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。     

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     

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.     

Propolis induces cell cycle arrest and apoptosis in human leukemic U937 cells through Bcl-2/Bax regulation

We investigated mechanism(s) where propolis induces apoptosis in human leukemic U937 cells. Propolis inhibited the proliferation of U937 cells in a dose-dependent manner by inducing apoptosis and blocking cell cycle progression in the G2/M phase. Western blot analysis showed that propolis increases the expression of p21 and p27 proteins, and decreases the levels of cyclin B1, cyclin A, Cdk2 and Cdc2, thereby contributing to cell cycle arrest. DAPI staining assay revealed typical morphology features of apoptotic cells. Propolis-induced apoptosis was also confirmed by assays with annexin V-FITC, PI-labeling and DNA fragmentation assay. The increase in apoptosis level induced by propolis was associated with down-regulation of Bcl-2 and activation of caspase-3, but not with Bax. These results suggests that propolis-induced apoptosis is related to the selective activation of caspase-3 and induction of Bcl-2/Bax regulation.     

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.     

A novel adenosine analog, thio-Cl-IB-MECA, induces G0/G1 cell cycle arrest and apoptosis in human promyelocytic leukemia HL-60 cells

Human A3 adenosine receptor (A3AR) agonists have been shown to play important roles in several physiological and pathological processes, including growth inhibition of human cancer cells. On this line, we recently found that a novel adenosine analog, 2-chloro-N6-(3-iodobenzyl)-4'-thioadenosine-5'-N-methyluronamide (thio-Cl-IB-MECA) was a potent human A3AR agonist, and is superior to a known agonist Cl-IB-MECA [Jeong LS, Jin DZ, Kim HO, Shin DH, Moon HR, Gunaga P, et al. J Med Chem 2003;46:3775]. Here, we report that a novel A3AR agonist, thio-Cl-IB-MECA inhibited the growth of human promyelocytic leukemia HL-60 cells by arresting cell cycle and induction of apoptosis. Thio-Cl-IB-MECA induced the cell cycle arrest of G0/G1 in the early time and at lower concentration (up to 25 microM). At higher concentration (50 microM), the apoptotic cell deaths were manifested by observation of the increase of sub-G0 phase of cell cycle distribution, DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage. In addition, the down-regulation of checkpoint protein cyclin D1 and c-myc by thio-Cl-IB-MECA was well correlated with the arrest of cell cycle transition of G1 to S phase. Further study revealed that the growth inhibitory activity of thio-Cl-IB-MECA is also related with the modulation of Wnt signaling pathway. The levels of beta-catenin, phosphorylated forms of GSK-beta and Akt were down-regulated by the treatment of thio-Cl-IB-MECA (10 nM) in a time-dependent manner, providing one of plausible mechanistic evidence for the involvement of the Wnt signaling pathway in the HL-60 cell growth inhibitory effects by thio-Cl-IB-MECA. These results suggest that a novel A3AR agonist, thio-Cl-IB-MECA can down-regulate Wnt signaling, inhibit proliferation and induce apoptosis in HL-60 leukemia cells, and thus provide the possibility of this compound in the potential therapeutic value of the treatment of leukemia.     

大黄素可能通过抑制Akt信号通路诱导HL-60细胞凋亡

为研究大黄素(emodin)对人髓系白血病细胞株HL-60细胞增殖、凋亡的影响及Akt信号通路在其中的作用, 应用MTT法检测大黄素对HL-60细胞增殖的影响；细胞周期分析、线粒体细胞凋亡流式检测法分析细胞周期变化及细胞凋亡；Western blotting检测Akt信号通路蛋白表达水平的变化。结果显示，大黄素能有效抑制HL-60细胞的增殖，作用48 h的IC₅₀约为20 μmol·L^-1， 并能诱导其凋亡, 随药物作用浓度的增加， 凋亡率也逐渐上升； 大黄素作用后， HL-60细胞G_0/G1期细胞增多， 而S期及G₂期的细胞减少； Western blotting检测结果显示， 大黄素下调HL-60细胞Akt、p-Akt、IκB-α、p-IκB-α、p65、p-p65、mTOR及p-mTOR蛋白的表达。因此,大黄素能有效抑制HL-60细胞增殖，将细胞阻滞于G_0/G1期，诱导其凋亡；Akt信号通路可能参与了大黄素抑制HL-60细胞增殖、诱导凋亡的过程。     

Induction of apoptosis by yomogin in human promyelocytic leukemic HL-60 cells

Yomogin is an active compound isolated from Artemisia princep, a traditional Oriental medicinal herb, which has been shown to inhibit tumor cell proliferation. In this study, we investigated the effects of yomogin on the cytotoxicity, induction of apoptosis, and putative pathways of its actions in human promyelocytic leukemia cells. Yomogin-treated HL-60 cells displayed several features of apoptosis, including DNA fragmentation, formation of DNA ladders in agarose gel electrophoresis, and externalization of annexin-V targeted phosphatidylserine residues. We observed that yomogin caused activation of caspase-8, caspase-9, and caspase-3. A general caspase inhibitor (z-VAD-fmk), caspase-8 inhibitor (z-IETD-fmk) and caspase-3 inhibitor (z-DEVD-fmk), almost completely suppressed the yomogin-induced DNA fragmentation. We further demonstrated that yomogin induced Bid cleavage, mitochondrial translocation of Bax from the cytosol, and cytochrome c release from mitochondria in a caspase-8-dependent manner. Taken together, our data indicate that yomogin is a potent inducer of apoptosis and facilitates its activity via caspase-8 activation, Bid cleavage, Bax translocation to mitochondria, and subsequent release of cytochrome c into the cytoplasm, providing a potential mechanism for the anticancer activity of yomogin.     

Tebufenozide induces G1/S cell cycle arrest and apoptosis in human cells

Tebufenozide is a non-steroidal insect growth regulator and is extensively used to control pests, although it is considered to be safe for mammals and environmentally friendly. However, previous studies have found that tebufenozide is cytotoxic to man, although the exact mechanism remains elusive. This study will investigate the apoptotic molecular mechanisms which result from tebufenozide-induced DNA damage in HeLa cells. Our results demonstrate that tebufenozide could trigger arrest in G1/S phase related to a downregulation of cyclin E and cyclin-dependent kinase (CDK) 2 protein. In addition, Western blotting showed apoptosis was associated with the upregulation of p53, Bax and cleaved-PARP, as well as downregulation of Bcl-2 and PARP. Tebufenozide also regulated changes in mitochondrial permeability and reduced mitochondrial number and intracellular ATP production. Briefly, these results suggest that tebufenozide- induces cell cycle arrest and apoptosis through activating p53 protein in a Bax- and Bcl-2-triggered mitochondrial pathway. This work provides some scientific context for the safe use of tebufenozide in agriculture.     

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     

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.     

Anti-proliferation,cell cycle arrest and apoptosis induced by a natural xanthone from Gentianopsis paludosa Ma,in human promyelocytic leukemia cell line HL-60 cells

1-hydroxy-3,7,8-trimethoxyxanthone (xanthone 1) was isolated from Gentianopsis paludosa Ma and identified by MS and NMR in our laboratory. In this study, the results showed that xanthone 1 is a potent inducer of anti-proliferation and apoptosis in HL-60 cells. When the cells treated with lower concentrations of xanthone 1 (12.4–74.4 μM), significant proliferation inhibition was detected by cell viability assay and morphological analyses, and conspicuous G1 and G2/M cell cycle arrest were observed by flow cytometric (FCM) analysis. However, when the cells treated with higher doses of xanthone 1 (82.7–330.8 μM), significant apoptosis was observed by double sequential AO/EB staining, DNA fragmentation assay and FCM analysis. In addition, conspicuous DNA damage was detected by comet assay. In short, all the results showed that xanthone 1 had a significant cytotoxic effect and could induce proliferation inhibition and apoptosis in HL-60 cells in a time- and dose-dependent manner. It was possible that xanthone 1 could induce DNA damage in HL-60 cells, which resulted in G1 phase arrest at the lower concentrations and G2/M phase arrest at the higher concentrations, thus inhibiting the cell proliferation, and irreparable DNA damage at the higher concentrations might be responsible for the occurrence of apoptosis.     

Novel dichlorophenyl urea compounds inhibit proliferation of human leukemia HL-60 cells by inducing cell cycle arrest, differentiation and apoptosis

Two novel dichlorophenyl urea compounds, SR4 and SR9, were synthesized in our laboratory and evaluated for anti-cancer activities. Specifically, we investigated the antiproliferative properties of these new compounds on promyelocytic HL-60 leukemia cells by analyzing their effects on cell differentiation, cell cycle progression and apoptosis. SR4 and SR9 were both cytotoxic to HL-60 cells in a dose-and time-dependent manner, with IC(50) of 1.2 μM and 2.2 μM, respectively, after 72 h treatment. Both compounds strongly suppressed growth of HL-60 cells by promoting cell cycle arrest at the G0/G1 transition, with concomitant decrease in protein levels of cyclins D1 and E2 and cyclin-dependent kinases (CDK 2 and CDK 4), and increased protein expression of CDK inhibitors p21(WAF1/Cip1) and p27(Kip1). In addition, either compounds induce cell differentiation as detected by increased NBT staining and expression of CD11b and CD14. Treatment with SR compounds also promoted mitochondrial-dependent apoptosis as confirmed by Annexin V-FITC double staining, DNA fragmentation, increased expression of caspase 3, 7 and 9, cytochrome c release, PARP degradation, and collapse in mitochondrial membrane potential (ΔΨ(MT)). Collectively, these results provide evidence that SR4 and SR9 have the potential for the treatment of human leukemia and merit further investigation as therapeutic agents against other types of cancer.     

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.     

Induction of apoptosis and G2/M cell cycle arrest by Gleditsioside E from Gleditsia sinensis in HL-60 cells

Gleditsioside E, a triterpene saponin isolated from Gleditsia sinensis, showed significant cytotoxicity against Bel-7402, BGC-823, HeLa, HL-60 and MCF-7 cell lines. The results of flow cytometry with annexin V-FITC/PI double staining proved that gleditsioside E mainly induced early apoptosis in HL-60 cells. Gleditsioside E treatment resulted in a prominent increase of the G 2 /M population in HL-60 cells, and an accumulation of the sub-G 1 (hypoploid) peak was observed.     

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.