scholarly journals Detection of Apoptosis Initiated in Treated HepG2 Cells with t-BHP: The Role of Phytochemicals to Reduce Toxicity and Stop Apoptosis

2021 ◽  
Vol 2 (9) ◽  
pp. 745-767
Author(s):  
Maha J Hashim
Keyword(s):  
Lipid Peroxidation ◽  
Cell Death ◽  
Hepg2 Cells ◽  
Caspase 3 ◽  
Physiological Mechanism ◽  
Oxygen Species ◽  
Caspase Activation ◽  
Apoptosis Detection ◽  
Infected Cells

Apoptosis or programmed cell death is a standard physiological mechanism. It is essential to control the number of cells, balance cell division and cell death, regulate the immune system, and eliminate pathogen-infected cells. Apoptosis entailed a different investigation to determine related biochemical reactions such as activated caspase, Reactive Oxygen Species (ROS), Lipid Peroxidation (LPO), and Evaluation of Glutathione Content (GSH) by using different techniques. HepG2 cells were exposed to +/- 0.4 and 0.8 mM t-BHP for specific times to induce toxicity for apoptosis detection. We aim to investigate the mechanism of cell death in treated HepG2 with t-BHP under consideration of the conditions of the cytoprotection assay. Results showed no strong evidence for apoptosis, although caspase-3 activity increased significantly (p ≤ 0.05) in treated HpG2 cells with 0.8 mM t-BHP at 150 minutes. The weak proof for apoptosis may attribute to the participation of Calpain through the cross-talk in blocking the caspase- activation. Similarly, we obtained significant ROS and lipid peroxidation increases in treated HepG2 cells with 0.8 mM t-BHP (p ≤ 0.05 and 0.01 respectively) at 150 minutes. Moreover, reported a (non-significant) decline in GSH amounts. Treatment of the cells with Q and I3C under the conditions used in the cytoprotection study prevented the weak activation of caspase-3 identified by western blot.

Reactive oxygen species and caspase activation mediate silica-induced apoptosis in alveolar macrophages

2001 ◽  
Vol 280 (1) ◽  
pp. L10-L17 ◽  
Author(s):  
Han-Ming Shen ◽  
Zhuo Zhang ◽  
Qi-Feng Zhang ◽  
Choon-Nam Ong
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Death ◽  
Alveolar Macrophages ◽  
Caspase 3 ◽  
Reactive Oxygen ◽  
Parp Cleavage ◽  
Oxygen Species ◽  
Caspase Activation ◽  
Caspase 9 ◽  
Induced Apoptosis

Alveolar macrophages (AMs) are the principal target cells of silica and occupy a key position in the pathogenesis of silica-related diseases. Silica has been found to induce apoptosis in AMs, whereas its underlying mechanisms involving the initiation and execution of apoptosis are largely unknown. The main objective of the present study was to examine the form of cell death caused by silica and the mechanisms involved. Silica-induced apoptosis in AMs was evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay and cell cycle/DNA content analysis. The elevated level of reactive oxygen species (ROS), caspase-9 and caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) cleavage in silica-treated AMs were also determined. The results showed that there was a temporal pattern of apoptotic events in silica-treated AMs, starting with ROS formation and followed by caspase-9 and caspase-3 activation, PARP cleavage, and DNA fragmentation. Silica-induced apoptosis was significantly attenuated by a caspase-3 inhibitor, N-acetyl-Asp-Glu-Val-Asp aldehyde, and ebselen, a potent antioxidant. These findings suggest that apoptosis is an important form of cell death caused by silica exposure in which the elevated ROS level that results from silica exposure may act as an initiator, leading to caspase activation and PARP cleavage to execute the apoptotic process.

scholarly journals Essential Role of STAT1 in Caspase-Independent Cell Death of Activated Macrophages through the p38 Mitogen-Activated Protein Kinase/STAT1/Reactive Oxygen Species Pathway

2005 ◽  
Vol 25 (15) ◽  
pp. 6821-6833 ◽  
Author(s):  
Hun Sik Kim ◽  
Myung-Shik Lee
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Death ◽  
P38 Mapk ◽  
Serine Phosphorylation ◽  
Mitogen Activated Protein Kinase ◽  
Oxygen Species ◽  
Activated Macrophages ◽  
Caspase Activation ◽  
Mitogen Activated Protein

ABSTRACT Unlike other immune cells, activation of macrophages by stimulating agents, such as lipopolysaccharide (LPS), confers significant resistance to many apoptotic stimuli, but the underlying mechanism of this phenomenon remains largely unknown. Here, we demonstrate that LPS-induced early caspase activation is essential for macrophage survival because blocking caspase activation with a pancaspase inhibitor (zVAD [benzyloxycarbonyl-Val-Ala-Asp]) rapidly induced death of activated macrophages. This type of death process by zVAD/LPS was principally mediated by intracellular generation of superoxide. STAT1 knockout macrophages demonstrated profoundly decreased superoxide production and were resistant to treatment with zVAD/LPS, indicating the crucial involvement of STAT1 in macrophage death by zVAD/LPS. STAT1 level and activity were reciprocally regulated by caspase activation and were associated with cell death. Activation of STAT1 was critically dependent upon serine phosphorylation induced by p38 mitogen-activated protein kinase (MAPK) because a p38 MAPK inhibitor nullified STAT1 serine phosphorylation, reactive oxygen species (ROS) production, and macrophage death by zVAD/LPS. Conversely, p38 MAPK activation was dependent upon superoxide and was also nullified in STAT1 knockout macrophages, probably due to impaired generation of superoxide. Our findings collectively indicate that STAT1 signaling modulates intracellular oxidative stress in activated macrophages through a positive-feedback mechanism involving the p38 MAPK/STAT1/ROS pathway, which is interrupted by caspase activation. Furthermore, our study may provide significant insights in regards to the unanticipated critical role of STAT1 in the caspase-independent death pathway.

scholarly journals Knock Out of Cell Death Pathway Components Results in Differential Caspase Expression in Response to HCV Infection

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 144
Author(s):  
Hannah L. Wallace ◽  
Lingyan Wang ◽  
Cassandra Davidson ◽  
Vipin Chelakkot ◽  
Michael Grant ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Cross Talk ◽  
Caspase 3 ◽  
Caspase Activation ◽  
Knock Out ◽  
Caspase 1 ◽  
Cell Death Pathway ◽  
Infected Cells ◽  
Active Caspase

Introduction: Pyroptosis (inflammatory programmed cell death) is induced after the activation of an inflammasome, ultimately resulting in pore formation and cell lysis. One factor in the pathology associated with chronic hepatitis C virus (HCV) infection is non-inflammatory caspase-3-mediated apoptosis. Our lab has found both apoptosis and pyroptosis occurring in HCV-infected Huh-7.5 cells. In the context of some viral infections, pyroptosis is beneficial to the virus; for others, pyroptosis is believed to represent an innate antiviral response. This study aimed to test the effects of knocking out components of the inflammasome pathway on caspase activation in HCV-infected cells. Methods: FAM-FLICA (Carboxyfluorescein - Fluorochrome Inhibitor of Caspases) probes or antibodies were used to visualize active caspase-1 and active caspase-3 in vitro. Huh-7.5 cells with components of the pyroptotic or apoptotic pathways knocked out (NLRP3, GSDM-D or caspase-3) were used to determine the effects of their absence on the virus and caspase activation using confocal microscopy and flow cytometry. Results: Increased levels of caspase-1 were consistently observed in HCV-infected cells compared to those in uninfected cells, and these levels increased with subsequent days post-infection. The inhibition of inflammasome activation using knock out cell lines induced the differential activation of caspase-1 and caspase-3, with the inhibition of pyroptosis, resulting in a trend towards greater expression of caspase-3, indicative of apoptosis. The inhibition of NLRP3 did not fully stop caspase-1 activation, but it was decreased. The flow cytometry results revealed a small sub-set of cells positive for both caspase-1 and caspase-3. Conclusions: These data confirm the occurrence of pyroptosis in HCV-infected cells and demonstrate the involvement of the NLRP3 inflammasome, although other inflammasome sensors might be involved. Since the inhibition of one cell death pathway resulted in the increased activation of the other, along with the presence of double-positive cells, there may be cross-talk between apoptotic and pyroptotic pathways; the role of this cross-talk during infection remains to be elucidated.

scholarly journals African Swine Fever Virus IAP Homologue Inhibits Caspase Activation and Promotes Cell Survival in Mammalian Cells

2001 ◽  
Vol 75 (6) ◽  
pp. 2535-2543 ◽  
Author(s):  
Marı́a L. Nogal ◽  
Gonzalo González de Buitrago ◽  
Clara Rodrı́guez ◽  
Beatriz Cubelos ◽  
Angel L. Carrascosa ◽  
...  
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Vero Cells ◽  
Proteolytic Processing ◽  
Fever Virus ◽  
Caspase Activation ◽  
Apoptosis Protein ◽  
Infected Cells

ABSTRACT African swine fever virus (ASFV) A224L is a member of the inhibitor of apoptosis protein (IAP) family. We have investigated the antiapoptotic function of the viral IAP both in stably transfected cells and in ASFV-infected cells. A224L was able to substantially inhibit caspase activity and cell death induced by treatment with tumor necrosis factor alpha and cycloheximide or staurosporine when overexpressed in Vero cells by gene transfection. We have also observed that ASFV infection induces caspase activation and apoptosis in Vero cells. Furthermore, using a deletion mutant of ASFV lacking the A224L gene, we have shown that the viral IAP modulates the proteolytic processing of the effector cell death protease caspase-3 and the apoptosis which are induced in the infected cells. Our findings indicate that A224L interacts with the proteolytic fragment of caspase-3 and inhibits the activity of this protease during ASFV infection. These observations could indicate a conserved mechanism of action for ASFV IAP and other IAP family members to suppress apoptosis.

scholarly journals Mitochondrion-Dependent Cell Death in TDP-43 Proteinopathies

Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 376
Author(s):  
Chantal B. Lucini ◽  
Ralf J. Braun
Keyword(s):  
Cell Death ◽  
Oxygen Species ◽  
In Vivo Models ◽  
Dependent Cell ◽  
Cell Death Mechanisms ◽  
Sting Pathway ◽  
Mitochondrial Membrane Permeabilization

In the last decade, pieces of evidence for TDP-43-mediated mitochondrial dysfunction in neurodegenerative diseases have accumulated. In patient samples, in vitro and in vivo models have shown mitochondrial accumulation of TDP-43, concomitantly with hallmarks of mitochondrial destabilization, such as increased production of reactive oxygen species (ROS), reduced level of oxidative phosphorylation (OXPHOS), and mitochondrial membrane permeabilization. Incidences of TDP-43-dependent cell death, which depends on mitochondrial DNA (mtDNA) content, is increased upon ageing. However, the molecular pathways behind mitochondrion-dependent cell death in TDP-43 proteinopathies remained unclear. In this review, we discuss the role of TDP-43 in mitochondria, as well as in mitochondrion-dependent cell death. This review includes the recent discovery of the TDP-43-dependent activation of the innate immunity cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway. Unravelling cell death mechanisms upon TDP-43 accumulation in mitochondria may open up new opportunities in TDP-43 proteinopathy research.

scholarly journals Ribavirin and Alpha Interferon Enhance Death Receptor-Mediated Apoptosis and Caspase Activation in Human Hepatoma Cells

2003 ◽  
Vol 47 (6) ◽  
pp. 1912-1921 ◽  
Author(s):  
Stephan F. Schlosser ◽  
Markus Schuler ◽  
Christoph P. Berg ◽  
Kirsten Lauber ◽  
Klaus Schulze-Osthoff ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Death Receptor ◽  
Alpha Interferon ◽  
Target Cells ◽  
Clinical Effects ◽  
Caspase Activation ◽  
Human Hepatoma Cells ◽  
Promoting Effect

ABSTRACT The molecular mechanisms underlying the clinical effects of alpha interferon (IFN) and ribavirin are not understood. Elimination of infected cells occurs in part by cytotoxic T lymphocytes (CTLs) expressing CD95 ligand and thereby attacking target cells which are positive for the death receptor CD95. Since many viruses have evolved mechanisms to inhibit apoptosis, the opposite, namely, promotion of apoptosis, could be a strategy to strengthen the host antiviral response. In the present study, we have asked whether the antiviral substances IFN and ribavirin could support CD95-mediated apoptosis by interfering with the activation of caspases, a family of proteases known for their essential role in apoptosis. HepG2 cells, stimulated with the agonistic anti-CD95 antibody, served as a minimal model to mimic the CD95 stimulation ocurring during a CTL attack of target cells in vivo. Apoptosis was quantitated by flow cytometric detection of hypodiploid nuclei. Caspase activity was measured by cytofluorometry, immunocytochemistry, and immunoblot analysis. IFN and ribavirin sensitized HepG2 cells for CD95-mediated apoptosis. This effect was correlated with an increase in CD95-mediated caspase activation and enhanced cleavage of the caspase substrate poly(ADP-ribose) polymerase. Furthermore, the positive effect on CD95-mediated caspase activation by IFN and ribavirin was confirmed by immunocytochemistry for activated caspase-3 and by immunoblot detection of activated caspase-3, caspase-7, and caspase-8. Our data demonstrate that the antiviral substances IFN and ribavirin are able to sensitize for CD95-mediated apoptosis. IFN and ribavirin also enhance CD95-mediated caspase activation, which might in part be responsible for the apoptosis-promoting effect of these antiviral compounds.

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