Imperatorin,a Furanocoumarin from Angelica dahurica (Umbelliferae), Induces Cytochrome c‐Dependent Apoptosis in Human Promyelocytic Leukaemia,HL‐60 Cells

Abstract: Imperatorin, a biologically active furanocoumarin from the roots of Angelica dahurica (Umbelliferae), was found to induce apoptosis in human promyelocytic leukaemia, HL‐60 cells. DNA fragmentation assay, morphology‐based evaluation, and flow cytometric analysis demonstrated that imperatorin at micromolar concentrations was able to trigger apoptosis of HL‐60 cells. Neither necrosis nor differentiation was observed at cytotoxic micromolar concentrations of imperatorin. Further studies showed that the cytochrome c/caspase‐9 pathway was responsible for imperatorin‐induced apoptosis; i.e., mitochondrial membrane was depolarized, Bcl‐2 was down‐regulated, cytochrome c was released from mitochondria, caspase‐9 and caspase‐3 were activated, and poly(ADP‐ribose) polymerase was cleaved. Furthermore, imperatorin‐induced apoptosis was significantly blocked by Z‐VAD‐FMK (a broad spectrum caspase inhibitor), Z‐LEHD‐FMK (a caspase‐9 inhibitor) and Ac‐DMQD‐CHO (a caspase‐3 inhibitor), but not by Z‐IEDT‐FMK (a caspase‐8 inhibitor).     

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.     

Synthetic 1,4-anthracenedione analogs induce cytochrome c release, caspase-9, -3, and -8 activities, poly(ADP-ribose) polymerase-1 cleavage and internucleosomal DNA fragmentation in HL-60 cells by a mechanism which involves caspase-2 activation but not Fas signaling

Synthetic analogs of 1,4-anthraquinone (AQ code number), a compound that mimics the antiproliferative effects of daunorubicin (daunomycin) in the nanomolar range in vitro but has the advantage of blocking nucleoside transport and retaining its efficacy in multidrug-resistant tumor cells, were tested for their ability to induce apoptosis in the HL-60 cell system. AQ10 and, especially, the new lead antiproliferative compounds AQ8 and AQ9 reduce the growth and integrity of wild-type, drug-sensitive, HL-60-S cells more effectively than AQ1, suggesting that various methyl group substituents at C6 may enhance the bioactivity of the parent compound. Internucleosomal DNA fragmentation, a late marker of apoptosis, is similarly induced in a biphasic manner by increasing concentrations of AQ8 and AQ9 at 24 hr. Poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, an early event required for cells committed to apoptosis, is detected within 3-6 hr in HL-60-S cells treated with AQ9. In accord with the fact that the caspases 9 and 3 cascade is responsible for PARP-1 cleavage, the activities of initiator caspase-9 and effector caspase-3 are induced by AQ9 in the same time- and concentration-dependent manners and to the same maximal degrees in both the HL-60-S and multidrug-resistant HL-60-RV cell lines. Interestingly, a 1-hr pulse treatment is sufficient for AQ8 and AQ9 to maximally induce caspase-9 and -3 activities at 6 hr. The release of mitochondrial cytochrome c (Cyt c) is also detected within 3-6hr in HL-60-S cells treated with AQ9, a finding consistent with the fact that Cyt c is the apoptotic trigger that activates caspase-9. Moreover, AQ analogs induce Cyt c release, caspase-9 and -3 activities and PARP-1 cleavage in relation with their abilities to decrease tumor cell growth and integrity, AQ8 and AQ9 being consistently the most effective. Since apical caspases 2 and 8 may both act upstream of mitochondria to promote Cyt c release, it is significant to show that AQ9 maximally induces caspase-2 and -8 activities at 6 and 9 hr, respectively. During AQ8 treatment, the caspase-2 inhibitor benzyloxycarbonyl (z)-Val-Asp-Val-Ala-Asp (VDVAD)-fluoromethyl ketone (fmk) totally blocks caspase-9, -3, and -8 activations, whereas the caspase-8 inhibitor z-Ile-Glu-Thr-Asp-(IETD)-fmk does not prevent caspase-2, -9, and -3 activations, suggesting that AQ-induced caspase-2 activity is an upstream event critical for the activation of the downstream caspases 9 and 3 cascade, including the mitochondrial amplification loop through caspase-8. However, these caspase-2 and -8 inhibitors fail to alter AQ8-induced Cyt c release, suggesting that AQs might also target mitochondria independently from caspase activation. Furthermore, the antagonistic anti-Fas DX2 and ZB4 monoclonal antibodies (mAbs), which block the induction of Cyt c release and caspase-2, -8, and -9 activities by the agonistic anti-Fas CH11 mAb, and the neutralizing anti-Fas ligand (FasL) NOK-1 mAb all fail to inhibit AQ9-induced Cyt c release and caspase-2, -8, and -9 activities, suggesting that the FasL/Fas signaling pathway is not involved in the mechanism by which antiproliferative AQ analogs trigger apoptosis in HL-60 cells.     

DNA引物酶抑制剂碘化-3,3′-二乙基-9-甲基-硫杂羰花青诱导人白血病HL-60细胞凋亡

目的研究DNA引物酶抑制剂碘化-3,3′-二乙基-9-甲基-硫杂羰花青(DMTCCI)诱导人粒细胞性白血病HL-60细胞凋亡并探索其机制。方法分别采用不同浓度的DMTCCI处理培养于RPMI-1640培养基的HL-60细胞。采用MTT法检测DMTCCI对HL-60细胞的生长抑制作用。采用流式细胞仪和DNA琼脂糖凝胶电泳方法检测细胞凋亡。采用蛋白免疫印迹(Western blotting)法观察凋亡相关蛋白survivin， Bcl-xL， Bad， Bax， Bcl-2， caspase-9， caspase-3， caspase-6， PARP， DFF45和lamin B的表达。采用ApoAlert Caspase-3分析试剂盒检测caspase-3的活性。结果DMTCCI具有抑制人白血病HL-60细胞增殖的作用，其IC₅₀值为0.24 μmol·L^-1。流式细胞仪和DNA琼脂糖凝胶电泳结果显示，DMTCCI可诱导HL-60细胞凋亡。在经DMTCCI处理的HL-60细胞中，survivin和Bcl-xL蛋白的表达水平下调，Bad和Bax蛋白的表达水平上调，Bcl-2蛋白的表达水平无变化，caspase-9，caspase-3，caspase-6，PARP，DFF45和lamin B被分别裂解，产生相应裂解产物。在HL-60细胞中，caspase-3的活性在1 μmol·L^-1 DMTCCI处理3 h时明显升高，在处理12 h时达到最高峰。结论DMTCCI可抑制人白血病HL-60细胞的增殖并诱导其发生细胞凋亡。Bcl-2家族蛋白、survivin和caspases家族蛋白可能参与了上述诱导HL-60细胞凋亡的过程。     

Mercuric chloride induces apoptosis via a mitochondrial-dependent pathway in human leukemia cells

Mercurial compounds modulate immunologic functions by inducing cytotoxicity. Although mercury chloride (HgCl(2)) is known to induce apoptosis in various immune system cells, the mechanism of the induction of apoptosis is poorly understood. In this study, we examined the activation of caspase-3, an important cysteine aspartic protease, during HgCl(2)-induced apoptosis in a human leukemia cell line (HL-60 cells). Both DNA fragmentation, a characteristic of apoptotic cells, and proteolysis of poly(ADP-ribose) polymerase (PARP), a substrate of caspase-3, occurred at 6 h after HgCl(2) treatment in HL-60 cells. These results suggest that the activation of caspase-3 was involved in HgCl(2)-induced apoptosis. The release of cytochrome c (Cyt c) from mitochondria into the cytosol, which is an initiator of the activation of caspase cascades, was also observed in HgCl(2)-treated HL-60 cells. Moreover, the release of Cyt c from mitochondria was observed in HgCl(2)-treated mitochondria isolated from mice liver, and this was followed by mitochondrial permeability transition (PT). The PT was inhibited by cyclosporin A (CsA), a potent inhibitor of PT. CsA also suppressed the occurrence of DNA fragmentation induced by HgCl(2) treatment in HL-60 cells. Taken together, these findings indicate that HgCl(2) is a potent inducer of apoptosis via Cyt c release from the mitochondria in HL-60 cells.     

Intrinsic pathway of hydroquinone induced apoptosis occurs via both caspase-dependent and caspase-independent mechanisms

The role of mitochondria and apical caspases in apoptosis induced by the benzene metabolite hydroquinone (HQ) remains to be elucidated. Here, we investigated the involvement of mitochondria and activation of the apical caspases-8 and -9 in HQ induced apoptosis in myeloperoxidase (MPO)-rich HL-60 and MPO-deficient Jurkat T cells. Treatment of HL-60 and Jurkat cells with HQ resulted in apoptosis as assessed by phosphatidyl serine (PS) exposure, loss of mitochondrial transmembrane potential (MTP), release of cytochrome c, and processing of apical caspases-8 and -9 and executioner caspase-3. In HQ-treated HL-60 cells, pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (ZVAD), which did not inhibit PS exposure, also failed to abrogate the loss of MTP and release of cytochrome c. However, complete processing of caspase-9 was inhibited in the presence of ZVAD. In marked contrast, in HQ-treated Jurkat cells, ZVAD significantly abrogated PS exposure, loss of MTP, and caspase-9 processing but not release of cytochrome c. Although ZVAD-sensitive caspase-8 processing occurred in both cell types, pretreatment with either fas-receptor blocking ZB4 or fas-ligand NOK1 neutralizing antibodies did not inhibit HQ-induced apoptosis. In conclusion, our results demonstrate that HQ induced apoptosis in Jurkat cells occurs via a ZVAD-inhibitable, caspase-dependent process, while in HL-60 cells, apoptosis occurs predominantly via caspase-independent mechanisms. Our results emphasize that both caspase-dependent and independent mechanisms should be considered in the intrinsic apoptotic pathway induced by HQ.     

DNA引物酶抑制剂碘化-3,3''-二乙基-9-甲基-硫杂羰花青诱导人白血病HL-60细胞凋亡

目的 研究DNA引物酶抑制剂碘化-3,3'-二乙基-9-甲基-硫杂羰花青(DMTCCI)诱导人粒细胞性白血病HL-60细胞凋亡并探索其机制.方法 分别采用不同浓度的DMTCCI处理培养于RPMI-1640培养基的HL-60细胞.采用MTT法检测DMTCCI对HL-60细胞的生长抑制作用.采用流式细胞仪和DNA琼脂糖凝胶电泳方法检测细胞凋亡.采用蛋白免疫印迹(Western blotting)法观察捌亡相关蛋白survivin,Bcl-xL,Bad,Bax,Bcl-2,caspase-9,caspase-3,caspase-6,PARP,DFF45和lamin B的表达.采用ApoAlert Caspase-3分析试剂盒检测caspase-3的活性.结果 DMTCCI具有抑制人白血病HL-60细胞增殖的作用,其IC50值为0.24μmol·L-1.流式细胞仪和DNA琼脂糖凝胶电泳结果显示,DMTCCI可诱导HL-60细胞凋亡.在经DMTCCI处理的HL-60细胞中,survivin和Bcl-xL蛋白的表达水平下调,Bad和Bax蛋白的表达水平上调,Bcl-2蛋白的表达水平无变化,caspase-9,caspase-3,caspase-6,PARP, DFF45和lamin B被分别裂解,产生相应裂解产物.在HL-60细胞中,caspase-3的活性在1μmol·L-1 DMTCCI处理3 h时明显升高,在处理12 h时达到最高峰.结论 DMTCCI可抑制人白血病HL-60细胞的增殖并诱导其发生细胞凋亡.Bcl-2家族蛋白、survivin和caspases家族蛋白可能参与了上述诱导HL-60细胞凋亡的过程.     

Antitumor triptycene bisquinones induce a caspase-independent release of mitochondrial cytochrome c and a caspase-2-mediated activation of initiator caspase-8 and -9 in HL-60 cells by a mechanism which does not involve Fas signaling

Synthetic triptycene analogs (TT code number) mimic the antitumor effects of daunorubicin (DAU) in vitro, but have the advantage of blocking nucleoside transport, inhibiting both DNA topoisomerase I and II activities, and retaining their efficacy in multidrug-resistant (MDR) tumor cells. Since TT bisquinones induce poly(ADP-ribose) polymerase-1 (PARP-1) cleavage at 6 h and internucleosomal DNA fragmentation at 24 h, which are, respectively, early and late markers of apoptosis, these antitumor drugs were tested for their ability to trigger the release of mitochondrial cytochrome c (Cyt c) and the caspase activation cascade in the HL-60 cell system. Based on their ability to reduce the viability of wild-type, drug-sensitive HL-60-S cells in the nanomolar range, six lead antitumor TT bisquinones have been identified so far: TT2, TT13, TT16, TT19, TT24 and TT26. In accord with the fact that effector caspase-3 is responsible for PARP-1 cleavage, 4 microM concentrations of DAU and these TT bisquinones all maximally induce caspase-3 activity at 6 h in HL-60-S cells, an effect which persists when the drugs are removed after a 1-h pulse treatment. Since caspase-3 may be activated by initiator caspase-9 and -8, it is significant to show that such caspase activation cascade is induced by 4 microM DAU and TT bisquinones at 6 h in HL-60-S cells. Although the relationship is not perfect, the ability of TT analogs to induce caspase-3, -8 and -9 activities may be linked to their quinone functionality and cytotoxicity. Interestingly, 4 microM concentrations of TT bisquinones retain their ability to induce caspase-3, -8 and -9 activities at 6 h in the MDR HL-60-RV cell line where 4 microM DAU becomes totally ineffective. The release of mitochondrial Cyt c is also detected within 6 h in HL-60-S cells treated with 4 microM DAU or TT bisquinones, a finding consistent with the fact that Cyt c is the apoptotic trigger that activates caspase-9. Caspase-2 and -8 may both act upstream of mitochondria to promote Cyt c release, but caspase-2 is already maximally activated 6 h after 4 microM DAU or TT13 treatments, whereas DAU- or TT-induced caspase-8 and -9 activities peak at 9 h. Pre-treatments with 15 microM of the caspase-2 inhibitor benzyloxycarbonyl (z)-Val-Asp-Val-Ala-Asp (VDVAD)-fluoromethyl ketone (fmk) totally block DAU- and TT13-induced caspase-2, -8 and -9 activities, whereas pre-treatments with 15 microM of the caspase-8 inhibitor z-Ile-Glu-Thr-Asp (IETD)-fmk prevent DAU and TT13 from inducing caspase-8 activities without affecting their caspase-2- and -9-inducing activities, suggesting that the induction of apical caspase-2 activity by these drugs may be a critical upstream event required for the activation of other downstream caspases, including caspase-9 and the mitochondrial amplification loop through caspase-8. However, the mechanisms by which DAU and TT13 induce the release of mitochondrial Cyt c appear to be caspase-independent since they are both insensitive to similar pre-treatments with 100 microM of these specific caspase-2 and -8 inhibitors. Moreover, pre-treatments with 10 microg/ml of the antagonistic anti-Fas DX2 and ZB4 monoclonal antibodies (mAbs), and the neutralizing anti-Fas ligand (FasL) NOK-1 mAb are all unable to prevent DAU and TT13 from inducing Cyt c release and caspase-2, -8 and -9 activities, suggesting that the Fas-FasL signaling pathway is not involved in the mechanism by which these quinone antitumor drugs trigger apoptosis in HL-60 cells.     

Beta-D-xylopyranosyl-(1-->3)-beta-D-glucuronopyranosyl echinocystic acid isolated from the roots of Codonopsis lanceolata induces caspase-dependent apoptosis in human acute promyelocytic leukemia HL-60 cells

We previously demonstrated that beta-D-xylopyranosyl-(1-->3)-beta-D-glucuronopyranosyl echinocystic acid (codonoposide 1c), a biologically active compound isolated from the roots of Codonopsis lanceolata, is cytotoxic to cancer cells. In the present study, we investigated the effects of codonoposide 1c on the induction of apoptosis, and its putative action pathway in HL-60 human promyelocytic leukemia cells. Codonoposide 1c-treated HL-60 cells displayed several features of apoptosis, including DNA fragmentation, formation of DNA ladders by agarose gel electrophoresis, and externalization of annexin-V targeted phosphatidylserine (PS) residues. We observed that codonoposide 1c caused activation of caspase-8, caspase-9, and caspase-3. A broad caspase inhibitor (z-VAD-fmk), caspase-8 inhibitor (z-IETD-fmk), and caspase-3 inhibitor (z-DEVD-fmk) almost completely suppressed codonoposide 1c-induced DNA fragmentation. We further found that codonoposide 1c induces mitochondrial translocation of Bid from cytosol, reduction of cytosolic Bax, and cytochrome c release from mitochondria. Interestingly, codonoposide 1c also triggered the mitochondrial release of Smac/DIABLO (second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with a low isoelectric point) into cytosol, and a reduction in X-linked inhibitor of apoptosis protein (XIAP). Taken together, our data indicate that codonoposide 1c is a potent inducer of apoptosis and facilates its activity via Bid cleavage and translocation to mitochondria, Bax reduction in cytosol, release of cytochrome c and Smac/DIABLO into the cytosol, and subsequently caspase activation, providing a potential mechanism for the cytotoxic activity of codonoposide 1c.     

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.     

Induction of apoptosis by 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol from Isaria japonica Yasuda through intracellular reactive oxygen species formation and caspase-3 activation in human leukemia HL-60 cells.

Recently we have reported that the trichothecene mycotoxin 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol (AETD) from the fruiting bodies of Isaria japonica Yasuda is a potent inducer of apoptosis in human promyelocytic HL-60 cells. The present study aims to characterize the molecular events leading to AETD-induced apoptosis in HL-60 cells. The percentage of apoptotic cells (annexin-V-positive cell population) increased dose- and time-dependently after AETD exposure. Apoptosis of HL-60 cells by AETD was associated with the formation of intracellular reactive oxygen species (ROS), the depletion of intracellular glutathione (GSH) and the activation of caspase-3. Pretreating the cells with the antioxidant N-acetyl-L-cystein (NAC) and the caspase-3 inhibitor Z-DEVD-fmk abrogated AETD-induced apoptosis and caspase-3 activation. NAC blocked intracellular ROS formation and GSH depletion, but Z-DEVD-fmk did not. These results indicate that AETD induces apoptosis in HL-60 cells by causing intracellular ROS formation and GSH depletion followed by the downstream event of caspase-3 activation.     

Mechanism of apoptosis in HL-60 cells induced by n-3 and n-6 polyunsaturated fatty acids.

The biochemical properties and specificity of n-3 and n-6 polyunsaturated fatty acids (PUFAs) are not well known. Because PUFAs induce apoptosis of different cells, we studied the effect of various PUFAs, such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosapentaenoic acid (DPA), on the fate of cultured human promyelocytic leukemia cells (HL-60) to elucidate the mechanism of apoptosis and the difference in action between n-3 and n-6 PUFAs. Fairly low concentrations of PUFAs inhibited the growth of HL-60 cells and induced their apoptosis by a mechanism that is sensitive to DMSO, an antioxidant, and z-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor. PUFAs stimulated the generation of reactive oxygen species (ROS) and activated various types of caspase-like proteases, such as caspase-3, -6, -8, and -9, but not caspase-1. In addition, PUFAs triggered the reaction leading to the cleavage of Bid, a death agonist member of the Bcl-2 family, and also released cytochrome c from mitochondria into the cytosol. PUFAs also decreased the mitochondrial membrane potential of intact HL-60 cells. All of these actions of n-3 PUFAs were stronger than those of AA, an n-6 PUFA, although the mechanism is not known. PUFAs stimulate swelling and membrane depolarization of isolated mitochondria in a cyclosporin A-sensitive manner. The results indicated that PUFA-induced apoptosis of HL-60 cells may be caused, in part, by direct action on the cells and by activation of the caspase cascade through cytochrome c release coupled with mitochondrial membrane depolarization.     

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.     

Riccardin D, a novel macrocyclic bisbibenzyl, induces apoptosis of human leukemia cells by targeting DNA topoisomerase II

We studied the effect of riccardin D, a macrocyclic bisbibenzyl, which was isolated from the Chinese liverwort plant, on human leukemia cells and the underlying molecular mechanism. Riccardin D had a significant antiproliferative effect on human leukemia cell lines HL-60, K562 and its multidrug resistant (MDR) counterpart K562/A02 cells, but showed no effect on the topoisomerase-II-deficient HL-60/MX2 cells, as measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The pBR322 DNA relaxation assay revealed that riccardin D selectively inhibited the activity of topoisomerase II (topo II). The suppression of topo II activity by riccardin D was stronger than that of etoposide, a known topo II inhibitor. After treatment with riccardin D, nuclear extracts of leukemia K562 and K562/A02 cells left the majority of pBR322 DNA in a supercoiled form. Further examination showed that riccardin D effectively induced HL-60, K562 and K562/A02 apoptosis as evidenced by externalization of phosphatidylserine and formation of DNA ladder fragments. The activation of cytochrome c, caspase-9, caspase-3 and cleaved poly ADP-ribose polymerase (PARP) was also enhanced, as estimated by Western blot analysis. By contrast, riccardin D was unable to induce apoptosis in the topoisomerase-II-deficient HL-60/MX2 cells, indicating that the induction of apoptosis by riccardin D was due to the inhibition of topo II activity. In addition, riccardin D was able to significantly decrease P-glycoprotein (P-gp) expression in K562/A02 cells. Taken together, our data demonstrate that riccardin D is a novel DNA topo II inhibitor which can induce apoptosis of human leukemia cells and that it has therapeutic potential for both regular and MDR strains of leukemia cells.     

Trimidox induces apoptosis via cytochrome c release in NALM-6 human B cell leukaemia cells

Trimidox (3,4,5-trihydroxybenzamidoxime) has been shown to reduce the activity of ribonucleotide reductase accompanied by growth inhibition and the differentiation of mammalian cells. Here we examine the induction of apoptosis by trimidox in several human leukaemia cell lines, focusing on the release of cytochrome c and the activation of caspase proteases in the human B cell line NALM-6. Induction of apoptosis by trimidox (300 microM) was detected in NALM-6, HL-60 (premyelocytic leukaemia cells), MOLT-4 (an acute lymphoblastic leukaemia cells), Jurkat (a T-cell leukaemia cells), U937 (expressing many monocyte-like characteristics), and K562 (erythroleukaemia). NALM-6 was most affected by trimidox among leukaemia cells; therefore, we employed NALM-6 cells in the subsequent experiments. The cells showed a time-dependent increase in DNA damage after trimidox (250 microM) treatment. A significant increase in the amount of cytochrome c release was detected after treatment with trimidox. Bcl-2 and Bax protein expressions were not changed by trimidox. Caspase-3 and -9 were activated by incubation with trimidox, whereas caspase-8 was not. Furthermore, trimidox-induced apoptosis was prevented by a broad-spectrum caspase inhibitor, a caspase-3, and a caspase-9 inhibitor, but not by a caspase-8 inhibitor. Inhibition of c-Jun NH2-terminal kinase (JNK) by SP600125 appreciably protected cells from trimidox-induced apoptosis, but no effect inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580. In contrast, extracellular signal-regulated kinase (ERK) inhibitors U0126 and PD98059 strongly potentiated the apoptotic effect of trimidox. This report shows that the induction of apoptosis by trimidox occurs through a cytochrome c-dependent pathway, which sequentially activates caspase-3 and caspase-9.     

Induction of caspase-3-dependent apoptosis in human leukemia HL-60 cells by δ-elemene

δ-Elemene, an antitumor component, is a chemical compound isolated from Curcuma wenyujin, a Chinese traditional herb. We examined whether δ-elemene could inhibit cell growth and cell cycle progression and induce apoptosis in human leukemia HL-60 cells. The results demonstrated that δ-elemene induces significant apoptosis of HL-60 cells, as shown by MTT assay, annexin V (AnV) binding of externalized phosphatidylserine (PS), and the mitochondrial probe JC-1 using flow cytometry. HL-60 cells treated with δ-elemene showed high percentages in the early apoptotic and late apoptoctic/necrotic stages, as well as caspase-3 activation of HL-60 cells. By monitoring the changes in cell cycle profiles, we confirmed that δ-elemene could interfere with the cell cycle in the G2/M phase and induce apoptosis in HL-60 cells in a time-dependent manner. Caspase-3 plays a direct role in proteolytic cleavage of the cellular proteins responsible for progression to apoptosis. Therefore we examined apoptosis in HL-60 cells after exposure to δ-elemene and measured caspase-3 activities with or without Z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk, a broad-spectrum caspase inhibitor) pretreatment using flow cytometric analysis. The results showed that δ-elemene could induce caspase-3 activation as detected by the decrease in δ-elemene-induced caspase-3 activities after treatment with z-VAD-fmk. In the present study, δ-elemene activated typical caspase-dependent apoptosis in HL-60 cells, as demonstrated by an inhibitory effect of z-VAD-fmk on this cell death. During δ-elemene-induced apoptosis, cytochrome c and apoptosis-inducing factor were released into the cytosol and BAX was translocated from the cytosol to mitochondria. However, these were not prevented by z-VAD-fmk. In conclusion, our study demonstrated that δ-elemene could induce G2/M cell cycle transition and trigger apoptosis through a caspase-3-dependent pathway.     

Cytotoxic and apoptogenic effect of Swietenia mahagoni (L.) Jacq. leaf extract in human leukemic cell lines U937, K562 and HL-60

The apoptogenic activity of Swietenia mahagoni leaf extract (SMLE) was investigated against three human leukemic cell lines – U937, K562 and HL-60. SMLE inhibited cell growth and metabolic activity of the leukemic cells and showed characteristic features of apoptosis. Flow-cytometric analysis showed that SMLE arrested U937 and K562 cell populations in the G2-M phase and the HL-60 cell population in the G1 phase of cell cycle. SMLE induced apoptosis was found to be mediated through mitochondrial intrinsic pathway involving the release of cytochrome c into the cytosol and activation of caspase-9 and caspase-3. Two flavonoids, catechin and quercetin-3-O-glucoside, isolated from SMLE, were found to inhibit the growth and metabolic activity of U937, K562 and HL-60 cells at much lower concentrations thus indicating that these two flavonoids might be the active ingredients responsible for the anti-leukemic activity of SMLE.     

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.     

Apoptosis-inducing active components from Corbicula fluminea through activation of caspase-2 and production of reactive oxygen species in human leukemia HL-60 cells.

The anti-cancer effects and possible mechanisms of the freshwater clam (Corbicula fluminea Muller) and its active compounds (FME) on cell viability in human leukemia HL-60 cells were investigated. This study demonstrated that FME was able to inhibit cell proliferation in a concentration- and time-dependent manner. Treatment with FME caused induction of caspase-2, caspase-3, caspase-6, caspase-8, and caspase-9 activity in a time-dependent manner, but not affect caspase-1 activity; it induced the proteolysis of DNA fragmentation factor (DFF-45) and poly(ADP-ribose) polymerase (PARP). Induction of cell death by FME was completely prevented by a pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) and a caspase-2 inhibitor, Z-Val-Asp-Val-Ala-Asp-FMK (Z-VDVAD-FMK). Furthermore, treatment with FME caused a rapid loss of mitochondrial transmembrane potential, stimulation of generation of reactive oxygen species (ROS), release of mitochondrial cytochrome c into cytosol, and GSH depletion. Anti-oxidants such as N-acetylcysteine, catalase, superoxide dismutase, allopurinol, and pyrrolidine dithiocarbamate, but not diphenylene iodonium, significantly inhibited FME-induced cell death. In addition, the results showed that FME-induced apoptosis was accompanied by up-regulation of Bax and Bad, and down-regulation of Bcl-2 and Bcl-XL. Taken together, induction of apoptosis on HL-60 cells by FME was mainly associated with ROS production, GSH depletion, mitochondrial dysfunction, and caspase activation.