scholarly journals Salmonella-Induced Caspase-2 Activation in Macrophages

2000 ◽  
Vol 192 (7) ◽  
pp. 1035-1046 ◽  
Author(s):  
Veronika Jesenberger ◽  
Katarzyna J. Procyk ◽  
Junying Yuan ◽  
Siegfried Reipert ◽  
Manuela Baccarini
Keyword(s):  
Type Iii Secretion ◽  
Caspase 3 ◽  
Secretion System ◽  
Caspase 1 ◽  
Chemical Inhibition ◽  
Induction Of Apoptosis ◽  
Caspase 2 ◽  
Induced Apoptosis

The enterobacterial pathogen Salmonella induces phagocyte apoptosis in vitro and in vivo. These bacteria use a specialized type III secretion system to export a virulence factor, SipB, which directly activates the host's apoptotic machinery by targeting caspase-1. Caspase-1 is not involved in most apoptotic processes but plays a major role in cytokine maturation. We show that caspase-1–deficient macrophages undergo apoptosis within 4–6 h of infection with invasive bacteria. This process requires SipB, implying that this protein can initiate the apoptotic machinery by regulating components distinct from caspase-1. Invasive Salmonella typhimurium targets caspase-2 simultaneously with, but independently of, caspase-1. Besides caspase-2, the caspase-1–independent pathway involves the activation of caspase-3, -6, and -8 and the release of cytochrome c from mitochondria, none of which occurs during caspase-1–dependent apoptosis. By using caspase-2 knockout macrophages and chemical inhibition, we establish a role for caspase-2 in both caspase-1–dependent and –independent apoptosis. Particularly, activation of caspase-1 during fast Salmonella-induced apoptosis partially relies on caspase-2. The ability of Salmonella to induce caspase-1–independent macrophage apoptosis may play a role in situations in which activation of this protease is either prevented or uncoupled from the induction of apoptosis.

scholarly journals Pseudomonas aeruginosa Cytotoxin ExoU Is Injected into Phagocytic Cells during Acute Pneumonia

2010 ◽  
Vol 78 (4) ◽  
pp. 1447-1456 ◽  
Author(s):  
Maureen H. Diaz ◽  
Alan R. Hauser
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Immune Cells ◽  
Type Iii Secretion ◽  
Cell Types ◽  
Secretion System ◽  
Host Cells ◽  
Phagocytic Cells ◽  
Acute Pneumonia

ABSTRACT ExoU, a cytotoxin translocated into host cells via the type III secretion system of Pseudomonas aeruginosa, is associated with increased mortality and disease severity. We previously showed that impairment of recruited phagocytic cells allowed survival of ExoU-secreting P. aeruginosa in the lung. Here we analyzed types of cells injected with ExoU in vivo using translational fusions of ExoU with a β-lactamase reporter (ExoU-Bla). Cells injected with ExoU-Bla were detectable in vitro but not in vivo, presumably due to the rapid cytotoxicity induced by the toxin. Therefore, we used a noncytotoxic ExoU variant, designated ExoU(S142A)-Bla, to analyze injection in vivo. We determined that phagocytic cells in the lung were frequently injected with ExoU(S142A). Early during infection, resident macrophages constituted the majority of cells into which ExoU was injected, but neutrophils and monocytes became the predominant types of cells into which ExoU was injected upon recruitment into the lung. We observed a modest preference for injection into neutrophils over injection into other cell types, but in general the repertoire of injected immune cells reflected the relative abundance of these cells in the lung. Our results indicate that phagocytic cells in the lung are injected with ExoU and support the hypothesis that ExoU-mediated impairment of phagocytes has a role in the pathogenesis of pneumonia caused by P. aeruginosa.

scholarly journals A novel anti-proliferative role of HMGA2 in induction of apoptosis through caspase 2 in primary human fibroblast cells

2015 ◽  
Vol 35 (1) ◽  
Author(s):  
Xi Shi ◽  
Baoqing Tian ◽  
Wenlong Ma ◽  
Na Zhang ◽  
Yuehua Qiao ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Flow Cytometric Analysis ◽  
Ectopic Expression ◽  
High Mobility ◽  
Primary Human Fibroblast ◽  
Primary Cell Lines ◽  
Induction Of Apoptosis ◽  
Caspase 2 ◽  
Induced Apoptosis ◽  
High Level

The HMGA2 (high-mobility group AT-hook) protein has previously been shown as an oncoprotein, whereas ectopic expression of HMGA2 is found to induce growth arrest in primary cells. The precise mechanisms underlying this phenomenon remain to be unravelled. In the present study, we determined that HMGA2 was able to induce apoptosis in WI38 primary human cells. We show that WI38 cells expressing high level of HMGA2 were arrested at G2/M phase and exhibited apoptotic nuclear phenotypes. Meanwhile, the cleaved caspase 3 (cysteine aspartic acid-specific protease 3) was detected 8 days after HMGA2 overexpression. Flow cytometric analysis confirmed that the ratio of cells undergoing apoptosis increased dramatically. Concurrently, other major apoptotic markers were also detected, including the up-regulation of p53, Bax and cleaved caspase 9, down-regulation of Bcl-2; as well as release of cytochrome c from the mitochondria. We further demonstrate that the shRNA (small-hairpin RNA)-mediated Apaf1 (apoptotic protease activating factor 1) silencing partially rescued the HMGA2-induced apoptosis, which was accompanied by the decrease of cleaved caspase-3 level and a decline of cell death ratio. Our results also reveal that γH2A was accumulated in nuclei during the HMGA2-induced apoptosis along with the up-regulation of cleaved caspase 2, suggesting that the HMGA2-induced apoptosis was dependent on the pathway of DNA damage. Overall, the present study unravelled a novel function of HMGA2 in induction of apoptosis in human primary cell lines, and provided clues for clarification of the mechanistic action of HMGA2 in addition to its function as an oncoprotein.

scholarly journals Voruciclib, an Oral, Selective CDK9 Inhibitor, Enhances Cell Death Induced By the Bcl-2 Selective Inhibitor Venetoclax in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1361-1361 ◽  
Author(s):  
Daniel A Luedtke ◽  
Yongwei Su ◽  
Holly Edwards ◽  
Lisa Polin ◽  
Juiwanna Kushner ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Caspase 3 ◽  
Combination Index ◽  
Survival Rates ◽  
Annexin V ◽  
Antileukemic Activity ◽  
Induction Of Apoptosis ◽  
Induced Apoptosis

Abstract Introduction: Patients with acute myeloid leukemia (AML) face overall 5-year survival rates of 65% and 27% for children and adults, respectively, leaving significant room for improvement. Relapse remains a major contributor to such low overall survival rates, and leukemic stem cells (LSCs) that survive treatment are believed to be responsible for AML relapse. The anti-apoptotic protein Bcl-2 is overexpressed in bulk AML cells and LSCs and is associated with poor clinical outcomes. Thus, Bcl-2 represents a promising therapeutic target for the treatment of AML. Venetoclax (ABT-199) is a selective Bcl-2 inhibitor that has shown great potential for treating a number of malignancies, including AML. Venetoclax inhibits Bcl-2, preventing it from sequestering pro-apoptotic Bcl-2 family protein Bim, leading to Bim activated Bax/Bak, resulting in apoptosis. However, Mcl-1 can also sequester Bim and prevent apoptosis. We previously showed that directly targeting Mcl-1 can enhance the antileukemic activity of venetoclax (Luedtke DA, et al. Signal Transduct Target Ther. Apr 2017). Alternatively, we proposed that indirect targeting of Mcl-1 may preserve or enhance the antileukemic activity of venetoclax, and prevent resistance resulting from Mcl-1. It has been reported that inhibition of CDK9 can downregulate cell survival genes regulated by superenhancers, including Mcl-1, MYC, and Cyclin D1. One CDK9 inhibitor in clinical development, flavopiridol (alvocidib), has progressed to phase II clinical trials in AML. However, off target effects and dose-limiting toxicities remain a concern. Voruciclib is an oral, selective CDK inhibitor differentiated by its potent inhibition of CDK9 as compared to other CDK inhibitors. This selectivity may potentially circumvent toxicities resulting from inhibition of non-CDK targets like MAK and ICK that are inhibited by flavopiridol. Voruciclib has been shown in vitro to promote apoptosis and decrease Mcl-1 expression levels in chronic lymphocytic leukemia (CLL) cells (Paiva C, et al. PLOS One. Nov 2015) and inhibit tumor growth in mouse xenograft models of diffuse large B-cell lymphoma (DLBCL) in combination with venetoclax (Dey J. et al Scientific Reports. Dec 2017). Based on these data, voruciclib may downregulate Mcl-1 in AML cells and therefore synergistically enhance the antileukemic activity of venetoclax. Methods/Results: Culturing AML cell lines (THP-1, U937, MOLM-13, MV4-11, and OCI-AML3) and primary patient samples with various concentrations of voruciclib resulted in a concentration-dependent increase in Annexin V+ cells (2 μM voruciclib induced 13.8-55.8% Annexin V+ cells) along with increased levels of cleaved caspase 3 and PARP, demonstrating that voruciclib induces apoptosis in AML cells. Next, we tested the combination of voruciclib and venetoclax in AML cell lines and primary AML patient samples at clinically achievable concentrations. Annexin V/PI staining, flow cytometry analysis, and combination index calculation (using CalcuSyn software) revealed synergistic induction of apoptosis by voruciclib and venetoclax combination (combination index values for MV4-11, U937, THP-1, and MOLM-13 cells were <0.73; treatment with 2 µM voruciclib and venetoclax for 24 h resulted in >80% apoptosis). Importantly, synergy was observed in both venetoclax sensitive and resistant cell lines. This was accompanied by increased cleavage of caspase 3 and PARP. Lentiviral shRNA knockdown of Bak and Bax partially rescued AML cells from voruciclib-induced apoptosis, showing that voruciclib induces apoptosis at least partially through the intrinsic apoptosis pathway. However, Bak and Bax knockdown had little to no effect on induction of apoptosis by the combination treatment, indicating that there might be other molecular mechanisms underlying the synergistic interaction between the two agents. Treatment with the pan-caspase inhibitor Z-VAD-FMK partially rescued cells from combination treatment induced-apoptosis. Discussion: Collectively, these results demonstrate that voruciclib and venetoclax synergistically induce apoptosis in AML cells in vitro and reverse venetoclax resistance. Further studies to determine the mechanism of action and in vivo efficacy of this promising combination in AML xenografts and PDX models are underway. Disclosures Ge: MEI Pharma: Research Funding.

Abstract 1950: Role of Superoxide, Nitric Oxide and Peroxynitrite in Doxorubicin-induced Cell Death in vitro and in vivo

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Partha Mukhopadhyay ◽  
Mohanraj Rajesh ◽  
Sandor Bátkai ◽  
György Haskó ◽  
Csaba Szabo ◽  
...  
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Superoxide Generation ◽  
Cytochrome C Release ◽  
Mitochondrial Superoxide ◽  
Induced Apoptosis ◽  
Peroxynitrite Scavengers

Although doxorubicin (DOX) is one of the most potent antitumor agents available, its clinical use is limited because of the risk of severe cardiotoxicity often leading to irreversible congestive heart failure. Apoptotic cell death is a key component in DOX-induced cardiotoxicity, but its trigger(s) and mechanisms are poorly understood. Here, we explore the role of peroxynitrite (a reactive oxidant produced from the diffusion-controlled reaction between nitric oxide and superoxide anion) in DOX-induced cell death. Using a well-established in vivo mouse model of DOX-induced acute heart failure, we demonstrate marked increases in myocardial apoptosis (caspase-3 and 9 gene expression, caspase 3 activity, cytochrome-c release, and TUNEL), iNOS but not eNOS and nNOS expression, 3-nitrotyrosine formation and a decrease in myocardial contractility following DOX treatment. Pre-treatment of mice with peroxynitrite scavengers markedly attenuated DOX-induced myocardial cell death and dysfunction without affecting iNOS expression. DOX induced increased superoxide generation and nitrotyrosine formation in the mitochondria, dissipation of mitochondrial membrane potential, apoptosis (cytochrome-C release, annexin V staining, caspase activation, nuclear fragmentation), and disruption of actin cytoskeleton structure in cardiac-derived H9c2 cells. Selective iNOS inhibitors attenuated DOX-induced apoptosis, without affecting increased mitochondrial superoxide generation, whereas NO donors increased DOX-induced cell death in vitro . The peroxynitrite scavengers FeTMPyP and MnTMPyP markedly reduced both DOX- or peroxynitrite-induced nitrotyrosine formation and cell death in vitro , without affecting DOX-induced increased mitochondrial superoxide formation. Thus, peroxynitrite is a major trigger of DOX-induced apoptosis, and its effective neutralization can be of significant therapeutic benefit.

scholarly journals Identification and analysis of bacterial virulence genes in vivo

2000 ◽  
Vol 355 (1397) ◽  
pp. 613-622 ◽  
Author(s):  
Kate E. Unsworth ◽  
David W. Holden
Keyword(s):  
Type Iii Secretion ◽  
Virulence Genes ◽  
Screening Method ◽  
Secretion System ◽  
Bacterial Virulence ◽  
Microbial Pathogens ◽  
Temperature Sensitive ◽  
Approaches To Study

Signature–tagged mutagenesis is a mutation–based screening method for the identification of virulence genes of microbial pathogens. Genes isolated by this approach fall into three classes: those with known biochemical function, those of suspected function and some whose functions cannot be predicted from database searches. A variety of in vitro and in vivo methods are available to elucidate the function of genes of the second and third classes. We describe the use of some of these approaches to study the function of the Salmonella pathogenicity island 2 type III secretion system of Salmonella typhimurium . This virulence determinant is required for intracellular survival. Secretion by this system is induced by an acidic pH, and its function may be to alter trafficking of the Salmonella –containing vacuole. Use of a temperature–sensitive non–replicating plasmid and competitive index tests with other genes show that in vivo phenotypes do not always correspond to those predicted from in vitro studies.

Indole-3-Carbinol Inhibits Nasopharyngeal Carcinoma

2010 ◽  
Vol 29 (2) ◽  
pp. 185-192 ◽  
Author(s):  
Wei Zhu ◽  
Wenxue Li ◽  
Guangyu Yang ◽  
Quanxin Zhang ◽  
Ming Li ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Nasopharyngeal Carcinoma ◽  
Glutathione Peroxidase ◽  
Tumor Growth ◽  
Caspase 3 ◽  
Caspase 9 ◽  
Human Nasopharyngeal Carcinoma ◽  
Induced Apoptosis

This study explored the effects of indole-3-carbinol on the proliferation of human nasopharyngeal carcinoma, both in vitro and in vivo, and the underlying mechanisms in inducing apoptosis of CNE1 cells. Proliferation, apoptosis, malondialdehyde, superoxide dismutase, glutathione peroxidase, expressions of caspase-9, and caspase-3 in human nasopharyngeal carcinoma cells CNE1 were examined. Indole-3-carbinol suppressed proliferation, induced apoptosis, decreased malondialdehyde level, increased the activity of superoxide dismutase and glutathione peroxidase, and up-regulated the expression of active fragments of caspase-9 and caspase-3 both in vitro and in vivo. It was concluded that indole-3-carbinol could inhibit proliferation and induce apoptosis of CNE1 cells and inhibit tumor growth in mice. Increased activity of superoxide dismutase and glutathione peroxidase and activated expression of caspase-9 and caspase-3 were also observed in indole-3-carbinol–treated tumors or tumor cells, suggesting that stress- and apoptosis-related molecules are involved in the indole-3-carbinol–induced apoptosis and inhibition of tumor growth.

scholarly journals Differential expression of Salmonella type III secretion system factors InvJ, PrgJ, SipC, SipD, SopA and SopB in cultures and in mice

Microbiology ◽  
2010 ◽  
Vol 156 (1) ◽  
pp. 116-127 ◽  
Author(s):  
Hao Gong ◽  
Gia-Phong Vu ◽  
Yong Bai ◽  
Edward Yang ◽  
Fenyong Liu ◽  
...  
Keyword(s):  
Differential Expression ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Systemic Infection ◽  
Secretion System ◽  
Flag Epitope ◽  
Type Iii ◽  
Late Stages

The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI-1) is important for the invasion of epithelial cells during development of Salmonella-associated enterocolitis. It has been suggested that the level and timing of the expression of the SPI-1 T3SS proteins and effectors dictate the consequences of bacterial infection and pathogenesis. However, the expression of these proteins has not been extensively studied in vivo, especially during the later stages of salmonellosis when the infection is established. We have constructed recombinant Salmonella strains that contain a FLAG epitope inserted in-frame to genes invJ, prgJ, sipC, sipD, sopA and sopB, and investigated the expression of the tagged proteins both in vitro and in vivo during murine salmonellosis. Mice were inoculated intraperitoneally or intragastrically with the tagged Salmonella strains. At different time points post-infection, bacteria were recovered from various organs, and the expression of the tagged proteins was determined. Our results provide direct evidence that PrgJ and SipD are expressed in Salmonella colonizing the liver and ileum of infected animals at both the early and late stages of infection. Furthermore, our study has shown that the InvJ protein is expressed preferentially in Salmonella colonizing the ileum but not the liver, while SipC is expressed preferentially in Salmonella colonizing the liver but not the ileum. Thus, Salmonella appears to express different SPI-1 proteins and effectors when colonizing specific tissues. Our results suggest that differential expression of these proteins may be important for tissue-specific aspects of bacterial pathogenesis such as gastroenterititis in the ileum and systemic infection in the liver.

Novel irreversible caspase-1 inhibitor attenuates the maturation of intracellular interleukin-1β

2007 ◽  
Vol 85 (1) ◽  
pp. 56-65 ◽  
Author(s):  
Xue-Qin Ma ◽  
Hua-Jie Zhang ◽  
Ya-Hui Zhang ◽  
Yi-Hua Chen ◽  
Fang Wu ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Proinflammatory Cytokines ◽  
Caspase 3 ◽  
Inflammatory Diseases ◽  
Interleukin 1Β ◽  
Interleukin 18 ◽  
Caspase 1

. Caspase-1, the most efficient enzyme in processing the proinflammatory cytokines interleukin 1β and interleukin 18 in humans, is associated with inflammatory diseases such as rheumatoid arthritis, osteoarthritis, and some neuronal diseases. We previously reported that isoquinoline-1,3,4-trione and its derivatives are novel caspase-3 inhibitors that could attenuate apoptosis in vitro and in vivo. Here we report a novel derivative of isoquinoline-1,3,4-trione that is highly potent in inhibiting caspase-1 activity in an irreversible and slow-binding manner, thus inhibiting cellular caspase-1 activity and the maturation of interleukin 1β in U-937 cells.

scholarly journals Hexarelin Modulation of MAPK and PI3K/Akt Pathways in Neuro-2A Cells Inhibits Hydrogen Peroxide—Induced Apoptotic Toxicity

2021 ◽  
Vol 14 (5) ◽  
pp. 444
Author(s):  
Ramona Meanti ◽  
Laura Rizzi ◽  
Elena Bresciani ◽  
Laura Molteni ◽  
Vittorio Locatelli ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Cell Viability ◽  
Caspase 3 ◽  
Mrna Levels ◽  
Protective Effects ◽  
Neuroprotective Effects ◽  
Caspase 7 ◽  
Induced Apoptosis

Hexarelin, a synthetic hexapeptide, exerts cyto-protective effects at the mitochondrial level in cardiac and skeletal muscles, both in vitro and in vivo, may also have important neuroprotective bioactivities. This study examined the inhibitory effects of hexarelin on hydrogen peroxide (H2O2)-induced apoptosis in Neuro-2A cells. Neuro-2A cells were treated for 24 h with various concentrations of H2O2 or with the combination of H2O2 and hexarelin following which cell viability and nitrite (NO2−) release were measured. Cell morphology was also documented throughout and changes arising were quantified using Image J skeleton and fractal analysis procedures. Apoptotic responses were evaluated by Real-Time PCR (caspase-3, caspase-7, Bax, and Bcl-2 mRNA levels) and Western Blot (cleaved caspase-3, cleaved caspase-7, MAPK, and Akt). Our results indicate that hexarelin effectively antagonized H2O2-induced damage to Neuro-2A cells thereby (i) improving cell viability, (ii) reducing NO2− release and (iii) restoring normal morphologies. Hexarelin treatment also reduced mRNA levels of caspase-3 and its activation, and modulated mRNA levels of the BCL-2 family. Moreover, hexarelin inhibited MAPKs phosphorylation and increased p-Akt protein expression. In conclusion, our results demonstrate neuroprotective and anti-apoptotic effects of hexarelin, suggesting that new analogues could be developed for their neuroprotective effects.

scholarly journals 12-Epi-Napelline Inhibits Leukemia Cell Proliferation via the PI3K/AKT Signaling Pathway In Vitro and In Vivo

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Jia Han ◽  
Wei Hou ◽  
Bi-qing Cai ◽  
Fan Zhang ◽  
Jian-cai Tang
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Caspase 3 ◽  
Animal Experiments ◽  
Leukemia Cell ◽  
Tumor Model ◽  
Inhibitory Effect ◽  
Induced Apoptosis

This study aimed to investigate the inhibitory effect of 12-epi-napelline on leukemia cells and its possible mechanisms. The inhibitory effects of 12-epi-napelline on K-562 and HL-60 cells were evaluated using the CCK-8 assay, cell cycle arrest and apoptosis were detected by flow cytometry, and the expression of related proteins was measured by western blot. A K-562 tumor model was established to evaluate the antitumor effect of 12-epi-napelline in vivo. A reduction in leukemia cell viability was observed after treatment with 12-epi-napelline. It was determined that the cell cycle was arrested in the G0/G1 phase, and the cell apoptosis rate was increased. Moreover, caspase-3 and Bcl-2 were downregulated, whereas cleaved caspase-3 and caspase-9 were upregulated. Further study revealed that 12-epi-napelline could suppress the expression of PI3K, AKT, p-AKT, and mTOR. Insulin-like growth factor 1 (IGF-1) attenuated 12-epi-napelline-induced apoptosis and ameliorated the repression of PI3K, AKT, p-AKT, and mTOR by 12-epi-napelline. Animal experiments clearly showed that 12-epi-napelline inhibited tumor growth. In conclusion, 12-epi-napelline restrained leukemia cell proliferation by suppressing the PI3K/AKT/mTOR pathway in vitro and in vivo.

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