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Aim: To investigate the effect of proteasome inhibitor MG132 on the proliferation and apoptosis of acute myeloid leukemia cells. Methods: qRT-PCR was used to detect the expression of ADRM1 mRNA in nine blood tumor cell lines. The expression of ADRM1 in HL60 cells was interfered by shRNA; HL60 cells before and after ADRM1 interfered were treated with different MG132 concentrations for 24 h; Then, the cell proliferation and viability were measured with CCK-8 by microplate reader. Meanwhile, the expressions of ADRM1 and UCH37 protein were detected by Western blot. Apoptosis of HL60 and NB4 cells treated with different MG132 concentrations was analyzed by flow cytometry. Results: ADRM1 mRNA was up-regulated in blood tumor cell lines. ADRM1 shRNA and scrambled shRNA HL60 cells were successfully constructed. Cell proliferation and viability were inhibited by AD-RM1 shRNA interference or decreased with the increase of MG132 concentration; meanwhile, ADRM1 and UCH37 protein expressions were down-regulated. The apoptosis of HL60 and NB4 cells increased with the increase of MG132 concentrations. The apoptotic effect of MG132 on HL60 cells was stronger than that of NB4 cells. Conclusions: ADRM1 mRNA is overexpressed in blood tumor cell lines; ADRM1 down-regulation induces UCH37 protein decrease and cell proliferation inhibition. MG132 induces AML cell apoptosis and restrains the proliferation and viability through down-regulating the expression of ADRM1 and UCH37 protein. The apoptotic effect of MG132 on different types of AML cells exists individual differences.
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Objective There are few studies on the in vitro induction of human leukemia cell line THP-1 macrophages by Staphylococcus aureus PV leukin S component (LukS-PV). This study aims to investigate whether LukS-PV could induce the polarization of Human THP-1 derived macrophage in vitro. Methods THP-1 monocytes were induced to macrophages by Phorbol myristate acetate (PMA). The cell surface markers CD11b and CD14 were detected by flow cytometry at 48h and 72h after the induction, respectively. Using 0.1, 0.2 and 0.4 μmol/LlukS-PV treated THP-1 macrophages as the 0.1 μmol/LLukS-PV group, 0.2 μmol/LLukS-PV group, and 0.4 μmol/LLukS-PV group, respectively. The THP-1 driven macrophage induced by PMA was used as the control group. Flow cytometry was used to detect the expression of CD80 and CD206 in each group at 24h and 48h. The mRNA expression levels of IL-12, TNF-α and TGF-β were detected by real-time fluorescence-based quantitative PCR (qRT-PCR). Results The expression of CD80 on the cell surface in the 0.1 μmol/LLukS-PV group, 0.2 μmol/LLukS-PV group and 0.4 μmol/LLukS-PV significantly increased (P <0.01), with the highest expression in the 0.2 μM LukS-PV group (P<0.01), when comparing to that of the control group. The mRNA expressions of IL-12 and TNF-α in the 0.2 μM LukS-PV group significantly increased (7.32±0.91 and 5.29±0.51, respectively) at 24h (P <0.01), and raised in 0.2 μM LukS-PV group (11.16±0.78、6.70±0.68) at 48h (P <0.01), when comparing to those of control group (0.71±0.11 and 0.73±0.14, respectively). Compared with the control group, expression of CD80 on cell surface in the 0.2 μM LukS-PV group significantly increased at 24h and 48h (P <0.000). Conclusion LukS-PV could stimulate the polarization of human THP-1 macrophages toward M1.
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Objective To explore the role of murine double minute 2 (MDM2) in apoptosis of acute lymphoblastic leukemia EU-4 cells induced by berberine.Methods EU-4 cells at logarithm growth phase were chosen to carry out the experiments.EU-4 cells were incubated with different doses of berberine for 72 hours,and the final doses of berberine were 0,1,10 and 100 μmoL/L,respectively.Then the apoptosis rate of EU-4 cells were detected by flow cytometry,and the expression levels of MDM2 protein were tested by Western blot.The levels of MDM2 gene were examined by means of real-time PCR at 0,24,48 and 72 hours after incubation of EU-4 cells with 100 μmol/L berberine,respectively.EU-4 cells in MDM2 small interfering RNA(siRNA) group were transfected with 150 nmol/L MDM2 siRNA,and the control siRNA group were transfected with 150 nmol/L control siRNA.The apoptosis rates of EU-4 cells were detected 48 hours after transfection by flow cytometry.Results The apoptosis rates of EU-4 cells were increased in a dosedependent manner,and the highest apoptosis rate was up to(60.13 ± 4.21)％,which was incubated with 100 μmol/L berberine.Significant differences were found in all groups (F =280.56,P ＜ 0.05).Berberine treatment suppressed the protein expression of MDM2 in a dose-dependent manner(F =73.82,P ＜ 0.01).The mRNA expression of MDM2 was inhibited in a time-dependent manner by 100 μmol/L berberine(F =45.37,P ＜0.01).The apoptosis rates of control siRNA group and MDM2 siRNA group were(11.09 ± 1.63) ％ and (29.84 ± 1.75) ％,respectively.Significant difference was found between the 2 groups (t =-13.57,P ＜ 0.01).Conclusions Berberine can inhibit the expression of MDM2 in a dose-and time-dependent manner,which may be one of the mechanisms of apoptosis induced by berberine in leukemia cells.
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Objective To investigate the effect and the mechanism of different G-CSF-priming protocols on leukemia cell lines (HL-60 and U937) in vitro and provide the clinical guidance to clinical treatment of acute leukemia.Methods The leukemia cell lines HL-60 and U937 were used as model to detect the effects of three drugs alone and combined two drugs (HA) or three drugs (HAG) respectively.Cell viability and cell growth inhibition were performed by cell count kit-8 (CCK-8) assay.Apoptotic marker AnnexinV/PI,cell membrane surface antigen CD11b,cell cycle,mitochondrial membrane potential (JC-1) and Caspase-3 were determined by flow cytometry.Results After using of HAG for 48 h,HL-60 and U937 cells counts were decreased significantly and the apoptotic marker Annexin V was significantly increased. To compare the single drug group with two drug combination group,the result was significantly different (P ＜0.05),and the apoptosis of U937 cells was higher than HL-60 cell line.CD11b expression among the three groups did not change (P ＞ 0.05).Using of CAG and MAG,the mitochondrial nembrane potential of HL-60 and U937cells was increased,the three-drug combination group was significantly higher than single-drug group and control group (P ＜0.05); Caspase-3 was activated,the fluorescence intensities of Caspase-3 of the three-drug combination group and single drug group were significantly higher (P ＜0.05) comparing with the control group.Conclusion HAG regimen could induce leukemia cells to apoptosis through the reduction of mitochondrial membrane potential and the activation of Caspase-3 to induce apoptosis of leukemia cells.
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Objeetive To observe NB4 cells that is treated with both ATO and BOR.Methods MTT assay demonstrated that NB4 cell lines,growth inhibiting effect after interfered with BOR alone or BOR plus ATO.Flow cytometry detect cell cycle and apoptosis.RT-PCR method detect the expression of survivin mRNA.Results MTT assay showed depressant effect by BOR.BOR can strengthen cells,lethal effect by ATO.Flow cytometry investigate obvious apoptosis and cycle blockage of G2/M stage.RT-PCR discover that ATO or BOR can downregulate the expression of survivin mRNA alone,there is an additional effect if used them at the same time.Conclusion Combination of BOR and ATO have stronger inflictive and apoptosisinducing activity.The blockage of G2/M stage and degression of survivin mRNA is a possible mechanism that leads to cell apoptosis treated with BOR and ATO.
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Objective To optimize experimental parameters for the photosensitization of 5-aminolevulinic acid (ALA) in promyelocytic leukemia cell HL60 and compare them with normal human peripheral blood mononuclear cell (PBMC). Methods ALA incubation time, wavelength applied to irradiate, concentration of ALA incubated, irradiation fluence may modulate the effect of 5-aminolevulinic acid based Photodynamic Therapy (ALA-PDT).The high-pressure mercury lamps of 400W served as light source, the interference filter of 410nm, 432nm, 545nm, 577nm were used to select the specific wavelength. Fluorescence microscope was used to detect the fluorescence intensity and location of protoporphyrin IX (PpIX) endogenously produced by ALA. MTT assay was used to measure the survival of cell. Flow cytometry with ANNEXIN V FITC kit (contains annexin V FITC, binding buffer and PI) was used to detect the mode of cell death. Results ① 1mmol/L ALA incubated 1×105/mL HL60 cell line for 4 hours, the maximum fluorescence of ALA induced PpIX was detected in cytomembrane. ② Irradiated with 410nm for 14.4J/cm2 can result in the minimum survivability of HL60 cell. ③ The main mode of HL60 cell death caused by ALA-PDT is necrosis. Conclusion ALA for 1mmol/L, 4 hours for dark incubation time, 410nm for irradiation wavelength, 14.4J/cm2 for irradiation fluence were the optimal parameters to selectively eliminate promyelocytic leukemia cell HL60 by ALA based PDT. The photosensitization of ALA based PDT caused the necrosis of HL60 cell, so it could be used for inactivation of certain leukemia cells.
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Objective To explore the silence of B-cell specific Moloney murine leukaemia virus insertion site 1(Bmi-1)by RNA interference on the proliferation of human leukemia cell line K562 and its mechanisms.Methods Small interfering RNA(siRNA)targeting Bmi-1 gene was designed and double-stranded siRNA was chemically synthesized.After double-stranded siRNA was transfected into K562 cells with Lipofectamine 2000,the proliferation of K562 cells was detected by MTT colorimetry,cell cycle was determined by flow cytometry,and the expression of Bmi-1 and P16 were analyzed by Western blotting.Results The siRNA targeting human Bmi-1 gene effectively prolonged the double time of K562 cells,increased the percentage of cells at G1 phase,and the expression of Bmi-1 was significantly down-regulated but the expression of P16 was up-regulated.Conclusion The siRNA targeting human Bmi-1 gene inhibits the proliferation of K562 cells,and up-regulates the expression of P16 in the cells.
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Purpose: To study new treatment of leukemia with both bcr-abl and mdrl positive. Methods: We detected the effect of STI571 ( 1 ?mol/L), an inhibitor of tyrosine kinase, in combination respectively with vincristine (VCR), daunorubicin (DNR), homoharringtonin( HHT) ( 10-4,10-5, 10-6, 10-7,10-8 mol/L) and DNR + arabinoside cytosine (Ara-C) of 1 mmol/L on a high tumorigenicity in nude mice multi drug-resistant leukemia cell line ( K562-n/VCR) by MTT method. Results: IC50 of VCR and VCR + STI571 were 127.28 ?mol/L, 1. 37 ?mol/L respectively, and synergistic interaction on K562-n/VCR cells was 93. 04-fold. IC50 of DNR and DNR + STI571 were 6. 96?mol/L, 0. 30?mol/L respectively, synergistic interaction was 23. 35-fold. IC50 of HHT and HHT + STI571 were 156.70?mol/L, 7.916?mol/L respectively, synergistic interaction was 19. 80-fold. IC50 of DNR + Ara-C and DNR + Ara-C + STI571 were 0. 10 ?mol/L, 0. 015 ?mol/L respectively, the synergistic interaction was 464-fold. Chemotheraputic agents have not intensive cytotoxic effect on K562-n/VCR cells, but the cytotoxic effect became greater when combined with STI571. Conclusions: Combination of STI571 with DNR, VCR, HHT and DNR + Ara-C had a greater synergistic inhibiting effect on K562-n/ VCR cells . The combinations of STI571 and these Chemotheraputic agents would display synergistic activity in bcr-abl and mdrl positive leukemia cells.
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PURPOSE To sutdy the effects of dibutyl pathalate (DBP) on the proliferation and apoptosis of leukemic cells from patients suffering from acute leukemia. METHODS The cell culture in vitro was used to study the effect of DBP on the proliferation of leukemic cells, while agrose gel electrophoresis of DNA fragments was used to study the effect of DBP on apoptosis of leukemic cells. RESULTS DBP could inhibit the proliferation of leukemic cells got from patients suffering from acute leukemia in vitro. The percentage of inhibition were 24.24% and 36.33% respectively when leukemic cells were exposed to 50?g/ml and 200?g/ml DBP for 24 hours. Leukemic cells from acute myelogenous leukemia were more susceptive to DBP than those from acute lymphocytic leukemia. DBP also induced the leukemic cells from patients to die via apoptosis and showed typical "ladder" of DNA fragments for apoptotic cells in agrose gel electrophoresis. CONCLUSION DBP could clear the leukemic cells in bone marrows of patients suffering from acute leukemia by suppressing the growth of leukemic cells and inducing them to die via apoptosis.
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Background and purpose:Drug-resistance is the main obstacle in terms of efficacy of chemotherapy for leukemia, RNA interference(RNAi) strategy possesses the characteristics of specilization, high-efficiency and low-toxicity, and can effectively and specifically inhibit the overexpression of given gene. This study was designed to investigate the effect of small interfering RNA (siRNA) on expression of mdr1 gene and drug-resistance in multidrug-resistant human leukemia K562/ADM cell.Methods:Human multidrug-resistant leukemia cell line K562/ADM over-expressing mdr1 gene was used as the target cells, Two siRNAs (si-mdr1-1 and si-mdr1-2) targeted mdr1 gene were chemically synthesized and transfected into K562/ADM cells. Expression of mdr1 mRNA was determined by RT-PCR, P-glycoprotein (P-gp) expression was measured using flow cytometry (FCM), and the sensitivity of K562/ADM cells to adriamycin was assessed with a MTT colorimetric assay.Results:Two siRNAs (si-mdr1-1 and si-mdr1-2) specially designed in this study could markedly down-regulate the expression of mdr1 mRNA and its product P-gp in K562/ADM cells. After cells transfected with si-mdr1-1 or si-mdr1-2 for 24h and 48h, the inhibition of mdr1 mRNA expression in the cells for si-mdr1-1 was 55.5% and 22.5%; and for si-mdr1-2, 16.0% and 57.6%, respectively. Treated with siRNA for 72h, the expression intensity of P-gp in the two transfected cell lines decreased 74% and 85%, respectively. Both si-mdr1-1 and si-mdr1-2 significantly enhanced the sensitivity of K562/ADM cells to adriamycin and reversed their drug-resistance, the reversal efficiency was 2.52-folds and 1.96-folds, respectively.Conclusions:The siRNA could effectively and specifically silence the expression of mdr1 gene and overcome the drug-resistance mediated by P-gp in K562/ADM cells.
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Objective: To study the effect of HSVtk/GCV system on ascites tumors. Methods: Using the methods of modified XTT assay, animal experiment, transmission electron microscopy (TEM ) and flow cytometric ( FCM) assay. Results: The sensitivity of P388tk cell was about 37-fold than that of their patental P388 cell. Furthermore we found P388tk cells exhibited potent bystander killing. In vivo , the growth of tumors, which were produced by injecting P388tk cells or a mixture of 50% P388tk cells and 50% P388 cells into DBA/2 mice, was inhibited , furthermore mice survival periods were prolonged contrasted to control groups. Conclusion: HSVtk/GCV suicide gene system could effectively killed the HSVtk-gene-positive P388tk cells and nearby HSVtk-gene-negative cells by the bystander effect in vitro and in vivo.
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Objective: To investigate the effect of BSO on DAAO/D-Ala system killing K562e cells. Methods: KDFGC cell stably expressing DAAO was obtained by retrovirus transfection. The integration and expression of DAAO gene in KDFGC cells were identified by PCR and in situ hybridization. The killing effects of D-Ala on KDfGC cells treating with Immol/L BSO were observed. Results: PCR and in situ hybridization analysis confirmed integration of DAAO gene in positive clone and expression of it at mRNA level. The IC50 of KDFGC and KDFGC + BSO treating with D-Ala for 24 hours were 10.21 mmol/L and 7.92 mmol/L, respectively. The 95% confidence limits of them were different. Conclusion: BSO can enhance the killing effect of DAAO/D-Ala system on K652e cells.
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Objective: To construct retroviral vector pLDAAOSN and observe the function of DAAO gene. Methods: With recombinant DNA technology, DAAO cDNA was cloned into retroviral vector (pLDAAOSN). The vector was transfected into ?XNA cells by CaPO4 method, and the DAAO cDNA was transferred by recombinant retroviral vector into leukemia cell line K562. The positive clones were obtained after G418 selection and termed K DAAO. PCR and in situ hybridization were used to identify the integration and expression of DAAO gene in K DAAO. In order to observe the function of DAAO, K DAAO was treated with 50 mm/L D-Ala. Results: Results of plasmid pLDAAOSN digested with KpnⅠ and the sequence determination confirmed pLDAAOSN contains full-length DAAO cDNA. Infectious titer generated by the packaging cells was 5.2?10 6 CFU/ml. PCR and in situ hybridization analysis showed integration of DAAO gene in K DAAO and expression of DAAO gene at mRNA level. Preliminary observation suggested that D-Ala could effectively kill K DAAO. Conclusion: Retroviral vector pLDAAOSN may be useful for futher study of gene therapy in cancer.
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Objective To investigate the effects of the folic acid on human T lymphoid leukemia cell line CEM cells. Methods 1. MTT method was used to detect the proliferation of CEM cells co- cultured with folic acid of different concentrations and time;2. E-xamine the changes of morphology by light microscopy with Giemsa stain;3. Detect the percentage of apoptosis and cell cycle distribution as well as the expression of the apoptosis protein(Bcl- 2,C- myc) by flow cytometry;4. Detect DNA fragments by Agaiose elec-trophoresis;5. Detect the influence of folic acid to the anticancer effects of methotrexatc(MTX) by MTT methods. Results 1 Folic acid could inhibit the proliferation of CEM cells, and the optimal inhibitive concentrations range from 0. 4 ? 10-4 ?g/L to 3. 0 ? 10 -4 ?g/L,the inhibition rate was about 30% - 40% ; 2. Co - cultured with folic acid at 24,48, 72 hours, examined by light microscopy with Giemsa stain, apoptosis cells were found in all study groups but the higher apoptosis rate was found co - cultured with folic acid at concentration of (0.4 - 3.0) ? 10-4?g/L;3.The highest apoptosis rate was 6. 19% found at the concentration of 3 ? 10-4 ?g/L, but the cell cycle distribution had no statistical difference with control group, the expression of apoptosis related protein Bcl - 2 and C-myc was decreased;4 DNA was extracted from CEM cells co - cultured with 0.4? 10 -4 ?g/L and 3 ? 10-4 ?g/L folic acid for 48 hours. UNA ladders were visible by agarose electrophoresis of DNA fragments; 5. Folic acid did not affect the antitumor effect of MTX at the concentration from 0 2? 10-4 ?g/L to 12.0?10-4 ?g/L. Conclusion Folic acid may suppress proliferation and induce apoptosis of CEM cell
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AIM The antisense drug design will be optimized based on bcl 2 mRNA secondary structure simulated with computer. METHODS bcl 2 mRNA second structures were simulated with computer and Mfold software, and the unstable zones on the second structure, as designing antisense zones, were selected. RESULTS Five antisense deoxynucletides were studied and evaluated with experiments of HL 60 and K562 leukemic cells. Two of them exist significant effect of inhibiting grow of HL 60 and K562 leukemic cells with dose of 10 ?mol?L -1 or more. CONCLUSION The designs with computer and corresponding software will be usefully efficient way to look for antisense drugs.
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Immunosuppressive activities have been found in the culture supernatant of four leukemiacell lines and in the sera of four patients with leukemia. The inhibitors suppressed PHA-p in-duced lymphocytes proliferation and IL-2 production and responsiveness of T cells to IL-2, butno inhibitory activity was observed in the culture supernatant of 2BS cell line or in the sera ofnormal humam. The culture supernatant of tumor cell lines was not cytoxic. The result of PAGEshows: the molecular weight of TDSF from HL-60 human promyelocytic leukemia is about87KD. It is protein in chemical nature. The synthesis and sercretion of TDSF were partially in-hibited by drugs of anti-acute non-lymphocytic leukemia.
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Based on the high level of human granulocyte—macrophage colony—stimulating factor(HGM—CSF)re-ceptor of HL60 cell line upon induction by DMSO,we developed a hioassay method for HGM—CSF with ahigher specificity and sensitivity.The sensitivity of this assay reached to 0.3ng/ml by selecting the optimalHL60 cell—induced time,cell density and GM—CSF—stimulated time.With this assay,we are able to quantifythe level of rHuGM—CSF as well as conditioned media from human bladder carcinoma cell line U5637 screatinga high level of GM—CSF.