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Objective To investigate the effect of 2-methoxyestradiol(2-ME) on U937 myeloid leukemia cell line and its mechanism.Methods The experiment was divided into control group(myeloid leukemia U937 cell in RPMI 1640 culture medium with equal DMSO),2-ME-treated group,NAC-treated group,and 2-ME+NAC-treated group.The cytotoxicity was analyzed by MTT assay.Apoptosis and cellular nitric oxide(NO) were detected by flow cytometry using annexin V and NO sensor dye.Superoxide anion was measured with a fluorescent plate reader by DHE.Results Viabilities of U937 cells treated with 2-ME(0.25,0.50,1.00,and 2.00 ?mol?L-1) for 48 h were gradually reduced to 0.68?0.05,0.28?0.07,0.18?0.07,and 0.11?0.04,respectively.The differences were significant compared with control group(1.00?0.05)(P0.05).2.00 ?mol?L1 2-ME also significantly increased the mean fluorescence of NO sensor dye and superoxide anions in U937 cells compared with control group(P0.05).An markedly increase in apoptotic cells was detected after 2-ME treatment for 3 h(6.78%?1.01% vs 1.59%?0.12%,P
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Objective To investigate the different biological characteristics of high-and low-expression of ATP-binding cassette transporter(ABCG2)in human multi-drug resistant nasopharyngeal carcinoma CNE2/DDP cell line(abbreviated as ABCG2High cell and ABCG2Low cell),and to analyze drug-resistant characteristics of the two cell lines.Methods ABCG2High and ABCG2Low cells were isolated by flow cytometry,while their cloning efficiency,cell cycle distribution,and the expression of ABCG2 before and after the 5th passage were examined as well.mRNA expressions of drug-resistant genes,such as ABCG2,Bcl-2,MDR1,MRP and MGMT,were detected by reversed transcriptive polymerase chain reaction(RT-PCR).MTT assay was utilized to detect the light absorbance(A)from day 1 to 14 to determine growth kinetics.Moreover,the drug sensitivity of the two cell lines to fluorouracil,cisplatin,vincristine,carboplatin,epirubicin,daunorubicin,paclitaxel and mitomycin were detected by MTT assay.Results The cloning efficiency of ABCG2High and ABCG2Low cells was 25.24%?1.18% and 16.28%?1.11%,respectively,on day 7.ABCG2High cells in S phase accounted for 41.5%,while the ABCG2Low cells in the G1/M0 phase went up to as high as 63.9%.The expression of ABCG2 in original ABCG2HighCNE2/DDP cells was 98%,while in ABCG2Low CNE2/DDP cells was 2%.However,at the 5th passage,the expression of ABCG2 decreased to 75% in the former cells and increased to 20% in the latter ones.The expression of ABCG2 mRNA in ABCG2High cells was obviously higher than that in ABCG2Low cells,while there was no difference in expression of other drug resistance genes between two cell lines.The growth velocity of ABCG2High cells exceeded that of ABCG2Low cells in early stage,but it slowed down 7 days later.MTT assay showed that IC50 of ABCG2High cells to 8 kinds of anti-tumor drugs were twofold higher than that of ABCG2Low cells,with statistically significant difference between them(P
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Aim To investigate the effects of Manumycin on HL-60 myeloid leukemia cell line,and to explore the mechanism,major in investigating changes in the mitochondria of leukemia cell line in response to Manumycin.Methods Human myeloid leukemia cells HL-60 were used.The cytotoxicity was analyzed by MTT assay.Apoptosis and cellular nitric oxide(NO) were detected by flow cytometry using Annexin V andNO sensor dye.Superoxide anion was measured with a fluorescent plate reader by DHE.GSH was assayed by fluorescent Monochlorobimane.The SOD activities were assayed by colorimetric methods using WST.ATP content was measured by luciferin-luciferase bioluminescence assay.The cytosolic proteins were extracted from the cells using a digitonin buffer.The protein expression of cytochrome C and Mn-SOD were determined by Western blot.Results Manumycin resulted in viability decrease in a dose-dependent manner,and induced the generation of ROS:NO and superoxide anions.Manumycin reduced intracellular glutathione.Manumycin induced mitochondria swollen,intracellular ATP content decrease and cytochrome C release from mitochondria to cytosol.Manumycin-induced apoptosis correlated with increase in ROS.Quenching of ROS with N-acetyl-L-cysteine protected leukemia cells from the cytotoxicity of Manumycin and prevented apoptosis induction by Manumycin.Conclusions Cellular ROS generation plays an important role in the cytotoxic effect of Manumycin.Manumycin induced apoptosis through mitochondria-mediated pathway that required upstream ROS generation,change of mitochondria,and cytochrome C release.
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Objective To investigate the photodynamic therapy(PDT)of human erythroleukemia cell line K562 and drug-resistance cell line K562/ADM using hexyl 5-aminolevulinate(He-ALA)in vitro.Methods Four groups were set:PDT group(photosensitizer with light irradiation),laser group(light irradiation only),dark-cytoxocity group(photosensitizer only)and normal control group(neither photosensitizer nor light irradiation).K562 cells and K562/ADM cells cultured were incubated with various concentrations of He-ALA(0.025,0.1,0.4 and 1.6mmol/L)for 4 hours,then illuminated with different light doses(4.5,9,18 and 36 J/cm2)using a laser with a wave length of 630nm.After 12 hours incubation,Wright's staining method was used to observe the cell's morphological changes,the survival rates of cells were analyzed by CCK8 assay,and the changes in colony-forming abilities of cells were analyzed by methylcellulose colony-forming assay.Results He-ALA or light irradiation alone had no evident cytotoxicity,while the application of He-ALA plus light irradiation effectively killed the leukemia cells.With the increase of the concentrations of He-ALA and the dosages of irradiation,the viability of cells decreased.Under the same conditions,the survival rate of K562 was significantly lower than that of K562/ADM(P