Analysis of the Differential Expression of circRNA in Acute Myeloid Leukemia by GEO Database / 中国实验血液学杂志

OBJECTIVE@#To investigate the difference expression of circular RNA (circRNA) in acute myeloid leukemia (AML) by using bioinformatics method.@*METHODS@#The microarray chip data of AML was searched and downloaded from the Gene Expression Omnibus (GEO) of the National Center for Bioinformatics (NCBI). The differences between AML samples and control samples were analyzed by R software. The interaction between deregulated circRNA, miRNA and mRNA were predicted by miranda software and miRTarBase software. The circRNA-miRNA-mRNA regulatory network was constructed by using the cytoHubba plugin based on the Cytoscape software.@*RESULTS@#A total of 203 differential expression of circRNAs were finally collected, including down-regulated 161 circRNAs and up-regulated 42 circRNAs. CircRNA/miRNA/mRNA interaction network was constructed through software prediction. hsa_circ_0001080, hsa_circ_0004511, hsa_circ_0054211, hsa_circ_0001944 may be positively regulated the gene expression in AML.@*CONCLUSION@#Abnormal expression of circRNA in AML may become a new target for AML treatment.

Analysis of RUNX1 Gene Mutation in Patients with Myelodysplastic Syndrome / 中国实验血液学杂志

OBJECTIVE@#To investigate the mutation of RUNX1 gene in patients with myelodysplastic syndrome (MDS) and its correlation with other gene mutations and some clinical parameters.@*METHODS@#The mutations of RUNX1, DNMT3A, TET2, IDH1/2, NPM1, FLT3-ITD and C-KIT in 170 patients with MDS were detected by direct and indirect sequencing of genomic DNA-PCR amplification products.@*RESULTS@#The RUNX1 mutation was found in 23 patients (13.5 %, 23/170). Among the 170 patients, other most frequent mutation was TET2 (11.2%, 19/170), followed by mutations in DNMT3A (9.4%, 16/170), NPM1 (8.2%, 14/170), IDH2 (4.1%, 7/170)、FLT3-ITD (2.9%, 5/170), IDH1 (1.7%, 3/170) and c-KIT (0.58%, 1/170). The most common coexisting mutations were TET2 (5/23). The RUNX1-mutated group showed significantly higher leukocyte levels, higher percentages of blast cells, higher incidences of leukemia transformation and lower platelet counts in comparison with RUNX1 non-mutation group (P＜0.05). whereas there were no statistically significant difference in age, MDS subtype, karyotype and hemoglobin level between 2 groups (P＞0.05). Seventeen patients harboring RUNX1 mutations were followed up and almost 47.05% (8/17) of the patients progressed into acute myeloid leukemia (AML). The rates of transformation into AML in ASXL1-mutation group was significantly higher than that in ASXLL- non-mutation group (47.05% vs 11.7%) (P=0.001).@*CONCLUSION@#The incidence of RUNX1 mutation is high in MDS patients. The RUNX1-mutated patients have higher leukocyte level, higher percentages of blast cells, higher incidences of leukemia transformation and lower platelet count.

Mechanism of Anti Apoptosis and Immune Evasion in Drug-Resistant Leukemia Cells Mediated by STAT3 / 中国实验血液学杂志

OBJECTIVE@#To investigate the mechanisms of anti-apoptosis and immune evasion in drug-resistant leukemia cells mediated by STAT3, further to explore the possible mechanism of leukemia relapse caused by minimal residual.@*METHODS@#Drug-resistance leukemia cell line was established by transfecting pcDNA3.1-STAT3 into K562 cells (K562/STAT3). The expression of STAT3, BAX and NKG2D ligands (MICA and ULBP1) in K562/-cells, K562/STAT3 were detected by Western blot and/or RQ-PCR. Cells apoptosis and the killing effect of NK cells on leukemia cells were detected by flow cytometry.@*RESULTS@#The expression of the total STAT3, STAT3 phosphorylation in K562/STAT3 was significantly increased, and P-gp mRNA expression was increased also significantly (P<0.005). In K562/STAT3 cells, the expression of pro-apoptotic BAX (P=0.005) was significantly lower, and the number of apoptotic cells (P=0.002) induced by adriamycin was significantly decreased as compared with those in K562/- cells. After K562/STAT3 cells were treated by STAT3 inhibitor (SH-4-54), the expression of BAX mRNA (P=0.017) was significantly higher and the number of apoptotic cells (P=0.005) was significantly increased. The MICA and ULBP1 mRNA expression in K562/STAT3 cells was significantly lower than that in K562/- cells, and also for MICA and ULBP1 protein (MICA and ULPB1 mRNA: P<0.0001, MICA protein: P=0.001, ULPB1 protein: P=0.022). After K562/STAT3 cells were treated with STAT3 inhibitor (SH-4-54), the expression of MICA mRNA and protein was increased (mRNA: P=0.001, protein: P=0.002), but ULBP1 mRNA and protein showed no significantly change (mRNA: P=0.137, protein: P=0.1905). The cytotoxicity of NK cells to K562/STAT3 cells was susceptible as compared with K562/- (P=0.002), but the cytotoxicity of K562/STAT3 cells to NK cell could be recovered by STAT3 inhibitor (P=0.006).@*CONCLUSION@#STAT3 phosphorylation can inhibits cell apoptosis and promotes cell immune escape. STAT3 inhibitors can promote the apoptosis of leukemia cells and increase their sensitivity to NK cells.

Coexisting Mutations in IDH1/2-Mutated Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To explore the coexisting mutations in IDH-mutated acute myeloid leukemia(AML) and its relation with partial clinical parametrs.@*METHODS@#The exon 4 mutation of IDH1/2 gene was screened by using genome DNA-PCR combined with sanger sequencing, 51 targeted gene mutations in the patients with IDH1/2 mutation were detected by using high throughput DNA sequencing combined with sanger sequencing.@*RESULTS@#Among 358 patients, the IDH1/2 mutation was found in 46 cases including IDH1 mutation in 35 cases and IDH2 mutation in 11 cases, 97.87%(45/46) patients with IDH1/2 mutation simultaneously carried other gene mutations including 8(17.8%) cases with mutation of double gene, 17(37.8%) cases with mutation of 3 genes and 20(44.4%) cases with mutation of ≥ 4 genes. The mutation frequency of each patient averaged 3.52 times. In mutation of accompanied genes, the common genes were NPM1(n=29, 63.0%), next DNMT3A(n=25, 54.3%), FLT3-ITD(n=7, 15.2%), TET2(n=5, 10.9%) and NRAS(n=5, 10.9%). The average WBC level of patients with NPM1 mutation in IDH1 mutation group was higher than that of patients in wild type group(P<0.05). The complete remission (CR) rate of patients with DNMT3A mutation was significant lower than that of patients with wild type (30% vs 80%, P<0.01). The presence of ≥ 4 mutations was found to be significantly associated with higher white blood level than that in the patients with double mutations(P<0.05).@*CONCLUSION@#More than 95% AML patients with IDH1/2 mutation commonly show additional mutations. The number and the type of IDH coexisting mutations have certain effect on the clinical features and CR rate.

Effects of Garcinia Acid Combined with Daunorubicin on Expression of Pregnane X Receptor in Leukemia Cell Line K562/A02 / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the expression of PXR (Pregnane X receptor) in several malignant hematological cell lines, and to investigate the reversal effect of Gambogic acid (GA) on multi-drug resistance (MDR) of K562/A02 cell line and its reversal mechanism.</p><p><b>METHODS</b>Transcription of PXR was detected by real-time PCR in several malignant hematological cell lines. The growth inhibition rate of K562/A02 in different experimental groups was assayed by MTT method, and the expression of PXR protein was measured by Western blot.</p><p><b>RESULTS</b>PXR gene transcription could be detected in several hematological malignancy cell lines, and it was significantly higher in K562/A02 cell line, compared with the other cell lines used in this experiment. Low-dose GA could enhance cell growth inhibition rate, increasing the effect of chemotherapy, which may be associated with down-regulation of PXR expression. PXR gene transcription and protein expression in GA and DNR+GA groups decreased as compared with control group and the DNR group, suggesting that low-dose GA can down-regulate PXR gene transcription and protein expression.</p><p><b>CONCLUSION</b>PXR gene transcription can be detected in several hematological malignancy cell line, which is significantly higher in K562/A02 cell line, as compared with the other cell lines used in this experiment. Low-dose GA can enhance cell growth inhibition rate, increasing the effect of chemotherapy, which may be associated with down-regulation of PXR expression.</p>

Influence of Fe₃O₄Magnetic Nanoparticles Combined with As2O3 and Adriamycin on Raji Cell Apoptosis and Autophagy / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of magnetic iron nanoparticles ( Fe₃O₄- MNP) in combination with arsenic trioxide and adriamycin on apoptosis and autophagy of Raji cells, a non-Hodgkin's lymphoma (NHL) cell line.</p><p><b>METHODS</b>The growth inhibition rate of Raji cells was analyzed by MTT assay, the cells apoptosis and intracellular concentration of ADM were determined by flow cytometry (FCM), the expression levels of apoptosis-related proteins such as BCL-2, NFκB, Survivin, BAX, P53, and Caspase-3, and related to autophagy-proteins, such as LC3, Beclin-1, and P62/SQSTM1 were detected by Western blot.</p><p><b>RESULTS</b>The growth inhibition of Raji cells in the group of ADM + As₂O₃were higher than that in the group of ADM or As₂O₃alone, however, lower than that in the group of Fe₃O₄- MNP combined with ADM and As₂O₃(ADM+As₂O₃+ MNP) (P < 0.05). The apoptotic rate and accumulation of intracellular ADM in the group of Fe₃O₄- MNP combined with ADM and As₂O₃were significantly higher than those in control, ADM, As₂O₃, and ADM plus As₂O₃groups (P < 0.05). The upregulation of BAX, P53 and Caspase-3 expression and the down regulation of BCL-2, NFκB, and Survivin expression at protein level were more remarkable in the group of ADM+As₂O₃ + MNP, compared with the other groups (P < 0.05). Moreover, the expressions of LC3 and Beclin-1 proteins in the group of ADM+As₂O₃+ MNP were higher, while the expression of P62/SQSTM1 was lower than that in other groups (P < 0.05).</p><p><b>CONCLUSION</b>The Fe3O4 - MNP combined with ADM and As₂O₃can increase the antitumor efficacy on Raji cells by promoting apoptosis and inducing autophagy. It would be a promising strategy for malignant lymphoma therapy.</p>

Anticancer and multidrug-resistance reversing potential of traditional medicinal plants and their bioactive compounds in leukemia cell lines / 中国天然药物

Multidrug resistance remains a serious clinical problem in the successful therapy of malignant diseases. It occurs in cultured tumor cell lines, as well as in human cancers. Therefore, it is critical to develop novel anticancer drugs with multidrug-resistance modulating potential to increase the survival rate of leukemia patients. Plant-derived natural products have been used for the treatment of various diseases for thousands of years. This review summarizes the anticancer and multidrug-resistance reversing properties of the extracts and bioactive compounds from traditional medicinal plants in different leukemia cell lines. Further mechanistic studies will pave the road to establish the anticancer potential of plant-derived natural compounds.

Cytotoxic effects of mono-(2-ethylhexyl) phthalate on human embryonic stem cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Mono-(2-ethylhexyl) phthalate (MEHP), the metabolite of di-(2-ethylhexyl) phthalate (DEHP), was suspected to be toxic to human embryos. This study contributes to investigating its toxic effects by an embryonic stem cell test (EST) based on two human embryonic stem cell (hESCs) lines.</p><p><b>METHODS</b>CH1 established in our own lab and H1, a federally registered cell line were two human embryonic stem cell lines used in this test. Four endpoint measurements were performed consisting of cell viability, proliferation ability, apoptosis as well as changes of gene expression patterns after spontaneous differentiation were determined. For measuring effects on the first three endpoints, the cells were treated with various concentrations of MEHP dissolved in dimethyl sulfoxide (DMSO) and only with DMSO which served as control and harvested after 5 days. For measuring effects during spontaneous differentiation, the RNA of embryoid bodies (EBs) formed after 8 days' MEHP exposure was collected and changes in differentiation specific gene expression patterns were analyzed by quantitative real time RT-PCR.</p><p><b>RESULTS</b>As a result the viability and proliferation ability of both cell lines decreased significantly at 1000 µmol/L MEHP, while there was no effect on apoptosis or cell morphology. In addition MEHP also changed the gene expression pattern in the EBs of both cell lines.</p><p><b>CONCLUSION</b>MEHP in a high dose was cytotoxic and affected the development of hESCs, which indicates its embryo toxicity in human embryos.</p>

Roles of NKG2D in cytokine-induced killer (CIK) against hematological malignant cells lines / 中国实验血液学杂志

This study was purposed to investigate the CIK cell cytotoxicity to hematological malignant cell lines by interaction NKG2D receptors and corresponding ligands. The CIK cells was expanded from healthy individual with interferon (IFN)γ, CD3 monoclonal antibodies (mAb) and interleukin-2 (IL-2). The subset of lymphocyte and the expression of NK cell receptors on CIK cells was detected by flow cytometry; NKG2D ligand expression on hematological malignant cell lines was also analyzed by flow cytometry, the calcein acetoxymethyl ester (CAM) was used for labeling target cells, then the cytotoxicity of CIK cells to hematological malignant cell lines was detected by flow cytometry. The results showed that most of CIK cells expressed CD3 (97.85 ± 1.95%) , CD3(+)CD8(+) cells and CD3(+)CD56(+) cells increased significantly as compared with un-cultured cells (P < 0.001;P = 0.033). About 86% CIK cells expressed NKG2D receptor but no other NK receptors such as CD158a, CD158b and NCR. Different levels of NKG2D ligands were detected in hematological malignant cell lines U266, K562 and Daudi. CIK cells showed high cytotoxicity to these three different cell lines, and this cytotoxicity was partially blocked by treating CIK cells with anti-NKG2D antibody (U266 52.67 ± 4.63% vs 32.67 ± 4.81%, P = 0.008;K562 71.67 ± 4.91% vs 50.33 ± 4.91%, P = 0.007;Daudi 68.67 ± 5.04 vs 52.67 ± 2.60%, P = 0.024) . It is concluded that most of CIK cells express NKG2D receptor, interaction of NKG2D-NKG2D ligands may be one of the mechanisms, by which CIK cells kill hematological malignant cells.

NKG2D-mediated natural killer cell cytotoxicity against myeloid leukemia cells OUN-1 / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate NK cell cytotoxicity to leukemic cell by NKG2D receptors and NKG2D ligands interaction upregulated by hydroxyurea (HU).</p><p><b>METHODS</b>Leukemic cell lines OUN-1 and primary leukemic cells were cultured for 24 hours in the presence of HU, then the NKG2D ligands expressions were analyzed by flow cytometry (FCM). Isolated NK cells from healthy individual cultured for 72 hours in presence of IL-2 were used as effect cell, and leukemic cell line OUN-1 treated with HU was used as target cell, NK cell cytotoxicity against leukemic cell line was assessed using chromium-51 release assay.</p><p><b>RESULTS</b>Leukemic cell lines showed upregulation of MIC A/B (MFI: 8.9 ± 0.9 vs 23.5 ± 3.4, P = 0.01) and ULBP2 (MFI: 14.5 ± 0.6 vs 33.5 ± 4.8, P = 0.03) following incubation with HU. HU also upregulated the NKG2DLs on primary leukemia cells from patients with acute myeloid leukemia. Treatment of OUN-1 with HU significantly increased the cytotoxicity of NK cells isolated from healthy individual \[(62.0 ± 5.6)% vs (76.0 ± 5.3)%, P = 0.02\], and the enhancing effect of HU was partly blocked by anti-NKG2D Abs \[(76.0 ± 5.3)% vs (46.0 ± 4.5)%, P = 0.00\].</p><p><b>CONCLUSION</b>HU selectively upregulated NKG2D ligand expression on leukemic cell lines, and enhanced NK cell cytotoxicity against leukemic cells through NKG2D receptors and NKG2D ligands interaction.</p>

Reversal effect of gambogic acid on multidrug resistance of K562/A02 cell line / 中国实验血液学杂志

This study was purposed to investigate the reversal effect of gambogic acid (GA) on multidrug resistance of K562/A02 cells and its mechanism. The IC(50) (half maximal inhibitory concentration) of adriamycin (ADM) was evaluated by MTT. Cell apoptosis was detected by flow cytometry. Morphological changes of K562/A02 cells were observed by fluorescent microscopy with DAPI staining. The expressions of Survivin and P-gp were determined by Western blot. The results showed that the IC(50) of ADM on K562 and K562/A02 cell proliferation were (1.42 ± 0.07) µg/ml and (28.42 ± 1.40) µg/ml respectively. GA ≤ 0.0625 µmol/L had no inhibitory effect on proliferation of K562 and K562/A02. 0.0625 µmol/L GA could enhance the sensitivity of K562/A02 cells to ADM (P < 0.05) and the reversal multiples was 1.53. The apoptotic rate was raised after treating with ADM combined with 0.0625 µmol/L GA for 48 h (P < 0.05). Morphological differences were typical and obvious between cells of control and treated groups under fluorescence microscopy using DAPI staining. After treating K562/A02 cells with ADM combined with 0.0625 µmol/L GA for 48 h, the expressions of Survivin and P-gp were down-regulated at protein levels. It is concluded that GA can enhance the sensitivity of K562/A02 cells to ADM, which may be related to increasing cell apoptosis and down-regulating expressions of Survivin and P-gp.

Mechanisms of tetrandrine and 5-bromotetrandrine in reversing multidrug resistance may relate to down-regulation of multidrug resistance associated protein 7 expression / 中国实验血液学杂志

Both tetrandrine (Tet) and 5-bromotetrandrine (BrTet) can effectively reverse P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). The structure of multidrug resistance associated protein 7 (MRP7) has its own specificity and difference compared with other members of the MRP family. This study was aimed to investigate whether Tet and BrTet can inhibit the expression level of MRP7 so as to further look into the mechanisms of the reversal effects of Tet and BrTet on MDR. The inhibitory effects of daunorubicin (DNR) used alone on the proliferation of K562 and K562/A02 cells were evaluated by MTT assay, the IC(50) of DNR and drug resistant folds were calculated. The mRNA level of MRP7 was tested by real-time PCR, and the protein levels of MRP7 and P-gp were tested by Western blot. The DNR accumulation was analyzed by flow cytometry (FCM). The results showed that the resistance of K562/A02 cells to DNR was 23.65-folds of that of K562 cells. After administration of 1 µmol/L Tet or 2 µmol/L BrTet, the mRNA level of MRP7 in the K562/A02 cells decreased to 2% and 12% respectively, and the protein level of MRP7 decreased by 53.2% and 83.7% respectively. The protein level of P-gp decreased by 58.47% and 52.20% in the 1 µmol/L Tet and 2 µmol/L BrTet groups. FCM detection showed that 1 µmol/L Tet and 2 µmol/L BrTet significantly increased the accumulation of DNR in K562/A02 cells by 94.32% and 271% respectively. It is concluded that Tet and BrTet both can reverse MDR in vitro. The mechanisms may be related to the inhibition of MRP7 overexpression and the increase of anticancer drug concentration in cells. At the same molar concentration, the effects of Tet and BrTet in inhibiting the protein level of MRP7 expression do not show significant difference.

Effects of 5-bromotetrandrine and daunorubicin on apoptosis and expression of survivin in K562/A02 cells / 中国实验血液学杂志

The aim of this study was to investigate the potential benefit of combination therapy with 5-bromotetrandrine (5-BrTet) and daunorubicin (DNR) on chronic leukemia. The apoptosis of K562/A02 cells treated by DNA, BrTet and BrTet combined with DNR for 48 hours was detected by flow cytometry; the expressions levels of survivin mRNA and protein K562/A02 cells treated by DNR, BrTet and BrTet combined with DNR and in untreated K562 cells for 48 hours were measured by RT-PCR and Western blot respectively. The results showed that the combination of BrTet with DNR increased apoptotic rate of K562/A02, down-regulated the expression levels of survivin mRNA and protein in K562/A02 cells as compared with blank control and cells treated by BrTet or DNR alone, the survivin expression in K562/A02 cells was higher than that in K562 cells. It is concluded that the combination of BrTet with DNR can effectively reverse the multidrug resistance of K562/A02 cells, promote the apoptosis of K562/A02 cells, the mechanism of which may be related with down-regulation of survivin expression. Survivin may be a target for the treatment of MDR in hematopoietic malignancies.

Analgesic effect of Corydalis yanhusuo in a rat model of trigeminal neuropathic pain / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the analgesic effect of Corydalis yanhusuo on trigeminal neuropathic pain.in a rat model.</p><p><b>METHOD</b>Rat model of trigeminal neuralgia pain were established by inducing chronic constriction injury (CCI) of the infraorbital branch of the trigeminal nerve (ION). The effect of Corydalis yanhusuo, a traditional Chinese medicine, in ameliorating the pain was tested. Western blotting was performed to investigate the change of cannabinoid CB1 receptors in the Vc the injury of the infraorbital branch of the trigeminal nerve (ION-CCI). CB1 receptor antagonist AM 251 was applied to observe its effect on the analgesic effect of Yanhusuo.</p><p><b>RESULT</b>Administration of dl-THP (2 mg/kg) intraperitoneally increased the response threshold and the cut-off threshold to the mechanical stimulation in ION-CCI rat models. ION-CCI induced an upregulation of cannabinoid CB1 receptors within the ipsilateral of Vc. The effect of Yanhusuo was antagonized by the application of AM 251.</p><p><b>CONCLUSION</b>The analgesic effect of Yanhusuo involves the participation of CB1 receptors, suggesting that Yanhusuo may offer a useful therapeutic approach for trigeminal neuropathic pain.</p>

Determination of guanfu base A hydrochloride in plasma by LC-MS method and its pharmacokinetics in dogs / 药学学报

<p><b>AIM</b>To establish an analytical method for determination of guanfu base A (GFA) concentration in plasma and to study its pharmacokinetic profile in dogs.</p><p><b>METHODS</b>Six dogs were given a 7.56 mg.kg-1 dose intravenously. Blood samples were collected at various time-points after drug administration. Analytical method based on liquid chromatography-mass spectrometry (LC-MS) was established to determine the plasma concentration of GFA. Pharmacokinetic evaluation was carried out using the 3P87 program.</p><p><b>RESULTS</b>The calibration curves were linear over the concentration range from 0.42 microgram.mL-1 to 21.2 micrograms.mL-1 (gamma = 0.9994). The intra-day and inter-day precisions were generally good (< 15%) at low, medium and high concentrations. The overall recovery of the analytes was more than 80%. Six dogs were given an i.v. dose of 7.56 mg.kg-1 of GFA hydrochloride, an open three compartment model best described the concentration-time profiles for GFA. The half-lives for the rapid and slow distribution phase and terminal elimination phase (T1/2 pi, T1/2 alpha and T1/2 beta) were 0.07 h, 1.5 h, and 13.5 h, respectively. The total area under the plasma concentration-time curve (AUC), the volume of the central compartment (Vc), and plasma clearance (CLs) were 61.43 micrograms.h.mL-1, 0.37 L.kg-1 and 0.14 L.kg-1.h-1, respectively.</p><p><b>CONCLUSION</b>The analytical method was shown to be sensitive, specific, rapid and reproducible, and was suitable for pharmacokinetic studies of GFA.</p>