Analyses of cell death mechanisms related to amino acid substitution at position 95 in the rabies virus matrix protein

We previously reported that the avirulent fixed rabies virus strain Ni-CE induces a clear cytopathic effect in mouse neuroblastoma cells, whereas its virulent progenitor, the Nishigahara strain, does not. Infection with Nishigahara and Ni-CE mutants containing a single amino acid substitution in the matrix protein (M) demonstrated that the amino acid at position 95 of M (M95) is a cytopathic determinant. The characteristics of cell death induced by Ni-CE infection resemble those of apoptosis (rounded and shrunken cells, DNA fragmentation), but the intracellular signalling pathway for this process has not been fully investigated. In this study, we aimed to elucidate the mechanism by which M95 affects cell death induced by human neuroblastoma cell infection with the Nishigahara, Ni-CE and M95-mutated strains. We demonstrated that the Ni-CE strain induced DNA fragmentation, cell membrane disruption, exposure of phosphatidylserine (PS), activation of caspase-3/7 and anti-poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, an early apoptosis indicator, whereas the Nishigahara strain did not induce DNA fragmentation, caspase-3/7 activation, cell membrane disruption, or PARP-1 cleavage, but did induce PS exposure. We also demonstrated that these characteristics were associated with M95 using M95-mutated strains. However, we found that Ni-CE induced cell death despite the presence of a caspase inhibitor, Z-VAD-FMK. In conclusion, our data suggest that M95 mutation-related cell death is caused by both the caspase-dependent and -independent pathways.

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Hypersensitivity of mtDNA-depleted cells to staurosporine-induced apoptosis: roles of Bcl-2 downregulation and cathepsin B

AJP Cell Physiology ◽

10.1152/ajpcell.00037.2010 ◽

2011 ◽

Vol 300 (5) ◽

pp. C1090-C1106 ◽

Cited By ~ 10

Author(s):

Guillaume Rommelaere ◽

Sébastien Michel ◽

Ludovic Mercy ◽

Antoine Fattaccioli ◽

Catherine Demazy ◽

...

Keyword(s):

Cell Death ◽

Cytochrome C ◽

B Cell ◽

Dna Fragmentation ◽

Cathepsin B ◽

Caspase 3 ◽

B Cell Lymphoma ◽

Parental Cell Line ◽

Induced Apoptosis ◽

Parp 1

We show that mitochondrial DNA (mtDNA)-depleted 143B cells are hypersensitive to staurosporine-induced cell death as evidenced by a more pronounced DNA fragmentation, a stronger activation of caspase-3, an enhanced poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, and a more dramatic cytosolic release of cytochrome c. We also show that B-cell CLL/lymphoma-2 (Bcl-2), B-cell lymphoma extra large (Bcl-XL), and myeloid cell leukemia-1 (Mcl-1) are constitutively less abundant in mtDNA-depleted cells, that the inhibition of Bcl-2 and Bcl-XL can sensitize the parental cell line to staurosporine-induced apoptosis, and that overexpression of Bcl-2 or Bcl-XL can prevent the activation of caspase-3 in ρ0143B cells treated with staurosporine. Moreover, the inactivation of cathepsin B with CA074-Me significantly reduced cytochrome c release, caspase-3 activation, PARP-1 cleavage, and DNA fragmentation in mtDNA-depleted cells, whereas the pan-caspase inhibitor failed to completely prevent PARP-1 cleavage and DNA fragmentation in these cells, suggesting that caspase-independent mechanisms are responsible for cell death even if caspases are activated. Finally, we show that cathepsin B is released in the cytosol of ρ0 cells in response to staurosporine, suggesting that the absence of mitochondrial activity leads to a facilitated permeabilization of lysosomal membranes in response to staurosporine.

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Quercetin in attenuation of ischemic/reperfusion injury: A review

Current Molecular Pharmacology ◽

10.2174/1874467213666201217122544 ◽

2020 ◽

Vol 13 ◽

Author(s):

Milad Ashrafizadeh ◽

Saeed Samarghandian ◽

Kiavash Hushmandi ◽

Amirhossein Zabolian ◽

Md Shahinozzaman ◽

...

Keyword(s):

Cell Death ◽

Cell Membrane ◽

Blood Supply ◽

Apoptotic Cell ◽

Ischemia Reperfusion ◽

Membrane Integrity ◽

Therapeutic Effects ◽

Molecular Pathways ◽

Cell Membrane Integrity ◽

Cell Membrane Disruption

Background: Ischemia/reperfusion (I/R) injury is a serious pathologic event that occurs due to restriction in blood supply to an organ, followed by hypoxia. This condition leads to enhanced levels of pro-inflammatory cytokines such as IL-6 and TNF-, and stimulation of oxidative stress via enhancing reactive oxygen species (ROS) levels. Upon reperfusion, blood supply increases, but it deteriorates condition, and leads to generation of ROS, cell membrane disruption and finally, cell death. Plant derived-natural compounds are well-known due to their excellent antioxidant and anti-inflammatory activities. Quercetin is a flavonoid exclusively found in different vegetables, herbs, and fruits. This naturally occurring compound possesses different pharmacological activities making it appropriate option in disease therapy. Quercetin can also demonstrate therapeutic effects via affecting molecular pathways such as NF-B, PI3K/Akt and so on. Methods: In the present review, we demonstrate that quercetin administration is beneficial in ameliorating I/R injury via reducing ROS levels, inhibition of inflammation, and affecting molecular pathways such as TLR4/NF-B, MAPK and so on. Results and conclusion: Quercetin can improve cell membrane integrity via decreasing lipid peroxidation. Apoptotic cell death is inhibited by quercetin via down-regulation of Bax, and caspases, and upregulation of Bcl-2. Quercetin is able to modulate autophagy (inhibition/induction) in decreasing I/R injury. Nanoparticles have been applied for delivery of quercetin, enhancing its bioavailability and efficacy in alleviation of I/R injury. Noteworthy, clinical trials have also confirmed the capability of quercetin in reducing I/R injury.

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Development of Antimicrobial Stapled Peptides Based on Magainin 2 Sequence

Molecules ◽

10.3390/molecules26020444 ◽

2021 ◽

Vol 26 (2) ◽

pp. 444

Author(s):

Motoharu Hirano ◽

Chihiro Saito ◽

Hidetomo Yokoo ◽

Chihiro Goto ◽

Ryuji Kawano ◽

...

Keyword(s):

Antimicrobial Activity ◽

Amino Acid ◽

Hemolytic Activity ◽

Helical Structure ◽

Antimicrobial Activities ◽

Side Chain ◽

Membrane Disruption ◽

Amino Acid Residues ◽

Electrophysiological Measurements ◽

Cell Membrane Disruption

Magainin 2 (Mag2), which was isolated from the skin of the African clawed frog, is a representative antimicrobial peptide (AMP) that exerts antimicrobial activity via microbial membrane disruption. It has been reported that the helicity and amphipathicity of Mag2 play important roles in its antimicrobial activity. We investigated and recently reported that 17 amino acid residues of Mag2 are required for its antimicrobial activity, and accordingly developed antimicrobial foldamers containing α,α-disubstituted amino acid residues. In this study, we further designed and synthesized a set of Mag2 derivatives bearing the hydrocarbon stapling side chain for helix stabilization. The preferred secondary structures, antimicrobial activities, and cell-membrane disruption activities of the synthesized peptides were evaluated. Our analyses revealed that hydrocarbon stapling strongly stabilized the helical structure of the peptides and enhanced their antimicrobial activity. Moreover, peptide 2 stapling between the first and fifth position from the N-terminus showed higher antimicrobial activity than that of Mag2 against both gram-positive and gram-negative bacteria without exerting significant hemolytic activity. To investigate the modes of action of tested peptides 2 and 8 in antimicrobial and hemolytic activity, electrophysiological measurements were performed.

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Two Overlapping Domains of a Lyssavirus Matrix Protein That Acts on Different Cell Death Pathways

Journal of Virology ◽

10.1128/jvi.00761-10 ◽

2010 ◽

Vol 84 (19) ◽

pp. 9897-9906 ◽

Cited By ~ 19

Author(s):

Florence Larrous ◽

Alireza Gholami ◽

Shahul Mouhamad ◽

Jérôme Estaquier ◽

Hervé Bourhy

Keyword(s):

Cell Death ◽

Amino Acid ◽

Matrix Protein ◽

Full Length ◽

M Protein ◽

Genotype 3 ◽

Cell Death Pathways ◽

Acid Fragment ◽

M Proteins ◽

Cellular Apoptosis

ABSTRACT The lyssavirus matrix (M) protein induces apoptosis. The regions of the M protein that are essential for triggering cell death pathways are not yet clearly defined. We therefore compared the M proteins from two viruses that have contrasting characteristics in terms of cellular apoptosis: a genotype 3 lyssavirus, Mokola virus (MOK), and a genotype 1 rabies virus isolated from a dog from Thailand (THA). We identified a 20-amino-acid fragment (corresponding to positions 67 to 86) that retained the cell death activities of the full-length M protein from MOK via both the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and inhibition of cytochrome c oxidase (CcO) activity. We found that the amino acids at positions 77 and 81 have an essential role in triggering these two cell death pathways. Directed mutagenesis demonstrated that the amino acid at position 77 affects CcO activity, whereas the amino acid at position 81 affects TRAIL-dependent apoptosis. Mutations in the full-length M protein that compromised induction of either of these two pathways resulted in delayed apoptosis compared with the time to apoptosis for the nonmutated control.

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Amino Acid Substitution at Position 95 in Rabies Virus Matrix Protein Affects Viral Pathogenicity

Journal of Veterinary Medical Science ◽

10.1292/jvms.11-0151 ◽

2011 ◽

Vol 73 (10) ◽

pp. 1363-1366 ◽

Cited By ~ 7

Author(s):

Naoto ITO ◽

Tetsuo MITA ◽

Kenta SHIMIZU ◽

Yuki ITO ◽

Tatsunori MASATANI ◽

...

Keyword(s):

Amino Acid ◽

Rabies Virus ◽

Amino Acid Substitution ◽

Matrix Protein

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Anticancer effects of mifepristone on human uveal melanoma cells

Cancer Cell International ◽

10.1186/s12935-021-02306-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Prisca Bustamante Alvarez ◽

Alexander Laskaris ◽

Alicia A. Goyeneche ◽

Yunxi Chen ◽

Carlos M. Telleria ◽

...

Keyword(s):

Cell Death ◽

Cell Growth ◽

Progesterone Receptor ◽

Dna Fragmentation ◽

Cell Lines ◽

Caspase 3 ◽

Annexin V ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Free Dna

Abstract Background Uveal melanoma (UM), the most prevalent intraocular tumor in adults, is a highly metastatic and drug resistant lesion. Recent studies have demonstrated cytotoxic and anti-metastatic effects of the antiprogestin and antiglucocorticoid mifepristone (MF) in vitro and in clinical trials involving meningioma, colon, breast, and ovarian cancers. Drug repurposing is a cost-effective approach to bring approved drugs with good safety profiles to the clinic. This current study assessed the cytotoxic effects of MF in human UM cell lines of different genetic backgrounds. Methods The effects of incremental concentrations of MF (0, 5, 10, 20, or 40 μM) on a panel of human UM primary (MEL270, 92.1, MP41, and MP46) and metastatic (OMM2.5) cells were evaluated. Cells were incubated with MF for up to 72 h before subsequent assays were conducted. Cellular functionality and viability were assessed by Cell Counting Kit-8, trypan blue exclusion assay, and quantitative label-free IncuCyte live-cell analysis. Cell death was analyzed by binding of Annexin V-FITC and/or PI, caspase-3/7 activity, and DNA fragmentation. Additionally, the release of cell-free DNA was assessed by droplet digital PCR, while the expression of progesterone and glucocorticoid receptors was determined by quantitative real-time reverse transcriptase PCR. Results MF treatment reduced cellular proliferation and viability of all UM cell lines studied in a concentration-dependent manner. A reduction in cell growth was observed at lower concentrations of MF, with evidence of cell death at higher concentrations. A significant increase in Annexin V-FITC and PI double positive cells, caspase-3/7 activity, DNA fragmentation, and cell-free DNA release suggests potent cytotoxicity of MF. None of the tested human UM cells expressed the classical progesterone receptor in the absence or presence of MF treatment, suggesting a mechanism independent of the modulation of the cognate nuclear progesterone receptor. In turn, all cells expressed non-classical progesterone receptors and the glucocorticoid receptor. Conclusion This study demonstrates that MF impedes the proliferation of UM cells in a concentration-dependent manner. We report that MF treatment at lower concentrations results in cell growth arrest, while increasing the concentration leads to lethality. MF, which has a good safety profile, could be a reliable adjuvant of a repurposing therapy against UM.

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