Induction of apoptosis by penta-O-galloyl-beta-D-glucose through activation of caspase-3 in human leukemia HL-60 cells.

Penta-O-galloyl-beta-D-glucose is structurally related to (-)-epigallocatechin gallate and is isolated from hydrolyzed tannin. Penta-O-galloyl-beta-D-glucose can inhibit tumor promotion by teleocidin. We investigated the effects of penta-O-galloyl-beta-D-glucose and various tea polyphenols on cell viability in human leukemia HL-60 cells. In this study, we demonstrated that penta-O-galloyl-beta-D-glucose was able to induce apoptosis in a concentration- and time-dependent manner; however, other polyphenols were less effective. We further investigated the molecular mechanisms of penta-O-galloyl-beta-D-glucose-induced apoptosis. Treatment with penta-O-galloyl-beta-D-glucose caused induction of caspase-3/CPP32 activity in dose- and time-dependent manner, but not caspase-1 activity, and induced the degradation of poly-(ADP-ribose) polymerase. Pretreatment with acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and Z-Val-Ala-Asp-fluoromethyl-ketone (Z-VAD-FMK) inhibited penta-O-galloyl-beta-D-glucose-induced DNA fragmentation. Furthermore, treatment with penta-O-galloyl-beta-D-glucose (50 microM) caused a rapid loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into cytosol, and subsequent induction of procaspase-9 processing. Our results indicate that penta-O-galloyl-beta-D-glucose allows caspase-activated deoxyribonuclease to enter the nucleus and degrade chromosomal DNA, and induces DFF-45 (DNA fragmentation factor) degradation. These results lead to a working hypothesis that penta-O-galloyl-beta-D-glucose-induced apoptosis is triggered by the release of cytochrome c into the cytosol, procaspase-9 processing, activation of caspase-3, degradation of poly-(ADP-ribose) polymerase, and DNA fragmentation caused by the caspase-activated deoxyribonuclease through the digestion of DFF-45. The induction of apoptosis by penta-O-galloyl-beta-D-glucose may provide a pivotal mechanism for its cancer chemopreventive action.     

冬凌草甲素诱导HL-60细胞凋亡

目的　研究冬凌草甲素诱导人白血病HL 6 0细胞凋亡的作用。方法　形态学观察 ,DNA凝胶电泳及流式细胞术。结果　冬凌草甲素能显著地诱导HL 6 0细胞发生凋亡 ,其作用呈明显的浓度效应关系和时间依赖性。形态学观察可见凋亡小体的形成 ,琼脂糖凝胶电泳可见明显的DNA梯带 ;流式细胞仪检测到G1亚峰。结论　冬凌草甲素能诱导HL 6 0细胞凋亡 ,并与其细胞杀伤活性相互平行 ,提示冬凌草甲素的抗癌活性与诱导肿瘤细胞凋亡相关     

毛兰素诱导人白血病HL—60细胞的凋亡

目的：研究毛兰素对HL－60细胞增殖的抑制作用,探讨其诱导细胞凋亡的机制。方法：用MTT比色法测定了毛兰素对HL－60细胞增殖的抑制作用：应用荧光显微镜、透射电镜、DNA电泳及流式细胞仪观察了药物对细胞凋亡的诱导作用,并用免疫组化的方法从基因水平阐述了凋亡的发生。结果：毛兰素20－81．9nmol/L在72h内显著抑制HL－60细胞增殖,作用24h后,对HL－60细胞的IC50为38nmol/L,而阳性对照药长春新碱对HL－60细胞的IC50为101nmol/L,前者明显优于后者;形态学观察可见凋亡的特征性改变;琼脂糖电泳出现典型的DNA“ladder”;流式细胞仪结果表明细胞被阻滞于G2／M期;免疫组化可见bcl-2表达下降,bax表达升高。结论：毛兰素显著抑制HL－60细胞的生长,该抑制作用可能是通过诱导细胞凋亡和改变HL－60细胞bcl-2和bax基因的表达而实现的。     

Induction of apoptosis by Cordyceps militaris through activation of caspase-3 in leukemia HL-60 cells

Cordyceps militaris is a traditional herbal ingredient frequently used for tonic and medicinal purposes in eastern Asia. The hot water extract of its cultivated fruiting bodies demonstrated a potent cytotoxic effect against the proliferation of the human premyelocytic leukemia cell HL-60, with an IC50 of 0.8 mg/ml for a 12-h treatment. It induced the characteristic apoptotic symptoms in the HL-60 cells, including DNA fragmentation and chromatin condensation, occurring within 12-16 h of treatment at a dose of 1 mg/ml. The activation of caspase-3 and the specific proteolytic cleavage of poly (ADP-ribose) polymerase were detected during the course of apoptosis induction. These results indicate that the hot water extract of Cordyceps militaris fruiting bodies inhibited cancer cell proliferation by inducing cell apoptosis through the activation of caspase-3, and that the Cordyceps militaris extract may therefore have therapeutic potential against human leukemia.     

Cadmium induces apoptosis partly via caspase-9 activation in HL-60 cells.

Cadmium (Cd), a potent immunotoxic metal, induces apoptosis both in vitro and in vivo. However, the mode of action remains unclear. We previously reported that Cd-induced apoptosis was partly dependent on mitochondria. In the present study, we investigated the involvement of caspase-9, which is the apex caspase in the mitochondoria-dependent apoptosis pathway, in Cd-induced apoptosis in human promyelocytic leukemia HL-60 cells. A specific inhibitor of caspase-9, Z-LEHD-FMK, partly inhibited DNA fragmentation induced by Cd treatment in HL-60 cells. Moreover, treatment of HL-60 cells with Cd resulted in the appearance of Cytochrome c (Cyt c), a potent activator of caspase-9, in the cytosol at 3 h, which closely paralleled the activation of caspase-9. Caspase-9 is an initiator caspase that is a potent activator of downstream effector caspases such as caspase-3. Caspase-3 activation was subsequent to the Cyt c release at 6 h. DNA fragmentation, an index of induction of apoptosis, also appeared 6 h after Cd treatment. The effects were more pronounced at 9 h after Cd addition. A broad-specificity inhibitor of caspases, Z-Asp-CH(2)-DCB, inhibited caspase-3 activation and DNA fragmentation induced by Cd in a dose-dependent fashion. The results suggest that Cd-induced apoptosis is partly caused by caspase-9 activation triggered by Cyt c.     

Induction of apoptosis by Korean medicine Gagam-whanglyun-haedoktang through activation of caspase-3 in human leukemia cell line, HL-60 cells

Gagam-whanglyun-haedoktang (GWH) is a newly designed herbal drug formula based on the traditional oriental pharmacological knowledge for the purpose of treating tumorous diseases. Apoptosis is an evolutionarily conserved suicide program residing in cells. In the present study, apoptosis inducing activities of the decocted water extract of GWH were studied. Results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that GWH had a strong cytotoxic effect on HL-60 cells. The number of live cells was less than 20% after exposure to 1 mg/ml GWH for 48 h. GWH increased cytotoxicity of HL-60 cells in a dose- and time-dependent manner. The percentage of apoptotic cells by flow cytometric analysis of the DNA-stained cells increased to 28%, 31%, and 37% at 24 h and to 37%, 44%, and 81% at 48 h after treatment with 0.01, 0.1, and 1 mg/ml GWH, respectively. DNA fragmentation also occurred in apoptosis and was characterized by a ladder pattern on agarose gel. In addition, GWH increased the secretion of tumor necrosis factor-alpha. GWH-induced apoptosis was accompanied by activation of caspase-3. These results suggest that GWH induces activation of caspase-3 and eventually leads to apoptosis.     

Hypericin induces both differentiation and apoptosis in human promyelocytic leukemia HL-60 cells

Hypericin is a unique photosensitizing plant pigment and has been separately reported to induce differentiation and apoptosis in neoplastic cells. In this study, we examined the relationship between activities to induce differentiation and apoptosis in human promyelocytic leukemia HL-60 cells, at a concentration range of 0.15 to 0.2 microM. When treated with hypericin, the cell ratio reducible of nitroblue tetrazolium was significantly increased and the cell size was enlarged by flow cytometry analysis. Hypericin also significantly increased the ratio of the cells, which were of positive alpha-naphthyl acetate esterase activity and phagocytic activity, whereas it hardly influenced the naphthol AS-D chloroacetate esterase activity in the cells, as well as 1 alpha, 25(OH)2D3 (10 nM). In addition, hypericin increased hypodiploid nuclei and caused a nucleosomal ladder. These results indicate that hypericin induces both differentiation toward monocyte/macrophage lineage and apoptosis in HL-60 cells.     

Glaucocalyxin A induces apoptosis in human leukemia HL-60 cells through mitochondria-mediated death pathway

Glaucocalyxin A (GLA) is a biologically active ent-kauranoid diterpenoid isolated from Rabdosia japonica var. glaucocalyx, a traditional Chinese medicinal herb, which has been shown to inhibit tumor cell proliferation. However, the mechanism underlying GLA-induced cytotoxicity remains unclear. In this study, we focused on the effect of GLA induction on apoptosis, the mitochondria-mediated death pathway and the accumulation of reactive oxygen species (ROS) in human leukemia cells (HL-60). GLA could induce a dose-dependent apoptosis in HL-60 cells as characterized by cell morphology, DNA fragmentation, activation of caspase-3, -9 and an increased expression ratio of Bax/Bcl-2. The mitochondrial membrane potential (Δψ_m) loss and cytochrome c release from mitochondria to cytosol were observed during the induction. Moreover, GLA caused a time- and dose-dependent elevation of intracellular ROS level in HL-60 cells, and N-acetyl-l-cysteine (NAC, a well-known antioxidant) could block GLA-induced ROS generation and apoptosis. These data suggest that GLA induces apoptosis in HL-60 cells through ROS-dependent mitochondrial dysfunction pathway.     

Fucoxanthin induces apoptosis in human leukemia HL-60 cells through a ROS-mediated Bcl-xL pathway

Fucoxanthin, a natural biologically active substance isolated from Ishige okamurae, evidences antitumor activity in human leukemia cell HL-60 cells via the induction of apoptosis. However, the mechanism underlying fucoxanthin-induced apoptosis in HL-60 cells remains unclear. In this study, we focused on the effect of fucoxanthin induction on the accumulation of reactive oxygen species (ROS), and on the triggering of Bcl-xL signaling pathway in HL-60 cells. We determined that ROS are generated during fucoxanthin-induced cytotoxicity and apoptosis in HL-60 cells, and that N-acetylcysteine (NAC), a ROS scavenger, suppressed fucoxanthin-induced cytotoxicity and apoptosis. Moreover, fucoxanthin-induced the cleavage of caspases -3 and -7, and poly-ADP-ribose polymerase (PARP) and a decrease of Bcl-xL levels, whereas NAC pre-treatment significantly inhibited caspase-3, -7, and PARP cleavage and the reduction in Bcl-xL levels. In this study, it was demonstrated for the first time that fucoxanthin generated ROS and that the accumulation of ROS performed a crucial role in the fucoxanthin-induced Bcl-xL signaling pathway.     

Costunolide induces differentiation of human leukemia HL-60 cells

Costunolide has been reported to be a cytotoxic and chemopreventive agent. This work investigated the mechanism of the antiproliferative effect of costunolide and determined that it induced differentiation of the human leukemia cell line HL-60. Costunolide exhibited a potent antiproliferative activity against HL-60 cells. It was also found to be a potent inducer of differentiation in human leukemia derived HL-60 cells through the examination of differentiation markers, as assessed by the reduction of nitroblue-tetrazolium, the increase in esterase activities and phagocytic activity, morphology change and the expression of CD14 and CD66b surface antigens. These results, accompanied by a decline in the expression of c-myc protein, suggest that costunolide induces differentiation of human leukemia cells to granulocytes and monocytes/macrophages lineage.     

Naringenin induces apoptosis through downregulation of Akt and caspase-3 activation in human leukemia THP-1 cells

Naringenin (NGEN), one of the most abundant flavonoids in citrus fruits, has been shown to inhibit in vitro growth of in human cancer cells, although the mechanism of action is poorly understood. Herein, we investigated NEGN’s pro-apoptotic effect on human leukemia THP-1 cells. NGEN treatment inhibited THP-1 cells’ growth a concentration-dependent manner by inducing apoptosis, as evidenced by the formation of apoptotic bodies and the accumulation of cells in the sub-G1 phase. NGEN-induced apoptosis was accompanied by increased hyperpolarization of the mitochondrial membrane potential, downregulation of Bcl-2, upregulation of Bax, activation of caspases and subsequent poly(ADP-ribose)polymerase (PARP) cleavages. z-DEVD-fmk, a caspase-3 inhibitor, significantly inhibited both the cytotoxic effect and apoptotic characteristics induced by NGEN treatment demonstrating caspase-3’s important role in the observed cytotoxic effect. The induction of apoptosis was also associated with the inactivation of phosphatidylinositol 3-kinase (PI3K)/Akt, and PI3K inhibitor LY29004 significantly increases NGEN-induced cell death. These findings provide evidence that NEGN’s pro-apoptotic effect is mediated by the activation of caspases and mitochondria dysfunctions that correlate with the inactivation of the PI3K/Akt pathway in THP-1 cells. Therefore, NGEN has a strong potential as a therapeutic agent for preventing cancers such as leukemia.     

Induction of apoptosis by lovastatin through activation of caspase-3 and DNase II in leukaemia HL-60 cells

Lovastatin, an HMG-CoA reductase inhibitor, was found to suppress growth and induce apoptosis in culture human promyelocytic leukaemic cell, HL-60. However, the mechanisms of lovastatin-induced apoptosis are still unclear. In this study, we attempted to elucidate the signal transduction pathway for lovastatin-induced apoptosis in HL-60 cells in a dose- and time-dependent manner. The features of this apoptosis were attenuated by the presence of mevalonate, a metabolic intermediate of cholesterol synthesis. Treatment of lovastatin caused a rapid release of mitochondrial cytochrome c into cytosol and subsequent induction of caspase-3, but not caspase-1 activity. Lovastatin also stimulated proteolytic cleavage of poly-(ADP-ribose) polymerase (PARP), and followed by the appearance of caspase activity and DNA fragmentation. Pretreatment with caspase-3 inhibitors, Ac-DEVD-CHO and Z-VAD-FMK, inhibited lovastatin induced caspase-3 activity and DNA fragmentation. Furthermore, we demonstrated that DNase II was involved in the DNA fragmentation induced by lovastatin. These results suggested that the mechanism of lovastatin induced HL-60 cells apoptosis through activation of caspase-3 and DNase II activities.     

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     

Caspases家族成员促进DADAG诱导的人白血病HL—60细胞凋亡

目的:研究caspases家族成员在二乙酰二脱水卫矛醇(DADAG)诱导人白血病HL-60细胞凋亡中的作用.方法:MTT法观察DADAG的体外抗增殖作用;透射电镜、DNA梯形条带和流式细胞仪检测HL-60细胞凋亡;caspase-3检测试剂盒和Western blot法分析caspases家族成员.结果:DADAG明显抑制HL-60细胞增殖和诱导细胞凋亡.DADAG处理HL-60细胞24h后,caspase-3酶活性达峰值,同时聚腺苷二磷酸核糖聚合酶(PARP)、lamin B和DFF45蛋白开始出现断裂片段.Caspase-3抑制剂z-DEVD·fmk可部分逆转DADAG诱导的HL-60细胞凋亡,而caspases广谱抑制剂z-VAD·fmk可完全逆转此作用.结论:Caspases在DADAG诱导HL-60细胞凋亡中起重要作用,它们通过酶解底物PARP、DFF45和lamin B促进细胞凋亡.     

Altholactone, a novel styryl-lactone induces apoptosis via oxidative stress in human HL-60 leukemia cells

Plant styryl-lactone derivatives isolated from Goniothalamus sp. are potential compounds for cancer chemotherapy. In this study, we have examined the mechanisms of apoptosis induced by altholactone, a stryl-lactone isolated from the Malaysian plant G. malayanus on human HL-60 promyelocytic leukemia cells. Flow cytometric analysis of the externalization of phosphatidylserine (PS) using the annexin V/PI method on altholactone treated HL-60 cells showed a concentration-dependent increase of apoptosis from concentrations ranging from 10.8 (2.5 microg/ml) to 172.4 microM (40 microg/ml). Pre-treatment with the antioxidant N-acetylcysteine (1 mM) completely abrogated apoptosis induced by altholactone, suggesting for the involvement of oxidative stress. Further flow cytometric assessment of the level of intracellular peroxides using the fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) confirmed that altholactone induced an increase in cellular oxidative stress in HL-60 cells which was suppressed by N-acetylcysteine. In summary, our results demonstrate for the first time that altholactone induced apoptosis in HL-60 cells occurs via oxidative stress.     

Saucernetin-7 isolated from Saururus chinensis induces caspase-dependent apoptosis in human promyelocytic leukemia HL-60 cells

In the present study, we investigated the effect of saucernetin-7 (a biologically active compound isolated from the underground parts of Saururus chinensi) on the induction of apoptosis and the putative pathways of its action in HL-60 human promyelocytic leukemia cells. Saucernetin-7-treated HL-60 cells displayed several features of apoptosis, including DNA fragmentation, DNA laddering by agarose gel electrophoresis, and externalization of annexin-V targeted phosphatidylserine (PS) residues. z-VAD-fmk (a broad-caspase inhibitor) almost completely suppressed saucernetin-7-induced DNA ladder formation, thereby implicating the caspase cascade in the apoptotic process. We also observed that saucernetin-7 caused the activations of caspase-3, -8 and -9, and that it induced Bid cleavage, the mitochondrial translocation of Bax from the cytosol, and cytochrome c release from mitochondria, but it had no effect on Bcl-2 and Bcl-xL levels. Taken together, the present study demonstrates that saucernetin-7 is a potent inducer of apoptosis and that its activity is facilitated by caspase-8 activation, Bid cleavage, Bax translocation to mitochondria, release of cytochrome c into cytoplasm, and subsequently caspase-3 activation, which offers a potential mechanism for the apoptosis-inducing activity of saucernetin-7.     

Effects of glycolic acid on the induction of apoptosis via caspase-3 activation in human leukemia cell line (HL-60).

Apoptosis is a particular process that leads to the programmed cell death, and it has been a potentially therapeutic target of cancer. In this study, we evaluated the possible apoptotic effects of glycolic acid on human leukemia cell line (HL-60) in vitro. The morphological changes, cell viability, apoptosis induction, and caspase-3 activity were measured by phase microscopy, flow cytometry, and Western blot analysis. Morphological changes including shrinkage of cells were clearly demonstrated in HL-60 cells treated with increasing concentrations of glycolic acid. Cell viability was significantly affected by glycolic acid treatment in a dose- and time-dependent manner. In comparison to the control group, glycolic acid treatment had a profound effect in the induction of apoptosis by flow cytometric assays. In the cell cycle analysis, glycolic acid caused the increased percentage of cells in G2/M phase and the decreased expression of the cyclin A and cyclin B1, suggesting the induction of G2/M arrest of cell cycle by glycolic acid. Moreover, glycolic acid treatment promoted caspase-9 and -3 activity in a dose-dependent manner, but caspse-8 activity was not affected during the same process. Glycolic acid co-administrated with broad-spectrum caspase inhibitor, z-VAD-fmk, caspase-3 activity was blunted and apoptosis was also markedly blocked in HL-60 cells. In conclusion, glycolic acid-induced apoptosis in HL-60 cells may be through the activation of caspase-3. Future studies focusing on cell signaling and biological significance of glycolic acid-induced apoptosis would lead to exploring the mechanisms of chemotherapeutic potency of glycolic acid in human cancers.     

Dracorhodin perchlorate induces apoptosis in HL-60 cells

Dracorhodin perchlorate, an anthocyanin red pigment, induces human premyelocytic leukemia HL-60 cell death through apoptotic pathway. Caspase -1, -3, -8, -9, and -10 inhibitors partially reversed the cell death induced by dracorhodin perchlorate. Caspase-3 and -8 were activated followed to the degradation of caspase-3 substrates, inhibitor of caspase-activated DNase (ICAD) and poly-(ADP-ribose) polymerase (PARP). Dracorhodin perchlorate up-regulated the expression ratio of mitochondrial proteins, Bax/Bcl-X_L. The cell death was accompanied with phosphorylation of ERK, JNK and p38 MAPK and partially reduced by MEK inhibitor (PD98059), JNK MAPK inhibitor (SP600125) and p38 MAPK inhibitor (SB 203580). Taken together, dracorhodin perchlorate-induced apoptosis in HL-60 cells via up-regulation of Bax, activation of caspases and ERK/p38/JNK MAPKs.