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Results for “Apoptosis”

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PubMed2000

TNF-related apoptosis-inducing ligand-induced apoptosis of melanoma is associated with changes in mitochondrial membrane potential and perinuclear clustering of mitochondria.

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.

Amino Acid Chloromethyl Ketones↗

PubMed2002

Kinetics of HL-60 cell entry to apoptosis during treatment with TNF-alpha or camptothecin assayed by the stathmo-apoptosis method.

BACKGROUND: Duration of apoptosis, from onset to final disintegration of the cell, is often short and variable. The apoptotic index (AI), as a snapshot of a transient event of variable length, does not truly represent incidence of apoptosis in the studied cell population. We recently proposed to estimate the cumulative apoptotic index (CAI) by inducing stathmo-apoptosis. A fluorescent inhibitor of caspases (FLICA) FAM-VAD-FMK is used to arrest the process of apoptosis and thereby prevent cell disintegration. Simultaneously, the arrested/apoptotic cells become FLICA-labeled. In the present study, this approach was applied to measure kinetics of HL-60 cell entrance into apoptosis induced via cell surface death receptor or a mitochondria-initiated pathway. Materials and Methods Cultures of HL-60 cells were treated with either TNF-alpha or camptothecin (CPT) in the absence or constant presence of 10-50 microM FLICA. The CAI was measured at different time points for up to 48 h by flow cytometry. Bivariate analysis of DNA content and cell labeling with FLICA was used to correlate apoptosis with the cell-cycle position. RESULTS: Selective loss of apoptotic cells seen in HL-60 cell cultures exposed to either TNF-alpha or CPT alone was prevented in cultures containing FLICA. Addition of FLICA alone had no effect on cell viability. The percentage of FLICA-labeled cells was plotted as a function of time after addition of TNF-alpha or CPT. The rate of cell entry to apoptosis was subsequently estimated from the slopes of the stathmo-apoptotic plot. The slopes revealed that the TNF-alpha or CPT-treated cells asynchronously underwent apoptosis with a stochastic-like kinetics and at two different rates. About 50% of cells in the TNF-alpha-treated cultures underwent apoptosis during the initial 6 h at a rate of approximately 8% of cells per hour; the remaining cells were undergoing apoptosis at a rate of approximately 2.5% of cells per hour for up to 24 h. Also, about 50% of the CPT-treated cells, predominantly those in S phase of the cell cycle, underwent apoptosis within the initial 8 h of CPT exposure, at a rate of approximately 7% of cells per hour. Remaining cells were undergoing apoptosis at a rate of approximately 1% of cells per hour during up to 48 h exposure to CPT. Spontaneous apoptosis in the untreated cultures occurred at a rate of 0.2% of cells per hour. CONCLUSIONS: This approach provides a means for measuring the kinetics of cell entrance to apoptosis (caspase activation) in large populations of cells in relation to the cell-cycle position.

Apoptosis↗

PubMed2000

The correlation between spontaneous and radiation-induced apoptosis in T3B bladder cancer (histological grade G3), and the precedence between the two kinds of apoptosis for predicting clinical prognosis.

PURPOSE: The correlation between the frequency of spontaneous and radiation-induced apoptosis, and the precedence between those for predicting prognosis were studied at clinical level. METHODS AND MATERIALS: Twenty-one patients (mean age, 65.8 years; 16 men and 5 women) with bladder cancer (transitional cell carcinoma Grade 3, T3bN0M0, Stage IIIb) underwent intraoperative radiotherapy: single 30-Gy 12-MV electron beam irradiation to bladder, followed by total cystectomy 6 h after irradiation. The specimens of pretreatment and irradiated bladder cancer were assayed for apoptosis, using TUNEL staining with counter staining of hematoxylin. The apoptotic index (AI) was calculated by dividing the number of apoptotic cells by the total number of cells and multiplying by 100. The Pearson's linear fitting was used to test the correlation between spontaneous and radiation-induced apoptosis. The Kaplan-Meier product-limit estimation was used for overall survival (OS) and freedom from recurrence (FFR). The precedence between spontaneous and radiation-induced apoptosis for predicting the clinical prognosis was estimated using the proportional hazard regression. RESULTS: The mean AI of spontaneous and radiation-induced apoptosis was 1.18 +/- 0.16 and 2.63 +/- 0.45, respectively, which was significantly different. There was strong correlation between spontaneous and radiation-induced apoptosis (r(2) = 0.864, adjusted r(2) = 0.857). Radiation-induced apoptosis was estimated by equation: y (radiation-induced apoptosis) = 2.67 x (spontaneous apoptosis) -0.52. However, the proportional hazard regression test indicated that only spontaneous apoptosis was significant for predicting OS and FFR (&z.sfnc;t&z.sfnc; > 0.2), but radiation-induced apoptosis was not. CONCLUSION: Estimating AI in radiation-induced apoptosis from AI in spontaneous apoptosis is possible. However, spontaneous apoptosis is more accurate in predicting clinical prognosis.

Aged↗

PubMed2000

Different mechanisms between premitotic apoptosis and postmitotic apoptosis in X-irradiated U937 cells.

PURPOSE: Apoptosis is currently being evaluated for its importance as a pathway of radiation-induced cell death. However, the difference in the mechanisms between premitotic and postmitotic apoptosis following X-irradiation remains not well understood. We show here that the human monoblastoid cell line U937 can be induced to undergo these two different types of apoptosis. METHODS AND MATERIALS: U937 cells were irradiated at a dose of 5 or 20 Gy, and the DNA fragmentation rate was measured by both flow cytometric analysis and gel electrophoresis. Activation of caspase-3 was detected by Western blot analysis and fluorogenic assay using acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl-coumarin (Ac-DEVD-AMC). Detection of mitochondrial transmembrane potential (DeltaPsi) was performed by using Rho123. Chasing of S-phase fraction following X-irradiation was performed after labeling with 5-bromo-2'-deoxyuridine (BrdU). Thymidine was used for synchronization of the cells. Inhibition of caspase-3 activity was achieved by Acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO). RESULTS: Time courses of the apoptotic rates, caspase activation, and DeltaPsi indicated that two different types of cell death were induced by the different X-ray doses. High-dose X-ray (20 Gy) induced a rapid and strong apoptosis, whereas low-dose X-ray (5 Gy) induced a slow and mild apoptosis. Cell-cycle analyses revealed that there was cell death before cell division in the former apoptosis but the cells must be dying after cell division in the latter apoptosis. By means of cell-cycle synchronization, the S-phase cells proved to be the most sensitive fraction to premitotic apoptosis, but an obvious difference in the susceptibility to cell death among the cell-cycle phases was not observed in postmitotic apoptosis. Ac-DEVD-CHO treatment effectively blocked caspase activity and premitotic apoptosis, but it failed to block postmitotic apoptosis. CONCLUSIONS: Irradiation of U937 cells at different X-ray doses induced two different types of apoptotic cell death, premitotic apoptosis and postmitotic apoptosis, which are characterized by the time course and cell-cycle specificity. Decision concerning these two types of apoptotic cell death may be made by the difference in the magnitude of cell damage following X-irradiation.

Apoptosis↗

PubMed2006

Mechanism of ASC-mediated apoptosis: bid-dependent apoptosis in type II cells.

Apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor molecule that mediates apoptotic and inflammatory signals, and implicated in tumor suppression. However, the mechanism of ASC-mediated apoptosis has not been well elucidated. Here, we investigated the molecular mechanisms of ASC-mediated apoptosis in several cell lines using a caspase recruitment domain 12-Nod2 chimeric protein that transduces the signal from muramyl dipeptide into ASC-mediated apoptosis. Experiments using dominant-negative mutants, small-interfering RNAs and peptide inhibitors for caspases indicated that caspase-8 was generally required for ASC-mediated apoptosis, whereas a requirement for caspase-9 depended on the cell type. In addition, caspase-like apoptosis-regulatory protein (CLARP)/Fas-like inhibitor protein, a natural caspase-8 inhibitor, suppressed ASC-mediated apoptosis, and Clarp-/- mouse embryonic fibroblasts were highly sensitive to ASC-mediated apoptosis. Bax-deficient HCT116 cells were resistant to ASC-mediated apoptosis as reported previously, although we failed to observe colocalization of ASC and Bax in cells. Like Fas-ligand-induced apoptosis, the ASC-mediated apoptosis was inhibited by Bcl-2 and/or Bcl-XL in type-II but not type-I cell lines. Bid was cleaved upon ASC activation, and suppression of endogenous Bid expression using small-interfering RNAs in type-II cells reduced the ASC-mediated apoptosis. These results indicate that ASC, like death receptors, mediates two types of apoptosis depending on the cell type, in a manner involving caspase-8.

Animals↗

PubMed2001

Stathmo-apoptosis: arresting apoptosis by fluorochrome-labeled inhibitor of caspases.

Apoptosis, like mitosis, is a kinetic event. The entire duration of apoptosis, from its onset to total disintegration of the cell, is often short and may be of variable duration. The time-window through which individual apoptotic cells display their characteristic features that serve to identify them varies depending on: a) the assay that is used, b) the cell type, c) the nature of the inducer of apoptosis, and d) the environmental factors the cell is exposed to that may shorten or prolong apoptosis. Thus, because the apoptotic index (AI) does not accurately represent incidence of apoptosis it is desirable to estimate the rate of cell death in analogy to the cell birth rate which is assessed by the stathmo-kinetic approach by arresting cells in mitosis. In this study the fluorescent caspase inhibitor FAM-VAD-FMK was used for dual purposes: a) to arrest the process of apoptosis (stathmo-apoptosis), and b) to have the arrested cells labeled with fluorochrome. Apoptosis of HL-60 and MCF-7 cells was induced by DNA topoisomerase I inhibitor camptothecin (CPT) and FAM-VAD-FMK was added at the same time as the inducer. While the cells become progressively labeled with FAM-VAD-FMK, their disintegration, loss of the phase-contrast and loss of the capability to bind the inhibitor, and in the case of MCF-7 cells, detachment from the slides, all were prevented for up to 48 h. The percentage of FAM-VAD-FMK labeled HL-60 cells was plotted as a function of time after addition of CPT and the rate of cell entrance to apoptosis was estimated from the slopes of the stathmo-apoptotic plot at different time after administration of CPT. The plot revealed the presence of two distinct subpopulations: during the initial 8 h of the treatment with CPT the cells of the first subpopulation, predominantly the S-phase cells, were entering apoptosis at a rate of about 7% of cells per hour. The remaining cells were stochastically entering apoptosis between 8 and 48 h at a rate 1% of cells per hour. The present approach offers a unique capability to accurately estimate the kinetics of cell transition to apoptosis, revealing the unbiased cumulative apoptotic index over a long time span after induction of apoptosis.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Regulation of apoptosis in mouse hepatocytes and alteration of apoptosis by nongenotoxic carcinogens.

Regulation of apoptosis is an important component of multistage hepatocarcinogenesis. The objectives of the present study were to characterize apoptosis regulation in primary mouse hepatocytes and to determine whether nongenotoxic carcinogens alter apoptosis regulation. Bleomycin-induced apoptosis was accompanied by decreases in bcl-2 and bcl-xl and increases in p53, bak, and bax protein levels. Transforming growth factor (TGF)-beta-induced apoptosis was accompanied by decreased bcl-xL and increased bak. Bleomycin-induced apoptosis was partially dependent on p53, whereas TGF-beta-induced apoptosis was independent of p53. Phenobarbital inhibited both TGF-beta and bleomycin-induced apoptosis and the normal regulation of p53, bcl-2, and bax. Nafenopin inhibited apoptosis through a mechanism dependent on PPAR-alpha and inhibited the normal regulation of bcl-2 and bak. 2,3,7,8-Tetrachlorodibenzo-p-dioxin did not alter apoptosis or its regulation. Apoptosis was increased in hepatocytes from bcl-2-null mice, which indicated that the bcl-2 family contributes to hepatocyte apoptosis regulation. This study demonstrated that apoptosis regulation in mouse hepatocytes involves distinct pathways and that diverse nongenotoxic carcinogens differentially alter molecular pathways that represent targets for hepatocarcinogenesis.

Animals↗

PubMed2006

Human soluble TRAIL/Apo2L induces apoptosis in a subpopulation of chemotherapy refractory nodal diffuse large B-cell lymphomas, determined by a highly sensitive in vitro apoptosis assay.

Resistance to chemotherapy in therapy-refractory diffuse large B-cell lymphomas (DLBCL) is related to inhibition of the intrinsic apoptosis pathway. Human soluble tumour necrosis factor (TNF)-related apoptosis-inducing ligand (hsTRAIL/Apo2L) induces apoptosis via the alternative, death-receptor mediated apoptosis pathway and might be an effective alternative form of therapy for these lymphomas. This study investigated whether hsTRAIL/Apo2L could actually induce apoptosis in isolated lymphoma cells of DLBCL biopsies of patients with chemotherapy-refractory DLBCL. Twelve out of a total of 22 DLBCL samples were sensitive to hsTRAIL/Apo2L. These sensitive lymphomas included seven clinically chemotherapy-refractory lymphomas. Furthermore, hsTRAIL/Apo2L induced apoptosis in DLBCL cells and in B-cell lines that showed high expression levels of inhibitors of the intrinsic apoptosis pathway: Bcl-2 and/or X-linked inhibitor of apoptosis (XIAP). hsTRAIL/Apo2L-sensitive lymphoma cells showed expression of the TRAIL receptors R1 and/or R2 and absence of R3 and R4. We conclude that hsTRAIL/Apo2L induced apoptosis in a subpopulation of chemotherapy-refractory nodal DLBCL and that disruption of the intrinsic apoptosis-mediated pathway and expression of Bcl-2 and XIAP did not confer resistance to hsTRAIL/Apo2L-induced apoptosis in DLBCL. Thus, based on our results, further exploration of hsTRAIL/Apo2L as an alternative treatment for patients with chemotherapy-refractory DLBCL should be considered.

Apoptosis↗

PubMed2004

Gene expression profile of inflammatory neutrophils: alterations in the inhibitors of apoptosis proteins during spontaneous and delayed apoptosis.

Inflammatory mediators delay neutrophil apoptosis, which contributes to the persistence of inflammation. The mechanisms responsible for this delay and resistance to Fas antibody-induced apoptosis are unknown but are dependent on protein synthesis. These proteins have been shown to inhibit caspase activity central to the induction of apoptosis. The inhibitors of apoptosis proteins have been shown to inhibit caspase activity and prevent apoptosis in a number of cellular systems. We hypothesize that the regulation of neutrophil apoptosis is dependent on the expression of the IAPs. c-IAP-1, c-IAP-2, and XIAP are expressed in the neutrophil at both the mRNA and protein level, but their relative protein expression is low compared with other cell types. The in vitro aging of human neutrophils results in their induction of apoptosis, which is associated with the loss of c-IAP-1 expression. The pancaspase inhibitor (zVAD-FMK) and LPS, which delay spontaneous apoptosis, also prevented this loss of c-IAP-1. Gene chip microarrays have shown that LPS increases c-IAP-1 and c-IAP-2 mRNA expression in neutrophils. However, this does not correspond to an increase in protein. Neutrophils from septic patients with delayed apoptosis show an increase in XIAP, with no change in cIAP-1 or cIAP-2 mRNA, demonstrating that different mechanisms contribute to the delay in neutrophil apoptosis. This study demonstrates that the loss of IAP expression may facilitate the induction of neutrophil apoptosis, and preventing this loss of IAP expression may represent a more significant contribution to delayed apoptosis rather than an increase in their expression.

Amino Acid Chloromethyl Ketones↗

PubMed2001

Apoptosis-inducing protein, AIP, from parasite-infected fish induces apoptosis in mammalian cells by two different molecular mechanisms.

AIP (apoptosis-inducing protein) is a protein purified and cloned from Chub mackerel infected with the larval nematode, Anisakis simplex, which induces apoptosis in various mammalian cells including human tumor cell lines. AIP has shown structural and functional homology to L-amino acid oxidase (LAO) which oxidizes several L-amino acids including L-lysine and AIP-induced apoptosis has been suggested to be mediated by H2O2 generated by LAO activity of AIP. In this study, we confirmed that recombinant AIP generated enough H2O2 in culture medium to induce rapid apoptosis in cells and this apoptosis was clearly inhibited by co-cultivation with antioxidants such as catalase and N-acetyl-cysteine. Surprisingly, however, we found that AIP still could induce H2O2-independent apoptosis more slowly than H2O2-dependent one in HL-60 cells even in the presence of antioxidants. In addition, the HL-60-derived cell line HP100-1, which is a H2O2-resistant variant, underwent apoptosis on treatment with AIP with a similar delayed time course. The latter apoptosis was completely blocked by addition of L-lysine to the culture medium, which is the best substrate of AIP as LAO, indicating that decreased concentration of L-lysine in the culture medium by AIP-treatment induced apoptosis. We also showed that the both apoptosis by AIP were associated with the release of cytochrome c from mitochondria and activation of caspase-9, and overexpressed Bcl-2 could inhibit both of the AIP-induced apoptosis. These results indicate that AIP induces apoptosis in cells by two distinct mechanisms; one rapid and mediated by H2O2, the other delayed and mediated by deprivation of L-lysine, both of which utilize caspase-9/cytochrome c system.

Animals↗

PubMed2004

Apoptosis in Gliomas: Molecular Mechanisms and Therapeutic Implications.

Understanding apoptosis is often considered a key to understand the genesis of tumors and to devise innovative strategies for their treatment. Similar to other types of cancer, essential pathways regulating apoptosis are also disrupted in malignant gliomas, notably the cell cycle control mechanisms regulated by the p53 and retinoblastoma (RB) proteins and their homologs. Moreover, cultured glioma cells appear not to activate the extrinsic death receptor-dependent apoptotic pathway in response to irradiation or cytotoxic drugs. A preferential expression of antiapoptotic rather than proapoptotic BCL-2 family proteins and high level expression of inhibitor-of-apoptosis proteins (IAP) may be responsible for the failure of glioma cells to activate caspases in response to apoptotic stimuli. Although apoptosis does occur spontaneously in malignant gliomas in vivo, there is little evidence that the current modes of non-surgical treatment, radiotherapy and chemotherapy, mediate their effects via induction of apoptosis, with the possible exception of anaplastic oligodendrogliomas which often show striking tumor regression on neuroimaging. Yet, the induction of apoptosis plays a conceptual role in the majority of novel experimental approaches to malignant glioma which are currently evaluated in cell culture and preclinical rodent models.

Journal Article↗

PubMed1999

Screening for inducers of apoptosis in apoptosis-resistant human carcinoma cells.

Many anticancer agents are known to induce apoptosis in cultured cells, but human solid tumor cells are often resistant to apoptosis induction. Human pancreatic adenocarcinoma AsPC-1 cells were found to be resistant to apoptosis. So, we screened microbial culture filtrates and synthetic chemicals for their ability to induce apoptosis in AsPC-1 cells. Polyoxypeptin A was isolated from a Streptomyces culture broth as a potent inducer of apoptosis. Its cyclic hexadepsipeptide structure contains a simple but hitherto unreported amino acid, (2S,3R)-3-hydroxy-3-methylproline. Polyoxypeptin A induced early cell death, apoptotic morphology, and internucleosomal DNA fragmentation in AsPC-1 cells at low concentrations. Polyoxypeptin A can induce caspase 3 activation in human T cell leukemia Jurkat cells but not in AsPC-1 cells. The mechanism of apoptosis in AsPC-1 cells has not been elucidated yet. Less toxic derivatives of polyoxypeptin A are being prepared. A macrocyclic lactam called BE-14106 was also isolated from Streptomyces as an inducer of apoptosis in AsPC-1 cells. Histidine-pyridine-histidine-3 (HPH-3) is an oxygen-activating ligand derived from the structure of bleomycin, and it also induced apoptosis in AsPC-1 cells. Induction of apoptosis by HPH-3 was inhibited by zinc and copper ions, indicating that chelation with ferrous ion is responsible for induction of apoptosis, as is chelation by bleomycin to cleave DNA. Bleomycin A2 and its portion having no DNA-binding region, glycopeptide-3(GP-3), did not induce apoptosis in AsPC-1 cells. Thus, among the actions of bleomycin-related compounds, the induction of apoptosis is a unique characteristic of HPH-3.

Anti-Bacterial Agents↗

PubMed1999

Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma.

Past studies have shown that apoptosis mediated by TNF-related apoptosis-inducing ligand (TRAIL) is regulated by the expression of two death receptors [TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2] and two decoy receptors (TRAIL-R3 and TRAIL-R4) that inhibit apoptosis. In previous studies, we have shown that TRAIL but not other members of the tumor necrosis factor family induce apoptosis in approximately two-thirds of melanoma cell lines. Here, we examined whether the expression of TRAIL-R at the mRNA and protein level in a panel of 28 melanoma cell lines and melanocytes correlated with their sensitivity to TRAIL-induced apoptosis. We report that at least three factors appear to underlie the variability in TRAIL-induced apoptosis. (a) Four of nine cell lines that were insensitive to TRAIL-induced apoptosis failed to express death receptors, and in two instances, lines were devoid of all TRAIL-Rs. Southern analysis suggested this was due to loss of the genes for the death receptors. (b) Despite the presence of mRNA for the TRAIL-R, some of the lines failed to express TRAIL-R protein on their surface. This was evident for TRAIL-R1 and more so for the TRAIL decoy receptors TRAIL-R3 and -R4. Studies on permeabilized cells revealed that the receptors were located within the cytoplasm and redistribution from the cytoplasm may represent a posttranslational control mechanism. (c) Surface expression of TRAIL-R1 and -R2 (but not TRAIL-R3 and -R4) showed an overall correlation with TRAIL-induced apoptosis. However, certain melanoma cell lines and clones were relatively resistant to TRAIL-induced apoptosis despite the absence of decoy receptors and moderate levels of TRAIL-R1 and -R2 expression. This may indicate the presence of inhibitors within the cells, but resistance to apoptosis could not be correlated with expression of the caspase inhibitor FLICE-inhibitory protein. mRNA for another TRAIL receptor, osteoprotegerin, was expressed in 22 of the melanoma lines but not on melanocytes. Its role in induction of apoptosis remains to be studied. These results appear to have important implications for future clinical studies on TRAIL.

Apoptosis↗

PubMed2000

Tumor necrosis factor-related apoptosis-inducing ligand retains its apoptosis-inducing capacity on Bcl-2- or Bcl-xL-overexpressing chemotherapy-resistant tumor cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family and has recently been shown to exert tumoricidal activity in vivo in the absence of any observable toxicity. The signaling pathways triggered by TRAIL stimulation and the mechanisms involved in resistance against TRAIL-mediated apoptosis are still poorly defined. We show here that TRAIL-induced apoptosis involves late dissipation of mitochondrial membrane potential (delta psi(m)) and cytochrome c release. These events follow activation of caspase-8 and caspase-3 and induction of DNA fragmentation. In addition, caspase-8-deficient cells are resistant against TRAIL-induced apoptosis, and inhibition of caspase-8 but not caspase-9 prevents mitochondrial permeability transition and apoptosis. In contrast, various Bcl-2- or Bcl-xL-overexpressing tumor cell lines are sensitive to TRAIL-induced apoptosis; however, they show a delay in TRAIL-induced mitochondrial permeability transition compared with control transfectants. This indicates that TRAIL-induced apoptosis depends on caspase-8 activation rather than on the disruption of mitochondrial integrity. Because most chemotherapeutic drugs used in the treatment of malignancies lead to apoptosis primarily by engagement of the mitochondrial proapoptotic machinery, we tested whether drug-resistant tumor cells retain sensitivity for TRAIL-induced apoptosis. Tumor cells overexpressing Bcl-2 or Bcl-xL become resistant to apoptosis induced by the chemotherapeutic drug etoposide. However, these cells are not protected or are only marginally protected against TRAIL-induced apoptosis. Thus, TRAIL may still kill tumors that have acquired resistance to chemotherapeutic drugs by overexpression of Bcl-2 or Bcl-xL. These data will influence future treatment strategies involving TRAIL.

Antineoplastic Agents, Phytogenic↗

PubMed2001

Failure of Bcl-2 to block mitochondrial dysfunction during TRAIL-induced apoptosis. Tumor necrosis-related apoptosis-inducing ligand.

Tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines that promotes apoptosis. TRAIL induces apoptosis in a wide variety of tumor cells but not in normal cells. Oncogene Bcl-2 can protect cells from apoptosis induced by various stress stimuli. However, it is not clear whether Bcl-2 can regulate TRAIL-induced apoptosis. The objective of this study was to investigate whether Bcl-2 can regulate apoptosis induced by TRAIL. TRAIL initiates the activation of caspases, the loss of mitochondrial transmembrane potential (Delta psi(m)), and the redistribution of mitochondrial cytochrome c. TRAIL has no effect on Delta psi(m) and apoptosis in Jurkat cells deficient in either FADD or caspase-8, suggesting both FADD and caspase-8 are required for TRAIL signaling. Overexpression of Bcl-2 delays, but does not inhibit, TRAIL-induced Delta psi(m), cytochrome c release from mitochondria and apoptosis, whereas etoposide-induced apoptosis is blocked by Bcl-2. XIAP, cowpox virus CrmA and baculovirus p35 inhibits TRAIL-induced apoptosis. These data suggest that TRAIL can be used to kill Bcl-2 positive cells that can not be killed by other class of chemotherapeutic drugs.

Apoptosis↗

PubMed2000

Differential regulation of Fas-mediated apoptosis of rheumatoid synoviocytes by tumor necrosis factor alpha and basic fibroblast growth factor is associated with the expression of apoptosis-related molecules.

OBJECTIVE: Fas-mediated apoptosis is associated with the pathophysiology of rheumatoid arthritis (RA). However, the molecular mechanisms of this process remain to be elucidated in rheumatoid synovium. We investigated the behavior of intracellular signaling molecules that regulate Fas-mediated apoptosis in RA synoviocytes. METHODS: Anti-Fas monoclonal antibody (mAb) was added to RA synoviocytes after treatment with tumor necrosis factor alpha (TNFalpha) or basic fibroblast growth factor (bFGF) for 5 days. The cytotoxic activity was measured using a lactate dehydrogenase-release assay. The expression of apoptosis-related molecules in RA synoviocytes was examined by immunoblot analysis. The enzymatic activities of caspases 3 and 8 under Fas ligation were examined. Transfer of the FADD (Fas-associated death domain) protein and the FLIP(L) (long form of the FLICE [FADD-like interleukin-1beta-converting enzyme]-inhibitory protein) gene into RA synoviocytes was performed using adenoviral vectors. RESULTS: Following a 5-day treatment with TNFalpha or bFGF, Fas ligation with its agonistic mAb induced apoptosis of almost all TNFalpha-treated RA synoviocytes but only showed a weak apoptotic activity in bFGF-treated synoviocytes. Although there was no correlation between the induction of Fas-mediated apoptosis and the expression of apoptosis-related molecules among these cells, a high enzymatic activity of caspases 3 and 8 was observed only in the TNFalpha-treated RA synoviocytes after Fas ligation. The bFGF-treated RA synoviocytes were relatively resistant to apoptosis induced by FADD gene transfection, as compared with the TNFalpha-treated synoviocytes. In addition, the expression of FLIP(L), an inhibitory molecule of Fas-mediated apoptosis, was reduced in TNFalpha-treated RA synoviocytes, and the expression of FLIP43 was augmented in bFGF-treated RA synoviocytes. Moreover, Fas-mediated apoptosis in TNFalpha-treated RA synoviocytes was partially inhibited by FLIP(L) gene transfection. CONCLUSION: Our results suggest that Fas-mediated apoptosis of RA synoviocytes is differentially regulated by TNFalpha and bFGF. In addition, the regulatory mechanisms of apoptosis involve the formation of the death-inducing signaling complex, especially at the level of caspase 8 activation, and this process may be partly associated with FLIP expression.

Antibodies, Monoclonal↗

PubMed2006

Osteoprotegerin (OPG) binds with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL): suppression of TRAIL-induced apoptosis in ameloblastomas.

Osteoprotegerin (OPG) is a useful receptor in inhibiting Receptor Activator of NFkappaB Ligand (RANKL) in inducing osteoclastogenesis. Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) is a potent apoptosis-inducing ligand in ameloblastomas. Since OPG has been reported to bind to TRAIL as well, the effect of OPG in TRAIL's function in inducing apoptosis should also be investigated. To investigate on the expression of OPG in ameloblastomas, immuhistochemistry, immunofluorescence and Western blot were performed. From the immunohistochemistry results, we found that OPG was expressed in ameloblastoma tissues. Expression of OPG was clearly seen in AM-1 cells by immunofluorescence as well. Additionally, Western blot analysis confirmed OPG expression in ameloblastoma tissues and AM-1 cells. To investigate on the potential of TNFalpha, TRAIL and RANKL in inducing apoptosis, we performed an apoptosis assay. From the apoptosis assay, we found that TRAIL had the highest potential in inducing apoptosis compared to TNFalpha and RANKL. To investigate the binding of OPG with RANKL and TRAIL, we performed a binding assay. We noticed that OPG preferably bind with RANKL than TRAIL. However, at low levels of RANKL, marked binding of OPG with TRAIL was seen. As we suspected that the binding of OPG and TRAIL might cause the effect of TRAIL in inducing apoptosis in ameloblastomas, we combined the treatment of OPG and TRAIL in AM-1 cells. From the apoptosis assay, we found that under treatment of OPG, TRAIL's function in inducing apoptosis was suppressed. These data suggest that by binding with TRAIL, OPG suppressed TRAIL's function in inducing apoptosis in ameloblastomas.

Ameloblastoma↗

PubMed2005

The lysosome-associated apoptosis-inducing protein containing the pleckstrin homology (PH) and FYVE domains (LAPF), representative of a novel family of PH and FYVE domain-containing proteins, induces caspase-independent apoptosis via the lysosomal-mitochondrial pathway.

Lysosomes have recently been identified as important apoptotic signal integrators in response to various stimuli. Here we report the functional characterization of LAPF, a novel lysosome-associated apoptosis-inducing protein containing PH and FYVE domains. LAPF is a representative of a new protein family, the Phafins (protein containing both PH and FYVE domains), which consists of 14 unidentified proteins from various species. Overexpression of LAPF in L929 cells induces apoptosis and also increases cell sensitivity to TNFalpha-induced apoptosis, concomitant with its translocation to lysosomes. Two mutants of LAPF, either lacking the PH or FYVE domain, failed to induce cell death and translocate to lysosomes, suggesting that both domains are required for its apoptosis-inducing activity and relocation. We demonstrate that LAPF may induce apoptosis via the following steps: LAPF translocation to lysosomes, lysosomal membrane permeabilization (LMP), release of cathepsin (cath) D and L, mitochondrial membrane permeabilization (MMP), release of apoptosis-inducing factor (AIF), and caspase-independent apoptosis. The cath D-specific inhibitor attenuates LAPF-induced apoptosis, indicating a pivotal role of lysosomes in LAPF-initiated apoptosis. We also demonstrate that the lysosomal pathway was employed in the typical apoptotic model in which high dose TNFalpha was used to stimulate L929 cells. Silencing of LAPF expression by small RNA interference protected L929 cells from hTNFalpha-induced apoptosis by impairing hTNFalpha-triggered LMP and MMP. Therefore, LAPF may launch caspase-independent apoptosis through the lysosomal-mitochondrial pathway.

Amino Acid Sequence↗