benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Leukemia--Myeloid

Ethacrynic acid, a diuretic, inhibits glutathione S-transferase P1-1 (GSTP1-1) activity and induces cell death in malignant cells at high concentrations. To improve ethacrynic acid activity, ethacrynic acid oxadiazole analogs 6s and 6u were synthesized. Although both compounds have greater antiproliferative effects than ethacrynic acid in human HL-60 cells, 6u has a reduced ability to inhibit GSTP1-1 activity. The mechanisms of both 6s- and 6u-induced cell death as well as the role of GSTP1-1 in their actions were studied. Both 6s and 6u equally induced apoptosis in HL-60 cells due to the activation of caspase-3, -9, and -8, which was correlated with the downregulation of antiapoptotic proteins c-FLIP, Mcl-1, and XIAP. The caspase inhibitor Z-VAD-FMK blocked the reduction of XIAP, but not of c-FLIP and Mcl-1, in 6s-treated cells. The reduction of c-FLIP and Mcl-1 by 6s was not blocked by the proteasomal inhibitor MG132, but was correlated with inhibition of the phosphorylation of extracellular signal-regulated kinase (ERK) and eIF4E. Both 6s and 6u decreased the intracellular glutathione (GSH) levels. N-acetylcysteine blocked reduction in the levels of Mcl-1, c-FLIP, and intracellular GSH as well as apoptosis in HL-60 cells treated by either compound. Silencing of GSTP1-1 in K562 cells sensitized, but overexpression of GSTP1-1 in Raji cells blocked, apoptosis induction by either compound. GSH conjugation at the methylene group abrogated the ability of inducing apoptosis. These data suggest that the methylene group plays an important role in the downregulation of c-FLIP and Mcl-1 proteins and apoptosis induction, which is inactivated by GSTP1-1 by forming GSH conjugates.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspases; Cell Line, Tumor; Enzyme Inhibitors; Ethacrynic Acid; Gene Expression Regulation, Leukemic; Glutathione; Glutathione S-Transferase pi; HL-60 Cells; Humans; Jurkat Cells; K562 Cells; Leukemia, Myeloid; Leupeptins; Myeloid Cell Leukemia Sequence 1 Protein; Oxadiazoles; Signal Transduction; X-Linked Inhibitor of Apoptosis Protein

Evidence suggests that mistletoe extract has the potential to be used as an anticancer agent. Abnobaviscum F® is a European mistletoe extract from the host tree Fraxinus. We investigated the effect of Abnobaviscum F on the growth and survival of different leukemia cell lines. Abnobaviscum F treatment strongly reduced survival and induced apoptosis of K562 (human myeloid leukemia), RPMI-8226 (human plasmacytoma) and L1210 (murine lymphocytic leukemia) cells in culture. Using K562 cells to further investigate the mechanism of action of Abnobaviscum F, we showed that Abnobaviscum F-induced cell death was associated with the activation of caspase-9, JNK-1/2 and p38 MAPK, as well as with the downregulation of Mcl-1, and inhibition of ERK-1/2 and PKB phosphorylation. Moreover, Abnobaviscum F treatment led to both a reduction of cellular glutathione (GSH) and the induction of ER stress (GRP78 and CHOP induction and eIF-2α phosphorylation). By contrast, Abnobaviscum F did not impact the expression of the DR4 and DR5 death receptors. The Abnobaviscum F-induced apoptosis of K562 cells was blocked by pretreatment with either GSH, z-VAD-fmk or SP600125. Our results, therefore, show that Abnobaviscum F induces apoptosis of K562 cells through the activation of the intrinsic caspase pathway, the phosphorylation of JNK-1, the reduction of cellular GSH, and the induction of ER stress.

Topics: Amino Acid Chloromethyl Ketones; Animals; Anthracenes; Apoptosis; Caspase 9; Cell Line, Tumor; DNA Fragmentation; Down-Regulation; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Extracellular Signal-Regulated MAP Kinases; Glutathione; Heat-Shock Proteins; Humans; JNK Mitogen-Activated Protein Kinases; K562 Cells; Leukemia, Lymphoid; Leukemia, Myeloid; Mice; Myeloid Cell Leukemia Sequence 1 Protein; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Plant Extracts; Plasmacytoma; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Receptors, TNF-Related Apoptosis-Inducing Ligand; Transcription Factor CHOP; Viscum album

Topics: Acute Disease; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Benzamides; Caspases; Cell Death; Cell Line, Tumor; Diphtheria Toxin; Drug Delivery Systems; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Activation; Humans; Interleukin-3; Leukemia, Myeloid; Neoplasm Proteins; Pyridines; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Receptors, Interleukin-3; Recombinant Fusion Proteins; TNF-Related Apoptosis-Inducing Ligand; U937 Cells

The mechanism of the cytotoxic effect of boswellic acid acetate, a 1:1 mixture of alpha-boswellic acid acetate and beta-boswellic acid acetate, isolated from Boswellia carterri Birdw on myeloid leukemia cells was investigated in six human myeloid leukemia cell lines (NB4, SKNO-1, K562, U937, ML-1, and HL-60 cells). Morphologic and DNA fragmentation assays indicated that the cytotoxic effect of boswellic acid acetate was mediated by induction of apoptosis. More than 50% of the cells underwent apoptosis after treatment with 20 mug/mL boswellic acid for 24 hours. This apoptotic process was p53 independent. The levels of apoptosis-related proteins Bcl-2, Bax, and Bcl-XL were not modulated by boswellic acid acetate. Boswellic acid acetate induced Bid cleavage and decreased mitochondrial membrane potential without production of hydrogen peroxide. A general caspase inhibitor (Z-VAD-FMK) and a specific caspase-8 inhibitor II (Z-IETD-FMK) blocked boswellic acid acetate-induced apoptosis. The mRNAs of death receptors 4 and 5 (DR4 and DR5) were induced in leukemia cells undergoing apoptosis after boswellic acid acetate treatment. These data taken together suggest that boswellic acid acetate induces myeloid leukemia cell apoptosis through activation of caspase-8 by induced expression of DR4 and DR5, and that the activated caspase-8 either directly activates caspase-3 by cleavage or indirectly by cleaving Bid, which in turn decreases mitochondria membrane potential.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Northern; Blotting, Western; Caspase 3; Caspases; Cell Line, Tumor; Cell Proliferation; DNA Fragmentation; HL-60 Cells; Humans; Hydrogen Peroxide; Inhibitory Concentration 50; K562 Cells; Leukemia, Myeloid; Membrane Potentials; Mitochondria; Models, Biological; Models, Chemical; Oligopeptides; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; RNA, Messenger; Triterpenes; U937 Cells

We reported previously that alpha-tocopheryl-succinate (VES) induced apoptosis of cultured human promyelocytic leukemia cells (HL-60) (Free Radic Res 2000;33:407-18). We have now studied the effect of cholesteryl-hemisuccinate (CS) on the fate of HL-60 cells to clarify whether CS has an effect similar to that of VES. CS inhibited the growth of HL-60 cells without differentiation to granulocytes and induced DNA fragmentation and ladder formation. CS inhibited the phosphorylation of pleckstrin homology domain-containing protein kinase B (Akt) and initiated the activation of a caspase cascade. CS triggered the reaction leading to the cleavage of Bid and also released cytochrome c (Cyt. c) from mitochondria. In addition, CS induced mitochondrial membrane depolarization and translocation of Bax to mitochondria in HL-60 cells. However, CS did not induce an increase in the concentration of intracellular calcium ions in HL-60 cells. The membrane depolarization, Cyt. c release, and DNA fragmentation were inhibited by z-VAD-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor, but not by cyclosporin A, an inhibitor of membrane permeability transition. These results suggested that CS-induced apoptosis of HL-60 cells might be caused by inhibiting Akt phosphorylation following cleavage of Bid through caspase-8 activation and subsequently via an Apaf complex-caspase cascade pathway.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Calcium; Carrier Proteins; Caspases; Cell Cycle; Cell Differentiation; Cell Division; Cholesterol Esters; Cyclic AMP; Cyclosporine; Cytochrome c Group; DNA Fragmentation; Enzyme Activation; Enzyme Inhibitors; HL-60 Cells; Humans; Leukemia, Myeloid; Membrane Potentials; Mitochondria; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2

The mechanisms of As(2)O(3)-induced apoptosis are very complex. In the present study, we investigated the molecular mechanism of As(2)O(3) in vitro at low concentration (0.25-2.0 micro M) on three human leukemia/lymphoma cell lines: HL-60, RL and K562. As(2)O(3) inhibited the growth of these cell lines significantly. During As(2)O(3) treatment, two forms of cell death, apoptosis in HL-60 and RL and oncosis in K562, were found by morphological study. In HL-60 and RL, cell cycle analysis showed, at a distinct SubG1 region, that CD95 and CD95 ligand (CD95L) expression was upregulated, caspase 8 and caspase 3 were activated, and Bcl-2 protein expression was downregulated. On the other hand, in K562, the cell cycle was arrested at the G2+M phase, CD95/CD95L expression was upregulated, caspase 8 and caspase 3 were activated, but Bcl-2 expression was not changed as compared with untreated cells. These findings suggest that the CD95/CD95L pathway is involved in cell killing by As(2)O(3). Using anti-CD95 IgG monoclonal antibody (anti-CD95 MoAb) or specific caspase inhibitor ZVAD-fmk to block the CD95 pathway, the cell death induced by As(2)O(3) was partially blocked in each cell line. These results suggest that As(2)O(3) inhibits the growth of these leukemia/lymphoma cell lines by inducing apoptosis or oncosis that is partially mediated by the CD95/CD95L pathway.

Topics: Acute Disease; Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Division; Cysteine Proteinase Inhibitors; Down-Regulation; Fas Ligand Protein; fas Receptor; HL-60 Cells; Humans; K562 Cells; Leukemia, Myeloid; Membrane Glycoproteins; Oxides; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Up-Regulation

Human myeloid leukemia cells respond to 12-tetradecanoylphorbol-13-acetate (TPA) and other activators of protein kinase C (PKC) with the induction of terminal monocytic differentiation. The present studies demonstrate that TPA treatment of U-937 leukemia cells is associated with release of mitochondrial cytochrome c, activation of caspase-3 and induction of internucleosomal DNA fragmentation. By contrast, the TUR cell variant, which is deficient in PKCbeta, failed to respond to TPA with release of cytochrome c and induction of the caspase-3 cascade. Moreover, stable overexpression of PKCbeta in TUR cells reconstituted sensitivity to TPA-induced cytochrome c release and activation of caspase-3. The results also demonstrate that treatment of cells with the caspase inhibitor Z-VAD-fmk blocks both TPA-induced apoptosis and monocytic differentiation. Similar results were obtained in U-937 cells stably expressing the CrmA caspase inhibitor. These findings demonstrate that TPA induces cytochrome c release by a PKCbeta-dependent mechanism and that activation of caspase-mediated signaling is required for induction of the differentiated monocytic phenotype.

Topics: Amino Acid Chloromethyl Ketones; Caspase 1; Caspase 3; Caspase Inhibitors; Caspases; Cell Differentiation; Cysteine Proteinase Inhibitors; Cytochrome c Group; Enzyme Activation; Humans; Isoenzymes; Leukemia, Myeloid; Mitochondria; Protein Kinase C; Protein Kinase C beta; Serpins; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Viral Proteins

Nonsteroidal antiinflammatory agents (NSAIA) have been shown to exert potent chemopreventive activity against colon, lung, and breast cancers. In this study, we show that at pharmacological concentrations (1 to 3 mmol/L) sodium salicylate (Na-Sal) can potently induce programmed cell death in several human myeloid leukemia cell lines, including TF-1, U937, CMK-1, HL-60, and Mo7e. TF-1 cells undergo rapid apoptosis on treatment with Na-Sal, as indicated by increased annexin V binding capacity, cpp-32 (caspase-3) activation, and cleavage of poly (ADP-ribose) polymerase (PARP) and gelsolin. In addition, the expression of MCL-1, an antiapoptotic member of the BCL-2 family, is downregulated during Na-Sal-induced cell death, whereas the expression of BCL-2, BAX, and BCL-XL is unchanged. Z-VAD, a potent caspase inhibitor, prevents the cleavage of PARP and gelsolin and rescues cells from Na-Sal-induced apoptosis. In addition, we show that Na-Sal accelerates growth factor withdrawal-induced apoptosis and synergizes with daunorubicin to induce apoptosis in TF-1 cells. Thus, our data provide a potential mechanism for the chemopreventive activity of NSAIA and suggest that salicylates may have therapeutic potential for the treatment of human leukemia.

Topics: Amino Acid Chloromethyl Ketones; Annexin A5; Anti-Inflammatory Agents, Non-Steroidal; Anticarcinogenic Agents; Apoptosis; Caspase 3; Caspases; Cysteine Proteinase Inhibitors; Daunorubicin; Drug Synergism; Enzyme Activation; Gelsolin; Gene Expression Regulation, Leukemic; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cells; Humans; K562 Cells; Leukemia, Myeloid; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Poly(ADP-ribose) Polymerases; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-bcl-2; Sodium Salicylate; Tumor Cells, Cultured

The primary objective of this study was to determine whether caspases are involved in arsenic trioxide(ATO)-induced apoptosis of human myeloid leukemia cells. A secondary objective was to determine whether apoptosis induced by ATO compared with VP-16 is differentially affected by an activator of protein kinase C (PKC), phorbol 12-myristate 13-acetate (PMA), which has been reported to inhibit apoptosis induced by some chemotherapeutic agents. NB4 and HL60 cells were incubated with ATO in the presence and absence of the caspase protease inhibitors Z-VAD.fmk or Y-VAD.cho. Apoptosis was assessed by morphology, DNA laddering and flow cytometry. Poly (ADP-ribose) polymerase (PARP) cleavage was used as a marker for the activation of caspases. PARP cleavage occurred during ATO-induced apoptosis in both NB4 and HL60 cells. Z-VAD.fmk, a broad-spectrum inhibitor, could block ATO-induced apoptosis and PARP cleavage, whilst Y-VAD.cho, a selective inhibitor of caspase 1, had no such effect. PMA pre-incubation for up to 8 hours under conditions known to activate PKC had no effect on either ATO- or VP-16-induced apoptosis. We conclude that in cultured myeloid leukemia cells ATO-induced apoptosis is executed by caspases from the distal, PARP-cleaving part of the activation cascade and that PKC activation has no effect on apoptosis induced by either ATO or VP-16 in these cells.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Caspase Inhibitors; Caspases; Enzyme Activation; Etoposide; Humans; Leukemia, Myeloid; Oxides; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

P39/Tsugane is a myelomonocytoid cell line derived from a patient with myelodysplastic syndrome (MDS). The cells readily undergo apoptosis in response to various agents, and the cell line has been suggested as a useful model to study apoptosis in MDS. The aims of the present study were to assess differentiation and apoptosis induced with all-trans retinoic acid (ATRA) and etoposide, to characterize the mode of apoptosis in these two model systems, and to assess the influence of granulocyte colony-stimulating factor (G-CSF), which in combination with erythropoietin has been shown to inhibit apoptosis in MDS. ATRA induced differentiation and apoptosis in a concentration- and time-dependent manner. Differentiated cells were partially rescued (by 50%) from apoptosis with G-CSF. Etoposide induced apoptosis in a concentration- and time-dependent manner, but no signs of preceding maturation or G-CSF rescue were detected. ATRA- and etoposide-induced apoptosis were both mediated through the caspase pathway and were partially blocked with the general caspase inhibitor zVAD-fmk. Simultaneous treatment with G-CSF and zVAD-fmk additively blocked ATRA-induced apoptosis. However, the two pathways differed in terms of substrate cleavage during apoptosis. ATRA-induced apoptosis caused actin cleavage, which was not affected by G-CSF, and Bcl-2 downregulation. Etoposide induced a caspase-dependent cleavage of Bcl-2, while actin remained intact. The Fas system did not seem to play a major role in any of these apoptotic pathways. Our results may provide new tools to study the mechanisms of apoptosis in MDS.

Topics: Actins; Acute Disease; Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Apoptosis; Blast Crisis; Caspase Inhibitors; Caspases; Cell Differentiation; Cysteine Proteinase Inhibitors; Cytoskeleton; Erythropoietin; Etoposide; fas Receptor; Granulocyte Colony-Stimulating Factor; Humans; Leukemia, Myeloid; Myelodysplastic Syndromes; Neoplasm Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tretinoin; Tumor Cells, Cultured