Caspase 3 is Activated through Caspase 8 instead of Caspase 9 during H<sub>2</sub>O<sub>2</sub>-induced Apoptosis in HeLa Cells

Abstract Abstract 2177 The antiapoptotic function of the inhibitors of apoptosis family of proteins (IAPs) is antagonized by mitochondria-released SMAC protein. The IAP-member XIAP suppresses apoptosis by directly binding and inhibiting caspase-9 and caspase-3, while cIAP1, a component of the cytoplasmic signaling complex containing TNF receptor associated factors, suppresses apoptosis via the caspase-8-mediated pathway. BV-6 (Genentech) is a bivalent SMAC-mimetic and has been shown to promote cell death by inducing cIAP autoubiquitination, NF-κB activation, and TNFα-dependent apoptosis. We examined its effect on leukemic cells and found that BV-6 only moderately induced apoptosis. The EC50 was found to be 15.3±5.1 μM at 48 hours in OCI-AML3 cells which are relatively sensitive. We then determined whether BV-6 sensitizes leukemic cells to the HDM2-inhibitor nutlin-3a and to Ara-C. p53 modulates the expression and activity of Bcl-2 family proteins and promotes the mitochondrial-mediated apoptosis. We showed previously that activation of p53 by nutlin-3a sensitizes AML cells to XIAP inhibition induced-death in part by promoting the release of SMAC from mitochondrion (Carter BZ et al., Blood 2010). We treated OCI-AML3 cells with BV-6, nutlin-3a or Ara-C, and BV-6+nutlin-3a or BV-6+Ara-C and found that the combination of BV-6 and nutlin-3a or BV-6 and Ara-C synergistically induced cell death in OCI-AML3 cells with a combination index (CI) of 0.27±0.11 and 0.22±0.05 (48 hours), respectively. To demonstrate that p53 activation is essential for the synergism of BV-6+nutlin-3a combination, we treated OCI-AML3 vector control and p53 knockdown cells with these two agents and found that the combination synergistically promoted cell death in the vector control (CI=0.47±0.15) but not in the p53 knockdown cells, as expected, while BV6+Ara-C was synergistic in both vector control and p53 knockdown cells (CI=0.15±0.03 and 0.08±0.03, respectively, 48 hours). BV-6 induced activation of caspase-8, caspase-9, and caspase-3 and decreased XIAP levels, but did not cause rapid cIAP1 degradation, as reported by others. To assess the contribution of death receptor-mediated apoptosis in BV-6-induced cell death, we treated Jurkat and caspase-8 mutated Jurkat cells (JurkatI9.2) with BV-6 and found that BV-6 induced cell death and significantly potentiated TRAIL-induced apoptosis in Jurkat cells (CI=0.14±0.08, 48 hours). Caspase-8 mutated JurkatI9.2 cells were significantly less sensitive to BV-6 than Jurkat cells and as expected, JurkatI9.2 was completely resistant to TRAIL. Collectively, we showed that the bivalent SMAC-mimetic BV-6 potentiates p53 activation-, chemotherapy-, and TRAIL-induced cell death, but has only minimal activity by itself in leukemic cells. SMAC-mimetics could be useful in enhancing the efficacy of different classes of therapeutic agents used in AML therapy. Disclosures: No relevant conflicts of interest to declare.

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NeosedumosideIII induced Apoptosis of Human Hepatocellular Carcinoma HepG2 cells and SMMC-7721 Cells and Related Mechanism

10.21203/rs.3.rs-396990/v1 ◽

2021 ◽

Author(s):

Xin-Yu Li ◽

Xin Zhou ◽

Yu- Liu ◽

Feng Qiu ◽

Qing-Qing Zhao

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Cycle ◽

Hepg2 Cells ◽

Caspase 3 ◽

Human Hepatocellular Carcinoma ◽

Caspase 8 ◽

Dependent Manner ◽

Caspase 9 ◽

Induced Apoptosis

Abstract Purpose: NeosedumosideIII (Neo) is a megastigmanes and belongs to monocyclic sesquiterpenoids compound with antioxidant, anti-inflammatory and other pharmacological activities. In order to explore the anti-cancer effect and possible mechanism of Neo, the study examined the anti-proliferation and apoptosis effect of Neo against human hepatocellular carcinoma HepG2 cells and SMMC-772 cells and related mechanism in vitro. Methods ：The anti-proliferation effect of Neo was detected on HepG2 cells and SMMC-772 cells by MTT assay and IC50 with increasing dose and time. Cell cycle and apoptosis were detected by flow cytometer. The changes of Bcl-2, Bax, Caspase-3, Caspase-8 and Caspase-9 proteins were detected by western blotting.Results ：The results indicated that Neo could inhibited proliferation of HepG2 cells and SMMC-772 cells in vitro and promoted apoptosis, it significantly induced apoptosis of HepG2 cells and SMMC-772 cells arrested cell cycle at G0/G1 phase in a dose-dependent manner, reduce the expression of Bcl-2 protein, and increase the expression of Bax and Caspase-3, Caspase-8 and Caspase-9 proteins. Conclusion：Neo could inhibit proliferation and induce apoptosis of HepG2 cells and SMMC-7721 cells in vivo which suggested that it might be served as a promising candidate for the treatment of liver cancer.

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Activation of caspase-8 in 3-deazaadenosine-induced apoptosis of U-937 cells occurs downstream of caspase-3 and caspase-9 without Fas receptor-ligand interaction

Experimental & Molecular Medicine ◽

10.1038/emm.2001.46 ◽

2001 ◽

Vol 33 (4) ◽

pp. 284-292 ◽

Cited By ~ 20

Author(s):

Yeo-Jin Chae ◽

Ho-Shik Kim ◽

Hyangshuk Rhim ◽

Bo-Eun Kim ◽

Seong-Whan Jeong ◽

...

Keyword(s):

Caspase 3 ◽

Caspase 8 ◽

Caspase 9 ◽

Ligand Interaction ◽

Receptor Ligand ◽

Fas Receptor ◽

Induced Apoptosis

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Rapid Apoptosis Induced by Shiga Toxin in HeLa Cells

Infection and Immunity ◽

10.1128/iai.71.5.2724-2735.2003 ◽

2003 ◽

Vol 71 (5) ◽

pp. 2724-2735 ◽

Cited By ~ 58

Author(s):

Jun Fujii ◽

Takashi Matsui ◽

Daniel P. Heatherly ◽

Kailo H. Schlegel ◽

Peter I. Lobo ◽

...

Keyword(s):

Dna Fragmentation ◽

Hela Cells ◽

Shiga Toxin ◽

Specific Inhibitor ◽

Mitochondrial Pathway ◽

Caspase 8 ◽

Dependent Manner ◽

Mitochondrial Membranes ◽

Caspase 9 ◽

Induced Apoptosis

ABSTRACT Apoptosis was induced rapidly in HeLa cells after exposure to bacterial Shiga toxin (Stx1 and Stx2; 10 ng/ml). Approximately 60% of HeLa cells became apoptotic within 4 h as detected by DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and electron microscopy. Stx1-induced apoptosis required enzymatic activity of the Stx1A subunit, and apoptosis was not induced by the Stx2B subunit alone or by the anti-globotriaosylceramide antibody. This activity was also inhibited by brefeldin A, indicating the need for toxin processing through the Golgi apparatus. The intracellular pathway leading to apoptosis was further defined. Exposure of HeLa cells to Stx1 activated caspases 3, 6, 8, and 9, as measured both by an enzymatic assay with synthetic substrates and by detection of proteolytically activated forms of these caspases by Western immunoblotting. Preincubation of HeLa cells with substrate inhibitors of caspases 3, 6, and 8 protected the cells against Stx1-dependent apoptosis. These results led to a more detailed examination of the mitochondrial pathway of apoptosis. Apoptosis induced by Stx1 was accompanied by damage to mitochondrial membranes, measured as a reduced mitochondrial membrane potential, and increased release of cytochrome c from mitochondria at 3 to 4 h. Bid, an endogenous protein known to permeabilize mitochondrial membranes, was activated in a Stx1-dependent manner. Caspase-8 is known to activate Bid, and a specific inhibitor of caspase-8 prevented the mitochondrial damage. Although these data suggested that caspase-8-mediated cleavage of Bid with release of cytochrome c from mitochondria and activation of caspase-9 were responsible for the apoptosis, preincubation of HeLa cells with a specific inhibitor of caspase-9 did not protect against apoptosis. These results were explained by the discovery of a simultaneous Stx1-dependent increase in endogenous XIAP, a direct inhibitor of caspase-9. We conclude that the primary pathway of Stx1-induced apoptosis and DNA fragmentation in HeLa cells is unique and includes caspases 8, 6, and 3 but is independent of events in the mitochondrial pathway.

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Involvement of the Mitochondria-Caspase Pathway in HeLa Cell Death Induced by 2-Ethanolamino-2-Demethoxy-17-Ethanolimino-Hypocrellin B (EAHB)-Mediated Photodynamic Therapy

International Journal of Toxicology ◽

10.1177/1091581812460696 ◽

2012 ◽

Vol 31 (5) ◽

pp. 483-492 ◽

Cited By ~ 1

Author(s):

Zhixiang Zhou ◽

Chen Zhao ◽

Wei Liu ◽

Qianqian Li ◽

Lina Zhang ◽

...

Keyword(s):

Photodynamic Therapy ◽

Hela Cells ◽

Caspase 3 ◽

Caspase 9 ◽

Cytochrome C Release ◽

Hypocrellin B ◽

Confocal Fluorescence ◽

Cellular Apoptosis ◽

Induced Apoptosis ◽

Dependent Mechanism

In order to elucidate the mechanism of cytotoxicity photoinduced by 2-ethanolamino-2-demethoxy-17-ethanolimino-hypocrellin B (EAHB), a derivative of hypocrellin B (HB), cellular uptake, subcellular localization as well as photodynamic therapy (PDT) efficiency of EAHB, and cell apoptosis photoinduced by EAHB were investigated in HeLa cells by laser confocal fluorescence microscopy, 3-(4,5-Dimethylthiazol-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, flow cytometry, DNA fragmentation on agarose gel, and Western blot. The results showed EAHB was distributed throughout the cytoplasm of the cell, with no detectable penetration into the nucleus. The proportion of dead cells increased with increases in both the dosage of light and the concentration of EAHB. Its phototoxicity to HeLa cells proceeded via apoptosis. The EAHB-PDT treatment induced a cytochrome c release from the mitochondria into the cytosol followed by the activation of both caspase 3 and caspase 9 in HeLa cells. The results suggested that EAHB-PDT treatment induced apoptosis in HeLa cells, and the cellular apoptosis involved a mitochondria-/caspase-dependent mechanism.

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Japanese encephalitis virus infection activates caspase-8 and -9 in a FADD-independent and mitochondrion-dependent manner

Journal of General Virology ◽

10.1099/vir.0.2008/000182-0 ◽

2008 ◽

Vol 89 (8) ◽

pp. 1930-1941 ◽

Cited By ~ 22

Author(s):

Chang-Huei Tsao ◽

Hong-Lin Su ◽

Yi-Ling Lin ◽

Han-Pang Yu ◽

Shu-Ming Kuo ◽

...

Keyword(s):

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Caspase 3 ◽

Encephalitis Virus ◽

Dominant Negative Mutant ◽

Caspase 8 ◽

Dependent Manner ◽

Caspase 9 ◽

Effector Caspase ◽

Induced Apoptosis

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, replicates primarily at the endoplasmic reticulum and thereby triggers apoptosis of infected cells. This study investigated the hierarchical activation of the caspase network induced by JEV infection. It was found that JEV activated the initiators caspase-8 and -9, as well as effector caspase-3, in infected baby hamster kidney and mouse neuroblastoma (N18) cells. In neuronal N18 cells, JEV infection triggered cytochrome c release from mitochondria, which in turn activated caspase-9 and -3. Treatment of JEV-infected N18 cells with cyclosporin A or ruthenium red, which attenuate mitochondrial injuries, blocked activation of caspase-9 or -3, typifying that, in neuronal cells, this apoptosis involves the mitochondrial pathway. Alternatively, in caspase-3-deficient MCF-7 cells, JEV persisted and readily triggered a typical apoptotic response, including cytochrome c release and full activation of caspase-9 and -8 along with caspase-6, indicating that JEV did not require caspase-3 to manifest caspase-8 activation and apoptosis. Interestingly, a Fas-associated death-domain-containing protein (FADD) dominant-negative mutant, which interfered with transmission of the extracellular death signals into cells through the Fas/tumour necrosis factor (TNF) receptor, failed to block JEV-induced apoptosis and caspase-8 activation, implying that receptor oligomerization of the Fas/TNF pathway might not participate in JEV-induced apoptosis. Taken together, these results illustrate that JEV infection triggers caspase cascades involving the initiators caspase-8 and -9, probably through FADD-independent but mitochondrion-dependent pathways.

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Smac Mimetic-Mediated Sensitization Of Childhood ALL Cells For Drug-Induced Apoptosis Independent Of TNFα and NFκB Signaling Pathways

Blood ◽

10.1182/blood.v122.21.2911.2911 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2911-2911

Author(s):

Karin Schmelz ◽

Nina Weichert ◽

Jutta Proba ◽

Marie-Sophie Erdmann ◽

Patrick Hundsdoerfer

Keyword(s):

Signaling Pathways ◽

Tumor Cells ◽

Caspase 3 ◽

Caspase 8 ◽

Apoptosis Induction ◽

Caspase 9 ◽

Drug Induced ◽

Childhood All ◽

Apoptosis Pathway ◽

Induced Apoptosis

Abstract Targeting inhibitor of apoptosis proteins (IAPs) using small molecular Smac mimetics (SM) has been shown to offer a novel promising treatment strategy for resistant malignant diseases including childhood acute lymphoblastic leukemia (ALL). The effect of SM alone has been shown to be associated with endogenous TNFα expression, therefore tumor cells can be classified into sensitive or resistant against apoptosis induction by SM alone. In SM sensitive tumor cells the effect of SM has been shown to be mediated mainly by degradation of cellular IAP (cIAP) and activation of TNFα and NFκB signaling pathways but not inhibition of XIAP. We show here, that sensitivity of ALL cells to SM alone (as well as TNFα expression) is highly variable. Nevertheless even in ALL cells resistant against SM alone, treatment with SM resulted in significant sensitization for drugs used within standard induction therapy for childhood ALL. Sensitization for drug-induced apoptosis by SM was not only mediated by activation of the intrinsic (cleavage of caspase 9) but also extrinsic apoptosis pathway (cleavage of caspase 8). Surprisingly, SM-induced cIAP degradation alone was not sufficient for caspase 8 activation and apoptosis induction. Consistently, SM-mediated sensitization for drug-induced apoptosis was independent of TNFα and NFκB signaling pathways. We demonstrate that caspase 8 activation by combined treatment with SM and cytostatic drugs is blocked by inhibition of caspase 3 and caspase 9 and therefore occurs downstream of intrinsic apoptosis pathway activation. In conclusion, our data argue for a model comprising inhibition of XIAP-mediated blockade of caspase 3/9 as the central effect of SM in chemo-sensitization of childhood ALL cells resistant against SM-alone. Disclosures: No relevant conflicts of interest to declare.

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Dexamethasone-induced apoptosis of thymocytes: role of glucocorticoid receptor–associated Src kinase and caspase-8 activation

Blood ◽

10.1182/blood-2002-06-1779 ◽

2003 ◽

Vol 101 (2) ◽

pp. 585-593 ◽

Cited By ~ 83

Author(s):

Maria Cristina Marchetti ◽

Barbara Di Marco ◽

Grazia Cifone ◽

Graziella Migliorati ◽

Carlo Riccardi

Keyword(s):

Cytochrome C ◽

Glucocorticoid Receptor ◽

Caspase 3 ◽

Kinase Inhibitor ◽

Src Kinase ◽

Caspase 8 ◽

Caspase 9 ◽

Cytochrome C Release ◽

Sequence Of Events ◽

Induced Apoptosis

Glucocorticoid hormones (GCHs) regulate normal and neoplastic lymphocyte development by exerting antiproliferative and/or apoptotic effects. We have previously shown that dexamethasone (DEX)–activated thymocyte apoptosis requires a sequence of events including interaction with the glucocorticoid receptor (GR), phosphatidylinositol-specific phospholipase C (PI-PLC), and acidic sphingomyelinase (aSMase) activation. We analyzed the mechanisms of GCH-activated apoptosis by focusing on GR-associated Src kinase, cytochrome c release, and caspase-8, -9, and -3 activation. We show here that PI-PLC binds to GR-associated Src kinase, as indicated by coimmunoprecipitation experiments. Moreover, DEX treatment induces PI-PLC phosphorylation and activation. DEX-induced PI-PLC phosphorylation, activation, and apoptosis are inhibited by PP1, a Src kinase inhibitor, thus suggesting that Src-mediated PI-PLC activation is involved in DEX-induced apoptosis. Caspase-9, -8, and -3 activation and cytochrome c release can be detected 1 to 2 hours after DEX treatment. Caspase-9 inhibition does not counter cytochrome crelease, caspase-8 and caspase-3 activation, and apoptosis. Caspase-8 inhibition counters cytochrome c release, caspase-9 and caspase-3 activation, and apoptosis, thus suggesting that caspase-8 inhibitor can directly inhibit caspase-9 and/or that DEX-induced caspase-8 activation is upstream to mitochondria and can regulate caspase-3 directly or through cytochrome c release and the consequent caspase-9/caspase-3 activation. DEX-induced caspase-8 activation, like ceramide-induced caspase-8 activation, correlates with the formation of Fas-associated death domain protein (FADD)/caspase-8 complex. Caspase-8 activation is countered by the inhibition of macromolecular synthesis and of Src kinase, PI-PLC, and aSMase activation, suggesting it is downstream in the DEX-activated apoptotic pathway of thymocytes.

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Molecular mechanism of satratoxin-induced apoptosis in HL-60 cells: activation of caspase-8 and caspase-9 is involved in activation of caspase-3

Immunology Letters ◽

10.1016/s0165-2478(02)00127-x ◽

2002 ◽

Vol 84 (1) ◽

pp. 23-27 ◽

Cited By ~ 19

Author(s):

Masahiro Nagase ◽

Tetsuya Shiota ◽

Akiko Tsushima ◽

Mohammad Murshedul Alam ◽

Satoshi Fukuoka ◽

...

Keyword(s):

Molecular Mechanism ◽

Caspase 3 ◽

Caspase 8 ◽

Caspase 9 ◽

Induced Apoptosis

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Leptospira interrogans Induces Apoptosis in Macrophages via Caspase-8- and Caspase-3-Dependent Pathways

Infection and Immunity ◽

10.1128/iai.00914-08 ◽

2008 ◽

Vol 77 (2) ◽

pp. 799-809 ◽

Cited By ~ 51

Author(s):

Dandan Jin ◽

David M. Ojcius ◽

Dexter Sun ◽

Haiyan Dong ◽

Yihui Luo ◽

...

Keyword(s):

Molecular Mechanisms ◽

Caspase 3 ◽

Host Cells ◽

Caspase 8 ◽

Lamin A ◽

Leptospira Interrogans ◽

Dependent Manner ◽

Caspase 9 ◽

Caspase 6 ◽

Induced Apoptosis

ABSTRACT Apoptosis of host cells plays an important role in modulating the pathogenesis of many infectious diseases. It has been reported that Leptospira interrogans, the causal agent of leptospirosis, induces apoptosis in macrophages and hepatocytes. However, the molecular mechanisms responsible for host cell death remained largely unknown. Here we demonstrate that L. interrogans induced apoptosis in a macrophage-like cell line, J774A.1, and primary murine macrophages in a time- and dose-dependent manner. Apoptosis was associated with the activation of cysteine aspartic acid-specific proteases (caspase-3, caspase-6, and caspase-8), the increased expression of Fas-associated death domain (FADD), and the cleavage of the caspase substrates poly(ADP-ribose) polymerase (PARP) and nuclear lamina protein (lamin A and lamin C). Caspase-9 was activated to a lesser extent, whereas no release of cytochrome c from mitochondria was detectable. Inhibition of caspase-8 impaired L. interrogans-induced caspase-3 and -6 activation, as well as PARP and lamin A/C cleavage and apoptosis, suggesting that apoptosis is initiated via caspase-8 activation. Furthermore, caspase-3 was required for the activation of caspase-6 and seemed to be involved in caspase-9 activation through a feedback amplification loop. These data indicate that L. interrogans-induced apoptosis in macrophages is mediated by caspase-3 and -6 activation through a FADD-caspase-8-dependent pathway, independently of mitochondrial cytochrome c-caspase-9-dependent signaling.

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