[Inhibitory Effect of Metformin and Arsenic Trioxide on KG1a Cell Proliferation].

OBJECTIVE: To investigate the effect of metformin and arsenic trioxide on KG1a cells proliferation of acute myeloid leukemia and its possible mechanism. METHODS: CCK-8 method was used to detect the killing effect of metformin, arsenic trioxide and combined application on KG1a cells. Annexin V-FITC/PI Dual Stain Flow Cytometry was used to detect the effect of combined application on apoptosis of KG1a cells. Western blot was used to detect the expression of intracellular apoptosis-,autophagy-related protein. RESULTS: Metformin and arsenic trioxide alone or in combination could inhibit the proliferation of KG1a cells and induce apoptosis of KG1a cells, and the proliferation inhibition rate and apoptosis rate in the combined drug group were higher than those in the drug group alone(P <0.05). The combination of drugs induced upregulation of Caspase 8 protein and P62 protein expression and was higher than that in the drug group alone(P <0.05). CONCLUSION: Metformin can synergize with arsenic trioxide to kill KG1a cells, and its mechanism of action may be related to inducing apoptosis and enhancing autophagy.

Neuroprotective effect of meglumine cyclic adenylate against ischemia/reperfusion injury via STAT3-Ser727 phosphorylation.

OBJECTIVES: Ischemia/reperfusion can induce neuronal apoptosis in the brain and lead to function deficits. The activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is neuroprotective against transient cerebral ischemia. The neuroprotective mechanisms of PKA mainly involve the regulation of gene transcription via the PKA/CREB pathway. The present study aims to investigate the neuroprotective effect of meglumine cyclic adenylate, an activator of PKA, under a rat model of global cerebral ischemia/reperfusion and to reveal the underlying mechanism involving signal transducer and activator of transcription 3 (STAT3)-Ser727 phosphorylation and mitochondrion modulation. MATERIALS AND METHODS: Male Sprague-Dawley rats were subjected to 15 min global cerebral ischemia, and meglumine cyclic adenylate was treated through tail intravenous injection 30 min before ischemia. Cresyl violet staining was used to evaluate neuron injury at 5 d of reperfusion. Western blotting was used to detect p-Ser727-STAT3, total STAT3, cytochrome c (Cyt c) and active caspase-3 in the tissues of hippocampal CA1 region at 6 h of reperfusion. STAT3-S727A was overexpressed in HT22 cells to reveal the significance of STAT3-Ser727 phosphorylation in the neuroprotective effect of meglumine cyclic adenylate. RESULTS: Pretreatment with meglumine cyclic adenylate not only significantly ameliorated neuron loss in CA1 region after global cerebral ischemia but also enhanced STAT3-Ser727 phosphorylation, increased mitochondrial STAT3, and decreased cytosolic Cyt c and active caspase-3. Overexpression of STAT3-S727A in HT22 cells eliminated meglumine cyclic adenylate-induced increase of p-Ser727-STAT3, mitochondrial STAT3, cytosolic Cyt c and active caspase-3. CONCLUSION: Meglumine cyclic adenylate protects neurons against ischemia/reperfusion injury via promoting p-Ser727-STAT3-associated mitochondrion modulation and inhibiting apoptosis pathway.

[Effects of manumycin combined with methoxyamine on apoptosis in myeloid leukemia U937 cells].

BACKGROUND & OBJECTIVE: Repair of DNA damage is important to cell survival. Our previous study showed DNA damage response induced by manumycin in cancer cells. We hypothesized that methoxyamine, an inhibitor of base-excision repair, can enhance the antineoplastic effect of manumycin. This study was to investigate apoptosis induced by manumycin combined with methoxyamine in myeloid leukemia cell line U937, and to explore the role of mitochondrial apoptotic pathway in apoptosis induction of the two drugs. METHODS: U937 cells were treated with various concentrations of manumycin and/or methoxyamine for 48 h. The cell viability was analyzed by MTT assay. Colony formation was evaluated by soft agar clonogenic assay. Cell apoptosis was investigated by flow cytometry. Protein expressions of cytochrome c, caspase-9, and poly ADP-ribose polymerase (PARP) were determined by Western blot. RESULTS: The dose-response curve of manumycin was shifted to the left after addition of methoxyamine. The combination index (CI) was less than 1 (P<0.05) in U937 cells (P<0.05), indicating a synergistic effect of manumycin and methoxyamine. Rates of colony formation of U937 cells treated with 1 micromol/L manumycin, or 5 mmol/L methoxyamine, or the combination of the two were 0.3641+/-0.0463, 0.7541+/-0.0379, and 0.0473+/-0.0024, respectively compared with that of control cells (P<0.05). Moreover, the drug combination resulted in enhanced apoptosis in U937 cells. The apoptotic rates of the control, manumycin, methoxyamine and combination group were (2.34+/-0.30)%, (8.80+/-0.95)%, (2.21+/-0.19)%, and (13.37+/-0.91)%, respectively. The combination of manumycin with methoxyamine also promoted the release of cytochrome c from mitochondria into the cytosol, activated caspase-9, and led appearance of specific cleavage of PARP in U937 cells. CONCLUSION: Methoxyamine enhances manumycin-induced apoptosis in U937 myeloid leukemia cells.

[Cytotoxicity of allogenetic natural killer cells against CD34+ acute myelogenous leukemia cells].

OBJECTIVE: To study the cytotoxic effect of allogenetic natural killer (NK) cells in vitro on human CD34+ acute myelogenous leukemia cells. METHODS: CD34 expression on acute myelogenous leukemia KG1a cells was detected by flow cytometry. KG1a cells were co-cultured at different effector-to-target (E:T) ratios with NK cells isolated from 5 healthy individuals using magnetic cell sorting. Lactate dehydrogenase (LDH) release assay was employed to examine the cytolysis of KG1a cells in the co-culture, and the inhibition rate of the KG1a cell colony formation in methylcellulose was determined with K562 cells sensitive to NK cells as the control. RESULTS: A expression rate as much as (98.0-/+1.1)% was detected for CD34 antigen on KG1a cells, and the isolated NK cells (CD3(-)CD16+CD56+ cells) had a purity of (93.2-/+3.7)% after magnetic cell sorting. Allogenetic NK cells exhibited obvious cytotoxicity and colony inhibition in vitro against KG1a cells at different E:T ratios, and the effects were significantly enhanced as the E:T ratios increased (P<0.05). At the same E:T ratio, the cytotoxicity and colony inhibition rate of allogenetic NK cells against KG1a cells was lower than those against K562 cells (P<0.05). CONCLUSION: Allogenetic NK cells exhibit obvious cytotoxicity and colony formation against CD34+ acute myelogenous leukemia cells.

[Involvement of mitochondria apoptotic pathway in the manumycin inducing apoptosis of U937 and HL-60].

OBJECTIVE: To investigate the apoptosis induced by manumycin in U937 and HL-60 cell lines, and to explore the role of mitochondria apoptotic pathway in manumycin-inducing apoptosis. METHODS: Leukemic cells line U937 and HL-60 were treated by manumycin at 2 micromol/L for different time. Apoptosis of leukemia cells was detected by flow cytometry. The cytosolic proteins were extracted using a digitonin buffer. The protein expression of cytochrome C, caspase-9, caspase-8, and caspase-3 were determined by western blot. Mitochondrial membrane potential was detected by JC-1. RESULTS: In U937 and HL-60 cells, manumycin induced mitochondrial depolarization after 6 h treatment. The average red/green fluorescence ratios at 6 h were significantly (P < 0.01) lower than those at time 0, being 0.51 +/- 0.07 and 0.41 +/- 0.06 for control group respectively. Manumycin induced cytochrome C release from the mitochondria into the cytosol after 6 h treatment, and activated caspase-9, caspase-8, and caspase-3 after a 16h treatment. The broad-spectrum caspase-inhibitor Z-VAD-fmk at 50 micromol/L was able to inhibit caspase cleavage completely, but only reduced the manumycin-induced apoptosis rates by 51.69% and 56.47% in U937 and HL-60, respectively. CONCLUSION: Manumycin induced apoptosis in U937 and HL-60 cell lines via mitochondria apoptotic pathway.

[Effect of hyperthermia in combination with chemotherapy on K562/AO2 cells in vitro].

This study was purposed to investigate the inhibitory effect, apoptosis, Bcl-2 and P-gp expression of K562/AO2 cells by hyperthermia combined with adriamycin. The working concentration of adriamycin against K562/AO2 was determined by MTT assay. The hyperthermia and chemotherapy were used alone or in combination, then the cell survival rate was detected at 48 hours. The inhibitory effect was evaluated by MTT assay. The apoptosis rate, Bcl-2 and P-gp expression of K562/AO2 were determined by flow cytometry. The concentration of adriamycin in the experiment was defined as its IC(50) at 48 hours action. The results indicated that the hyperthermia at 40, 41 and 42 degrees C for 60 minutes showed obvious inhibitory effect on K562/AO2 cells (p < 0.01). Adriamycin chemotherapy combined with hyperthermia showed more obvious inhibitory effect on K562/AO2. According to flow cytometric analysis, the hyperthermia and adriamycin used alone or in combination could obviously increase the apoptosis rate and down-regulate Bcl-2 and P-gp expression of K562/AO2 cells (p < 0.01). It is concluded that the adriamycin chemotherapy combined with hyperthermia for 60 minutes shows obvious inhibitory effect on K562/AO2 cells, which increases the apoptosis rate and down-regulates expression of Bcl-2 and P-gp.

Requirement of reactive oxygen species generation in apoptosis of leukemia cells induced by 2-methoxyestradiol.

AIM: To investigate the effects of 2-methoxyestradiol (2-ME) on 2 myeloid leukemia cell lines HL-60 and U937, and to explore its mechanisms. METHODS: Human myeloid leukemia cells HL-60 and U937 were used. Measurement of mitochondrial membrane potential (Dym) was performed using 5,5',6,6'-Tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide ( JC-1). 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 dihydroethidium (DHE). Cytotoxicity was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay. RESULTS: 2-ME resulted in viability decrease in a dose-dependent manner. 2-ME treatment also generated reactive oxygen species (ROS), including NO and superoxide anions, which resulted in mitochondria damage. 2-ME-induced apoptosis was correlated with an increase in ROS. The quenching of ROS with N-acetyl-L-cysteine protected leukemia cells from 2-ME cytotoxicity and prevented apoptosis induction by 2-ME. Furthermore, the addition of manumycin, a farnesyltransferase inhibitor, significantly enhanced apoptosis induced by 2-ME. CONCLUSION: Cellular ROS generation plays an important role in the cytotoxic effect of 2-ME. It is possible to use ROS generation agents, such as manumycin, to enhance the antileukemic effect. The combination strategy needs further in vivo justification and may have potential clinical application.

[Expression of HLA class I molecules and MHC class I chain-related molecules A/B in K562 and K562/AO2 cell lines and their effects on cytotoxicity of NK cells].

The study was aimed to investigate the expression of HLA class I molecules and MHC class I chain-related molecules A/B (MICA/MICB) in K562 and adriamycin (ADM)-resistant K562 cell lines (K562/AO2) and their effect on cytotoxicity of NK cells. Expression of HLA class I molecules and MICA/MICB on the surface of K562 and K562/AO2 cell lines were analyzed by flow cytometry. Cytotoxicity of NK cells (isolated from 3 healthy persons) against K562 and K562/AO2 cells were detected by LDH releasing assay at different effect-to-target cell ratios (E:T). In blocking experiments, anti-MHC class I monoclonal antibody (McAb) (W6/32, a pan anti-HLA class I antibody) and anti-MHC class I chain-related molecules McAb (BAMO-1, specifically against MICA and MICB) were added to the target cells at E:T of 10:1. The results showed that the expression of MHC class I chain-related molecules on K562 was higher than that on K562/AO2 (P=0.000), and HLA class I molecules were not detectable on both cells. Cytotoxicities of NK cells against K562 and K562/AO2 cells were (29.32 +/- 0.12)%, (45.33 +/- 0.78)%, (58.37 +/- 0.87)%, (72.37 +/- 0.96)% and (12.47 +/- 0.91)%, (24.36 +/- 1.11)%, (33.29 +/- 1.03)%, (53.87 +/- 1.27)% at E:T ratios of 5:1, 10:1, 20:1 and 30:1 respectively (P=0.000), the cytotoxicity of NK cells on K562 cells was significantly higher than that on K562/A02 cells at different E:T ratios. Blocking experiments confirmed that at E:T of 10:1 killing of NK cells against K562 and K562/AO2 cells was efficiently inhibited by BAMO-1, whereas W6/32 had no effect on K562 and K562/AO2 cells. It is concluded that the expression of MHC class I chain-related molecules on K562 and K562/AO2 cells is correlated with NK cell-mediated lysis. NK cells display higher cytotoxicity against parental K562 cells than multi-drug resistant K562/AO2 cells. Down-regulation of MICA/B in multi-drug resistant tumor cell lines leads to reduction of susceptibility to NK lysis.