Role of extracellular regulated protein kinases in FTY720-induced apoptosis of leukemia cell lines HL-60 and U937

Summary The effects of a novel immunosuppressive agent FTY720 on proliferation inhibition and apoptosis of acute leukemia cell lines HL-60 and U937, and the role of extracellular regulated protein kinase (ERK) in the course of proliferation inhibition and apoptosis induced by FTY720 were studied. The proliferation inhibition rate of HL-60 and U937 cells by various concentrations of FTY720 was detected by MTT assay. Cell apoptosis was detected by DNA fragment analysis and flow cytometry. The phosphorylated ERK1/2 protein expression was observed by Western blotting. The change of intracellular distribution of ERK1/2 protein was identified by SP immunohistochemical staining. The results showed that FTY720 could inhibit the growth of HL-60 and U937 cells effectively in a dose-dependent manner. After incubation with FTY720 for 24 h, apoptosis was observed in HL-60 and U937 cells. The intracellular expression of phosphorylated ERK1/2 protein was also down-regulated and the distribution of ERK1/2 protein in cell nuclear was reduced during FTY720-induced apoptosis. So, that FTY720 inhibited ERK1/2 phosphorylation might mediate the role of FTY720-induced apoptosis and proliferation inhibition of leukemia cells. LI Dengju, male, born in 1972. Doctor in Charge.     

Regulative Function of Telomerase and Extracelluar Regulated Protein Kinases to Leukemic Cell Apoptosis

In order to investigate the regulative function of telomerase and phosphorylated (activated) extracelluar regulated protein kinase (ERK) 1 and 2 in the leukemic cell lines HL-60 and K562 proliferation inhibition and apoptosis,three chemotherapeutic drugs Harringtonine (HRT),Vincristine (VCR) and Etoposide(Vp16) were selected as inducers.The proliferation inhibition rate was detected by MTT method,the cell cycle and cell apoptosis was analyzed by flow cytometry and the telomerase activity was detected by the telomeric repeat amplification protocol (TRAP) assay and bioluminescence analysis method.The phosphorylated ERK 1/2 protein expression was detected by western blot method.The results showed that HRT,VCR and Vp16 could inhibit cell proliferation,induce apoptosis,inhibit telomerase activity and down-regulate the protein expression of phosphorylated ERK.It was suggested that ERK signal transduction pathway was involved in the down-regulation of telomerase activity and the onset of apoptosis in the leukemic cells treated by HRT,VCR and Vp16.     

抑制细胞外调节蛋白激酶增强Jurkat淋巴瘤细胞对三尖杉酯碱的敏感性

目的 研究细胞外调节蛋白激酶（EPK）在三尖杉酯碱（HRT）诱导Jurkat淋巴瘤细胞调亡中的作用。方法 用四甲基偶氮唑盐（MTT）法、流式细胞术和Western印迹检测法分别检测细胞增殖抑制率，细胞凋亡和磷酸化ERK蛋白的表达。结果 研究发现下调磷酸化状态的细胞外调节蛋白激酶的表达可以增强淋巴瘤细胞系Jurkat对三尖杉酯碱的敏感性。结论 ERK通路对维系Jurkat淋巴瘤生存有重要作用，该通路的抑制可以增强细胞对凋亡诱导剂的敏感性。     

Regulative function of telomerase and extracellular regulated protein kinases to leukemic cell apoptosis.

In order to investigate the regulative function of telomerase and phosphorylated (activated) extracellular regulated protein kinase (ERK) 1 and 2 in the leukemic cell lines HL-60 and K562 proliferation inhibition and apoptosis, three chemotherapeutic drugs Harringtonine (HRT), Vincristine (VCR) and Etoposide (Vp16) were selected as inducers. The proliferation inhibition rate was detected by MTT method, the cell cycle and cell apoptosis was analyzed by flow cytometry and the telomerase activity was detected by the telomeric repeat amplification protocol (TRAP) assay and bioluminescence analysis method. The phosphorylated ERK1/2 protein expression was detected by western blot method. The results showed that HRT, VCR and Vp16 could inhibit cell proliferation, induce apoptosis, inhibit telomerase activity and down-regulate the protein expression of phosphorylated ERK. It was suggested that ERK signal transduction pathway was involved in the down-regulation of telomerase activity and the onset of apoptosis in the leukemic cells treated by HRT, VCR and Vp16.     

新型免疫抑制剂FTY720诱导K562细胞凋亡及其机制

目的：探讨新型免疫抑制剂FTY720诱导K562细胞凋亡的效应及其机制,为临床白血病的疗提供实验依据。方法：体外培养人白血病K562细胞,分为空白对照组和FTY720处理组,FTY720处理组细胞分别采用6 μmol•L^-1 FTY720诱导3、6和12 h,或采用2、4、6和8 μmol•L^-1 FTY720处理24 h。采用流式细胞术分析细胞凋亡百分率、活性氧（ROS）水平、线粒体膜电位（MMP）和细胞周期。采用WST-1 还原法检测FTY720作用下K562细胞增殖抑制率。结果：流式细胞术检测,与空白对照组比较,6 μmol?L-1 FTY720诱导细胞3、6和12 h后,细胞凋亡率随时间延长而升高（P<0.01）;与空白对照组比较,2、4、6和8 μmol•L^-1 FTY720诱导细胞24 h 后,细胞凋亡率随药物浓度增加而升高（P<0.01）。与空白对照组比较,随FTY720浓度增加,K562细胞内ROS水平增加（P<0.01）,同时其MMP下降（P<0.01）。细胞周期分析,与空白对照组比较,随FTY720浓度增加,G0/G1期细胞百分率增加（P<0.05）,而S期和G2/M期细胞百分率减少（P<0.05）。WST-1 还原法分析,与空白对照组比较,FTY720处理72 h后,2、4、6和8 μmol?L-1FTY720处理组K562细胞增殖抑制率［（24.0±4.1）%、(46.4±3.9)%、(67.0±4.8)%和（88.2±5.6）%］明显升高（P<0.01）,FTY720对K562细胞的半数抑制浓度（IC50）为5.5　μmol•L^-1。结论：FTY720可通过导致细胞周期阻滞和激发ROS而诱导细胞凋亡来发挥抗肿瘤作用。     

三七总皂苷诱导U937细胞凋亡及对Caspase-3表达的影响

目的观察三七总皂苷（PNS）对人急性单核细胞白血病细胞系U937细胞的增殖、凋亡及Caspase-3表达的影响。方法用不同浓度PNS作用于U937细胞,采用MTT法检测细胞增殖活性,用流式细胞术检测细胞周期和凋亡,Western-blot法检测Caspase-3蛋白表达。结果 PNS抑制U943细胞生长并呈时间和剂量依赖性;分别予100、200、400、800mg/L处理U937细胞48h,细胞凋亡率分别为（5.93±0.15）%、（7.43±0.70）%、（12.3±0.38）%和（20.4±0.91）%,对照组细胞凋亡率为（1.73±1.50）%（P〈0.01）,并诱导细胞G1期阻滞;Western-blot结果显示,PNS使Caspase-3蛋白表达上调。结论 PNS能抑制U937细胞增殖,并可能通过上调Caspase-3蛋白表达诱导细胞凋亡。     

蛋白酶体抑制剂硼替佐米诱导髓系白血病细胞株HL60凋亡的机制研究

目的 探讨蛋白酶体抑制剂硼替佐米诱导髓系白血病细胞HL60凋亡的机制。方法 四甲基偶氮唑盐（MTT）法、Hoechst33342染色形态学观察及流式细胞仪证实细胞凋亡,Western印迹检测B细胞淋巴瘤／白血病-2（Bcl-2）、Caspase-9、Caspase-3、多聚ADP-核糖聚合酶（PARP）蛋白表达。结果 硼替佐米对HL60细胞的增殖抑制作用呈浓度和时间依赖关系,30nmol/L的硼替佐米作用24h能明显抑制HL60细胞增殖,抑制率为76％,形态学观察可见明显的胞核凝聚、固缩及碎裂;流式细胞仪检测可见明显的凋亡峰凋亡率为62．6％;Western印迹检测显示Bcl-2表达下降,Caspase-9、Caspase-3、PARP蛋白均裂解激活。结论 硼替佐米能诱导髓系白血病细胞HL60凋亡,其机制与Bcl-2蛋白的抑制及Caspase凋亡信号途径的激活有关。     

FTY720对胆管癌细胞系QBC939增殖及侵袭的影响

目的：探讨FTY720对体外培养QBC939细胞系增殖及侵袭的影响。方法：体外培养QBC939细胞系,将其分为对照组、FTY720不同浓度给药组。通过MTT法、流式细胞仪检测周期和凋亡、细胞侵袭等实验观察FTY720对QBC939细胞系增殖及侵袭的影响。结果：当FTY720浓度为2400 ng/ml时,体外培养的QBC939细胞系的增殖受到明显抑制,抑制率为65．4％（ P〈0．05）;流式细胞仪检测提示QBC939细胞系被阻滞于G1期,并促进其发生凋亡;侵袭实验显示FTY720可以明显抑制QBC939细胞系的侵袭,当FTY720浓度为2400 ng/ml时,侵袭抑制率可达80％。结论：FTY720可抑制体外培养QBC939细胞系的增殖,诱导该肿瘤细胞凋亡,并且抑制其侵袭。     

FTY720联合吉西他滨对非小细胞肺癌肿瘤相关细胞株增殖和凋亡作用的研究

目的 探讨FTY720与吉西他滨用药对人非小细胞肺癌(NSCLC)肿瘤细胞株A549和H520增殖和凋亡的影响作用.方法 分别选取0、2、4、6、8、10 μmol/L浓度的FTY720对体外培养得到NSCLC细胞株A549及H520做干预影响,于培养24、48、72 h后分别检测吸光度值,观察各浓度条件下FTY720对A549及H520的抑增殖作用;对A549及H520细胞株加分别单独加7 μmol/L FTY720和0.2 μmol/L吉西他滨及37 μmol/L FTY720结合0.2μmol/L吉西他滨,观察各组细胞培养48 h后抑制和凋亡差异.结果 不同浓度FTY270抑制NSCLC细胞株A549、H520增殖,差异有统计学意义(P＜0.05),FTY720对NSCLC细胞株A549、H520的增殖抑制作用和浓度、时间具有相关性,FTY720联合吉西他滨抑制A549、H520细胞和凋亡率显著强于两种药品的单独使用(P＜0.05).结论 FTY720联合吉西他滨可显著抑制人NSCLC细胞株A549及H520的增殖,并可有效促进癌细胞凋亡的作用,临床价值较高.     

和厚朴酚对人白血病细胞系U937细胞增殖和凋亡的影响

 【目的】 探讨和厚朴酚(HNK)对白血病细胞系U937细胞增殖和凋亡的影响&#65377;【方法】 将U937细胞和正常外周血单核细胞分别与HNK共培养,Hoechst33342荧光染色检测细胞凋亡形态,用噻唑蓝比色法(MTT)检测HNK对细胞增殖的抑制作用;流式细胞仪检测凋亡;罗丹明123检测线粒体膜电位;Caspase3/7活性检测试剂盒检测Caspase3/7活性;荧光定量PCR(FQ-PCR)检测Caspase-3&#65380;Caspase-7 mRNA水平变化&#65377;【结果】 HNK对U937的体外增殖有显著抑制作用,48 h半数抑制浓度(IC50)为11.8 μg/mL,而对正常外周血单核细胞的IC50为40.3 μg/mL,Annexin V/PI双标染色显示,细胞凋亡与和厚朴酚呈浓度和时间依赖相关&#65377;HNK明显降低U937细胞线粒体膜电位,增加Caspase3/7活性及mRNA表达水平,但对正常外周血单核细胞的增殖抑制和凋亡作用不明显&#65377;【结论】 HNK可能通过激活caspase-3/7抑制U937细胞增殖&#65380;诱导U937细胞凋亡&#65377;     

4-1BBL分子在人恶性血液病细胞系的表达

目的:研究共刺激分子4-1BBL分子在人恶性血液病细胞系的表达及其生物学意义。方法:流式细胞仪检测4-1BBL分子在不同的人恶性血液病细胞系的表达;用可介导4-1BBL逆向信号的鼠抗人4-1BBL单抗(mAb1F1)作用于8个不同的人恶性血液病细胞系,细胞计数法和3H-TdR掺入法评价细胞系的体外生长和增殖。结果:FACS结果显示4-1BBL分子在人恶性血液病细胞系上有不同程度的表达,其中髓性白血病细胞系U937、HL60及B淋巴瘤细胞系Raji、Daudi上呈现较高水平的表达。细胞计数和3H-TdR掺入实验结果表明mAb1F1对髓性白血病细胞系U937和HL60,具有较强的促增殖效应。结论:共刺激分子4-1BBL分子在髓性白血病细胞系和B淋巴瘤细胞系上表达率较高;4-1BBL分子可通过介导逆向信号促进髓性白血病细胞系U937和HL60的生长和增殖。     

丙戊酸钠诱导髓系白血病细胞分化、凋亡作用及其机制研究

目的研究组蛋白去乙酰化酶抑制剂丙戊酸钠(VPA)诱导髓系白血病细胞体外分化和凋亡作用及其机制。方法采用四甲基偶氮唑盐(MTT)微量酶反应比色法增殖抑制实验和锥虫蓝拒染法检测VPA对细胞增殖的抑制,流式细胞仪检测细胞表面分化抗原的表达和细胞凋亡,Western印迹法检测组蛋白H3的9位赖氨酸残基(H3-Ac-lys9)的乙酰化水平变化。结果VPA抑制U937和K562髓系白血病细胞增殖,并呈时间-剂量依赖效应。在VPA较低浓度下(0.5～1mmol/L)作用6d后,VPA诱导U937细胞分化,其细胞表面髓系分化抗原CD11b表达明显上调,细胞形态呈终末分化改变。VPA与全反式维甲酸和(或)粒细胞集落刺激因子联合应用,诱导分化作用更为显著。在VPA较高浓度下(2mmol/L),VPA诱导U937细胞磷脂结合蛋白结合力明显上升,细胞呈现凋亡状态。而同样条件下VPA不能诱导K562细胞出现显著的分化和凋亡。U937和K562细胞在H3-Ac-lys9的乙酰化水平存在差异。VPA能够上调U937细胞H3-Ac-Lys9乙酰化,而K562细胞未观察到类似调变作用。结论VPA诱导U937细胞分化和凋亡,其作用与U937细胞的H3-Ac-lys9乙酰化水平及其对VPA处理后的上调有关。     

抑制 PKD2介导8-硝基白杨素诱导 U937细胞凋亡

目的：研究8-硝基白杨素（ NOC）诱导单核细胞白血病U937细胞凋亡作用是否涉及调控蛋白激酶D2（ PKD2）活性。方法体外培养U937细胞系细胞。碘化丙啶（PI）染色流式细胞术（FCM）分析细胞凋亡率。 DNA琼脂糖凝胶电泳检定细胞DNA梯形条带图谱。 Western blot分析PDK2磷酸化蛋白表达。结果 NOC（2.5、5.0、10.0μmol/L）增高U937细胞凋亡率,呈浓度依赖性（ P ＜0.05）。 NOC（5.0和10.0μmol/L）处理24 h,U937细胞呈现典型DNA梯形条带图谱。同时,NOC有效下调磷酸化PDK2蛋白表达;并与PKD2特异性抑制剂Goe6976协同增高U937细胞凋亡率。结论 NOC诱导U937细胞凋亡作用与其抑制PKD2激酶活性相关。     

青蒿琥酯对不同急性白血病细胞株凋亡抑制的实验研究

[目的]观察青蒿琥酯对不同急性白血病细胞株的增殖抑制作用及凋亡机制探讨。[方法]应用不同浓度青蒿琥酯作用U937、HL60、Jurkat细胞株,MTT法检测抑制率;细胞形态学、DNA琼脂糖凝胶电泳、流式细胞术检测细胞凋亡;Western-blot法检测细胞凋亡过程中Bcl-2、Bax、Caspase 8和ICAD蛋白表达的变化。[结果]青蒿琥酯能够明显抑制U937、HL60、Jurkat细胞增殖;8μg/ml以上浓度青蒿琥酯作用三种细胞24h、48h、72h抑制率均为Jurkat〉HL60〉U937(均P<0.01);其增殖抑制作用与诱导细胞凋亡有关;随药物浓度的增高Bcl-2和ICAD蛋白表达量下降,而Bax蛋白表达上调,Caspase8蛋白出现明显的剪切激活带。[结论]青蒿琥酯既能通过线粒体途径又能通过外源性途径诱导急性白血病细胞凋亡,对Jurkat的抑制最强。     

酸对人正常食管粘膜上皮细胞增殖和ERK表达水平的影响

目的 观察酸对人正常食管粘膜上皮细胞的增殖和细胞外信号调节蛋白激酶（ERK）表达水平的影响。方法 体外培养人正常食管粘膜上皮细胞，分别在pH4．0～6．5的条件下培养3～60min并设对照（pH7．3）进行比较。采用MTT法和流式细胞技术测定细胞增殖状态。免疫印迹法测定磷酸化ERK1／2蛋白表达。结果 酸的短时间（3min）作用使细胞增殖比大于1，S期细胞比率升高，磷酸化ERK1／2蛋白表达增加。当酸作用时间大于6min时，细胞增殖比小于1，S期细胞比率下降。ERK抑制剂U0126可阻断酸诱导的细胞增殖。结论 酸的短时间刺激可促进人正常食管细胞的增殖，与ERK表达上调有关。     

苦参碱对急性白血病早幼粒细胞 HL-60的抑制细胞增殖与诱导凋亡作用

目的：探讨苦参碱对急性白血病早幼粒细胞 HL-60的抑制细胞增殖与诱导凋亡作用。方法：以早幼粒急性白血病细胞系（HL-60细胞）为实验对象,采用体外培养技术,试验检测不同浓度苦参碱对HL-60细胞活力、细胞周期以及细胞凋亡的作用。结果：随着苦参碱浓度（12.5~50μg/ ml）的增加,苦参碱对 HL-60细胞增殖的作用不断增强;苦参碱处理72 h 后的 HL-60细胞与空白对照组比较,G0/ G1期细胞明显增多,S 期细胞明显减少,但比较差异无统计学意义（P〉0.05）;细胞凋亡率均明显高于空白对照组（P〈0.01）。结论：苦参碱对于急性白血病早幼粒细胞 HL-60的增殖具有明显的抑制作用,并能促进其凋亡。     

FTY720对体外混合培养淋巴细胞增殖和凋亡的影响

目的观察FTY720对体外混合培养小鼠脾脏淋巴细胞的增殖、分化和凋亡的影响。方法将C57BL/6J小鼠与BALB/c小鼠的脾脏淋巴细胞体外混合培养5 d,培养液中加入不同浓度的FTY720。分别运用MTT法和流式细胞仪检测在不同浓度FTY720作用下,混合培养淋巴细胞的增殖状况、细胞表型和凋亡率。结果当FTY720为400 ng/mL时,体外混合培养淋巴细胞的增殖即受到明显抑制,抑制率为47.6%(P<0.05);当FTY720为3 200 ng/mL时,其抑制率达到为51.6%,呈剂量依赖性。FTY720为1 600 ng/mL时,混合培养淋巴细胞中的CD4 CD25 T细胞比例显著增加(P<0.05)。FTY720在200 ng/mL时,混合培养淋巴细胞的细胞凋亡率即显著增加(P<0.05)。结论在小鼠混合淋巴细胞培养体系中加入高浓度的FTY720,能显著抑制淋巴细胞增殖能力,增加调节性细胞的比例并促进细胞凋亡。     

Antitumor Effect of Interferon-α on U937 Human Acute Leukemia Cells in vitro and Its Molecular Mechanism

In order to investigate the antitumor effect and molecular mechanism of interferon-α(IFN-α) on human acute myeloid leukemia cell line U937 cells in vitro, the proliferation of U937 cells was determined by MTT assay, the apoptosis rate was analyzed by flow cytometry (FCM), and the mRNA expression of cell cycle regulatory protein cyclin E was detected by RT-PCR. The results showed that IFN-α could inhibit the proliferation of U937 cells significantly in a dose- and time-dependent way (P<0.01), and induce the apoptosis of U937 cells also in a dose- and time-dependent manner at the concentration of 1000-4000 U/L (P<0.01). The apoptosis rate of U937 cells was even over 50% when cultured with IFN-α for 36-48 h at the concentration of 2000-4000 U/L. Moreover, the expression of cyclin E mRNA was markedly inhibited by the addition of IFN-α, and the inhibition was time-dependent (P<0.01). It was concluded that the anti-leukemia mechanism of IFN-α might be correlated with its antiproliferative and apoptotic inducing effects, and the down-regnlation of the cyclin E expression might be one of its molecular mechanisms.     

Resveratrol Inhibits the Secretion of Vascular Endothelial Growth Factor and Subsequent Proliferation in Human Leukemia U937 Cells

This study examined the effect of resveratrol on the secretion of vascular endothelial growth factor(VEGF) and subsequent proliferation of human leukemia U937 cells,and explored the mechanisms involved.Human leukemia U937 cells were treated with resveratrol of different concentrations(12.5-200 μmol/L) for different time lengths(12-48 h).The proliferation of the U937 leukemic cells was determined by MTT assay.Apoptosis was observed by Annexin-Ⅴ-FIFC/PI double staining and flow cytometry(FCM).Cells cycle was analyzed by PI staining and FCM.The content of VEGF was determined by ELISA.Human umbibical vein endothelial cells were examined for vasoformation in vitro after exposures to resveratrol of various concetrations.The results showed that resveratrol inhibited the proliferation of U937 leukemia cells in a dose-and time-dependent manner.Resveratrol induced apoptosis and S-phase cell cycle arrest in human leukemic U937 cells.Resveratrol inhibited the secretion of VEGF in U937 cells.Resveratrol inhibited the vasoformation of human vein endothelial cells in a dose-dependent manner.It was concluded that resveratrol could down-regulate the secretion of VEGF,induce apoptosis and suppress the proliferation of U937 cells.     

急性髓系白血病细胞株APP表达及APP—siRNA对HL60细胞的沉默作用

目的：研究淀粉样前体蛋白（APP）基因在HL60、HL60／ADM、U937和kasumi-1细胞株中的表达及筛选针对APP基因的有效干扰片段。方法：采用荧光定量PCR和Western blot方法分别检测APP基因在4种细胞株中的mRNA和蛋白表达。用化学法合成4条针对APP的干扰片段和一条阴性对照,脂质体法转染HL60细胞,分别用定量PCR和Western blot检测APP表达。结果：在4种细胞株中,APP mRNA和蛋白表达由高到低依次为：kasumi-1、HL60、HL60／ADM和U937,kasumi-1和HL60均显著高于HL60／ADM、U937（P〈0．05）。同阴性对照相比较,4条干扰片段均能不同程度地抑制APP表达,其中siRNA-4效果最佳,APP mRNA和蛋白分别下降了约64％和65％。结论：APP基因在不同AML细胞株中表达差异显著。siRNA-4的干扰效果最佳。