cytochrome-c-t and Leukemia--T-Cell

Ceramide, a biologically active sphingolipid in cell death signaling, accumulates upon CD95L treatment, concomitantly to apoptosis induction in Jurkat leukemia T cells. Herein, we show that ceramide did not increase in caspase-8 and -10-doubly deficient Jurkat cells in response to CD95L, indicating that apical caspases are essential for CD95L-triggered ceramide formation. Jurkat cells are typically defined as type 2 cells, which require the activation of the mitochondrial pathway for efficient apoptosis induction in response to CD95L. Caspase-9-deficient Jurkat cells significantly resisted CD95L-induced apoptosis, despite ceramide accumulation. Knock-down of sphingomyelin synthase 1, which metabolizes ceramide to sphingomyelin, enhanced (i) CD95L-triggered ceramide production, (ii) cytochrome c release from the mitochondria and (iii) caspase-9 activation. Exogenous ceramide-induced caspase-3 activation and apoptosis were impaired in caspase-9-deficient Jurkat cells. Conversely, caspase-9 re-expression in caspase-9-deficient Jurkat cells restored caspase-3 activation and apoptosis upon exogenous ceramide treatment. Collectively, our data provide genetic evidence that CD95L-triggered endogenous ceramide increase in Jurkat leukemia T cells (i) is not a mere consequence of cell death and occurs mainly in a caspase-9-independent manner, (ii) is likely involved in the pro-apoptotic mitochondrial pathway leading to caspase-9 activation.

Topics: Apoptosis; Blotting, Western; Caspase 9; Cell Survival; Ceramides; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Fas Ligand Protein; Flow Cytometry; Gene Knockdown Techniques; Humans; Jurkat Cells; Leukemia, T-Cell; Membrane Proteins; Microscopy, Fluorescence; Mutation; Nerve Tissue Proteins; Transferases (Other Substituted Phosphate Groups)

The dried spikes of Prunella vulgaris var. lilacina (Labiatae) have been used for traditional herbal medicine to treat fever, inflammation, dropsy, gonorrhea and cancer in Korea, Japan and China. The present study evaluated the apoptotic effect of 2α,3α-dihydroxyurs-12-en-28-oic acid (DHURS), purified from the dried spikes on human acute leukemia Jurkat T cells.. Cell viability was assessed by MTT assay. Mitochondrial membrane potential (Δψm) loss, apoptotic change of the cell cycle, and apoptotic cells were measured by flow cytometric analysis. Mitochondrial cytochrome c release and activation of caspase cascade were determined by Western blot analysis. Caspase-12 activity and caspase-3 activity were assayed using the Fluorometric Assay Kit and the Colorimetric Assay Kit, respectively.. Treatment of Jurkat T cells with DHURS (20-25 μg/ml) caused cytotoxicity and apoptotic DNA fragmentation along with Δψm loss, mitochondrial cytochrome c release, activation of caspase-9, -7, -3, and -8, and PARP degradation. However, these apoptotic events were abrogated by overexpression of Bcl-2. Pretreatment of the cells with the pan-caspase inhibitor (z-VAD-fmk), the caspase-9 inhibitor (z-LEHD-fmk) or the caspase-3 inhibitor (z-DEVD-fmk) to prevent DHURS-induced apoptosis could block the activation of caspase-7 and -8, and PARP degradation, but not the Δψm loss, activation of caspase-9 and -3. Both FADD- and caspase-8-positive wild-type Jurkat clone A3, FADD-deficient Jurkat clone I2.1, and caspase-8-deficient Jurkat clone I9.2 exhibited similar susceptibilities to the cytotoxicity of DHURS, excluding an involvement of Fas/FasL system in triggering the apoptosis. The IC(50) value for Jurkat T cells was ∼22 μg/ml, whereas that for human peripheral T cells was 25 μg/ml.. These results indicate that DHURS-induced apoptogenic activity in Jurkat T cells, which was less potent in normal peripheral T cells, was mediated by Δψm loss, mitochondrial cytochrome c release, and subsequent activation of caspase-9 and -3, leading to activation of caspase-7 and -8, which could be regulated by Bcl-2.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspases; Cytochromes c; DNA Fragmentation; Enzyme Inhibitors; Fas-Associated Death Domain Protein; Humans; Inflorescence; Inhibitory Concentration 50; Jurkat Cells; Leukemia, T-Cell; Membrane Potential, Mitochondrial; Phytotherapy; Plant Extracts; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Prunella; Saponins; Signal Transduction; T-Lymphocytes

Defects in apoptosis signaling contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL), and overexpression of antiapoptotic Bcl-2 (Bcl-2 and Bcl-X(L)) family proteins has been observed in ALL. ABT-737 is a small-molecule BH3-mimetic that inhibits the antiapoptotic Bcl-2 family proteins. We evaluated the cytotoxicity of ABT-737 in combination with vincristine, dexamethasone, and L-asparaginase (VXL) in 7 ALL cell lines. Multilog synergistic cytotoxicity was observed in all 7 cell lines with ABT-737 plus L-asparaginase or vincristine, and in 5 of 7 cell lines with ABT-737 plus dexamethasone or VXL. In leukemia cells, but not in normal lymphocytes, ABT-737 plus L-asparaginase induced greater mitochondrial depolarization (JC-1 staining); mitochondrial cytochrome c release; activation of Bax, Bid, and caspases (immunoblotting); and eventually apoptosis (annexin V staining) than did either drug alone. In mouse xenografts derived from patients with ALL at diagnosis (ALL-7) or at relapse (ALL-19), event-free survival (EFS) was significantly enhanced with ABT-737 plus VXL relative to VXL or ABT-737 alone (P

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Apoptosis; Asparaginase; Biphenyl Compounds; Blotting, Western; Cell Line, Tumor; Child; Cytochromes c; Dexamethasone; Drug Therapy, Combination; Female; Flow Cytometry; Humans; In Vitro Techniques; Leukemia, T-Cell; Membrane Potential, Mitochondrial; Mice; Mice, Inbred NOD; Mice, SCID; Mitochondria; Nitrophenols; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Survival Rate; Vincristine

Photodynamic therapy (PDT) is a cancer treatment based on the interaction of a photosensitizer, light and oxygen. PDT with the endogenous photosensitizer, protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) or its derivatives is a modification of this treatment modality with successful application in dermatology. However, the mechanism of cell destruction by ALA-PDT has not been elucidated. In this study a human T-cell lymphoma Jurkat cell line was treated with PDT using hexaminolevulinate (HAL, hexylester of ALA). Four hours following treatment nearly 80% of the cells exhibited typical apoptotic features. Mitochondrial pro-apoptotic proteins were evaluated by Western blots in subcellular fractionated samples. PDT caused cytosolic translocation of cytochrome c and nuclear redistribution of apoptosis-inducing factor (AIF), but the release of mitochondrial Smac/DIABLO, Omi/HtrA2 and EndoG was not observed. The release of cytochrome c was followed by the cleavage of caspase-9 and caspase-3 as well as its downstream substrates, together with oligonucleosomal DNA fragmentation. The pan-caspases inhibitor, z-VAD.fmk, prevented oligonucleosomal DNA fragmentation, but failed to inhibit PDT-mediated apoptosis. The apoptotic induction by AIF-mediated caspase-independent pathway was also found after HAL-PDT with large-scale DNA fragmentation in the presence of z-VAD.fmk. These results demonstrate that cytochrome c-mediated caspase-dependent pathway and AIF-induced caspase-independent pathway are simultaneously involved in the apoptotic induction by PDT. When the cytochrome c-induced caspase-dependent pathway is blocked, the cells go into apoptosis via AIF-mediated pathway, clearly demonstrating that the cytochrome c-mediated caspase-dependent pathway is not required for such apoptotic induction. This finding may have an impact on improved PDT effectiveness.

Topics: Amino Acid Chloromethyl Ketones; Aminolevulinic Acid; Apoptosis; Apoptosis Inducing Factor; Caspases; Cytochromes c; Enzyme Inhibitors; Humans; Jurkat Cells; Leukemia, T-Cell; Membrane Potential, Mitochondrial; Mitochondria; Photochemotherapy; Protoporphyrins

Targeting cannabinoid receptors has recently been shown to trigger apoptosis and offers a novel treatment modality against malignancies of the immune system. However, the precise mechanism of apoptosis in such cancers has not been previously addressed. In this study, we used human Jurkat leukemia cell lines with defects in intrinsic and extrinsic signaling pathways to elucidate the mechanism of apoptosis induced by Delta9-tetrahydrocannabinol (THC). We observed that Jurkat cells deficient in FADD or caspase-8 were partially resistant to apoptosis, while dominant-negative caspase-9 mutant cells were completely resistant to apoptosis. Use of caspase inhibitors confirmed these results. Furthermore, overexpression of Bcl-2 rendered the cells resistant to THC at early time points but not upon prolonged exposure. THC treatment led to loss of Deltapsi(m), in both wild-type and FADD-deficient Jurkat cells thereby suggesting that THC-induced intrinsic pathway was independent of FADD. THC treatment of wild-type Jurkat cells caused cytochrome c release, and cleavage of caspase-8, -9, -2, -10, and Bid. Caspase-2 inhibitor blocked THC-induced caspase-3 in wild-type Jurkat cells but not loss of Deltapsi(m). Together, these data suggest that the intrinsic pathway plays a more critical role in THC-induced apoptosis while the extrinsic pathway may facilitate apoptosis via cross-talk with the intrinsic pathway.

Topics: Adolescent; Antineoplastic Agents; Apoptosis; Caspase 2; Caspase 3; Caspase 7; Caspases; Cell Line, Tumor; Cytochromes c; Cytosol; Dronabinol; Humans; Jurkat Cells; Leukemia, T-Cell; Male; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Receptors, Cannabinoid; Signal Transduction

Lymphoid malignancies can escape from DNA-damaging anti-cancer drugs and gamma-radiation by blocking apoptosis-signaling pathways. How these regimens induce apoptosis is incompletely defined, especially in cells with nonfunctional p53. We report here that the BH3-only Bcl-2 family member Bid is required for mitochondrial permeabilization and apoptosis induction by etoposide and gamma-radiation in p53 mutant T leukemic cells. Bid is not transcriptionally up-regulated in response to these stimuli but is activated by cleavage on aspartate residues 60 and/or 75, which are the targets of caspase-8 and granzyme B. Bid activity is not inhibitable by c-Flip(L), CrmA, or dominant negative caspase-9 and therefore is independent of inducer caspase activation by death receptors or the mitochondria. Caspase-2, which has been implicated as inducer caspase in DNA damage pathways, appeared to be processed in response to etoposide and gamma-radiation but downstream of caspase-9. Knock down of caspase-2 by short interfering RNA further excluded its role in Bid activation by DNA damage. Caspase-2 was implicated in the death receptor pathway however, where it contributed to effector caspase processing downstream of inducer caspases. Granzyme B-specific serpins could not block DNA damage-induced apoptosis, excluding a role for granzyme B in the generation of active Bid. We conclude that Bid, cleaved by an undefined aspartate-specific protease, can be a key mediator of the apoptotic response to DNA-damaging anticancer regimens.

Topics: Animals; Antineoplastic Agents; Apoptosis; Aspartic Acid; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspase 2; Caspase 8; Caspase 9; Caspases; Cell Line; Cytochromes c; Cytoplasm; DNA Damage; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Etoposide; Flow Cytometry; Gamma Rays; Genes, Dominant; Granzymes; Humans; Immunoblotting; Jurkat Cells; Leukemia, T-Cell; Liver; Mice; Mitochondria; Models, Biological; Mutation; Retroviridae; RNA, Small Interfering; Serine Endopeptidases; Signal Transduction; Tumor Suppressor Protein p53; Up-Regulation

Previous studies have shown that the lymphoblastic leukemia CEM cell line is resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis because of a low expression of caspase-8. Bcl-2 inhibitors, BH3I-2' and HA14-1, are small cell-permeable nonpeptide compounds, are able to induce apoptosis by mediating cytochrome c release, and also lead to dissipation of the mitochondrial membrane potential (DeltaPsim). This study aimed to use the Bcl-2 inhibitors to sensitize CEM cells to TRAIL-induced apoptosis by switching on the mitochondrial apoptotic pathway. We found that a low dose of BH3I-2' or HA14-1, which did not induce cytochrome c release, greatly sensitized CEM cells to TRAIL-induced apoptosis. In a similar manner to the classical uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), both BH3I-2' and HA14-1 induced a reduction in DeltaPsim, a generation of reactive oxygen species (ROS), an increased mitochondrial respiration, and a decreased ATP synthesis. This uncoupling function of the Bcl-2 inhibitors was responsible for the synergy with TRAIL-induced apoptosis. CCCP per se did not induce apoptosis but again sensitized CEM cells to TRAIL-induced apoptosis by uncoupling mitochondrial respiration. The uncoupling effect facilitated TRAIL-induced Bax conformational change and cytochrome c release from mitochondria. Inhibition of caspases failed to block TRAIL-mediated cell death when mitochondrial respiration was uncoupled. We observed that BH3I-2', HA14-1, or CCCP can overcome resistance to TRAIL-induced apoptosis in TRAIL-resistant cell lines, such as CEM, HL-60, and U937. Our results suggest that the uncoupling of mitochondrial respiration can sensitize leukemic cells to TRAIL-induced apoptosis. However, caspase activation per se does not represent an irreversible point of commitment to TRAIL-induced cell death when mitochondrial respiration is uncoupled.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Benzamides; Benzopyrans; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cytochromes c; Drug Synergism; HL-60 Cells; Humans; K562 Cells; Leukemia, T-Cell; Membrane Glycoproteins; Mitochondria; Nitriles; Oxidative Phosphorylation; Oxygen Consumption; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Uncoupling Agents