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PubMed2006

Silibinin activates p53-caspase 2 pathway and causes caspase-mediated cleavage of Cip1/p21 in apoptosis induction in bladder transitional-cell papilloma RT4 cells: evidence for a regulatory loop between p53 and caspase 2.

Silibinin, a natural flavonolignan, induces apoptosis in human bladder transitional-cell papilloma RT4 cells both in vitro and in vivo; however, mechanisms of such efficacy are not completely identified. Here, we studied the mechanisms involved in silibinin-induced apoptosis of RT4 cells having intact p53. Silibinin increased p53 protein level together with its increased phosphorylation at serine 15, activated caspase cascade and caused Bid cleavage for apoptosis. Silibinin-caused p53 activation was mediated via ATM-Chk2 pathway, which in turn induced caspase 2-mediated apoptosis. Pifithrin-alpha, a p53 inhibitor, reversed silibinin-induced caspase activation including caspase 2; however, caspase 2 inhibitor also reversed p53 phosphorylation suggesting a bidirectional regulation between them. Further, silibinin caused a rapid translocation of p53 and Bid into mitochondria leading to increased permeabilization of mitochondrial membrane and cytochrome c release into the cytosol. JNK1/2 activation was observed as a connecting link for p53-mediated caspase 2 activation. Interestingly, silibinin-induced apoptosis was mediated, in part, via Cip1/p21 cleavage by caspase, which was reversed by Cip1/p21 siRNA. Together, these results suggested the novel mechanisms for apoptosis induction by silibinin involving p53-caspase 2 activation and caspase-mediated cleavage of Cip1/p21.

Apoptosis↗

PubMed2003

Caspase-independent cell death in AML: caspase inhibition in vitro with pan-caspase inhibitors or in vivo by XIAP or Survivin does not affect cell survival or prognosis.

Survivin and XIAP, members of the protein family known as the inhibitors of apoptosis, interfere with the activation of caspases, called the "cell death executioners." We examined Survivin (n = 116) and XIAP (n = 172) expression in primary acute myeloid leukemia (AML) blasts and assessed the impact of their expression on prognosis. They were detected in all samples analyzed. However, no correlation was observed with cytogenetics, remission attainment, or overall survival of patients with AML. To investigate the importance of caspases in chemotherapy-induced apoptosis in AML, we treated OCI-AML3 cells with Ara-C, doxorubicin, vincristine, and paclitaxel, which induced caspase cleavage and apoptosis. Blocking of caspase activation by pan-caspase inhibitor abolished poly(adenosine diphosphate [ADP]-ribose) polymerase cleavage and DNA fragmentation but did not prevent chemotherapy-induced cell death and did not inhibit, or only partially inhibited, mitochondrial release of cytochrome c, Smac, apoptosis-inducing factor (AIF), or loss of mitochondrial membrane potential. Caspase inhibition also did not protect AML blasts from chemotherapy-induced cell death in vitro. These results suggest that expression levels of Survivin or XIAP have no prognostic impact in AML patients. Although anticancer drugs induced caspase cleavage and apoptosis, cell killing was caspase independent. This may partially explain the lack of prognostic impact of XIAP and Survivin and may suggest caspase-independent mechanisms of cell death in AML.

Acute Disease↗

PubMed1999

DECAY, a novel Drosophila caspase related to mammalian caspase-3 and caspase-7.

Caspases are key effectors of programmed cell death in metazoans. In Drosophila, four caspases have been described so far. Here we describe the identification and characterization of the fifth Drosophila caspase, DECAY. DECAY shares a high degree of homology with the members of the mammalian caspase-3 subfamily, particularly caspase-3 and caspase-7. DECAY lacks a long prodomain and thus appears to be a class II effector caspase. Ectopic expression of DECAY in cultured cells induces apoptosis. Recombinant DECAY exhibited substrate specificity similar to the mammalian caspase-3 subfamily. Low levels of decay mRNA are ubiquitously expressed in Drosophila embryos during early stages of development but its expression becomes somewhat spatially restricted in some tissues. During oogenesis decay mRNA was detected in egg chambers of all stages consistent with a role for DECAY in apoptosis of nurse cells. Relatively high levels of decay mRNA are expressed in larval salivary glands and midgut, two tissues which undergo histolysis during larval/pupal metamorphosis, suggesting that DECAY may play a role in developmentally programmed cell death in Drosophila.

Amino Acid Sequence↗

PubMed2003

Mitochondrially localized active caspase-9 and caspase-3 result mostly from translocation from the cytosol and partly from caspase-mediated activation in the organelle. Lack of evidence for Apaf-1-mediated procaspase-9 activation in the mitochondria.

Active caspase-9 and caspase-3 have been observed in the mitochondria, but their origins are unclear. Theoretically, procaspase-9 might be activated in the mitochondria in a cytochrome c/Apaf-1-dependent manner, or activated caspase-9 and -3 may translocate to the mitochondria, or the mitochondrially localized procaspases may be activated by the translocated active caspases. Here we present evidence that the mitochondrially localized active caspase-9 and -3 result mostly from translocation from the cytosol (into the intermembrane space) and partly from caspase-mediated activation in the organelle rather than from the Apaf-1-mediated activation. Apaf-1 localizes exclusively in the cytosol and, upon apoptotic stimulation, translocates to the perinuclear area but not to the mitochondria. In most cases, the mitochondrially localized procaspase-9 and -3 are released early during apoptosis and translocate to the cytosol and/or perinuclear area. Cytochrome c and the mitochondrial matrix protein Hsp60 are also rapidly released to the cytosol early during apoptosis. Both the early release of proteins like cytochrome c and Hsp60 from the mitochondria as well as the later translocation of the active caspase-9/-3 are partially inhibited by cyclosporin A, an inhibitor of mitochondrial membrane permeabilization. The mitochondrial active caspases may function as a positive feedback mechanism to further activate other or residual mitochondrial procaspases, degrade mitochondrial constituents, and disintegrate mitochondrial functions.

Apoptosis↗

PubMed2002

Role of Caspase-1 in experimental pneumococcal meningitis: Evidence from pharmacologic Caspase inhibition and Caspase-1-deficient mice.

Caspase 1 plays a pivotal role in generating mature cytokine interleukin-1beta. Interleukin-1beta is implicated as a mediator of pneumococcal meningitis, both in experimental models and in humans. We demonstrated here that (1) Caspase 1 mRNA and protein expression is upregulated in the brain during experimental pneumococcal meningitis, and (2) Caspase 1 levels are elevated in the cerebrospinal fluid of patients with acute bacterial meningitis. The upregulation/activation of Caspase 1 was associated with increased levels of interleukin-1beta. Depletion of the Caspase 1 gene and pharmacologic blockade of Caspase 1 significantly attenuated the meningitis-induced increase in interleukin-1beta. This was paralleled by a significantly diminished inflammatory host response to pneumococci. The antiinflammatory effect of Caspase 1 depletion or blockade was associated with a marked reduction of meningitis-induced intracranial complications, thus leading to an improved clinical status. In humans, cerebrospinal fluid Caspase 1 levels correlated with the clinical outcome. Thus, pharmacologic inhibition may provide an efficient adjuvant therapeutic strategy in this disease.

Amino Acid Chloromethyl Ketones↗

PubMed2002

CDP-choline reduces pro-caspase and cleaved caspase-3 expression, nuclear DNA fragmentation, and specific PARP-cleaved products of caspase activation following middle cerebral artery occlusion in the rat.

Citicoline has been demonstrated to be beneficial in several models of cerebral ischaemia. We tested the hypothesis that citicoline may provide apoptotic pathways following focal cerebral ischaemia. Focal cerebral ischaemia was produced by distal, permanent middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. The animals were randomised into four groups: (B+A) Citicoline 500 mg/kg IP 24 and 1 h before MCAO, and 23 h after MCAO; (A) citicoline 500 mg/kg IP, within 30 min after MCAO, and 23 h after MCAO; (C) vehicle IP; and (D) sham-operated. The animals were sacrificed at 12 h (n=8 per group) and 24 h (n=8 per group) after MCAO. Immunohistochemistry was performed on free-floating tissue sections with goat polyclonal antibodies to procaspase-1, -2, -3, -6 and -8, and in paraffin-embedded sections processed for cleaved caspase-3 (17 kDa) immunohistochemistry. Finally, some sections were stained with the method of in situ end-labelling of nuclear DNA fragmentation. For gel electrophoresis and Western blotting, antibodies to poly (ADP-ribose) polymerase (PARP) products of 89 kDa were used to reveal specific cleavage substrates of caspases. MCAO induced the expression of all procaspases and the expression of PARP products of 89 kDa, as well as cells with nuclear DNA fragmentation, at 12 and 24 h, in the infarcted core and penumbra. Citicoline reduced the expression of all procaspases at 12 and 24 h after MCAO, as well as the expression of cleaved caspase-3 in cells in the penumbra area. This was accompanied by a reduction in the number of cells bearing nuclear DNA fragments. The expression of caspase-cleaved products of PARP (PARP 89 kDa) was reduced in citicoline-treated ischaemic rats. These results show that citicoline inhibits the expression of proteins involved in apoptosis following MCAO.

Animals↗

PubMed2003

Teratogen-induced activation of caspase-6 and caspase-7 in early postimplantation mouse embryos.

BACKGROUND: Previous work has shown that teratogens such as hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine (ST) induce cell death in day 9 mouse embryos by activating the mitochondrial apoptotic pathway. Key to the activation of this pathway is the activation of a caspase cascade involving the cleavage-induced activation of an initiator procaspase, caspase-9, and the downstream effector procaspase, caspase-3. For example, procaspase-3, an inactive proenzyme of 32 kDa is cleaved by activated caspase-9 to generate a large subunit of approximately 17 kDa and a small subunit of approximately 10 kDa. In turn, caspase-3 is known to target a variety of cellular proteins for proteolytic cleavage as part of the process by which dying cells are eliminated. Previous work has also shown that neuroepithelial cells are sensitive to teratogen-induced activation of this pathway and subsequent cell death whereas cells of the heart are resistant. Although caspase-3 is a key effector caspase activated by teratogens, two other effector caspases, caspase-6 and caspase-7, are known; however, their role in teratogen-induced cell death is unknown. METHODS: Because cleavage-induced generation of specific subunits is the most specific assay for activation of caspases, we have used antibodies that recognize the procaspase and one of its active subunits and a Western blot approach to assess the activation of caspase-6 and caspase-7 in day 9 mouse embryos (or heads, hearts and trunks isolated from whole embryos) exposed to HS, 4CP, and ST. To probe the relationship between teratogen-induced activation of caspase-9/caspase-3 and the activation of caspase-6/caspase-7, we used a mitochondrial-free embryo lysate with or without the addition of cytochrome c, recombinant active caspase-3, or recombinant active caspase-9. RESULTS: Western blot analyses show that these three teratogens, HS, 4CP, and ST, induce the activation of procaspase-6 (appearance of the 13 kDa subunit, p13) and caspase-7 (appearance of the 19 kDa subunit, p19) in day 9 mouse embryos. In vitro studies showed that both caspase-6 and caspase-7 could be activated by the addition of cytochrome c to a lysate prepared from untreated embryos. In addition, caspase-6 could be activated by the addition of either recombinant caspase-3 or caspase-9 to a lysate prepared from untreated embryos. In contrast, caspase-7 could be activated by addition of recombinant caspase-3 but only minimally by recombinant caspase-9. Like caspase-9/caspase-3, caspase-6 and caspase-7 were not activated in hearts isolated from embryos exposed to these three teratogens. CONCLUSIONS: HS, 4CP and ST induce the cleavage-dependent activation of caspase-6 and caspase-7 in day 9 mouse embryos. Results using DEVD-CHO, a caspase-3 inhibitor, suggest that teratogen-induced activation of caspase-6 is mediated by caspase-3. In addition, our data suggest that caspase-7 is activated primarily by caspase-3; however, we cannot rule out the possibility that this caspase is also activated by caspase-9. Finally, we also show that teratogen-induced activation of caspase-6 and caspase-7 are blocked in the heart, a tissue resistant to teratogen-induced cell death.

Animals↗

PubMed2003

ER stress induces caspase-8 activation, stimulating cytochrome c release and caspase-9 activation.

Excess ER stress induces caspase-12 activation and/or cytochrome c release, causing caspase-9 activation. Little is known about their relationship during ER stress-mediated cell death. Upon ER stress, P19 embryonal carcinoma (EC) cells showed activation of various caspases, including caspase-3, caspase-8, caspase-9, and caspase-12, and extensive DNA fragmentation. We examined the relationship between ER stress-mediated cytochrome c/caspase-9 and caspase-12 activation by using caspase-9- and caspase-8-deficient mouse embryonic fibroblasts and a P19 EC cell clone [P19-36/12 (-) cells] lacking expression of caspase-12. Caspase-9 and caspase-8 deficiency inhibited and delayed the onset of DNA fragmentation but did not inhibit caspase-12 processing induced by ER stress. P19-36/12 (-) cells underwent apoptosis upon ER stress, with cytochrome c release and caspase-8 and caspase-9 activation. The dominant negative form of FADD and z-VAD-fmk inhibited caspase-8, caspase-9, Bid processing, cytochrome c release, and DNA fragmentation induced by ER stress, suggesting that caspase-8 and caspase-9 are the main caspases involved in ER stress-mediated apoptosis of P19-36/12 (-) cells. Caspase-8 deficiency also inhibited the cytochrome c release induced by ER stress. Thus, in parallel with the caspase-12 activation, ER stress triggers caspase-8 activation, resulting in cytochrome c/caspase-9 activation via Bid processing.

Animals↗

PubMed1998

Identification of a new caspase homologue: caspase-14.

Caspases are cysteinyl aspartate-specific proteinases, many of which play a central role in apoptosis. Here, we report the identification of a new murine caspase homologue, viz. caspase-14. It is most related to human/murine caspase-2 and human caspase-9, possesses all the typical amino acid residues of the caspases involved in catalysis, including the QACRG box, and contains no or only a very short prodomain. Murine caspase-14 shows 83% similarity to human caspase-14. Human caspase-14 is assigned to chromosome 19p13.1. Northern blot analysis revealed that mRNA expression of caspase-14 is undetectable in all mouse adult tissues examined except for skin, while it is abundantly expressed in mouse embryos. In contrast to many other caspase family members, murine caspase-14 is not cleaved by granzyme B, caspase-1, caspase-2, caspase-3, caspase-6, caspase-7 or caspase-11, but is weakly processed into p18 and p11 subunits by murine caspase-8. No aspartase activity of murine caspase-14 could be generated by bacterial or yeast expression. Transient overexpression of murine caspase-14 in mammalian cells did not elicit cell death and did not interfere with caspase-8-induced apoptosis. In conclusion, caspase-14 is a member of the caspase family but no proteolytic or biological activities have been identified so far. The high constitutive expression levels in embryos and specific expression in adult skin suggest a role in ontogenesis and skin physiology.

Amino Acid Sequence↗

PubMed1998

Caspase-2 (Nedd-2) processing and death of trophic factor-deprived PC12 cells and sympathetic neurons occur independently of caspase-3 (CPP32)-like activity.

We have previously shown that caspase-2 (Nedd-2) is required for apoptosis induced by withdrawal of trophic support from PC12 cells and sympathetic neurons. Here, we examine the relationship of caspase-2 processing and cell death to induction of caspase-3 (CPP32)-like activity in PC12 cells. Caspase-2 processing, at a site tentatively identified as D333, led to the formation of an N-terminal 37 kDa product. This processing correlated temporally with induction of caspase-3-like activity. Agents previously shown to inhibit caspase-3-like activation, such as bcl-2 and the Cdk inhibitor flavopiridol, also acted upstream of caspase-2 processing. The general caspase inhibitors BAF and zVAD-FMK inhibited N-terminal caspase-2 processing. In contrast, the more selective caspase inhibitor DEVD-FMK inhibited the induction of caspase-3-like activity but did not affect caspase-2 processing or significantly suppress death in PC12 cells or sympathetic neurons. This indicates that caspase-3-like activity is not required for either caspase-2 processing or apoptosis in this paradigm. An antisense oligonucleotide to caspase-2 inhibited cell death but did not affect caspase-3-like activity, indicating that caspase-2 is not upstream of this activity and that activation of caspase-3-like caspases is not sufficient for death. Thus, in our paradigm, caspase-2 processing and caspase-3-like activity are induced independently of each other. Moreover, although death requires caspase-2, caspase-3-like activity is neither necessary nor sufficient for death.

Amino Acid Chloromethyl Ketones↗

PubMed2006

Caspase-3 mediated feedback activation of apical caspases in doxorubicin and TNF-alpha induced apoptosis.

Aberrant apoptosis has been associated with the development and therapeutic resistance of cancer. Recent studies suggest that caspase deficiency/downregulation is frequently detected in different cancers. We have previously shown that caspase-3 reconstitution significantly sensitized MCF-7 cells to doxorubicin and etoposide. In contrast to the well established role of caspase-3 as an effector caspase, the focus of this study is to delineate caspase-3 induced feedback activation of the apical caspases-2, -8, -9 and -10A in doxorubicin and TNF-alpha induced apoptosis. Using cell-free systems we show that caspases-9 and 2 are the most sensitive, caspase-8 is less sensitive and caspase-10A is the least sensitive to caspase-3 mediated-cleavage. When apoptosis is induced by doxorubicin or TNF-alpha in an intact cell model, cleavage of caspases-8 and -9, but not caspase-2, was markedly enhanced by caspase-3. Caspase-3 mediated-feedback and activation of caspase-8 and -9 in MCF-7/C3 cells is further supported by an increase in the cleavage of caspase-8 and 9 substrates and cytochrome c release. These data indicate that, in addition to its function as an effector caspase, caspase-3 plays an important role in maximizing the activation of apical caspases and crosstalk between the two major apoptotic pathways. The significant impact of caspase-3 on both effector and apical caspases suggests that modulation of caspase-3 activity would be a useful approach to overcome drug resistance in clinical oncology.

Antibiotics, Antineoplastic↗

PubMed2001

Death receptor recruitment of endogenous caspase-10 and apoptosis initiation in the absence of caspase-8.

Caspase-8 is believed to play an obligatory role in apoptosis initiation by death receptors, but the role of its structural relative, caspase-10, remains controversial. Although earlier evidence implicated caspase-10 in apoptosis signaling by CD95L and Apo2L/TRAIL, recent studies indicated that these death receptor ligands recruit caspase-8 but not caspase-10 to their death-inducing signaling complex (DISC) even in presence of abundant caspase-10. We characterized a series of caspase-10-specific antibodies and found that certain commercially available antibodies cross-react with HSP60, shedding new light on previous results. The majority of 55 lung and breast carcinoma cell lines expressed mRNA for both caspase-8 and -10; however, immunoblot analysis revealed that caspase-10 protein expression was more frequently absent than that of caspase-8, suggesting a possible selective pressure against caspase-10 production in cancer cells. In nontransfected cells expressing both caspases, CD95L and Apo2L/TRAIL recruited endogenous caspase-10 as well as caspase-8 to their DISC, where both enzymes were proteolytically processed with similar kinetics. Caspase-10 recruitment required the adaptor FADD/Mort1, and caspase-10 cleavage in vitro required DISC assembly, consistent with the processing of an apoptosis initiator. Cells expressing only one of the caspases underwent ligand-induced apoptosis, indicating that each caspase can initiate apoptosis independently of the other. Thus, apoptosis signaling by death receptors involves not only caspase-8 but also caspase-10, and both caspases may have equally important roles in apoptosis initiation.

Amino Acid Sequence↗

PubMed2000

Frequent nuclear localization of ICAD and cytoplasmic co-expression of caspase-8 and caspase-3 in human lymphomas.

Lymphoma cells often display in vitro resistance to FAS-induced apoptosis, in which caspases act as crucial cell death effectors. Following FAS stimulation, caspase-8 activates caspase-3, which in turn activates the caspase-activated DNAse (CAD) by proteolysis of its inhibitor (ICAD). To investigate the mechanism of FAS resistance, the expression of caspase-8 was analysed by immunohistochemistry, together with that of the substrates caspase-3 and ICAD, in 52 representative samples from non Hodgkin's lymphoma (NHL), 12 from Hodgkin's disease (HD), and eight benign lymphoid tissues. In benign tissues, caspase-8 was co-expressed with caspase-3 in the cytoplasm in germinal centre (GC) cells and was co-expressed with ICAD in the nuclei of the mantle and marginal zone cells. ICAD expression was weak or absent in GC cells. Cytoplasmic staining for both caspase-8 and caspase-3 was present in 11/12 cases of diffuse large cell B-NHL. Caspase-8 positivity was nuclear and cytoplasmic in 9/9 follicular NHLs, in 5/5 mantle cell NHLs and in 6/6 marginal zone NHLs. Five out of six peripheral T-cell NHLs expressed cytoplasmic caspase-8. Ten out of the 12 HD cases lacked significant cytoplasmic staining for caspase-3 and caspase-8 in the majority of Reed-Sternberg cells. All lymphoma cases exhibited predominant nuclear ICAD positivity. Subcellular fractionation analysis of three lymphoma samples and normal mantle zone cells confirmed that ICAD and caspase-8 were at least partly localized in the nucleus. These results show that the profile of caspase-8 expression is correlated with histological lymphoma subtypes; that caspase-8 is co-expressed with caspase-3 in GC cells and their neoplastic counterparts; that ICAD has an immunohistochemical nuclear localization in vivo; and that caspase-8 and ICAD can be co-expressed in the nuclei of mantle zone and marginal zone cells; their unexpected nuclear localization allows a reappraisal of the biochemical cascade of caspase activation.

Apoptosis Regulatory Proteins↗

PubMed2004

Synthetic 1,4-anthracenedione analogs induce cytochrome c release, caspase-9, -3, and -8 activities, poly(ADP-ribose) polymerase-1 cleavage and internucleosomal DNA fragmentation in HL-60 cells by a mechanism which involves caspase-2 activation but not Fas signaling.

Synthetic analogs of 1,4-anthraquinone (AQ code number), a compound that mimics the antiproliferative effects of daunorubicin (daunomycin) in the nanomolar range in vitro but has the advantage of blocking nucleoside transport and retaining its efficacy in multidrug-resistant tumor cells, were tested for their ability to induce apoptosis in the HL-60 cell system. AQ10 and, especially, the new lead antiproliferative compounds AQ8 and AQ9 reduce the growth and integrity of wild-type, drug-sensitive, HL-60-S cells more effectively than AQ1, suggesting that various methyl group substituents at C6 may enhance the bioactivity of the parent compound. Internucleosomal DNA fragmentation, a late marker of apoptosis, is similarly induced in a biphasic manner by increasing concentrations of AQ8 and AQ9 at 24 hr. Poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, an early event required for cells committed to apoptosis, is detected within 3-6 hr in HL-60-S cells treated with AQ9. In accord with the fact that the caspases 9 and 3 cascade is responsible for PARP-1 cleavage, the activities of initiator caspase-9 and effector caspase-3 are induced by AQ9 in the same time- and concentration-dependent manners and to the same maximal degrees in both the HL-60-S and multidrug-resistant HL-60-RV cell lines. Interestingly, a 1-hr pulse treatment is sufficient for AQ8 and AQ9 to maximally induce caspase-9 and -3 activities at 6 hr. The release of mitochondrial cytochrome c (Cyt c) is also detected within 3-6hr in HL-60-S cells treated with AQ9, a finding consistent with the fact that Cyt c is the apoptotic trigger that activates caspase-9. Moreover, AQ analogs induce Cyt c release, caspase-9 and -3 activities and PARP-1 cleavage in relation with their abilities to decrease tumor cell growth and integrity, AQ8 and AQ9 being consistently the most effective. Since apical caspases 2 and 8 may both act upstream of mitochondria to promote Cyt c release, it is significant to show that AQ9 maximally induces caspase-2 and -8 activities at 6 and 9 hr, respectively. During AQ8 treatment, the caspase-2 inhibitor benzyloxycarbonyl (z)-Val-Asp-Val-Ala-Asp (VDVAD)-fluoromethyl ketone (fmk) totally blocks caspase-9, -3, and -8 activations, whereas the caspase-8 inhibitor z-Ile-Glu-Thr-Asp-(IETD)-fmk does not prevent caspase-2, -9, and -3 activations, suggesting that AQ-induced caspase-2 activity is an upstream event critical for the activation of the downstream caspases 9 and 3 cascade, including the mitochondrial amplification loop through caspase-8. However, these caspase-2 and -8 inhibitors fail to alter AQ8-induced Cyt c release, suggesting that AQs might also target mitochondria independently from caspase activation. Furthermore, the antagonistic anti-Fas DX2 and ZB4 monoclonal antibodies (mAbs), which block the induction of Cyt c release and caspase-2, -8, and -9 activities by the agonistic anti-Fas CH11 mAb, and the neutralizing anti-Fas ligand (FasL) NOK-1 mAb all fail to inhibit AQ9-induced Cyt c release and caspase-2, -8, and -9 activities, suggesting that the FasL/Fas signaling pathway is not involved in the mechanism by which antiproliferative AQ analogs trigger apoptosis in HL-60 cells.

Anthraquinones↗

PubMed1997

Caspase-4 and caspase-5, members of the ICE/CED-3 family of cysteine proteases, are CrmA-inhibitable proteases.

Proteases of the caspase family are implicated in mammalian apoptosis and constitute a protease cascade. We characterized caspase-4 (TX/ICH-2/ICErelII) and caspase-5 (ICErelIII/TY), which are most closely related to caspase-1 (ICE) among the caspase family. Although overexpression of caspase-4 and caspase-5 induced apoptosis, confirming previous observations, this apoptosis was not inhibited by a caspase-1-specific tetrapeptide inhibitor (Ac-YVAD-CHO), suggesting that caspase-4 and caspase-5 have different substrate specificities from caspase-1 and also that caspase-4- and caspase-5-induced apoptosis is not mediated by caspase-1. CrmA, a cowpox virus-derived caspase-1 inhibitor that prevents apoptosis induced by various stimuli, was cleaved by caspase-4 and caspase-5, and inhibited their proteolytic activity as assessed by cleavage of pro-caspase-3 (pro-CPP32/Yama/apopain). Thus, caspase-4 and caspase-5 are CrmA-inhibitable proteases like caspase-1 and might be involved in apoptosis.

Journal Article↗

PubMed2001

Caspase-9 activation results in downstream caspase-8 activation and bid cleavage in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease.

Parkinson's disease (PD) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity are both associated with dopaminergic neuron death in the substantia nigra (SN). Apoptosis has been implicated in this cell loss; however, whether or not it is a major component of disease pathology remains controversial. Caspases are a major class of proteases involved in the apoptotic process. To evaluate the role of caspases in PD, we analyzed caspase activation in MPTP-treated mice, in cultured dopaminergic cells, and in postmortem PD brain tissue. MPTP was found to elicit not only the activation of the effector caspase-3 but also the initiators caspase-8 and caspase-9, mitochondrial cytochrome c release, and Bid cleavage in the SN of wild-type mice. These changes were attenuated in transgenic mice neuronally expressing the general caspase inhibitor protein baculoviral p35. These mice also displayed increased resistance to the cytotoxic effects of the drug. MPTP-associated toxicity in culture was found temporally to involve cytochrome c release, activation of caspase-9, caspase-3, and caspase-8, and Bid cleavage. Caspase-9 inhibition prevented the activation of both caspase-3 and caspase-8 and also inhibited Bid cleavage, but not cytochrome c release. Activated caspase-8 and caspase-9 were immunologically detectable within MPP(+)-treated mesencephalic dopaminergic neurons, dopaminergic nigral neurons from MPTP-treated mice, and autopsied Parkinsonian tissue from late-onset sporadic cases of the disease. These data demonstrate that MPTP-mediated activation of caspase-9 via cytochrome c release results in the activation of caspase-8 and Bid cleavage, which we speculate may be involved in the amplification of caspase-mediated dopaminergic cell death. These data suggest that caspase inhibitors constitute a plausible therapeutic for PD.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2004

Sequential caspase-2 and caspase-8 activation upstream of mitochondria during ceramideand etoposide-induced apoptosis.

Recently, caspase-2 was shown to act upstream of mitochondria in stress-induced apoptosis. Activation of caspase-8, a key event in death receptor-mediated apoptosis, also has been demonstrated in death receptor-independent apoptosis. The regulation of these initiator caspases, which trigger the mitochondrial apoptotic pathway, is unclear. Here we report a potential regulatory role of caspase-2 on caspase-8 during ceramide-induced apoptosis. Our results demonstrate the sequential events of initiator caspase-2 and caspase-8 activation, Bid cleavage and translocation, and mitochondrial damage followed by downstream caspase-9 and -3 activation and cell apoptosis after ceramide induction in T cell lines. The expression of truncated Bid (tBid) and the reduction in mitochondrial transmembrane potential were blocked by caspase-2 or caspase-8, but not caspase-3, knockdown using an RNA interference technique. Ceramide-induced caspase-8 activation, mitochondrial damage, and apoptosis were blocked in caspase-2 short interfering RNA-expressing cells. Therefore, caspase-2 acts upstream of caspase-8 during ceramide-induced mitochondrial apoptosis. Similarly, sequential caspase-2 and caspase-8 activation upstream of mitochondria was also observed in etoposide-induced apoptosis. These data suggest sequential initiator caspase-2 and caspase-8 activation in the mitochondrial apoptotic pathway induced by ceramide or etoposide.

Animals↗

PubMed1998

Caspase 2 and caspase 3 protein levels as predictors of survival in acute myelogenous leukemia.

Because caspase activation is an essential step in programmed cell death (apoptosis) and cytotoxic drug-induced apoptosis is mediated by caspase 2 and caspase 3, we hypothesized that caspase 2 and 3 levels predict clinical outcome in acute myelogenous leukemia (AML). Using quantitative Western blot analysis, we studied the levels of nonactivated (uncleaved) caspase 2 and 3 in peripheral blood low-density cells from 185 patients with newly diagnosed AML. We also measured the level of activated (cleaved) caspase 3 in 41 randomly selected samples from the 185 patients. Finally, we analyzed the effect of caspase 2 and 3 levels and other prognostic variables on patient survival using a multivariate Cox model. We found that median levels of nonactivated caspase 2 and 3 were higher in AML than in normal peripheral blood cells (P < .001 and P <.02, respectively). There was no association between caspase level and either the percentage of peripheral blasts or any specific type of leukemia cell cytogenetic abnormalities. When the effect of each uncleaved caspase was considered individually, a high level of uncleaved caspase 3 (P = .04), but not of caspase 2 (P = .16), was associated with decreased survival. Conversely, a high level of cleaved caspase 3 denoted improved survival and correlated with the inactivation of the DNA-repair enzyme poly(ADP-ribose) polymerase. Thus, cleaved caspase 3 could stimulate the apoptotic cascade further, and lack of its activation likely caused an accumulation of the uncleaved caspase. Although uncleaved caspase 2 level per se had no prognostic significance, the interactive effect of high levels of both uncleaved caspase 2 and 3 denoted very poor survival (P < .001) and had the largest effect of all prognostic variables (P < .001; estimated relative risk, 2.49; 95% confidence interval, 1.59 to 3. 90). Taken together, caspase 2 and caspase 3 protein levels obtained at diagnosis may constitute a reliable prognostic factor in AML.

Acute Disease↗