Activation of plasma membrane reduced glutathione transport in death receptor apoptosis of HepG2 cells

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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Expression cloning of the cDNA for a polypeptide associated with rat hepatic sinusoidal reduced glutathione transport: characteristics and comparison with the canalicular transporter.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.92.5.1495 ◽

1995 ◽

Vol 92 (5) ◽

pp. 1495-1499 ◽

Cited By ~ 19

Author(s):

J. R. Yi ◽

S. Lu ◽

J. Fernandez-Checa ◽

N. Kaplowitz

Keyword(s):

Reduced Glutathione ◽

Expression Cloning ◽

Glutathione Transport

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In Vitro Protective Effect of Paste and Sauce Extract Made with Protaetia brevitarsis Larvae on HepG2 Cells Damaged by Ethanol

Insects ◽

10.3390/insects11080494 ◽

2020 ◽

Vol 11 (8) ◽

pp. 494 ◽

Cited By ~ 1

Author(s):

Dooseon Hwang ◽

Tae-Won Goo ◽

Eun-Young Yun

Keyword(s):

Hepg2 Cells ◽

Tumor Necrosis ◽

Reduced Glutathione ◽

Fermented Food ◽

Inhibition Rate ◽

Tnf Α ◽

Antioxidative Effects ◽

Protein Rich ◽

Necrosis Factor

We made paste and sauce using protein-rich Protaetia brevitarsis larvae (PBL) and evaluated their fermentation levels. After pretreatment with the paste and sauce extracts, HepG2 cells were damaged with ethanol (EtOH), and then the effects of the paste and sauce were evaluated. As a result, we confirmed that the PBL paste and sauce extracts reduced the aspartate aminotransferase (AST) and alanine aminotransaminase (ALT) content in the medium as compared to soybean (Glycine max) sauce and paste extracts. In addition, the PBL paste and sauce extracts significantly lowered the level of tumor necrosis factor (TNF)-α and interleukin (IL)-6, which are biomarkers of inflammation, and significantly increased the inhibition rate of superoxide dismutase (SOD) and reduced glutathione (GSH), which are antioxidative indicators, in proportion to the amount of PBL added to the paste and sauce. These results suggest that an intake of PBL paste and sauce, a novel type of fermented food made from insects, may be effective for liver protection through anti-inflammatory and antioxidative effects against hepatocyte injury caused by EtOH.

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Decrease in Multidrug Resistance-associated Protein 2 Activities by Knockdown of Phosphatidylinositol 4-phosphate 5-kinase in Hepatocytes and Cancer Cells

Journal of Pharmacy & Pharmaceutical Sciences ◽

10.18433/jpps30444 ◽

2019 ◽

Vol 22 ◽

pp. 576-584 ◽

Cited By ~ 1

Author(s):

Atsushi Kawase ◽

Yuta Inoue ◽

Miho Hirosoko ◽

Yuka Sugihara ◽

Hiroaki Shimada ◽

...

Keyword(s):

Plasma Membrane ◽

Multidrug Resistance ◽

Cancer Cells ◽

Hepg2 Cells ◽

Carcinoma Cells ◽

Efflux Transporters ◽

Membrane Localization ◽

Intracellular Accumulation ◽

Primary Hepatocytes ◽

Phosphatidylinositol 4

Purpose: The plasma membrane localization and transport activity of multidrug resistance-associated protein 2 (MRP2/ABCC2) and P-glycoprotein (P-gp/ABCB1) efflux transporters are governed by transporter-associated proteins. Phosphatidylinositol 4,5-bisphosphate (PIP2) formed by phosphatidylinositol 4-phosphate 5-kinase type 1 (PIP5K1) activates the linker function of radixin for efflux transporters. Radixin is involved in the plasma membrane localization of efflux transporters. We examined whether PIP5K1 could be a target for the modulation of transporter activities in hepatocytes and cancer cells. Methods: The effects of PIP5K1 depletion by siRNA in mouse primary hepatocytes, PANC1 human pancreatic carcinoma cells, and HepG2 human hepatocellular carcinoma cells on the intracellular accumulation of MRP2 and P-gp substrates were examined. Results: PIP5K1A depletion resulted in increased intracellular accumulation of carboxydichlorofluorescein, a MRP2 fluorescent substrate, in mouse primary hepatocytes, PANC1 cells, and HepG2 cells. In PANC1 and HepG2 cells, the transport activities of MRP2 were significantly decreased by PIP5K1C depletion. However, the transport activities of P-gp were unchanged by PIP5K1 depletion. PIP2 levels were unchanged between control and PIP5K1A- or PIP5K1C-depleted HepG2 cells. MRP2 mRNA levels showed few changes in HepG2 cells following PIP5K1A or PIP5K1C depletion. The expression of phosphorylated radixin was decreased by PIP5K1A and PIP5K1C depletion, although total radixin levels were unchanged. Conclusions: These data suggest that PIP5K1A and PIP5K1C could be target proteins for modulating MRP2 function, partly because of the resulting changes of the linker function of radixin.

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MRP2-Mediated Reduced Glutathione Transport in Protection Against Oxidant Stress

Comments on Toxicology ◽

10.1080/08865140390427445 ◽

2003 ◽

Vol 9 (3-4) ◽

pp. 215-228 ◽

Cited By ~ 1

Author(s):

Nazzareno Ballatori ◽

Christine L. Hammond ◽

Jennifer B. Cunningham

Keyword(s):

Reduced Glutathione ◽

Oxidant Stress ◽

Glutathione Transport

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Molecular Mode of Action of TRAIL Receptor Agonists—Common Principles and Their Translational Exploitation

Cancers ◽

10.3390/cancers11070954 ◽

2019 ◽

Vol 11 (7) ◽

pp. 954 ◽

Cited By ~ 7

Author(s):

Wajant

Keyword(s):

Cell Death ◽

Plasma Membrane ◽

Clinical Studies ◽

Molecular Mechanisms ◽

Death Receptor ◽

Death Receptors ◽

Maximum Activity ◽

Receptor Agonists ◽

Receptor Antibodies ◽

Trail Death Receptor

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors TRAILR1/death receptor 4 (DR4) and TRAILR2/DR5 trigger cell death in many cancer cells but rarely exert cytotoxic activity on non-transformed cells. Against this background, a variety of recombinant TRAIL variants and anti-TRAIL death receptor antibodies have been developed and tested in preclinical and clinical studies. Despite promising results from mice tumor models, TRAIL death receptor targeting has failed so far in clinical studies to show satisfying anti-tumor efficacy. These disappointing results can largely be explained by two issues: First, tumor cells can acquire TRAIL resistance by several mechanisms defining a need for combination therapies with appropriate sensitizing drugs. Second, there is now growing preclinical evidence that soluble TRAIL variants but also bivalent anti-TRAIL death receptor antibodies typically require oligomerization or plasma membrane anchoring to achieve maximum activity. This review discusses the need for oligomerization and plasma membrane attachment for the activity of TRAIL death receptor agonists in view of what is known about the molecular mechanisms of how TRAIL death receptors trigger intracellular cell death signaling. In particular, it will be highlighted which consequences this has for the development of next generation TRAIL death receptor agonists and their potential clinical application.

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Comparative cytotoxicity and apoptotic pathways induced by nanosilver in human liver HepG2 and L02 cells

Human & Experimental Toxicology ◽

10.1177/0960327118769718 ◽

2018 ◽

Vol 37 (12) ◽

pp. 1293-1309 ◽

Cited By ~ 12

Author(s):

Y Xue ◽

J Wang ◽

Y Huang ◽

X Gao ◽

L Kong ◽

...

Keyword(s):

Cell Line ◽

Hepg2 Cells ◽

Molecular Mechanisms ◽

Caspase 3 ◽

Hepatoma Cell ◽

Death Receptor ◽

Hepatoma Cell Line ◽

Dependent Manner ◽

Death Receptor Pathway ◽

L02 Cells

Silver nanoparticles are used in many commercial products in daily life. Exposure to nanosilver has hepatotoxic effects in animals. This study investigated the cytotoxicity associated with polyvinylpyrrolidone-coated nanosilver (23.44 ± 4.92 nm in diameter) exposure in the human hepatoma cell line (HepG2) and normal hepatic cell line (L02), and the molecular mechanisms induced by nanosilver in HepG2 cells. Nanosilver, in doses of 20–160 μg mL−1 for 24 and 48 h, reduced cell viability in a dose- and time-dependent manner and induced cell membrane leakage and mitochondria injury in both cell lines; these effects were more pronounced in HepG2 cells than in L02 cells. Intracellular oxidative stress was documented by reactive oxygen species (ROS) being generated in HepG2 cells but not in L02 cells, an effect possibly due to differential uptake of nanosilver by cancer cells and normal cells. In HepG2 cells, apoptosis was documented by finding that ROS triggered a decrease in mitochondrial membrane potential, an increase in cytochrome c release, activation of caspase 3 and caspase 9, and a decrease in the ratio of Bcl-2/Bax. Furthermore, nanosilver activated the Fas death receptor pathway by downregulation of nuclear factor-κB and activation of caspase 8 and caspase 3. These results suggest that apoptosis induced by nanosilver in HepG2 cells is mediated via a mitochondria-dependent pathway and the Fas death receptor pathway. These findings provide toxicological and mechanistic information that can help in assessing the effects of nanosilver in biological systems, including the potential for anticancer activities.

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