Fluoride induces apoptosis by caspase-3 activation in human leukemia HL-60 cells

Even though fluoride toxicity is increasingly being considered to be important, very little information is available on the mechanism of action of fluoride. In the present study, the toxicity of fluoride on human leukemia (HL-60) cells was investigated and the involvement of caspase-3 was also studied. Fluoride induced apoptosis in HL-60 cells in a dose- and time-dependent manner. Annexin staining and DNA ladder formation on agarose gel electrophoresis further revealed that HL-60 cells underwent apoptosis on exposure to 2-5 mM fluoride. Western blotting using polyclonal anti-caspase-3 antibody and mouse anti-human poly(ADP-ribose) polymerase (PARP) monoclonal antibody was performed to investigate caspase-3 and PARP activity. Fluoride led to the activation of caspase-3 which was evident by the loss of the 32 kDa precursor and appearance of the 17 kDa subunit. Furthermore, intact 116 kDa PARP was cleaved by fluoride treatment as shown by the appearance of a cleaved 89 kDa fragment. The results clearly suggest that fluoride causes cell death in HL-60 cells by causing the activation of caspase-3 which in turn cleaves PARP leading to DNA damage and ultimately cell death.     

Citrinin induces apoptosis in HL-60 cells via activation of the mitochondrial pathway

The mycotoxin citrinin (CTN), a frequent natural contaminants of certain food and feeds, is known to be cytotoxic and genotoxic to various mammalian cells. To investigate the death mode of cells exposed to CTN, human promyelocytic leukemia (HL-60) cells were chosen to identify the apoptotic process induced by CTN. Morphological evidence of apoptosis, including nuclei fragmentation and DNA laddering formation, was clearly observed 24h after exposure to CTN. Flow cytometry analysis revealed that apoptotic cells in the hypodiploid region dramatically increased in cultures treated with CTN at concentrations above 50muM. Results of Western blotting showed that CTN induced the formation of processed caspase-3, -6, -7, -9, but not caspase-8, in a dose-dependent manner; CTN also induced a time-dependent increase in caspase-3 catalytic activity. The apoptosis triggered by CTN in HL-60 was accompanied by the cytochrome c release from mitochondria to cytoplasm. The presence of antioxidants in cultures did not effectively suppress CTN-induced cytotoxicity and caspase-3 activity. These findings suggest that CTN induces apoptosis in HL-60 cells by stimulating cytochrome c release followed by activation of multiple caspases, but oxidative stress may not play a role in the apoptotic process.     

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.     

金喜素诱导HL-60细胞凋亡依赖caspase活化

目的　探讨金喜素诱导急性白血病细胞凋亡的生化机制。方法　采用MTT法测定金喜素对HL 6 0细胞的杀伤作用 ;通过形态学、DNA凝胶电泳及AnnexinVFITC染色 ,研究金喜素对靶细胞的促凋亡活性 ;采用caspase 8、3特异性抑制剂IETD fmk、DEVD CHO ,分析金喜素介导的靶细胞凋亡与caspase活化的关系。结果　经 0 1μmol·L-1金喜素处理至 8、12及 16h时 ,HL 6 0细胞的存活率依次降至6 2 %± 12 %、、43%± 15 %及 32 %± 10 % ,低于对照 (P <0 0 5 ) ,同时 ,靶细胞逐渐出现磷脂酰丝氨酸外化 ,并产生梯状DNA及凋亡小体 ;经caspase抑制剂与金喜素联合处理12、16h时 ,HL 6 0细胞的存活率高于单用金喜素组 ,分别为77%± 14%、6 5 %± 16 % (IETD fmk组 )与 74%± 12 %、6 0 %± 11% (DEVD CHO组 ) ,同时 ,靶细胞的梯状DNA与凋亡小体的诱生也明显受抑。结论　金喜素对HL 6 0细胞具有较强的促凋亡作用 ,该过程可能依赖caspase 8、caspase 3的活化     

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。     

Trichothecin induces apoptosis of HepG2 cells via caspase-9 mediated activation of the mitochondrial death pathway

Trichothecin, one of fungal toxins which were encountered in food and in the environment, seriously threatens human and animal health. It has been shown that trichothecin changed the morphology of cellular mitochondria. However, the molecular mechanism remains unknown. Here we found that cell viability was attenuated by trichothecin. Features of apoptosis such as homosomal condensation and inter nucleosomal fragmentation were observed. In consistence with the elevated apoptosis rate, expression of anti-apoptotic protein Bcl-2 was diminished and expression of proapoptotic protein Bax was enhanced at mRNA levels. Furthermore, expression of caspase-9 and activity of caspase-3 were increased after the treatment of trichothecin. Accordingly, the mitochondrial membrane potential (?Ψm) was decreased in a dose-dependent manner. And Ca²⁺ overload was induced by trichothecin, followed by the generation of reactive oxygen species (ROS). Collectedly, our results suggested that apoptosis induced by trichothecin is mediated by caspase-9 activation and the decrement of mitochondrial function resulted from the overloaded calcium and ROS production.     

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.     

Loss of mitochondrial transmembrane potential and caspase-9 activation during apoptosis induced by the novel styryl-lactone goniothalamin in HL-60 leukemia cells.

Styryl-lactones such as goniothalamin represent a new class of compounds with potential anti-cancer properties. In this study, we investigated the mechanisms of goniothalamin (GTN), a plant styryl-lactone induced apoptosis in human promyelocytic leukemia HL-60 cells. This plant extract resulted in apoptosis in HL-60 cells as assessed by the externalisation of phosphatidylserine. Using the mitochondrial membrane dye (DIOC(6)) in conjunction with flow cytometry, we found that GTN treated HL-60 cells demonstrated a loss of mitochondrial transmembrane potential (Deltapsi(m)). Further immunoblotting on these cells showed activation of initiator caspase-9 and the executioner caspases-3 and -7. Pretreatment with the pharmacological caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) abrogated apoptosis as assessed by all of the apoptotic features in this study. In summary, our results demonstrate that goniothalamin-induced apoptosis occurs via the mitochondrial pathway in a caspase dependent manner.     

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.     

Organic solvent-induced proximal tubular cell apoptosis via caspase-9 activation

Long-term exposure to solvents is associated with apoptosis, which is implicated in the development and progression of tubulo-interstitial fibrosis and chronic renal failure. In our previous study, we demonstrated that toluene and p-xylene as the most commonly used organic solvents induced proximal tubular cells apoptosis. This study was conducted to assess the apoptotic pathway of toluene and p-xylene induced proximal tubular apoptosis. This was assessed by measuring the caspase-9 activity LLC-PK1 cells exposed to both compounds. A model of proximal tubular cell (LLC-PK1) cytotoxicity exposed to 1 mM of either p-xylene or toluene was compared to untreated control for caspase-9 activity and Bax/Bcl-2 protein level. Furthermore, DNA fragmentation in the presence of caspase-9 inhibitor (Z-LEHD-FMK) in a dose-dependent manner was assessed. Both compounds induced caspase-9 activity, which was accompanied by up-regulation of Bax, whereas Bcl-2 level did not change. DNA fragmentation induced by both solvents was inhibited by caspase-9 inhibitor in dose-dependent manner. This data suggest that p-xylene or toluene induces nephrotoxicity via mitochondrial caspase-9 pathway. This mechanism involves up-regulation of the apoptotic protein, Bax.     

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.     

Sucutinirane-diterpene derivatives induce apoptosis via oxidative stress in HL-60 cells

Previously, we reported the isolation of cassane-type diterpenes, sucutiniranes A–F, from the seeds of Bowdichia nitida. In this study, a series of sucutinirane derivatives was prepared, and their in vitro toxicity in the HL-60 cell line was evaluated. Then the action mechanism of a representative compound that induces cell death was investigated. Whereas C-6 or C-7 diol esters and ether decreased the activity against the HL-60 cell line, furan-oxidized derivatives 12 and 13 showed improvement or retention of the activity compared with those of the natural products sucutinirane A (11), E (1), and F (2). Treatment with sucutinirane derivative 13 elevated caspase 3/7 activity and also decreased expression of Bcl-2 family proteins, Mcl-1, and Bid. Derivative 13 generated reactive oxygen species in HL-60 cells, whose apoptotic effects were attenuated by the addition of an antioxidant, N-acetyl-l-cysteine. These results suggest that cassane butenolide 13 induces apoptosis in HL-60 via its oxidative effects.     

Pisosterol induces monocytic differentiation in HL-60 cells.

The aim of this study was to determine whether the antiproliferative effects observed for pisosterol, a cytotoxic triterpene isolated from Pisolithus tinctorius, are related to cell differentiation induction using HL-60 cell line as a model. Also, the effects of pisosterol on normal human cells were examined in peripheral blood mononuclear cells (PBMC). The effects on cell viability and morphological changes were the first indications showing that pisosterol induces HL-60 differentiation. The demonstration of blue tetrazolium reduction in HL-60 cells exposed to pisosterol demonstrated differentiation in a dose- and time-dependent manner, reaching a maximum effect after 72 h incubation at 5 microg/mL. Assays for alpha-naphthyl acetate esterase activity indicated that pisosterol triggers differentiation towards a monocytic cell-like pathway. The antiproliferative effect of pisosterol was determined by inhibition of DNA synthesis based on BrdU incorporation into HL-60 proliferating cells. It appears that pisosterol-treated cells, despite displaying a differentiated phenotype, continued to proliferate at all doses tested after 72 h, with a slightly decrease at 5 microg/mL. Apoptosis was observed in pisosterol-treated cells in a dose-dependent way. Nevertheless, after the same period of incubation, no cytotoxicity was detected in PBMC in the presence of pisosterol even at 25 microg/mL, providing some evidence that pisosterol may be selective for tumor cells. The mechanisms underlying the effect of pisosterol in leukemia cells indicates the induction of a monocytic cell-like differentiation, suggesting that this compound could be used in the development of new pharmacological tools with potential therapeutic value in the management of leukemia with fewer side effects.     

Modulation of 9-nitrocamptothecin-induced apoptosis by hyperthermia in human leukemia HL-60 cells.

We have investigated whether hyperthermia (HT) treatment at 43 degrees C for 15-60 min can affect the extent of apoptosis induced in human leukemia HL-60 cells by the anticancer drug 9-nitrocamptothecin (9NC). Quantitative changes in the apoptotic (Ap) fraction in the cell cultures were monitored by flow cytometry. The results showed that (i) heating for 15 min prior to or concurrently with 9NC exposure had no effect on the Ap fraction generated by the drug alone, whereas 60 min heating resulted in an increase in the Ap fraction; and (ii) heating of the cells at 6-24 h after exposure to the drug enhanced the Ap fraction. These results indicate that appropriate scheduling of HT and 9NC treatments may lead to thermochemotherapy protocols that will result in increased 9NC-induced death of human leukemia cells.     

3-Hydrogenkwadaphnin from Dendrostellera lessertii induces differentiation and apoptosis in HL-60 cells

3-Hydrogenkwadaphnin (3-HK) is a new diterpene ester, recently isolated from the leaves of Dendrostellera lessertii with potent anti-tumoral and anti-metastatic activities. Herein, we report that 3-HK induces differentiation and apoptosis in HL-60 cells. The drug at 2.5 - 40 nM inhibited proliferation of HL-60 cells after 24 - 96 h of treatment. Cell viability was also decreased by 57 % after 96 h treatment with the drug. NBT reducing assay and phagocytic activity revealed that the inhibition of proliferation is associated with differentiation especially toward macrophages-like morphology. Indeed, the drug at 2.5 - 10 nM induced differentiation by 5 - 49 % in HL-60 cells. Acridine orange/ethidium bromide (AO/EtBr) double staining and DNA fragmentation assays revealed that apoptosis occurred after differentiation of HL-60 cells. Guanosine at 50 microM decreased the apoptotic cell death and the differentiation caused by the drug. Therefore, GTP depletion, as a result of inhibition of inosine monophosphate dehydrogenase (IMPDH), is considered as one of the main reasons for differentiation and apoptotic cell death by this new drug.     

Toosendanin induces apoptosis through suppression of JNK signaling pathway in HL-60 cells

Toosendanin (TSN), a triterpenoid isolated from Melia toosendan Sieb. et Zucc., has been found to suppress proliferation and induce apoptosis in a variety of human cancer cells. However, the mechanism how TSN induces apoptosis remains poorly understood. In this study, we examined the effects of TSN on the growth, cell cycle arrest, induction of apoptosis and the involved signaling pathway in human promyelocytic leukemia HL-60 cells. Proliferation of HL-60 cells was inhibited in a dose-dependent manner with the IC_{50 (48h)} of 28 ng/mL. The growth inhibition was due primarily to the S phase arrest and cell apoptosis. Cell apoptosis induced by TSN was confirmed by Annexin V-FITC/propidium iodide staining. The increase of the pro-apoptotic protein Bax, cleaved PARP and caspase-3, and the decrease of anti-apoptotic protein Bcl-2 were observed. Western blot analysis indicated that TSN inhibits the CDC42/MEKK1/JNK pathway. Taken together, our study suggested, for the first time, that the pro-apoptotic effects of TSN on HL-60 cells were mediated through JNK signaling pathway.     

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.