Baicalein induces cell death in murine T cell lymphoma via inhibition of thioredoxin system.

Science.gov (United States)

Patwardhan, Raghavendra S; Pal, Debojyoti; Checker, Rahul; Sharma, Deepak; Sandur, Santosh K

2017-10-01

We have earlier demonstrated the radioprotective potential of baicalein using murine splenic lymphocytes. Here, we have studied the effect of baicalein on murine T cell lymphoma EL4 cells and investigated the underlying mechanism of action. We observed that baicalein induced a dose dependent cell death in EL4 cells in vitro and significantly reduced the frequency of cancer stem cells. Previously, we have reported that murine and human T cell lymphoma cells have increased oxidative stress tolerance capacity due to active thioredoxin system. Hence, we monitored the effect of baicalein on thioredoxin system in EL4 cells. Docking studies revealed that baicalein could bind to the active site of thioredoxin reductase. Baicalein treatment led to significant reduction in the activity of thioredoxin reductase and nuclear levels of thioredoxin-1 thereby increasing ASK1 levels and caspase-3 activity. Interestingly, CRISPR-Cas9 based knock-out of ASK1 or over-expression of thioredoxin-1 abolished anti-tumor effects of baicalein in EL4 cells. Further, baicalein administration significantly reduced intra-peritoneal tumor burden of EL4 cells in C57BL/6 mice. Thus, our study describes anti-tumor effects of baicalein in EL4 cells via inhibition of thioredoxin system. Copyright © 2017 Elsevier Ltd. All rights reserved.

Resistance of activated human T(h)2 cells to NO-induced apoptosis is mediated by gamma-glutamyltranspeptidase

NARCIS (Netherlands)

Roozendaal, R; Vellenga, E; de Jong, MA; Traanberg, KF; Postma, DS; de Monchy, JGR; Kauffman, HF

Activation-induced death of inflammatory cells (AICD) has an important function in immune maintenance, Type 1 T-h cells are known to be more susceptible to AICD than T(h)2 cells. In the current study we examined whether NO-induced apoptosis also preferentially eliminates T(h)1 cells over Th2 cells.

Group I mGlu receptor stimulation inhibits activation-induced cell death of human T lymphocytes

Science.gov (United States)

Chiocchetti, Annalisa; Miglio, Gianluca; Mesturini, Riccardo; Varsaldi, Federica; Mocellin, Marco; Orilieri, Elisabetta; Dianzani, Chiara; Fantozzi, Roberto; Dianzani, Umberto; Lombardi, Grazia

2006-01-01

The effects of L-glutamate on activation-induced cell death (AICD) of human activated (1 μg ml−1 phytohemagglutinin plus 2 U ml−1 interleukin-2; 8 days) T lymphocytes were studied by measuring anti-CD3 monoclonal antibody (10 μg ml−1; 18 h)-induced cell apoptosis (Annexin V and propidium iodide staining). L-Glutamate (1 × 10−8–1 × 10−4 M) significantly (P⩽0.01) inhibited AICD in a concentration-dependent manner (EC50=6.3 × 10−8 M; maximum inhibition 54.8±6.3% at 1 × 10−6 M). The L-glutamate inhibitory effect was pharmacologically characterized as mediated by group I mGlu receptors, since mGlu receptor agonists reproduced this effect. The EC50 values were: 3.2 × 10−7 M for (1S,3R)-ACPD; 4.5 × 10−8 M for quisqualate; 1.0 × 10−6 M for (S)-3,5-DHPG; 2.0 × 10−5 M for CHPG. Group I mGlu receptor antagonists inhibited the effects of quisqualate 1.0 × 10−6 M. The IC50 values calculated were: 8.7 × 10−5, 4.3 × 10−6 and 6.3 × 10−7 M for AIDA, LY 367385 and MPEP, respectively. L-Glutamate (1 × 10−6 M; 18 h) significantly (P⩽0.05) inhibited FasL expression (40.8±11.3%) (cytofluorimetric analysis), whereas it did not affect Fas signalling. Expression of both mGlu1 and mGlu5 receptor mRNA by T lymphocytes and T-cell lines, as demonstrated by reverse transcriptase–PCR analysis, suggests that L-glutamate-mediated inhibition of AICD was exerted on T cells. These data depict a novel role for L-glutamate in the regulation of the immune response through group I mGlu receptor-mediated mechanisms. PMID:16751798

Human retinal pigment epithelial cell-induced apoptosis in activated T cells

DEFF Research Database (Denmark)

Jørgensen, A; Wiencke, A K; la Cour, M

1998-01-01

human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

Activation of AMP-activated protein kinase by tributyltin induces neuronal cell death

International Nuclear Information System (INIS)

Nakatsu, Yusuke; Kotake, Yaichiro; Hino, Atsuko; Ohta, Shigeru

2008-01-01

AMP-activated protein kinase (AMPK), a member of the metabolite-sensing protein kinase family, is activated by energy deficiency and is abundantly expressed in neurons. The environmental pollutant, tributyltin chloride (TBT), is a neurotoxin, and has been reported to decrease cellular ATP in some types of cells. Therefore, we investigated whether TBT activates AMPK, and whether its activation contributes to neuronal cell death, using primary cultures of cortical neurons. Cellular ATP levels were decreased 0.5 h after exposure to 500 nM TBT, and the reduction was time-dependent. It was confirmed that most neurons in our culture system express AMPK, and that TBT induced phosphorylation of AMPK. Compound C, an AMPK inhibitor, reduced the neurotoxicity of TBT, suggesting that AMPK is involved in TBT-induced cell death. Next, the downstream target of AMPK activation was investigated. Nitric oxide synthase, p38 phosphorylation and Akt dephosphorylation were not downstream of TBT-induced AMPK activation because these factors were not affected by compound C, but glutamate release was suggested to be controlled by AMPK. Our results suggest that activation of AMPK by TBT causes neuronal death through mediating glutamate release

FOXP3 renders activated human regulatory T cells resistant to restimulation-induced cell death by suppressing SAP expression.

Science.gov (United States)

Katz, Gil; Voss, Kelsey; Yan, Toria F; Kim, Yong Chan; Kortum, Robert L; Scott, David W; Snow, Andrew L

2018-05-01

Restimulation-induced cell death (RICD) is an apoptotic program that regulates effector T cell expansion, triggered by repeated stimulation through the T cell receptor (TCR) in the presence of interleukin-2 (IL-2). Although CD4 + regulatory T cells (Tregs) consume IL-2 and experience frequent TCR stimulation, they are highly resistant to RICD. Resistance in Tregs is dependent on the forkhead box P3 (FOXP3) transcription factor, although the mechanism remains unclear. T cells from patients with X-linked lymphoproliferative disease (XLP-1), that lack the adaptor molecule SLAM-associated protein (SAP), are also resistant to RICD. Here we demonstrate that normal Tregs express very low levels of SAP compared to conventional T cells. FOXP3 reduces SAP expression by directly binding to and repressing the SH2D1A (SAP) promoter. Indeed, ectopic SAP expression restores RICD sensitivity in human FOXP3 + Tregs. Our findings illuminate the mechanism behind FOXP3-mediated RICD resistance in Tregs, providing new insight into their long-term persistence. Published by Elsevier Inc.

DNA fragmentation and cell death mediated by T cell antigen receptor/CD3 complex on a leukemia T cell line.

Science.gov (United States)

Takahashi, S; Maecker, H T; Levy, R

1989-10-01

An anti-T cell receptor (TcR) monoclonal antibody (mAb), LC4, directed against a human leukemic T cell line, SUP-T13, caused DNA fragmentation ("apoptosis") and cell death upon binding to this cell line. Cross-linking of receptor molecules was necessary for this effect since F(ab')2, but not Fab', fragments of LC4 could induce cell death. Five anti-CD3 mAb tested also caused apoptosis, but only when they were presented on a solid phase. Interestingly, soluble anti-CD3 mAb induced calcium flux and had an additive effect on the calcium flux and interleukin 2 receptor expression induced by LC4, but these anti-CD3 mAb reversed the growth inhibition and apoptosis caused by LC4. The calcium ionophore A23187, but not the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), also induced apoptosis, suggesting that protein kinase C activation alone does not cause apoptosis, although PMA is growth inhibitory. These results suggest that two distinct biological phenomena can accompany stimulation of the TcR/CD3 complex. In both cases, calcium flux and interleukin 2 receptor expression is induced, but only in one case is apoptosis and cell death seen. The signal initiating apoptosis can be selectively prevented by binding CD3 portion of the receptor in this cell line. This difference in signals mediated by the TcR/CD3 complex may be important in explaining the process of thymic selection, as well as in choosing anti-TcR mAb for therapeutic use.

Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

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Tongfang Tang

Full Text Available BACKGROUND: Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD through alternation of liver innate immune response. AIMS: The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. METHODS: Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. RESULTS: High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4 expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. CONCLUSION: High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

Science.gov (United States)

Tang, Tongfang; Sui, Yongheng; Lian, Min; Li, Zhiping; Hua, Jing

2013-01-01

Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD) through alternation of liver innate immune response. The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4) expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

Elevated p21-Activated Kinase 2 Activity Results in Anchorage-Independent Growth and Resistance to Anticancer Drug–Induced Cell Death

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Jerry W. Marlin

2009-03-01

Full Text Available p21-Activated kinase 2 (PAK-2 seems to be a regulatory switch between cell survival and cell death signaling. We have shown previously that activation of full-length PAK-2 by Rac or Cdc42 stimulates cell survival, whereas caspase activation of PAK-2 to the proapoptotic PAK-2p34 fragment is involved in the cell death response. In this study, we present a role of elevated activity of full-length PAK-2 in anchorage-independent growth and resistance to anticancer drug–induced apoptosis of cancer cells. Hs578T human breast cancer cells that have low levels of PAK-2 activity were more sensitive to anticancer drug–induced apoptosis and showed higher levels of caspase activation of PAK-2 than MDA-MB435 and MCF-7 human breast cancer cells that have high levels of PAK-2 activity. To examine the role of elevated PAK-2 activity in breast cancer, we have introduced a conditionally active PAK-2 into Hs578T human breast cells. Conditional activation of PAK-2 causes loss of contact inhibition and anchorage-independent growth of Hs578T cells. Furthermore, conditional activation of PAK-2 suppresses activation of caspase 3, caspase activation of PAK-2, and apoptosis of Hs578T cells in response to the anticancer drug cisplatin. Our data suggest a novel mechanism by which full-length PAK-2 activity controls the apoptotic response by regulating levels of activated caspase 3 and thereby its own cleavage to the proapoptotic PAK-2p34 fragment. As a result, elevated PAK-2 activity interrupts the apoptotic response and thereby causes anchorage-independent survival and growth and resistance to anticancer drug–induced apoptosis.

Ouabain exacerbates activation-induced cell death in human peripheral blood lymphocytes

OpenAIRE

Esteves Mabel B.; Marques-Santos Luis F.; Affonso-Mitidieri Ottília R.; Rumjanek Vivian M.

2005-01-01

Lymphocytes activated by mitogenic lectins display changes in transmembrane potential, an elevation in the cytoplasmic Ca2+ concentrations, proliferation and/or activation induced cell death. Low concentrations of ouabain (an inhibitor of Na+,K+-ATPase) suppress mitogen-induced proliferation and increases cell death. To understand the mechanisms involved, a number of parameters were analyzed using fluorescent probes and flow cytometry. The addition of 100nM ouabain to cultures of peripheral b...

The cathepsin B inhibitor z-FA-CMK induces cell death in leukemic T cells via oxidative stress.

Science.gov (United States)

Liow, K Y; Chow, Sek C

2018-01-01

The cathepsin B inhibitor benzyloxycarbonyl-phenylalanine-alanine-chloromethyl ketone (z-FA-CMK) was recently found to induce apoptosis at low concentrations in Jurkat T cells, while at higher concentrations, the cells die of necrosis. In the present study, we showed that z-FA-CMK readily depletes intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) generation. The toxicity of z-FA-CMK in Jurkat T cells was completely abrogated by N-acetylcysteine (NAC), suggesting that the toxicity mediated by z-FA-CMK is due to oxidative stress. We found that L-buthionine sulfoximine (BSO) which depletes intracellular GSH through the inhibition of GSH biosynthesis in Jurkat T cells did not promote ROS increase or induce cell death. However, NAC was still able to block z-FA-CMK toxicity in Jurkat T cells in the presence of BSO, indicating that the protective effect of NAC does not involve GSH biosynthesis. This is further corroborated by the protective effect of the non-metabolically active D-cysteine on z-FA-CMK toxicity. Furthermore, in BSO-treated cells, z-FA-CMK-induced ROS increased which remains unchanged, suggesting that the depletion of GSH and increase in ROS generation mediated by z-FA-CMK may be two separate events. Collectively, our results demonstrated that z-FA-CMK toxicity is mediated by oxidative stress through the increase in ROS generation.

High susceptibility of activated lymphocytes to oxidative stress-induced cell death

Directory of Open Access Journals (Sweden)

Giovanna R. Degasperi

2008-03-01

Full Text Available The present study provides evidence that activated spleen lymphocytes from Walker 256 tumor bearing rats are more susceptible than controls to tert-butyl hydroperoxide (t-BOOH-induced necrotic cell death in vitro. The iron chelator and antioxidant deferoxamine, the intracellular Ca2+ chelator BAPTA, the L-type Ca2+ channel antagonist nifedipine or the mitochondrial permeability transition inhibitor cyclosporin A, but not the calcineurin inhibitor FK-506, render control and activated lymphocytes equally resistant to the toxic effects of t-BOOH. Incubation of activated lymphocytes in the presence of t-BOOH resulted in a cyclosporin A-sensitive decrease in mitochondrial membrane potential. These results indicate that the higher cytosolic Ca2+ level in activated lymphocytes increases their susceptibility to oxidative stress-induced cell death in a mechanism involving the participation of mitochondrial permeability transition.O presente estudo demonstra que linfócitos ativados de baço de ratos portadores do tumor de Walker 256 são mais susceptíveis à morte celular necrótica induzida por tert-butil hidroperóxido (t-BOOH in vitro quando comparados aos controles. O quelante de ferro e antioxidante deferoxamina, o quelante intracelular de Ca2+ BAPTA, o antagonista de canal de Ca2+ nifedipina ou o inibidor da transição de permeabilidade mitocondrial ciclosporina-A, mas não o inibidor de calcineurina FK-506, inibiram de maneira similar a morte celular induzida por t-BOOH em linfócitos ativados e controles. Os linfócitos ativados apresentaram redução do potencial de membrana mitocondrial induzida por t-BOOH num mecanismo sensível a ciclosporina-A. Nossos resultados indicam que o aumento da concentração de Ca2+ citosólico em linfócitos ativados aumenta a susceptibilidade dos mesmos à morte celular induzida por estresse oxidativo, num mecanismo envolvendo a participação do poro de transição de permeabilidade mitocondrial.

Tissue Plasminogen Activator (tPA) Mediates Neurotoxin-Induced Cell Death and Microglial Activation

National Research Council Canada - National Science Library

Tsirka, Styliani-Anna

2000-01-01

.... In mice lacking tPA (tPA-/-), neurons are resistant to neurotoxic death. Delivery of tPA into tPA mice restores susceptibility to neuronal death, indicating that tPA is neurotoxic in the context of excitotoxic injury...

Tissue Plasminogen Activator (tPA) Mediates Neurotoxin-Induced Cell Death and Microglial Activation

National Research Council Canada - National Science Library

Tsirka, Styliani-Anna

2001-01-01

.... In mice lacking tPA (tPA-/1), neurons are resistant to neurotoxic death. Delivery of tPA into tpA-/- mice restores susceptibility to neuronal death, indicating that tPA is neurotoxic in the context of excitotoxic injury...

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion.

Science.gov (United States)

Li, Peng; Tian, Mingxing; Bao, Yanqing; Hu, Hai; Liu, Jiameng; Yin, Yi; Ding, Chan; Wang, Shaohui; Yu, Shengqing

2017-01-01

Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant Δ rfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion

Directory of Open Access Journals (Sweden)

Peng Li

2017-09-01

Full Text Available Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant ΔrfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the

The cathepsin B inhibitor, z-FA-CMK is toxic and readily induced cell death in human T lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Liow, K.Y.; Chow, S.C., E-mail: chow.sek.chuen@monash.edu

2013-11-01

The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) was found to be toxic and readily induced cell death in the human T cell line, Jurkat, whereas two other analogs benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were not toxic. The toxicity of z-FA-CMK requires not only the CMK group, but also the presence of alanine in the P1 position and the benzyloxycarbonyl group at the N-terminal. Dose–response studies showed that lower concentrations of z-FA-CMK induced apoptosis in Jurkat T cells whereas higher concentrations induced necrosis. In z-FA-CMK-induced apoptosis, both initiator caspases (-8 and -9) and effector caspases (-3, -6 and -7) were processed to their respective subunits in Jurkat T cells. However, only the pro-form of the initiator caspases were reduced in z-FA-CMK-induced necrosis and no respective subunits were apparent. The caspase inihibitor benzyloxycarbonyl-valine-alanine-aspartic acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) inhibits apoptosis and caspase processing in Jurkat T cells treated with low concentration of z-FA-CMK but has no effect on z-FA-CMK-induced necrosis and the loss of initiator caspases. This suggests that the loss of initiator caspases in Jurkat T cells during z-FA-CMK-induced necrosis is not a caspase-dependent process. Taken together, we have demonstrated that z-FA-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. - Highlights: • z-FA-CMK is toxic and induce cell death in the human T cells. • z-FA-CMK toxicity requires the CMK group, alanine and the benzyloxycarbonyl group. • z-FA-CMK induced apoptosis at low concentration and necrosis at high concentration.

The cathepsin B inhibitor, z-FA-CMK is toxic and readily induced cell death in human T lymphocytes

International Nuclear Information System (INIS)

Liow, K.Y.; Chow, S.C.

2013-01-01

The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) was found to be toxic and readily induced cell death in the human T cell line, Jurkat, whereas two other analogs benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were not toxic. The toxicity of z-FA-CMK requires not only the CMK group, but also the presence of alanine in the P1 position and the benzyloxycarbonyl group at the N-terminal. Dose–response studies showed that lower concentrations of z-FA-CMK induced apoptosis in Jurkat T cells whereas higher concentrations induced necrosis. In z-FA-CMK-induced apoptosis, both initiator caspases (-8 and -9) and effector caspases (-3, -6 and -7) were processed to their respective subunits in Jurkat T cells. However, only the pro-form of the initiator caspases were reduced in z-FA-CMK-induced necrosis and no respective subunits were apparent. The caspase inihibitor benzyloxycarbonyl-valine-alanine-aspartic acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) inhibits apoptosis and caspase processing in Jurkat T cells treated with low concentration of z-FA-CMK but has no effect on z-FA-CMK-induced necrosis and the loss of initiator caspases. This suggests that the loss of initiator caspases in Jurkat T cells during z-FA-CMK-induced necrosis is not a caspase-dependent process. Taken together, we have demonstrated that z-FA-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. - Highlights: • z-FA-CMK is toxic and induce cell death in the human T cells. • z-FA-CMK toxicity requires the CMK group, alanine and the benzyloxycarbonyl group. • z-FA-CMK induced apoptosis at low concentration and necrosis at high concentration

Activation-induced cell death of dendritic cells is dependent on sphingosine kinase 1

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Anja eSchwiebs

2016-04-01

Full Text Available Sphingosine 1-phosphate (S1P is an immune modulatory lipid mediator and has been implicated in numerous pathophysiological processes. S1P is produced by sphingosine kinase 1 (Sphk1 and Sphk2. Dendritic cells (DCs are central for the direction of immune responses and crucially involved in autoimmunity and cancerogenesis. In this study we examined the function and survival of bone marrow-derived DCs under long-term inflammatory stimulation. We observed that differentiated cells undergo activation-induced cell death upon LPS stimulation with an increased metabolic activity shortly after stimulation, followed by a rapid activation of caspase 3 and subsequent augmented apoptosis. Importantly, we highlight a profound role of Sphk1 in secretion of inflammatory cytokines and survival of dendritic cells that might be mediated by a change in sphingolipid levels as well as by a change in STAT3 expression. Cell growth during differentiation of Sphk1-deficient cells treated with the functional S1P receptor antagonist FTYP was reduced. Importantly, in dendritic cells we did not observe a compensatory regulation of Sphk2 mRNA in Sphk1-deficient cells. Instead, we discovered a massive increase in Sphk1 mRNA concentration upon long-term stimulation with LPS in wild type cells that might function as an attempt to rescue from inflammation-caused cell death. Taken together, in this investigation we describe details of a crucial involvement of sphingolipids and Sphk1 in activation-induced cell death during long-term immunogenic activity of DCs that might play an important role in autoimmunity and might explain the differences in immune response observed in in vivo studies of Sphk1 modulation.

Human retinal pigment epithelial cell-induced apoptosis in activated T cells

DEFF Research Database (Denmark)

Jørgensen, A; Wiencke, A K; la Cour, M

1998-01-01

PURPOSE: The immune privilege of the eye has been thought to be dependent on physical barriers and absence of lymphatic vessels. However, the immune privilege may also involve active immunologic processes, as recent studies have indicated. The purpose of the present study was to investigate whether...... human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...

Networked T cell death following macrophage infection by Mycobacterium tuberculosis.

Directory of Open Access Journals (Sweden)

Stephen H-F Macdonald

Full Text Available BACKGROUND: Depletion of T cells following infection by Mycobacterium tuberculosis (Mtb impairs disease resolution, and interferes with clinical test performance that relies on cell-mediated immunity. A number of mechanisms contribute to this T cell suppression, such as activation-induced death and trafficking of T cells out of the peripheral circulation and into the diseased lungs. The extent to which Mtb infection of human macrophages affects T cell viability however, is not well characterised. METHODOLOGY/PRINCIPAL FINDINGS: We found that lymphopenia (<1.5 × 10(9 cells/l was prevalent among culture-positive tuberculosis patients, and lymphocyte counts significantly improved post-therapy. We previously reported that Mtb-infected human macrophages resulted in death of infected and uninfected bystander macrophages. In the current study, we sought to examine the influence of infected human alveolar macrophages on T cells. We infected primary human alveolar macrophages (the primary host cell for Mtb or PMA-differentiated THP-1 cells with Mtb H37Ra, then prepared cell-free supernatants. The supernatants of Mtb-infected macrophages caused dose-dependent, caspase-dependent, T cell apoptosis. This toxic effect of infected macrophage secreted factors did not require TNF-α or Fas. The supernatant cytotoxic signal(s were heat-labile and greater than 50 kDa in molecular size. Although ESAT-6 was toxic to T cells, other Mtb-secreted factors tested did not influence T cell viability; nor did macrophage-free Mtb bacilli or broth from Mtb cultures. Furthermore, supernatants from Mycobacterium bovis Bacille de Calmette et Guerin (BCG- infected macrophages also elicited T cell death suggesting that ESAT-6 itself, although cytotoxic, was not the principal mediator of T cell death in our system. CONCLUSIONS: Mtb-Infected macrophages secrete heat-labile factors that are toxic to T cells, and may contribute to the immunosuppression seen in tuberculosis as well as

Phosphoinositide 3–kinase γ participates in T cell receptor–induced T cell activation

Science.gov (United States)

Alcázar, Isabela; Marqués, Miriam; Kumar, Amit; Hirsch, Emilio; Wymann, Matthias; Carrera, Ana C.; Barber, Domingo F.

2007-01-01

Class I phosphoinositide 3–kinases (PI3Ks) constitute a family of enzymes that generates 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (Tyr) kinase–associated receptors or G protein–coupled receptors (GPCRs). The class I PI3Ks are divided into two types: class IA p85/p110 heterodimers, which are activated by Tyr kinases, and the class IB p110γ isoform, which is activated by GPCR. Although the T cell receptor (TCR) is a protein Tyr kinase–associated receptor, p110γ deletion affects TCR-induced T cell stimulation. We examined whether the TCR activates p110γ, as well as the consequences of interfering with p110γ expression or function for T cell activation. We found that after TCR ligation, p110γ interacts with Gαq/11, lymphocyte-specific Tyr kinase, and ζ-associated protein. TCR stimulation activates p110γ, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. We show that TCR-stimulated p110γ controls RAS-related C3 botulinum substrate 1 activity, F-actin polarization, and the interaction between T cells and antigen-presenting cells, illustrating a crucial role for p110γ in TCR-induced T cell activation. PMID:17998387

T315 Decreases Acute Myeloid Leukemia Cell Viability through a Combination of Apoptosis Induction and Autophagic Cell Death

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Chang-Fang Chiu

2016-08-01

Full Text Available T315, an integrin-linked kinase (ILK inhibitor, has been shown to suppress the proliferation of breast cancer, stomach cancer and chronic lymphocytic leukemia cells. Here we demonstrate that T315 decreases cell viability of acute myeloid leukemia (AML cell lines (HL-60 and THP-1 and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells are less sensitive than leukemia cells to T315. T315 down regulates protein kinase B (Akt and p-Akt and induces caspase activation, poly-ADP-ribose polymerase (PARP cleavage, apoptosis and autophagy through an ILK-independent manner. Interestingly, pretreatment with autophagy inhibitors rescues cells from apoptosis and concomitant PARP cleavage, which implicates a key role of autophagic cell death in T315-mediated cytotoxicity. T315 also demonstrates efficacy in vivo, suppressing the growth of THP-1 xenograft tumors in athymic nude mice when administered intraperitoneally. This study shows that autophagic cell death and apoptosis cooperatively contribute to the anticancer activity of T315 in AML cells. In conclusion, the complementary roles of apoptotic and autophagic cell death should be considered in the future assessment of the translational value of T315 in AML therapy.

TRPV2 activation induces apoptotic cell death in human T24 bladder cancer cells: a potential therapeutic target for bladder cancer.

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Yamada, Takahiro; Ueda, Takashi; Shibata, Yasuhiro; Ikegami, Yosuke; Saito, Masaki; Ishida, Yusuke; Ugawa, Shinya; Kohri, Kenjiro; Shimada, Shoichi

2010-08-01

To investigate the functional expression of the transient receptor potential vanilloid 2 (TRPV2) channel protein in human urothelial carcinoma (UC) cells and to determine whether calcium influx into UC cells through TRPV2 is involved in apoptotic cell death. The expression of TRPV2 mRNA in bladder cancer cell lines (T24, a poorly differentiated UC cell line and RT4, a well-differentiated UC cell line) was analyzed using reverse transcriptase-polymerase chain reaction. The calcium permeability of TRPV2 channels in T24 cells was investigated using a calcium imaging assay that used cannabidiol (CBD), a relatively selective TRPV2 agonist, and ruthenium red (RuR), a nonselective TRPV channel antagonist. The death of T24 or RT4 cells in the presence of CBD was evaluated using a cellular viability assay. Apoptosis of T24 cells caused by CBD was confirmed using an annexin-V assay and small interfering RNA (siRNA) silencing of TRPV2. TRPV2 mRNA was abundantly expressed in T24 cells. The expression level in UC cells was correlated with high-grade disease. The administration of CBD increased intracellular calcium concentrations in T24 cells. In addition, the viability of T24 cells progressively decreased with increasing concentrations of CBD, whereas RT4 cells were mostly unaffected. Cell death occurred via apoptosis caused by continuous influx of calcium through TRPV2. TRPV2 channels in UC cells are calcium-permeable and the regulation of calcium influx through these channels leads directly to the death of UC cells. TRPV2 channels in UC cells may be a potential new therapeutic target, especially in higher-grade UC cells. Copyright 2010 Elsevier Inc. All rights reserved.

Increased anion channel activity is an unavoidable event in ozone-induced programmed cell death.

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Takashi Kadono

Full Text Available BACKGROUND: Ozone is a major secondary air pollutant often reaching high concentrations in urban areas under strong daylight, high temperature and stagnant high-pressure systems. Ozone in the troposphere is a pollutant that is harmful to the plant. PRINCIPAL FINDINGS: By exposing cells to a strong pulse of ozonized air, an acute cell death was observed in suspension cells of Arabidopsis thaliana used as a model. We demonstrated that O(3 treatment induced the activation of a plasma membrane anion channel that is an early prerequisite of O(3-induced cell death in A. thaliana. Our data further suggest interplay of anion channel activation with well known plant responses to O(3, Ca(2+ influx and NADPH-oxidase generated reactive oxygen species (ROS in mediating the oxidative cell death. This interplay might be fuelled by several mechanisms in addition to the direct ROS generation by O(3; namely, H(2O(2 generation by salicylic and abscisic acids. Anion channel activation was also shown to promote the accumulation of transcripts encoding vacuolar processing enzymes, a family of proteases previously reported to contribute to the disruption of vacuole integrity observed during programmed cell death. SIGNIFICANCE: Collectively, our data indicate that anion efflux is an early key component of morphological and biochemical events leading to O(3-induced programmed cell death. Because ion channels and more specifically anion channels assume a crucial position in cells, an understanding about the underlying role(s for ion channels in the signalling pathway leading to programmed cell death is a subject that warrants future investigation.

Triglyceride-induced macrophage cell death is triggered by caspase-1.

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Son, Sin Jee; Rhee, Ki-Jong; Lim, Jaewon; Kim, Tae Ue; Kim, Tack-Joong; Kim, Yoon Suk

2013-01-01

Triglyceride (TG) induces macrophage cell death which contributes to the development of atherosclerosis. We confirmed that exogenous TG accumulates in human THP-1 macrophages and causes cell death. TG treated THP-1 macrophages exhibited no change in tumor necrosis factor (TNF)-α, interleukin (IL)-18, macrophage inflammatory protein (MIP)-1α, and IL-1R1 receptor mRNA expression. However, there was a marked decrease in IL-1β mRNA expression but an increase in IL-1β protein secretion. Decreased expression of IL-1β mRNA and increased secretion of IL-1β protein was not the direct cause of cell death. Until now, TG was assumed to induce necrotic cell death in macrophages. Since caspase-1 is known to be involved in activation and secretion of IL-1β protein and pyroptotic cell death, next we determined whether caspase-1 is associated with TG-induced macrophage cell death. We found an increase in caspase-1 activity in TG-treated THP-1 macrophages and inhibition of caspase-1 activity using a specific inhibitor partially rescued cell death. These results suggest activation of the pyroptotic pathway by TG. This is the first report implicating the activation of caspase-1 and the triggering of the pyroptosis pathway in TG-induced macrophage cell death.

Inhibition of Autophagy Potentiates Atorvastatin-Induced Apoptotic Cell Death in Human Bladder Cancer Cells in Vitro

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Kang, Minyong; Jeong, Chang Wook; Ku, Ja Hyeon; Kwak, Cheol; Kim, Hyeon Hoe

2014-01-01

Statins are cholesterol reduction agents that exhibit anti-cancer activity in several human cancers. Because autophagy is a crucial survival mechanism for cancer cells under stress conditions, cooperative inhibition of autophagy acts synergistically with other anti-cancer drugs. Thus, this study investigates whether combined treatment of atorvastatin and autophagy inhibitors results in enhancing the cytotoxic effects of atorvastatin, upon human bladder cancer cells, T24 and J82, in vitro. To measure cell viability, we performed the EZ-Cytox cell viability assay. We examined apoptosis by flow cytometry using annexin-V/propidium iodide (PI and western blot using procaspase-3 and poly (ADP-ribose) polymerase (PARP) antibodies. To examine autophagy activation, we evaluated the co-localization of LC3 and LysoTracker by immunocytochemistry, as well as the expression of LC3 and p62/sequestosome-1 (SQSTM1) by western blot. In addition, we assessed the survival and proliferation of T24 and J82 cells by a clonogenic assay. We found that atorvastatin reduced the cell viability of T24 and J82 cells via apoptotic cell death and induced autophagy activation, shown by the co-localization of LC3 and LysoTracker. Moreover, pharmacologic inhibition of autophagy significantly enhanced atorvastatin-induced apoptosis in T24 and J82 cells. In sum, inhibition of autophagy potentiates atorvastatin-induced apoptotic cell death in human bladder cancer cells in vitro, providing a potential therapeutic approach to treat bladder cancer. PMID:24815071

Sulforaphane Prevents Angiotensin II-Induced Testicular Cell Death via Activation of NRF2

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Yonggang Wang

2017-01-01

Full Text Available Although angiotensin II (Ang II was reported to facilitate sperm motility and intratesticular sperm transport, recent findings shed light on the efficacy of Ang II in stimulating inflammatory events in testicular peritubular cells, effect of which may play a role in male infertility. It is still unknown whether Ang II can induce testicular apoptotic cell death, which may be a more direct action of Ang II in male infertility. Therefore, the present study aims to determine whether Ang II can induce testicular apoptotic cell death and whether this action can be prevented by sulforaphane (SFN via activating nuclear factor (erythroid-derived 2-like 2 (NRF2, the governor of antioxidant-redox signalling. Eight-week-old male C57BL/6J wild type (WT and Nrf2 gene knockout mice were treated with Ang II, in the presence or absence of SFN. In WT mice, SFN activated testicular NRF2 expression and function, along with a marked attenuation in Ang II-induced testicular oxidative stress, inflammation, endoplasmic reticulum stress, and apoptotic cell death. Deletion of the Nrf2 gene led to a complete abolishment of these efficacies of SFN. The present study indicated that Ang II may result in testicular apoptotic cell death, which can be prevented by SFN via the activation of NRF2.

Selective tumor cell death induced by irradiated riboflavin through recognizing DNA G-T mismatch.

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Yuan, Yi; Zhao, Yongyun; Chen, Lianqi; Wu, Jiasi; Chen, Gangyi; Li, Sheng; Zou, Jiawei; Chen, Rong; Wang, Jian; Jiang, Fan; Tang, Zhuo

2017-09-06

Riboflavin (vitamin B2) has been thought to be a promising antitumoral agent in photodynamic therapy, though the further application of the method was limited by the unclear molecular mechanism. Our work reveals that riboflavin was able to recognize G-T mismatch specifically and induce single-strand breaks in duplex DNA targets efficiently under irradiation. In the presence of riboflavin, the photo-irradiation could induce the death of tumor cells that are defective in mismatch repair system selectively, highlighting the G-T mismatch as potential drug target for tumor cells. Moreover, riboflavin is a promising leading compound for further drug design due to its inherent specific recognition of the G-T mismatch. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Titanium dioxide induces apoptotic cell death through reactive oxygen species-mediated Fas upregulation and Bax activation

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Yoon TH

2012-03-01

Full Text Available Ki-Chun Yoo1, Chang-Hwan Yoon1, Dongwook Kwon2, Kyung-Hwan Hyun1, Soo Jung Woo1, Rae-Kwon Kim1, Eun-Jung Lim1, Yongjoon Suh1, Min-Jung Kim1, Tae Hyun Yoon2, Su-Jae Lee11Laboratory of Molecular Biochemistry, 2Laboratory of Nanoscale Characterization and Environmental Chemistry, Department of Chemistry, Hanyang University, Seoul, Republic of KoreaBackground: Titanium dioxide (TiO2 has been widely used in many areas, including biomedicine, cosmetics, and environmental engineering. Recently, it has become evident that some TiO2 particles have a considerable cytotoxic effect in normal human cells. However, the molecular basis for the cytotoxicity of TiO2 has yet to be defined.Methods and results: In this study, we demonstrated that combined treatment with TiO2 nanoparticles sized less than 100 nm and ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-dependent upregulation of Fas and conformational activation of Bax in normal human cells. Treatment with P25 TiO2 nanoparticles with a hydrodynamic size distribution centered around 70 nm (TiO2P25–70 together with ultraviolet A irradiation-induced caspase-dependent apoptotic cell death, accompanied by transcriptional upregulation of the death receptor, Fas, and conformational activation of Bax. In line with these results, knockdown of either Fas or Bax with specific siRNA significantly inhibited TiO2-induced apoptotic cell death. Moreover, inhibition of reactive oxygen species with an antioxidant, N-acetyl-L-cysteine, clearly suppressed upregulation of Fas, conformational activation of Bax, and subsequent apoptotic cell death in response to combination treatment using TiO2P25–70 and ultraviolet A irradiation.Conclusion: These results indicate that sub-100 nm sized TiO2 treatment under ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-mediated upregulation of the death receptor, Fas, and activation of the preapoptotic protein

The hydroxyflavone, fisetin, suppresses mast cell activation induced by interaction with activated T cell membranes

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Nagai, K; Takahashi, Y; Mikami, I; Fukusima, T; Oike, H; Kobori, M

2009-01-01

Background and purpose: Cell-to-cell interactions between mast cells and activated T cells are increasingly recognized as a possible mechanism in the aetiology of allergic or non-allergic inflammatory disorders. To determine the anti-allergic effect of fisetin, we examined the ability of fisetin to suppress activation of the human mast cell line, HMC-1, induced by activated Jurkat T cell membranes. Experimental approach: HMC-1 cells were incubated with or without fisetin for 15 min and then co-cultured with Jurkat T cell membranes activated by phorbol-12-myristate 13-acetate for 16 h. We determined gene expression in activated HMC-1 cells by DNA microarray and quantitative reverse transcription (RT)-PCR analysis. We also examined activation of the transcription factor NF-κB and MAP kinases (MAPKs) in activated HMC-1 cells. Key results: Fisetin suppresses cell spreading and gene expression in HMC-1 cells stimulated by activated T cell membranes. Additionally, we show that these stimulated HMC-1 cells expressed granzyme B. The stimulatory interaction also induced activation of NF-κB and MAPKs; these activations were suppressed by fisetin. Fisetin also reduced the amount of cell surface antigen CD40 and intercellular adhesion molecule-1 (ICAM-1) on activated HMC-1 cells. Conclusions and implications: Fisetin suppressed activation of HMC-1 cells by activated T cell membranes by interfering with cell-to-cell interaction and inhibiting the activity of NF-κB and MAPKs and thereby suppressing gene expression. Fisetin may protect against the progression of inflammatory diseases by limiting interactions between mast cells and activated T cells. PMID:19702784

Oxidative stress activates the TRPM2-Ca2+-CaMKII-ROS signaling loop to induce cell death in cancer cells.

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Wang, Qian; Huang, Lihong; Yue, Jianbo

2017-06-01

High intracellular levels of reactive oxygen species (ROS) cause oxidative stress that results in numerous pathologies, including cell death. Transient potential receptor melastatin-2 (TRPM2), a Ca 2+ -permeable cation channel, is mainly activated by intracellular adenosine diphosphate ribose (ADPR) in response to oxidative stress. Here we studied the role and mechanisms of TRPM2-mediated Ca 2+ influx on oxidative stress-induced cell death in cancer cells. We found that oxidative stress activated the TRPM2-Ca 2+ -CaMKII cascade to inhibit early autophagy induction, which ultimately led to cell death in TRPM2 expressing cancer cells. On the other hand, TRPM2 knockdown switched cells from cell death to autophagy for survival in response to oxidative stress. Moreover, we found that oxidative stress activated the TRPM2-CaMKII cascade to further induce intracellular ROS production, which led to mitochondria fragmentation and loss of mitochondrial membrane potential. In summary, our data demonstrated that oxidative stress activates the TRPM2-Ca 2+ -CaMKII-ROS signal loop to inhibit autophagy and induce cell death. Copyright © 2016 Elsevier B.V. All rights reserved.

Activation of intracellular angiotensin AT2 receptors induces rapid cell death in human uterine leiomyosarcoma cells

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Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen

2015-01-01

The presence of AT2 receptors in mitochondria and their role in NO generation and cell aging were recently demonstrated in various human and mouse non-tumour cells. We investigated the intracellular distribution of AT2 receptors including their presence in mitochondria and the role in the induction...... agonist, Compound 21 (C21) penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT2 receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped from the cell death, displayed activation of the mitochondrial apoptotic pathway, i...

Caspase-2-dependent dendritic cell death, maturation, and priming of T cells in response to Brucella abortus infection.

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Xinna Li

Full Text Available Smooth virulent Brucella abortus strain 2308 (S2308 causes zoonotic brucellosis in cattle and humans. Rough B. abortus strain RB51, derived from S2308, is a live attenuated cattle vaccine strain licensed in the USA and many other countries. Our previous report indicated that RB51, but not S2308, induces a caspase-2-dependent apoptotic and necrotic macrophage cell death. Dendritic cells (DCs are professional antigen presenting cells critical for bridging innate and adaptive immune responses. In contrast to Brucella-infected macrophages, here we report that S2308 induced higher levels of apoptotic and necrotic cell death in wild type bone marrow-derived DCs (WT BMDCs than RB51. The RB51 and S2308-induced BMDC cell death was regulated by caspase-2, indicated by the minimal cell death in RB51 and S2308-infected BMDCs isolated from caspase-2 knockout mice (Casp2KO BMDCs. More S2308 bacteria were taken up by Casp2KO BMDCs than wild type BMDCs. Higher levels of S2308 and RB51 cells were found in infected Casp2KO BMDCs compared to infected WT BMDCs at different time points. RB51-infected wild type BMDCs were mature and activated as shown by significantly up-regulated expression of CD40, CD80, CD86, MHC-I, and MHC-II. RB51 induced the production of cytokines TNF-α, IL-6, IFN-γ and IL12/IL23p40 in infected BMDCs. RB51-infected WT BMDCs also stimulated the proliferation of CD4(+ and CD8(+ T cells compared to uninfected WT BMDCs. However, the maturation, activation, and cytokine secretion are significantly impaired in Casp2KO BMDCs infected with RB51 or Salmonella (control. S2308-infected WT and Casp2KO BMDCs were not activated and could not induce cytokine production. These results demonstrated that virulent smooth strain S2308 induced more apoptotic and necrotic dendritic cell death than live attenuated rough vaccine strain RB51; however, RB51, but not its parent strain S2308, induced caspase-2-mediated DC maturation, cytokine production, antigen

Immunogenic Cell Death Induced by Ginsenoside Rg3: Significance in Dendritic Cell-based Anti-tumor Immunotherapy.

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Son, Keum-Joo; Choi, Ki Ryung; Lee, Seog Jae; Lee, Hyunah

2016-02-01

Cancer is one of the leading causes of morbidity and mortality worldwide; therefore there is a need to discover new therapeutic modules with improved efficacy and safety. Immune-(cell) therapy is a promising therapeutic strategy for the treatment of intractable cancers. The effectiveness of certain chemotherapeutics in inducing immunogenic tumor cell death thus promoting cancer eradication has been reported. Ginsenoside Rg3 is a ginseng saponin that has antitumor and immunomodulatory activity. In this study, we treated tumor cells with Rg3 to verify the significance of inducing immunogenic tumor cell death in antitumor therapy, especially in DC-based immunotherapy. Rg3 killed the both immunogenic (B16F10 melanoma cells) and non-immunogenic (LLC: Lewis Lung Carcinoma cells) tumor cells by inducing apoptosis. Surface expression of immunogenic death markers including calreticulin and heat shock proteins and the transcription of relevant genes were increased in the Rg3-dying tumor. Increased calreticulin expression was directly related to the uptake of dying tumor cells by dendritic cells (DCs): the proportion of CRT(+) CD11c(+) cells was increased in the Rg3-treated group. Interestingly, tumor cells dying by immunogenic cell death secreted IFN-γ, an effector molecule for antitumor activity in T cells. Along with the Rg3-induced suppression of pro-angiogenic (TNF-α) and immunosuppressive cytokine (TGF-β) secretion, IFN-γ production from the Rg3-treated tumor cells may also indicate Rg3 as an effective anticancer immunotherapeutic strategy. The data clearly suggests that Rg3-induced immunogenic tumor cell death due its cytotoxic effect and its ability to induce DC function. This indicates that Rg3 may be an effective immunotherapeutic strategy.

Mycobacterium tuberculosis infection induces non-apoptotic cell death of human dendritic cells

LENUS (Irish Health Repository)

Ryan, Ruth CM

2011-10-24

Abstract Background Dendritic cells (DCs) connect innate and adaptive immunity, and are necessary for an efficient CD4+ and CD8+ T cell response after infection with Mycobacterium tuberculosis (Mtb). We previously described the macrophage cell death response to Mtb infection. To investigate the effect of Mtb infection on human DC viability, we infected these phagocytes with different strains of Mtb and assessed viability, as well as DNA fragmentation and caspase activity. In parallel studies, we assessed the impact of infection on DC maturation, cytokine production and bacillary survival. Results Infection of DCs with live Mtb (H37Ra or H37Rv) led to cell death. This cell death proceeded in a caspase-independent manner, and without nuclear fragmentation. In fact, substrate assays demonstrated that Mtb H37Ra-induced cell death progressed without the activation of the executioner caspases, 3\\/7. Although the death pathway was triggered after infection, the DCs successfully underwent maturation and produced a host-protective cytokine profile. Finally, dying infected DCs were permissive for Mtb H37Ra growth. Conclusions Human DCs undergo cell death after infection with live Mtb, in a manner that does not involve executioner caspases, and results in no mycobactericidal effect. Nonetheless, the DC maturation and cytokine profile observed suggests that the infected cells can still contribute to TB immunity.

Tumor cell-released TLR4 ligands stimulate Gr-1+CD11b+F4/80+ cells to induce apoptosis of activated T cells.

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Liu, Yan-Yan; Sun, Ling-Cong; Wei, Jing-Jing; Li, Dong; Yuan, Ye; Yan, Bin; Liang, Zhi-Hui; Zhu, Hui-Fen; Xu, Yong; Li, Bo; Song, Chuan-Wang; Liao, Sheng-Jun; Lei, Zhang; Zhang, Gui-Mei; Feng, Zuo-Hua

2010-09-01

Gr-1(+)CD11b(+)F4/80(+) cells play important roles in tumor development and have a negative effect on tumor immunotherapy. So far, the mechanisms underlying the regulation of their immunosuppressive phenotype by classical and alternative macrophage activation stimuli are not well elucidated. In this study, we found that molecules from necrotic tumor cells (NTC-Ms) stimulated Gr-1(+)CD11b(+)F4/80(+) cells to induce apoptosis of activated T cells but not nonstimulated T cells. The apoptosis-inducing capacity was determined by higher expression levels of arginase I and IL-10 relative to those of NO synthase 2 and IL-12 in Gr-1(+)CD11b(+)F4/80(+) cells, which were induced by NTC-Ms through TLR4 signaling. The apoptosis-inducing capacity of NTC-Ms-stimulated Gr-1(+)CD11b(+)F4/80(+) cells could be enhanced by IL-10. IFN-gamma may reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells only if their response to IFN-gamma was not attenuated. However, the potential of Gr-1(+)CD11b(+)F4/80(+) cells to express IL-12 in response to IFN-gamma could be attenuated by tumor, partially due to the existence of active STAT3 in Gr-1(+)CD11b(+)F4/80(+) cells and NTC-Ms from tumor. In this situation, IFN-gamma could not effectively reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells. Tumor immunotherapy with 4-1BBL/soluble programmed death-1 may significantly reduce, but not abolish the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells in local microenvironment. Blockade of TLR4 signaling could further reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and enhance the suppressive effect of 4-1BBL/soluble form of programmed death-1 on tumor growth. These findings indicate the relationship of distinct signaling pathways with apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and emphasize the importance of blocking TLR4 signaling to prevent the induction of T cell apoptosis by Gr-1(+)CD11b(+)F4/80(+) cells.

Tumor-Induced CD8+ T-Cell Dysfunction in Lung Cancer Patients

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Heriberto Prado-Garcia

2012-01-01

Full Text Available Lung cancer is the leading cause of cancer deaths worldwide and one of the most common types of cancers. The limited success of chemotherapy and radiotherapy regimes have highlighted the need to develop new therapies like antitumor immunotherapy. CD8+ T-cells represent a major arm of the cell-mediated anti-tumor response and a promising target for developing T-cell-based immunotherapies against lung cancer. Lung tumors, however, have been considered to possess poor immunogenicity; even so, lung tumor-specific CD8+ T-cell clones can be established that possess cytotoxicity against autologous tumor cells. This paper will focus on the alterations induced in CD8+ T-cells by lung cancer. Although memory CD8+ T-cells infiltrate lung tumors, in both tumor-infiltrating lymphocytes (TILs and malignant pleural effusions, these cells are dysfunctional and the effector subset is reduced. We propose that chronic presence of lung tumors induces dysfunctions in CD8+ T-cells and sensitizes them to activation-induced cell death, which may be associated with the poor clinical responses observed in immunotherapeutic trials. Getting a deeper knowledge of the evasion mechanisms lung cancer induce in CD8+ T-cells should lead to further understanding of lung cancer biology, overcome tumor evasion mechanisms, and design improved immunotherapeutic treatments for lung cancer.

Free cholesterol accumulation impairs antioxidant activities and aggravates apoptotic cell death in menadione-induced oxidative injury.

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Lee, Waisin; Xu, Mingjing; Li, Yue; Gu, Yong; Chen, Jianping; Wong, Derek; Fung, Peter C W; Shen, Jiangang

2011-10-01

Although the relationship between hypercholesterolemia and oxidative stress has been extensively investigated, direct evidence regarding to the roles of cholesterol accumulation in the generations of reactive oxygen species (ROS) and apoptotic cell death under oxidative stress is lack. In this study, we investigated productions of superoxide anions (O(2)(-)) and nitric oxide (NO), and apoptotic cell death in wild type Chinese hamster ovary (CHO) cells and cholesterol accumulated CHO cells genetically and chemically. Oxidative stress was induced by menadione challenge. The results revealed that abundance of free cholesterol (FC) promoted menadione-induced O(2)(-) and NO productions. FC accumulation down-regulated eNOS expression but up-regulated NADPH oxidases, and inhibited the activities of superoxide dismutase (SOD) and catalase. Treatment of menadione increased the expressions of iNOS and qp91 phox, enhanced the activities of SOD and catalase in the wild-type CHO cells but inhibited the activity of glutathione peroxidase in the cholesterol accumulated CHO cells. Moreover, FC abundance promoted apoptotic cell death in these cells. Taken together, those results suggest that free cholesterol accumulation aggravates menadione-induced oxidative stress and exacerbates apoptotic cell death. Copyright © 2011 Elsevier Inc. All rights reserved.

PKC activation induces inflammatory response and cell death in human bronchial epithelial cells.

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Hyunhee Kim

Full Text Available A variety of airborne pathogens can induce inflammatory responses in airway epithelial cells, which is a crucial component of host defence. However, excessive inflammatory responses and chronic inflammation also contribute to different diseases of the respiratory system. We hypothesized that the activation of protein kinase C (PKC is one of the essential mechanisms of inflammatory response in airway epithelial cells. In the present study, we stimulated human bronchial lung epithelial (BEAS-2B cells with the phorbol ester Phorbol 12, 13-dibutyrate (PDBu, and examined gene expression profile using microarrays. Microarray analysis suggests that PKC activation induced dramatic changes in gene expression related to multiple cellular functions. The top two interaction networks generated from these changes were centered on NFκB and TNF-α, which are two commonly known pathways for cell death and inflammation. Subsequent tests confirmed the decrease in cell viability and an increase in the production of various cytokines. Interestingly, each of the increased cytokines was differentially regulated at mRNA and/or protein levels by different sub-classes of PKC isozymes. We conclude that pathological cell death and cytokine production in airway epithelial cells in various situations may be mediated through PKC related signaling pathways. These findings suggest that PKCs can be new targets for treatment of lung diseases.

SB225002 Induces Cell Death and Cell Cycle Arrest in Acute Lymphoblastic Leukemia Cells through the Activation of GLIPR1

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Leal, Paulo C.; Bhasin, Manoj K.; Zenatti, Priscila Pini; Nunes, Ricardo J.; Yunes, Rosendo A.; Nowill, Alexandre E.; Libermann, Towia A.; Zerbini, Luiz Fernando; Yunes, José Andrés

2015-01-01

Acute Lymphoblastic Leukemia (ALL) is the most frequent childhood malignancy. In the effort to find new anti-leukemic agents, we evaluated the small drug SB225002 (N-(2-hydroxy-4-nitrophenyl)-N’-(2-bromophenyl)urea). Although initially described as a selective antagonist of CXCR2, later studies have identified other cellular targets for SB225002, with potential medicinal use in cancer. We found that SB225002 has a significant pro-apoptotic effect against both B- and T-ALL cell lines. Cell cycle analysis demonstrated that treatment with SB225002 induces G2-M cell cycle arrest. Transcriptional profiling revealed that SB225002-mediated apoptosis triggered a transcriptional program typical of tubulin binding agents. Network analysis revealed the activation of genes linked to the JUN and p53 pathways and inhibition of genes linked to the TNF pathway. Early cellular effects activated by SB225002 included the up-regulation of GLIPR1, a p53-target gene shown to have pro-apoptotic activities in prostate and bladder cancer. Silencing of GLIPR1 in B- and T-ALL cell lines resulted in increased resistance to SB225002. Although SB225002 promoted ROS increase in ALL cells, antioxidant N-Acetyl Cysteine pre-treatment only modestly attenuated cell death, implying that the pro-apoptotic effects of SB225002 are not exclusively mediated by ROS. Moreover, GLIPR1 silencing resulted in increased ROS levels both in untreated and SB225002-treated cells. In conclusion, SB225002 induces cell cycle arrest and apoptosis in different B- and T-ALL cell lines. Inhibition of tubulin function with concurrent activation of the p53 pathway, in particular, its downstream target GLIPR1, seems to underlie the anti-leukemic effect of SB225002. PMID:26302043

Akebia saponin PA induces autophagic and apoptotic cell death in AGS human gastric cancer cells.

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Xu, Mei-Ying; Lee, Dong Hwa; Joo, Eun Ji; Son, Kun Ho; Kim, Yeong Shik

2013-09-01

In this study, we investigated the anticancer mechanism of akebia saponin PA (AS), a natural product isolated from Dipsacus asperoides in human gastric cancer cell lines. It was shown that AS-induced cell death is caused by autophagy and apoptosis in AGS cells. The apoptosis-inducing effect of AS was characterized by annexin V/propidium (PI) staining, increase of sub-G1 phase and caspase-3 activation, while the autophagy-inducing effect was indicated by the formation of cytoplasmic vacuoles and microtubule-associated protein 1 light chain-3 II (LC3-II) conversion. The autophagy inhibitor bafilomycin A1 (BaF1) decreased AS-induced cell death and caspase-3 activation, but caspase-3 inhibitor Ac-DEVD-CHO did not affect LC3-II accumulation or AS-induced cell viability, suggesting that AS induces autophagic cell death and autophagy contributes to caspase-3-dependent apoptosis. Furthermore, AS activated p38/c-Jun N-terminal kinase (JNK), which could be inhibited by BaF1, and caspase-3 activation was attenuated by both SB202190 and SP600125, indicating that AS-induced autophagy promotes mitogen-activated protein kinases (MAPKs)-mediated apoptosis. Taken together, these results demonstrate that AS induces autophagic and apoptotic cell death and autophagy plays the main role in akebia saponin PA-induced cell death. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cytotoxic T lymphocyte-mediated cytolysis: an example of programmed cell death in the immune system

International Nuclear Information System (INIS)

Duke, R.C.

1985-01-01

Target cells are programmed to die following interaction with cytotoxic T lymphocytes (CTLs). Within minutes of exposure to CTL the target cell's nuclear DNA is fragmented. Target cell lysis, as measured by 51 Cr release, occurs about 60 minutes after induction of DNA fragmentation. DNA fragmentation results from the action of an endonuclease which cleaves DNA in the linker region between nucleosomes. The origin of this nuclease, whether transferred to the target by the CTL or endogenous to the target cell, has not been resolved. DNA fragmentation occurs only when appropriately sensitized CTL are used and is not merely the result of cell death because killing of target cells by extreme deviation from homeostasis, by interruption of energy production, or by lysis with antibody and complement does not induce DNA cleavage. When Triton X-100 is added to target cells which have interacted with CTL, the DNA fragments do not remain in association with the nucleus. This observation suggests that breakdown of overall nuclear structure is induced concomitantly with DNA fragmentation. Morphologically, disruption of nuclear structure and DNA fragmentation are observed as widespread chromatin condensation (apoptosis). Apoptosis is observed in metabolically active target cells and is not a consequence of cell death. A cell whose DNA is extensively fragmented is condemmed to die. Induction of oligonucleosome-sized DNA is also an early event in glucocorticoid-induced thymocyte death and death of T cells upon removal of growth factor. Several similarities exist between these systems and CTL-mediated cytolysis suggesting a final common biochemical pathway for all three types of cell death

Ionizing radiation-induced cell death

International Nuclear Information System (INIS)

Szumiel, I.

1994-01-01

Selected aspects of radiation-induced cell death, connected with signal transduction pathways are reviewed. Cell death is defined as insufficiency of the cellular signal transducing system to maintain the cell's physiological functions. The insufficiency may be due to impaired signal reception and/or transduction, lack or erroneous transcription activation, and eventual cellular ''misexpression'' of the signal. The molecular basis of this insufficiency would be damage to genomic (but also other cellular) structures and closing of specific signalling pathways or opening of others (like those leading to apoptosis). I describe experimental data that suggest an important role of RAS/NFI and p53/p105 Rb proteins in cell cycle control-coupled responses to DNA damage. (Author)

Preferential killing of cancer cells and activated human T cells using ZnO nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Hanley, Cory; Layne, Janet; Feris, Kevin; Wingett, Denise [Department of Biological Sciences, Boise State University, Boise, ID 83725 (United States); Punnoose, Alex; Reddy, K M; Coombs, Isaac; Coombs, Andrew [Department of Physics, Boise State University, Boise, ID 83725 (United States)], E-mail: denisewingett@boisestate.edu

2008-07-23

Nanoparticles are increasingly being recognized for their potential utility in biological applications including nanomedicine. Here we examine the response of normal human cells to ZnO nanoparticles under different signaling environments and compare it to the response of cancerous cells. ZnO nanoparticles exhibit a strong preferential ability to kill cancerous T cells ({approx}28-35 x) compared to normal cells. Interestingly, the activation state of the cell contributes toward nanoparticle toxicity, as resting T cells display a relative resistance while cells stimulated through the T cell receptor and CD28 costimulatory pathway show greater toxicity in direct relation to the level of activation. Mechanisms of toxicity appear to involve the generation of reactive oxygen species, with cancerous T cells producing higher inducible levels than normal T cells. In addition, nanoparticles were found to induce apoptosis and the inhibition of reactive oxygen species was found to be protective against nanoparticle induced cell death. The novel findings of cell selective toxicity, towards potential disease causing cells, indicate a potential utility of ZnO nanoparticles in the treatment of cancer and/or autoimmunity.

Preferential killing of cancer cells and activated human T cells using ZnO nanoparticles

International Nuclear Information System (INIS)

Hanley, Cory; Layne, Janet; Feris, Kevin; Wingett, Denise; Punnoose, Alex; Reddy, K M; Coombs, Isaac; Coombs, Andrew

2008-01-01

Nanoparticles are increasingly being recognized for their potential utility in biological applications including nanomedicine. Here we examine the response of normal human cells to ZnO nanoparticles under different signaling environments and compare it to the response of cancerous cells. ZnO nanoparticles exhibit a strong preferential ability to kill cancerous T cells (∼28-35 x) compared to normal cells. Interestingly, the activation state of the cell contributes toward nanoparticle toxicity, as resting T cells display a relative resistance while cells stimulated through the T cell receptor and CD28 costimulatory pathway show greater toxicity in direct relation to the level of activation. Mechanisms of toxicity appear to involve the generation of reactive oxygen species, with cancerous T cells producing higher inducible levels than normal T cells. In addition, nanoparticles were found to induce apoptosis and the inhibition of reactive oxygen species was found to be protective against nanoparticle induced cell death. The novel findings of cell selective toxicity, towards potential disease causing cells, indicate a potential utility of ZnO nanoparticles in the treatment of cancer and/or autoimmunity

Crystalline structure of pulverized dental calculus induces cell death in oral epithelial cells.

Science.gov (United States)

Ziauddin, S M; Yoshimura, A; Montenegro Raudales, J L; Ozaki, Y; Higuchi, K; Ukai, T; Kaneko, T; Miyazaki, T; Latz, E; Hara, Y

2018-06-01

Dental calculus is a mineralized deposit attached to the tooth surface. We have shown that cellular uptake of dental calculus triggers nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation, leading to the processing of the interleukin-1β precursor into its mature form in mouse and human phagocytes. The activation of the NLRP3 inflammasome also induced a lytic form of programmed cell death, pyroptosis, in these cells. However, the effects of dental calculus on other cell types in periodontal tissue have not been investigated. The aim of this study was to determine whether dental calculus can induce cell death in oral epithelial cells. HSC-2 human oral squamous carcinoma cells, HOMK107 human primary oral epithelial cells and immortalized mouse macrophages were exposed to dental calculus or 1 of its components, hydroxyapatite crystals. For inhibition assays, the cells were exposed to dental calculus in the presence or absence of cytochalasin D (endocytosis inhibitor), z-YVAD-fmk (caspase-1 inhibitor) or glyburide (NLRP3 inflammasome inhibitor). Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release and staining with propidium iodide. Tumor necrosis factor-α production was quantified by enzyme-linked immunosorbent assay. Oral epithelial barrier function was examined by permeability assay. Dental calculus induced cell death in HSC-2 cells, as judged by LDH release and propidium iodide staining. Dental calculus also induced LDH release from HOMK107 cells. Following heat treatment, dental calculus lost its capacity to induce tumor necrosis factor-α in mouse macrophages, but could induce LDH release in HSC-2 cells, indicating a major role of inorganic components in cell death. Hydroxyapatite crystals also induced cell death in both HSC-2 and HOMK107 cells, as judged by LDH release, indicating the capacity of crystal particles to induce cell death. Cell death induced by dental

Jasmonic acid signaling modulates ozone-induced hypersensitive cell death.

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Rao, M V; Lee, H; Creelman, R A; Mullet, J E; Davis, K R

2000-09-01

Recent studies suggest that cross-talk between salicylic acid (SA)-, jasmonic acid (JA)-, and ethylene-dependent signaling pathways regulates plant responses to both abiotic and biotic stress factors. Earlier studies demonstrated that ozone (O(3)) exposure activates a hypersensitive response (HR)-like cell death pathway in the Arabidopsis ecotype Cvi-0. We now have confirmed the role of SA and JA signaling in influencing O(3)-induced cell death. Expression of salicylate hydroxylase (NahG) in Cvi-0 reduced O(3)-induced cell death. Methyl jasmonate (Me-JA) pretreatment of Cvi-0 decreased O(3)-induced H(2)O(2) content and SA concentrations and completely abolished O(3)-induced cell death. Cvi-0 synthesized as much JA as did Col-0 in response to O(3) exposure but exhibited much less sensitivity to exogenous Me-JA. Analyses of the responses to O(3) of the JA-signaling mutants jar1 and fad3/7/8 also demonstrated an antagonistic relationship between JA- and SA-signaling pathways in controlling the magnitude of O(3)-induced HR-like cell death.

Docetaxel-induced prostate cancer cell death involves concomitant activation of caspase and lysosomal pathways and is attenuated by LEDGF/p75

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Leoh Lai

2009-08-01

Full Text Available Abstract Background Hormone-refractory prostate cancer (HRPC is characterized by poor response to chemotherapy and high mortality, particularly among African American men when compared to other racial/ethnic groups. It is generally accepted that docetaxel, the standard of care for chemotherapy of HRPC, primarily exerts tumor cell death by inducing mitotic catastrophe and caspase-dependent apoptosis following inhibition of microtubule depolymerization. However, there is a gap in our knowledge of mechanistic events underlying docetaxel-induced caspase-independent cell death, and the genes that antagonize this process. This knowledge is important for circumventing HRPC chemoresistance and reducing disparities in prostate cancer mortality. Results We investigated mechanistic events associated with docetaxel-induced death in HRPC cell lines using various approaches that distinguish caspase-dependent from caspase-independent cell death. Docetaxel induced both mitotic catastrophe and caspase-dependent apoptosis at various concentrations. However, caspase activity was not essential for docetaxel-induced cytotoxicity since cell death associated with lysosomal membrane permeabilization still occurred in the presence of caspase inhibitors. Partial inhibition of docetaxel-induced cytotoxicity was observed after inhibition of cathepsin B, but not inhibition of cathepsins D and L, suggesting that docetaxel induces caspase-independent, lysosomal cell death. Simultaneous inhibition of caspases and cathepsin B dramatically reduced docetaxel-induced cell death. Ectopic expression of lens epithelium-derived growth factor p75 (LEDGF/p75, a stress survival autoantigen and transcription co-activator, attenuated docetaxel-induced lysosomal destabilization and cell death. Interestingly, LEDGF/p75 overexpression did not protect cells against DTX-induced mitotic catastrophe, and against apoptosis induced by tumor necrosis factor related apoptosis inducing ligand (TRAIL

Probiotic Lactobacilli Modulate Staphylococcus aureus-Induced Activation of Conventional and Unconventional T cells and NK cells

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Maria A Johansson

2016-07-01

Full Text Available Lactobacilli are probiotic commensal bacteria and potent modulators of immunity. When present in the gut or supplemented as probiotics, they beneficially modulate ex vivo immune responsiveness. Further, factors derived from several lactobacilli strains act immune regulato-ry in vitro. In contrast, Staphylococcus aureus (S. aureus is known to induce excessive T cell activation. In this study we aimed to investigate S. aureus-induced activation of human muco-sal associated invariant T cells (MAIT cells, γδ T cells, NK cells, as well as of conventional CD4+ and CD8+ T cells in vitro. Further, we investigated if lactobacilli-derived factors could modulate their activation.PBMC were cultured with S. aureus 161:2 cell free supernatant (CFS, staphylococcal en-terotoxin A or CD3/CD28-beads alone or in combination with Lactobacillus rhamnosus (L. rhamnosus GG-CFS or Lactobacillus reuteri (L. reuteri DSM 17938-CFS, and activation of T and NK cells was evaluated. S. aureus-CFS induced IFN-γ and CD107a expression as well as proliferation. Co-stimulation with lactobacilli-CFS dampened lymphocyte activation in all cell types analysed. Pre-incubation with lactobacilli-CFS was enough to reduce subsequent activation and the ab-sence of APC or APC-derived IL-10 did not prevent lactobacilli-mediated dampening. Final-ly, lactate selectively dampened activation of unconventional T cells and NK cells. In summary, we show that molecules present in the lactobacilli-CFS are able to directly dampen in vitro activation of conventional and unconventional T cells and of NK cells. This study provides novel insights on the immune modulatory nature of probiotic lactobacilli and suggests a role for lactobacilli in modulation of induced T and NK cell activation.

Chemoprotective effect of insulin-like growth factor I against acetaminophen-induced cell death in Chang liver cells via ERK1/2 activation

International Nuclear Information System (INIS)

Hwang, Hye-Jung; Kwon, Mi-Jin; Nam, Taek-Jeong

2007-01-01

The insulin-like growth factor (IGF) system and type-I IGF receptor (IGF-IR) signaling are involved in protecting against chemotherapeutic drug-induced cell death in human hepatoma cells. Acetaminophen (AAP) hepatotoxicity is the leading cause of liver failure, and the prevention of AAP-induced cell death has been the focus of many studies. We determined whether IGF-I could protect against AAP-induced cell death in Chang liver cells and investigated the protective mechanism. Based on the results of MTS assays, LDH release assays, Hoechst 33342 cell staining, and DNA fragmentation experiments, AAP induced cell death in a dose-dependent manner. According to Western blot analysis, treatment with AAP increased the level of poly(ADP-ribose) polymerase (PARP) fragments in cells compared with that in control cells; however, caspase-3, a critical signaling molecule in apoptosis, was not activated after AAP overdose. Moreover, combined treatment with AAP and IGF-I inhibited PARP cleavage, which was consistent with the ability of IGF-I to restore the level of glutathione (GSH) and cell viability in GSH and MTS assays, respectively. We investigated whether the protective effect of IGF-I against AAP cytotoxicity is related to the extracellular signal-related kinase ERK1/2, which is generally activated by mitogenic and proliferative stimuli such as growth factors. Compared with AAP treatment alone, IGF-I and AAP co-treatment increased ERK1/2 phosphorylation but inhibited PARP cleavage. Thus ERK1/2 activation is instrumental in the protective effect of IGF-I against AAP-induced cell death in Chang liver cells

Blockade of the ERK pathway markedly sensitizes tumor cells to HDAC inhibitor-induced cell death

International Nuclear Information System (INIS)

Ozaki, Kei-ichi; Minoda, Ai; Kishikawa, Futaba; Kohno, Michiaki

2006-01-01

Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway is associated with the neoplastic phenotype of a large number of human tumor cells. Although specific blockade of the ERK pathway by treating such tumor cells with potent mitogen-activated protein kinase/ERK kinase (MEK) inhibitors completely suppresses their proliferation, it by itself shows only a modest effect on the induction of apoptotic cell death. However, these MEK inhibitors markedly enhance the efficacy of histone deacetylase (HDAC) inhibitors to induce apoptotic cell death: such an enhanced cell death is observed only in tumor cells in which the ERK pathway is constitutively activated. Co-administration of MEK inhibitor markedly sensitizes tumor cells to HDAC inhibitor-induced generation of reactive oxygen species, which appears to mediate the enhanced cell death induced by the combination of these agents. These results suggest that the combination of MEK inhibitors and HDAC inhibitors provides an efficient chemotherapeutic strategy for the treatment of tumor cells in which the ERK pathway is constitutively activated

Black rice extract protected HepG2 cells from oxidative stress-induced cell death via ERK1/2 and Akt activation

Science.gov (United States)

Yoon, Jaemin; Ham, Hyeonmi; Sung, Jeehye; Kim, Younghwa; Choi, Youngmin; Lee, Jeom-Sig; Jeong, Heon-Sang; Lee, Junsoo

2014-01-01

BACKGROUND/OBJECTIVES The objective of this study was to evaluate the protective effect of black rice extract (BRE) on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells. MATERIALS/METHODS Methanolic extract from black rice was evaluated for the protective effect on TBHP-induced oxidative injury in HepG2 cells. Several biomarkers that modulate cell survival and death including reactive oxygen species (ROS), caspase-3 activity, and related cellular kinases were determined. RESULTS TBHP induced cell death and apoptosis by a rapid increase in ROS generation and caspase-3 activity. Moreover, TBHP-induced oxidative stress resulted in a transient ERK1/2 activation and a sustained increase of JNK1/2 activation. While, BRE pretreatment protects the cells against oxidative stress by reducing cell death, caspase-3 activity, and ROS generation and also by preventing ERKs deactivation and the prolonged JNKs activation. Moreover, pretreatment of BRE increased the activation of ERKs and Akt which are pro-survival signal proteins. However, this effect was blunted in the presence of ERKs and Akt inhibitors. CONCLUSIONS These results suggest that activation of ERKs and Akt pathway might be involved in the cytoprotective effect of BRE against oxidative stress. Our findings provide new insights into the cytoprotective effects and its possible mechanism of black rice against oxidative stress. PMID:24741394

Annonaceous acetogenin mimic AA005 induces cancer cell death via apoptosis inducing factor through a caspase-3-independent mechanism.

Science.gov (United States)

Han, Bing; Wang, Tong-Dan; Shen, Shao-Ming; Yu, Yun; Mao, Chan; Yao, Zhu-Jun; Wang, Li-Shun

2015-03-18

Annonaceous acetogenins are a family of natural products with antitumor activities. Annonaceous acetogenin mimic AA005 reportedly inhibits mammalian mitochondrial NADH-ubiquinone reductase (Complex I) and induces gastric cancer cell death. However, the mechanisms underlying its cell-death-inducing activity are unclear. We used SW620 colorectal adenocarcinoma cells to study AA005 cytotoxic activity. Cell deaths were determined by Trypan blue assay and flow cytometry, and related proteins were characterized by western blot. Immunofluorescence and subcellular fractionation were used to evaluate AIF nuclear translocation. Reactive oxygen species were assessed by using redox-sensitive dye DCFDA. AA005 induces a unique type of cell death in colorectal adenocarcinoma cells, characterized by lack of caspase-3 activation or apoptotic body formation, sensitivity to poly (ADP-ribose) polymerase inhibitor Olaparib (AZD2281) but not pan-caspase inhibitor Z-VAD.fmk, and dependence on apoptosis-inducing factor (AIF). AA005 treatment also reduced expression of mitochondrial Complex I components, and leads to accumulation of intracellular reactive oxygen species (ROS) at the early stage. Blocking ROS formation significantly suppresses AA005-induced cell death in SW620 cells. Moreover, blocking activation of RIP-1 by necroptosis inhibitor necrotatin-1 inhibits AIF translocation and partially suppresses AA005-induced cell death in SW620 cells demonstrating that RIP-1 protein may be essential for cell death. AA005 may trigger the cell death via mediated by AIF through caspase-3 independent pathway. Our work provided new mechanisms for AA005-induced cancer cell death and novel clues for cancer treatment via AIF dependent cell death.

Morphologic categorization of cell death induced by mild hyperthermia and comparison with death induced by ionizing radiation and cytotoxic drugs

International Nuclear Information System (INIS)

Allan, D.J.; Harmon, B.V.

1986-01-01

This paper presents a summary of the morphological categorization of cell death, results of two in vivo studies on the cell death induced by mild hyperthermia in rat small intestine and mouse mastocytoma, and a comparison of the cell death induced by hyperthermia, radiation and cytotoxic drugs. Two distinct forms of cell death, apoptosis and necrosis, can be recognized on morphologic grounds. Apoptosis appears to be a process of active cellular self-destruction to which a biologically meaningful role can usually be attributed, whereas necrosis is a passive degenerative phenomenon that results from irreversible cellular injury. Light and transmission electron microscopic studies showed that lower body hyperthermia (43 degrees C for 30 min) induced only apoptosis of intestinal epithelial cells, and of lymphocytes, plasma cells, and eosinophils. In the mastocytoma, hyperthermia (43 degrees C for 15 min) produced widespread tumor necrosis and also enhanced apoptosis of tumor cells. Ionizing radiation and cytotoxic drugs are also known to induce apoptosis in a variety of tissues. It is attractive to speculate that DNA damage by each agent is the common event which triggers the same process of active cellular self-destruction that characteristically effects selective cell deletion in normal tissue homeostasis

Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection

Science.gov (United States)

Doitsh, Gilad; Galloway, Nicole L. K.; Geng, Xin; Yang, Zhiyuan; Monroe, Kathryn M.; Zepeda, Orlando; Hunt, Peter W.; Hatano, Hiroyu; Sowinski, Stefanie; Muñoz-Arias, Isa; Greene, Warner C.

2014-01-01

The pathway causing CD4 T-cell death in HIV-infected hosts remains poorly understood although apoptosis has been proposed as a key mechanism. We now show that caspase-3-mediated apoptosis accounts for the death of only a small fraction of CD4 T cells corresponding to those that are both activated and productively infected. The remaining over 95% of quiescent lymphoid CD4 T cells die by caspase-1-mediated pyroptosis triggered by abortive viral infection. Pyroptosis corresponds to an intensely inflammatory form of programmed cell death in which cytoplasmic contents and pro-inflammatory cytokines, including IL-1β, are released. This death pathway thus links the two signature events in HIV infection--CD4 T-cell depletion and chronic inflammation--and creates a pathogenic vicious cycle in which dying CD4 T cells release inflammatory signals that attract more cells to die. This cycle can be broken by caspase 1 inhibitors shown to be safe in humans, raising the possibility of a new class of `anti-AIDS' therapeutics targeting the host rather than the virus.

Bee Venom Protects against Rotenone-Induced Cell Death in NSC34 Motor Neuron Cells

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So Young Jung

2015-09-01

Full Text Available Rotenone, an inhibitor of mitochondrial complex I of the mitochondrial respiratory chain, is known to elevate mitochondrial reactive oxygen species and induce apoptosis via activation of the caspase-3 pathway. Bee venom (BV extracted from honey bees has been widely used in oriental medicine and contains melittin, apamin, adolapin, mast cell-degranulating peptide, and phospholipase A2. In this study, we tested the effects of BV on neuronal cell death by examining rotenone-induced mitochondrial dysfunction. NSC34 motor neuron cells were pretreated with 2.5 μg/mL BV and stimulated with 10 μM rotenone to induce cell toxicity. We assessed cell death by Western blotting using specific antibodies, such as phospho-ERK1/2, phospho-JNK, and cleaved capase-3 and performed an MTT assay for evaluation of cell death and mitochondria staining. Pretreatment with 2.5 μg/mL BV had a neuroprotective effect against 10 μM rotenone-induced cell death in NSC34 motor neuron cells. Pre-treatment with BV significantly enhanced cell viability and ameliorated mitochondrial impairment in rotenone-treated cellular model. Moreover, BV treatment inhibited the activation of JNK signaling and cleaved caspase-3 related to cell death and increased ERK phosphorylation involved in cell survival in rotenone-treated NSC34 motor neuron cells. Taken together, we suggest that BV treatment can be useful for protection of neurons against oxidative stress or neurotoxin-induced cell death.

8-C-(E-phenylethenyl)quercetin from onion/beef soup induces autophagic cell death in colon cancer cells through ERK activation.

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Zhao, Yueliang; Fan, Daming; Zheng, Zong-Ping; Li, Edmund T S; Chen, Feng; Cheng, Ka-Wing; Wang, Mingfu

2017-02-01

Quercetin, a flavonoid, widely distributed in edible fruits and vegetables, was reported to effectively inhibit 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP) formation in a food model (roast beef patties) with itself being converted into a novel compound 8-C-(E-phenylethenyl)quercetin (8-CEPQ). Here we investigated whether 8-CEPQ could be formed in a real food system, and tested its anticancer activity in human colon cancer cell lines. LC-MS was applied for the determination of 8-CEPQ formation in onion/beef soup. Anticancer activity of 8-CEPQ was evaluated by using cell viability assay and flow cytometry. Results showed that 8-CEPQ suppressed proliferation and caused G 2 phase arrest in colon cancer cells. Based on immunofluorescent staining assay, western blot assay, and RNA knockdown data, we found that 8-CEPQ did not cause apoptotic cell death. Instead, it induced autophagic cell death. Moreover, treatment with 8-CEPQ induced phosphorylation of extracellular signal-regulated kinase (ERK). Inhibition of ERK phosphorylation by the mitogen-activated protein kinase kinase (MEK)/ERK inhibitor U0126 attenuated 8-CEPQ-induced autophagy and reversed 8-CEPQ-mediated cell growth inhibition. Our results demonstrate that 8-CEPQ, a novel quercetin derivative, could be formed in onion/beef soup. 8-CEPQ inhibited colon cancer cell growth by inducing autophagic cell death through ERK activation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transient Receptor Potential Vanilloid 1 Expression Mediates Capsaicin-Induced Cell Death

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Ricardo Ramírez-Barrantes

2018-06-01

Full Text Available The transient receptor potential (TRP ion channel family consists of a broad variety of non-selective cation channels that integrate environmental physicochemical signals for dynamic homeostatic control. Involved in a variety of cellular physiological processes, TRP channels are fundamental to the control of the cell life cycle. TRP channels from the vanilloid (TRPV family have been directly implicated in cell death. TRPV1 is activated by pain-inducing stimuli, including inflammatory endovanilloids and pungent exovanilloids, such as capsaicin (CAP. TRPV1 activation by high doses of CAP (>10 μM leads to necrosis, but also exhibits apoptotic characteristics. However, CAP dose–response studies are lacking in order to determine whether CAP-induced cell death occurs preferentially via necrosis or apoptosis. In addition, it is not known whether cytosolic Ca2+ and mitochondrial dysfunction participates in CAP-induced TRPV1-mediated cell death. By using TRPV1-transfected HeLa cells, we investigated the underlying mechanisms involved in CAP-induced TRPV1-mediated cell death, the dependence of CAP dose, and the participation of mitochondrial dysfunction and cytosolic Ca2+ increase. Together, our results contribute to elucidate the pathophysiological steps that follow after TRPV1 stimulation with CAP. Low concentrations of CAP (1 μM induce cell death by a mechanism involving a TRPV1-mediated rapid and transient intracellular Ca2+ increase that stimulates plasma membrane depolarization, thereby compromising plasma membrane integrity and ultimately leading to cell death. Meanwhile, higher doses of CAP induce cell death via a TRPV1-independent mechanism, involving a slow and persistent intracellular Ca2+ increase that induces mitochondrial dysfunction, plasma membrane depolarization, plasma membrane loss of integrity, and ultimately, cell death.

Mechanism of neem limonoids-induced cell death in cancer: Role of oxidative phosphorylation.

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Yadav, Neelu; Kumar, Sandeep; Kumar, Rahul; Srivastava, Pragya; Sun, Leimin; Rapali, Peter; Marlowe, Timothy; Schneider, Andrea; Inigo, Joseph R; O'Malley, Jordan; Londonkar, Ramesh; Gogada, Raghu; Chaudhary, Ajay K; Yadava, Nagendra; Chandra, Dhyan

2016-01-01

We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition pore, and mitochondrial fragmentation. Neem enhanced mitochondrial DNA and mitochondrial biomass. While oxidative phosphorylation (OXPHOS) Complex-I activity was decreased, the activities of other OXPHOS complexes including Complex-II and -IV were unaltered. Increased reactive oxygen species (ROS) levels were associated with an increase in mitochondrial biomass and apoptosis upon neem exposure. Complex-I deficiency due to the loss of Ndufa1-encoded MWFE protein inhibited neem-induced caspase activation and apoptosis, but cell death induction was enhanced. Complex II-deficiency due to the loss of succinate dehydrogenase complex subunit C (SDHC) robustly decreased caspase activation, apoptosis, and cell death. Additionally, the ablation of Complexes-I, -III, -IV, and -V together did not inhibit caspase activation. Together, we demonstrate that neem limonoids target OXPHOS system to induce cancer cell death, which does not require upregulation or activation of proapoptotic Bcl-2 family proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Semiallogenic fusions of MSI+ tumor cells and activated B cells induce MSI-specific T cell responses

International Nuclear Information System (INIS)

Garbe, Yvette; Klier, Ulrike; Linnebacher, Michael

2011-01-01

Various strategies have been developed to transfer tumor-specific antigens into antigen presenting cells in order to induce cytotoxic T cell responses against tumor cells. One approach uses cellular vaccines based on fusions of autologous antigen presenting cells and allogeneic tumor cells. The fusion cells combine antigenicity of the tumor cell with optimal immunostimulatory capacity of the antigen presenting cells. Microsatellite instability caused by mutational inactivation of DNA mismatch repair genes results in translational frameshifts when affecting coding regions. It has been shown by us and others that these mutant proteins lead to the presentation of immunogenic frameshift peptides that are - in principle - recognized by a multiplicity of effector T cells. We chose microsatellite instability-induced frameshift antigens as ideal to test for induction of tumor specific T cell responses by semiallogenic fusions of microsatellite instable carcinoma cells with CD40-activated B cells. Two fusion clones of HCT116 with activated B cells were selected for stimulation of T cells autologous to the B cell fusion partner. Outgrowing T cells were phenotyped and tested in functional assays. The fusion clones expressed frameshift antigens as well as high amounts of MHC and costimulatory molecules. Autologous T cells stimulated with these fusions were predominantly CD4 + , activated, and reacted specifically against the fusion clones and also against the tumor cell fusion partner. Interestingly, a response toward 6 frameshift-derived peptides (of 14 tested) could be observed. Cellular fusions of MSI + carcinoma cells and activated B cells combine the antigen-presenting capacity of the B cell with the antigenic repertoire of the carcinoma cell. They present frameshift-derived peptides and can induce specific and fully functional T cells recognizing not only fusion cells but also the carcinoma cells. These hybrid cells may have great potential for cellular immunotherapy and

Ayanin diacetate-induced cell death is amplified by TRAIL in human leukemia cells

International Nuclear Information System (INIS)

Marrero, María Teresa; Estévez, Sara; Negrín, Gledy; Quintana, José; López, Mariana; Pérez, Francisco J.; Triana, Jorge; León, Francisco; Estévez, Francisco

2012-01-01

Highlights: ► Ayanin diacetate as apoptotic inducer in leukemia cells. ► Cell death was prevented by caspase inhibitors and by the overexpression of Bcl-x L . ► The intrinsic and the extrinsic pathways are involved in the mechanism of action. ► Death receptors are up-regulated and TRAIL enhances apoptotic cell death. -- Abstract: Here we demonstrate that the semi-synthetic flavonoid ayanin diacetate induces cell death selectively in leukemia cells without affecting the proliferation of normal lymphocytes. Incubation of human leukemia cells with ayanin diacetate induced G 2 -M phase cell cycle arrest and apoptosis which was prevented by the non-specific caspase inhibitor z-VAD-fmk and reduced by the overexpression of Bcl-x L . Ayanin diacetate-induced cell death was found to be associated with: (i) loss of inner mitochondrial membrane potential, (ii) the release of cytochrome c, (iii) the activation of multiple caspases, (iv) cleavage of poly(ADP-ribose) polymerase and (v) the up-regulation of death receptors for TRAIL, DR4 and DR5. Moreover, the combined treatment with ayanin diacetate and TRAIL amplified cell death, compared to single treatments. These results provide a basis for further exploring the potential applications of this combination for the treatment of cancer.

UV-Induced Cell Death in Plants

Science.gov (United States)

Nawkar, Ganesh M.; Maibam, Punyakishore; Park, Jung Hoon; Sahi, Vaidurya Pratap; Lee, Sang Yeol; Kang, Chang Ho

2013-01-01

Plants are photosynthetic organisms that depend on sunlight for energy. Plants respond to light through different photoreceptors and show photomorphogenic development. Apart from Photosynthetically Active Radiation (PAR; 400–700 nm), plants are exposed to UV light, which is comprised of UV-C (below 280 nm), UV-B (280–320 nm) and UV-A (320–390 nm). The atmospheric ozone layer protects UV-C radiation from reaching earth while the UVR8 protein acts as a receptor for UV-B radiation. Low levels of UV-B exposure initiate signaling through UVR8 and induce secondary metabolite genes involved in protection against UV while higher dosages are very detrimental to plants. It has also been reported that genes involved in MAPK cascade help the plant in providing tolerance against UV radiation. The important targets of UV radiation in plant cells are DNA, lipids and proteins and also vital processes such as photosynthesis. Recent studies showed that, in response to UV radiation, mitochondria and chloroplasts produce a reactive oxygen species (ROS). Arabidopsis metacaspase-8 (AtMC8) is induced in response to oxidative stress caused by ROS, which acts downstream of the radical induced cell death (AtRCD1) gene making plants vulnerable to cell death. The studies on salicylic and jasmonic acid signaling mutants revealed that SA and JA regulate the ROS level and antagonize ROS mediated cell death. Recently, molecular studies have revealed genes involved in response to UV exposure, with respect to programmed cell death (PCD). PMID:23344059

alpha-Toxin is a mediator of Staphylococcus aureus-induced cell death and activates caspases via the intrinsic death pathway independently of death receptor signaling

NARCIS (Netherlands)

Bantel, H; Sinha, B; Domschke, W; Peters, Georg; Schulze-Osthoff, K; Jänicke, R U

2001-01-01

Infections with Staphylococcus aureus, a common inducer of septic and toxic shock, often result in tissue damage and death of various cell types. Although S. aureus was suggested to induce apoptosis, the underlying signal transduction pathways remained elusive. We show that caspase activation and

Drosophila Ninjurin A induces nonapoptotic cell death.

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Sarah Broderick

Full Text Available Ninjurins are conserved transmembrane proteins that are upregulated across species in response to injury and stress. Their biological functions are not understood, in part because there have been few in vivo studies of their function. We analyzed the expression and function of one of three Drosophila Ninjurins, NijA. We found that NijA protein is redistributed to the cell surface in larval immune tissues after septic injury and is upregulated by the Toll pathway. We generated a null mutant of NijA, which displayed no detectable phenotype. In ectopic expression studies, NijA induced cell death, as evidenced by cell loss and acridine orange staining. These dying cells did not display hallmarks of apoptotic cells including TUNEL staining and inhibition by p35, indicating that NijA induced nonapoptotic cell death. In cell culture, NijA also induced cell death, which appeared to be cell autonomous. These in vivo studies identify a new role for the Ninjurin family in inducing nonapoptotic cell death.

Ouabain exacerbates activation-induced cell death in human peripheral blood lymphocytes

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Mabel B. Esteves

2005-06-01

Full Text Available Lymphocytes activated by mitogenic lectins display changes in transmembrane potential, an elevation in the cytoplasmic Ca2+ concentrations, proliferation and/or activation induced cell death. Low concentrations of ouabain (an inhibitor of Na+,K+-ATPase suppress mitogen-induced proliferation and increases cell death. To understand the mechanisms involved, a number of parameters were analyzed using fluorescent probes and flow cytometry. The addition of 100nM ouabain to cultures of peripheral blood lymphocytes activated with 5µg/ml phytohemagglutinin (PHA did not modify the increased expression of the Fas receptor or its ligand FasL induced by the mitogen. However, treatment with ouabain potentiated apoptosis induced by an anti-Fas agonist antibody. A synergy between ouabain and PHA was also observed with regard to plasma membrane depolarization. PHA per se did not induce dissipation of mitochondrial membrane potential but when cells were also exposed to ouabain a marked depolarization could be observed, and this was a late event. It is possible that the inhibitory effect of ouabain on activated peripheral blood lymphocytes involves the potentiation of some of the steps of the apoptotic process and reflects an exacerbation of the mechanism of activation-induced cell death.Quando linfócitos são ativados por lectinas mitogênicas apresentam mudanças do potencial de membrana, elevação das concentrações citoplasmáticas de cálcio, proliferação e/ou morte celular induzida por ativação (AICD. Concentrações baixas de ouabaína (um inibidor da Na,K-ATPase suprimem a proliferação induzida por mitógenos e aumentam a morte celular. Para entender os mecanismos envolvidos, uma série de parâmetros foram avaliados usando sondas fluorescentes e citometria de fluxo. A adição de 100nM de ouabaína para culturas de linfócitos de sangue periférico ativadas por fitohemaglutinina (PHA não modificou o aumento de expressão do receptor Fas ou de

Ayanin diacetate-induced cell death is amplified by TRAIL in human leukemia cells

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Marrero, Maria Teresa; Estevez, Sara; Negrin, Gledy; Quintana, Jose [Departamento de Bioquimica, Unidad Asociada al Consejo Superior de Investigaciones Cientificas, Universidad de Las Palmas de Gran Canaria, Plaza Dr. Pasteur s/n, 35016 Las Palmas de Gran Canaria (Spain); Lopez, Mariana; Perez, Francisco J.; Triana, Jorge [Departamento de Quimica, Universidad de Las Palmas de Gran Canaria, Instituto Canario de Investigacion del Cancer, 35017 Las Palmas de Gran Canaria (Spain); Leon, Francisco [Instituto de Productos Naturales y Agrobiologia, Consejo Superior de Investigaciones Cientificas, Avda. Astrofisico F. Sanchez 3, 38206 La Laguna, Tenerife (Spain); Estevez, Francisco, E-mail: festevez@dbbf.ulpgc.es [Departamento de Bioquimica, Unidad Asociada al Consejo Superior de Investigaciones Cientificas, Universidad de Las Palmas de Gran Canaria, Plaza Dr. Pasteur s/n, 35016 Las Palmas de Gran Canaria (Spain)

2012-11-09

Highlights: Black-Right-Pointing-Pointer Ayanin diacetate as apoptotic inducer in leukemia cells. Black-Right-Pointing-Pointer Cell death was prevented by caspase inhibitors and by the overexpression of Bcl-x{sub L}. Black-Right-Pointing-Pointer The intrinsic and the extrinsic pathways are involved in the mechanism of action. Black-Right-Pointing-Pointer Death receptors are up-regulated and TRAIL enhances apoptotic cell death. -- Abstract: Here we demonstrate that the semi-synthetic flavonoid ayanin diacetate induces cell death selectively in leukemia cells without affecting the proliferation of normal lymphocytes. Incubation of human leukemia cells with ayanin diacetate induced G{sub 2}-M phase cell cycle arrest and apoptosis which was prevented by the non-specific caspase inhibitor z-VAD-fmk and reduced by the overexpression of Bcl-x{sub L}. Ayanin diacetate-induced cell death was found to be associated with: (i) loss of inner mitochondrial membrane potential, (ii) the release of cytochrome c, (iii) the activation of multiple caspases, (iv) cleavage of poly(ADP-ribose) polymerase and (v) the up-regulation of death receptors for TRAIL, DR4 and DR5. Moreover, the combined treatment with ayanin diacetate and TRAIL amplified cell death, compared to single treatments. These results provide a basis for further exploring the potential applications of this combination for the treatment of cancer.

Docosahexaenoic Acid Induces Cell Death in Human Non-Small Cell Lung Cancer Cells by Repressing mTOR via AMPK Activation and PI3K/Akt Inhibition

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Nayeong Kim

2015-01-01

Full Text Available The anticancer properties and mechanism of action of omega-3 polyunsaturated fatty acids (ω3-PUFAs have been demonstrated in several cancers; however, the mechanism in lung cancer remains unclear. Here, we show that docosahexaenoic acid (DHA, a ω3-PUFA, induced apoptosis and autophagy in non-small cell lung cancer (NSCLC cells. DHA-induced cell death was accompanied by AMP-activated protein kinase (AMPK activation and inactivated phosphatidylinositol 3-kinase (PI3K/Akt/mammalian target of rapamycin (mTOR signaling. Knocking down AMPK and overexpressing Akt increased mTOR activity and attenuated DHA-induced cell death, suggesting that DHA induces cell death via AMPK- and Akt-regulated mTOR inactivation. This was confirmed in Fat-1 transgenic mice, which produce ω3-PUFAs. Lewis lung cancer (LLC tumor cells implanted into Fat-1 mice showed slower growth, lower phospho-Akt levels, and higher levels of apoptosis and autophagy than cells implanted into wild-type mice. Taken together, these data suggest that DHA-induced apoptosis and autophagy in NSCLC cells are associated with AMPK activation and PI3K/Akt inhibition, which in turn lead to suppression of mTOR; thus ω3-PUFAs may be utilized as potential therapeutic agents for NSCLC treatment.

Suppression of T cell-induced osteoclast formation

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Karieb, Sahar; Fox, Simon W., E-mail: Simon.fox@plymouth.ac.uk

2013-07-12

Highlights: •Genistein and coumestrol prevent activated T cell induced osteoclast formation. •Anti-TNF neutralising antibodies prevent the pro-osteoclastic effect of activated T cells. •Phytoestrogens inhibit T cell derived TNF alpha and inflammatory cytokine production. •Phytoestrogens have a broader range of anti-osteoclastic actions than other anti-resorptives. -- Abstract: Inhibition of T cell derived cytokine production could help suppress osteoclast differentiation in inflammatory skeletal disorders. Bisphosphonates are typically prescribed to prevent inflammatory bone loss but are not tolerated by all patients and are associated with an increased risk of osteonecrosis of the jaw. In light of this other anti-resorptives such as phytoestrogens are being considered. However the effect of phytoestrogens on T cell-induced osteoclast formation is unclear. The effect of genistein and coumestrol on activated T cell-induced osteoclastogenesis and cytokine production was therefore examined. Concentrations of genistein and coumestrol (10{sup −7} M) previously shown to directly inhibit osteoclast formation also suppressed the formation of TRAP positive osteoclast induced by con A activated T cells, which was dependent on inhibition of T cell derived TNF-α. While both reduced osteoclast formation their mechanism of action differed. The anti-osteoclastic effect of coumestrol was associated with a dual effect on con A induced T cell proliferation and activation; 10{sup −7} M coumestrol significantly reducing T cell number (0.36) and TNF-α (0.47), IL-1β (0.23) and IL-6 (0.35) expression, whereas genistein (10{sup −7} M) had no effect on T cell number but a more pronounced effect on T cell differentiation reducing expression of TNF-α (0.49), IL-1β (0.52), IL-6 (0.71) and RANKL (0.71). Phytoestrogens therefore prevent the pro-osteoclastic action of T cells suggesting they may have a role in the control of inflammatory bone loss.

Mechanisms underlying 3-bromopyruvate-induced cell death in colon cancer.

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Sun, Yiming; Liu, Zhe; Zou, Xue; Lan, Yadong; Sun, Xiaojin; Wang, Xiu; Zhao, Surong; Jiang, Chenchen; Liu, Hao

2015-08-01

3-Bromopyruvate (3BP) is an energy-depleting drug that inhibits Hexokinase II activity by alkylation during glycolysis, thereby suppressing the production of ATP and inducing cell death. As such, 3BP can potentially serve as an anti-tumorigenic agent. Our previous research showed that 3BP can induce apoptosis via AKT /protein Kinase B signaling in breast cancer cells. Here we found that 3BP can also induce colon cancer cell death by necroptosis and apoptosis at the same time and concentration in the SW480 and HT29 cell lines; in the latter, autophagy was also found to be a mechanism of cell death. In HT29 cells, combined treatment with 3BP and the autophagy inhibitor 3-methyladenine (3-MA) exacerbated cell death, while viability in 3BP-treated cells was enhanced by concomitant treatment with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (z-VAD-fmk) and the necroptosis inhibitor necrostatin (Nec)-1. Moreover, 3BP inhibited tumor growth in a SW480 xenograft mouse model. These results indicate that 3BP can suppress tumor growth and induce cell death by multiple mechanisms at the same time and concentration in different types of colon cancer cell by depleting cellular energy stores.

Induction of cell death by graphene in Arabidopsis thaliana (Columbia ecotype) T87 cell suspensions

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Begum, Parvin; Fugetsu, Bunshi

2013-01-01

Highlights: • This study was set up to explore potential influence of graphene on T87 cells. • Fragmented nuclei, membrane damage, mitochondrial dysfunction were observed. • ROS increased, ROS are key mediators in the cell death signaling pathway. • Translocation of graphene into cells and an endocytosis-like structure was observed. • Graphene entering into the cells by endocytosis. -- Abstract: The toxicity of graphene on suspensions of Arabidopsis thaliana (Columbia ecotype) T87 cells was investigated by examining the morphology, mitochondrial dysfunction, reactive oxygen species generation (ROS), and translocation of graphene as the toxicological endpoints. The cells were grown in Jouanneau and Péaud-Lenoel (JPL) media and exposed to graphene at concentrations 0–80 mg/L. Morphological changes were observed by scanning electron microscope and the adverse effects such as fragmented nuclei, membrane damage, mitochondrial dysfunction was observed with fluorescence microscopy by staining with Hoechst 33342/propidium iodide and succinate dehydrogenase (mitochondrial bioenergetic enzyme). Analysis of intracellular ROS by 2′,7′-dichlorofluorescein diacetate demonstrated that graphene induced a 3.3-fold increase in ROS, suggesting that ROS are key mediators in the cell death signaling pathway. Transmission electron microscopy verified the translocation of graphene into cells and an endocytosis-like structure was observed which suggested graphene entering into the cells by endocytosis. In conclusion, our results show that graphene induced cell death in T87 cells through mitochondrial damage mediated by ROS

Restimulation-induced T-cell death through NTB-A/SAP signaling pathway is impaired in tuberculosis patients with depressed immune responses.

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Hernández Del Pino, Rodrigo E; Pellegrini, Joaquín M; Rovetta, Ana I; Peña, Delfina; Álvarez, Guadalupe I; Rolandelli, Agustín; Musella, Rosa M; Palmero, Domingo J; Malbran, Alejandro; Pasquinelli, Virginia; García, Verónica E

2017-09-01

Production of IFN-γ contributes to host defense against Mycobacterium tuberculosis (Mtb) infection. We previously demonstrated that Signaling lymphocytic activation molecule-associated protein (SAP) expression on cells from tuberculosis (TB) patients was inversely correlated with IFN-γ production. Here we first investigated the role of NK, T- and B-cell antigen (NTB-A)/SAP pathway in the regulation of Th1 response against Mtb. Upon antigen stimulation, NTB-A phosphorylation rapidly increases and afterwards modulates IFN-γ and IL-17 secretion. To sustain a healthy immune system, controlled expansion and contraction of lymphocytes, both during and after an adaptive immune response, is essential. Besides, restimulation-induced cell death (RICD) results in an essential homeostatic mechanism for precluding excess T-cell accumulation and associated immunopathology during the course of certain infections. Accordingly, we found that the NTB-A/SAP pathway was required for RICD during active tuberculosis. In low responder (LR) TB patients, impaired RICD was associated with diminished FASL levels, IL-2 production and CD25 high expression after cell-restimulation. Interestingly, we next observed that SAP mediated the recruitment of the Src-related kinase FYNT, only in T cells from LR TB patients that were resistant to RICD. Together, we showed that the NTB-A/SAP pathway regulates T-cell activation and RICD during human TB. Moreover, the NTB-A/SAP/FYNT axis promotes polarization to an unfavorable Th2-phenotype.

mTOR inhibition sensitizes ONC201-induced anti-colorectal cancer cell activity.

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Jin, Zhe-Zhu; Wang, Wei; Fang, Di-Long; Jin, Yong-Jun

2016-09-30

We here tested the anti-colorectal cancer (CRC) activity by a first-in-class small molecule TRAIL inducer ONC201. The potential effect of mTOR on ONC201's actions was also examined. ONC201 induced moderate cytotoxicity against CRC cell lines (HT-29, HCT-116 and DLD-1) and primary human CRC cells. Significantly, AZD-8055, a mTOR kinase inhibitor, sensitized ONC201-induced cytotoxicity in CRC cells. Meanwhile, ONC201-induced TRAIL/death receptor-5 (DR-5) expression, caspase-8 activation and CRC cell apoptosis were also potentiated with AZD-8055 co-treatment. Reversely, TRAIL sequestering antibody RIK-2 or the caspase-8 specific inhibitor z-IETD-fmk attenuated AZD-8055 plus ONC201-induced CRC cell death. Further, mTOR kinase-dead mutation (Asp-2338-Ala) or shRNA knockdown significantly sensitized ONC201's activity in CRC cells, leading to profound cell death and apoptosis. On the other hand, expression of a constitutively-active S6K1 (T389E) attenuated ONC201-induced CRC cell apoptosis. For the mechanism study, we showed that ONC201 blocked Akt, but only slightly inhibited mTOR in CRC cells. Co-treatment with AZD-8055 also concurrently blocked mTOR activation. These results suggest that mTOR could be a primary resistance factor of ONC201 in CRC cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Glucose Metabolism and AMPK Signaling Regulate Dopaminergic Cell Death Induced by Gene (α-Synuclein)-Environment (Paraquat) Interactions.

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Anandhan, Annadurai; Lei, Shulei; Levytskyy, Roman; Pappa, Aglaia; Panayiotidis, Mihalis I; Cerny, Ronald L; Khalimonchuk, Oleh; Powers, Robert; Franco, Rodrigo

2017-07-01

While environmental exposures are not the single cause of Parkinson's disease (PD), their interaction with genetic alterations is thought to contribute to neuronal dopaminergic degeneration. However, the mechanisms involved in dopaminergic cell death induced by gene-environment interactions remain unclear. In this work, we have revealed for the first time the role of central carbon metabolism and metabolic dysfunction in dopaminergic cell death induced by the paraquat (PQ)-α-synuclein interaction. The toxicity of PQ in dopaminergic N27 cells was significantly reduced by glucose deprivation, inhibition of hexokinase with 2-deoxy-D-glucose (2-DG), or equimolar substitution of glucose with galactose, which evidenced the contribution of glucose metabolism to PQ-induced cell death. PQ also stimulated an increase in glucose uptake, and in the levels of glucose transporter type 4 (GLUT4) and Na + -glucose transporters isoform 1 (SGLT1) proteins, but only inhibition of GLUT-like transport with STF-31 or ascorbic acid reduced PQ-induced cell death. Importantly, while autophagy protein 5 (ATG5)/unc-51 like autophagy activating kinase 1 (ULK1)-dependent autophagy protected against PQ toxicity, the inhibitory effect of glucose deprivation on cell death progression was largely independent of autophagy or mammalian target of rapamycin (mTOR) signaling. PQ selectively induced metabolomic alterations and adenosine monophosphate-activated protein kinase (AMPK) activation in the midbrain and striatum of mice chronically treated with PQ. Inhibition of AMPK signaling led to metabolic dysfunction and an enhanced sensitivity of dopaminergic cells to PQ. In addition, activation of AMPK by PQ was prevented by inhibition of the inducible nitric oxide syntase (iNOS) with 1400W, but PQ had no effect on iNOS levels. Overexpression of wild type or A53T mutant α-synuclein stimulated glucose accumulation and PQ toxicity, and this toxic synergism was reduced by inhibition of glucose metabolism

Active ras triggers death in glioblastoma cells through hyperstimulation of macropinocytosis.

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Overmeyer, Jean H; Kaul, Aparna; Johnson, Erin E; Maltese, William A

2008-06-01

Expression of activated Ras in glioblastoma cells induces accumulation of large phase-lucent cytoplasmic vacuoles, followed by cell death. This was previously described as autophagic cell death. However, unlike autophagosomes, the Ras-induced vacuoles are not bounded by a double membrane and do not sequester organelles or cytoplasm. Moreover, they are not acidic and do not contain the autophagosomal membrane protein LC3-II. Here we show that the vacuoles are enlarged macropinosomes. They rapidly incorporate extracellular fluid-phase tracers but do not sequester transferrin or the endosomal protein EEA1. Ultimately, the cells expressing activated Ras detach from the substratum and rupture, coincident with the displacement of cytoplasm with huge macropinosome-derived vacuoles. These changes are accompanied by caspase activation, but the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone does not prevent cell death. Moreover, the majority of degenerating cells do not exhibit chromatin condensation typical of apoptosis. These observations provide evidence for a necrosis-like form of cell death initiated by dysregulation of macropinocytosis, which we have dubbed "methuosis." An activated form of the Rac1 GTPase induces a similar form of cell death, suggesting that Ras acts through Rac-dependent signaling pathways to hyperstimulate macropinocytosis in glioblastoma. Further study of these signaling pathways may lead to the identification of other chemical and physiologic triggers for this unusual form of cell death.

Annonaceous acetogenin mimic AA005 induces cancer cell death via apoptosis inducing factor through a caspase-3-independent mechanism

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Han, Bing; Wang, Tong-Dan; Shen, Shao-Ming; Yu, Yun; Mao, Chan; Yao, Zhu-Jun; Wang, Li-Shun

2015-01-01

Background Annonaceous acetogenins are a family of natural products with antitumor activities. Annonaceous acetogenin mimic AA005 reportedly inhibits mammalian mitochondrial NADH-ubiquinone reductase (Complex I) and induces gastric cancer cell death. However, the mechanisms underlying its cell-death-inducing activity are unclear. Methods We used SW620 colorectal adenocarcinoma cells to study AA005 cytotoxic activity. Cell deaths were determined by Trypan blue assay and flow cytometry, and rel...

Porcine circovirus-2 capsid protein induces cell death in PK15 cells

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Walia, Rupali; Dardari, Rkia, E-mail: rdardari@ucalgary.ca; Chaiyakul, Mark; Czub, Markus

2014-11-15

Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathways involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis. - Highlights: • IFN-γ enhances PCV2 replication that leads to cell death in PK15 cells. • IFN-γ enhances nuclear localization of the PCV2 Capsid protein. • Transient PCV2a and 2b-Capsid protein expression induces cell death. • Cell death is not dictated by specific Capsid protein sub-localization.

The anti-cell death FNK protein protects cells from death induced by freezing and thawing

International Nuclear Information System (INIS)

Sudo, Kentaro; Asoh, Sadamitsu; Ohsawa, Ikuroh; Ozaki, Daiya; Yamagata, Kumi; Ito, Hiromoto; Ohta, Shigeo

2005-01-01

The FNK protein, constructed from anti-apoptotic Bcl-x L with enhanced activity, was fused with the protein transduction domain (PTD) of the HIV/Tat protein to mediate the delivery of FNK into cells. The fusion protein PTD-FNK was introduced into chondrocytes in isolated articular cartilage-bone sections, cultured neurons, and isolated bone marrow mononuclear cells to evaluate its ability to prevent cell death induced by freezing and thawing. PTD-FNK protected the cells from freeze-thaw damage in a concentration-dependent manner. Addition of PTD-FNK with conventional cryoprotectants (dimethyl sulfoxide and hydroxyethyl starch) increased surviving cell numbers around 2-fold compared with controls treated only with the cryoprotectants. Notably, PTD-FNK allowed CD34 + cells among bone marrow mononuclear cells to survive more efficiently (12-fold more than the control cells) from two successive freeze-thaw cycles. Thus, PTD-FNK prevented cell death induced by freezing and thawing, suggesting that it provides for the successful cryopreservation of biological materials

Association of Marek's Disease induced immunosuppression with activation of a novel regulatory T cells in chickens.

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Angila Gurung

2017-12-01

Full Text Available Marek's Disease Virus (MDV is an alphaherpesvirus that infects chickens, transforms CD4+ T cells and causes deadly lymphomas. In addition, MDV induces immunosuppression early during infection by inducing cell death of the infected lymphocytes, and potentially due to activation of regulatory T (Treg-cells. Furthermore, immunosuppression also occurs during the transformation phase of the disease; however, it is still unknown how the disease can suppress immune response prior or after lymphoma formation. Here, we demonstrated that chicken TGF-beta+ Treg cells are found in different lymphoid tissues, with the highest levels found in the gut-associated lymphoid tissue (cecal tonsil: CT, fostering an immune-privileged microenvironment exerted by TGF-beta. Surprisingly, significantly higher frequencies of TGF-beta+ Treg cells are found in the spleens of MDV-susceptible chicken lines compared to the resistant line, suggesting an association between TGF-beta+ Treg cells and host susceptibility to lymphoma formation. Experimental infection with a virulent MDV elevated the levels of TGF-beta+ Treg cells in the lungs as early as 4 days post infection, and during the transformation phase of the disease in the spleens. In contrast to TGF-beta+ Treg cells, the levels of CD4+CD25+ T cells remained unchanged during the infection and transformation phase of the disease. Furthermore, our results demonstrate that the induction of TGF-beta+ Treg cells is associated with pathogenesis of the disease, as the vaccine strain of MDV did not induce TGF-beta+ Treg cells. Similar to human haematopoietic malignant cells, MDV-induced lymphoma cells expressed high levels of TGF-beta but very low levels of TGF-beta receptor I and II genes. The results confirm that COX-2/ PGE2 pathway is involved in immunosuppression induced by MDV-lymphoma cells. Taken together, our results revealed a novel TGF-beta+ Treg subset in chickens that is activated during MDV infection and tumour

Taxifolin synergizes Andrographolide-induced cell death by attenuation of autophagy and augmentation of caspase dependent and independent cell death in HeLa cells.

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Mazen Alzaharna

Full Text Available Andrographolide (Andro has emerged recently as a potential and effective anticancer agent with induction of apoptosis in some cancer cell lines while induction of G2/M arrest with weak apoptosis in others. Few studies have proved that Andro is also effective in combination therapy. The flavonoid Taxifolin (Taxi has showed anti-oxidant and antiproliferative effects against different cancer cells. Therefore, the present study investigated the cytotoxic effects of Andro alone or in combination with Taxi on HeLa cells. The combination of Andro with Taxi was synergistic at all tested concentrations and combination ratios. Andro alone induced caspase-dependent apoptosis which was enhanced by the combination with Taxi and attenuated partly by using Z-Vad-Fmk. Andro induced a protective reactive oxygen species (ROS-dependent autophagy which was attenuated by Taxi. The activation of p53 was involved in Andro-induced autophagy where the use of Taxi or pifithrin-α (PFT-α decreased it while the activation of JNK was involved in the cell death of HeLa cells but not in the induction of autophagy. The mitochondrial outer-membrane permeabilization (MOMP plays an important role in Andro-induced cell death in HeLa cells. Andro alone increased the MOMP which was further increased in the case of combination. This led to the increase in AIF and cytochrome c release from mitochondria which consequently increased caspase-dependent and independent cell death. In conclusion, Andro induced a protective autophagy in HeLa cells which was reduced by Taxi and the cell death was increased by increasing the MOMP and subsequently the caspase-dependent and independent cell death.

Taxifolin synergizes Andrographolide-induced cell death by attenuation of autophagy and augmentation of caspase dependent and independent cell death in HeLa cells

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Alzaharna, Mazen; Alqouqa, Iyad; Cheung, Hon-Yeung

2017-01-01

Andrographolide (Andro) has emerged recently as a potential and effective anticancer agent with induction of apoptosis in some cancer cell lines while induction of G2/M arrest with weak apoptosis in others. Few studies have proved that Andro is also effective in combination therapy. The flavonoid Taxifolin (Taxi) has showed anti-oxidant and antiproliferative effects against different cancer cells. Therefore, the present study investigated the cytotoxic effects of Andro alone or in combination with Taxi on HeLa cells. The combination of Andro with Taxi was synergistic at all tested concentrations and combination ratios. Andro alone induced caspase-dependent apoptosis which was enhanced by the combination with Taxi and attenuated partly by using Z-Vad-Fmk. Andro induced a protective reactive oxygen species (ROS)-dependent autophagy which was attenuated by Taxi. The activation of p53 was involved in Andro-induced autophagy where the use of Taxi or pifithrin-α (PFT-α) decreased it while the activation of JNK was involved in the cell death of HeLa cells but not in the induction of autophagy. The mitochondrial outer-membrane permeabilization (MOMP) plays an important role in Andro-induced cell death in HeLa cells. Andro alone increased the MOMP which was further increased in the case of combination. This led to the increase in AIF and cytochrome c release from mitochondria which consequently increased caspase-dependent and independent cell death. In conclusion, Andro induced a protective autophagy in HeLa cells which was reduced by Taxi and the cell death was increased by increasing the MOMP and subsequently the caspase-dependent and independent cell death. PMID:28182713

Chemical chaperones reduce ionizing radiation-induced endoplasmic reticulum stress and cell death in IEC-6 cells

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Lee, Eun Sang; Lee, Hae-June; Lee, Yoon-Jin [Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Jeong, Jae-Hoon [Division of Radiotherapy, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Kang, Seongman [Division of Life Sciences, Korea University, Seoul 136-701 (Korea, Republic of); Lim, Young-Bin, E-mail: yblim@kirams.re.kr [Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of)

2014-07-25

Highlights: • UPR activation precedes caspase activation in irradiated IEC-6 cells. • Chemical ER stress inducers radiosensitize IEC-6 cells. • siRNAs that targeted ER stress responses ameliorate IR-induced cell death. • Chemical chaperons prevent cell death in irradiated IEC-6 cells. - Abstract: Radiotherapy, which is one of the most effective approaches to the treatment of various cancers, plays an important role in malignant cell eradication in the pelvic area and abdomen. However, it also generates some degree of intestinal injury. Apoptosis in the intestinal epithelium is the primary pathological factor that initiates radiation-induced intestinal injury, but the mechanism by which ionizing radiation (IR) induces apoptosis in the intestinal epithelium is not clearly understood. Recently, IR has been shown to induce endoplasmic reticulum (ER) stress, thereby activating the unfolded protein response (UPR) signaling pathway in intestinal epithelial cells. However, the consequences of the IR-induced activation of the UPR signaling pathway on radiosensitivity in intestinal epithelial cells remain to be determined. In this study, we investigated the role of ER stress responses in IR-induced intestinal epithelial cell death. We show that chemical ER stress inducers, such as tunicamycin or thapsigargin, enhanced IR-induced caspase 3 activation and DNA fragmentation in intestinal epithelial cells. Knockdown of Xbp1 or Atf6 with small interfering RNA inhibited IR-induced caspase 3 activation. Treatment with chemical chaperones prevented ER stress and subsequent apoptosis in IR-exposed intestinal epithelial cells. Our results suggest a pro-apoptotic role of ER stress in IR-exposed intestinal epithelial cells. Furthermore, inhibiting ER stress may be an effective strategy to prevent IR-induced intestinal injury.

A Lactose-Binding Lectin from the Marine Sponge Cinachyrella Apion (Cal) Induces Cell Death in Human Cervical Adenocarcinoma Cells

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Rabelo, Luciana; Monteiro, Norberto; Serquiz, Raphael; Santos, Paula; Oliveira, Ruth; Oliveira, Adeliana; Rocha, Hugo; Morais, Ana Heloneida; Uchoa, Adriana; Santos, Elizeu

2012-01-01

Cancer represents a set of more than 100 diseases, including malignant tumors from different locations. Strategies inducing differentiation have had limited success in the treatment of established cancers. Marine sponges are a biological reservoir of bioactive molecules, especially lectins. Several animal and plant lectins were purified with antitumor activity, mitogenic, anti-inflammatory and antiviral, but there are few reports in the literature describing the mechanism of action of lectins purified from marine sponges to induce apoptosis in human tumor cells. In this work, a lectin purified from the marine sponge Cinachyrella apion (CaL) was evaluated with respect to its hemolytic, cytotoxic and antiproliferative properties, besides the ability to induce cell death in tumor cells. The antiproliferative activity of CaL was tested against HeLa, PC3 and 3T3 cell lines, with highest growth inhibition for HeLa, reducing cell growth at a dose dependent manner (0.5–10 µg/mL). Hemolytic activity and toxicity against peripheral blood cells were tested using the concentration of IC50 (10 µg/mL) for both trials and twice the IC50 for analysis in flow cytometry, indicating that CaL is not toxic to these cells. To assess the mechanism of cell death caused by CaL in HeLa cells, we performed flow cytometry and western blotting. Results showed that lectin probably induces cell death by apoptosis activation by pro-apoptotic protein Bax, promoting mitochondrial membrane permeabilization, cell cycle arrest in S phase and acting as both dependent and/or independent of caspases pathway. These results indicate the potential of CaL in studies of medicine for treating cancer. PMID:22690140

A Lactose-Binding Lectin from the Marine Sponge Cinachyrella Apion (Cal Induces Cell Death in Human Cervical Adenocarcinoma Cells

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Adriana Uchoa

2012-03-01

Full Text Available Cancer represents a set of more than 100 diseases, including malignant tumors from different locations. Strategies inducing differentiation have had limited success in the treatment of established cancers. Marine sponges are a biological reservoir of bioactive molecules, especially lectins. Several animal and plant lectins were purified with antitumor activity, mitogenic, anti-inflammatory and antiviral, but there are few reports in the literature describing the mechanism of action of lectins purified from marine sponges to induce apoptosis in human tumor cells. In this work, a lectin purified from the marine sponge Cinachyrella apion (CaL was evaluated with respect to its hemolytic, cytotoxic and antiproliferative properties, besides the ability to induce cell death in tumor cells. The antiproliferative activity of CaL was tested against HeLa, PC3 and 3T3 cell lines, with highest growth inhibition for HeLa, reducing cell growth at a dose dependent manner (0.5–10 µg/mL. Hemolytic activity and toxicity against peripheral blood cells were tested using the concentration of IC50 (10 µg/mL for both trials and twice the IC50 for analysis in flow cytometry, indicating that CaL is not toxic to these cells. To assess the mechanism of cell death caused by CaL in HeLa cells, we performed flow cytometry and western blotting. Results showed that lectin probably induces cell death by apoptosis activation by pro-apoptotic protein Bax, promoting mitochondrial membrane permeabilization, cell cycle arrest in S phase and acting as both dependent and/or independent of caspases pathway. These results indicate the potential of CaL in studies of medicine for treating cancer.

Involvement of ERK-Nrf-2 signaling in ionizing radiation induced cell death in normal and tumor cells.

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Raghavendra S Patwardhan

Full Text Available Prolonged oxidative stress favors tumorigenic environment and inflammation. Oxidative stress may trigger redox adaptation mechanism(s in tumor cells but not normal cells. This may increase levels of intracellular antioxidants and establish a new redox homeostasis. Nrf-2, a master regulator of battery of antioxidant genes is constitutively activated in many tumor cells. Here we show that, murine T cell lymphoma EL-4 cells show constitutive and inducible radioresistance via activation of Nrf-2/ERK pathway. EL-4 cells contained lower levels of ROS than their normal counterpart murine splenic lymphocytes. In response to radiation, the thiol redox circuits, GSH and thioredoxin were modified in EL-4 cells. Pharmacological inhibitors of ERK and Nrf-2 significantly enhanced radiosensitivity and reduced clonogenic potential of EL-4 cells. Unirradiated lymphoma cells showed nuclear accumulation of Nrf-2, upregulation of its dependent genes and protein levels. Interestingly, MEK inhibitor abrogated its nuclear translocation suggesting role of ERK in basal and radiation induced Nrf-2 activation in tumor cells. Double knockdown of ERK and Nrf-2 resulted in higher sensitivity to radiation induced cell death as compared to individual knockdown cells. Importantly, NF-kB which is reported to be constitutively active in many tumors was not present at basal levels in EL-4 cells and its inhibition did not influence radiosensitivity of EL-4 cells. Thus our results reveal that, tumor cells which are subjected to heightened oxidative stress employ master regulator cellular redox homeostasis Nrf-2 for prevention of radiation induced cell death. Our study reveals the molecular basis of tumor radioresistance and highlights role of Nrf-2 and ERK.

Tetrandrine, an Activator of Autophagy, Induces Autophagic Cell Death via PKC-α Inhibition and mTOR-Dependent Mechanisms

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Vincent Kam Wai Wong

2017-06-01

Full Text Available Emerging evidence suggests the therapeutic role of autophagic modulators in cancer therapy. This study aims to identify novel traditional Chinese medicinal herbs as potential anti-tumor agents through autophagic induction, which finally lead to autophagy mediated-cell death in apoptosis-resistant cancer cells. Using bioactivity-guided purification, we identified tetrandrine (Tet from herbal plant, Radix stephaniae tetrandrae, as an inducer of autophagy. Across a number of cancer cell lines, we found that breast cancer cells treated with tetrandrine show an increase autophagic flux and formation of autophagosomes. In addition, tetrandrine induces cell death in a panel of apoptosis-resistant cell lines that are deficient for caspase 3, caspase 7, caspase 3 and 7, or Bax-Bak respectively. We also showed that tetrandrine-induced cell death is independent of necrotic cell death. Mechanistically, tetrandrine induces autophagy that depends on mTOR inactivation. Furthermore, tetrandrine induces autophagy in a calcium/calmodulin-dependent protein kinase kinase-β (CaMKK-β, 5′ AMP-activated protein kinase (AMPK independent manner. Finally, by kinase profiling against 300 WT kinases and computational molecular docking analysis, we showed that tetrandrine is a novel PKC-α inhibitor, which lead to autophagic induction through PKC-α inactivation. This study provides detailed insights into the novel cytotoxic mechanism of an anti-tumor compound originated from the herbal plant, which may be useful in promoting autophagy mediated- cell death in cancer cell that is resistant to apoptosis.

Caffeine Induces Cell Death via Activation of Apoptotic Signal and Inactivation of Survival Signal in Human Osteoblasts

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Wen-Hsiung Chan

2008-05-01

Full Text Available Caffeine consumption is a risk factor for osteoporosis, but the precise regulatory mechanisms are currently unknown. Here, we show that cell viability decreases in osteoblasts treated with caffeine in a dose-dependent manner. This cell death is attributed primarily to apoptosis and to a smaller extent, necrosis. Moreover, caffeine directly stimulates intracellular oxidative stress. Our data support caffeine-induced apoptosis in osteoblasts via a mitochondria-dependent pathway. The apoptotic biochemical changes were effectively prevented upon pretreatment with ROS scavengers, indicating that ROS plays a critical role as an upstream controller in the caffeine-induced apoptotic cascade. Additionally, p21-activated protein kinase 2 (PAK2 and c-Jun N-terminal kinase (JNK were activated in caffeine-treated osteoblasts. Experiments further found that PAK2 activity is required for caffeine-induced JNK activation and apoptosis. Importantly, our data also show that caffeine triggers cell death via inactivation of the survival signal, including the ERK- and Akt-mediated anti-apoptotic pathways. Finally, exposure of rats to dietary water containing 10~20 μM caffeine led to bone mineral density loss. These results demonstrate for the first time that caffeine triggers apoptosis in osteoblasts via activation of mitochondria-dependent cell death signaling and inactivation of the survival signal, and causes bone mineral density loss in vivo.

Mechanisms of Betulinic acid‐induced cell death

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Potze, L.

2015-01-01

The scope of this thesis was to investigate the mechanisms by which BetA induces cell death in cancer cells in more detail. At the start of the studies described in this thesis several questions urgently needed an answer. Although BetA induces cell death via apoptosis, when blocking this form of

Caspase-independent cell death mediated by apoptosis-inducing factor (AIF) nuclear translocation is involved in ionizing radiation induced HepG2 cell death

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Sun, Hengwen [Department of Radiation, Cancer Center of Guangdong General Hospital (Guangdong Academy of Medical Science), Guangzhou, 510080, Guangdong (China); Yang, Shana; Li, Jianhua [Department of Physiology, Guangzhou Medical University, Guangzhou, 510182, Guangdong (China); Zhang, Yajie [Department of Pathology, Guangzhou Medical University, Guangzhou, 510182, Guangdong (China); Gao, Dongsheng [Department of Oncology, Guangdong Medical College Affiliated Pengpai Memorial Hospital, Hai Feng, 516400, Gungdong (China); Zhao, Shenting, E-mail: zhaoshenting@126.com [Department of Physiology, Guangzhou Medical University, Guangzhou, 510182, Guangdong (China)

2016-03-25

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. The aim of radiotherapy is to eradicate cancer cells with ionizing radiation. Except for the caspase-dependent mechanism, several lines of evidence demonstrated that caspase-independent mechanism is directly involved in the cell death responding to irradiation. For this reason, defining the contribution of caspase-independent molecular mechanisms represents the main goal in radiotherapy. In this study, we focused on the role of apoptosis-inducing factor (AIF), the caspase-independent molecular, in ionizing radiation induced hepatocellular carcinoma cell line (HepG2) cell death. We found that ionizing radiation has no function on AIF expression in HepG2 cells, but could induce AIF release from the mitochondria and translocate into nuclei. Inhibition of AIF could reduce ionizing radiation induced HepG2 cell death. These studies strongly support a direct relationship between AIF nuclear translocation and radiation induced cell death. What's more, AIF nuclear translocation is caspase-independent manner, but not caspase-dependent manner, in this process. These new findings add a further attractive point of investigation to better define the complex interplay between caspase-independent cell death and radiation therapy. - Highlights: • AIF nuclear translocation is involved in ionizing radiation induced hepatocellular carcinoma cell line HepG2 cell death. • AIF mediated cell death induced by ionizing radiation is caspase-independent. • Caspase-independent pathway is involved in ionzing radiation induced HepG2 cell death.

Caspase-independent cell death mediated by apoptosis-inducing factor (AIF) nuclear translocation is involved in ionizing radiation induced HepG2 cell death

International Nuclear Information System (INIS)

Sun, Hengwen; Yang, Shana; Li, Jianhua; Zhang, Yajie; Gao, Dongsheng; Zhao, Shenting

2016-01-01

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. The aim of radiotherapy is to eradicate cancer cells with ionizing radiation. Except for the caspase-dependent mechanism, several lines of evidence demonstrated that caspase-independent mechanism is directly involved in the cell death responding to irradiation. For this reason, defining the contribution of caspase-independent molecular mechanisms represents the main goal in radiotherapy. In this study, we focused on the role of apoptosis-inducing factor (AIF), the caspase-independent molecular, in ionizing radiation induced hepatocellular carcinoma cell line (HepG2) cell death. We found that ionizing radiation has no function on AIF expression in HepG2 cells, but could induce AIF release from the mitochondria and translocate into nuclei. Inhibition of AIF could reduce ionizing radiation induced HepG2 cell death. These studies strongly support a direct relationship between AIF nuclear translocation and radiation induced cell death. What's more, AIF nuclear translocation is caspase-independent manner, but not caspase-dependent manner, in this process. These new findings add a further attractive point of investigation to better define the complex interplay between caspase-independent cell death and radiation therapy. - Highlights: • AIF nuclear translocation is involved in ionizing radiation induced hepatocellular carcinoma cell line HepG2 cell death. • AIF mediated cell death induced by ionizing radiation is caspase-independent. • Caspase-independent pathway is involved in ionzing radiation induced HepG2 cell death.

Tumor-specific CD4+ T cells develop cytotoxic activity and eliminate virus-induced tumor cells in the absence of regulatory T cells.

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Akhmetzyanova, Ilseyar; Zelinskyy, Gennadiy; Schimmer, Simone; Brandau, Sven; Altenhoff, Petra; Sparwasser, Tim; Dittmer, Ulf

2013-02-01

The important role of tumor-specific cytotoxic CD8(+) T cells is well defined in the immune control of the tumors, but the role of effector CD4(+) T cells is poorly understood. In the current research, we have used a murine retrovirus-induced tumor cell line of C57BL/6 mouse origin, namely FBL-3 cells, as a model to study basic mechanisms of immunological control and escape during tumor formation. This study shows that tumor-specific CD4(+) T cells are able to protect against virus-induced tumor cells. We show here that there is an expansion of tumor-specific CD4(+) T cells producing cytokines and cytotoxic molecule granzyme B (GzmB) in the early phase of tumor growth. Importantly, we demonstrate that in vivo depletion of regulatory T cells (Tregs) and CD8(+) T cells in FBL-3-bearing DEREG transgenic mice augments IL-2 and GzmB production by CD4(+) T cells and increases FV-specific CD4(+) T-cell effector and cytotoxic responses leading to the complete tumor regression. Therefore, the capacity to reject tumor acquired by tumor-reactive CD4(+) T cells largely depends on the direct suppressive activity of Tregs. We suggest that a cytotoxic CD4(+) T-cell immune response may be induced to enhance resistance against oncovirus-associated tumors.

Ebola virus glycoprotein directly triggers T lymphocyte death despite of the lack of infection.

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Iampietro, Mathieu; Younan, Patrick; Nishida, Andrew; Dutta, Mukta; Lubaki, Ndongala Michel; Santos, Rodrigo I; Koup, Richard A; Katze, Michael G; Bukreyev, Alexander

2017-05-01

Fatal outcomes of Ebola virus (EBOV) infections are typically preceded by a 'sepsis-like' syndrome and lymphopenia despite T cells being resistant to Ebola infection. The mechanisms that lead to T lymphocytes death remain largely unknown; however, the degree of lymphopenia is highly correlative with fatalities. Here we investigated whether the addition of EBOV or its envelope glycoprotein (GP) to isolated primary human CD4+ T cells induced cell death. We observed a significant decrease in cell viability in a GP-dependent manner, which is suggestive of a direct role of GP in T cell death. Using immunoprecipitation assays and flow cytometry, we demonstrate that EBOV directly binds to CD4+ T cells through interaction of GP with TLR4. Transcriptome analysis revealed that the addition of EBOV to CD4+ T cells results in the significant upregulation of pathways associated with interferon signaling, pattern recognition receptors and intracellular activation of NFκB signaling pathway. Both transcriptome analysis and specific inhibitors allowed identification of apoptosis and necrosis as mechanisms associated with the observed T cell death following exposure to EBOV. The addition of the TLR4 inhibitor CLI-095 significantly reduced CD4+ T cell death induced by GP. EBOV stimulation of primary CD4+ T cells resulted in a significant increase in secreted TNFα; inhibition of TNFα-mediated signaling events significantly reduced T cell death while inhibitors of both necrosis and apoptosis similarly reduced EBOV-induced T cell death. Lastly, we show that stimulation with EBOV or GP augments monocyte maturation as determined by an overall increase in expression levels of markers of differentiation. Subsequently, the increased rates of cellular differentiation resulted in higher rates of infection further contributing to T cell death. These results demonstrate that GP directly subverts the host's immune response by increasing the susceptibility of monocytes to EBOV infection and

Ursodeoxycholic Acid Induces Death Receptor-mediated Apoptosis in Prostate Cancer Cells

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Lee, Won Sup; Jung, Ji Hyun; Panchanathan, Radha; Yun, Jeong Won; Kim, Dong Hoon; Kim, Hye Jung; Kim, Gon Sup; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan; Choi, Yung Hyun; Jung, Jin-Myung

2017-01-01

Background Bile acids have anti-cancer properties in a certain types of cancers. We determined anticancer activity and its underlying molecular mechanism of ursodeoxycholic acid (UDCA) in human DU145 prostate cancer cells. Methods Cell viability was measured with an MTT assay. UDCA-induced apoptosis was determined with flow cytometric analysis. The expression levels of apoptosis-related signaling proteins were examined with Western blotting. Results UDCA treatment significantly inhibited cell growth of DU145 in a dose-dependent manner. It induced cellular shrinkage and cytoplasmic blebs and accumulated the cells with sub-G1 DNA contents. Moreover, UDCA activated caspase 8, suggesting that UDCA-induced apoptosis is associated with extrinsic pathway. Consistent to this finding, UDCA increased the expressions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, death receptor 4 (DR4) and death receptor 5 (DR5), and TRAIL augmented the UDCA-induced cell death in DU145 cells. In addition, UDCA also increased the expressions of Bax and cytochrome c and decreased the expression of Bcl-xL in DU145 cells. This finding suggests that UDCA-induced apoptosis may be involved in intrinsic pathway. Conclusions UDCA induces apoptosis via extrinsic pathway as well as intrinsic pathway in DU145 prostate cancer cells. UDCA may be a promising anti-cancer agent against prostate cancer. PMID:28382282

Apoptotic Cell Death Induced by Resveratrol Is Partially Mediated by the Autophagy Pathway in Human Ovarian Cancer Cells.

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Fangfang Lang

Full Text Available Resveratrol (trans-3,4,5'-trihydroxystilbene is an active compound in food, such as red grapes, peanuts, and berries. Resveratrol exhibits an anticancer effect on various human cancer cells. However, the mechanism of resveratrol-induced anti-cancer effect at the molecular level remains to be elucidated. In this study, the mechanism underlying the anti-cancer effect of resveratrol in human ovarian cancer cells (OVCAR-3 and Caov-3 was investigated using various molecular biology techniques, such as flow cytometry, western blotting, and RNA interference, with a major focus on the potential role of autophagy in resveratrol-induced apoptotic cell death. We demonstrated that resveratrol induced reactive oxygen species (ROS generation, which triggers autophagy and subsequent apoptotic cell death. Resveratrol induced ATG5 expression and promoted LC3 cleavage. The apoptotic cell death induced by resveratrol was attenuated by both pharmacological and genetic inhibition of autophagy. The autophagy inhibitor chloroquine, which functions at the late stage of autophagy, significantly reduced resveratrol-induced cell death and caspase 3 activity in human ovarian cancer cells. We also demonstrated that targeting ATG5 by siRNA also suppressed resveratrol-induced apoptotic cell death. Thus, we concluded that a common pathway between autophagy and apoptosis exists in resveratrol-induced cell death in OVCAR-3 human ovarian cancer cells.

N-acetylcysteine attenuates hexavalent chromium-induced hypersensitivity through inhibition of cell death, ROS-related signaling and cytokine expression.

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Yu-Hsuan Lee

Full Text Available Chromium hypersensitivity (chromium-induced allergic contact dermatitis is an important issue in occupational skin disease. Hexavalent chromium (Cr (VI can activate the Akt, Nuclear factor κB (NF-κB, and Mitogen-activated protein kinase (MAPK pathways and induce cell death, via the effects of reactive oxygen species (ROS. Recently, cell death stimuli have been proposed to regulate the release of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α and interleukin-1 (IL-1. However, the exact effects of ROS on the signaling molecules and cytotoxicity involved in Cr(VI-induced hypersensitivity have not yet been fully demonstrated. N-acetylcysteine (NAC could increase glutathione levels in the skin and act as an antioxidant. In this study, we investigated the effects of NAC on attenuating the Cr(VI-triggered ROS signaling in both normal keratinocyte cells (HaCaT cells and a guinea pig (GP model. The results showed the induction of apoptosis, autophagy and ROS were observed after different concentrations of Cr(VI treatment. HaCaT cells pretreated with NAC exhibited a decrease in apoptosis and autophagy, which could affect cell viability. In addition, Cr (VI activated the Akt, NF-κB and MAPK pathways thereby increasing IL-1α and TNF-α production. However, all of these stimulation phenomena could be inhibited by NAC in both of in vitro and in vivo studies. These novel findings indicate that NAC may prevent the development of chromium hypersensitivity by inhibiting of ROS-induced cell death and cytokine expression.

Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis.

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Loor, Gabriel; Kondapalli, Jyothisri; Schriewer, Jacqueline M; Chandel, Navdeep S; Vanden Hoek, Terry L; Schumacker, Paul T

2010-12-15

Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, whereas other studies implicate the activation of the mitochondrial permeability transition pore as the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, whereas it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetylcysteine and exogenous glutathione or by overexpression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells overexpressing Cu,Zn-SOD or Mn-SOD. Overexpression of antiapoptotic Bcl-X(L) protected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D, or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochrome c, Bax/Bak, caspase-9, and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them. Copyright © 2010 Elsevier Inc. All rights reserved.

NF-κB Protects NKT Cells from Tumor Necrosis Factor Receptor 1-induced Death.

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Kumar, Amrendra; Gordy, Laura E; Bezbradica, Jelena S; Stanic, Aleksandar K; Hill, Timothy M; Boothby, Mark R; Van Kaer, Luc; Joyce, Sebastian

2017-11-15

Semi-invariant natural killer T (NKT) cells are innate-like lymphocytes with immunoregulatory properties. NKT cell survival during development requires signal processing by activated RelA/NF-κB. Nonetheless, the upstream signal(s) integrated by NF-κB in developing NKT cells remains incompletely defined. We show that the introgression of Bcl-x L -coding Bcl2l1 transgene into NF-κB signalling-deficient IκBΔN transgenic mouse rescues NKT cell development and differentiation in this mouse model. We reasoned that NF-κB activation was protecting developing NKT cells from death signals emanating either from high affinity agonist recognition by the T cell receptor (TCR) or from a death receptor, such as tumor necrosis factor receptor 1 (TNFR1) or Fas. Surprisingly, the single and combined deficiency in PKC-θ or CARMA-1-the two signal transducers at the NKT TCR proximal signalling node-only partially recapitulated the NKT cell deficiency observed in IκBΔN tg mouse. Accordingly, introgression of the Bcl2l1 transgene into PKC-θ null mouse failed to rescue NKT cell development. Instead, TNFR1-deficiency, but not the Fas-deficiency, rescued NKT cell development in IκBΔN tg mice. Consistent with this finding, treatment of thymocytes with an antagonist of the inhibitor of κB kinase -which blocks downstream NF-κB activation- sensitized NKT cells to TNF-α-induced cell death in vitro. Hence, we conclude that signal integration by NF-κB protects developing NKT cells from death signals emanating from TNFR1, but not from the NKT TCR or Fas.

Death Receptor-Induced Apoptosis Signalling Regulation by Ezrin Is Cell Type Dependent and Occurs in a DISC-Independent Manner in Colon Cancer Cells

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Iessi, Elisabetta; Zischler, Luciana; Etringer, Aurélie; Bergeret, Marion; Morlé, Aymeric; Jacquemin, Guillaume; Morizot, Alexandre; Shirley, Sarah; Lalaoui, Najoua; Elifio-Esposito, Selene L.; Fais, Stefano; Garrido, Carmen; Solary, Eric; Micheau, Olivier

2015-01-01

Ezrin belongs to the ERM (ezrin-radixin-moesin) protein family and has been demonstrated to regulate early steps of Fas receptor signalling in lymphoid cells, but its contribution to TRAIL-induced cell death regulation in adherent cancer cells remains unknown. In this study we report that regulation of FasL and TRAIL-induced cell death by ezrin is cell type dependant. Ezrin is a positive regulator of apoptosis in T-lymphoma cell line Jurkat, but a negative regulator in colon cancer cells. Using ezrin phosphorylation or actin-binding mutants, we provide evidence that negative regulation of death receptor-induced apoptosis by ezrin occurs in a cytoskeleton- and DISC-independent manner, in colon cancer cells. Remarkably, inhibition of apoptosis induced by these ligands was found to be tightly associated with regulation of ezrin phosphorylation on serine 66, the tumor suppressor gene WWOX and activation of PKA. Deficiency in WWOX expression in the liver cancer SK-HEP1 or the pancreatic Mia PaCa-2 cell lines as well as WWOX silencing or modulation of PKA activation by pharmacological regulators, in the colon cancer cell line SW480, abrogated regulation of TRAIL signalling by ezrin. Altogether our results show that death receptor pro-apoptotic signalling regulation by ezrin can occur downstream of the DISC in colon cancer cells. PMID:26010871

Death Receptor-Induced Apoptosis Signalling Regulation by Ezrin Is Cell Type Dependent and Occurs in a DISC-Independent Manner in Colon Cancer Cells.

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Elisabetta Iessi

Full Text Available Ezrin belongs to the ERM (ezrin-radixin-moesin protein family and has been demonstrated to regulate early steps of Fas receptor signalling in lymphoid cells, but its contribution to TRAIL-induced cell death regulation in adherent cancer cells remains unknown. In this study we report that regulation of FasL and TRAIL-induced cell death by ezrin is cell type dependant. Ezrin is a positive regulator of apoptosis in T-lymphoma cell line Jurkat, but a negative regulator in colon cancer cells. Using ezrin phosphorylation or actin-binding mutants, we provide evidence that negative regulation of death receptor-induced apoptosis by ezrin occurs in a cytoskeleton- and DISC-independent manner, in colon cancer cells. Remarkably, inhibition of apoptosis induced by these ligands was found to be tightly associated with regulation of ezrin phosphorylation on serine 66, the tumor suppressor gene WWOX and activation of PKA. Deficiency in WWOX expression in the liver cancer SK-HEP1 or the pancreatic Mia PaCa-2 cell lines as well as WWOX silencing or modulation of PKA activation by pharmacological regulators, in the colon cancer cell line SW480, abrogated regulation of TRAIL signalling by ezrin. Altogether our results show that death receptor pro-apoptotic signalling regulation by ezrin can occur downstream of the DISC in colon cancer cells.

Virus-induced polyclonal T cell activation is followed by apoptosis: partitioning of CD8+ T cells based on alpha 4 integrin expression

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Christensen, Jan Pravsgaard; Röpke, C; Thomsen, Allan Randrup

1996-01-01

that LCMV-induced activation of T cells is followed by apoptosis of many of the activated cells. Those CD8+VLA-4hi cells which do persist in LCMV immune mice are more sensitive to treatment with the cell-cycle-specific drug hydroxyurea than are phenotypically naive T cells. Our results therefore indicate...

Eclalbasaponin II induces autophagic and apoptotic cell death in human ovarian cancer cells

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Yoon Jin Cho

2016-09-01

Full Text Available Triterpenoids echinocystic acid and its glycosides, isolated from several Eclipta prostrata, have been reported to possess various biological activities such as anti-inflammatory, anti-bacterial, and anti-diabetic activity. However, the cytotoxicity of the triterpenoids in human cancer cells and their molecular mechanism of action are poorly understood. In the present study, we found that eclalbasaponin II with one glucose moiety has potent cytotoxicity in three ovarian cancer cells and two endometrial cancer cells compared to an aglycone echinocystic acid and eclalbasaponin I with two glucose moiety. Eclalbasaponin II treatment dose-dependently increased sub G1 population. Annexin V staining revealed that eclalbasaponin II induced apoptosis in SKOV3 and A2780 ovarian cancer cells. In addition, eclalbasaponin II-induced cell death was associated with characteristics of autophagy; an increase in acidic vesicular organelle content and elevation of the levels of LC3-II. Interestingly, autophagy inhibitor BaF1 suppressed the eclalbasaponin II-induced apoptosis. Moreover, eclalbasaponin II activated JNK and p38 signaling and inhibited the mTOR signaling. We further demonstrated that pre-treatment with a JNK and p38 inhibitor and mTOR activator attenuated the eclalbasaponin II-induced autophagy. This suggests that eclalbasaponin II induces apoptotic and autophagic cell death through the regulation of JNK, p38, and mTOR signaling in human ovarian cancer cells.

Staurosporine induces different cell death forms in cultured rat astrocytes

International Nuclear Information System (INIS)

Simenc, Janez; Lipnik-Stangelj, Metoda

2012-01-01

Astroglial cells are frequently involved in malignant transformation. Besides apoptosis, necroptosis, a different form of regulated cell death, seems to be related with glioblastoma genesis, proliferation, angiogenesis and invasion. In the present work we elucidated mechanisms of necroptosis in cultured astrocytes, and compared them with apoptosis, caused by staurosporine. Cultured rat cortical astrocytes were used for a cell death studies. Cell death was induced by different concentrations of staurosporine, and modified by inhibitors of apoptosis (z-vad-fmk) and necroptosis (nec-1). Different forms of a cell death were detected using flow cytometry. We showed that staurosporine, depending on concentration, induces both, apoptosis as well as necroptosis. Treatment with 10 −7 M staurosporine increased apoptosis of astrocytes after the regeneration in a staurosporine free medium. When caspases were inhibited, apoptosis was attenuated, while necroptosis was slightly increased. Treatment with 10 −6 M staurosporine induced necroptosis that occurred after the regeneration of astrocytes in a staurosporine free medium, as well as without regeneration period. Necroptosis was significantly attenuated by nec-1 which inhibits RIP1 kinase. On the other hand, the inhibition of caspases had no effect on necroptosis. Furthermore, staurosporine activated RIP1 kinase increased the production of reactive oxygen species, while an antioxidant BHA significantly attenuated necroptosis. Staurosporine can induce apoptosis and/or necroptosis in cultured astrocytes via different signalling pathways. Distinction between different forms of cell death is crucial in the studies of therapy-induced necroptosis

Cytotoxic Vibrio T3SS1 Rewires Host Gene Expression to Subvert Cell Death Signaling and Activate Cell Survival Networks

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De Nisco, Nicole J.; Kanchwala, Mohammed; Li, Peng; Fernandez, Jessie; Xing, Chao; Orth, Kim

2017-01-01

Bacterial effectors are potent manipulators of host signaling pathways. The marine bacterium Vibrio parahaemolyticus (V. para), delivers effectors into host cells through two type three secretion systems (T3SS). The ubiquitous T3SS1 is vital for V. para survival in the environment, whereas T3SS2 causes acute gastroenteritis in human hosts. Although the natural host is undefined, T3SS1 effectors attack highly conserved cellular processes and pathways to orchestrate non-apoptotic cell death. Much is known about how T3SS1 effectors function in isolation, but we wanted to understand how their concerted action globally affects host cell signaling. To assess the host response to T3SS1, we compared gene expression changes over time in primary fibroblasts infected with V. para that have a functional T3SS1 (T3SS1+) to those in cells infected with V. para lacking T3SS1 (T3SS1−). Overall, the host transcriptional response to both T3SS1+ and T3SS1− V. para was rapid, robust, and temporally dynamic. T3SS1 re-wired host gene expression by specifically altering the expression of 398 genes. Although T3SS1 effectors target host cells at the posttranslational level to cause cytotoxicity, network analysis indicated that V. para T3SS1 also precipitates a host transcriptional response that initially activates cell survival and represses cell death networks. The increased expression of several key pro-survival transcripts mediated by T3SS1 was dependent on a host signaling pathway that is silenced later in infection by the posttranslational action of T3SS1. Taken together, our analysis reveals a complex interplay between roles of T3SS1 as both a transcriptional and posttranslational manipulator of host cell signaling. PMID:28512145

Lytic cell death induced by melittin bypasses pyroptosis but induces NLRP3 inflammasome activation and IL-1β release.

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Martín-Sánchez, Fátima; Martínez-García, Juan José; Muñoz-García, María; Martínez-Villanueva, Miriam; Noguera-Velasco, José A; Andreu, David; Rivas, Luís; Pelegrín, Pablo

2017-08-10

The nucleotide-binding domain and leucine-rich repeat-containing receptor with a pyrin domain 3 (NLRP3) inflammasome is a sensor for different types of infections and alterations of homeostatic parameters, including abnormally high levels of the extracellular nucleotide ATP or crystallization of different metabolites. All NLRP3 activators trigger a similar intracellular pathway, where a decrease in intracellular K + concentration and permeabilization of plasma membrane are key steps. Cationic amphipathic antimicrobial peptides and peptide toxins permeabilize the plasma membrane. In fact, some of them have been described to activate the NLRP3 inflammasome. Among them, the bee venom antimicrobial toxin peptide melittin is known to elicit an inflammatory reaction via the NLRP3 inflammasome in response to bee venom. Our study found that melittin induces canonical NLRP3 inflammasome activation by plasma membrane permeabilization and a reduction in the intracellular K + concentration. Following melittin treatment, the apoptosis-associated speck-like protein, an adaptor protein with a caspase recruitment domain (ASC), was necessary to activate caspase-1 and induce IL-1β release. However, cell death induced by melittin prevented the formation of large ASC aggregates, amplification of caspase-1 activation, IL-18 release and execution of pyroptosis. Therefore, melittin-induced activation of the NLRP3 inflammasome results in an attenuated inflammasome response that does not result in caspase-1 dependent cell death.

An extensive microarray analysis of AAL-toxin-induced cell death in Arabidopsis thaliana brings new insights into the complexity of programmed cell death in plants

NARCIS (Netherlands)

Gechev, T.S.; Gadjev, I.Z.; Hille, J.

2004-01-01

A T-DNA knockout of the Arabidopsis homologue of the tomato disease resistance gene Asc was obtained. The asc gene renders plants sensitive to programmed cell death (PCD) triggered by the fungal AAL toxin. To obtain more insights into the nature of AAL-toxin-induced cell death and to identify genes

Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

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Lee, Dae-Hee, E-mail: leedneo@gmail.com [Departments of Surgery and Pharmacology and Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA (United States); Kim, Dong-Wook [Department of Microbiology, Immunology, and Cancer Biology, University of VA (United States); Jung, Chang-Hwa [Division of Metabolism and Functionality Research, Korea Food Research Institute (Korea, Republic of); Lee, Yong J. [Departments of Surgery and Pharmacology and Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA (United States); Park, Daeho, E-mail: daehopark@gist.ac.kr [School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712 (Korea, Republic of)

2014-09-15

Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. - Highlights: • Most GBM cells have been reported to be resistant to TRAIL-induced apoptosis. • Gingerol enhances the expression level of anti-apoptotic proteins by ROS. • Gingerol enhances TRAIL-induced apoptosis through actions on the ROS–Bcl2 pathway.

Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

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Lee, Dae-Hee; Kim, Dong-Wook; Jung, Chang-Hwa; Lee, Yong J.; Park, Daeho

2014-01-01

Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. - Highlights: • Most GBM cells have been reported to be resistant to TRAIL-induced apoptosis. • Gingerol enhances the expression level of anti-apoptotic proteins by ROS. • Gingerol enhances TRAIL-induced apoptosis through actions on the ROS–Bcl2 pathway

Alterations in protein kinase C activity and processing during zinc-deficiency-induced cell death.

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Chou, Susan S; Clegg, Michael S; Momma, Tony Y; Niles, Brad J; Duffy, Jodie Y; Daston, George P; Keen, Carl L

2004-10-01

Protein kinases C (PKCs) are a family of serine/threonine kinases that are critical for signal transduction pathways involved in growth, differentiation and cell death. All PKC isoforms have four conserved domains, C1-C4. The C1 domain contains cysteine-rich finger-like motifs, which bind two zinc atoms. The zinc-finger motifs modulate diacylglycerol binding; thus, intracellular zinc concentrations could influence the activity and localization of PKC family members. 3T3 cells were cultured in zinc-deficient or zinc-supplemented medium for up to 32 h. Cells cultured in zinc-deficient medium had decreased zinc content, lowered cytosolic classical PKC activity, increased caspase-3 processing and activity, and reduced cell number. Zinc-deficient cytosols had decreased activity and expression levels of PKC-alpha, whereas PKC-alpha phosphorylation was not altered. Inhibition of PKC-alpha with Gö6976 had no effect on cell number in the zinc-deficient group. Proteolysis of the novel PKC family member, PKC-delta, to its 40-kDa catalytic fragment occurred in cells cultured in the zinc-deficient medium. Occurrence of the PKC-delta fragment in mitochondria was co-incident with caspase-3 activation. Addition of the PKC-delta inhibitor, rottlerin, or zinc to deficient medium reduced or eliminated proteolysis of PKC-delta, activated caspase-3 and restored cell number. Inhibition of caspase-3 processing by Z-DQMD-FMK (Z-Asp-Gln-Met-Asp-fluoromethylketone) did not restore cell number in the zinc-deficient group, but resulted in processing of full-length PKC-delta to a 56-kDa fragment. These results support the concept that intracellular zinc concentrations influence PKC activity and processing, and that zinc-deficiency-induced apoptosis occurs in part through PKC-dependent pathways.

Mechanisms of cell death induced by infusion sets leachables in in vitro experimental settings.

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Kozlovskaya, Luba; Stepensky, David

2015-01-30

Leachable materials that are released from infusion sets during their use can induce local and systemic toxic effects. We studied the mechanisms and kinetics of cell death induced by infusion sets leachates in vitro using L-929 and bEnd. 3 cells. Changes in cell morphology and metabolic activity were determined using light microscopy and the MTT test, respectively. Detailed analysis of the mechanisms of cell death was performed using membrane integrity and caspases 3 and 7 activity tests, annexin V-FITC/7-AAD analysis by FACS, and DAPI nuclear staining followed by confocal microscopy. Infusion sets released toxic leachables and induced toxic effects. Latex flashball was the most toxic part of the studied infusion sets, and it potently induced cell oncosis via increased permeability of the cell membrane. Latex-induced decrease in cells metabolic activity and cell death were not accompanied by activation of caspases 3 and 7, changes in nuclear morphology, or substantial annexin V-FITC cell staining. Leachables from the tube part of the infusion sets were less toxic, and induced some biochemical changes without altering the cells morphology. Further studies are needed to reveal the in vivo toxicity of infusion sets and its correlation with the results of in vitro toxicity studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Collagen gel protects L929 cells from TNFα-induced death by activating NF-κB.

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Wang, Hong-Ju; Li, Meng-Qi; Liu, Wei-Wei; Hayashi, Toshihiko; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2017-09-01

Type I collagen is one of the most abundant components of extracellular matrix. We previously illustrated that murine fibrosarcoma L929 cells grew well on type I collagen gel and escaped from TNFα-induced cell death. In this study, we investigated the mechanism underlying the protective effect of collagen gel. We used western blot, confocal microscopy, MTT assay and flow cytometry by introducing fluorescence staining to determine the expression levels of nuclear factor kappa B (NF-κB), inhibitory ratio and autophagy. L929 cells on collagen gel showed higher expression of NF-κB in the nucleus. Inhibition of NF-κB with pyrrolidine dithiocarbamate hydrochloride (PDTC) or knockdown by NF-κB-siRNA canceled the protective effect of collagen gel on L929 cells from TNFα-induced death, suggesting for the role of NF-κB in the protection from cell death. We found a new aspect of the effect of PDTC on L929 cells cultured on collagen gel. PDTC alone without TNFα induced apoptosis in the L929 cells cultured on collagen gel but not the cells on plastic dish. The apoptosis induction of the L929 cells cultured on collagen gel with PDTC was repressed by inhibiting autophagy with chloroquine, an autophagy inhibitor, suggesting that autophagy contributes to the death induced by the treatment with PDTC. Possible underlying mechanism of this finding is discussed. NF-κB played an important role in protecting the L929 cells cultured on collagen gel from TNFα-induced death.

Sorafenib-induced defective autophagy promotes cell death by necroptosis.

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Kharaziha, Pedram; Chioureas, Dimitris; Baltatzis, George; Fonseca, Pedro; Rodriguez, Patricia; Gogvadze, Vladimir; Lennartsson, Lena; Björklund, Ann-Charlotte; Zhivotovsky, Boris; Grandér, Dan; Egevad, Lars; Nilsson, Sten; Panaretakis, Theocharis

2015-11-10

Autophagy is one of the main cytoprotective mechanisms that cancer cells deploy to withstand the cytotoxic stress and survive the lethal damage induced by anti-cancer drugs. However, under specific conditions, autophagy may, directly or indirectly, induce cell death. In our study, treatment of the Atg5-deficient DU145 prostate cancer cells, with the multi-tyrosine kinase inhibitor, sorafenib, induces mitochondrial damage, autophagy and cell death. Molecular inhibition of autophagy by silencing ULK1 and Beclin1 rescues DU145 cells from cell death indicating that, in this setting, autophagy promotes cell death. Re-expression of Atg5 restores the lipidation of LC3 and rescues DU145 and MEF atg5-/- cells from sorafenib-induced cell death. Despite the lack of Atg5 expression and LC3 lipidation, DU145 cells form autophagosomes as demonstrated by transmission and immuno-electron microscopy, and the formation of LC3 positive foci. However, the lack of cellular content in the autophagosomes, the accumulation of long-lived proteins, the presence of GFP-RFP-LC3 positive foci and the accumulated p62 protein levels indicate that these autophagosomes may not be fully functional. DU145 cells treated with sorafenib undergo a caspase-independent cell death that is inhibited by the RIPK1 inhibitor, necrostatin-1. Furthermore, treatment with sorafenib induces the interaction of RIPK1 with p62, as demonstrated by immunoprecipitation and a proximity ligation assay. Silencing of p62 decreases the RIPK1 protein levels and renders necrostatin-1 ineffective in blocking sorafenib-induced cell death. In summary, the formation of Atg5-deficient autophagosomes in response to sorafenib promotes the interaction of p62 with RIPK leading to cell death by necroptosis.

Role of non-canonical Beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells.

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Scarlatti, F; Maffei, R; Beau, I; Codogno, P; Ghidoni, R

2008-08-01

Resveratrol, a polyphenol found in grapes and other fruit and vegetables, is a powerful chemopreventive and chemotherapeutic molecule potentially of interest for the treatment of breast cancer. The human breast cancer cell line MCF-7, which is devoid of caspase-3 activity, is refractory to apoptotic cell death after incubation with resveratrol. Here we show that resveratrol arrests cell proliferation, triggers death and decreases the number of colonies of cells that are sensitive to caspase-3-dependent apoptosis (MCF-7 casp-3) and also those that are unresponsive to it (MCF-7vc). We demonstrate that resveratrol (i) acts via multiple pathways to trigger cell death, (ii) induces caspase-dependent and caspase-independent cell death in MCF-7 casp-3 cells, (iii) induces only caspase-independent cell death in MCF-7vc cells and (iv) stimulates macroautophagy. Using BECN1 and hVPS34 (human vacuolar protein sorting 34) small interfering RNAs, we demonstrate that resveratrol activates Beclin 1-independent autophagy in both cell lines, whereas cell death via this uncommon form of autophagy occurs only in MCF-7vc cells. We also show that this variant form of autophagic cell death is blocked by the expression of caspase-3, but not by its enzymatic activity. In conclusion, this study reveals that non-canonical autophagy induced by resveratrol can act as a caspase-independent cell death mechanism in breast cancer cells.

Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

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Pan, Mu-Yun; Shen, Yuh-Chiang; Lu, Chien-Hsing; Yang, Shu-Yi; Ho, Tsing-Fen; Peng, Yu-Ta; Chang, Chia-Che

2012-01-01

Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming prodigiosin as an ER stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered prodigiosin's capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in prodigiosin-induced cytotoxic ER stress response. In addition, prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked prodigiosin-induced PARP cleavage and greatly enhanced the survival of prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2α phosphorylation impaired prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of prodigiosin's tumoricidal effect. Mechanistically, prodigiosin engages the IRE1–JNK and PERK–eIF2α branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death. Highlights: ► Prodigiosin is a bacterial tripyrrole pigment with potent anticancer effect. ► Prodigiosin is herein identified as an

Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

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Pan, Mu-Yun [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Shen, Yuh-Chiang [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); National Research Institute of Chinese Medicine, Taipei, Taiwan (China); Lu, Chien-Hsing [Department of Obstetrics and Gynecology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Obstetrics and Gynecology, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Yang, Shu-Yi [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Ho, Tsing-Fen [Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan (China); Peng, Yu-Ta [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Chang, Chia-Che, E-mail: chia_che@dragon.nchu.edu.tw [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China)

2012-12-15

Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming prodigiosin as an ER stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered prodigiosin's capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in prodigiosin-induced cytotoxic ER stress response. In addition, prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked prodigiosin-induced PARP cleavage and greatly enhanced the survival of prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2α phosphorylation impaired prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of prodigiosin's tumoricidal effect. Mechanistically, prodigiosin engages the IRE1–JNK and PERK–eIF2α branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death. Highlights: ► Prodigiosin is a bacterial tripyrrole pigment with potent anticancer effect. ► Prodigiosin is herein identified

Impaired Autophagy and Defective T Cell Homeostasis in Mice with T Cell-Specific Deletion of Receptor for Activated C Kinase 1

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Guihua Qiu

2017-05-01

Full Text Available Autophagy plays a central role in maintaining T cell homeostasis. Our previous study has shown that hepatocyte-specific deficiency of receptor for activated C kinase 1 (RACK1 leads to lipid accumulation in the liver, accompanied by impaired autophagy, but its in vivo role in T cells remains unclear. Here, we report that mice with T cell-specific deletion of RACK1 exhibit normal intrathymic development of conventional T cells and regulatory T (Treg cells but reduced numbers of peripheral CD4+ and CD8+ T cells. Such defects are cell intrinsic with impaired mitochondrial clearance, increased sensitivity to cell death, and decreased proliferation that could be explained by impaired autophagy. Furthermore, RACK1 is essential for invariant natural T cell development. In vivo, T cell-specific loss of RACK1 dampens concanavalin A-induced acute liver injury. Our data suggest that RACK1 is a key regulator of T cell homeostasis.

Granzyme B of cytotoxic T cells induces extramitochondrial reactive oxygen species production via caspase-dependent NADPH oxidase activation.

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Aguiló, Juan I; Anel, Alberto; Catalán, Elena; Sebastián, Alvaro; Acín-Pérez, Rebeca; Naval, Javier; Wallich, Reinhard; Simon, Markus M; Pardo, Julián

2010-07-01

Induction of reactive oxygen species (ROS) is a hallmark of granzyme B (gzmB)-mediated pro-apoptotic processes and target cell death. However, it is unclear to what extent the generated ROS derive from mitochondrial and/or extra-mitochondrial sources. To clarify this point, we have produced a mutant EL4 cell line, termed EL4-rho(0), which lacks mitochondrial DNA, associated with a decreased mitochondrial membrane potential and a defective ROS production through the electron transport chain of oxidative phosphorylation. When incubated with either recombinant gzmB plus streptolysin or ex vivo gzmB(+) cytotoxic T cells, EL4-rho(0) cells showed phosphatydylserine translocation, caspase 3 activation, Bak conformational change, cytochrome c release and apoptotic morphology comparable to EL4 cells. Moreover, EL4-rho(0) cells produced ROS at levels similar to EL4 under these conditions. GzmB-mediated ROS production was almost totally abolished in both cell lines by the pan-caspase inhibitor, Z-VAD-fmk. However, addition of apocynin, a specific inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, led to a significant reduction of ROS production and cell death only in EL4-rho(0) but not EL4 cells. These data suggest that gzmB-induced cell death is accompanied by a caspase-dependent pathway of extra-mitochondrial ROS production, most probably through activation of NADPH oxidase.

Apoptosis-Inducing-Factor-Dependent Mitochondrial Function Is Required for T Cell but Not B Cell Function.

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Milasta, Sandra; Dillon, Christopher P; Sturm, Oliver E; Verbist, Katherine C; Brewer, Taylor L; Quarato, Giovanni; Brown, Scott A; Frase, Sharon; Janke, Laura J; Perry, S Scott; Thomas, Paul G; Green, Douglas R

2016-01-19

The role of apoptosis inducing factor (AIF) in promoting cell death versus survival remains controversial. We report that the loss of AIF in fibroblasts led to mitochondrial electron transport chain defects and loss of proliferation that could be restored by ectopic expression of the yeast NADH dehydrogenase Ndi1. Aif-deficiency in T cells led to decreased peripheral T cell numbers and defective homeostatic proliferation, but thymic T cell development was unaffected. In contrast, Aif-deficient B cells developed and functioned normally. The difference in the dependency of T cells versus B cells on AIF for function and survival correlated with their metabolic requirements. Ectopic Ndi1 expression rescued homeostatic proliferation of Aif-deficient T cells. Despite its reported roles in cell death, fibroblasts, thymocytes and B cells lacking AIF underwent normal death. These studies suggest that the primary role of AIF relates to complex I function, with differential effects on T and B cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Blockage of NOX2/MAPK/NF-κB Pathway Protects Photoreceptors against Glucose Deprivation-Induced Cell Death

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Bin Fan

2017-01-01

Full Text Available Acute energy failure is one of the critical factors contributing to the pathogenic mechanisms of retinal ischemia. Our previous study demonstrated that glucose deprivation can lead to a caspase-dependent cell death of photoreceptors. The aim of this study was to decipher the upstream signal pathway in glucose deprivation- (GD- induced cell death. We mimicked acute energy failure by using glucose deprivation in photoreceptor cells (661W cells. GD-induced oxidative stress was evaluated by measuring ROS with the DCFH-DA assay and HO-1 expression by Western blot analysis. The activation of NOX2/MAPK/NF-κB signal was assessed by Western blot and immunohistochemical assays. The roles of these signals in GD-induced cell death were measured by using their specific inhibitors. Inhibition of Rac-1 and NOX2 suppressed GD-induced oxidative stress and protected photoreceptors against GD-induced cell death. NOX2 was an upstream signal in the caspase-dependent cell death cascade, yet the downstream MAPK pathways were activated and blocking MAPK signals rescued 661W cells from GD-induced death. In addition, GD caused the activation of NF-κB signal and inhibiting NF-κB significantly protected 661W cells. These observations may provide insights for treating retinal ischemic diseases and protecting retinal neurons from ischemia-induced cell death.

Protective effect of aqueous extract from Spirulina platensis against cell death induced by free radicals

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Radhakrishnan Ammu

2010-09-01

Full Text Available Abstract Background Spirulina is a commercial alga well known to contain various antioxidants, especially phycocyanin. Apart from being sold as a nutraceutical, Spirulina is incorporated as a functional ingredient in food products and beverages. Most of the previous reports on antioxidant activity of Spirulina were based on chemical rather than cell-based assays. The primary objective of this study was to assess the antioxidant activity of aqueous extract from Spirulina based on its protective effect against cell death induced by free radicals. Methods The antioxidant activity of the cold water extract from food-grade Spirulina platensis was assessed using both chemical and cell-based assays. In the cell-based assay, mouse fibroblast cells (3T3 cells were incubated for 1 h in medium containing aqueous extract of Spirulina or vitamin C (positive control at 25, 125 and 250 μg/mL before the addition of 50 μM 1,1-diphenyl-2-picrylhydrazyl (DPPH or 3-ethylbenzothiazoline-6-sulfonic acid (ABTS. The cells were incubated for another 24 h before being assessed for cell death due to apoptosis using the Cell Death Detection ELISA Kit. Spectrophotometric assays based on DPPH and ABTS were also used to assess the antioxidant activity of the extract compared to vitamin C and vitamin E (positive controls. Results Spirulina extract did not cause cytotoxic effect on 3T3 cells within the range of concentrations tested (0 - 250 μg/mL. The extract reduced significantly (p Conclusions The results showed that aqueous extract of Spirulina has a protective effect against apoptotic cell death due to free radicals. The potential application of incorporating Spirulina into food products and beverages to enhance their antioxidant capacity is worth exploring.

Autophagy protects against neural cell death induced by piperidine alkaloids present in Prosopis juliflora (Mesquite).

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Silva, Victor D A; Cuevas, Carlos; Muñoz, Patricia; Villa, Monica; Ahumada-Castro, Ulises; Huenchuguala, Sandro; Santos, Cleonice C Dos; Araujo, Fillipe M DE; Ferreira, Rafael S; Silva, Vanessa B DA; Silva, Juliana H C E; Soares, Érica N; Velozo, Eudes S; Segura-Aguilar, Juan; Costa, Silvia L

2017-01-01

Prosopis juliflora is a shrub that has been used to feed animals and humans. However, a synergistic action of piperidine alkaloids has been suggested to be responsible for neurotoxic damage observed in animals. We investigated the involvement of programmed cell death (PCD) and autophagy on the mechanism of cell death induced by a total extract (TAE) of alkaloids and fraction (F32) from P. juliflora leaves composed majoritary of juliprosopine in a model of neuron/glial cell co-culture. We saw that TAE (30 µg/mL) and F32 (7.5 µg/mL) induced reduction in ATP levels and changes in mitochondrial membrane potential at 12 h exposure. Moreover, TAE and F32 induced caspase-9 activation, nuclear condensation and neuronal death at 16 h exposure. After 4 h, they induced autophagy characterized by decreases of P62 protein level, increase of LC3II expression and increase in number of GFP-LC3 cells. Interestingly, we demonstrated that inhibition of autophagy by bafilomycin and vinblastine increased the cell death induced by TAE and autophagy induced by serum deprivation and rapamycin reduced cell death induced by F32 at 24 h. These results indicate that the mechanism neural cell death induced by these alkaloids involves PCD via caspase-9 activation and autophagy, which seems to be an important protective mechanism.

Autophagy protects against neural cell death induced by piperidine alkaloids present in Prosopis juliflora (Mesquite

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VICTOR D.A. SILVA

Full Text Available ABSTRACT Prosopis juliflora is a shrub that has been used to feed animals and humans. However, a synergistic action of piperidine alkaloids has been suggested to be responsible for neurotoxic damage observed in animals. We investigated the involvement of programmed cell death (PCD and autophagy on the mechanism of cell death induced by a total extract (TAE of alkaloids and fraction (F32 from P. juliflora leaves composed majoritary of juliprosopine in a model of neuron/glial cell co-culture. We saw that TAE (30 µg/mL and F32 (7.5 µg/mL induced reduction in ATP levels and changes in mitochondrial membrane potential at 12 h exposure. Moreover, TAE and F32 induced caspase-9 activation, nuclear condensation and neuronal death at 16 h exposure. After 4 h, they induced autophagy characterized by decreases of P62 protein level, increase of LC3II expression and increase in number of GFP-LC3 cells. Interestingly, we demonstrated that inhibition of autophagy by bafilomycin and vinblastine increased the cell death induced by TAE and autophagy induced by serum deprivation and rapamycin reduced cell death induced by F32 at 24 h. These results indicate that the mechanism neural cell death induced by these alkaloids involves PCD via caspase-9 activation and autophagy, which seems to be an important protective mechanism.

Ultra-violet B (UVB)-induced skin cell death occurs through a cyclophilin D intrinsic signaling pathway.

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Ji, Chao; Yang, Bo; Yang, Zhi; Tu, Ying; Yang, Yan-li; He, Li; Bi, Zhi-Gang

2012-09-07

UVB-induced skin cell damage involves the opening of mitochondrial permeability transition pore (mPTP), which leads to both apoptotic and necrotic cell death. Cyclophilin D (Cyp-D) translocation to the inner membrane of mitochondrion acts as a key component to open the mPTP. Our Western-Blot results in primary cultured human skin keratinocytes and in HaCaT cell line demonstrated that UVB radiation and hydrogen peroxide (H(2)O(2)) induced Cyp-D expression, which was inhibited by anti-oxidant N-acetyl cysteine (NAC). We created a stable Cyp-D deficiency skin keratinocytes by expressing Cyp-D-shRNA through lentiviral infection. Cyp-D-deficient cells were significantly less susceptible than their counterparts to UVB- or H(2)O(2)-induced cell death. Further, cyclosporine A (Cs-A), a Cyp-D inhibitor, inhibited UVB- or H(2)O(2)-induced keratinocytes cell death. Reversely, over-expression of Cyp-D in primary keratinocytes caused spontaneous keratinocytes cell death. These results suggest Cyp-D's critical role in UVB/oxidative stress-induced skin cell death. Copyright © 2012 Elsevier Inc. All rights reserved.

Expression of Peroxisome Proferator-Activated Receptor γ (PPARγ in Human Transitional Bladder Cancer and its Role in Inducing Cell Death

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You-Fei Guan

1999-10-01

Full Text Available The present study examined the expression and role of the thiazolidinedione (TZD-activated transcription factor, peroxisome proliferator-activated receptor γ (PPARγ, in human bladder cancers. In situ hybridization shows that PPARγ mRNA is highly expressed in all human transitional epithelial cell cancers (TCCa's studied (n=11. PPARγ was also expressed in five TCCa cell lines as determined by RNase protection assays and immunoblot. Retinoid X receptor α (RXRα, a 9-cis-retinoic acid stimulated (9-cis-RA heterodimeric partner of PPARγ, was also co-expressed in all TCCa tissues and cell lines. Treatment of the T24 bladder cancer cells with the TZD PPARγ agonist troglitazone, dramatically inhibited 3H-thymidine incorporation and induced cell death. Addition of the RXRα ligands, 9-cis-RA or LG100268, sensitized T24 bladder cancer cells to the lethal effect of troglitazone and two other PPARγ activators, ciglitazone and 15-deoxy-Δ12,14-PGJ2 (15dPGJ2. Troglitazone treatment increased expression of two cyclin-dependent kinase inhibitors, p21wAF1/CIP1 and p16INK4, reduced cyclin D1 expression, consistent with G1 arrest. Troglitazone also induced an endogenous PPARγ target gene in T24 cells, adipocyte-type fatty acid binding protein (A-FABP, the expression of which correlates with bladder cancer differentiation. In situ hybridization shows that A-FABP expression is localized to normal uroepithelial cells as well as some TCCa's. Taken together, these results demonstrate that PPARγ is expressed in human TCCa where it may play a role in regulating TCCa differentiation and survival, thereby providing a potential target for therapy of uroepithelial cancers.

Up-regulation of Kir2.1 by ER stress facilitates cell death of brain capillary endothelial cells

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Kito, Hiroaki; Yamazaki, Daiju; Ohya, Susumu; Yamamura, Hisao; Asai, Kiyofumi; Imaizumi, Yuji

2011-01-01

Highlights: → We found that application of endoplasmic reticulum (ER) stress with tunicamycin to brain capillary endothelial cells (BCECs) induced cell death. → The ER stress facilitated the expression of inward rectifier K + channel (K ir 2.1) and induced sustained membrane hyperpolarization. → The membrane hyperpolarization induced sustained Ca 2+ entry through voltage-independent nonspecific cation channels and consequently facilitated cell death. → The K ir 2.1 up-regulation by ER stress is, at least in part, responsible for cell death of BCECs under pathological conditions. -- Abstract: Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cell turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K + channel (K ir 2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K ir channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca 2+ concentration due to Ca 2+ influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K ir 2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.

Regulatory T cells ameliorate tissue plasminogen activator-induced brain haemorrhage after stroke.

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Mao, Leilei; Li, Peiying; Zhu, Wen; Cai, Wei; Liu, Zongjian; Wang, Yanling; Luo, Wenli; Stetler, Ruth A; Leak, Rehana K; Yu, Weifeng; Gao, Yanqin; Chen, Jun; Chen, Gang; Hu, Xiaoming

2017-07-01

Delayed thrombolytic treatment with recombinant tissue plasminogen activator (tPA) may exacerbate blood-brain barrier breakdown after ischaemic stroke and lead to lethal haemorrhagic transformation. The immune system is a dynamic modulator of stroke response, and excessive immune cell accumulation in the cerebral vasculature is associated with compromised integrity of the blood-brain barrier. We previously reported that regulatory T cells, which function to suppress excessive immune responses, ameliorated blood-brain barrier damage after cerebral ischaemia. This study assessed the impact of regulatory T cells in the context of tPA-induced brain haemorrhage and investigated the underlying mechanisms of action. The number of circulating regulatory T cells in stroke patients was dramatically reduced soon after stroke onset (84 acute ischaemic stroke patients with or without intravenous tPA treatment, compared to 115 age and gender-matched healthy controls). Although stroke patients without tPA treatment gradually repopulated the numbers of circulating regulatory T cells within the first 7 days after stroke, post-ischaemic tPA treatment led to sustained suppression of regulatory T cells in the blood. We then used the murine suture and embolic middle cerebral artery occlusion models of stroke to investigate the therapeutic potential of adoptive regulatory T cell transfer against tPA-induced haemorrhagic transformation. Delayed administration of tPA (10 mg/kg) resulted in haemorrhagic transformation in the ischaemic territory 1 day after ischaemia. When regulatory T cells (2 × 106/mouse) were intravenously administered immediately after delayed tPA treatment in ischaemic mice, haemorrhagic transformation was significantly decreased, and this was associated with improved sensorimotor functions. Blood-brain barrier disruption and tight junction damages were observed in the presence of delayed tPA after stroke, but were mitigated by regulatory T cell transfer. Mechanistic

Singapore grouper iridovirus, a large DNA virus, induces nonapoptotic cell death by a cell type dependent fashion and evokes ERK signaling.

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Huang, Xiaohong; Huang, Youhua; Ouyang, Zhengliang; Xu, Lixiao; Yan, Yang; Cui, Huachun; Han, Xin; Qin, Qiwei

2011-08-01

Virus induced cell death, including apoptosis and nonapoptotic cell death, plays a critical role in the pathogenesis of viral diseases. Singapore grouper iridovirus (SGIV), a novel iridovirus of genus Ranavirus, causes high mortality and heavy economic losses in grouper aquaculture. Here, using fluorescence microscopy, electron microscopy and biochemical assays, we found that SGIV infection in host (grouper spleen, EAGS) cells evoked nonapoptotic programmed cell death (PCD), characterized by appearance of cytoplasmic vacuoles and distended endoplasmic reticulum, in the absence of DNA fragmentation, apoptotic bodies and caspase activation. In contrast, SGIV induced typical apoptosis in non-host (fathead minnow, FHM) cells, as evidenced by caspase activation and DNA fragmentation, suggesting that SGIV infection induced nonapoptotic cell death by a cell type dependent fashion. Furthermore, viral replication was essential for SGIV induced nonapoptotic cell death, but not for apoptosis. Notably, the disruption of mitochondrial transmembrane potential (ΔΨm) and externalization of phosphatidylserine (PS) were not detected in EAGS cells but in FHM cells after SGIV infection. Moreover, the extracellular signal-regulated kinase (ERK) signaling was involved in SGIV infection induced nonapoptotic cell death and viral replication. This is a first demonstration of ERK-mediated nonapoptotic cell death induced by a DNA virus. These findings contribute to understanding the mechanisms of iridovirus pathogenesis.

Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

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Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

2013-10-01

Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

BAX/BAK–Independent Mitoptosis during Cell Death Induced by Proteasome Inhibition?

OpenAIRE

Lomonosova, Elena; Ryerse, Jan; Chinnadurai, G.

2009-01-01

Proteasome inhibitors induce rapid death of cancer cells. We show that in epithelial cancer cells, such death is associated with dramatic and simultaneous up-regulation of several BH3-only proteins, including BIK, BIM, MCL-1S, NOXA, and PUMA, as well as p53. Elevated levels of these proteins seem to be the result of direct inhibition of their proteasomal degradation, induction of transcription, and active translation. Subsequent cell death is independent of BAX, and probably BAK, and proceeds...

Fas (CD95) expression and death-mediating function are induced by CD4 cross-linking on CD4+ T cells.

OpenAIRE

Desbarats, J; Freed, J H; Campbell, P A; Newell, M K

1996-01-01

The CD4 receptor contributes to T-cell activation by coligating major histocompatibility complex class II on antigen presenting cells with the T-cell receptor (TCR)/CD3 complex, and triggering a cascade of signaling events including tyrosine phosphorylation of intracellular proteins. Paradoxically, CD3 cross-linking prior to TCR stimulation results in apoptotic cell death, as does injection of anti-CD4 antibodies in vivo of CD4 ligation by HIV glycoprotein (gp) 120. In this report we investig...

Ultra-violet B (UVB)-induced skin cell death occurs through a cyclophilin D intrinsic signaling pathway

International Nuclear Information System (INIS)

Ji, Chao; Yang, Bo; Yang, Zhi; Tu, Ying; Yang, Yan-li; He, Li; Bi, Zhi-Gang

2012-01-01

Highlights: ► UVB radiated skin keratinocytes show cyclophilin D (Cyp-D) upregulation. ► NAC inhibits UVB induced Cyp-D expression, while H 2 O 2 facilitates it. ► Cyp-D-deficient cells are significantly less susceptible to UVB induced cell death. ► Over-expression of Cyp-D causes spontaneous keratinocytes cell death. -- Abstract: UVB-induced skin cell damage involves the opening of mitochondrial permeability transition pore (mPTP), which leads to both apoptotic and necrotic cell death. Cyclophilin D (Cyp-D) translocation to the inner membrane of mitochondrion acts as a key component to open the mPTP. Our Western-Blot results in primary cultured human skin keratinocytes and in HaCaT cell line demonstrated that UVB radiation and hydrogen peroxide (H 2 O 2 ) induced Cyp-D expression, which was inhibited by anti-oxidant N-acetyl cysteine (NAC). We created a stable Cyp-D deficiency skin keratinocytes by expressing Cyp-D-shRNA through lentiviral infection. Cyp-D-deficient cells were significantly less susceptible than their counterparts to UVB- or H 2 O 2 -induced cell death. Further, cyclosporine A (Cs-A), a Cyp-D inhibitor, inhibited UVB- or H 2 O 2 -induced keratinocytes cell death. Reversely, over-expression of Cyp-D in primary keratinocytes caused spontaneous keratinocytes cell death. These results suggest Cyp-D’s critical role in UVB/oxidative stress-induced skin cell death.

Triptolide induces lysosomal-mediated programmed cell death in MCF-7 breast cancer cells

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Owa C

2013-09-01

Full Text Available Chie Owa, Michael E Messina Jr, Reginald HalabyDepartment of Biology, Montclair State University, Montclair, NJ, USABackground: Breast cancer is a major cause of death; in fact, it is the most common type, in order of the number of global deaths, of cancer in women worldwide. This research seeks to investigate how triptolide, an extract from the Chinese herb Tripterygium wilfordii Hook F, induces apoptosis in MCF-7 human breast cancer cells. Accumulating evidence suggests a role for lysosomal proteases in the activation of apoptosis. However, there is also some controversy regarding the direct participation of lysosomal proteases in activation of key apoptosis-related caspases and release of mitochondrial cytochrome c. In the present study, we demonstrate that triptolide induces an atypical, lysosomal-mediated apoptotic cell death in MCF-7 cells because they lack caspase-3.Methods: MCF-7 cell death was characterized via cellular morphology, chromatin condensation, 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide colorimetric cell growth inhibition assay and the expression levels of proapoptotic proteins. Acridine orange and LysoTracker® staining were performed to visualize lysosomes. Lysosomal enzymatic activity was monitored using an acid phosphatase assay and western blotting of cathepsin B protein levels in the cytosolic fraction, which showed increased enzymatic activity in drug-treated cells.Results: These experiments suggest that triptolide-treated MCF-7 cells undergo atypical apoptosis and that, during the early stages, lysosomal enzymes leak into the cytosol, indicating lysosomal membrane permeability.Conclusion: Our results suggest that further studies are warranted to investigate triptolide's potential as an anticancer therapeutic agent.Keywords: triptolide, MCF-7 breast cancer cells, apoptosis, lysosomes, lysosomal membrane permeabilization (LMP

Cytoprotective dibenzoylmethane derivatives protect cells from oxidative stress-induced necrotic cell death.

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Hegedűs, Csaba; Lakatos, Petra; Kiss-Szikszai, Attila; Patonay, Tamás; Gergely, Szabolcs; Gregus, Andrea; Bai, Péter; Haskó, György; Szabó, Éva; Virág, László

2013-06-01

Screening of a small in-house library of 1863 compounds identified 29 compounds that protected Jurkat cells from hydrogen peroxide-induced cytotoxicity. From the cytoprotective compounds eleven proved to possess antioxidant activity (ABTS radical scavenger effect) and two were found to inhibit poly(ADP-ribosyl)ation (PARylation), a cytotoxic pathway operating in severely injured cells. Four cytoprotective dibenzoylmethane (DBM) derivatives were investigated in more detail as they did not scavenge hydrogen peroxide nor did they inhibit PARylation. These compounds protected cells from necrotic cell death while caspase activation, a parameter of apoptotic cell death was not affected. Hydrogen peroxide activated extracellular signal regulated kinase (ERK1/2) and p38 MAP kinases but not c-Jun N-terminal kinase (JNK). The cytoprotective DBMs suppressed the activation of Erk1/2 but not that of p38. Cytoprotection was confirmed in another cell type (A549 lung epithelial cells), indicating that the cytoprotective effect is not cell type specific. In conclusion we identified DBM analogs as a novel class of cytoprotective compounds inhibiting ERK1/2 kinase and protecting from necrotic cell death by a mechanism independent of poly(ADP-ribose) polymerase inhibition. Copyright © 2013 Elsevier Ltd. All rights reserved.

α-Hispanolol sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis via death receptor up-regulation

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Mota, Alba, E-mail: amota@iib.uam.es [Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid (Spain); Jiménez-Garcia, Lidia, E-mail: ljimenez@isciii.es [Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid (Spain); Herránz, Sandra, E-mail: sherranz@isciii.es [Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid (Spain); Heras, Beatriz de las, E-mail: lasheras@ucm.es [Departamento de Farmacología, Facultad de Farmacia, Universidad Complutense de Madrid (UCM), Madrid (Spain); Hortelano, Sonsoles, E-mail: shortelano@isciii.es [Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid (Spain)

2015-08-01

Hispanolone derivatives have been previously described as anti-inflammatory and antitumoral agents. However, their effects on overcoming Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance remain to be elucidated. In this study, we analyzed the cytotoxic effects of the synthetic hispanolone derivative α-hispanolol (α-H) in several tumor cell lines, and we evaluated the induction of apoptosis, as well as the TRAIL-sensitizing potential of α-H in the hepatocellular carcinoma cell line HepG2. Our data show that α-H decreased cell viability in a dose-dependent manner in HeLa, MDA-MB231, U87 and HepG2 cell lines, with a more prominent effect in HepG2 cells. Interestingly, α-H had no effect on non-tumoral cells. α-H induced activation of caspase-8 and caspase-9 and also increased levels of the proapoptotic protein Bax, decreasing antiapoptotic proteins (Bcl-2, X-IAP and IAP-1) in HepG2 cells. Specific inhibition of caspase-8 abrogated the cascade of caspase activation, suggesting that the extrinsic pathway has a critical role in the apoptotic events induced by α-H. Furthermore, combined treatment of α-H with TRAIL enhanced apoptosis in HepG2 cells, activating caspase-8 and caspase-9. This correlated with up-regulation of both the TRAIL death receptor DR4 and DR5. DR4 or DR5 neutralizing antibodies abolished the effect of α-H on TRAIL-induced apoptosis, suggesting that sensitization was mediated through the death receptor pathway. Our results demonstrate that α-H induced apoptosis in the human hepatocellular carcinoma cell line HepG2 through activation of caspases and induction of the death receptor pathway. In addition, we describe a novel function of α-H as a sensitizer on TRAIL-induced apoptotic cell death in HepG2 cells. - Highlights: • α-Hispanolol induced apoptosis in the human hepatocellular carcinoma cell line HepG2. • α-Hispanolol induced activation of caspases and the death receptor pathway. • α-Hispanolol enhanced

α-Hispanolol sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis via death receptor up-regulation

International Nuclear Information System (INIS)

Mota, Alba; Jiménez-Garcia, Lidia; Herránz, Sandra; Heras, Beatriz de las; Hortelano, Sonsoles

2015-01-01

Hispanolone derivatives have been previously described as anti-inflammatory and antitumoral agents. However, their effects on overcoming Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance remain to be elucidated. In this study, we analyzed the cytotoxic effects of the synthetic hispanolone derivative α-hispanolol (α-H) in several tumor cell lines, and we evaluated the induction of apoptosis, as well as the TRAIL-sensitizing potential of α-H in the hepatocellular carcinoma cell line HepG2. Our data show that α-H decreased cell viability in a dose-dependent manner in HeLa, MDA-MB231, U87 and HepG2 cell lines, with a more prominent effect in HepG2 cells. Interestingly, α-H had no effect on non-tumoral cells. α-H induced activation of caspase-8 and caspase-9 and also increased levels of the proapoptotic protein Bax, decreasing antiapoptotic proteins (Bcl-2, X-IAP and IAP-1) in HepG2 cells. Specific inhibition of caspase-8 abrogated the cascade of caspase activation, suggesting that the extrinsic pathway has a critical role in the apoptotic events induced by α-H. Furthermore, combined treatment of α-H with TRAIL enhanced apoptosis in HepG2 cells, activating caspase-8 and caspase-9. This correlated with up-regulation of both the TRAIL death receptor DR4 and DR5. DR4 or DR5 neutralizing antibodies abolished the effect of α-H on TRAIL-induced apoptosis, suggesting that sensitization was mediated through the death receptor pathway. Our results demonstrate that α-H induced apoptosis in the human hepatocellular carcinoma cell line HepG2 through activation of caspases and induction of the death receptor pathway. In addition, we describe a novel function of α-H as a sensitizer on TRAIL-induced apoptotic cell death in HepG2 cells. - Highlights: • α-Hispanolol induced apoptosis in the human hepatocellular carcinoma cell line HepG2. • α-Hispanolol induced activation of caspases and the death receptor pathway. • α-Hispanolol enhanced

Complementary roles of Fas-associated death domain (FADD) and receptor interacting protein kinase-3 (RIPK3) in T-cell homeostasis and antiviral immunity.

Science.gov (United States)

Lu, Jennifer V; Weist, Brian M; van Raam, Bram J; Marro, Brett S; Nguyen, Long V; Srinivas, Prathna; Bell, Bryan D; Luhrs, Keith A; Lane, Thomas E; Salvesen, Guy S; Walsh, Craig M

2011-09-13

Caspase-8 (casp8) is required for extrinsic apoptosis, and mice deficient in casp8 fail to develop and die in utero while ultimately failing to maintain the proliferation of T cells, B cells, and a host of other cell types. Paradoxically, these failures are not caused by a defect in apoptosis, but by a presumed proliferative function of this protease. Indeed, following mitogenic stimulation, T cells lacking casp8 or its adaptor protein FADD (Fas-associated death domain protein) develop a hyperautophagic morphology, and die a programmed necrosis-like death process termed necroptosis. Recent studies have demonstrated that receptor-interacting protein kinases (RIPKs) RIPK1 and RIPK3 together facilitate TNF-induced necroptosis, but the precise role of RIPKs in the demise of T cells lacking FADD or casp8 activity is unknown. Here we demonstrate that RIPK3 and FADD have opposing and complementary roles in promoting T-cell clonal expansion and homeostasis. We show that the defective proliferation of T cells bearing an interfering form of FADD (FADDdd) is rescued by crossing with RIPK3(-/-) mice, although such rescue ultimately leads to lymphadenopathy. Enhanced recovery of these double-mutant T cells following stimulation demonstrates that FADD, casp8, and RIPK3 are all essential for clonal expansion, contraction, and antiviral responses. Finally, we demonstrate that caspase-mediated cleavage of RIPK1-containing necrosis inducing complexes (necrosomes) is sufficient to prevent necroptosis in the face of death receptor signaling. These studies highlight the "two-faced" nature of casp8 activity, promoting clonal expansion in some situations and apoptotic demise in others.

Mechanisms of palmitate-induced cell death in human osteoblasts

Science.gov (United States)

Gunaratnam, Krishanthi; Vidal, Christopher; Boadle, Ross; Thekkedam, Chris; Duque, Gustavo

2013-01-01

Summary Lipotoxicity is an overload of lipids in non-adipose tissues that affects function and induces cell death. Lipotoxicity has been demonstrated in bone cells in vitro using osteoblasts and adipocytes in coculture. In this condition, lipotoxicity was induced by high levels of saturated fatty acids (mostly palmitate) secreted by cultured adipocytes acting in a paracrine manner. In the present study, we aimed to identify the underlying mechanisms of lipotoxicity in human osteoblasts. Palmitate induced autophagy in cultured osteoblasts, which was preceded by the activation of autophagosomes that surround palmitate droplets. Palmitate also induced apoptosis though the activation of the Fas/Jun kinase (JNK) apoptotic pathway. In addition, osteoblasts could be protected from lipotoxicity by inhibiting autophagy with the phosphoinositide kinase inhibitor 3-methyladenine or by inhibiting apoptosis with the JNK inhibitor SP600125. In summary, we have identified two major molecular mechanisms of lipotoxicity in osteoblasts and in doing so we have identified a new potential therapeutic approach to prevent osteoblast dysfunction and death, which are common features of age-related bone loss and osteoporosis. PMID:24285710

Silibinin induces apoptosis via calpain-dependent AIF nuclear translocation in U87MG human glioma cell death

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Kim Yong K

2011-04-01

Full Text Available Abstract Background Silibinin, a natural polyphenolic flavonoid, has been reported to induce cell death in various cancer cell types. However, the molecular mechanism is not clearly defined. Our previous study showed that silibinin induces glioma cell death and its effect was effectively prevented by calpain inhibitor. The present study was therefore undertaken to examine the role of calpain in the silibinin-induced glioma cell death. Methods U87MG cells were grown on well tissue culture plates and cell viability was measured by MTT assay. ROS generation and △ψm were estimated using the fluorescence dyes. PKC activation and Bax expression were measured by Western blot analysis. AIF nuclear translocation was determined by Western blot and immunocytochemistry. Results Silibinin induced activation of calpain, which was blocked by EGTA and the calpain inhibitor Z-Leu-Leu-CHO. Silibinin caused ROS generation and its effect was inhibited by calpain inhibitor, the general PKC inhibitor GF 109203X, the specific PKCδ inhibitor rottlerin, and catalase. Silibinin-induce cell death was blocked by calpain inhibitor and PKC inhibitors. Silibinin-induced PKCδ activation and disruption of △ψm were prevented by the calpain inhibitor. Silibinin induced AIF nuclear translocation and its effect was prevented by calpain inhibitor. Transfection of vector expressing microRNA of AIF prevented the silibinin-induced cell death. Conclusions Silibinin induces apoptotic cell death through a calpain-dependent mechanism involving PKC, ROS, and AIF nuclear translocation in U87MG human glioma cells.

Characterization of a serine protease-mediated cell death program activated in human leukemia cells

International Nuclear Information System (INIS)

O'Connell, A.R.; Holohan, C.; Torriglia, A.; Lee, B.F.; Stenson-Cox, C.

2006-01-01

Tightly controlled proteolysis is a defining feature of apoptosis and caspases are critical in this regard. Significant roles for non-caspase proteases in cell death have been highlighted. Staurosporine causes a rapid induction of apoptosis in virtually all mammalian cell types. Numerous studies demonstrate that staurosporine can activate cell death under caspase-inhibiting circumstances. The aim of this study was to investigate the proteolytic mechanisms responsible for cell death under these conditions. To that end, we show that inhibitors of serine proteases can delay cell death in one such system. Furthermore, through profiling of proteolytic activation, we demonstrate, for the first time, that staurosporine activates a chymotrypsin-like serine protease-dependent cell death in HL-60 cells independently, but in parallel with the caspase controlled systems. Features of the serine protease-mediated system include cell shrinkage and apoptotic morphology, regulation of caspase-3, altered nuclear morphology, generation of an endonuclease and DNA degradation. We also demonstrate a staurosporine-induced activation of a putative 16 kDa chymotrypsin-like protein during apoptosis

Retinal Cell Death Caused by Sodium Iodate Involves Multiple Caspase-Dependent and Caspase-Independent Cell-Death Pathways

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Jasmin Balmer

2015-07-01

Full Text Available Herein, we have investigated retinal cell-death pathways in response to the retina toxin sodium iodate (NaIO3 both in vivo and in vitro. C57/BL6 mice were treated with a single intravenous injection of NaIO3 (35 mg/kg. Morphological changes in the retina post NaIO3 injection in comparison to untreated controls were assessed using electron microscopy. Cell death was determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL staining. The activation of caspases and calpain was measured using immunohistochemistry. Additionally, cytotoxicity and apoptosis in retinal pigment epithelial (RPE cells, primary retinal cells, and the cone photoreceptor (PRC cell line 661W were assessed in vitro after NaIO3 treatment using the ApoToxGlo™ assay. The 7-AAD/Annexin-V staining was performed and necrostatin (Nec-1 was administered to the NaIO3-treated cells to confirm the results. In vivo, degenerating RPE cells displayed a rounded shape and retracted microvilli, whereas PRCs featured apoptotic nuclei. Caspase and calpain activity was significantly upregulated in retinal sections and protein samples from NaIO3-treated animals. In vitro, NaIO3 induced necrosis in RPE cells and apoptosis in PRCs. Furthermore, Nec-1 significantly decreased NaIO3-induced RPE cell death, but had no rescue effect on treated PRCs. In summary, several different cell-death pathways are activated in retinal cells as a result of NaIO3.

The cell biology of T-dependent B cell activation

DEFF Research Database (Denmark)

Owens, T; Zeine, R

1989-01-01

The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells...... includes multipoint intermolecular interactions that probably involve aggregation of both polymorphic and monomorphic T cell surface molecules. Such aggregations have been shown in vitro to markedly enhance and, in some cases, induce T cell activation. The production of T-derived lymphokines that have been...... implicated in B cell activation is dependent on the T cell receptor for antigen and its associated CD3 signalling complex. T-dependent help for B cell activation is therefore similarly MHC-restricted and involves T-B intercellular interaction. Recent reports that describe antigen-independent B cell...

Protective effect of aqueous extract from Spirulina platensis against cell death induced by free radicals.

Science.gov (United States)

Chu, Wan-Loy; Lim, Yen-Wei; Radhakrishnan, Ammu Kutty; Lim, Phaik-Eem

2010-09-21

Spirulina is a commercial alga well known to contain various antioxidants, especially phycocyanin. Apart from being sold as a nutraceutical, Spirulina is incorporated as a functional ingredient in food products and beverages. Most of the previous reports on antioxidant activity of Spirulina were based on chemical rather than cell-based assays. The primary objective of this study was to assess the antioxidant activity of aqueous extract from Spirulina based on its protective effect against cell death induced by free radicals. The antioxidant activity of the cold water extract from food-grade Spirulina platensis was assessed using both chemical and cell-based assays. In the cell-based assay, mouse fibroblast cells (3T3) cells were incubated for 1 h in medium containing aqueous extract of Spirulina or vitamin C (positive control) at 25, 125 and 250 μg/mL before the addition of 50 μM 1,1-diphenyl-2-picrylhydrazyl (DPPH) or 3-ethylbenzothiazoline-6-sulfonic acid (ABTS). The cells were incubated for another 24 h before being assessed for cell death due to apoptosis using the Cell Death Detection ELISA Kit. Spectrophotometric assays based on DPPH and ABTS were also used to assess the antioxidant activity of the extract compared to vitamin C and vitamin E (positive controls). Spirulina extract did not cause cytotoxic effect on 3T3 cells within the range of concentrations tested (0 - 250 μg/mL). The extract reduced significantly (p assay, the radical scavenging activity of the extract was higher than phycocyanin and was at least 50% of vitamin C and vitamin E. Based on the ABTS assay, the antioxidant activity of the extract at 50 μmug/mL was as good as vitamin C and vitamin E. The results showed that aqueous extract of Spirulina has a protective effect against apoptotic cell death due to free radicals. The potential application of incorporating Spirulina into food products and beverages to enhance their antioxidant capacity is worth exploring.

The process and promotion of radiation-induced cell death

International Nuclear Information System (INIS)

Sasaki, Hiroshi

1998-01-01

Radiation-induced cell death is divided into reproductive and interphase death, whose process can be revealed by time-lapse observations. Pedigree analyses of progenies derived from a surviving progenitor cell have shown that moribund cells appear in clusters among cells which are apparently undamaged (lethal sectoring). Sister cell fusion, which likely results from chromosome bridge, is the most frequently observed cell abnormality leading to reproductive death. While interphase death does not occur unless the dose exceeds 10 Gy for low LET radiation such as X-rays, high-LET radiation is very effective at inducing interphase death (RBE: ≅3 at 230 keV/μm). Expression or fixation of potentially lethal damage (PLD) is closely associated with cell cycle events and enhanced by inducing premature chromosome condensation (PCC) at a nonpermissive temperature in tsBN2 cells with a ts-defect in RCC1 protein (a regulator of chromatin condensation) which monitors the completion of DNA replication. Furthermore, higher-order structural changes in nuclear matrix such as induced by leptomycin B, an inhibitor of CRM1 (chromosome region maintenance) protein, also play an important role in the fixation of PLD. (author)

CD8+ T cells induce thyroid epithelial cell hyperplasia and fibrosis.

Science.gov (United States)

Yu, Shiguang; Fang, Yujiang; Sharav, Tumenjargal; Sharp, Gordon C; Braley-Mullen, Helen

2011-02-15

CD8(+) T cells can be important effector cells in autoimmune inflammation, generally because they can damage target cells by cytotoxicity. This study shows that activated CD8(+) T cells induce thyroid epithelial cell hyperplasia and proliferation and fibrosis in IFN-γ(-/-) NOD.H-2h4 SCID mice in the absence of CD4(+) T cells. Because CD8(+) T cells induce proliferation rather than cytotoxicity of target cells, these results describe a novel function for CD8(+) T cells in autoimmune disease. In contrast to the ability of purified CD8(+) T cells to induce thyrocyte proliferation, CD4(+) T cells or CD8 T cell-depleted splenocytes induced only mild thyroid lesions in SCID recipients. T cells in both spleens and thyroids highly produce TNF-α. TNF-α promotes proliferation of thyrocytes in vitro, and anti-TNF-α inhibits development of thyroid epithelial cell hyperplasia and proliferation in SCID recipients of IFN-γ(-/-) splenocytes. This suggests that targeting CD8(+) T cells and/or TNF-α may be effective for treating epithelial cell hyperplasia and fibrosis.

Cytoprotective effects of fisetin against hypoxia-induced cell death in PC12 cells.

Science.gov (United States)

Chen, Pei-Yi; Ho, Yi-Ru; Wu, Ming-Jiuan; Huang, Shun-Ping; Chen, Po-Kong; Tai, Mi-Hsueh; Ho, Chi-Tang; Yen, Jui-Hung

2015-01-01

Fisetin (3,7,3',4'-tetrahydroxyflavone), a flavonol compound of flavonoids, exhibits a broad spectrum of biological activities including anti-oxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The aim of this study is to investigate the cytoprotective effect of fisetin and the underlying molecular mechanism against hypoxia-induced cell death in PC12 cells. The results of this study showed that fisetin significantly restored the cell viability of PC12 cells under both cobalt chloride (CoCl₂)- and low oxygen-induced hypoxic conditions. Treatment with fisetin successfully reduced the CoCl₂-mediated reactive oxygen species (ROS) production, which was accompanied by an increase in the cell viability of PC12 cells. Furthermore, we found that treatment of PC12 cells with fisetin markedly upregulated hypoxia-inducible factor 1α (HIF-1α), its nuclear accumulation and the hypoxia-response element (HRE)-driven transcriptional activation. The fisetin-mediated cytoprotection during CoCl₂ exposure was significantly attenuated through the administration of HIF-1α siRNA. Moreover, we demonstrated that MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK and phosphatidylinositol 3-kinase (PI3 K) inhibitors significantly blocked the increase in cell survival that was induced by fisetin treatment under hypoxic conditions. Consistently, increased phosphorylation of ERK, p38 and Akt proteins was observed in PC12 cells treated with fisetin. However, the fisetin-induced HRE-driven transcription was not affected by inhibition of these kinase signaling pathways. Current results reveal for the first time that fisetin promotes cell survival and protects against hypoxia-induced cell death through ROS scavenging and the activation of HIF1α-, MAPK/ERK-, p38 MAPK- and PI3 K/Akt-dependent signaling pathways in PC12 cells.

cGMP-Phosphodiesterase Inhibition Prevents Hypoxia-Induced Cell Death Activation in Porcine Retinal Explants.

Directory of Open Access Journals (Sweden)

Lorena Olivares-González

Full Text Available Retinal hypoxia and oxidative stress are involved in several retinal degenerations including diabetic retinopathy, glaucoma, central retinal artery occlusion, or retinopathy of prematurity. The second messenger cyclic guanosine monophosphate (cGMP has been reported to be protective for neuronal cells under several pathological conditions including ischemia/hypoxia. The purpose of this study was to evaluate whether the accumulation of cGMP through the pharmacological inhibition of phosphodiesterase (PDE with Zaprinast prevented retinal degeneration induced by mild hypoxia in cultures of porcine retina. Exposure to mild hypoxia (5% O2 for 24h reduced cGMP content and induced retinal degeneration by caspase dependent and independent (PARP activation mechanisms. Hypoxia also produced a redox imbalance reducing antioxidant response (superoxide dismutase and catalase activities and increasing superoxide free radical release. Zaprinast reduced mild hypoxia-induced cell death through inhibition of caspase-3 or PARP activation depending on the cell layer. PDE inhibition also ameliorated the effects of mild hypoxia on antioxidant response and the release of superoxide radical in the photoreceptor layer. The use of a PKG inhibitor, KT5823, suggested that cGMP-PKG pathway is involved in cell survival and antioxidant response. The inhibition of PDE, therefore, could be useful for reducing retinal degeneration under hypoxic/ischemic conditions.

Simultaneous activation of mitophagy and autophagy by staurosporine protects against dopaminergic neuronal cell death.

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Ha, Ji-Young; Kim, Ji-Soo; Kim, Seo-Eun; Son, Jin H

2014-02-21

Abnormal autophagy is frequently observed during dopaminergic neurodegeneration in Parkinson's disease (PD). However, it is not yet firmly established whether active autophagy is beneficial or pathogenic with respect to dopaminergic cell loss. Staurosporine, a common inducer of apoptosis, is often used in mechanistic studies of dopaminergic cell death. Here we report that staurosporine activates both autophagy and mitophagy simultaneously during dopaminergic neuronal cell death, and evaluate the physiological significance of these processes during cell death. First, staurosporine treatment resulted in induction of autophagy in more than 75% of apoptotic cells. Pharmacological inhibition of autophagy by bafilomycin A1 decreased significantly cell viability. In addition, staurosporine treatment resulted in activation of the PINK1-Parkin mitophagy pathway, of which deficit underlies some familial cases of PD, in the dopaminergic neuronal cell line, SN4741. The genetic blockade of this pathway by PINK1 null mutation also dramatically increased staurosporine-induced cell death. Taken together, our data suggest that staurosporine induces both mitophagy and autophagy, and that these pathways exert a significant neuroprotective effect, rather than a contribution to autophagic cell death. This model system may therefore be useful for elucidating the mechanisms underlying crosstalk between autophagy, mitophagy, and cell death in dopaminergic neurons. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Ultra-violet B (UVB)-induced skin cell death occurs through a cyclophilin D intrinsic signaling pathway

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Ji, Chao [Department of Dermatology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210024, Jiangsu (China); Yang, Bo [Department of Dermatology, Huashan Hospital, Fudan University, Shanghai 200040 (China); Yang, Zhi; Tu, Ying [Department of Dermatology, The First Affiliated Hospital of Kunming Medical University, Yunnan Provincial Institute of Dermatology, Kunming 650032, Yunnan (China); Yang, Yan-li [Department of Otorhinolaryngology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210024, Jiangsu (China); He, Li, E-mail: heli2662@yahoo.com.cn [Department of Otorhinolaryngology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210024, Jiangsu (China); Bi, Zhi-Gang, E-mail: eltonbibenqhospital@yahoo.com.cn [Department of Dermatology, BenQ Medical Center, Nanjing Medical University, Nanjing 210019, Jiangsu (China)

2012-09-07

Highlights: Black-Right-Pointing-Pointer UVB radiated skin keratinocytes show cyclophilin D (Cyp-D) upregulation. Black-Right-Pointing-Pointer NAC inhibits UVB induced Cyp-D expression, while H{sub 2}O{sub 2} facilitates it. Black-Right-Pointing-Pointer Cyp-D-deficient cells are significantly less susceptible to UVB induced cell death. Black-Right-Pointing-Pointer Over-expression of Cyp-D causes spontaneous keratinocytes cell death. -- Abstract: UVB-induced skin cell damage involves the opening of mitochondrial permeability transition pore (mPTP), which leads to both apoptotic and necrotic cell death. Cyclophilin D (Cyp-D) translocation to the inner membrane of mitochondrion acts as a key component to open the mPTP. Our Western-Blot results in primary cultured human skin keratinocytes and in HaCaT cell line demonstrated that UVB radiation and hydrogen peroxide (H{sub 2}O{sub 2}) induced Cyp-D expression, which was inhibited by anti-oxidant N-acetyl cysteine (NAC). We created a stable Cyp-D deficiency skin keratinocytes by expressing Cyp-D-shRNA through lentiviral infection. Cyp-D-deficient cells were significantly less susceptible than their counterparts to UVB- or H{sub 2}O{sub 2}-induced cell death. Further, cyclosporine A (Cs-A), a Cyp-D inhibitor, inhibited UVB- or H{sub 2}O{sub 2}-induced keratinocytes cell death. Reversely, over-expression of Cyp-D in primary keratinocytes caused spontaneous keratinocytes cell death. These results suggest Cyp-D's critical role in UVB/oxidative stress-induced skin cell death.

EBV induces persistent NF-κB activation and contributes to survival of EBV-positive neoplastic T- or NK-cells.

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Honami Takada

Full Text Available Epstein-Barr virus (EBV has been detected in several T- and NK-cell neoplasms such as extranodal NK/T-cell lymphoma nasal type, aggressive NK-cell leukemia, EBV-positive peripheral T-cell lymphoma, systemic EBV-positive T-cell lymphoma of childhood, and chronic active EBV infection (CAEBV. However, how this virus contributes to lymphomagenesis in T or NK cells remains largely unknown. Here, we examined NF-κB activation in EBV-positive T or NK cell lines, SNT8, SNT15, SNT16, SNK6, and primary EBV-positive and clonally proliferating T/NK cells obtained from the peripheral blood of patients with CAEBV. Western blotting, electrophoretic mobility shift assays, and immunofluorescent staining revealed persistent NF-κB activation in EBV-infected cell lines and primary cells from patients. Furthermore, we investigated the role of EBV in infected T cells. We performed an in vitro infection assay using MOLT4 cells infected with EBV. The infection directly induced NF-κB activation, promoted survival, and inhibited etoposide-induced apoptosis in MOLT4 cells. The luciferase assay suggested that LMP1 mediated NF-κB activation in MOLT4 cells. IMD-0354, a specific inhibitor of NF-κB that suppresses NF-κB activation in cell lines, inhibited cell survival and induced apoptosis. These results indicate that EBV induces NF-κB-mediated survival signals in T and NK cells, and therefore, may contribute to the lymphomagenesis of these cells.

Lico A Enhances Nrf2-Mediated Defense Mechanisms against t-BHP-Induced Oxidative Stress and Cell Death via Akt and ERK Activation in RAW 264.7 Cells

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Hongming Lv

2015-01-01

Full Text Available Licochalcone A (Lico A exhibits various biological properties, including anti-inflammatory and antioxidant activities. In this study, we investigated the antioxidative potential and mechanisms of Lico A against tert-butyl hydroperoxide- (t-BHP- induced oxidative damage in RAW 264.7 cells. Our results indicated that Lico A significantly inhibited t-BHP-induced cytotoxicity, apoptosis, and reactive oxygen species (ROS generation and reduced glutathione (GSH depletion but increased the glutamate-cysteine ligase modifier (GCLM subunit and the glutamate-cysteine ligase catalytic (GCLC subunit genes expression. Additionally, Lico A dramatically upregulated the antioxidant enzyme heme oxygenase 1 (HO-1 and nuclear factor erythroid 2-related factor 2 (Nrf2, which were associated with inducing Nrf2 nuclear translocation, decreasing Keap1 protein expression and increasing antioxidant response element (ARE promoter activity. Lico A also obviously induced the activation of serine/threonine kinase (Akt and extracellular signal-regulated kinase (ERK, but PI3K/Akt and ERK inhibitors treatment displayed clearly decreased levels of LicoA-induced Nrf2 nuclear translocation and HO-1 expression, respectively. Furthermore, Lico A treatment markedly attenuated t-BHP-induced oxidative damage, which was reduced by treatment with PI3K/Akt, ERK, and HO-1 inhibitors. Therefore, Lico A might have a protective role against t-BHP-induced cytotoxicity by modulating HO-1 and by scavenging ROS via the activation of the PI3K/Akt and ERK/Nrf2 signaling pathways.

Sorafenib-induced defective autophagy promotes cell death by necroptosis

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Kharaziha, Pedram; Chioureas, Dimitris; Baltatzis, George; Fonseca, Pedro; Rodriguez, Patricia; Gogvadze, Vladimir; Lennartsson, Lena; Bj?rklund, Ann-Charlotte; Zhivotovsky, Boris; Grand?r, Dan; Egevad, Lars; Nilsson, Sten; Panaretakis, Theocharis

2015-01-01

Autophagy is one of the main cytoprotective mechanisms that cancer cells deploy to withstand the cytotoxic stress and survive the lethal damage induced by anti-cancer drugs. However, under specific conditions, autophagy may, directly or indirectly, induce cell death. In our study, treatment of the Atg5-deficient DU145 prostate cancer cells, with the multi-tyrosine kinase inhibitor, sorafenib, induces mitochondrial damage, autophagy and cell death. Molecular inhibition of autophagy by silencin...

Intracellular cholesterol level regulates sensitivity of glioblastoma cells against temozolomide-induced cell death by modulation of caspase-8 activation via death receptor 5-accumulation and activation in the plasma membrane lipid raft.

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Yamamoto, Yutaro; Tomiyama, Arata; Sasaki, Nobuyoshi; Yamaguchi, Hideki; Shirakihara, Takuya; Nakashima, Katsuhiko; Kumagai, Kosuke; Takeuchi, Satoru; Toyooka, Terushige; Otani, Naoki; Wada, Kojiro; Narita, Yoshitaka; Ichimura, Koichi; Sakai, Ryuichi; Namba, Hiroki; Mori, Kentaro

2018-01-01

Development of resistance against temozolomide (TMZ) in glioblastoma (GBM) after continuous treatment with TMZ is one of the critical problems in clinical GBM therapy. Intracellular cholesterol regulates cancer cell biology, but whether intracellular cholesterol is involved in TMZ resistance of GBM cells remains unclear. The involvement of intracellular cholesterol in acquired resistance against TMZ in GBM cells was investigated. Intracellular cholesterol levels were measured in human U251 MG cells with acquired TMZ resistance (U251-R cells) and TMZ-sensitive control U251 MG cells (U251-Con cells), and found that the intracellular cholesterol level was significantly lower in U251-R cells than in U251-Con cells. In addition, treatment by intracellular cholesterol remover, methyl-beta cyclodextrin (MβCD), or intracellular cholesterol inducer, soluble cholesterol (Chol), regulated TMZ-induced U251-Con cell death in line with changes in intracellular cholesterol level. Involvement of death receptor 5 (DR5), a death receptor localized in the plasma membrane, was evaluated. TMZ without or with MβCD and/or Chol caused accumulation of DR5 into the plasma membrane lipid raft and formed a complex with caspase-8, an extrinsic caspase cascade inducer, reflected in the induction of cell death. In addition, treatment with caspase-8 inhibitor or knockdown of DR5 dramatically suppressed U251-Con cell death induced by combination treatment with TMZ, MβCD, and Chol. Combined treatment of Chol with TMZ reversed the TMZ resistance of U251-R cells and another GBM cell model with acquired TMZ resistance, whereas clinical antihypercholesterolemia agents at physiological concentrations suppressed TMZ-induced cell death of U251-Con cells. These findings suggest that intracellular cholesterol level affects TMZ treatment of GBM mediated via a DR5-caspase-8 mechanism. Copyright © 2017 Elsevier Inc. All rights reserved.

Radiation-induced cell death in embryogenic cells of coniferous plants

International Nuclear Information System (INIS)

Watanabe, Yoshito; Homma-Takeda, Shino; Yukawa, Masae; Nishimura, Yoshikazu; Sasamoto, Hamako; Takahagi, Masahiko

2004-01-01

Reproductive processes are particularly radiosensitive in plant development, which was clearly illustrated in reduction of seed formation in native coniferous plants around Chernobyl after the nuclear accident. For the purpose to investigate the effects of ionizing radiation on embryonic formation in coniferous plants, we used an embryo-derived embryogenic cell culture of a Japanese native coniferous plant, Japanese cedar (Cryplomeria japonica). The embryogenic cells were so radiosensitive that most of the cells died by X-ray irradiation of 5 Gy. This indicated that the embryogenic cells are as radiosensitive as some mammalian cells including lymphocytes. We considered that this type of radiosensitive cell death in the embryogenic cells should be responsible for reproductive damages of coniferous plants by low dose of ionizing radiation. The cell death of the embryogenic cells was characteristic of nuclear DNA fragmentation, which is typically observed in radiation-induced programmed cell death, i.e. apoptosis, in mammalian cells. On the other hand, cell death with nuclear DNA fragmentation did not develop by X-ray irradiation in vegetative cells including meristematic cells of Japanese cedar. This suggests that an apoptosis-like programmed cell death should develop cell-specifically in embryogenic cells by ionizing radiation. The abortion of embryogenic cells may work to prevent transmission of radiation-induced genetic damages to the descendants. (author)

DRAM Triggers Lysosomal Membrane Permeabilization and Cell Death in CD4+ T Cells Infected with HIV

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Laforge, Mireille; Limou, Sophie; Harper, Francis; Casartelli, Nicoletta; Rodrigues, Vasco; Silvestre, Ricardo; Haloui, Houda; Zagury, Jean-Francois; Senik, Anna; Estaquier, Jerome

2013-01-01

Productive HIV infection of CD4+ T cells leads to a caspase-independent cell death pathway associated with lysosomal membrane permeabilization (LMP) and cathepsin release, resulting in mitochondrial outer membrane permeabilization (MOMP). Herein, we demonstrate that HIV infection induces damage-regulated autophagy modulator (DRAM) expression in a p53-dependent manner. Knocking down the expression of DRAM and p53 genes with specific siRNAs inhibited autophagy and LMP. However, inhibition of Atg5 and Beclin genes that prevents autophagy had a minor effect on LMP and cell death. The knock down of DRAM gene inhibited cytochrome C release, MOMP and cell death. However, knocking down DRAM, we increased viral infection and production. Our study shows for the first time the involvement of DRAM in host-pathogen interactions, which may represent a mechanism of defense via the elimination of infected cells. PMID:23658518

Effects of epigallocatechin gallate on ultra-violet-induced cell death in PC12 cells

International Nuclear Information System (INIS)

Takahashi, Hideo; Seki, Sakiko; Sakamoto, Naotaka; Nakagawa, Shigeki

2002-01-01

We examined the effects of catechin on ultra-violet-induced cell death in PC12 cells. PC12 cells were irradiated by ultra-violet C (254 nm) (UVC). We found that the lactate dehydrogenase (LDH) activities in culture media and lipid peroxide in PC12 cells, which indicate cell death and cell membrane damage, respectively, were increased by UVC irradiation in a time-dependent manner. Cell death was gradually stimulated for 9 hours of cultivation after a UVC irradiation period of 10 or 30 min. Epigallocatechin gallate (EGCG), which is one of the main catechins found in green tea, suppressed the increase in LDH activity in culture medium and also inhibited the formation of lipid peroxide. IκB, a member of the cell death signaling system, was phosphorylated at 1 hour after 10 min of UVC irradiation. Stimulation of phosphorylation of IκB by UVC was suppressed by the addition of EGCG. We concluded that EGCG protects the PC12 cell from cell damage caused by UVC irradiation. (author)

Cutting edge: CD95 maintains effector T cell homeostasis in chronic immune activation

NARCIS (Netherlands)

Arens, Ramon; Baars, Paul A.; Jak, Margot; Tesselaar, Kiki; van der Valk, Martin; van Oers, Marinus H. J.; van Lier, René A. W.

2005-01-01

The elimination of activated T cells is important to maintain homeostasis and avoid immunopathology. CD95 (Fas/APO-1) has been identified as a death mediator for activated T cells in vitro but the function of CD95 in death of mature T cells in vivo is still controversial. Here we show that

TRAIL enhances paracetamol-induced liver sinusoidal endothelial cell death in a Bim- and Bid-dependent manner

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Badmann, A; Langsch, S; Keogh, A; Brunner, T; Kaufmann, T; Corazza, N

2012-01-01

Paracetamol (acetaminophen, APAP) is a universally used analgesic and antipyretic agent. Considered safe at therapeutic doses, overdoses cause acute liver damage characterized by centrilobular hepatic necrosis. One of the major clinical problems of paracetamol-induced liver disease is the development of hemorrhagic alterations. Although hepatocytes represent the main target of the cytotoxic effect of paracetamol overdose, perturbations within the endothelium involving morphological changes of liver sinusoidal endothelial cells (LSECs) have also been described in paracetamol-induced liver disease. Recently, we have shown that paracetamol-induced liver damage is synergistically enhanced by the TRAIL signaling pathway. As LSECs are constantly exposed to activated immune cells expressing death ligands, including TRAIL, we investigated the effect of TRAIL on paracetamol-induced LSEC death. We here demonstrate for the first time that TRAIL strongly enhances paracetamol-mediated LSEC death with typical features of apoptosis. Inhibition of caspases using specific inhibitors resulted in a strong reduction of cell death. TRAIL appears to enhance paracetamol-induced LSEC death via the activation of the pro-apoptotic BH3-only proteins Bid and Bim, which initiate the mitochondrial apoptotic pathway. Taken together this study shows that the liver endothelial layer, mainly LSECs, represent a direct target of the cytotoxic effect of paracetamol and that activation of TRAIL receptor synergistically enhances paracetamol-induced LSEC death via the mitochondrial apoptotic pathway. TRAIL-mediated acceleration of paracetamol-induced cell death may thus contribute to the pathogenesis of paracetamol-induced liver damage. PMID:23254290

A Petiveria alliacea standardized fraction induces breast adenocarcinoma cell death by modulating glycolytic metabolism.

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Hernández, John Fredy; Urueña, Claudia Patricia; Cifuentes, Maria Claudia; Sandoval, Tito Alejandro; Pombo, Luis Miguel; Castañeda, Diana; Asea, Alexzander; Fiorentino, Susana

2014-05-14

Folk medicine uses aqueous and alcoholic extracts from Petiveria alliacea (Phytolaccaceae) in leukemia and breast cancer treatment in the Caribbean, Central and South America. Herein, we validated the biological activity of a Petiveria alliacea fraction using a metastatic breast adenocarcinoma model (4T1). Petiveria alliacea fraction biological activity was determined estimating cell proliferation, cell colony growth capacity and apoptosis (caspase-3 activity, DNA fragmentation and mitochondrial membrane potential) in 4T1 cells. Petiveria alliacea was used at IC₅₀ concentration (29 µg/mL) and 2 dilutions below, doxorubicin at 0.27 µg/mL (positive control) and dibenzyl disulfide at 2.93 µg/mL (IC50 fraction marker compound). Proteomic estimations were analyzed by LC-MS-MS. Protein level expression was confirmed by RT-PCR. Glucose and lactate levels were measured by enzymatic assays. LD50 was established in BALB/c mice and antitumoral activity evaluated in mice transplanted with GFP-tagged 4T1 cells. Mice were treated with Petiveria alliacea fraction via I.P (182 mg/kg corresponding to 1/8 of LD₅₀ and 2 dilutions below). Petiveria alliacea fraction in vitro induces 4T1 cells apoptosis, caspase-3 activation, DNA fragmentation without mitochondria membrane depolarization, and decreases cell colony growth capacity. Also, changes in glycolytic enzymes expression cause a decrease in glucose uptake and lactate production. Fraction also promotes breast primary tumor regression in BALB/c mice transplanted with GFP-tagged 4T1 cells. A fraction of Petiveria alliacea leaves and stems induces in vitro cell death and in vivo tumor regression in a murine breast cancer model. Our results validate in partly, the traditional use of Petiveria alliacea in breast cancer treatment, revealing a new way of envisioning Petiveria alliacea biological activity. The fraction effect on the glycolytic pathway enzymes contributes to explain the antiproliferative and antitumor activities

CD8+ T Cells Induce Fatal Brainstem Pathology during Cerebral Malaria via Luminal Antigen-Specific Engagement of Brain Vasculature.

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Phillip A Swanson

2016-12-01

Full Text Available Cerebral malaria (CM is a severe complication of Plasmodium falciparum infection that results in thousands of deaths each year, mostly in African children. The in vivo mechanisms underlying this fatal condition are not entirely understood. Using the animal model of experimental cerebral malaria (ECM, we sought mechanistic insights into the pathogenesis of CM. Fatal disease was associated with alterations in tight junction proteins, vascular breakdown in the meninges / parenchyma, edema, and ultimately neuronal cell death in the brainstem, which is consistent with cerebral herniation as a cause of death. At the peak of ECM, we revealed using intravital two-photon microscopy that myelomonocytic cells and parasite-specific CD8+ T cells associated primarily with the luminal surface of CNS blood vessels. Myelomonocytic cells participated in the removal of parasitized red blood cells (pRBCs from cerebral blood vessels, but were not required for the disease. Interestingly, the majority of disease-inducing parasite-specific CD8+ T cells interacted with the lumen of brain vascular endothelial cells (ECs, where they were observed surveying, dividing, and arresting in a cognate peptide-MHC I dependent manner. These activities were critically dependent on IFN-γ, which was responsible for activating cerebrovascular ECs to upregulate adhesion and antigen-presenting molecules. Importantly, parasite-specific CD8+ T cell interactions with cerebral vessels were impaired in chimeric mice rendered unable to present EC antigens on MHC I, and these mice were in turn resistant to fatal brainstem pathology. Moreover, anti-adhesion molecule (LFA-1 / VLA-4 therapy prevented fatal disease by rapidly displacing luminal CD8+ T cells from cerebrovascular ECs without affecting extravascular T cells. These in vivo data demonstrate that parasite-specific CD8+ T cell-induced fatal vascular breakdown and subsequent neuronal death during ECM is associated with luminal, antigen

Lapatinib induces autophagic cell death and differentiation in acute myeloblastic leukemia

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Chen YJ

2016-07-01

Full Text Available Yu-Jen Chen,1–4 Li-Wen Fang,5 Wen-Chi Su,6,7 Wen-Yi Hsu,1 Kai-Chien Yang,1 Huey-Lan Huang8 1Department of Medical Research, 2Department of Radiation Oncology, Mackay Memorial Hospital, 3Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, 4Institute of Pharmacology, Taipei Medical University, Taipei, 5Department of Nutrition, I-Shou University, Kaohsiung, 6Research Center for Emerging Viruses, China Medical University Hospital, 7Graduate Institute of Clinical Medical Science, China Medical University, Taichung, 8Department of Bioscience Technology, College of Health Science, Chang Jung Christian University, Tainan, Taiwan, Republic of China Abstract: Lapatinib is an oral-form dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR or ErbB/Her superfamily members with anticancer activity. In this study, we examined the effects and mechanism of action of lapatinib on several human leukemia cells lines, including acute myeloid leukemia (AML, chronic myeloid leukemia (CML, and acute lymphoblastic leukemia (ALL cells. We found that lapatinib inhibited the growth of human AML U937, HL-60, NB4, CML KU812, MEG-01, and ALL Jurkat T cells. Among these leukemia cell lines, lapatinib induced apoptosis in HL-60, NB4, and Jurkat cells, but induced nonapoptotic cell death in U937, K562, and MEG-01 cells. Moreover, lapatinib treatment caused autophagic cell death as shown by positive acridine orange staining, the massive formation of vacuoles as seen by electronic microscopy, and the upregulation of LC3-II, ATG5, and ATG7 in AML U937 cells. Furthermore, autophagy inhibitor 3-methyladenine and knockdown of ATG5, ATG7, and Beclin-1 using short hairpin RNA (shRNA partially rescued lapatinib-induced cell death. In addition, the induction of phagocytosis and ROS production as well as the upregulation of surface markers CD14 and CD68 was detected in lapatinib-treated U937 cells, suggesting the induction of

Serratia marcescens Induces Apoptotic Cell Death in Host Immune Cells via a Lipopolysaccharide- and Flagella-dependent Mechanism*

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Ishii, Kenichi; Adachi, Tatsuo; Imamura, Katsutoshi; Takano, Shinya; Usui, Kimihito; Suzuki, Kazushi; Hamamoto, Hiroshi; Watanabe, Takeshi; Sekimizu, Kazuhisa

2012-01-01

Injection of Serratia marcescens into the blood (hemolymph) of the silkworm, Bombyx mori, induced the activation of c-Jun NH2-terminal kinase (JNK), followed by caspase activation and apoptosis of blood cells (hemocytes). This process impaired the innate immune response in which pathogen cell wall components, such as glucan, stimulate hemocytes, leading to the activation of insect cytokine paralytic peptide. S. marcescens induced apoptotic cell death of silkworm hemocytes and mouse peritoneal macrophages in vitro. We searched for S. marcescens transposon mutants with attenuated ability to induce apoptosis of silkworm hemocytes. Among the genes identified, disruption mutants of wecA (a gene involved in lipopolysaccharide O-antigen synthesis), and flhD and fliR (essential genes in flagella synthesis) showed reduced motility and impaired induction of mouse macrophage cell death. These findings suggest that S. marcescens induces apoptosis of host immune cells via lipopolysaccharide- and flagella-dependent motility, leading to the suppression of host innate immunity. PMID:22859304

Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

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Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Sun, Kaiji; Zhang, Qing; Wu, Guoyao

2016-01-01

High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P L-cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P L-cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

Streptococcus sanguinis induces foam cell formation and cell death of macrophages in association with production of reactive oxygen species.

Science.gov (United States)

Okahashi, Nobuo; Okinaga, Toshinori; Sakurai, Atsuo; Terao, Yutaka; Nakata, Masanobu; Nakashima, Keisuke; Shintani, Seikou; Kawabata, Shigetada; Ooshima, Takashi; Nishihara, Tatsuji

2011-10-01

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, is a common streptococcal species implicated in infective endocarditis. Herein, we investigated the effects of infection with S. sanguinis on foam cell formation and cell death of macrophages. Infection with S. sanguinis stimulated foam cell formation of THP-1, a human macrophage cell line. At a multiplicity of infection >100, S. sanguinis-induced cell death of the macrophages. Viable bacterial infection was required to trigger cell death because heat-inactivated S. sanguinis did not induce cell death. The production of cytokines interleukin-1β and tumor necrosis factor-α from macrophages was also stimulated during bacterial infection. Inhibition of the production of reactive oxygen species (ROS) resulted in reduced cell death, suggesting an association of ROS with cell death. Furthermore, S. sanguinis-induced cell death appeared to be independent of activation of inflammasomes, because cleavage of procaspase-1 was not evident in infected macrophages. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

The mechanism of radiation-induced interphase death of lymphoid cells: A new hypothesis

International Nuclear Information System (INIS)

Eidus, L.K.; Korystov, Yu.N.; Dobrovinskaja, O.R.; Shaposhnikova, V.V.

1990-01-01

The interphase death of irradiated rat thymocytes depends on their concentration during postirradiation incubation. The kinetics of pycnosis and cell death determined with the trypan blue exclusion test in the samples with the highest cell concentration (1-2 x 10(7) cells/ml) is consistent with the data available in the literature, whereas the samples with the lowest concentration (2 x 10(5) cells/ml) undergo almost no pycnosis and death after irradiation with doses up to 50 Gy. On the basis of these results, we suggest a new mechanism of interphase death involving an interaction between irradiated thymocytes and the fraction of thymus cells possessing cytocidal activity. The observed correlation between the cytocidal activity and interphase death of thymocytes from animals of different ages favors our mechanism. It was found that the inhibitors which prevent the conjugation of killer cells and their targets do not influence interphase death, while the substances which block the secretion of cytotoxic factors or their action on the target membrane do protect from interphase death. Thus we suggest that the irradiation activates the killer cells to secrete some cytotoxic factors which induce pycnosis and interphase death of thymocytes

DRAM triggers lysosomal membrane permeabilization and cell death in CD4(+ T cells infected with HIV.

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Mireille Laforge

Full Text Available Productive HIV infection of CD4(+ T cells leads to a caspase-independent cell death pathway associated with lysosomal membrane permeabilization (LMP and cathepsin release, resulting in mitochondrial outer membrane permeabilization (MOMP. Herein, we demonstrate that HIV infection induces damage-regulated autophagy modulator (DRAM expression in a p53-dependent manner. Knocking down the expression of DRAM and p53 genes with specific siRNAs inhibited autophagy and LMP. However, inhibition of Atg5 and Beclin genes that prevents autophagy had a minor effect on LMP and cell death. The knock down of DRAM gene inhibited cytochrome C release, MOMP and cell death. However, knocking down DRAM, we increased viral infection and production. Our study shows for the first time the involvement of DRAM in host-pathogen interactions, which may represent a mechanism of defense via the elimination of infected cells.

Pro- and anti-apoptotic CD95 signaling in T cells

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Janssen Ottmar

2011-04-01

Full Text Available Abstract The TNF receptor superfamily member CD95 (Fas, APO-1, TNFRSF6 is known as the prototypic death receptor in and outside the immune system. In fact, many mechanisms involved in apoptotic signaling cascades were solved by addressing consequences and pathways initiated by CD95 ligation in activated T cells or other "CD95-sensitive" cell populations. As an example, the binding of the inducible CD95 ligand (CD95L to CD95 on activated T lymphocytes results in apoptotic cell death. This activation-induced cell death was implicated in the control of immune cell homeostasis and immune response termination. Over the past years, however, it became evident that CD95 acts as a dual function receptor that also exerts anti-apoptotic effects depending on the cellular context. Early observations of a potential non-apoptotic role of CD95 in the growth control of resting T cells were recently reconsidered and revealed quite unexpected findings regarding the costimulatory capacity of CD95 for primary T cell activation. It turned out that CD95 engagement modulates TCR/CD3-driven signal initiation in a dose-dependent manner. High doses of immobilized CD95 agonists or cellular CD95L almost completely silence T cells by blocking early TCR-induced signaling events. In contrast, under otherwise unchanged conditions, lower amounts of the same agonists dramatically augment TCR/CD3-driven activation and proliferation. In the present overview, we summarize these recent findings with a focus on the costimulatory capacity of CD95 in primary T cells and discuss potential implications for the T cell compartment and the interplay between T cells and CD95L-expressing cells including antigen-presenting cells.

Corosolic Acid Induces Non-Apoptotic Cell Death through Generation of Lipid Reactive Oxygen Species Production in Human Renal Carcinoma Caki Cells

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Seon Min Woo

2018-04-01

Full Text Available Corosolic acid is one of the pentacyclic triterpenoids isolated from Lagerstroemia speciose and has been reported to exhibit anti-cancer and anti-proliferative activities in various cancer cells. In the present study, we investigated the molecular mechanisms of corosolic acid in cancer cell death. Corosolic acid induces a decrease of cell viability and an increase of cell cytotoxicity in human renal carcinoma Caki cells. Corosolic acid-induced cell death is not inhibited by apoptosis inhibitor (z-VAD-fmk, a pan-caspase inhibitor, necroptosis inhibitor (necrostatin-1, or ferroptosis inhibitors (ferrostatin-1 and deferoxamine (DFO. Furthermore, corosolic acid significantly induces reactive oxygen species (ROS levels, but antioxidants (N-acetyl-l-cysteine (NAC and trolox do not inhibit corosolic acid-induced cell death. Interestingly, corosolic acid induces lipid oxidation, and α-tocopherol markedly prevents corosolic acid-induced lipid peroxidation and cell death. Anti-chemotherapeutic effects of α-tocopherol are dependent on inhibition of lipid oxidation rather than inhibition of ROS production. In addition, corosolic acid induces non-apoptotic cell death in other renal cancer (ACHN and A498, breast cancer (MDA-MB231, and hepatocellular carcinoma (SK-Hep1 and Huh7 cells, and α-tocopherol markedly inhibits corosolic acid-induced cell death. Therefore, our results suggest that corosolic acid induces non-apoptotic cell death in cancer cells through the increase of lipid peroxidation.

Dying cells protect survivors from radiation-induced cell death in Drosophila.

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Amber Bilak

2014-03-01

Full Text Available We report a phenomenon wherein induction of cell death by a variety of means in wing imaginal discs of Drosophila larvae resulted in the activation of an anti-apoptotic microRNA, bantam. Cells in the vicinity of dying cells also become harder to kill by ionizing radiation (IR-induced apoptosis. Both ban activation and increased protection from IR required receptor tyrosine kinase Tie, which we identified in a genetic screen for modifiers of ban. tie mutants were hypersensitive to radiation, and radiation sensitivity of tie mutants was rescued by increased ban gene dosage. We propose that dying cells activate ban in surviving cells through Tie to make the latter cells harder to kill, thereby preserving tissues and ensuring organism survival. The protective effect we report differs from classical radiation bystander effect in which neighbors of irradiated cells become more prone to death. The protective effect also differs from the previously described effect of dying cells that results in proliferation of nearby cells in Drosophila larval discs. If conserved in mammals, a phenomenon in which dying cells make the rest harder to kill by IR could have implications for treatments that involve the sequential use of cytotoxic agents and radiation therapy.

Nitric oxide production is not required for dihydrosphingosine-induced cell death in tobacco BY-2 cells.

Science.gov (United States)

Da Silva, Daniel; Lachaud, Christophe; Cotelle, Valérie; Brière, Christian; Grat, Sabine; Mazars, Christian; Thuleau, Patrice

2011-05-01

Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid Long Chain Base (LCB) in plants, is known to induce a calcium dependent programmed cell death (PCD) in tobacco BY-2 cells. In addition, we have recently shown that DHS triggers a production of H2O2, via the activation of NADPH oxidase(s). However, this production of H2O2 is not correlated with the DHS-induced cell death but would rather be associated with basal cell defense mechanisms. In the present study, we extend our current knowledge of the DHS signaling pathway, by demonstrating that DHS also promotes a production of nitric oxide (NO) in tobacco BY-2 cells. As for H2O2, this NO production is not necessary for cell death induction. 

Effects of intracellular iron overload on cell death and identification of potent cell death inhibitors.

Science.gov (United States)

Fang, Shenglin; Yu, Xiaonan; Ding, Haoxuan; Han, Jianan; Feng, Jie

2018-06-11

Iron overload causes many diseases, while the underlying etiologies of these diseases are unclear. Cell death processes including apoptosis, necroptosis, cyclophilin D-(CypD)-dependent necrosis and a recently described additional form of regulated cell death called ferroptosis, are dependent on iron or iron-dependent reactive oxygen species (ROS). However, whether the accumulation of intracellular iron itself induces ferroptosis or other forms of cell death is largely elusive. In present study, we study the role of intracellular iron overload itself-induced cell death mechanisms by using ferric ammonium citrate (FAC) and a membrane-permeable Ferric 8-hydroxyquinoline complex (Fe-8HQ) respectively. We show that FAC-induced intracellular iron overload causes ferroptosis. We also identify 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor GSK2334470 as a potent ferroptosis inhibitor. Whereas, Fe-8HQ-induced intracellular iron overload causes unregulated necrosis, but partially activates PARP-1 dependent parthanatos. Interestingly, we identify many phenolic compounds as potent inhibitors of Fe-8HQ-induced cell death. In conclusion, intracellular iron overload-induced cell death form might be dependent on the intracellular iron accumulation rate, newly identified cell death inhibitors in our study that target ferroptosis and unregulated oxidative cell death represent potential therapeutic strategies against iron overload related diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

Canthin-6-one induces cell death, cell cycle arrest and differentiation in human myeloid leukemia cells.

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Vieira Torquato, Heron F; Ribeiro-Filho, Antonio C; Buri, Marcus V; Araújo Júnior, Roberto T; Pimenta, Renata; de Oliveira, José Salvador R; Filho, Valdir C; Macho, Antonio; Paredes-Gamero, Edgar J; de Oliveira Martins, Domingos T

2017-04-01

Canthin-6-one is a natural product isolated from various plant genera and from fungi with potential antitumor activity. In the present study, we evaluate the antitumor effects of canthin-6-one in human myeloid leukemia lineages. Kasumi-1 lineage was used as a model for acute myeloid leukemia. Cells were treated with canthin-6-one and cell death, cell cycle and differentiation were evaluated in both total cells (Lin + ) and leukemia stem cell population (CD34 + CD38 - Lin -/low ). Among the human lineages tested, Kasumi-1 was the most sensitive to canthin-6-one. Canthin-6-one induced cell death with apoptotic (caspase activation, decrease of mitochondrial potential) and necrotic (lysosomal permeabilization, double labeling of annexin V/propidium iodide) characteristics. Moreover, canthin-6-one induced cell cycle arrest at G 0 /G 1 (7μM) and G 2 (45μM) evidenced by DNA content, BrdU incorporation and cyclin B1/histone 3 quantification. Canthin-6-one also promoted differentiation of Kasumi-1, evidenced by an increase in the expression of myeloid markers (CD11b and CD15) and the transcription factor PU.1. Furthermore, a reduction of the leukemic stem cell population and clonogenic capability of stem cells were observed. These results show that canthin-6-one can affect Kasumi-1 cells by promoting cell death, cell cycle arrest and cell differentiation depending on concentration used. Canthin-6-one presents an interesting cytotoxic activity against leukemic cells and represents a promising scaffold for the development of molecules for anti-leukemic applications, especially by its anti-leukemic stem cell activity. Copyright © 2017 Elsevier B.V. All rights reserved.

Up-regulation of K{sub ir}2.1 by ER stress facilitates cell death of brain capillary endothelial cells

Energy Technology Data Exchange (ETDEWEB)

Kito, Hiroaki [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Yamazaki, Daiju [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Department of Biological Chemistry, Kyoto University, Graduate School of Pharmaceutical Sciences, Kyoto (Japan); Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Ohya, Susumu; Yamamura, Hisao [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Asai, Kiyofumi [Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Imaizumi, Yuji, E-mail: yimaizum@phar.nagoya-cu.ac.jp [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan)

2011-07-29

Highlights: {yields} We found that application of endoplasmic reticulum (ER) stress with tunicamycin to brain capillary endothelial cells (BCECs) induced cell death. {yields} The ER stress facilitated the expression of inward rectifier K{sup +} channel (K{sub ir}2.1) and induced sustained membrane hyperpolarization. {yields} The membrane hyperpolarization induced sustained Ca{sup 2+} entry through voltage-independent nonspecific cation channels and consequently facilitated cell death. {yields} The K{sub ir}2.1 up-regulation by ER stress is, at least in part, responsible for cell death of BCECs under pathological conditions. -- Abstract: Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cell turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K{sup +} channel (K{sub ir}2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K{sub ir} channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca{sup 2+} concentration due to Ca{sup 2+} influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K{sub ir}2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.

Leptin suppresses non-apoptotic cell death in ischemic rat cardiomyocytes by reduction of iPLA2 activity

International Nuclear Information System (INIS)

Takatani-Nakase, Tomoka; Takahashi, Koichi

2015-01-01

Caspase-independent, non-apoptotic cell death is an important therapeutic target in myocardial ischemia. Leptin, an adipose-derived hormone, is known to exhibit cytoprotective effects on the ischemic heart, but the mechanisms are poorly understood. In this research, we found that pretreatment of leptin strongly suppressed ischemic-augmented nuclear shrinkage and non-apoptotic cell death on cardiomyocytes. Leptin was also shown to significantly inhibit the activity of iPLA 2 , which is considered to play crucial roles in non-apoptotic cell death, resulting in effective prevention of ischemia-induced myocyte death. These findings provide the first evidence of a protective mechanism of leptin against ischemia-induced non-apoptotic cardiomyocyte death. - Highlights: • Myocardial ischemia-model induces in caspase-independent, non-apoptotic cell death. • Leptin strongly inhibits ischemic-augmented non-apoptotic cell death. • Leptin reduces iPLA 2 activity, leading to avoidance of non-apoptotic cell death

Effects of aromatic amino acids on glutamate-induced neuronal cell death

International Nuclear Information System (INIS)

Zafar, Z.; Sumners, C.

2005-01-01

Glutamate accumulation is believed to lead to overstimulation of glutamate receptors which results in neuronal death. The protective effects of aromatic amino acids on glutamate induced neuronal cell death were examined using rat cerebral cortical neurons. Neuronal death is quantified by measuring lactate dehydrogenase (LDH) using a spectrophotometric microtiter plate reader (ELISA reader). Neuronal cells were incubated with varying doses of glutamate plus or minus the aromatic amino acid D-Phenylalanine (D-Phe) for different time periods to observe protection against cytotoxicity. Percent cytotoxicity was seen to follow a dose dependent rise with increasing concentrations of glutamate, reaching a plateau at around 100 -500 uM glutamate. Lower levels of cytotoxicity were achieved with cell exposed to D-Phe and Dibromo tyrosine (DBrT). 48-hour experimental runs were also carried out to further investigate the mode of action of D-Phe. It was found that the difference between cytotoxicity levels of control cells and protected cells was higher over longer time. (author)

Phytol isolated from watermelon (Citrullus lanatus) sprouts induces cell death in human T-lymphoid cell line Jurkat cells via S-phase cell cycle arrest.

Science.gov (United States)

Itoh, Tomohiro; Ono, Akito; Kawaguchi, Kaori; Teraoka, Sayaka; Harada, Mayo; Sumi, Keitaro; Ando, Masashi; Tsukamasa, Yasuyuki; Ninomiya, Masayuki; Koketsu, Mamoru; Hashizume, Toshiharu

2018-05-01

The phytol isolated from watermelon (Citrullus lanatus) sprouts inhibited the growth of a human T-cell leukemia line Jurkat cell and suppressed tumor progression in a xenograft model of human lung adenocarcinoma epithelial cell line A549 in nude mice. To elucidate the mechanisms underlying the phytol-induced cell death in the present study, we examined the changes in cell morphology, DNA fragmentation, and intracellular reactive oxygen species (ROS) levels and performed flow cytometric analysis to evaluate cell cycle stage. There were no significant changes in apoptosis, autophagy, and necrosis marker in cells treated with the phytol. But, we found, for the first time, that phytol remarkably induced S-phase cell cycle arrest accompanied with intracellular ROS production. Western blot analyses showed that phytolinduced S-phase cell cycle arrest was mediated through the decreased expression of cyclins A and D and the downregulations of MAPK and PI3K/Akt. The tumor volume levels in mice treated with phytol were lower than those of non-treatment groups, and it showed very similar suppression compared with those of mice treated with cyclophosphamide. Based on the data of in vitro and in vivo studies and previous studies, we suggest phytol as a potential therapeutic compound for cancer. Copyright © 2018 Elsevier Ltd. All rights reserved.

Novel oxindole derivatives prevent oxidative stress-induced cell death in mouse hippocampal HT22 cells.

Science.gov (United States)

Hirata, Yoko; Yamada, Chika; Ito, Yuki; Yamamoto, Shotaro; Nagase, Haruna; Oh-Hashi, Kentaro; Kiuchi, Kazutoshi; Suzuki, Hiromi; Sawada, Makoto; Furuta, Kyoji

2018-03-15

The current medical and surgical therapies for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease offer symptomatic relief but do not provide a cure. Thus, small synthetic compounds that protect neuronal cells from degeneration are critically needed to prevent and treat these. Oxidative stress has been implicated in various pathophysiological conditions, including neurodegenerative diseases. In a search for neuroprotective agents against oxidative stress using the murine hippocampal HT22 cell line, we found a novel oxindole compound, GIF-0726-r, which prevented oxidative stress-induced cell death, including glutamate-induced oxytosis and erastin-induced ferroptosis. This compound also exerted a protective effect on tunicamycin-induced ER stress to a lesser extent but had no effect on campthothecin-, etoposide- or staurosporine-induced apoptosis. In addition, GIF-0726-r was also found to be effective after the occurrence of oxidative stress. GIF-0726-r was capable of inhibiting reactive oxygen species accumulation and Ca 2+ influx, a presumed executor in cell death, and was capable of activating the antioxidant response element, which is a cis-acting regulatory element in promoter regions of several genes encoding phase II detoxification enzymes and antioxidant proteins. These results suggest that GIF-0726-r is a low-molecular-weight compound that prevents neuronal cell death through attenuation of oxidative stress. Among the more than 200 derivatives of the GIF-0726-r synthesized, we identified the 11 most potent activators of the antioxidant response element and characterized their neuroprotective activity in HT22 cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Interaction of CD154 with the α5β1 Integrin Inhibits Fas-Induced T Cell Death.

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Bachsais, Meriem; Naddaf, Nadim; Yacoub, Daniel; Salti, Suzanne; Alaaeddine, Nada; Aoudjit, Fawzi; Hassan, Ghada S; Mourad, Walid

2016-01-01

CD154, a critical regulator of the immune response, is usually associated with chronic inflammatory, autoimmune diseases as well as malignant disorders. In addition to its classical receptor CD40, CD154 is capable of binding other receptors, members of the integrin family, the αIIbβ3, αMβ2 and α5β1. Given the role attributed to integrins and particularly the β1 integrins in inhibiting apoptotic events in normal as well as malignant T cells, we were highly interested in investigating the role of the CD154/α5β1 interaction in promoting survival of malignant T cells contributing as such to tumor development and/or propagation. To support our hypothesis, we first show that soluble CD154 binds to the T-cell acute lymphoblastic leukemia cell line, Jurkat E6.1 in a α5β1-dependent manner. Binding of soluble CD154 to α5β1 integrin of Jurkat cells leads to the activation of key survival proteins, including the p38 and ERK1/2 mitogen-activated protein kinases (MAPKs), phosphoinositide 3 kinase (PI-3K), and Akt. Interestingly, soluble CD154 significantly inhibits Fas-mediated apoptosis in T cell leukemia-lymphoma cell lines, Jurkat E6.1 and HUT78 cells, an important hallmark of T cell survival during malignancy progression. These anti-apoptotic effects were mainly mediated by the activation of the PI-3K/Akt pathway but also involved the p38 and the ERK1/2 MAPKs cascades. Our data also demonstrated that the CD154-triggered inhibition of the Fas-mediated cell death response was dependent on a suppression of caspase-8 cleavage, but independent of de novo protein synthesis or alterations in Fas expression on cell surface. Together, our results highlight the impact of the CD154/α5β1 interaction in T cell function/survival and identify novel targets for the treatment of malignant disorders, particularly of T cell origin.

The Interaction of CD154 with the α5β1 Integrin Inhibits Fas-Induced T Cell Death.

Directory of Open Access Journals (Sweden)

Meriem Bachsais

Full Text Available CD154, a critical regulator of the immune response, is usually associated with chronic inflammatory, autoimmune diseases as well as malignant disorders. In addition to its classical receptor CD40, CD154 is capable of binding other receptors, members of the integrin family, the αIIbβ3, αMβ2 and α5β1. Given the role attributed to integrins and particularly the β1 integrins in inhibiting apoptotic events in normal as well as malignant T cells, we were highly interested in investigating the role of the CD154/α5β1 interaction in promoting survival of malignant T cells contributing as such to tumor development and/or propagation. To support our hypothesis, we first show that soluble CD154 binds to the T-cell acute lymphoblastic leukemia cell line, Jurkat E6.1 in a α5β1-dependent manner. Binding of soluble CD154 to α5β1 integrin of Jurkat cells leads to the activation of key survival proteins, including the p38 and ERK1/2 mitogen-activated protein kinases (MAPKs, phosphoinositide 3 kinase (PI-3K, and Akt. Interestingly, soluble CD154 significantly inhibits Fas-mediated apoptosis in T cell leukemia-lymphoma cell lines, Jurkat E6.1 and HUT78 cells, an important hallmark of T cell survival during malignancy progression. These anti-apoptotic effects were mainly mediated by the activation of the PI-3K/Akt pathway but also involved the p38 and the ERK1/2 MAPKs cascades. Our data also demonstrated that the CD154-triggered inhibition of the Fas-mediated cell death response was dependent on a suppression of caspase-8 cleavage, but independent of de novo protein synthesis or alterations in Fas expression on cell surface. Together, our results highlight the impact of the CD154/α5β1 interaction in T cell function/survival and identify novel targets for the treatment of malignant disorders, particularly of T cell origin.

Curcumin induces autophagic cell death in Spodoptera frugiperda cells.

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Veeran, Sethuraman; Shu, Benshui; Cui, Gaofeng; Fu, Shengjiao; Zhong, Guohua

2017-06-01

The increasing interest in the role of autophagy (type II cell death) in the regulation of insect toxicology has propelled study of investigating autophagic cell death pathways. Turmeric, the rhizome of the herb Curcuma longa (Mañjaḷ in Tamil, India and Jiānghuáng in Chinese) have been traditionally used for the pest control either alone or combination with other botanical pesticides. However, the mechanisms by which Curcuma longa or curcumin exerts cytotoxicity in pests are not well understood. In this study, we investigated the potency of Curcuma longa (curcumin) as a natural pesticide employing Sf9 insect line. Autophagy induction effect of curcumin on Spodoptera frugiperda (Sf9) cells was investigated using various techniques including cell proliferation assay, morphology analysis with inverted phase contrast microscope and Transmission Electron Microscope (TEM) analysis. Autophagy was evaluated using the fluorescent dye monodansylcadaverine (MDC). Cell death measurement was examined using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) within the concentrations of 5-15μg/mL. Curcumin inhibited the growth of the Sf9 cells and induced autophagic cell death in a time and dose dependent manner. Staining the cells with MDC showed the presence of autophagic vacuoles while increased in a dose and time dependent manner. At the ultrastructural level transmission electron microscopy, cells revealed massive autophagy vacuole accumulation and absence of chromatin condensation. Protein expression levels of ATG8-I and ATG8-II, well-established markers of autophagy related protein were elevated in a time dependent manner after curcumin treatment. The present study proves that curcumin induces autophagic cell death in Sf9 insect cell line and this is the first report of cytotoxic effect of curcumin in insect cells and that will be utilized as natural pesticides in future. Copyright © 2017. Published by Elsevier Inc.

Dioscin induces caspase-independent apoptosis through activation of apoptosis-inducing factor in breast cancer cells.

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Kim, Eun-Ae; Jang, Ji-Hoon; Lee, Yun-Han; Sung, Eon-Gi; Song, In-Hwan; Kim, Joo-Young; Kim, Suji; Sohn, Ho-Yong; Lee, Tae-Jin

2014-07-01

Dioscin, a saponin extracted from the roots of Polygonatum zanlanscianense, shows several bioactivities such as antitumor, antifungal, and antiviral properties. Although, dioscin is already known to induce cell death in variety cancer cells, the molecular basis for dioscin-induced cell death was not definitely known in cancer cells. In this study, we found that dioscin treatment induced cell death in dose-dependent manner in breast cancer cells such as MDA-MB-231, MDA-MB-453, and T47D cells. Dioscin decreased expressions of Bcl-2 and cIAP-1 proteins, which were down-regulated at the transcriptional level. Conversely, Mcl-1 protein level was down-regulated by facilitating ubiquitin/proteasome-mediated Mcl-1 degradation in dioscin-treated cells. Pretreatment with z-VAD fails to attenuate dioscin-induced cell death as well as caspase-mediated events such as cleavages of procaspase-3 and PARP. In addition, dioscin treatment increased the population of annexin V positive cells and induced DNA fragmentation in a dose-dependent manner in MDA-MB-231 cells. Furthermore, apoptosis inducing factor (AIF) was released from the mitochondria and translocated to the nucleus. Suppression in AIF expression by siRNA reduced dioscin-induced apoptosis in MDA-MB-231 cells. Taken together, our results demonstrate that dioscin-induced cell death was mediated via AIF-facilitating caspase-independent pathway as well as down-regulating anti-apoptotic proteins such as Bcl-2, cIAP-1, and Mcl-1 in breast cancer cells.

Inducible cell death in plant immunity

DEFF Research Database (Denmark)

Hofius, Daniel; Tsitsigiannis, Dimitrios I; Jones, Jonathan D G

2006-01-01

Programmed cell death (PCD) occurs during vegetative and reproductive plant growth, as typified by autumnal leaf senescence and the terminal differentiation of the endosperm of cereals which provide our major source of food. PCD also occurs in response to environmental stress and pathogen attack......, and these inducible PCD forms are intensively studied due their experimental tractability. In general, evidence exists for plant cell death pathways which have similarities to the apoptotic, autophagic and necrotic forms described in yeast and metazoans. Recent research aiming to understand these pathways...

Insulin receptor substrate-1 prevents autophagy-dependent cell death caused by oxidative stress in mouse NIH/3T3 cells

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Chan Shih-Hung

2012-07-01

Full Text Available Abstract Background Insulin receptor substrate (IRS-1 is associated with tumorigenesis; its levels are elevated in several human cancers. IRS-1 protein binds to several oncogene proteins. Oxidative stress and reactive oxygen species (ROS are involved in the initiation and progression of cancers. Cancer cells produce greater levels of ROS than normal cells do because of increased metabolic stresses. However, excessive production of ROS kills cancer cells. Autophagy usually serves as a survival mechanism in response to stress conditions, but excessive induction of autophagy results in cell death. In addition to inducing necrosis and apoptosis, ROS induces autophagic cell death. ROS inactivates IRS-1 mediated signaling and reduces intracellular IRS-1 concentrations. Thus, there is a complex relationship between IRS-1, ROS, autophagy, and cancer. It is not fully understood how cancer cells grow rapidly and survive in the presence of high ROS levels. Methods and results In this study, we established mouse NIH/3T3 cells that overexpressed IRS-1, so mimicking cancers with increased IRS-1 expression levels; we found that the IRS-1 overexpressing cells grow more rapidly than control cells do. Treatment of cells with glucose oxidase (GO provided a continuous source of ROS; low dosages of GO promoted cell growth, while high doses induced cell death. Evidence for GO induced autophagy includes increased levels of isoform B-II microtubule-associated protein 1 light chain 3 (LC3, aggregation of green fluorescence protein-tagged LC3, and increased numbers of autophagic vacuoles in cells. Overexpression of IRS-1 resulted in inhibition of basal autophagy, and reduced oxidative stress-induced autophagy and cell death. ROS decreased the mammalian target of rapamycin (mTOR/p70 ribosomal protein S6 kinase signaling, while overexpression of IRS-1 attenuated this inhibition. Knockdown of autophagy-related gene 5 inhibited basal autophagy and diminished oxidative stress-induced

Mechanisms of Virus-Induced Neural Cell Death

National Research Council Canada - National Science Library

Tyler, Kenneth

2002-01-01

Virtually all known neurotropic viruses are capable of killing infected cells by inducing a specific pattern of cell death known as apoptosis, yet the mechanism by which this occurs and its relevance...

A noncognate interaction with anti-receptor antibody-activated helper T cells induces small resting murine B cells to proliferate and to secrete antibody

DEFF Research Database (Denmark)

Owens, T

1988-01-01

on resting B cells (even in the presence of intact F23.1 antibody), but could induce antibody secretion by anti-Ig-preactivated B cells. Both F23.1+ clones (E9.D4 and 4.35F2) and one F23.1- clone (D2.2) could synergize with supernatants from activated E9.D4 T cells to induce B cell activation. F(ab')2......Culture of small resting allogeneic B cells (of an irrelevant haplotype) with two clones of T helper (Th) cells that were activated by the F23.1 anti-T cell receptor antibody led to the activation of B cells to proliferate and to secrete antibody. Th cell supernatants by themselves had no effect...... fragments of F23.1 induced E9.D4 to activate B cells as efficiently as intact F23.1 and B cell populations that had been incubated with F23.1 were not activated when cultured with E9.D4, although T cells recognized cell-presented F23.1 and were weakly activated. Reduction of the density of F23.1 adsorbed...

Combined treatment with cotylenin A and phenethyl isothiocyanate induces strong antitumor activity mainly through the induction of ferroptotic cell death in human pancreatic cancer cells.

Science.gov (United States)

Kasukabe, Takashi; Honma, Yoshio; Okabe-Kado, Junko; Higuchi, Yusuke; Kato, Nobuo; Kumakura, Shunichi

2016-08-01

The treatment of pancreatic cancer, one of the most aggressive gastrointestinal tract malignancies, with current chemotherapeutic drugs has had limited success due to its chemoresistance and poor prognosis. Therefore, the development of new drugs or effective combination therapies is urgently needed. Cotylenin A (CN-A) (a plant growth regulator) is a potent inducer of differentiation in myeloid leukemia cells and exhibits potent antitumor activities in several cancer cell lines. In the present study, we demonstrated that CN-A and phenethyl isothiocyanate (PEITC), an inducer of reactive oxygen species (ROS) and a dietary anticarcinogenic compound, synergistically inhibited the proliferation of MIAPaCa-2, PANC-1 and gemcitabine-resistant PANC-1 cells. A combined treatment with CN-A and PEITC also effectively inhibited the anchorage-independent growth of these cancer cells. The combined treatment with CN-A and PEITC strongly induced cell death within 1 day at concentrations at which CN-A or PEITC alone did not affect cell viability. A combined treatment with synthetic CN-A derivatives (ISIR-005 and ISIR-042) or fusicoccin J (CN-A-related natural product) and PEITC did not have synergistic effects on cell death. The combined treatment with CN-A and PEITC synergistically induced the generation of ROS. Antioxidants (N-acetylcysteine and trolox), ferroptosis inhibitors (ferrostatin-1 and liproxstatin), and the lysosomal iron chelator deferoxamine canceled the synergistic cell death. Apoptosis inhibitors (Z-VAD-FMK and Q-VD-OPH) and the necrosis inhibitor necrostatin-1s did not inhibit synergistic cell death. Autophagy inhibitors (3-metyladenine and chloroquine) partially prevented cell death. These results show that synergistic cell death induced by the combined treatment with CN-A and PEITC is mainly due to the induction of ferroptosis. Therefore, the combination of CN-A and PEITC has potential as a novel therapeutic strategy against pancreatic cancer.

Drug-Induced Liver Injury: Cascade of Events Leading to Cell Death, Apoptosis or Necrosis

Directory of Open Access Journals (Sweden)

Andrea Iorga

2017-05-01

Full Text Available Drug-induced liver injury (DILI can broadly be divided into predictable and dose dependent such as acetaminophen (APAP and unpredictable or idiosyncratic DILI (IDILI. Liver injury from drug hepatotoxicity (whether idiosyncratic or predictable results in hepatocyte cell death and inflammation. The cascade of events leading to DILI and the cell death subroutine (apoptosis or necrosis of the cell depend largely on the culprit drug. Direct toxins to hepatocytes likely induce oxidative organelle stress (such as endoplasmic reticulum (ER and mitochondrial stress leading to necrosis or apoptosis, while cell death in idiosyncratic DILI (IDILI is usually the result of engagement of the innate and adaptive immune system (likely apoptotic, involving death receptors (DR. Here, we review the hepatocyte cell death pathways both in direct hepatotoxicity such as in APAP DILI as well as in IDILI. We examine the known signaling pathways in APAP toxicity, a model of necrotic liver cell death. We also explore what is known about the genetic basis of IDILI and the molecular pathways leading to immune activation and how these events can trigger hepatotoxicity and cell death.

Semi-allogeneic dendritic cells can induce antigen-specific T-cell activation, which is not enhanced by concurrent alloreactivity.

Science.gov (United States)

Wells, James W; Cowled, Chris J; Darling, David; Guinn, Barbara-Ann; Farzaneh, Farzin; Noble, Alistair; Galea-Lauri, Joanna

2007-12-01

Alloreactive T-cell responses are known to result in the production of large amounts of proinflammatory cytokines capable of activating and maturing dendritic cells (DC). However, it is unclear whether these allogeneic responses could also act as an adjuvant for concurrent antigen-specific responses. To examine effects of simultaneous alloreactive and antigen-specific T-cell responses induced by semi-allogeneic DC. Semi-allogeneic DC were generated from the F(1) progeny of inbred strains of mice (C57BL/6 and C3H, or C57BL/6 and DBA). We directly primed antigen-specific CD8(+) and CD4(+) T-cells from OT-I and OT-II mice, respectively, in the absence of allogeneic responses, in vitro, and in the presence or absence of alloreactivity in vivo. In vitro, semi-allogeneic DC cross-presented ovalbumin (OVA) to naïve CD8(+) OT-I transgenic T-cells, primed naïve CD4(+) OT-II transgenic T-cells and could stimulate strong alloreactive T-cell proliferation in a primary mixed lymphocyte reaction (MLR). In vivo, semi-allogeneic DC migrated efficiently to regional lymph nodes but did not survive there as long as autologous DC. In addition, they were not able to induce cytotoxic T-lymphocyte (CTL) activity to a target peptide, and only weakly stimulated adoptively transferred OT-II cells. The CD4(+) response was unchanged in allo-tolerized mice, indicating that alloreactive T-cell responses could not provide help for concurrently activated antigen-specific responses. In an EL4 tumour-treatment model, vaccination with semi-allogeneic DC/EL4 fusion hybrids, but not allogeneic DC/EL4 hybrids, significantly increased mouse survival. Expression of self-Major histocompatibility complex (MHC) by semi-allogeneic DC can cause the induction of antigen-specific immunity, however, concurrently activated allogeneic bystander responses do not provide helper or adjuvant effects.

miR-145 induces caspase-dependent and -independent cell death in urothelial cancer cell lines with targeting of an expression signature present in Ta bladder tumors

DEFF Research Database (Denmark)

Ostenfeld, Marie Stampe; Bramsen, Jesper Bertram; Lamy, Philippe

2010-01-01

hybridization. Ectopic expression of miR-145 induced extensive apoptosis in urothelial carcinoma cell lines (T24 and SW780) as characterized by caspase activation, nuclear condensation and fragmentation, cellular shrinkage, and detachment. However, cell death also proceeded upon caspase inhibition...... sites. Among these, direct targeting of CBFB, PPP3CA, and CLINT1 was confirmed by a luciferase reporter assay. Notably, a 22-gene signature targeted on enforced miR-145 expression in T24 cells was significantly (P

Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

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Morotomi-Yano, Keiko; Akiyama, Hidenori [Institute of Pulsed Power Science, Kumamoto University, Kumamoto 860-8555 (Japan); Yano, Ken-ichi, E-mail: yanoken@kumamoto-u.ac.jp [Priority Organization for Innovation and Excellence, Kumamoto University, Kumamoto 860-8555 (Japan)

2013-08-30

Highlights: •Nanosecond pulsed electric field (nsPEF) is a new and unique means for life sciences. •Apoptosis was induced by nsPEF exposure in Jurkat cells. •No signs of apoptosis were detected in HeLa S3 cells exposed to nsPEFs. •Formation of poly(ADP-ribose) was induced in nsPEF-exposed HeLa S3 cells. •Two distinct modes of cell death were activated by nsPEF in a cell-dependent manner. -- Abstract: Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs.

Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

International Nuclear Information System (INIS)

Morotomi-Yano, Keiko; Akiyama, Hidenori; Yano, Ken-ichi

2013-01-01

Highlights: •Nanosecond pulsed electric field (nsPEF) is a new and unique means for life sciences. •Apoptosis was induced by nsPEF exposure in Jurkat cells. •No signs of apoptosis were detected in HeLa S3 cells exposed to nsPEFs. •Formation of poly(ADP-ribose) was induced in nsPEF-exposed HeLa S3 cells. •Two distinct modes of cell death were activated by nsPEF in a cell-dependent manner. -- Abstract: Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs

Vacquinol-1 inducible cell death in glioblastoma multiforme is counter regulated by TRPM7 activity induced by exogenous ATP.

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Sander, Philip; Mostafa, Haouraa; Soboh, Ayman; Schneider, Julian M; Pala, Andrej; Baron, Ann-Kathrin; Moepps, Barbara; Wirtz, C Rainer; Georgieff, Michael; Schneider, Marion

2017-05-23

Glioblastomas (GBM) are the most malignant brain tumors in humans and have a very poor prognosis. New therapeutic options are urgently needed. A novel drug, Vacquinol-1 (Vac), a quinolone derivative, displays promising properties by inducing rapid cell death in GBM but not in non-transformed tissues. Features of this type of cell death are compatible with a process termed methuosis. Here we tested Vac on a highly malignant glioma cell line observed by long-term video microscopy. Human dental-pulp stem cells (DPSCs) served as controls. A major finding was that an exogenous ATP concentration of as little as 1 μM counter regulated the Vac-induced cell death. Studies using carvacrol, an inhibitor of transient receptor potential cation channel, subfamily M, member 7 (TRPM7), demonstrated that the ATP-inducible inhibitory effect is likely to be via TRPM7. Exogenous ATP is of relevance in GBM with large necrotic areas. Our results support the use of GBM cultures with different grades of malignancy to address their sensitivity to methuosis. The video-microscopy approach presented here allows decoding of signaling pathways as well as mechanisms of chemotherapeutic resistance by long-term observation. Before implementing Vac as a novel therapeutic drug in GBM, cells from each individual patient need to be assessed for their ATP sensitivity. In summary, the current investigation supports the concept of methuosis, described as non-apoptotic cell death and a promising approach for GBM treatment. Tissue-resident ATP/necrosis may interfere with this cell-death pathway but can be overcome by a natural compound, carvacrol that even penetrates the blood-brain barrier.

The calcimimetic R-568 induces apoptotic cell death in prostate cancer cells

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Cheng Guangming

2009-07-01

Full Text Available Abstract Background Increased serum level of parathyroid hormone (PTH was found in metastatic prostate cancers. Calcimimetic R-568 was reported to reduce PTH expression, to suppress cell proliferation and to induce apoptosis in parathyroid cells. In this study, we investigated the effect of R-568 on cellular survival of prostate cancer cells. Methods Prostate cancer cell lines LNCaP and PC-3 were used in this study. Cellular survival was determined with MTT, trypan blue exclusion and fluorescent Live/Death assays. Western blot assay was utilized to assess apoptotic events induced by R-568 treatment. JC-1 staining was used to evaluate mitochondrial membrane potential. Results In cultured prostate cancer LNCaP and PC-3 cells, R-568 treatment significantly reduced cellular survival in a dose- and time-dependent manner. R-568-induced cell death was an apoptotic event, as evidenced by caspase-3 processing and PARP cleavage, as well as JC-1 color change in mitochondria. Knocking down calcium sensing receptor (CaSR significantly reduced R-568-induced cytotoxicity. Enforced expression of Bcl-xL gene abolished R-568-induced cell death, while loss of Bcl-xL expression led to increased cell death in R-568-treated LNCaP cells,. Conclusion Taken together, our data demonstrated that calcimimetic R-568 triggers an intrinsic mitochondria-related apoptotic pathway, which is dependent on the CaSR and is modulated by Bcl-xL anti-apoptotic pathway.

The Alpha-Melanocyte Stimulating Hormone Induces Conversion of Effector T Cells into Treg Cells

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Andrew W. Taylor

2011-01-01

Full Text Available The neuropeptide alpha-melanocyte stimulating hormone (α-MSH has an important role in modulating immunity and homeostasis. The production of IFN-γ by effector T cells is suppressed by α-MSH, while TGF-β production is promoted in the same cells. Such α-MSH-treated T cells have immune regulatory activity and suppress hypersensitivity, autoimmune diseases, and graft rejection. Previous characterizations of the α-MSH-induced Treg cells showed that the cells are CD4+ T cells expressing the same levels of CD25 as effector T cells. Therefore, we further analyzed the α-MSH-induced Treg cells for expression of effector and regulatory T-cell markers. Also, we examined the potential for α-MSH-induced Treg cells to be from the effector T-cell population. We found that the α-MSH-induced Treg cells are CD25+  CD4+ T cells that share similar surface markers as effector T cells, except that they express on their surface LAP. Also, the α-MSH treatment augments FoxP3 message in the effector T cells, and α-MSH induction of regulatory activity was limited to the effector CD25+ T-cell population. Therefore, α-MSH converts effector T cells into Treg cells, which suppress immunity targeting specific antigens and tissues.

Spirulina maxima extract prevents cell death through BDNF activation against amyloid beta 1-42 (Aβ1-42) induced neurotoxicity in PC12 cells.

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Koh, Eun-Jeong; Kim, Kui-Jin; Choi, Jia; Kang, Do-Hyung; Lee, Boo-Yong

2018-04-23

Spirulina maxima is a blue-green micro alga that contains abundant amounts of proteins (60-70%), vitamins, chlorophyll a, and C-phycocyanin (C-PC). It has been shown to reduce oxidative stress, and prevent diabetes and non-alcoholic fatty liver disease. However, it is unclear whether Spirulina maxima 70% ethanol extract (SM70EE), chlorophyll a, and C-PC prevent Aβ 1-42 -induced neurotoxicity in PC12 cells. The aim of this study was to investigate whether SM70EE, chlorophyll a, and C-PC prevent Aβ 1-42 -induced cell death. SM70EE, chlorophyll a, and C-PC suppressed the Aβ 1-42 -induced increase in poly-ADP ribose polymerase-1 (PARP-1) cleavage and reduced Aβ 1-42 -induced decreases in glutathione and its associated factors. The level of brain-derived neurotrophic factor (BDNF), which plays a critical role in neuronal survival and neuroprotection, was increased by SM70EE, chlorophyll a, and C-PC in Aβ 1-42 -treated cells. SM70EE treatment decreased oxidative stress and cell death in response to Aβ 1-42 treatment, while simultaneously suppressing PARP cleavage and increasing the levels of glutathione (GSH) and its associated factors. Moreover, SM70EE lowered the levels of APP and BACE1, two major factors involved in APP processing, and increased BDNF expression during Aβ 1-42 -induced neurotoxicity in PC12 cells. We suggest that SM70EE prevents cell death caused by Aβ 1-42 -induced neurotoxicity via the activation of BDNF signaling. Copyright © 2018 Elsevier B.V. All rights reserved.

Tax Protein-induced Expression of Antiapoptotic Bfl-1 Protein Contributes to Survival of Human T-cell Leukemia Virus Type 1 (HTLV-1)-infected T-cells*♦

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Macaire, Héloïse; Riquet, Aurélien; Moncollin, Vincent; Biémont-Trescol, Marie-Claude; Duc Dodon, Madeleine; Hermine, Olivier; Debaud, Anne-Laure; Mahieux, Renaud; Mesnard, Jean-Michel; Pierre, Marlène; Gazzolo, Louis; Bonnefoy, Nathalie; Valentin, Hélène

2012-01-01

Human T lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). ATLL is a severe malignancy with no effective treatment. HTLV-1 regulatory proteins Tax and HTLV-1 basic leucine zipper factor (HBZ) play a major role in ATLL development, by interfering with cellular functions such as CD4+ T-cell survival. In this study, we observed that the expression of Bfl-1, an antiapoptotic protein of the Bcl-2 family, is restricted to HTLV-1-infected T-cell lines and to T-cells expressing both Tax and HBZ proteins. We showed that Tax-induced bfl-1 transcription through the canonical NF-κB pathway. Moreover, we demonstrated that Tax cooperated with c-Jun or JunD, but not JunB, transcription factors of the AP-1 family to stimulate bfl-1 gene activation. By contrast, HBZ inhibited c-Jun-induced bfl-1 gene activation, whereas it increased JunD-induced bfl-1 gene activation. We identified one NF-κB, targeted by RelA, c-Rel, RelB, p105/p50, and p100/p52, and two AP-1, targeted by both c-Jun and JunD, binding sites in the bfl-1 promoter of T-cells expressing both Tax and HBZ. Analyzing the potential role of antiapoptotic Bcl-2 proteins in HTLV-1-infected T-cell survival, we demonstrated that these cells are differentially sensitive to silencing of Bfl-1, Bcl-xL, and Bcl-2. Indeed, both Bfl-1 and Bcl-xL knockdowns decreased the survival of HTLV-1-infected T-cell lines, although no cell death was observed after Bcl-2 knockdown. Furthermore, we demonstrated that Bfl-1 knockdown sensitizes HTLV-1-infected T-cells to ABT-737 or etoposide treatment. Our results directly implicate Bfl-1 and Bcl-xL in HTLV-1-infected T-cell survival and suggest that both Bfl-1 and Bcl-xL represent potential therapeutic targets for ATLL treatment. PMID:22553204

Analysis of epothilone B-induced cell death in normal ovarian cells.

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Rogalska, Aneta; Gajek, Arkadiusz; Marczak, Agnieszka

2013-12-01

We have investigated the mode of cell death induced by a new microtubule-stabilizing agent, epothilone B (EpoB, patupilone), and a clinically used medicine, paclitaxel (PTX), in normal ovarian cells. Using fluorescence microscopy, polyacrylamide gel electrophoresis preceding Western blot analysis, as well as spectrofluorimetric and colorimetric detection, we demonstrate that, compared to EpoB, PTX induced high time-dependent morphological and biochemical changes typical of apoptosis. Induction of apoptosis followed an early increase in p53 levels. Apoptosis reached its maximum at 24-48 h. At the same time, there was a significant increase in caspase-9 and -3 activity and PARP fragmentation, which suggests that an intrinsic path was involved. Apoptosis in MM14 cells was increased more by PTX than EpoB, and also induced more necrosis responsible for inflammation (1.4-fold) than EpoB. © 2013 International Federation for Cell Biology.

Analysis of porcine granulosa cell death signaling pathways induced by vinclozolin.

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Knet, Malgorzata; Wartalski, Kamil; Hoja-Lukowicz, Dorota; Tabarowski, Zbigniew; Slomczynska, Maria; Duda, Malgorzata

2015-10-01

Recent studies suggest that disturbing androgen-signaling pathways in porcine ovarian follicles may cause granulosa cell (GC) death. For this reason, we investigated which apoptotic pathway is initiated after GC exposure to an environmental antiandrogen, vinclozolin (Vnz), in vitro. Immunocytochemistry, Western blots, and fluorometric assays were used to quantify caspase-3 and -9 expression and activity. To elucidate the specific mechanism of Vnz action and toxicity, GCs were assessed for viability, cytotoxicity, and apoptotic activity using the ApoTox-Glo Triplex Assay. To further determine the mechanism of GC death induced by Vnz, we used the Apoptosis Antibody Array Kit. In response to Vnz stimulus, we found an increased level of caspase-3 protein expression (P ≤ 0.001) and an increase in caspase-3 proteolytic activity (P ≤ 0.001), confirming that Vnz is a potent proapoptotic factor. The strong immunoreaction of caspase-9 after Vnz treatment (P ≤ 0.001) suggests that intrinsic mitochondrial apoptosis pathway was activated during GC death. On the other hand, caspase-8, being a part of the extrinsic receptor pathway, was also activated (P ≤ 0.001). Therefore, it is possible that Vnz induces porcine granulosal apoptosis also through a parallel pathway. Activation of these two pathways was confirmed by the Apoptosis Antibody Array Kit. In conclusion, it is possible that the intrinsic signaling pathway may not act as an initial trigger for GC apoptosis but might contribute to the amplification and propagation of apoptotic cell death in the granulosa layer after treatment with this antiandrogen. Moreover, Vnz disturbs the physiological process of programmed cell death. Consequently, this could explain why atretic follicles are rapidly removed and suggests that normal function of the ovarian follicle may be destroyed. Copyright © 2015 Elsevier Inc. All rights reserved.

γδ T Cells Support Pancreatic Oncogenesis by Restraining αβ T Cell Activation.

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Daley, Donnele; Zambirinis, Constantinos Pantelis; Seifert, Lena; Akkad, Neha; Mohan, Navyatha; Werba, Gregor; Barilla, Rocky; Torres-Hernandez, Alejandro; Hundeyin, Mautin; Mani, Vishnu Raj Kumar; Avanzi, Antonina; Tippens, Daniel; Narayanan, Rajkishen; Jang, Jung-Eun; Newman, Elliot; Pillarisetty, Venu Gopal; Dustin, Michael Loran; Bar-Sagi, Dafna; Hajdu, Cristina; Miller, George

2016-09-08

Inflammation is paramount in pancreatic oncogenesis. We identified a uniquely activated γδT cell population, which constituted ∼40% of tumor-infiltrating T cells in human pancreatic ductal adenocarcinoma (PDA). Recruitment and activation of γδT cells was contingent on diverse chemokine signals. Deletion, depletion, or blockade of γδT cell recruitment was protective against PDA and resulted in increased infiltration, activation, and Th1 polarization of αβT cells. Although αβT cells were dispensable to outcome in PDA, they became indispensable mediators of tumor protection upon γδT cell ablation. PDA-infiltrating γδT cells expressed high levels of exhaustion ligands and thereby negated adaptive anti-tumor immunity. Blockade of PD-L1 in γδT cells enhanced CD4(+) and CD8(+) T cell infiltration and immunogenicity and induced tumor protection suggesting that γδT cells are critical sources of immune-suppressive checkpoint ligands in PDA. We describe γδT cells as central regulators of effector T cell activation in cancer via novel cross-talk. Copyright © 2016 Elsevier Inc. All rights reserved.