Arsenic Trioxide ($As_2O_3$) Induced Apoptosis in NB4 Cell lines
Kyeong-Bae ParkDae-Sik HongWon-Suk SeoNam-Soo LeeSung‐Gyu ParkJong-Ho WonHeejung JungSook-Ja KimYoung-Seon HongJong‐Youl JinHee-Sook ParkSang-Man Shin
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Background : Recently, inorganic arsenic trioxide () was reported to induce complete remiss ion in a high proportion of patients with refractory acute promyelocytic leukemia (APL). To illustrate cellular and molecular mechanisms of in the treatment of APL, many experimental studies were performed on APL- derived cell lines in vitro. Previous studies showed that inhibited proliferation and induced apoptosis in the APL- derived cell lines. This study was done to clarify the in vitro mechanisms of - induced apoptosis in APL- derived NB4 cell lines. Methods : To determine the effects of in the various concentrations, NB4 cells were cultured with 0.1 to 2 M/L of . To assay the apoptosis in NB4 cell lines, DNA fragmentation assay and TUNEL were performed. To find out the molecular change of - induced apoptotic NB4 cell lines, RT-PCR and Western blot analysis for PML- RAR chimeric protein expression and flow cytometry for bcl- 2/bax expression were performed. To clarify the caspase activation pathway, Western blot analysis and flow cytometry for procaspase express ion were performed. Results : induces apoptosis on NB4 cells in relatively high concentration (0.5 to 2 M/ L) for 2 days. After 2 days of culture the PML-RAR chimeric protein expression decreased rapidly by Western blot and RT-PCR analysis and bcl-2 expression also decreased by flow cytometry. The express ion of bax by flow cytometry showed a marked increase in high concentration (2M/ L) but there was no change in low concentration (0.5M/ L). In the Western blot analysis, the amount of pro-`enzyme of caspase-3 was significantly decreased in the cells with high concentration (2M/ L) compared with that in the cells with low concentration (0.5M/L). induces proteolytic processing of pro-caspase 7 but not pro-caspase 9 and 8. Conclusion : Apoptosis of APL- derived NB4 cell lines was induced by and progressed rapidly in higher concentrations. During apoptosis, activation of caspase- 7 pathway and degradation of PML-RAR chimeric protein, decrease in bcl- 2 and increase in bax were shown. (Korean J Hematol 2002;37: 200 2 11)Keywords:
Arsenic Trioxide
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Arsenic Trioxide
Survivin
XIAP
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Aim: To investigate the apoptosis and c-myc mRNA expression induced by arsenic trioxide in K562 cells in vitro and to explore its cellular and molecular mechanisms. Methods: Cell proliferation and viability were analyzed by MTT assay and trypan blue exclusion assay. Cell morphological analysis, DNA electrophoresis and flow cytometry were performed to detect whether 0 μmol/L,0.5 μmol/L,1 μmol/L,2 μmol/L,4 μmol/L,8 μmol/L As_2O_3 for 24 h,48 h,and 72 h could induce apoptosis of K562 cell in virto. RT-PCR and immunohistochemistry techniques were used to detect c-myc mRNA and protein expression in K562 cells. Results: As_2O_3 induced chromatin condensation, nuclear fragmentation and the formation of apoptotic body at different concentrations. Cell viability decreased with raised As_2O_3 concentration and prolonged treatment within a certain range. Flow cytometry analysis manifested that As_2O_3 induced K562 cells apoptosis and affected the cell cycle progression. Most of cells were arrested at G_2/M phase. After As_2O_3 treatment c-myc mRNA and protein expression was up-regulated firstly then down-regulated to a lower level. Conclusion: As_2O_3 can inhibit the proliferation and induce apoptosis in K562 cells through blocking the cell cycle at G_2/M phase. The c-myc gene may be involved in the gene regulation of As_2O_3-induced apoptosis.
Arsenic Trioxide
Viability assay
K562 cells
MTT assay
Apoptotic body
Trypan blue
Fragmentation
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AIM To study the expression and the effects of Bcl 2 and Bax protein in apoptosis of K562 cell induced by arsenic sulfide. METHODS After being incubated in culture medium containing variable levels of arsenic sulfide, cell growth and proliferation of K562 cell was analyzed with MTT assay; apoptosis was detected with cell morphology by light and transmission electron microscopy. The expression of Bcl 2 and Bax protein was determined with S P immunohistochemical stainning and imaging analysis. RESULTS After the treatment, cell growth and proliferation of K562 cell was inhibited in a dose and time dependent manner. Morphologically, after treatment with 175 to 1400 μg·L -1 of arsenic sulfide for 18 to 144 h, K562 cells presented some features of apoptosis. The baseline Bcl 2 expression of K562 cell was very low ( A : 0.152±0.023) while the absorbance (optical density) value ( A ) of negative control was 0.108±0.012. No significant difference was found after the treatment ( A : 0.138±0.028) ( P 0.05), but the expression of Bax protein in K562 cells co cultured with arsenic sulfide ( A : 0.316 ±0.021) was increased, compared with the untreated ( A : 0.240±0.013) ( P 0.01). CONCLUSION Arsenic sulfide could inhibit growth and proliferation of K562 cell and induce its apoptosis. These effects may be correlated with the up regulation of Bax protein.
K562 cells
BAX Protein
Arsenic Trioxide
Growth inhibition
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Agarose gel electrophoresis
Viability assay
MTT assay
Cell counting
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To determine effects of berbamine on the growth of leukemia cell line NB4 and explore its possible mechanisms.The growth of NB4 cells was examined with MTT assay. Morphological analysis and DNA agarose electrophoresis were used to detect apoptosis in NB4 cells, and the apoptosis rate was measured by flow cytometry. The PML/RAR alpha mRNA was determined by nested-PCR, and the Survivin mRNA was tested by RT-PCR. The expression of caspase 3 protein in NB4 cells was evaluated by flow cytometry.The growth of NB4 cells was inhibited significantly after treated with berbamine at different concentrations for different time points, the IC(50)value was 3.860 microg/ml at 48 hours. Morphology analysis showed the characteristics of apoptosis, and the DNA agarose electrophoresis showed the typical DNA ladder. The apoptosis rate increased from 2.83% to 58.44% after treated with berbamine at 12 microg/ml for 48 hours. The expression of PML/RAR alpha mRNA presented no significant changes, however, Survivin mRNA was decreased dramatically. The protein expression of Caspase 3 increased significantly from 2.06% to 70.89% after treated with berberine at a concentration of 12 mug/ml for 48 hours.Berbamine could inhibit the growth of leukemia cell line NB4. The induction of cell apoptosis may be one of the mechanisms for suppressing the growth of leukemia cell line NB4. Inhibition of Survivin mRNA and upregulation of Caspase 3 protein might be also involved in cell apoptosis.
Survivin
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Growth inhibition
MTT assay
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Cinobufocini injection is a preparation containing water-soluble components of the toad skin. The aim of the present study was to investigate the apoptosis of human lung adenocarcinoma cell line A 549 induced by cinobufocini. A 549 or HLF-1(human lung fibroblast) cells were treated with cinobufocini at different concentrations for 24 and 48 h, respectively. The proliferation of cells was detected with the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Morphology of cells was carried out with scanning electronic microscopy (SEM) and Hoechst 33258 staining. The apoptosis rate was examined by flow cytometry. The expression of survivin was examined with RT-PCR and Western blot assay. The caspase-3 and caspase-7 activities were detected with caspase colorimetric protease assay. We found that cinobufocini significantly inhibited tumor growth of A 549 cells in a dose- and time-dependent manner without damaging non-cancerous cells (HLF-1) and induced granular apoptotic bodies of A 549 cells. Next, cinobufocini increased the percentage of cells in G1 phase and decreased the percentage of cells in S phase in A 549 cells. Furthermore, cinobufocini downregulated the expression of survivin mRNA and protein. Finally, cinobufocini upregulated caspase-3 activity. We concluded that cinobufocini induces apoptosis of A 549 cells, which is associated with the decreasing expression of survivin mRNA and protein, increasing caspase-3 activity of A 549 cells.
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Objective To investigate the apoptosis of MCF-7 cells induced by different concentrations of As2O3 and expressions of FoxO3a and Caspase-3 protein.Metheds The apoptosis of human breast carcinoma MCF-7 cells treated for 24 hours with 2.0,4.0,8.0 μmol/L As2O3 respectively was detected by flow cytometry.The activities of FoxO3a and Caspase-3 proteins were examined by immunocytochemistry and Western blot in untreated or treated by As2O3.Results The rate of the apoptosis in MCF-7 cells treated by 2.0,4.0,8.0 μmol/L of As2O3 were(6.26±0.94)%,(9.30±1.63)% and(13.02±3.82)%,respectively.There were obvious dose-effect correlations between As2O3 and apoptosis(rs=0.949,P0.01).As2O3 promoted the expression of nucleus-FoxO3a and Caspase-3 protein.Conclusion The results suggested that As2O3 could induce apoptosis of MCF-7 cells via FoxO3a protein activation and Caspase-3 protein up-regulation.
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MCF-7
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This study was purposed to explore the inhibitory effect of Curcumin on growth of retinoic acid-resistant acute promyelocytic leukemia (APL) cells and its mechanism. The NB4-R1, an APL cell line resistant to retinoic acid, was used as a model. The growth level of NB4-R1 was detected by MTT assay, the morphologic features of cells were observed by light microscopy, the mitochondrial transmembrane potential was determined by flow cytometry, the expressions of apoptosis-related proteins procaspase 3, caspase 3, PARP and BCL-XL were measured by Western blot. The results indicated that the sensitivity of NB4-R1 to Curcumin was consistent with NB4 though NB4-R1 was resistant to retinoic acid, Curcumin displayed inhibitory effect on growth of NB4-R1 in time-and concentration-dependent manners. The morphologic observation showed existence of apoptotic bodies in NB4-R1 cells treated with 20 micromol/L of Curcumin. The flow cytometry indicated that the mitochondrial transmembrane potential in NB4-R1 cells treated with 20 micromol/L of Curcumin obviously decreased. The Western blot detection revealed that expressions of pro-caspase 3 and BCL-XL were down-regulated, expressions of caspase 3 and sheared PAPP were up-regulated in NB4-R1 cells treated with 20 micromol/L of Curcumin. It is concluded that the Curcumin can inhibit the growth and induce the apoptosis of NB4-R1.
MTT assay
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To explore the effects of vascular endothelial growth factor (VEGF) on the mechanisms of CML pathogenesis, the effect of VEGF on K562 cell apoptosis induced by As(2)O(3) was analyzed through morphologic observation, DNA fragmentation agarose gel electrophoresis and DNA ploidy flow cytometry analysis, and the effect of VEGF on the expression of bcl-X(L), Bax and caspase-3 in K562 cells was determined by Western blot, meanwhile the expression difference between bcl-X(L) and Bax mRNA in above conditions was detected by RT-PCR. The results showed that after VEGF added, the apoptosis of K562 cells reduced, however, there was no significant changes in cell cycle distribution (P > 0.05). At the same time, following the increasing of the concentration of VEGF, expression of mRNA and protein of bcl-X(L) was up-regulated and the expression of Bax protein was down-regulated in K562 cells, and the activation of pro-caspase-3 into caspase-3 was inhibited or reduced. It is concluded that VEGF may suppress the apoptosis of K562 cells through its influence on the bcl-X(L)/Bax expression ratio in K562 cells.
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Objective To investigate the molecular pathway of apoptosis of U937 cells in myelocytic leukemia cell lines induced by trichostatin A(TSA).Method Cultured U937 cells were treated with TSA at different concentration of(50,100,200,300 and 400)nmol/L and 300 nmol/L for varied times(12,24,36,48 and 60 h).Flow cytometer was used to determine the rate of apoptosis of cells and the expression of Fas antigen(CD95)and Western blot was performed to determine the expression of caspase 6,caspase 8 and caspase 9.Rusult The apoptosis induced by the effect of TSA on U937 cells showed a time and dose dependent manner,with more than 50% of apoptosis at IC 50 of TSA for 24 h.The expression of Fas elevated significantly with 300 nmol/L TSA for 24 h(P0.01).The expression of proteins increased significantly treated with 300 nmol/L of TSA for 24 h on apoptosis proteins caspase 6,caspase 8 and caspase 9(P0.05).Conclusion Both TSA and endogenous mitochondria were involved in molecular pathway of apoptosis of U937 cells in myelocytic leukemia cell lines.
Trichostatin A
U937 cell
Caspase-9
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