scholarly journals Viral Nonstructural Protein 1 Induces Mitochondrion-Mediated Apoptosis in Mink Enteritis Virus Infection

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Peng Lin ◽  
Yuening Cheng ◽  
Shanshan Song ◽  
Jianming Qiu ◽  
Li Yi ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Nonstructural Protein ◽  
Death Receptor ◽  
Ns1 Protein ◽  
Apoptotic Pathway ◽  
Mink Enteritis Virus ◽  
Hemorrhagic Enteritis ◽  
Enteritis Virus ◽  
Induced Apoptosis

ABSTRACT Mink enteritis virus (MEV), an autonomous parvovirus, causes acute hemorrhagic enteritis in minks. The molecular pathogenesis of MEV infection has not been fully understood. In this study, we observed significantly increased apoptosis in the esophagus, small intestine, mesenteric lymph nodes, and kidney in minks experimentally infected with strain MEVB. In vitro infection of feline F81 cells with MEVB decreased cell viability and induced cell cycle arrest at G1 phase and apoptosis. By screening MEV nonstructural proteins (NS1 and NS2) and structural proteins (VP1 and VP2), we demonstrated that the MEV NS1 induced apoptosis in both F81 and human embryonic kidney 293T (HEK293T) cells, similar to that induced during MEV infection in minks. We found that the NS1 protein-induced apoptosis in HEK293T cells was mediated not by the death receptor but by the mitochondrial pathway, as demonstrated by mitochondrial depolarization, opening of mitochondrial transition pore, release of cytochrome c, and activation of caspase-9 and -3. Moreover, in NS1-transfected cells, we observed an increase of Bax expression and its translocation to the mitochondria, as well as an increased ratio of the Bax/Bcl-2, reactive oxygen species (ROS) production, and activated p38 mitogen-activated protein kinase (MAPK) and p53. Taken together, our results demonstrated that MEV induces apoptosis through activation of p38 MAPK and the p53-mediated mitochondrial apoptotic pathway induced by NS1 protein, which sheds light on the molecular pathogenesis of MEV infection. IMPORTANCE MEV causes fatal hemorrhagic enteritis in minks. Apoptosis is a cellular mechanism that effectively sacrifices virus-infected cells to maintain homeostasis between the virus and host. In this study, we demonstrated that MEV induces apoptosis both in vivo and in vitro. Mechanistically, the viral large nonstructural protein NS1 activates p38 MAPK, which leads p53 phosphorylation to mediate the mitochondrial apoptotic pathway but not the death receptor-mediated apoptotic pathway. This is the first report to uncover the mechanism underlying MEV-induced apoptosis.

scholarly journals Apigenin Suppresses Angiogenesis by Inhibiting Tube Formation and Inducing Apoptosis

2016 ◽  
Vol 11 (10) ◽  
pp. 1934578X1601101
Author(s):  
Hyun Ju Kim ◽  
Mok-Ryeon Ahn
Keyword(s):  
Umbilical Vein ◽  
Chorioallantoic Membrane ◽  
Tube Formation ◽  
Anticancer Activities ◽  
Apoptotic Pathway ◽  
Inhibition Of Angiogenesis ◽  
Induced Apoptosis ◽  
Umbilical Vein Endothelial Cells

Apigenin has been reported to exert angiogenic and anticancer activities in vitro. The mechanism of inhibition of angiogenesis by apigenin, however, has not been well-established. In this study, we investigated whether apigenin not only inhibited tube formation but also induced apoptosis in human umbilical vein endothelial cells (HUVECs). Furthermore, strong antiangiogenic activity of apigenin was observed in the in vivo assay using chick embryo chorioallantoic membrane (CAM). We also analyzed changes in survival signals and the apoptotic pathway through Western blotting. The results indicate that apigenin exerts its antiangiogenic effects through induction of endothelial apoptosis.

scholarly journals Mutations in the NS1 Protein of Swine Influenza Virus Impair Anti-Interferon Activity and Confer Attenuation in Pigs

2005 ◽  
Vol 79 (12) ◽  
pp. 7535-7543 ◽  
Author(s):  
Alicia Solórzano ◽  
Richard J. Webby ◽  
Kelly M. Lager ◽  
Bruce H. Janke ◽  
Adolfo García-Sastre ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Nonstructural Protein ◽  
Swine Influenza Virus ◽  
Swine Influenza ◽  
Influenza Viruses ◽  
Model Systems ◽  
Ns1 Protein ◽  
Wild Type ◽  
Virulence Attenuation

ABSTRACT It has been shown previously that the nonstructural protein NS1 of influenza virus is an alpha/beta interferon (IFN-α/β) antagonist, both in vitro and in experimental animal model systems. However, evidence of this function in a natural host has not yet been obtained. Here we investigated the role of the NS1 protein in the virulence of a swine influenza virus (SIV) isolate in pigs by using reverse genetics. The virulent wild-type A/Swine/Texas/4199-2/98 (TX/98) virus and various mutants encoding carboxy-truncated NS1 proteins were rescued. Growth properties of TX/98 viruses with mutated NS1, induction of IFN in tissue culture, and virulence-attenuation in pigs were analyzed and compared to those of the recombinant wild-type TX/98 virus. Our results indicate that deletions in the NS1 protein decrease the ability of the TX/98 virus to prevent IFN-α/β synthesis in pig cells. Moreover, all NS1 mutant viruses were attenuated in pigs, and this correlated with the amount of IFN-α/β induced in vitro. These data suggest that the NS1 protein of SIV is a virulence factor. Due to their attenuation, NS1-mutated swine influenza viruses might have a great potential as live attenuated vaccine candidates against SIV infections of pigs.

scholarly journals A Novel High-Throughput Technique for Identifying Monoclonal Antibodies capable of Death Receptor Induced Apoptosis

2009 ◽  
Vol 2 ◽  
pp. JCD.S3660
Author(s):  
Hang Fai Kwok ◽  
Julie A. Gormley ◽  
Christopher J. Scott ◽  
James A. Johnston ◽  
Shane A. Olwill
Keyword(s):  
Cell Death ◽  
High Throughput ◽  
High Throughput Screening ◽  
False Negative ◽  
Death Receptor ◽  
Annexin V ◽  
Fas Receptor ◽  
Induced Apoptosis

The study of death receptor family induced apoptosis has gained momentum in recent years with the knowledge that therapeutic antibodies targeting DR4 and DR5 (death receptor's 4 and 5) have proved efficacious in multiple clinical trials. The therapeutic rationale is based on targeting and amplifying a tumour tissues normal cell death programme (apoptosis). While advances in the targeting of DR4 and DR5 have been successful the search for an agonistic antibody to another family member, the Fas receptor, has proven more elusive. This is partly due to the differing in vitro and in vivo characteristics of individual antibodies. In order to induce Fas targeted cell death an antibody must be capable of binding to and trimerising the receptor. It has been shown that antibodies capable of performing this function in vivo, with the assistance of tumour associated cells, do not always induce apoptosis in vitro. As a result the use of current methodologies to detect functional antibodies in vitro may have dismissed potential therapeutic candidates ('false negative'). Here we report a novel high throughput screening technique which artificially cross-links antibodies bound to the Fas receptor. By combining this process with Annexin-V and Prodidium Iodide (PI) staining we can select for antibodies which have the potential to induce apoptosis in vivo.

Azacytidine Suppressors Autocrine IL-6 Secretion and Demonstrates In Vitro and In Vivo Activity Against Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1566-1566
Author(s):  
Tiffany Khong ◽  
Janelle Sharkey ◽  
Andrew Spencer
Keyword(s):  
Multiple Myeloma ◽  
Murine Model ◽  
Dna Methyltransferase ◽  
Myeloma Cell ◽  
Apoptotic Pathway ◽  
P16 Gene ◽  
Induced Apoptosis ◽  
The Impact

Abstract Azacytidine (AZA), a DNA methyltransferase inhibitor, has been shown to inhibit cell growth and induce apoptosis in some cancer cells. We determined the impact of AZA on a panel of human myeloma cell lines (HMCL); KMS 12PE, KMS 18, LP-1, NCI-H929, OPM-2, RPMI-8226 and U266 and in an in vivo murine model of multiple myeloma (5T33 model). Dose responsiveness to AZA was determined via MTS assays with a range of AZA doses (1–10mM) for 72 hours. FACS and cell cycle analysis were used to evaluate the profile of the cells after exposure to AZA for 72 hours. MTS assays demonstrated a dose and time dependent AZA-induced inhibition of HMCL viability with effective concentrations of AZA ranging from 1–10 mM. This was associated with accumulation of cells in the Go/G1 phase with decreasing number of cells in the S and G2/M phases. Western Blot analysis using antibodies against caspases 3,8,10, PARP, phospho-ERK, ERK, Stat3 and phospho -Stat3 were performed to help characterize the mechanism(s) of cell killing. Cleavage of caspases 3,8,10 and PARP within 24 hours of AZA treatment confirmed early AZA-induced HMCL apoptosis. phospho-ERK which was absent in untreated U266 appeared after 48 hours exposure to 5mM AZA. Similarly inhibitors of caspases 3,8 and 9 were used to determine which apoptotic pathway was being preferentially activated by AZA. Inhibitors of both caspase 3 and 9 effectively abrogated AZA-induced apoptosis in U266 and NCI-H929. In contrast caspase 8 inhibitor was less effective which is consistent with AZA acting via the mitochondrial apoptotic pathway. Reactivation of p16 gene by AZA-induced hypomethylation was assessed with methylation specific PCR. MSP-PCR of the p16 gene indicated a loss of methylation and up-regulated transcription after 48 hours treatment with 5 mM AZA. The level of IL-6 in conditioned media from U266 cells treated with AZA was determined by ELISA assay and demonstrated a rapid fall in autocrine IL-6 production. RT-PCR demonstrated rapid AZA-induced cessation of IL-6 transcription temporarily associated with the disappearance of upstream phospho -Stat3. Addition of exogenous IL-6 did not rescue U266 from AZA-induced apoptosis. AZA was also administered to a 5T33 murine model of multiple myeloma at increasing concentrations (1, 3, 10 mg/kg). At 10 mg/kg the median survival of vehicle versus AZA treated mice was 28 days versus 30+ days (p=0.003). These findings justify further evaluation of AZA as a potential therapeutic agent for multiple myeloma.

Acquired Resistance to Tumor Necrosis Factor-Related Apoptosis- Inducing Ligand (TRAIL): Histone Deacetylase Inhibitors Resensitize TRAIL-Resistant Jurkat Acute Lymphocytic Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5032-5032
Author(s):  
Pavel Klener ◽  
Jan Molinsky ◽  
Tereza Simonova ◽  
Emanuel Necas ◽  
Ladislav Andera ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Fas Ligand ◽  
Death Receptor ◽  
Lymphocytic Leukemia ◽  
Jurkat Cells ◽  
Tnf Alpha ◽  
Apoptotic Pathway ◽  
Induced Apoptosis ◽  
Disc Formation

Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a death-ligand from the TNF family. TRAIL induces programmed cell death by the cell-extrinsic p53-independent apoptotic pathway. A potential of TRAIL as cancer-specific therapeutic agent has been proposed and is preclinically and clinically tested. Development of TRAIL-resistant clones in the TRAIL-sensitive tumor cells may be a serious complication of TRAIL based cancer therapy. Jurkat acute lymphocytic leukemia cells are sensitive to TRAIL-induced apoptosis, as well as other apoptosis inducing ligands from TNF family, Fas and TNF-alpha. Jurkat cells express only one of the four receptors for TRAIL, death receptor 5 (DR5). Prolonged exposure of TRAIL-sensitive Jurkat cells to recombinant soluble TRAIL (1000 ng/mL) resulted in the establishment of three TRAIL-resistant (TR) Jurkat cell subclones, Jurkat TR1, TR2, and TR3. The Jurkat TR subclones were also resistant to TNF-alpha and Fas ligand, suggesting disruption of the extrinsic apoptotic pathway. TRAIL-resistant subclone TR1, but not TR2 and TR3, demonstrated decreased susceptibility to undergo apoptosis in response to histone-deacetylase inhibitors, valproic acid (VA), sodium butyrate (SB) and suberoylanilide hydroxamic acid (SAHA) and was resistant to fludarabine. Flow cytometry analysis showed Jurkat TR subclones had unchanged expression of cell surface death receptor DR5, Fas, and receptors for TNF-alpha, TNF-R1 and TNF-R2, compared to TRAIL-sensitive Jurkat cells. Analysis of death-inducing signaling complex (DISC) formation by immunoprecipitation (anti-TRAIL, anti-DR5) and subsequent western blotting (anti-caspase 8, anti-FADD) clearly demonstrated that the DISC formation in response to TRAIL binding to DR5 was significantly decreased in subclones TR2 and TR3, but remained unchanged in subclone TR1 compared to TRAIL-sensitive Jurkat cells. To gain further insight into potential molecular aletarations associated with acquired TRAIL resistance of Jurkat subclones, we measured gene expression of several key apoptotic regulators, including receptors for TRAIL, cFLIP, BCL2 family, IAP family, HSP family members in TRAIL-resistant and TRAIL-sensitive Jurkat cells and did not detect any significant (>2-fold) change. These results suggest acquired TRAIL resistance of Jurkat cells might be mediated by changes on the protein rather than mRNA level. We analyzed whether the TRAIL-resistant Jurkat cells could be resensitized to TRAIL-induced apoptosis by pretreatment with diverse inhibitors of important prosurvival pathways, including inhibitors of proteosynthesis (cycloheximid), inhibitors of transcription (actinomycin D), NFkB inhibitors (bortezomib, SN-50), PI3K-Akt-mTOR inhibitors (rapamycine, LY294002, Hsp90 inhibitor (17-AAG), cyclin-dependent kinase inhibitors (roscovitine), casein kinase II inhibitors (DRB), or histone deacetylase inhibitors (HDACi: SAHA, VA, SB). Pretreatment with HDAC inhibitors for 12 hour was able to resensitize all three TRAIL-resistant Jurkat subclones to TRAIL-induced apoptosis. The percentage of apoptotic cells of HDACi-pretreated subclones was 70–95% 24 h after the exposure to TRAIL compared to 5–15% apoptosis for HDACi-untreated TRAIL-exposed controls, and to 10–15% apoptosis for HDACi-treated TRAIL unexposed controls. We established TRAIL-resistant subclones from the original TRAIL-sensitive Jurkat cells. Acquired resistance to TRAIL was not mediated by downregulation of TRAIL death receptor DR5 and was associated with (cross)resistance to TNFa and Fas ligand, suggesting disruption of cell-extrinsic apoptotic pathway. We assume diverse molecular mechanisms were involved in the development of TRAIL-resistant subclones upon exposure to TRAIL, as exemplified by disrupted formation of DISC in case of subclones TR2 and TR3 and normal DISC formation and fludarabine resistance in subclone TR1, suggesting deregulated apoptotic pathway downstream of DISC. Finally, we observed that HDACi resensitized the TRAIL-resistant subclones to TRAIL. The results provide substantiation for combinatorial approaches in the potential TRAIL-based therapies of hematological malignancies.

scholarly journals Dexmedetomidine protects against oxygen-glucose deprivation/reoxygenation injury-induced apoptosis via the p38 MAPK/ERK signalling pathway

2017 ◽  
Vol 46 (2) ◽  
pp. 675-686 ◽  
Author(s):  
Ke Wang ◽  
Yuekun Zhu
Keyword(s):  
Cell Survival ◽  
P38 Mapk ◽  
Glucose Deprivation ◽  
Signalling Pathway ◽  
Cell Counting ◽  
Oxygen Glucose Deprivation ◽  
Protective Effects ◽  
N2a Cells ◽  
Induced Apoptosis

Objective To investigate the protective effects of dexmedetomidine (DEX) in oxygen-glucose deprivation/reoxygenation (OGD/R) injury, which is involved in a number of ischaemic diseases. Methods An in vitro OGD/R injury model was generated using mouse Neuro 2A neuroblastoma (N2A) cells. Different concentrations of DEX were administrated to OGD/R cells. CV-65 was used to inhibit p38 microtubule associated protein kinase/extracellular signal-regulated kinases (MAPK/ERK) signalling. Cell proliferation, cell cycle, apoptosis, and the levels of proteins related to p38 MAPK/ERK signalling and apoptosis were evaluated using Cell Counting Kit-8, flow cytometry, TdT-UTP nick end labelling and Western blot analysis, respectively. Results DEX treatment of OGD/R cells promoted cell survival and attenuated OGD/R-induced cell apoptosis. It also activated the p38 MAPK/ERK signalling pathway, increased the levels of Bcl-2, and decreased the levels of Bax and cleaved caspase-3. Treatment with the p38 MAPK/ERK inhibitor CV-65 inhibited the activation of p38 MAPK/ERK and abrogated the DEX-induced effects on cell survival and apoptosis. Conclusions DEX protects N2A cells from OGD/R-induced apoptosis via the activation of the p38 MAPK/ERK signalling pathway. DEX might be an effective agent for the treatment of ischaemic diseases.

scholarly journals Time Dependent Appearance of Selected Apoptotic Markers and Usefulness of Their Detection In vitro

2002 ◽  
Vol 45 (4) ◽  
pp. 135-144 ◽  
Author(s):  
Emil Rudolf ◽  
Miroslav Červinka
Keyword(s):  
Caspase 3 ◽  
Potassium Chromate ◽  
Experimental Setting ◽  
Apoptotic Pathway ◽  
Detection Systems ◽  
Chemically Induced ◽  
Cell Blebbing ◽  
Temporary Decrease ◽  
Induced Apoptosis

Many experiments have demonstrated that some cell lines are resistant to chemically induced apoptosis in vitro, and that apoptosis itself is far from being a homogenous phenomenon. Here we show that 10 μg/ml etoposide elicited only minor changes in Bowes human melanoma cells (temporary decrease in cell viability and proliferation, transient phospatidylserine externalization and caspase-3 activation), which weren’t clearly capable to start apoptotic pathway in the entire treated population. On the other hand, potassium chromate at concentration of 150 μg/ml executed cell death bearing some features of apoptosis (cell blebbing, caspase-3 activation and cytoskeletal changes) but lacking or showing weakly others (DNA fragmentation and phospatidylserine externalization). Our results suggest that in detecting apoptosis several faultproof detection systems are to be used to avoid misleading results and conclusions in each experimental setting.

scholarly journals MicroRNA-182-5p protects human lens epithelial cells against oxidative stress-induced apoptosis by inhibiting NOX4 and p38 MAPK signalling

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Zhao-Na Li ◽  
Ming-Xu Ge ◽  
Zhong-Fang Yuan
Keyword(s):  
Epithelial Cells ◽  
P38 Mapk ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Human Lens ◽  
Lens Epithelial Cells ◽  
Luciferase Reporter Gene ◽  
Age Related ◽  
Induced Apoptosis

Abstract Background MicroRNAs (miRNAs) are abnormally expressed in various ocular diseases, including age-related cataract. However, the role of miR-182-5p in the progression of age-related cataract remains unclear. Methods The expression of miR-182-5p in HLE-B3 cells was detected by qRT-PCR. HLE-B3 cells were transfected with miR-182-5p mimics. CCK-8, EdU, flow cytometry, 2′,7′-dichlorodihydrofluorescein diacetate, JC-1 kit, and western blot were used to assess the cell viability, proliferation, apoptosis, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro. The relationship between miR-182-5p and NOX4 was confirmed using the dual-luciferase reporter gene analysis. Results We found that miR-182-5p expression was significantly decreased by the H2O2 exposure. Overexpression of miR-182-5p promoted cell proliferation and inhibited ROS production and apoptosis in H2O2-induced HLE-B3 cells. Moreover, p-p-38, p-ERK, and p-JNK were up-regulated in H2O2-treated HLE-B3 cells, and overexpression of miR-182-5p reversed the effects of H2O2 on HLE-B3 cells. In addition, dual-luciferase reporter assay substantiated that NOX4 was a direct target and downregulated by miR-182-5p. Conclusions We concluded that miR-182-5p inhibited lens epithelial cells apoptosis through regulating NOX4 and p38 MAPK signaling, providing a novel biomarker for treatment of age-related cataract.

scholarly journals The Chaperone Function of hsp70 Is Required for Protection against Stress-Induced Apoptosis

2000 ◽  
Vol 20 (19) ◽  
pp. 7146-7159 ◽  
Author(s):  
Dick D. Mosser ◽  
Antoine W. Caron ◽  
Lucie Bourget ◽  
Anatoli B. Meriin ◽  
Michael Y. Sherman ◽  
...  
Keyword(s):  
Protective Effects ◽  
Atpase Domain ◽  
Caspase Activation ◽  
Apoptotic Pathway ◽  
Mutant Proteins ◽  
Chaperone Function ◽  
Induced Apoptosis ◽  
Jnk Activation ◽  
In Cells

ABSTRACT Cellular stress can trigger a process of self-destruction known as apoptosis. Cells can also respond to stress by adaptive changes that increase their ability to tolerate normally lethal conditions. Expression of the major heat-inducible protein hsp70 protects cells from heat-induced apoptosis. hsp70 has been reported to act in some situations upstream or downstream of caspase activation, and its protective effects have been said to be either dependent on or independent of its ability to inhibit JNK activation. Purified hsp70 has been shown to block procaspase processing in vitro but is unable to inhibit the activity of active caspase 3. Since some aspects of hsp70 function can occur in the absence of its chaperone activity, we examined whether hsp70 lacking its ATPase domain or the C-terminal EEVD sequence that is essential for peptide binding was required for the prevention of apoptosis. We generated stable cell lines with tetracycline-regulated expression of hsp70, hsc70, and chaperone-defective hsp70 mutants lacking the ATPase domain or the C-terminal EEVD sequence or containing AAAA in place of EEVD. Overexpression of hsp70 or hsc70 protected cells from heat shock-induced cell death by preventing the processing of procaspases 9 and 3. This required the chaperone function of hsp70 since hsp70 mutant proteins did not prevent procaspase processing or provide protection from apoptosis. JNK activation was inhibited by both hsp70 and hsc70 and by each of the hsp70 domain mutant proteins. The chaperoning activity of hsp70 is therefore not required for inhibition of JNK activation, and JNK inhibition was not sufficient for the prevention of apoptosis. Release of cytochrome c from mitochondria was inhibited in cells expressing full-length hsp70 but not in cells expressing the protein with ATPase deleted. Together with the recently identified ability of hsp70 to inhibit cytochromec-mediated procaspase 9 processing in vitro, these data demonstrate that hsp70 can affect the apoptotic pathway at the levels of both cytochrome c release and initiator caspase activation and that the chaperone function of hsp70 is required for these effects.

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