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Objective To investigate the effects of artesunate combined with arsenic trioxide (ATO) on the proliferation and apoptosis of NB4 cells.Methods The NB4 cells were treated with different concentrations of artesunate and arsenic trioxide respectively for 48 h.The cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) method.The cells were divided into 4 groups:control group,artesunate group,arsenic trioxide group,and the combination of artesunate and arsenic trioxide group.The cell cycle and apoptosis were detected by flow cytometry (FCM).The protein expression levels of Bcl-2 and Bax were detected by Western blot.Results The MTT results showed that compared with the control group,the proliferation inhibition rates of 0.25,0.50,1.00,2.00,4.00 μmol/L artesunate group (19.26％ ± 3.59％,36.53％ ± 2.67％,61.32％ ± 2.50％,70.30％ ± 3.15％,86.92 ± 0.02％) significantly increased (P＜0.05);the proliferation inhibition rates of 1,2,4,8,16 μmol/L arsenic trioxide group (12.69％ ± 2.43％,64.26％ ± 2.02％,85.10％ ± 2.67％,92.06％ ± 2.21％,93.67％ ± 3.36％) significantly increased (P＜0.05);and the proliferation inhibition rate (40.17％ ± 5.49％ vs.32.23％ ± 3.52％) of combination of artesunate and arsenic trioxide group significantly higher than the arsenic trioxide group (P＜0.05).Compared with the arsenic trioxide group,the percentage of G0/G1 phase cells (74.20％ ± 1.43％ vs.66.14％ ± 1.78％),the apoptosis rate (58.00％ ± 2.41％ vs.34.57％ ± 1.22％),and the expression level of Bax protein (1.35 ± 0.09 vs.1.13 ± 0.09) in the combination of artesunate and arsenic trioxide group significantly increased (P＜0.05),the expression level of Bcl-2 protein (0.45 ± 0.09 vs.1.03 ± 0.10) in the combination of artesunate and arsenic trioxide group significantly decreased (P＜0.05).Conclusions Artesunate can significantly enhance the proliferation inhibition and apoptosis induced by arsenic trioxide on NB4 cells.The possible mechanism of proliferation inhibition and apoptosis of NB4 cells by artesunate combined with arsenic trioxide may be related to reduce the expression of anti-apoptotic protein Bcl-2 and increase the expression of apoptotic protein Bax.
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Objective To investigate the expression of SP 100 protein in ATRA-treated NB4 cells and its effect on pro-liferation in NB4 cells.Methods Q-PCR was employed to measure the expression of SP 100 mRNA;Western blot was used to detect the expression of SP 100 protein; Immunofluorescence was adopted to determine the location of SP100;Cell viability was analyzed by CCK 8;Flow cytometry was used for cell cycle analysis .Results ATRA may induce the expression of mRNA and protein of SP 100.ATRA changes the location of SP100 from a micro-punctate pattern into a punctate nuclear pattern in NB 4 cells.SP100-shRNA promotes the proliferation of NB 4 cells and in-creased the cells in G2/M phase.Conclusions The expression of SP100 was significantly increased in ATRA-treated NB4 cells, and SP100 may be involved in the regulation of proliferation activity of NB 4 cells.
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of NB4 cells at S phase was lower than those in control group (P <0.05),and the number of NB4 cells at G1 phase was increased (P <0.05).The expression levels of Bcl-2 in ICA groups were significantly lower than that in control group (P < 0.05 ), while the expression levels of Bax were higher than that in control group (P < 0.05 ). Conclusion ICA can induce the apoptosis of NB4 cells via inhibiting the expression level of Bcl-2 and up-regulating the expression level of Bax.
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Objective To establish a nude mice model for NB4/ShHMGA2 xenograft and explore the effect of HMGA2 knockdown on hematological malignancies. Methods NB4/ShHMGA2 or NB4/ShControl cell lines were established by transfecting the recombinant Lentivirus-HMGA2shRNA and the vacant Lentivirus-NC-marked into NB4 cells. The knockdown of HMGA2 was identified by RT-PCR and Western blot. Ten male BALB/c nude mice aged 4 ~ 5 weeks were equally divided into two groups. The mice irradiated by 4 Gy 60 Co were subcutaneously injected with 8 × 106 NB4/ShHMGA2 or NB4/ShControl cells into one side of axilla. The volumes of xenograft tumor were evaluated using the equation volume (mm3) = (L × W2)/2. The xenograft tumor section was detected by IHC with Ki-67 antibody. Results NB4 cell xenograft tumors developed in all mice of both the two groups. The NB4/ShHMGA2 cells in the nude mice grew at a lower rate than those in the controls. There were statistically significant differences in the volume and weight of xenograft tumor between the two groups [(1 484.25 ± 156.342)mm3 vs (3 228.674 ± 285.64)mm3, P < 0.05] and [(2 135.33 ± 198.05) mg vs (650.46 ± 85.12)mg, P < 0.05]. The Ki-67 protein level in NB4/ShHMGA2 cells xenografts was lower than that in the controls. Conclusion The knockdown of HMGA2 could inhibit proliferation of NB4 cells in NB4 cells xenograft tumor.
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Objective To investigate the effect of TRAIL on NB4 and K562 cell lines, and its relationship with TRAIL releptors.Methods Jurkat cells were used as positive control,NB4 and K562 cells were treated with different concentrations of TRAIL. Cell morphologic changes were monitored. The cell proliferation was evaluated by MTT assay. The expression of TRAIL receptor were determined by flow cytometry.Results MTT assay showed that TRAIL inhibited the growth of NB4 and Jurkat cells in vitro in a dose-and time-dependent manner,but the effect of TRAIL on Jurkat cells was stronger than that on NB4 cells.However, the growth of K562 was not inhibited. Flow cytometry analysis revealed that DR4,DR5 and DcR1 were expressed higher in NB4 and K562 cells, but the levels of DR4 and DcR1 were very low in K562 cells.DR5 was expressed in Jurakat cells with low level. No DcR2 was detected on the surface of all the three cell lines.Conclusion NB4 cell line is moderately sensitive to TRAIL,and K562 cell line is resistant to TRAIL.The sensitivity of NB4 cells to TRAIL may be associated with the expression of DcR1, but the sensitivity of K562 cells have nothing to do with the expression of TRAIL receptors.
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Objective To investigate the effect of methylation inhibitor decitabine (DAC) alone and combination with As2O3 on apoptosis of NB4 cells. Methods NB4 cells were treated with DAC, As2O3 and the combination of them in different concentrations. The cell proliferation was analyzed by MTT assay and the apoptosis of NB4 cells was detected by flow cytometry. Results Both DAC and As2O3 induced time and concentration-dependent cell death, in which the inhabitation rate were 12.18 %, 22.72 %, 35.54 %, respectively, after 24 h, 48 h, 72 h on treatment by DAC at 1 μmol/L and the inhibition rates were increased to 22.14 %, 31.18 %, 45.21 % by DAC at 1 μmol/L. The inhibition rates were 21.09 %, 32.43 %, 44.93 %, respectively, by treating with As2O3 0.5 μmol/L after 24 h, 48 h, 72 h, which were increased to 31.69 %, 41.12 % and 54.27 %, respectively after 24 h, 48 h, 72 h. The inhibition rates were significantly increased by using both DAC and As2O3 with significant differences (P <0.05). DAC and As2O3 in combination produced a greater inhibition of growth against NB4 cells (by treating with DAC 1 μmol/L + As2O3 0.5 μmol/L after 24 h, 48 h, 72 h, the inhibition rates were 42.10 %, 48.75 %, 60.78 %) (P <0.05). In each concentration group and control group the differences were statistically significant (P <0.05). The incubation for 48 h with As2O3 1 μmol/L alone or combined with DAC 2 μmol/L showed apoptosis cells by 5.8 % and 17.3 %. Conclusion Decitabine can significantly inhibit the proliferation of NB4 cells and the apoptosis with synergistic effectiveness can be found when Decitabine combination with As2O3.
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Objective To investigated the effects of combined arsenic trioxide(ATO) and resveratrol(Res)on the viability of NB4 human leukemia cells. Methods NB4 human leukemia cell was used in this experiment.Cells were cultured in ATO (0,0.1875,0.3750,0.7500, 1.1250, 1.5000,2.2500,3.0000,5.0000 μmol/L) and Res (0, 1.5625,3.1250,6.2500, 12.5000, 18.7500,25.0000,37.5000,50.0000 μmol/L). Cell viabilities were measured by MTT in different treatment groups. Half inhibitory concentration(IC50) was calculated. The ratio of concentration of ATO and Res 1.5∶ 18,1.5∶ 25,1.5∶ 35 was added to cells, and the combination index(CI) was calculated. The level of ROS in control, ATO( 1.5000 μmol/L), Res(25.0000 μmol/L) and ATO(0.9000 μmol/L) + Res( 12.5000μmol/L) groups was measured by chemiluminescence assay. Results ①ATO( ≥0.7500 μmol/L) reduced the viability of NB4 cells in a concentration-dependent manner(P ＜ 0.05 ), and IC50 was (1.78 ± 0.11 )μmol/L. ②)Res (≥18.7500 μ mol/L) dose-dependently decreased the viability of NB4 cells (P ＜ 0.05 ), and IC50 was ( 18.71 ±0.18)μ mol/L. ③Combination of ATO and Res showed an antagonistic effect on NB4 cells viability. ④The ROS in Res group( 1670.55 ± 13.97) was significantly lower than that in control group(2345.88 ± 14.48,P ＜ 0.05). The ROS in ATO group (3092.42 ± 94.84) was significantly higher than that in control group(P ＜ 0.05). The ROS in ATO + Res group (1860.27 ± 15.99) was significantly lower than that in ATO group(P ＜ 0.05). Conclusions NB4 cell survival rate can be decreased by ATO and Res. The combination of arsenic trioxide and Res presents an antagonistic effect on NB4 cell viability, in part by reducing intracellular ROS formation.
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Objective To explore the effects of psoralen (PSO) and long wave ultraviolet A (PUVA) on apoptosis and expression of Fas in HL-60,K562 and NB4 leukemia cells.Methods The cells were incubated with PSO in different concentrations irradiated with or without UVA.The changes of ultrastructure of cells were observed under the electron microscope.The expression of Fas gene was detected by fluorescent quantitation PCR.The apoptosis ratio and the expression of Fas protein were detected through the flow cytometry.The factorial design and analysis of variance were used to analyze the interaction among the factors.Results There were obvious ultrastructure changes about apoptosis in leukemia cells after treated with PUVA.PSO,UVA and PUVA all increased the apoptosis ratio and expression of Fas gene and protein,and the effects of PUVA were stronger than the other two (P
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Objective To investigate the effects and the gene expression of mitochondria on arsenic trioxide-induced apoptosis of acute promyelocytic leukemia (APL) NB4 cells. Methods NB4 cells were treated with As2O3. Fluorescent microscopy and flow cytometry were used and mitochondria membrane potential detection and RT-PCR were performed to observe the NB4 cell apoptosis, growth inhibitory effect and mitochondrial transmembrane potentials. Mitochondria genome primers were designed, and the expression of mitochondria genome at gene and protein levels was studied. Results Induced by As2O3, the NB4 cells showed typical morphological changes of apoptosis with a significant growth inhibitory effect. The ratios of NB4 cells apoptosis were 5.02%, 6.40%, 28.40% and 33.34%, respectively, when treated with As2O3 in concentrations of 0.5?mol/L, 1?mol/L, 2?mol/L and 3?mol/L. When treated with As2O3 in 0.5?mol/L, 1?mol/L, 2?mol/L, 4?mol/L and 8?mol/L at 48h, the mitochondria potential of NB4 cells was decreased by 12.8%, 21.6%, 66.9%, 83.7% and 83.8%, respectively. After the NB4 cell apoptosis was induced by As2O3, RT-PCR assay was used to detect the expression of 13 genes of mitochondria genome. The expression of COX2 gene was down-regulated in this process, while no change was found in the expression of other 12 genes. Conclusions As2O3 has shown to exert a significant effect on promoting apoptosis and growth inhibition on APL cells. The apoptotic effect induced by As2O3 on NB4 cells is closely related to a decrease of mitochondrial membrane potential. The expression change in the mitochondria gene COX2 is involved in the As2O3-induced apoptosis of NB4 cells.
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AIM: To transfer 4 full-length WT1 isoforms cDNA into the leukemia cell line NB_4 so as to provide a cell model for studying the WT-1 gene function. METHODS: The eukaryotic expression recombinant vectors for WT1 isoforms (pCB6+/WT1) were introduced into the leukemia cell line NB_4 by electroporation. The positive cell clones were screened by G418 culture. The integration of WT1 gene isoforms in NB_4 cells as confirmed by PCR. The mRNA and protein of WT1 were detected by RT-PCR and Western blotting. RESULTS: WT1 gene isoforms were successfully transferred into NB_4 cells. WT1 mRNA and protein expression in the G418-selected cells increased remarkably compared with the control. CONCLUSION: WT1 gene isoforms were effectively transferred into NB_4 cells by electroporation and stably expressed in the transfected cells.
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Objective To explore the effects of psoralen(PSO)and long-wave ultraviolet chemical therapy(PUVA) on cell apoptosis and expressions of FasL in HL-60,K562,and NB4 leukemia cells.Methods The cells were taken as the studying objects and their apoptosis ratios,ultrastructure changes and the expressions of FasL were detected in or- der to observe the effects of PSO extracted from Chinese medicine and ultraviolet at 360 nm on human leukemia cells.The factor design and analysis of variance were used to analyze the interaction among the factors.Results(1)PSO,UVA and PUVA all induced the apoptosis and the effects of PUVA were stronger than the other two.(2)Obvious changes of ultra- structure of leukemia cells were found under electron microscope after treatment with PUVA.(3)PSO,UVA and PUVA down-regulated FasL gene and protein expression levels,and the effects of PUVA are the strongest.Conclusion PUVA can induce the apoptosis of human leukemia cells and its effects are the strongest.One of the pathway of PUVA to induce apoptosis is by down-regulating the expression of FasL gene.
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Objective Gene microarray technology was used to investigate the differential gene expression of apoptosis related genes in NB4 cells induced by arsenic trioxide. Methods The databases of Evntrez and Human IPI were searched with "apoptosis or apoptotic" as the key words, and 1384 apoptosis related genes were found after the redundant genes were eliminated by chromosomal localization. The probes of these genes were designed using OligoArray 2.0, and then analyzed by BLAST. All the probes were immobilized on the glass slide, which were used as oligonucleotide microarray. After NB4 cells were treated with 2umol/L As2O3 for 48h, the total RNA were extracted. cDNAs of control group and test group were fluorescently labeled with Cy3 and Cy5, respectively, by RT-PCR. The fluorescent samples were hybridized with an oligonucleotide microarray containing 1384 apoptosis related genes to search for the differentially expressed genes in the cells with or without As2O3 treatment. The hybridization signals were scanned by oligonucleotide microarray, and then the fluorescent intensity of Cy3 and Cy5 and the ratio of two fluoresceins were analyzed using certain software. Then the differential expressed genes were analyzed after As2O3 treatment, in which the most distinctly differential expressed genes were chosen as targets, and through PCR amplification and gel electrophoresis the above genes were verified. Results There are 4 genes up-regulated and 12 genes down-regulated in expression in NB4 cells after 48h treatment with 2umol/L As2O3, which were in accordance with the results of RT-PCR and oligonucleotide microarray. Conclusion Differential gene expression in NB4 cells was induced by As2O3 treatment. These differentially expressed genes, with relation to signal transduction, transcription regulation, cell cycles, oxidation response, protein translation and cell differentiation, may play an important role in NB4 cell apoptosis.