羊栖菜多糖诱导HL-60细胞凋亡的研究

用MTT法观察羊栖莱多糖(SFPS)在体外抗人白血病HL-60细胞增殖作用;扫描电镜、透射电镜、DNA电泳和流式细胞仪检测HL-60细胞凋亡。结果表明SFPS对HL-60细胞具有显著生长抑制作用,并呈量效和时效关系,药物作用24,36,48,72h的IC50分别为390,362,402,421mg／L;药物浓度为300mg／L和500mg／L作用HL-60细胞后,琼脂糖凝胶电泳显示有凋亡细胞特有的DNA梯状条带,细胞微绒毛减少、染色质固缩、边集,凋亡小体形成;DNA直方图出现亚G1峰。在一定浓度范围内,SFPS诱导细胞凋亡的作用呈现浓度和时间依赖性,同时G2／M期细胞比例增多。因此,SFPS抗肿瘤作用与诱导细胞凋亡和G2／M期细胞阻滞有关。     

三氧化二砷通过线粒体途径诱导人白血病HL-60细胞凋亡

目的:研究三氧化二砷(Arsenic trioxide,ATO)对人白血病HL-60细胞凋亡的影响,并以线粒体通路为靶点探讨其可能的机制。方法:采用1μg/m L、5μg/m L及10μg/m LATO处理HL-60细胞24小时后,采用流式细胞术检测细胞凋亡情况,通过细胞内MDA与GSH含量检测反映氧化应激水平,采用免疫印迹法检测凋亡相关分子表达,并通过免疫荧光染色检测细胞线粒体膜电位(mitochondrial membrane potential,MMP)水平。结果:5μg/m L及10μg/m L ATO可显著诱导人白血病HL-60细胞凋亡,并显著增加其氧化应激水平,增加促凋亡分子Bax和Caspase-3的表达,而抑制抗凋亡分子Bcl-2的表达,降低HL-60细胞线粒体膜电位的水平。结论:一定剂量的ATO可诱导人白血病HL-60细胞凋亡,而这一作用可能是通过诱导线粒体相关性凋亡信号通路激活实现。     

羊栖菜多糖诱导HL-60细胞凋亡的研究

用MTT法观察羊栖菜多糖(SFPS)在体外抗人白血病HL-60细胞增殖作用;扫描电镜、透射电镜、DNA电泳和流式细胞仪检测HL-60细胞凋亡。结果表明SFPS对HL-60细胞具有显著生长抑制作用,并呈量效和时效关系,药物作用24,36,48,72h的IC_(50)分别为390,362,402,421mg/L;药物浓度为300mg/L和500mg/L作用HL-60细胞后,琼脂糖凝胶电泳显示有凋亡细胞特有的DNA梯状条带,细胞微绒毛减少、染色质固缩、过集,凋亡小体形成;DNA直方图出现亚G_1峰。在一定浓度范围内,SFPS诱导细胞凋亡的作用呈现浓度和时间依赖性,同时G_2/M期细胞比例增多。因此,SFPS抗肿瘤作用与诱导细胞凋亡和G_2/M期细胞阻滞有关。     

2'-羟基二氢黄酮诱导白血病HL-60细胞凋亡过程中抗氧化酶活性的变化

目的:观察2'-羟基二氢黄酮诱导白血病细胞HL-60凋亡过程中胞内抗氧化酶活性的变化,并探讨其抗肿瘤作用机制.方法:采用CASY-TT亚流式细胞术测定2′-羟基二氢黄酮对HL-60细胞存活率的影响;Annexin V/PI双染流式细胞仪检测分析细胞凋亡变化;化学比色法测定2′-羟基二氢黄酮作用后,HL-60细胞胞内抗氧化酶SOD、CAT、GSH-Px的活性变化.结果:2′-羟基二氢黄酮显著降低HL-60细胞存活率,其作用呈剂量和时间依赖性;凋亡分析结果显示,20 μM的2′-羟基二氢黄酮作用HL-60细胞后,细胞凋亡率逐渐升高,并在12 h后显著高于作用前水平;酶活性检测表明,20 μM 2′-羟基二氢黄酮作用后,HL-60细胞胞内抗氧化酶SOD、CAT、GSH-Px活性均显著降低.脂质过氧化产物MDA含量显著升高.结论:2′-羟基二氢黄酮显著降低白血病HL-60细胞存活率并诱导细胞凋亡,其中伴随着胞内抗氧化酶活性的显著降低.     

2’-羟基二氢黄酮诱导白血病HL-60细胞凋亡过程中抗氧化酶活性的变化

目的：观察2’-羟基二氢黄酮诱导白血病细胞HL-60凋亡过程中胞内抗氧化酶活性的变化,并探讨其抗肿瘤作用机制。方法：采用CASY-TT亚流式细胞术测定2’-羟基二氢黄酮对HL-60细胞存活率的影响;Annexin V／PI双染流式细胞仪检测分析细胞凋亡变化;化学比色法测定2’-羟基二氢黄酮作用后,HL-60细胞胞内抗氧化酶SOD、CAT、GSH—Px的活性变化。结果：2’-羟基二氢黄酮显著降低HL-60细胞存活率,其作用呈剂量和时间依赖性;凋亡分析结果显示,20μM的2’一羟基二氢黄酮作用HL-60细胞后,细胞凋亡率逐渐升高,并在12h后显著高于作用前水平;酶活性检测表明,20μM2’一羟基二氢黄酮作用后,HL-60细胞胞内抗氧化酶SOD、CAT、GSH．Px活性均显著降低,脂质过氧化产物MDA含量显著升高。结论：2’-羟基二氢黄酮显著降低白血病HL广60细胞存活率并诱导细胞凋亡,其中伴随着胞内抗氧化酶活性的显著降低。     

三氧化二砷通过激活Drp-1增加人白血病HL-60细胞的放疗敏感性

目的:研究三氧化二砷(Arsenic trioxide,ATO)对人白血病HL-60细胞放疗敏感性的影响,并以动力相关蛋白-1(dynamin-related protein 1,Drp-1)为靶点探讨其可能的机制。方法:1μg/m L浓度的ATO处理HL-60细胞后使用20Gy强度进行放疗,采用MTT法检测细胞活力,流式细胞术检测细胞凋亡,通过线粒体钙离子缓冲能力检测线粒体钙离子代谢障碍,采用免疫印迹法检测Drp-1蛋白表达,并通过Drp-1阻滞剂mdivi-1预处理的方法检测Drp-1在实验中的作用。结果:1μg/m L浓度的ATO对HL-60细胞活力无影响,但可显著增加20Gy放疗所致的细胞活力下降、细胞凋亡和线粒体钙离子代谢障碍;1μg/m L浓度的ATO可显著增加Drp-1蛋白表达,使用mdivi-1预处理可部分逆转ATO的放疗增敏作用。结论:一定剂量的ATO可增加人白血病HL-60细胞的放疗敏感性,而这一作用可能是通过激活Drp-1蛋白表达而实现。     

HL-60细胞诱导分化与细胞膜流动性变化研究

 本实验以二甲基亚砜(Dimethyl Sulfoxide,DMSO)为诱导剂,诱导人早幼粒白血病细胞系HL-60沿粒系统成熟分化。动态观察了诱导后1—6天HL-60的形态、功能成熟度改变、膜流动性和增殖活性的变化。结果表明,早幼粒细胞由诱导前的78％降至5％,杆核与分叶核细胞由6％和1.5％分别增加至32.5％和5.5％。NBT(Nitroblue tetrazolium,四唑氮蓝)还原试验显示其功能亦渐超成熟。诱导后HL-60 DNA合成速率明显降低(降低40～75％),膜脂流动度亦显著降低(降低30％,P<0.01)。提示诱导分化后的HL-60细胞不仅获得了与正常成熟粒细胞相似的形态功能特征,同时还失去了膜流动性增高及快速繁殖增生的恶性特征。     

黄色黄素抑制人白血病HL-60细胞内蛋白激酶CK2的实验研究

蛋白激酶CK2在寻找抗肿瘤及抗病毒药物方面是一个具有吸引力的分子靶点,其特异性抑制剂具有潜在的临床应用价值.通过测定药物作用后转移到CK2底物上[γ-32P]ATP的放射性活度,探讨黄色黄素对重组人CK2全酶以及细胞内CK2活性的影响;采用多重RT-PCR检测CK2α、α'和β亚基的mRNA表达水平;Lineweaver-Burk作图法分析CK2的酶动力学机制.发现黄色黄素能显著抑制重组人CK2全酶(IC50=0.86μmol/L)以及HL-60细胞内的CK2活性,其作用效果均强于阳性对照TBB.另外,黄色黄素作用2h可使CK2α和α'亚基的mRNA表达下降,对β亚基则无明显的影响.酶动力学分析表明,黄色黄素与ATP呈竞争性抑制CK2的活性,与酪蛋白则呈混合性抑制CK2的活性.研究说明,黄色黄素是一种有效的细胞内蛋白激酶CK2抑制剂.     

新生牛肝中三种低分子化合物牛磺酸、鸟氨酸、肌肽对HL-60细胞凋亡的诱导作用

目的:研究三种新生牛肝源低分子化合物:牛磺酸、鸟氨酸、肌肽对HL-60白血病细胞增殖的抑制作用并探讨其调控机理.方法:用MTT法分别检测三种活性成分作用后HL-60细胞和正常人淋巴细胞的存活率.分别用琼脂糖凝胶电泳,顺磁共振ESR技术,免疫组化法测定其对HL-60细胞的核DNA、氧自由基活性和细胞周期蛋白水平的影响.结果:三种化合物能够有效地抑制HL-60细胞的增殖,而对正常的人淋巴细胞的生长没有抑制作用;三种化合物使HL-60细胞的核DNA产生30 kb片段,使HL-60细胞内的氧自由基活性降至痕量水平,并下调HL-60的p45/skp2的水平,而上调p27/kip的水平.结论:牛磺酸、鸟氨酸、肌肽能够通过调控细胞周期蛋白的水平而选择性的抑制肿瘤细胞的增殖.     

柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡及其机理研究

柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡从而抑制其细胞生长.为了研究该过程的作用机理,我们研究了丝裂原活化蛋白激酶(MAPKs),包括胞外信号调节激酶(ERK1/2),c-jun氨基末端蛋白激酶(JNK)和p38丝裂原活化蛋白激酶(MAPK),在该过程中的磷酸化特征与动态变化.结果表明,柴胡提取物显著的增加了p38丝裂原活化蛋白激酶和胞外信号调节激酶(ERK1/2)的磷酸化作用,其增加值在测试范围内与测试剂量和作用时间成正相关,但在柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡过程中,没有发现对氨基末端蛋白激酶(JNK)表现出磷酸化活性.柴胡提取物诱导白血病HL-60的细胞凋亡部分归结于对p38丝裂原活化蛋白激酶的上调节作用,这种上调节作用能够受到p38 MAPK特异性的抑制剂SB203580的部分逆转,而MEK的抑制剂U0126则对柴胡提取物诱导HL-60细胞凋亡过程中的胞外信号调节激酶(ERK1/2)的磷酸化具有显著的协同效应.这是首次报道柴胡提取物在诱导人白血病细胞HL-60细胞凋亡过程中参与p38丝裂原活化蛋白激酶的磷酸化,同时柴胡提取物作为胞外信号调节激酶(ERK1/2)抑制剂的协同作用物具有相应的药物学功能.     

Molecular Mechanisms of Apoptosis in HL-60 Cells Induced by a Nitric Oxide-Releasing Compound

Nitric oxide (NO) generated from 1-hydroxy-2-0×0-3, 3-bis(2-aminoethyl)-l-triazene (NOC 18), an NO-releasing compound, induced monocytic differentiation of human promyelocytic leukemia HL-60 cells as assessed by expression of nonspecific esterases and morphologic maturation. Simultaneously, DNA fragmentation and morphological alterations typical of apoptosis were also induced. To investigate the mechanisms of apoptosis during differentiation of HL-60 cells induced by NO, the endogenous levels of Bcl-2 and Bax were assessed by immunoblotting. Treatment of cells with NOC 18 slightly reduced the level of Bcl-2 followed by Bax. These changes might be involved in the induction of apoptosis. The involvement of the activation of the interleukin-lβ converting enzyme (ICE) family of proteases (caspases), such as ICE and CPP32, in the pathways was also investigated. CPP32, but not ICE, was strongly activated in response to NOC 18 stimulation, thereby implicating CPP32-like activity in the induction of apoptosis. Moreover, the possible involvement of tyrosine phosphorylation in apoptosis was investigated. Pretreatment of cells with herbimycin A, an inhibitor of tyrosine kinases, suppressed DNA fragmentation and CPP32-like activity, whereas pretreatment with vanadate, an inhibitor of tyrosine phosphatases, enhanced both parameters, suggesting that tyrosine phosphorylation might be involved in the pathways of apoptosis in HL-60 cells induced by NO.     

Effect of Dimethyl Sulfides on the Induction of Apoptosis in Human Leukemia Jurkat Cells and HL-60 Cells

Organosulfur compounds have been established to possess anticancer effects. To provide a better understanding of the biological function of dimethyl sulfides, dimethyl monosulfide (Me₂S), dimethyl disulfide (Me₂S₂), dimethyl trisulfide (Me₂S₃) and dimethyl tetrasulfide (Me₂S₄) were used as experimental materials to investigate their effects on apoptosis induction in human leukemia Jurkat cells and HL-60 cells. Treatment with 20 μM dimethyl sulfides for 24 h decreased the viability of both cells. The cell viability-reducing effect of these sulfides was in the following order: Me₂S₄ ≈ Me₂S₃ > Me₂S₂ ≈ Me₂S for Jurkat cells and Me₂S₄ > Me₂S₃ > Me₂S₂ ≈ Me₂S for HL-60 cells. Me₂S₃ and Me₂S₄ significantly induced DNA fragmentation and caspase-3 activation. The addition of GSH or NAC completely suppressed the sulfide-induced apoptosis. Our results indicate that dimethyl sulfides with a larger number of sulfur atoms more strongly induced apoptosis in both human leukemia cells via ROS production and caspase-3 activation.     

Inhibition of Phosphatidylinositol 3-Kinase Causes Apoptosis in Retinoic Acid-Differentiated HL-60 Leukemia Cells

Phosphatidylinositol 3-kinase (PI3-K) signaling may inhibit apoptosis in neoplastic cells. The PI-3K inhibitor wortmannin renders cells apoptosis-prone. Inducers of differentiation may also cause apoptosis. To detect the effect of wortmannin on the survival of differentiated human acute promyeloid leukemia cells, HL-60 cells were induced to differentiation with treatment of all trans-retinoic acid (ATRA) followed by treatment with wortmannin. Results showed that apoptosis occurred in cells that underwent differentiation, but not in undifferentiated HL-60 cells. The pro-apoptotic molecule, Bad, played a role in this apoptotic mechanism. Thus, the survival of differentiated HL-60 cells induced by ATRA depends on the ability of the PI3-K pathway to transduce survival signals; the PI3-K inhibitor, wortmannin, can induce apoptosis of differentiated HL-60 cells. These results may indicate a novel method for treating cancer with differentiation induction and signal pathway regulation.     

外泌体分泌在1,4-苯醌诱导的HL-60细胞凋亡中起保护作用

慢性苯暴露损害造血系统,可引起再生障碍性贫血,甚至白血病。外泌体(exosomes)是细胞分泌的纳米级膜泡,在许多生理和病理过程中发挥重要作用。然而,苯及其代谢产物对外泌体分泌的影响仍不清楚。本研究旨在观察苯的活性代谢产物1,4-苯醌(1,4-benzoquinone,1,4-BQ)能否引起人早幼粒白血病细胞HL-60外泌体分泌量的变化以及外泌体释放在1,4-BQ诱导的细胞凋亡中的作用。应用不同浓度1,4-BQ处理细胞24 h,超高速离心法提取细胞培养基中的外泌体,结果发现1,4-BQ能促进外泌体分泌,呈剂量反应关系。进一步应用外泌体抑制剂GW4869抑制外泌体分泌,流式细胞仪检测细胞凋亡率、蛋白质免疫印迹法检测抑凋亡蛋白质Bcl-2、凋亡通路关键蛋白质cleaved caspase-9和cleaved caspase-3的表达,探讨外泌体分泌对1,4-BQ所致细胞凋亡的影响。结果显示:与对照组相比,1,4-BQ单独处理组的凋亡率、Bcl-2、cleaved caspase-9和cleaved caspase-3的表达均显著增高(P<0.05),而1,4-BQ+GW4869组的凋亡率及凋亡相关蛋白质的表达均显著高于1,4-BQ单独处理组(P<0.05),表明抑制外泌体分泌可增加1,4-BQ诱导的细胞凋亡。综上表明,1,4-BQ能促进外泌体分泌,并在1,4-BQ诱导的细胞凋亡中起保护作用。本研究为了解苯的毒性效应和毒性机制提供了新的实验证据。     

GPI-PLD调节CEA的释放对白血病HL-60细胞的增殖和凋亡的影响

糖基化磷脂酰肌醇特异性磷脂酶D(glycosyl phosphatidyl inositol specific phospholipase D,GPI-PLD)是人体内唯一可水解细胞膜表面GPI结构、调节GPI锚定蛋白释放的酶.将GPI-PLD转染入急性粒细胞白血病(AGL)的HL-60细胞株,采用实时荧光定量PCR法和Western blot法确定转染后HL-60细胞内GPI-PLD的表达水平;并检测GPI-PLD活性;噻唑蓝(MTT)检测HL-60细胞的增殖;流式细胞仪检测HL-60细胞的凋亡.ELISA检测GPI锚定癌胚抗原(CEA)的表达和释放情况.转染GPI-PLD后,HL-60细胞株中GPI-PLD表达量与活性增加;MTT检测显示,GPI-PLD过表达后HL-60细胞株增殖生长受到抑制;流式检测证实HL-60细胞凋亡增加;且GPI锚定的蛋白质CEA释放增加.该结果提示GPI-PLD基因有抗肿瘤的作用,过表达GPI-PLD后能抑制HL-60细胞增殖且促进其凋亡,所涉机制可能与GPI-PLD释放GPI锚定蛋白,增强白血病细胞对补体杀伤的敏感性有关.     

青蒿素诱导人白血病细胞K562凋亡的线粒体机制

目的:探讨青蒿素诱导人白血病细胞K562凋亡的线粒体机制.方法:用青蒿素处理K562细胞.通过MTT比色法检别细胞增殖抑制的效果;荧光显微镜观察细胞的凋亡;流式细胞术(flow cytometry,FCM)进行细胞周期分析;Western-blotting测定药物作用前后线粒体、细胞浆细胞色素C的表达.结果:青蒿素抑制K562细胞的增殖,IC5D为1.5× 10-5mol·L-1;Hoechst33342/PI双荧光染色可观察到明显的核浓缩、凝集等细胞凋亡表现;流式细胞仪检测G2期细胞比例增高,S期减少;Western-blotting检测药物处理细胞后线粒体细胞色素C表达水平下调,细胞浆出现明显细胞色素C蛋白条带.结论:青蒿素可能通过线粒体细胞色素C途径诱导K562细胞凋亡.     

A Lichen Substance as an Antiproliferative Compound against HL-60 Human Leukemia Cells: 16-O-Acetyl-leucotylic Acid Isolated from Myelochroa aurulenta

The lichen substance, 16-O-acetyl-leucotylic acid (1), was isolated from an acetone extract of Myelochroa aurulenta and found to exhibit antiproliferative activity against HL-60 human leukemia cells. This is the first report on its anti-leukemia activity (EC₅₀=21 μM) which is greater than that of leucotylic acid (2) and the structurally related anti-tumor agent, betulinic acid (4).     

The Synthetic Tryptanthrin Analogue Suppresses STAT3 Signaling and Induces Caspase Dependent Apoptosis via ERK Up Regulation in Human Leukemia HL-60 Cells

Tryptanthrin is a natural product which has been reported to have several medicinal properties. In this study, we tried to investigate the detailed molecular mechanism of its bromo analogue (TBr), a potent cytotoxic agent in the induction of cancer cell death. It was found that TBr primarily targets STAT3 and ERK signaling during the induction of apoptosis in several human leukemia cell lines. In HL-60 cells, TBr treatment caused early down regulation of p-STAT3 with concomitant up regulation of p-ERK which led to the activation of intrinsic and extrinsic pathways of apoptosis. The mechanism of TBr mediated inhibition of p-STAT3 was found to be due to the activation of ubiquitin dependent degradation of tyrosine 705 and serine 727 p-STAT3. As IL-6 is the main driver of the STAT3 pathway, the effect of TBr on cell death was subdued when treated in the combination with IL-6 in HL60 cells. Interestingly, PD98059 significantly reduced the apoptotic effects of TBr, thus showing the direct involvement of p-ERK in TBr mediated cell death. It was further shown that apoptotic protein Bax silencing in HL-60 cells resists TBr mediated ERK dependent apoptosis. In summary, for the first time we report the mechanism of TBr mediated cell death in human leukemia cell lines by targeting STAT3 and ERK pathways.