Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.62 (caspase-9)
 7,507 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent studies have demonstrated that Apaf-1 is the adaptor molecule which in the presence of cytosolic cytochrome c (cyt c) and dATP interacts with procaspase-9, resulting in the sequential cleavage and activity of caspase-9 and caspase-3, followed by apoptosis. In the present studies, we determined the effect of enforced overexpression of Apaf-1 on the apoptotic threshold in the human myeloid leukemia HL-60 cells. Our findings demonstrate that both transient and stable transfections resulted in a 2.5-fold higher expression of Apaf-1, which was associated with approximately a 5-fold increase in the percentage of apoptosis in the transfectants (HL-60/Apaf-1) as compared with the control HL-60/neo cells. In cells overexpressing either Bcl-2 or Bcl-xL, transient overexpression of Apaf-1 did not induce apoptosis. Stably overexpressing Apaf-1 levels significantly sensitized HL-60/Apaf-1 cells to apoptosis induced by clinically achievable concentrations of paclitaxel or etoposide (P < 0.01). This increase in paclitaxel- or etoposide-induced apoptosis of HL-60/Apaf-1 cells was not associated with any significant alterations in Bcl-2, Bcl-xL, Bax, Fas, or Fas ligand expression. It was, however, clearly associated with caspase-9 cleavage, as well as the poly(ADP-ribose) polymerase and DFF45 cleavage activity of caspase-3. Coexpression of the catalytically inactive, dominant-negative, mutant caspase-9, XIAP, or treatment with the caspase inhibitor, zVAD, significantly inhibited the increase in apoptosis of HL-60/Apaf-1 cells (P < 0.01). These data indicate that the intracellular levels of Apaf-1 is an important molecular determinant of the threshold for apoptosis induced by paclitaxel and etoposide.
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PMID:Overexpression of Apaf-1 promotes apoptosis of untreated and paclitaxel- or etoposide-treated HL-60 cells. 978 1

Epidermal growth factor (EGF), a hormone that stimulates proliferation of many cell types, induces apoptosis in some cell lines that overexpress the EGF receptor. To evaluate the mechanism of EGF-induced apoptosis, MDA-MB-468 breast cancer cells were examined by microscopy, flow cytometry, immunoblotting, enzyme assays, and affinity labeling after treatment with EGF, paclitaxel, or 5-fluoro-2'-deoxyuridine (5FUdR). Apoptosis induced by all three agents was accompanied by activation of caspases-3, -6, and -7, as indicated by disappearance of the corresponding zymogens from immunoblots, cleavage of substrate polypeptides in situ, and detection of active forms of these caspases in cytosol and nuclei using fluorogenic assays and affinity labeling. Further analysis indicated involvement of the cytochrome c/Apaf-1/caspase-9 pathway of caspase activation, but not the Fas/Fas ligand pathway. Interestingly, caspase activation was consistently lower after EGF treatment than after paclitaxel or 5FUdR treatment. Additional experiments revealed that the majority of cells detaching from the substratum after EGF (but not paclitaxel or 5FUdR) were morphologically normal and retained the capacity to readhere, suggesting that EGF-induced apoptosis involves cell detachment followed by anoikis. These observations not only indicate that EGF- and chemotherapy-induced apoptosis in this cell line involve the same downstream pathways but also suggest that detachment-induced apoptosis is responsible for the paradoxical antiproliferative effects of EGF.
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PMID:Comparison of paclitaxel-, 5-fluoro-2'-deoxyuridine-, and epidermal growth factor (EGF)-induced apoptosis. Evidence for EGF-induced anoikis. 1033 99

Transforming growth factor-beta1 (TGF-beta1) has been shown to induce apoptosis in normal or transformed hepatocytes. To elucidate the biochemical pathways leading to apoptosis induced by TGF-beta1 in human hepatoma cells (HuH-7), we examined the expression of Bcl-2-related proteins and X-chromosome-linked inhibitor of apoptosis (XIAP), and activation of the caspase cascade following TGF-beta1 treatment. Bcl-xL expression began to decline at 12 hours after TGF-beta1 treatment and progressively decreased to very low levels in a time-dependent manner. Bax expression showed a little change throughout the experiment. On the other hand, activation of caspase-8 was clearly observed at 36 hours after TGF-beta1 treatment, followed by activation of caspase-9, and caspase-3 was activated at 48 hours after treatment at which time apoptosis of HuH-7 cells was observed. TGF-beta1 significantly decreased XIAP expression in HuH-7 cells. Addition of an inhibitor of caspase-8 or caspase-3 (IETD-FMK or DEVD-CHO) markedly inhibited TGF-beta1-induced apoptosis of HuH-7 cells. Fas/Fas ligand (FasL) interactions in HuH-7 cells were not involved in the apoptotic process. Furthermore, epidermal growth factor (EGF) also completely inhibited TGF-beta1-induced apoptosis of HuH-7 cells by inhibiting activation of the caspase cascade. Our results suggested that activation of caspase-3 initiated through caspase-8 activation is involved in the apoptotic process induced by TGF-beta1 in HuH-7 cells. Our results also showed that down-regulation of the expression of Bcl-xL and XIAP by TGF-beta1 may facilitate activation of caspase-3 in these cells.
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PMID:Activation of caspase-8 in transforming growth factor-beta-induced apoptosis of human hepatoma cells. 1053 43

alpha-Fetoprotein (AFP) is an oncoembryonal protein with multiple cell growth regulating, differentiating and immunosuppressive activities. Previous studies have shown that treatment of tumor cells in vitro with 1-10 microM AFP produces significant suppression of tumor cell growth by inducing dose-dependent cytotoxicity, but the molecular mechanisms underlying these AFP functions are obscure. Here, we show that AFP cytotoxicity is closely related to apoptosis, as shown by cell morphology, nuclear DNA fragmentation and caspase-3-like activity resulting in cleavage of poly(ADP-ribose) polymerase. Apoptosis was significantly inhibited by a CPP32 family protease inhibitor whereas a general caspase inhibitor had no inhibitory effect, showing some enhancement of AFP-mediated cell death. Using fluorogenic caspase substrates, we found that caspase-3-like proteases were activated as early as 4 h after treatment of Raji cells with 15 microM AFP, whereas caspase-1, caspase-8, and caspase-9-like activity was not detected during the time interval 0.5-17 h. AFP treatment of Raji cells increased Bcl-2 protein, showing that AFP-induced apoptosis is not explained by downregulation of the Bcl-2 gene. This also suggests that AFP operates downstream of the Bcl-2-sensitive step. AFP notably decreased basal levels of soluble and membrane-bound Fas ligand. Incubation of AFP-sensitive tumor cells (HepG2, Raji) with neutralizing anti-Fas, anti-tumor necrosis factor receptor (TNFR)1 or anti-TNFR2 mAb did not prevent AFP-induced apoptosis, demonstrating its independence of Fas-dependent and TNFR-dependent signaling. In addition, it was found that cells resistant to TNF-induced (Raji) or Fas-induced (MCF-7) apoptosis are, nevertheless, sensitive to AFP-mediated cell death. In contrast, cells sensitive to Fas-mediated cell death (Jurkat) are completely resistant to AFP. Taken as a whole, our data demonstrate that: (a) AFP induces apoptosis in tumor cells independently of Fas/Fas ligand or TNFR/TNF signaling pathways, and (b) AFP-mediated cell death involves activation of the effector caspase-3-like proteases, but is independent of upstream activation of the initiator caspase-1, caspase-8, and caspase-9-like proteases.
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PMID:alpha-fetoprotein causes apoptosis in tumor cells via a pathway independent of CD95, TNFR1 and TNFR2 through activation of caspase-3-like proteases. 1058 68

Ceramide is characterized as a second messenger of apoptosis induced by various agents such as tumor necrosis factor (TNF-alpha), Fas ligand, hydrogen peroxide, heat shock and ionizing radiation. In this study, we investigated the mechanism of ceramide-induced apoptosis using a human neuroblastoma cell line, SK-N-MC. N-Acetyl-sphingosine (C2-ceramide), a cell-permeable ceramide analogue, was able to induce apoptosis in SK-N-MC cells as estimated by DNA fragmentation and chromatin condensation. C2-ceramide-induced DNA fragmentation was blocked by caspase inhibitor (Z-Asp-CH(2)-DCB). An increase in caspase-3 (CPP32)-like protease activity was evident during C2-ceramide-induced apoptosis, suggesting that caspases are involved in this apoptosis. Moreover, enzymatic cleavage of VDVAD-AFC and LEHD-AFC (specific substrates for caspase-2 and -9, respectively) was increased by treatment with C2-ceramide. To elucidate which types of caspase are activated in C2-ceramide-treated cells, we performed Western blot analysis using antibodies against each isoform. Both proforms of caspase-2 and -3 were decreased in response to C2-ceramide in a time-dependent manner. Mitochondrial cytochrome c is also time-dependently released into the cytosol in response to treatment with C2-ceramide. Addition of cytochrome c into the S-100 fractions prepared from SK-N-MC cells could activate caspase-2 in cell-free systems. These results suggest the possibility that cytochrome c released to the cytosol can activate caspases (caspase-9, -3, and -2) during C2-ceramide-induced apoptosis of SK-N-MC cells.
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PMID:Possible involvement of cytochrome c release and sequential activation of caspases in ceramide-induced apoptosis in SK-N-MC cells. 1059 Mar 15

Multidrug resistance (MDR) is often characterized by the expression of P-glycoprotein (P-gp), a 170-kd ATP-dependent drug efflux protein. As well as effluxing xenotoxins, functional P-gp can confer resistance to caspase-dependent apoptosis induced by a range of different stimuli, including Fas ligand, tumor necrosis factor, UV irradiation, and serum starvation. However, P-gp-positive cells remain sensitive to caspase-independent death induced by cytotoxic T-cell granule proteins, perforin, and granzyme B. It is, therefore, possible that agents that induce cell death in a caspase-independent manner might circumvent P-gp-mediated MDR. We demonstrated here that hexamethylene bisacetamide (HMBA) induced equivalent caspase-independent cell death in both P-gp-positive and -negative cell lines at concentrations of 10 mmol/L and above. The HMBA-induced death pathway was marked by release of cytochrome c from the mitochondria and reduction of Bcl-2 protein levels. In addition, we show that functional P-gp specifically inhibits the activation of particular caspases, such as caspases-8 and -3, whereas others, such as caspase-9, remain unaffected. These studies greatly enhance our understanding of the molecular cell death events that can be regulated by functional P-gp and highlight the potential clinical use of drugs that function via a caspase-independent pathway for the treatment of MDR tumors.
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PMID:HMBA induces activation of a caspase-independent cell death pathway to overcome P-glycoprotein-mediated multidrug resistance. 1073 10

Cytotoxic endoribonucleases (RNases) possess a potential for use in cancer therapy. However, the molecular determinants of RNase-induced cell death are not well understood. In this work, we identify such determinants of the cytotoxicity induced by onconase, an amphibian cytotoxic RNase. Onconase displayed a remarkable specificity for tRNA in vivo, leaving rRNA and mRNA apparently undamaged. Onconase-treated cells displayed apoptosis-associated cell blebbing, nuclear pyknosis and fragmentation (karyorrhexis), DNA fragmentation, and activation of caspase-3-like activity. The cytotoxic action of onconase correlated with inhibition of protein synthesis; however, we present evidence for the existence of a mechanism of onconase-induced apoptosis that is independent of inhibition of protein synthesis. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone (zVADfmk), at concentrations that completely prevent apoptosis and caspase activation induced by ligation of the death receptor Fas, had only a partial protective effect on onconase-induced cell death. The proapoptotic activity of the p53 tumor suppressor protein and the Fas ligand/Fas/Fas-associating protein with death domain (FADD)/caspase-8 proapoptotic cascade were not required for onconase-induced apoptosis. Procaspases-9, -3, and -7 were processed in onconase-treated cells, suggesting the involvement of the mitochondrial apoptotic machinery in onconase-induced apoptosis. However, the onconase-induced activation of the caspase-9/caspase-3 cascade correlated with atypically little release of cytochrome c from mitochondria. In turn, the low levels of cytochrome c released from mitochondria correlated with a lack of detectable translocation of proapoptotic Bax from the cytosol onto mitochondria in response to onconase. This suggests the possibility of involvement of a different, potentially Bax- and cytochrome c-independent mechanism of caspase-9 activation in onconase-treated cells. As one possible mechanism, we demonstrate that procaspase-9 is released from mitochondria in onconase-treated cells. A detailed understanding of the molecular determinants of the cytotoxic action of onconase could provide means of positive or negative therapeutic modulation of the activity of this potent anticancer agent.
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PMID:Molecular determinants of apoptosis induced by the cytotoxic ribonuclease onconase: evidence for cytotoxic mechanisms different from inhibition of protein synthesis. 1076 89

The BRCA1 tumor suppressor gene has previously been implicated in induction of high levels of apoptosis in osteocarcinoma cell lines. Overexpression of BRCA1 was shown to induce an apoptotic signaling pathway involving the c-Jun N-terminal kinase (JNK), but the signaling steps upstream and downstream of JNK were not delineated. To better understand the role of BRCA1 in apoptosis, we examined the effect of wild-type and C-terminal-truncated dominant negative BRCA1 on breast and ovarian cancer cell lines subjected to a number of different pro-apoptotic stimuli, including growth factor withdrawal, substratum detachment, ionizing radiation, and treatment with anticancer agents. All of these treatments were found to induce substantial levels of apoptosis in the presence of wild-type BRCA1, whereas dominant negative BRCA1 truncation mutants diminished the apoptotic response. Subsequent mapping of the apoptotic pathway induced by growth factor withdrawal demonstrated that BRCA1 enhanced signaling through a pathway that sequentially involved H-Ras, MEKK4, JNK, Fas ligand/Fas interactions, and caspase-9 activation. In addition, the pathway functioned independently of the p53 tumor suppressor. These data suggest that BRCA1 is an important modulator of the response to cellular stress and that loss of this apoptotic potential due to BRCA1 mutations may contribute to tumor development.
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PMID:BRCA1 facilitates stress-induced apoptosis in breast and ovarian cancer cell lines. 1093 85

In the therapy of various kinds of tumors, methylating agents generating O6-methylguanine (O6MeG) in DNA are used. We studied the molecular mechanism of cell death induced by these agents by comparing isogenic cell lines proficient (MGMT+) and deficient (MGMT-) for the DNA repair protein alkyltransferase and exhibiting the tolerance phenotype. Hypersensitivity to methylation-induced cell killing of MGMT- cells is attributable to the potent induction of apoptosis. We show that apoptosis is a late event occurring >48 h after methylation. It was preceded by decrease in Bcl-2 protein level and accompanied by activation of caspase-9 and caspase-3. We also observed cytochrome c release and hypophosphorylation of Bad. Other members of the Bcl-2 family (Bag-1, Bak, Bax, and Bcl-xL) were not altered in expression. Transfection of MGMT- cells with bcl-2 protected against methylation-induced apoptosis, indicating that Bcl-2 plays a key role in the response. Induction of apoptosis in MGMT- cells was not triggered by Fas and Fas ligand (CD95, Apo-1) because both proteins remained unaltered in expression and receptor-proximal caspase-8 was not activated after methylation. Also, inhibition of caspase-8 was ineffective in modifying the apoptotic response, whereas inhibition of caspase-3 and caspase-9 blocked apoptosis. Tolerant cells that are unable to repair O6MeG and are impaired in mismatch repair were less sensitive regarding the induction of apoptosis and Bcl-2 decline, supporting the view that O6MeG-induced apoptosis requires mismatch repair. The ultimate O6MeG-derived lesions triggering the apoptotic pathway are likely to be DNA double-strand breaks, which were significantly formed in MGMT- but not in MGMT+ and tolerant cells and which preceded apoptosis. Overall, the data indicate that O6MeG induces apoptosis via secondary lesions that trigger Bcl-2 decline, cytochrome c release, and caspase-9 and caspase-3 activation independently of Fas/Fas ligand and p53, for which the cells are mutated.
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PMID:Apoptosis induced by DNA damage O6-methylguanine is Bcl-2 and caspase-9/3 regulated and Fas/caspase-8 independent. 1105 78

Past studies have shown that TNF-related apoptosis-inducing ligand (TRAIL) induced apoptosis in a high proportion of cultured melanoma by caspase-dependent mechanisms. In the present studies we have examined whether TRAIL-induced apoptosis of melanoma was mediated by direct activation of effector caspases or whether apoptosis was dependent on changes in mitochondrial membrane potential (MMP) and mitochondrial-dependent pathways of apoptosis. Changes in MMP were measured by fluorescent emission from rhodamine 123 in mitochondria. TRAIL, but not TNF-alpha or Fas ligand, was shown to induce marked changes in MMP in melanoma, which showed a high correlation with TRAIL-induced apoptosis. This was associated with activation of proapoptotic protein Bid and release of cytochrome c into the cytosol. Overexpression of B cell lymphoma gene 2 (Bcl-2) inhibited TRAIL-induced release of cytochrome c, changes in MMP, and apoptosis. The pan caspase inhibitor z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and the inhibitor of caspase-8 (z-Ile-Glu-Thr-Asp-fluoromethylketone; zIETD-fmk) blocked changes in MMP and apoptosis, suggesting that the changes in MMP were dependent on activation of caspase-8. Activation of caspase-9 also appeared necessary for TRAIL-induced apoptosis of melanoma. In addition, TRAIL, but not TNF-alpha or Fas ligand, was shown to induce clustering of mitochondria around the nucleus. This process was not essential for apoptosis but appeared to increase the rate of apoptosis. Taken together, these results suggest that TRAIL induces apoptosis of melanoma cells by recruitment of mitochondrial pathways to apoptosis that are dependent on activation of caspase-8. Therefore, factors that regulate the mitochondrial pathway may be important determinants of TRAIL-induced apoptosis of melanoma.
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PMID:TNF-related apoptosis-inducing ligand-induced apoptosis of melanoma is associated with changes in mitochondrial membrane potential and perinuclear clustering of mitochondria. 1106 17


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