维胺酯和维胺酸对人早幼粒白血病细胞(HL-60)的分化诱导作用

本文研究了新维甲酸衙生物——维胺酯(RⅠ)和维胺酸(RⅡ)对人早幼粒白血病细胞(HL-60)的分化诱导作用,并与维甲酸(RA)和二甲基亚砜(DMSO)进行了比较。HL-60细胞经RⅠ和RⅡ处理后,细胞对NBT染料还原能力增强,细胞表面出现C₃补体受体等中性粒细胞的分化特征,但作用比RA弱。DMSO诱导上述分化性状出现时间不同,提示维甲酸类药物与DMSO诱导分化的机制可能不同。上述药物均可使HL-60细胞形态按粒系途径向较成熟的方向发展,但变化出现较晚。     

放线菌素23—21对人低分化鼻咽癌细胞系裸小鼠移植瘤的抑制作用

采用人低分化鼻咽癌细胞系裸小鼠移植瘤模型,研究了放线菌素23-21的抗瘤作用。结果表明放线菌素23-21的50μg/kg(LD_(50) 1/10)抑瘤率为58％(p<0.05)。光电镜检查,给药组瘤细胞严重变性坏死,且引起核仁分离和微球体形成的超微结构变化。说明放线菌素23-21能抑制人低分化鼻咽癌细胞系裸小鼠移植瘤的生长,提示放线菌素23-21可能有抑制核仁rRNA合成的作用。     

全反式维甲酸联合丙戊酸钠诱导白血病细胞分化过程中BRD4表达变化及其作用的研究

目的探讨全反式维甲酸(ATRA)联合丙戊酸钠(VPA)诱导HL-60细胞分化过程中BRD4表达调控的分子机制。方法建立ATRA联合VPA诱导白血病细胞株HL-60的分化模型,瑞氏-吉姆萨(Wright-Gimesa)染色观察HL-60细胞形态,流式细胞术检测细胞表面分化抗原的表达,蛋白质印记从蛋白水平检测HL-60细胞经药物诱导24、48、72h后BRD4表达的变化。结果单独应用ATRA或VPA均能够明显抑制白血病细胞生长,诱导细胞分化,倒置显微镜下可观察到HL-60细胞向成熟粒细胞方向分化,细胞表面的分化抗原表达升高,联合诱导分化模型中细胞表面分化抗原表达升高更明显,两者均有统计学意义,在蛋白水平BRD4的表达逐渐降低,联合诱导分化模型中降低更明显,两者有统计学意义。结论 VPA可以明显的促进ATRA诱导白血病细胞的分化作用,BRD4基因的表达在随着分化程度的加强出现逐渐降低的趋势。     

小剂量化疗药物与维甲酸对原代培养人白血病细胞诱导分化的研究(摘要)

根据细胞动力学的研究,急性白血病的发生是由于细胞增殖/分化的调控失常,以分化阻滞而导致大量原幼细胞积聚是急性白血病发病的机理。近十余年来开展了大量白血病细胞建株(HL-60,ML-3U-937)等的体外诱导分化试验成功,相继发现小剂量化疗药物如阿糖胞苷(Ara-C)、阿霉素、柔红霉素、放线菌素D等亦     

猪胆酸钠对人早幼粒白血病细胞系(HL-60)作用机理的初步探讨

观察了猪胆酸钠和维甲酸对人早幼粒白血病细胞系(HL-60)体外诱导分化过程中细胞膜脂肪酸的组成、细胞蛋白激酶C(PKC)活性及c-myc癌基因表达的变化,初步探讨猪胆酸钠对HL-60细胞作用的分子机理,结果表明,经100～400μg·ml~(-1)猪胆酸钠处理3d的HL-60细胞,其PKC活性和c-myc表达与对照组比较均明显下调;1μmol·L~(-1)维甲酸诱导3 d的细胞,PKC活性未见明显变化,而癌基因c-myc表达则降至对照组的17％;无论是猪胆酸钠(400 μg·L~(-1))还是维甲酸(1μmol·L~(-1))诱导3d的HL-60细胞,其细胞膜脂肪酸组成均无明显改变。     

四嗪二甲酰胺对白血病细胞株HL-60体外作用的研究

目的:观察四嗪二甲酰胺(ZGDHu-1)体外抑制白血病细胞株HL-60增殖,诱导细胞分化和凋亡作用,并对其作用机制进行初步探讨。方法:将不同浓度的ZGDHu-1与HL-60细胞在体外培养,台盼蓝染色、MTT比色法观察对HL-60细胞增殖的抑制作用;用细胞形态学、DNA凝胶电泳、DNA含量及细胞周期分析、Annexin-V/PI双标记和DNA片段原位末端标记法等分析细胞凋亡。通过NBT试验、细胞表面抗原CD11b、CD13、CD14、CD64和细胞形态学检测ZGDHu-1对HL-60细胞的分化作用。用Rh123/PI测定线粒体跨膜电位(ΔΨm),用流式细胞术检测Bcl-2、Bax、P53、Fas和线粒体膜蛋白的表达变化。结果:ZGDHu-1呈现作用时间和剂量的量效性抑制HL-60细胞增殖和活力,10 ng.ml-1ZGDHu-1作用HL-60细胞24~72 h后,MTT法检测细胞增殖抑制率分别为(7.3±0.3)%、(15.8±1.0)%和(21.3±1.2)%,均显著高于对照组(P<0.01);48 h、72 h的IC50分别为180、180 ng.ml-1。HL-60细胞经ZGDHu-1作用后,大部分细胞阻滞于G2-M期,G0/1期细胞减少;出现典型的细胞形态改变,DNA片端化,经50~1 000 ng.ml-1的ZGDHu-1作用后,SubG1由(3.2±1.6)%上升至(67.7±5.9)%,与对照组(0.7±0.5)%比较,具有显著性差异(P<0.05),Annexin-V/PI标记升高,TUNEL染色后的凋亡细胞的特征性改变等均证实ZGDHu-1能诱导HL-60细胞凋亡。HL-60细胞经10~100 ng.ml-1ZGDHu-1作用3 d后,细胞发生部分分化,NBT阳性率增加,细胞表面CD11b、CD13、CD14、CD64表达增加。ZGDHu-1诱导HL-60细胞凋亡和分化过程中,Bcl-2表达无变化,但Bax表达显著增加,Bax/Bcl-2的比值升高,Fas表达增强,而P53表达无变化。随ZGDHu-1作用的浓度增加,ΔΨm下降、而线粒体膜蛋白表达显著升高。结论:ZGDHu-1能抑制HL-60细胞增殖和细胞活力,低浓度时诱导HL-60细胞向粒单系分化,高浓度时促进细胞凋亡,其机制可能与Bax表达上调,线粒体膜电位下降等有关。     

二甲基氨氯吡咪诱导HL-60细胞分化(英文)

目的:研究阿米洛利衍生物二甲基阿米洛利(DMA)对培养的HL-60细胞增殖和分化的影响。方法:MTT法测定细胞增殖,细胞染色和NBT还原试验测定细胞分化。结果:DMA以浓度依赖方式抑制HL-60细胞增殖,药物作用96小时测得半数抑制浓度(IC_(50))为31.7(95％可信限为6.3-57.1)μmol·L~(-1)。DMA诱导HL-60细胞向粒细胞系分化。加入DMA100μmol·L~(-1)作用3天,细胞分化率从6.5％升至70％。阿米洛利及阿米洛利的另二个衍生物对细胞分化无明显作用。结论:二甲基阿米洛利抑制HL-60细胞增殖和诱导HL-60细胞分化。     

氢醌抑制HL-60细胞分化上调过氧化物酶2-Cys过氧化物氧还蛋白表达

目的研究氢醌(HQ)对HL-60细胞向单核系、粒系分化的影响。方法 HQ 1,5和50μmo·lL-1分别与豆蔻酰佛波醇乙酯(PMA)20 nmol·L-1或1.25%DMSO共同处理细胞,分别于48和96 h收集细胞。通过观察细胞形态、硝基四氮唑蓝还原反应鉴定细胞分化;CCK-8检测细胞增殖,荧光定量PCR检测2-Cys过氧化物氧还蛋白(Prxs)基因表达的变化;Western印迹检测2-Cys Prxs蛋白表达的变化。结果在HQ1~50μmol·L-1作用下,HL-60细胞向单核系分化受到抑制;HQ1和5μmol·L-1对DMSO诱导的粒系分化无影响,但HQ50μmol·L-1可抑制细胞向粒系分化。HQ5和50μmol·L-1与诱导剂的共同作用可以抑制HL-60细胞增殖。与正常对照组比较,PMA和DMSO组2-Cys Prxs基因表达水平均有降低的趋势,HQ1,5和50μmol.L-1+PMA20 nmol·L-1组PrxⅠ,PrxⅢ和PrxⅣ各个基因表达水平与PMA组比较均有增高的趋势;DMSO诱导分化组中仅HQ 50μmol·L-1+1.25%DMSO组PrxⅠ和PrxⅢ基因表达水平与单独DMSO组比较显著增高(P<0.05)。结论 HQ1和5μmol.L-1可以抑制HL-60细胞向单核系分化,HQ50μmol·L-1可抑制细胞向粒系分化,并上调PrxⅠ和PrxⅢ基因的表达。     

二烯丙基二硫下调DJ-1抑制人白血病HL-60细胞增殖及诱导分化

目的研究二烯丙基二硫(DADS)通过下调DJ-1对人白血病细胞系HL-60增殖抑制及诱导分化的影响。方法通过细胞形态法观察细胞分化;Western blot及免疫细胞化学检测DADS对HL-60细胞DJ-1表达的影响;针对DJ-1mRNA序列设计合成siRNA转染人白血病细胞HL-60,Western blot检测基因干扰效果;利用MTT法及间接免疫荧光检测DADS及DJ-1沉默对HL-60细胞的生长及细胞分化的影响。结果 DADS可以诱导HL-60细胞向成熟粒细胞系样分化。DADS呈时间依赖性抑制DJ-1的表达(P<0.05),DJ-1干扰组细胞生长减慢(P<0.05),与DADS共同作用后可诱导HL-60细胞分化。检测细胞生长情况发现干扰组细胞生长减慢(P<0.05)。结论 DADS通过下调DJ-1抑制细胞增殖并诱导HL-60细胞分化;干扰DJ-1基因可以增强DADS对HL-60细胞的增殖抑制诱导分化作用。     

阿糖胞苷、二甲基亚砜对HL-60细胞周期动力学的影响

本文应用流式细胞仪技术研究了二甲基亚砜(DMSO)、阿糖胞苷(Ara-c)诱导HL-60分化成熟过程中,细胞周期动力学的变化。结果表明DMSO、Ara-c作用3天后,具有S、G_2期DNA含量的细胞占全部细胞的百分率,在DMSO组减小,而Ara-c组略有增加。     

Intermedeol isolated from the leaves of Ligularia fischeri var. spiciformis induces the differentiation of human acute promyeocytic leukemia HL-60 Cells

The present work was performed to investigate the effects of intermedeol on proliferation and differentiation of human leukemia-derived HL-60 cells as well as the underlying mechanisms for these effects. Intermedeol exhibited a potent antiproliferative activity against HL-60 cells. In addition, this compound was found to be a potent inducer for HL-60 cell differentiation as assessed by nitroblue tetrazolium reduction test, esterase activity assay, phagocytic activity assay, morphology change, and expression of CD14 and CD66b surface antigens. These results suggest that intermedeol induces differentiation of human leukemia cells to granulocytes and monocytes/macrophage lineage. Moreover, the expression level of c-myc was down-regulated during intermedeol-dependent HL-60 cell differentiation, whereas p21(CIP1) was up-regulated. Taken together, our results suggest that intermedeol may have potential as a therapeutic agent in human leukemia.     

Induction of differentiation in human myeloid leukemic cells by T-2 toxin and other trichothecenes

Trichothecenes are sesquiterpen mycotoxins characterized by the tetracyclic 12,13-epoxytrichothec-9-ene skeleton. We determined the effect of these mycotoxins on the growth and differentiation of the human acute promyelocytic leukemia (HL-60) cell line. Sixteen natural and semisynthetic trichothecenes were tested at concentrations of 0.2-60,000 ng/ml. The cytotoxicity exerted by these compounds varied: e.g., roridin A was found to be toxic at 1 ng/ml, whereas T-2 palmitate was not toxic even at 1 microgram/ml. These compounds varied also in their potential to induce differentiation: 9,10-epoxy T-2 toxin and T-2 toxin induced differentiation at concentrations of 2-5 ng/ml, while 9,10-dihydro T-2 toxin was effective only at 100 ng/ml. Other trichothecenes (e.g., verrucarin A and verrucarol) did not induce differentiation at either subtoxic or toxic concentrations. Cell differentiation was always associated with cytotoxicity; optimal concentrations for induction of differentiation were usually 30-60% of the toxic concentrations. The HL-60 cell population was found to be heterogenous with respect to the ability to differentiate in response to trichothecenes, while in some clones up to 70% of the cells underwent differentiation, and other clones were completely resistant. The latter clones could, however, be induced to differentiate by other agents such as retinoic acid, dimethyl sulfoxide and 12-O-tetradecanoylphorbol-13-acetate. Some of the inducible clones differentiated into neutrophilic granulocytes while others into mature macrophages. Thus, a single trichothecene could induce differentiation into either cell types, depending on the clone used. This study presents a new group of differentiation inducers. Further investigation is required to evaluate their possible therapeutic application.     

Gs alpha availability to cholera toxin-catalysed ADP-ribosylation is decreased in membranes of retinoic acid-treated leukemic cell lines HL-60 and THP-1. A posttranslational effect.

Retinoic acid (RA) induces HL-60 and THP-1 leukemic cell lines to differentiate into granulocyte-like and monocyte-like cells. Limited data are available concerning the effects of RA on components of the cyclic AMP pathway in human myeloid leukemic cells. We showed previously a decrease in adenylate cyclase activity in the presence of histamine, prostaglandin E1 and forskolin in RA-treated HL-60 cells as compared to untreated cells. We examined the elements of the signal transduction pathway utilized by RA in the human myeloid cell line HL-60 and the human monocytic cell line THP-1. We therefore studied the effect of RA on the activity of the stimulatory G-protein (Gs). We demonstrate that addition of RA to two human myeloid leukemia cell lines, HL-60 and THP-1, does not induce a reduction of the 2 subunit of Gs (Gs alpha) RNA or Gs alpha protein in the plasma membrane but leads to a rapid decrease in the cholera toxin (CTX)-catalysed ADP-ribosylation of Gs alpha. In addition, this effect seems to be specific to RA, since there was no modification in Gs alpha ADP-ribosylation in the membranes of cells treated with dimethyl sulfoxide (DMSO), another inducer of differentiation in HL-60 cells.     

Pyranocoumarins isolated from Peucedanum praeruptorum as differentiation inducers in human leukemic HL-60 cells

Differentiation therapy for myeloid leukemia offers great potential as a supplement to the current treatment modalities. In the present report, we investigated if the pyranocoumarins, (+/-)-4'- O-acetyl-3'- O-angeloyl- cis-khellactone (or angular pyranocoumarin, APC) isolated from the medicinal plant Peucedanum praeruptorum Dunn, could induce human acute myeloid leukemic HL-60 cells to differentiate and elucidated the molecular mechanism(s) involved. The ability of HL-60 cells to reduce nitroblue tetrazolium (NBT) was significantly increased after APC treatment for 72 h. In these differentiating HL-60 cells, cell surface differentiation markers CD11b (for myeloid cells) and CD14 (for monocytic cells) were detected in 90.3 % and 70.1 % of the cells, respectively. The differentiation inducing effect of APC was time- and dose-dependent. Treatment with 20 microg/mL APC for 72 h inhibited cell growth by 90 % and cell cycle analysis revealed an increase in the proportion of G1 phase cells. In these growth-inhibited cells the expression of the cyclin-dependent kinase inhibitor p27 kip1, but not p21 WAF1, was up-regulated as shown by Western blotting. Differentiation inducing signal pathways were investigated and it was shown that phospho-MEK and phospho-ERK were elevated shortly after the addition of APC. Pre-incubation of the cells with MEK1 inhibitor PD98059 blocked this APC-induced differentiation. Our results suggest that APC are potent inducers of HL-60 cell differentiation along both the myelocytic and monocytic lineages and are potential agents for differentiation-treatment of leukemia.     

Effect of neplanocin A on differentiation, nucleic acid methylation, and c-myc mRNA expression in human promyelocytic leukemia cells

The effect of the cyclopentenyl adenosine analog neplanocin A (NPC) on cell growth and differentiation was examined in the human promyelocytic leukemia cell line HL-60. Continuous exposure of HL-60 cells to 0.1-3.3 microM NPC resulted in a progressive reduction in cell growth which was accompanied by an increase in differentiation to cells with a myelocyte and neutrophil morphology. The latter effect was expressed as an increase in the capacity of cells to reduce nitro blue tetrazolium and reached a level of 40% of the total cell population. Preceding the phenotypic changes was the preferential inhibition of RNA and DNA methylation in comparison to inhibition of their synthesis which coincided with the formation of a metabolite of NPC with the chromatographic characteristics of S-adenosyl-L-methionine (AdoMet). In addition, c-myc mRNA expression, which is amplified in HL-60 cells, was markedly reduced following NPC treatment. These results indicate that NPC is an effective inhibitor of RNA and DNA methylation resulting from its conversion to an analog of AdoMet, and that these effects appear to be responsible for reduced c-myc RNA expression and the induction of myeloid differentiation in this cell line.     

DADS抑制JAK1/STAT3信号通路诱导人白血病HL-60细胞分化

目的探讨JAKs/STATs信号转导通路在二烯丙基二硫(DADS)诱导人白血病HL60细胞分化中的变化及其调控机制。方法将HL60细胞与DADS或JAKs/STATs信号通路的激酶抑制剂AG490在体外共同培养,观察细胞形态变化,检测药物作用前后细胞NBT还原能力及细胞表面分化抗原CD11b的改变;用Westernblot检测JAKs,STATs各家族成员在DADS诱导HL60细胞分化中的改变;并用免疫细胞化学法检测核转录基因STATs,cmyc,cfos,cjun的表达变化。结果DADS和AG490均可诱导HL60细胞向成熟粒系分化,且DADS在1.25mg·L-1时诱导分化作用达峰值;Westernblot检测JAK1,STAT3的酪氨酸激酶发生了磷酸化改变;免疫细胞化学示STAT3与cmyc基因蛋白核内表达下降,cjun,cfos基因蛋白核内表达上升。结论JAK1,STAT3酪氨酸激酶的磷酸化抑制参与了DADS诱导HL60细胞分化的调控,其机制可能通过调控与HL60细胞增殖分化相关的基因表达,抑制细胞DNA合成,从而抑制细胞增殖,诱导分化。DADS的作用相当于JAK1/STAT3信号通路的阻断剂。     

广东省凤尾蕨属植物抗白血病药物研究

目的探讨广东地区5种常见凤尾蕨属植物半边旗、剑叶凤尾蕨、刺齿凤尾蕨、蜈蚣草和井栏边草对白血病细胞生长的抑制作用。方法用乙醇经索氏提取器分别提取5种植物的有效成分,以二甲基亚砜分别配制供试品溶液;将供试品溶液加入对数生长期的人早幼粒白血病HL-60细胞株(简称HL-60细胞)和人慢性髓样白血病K562细胞株(简称K562细胞)中,采用台盼蓝染色法染色,统计细胞存活率。结果半边旗、剑叶凤尾蕨、刺齿凤尾蕨、蜈蚣草和井栏边草对HL-60细胞和K562细胞的生长均有抑制作用,其中半边旗和刺齿凤尾蕨对HL-60细胞生长的抑制作用较强,4.0 g/L半边旗和刺齿凤尾蕨乙醇提取物中,HL-60细胞的存活率均为0;半边旗和蜈蚣草对K562细胞生长的抑制作用较强,4.0 g/L半边旗和蜈蚣草乙醇提取物中,K562细胞的存活率分别为0和7.70%。结论 5种凤尾蕨属植物对HL-60细胞的生长抑制作用强于K562细胞;除半边旗外,刺齿凤尾蕨和蜈蚣草可能是潜在的抗白血病药物。     

Taraxinic acid,a hydrolysate of sesquiterpene lactone glycoside from the Taraxacum coreanum NAKAI,induces the differentiation of human acute promyelocytic leukemia HL-60 cells

The present work was performed to elucidate the active moiety of a sesquiterpene lactone, taraxinic acid-1'-O-beta-D-glucopyranoside (1). from Taraxacum coreanum NAKAI on the cytotoxicity of various cancer cells. Based on enzymatic hydrolysis and MTT assay, the active moiety should be attributed to the aglycone taraxinic acid (1a). rather than the glycoside (1). Taraxinic acid exhibited potent antiproliferative activity against human leukemia-derived HL-60. In addition, this compound was found to be a potent inducer of HL-60 cell differentiation as assessed by a nitroblue tetrazolium reduction test, esterase activity assay, phagocytic activity assay, morphology change, and expression of CD 14 and CD 66 b surface antigens. These results suggest that taraxinic acid induces the differentiation of human leukemia cells to monocyte/macrophage lineage. Moreover, the expression level of c-myc was down-regulated during taraxinic acid-dependent HL-60 cell differentiation, whereas p21(CIP1) and p27(KIP1) were up-regulated. Taken together, our results suggest that taraxinic acid may have potential as a therapeutic agent in human leukemia.     

Induction of monocytic differentiation by calcitriol (1,25-dihydroxyvitamin D3) in the human promyelocytic leukemic cell line (HL-60) in serum-free medium

The effect of calcitriol on the induction of differentiation in human promyelocytic leukemic cell line (HL-60) cultured in serum-free chemically defined medium (SFM) was investigated. The utilization of SFM containing RPMI-1640 basal medium supplemented with insulin (5 micrograms/ml), transferrin (5 micrograms/ml), sodium selenite (5 ng/ml), and bovine serum albumin (0.5 micrograms/ml), transferrin examination of the cellular/molecular mechanism of calcitriol's action in HL-60 cell differentiation without interference of components present in serum. HL-60 cells grown in SFM were induced to differentiate into monocytes/macrophages by calcitriol as indicated by induction of differentiation-associated biological and biochemical parameters: chemiluminescent (CL) responsiveness, lysozyme activity, nonspecific esterase, expression of cell surface antigens, and reduced proliferation. The exposure of HL-60 cells in SFM to calcitriol (from 10(-10) to 10(-8)M) resulted in dose-dependent induction of these parameters, which was similar to those obtained with cells grown in 10% fetal calf serum containing medium (10% SCM). However, calcitriol was 5-fold more potent for HL-60 cells cultured in SFM than those cultured in 10% SCM as indicated by shifts in dose-response curves for induction of CL responsiveness and lysozyme activity. The effect of calcitriol on the proliferation and acquisition of several monocyte-associated cell surface antigens was also more sensitive for HL-60 cells cultured in SFM than for cells grown in 10% SCM. We characterized and quantitated calcitriol receptors in HL-60 cells cultured in SFM in comparison to those in 10% SCM after exposing intact cells to radiolabeled calcitriol. Cells cultured in either SFM or 10% SCM exhibited calcitriol receptors that migrated at 3.4S as a single peak on sucrose gradients and elicited inherent DNA binding ability. There was essentially no difference in the apparent dissociation constants (Kd) nor in the number of calcitriol binding sites per HL-60 cell, that is approximately 6.0 X 10(-11) M and approximately 3000 binding sites/cell respectively. It is concluded that culturing HL-60 cells in SFM results in full expression of calcitriol-induced phenotypic changes excluding the possibility that such changes result from the indirect effect of calcitriol mediated by identified and/or unidentified components present in serum.