF-CoMMpass data study was verified.@*RESULTS@#44 up-regulated and 46 down-regulated DEGs were identified. Top 10 hub genes (CCND1, CXCR4, HGF, PECAM1, ID1, HEY1, TCF4, HIST1H4J, HIST1H2BD and HIST1H2BH) were identified via Protein-protein interaction (PPI) network analysis. The CoMMpass data showed that high CXCR4 expression showed correlation to relative higher relapse and progress rates and the overall survival was significant decreased in high CXCR4 patients (P=0.013).@*CONCLUSION@#CXCR4 perhaps plays a crucial role in CFZ acquired resistance, which might help identifying potential CFZ-sensitive patients before treatment and providing a new therapeutic target in CFZ-resistant MM.

Effect of Hypoxia Inducible Factor-1α on Chemosensitivity of B-ALL Cells to Vincristine and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of hypoxia on the chemosensitivity of B-acute lymphoblastic leukemia （B-ALL） cells to Vincristine (VCR) and the mechanisms.@*METHODS@#B-ALL cells SUP-B15, Nalm-6 and RS4;11 were selected as the research objects. The cells were divided into the control group and the hypoxia mimic group (CoCl2 pretreatment). The two groups were treated with VCR at different concentrations for 24 hours, CCK-8 was used to detect cell viability, flow cytometry was used to detect cell apoptosis, and Western bolt method was used to detect hypoxia inducible factor (HIF-1α), BAX, Bcl-2 and β-actin protein expression. Quantitative real-time fluorescent PCR (qRT-PCR) was used to detect BAX and β-actin mRNA levels.@*RESULTS@#CoCl2 could simulate hypoxic environment to induce the expression of HIF-1α. The cells SUP-B15 and RS4;11 of the hypoxia mimic group were lower sensitivity to VCR as compared with the control group; the apoptosis rate of the hypoxia mimic group was lower than that of the control group after 80 nmol/L VCR treatment. The expression levels of BAX protein and mRNA in the hypoxia mimic group were lower than those of the control group, and there was no significant difference in the expression levels of Bcl-2 protein between two groups.@*CONCLUSION@#Under hypoxic conditions, HIF-1α may mediate VCR resistance in B-ALL cells by downregulating the pro-apoptotic protein BAX.

Significance of Targeted Sequencing Assay for Patients with Suspected Myeloid Malignancies / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical significance of the targeted next-generation sequencing assay for patients with suspected myeloid malignancies.@*METHODS@#A total of 39 hematopenia patients with suspected myeloid malignamies in Department of Hematology of The Affiliated Huai'an No.1 People's Hospital of Nanjing Medical University from January 2018 to April 2019 were treated, 20 hot spot genes of myelodysplastic syndrome (MDS) were detected.@*RESULTS@#Regarding the diagnostic type, there were 7 cases of idiopathic cytopenia of undetermined significance (ICUS), 8 cases of clonal cytopenias of undetermined significance (CCUS) and 24 cases of myeloid myeloid malignancies which included 18 cases of MDS, 4 cases of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and 2 cases of acute myeloid leukemia. Positive mutation was detected in 70.8% (17/24) of myeloid malignancy patients , and 72.7% (16/22) in MDS and MDS/MPN patients. The main mutation types were ASXL1, TET2 and RUNX1. Compared with gene negative group, there were no significant differences in sex, age (＜60 years old or ≥60 years old), proportion of bone marrow blast cells (＜5% or≥5%) and cytogenetics (good, medium and poor) (P＞0.05). Furthermore, all 8 CCUS patients showed positive mutation, and the incidence of double or multiple mutation in CCUS group was significantly lower than that of the MDS and MDS/MPN group (37.5% vs 54.5%) (P=0.002). The mutation types between the two groups were similar, and there was no significant difference in variant allele frequency (P＞0.05).@*CONCLUSION@#Our results suggest that there are high rates of double or multiple mutations in myeloid malignancies, especially in patients with MDS and MDS/MPN. Targeted sequencing assay can improve the diagnosis of myeloid malignancies, and guide clinical treatment.

Clinical Analysis of 66 Patients with Essential Thrombocytopenia / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical characteristics of essential thrombocytopenia (ET) patients with positive mutations including JAK2, CALR, MPL, or negative mutations.@*METHODS@#A total of 66 newly diagnosed ET cases from January 2016 to December 2018 in Department of Hematology, Huaian No.1 People's Hospital affiliated to Nanjing Medical University were analyzed. Statistical analysis data included the patient's sex, age, symptoms, thrombosis and embolism events, spleen omegaly, platelet count (Plt), leukocyte (WBC) count, hemoglobin (Hb), fibrinogen (FIB), thrombus elastic diagram (TEG), serum potassium, blood glucose (GLU), lactate dehydrogenase (LDH), JAK2, CALR and MPL mutations, treatment options, and efficacy.@*RESULTS@#All the patients were not MPL-positive, and divided in three groups: JAK2 mutation (46 cases, 69.7%), CALR mutation (9 cases, 13.6%) and gene negative mutation (11 cases, 16.7%) group. The average age of patients in the JAK2 mutation group was 63.2 years old, and significantly higher than that in the CALR mutation group (51.8 year) and gene negative group (50.2 year) (P＜0.05). Compared with the JAK2 mutation group and gene negative group, the CALR mutation group had lower WBC count (6.3×10/L vs 13.79×10/L) (P=0.003) (6.3×10/L vs 9.70×10/L) (P=0.009). Also the Hb level of patients in CALR mutation group was lower than the JAK2 mutation group (121.22 g/L vs 136.2 g/L) (P=0.036). However, there was higher tumor burden in the CALR mutation group, compared with the gene negative mutation group (300.11 U/L vs 227.4 U/L) (P=0. 033). There was no significant difference among the three groups, such as the Plt counts, serum potassium level, GLU level and FIB level (P＞0.05). In addition, thrombus and embolism appeared in 30.3% (20/66) cases. 18.2% (12/66) cases were complicated with hyperkalemia, which significantly correlated with Plt counts (r=0.518). TEG was performed in 34 patients, of which 41.2% (14/34) had abnormal TEG and 55.9% (19/34) were accompanied by Plt count ＞ 1 000 ×10/L, but there was no significant correlation between them (r=0.134). After routine clinical treatment, all the 66 cases achieved partial or complete hematological remission, but the disease usually repeated. Until now 4.5% (3/66) cases had been converted to myelofibrosis (MF) all with JAK2 mutation, but without advancing to acute myeloid leukemia.@*CONCLUSION@#ET patients with JAK2 mutation have higher incidence, moreover were in older age. However, the patients with CALR mutations display lower WBC count and Hb level, but higher tumor burden. In short, the multiple gene mutations of ET showed different clinical features closely relates with the prognosis, thus providing guidance for the clinical diagnosis and treatment.

Therapeutic Response and Prognosis of Adult Acute Myeloid Leukemia with Chromosome Karyotype Abnormalities / 中国实验血液学杂志

OBJECTIVE@#To investigate the efficacy and prognosis of acute myeloid leukemia (AML) patients with chromosome karyotype abnormalities.@*METHODS@#The clinical features and treatment responses of 91 patients with AML were collected and analyzed retrospectively. The efficacy and survival rate of the AML patients with normal and abnormal chromosome karyotype were compared.@*RESULTS@#Chromosome translocations and monosomal karyotypes were the main heterogeneity of AML. There was no significant difference in complete remission rate and overall response rate between the normal and abnormal karyotype groups, but the recurrence rate was higher in abnormal karyotype group. There was no significant difference in response of AML patients received the standard "3+7 regimen" and pre-excitation chemotherapy in the treatment of normal and abnormal karyotype groups. The relapse free survival time (RFS) was longer in the normal karyotype group, but there was no significant difference in overall survival time (OS).@*CONCLUSION@#The abnormal karyotype of AML is an independent prognostic factor, monosomal karyotype shows a poor prognosis, and the recurrence rate in AML patients with monosomal karyotype is higher.

Differential Regulation of Bruton's Tyrosine Kinase by the Ubiquitin Pathway / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the regulation of Bruton's tyrosine kinase (Btk) and to explore its possible mechanism.</p><p><b>METHODS</b>After treatment with the proteasome inhibitors and/or phorbol esters (PMA), the mRNA and protein expression level of Btk was detected by RT-PCR and Western blot, respectively. The ubiquitination level of Btk in B lymphoblastoid A20 cells was estimated after stimulation via the crosslinking of BCR with anti-IgM antibody. The cotransfection of COS-7 cell with Btk, ubiquitin and Cbl was performed, then the ubiquitination level of Btk was measured. The Btk ubiquitination level was detected after ectopic expression of ubiquitin transfected with the wild type or triple mutant of Ub (K29R, K48R, K63R) . Mono-ubiquitination of Btk was detected with antibodies preferentially against monovalent ubiquitin; in addition, the protein expression levels of chloroquine-treated stably transfected cells expressing Btk-GFP were detected by Western blot, and quantified with the strength of GFP fluorescence.</p><p><b>RESULTS</b>In the presence of proteasome-specific inhibitors and/or PMA, steady-state levels of Btk protein were reduced due to decrease of transcription. Posttranslational modification of Btk by ubiquitination was observed, which was related with the level of Btk expression and activation. The E3 ubiquitin ligase Cbl, which binds to Btk, was also found to ubiquitinate this kinase. Altogether, the data of this study strongly suggest that Btk is regulated by poly- and/or mono-ubiquitination events.</p><p><b>CONCLUSION</b>The Btk protein is dictated by its expression level through the ubiquitination pathway.</p>

Effect of Homoharringtonine Combined with Imatinib on the K562/G01 Cells and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of homoharringtonine(HHT) combined with imatinib(IM) on proliferation and apoptosis of K562/G01 cells and its potential mechanism.</p><p><b>METHODS</b>K562/G01 cells were cultured with HHT and/or IM. CCK-8 assay was used to detect cell proliferation. Cell apoptosis and phosphorylated tyrosine levels were analyzed by flow cytometry. The expression levels of p210, PI3K, p-Akt and Akt protein were determined by Western blot.</p><p><b>RESULTS</b>Compared with HHT or IM alone, drug combination significantly inhibited cell proliferation and induced apoptosis of K562/G01 cells (both P< 0.05). HHT combined with IM could inhibit the levels of phosphorylated tyrosine and phosphorylated Crkl and downregulate the expressions of p210, PI3K and p-Akt in K562/G01 cells.</p><p><b>CONCLUSION</b>HHT combined with IM can synergistically inhibit proliferation and induce apoptosis of K562/G01 cells by suppressing the p210 expression and its kinase activity.</p>

Effect of Autophagy on Homoharringtonine-treated K562 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the effect of homoharringtonine (HHT) alone or combined with 3-methyladenine (3-MA) , an autophagy inhibitor, on the apoptosis and autophagy of K562 cells.</p><p><b>METHODS</b>K562 cells were treated with HHT(10 ng/ml) or HHT(10 ng/ml) combined with 3-MA (1.5 mmol/L) for 1 to 8 days. The apoptosis of treated cells was tested by means of flow cytometry(FCM), and the autophagy levels were tested with RT-PCR, Western blot and electron microscopy.</p><p><b>RESULTS</b>In the early stage of HHT-treated group, the apoptosis rate increased and decreased later. Beclin1 mRNA expression level and the LC3II/I ratio were declined firstly and increased later in HHT group. While combining with autophagy inhibitor 3-MA, both the Beclin1 mRNA expression level and the LC3II/I ratio were declined continually during the treated period. The activated caspase-3 protein expression level was also raised sustainability during both HHT and 3-MA cultured period.</p><p><b>CONCLUSIONS</b>HHT can induce apoptosis of K562 cells, but the sustaining effect of HHT can induc autophagy of K562 cells, the combination of HHT with 3-MA may enhance the cytotoxicitic effect of HHT on K562 cells.</p>

Acadesine Inhibits the Proliferation of K562 Cells and Enhances their Sensitivity to Imatinib / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of AMPK agonist Acadesine (AICAR) on growth inhibition of K562 cells and their sensitivity to imatinib (IM).</p><p><b>METHODS</b>K562 cells were cultured with different concentrations of AICAR alone or its combination with IM for 48 hours, the CCK-8 assay was used to detect cell proliferation, the cell cycle distribution and apoptosis were analyzed by flow cytometry. The expression levels of Cyclin D1, Cyclin E1 and Caspase 3 protein were determined by Western blot.</p><p><b>RESULTS</b>AICAR inhibited the proliferation of K562 cells in dose-dependent manner, and their IC50 value was 0.45 mmol/L at 48 hours. AICAR could induce arrest of K562 cells in G1 phase and down-regulated the protein expression levels of Cyclin D1 and Cyclin E1; whereas it didn't influence the cell apoptosis. Additionally, the growth inhibition of cells induced by IM was enhanced by AICAR.</p><p><b>CONCLUSION</b>AICAR can inhibit the proliferation of K562 cells by arresting the cell cycle and enhancing the sensitivity of K562 cells to IM.</p>

Effect of homoharringtonine on expression of NF-κB and BCL-2 proteins in K562 cells / 中国实验血液学杂志

This study was aimed to investigate the effect of homoharringtonine (HHT) on K562 cell proliferation, apoptosis and expression of BCL-2 and NF-κB proteins. The cells proliferation was assayed with MTT method, the cell apoptosis, cell cycle and BCL-2 expression were analyzed with flow cytometry, NF-κB protein expression was detected with Western blot. The results showed that HHT concentration-dependently inhibited proliferation of K562 cells, the IC50 at 48 h was 43.89 ng/ml. Treated with HHT 10 ng/ml for 48 h, K562 cell apoptosis significantly increased, cell cycle was blocked at G0/G1, the expression level of BCL-2 and NF-κB proteins was lower than that in control group (P < 0.05). It is concluded that HHT may inhibit the proliferation of K562 cells, and down-regulating expression levels of BCL-2 and NF-κB may be one of its anti-CML mechanisms.

Effects of interferon-α combined with homoharringtonine on K562 cell proliferation and β-catenin expression / 中国实验血液学杂志

The study was aimed to investigate the synergistically effect of interferon-α (IFN-α) and homoharringtonine (HHT) on the proliferation, apoptosis, cell cycle of K562 cells and the expression of β-catenin. The proliferation, apoptosis, cell cycle and β-catenin mRNA expression of K562 cells treated with IFN-α and/or HHT were assayed with MTT, flow cytometry or RT-PCR respectively. The results showed that HHT alone, but not IFN-α alone, displayed a proliferation inhibition, apoptosis induction, G(0)/G(1) phase block and down-regulation of β-catenin expression in K562 cells with concentration- and time-dependent manners. The expression level of β-catenin mRNA after being treated with HHT was 0.5576 ± 0.0373, which were lower than that in control group (0.9369 ± 0.0142). The down-regulation of β-catenin expression in group of IFN-α combined with HHT was higher significantly than that in HHT group (0.3737 ± 0.0529 vs 0.5576 ± 0.0373, P < 0.05). Otherwise, HHT combined with IFN-α did not demonstrate obvious toxicologic effect on bone marrow mononuclear cells. It is concluded that IFN-α combined with HHT can enhance the cytotoxic effect of HHT on K562 cells, which may be associated with down-regulation of β-catenin expression.