Objective:To discuss the effect and mechanism of autophagy inhibitor 3-MA on arsenic trioxide inducing apoptosis of acute T-cell leukemia cell line Jurkat cells.Methods:Proliferation inhibition of Jurkat cells treated with arsenic trioxide was detected by XTT.Morphological characteristics of Jurkat cells treated with different concentrations arsenic trioxide were observed by electron mi-croscope.Microtubule-associated protein 1 light chain 3B (LC-3B) protein expression was detected by Western blot and flow cytome-try.Apoptosis rates of Jurkat cells treated with 3-MA combining arsenic trioxide were detected by flow cytometry using AnnexinV-FITC/PI double staining.Results:Arsenic trioxide inhibited the growth of Jurkat cells in a dose and time dependence.We observed different morphological characteristics of autophagy , apoptosis and necrosis accompanying more autophagosomes in Jurkat cells which were treated with arsenic trioxide 2.5,5,10 μmol/L after 24 h.LC3B mean fluorescence intensity (MFI)relative multiples were(3.1±0.2) fold,(4.6±0.31)fold,(34.2±4.5)fold with 5 μmol/L arsenic trioxide treated Jurkat cells 0,24,48 h,and the P values between each of the two groups were less than 0.05,which increased depending time consistently with the growth inhibition rates.LC-3B protein expression gradually increased treated Jurkat cells with arsenic trioxide after 24 h,48 h.The growth inhibition rate (60.6±8.3)%was significantly different treated with arsenic trioxide combining 3-methyl adenine ( 3-MA ) while it was ( 33.4 ±9.1 )% treated with arsenic trioxide alone, however, LC-3B protein expression gradually decreased.Jurkat cell apoptosis rate ( 44.96 ±3.60 )% was significantly increased treated with arsenic trioxide combining autophagy inhibitor(3-MA) while it was (2.94±0.26)% treated with arsenic trioxide alone, and this difference was statistically significant.Conclusion: 3-MA increased apoptosis rates of Jurkat cells inducing by Arsenic trioxide and it may be related with inhibition of autophagy and induction of apoptosis.

Objective To investigate the role of renal nerve in cardioprotection provided by renal ischemic preconditioning(RIP).Methods The effects of ischemia-reperfusion and RIP on the hemodynamics, myocardial oxygen consumption, epicardial electrography and infarct size were examined in anesthetized rabbit.Results During the 45 min of myocardial ischemia and 180 min of reperfusion, all hemodynamic parameters and myocardial oxygen consumption decreased progressively significantly. Epicardial electrographic ST-segment was elevated significantly during myocardial ischemia and return to baseline progressively in the course of reperfusion. The myocardial infarct size occupied 55.80±1.25% of area at risk,and RIP significantly reduced the myocardial infarct size to 36.51±2.80%(P＜0.01). The renal nerve section (RNS) per se didn't affect myocardial infarct size produced by ischemia-reperfusion, while cardioprotection afforded by RIP was completely abolished by RNS.Conlusion RIP have the protective effect on heart, and activation of renal afferents by transient ischemia-reperfusion play an important role in such a cardioprotection.

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

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.

Objective:To observe the effect of arsenic trioxide (ATO) on the expression of NKG2D ligand UL16 binding protein 1(ULBP1) in acute myeloid leukemia KG1a cells, and explore the molecular mechanism for its regulation of ULBP1 expression.Methods:KG1a cells were cultured in vitro.Then, the inhibition of KG1a cell proli-feration by different concentrations of ATO was detected by cell counting kit-8(CCK8) assay, and the expression of ULBP1 mRNA and surface protein in KG1a cells were examined by real-time RT-PCR and flow cytometry, respectively.After that, the blocking effects of ataxia telangiectasia mutated and RAD3-related kinase (ATM/ATR) inhibitor caffeine on ATO-upregulated expression of ULBP1 mRNA and surface protein expressions were investigated, and the effects of ATO on the expression of CHK1 and CHK2 proteins and their phosphorylation in KG1a cells were observed by Western blot method. Results:Different concentrations (1, 2, 3, 4, 5 μmol/L) of ATO could inhibit the proliferation of KG1a cells, which was concentration dependent, and the half inhibitory (IC 50) concentration to KG1a cells was 2.7 μmol/L.The expression of ULBP1 mRNA on KG1a cells were increased when incubated with ATO at concentration 1, 2, 3, 4, 5 μmol/L, compared without ATO group, ULBP1 mRNA expression level relatively increased respectively to (1.86±0.30) times, (3.02±0.71) times, (3.16±0.75) times, (4.80±0.70) times and (3.70±0.89) times, and the differences were statistically significant (all P<0.05). Furthermore, ATO (1, 2, 3, 4 and 5 μmol/L) upregulated ULBP1 protein expression on KG1a cells compared with that in the group without caffeine, and the differences were statistically significant (all P<0.05). After caffeine pretreat KG1a cell 2 h and ATO incubate KG1a cell 24 h, ULBP1 mRNA and protein expression levels were significantly reduced.When caffeine concentration was 8 mmol/L, ULBP1 mRNA expression level relatively reduces from (9.55±0.38) times to (6.36±0.93) times compared with that in the group without caffeine, and the difference was statistically significant ( P<0.05). When caffeine concentration was 2, 4 and 8 mmol/L respectively, the expression of ULBP1 protein was reduced from that in the group without caffein treatment (3.50±0.08) times to (2.17±0.07) times, (2.02±0.06) times and (1.75±0.06) times, respectively, and the differences were statistically significant (all P<0.05). The expression of CHK1 and CHK2 proteins decreased with the increase of ATO concentration, while p-CHK1 and p-CHK2 are increased as ATO. Conclusions:ATO upregulate the expression of ULBP1 mRNA and protein in KG1a cells, and the ATM/ATR-CHK1/CHK2 pathway may be involved in it.