Induction of apoptosis by diallyl disulfide through activation of caspase-3 in human leukemia HL-60 cells.

Diallyl disulfide (DADS), a component of garlic (Allium sativum), has been known to exert potent chemopreventative activity against colon, lung, and skin cancers. However, its molecular mechanism of action is still obscure. The present study demonstrated that DADS induces apoptosis of human leukemia HL-60 cells in a concentration- and time-dependent manner with an IC50 for cell viability of less than 25 microM. DADS activated caspase-3 as evidenced by both the proteolytic cleavage of the proenzyme and increased protease activity. Activation of caspase-3 was maximal at 3 hr and led to the cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP), resulting in the accumulation of an 85 kDa cleavage product. Both activation of caspase-3 and cleavage of PARP were blocked by pretreatment with either antioxidants or a caspase-3 inhibitor, but not a caspase-1 inhibitor. DADS increased the production of intracellular hydrogen peroxide, which was blocked by preincubation with catalase. These results indicate that DADS-induced apoptosis is triggered by the generation of hydrogen peroxide, activation of caspase-3, degradation of PARP, and fragmentation of DNA. The induction of apoptosis by DADS may be the pivotal mechanism by which its chemopreventative action against cancer is based.     

二烯丙基二硫对人白血病HL-60细胞株survivin表达的影响

目的:观察二烯丙基二硫(diallyl disulfide,DADS)作用于人白血病HL-60细胞株后,细胞增殖及survivin蛋白表达的变化,探讨DADS对HL-60细胞增殖的影响及其机制。方法:DADS作用HL-60后,MTT法检测细胞增殖情况,SP-免疫组化法检测细胞survivin蛋白表达的变化。结果:DADS可以呈时间-浓度依赖性抑制HL-60细胞增殖并下调survivin蛋白的表达。结论:DADS能通过下调survivin蛋白的表达而抑制HL-60细胞的增殖。     

p42/44 MAPK激酶抑制剂增强二烯丙基二硫化物诱导CNE2细胞凋亡

目的 :探讨二烯丙基二硫化物 (DADS)诱导CNE2细胞凋亡及p4 2 4 4MAPK信号转导通路对此过程的作用。方法 :DADS处理CNE2细胞 2 4h后 ,荧光显微镜下观察形态学变化及凋亡细胞计数 ,四甲基偶氮唑盐微量酶反应比色法测定细胞活性 ,流式细胞仪检测凋亡细胞 ,蛋白质印迹法检测磷酸化p4 2 4 4MAPK表达。结果 :DADS(5 0～ 15 0 μmol·L-1)作用 2 4h后 ,诱导CNE2细胞产生典型的凋亡细胞形态学变化 (核浓染 核碎裂 ) ,流式细胞仪结果显示 ,随着DADS给药浓度增加 ,细胞凋亡率呈浓度依赖性逐渐升高 ,细胞周期中各期细胞所占百分率的变化无规律 ,DADS(5 0～ 15 0 μmol·L-1)浓度依赖性刺激磷酸化p4 2 4 4MAPK的表达 ,p4 2 4 4MAPK抑制剂U0 12 6显著增强DADS致凋亡作用。结论 :DADS诱导CNE2细胞凋亡时激活磷酸化p4 2 4 4MAPK表达 ,磷酸化p4 2 4 4MAPK抑制剂能增强DADS诱导CNE2细胞凋亡效应。     

p38 MAPK激酶抑制剂增强二烯丙基二硫化物诱导CNE2细胞凋亡

目的　研究二烯丙基二硫化物 (DADS)诱导CNE2细胞凋亡及 p38MAPK信号转导通路对此过程的作用。 方法　DADS处理CNE2细胞 2 4h后 ,荧光显微镜下观察形态学变化及凋亡细胞计数 ,MTT法测定细胞活性 ,流式细胞仪检测凋亡细胞 ,蛋白质印迹法检测磷酸化p38MAPK表达。结果　在培养的CNE2细胞中 ,DADS(50～ 1 50 μmol·L- 1 )作用 2 4h后 ,DADS诱导CNE2细胞产生典型的凋亡细胞形态学变化 (核浓染 ,核碎裂 ) ,流式细胞仪结果显示 ,随着DADS给药剂量增加 ,细胞周期中各期细胞所占百分率的变化无规律 ,细胞凋亡呈剂量依赖性 ,DADS(50～ 1 50 μmol·L- 1 )浓度依赖性刺激磷酸化p38MAPK的表达 ,p38MAPK抑制剂SB2 0 3580明显增强DADS致凋亡作用。结论　DADS诱导CNE2细胞凋亡时激活磷酸化 p38MAPK表达 ,磷酸化p38MAPK抑制剂增强DADS诱导CNE2细胞凋亡效应     

二烯丙基二硫诱导人白血病细胞系HL-60细胞分化的实验研究

目的　探讨二烯丙基二硫 (DADS)对人急性髓性白血病细胞系HL 6 0细胞增殖抑制及诱导分化作用的影响。方法　采用MTT法检测对细胞增殖的影响 ,通过观察细胞形态、硝基四氮唑蓝 (NBT)还原反应、流式细胞仪测定鉴定细胞分化。结果　HL 6 0细胞经DADS处理后 ,细胞增殖受抑 ,呈剂量效应关系。 0 6 2 5～ 1 2 5mg·L-1时NBT还原反应增强 (P <0 0 1或 <0 0 5 ) ,流式细胞术显示可诱导表面分化抗原CD11b表达升高及细胞周期阻滞于G1期。结论　小剂量DADS长时间作用对HL 6 0细胞具有诱导分化作用 ,其作用相当于全反式维甲酸 ,对于白血病的治疗具有潜在的应用价值     

Molecular mechanisms for the anti-cancer effects of diallyl disulfide

Considerable evidence in recent years suggests that garlic has anti-proliferative effects against various types of cancer. Garlic contains water-soluble and oil-soluble sulfur compounds. Oil-soluble compounds such as diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS) and ajoene are more effective than water-soluble compounds in protection against cancer. DADS, a major organosulfur compound derived from garlic, can decrease carcinogen-induced cancers in experimental animals and inhibit the proliferation of various types of cancer cells. Its mechanisms of action include: the activation of metabolizing enzymes that detoxify carcinogens; suppression of the formation of DNA adducts; antioxidant effects; regulation of cell-cycle arrest; induction of apoptosis and differentiation; histone modification; and inhibition of angiogenesis and invasion. These topics are discussed in depth in this review.     

二烯丙基二硫诱导K562细胞凋亡及与G2 / M期阻滞关系研究

目的探讨二烯丙基二硫（DADS）对体外培养的人白血病细胞系K562细胞增殖、凋亡和细胞周期的影响。方法采用细胞计数法、形态学观察、MTT分析法、AO／EB染色、流式细胞术探讨DADS对K562细胞增殖、凋亡和细胞周期的影响。结果1．DADS在10-80mg-L。范围内,对K562细胞的抑制作用呈剂量一时间依赖效应。2．DADS浓度在10-80mg．L-1。时作用K562细胞24h后,凋亡率逐渐升高,差异有统计学意义（p〈0．05或P〈0．01）。3．不同浓度DADS作用于K562细胞24h后,K562细胞阻滞于G2／M期。结论DADS有抑制K562细胞增殖和促进K562细胞凋亡的作用,其作用的可能机制与k562细胞阻滞于G2／M期有关。     

Induction of apoptosis and transient increase of phosphorylated MAPKs by diallyl disulfide treatment in human nasopharyngeal carcinoma CNE2 cells

This study was undertaken to elucidate the effect of diallyl disulfide (DADS), an oil-soluble organosulfur compound found in garlic, in suppressing human nasopharyngeal carcinoma cells. A potent increase (of at least 9-fold) in apoptotic cells has accompanied 1) a decrease in cell viability, 2) a increase of the fraction of S-phase cells by up to 63.8%, and 3) a transient increase of the phospho-p38 and phospho-p42/44 (phosphorylated p38 MAPK and phosphorylated p42/44 MAPK) in a time- and concentration-dependent manner. These results indicate that DADS can induce apoptosis in human nasopharyngeal carcinoma cells via, at least partly, S-phase block of the cell cycle, related to a rise in MAPK phosphorylation.     

Effect of diallyl disulfide on the hepatic glutathione s-transferase activity in rat: Diallyl disulfide effect on the glutathione S-transferase

Glutathione s-transferase is thought to play a key role in initiating the detoxication of potential alkylating agents, including pharmacologically active compounds. It is widely accepted that garlic contains allin which is converted to allicin by alliinase. Allicin is easily degraded to diallyl disulfide and other components. This report attempted to observe the effect of diallyl disulfide on some biological activities. It was observed that the activity of serum transaminase was not changed by the treatment of diallyl disulfide. The liver cytosolic glutathione s-transferase was significantly increased. whereas the microsomal glutathione s-transferase was not increased.     

小剂量二烯丙基二硫联合全反式维甲酸对HL-60细胞的生长抑制和诱导分化作用研究

二烯丙基二硫(diallyl disulfide,DADS)具有抑制肿瘤细胞增殖,调控细胞周期依赖素激酶、信号转导,诱导肿瘤细胞分化、凋亡及影响癌基因与抑癌基因表达的作用。小剂量DADS(1·25mg·L-1)可以抑制JAK1/STAT3信号通路,将HL-60细胞阻滞在G0/G1期,诱导HL-60细胞向粒系分化,其作用与全反式维甲酸(ATRA)相当[1~3]。本实验将探讨小剂量DADS与ATRA联合应用对HL-60细胞的生长抑制及诱导分化效应。1材料和方法参见文献2。2结果2.1DADS与ATRA单独或联合用药对HL-60细胞生长的影响1·25mg·L-1DADS或ATRA对体外培养的HL-60细胞均…     

Chromene induces apoptosis via caspase-3 activation in human leukemia HL-60 cells

In this study, the potent anti-tumor effects of brown algae on human leukemia HL-60 cells were investigated. The Sargassum siliquastrum extract among the 14 species of brown algae exhibited profound growth inhibitory effect on HL-60 cells in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, therefore, S. siliquastrum was selected for use in further experiments. The highest inhibitory activity of S. siliquastrum on HL-60 cells was detected in the chloroform fraction, and the active compound was identified as a kind of chromene, sargachromanol E (SE). SE treatment showed significant growth inhibitory effects on HL-60 cells in a dose-dependent manner by inducing apoptosis, as evidenced by the formation of apoptotic bodies, fragmented DNA ladder, and the accumulation of DNA in the sub-G₁ phase of cell cycle. SE induced apoptosis was accompanied by downregulation of Bcl-xL, upregulation of Bax, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP). Moreover, z-DEVD-fmk, a caspase-3 inhibitor, significantly inhibited cell cytotoxicity, apoptotic characteristics such as apoptotic bodies, sub-G₁ DNA content, and cleavage of PARP induced by SE. These results suggest that SE exerts its growth inhibitory effects on HL-60 cells through caspase-3-mediated induction of apoptosis. Therefore, SE offers promising chemotherapeuric potential to prevent cancers such as human leukemia.     

槲皮素诱导人白血病HL-60细胞凋亡(英文)

目的:研究槲皮素是否能诱导人白血病HL-60细胞凋亡.方法:应用琼脂糖凝胶电泳法观察DNA碎片;采用流式细胞仪检测DNA断裂;电镜技术观察凋亡的形态学改变,用MTT测定法测定细胞增殖.结果:槲皮素15-120 μmol·L~(-1)诱导HL-60细胞凋亡,电镜观察到典型的形态学改变,电泳显示梯状条带,槲皮素能剂量依赖性地触发DNA降解及抑制细胞增生(IG_(50)和95％可信区间分别为43(30-61)μmol·L~(-1).结论:槲皮素诱导人白血病HL-60细胞凋亡.     

二烯丙基二硫诱导人白血病HL-60细胞凋亡及机制

目的　探讨二烯丙基二硫 (DADS)诱导人白血病HL 6 0细胞凋亡的生物学效应及抗肿瘤机制。方法　通过MTT还原法检测DADS对该细胞系生长的影响 ;用电镜、荧光显微镜、流式细胞仪和细胞凋亡原位检测 (TUNEL)研究DADS诱导的细胞凋亡。SP免疫组化法检测细胞内Bcl 2、Bax蛋白的表达。结果　DADS能抑制HL 6 0细胞的生长 ,DADS处理HL 6 0细胞后 ,电镜下细胞呈凋亡特征性改变 :体积变小 ,核浓缩 ,凋亡小体形成 ,流式细胞仪示不同浓度DADS作用于HL 6 0细胞 ,亚G1期细胞明显增多 ,TUNEL测凋亡指数增加。SP免疫组化结果表明 10mg·L-1DADS处理细胞 2 4h后 ,Bax蛋白表达升高 ,Bcl 2蛋白表达下降。结论　DADS有诱导人白血病HL 6 0细胞凋亡的作用 ,其机制与Bcl 2 /Bax比例下降有关。     

一叶秋碱诱导人白血病HL—60细胞凋亡

目的 研究一叶秋碱能否诱导ＨＬ－６０细胞凋亡,方法 用ＭＴＴ法检测一叶秋碱对细胞增殖影响;应用流式细胞仪检测凋亡细胞数;采用琼脂糖凝胶电泳法观测ＤＮＡ碎片,透射电镜观察凋亡的形态改变,结果：一叶秋碱５－８０ｍｇ．Ｌ＾－１能诱导ＨＬ－６０细胞凋亡,电镜观察到典型的凋亡形态学改变,电泳呈现出阶梯状条带,流式细胞仪检测到凋亡率随剂量的增高而升高,ＭＴＴ法示一叶秋碱抑制ＨＬ－６０细胞增殖,并且呈时间,剂量     

Effect of diallyl disulfide on Ca movement and viability in PC3 human prostate cancer cells

The effect of diallyl disulfide (DADS) on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells is unclear. This study explored whether DADS changed [Ca²⁺]_i in PC3 cells by using fura-2. DADS at 50-1000 μM increased [Ca²⁺]_i in a concentration-dependent manner. The signal was reduced by removing Ca²⁺. DADS-induced Ca²⁺ influx was not inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators; but was inhibited by aristolochic acid. In Ca²⁺-free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) nearly abolished DADS-induced [Ca²⁺]_i rise. Incubation with DADS inhibited thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not alter DADS-induced [Ca²⁺]_i rise. At 500-1000 μM, DADS killed cells in a concentration-dependent manner. The cytotoxic effect of DADS was partly reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Propidium iodide staining suggests that DADS (500 μM) induced apoptosis in a Ca²⁺-independent manner. Annexin V/PI staining further shows that 10 μM and 500 μM DADS both evoked apoptosis. DADS also increased reactive oxygen species (ROS) production. Collectively, in PC3 cells, DADS induced [Ca²⁺]_i rise probably by causing phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A₂-sensitive channels. DADS induced Ca²⁺-dependent cell death, ROS production, and Ca²⁺-independent apoptosis.     

生存素在VP16诱导HL-60白血病细胞凋亡中的作用

目的探讨生存素在化疗药物足叶乙甙（VP16）诱导白血病细胞凋亡中的作用。方法用HL-60白血病细胞株进行传代培养,MTT试验检测VP16不同药物浓度对白血病细胞增殖的影响。通过光镜下观察细胞形态学变化和DNA凝胶电泳定性检测细胞凋亡,用流式细胞术（FCM）定量检测细胞凋亡及周期变化,用RT-PCR检测生存素基因表达,免疫组化检测其蛋白表达。结果各种浓度的VP16均可诱导HL-60细胞凋亡,下调生存素表达,阻滞细胞周期,具有明显时间和浓度依赖性。结论VP16诱导白血病细胞凋亡可能与其抑制生存素表达有关。     

Proliferation inhibition, cell cycle arrest and apoptosis induced in HL-60 cells by a natural diterpene ester from Daphne mucronata

Background and the purpose of the study

Gnidilatimonoein (Gn), a new diterpene ester from Daphne mucronata, possesses strong anti-metastasis and anti-tumor activities. In this study, its apoptosis and differentiation capabilities were evaluated by using the leukemia HL-60 cell line.

Material and methods

Cell prolifaration inhibition was estimated by MTT assay. The occurrence of apoptosis was evaluated by EtBr/AO double staining technique, cell cycle analyses and detection of apoptotic cells by Annexin V-FITC and propodium iodide (PI). Differentiation of the cells was determined by NBT reduction assay and the expression of specific cell surface markers such as CD14 and CD11b, were analyzed by flow cytometry.

Results

The drug decreased the growth of the cells dose- and time-dependently and the IC₅₀ was found to be 1.3 µM. Our data suggested that Gn induced both monocytic differentiation and apoptosis among HL-60 cells. In addition, cell cycle analyses showed an increase in G1 phase population by 24 hrs, which was gradually replaced by Sub-G1 cell population (apoptotic cells) by 72 hrs.

Conclusion

Based on these data, the Gn-treated HL-60 cells displayed differentiation-dependent apoptosis. Thus, Gn might be a good candidate for differentiation therapy of leukemia, pending full biological evaluation of the compound among the wide array of leukemia cells.     

Inhibition by ascorbic acid of apoptosis induced by oxidative stress in HL-60 myeloid leukemia cells

The human myeloid leukemia cell line HL-60 transports the oxidized form of ascorbic acid, dehydroascorbic acid (DHA), and accumulates reduced ascorbic acid. We studied the effect of ascorbic acid loading on apoptosis induced by serum- and glucose-free culture and by oxidative stress induced by H2O2. Uptake accumulation studies indicated that incubation of HL-60 cells with DHA resulted in the accumulation of intracellular ascorbic acid which decreased with time when cells were incubated in DHA-free medium. Exposure of HL-60 cells to increasing concentrations of H2O2 resulted in dose-dependent intracellular accumulation of peroxides, as determined by the use of the oxidation-sensitive fluorescent probe 2',7'-dichlorofluorescin-diacetate (DCFH-DA), which was accompanied by a decrease in intracellular ascorbic acid and an increase in apoptosis. A dramatic decrease in intracellular ascorbic acid was noted when preloaded HL-60 cells were exposed to 150 microM H2O2 (the concentration dropped from 5.2 +/- 0.6 mM to 3.6 +/- 0.1 mM in cells preincubated with 150 microM DHA). A dose-dependent protective effect of DHA was observed. Ascorbic acid loading also provided strong protection from apoptosis associated with serum- and glucose-free culture. Flow cytometry studies showed that exposure of HL-60 cells to 150 microM H2O2 resulted in decreased Bcl-2 expression that was associated with enhanced apoptosis (up to 33.6 +/- 2.6%). No significant variation of Bcl-2 expression was measured following exposure of HL-60 cells, loaded with ascorbic acid, to 150 microM H2O2 and only a slight increase (up to 10.1 +/- 3.1%) in apoptosis. These findings indicate that ascorbic acid can inhibit apoptosis induced by oxidative stress in HL-60 cells.