Sensitization for death receptor- or drug-induced apoptosis by re-expression of caspase-8 through demethylation or gene transfer

Apoptosis is induced by different stimuli, among them triggering of the death receptor CD95, staurosporine, and chemotherapeutic drugs. In all cases, apoptosis is mediated by caspases, although it is unclear how these diverse apoptotic stimuli cause protease activation. Two regulatory pathways have been recently identified, but it remains unknown whether they are functionally independent or linked to each other. One is mediated by recruitment of the proximal regulator caspase-8 to the death receptor complex. The other pathway is controlled by the release of cytochrome c from mitochondria and the subsequent ATP-dependent activation of the death regulator apoptotic protease-activating factor 1 (Apaf-1). Here, we report that both pathways can be dissected by depletion of intracellular ATP. Prevention of ATP production completely inhibited caspase activation and apoptosis in response to chemotherapeutic drugs and staurosporine. Interestingly, caspase-8, whose function appeared to be restricted to death receptors, was also activated by these drugs under normal conditions, but not after ATP depletion. In contrast, inhibition of ATP production did not affect caspase activation after triggering of CD95. These results suggest that chemotherapeutic drug–induced caspase activation is entirely controlled by a receptor-independent mitochondrial pathway, whereas CD95-induced apoptosis can be regulated by a separate pathway not requiring Apaf-1 function.

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Anticancer Drugs Induce Caspase-8/FLICE Activation and Apoptosis in the Absence of CD95 Receptor/Ligand Interaction

Blood ◽

10.1182/blood.v93.9.3053 ◽

1999 ◽

Vol 93 (9) ◽

pp. 3053-3063 ◽

Cited By ~ 177

Author(s):

Sebastian Wesselborg ◽

Ingo H. Engels ◽

Evi Rossmann ◽

Marek Los ◽

Klaus Schulze-Osthoff

Keyword(s):

Anticancer Drugs ◽

De Novo ◽

Death Receptor ◽

Dominant Negative ◽

Caspase 8 ◽

Drug Induced ◽

Ligand Interaction ◽

Caspase Family ◽

Death Receptor Signaling ◽

Induced Apoptosis

Abstract Proteases of the caspase family are the critical executioners of apoptosis. Their activation has been mainly studied upon triggering of death receptors, such as CD95 (Fas/APO-1) and tumor necrosis factor-R1, which recruit caspase-8/FLICE as the most proximal effector to the receptor complex. Because apoptosis induced by anticancer drugs has been proposed to involve CD95/CD95 ligand interaction, we investigated the mechanism of caspase activation by daunorubicin, doxorubicin, etoposide, and mitomycin C. In Jurkat leukemic T cells, all drugs induced apoptosis and the cleavage of procaspase-8 to its active p18 subunit. However, cells resistant to CD95 were equally susceptible to anticancer drugs and activated caspase-8 with a similar kinetic and dose response as CD95-sensitive cells. The broad caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone prevented apoptosis and caspase-8 activation in response to CD95 and drug treatment, whereas a neutralizing CD95 decoy as well as a dominant-negative FADD construct selectively abrogated CD95, but not drug-induced effects. A potent activation of caspase-8 was also induced by cycloheximide, indicating that it was independent of protein synthesis. Our data, therefore, show that (1) anticancer drug-induced apoptosis does not require de novo synthesis of death ligands or CD95 interaction, and (2) that caspase-8 can be activated in the absence of a death receptor signaling.

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Anticancer Drugs Induce Caspase-8/FLICE Activation and Apoptosis in the Absence of CD95 Receptor/Ligand Interaction

Blood ◽

10.1182/blood.v93.9.3053.409a33_3053_3063 ◽

1999 ◽

Vol 93 (9) ◽

pp. 3053-3063 ◽

Cited By ~ 43

Author(s):

Sebastian Wesselborg ◽

Ingo H. Engels ◽

Evi Rossmann ◽

Marek Los ◽

Klaus Schulze-Osthoff

Keyword(s):

Anticancer Drugs ◽

De Novo ◽

Death Receptor ◽

Dominant Negative ◽

Caspase 8 ◽

Drug Induced ◽

Ligand Interaction ◽

Caspase Family ◽

Death Receptor Signaling ◽

Induced Apoptosis

Proteases of the caspase family are the critical executioners of apoptosis. Their activation has been mainly studied upon triggering of death receptors, such as CD95 (Fas/APO-1) and tumor necrosis factor-R1, which recruit caspase-8/FLICE as the most proximal effector to the receptor complex. Because apoptosis induced by anticancer drugs has been proposed to involve CD95/CD95 ligand interaction, we investigated the mechanism of caspase activation by daunorubicin, doxorubicin, etoposide, and mitomycin C. In Jurkat leukemic T cells, all drugs induced apoptosis and the cleavage of procaspase-8 to its active p18 subunit. However, cells resistant to CD95 were equally susceptible to anticancer drugs and activated caspase-8 with a similar kinetic and dose response as CD95-sensitive cells. The broad caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone prevented apoptosis and caspase-8 activation in response to CD95 and drug treatment, whereas a neutralizing CD95 decoy as well as a dominant-negative FADD construct selectively abrogated CD95, but not drug-induced effects. A potent activation of caspase-8 was also induced by cycloheximide, indicating that it was independent of protein synthesis. Our data, therefore, show that (1) anticancer drug-induced apoptosis does not require de novo synthesis of death ligands or CD95 interaction, and (2) that caspase-8 can be activated in the absence of a death receptor signaling.

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Activation of caspase-8 in drug-induced apoptosis of B-lymphoid cells is independent of CD95/Fas receptor-ligand interaction and occurs downstream of caspase-3

Blood ◽

10.1182/blood.v97.5.1378 ◽

2001 ◽

Vol 97 (5) ◽

pp. 1378-1387 ◽

Cited By ~ 168

Author(s):

Thomas Wieder ◽

Frank Essmann ◽

Aram Prokop ◽

Karin Schmelz ◽

Klaus Schulze-Osthoff ◽

...

Keyword(s):

Caspase 3 ◽

Death Receptor ◽

Lymphoid Cells ◽

Caspase 8 ◽

Lymphoma Cells ◽

Drug Induced ◽

Ligand Interaction ◽

B Lymphoma ◽

B Lymphoma Cells ◽

Induced Apoptosis

The activation of caspase-8, a crucial upstream mediator of death receptor signaling, was investigated in epirubicin- and Taxol-induced apoptosis of B-lymphoma cells. This study was performed because the CD95/Fas receptor-ligand interaction, recruitment of the Fas-associated death domain (FADD) adaptor protein, and subsequent activation of procaspase-8 have been implicated in the execution of drug-induced apoptosis in other cell types. Indeed, active caspase-8 was readily detected after treatment of mature and immature B-lymphoid cells with epirubicin or Taxol. However, neither constitutive nor drug-induced expression of the CD95/Fas ligand was detectable in B-lymphoma cells. Furthermore, overexpression of a dominant-negative FADD mutant (FADDdn) did not block caspase-8 processing and subsequent DNA fragmentation, indicating that drug-induced caspase-8 activation was mediated by a CD95/Fas-independent mechanism. Instead, caspase-8 cleavage was slightly preceded by activation of caspase-3, suggesting that drug-induced caspase-8 activation in B-lymphoma cells is a downstream event mediated by other caspases. This assumption was confirmed in 2 experimental systems—zDEVD-fmk, a cell-permeable inhibitor of caspase-3–like activity, blocked drug-induced caspase-8 cleavage, and depletion of caspase-3 from cell extracts impaired caspase-8 cleavage after in vitro activation with dATP and cytochrome c. Thus, these data indicate that drug-induced caspase-8 activation in B-lymphoma cells is independent of death receptor signaling and is mediated by postmitochondrial caspase-3 activation.

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Faculty Opinions recommendation of Caspase-8 can be activated by interchain proteolysis without receptor-triggered dimerization during drug-induced apoptosis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1023131.266647 ◽

2005 ◽

Author(s):

David Wallach

Keyword(s):

Caspase 8 ◽

Drug Induced ◽

Induced Apoptosis

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Caspase-8 deficiency induces a switch from TLR3 induced apoptosis to lysosomal cell death in neuroblastoma

Scientific Reports ◽

10.1038/s41598-021-89793-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marie-Anaïs Locquet ◽

Gabriel Ichim ◽

Joseph Bisaccia ◽

Aurelie Dutour ◽

Serge Lebecque ◽

...

Keyword(s):

Cell Death ◽

Cancer Cells ◽

Neuroblastoma Cell ◽

Death Receptor ◽

Neuroblastoma Cell Line ◽

Caspase 8 ◽

Caspase Activation ◽

Extrinsic Pathway ◽

Lysosomal Membrane Permeabilization ◽

Induced Apoptosis

AbstractIn cancer cells only, TLR3 acquires death receptor properties by efficiently triggering the extrinsic pathway of apoptosis with Caspase-8 as apical protease. Here, we demonstrate that in the absence of Caspase-8, activation of TLR3 can trigger a form of programmed cell death, which is distinct from classical apoptosis. When TLR3 was activated in the Caspase-8 negative neuroblastoma cell line SH-SY5Y, cell death was accompanied by lysosomal permeabilization. Despite caspases being activated, lysosomal permeabilization as well as cell death were not affected by blocking caspase-activity, positioning lysosomal membrane permeabilization (LMP) upstream of caspase activation. Taken together, our data suggest that LMP with its deadly consequences represents a “default” death mechanism in cancer cells, when Caspase-8 is absent and apoptosis cannot be induced.

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Dexamethasone induces pancreatic β-cell apoptosis through upregulation of TRAIL death receptor

Journal of Molecular Endocrinology ◽

10.1530/jme-20-0238 ◽

2021 ◽

Author(s):

Kanchana Suksri ◽

Namoiy Semprasert ◽

Mutita Junking ◽

Suchanoot Kutpruek ◽

Thawornchai Limjindaporn ◽

...

Keyword(s):

Cell Apoptosis ◽

Molecular Mechanisms ◽

Cultured Cells ◽

Death Receptor ◽

Caspase 8 ◽

Pancreatic Β Cell ◽

Β Cell ◽

Apoptotic Protein ◽

Induced Apoptosis ◽

Trail Death Receptor

Long-term medication with dexamethasone (a synthetic glucocorticoid (GC) drug) results in hyperglycemia, or steroid-induced diabetes. Although recent studies revealed dexamethasone directly induces pancreatic β-cell apoptosis, its molecular mechanisms remain unclear. In our initial analysis of mRNA transcripts, we discovered the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway may be involved in dexamethasone-induced pancreatic β-cell apoptosis. In the present study, a mechanism of dexamethasone-induced pancreatic β-cell apoptosis through the TRAIL pathway was investigated in cultured cells and isolated mouse islets. INS-1 cells were cultured with and without dexamethasone in the presence or absence of a glucocorticoid receptor (GR) inhibitor, RU486. We found that dexamethasone induced pancreatic β-cell apoptosis in association with the upregulation of TRAIL mRNA and protein expression. Moreover, dexamethasone upregulated the TRAIL death receptor (DR5) protein but suppressed the decoy receptor (DcR1) protein. Similar findings were observed in mouse isolated islets: dexamethasone increased TRAIL and DR5 compared to that of control mice. Furthermore, dexamethasone stimulated pro-apoptotic signaling including superoxide production, caspase-8, -9, and -3 activities, NF-B, and Bax, but repressed the anti-apoptotic protein, Bcl-2. All these effects were inhibited by the GR-inhibitor, RU486. Furthermore, knock down DR5 decreased dexamethasone-induced caspase 3 activity. Caspase-8 and caspase-9 inhibitors protected pancreatic β-cells from dexamethasone-induced apoptosis. Taken together, dexamethasone induced pancreatic β-cell apoptosis by binding to the GR and inducing DR5 and TRAIL pathway.

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RIPK1 promotes death receptor-independent caspase-8-mediated apoptosis under unresolved ER stress conditions

Cell Death and Disease ◽

10.1038/cddis.2014.523 ◽

2014 ◽

Vol 5 (12) ◽

pp. e1555-e1555 ◽

Cited By ~ 23

Author(s):

Y Estornes ◽

M A Aguileta ◽

C Dubuisson ◽

J De Keyser ◽

V Goossens ◽

...

Keyword(s):

Er Stress ◽

Molecular Mechanisms ◽

Death Receptor ◽

Caspase 8 ◽

Interacting Protein ◽

Life And Death ◽

Unfolded Protein ◽

Induced Apoptosis ◽

The Unfolded Protein Response ◽

Protein Response

Abstract Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and results in the activation of the unfolded protein response (UPR), which aims at restoring ER homeostasis. However, when the stress is too severe the UPR switches from being a pro-survival response to a pro-death one, and the molecular mechanisms underlying ER stress-mediated death have remained incompletely understood. In this study, we identified receptor interacting protein kinase 1 (RIPK1)—a kinase at the crossroad between life and death downstream of various receptors—as a new regulator of ER stress-induced death. We found that Ripk1-deficient MEFs are protected from apoptosis induced by ER stressors, which is reflected by reduced caspase activation and PARP processing. Interestingly, the pro-apoptotic role of Ripk1 is independent of its kinase activity, is not regulated by its cIAP1/2-mediated ubiquitylation, and does not rely on the direct regulation of JNK or CHOP, two reportedly main players in ER stress-induced death. Instead, we found that ER stress-induced apoptosis in these cells relies on death receptor-independent activation of caspase-8, and identified Ripk1 upstream of caspase-8. However, in contrast to RIPK1-dependent apoptosis downstream of TNFR1, we did not find Ripk1 associated with caspase-8 in a death-inducing complex upon unresolved ER stress. Our data rather suggest that RIPK1 indirectly regulates caspase-8 activation, in part via interaction with the ER stress sensor inositol-requiring protein 1 (IRE1).

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Functional CD95 ligand and CD95 death-inducing signaling complex in activation-induced cell death and doxorubicin-induced apoptosis in leukemic T cells

Blood ◽

10.1182/blood.v95.1.301 ◽

2000 ◽

Vol 95 (1) ◽

pp. 301-308 ◽

Cited By ~ 90

Author(s):

Simone Fulda ◽

Gudrun Strauss ◽

Eric Meyer ◽

Klaus-Michael Debatin

Keyword(s):

T Cells ◽

Cell Death ◽

Leukemia Cell Line ◽

Receptor Interaction ◽

Caspase 8 ◽

Drug Induced ◽

Signaling Complex ◽

Cd95 Ligand ◽

Activation Induced Cell Death ◽

Induced Apoptosis

Abstract Activation-induced cell death (AICD) in T cells is mediated by CD95 ligand (CD95L)/receptor interaction, which has also been implicated in apoptosis induction by some anticancer agents. In this article we show that both anti-CD3-triggering (AICD) and doxorubicin treatment led to the production of a functionally active CD95L in the CD3+/T-cell receptor-positive (TCR+) T leukemia cell line H9. CD95L-expressing H9 cells killed CD95-sensitive J16 or CEM target cells, but not CD95-resistant CEM or J16 cells overexpressing dominant negative FADD (J16/FADD-DN). By immunoprecipitation, CD95L was physically bound to CD95, suggesting that AICD and doxorubicin-induced apoptosis involve CD95L-mediated CD95 aggregation, thereby triggering the CD95 death pathway. CD95 aggregation was associated with the recruitment of FADD and caspase-8 to the CD95 receptor to form the CD95 death-inducing signaling complex (DISC), resulting in caspase-8 activation and cleavage of the effector caspase-3 and PARP. Blocking of the CD95L/receptor interaction by antagonistic antibodies to CD95 or to CD95L also blocked AICD and inhibited the early phase of doxorubicin-induced apoptosis, though cell death induced by doxorubicin eventually proceeded in a CD95-independent manner. These findings may explain some conflicting data on the role of death receptor systems in drug-induced apoptosis. Thus, in cells with an inducible CD95 receptor/ligand system, drug-induced apoptosis may be mediated by CD95L-initiated DISC formation and activation of downstream effector programs similar to AICD in T cells. (Blood. 2000;95:301-308)

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Death Receptor-induced Apoptosis Reveals a Novel Interplay between the Chromosomal Passenger Complex and CENP-C during Interphase

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0409 ◽

2007 ◽

Vol 18 (4) ◽

pp. 1337-1347 ◽

Cited By ~ 12

Author(s):

Alison J. Faragher ◽

Xiao-Ming Sun ◽

Michael Butterworth ◽

Nick Harper ◽

Mike Mulheran ◽

...

Keyword(s):

Death Receptor ◽

Caspase 8 ◽

Cleavage Sites ◽

Caspase Activation ◽

Functional Link ◽

Chromosomal Passenger Complex ◽

Caspase Cleavage ◽

Chromosomal Passenger ◽

Caspase 7 ◽

Induced Apoptosis

Despite the fact that the chromosomal passenger complex is well known to regulate kinetochore behavior in mitosis, no functional link has yet been established between the complex and kinetochore structure. In addition, remarkably little is known about how the complex targets to centromeres. Here, in a study of caspase-8 activation during death receptor-induced apoptosis in MCF-7 cells, we have found that cleaved caspase-8 rapidly translocates to the nucleus and that this translocation is correlated with loss of the centromere protein (CENP)-C, resulting in extensive disruption of centromeres. Caspase-8 activates cytoplasmic caspase-7, which is likely to be the primary caspase responsible for cleavage of CENP-C and INCENP, a key chromosomal passenger protein. Caspase-mediated cleavage of CENP-C and INCENP results in their mislocalization and the subsequent mislocalization of Aurora B kinase. Our results demonstrate that the chromosomal passenger complex is displaced from centromeres as a result of caspase activation. Furthermore, mutation of the primary caspase cleavage sites of INCENP and CENP-C and expression of noncleavable CENP-C or INCENP prevent the mislocalization of the passenger complex after caspase activation. Our studies provide the first evidence for a functional interplay between the passenger complex and CENP-C.

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