benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Leukemia--Promyelocytic--Acute

In our quest to develop an isoindigo with improved efficacy and bioavailability, we recently synthesized a series of novel substituted pyridone-annelated isoindigo and evaluated their antiproliferative effects. We identified the compound [(E)-1-(5'-Chloro-2'-oxoindolin-3'-ylidene)-6-ethyl-2,3,6,9-tetrahydro-2,9-dioxo-1H-pyrrolo[3,2-f] quinoline-8-carboxylic acid], abbreviated as 5'-Cl, which shows selective antiproliferative activities against various cancer cell lines mediated through apoptosis. Here we have investigated the molecular mechanisms underlying the apoptotic activity of 5'-Cl in the human promyelocytic leukemia HL-60 cells.. We employed different methods to determine the apoptotic pathways triggered by 5'-Cl in HL-60 cells, using flow cytometry, nuclear staining, caspases activation, mitochondria functioning, generation of reactive oxygen species (ROS) and Western blotting techniques.. Low concentrations (1-8 µM) of 5'-Cl inhibited the growth of HL-60 cells in a dose and time-dependent manner. Cytotoxicity of this compound is found to be mediated by a caspase-dependent apoptosis. Also, there were indications of caspase independent apoptosis as z-VAD-FMK failed to fully rescue the cells from 5'-Cl-induced apoptosis. In addition, the compound triggered generation of Reactive Oxygen Species (ROS), caused depolarization of the mitochondrial inner membrane, decreased the level of cellular ATP, modulated the expression and phosphorylation of Bcl-2 leading to loss of its association with Bax and increased the release of cytochrome c to the cytosol of treated cells. The effects of 5'-Cl on mitochondria and apoptosis were substantially blocked in the presence of a combination between z-VAD-FMK and either of the ROS scavenger N-acetyl-L-cysteine (NAC) or pyrrolidine dithiocarbamate (PDTC).. We demonstrated that the growth inhibitory effects of 5'-Cl in HL-60 cells involve multiple pathways of apoptosis and dysregulation of mitochondrial functions.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cytochromes c; HL-60 Cells; Humans; Indoles; Leukemia, Promyelocytic, Acute; Membrane Potential, Mitochondrial; Mitochondria; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Pyridones; Pyrrolidines; Reactive Oxygen Species; Thiocarbamates

The block of hematopoietic differentiation program in acute myeloid leukemia cells can be overcome by differentiating agent like retinoic acid, but it has several side effects. A study of other differentiation signaling pathways is therefore useful to predict potential targets of anti-leukemic therapy. We demonstrated previously that the co-treatment of HL-60 cells with Tumor necrosis factor-alpha (TNF-alpha) (1 ng/mL) and inhibitor of 5-lipoxygenase MK886 (5 microm) potentiated both monocytic differentiation and apoptosis. In this study, we detected enhanced activation of three main types of mitogen-activated protein kinases (MAPKs) (p38, c-Jun amino-terminal kinase [JNK], extracellular signal-regulated kinase [ERK]), so we assessed their role in differentiation using appropriate pharmacologic inhibitors. The inhibition of pro-apoptotic MAPKs (p38 and JNK) suppressed the effect of MK886 + TNF-alpha co-treatment. On the other hand, down-regulation of pro-survival ERK pathway led to increased differentiation. Those effects were accompanied by increased activation of caspases in cells treated by MK886 + TNF-alpha. Pan-caspase inhibitor ZVAD-fmk significantly decreased both number of apoptotic and differentiated cells. The same effect was observed after inhibition of caspase 9, but not caspase 3 and 8. To conclude, we evidenced that the activation of apoptotic processes and pathways supporting apoptosis (p38 and JNK MAPKs) is required for the monocytic differentiation of HL-60 cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Caspases; Cell Differentiation; Cysteine Proteinase Inhibitors; Enzyme Activation; HL-60 Cells; Humans; Indoles; Leukemia, Promyelocytic, Acute; Lipoxygenase Inhibitors; MAP Kinase Signaling System; Monocytes; NF-kappa B; Tumor Necrosis Factor-alpha

Patulin (PAT) is a fungal secondary metabolite that exhibits potential cellular and animal toxicities. In this study, human promyelocytic leukemia (HL-60) cells were used to elucidate the mechanism and death mode associated with PAT. Morphological evidence of apoptosis, including membrane blebbing, nuclei fragmentation and DNA laddering formation was clearly observed 6h after exposure to PAT. The results of Western blotting indicated that PAT activated various processed caspases, and cleaved DFF45 and poly (ADP-ribose) polymerase (PARP) in a dose-dependent manner; it also induced a time-dependent increase in caspase 3 and 9 catalytic activities. The apoptosis mediated by PAT in HL-60 was accompanied with cytochrome c release from mitochondria and Bcl-2 expression decrease. The presence of thiol-containing compounds with PAT dramatically reduced the caspase 3 activity that was triggered by PAT; the addition of antioxidants, including mannitol and Tiron, had a similar effect. However, the suppression of p53 protein expression by RNA interference (RNAi) in human embryonic kidney (HEK293) cells did not significantly modify PAT-elicited caspase 3 activity. These findings suggest that PAT-induced apoptosis is mediated through the mitochondrial pathway without the involvement of p53; the interaction with sulfhydryl groups of macromolecules by PAT and the subsequent generation of reactive oxygen species (ROS) plays a primary role in the apoptotic process.

Topics: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Caspase 3; Caspase 9; Caspase Inhibitors; Cell Survival; Cysteine Proteinase Inhibitors; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Flow Cytometry; Free Radical Scavengers; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Mannitol; Microscopy, Phase-Contrast; Molecular Structure; Mycotoxins; Patulin; Time Factors; Tumor Suppressor Protein p53

We have investigated the effects of glycyrrhizin (GL) on cell proliferations of human stomach cancer KATO III and promyelotic leukemia HL-60 cells, and on DNA of those cell lines. GL displayed growth inhibitory effect against KATO III and HL-60 cells. Morphological change showing apoptotic bodies was observed in the KATO III and HL-60 cells treated with GL. The fragmentation of DNA by GL to oligonucleosomal-sized fragments that is a characteristic of apoptosis was observed to be concentration- and time-dependent in both cell lines. Caspase inhibitors such as Z-VAD-FMK and Z-Asp-CH2-DCB suppressed the DNA fragmentation induced by GL. The data of the present study show that the suppression of KATO III and HL-60 cell-growth by GL results from the induction of apoptosis by GL, and that caspase is involved in the induction of apoptosis by GL in these cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Aspartic Acid; Caspase Inhibitors; Cell Line, Tumor; Cysteine Proteinase Inhibitors; DNA Fragmentation; Dose-Response Relationship, Drug; Glycyrrhizic Acid; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Oligopeptides; Stomach Neoplasms; Time Factors

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in a variety of cells, but the mechanism of this effect has not been fully elucidated. We report that diclofenac, a NSAID, induces growth inhibition and apoptosis of HL-60 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS), Akt, caspase-8, and Bid. ROS generation occurs in an early stage of diclofenac-induced apoptosis preceding cytochrome c release, caspase activation, and DNA fragmentation. N-Acetyl-L-cysteine, an antioxidant, suppresses ROS generation, Akt inactivation, caspase-8 activation, and DNA fragmentation. Cyclic AMP, an inducer of Akt phosphorylation, suppresses Akt inactivation, Bid cleavage, and DNA fragmentation. LY294002, a PI3 kinase inhibitor, enhances Akt inactivation and DNA fragmentation. Ac-IETD-CHO, a caspase-8 inhibitor, suppresses Bid cleavage and DNA fragmentation. z-VAD-fmk, a universal caspase inhibitor, but not cyclosporin A (CsA), an inhibitor of mitochondrial membrane permeability transition, suppresses DNA fragmentation. These results suggest the sequential mechanism of diclofenac-induced apoptosis of HL-60 cells: ROS generation suppresses Akt activity, thereby activating caspase-8, which stimulates Bid cleavage and induces cytochrome c release and the activation of caspase-9 and-3 in a CsA-insensitive mechanism. Furthermore, we found that 2-methoxyestradiol (2-ME), a superoxide dismutase inhibitor, significantly enhances diclofenac-induced apoptosis; that is, diclofenac combined with 2-ME may have therapeutic potential in the treatment of human leukemia.

Topics: 2-Methoxyestradiol; Acetylcysteine; Amino Acid Chloromethyl Ketones; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspases; Chromones; Cyclic AMP; Cyclosporine; Cysteine Proteinase Inhibitors; Cytochromes c; Diclofenac; DNA Fragmentation; DNA, Neoplasm; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Estradiol; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Morpholines; Oligopeptides; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; Superoxide Dismutase

A metal chelator, diphenylthiocarbazone (dithizone), has been reported to induce differentiation and apoptosis of the human myeloid leukemia cell line HL-60, however, very little is known about the mechanism of dithizone-induced apoptosis. Here, we report for the first time that dithizone can induce inhibition of cellular growth of retinoic acid (RA)-sensitive NB4 and RA-resistant UF-1 APL cells via induction of apoptosis but not differentiation. Treatment of NB4 cells with dithizone markedly-induced apoptosis, which was associated with the loss of mitochondrial transmembrane potentials (Delta Psi(m)) and activation of caspase-3 and -9. Further investigation of the RA-resistant UF-1 APL cells showed that dithizone-induced apoptosis to a lesser extent. However, neither dithizone alone nor in combination with all-trans RA induced the expression of myeloid differentiation antigen CD11b. Concomitantly, the degradation of PML/RARalpha fusion protein was not observed after treatment with dithizone alone, and the degradation was not enhanced by the combination of dithizone and all-trans RA. We conclude that dithizone, a metal chelator, induced apoptosis without differentiation in APL cells in association with Delta Psi(m) collapse and caspase-3 and -9 activation.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cell Differentiation; Chelating Agents; Cysteine Proteinase Inhibitors; Dithizone; Drug Interactions; Drug Resistance, Neoplasm; Enzyme Activation; Intracellular Membranes; Leukemia, Promyelocytic, Acute; Macrophage-1 Antigen; Membrane Potentials; Mitochondria; Neoplasm Proteins; Neoplastic Stem Cells; Oncogene Proteins, Fusion; Signal Transduction; Tretinoin; Tumor Cells, Cultured

Selective induction of apoptosis in tumor cells is important for treating patients with cancer. Because oxidative stress plays an important role in the process of apoptosis, we studied the effect of alpha-tocopheryl succinate (VES) on the fate of cultured human promyelocytic leukemia cells (HL-60). The presence of fairly low concentrations of VES inhibited the growth and DNA synthesis of HL-60 cells, and also induced their apoptosis via a mechanism that was inhibited by z-VAD-fluoromethylketone (z-VAD-fmk), an inhibitor of pan-caspases. VES activated various types of caspases, including caspase-3, 6, 8, and 9, but not caspase-1. VES triggered the reaction leading to the cleavage of Bid, a member of the death agonist Bcl-2 family, and released cytochrome c (Cyt.c) from the mitochondria into the cytosol by a z-VAD-fmk-inhibitable mechanism. VES transiently increased the intracellular calcium level [Ca2+]i and stimulated the release of Cyt.c in the presence of inorganic phosphate (Pi). However, high concentrations of VES (approximately 100 microM) hardly induced swelling of isolated mitochondria but depolarized the mitochondrial membrane potential by a cyclosporin A (CsA)-insensitive mechanism. These results indicate that VES-induced apoptosis of HL-60 cells might be caused by activation of the caspase cascade coupled with modulation of mitochondrial membrane function.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Caspases; Cell Differentiation; Cell Division; Cysteine Proteinase Inhibitors; DNA; DNA Fragmentation; Enzyme Activation; Humans; Leukemia, Promyelocytic, Acute; Male; Mitochondria; Rats; Rats, Wistar; Reactive Oxygen Species; Tocopherols; Tumor Cells, Cultured; Vitamin E

The synthetic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), which was originally developed as an retinoic acid receptor (RAR)-gamma agonist, induces rapid apoptosis in all-trans retinoic acid (ATRA)-sensitive and ATRA-resistant clones of the NB4 cell line, a widely used experimental model of acute promyelocytic leukemia (APL). In addition, the compound is apoptogenic in primary cultures of freshly isolated APL blasts obtained from a newly diagnosed case and an ATRA-resistant relapsed patient. NB4 cells in the S-phase of the cycle are most sensitive to CD437-triggered apoptosis. CD437-dependent apoptosis does not require de novo protein synthesis and activation of RAR-gamma or any of the other nuclear retinoic acid receptors. The process is preceded by rapid activation of a caspase-like enzymatic activity capable of cleaving the fluorogenic DEVD but not the fluorogenic YVAD tetrapeptide. Increased caspase activity correlates with caspase-3 and caspase-7 activation. Inhibition of caspases by z-VAD suppresses the nuclear DNA degradation observed in NB4 cells treated with CD437, as well as the degradation of pro-caspase-3 and pro-caspase-7. CD437-dependent activation of caspases is preceded by release of cytochrome c from the mitochondria into the cytosol of treated cells. Leakage of cytochrome c lays upstream of caspase activation, because the phenomenon is left unaffected by pretreatment of NB4 cells with z-VAD. Treatment of APL cells with CD437 is associated with a caspase-dependent degradation of promyelocytic leukemia-RAR-alpha, which can be completely inhibited by z-VAD.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Caspases; Cytochrome c Group; Cytosol; Enzyme Activation; Enzyme Inhibitors; Humans; Leukemia, Promyelocytic, Acute; Mitochondria; Neoplasm Proteins; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Retinoids; S Phase; Signal Transduction; Tretinoin

We have investigated whether the increases in ceramide levels that occur during apoptosis in SKW 6.4 cells induced by anti-Fas antibody depend on the activation of caspases. Using cells prelabelled to equilibrium with [14C]acetate, it was shown that the amount of ceramide approximately doubled after 24 h incubation with anti-Fas, but the time course of ceramide changes was slower than that of anti-Fas-induced cell death. Complete inhibition of the effects of anti-Fas on cell death and on ceramide production was observed when the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethane (zVAD.fmk) was added together with anti-Fas, but N-benzyloxycarbonyl-Phe-Ala-fluoromethane (a structurally similar cathepsin B inhibitor) had no effect. Treatment of cells with the Ca2+-ionophore A23187 also doubled ceramide levels, but in this case the effect was complete within 2 h, was not blocked by zVAD.fmk and was not associated with increases in nuclear fragmentation. These results suggest that ceramide is not an upstream messenger in Fas-mediated apoptosis and may instead be produced as a consequence of processes downstream of the activation of caspases and increases in cytosolic calcium concentration.

Topics: Acetates; Amino Acid Chloromethyl Ketones; Antibodies; Apoptosis; Calcimycin; Calcium; Ceramides; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; fas Receptor; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Lymphoma, B-Cell; Signal Transduction; Time Factors; Tumor Cells, Cultured