Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.22.56 (caspase-3)
 35,750 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Peroxisomicine A(1) (T-514) is a dimeric anthracenone first isolated from the plant Karwinskia humboldtiana. The compound presents a high and selective toxicity toward liver and skin cell cultures and is currently the subject of preclinical studies as an antitumor drug. To date, the molecular basis for its diverse biological effects remains poorly understood. To elucidate its mechanism of action, we studied its interaction with DNA and its effects on human DNA topoisomerases. Practically no interaction with DNA was detected. Peroxisomicine was found to inhibit topoisomerase II but not topoisomerase I. DNA relaxation and decatenation assays indicated that the drug interferes with the catalytic activity of topoisomerase II but does not stimulate DNA cleavage, in contrast to conventional topoisomerase poisons such as etoposide. Two human leukemia cell lines sensitive or resistant to mitoxantrone were used to assess the cytotoxicity of the toxin and its effect on the cell cycle. In both cases, peroxisomicine treatment was associated with a loss of cells from every phase of the cell cycle and was accompanied by a large increase in the sub-G1 region which is characteristic of apoptotic cells. The cell cycle changes were more pronounced with the sensitive HL-60 cells than with the resistant HL-60/MX2 cells (with reduced topoisomerase II activity), in agreement with the cytotoxicity measurements. Treatment of HL-60 cells with T-514 stimulated the cleavage of the poly(ADP-ribose) polymerase by intracellular proteases such as caspase-3. The cytometry and Western blot analyses reveal that peroxisomicine induces apoptosis in leukemia cells. In addition, we characterized a catabolite of peroxisomicine, named T-510R, in the form of a highly stable radical metabolite. The electron spin resonance and mass spectrometry data are consistent with the formation of an anionic semiquinonic radical. The oxidized product T-510R inhibits topoisomerase II with a reduced efficiency compared to the parent toxin and was found to be about 3-4 times less toxic to both the sensitive and resistant leukemia cell lines than T-514. Collectively, the results suggest that topoisomerase II inhibition plays a role in the cytotoxicity of the plant toxin peroxisomicine. Inhibition of topoisomerase II may serve as an inducing signal triggering the apoptotic cell death of leukemia cells exposed to the toxin. The dihydroxyanthracenone unit may represent a useful chemotype for the preparation of topoisomerase II-targeted anticancer agents.
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PMID:DNA topoisomerase II inhibition by peroxisomicine A(1) and its radical metabolite induces apoptotic cell death of HL-60 and HL-60/MX2 human leukemia cells. 1117 May 4

We studied morphological changes of the nucleoli in HeLa cells treated with cisplatin and compared them with induction of markers of programmed cell death and TUNEL staining. We used different light microscopic nucleolar staining methods allowing us to visualize not only nucleolar proteins but also nucleolar RNA. Our results show predominantly compact, centrally localized nucleoli in intact control HeLa cells. In cisplatin-treated HeLa cells, we found an early onset of nucleolar segregation of proteins detected by argyrophilic nucleolar organizer regions and anti-nucleolar monoclonal antibody as well as an increased immunoreactivity for activated caspase-3 after 6 hours. Staining with Toluidine Blue and Methyl-green Pyronine revealed segregated nucleoli 12 hours after the treatment with cisplatin. TUNEL positivity in cisplatin-treated HeLa cells was accompanied by the aggregation of the argyrophilic proteins in the central portion of nucleus, disappearance of nucleolar RNA and shrinkage of the nucleus after 24 hours. Monitoring of the biochemical changes by immunoblotting revealed that activation of distinct caspases and degradation of their downstream protein substrates is executed in two phases. During an early apoptotic stage beginning 4.5 hours post treatment an activation of caspase-9 and caspase-3 was observed. This was accompanied by proteolytic cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). The caspase-9 activation seems to be mediated by recruitment by the activating factor Apaf-1 because the increased accumulation of Apaf-1 and cytochrome C in cytosol preceded the generation of mature caspase-9 form. A second phase of apoptosis occurring between 10 and 15 hours post treatment was characterized by degradation of other nucleolar and nuclear proteins such as nuclear lamins, topoisomerase I and B23. In conclusion, remarkable segregation of nucleolar argyrophilic proteins, nucleolar RNA and a simultaneous activation of the cascade of caspases markedly preceded the TUNEL positivity in cisplatin-treated HeLa cells thereby substantiating the hypothesis that the nucleolus is a preferred target for caspase-3-dependent proteolysis in cisplatin-treated HeLa cells.
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PMID:Segregation of nucleolar components coincides with caspase-3 activation in cisplatin-treated HeLa cells. 1117 71

The antitumor drug NB-506 is a glycosylated indolocarbazole derivative targeting topoisomerase I. This DNA-intercalating agent, which is currently undergoing phase I/II clinical trials, was shown to induce apoptosis in HL-60 human leukemia cells. We compared the cellular dysfunctions induced by NB-506 and the reference topoisomerase I poison camptothecin (CPT) at the nuclear, mitochondrial, and cytoplasmic levels. The two drugs NB-506 and CPT were almost equally toxic to HL-60 cells and produced similar cell cycle changes with a considerable increase in the fraction of cells with DNA content less than G1. The sub-G1 fraction, which can be considered as the apoptotic cell population, appeared more rapidly with CPT than with NB-506 but in both cases, the cell cycle perturbation was accompanied by a marked decrease in the mitochondrial transmembrane potential and the intracellular pH. In contrast, no change in the intracellular calcium concentration was detected. Treatment of HL-60 cells with NB-506 resulted in an increase in the activity of the intracellular protease caspase-3, as determined by a DEVD-based colorimetric assay and direct monitoring of poly(ADP-ribose) polymerase (PARP) cleavage by Western blot analysis. The initiator caspase-8 was also stimulated by NB-506 but, as for caspase-3, the extent of the caspase activation was weaker with NB-506 compared to CPT. With both drugs, the protease activation resulted in DNA degradation, as independently confirmed via the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and characterization of internucleosomal DNA fragmentation. Collectively, these findings identify some of the molecular events leading to NB-506-induced apoptosis and as such, provide important mechanistic insights into the mode of action of topoisomerase I-targeted indolocarbazole antitumor drugs.
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PMID:Apoptotic response of HL-60 human leukemia cells to the antitumor drug NB-506, a glycosylated indolocarbazole inhibitor of topoisomerase 1. 1117 34

The present study explored the role of the cell surface receptor Fas (CD95/APO-1) in apoptosis induced by camptothecin (CPT) in the HT29 colon carcinoma cell line. CPT-induced apoptosis was associated with high molecular weight DNA fragmentation as measured by filter elution. This fragmentation was inhibited by the caspase inhibitor, z-VAD-fmk and by cycloheximide, which also prevented proteolytic activation of caspase-3 and poly(ADP-ribose)polymerase cleavage. Under such conditions, Fas, Fas ligand, Bax, and p21 expression were increased and Fas recruited the FADD adaptor. Fas expression increase was blocked by cycloheximide but not by z-VAD-fmk, consistent with caspase activation downstream from Fas. Treatment of HT29 cells with FasL or with the CH-11 agonistic anti-Fas antibody potentiated the apoptotic response of cells treated with CPT. The anti-Fas blocking antibody ZB4 and the Fas-ligand inhibitor failed to protect HT29 cells from CPT-induced apoptosis. Such a protection was obtained by transient expression of constructs encoding a dominant-negative mutant of FADD, FADD in an antisense orientation and E8 or MC159 viral proteins that inhibit Fas-induced apoptosis at the level of FADD and procaspase-8, respectively. Together, these data show that topoisomerase I-mediated DNA damage-induced apoptosis involves activation of the Fas pathway without detectable Fas-ligand requirement in CPT-treated cells.
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PMID:Activation of the Fas pathway independently of Fas ligand during apoptosis induced by camptothecin in p53 mutant human colon carcinoma cells. 1131 33

Our results demonstrate that sodium phenylbutyrate, a compound with a low degree of toxicity, exerted a cytotoxic effect on human retinoblastoma Y79 cells in a time- and dose-dependent manner. Treatment of Y79 cells for 72 h with phenylbutyrate reduced cell viability by 63% at 2 mM and 90% at 4 mM. Cell death caused by phenylbutyrate exhibited the typical features of apoptosis, as shown by light and fluorescent microscopy. Western blot analysis demonstrated that exposure of Y79 cells to phenylbutyrate decreased the level of the antiapoptotic factor Bcl-2 and induced the activation of caspase-3, a key enzyme in the execution phase of apoptosis. Moreover, treatment with phenylbutyrate markedly increased the level of acetylated histone-H3. Combined treatment with phenylbutyrate and topotecan, a topoisomerase I-inhibitor, resulted in a clear synergistic effect. We suggest that the effects exerted by phenylbutyrate on Y79 cells essentially depend on modifications of gene expression consequent to histone hyperacetylation.
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PMID:Sodium phenylbutyrate induces apoptosis in human retinoblastoma Y79 cells: the effect of combined treatment with the topoisomerase I-inhibitor topotecan. 1135 Dec 56

This paper studies the cytotoxic effect induced by the topoisomerase I inhibitor camptothecin in human osteosarcoma Saos-2 cells, which lack p53 and contain a non-functional form of the product of the retinoblastoma gene, pRb. Cytotoxicity induced by camptothecin was dose- and time-dependent; the treatment with 100 nM camptothecin reduced cell viability by 50% at 32 h and by 75% at 72 h of exposure. The cytotoxic effect was caused by apoptosis, as ascertained by morphological evidence, acridine orange-ethidium bromide staining and flow cytometric analysis. Apoptosis was accompanied by both the activation of caspase-3 and the fragmentation of poly(ADP-ribose) polymerase. Treatment with camptothecin caused a threefold increase in the activity of c-Jun N-terminal kinase (JNK) and an eightfold increase in the level of phosphorylated c-Jun. The introduction of the RB gene into Saos-2 cells reduced the rate of cell growth. Moreover, stable clones of transfected cells were resistant to camptothecin. Exposure to 100 nM camptothecin for 72 h reduced the viability of transfected cells by only 10%; moreover, very modest effects were observed on the activity of JNK as well as on the level of phosphorylated c-Jun. The results reported in this paper support the conclusion that the expression of wild-type pRb in Saos-2 cells exerts an anti-apoptotic influence through the control of JNK activity.
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PMID:pRb suppresses camptothecin-induced apoptosis in human osteosarcoma Saos-2 cells by inhibiting c-Jun N-terminal kinase. 1141 38

The homocamptothecin (hCPT) derivative BN80915 containing a seven-membered lactone ring represents one of the most potent topoisomerase I inhibitors described. This anticancer agent, currently undergoing phase I clinical trials, has been shown to produce a greater number of DNA strand breaks than conventional camptothecins with a six-membered lactone ring. To shed light on the mechanism of action of hCPT at the cellular level, we compared the effects of BN80915 and the classic camptothecin SN-38, the active metabolite of irinotecan, on HL-60 human promyelocytic cancer cells. A variety of biochemical events, at both the mitochondrial and the nuclear levels, were characterized to determine how and to what extent the hCPT derivative can induce apoptotic cell death. The use of cytometry, Western blot analysis, confocal microscopy, and different colorimetric assays enabled us to demonstrate that BN80915 is a potent inducer of apoptosis in HL-60 cells. This induction of apoptosis is associated with cell cycle changes, a marked decrease of intracellular pH, activation of caspase-3 and -8, DNA fragmentation, and externalization of phosphatidylserine lipids but no significant changes of the mitochondrial membrane potential or the expression of Bcl-2. The interconnections between these different events are discussed. Collectively, the results indicate that the superior activity expressed at the topoisomerase I level leads to a more pronounced induction of apoptosis by BN80915 compared with SN-38. The study identifies and delineates signaling factors involved in BN80915-induced apoptosis in HL-60 cells.
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PMID:Apoptosis induced by the homocamptothecin anticancer drug BN80915 in HL-60 cells. 1150 75

MCF-7 human breast cancer cells are widely utilized to study apoptotic processes. Recent studies demonstrated that these cells lack procaspase-3. In the present study, caspase activation and activity were examined in this cell line after treatment with the microtubule poison paclitaxel. When cells were harvested 72 h after the start of a 24-h treatment with 100 nm paclitaxel, 37 +/- 5% of the cells were nonadherent and displayed apoptotic morphological changes. Although mitochondrial cytochrome c release and caspase-9 cleavage were detectable by immunoblotting, assays of cytosol and nuclei prepared from the apoptotic cells failed to demonstrate the presence of activity that cleaved the synthetic caspase substrates LEHD-7-amino-4-trifluoromethylcoumarin (LEHD-AFC), DEVD-AFC, and VEID-AFC. Likewise, the paclitaxel-treated MCF-7 cells failed to cleave a variety of caspase substrates, including lamin A, beta-catenin, gelsolin, protein kinase Cdelta, topoisomerase I, and procaspases-6, -8, and -10. Transfection of MCF-7 cells with wild type procaspase-3 partially restored cleavage of these polypeptides but did not result in detectable activities that could cleave the synthetic caspase substrates. Immunoblotting revealed that caspase-9, and -3, which were proteolytically cleaved in paclitaxel-treated MCF-7/caspase-3 cells, were sequestered in a salt-resistant sedimentable fraction rather than released to the cytosol. Immunofluorescence indicated large cytoplasmic aggregates containing cleaved caspase-3 in these apoptotic cells. These observations suggest that sequestration of caspases can occur in some model systems, causing tetrapeptide-based activity assays to underestimate the amount of caspase activation that has occurred in situ.
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PMID:Lack of correlation between caspase activation and caspase activity assays in paclitaxel-treated MCF-7 breast cancer cells. 1167 38

Because apoptosis is deregulated in most cancers, apoptosis-modulating approaches offer an attractive opportunity for clinical therapy of many tumors, including that of the prostate. LNCaP-derived C4-2 human prostate cancer cells are quite resistant to treatment with Apo2 ligand (Apo2L) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), when using a nontagged, Zn-bound recombinant trimeric version that is devoid of any exogeneous sequences and therefore least likely to be immunogenic in human patients and that has been optimized for maximum efficacy and minimum toxicity. When combined with the topoisomerase I inhibitor CPT-11 (irinotecan), Apo2L/TRAIL exhibits enhanced apoptotic activity in C4-2 cells cultured in vitro as well as xenografted as tumors in vivo. Apoptosis both in vitro and in vivo was characterized by two major molecular events. First, apoptosis induction was accompanied by changes in expression levels of the Bcl-2 family genes and their products. However, whereas combination treatment applied to in vitro cell culture was characterized by a significant up-regulation and activation of Bax and down-regulation of Bcl-xL, the treatment applied to tumors induced Bak and Bcl-xS, whereas Bcl-omega and Bcl-xL were down-regulated. Because there are multiple members of the Bcl-2 family (24 members to date), these data indicate that, under different biological conditions, different proteins may be responsible for activating apoptosis and provide evidence for a differential regulation of the multidomain Bcl-2 protein-encoding genes, bax and bak. Increased Bax expression led to its activation, translocation to the mitochondria, and release of cytochrome c. In addition, this combination treatment induced apoptosis through potent activation of caspase-8 and the proapoptotic protein Bid, resulting in activation of effector caspase-3 and cleavage of its cellular target protein, poly(ADP-ribose) polymerase (PARP), events blocked by the pan-caspase inhibitor N-tert-butoxy-carbonyl-Val-Ala-Asp-fluoro methylketone (zVAD-fmk). Activation of multiple caspases and PARP cleavage were also observed in the C4-2 tumors treated with doses resulting in effective tumor control at 42 days after Apo2L/TRAIL plus CPT-11 treatment. Down-regulation of Bax by small interference (RNA) (siRNA) in C4-2 cells significantly prevented PARP cleavage and apoptosis. Strikingly, similar experiments in cells stably expressing a dominant-negative death receptor DR5 led to complete ablation of PARP cleavage and apoptosis, indicating the essential role of both mitochondrial and receptor-mediated apoptotic pathways. Our data indicate that the combined treatment of Apo2L/TRAIL and CPT-11 achieves tumor control in prostate cancer tumors through regulation of Bcl-2 family proteins and potent activation of caspases.
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PMID:Apoptosis induction in prostate cancer cells and xenografts by combined treatment with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand and CPT-11. 1290 54

Poly(ADP-ribose) polymerase-1 is a highly abundant nuclear enzyme implicated in transcription, DNA replication, and DNA repair through binding of nascent RNA and interactions with various factors. We found that purified fractions of recombinant human poly(ADP-ribose) polymerase-1 expressed in Escherichia coli possess yet another activity, a Mg(2+)-dependent DNA supercoil relaxation activity. Cleavage of recombinant poly(ADP-ribose) polymerase-1 by caspase-3, an apoptotic protease, reduced this activity, as did the removal of either of the two zinc finger motifs located in the N-terminal DNA-binding domain of poly(ADP-ribose) polymerase-1. In addition, this activity was separated from E. coli topoisomerase I by gel-filtration column chromatography, suggesting that this activity is specifically associated with poly(ADP-ribose) polymerase-1. Because this relaxation activity did not require ATP and was resistant to VP16, a topoisomerase II inhibitor, this activity is closer to that of topoisomerase I. However, the supercoiled DNA relaxation activity associated with poly(ADP-ribose) polymerase-1 is distinct from that of human or E. coli topoisomerase I, as this activity could not completely remove superhelical tensions from plasmid DNA. Thus, we referred to this activity as topoisomerase I-like activity. This Mg(2+)-dependent DNA supercoil relaxation activity was found to be sensitive to camptothecin, a mammalian topoisomerase I inhibitor.
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PMID:Camptothecin-sensitive relaxation of supercoiled DNA by the topoisomerase I-like activity associated with poly(ADP-ribose) polymerase-1. 1471 57


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