Investigation of the apoptotic effect of curcumin in human leukemia HL-60 cells by using flow cytometry

Curcumin (diferuloylmethane), the major yellow pigment isolated from the turmeric (Curcuma longa), has received much attention due to several biological properties. Curcumin exhibits a variety of pharmacological effects including antitumor, anti-inflammatory, and anti-infectious activities. In the present study, the effects of curcumin on apoptosis in the acute promyelocytic human leukemia (HL-60) cells was evaluated. Cytotoxic effects of curcumin on HL-60 cells were determined by MTT. HL-60 cells underwent apoptosis on treatment with curcumin, as indicated by increased annexin V-binding capacity and caspase-3 activation with flow cytometric analysis. Concentrations of 15, 20, and 40?μM curcumin significantly reduced cell proliferations. When HL-60 cells were treated with 10, 15, 20, and 40?μM concentration of curcumin, apoptotic rates were determined as 1.2, 81.1, 84.5, and 88.6%, respectively. On the incubations with the concentrations of curcumin, caspase-3 expressions (+) were found to be elevated by 8.5, 18.6, 91.2, and 92.4%, respectively. It was shown that curcumin had significant cytotoxic and apoptotic effects on HL-60 cells. It was suggested that curcumin may have a potential therapeutic role for human leukemia.     

PARP抑制剂olaparib对急性髓系白血病细胞HL-60抑制作用研究

目的 研究PARP抑制剂olaparib对人急性髓系白血病细胞株HL-60细胞的抑制作用。方法 对数生长期HL-60细胞经不同浓度(0、1.25、2.5、5、10 μmol/L)olaparib作用不同时间后,CCK-8法检测olaparib对HL-60细胞的增殖抑制作用;Annexin-V/PI双标法检测HL-60细胞凋亡水平,Western blot检测HL-60细胞内相关信号蛋白(PARP-1、Caspase-3)表达变化。结果 与对照组相比,经不同浓度(1.25、2.5、5、10 μmol/L)olaparib作用48 h后的HL-60细胞出现增殖抑制,并且随着作用时间的延长,抑制率逐渐增加;同时发现olaparib诱导HL-60细胞发生凋亡,并显示出剂量依赖效应;Western blot结果显示,olaparib处理后的HL-60细胞内PARP活性受到抑制,Caspase-3活化。结论 PARP抑制剂olaparib对HL-60细胞不仅具有增殖抑制作用,同时可通过激活Caspase-3,抑制PARP活性,诱导HL-60细胞凋亡。     

二烯丙基二硫作用于HL-60细胞后凋亡相关基因的差异表达

背景与目的:二烯丙基二硫(diallyl disulfide,DADS)对多种肿瘤细胞有促凋亡作用,白血病是儿童最常见的恶性肿瘤,近期研究提示DADS能够在体外诱导人白血病细胞发生凋亡,但其具体作用机制尚不清楚.本实验旨在研究DADS诱导人白血病细胞HL-60凋亡的生物学效应,并探讨其分子机制.方法:运用DNA含量分析、Annexin V/PI双标记流式细胞仪、DNA琼脂糖凝胶电泳以及透射电子显微镜形态学观察等方法来证实细胞凋亡.通过基因芯片检测60 μmol/L DADS作用于HL-60细胞4 h后凋亡相关基因的表达谱,采用RT-PCR技术验证上调基因Fas-L和下调基因Bag-1.结果:15、30、60和120 μmol/L DADS作用于HL-60细胞24 h后,DNA含量分析出现了明显的亚二倍体峰;60 μmol/L DADS作用4、8、12、24 h后,Annexin V/PI双标流式细胞仪检测表明早期凋亡细胞显著增加.60 μmol/L DADS作用24 h后,在DNA琼脂糖凝胶上可见特征性的梯形条带.电子显微镜观察到细胞体积缩小,核浓缩和凋亡小体形成等典型的形态学改变.通过基因芯片检测发现,60 μmol/L DADS作用4 h后有8个凋亡相关基因表达差异显著,选择其中Fas-L和Bag-1两个基因运用RT-PCR技术进行验证,其结果与基因芯片结果一致.结论:DADS能够诱导人白血病细胞HL-60凋亡,这可能是多个基因和多条信号转导通路共同作用的结果.     

Curcumin-induced Apoptosis in HL-60 Human Leukemic Cells

Curcumin is the main biologically active phytochemical compound in turmeric. It has been shown to have ‍anticarcinogenic activity. The aims of the study were to identify the mechanism of apoptosis of HL-60 human ‍promyelocytic leukemic cells induced by curcumin and to determine the effects of water-soluble antioxidants, ascorbic ‍acid, Trolox (a water-soluble form of vitamin E), glutathione (GSH) and N-acetylcysteine (NAC) on this process. ‍HL-60 cells were incubated with curcumin for 24 h and apoptotic cells were quantitated by flow cytometry following ‍staining with annexin V-FITC and propidium iodide. Curcumin-treated HL-60 cells produced reactive oxygen ‍species as detected by the dichlorofluorescein fluorescent assay. Apoptosis occurred via the mitochondria pathway ‍as curcumin reduced mitochondrial membrane potential in a dose-dependent manner. In the presence of 10 iM ‍curcumin, vitamin C (56 nM – 5.6 iM) inhibited apoptosis of HL-60 cells; GSH at low concentration (1 iM) reduced ‍apoptosis but had no effect at higher concentrations (10, 100 iM); and Trolox and NAC at 10 and 100 iM, respectively, ‍enhanced apoptosis, but this effect was abolished at higher concentration (1 mM) of NAC. MAPKK/MEK inhibitor ‍PD98059, enhanced curcumin-induced HL-60 apoptotic cell death.     

Baicalin induces apoptosis in leukemia HL-60/ADR cells via possible down-regulation of the PI3K/Akt signaling pathway

Background: The effect and possible mechanism of traditional Chinese medicine, baicalin, on the PI3K/Akt signaling pathway in drug-resistant human myeloid leukemia HL-60/ADR cells have been investigated inthis current study. Methods: HL-60/ADR cells were treated by 20, 40, 80 μmol/L baicalin followed by cell cycleanalysis at 24h. The mRNA expression level of the apoptosis related gene, Bcl-2 and bad, were measured byRT-PCR on cells treated with 80 μmol/L baicalin at 12, 24 and 48hr. Western blot was performed to detect thechanges in the expression of the proteins related to HL-60/ADR cell apoptosis and the signaling pathway beforeand after baicalin treatment, including Bcl-2, PARP, Bad, Caspase 3, Akt, p-Akt, NF-κB, p-NF-κB, mTOR andp-mTOR. Results: Sub-G1 peak of HL-60/ADR cells appeared 24 h after 20 μmol/L baicalin treatment, andthe ratio increased as baicalin concentration increased. Cell cycle analysis showed 44.9% G0/G1 phase cells24 h after baicalin treatment compared to 39.6% in the control group. Cells treated with 80 μmol/L baicalindisplayed a trend in decreasing of Bcl-2 mRNA expression over time. Expression level of the Bcl-2 and PARPproteins decreased significantly while that of the PARP, Caspase-3, and Bad proteins gradually increased. Nosignificant difference in Akt expression was observed between treated and the control groups. However, theexpression levels of p-Akt, NF-κB, p-NF-κB, mTOR and p-mTOR decreased significantly in a time-dependentmanner. Conclusions: We conclude that baicalin may induce HL-60/ADR cell apoptosis through the PI3K/AKTsignaling pathway.     

Coriolus versicolor (Yunzhi) extract attenuates growth of human leukemia xenografts and induces apoptosis through the mitochondrial pathway

Coriolus versicolor (CV), also called Yunzhi, has been demonstrated to exert anti-tumor effects on various types of cancer cells. Our previous studies have demonstrated that a standardized aqueous ethanol extract prepared from CV inhibited the proliferation of human leukemia cells via induction of apoptosis. The present study aimed to evaluate the underlying mechanisms of apoptosis through modulation of Bax, Bcl-2 and cytochrome c protein expressions in a human pro-myelocytic leukemia (HL-60) cell line, as well as the potential of the CV extract as anti-leukemia agent using the athymic mouse xenograft model. Our results demonstrated that the CV extract dose-dependently suppressed the proliferation of HL-60 cells (IC50 = 150.6 microg/ml), with increased nucleosome production from apoptotic cells. Expression of pro-apoptotic protein Bax was significantly up-regulated in HL-60 cells treated with the CV extract, especially after 16 and 24 h. Meanwhile, expression of anti-apoptotic protein Bcl-2 was concomitantly down-regulated, as reflected by the increased Bax/Bcl-2 ratio. The CV extract markedly, but transiently, promoted the release of cytochrome c from mitochondria to cytosol after 24-h incubation. In vivo studies in the athymic nude mouse xenograft model also confirmed the growth-inhibitory activity of the CV extract on human leukemia cells. In conclusion, the CV extract attenuated the human leukemia cell proliferation in vivo, and in vitro possibly by inducing apoptosis through the mitochondrial pathway. The CV extract is likely to be valuable for the treatment of some forms of human leukemia.     

hTERT反义寡核苷酸对HL-60细胞凋亡及细胞周期的影响

目的：研究hTERT基因反义寡核苷酸（ASODN）对HL-60细胞诱导凋亡作用及细胞周期的影响.方法：应用hTERT ASODN封闭HL-60细胞hTERT基因,RT-PCR方法检测目的基因的表达;流式细胞仪检测细胞凋亡及细胞周期分析,TUNEL方法检测细胞凋亡,琼脂糖凝胶电泳检测凋亡细胞DNA梯带.结果：hTERT ASODN作用细胞72h后,hTERT基因表达明显受到抑制（P＜0.01）.流式细胞仪检测显示：ASODN组细胞阻滞于G0/G1期,S、G2/M期细胞减少（P＜0.05）;细胞增殖指数（PI）明显降低（P＜0.05）;检测出早期凋亡峰.Annexin V/PI检测及TUNEL检测均显示：ASODN组凋亡细胞阳性率明显高于SODN组（P＜0.01）.琼脂糖凝胶电泳显示：ASODN组出现凋亡DNA梯带.结论：hTERT ASODN能够封闭目的基因表达,阻滞HL-60细胞于G0/G1期,并诱导细胞凋亡,对治疗白血病具有潜在应用价值.     

The novel synthesized 2-(3-(methylamino)phenyl)-6-(pyrrolidin-1-yl)quinolin-4-one (Smh-3) compound induces G2/M phase arrest and mitochondrial-dependent apoptotic cell death through inhibition of CDK1 and AKT activity in HL-60 human leukemia cells

2-Phenyl-4-quinolone series compounds have exhibited growth inhibitory influence on several human cancer cell lines. In this study, we investigated the effects of 2-(3-(methylamino)phenyl)-6-(pyrrolidin-1-yl)quinolin-4-one (Smh-3) on viability, cell cycle and apoptotic cell death which occurred in different leukemia cell lines (HL-60, U937 and K562) in a dose- and time-dependent manner, but which did not obviously impair the viability of normal human umbilical vein endothelial cells (HUVEC) in vitro. The approximate IC50 was 103.26 ± 4.59 nM for a 48 h treatment in HL-60 cells. Cell cycle analysis showed that 100 nM Smh-3 induced signi-ficant G2/M arrest in examined cells. Within 0, 12, 24 and 48 h of treatment, Smh-3 inhibited CDK1 activity and decreased protein levels of CDK1, cyclin A and cyclin B. Smh-3-induced chromatin condensation and DNA fragmentation were determined by DAPI and TUNEL staining. Cell apoptosis was significantly reduced after pretreatment with a pan-caspase inhibitor (Z-VAD-fmk) and results indicated that Smh-3-induced apoptosis was mainly mediated by activation of the caspase cascade in HL-60 cells. Results from colorimetric assays and Western blot analysis indicated that activities of caspase-9, -7 and -3 were promoted in Smh-3-treated HL-60 cells during cell apoptosis. Smh-3-induced apoptosis in HL-60 cells was accompanied by an apparent increase in ROS production, and protein levels of cytosolic cytochrome c, apoptotic protease activating factor-1 (Apaf-1) and apoptosis-inducing factor (AIF). Strikingly, Smh-3 induced apoptosis in HL-60 cells by simultaneously suppressing protein levels of AKT, p-AKT, p-mTOR and p-BAD and inducing BAD protein levels. Taken together, we conclude that Smh-3 acts against leukemia cells in vitro via G2/M phase arrest, down-regulation of AKT activity and induction of mitochondrial-dependent apoptotic pathways.     

氨基酸转运蛋白LAT4调控髓系白血病细胞自噬的机制探讨

目的:探究氨基酸转运蛋白LAT4调控髓系白血病细胞自噬的机制。方法:通过RNAi技术敲低HL-60细胞中LAT4的表达，CCK-8检测细胞体外增殖能力的变化，免疫荧光检测自噬标志物LC3Ⅱ的表达变化。Western Blot检测给予LAT4抑制剂BCH梯度浓度抑制后细胞内自噬相关蛋白的表达差异。谷氨酰胺剥夺诱导自噬后加入最适剂量BCH抑制LAT4，采用液滴计数仪检测细胞对3H-Leucine摄取率的变化。结果:干扰LAT4表达能显著降低HL-60细胞体外增殖能力并促进自噬蛋白LC3Ⅱ的表达。BCH梯度抑制LAT4活性后LAT4和pS6K蛋白表达受到显著抑制，LC3Ⅱ蛋白表达量增加，50 μmol/L BCH的LAT4活性抑制效果最佳。3H-Leucine摄取率随BCH抑制LAT4时间增加而降低，BCH作用8 h后3H-Leucine摄取率分别较0 h、4 h降低47.64%和27.05%（P＜0.001）。结论:LAT4通过转运亮氨酸激活HL-60细胞内pS6K表达，抑制自噬发生。     

Knockdown of IARS2 Inhibited Proliferation of Acute Myeloid Leukemia Cells by Regulating p53/p21/PCNA/eIF4E Pathway

IARS2 encodes mitochondrial isoleucine-tRNA synthetase, which mutation may cause multiple diseases. However, the biological function of IARS2 on acute myeloid leukemia (AML) has not yet been identified. In the present study, qRT-PCR was used to determine the expression of IARS2 in K562, THP1, and HL-60 leukemia cells. Additionally the mRNA levels of IARS2 in CD34 cells and AML cells obtained from patients were detected by qRT-PCR. IARS2-shRNA lentiviral vector was established and used to infect acute myeloid leukemia HL-60 cells. qRT-PCR and Western blot analysis were employed to assess the knockdown effect of IARS2. The proliferation rate and cell cycle phase of HL-60 cells after IARS2 knockdown were evaluated by CCK-8 assay and flow cytometry. The PathScan Antibody Array was used to determine the expression of cell cycle-related proteins in HL-60 cells after IARS2 knockdown. The expression of proliferation-related proteins in HL-60 cells after IARS2 knockdown was determined by Western blot analysis. Results showed that IARS2 expression was stable and much higher in HL-60, THP-1, and K562 leukemia cells and AML cells obtained from patients than that of human CD34 cells. Compared with cells of the shCtrl group, IARS2 was markedly knocked down in cells that were transfected with lentivirus encoding shRNA of IARS2 in HL-60 cells (p<0.05). IARS2 knockdown significantly inhibited the proliferation and induced cycle arrest at the G₁ phase in HL-60 cells. Additionally IARS2 knockdown significantly increased the expression of p53 and p21, and decreased the expression of PCNA and eIF4E in HL-60 cells. In conclusion, IARS2 knockdown can inhibit acute myeloid leukemia HL-60 cell proliferation and cause cell cycle arrest at the G₁ phase by regulating the p53/p21/PCNA/eIF4E pathways.     

血管内皮细胞生长因子影响难治性急性髓系白血病发生发展的作用研究

背景和目的:难治性白血病的发生发展机制非常复杂,近年来发现白血病患者骨髓过度表达血管内皮细胞生长因子(vascularendothelialgrowthfactor,VEGF),但VEGF如何影响难治性白血病尚未明确。本研究探讨高表达VEGF对人急性髓系白血病细胞株HL-60增殖及三尖杉酯碱诱导HL-60细胞凋亡影响;观察难治性急性髓系白血病(acutemyeloidleukemia,AML)中VEGF蛋白表达与疾病发展的相关性。方法:采用脂质体转染VEGF165cDNA入HL-60细胞,通过RT-PCR及ELISA方法鉴定转染克隆HL-60/VEGF165细胞中VEGFmRNA及蛋白的表达,MTT法、集落形成实验比较HL-60/VEGF165及转染pcDNA3.1-neo空载体的HL-60/neo细胞增殖活性,流式细胞仪观察三尖杉酯碱诱导的细胞凋亡。用ELISA法检测难治性与非难治性AML患者血浆中VEGF蛋白浓度。结果:HL-60/VEGF165细胞培养上清中VEGF蛋白浓度为(399.07±12.45)ng/L,高于HL-60/neo细胞(184.45±10.53)ng/L(P<0.01)。HL-60/VEGF165细胞与HL-60/neo细胞相比生长速度明显加快,集落形成能力明显增加,集落数分别为(157.00±17.00)/500细胞和(110.00±12.90)/500细胞(P<0.05);而细胞凋亡率减少,分别为(3.18±0.33)%和(6.61±0.50)%,三尖杉酯碱诱导HL-60/VEGF165细胞凋亡率低于HL-60/neo细胞。难治性AML患者血浆中VE     

正常人骨髓成纤维样基质细胞对HL-60/VCR耐药细胞增殖的影响

目的观察正常人骨髓成纤维样细胞系HFCL对白血病多药耐药细胞HL-60/VCR增殖和分化的影响.方法采用四唑氮蓝(MTT)法进行HL-60/VCR细胞药物敏感实验.建立HL-60/VCR细胞和HFCL细胞共培养体系,苔盼蓝拒染法测定生长曲线;硝基四氮唑蓝(NBT)确定细胞分化;流式细胞仪检测细胞周期和CD11b、CD13、CD14、CD33细胞表面抗原进一步鉴定细胞分化;Western blot检测增殖细胞核抗原(Proliferating Cell Nuclear Antigen,PCNA)和P糖蛋白(P-glycoprotein,P-gp).结果HL-60/VCR细胞对多种药物耐药.与HFCL细胞共培养后,HL-60/VCR细胞生长受抑,且与HFCL细胞直接接触组的抑制作用＞用transwell组.同时发现HL-60/VCR细胞与HFCL细胞共培养后,G1期细胞增多,S期细胞减少;同时CD11b和CD14表达增高,CD13和CD33变化不大;且NBT阳性细胞轻度增多.Western blot检测结果显示,PCNA表达下调,以直接接触组为甚.但是P-gp表达无变化.结论正常人骨髓成纤维样细胞HFCL能抑制白血病MDR细胞HL-60/VCR的增殖,抑制PCNA的表达,出现G1期阻滞,并部分向单核细胞分化.     

Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID cleavage and cytochrome c release: its suppression by ectopic expression of Bcl-2 and Bcl-xl.

Pharmacologically safe compounds that can inhibit the proliferation of tumor cells have potential as anticancer agents. Curcumin, a diferuloylmethane, is a major active component of the food flavor turmeric (Curcuma longa) that has been shown to inhibit the proliferation of a wide variety of tumor cells. The apoptotic intermediates through which curcumin exhibits its cytotoxic effects against tumor cells are not known, and the participation of antiapoptotic proteins Bcl-2 or Bcl-xl in the curcumin-induced apoptosis pathway is not established. In the present report we investigated the effect of curcumin on the activation of the apoptotic pathway in human acute myelogenous leukemia HL-60 cells and in established stable cell lines expressing Bcl-2 and Bcl-xl. Curcumin inhibited the growth of HL-60 cells (neo) in a dose- and time-dependent manner, whereas Bcl-2 and Bcl-xl-transfected cells were relatively resistant. Curcumin activated caspase-8 and caspase-3 in HL-60 neo cells but not in Bcl-2 and Bcl-xl-transfected cells. Similarly, time-dependent poly(ADP)ribose polymerase (PARP) cleavage by curcumin was observed in neo cells but not in Bcl-2 and Bcl-xl-transfected cells. Curcumin treatment also induced BID cleavage and mitochondrial cytochrome c release in neo cells but not in Bcl-2 and Bcl-xl-transfected cells. In neo HL-60 cells, curcumin also downregulated the expression of cyclooxygenase-2. Because DN-FLICE blocked curcumin-induced apoptosis, caspase-8 must play a critical role. Overall, our results indicate that curcumin induces apoptosis through mitochondrial pathway involving caspase-8, BID cleavage, cytochrome c release, and caspase-3 activation. Our results also suggest that Bcl-2 and Bcl-xl are critical negative regulators of curcumin-induced apoptosis.     

Rotenone, a mitochondrial NADH dehydrogenase inhibitor, induces cell surface expression of CD 13 and CD38 and apoptosis in HL-60 cells

We previously demonstrated that the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene was overexpressed in human acute myelogenous leukemia (AML) cells. Since this finding suggested that ND2 gene expression was related to myeloid differentiation, we here investigated the effects of rotenone, a specific NADH dehydrogenase inhibitor, on HL-60 cell growth, differentiation and death. Fifty nM rotenone inhibited the growth of HL-60 cells and caused an increase in the cell population in the Gz +M phase. In the quantitative comparison of myeloid antigen, the expression of CD13 and CD38 were relatively increased in the rotenone-treated cells. These findings suggest that the inhibition of NADH dehydrogenase changes the cell cycle and induces some specific surface antigens of HL-60 cells. On the other hand, the expression of ND2 gene remained unchanged after the rotenone treatment, suggesting the rotenone-mediated mitochondrial inhibition did not affect the mitochondrial gene expression. Five pM rotenone strongly inhibited the cellular proliferation. Electron microscopy and an electrophoretic analysis of DNA showed that the majority of the HL-60 cells were induced into typical apoptosis within 24-48 hours. On the basis of this and other studies, we believe that mitochondrial function is directly involved in both cellular differentiation and apoptotic cell death.     

The newly synthesized 2-(3-hydroxy-5-methoxyphenyl)-6,7-methylenedioxyquinolin-4-one triggers cell apoptosis through induction of oxidative stress and upregulation of the p38 MAPK signaling pathway in HL-60 human leukemia cells

The aim of the present study was to discover the signaling pathways associated with 2-(3-hydroxy-5-methoxy-phenyl)-6,7-methylenedioxyquinolin-4-one (YYK1)-induced apoptosis in HL-60 human leukemia cells. YYK1 induced cytotoxic effects, cell morphological changes, decreased the cell number and increased reactive oxygen species (ROS) production and loss of mitochondrial membrane potential (ΔΨm) in HL-60 cells. YYK1-induced apoptosis was confirmed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Results from colorimetric assays and western blot analysis indicated that activities of caspase-7/-3, caspase-8 and caspase-9 were increased in YYK1-treated HL-60 cells. Western blot analysis showed that the protein levels of extrinsic apoptotic proteins (Fas/CD95, FasL and FADD), intrinsic related proteins (cytochrome c, Apaf-1, AIF and Endo G), the ratio of Bax/Bcl-2 and phosphorylated p38 MAPK were increased in HL-60 cells after YYK1 treatment. Cell apoptosis was significantly reduced after pre-treatment with N-acetylcysteine (NAC; a ROS scavenger) or diphenyleneiodonium chloride (DPI; a NADPH oxidase inhibitor). Blockage of p38 MAPK signaling by SB202190 abolished YYK1-induced Fas/CD95 upregulation and apoptosis in HL-60 cells. We conclude that YYK1 induces both of extrinsic and intrinsic apoptotic pathways via ROS-mediated activation of p38 MAPK signaling in HL-60 human leukemia cells in?vitro.     

藤黄酸调节急性白血病细胞HL-60核孔蛋白Nup88的意义

目的 观察藤黄酸对白血病细胞株HL-60增殖和凋亡的影响及其对核孔蛋白Nup88的调控作用,探讨二者间的相互关系.方法 采用二苯基溴化四氮唑蓝(MTT)比色法检测细胞增殖活性;Annexin-V FITC/PI双标法和Hoechst33258染色法分析细胞凋亡的改变;流式细胞术分析细胞周期和细胞内核孔蛋白Nup88的改变;逆转录聚合酶链反应(RT-PCR)检测藤黄酸对白血病细胞内Nup88基因表达的影响;共聚焦显微镜下观察Nup88蛋白的分布情况.结果 藤黄酸能明显抑制HL-60细胞的增殖,其抑制作用呈时间、剂量依赖性,其12 h的IC50Molecule targetecl therapy; Response evaluation criteria为1.797 μmol/L.藤黄酸具有较强的诱导白血病细胞凋亡的效应,0.4 μmol/L藤黄酸即能诱导15.1%的HL-60细胞发生凋亡.当藤黄酸浓度达到1.6 μmol/L时,总凋亡率为79.0%,且该效应并不依赖于细胞周期阻滞作用.核孔蛋白Nup88弥漫分布于白血病细胞的核浆之间,以细胞浆和核膜为主,经藤黄酸干预后,Nup88蛋白和mRNA的表达水平明显下降,主要集中于核膜的胞浆面,偶见胞浆中表达.结论 藤黄酸能明显抑制HL-60白血病细胞的增殖,并诱导其凋亡;藤黄酸诱导的核孔蛋白Nup88的重新分布和表达量的下调可能参与了其诱导凋亡作用.     

In Vitro Anticancer Activities of Anogeissus latifolia,Terminalia bellerica,Acacia catechu and Moringa oleiferna Indian Plants

The present study was designed to evaluate in vitro anti-proliferative potential of extracts from four Indianmedicinal plants, namely Anogeissus latifolia, Terminalia bellerica, Acacia catechu and Moringa oleiferna. Theircytotoxicity was tested in nine human cancer cell lines, including cancers of lung (A549), prostate (PC-3), breast(T47D and MCF-7), colon (HCT-16 and Colo-205) and leukemia (THP-1, HL-60 and K562) by using SRB andMTT assays. The findings showed that the selected plant extracts inhibited the cell proliferation of nine humancancer cell lines in a concentration dependent manner. The extracts inhibited cell viability of leukemia HL-60and K562 cells by blocking G0/G1 phase of the cell cycle. Interestingly, A. catechu extract at 100 μg/mL inducedG2/M arrest in K562 cells. DNA fragmentation analysis displayed the appearance of a smear pattern of cellnecrosis upon agarose gel electrophoresis after incubation of HL-60 cells with these extracts for 24h.     

Monoclonal antibody to human chronic myeloid leukemia cell line MOLM-7 specifically reacts with an antigen of apoptotic cells.

Monoclonal antibody 2E12 was prepared by immunization of mice with fresh cells of chronic myeloid leukemia cell line MOLM-7. A panel of 15 leukemic cell lines (myeloid, promyelocytic, erythroid, B and T lymphoid) and numerous cultured patient's leukemia and myeloma cells were tested for reactivity with 2E12 antibody. A subset of cells in all cell lines and various number of patient's cells cultivated for 10 days or more were 2E12 positive. KG-1 and HL-60 cell lines were treated by camptothecin (CAM) (5 microg/ml, 4 h), washed and further cultivated without CAM. After 24 and 48 h in culture a considerable increase of 2E12 positivity was detected both in KG-1 and HL-60 cells, which well correlated with the increase of APO2.7 positivity and the sub-G1 peaks. The 2E12 positive cells were morphologically the same as cells in PCD, possibly apoptosis. We suggest that the 2E12 antibody detects a strong antigen on apoptotic cells which could be a part of the signaling process for ingestion by phagocytes.