Cleavage of the death domain kinase RIP by Caspase-8 prompts TNF-induced apoptosis

LIGHT is a member of tumor necrosis factor (TNF) superfamily, and its receptors have been identified as lymphotoxin-β receptor (LTβR) and the herpesvirus entry mediator (HVEM)/ATAR/TR2, both of which lack the cytoplasmic sequence termed the “death domain.” The present study has demonstrated that LIGHT inhibits TNFα-mediated apoptosis of human primary hepatocytes sensitized by actinomycin D (ActD), but not Fas- or TRAIL-mediated apoptosis. Furthermore, LIGHT does not prevent some cell lines such as HepG2 or HeLa from undergoing ActD/TNFα-induced apoptosis. This protective effect requires LIGHT pretreatment at least 3 h prior to ActD sensitization. LIGHT stimulates nuclear factor-κB (NF-κB)-dependent transcriptional activity in human hepatocytes like TNFα. The time course of NF-κB activation after LIGHT administration is similar to that of the pretreatment required for the anti-apoptotic effect of LIGHT. LIGHT inhibits caspase-3 processing on the apoptotic protease cascade in TNFα-mediated apoptosis but not Fas-mediated apoptosis. In addition, increased caspase-3 and caspase-8 activities in ActD/TNFα-treated cells are effectively blocked by LIGHT pretreatment. However, LIGHT does not change the expression of TNFRp55, TNFRp75, and Fas. These results indicate that LIGHT may act as an anti-apoptotic agent against TNFα-mediated liver injury by blocking the activation of both caspase-3 and caspase-8.

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Evidence for a Novel, Caspase-8-Independent, Fas Death Domain-Mediated Apoptotic Pathway

Journal of Biomedicine and Biotechnology ◽

10.1155/s1110724304308041 ◽

2004 ◽

Vol 2004 (1) ◽

pp. 41-51 ◽

Cited By ~ 14

Author(s):

Hanping Feng ◽

Yi Zeng ◽

Michael W. Graner ◽

Luke Whitesell ◽

Emmanuel Katsanis

Keyword(s):

Transmembrane Potential ◽

Caspase 8 ◽

Death Domain ◽

Caspase Inhibitor ◽

Caspase Activation ◽

Null Cell ◽

Apoptotic Pathway ◽

Induced Apoptosis ◽

Mitochondrial Transition Pore ◽

Pore Permeability

Certain caspase-8 null cell lines demonstrate resistance to Fas-induced apoptosis, indicating that the Fas/FasL apoptotic pathway may be caspase-8-dependent. Some reports, however, have shown that Fas induces cell death independent of caspase-8. Here we provide evidence for an alternative, caspase-8-independent, Fas death domain-mediated apoptotic pathway. Murine 12B1-D1 cells express procaspase-3, -8, and -9, which were activated upon the dimerization of Fas death domain. Bid was cleaved and mitochondrial transmembrane potential was disrupted in this apoptotic process. All apoptotic events were completely blocked by the broad-spectrum caspase inhibitor Z-VAD-FMK, but not by other peptide caspase inhibitors. Cyclosporin A (CsA), which inhibits mitochondrial transition pore permeability, blocked neither pore permeability disruption nor caspase activation. However, CsA plus caspase-8 inhibitor blocked all apoptotic events of 12B1-D1 induced by Fas death domain dimerization. Our data therefore suggest that there is a novel, caspase-8-independent, Z-VAD-FMK-inhibitable, apoptotic pathway in 12B1-D1 cells that targets mitochondria directly.

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Calmodulin inhibition contributes to sensitize TRAIL-induced apoptosis in human lung cancer H1299 cells

Biochemistry and Cell Biology ◽

10.1139/o09-058 ◽

2009 ◽

Vol 87 (6) ◽

pp. 919-926 ◽

Cited By ~ 8

Author(s):

Mi-kyung Hwang ◽

Yong Ki Min ◽

Seong Hwan Kim

Keyword(s):

Lung Cancer ◽

Enzyme Inhibitors ◽

Human Lung ◽

Therapeutic Potential ◽

Human Lung Cancer ◽

Caspase 8 ◽

Death Domain ◽

Akt Phosphorylation ◽

Apoptosis Protein ◽

Induced Apoptosis

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) preferentially triggers apoptosis in tumor cells versus normal cells. However, TRAIL alone is not effective in treating TRAIL-resistant tumors. We evaluated the effect of 180 enzyme inhibitors on TRAIL-induced apoptosis in human lung cancer H1299 cells, and found fluphenazine-N-2-chloroethane (a calmodulin (CaM) antagonist) sensitized TRAIL-induced apoptosis. Interestingly, in the presence of TRAIL, it increased caspase-8 binding to the Fas-associated death domain (FADD), but decreased binding of FADD-like interleukin-1β-converting enzyme inhibitory proteins (FLIPs). Additionally, its combination with TRAIL inhibited Akt phosphorylation. These results were consistently observed in cells treated with CaM siRNA. We suggested the blockade of CaM could sensitize lung cancer cells to TRAIL-induced apoptosis in at least 2 ways: (i) it can activate death-inducing signaling complex mediated apoptosis by inhibiting TRAIL-induced binding of FLIP and TRAIL-enhanced binding of caspase-8 to FADD; (ii) it can inhibit Akt phosphorylation, consequently leading to decreased expression of anti-apoptotic molecules such as FLIP and members of the inhibitor of apoptosis protein family. This study suggests the combination of CaM antagonists with TRAIL may have the therapeutic potential to overcome the resistance of lung cancers to apoptosis.

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Albumin overload induces apoptosis in LLC-PK1cells

AJP Renal Physiology ◽

10.1152/ajprenal.2001.280.6.f1107 ◽

2001 ◽

Vol 280 (6) ◽

pp. F1107-F1114 ◽

Cited By ~ 69

Author(s):

Elif Erkan ◽

Maryely De Leon ◽

Prasad Devarajan

Keyword(s):

Dna Cleavage ◽

Morphological Changes ◽

Prognostic Indicator ◽

Caspase 8 ◽

Death Domain ◽

Tubulointerstitial Injury ◽

Nuclear Condensation ◽

Glomerular Diseases ◽

Induced Apoptosis

The degree of albuminuria is a well-known adverse prognostic indicator in human glomerular diseases. However, the mechanisms by which albuminuria by itself contributes to tubulointerstitial injury and progression of renal disease remain unclear. We tested the hypothesis that apoptosis may represent one of the mechanisms by which tubule epithelial cells are damaged after albumin overload in vitro. Cultured LLC-PK1 proximal tubule cells were incubated with varying concentrations of BSA. This resulted in a dose- and duration-dependent induction of apoptosis, as evidenced by internucleosomal DNA cleavage (DNA laddering and nick-end labeling), externalization of plasma membrane phosphatidylserine (annexin labeling), and characteristic morphological changes (cell shrinkage and nuclear condensation). Albumin overload also resulted in a dose-dependent upregulation of Fas and Fas-associated protein with death domain (FADD), and activation of caspase 8. Incubation with the caspase 8 inhibitor IETD ameliorated the albumin-induced apoptosis. Collectively, our results indicate that albumin overload induces apoptosis of cultured LLC-PK1 cells, mediated at least in part by the Fas-FADD-caspase 8 pathway.

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C5, A Cassaine Diterpenoid Amine, Induces Apoptosis via the Extrinsic Pathways in Human Lung Cancer Cells and Human Lymphoma Cells

International Journal of Molecular Sciences ◽

10.3390/ijms21041298 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1298 ◽

Cited By ~ 1

Author(s):

Hyo-Jin Kim ◽

Bo-Gyeong Seo ◽

Kwang Dong Kim ◽

Jiyun Yoo ◽

Joon-Hee Lee ◽

...

Keyword(s):

Cancer Cells ◽

Death Receptor ◽

B Cell Lymphoma ◽

Human Lung Cancer ◽

Caspase 8 ◽

Death Domain ◽

Extrinsic Pathway ◽

Inhibitory Protein ◽

Apoptosis Pathways ◽

Induced Apoptosis

Apoptosis pathways in cells are classified into two pathways: the extrinsic pathway, mediated by binding of the ligand to a death receptor and the intrinsic pathway, mediated by mitochondria. Apoptosis is regulated by various proteins such as Bcl-2 (B-cell lymphoma 2) family and cellular FLICE (Fas-associated Death Domain Protein Interleukin-1β-converting enzyme)-inhibitory protein (c-FLIP), which have been reported to inhibit caspase-8 activity. In this study, it was found that C5 (3β-Acetyl-nor-erythrophlamide), a compound of cassaine diterpene amine from Erythrophleum fordii, induced cell apoptosis in a variety of types of cancer cells. Induction of apoptosis in cancer cells by C5 was inversely related to the level of Bcl-2 expression. Overexpression of Bcl-2 into cancer cells significantly decreased C5-induced apoptosis. It was also found that treatment of cancer cells with a caspase-8 inhibitor significantly suppressed C5-induced apoptosis; however, treatment with caspase-9 inhibitors did not affect C5-induced apoptosis, suggesting that C5 may induce apoptosis via the extrinsic pathway by activating caspase-8. It was confirmed that treatment with C5 alone induced an association of FADD with procaspase-8; however, overexpression of c-FLIP decreased C5-induced caspase-8 activation. In conclusion, C5 could be utilized as a new useful lead compound for the development of an anti-cancer agent that has the goal of apoptosis.

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A New Caspase-8 Isoform Caspase-8s Increased Sensitivity to Apoptosis in Jurkat Cells

Journal of Biomedicine and Biotechnology ◽

10.1155/2009/930462 ◽

2009 ◽

Vol 2009 ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

Zhifang Xu ◽

Kejing Tang ◽

Min Wang ◽

Qing Rao ◽

Bolin Liu ◽

...

Keyword(s):

Death Receptor ◽

Adaptor Protein ◽

Jurkat Cells ◽

Caspase 8 ◽

Death Domain ◽

Binding Assays ◽

Increased Sensitivity ◽

Death Effector ◽

Induced Apoptosis

Caspase-8 is a key initiator of death receptor-induced apoptosis. Here we report a novel short isoform of caspase-8 (caspase-8s), which encodes the first (Death Effector Domain) DED and part of the second DED, missing the C-terminal caspase domain. In vivo binding assays showed that transfected caspase-8s bound to (Fas-associated death domain protein) FADD, the adaptor protein in (death-induced signal complex) DISC. To investigate the potential effects of caspase-8s on cell apoptosis, Jurkat cells were stably transfected with caspase-8s. Overexpression of caspase-8s increased sensitivity to the apoptotic stimuli, Fas-agonistic antibody CH11. These results suggest that caspase-8s may act as a promoter of apoptosis through binding to FADD and is involved in the regulation of apoptosis. In addition, the results also indicate that the first DED was an important structure mediating combination between caspase-8 and FADD.

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Binding of FADD and Caspase-8 to Molluscum Contagiosum Virus MC159 v-FLIP Is Not Sufficient for Its Antiapoptotic Function

Journal of Virology ◽

10.1128/jvi.76.2.697-706.2002 ◽

2002 ◽

Vol 76 (2) ◽

pp. 697-706 ◽

Cited By ~ 61

Author(s):

Tara L. Garvey ◽

John Bertin ◽

Richard M. Siegel ◽

Guang-hua Wang ◽

Michael J. Lenardo ◽

...

Keyword(s):

Amino Acid ◽

Protein Interactions ◽

Molluscum Contagiosum ◽

Site Directed Mutagenesis ◽

Caspase 8 ◽

Death Domain ◽

Molluscum Contagiosum Virus ◽

Hydrophobic Patch ◽

Death Effector ◽

Induced Apoptosis

ABSTRACT Molluscum contagiosum virus (MCV), a member of the human poxvirus family, encodes the MC159 protein that inhibits Fas-, tumor necrosis factor (TNF)-, and TNF-related apoptosis-inducing ligant (TRAIL)-induced apoptosis. We used site-directed mutagenesis to change charged or hydrophobic amino acid residues to alanines to identify regions of MC159 that are critical for protection from apoptosis and for protein-protein interactions. Surprisingly, while MC159 is thought to block apoptosis by binding to Fas-associated death domain (FADD) or caspase-8, several mutants that lost apoptosis blocking activity still bound to both FADD and caspase-8. Mutations in the predicted hydrophobic patch 1 and α2 regions of both death effector domains (DEDs) within MC159 resulted in loss of the ability to bind to FADD or caspase-8 and to block apoptosis. Amino acid substitutions in the RXDL motif located in the α6 region of either DED resulted in loss of protection from apoptosis induced by Fas, TNF, and TRAIL and abolished the ability of MC159 to block death effector filament formation. Thus, charged or hydrophobic amino acids in three regions of the MC159 DEDs (hydrophobic patch 1, α2, and α6) are critical for the protein’s ability to interact with cellular proteins and to block apoptosis.

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NPI-0052, a novel proteasome inhibitor, induces caspase-8 and ROS-dependent apoptosis alone and in combination with HDAC inhibitors in leukemia cells

Blood ◽

10.1182/blood-2006-03-013128 ◽

2007 ◽

Vol 110 (1) ◽

pp. 267-277 ◽

Cited By ~ 147

Author(s):

Claudia P. Miller ◽

Kechen Ban ◽

Melanie E. Dujka ◽

David J. McConkey ◽

Mark Munsell ◽

...

Keyword(s):

Proteasome Inhibitor ◽

Histone Deacetylase Inhibitors ◽

Mononuclear Cells ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Jurkat Cells ◽

Model Systems ◽

Caspase 8 ◽

Death Domain ◽

Induced Apoptosis

The proteasome has been successfully targeted for the treatment of multiple myeloma and mantle cell lymphoma; however, in other hematologic malignancies, bortezomib has been less effective as a single agent. Here, we describe effects of NPI-0052, a novel proteasome inhibitor, in leukemia model systems. In cell lines, NPI-0052 inhibits all 3 proteolytic activities associated with the proteasome: chymotrypsin-, trypsin-, and caspase-like. NPI-0052 also induces DNA fragmentation in leukemia lines and in mononuclear cells from a Ph + acute lymphoblastic leukemia (ALL) patient. Caspase-3 activation by NPI-0052 was seen in wild-type Jurkat cells, but was significantly lessened in Fas-associated death domain (FADD)–deficient or caspase-8–deficient counterparts. NPI-0052–induced apoptosis was further probed using caspase-8 inhibitors, which were more protective than caspase-9 inhibitors. N-acetyl cysteine (NAC) also conferred protection against NPI-0052–induced apoptosis, indicating a role for oxidative stress by NPI-0052. In support of the drug's in vitro activities, biweekly treatment with NPI-0052 lessened total white blood cell (WBC) burden over 35 days in leukemic mice. Interestingly, combining NPI-0052 with either MS-275 or valproic acid (VPA) induced greater levels of cell death than the combination of bortezomib with these histone deacetylase inhibitors (HDACi). These effects of NPI-0052, alone and in combination with HDACi, warrant further testing to determine the compound's clinical efficacy in leukemia.

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