Dihydroartemisinin Induces Apoptosis of Human Acute T Lymphocytic Leukemia Cells by Activating Oxidative Stress / 中国实验血液学杂志

OBJECTIVE@#To investigate the effects of dihydroartemisinin (DHA) on the proliferation and apoptosis of human T-cell acute lymphoblastic leukemia (T-ALL) Jurkat cell.@*METHODS@#The effects of DHA on the proliferation of Jurkat cells and the recovery of DHA-inhibited cell viability by N-acetyl-L-cysteine (NAC) were examined by CCK-8 assay. Flow cytometry was performed to analyze the cell apoptosis and generation of reactive oxygen species (ROS). Western-blot was used to detected protein expression of DNA damage-related genes, as well as apoptosis-associated genes, respectively.@*RESULTS@#DHA inhibited the proliferation of Jurkat cells, and shows a concentration-dependent manner(r =0.936), and NAC could partially restore the activity of DHA on cell proliferation inhibition. With the increase of drug concentration, the apoptosis rate (r =0.946) and ROS accumulation was increased (r =0.965). Western blot showed that the protein expressions of DNA damage-related gene γ-H2AX and apoptosis-related genes p53, c-Caspase3, BAX and cPARP were significantly increased, and BCL-2 protein expression was decreased.@*CONCLUSION@#DHA can induce ROS production in Jurkat cells, which can cause DNA damage, activate the P53 apoptotic pathway, and promote apoptosis of cells.

Dihydroartemisinin Induces AML cell Apoptosis by Inhibition of PTEN/AKT pathway / 中国实验血液学杂志

OBJECTIVE@#To study the effects of dihydroartemisinin (DHA) on the proliferation and apoptosis of acute myeloid leukemia (AML) cells.@*METHODS@#The effects of DHA on the proliferation of acute myeloid leukemia cells and the inhibitory effect of Z-VAD-FMK on the DHA-induced cell apoptosis were detected by CCK-8 assay. The expression level of cleaved-caspased 3 was detected by indirect immunofluorescence. Western blot was used to quantify the protein expression of PTEN, p-Akt, AKT, β-actin, and the apoptosis-associated proteins, such as C-PARP, Cleaved-caspase3 and Caspase3 respectively.@*RESULTS@#DHA induced the AML cell apoptosis with concentration-dependent manner (r=-0.959, r=-0.956). The DHA could induce the accumulation of cleaved-caspase 3 and C-PARP in AML cells, activate PTEN gene and inhibited Akt phosphorylation. Apoptosis inhibitor Z-VAD-FMK could partially restored the activity of DHA-inhibited cell proliferation.@*CONCLUSION@#Dihydroartemisinin induces AML cell apoptosis by inhibition of PTEN/AKT pathway. Dihydroartemisinin is expected to be a safe and effective drug for treatment of acute myeloid leukemia.

Mechanism of Sorafenib and Decitabine Inducing Apoptosis of Diffuse Large B-Cell Lymphoma Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of sorafenib combined with decitabine on the viability and apoptosis of diffuse large B-cell lymphoma cell line OCI-LY1 and its mechanism.@*METHODS@#Sorafenib at 1.5μmol/L or decitabine at 25μmol/L was used to treat the cells alone or in combination. The viability of OCI-LY1 cells was detected by CCK8 assay; the PI positive cells were observed by fluorescence microscopy; the cell proliferation and ROS levels were measured by flow cytometry; The expression levels of proteins related to apoptosis were detected by Western blot.@*RESULTS@#Compared with the control group, treatment with sorafenib and decitabine alone or in combination inhibited the cell proliferation, activated ROS formation and induced apoptosis finally. Sorafenib in combination with decitabine produced a synergistic effect. Western blot analysis showed that sorafenib combined with decitabine significantly up-regulated the levels of Bax/Bcl-2, P53, C-Caspase3 and C-PARP and activated apoptosis by inhibiting PI3K-AKT pathway.@*CONCLUSION@#Sorafenib combined with decitabine induces the apoptosis of diffuse large B-cell lymphoma cell line OCI-LY1 by inhibiting PI3K-AKT pathway and activating P53.

Study of RON mediated invasion of Raji cell line and drug-target effects / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the proto-oncogene RON mediated aggression of Raji cells and the inhibitory effects by monoclonal antibody Zt/f2 (2f2).</p><p><b>METHODS</b>The effects of RON ligand macrophage stimulating protein (MSP) (2.0 nmol/L) and inhibitory Zt/f2 (2F2) (2.0 nmol/L) antibody on proliferation of RON positive Raji cells after treatment for 24 and72 hours were detected by MTT method, colony formation units (CFU) of Raji cells by methylcellulose semi solid culture, Raji cells apoptosis and cell cycle analysis by AnnexinV/PI double staining, expression of RON, apoptosis-related proteins, and cyclins by Western blot.</p><p><b>RESULTS</b>(1)Compared with the cell viability (1.0) and counts of CFU (103.6±7.0) in control group, Raji cells after MSP treatment had better viability (1.35±0.20) and CFU counts (133.7±10.4) (P<0.05), but worse viability (0.68±0.11) and CFU counts (66.3±6.1) after Zt/f2 (2F2) treatment (P<0.05). (2)Percentage of Raji cells apoptosis after Zt/f2 (2F2) antibody treatment (12.16±2.33)% was significantly increased than the control (2.89±1.03)% (P<0.05). The percentage of Raji cells arrested in G0/G1 phase was increased after Zt/f2 (2F2) antibody treatment as compared to the control [ (54.96 ±3.70)% vs (39.10±2.30)%, (P<0.05) ]. (3) High-level of RON phosphorylation and β-catenin expression activated by MSP could be inhibited significantly by Zt/f2 (2F2), which also up-regulated the expression of caspase-3, caspase-8, caspase-9 and PARP and down-regulated anti-apoptotic MCL-1 gene and inhibitor of apoptosis protein XIAP expression, accompanied with G1 phase protein changes accordingly.</p><p><b>CONCLUSION</b>MSP could aggravate Raji cells proliferation. Inversely, Zt/f2 (2F2) could inhibit proliferation and induce apoptosis by inhibition of RON phosphorylation and up-regulation of apoptosis related proteins.</p>

Cordyceps sinensis polysaccharide enhances apoptosis of HL-60 cells induced by triptolide / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of polysaccharide fraction of Cordyceps sinensis (PSCS) on triptolide (TPL)-induced apoptosis in the HL-60 cells and the involved molecular mechanism.</p><p><b>METHODS</b>The cultured leukemia HL-60 cells were divided into three groups: control group, TPL group (cells were treated with 5 ng/ml TPL only), and PSCS+TPL cells group (cells treated with 5 ng/ml TPL and 100 microg/ml or 200 microg/ml PSCS for 18 h). Cell viability was tested by MTT assay and apoptotic cells were quantitatively measured by flow cytometry with Annexin V/PI double stain.The expressions of Caspase-3, 6, 7, 9 and NF-kappa B proteins were tested by Western blot.</p><p><b>RESULT</b>MTT assay showed that different concentrations of PSCS inhibited the cell viability. Flow cytometry indicated that TPL markedly increased the apoptosis rate of the HL-60 cells, and PSCS enhanced the apoptosis in a dose-dependent manner. Western blot showed that TPL did not inhibit the expression of the Caspase-3, 6, 7, 9 and NF-kappa B proteins, and when cells were treated with PSCS, the expression of proteins decreased with the PSCS concentration rising.</p><p><b>CONCLUSION</b>PSCS can enhance TPL-induced apoptosis in HL-60 cells and inhibit the expression of NF-kappa B and Caspase 3,6,7,9,which might be the possible signaling pathway of inducing apoptosis.</p>

beta-elemene enhances aclarubicin-induced apoptotic effect in HL-60 cells and its mechanism / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effects of beta-elemene combined with aclarubicin on the induction of HL-60 cell apoptosis and its mechanisms in antileukemia therapy.</p><p><b>METHODS</b>HL-60 cells were treated for 20 hours with different dose of aclarubicin (0.05, 0.10, 0.25 microg/ml) or with different concentrations of beta-elemene (10, 20, 40 microg/ml) in the presence or absence of aclarubicin (0.10 microg/m). The apoptotic rate was analyzed by flow cytometry (FCM), the productions of PGE2 in culture supernatants was detected by competitive ELISA and the expressions of COX-2 and NF-kappaB activity in HL-60 cells by Western blot.</p><p><b>RESULTS</b>Lower concentration of aclarubicin (0.05, 0.10 microg/ml) didn't affect apoptotic rate, and COX-2, NF-kappa B and PGE2 expression on HL-60 cells. Combined treatment of beta-elemene and aclarubicin (0.10 microg/ml) enhanced the apoptotic effect and down-regulated COX-2, NF-kappaB and PGE2 expressions. There was a positive correlation between the effects and beta-elemene concentrations.</p><p><b>CONCLUSION</b>beta-elemene enhances aclarubicin-mediated apoptotic effect, down-regulation of COX-2 and their inducing products PGE2 in HL-60 cells by suppressing activitation of NF-kappaB.</p>

A study of CXCR4/SDF-1 in hepatocellular carcinoma and liver cirrhosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the expressions of stromal cell-derived factor-1 (SDF-1) and CX chemokine receptor-4 (CXCR-4) in patients with hepatocellular carcinoma (HC) and liver cirrhosis.</p><p><b>METHODS</b>Peripheral blood and/or ascites fluid were collected from 39 hepatocellular carcinoma patients, 16 patients with liver cirrhosis, 12 with hepatitis and 12 healthy donors. The SDF-1 expression was assayed by ELISA and CXCR-4 was measured by immunohistochemical methods.</p><p><b>RESULTS</b>The level of SDF-1 expression in the carcinoma patients was higher than that of the liver cirrhosis, hepatitis patients and healthy donors, but there was no significant difference between those of the healthy donors and hepatitis patients or liver cirrhosis patients. The levels of CXCR-4 expression were closely related to the tumor differentiation.</p><p><b>CONCLUSION</b>The expression of SDF-1 in the peripheral blood and the CXCR4 expression in the HCC tissues of the HC patients may be regarded as markers of HC and they may have a positive relationship with the differentiation and metastasis of HC.</p>

Construction of expression vector of hTERT/hIL-18 fusion gene in eukaryotic cells and its function / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct expression vector of hTERT-hIL-18 fusion gene in eukaryotic cells and to study its biological function.</p><p><b>METHODS</b>hIL-18 gene was amplified by RT-PCR, then T-A cloned and inserted into PCDNA3.1(+)/hTERT vector. The sequence of fusion gene was examined by enzyme incision and DNA sequencing. The vector with fusion gene was transformed into 3T3 cells by the method of lipofecting, and proved by Western blot. The secretion gamma-interferon was measured with ELISA and cell apoptosis was detected with flow cytometry.</p><p><b>RESULT</b>Expression vector PCDNA3.1(+) of hTERT/hIL-18 fusion gene was constructed successfully. The correct sequence was proved by enzyme incision and sequencing and there was a correct open reading frame. Fusion protein of hTERT/hIL-18 was effectively expressed in eukaryotic cells and was proved by Western blot and immunofluorescence stain. The fusion protein stimulated KG-1 cells to secrete gamma-interferon and had anti-apoptosis effect.</p><p><b>CONCLUSION</b>Fusion protein hTERT-hIL-18 is highly effectively expressed in eukaryotic cells and is biologically active.</p>

Effects of ST1571 on the development of dendritic cells derived from bone marrow mononuclear cells in patients with chronic myeloid leukemia / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effects of ST1571 on the development of dendritic cells (DC) derived from bone marrow mononuclear cells of patients with chronic myeloid leukemia (CML).</p><p><b>METHODS</b>Bone marrow mononuclear cells (BMMNC) from CML patients and healthy volunteers were cultured initially using multiple cytokine combinations as follows: recombinant human granulocyte/ macrophage colony-stimulating-factor (rhGM-CSF) plus recombinant human interleukin-4 (rhIL-4) as CML and normal control groups, rhGM-CSF plus rhIL-4 and ST1571 as CML experimental groups, and from day 8 recombinant human tumor necrosis factor-alpha ( rhTNF-alpha) was added to stimulate DC maturation. The morphologic features of cells were observed by Wright's staining and phenotypes were assessed by flow cytometry. Cytogenetic analysis was performed by fluorescence in-situ hybridization (FISH), and the antigen-presenting function was assayed by mixed lymphocyte reaction (MLR). The concentration of VEGF was detected by ELISA.</p><p><b>RESULTS</b>CML experimental groups treated with STI571 displayed morphological features similar to those of control groups with delicate membrane projections. However, in comparison with the CML control groups, the CML experimental groups showed an increased expression of CD80, CD86, CD83 and HLA-DR and showed more intense abilities of allogeneic antigen presentation, which were similar to those of normal control groups. FISH confirmed that DCs of both CML, groups were of leukemic origin. The concentration of VEGF was dramatically reduced in CML experimental groups.</p><p><b>CONCLUSION</b>In vitro, STI571 promotes the activation/maturation of DCs derived from BMMNCs of patients with CMI, and decreases VEGF production by the leukemic cells. The promotion of DC maturation may be partially due to decreased inhibitory effect of VEGF.</p>

Biological features of dendritic cells derived from chronic myeloid leukemia cells in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To induce primary chronic myeloid leukemia (CML) cells into dendritic cells (DCs).</p><p><b>METHODS</b>Bone marrow mononuclear cells (MNCs) were isolated from 13 CML patients and peripheral blood MNCs from 5 healthy donors. The isolated MNCs were co-cultured with rhGM-CSF 1,000 U/ml, rhIL- 4,500 U/ml and TNF-alpha 50 U/ml for 10 days. The morphological features were observed by Wright's staining,inverted microscope and electron microscope. CD(80), CD(86), CD(83), CD(1a) and HLA-DR expression were assayed by flow cytometry, cytogenetic analysis was performed by fluorescence in-situ hybridization(FISH). The concentration of IL-12 was measured by ELISA and the function of antigen presenting was tested by mixed lymphocyte reaction (MLR).</p><p><b>RESULT</b>After being cultured with cytokines, the typical dendritic appearance with delicate membrane projections was observed. The CD(80), CD(86), CD(83), CD(1a) and HLA-DR markers and capacity of stimulating allogeneic T cells were upregulated significantly. FISH confirmed that the DCs were generated from leukemic origin and CML DCs could secrete higher level of IL-12 than CML MNCs. There were no differences in morphology and immunophenotype expression between DCs derived from CML and those from normal individuals. However, DCs from CML patients displayed weaker activity than that of normal individuals when tested in MLR.</p><p><b>CONCLUSION</b>CML cells could be induced into leukemia-DCs by co-culture with cytokines.</p>