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

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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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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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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Human MxB Inhibits the Replication of Hepatitis C Virus

Journal of Virology ◽

10.1128/jvi.01285-18 ◽

2018 ◽

Vol 93 (1) ◽

Cited By ~ 7

Author(s):

Dong-Rong Yi ◽

Ni An ◽

Zhen-Long Liu ◽

Feng-Wen Xu ◽

Kavita Raniga ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Dengue Virus ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Rna Replication ◽

Encephalitis Virus ◽

Common Mechanism ◽

Type I ◽

Dependent Manner

ABSTRACTType I interferon (IFN) inhibits viruses by inducing the expression of antiviral proteins. The IFN-induced myxovirus resistance B (MxB) protein has been reported to inhibit a limited number of viruses, including HIV-1 and herpesviruses, but its antiviral coverage remains to be explored further. Here we show that MxB interferes with RNA replication of hepatitis C virus (HCV) and significantly inhibits viral replication in a cyclophilin A (CypA)-dependent manner. Our data further show that MxB interacts with the HCV protein NS5A, thereby impairing NS5A interaction with CypA and NS5A localization to the endoplasmic reticulum, two events essential for HCV RNA replication. Interestingly, we found that MxB significantly inhibits two additional CypA-dependent viruses of theFlaviviridaefamily, namely, Japanese encephalitis virus and dengue virus, suggesting a potential link between virus dependence on CypA and virus susceptibility to MxB inhibition. Collectively, these data have identified MxB as a key factor behind IFN-mediated suppression of HCV infection, and they suggest that other CypA-dependent viruses may also be subjected to MxB restriction.IMPORTANCEViruses of theFlaviviridaefamily cause major illness and death around the world and thus pose a great threat to human health. Here we show that IFN-inducible MxB restricts several members of theFlaviviridae, including HCV, Japanese encephalitis virus, and dengue virus. This finding not only suggests an active role of MxB in combating these major pathogenic human viruses but also significantly expands the antiviral spectrum of MxB. Our study further strengthens the link between virus dependence on CypA and susceptibility to MxB restriction and also suggests that MxB may employ a common mechanism to inhibit different viruses. Elucidating the antiviral functions of MxB advances our understanding of IFN-mediated host antiviral defense and may open new avenues to the development of novel antiviral therapeutics.

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Japanese Encephalitis Virus Infected Microglial Cells Secrete Exosomes Containing let-7a/b that Facilitate Neuronal Damage via Caspase Activation

10.1101/324715 ◽

2018 ◽

Author(s):

Sriparna Mukherjee ◽

Irshad Akbar ◽

Bharti Kumari ◽

Sudhanshu Vrati ◽

Anirban Basu ◽

...

Keyword(s):

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Neuronal Damage ◽

Cell Communication ◽

Notch Signaling Pathway ◽

Encephalitis Virus ◽

Microglial Cells ◽

Dependent Manner ◽

Caspase Activation ◽

Hek 293

AbstractExtracellular microRNAs (miRNAs) are essential for the cell to cell communication in the healthy and diseased brain. MicroRNAs released from the activated microglia upon neurotropic virus infection may exacerbate CNS damage. Here, we identified let-7a and let-7b (let-7a/b) as the overexpressed miRNAs in Japanese Encephalitis virus (JEV) infected microglia and assessed their role in JEV pathogenesis. We measured the let-7a/b expressions in JEV infected post-mortem human brains, mice brains and in mouse microglial N9 cells by the qRT-PCR and in situ hybridization assay. The interaction between let-7a/b and NOTCH signaling pathway further examined in Toll-like receptor 7 knockdown (TLR7 KD) mice to assess the functions. Exosomes released from JEV infected or let-7a/b mimic transfected N9, and HEK-293 cells were isolated and evaluated their function. We observed an upregulation of let-7a/b in the infected brains as well as in microglia. Knockdown of TLR7 or Inhibition of let-7a/b suppressed the JEV induced NOTCH activation possibly via NF-κB dependent manner and subsequently, attenuated JEV induced TNFα production in microglial cells. Further, exosomes secreted from JEV-infected microglial cells specifically contained let-7a/b. Exosomes overexpressed with let-7a/b were injected into BALB/c mice as well as co-incubated with mouse neuronal (Neuro2a) cells, or primary cortical neuron resulted in caspase activation leading to neuronal damage in the brain. Thus, our results provide evidence for the multifaceted role of let-7a/b miRNAs and unravel the exosomes mediated mechanism for JEV induced pathogenesis.

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Japanese Encephalitis Virus Induces Apoptosis and Encephalitis by Activating the PERK Pathway

Journal of Virology ◽

10.1128/jvi.00887-19 ◽

2019 ◽

Vol 93 (17) ◽

Cited By ~ 6

Author(s):

Qianruo Wang ◽

Xiu Xin ◽

Ting Wang ◽

Jiawu Wan ◽

Yangtao Ou ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Encephalitis Virus ◽

Protein Kinase R ◽

Apoptosis Pathway ◽

Stress Sensor ◽

Sensor Protein ◽

Induced Apoptosis

ABSTRACTAccumulated evidence demonstrates that Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, the precise role of PERK in JEV-induced apoptosis and encephalitis remains unknown. Here, we report that JEV infection activates the PERK-ATF4-CHOP apoptosis pathway bothin vitroandin vivo. PERK activation also promotes the formation of stress granule, which in turn represses JEV-induced apoptosis. However, PERK inhibitor reduces apoptosis, indicating that JEV-activated PERK predominantly induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway. Among JEV proteins that have been reported to induce ER stress, only JEV NS4B can induce PERK activation. PERK has been reported to form an active molecule by dimerization. The coimmunoprecipitation assay shows that NS4B interacts with PERK. Moreover, glycerol gradient centrifugation shows that NS4B induces PERK dimerization. Both the LIG-FHA and the LIG-WD40 domains within NS4B are required to induce PERK dimerization, suggesting that JEV NS4B pulls two PERK molecules together by simultaneously interacting with them via different motifs. PERK deactivation reduces brain cell damage and encephalitis during JEV infection. Furthermore, expression of JEV NS4B is sufficient to induce encephalitis via PERK in mice, indicating that JEV activates PERK primarily via its NS4B to cause encephalitis. Taken together, our findings provide a novel insight into JEV-caused encephalitis.IMPORTANCEJapanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, whether the PERK pathway of ER stress response plays important roles in JEV-induced apoptosis and encephalitis remains unknown. Here, we found that JEV infection activates ER stress sensor PERK in neuronal cells and mouse brains. PERK activation induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway upon JEV infection. Among the JEV proteins prM, E, NS1, NS2A, NS2B, and NS4B, only NS4B activates PERK. Moreover, activated PERK participates in apoptosis and encephalitis induced by JEV and NS4B. These findings provide a novel therapeutic approach for JEV-caused encephalitis.

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SMAC-Mimetic BV-6 Sensitizes Therapeutic Agents-Induced Apoptosis In AML Cells

Blood ◽

10.1182/blood.v116.21.2177.2177 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2177-2177

Author(s):

Duncan H Mak ◽

Christa Manton ◽

Michael Andreeff ◽

Bing Z Carter

Keyword(s):

Cell Death ◽

Vector Control ◽

Caspase 3 ◽

Jurkat Cells ◽

Leukemic Cells ◽

Caspase 8 ◽

Caspase 9 ◽

Smac Mimetic ◽

P53 Activation ◽

Induced Apoptosis

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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Arsenic trioxide–induced apoptosis in myeloma cells: p53-dependent G1 or G2/M cell cycle arrest, activation of caspase-8 or caspase-9, and synergy with APO2/TRAIL

Blood ◽

10.1182/blood-2002-10-3231 ◽

2003 ◽

Vol 101 (10) ◽

pp. 4078-4087 ◽

Cited By ~ 138

Author(s):

Qun Liu ◽

Susan Hilsenbeck ◽

Yair Gazitt

Keyword(s):

Cell Cycle ◽

Arsenic Trioxide ◽

Cell Lines ◽

Myeloma Cell ◽

Caspase 8 ◽

Dependent Manner ◽

Caspase 9 ◽

Myeloma Cells ◽

Induced Apoptosis ◽

In Cells

Abstract Arsenic trioxide (ATO) has been shown to induce differentiation and apoptosis in acute promyelocytic leukemia (APL) cells concomitant with down-regulation of the PML-RARα fusion protein, a product of the t(15:17) translocation characteristic of APL leukemic cells. However, ATO is also a potent inducer of apoptosis in a number of other cancer cells lacking the t(15:17) translocation. The exact mechanism of ATO-induced apoptosis in these cells is not yet clear. We tested the effect of ATO on 7 myeloma cell lines with varying p53 status and report that in cells with mutated p53, ATO induced rapid and extensive (more than 90%) apoptosis in a time- and dose-dependent manner concomitant with arrest of cells in G2/M phase of the cell cycle. Myeloma cells with wild-type (wt) p53 were relatively resistant to ATO with maximal apoptosis of about 40% concomitant with partial arrest of cells in G1 and up-regulation of p21. The use of caspase blocking peptides, fluorescence-tagged caspase-specific substrate peptides, and Western immunoblotting confirmed the involvement of primarily caspase-8 and -3 in ATO-induced apoptosis in myeloma cells with mutated p53 and primarily caspase-9 and -3 in cells expressing wt p53. We also observed up-regulation by ATO of R1 and R2 APO2/TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) receptors. Most important, however, we observed a synergy between ATO and APO2/TRAIL in the induction of apoptosis in the partially resistant myeloma cell lines and in myeloma cells freshly isolated from myeloma patients. Our results justify the use of the combination of these 2 drugs in clinical setting in myeloma patients.

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Apoptotic Signaling Pathway Activated byHelicobacter pylori Infection and Increase of Apoptosis-Inducing Activity under Serum-Starved Conditions

Infection and Immunity ◽

10.1128/iai.69.5.3181-3189.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 3181-3189 ◽

Cited By ~ 37

Author(s):

Keigo Shibayama ◽

Yohei Doi ◽

Naohiro Shibata ◽

Tetsuya Yagi ◽

Toshi Nada ◽

...

Keyword(s):

Membrane Fraction ◽

Proteinase K ◽

Caspase 8 ◽

Peptic Ulcers ◽

Caspase 9 ◽

Effector Caspase ◽

Epithelial Cell Apoptosis ◽

Apoptotic Activity ◽

H Pylori ◽

Induced Apoptosis

ABSTRACT The enhanced gastric epithelial cell apoptosis observed during infection with Helicobacter pylori has been suggested to be of significance in the etiology of gastritis, peptic ulcers, and neoplasia. To investigate the cell death signaling induced by H. pylori infection, human gastric epithelial cells were incubated with H. pylori for up to 72 h. H. pyloriinfection induced the activation of caspase -8, -9, and -3 and the expression of the proapoptotic Bcl-2 family proteins Bad and Bid. The peak of the activity of the caspases occurred at 24 h. At this time, the inhibition of caspase-8 or -9 almost completely suppressedH. pylori-induced apoptosis. Inhibition of caspase-8 suppressed the expression of Bad and Bid and the subsequent activation of caspase-9 and -3. These observations indicate that H. pylori induces apoptosis through a pathway involving the sequential induction of apical caspase-8 activity, the proapoptotic proteins Bad and Bid, caspase-9 activity, and effector caspase-3 activity. Activation of the pathway was independent of CagA or vacuolating toxin. A membrane fraction of H. pylori was sufficient to activate this pathway, and treatment with proteinase K eliminated the activity. Apoptotic activity of the membrane fraction was significantly increased by incubating the bacteria under serum-starved conditions for 24 h. These observations suggest that environmental conditions in the human stomach could induce H. pylori-mediated pathogenesis, leading to a variety of clinical outcomes.

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Role of mitochondria in aspirin-induced apoptosis in human gastric epithelial cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00150.2005 ◽

2005 ◽

Vol 289 (4) ◽

pp. G731-G738 ◽

Cited By ~ 24

Author(s):

Maria J. Redlak ◽

Jacinda J. Power ◽

Thomas A. Miller

Keyword(s):

Epithelial Cells ◽

Complex Formation ◽

Oxidative Phosphorylation ◽

Caspase 8 ◽

Mucosal Cell ◽

Dependent Manner ◽

Gastric Epithelial Cells ◽

Caspase 9 ◽

Mitochondrial Fractions ◽

Induced Apoptosis

This study was undertaken to determine whether the Bcl-2 family proteins and Smac are regulators of aspirin-mediated apoptosis in a gastric mucosal cell line known as AGS cells. Cells were incubated with varying concentrations of acetylsalicylic acid (ASA; 2–40 mM), with or without preincubation of caspase inhibitors. Apoptosis was characterized by Hoechst staining and DNA-histone-associated complex formation. Antiapoptotic Bcl-2, proapoptotic Bax and Bid, Smac, and cytochrome- c oxidase (COX IV) were analyzed by Western blot analyses from cytosol and mitochondrial fractions. ASA downregulated Bcl-2 protein expression and induced Bax translocation into the mitochondria and cleavage of Bid. In contrast, expression of Smac was significantly decreased in mitochondrial fractions of ASA-treated cells. Bax and Bid involvement in apoptosis regulation was dependent on caspase activation, because caspase-8 inhibition suppressed Bax translocation and Bid processing. Caspase-9 inhibition prevented Smac release from mitochondria. Additionally, increased expression of the oxidative phosphorylation enzyme COX IV was observed in mitochondrial fractions exposed to ASA at concentrations >5 mM. Although caspase-8 inhibition had no effect on aspirin-induced apoptosis and DNA-histone complex formation, caspase-9 inhibition significantly decreased both of these events. We conclude that Bcl-2 protein family members and Smac regulate the apoptotic pathway in a caspase-dependent manner. Our results indicate also that mitochondrial integration and oxidative phosphorylation play a critical role in the pathogenesis of apoptosis in human gastric epithelial cells.

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