Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P42574 (caspase-3)
 45,978 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent evidence supports a role for heat-shock protein 70 (hsp70) and the 26 S proteasome in regulating apoptosis, although the precise nature of their involvement is not known. In the present study, control and Bcl-x(L)-overexpressing, interleukin-3-dependent FL5.12 cell lines were treated with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). Basal proteasome activity appeared to be approximately 30% lower in bcl-x(L) cells compared with control cells using a substrate for the chymotrypsin-like activity. However, no difference in proteasome activity was detected using substrates for the trypsin-like or peptidylglutamyl peptide-hydrolysing activities. In addition, protein levels of the 20 S proteasome beta-subunit, as determined by Western blot analyses, were similar in control and bcl-x(L) cells, leading to the conclusion that proteasome activities were the same in these two cell lines. At 24 h after treatment with 500 nM MG132, apoptosis in bcl-x(L) cells (22%) was less than that observed in control cells (34%). Concomitantly, caspase activity in control cells, as assessed by N-acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspartyl-7-amino-4-methylcou marin (Ac-DEVD-AMC), was twice that observed in bcl-x(L) cells. By 48 h after MG132 treatment, apoptosis and caspase activity in bcl-x(L) cells were similar to those observed in control cells at 24 h. Proteasome inhibition stimulated increases in hsp70 protein levels in control and bcl-x(L) cells by 12 h, although the maximal increases found in bcl-x(L) cells were less. Blocking this induction with hsp70 antisense oligonucleotides potentiated apoptosis after treatment with MG132. Inhibiting caspase activity with a broad-spectrum caspase inhibitor, t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone, prevented MG132-induced apoptosis. The more specific caspase-3 inhibitor, Ac-DEVD-aldehyde, afforded less protection, although both inhibitors completely inhibited Ac-DEVD-AMC cleavage. These data indicate that both hsp70 and Bcl-x(L) provide some protection against proteasome inhibitor-induced apoptosis.
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PMID:Heat-shock protein 70 antisense oligomers enhance proteasome inhibitor-induced apoptosis. 1056 31

beta-Lapachone (beta-lap) effectively killed MCF-7 and T47D cell lines via apoptosis in a cell-cycle-independent manner. However, the mechanism by which this compound activated downstream proteolytic execution processes were studied. At low concentrations, beta-lap activated the caspase-mediated pathway, similar to the topoisomerase I poison, topotecan; apoptotic reactions caused by both agents at these doses were inhibited by zVAD-fmk. However at higher doses of beta-lap, a novel non-caspase-mediated "atypical" cleavage of PARP (i.e., an approximately 60-kDa cleavage fragment) was observed. Atypical PARP cleavage directly correlated with apoptosis in MCF-7 cells and was inhibited by the global cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. This cleavage was insensitive to inhibitors of caspases, granzyme B, cathepsins B and L, trypsin, and chymotrypsin-like proteases. The protease responsible appears to be calcium-dependent and the concomitant cleavage of PARP and p53 was consistent with a beta-lap-mediated activation of calpain. beta-Lap exposure also stimulated the cleavage of lamin B, a putative caspase 6 substrate. Reexpression of procaspase-3 into caspase-3-null MCF-7 cells did not affect this atypical PARP proteolytic pathway. These findings demonstrate that beta-lap kills cells through the cell-cycle-independent activation of a noncaspase proteolytic pathway.
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PMID:Activation of a cysteine protease in MCF-7 and T47D breast cancer cells during beta-lapachone-mediated apoptosis. 1069 31

MNEI (monocyte/neutrophil elastase inhibitor) is a 42 kDa serpin superfamily protein characterized initially as a fast-acting inhibitor of neutrophil elastase. Here we show that MNEI has a broader specificity, efficiently inhibiting proteases with elastase- and chymotrypsin-like specificities. Reaction of MNEI with neutrophil proteinase-3, an elastase-like protease, and porcine pancreatic elastase demonstrated rapid inhibition rate constants >10(7) M(-1) s(-1), similar to that observed for neutrophil elastase. Reactions of MNEI with chymotrypsin-like proteases were also rapid: cathepsin G from neutrophils (>10(6) M(-1) s(-1)), mast cell chymase (>10(5) M(-1) s(-1)), chymotrypsin (>10(6) M(-1) s(-1)), and prostate-specific antigen (PSA), which had the slowest rate constant at approximately 10(4) M(-1) s(-1). Inhibition of trypsin-like (plasmin, granzyme A, and thrombin) and caspase-like (granzyme B) serine proteases was not observed or highly inefficient (trypsin), nor was inhibition of proteases from the cysteine (caspase-1 and caspase-3) and metalloprotease (macrophage elastase, MMP-12) families. The stoichiometry of inhibition for all inhibitory reactions was near 1, and inhibitory complexes were resistant to dissociation by SDS, further indicating the specificity of MNEI for elastase- and chymotrypsin-like proteases. Determination of the reactive site of MNEI by N-terminal sequencing and mass analysis of reaction products identified two reactive sites, each with a different specificity. Cys(344), which corresponds to Met(358), the P(1) site of alpha1-antitrypsin, was the inhibitory site for elastase-like proteases and PSA, while the preceding residue, Phe(343), was the inhibitory site for chymotrypsin-like proteases. This study demonstrates that MNEI has two functional reactive sites corresponding to the predicted P(1) and P(2) positions of the reactive center loop. The data suggest that MNEI plays a regulatory role at extravascular sites to limit inflammatory damage due to proteases of cellular origin.
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PMID:The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites. 1174 53

The proteasome is a multi-subunit protease complex that is involved in intracellular protein degradation in eukaryotes. Previously, we have reported that selective, synthetic chymotryptic proteasome inhibitors inhibit A-NK cell-mediated cytotoxicity by approximately 50%; however, the exact role of the proteasome in NK cell-mediated cytotoxicity remains unknown. Herein, we report that proteasome inhibitors, MG115 and MG132, decreased the proteasome chymotrypsin-like activity in the rat natural killer cell line RNK16 by 85% at a concentration of 5 microM. The viability of RNK16 cells was also reduced in the presence of these inhibitors. Both inhibitors induced the apoptosis of RNK16 cells, as shown by DNA fragmentation, caspase-3 activation and the appearance of sub-G-cell populations. An increase in the fraction of apoptotic cells was observed in a dose- and time-dependent manner in our studies. In addition, the activity of caspase-1, -2, -6, -7, -8, and -9, was increased following the treatment of RNK16 cells with these inhibitors. Further investigation revealed that the expression of Fas (CD95) protein on the RNK16 cell surface was increased after the treatment by MG115 or MG132, indicating that apoptosis induced by proteasome inhibitors in RNK16 cells might be mediated through the Fas (CD95)-mediated death pathway as well. Our studies indicate, for the first time, that proteasomal chymotryptic inhibitors can reduce natural killer cell viability and therefore indirectly inhibit cell-mediated cytotoxicity via the apoptosis-inducing properties of these agents.
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PMID:Activation of multiple caspases and modification of cell surface fas (CD95) in proteasome inhibitor-induced apoptosis of rat natural killer cells. 1253 25

Treatment of C(2)C(12) myotubes with a tumour-derived proteolysis-inducing factor (PIF) at concentrations between 1 and 10 nM was shown to stimulate the activity of the apoptotic initiator caspases-8 and -9 and the apoptotic effector caspases-2, -3 and -6. This increased caspase activity was attenuated in myotubes pretreated with 50 microM eicosapentaenoic acid (EPA). At least part of the increase in caspase activity may be related to the increased proteasome proteolytic activity, since a caspase-3 inhibitor completely attenuated the PIF-induced increase in 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the proteasome. However, Western blot analysis showed that PIF induced an increase in expression of the active form of caspase-3, which was also attenuated by EPA. Further Western blot analysis showed PIF increased the cytosolic content of cytochrome c, as well as expression of the pro-apoptotic protein bax but not the anti-apoptotic protein bcl-2, which were both attenuated by 50 microM EPA. Induction of apoptosis by PIF in murine myotubes was confirmed by an increase in free nucleasomes formation and increased DNA fragmentation evidenced by a nucleasomal ladder typical of apoptotic cells. This process was again inhibited by pre-incubation with EPA. These results suggest that in addition to activating the proteasome, PIF induces apoptosis in C(2)C(12) myotubes, possibly through the common intermediate arachidonic acid. Both of these processes would contribute to the loss of skeletal muscle in cancer cachexia.
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PMID:Induction of apoptosis by a cachectic-factor in murine myotubes and inhibition by eicosapentaenoic acid. 1276 76

There is a large amount of scientific evidence showing that fruits and vegetables lower the risk of cancer. However, the responsible molecular mechanisms remain poorly understood. Our previous studies have demonstrated that inhibition of proteasomal chymotrypsin-like activity is associated with cancer cell apoptosis, which may also be the major mechanism responsible for the anticancer effects of green tea polyphenols. In the current study, we tested the hypothesis that some fruits and vegetables inhibit tumor cell proteasome activity and that this inhibition contributes to their cancer-preventative activities. We report that the extracts of apple and grape are more potent than onion, tomato and celery in: (i) inhibiting the proteasomal chymotrypsin-like activity in leukemia Jurkat T cell extract; (ii) accumulating the polyubiquitinated proteins in intact Jurkat T cells; (iii) inducing activation of caspase-3/-7 and cleavage of poly(ADP-ribose) polymerase in intact Jurkat T cells; and (iv) inducing the appearance of spherical cells preferentially in prostate cancer PC-3 over the normal NIH 3T3 cell line. We also found that strawberry extract had some effect on Jurkat T cell extract and the prostate PC-3 cell line but not on intact Jurkat T cells. Our findings suggest that the proteasome is a cancer-related molecular target for, at least, the extracts of apple, grape and onion, and that the inhibition of proteasome activity by these fruits or vegetable may contribute to their cancer-preventative effects, although other molecular mechanisms may also be involved.
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PMID:Inhibition of proteasome activity by various fruits and vegetables is associated with cancer cell death. 1501 55

The irreversible inhibitor of chymotrypsin-like serine proteases, N-tosyl-L-phenylalanine chloromethylketone (TPCK), was shown to prevent internucleosomal DNA cleavage caused by inducers of apoptosis. The pro-apoptotic properties of TPCK have been studied less thoroughly. The aim of the present study was to investigate the pro- and anti-apoptotic activities of TPCK on HL-60 cells and compare them with the actions of the mitochondrial electron transport inhibitor antimycin A (AMA). The results showed that TPCK alone caused activation of cell cycle checkpoints, mitochondrial cytochrome c release, caspase-3 activation, and chromatin condensation. Caspase-8 was not required for cytochrome c release but was crucial to caspase-3 activation. TPCK synergistically enhanced AMA-induced cytochrome c release and caspase-3 activation while completely blocking AMA-induced internucleosomal DNA fragmentation for at least 8 hours. Rather than blocking AMA-induced DNA fragmentation, the general serine protease inhibitor 4-(2-aminoethyl)-benzenesulphonyl fluoride (AEBSF) actually enhanced it. The pro-apoptotic effect of TPCK may be due to activation of cell cycle checkpoints via inhibition of the proteasome. The apoptotic pathways activated by TPCK and AMA probably converge at the level of the mitochondria. The mode by which TPCK prevents internucleosomal DNA fragmentation is probably not through serine protease inhibition.
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PMID:Pro- and anti-apoptotic effects of an inhibitor of chymotrypsin-like serine proteases. 1553 54

It has been shown that proteasome activity is required for cancer cell survival and consumption of fruits and vegetables is associated with decreased cancer risk. Previously, we reported that grape extract could inhibit proteasome activity and induce apoptosis in tumor cells. In this study, we examined the flavonoids apigenin, quercetin, kaempferol and myricetin for their proteasome-inhibitory and apoptosis-inducing abilities in human tumor cells. We report that apigenin and quercetin are much more potent than kaempferol and myricetin at: (i) inhibiting chymotrypsin-like activity of purified 20S proteasome and of 26S proteasome in intact leukemia Jurkat T cells; (ii) accumulating putative ubiquitinated forms of two proteasome target proteins, Bax and Inhibitor of nuclear factor kappabeta-alpha in Jurkat T cells and (iii) inducing activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase in Jurkat T cells. The proteasome-inhibitory abilities of these compounds correlated with their apoptosis-inducing potencies. Results from computational modeling of the potential interactions of these flavonoids to the chymotrypsin site (beta5 subunit) of the proteasome were consistent with the obtained proteasome-inhibitory activities. We found that the C(4) carbon may be a site of nucleophilic attack by the OH group of N-terminal threonine of proteasomal beta5 subunit and that the C(3) hydroxyl may alter the ability of these flavonoids to inhibit the proteasome. Finally, apigenin neither effectively inhibited the proteasome activity nor induced apoptosis in non-transformed human natural killer cells. Our results suggested that the proteasome may be a target of these dietary flavonoids in human tumor cells and that inhibition of the proteasome by flavonoids may be one of the mechanisms responsible for their cancer-preventive effects.
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PMID:Dietary flavonoids as proteasome inhibitors and apoptosis inducers in human leukemia cells. 1585 6

Either a single (acute) or repeated daily (chronic) injections (1 injection/day) of 20 mg/kg cocaine for 10 days to rats was found to increase reactive oxygen species production in two dopaminergic brain structures, the frontal cortex and the striatum. We found that the mitochondrial genome was down-regulated after acute cocaine injection. Hydroperoxide and lipid peroxide generation was correlated with an increase in mitochondrial hydrogen peroxide generation and with a reduced functioning of mitochondrial complex I in response to cocaine. As judged from the measurement of caspase-3 activity and TUNEL labeling, neither acute nor chronic cocaine treatment has been found to induce apoptosis in any of the structures examined. This differs dramatically from what has been described for methamphetamine. Cocaine-induced radical formation was accompanied by the induction of the antioxidant enzymes superoxide dismutase and glutathione peroxidase, after both acute and chronic cocaine treatment. In addition, proteasome chymotrypsin-like activity was enhanced following a single cocaine injection in both cortex and striatum. It is proposed that the compensatory mechanisms to oxidative stress occurring in response to cocaine were effective in scavenging reactive oxygen species and in preventing subsequent cellular damage, thus explaining why no significant cell death was found in these brain structures.
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PMID:Acute or repeated cocaine administration generates reactive oxygen species and induces antioxidant enzyme activity in dopaminergic rat brain structures. 1585 23

Herein, we report differential effects of various proteasome inhibitors including clasto-lactacystin-beta-lactone, (-)-epigallocatechin gallate (EGCG) and N-Acetyl-Leu-Leu-Norleu-al (LLnL) on proteasomal activities of YT and Jurkat cells, human natural killer (NK) and T cell lines, respectively. The inhibitory rates of these inhibitors on the purified 20S proteasomal and 26S proteasomal chymotrypsin-like activity in whole cell extracts and intact cells did not show significant differences between the two cell lines. The viability of both cell lines was reduced in the presence of LLnL. Subsequent studies revealed a reduction of the mitochondrial membrane potential and caspase-3 activation in these two cell lines upon treatment with proteasome inhibitors; however, caspase-3 activation occurred much earlier in Jurkat cells. Cell cycle analysis indicated a sub-G(1) apoptotic cell population in Jurkat cells and G(2)/M arrest in YT cells after they were treated by proteasome inhibitors. Moreover, pretreatment of YT cells by a caspase inhibitor followed by a proteasome inhibitor did not increase the percentage of G(2)/M phase cells. In addition, accumulation of p27 and IkappaB-alpha was detected only in Jurkat cells, but not YT cells. In summary, proteasome inhibitors may act differentially in cell cycle arrest and apoptosis of tumors of NK and T cell origin, and may have similar effects on normal NK and T cells.
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PMID:Differential effects of proteasome inhibitors on cell cycle and apoptotic pathways in human YT and Jurkat cells. 1617 95


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