Mechanism of Proliferation and Apoptosis of Acute Promyelocytic Leukemia Cell Line NB4 Induced by TPA / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of phorbol-12-myristate-13-ace-tate (TPA) on the proliferation and apoptosis of acute promyelocytic leukemia cell line NB4 and its molecular mechanism.@*METHODS@#The effect of different concentrations of TPA on the proliferation of NB4 cells at different time points was detected by CCK-8 assay. The morphological changes of NB4 cells were observed by Wright-Giemsa staining. The cell cycle and apoptosis of NB4 cells after TPA treatment were detected by flow cytometry. The mRNA expressions of NB4 cells after TPA treatment were analyzed by high-throughput microarray analysis and real-time quantitative PCR. Western blot was used to detect the protein expression of CDKN1A, CDKN1B, CCND1, MYC, Bax, Bcl-2, c-Caspase 3, c-Caspase 9, PIK3R6, AKT and p-AKT.@*RESULTS@#Compared with the control group, TPA could inhibit the proliferation of NB4 cells, induce the cells to become mature granulocyte-monocyte differentiation, and also induce cell G1 phase arrest and apoptosis. Differentially expressed mRNAs were significantly enriched in PI3K/AKT pathway. TPA treatment could increase the mRNA levels of CCND1, CCNA1, and CDKN1A, while decrease the mRNA level of MYC. It could also up-regulate the protein levels of CDKN1A, CDKN1B, CCND1, Bax, c-Caspase 3, c-Caspase 9, and PIK3R6, while down-regulate MYC, Bcl-2, and p-AKT in NB4 cells.@*CONCLUSION@#TPA induces NB4 cell cycle arrest in G1 phase and promotes its apoptosis by regulating PIK3/AKT signaling pathway.

Effect of a Novel Dihydroartemisinin Dimer Containing Nitrogen Atoms SM 1044 on Apoptosis of Human Leukemia Cell Line NB4-R1 / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of a water-soluble novel dihydroartemisinin dimer containing nitrogen atoms SM 1044 on the apoptosis of all-trans retinoic acid (ATRA) resistant acute promyelocytic leukemia (APL) NB4-R1 cells and its potential mechanism.@*METHODS@#The effects of SM 1044 on cell apoptosis, mitochondrial transmembrane potential, and the level of reactive oxygen species (ROS) were assessed by flow cytometry. Expressions of apoptosis-related proteins were determined by Western blot. The effects of SM 1044 on MAPK (ERK, JNK) signaling pathway, PML/RARα fusion protein, and expressions of apoptosis-related proteins were detected by Western blot.@*RESULTS@#SM 1044 could significantly induce apoptosis and the loss of mitochondrial transmembrane potential in NB4-R1 cells, and activate apoptosis-related proteins caspase-3, caspase-8, caspase-9 and poly (ADP-ribose) polymerase (PARP). SM 1044 could also induce NB4-R1 cells to produce ROS. Western blot showed that SM 1044 activated the phosphorylation of MAPK (ERK, JNK) signaling pathway and down-regulated the expression of PML/RARα fusion protein.@*CONCLUSION@#SM 1044 can induce apoptosis of ATRA resistant APL NB4-R1 cells, which may be related to ROS/ERK and ROS/JNK signaling pathway, and can also induce by down-regulating PML/RARα fusion protein.

Effects of Monoammonium Glycyrrhizinate on Stem Cell-like Characteristics, Oxidative Stress and Mitochondrial Function of Acute Promyelocytic Leukemia NB4 Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of monoammonium glycyrrhizinate on the stem cell-like characteristics, oxidative stress and mitochondrial function of acute promyelocytic leukemia cells NB4.@*METHODS@#CCK-8 method was used to detect the viability of acute promyelocytic leukemia cells NB4, and the appropriate dose was screened; Cloning method was used to detect the proliferation rate of NB4 cell; Western blot was used to detect the expression of cell cycle-related protein; flow cytometry was used to detect cell apoptosis and sort NB4 stem cells positive (CD133+); Stem cell markers (Oct4, ABCG2, Dclk1) were detected by RT-PCR; ROS was detected by fluorescence; The kit was used to detect the level of oxidative stress markers (MDA); The flow cytometry was used to detect the change of mitochondrial membrane potential; Western blot was used to detect the expression of mitochondrial damage index-related proteins (Bax/BCL-2).@*RESULTS@#Compared with the control group, if the concentration of MAG was less than 5 μmol/L, the cell NB4 viability showed no significant difference; if the concentration was higher than 5 μmol/L, the inhibitory effect on the growth of cell NB4 increased and showed significant difference (P<0.05), according to the results of CCK-8 experiment, four groups were set based on the concentration of MAG 0 μmol/L, MAG 5 μmol/L, MAG 10 μmol/L, and MAG 20 μmol/L; compared with the control group (MAG 0 μmol/L), the cells in MAG 5 μmol/L group showed no significant difference, while the proliferation rate, cyclin expression, mitochondrial membrane potential, stem cell CD133+ ratio, and marker mRNA level ( Oct4, ABCG2, Dclk1) of NB4 cell were significantly reduced (P<0.05); the apoptosis rate, reactive oxygen species, MDA content and Bax/BCL-2 expression of NB4 cell significantly increased (P<0.05).@*CONCLUSION@#Monoammonium glycyrrhizinate has a significant inhibitory effect on acute promyelocytic leukemia cells NB4, which may be related to the regulation of stem cell-like characteristics, oxidative stress and mitochondrial function.

Effect of combination of artesunate and arsenic trioxide on the proliferation and apoptosis of NB4 cells / 国际中医中药杂志

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.

Establishment of cell-based screening system for compound regulating the stability of retinoic acid receptors / 上海交通大学学报（医学版）

Objective·To establish a cell-based screening system for identification of compounds with activity in regulating retinoic acid receptor (RARα) stability. Methods·The modified pMSCV plasmid constructs, named as RARα-EGFP-IRES-DsRed, consists of enhanced green fluorescent protein (EGFP) fusing to RARα and red fluorescent protein (DsRed) as internal references incorporating the internal ribosome entry site (IRES) as interval sequence. The RARα-EGFP-IRES-DsRed plasmid was stably transfected into NB4 cells which were named as NB4-pMGIR-RARα. Fluorescence signals of EGFP and DsRed indirectly reflecting the expression of RARα, were detected by flow cytometry in cells that were treated with all-trans retinoic acid, sodium valproate, cytarabine, lenalidomide, etoposide, montelukast and gambogic acid, respectively. Effects of these compounds on the expression of RARα protein were further examined by Western blotting. Results·A double fluorescence reporter system for screening compounds that can increase the stability of RARα protein was successfully established, and sodium valproate was identified as a potent compound to promote the stability of RARα. Conclusion·The double fluorescence reporter system can be used to screen compounds regulating the stability of RARα protein, which can be further used to identify compounds regulating the stability of other proteins.

Expression of SP100 protein induced by ATRA and its effect on proliferation in NB 4 cells / 基础医学与临床

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.

Preparation of CD123 mono-antibody modified tanshinone ⅡA loaded immunoliposome and its in vitro evaluation / 中国中药杂志

In the study, we developed a novel formulation, CD123 mono-antibody (mAb) modified tanshinone ⅡA loaded immunoliposome (CD123-TanⅡA-ILP) to achieve the targeted drug delivery for leukemia cells. Orthogonal test was used to optimize liposome preparation, and the TanⅡA-loaded PEGylated liposomes (TanⅡA-LP) of S100PC-Chol-(mPEG2000-DSPE)-TanⅡA at 19∶5∶1∶1 molar ratio were prepared by the thin film hydration-probe ultrasonic method. A post-insertion method was applied to prepare CD123-TanⅡA-ILP via thiolated mAb conjugated to the terminal of maleimide-PEG2000-DSPE. The cellular uptake assay was measured by flow cytometry, and the inhibitory effect of CD123-TanⅡA-ILP on NB4 cells proliferation was tested by using MTT assay. The results of cellular uptake assay showed that CD123-ILP could significantly increase the drug uptake of NB4 cells as compared with free drugs and LP. The IC₅₀ values at 48 h incubation were 20.87, 11.71, 7.17 μmol•L⁻¹ respectively for TanⅡA，TanⅡA-LP and CD123-TanⅡA-ILP. CD123-ILP demonstrated a potential and promising targeted drug delivery strategy for acute myelogenous leukemia (AML) treatment.

Influence of icaritin in proliferation and apoptosis of NB4 leukemia cells and its mechanism / 吉林大学学报(医学版)

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.

Influence of targeted silence HOXA10 gene on leukemia NB4 cells / 白血病·淋巴瘤

Objective To explore the effects of lentivirus-mediated RNA interference targeting HOXA10 gene on the proliferation, apoptosis and drug resistance of leukemic cell line NB4.Methods NB4 cells were divided into three groups: interference group, negative control group and untreated group.The infection efficiency of lentivirus for NB4 cells was detected by flow cytometry, and the expression of HOXA10 gene of NB4 cells at mRNA and protein level was detected by real-time PCR and Western blot.Cell survival was determined by MTF assay, and apoptosis and necrosis rates were detected by flow cytometry.Western blot was used to detect the influence of down-regulation HOXA10 gene on the multi-drug resistance-1 (MDR-1) protein.Results The ratio of GFP positive cells was up to 90 ％.HOXA10 gene mRNA and protein levels were decreased in interference group compared with control group.The inhibition rate of interference group was (52.12±4.02) ％, the apoptosis rate of interference group was (30.0±2.7) ％, and their differences in the interference group and in control groups (negative control group and untreated group) were significant (P ＜ 0.05).Western blot results showed that interfering HOXA10 gene significantly reduced the resistance gene MDR-1 expression level and reverse the drug-resistant of leukemia cells.Conclusions Lentivirns-shHOXA10 can steadily reduce the expression level of HOXA10, inhibit the leukemic cells proliferation, promote apoptosis and reverse drug-resistant.HOXA10 gene is expected to become a new target for reversing leukemia drug resistance.

Effect of HMGA2 knockdown on cell growth in NB4 cell xenograft tumor / 实用医学杂志

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.

Regulatory effects of CD44 antibody-A3 D8 on IL-3 Rαand downstream PI3 K/Akt pathway in NB4 cells / 中国药理学通报

Aim To investigate the effect of CD44 anti-body-A3 D8 on the expression of IL-3 Rα and down-stream PI3K/Akt in NB4 cells. Methods The ex-pression of IL-3 Rα mRNA was detected by real-time quantitative RT-PCR, the IL-3Rα protein expression and changes of PI3 K/Akt signal pathway in NB4 cells treated with A3D8 were analyzed by Western blot. An-nexin-V-FITC/PI double staining flow cytometry was u-tilized to detect the apoptotic cells. The inhibitor of PI3 K/Akt signaling LY294002 combined with A3 D8 was used to inhibit the PI3K/Akt in NB4 cells. Re-sults After treated with A3 D8 , both the transcription-al level and translational level of IL-3 Rα were remark-ably reduced, and the PI3K/Akt pathway was inhibi-ted. LY294002 improved the inhibitory and apoptotic effects of A3D8 on NB4 cells. Conclusion CD44 antibody A3 D8 can downregulate the expression of IL-3Rα and inhibit the downstream PI3K/Akt pathway.

Quercetin inhibits leukemia cells by regulating PI3k/Akt pathway in vitro / 白血病·淋巴瘤

Objective To study the anti-tumor activity of quercetin in NB4 leukemia cells and the roles of PI3K/Akt,bcl-2,and Bax on the quercetin-induced apoptosis,and to investigate the potential underlying mechanism.Methods MTT assay was used to monitor cell proliferation,Hoechst 33258 fluorescent staining and flow cytometry were employed to detect apoptosis in NB4 cells.Western blot was used to detect the expression changes of related proteins in quercetin treated NB4 cells.Confocal laser microscopy was used to test the distributional variation of Akt between cytoplasm and nucleus.Results Quercetin significantly inhibited the NB4 cell proliferation in a dose-and time-dependent manner (20-160 μ mol/L).In addition,treated by 20,40 80 μmol/L quercetin,the rates of apoptosis were (9.25±0.11) ％,(20.83±2.10) ％and (41.43±2.90) ％,there were statistical difference compared with blank cells (t were 4.14,6.56 and 7.02,all P ＜ 0.05).This was concentration dependent and accompanied by morphological changes characteristic of apoptosis.Further,quercetin induced a G~M arrest,which might account for its cytotoxic effects.Quercetin decreased PI3k/Akt expression and caused an inhibition of the anti-apoptotic protein bcl-2,while increasing the expression of Bax.Quercetin had no effects on total Akt,but it promoted Akt translocation from cell nucleus to the cytoplasm (F =15.12,P ＜ 0.05).Conclusion Quercetin induces the leukemia NB4 cell apoptosis by affecting multiple signal pathways and plays a strong anti-leukemia effect.In addition,our results suggest that PI3K/Akt pathway could be a novel target for the leukemia chemotherapy.

Induction of acute myelogenous leukemia cell apoptosis by bortezomib and its effect on SALL4 gene expression / 中国药学杂志

OBJECTIVE: To investigate the proteasome inhibitor bortezomib induces acute myelogenous leukemia cell lines TF1 and NB4 apoptosis and its effect on SALL4 gene expression. METHODS: Cell proliferations were analyzed by MTT assay. Flow cytometry was used to analyze cell apoptosis rate. SALL4 protein was detected by immunocytochemistry. The expressions of SALL4 gene were detected by Real-time PCR. SALL4 proteins in two cell lines were detected by Western Blotting. RESULTS: MTT assay showed bortezomib inhibited the proliferations of two cell lines in a time-and-does manner. TF1 and NB4 cells' 48 h IC50 were (29.15 ± 0.55) and (30.55 ± 0.74) nmol · L-1 respectively. Flow cytometry showed bortezomib could induce apoptosis of two cell lines in a dose-dependent manner. Immunocytochemistry analysis revealed that both of two cell lines expressed SALL4 proteins which located in cell nucleus. Real-time PCR demonstrated that SALL4 genes were down-regulated after cells were treated by different concentrations(10, 30, 50 nmol · L-1) of bortezomib for 24 h, and bortezomib 50 nmol · L-1 groups' genes were down-regulated to 45.11% (TF1) and 69.77% (NB4) respectively comparing with the control groups (P < 0.05). Western blotting revealed that both of the cell lines expressed SALL4B proteins and which could be inhibited by bortezomib in a time-and-does manner. CONCLUSION: Bortezomib can significantly inhibit two cell lines proliferation and induce apoptosis, meanwhile down-regulate the expressions of SALL4 gene. Copyright 2012 by the Chinese Pharmaceutical Association.

Effect of TRAIL on NB4 and K562 cell lines and its relationship with TRAIL receptors expression / 白血病·淋巴瘤

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.

Induction of apoptosis in NB4 cells by decitabine alone and combination with As2O3 / 白血病·淋巴瘤

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.

Effects of combined arsenic trioxide and resveratrol on the viability of human acute promyelocytic leukemia cell line NB4 cells / 中国地方病学杂志

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.

Effects of simvastatin on differentiation and apoptosis of human promyelocytic leukemia cell line NB4 / 白血病·淋巴瘤

Objective To investigate the effects of simvastatin (SV) on the proliferation,differentiation and apoptosis of human promyelocytic leukemia cell line NB4.Methods NB4 cells were incubated with SV at different concentration with or without all-trans retinoic acid (ATRA),and NB4 cells without any treatment were taken as normal control.Cells of different groups were collected at 24 h,48 h and 72 h after incubation for further detection.Morphological changes by Wright stain were performed.MTT method was used to assay the growth inhibition rate and flow cytometry was used to detect the surface CD11b expression levels,the early stage apoptosis ratio and cell necrosis ratio.Results Treated with 15 μ mol/L SV,10 μ mol/L SV and 5 μ mol/L SV respectively,with the NB4 cells growth,the cell inhibition rates gradually increased (F =7.15,P =0.000),as well as CD11b expression levels (F =3.41,P =0.014) and AnnexinVexpression levels (F =43.38,P =0.000).Furthermore the NB4 cells treated with 15 μ mol/L SV exhibited the most significant changes with cell inhibition rate of 0.96±0.02,CD11b expression level increased to (62.41±6.37) ％ and AnnexinV expression level increased to (87.38±2.94) ％ after 72 h incubation.Combination of 15 μmol/L SV with 0.5 μmol/L ATRA displayed obvious interaction for increasing CD11b expression levels (F =4.093,P =0.025),while no significant interaction for cell inhibition rates and Annexin V expression levels were observed.After 72 h incubation,the CD11b expression levels (89.46±9.13) ％ in NB4 cells treated with 15 μ mol/L SV in combination with 0.5 μ mol/L ATRA were significantly higher than those treated with ATRA (71.27±7.27) ％ and SV (62.41±6.37) ％ (t =2.71,P =0.054; t =4.37,P =0.017)' solely.Conclusion Simvastatin in vitro inhibits NB4 cell proliferation,promotes cell apoptosis,and synergistically induces cell differentiation with ATRA dose-dependently in vitro,which indicates that SV may have the effect of synergistic anti-promyelocytic potency with ATRA.

Apoptosis of leukemic cells induced by flavonoids of puerarin and its molecular mechanisms / 白血病·淋巴瘤

Objective To explore the effects and the possible molecular mechanism of flavonoids of puerarin (PR) on chronic myelogenous leukemia (CML) cell line K562 and acute promyelocytic leukemia (APL) cell line NB4 in vitro. Methods MTT assays were used to detect the inhibitory effects of cell proliferation. The apoptosis of K562 and NB4 cells was detected by flow cytometry marked with Annexin V/PI. The expression of bcr-abl, p53, bcl-2, Fas/FasL in K562 cells and JNK, PARP, bcl-2 and Caspase 3 in NB4 cells at protein level was detected by Western blot. Results PR could inhibit the proliferation of K562 and NB4 cells in a time-dose dependent manner. The expression of protein levels of bcr-abl fusion gene declined, while the p53 protein otherwise increased, and both were in a dose-dependent manner (F = 18.74, P <0.05). The application of PR had no effect on bcl-2 and Fas/FasL protein expression in K562 cells. The JNK, PARP and Caspase3 proteins were upregulated in NB4 cells, while bcl-2 was downregulated with the increasing concentrations of PR (F=42.32, P <0.05). Conclusion PR could inhibit leukemic cell proliferation, induce cell cycle block, and increase cell apoptosis through different molecular mechanisms. It suggestes that PR might potentially be a kind of broad spectrum anti-leukemia agent.

Inhibition effects of Fuerkang Injection and its ingredients on SMMC-7721 and NB4 cell lines / 中成药

AIM: To study the inhibitiory effects of Fuerkang Injection (sweet worm wood oil, glycyrrhetate and Vitamin A acetate). and its ingredients on SMMC-7721 and NB4 cell lines, in order to understand its antitumour spectrum and reasonability of its composition. METHODS: SMMC-7721 and NB4 cell lines were treated with Fuerkang Injection and its ingredients under the same concentration for 48 h, adfiamycin was used as standard com-parison,inhibitiory effect on growth of two cell lines was detected by MTT method. RESULTS: At 1.260 μg/mL, the inhibition rate of Fuerkang Injection and its decomposed recipes preparations 1,2,3 to SMMC-7721 cell lines were 54.78%, 55.49%, 53.18%, 53.79%, hemi-inhibitory concentration IC_(50) were 0.415,0.376,0.715 and 0.636 μg/mL,respectively. The inhibition rate of NB4 cell lines were 88.6% ,82.5% ,77.9% and 76.9% ,hemi-inhibitory concentration IC_(50) were 0.091,0.108 1,0.084 and 0.120 μg/mL. CONCLUSION: Fuerkang Injection and its ingredients have significant inhibitory action on SMMC-7721 and NB4 cell lines,each component herb in the compound preparation has synergetic effects.

Comparison of the effect of arsenic sulfide on apoptosis and hTERT-mRNA expression in two leukemia cell lines / 细胞与分子免疫学杂志

AIM: To explore the different effect and mechanism of arsenic sulfide on telemorase activity and hTERT-mRNA expression in CML cell lines-KS62 and APL cell lines-NB4. METHODS: Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). The expression of hTERT-mRNA was analyzed by semi-quantitative RT-PCR. Flow cytometry was used to analyze the cell cycle and apoptosis. RESULTS: 0.15-0.6 mg/L arsenic sulfide (72 h)can induce apoptosis and inhibit telomerase activity and hTERT-mRNA expression in NB4 cell. The concentration of arsenic sulfide with the same effect on K562 cell was 0.3-3 mg/U 0.3 mg/L arsenic sulfide (72 h) can cause the proportion of the NB4 cell in G2/M phase increased, but for K562 cell, The concentration of arsenic sulfide was 1.5 mg/L. CONCLUSION: Telomerase system may be one.of the pathway for arsenic sulfide inducing apoptosis of NB4 and K562 cell; G2/M phrase arrest may have correlation with decrease of telomerase activity; The sensitivity of NB4 and K562 call for arsenic sulfide is different, the mechanism of it need to study more.