Autophagy as a trigger for cell death: autophagic degradation of inhibitor of apoptosis dBruce controls DNA fragmentation during late oogenesis in Drosophila.

PubMed

Nezis, Ioannis P; Shravage, Bhupendra V; Sagona, Antonia P; Johansen, Terje; Baehrecke, Eric H; Stenmark, Harald

2010-11-01

Autophagy has been reported to contribute to cell death, but the underlying mechanisms remain largely unknown and controversial. We have: been studying oogenesis in Drosophila melanogaster as a model system to understand the interplay between autophagy and cell death. Using a novel autophagy reporter we found that autophagy occurs during developmental cell death of nurse cells in late oogenesis. Genetic inhibition: of autophagy-related genes atg1, atg13 and vps34 results in late-stage egg chambers containing persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. We found that Drosophila inhibitor of apoptosis dBruce is degraded by autophagy and this degradation promotes DNA fragmentation and subsequent nurse cell death. These studies demonstrate that autophagic degradation of an inhibitor: of apoptosis is a novel mechanism of triggering cell death.

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

PubMed

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 triggering lymphopenia through direct and indirect mechanisms.

The engulfment receptor Draper is required for autophagy during cell death.

PubMed

McPhee, Christina K; Baehrecke, Eric H

2010-11-01

Autophagy is a process to degrade and recycle cytoplasmic contents. Autophagy is required for survival in response to starvation, but has also been associated with cell death. How autophagy functions during cell survival in some contexts and cell death in others is unknown. Drosophila larval salivary glands undergo programmed cell death requiring autophagy genes, and are cleared in the absence of known phagocytosis. Recently, we demonstrated that Draper (Drpr), the Drosophila homolog of C. elegans engulfment receptor CED-1, is required for autophagy induction: during cell death, but not during cell survival. drpr mutants fail to clear salivary glands. drpr knockdown in salivary glands prevents the induction of autophagy, and Atg1 misexpression in drpr null mutants suppresses salivary gland persistence. Surprisingly, drpr knockdown cell-autonomously prevents autophagy induction in dying salivary gland cells, but not in larval fat body cells following starvation. This is the first engulfment factor shown to function in cellular self-clearance, and the first report of a cell-death-specific autophagy regulator.

Ultrastructural aspects of autoschizis: a new cancer cell death induced by the synergistic action of ascorbate/menadione on human bladder carcinoma cells.

PubMed

Gilloteaux, J; Jamison, J M; Arnold, D; Taper, H S; Summers, J L

2001-01-01

Scanning and transmission electron microscopy were employed to further characterize the cytotoxic effects of a ascorbic acid/menadione (or vitamin C/vitamin K3) combination on a human bladder carcinoma T24 cell line. Following 1-h treatment T24 cells display membrane and mitochondrial defects as well as excision of cytoplasmic fragments that contain no organelles. These continuous self-excisions reduce the cell size. Concomitant, nuclear changes, chromatin disassembly, nucleolar condensation and fragmentation, and decreased nuclear volume lead to cell death via a process similar to karyorrhexis and karyolysis. Because this cell death is achieved through a progressive loss of cytoplasm due to self-morsellation, the authors named this mode of cell death autoschizis (from the Greek autos, self, and schizein, to split, as defined in Scanning. 1998; 20: 564-575). This morphological characterization of autoschizic cell death confirms and extends the authors previous reports and demonstrates that this cell death is distinct from apoptosis.

Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line.

PubMed

Thomas, Andrew J; Hailey, Dale W; Stawicki, Tamara M; Wu, Patricia; Coffin, Allison B; Rubel, Edwin W; Raible, David W; Simon, Julian A; Ou, Henry C

2013-03-06

Cisplatin, one of the most commonly used anticancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analog of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line.

ZBP1/DAI ubiquitination and sensing of influenza vRNPs activate programmed cell death

PubMed Central

Kuriakose, Teneema; Malireddi, R.K. Subbarao; Mishra, Ashutosh

2017-01-01

Innate sensing of influenza virus infection induces activation of programmed cell death pathways. We have recently identified Z-DNA–binding protein 1 (ZBP1) as an innate sensor of influenza A virus (IAV). ZBP1-mediated IAV sensing is critical for triggering programmed cell death in the infected lungs. Surprisingly, little is known about the mechanisms regulating ZBP1 activation to induce programmed cell death. Here, we report that the sensing of IAV RNA by retinoic acid inducible gene I (RIG-I) initiates ZBP1-mediated cell death via the RIG-I–MAVS–IFN-β signaling axis. IAV infection induces ubiquitination of ZBP1, suggesting potential regulation of ZBP1 function through posttranslational modifications. We further demonstrate that ZBP1 senses viral ribonucleoprotein (vRNP) complexes of IAV to trigger cell death. These findings collectively indicate that ZBP1 activation requires RIG-I signaling, ubiquitination, and vRNP sensing to trigger activation of programmed cell death pathways during IAV infection. The mechanism of ZBP1 activation described here may have broader implications in the context of virus-induced cell death. PMID:28634194

Cell death and cell lysis are separable events during pyroptosis

PubMed Central

DiPeso, Lucian; Ji, Daisy X; Vance, Russell E; Price, Jordan V

2017-01-01

Although much insight has been gained into the mechanisms by which activation of the inflammasome can trigger pyroptosis in mammalian cells, the precise kinetics of the end stages of pyroptosis have not been well characterized. Using time-lapse fluorescent imaging to analyze the kinetics of pyroptosis in individual murine macrophages, we observed distinct stages of cell death and cell lysis. Our data demonstrate that cell membrane permeability resulting from gasdermin D pore formation is coincident with the cessation of cell movement, loss of mitochondrial activity, and cell swelling, events that can be uncoupled from cell lysis. We propose a model of pyroptosis in which cell death can occur independently of cell lysis. The uncoupling of cell death from cell lysis may allow for better control of cytosolic contents upon activation of the inflammasome. PMID:29147575

The natural product peiminine represses colorectal carcinoma tumor growth by inducing autophagic cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lyu, Qing; Key Lab in Healthy Science and Technology, Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen, 518055; Tou, Fangfang

Autophagy is evolutionarily conservative in eukaryotic cells that engulf cellular long-lived proteins and organelles, and it degrades the contents through fusion with lysosomes, via which the cell acquires recycled building blocks for the synthesis of new molecules. In this study, we revealed that peiminine induces cell death and enhances autophagic flux in colorectal carcinoma HCT-116 cells. We determined that peiminine enhances the autophagic flux by repressing the phosphorylation of mTOR through inhibiting upstream signals. Knocking down ATG5 greatly reduced the peiminine-induced cell death in wild-type HCT-116 cells, while treating Bax/Bak-deficient cells with peiminine resulted in significant cell death. In summary,more » our discoveries demonstrated that peiminine represses colorectal carcinoma cell proliferation and cell growth by inducing autophagic cell death. - Highlights: • Peiminine induces autophagy and upregulates autophagic flux. • Peiminine represses colorectal carcinoma tumor growth. • Peiminine induces autophagic cell death. • Peiminine represses mTOR phosphorylation by influencing PI3K/Akt and AMPK pathway.« less

NF-κB p65 repression by the sesquiterpene lactone, Helenalin, contributes to the induction of autophagy cell death

PubMed Central

2012-01-01

Background Numerous studies have demonstrated that autophagy plays a vital role in maintaining cellular homeostasis. Interestingly, several anticancer agents were found to exert their anticancer effects by triggering autophagy. Emerging data suggest that autophagy represents a novel mechanism that can be exploited for therapeutic benefit. Pharmacologically active natural compounds such as those from marine, terrestrial plants and animals represent a promising resource for novel anticancer drugs. There are several prominent examples from the past proving the success of natural products and derivatives exhibiting anticancer activity. Helenalin, a sesquiterpene lactone has been demonstrated to have potent anti-inflammatory and antitumor activity. Albeit previous studies demonstrating helenalin’s multi modal action on cellular proliferative and apoptosis, the mechanisms underlying its action are largely unexplained. Methods To deduce the mechanistic action of helenalin, cancer cells were treated with the drug at various concentrations and time intervals. Using western blot, FACS analysis, overexpression and knockdown studies, cellular signaling pathways were interrogated focusing on apoptosis and autophagy markers. Results We show here that helenalin induces sub-G1 arrest, apoptosis, caspase cleavage and increases the levels of the autophagic markers. Suppression of caspase cleavage by the pan caspase inhibitor, Z-VAD-fmk, suppressed induction of LC3-B and Atg12 and reduced autophagic cell death, indicating caspase activity was essential for autophagic cell death induced by helenalin. Additionally, helenalin suppressed NF-κB p65 expression in a dose and time dependent manner. Exogenous overexpression of p65 was accompanied by reduced levels of cell death whereas siRNA mediated suppression led to augmented levels of caspase cleavage, autophagic cell death markers and increased cell death. Conclusions Taken together, these results show that helenalin mediated autophagic cell death entails inhibition of NF-κB p65, thus providing a promising approach for the treatment of cancers with aberrant activation of the NF-κB pathway. PMID:22784363

Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line

PubMed Central

Thomas, Andrew J.; Hailey, Dale W.; Stawicki, Tamara M.; Wu, Patricia; Coffin, Allison B.; Rubel, Edwin W.; Raible, David W.; Simon, Julian A.; Ou, Henry C.

2013-01-01

Cisplatin, one of the most commonly used anti-cancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analogue of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line. PMID:23467357

In Situ Gelation-Induced Death of Cancer Cells Based on Proteinosomes.

PubMed

Zhou, Yuting; Song, Jianmin; Wang, Lei; Xue, Xuting; Liu, Xiaoman; Xie, Hui; Huang, Xin

2017-08-14

Hydrogels are an excellent type of material that can be utilized as a platform for cell culture. However, when a bulky hydrogel forms on the inside of cancer cells, the result would be different. In this study, we demonstrate a method for in situ gelation inside cancer cells that can efficiently induce cell death. Glutathione-responsive proteinosomes with good biocompatibility were prepared as carriers for sodium alginate to be endocytosed by cancer cells, where the chelation between sodium alginate and free calcium ions in the culture medium occurs during the diffusion process. The uptake of the hydrogel-loaded proteinosomes into the cancer cells, and then the triggered release of hydrogel with concomitant aggregation, was well-confirmed by monitoring the change of the Young's modulus of the cells based on AFM force measurements. Accordingly, when a large amount of hydrogel formed in cells, the cell viability would be inhibited by ∼90% by MTT assay at a concentration of 5.0 μM of hydrogel-loaded proteinosomes after 48 h incubation, which clearly proves the feasibility of the demonstrated method for killing cancer cells. Although more details regarding the mechanism of cell death should be conducted in the near future, such a demonstrated method of in situ gelation inside cells provides another choice for killing cancer cells.

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

PubMed

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.

Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells.

PubMed

Yang, Jianhong; Zhou, Yongzhao; Cheng, Xia; Fan, Yi; He, Shichao; Li, Shucai; Ye, Haoyu; Xie, Caifeng; Wu, Wenshuang; Li, Chunyan; Pei, Heying; Li, Luyuan; Wei, Zhe; Peng, Aihua; Wei, Yuquan; Li, Weimin; Chen, Lijuan

2015-01-09

To overcome drug resistance caused by apoptosis deficiency in patients with non-small cell lung carcinoma (NSCLC), there is a need to identify other means of triggering apoptosis-independent cancer cell death. We are the first to report that isogambogenic acid (iso-GNA) can induce apoptosis-independent autophagic cell death in human NSCLC cells. Several features of the iso-GNA-treated NSCLC cells indicated that iso-GNA induced autophagic cell death. First, there was no evidence of apoptosis or cleaved caspase 3 accumulation and activation. Second, iso-GNA treatment induced the formation of autophagic vacuoles, increased LC3 conversion, caused the appearance of autophagosomes and increased the expression of autophagy-related proteins. These findings provide evidence that iso-GNA induces autophagy in NSCLC cells. Third, iso-GNA-induced cell death was inhibited by autophagic inhibitors or by selective ablation of Atg7 and Beclin 1 genes. Furthermore, the mTOR inhibitor rapamycin increased iso-GNA-induced cell death by enhancing autophagy. Finally, a xenograft model provided additional evidence that iso-GNA exhibited anticancer effect through inducing autophagy-dependent cell death in NSCLC cells. Taken together, our results demonstrated that iso-GNA exhibited an anticancer effect by inducing autophagy-dependent cell death in NSCLC cells, which may be an effective chemotherapeutic agent that can be used against NSCLC in a clinical setting.

Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells

PubMed Central

Yang, Jianhong; Zhou, Yongzhao; Cheng, Xia; Fan, Yi; He, Shichao; Li, Shucai; Ye, Haoyu; Xie, Caifeng; Wu, Wenshuang; Li, Chunyan; Pei, Heying; Li, Luyuan; Wei, Zhe; Peng, Aihua; Wei, Yuquan; Li, Weimin; Chen, Lijuan

2015-01-01

To overcome drug resistance caused by apoptosis deficiency in patients with non-small cell lung carcinoma (NSCLC), there is a need to identify other means of triggering apoptosis-independent cancer cell death. We are the first to report that isogambogenic acid (iso-GNA) can induce apoptosis-independent autophagic cell death in human NSCLC cells. Several features of the iso-GNA-treated NSCLC cells indicated that iso-GNA induced autophagic cell death. First, there was no evidence of apoptosis or cleaved caspase 3 accumulation and activation. Second, iso-GNA treatment induced the formation of autophagic vacuoles, increased LC3 conversion, caused the appearance of autophagosomes and increased the expression of autophagy-related proteins. These findings provide evidence that iso-GNA induces autophagy in NSCLC cells. Third, iso-GNA-induced cell death was inhibited by autophagic inhibitors or by selective ablation of Atg7 and Beclin 1 genes. Furthermore, the mTOR inhibitor rapamycin increased iso-GNA-induced cell death by enhancing autophagy. Finally, a xenograft model provided additional evidence that iso-GNA exhibited anticancer effect through inducing autophagy-dependent cell death in NSCLC cells. Taken together, our results demonstrated that iso-GNA exhibited an anticancer effect by inducing autophagy-dependent cell death in NSCLC cells, which may be an effective chemotherapeutic agent that can be used against NSCLC in a clinical setting. PMID:25571970

Glutathione Efflux and Cell Death

PubMed Central

2012-01-01

Abstract Significance: Glutathione (GSH) depletion is a central signaling event that regulates the activation of cell death pathways. GSH depletion is often taken as a marker of oxidative stress and thus, as a consequence of its antioxidant properties scavenging reactive species of both oxygen and nitrogen (ROS/RNS). Recent Advances: There is increasing evidence demonstrating that GSH loss is an active phenomenon regulating the redox signaling events modulating cell death activation and progression. Critical Issues: In this work, we review the role of GSH depletion by its efflux, as an important event regulating alterations in the cellular redox balance during cell death independent from oxidative stress and ROS/RNS formation. We discuss the mechanisms involved in GSH efflux during cell death progression and the redox signaling events by which GSH depletion regulates the activation of the cell death machinery. Future Directions: The evidence summarized here clearly places GSH transport as a central mechanism mediating redox signaling during cell death progression. Future studies should be directed toward identifying the molecular identity of GSH transporters mediating GSH extrusion during cell death, and addressing the lack of sensitive approaches to quantify GSH efflux. Antioxid. Redox Signal. 17, 1694–1713. PMID:22656858

The antihypertensive drug hydralazine activates the intrinsic pathway of apoptosis and causes DNA damage in leukemic T cells

PubMed Central

Ruiz-Magaña, María J.; Martínez-Aguilar, Rocío; Lucendo, Estefanía; Campillo-Davo, Diana; Schulze-Osthoff, Klaus; Ruiz-Ruiz, Carmen

2016-01-01

Epigenetic therapies have emerged as promising anticancer approaches, since epigenetic modifications play a major role in tumor initiation and progression. Hydralazine, an approved vasodilator and antihypertensive drug, has been recently shown to act as a DNA methylation inhibitor. Even though hydralazine is already tested in clinical cancer trials, its mechanism of antitumor action remains undefined. Here, we show that hydralazine induced caspase-dependent apoptotic cell death in human p53-mutant leukemic T cells. Moreover, we demonstrate that hydralazine triggered the mitochondrial pathway of apoptosis by inducing Bak activation and loss of the mitochondrial membrane potential. Hydralazine treatment further resulted in the accumulation of reactive oxygen species, whereas a superoxide dismutase mimetic inhibited hydralazine-induced cell death. Interestingly, caspase-9-deficient Jurkat cells or Bcl-2- and Bcl-xL-overexpressing cells were strongly resistant to hydralazine treatment, thereby demonstrating the dependence of hydralazine-induced apoptosis on the mitochondrial death pathway. Furthermore, we demonstrate that hydralazine treatment triggered DNA damage which might contribute to its antitumor effect. PMID:26942461

Anti-Yo antibody uptake and interaction with its intracellular target antigen causes Purkinje cell death in rat cerebellar slice cultures: a possible mechanism for paraneoplastic cerebellar degeneration in humans with gynecological or breast cancers.

PubMed

Greenlee, John E; Clawson, Susan A; Hill, Kenneth E; Wood, Blair; Clardy, Stacey L; Tsunoda, Ikuo; Carlson, Noel G

2015-01-01

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation.

Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

PubMed Central

Greenlee, John E.; Clawson, Susan A.; Hill, Kenneth E.; Wood, Blair; Clardy, Stacey L.; Tsunoda, Ikuo; Carlson, Noel G.

2015-01-01

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation. PMID:25885452

Nitric oxide released from JS-K induces cell death by mitotic catastrophe as part of necrosis in glioblastoma multiforme

PubMed Central

Günzle, Jessica; Osterberg, Nadja; Saavedra, Joseph E; Weyerbrock, Astrid

2016-01-01

The nitric oxide (NO) donor JS-K is specifically activated by glutathione S-transferases (GSTs) in GST-overexpressing cells. We have shown the induction of cell death in glioblastoma multiforme (GBM) cells at high JS-K doses but the mechanism remains unclear. The aim of this study was to determine whether NO-induced cell death is triggered by induction of apoptotic or necrotic pathways. For the first time, we demonstrate that NO induces cell death via mitotic catastrophe (MC) with non-apoptotic mechanisms in GBM cells. Moreover, the level of morphological changes indicating MC correlates with increased necrosis. Therefore, we conclude that MC is the main mechanism by which GBM cells undergo cell death after treatment with JS-K associated with necrosis rather than apoptosis. In addition, we show that PARP1 is not an exclusive marker for late apoptosis but is also involved in MC. Activating an alternative way of cell death can be useful for the multimodal cancer therapy of GBM known for its strong anti-apoptotic mechanisms and drug resistance. PMID:27584787

Nitric oxide released from JS-K induces cell death by mitotic catastrophe as part of necrosis in glioblastoma multiforme.

PubMed

Günzle, Jessica; Osterberg, Nadja; Saavedra, Joseph E; Weyerbrock, Astrid

2016-09-01

The nitric oxide (NO) donor JS-K is specifically activated by glutathione S-transferases (GSTs) in GST-overexpressing cells. We have shown the induction of cell death in glioblastoma multiforme (GBM) cells at high JS-K doses but the mechanism remains unclear. The aim of this study was to determine whether NO-induced cell death is triggered by induction of apoptotic or necrotic pathways. For the first time, we demonstrate that NO induces cell death via mitotic catastrophe (MC) with non-apoptotic mechanisms in GBM cells. Moreover, the level of morphological changes indicating MC correlates with increased necrosis. Therefore, we conclude that MC is the main mechanism by which GBM cells undergo cell death after treatment with JS-K associated with necrosis rather than apoptosis. In addition, we show that PARP1 is not an exclusive marker for late apoptosis but is also involved in MC. Activating an alternative way of cell death can be useful for the multimodal cancer therapy of GBM known for its strong anti-apoptotic mechanisms and drug resistance.

Tanshinone IIA protects H9c2 cells from oxidative stress-induced cell death via microRNA-133 upregulation and Akt activation.

PubMed

Gu, Yunfei; Liang, Zhuo; Wang, Haijun; Jin, Jun; Zhang, Shouyan; Xue, Shufeng; Chen, Jianfeng; He, Huijuan; Duan, Kadan; Wang, Jing; Chang, Xuewei; Qiu, Chunguang

2016-08-01

The aim of the present study was to investigate the cardioprotective effect of tanshinone IIA and the underlying molecular mechanisms. An in vitro model of oxidative stress injury was established in cardiac H9c2 cells, and the effects of tanshinone IIa were investigated using cell viability, reverse transcription-quantitative polymerase chain reaction and western blotting assays. The results demonstrated that tanshinone IIA protects H9c2 cells from H 2 O 2 -induced cell death in a concentration-dependent manner, via a mechanism involving microRNA-133 (miR-133), and that treatment with TIIA alone exerted no cytotoxic effects on H9c2. In order to further elucidate the mechanisms underlying the actions of TIIA, reverse transcription-quantitative polymease chain reaction and western blot analysis were performed. Reductions in miR-133 expression levels induced by increasing concentrations of H 2 O 2 were reversed by treatment with tanshinone IIA. In addition, the inhibition of miR-133 by transfection with an miR-133 inhibitor abolished the cardioprotective effects of tanshinone IIA against H 2 O 2 -induced cell death. Furthermore, western blot analysis demonstrated that tanshinone IIA activated Akt kinase via the phosphorylation of serine 473. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by pretreatment with the PI3K specific inhibitors wortmannin and LY294002 also eliminated the cardioprotective effects of tanshinone IIA against H 2 O 2 -induced cell death. Western blot analysis demonstrated that H 2 O 2 -induced reductions in B cell lymphoma 2 (Bcl-2) expression levels were reversed by tanshinone IIA. In addition, the effect of tanshinone IIA on Bcl-2 protein expression level in an oxidative environment was suppressed by a PI3K inhibitor, wortmannin, indicating that tanshinone IIA exerts cardioprotective effects against H 2 O 2 -induced cell death via the activation of the PI3K/Akt signal transduction pathway and the consequent upregulation of Bcl-2. In conclusion, the present study demonstrates that TIIA is able to protcet H9c2 cells from oxidative stress-induced cell death through signalling pathways involving miR-133 and Akt, and that tanshinone IIA is a promising natural cardioprotective agent.

The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise.

PubMed

Reuther, C; Ganjam, G K; Dolga, A M; Culmsee, C

2014-11-01

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.

Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death

PubMed Central

Graham, Nicholas A; Tahmasian, Martik; Kohli, Bitika; Komisopoulou, Evangelia; Zhu, Maggie; Vivanco, Igor; Teitell, Michael A; Wu, Hong; Ribas, Antoni; Lo, Roger S; Mellinghoff, Ingo K; Mischel, Paul S; Graeber, Thomas G

2012-01-01

The altered metabolism of cancer can render cells dependent on the availability of metabolic substrates for viability. Investigating the signaling mechanisms underlying cell death in cells dependent upon glucose for survival, we demonstrate that glucose withdrawal rapidly induces supra-physiological levels of phospho-tyrosine signaling, even in cells expressing constitutively active tyrosine kinases. Using unbiased mass spectrometry-based phospho-proteomics, we show that glucose withdrawal initiates a unique signature of phospho-tyrosine activation that is associated with focal adhesions. Building upon this observation, we demonstrate that glucose withdrawal activates a positive feedback loop involving generation of reactive oxygen species (ROS) by NADPH oxidase and mitochondria, inhibition of protein tyrosine phosphatases by oxidation, and increased tyrosine kinase signaling. In cells dependent on glucose for survival, glucose withdrawal-induced ROS generation and tyrosine kinase signaling synergize to amplify ROS levels, ultimately resulting in ROS-mediated cell death. Taken together, these findings illustrate the systems-level cross-talk between metabolism and signaling in the maintenance of cancer cell homeostasis. PMID:22735335

CDIP, a novel pro-apoptotic gene, regulates TNFalpha-mediated apoptosis in a p53-dependent manner.

PubMed

Brown, Lauren; Ongusaha, Pat P; Kim, Hyung-Gu; Nuti, Shanthy; Mandinova, Anna; Lee, Ji Won; Khosravi-Far, Roya; Aaronson, Stuart A; Lee, Sam W

2007-07-25

We have identified a novel pro-apoptotic p53 target gene named CDIP (Cell Death Involved p53-target). Inhibition of CDIP abrogates p53-mediated apoptotic responses, demonstrating that CDIP is an important p53 apoptotic effector. CDIP itself potently induces apoptosis that is associated with caspase-8 cleavage, implicating the extrinsic cell death pathway in apoptosis mediated by CDIP. siRNA-directed knockdown of caspase-8 results in a severe impairment of CDIP-dependent cell death. In investigating the potential involvement of extrinsic cell death pathway in CDIP-mediated apoptosis, we found that TNF-alpha expression tightly correlates with CDIP expression, and that inhibition of TNF-alpha signaling attenuates CDIP-dependent apoptosis. We also demonstrate that TNF-alpha is upregulated in response to p53 and p53 inducing genotoxic stress, in a CDIP-dependent manner. Consistently, knockdown of TNF-alpha impairs p53-mediated stress-induced apoptosis. Together, these findings support a novel p53 --> CDIP --> TNF-alpha apoptotic pathway that directs apoptosis after exposure of cells to genotoxic stress. Thus, CDIP provides a new link between p53-mediated intrinsic and death receptor-mediated extrinsic apoptotic signaling, providing a novel target for cancer therapeutics aimed at maximizing the p53 apoptotic response of cancer cells to drug therapy.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Hengwen; Yang, Shana; Li, Jianhua

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 expressionmore » 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.« less

Role of Hsp-70 in triptolide-mediated cell death of neuroblastoma.

PubMed

Antonoff, Mara B; Chugh, Rohit; Skube, Steven J; Dudeja, Vikas; Borja-Cacho, Daniel; Clawson, Kimberly A; Vickers, Selwyn M; Saluja, Ashok K

2010-09-01

Our recent work demonstrated that treatment of neuroblastoma with triptolide causes apoptotic cell death in vitro and decreases tumor size in vivo. Triptolide therapy has been associated with reduced expression of Hsp-70, suggesting a mechanism of cell killing involving Hsp-70 inhibition. The principal objective of this study was to investigate the role of Hsp-70 in triptolide-mediated cell death in neuroblastoma. Neuroblastoma cells were transfected with Hsp-70-specific siRNA. Viability, caspase activity, and phosphatidylserine externalization were subsequently measured. An orthotopic, syngeneic murine tumor model was developed, and randomized mice received daily injections of triptolide or vehicle. At 21 d, mice were sacrificed. Immunohistochemisty was used to characterize Hsp-70 levels in residual tumors, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to identify cells undergoing apoptosis. Targeted silencing of Hsp-70 with siRNA significantly decreased cellular viability, augmented caspase-3 activity, and resulted in increased annexin-V staining. These effects parallel those findings obtained following treatment with triptolide. Residual tumors from triptolide-treated mice showed minimal staining with Hsp-70 immunohistochemistry, while control tumors stained prominently. Tumors from treated mice demonstrated marked staining with the TUNEL assay, while control tumors showed no evidence of apoptosis. Use of siRNA to suppress Hsp-70 expression in neuroblastoma resulted in apoptotic cell death, similar to the effects of triptolide. Residual tumors from triptolide-treated mice expressed decreased levels of Hsp-70 and demonstrated significant apoptosis. These findings support the hypothesis that Hsp-70 inhibition plays a significant role in triptolide-mediated neuroblastoma cell death. Copyright 2010 Elsevier Inc. All rights reserved.

Autophagy modulates endoplasmic reticulum stress-induced cell death in podocytes: A protective role

PubMed Central

Cheng, Yu-Chi; Chang, Jer-Ming; Chen, Chien-An

2015-01-01

Endoplasmic reticulum stress occurs in a variety of patho-physiological mechanisms and there has been great interest in managing this pathway for the treatment of clinical diseases. Autophagy is closely interconnected with endoplasmic reticulum stress to counteract the possible injurious effects related with the impairment of protein folding. Studies have shown that glomerular podocytes exhibit high rate of autophagy to maintain as terminally differentiated cells. In this study, podocytes were exposed to tunicamycin and thapsigargin to induce endoplasmic reticulum stress. Thapsigargin/tunicamycin treatment induced a significant increase in endoplasmic reticulum stress and of cell death, represented by higher GADD153 and GRP78 expression and propidium iodide flow cytometry, respectively. However, thapsigargin/tunicamycin stimulation also enhanced autophagy development, demonstrated by monodansylcadaverine assay and LC3 conversion. To evaluate the regulatory effects of autophagy on endoplasmic reticulum stress-induced cell death, rapamycin (Rap) or 3-methyladenine (3-MA) was added to enhance or inhibit autophagosome formation. Endoplasmic reticulum stress-induced cell death was decreased at 6 h, but was not reduced at 24 h after Rap+TG or Rap+TM treatment. In contrast, endoplasmic reticulum stress-induced cell death increased at 6 and 24 h after 3-MA+TG or 3-MA+TM treatment. Our study demonstrated that thapsigargin/tunicamycin treatment induced endoplasmic reticulum stress which resulted in podocytes death. Autophagy, which counteracted the induced endoplasmic reticulum stress, was simultaneously enhanced. The salvational role of autophagy was supported by adding Rap/3-MA to mechanistically regulate the expression of autophagy and autophagosome formation. In summary, autophagy helps the podocytes from cell death and may contribute to sustain the longevity as a highly differentiated cell lineage. PMID:25322957

Upregulation of NAD(P)H:Quinone Oxidoreductase By Radiation Potentiates the Effect of Bioreductive β-Lapachone on Cancer Cells1

PubMed Central

Choi, Eun K; Terai, Kaoru; Ji, In-Mi; Kook, Yeon H; Park, Kyung H; Oh, Eun T; Griffin, Robert J; Lim, Byung U; Kim, Jin-Seok; Lee, Doo S; Boothman, David A; Loren, Melissa; Song, Chang W; Park, Heon Joo

2007-01-01

We found that β-lapachone (β-lap), a novel bioreductive drug, caused rapid apoptosis and clonogenic cell death in A549 human lung epithelial cancer cells in vitro in a dose-dependent manner. The clonogenic cell death caused by β-lap could be significantly inhibited by dicoumarol, an inhibitor of NAD(P)H:quinone oxido-reductase (NQO1), and also by siRNA for NQO1, demonstrating that NQO1-induced bioreduction of β-lap is an essential step in β-lap-induced cell death. Irradiation of A549 cells with 4 Gy caused a long-lasting upregulation of NQO1, thereby increasing NQO1-mediated β-lap-induced cell deaths. Although the direct cause of β-lap-induced apoptosis is not yet clear, β-lap treatment reduced the expression of p53 and NF-κB, whereas it increased cytochrome C release, caspase-3 activity, and γH2AX foci formation. Importantly, β-lap treatment immediately after irradiation enhanced radiation-induced cell death, indicating that β-lap sensitizes cancer cells to radiation, in addition to directly killing some of the cells. The growth of A549 tumors induced in immunocompromised mice could be markedly suppressed by local radiation therapy when followed by β-lap treatment. This is the first study to demonstrate that combined radiotherapy and β-lap treatment can have a significant effect on human tumor xenografts. PMID:17786182

RSL3 and Erastin differentially regulate redox signaling to promote Smac mimetic-induced cell death

PubMed Central

Dächert, Jasmin; Schoeneberger, Hannah; Rohde, Katharina; Fulda, Simone

2016-01-01

Redox mechanisms play an important role in the control of various signaling pathways. Here, we report that Second mitochondrial activator of caspases (Smac) mimetic-induced cell death is regulated by redox signaling. We show that RSL3, a glutathione (GSH) peroxidase (GPX) 4 inhibitor, or Erastin, an inhibitor of the cystine/glutamate antiporter, cooperate with the Smac mimetic BV6 to induce reactive oxygen species (ROS)-dependent cell death in acute lymphoblastic leukemia (ALL) cells. Addition of the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to rescue ROS-induced cell death, demonstrating that RSL3/BV6- or Erastin/BV6-induced cell death occurs in a caspase-independent manner. Interestingly, the iron chelator Deferoxamine (DFO) significantly inhibits RSL3/BV6-induced cell death, whereas it is unable to rescue cell death by Erastin/BV6, showing that RSL3/BV6-, but not Erastin/BV6-mediated cell death depends on iron. ROS production is required for both RSL3/BV6- and Erastin/BV6-induced cell death, since the ROS scavenger α-tocopherol (α-Toc) rescues RSL3/BV6- and Erastin/BV6-induced cell death. By comparison, genetic or pharmacological inhibition of lipid peroxidation by GPX4 overexpression or ferrostatin (Fer)-1 significantly decreases RSL3/BV6-, but not Erastin/BV6-induced cell death, despite inhibition of lipid peroxidation upon exposure to RSL3/BV6 or Erastin/BV6. Of note, inhibition of lipid peroxidation by Fer-1 protects from RSL3/BV6-, but not from Erastin/BV6-stimulated ROS production, indicating that other forms of ROS besides lipophilic ROS occur during Erastin/BV6-induced cell death. Taken together, RSL3/BV6 and Erastin/BV6 differentially regulate redox signaling and cell death in ALL cells. While RSL3/BV6 cotreatment induces ferroptotic cell death, Erastin/BV6 stimulates oxidative cell death independently of iron. These findings have important implications for the therapeutic targeting of redox signaling to enhance Smac mimetic-induced cell death in ALL. PMID:27588473

RSL3 and Erastin differentially regulate redox signaling to promote Smac mimetic-induced cell death.

PubMed

Dächert, Jasmin; Schoeneberger, Hannah; Rohde, Katharina; Fulda, Simone

2016-09-27

Redox mechanisms play an important role in the control of various signaling pathways. Here, we report that Second mitochondrial activator of caspases (Smac) mimetic-induced cell death is regulated by redox signaling. We show that RSL3, a glutathione (GSH) peroxidase (GPX) 4 inhibitor, or Erastin, an inhibitor of the cystine/glutamate antiporter, cooperate with the Smac mimetic BV6 to induce reactive oxygen species (ROS)-dependent cell death in acute lymphoblastic leukemia (ALL) cells. Addition of the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to rescue ROS-induced cell death, demonstrating that RSL3/BV6- or Erastin/BV6-induced cell death occurs in a caspase-independent manner. Interestingly, the iron chelator Deferoxamine (DFO) significantly inhibits RSL3/BV6-induced cell death, whereas it is unable to rescue cell death by Erastin/BV6, showing that RSL3/BV6-, but not Erastin/BV6-mediated cell death depends on iron. ROS production is required for both RSL3/BV6- and Erastin/BV6-induced cell death, since the ROS scavenger α-tocopherol (α-Toc) rescues RSL3/BV6- and Erastin/BV6-induced cell death. By comparison, genetic or pharmacological inhibition of lipid peroxidation by GPX4 overexpression or ferrostatin (Fer)-1 significantly decreases RSL3/BV6-, but not Erastin/BV6-induced cell death, despite inhibition of lipid peroxidation upon exposure to RSL3/BV6 or Erastin/BV6. Of note, inhibition of lipid peroxidation by Fer-1 protects from RSL3/BV6-, but not from Erastin/BV6-stimulated ROS production, indicating that other forms of ROS besides lipophilic ROS occur during Erastin/BV6-induced cell death. Taken together, RSL3/BV6 and Erastin/BV6 differentially regulate redox signaling and cell death in ALL cells. While RSL3/BV6 cotreatment induces ferroptotic cell death, Erastin/BV6 stimulates oxidative cell death independently of iron. These findings have important implications for the therapeutic targeting of redox signaling to enhance Smac mimetic-induced cell death in ALL.

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

PubMed

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. 

Integrative functional transcriptomic analyses implicate specific molecular pathways in pulmonary toxicity from exposure to aluminum oxide nanoparticles.

PubMed

Li, Xiaobo; Zhang, Chengcheng; Bian, Qian; Gao, Na; Zhang, Xin; Meng, Qingtao; Wu, Shenshen; Wang, Shizhi; Xia, Yankai; Chen, Rui

2016-09-01

Gene expression profiling has developed rapidly in recent years and it can predict and define mechanisms underlying chemical toxicity. Here, RNA microarray and computational technology were used to show that aluminum oxide nanoparticles (Al2O3 NPs) were capable of triggering up-regulation of genes related to the cell cycle and cell death in a human A549 lung adenocarcinoma cell line. Gene expression levels were validated in Al2O3 NPs exposed A549 cells and mice lung tissues, most of which showed consistent trends in regulation. Gene-transcription factor network analysis coupled with cell- and animal-based assays demonstrated that the genes encoding PTPN6, RTN4, BAX and IER play a role in the biological responses induced by the nanoparticle exposure, which caused cell death and cell cycle arrest in the G2/S phase. Further, down-regulated PTPN6 expression demonstrated a core role in the network, thus expression level of PTPN6 was rescued by plasmid transfection, which showed ameliorative effects of A549 cells against cell death and cell cycle arrest. These results demonstrate the feasibility of using gene expression profiling to predict cellular responses induced by nanomaterials, which could be used to develop a comprehensive knowledge of nanotoxicity.

Cutting Edge: Eosinophils Undergo Caspase-1-Mediated Pyroptosis in Response to Necrotic Liver Cells.

PubMed

Palacios-Macapagal, Daphne; Connor, Jane; Mustelin, Tomas; Ramalingam, Thirumalai R; Wynn, Thomas A; Davidson, Todd S

2017-08-01

Many chronic liver disorders are characterized by dysregulated immune responses and hepatocyte death. We used an in vivo model to study the immune response to necrotic liver injury and found that necrotic liver cells induced eosinophil recruitment. Necrotic liver induced eosinophil IL-1β and IL-18 secretion, degranulation, and cell death. Caspase-1 inhibitors blocked all of these responses. Caspase-1-mediated cell death with accompanying cytokine release is the hallmark of a novel form of cell death termed pyroptosis. To confirm this response in a disease model, we isolated eosinophils from the livers of Schistosoma mansoni -infected mice. S. mansoni eggs lodge in the hepatic sinusoids of infected mice, resulting in hepatocyte death, inflammation, and progressive liver fibrosis. This response is typified by massive eosinophilia, and we were able to confirm pyroptosis in the infiltrating eosinophils. This demonstrated that pyroptosis is a cellular pathway used by eosinophils in response to large-scale hepatic cell death. Copyright © 2017 by The American Association of Immunologists, Inc.

Dual Roles of Reactive Oxygen Species and NADPH Oxidase RBOHD in an Arabidopsis-Alternaria Pathosystem1[W

PubMed Central

Pogány, Miklós; von Rad, Uta; Grün, Sebastian; Dongó, Anita; Pintye, Alexandra; Simoneau, Philippe; Bahnweg, Günther; Kiss, Levente; Barna, Balázs; Durner, Jörg

2009-01-01

Arabidopsis (Arabidopsis thaliana) NADPH oxidases have been reported to suppress the spread of pathogen- and salicylic acid-induced cell death. Here, we present dual roles of RBOHD (for respiratory burst oxidase homolog D) in an Arabidopsis-Alternaria pathosystem, suggesting either initiation or prevention of cell death dependent on the distance from pathogen attack. Our data demonstrate that a rbohD knockout mutant exhibits increased spread of cell death at the macroscopic level upon inoculation with the fungus Alternaria brassicicola. However, the cellular patterns of reactive oxygen species accumulation and cell death are fundamentally different in the AtrbohD mutant compared with the wild type. Functional RBOHD causes marked extracellular hydrogen peroxide accumulation as well as cell death in distinct, single cells of A. brassicicola-infected wild-type plants. This single cell response is missing in the AtrbohD mutant, where infection triggers spreading-type necrosis preceded by less distinct chloroplastic hydrogen peroxide accumulation in large clusters of cells. While the salicylic acid analog benzothiadiazole induces the action of RBOHD and the development of cell death in infected tissues, the ethylene inhibitor aminoethoxyvinylglycine inhibits cell death, indicating that both salicylic acid and ethylene positively regulate RBOHD and cell death. Moreover, A. brassicicola-infected AtrbohD plants hyperaccumulate ethylene and free salicylic acid compared with the wild type, suggesting negative feedback regulation of salicylic acid and ethylene by RBOHD. We propose that functional RBOHD triggers death in cells that are damaged by fungal infection but simultaneously inhibits death in neighboring cells through the suppression of free salicylic acid and ethylene levels. PMID:19726575

Lysosomal membrane permeabilization in cell death: new evidence and implications for health and disease.

PubMed

Serrano-Puebla, Ana; Boya, Patricia

2016-05-01

Recent studies have demonstrated that, in addition to their central role in cellular catabolic reactions, lysosomes are implicated in many cellular processes, including metabolism, membrane repair, and cell death. Lysosomal membrane permeabilization (LMP) has emerged as a pathway by which cell demise is regulated under physiological conditions and contributes to cell death in many pathological situations. Here, we review the latest evidence on LMP-mediated cell death, the upstream and downstream signals involved, and the role of LMP in the normal physiology of organisms. We also discuss the contributions of lysosomal damage and LMP to the pathogenic features of several disease states, such as lysosomal storage disorders and other neurodegenerative conditions. © 2015 New York Academy of Sciences.

Rip3 knockdown rescues photoreceptor cell death in blind pde6c zebrafish.

PubMed

Viringipurampeer, I A; Shan, X; Gregory-Evans, K; Zhang, J P; Mohammadi, Z; Gregory-Evans, C Y

2014-05-01

Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6c(w59) mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6c(w59) embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed. Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6c(w59) mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marrero, Maria Teresa; Estevez, Sara; Negrin, Gledy

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 inhibitormore » 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.« less

METACASPASE9 modulates autophagy to confine cell death to the target cells during Arabidopsis vascular xylem differentiation

PubMed Central

Escamez, Sacha; André, Domenique; Zhang, Bo; Bollhöner, Benjamin; Pesquet, Edouard; Tuominen, Hannele

2016-01-01

ABSTRACT We uncovered that the level of autophagy in plant cells undergoing programmed cell death determines the fate of the surrounding cells. Our approach consisted of using Arabidopsis thaliana cell cultures capable of differentiating into two different cell types: vascular tracheary elements (TEs) that undergo programmed cell death (PCD) and protoplast autolysis, and parenchymatic non-TEs that remain alive. The TE cell type displayed higher levels of autophagy when expression of the TE-specific METACASPASE9 (MC9) was reduced using RNAi (MC9-RNAi). Misregulation of autophagy in the MC9-RNAi TEs coincided with ectopic death of the non-TEs, implying the existence of an autophagy-dependent intercellular signalling from within the TEs towards the non-TEs. Viability of the non-TEs was restored when AUTOPHAGY2 (ATG2) was downregulated specifically in MC9-RNAi TEs, demonstrating the importance of autophagy in the spatial confinement of cell death. Our results suggest that other eukaryotic cells undergoing PCD might also need to tightly regulate their level of autophagy to avoid detrimental consequences for the surrounding cells. PMID:26740571

Glioblastoma cells deficient in DNA-dependent protein kinase are resistant to cell death.

PubMed

Chen, George G; Sin, Fanny L F; Leung, Billy C S; Ng, Ho K; Poon, Wai S

2005-04-01

DNA-dependent protein kinase (DNA-PK), a nuclear serine/threonine kinase, is responsible for the DNA double-strand break repair. Cells lacking or with dysfunctional DNA-PK are often associated with mis-repair, chromosome aberrations, and complex exchanges, all of which are known to contribute to the development of human cancers including glioblastoma. Two human glioblastoma cell lines were used in the experiment, M059J cells lacking the catalytic subunit of DNA-PK, and their isogenic but DNA-PK proficient counterpart, M059K. We found that M059K cells were much more sensitive to staurosporine (STS) treatment than M059J cells, as demonstrated by MTT assay, TUNEL detection, and annexin-V and propidium iodide (PI) staining. A possible mechanism responsible for the different sensitivity in these two cell lines was explored by the examination of Bcl-2, Bax, Bak, and Fas. The cell death stimulus increased anti-apoptotic Bcl-2 and decreased pro-apoptotic Bcl-2 members (Bak and Bax) and Fas in glioblastoma cells deficient in DNA-PK. Activation of DNA-PK is known to promote cell death of human tumor cells via modulation of p53, which can down-regulate the anti-apoptotic Bcl-2 member proteins, induce pro-apoptotic Bcl-2 family members and promote a Bax-Bak interaction. Our experiment also demonstrated that the mode of glioblastoma cell death induced by STS consisted of both apoptosis and necrosis and the percentage of cell death in both modes was similar in glioblastoma cell lines either lacking DNA-PK or containing intact DNA-PK. Taken together, our findings suggest that DNA-PK has a positive role in the regulation of apoptosis in human glioblastomas. The aberrant expression of Bcl-2 family members and Fas was, at least in part, responsible for decreased sensitivity of DNA-PK deficient glioblastoma cells to cell death stimuli. 2004 Wiley-Liss, Inc.

Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death.

PubMed

Clark, Amy L; Kanekura, Kohsuke; Lavagnino, Zeno; Spears, Larry D; Abreu, Damien; Mahadevan, Jana; Yagi, Takuya; Semenkovich, Clay F; Piston, David W; Urano, Fumihiko

2017-07-17

Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

Mechanical Stress Promotes Cisplatin-Induced Hepatocellular Carcinoma Cell Death

PubMed Central

Riad, Sandra; Bougherara, Habiba

2015-01-01

Cisplatin (CisPt) is a commonly used platinum-based chemotherapeutic agent. Its efficacy is limited due to drug resistance and multiple side effects, thereby warranting a new approach to improving the pharmacological effect of CisPt. A newly developed mathematical hypothesis suggested that mechanical loading, when coupled with a chemotherapeutic drug such as CisPt and immune cells, would boost tumor cell death. The current study investigated the aforementioned mathematical hypothesis by exposing human hepatocellular liver carcinoma (HepG2) cells to CisPt, peripheral blood mononuclear cells, and mechanical stress individually and in combination. HepG2 cells were also treated with a mixture of CisPt and carnosine with and without mechanical stress to examine one possible mechanism employed by mechanical stress to enhance CisPt effects. Carnosine is a dipeptide that reportedly sequesters platinum-based drugs away from their pharmacological target-site. Mechanical stress was achieved using an orbital shaker that produced 300 rpm with a horizontal circular motion. Our results demonstrated that mechanical stress promoted CisPt-induced death of HepG2 cells (~35% more cell death). Moreover, results showed that CisPt-induced death was compromised when CisPt was left to mix with carnosine 24 hours preceding treatment. Mechanical stress, however, ameliorated cell death (20% more cell death). PMID:25685789

Lipoic acid induces p53-independent cell death in colorectal cancer cells and potentiates the cytotoxicity of 5-fluorouracil.

PubMed

Dörsam, Bastian; Göder, Anja; Seiwert, Nina; Kaina, Bernd; Fahrer, Jörg

2015-10-01

Alpha-lipoic acid (LA), which plays a pivotal role in mitochondrial energy metabolism, is an endogenous dithiol compound with an array of antioxidative functions. It has been shown that LA triggers cell death in tumor cell lines, whereas non-transformed cells are hardly affected. In the present study, we analyzed the cytotoxicity of LA on colorectal cancer (CRC) cells differing in their p53 status and investigated a putative synergistic effect with the anticancer drug 5-fluorouracil (5-FU). We show that LA induces a dose-dependent decrease in cell viability, which was independent of the p53 status as attested in isogenic p53-proficient and p53-deficient cell lines. This effect was largely attributable to cell death induction as revealed by Annexin-V/PI staining. LA-treated HCT116 cells underwent caspase-dependent and caspase-independent cell death, which was blocked by the pan-caspase inhibitor zVAD and the RIP-kinase inhibitor Necrostatin-1, respectively. In CaCO-2 and HT29 cells, LA induced caspase-dependent cell demise via activation of caspase-9, caspase-3 and caspase-7 with subsequent PARP-1 cleavage as demonstrated by immunoblot analysis, activity assays and pan-caspase inhibition. Interestingly, LA treatment did neither activate p53 nor induced genotoxic effects as shown by lack of DNA strand breaks and phosphorylation of histone 2AX. Finally, we provide evidence that LA increases the cytotoxic effect induced by the anticancer drug 5-FU as revealed by significantly enhanced cell death rates in HCT116 and CaCO-2 cells. Collectively, these findings demonstrate that LA induces CRC cell death independent of their p53 status and potentiates the cytotoxicity of 5-FU without causing DNA damage on its own, which makes it a candidate for tumor therapy.

MEK inhibitor U0126 interferes with immunofluorescence analysis of apoptotic cell death.

PubMed

Blank, Norbert; Burger, Renate; Duerr, Birgit; Bakker, Frank; Wohlfarth, Anika; Dumitriu, Ingrid; Kalden, Joachim R; Herrmann, Martin

2002-08-01

Binding of extracellular growth factors to cell surface receptors often results in activation of the mitogen-activated protein kinase (MAPK). MAPK is regulated by MAPK kinase, also called MEK. Deprivation of growth factors during cell culture or intracellular MEK inhibition leads to inhibition of proliferation and apoptotic cell death. Besides other techniques, apoptotic cells can be identified by phosphatidylserine (PS) exposure and exclusion of membrane-impermeant propidium iodide (PI). We investigated the limitations of detection of apoptotic cell death and cytofluorometry in cells cultured in the presence of the MEK inhibitor U0126. Apoptotic cell death was induced in the plasmacytoma cell line INA-6, in peripheral blood mononuclear cells (PBMC), and in cultured T lymphoblasts by deprivation of interleukin-6 (IL-6) or by incubation with the MEK inhibitor U0126. Apoptotic cell death was quantified by flow cytometry using annexin V/propidium iodide (AxV/PI) double staining. U0126-treated cells dramatically changed their fluorescence pattern during cell culture. If AxV/PI staining is employed to detect apoptotic cell death, the background fluorescence mimicks PS exposure on viable cells. The compound itself has no intrinsic fluorescence in vitro but develops an intensive fluorescence during cell culture which can be observed in all fluorescence channels with a predominance in the FL1 channel (525 nm). We further demonstrate that at least some of the U0126-induced background fluorescence is dependent on cellular uptake and intracellular modifications or cellular responses. These results demonstrate that appropriate controls for every single time point are necessary if fluorescence analyses are performed in the presence of chemical enzyme inhibitors. In the case of MEK inhibitors, either the use of PD098059 or PD184352 as an alternative for U0126 or nonfluorometric methods for detection of apoptosis should be considered. Copyright 2002 Wiley-Liss, Inc.

Identification and characterization of cannabinoids that induce cell death through mitochondrial permeability transition in Cannabis leaf cells.

PubMed

Morimoto, Satoshi; Tanaka, Yumi; Sasaki, Kaori; Tanaka, Hiroyuki; Fukamizu, Tomohide; Shoyama, Yoshinari; Shoyama, Yukihiro; Taura, Futoshi

2007-07-13

Cannabinoids are secondary metabolites stored in capitate-sessile glands on leaves of Cannabis sativa. We discovered that cell death is induced in the leaf tissues exposed to cannabinoid resin secreted from the glands, and identified cannabichromenic acid (CBCA) and Delta(1)-tetrahydrocannabinolic acid (THCA) as unique cell death mediators from the resin. These cannabinoids effectively induced cell death in the leaf cells or suspension-cultured cells of C. sativa, whereas pretreatment with the mitochondrial permeability transition (MPT) inhibitor cyclosporin A suppressed this cell death response. Examinations using isolated mitochondria demonstrated that CBCA and THCA mediate opening of MPT pores without requiring Ca(2+) and other cytosolic factors, resulting in high amplitude mitochondrial swelling, release of mitochondrial proteins (cytochrome c and nuclease), and irreversible loss of mitochondrial membrane potential. Therefore, CBCA and THCA are considered to cause serious damage to mitochondria through MPT. The mitochondrial damage was also confirmed by a marked decrease of ATP level in cannabinoid-treated suspension cells. These features are in good accord with those of necrotic cell death, whereas DNA degradation was also observed in cannabinoid-mediated cell death. However, the DNA degradation was catalyzed by nuclease(s) released from mitochondria during MPT, indicating that this reaction was not induced via a caspase-dependent apoptotic pathway. Furthermore, the inhibition of the DNA degradation only slightly blocked the cell death induced by cannabinoids. Based on these results, we conclude that CBCA and THCA have the ability to induce necrotic cell death via mitochondrial dysfunction in the leaf cells of C. sativa.

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

PubMed

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.

Mitochondrial calcium uniporter silencing potentiates caspase-independent cell death in MDA-MB-231 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Curry, Merril C.; Peters, Amelia A.; Kenny, Paraic A.

Highlights: •Some clinical breast cancers are associated with MCU overexpression. •MCU silencing did not alter cell death initiated with the Bcl-2 inhibitor ABT-263. •MCU silencing potentiated caspase-independent cell death initiated by ionomycin. •MCU silencing promoted ionomycin-mediated cell death without changes in bulk Ca{sup 2+}. -- Abstract: The mitochondrial calcium uniporter (MCU) transports free ionic Ca{sup 2+} into the mitochondrial matrix. We assessed MCU expression in clinical breast cancer samples using microarray analysis and the consequences of MCU silencing in a breast cancer cell line. Our results indicate that estrogen receptor negative and basal-like breast cancers are characterized by elevated levelsmore » of MCU. Silencing of MCU expression in the basal-like MDA-MB-231 breast cancer cell line produced no change in proliferation or cell viability. However, distinct consequences of MCU silencing were seen on cell death pathways. Caspase-dependent cell death initiated by the Bcl-2 inhibitor ABT-263 was not altered by MCU silencing; whereas caspase-independent cell death induced by the calcium ionophore ionomycin was potentiated by MCU silencing. Measurement of cytosolic Ca{sup 2+} levels showed that the promotion of ionomycin-induced cell death by MCU silencing occurs independently of changes in bulk cytosolic Ca{sup 2+} levels. This study demonstrates that MCU overexpression is a feature of some breast cancers and that MCU overexpression may offer a survival advantage against some cell death pathways. MCU inhibitors may be a strategy to increase the effectiveness of therapies that act through the induction of caspase-independent cell death pathways in estrogen receptor negative and basal-like breast cancers.« less

Oxidative Stress Facilitates IFN-γ-Induced Mimic Extracellular Trap Cell Death in A549 Lung Epithelial Cancer Cells.

PubMed

Lin, Chiou-Feng; Chen, Chia-Ling; Chien, Shun-Yi; Tseng, Po-Chun; Wang, Yu-Chih; Tsai, Tsung-Ting

2016-01-01

We previously demonstrated that IFN-γ induces an autophagy-regulated mimic extracellular trap cell death (ETosis) in A549 human lung cancer cells. Regarding reactive oxygen species (ROS) are involved in ETosis, this study investigated the role of oxidative stress. After IFN-γ stimulation, a necrosis-like cell death mimic ETosis occurred accompanied by the inhibition of cell growth, aberrant nuclear staining, and nucleosome release. ROS were generated in a time-dependent manner with an increase in NADPH oxidase component protein expression. STAT1-mediated IFN regulatory factor-1 activation was essential for upregulating ROS production. By genetically silencing p47phox, IFN-γ-induced ROS and mimic ETosis were significantly attenuated. This mechanistic study indicated that ROS may mediate DNA damage followed by histone H3 citrullination. Furthermore, ROS promoted IFN-γ-induced mimic ETosis in cooperation with autophagy. These findings further demonstrate that ROS regulates IFN-γ-induced mimic ETosis in lung epithelial malignancy.

Oxidative Stress Facilitates IFN-γ-Induced Mimic Extracellular Trap Cell Death in A549 Lung Epithelial Cancer Cells

PubMed Central

Lin, Chiou-Feng; Chen, Chia-Ling; Chien, Shun-Yi; Tseng, Po-Chun; Wang, Yu-Chih; Tsai, Tsung-Ting

2016-01-01

We previously demonstrated that IFN-γ induces an autophagy-regulated mimic extracellular trap cell death (ETosis) in A549 human lung cancer cells. Regarding reactive oxygen species (ROS) are involved in ETosis, this study investigated the role of oxidative stress. After IFN-γ stimulation, a necrosis-like cell death mimic ETosis occurred accompanied by the inhibition of cell growth, aberrant nuclear staining, and nucleosome release. ROS were generated in a time-dependent manner with an increase in NADPH oxidase component protein expression. STAT1-mediated IFN regulatory factor-1 activation was essential for upregulating ROS production. By genetically silencing p47phox, IFN-γ-induced ROS and mimic ETosis were significantly attenuated. This mechanistic study indicated that ROS may mediate DNA damage followed by histone H3 citrullination. Furthermore, ROS promoted IFN-γ-induced mimic ETosis in cooperation with autophagy. These findings further demonstrate that ROS regulates IFN-γ-induced mimic ETosis in lung epithelial malignancy. PMID:27575372

Hydrogen peroxide production and mitochondrial dysfunction contribute to the fusaric acid-induced programmed cell death in tobacco cells.

PubMed

Jiao, Jiao; Sun, Ling; Zhou, Benguo; Gao, Zhengliang; Hao, Yu; Zhu, Xiaoping; Liang, Yuancun

2014-08-15

Fusaric acid (FA), a non-specific toxin produced mainly by Fusarium spp., can cause programmed cell death (PCD) in tobacco suspension cells. The mechanism underlying the FA-induced PCD was not well understood. In this study, we analyzed the roles of hydrogen peroxide (H2O2) and mitochondrial function in the FA-induced PCD. Tobacco suspension cells were treated with 100 μM FA and then analyzed for H2O2 accumulation and mitochondrial functions. Here we demonstrate that cells undergoing FA-induced PCD exhibited H2O2 production, lipid peroxidation, and a decrease of the catalase and ascorbate peroxidase activities. Pre-treatment of tobacco suspension cells with antioxidant ascorbic acid and NADPH oxidase inhibitor diphenyl iodonium significantly reduced the rate of FA-induced cell death as well as the caspase-3-like protease activity. Moreover, FA treatment of tobacco cells decreased the mitochondrial membrane potential and ATP content. Oligomycin and cyclosporine A, inhibitors of the mitochondrial ATP synthase and the mitochondrial permeability transition pore, respectively, could also reduce the rate of FA-induced cell death significantly. Taken together, the results presented in this paper demonstrate that H2O2 accumulation and mitochondrial dysfunction are the crucial events during the FA-induced PCD in tobacco suspension cells. Copyright © 2014 Elsevier GmbH. All rights reserved.

Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells

PubMed Central

Mahamed, Deeqa; Boulle, Mikael; Ganga, Yashica; Mc Arthur, Chanelle; Skroch, Steven; Oom, Lance; Catinas, Oana; Pillay, Kelly; Naicker, Myshnee; Rampersad, Sanisha; Mathonsi, Colisile; Hunter, Jessica; Wong, Emily B; Suleman, Moosa; Sreejit, Gopalkrishna; Pym, Alexander S; Lustig, Gila; Sigal, Alex

2017-01-01

A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states. DOI: http://dx.doi.org/10.7554/eLife.22028.001 PMID:28130921

Anti-cancer Effect of Luminacin, a Marine Microbial Extract, in Head and Neck Squamous Cell Carcinoma Progression via Autophagic Cell Death.

PubMed

Shin, Yoo Seob; Cha, Hyun Young; Lee, Bok-Soon; Kang, Sung Un; Hwang, Hye Sook; Kwon, Hak Cheol; Kim, Chul-Ho; Choi, Eun Chang

2016-04-01

The purpose of this study is to determine whether luminacin, a marine microbial extract from the Streptomyces species, has anti-tumor effects on head and neck squamous cell carcinoma (HNSCC) cell lines via autophagic cell death. Inhibition of cell survival and increased cell death was measured using cell viability, colony forming, and apoptosis assays. Migration and invasion abilities of head and cancer cells were evaluated using wound healing, scattering, and invasion assays. Changes in the signal pathway related to autophagic cell death were investigated. Drug toxicity of luminacin was examined in in vitro HaCaT cells and an in vivo zebrafish model. Luminacin showed potent cytotoxicity in HNSCC cells in cell viability, colony forming, and fluorescence-activated cell sorting analysis. In vitro migration and invasion of HNSCC cells were attenuated by luminacin treatment. Combined with Beclin-1 and LC3B, Luminacin induced autophagic cell death in head and neck cancer cells. In addition, in a zebrafish model and human keratinocyte cell line used for toxicity testing, luminacin treatment with a cytotoxic concentration to HNSCC cells did not cause toxicity. Taken together, these results demonstrate that luminacin induces the inhibition of growth and cancer progression via autophagic cell death in HNSCC cell lines, indicating a possible alternative chemotherapeutic approach for treatment of HNSCC.

Cell death in neural precursor cells and neurons before neurite formation prevents the emergence of abnormal neural structures in the Drosophila optic lobe.

PubMed

Hara, Yusuke; Sudo, Tatsuya; Togane, Yu; Akagawa, Hiromi; Tsujimura, Hidenobu

2018-04-01

Programmed cell death is a conserved strategy for neural development both in vertebrates and invertebrates and is recognized at various developmental stages in the brain from neurogenesis to adulthood. To understand the development of the central nervous system, it is essential to reveal not only molecular mechanisms but also the role of neural cell death (Pinto-Teixeira et al., 2016). To understand the role of cell death in neural development, we investigated the effect of inhibition of cell death on optic lobe development. Our data demonstrate that, in the optic lobe of Drosophila, cell death occurs in neural precursor cells and neurons before neurite formation and functions to prevent various developmental abnormalities. When neuronal cell death was inhibited by an effector caspase inhibitor, p35, multiple abnormal neuropil structures arose during optic lobe development-e.g., enlarged or fused neuropils, misrouted neurons and abnormal neurite lumps. Inhibition of cell death also induced morphogenetic defects in the lamina and medulla development-e.g., failures in the separation of the lamina and medulla cortices and the medulla rotation. These defects were reproduced in the mutant of an initiator caspase, dronc. If cell death was a mechanism for removing the abnormal neuropil structures, we would also expect to observe them in mutants defective for corpse clearance. However, they were not observed in these mutants. When dead cell-membranes were visualized with Apoliner, they were observed only in cortices and not in neuropils. These results suggest that the cell death occurs before mature neurite formation. Moreover, we found that inhibition of cell death induced ectopic neuroepithelial cells, neuroblasts and ganglion mother cells in late pupal stages, at sites where the outer and inner proliferation centers were located at earlier developmental stages. Caspase-3 activation was observed in the neuroepithelial cells and neuroblasts in the proliferation centers. These results indicate that cell death is required for elimination of the precursor cells composing the proliferation centers. This study substantiates an essential role of early neural cell death for ensuring normal development of the central nervous system. Copyright © 2018 Elsevier Inc. All rights reserved.

Cullin-4 regulates Wingless and JNK signaling-mediated cell death in the Drosophila eye

PubMed Central

Tare, Meghana; Sarkar, Ankita; Bedi, Shimpi; Kango-Singh, Madhuri; Singh, Amit

2016-01-01

In all multicellular organisms, the fundamental processes of cell proliferation and cell death are crucial for growth regulation during organogenesis. Strict regulation of cell death is important to maintain tissue homeostasis by affecting processes like regulation of cell number, and elimination of unwanted/unfit cells. The developing Drosophila eye is a versatile model to study patterning and growth, where complex signaling pathways regulate growth and cell survival. However, the molecular mechanisms underlying regulation of these processes is not fully understood. In a gain-of-function screen, we found that misexpression of cullin-4 (cul-4), an ubiquitin ligase, can rescue reduced eye mutant phenotypes. Previously, cul-4 has been shown to regulate chromatin remodeling, cell cycle and cell division. Genetic characterization of cul-4 in the developing eye revealed that loss-of-function of cul-4 exhibits a reduced eye phenotype. Analysis of twin-spots showed that in comparison with their wild-type counterparts, the cul-4 loss-of-function clones fail to survive. Here we show that cul-4 clones are eliminated by induction of cell death due to activation of caspases. Aberrant activation of signaling pathways is known to trigger cell death in the developing eye. We found that Wingless (Wg) and c-Jun-amino-terminal-(NH2)-Kinase (JNK) signaling are ectopically induced in cul-4 mutant clones, and these signals co-localize with the dying cells. Modulating levels of Wg and JNK signaling by using agonists and antagonists of these pathways demonstrated that activation of Wg and JNK signaling enhances cul-4 mutant phenotype, whereas downregulation of Wg and JNK signaling rescues the cul-4 mutant phenotypes of reduced eye. Here we present evidences to demonstrate that cul-4 is involved in restricting Wg signaling and downregulation of JNK signaling-mediated cell death during early eye development. Overall, our studies provide insights into a novel role of cul-4 in promoting cell survival in the developing Drosophila eye. PMID:28032862

Adoptive Transfer of Dying Cells Causes Bystander-Induced Apoptosis

PubMed Central

Schwulst, Steven J.; Davis, Christopher G.; Coopersmith, Craig M.; Hotchkiss, Richard S.

2009-01-01

The anti-apoptotic Bcl-2 protein has the remarkable ability to prevent cell death from several noxious stimuli. Intriguingly, Bcl-2 overexpression in one cell type has been reported to protect against cell death in neighboring non-Bcl-2 overexpressing cell types. The mechanism of this “trans” protection has been speculated to be secondary to the release of a cytoprotective factor by Bcl-2 overexpressing cells. We employed a series of adoptive transfer experiments in which lymphocytes that overexpress Bcl-2 were administered to either wild type mice or mice lacking mature T and B cells (Rag-1-/-) to detect the presence or absence of the putative protective factor. We were unable to demonstrate “trans” protection. However, adoptive transfer of apoptotic or necrotic cells exacerbated the degree of apoptotic death in neighboring non-Bcl-2 overexpressing cells (p≤0.05). Therefore, this data suggests that dying cells emit signals triggering cell death in neighboring non-Bcl-2 overexpressing cells, i.e. a “trans” destructive effect. PMID:17194455

Do antioxidants inhibit oxidative-stress-induced autophagy of tenofibroblasts?

PubMed

Kim, Ra-Jeong; Hah, Young-Sool; Sung, Chang-Meen; Kang, Jae-Ran; Park, Hyung Bin

2014-07-01

Recent research on tendinopathy has focused on its relationship to programmed cell death. Increased autophagy has been observed in ruptured rotator cuff tendon tissues, suggesting a causal relationship. We investigated whether autophagy occurs in human rotator cuff tenofibroblast death induced by oxidative stress and whether antioxidants protect against autophagic cell death. We used H2 O2 (0.75 mM) as oxidative stressor, cyanidin (100 µg/ml) as antioxidant, zVAD (20 µM) as apoptosis inhibitor, and 3-MA (10 mM) as autophagy inhibitor. We evaluated cell viability and known autophagic markers: LC3-II expression, GFP-LC3 puncta formation, autolysosomes, and Atg5-12 and Beclin 1 expression. H2 O2 exposure increased the rates of cell death, LC3-II expression, GFP-LC3 puncta formation, and autolysosomes. After we induced apoptosis arrest using zVAD, H2 O2 exposure still induced cell death, LC3-II expression, and GFP-LC3 puncta formation. H2 O2 exposure also increased Atg5-12 and Beclin 1 expressions, indicating autophagic cell death. However, cyanidin treatment reduced H2 O2 -induced cell death, LC3-II expression, GFP-LC3 puncta formation, and autolysosomes. Cyanidin and 3-MA similarly reduced the cell-death rate, and Atg5-12 and Beclin 1 expression. This study demonstrated that H2 O2 , an oxidative stressor, induces autophagic cell death in rotator cuff tenofibroblasts, and that cyanidin, a natural antioxidant, inhibits autophagic cell death. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

CDIP, a novel pro-apoptotic gene, regulates TNFα-mediated apoptosis in a p53-dependent manner

PubMed Central

Brown, Lauren; Ongusaha, Pat P; Kim, Hyung-Gu; Nuti, Shanthy; Mandinova, Anna; Lee, Ji Won; Khosravi-Far, Roya; Aaronson, Stuart A; Lee, Sam W

2007-01-01

We have identified a novel pro-apoptotic p53 target gene named CDIP (Cell Death Involved p53-target). Inhibition of CDIP abrogates p53-mediated apoptotic responses, demonstrating that CDIP is an important p53 apoptotic effector. CDIP itself potently induces apoptosis that is associated with caspase-8 cleavage, implicating the extrinsic cell death pathway in apoptosis mediated by CDIP. siRNA-directed knockdown of caspase-8 results in a severe impairment of CDIP-dependent cell death. In investigating the potential involvement of extrinsic cell death pathway in CDIP-mediated apoptosis, we found that TNF-α expression tightly correlates with CDIP expression, and that inhibition of TNF-α signaling attenuates CDIP-dependent apoptosis. We also demonstrate that TNF-α is upregulated in response to p53 and p53 inducing genotoxic stress, in a CDIP-dependent manner. Consistently, knockdown of TNF-α impairs p53-mediated stress-induced apoptosis. Together, these findings support a novel p53 → CDIP → TNF-α apoptotic pathway that directs apoptosis after exposure of cells to genotoxic stress. Thus, CDIP provides a new link between p53-mediated intrinsic and death receptor-mediated extrinsic apoptotic signaling, providing a novel target for cancer therapeutics aimed at maximizing the p53 apoptotic response of cancer cells to drug therapy. PMID:17599062

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

PubMed Central

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

3-Decylcatechol induces autophagy-mediated cell death through the IRE1α/JNK/p62 in hepatocellular carcinoma cells

PubMed Central

Kim, Jin-A; Jo, In-Hwa; Han, Yeon Soo; Jo, Yong Hun; Kim, Kwang-Youn; Seo, Young-Kyo; Moon, Jae-Hak; Jung, Chang Hwa; Jeon, Tae-Il

2017-01-01

The natural, phenolic lipid urushiol exhibits both antioxidant and anticancer activities; however, its biological activity on hepatocellular carcinoma (HCC) has not been previously investigated. Here, we demonstrate that an urushiol derivative, 3-decylcatechol (DC), induces human HCC Huh7 cell death by induction of autophagy. DC initiates the autophagic process by activation of the mammalian target of rapamycin signaling pathway via Unc-51-like autophagy activating kinase 1, leading to autophagosome formation. The autophagy inhibitor, chloroquine, suppressed autolysosome formation and cell death induction by DC, indicating an autophagic cell death. Interestingly, DC also activated the endoplasmic reticulum (ER) stress response that promotes autophagy via p62 transcriptional activation involving the inositol-requiring enzyme 1α/c-Jun N-terminal kinase/c-jun pathway. We also show that cytosolic calcium mobilization is necessary for the ER stress response and autophagy induction by DC. These findings reveal a novel mechanism by which this urushiol derivative induces autophagic cell death in HCC. PMID:28938597

Thiol-redox signaling, dopaminergic cell death, and Parkinson's disease.

PubMed

Garcia-Garcia, Aracely; Zavala-Flores, Laura; Rodriguez-Rocha, Humberto; Franco, Rodrigo

2012-12-15

Parkinson's disease (PD) is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta, which has been widely associated with oxidative stress. However, the mechanisms by which redox signaling regulates cell death progression remain elusive. Early studies demonstrated that depletion of glutathione (GSH), the most abundant low-molecular-weight thiol and major antioxidant defense in cells, is one of the earliest biochemical events associated with PD, prompting researchers to determine the role of oxidative stress in dopaminergic cell death. Since then, the concept of oxidative stress has evolved into redox signaling, and its complexity is highlighted by the discovery of a variety of thiol-based redox-dependent processes regulating not only oxidative damage, but also the activation of a myriad of signaling/enzymatic mechanisms. GSH and GSH-based antioxidant systems are important regulators of neurodegeneration associated with PD. In addition, thiol-based redox systems, such as peroxiredoxins, thioredoxins, metallothioneins, methionine sulfoxide reductases, transcription factors, as well as oxidative modifications in protein thiols (cysteines), including cysteine hydroxylation, glutathionylation, and nitrosylation, have been demonstrated to regulate dopaminergic cell loss. In this review, we summarize major advances in the understanding of the role of thiol-redox signaling in dopaminergic cell death in experimental PD. Future research is still required to clearly understand how integrated thiol-redox signaling regulates the activation of the cell death machinery, and the knowledge generated should open new avenues for the design of novel therapeutic approaches against PD.

Impairment of Atg5-Dependent Autophagic Flux Promotes Paraquat- and MPP+-Induced Apoptosis But Not Rotenone or 6-Hydroxydopamine Toxicity

PubMed Central

Franco, Rodrigo

2013-01-01

Controversial reports on the role of autophagy as a survival or cell death mechanism in dopaminergic cell death induced by parkinsonian toxins exist. We investigated the alterations in autophagic flux and the role of autophagy protein 5 (Atg5)-dependent autophagy in dopaminergic cell death induced by parkinsonian toxins. Dopaminergic cell death induced by the mitochondrial complex I inhibitors 1-methyl-4-phenylpyridinium (MPP+) and rotenone, the pesticide paraquat, and the dopamine analog 6-hydroxydopamine (6-OHDA) was paralleled by increased autophagosome accumulation. However, when compared with basal autophagy levels using chloroquine, autophagosome accumulation was a result of impaired autophagic flux. Only 6-OHDA induced an increase in autophagosome formation. Overexpression of a dominant negative form of Atg5 increased paraquat- and MPP+-induced cell death. Stimulation of mammalian target of rapamycin (mTOR)-dependent signaling protected against cell death induced by paraquat, whereas MPP+-induced toxicity was enhanced by wortmannin, a phosphoinositide 3-kinase class III inhibitor, rapamycin, and trehalose, an mTOR-independent autophagy activator. Modulation of autophagy by either pharmacological or genetic approaches had no effect on rotenone or 6-OHDA toxicity. Cell death induced by parkinsonian neurotoxins was inhibited by the pan caspase inhibitor (Z-VAD), but only caspase-3 inhibition was able to decrease MPP+-induced cell death. Finally, inhibition of the lysosomal hydrolases, cathepsins, increased the toxicity by paraquat and MPP+, supporting a protective role of Atg5-dependent autophagy and lysosomes degradation pathways on dopaminegic cell death. These results demonstrate that in dopaminergic cells, Atg5-dependent autophagy acts as a protective mechanism during apoptotic cell death induced by paraquat and MPP+ but not during rotenone or 6-OHDA toxicity. PMID:23997112

Colorectal cancer chemoprevention: the potential of a selective approach.

PubMed

Ben-Amotz, Oded; Arber, Nadir; Kraus, Sarah

2010-10-01

Colorectal cancer (CRC) is a leading cause of cancer death, and therefore demands special attention. Novel recent approaches for the chemoprevention of CRC focus on selective targeting of key pathways. We review the study by Zhang and colleagues, evaluating a selective approach targeting APC-deficient premalignant cells using retinoid-based therapy and TNF-related apoptosis-inducing ligand (TRAIL). This study demonstrates that induction of TRAIL-mediated death signaling contributes to the chemopreventive value of all-trans-retinyl acetate (RAc) by sensitizing premalignant adenoma cells for apoptosis without affecting normal cells. We discuss these important findings, raise few points that deserve consideration, and may further contribute to the development of RAc-based combination therapies with improved efficacy. The authors clearly demonstrate a synergistic interaction between TRAIL, RAc and APC, which leads to the specific cell death of premalignant target cells. The study adds to the growing body of literature related to CRC chemoprevention, and provides solid data supporting a potentially selective approach for preventing CRC using RAc and TRAIL.

Combining chemotherapy with PD-1 blockade in NSCLC.

PubMed

Mathew, Matthen; Enzler, Thomas; Shu, Catherine A; Rizvi, Naiyer A

2018-06-01

Antitumor immunity relies on the ability of the immune system to recognize tumor cells as foreign and eliminate them. An effective immune response in this setting is due to surveillance of tumor-specific antigens that induce an adaptive immune response resulting in T-cell mediated cytotoxicity. Immune checkpoint inhibitors, specifically those targeting the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis, have demonstrated promising activity in non-small cell lung cancer (NSCLC). However, there remains a crucial need for better treatment strategies for the majority of patients with advanced NSCLC, particularly in the frontline setting. Chemotherapy can increase antigenicity via immunogenic cell death (ICD) of tumor cells as well as also reduce "off target" immunosuppression in the tumor microenvironment (TME). Combining chemotherapy with PD-1 blockade harnesses the potential synergy between these agents and has led to encouraging results in the up-front treatment of NSCLC. In this review, we summarize the preclinical rationale behind these combinations and review recent trial data demonstrating their efficacy. Copyright © 2018. Published by Elsevier Inc.

Susceptibility of Mycobacterium tuberculosis-infected host cells to phospho-MLKL driven necroptosis is dependent on cell type and presence of TNFα.

PubMed

Butler, Rachel E; Krishnan, Nitya; Garcia-Jimenez, Waldo; Francis, Robert; Martyn, Abbe; Mendum, Tom; Felemban, Shaza; Locker, Nicolas; Salguero, Francisco J; Robertson, Brian; Stewart, Graham R

2017-11-17

An important feature of Mycobacterium tuberculosis pathogenesis is the ability to control cell death in infected host cells, including inhibition of apoptosis and stimulation of necrosis. Recently an alternative form of programmed cell death, necroptosis, has been described where necrotic cell death is induced by apoptotic stimuli under conditions where apoptotic execution is inhibited. We show for the first time that M. tuberculosis and TNFα synergise to induce necroptosis in murine fibroblasts via RIPK1-dependent mechanisms and characterized by phosphorylation of Ser345 of the MLKL necroptosis death effector. However, in murine macrophages M. tuberculosis and TNFα induce non-necroptotic cell death that is RIPK1-dependent but independent of MLKL phosphorylation. Instead, M. tuberculosis-infected macrophages undergo RIPK3-dependent cell death which occurs both in the presence and absence of TNFα and involves the production of mitochondrial ROS. Immunocytochemical staining for MLKL phosphorylation further demonstrated the occurrence of necroptosis in vivo in murine M. tuberculosis granulomas. Phosphorylated-MLKL immunoreactivity was observed associated with the cytoplasm and nucleus of fusiform cells in M. tuberculosis lesions but not in proximal macrophages. Thus whereas pMLKL-driven necroptosis does not appear to be a feature of M. tuberculosis-infected macrophage cell death, it may contribute to TNFα-induced cytotoxicity of the lung stroma and therefore contribute to necrotic cavitation and bacterial dissemination.

A new lactoferrin- and iron-dependent lysosomal death pathway is induced by benzo[a]pyrene in hepatic epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gorria, Morgane; Tekpli, Xavier; Rissel, Mary

2008-04-15

While lysosomal disruption seems to be a late step of necrosis, a moderate lysosomal destabilization has been suggested to participate early in the apoptotic cascade. The origin of lysosomal dysfunction and its precise role in apoptosis or apoptosis-like process still needs to be clarified, especially upon carcinogen exposure. In this study, we focused on the implication of lysosomes in cell death induced by the prototype carcinogen benzo[a]pyrene (B[a]P; 50 nM) in rat hepatic epithelial F258 cells. We first demonstrated that B[a]P affected lysosomal morphology (increase in size) and pH (alkalinization), and that these changes were involved in caspase-3 activation andmore » cell death. Subsequently, we showed that lysosomal modifications were partly dependent on mitochondrial dysfunction, and that lysosomes together with mitochondria participate in B[a]P-induced oxidative stress. Using two iron chelators (desferrioxamine and deferiprone) and siRNA targeting the lysosomal iron-binding protease lactoferrin, we further demonstrated that both lysosomal iron content and lactoferrin were required for caspase-3 activation and apoptosis-like cell death.« less

The impairment of HCCS leads to MLS syndrome by activating a non-canonical cell death pathway in the brain and eyes

PubMed Central

Indrieri, Alessia; Conte, Ivan; Chesi, Giancarlo; Romano, Alessia; Quartararo, Jade; Tatè, Rosarita; Ghezzi, Daniele; Zeviani, Massimo; Goffrini, Paola; Ferrero, Ileana; Bovolenta, Paola; Franco, Brunella

2013-01-01

Mitochondrial-dependent (intrinsic) programmed cell death (PCD) is an essential homoeostatic mechanism that selects bioenergetically proficient cells suitable for tissue/organ development. However, the link between mitochondrial dysfunction, intrinsic apoptosis and developmental anomalies has not been demonstrated to date. Now we provide the evidence that non-canonical mitochondrial-dependent apoptosis explains the phenotype of microphthalmia with linear skin lesions (MLS), an X-linked developmental disorder caused by mutations in the holo-cytochrome c-type synthase (HCCS) gene. By taking advantage of a medaka model that recapitulates the MLS phenotype we demonstrate that downregulation of hccs, an essential player of the mitochondrial respiratory chain (MRC), causes increased cell death via an apoptosome-independent caspase-9 activation in brain and eyes. We also show that the unconventional activation of caspase-9 occurs in the mitochondria and is triggered by MRC impairment and overproduction of reactive oxygen species (ROS). We thus propose that HCCS plays a key role in central nervous system (CNS) development by modulating a novel non-canonical start-up of cell death and provide the first experimental evidence for a mechanistic link between mitochondrial dysfunction, intrinsic apoptosis and developmental disorders. PMID:23239471

Aging, mortality, and the fast growth trade-off of Schizosaccharomyces pombe

PubMed Central

Nakaoka, Hidenori; Wakamoto, Yuichi

2017-01-01

Replicative aging has been demonstrated in asymmetrically dividing unicellular organisms, seemingly caused by unequal damage partitioning. Although asymmetric segregation and inheritance of potential aging factors also occur in symmetrically dividing species, it nevertheless remains controversial whether this results in aging. Based on large-scale single-cell lineage data obtained by time-lapse microscopy with a microfluidic device, in this report, we demonstrate the absence of replicative aging in old-pole cell lineages of Schizosaccharomyces pombe cultured under constant favorable conditions. By monitoring more than 1,500 cell lineages in 7 different culture conditions, we showed that both cell division and death rates are remarkably constant for at least 50–80 generations. Our measurements revealed that the death rate per cellular generation increases with the division rate, pointing to a physiological trade-off with fast growth under balanced growth conditions. We also observed the formation and inheritance of Hsp104-associated protein aggregates, which are a potential aging factor in old-pole cell lineages, and found that these aggregates exhibited a tendency to preferentially remain at the old poles for several generations. However, the aggregates were eventually segregated from old-pole cells upon cell division and probabilistically allocated to new-pole cells. We found that cell deaths were typically preceded by sudden acceleration of protein aggregation; thus, a relatively large amount of protein aggregates existed at the very ends of the dead cell lineages. Our lineage tracking analyses, however, revealed that the quantity and inheritance of protein aggregates increased neither cellular generation time nor cell death initiation rates. Furthermore, our results demonstrated that unusually large amounts of protein aggregates induced by oxidative stress exposure did not result in aging; old-pole cells resumed normal growth upon stress removal, despite the fact that most of them inherited significant quantities of aggregates. These results collectively indicate that protein aggregates are not a major determinant of triggering cell death in S. pombe and thus cannot be an appropriate molecular marker or index for replicative aging under both favorable and stressful environmental conditions. PMID:28632741

The Cytolytic Amphipathic β(2,2)-Amino Acid LTX-401 Induces DAMP Release in Melanoma Cells and Causes Complete Regression of B16 Melanoma.

PubMed

Eike, Liv-Marie; Mauseth, Brynjar; Camilio, Ketil André; Rekdal, Øystein; Sveinbjørnsson, Baldur

2016-01-01

In the present study we examined the ability of the amino acid derivative LTX-401 to induce cell death in cancer cell lines, as well as the capacity to induce regression in a murine melanoma model. Mode of action studies in vitro revealed lytic cell death and release of danger-associated molecular pattern molecules, preceded by massive cytoplasmic vacuolization and compromised lysosomes in treated cells. The use of a murine melanoma model demonstrated that the majority of animals treated with intratumoural injections of LTX-401 showed complete and long-lasting remission. Taken together, these results demonstrate the potential of LTX-401 as an immunotherapeutic agent for the treatment of solid tumors.

Berberine-induced autophagic cell death by elevating GRP78 levels in cancer cells.

PubMed

La, Xiaoqin; Zhang, Lichao; Li, Zhuoyu; Yang, Peng; Wang, Yingying

2017-03-28

Berberine, an isoquinoline alkaloid extracted from Coptidis Rhizoma, has been shown to induce cancer cell autophagic death. Glucose regulated protein 78 (GRP78) is associated with stress-induced autophagy. However, the related mechanisms between berberine-induced cancer cell autophagy and GRP78 remain to be elucidated. Here, we report that berberine can induce autophagic cancer cell death by elevating levels of GRP78. These results further demonstrated that berberine enhanced GRP78 by suppression of ubiquitination / proteasomal degradation of GRP78 and activation of ATF6. Moreover, fluorescence spectrum assay revealed that berberine could bind to GRP78 and form complexes. Finally, co-IP analysis showed that the ability of GRP78 to bind to VPS34 was increased with berberine treatment. Taken together, our results suggest that berberine induces autophagic cancer cell death via enhancing GRP78 levels and the ability of GRP78 to bind to VPS34.

Berberine-induced autophagic cell death by elevating GRP78 levels in cancer cells

PubMed Central

Li, Zhuoyu; Yang, Peng; Wang, Yingying

2017-01-01

Berberine, an isoquinoline alkaloid extracted from Coptidis Rhizoma, has been shown to induce cancer cell autophagic death. Glucose regulated protein 78 (GRP78) is associated with stress-induced autophagy. However, the related mechanisms between berberine-induced cancer cell autophagy and GRP78 remain to be elucidated. Here, we report that berberine can induce autophagic cancer cell death by elevating levels of GRP78. These results further demonstrated that berberine enhanced GRP78 by suppression of ubiquitination / proteasomal degradation of GRP78 and activation of ATF6. Moreover, fluorescence spectrum assay revealed that berberine could bind to GRP78 and form complexes. Finally, co-IP analysis showed that the ability of GRP78 to bind to VPS34 was increased with berberine treatment. Taken together, our results suggest that berberine induces autophagic cancer cell death via enhancing GRP78 levels and the ability of GRP78 to bind to VPS34. PMID:28157699

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ji, Chao; Yang, Bo; Yang, Zhi

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 HaCaTmore » 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.« less

Hyperglycemia potentiates a shift from apoptosis to RIP1-dependent necroptosis.

PubMed

McCaig, William D; Patel, Payal S; Sosunov, Sergey A; Shakerley, Nicole L; Smiraglia, Tori A; Craft, Miranda M; Walker, Katharine M; Deragon, Matthew A; Ten, Vadim S; LaRocca, Timothy J

2018-01-01

Apoptosis and necroptosis are the primary modes of eukaryotic cell death, with apoptosis being non-inflammatory while necroptosis is highly inflammatory. We previously demonstrated that, once activated, necroptosis is enhanced by hyperglycemia in several cell types. Here, we determine if hyperglycemia affects apoptosis similarly. We show that hyperglycemia does not enhance extrinsic apoptosis but potentiates a shift to RIP1-dependent necroptosis. This is due to increased levels and activity of RIP1, RIP3, and MLKL, as well as decreased levels and activity of executioner caspases under hyperglycemic conditions following stimulation of apoptosis. Cell death under hyperglycemic conditions was classified as necroptosis via measurement of markers and involvement of RIP1, RIP3, and MLKL. The shift to necroptosis was driven by RIP1, as mutation of this gene using CRISPR-Cas9 caused cell death to revert to apoptosis under hyperglycemic conditions. The shift of apoptosis to necroptosis depended on glycolysis and production of mitochondrial ROS. Importantly, the shift in PCD was observed in primary human T cells. Levels of RIP1 and MLKL increased, while executioner caspases and PARP1 cleavage decreased, in cerebral tissue from hyperglycemic neonatal mice that underwent hypoxia-ischemia (HI) brain injury, suggesting that this cell death shift occurs in vivo . This is significant as it demonstrates a shift from non-inflammatory to inflammatory cell death which may explain the exacerbation of neonatal HI-brain injury during hyperglycemia. These results are distinct from our previous findings where hyperglycemia enhanced necroptosis under conditions where apoptosis was inhibited artificially. Here we demonstrate a shift from apoptosis to necroptosis under hyperglycemic conditions while both pathways are fully active. Therefore, while our previous work documented that intensity of necroptosis is responsive to glucose, this work sheds light on the molecular balance between apoptosis and necroptosis and identifies hyperglycemia as a condition that pushes cells to undergo necroptosis despite the initial activation of apoptosis.

Synergistic interaction of the cannabinoid and death receptor systems - a potential target for future cancer therapies?

PubMed

Keresztes, Attila; Streicher, John M

2017-10-01

Cannabinoid receptors have been shown to interact with other receptors, including tumor necrosis factor receptor superfamily (TNFRS) members, to induce cancer cell death. When cannabinoids and death-inducing ligands (including TNF-related apoptosis-inducing ligand) are administered together, they have been shown to synergize and demonstrate enhanced antitumor activity in vitro. Certain cannabinoid ligands have been shown to sensitize cancer cells and synergistically interact with members of the TNFRS, thus suggesting that the combination of cannabinoids with death receptor (DR) ligands induces additive or synergistic tumor cell death. This review summarizes recent findings on the interaction of the cannabinoid and DR systems and suggests possible clinical co-application of cannabinoids and DR ligands in the treatment of various malignancies. © 2017 Federation of European Biochemical Societies.

Hepatic leukemia factor promotes resistance to cell death: Implications for therapeutics and chronotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J., E-mail: Thomas.Weber@pnl.gov

Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional programmore » encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation. - Highlights: ► Circadian-dependent physiological variation impacts therapeutic efficacy. ► Hepatic leukemia factor inhibits cell death and is a candidate circadian factor. ► Hepatic leukemia factor anti-death program is conserved in murine and human cells. ► Transcriptomics indicates the anti-death program results from a systems response.« less

Yersinia YopP-induced apoptotic cell death in murine dendritic cells is partially independent from action of caspases and exhibits necrosis-like features.

PubMed

Gröbner, Sabine; Autenrieth, Stella E; Soldanova, Irena; Gunst, Dani S J; Schaller, Martin; Bohn, Erwin; Müller, Steffen; Leverkus, Martin; Wesselborg, Sebastian; Autenrieth, Ingo B; Borgmann, Stefan

2006-11-01

Yersinia outer protein P (YopP) is a virulence factor of Yersinia enterocolitica that is injected into the cytosol of host cells where it targets MAP kinase kinases (MKKs) and inhibitor of kappaB kinase (IKK)-beta resulting in inhibition of cytokine production as well as induction of apoptosis in murine macrophages and dendritic cells (DC). Here we show that DC death was only partially prevented by the broad spectrum caspase inhibitor zVAD-fmk, indicating simultaneous caspase-dependent and caspase-independent mechanisms of cell death induction by YopP. Microscopic analyses and measurement of cell size demonstrated necrosis-like morphology of caspase-independent cell death. Application of zVAD-fmk prevented cleavage of procaspases and Bid, decrease of the inner transmembrane mitochondrial potential DeltaPsi(m) and mitochondrial release of cytochrome c. From these data we conclude that YopP-induced activation of the mitochondrial death pathway is mediated upstream via caspases. In conclusion, our results suggest that YopP simultaneously induces caspase-dependent apoptotic and caspase-independent necrosis-like death in DC. However, it has to be resolved if necrosis-like DC death occurs independently from apoptotic events or as an apoptotic epiphenomenon.

α-Secretase-derived Fragment of Cellular Prion, N1, Protects against Monomeric and Oligomeric Amyloid β (Aβ)-associated Cell Death*

PubMed Central

Guillot-Sestier, Marie-Victoire; Sunyach, Claire; Ferreira, Sergio T.; Marzolo, Maria-Paz; Bauer, Charlotte; Thevenet, Aurélie; Checler, Frédéric

2012-01-01

In physiological conditions, both β-amyloid precursor protein (βAPP) and cellular prion (PrPc) undergo similar disintegrin-mediated α-secretase cleavage yielding N-terminal secreted products referred to as soluble amyloid precursor protein-α (sAPPα) and N1, respectively. We recently demonstrated that N1 displays neuroprotective properties by reducing p53-dependent cell death both in vitro and in vivo. In this study, we examined the potential of N1 as a neuroprotector against amyloid β (Aβ)-mediated toxicity. We first show that both recombinant sAPPα and N1, but not its inactive parent fragment N2, reduce staurosporine-stimulated caspase-3 activation and TUNEL-positive cell death by lowering p53 promoter transactivation and activity in human cells. We demonstrate that N1 also lowers toxicity, cell death, and p53 pathway exacerbation triggered by Swedish mutated βAPP overexpression in human cells. We designed a CHO cell line overexpressing the London mutated βAPP (APPLDN) that yields Aβ oligomers. N1 protected primary cultured neurons against toxicity and cell death triggered by oligomer-enriched APPLDN-derived conditioned medium. Finally, we establish that N1 also protects neurons against oligomers extracted from Alzheimer disease-affected brain tissues. Overall, our data indicate that a cellular prion catabolite could interfere with Aβ-associated toxicity and that its production could be seen as a cellular protective mechanism aimed at compensating for an sAPPα deficit taking place at the early asymptomatic phase of Alzheimer disease. PMID:22184125

Vitamin K3 triggers human leukemia cell death through hydrogen peroxide generation and histone hyperacetylation.

PubMed

Lin, Changjun; Kang, Jiuhong; Zheng, Rongliang

2005-10-01

Vitamin K3 (VK3) is a well-known anticancer agent, but its mechanism remains elusive. In the present study, VK3 was found to simultaneously induce cell death, reactive oxygen species (ROS) generation, including superoxide anion (O2*-) and hydrogen peroxide (H2O2) generation, and histone hyperacetylation in human leukemia HL-60 cells in a concentration- and time-dependent manner. Catalase (CAT), an antioxidant enzyme that specifically scavenges H2O2, could significantly diminish both histone acetylation increase and cell death caused by VK3, whereas superoxide dismutase (SOD), an enzyme that specifically eliminates O2*-, showed no effect on both of these, leading to the conclusion that H2O2 generation, but not O2*- generation, contributes to VK3-induced histone hyperacetylation and cell death. This conclusion was confirmed by the finding that enhancement of VK3-induced H2O2 generation by vitamin C (VC) could significantly promote both the histone hyperacetylation and cell death. Further studies suggested that histone hyperacetylation played an important role in VK3-induced cell death, since sodium butyrate, a histone deacetylase (HDAC) inhibitor, showed no effect on ROS generation, but obviously potentiated VK3-induced histone hyperacetylation and cell death. Collectively, these results demonstrate a novel mechanism for the anticancer activity of VK3, i.e., VK3 induced tumor cell death through H2O2 generation, which then further induced histone hyperacetylation.

Novel smac mimetic APG-1387 elicits ovarian cancer cell killing through TNF-alpha, Ripoptosome and autophagy mediated cell death pathway.

PubMed

Li, Bao-Xia; Wang, Heng-Bang; Qiu, Miao-Zhen; Luo, Qiu-Yun; Yi, Han-Jie; Yan, Xiang-Lei; Pan, Wen-Tao; Yuan, Lu-Ping; Zhang, Yu-Xin; Xu, Jian-Hua; Zhang, Lin; Yang, Da-Jun

2018-03-12

Ovarian cancer is a deadly disease. Inhibitors of apoptosis proteins (IAPs) are key regulators of apoptosis and are frequently dysregulated in ovarian cancer. Overexpression of IAPs proteins has been correlated with tumorigenesis, treatment resistance and poor prognosis. Reinstalling functional cell death machinery by pharmacological inhibition of IAPs proteins may represent an attractive therapeutic strategy for treatment of ovarian cancer. CCK-8 and colony formation assay was performed to examine cytotoxic activity. Apoptosis was analyzed by fluorescence microscopy, flow cytometry and TUNEL assay. Elisa assay was used to determine TNFα protein. Caspase activity assay was used for caspase activation evaluation. Immunoprecipitation and siRNA interference were carried out for functional analysis. Western blotting analysis were carried out to test protein expression. Ovarian cancer cell xenograft nude mice model was used for in vivo efficacy evaluation. APG-1387 demonstrated potent inhibitory effect on ovarian cancer cell growth and clonogenic cell survival. APG-1387 induced RIP1- and TNFα-dependent apoptotic cell death in ovarian cancer through downregulation of IAPs proteins and induction of caspase-8/FADD/RIP1 complex, which drives caspase-8 activation. NF-κB signaling pathway was activated upon APG-1387 treatment and RIP1 contributed to NF-κB activation. APG-1387 induced cytoprotective autophagy while triggering apoptosis in ovarian cancer cells and inhibition of autophagy enhanced APG-1387-induced apoptotic cell death. APG-1387 exhibited potent antitumor activity against established human ovarian cancer xenografts. Our results demonstrate that APG-1387 targets IAPs proteins to potently elicit apoptotic cell death in vitro and in vivo, and provide mechanistic and applicable rationale for future clinical evaluation of APG-1387 in ovarian cancer.

Cigarette smoke-induced cell death of a spermatocyte cell line can be prevented by inactivating the Aryl hydrocarbon receptor

PubMed Central

Esakky, P; Hansen, D A; Drury, A M; Cusumano, A; Moley, K H

2015-01-01

Cigarette smoke exposure causes germ cell death during spermatogenesis. Our earlier studies demonstrated that cigarette smoke condensate (CSC) causes spermatocyte cell death in vivo and growth arrest of the mouse spermatocyte cell line (GC-2spd(ts)) in vitro via the aryl hydrocarbon receptor (AHR). We hypothesize here that inactivation of AHR could prevent the CSC-induced cell death in spermatocytes. We demonstrate that CSC exposure generates oxidative stress, which differentially regulates mitochondrial apoptosis in GC-2spd(ts) and wild type (WT) and AHR knockout (AHR-KO) mouse embryonic fibroblasts (MEFs). SiRNA-mediated silencing of Ahr augments the extent of CSC-mediated cellular damage while complementing the AHR-knockout condition. Pharmacological inhibition using the AHR-antagonist (CH223191) modulates the CSC-altered expression of apoptotic proteins and significantly abrogates DNA fragmentation though the cleavage of PARP appears AHR independent. Pretreatment with CH223191 at concentrations above 50 μM significantly prevents the CSC-induced activation of caspase-3/7 and externalization of phosphatidylserine in the plasma membrane. However, MAPK inhibitors alone or together with CH223191 could not prevent the membrane damage upon CSC addition and the caspase-3/7 activation and membrane damage in AHR-deficient MEF indicates the interplay of multiple cell signaling and cytoprotective ability of AHR. Thus the data obtained on one hand signifies the protective role of AHR in maintaining normal cellular homeostasis and the other, could be a potential prophylactic therapeutic target to promote cell survival and growth under cigarette smoke exposed environment by receptor antagonism via CH223191-like mechanism. Antagonist-mediated inactivation of the aryl hydrocarbon receptor blocks downstream events leading to cigarette smoke-induced cell death of a spermatocyte cell line. PMID:27551479

Necroptosis: an alternative cell death program defending against cancer

PubMed Central

Chen, Dongshi; Yu, Jian; Zhang, Lin

2016-01-01

One of the hallmarks of cancer is resistance to programmed cell death, which maintains the survival of cells en route to oncogenic transformation and underlies therapeutic resistance. Recent studies demonstrate that programmed cell death is not confined to caspase-dependent apoptosis, but includes necroptosis, a form of necrotic death governed by Receptor-Interacting Protein 1 (RIP1), RIP3, and Mixed Lineage Kinase Domain-Like (MLKL). Necroptosis serves as a critical cell-killing mechanism in response to severe stress and blocked apoptosis, and can be induced by inflammatory cytokines or chemotherapeutic drugs. Genetic or epigenetic alterations of necroptosis regulators such as RIP3 and cylindromatosis (CYLD), are frequently found in human tumors. Unlike apoptosis, necroptosis elicits a more robust immune response that may function as a defensive mechanism by eliminating tumor-causing mutations and viruses. Furthermore, several classes of anticancer agents currently under clinical development, such as SMAC and BH3 mimetics, can promote necroptosis in addition to apoptosis. A more complete understanding of the interplay among necroptosis, apoptosis, and other cell death modalities is critical for developing new therapeutic strategies to enhance killing of tumor cells. PMID:26968619

Cell Death Pathways in Mutant Rhodopsin Rat Models Identifies Genotype-Specific Targets Controlling Retinal Degeneration.

PubMed

Viringipurampeer, Ishaq A; Gregory-Evans, Cheryl Y; Metcalfe, Andrew L; Bashar, Emran; Moritz, Orson L; Gregory-Evans, Kevin

2018-06-18

Retinitis pigmentosa (RP) is a group of inherited neurological disorders characterized by rod photoreceptor cell death, followed by secondary cone cell death leading to progressive blindness. Currently, there are no viable treatment options for RP. Due to incomplete knowledge of the molecular signaling pathways associated with RP pathogenesis, designing therapeutic strategies remains a challenge. In particular, preventing secondary cone photoreceptor cell loss is a key goal in designing potential therapies. In this study, we identified the main drivers of rod cell death and secondary cone loss in the transgenic S334ter rhodopsin rat model, tested the efficacy of specific cell death inhibitors on retinal function, and compared the effect of combining drugs to target multiple pathways in the S334ter and P23H rhodopsin rat models. The primary driver of early rod cell death in the S334ter model was a caspase-dependent process, whereas cone cell death occurred though RIP3-dependent necroptosis. In comparison, rod cell death in the P23H model was via necroptotic signaling, whereas cone cell loss occurred through inflammasome activation. Combination therapy of four drugs worked better than the individual drugs in the P23H model but not in the S334ter model. These differences imply that treatment modalities need to be tailored for each genotype. Taken together, our data demonstrate that rationally designed genotype-specific drug combinations will be an important requisite to effectively target primary rod cell loss and more importantly secondary cone survival.

Loss of Atrx Sensitizes Cells to DNA Damaging Agents through p53-Mediated Death Pathways

PubMed Central

Conte, Damiano; Huh, Michael; Goodall, Emma; Delorme, Marilyne; Parks, Robin J.; Picketts, David J.

2012-01-01

Prevalent cell death in forebrain- and Sertoli cell-specific Atrx knockout mice suggest that Atrx is important for cell survival. However, conditional ablation in other tissues is not associated with increased death indicating that diverse cell types respond differently to the loss of this chromatin remodeling protein. Here, primary macrophages isolated from Atrx f/f mice were infected with adenovirus expressing Cre recombinase or β-galactosidase, and assayed for cell survival under different experimental conditions. Macrophages survive without Atrx but undergo rapid apoptosis upon lipopolysaccharide (LPS) activation suggesting that chromatin reorganization in response to external stimuli is compromised. Using this system we next tested the effect of different apoptotic stimuli on cell survival. We observed that survival of Atrx-null cells were similar to wild type cells in response to serum withdrawal, anti-Fas antibody, C2 ceramide or dexamethasone treatment but were more sensitive to 5-fluorouracil (5-FU). Cell survival could be rescued by re-introducing Atrx or by removal of p53 demonstrating the cell autonomous nature of the effect and its p53-dependence. Finally, we demonstrate that multiple primary cell types (myoblasts, embryonic fibroblasts and neurospheres) were sensitive to 5-FU, cisplatin, and UV light treatment. Together, our results suggest that cells lacking Atrx are more sensitive to DNA damaging agents and that this may result in enhanced death during development when cells are at their proliferative peak. Moreover, it identifies potential treatment options for cancers associated with ATRX mutations, including glioblastoma and pancreatic neuroendocrine tumors. PMID:23284920

Loss of Atrx sensitizes cells to DNA damaging agents through p53-mediated death pathways.

PubMed

Conte, Damiano; Huh, Michael; Goodall, Emma; Delorme, Marilyne; Parks, Robin J; Picketts, David J

2012-01-01

Prevalent cell death in forebrain- and Sertoli cell-specific Atrx knockout mice suggest that Atrx is important for cell survival. However, conditional ablation in other tissues is not associated with increased death indicating that diverse cell types respond differently to the loss of this chromatin remodeling protein. Here, primary macrophages isolated from Atrx(f/f) mice were infected with adenovirus expressing Cre recombinase or β-galactosidase, and assayed for cell survival under different experimental conditions. Macrophages survive without Atrx but undergo rapid apoptosis upon lipopolysaccharide (LPS) activation suggesting that chromatin reorganization in response to external stimuli is compromised. Using this system we next tested the effect of different apoptotic stimuli on cell survival. We observed that survival of Atrx-null cells were similar to wild type cells in response to serum withdrawal, anti-Fas antibody, C2 ceramide or dexamethasone treatment but were more sensitive to 5-fluorouracil (5-FU). Cell survival could be rescued by re-introducing Atrx or by removal of p53 demonstrating the cell autonomous nature of the effect and its p53-dependence. Finally, we demonstrate that multiple primary cell types (myoblasts, embryonic fibroblasts and neurospheres) were sensitive to 5-FU, cisplatin, and UV light treatment. Together, our results suggest that cells lacking Atrx are more sensitive to DNA damaging agents and that this may result in enhanced death during development when cells are at their proliferative peak. Moreover, it identifies potential treatment options for cancers associated with ATRX mutations, including glioblastoma and pancreatic neuroendocrine tumors.

Macrophage-mediated trogocytosis leads to death of antibody-opsonized tumor cells

PubMed Central

Velmurugan, Ramraj; Challa, Dilip K.; Ram, Sripad; Ober, Raimund J.; Ward, E. Sally

2016-01-01

Understanding the complex behavior of effector cells such as monocytes or macrophages in regulating cancerous growth is of central importance for cancer immunotherapy. Earlier studies using CD20-specific antibodies have demonstrated that the Fcγ receptor (FcγR)-mediated transfer of the targeted receptors from tumor cells to these effector cells through trogocytosis can enable escape from antibody therapy, leading to the viewpoint that this process is pro-tumorigenic. In the current study we demonstrate that persistent trogocytic attack results in the killing of HER2-overexpressing breast cancer cells. Further, antibody engineering to increase FcγR interactions enhances this tumoricidal activity. These studies extend the complex repertoire of activities of macrophages to trogocytic-mediated cell death of HER2-overexpressing target cells and have implications for the development of effective antibody-based therapies. PMID:27226489

Live-cell visualization of gasdermin D-driven pyroptotic cell death.

PubMed

Rathkey, Joseph K; Benson, Bryan L; Chirieleison, Steven M; Yang, Jie; Xiao, Tsan S; Dubyak, George R; Huang, Alex Y; Abbott, Derek W

2017-09-01

Pyroptosis is a form of cell death important in defenses against pathogens that can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, GSDMD (gasdermin D), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and -11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis but also enable its direct visualization. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Arctigenin preferentially induces tumor cell death under glucose deprivation by inhibiting cellular energy metabolism.

PubMed

Gu, Yuan; Qi, Chunting; Sun, Xiaoxiao; Ma, Xiuquan; Zhang, Haohao; Hu, Lihong; Yuan, Junying; Yu, Qiang

2012-08-15

Selectively eradicating cancer cells with minimum adverse effects on normal cells is a major challenge in the development of anticancer therapy. We hypothesize that nutrient-limiting conditions frequently encountered by cancer cells in poorly vascularized solid tumors might provide an opportunity for developing selective therapy. In this study, we investigated the function and molecular mechanisms of a natural compound, arctigenin, in regulating tumor cell growth. We demonstrated that arctigenin selectively promoted glucose-starved A549 tumor cells to undergo necrosis by inhibiting mitochondrial respiration. In doing so, arctigenin elevated cellular level of reactive oxygen species (ROS) and blocked cellular energy metabolism in the glucose-starved tumor cells. We also demonstrated that cellular ROS generation was caused by intracellular ATP depletion and played an essential role in the arctigenin-induced tumor cell death under the glucose-limiting condition. Furthermore, we combined arctigenin with the glucose analogue 2-deoxyglucose (2DG) and examined their effects on tumor cell growth. Interestingly, this combination displayed preferential cell-death inducing activity against tumor cells compared to normal cells. Hence, we propose that the combination of arctigenin and 2DG may represent a promising new cancer therapy with minimal normal tissue toxicity. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

Cell death sensitization of leukemia cells by opioid receptor activation

PubMed Central

Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich

2013-01-01

Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

The antioxidant N-acetyl cysteine suppresses lidocaine-induced intracellular reactive oxygen species production and cell death in neuronal SH-SY5Y cells.

PubMed

Okamoto, Akihisa; Tanaka, Masahiro; Sumi, Chisato; Oku, Kanako; Kusunoki, Munenori; Nishi, Kenichiro; Matsuo, Yoshiyuki; Takenaga, Keizo; Shingu, Koh; Hirota, Kiichi

2016-10-24

The local anesthetic lidocaine can affect intra- and extra-cellular signaling pathways in both neuronal and non-neuronal cells, resulting in long-term modulation of biological functions, including cell growth and death. Indeed, lidocaine was shown to induce necrosis and apoptosis in vitro. While several studies have suggested that lidocaine-induced apoptosis is mitochondrial pathway-dependent, it remains unclear whether reactive oxygen species (ROS) are involved in this process and whether the observed cell death can be prevented by antioxidant treatment. The effects of lidocaine and antioxidants on cell viability and death were evaluated using SH-SY5Y cells, HeLa cells, and HeLa cell derivatives. Cell viability was examined via MTS/PES ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]/phenazine ethosulfate) assay. Meanwhile, cell apoptosis and necrosis were evaluated using a cell death detection assay with Annexin V-FITC and PI staining, as well as by assaying for caspase-3/7 and caspase-9 activity, and by measuring the release of lactate dehydrogenase, respectively. Mitochondrial transmembrane potential (ΔΨm) was assessed using the fluorescent probe tetramethylrhodamine ethyl ester. Lidocaine treatment resulted in suppression of the mitochondrial electron transport chain and subsequent attenuation of mitochondrial membrane potential, as well as enhanced ROS production, activation of caspase-3/7 and caspase-9, and induction of apoptosis and necrosis in SH-SY5Y cells in a dose- and time-dependent manner. Likewise, the anesthetics mepivacaine and bupivacaine also induced apoptosis in SH-SY5Y cells. Notably, the antioxidants N-acetyl cysteine (NAC) and Trolox successfully scavenged the mitochondria-derived ROS and suppressed local lidocaine-induced cell death. Our findings demonstrate that the local anesthetics lidocaine, mepivacaine, and bupivacaine inhibited the activity of mitochondria and induced apoptosis and necrosis in a dose-dependent manner. Furthermore, they demonstrate that treatment with the antioxidants NAC, Trolox, and GGA resulted in preservation of mitochondrial voltage and inhibition of apoptosis via suppression of caspase activation.

Phycocyanin Inhibits Tumorigenic Potential of Pancreatic Cancer Cells: Role of Apoptosis and Autophagy

PubMed Central

Liao, Gaoyong; Gao, Bing; Gao, Yingnv; Yang, Xuegan; Cheng, Xiaodong; Ou, Yu

2016-01-01

Pancreatic adenocarcinoma (PDA) is one of the most lethal human malignancies, and unresponsive to current chemotherapies. Here we investigate the therapeutic potential of phycocyanin as an anti-PDA agent in vivo and in vitro. Phycocyanin, a natural product purified from Spirulina, effectively inhibits the pancreatic cancer cell proliferation in vitro and xenograft tumor growth in vivo. Phycocyanin induces G2/M cell cycle arrest, apoptotic and autophagic cell death in PANC-1 cells. Inhibition of autophagy by targeting Beclin 1 using siRNA significantly suppresses cell growth inhibition and death induced by phycocyanin, whereas inhibition of both autophagy and apoptosis rescues phycocyanin-mediated cell death. Mechanistically, cell death induced by phycocyanin is the result of cross-talk among the MAPK, Akt/mTOR/p70S6K and NF-κB pathways. Phycocyanin is able to induce apoptosis of PANC-1 cell by activating p38 and JNK signaling pathways while inhibiting Erk pathway. On the other hand, phycocyanin promotes autophagic cell death by inhibiting PI3/Akt/mTOR signaling pathways. Furthermore, phycocyanin promotes the activation and nuclear translocation of NF-κB, which plays an important role in balancing phycocyanin-mediated apoptosis and autosis. In conclusion, our studies demonstrate that phycocyanin exerts anti-pancreatic cancer activity by inducing apoptotic and autophagic cell death, thereby identifying phycocyanin as a promising anti-pancreatic cancer agent. PMID:27694919

Immotile Active Matter: Activity from Death and Reproduction.

PubMed

Kalziqi, Arben; Yanni, David; Thomas, Jacob; Ng, Siu Lung; Vivek, Skanda; Hammer, Brian K; Yunker, Peter J

2018-01-05

Unlike equilibrium atomic solids, biofilms-soft solids composed of bacterial cells-do not experience significant thermal fluctuations at the constituent level. However, living cells stochastically reproduce and die, provoking a mechanical response. We investigate the mechanical consequences of cellular death and reproduction by measuring surface-height fluctuations of biofilms containing two mutually antagonistic strains of Vibrio cholerae that kill one another on contact via the type VI secretion system. While studies of active matter typically focus on activity via constituent mobility, here, activity is mediated by reproduction and death events in otherwise immobilized cells. Biofilm surface topography is measured in the nearly homeostatic limit via white light interferometry. Although biofilms are far from equilibrium systems, measured surface-height fluctuation spectra resemble the spectra of thermal permeable membranes but with an activity-mediated effective temperature, as predicted by Risler, Peilloux, and Prost [Phys. Rev. Lett. 115, 258104 (2015)PRLTAO0031-900710.1103/PhysRevLett.115.258104]. By comparing the activity of killer strains of V. cholerae with that of genetically modified strains that cannot kill each other and validating with individual-based simulations, we demonstrate that extracted effective temperatures increase with the amount of death and reproduction and that death and reproduction can fluidize biofilms. Together, these observations demonstrate the unique physical consequences of activity mediated by death and reproduction events.

Characterization of Puma-Dependent and Puma-Independent Neuronal Cell Death Pathways following Prolonged Proteasomal Inhibition▿

PubMed Central

Tuffy, Liam P.; Concannon, Caoimhín G.; D'Orsi, Beatrice; King, Matthew A.; Woods, Ina; Huber, Heinrich J.; Ward, Manus W.; Prehn, Jochen H. M.

2010-01-01

Proteasomal stress and the accumulation of polyubiquitinated proteins are key features of numerous neurodegenerative disorders. Previously we demonstrated that stabilization of p53 and activation of its target gene, puma (p53-upregulated mediator of apoptosis), mediated proteasome inhibitor-induced apoptosis in cancer cells. Here we demonstrated that Puma also contributed to proteasome inhibitor-induced apoptosis in mouse neocortical neurons. Although protection afforded by puma gene deletion was incomplete, we found little evidence indicating contributions from other proapoptotic BH3-only proteins. Attenuation of bax expression did not further reduce Puma-independent apoptosis, suggesting that pathways other than the mitochondrial apoptosis pathway were activated. Real-time imaging experiments in wild-type and puma-deficient neurons using a fluorescence resonance energy transfer (FRET)-based caspase sensor confirmed the involvement of a second cell death pathway characterized by caspase activation prior to mitochondrial permeabilization and, more prominently, a third, caspase-independent and Puma-independent pathway characterized by rapid cell shrinkage and nuclear condensation. This pathway involved lysosomal permeabilization in the absence of autophagy activation and was sensitive to cathepsin but not autophagy inhibition. Our data demonstrate that proteasomal stress activates distinct cell death pathways in neurons, leading to both caspase-dependent and caspase-independent apoptosis, and demonstrate independent roles for Puma and lysosomal permeabilization in this model. PMID:20921277

A crucial role for the phosphorylation of STRAP at Ser188 by MPK38 in STRAP-dependent cell death through ASK1, TGF-β, p53, and PI3K/PDK1 signaling pathways

PubMed Central

Seong, Hyun-A; Manoharan, Ravi; Ha, Hyunjung

2014-01-01

Serine-threonine kinase receptor-associated protein (STRAP) is a TGF-β receptor-interacting protein that participates in the regulation of cell proliferation and cell death in response to various stresses. Here, we demonstrate that STRAP phosphorylation plays an important role in determining the pro- or anti-apoptotic function of STRAP. Murine protein serine/threonine kinase 38 (MPK38) phosphorylates STRAP at Ser188 via direct interaction. Complex formation between STRAP and MPK38 is mediated by Cys152 and Cys270 of STRAP and Cys339 and Cys377 of MPK38, suggesting the redox dependency of this interaction. MPK38-mediated STRAP Ser188 phosphorylation contributes to the pro-apoptotic function of STRAP by modulating key steps in STRAP-dependent ASK1, TGF-β, p53, and PI3K/PDK1 signaling pathways. Moreover, knockdown of endogenous MPK38 using an inducible MPK38 shRNA system and in vivo activation of MPK38 by treatment of HEK293 and STRAP-null MEF cells with 1-chloro-2,4-dinitrobenzene (DNCB), a specific inhibitor of Trx reductase, provide evidence that STRAP Ser188 phosphorylation plays a key role in STRAP-dependent cell death. Adenoviral delivery of MPK38 in mice also demonstrates that STRAP Ser188 phosphorylation in the liver is tightly associated with cell death and proliferation through ASK1, TGF-β, p53, and PI3K/PDK1 pathways, resulting in apoptotic cell death. PMID:25485581

A crucial role for the phosphorylation of STRAP at Ser(188) by MPK38 in STRAP-dependent cell death through ASK1, TGF-β, p53, and PI3K/PDK1 signaling pathways.

PubMed

Seong, Hyun-A; Manoharan, Ravi; Ha, Hyunjung

2014-01-01

Serine-threonine kinase receptor-associated protein (STRAP) is a TGF-β receptor-interacting protein that participates in the regulation of cell proliferation and cell death in response to various stresses. Here, we demonstrate that STRAP phosphorylation plays an important role in determining the pro- or anti-apoptotic function of STRAP. Murine protein serine/threonine kinase 38 (MPK38) phosphorylates STRAP at Ser(188) via direct interaction. Complex formation between STRAP and MPK38 is mediated by Cys(152) and Cys(270) of STRAP and Cys(339) and Cys(377) of MPK38, suggesting the redox dependency of this interaction. MPK38-mediated STRAP Ser(188) phosphorylation contributes to the pro-apoptotic function of STRAP by modulating key steps in STRAP-dependent ASK1, TGF-β, p53, and PI3K/PDK1 signaling pathways. Moreover, knockdown of endogenous MPK38 using an inducible MPK38 shRNA system and in vivo activation of MPK38 by treatment of HEK293 and STRAP-null MEF cells with 1-chloro-2,4-dinitrobenzene (DNCB), a specific inhibitor of Trx reductase, provide evidence that STRAP Ser(188) phosphorylation plays a key role in STRAP-dependent cell death. Adenoviral delivery of MPK38 in mice also demonstrates that STRAP Ser(188) phosphorylation in the liver is tightly associated with cell death and proliferation through ASK1, TGF-β, p53, and PI3K/PDK1 pathways, resulting in apoptotic cell death.

NEMO Inhibits Programmed Necrosis in an NFκB-Independent Manner by Restraining RIP1

PubMed Central

Legarda, Diana; Ting, Adrian T.

2012-01-01

TNF can trigger two opposing responses: cell survival and cell death. TNFR1 activates caspases that orchestrate apoptosis but some cell types switch to a necrotic death when treated with caspase inhibitors. Several genes that are required to orchestrate cell death by programmed necrosis have been identified, such as the kinase RIP1, but very little is known about the inhibitory signals that keep this necrotic cell death pathway in check. We demonstrate that T cells lacking the regulatory subunit of IKK, NFκB essential modifier (NEMO), are hypersensitive to programmed necrosis when stimulated with TNF in the presence of caspase inhibitors. Surprisingly, this pro-survival activity of NEMO is independent of NFκB-mediated gene transcription. Instead, NEMO inhibits necrosis by binding to ubiquitinated RIP1 to restrain RIP1 from engaging the necrotic death pathway. In the absence of NEMO, or if ubiquitination of RIP1 is blocked, necrosis ensues when caspases are blocked. These results indicate that recruitment of NEMO to ubiquitinated RIP1 is a key step in the TNFR1 signaling pathway that determines whether RIP1 triggers a necrotic death response. PMID:22848449

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

PubMed Central

Yadav, Neelu; Kumar, Sandeep; Kumar, Rahul; Srivastava, Pragya; Sun, Leimin; Rapali, Peter; Marlowe, Timothy; Schneider, Andrea; Inigo, Joseph; O’Malley, Jordan; Londonkar, Ramesh; Gogada, Raghu; Chaudhary, Ajay; 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. PMID:26627937

Hedgehog inhibition promotes a switch from Type II to Type I cell death receptor signaling in cancer cells.

PubMed

Kurita, Satoshi; Mott, Justin L; Cazanave, Sophie C; Fingas, Christian D; Guicciardi, Maria E; Bronk, Steve F; Roberts, Lewis R; Fernandez-Zapico, Martin E; Gores, Gregory J

2011-03-31

TRAIL is a promising therapeutic agent for human malignancies. TRAIL often requires mitochondrial dysfunction, referred to as the Type II death receptor pathway, to promote cytotoxicity. However, numerous malignant cells are TRAIL resistant due to inhibition of this mitochondrial pathway. Using cholangiocarcinoma cells as a model of TRAIL resistance, we found that Hedgehog signaling blockade sensitized these cancer cells to TRAIL cytotoxicity independent of mitochondrial dysfunction, referred to as Type I death receptor signaling. This switch in TRAIL requirement from Type II to Type I death receptor signaling was demonstrated by the lack of functional dependence on Bid/Bim and Bax/Bak, proapoptotic components of the mitochondrial pathway. Hedgehog signaling modulated expression of X-linked inhibitor of apoptosis (XIAP), which serves to repress the Type I death receptor pathway. siRNA targeted knockdown of XIAP mimics sensitization to mitochondria-independent TRAIL killing achieved by Hedgehog inhibition. Regulation of XIAP expression by Hedgehog signaling is mediated by the glioma-associated oncogene 2 (GLI2), a downstream transcription factor of Hedgehog. In conclusion, these data provide additional mechanisms modulating cell death by TRAIL and suggest Hedgehog inhibition as a therapeutic approach for TRAIL-resistant neoplasms.

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

PubMed

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.

Overexpression of BAX INHIBITOR-1 Links Plasma Membrane Microdomain Proteins to Stress.

PubMed

Ishikawa, Toshiki; Aki, Toshihiko; Yanagisawa, Shuichi; Uchimiya, Hirofumi; Kawai-Yamada, Maki

2015-10-01

BAX INHIBITOR-1 (BI-1) is a cell death suppressor widely conserved in plants and animals. Overexpression of BI-1 enhances tolerance to stress-induced cell death in plant cells, although the molecular mechanism behind this enhancement is unclear. We recently found that Arabidopsis (Arabidopsis thaliana) BI-1 is involved in the metabolism of sphingolipids, such as the synthesis of 2-hydroxy fatty acids, suggesting the involvement of sphingolipids in the cell death regulatory mechanism downstream of BI-1. Here, we show that BI-1 affects cell death-associated components localized in sphingolipid-enriched microdomains of the plasma membrane in rice (Oryza sativa) cells. The amount of 2-hydroxy fatty acid-containing glucosylceramide increased in the detergent-resistant membrane (DRM; a biochemical counterpart of plasma membrane microdomains) fraction obtained from BI-1-overexpressing rice cells. Comparative proteomics analysis showed quantitative changes of DRM proteins in BI-1-overexpressing cells. In particular, the protein abundance of FLOTILLIN HOMOLOG (FLOT) and HYPERSENSITIVE-INDUCED REACTION PROTEIN3 (HIR3) markedly decreased in DRM of BI-1-overexpressing cells. Loss-of-function analysis demonstrated that FLOT and HIR3 are required for cell death by oxidative stress and salicylic acid, suggesting that the decreased levels of these proteins directly contribute to the stress-tolerant phenotypes in BI-1-overexpressing rice cells. These findings provide a novel biological implication of plant membrane microdomains in stress-induced cell death, which is negatively modulated by BI-1 overexpression via decreasing the abundance of a set of key proteins involved in cell death. © 2015 American Society of Plant Biologists. All Rights Reserved.

Protective mechanisms of p53-p21-pRb proteins against DNA damage-induced cell death.

PubMed

Garner, Elizabeth; Raj, Kenneth

2008-02-01

There have been innumerate demonstrations of p53's activity as a tumour suppressor protein with the ability to stimulate cell signalling that can lead to cell cycle arrest and cell death in the event of DNA damage. Despite the solid body of evidence to support these properties of p53, reports have emerged that suggest a role for p53 in protecting cells from cell death. Our recent report highlighted a mechanism by which p53 activity can promote cell survival in the event of DNA damage. Here we present the various mechanisms that are activated by p53 signalling that can confer protection to cells with damaged DNA and emphasise the practical and clinical implications of a more balanced and context-dependent understanding of p53's pro-apoptotic and pro-survival activities.

Apoptosome activation, an important molecular instigator in 6-mercaptopurine induced Leydig cell death

PubMed Central

Morgan, Jessica A.; Lynch, John; Panetta, John C.; Wang, Yao; Frase, Sharon; Bao, Ju; Zheng, Jie; Opferman, Joseph T.; Janke, Laura; Green, Daniel M.; Chemaitilly, Wassim; Schuetz, John D.

2015-01-01

Leydig cells are crucial to the production of testosterone in males. It is unknown if the cancer chemotherapeutic drug, 6-mercaptopurine (6 MP), produces Leydig cell failure among adult survivors of childhood acute lymphoblastic leukemia. Moreover, it is not known whether Leydig cell failure is due to either a loss of cells or an impairment in their function. Herein, we show, in a subset of childhood cancer survivors, that Leydig cell failure is related to the dose of 6 MP. This was extended, in a murine model, to demonstrate that 6 MP exposure induced caspase 3 activation, and the loss of Leydig cells was independent of Bak and Bax activation. The death of these non-proliferating cells was triggered by 6 MP metabolism, requiring formation of both cytosolic reactive oxygen species and thiopurine nucleotide triphosphates. The thiopurine nucleotide triphosphates (with physiological amounts of dATP) uniquely activated the apoptosome. An ABC transporter (Abcc4/Mrp4) reduced the amount of thiopurines, thereby providing protection for Leydig cells. The studies reported here demonstrate that the apoptosome is uniquely activated by thiopurine nucleotides and suggest that 6 MP induced Leydig cell death is likely a cause of Leydig cell failure in some survivors of childhood cancer. PMID:26576726

Apoptosome activation, an important molecular instigator in 6-mercaptopurine induced Leydig cell death.

PubMed

Morgan, Jessica A; Lynch, John; Panetta, John C; Wang, Yao; Frase, Sharon; Bao, Ju; Zheng, Jie; Opferman, Joseph T; Janke, Laura; Green, Daniel M; Chemaitilly, Wassim; Schuetz, John D

2015-11-18

Leydig cells are crucial to the production of testosterone in males. It is unknown if the cancer chemotherapeutic drug, 6-mercaptopurine (6 MP), produces Leydig cell failure among adult survivors of childhood acute lymphoblastic leukemia. Moreover, it is not known whether Leydig cell failure is due to either a loss of cells or an impairment in their function. Herein, we show, in a subset of childhood cancer survivors, that Leydig cell failure is related to the dose of 6 MP. This was extended, in a murine model, to demonstrate that 6 MP exposure induced caspase 3 activation, and the loss of Leydig cells was independent of Bak and Bax activation. The death of these non-proliferating cells was triggered by 6 MP metabolism, requiring formation of both cytosolic reactive oxygen species and thiopurine nucleotide triphosphates. The thiopurine nucleotide triphosphates (with physiological amounts of dATP) uniquely activated the apoptosome. An ABC transporter (Abcc4/Mrp4) reduced the amount of thiopurines, thereby providing protection for Leydig cells. The studies reported here demonstrate that the apoptosome is uniquely activated by thiopurine nucleotides and suggest that 6 MP induced Leydig cell death is likely a cause of Leydig cell failure in some survivors of childhood cancer.

The sirtuin 1/2 inhibitor tenovin-1 induces a nonlinear apoptosis-inducing factor-dependent cell death in a p53 null Ewing's sarcoma cell line.

PubMed

Marx, Christian; Marx-Blümel, Lisa; Lindig, Nora; Thierbach, René; Hoelzer, Doerte; Becker, Sabine; Wittig, Susan; Lehmann, Roland; Slevogt, Hortense; Heinzel, Thorsten; Wang, Zhao-Qi; Beck, James F; Sonnemann, Jürgen

2018-06-01

The sirtuin 1/2 inhibitor tenovin-1 activates p53 and may have potential in the management of cancer. Here, we investigated the responsiveness of Ewing's sarcoma cells to tenovin-1. We examined its effects in two Ewing's sarcoma cell lines with different p53 status, i.e. in p53 wild-type and p53 null cells. Effects were assessed by flow cytometric analyses of cell death, mitochondrial membrane depolarization and reactive oxygen species (ROS) generation, by caspase 3/7 activity measurement, by mRNA expression profiling and by immunoblotting. Tenovin-1 elicited caspase-mediated cell death in p53 wild-type cells, but caspase-independent cell death in p53 null cells. Remarkably, it induced a nonlinear concentration response in the latter: low concentrations of tenovin-1 were much more effective than were higher concentrations. Tenovin-1's effects in p53 null cells involved gene expression changes of Bcl-2 family members, mitochondrial membrane depolarization, nuclear translocation of apoptosis-inducing factor, ROS formation and DNA damage; all these effects followed a bell-shaped pattern. In conclusion, our results provide new insights into tenovin-1's mode of action by demonstrating that it can induce different pathways of cell death.

N-Desmethyldauricine Induces Autophagic Cell Death in Apoptosis-Defective Cells via Ca2+ Mobilization.

PubMed

Law, Betty Y K; Mok, Simon W F; Chen, Juan; Michelangeli, Francesco; Jiang, Zhi-Hong; Han, Yu; Qu, Yuan Q; Qiu, Alena C L; Xu, Su-Wei; Xue, Wei-Wei; Yao, Xiao-Jun; Gao, Jia Y; Javed, Masood-Ul-Hassan; Coghi, Paolo; Liu, Liang; Wong, Vincent K W

2017-01-01

Resistance of cancer cells to chemotherapy remains a significant problem in oncology. Mechanisms regulating programmed cell death, including apoptosis, autophagy or necrosis, in the treatment of cancers have been extensively investigated over the last few decades. Autophagy is now emerging as an important pathway in regulating cell death or survival in cancer therapy. Recent studies demonstrated variety of natural small-molecules could induce autophagic cell death in apoptosis-resistant cancer cells, therefore, discovery of novel autophagic enhancers from natural products could be a promising strategy for treatment of chemotherapy-resistant cancer. By computational virtual docking analysis, biochemical assays, and advanced live-cell imaging techniques, we have identified N -desmethyldauricine (LP-4), isolated from rhizoma of Menispermum dauricum DC as a novel inducer of autophagy. LP-4 was shown to induce autophagy via the Ulk-1-PERK and Ca 2+ /Calmodulin-dependent protein kinase kinase β (CaMKKβ)-AMPK-mTOR signaling cascades, via mobilizing calcium release through inhibition of SERCA, and importantly, lead to autophagic cell death in a panel of cancer cells, apoptosis-defective and apoptosis-resistant cells. Taken together, this study provides detailed insights into the cytotoxic mechanism of a novel autophagic compound that targeting the apoptosis resistant cancer cells, and new implication on drug discovery from natural products for drug resistant cancer therapy.

Getting the “kill” into “shock and kill”: strategies to eliminate latent HIV

PubMed Central

Kim, Youry; Anderson, Jenny L.; Lewin, Sharon R.

2018-01-01

Despite the success of antiretroviral therapy (ART), there is currently no HIV cure and treatment is lifelong. HIV persists during ART due to long lived and proliferating latently infected CD4+ T-cells. One strategy to eliminate latency is to activate virus production using latency reversing agents (LRAs) with the goal of triggering cell death through virus-induced cytolysis or immune-mediated clearance. However, multiple studies have demonstrated that activation of viral transcription alone is insufficient to induce cell death and some LRAs may counteract cell death by promoting cell survival. Here, we review new approaches to induce death of latently infected cells through apoptosis and inhibition of pathways critical for cell survival, which are often hijacked by HIV proteins. Given advances in the commercial development of compounds that induce apoptosis in cancer chemotherapy, these agents could move rapidly into clinical trials, either alone or in combination with LRAs, to eliminate latent HIV infection. PMID:29324227

The Cytolytic Amphipathic β(2,2)-Amino Acid LTX-401 Induces DAMP Release in Melanoma Cells and Causes Complete Regression of B16 Melanoma

PubMed Central

Eike, Liv-Marie; Mauseth, Brynjar; Camilio, Ketil André; Rekdal, Øystein; Sveinbjørnsson, Baldur

2016-01-01

In the present study we examined the ability of the amino acid derivative LTX-401 to induce cell death in cancer cell lines, as well as the capacity to induce regression in a murine melanoma model. Mode of action studies in vitro revealed lytic cell death and release of danger-associated molecular pattern molecules, preceded by massive cytoplasmic vacuolization and compromised lysosomes in treated cells. The use of a murine melanoma model demonstrated that the majority of animals treated with intratumoural injections of LTX-401 showed complete and long-lasting remission. Taken together, these results demonstrate the potential of LTX-401 as an immunotherapeutic agent for the treatment of solid tumors. PMID:26881822

Combined treatment with fenretinide and indomethacin induces AIF-mediated, non-classical cell death in human acute T-cell leukemia Jurkat cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hojka-Osinska, Anna, E-mail: hojka@immuno.iitd.pan.wroc.pl; Ziolo, Ewa, E-mail: ziolo@immuno.iitd.pan.wroc.pl; Rapak, Andrzej, E-mail: rapak@immuno.iitd.pan.wroc.pl

Highlights: Black-Right-Pointing-Pointer The combination of fenretinide and indomethacin induces a high level of cell death. Black-Right-Pointing-Pointer Apoptotic pathway is caspase-independent. Black-Right-Pointing-Pointer Jurkat cells undergo AIF-mediated cell death. -- Abstract: Currently used cytotoxic drugs in cancer therapy have a similar mechanism of action and low specificity. Applied simultaneously, they show an additive effect with strong side effects. Clinical trials with the use of different agents in cancer therapy show that the use of these compounds alone is not very effective in fighting cancer. An alternative solution could be to apply a combination of these agents, because their combination has a synergisticmore » effect on some cancer cells. Therefore, in our investigations we examined the effects of a synthetic retinoid-fenretinide when combined with a non-steroidal anti-inflammatory drug-indomethacin on the process of apoptosis in the acute human T-cell leukemia cell line Jurkat. We demonstrate that treatment with the combination of the tested compounds induces the death of cells, that is peculiar and combines features of apoptosis as well as non-apoptotic cell death. In detail we observed, cell membrane permeabilization, phosphatydylserine exposure, no oligonucleosomal DNA fragmentation, no caspase-3 activation, but apoptosis inducing factor (AIF) nuclear translocation. Taken together these results indicate, that Jurkat cells after treatment with a combination of fenretinide and indomethacin undergo AIF-mediated programmed cell death.« less

AtPDCD5 Plays a Role in Programmed Cell Death after UV-B Exposure in Arabidopsis1[OPEN

PubMed Central

Falcone Ferreyra, María Lorena; D’Andrea, Lucio; AbdElgawad, Hamada

2016-01-01

DNA damage responses have evolved to sense and react to DNA damage; the induction of DNA repair mechanisms can lead to genomic restoration or, if the damaged DNA cannot be adequately repaired, to the execution of a cell death program. In this work, we investigated the role of an Arabidopsis (Arabidopsis thaliana) protein, AtPDCD5, which is highly similar to the human PDCD5 protein; it is induced by ultraviolet (UV)-B radiation and participates in programmed cell death in the UV-B DNA damage response. Transgenic plants expressing AtPDCD5 fused to GREEN FLUORESCENT PROTEIN indicate that AtPDCD5 is localized both in the nucleus and the cytosol. By use of pdcd5 mutants, we here demonstrate that these plants have an altered antioxidant metabolism and accumulate higher levels of DNA damage after UV-B exposure, similar to levels in ham1ham2 RNA interference transgenic lines with decreased expression of acetyltransferases from the MYST family. By coimmunoprecipitation and pull-down assays, we provide evidence that AtPDCD5 interacts with HAM proteins, suggesting that both proteins participate in the same pathway of DNA damage responses. Plants overexpressing AtPDCD5 show less DNA damage but more cell death in root tips upon UV-B exposure. Finally, we here show that AtPDCD5 also participates in age-induced programmed cell death. Together, the data presented here demonstrate that AtPDCD5 plays an important role during DNA damage responses induced by UV-B radiation in Arabidopsis and also participates in programmed cell death programs. PMID:26884483

Oxaliplatin triggers necrosis as well as apoptosis in gastric cancer SGC-7901 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Ping; Zhu, Xueping; Jin, Wei

Intrinsic apoptotic pathway is considered to be responsible for cell death induced by platinum anticancer drugs. While in this study, we found that, necrosis is an indispensable pathway besides apoptosis in oxaliplatin-treated gastric cancer SGC-7901 cells. Upon exposure to oxaliplatin, both apoptotic and necrotic features were observed. The majority of dead cells were double positive for Annexin V and propidium iodide (PI). Moreover, mitochondrial membrane potential collapsed and caspase cascades were activated. However, ultrastructural changes under transmission electron microscope, coupled with the release of cellular contents, demonstrated the rupture of the plasma membrane. Oxaliplatin administration did not stimulate reactive oxygenmore » species (ROS) production and autophagy, but elevated the protein level of Bmf. In addition, receptor interacting protein 1 (RIP1), but not receptor interacting protein 3 (RIP3) and its downstream components participated in this death process. Necrostatin-1 (Nec-1) blocked oxaliplatin-induced cell death nearly completely, whereas z-VAD-fmk could partially suppress cell death. Oxaliplatin treatment resulted in poly(ADP-ribose) polymerase-1 (PARP-1) overactivation, as indicated by the increase of poly(ADP-ribose) (PAR), which led to NAD{sup +} and ATP depletion. PARP-1 inhibitor, olaparib, could significantly block oxaliplatin-induced cell death, thus confirming that PARP-1 activation is mainly responsible for the cytotoxicity of oxaliplatin. Phosphorylation of H2AX at Ser139 and translocalization of apoptosis-inducing factor (AIF) are critical for this death process. Taken together, these results indicate that oxaliplatin can bypass canonical cell death pathways to kill gastric cancer cells, which may be of therapeutic advantage in the treatment of gastric cancer. - Highlights: • Oxaliplatin induces apoptotic and necrotic cell death. • Nec-1 can inhibit oxaliplatin-induced cell death nearly completely. • RIP3 and its downstream components are not involved in this process. • PARP-1 overactivation-mediated energy depletion, H2AX phosphorylation and AIF translocation are crucial for this cell death.« less

RNA silencing of Mcl-1 enhances ABT-737-mediated apoptosis in melanoma: role for a caspase-8-dependent pathway.

PubMed

Keuling, Angela M; Felton, Kathleen E A; Parker, Arabesque A M; Akbari, Majid; Andrew, Susan E; Tron, Victor A

2009-08-17

Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma's striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance. Here we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10) activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity. Our findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity.

ADAM28 is expressed by epithelial cells in human normal tissues and protects from C1q-induced cell death.

PubMed

Miyamae, Yuka; Mochizuki, Satsuki; Shimoda, Masayuki; Ohara, Kentaro; Abe, Hitoshi; Yamashita, Shuji; Kazuno, Saiko; Ohtsuka, Takashi; Ochiai, Hiroki; Kitagawa, Yuko; Okada, Yasunori

2016-05-01

ADAM28 (disintegrin and metalloproteinase 28), which was originally reported to be lymphocyte-specific, is over-expressed by carcinoma cells and plays a key role in cell proliferation and progression in human lung and breast carcinomas. We studied ADAM28 expression in human normal tissues and examined its biological function. By using antibodies specific to ADAM28, ADAM28 was immunolocalized mainly to epithelial cells in several tissues, including epididymis, bronchus and stomach, whereas lymphocytes in lymph nodes and spleen were negligibly immunostained. RT-PCR, immunoblotting and ELISA analyses confirmed the expression in these tissues, and low or negligible expression by lymphocytes was found in the lymph node and spleen. C1q was identified as a candidate ADAM28-binding protein from a human lung cDNA library by yeast two-hybrid system, and specific binding was demonstrated by binding assays, immunoprecipitation and surface plasmon resonance. C1q treatment of normal bronchial epithelial BEAS-2B and NHBE cells, both of which showed low-level expression of ADAM28, caused apoptosis through activation of p38 and caspase-3, and cell death with autophagy through accumulation of LC3-II and autophagosomes, respectively. C1q-induced cell death was attenuated by treatment of the cells with antibodies against the C1q receptor gC1qR/p33 or cC1qR/calreticulin. Treatment of C1q with recombinant ADAM28 prior to addition to culture media reduced C1q-induced cell death, and knockdown of ADAM28 using siRNAs increased cell death. These data demonstrate that ADAM28 is expressed by epithelial cells of several normal organs, and suggest that ADAM28 plays a role in cell survival by suppression of C1q-induced cytotoxicity in bronchial epithelial cells. © 2016 Federation of European Biochemical Societies.

The sweet taste of death: glucose triggers apoptosis during yeast chronological aging.

PubMed

Ruckenstuhl, Christoph; Carmona-Gutierrez, Didac; Madeo, Frank

2010-10-01

As time goes by, a postmitotic cell ages following a degeneration process ultimately ending in cell death. This phenomenon is evolutionary conserved and present in unicellular eukaryotes as well, making the yeast chronological aging system an appreciated model. Here, single cells die in a programmed fashion (both by apoptosis and necrosis) for the benefit of the whole population. Besides its meaning for aging and cell death research, age-induced programmed cell death represents the first experimental proof for the so-called group selection theory: Apoptotic genes became selected during evolution because of the benefits they might render to the whole cell culture and not to the individual cell. Many anti‐aging stimuli have been discovered in the yeast chronological aging system and have afterwards been confirmed in higher cells or organisms. New work from the Burhans group (this issue) now demonstrates that glucose signaling has a progeriatric effect on chronologically aged yeast cells: Glucose administration results in a diminished efficacy of cells to enter quiescence, finally causing superoxide‐mediated replication stress and apoptosis.

bis-Dehydroxy-Curcumin triggers mitochondrial-associated cell death in human colon cancer cells through ER-stress induced autophagy.

PubMed

Basile, Valentina; Belluti, Silvia; Ferrari, Erika; Gozzoli, Chiara; Ganassi, Sonia; Quaglino, Daniela; Saladini, Monica; Imbriano, Carol

2013-01-01

The activation of autophagy has been extensively described as a pro-survival strategy, which helps to keep cells alive following deprivation of nutrients/growth factors and other stressful cellular conditions. In addition to cytoprotective effects, autophagy can accompany cell death. Autophagic vacuoles can be observed before or during cell death, but the role of autophagy in the death process is still controversial. A complex interplay between autophagy and apoptosis has come to light, taking into account that numerous genes, such as p53 and Bcl-2 family members, are shared between these two pathways. In this study we showed a potent and irreversible cytotoxic activity of the stable Curcumin derivative bis-DeHydroxyCurcumin (bDHC) on human colon cancer cells, but not on human normal cells. Autophagy is elicited by bDHC before cell death as demonstrated by increased autophagosome formation -measured by electron microscopy, fluorescent LC3 puncta and LC3 lipidation- and autophagic flux -measured by interfering LC3-II turnover. The accumulation of poly-ubiquitinated proteins and ER-stress occurred upstream of autophagy induction and resulted in cell death. Cell cycle and Western blot analyses highlighted the activation of a mitochondrial-dependent apoptosis, which involves caspase 7, 8, 9 and Cytochrome C release. Using pharmacological inhibitions and RNAi experiments, we showed that ER-stress induced autophagy has a major role in triggering bDHC-cell death. Our findings describe the mechanism through which bDHC promotes tumor selective inhibition of proliferation, providing unequivocal evidence of the role of autophagy in contrasting the proliferation of colon cancer cells.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Jing-Ping; Lin, Kai-Han; Liu, Chun-Yen

In this work, we demonstrated that the growth of human non-small-cell-lung-cancer cells H460 and A549 cells can be inhibited by low concentrations of an epoxide derivative, teroxirone, in both in vitro and in vivo models. The cytotoxicity was mediated by apoptotic cell death through DNA damage. The onset of ultimate apoptosis is dependent on the status of p53. Teroxirone caused transient elevation of p53 that activates downstream p21 and procaspase-3 cleavage. The presence of caspase-3 inhibitor reverted apoptotic phenotype. Furthermore, we showed the cytotoxicity of teroxirone in H1299 cells with stable ectopic expression of p53, but not those of mutantmore » p53. A siRNA-mediated knockdown of p53 expression attenuated drug sensitivity. The in vivo experiments demonstrated that teroxirone suppressed growth of xenograft tumors in nude mice. Being a potential therapeutic agent by restraining cell growth through apoptotic death at low concentrations, teroxirone provides a feasible perspective in reversing tumorigenic phenotype of human lung cancer cells. - Highlights: • Teroxirone repressed tumor cell growth in nude mice of human lung cancer cells. • The apoptotic cell death reverted by caspase-3 inhibitor is related to p53 status. • Teroxirone provides a good candidate for lung cancer treatment.« less

Induction of morphological changes in death-induced cancer cells monitored by holographic microscopy.

PubMed

El-Schich, Zahra; Mölder, Anna; Tassidis, Helena; Härkönen, Pirkko; Falck Miniotis, Maria; Gjörloff Wingren, Anette

2015-03-01

We are using the label-free technique of holographic microscopy to analyze cellular parameters including cell number, confluence, cellular volume and area directly in the cell culture environment. We show that death-induced cells can be distinguished from untreated counterparts by the use of holographic microscopy, and we demonstrate its capability for cell death assessment. Morphological analysis of two representative cell lines (L929 and DU145) was performed in the culture flasks without any prior cell detachment. The two cell lines were treated with the anti-tumour agent etoposide for 1-3days. Measurements by holographic microscopy showed significant differences in average cell number, confluence, volume and area when comparing etoposide-treated with untreated cells. The cell volume of the treated cell lines was initially increased at early time-points. By time, cells decreased in volume, especially when treated with high doses of etoposide. In conclusion, we have shown that holographic microscopy allows label-free and completely non-invasive morphological measurements of cell growth, viability and death. Future applications could include real-time monitoring of these holographic microscopy parameters in cells in response to clinically relevant compounds. Copyright © 2015 Elsevier Inc. All rights reserved.

Nuclear DAMP complex-mediated RAGE-dependent macrophage cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Ruochan; Department of Infectious Diseases and State Key Lab of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, Hunan 410008; Fu, Sha

High mobility group box 1 (HMGB1), histone, and DNA are essential nuclear components involved in the regulation of chromosome structure and function. In addition to their nuclear function, these molecules act as damage-associated molecular patterns (DAMPs) alone or together when released extracellularly. The synergistic effect of these nuclear DNA-HMGB1-histone complexes as DAMP complexes (nDCs) on immune cells remains largely unexplored. Here, we demonstrate that nDCs limit survival of macrophages (e.g., RAW264.7 and peritoneal macrophages) but not cancer cells (e.g., HCT116, HepG2 and Hepa1-6). nDCs promote production of inflammatory tumor necrosis factor α (TNFα) release, triggering reactive oxygen species-dependent apoptosis andmore » necrosis. Moreover, the receptor for advanced glycation end products (RAGE), but not toll-like receptor (TLR)-4 and TLR-2, was required for Akt-dependent TNFα release and subsequent cell death following treatment with nDCs. Genetic depletion of RAGE by RNAi, antioxidant N-Acetyl-L-cysteine, and TNFα neutralizing antibody significantly attenuated nDC-induced cell death. These findings provide evidence supporting novel signaling mechanisms linking nDCs and inflammation in macrophage cell death. - Highlights: • Nuclear DAMP complexes (nDCs) selectively induce cell death in macrophages, but not cancer cells. • TNFα-mediated oxidative stress is required for nDC-induced death. • RAGE-mediated Akt activation is required for nDC-induced TNFα release. • Blocking RAGE and TNFα inhibits nDC-induced macrophage cell death.« less

Systems analysis of apoptosis protein expression allows the case-specific prediction of cell death responsiveness of melanoma cells

PubMed Central

Passante, E; Würstle, M L; Hellwig, C T; Leverkus, M; Rehm, M

2013-01-01

Many cancer entities and their associated cell line models are highly heterogeneous in their responsiveness to apoptosis inducers and, despite a detailed understanding of the underlying signaling networks, cell death susceptibility currently cannot be predicted reliably from protein expression profiles. Here, we demonstrate that an integration of quantitative apoptosis protein expression data with pathway knowledge can predict the cell death responsiveness of melanoma cell lines. By a total of 612 measurements, we determined the absolute expression (nM) of 17 core apoptosis regulators in a panel of 11 melanoma cell lines, and enriched these data with systems-level information on apoptosis pathway topology. By applying multivariate statistical analysis and multi-dimensional pattern recognition algorithms, the responsiveness of individual cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or dacarbazine (DTIC) could be predicted with very high accuracy (91 and 82% correct predictions), and the most effective treatment option for individual cell lines could be pre-determined in silico. In contrast, cell death responsiveness was poorly predicted when not taking knowledge on protein–protein interactions into account (55 and 36% correct predictions). We also generated mathematical predictions on whether anti-apoptotic Bcl-2 family members or x-linked inhibitor of apoptosis protein (XIAP) can be targeted to enhance TRAIL responsiveness in individual cell lines. Subsequent experiments, making use of pharmacological Bcl-2/Bcl-xL inhibition or siRNA-based XIAP depletion, confirmed the accuracy of these predictions. We therefore demonstrate that cell death responsiveness to TRAIL or DTIC can be predicted reliably in a large number of melanoma cell lines when investigating expression patterns of apoptosis regulators in the context of their network-level interplay. The capacity to predict responsiveness at the cellular level may contribute to personalizing anti-cancer treatments in the future. PMID:23933815

UPREGULATION OF BNIP3 AND TRANSLOCATION TO MITOCHONDRIA MEDIATES CYANIDE-INDUCED APOPTOSIS IN CORTICAL CELLS

PubMed Central

Prabhakaran, K.; Li, L.; Zhang, L.; Borowitz, J.L.; Isom, G.E.

2008-01-01

BNIP3, a BH3 domain only Bcl-2 protein, has been identified as a mitochrondrial mediator of hypoxia-induced cell death. Since cyanide produces histotoxic anoxia (chemical hypoxia), the present study was undertaken in primary cortical cells to determine involvement of the BNIP3 signaling pathway in cyanide-induced death. Over a 20 h exposure KCN increased BNIP3 expression, followed by a concentration-related apoptotic death. To determine if BNIP3 plays a role in the cell death, expression was either overexpressed with BNIP3 cDNA (BNIP3+) or knocked down with small interfering RNA (RNAi). In BNIP3+ cells, cyanide-induced apoptotic death was markedly enhanced and preceded by reduction of mitochondrial membrane potential (Δψm), release of cytochrome c from mitochondria and elevated caspase 3 and 7 activity. Pretreatment with the pan caspase inhibitor zVAD-fmk suppressed BNIP3+-mediated cell death, thus confirming a caspase-dependent apoptosis. On the other hand, BNIP3 knock down by RNAi or antagonism of BNIP3 by a transmembrane-deleted dominant-negative mutant (BNIP3ΔTM) markedly reduced cell death. Immunohistochemical imaging showed that cyanide stimulated translocation of BNIP3 from cytosol to mitochondria and displacement studies with BNIP3ΔTM showed that integration of BNIP3 into the mitochondrial outer membrane was necessary for the cell death. In BNIP3+ cells, cyclosporin-A, an inhibitor of mitochondrial pore transition, blocked the cyanide-induced reduction of Δψm and decreased the apoptotic death. These results demonstrate in cortical cells that cyanide induces a rapid upregulation of BNIP3 expression, followed by translocation to the mitochondrial outer membrane to reduceΔψm This was followed by mitochondrial release of cytochrome c to execute a caspase-dependent cell death. PMID:17980495

Experimental Proof for the Role of Nonlinear Photoionization in Plasmonic Phototherapy.

PubMed

Minai, Limor; Zeidan, Adel; Yeheskely-Hayon, Daniella; Yudovich, Shimon; Kviatkovsky, Inna; Yelin, Dvir

2016-07-13

Targeting individual cells within a heterogeneous tissue is a key challenge in cancer therapy, encouraging new approaches for cancer treatment that complement the shortcomings of conventional therapies. The highly localized interactions triggered by focused laser beams promise great potential for targeting single cells or small cell clusters; however, most laser-tissue interactions often involve macroscopic processes that may harm healthy nearby tissue and reduce specificity. Specific targeting of living cells using femtosecond pulses and nanoparticles has been demonstrated promising for various potential therapeutic applications including drug delivery via optoporation, drug release, and selective cell death. Here, using an intense resonant femtosecond pulse and cell-specific gold nanorods, we show that at certain irradiation parameters cell death is triggered by nonlinear plasmonic photoionization and not by thermally driven processes. The experimental results are supported by a physical model for the pulse-particle-medium interactions. A good correlation is found between the calculated total number and energy of the generated free electrons and the observed cell death, suggesting that femtosecond photoionization plays the dominant role in cell death.

PMCA2 silencing potentiates MDA-MB-231 breast cancer cell death initiated with the Bcl-2 inhibitor ABT-263.

PubMed

Curry, Merril; Roberts-Thomson, Sarah J; Monteith, Gregory R

2016-09-30

PMCA2 overexpression in some breast cancers suggests that this calcium pump isoform may play a role in breast pathophysiology. To investigate PMCA2 as a potential drug target for breast cancer therapy, we assessed the functional consequence of PMCA2 silencing on cell death pathways and calcium signals in the basal-like MDA-MB-231 breast cancer cell line. Silencing PMCA2 expression alone has no effect on MDA-MB-231 cell viability, however, PMCA2 silencing promotes calcium-induced cell death initiated with the calcium ionophore ionomycin. Assessment of cytoplasmic calcium responses generated with various agents including ionomycin demonstrates that in MDA-MB-231 cells, PMCA2 does not play a major role in shaping global calcium signals. We also examined the ability of PMCA2 silencing to modulate caspase-dependent cell death triggered by a Bcl-2 inhibitor that is in clinical development for the treatment of various cancers, ABT-263 (Navitoclax). Despite the lack of effect on global calcium responses, PMCA2 silencing augmented Bcl-2 inhibitor (ABT-263)-mediated MDA-MB-231 breast cancer cell death. These studies provide evidence that PMCA2 inhibitors could sensitize PMCA2-positive breast cancers to cell death initiators that work through mechanisms involving the Bcl-2 survival pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

The CNGRC-GG-D(KLAKLAK)2 peptide induces a caspase-independent, Ca2+-dependent death in human leukemic myeloid cells by targeting surface aminopeptidase N/CD13

PubMed Central

Bouchet, Sandrine; Tang, Ruoping; Fava, Fanny; Legrand, Ollivier; Bauvois, Brigitte

2016-01-01

The CD13 antigen's binding site for the Asn-Gly-Arg (NGR) motif enables NGR-containing chemotherapeutic drugs to be delivered to CD13-positive tumours. Human CD13-positive acute myeloid leukemia (AML) cells proliferate abnormally and escape death. Here, we show that the CNGRC-GG-D(KLAKLAK)2 peptide induces death in AML cell lines (U937, THP-1, NB4, HL-60) and primary blood cells from AML patients. Cell death was characterized as a caspase-independent mechanism, without DNA fragmentation, but phosphatidylserine externalization and membrane disruption. Our results demonstrate in U937 cells that (i) the NGR-peptide triggers the loss of mitochondrial potential(ΔΨm) and generates superoxide anion (O2−), (ii) N-acetyl-L-cysteine (NAC) and extra/intracellular Ca2+ chelators (BAPTA) prevent both O2− production and cell death, (iii) the Ca2+-channel blocker nifedipine prevents cell death (indicating that Ca2+ influx is the initial death trigger), and (iv) BAPTA, but not NAC, prevents ΔΨm loss (suggesting O2− is a mitochondrial downstream effector). AML cell lines and primary blasts responding to the lethal action of NGR-peptide express promatrix metalloproteinase-12 (proMMP-12) and its substrate progranulin (an 88 kDa cell survival factor). A cell-free assay highlighted proMMP-12 activation by O2−. Accordingly, NGR-peptide's downregulation of 88 kDa progranulin protein was prevented by BAPTA and NAC. Conversely, AML blast resistance to NGR-peptide is associated with the expression of a distinct, 105 kDa progranulin isoform. These results indicate that CNGRC-GG-D(KLAKLAK)2 induces death in AML cells through the Ca2+-mitochondria-O2.-pathway, and support the link between proMMP-12 activation and progranulin cleavage during cell death. Our findings may have implications for the understanding of tumour biology and treatment. PMID:26655501

The CNGRC-GG-D(KLAKLAK)2 peptide induces a caspase-independent, Ca2+-dependent death in human leukemic myeloid cells by targeting surface aminopeptidase N/CD13.

PubMed

Bouchet, Sandrine; Tang, Ruoping; Fava, Fanny; Legrand, Ollivier; Bauvois, Brigitte

2016-04-12

The CD13 antigen's binding site for the Asn-Gly-Arg (NGR) motif enables NGR-containing chemotherapeutic drugs to be delivered to CD13-positive tumours. Human CD13-positive acute myeloid leukemia (AML) cells proliferate abnormally and escape death. Here, we show that the CNGRC-GG-D(KLAKLAK)2 peptide induces death in AML cell lines (U937, THP-1, NB4, HL-60) and primary blood cells from AML patients. Cell death was characterized as a caspase-independent mechanism, without DNA fragmentation, but phosphatidylserine externalization and membrane disruption. Our results demonstrate in U937 cells that (i) the NGR-peptide triggers the loss of mitochondrial potential(ΔΨm) and generates superoxide anion (O2-), (ii) N-acetyl-L-cysteine (NAC) and extra/intracellular Ca2+ chelators (BAPTA) prevent both O2- production and cell death, (iii) the Ca2+-channel blocker nifedipine prevents cell death (indicating that Ca2+ influx is the initial death trigger), and (iv) BAPTA, but not NAC, prevents ΔΨm loss (suggesting O2- is a mitochondrial downstream effector). AML cell lines and primary blasts responding to the lethal action of NGR-peptide express promatrix metalloproteinase-12 (proMMP-12) and its substrate progranulin (an 88 kDa cell survival factor). A cell-free assay highlighted proMMP-12 activation by O2-. Accordingly, NGR-peptide's downregulation of 88 kDa progranulin protein was prevented by BAPTA and NAC. Conversely, AML blast resistance to NGR-peptide is associated with the expression of a distinct, 105 kDa progranulin isoform. These results indicate that CNGRC-GG-D(KLAKLAK)2 induces death in AML cells through the Ca2+-mitochondria-O2.-pathway, and support the link between proMMP-12 activation and progranulin cleavage during cell death. Our findings may have implications for the understanding of tumour biology and treatment.

Monte Carlo Study Elucidates the Type 1/Type 2 Choice in Apoptotic Death Signaling in Healthy and Cancer Cells

PubMed Central

Raychaudhuri, Subhadip; Raychaudhuri, Somkanya C

2013-01-01

Apoptotic cell death is coordinated through two distinct (type 1 and type 2) intracellular signaling pathways. How the type 1/type 2 choice is made remains a central problem in the biology of apoptosis and has implications for apoptosis related diseases and therapy. We study the problem of type 1/type 2 choice in silico utilizing a kinetic Monte Carlo model of cell death signaling. Our results show that the type 1/type 2 choice is linked to deterministic versus stochastic cell death activation, elucidating a unique regulatory control of the apoptotic pathways. Consistent with previous findings, our results indicate that caspase 8 activation level is a key regulator of the choice between deterministic type 1 and stochastic type 2 pathways, irrespective of cell types. Expression levels of signaling molecules downstream also regulate the type 1/type 2 choice. A simplified model of DISC clustering elucidates the mechanism of increased active caspase 8 generation and type 1 activation in cancer cells having increased sensitivity to death receptor activation. We demonstrate that rapid deterministic activation of the type 1 pathway can selectively target such cancer cells, especially if XIAP is also inhibited; while inherent cell-to-cell variability would allow normal cells stay protected. PMID:24709706

Fluoxetine induces autophagic cell death via eEF2K-AMPK-mTOR-ULK complex axis in triple negative breast cancer.

PubMed

Sun, Dejuan; Zhu, Lingjuan; Zhao, Yuqian; Jiang, Yingnan; Chen, Lixia; Yu, Yang; Ouyang, Liang

2018-04-01

Triple negative breast cancer (TNBC) is a complex and intrinsically aggressive tumour with poor prognosis, and the discovery of targeted small-molecule drugs for TNBC treatment still remains in its infancy. In this study, we aimed to discover a small-molecule agent for TNBC treatment and illuminate its potential mechanisms. Cell viability was detected by using methylthiazoltetrazolium (MTT) assay. Electron microscopy, GFP-LC3 transfection, monodansylcadaverine staining and apoptosis assay were performed to determine Fluoxetine-induced autophagy and apoptosis. Western blotting and siRNA transfection were carried out to investigate the mechanisms of Fluoxetine-induced autophagy. iTRAQ-based proteomics analysis was used to explore the underlying mechanisms. We have demonstrated that Fluoxetine had remarkable anti-proliferative activities and induced autophagic cell death in MDA-MB-231 and MDA-MB-436 cells. The mechanism for Fluoxetine-induced autophagic cell death was associated with inhibition of eEF2K and activation of AMPK-mTOR-ULK complex axis. Further iTRAQ-based proteomics and network analyses revealed that Fluoxetine-induced mechanism was involved in BIRC6, BNIP1, SNAP29 and Bif-1. These results demonstrate that Fluoxetine induces apoptosis and autophagic cell death in TNBC, which will hold a promise for the future TNBC therapy. © 2017 John Wiley & Sons Ltd.

Oncolytic vesicular stomatitis virus induces apoptosis in U87 glioblastoma cells by a type II death receptor mechanism and induces cell death and tumor clearance in vivo.

PubMed

Cary, Zachary D; Willingham, Mark C; Lyles, Douglas S

2011-06-01

Vesicular stomatitis virus (VSV) is a potential oncolytic virus for treating glioblastoma multiforme (GBM), an aggressive brain tumor. Matrix (M) protein mutants of VSV have shown greater selectivity for killing GBM cells versus normal brain cells than VSV with wild-type M protein. The goal of this research was to determine the contribution of death receptor and mitochondrial pathways to apoptosis induced by an M protein mutant (M51R) VSV in U87 human GBM tumor cells. Compared to controls, U87 cells expressing a dominant negative form of Fas (dnFas) or overexpressing Bcl-X(L) had reduced caspase-3 activation following infection with M51R VSV, indicating that both the death receptor pathway and mitochondrial pathways are important for M51R VSV-induced apoptosis. Death receptor signaling has been classified as type I or type II, depending on whether signaling is independent (type I) or dependent on the mitochondrial pathway (type II). Bcl-X(L) overexpression inhibited caspase activation in response to a Fas-inducing antibody, similar to the inhibition in response to M51R VSV infection, indicating that U87 cells behave as type II cells. Inhibition of apoptosis in vitro delayed, but did not prevent, virus-induced cell death. Murine xenografts of U87 cells that overexpress Bcl-X(L) regressed with a time course similar to that of control cells following treatment with M51R VSV, and tumors were not detectable at 21 days postinoculation. Immunohistochemical analysis demonstrated similar levels of viral antigen expression but reduced activation of caspase-3 following virus treatment of Bcl-X(L)-overexpressing tumors compared to controls. Further, the pathological changes in tumors following treatment with virus were quite different in the presence versus the absence of Bcl-X(L) overexpression. These results demonstrate that M51R VSV efficiently induces oncolysis in GBM tumor cells despite deregulation of apoptotic pathways, underscoring its potential use as a treatment for GBM.

Zebrafish hair cell mechanics and physiology through the lens of noise-induced hair cell death

NASA Astrophysics Data System (ADS)

Coffin, Allison B.; Xu, Jie; Uribe, Phillip M.

2018-05-01

Hair cells are exquisitely sensitive to auditory stimuli, but also to damage from a variety of sources including noise trauma and ototoxic drugs. Mammals cannot regenerate cochlear hair cells, while non-mammalian vertebrates exhibit robust regenerative capacity. Our research group uses the lateral line system of larval zebrafish to explore the mechanisms underlying hair cell damage, identify protective therapies, and determine molecular drivers of innate regeneration. The lateral line system contains externally located sensory organs called neuromasts, each composed of ˜8-20 hair cells. Lateral line hair cells are homologous to vertebrate inner ear hair cells and share similar susceptibility to ototoxic damage. In the last decade, the lateral line has emerged as a powerful model system for understanding hair cell death mechanisms and for identifying novel protective compounds. Here we demonstrate that the lateral line is a tractable model for noise-induced hair cell death. We have developed a novel noise damage system capable of inducing over 50% loss of lateral line hair cells, with hair cell death occurring in a dose- and time-dependent manner. Cell death is greatest 72 hours post-exposure. However, early signs of hair cell damage, including changes in membrane integrity and reduced mechanotransduction, are apparent within hours of noise exposure. These features, early signs of damage followed by delayed hair cell death, are consistent with mammalian data, suggesting that noise acts similarly on zebrafish and mammalian hair cells. In our future work we will use our new model system to investigate noise damage events in real time, and to develop protective therapies for future translational research.

Long-term treatment of anterior pituitary cells with nitric oxide induces programmed cell death.

PubMed

Velardez, Miguel Omar; Poliandri, Ariel Hernán; Cabilla, Jimena Paula; Bodo, Cristian Carlos Armando; Machiavelli, Leticia Inés; Duvilanski, Beatriz Haydeé

2004-04-01

Nitric oxide (NO) plays a complex role in modulating programmed cell death. It can either protect the cell from apoptotic death or mediate apoptosis, depending on its concentration and the cell type and/or status. In this study, we demonstrate that long-term exposition to NO induces cell death of anterior pituitary cells from Wistar female rats. DETA NONOate (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, 1 mm], a NO donor that releases NO for an extended period of time, decreased cellular viability and prolactin release from primary cultures of anterior pituitary cells. Morphological studies showed an increase in the number of cells with chromatin condensation and nuclear fragmentation at 24 and 48 h after DETA/NO exposure. DNA internucleosomal fragmentation was also observed at the same time. Reversibility of the NO effect on cellular viability and prolactin release was observed only when the cells were incubated with DETA/NO for less than 6 h. Most apoptotic cells were immunopositive for prolactin, suggesting a high susceptibility of lactotrophs to the effect of NO. The cytotoxic effect of NO is dependent of caspase-9 and caspase-3, but seems to be independent of oxidative stress or nitrosative stress. Our results show that the exposition of anterior pituitary cells to NO for long periods induces programmed cell death of anterior pituitary cells.

Activation of the endoplasmic reticulum stress response by the amyloid-beta 1-40 peptide in brain endothelial cells.

PubMed

Fonseca, Ana Catarina R G; Ferreiro, Elisabete; Oliveira, Catarina R; Cardoso, Sandra M; Pereira, Cláudia F

2013-12-01

Neurovascular dysfunction arising from endothelial cell damage is an early pathogenic event that contributes to the neurodegenerative process occurring in Alzheimer's disease (AD). Since the mechanisms underlying endothelial dysfunction are not fully elucidated, this study was aimed to explore the hypothesis that brain endothelial cell death is induced upon the sustained activation of the endoplasmic reticulum (ER) stress response by amyloid-beta (Aβ) peptide, which deposits in the cerebral vessels in many AD patients and transgenic mice. Incubation of rat brain endothelial cells (RBE4 cell line) with Aβ1-40 increased the levels of several markers of ER stress-induced unfolded protein response (UPR), in a time-dependent manner, and affected the Ca(2+) homeostasis due to the release of Ca(2+) from this intracellular store. Finally, Aβ1-40 was shown to activate both mitochondria-dependent and -independent apoptotic cell death pathways. Enhanced release of cytochrome c from mitochondria and activation of the downstream caspase-9 were observed in cells treated with Aβ1-40 concomitantly with caspase-12 activation. Furthermore, Aβ1-40 activated the apoptosis effectors' caspase-3 and promoted the translocation of apoptosis-inducing factor (AIF) to the nucleus demonstrating the involvement of caspase-dependent and -independent mechanisms during Aβ-induced endothelial cell death. In conclusion, our data demonstrate that ER stress plays a significant role in Aβ1-40-induced apoptotic cell death in brain endothelial cells suggesting that ER stress-targeted therapeutic strategies might be useful in AD to counteract vascular defects and ultimately neurodegeneration. © 2013.

NF-κB Regulates Caspase-4 Expression and Sensitizes Neuroblastoma Cells to Fas-Induced Apoptosis

PubMed Central

Yang, Hai-Jie; Wang, Mian; Wang, Lei; Cheng, Bin-Feng; Lin, Xiao-Yu; Feng, Zhi-Wei

2015-01-01

Found in neurons and neuroblastoma cells, Fas-induced apoptosis and accompanied activation of NF-κB signaling were thought to be associated with neurodegenerative diseases. However, the detailed functions of NF-κB activation in Fas killing and the effect of NF-κB activation on its downstream events remain unclear. Here, we demonstrated that agonistic Fas antibody induces cell death in a dose-dependent way and NF-κB signaling is activated as well, in neuroblastoma cells SH-EP1. Unexpectedly, NF-κB activation was shown to be pro-apoptotic, as suggested by the reduction of Fas-induced cell death with either a dominant negative form of IκBα (DN-IκBα) or an IκB kinase-specific inhibitor. To our interest, when analyzing downstream events of NF-κB signaling, we found that DN-IκBα only suppressed the expression of caspase-4, but not other caspases. Vice versa, enhancement of NF-κB activity by p65 (RelA) overexpression increased the expression of caspase-4 at both mRNA and protein levels. More directly, results from dual luciferase reporter assay demonstrated the regulation of caspase-4 promoter activity by NF-κB. When caspase-4 activity was blocked by its dominant negative (DN) form, Fas-induced cell death was substantially reduced. Consistently, the cleavage of PARP and caspase-3 induced by Fas was also reduced. In contrast, the cleavage of caspase-8 remained unaffected in caspase-4 DN cells, although caspase-8 inhibitor could rescue Fas-induced cell death. Collectively, these data suggest that caspase-4 activity is required for Fas-induced cell apoptosis and caspase-4 may act upstream of PARP and caspase-3 and downstream of caspase-8. Overall, we demonstrate that NF-κB can mediate Fas-induced apoptosis through caspase-4 protease, indicating that caspase-4 is a new mediator of NF-κB pro-apoptotic pathway in neuroblastoma cells. PMID:25695505

The Roles of 4β-Hydroxywithanolide E from Physalis peruviana on the Nrf2-Anti-Oxidant System and the Cell Cycle in Breast Cancer Cells.

PubMed

Peng, Chieh Yu; You, Bang Jau; Lee, Chia Lin; Wu, Yang Chang; Lin, Wen Hsin; Lu, Te Ling; Chang, Fei-Ching; Lee, Hong Zin

2016-01-01

4[Formula: see text]-Hydroxywithanolide E is an active component of the extract of Physalis peruviana that has been reported to exhibit antitumor effects. Although the involvement of reactive oxygen species (ROS) production and the ataxia-telangiectasia mutated protein (ATM)-dependent DNA damage signaling pathway in 4[Formula: see text]-hydroxywithanolide E-induced apoptosis of breast cancer MCF-7 cells was demonstrated in our previous study, the relationship between ROS production and the cellular defense system response in 4[Formula: see text]-hydroxywithanolide E-induced cell death requires further verification. The present study suggests that ROS play an important role in 4[Formula: see text]-hydroxywithanolide E-induced MCF-7 cell death in which anti-oxidants, such as glutathione or N-acetylcysteine, can resist the 4[Formula: see text]-hydroxywithanolide E-induced accumulation of ROS and cell death. Furthermore, N-acetylcysteine or glutathione can reverse the 4[Formula: see text]-hydroxywithanolide E-induced changes in the cell cycle distribution and the expression of cell cycle regulators. We found that the 4[Formula: see text]-hydroxywithanolide E-induced ROS accumulation was correlated with the upregulation of Nrf2 and Nrf2-downstream genes, such as antioxidative defense enzymes. In general, the activity of Nrf2 is regulated by the Ras signalling pathway. However, we demonstrated that Nrf2 was activated during 4[Formula: see text]-hydroxywithanolide E-induced MCF-7 cell death in spite of the 4[Formula: see text]-hydroxywithanolide E-induced inhibition of the Ras/Raf/ERK pathway. The activity and protein expression of superoxide dismutase and catalase were involved in the 4[Formula: see text]-hydroxywithanolide E-induced ROS production in MCF-7 cells. Furthermore, 4[Formula: see text]-hydroxywithanolide E was demonstrated to significantly reduce the sizes of the tumor nodules in the human breast cancer MDA-MB231 xenograft tumor model.

Necroptosis: an alternative cell death program defending against cancer.

PubMed

Chen, Dongshi; Yu, Jian; Zhang, Lin

2016-04-01

One of the hallmarks of cancer is resistance to programmed cell death, which maintains the survival of cells en route to oncogenic transformation and underlies therapeutic resistance. Recent studies demonstrate that programmed cell death is not confined to caspase-dependent apoptosis, but includes necroptosis, a form of necrotic death governed by Receptor-Interacting Protein 1 (RIP1), RIP3, and Mixed Lineage Kinase Domain-Like (MLKL) protein. Necroptosis serves as a critical cell-killing mechanism in response to severe stress and blocked apoptosis, and can be induced by inflammatory cytokines or chemotherapeutic drugs. Genetic or epigenetic alterations of necroptosis regulators such as RIP3 and cylindromatosis (CYLD), are frequently found in human tumors. Unlike apoptosis, necroptosis elicits a more robust immune response that may function as a defensive mechanism by eliminating tumor-causing mutations and viruses. Furthermore, several classes of anticancer agents currently under clinical development, such as SMAC and BH3 mimetics, can promote necroptosis in addition to apoptosis. A more complete understanding of the interplay among necroptosis, apoptosis, and other cell death modalities is critical for developing new therapeutic strategies to enhance killing of tumor cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Killing to Fluctuate, or: How Death and Reproduction Drive a Fluctuation-Response Relation in Biofilms

NASA Astrophysics Data System (ADS)

Kalziqi, Arben; Yunker, Peter; Thomas, Jacob

Unlike equilibrium atomic solids, biofilms do not experience significant thermal fluctuations at the constituent level. However, cells inside the biofilm stochastically die and reproduce, provoking a mechanical response. We investigate the mechanical response of biofilms to the death and reproduction of cells by measuring surface-height fluctuations of biofilms with two mutual predator strains of Vibrio cholerae which kill one another on contact via the Type VI Secretion System. Biofilm surface topography is measured in the homeostatic limit, wherein cell division and death occur at roughly the same rate, via white light interferometry. Although biofilms are far from equilibrium systems, measured height correlation functions line up with expectations from a generalized fluctuation-response relation derived from replication and death events, as predicted by Risler et al. (PRL 2015). Using genetically modified strains of V. cholerae which cannot kill, we demonstrate that extracted effective temperatures increase with the amount of death and reproduction. Thus, high-precision measurement of surface topography reveals the physical consequences of death and reproduction within a biofilm, providing a new approach to studying interactions between bacteria and cells.

The NRF2 Activation and Antioxidative Response Are Not Impaired Overall during Hyperoxia-Induced Lung Epithelial Cell Death

PubMed Central

Potteti, Haranatha R.; Reddy, Narsa M.; Hei, Tom K.; Kalvakolanu, Dhananjaya V.; Reddy, Sekhar P.

2013-01-01

Lung epithelial and endothelial cell death caused by pro-oxidant insults is a cardinal feature of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) patients. The NF-E2-related factor 2 (NRF2) activation in response to oxidant exposure is crucial to the induction of several antioxidative and cytoprotective enzymes that mitigate cellular stress. Since prolonged exposure to hyperoxia causes cell death, we hypothesized that chronic hyperoxia impairs NRF2 activation, resulting in cell death. To test this hypothesis, we exposed nonmalignant small airway epithelial cells (AECs) to acute (1–12 h) and chronic (36–48 h) hyperoxia and evaluated cell death, NRF2 nuclear accumulation and target gene expression, and NRF2 recruitment to the endogenous HMOX1 and NQO1 promoters. As expected, hyperoxia gradually induced death in AECs, noticeably and significantly by 36 h; ~60% of cells were dead by 48 h. However, we unexpectedly found increased expression levels of NRF2-regulated antioxidative genes and nuclear NRF2 in AECs exposed to chronic hyperoxia as compared to acute hyperoxia. Chromatin Immunoprecipitation (ChIP) assays revealed an increased recruitment of NRF2 to the endogenous HMOX1 and NQO1 promoters in AECs exposed to acute or chronic hyperoxia. Thus, our findings demonstrate that NRF2 activation and antioxidant gene expression are functional during hyperoxia-induced lung epithelial cell death and that chronic hyperoxia does not impair NRF2 signaling overall. PMID:23738042

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

PubMed Central

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

Beta sitosterol and Daucosterol (phytosterols identified in Grewia tiliaefolia) perturbs cell cycle and induces apoptotic cell death in A549 cells.

PubMed

Rajavel, Tamilselvam; Mohankumar, Ramar; Archunan, Govindaraju; Ruckmani, Kandasamy; Devi, Kasi Pandima

2017-06-13

Lung cancer is the leading cause of cancer related deaths both in developed and developing countries. Since majority of the existing therapeutic methods harms both normal and malignant cells, a viable alternative is to switch into safe and beneficial traditional medicinal plants. Hence the present study was framed to identify selective anti-lung cancer agents from the medicinal plant Grewia tiliaefolia (GT). Cell viability experiments showed that benzene extract of GT (BGT) leaf effectively inhibited the growth of A549 cells, while being non-toxic to normal human lung L132 and PBMC cells. Ames and comet assays demonstrated that BGT is of non-mutagenic and non-genotoxic nature in untransformed cells. The hematological and histopathological profiles of the in vivo acute and sub-acute toxicity studies demonstrated that BGT is safe and tolerable. Importantly, western blot analysis and Annexin V-FITC staining confirmed that BGT promotes mitochondrial dependent apoptotic cell death in A549 cells by arresting cell cycle at G2/M phase. Bio-assay guided fractionation revealed the presence of phytosteols (β-sitosterol and daucosterol) which significantly inhibited the growth of A549 cells both alone and in combination. This study warrants that these phytosterols in alone or in combination can be considered as safe and potential drug candidates for lung cancer treatment.

Novel Mechanisms of Target Cell Death and Survival and of Therapeutic Action of IVIg in Pemphigus

PubMed Central

Arredondo, Juan; Chernyavsky, Alexander I.; Karaouni, Ali; Grando, Sergei A.

2005-01-01

Pemphigus vulgaris (PV) is a potentially lethal mucocutaneous blistering disease characterized by cell-cell detachment within the stratified epithelium (acantholysis) caused by IgG autoantibodies. Intravenous immunoglobulin (IVIg) therapy effectively treats PV, but the mechanism is not fully understood. To further understand acantholysis and the efficacy of IVIg, we measured effects of IgG fractions from PV patients on keratinocyte death processes. Using IgGs from representative PV patients who improved with IVIg, we identified apoptotic and oncotic signaling pathways in in vitro and in vivo PV models. We identified two groups of PV patients, each producing autoantibodies activating predominantly either apoptotic or oncotic cell death pathway. Experimental treatments with caspase 3 or calpain inhibitors demonstrated that PV IgGs induced acantholysis through both pathways. Upstream, the apoptotic signaling involved activation of caspases 8 and 3 and up-regulation of Fas ligand mRNA, whereas calpain-mediated cell death depended on elevated intracellular free Ca2+. IVIg reduced PV IgG-mediated acantholysis and cell death and up-regulated the caspase inhibitor FLIP and the calpain inhibitor calpastatin. These results indicate that in different PV patients, IgG-induced acantholysis proceeds predominantly via distinct, yet complementary, pathways of programmed cell death differentially mediated by apoptosis and oncosis effectors, with IVIg protecting target cells by up-regulating endogenous caspase and calpain inhibitors. PMID:16314468

Effect of vitamin E on 24(S)-hydroxycholesterol-induced necroptosis-like cell death and apoptosis.

PubMed

Nakazawa, Takaya; Miyanoki, Yuta; Urano, Yasuomi; Uehara, Madoka; Saito, Yoshiro; Noguchi, Noriko

2017-05-01

24(S)-Hydroxycholesterol (24S-OHC) has diverse physiological and pathological functions. In particular, cytotoxic effects of 24S-OHC in neuronal cells are important in development of neurodegenerative diseases. 24S-OHC induces necroptosis-like cell death in SH-SY5Y cells expressing little caspase-8. In the present study, 24S-OHC was found to induce apoptosis as determined by caspase-3 activation in all-trans-retinoic acid (atRA)-treated SH-SY5Y cells in which expression of caspase-8 was induced. 24S-OHC-induced cell death was inhibited by α-tocopherol (α-Toc) but not by α-tocotrienol (α-Toc3) in SH-SY5Y cells regardless of whether cells were treated with atRA. In contrast, cumene hydroperoxide (CumOOH)-induced cell death was significantly inhibited by α-Toc and α-Toc3. In atRA-treated SH-SY5Y cells, generation of reactive oxygen species (ROS) was induced by stimulation with CumOOH but was not induced by stimulation with 24S-OHC. These results suggest that inhibition of 24S-OHC-induced cell death by α-Toc cannot be explained by its radical scavenging antioxidant activity. Esterification of 24S-OHC followed by lipid droplet (LD) formation due to acyl-CoA:cholesterol acyltransferase 1 (ACAT1) are key events in 24S-OHC-induced cell death in atRA-treated SH-SY5Y cells as demonstrated by inhibition of cell death by ACAT1 inhibitor. LD number was not changed by treatment with either α-Toc or α-Toc3. The different physical properties of α-Toc and α-Toc3 may account for their different inhibitory effects on 24S-OHC-induced cell death. Copyright © 2016 Elsevier Ltd. All rights reserved.

Patient-derived glioblastoma cells show significant heterogeneity in treatment responses to the inhibitor-of-apoptosis-protein antagonist birinapant

PubMed Central

Zakaria, Z; Tivnan, A; Flanagan, L; Murray, D W; Salvucci, M; Stringer, B W; Day, B W; Boyd, A W; Kögel, D; Rehm, M; O'Brien, D F; Byrne, A T; Prehn, J H M

2016-01-01

Background: Resistance to temozolomide (TMZ) greatly limits chemotherapeutic effectiveness in glioblastoma (GBM). Here we analysed the ability of the Inhibitor-of-apoptosis-protein (IAP) antagonist birinapant to enhance treatment responses to TMZ in both commercially available and patient-derived GBM cells. Methods: Responses to TMZ and birinapant were analysed in a panel of commercial and patient-derived GBM cell lines using colorimetric viability assays, flow cytometry, morphological analysis and protein expression profiling of pro- and antiapoptotic proteins. Responses in vivo were analysed in an orthotopic xenograft GBM model. Results: Single-agent treatment experiments categorised GBM cells into TMZ-sensitive cells, birinapant-sensitive cells, and cells that were insensitive to either treatment. Combination treatment allowed sensitisation to therapy in only a subset of resistant GBM cells. Cell death analysis identified three principal response patterns: Type A cells that readily activated caspase-8 and cell death in response to TMZ while addition of birinapant further sensitised the cells to TMZ-induced cell death; Type B cells that readily activated caspase-8 and cell death in response to birinapant but did not show further sensitisation with TMZ; and Type C cells that showed no significant cell death or moderately enhanced cell death in the combined treatment paradigm. Furthermore, in vivo, a Type C patient-derived cell line that was TMZ-insensitive in vitro and showed a strong sensitivity to TMZ and TMZ plus birinapant treatments. Conclusions: Our results demonstrate remarkable differences in responses of patient-derived GBM cells to birinapant single and combination treatments, and suggest that therapeutic responses in vivo may be greatly affected by the tumour microenvironment. PMID:26657652

The effector SPRYSEC-19 of Globodera rostochiensis suppresses CC-NB-LRR-mediated disease resistance in plants.

PubMed

Postma, Wiebe J; Slootweg, Erik J; Rehman, Sajid; Finkers-Tomczak, Anna; Tytgat, Tom O G; van Gelderen, Kasper; Lozano-Torres, Jose L; Roosien, Jan; Pomp, Rikus; van Schaik, Casper; Bakker, Jaap; Goverse, Aska; Smant, Geert

2012-10-01

The potato cyst nematode Globodera rostochiensis invades roots of host plants where it transforms cells near the vascular cylinder into a permanent feeding site. The host cell modifications are most likely induced by a complex mixture of proteins in the stylet secretions of the nematodes. Resistance to nematodes conferred by nucleotide-binding-leucine-rich repeat (NB-LRR) proteins usually results in a programmed cell death in and around the feeding site, and is most likely triggered by the recognition of effectors in stylet secretions. However, the actual role of these secretions in the activation and suppression of effector-triggered immunity is largely unknown. Here we demonstrate that the effector SPRYSEC-19 of G. rostochiensis physically associates in planta with the LRR domain of a member of the SW5 resistance gene cluster in tomato (Lycopersicon esculentum). Unexpectedly, this interaction did not trigger defense-related programmed cell death and resistance to G. rostochiensis. By contrast, agroinfiltration assays showed that the coexpression of SPRYSEC-19 in leaves of Nicotiana benthamiana suppresses programmed cell death mediated by several coiled-coil (CC)-NB-LRR immune receptors. Furthermore, SPRYSEC-19 abrogated resistance to Potato virus X mediated by the CC-NB-LRR resistance protein Rx1, and resistance to Verticillium dahliae mediated by an unidentified resistance in potato (Solanum tuberosum). The suppression of cell death and disease resistance did not require a physical association of SPRYSEC-19 and the LRR domains of the CC-NB-LRR resistance proteins. Altogether, our data demonstrated that potato cyst nematodes secrete effectors that enable the suppression of programmed cell death and disease resistance mediated by several CC-NB-LRR proteins in plants.

The Effector SPRYSEC-19 of Globodera rostochiensis Suppresses CC-NB-LRR-Mediated Disease Resistance in Plants1[C][W][OA

PubMed Central

Postma, Wiebe J.; Slootweg, Erik J.; Rehman, Sajid; Finkers-Tomczak, Anna; Tytgat, Tom O.G.; van Gelderen, Kasper; Lozano-Torres, Jose L.; Roosien, Jan; Pomp, Rikus; van Schaik, Casper; Bakker, Jaap; Goverse, Aska; Smant, Geert

2012-01-01

The potato cyst nematode Globodera rostochiensis invades roots of host plants where it transforms cells near the vascular cylinder into a permanent feeding site. The host cell modifications are most likely induced by a complex mixture of proteins in the stylet secretions of the nematodes. Resistance to nematodes conferred by nucleotide-binding-leucine-rich repeat (NB-LRR) proteins usually results in a programmed cell death in and around the feeding site, and is most likely triggered by the recognition of effectors in stylet secretions. However, the actual role of these secretions in the activation and suppression of effector-triggered immunity is largely unknown. Here we demonstrate that the effector SPRYSEC-19 of G. rostochiensis physically associates in planta with the LRR domain of a member of the SW5 resistance gene cluster in tomato (Lycopersicon esculentum). Unexpectedly, this interaction did not trigger defense-related programmed cell death and resistance to G. rostochiensis. By contrast, agroinfiltration assays showed that the coexpression of SPRYSEC-19 in leaves of Nicotiana benthamiana suppresses programmed cell death mediated by several coiled-coil (CC)-NB-LRR immune receptors. Furthermore, SPRYSEC-19 abrogated resistance to Potato virus X mediated by the CC-NB-LRR resistance protein Rx1, and resistance to Verticillium dahliae mediated by an unidentified resistance in potato (Solanum tuberosum). The suppression of cell death and disease resistance did not require a physical association of SPRYSEC-19 and the LRR domains of the CC-NB-LRR resistance proteins. Altogether, our data demonstrated that potato cyst nematodes secrete effectors that enable the suppression of programmed cell death and disease resistance mediated by several CC-NB-LRR proteins in plants. PMID:22904163

Different ways to die: cell death modes of the unicellular chlorophyte Dunaliella viridis exposed to various environmental stresses are mediated by the caspase-like activity DEVDase.

PubMed

Jiménez, Carlos; Capasso, Juan M; Edelstein, Charles L; Rivard, Christopher J; Lucia, Scott; Breusegem, Sophia; Berl, Tomás; Segovia, María

2009-01-01

Programmed cell death is necessary for homeostasis in multicellular organisms and it is also widely recognized to occur in unicellular organisms. However, the mechanisms through which it occurs in unicells, and the enzymes involved within the final response is still the subject of heated debate. It is shown here that exposure of the unicellular microalga Dunaliella viridis to several environmental stresses, induced different cell death morphotypes, depending on the stimulus received. Senescent cells demonstrated classical and unambiguous apoptotic-like characteristics such as chromatin condensation, DNA fragmentation, intact organelles, and blebbing of the cell membrane. Acute heat shock caused general swelling and altered plasma membrane, but the presence of chromatin clusters and DNA strand breaks suggested a necrotic-like event. UV irradiated cells presented changes typical for necrosis, together with apoptotic characteristics resembling an intermediate cell-death phenotype termed aponecrosis-like. Cells subjected to hyperosmotic shock revealed chromatin spotting without DNA fragmentation, and extensive cytoplasmic swelling and vacuolization, comparable to a paraptotic-like cell death phenotype. Nitrogen-starved cells showed pyknosis, blebbing, and cytoplasmic consumption, indicating a similarity to autophagic/vacuolar-like cell death. The caspase-like activity DEVDase was measured by using the fluorescent substrate Ac-DEVD-AMC and antibodies against the human caspase-3 active enzyme cross-reacted with bands, the intensity of which paralleled the activity. All the environmental stresses tested produced a substantial increase in both DEVDase activity and protein levels. The irreversible caspase-3 inhibitor Z-DEVD-FMK completely inhibited the enzymatic activity whereas serine and aspartyl proteases inhibitors did not. These results show that cell death in D. viridis does not conform to a single pattern and that environmental stimuli may produce different types of cell death depending on the type and intensity of the stimulus, all of which help to understand the cell death-dependent and cell death-independent functions of caspase-like proteins. Hence, these data support the theory that alternative, non-apoptotic programmed cell death (PCDs), exist either in parallel or in an independent manner with apoptosis and were already present in single-celled organisms that evolved some 1.2-1.6 billion years ago.

Different ways to die: cell death modes of the unicellular chlorophyte Dunaliella viridis exposed to various environmental stresses are mediated by the caspase-like activity DEVDase

PubMed Central

Jiménez, Carlos; Capasso, Juan M.; Edelstein, Charles L.; Rivard, Christopher J.; Lucia, Scott; Breusegem, Sophia; Berl, Tomás; Segovia, María

2009-01-01

Programmed cell death is necessary for homeostasis in multicellular organisms and it is also widely recognized to occur in unicellular organisms. However, the mechanisms through which it occurs in unicells, and the enzymes involved within the final response is still the subject of heated debate. It is shown here that exposure of the unicellular microalga Dunaliella viridis to several environmental stresses, induced different cell death morphotypes, depending on the stimulus received. Senescent cells demonstrated classical and unambiguous apoptotic-like characteristics such as chromatin condensation, DNA fragmentation, intact organelles, and blebbing of the cell membrane. Acute heat shock caused general swelling and altered plasma membrane, but the presence of chromatin clusters and DNA strand breaks suggested a necrotic-like event. UV irradiated cells presented changes typical for necrosis, together with apoptotic characteristics resembling an intermediate cell-death phenotype termed aponecrosis-like. Cells subjected to hyperosmotic shock revealed chromatin spotting without DNA fragmentation, and extensive cytoplasmic swelling and vacuolization, comparable to a paraptotic-like cell death phenotype. Nitrogen-starved cells showed pyknosis, blebbing, and cytoplasmic consumption, indicating a similarity to autophagic/vacuolar-like cell death. The caspase-like activity DEVDase was measured by using the fluorescent substrate Ac-DEVD-AMC and antibodies against the human caspase-3 active enzyme cross-reacted with bands, the intensity of which paralleled the activity. All the environmental stresses tested produced a substantial increase in both DEVDase activity and protein levels. The irreversible caspase-3 inhibitor Z-DEVD-FMK completely inhibited the enzymatic activity whereas serine and aspartyl proteases inhibitors did not. These results show that cell death in D. viridis does not conform to a single pattern and that environmental stimuli may produce different types of cell death depending on the type and intensity of the stimulus, all of which help to understand the cell death-dependent and cell death-independent functions of caspase-like proteins. Hence, these data support the theory that alternative, non-apoptotic programmed cell death (PCDs), exist either in parallel or in an independent manner with apoptosis and were already present in single-celled organisms that evolved some 1.2-1.6 billion years ago. PMID:19251986

Effect of blue light radiation on curcumin-induced cell death of breast cancer cells

NASA Astrophysics Data System (ADS)

Zeng, X. B.; Leung, A. W. N.; Xia, X. S.; Yu, H. P.; Bai, D. Q.; Xiang, J. Y.; Jiang, Y.; Xu, C. S.

2010-06-01

In the present study, we have successfully set up a novel blue light source with the power density of 9 mW/cm2 and the wavelength of 435.8 nm and then the novel light source was used to investigate the effect of light radiation on curcumin-induced cell death. The cytotoxicity was investigated 24 h after the treatment of curcumin and blue light radiation together using MTT reduction assay. Nuclear chromatin was observed using a fluorescent microscopy with Hoechst33258 staining. The results showed blue light radiation could significantly enhance the cytotoxicity of curcumin on the MCF-7 cells and apoptosis induction. These findings demonstrated that blue light radiation could enhance curcumin-induced cell death of breast cancer cells, suggesting light radiation may be an efficient enhancer of curcumin in the management of breast cancer.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rawal, Nina; Corti, Olga; CNRS, UMR 7225, Paris

Parkinson's disease (PD) is caused by degeneration of the dopaminergic (DA) neurons of the substantia nigra but the molecular mechanisms underlying the degenerative process remain elusive. Several reports suggest that cell cycle deregulation in post-mitotic neurons could lead to neuronal cell death. We now show that Parkin, an E3 ubiquitin ligase linked to familial PD, regulates {beta}-catenin protein levels in vivo. Stabilization of {beta}-catenin in differentiated primary ventral midbrain neurons results in increased levels of cyclin E and proliferation, followed by increased levels of cleaved PARP and loss of DA neurons. Wnt3a signaling also causes death of post-mitotic DA neuronsmore » in parkin null animals, suggesting that both increased stabilization and decreased degradation of {beta}-catenin results in DA cell death. These findings demonstrate a novel regulation of Wnt signaling by Parkin and suggest that Parkin protects DA neurons against excessive Wnt signaling and {beta}-catenin-induced cell death.« less

Chloroquine synergizes with FTS to enhance cell growth inhibition and cell death

PubMed Central

Schmukler, Eran; Wolfson, Eya; Haklai, Roni; Elad-Sfadia, Galit; Kloog, Yoel; Pinkas-Kramarski, Ronit

2014-01-01

The Ras family of small GTPases transmits extracellular signals that regulate cell growth, differentiation, motility and death. Ras signaling is constitutively active in a large number of human cancers. Ras can also regulate autophagy by affecting several signaling pathways including the mTOR pathway. Autophagy is a process that regulates the balance between protein synthesis and protein degradation. It is important for normal growth control, but may be defective in diseases. Previously, we have shown that Ras inhibition by FTS induces autophagy, which partially protects cancer cells and may limit the use of FTS as an anti-cancer drug. Since FTS is a non toxic drug we hypothesized that FTS and chloroquine (an autophagy inhibitor) will synergize in cell growth inhibition and cell death. Thus, in the present study, we explored the mechanism of each individual drug and their combined action. Our results demonstrate that in HCT-116 and in Panc-1 cells, FTS induces autophagy, which can be inhibited by chloroquine. Furthermore, the combined treatment synergistically decreased the number of viable cells. Interestingly, the combined treatment enhanced apoptotic cell death as indicated by increased sub-G1 cell population, increased Hoechst staining, activation of caspase 3, decrease in survivin expression and release of cytochrome c. Thus, chloroquine treatment may promote FTS-mediated inhibition of tumor cell growth and may stimulate apoptotic cell death. PMID:24368422

Violacein Induces Death of Resistant Leukaemia Cells via Kinome Reprogramming, Endoplasmic Reticulum Stress and Golgi Apparatus Collapse

PubMed Central

Queiroz, Karla C. S.; Milani, Renato; Ruela-de-Sousa, Roberta R.; Fuhler, Gwenny M.; Justo, Giselle Z.; Zambuzzi, Willian F.; Duran, Nelson; Diks, Sander H.; Spek, C. Arnold; Ferreira, Carmen V.; Peppelenbosch, Maikel P.

2012-01-01

It is now generally recognised that different modes of programmed cell death (PCD) are intimately linked to the cancerous process. However, the mechanism of PCD involved in cancer chemoprevention is much less clear and may be different between types of chemopreventive agents and tumour cell types involved. Therefore, from a pharmacological view, it is crucial during the earlier steps of drug development to define the cellular specificity of the candidate as well as its capacity to bypass dysfunctional tumoral signalling pathways providing insensitivity to death stimuli. Studying the cytotoxic effects of violacein, an antibiotic dihydro-indolone synthesised by an Amazon river Chromobacterium, we observed that death induced in CD34+/c-Kit+/P-glycoprotein+/MRP1+ TF1 leukaemia progenitor cells is not mediated by apoptosis and/or autophagy, since biomarkers of both types of cell death were not significantly affected by this compound. To clarify the working mechanism of violacein, we performed kinome profiling using peptide arrays to yield comprehensive descriptions of cellular kinase activities. Pro-death activity of violacein is actually carried out by inhibition of calpain and DAPK1 and activation of PKA, AKT and PDK, followed by structural changes caused by endoplasmic reticulum stress and Golgi apparatus collapse, leading to cellular demise. Our results demonstrate that violacein induces kinome reprogramming, overcoming death signaling dysfunctions of intrinsically resistant human leukaemia cells. PMID:23071514

Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Mi Na; Lee, Kyung Eun; Hong, Jung Yeon

Highlights: Black-Right-Pointing-Pointer Hyperoxia induces apoptosis and chitinase 3-like 1 expression in human airway epithelial cells. Black-Right-Pointing-Pointer Presence of chitinase 3-like 1 affects airway epithelial cell death after hyperoxic exposure. Black-Right-Pointing-Pointer Silencing chitinase 3-like 1 manipulate the phosphorylation of ERK, p38 and Akt. -- Abstract: Background: Exposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associatedmore » with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood. Purpose: In this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells. Method: The human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process. Results: Hyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally, phosphorylation of ERK1/2, p38, and Akt were affected by CHI3L1 knockdown. Conclusion: This study indicates that CHI3L1 is involved in hyperoxia-induced cell death, suggesting that CHI3L1 may be one of several cell death regulators influencing the MAPK and PI3K pathways during oxidative stress in human airway epithelial cells.« less

Distinct regions of the Phytophthora essential effector Avh238 determine its function in cell death activation and plant immunity suppression.

PubMed

Yang, Bo; Wang, Qunqing; Jing, Maofeng; Guo, Baodian; Wu, Jiawei; Wang, Haonan; Wang, Yang; Lin, Long; Wang, Yan; Ye, Wenwu; Dong, Suomeng; Wang, Yuanchao

2017-04-01

Phytophthora pathogens secrete effectors to manipulate host innate immunity, thus facilitating infection. Among the RXLR effectors highly induced during Phytophthora sojae infection, Avh238 not only contributes to pathogen virulence but also triggers plant cell death. However, the detailed molecular basis of Avh238 functions remains largely unknown. We mapped the regions responsible for Avh238 functions in pathogen virulence and plant cell death induction using a strategy that combines investigation of natural variation and large-scale mutagenesis assays. The correlation between cellular localization and Avh238 functions was also evaluated. We found that the 79 th residue (histidine or leucine) of Avh238 determined its cell death-inducing activity, and that the 53 amino acids in its C-terminal region are responsible for promoting Phytophthora infection. Transient expression of Avh238 in Nicotiana benthamiana revealed that nuclear localization is essential for triggering cell death, while Avh238-mediated suppression of INF1-triggered cell death requires cytoplasmic localization. Our results demonstrate that a representative example of an essential Phytophthora RXLR effector can evolve to escape recognition by the host by mutating one nucleotide site, and can also retain plant immunosuppressive activity to enhance pathogen virulence in planta. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Cathepsin-mediated Necrosis Controls the Adaptive Immune Response by Th2 (T helper type 2)-associated Adjuvants*

PubMed Central

Jacobson, Lee S.; Lima, Heriberto; Goldberg, Michael F.; Gocheva, Vasilena; Tsiperson, Vladislav; Sutterwala, Fayyaz S.; Joyce, Johanna A.; Gapp, Bianca V.; Blomen, Vincent A.; Chandran, Kartik; Brummelkamp, Thijn R.; Diaz-Griffero, Felipe; Brojatsch, Jürgen

2013-01-01

Immunologic adjuvants are critical components of vaccines, but it remains unclear how prototypical adjuvants enhance the adaptive immune response. Recent studies have shown that necrotic cells could trigger an immune response. Although most adjuvants have been shown to be cytotoxic, this activity has traditionally been considered a side effect. We set out to test the role of adjuvant-mediated cell death in immunity and found that alum, the most commonly used adjuvant worldwide, triggers a novel form of cell death in myeloid leukocytes characterized by cathepsin-dependent lysosome-disruption. We demonstrated that direct lysosome-permeabilization with a soluble peptide, Leu-Leu-OMe, mimics the alum-like form of necrotic cell death in terms of cathepsin dependence and cell-type specificity. Using a combination of a haploid genetic screen and cathepsin-deficient cells, we identified specific cathepsins that control lysosome-mediated necrosis. We identified cathepsin C as critical for Leu-Leu-OMe-induced cell death, whereas cathepsins B and S were required for alum-mediated necrosis. Consistent with a role of necrotic cell death in adjuvant effects, Leu-Leu-OMe replicated an alum-like immune response in vivo, characterized by dendritic cell activation, granulocyte recruitment, and production of Th2-associated antibodies. Strikingly, cathepsin C deficiency not only blocked Leu-Leu-OMe-mediated necrosis but also impaired Leu-Leu-OMe-enhanced immunity. Together our findings suggest that necrotic cell death is a powerful mediator of a Th2-associated immune response. PMID:23297415

Tumor suppressor Spred2 interaction with LC3 promotes autophagosome maturation and induces autophagy-dependent cell death.

PubMed

Jiang, Ke; Liu, Min; Lin, Guibin; Mao, Beibei; Cheng, Wei; Liu, Han; Gal, Jozsef; Zhu, Haining; Yuan, Zengqiang; Deng, Wuguo; Liu, Quentin; Gong, Peng; Bi, Xiaolin; Meng, Songshu

2016-05-03

The tumor suppressor Spred2 (Sprouty-related EVH1 domain-2) induces cell death in a variety of cancers. However, the underlying mechanism remains to be elucidated. Here we show that Spred2 induces caspase-independent but autophagy-dependent cell death in human cervical carcinoma HeLa and lung cancer A549 cells. We demonstrate that ectopic Spred2 increased both the conversion of microtubule-associated protein 1 light chain 3 (LC3), GFP-LC3 puncta formation and p62/SQSTM1 degradation in A549 and HeLa cells. Conversely, knockdown of Spred2 in tumor cells inhibited upregulation of autophagosome maturation induced by the autophagy inducer Rapamycin, which could be reversed by the rescue Spred2. These data suggest that Spred2 promotes autophagy in tumor cells. Mechanistically, Spred2 co-localized and interacted with LC3 via the LC3-interacting region (LIR) motifs in its SPR domain. Mutations in the LIR motifs or deletion of the SPR domain impaired Spred2-mediated autophagosome maturation and tumor cell death, indicating that functional LIR is required for Spred2 to trigger tumor cell death. Additionally, Spred2 interacted and co-localized with p62/SQSTM1 through its SPR domain. Furthermore, the co-localization of Spred2, p62 and LAMP2 in HeLa cells indicates that p62 may be involved in Spred2-mediated autophagosome maturation. Inhibition of autophagy using the lysosomal inhibitor chloroquine, reduced Spred2-mediated HeLa cell death. Silencing the expression of autophagy-related genes ATG5, LC3 or p62 in HeLa and A549 cells gave similar results, suggesting that autophagy is required for Spred2-induced tumor cell death. Collectively, these data indicate that Spred2 induces tumor cell death in an autophagy-dependent manner.

Tumor suppressor Spred2 interaction with LC3 promotes autophagosome maturation and induces autophagy-dependent cell death

PubMed Central

Lin, Guibin; Mao, Beibei; Cheng, Wei; Liu, Han; Gal, Jozsef; Zhu, Haining; Yuan, Zengqiang; Deng, Wuguo; Liu, Quentin; Gong, Peng; Bi, Xiaolin; Meng, Songshu

2016-01-01

The tumor suppressor Spred2 (Sprouty-related EVH1 domain-2) induces cell death in a variety of cancers. However, the underlying mechanism remains to be elucidated. Here we show that Spred2 induces caspase-independent but autophagy-dependent cell death in human cervical carcinoma HeLa and lung cancer A549 cells. We demonstrate that ectopic Spred2 increased both the conversion of microtubule-associated protein 1 light chain 3 (LC3), GFP-LC3 puncta formation and p62/SQSTM1 degradation in A549 and HeLa cells. Conversely, knockdown of Spred2 in tumor cells inhibited upregulation of autophagosome maturation induced by the autophagy inducer Rapamycin, which could be reversed by the rescue Spred2. These data suggest that Spred2 promotes autophagy in tumor cells. Mechanistically, Spred2 co-localized and interacted with LC3 via the LC3-interacting region (LIR) motifs in its SPR domain. Mutations in the LIR motifs or deletion of the SPR domain impaired Spred2-mediated autophagosome maturation and tumor cell death, indicating that functional LIR is required for Spred2 to trigger tumor cell death. Additionally, Spred2 interacted and co-localized with p62/SQSTM1 through its SPR domain. Furthermore, the co-localization of Spred2, p62 and LAMP2 in HeLa cells indicates that p62 may be involved in Spred2-mediated autophagosome maturation. Inhibition of autophagy using the lysosomal inhibitor chloroquine, reduced Spred2-mediated HeLa cell death. Silencing the expression of autophagy-related genes ATG5, LC3 or p62 in HeLa and A549 cells gave similar results, suggesting that autophagy is required for Spred2-induced tumor cell death. Collectively, these data indicate that Spred2 induces tumor cell death in an autophagy-dependent manner. PMID:27028858

3,3′-Diindolylmethane Ameliorates Staphylococcal Enterotoxin B–Induced Acute Lung Injury through Alterations in the Expression of MicroRNA that Target Apoptosis and Cell-Cycle Arrest in Activated T Cells

PubMed Central

Elliott, David M.; Nagarkatti, Mitzi

2016-01-01

3,3′-Diindolylmethane (DIM), a natural indole found in cruciferous vegetables, has significant anti-cancer and anti-inflammatory properties. In this current study, we investigated the effects of DIM on acute lung injury (ALI) induced by exposure to staphylococcal enterotoxin B (SEB). We found that pretreatment of mice with DIM led to attenuation of SEB-induced inflammation in the lungs, vascular leak, and IFN-γ secretion. Additionally, DIM could induce cell-cycle arrest and cell death in SEB-activated T cells in a concentration-dependent manner. Interestingly, microRNA (miRNA) microarray analysis uncovered an altered miRNA profile in lung-infiltrating mononuclear cells after DIM treatment of SEB-exposed mice. Moreover, computational analysis of miRNA gene targets and regulation networks indicated that DIM alters miRNA in the cell death and cell-cycle progression pathways. Specifically, DIM treatment significantly downregulated several miRNA and a correlative increase associated gene targets. Furthermore, overexpression and inhibition studies demonstrated that DIM-induced cell death, at least in part, used miR-222. Collectively, these studies demonstrate for the first time that DIM treatment attenuates SEB-induced ALI and may do so through the induction of microRNAs that promote apoptosis and cell-cycle arrest in SEB-activated T cells. PMID:26818958

RNA Silencing of Mcl-1 Enhances ABT-737-Mediated Apoptosis in Melanoma: Role for a Caspase-8-Dependent Pathway

PubMed Central

Keuling, Angela M.; Felton, Kathleen E. A.; Parker, Arabesque A. M.; Akbari, Majid; Andrew, Susan E.; Tron, Victor A.

2009-01-01

Background Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma's striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance. Methodology/Principal Findings Here we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10) activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity. Conclusions/Significance Our findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity. PMID:19684859

Cold atmospheric-pressure air plasma treatment of C6 glioma cells: effects of reactive oxygen species in the medium produced by the plasma on cell death

NASA Astrophysics Data System (ADS)

Wang, Yuyang; Cheng, Cheng; Gao, Peng; Li, Shaopeng; Shen, Jie; Lan, Yan; Yu, Yongqiang; Chu, Paul K.

2017-02-01

An atmospheric-pressure air plasma is employed to treat C6 glioma cells in vitro. To elucidate on the mechanism causing cell death and role of reactive species (RS) in the medium produced by the plasma, the concentration of the long-lived RS such as hydrogen peroxide, nitrate, and ozone in the plasma-treated liquid (phosphate-buffered saline solution) is measured. When vitamin C is added to the medium as a ROS quencher, the viability of C6 glioma cells after the plasma treatment is different from that without vitamin C. The results demonstrate that reactive oxygen species (ROS) such as H2O2, and O3 constitute the main factors for inactivation of C6 glioma cells and the reactive nitrogen species (RNS) may only play an auxiliary role in cell death.

Induction of cell death in renal cell carcinoma with combination of D-fraction and vitamin C.

PubMed

Alexander, Bobby; Fishman, Andrew I; Eshghi, Majid; Choudhury, Muhammad; Konno, Sensuke

2013-09-01

Although several conventional therapeutic options for advanced renal cell carcinoma (RCC) are currently available, the unsatisfactory outcomes demand establishing more effective interventions. D-fraction (PDF), a bioactive proteoglucan of Maitake mushroom, demonstrates anticancer and immunomodulatory activities, which are also shown to be potentiated by vitamin C (VC). We thus hypothesized that a combination of PDF and VC (PDF + VC) could be an alternative approach to more effectively inhibit the growth of RCC. We examined the dose-dependent effects of PDF + VC on RCC cell viability and also performed biochemical assays to explore the growth regulatory mechanism. Human RCC, ACHN cell line, was employed and exposed to varying concentrations of PDF or VC and their combinations. Cell viability at specified times was determined by MTT assay. Lipid peroxidation assay, cell cycle analysis, and Western blot analysis were also performed. PDF or VC alone led to the significant reduction in cell viability at 72 hours with PDF >500 µg/mL and VC ≥300 µM. When various combinations of PDF and VC were tested, the combination of the ineffective concentrations of PDF (300 µg/mL) and VC (200 µM) resulted in ~90% cell death in 24 hours. Lipid peroxidation assay then indicated significantly (~2.5 fold) elevated oxidative stress with this PDF + VC. Cell cycle analysis also indicated a G1 cell cycle arrest following a 6-hour PDF + VC treatment. Western blots further revealed a downregulation of Bcl2, an upregulation of Bax, and proteolytic activation of PARP (poly[ADP-ribose] polymerase) in PDF + VC-treated cells, indicating induction of apoptosis. The present study demonstrates that the combination of PDF and VC can become highly cytotoxic, inducing severe cell death in ACHN cells. This cytotoxic mechanism appears to be primarily attributed to oxidative stress, accompanied by a G1 cell cycle arrest. Such cell death induced by PDF + VC could be more likely linked to apoptosis, as indicated by the modulation of apoptosis regulators (Bcl2, Bax, and PARP). Therefore, as PDF and VC may work synergistically to induce apoptotic cell death, they may have clinical implications in an alternative, improved therapeutic modality for advanced RCC.

Latent KSHV Infected Endothelial Cells Are Glutamine Addicted and Require Glutaminolysis for Survival

PubMed Central

Sanchez, Erica L.; Carroll, Patrick A.; Thalhofer, Angel B.; Lagunoff, Michael

2015-01-01

Kaposi’s Sarcoma-associated Herpesvirus (KSHV) is the etiologic agent of Kaposi’s Sarcoma (KS). KSHV establishes a predominantly latent infection in the main KS tumor cell type, the spindle cell, which is of endothelial cell origin. KSHV requires the induction of multiple metabolic pathways, including glycolysis and fatty acid synthesis, for the survival of latently infected endothelial cells. Here we demonstrate that latent KSHV infection leads to increased levels of intracellular glutamine and enhanced glutamine uptake. Depletion of glutamine from the culture media leads to a significant increase in apoptotic cell death in latently infected endothelial cells, but not in their mock-infected counterparts. In cancer cells, glutamine is often required for glutaminolysis to provide intermediates for the tri-carboxylic acid (TCA) cycle and support for the production of biosynthetic and bioenergetic precursors. In the absence of glutamine, the TCA cycle intermediates alpha-ketoglutarate (αKG) and pyruvate prevent the death of latently infected cells. Targeted drug inhibition of glutaminolysis also induces increased cell death in latently infected cells. KSHV infection of endothelial cells induces protein expression of the glutamine transporter, SLC1A5. Chemical inhibition of SLC1A5, or knockdown by siRNA, leads to similar cell death rates as glutamine deprivation and, similarly, can be rescued by αKG. KSHV also induces expression of the heterodimeric transcription factors c-Myc-Max and related heterodimer MondoA-Mlx. Knockdown of MondoA inhibits expression of both Mlx and SLC1A5 and induces a significant increase in cell death of only cells latently infected with KSHV, again, fully rescued by the supplementation of αKG. Therefore, during latent infection of endothelial cells, KSHV activates and requires the Myc/MondoA-network to upregulate the glutamine transporter, SLC1A5, leading to increased glutamine uptake for glutaminolysis. These findings expand our understanding of the required metabolic pathways that are activated during latent KSHV infection of endothelial cells, and demonstrate a novel role for the extended Myc-regulatory network, specifically MondoA, during latent KSHV infection. PMID:26197457

Combined pitavastatin and dacarbazine treatment activates apoptosis and autophagy resulting in synergistic cytotoxicity in melanoma cells.

PubMed

Al-Qatati, Abeer; Aliwaini, Saeb

2017-12-01

Melanoma is an aggressive skin cancer and its incidence is increasing faster than any other type of cancer. Whilst dacarbazine (DTIC) is the standard chemotherapy for metastatic melanoma, it has limited success. Statins, including pitavastatin, have been demonstrated to have a range of anti-cancer effects in a number of human cancer cell lines. The present study therefore explored the anti-cancer activity of combined DTIC and pitavastatin in A375 and WM115 human melanoma cells. Cell survival assays demonstrated that combined DTIC and pitavastatin treatment resulted in synergistic cell death. Cell cycle analyses further revealed that this combined treatment resulted in a G1 cell cycle arrest, as well as a sub-G1 population, indicative of apoptosis. Activation of apoptosis was confirmed by Annexin V-fluorescein isothiocyanate/propidium iodide double-staining and an increase in the levels of active caspase 3 and cleaved poly (ADP-ribose) polymerase. Furthermore, it was demonstrated that apoptosis occurs through the intrinsic pathway, evident from the release of cytochrome c. Finally, combined DTIC and pitavastatin treatment was demonstrated to also activate autophagy as part of a cell death mechanism. The present study provides novel evidence to suggest that the combined treatment of DTIC and pitavastatin may be effective in the treatment of melanoma.

Combined pitavastatin and dacarbazine treatment activates apoptosis and autophagy resulting in synergistic cytotoxicity in melanoma cells

PubMed Central

Al-Qatati, Abeer; Aliwaini, Saeb

2017-01-01

Melanoma is an aggressive skin cancer and its incidence is increasing faster than any other type of cancer. Whilst dacarbazine (DTIC) is the standard chemotherapy for metastatic melanoma, it has limited success. Statins, including pitavastatin, have been demonstrated to have a range of anti-cancer effects in a number of human cancer cell lines. The present study therefore explored the anti-cancer activity of combined DTIC and pitavastatin in A375 and WM115 human melanoma cells. Cell survival assays demonstrated that combined DTIC and pitavastatin treatment resulted in synergistic cell death. Cell cycle analyses further revealed that this combined treatment resulted in a G1 cell cycle arrest, as well as a sub-G1 population, indicative of apoptosis. Activation of apoptosis was confirmed by Annexin V-fluorescein isothiocyanate/propidium iodide double-staining and an increase in the levels of active caspase 3 and cleaved poly (ADP-ribose) polymerase. Furthermore, it was demonstrated that apoptosis occurs through the intrinsic pathway, evident from the release of cytochrome c. Finally, combined DTIC and pitavastatin treatment was demonstrated to also activate autophagy as part of a cell death mechanism. The present study provides novel evidence to suggest that the combined treatment of DTIC and pitavastatin may be effective in the treatment of melanoma. PMID:29344241

Interferon-γ Reduces the Proliferation of Primed Human Renal Tubular Cells.

PubMed

García-Sánchez, Omar; López-Novoa, José Miguel; López-Hernández, Francisco J

2014-01-01

Chronic kidney disease (CKD) is a progressive deterioration of the kidney function, which may eventually lead to renal failure and the need for dialysis or kidney transplant. Whether initiated in the glomeruli or the tubuli, CKD is characterized by progressive nephron loss, for which the process of tubular deletion is of key importance. Tubular deletion results from tubular epithelial cell death and defective repair, leading to scarring of the renal parenchyma. Several cytokines and signaling pathways, including transforming growth factor-β (TGF-β) and the Fas pathway, have been shown to participate in vivo in tubular cell death. However, there is some controversy about their mode of action, since a direct effect on normal tubular cells has not been demonstrated. We hypothesized that epithelial cells would require specific priming to become sensitive to TGF-β or Fas stimulation and that this priming would be brought about by specific mediators found in the pathological scenario. Herein we studied whether the combined effect of several stimuli known to take part in CKD progression, namely TGF-β, tumor necrosis factor-α, interferon-γ (IFN-γ), and Fas stimulation, on primed resistant human tubular cells caused cell death or reduced proliferation. We demonstrate that these cytokines have no synergistic effect on the proliferation or viability of human kidney (HK2) cells. We also demonstrate that IFN-γ, but not the other stimuli, reduces the proliferation of cycloheximide-primed HK2 cells without affecting their viability. Our results point at a potentially important role of IFN-γ in defective repair, leading to nephron loss during CKD.

Interferon-γ Reduces the Proliferation of Primed Human Renal Tubular Cells

PubMed Central

García-Sánchez, Omar; López-Novoa, José Miguel; López-Hernández, Francisco J.

2014-01-01

Background/Aims Chronic kidney disease (CKD) is a progressive deterioration of the kidney function, which may eventually lead to renal failure and the need for dialysis or kidney transplant. Whether initiated in the glomeruli or the tubuli, CKD is characterized by progressive nephron loss, for which the process of tubular deletion is of key importance. Tubular deletion results from tubular epithelial cell death and defective repair, leading to scarring of the renal parenchyma. Several cytokines and signaling pathways, including transforming growth factor-β (TGF-β) and the Fas pathway, have been shown to participate in vivo in tubular cell death. However, there is some controversy about their mode of action, since a direct effect on normal tubular cells has not been demonstrated. We hypothesized that epithelial cells would require specific priming to become sensitive to TGF-β or Fas stimulation and that this priming would be brought about by specific mediators found in the pathological scenario. Methods Herein we studied whether the combined effect of several stimuli known to take part in CKD progression, namely TGF-β, tumor necrosis factor-α, interferon-γ (IFN-γ), and Fas stimulation, on primed resistant human tubular cells caused cell death or reduced proliferation. Results We demonstrate that these cytokines have no synergistic effect on the proliferation or viability of human kidney (HK2) cells. We also demonstrate that IFN-γ, but not the other stimuli, reduces the proliferation of cycloheximide-primed HK2 cells without affecting their viability. Conclusion Our results point at a potentially important role of IFN-γ in defective repair, leading to nephron loss during CKD. PMID:24575118

Catalytically active Yersinia outer protein P induces cleavage of RIP and caspase-8 at the level of the DISC independently of death receptors in dendritic cells.

PubMed

Gröbner, Sabine; Adkins, Irena; Schulz, Sebastian; Richter, Kathleen; Borgmann, Stefan; Wesselborg, Sebastian; Ruckdeschel, Klaus; Micheau, Olivier; Autenrieth, Ingo B

2007-10-01

Yersinia outer protein P (YopP) is injected by Y. enterocolitica into host cells thereby inducing apoptotic and necrosis-like cell death in dendritic cells (DC). Here we show the pathways involved in DC death caused by the catalytic activity of YopP. Infection with Yersinia enterocolitica, translocating catalytically active YopP into DC, triggered procaspase-8 cleavage and c-FLIPL degradation. YopP-dependent caspase-8 activation was, however, not mediated by tumor necrosis factor (TNF) receptor family members since the expression of both CD95/Fas/APO-1 and TRAIL-R2 on DC was low, and DC were resistant to apoptosis induced by agonistic anti-CD95 antibodies or TNF-related apoptosis-inducing ligand (TRAIL). Moreover, DC from TNF-Rp55-/- mice were not protected against YopP-induced cell death demonstrating that TNF-R1 is also not involved in this process. Activation of caspase-8 was further investigated by coimmunoprecitation of FADD from Yersinia-infected DC. We found that both cleaved caspase-8 and receptor interacting protein 1 (RIP1) were associated with the Fas-associated death domain (FADD) indicating the formation of an atypical death-inducing signaling complex (DISC). Furthermore, degradation of RIP mediated by the Hsp90 inhibitor geldanamycin significantly impaired YopP-induced cell death. Altogether our findings indicate that Yersinia-induced DC death is independent of death domain containing receptors, but mediated by RIP and caspase-8 at the level of DISC.

The role of tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) in mediating autophagy in myositis skeletal muscle: A potential non-immune mechanism of muscle damage

PubMed Central

Alger, Heather M.; Raben, Nina; Pistilli, Emidio; Francia, Dwight; Rawat, Rashmi; Getnet, Derese; Ghimbovschi, Svetlana; Chen, Yi-Wen; Lundberg, Ingrid E.; Nagaraju, Kanneboyina

2011-01-01

Objective Multinucleated cells are relatively resistant to classical apoptosis, and the factors initiating cell-death and damage in myositis are not well defined. We hypothesized that non-immune autophagic cell death may play a role in muscle fiber damage. Recent literature indicates that tumor necrosis factor-alpha-related apoptosis inducing ligand (TRAIL) may induce both NFκB (nuclear factor kappa-light chain enhancer of activated B cells) activation and autophagic cell death in other systems. Here, we have investigated its role in cell death and pathogenesis in vitro and in vivo using myositis (human and mouse) muscle tissues. Methods Gene expression profiling indicated that expression of TRAIL and several autophagy markers was specifically upregulated in myositis muscle tissue; these results were confirmed by immunohistochemistry and immunoblotting. We also analyzed TRAIL-induced cell death (apoptosis and autophagy) and NFκB activation in vitro in cultured cells. Results TRAIL was expressed predominantly in muscle fibers of myositis, but not in biopsies from normal or other dystrophic-diseased muscle. Autophagy markers were upregulated in human and mouse models of myositis. TRAIL expression was restricted to regenerating/atrophic areas of muscle fascicles, blood vessels, and infiltrating lymphocytes. TRAIL induced NFκB activation and IκB degradation in cultured cells that are resistant to TRAIL-induced apoptosis but undergo autophagic cell death. Conclusion Our data demonstrate that TRAIL is expressed in myositis muscle and may mediate both activation of NFκB and autophagic cell death in myositis. Thus, this non-immune pathway may be an attractive target for therapeutic intervention in myositis. PMID:21769834

Doxorubicin resistance mediated by cytoplasmic macrophage colony-stimulating factor is associated with switch from apoptosis to autophagic cell death in MCF-7 breast cancer cells

PubMed Central

Zhang, Mengxia; Zhang, Hailiang; Tang, Fan; Wang, Yuhua; Mo, Zhongcheng; Lei, Xiaoyong

2016-01-01

Macrophage colony-stimulating factor is a vital factor in maintaining the biological function of monocyte–macrophage lineage. It is expressed in many tumor tissues and cancer cells. Recent findings indicate that macrophage colony-stimulating factor might contribute to chemoresistance, but the precise mechanisms are unclear. This study was to explore the effect of macrophage colony-stimulating factor on doxorubicin resistance in MCF-7 breast cancer cells and the possible mechanism. In the study, the human breast cancer cells, MCF-7, were transfected with macrophage colony-stimulating factor. We document that cytoplasmic macrophage colony-stimulating factor induces doxorubicin resistance and inhibits apoptosis in MCF-7 cells. Further studies demonstrated that cytoplasmic macrophage colony-stimulating factor-mediated apoptosis inhibition was dependent on the activation of PI3K/Akt/Survivin pathway. More importantly, we found that macrophage colony-stimulating factor-induced autophagic cell death in doxorubicin-treated MCF-7 cells. Taken together, we show for the first time that macrophage colony-stimulating factor-induced doxorubicin resistance is associated with the changes in cell death response with defective apoptosis and promotion of autophagic cell death. PMID:27439542

Hepatic Leukemia Factor Promotes Resistance To Cell Death: Implications For Therapeutics and Chronotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J.

Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional programmore » encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation.« less

Molecular mechanisms of programmed cell death-1 dependent T cell suppression: relevance for immunotherapy

PubMed Central

Zuazo, Miren; Gato-Cañas, Maria; Llorente, Noelia; Ibañez-Vea, María; Arasanz, Hugo

2017-01-01

Programmed cell death-1 (PD1) has become a significant target for cancer immunotherapy. PD1 and its receptor programmed cell death 1 ligand 1 (PDL1) are key regulatory physiological immune checkpoints that maintain self-tolerance in the organism by regulating the degree of activation of T and B cells amongst other immune cell types. However, cancer cells take advantage of these immunosuppressive regulatory mechanisms to escape T and B cell-mediated immunity. PD1 engagement on T cells by PDL1 on the surface of cancer cells dramatically interferes with T cell activation and the acquisition of effector capacities. Interestingly, PD1-targeted therapies have demonstrated to be highly effective in rescuing T cell anti-tumor effector functions. Amongst these the use of anti-PD1/PDL1 monoclonal antibodies are particularly efficacious in human therapies. Furthermore, clinical findings with PD1/PDL1 blockers over several cancer types demonstrate clinical benefit. Despite the successful results, the molecular mechanisms by which PD1-targeted therapies rescue T cell functions still remain elusive. Therefore, it is a key issue to uncover the molecular pathways by which these therapies exert its function in T cells. A profound knowledge of PDL1/PD1 mechanisms will surely uncover a new array of targets susceptible of therapeutic intervention. Here, we provide an overview of the molecular events underlying PD1-dependent T cell suppression in cancer. PMID:29114543

Inhibition of paraquat-induced autophagy accelerates the apoptotic cell death in neuroblastoma SH-SY5Y cells.

PubMed

González-Polo, Rosa A; Niso-Santano, Mireia; Ortíz-Ortíz, Miguel A; Gómez-Martín, Ana; Morán, José M; García-Rubio, Lourdes; Francisco-Morcillo, Javier; Zaragoza, Concepción; Soler, Germán; Fuentes, José M

2007-06-01

Autophagy is a degradative mechanism involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. This phenomenon of autophagy has been observed in neurons from patients with Parkinson's disease (PD), suggesting a functional role for autophagy in neuronal cell death. On the other hand, it has been demonstrated that exposure to pesticides can be a risk factor in the incidence of PD. In this sense, paraquat (PQ) (1,1'-dimethyl-4,4'-bipyridinium dichloride), a widely used herbicide that is structurally similar to the known dopaminergic neurotoxicant MPP(+) (1-methyl-4-phenyl-pyridine), has been suggested as a potential etiologic factor for the development of PD. The current study shows, for the first time, that low concentrations of PQ induce several characteristics of autophagy in human neuroblastoma SH-SY5Y cells. In this way, PQ induced the accumulation of autophagic vacuoles (AVs) in the cytoplasm and the recruitment of a LC3-GFP fusion protein to AVs. Furthermore, the cells treated with PQ showed an increase of the long-lived protein degradation which is blocked in the presence of the autophagy inhibitor 3-methyladenine and regulated by the mammalian target of rapamycin (mTOR) signaling. Finally, the cells succumbed to cell death with hallmarks of apoptosis such as phosphatidylserine exposure, caspase activation, and chromatin condensation. While caspase inhibition retarded cell death, autophagy inhibition accelerated the apoptotic cell death induced by PQ. Altogether, these findings show the relationship between autophagy and apoptotic cell death in human neuroblastoma cells treated with PQ.

Synthesis of cytochrome C oxidase 2: a p53-dependent metabolic regulator that promotes respiratory function and protects glioma and colon cancer cells from hypoxia-induced cell death.

PubMed

Wanka, C; Brucker, D P; Bähr, O; Ronellenfitsch, M; Weller, M; Steinbach, J P; Rieger, J

2012-08-16

P53 has an important role in the processing of starvation signals. P53-dependent molecular mediators of the Warburg effect reduce glucose consumption and promote mitochondrial function. We therefore hypothesized that the retention of wild-type p53 characteristic of primary glioblastomas limits metabolic demands induced by deregulated signal transduction in the presence of hypoxia and nutrient depletion. Here we report that short hairpin RNA-mediated gene suppression of wild-type p53 or ectopic expression of mutant temperature-sensitive dominant-negative p53(V135A) increased glucose consumption and lactate production, decreased oxygen consumption and enhanced hypoxia-induced cell death in p53 wild-type human glioblastoma cells. Similarly, genetic knockout of p53 in HCT116 colon carcinoma cells resulted in reduced respiration and hypersensitivity towards hypoxia-induced cell death. Further, wild-type p53 gene silencing reduced the expression of synthesis of cytochrome c oxidase 2 (SCO2), an effector necessary for respiratory chain function. An SCO2 transgene reverted the metabolic phenotype and restored resistance towards hypoxia in p53-depleted and p53 mutant glioma cells in a rotenone-sensitive manner, demonstrating that this effect was dependent on intact oxidative phosphorylation. Supplementation with methyl-pyruvate, a mitochondrial substrate, rescued p53 wild-type but not p53 mutant cells from hypoxic cell death, demonstrating a p53-mediated selective aptitude to metabolize mitochondrial substrates. Further, SCO2 gene silencing in p53 wild-type glioma cells sensitized these cells towards hypoxia. Finally, lentiviral gene suppression of SCO2 significantly enhanced tumor necrosis in a subcutaneous HCT116 xenograft tumor model, compatible with impaired energy metabolism in these cells. These findings demonstrate that glioma and colon cancer cells with p53 wild-type status can skew the Warburg effect and thereby reduce their vulnerability towards tumor hypoxia in an SCO2-dependent manner. Targeting SCO2 may therefore represent a valuable strategy to enhance sensitivity towards hypoxia and may complement strategies targeting glucose metabolism.

Apigenin potentiates TRAIL therapy of non-small cell lung cancer via upregulating DR4/DR5 expression in a p53-dependent manner

PubMed Central

Chen, Minghui; Wang, Xueshi; Zha, Daolong; Cai, Fangfang; Zhang, Wenjing; He, Yan; Huang, Qilai; Zhuang, Hongqin; Hua, Zi-Chun

2016-01-01

Apigenin (APG) is an edible plant-derived flavonoid that shows modest antitumor activities in vitro and in vivo. APG treatment results in cell growth arrest and apoptosis in various types of tumors by modulating several signaling pathways. In the present study, we evaluated interactions between APG and TRAIL in non-small cell lung cancer (NSCLC) cells. We observed a synergistic effect between APG and TRAIL on apoptosis of NSCLC cells. A549 cells and H1299 cells were resistant to TRAIL treatment alone. The presence of APG sensitized NSCLC cells to TRAIL-induced apoptosis by upregulating the levels of death receptor 4 (DR4) and death receptor 5 (DR5) in a p53-dependent manner. Consistently, the pro-apoptotic proteins Bad and Bax were upregulated, while the anti-apoptotic proteins Bcl-xl and Bcl-2 were downregulated. Meanwhile, APG suppressed NF-κB, AKT and ERK activation. Treatment with specific small-molecule inhibitors of these pathways enhanced TRAIL-induced cell death, mirroring the effect of APG. Furthermore, using a mouse xenograft model, we demonstrated that the combined treatment completely suppressed tumor growth as compared with APG or TRAIL treatment alone. Our results demonstrate a novel strategy to enhance TRAIL-induced antitumor activity in NSCLC cells by APG via inhibition of the NF-κB, AKT and ERK prosurvival regulators. PMID:27752089

Apigenin potentiates TRAIL therapy of non-small cell lung cancer via upregulating DR4/DR5 expression in a p53-dependent manner.

PubMed

Chen, Minghui; Wang, Xueshi; Zha, Daolong; Cai, Fangfang; Zhang, Wenjing; He, Yan; Huang, Qilai; Zhuang, Hongqin; Hua, Zi-Chun

2016-10-18

Apigenin (APG) is an edible plant-derived flavonoid that shows modest antitumor activities in vitro and in vivo. APG treatment results in cell growth arrest and apoptosis in various types of tumors by modulating several signaling pathways. In the present study, we evaluated interactions between APG and TRAIL in non-small cell lung cancer (NSCLC) cells. We observed a synergistic effect between APG and TRAIL on apoptosis of NSCLC cells. A549 cells and H1299 cells were resistant to TRAIL treatment alone. The presence of APG sensitized NSCLC cells to TRAIL-induced apoptosis by upregulating the levels of death receptor 4 (DR4) and death receptor 5 (DR5) in a p53-dependent manner. Consistently, the pro-apoptotic proteins Bad and Bax were upregulated, while the anti-apoptotic proteins Bcl-xl and Bcl-2 were downregulated. Meanwhile, APG suppressed NF-κB, AKT and ERK activation. Treatment with specific small-molecule inhibitors of these pathways enhanced TRAIL-induced cell death, mirroring the effect of APG. Furthermore, using a mouse xenograft model, we demonstrated that the combined treatment completely suppressed tumor growth as compared with APG or TRAIL treatment alone. Our results demonstrate a novel strategy to enhance TRAIL-induced antitumor activity in NSCLC cells by APG via inhibition of the NF-κB, AKT and ERK prosurvival regulators.

BAG3 protects bovine papillomavirus type 1-transformed equine fibroblasts against pro-death signals.

PubMed

Cotugno, Roberta; Gallotta, Dario; d'Avenia, Morena; Corteggio, Annunziata; Altamura, Gennaro; Roperto, Franco; Belisario, Maria Antonietta; Borzacchiello, Giuseppe

2013-07-22

In human cancer cells, BAG3 protein is known to sustain cell survival. Here, for the first time, we demonstrate the expression of BAG3 protein both in equine sarcoids in vivo and in EqS04b cells, a sarcoid-derived fully transformed cell line harbouring bovine papilloma virus (BPV)-1 genome. Evidence of a possible involvement of BAG3 in equine sarcoid carcinogenesis was obtained by immunohistochemistry analysis of tumour samples. We found that most tumour samples stained positive for BAG3, even though to a different grade, while normal dermal fibroblasts from healthy horses displayed very weak staining pattern for BAG3 expression. By siRNA technology, we demonstrate in EqS04b the role of BAG3 in counteracting basal as well as chemical-triggered pro-death signals. BAG3 down-modulation was indeed shown to promote cell death and cell cycle arrest in G0/G1. In addition, we found that BAG3 silencing sensitized EqS04b cells to phenethylisothiocyanate (PEITC), a promising cancer chemopreventive/chemotherapeutic agent present in edible cruciferous vegetables. Notably, such a pro-survival role of BAG3 was less marked in E. Derm cells, an equine BPV-negative fibroblast cell line taken as a normal counterpart. Altogether our findings might suggest a mutual cooperation between BAG3 and viral oncoproteins to sustain cell survival.

BAG3 protects Bovine Papillomavirus type 1-transformed equine fibroblasts against pro-death signals

PubMed Central

2013-01-01

In human cancer cells, BAG3 protein is known to sustain cell survival. Here, for the first time, we demonstrate the expression of BAG3 protein both in equine sarcoids in vivo and in EqS04b cells, a sarcoid-derived fully transformed cell line harbouring bovine papilloma virus (BPV)-1 genome. Evidence of a possible involvement of BAG3 in equine sarcoid carcinogenesis was obtained by immunohistochemistry analysis of tumour samples. We found that most tumour samples stained positive for BAG3, even though to a different grade, while normal dermal fibroblasts from healthy horses displayed very weak staining pattern for BAG3 expression. By siRNA technology, we demonstrate in EqS04b the role of BAG3 in counteracting basal as well as chemical-triggered pro-death signals. BAG3 down-modulation was indeed shown to promote cell death and cell cycle arrest in G0/G1. In addition, we found that BAG3 silencing sensitized EqS04b cells to phenethylisothiocyanate (PEITC), a promising cancer chemopreventive/chemotherapeutic agent present in edible cruciferous vegetables. Notably, such a pro-survival role of BAG3 was less marked in E. Derm cells, an equine BPV-negative fibroblast cell line taken as a normal counterpart. Altogether our findings might suggest a mutual cooperation between BAG3 and viral oncoproteins to sustain cell survival. PMID:23876161

Autophagy induced by baicalin involves downregulation of CD147 in SMMC-7721 cells in vitro

PubMed Central

ZHANG, XIANJIAO; TANG, XU; LIU, HANQIANG; LI, LIANXIANG; HOU, QIAN; GAO, JIANMIN

2012-01-01

Baicalin has been demonstrated to exert anticancer effects mainly through induction of tumor cell apoptosis and cell cycle arrest. However, the precise mechanisms underlying its anticancer role remain to be elucidated. In the present study, we investigated whether autophagy was involved in the anticancer activity of baicalin in the human hepatocellular carcinoma (HCC) cell line SMMC-7721 and the possible molecular mechanisms. Our data showed that the viability of SMMC-7721 cells was significantly inhibited by baicalin in a dose- and time-dependent manner. Alongside apoptosis, autophagy was also induced by baicalin dose- and time-dependently with the involvement of the autophagy-associated protein Beclin 1. Moreover, we demonstrated that cell death induced by baicalin was significantly inhibited by the apoptosis inhibitor z-DEVD-fmk or the autophagy inhibitor 3-MA, respectively. In addition, we found that CD147, a key molecule related both to apoptosis and autophagy, was markedly downregulated at the protein level in SMMC-7721 cells treated with baicalin. Collectively, this is the first study to suggest that baicalin induces autophagic cell death in SMMC-7721 cells, which involves the downregulation of CD147. Our study reveals a new mechanism for the anticancer effects of baicalin and puts forward a potential crucial role of CD147 in baicalin-induced cancer cell death. PMID:22200845

Impairment of Fas-ligand-caveolin-1 interaction inhibits Fas-ligand translocation to rafts and Fas-ligand-induced cell death.

PubMed

Glukhova, Xenia A; Trizna, Julia A; Proussakova, Olga V; Gogvadze, Vladimir; Beletsky, Igor P

2018-01-22

Fas-ligand/CD178 belongs to the TNF family proteins and can induce apoptosis through death receptor Fas/CD95. The important requirement for Fas-ligand-dependent cell death induction is its localization to rafts, cholesterol- and sphingolipid-enriched micro-domains of membrane, involved in regulation of different signaling complexes. Here, we demonstrate that Fas-ligand physically associates with caveolin-1, the main protein component of rafts. Experiments with cells overexpressing Fas-ligand revealed a FasL N-terminal pre-prolin-rich region, which is essential for the association with caveolin-1. We found that the N-terminal domain of Fas-ligand bears two caveolin-binding sites. The first caveolin-binding site binds the N-terminal domain of caveolin-1, whereas the second one appears to interact with the C-terminal domain of caveolin-1. The deletion of both caveolin-binding sites in Fas-ligand impairs its distribution between cellular membranes, and attenuates a Fas-ligand-induced cytotoxicity. These results demonstrate that the interaction of Fas-ligand and caveolin-1 represents a molecular basis for Fas-ligand translocation to rafts, and the subsequent induction of Fas-ligand-dependent cell death. A possibility of a similar association between other TNF family members and caveolin-1 is discussed.

Necroptosis in cardiovascular disease - a new therapeutic target.

PubMed

Gupta, Kartik; Phan, Noel; Wang, Qiwei; Liu, Bo

2018-05-01

Contrary to the apoptosis-necrosis binary view of cell death, recent experimental evidence demonstrates that several forms of necrosis, represented by necroptosis, are regulated or programmed in nature. Multiple death stimuli known to be associated with cardiovascular disease are capable of causing either apoptosis or necroptosis. Whether a cell dies from apoptosis or necroptosis has distinct consequences on inflammation. It is known that apoptosis, a non-lytic form of death mediated by the caspase family of proteases, does not generally evoke an immune response. Necroptosis, on the other hand, is a lytic form of cell death. Due to the rapid loss of plasma membrane integrity, cells dying from necroptosis release proinflammatory intracellular contents and subsequently cause inflammation. Our review delineates various genetic and biochemical evidence that demonstrates a compelling role of necroptosis in the pathogenesis and/or progression of cardiovascular disease including myocardial infarction, atherosclerosis, and aortic aneurysm. Through recent studies of necroptosis in cardiovascular diseases, we attempt to discuss the role of necroptosis in vascular inflammation as well as the potential of necroptosis inhibitors in future clinical management of cardiovascular events. Inhibiting necroptosis in the vasculature has an overall protective role and necroptosis may represent a new therapeutic target to prevent the development and progression of cardiovascular diseases. Copyright © 2018 Elsevier Ltd. All rights reserved.

Synthesis and preliminary evaluation of radiolabeled bis(zinc(II)-dipicolylamine) coordination complexes as cell death imaging agents

PubMed Central

wyffels, Leonie; Gray, Brian D.; Barber, Christy; Woolfenden, James M.; Pak, Koon Y.; Liu, Zhonglin

2011-01-01

The aim of this study was the development of 99mTc labeled bis(zinc(II)-dipicolylamine) (Zn2+-DPA) coordination complexes, and the in vivo evaluation of their usefulness as radiotracers for the detection of cell death. DPA ligand 1 was labeled with 99mTc via the 99mTc-tricarbonyl core ([99mTc(CO)3-1]3+) or via HYNIC (99mTc-HYNIC-1) in good radiochemical yields. Highest in vitro stabilities were demonstrated for [99mTc(CO)3-1]3+. A mouse model of hepatic apoptosis (anti-Fas mAb) was used to demonstrate binding to apoptotic cells. 99mTc-HYNIC-1 showed the best targeting of apoptotic hepatic tissue with a 2.2 times higher liver uptake in anti-Fas treated mice as compared to healthy animals. A rat model of ischemia-reperfusion injury was used to further explore the ability of the 99mTc-labeled Zn2+-DPA coordination complexes to target cell death. Selective accumulation could be detected for both tracers in the area at risk, correlating with histological proof of cell death. Area at risk to normal tissue uptake ratios were 3.82 for [99mTc(CO)3-1]3+ and 5.45 for 99mTc-HYNIC-1. PMID:21570306

Elevated extracellular [K+] inhibits death-receptor- and chemical-mediated apoptosis prior to caspase activation and cytochrome c release.

PubMed Central

Thompson, G J; Langlais, C; Cain, K; Conley, E C; Cohen, G M

2001-01-01

Efflux of intracellular K(+) and cell shrinkage are features of apoptosis in many experimental systems, and a regulatory role has been proposed for cytoplasmic [K(+)] in initiating apoptosis. We have investigated this in both death-receptor-mediated and chemical-induced apoptosis. Using Jurkat T cells pre-loaded with the K(+) ion surrogate (86)Rb(+), we have demonstrated an efflux of intracellular K(+) during apoptosis that was concomitant with, but did not precede, other apoptotic changes, including phosphatidylserine externalization, mitochondrial depolarization and cell shrinkage. To further clarify the role of K(+) ions in apoptosis, cytoprotection by elevated extracellular [K(+)] was studied. Induction of apoptosis by diverse death-receptor and chemical stimuli in two cell lines was inhibited prior to phosphatidylserine externalization, mitochondrial depolarization, cytochrome c release and caspase activation. Using a cell-free system, we have demonstrated a novel mechanism by which increasing [K(+)] inhibited caspase activation. In control dATP-activated lysates, Apaf-1 oligomerized to a biologically active caspase processing approximately 700 kDa complex and an inactive approximately 1.4 MDa complex. Increasing [K(+)] inhibited caspase activation by preventing formation of the approximately 700 kDa complex, but not of the inactive complex. Thus intracellular and extracellular [K(+)] markedly affect caspase activation and the initiation of apoptosis induced by both death-receptor ligation and chemical stress. PMID:11415444

The Possible Mechanism of Advanced Glycation End Products (AGEs) for Alzheimer’s Disease

PubMed Central

Ko, Shun-Yao; Ko, Hshin-An; Chu, Kuo-Hsiung; Shieh, Tzong-Ming; Chi, Tzong-Cherng; Chen, Hong-I; Chang, Weng-Cheng; Chang, Shu-Shing

2015-01-01

Amyloid precursor protein (APP) has been modified by β and γ-secretase that cause amyloid deposits (plaques) in neuronal cells. Glyceraldhyde-derived AGEs has been identified as a major source of neurotoxicity in Alzheimer’s disease (AD). In a previous study, we demonstrated that glyceraldehyde-derived AGEs increase APP and Aβ via ROS. Furthermore, the combination of AGEs and Aβ has been shown to enhance neurotoxicity. In mice, APP expression is increased by tail vein injection of AGEs. This evidence suggests a correlation between AGEs and the development of AD. However, the role played by AGEs in the pathogenesis of AD remains unclear. In this report, we demonstrate that AGEs up-regulate APP processing protein (BACE and PS1) and Sirt1 expression via ROS, but do not affect the expression of downstream antioxidant genes HO-1 and NQO-1. Moreover, we found that AGEs increase GRP78 expression and enhance the cell death-related pathway p53, bcl-2/bax ratio, caspase 3. These results indicate that AGEs impair the neuroprotective effects of Sirt1 and lead to neuronal cell death via ER stress. Our findings suggest that AGEs increase ROS production, which stimulates downstream pathways related to APP processing, Aβ production, Sirt1, and GRP78, resulting in the up-regulation of cell death related pathway. This in-turn enhances neuronal cell death, which leads to the development of AD. PMID:26587989

The Possible Mechanism of Advanced Glycation End Products (AGEs) for Alzheimer's Disease.

PubMed

Ko, Shun-Yao; Ko, Hshin-An; Chu, Kuo-Hsiung; Shieh, Tzong-Ming; Chi, Tzong-Cherng; Chen, Hong-I; Chang, Weng-Cheng; Chang, Shu-Shing

2015-01-01

Amyloid precursor protein (APP) has been modified by β and γ-secretase that cause amyloid deposits (plaques) in neuronal cells. Glyceraldhyde-derived AGEs has been identified as a major source of neurotoxicity in Alzheimer's disease (AD). In a previous study, we demonstrated that glyceraldehyde-derived AGEs increase APP and Aβ via ROS. Furthermore, the combination of AGEs and Aβ has been shown to enhance neurotoxicity. In mice, APP expression is increased by tail vein injection of AGEs. This evidence suggests a correlation between AGEs and the development of AD. However, the role played by AGEs in the pathogenesis of AD remains unclear. In this report, we demonstrate that AGEs up-regulate APP processing protein (BACE and PS1) and Sirt1 expression via ROS, but do not affect the expression of downstream antioxidant genes HO-1 and NQO-1. Moreover, we found that AGEs increase GRP78 expression and enhance the cell death-related pathway p53, bcl-2/bax ratio, caspase 3. These results indicate that AGEs impair the neuroprotective effects of Sirt1 and lead to neuronal cell death via ER stress. Our findings suggest that AGEs increase ROS production, which stimulates downstream pathways related to APP processing, Aβ production, Sirt1, and GRP78, resulting in the up-regulation of cell death related pathway. This in-turn enhances neuronal cell death, which leads to the development of AD.

Image Analysis Algorithms for Immunohistochemical Assessment of Cell Death Events and Fibrosis in Tissue Sections

PubMed Central

Krajewska, Maryla; Smith, Layton H.; Rong, Juan; Huang, Xianshu; Hyer, Marc L.; Zeps, Nikolajs; Iacopetta, Barry; Linke, Steven P.; Olson, Allen H.; Reed, John C.; Krajewski, Stan

2009-01-01

Cell death is of broad physiological and pathological importance, making quantification of biochemical events associated with cell demise a high priority for experimental pathology. Fibrosis is a common consequence of tissue injury involving necrotic cell death. Using tissue specimens from experimental mouse models of traumatic brain injury, cardiac fibrosis, and cancer, as well as human tumor specimens assembled in tissue microarray (TMA) format, we undertook computer-assisted quantification of specific immunohistochemical and histological parameters that characterize processes associated with cell death. In this study, we demonstrated the utility of image analysis algorithms for color deconvolution, colocalization, and nuclear morphometry to characterize cell death events in tissue specimens: (a) subjected to immunostaining for detecting cleaved caspase-3, cleaved poly(ADP-ribose)-polymerase, cleaved lamin-A, phosphorylated histone H2AX, and Bcl-2; (b) analyzed by terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay to detect DNA fragmentation; and (c) evaluated with Masson's trichrome staining. We developed novel algorithm-based scoring methods and validated them using TMAs as a high-throughput format. The proposed computer-assisted scoring methods for digital images by brightfield microscopy permit linear quantification of immunohistochemical and histochemical stainings. Examples are provided of digital image analysis performed in automated or semiautomated fashion for successful quantification of molecular events associated with cell death in tissue sections. (J Histochem Cytochem 57:649–663, 2009) PMID:19289554

Improving Accuracy in Arrhenius Models of Cell Death: Adding a Temperature-Dependent Time Delay.

PubMed

Pearce, John A

2015-12-01

The Arrhenius formulation for single-step irreversible unimolecular reactions has been used for many decades to describe the thermal damage and cell death processes. Arrhenius predictions are acceptably accurate for structural proteins, for some cell death assays, and for cell death at higher temperatures in most cell lines, above about 55 °C. However, in many cases--and particularly at hyperthermic temperatures, between about 43 and 55 °C--the particular intrinsic cell death or damage process under study exhibits a significant "shoulder" region that constant-rate Arrhenius models are unable to represent with acceptable accuracy. The primary limitation is that Arrhenius calculations always overestimate the cell death fraction, which leads to severely overoptimistic predictions of heating effectiveness in tumor treatment. Several more sophisticated mathematical model approaches have been suggested and show much-improved performance. But simpler models that have adequate accuracy would provide useful and practical alternatives to intricate biochemical analyses. Typical transient intrinsic cell death processes at hyperthermic temperatures consist of a slowly developing shoulder region followed by an essentially constant-rate region. The shoulder regions have been demonstrated to arise chiefly from complex functional protein signaling cascades that generate delays in the onset of the constant-rate region, but may involve heat shock protein activity as well. This paper shows that acceptably accurate and much-improved predictions in the simpler Arrhenius models can be obtained by adding a temperature-dependent time delay. Kinetic coefficients and the appropriate time delay are obtained from the constant-rate regions of the measured survival curves. The resulting predictions are seen to provide acceptably accurate results while not overestimating cell death. The method can be relatively easily incorporated into numerical models. Additionally, evidence is presented to support the application of compensation law behavior to the cell death processes--that is, the strong correlation between the kinetic coefficients, ln{A} and E(a), is confirmed.

DISTINCT FUNCTIONS OF JNK AND C-JUN IN OXIDANT-INDUCED HEPATOCYTE DEATH

PubMed Central

Amir, Muhammad; Liu, Kun; Zhao, Enpeng; Czaja, Mark J.

2013-01-01

Overactivation of c-Jun N-terminal kinase (JNK)/c-Jun signaling is a central mechanism of hepatocyte injury and death including that from oxidative stress. However, the functions of JNK and c-Jun are still unclear, and this pathway also inhibits hepatocyte death. Previous studies of menadione-induced oxidant stress demonstrated that toxicity resulted from sustained JNK/c-Jun activation as death was blocked by the c-Jun dominant negative TAM67. To further delineate the function of JNK/c-Jun signaling in hepatocyte injury from oxidant stress, the effects of direct JNK inhibition on menadione-induced death were examined. In contrast to the inhibitory effect of TAM67, pharmacological JNK inhibition by SP600125 sensitized the rat hepatocyte cell line RALA255-10G to death from menadione. SP600125 similarly sensitized mouse primary hepatocytes to menadione toxicity. Death from SP600125/menadione was c-Jun dependent as it was blocked by TAM67, but independent of c-Jun phosphorylation. Death occurred by apoptosis and necrosis and activation of the mitochondrial death pathway. Short hairpin RNA knockdowns of total JNK or JNK2 sensitized to death from menadione, whereas a jnk1 knockdown was protective. Jnk2 null mouse primary hepatocytes were also sensitized to menadione death. JNK inhibition magnified decreases in cellular ATP content and β-oxidation induced by menadione. This effect mediated cell death as chemical inhibition of β-oxidation also sensitized cells to death from menadione, and supplementation with the β-oxidation substrate oleate blocked death. Components of the JNK/c-Jun signaling pathway have opposing functions in hepatocyte oxidant stress with JNK2 mediating resistance to cell death and c-Jun promoting death. PMID:22644775

INF-γ sensitizes head and neck squamous cell carcinoma cells to chemotherapy-induced apoptosis and necroptosis through up-regulation of Egr-1.

PubMed

Xu, Bei; Shu, Yongqian; Liu, Peng

2014-11-01

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Acquired resistance to standard chemotherapy accounts for most of treatment failure. Here we demonstrate that Interferon-γ (INF-γ) may up-regulate Egr-1 gene expression in HNSCC cell line SCC-25. Forced expression of Egr-1 sensitizes SCC-25 cells to chemotherapy-induced apoptosis and necroptosis, a novel form of programmed cell death. Egr-1 up-regulation also significantly increases the production of Thrombospondin-1 (TSP-1), a matricellular glycoprotein which has been described to induce cell death in HNSCC. Moreover, INF-γ-induced sensitization of cells to chemotherapy-mediated cell death and TSP-1 production could be markedly abolished by Egr-1 silencing. The present investigation provides the first evidence that INF-γ may sensitize HNSCC cells to chemotherapy-induced apoptosis and necroptosis through up-regulation of Egr-1. These data support the combination use of INF-γ and cytotoxic drugs for HNSCC Therapy.

Efavirenz and 8-hydroxyefavirenz induce cell death via a JNK- and BimEL-dependent mechanism in primary human hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bumpus, Namandje N., E-mail: nbumpus1@jhmi.edu

Chronic use of efavirenz (EFV) has been linked to incidences of hepatotoxicity in patients receiving EFV to treat HIV-1. While recent studies have demonstrated that EFV stimulates hepatic cell death a role for the metabolites of efavirenz in this process has yet to be examined. In the present study, incubation of primary human hepatocytes with synthetic 8-hydroxyEFV (8-OHEFV), which is the primary metabolite of EFV, resulted in cell death, caspase-3 activation and reactive oxygen species formation. The metabolite exerted these effects at earlier time points and using lower concentrations than were required for the parent compound. In addition, pharmacological inhibitionmore » of cytochrome P450-dependent metabolism of EFV using 1-aminobenzotriazole markedly decreased reactive oxygen species formation and cell death. Treatment of primary human hepatocytes with EFV and 8-OHEFV also stimulated phosphorylation of c-Jun N-terminal kinase (JNK) as well as phosphorylation of the JNK substrate c-Jun. Further, the mRNA and protein expression of an isoform of Bim (Bcl-2 interacting mediator of cell death) denoted as BimEL, which is proapoptotic and has been shown to be modulated by JNK, was increased. Inhibition of JNK using SP600125 prevented the EFV- and 8-OHEFV-mediated cell death. Silencing of Bim using siRNA transfected into hepatocytes also prevented cell death resulting from 8-OHEFV-treatment. These data suggest that the oxidative metabolite 8-OHEFV is a more potent inducer of hepatic cell death than the parent compound EFV. Further, activation of the JNK signaling pathway and BimEL mRNA expression appear to be required for EFV- and 8-OHEFV-mediated hepatocyte death. -- Highlights: Black-Right-Pointing-Pointer 8-Hydroxyefavirenz is a more potent stimulator of cell death than efavirenz. Black-Right-Pointing-Pointer Efavirenz and 8-hydroxyefavirenz increase JNK activity and BimEL mRNA expression. Black-Right-Pointing-Pointer JNK and Bim are required for efavirenz- and 8-hydroxyefavirenz-mediated cell death. Black-Right-Pointing-Pointer Efavirenz and 8-hydroxyefavirenz may be novel modulators of Bim.« less

Cordycepin-induced apoptosis and autophagy in breast cancer cells are independent of the estrogen receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Sunga; Lim, Mi-Hee; Kim, Ki Mo

2011-12-15

Cordycepin (3-deoxyadenosine), found in Cordyceps spp., has been known to have many therapeutic effects including immunomodulatory, anti-inflammatory, antimicrobial, and anti-aging effects. Moreover, anti-tumor and anti-metastatic effects of cordycepin have been reported, but the mechanism causing cancer cell death is poorly characterized. The present study was designed to investigate whether the mechanisms of cordycepin-induced cell death were associated with estrogen receptor in breast cancer cells. Exposure of both MDA-MB-231 and MCF-7 human breast cancer cells to cordycepin resulted in dose-responsive inhibition of cell growth and reduction in cell viability. The cordycepin-induced cell death in MDA-MB-231 cells was associated with several specificmore » features of the mitochondria-mediated apoptotic pathway, which was confirmed by DNA fragmentation, TUNEL, and biochemical assays. Cordycepin also caused a dose-dependent increase in mitochondrial translocation of Bax, triggering cytosolic release of cytochrome c and activation of caspases-9 and -3. Interestingly, MCF-7 cells showed autophagy-associated cell death, as observed by the detection of an autophagosome-specific protein and large membranous vacuole ultrastructure morphology in the cytoplasm. Cordycepin-induced autophagic cell death has applications in treating MCF-7 cells with apoptotic defects, irrespective of the ER response. Although autophagy has a survival function in tumorigenesis of some cancer cells, autophagy may be important for cordycepin-induced MCF-7 cell death. In conclusion, the results of our study demonstrate that cordycepin effectively kills MDA-MB-231 and MCF-7 human breast cancer cell lines in culture. Hence, further studies should be conducted to determine whether cordycepin will be a clinically useful, ER-independent, chemotherapeutic agent for human breast cancer. -- Highlights: Black-Right-Pointing-Pointer We studied the mechanism which cordycepin-induced cell death association with estrogen receptor (ER) in breast cancer cells, MDA-MB-231 and MCF-7. Black-Right-Pointing-Pointer The cordycepin-induced cell death in MDA-MB-231 cells was associated with the mitochondria-mediated apoptotic pathway. Black-Right-Pointing-Pointer Cordycepin treatment also resulted in autophagy in MCF-7 cells, associated with induction of autophagosome formation. Black-Right-Pointing-Pointer The different cordycepin-mediated cell death pathways are irrespective of the ER response. Black-Right-Pointing-Pointer Cordycepin proves a clinically useful, ER-independent chemotherapeutic agent for human breast cancer cells.« less

Isolated primary blast alters neuronal function with minimal cell death in organotypic hippocampal slice cultures.

PubMed

Effgen, Gwen B; Vogel, Edward W; Lynch, Kimberly A; Lobel, Ayelet; Hue, Christopher D; Meaney, David F; Bass, Cameron R Dale; Morrison, Barclay

2014-07-01

An increasing number of U.S. soldiers are diagnosed with traumatic brain injury (TBI) subsequent to exposure to blast. In the field, blast injury biomechanics are highly complex and multi-phasic. The pathobiology caused by exposure to some of these phases in isolation, such as penetrating or inertially driven injuries, has been investigated extensively. However, it is unclear whether the primary component of blast, a shock wave, is capable of causing pathology on its own. Previous in vivo studies in the rodent and pig have demonstrated that it is difficult to deliver a primary blast (i.e., shock wave only) without rapid head accelerations and potentially confounding effects of inertially driven TBI. We have previously developed a well-characterized shock tube and custom in vitro receiver for exposing organotypic hippocampal slice cultures to pure primary blast. In this study, isolated primary blast induced minimal hippocampal cell death (on average, below 14% in any region of interest), even for the most severe blasts tested (424 kPa peak pressure, 2.3 ms overpressure duration, and 248 kPa*ms impulse). In contrast, measures of neuronal function were significantly altered at much lower exposures (336 kPa, 0.84 ms, and 86.5 kPa*ms), indicating that functional changes occur at exposures below the threshold for cell death. This is the first study to investigate a tolerance for primary blast-induced brain cell death in response to a range of blast parameters and demonstrate functional deficits at subthreshold exposures for cell death.

Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions.

PubMed

Deegan, Shane; Saveljeva, Svetlana; Logue, Susan E; Pakos-Zebrucka, Karolina; Gupta, Sanjeev; Vandenabeele, Peter; Bertrand, Mathieu J M; Samali, Afshin

2014-01-01

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.

Induction of cyto-protective autophagy by paramontroseite VO2 nanocrystals

NASA Astrophysics Data System (ADS)

Zhou, Wei; Miao, Yanyan; Zhang, Yunjiao; Liu, Liang; Lin, Jun; Yang, James Y.; Xie, Yi; Wen, Longping

2013-04-01

A variety of inorganic nanomaterials have been shown to induce autophagy, a cellular degradation process critical for the maintenance of cellular homeostasis. The overwhelming majority of autophagic responses elicited by nanomaterials were detrimental to cell fate and contributed to increased cell death. A widely held view is that the inorganic nanoparticles, when encapsulated and trapped by autophagosomes, may compromise the normal autophagic process due to the inability of the cells to degrade these materials and thus they manifest a detrimental effect on the well-being of a cell. Here we show that, contrary to this notion, nano-sized paramontroseite VO2 nanocrystals (P-VO2) induced cyto-protective, rather than death-promoting, autophagy in cultured HeLa cells. P-VO2 also caused up-regulation of heme oxygenase-1 (HO-1), a cellular protein with a demonstrated role in protecting cells against death under stress situations. The autophagy inhibitor 3-methyladenine significantly inhibited HO-1 up-regulation and increased the rate of cell death in cells treated with P-VO2, while the HO-1 inhibitor protoporphyrin IX zinc (II) (ZnPP) enhanced the occurrence of cell death in the P-VO2-treated cells while having no effect on the autophagic response induced by P-VO2. On the other hand, Y2O3 nanocrystals, a control nanomaterial, induced death-promoting autophagy without affecting the level of expression of HO-1, and the pro-death effect of the autophagy induced by Y2O3. Our results represent the first report on a novel nanomaterial-induced cyto-protective autophagy, probably through up-regulation of HO-1, and may point to new possibilities for exploiting nanomaterial-induced autophagy for therapeutic applications.

Protein functionalization of ZnO nanostructure exhibits selective and enhanced toxicity to breast cancer cells through oxidative stress-based cell death mechanism.

PubMed

Mahanta, Sailendra; Prathap, S; Ban, Deependra Kumar; Paul, Subhankar

2017-08-01

Zinc oxide nanostructure (ZnONS) was chemically synthesized and functionalized (FZnONS BLA ) with a small protein bovine α-lactalbumin (BLA) by chemical cross-linking methods. Both nano-structures were characterized using various techniques such as electron microscopy, dynamic light scattering (DLS), UV-Vis spectroscopy, FT-IR, photo-luminescence and X-ray diffraction. Electron microscopy and DLS analysis revealed their (ZnONS and FZnONS BLA ) average size of 200nm and 450nm, respectively. When cytotoxicity of both the nanostructures were assessed in breast cancer cells MCF-7 and MDAMB231 by MTT assay and PI/Annexin V staining (FACS), FZnONS BLA demonstrated higher cell death than ZnONS primarily due to generation of intracellular reactive oxygen species (ROS). Our experimental results also suggested that such enhanced toxicity was due to the lethal structural variant of BLA in FZnONS BLA as well as higher cellular uptake than ZnONS by cancer cells. The death kinetics study with time in cancer cells further proved that FZnONS BLA caused toxicity much faster than ZnONS, thus suggested a strong role of lethal variant of BLA in FZnONS BLA as a cytotoxic agent in cancer cells. Furthermore, FZnONS BLA demonstrated excellent cytocompatibility (normal cells) and hemocompatibility compared to ZnONS. Hence, considering the biodegradable nature of ZnO nonmaterial, our results demonstrated that BLA functionalized ZnONS could be used to develop a suitable therapeutic strategy in cancer. Copyright © 2017. Published by Elsevier B.V.

Time resolved study of cell death mechanisms induced by amine-modified polystyrene nanoparticles

NASA Astrophysics Data System (ADS)

Wang, Fengjuan; Bexiga, Mariana G.; Anguissola, Sergio; Boya, Patricia; Simpson, Jeremy C.; Salvati, Anna; Dawson, Kenneth A.

2013-10-01

Positively charged polymers and nanoparticles (NPs) can be toxic to cells in various systems. Using human astrocytoma cells, we have previously shown that 50 nm amine-modified polystyrene NPs damage mitochondria and induce cell death by apoptosis. Here we provide comprehensive details of the cellular events occurring after exposure to the NPs in a time-resolved manner. We demonstrate that the accumulation of NPs in lysosomes plays a central role in the observed cell death, leading to swelling of the lysosomes and release of cathepsins into the cytosol, which ultimately propagates the damage to the mitochondria with subsequent activation of apoptosis. This is accompanied and sustained by other events, such as increasing ROS levels and autophagy. Using various inhibitors, we also show the interplay between apoptosis and autophagy as a response to NP accumulation in lysosomes.Positively charged polymers and nanoparticles (NPs) can be toxic to cells in various systems. Using human astrocytoma cells, we have previously shown that 50 nm amine-modified polystyrene NPs damage mitochondria and induce cell death by apoptosis. Here we provide comprehensive details of the cellular events occurring after exposure to the NPs in a time-resolved manner. We demonstrate that the accumulation of NPs in lysosomes plays a central role in the observed cell death, leading to swelling of the lysosomes and release of cathepsins into the cytosol, which ultimately propagates the damage to the mitochondria with subsequent activation of apoptosis. This is accompanied and sustained by other events, such as increasing ROS levels and autophagy. Using various inhibitors, we also show the interplay between apoptosis and autophagy as a response to NP accumulation in lysosomes. Electronic supplementary information (ESI) available: additional analysis of flow cytometry results, western blots and experiments with cathepsin inhibitors. See DOI: 10.1039/c3nr03249c

Oxygenated monoterpenes citral and carvacrol cause oxidative damage in Escherichia coli without the involvement of tricarboxylic acid cycle and Fenton reaction.

PubMed

Chueca, Beatriz; Pagán, Rafael; García-Gonzalo, Diego

2014-10-17

Oxygenated monoterpenes citral and carvacrol are common constituents of many essential oils (EOs) that have been extensively studied as antimicrobial agents but whose mechanisms of microbial inactivation have not been totally elucidated. A recent study described a mechanism of Escherichia coli death for (+)-limonene, a hydrocarbon monoterpene also frequently present in EOs, similar to the common mechanism proposed for bactericidal antibiotics. This mechanism involves the formation of Fenton-mediated hydroxyl radical, a reactive oxygen species (ROS), via tricarboxylic acid (TCA) cycle, which would ultimately inactivate cells. Our objective was to determine whether E. coli MG1655 inactivation by citral and carvacrol follows a similar mechanism of cell death. Challenging experiments with 300μL/L citral and 100μL/L carvacrol inactivated at least 2.5log10cycles of exponentially growing cells in 3h under aerobic conditions. The presence of thiourea (an ROS scavenger) reduced cell inactivation in 2log10cycles, demonstrating the role of ROS in cell death. Decreased resistance of a ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) indicated that citral and carvacrol caused oxidative damage to DNA. Although the mechanism of E. coli inactivation by carvacrol and citral was similarly mediated by ROS, their formation did not follow the same pathways described for (+)-limonene and bactericidal drugs because neither Fenton reaction nor NADH production via the TCA cycle was involved in cell death. Moreover, further experiments demonstrated antimicrobial activity of citral and carvacrol in anaerobic environments without the involvement of ROS. As a consequence, cell death by carvacrol and citral in anaerobiosis follows a different mechanism than that observed under aerobic conditions. These results demonstrated a different mechanism of inactivation by citral and carvacrol with regard to (+)-limonene and bactericidal antibiotics, indicating the complexity of the mechanisms of bacterial inactivation among EO constituents. Advancements in the description of these mechanisms will help in extending and improving the use of these compounds as natural antimicrobials. Copyright © 2014 Elsevier B.V. All rights reserved.

Ergosterol Peroxide Isolated from Ganoderma lucidum Abolishes MicroRNA miR-378-Mediated Tumor Cells on Chemoresistance

PubMed Central

Li, Xiang-Min; Yang, Weining; Jiao, Chun-Wei; Fang, Ling; Li, Sen-Zhu; Pan, Hong-Hui; Yee, Albert J.; Lee, Daniel Y.; Li, Chong; Zhang, Zhi; Guo, Jun; Yang, Burton B.

2012-01-01

Due to an altered expression of oncogenic factors and tumor suppressors, aggressive cancer cells have an intrinsic or acquired resistance to chemotherapeutic agents. This typically contributes to cancer recurrence after chemotherapy. microRNAs are short non-coding RNAs that are involved in both cell self-renewal and cancer development. Here we report that tumor cells transfected with miR-378 acquired properties of aggressive cancer cells. Overexpression of miR-378 enhanced both cell survival and colony formation, and contributed to multiple drug resistance. Higher concentrations of chemotherapeutic drugs were needed to induce death of miR-378-transfected cells than to induce death of control cells. We found that the biologically active component isolated from Ganoderma lucidum could overcome the drug-resistance conferred by miR-378. We purified and identified the biologically active component of Ganoderma lucidum as ergosterol peroxide. We demonstrated that ergosterol peroxide produced greater activity in inducing death of miR-378 cells than the GFP cells. Lower concentrations of ergosterol peroxide were needed to induce death of the miR-378-transfected cells than in the control cells. With further clinical development, ergosterol peroxide represents a promising new reagent that can overcome the drug-resistance of tumor cells. PMID:22952996

Knockdown of TWIST1 enhances arsenic trioxide- and ionizing radiation-induced cell death in lung cancer cells by promoting mitochondrial dysfunction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seo, Sung-Keum; Kim, Jae-Hee; Choi, Ha-Na

Highlights: • Knockdown of TWIST1 enhanced ATO- and IR-induced cell death in NSCLCs. • Intracellular ROS levels were increased in cells treated with TWIST1 siRNA. • TWIST1 siRNA induced MMP loss and mitochondrial fragmentation. • TWIST1 siRNA upregulated the fission-related proteins FIS1 and DRP1. - Abstract: TWIST1 is implicated in the process of epithelial mesenchymal transition, metastasis, stemness, and drug resistance in cancer cells, and therefore is a potential target for cancer therapy. In the present study, we found that knockdown of TWIST1 by small interfering RNA (siRNA) enhanced arsenic trioxide (ATO)- and ionizing radiation (IR)-induced cell death in non-small-cellmore » lung cancer cells. Interestingly, intracellular reactive oxygen species levels were increased in cells treated with TWIST1 siRNA and further increased by co-treatment with ATO or IR. Pretreatment of lung cancer cells with the antioxidant N-acetyl-cysteine markedly suppressed the cell death induced by combined treatment with TWIST1 siRNA and ATO or IR. Moreover, treatment of cells with TWIST1 siRNA induced mitochondrial membrane depolarization and significantly increased mitochondrial fragmentation (fission) and upregulated the fission-related proteins FIS1 and DRP1. Collectively, our results demonstrate that siRNA-mediated TWIST1 knockdown induces mitochondrial dysfunction and enhances IR- and ATO-induced cell death in lung cancer cells.« less

Quantitative Differences in a Single Maternal Factor Determine Survival Probabilities among Drosophila Germ Cells.

PubMed

Slaidina, Maija; Lehmann, Ruth

2017-01-23

Germ cell death occurs in many species [1-3] and has been proposed as a mechanism by which the fittest, strongest, or least damaged germ cells are selected for transmission to the next generation. However, little is known about how the choice is made between germ cell survival and death. Here, we focus on the mechanisms that regulate germ cell survival during embryonic development in Drosophila. We find that the decision to die is a germ cell-intrinsic process linked to quantitative differences in germ plasm inheritance, such that higher germ plasm inheritance correlates with higher primordial germ cell (PGC) survival probability. We demonstrate that the maternal factor lipid phosphate phosphatase Wunen-2 (Wun2) regulates PGC survival in a dose-dependent manner. Since wun2 mRNA levels correlate with the levels of other maternal determinants at the single-cell level, we propose that Wun2 is used as a readout of the overall germ plasm quantity, such that only PGCs with the highest germ plasm quantity survive. Furthermore, we demonstrate that Wun2 and p53, another regulator of PGC survival, have opposite yet independent effects on PGC survival. Since p53 regulates cell death upon DNA damage and various cellular stresses, we hypothesize that together they ensure selection of the PGCs with highest germ plasm quantity and least cellular damage. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cisplatin or LA-12 enhance killing effects of TRAIL in prostate cancer cells through Bid-dependent stimulation of mitochondrial apoptotic pathway but not caspase-10

PubMed Central

Herůdková, Jarmila; Krkoška, Martin; Tománková, Silvie; Kahounová, Zuzana; Anděra, Ladislav; Bouchal, Jan; Kharaishvili, Gvantsa; Král, Milan; Sova, Petr; Kozubík, Alois

2017-01-01

Searching for new strategies for effective elimination of human prostate cancer cells, we investigated the cooperative cytotoxic action of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and two platinum-based complexes, cisplatin or LA-12, and related molecular mechanisms. We demonstrated a notable ability of cisplatin or LA-12 to enhance the sensitivity of several human prostate cancer cell lines to TRAIL-induced cell death via an engagement of mitochondrial apoptotic pathway. This was accompanied by augmented Bid cleavage, Bak activation, loss of mitochondrial membrane potential, activation of caspase-8, -10, -9, and -3, and XIAP cleavage. RNAi-mediated silencing of Bid or Bak in Bax-deficient DU 145 cells suppressed the drug combination-induced cytotoxicity, further underscoring the involvement of mitochondrial signaling. The caspase-10 was dispensable for enhancement of cisplatin/LA-12 and TRAIL combination-induced cell death and stimulation of Bid cleavage. Importantly, we newly demonstrated LA-12-mediated enhancement of TRAIL-induced cell death in cancer cells derived from human patient prostate tumor specimens. Our results provide convincing evidence that employing TRAIL combined with cisplatin/LA-12 could contribute to more effective killing of prostate cancer cells compared to the individual action of the drugs, and offer new mechanistic insights into their cooperative anticancer action. PMID:29182622

Adipose Fatty Acid Binding Protein Promotes Saturated Fatty Acid-induced Macrophage Cell Death through Enhancing Ceramide Production

PubMed Central

Zhang, Yuwen; Rao, Enyu; Zeng, Jun; Hao, Jiaqing; Sun, Yanwen; Liu, Shujun; Sauter, Edward R.; Bernlohr, David A.; Cleary, Margot P.; Suttles, Jill; Li, Bing

2016-01-01

Macrophages play a critical role in obesity-associated chronic inflammation and disorders. However, the molecular mechanisms underlying the response of macrophages to elevated fatty acids (FAs) and their contribution to metabolic inflammation in obesity remain to be fully elucidated. Here, we report a new mechanism by which dietary FAs, in particular saturated FAs, are able to directly trigger macrophage cell death. We demonstrated that excess saturated FAs, but not unsaturated FAs, induced the production of cytotoxic ceramides in macrophage cell lines. Most importantly, expression of adipose fatty acid binding protein (A-FABP) in macrophages facilitated metabolism of excess saturated FAs for ceramide synthesis. Inhibition or deficiency of A-FABP in macrophage cell lines decreased saturated FA-induced ceramide production, thereby resulting in reduced cell death. Furthermore, we validated the role of A-FABP in promoting saturated FA-induced macrophage cell death with primary bone-marrow derived macrophages and high-fat diet-induced obese mice. Altogether, our data reveal that excess dietary saturated FAs may serve as direct triggers in induction of ceramide production and macrophage cell death through elevated expression of A-FABP, thus establishing A-FABP as a new molecular sensor in triggering macrophage-associated sterile inflammation in obesity. PMID:27920274

Modulating cell-to-cell variability and sensitivity to death ligands by co-drugging

NASA Astrophysics Data System (ADS)

Flusberg, Deborah A.; Sorger, Peter K.

2013-06-01

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) holds promise as an anti-cancer therapeutic but efficiently induces apoptosis in only a subset of tumor cell lines. Moreover, even in clonal populations of responsive lines, only a fraction of cells dies in response to TRAIL and individual cells exhibit cell-to-cell variability in the timing of cell death. Fractional killing in these cell populations appears to arise not from genetic differences among cells but rather from differences in gene expression states, fluctuations in protein levels and the extent to which TRAIL-induced death or survival pathways become activated. In this study, we ask how cell-to-cell variability manifests in cell types with different sensitivities to TRAIL, as well as how it changes when cells are exposed to combinations of drugs. We show that individual cells that survive treatment with TRAIL can regenerate the sensitivity and death-time distribution of the parental population, demonstrating that fractional killing is a stable property of cell populations. We also show that cell-to-cell variability in the timing and probability of apoptosis in response to treatment can be tuned using combinations of drugs that together increase apoptotic sensitivity compared to treatment with one drug alone. In the case of TRAIL, modulation of cell-to-cell variability by co-drugging appears to involve a reduction in the threshold for mitochondrial outer membrane permeabilization.

Tumour Vascular Shutdown and Cell Death Following Ultrasound-Microbubble Enhanced Radiation Therapy

PubMed Central

El Kaffas, Ahmed; Gangeh, Mehrdad J.; Farhat, Golnaz; Tran, William Tyler; Hashim, Amr; Giles, Anoja; Czarnota, Gregory J.

2018-01-01

High-dose radiotherapy effects are regulated by acute tumour endothelial cell death followed by rapid tumour cell death instead of canonical DNA break damage. Pre-treatment with ultrasound-stimulated microbubbles (USMB) has enabled higher-dose radiation effects with conventional radiation doses. This study aimed to confirm acute and longitudinal relationships between vascular shutdown and tumour cell death following radiation and USMB in a wild type murine fibrosarcoma model using in vivo imaging. Methods: Tumour xenografts were treated with single radiation doses of 2 or 8 Gy alone, or in combination with low-/high-concentration USMB. Vascular changes and tumour cell death were evaluated at 3, 24 and 72 h following therapy, using high-frequency 3D power Doppler and quantitative ultrasound spectroscopy (QUS) methods, respectively. Staining using in situ end labelling (ISEL) and cluster of differentiation 31 (CD31) of tumour sections were used to assess cell death and vascular distributions, respectively, as gold standard histological methods. Results: Results indicated a decrease in the power Doppler signal of up to 50%, and an increase of more than 5 dBr in cell-death linked QUS parameters at 24 h for tumours treated with combined USMB and radiotherapy. Power Doppler and quantitative ultrasound results were significantly correlated with CD31 and ISEL staining results (p < 0.05), respectively. Moreover, a relationship was found between ultrasound power Doppler and QUS results, as well as between micro-vascular densities (CD31) and the percentage of cell death (ISEL) (R2 0.5-0.9). Conclusions: This study demonstrated, for the first time, the link between acute vascular shutdown and acute tumour cell death using in vivo longitudinal imaging, contributing to the development of theoretical models that incorporate vascular effects in radiation therapy. Overall, this study paves the way for theranostic use of ultrasound in radiation oncology as a diagnostic modality to characterize vascular and tumour response effects simultaneously, as well as a therapeutic modality to complement radiation therapy. PMID:29290810

Expression of animal anti-apoptotic gene Ced-9 enhances tolerance during Glycine max L.-Bradyrhizobium japonicum interaction under saline stress but reduces nodule formation.

PubMed

Robert, Germán; Muñoz, Nacira; Melchiorre, Mariana; Sánchez, Federico; Lascano, Ramiro

2014-01-01

The mechanisms by which the expression of animal cell death suppressors in economically important plants conferred enhanced stress tolerance are not fully understood. In the present work, the effect of expression of animal antiapoptotic gene Ced-9 in soybean hairy roots was evaluated under root hairs and hairy roots death-inducing stress conditions given by i) Bradyrhizobium japonicum inoculation in presence of 50 mM NaCl, and ii) severe salt stress (150 mM NaCl), for 30 min and 3 h, respectively. We have determined that root hairs death induced by inoculation in presence of 50 mM NaCl showed characteristics of ordered process, with increased ROS generation, MDA and ATP levels, whereas the cell death induced by 150 mM NaCl treatment showed non-ordered or necrotic-like characteristics. The expression of Ced-9 inhibited or at least delayed root hairs death under these treatments. Hairy roots expressing Ced-9 had better homeostasis maintenance, preventing potassium release; increasing the ATP levels and controlling the oxidative damage avoiding the increase of reactive oxygen species production. Even when our results demonstrate a positive effect of animal cell death suppressors in plant cell ionic and redox homeostasis under cell death-inducing conditions, its expression, contrary to expectations, drastically inhibited nodule formation even under control conditions.

Analysis of Early Death in Japanese Patients With Advanced Non-small-cell Lung Cancer Treated With Nivolumab.

PubMed

Inoue, Takako; Tamiya, Motohiro; Tamiya, Akihiro; Nakahama, Kenji; Taniguchi, Yoshihiko; Shiroyama, Takayuki; Isa, Shin-Ichi; Nishino, Kazumi; Kumagai, Toru; Kunimasa, Kei; Kimura, Madoka; Suzuki, Hidekazu; Hirashima, Tomonori; Atagi, Shinji; Imamura, Fumio

2018-03-01

The increased risk for early death owing to anti-programmed cell death 1 inhibitors is a major disadvantage that requires special management. We evaluated the frequency, causes, and risk factors of early death during nivolumab treatment for non-small cell lung cancer (NSCLC) in a Japanese clinical setting. The medical records of patients with NSCLC who started receiving nivolumab between December 17, 2015 and July 31, 2016 in 3 Japanese institutes were collected. Early death was defined as any death within 3 months from the start of nivolumab treatment, irrespective of its cause. Treatment response was evaluated using the Response Evaluation Criteria In Solid Tumors criteria, version 1.1. A total of 201 patients with NSCLC were enrolled, and 38 (18.9%) died within the first 3 months. Thirty-one (81.6%) patients who experienced early death developed progressive disease, whereas 14 (36.8%) patients who experienced early death demonstrated nivolumab-induced immune-related adverse events, which required corticosteroid intervention, including interstitial lung disease in 7 (18.4%) patients. Multivariate logistic regression demonstrated that an Eastern Cooperative Oncology Group performance status score ≥ 2 (odds ratio [OR], 5.66; 95% confidence interval [CI], 2.01-15.61; P < .001), C-reactive protein-to-albumin ratio > 0.3 (OR, 10.56; 95% CI, 3.61-30.86; P < .001), and the response to prior treatment (OR, 2.07; 95% CI, 1.03-4.14; P = .041) were independent predictors for early death. Disease progression and immune-related adverse events are 2 major causes of early death with nivolumab in patients with NSCLC. An Eastern Cooperative Oncology Group performance status score ≥ 2, pretreatment C-reactive protein-to-albumin ratio > 0.3, and poor response to prior treatment were associated with early death. Copyright © 2017 Elsevier Inc. All rights reserved.

A simple and powerful approach for isolation of Arabidopsis mutants with increased tolerance to H2O2-induced cell death.

PubMed

Gechev, Tsanko; Mehterov, Nikolay; Denev, Iliya; Hille, Jacques

2013-01-01

A genetic approach is described to isolate mutants more tolerant to oxidative stress. A collection of T-DNA activation tag Arabidopsis thaliana mutant lines was screened for survivors under conditions that trigger H2O2-induced cell death. Oxidative stress was induced by applying the catalase (CAT) inhibitor aminotriazole (AT) in the growth media, which results in decrease in CAT enzyme activity, H2O2 accumulation, and subsequent plant death. One mutant was recovered from the screening and named oxr1 (oxidative stress resistant 1). The location of the T-DNA insertion was identified by TAIL-PCR. Oxr1 exhibited lack of cell death symptoms and more fresh weight and chlorophyll content compared to wild type. The lack of cell death correlated with more prominent induction of anthocyanins synthesis in oxr1. These results demonstrate the feasibility of AT as a screening agent for the isolation of oxidative stress-tolerant mutants and indicate a possible protective role for anthocyanins against AT-induced cell death. The chapter includes protocols for ethyl methanesulfonate mutagenesis, mutant screening using AT, T-DNA identification by TAIL-PCR, CAT activity measurements, and determination of malondialdehyde, chlorophyll, and anthocyanins. Copyright © 2013 Elsevier Inc. All rights reserved.

PI3K and Inhibitor of Apoptosis Proteins Modulate Gentamicin- Induced Hair Cell Death in the Zebrafish Lateral Line

PubMed Central

Wiedenhoft, Heather; Hayashi, Lauren; Coffin, Allison B.

2017-01-01

Inner ear hair cell death leads to sensorineural hearing loss and can be a direct consequence of aminoglycoside antibiotic treatment. Aminoglycosides such as gentamicin are effective therapy for serious Gram-negative bacterial infections such as some forms of meningitis, pneumonia, and sepsis. Aminoglycosides enter hair cells through mechanotransduction channels at the apical end of hair bundles and initiate intrinsic cell death cascades, but the precise cell signaling that leads to hair cell death is incompletely understood. Here, we examine the cell death pathways involved in aminoglycoside damage using the zebrafish (Danio rerio). The zebrafish lateral line contains hair cell-bearing organs called neuromasts that are homologous to hair cells of the mammalian inner ear and represents an excellent model to study ototoxicity. Based on previous research demonstrating a role for p53, Bcl2 signaling, autophagy, and proteasomal degradation in aminoglycoside-damaged hair cells, we used the Cytoscape GeneMANIA Database to identify additional proteins that might play a role in neomycin or gentamicin ototoxicity. Our bioinformatics analysis identified the pro-survival proteins phosphoinositide-dependent kinase-1 (PDK1) and X-linked inhibitor of apoptosis protein (Xiap) as potential mediators of gentamicin-induced hair cell damage. Pharmacological inhibition of PDK1 or its downstream mediator protein kinase C facilitated gentamicin toxicity, as did Xiap mutation, suggesting that both PI3K and endogenous Xiap confer protection. Surprisingly, aminoglycoside-induced hair cell death was highly attenuated in wild type Tupfel long-fin (TL fish; the background strain for the Xiap mutant line) compared to wild type ∗AB zebrafish. Pharmacologic manipulation of p53 suggested that the strain difference might result from decreased p53 in TL hair cells, allowing for increased hair cell survival. Overall, our studies identified additional steps in the cell death cascade triggered by aminoglycoside damage, suggesting possible drug targets to combat hearing loss resulting from aminoglycoside exposure. PMID:29093665

Ursodeoxycholic acid effectively kills drug-resistant gastric cancer cells through induction of autophagic death.

PubMed

Lim, Sung-Chul; Han, Song Iy

2015-09-01

Carcinoma cells that have acquired drug resistance often exhibit cross-resistance to various other cytotoxic stimuli. Here, we investigated the effects of ursodeoxycholic acid (UDCA), a gastrointestinal tumor-suppressor, on a cisplatin‑resistant SNU601 gastric cancer subline (SNU601/R). While other anticancer drugs, including L-OHP, etoposide, and death ligand TRAIL, had minimal effects on the viability of these resistant cells, they were sensitive to UDCA. The UDCA‑induced reduction in the viability of the SNU601/R cells was accomplished through autophagy while the primary means of cell death in the parental SNU601 cells (SNU601/WT) was apoptosis. Previously, we demonstrated that the UDCA-triggered apoptosis of gastric cancer cells was regulated by a cell surface death receptor, TRAIL-R2/DR5, which was upregulated and re-distributed on lipid rafts. The UDCA stimulation of TRAIL-R2/DR5 also occurred in the SNU601/R cells despite the lack of apoptosis. In the present study, we found that CD95/Fas, another cell surface death receptor, was also translocated into lipid rafts in response to UDCA although it was not involved in the decrease in cell viability. Specifically, raft relocalization of CD95/Fas was triggered by UDCA in the SNU601/WT cells in which apoptosis occurred, but not in the SNU601/R cells where autophagic death occurred. Notably, UDCA reduced ATG5 levels, an essential component of autophagy, in the SNU601/WT, but not in the SNU601/R cell line. Moreover, in CD95/Fas-silenced SNU601/WT cells, UDCA did not decrease ATG5 levels and induced autophagic cell death rather than apoptosis. These results imply that raft‑distributed CD95/Fas may support UDCA-induced apoptosis via downregulation of ATG5 levels, preventing the autophagic pathway. Taken together, these results suggest that UDCA induces both apoptotic and autophagic cell death depending on the intracellular signaling environment, thereby conferring the advantage to overcome drug resistance through apoptotic defects.

Bok Is Not Pro-Apoptotic But Suppresses Poly ADP-Ribose Polymerase-Dependent Cell Death Pathways and Protects against Excitotoxic and Seizure-Induced Neuronal Injury.

PubMed

D'Orsi, Beatrice; Engel, Tobias; Pfeiffer, Shona; Nandi, Saheli; Kaufmann, Thomas; Henshall, David C; Prehn, Jochen H M

2016-04-20

Bok (Bcl-2-related ovarian killer) is a Bcl-2 family member that, because of its predicted structural homology to Bax and Bak, has been proposed to be a pro-apoptotic protein. In this study, we demonstrate that Bok is highly expressed in neurons of the mouse brain but that bok was not required for staurosporine-, proteasome inhibition-, or excitotoxicity-induced apoptosis of cultured cortical neurons. On the contrary, we found that bok-deficient neurons were more sensitive to oxygen/glucose deprivation-induced injury in vitro and seizure-induced neuronal injury in vivo Deletion of bok also increased staurosporine-, excitotoxicity-, and oxygen/glucose deprivation-induced cell death in bax-deficient neurons. Single-cell imaging demonstrated that bok-deficient neurons failed to maintain their neuronal Ca(2+)homeostasis in response to an excitotoxic stimulus; this was accompanied by a prolonged deregulation of mitochondrial bioenergetics.bok deficiency led to a specific reduction in neuronal Mcl-1 protein levels, and deregulation of both mitochondrial bioenergetics and Ca(2+)homeostasis was rescued by Mcl-1 overexpression. Detailed analysis of cell death pathways demonstrated the activation of poly ADP-ribose polymerase-dependent cell death in bok-deficient neurons. Collectively, our data demonstrate that Bok acts as a neuroprotective factor rather than a pro-death effector during Ca(2+)- and seizure-induced neuronal injury in vitro and in vivo Bcl-2 proteins are essential regulators of the mitochondrial apoptosis pathway. The Bcl-2 protein Bok is highly expressed in the CNS. Because of its sequence similarity to Bax and Bak, Bok has long been considered part of the pro-apoptotic Bax-like subfamily, but no studies have yet been performed in neurons to test this hypothesis. Our study provides important new insights into the functional role of Bok during neuronal apoptosis and specifically in the setting of Ca(2+)- and seizure-mediated neuronal injury. We show that Bok controls neuronal Ca(2+)homeostasis and bioenergetics and, contrary to previous assumptions, exerts neuroprotective activities in vitro and in vivo Our results demonstrate that Bok cannot be placed unambiguously into the Bax-like Bcl-2 subfamily of pro-apoptotic proteins. Copyright © 2016 the authors 0270-6474/16/364564-15$15.00/0.

Induction of non-apoptotic cell death by morphinone in human promyelocytic leukemia HL-60 cells.

PubMed

Takeuchi, Risa; Hoshijima, Hiroshi; Nagasaka, Hiroshi; Chowdhury, Shahead Ali; Kikuchi, Hirotaka; Kanda, Yumiko; Kunii, Shiro; Kawase, Masami; Sakagami, Hiroshi

2006-01-01

As previously suggested, codeinone (oxidation product of codeine) induces non-apoptotic cell death, characterized by marginal caspase activation and the lack of DNA fragmentation in HL-60 human promyelocytic leukemia cells, which was inhibited by N-acetyl-L-cysteine. Whether, morphinone, an oxidative metabolite of morphine, also induced a similar type of cell death in HL-60 cells was investigated. Morphinone showed slightly higher cytotoxic activity against human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, HSC-4, NA, Ca9-22, promyelocytic leukemia HL-60, cervical carcinoma HeLa) than against normal oral human cells (gingival fibroblast HGF, pulp cells HPC, periodontal ligament fibroblast HPLF). Morphinone also induced an almost undetectable level of internucleosomal DNA fragmentation in the HL-60 cells. Morphinone did not activate caspase-8 or -9 in these cells. Morphinone dose-dependently activated caspase-3 in both HL-60 and HSC-2 cell lines, but to a much lesser extent than actinomycin D. Electron microscopy demonstrated that morphinone induced mitochondrial shrinkage, vacuolization and production of autophagosome and the loss of cell surface microvilli, without destruction of cell surface and nuclear membranes in the HL-60 cells. The autophagy inhibitor 3-methyladenine (0.3-10 mM) slightly inhibited the morphinone-induced cytotoxicity, when corrected for its own cytotoxicity. These data suggest that morphinone induces non-apoptotic cell death in HL-60 cells.

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

PubMed Central

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

Stathmin Mediates Hepatocyte Resistance to Death from Oxidative Stress by down Regulating JNK

PubMed Central

Zhao, Enpeng; Amir, Muhammad; Lin, Yu; Czaja, Mark J.

2014-01-01

Stathmin 1 performs a critical function in cell proliferation by regulating microtubule polymerization. This proliferative function is thought to explain the frequent overexpression of stathmin in human cancer and its correlation with a bad prognosis. Whether stathmin also functions in cell death pathways is unclear. Stathmin regulates microtubules in part by binding free tubulin, a process inhibited by stathmin phosphorylation from kinases including c-Jun N-terminal kinase (JNK). The involvement of JNK activation both in stathmin phosphorylation, and in hepatocellular resistance to oxidative stress, led to an examination of the role of stathmin/JNK crosstalk in oxidant-induced hepatocyte death. Oxidative stress from menadione-generated superoxide induced JNK-dependent stathmin phosphorylation at Ser-16, Ser-25 and Ser-38 in hepatocytes. A stathmin knockdown sensitized hepatocytes to both apoptotic and necrotic cell death from menadione without altering levels of oxidant generation. The absence of stathmin during oxidative stress led to JNK overactivation that was the mechanism of cell death as a concomitant knockdown of JNK1 or JNK2 blocked death. Hepatocyte death from JNK overactivation was mediated by the effects of JNK on mitochondria. Mitochondrial outer membrane permeabilization occurred in stathmin knockdown cells at low concentrations of menadione that triggered apoptosis, whereas mitochondrial β-oxidation and ATP homeostasis were compromised at higher, necrotic menadione concentrations. Stathmin therefore mediates hepatocyte resistance to death from oxidative stress by down regulating JNK and maintaining mitochondrial integrity. These findings demonstrate a new mechanism by which stathmin promotes cell survival and potentially tumor growth. PMID:25285524

Combined effects of starvation and butyrate on autophagy-dependent gingival epithelial cell death.

PubMed

Evans, M; Murofushi, T; Tsuda, H; Mikami, Y; Zhao, N; Ochiai, K; Kurita-Ochiai, T; Yamamoto, M; Otsuka, K; Suzuki, N

2017-06-01

Bacteria in the dental biofilm surrounding marginal gingival grooves cause periodontal diseases. Numerous bacteria within the biofilm consume nutrients from the gingival crevicular fluid. Furthermore, some gram-negative bacteria in mature dental biofilms produce butyrate. Thus, gingival epithelial cells in close proximity to mature dental biofilms are at risk of both starvation and exposure to butyrate. In the present study, we determined the combined effects of starvation and butyrate exposure on gingival epithelial cell death and the underlying mechanisms. The Ca9-22 cell line was used as an in vitro counterpart of gingival epithelial cells. Cell death was measured as the amount of total DNA in the dead cells using SYTOX Green dye, which penetrates through membranes of dead cells and emits fluorescence when it intercalates into double-stranded DNA. AMP-activated protein kinase (AMPK) activity, the amount of autophagy, and acetylation of histone H3 were determined using western blot. Gene expression levels of microtubule-associated protein 1 light chain 3b (lc3b) were determined using quantitative reverse transcription-polymerase chain reaction. Butyrate-induced cell death occurred in a dose-dependent manner whether cells were starved or fed. However, the induction of cell death was two to four times higher when cells were placed under starvation conditions compared to when they were fed. Moreover, both starvation and butyrate exposure induced AMPK activity and autophagy. While AMPK inactivation resulted in decreased autophagy and butyrate-induced cell death under conditions of starvation, AMPK activation resulted in butyrate-induced cell death when cells were fed. Combined with the results of our previous report, which demonstrated butyrate-induced autophagy-dependent cell death, the results of this study suggest that the combination of starvation and butyrate exposure activates AMPK inducing autophagy and subsequent cell death. Notably, this combination markedly induced LC3B production and the induction was attenuated by AMPK inhibition. LC3B knockdown, in turn, significantly decreased butyrate-induced cell death. Therefore, AMPK-dependent LC3B induction apparently plays an important role in butyrate-induced cell death. There was a lack of correspondence between the levels of AMPK activation and LC3B induction; this may reflect the histone deacetylase-inhibitory capacity of butyrate on histone proteins. Taken together, starvation and butyrate exposure promote autophagy via AMPK signaling, while the histone deacetylase-inhibitory effects of butyrate alter chromatin to transcriptionally active state, resulting in strong LC3B induction and subsequent cell death. These findings may help improve the understanding of the cellular processes underlying periodontal disease initiation. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Curcumin Induces Pancreatic Adenocarcinoma Cell Death via Reduction of the Inhibitors of Apoptosis

PubMed Central

Osterman, Carlos J. Díaz; Gonda, Amber; Stiff, TessaRae; Sigaran, Ulysses; Valenzuela, Malyn May Asuncion; Bennit, Heather R. Ferguson; Moyron, Ron B.; Khan, Salma; Wall, Nathan R.

2015-01-01

Objectives The inhibitor of apoptosis (IAP) proteins are critical modulators of chemotherapeutic resistance in various cancers. To address the alarming emergence of chemotherapeutic resistance in pancreatic cancer, we investigated the efficacy of the turmeric derivative curcumin in reducing IAP protein and mRNA expression resulting in pancreatic cancer cell death. Methods The pancreatic adenocarcinoma cell line PANC-1 was used to assess curcumin’s effects in pancreatic cancer. Curcumin uptake was measured by spectral analysis and fluorescence microscopy. AlamarBlue and Trypan blue exclusion assays were used to determine PANC-1 cell viability following curcumin treatment. Visualization of PANC-1 cell death was performed using Hoffman Modulation Contrast microscopy. Western blot and PCR analyses were used to evaluate curcumin’s effects on IAP protein and mRNA expression. Results Curcumin enters PANC-1 cells and is ubiquitously present within the cell following treatment. Furthermore, curcumin reduces cell viability and induces morphological changes characteristic of cell death. Additionally, curcumin decreases IAP protein and mRNA expression in PANC-1 cells. Conclusions These data demonstrate that PANC-1 cells are sensitive to curcumin treatment. Furthermore, curcumin as a potential therapeutic tool for overcoming chemotherapeutic resistance mediated by IAPs, supports a role for curcumin as part of the therapeutic approach for pancreatic cancer. PMID:26348467

Recombinant Vaccinia Viruses Coding Transgenes of Apoptosis-Inducing Proteins Enhance Apoptosis But Not Immunogenicity of Infected Tumor Cells

PubMed Central

Tkachenko, Anastasiya; Richter, Vladimir

2017-01-01

Genetic modifications of the oncolytic vaccinia virus (VV) improve selective tumor cell infection and death, as well as activation of antitumor immunity. We have engineered a double recombinant VV, coding human GM-CSF, and apoptosis-inducing protein apoptin (VV-GMCSF-Apo) for comparing with the earlier constructed double recombinant VV-GMCSF-Lact, coding another apoptosis-inducing protein, lactaptin, which activated different cell death pathways than apoptin. We showed that both these recombinant VVs more considerably activated a set of critical apoptosis markers in infected cells than the recombinant VV coding GM-CSF alone (VV-GMCSF-dGF): these were phosphatidylserine externalization, caspase-3 and caspase-7 activation, DNA fragmentation, and upregulation of proapoptotic protein BAX. However, only VV-GMCSF-Lact efficiently decreased the mitochondrial membrane potential of infected cancer cells. Investigating immunogenic cell death markers in cancer cells infected with recombinant VVs, we demonstrated that all tested recombinant VVs were efficient in calreticulin and HSP70 externalization, decrease of cellular HMGB1, and ATP secretion. The comparison of antitumor activity against advanced MDA-MB-231 tumor revealed that both recombinants VV-GMCSF-Lact and VV-GMCSF-Apo efficiently delay tumor growth. Our results demonstrate that the composition of GM-CSF and apoptosis-inducing proteins in the VV genome is very efficient tool for specific killing of cancer cells and for activation of antitumor immunity. PMID:28951871

Mouse lung fibroblasts are highly susceptible to necroptosis in a reactive oxygen species-dependent manner.

PubMed

Hussain, Muadh; Zimmermann, Vanessa; van Wijk, Sjoerd J L; Fulda, Simone

2018-07-01

Mouse embryonic fibroblasts (MEFs) have extensively been used to study necroptosis, a recently identified form of programmed cell death. However, very little is yet known about the role of necroptosis and its regulation by reactive oxygen species (ROS) in cell types naturally exposed to high oxygen levels such as mouse lung fibroblasts (MLFs). Here, we discover that MLFs are highly susceptible to undergo necroptosis in a ROS-dependent manner upon exposure to a prototypic death receptor-mediated necroptotic stimulus, i.e. cotreatment with tumor necrosis factor (TNF)α, Smac mimetic and the caspase inhibitor zVAD.fmk (TSZ). Kinetic analysis revealed that TSZ rapidly induces cell death in MLFs. Pharmacological inhibition of receptor-interacting protein kinase (RIPK)1 by necrostatin-1 (Nec-1) or RIPK3 by GSK'872 significantly rescues TSZ-stimulated cell death. Also, genetic silencing of RIPK3 or mixed lineage kinase domain-like pseudokinase (MLKL) significantly protects MLFs from TSZ-mediated cell death. Prior to cell death, TSZ significantly increases production of ROS. Importantly, addition of radical scavengers such as butylated hydroxyanisole (BHA) or α-Tocopherol (α-Toc) significantly suppresses TSZ-induced cell death in parallel with a significant reduction of ROS generation. Consistently, BHA prevented TSZ-triggered phosphorylation of MLKL similar to the addition of GSK'872. Thus, our study demonstrates for the first time that MLFs are prone to undergo necroptosis in response to a prototypic necroptotic stimulus and identifies ROS as important mediators of TSZ-triggered necroptosis. Copyright © 2018 Elsevier Inc. All rights reserved.

Fluoxetine Prevents Oligodendrocyte Cell Death by Inhibiting Microglia Activation after Spinal Cord Injury

PubMed Central

Lee, Jee Y.; Kang, So R.

2015-01-01

Abstract Oligodendrocyte cell death and axon demyelination after spinal cord injury (SCI) are known to be important secondary injuries contributing to permanent neurological disability. Thus, blocking oligodendrocyte cell death should be considered for therapeutic intervention after SCI. Here, we demonstrated that fluoxetine, an antidepressant drug, alleviates oligodendrocyte cell death by inhibiting microglia activation after SCI. After injury at the T9 level with a Precision Systems and Instrumentation (Lexington, KY) device, fluoxetine (10 mg/kg, intraperitoneal) was administered once a day for the indicated time points. Immunostaining with CD11b (OX-42) antibody and quantification analysis showed that microglia activation was significantly inhibited by fluoxetine at 5 days after injury. Fluoxetine also significantly inhibited activation of p38 mitogen-activated protein kinase (p38-MAPK) and expression of pro-nerve growth factor (pro-NGF), which is known to mediate oligodendrocyte cell death through the p75 neurotrophin receptor after SCI. In addition, fluoxetine attenuated activation of Ras homolog gene family member A and decreased the level of phosphorylated c-Jun and, ultimately, alleviated caspase-3 activation and significantly reduced cell death of oligodendrocytes at 5 days after SCI. Further, the decrease of myelin basic protein, myelin loss, and axon loss in white matter was also significantly blocked by fluoxetine, as compared to vehicle control. These results suggest that fluoxetine inhibits oligodendrocyte cell death by inhibiting microglia activation and p38-MAPK activation, followed by pro-NGF production after SCI, and provide a potential usage of fluoxetine for a therapeutic agent after acute SCI in humans. PMID:25366938

Autophagy protein p62/SQSTM1 is involved in HAMLET-induced cell death by modulating apotosis in U87MG cells

PubMed Central

Zhang, Y-B; Gong, J-L; Xing, T-Y; Zheng, S-P; Ding, W

2013-01-01

HAMLET is a complex of oleic acids and decalcified α-lactalbumin that was discovered to selectively kill tumor cells both in vitro and in vivo. Autophagy is an important cellular process involved in drug-induced cell death of glioma cells. We treated U87MG human glioma cells with HAMLET and found that the cell viability was significantly decreased and accompanied with the activation of autophagy. Interestingly, we observed an increase in p62/SQSTM1, an important substrate of autophagosome enzymes, at the protein level upon HAMLET treatment for short periods. To better understand the functionality of autophagy and p62/SQSTM1 in HAMLET-induced cell death, we modulated the level of autophagy or p62/SQSTM1 with biochemical or genetic methods. The results showed that inhibition of autophagy aggravated HAMLET-induced cell death, whereas activation of authophagy attenuated this process. Meanwhile, we found that overexpression of wild-type p62/SQSTM1 was able to activate caspase-8, and then promote HAMLET-induced apoptosis, whereas knockdown of p62/SQSTM1 manifested the opposite effect. We further demonstrated that the function of p62/SQSTM1 following HAMLET treatment required its C-terminus UBA domain. Our results indicated that in addition to being a marker of autophagy activation in HAMLET-treated glioma cells, p62/SQSTM1 could also function as an important mediator for the activation of caspase-8-dependent cell death. PMID:23519119

Autophagy protein p62/SQSTM1 is involved in HAMLET-induced cell death by modulating apotosis in U87MG cells.

PubMed

Zhang, Y-B; Gong, J-L; Xing, T-Y; Zheng, S-P; Ding, W

2013-03-21

HAMLET is a complex of oleic acids and decalcified α-lactalbumin that was discovered to selectively kill tumor cells both in vitro and in vivo. Autophagy is an important cellular process involved in drug-induced cell death of glioma cells. We treated U87MG human glioma cells with HAMLET and found that the cell viability was significantly decreased and accompanied with the activation of autophagy. Interestingly, we observed an increase in p62/SQSTM1, an important substrate of autophagosome enzymes, at the protein level upon HAMLET treatment for short periods. To better understand the functionality of autophagy and p62/SQSTM1 in HAMLET-induced cell death, we modulated the level of autophagy or p62/SQSTM1 with biochemical or genetic methods. The results showed that inhibition of autophagy aggravated HAMLET-induced cell death, whereas activation of authophagy attenuated this process. Meanwhile, we found that overexpression of wild-type p62/SQSTM1 was able to activate caspase-8, and then promote HAMLET-induced apoptosis, whereas knockdown of p62/SQSTM1 manifested the opposite effect. We further demonstrated that the function of p62/SQSTM1 following HAMLET treatment required its C-terminus UBA domain. Our results indicated that in addition to being a marker of autophagy activation in HAMLET-treated glioma cells, p62/SQSTM1 could also function as an important mediator for the activation of caspase-8-dependent cell death.

Quercetin induced apoptosis of human oral cancer SAS cells through mitochondria and endoplasmic reticulum mediated signaling pathways.

PubMed

Ma, Yi-Shih; Yao, Chien-Ning; Liu, Hsin-Chung; Yu, Fu-Shun; Lin, Jen-Jyh; Lu, Kung-Wen; Liao, Ching-Lung; Chueh, Fu-Shin; Chung, Jing-Gung

2018-06-01

Oral cancer is a cause of cancer-associated mortality worldwide and the treatment of oral cancer includes radiation, surgery and chemotherapy. Quercetin is a component from natural plant products and it has been demonstrated that quercetin is able to induce cytotoxic effects through induction of cell apoptosis in a number of human cancer cell lines. However, there is no available information to demonstrate that quercetin is able to induce apoptosis in human oral cancer cells. In the present study, the effect of quercetin on the cell death via the induction of apoptosis in human oral cancer SAS cells was investigated using flow cytometry, Annexin V/propidium iodide (PI) double staining, western blotting and confocal laser microscopy examination, to test for cytotoxic effects at 6-48 h after treatment with quercetin. The rate of cell death increased with the duration of quercetin treatment based on the results of a cell viability assay, increased Annexin V/PI staining, increased reactive oxygen species and Ca 2+ production, decreased the levels of mitochondrial membrane potential (ΔΨ m ), increased proportion of apoptotic cells and altered levels of apoptosis-associated protein expression in SAS cells. The results from western blotting revealed that quercetin increased Fas, Fas-Ligand, fas-associated protein with death domain and caspase-8, all of which associated with cell surface death receptor. Furthermore, quercetin increased the levels of activating transcription factor (ATF)-6α, ATF-6β and gastrin-releasing peptide-78 which indicated an increase in endoplasm reticulum stress, increased levels of the pro-apoptotic protein BH3 interacting-domain death antagonist, and decreased levels of anti-apoptotic proteins B-cell lymphoma (Bcl) 2 and Bcl-extra large which may have led to the decreases of ΔΨ m . Additionally, confocal microscopy suggested that quercetin was able to increase the expression levels of cytochrome c , apoptosis-inducing factor and endonuclease G, which are associated with apoptotic pathways. Therefore, it is hypothesized that quercetin may potentially be used as a novel anti-cancer agent for the treatment of oral cancer in future.

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.

PubMed

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.

Akt-phosphorylated Mitogen-activated Kinase-activating Death Domain Protein (MADD) Inhibits TRAIL-induced Apoptosis by Blocking Fas-associated Death Domain (FADD) Association with Death Receptor 4*

PubMed Central

Li, Peifeng; Jayarama, Shankar; Ganesh, Lakshmy; Mordi, David; Carr, Ryan; Kanteti, Prasad; Hay, Nissim; Prabhakar, Bellur S.

2010-01-01

MADD plays an essential role in cancer cell survival. Abrogation of endogenous MADD expression results in significant spontaneous apoptosis and enhanced susceptibility to tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, the regulation of MADD function is largely unknown. Here, we demonstrate that endogenous MADD is phosphorylated at three highly conserved sites by Akt, and only the phosphorylated MADD can directly interact with the TRAIL receptor DR4 thereby preventing Fas-associated death domain recruitment. However, in cells susceptible to TRAIL treatment, TRAIL induces a reduction in MADD phosphorylation levels resulting in MADD dissociation from, and Fas-associated death domain association with DR4, which allows death-inducing signaling complex (DISC) formation leading to apoptosis. Thus, the pro-survival function of MADD is dependent upon its phosphorylation by Akt. Because Akt is active in most cancer cells and phosphorylated MADD confers resistance to TRAIL-induced apoptosis, co-targeting Akt-MADD axis is likely to increase efficacy of TRAIL-based therapies. PMID:20484047

Apoptosis is an innate defense function of macrophages against Mycobacterium tuberculosis

PubMed Central

Behar, SM; Martin, CJ; Booty, MG; Nishimura, T; Zhao, X; Gan, H; Divangahi, M; Remold, HG

2011-01-01

Two different forms of death are commonly observed when Mycobacterium tuberculosis (Mtb)-infected macrophages die: (i) necrosis, a death modality defined by cell lysis and (ii) apoptosis, a form of death that maintains an intact plasma membrane. Necrosis is a mechanism used by bacteria to exit the macrophage, evade host defenses, and spread. In contrast, apoptosis of infected macrophages is associated with diminished pathogen viability. Apoptosis occurs when tumor necrosis factor activates the extrinsic death domain pathway, leading to caspase-8 activation. In addition, mitochondrial outer membrane permeabilization leading to activation of the intrinsic apoptotic pathway is required. Both pathways lead to caspase-3 activation, which results in apoptosis. We have recently demonstrated that during mycobacterial infection, cell death is regulated by the eicosanoids, prostaglandin E2 (proapoptotic) and lipoxin (LX)A4 (pronecrotic). Although PGE2 protects against necrosis, virulent Mtb induces LXA4 and inhibits PGE2 production. Under such conditions, mitochondrial inner membrane damage leads to macrophage necrosis. Thus, virulent Mtb subverts eicosanoid regulation of cell death to foil innate defense mechanisms of the macrophage. PMID:21307848

Apoptosis is an innate defense function of macrophages against Mycobacterium tuberculosis.

PubMed

Behar, S M; Martin, C J; Booty, M G; Nishimura, T; Zhao, X; Gan, H-X; Divangahi, M; Remold, H G

2011-05-01

Two different forms of death are commonly observed when Mycobacterium tuberculosis (Mtb)-infected macrophages die: (i) necrosis, a death modality defined by cell lysis and (ii) apoptosis, a form of death that maintains an intact plasma membrane. Necrosis is a mechanism used by bacteria to exit the macrophage, evade host defenses, and spread. In contrast, apoptosis of infected macrophages is associated with diminished pathogen viability. Apoptosis occurs when tumor necrosis factor activates the extrinsic death domain pathway, leading to caspase-8 activation. In addition, mitochondrial outer membrane permeabilization leading to activation of the intrinsic apoptotic pathway is required. Both pathways lead to caspase-3 activation, which results in apoptosis. We have recently demonstrated that during mycobacterial infection, cell death is regulated by the eicosanoids, prostaglandin E(2) (proapoptotic) and lipoxin (LX)A(4) (pronecrotic). Although PGE(2) protects against necrosis, virulent Mtb induces LXA(4) and inhibits PGE(2) production. Under such conditions, mitochondrial inner membrane damage leads to macrophage necrosis. Thus, virulent Mtb subverts eicosanoid regulation of cell death to foil innate defense mechanisms of the macrophage.

Programmed cell death during development of cowpea (Vigna unguiculata (L.) Walp.) seed coat.

PubMed

Lima, Nathália Bastos; Trindade, Fernanda Gomes; da Cunha, Maura; Oliveira, Antônia Elenir Amâncio; Topping, Jennifer; Lindsey, Keith; Fernandes, Kátia Valevski Sales

2015-04-01

The seed coat develops primarily from maternal tissues and comprises multiple cell layers at maturity, providing a metabolically dynamic interface between the developing embryo and the environment during embryogenesis, dormancy and germination of seeds. Seed coat development involves dramatic cellular changes, and the aim of this research was to investigate the role of programmed cell death (PCD) events during the development of seed coats of cowpea [Vigna unguiculata (L.) Walp.]. We demonstrate that cells of the developing cowpea seed coats undergo a programme of autolytic cell death, detected as cellular morphological changes in nuclei, mitochondria, chloroplasts and vacuoles, DNA fragmentation and oligonucleosome accumulation in the cytoplasm, and loss of membrane viability. We show for the first time that classes 6 and 8 caspase-like enzymes are active during seed coat development, and that these activities may be compartmentalized by translocation between vacuoles and cytoplasm during PCD events. © 2014 John Wiley & Sons Ltd.

Cytosolic Irradiation of Femtosecond Laser Induces Mitochondria-dependent Apoptosis-like Cell Death via Intrinsic Reactive Oxygen Cascades

PubMed Central

Yoon, Jonghee; Ryu, Seung-wook; Lee, Seunghee; Choi, Chulhee

2015-01-01

High-intensity femtosecond lasers have recently been used to irreversibly disrupt nanoscale structures, such as intracellular organelles, and to modify biological functions in a reversible manner: so-called nanosurgery and biophotomodulation. Femtosecond laser pulses above the threshold intensity sufficient for reversible biophotomodulation can cause irreversible changes in the irradiated cell, eventually leading to cell death. Here, we demonstrated that cytosolic irradiation with a femtosecond laser produced intrinsic cascades of reactive oxygen species (ROS), which led to rapid apoptosis-like cell death via a caspase and poly (ADP-ribose) polymerase 1 (PARP-1) signaling pathway. We further showed that cells with enhanced mitochondrial fusion activity are more resilient to laser-induced stress compared to those with enforced mitochondrial fission. Taken together, these findings provide fundamental insight into how optical stimulation intervenes in intrinsic cellular signaling pathways and functions. PMID:25648455

Cytosolic irradiation of femtosecond laser induces mitochondria-dependent apoptosis-like cell death via intrinsic reactive oxygen cascades.

PubMed

Yoon, Jonghee; Ryu, Seung-Wook; Lee, Seunghee; Choi, Chulhee

2015-02-04

High-intensity femtosecond lasers have recently been used to irreversibly disrupt nanoscale structures, such as intracellular organelles, and to modify biological functions in a reversible manner: so-called nanosurgery and biophotomodulation. Femtosecond laser pulses above the threshold intensity sufficient for reversible biophotomodulation can cause irreversible changes in the irradiated cell, eventually leading to cell death. Here, we demonstrated that cytosolic irradiation with a femtosecond laser produced intrinsic cascades of reactive oxygen species (ROS), which led to rapid apoptosis-like cell death via a caspase and poly (ADP-ribose) polymerase 1 (PARP-1) signaling pathway. We further showed that cells with enhanced mitochondrial fusion activity are more resilient to laser-induced stress compared to those with enforced mitochondrial fission. Taken together, these findings provide fundamental insight into how optical stimulation intervenes in intrinsic cellular signaling pathways and functions.

Reduction of conventional dendritic cells during Plasmodium infection is dependent on activation induced cell death by type I and II interferons.

PubMed

Tamura, Takahiko; Kimura, Kazumi; Yui, Katsuyuki; Yoshida, Shigeto

2015-12-01

Dendritic cells (DCs) play critical roles in innate and adaptive immunity and in pathogenesis during the blood stage of malaria infection. The mechanisms underlying DC homeostasis during malaria infection are not well understood. In this study, the numbers of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) in the spleens after lethal rodent malaria infection were examined, and were found to be significantly reduced. Concomitant with up-regulation of maturation-associated molecules, activation of caspase-3 was significantly increased, suggesting induction of cell death. Studies using neutralizing antibody and gene-deficient mice showed that type I and II interferons were critically involved in activation induced cell death of cDCs during malaria infection. These results demonstrate that DCs rapidly disappeared following IFN-mediated DC activation, and that homeostasis of DCs was significantly impaired during malaria infection. Copyright © 2015 Elsevier Inc. All rights reserved.

Berberine induces autophagy in glioblastoma by targeting the AMPK/mTOR/ULK1-pathway

PubMed Central

Wang, Jiwei; Qi, Qichao; Feng, Zichao; Zhang, Xin; Huang, Bin; Chen, Anjing; Prestegarden, Lars; Li, Xingang; Wang, Jian

2016-01-01

There is an urgent need for new therapeutic strategies for patients with glioblastoma multiforme (GBM). Previous studies have shown that berberine (BBR), a natural plant alkaloid, has potent anti-tumor activity. However, the mechanisms leading to cancer cell death have not been clearly elucidated. In this study, we show that BBR has profound effects on the metabolic state of GBM cells, leading to high autophagy flux and impaired glycolytic capacity. Functionally, these alterations reduce the invasive properties, proliferative potential and induce apoptotic cell death. The molecular alterations preceding these changes are characterized by inhibition of the AMPK/mTOR/ULK1 pathway. Finally, we demonstrate that BBR significantly reduces tumor growth in vivo, demonstrating the potential clinical benefits for autophagy modulating plant alkaloids in cancer therapy. PMID:27557493

Coniferyl Aldehyde Attenuates Radiation Enteropathy by Inhibiting Cell Death and Promoting Endothelial Cell Function

PubMed Central

Son, Yeonghoon; Jang, Jun-Ho; Lee, Yoon-Jin; Kim, Sung-Ho; Ko, Young-Gyo; Lee, Yun-Sil; Lee, Hae-June

2015-01-01

Radiation enteropathy is a common complication in cancer patients. The aim of this study was to investigate whether radiation-induced intestinal injury could be alleviated by coniferyl aldehyde (CA), an HSF1-inducing agent that increases cellular HSP70 expression. We systemically administered CA to mice with radiation enteropathy following abdominal irradiation (IR) to demonstrate the protective effects of CA against radiation-induced gastrointestinal injury. CA clearly alleviated acute radiation-induced intestinal damage, as reflected by the histopathological data and it also attenuated sub-acute enteritis. CA prevented intestinal crypt cell death and protected the microvasculature in the lamina propria during the acute and sub-acute phases of damage. CA induced HSF1 and HSP70 expression in both intestinal epithelial cells and endothelial cells in vitro. Additionally, CA protected against not only the apoptotic cell death of both endothelial and epithelial cells but also the loss of endothelial cell function following IR, indicating that CA has beneficial effects on the intestine. Our results provide novel insight into the effects of CA and suggest its role as a therapeutic candidate for radiation-induced enteropathy due to its ability to promote rapid re-proliferation of the intestinal epithelium by the synergic effects of the inhibition of cell death and the promotion of endothelial cell function. PMID:26029925

Methamphetamine-induced neurotoxicity linked to UPS dysfunction and autophagy related changes that can be modulated by PKCδ in dopaminergic neuronal cells

PubMed Central

Lin, Mengshien; Shivalingappa, Prashanth Chandramani; Jin, Huajun; Ghosh, Anamitra; Anantharam, Vellareddy; Ali, Syed; Kanthasamy, Anumantha G.; Kanthasamy, Arthi

2012-01-01

A compromised protein degradation machinery has been implicated in methamphetamine (MA)-induced neurodegeneration. However, the signaling mechanisms that induce autophagy and UPS dysfunction are not well understood. The present study investigates the contributions of PKC delta (PKCδ) mediated signaling events in MA-induced autophagy, UPS dysfunction and cell death. Using an in vitro mesencephalic dopaminergic cell culture model, we demonstrate that MA-induced early induction of autophagy is associated with reduction in proteasomal function and concomitant dissipation of mitochondrial membrane potential (MMP), followed by significantly increased of PKCδ activation, caspase-3 activation, accumulation of ubiquitin positive aggregates and microtubule associated light chain-3 (LC3-II) levels. Interestingly, siRNA mediated knockdown of PKCδ or overexpression of cleavage resistant mutant of PKCδ dramatically reduced MA-induced autophagy, proteasomal function, and associated accumulation of ubiquitinated protein aggregates, which closely paralleled cell survival. Importantly, when autophagy was inhibited either pharmacologically (3-MA) or genetically (siRNA mediated silencing of LC3), the dopaminergic cells became sensitized to MA-induced apoptosis through caspase-3 activation. Conversely, overexpression of LC3 partially protected against MA-induced apoptotic cell death, suggesting a neuroprotective role for autophagy in MA-induced neurotoxicity. Notably, rat striatal tissue isolated from MA treated rats also exhibited elevated LC3-II, ubiquitinated protein levels, and PKCδ cleavage. Taken together, our data demonstrate that MA-induced autophagy serves as an adaptive strategy for inhibiting mitochondria mediated apoptotic cell death and degradation of aggregated proteins. Our results also suggest that the sustained activation of PKCδ leads to UPS dysfunction, resulting in the activation of caspase-3 mediated apoptotic cell death in the nigrostriatal dopaminergic system. PMID:22445524

Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner.

PubMed

Conos, Stephanie A; Chen, Kaiwen W; De Nardo, Dominic; Hara, Hideki; Whitehead, Lachlan; Núñez, Gabriel; Masters, Seth L; Murphy, James M; Schroder, Kate; Vaux, David L; Lawlor, Kate E; Lindqvist, Lisa M; Vince, James E

2017-02-07

Necroptosis is a physiological cell suicide mechanism initiated by receptor-interacting protein kinase-3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), which results in disruption of the plasma membrane. Necroptotic cell lysis, and resultant release of proinflammatory mediators, is thought to cause inflammation in necroptotic disease models. However, we previously showed that MLKL signaling can also promote inflammation by activating the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome to recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) and trigger caspase-1 processing of the proinflammatory cytokine IL-1β. Here, we provide evidence that MLKL-induced activation of NLRP3 requires (i) the death effector four-helical bundle of MLKL, (ii) oligomerization and association of MLKL with cellular membranes, and (iii) a reduction in intracellular potassium concentration. Although genetic or pharmacological targeting of NLRP3 or caspase-1 prevented MLKL-induced IL-1β secretion, they did not prevent necroptotic cell death. Gasdermin D (GSDMD), the pore-forming caspase-1 substrate required for efficient NLRP3-triggered pyroptosis and IL-1β release, was not essential for MLKL-dependent death or IL-1β secretion. Imaging of MLKL-dependent ASC speck formation demonstrated that necroptotic stimuli activate NLRP3 cell-intrinsically, indicating that MLKL-induced NLRP3 inflammasome formation and IL-1β cleavage occur before cell lysis. Furthermore, we show that necroptotic activation of NLRP3, but not necroptotic cell death alone, is necessary for the activation of NF-κB in healthy bystander cells. Collectively, these results demonstrate the potential importance of NLRP3 inflammasome activity as a driving force for inflammation in MLKL-dependent diseases.

Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner

PubMed Central

Conos, Stephanie A.; Hara, Hideki; Whitehead, Lachlan; Núñez, Gabriel; Masters, Seth L.; Murphy, James M.; Schroder, Kate; Vaux, David L.; Lawlor, Kate E.; Vince, James E.

2017-01-01

Necroptosis is a physiological cell suicide mechanism initiated by receptor-interacting protein kinase-3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), which results in disruption of the plasma membrane. Necroptotic cell lysis, and resultant release of proinflammatory mediators, is thought to cause inflammation in necroptotic disease models. However, we previously showed that MLKL signaling can also promote inflammation by activating the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome to recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) and trigger caspase-1 processing of the proinflammatory cytokine IL-1β. Here, we provide evidence that MLKL-induced activation of NLRP3 requires (i) the death effector four-helical bundle of MLKL, (ii) oligomerization and association of MLKL with cellular membranes, and (iii) a reduction in intracellular potassium concentration. Although genetic or pharmacological targeting of NLRP3 or caspase-1 prevented MLKL-induced IL-1β secretion, they did not prevent necroptotic cell death. Gasdermin D (GSDMD), the pore-forming caspase-1 substrate required for efficient NLRP3-triggered pyroptosis and IL-1β release, was not essential for MLKL-dependent death or IL-1β secretion. Imaging of MLKL-dependent ASC speck formation demonstrated that necroptotic stimuli activate NLRP3 cell-intrinsically, indicating that MLKL-induced NLRP3 inflammasome formation and IL-1β cleavage occur before cell lysis. Furthermore, we show that necroptotic activation of NLRP3, but not necroptotic cell death alone, is necessary for the activation of NF-κB in healthy bystander cells. Collectively, these results demonstrate the potential importance of NLRP3 inflammasome activity as a driving force for inflammation in MLKL-dependent diseases. PMID:28096356

Mitochondrial protection impairs BET bromodomain inhibitor-mediated cell death and provides rationale for combination therapeutic strategies.

PubMed

Lasorsa, E; Smonksey, M; Kirk, J S; Rosario, S; Hernandez-Ilizaliturri, F J; Ellis, L

2015-12-10

Inhibitors of the bromodomain and extraterminal domain family (BETI) have recently entered phase I clinical trials. In patients with advanced leukemia's, potent antileukemia activity was displayed with minimum dose-limiting toxicity. In preclinical models of hematological malignancies, including aggressive B-cell lymphomas, BETI induced cell-cycle arrest and apoptosis. However, the underlying cell death mechanisms are still not well understood. Dissecting the mechanisms required by BETI to mediate cell death would provide strong direction on how to best utilize BETI to treat patients with aggressive hematological malignancies. Herein, we provide understanding of the molecular mechanisms underlying BETI-mediated cell death using I-BET762. Induction of cell death occurred in primary murine and human B-cell lymphomas through apoptosis. Genetic dissection using Eμ-myc B-cell lymphoma compound mutants demonstrated that I-BET762-induced apoptosis does not require the p53 pathway. Furthermore, deletion of Apaf1, and thus the absence of a functional apoptosome, is associated with a delayed drug response but do not provide long-term resistance. Prolonged treatment of this model in fact fails to suppress the therapeutic efficacy of the drug and is associated with biochemical features of autophagy. However, lack of mitochondrial permeability completely inhibited I-BET762-mediated tumor cell death, indicating mitochondrial damage as key events for its activity. Combination of I-BET762 with BH3-only mimetics ABT-263 or obatoclax, restored sensitivity to I-BET762 lymphoma killing; however, success was determined by expression of Bcl-2 family antiapoptotic proteins. Our study provides critical insight for clinical decisions regarding the appropriate strategy for using BETI as a single agent or in combination to treat patients with aggressive B-cell lymphomas.

The Neuroprotective Potential of Cyanidin-3-glucoside Fraction Extracted from Mulberry Following Oxygen-glucose Deprivation.

PubMed

Bhuiyan, Mohammad Iqbal Hossain; Kim, Hyun-Bok; Kim, Seong Yun; Cho, Kyung-Ok

2011-12-01

In this study, cyanidin-3-glucoside (C3G) fraction extracted from the mulberry fruit (Morus alba L.) was investigated for its neuroprotective effects against oxygen-glucose deprivation (OGD) and glutamate-induced cell death in rat primary cortical neurons. Cell membrane damage and mitochondrial function were assessed by LDH release and MTT reduction assays, respectively. A time-course study of OGD-induced cell death of primary cortical neurons at 7 days in vitro (DIV) indicated that neuronal death was OGD duration-dependent. It was also demonstrated that OGD for 3.5 h resulted in approximately 50% cell death, as determined by the LDH release assay. Treatments with mulberry C3G fraction prevented membrane damage and preserved the mitochondrial function of the primary cortical neurons exposed to OGD for 3.5 h in a concentration-dependent manner. Glutamate-induced cell death was more pronounced in DIV-9 and DIV-11 cells than that in DIV-7 neurons, and an application of 50µM glutamate was shown to induce approximately 40% cell death in DIV-9 neurons. Interestingly, treatment with mulberry C3G fraction did not provide a protective effect against glutamate-induced cell death in primary cortical neurons. On the other hand, treatment with mulberry C3G fraction maintained the mitochondrial membrane potential (MMP) in primary cortical neurons exposed to OGD as assessed by the intensity of rhodamine-123 fluorescence. These results therefore suggest that the neuroprotective effects of mulberry C3G fraction are mediated by the maintenance of the MMP and mitochondrial function but not by attenuating glutamate-induced excitotoxicity in rat primary cortical neurons.

5-Benzylglycinyl-Amiloride Kills Proliferating and Nonproliferating Malignant Glioma Cells through Caspase-Independent Necroptosis Mediated by Apoptosis-Inducing Factor

PubMed Central

Pasupuleti, Nagarekha; Leon, Leonardo; Carraway, Kermit L.

2013-01-01

5′–Βenzylglycinyl-amiloride (UCD38B) and glycinyl-amiloride (UCD74A) are cell-permeant and cell-impermeant derivatives of amiloride, respectively, and used here to identify the cellular mechanisms of action underlying their antiglioma effects. UCD38B comparably kills proliferating and nonproliferating gliomas cells when cell cycle progression is arrested either by cyclin D1 siRNA or by acidification. Cell impermeant UCD74A inhibits plasmalemmal urokinase plasminogen activator (uPA) and the type 1 sodium-proton exchanger with potencies analogous to UCD38B, but is cytostatic. In contrast, UCD38B targets intracellular uPA causing mistrafficking of uPA into perinuclear mitochondria, reducing the mitochondrial membrane potential, and followed by the release of apoptotic inducible factor (AIF). AIF nuclear translocation is followed by a caspase-independent necroptotic cell death. Reduction in AIF expression by siRNA reduces the antiglioma cytotoxic effects of UCD38B, while not activating the caspase pathway. Ultrastructural changes shortly following treatment with UCD38B demonstrate dilation of endoplasmic reticulum (ER) and mitochondrial swelling followed by nuclear condensation within hours consistent with a necroptotic cell death differing from apoptosis and from autophagy. These drug mechanism of action studies demonstrate that UCD38B induces a cell cycle-independent, caspase-independent necroptotic glioma cell death that is mediated by AIF and independent of poly (ADP-ribose) polymerase and H2AX activation. PMID:23241369

miR-106a suppresses tumor cells death in colorectal cancer through targeting ATG7.

PubMed

Hao, Haibin; Xia, Guangfeng; Wang, Chao; Zhong, Fuping; Liu, Laipeng; Zhang, Dong

2017-06-01

Autophagy-related gene 7 (ATG7) and miR-106a play an important role in cancer cell autophagy and apoptosis, but the outcome of ATG7 and miR-106a in colorectal cancer (CRC) still remains not clear. In this study, we found that ATG7 and miR-106a expression were mutually related with cell death and prognosis in CRC patients. In addition, we also showed that ATG7 and miR-106a expression were changeable in colorectal cancer cell lines when compared with normal cell lines, but ATG7 and miR-106a mRNA level was negatively correlated. Furthermore, ATG7 protein and mRNA levels decreased after over-expression of miR-106a, whereas the suppression of ATG7 had the opposite effect. We confirmed that miR-106a down-regulated ATG7 mRNA level by binding the specific sequence of ATG7 mRNA 3'UTR region. Moreover, the over-expression of ATG7 induced CRC cells death both in vitro and in vivo. Taken together, our study data demonstrated that ATG7 aggravated the cell death of CRC, which was inhibited by miR-106a.

Immotile Active Matter: Activity from Death and Reproduction

NASA Astrophysics Data System (ADS)

Kalziqi, Arben; Yanni, David; Thomas, Jacob; Ng, Siu Lung; Vivek, Skanda; Hammer, Brian K.; Yunker, Peter J.

2018-01-01

Unlike equilibrium atomic solids, biofilms—soft solids composed of bacterial cells—do not experience significant thermal fluctuations at the constituent level. However, living cells stochastically reproduce and die, provoking a mechanical response. We investigate the mechanical consequences of cellular death and reproduction by measuring surface-height fluctuations of biofilms containing two mutually antagonistic strains of Vibrio cholerae that kill one another on contact via the type VI secretion system. While studies of active matter typically focus on activity via constituent mobility, here, activity is mediated by reproduction and death events in otherwise immobilized cells. Biofilm surface topography is measured in the nearly homeostatic limit via white light interferometry. Although biofilms are far from equilibrium systems, measured surface-height fluctuation spectra resemble the spectra of thermal permeable membranes but with an activity-mediated effective temperature, as predicted by Risler, Peilloux, and Prost [Phys. Rev. Lett. 115, 258104 (2015), 10.1103/PhysRevLett.115.258104]. By comparing the activity of killer strains of V. cholerae with that of genetically modified strains that cannot kill each other and validating with individual-based simulations, we demonstrate that extracted effective temperatures increase with the amount of death and reproduction and that death and reproduction can fluidize biofilms. Together, these observations demonstrate the unique physical consequences of activity mediated by death and reproduction events.

Defining external factors that determine neuronal survival, apoptosis and necrosis during excitotoxic injury using a high content screening imaging platform.

PubMed

Anilkumar, Ujval; Weisova, Petronela; Schmid, Jasmin; Bernas, Tytus; Huber, Heinrich J; Düssmann, Heiko; Connolly, Niamh M C; Prehn, Jochen H M

2017-01-01

Cell death induced by excessive glutamate receptor overactivation, excitotoxicity, has been implicated in several acute and chronic neurological disorders. While numerous studies have demonstrated the contribution of biochemically and genetically activated cell death pathways in excitotoxic injury, the factors mediating passive, excitotoxic necrosis are less thoroughly investigated. To address this question, we developed a high content screening (HCS) based assay to collect high volumes of quantitative cellular imaging data and elucidated the effects of intrinsic and external factors on excitotoxic necrosis and apoptosis. The analysis workflow consisted of robust nuclei segmentation, tracking and a classification algorithm, which enabled automated analysis of large amounts of data to identify and quantify viable, apoptotic and necrotic neuronal populations. We show that mouse cerebellar granule neurons plated at low or high density underwent significantly increased necrosis compared to neurons seeded at medium density. Increased extracellular Ca2+ sensitized neurons to glutamate-induced excitotoxicity, but surprisingly potentiated cell death mainly through apoptosis. We also demonstrate that inhibition of various cell death signaling pathways (including inhibition of calpain, PARP and AMPK activation) primarily reduced excitotoxic apoptosis. Excitotoxic necrosis instead increased with low extracellular glucose availability. Our study is the first of its kind to establish and implement a HCS based assay to investigate the contribution of external and intrinsic factors to excitotoxic apoptosis and necrosis.

Defining external factors that determine neuronal survival, apoptosis and necrosis during excitotoxic injury using a high content screening imaging platform

PubMed Central

Weisova, Petronela; Schmid, Jasmin; Bernas, Tytus; Huber, Heinrich J.; Düssmann, Heiko; Connolly, Niamh M. C.; Prehn, Jochen H. M.

2017-01-01

Cell death induced by excessive glutamate receptor overactivation, excitotoxicity, has been implicated in several acute and chronic neurological disorders. While numerous studies have demonstrated the contribution of biochemically and genetically activated cell death pathways in excitotoxic injury, the factors mediating passive, excitotoxic necrosis are less thoroughly investigated. To address this question, we developed a high content screening (HCS) based assay to collect high volumes of quantitative cellular imaging data and elucidated the effects of intrinsic and external factors on excitotoxic necrosis and apoptosis. The analysis workflow consisted of robust nuclei segmentation, tracking and a classification algorithm, which enabled automated analysis of large amounts of data to identify and quantify viable, apoptotic and necrotic neuronal populations. We show that mouse cerebellar granule neurons plated at low or high density underwent significantly increased necrosis compared to neurons seeded at medium density. Increased extracellular Ca2+ sensitized neurons to glutamate-induced excitotoxicity, but surprisingly potentiated cell death mainly through apoptosis. We also demonstrate that inhibition of various cell death signaling pathways (including inhibition of calpain, PARP and AMPK activation) primarily reduced excitotoxic apoptosis. Excitotoxic necrosis instead increased with low extracellular glucose availability. Our study is the first of its kind to establish and implement a HCS based assay to investigate the contribution of external and intrinsic factors to excitotoxic apoptosis and necrosis. PMID:29145487

Time-resolved, single-cell analysis of induced and programmed cell death via non-invasive propidium iodide and counterstain perfusion.

PubMed

Krämer, Christina E M; Wiechert, Wolfgang; Kohlheyer, Dietrich

2016-09-01

Conventional propidium iodide (PI) staining requires the execution of multiple steps prior to analysis, potentially affecting assay results as well as cell vitality. In this study, this multistep analysis method has been transformed into a single-step, non-toxic, real-time method via live-cell imaging during perfusion with 0.1 μM PI inside a microfluidic cultivation device. Dynamic PI staining was an effective live/dead analytical tool and demonstrated consistent results for single-cell death initiated by direct or indirect triggers. Application of this method for the first time revealed the apparent antibiotic tolerance of wild-type Corynebacterium glutamicum cells, as indicated by the conversion of violet fluorogenic calcein acetoxymethyl ester (CvAM). Additional implementation of this method provided insight into the induced cell lysis of Escherichia coli cells expressing a lytic toxin-antitoxin module, providing evidence for non-lytic cell death and cell resistance to toxin production. Finally, our dynamic PI staining method distinguished necrotic-like and apoptotic-like cell death phenotypes in Saccharomyces cerevisiae among predisposed descendants of nutrient-deprived ancestor cells using PO-PRO-1 or green fluorogenic calcein acetoxymethyl ester (CgAM) as counterstains. The combination of single-cell cultivation, fluorescent time-lapse imaging, and PI perfusion facilitates spatiotemporally resolved observations that deliver new insights into the dynamics of cellular behaviour.

Cell signaling pathways in the mechanisms of neuroprotection afforded by bergamot essential oil against NMDA-induced cell death in vitro.

PubMed

Corasaniti, M T; Maiuolo, J; Maida, S; Fratto, V; Navarra, M; Russo, R; Amantea, D; Morrone, L A; Bagetta, G

2007-06-01

The effects of bergamot essential oil (BEO; Citrus bergamia, Risso) on excitotoxic neuronal damage was investigated in vitro. The study was performed in human SH-SY5Y neuroblastoma cells exposed to N-methyl-D-aspartate (NMDA). Cell viability was measured by dye exclusion. Reactive oxygen species (ROS) and caspase-3 activity were measured fluorimetrically. Calpain I activity and the activation (phosphorylation) of Akt and glycogen synthase kinase-3beta (GSK-3beta) were assayed by Western blotting. NMDA induced concentration-dependent, receptor-mediated, death of SH-SY5Y cells, ranging from 11 to 25% (0.25-5 mM). Cell death induced by 1 mM NMDA (21%) was preceded by a significant accumulation of intracellular ROS and by a rapid activation of the calcium-activated protease calpain I. In addition, NMDA caused a rapid deactivation of Akt kinase and this preceded the detrimental activation of the downstream kinase, GSK-3beta. BEO (0.0005-0.01%) concentration dependently reduced death of SH-SY5Y cells caused by 1 mM NMDA. In addition to preventing ROS accumulation and activation of calpain, BEO (0.01%) counteracted the deactivation of Akt and the consequent activation of GSK-3beta, induced by NMDA. Results obtained by using specific fractions of BEO, suggested that monoterpene hydrocarbons were responsible for neuroprotection afforded by BEO against NMDA-induced cell death. Our data demonstrate that BEO reduces neuronal damage caused in vitro by excitotoxic stimuli and that this neuroprotection was associated with prevention of injury-induced engagement of critical death pathways.

Umbelliprenin is cytotoxic against QU-DB large cell lung cancer cell line but anti-proliferative against A549 adenocarcinoma cells

PubMed Central

2012-01-01

Background Umbelliprenin is a natural compound, belonging to the class of sesquiterpene coumarins. Recently, umbelliprenin has attracted the researchers' attention for its antitumor activities against skin tumors. Its effect on lung cancer is largely unknown. The aim of our study was to investigate the effects of this natural compound, which is expected to have low adverse effects, on lung cancer. Methods The QU-DB large cell and A549 adenocarcinoma lung cancer cell lines were treated with umbelliprenin. IC50 values were estimated using methyl thiazolely diphenyl-tetrazolium bromide (MTT) assay, in which a decrease in MTT reduction can occur as a result of cell death or cell proliferation inhibition. To quantify the rate of cell death at IC50 values, flow cytometry using Annexin V-FITC (for apoptotic cells), and propidium iodide (for necrotic cells) dyes were employed. Results Data from three independent MTT experiments in triplicate revealed that IC50 values for QU-DB and A549 were 47 ± 5.3 μM and 52 ± 1.97 μM, respectively. Annexin V/PI staining demonstrated that umbelliprenin treatment at IC50 induced 50% cell death in QU-DB cells, but produced no significant death in A549 cells until increasing the umbelliprenin concentration to IC80. The pattern of cell death was predominantly apoptosis in both cell lines. When peripheral blood mononuclear cells were treated with 50 μM and less concentrations of umbelliprenin, no suppressive effect was observed. Conclusions We found cytotoxic/anti-proliferative effects of umbelliprenin against two different types of lung cancer cell lines. PMID:23351548

Programming stress-induced altruistic death in engineered bacteria

PubMed Central

Tanouchi, Yu; Pai, Anand; Buchler, Nicolas E; You, Lingchong

2012-01-01

Programmed death is often associated with a bacterial stress response. This behavior appears paradoxical, as it offers no benefit to the individual. This paradox can be explained if the death is ‘altruistic': the killing of some cells can benefit the survivors through release of ‘public goods'. However, the conditions where bacterial programmed death becomes advantageous have not been unambiguously demonstrated experimentally. Here, we determined such conditions by engineering tunable, stress-induced altruistic death in the bacterium Escherichia coli. Using a mathematical model, we predicted the existence of an optimal programmed death rate that maximizes population growth under stress. We further predicted that altruistic death could generate the ‘Eagle effect', a counter-intuitive phenomenon where bacteria appear to grow better when treated with higher antibiotic concentrations. In support of these modeling insights, we experimentally demonstrated both the optimality in programmed death rate and the Eagle effect using our engineered system. Our findings fill a critical conceptual gap in the analysis of the evolution of bacterial programmed death, and have implications for a design of antibiotic treatment. PMID:23169002

Caspase Activation in Fetal Rat Brain Following Experimental Intrauterine Inflammation

PubMed Central

Sharangpani, Aditi; Takanohashi, Asako; Bell, Michael J.

2009-01-01

Intrauterine inflammation has been implicated in developmental brain injuries, including the development of periventricular leukomalacia (PVL) and cerebral palsy (CP). Previous studies in our rat model of intrauterine inflammation demonstrated apoptotic cell death in fetal brains within the first 5 days after lipopolysaccharide (LPS) administration to mothers and eventual dysmyelination. Cysteine-containing, aspartate-specific proteases, or caspases, are proteins involved with apoptosis through both intracellular (intrinsic pathway) and extracellular (extrinsic pathway) mechanisms. We hypothesized that cell death in our model would occur mainly via activation of the extrinsic pathway. We further hypothesized that Fas, a member of the tumor necrosis factor receptor (TNFR) superfamily, would be increased and the death inducing signaling complex (DISC) would be detectable. Pregnant rats were injected intracervically with LPS at E15 and immunoblotting, immunohistochemical and immunoprecipitation analyses were performed. The presence of the activated form of the effector caspase (caspase-3) was observed 24 h after LPS administration. Caspase activity assays demonstrated rapid increases in (i) caspases-9 and -10 within 1 h, (ii) caspase-8 at 2 h and (iii) caspase-3 at 4 h. At 24 h after LPS, activated caspase-3+/Fas+ cells were observed within the developing white matter. Lastly, the DISC complex (caspase-8, Fas and Fas-associated Death Domain (FADD)) was observed within 30 min by immunoprecipitation. Apoptosis in our model occurs via both extrinsic and intrinsic pathways, and activation of Fas may play a role. Understanding the mechanisms of cell death in models of intrauterine inflammation may affect development of future strategies to mitigate these injuries in children. PMID:18289516

Involvement of tumour necrosis factor-α-related apoptosis-inducing ligand in enhanced cytotoxicity of lipopolysaccharide-stimulated dendritic cells to activated T cells

PubMed Central

Yu, Yizhi; Liu, Shuxun; Wang, Wenya; Song, Wengang; Zhang, Minghui; Zhang, Weiping; Qin, Zhihai; Cao, Xuetao

2002-01-01

Dendritic cells (DC) are potent antigen-presenting cells (APC) specialized in T-cell mediated immune responses, and also play critical roles in the homeostasis of T cells for controlling immune responses. In the present study, we demonstrated that during mouse bone-marrow-derived DC activation of ovalbumin (OVA)-specific Ia-kb-restricted T hybridoma cells, MF2.2D9 and OVA257–264-specific H-2kb-restricted RF33.70 T cells, respectively, both hybridomas undergo cell death, partially mediated via apoptotic ligand–tumour necrosis factor-α (TNF-α)-related apoptosis-inducing ligand (TRAIL). Lipopolysaccharide enhanced the cytotoxic effect on the two activated T hybridoma cells, which was correlated with up-regulation of TRAIL-expression on DC to some extent. The activation of caspase-3 in activated T hybridoma cells cocultured with DC contributed to the programmed cell death pathway T cells underwent. Therefore, our results show that activation-induced cell death of T hybridoma cells can be influenced by DC, suggesting that DC may be involved in elimination of activated T cells at the end of primary immune responses. PMID:12100718

Involvement of tumour necrosis factor-alpha-related apoptosis-inducing ligand in enhanced cytotoxicity of lipopolysaccharide-stimulated dendritic cells to activated T cells.

PubMed

Yu, Yizhi; Liu, Shuxun; Wang, Wenya; Song, Wengang; Zhang, Minghui; Zhang, Weiping; Qin, Zhihai; Cao, Xuetao

2002-07-01

Dendritic cells (DC) are potent antigen-presenting cells (APC) specialized in T-cell mediated immune responses, and also play critical roles in the homeostasis of T cells for controlling immune responses. In the present study, we demonstrated that during mouse bone-marrow-derived DC activation of ovalbumin (OVA)-specific Ia-kb-restricted T hybridoma cells, MF2.2D9 and OVA257-264-specific H-2kb-restricted RF33.70 T cells, respectively, both hybridomas undergo cell death, partially mediated via apoptotic ligand-tumour necrosis factor-alpha (TNF-alpha)-related apoptosis-inducing ligand (TRAIL). Lipopolysaccharide enhanced the cytotoxic effect on the two activated T hybridoma cells, which was correlated with up-regulation of TRAIL-expression on DC to some extent. The activation of caspase-3 in activated T hybridoma cells cocultured with DC contributed to the programmed cell death pathway T cells underwent. Therefore, our results show that activation-induced cell death of T hybridoma cells can be influenced by DC, suggesting that DC may be involved in elimination of activated T cells at the end of primary immune responses.

Ca2+ is a key factor in α-synuclein-induced neurotoxicity

PubMed Central

Angelova, Plamena R.; Ludtmann, Marthe H. R.; Horrocks, Mathew H.; Negoda, Alexander; Cremades, Nunilo; Klenerman, David; Dobson, Christopher M.; Wood, Nicholas W.; Pavlov, Evgeny V.; Gandhi, Sonia

2016-01-01

ABSTRACT Aggregation of α-synuclein leads to the formation of oligomeric intermediates that can interact with membranes to form pores. However, it is unknown how this leads to cell toxicity in Parkinson's disease. We investigated the species-specific effects of α-synuclein on Ca2+ signalling in primary neurons and astrocytes using live neuronal imaging and electrophysiology on artificial membranes. We demonstrate that α-synuclein induces an increase in basal intracellular Ca2+ in its unfolded monomeric state as well as in its oligomeric state. Electrophysiology of artificial membranes demonstrated that α-synuclein monomers induce irregular ionic currents, whereas α-synuclein oligomers induce rare discrete channel formation events. Despite the ability of monomeric α-synuclein to affect Ca2+ signalling, it is only the oligomeric form of α-synuclein that induces cell death. Oligomer-induced cell death was abolished by the exclusion of extracellular Ca2+, which prevented the α-synuclein-induced Ca2+ dysregulation. The findings of this study confirm that α-synuclein interacts with membranes to affect Ca2+ signalling in a structure-specific manner and the oligomeric β-sheet-rich α-synuclein species ultimately leads to Ca2+ dysregulation and Ca2+-dependent cell death. PMID:26989132

Non-conventional apoptotic response to ionising radiation mediated by N-methyl D-aspartate receptors in immature neuronal cells

PubMed Central

SAMARI, NADA; DE SAINT-GEORGES, LOUIS; PANI, GIUSEPPE; BAATOUT, SARAH; LEYNS, LUC; BENOTMANE, MOHAMMED ABDERRAFI

2013-01-01

During cortical development, N-methyl D-aspartate (NMDA) receptors are highly involved in neuronal maturation and synapse establishment. Their implication in the phenomenon of excitotoxicity has been extensively described in several neurodegenerative diseases due to the permissive entry of Ca2+ ions and massive accumulation in the intracellular compartment, which is highly toxic to cells. Ionising radiation is also a source of stress to the cells, particularly immature neurons. Their capacity to induce cell death has been described for various cell types either by directly damaging the DNA or indirectly through the generation of reactive oxygen species responsible for the activation of a battery of stress response effectors leading in certain cases, to cell death. In this study, in order to determine whether a link exists between NMDA receptors-mediated excitotoxicity and radiation-induced cell death, we evaluated radiation-induced cell death in vitro and in vivo in maturing neurons during the fetal period. Cell death induction was assessed by TUNEL, caspase-3 activity and DNA ladder assays, with or without the administration of dizocilpine (MK-801), a non-competitive NMDA receptor antagonist which blocks neuronal Ca2+ influx. To further investigate the possible involvement of Ca2+-dependent enzyme activation, known to occur at high Ca2+ concentrations, we examined the protective effect of a calpain inhibitor on cell death induced by radiation. Doses ranging from 0.2 to 0.6 Gy of X-rays elicited a clear apoptotic response that was prevented by the injection of dizocilpine (MK-801) or calpain inhibitor. These data demonstrate the involvement of NMDA receptors in radiation-induced neuronal death by the activation of downstream effectors, including calpain-related pathways. An increased apoptotic process elicited by radiation, occurring independently of the normal developmental scheme, may eliminate post-mitotic but immature neuronal cells and deeply impair the establishment of the neuronal network, which in the case of cortical development is critical for cognitive capacities. PMID:23338045

High cell surface death receptor expression determines type I versus type II signaling.

PubMed

Meng, Xue Wei; Peterson, Kevin L; Dai, Haiming; Schneider, Paula; Lee, Sun-Hee; Zhang, Jin-San; Koenig, Alexander; Bronk, Steve; Billadeau, Daniel D; Gores, Gregory J; Kaufmann, Scott H

2011-10-14

Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression.

Glutamine-mediated protection from neuronal cell death depends on mitochondrial activity.

PubMed

Stelmashook, E V; Lozier, E R; Goryacheva, E S; Mergenthaler, P; Novikova, S V; Zorov, D B; Isaev, N K

2010-09-27

The specific aim of this study was to elucidate the role of mitochondria in a neuronal death caused by different metabolic effectors and possible role of intracellular calcium ions ([Ca(2+)](i)) and glutamine in mitochondria- and non-mitochondria-mediated cell death. Inhibition of mitochondrial complex I by rotenone was found to cause intensive death of cultured cerebellar granule neurons (CGNs) that was preceded by an increase in intracellular calcium concentration ([Ca(2+)](i)). The neuronal death induced by rotenone was significantly potentiated by glutamine. In addition, inhibition of Na/K-ATPase by ouabain also caused [Ca(2+)](i) increase, but it induced neuronal cell death only in the absence of glucose. Treatment with glutamine prevented the toxic effect of ouabain and decreased [Ca(2+)](i). Blockade of ionotropic glutamate receptors prevented neuronal death and significantly decreased [Ca(2+)](i), demonstrating that toxicity of rotenone and ouabain was at least partially mediated by activation of these receptors. Activation of glutamate receptors by NMDA increased [Ca(2+)](i) and decreased mitochondrial membrane potential leading to markedly decreased neuronal survival under glucose deprivation. Glutamine treatment under these conditions prevented cell death and significantly decreased the disturbances of [Ca(2+)](i) and changes in mitochondrial membrane potential caused by NMDA during hypoglycemia. Our results indicate that glutamine stimulates glutamate-dependent neuronal damage when mitochondrial respiration is impaired. However, when mitochondria are functionally active, glutamine can be used by mitochondria as an alternative substrate to maintain cellular energy levels and promote cell survival. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Label-free monitoring of cell death induced by oxidative stress in living human cells using terahertz ATR spectroscopy

PubMed Central

Zou, Yi; Liu, Qiao; Yang, Xia; Huang, Hua-Chuan; Li, Jiang; Du, Liang-Hui; Li, Ze-Ren; Zhao, Jian-Heng; Zhu, Li-Guo

2017-01-01

We demonstrated that attenuated total reflectance terahertz time-domain spectroscopy (ATR THz-TDS) is able to monitor oxidative stress response of living human cells, which is proven in this work that it is an efficient non-invasive, label-free, real-time and in situ monitoring of cell death. Furthermore, the dielectric constant and dielectric loss of cultured living human breast epithelial cells, and along with their evolution under oxidative stress response induced by high concentration of H2O2, were quantitatively determined in the work. Our observation and results were finally confirmed using standard fluorescence-labeled flow cytometry measurements and visible fluorescence imaging. PMID:29359084

Telomerase Is Involved in IL-7-Mediated Differential Survival of Naive and Memory CD4+ T Cells1

PubMed Central

Yang, Yinhua; An, Jie; Weng, Nan-ping

2008-01-01

IL-7 plays an essential role in T cell maintenance and survival. The survival effect of IL-7 is thought to be mediated through regulation of Bcl2 family proteins. After a comparative analysis of IL-7-induced growth and cell death of human naive and memory CD4+ T cells, we observed that more memory CD4+ T cells underwent cell division and proceeded to apoptosis than naive cells in response to IL-7. However, IL-7-induced expressions of Bcl2 family members (Bcl2, Bcl-xL, Bax, and Bad) were similar between naive and memory cells. Instead, we found that IL-7 induced higher levels of telomerase activity in naive cells than in memory cells, and the levels of IL-7-induced telomerase activity had a significant inverse correlation with cell death in CD4+ T cells. Furthermore, we showed that reducing expression of telomerase reverse transcriptase and telomerase activity significantly increased cell death of IL-7-cultured CD4+ T cells. Together, these findings demonstrate that telomerase is involved in IL-7-mediated differential survival of naive and memory CD4+ T cells. PMID:18322183

Lysozyme amyloid oligomers and fibrils induce cellular death via different apoptotic/necrotic pathways.

PubMed

Gharibyan, Anna L; Zamotin, Vladimir; Yanamandra, Kiran; Moskaleva, Olesya S; Margulis, Boris A; Kostanyan, Irina A; Morozova-Roche, Ludmilla A

2007-02-02

Among the newly discovered amyloid properties, its cytotoxicity plays a key role. Lysozyme is a ubiquitous protein involved in systemic amyloidoses in vivo and forming amyloid under destabilising conditions in vitro. We characterized both oligomers and fibrils of hen lysozyme by atomic force microscopy and demonstrated their dose (5-50 microM) and time-dependent (6-48 h) effect on neuroblastoma SH-SY5Y cell viability. We revealed that fibrils induce a decrease of cell viability after 6 h due to membrane damage shown by inhibition of WST-1 reduction, early lactate dehydrogenase release, and propidium iodide intake; by contrast, oligomers activate caspases after 6 h but cause the cell viability to decline only after 48 h, as shown by fluorescent-labelled annexin V binding to externalized phosphatidylserine, propidium iodide DNA staining, lactate dehydrogenase release, and by typical apoptotic shrinking of cells. We conclude that oligomers induce apoptosis-like cell death, while the fibrils lead to necrosis-like death. As polymorphism is a common property of an amyloid, we demonstrated that it is not a single uniform species but rather a continuum of cross-beta-sheet-containing amyloids that are cytotoxic. An abundance of lysozyme highlights a universal feature of this phenomenon, indicating that amyloid toxicity should be assessed in all clinical applications involving proteinaceous materials.

A Virulence Essential CRN Effector of Phytophthora capsici Suppresses Host Defense and Induces Cell Death in Plant Nucleus.

PubMed

Mafurah, Joseph Juma; Ma, Huifei; Zhang, Meixiang; Xu, Jing; He, Feng; Ye, Tingyue; Shen, Danyu; Chen, Yanyu; Rajput, Nasir Ahmed; Dou, Daolong

2015-01-01

Phytophthora capsici is a soil-borne plant pathogen with a wide range of hosts. The pathogen secretes a large array of effectors during infection of host plants, including Crinkler (CRN) effectors. However, it remains largely unknown on the roles of these effectors in virulence especially in P. capsici. In this study, we identified a cell death-inducing CRN effector PcCRN4 using agroinfiltration approach. Transient expression of PcCRN4 gene induced cell death in N. benthamiana, N. tabacum and Solanum lycopersicum. Overexpression of the gene in N. benthamiana enhanced susceptibility to P. capsici. Subcellular localization results showed that PcCRN4 localized to the plant nucleus, and the localization was required for both of its cell death-inducing activity and virulent function. Silencing PcCRN4 gene in P. capsici significantly reduced pathogen virulence. The expression of the pathogenesis-related gene PR1b in N. benthamiana was significantly induced when plants were inoculated with PcCRN4-silenced P. capsici transformant compared to the wilt-type. Callose deposits were also abundant at sites inoculated with PcCRN4-silenced transformant, indicating that silencing of PcCRN4 in P. capsici reduced the ability of the pathogen to suppress plant defenses. Transcriptions of cell death-related genes were affected when PcCRN4-silenced line were inoculated on Arabidopsis thaliana, suggesting that PcCRN4 may induce cell death by manipulating cell death-related genes. Overall, our results demonstrate that PcCRN4 is a virulence essential effector and it needs target to the plant nucleus to suppress plant immune responses.

ONC201 demonstrates anti-tumor effects in both triple negative and non-triple negative breast cancers through TRAIL-dependent and TRAIL-independent mechanisms

PubMed Central

Ralff, Marie D.; Kline, Christina L.B.; Küçükkase, Ozan C; Wagner, Jessica; Lim, Bora; Dicker, David T.; Prabhu, Varun V.; Oster, Wolfgang; El-Deiry, Wafik S.

2017-01-01

Breast cancer is a major cause of cancer-related death. TRAIL has been of interest as a cancer therapeutic, but only a subset of triple negative breast cancers (TNBC) is sensitive to TRAIL. The small molecule ONC201 induces expression of TRAIL and its receptor DR5. ONC201 has entered clinical trials in advanced cancers. Here we show that ONC201 is efficacious against both TNBC and non-TNBC cells (n=13). A subset of TNBC and non-TNBC cells succumb to ONC201-induced cell death. In 2/8 TNBC cell lines, ONC201 treatment induces caspase-8 cleavage and cell death that is blocked by TRAIL-neutralizing antibody RIK2. The pro-apoptotic effect of ONC201 translates to in vivo efficacy in the MDA-MB-468 xenograft model. In most TNBC lines tested (6/8) ONC201 has an anti-proliferative effect but does not induce apoptosis. ONC201 decreases cyclin D1 expression and causes an accumulation of cells in the G1 phase of the cell cycle. pRb expression is associated with sensitivity to the anti-proliferative effects of ONC201, and the compound synergizes with taxanes in less sensitive cells. All non-TNBC cells (n=5) are growth inhibited following ONC201 treatment, and unlike what has been observed with TRAIL, a subset (n=2) show PARP cleavage. In these cells, cell death induced by ONC201 is TRAIL-independent. Our data demonstrate that ONC201 has potent anti-proliferative and pro-apoptotic effects in a broad range of breast cancer subtypes, through TRAIL-dependent and TRAIL-independent mechanisms. These findings develop a pre-clinical rationale for developing ONC201 as a single agent and/or in combination with approved therapies in breast cancer. PMID:28424227

ONC201 Demonstrates Antitumor Effects in Both Triple-Negative and Non-Triple-Negative Breast Cancers through TRAIL-Dependent and TRAIL-Independent Mechanisms.

PubMed

Ralff, Marie D; Kline, Christina L B; Küçükkase, Ozan C; Wagner, Jessica; Lim, Bora; Dicker, David T; Prabhu, Varun V; Oster, Wolfgang; El-Deiry, Wafik S

2017-07-01

Breast cancer is a major cause of cancer-related death. TNF-related apoptosis-inducing ligand (TRAIL) has been of interest as a cancer therapeutic, but only a subset of triple-negative breast cancers (TNBC) is sensitive to TRAIL. The small-molecule ONC201 induces expression of TRAIL and its receptor DR5. ONC201 has entered clinical trials in advanced cancers. Here, we show that ONC201 is efficacious against both TNBC and non-TNBC cells ( n = 13). A subset of TNBC and non-TNBC cells succumbs to ONC201-induced cell death. In 2 of 8 TNBC cell lines, ONC201 treatment induces caspase-8 cleavage and cell death that is blocked by TRAIL-neutralizing antibody RIK2. The proapoptotic effect of ONC201 translates to in vivo efficacy in the MDA-MB-468 xenograft model. In most TNBC lines tested (6/8), ONC201 has an antiproliferative effect but does not induce apoptosis. ONC201 decreases cyclin D1 expression and causes an accumulation of cells in the G 1 phase of the cell cycle. pRb expression is associated with sensitivity to the antiproliferative effects of ONC201, and the compound synergizes with taxanes in less sensitive cells. All non-TNBC cells ( n = 5) are growth inhibited following ONC201 treatment, and unlike what has been observed with TRAIL, a subset ( n = 2) shows PARP cleavage. In these cells, cell death induced by ONC201 is TRAIL independent. Our data demonstrate that ONC201 has potent antiproliferative and proapoptotic effects in a broad range of breast cancer subtypes, through TRAIL-dependent and TRAIL-independent mechanisms. These findings develop a preclinical rationale for developing ONC201 as a single agent and/or in combination with approved therapies in breast cancer. Mol Cancer Ther; 16(7); 1290-8. ©2017 AACR . ©2017 American Association for Cancer Research.

RITA can induce cell death in p53-defective cells independently of p53 function via activation of JNK/SAPK and p38

PubMed Central

Weilbacher, A; Gutekunst, M; Oren, M; Aulitzky, W E; van der Kuip, H

2014-01-01

Significant advances have been made in the development of small molecules blocking the p53/MDM2 interaction. The Mdm2 inhibitor Nutlin-3 is restricted to tumors carrying wtp53. In contrast, RITA, a compound that binds p53, has recently been shown also to restore transcriptional functions of mtp53. As more than 50% of solid tumors carry p53 mutations, RITA promises to be a more effective therapeutic strategy than Nutlin-3. We investigated effects of RITA on apoptosis, cell cycle and induction of 45 p53 target genes in a panel of 14 cell lines from different tumor entities with different p53 status as well as primary lymphocytes and fibroblasts. Nine cell strains expressed wtp53, four harbored mtp53, and three were characterized by the loss of p53 protein. A significant induction of cell death upon RITA was observed in 7 of 16 cell lines. The nonmalignant cells in our panel were substantially less sensitive. We found that in contrast to Nultin-3, RITA is capable to induce cell death not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells. Importantly, whereas p53 has a central role for RITA-mediated effects in wtp53 cells, neither p53 nor p63 or p73 were essential for the RITA response in mtp53 or p53-null cells in our panel demonstrating that besides the known p53-dependent action of RITA in wtp53 cells, RITA can induce cell death also independently of p53 in cells harboring defective p53. We identified an important role of both p38 and JNK/SAPK for sensitivity to RITA in these cells leading to a typical caspase- and BAX/BAK-dependent mitochondrial apoptosis. In conclusion, our data demonstrate that RITA can induce apoptosis through p38 and JNK/SAPK not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells, making RITA an interesting tumor-selective drug. PMID:25010984

RITA can induce cell death in p53-defective cells independently of p53 function via activation of JNK/SAPK and p38.

PubMed

Weilbacher, A; Gutekunst, M; Oren, M; Aulitzky, W E; van der Kuip, H

2014-07-10

Significant advances have been made in the development of small molecules blocking the p53/MDM2 interaction. The Mdm2 inhibitor Nutlin-3 is restricted to tumors carrying wtp53. In contrast, RITA, a compound that binds p53, has recently been shown also to restore transcriptional functions of mtp53. As more than 50% of solid tumors carry p53 mutations, RITA promises to be a more effective therapeutic strategy than Nutlin-3. We investigated effects of RITA on apoptosis, cell cycle and induction of 45 p53 target genes in a panel of 14 cell lines from different tumor entities with different p53 status as well as primary lymphocytes and fibroblasts. Nine cell strains expressed wtp53, four harbored mtp53, and three were characterized by the loss of p53 protein. A significant induction of cell death upon RITA was observed in 7 of 16 cell lines. The nonmalignant cells in our panel were substantially less sensitive. We found that in contrast to Nultin-3, RITA is capable to induce cell death not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells. Importantly, whereas p53 has a central role for RITA-mediated effects in wtp53 cells, neither p53 nor p63 or p73 were essential for the RITA response in mtp53 or p53-null cells in our panel demonstrating that besides the known p53-dependent action of RITA in wtp53 cells, RITA can induce cell death also independently of p53 in cells harboring defective p53. We identified an important role of both p38 and JNK/SAPK for sensitivity to RITA in these cells leading to a typical caspase- and BAX/BAK-dependent mitochondrial apoptosis. In conclusion, our data demonstrate that RITA can induce apoptosis through p38 and JNK/SAPK not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells, making RITA an interesting tumor-selective drug.

Isocitrate dehydrogenase 1 mutant R132H sensitizes glioma cells to BCNU-induced oxidative stress and cell death.

PubMed

Mohrenz, Isabelle Vanessa; Antonietti, Patrick; Pusch, Stefan; Capper, David; Balss, Jörg; Voigt, Sophia; Weissert, Susanne; Mukrowsky, Alicia; Frank, Jan; Senft, Christian; Seifert, Volker; von Deimling, Andreas; Kögel, Donat

2013-11-01

Isocitrate dehydrogenase 1 (IDH1) decarboxylates isocitrate to α-ketoglutarate (α-KG) leading to generation of NADPH, which is required to regenerate reduced glutathione (GSH), the major cellular ROS scavenger. Mutation of R132 of IDH1 abrogates generation of α-KG and leads to conversion of α-KG to 2-hydroxyglutarate. We hypothesized that glioma cells expressing mutant IDH1 have a diminished antioxidative capacity and therefore may encounter an ensuing loss of cytoprotection under conditions of oxidative stress. Our study was performed with LN229 cells stably overexpressing IDH1 R132H and wild type IDH1 or with a lentiviral IDH1 knockdown. Quantification of GSH under basal conditions and following treatment with the glutathione reductase inhibitor BCNU revealed significantly lower GSH levels in IDH1 R132H expressing cells and IDH1 KD cells compared to their respective controls. FACS analysis of cell death and ROS production also demonstrated an increased sensitivity of IDH1-R132H-expressing cells and IDH1 KD cells to BCNU, but not to temozolomide. The sensitivity of IDH1-R132H-expressing cells and IDH1 KD cells to ROS induction and cell death was further enhanced with the transaminase inhibitor aminooxyacetic acid and under glutamine free conditions, indicating that these cells were more addicted to glutaminolysis. Increased sensitivity to BCNU-induced ROS production and cell death was confirmed in HEK293 cells inducibly expressing the IDH1 mutants R132H, R132C and R132L. Based on these findings we propose that in addition to its established pro-tumorigenic effects, mutant IDH1 may also limit the resistance of gliomas to specific death stimuli, therefore opening new perspectives for therapy.

Ongoing cell death and immune influences on regeneration in the vestibular sensory organs

NASA Technical Reports Server (NTRS)

Warchol, M. E.; Matsui, J. I.; Simkus, E. L.; Ogilive, J. M.

2001-01-01

Hair cells in the vestibular organs of birds have a relatively short life span. Mature hair cells appear to die spontaneously and are then quickly replaced by new hair cells that arise from the division of epithelial supporting cells. A similar regenerative mechanism also results in hair cell replacement after ototoxic damage. The cellular basis of hair cell turnover in the avian ear is not understood. We are investigating the signaling pathways that lead to hair cell death and the relationship between ongoing cell death and cell production. In addition, work from our lab and others has demonstrated that the avian inner ear contains a resident population of macrophages and that enhanced numbers of macrophages are recruited to sites of hair cells lesions. Those observations suggest that macrophages and their secretory products (cytokines) may be involved in hair cell regeneration. Consistent with that suggestion, we have found that treatment with the anti-inflammatory drug dexamethasone reduces regenerative cell proliferation in the avian ear, and that certain macrophage-secreted cytokines can influence the proliferation of vestibular supporting cells and the survival of statoacoustic neurons. Those results suggest a role for the immune system in the process of sensory regeneration in the inner ear.

Network analysis of genes involved in the enhancement of hyperthermia sensitivity by the knockdown of BAG3 in human oral squamous cell carcinoma cells.

PubMed

Yunoki, Tatsuya; Tabuchi, Yoshiaki; Hayashi, Atsushi; Kondo, Takashi

2016-07-01

BCL2-associated athanogene 3 (BAG3), a co-chaperone of the heat shock 70 kDa protein (HSPA) family of proteins, is a cytoprotective protein that acts against various stresses, including heat stress. The aim of the present study was to identify gene networks involved in the enhancement of hyperthermia (HT) sensitivity by the knockdown (KD) of BAG3 in human oral squamous cell carcinoma (OSCC) cells. Although a marked elevation in the protein expression of BAG3 was detected in human the OSCC HSC-3 cells exposed to HT at 44˚C for 90 min, its expression was almost completely suppressed in the cells transfected with small interfering RNA against BAG3 (siBAG) under normal and HT conditions. The silencing of BAG3 also enhanced the cell death that was increased in the HSC-3 cells by exposure to HT. Global gene expression analysis revealed many genes that were differentially expressed by >2-fold in the cells exposed to HT and transfected with siBAG. Moreover, Ingenuity® pathways analysis demonstrated two unique gene networks, designated as Pro-cell death and Anti-cell death, which were obtained from upregulated genes and were mainly associated with the biological functions of induction and the prevention of cell death, respectively. Of note, the expression levels of genes in the Pro-cell death and Anti-cell death gene networks were significantly elevated and reduced in the HT + BAG3-KD group compared to those in the HT control group, respectively. These results provide further insight into the molecular mechanisms involved in the enhancement of HT sensitivity by the silencing of BAG3 in human OSCC cells.

Myricetin Induces Pancreatic Cancer Cell Death via the Induction of Apoptosis and Inhibition of the Phosphatidylinositol 3-Kinase (PI3K) Signaling Pathway

PubMed Central

Phillips, P.A.; Sangwan, V.; Borja-Cacho, D.; Dudeja, V.; Vickers, S.M.; Saluja, A.K.

2011-01-01

Pancreatic cancer is a the four leading cause of cancer related deaths and is adisease with poor prognosis. It is refractory to standard chemotherapeutic drugs or to novel treatment modalities, making it imperative to find new treatments. In this study, using both primary and metastatic pancreatic cancer cell lines, we have demonstrated that the flavonoid myricetin induced pancreatic cancer cell death in vitro via apoptosis, and caused a decrease in PI3 kinase activity. In vivo, treatment of orthotopic pancreatic tumors with myricetin resulted in tumor regression and decreased metastatic spread. Importantly, myricetin was non-toxic, both in vitro and in vivo, underscoring its use as a therapeutic agent against pancreatic cancer. PMID:21676539

The anti-caspase inhibitor Q-VD-OPH prevents AIDS disease progression in SIV-infected rhesus macaques.

PubMed

Laforge, Mireille; Silvestre, Ricardo; Rodrigues, Vasco; Garibal, Julie; Campillo-Gimenez, Laure; Mouhamad, Shahul; Monceaux, Valérie; Cumont, Marie-Christine; Rabezanahary, Henintsoa; Pruvost, Alain; Cordeiro-da-Silva, Anabela; Hurtrel, Bruno; Silvestri, Guido; Senik, Anna; Estaquier, Jérôme

2018-04-02

Apoptosis has been proposed as a key mechanism responsible for CD4+ T cell depletion and immune dysfunction during HIV infection. We demonstrated that Q-VD-OPH, a caspase inhibitor, inhibits spontaneous and activation-induced death of T cells from SIV-infected rhesus macaques (RMs). When administered during the acute phase of infection, Q-VD-OPH was associated with (a) reduced levels of T cell death, (b) preservation of CD4+/CD8+ T cell ratio in lymphoid organs and in the gut, (c) maintenance of memory CD4+ T cells, and (d) increased specific CD4+ T cell response associated with the expression of cytotoxic molecules. Although therapy was limited to the acute phase of infection, Q-VD-OPH-treated RMs showed lower levels of both viral load and cell-associated SIV DNA as compared with control SIV-infected RMs throughout the chronic phase of infection, and prevented the development of AIDS. Overall, our data demonstrate that Q-VD-OPH injection in SIV-infected RMs may represent an adjunctive therapeutic agent to control HIV infection and delaying disease progression to AIDS.

Gecko proteins induce the apoptosis of bladder cancer 5637 cells by inhibiting Akt and activating the intrinsic caspase cascade.

PubMed

Kim, Geun-Young; Park, Soon Yong; Jo, Ara; Kim, Mira; Leem, Sun-Hee; Jun, Woo-Jin; Shim, Sang In; Lee, Sang Chul; Chung, Jin Woong

2015-09-01

Gecko proteins have long been used as anti-tumor agents in oriental medicine, without any scientific background. Although anti-tumor effects of Gecko proteins on several cancers were recently reported, their effect on bladder cancer has not been investigated. Thus, we explored the anti-tumor effect of Gecko proteins and its cellular mechanisms in human bladder cancer 5637 cells. Gecko proteins significantly reduced the viability of 5637 cells without any cytotoxic effect on normal cells. These proteins increased the Annexin-V staining and the amount of condensed chromatin, demonstrating that the Gecko proteinsinduced cell death was caused by apoptosis. Gecko proteins suppressed Akt activation, and the overexpression of constitutively active form of myristoylated Akt prevented Gecko proteins-induced death of 5637 cells. Furthermore, Gecko proteins activated caspase 9 and caspase 3/7. Taken together, our data demonstrated that Gecko proteins suppressed the Akt pathway and activated the intrinsic caspase pathway, leading to the apoptosis of bladder cancer cells. [BMB Reports 2015; 48(9): 531-536].

Sonic hedgehog is required for survival of both myogenic and chondrogenic somitic lineages.

PubMed

Teillet, M; Watanabe, Y; Jeffs, P; Duprez, D; Lapointe, F; Le Douarin, N M

1998-06-01

In vertebrates, the medial moieties of the somites give rise to the vertebrae and epaxial muscles, which develop in close relationship with the axial organs, neural tube and notochord. The lateral moieties contribute to the ribs and to limb and body wall muscles (hypaxial muscles) after a phase of lateral and ventral migration. Surgical ablation of the neural tube and notochord in the chick embryo during segmentation and early differentiation of the somites (day 2 of incubation) does not affect primary development of the hypaxial muscles, but leads to a complete absence of epaxial muscles, vertebrae and ribs, due to cell death in the somites. Here we demonstrate that cell death, which occurs within 24 hours of excision of the axial organs, affects both myogenic and chondrogenic cell lineages defined, respectively, by the expression of MyoD and Pax-1 genes. In contrast, Pax-3 transcripts, normally present in cells giving rise to hypaxial muscles, are preserved in the excised embryos. Backgrafting either the ventral neural tube or the notochord allows survival of MyoD- and Pax-1-expressing cells. Similarly, Sonic hedgehog-producing cells grafted in place of axial organs also rescue MyoD- and Pax-1-expressing cells from death and allow epaxial muscles, ribs and vertebrae to undergo organogenesis. These results demonstrate that the ventral neural tube and the notochord promote the survival of both myogenic and chondrogenic cell lineages in the somites and that this action is mediated by Sonic hedgehog.

Induction of Mitotic Cell Death by Overriding G2/M Checkpoint in Endometrial Cancer Cells with Non-functional p53

PubMed Central

Meng, Xiangbing; Laidler, Laura L.; Kosmacek, Elizabeth A.; Yang, Shujie; Xiong, Zhi; Zhu, Danlin; Wang, Xinjun; Dai, Donghai; Zhang, Yuping; Wang, Xiaofang; Brachova, Pavla; Albitar, Lina; Liu, Dawei; Ianzini, Fiorenza; Mackey, Michael A.; Leslie, Kimberly K.

2012-01-01

Objective Endometrial tumors with non-functional p53, such as serous uterine endometrial carcinomas, are aggressive malignancies with a poor outcome, yet they have an Achilles’ heel: due to loss of p53 function, these tumors may be sensitive to treatments which abrogate the G2/M checkpoint. Our objective was to exploit this weakness to induce mitotic cell death using two strategies: (1) EGFR inhibitor gefitinib combined with paclitaxel to arrest cells at mitosis, or (2) BI2536, an inhibitor of polo-like kinase 1 (PLK1), to block PLK1 activity. Methods We examined the impact of combining gefitinib and paclitaxel or PLK1 inhibitor on expression of G2/M checkpoint controllers, cell viability, and cell cycle progression in endometrial cancer cells with mutant p53. Results In cells lacking normal p53 activity, each treatment activated CDC25C and inactivated Wee1, which in turn activated cdc2 and sent cells rapidly through the G2/M checkpoint and into mitosis. Live cell imaging demonstrated irreversible mitotic arrest and eventual cell death. Combinatorial therapy with paclitaxel and gefitinib was highly synergistic and resulted in a 10-fold reduction in the IC50 for paclitaxel, from 14 nM as a single agent to 1.3 nM in the presence of gefitinib. However, BI2536 alone at low concentrations (5 nM) was the most effective treatment and resulted in massive mitotic cell death. In a xenograft mouse model with p53-deficient cells, low dose BI2536 significantly inhibited tumor growth. Conclusions These findings reveal induction of mitotic cell death as a therapeutic strategy for endometrial tumors lacking functional p53. PMID:23146687

Curcumin inhibits autocrine growth hormone-mediated invasion and metastasis by targeting NF-κB signaling and polyamine metabolism in breast cancer cells.

PubMed

Coker-Gurkan, Ajda; Celik, Merve; Ugur, Merve; Arisan, Elif-Damla; Obakan-Yerlikaya, Pinar; Durdu, Zeynep Begum; Palavan-Unsal, Narcin

2018-05-16

Curcumin is assumed to be a plant-derived therapeutic drug that triggers apoptotic cell death in vitro and in vivo by affecting different molecular targets such as NF-κB. Phase I/II trial of curcumin alone or with chemotherapeutic drugs has been accomplished in pancreatic, colon, prostate and breast cancer cases. Recently, autocrine growth hormone (GH) signaling-induced cell growth, metastasis and drug resistance have been demonstrated in breast cancer. In this study, our aim was to investigate the potential therapeutic effect of curcumin by evaluating the molecular machinery of curcumin-triggered apoptotic cell death via focusing on NF-κB signaling and polyamine (PA) metabolism in autocrine GH-expressing MCF-7, MDA-MB-453 and MDA-MB-231 breast cancer cells. For this purpose, a pcDNA3.1 (+) vector with a GH gene insert was transfected by a liposomal agent in all breast cancer cells and then selection was conducted in neomycin (G418) included media. Autocrine GH-induced curcumin resistance was overcome in a dose-dependent manner and curcumin inhibited cell proliferation, invasion-metastasis and phosphorylation of p65 (Ser536), and thereby partly prevented its DNA binding activity in breast cancer cells. Moreover, curcumin induced caspase-mediated apoptotic cell death by activating the PA catabolic enzyme expressions, which led to generation of toxic by-products such as H 2 O 2 in MCF-7, MDA-MB-453 and MDA-MB-231 GH+ breast cancer cells. In addition, transient silencing of SSAT prevented curcumin-induced cell viability loss and apoptotic cell death in each breast cancer cells. In conclusion, curcumin could overcome the GH-mediated resistant phenotype via modulating cell survival, death-related signaling routes and activating PA catabolic pathway.

The Btk-dependent PIP5K1γ lipid kinase activation by Fas counteracts FasL-induced cell death.

PubMed

Rossin, Aurélie; Lounnas, Nadia; Durivault, Jérôme; Miloro, Giorgia; Gagnoux-Palacios, Laurent; Hueber, Anne-Odile

2017-11-01

The Fas/FasL system plays a critical role in death by apoptosis and immune escape of cancer cells. The Fas receptor being ubiquitously expressed in tissues, its apoptotic-inducing function, initiated upon FasL binding, is tightly regulated by several negative regulatory mechanisms to prevent inappropriate cell death. One of them, involving the non-receptor tyrosine kinase Btk, was reported mainly in B cells and only poorly described. We report here that Btk negatively regulates, through its tyrosine kinase activity, the FasL-mediated cell death in epithelial cell lines from colon cancer origin. More importantly, we show that Btk interacts not only with Fas but also with the phosphatidylinositol-4-phosphate 5-kinase, PIP5K1γ, which, upon stimulation by Fas ligand, is responsible of a rapid and transient synthesis of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ). This production requires both the presence and the tyrosine kinase activity of Btk, and participates in the negative regulation of FasL-mediated cell death since knocking down PIP5K1γ expression significantly strengthens the apoptotic signal upon FasL engagement. Altogether, our data demonstrate the cooperative role of Btk and PIP5K1γ in a FasL-induced PI(4,5)P 2 production, both proteins participating to the threshold setting of FasL-induced apoptotic commitment in colorectal cell lines.

Human Cytomegalovirus Protein pUL38 Prevents Premature Cell Death by Binding to Ubiquitin-Specific Protease 24 and Regulating Iron Metabolism.

PubMed

Sun, Yamei; Bao, Qunchao; Xuan, Baoqin; Xu, Wenjia; Pan, Deng; Li, Qi; Qian, Zhikang

2018-07-01

Human cytomegalovirus (HCMV) protein pUL38 has been shown to prevent premature cell death by antagonizing cellular stress responses; however, the underlying mechanism remains unknown. In this study, we identified the host protein ubiquitin-specific protease 24 (USP24) as an interaction partner of pUL38. Mutagenesis analysis of pUL38 revealed that amino acids TFV at positions 227 to 230 were critical for its interaction with USP24. Mutant pUL38 TFV/AAA protein did not bind to USP24 and failed to prevent cell death induced by pUL38-deficient HCMV infection. Knockdown of USP24 suppressed the cell death during pUL38-deficient HCMV infection, suggesting that pUL38 achieved its function by antagonizing the function of USP24. We investigated the cellular pathways regulated by USP24 that might be involved in the cell death phenotype by testing several small-molecule compounds known to have a protective effect during stress-induced cell death. The iron chelators ciclopirox olamine and Tiron specifically protected cells from pUL38-deficient HCMV infection-induced cell death, thus identifying deregulated iron homeostasis as a potential mechanism. Protein levels of nuclear receptor coactivator 4 (NCOA4) and lysosomal ferritin degradation, a process called ferritinophagy, were also regulated by pUL38 and USP24 during HCMV infection. Knockdown of USP24 decreased NCOA4 protein stability and ferritin heavy chain degradation in lysosomes. Blockage of ferritinophagy by genetic inhibition of NCOA4 or Atg5/Atg7 prevented pUL38-deficient HCMV infection-induced cell death. Overall, these results support the hypothesis that pUL38 binds to USP24 to reduce ferritinophagy, which may then protect cells from lysosome dysfunction-induced cell death. IMPORTANCE Premature cell death is considered a first line of defense against various pathogens. Human cytomegalovirus (HCMV) is a slow-replicating virus that encodes several cell death inhibitors, such as pUL36 and pUL37x1, which allow it to overcome both extrinsic and intrinsic mitochondrion-mediated apoptosis. We previously identified HCMV protein pUL38 as another virus-encoded cell death inhibitor. In this study, we demonstrated that pUL38 achieved its activity by interacting with and antagonizing the function of the host protein ubiquitin-specific protease 24 (USP24). pUL38 blocked USP24-mediated ferritin degradation in lysosomes, which could otherwise be detrimental to the lysosome and initiate cell death. These novel findings suggest that iron metabolism is finely tuned during HCMV infection to avoid cellular toxicity. The results also provide a solid basis for further investigations of the role of USP24 in regulating iron metabolism during infection and other diseases. Copyright © 2018 American Society for Microbiology.

The influence of proteasome inhibitor MG132, external radiation and unlabeled antibody on the tumor uptake and biodistribution of 188Re-labeled anti-E6 C1P5 antibody in cervical cancer in mice

PubMed Central

Phaeton, Rébécca; Wang, Xing Guo; Einstein, Mark H.; Goldberg, Gary L.; Casadevall, Arturo; Dadachova, Ekaterina

2009-01-01

Background Human Papillomavirus (HPV) infection is considered a necessary step for the development of cervical cancer and >95% of all cervical cancers have detectable HPV sequences. We have recently demonstrated the efficacy of radioimmunotherapy (RIT) which targeted viral oncoprotein E6 in treatment of experimental cervical cancer We hypothesized that pre-treatment of tumor cells with various agents which cause cell death and/or elevation of E6 levels would increase the accumulation of radiolabeled antibodies to E6 in cervical tumors. Methods HPV-16 positive CasKi cells were treated in vitro with up to 6 Gy of external radiation, or proteasome inhibitor MG-132 or unlabeled anti-E6 antibody C1P5 and cell death was assessed. Biodistribution of 188Rhenium (188Re)-labeled C1P5 antibody was performed in both control and radiation MG-132 treated CasKi tumor-bearing nude mice. Results . 188Re-C1P5 antibody demonstrated tumor specificity and very low uptake and fast clearance from the major organs. The amount of tumor uptake was enhanced by MG-132 but was unaffected by pre-treatment with radiation. In addition, in vitro studies demonstrated an unanticipated effect of unlabeled antibody on the amount of cell death, a finding that was suggested by our previous in vivo studies in CasKi tumor model. Conclusion We demonstrated that pre-treatment of cervical tumors with proteasome inhibitor MG-132 and with unlabeled antibody to E6 can serve as a means to generate non-viable cancer cells and to elevate the levels of target oncoproteins in the cells for increasing the accumulation of targeted radiolabeled antibodies in tumors. These results favor further development of RIT of cervical cancers targeting viral antigens. PMID:20127955

Heat-shock protein 27 (HSP27, HSPB1) is synthetic lethal to cells with oncogenic activation of MET, EGFR and BRAF.

PubMed

Konda, John D; Olivero, Martina; Musiani, Daniele; Lamba, Simona; Di Renzo, Maria F

2017-06-01

The small heat-shock protein of 27 kDa (HSP27) is highly expressed in many cancers and is associated with aggressive tumour behaviour, metastasis, poor prognosis and resistance to chemotherapy. We aimed at assessing the role of HSP27 in modulating responses to target therapies. We selected several oncogene-addicted cancer cell lines, which undergo either cell cycle blockade or cell death in response to agents that target the specific oncogene. Surprisingly, HSP27 suppression alone resulted in the apoptotic death of MET-addicted EBC-1 lung cancer cells, epidermal growth factor receptor (EGFR)-addicted colorectal carcinoma (CRC) DiFi cells and BRAF-addicted CRC COLO205 and OXCO-1 and melanoma COLO741 cells, all of which also undergo death when treated with the specific targeted agent. In other cell lines, such as MET-addicted gastric carcinoma MKN45 and EGFR-addicted CRC SW48 lines, where oncogene inhibition only blocked proliferation, HSP27 knockdown made targeted agents switch from cytostatic to cytotoxic activity. Mechanistically, the more the cells were susceptible to HSP27 suppression, the more they were primed for death, as demonstrated by increased levels of mitochondrial outer membrane permeabilization. Priming for death was accompanied by the increase in pro-apoptotic proteins of the BCL2 family and of active caspase-3 and lamin B. Together, these data suggest that oncogene-addicted cells require HSP27 for survival and that HSP27 might interfere with the effectiveness of targeted agents. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Bone marrow stromal-B cell interactions in polycyclic aromatic hydrocarbon-induced pro/pre-B cell apoptosis.

PubMed

Allan, Lenka L; Mann, Koren K; Matulka, Raymond A; Ryu, Heui-Young; Schlezinger, Jennifer J; Sherr, David H

2003-12-01

Environmental polycyclic aromatic hydrocarbons (PAH) and related halogenated hydrocarbons are immunotoxic in a variety of systems. In a model system of B lymphopoiesis, PAH exposure rapidly induces apoptosis in CD43- pre-B and CD43+ pro/pre-B cells. Apoptosis induction by 7,12-dimethylbenzo[a]anthracene (DMBA) is dependent upon AhR+ bone marrow stromal cells and likely involves DMBA metabolism within the stromal cell. However, it is not known if PAH-treated stromal cells release free metabolites or soluble factors that may directly induce B cell death or if the effector death signal is delivered by stromal cell-B cell contact. Here, we demonstrate that supernatants from DMBA-treated bone marrow stromal cells contain an activity capable of inducing apoptosis in pro/pre-B cells cocultured with stromal cells. This activity (1) is not produced when stromal cells are cotreated with DMBA and alpha-naphthoflavone (alpha-NF), an aryl hydrocarbon receptor (AhR) and cytochrome P-450 inhibitor, (2) is > or = 50 kDa, (3) is trypsin and heat sensitive, and (4) is dependent on AhR+ stromal cells, which in turn deliver the effector death signal to pro/pre-B cells. The results (1) argue against a role for a soluble, stromal cell-derived cytokine as the effector of PAH-induced pro/pre-B cell death, (2) exclude the possibility of a free metabolite acting directly on AhR- pro/pre-B cell targets, and (3) suggest the elaboration by stromal cells of a relatively stable, DMBA metabolite-protein complex capable of acting on other stromal cells at some distance. Collectively, these studies suggest that, while stromal cell products, e.g., metabolite-protein complexes, may affect the function of distant stromal cells, the effector death signal delivered by stromal cells to bone marrow B cells is mediated by cell-cell contact.

Differential PKA activation and AKAP association determines cell fate in cancer cells

PubMed Central

2013-01-01

Background The dependence of malignant properties of colorectal cancer (CRC) cells on IGF1R signaling has been demonstrated and several IGF1R antagonists are currently in clinical trials. Recently, we identified a novel pathway in which cAMP independent PKA activation by TGFβ signaling resulted in the destabilization of survivin/XIAP complex leading to increased cell death. In this study, we evaluated the effect of IGF1R inhibition or activation on PKA activation and its downstream cell survival signaling mechanisms. Methods Small molecule IGF1R kinase inhibitor OSI-906 was used to test the effect of IGF1R inhibition on PKA activation, AKAP association and its downstream cell survival signaling. In a complementary approach, ligand mediated activation of IGF1R was performed and AKAP/PKA signaling was analyzed for their downstream survival effects. Results We demonstrate that the inhibition of IGF1R in the IGF1R-dependent CRC subset generates cell death through a novel mechanism involving TGFβ stimulated cAMP independent PKA activity that leads to disruption of cell survival by survivin/XIAP mediated inhibition of caspase activity. Importantly, ligand mediated activation of the IGF1R in CRC cells results in the generation of cAMP dependent PKA activity that functions in cell survival by inhibiting caspase activity. Therefore, this subset of CRC demonstrates 2 opposing pathways organized by 2 different AKAPs in the cytoplasm that both utilize activation of PKA in a manner that leads to different outcomes with respect to life and death. The cAMP independent PKA activation pathway is dependent upon mitochondrial AKAP149 for its apoptotic functions. In contrast, Praja2 (Pja2), an AKAP-like E3 ligase protein was identified as a key element in controlling cAMP dependent PKA activity and pro-survival signaling. Genetic manipulation of AKAP149 and Praja2 using siRNA KD had opposing effects on PKA activity and survivin/XIAP regulation. Conclusions We had identified 2 cytoplasmic pathways dependent upon the same enzymatic activity with opposite effects on cell fate in terms of life and death. Understanding the specific mechanistic functions of IGF1R with respect to determining the PKA survival functions would have potential for impact upon the development of new therapeutic strategies by exploiting the IGF1R/cAMP-PKA survival signaling in cancer. PMID:24083380

Neuroprotective effects of hydrogen sulfide on sodium azide‑induced autophagic cell death in PC12 cells.

PubMed

Shan, Haiyan; Chu, Yang; Chang, Pan; Yang, Lijun; Wang, Yi; Zhu, Shaohua; Zhang, Mingyang; Tao, Luyang

2017-11-01

Sodium azide (NaN3) is a chemical of rapidly growing commercial importance. It is very acutely toxic and inhibits cytochrome oxidase (COX) by binding irreversibly to the heme cofactor. A previous study from our group demonstrated that hydrogen sulfide (H2S), the third endogenous gaseous mediator identified, had protective effects against neuronal damage induced by traumatic brain injury (TBI). It is well‑known that TBI can reduce the activity of COX and have detrimental effects on the central nervous system metabolism. Therefore, in the present study, it was hypothesized that H2S may provide neuroprotection against NaN3 toxicity. The current results revealed that NaN3 treatment induced non‑apoptotic cell death, namely autophagic cell death, in PC12 cells. Expression of the endogenous H2S‑producing enzymes, cystathionine‑β‑synthase and 3‑mercaptopyruvate sulfurtransferase, decreased in a dose‑dependent manner following NaN3 treatment. Pretreatment with H2S markedly attenuated the NaN3‑induced cell viability loss and autophagic cell death in a dose‑dependent manner. The present study suggests that H2S‑based strategies may have future potential in the prevention and/or therapy of neuronal damage following NaN3 exposure.

Critical Involvement of Extracellular ATP Acting on P2RX7 Purinergic Receptors in Photoreceptor Cell Death

PubMed Central

Notomi, Shoji; Hisatomi, Toshio; Kanemaru, Takaaki; Takeda, Atsunobu; Ikeda, Yasuhiro; Enaida, Hiroshi; Kroemer, Guido; Ishibashi, Tatsuro

2011-01-01

Stressed cells release ATP, which participates in neurodegenerative processes through the specific ligation of P2RX7 purinergic receptors. Here, we demonstrate that extracellular ATP and the more specific P2RX7 agonist, 2′- and 3′-O-(4-benzoylbenzoyl)-ATP, both induce photoreceptor cell death when added to primary retinal cell cultures or when injected into the eyes from wild-type mice, but not into the eyes from P2RX7−/− mice. Photoreceptor cell death was accompanied by the activation of caspase-8 and -9, translocation of apoptosis-inducing factor from mitochondria to nuclei, and TUNEL-detectable chromatin fragmentation. All hallmarks of photoreceptor apoptosis were prevented by premedication or co-application of Brilliant Blue G, a selective P2RX7 antagonist that is already approved for the staining of internal limiting membranes during ocular surgery. ATP release is up-regulated by nutrient starvation in primary retinal cell cultures and seems to be an initializing event that triggers primary and/or secondary cell death via the positive feedback loop on P2RX7. Our results encourage the potential application of Brilliant Blue G as a novel neuroprotective agent in retinal diseases or similar neurodegenerative pathologies linked to excessive extracellular ATP. PMID:21983632

Protective effects of transduced Tat-DJ-1 protein against oxidative stress and ischemic brain injury.

PubMed

Jeong, Hoon Jae; Kim, Dae Won; Kim, Mi Jin; Woo, Su Jung; Kim, Hye Ri; Kim, So Mi; Jo, Hyo Sang; Hwang, Hyun Sook; Kim, Duk Soo; Cho, Sung Woo; Won, Moo Ho; Han, Kyu Hyung; Park, Jin Seu; Eum, Won Sik; Choi, Soo Young

2012-10-31

Reactive oxygen species (ROS) contribute to the development of a number of neuronal diseases including ischemia. DJ-1, also known to PARK7, plays an important role in transcriptional regulation, acting as molecular chaperone and antioxidant. In the present study, we investigated whether DJ-1 protein shows a protective effect against oxidative stress-induced neuronal cell death in vitro and in ischemic animal models in vivo. To explore DJ-1 protein's potential role in protecting against ischemic cell death, we constructed cell permeable Tat-DJ-1 fusion proteins. Tat-DJ-1 protein efficiently transduced into neuronal cells in a doseand time-dependent manner. Transduced Tat-DJ-1 protein increased cell survival against hydrogen peroxide (H2O2) toxicity and also reduced intracellular ROS. In addition, Tat-DJ-1 protein inhibited DNA fragmentation induced by H2O2. Furthermore, in animal models, immunohistochemical analysis revealed that Tat-DJ-1 protein prevented neuronal cell death induced by transient forebrain ischemia in the CA1 region of the hippocampus. These results demonstrate that transduced Tat-DJ-1 protein protects against cell death in vitro and in vivo, suggesting that the transduction of Tat-DJ-1 may be useful as a therapeutic agent for ischemic injuries related to oxidative stress.

Cell signaling pathways in the mechanisms of neuroprotection afforded by bergamot essential oil against NMDA-induced cell death in vitro

PubMed Central

Corasaniti, M T; Maiuolo, J; Maida, S; Fratto, V; Navarra, M; Russo, R; Amantea, D; Morrone, L A; Bagetta, G

2007-01-01

Background and purpose: The effects of bergamot essential oil (BEO; Citrus bergamia, Risso) on excitotoxic neuronal damage was investigated in vitro. Experimental approach: The study was performed in human SH-SY5Y neuroblastoma cells exposed to N-methyl-D-aspartate (NMDA). Cell viability was measured by dye exclusion. Reactive oxygen species (ROS) and caspase-3 activity were measured fluorimetrically. Calpain I activity and the activation (phosphorylation) of Akt and glycogen synthase kinase-3β (GSK-3β) were assayed by Western blotting. Key results: NMDA induced concentration-dependent, receptor-mediated, death of SH-SY5Y cells, ranging from 11 to 25% (0.25–5 mM). Cell death induced by 1 mM NMDA (21%) was preceded by a significant accumulation of intracellular ROS and by a rapid activation of the calcium-activated protease calpain I. In addition, NMDA caused a rapid deactivation of Akt kinase and this preceded the detrimental activation of the downstream kinase, GSK-3β. BEO (0.0005–0.01%) concentration dependently reduced death of SH-SY5Y cells caused by 1 mM NMDA. In addition to preventing ROS accumulation and activation of calpain, BEO (0.01%) counteracted the deactivation of Akt and the consequent activation of GSK-3β, induced by NMDA. Results obtained by using specific fractions of BEO, suggested that monoterpene hydrocarbons were responsible for neuroprotection afforded by BEO against NMDA-induced cell death. Conclusions and Implications: Our data demonstrate that BEO reduces neuronal damage caused in vitro by excitotoxic stimuli and that this neuroprotection was associated with prevention of injury-induced engagement of critical death pathways. PMID:17401440

TNF-alpha induction of GM2 expression on renal cell carcinomas promotes T cell dysfunction.

PubMed

Raval, Gira; Biswas, Soumika; Rayman, Patricia; Biswas, Kaushik; Sa, Gaurisankar; Ghosh, Sankar; Thornton, Mark; Hilston, Cynthia; Das, Tanya; Bukowski, Ronald; Finke, James; Tannenbaum, Charles S

2007-05-15

Previous studies from our laboratory demonstrated the role of tumor-derived gangliosides as important mediators of T cell apoptosis, and hence, as one mechanism by which tumors evade immune destruction. In this study, we report that TNF-alpha secreted by infiltrating inflammatory cells and/or genetically modified tumors augments tumor-associated GM2 levels, which leads to T cell death and immune dysfunction. The conversion of weakly apoptogenic renal cell carcinoma (RCC) clones to lines that can induce T cell death requires 3-5 days of TNF-alpha pretreatment, a time frame paralleling that needed for TNF-alpha to stimulate GM2 accumulation by SK-RC-45, SK-RC-54, and SK-RC-13. RCC tumor cell lines permanently transfected with the TNF-alpha transgene are similarly toxic for T lymphocytes, which correlates with their constitutively elevated levels of GM2. TNF-alpha increases GM2 ganglioside expression by enhancing the mRNA levels encoding its synthetic enzyme, GM2 synthase, as demonstrated by both RT-PCR and Southern analysis. The contribution of GM2 gangliosides to tumor-induced T cell death was supported by the finding that anti-GM2 Abs significantly blocked T cell apoptosis mediated by TNF-alpha-treated tumor cells, and by the observation that small interfering RNA directed against TNF-alpha abrogated GM2 synthase expression by TNF-transfected SK-RC-45, diminished its GM2 accumulation, and inhibited its apoptogenicity for T lymphocytes. Our results indicate that TNF-alpha signaling promotes RCC-induced killing of T cells by stimulating the acquisition of a distinct ganglioside assembly in RCC tumor cells.

Mitofusin 1 degradation is induced by a disruptor of mitochondrial calcium homeostasis, CGP37157: a role in apoptosis in prostate cancer cells.

PubMed

Choudhary, Vivek; Kaddour-Djebbar, Ismail; Alaisami, Rabei; Kumar, M Vijay; Bollag, Wendy B

2014-05-01

Mitochondria constantly divide (mitochondrial fission) and fuse (mitochondrial fusion) in a normal cell. Disturbances in the balance between these two physiological processes may lead to cell dysfunction or to cell death. Induction of cell death is the prime goal of prostate cancer chemotherapy. Our previous study demonstrated that androgens increase the expression of a mitochondrial protein involved in fission and facilitate an apoptotic response to CGP37157 (CGP), an inhibitor of mitochondrial calcium efflux, in prostate cancer cells. However, the regulation and role of mitochondrial fusion proteins in the death of these cells have not been examined. Therefore, our objective was to investigate the effect of CGP on a key mitochondrial fusion protein, mitofusin 1 (Mfn1), and the role of Mfn1 in prostate cancer cell apoptosis. We used various prostate cancer cell lines and western blot analysis, qRT-PCR, siRNA, M30 apoptosis assay and immunoprecipitation techniques to determine mechanisms regulating Mfn1. Treatment of prostate cancer cells with CGP resulted in selective degradation of Mfn1. Mfn1 ubiquitination was detected following immunoprecipitation of overexpressed Myc-tagged Mfn1 protein from CGP-treated cells, and treatment with the proteasomal inhibitor lactacystin, as well as siRNA-mediated knockdown of the E3 ubiquitin ligase March5, protected Mfn1 from CGP-induced degradation. These data indicate the involvement of the ubiquitin-proteasome pathway in CGP-induced degradation of Mfn1. We also demonstrated that downregulation of Mfn1 by siRNA enhanced the apoptotic response of LNCaP cells to CGP, suggesting a likely pro-survival role for Mfn1 in these cells. Our results suggest that manipulation of mitofusins may provide a novel therapeutic advantage in treating prostate cancer.

Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide

PubMed Central

Barquero-Calvo, Elías; Mora-Cartín, Ricardo; Arce-Gorvel, Vilma; de Diego, Juana L.; Chacón-Díaz, Carlos; Chaves-Olarte, Esteban; Guzmán-Verri, Caterina; Buret, Andre G.; Gorvel, Jean-Pierre; Moreno, Edgardo

2015-01-01

Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1β by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN. PMID:25946018

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mota, Alba, E-mail: amota@iib.uam.es; Jiménez-Garcia, Lidia, E-mail: ljimenez@isciii.es; Herránz, Sandra, E-mail: sherranz@isciii.es

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 hadmore » 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 TRAIL-induced apoptosis through upregulation of death receptors.« less

Cell death and renewal during prey capture and digestion in the carnivorous sponge Asbestopluma hypogea (Porifera: Poecilosclerida).

PubMed

Martinand-Mari, Camille; Vacelet, Jean; Nickel, Michael; Wörheide, Gert; Mangeat, Paul; Baghdiguian, Stephen

2012-11-15

The sponge Asbestopluma hypogea is unusual among sponges due to its peculiar carnivorous feeding habit. During various stages of its nutrition cycle, the sponge is subjected to spectacular morphological modifications. Starved animals are characterized by many elongated filaments, which are crucial for the capture of prey. After capture, and during the digestion process, these filaments actively regress before being regenerated during a subsequent period of starvation. Here, we demonstrate that these morphological events rely on a highly dynamic cellular turnover, implying a coordinated sequence of programmed cell death (apoptosis and autophagy), cell proliferation and cell migration. A candidate niche for cell renewal by stem cell proliferation and differentiation was identified at the base of the sponge peduncle, characterized by higher levels of BrdU/EdU incorporation. Therefore, BrdU/EdU-positive cells of the peduncle base are candidate motile cells responsible for the regeneration of the prey-capturing main sponge body, i.e. the dynamic filaments. Altogether, our results demonstrate that dynamics of cell renewal in sponge appear to be regulated by cellular mechanisms as multiple and complex as those already identified in bilaterian metazoans.

Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex

PubMed Central

Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.; James, Ho C. S.; Rydström, Anna; Ngassam, Viviane N.; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N.; Svanborg, Catharina

2015-01-01

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ‘’protein-centric” view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ‘’receptor independent” transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death. PMID:26561036

Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

PubMed

Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

2015-11-12

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

PD-1 regulates extrathymic regulatory T-cell differentiation

PubMed Central

Chen, Xiufen; Fosco, Dominick; Kline, Douglas E.; Meng, Liping; Nishi, Saki; Savage, Peter A.; Kline, Justin

2014-01-01

Regulatory T (Treg) cells and the programmed death-1/programmed death ligand-1 (PD-1/PD-L1) pathway are both critical for maintaining peripheral tolerance to self antigens. A significant subset of Treg cells constitutively expresses PD-1, which prompted an investigation into the role of PD-1/PD-L1 interactions in Treg-cell development, function and induction in vivo. The phenotype and abundance of Treg cells was not significantly altered in PD-1-deficient mice. The thymic development of polyclonal and monospecific Treg cells was not negatively impacted by PD-1 deficiency. The suppressive function of PD-1−/− Treg cells was similar to their PD-1+/+ counterparts both in vitro and in vivo. However, in three different in vivo experimental settings, PD-1−/− conventional CD4+ T cells demonstrated a strikingly diminished tendency toward differentiation into peripherally induced Treg (pTreg) cells. Our results demonstrate that PD-1 is dispensable for thymic (tTreg) Treg-cell development and suppressive function, but is critical for the extrathymic differentiation of pTreg cells in vivo. These data suggest that antibody blockade of the PD-1/PD-L1 pathway may augment T-cell responses by acting directly on conventional T cells, and also by suppressing the differentiation of pTreg cells. PMID:24975127

Gasdermin D: the long-awaited executioner of pyroptosis.

PubMed

Man, Si Ming; Kanneganti, Thirumala-Devi

2015-11-01

Inflammatory caspases drive a lytic form of cell death called pyroptosis in response to microbial infection and endogenous damage-associated signals. Two studies now demonstrate that cleavage of the substrate gasdermin D by inflammatory caspases necessitates eventual pyroptotic demise of a cell.

Antitumor agent 25-epi Ritterostatin GN1N induces endoplasmic reticulum stress and autophagy mediated cell death in melanoma cells.

PubMed

Riaz Ahmed, Kausar Begam; Kanduluru, Ananda Kumar; Feng, Li; Fuchs, Philip L; Huang, Peng

2017-05-01

Metastatic melanoma is the most aggressive of all skin cancers and is associated with poor prognosis owing to lack of effective treatments. 25-epi Ritterostatin GN1N is a novel antitumor agent with yet undefined mechanisms of action. We sought to delineate the antitumor mechanisms of 25-epi Ritterostatin GN1N in melanoma cells to determine the potential of this compound as a treatment for melanoma. Activation of the endoplasmic reticulum (ER) stress protein glucose-regulated protein 78 (GRP78) has been associated with increased melanoma progression, oncogenic signaling, drug resistance, and suppression of cell death. We found that 25-epi Ritterostatin GN1N induced cell death in melanoma cells at nanomolar concentrations, and this cell death was characterized by inhibition of GRP78 expression, increased expression of the ER stress marker CHOP, loss of mitochondrial membrane potential, and lipidation of the autophagy marker protein LC3B. Importantly, normal melanocytes exhibited limited sensitivity to 25-epi Ritterostatin GN1N. Subsequent in vivo results demonstrated that 25-epi Ritterostatin GN1N reduced melanoma growth in mouse tumor xenografts and did not affect body weight, suggesting minimal toxicity. In summary, our findings indicate that 25-epi Ritterostatin GN1N causes ER stress and massive autophagy, leading to collapse of mitochondrial membrane potential and cell death in melanoma cells, with minimal effects in normal melanocytes. Thus, 25-epi Ritterostatin GN1N is a promising anticancer agent that warrants further investigation.

RNA interference-mediated silencing of mutant superoxide dismutase rescues cyclosporin A-induced death in cultured neuroblastoma cells

PubMed Central

Maxwell, Michele M.; Pasinelli, Piera; Kazantsev, Aleksey G.; Brown, Robert H.

2004-01-01

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder resulting from selective death of motor neurons in the brain and spinal cord. In ≈25% of familial ALS cases, the disease is caused by dominantly acting point mutations in the gene encoding cytosolic Cu,Zn superoxide dismutase (SOD1). In cell culture and in rodent models of ALS, mutant SOD1 proteins exhibit dose-dependent toxicity; thus, agents that reduce mutant protein expression would be powerful therapeutic tools. A wealth of recent evidence has demonstrated that the mechanism of RNA-mediated interference (RNAi) can be exploited to achieve potent and specific gene silencing in vitro and in vivo. We have evaluated the utility of RNAi for selective silencing of mutant SOD1 expression in cultured cells and have identified small interfering RNAs capable of specifically inhibiting expression of ALS-linked mutant, but not wild-type, SOD1. We have investigated the functional effects of RNAi-mediated silencing of mutant SOD1 in cultured murine neuroblastoma cells. In this model, stable expression of mutant, but not wild-type, human SOD1 sensitizes cells to cytotoxic stimuli. We find that silencing of mutant SOD1 protects these cells against cyclosporin A-induced cell death. These results demonstrate a positive physiological effect caused by RNAi-mediated silencing of a dominant disease allele. The present study further supports the therapeutic potential of RNAi-based methods for the treatment of inherited human diseases, including ALS. PMID:14981234

Inositol pyrophosphates mediate the DNA-PK/ATM-p53 cell death pathway by regulating CK2 phosphorylation of Tti1/Tel2

PubMed Central

Rao, Feng; Cha, Jiyoung; Xu, Jing; Xu, Risheng; Vandiver, M. Scott; Tyagi, Richa; Tokhunts, Robert; Koldobskiy, Michael A.; Fu, Chenglai; Barrow, Roxanne; Wu, Mingxuan; Fiedler, Dorothea; Barrow, James C.; Snyder, Solomon H.

2014-01-01

The apoptotic actions of p53 require its phosphorylation by a family of phosphoinositide-3-kinase-related-kinases (PIKKs), which include DNA-PKcs and ATM. These kinases are stabilized by the TTT (Tel2, Tti1, Tti2) co-chaperone family, whose actions are mediated by CK2 phosphorylation. The inositol pyrophosphates, such as 5-diphosphoinositol pentakisphosphate (IP7), are generated by a family of inositol hexakisphosphate kinases (IP6Ks) of which IP6K2 has been implicated in p53-associated cell death. In the present study we report a novel apoptotic signaling cascade linking CK2, TTT, the PIKKs, and p53. We demonstrate that IP7, formed by IP6K2, binds CK2 to enhance its phosphorylation of the TTT complex thereby stabilizing DNA-PKcs and ATM. This process stimulates p53 phosphorylation at serine-15 to activate the cell death program in human cancer cells and in murine B cells. PMID:24657168

Bioenergetic metabolites regulate base excision repair dependent cell death in response to DNA damage

PubMed Central

Tang, Jiang-bo; Goellner, Eva M.; Wang, Xiao-hong; Trivedi, Ram N.; Croix, Claudette M. St; Jelezcova, Elena; Svilar, David; Brown, Ashley R.; Sobol, Robert W.

2009-01-01

Base excision repair (BER) protein expression is important for resistance to DNA damage-induced cytotoxicity. Conversely, BER imbalance (Polß deficiency or repair inhibition) enhances cytotoxicity of radiation and chemotherapeutic DNA-damaging agents. Whereas inhibition of critical steps in the BER pathway result in the accumulation of cytotoxic DNA double-strand breaks, we report that DNA damage-induced cytotoxicity due to deficiency in the BER protein Polß triggers cell death dependent on PARP activation yet independent of poly(ADP-ribose) (PAR)-mediated AIF nuclear translocation or PARG, suggesting that cytotoxicity is not from PAR or PAR-catabolite signaling. Cell death is rescued by the NAD+ metabolite NMN and is synergistic with inhibition of NAD+ biosynthesis, demonstrating that DNA damage-induced cytotoxicity mediated via BER inhibition is primarily dependent on cellular metabolite bioavailability. We offer a mechanistic justification for the elevated alkylation-induced cytotoxicity of Polß deficient cells, suggesting a linkage between DNA repair, cell survival and cellular bioenergetics. PMID:20068071

Environmentally induced amplitude death and firing provocation in large-scale networks of neuronal systems

NASA Astrophysics Data System (ADS)

Pankratova, Evgeniya V.; Kalyakulina, Alena I.

2016-12-01

We study the dynamics of multielement neuronal systems taking into account both the direct interaction between the cells via linear coupling and nondiffusive cell-to-cell communication via common environment. For the cells exhibiting individual bursting behavior, we have revealed the dependence of the network activity on its scale. Particularly, we show that small-scale networks demonstrate the inability to maintain complicated oscillations: for a small number of elements in an ensemble, the phenomenon of amplitude death is observed. The existence of threshold network scales and mechanisms causing firing in artificial and real multielement neural networks, as well as their significance for biological applications, are discussed.

18F-FDG PET/CT for Monitoring Response of Merkel Cell Carcinoma to the Novel Programmed Cell Death Ligand 1 Inhibitor Avelumab.

PubMed

Eshghi, Naghmehossadat; Lundeen, Tamara F; MacKinnon, Lea; Avery, Ryan; Kuo, Phillip H

2018-05-01

An 85-year-old man with stage IIIA Merkel cell carcinoma of the left arm was initially treated with local excision and axillary node dissection followed by radiation therapy. Eight months after surgery, whole-body FDG PET/CT demonstrated intensely hypermetabolic hepatic metastases and abdominal lymphadenopathy. Given his age and comorbidities, he was considered a poor candidate for chemotherapy, and therefore the novel programmed cell death ligand 1 inhibitor avelumab was initiated. FDG PET/CT after 4 cycles showed complete resolution of hepatic and nodal metastases. Whole-body FDG PET/CT can be used for monitoring response of multisystem metastases from Merkel cell carcinoma to active immunotherapy.

Cold PSM, but not TRAIL, triggers autophagic cell death: A therapeutic advantage of PSM over TRAIL.

PubMed

Ito, Tomohisa; Ando, Takashi; Suzuki-Karasaki, Miki; Tokunaga, Tomohiko; Yoshida, Yukihiro; Ochiai, Toyoko; Tokuhashi, Yasuaki; Suzuki-Karasaki, Yoshihiro

2018-05-21

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cold plasma-stimulated medium (PSM) are promising novel anticancer tools due to their strong anticancer activities and high tumor-selectivity. The present study demonstrated that PSM and TRAIL may trigger autophagy in human malignant melanoma and osteosarcoma cells. Live-cell imaging revealed that even under nutritional and stress-free conditions, these cells possessed a substantial level of autophagosomes, which were localized in the cytoplasm separately from tubular mitochondria. In response to cytotoxic levels of PSM, the mitochondria became highly fragmented, and aggregated and colocalized with the autophagosomes. The cytotoxic effects of PSM were suppressed in response to various pharmacological autophagy inhibitors, including 3-methyladenine (3-MA) and bafilomycin A1, thus indicating the induction of autophagic cell death (ACD). Lethal levels of PSM also resulted in non-apoptotic, non-autophagic cell death in a reactive oxygen species-dependent manner under certain circumstances. Furthermore, TRAIL exhibited only a modest cytotoxicity toward these tumor cells, and did not induce ACD and mitochondrial aberration. The combined use of TRAIL and subtoxic concentrations of 3-MA resulted in decreased basal autophagy, increased mitochondrial aberration, colocalization with autophagosomes and apoptosis. These results indicated that PSM may induce ACD, whereas TRAIL may trigger cytoprotective autophagy that compromises apoptosis. To the best of our knowledge, the present study is the first to demonstrate that PSM can induce ACD in human cancer cells. These findings provide a rationale for the advantage of PSM over TRAIL in the destruction of apoptosis-resistant melanoma and osteosarcoma cells.

Anticancer Effect of Nemopilema nomurai Jellyfish Venom on HepG2 Cells and a Tumor Xenograft Animal Model

PubMed Central

Bae, Seong Kyeong; Kim, Munki; Pyo, Min Jung; Kim, Minkyung; Yang, Sujeoung; Yoon, Won Duk; Han, Chang Hoon

2017-01-01

Various kinds of animal venoms and their components have been widely studied for potential therapeutic applications. This study evaluated whether Nemopilema nomurai jellyfish venom (NnV) has anticancer activity. NnV strongly induced cytotoxicity of HepG2 cells through apoptotic cell death, as demonstrated by alterations of chromatic morphology, activation of procaspase-3, and an increase in the Bax/Bcl-2 ratio. Furthermore, NnV inhibited the phosphorylation of PI3K, PDK1, Akt, mTOR, p70S6K, and 4EBP1, whereas it enhanced the expression of p-PTEN. Interestingly, NnV also inactivated the negative feedback loops associated with Akt activation, as demonstrated by downregulation of Akt at Ser473 and mTOR at Ser2481. The anticancer effect of NnV was significant in a HepG2 xenograft mouse model, with no obvious toxicity. HepG2 cell death by NnV was inhibited by tetracycline, metalloprotease inhibitor, suggesting that metalloprotease component in NnV is closely related to the anticancer effects. This study demonstrates, for the first time, that NnV exerts highly selective cytotoxicity in HepG2 cells via dual inhibition of the Akt and mTOR signaling pathways, but not in normal cells. PMID:28785288

Kir 4.1 inward rectifier potassium channel is upregulated in astrocytes in a murine multiple sclerosis model.

PubMed

Mercado, Francisco; Almanza, Angélica; Rubio, Nazario; Soto, Enrique

2018-06-11

Multiple sclerosis (MS) is a high prevalence degenerative disease characterized at the cellular level by glial and neuronal cell death. The causes of cell death during the disease course are not fully understood. In this work we demonstrate that in a MS model induced by Theiler's murine encephalomyelitis virus (TMEV) infection, the inward rectifier (Kir) 4.1 potassium channel subunit is overexpressed in astrocytes. In voltage clamp experiments the inward current density from TMEV-infected astrocytes was significantly larger than in mock-infected ones. The cRNA hybridization analysis from mock- and TMEV-infected cells showed an upregulation of a potassium transport channel coding sequence. We validated this mRNA increase by RT-PCR and quantitative PCR using Kir 4.1 specific primers. Western blotting experiments confirmed the upregulation of Kir 4.1, and alignment between sequences provided the demonstration that the over-expressed gene encodes for a Kir family member. Flow cytometry showed that the Kir 4.1 protein is located mainly in the cell membrane in mock and TMEV-infected astrocytes. Our results demonstrate an increase in K + inward current in TMEV-infected glial cells, this increment may reduce the neuronal depolarization, contributing to cell resilience mechanisms. Copyright © 2018 Elsevier B.V. All rights reserved.

HSP-70 mitigates LPS/SKI-induced cell damage by increasing sphingosine kinase 1 (SK1).

PubMed

Ding, Xuan Z; Feng, Xiao R; Borschel, Richard H; Nikolich, Mikeljon P; Feng, Jie; Li, Yan S; Hoover, David L

2010-06-01

Heat shock proteins (HSPs) are potent protectors of cellular integrity against environmental stresses, including toxic microbial products. To investigate the mechanism of HSP-70 cell protection against bacterial lipopolysaccharide (LPS), we established a stable HSP-70 gene-transfected RAW 264.7 murine macrophage model of LPS-induced cell death. Bacterial LPS increases the activity of sphingosine kinase 1 (SK1), which catalyzes formation of sphingosine-1-phosphate (S1P). S1P functions as a critical signal for initiation and maintenance of diverse aspects of immune cell activation and function. When mouse macrophages were incubated with Escherichia coli LPS (1 microg/ml) and sphingosine kinase inhibitor (SKI, 5 microM), 90% of cells died. Neither LPS nor SKI alone at these doses damaged the cells. The LPS/SKI-induced cell death was partially reversed by overexpression of HSP-70 in gene-transfected macrophages. The specificity of HSP-70 in this reversal was demonstrated by transfection of HSP-70-specific siRNA. Down-regulation of HSP-70 expression after transfection of siRNA specific for HSP-70 was associated with increased LPS/SKI-induced cell damage. Overexpression of human or murine HSP-70 (HSPA1A and Hspa1a, respectively) increased both cellular SK1 mRNA and protein levels. Cellular heat shock also increased SK1 protein. These studies confirm the importance of SK1 as a protective moiety in LPS-induced cell injury and demonstrate that HSP-70-mediated protection from cells treated with LPS/SKI is accompanied by upregulating expression of SK1. HSP-70-mediated increases in SK1 and consequent increased levels of S1P may also play a role in protection of cells from other processes that lead to programmed cell death. Published by Elsevier Inc.

Parkin elimination of mitochondria is important for maintenance of lens epithelial cell ROS levels and survival upon oxidative stress exposure.

PubMed

Brennan, Lisa; Khoury, Josef; Kantorow, Marc

2017-01-01

Age-related cataract is associated with oxidative stress and death of lens epithelial cells (LECs) whose survival is dependent on functional mitochondrial populations. Oxidative stress-induced depolarization/damage of LEC mitochondria results in increased reactive oxygen species (ROS) levels and cell death suggesting the need for a LEC mechanism to remove mitochondria depolarized/damaged upon oxidative stress exposure to prevent ROS release and LEC death. To date, a mechanism(s) for removal of depolarized/damaged LEC mitochondria has yet to be identified and the importance of eliminating oxidative stress-damaged mitochondria to prevent LEC ROS release and death has not been established. Here, we demonstrate that Parkin levels increase in LECs exposed to H 2 O 2 -oxidative stress. We establish that Parkin translocates to LEC mitochondria depolarized upon oxidative stress exposure and that Parkin recruits p62/SQSTM1 to depolarized LEC mitochondria. We demonstrate that translocation of Parkin results in the elimination of depolarized/damaged mitochondria and that Parkin clearance of LEC mitochondria is dependent on its ubiquitin ligase activity. Importantly, we demonstrate that Parkin elimination of damaged LEC mitochondria results in reduced ROS levels and increased survival upon oxidative stress exposure. These results establish that Parkin functions to eliminate LEC mitochondria depolarized/damaged upon oxidative stress exposure and that elimination of damaged mitochondria by Parkin is important for LEC homeostasis and survival. The data also suggest that mitochondrial quality control by Parkin could play a role in lens transparency. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Cytotoxic Activities of Physalis minima L. Chloroform Extract on Human Lung Adenocarcinoma NCI-H23 Cell Lines by Induction of Apoptosis

PubMed Central

Leong, Ooi Kheng; Muhammad, Tengku Sifzizul Tengku; Sulaiman, Shaida Fariza

2011-01-01

Physalis minima L. is reputed for having anticancer property. In this study, the chloroform extract of this plant exhibited remarkable cytotoxic activities on NCI-H23 (human lung adenocarcinoma) cell line at dose- and time-dependent manners (after 24, 48 and 72 h of incubation). Analysis of cell-death mechanism demonstrated that the extract exerted apoptotic programed cell death in NCI-H23 cells with typical DNA fragmentation, which is a biochemical hallmark of apoptosis. Morphological observation using transmission electron microscope (TEM) also displayed apoptotic characteristics in the treated cells, including clumping and margination of chromatins, followed by convolution of the nuclear and budding of the cells to produce membrane-bound apoptotic bodies. Different stages of apoptotic programed cell death as well as phosphatidylserine externalization were confirmed using annexin V and propidium iodide staining. Furthermore, acute exposure to the extract produced a significant regulation of c-myc, caspase-3 and p53 mRNA expression in this cell line. Due to its apoptotic effect on NCI-H23 cells, it is strongly suggested that the extract could be further developed as an anticancer drug. PMID:19541726

Pivotal Role of Receptor-Interacting Protein Kinase 1 and Mixed Lineage Kinase Domain-Like in Neuronal Cell Death Induced by the Human Neuroinvasive Coronavirus OC43

PubMed Central

Meessen-Pinard, Mathieu; Le Coupanec, Alain

2016-01-01

ABSTRACT Human coronaviruses (HCoV) are respiratory pathogens with neuroinvasive, neurotropic, and neurovirulent properties, highlighting the importance of studying the potential implication of these viruses in neurological diseases. The OC43 strain (HCoV-OC43) was reported to induce neuronal cell death, which may participate in neuropathogenesis. Here, we show that HCoV-OC43 harboring two point mutations in the spike glycoprotein (rOC/Us183–241) was more neurovirulent than the wild-type HCoV-OC43 (rOC/ATCC) in mice and induced more cell death in murine and human neuronal cells. To evaluate the role of regulated cell death (RCD) in HCoV-OC43-mediated neural pathogenesis, we determined if knockdown of Bax, a key regulator of apoptosis, or RIP1, a key regulator of necroptosis, altered the percentage of neuronal cell death following HCoV-OC43 infection. We found that Bax-dependent apoptosis did not play a significant role in RCD following infection, as inhibition of Bax expression mediated by RNA interference did not confer cellular protection against the cell death process. On the other hand, we demonstrated that RIP1 and MLKL were involved in neuronal cell death, as RIP1 knockdown and chemical inhibition of MLKL significantly increased cell survival after infection. Taken together, these results indicate that RIP1 and MLKL contribute to necroptotic cell death after HCoV-OC43 infection to limit viral replication. However, this RCD could lead to neuronal loss in the mouse CNS and accentuate the neuroinflammation process, reflecting the severity of neuropathogenesis. IMPORTANCE Because they are naturally neuroinvasive and neurotropic, human coronaviruses are suspected to participate in the development of neurological diseases. Given that the strain OC43 is neurovirulent in mice and induces neuronal cell death, we explored the neuronal response to infection by characterizing the activation of RCD. Our results revealed that classical apoptosis associated with the Bax protein does not play a significant role in HCoV-OC43-induced neuronal cell death and that RIP1 and MLKL, two cellular proteins usually associated with necroptosis (an RCD back-up system when apoptosis is not adequately induced), both play a pivotal role in the process. As necroptosis disrupts cellular membranes and allows the release of damage-associated molecular patterns (DAMP) and possibly induces the production of proinflammatory cytokines, it may represent a proinflammatory cell death mechanism that contributes to excessive neuroinflammation and neurodegeneration and eventually to neurological disorders after a coronavirus infection. PMID:27795420

Anticancer Effect of Ginger Extract against Pancreatic Cancer Cells Mainly through Reactive Oxygen Species-Mediated Autotic Cell Death

PubMed Central

Akimoto, Miho; Iizuka, Mari; Kanematsu, Rie; Yoshida, Masato; Takenaga, Keizo

2015-01-01

The extract of ginger (Zingiber officinale Roscoe) and its major pungent components, [6]-shogaol and [6]-gingerol, have been shown to have an anti-proliferative effect on several tumor cell lines. However, the anticancer activity of the ginger extract in pancreatic cancer is poorly understood. Here, we demonstrate that the ethanol-extracted materials of ginger suppressed cell cycle progression and consequently induced the death of human pancreatic cancer cell lines, including Panc-1 cells. The underlying mechanism entailed autosis, a recently characterized form of cell death, but not apoptosis or necroptosis. The extract markedly increased the LC3-II/LC3-I ratio, decreased SQSTM1/p62 protein, and enhanced vacuolization of the cytoplasm in Panc-1 cells. It activated AMPK, a positive regulator of autophagy, and inhibited mTOR, a negative autophagic regulator. The autophagy inhibitors 3-methyladenine and chloroquine partially prevented cell death. Morphologically, however, focal membrane rupture, nuclear shrinkage, focal swelling of the perinuclear space and electron dense mitochondria, which are unique morphological features of autosis, were observed. The extract enhanced reactive oxygen species (ROS) generation, and the antioxidant N-acetylcystein attenuated cell death. Our study revealed that daily intraperitoneal administration of the extract significantly prolonged survival (P = 0.0069) in a peritoneal dissemination model and suppressed tumor growth in an orthotopic model of pancreatic cancer (P < 0.01) without serious adverse effects. Although [6]-shogaol but not [6]-gingerol showed similar effects, chromatographic analyses suggested the presence of other constituent(s) as active substances. Together, these results show that ginger extract has potent anticancer activity against pancreatic cancer cells by inducing ROS-mediated autosis and warrants further investigation in order to develop an efficacious candidate drug. PMID:25961833

High-throughput drug screen identifies chelerythrine as a selective inducer of death in a TSC2-null setting.

PubMed

Medvetz, Doug; Sun, Yang; Li, Chenggang; Khabibullin, Damir; Balan, Murugabaskar; Parkhitko, Andrey; Priolo, Carmen; Asara, John M; Pal, Soumitro; Yu, Jane; Henske, Elizabeth P

2015-01-01

Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome associated with tumors of the brain, heart, kidney, and lung. The TSC protein complex inhibits the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Inhibitors of mTORC1, including rapamycin, induce a cytostatic response in TSC tumors, resulting in temporary disease stabilization and prompt regrowth when treatment is stopped. The lack of TSC-specific cytotoxic therapies represents an important unmet clinical need. Using a high-throughput chemical screen in TSC2-deficient, patient-derived cells, we identified a series of molecules antagonized by rapamycin and therefore selective for cells with mTORC1 hyperactivity. In particular, the cell-permeable alkaloid chelerythrine induced reactive oxygen species (ROS) and depleted glutathione (GSH) selectively in TSC2-null cells based on metabolic profiling. N-acetylcysteine or GSH cotreatment protected TSC2-null cells from chelerythrine's effects, indicating that chelerythrine-induced cell death is ROS dependent. Induction of heme-oxygenase-1 (HMOX1/HO-1) with hemin also blocked chelerythrine-induced cell death. In vivo, chelerythrine inhibited the growth of TSC2-null xenograft tumors with no evidence of systemic toxicity with daily treatment over an extended period of time. This study reports the results of a bioactive compound screen and the identification of a potential lead candidate that acts via a novel oxidative stress-dependent mechanism to selectively induce necroptosis in TSC2-deficient tumors. This study demonstrates that TSC2-deficient tumor cells are hypersensitive to oxidative stress-dependent cell death, and provide critical proof of concept that TSC2-deficient cells can be therapeutically targeted without the use of a rapalog to induce a cell death response. ©2014 American Association for Cancer Research.

Monitoring of thermal dose during ablation therapy using quantum dot-mediated fluorescence thermometry.

PubMed

Bensalah, Karim; Tuncel, Altug; Hanson, Willard; Stern, Joshua; Han, Bumsoo; Cadeddu, Jeffrey

2010-12-01

The objective of this study was to demonstrate the feasibility of quantum dot (QD)-mediated fluorescence thermometry to monitor thermal dose in an in-vitro thermal ablation zone generated by laser-heated gold nanoshells (LGNS). Hyperthermic cell death of human prostate cancer cell line (PC-3) was determined after various heating settings and correlated to the thermal conditions using an Arrhenius model prior to LGNS ablation. PC-3 cells with gold nanoshells (GNS) and QDs were exposed to a near-infrared laser and QD excitation light. When the cells were heated by GNS, local temperature was measured using the temperature-dependent fluorescence intensity of QDs. Using the predetermined Arrhenius model, the thermal dose (i.e., cell death of PC-3 cells) by LGNS was estimated with local temperatures measured with QD-mediated thermometry. The estimated thermal dose was confirmed with calcein-acetoxy-methylester viability assay. For PC-3 cell line, the activation energy and frequency factor of the Arrhenius model were 86.78 kcal/mol and 6.35 × 10(55) Hz, respectively. During LGNS ablation of PC-3 cells, QD-mediated temperature measurement showed that the temperature of the laser spot increased rapidly to ∼58 °C ± 4 °C. The estimated thermal dose showed that cell death reached to ∼90% in 120 seconds. The death cell zone observed after staining corresponded to a peak area of the temperature profile generated after analysis of the QD fluorescence intensity. This study shows that the QD fluorescence thermometry can accurately monitor the PC-3 cell death by LGNS ablation. This approach holds promises for a better monitoring of thermal ablation procedures in clinical practice.

JNK Controls the Onset of Mitosis in Planarian Stem Cells and Triggers Apoptotic Cell Death Required for Regeneration and Remodeling

PubMed Central

Almuedo-Castillo, María; Crespo, Xenia; Seebeck, Florian; Bartscherer, Kerstin; Salò, Emili; Adell, Teresa

2014-01-01

Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun–NH2–kinase (JNK) links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal. PMID:24922054

Estrogen attenuates glutamate-induced cell death by inhibiting Ca2+ influx through L-type voltage-gated Ca2+ channels

PubMed Central

Sribnick, Eric A.; Del Re, Angelo M.; Ray, Swapan K.; Woodward, John J.; Banik, Naren L.

2009-01-01

Estrogen-mediated neuroprotection is observed in neurodegenerative disease and neurotrauama models; however, determining a mechanism for these effects has been difficult. We propose that estrogen may limit cell death in the nervous system tissue by inhibiting increases in intracellular free Ca2+. Here, we present data using VSC 4.1 cell line, a ventral spinal motoneuron and neuroblastoma hybrid cell line. Treatment with 1 mM glutamate for 24 h induced apoptosis. When cells were pre-treated with 100 nM 17β-estradiol (estrogen) for 1 h and then co-treated with glutamate, apoptotic death was significantly attenuated. Estrogen also prevented glutamate-mediated changes in resting membrane potential and membrane capacitance. Treatment with either 17α-estradiol or cell impermeable estrogen did not mimic the findings seen with estrogen. Glutamate treatment significantly increased both intracellular free Ca2+ and the activities of downstream proteases such as calpain and caspase-3. Estrogen attenuated both the increases in intracellular free Ca2+ and protease activities. In order to determine the pathway responsible for estrogen-mediated inhibition of these increases in intracellular free Ca2+, cells were treated with several Ca2+ entry inhibitors, but only the L-type Ca2+ channel blocker nifedipine demonstrated cytoprotective effects comparable to estrogen. To expand these findings, cells were treated with the L-type Ca2+ channel agonist FPL 64176, which increased both cell death and intracellular free Ca2+, and estrogen inhibited both effects. From these observations, we conclude that estrogen limits glutamate-induced cell death in VSC 4.1 cells through effects on L-type Ca2+ channels, inhibiting Ca2+ influx as well as activation of the pro-apoptotic proteases calpain and caspase-3. PMID:19389388

Identification of tissue-specific cell death using methylation patterns of circulating DNA

PubMed Central

Lehmann-Werman, Roni; Neiman, Daniel; Zemmour, Hai; Moss, Joshua; Magenheim, Judith; Vaknin-Dembinsky, Adi; Rubertsson, Sten; Nellgård, Bengt; Blennow, Kaj; Zetterberg, Henrik; Spalding, Kirsty; Haller, Michael J.; Wasserfall, Clive H.; Schatz, Desmond A.; Greenbaum, Carla J.; Dorrell, Craig; Grompe, Markus; Zick, Aviad; Hubert, Ayala; Maoz, Myriam; Fendrich, Volker; Bartsch, Detlef K.; Golan, Talia; Ben Sasson, Shmuel A.; Zamir, Gideon; Razin, Aharon; Cedar, Howard; Shapiro, A. M. James; Glaser, Benjamin; Shemer, Ruth; Dor, Yuval

2016-01-01

Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics. PMID:26976580

Fenofibrate activates Nrf2 through p62-dependent Keap1 degradation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Jeong Su; Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752; Kang, Dong Hoon

Peroxisome proliferator-activated receptor α (PPARα) activates the β-oxidation of fatty acids in the liver. Fenofibrate is a potent agonist of PPARα and is used in the treatment of hyperlipidemia. Fenofibrate treatment often induces the production of intracellular reactive oxygen species (ROS), leading to cell death. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is an essential component of the defense mechanism against oxidative stress. However, the molecular mechanism underlying the regulation of the Nrf2-Keap1 pathway in fenofibrate-induced cell death is not known. In this study, we demonstrated that fenofibrate induces Keap1 degradation and Nrf2 activation.more » This fenofibrate-mediated Keap1 degradation is partly dependent on autophagy. Furthermore, fenofibrate-induced Keap1 degradation followed by Nrf2 activation is mainly mediated by p62, which functions as an adaptor protein in the autophagic pathway. Consistent with these findings, ablation of p62 increased fenofibrate-mediated apoptotic cell death associated with ROS accumulation. These results strongly suggest that p62 plays a crucial role in preventing fenofibrate-induced cell death. - Highlights: • Fenofibrate induces cell death by increasing ROS production. • The underlying defense mechanism against this effect is unknown. • Fenofibrate induces autophagy-dependent Keap1 degradation and Nrf2 activation. • This process is p62-dependent; lack of p62 enhanced fenofibrate-mediated apoptosis. • p62 plays a crucial role in preventing fenofibrate-induced cell death.« less

Neisseria gonorrhoeae–Induced Inflammatory Pyroptosis in Human Macrophages is Dependent on Intracellular Gonococci and Lipooligosaccharide

PubMed Central

Ritter, Jessica Leigh; Genco, Caroline Attardo

2018-01-01

Neisseria gonorrhoeae, the human obligate pathogen responsible for the sexually transmitted disease gonorrhea, has evolved several mechanisms to evade the host immune response. One such mechanism is the modulation of host cell death pathways. In this study, we defined cell death pathways induced by N gonorrhoeae in human monocyte-derived macrophages (MDMs). In a dose-dependent manner, N gonorrhoeae stimulation of MDMs resulted in caspase 1 and 4–dependent cell deaths, indicative of canonical and noncanonical pyroptosis, respectively. Internalization of bacteria or stimulation with lipooligosaccharide (LOS) specifically induced pyroptosis in MDMs and increased secretion of IL-1β. Collectively, our results demonstrate that N gonorrhoeae induces inflammatory pyroptosis in human macrophages due in part to intracellular LOS. We propose that this in turn may exacerbate inflammatory outcomes observed during mucosal infection. PMID:29434478

Numerical models of cell death in RF ablation with monopolar and bipolar probes

NASA Astrophysics Data System (ADS)

Bright, Benjamin M.; Pearce, John A.

2013-02-01

Radio frequency (RF) is used clinically to treat unresectible tumors. Finite element modeling has proven useful in treatment planning and applicator design. Typically isotherms in the middle 50s °C have been used as the parameter of assessment in these models. We compare and contrast isotherms for multiple known Arrhenius thermal damage predictors including collagen denaturation, vascular disruption, liver coagulation and cell death. Models for RITA probe geometries are included in the study. Comparison to isotherms is sensible when the activation time is held constant, but varies considerably when heating times vary. The purpose of this paper is to demonstrate the importance of looking at specific processes and keeping track of the methods used to derive the Arrhenius coefficients in order to study the extremely complex cell death processes due to thermal therapies.

Myeloid-derived suppressor cells modulate B-cell responses.

PubMed

Lelis, Felipe J N; Jaufmann, Jennifer; Singh, Anurag; Fromm, Katja; Teschner, Annkathrin Chiara; Pöschel, Simone; Schäfer, Iris; Beer-Hammer, Sandra; Rieber, Nikolaus; Hartl, Dominik

2017-08-01

Myeloid-derived suppressor cells (MDSCs) are key regulators of adaptive immunity by suppressing T-cell functions. However, their potential action on or interaction with B cells remained poorly understood. Here we demonstrate that human polymorphonuclear MDSCs differentially modulate B-cell function by suppressing B-cell proliferation and antibody production. We further demonstrate that this MDSC-mediated effect is cell contact dependent and involves established mediators such as arginase-1, nitric oxide (NO), reactive oxygen species (ROS) as well as B-cell death. Collectively, our studies provide novel evidence that human MDSCs modulate B cells, which could have future implications for immunotherapy approaches. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

The Akt signaling pathway is required for tissue maintenance and regeneration in planarians.

PubMed

Peiris, T Harshani; Ramirez, Daniel; Barghouth, Paul G; Oviedo, Néstor J

2016-04-11

Akt (PKB) is a serine threonine protein kinase downstream of the phosphoinositide 3-kinase (PI3K) pathway. In mammals, Akt is ubiquitously expressed and is associated with regulation of cellular proliferation, metabolism, cell growth and cell death. Akt has been widely studied for its central role in physiology and disease, in particular cancer where it has become an attractive pharmacological target. However, the mechanisms by which Akt signaling regulates stem cell behavior in the complexity of the whole body are poorly understood. Planarians are flatworms with large populations of stem cells capable of dividing to support adult tissue renewal and regeneration. The planarian ortholog Smed-Akt is molecularly conserved providing unique opportunities to analyze the function of Akt during cellular turnover and repair of adult tissues. Our findings abrogating Smed-Akt with RNA-interference in the planarian Schmidtea mediterranea led to a gradual decrease in stem cell (neoblasts) numbers. The reduced neoblast numbers largely affected the maintenance of adult tissues including the nervous and excretory systems and ciliated structures in the ventral epithelia, which impaired planarian locomotion. Downregulation of Smed-Akt function also resulted in an increase of cell death throughout the animal. However, in response to amputation, levels of cell death were decreased and failed to localize near the injury site. Interestingly, the neoblast mitotic response was increased around the amputation area but the regenerative blastema failed to form. We demonstrate Akt signaling is essential for organismal physiology and in late stages of the Akt phenotype the reduction in neoblast numbers may impair regeneration in planarians. Functional disruption of Smed-Akt alters the balance between cell proliferation and cell death leading to systemic impairment of adult tissue renewal. Our results also reveal novel roles for Akt signaling during regeneration, specifically for the timely localization of cell death near the injury site. Thus, Akt signaling regulates neoblast biology and mediates in the distribution of injury-mediated cell death during tissue repair in planarians.

Embryotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): Theembryonic vasculature is a physiological target for TCDD-induced DNA damage and apoptotic cell death in medaka (Orizias latipes)

USGS Publications Warehouse

Cantrell, Susannah M.; Lutz, Linda H.; Tillitt, Donald E.; Hannink, Mark

1996-01-01

Vertebrate embryos are extremely sensitive to environmental contaminants known as planar halogenated hydrocarbons (PHHs). The physiological targets that mediate PHH-induced embryotoxicity are not known. We have characterized embryotoxicity in medaka (Orizias latipes) caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototypic PHH. DNA degradation in cells of the embryonic vasculature and loss of functional integrity of the medial yolk vein were demonstrated in TCDD-exposed embryos. Pharmacological intervention with piperonyl butoxide inhibited TCDD-induced DNA degradation, restored the functional integrity of the medial yolk vein, and protected against the embryotoxicity of TCDD. Treatment of TCDD-exposed embryos with the antioxidant N-acetylcysteine also provided significant protection against the embryotoxicity of TCDD. These results demonstrate that DNA damage and consequent cell death in the embryonic vasculature are key physiological mediators of TCDD-induced embryotoxicity.

Fluvoxamine alleviates ER stress via induction of Sigma-1 receptor

PubMed Central

Omi, T; Tanimukai, H; Kanayama, D; Sakagami, Y; Tagami, S; Okochi, M; Morihara, T; Sato, M; Yanagida, K; Kitasyoji, A; Hara, H; Imaizumi, K; Maurice, T; Chevallier, N; Marchal, S; Takeda, M; Kudo, T

2014-01-01

We recently demonstrated that endoplasmic reticulum (ER) stress induces sigma-1 receptor (Sig-1R) expression through the PERK pathway, which is one of the cell's responses to ER stress. In addition, it has been demonstrated that induction of Sig-1R can repress cell death signaling. Fluvoxamine (Flv) is a selective serotonin reuptake inhibitor (SSRI) with a high affinity for Sig-1R. In the present study, we show that treatment of neuroblastoma cells with Flv induces Sig-1R expression by increasing ATF4 translation directly, through its own activation, without involvement of the PERK pathway. The Flv-mediated induction of Sig-1R prevents neuronal cell death resulting from ER stress. Moreover, Flv-induced ER stress resistance reduces the infarct area in mice after focal cerebral ischemia. Thus, Flv, which is used frequently in clinical practice, can alleviate ER stress. This suggests that Flv could be a feasible therapy for cerebral diseases caused by ER stress. PMID:25032855

Inhibitory effects of 3-bromopyruvate in human nasopharyngeal carcinoma cells.

PubMed

Zou, Xue; Zhang, Mengxiao; Sun, Yiming; Zhao, Surong; Wei, Yingmei; Zhang, Xudong; Jiang, Chenchen; Liu, Hao

2015-10-01

Tumor cells depend on aerobic glycolysis for adenosine triphosphate (ATP) production, which is therefore targeted by therapeutic agents. The compound 3-bromopyruvate (3-BrPA), a strong alkylating agent and hexokinase inhibitor, inhibits tumor cell glycolysis and the production of ATP, causing apoptosis. 3-BrPA induces apoptosis of nasopharyngeal carcinoma (NPC) cell lines HNE1 and CNE-2Z, which may be related to its molecular mechanisms. In the present study, we investigated the effects of 3-BrPA on the viability, reactive oxygen species (ROS), apoptosis and other types of programmed cell death in NPC cells in vitro and in vivo. PI staining showed significant apoptosis in NPC cells accompanied by the overproduction of ROS and downregulation of mitochondrial membrane potential (MMP, ΔΨm) by 3-BrPA. However, the ROS scavenger N-acetyl-L-cysteine (NAC) significantly reduced 3-BrPA-induced apoptosis by decreasing ROS and facilitating the recovery of MMP. We elucidated the molecular mechanisms underlying 3-BrPA activity and found that it caused mitochondrial dysfunction and ROS production, leading to necroptosis of NPC cells. We investigated the effects of the caspase inhibitor z-VAD-fmk, which inhibits apoptosis but promotes death domain receptor (DR)-induced NPC cell necrosis. Necrostatin-1 (Nec-1) inhibits necroptosis, apparently via a DR signaling pathway and thus abrogates the effects of z-VAD‑fmk. In addition, we demonstrated the effective attenuation of 3-BrPA-induced necrotic cell death by Nec-1. Finally, animal studies proved that 3-BrPA exhibited significant antitumor activity in nude mice. The present study is the first demonstration of 3-BrPA-induced non-apoptotic necroptosis and ROS generation in NPC cells and provides potential strategies for developing agents against apoptosis‑resistant cancers.

Resistance to Cell Death and Its Modulation in Cancer Stem Cells

PubMed Central

Safa, Ahmad R.

2017-01-01

Accumulating evidence has demonstrated that human cancers arise from various tissues of origin that initiate from cancer stem cells (CSCs) or cancer-initiating cells. The extrinsic and intrinsic apoptotic pathways are dysregulated in CSCs, and these cells play crucial roles in tumor initiation, progression, cell death resistance, chemo- and radiotherapy resistance, and tumor recurrence. Understanding CSC-specific signaling proteins and pathways is necessary to identify specific therapeutic targets that may lead to the development of more efficient therapies selectively targeting CSCs. Several signaling pathways—including the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), maternal embryonic leucine zipper kinase (MELK), NOTCH1, and Wnt/β-catenin—and expression of the CSC markers CD133, CD24, CD44, Oct4, Sox2, Nanog, and ALDH1A1 maintain CSC properties. Studying such pathways may help to understand CSC biology and lead to the development of potential therapeutic interventions to render CSCs more sensitive to cell death triggered by chemotherapy and radiation therapy. Moreover, recent demonstrations of dedifferentiation of differentiated cancer cells into CSC-like cells have created significant complexity in the CSCs hypothesis. Therefore, any successful therapeutic agent or combination of drugs for cancer therapy must eliminate not only CSCs but differentiated cancer cells and the entire bulk of tumor cells. This review article expands on the CSC hypothesis and paradigm with respect to major signaling pathways and effectors that regulate CSC apoptosis resistance. Moreover, selective CSC apoptotic modulators and their therapeutic potential for making tumors more responsive to therapy are discussed. The use of novel therapies, including small-molecule inhibitors of specific proteins in signaling pathways that regulate stemness, proliferation and migration of CSCs, immunotherapy, and noncoding microRNAs may provide better means of treating CSCs. PMID:27915972

Programmed death receptor-1/programmed death receptor ligand-1 blockade after transient lymphodepletion to treat myeloma.

PubMed

Kearl, Tyce J; Jing, Weiqing; Gershan, Jill A; Johnson, Bryon D

2013-06-01

Early phase clinical trials targeting the programmed death receptor-1/ligand-1 (PD-1/PD-L1) pathway to overcome tumor-mediated immunosuppression have reported promising results for a variety of cancers. This pathway appears to play an important role in the failure of immune reactivity to malignant plasma cells in multiple myeloma patients, as the tumor cells express relatively high levels of PD-L1, and T cells show increased PD-1 expression. In the current study, we demonstrate that PD-1/PD-L1 blockade with a PD-L1-specific Ab elicits rejection of a murine myeloma when combined with lymphodepleting irradiation. This particular combined approach by itself has not previously been shown to be efficacious in other tumor models. The antitumor effect of lymphodepletion/anti-PD-L1 therapy was most robust when tumor Ag-experienced T cells were present either through cell transfer or survival after nonmyeloablative irradiation. In vivo depletion of CD4 or CD8 T cells completely eliminated antitumor efficacy of the lymphodepletion/anti-PD-L1 therapy, indicating that both T cell subsets are necessary for tumor rejection. Elimination of myeloma by T cells occurs relatively quickly as tumor cells in the bone marrow were nearly nondetectable by 5 d after the first anti-PD-L1 treatment, suggesting that antimyeloma reactivity is primarily mediated by preactivated T cells, rather than newly generated myeloma-reactive T cells. Anti-PD-L1 plus lymphodepletion failed to improve survival in two solid tumor models, but demonstrated significant efficacy in two hematologic malignancy models. In summary, our results support the clinical testing of lymphodepletion and PD-1/PD-L1 blockade as a novel approach for improving the survival of patients with multiple myeloma.

Safety and efficacy of autologous cell therapy in critical limb ischemia: a systematic review.

PubMed

Benoit, Eric; O'Donnell, Thomas F; Patel, Amit N

2013-01-01

Researchers have accumulated a decade of experience with autologous cell therapy in the treatment of critical limb ischemia (CLI). We conducted a systematic review of clinical trials in the literature to determine the safety and efficacy of cell therapy in CLI. We searched the literature for clinical trials of autologous cell therapy in CLI, including observational series of five or more patients to accrue a large pool of patients for safety analysis. Safety analysis included evaluation of death, cancer, unregulated angiogenesis, and procedural adverse events such as bleeding. Efficacy analysis included the clinical endpoints amputation and death as well as functional and surrogate endpoints. We identified 45 clinical trials, including seven RCTs, and 1,272 patients who received cell therapy. The overall adverse event rate was low (4.2%). Cell therapy patients did not have a higher mortality rate than control patients and demonstrated no increase in cancer incidence when analyzed against population rates. With regard to efficacy, cell therapy patients had a significantly lower amputation rate than control patients (OR 0.36, p = 0.0004). Cell therapy also demonstrated efficacy in a variety of functional and surrogate outcomes. Clinical trials differed in the proportion of patients with risk factors for clinical outcomes, and these influenced rates of amputation and death. Cell therapy presents a favorable safety profile with a low adverse event rate and no increase in severe events such as mortality and cancer and treatment with cell therapy decreases the risk of amputation. Cell therapy has a positive benefit-to-risk ratio in CLI and may be a valuable treatment option, particularly for those challenging patients who cannot undergo arterial reconstruction.

6-Shogaol induces caspase-independent paraptosis in cancer cells via proteasomal inhibition.

PubMed

Nedungadi, Divya; Binoy, Anupama; Pandurangan, Nanjan; Pal, Sanjay; Nair, Bipin G; Mishra, Nandita

2018-03-15

An α, β-unsaturated carbonyl compound of ginger, 6-Shogaol (6S), induced extensive cytoplasmic vacuolation and cell death in breast cancer cell (MDA-MB-231) and non-small lung cancer (A549) cells. In the presence of autophagic inhibitors the cells continued to exhibit cytoplasmic vacuolation and cell death clearly distinguishing it from the classic autophagic process. 6S induced death did not exhibit the characteristic apoptotic features like caspase cleavage, phosphatidyl serine exposure and DNA fragmentation. The immunofluorescence with the Endoplasmic Reticulum (ER) resident protein, calreticulin indicated that the vacuoles were of ER origin, typical of paraptosis. This was supported by the increase in level of microtubule associated protein light chain 3B (LC3 I and LC3 II) and polyubiquitin binding protein, p62. The level of ER stress markers like polyubiquitinated proteins, Bip and CHOP also consistently increased. We have found that 6S inhibits the 26S proteasome. The proteasomal inhibitory activity was elucidated by a) molecular docking of 6S onto the active site of β5 subunit and b) reduced fluorescence by the fluorogenic substrate of the chymotrypsin-like subunit. In conclusion these studies demonstrate for the first time that proteasomal inhibition by 6S induces cell death via paraptosis. So 6-shogaol may act as a template for anti-cancer lead discovery against the apoptosis resistant cancer cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Hemeoxygenase-1 Mediates an Adaptive Response to Spermidine-Induced Cell Death in Human Endothelial Cells

PubMed Central

Yang, Hana; Lee, Seung Eun; Kim, Gun-Dong; Park, Hye Rim; Park, Yong Seek

2013-01-01

Spermidine (SPD) is a ubiquitous polycation that is commonly distributed in living organisms. Intracellular levels of SPD are tightly regulated, and SPD controls cell proliferation and death. However, SPD undergoes oxidation in the presence of serum, producing aldehydes, hydrogen peroxide, and ammonia, which exert cytotoxic effect on cells. Hemeoxygenase-1 (HO-1) is thought to have a protective effect against oxidative stress. Upregulation of HO-1 in endothelial cells is considered to be beneficial in the cardiovascular disease. In the present study, we demonstrate that the ubiquitous polyamine, SPD, induces HO-1 in human umbilical vein endothelial cells (HUVECs). SPD-induced HO-1 expression was examined by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Involvement of reactive oxygen species, serum amine oxidase, PI3K/Akt signaling pathway, and transcription factor Nrf2 in the induction of HO-1 by SPD was also investigated. Furthermore, small interfering RNA knockdown of Nrf2 or HO-1 and treatment with the specific HO-1 inhibitor ZnPP exhibited a noteworthy increase of death of SPD-stimulated HUVECs. In conclusion, these results suggest that SPD induces PI3K/Akt-Nrf2-mediated HO-1 expression in human endothelial cells, which may have a role in cytoprotection of the cells against oxidative stress-induced death. PMID:23983896

Potato NPH3/RPT2-Like Protein StNRL1, Targeted by a Phytophthora infestans RXLR Effector, Is a Susceptibility Factor.

PubMed

Yang, Lina; McLellan, Hazel; Naqvi, Shaista; He, Qin; Boevink, Petra C; Armstrong, Miles; Giuliani, Licida M; Zhang, Wei; Tian, Zhendong; Zhan, Jiasui; Gilroy, Eleanor M; Birch, Paul R J

2016-05-01

Plant pathogens deliver effectors to manipulate host processes. We know little about how fungal and oomycete effectors target host proteins to promote susceptibility, yet such knowledge is vital to understand crop disease. We show that either transient expression in Nicotiana benthamiana, or stable transgenic expression in potato (Solanum tuberosum), of the Phytophthora infestans RXLR effector Pi02860 enhances leaf colonization by the pathogen. Expression of Pi02860 also attenuates cell death triggered by the P. infestans microbe-associated molecular pattern INF1, indicating that the effector suppresses pattern-triggered immunity. However, the effector does not attenuate cell death triggered by Cf4/Avr4 coexpression, showing that it does not suppress all cell death activated by cell surface receptors. Pi02860 interacts in yeast two-hybrid assays with potato NPH3/RPT2-LIKE1 (NRL1), a predicted CULLIN3-associated ubiquitin E3 ligase. Interaction of Pi02860 in planta was confirmed by coimmunoprecipitation and bimolecular fluorescence complementation assays. Virus-induced gene silencing of NRL1 in N. benthamiana resulted in reduced P. infestans colonization and accelerated INF1-mediated cell death, indicating that this host protein acts as a negative regulator of immunity. Moreover, whereas NRL1 virus-induced gene silencing had no effect on the ability of the P. infestans effector Avr3a to suppress INF1-mediated cell death, such suppression by Pi02860 was significantly attenuated, indicating that this activity of Pi02860 is mediated by NRL1. Transient overexpression of NRL1 resulted in the suppression of INF1-mediated cell death and enhanced P. infestans leaf colonization, demonstrating that NRL1 acts as a susceptibility factor to promote late blight disease. © 2016 American Society of Plant Biologists. All Rights Reserved.

Zinc deficiency mediates alcohol-induced apoptotic cell death in the liver of rats through activating ER and mitochondrial cell death pathways

PubMed Central

Sun, Qian; Zhong, Wei; Zhang, Wenliang; Li, Qiong; Sun, Xiuhua; Tan, Xiaobing; Sun, Xinguo; Dong, Daoyin

2015-01-01

Hepatic zinc deficiency has been well documented in alcoholic patients, but the mechanisms by which zinc deficiency mediates cell death have not been well defined. The objectives of this study were to determine whether alcohol perturbs subcellular zinc homeostasis and how organelle zinc depletion may link with cell death pathways. Wistar rats were pair-fed with the Lieber-DeCarli control or ethanol diet for 5 mo. Chronic alcohol exposure significantly reduced zinc level in isolated hepatic endoplasmic reticulum (ER) and mitochondria. Among the detected zinc transporters, ER Zrt/Irt-like protein (ZIP)13 and mitochondrial ZIP8, which transport zinc from ER and mitochondria to cytosol, were significantly increased. Mitochondrial zinc transporter (ZnT) 4, which transports zinc from cytosol to mitochondria, was also increased. ER phosphorylated eukaryotic initiation factor 2α, activating transcription factor 4, and C/EBP homologous protein were significantly upregulated, and mitochondrial cytochrome c release and Bax insertion were detected in association with caspase-3 activation and apoptotic cell death. To define the role of zinc deficiency in ER and mitochondrial stress, H4IIEC3 cells were treated with 3 μM N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine for 6 h with or without supplementation with zinc or N-acetylcysteine (NAC). The results demonstrated that zinc deprivation induced caspase-3 activation and apoptosis in association with ER and mitochondria dysfunction, which were inhibited by zinc as low as 10 μM but not by 2 mM NAC. These results suggest that chronic ethanol exposure induced in ER and mitochondrial zinc deficiency might activate intrinsic cell death signaling pathway, which could not be effectively rescued by antioxidant treatment. PMID:25767260

Coxiella burnetii Avirulent Nine Mile Phase II Induces Caspase-1-Dependent Pyroptosis in Murine Peritoneal B1a B Cells.

PubMed

Schoenlaub, Laura; Cherla, Rama; Zhang, Yan; Zhang, Guoquan

2016-12-01

Our recent study demonstrated that virulent Coxiella burnetii Nine Mile phase I (NMI) is capable of infecting and replicating within peritoneal B1a cells and that B1a cells play an important role in host defense against C. burnetii infection in mice. However, it remains unknown if avirulent Nine Mile phase II (NMII) can infect and replicate in B1a cells and whether NMI and NMII can differentially interact with B1a cells. In this study, we examined if NMI and NMII can differentially modulate host cell apoptotic signaling in B1a cells. The results showed that NMII induced dose-dependent cell death in murine peritoneal B1a cells but NMI did not, suggesting that NMI and NMII may differentially activate host cell apoptotic signaling in B1a cells. Western blotting indicated that NMII-induced B1a cell death was not dependent on either caspase-3 or PARP-1 cleavage, but cleavage of caspase-1 was detected in NMII-infected B1a cells. In addition, inhibition or deficiency of caspase-1 activity blocked NMII-induced B1a cell death. These results suggest that NMII induces a caspase-1-dependent pyroptosis in murine peritoneal B1a cells. We also found that heat-killed NMII and type 4 secretion system (T4SS) mutant NMII were unable to induce B1a cell death and that NMII infection did not induce cell death in peritoneal B1a cells from Toll-like receptor 2 (TLR-2)- or NLRP3 inflammasome-deficient mice. These data suggest that NMII-induced caspase-1-dependent pyroptosis may require its T4SS and activation of the TLR-2 and NLRP3 signaling pathways. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Alpha Particles Induce Autophagy in Multiple Myeloma Cells.

PubMed

Gorin, Jean-Baptiste; Gouard, Sébastien; Ménager, Jérémie; Morgenstern, Alfred; Bruchertseifer, Frank; Faivre-Chauvet, Alain; Guilloux, Yannick; Chérel, Michel; Davodeau, François; Gaschet, Joëlle

2015-01-01

Radiation emitted by the radionuclides in radioimmunotherapy (RIT) approaches induce direct killing of the targeted cells as well as indirect killing through the bystander effect. Our research group is dedicated to the development of α-RIT, i.e., RIT using α-particles especially for the treatment of multiple myeloma (MM). γ-irradiation and β-irradiation have been shown to trigger apoptosis in tumor cells. Cell death mode induced by (213)Bi α-irradiation appears more controversial. We therefore decided to investigate the effects of (213)Bi on MM cell radiobiology, notably cell death mechanisms as well as tumor cell immunogenicity after irradiation. Murine 5T33 and human LP-1 MM cell lines were used to study the effects of such α-particles. We first examined the effects of (213)Bi on proliferation rate, double-strand DNA breaks, cell cycle, and cell death. Then, we investigated autophagy after (213)Bi irradiation. Finally, a coculture of dendritic cells (DCs) with irradiated tumor cells or their culture media was performed to test whether it would induce DC activation. We showed that (213)Bi induces DNA double-strand breaks, cell cycle arrest, and autophagy in both cell lines, but we detected only slight levels of early apoptosis within the 120 h following irradiation in 5T33 and LP-1. Inhibition of autophagy prevented (213)Bi-induced inhibition of proliferation in LP-1 suggesting that this mechanism is involved in cell death after irradiation. We then assessed the immunogenicity of irradiated cells and found that irradiated LP-1 can activate DC through the secretion of soluble factor(s); however, no increase in membrane or extracellular expression of danger-associated molecular patterns was observed after irradiation. This study demonstrates that (213)Bi induces mainly necrosis in MM cells, low levels of apoptosis, and autophagy that might be involved in tumor cell death.

The suppression of radiation-induced NF-{kappa}B activity by dexamethasone correlates with increased cell death in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nam, Seon Young; Chung, Hee-Yong

2005-10-21

In this study, we show that dexamethasone treatment increases ionizing radiation-induced cell death by inducing the inhibitory {kappa}B{alpha} (I{kappa}B{alpha}) pathway in mice. The effect of dexamethasone on radiation-induced cell death was assessed by changes in total spleen cellularity and bone marrow colony-forming unit-granulocyte-macrophage (CFU-GM) contents after total body irradiation. While in vivo treatment of mice with dexamethasone alone (1 mg/kg/day, for 2 days) failed to elicit cell death in spleen cells, the combined treatment with dexamethasone (1 mg/kg/day, for 2 days) and {gamma}-rays (1 or 5 Gy) caused a 50-80% reduction in total cellularity in spleen and CFU-GM contents inmore » bone marrow. These results demonstrate that dexamethasone has a synergistic effect on radiation-induced cellular damages in vivo. Immunoblot analysis showed that dexamethasone treatment significantly increases I{kappa}B{alpha} expression in the spleens of irradiated mice. In addition, the dexamethasone treatment significantly reduced radiation-induced nuclear translocation of the nucleus factor-{kappa}B in the spleens of irradiated mice. These results indicate that dexamethasone treatment in vivo may increase radiation-induced cell damages by increasing I{kappa}B{alpha} expression in hematopoietic organs such as spleen and bone marrow.« less

Neuroglobin protects astroglial cells from hydrogen peroxide-induced oxidative stress and apoptotic cell death.

PubMed

Amri, Fatma; Ghouili, Ikram; Amri, Mohamed; Carrier, Alice; Masmoudi-Kouki, Olfa

2017-01-01

Oxidative stress, resulting from accumulation of reactive oxygen species, plays a critical role in astroglial cell death occurring in diverse neuropathological conditions. Numerous studies indicate that neuroglobin (Ngb) promotes neuron survival, but nothing is known regarding the action of Ngb in astroglial cell survival. Thus, the purpose of this study was to investigate the potential glioprotective effect of Ngb on hydrogen peroxide (H 2 O 2 )-induced oxidative stress and apoptosis in cultured mouse astrocytes. Incubation of cells with subnanomolar concentrations of Ngb (10 -14 -10 -10  M) was found to prevent both H 2 O 2 -evoked reduction in surviving cells number and accumulation of reactive oxygen species in a concentration-dependent manner. Furthermore, Ngb treatment abolishes H 2 O 2 -induced increase in mitochondrial oxygen consumption rates. Concomitantly, Ngb treatment rescues H 2 O 2 -associated reduced expression of endogenous antioxidant enzymes (superoxide dismutases and catalase) and prevents the stimulation of the expression of pro-inflammatory genes (inducible nitric oxide synthase, cyclooxygenase-2, and interleukin (IL) IL-6 and IL-33). Moreover, Ngb blocks the stimulation of Bax (pro-apoptotic) and the inhibition of Bcl-2 (anti-apoptotic) gene expression induced by H 2 O 2 , which in turn abolishes caspase 3 activation. The protective effect of Ngb upon H 2 O 2 induced activation of caspase 3 activity and cell death can be accounted for by activation of protein kinase A and mitogen-activated protein kinase transduction cascade. Finally, we demonstrate that Ngb increases Akt phosphorylation and prevents H 2 O 2 -provoked inhibition of ERK and Akt phosphorylation. Taken together, these data demonstrate for the first time that Ngb is a glioprotective agent that prevents H 2 O 2 -induced oxidative stress and apoptotic astroglial cell death. Protection of astrocytes from oxidative insult may thus contribute to the neuroprotective effect of Ngb. © 2016 International Society for Neurochemistry.

Dexamethasone protection from TNF-alpha-induced cell death in MCF-7 cells requires NF-kappaB and is independent from AKT.

PubMed

Machuca, Catalina; Mendoza-Milla, Criselda; Córdova, Emilio; Mejía, Salvador; Covarrubias, Luis; Ventura, José; Zentella, Alejandro

2006-02-21

The biochemical bases for hormone dependence in breast cancer have been recognized as an important element in tumor resistance, proliferation and metastasis. On this respect, dexamethasone (Dex) dependent protection against TNF-alpha-mediated cell death in the MCF-7 cell line has been demonstrated to be a useful model for the study of this type of cancer. Recently, cytoplasmic signaling induced by steroid receptors has been described, such as the activation of the PI3K/Akt and NF-kappaB pathways. We evaluated their possible participation in the Dex-dependent protection against TNF-alpha-mediated cell death. Cellular cultures of the MCF-7 cell line were exposed to either, TNF-alpha or TNF-alpha and Dex, and cell viability was evaluated. Next, negative dominants of PI3K and IkappaB-alpha, designed to block the PI3K/Akt and NF-kappaB pathways, respectively, were transfected and selection and evaluation of several clones overexpressing the mutants were examined. Also, correlation with inhibitor of apoptosis proteins (IAPs) expression was examined. Independent inhibition of these two pathways allowed us to test their participation in Dex-dependent protection against TNF-alpha-cytotoxicity in MCF-7 cells. Expression of the PI3K dominant negative mutant did not alter the protection conferred by Dex against TNF-alpha mediated cell death. Contrariwise, clones expressing the IkappaB-alpha dominant negative mutant lost the Dex-conferred protection against TNF-alpha. In these clones degradation of c-IAP was accelerated, while that of XIAP was remained unaffected. NF-kappaB, but not PI3K/Akt activation, is required for the Dex protective effect against TNF-alpha-mediated cell death, and correlates with lack of degradation of the anti-apoptotic protein c-IAP1.

Polychlorinated biphenyl 138 exposure-mediated lipid droplet enlargement endows adipocytes with resistance to TNF-α-induced cell death.

PubMed

Kim, Yeon A; Kim, Hye Young; Oh, Yoo Jin; Kwon, Woo Young; Lee, Mi Hwa; Bae, Ju Yong; Woo, Min Seok; Kim, Jong-Min; Yoo, Young Hyun

2018-04-25

Although epidemiological reports have shown the association between polychlorinated biphenyls (PCBs) and obesity, the molecular mechanism of PCB-induced obesity is mostly unknown. The aim of the present study was to further dissect the significance of lipid droplet (LD) enlargement in PCB-induced obesity. For this aim, we hypothesized that PCB-induced LD enlargement endows adipocytes with resistance to cell death, inhibiting the natural loss of adipocytes. Four types of PCBs were screened, and the detailed molecular mechanism was investigated by using PCB-138. We observed that PCB-138-conferred cell death resistance to hypertrophic adipocytes with enlarged LDs. We further observed that PCB-138 prevents Tumour necrosis factor-α (TNF-α)-induced apoptosis and necroptosis in 3T3-L1 adipocytes and increases the expression of anti-apoptotic proteins, including survivin, in vitro and in vivo. In addition, we demonstrated that fat-specific protein 27 (Fsp27), perilipin, and survivin endow adipocytes with resistance to TNF-α-induced cell death through sustaining enlarged LDs. Thus, the present study suggests that PCB-138-induced LD enlargement endows adipocytes with resistance to TNF-α-induced cell death and that Fsp27, perilipin, and survivin, at least in part, help adipocytes to sustain enlarged LDs, contributing to the induction of obesity. Copyright © 2018 Elsevier B.V. All rights reserved.

Two host cytoplasmic effectors are required for pathogenesis of Phytophthora sojae by suppression of host defenses.

PubMed

Liu, Tingli; Ye, Wenwu; Ru, Yanyan; Yang, Xinyu; Gu, Biao; Tao, Kai; Lu, Shan; Dong, Suomeng; Zheng, Xiaobo; Shan, Weixing; Wang, Yuanchao; Dou, Daolong

2011-01-01

Phytophthora sojae encodes hundreds of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling- and necrosis-inducing proteins (CRN) or Crinkler. Their functions and mechanisms in pathogenesis are mostly unknown. Here, we identify a group of five P. sojae-specific CRN-like genes with high levels of sequence similarity, of which three are putative pseudogenes. Functional analysis shows that the two functional genes encode proteins with predicted nuclear localization signals that induce contrasting responses when expressed in Nicotiana benthamiana and soybean (Glycine max). PsCRN63 induces cell death, while PsCRN115 suppresses cell death elicited by the P. sojae necrosis-inducing protein (PsojNIP) or PsCRN63. Expression of CRN fragments with deleted signal peptides and FLAK motifs demonstrates that the carboxyl-terminal portions of PsCRN63 or PsCRN115 are sufficient for their activities. However, the predicted nuclear localization signal is required for PsCRN63 to induce cell death but not for PsCRN115 to suppress cell death. Furthermore, silencing of the PsCRN63 and PsCRN115 genes in P. sojae stable transformants leads to a reduction of virulence on soybean. Intriguingly, the silenced transformants lose the ability to suppress host cell death and callose deposition on inoculated plants. These results suggest a role for CRN effectors in the suppression of host defense responses.

Neem oil limonoids induces p53-independent apoptosis and autophagy

PubMed Central

Chandra, Dhyan

2012-01-01

Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells. PMID:22915764

Neem oil limonoids induces p53-independent apoptosis and autophagy.

PubMed

Srivastava, Pragya; Yadav, Neelu; Lella, Ravi; Schneider, Andrea; Jones, Anthony; Marlowe, Timothy; Lovett, Gabrielle; O'Loughlin, Kieran; Minderman, Hans; Gogada, Raghu; Chandra, Dhyan

2012-11-01

Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells.

Sensitization of vascular smooth muscle cell to TNF-{alpha}-mediated death in the presence of palmitate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rho, Mun-Chual; Ah Lee, Kyeong; Mi Kim, Sun

2007-05-01

Saturated free fatty acids (FFAs), including palmitate, can activate the intrinsic death pathway in cells. However, the relationship between FFAs and receptor-mediated death pathway is still unknown. In this study, we have investigated whether FFAs are able to trigger receptor-mediated death. In addition, to clarify the mechanisms responsible for the activation, we examined the biochemical changes in dying vascular smooth muscle cell (VSMC) and the effects of various molecules to the receptor-mediated VSMC death. Tumor necrosis factor (TNF)-{alpha}-mediated VSMC death occurred in the presence of sub-cytotoxic concentration of palmitate as determined by assessing viability and DNA degradation, while the cytokinemore » did not influence VSMC viability in the presence of oleate. The VSMC death was inhibited by the gene transfer of a dominant-negative Fas-associated death domain-containing protein and the baculovirus p35, but not by the bcl-xL or the c-Jun N-terminal kinase (JNK) binding domain of JNK-interacting protein-1, in tests utilizing recombinant adenoviruses. The VSMC death was also inhibited by a neutralizing anti-TNF receptor 1 antibody, the caspase inhibitor z-VAD, and the cathepsin B inhibitor CA074, a finding indicative of the role of both caspases and cathepsin B in this process. Consistent with this finding, caspase-3 activation and an increase in cytosolic cathepsin B activity were detected in the dying VSMC. Palmitate inhibited an increase of TNF-{alpha}-mediated nuclear factor kappa B (NF-{kappa}B) activity, the survival pathway activated by the cytokine, by hindering the translocation of the NF-{kappa}B subunit of p65 from the cytosol into the nucleus. The gene transfer of inhibitor of NF-{kappa}B predisposed VSMC to palmitate-induced cell death. To the best of our knowledge, this study is the first report to demonstrate the activation of TNF-{alpha}-mediated cell death in the presence of palmitate. The current study proposes that FFAs would take part in deleterious vascular consequences of such patients with elevated levels of FFAs as diabetics and obese individuals via the triggering of receptor-mediated death pathways of VSMC.« less

Cytotoxicity of Manganese (III) Complex in Human Breast Adenocarcinoma Cell Line Is Mediated by the Generation of Reactive Oxygen Species Followed by Mitochondrial Damage.

PubMed

Al-Anbaky, Qudes; Al-Karakooly, Zeiyad; Kilaparty, Surya P; Agrawal, Megha; Albkuri, Yahya M; RanguMagar, Ambar B; Ghosh, Anindya; Ali, Nawab

2016-11-01

Manganese (Mn) complexes are widely studied because of their important catalytic properties in synthetic and biochemical reactions. A Mn (III) complex of an amidoamine ligand was synthesized using a tetradentate amidoamine ligand. In this study, the Mn (III) complex was evaluated for its biological activity by measuring its cytotoxicity in human breast adenocarcinoma cell line (MCF-7). Cytotoxic effects of the Mn (III) complex were determined using established biomarkers in an attempt to delineate the mechanism of action and the utility of the complex as a potential anticancer drug. The Mn (III) complex induces cell death in a dose- and time-dependent manner as shown by microculture tetrazolium assay, a measure of cytotoxic cell death. Our results demonstrated that cytotoxic effects were significantly increased at higher concentrations of Mn (III) complex and with longer time of treatment. The IC 50 (Inhibitor concentration that results in 50% cell death) value of Mn (III) complex in MCF-7 cells was determined to be 2.5 mmol/L for 24 hours of treatment. In additional experiments, we determined the Mn (III) complex-mediated cell death was due to both apoptotic and nonspecific necrotic cell death mechanisms. This was assessed by ethidium bromide/acridine orange staining and flow cytometry techniques. The Mn (III) complex produced reactive oxygen species (ROS) triggering the expression of manganese superoxide dismutase 1 and ultimately damaging the mitochondrial function as is evident by a decline in mitochondrial membrane potential. Treatment of the cells with free radical scavenger, N, N-dimethylthiourea decreased Mn (III) complex-mediated generation of ROS and attenuated apoptosis. Together, these results suggest that the Mn (III) complex-mediated MCF-7 cell death utilizes combined mechanism involving apoptosis and necrosis perhaps due to the generation of ROS. © The Author(s) 2016.

Photochemical Targeting Of Phagocytic Trabecular Meshwork Cells Using Chlorin E6 Coupled Microspheres

NASA Astrophysics Data System (ADS)

Latina, M. A.; Kobsa, P. H.; Rakestraw, S. L.; Crean, E. A.; Hasan, T.; Yarmush, M. L.

1989-03-01

We have investigated a novel and efficient delivery system utilizing photosensitizer-coupled-latex microspheres to photochemically target and kill phagocytic trabecular meshwork (TM) cells. TM cells are the most actively phagocytic cells within the anterior chamber of the eye and are located within an optically accessible discrete band. This delivery system, along with the property of cell photocytosis, will achieve double selectivity by combining preferential localization of the photosensitizer to the target cells with spatial localization of illumination on the target cells. All experiments were performed with preconfluent bovine TM cells, 3rd to 4th passage, plated in 15 mm wells. Chlorin e6 monoethylene diamine monoamide was conjugated to the surface of 1.0 Am MX Duke Scientific fluorescent latex microspheres. Spectroscopic analysis revealed an average of 1.3 x 10 -17 moles of chlorin e6 per microsphere. TM cells were incubated for 18 hours with 5 x 10 7 microspheres/ml in MEM with 10% FCS, washed with MEM, and irradiated through fresh media using an argon-pumped dye laser emitting .2 W at 660 nm. A dose-survival study indicated that energy doses of 10 J/cm2 or greater resulted in greater than 95% cell death as determined by ethidium bromide exclusion. Cell death could be demonstrated as early as 4 hours post-irradiation. TM cells incubated with a solution of chlorin e6 at a concentration equal to that conjugated to the microspheres showed no cell death. Unirradiated controls also showed no cell death.

Involvement of miR17 pathway in glucocorticoid-induced cell death in pediatric acute lymphoblastic leukemia.

PubMed

Harada, Masako; Pokrovskaja-Tamm, Katja; Söderhäll, Stefan; Heyman, Mats; Grander, Dan; Corcoran, Martin

2012-10-01

Analysis of the microRNA transcriptome following dexa- methasone treatment of the acute lymphocytic leukemia (ALL) cell line RS4;11 showed a global down-regulation of microRNA levels. MIR17HG was rapidly down-regulated following treatment, with chromatin immunoprecipitation (ChIP) analysis demonstrating the promoter to be a direct target of glucocorticoid (GC)-transcriptional repression and revealing the miR17-92 cluster as a prime target for dexamethasone-induced repression. The loss of miR17 family expression and concomitant increases in the miR17 target Bim occurred in an additional ALL cell line SUP-B15 but not in the dexamethasone-resistant REH. Alteration of miR17 levels through up-regulation or inhibition resulted in an decrease and increase, respectively, in Bim protein levels and dexamethasone-induced cell death. Primary ex vivo ALL cells that underwent apoptosis induced by dexamethasone also down-regulated miR17 levels. Thus, down-regulation of miR17 plays an important role in glucocorticoid-induced cell death suggesting that targeting miR17 may improve the current ALL combination therapy.

Induction of MAPK- and ROS-dependent autophagy and apoptosis in gastric carcinoma by combination of romidepsin and bortezomib

PubMed Central

Hui, Kwai Fung; Yeung, Po Ling; Chiang, Alan K.S.

2016-01-01

Proteasome inhibitors and histone deacetylase (HDAC) inhibitors can synergistically induce apoptotic cell death in certain cancer cell types but their combinatorial effect on the induction of autophagy remains unknown. Here, we investigated the combinatorial effects of a proteasome inhibitor, bortezomib, and an HDAC inhibitor, romidepsin, on the induction of apoptotic and autophagic cell death in gastric carcinoma (GC) cells. Isobologram analysis showed that low nanomolar concentrations of bortezomib/romidepsin could synergistically induce killing of GC cells. The synergistic killing was due to the summative effect of caspase-dependent intrinsic apoptosis and caspase-independent autophagy. The autophagic cell death was dependent on the activation of MAPK family members (ERK1/2 and JNK), and generation of reactive oxygen species (ROS), but was independent of Epstein-Barr virus infection. In vivo, bortezomib/romidepsin also significantly induced apoptosis and autophagy in GC xenografts in nude mice. This is the first report demonstrating the potent effect of combination of HDAC and proteasome inhibitors on the induction of MAPK- and ROS-dependent autophagy in addition to caspase-dependent apoptosis in a cancer type. PMID:26683357

Induction of MAPK- and ROS-dependent autophagy and apoptosis in gastric carcinoma by combination of romidepsin and bortezomib.

PubMed

Hui, Kwai Fung; Yeung, Po Ling; Chiang, Alan K S

2016-01-26

Proteasome inhibitors and histone deacetylase (HDAC) inhibitors can synergistically induce apoptotic cell death in certain cancer cell types but their combinatorial effect on the induction of autophagy remains unknown. Here, we investigated the combinatorial effects of a proteasome inhibitor, bortezomib, and an HDAC inhibitor, romidepsin, on the induction of apoptotic and autophagic cell death in gastric carcinoma (GC) cells. Isobologram analysis showed that low nanomolar concentrations of bortezomib/romidepsin could synergistically induce killing of GC cells. The synergistic killing was due to the summative effect of caspase-dependent intrinsic apoptosis and caspase-independent autophagy. The autophagic cell death was dependent on the activation of MAPK family members (ERK1/2 and JNK), and generation of reactive oxygen species (ROS), but was independent of Epstein-Barr virus infection. In vivo, bortezomib/romidepsin also significantly induced apoptosis and autophagy in GC xenografts in nude mice. This is the first report demonstrating the potent effect of combination of HDAC and proteasome inhibitors on the induction of MAPK- and ROS-dependent autophagy in addition to caspase-dependent apoptosis in a cancer type.

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

PubMed

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.

Microbubble and ultrasound radioenhancement of bladder cancer

PubMed Central

Tran, W T; Iradji, S; Sofroni, E; Giles, A; Eddy, D; Czarnota, G J

2012-01-01

Background: Tumour vasculature is an important component of tumour growth and survival. Recent evidence indicates tumour vasculature also has an important role in tumour radiation response. In this study, we investigated ultrasound and microbubbles to enhance the effects of radiation. Methods: Human bladder cancer HT-1376 xenografts in severe combined immuno-deficient mice were used. Treatments consisted of no, low and high concentrations of microbubbles and radiation doses of 0, 2 and 8 Gy in short-term and longitudinal studies. Acute response was assessed 24 h after treatment and longitudinal studies monitored tumour response weekly up to 28 days using power Doppler ultrasound imaging for a total of 9 conditions (n=90 animals). Results: Quantitative analysis of ultrasound data revealed reduced blood flow with ultrasound-microbubble treatments alone and further when combined with radiation. Tumours treated with microbubbles and radiation revealed enhanced cell death, vascular normalisation and areas of fibrosis. Longitudinal data demonstrated a reduced normalised vascular index and increased tumour cell death in both low and high microbubble concentrations with radiation. Conclusion: Our study demonstrated that ultrasound-mediated microbubble exposure can enhance radiation effects in tumours, and can lead to enhanced tumour cell death. PMID:22790798

The selective Aurora B kinase inhibitor AZD1152 is a potential new treatment for multiple myeloma.

PubMed

Evans, Robert P; Naber, Claudia; Steffler, Tara; Checkland, Tamara; Maxwell, Christopher A; Keats, Jonathan J; Belch, Andrew R; Pilarski, Linda M; Lai, Raymond; Reiman, Tony

2008-02-01

Aurora kinases are potential targets for cancer therapy. Previous studies have validated Aurora kinase A as a therapeutic target in multiple myeloma (MM), and have demonstrated in vitro anti-myeloma effects of small molecule Aurora kinase inhibitors that inhibit both Aurora A and B. This study demonstrated that Aurora B kinase was strongly expressed in myeloma cell lines and primary plasma cells. The selective Aurora B inhibitor AZD1152-induced apoptotic death in myeloma cell lines at nanomolar concentrations, with a cell cycle phenotype consistent with that reported previously for Aurora B inhibition. In some cases, AZD1152 in combination with dexamethasone showed increased anti-myeloma activity compared with the use of either agent alone. AZD1152 was active against sorted CD138(+) BM plasma cells from myeloma patients but also, as expected, was toxic to CD138(-) marrow cells from the same patients. In a murine myeloma xenograft model, AZD1152-inhibited tumour growth at well-tolerated doses and induced cell death in established tumours, with associated mild, transient leucopenia. AZD1152 shows promise in these preclinical studies as a novel treatment for MM.

Apoptotic cell death in the central nervous system of Bufo arenarum tadpoles induced by cypermethrin.

PubMed

Casco, V H; Izaguirre, M F; Marín, L; Vergara, M N; Lajmanovich, R C; Peltzer, P; Soler, A Peralta

2006-05-01

Tadpoles of the toad Bufo arenarum treated with cypermethrin (CY) at concentrations above 39 mug CY/L showed dose-dependent apoptotic cell death in immature cells of the central nervous system as demonstrated by morphometric analysis, the TUNEL method, and DNA fragmentation assay. Light-and electron-microscopic studies showed structural alterations in the intermediate and marginal layers of the brain. Immature cerebral tissue showed cellular shrinkage, nuclear fragmentation and increase of intercellular spaces. In this study we demonstrated high toxicity of CY to larval stages of Bufo arenarum. Our results show that doses lower than those used in routine insecticide applications can cause massive apoptosis in the immature cells of the central nervous system. These results coincide with our previous studies in Physalaemus biligonigerus, confirming the severe toxic effects of CY to the central nervous system of anuran species from Argentina. This may increase the mortality index in wild animals and contribute to the loss of biodiversity in our agroecosystems. We postulate that CY induces apoptosis in central nervous system cells of Bufo arenarum tadpoles by specific neurotoxic mechanisms.

Mitochondria-derived reactive oxygen species drive GANT61-induced mesothelioma cell apoptosis.

PubMed

Lim, Chuan Bian; Prêle, Cecilia M; Baltic, Svetlana; Arthur, Peter G; Creaney, Jenette; Watkins, D Neil; Thompson, Philip J; Mutsaers, Steven E

2015-01-30

Gli transcription factors of the Hedgehog (Hh) pathway have been reported to be drivers of malignant mesothelioma (MMe) cell survival. The Gli inhibitor GANT61 induces apoptosis in various cancer cell models, and has been associated directly with Gli inhibition. However various chemotherapeutics can induce cell death through generation of reactive oxygen species (ROS) but whether ROS mediates GANT61-induced apoptosis is unknown. In this study human MMe cells were treated with GANT61 and the mechanisms regulating cell death investigated. Exposure of MMe cells to GANT61 led to G1 phase arrest and apoptosis, which involved ROS but not its purported targets, GLI1 or GLI2. GANT61 triggered ROS generation and quenching of ROS protected MMe cells from GANT61-induced apoptosis. Furthermore, we demonstrated that mitochondria are important in mediating GANT61 effects: (1) ROS production and apoptosis were blocked by mitochondrial inhibitor rotenone; (2) GANT61 promoted superoxide formation in mitochondria; and (3) mitochondrial DNA-deficient LO68 cells failed to induce superoxide, and were more resistant to apoptosis induced by GANT61 than wild-type cells. Our data demonstrate for the first time that GANT61 induces apoptosis by promoting mitochondrial superoxide generation independent of Gli inhibition, and highlights the therapeutic potential of mitochondrial ROS-mediated anticancer drugs in MMe.

DNA damage response in nephrotoxic and ischemic kidney injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Mingjuan; Tang, Chengyuan

DNA damage activates specific cell signaling cascades for DNA repair, cell cycle arrest, senescence, and/or cell death. Recent studies have demonstrated DNA damage response (DDR) in experimental models of acute kidney injury (AKI). In cisplatin-induced AKI or nephrotoxicity, the DDR pathway of ATR/Chk2/p53 is activated and contributes to renal tubular cell apoptosis. In ischemic AKI, DDR seems more complex and involves at least the ataxia telangiectasia mutated (ATM), a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, and p53; however, while ATM may promote DNA repair, p53 may trigger cell death. Targeting DDR for kidney protection in AKI therefore reliesmore » on a thorough elucidation of the DDR pathways in various forms of AKI.« less

In vitro model to study cocaine and its contaminants.

PubMed

Steinmetz, Aline; Steffens, Luiza; Morás, Ana Moira; Prezzi, Flávia; Braganhol, Elizandra; Saffi, Jenifer; Ortiz, Rafael Scorsatto; Barros, Helena M T; Moura, Dinara Jaqueline

2018-04-01

Cocaine is one of the most popular illicit drug worldwide. Due its great addictive potential, which leads to euphoria and hyperactivity, it is considered a public health concern. At the central nervous system, the drug acts inhibiting catecholamine re-uptake. It is now known that in addition to the toxicity of the drug itself, the contaminants present in the street drug have raised concern about the harmful effects on health. Toxicological in vivo and in vitro studies have demonstrated the toxic effects of cocaine correlated with the generation of reactive oxygen species (ROS), which in turn lead to oxidative damage to the cells. Therefore the aim of this work was to propose an in vitro model that reunites the main parameters of toxicity of the cocaine already observed in the literature so far, and we tested this model using cocaine and seizure cocaine sample (SCS), kindly provided by Federal Police of Brazil. For that, we used a C6 glioblastoma cells and evaluated cell death, oxygen reactive species induction, oxidation of macromolecules as membrane lipids and DNA and loss of mitochondrial membrane potential after cocaine exposure. The results showed that cocaine can decrease cellular viability in a dose-dependent way in the C6 cell immortalized and astrocytes primary culture. Cocaine also induced cellular death by apoptosis. However, in the seizure cocaine sample (SCS), the predominant cell death was due to necrosis. Using dichlorofluorescein (DCF) assay, we confirmed ROS production after cocaine exposition. In agreement with these findings, occurred an increasing in MDA production, as well as increased superoxide dismutase (SOD) and catalase (CAT) activity. The induction of DNA damage was observed after cocaine. Our results demonstrate the occurrence of mitochondrial dysfunction by depolarization of mitochondrial membrane as a consequence of cocaine treatment. In summary, these results demonstrated that cocaine can induce reactive oxygen species formation, leading to oxidative stress. As a consequence of this unbalance, DNA damage, lipidic peroxidation and loss of mitochondrial membrane occurred, which could be an answer to cell death observed. Copyright © 2018 Elsevier B.V. All rights reserved.

Compartmentalized oxidative stress in dopaminergic cell death induced by pesticides and complex I inhibitors: Distinct roles of superoxide anion and superoxide dismutases

PubMed Central

Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely; Pickett, Chillian; Sumin, Li; Jones, Jocelyn; Chen, Han; Webb, Brian; Choi, Jae; Zhou, You; Zimmerman, Matthew C.; Franco, Rodrigo

2013-01-01

The loss of dopaminergic neurons induced by the parkinsonian toxins paraquat, rotenone and 1-methyl-4-phenylpyridinium (MPP+) is associated with oxidative stress. However, controversial reports exist regarding the source/compartmentalization of reactive oxygen species (ROS) generation and its exact role in cell death. We aimed to determine in detail the role of superoxide anion (O2•−), oxidative stress and their subcellular compartmentalization in dopaminergic cell death induced by parkinsonian toxins. Oxidative stress and ROS formation was determined in the cytosol, intermembrane (IMS) and mitochondrial matrix compartments, using dihydroethidine derivatives, the redox sensor roGFP, as well as electron paramagnetic resonance spectroscopy. Paraquat induced an increase in ROS and oxidative stress in both the cytosol and mitochondrial matrix prior to cell death. MPP+ and rotenone primarily induced an increase in ROS and oxidative stress in the mitochondrial matrix. No oxidative stress was detected at the level of the IMS. In contrast to previous studies, overexpression of manganese superoxide dismutase (MnSOD) or copper/zinc SOD (CuZnSOD) had no effect on ROS steady state levels, lipid peroxidation, loss of mitochondrial membrane potential (ΔΨm) and dopaminergic cell death induced by MPP+ or rotenone. In contrast, paraquat-induced oxidative stress and cell death were selectively reduced by MnSOD overexpression, but not by CuZnSOD or manganese-porphyrins. However, MnSOD also failed to prevent ΔΨm loss. Finally, paraquat, but not MPP+ or rotenone, induced the transcriptional activation the redox-sensitive antioxidant response elements (ARE) and nuclear factor kappa-B (NF-κB). These results demonstrate a selective role of mitochondrial O2•− in dopaminergic cell death induced by paraquat, and show that toxicity induced by the complex I inhibitors rotenone and MPP+ does not depend directly on mitochondrial O2•− formation. PMID:23602909

A microplate assay for measuring cell death in C2C12 cells.

PubMed

Lima, Tanes; Silveira, Leonardo

2018-03-22

The main goal of this study was to develop a straightforward and rapid microplate assay for measuring propidium iodide (PI) in C2C12 cells. The PI method proves to be an efficient quantitative assay for analyzing cell viability through PI fluorescence analysis. Importantly, the protocol takes less than 30 minutes, and the results are reproducible. C2C12 cells were exposed to an increasing concentration of palmitate for a period of 24 hours to induce cell death, and the PI fluorescence increased in a concentration-dependent manner. Evaluation of mitochondrial function and reactive oxygen species production validated the deleterious effects of palmitate treatment. Also, the microplate PI assay demonstrated high sensitivity as indicated by the detection of modest fluctuations in cell viability in response to catalase overexpression in palmitate-treated cells. The microplate PI assay, therefore, offers an accurate method to be used for in vitro studies.

Novel role of TRPV2 in promoting the cytotoxicity of H2O2-mediated oxidative stress in human hepatoma cells.

PubMed

Ma, Wenbo; Li, Caiyue; Yin, Shikui; Liu, Jingxin; Gao, Chao; Lin, Zuoxian; Huang, Rongqi; Huang, Jufang; Li, Zhiyuan

2015-12-01

Oxidative stress is important for the initiation and progression of cancers, which confers the cells with a survival advantage by inducing oxidative adaption and drug resistance. Therefore, developing strategies to promote oxidative stress-induced cytotoxicity could be important for cancer therapy. Herein, we found that H2O2-mediated oxidative stress increases TRPV2 expression in human hepatoma (HepG2 and Huh-7) cells. This occurred at the mRNA and protein levels in a dose-dependent manner. The significance of TRPV2 in promoting H2O2-induced cell death was demonstrated in gain and loss of function studies with overexpression and knockdown of TRPV2, respectively. Mechanistically, H2O2-induced cell death involves inhibition of pro-survival signaling proteins (Akt, Nrf2) and activation of pro-death signaling proteins (p38, JNK1). Overexpression of TRPV2 in H2O2-treated hepatoma cells aggravates the inhibition of Akt and Nrf2, while it enhances the activation of p38 and JNK1 at the early stage of cell death. Interestingly, increased expression of TRPV2 in HepG2 cells improved the efficacy of stress-associated chemicals to induce cell death. Our findings suggest that TRPV2 acts as an important enhancer for H2O2-induced cytotoxicity. This process occurred by the inhibition of Akt and Nrf2 as well as the early activation of p38 and JNK1. These findings have important implications for inhibition of oxidative adaption and drug resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

Leukemia cell proliferation and death in chronic lymphocytic leukemia patients on therapy with the BTK inhibitor ibrutinib.

PubMed

Burger, Jan A; Li, Kelvin W; Keating, Michael J; Sivina, Mariela; Amer, Ahmed M; Garg, Naveen; Ferrajoli, Alessandra; Huang, Xuelin; Kantarjian, Hagop; Wierda, William G; O'Brien, Susan; Hellerstein, Marc K; Turner, Scott M; Emson, Claire L; Chen, Shih-Shih; Yan, Xiao-Jie; Wodarz, Dominik; Chiorazzi, Nicholas

2017-01-26

BACKGROUND. Ibrutinib is an effective targeted therapy for patients with chronic lymphocytic leukemia (CLL) that inhibits Bruton's tyrosine kinase (BTK), a kinase involved in B cell receptor signaling. METHODS. We used stable isotopic labeling with deuterated water ( 2 H 2 O) to measure directly the effects of ibrutinib on leukemia cell proliferation and death in 30 patients with CLL. RESULTS. The measured average CLL cell proliferation ("birth") rate before ibrutinib therapy was 0.39% of the clone per day (range 0.17%-1.04%); this decreased to 0.05% per day (range 0%-0.36%) with treatment. Death rates of blood CLL cells increased from 0.18% per day (average, range 0%-0.7%) prior to treatment to 1.5% per day (range 0%-3.0%) during ibrutinib therapy, and they were even higher in tissue compartments. CONCLUSIONS. This study provides the first direct in vivo measurements to our knowledge of ibrutinib's antileukemia actions, demonstrating profound and immediate inhibition of CLL cell proliferation and promotion of high rates of CLL cell death. TRIAL REGISTRATION. This trial was registered at clinicaltrials.gov (NCT01752426). FUNDING. This study was supported by a Cancer Center Support Grant (National Cancer Institute grant P30 CA016672), an NIH grant (CA081554) from the National Cancer Institute, MD Anderson's Moon Shots Program in CLL, and Pharmacyclics, an AbbVie company.

Critical involvement of extracellular ATP acting on P2RX7 purinergic receptors in photoreceptor cell death.

PubMed

Notomi, Shoji; Hisatomi, Toshio; Kanemaru, Takaaki; Takeda, Atsunobu; Ikeda, Yasuhiro; Enaida, Hiroshi; Kroemer, Guido; Ishibashi, Tatsuro

2011-12-01

Stressed cells release ATP, which participates in neurodegenerative processes through the specific ligation of P2RX7 purinergic receptors. Here, we demonstrate that extracellular ATP and the more specific P2RX7 agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP, both induce photoreceptor cell death when added to primary retinal cell cultures or when injected into the eyes from wild-type mice, but not into the eyes from P2RX7(-/-) mice. Photoreceptor cell death was accompanied by the activation of caspase-8 and -9, translocation of apoptosis-inducing factor from mitochondria to nuclei, and TUNEL-detectable chromatin fragmentation. All hallmarks of photoreceptor apoptosis were prevented by premedication or co-application of Brilliant Blue G, a selective P2RX7 antagonist that is already approved for the staining of internal limiting membranes during ocular surgery. ATP release is up-regulated by nutrient starvation in primary retinal cell cultures and seems to be an initializing event that triggers primary and/or secondary cell death via the positive feedback loop on P2RX7. Our results encourage the potential application of Brilliant Blue G as a novel neuroprotective agent in retinal diseases or similar neurodegenerative pathologies linked to excessive extracellular ATP. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Defective TFH Cell Function and Increased TFR Cells Contribute to Defective Antibody Production in Aging.

PubMed

Sage, Peter T; Tan, Catherine L; Freeman, Gordon J; Haigis, Marcia; Sharpe, Arlene H

2015-07-14

Defective antibody production in aging is broadly attributed to immunosenescence. However, the precise immunological mechanisms remain unclear. Here, we demonstrate an increase in the ratio of inhibitory T follicular regulatory (TFR) cells to stimulatory T follicular helper (TFH) cells in aged mice. Aged TFH and TFR cells are phenotypically distinct from those in young mice, exhibiting increased programmed cell death protein-1 expression but decreased ICOS expression. Aged TFH cells exhibit defective antigen-specific responses, and programmed cell death protein-ligand 1 blockade can partially rescue TFH cell function. In contrast, young and aged TFR cells have similar suppressive capacity on a per-cell basis in vitro and in vivo. Together, these studies reveal mechanisms contributing to defective humoral immunity in aging: an increase in suppressive TFR cells combined with impaired function of aged TFH cells results in reduced T-cell-dependent antibody responses in aged mice. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Collapsing Aged Culture of the Cyanobacterium Synechococcus elongatus Produces Compound(s) Toxic to Photosynthetic Organisms

PubMed Central

Cohen, Assaf; Sendersky, Eleonora; Carmeli, Shmuel; Schwarz, Rakefet

2014-01-01

Phytoplankton mortality allows effective nutrient cycling, and thus plays a pivotal role in driving biogeochemical cycles. A growing body of literature demonstrates the involvement of regulated death programs in the abrupt collapse of phytoplankton populations, and particularly implicates processes that exhibit characteristics of metazoan programmed cell death. Here, we report that the cell-free, extracellular fluid (conditioned medium) of a collapsing aged culture of the cyanobacterium Synechococcus elongatus is toxic to exponentially growing cells of this cyanobacterium, as well as to a large variety of photosynthetic organisms, but not to eubacteria. The toxic effect, which is light-dependent, involves oxidative stress, as suggested by damage alleviation by antioxidants, and the very high sensitivity of a catalase-mutant to the conditioned medium. At relatively high cell densities, S. elongatus cells survived the deleterious effect of conditioned medium in a process that required de novo protein synthesis. Application of conditioned medium from a collapsing culture caused severe pigment bleaching not only in S. elongatus cells, but also resulted in bleaching of pigments in a cell free extract. The latter observation indicates that the elicited damage is a direct effect that does not require an intact cell, and therefore, is mechanistically different from the metazoan-like programmed cell death described for phytoplankton. We suggest that S. elongatus in aged cultures are triggered to produce a toxic compound, and thus, this process may be envisaged as a novel regulated death program. PMID:24959874

Apigenin sensitizes colon cancer cells to anti-tumor activity of ABT-263

PubMed Central

Shao, Huanjie; Jing, Kai; Mahmoud, Esraa; Huang, Haihong; Fang, Xianjun; Yu, Chunrong

2013-01-01

Apigenin is an edible plant-derived flavonoid that shows modest anti-tumor activities in vitro and in vivo. Apigenin treatment resulted in cell growth arrest and apoptosis in various types of tumors by modulating several signaling pathways. In the present study, we evaluated interactions between apigenin and ABT-263 in colon cancer cells. We observed a synergistic effect between apigenin and ABT-263 on apoptosis of colon cancer cells. ABT-263 alone induced limited cell death while upregulating expression of Mcl-1, a potential mechanism for the acquired resistance to ABT-263. The presence of apigenin antagonized ABT-263-induced Mcl-1 upregulation and dramatically enhanced ABT-263-induced cell death. Meanwhile, apigenin suppressed AKT and ERK activation. Inactivation of either AKT or ERK by lentivirus-transduced shRNA or treatment with specific small molecule inhibitors of these pathways enhanced ABT-263-induced cell death, mirroring the effect of apigenin. Moreover, the combination response was associated with upregulation of Bim and activation of Bax. Downregulation of Bax eliminated the synergistic effect of apigenin and ABT-263 on cell death. Xenograft studies in SCID mice showed that the combined treatment with apigenin and ABT-263 inhibited tumor growth by up to 70% without obvious adverse effects, while either agent only inhibited around 30%. Our results demonstrate a novel strategy to enhance ABT-263 induced anti-tumor activity in human colon cancer cells by apigenin via inhibition of the Mcl-1, AKT and ERK pro-survival regulators. PMID:24126433

Fatty acids trigger mitochondrion-dependent necrosis.

PubMed

Rockenfeller, Patrick; Ring, Julia; Muschett, Vera; Beranek, Andreas; Buettner, Sabrina; Carmona-Gutierrez, Didac; Eisenberg, Tobias; Khoury, Chamel; Rechberger, Gerald; Kohlwein, Sepp D; Kroemer, Guido; Madeo, Frank

2010-07-15

Obesity is characterised by lipid accumulation in non-adipose tissues, leading to organ degeneration and a wide range of diseases, including diabetes, heart attack and liver cirrhosis. Free fatty acids (FFA) are believed to be the principal toxic triggers mediating the adverse cellular effects of lipids. Here, we show that various cooking oils used in human nutrition cause cell death in yeast in the presence of a triacylglycerol lipase, mimicking the physiological microenvironment of the small intestine. Combining genetic and cell death assays, we demonstrate that elevated FFA concentrations lead to necrotic cell death, as evidenced by loss of membrane integrity and release of nuclear HMGB1. FFA-mediated necrosis depends on functional mitochondria and leads to the accumulation of reactive oxygen species. We conclude that lipotoxicity is executed via a mitochondrial necrotic pathway, challenging the dogma that the adverse effects of lipid stress are exclusively apoptotic.

Methanolic extract of white asparagus shoots activates TRAIL apoptotic death pathway in human cancer cells and inhibits colon carcinogenesis in a preclinical model

PubMed Central

BOUSSEROUEL, SOUAD; LE GRANDOIS, JULIE; GOSSÉ, FRANCINE; WERNER, DALAL; BARTH, STEPHAN W.; MARCHIONI, ERIC; MARESCAUX, JACQUES; RAUL, FRANCIS

2013-01-01

Shoots of white asparagus are a popular vegetable dish, known to be rich in many bioactive phytochemicals reported to possess antioxidant, and anti-inflammatory and antitumor activities. We evaluated the anticancer mechanisms of a methanolic extract of Asparagus officinalis L. shoots (Asp) on human colon carcinoma cells (SW480) and their derived metastatic cells (SW620), and Asp chemopreventive properties were also assessed in a model of colon carcinogenesis. SW480 and SW620 cell proliferation was inhibited by 80% after exposure to Asp (80 μg/ml). We demonstrated that Asp induced cell death through the activation of TRAIL DR4/DR5 death receptors leading to the activation of caspase-8 and caspase-3 and to cell apoptosis. By specific blocking agents of DR4/DR5 receptors we were able to prevent Asp-triggered cell death confirming the key role of DR4/DR5 receptors. We found also that Asp (80 μg/ml) was able to potentiate the effects of the cytokine TRAIL on cell death even in the TRAIL-resistant metastatic SW620 cells. Colon carcinogenesis was initiated in Wistar rats by intraperitoneal injections of azoxymethane (AOM), once a week for two weeks. One week after (post-initiation) rats received daily Asp (0.01%, 14 mg/kg body weight) in drinking water. After 7 weeks of Asp-treatment the colon of rats exhibited a 50% reduction of the number of preneoplastic lesions (aberrant crypt foci). In addition Asp induced inhibition of several pro-inflammatory mediators, in association with an increased expression of host-defense mediators. In the colonic mucosa of Asp-treated rats we also confirmed the pro-apoptotic effects observed in vitro including the activation of the TRAIL death-receptor signaling pathway. Taken together, our data highlight the chemopreventive effects of Asp on colon carcinogenesis and its ability to promote normal cellular homeostasis. PMID:23754197

Activation of apoptosis signal-regulating kinase 1 is a key factor in paraquat-induced cell death: modulation by the Nrf2/Trx axis.

PubMed

Niso-Santano, Mireia; González-Polo, Rosa A; Bravo-San Pedro, José M; Gómez-Sánchez, Rubén; Lastres-Becker, Isabel; Ortiz-Ortiz, Miguel A; Soler, Germán; Morán, José M; Cuadrado, Antonio; Fuentes, José M

2010-05-15

Although oxidative stress is fundamental to the etiopathology of Parkinson disease, the signaling molecules involved in transduction after oxidant exposure to cell death are ill-defined, thus making it difficult to identify molecular targets of therapeutic relevance. We have addressed this question in human dopaminergic neuroblastoma SH-SY5Y cells exposed to the parkinsonian toxin paraquat (PQ). This toxin elicited a dose-dependent increase in reactive oxygen species and cell death that correlated with activation of ASK1 and the stress kinases p38 and JNK. The relevance of these kinases in channeling PQ neurotoxicity was demonstrated with the use of interference RNA for ASK1 and two well-established pharmaceutical inhibitors for JNK and p38. The toxic effect of PQ was substantially attenuated by preincubation with vitamin E, blocking ASK1 pathways and preventing oxidative stress and cell death. In a search for a physiological pathway that might counterbalance PQ-induced ASK1 activation, we analyzed the role of the transcription factor Nrf2, master regulator of redox homeostasis, and its target thioredoxin (Trx), which binds and inhibits ASK1. Trx levels were undetectable in Nrf2-deficient mouse embryo fibroblasts (MEFs), whereas they were constitutively high in Keap1-deficient MEFs as well as in SH-SY5Y cells treated with sulforaphane (SFN). Consistent with these data, Nrf2-deficient MEFs were more sensitive and Keap1-deficient MEFs and SH-SY5Y cells incubated with SFN were more resistant to PQ-induced cell death. This study identifies ASK1/JNK and ASK1/p38 as two critical pathways involved in the activation of cell death under oxidative stress conditions and identifies the Nrf2/Trx axis as a new target to block these pathways and protect from oxidant exposure such as that found in Parkinson and other neurodegenerative diseases. Copyright 2010 Elsevier Inc. All rights reserved.

Vitamin C in synergism with cisplatin induces cell death in cervical cancer cells through altered redox cycling and p53 upregulation.

PubMed

Leekha, Ankita; Gurjar, Bahadur S; Tyagi, Aakriti; Rizvi, Moshahid A; Verma, Anita K

2016-12-01

Cervical cancer is the second most prevalent cancer in women worldwide. Survival of patients has been improved by cisplatin-based chemotherapy, but its effectiveness is limited due to its adverse effects on many tissues, especially nephrotoxicity. To optimize the efficacy of CDDP, we propose a combination therapy using natural products with minimal side effects. Vitamin C being a natural antioxidant is capable of selectively targeting cancer cells at pharmacological concentrations. Vitamin C synergistically enhances the activity of chemotherapeutic agents without increasing toxicity to normal cells. Therefore, we exploited co-therapy with cisplatin and vitamin C to kill cervical cancer cells. We elucidated the role of CDDP and VC on cervical cancer cell line (SiHa) by using cell growth assays, DNA fragmentation analysis, comet assay, in vitro morphological assessment of apoptosis (AO/EB and DAPI staining), ROS analysis by DCFDA, flow cytometry, biochemical assays (GST, GSH, NO, catalase, TPA) and Western blotting. Our results clearly demonstrated that CDDP and VC treatment exhibited ameliorative effect on induction of cell death by p53 overexpression and generation of hydrogen peroxide in SiHa cells, thereby reducing the dosage of CDDP required to induce cell death in cancer cells. These studies provide novel approaches to combat cisplatin resistance in cervical cancer.

Protective Effect of Edaravone on Glutamate-Induced Neurotoxicity in Spiral Ganglion Neurons

PubMed Central

Bai, Xiaohui; Zhang, Chi; Chen, Aiping; Liu, Wenwen; Li, Jianfeng; Sun, Qian

2016-01-01

Glutamate is an important excitatory neurotransmitter in mammalian brains, but excessive amount of glutamate can cause “excitotoxicity” and lead to neuronal death. As bipolar neurons, spiral ganglion neurons (SGNs) function as a “bridge” in transmitting auditory information from the ear to the brain and can be damaged by excessive glutamate which results in sensorineural hearing loss. In this study, edaravone, a free radical scavenger, elicited both preventative and therapeutic effects on SGNs against glutamate-induced cell damage that was tested by MTT assay and trypan blue staining. Ho.33342 and PI double staining revealed that apoptosis as well as necrosis took place during glutamate treatment, and apoptosis was the main type of cell death. Oxidative stress played an important role in glutamate-induced cell damage but pretreatment with edaravone alleviated cell death. Results of western blot demonstrated that mechanisms underlying the toxicity of glutamate and the protection of edaravone were related to the PI3K pathway and Bcl-2 protein family. PMID:27957345

Antioxidant gene therapy against neuronal cell death

PubMed Central

Navarro-Yepes, Juliana; Zavala-Flores, Laura; Annadurai, Anandhan; Wang, Fang; Skotak, Maciej; Chandra, Namas; Li, Ming; Pappa, Aglaia; Martinez-Fong, Daniel; Razo, Luz Maria Del; Quintanilla-Vega, Betzabet; Franco, Rodrigo

2014-01-01

Oxidative stress is a common hallmark of neuronal cell death associated with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, as well as brain stroke/ischemia and traumatic brain injury. Increased accumulation of reactive species of both oxygen (ROS) and nitrogen (RNS) has been implicated in mitochondrial dysfunction, energy impairment, alterations in metal homeostasis and accumulation of aggregated proteins observed in neurodegenerative disorders, which lead to the activation/modulation of cell death mechanisms that include apoptotic, necrotic and autophagic pathways. Thus, the design of novel antioxidant strategies to selectively target oxidative stress and redox imbalance might represent important therapeutic approaches against neurological disorders. This work reviews the evidence demonstrating the ability of genetically encoded antioxidant systems to selectively counteract neuronal cell loss in neurodegenerative diseases and ischemic brain damage. Because gene therapy approaches to treat inherited and acquired disorders offer many unique advantages over conventional therapeutic approaches, we discussed basic research/clinical evidence and the potential of virus-mediated gene delivery techniques for antioxidant gene therapy. PMID:24333264

Manipulating the mitochondria activity in human hepatic cell line Huh7 by low-power laser irradiation

PubMed Central

Lynnyk, Anna; Lunova, Mariia; Jirsa, Milan; Egorova, Daria; Kulikov, Andrei; Kubinová, Šárka; Lunov, Oleg; Dejneka, Alexandr

2018-01-01

Low-power laser irradiation of red light has been recognized as a promising tool across a vast variety of biomedical applications. However, deep understanding of the molecular mechanisms behind laser-induced cellular effects remains a significant challenge. Here, we investigated mechanisms involved in the death process in human hepatic cell line Huh7 at a laser irradiation. We decoupled distinct cell death pathways targeted by laser irradiations of different powers. Our data demonstrate that high dose laser irradiation exhibited the highest levels of total reactive oxygen species production, leading to cyclophilin D-related necrosis via the mitochondrial permeability transition. On the contrary, low dose laser irradiation resulted in the nuclear accumulation of superoxide and apoptosis execution. Our findings offer a novel insight into laser-induced cellular responses, and reveal distinct cell death pathways triggered by laser irradiation. The observed link between mitochondria depolarization and triggering ROS could be a fundamental phenomenon in laser-induced cellular responses. PMID:29541521

The Boston Keratoprosthesis: Comparing Corneal Epithelial Cell Compatibility with Titanium and PMMA

PubMed Central

Ament, Jared D.; Spurr-Michaud, Sandra J.; Dohlman, Claes H.; Gipson, Ilene K.

2014-01-01

Purpose To determine in vitro whether titanium is superior in corneal cell compatibility to standard polymethyl-methacrylate (PMMA) for the Boston Keratoprosthesis (KPro). Methods Human corneal-limbal epithelial (HCLE) cells were cultured 24, 48, 72, 96, 120, 144, or 168 hours in culture plates alone (controls) or with PMMA or titanium discs. Experiments were performed in triplicate and repeated (final n = 6). To determine if a soluble, toxic factor is emitted from materials, concurrent experiments at 48 and 144 hours were performed with discs placed in Transwell Supports, with HCLE cells plated beneath. As an additional test for soluble factors, cells were incubated 24 hours with disc-conditioned media, and number of viable cells per well was quantified at each timepoint by proliferation assay. To determine if delayed cell proliferation was attributable to cell death, HCLE cell death was measured under all conditions and quantified at each timepoint by cytotoxicity assay. The effects of material on HCLE cell proliferation over time was determined by repeated measures ANOVA. P < 0.05 was statistically significant. Results HCLE cell proliferation was greater in wells with titanium discs compared to PMMA. Differences between the test discs and control non-disc cocultures were statistically significant over time for both cell proliferation (P = 0.001) and death (P = 0.0025). No significant difference was found using Transwells (P = 0.9836) or disc-conditioned media (P = 0.36). Conclusion This in vitro HCLE cell model demonstrates significantly increased cell proliferation and decreased cell death with cell/titanium contact compared to cell/PMMA contact. Moreover, differences are unlikely attributable to a soluble factor. Prospective in vivo analysis of the two KPro biomaterials is indicated. PMID:19574903

Antiproliferation and induction of cell death of Phaffia rhodozyma (Xanthophyllomyces dendrorhous) extract fermented by brewer malt waste on breast cancer cells.

PubMed

Teo, Ivy Tuang Ngo; Chui, Chung Hin; Tang, Johnny Cheuk On; Lau, Fung Yi; Cheng, Gregory Yin Ming; Wong, Raymond Siu Ming; Kok, Stanton Hon Lung; Cheng, Chor Hing; Chan, Albert Sun Chi; Ho, Kwok Ping

2005-11-01

Astaxanthin has been shown to have antiproliferative activity on breast cancer and skin cancer cells. However, the high cost of production, isolation and purification of purified astaxanthin from natural sources or chemically synthetic methods limit its usage on cancer therapy. We show that astaxanthin could be produced by fermentating the Phaffia rhodozyma (Xanthophyllomyces dendrorhous) yeast cells with brewer malt waste using a 20 L B. Braun fermentor. The percentage composition of astaxanthin from the P. rhodozyma was >70% of total pigment as estimated by the high performance liquid chromatographic analysis. Furthermore, the antiproliferative activity of this P. rhodozyma cell extract (PRE) was demonstrated on breast cancer cell lines including the MCF-7 (estrogen receptor positive) and MDA-MB231 (estrogen receptor negative) by using the [3-(4,5-dimethylthiazol-2-yl)-5-(3-arboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium] (MTS) assay. No apoptotic cell death, but growth inhibitory effect was induced after 48 h of PRE incubation as suggested by morphological investigation. Anchorage-dependent clonogenicity assay showed that PRE could reduce the colony formation potential of both breast cancer cell lines. Cell death was observed from both breast cancer cell lines after incubation with PRE for 6 days. Taken together, our results showed that by using an economic method of brewer malt waste fermentation, we obtained P. rhodozyma with a high yield of astaxanthin and the corresponding PRE could have short-term growth inhibition and long-term cell death activity on breast cancer cells.

Topological control of life and death in non-proliferative epithelia.

PubMed

Martinand-Mari, Camille; Maury, Benoit; Rousset, François; Sahuquet, Alain; Mennessier, Gérard; Rochal, Sergei; Lorman, Vladimir; Mangeat, Paul; Baghdiguian, Stephen

2009-01-01

Programmed cell death is one of the most fascinating demonstrations of the plasticity of biological systems. It is classically described to act upstream of and govern major developmental patterning processes (e.g. inter-digitations in vertebrates, ommatidia in Drosophila). We show here the first evidence that massive apoptosis can also be controlled and coordinated by a pre-established pattern of a specific 'master cell' population. This new concept is supported by the development and validation of an original model of cell patterning. Ciona intestinalis eggs are surrounded by a three-layered follicular organization composed of 60 elongated floating extensions made of as many outer and inner cells, and indirectly spread through an extracellular matrix over 1200 test cells. Experimental and selective ablation of outer and inner cells results in the abrogation of apoptosis in respective remaining neighbouring test cells. In addition incubation of outer/inner follicular cell-depleted eggs with a soluble extract of apoptotic outer/inner cells partially restores apoptosis to apoptotic-defective test cells. The 60 inner follicular cells were thus identified as 'apoptotic master' cells which collectively are induction sites for programmed cell death of the underlying test cells. The position of apoptotic master cells is controlled by topological constraints exhibiting a tetrahedral symmetry, and each cell spreads over and can control the destiny of 20 smaller test cells, which leads to optimized apoptosis signalling.

BCL2 and BCL(X)L selective inhibitors decrease mitochondrial ATP production in breast cancer cells and are synthetically lethal when combined with 2-deoxy-D-glucose.

PubMed

Lucantoni, Federico; Düssmann, Heiko; Llorente-Folch, Irene; Prehn, Jochen H M

2018-05-25

Cancer cells display differences regarding their engagement of glycolytic vs. mitochondrial oxidative phosphorylation (OXPHOS) pathway. Triple negative breast cancer, an aggressive form of breast cancer, is characterized by elevated glycolysis, while estrogen receptor positive breast cancer cells rely predominantly on OXPHOS. BCL2 proteins control the process of mitochondrial outer membrane permeabilization during apoptosis, but also regulate cellular bioenergetics. Because BCL2 proteins are overexpressed in breast cancer and targetable by selective antagonists, we here analysed the effect of BCL2 and BCL(X)L selective inhibitors, Venetoclax and WEHI-539, on mitochondrial bioenergetics and cell death. Employing single cell imaging using a FRET-based mitochondrial ATP sensor, we found that MCF7 breast cancer cells supplied with mitochondrial substrates reduced their mitochondrial ATP production when treated with Venetoclax or WEHI-539 at concentrations that per se did not induce cell death. Treatments with lower concentrations of both inhibitors also reduced the length of the mitochondrial network and the dynamics, as evaluated by quantitative confocal microscopy. We next tested the hypothesis that mitochondrial ATP production inhibition with BCL2 or BCL(X)L antagonists was synthetically lethal when combined with glycolysis inhibition. Treatment with 2-deoxy-D-glucose in combination with Venetoclax or WEHI-539 synergistically reduced the cellular bioenergetics of ER+ and TNBC breast cancer cells and abolished their clonogenic potential. Synthetic lethality was also observed when cultures were grown in 3D spheres. Our findings demonstrate that BCL2 antagonists exert potent effects on cancer metabolism independent of cell death-inducing effects, and demonstrate a synthetic lethality when these are applied in combination with glycolysis inhibitors.

BCL2 and BCL(X)L selective inhibitors decrease mitochondrial ATP production in breast cancer cells and are synthetically lethal when combined with 2-deoxy-D-glucose

PubMed Central

Lucantoni, Federico; Düssmann, Heiko; Llorente-Folch, Irene; Prehn, Jochen H.M.

2018-01-01

Cancer cells display differences regarding their engagement of glycolytic vs. mitochondrial oxidative phosphorylation (OXPHOS) pathway. Triple negative breast cancer, an aggressive form of breast cancer, is characterized by elevated glycolysis, while estrogen receptor positive breast cancer cells rely predominantly on OXPHOS. BCL2 proteins control the process of mitochondrial outer membrane permeabilization during apoptosis, but also regulate cellular bioenergetics. Because BCL2 proteins are overexpressed in breast cancer and targetable by selective antagonists, we here analysed the effect of BCL2 and BCL(X)L selective inhibitors, Venetoclax and WEHI-539, on mitochondrial bioenergetics and cell death. Employing single cell imaging using a FRET-based mitochondrial ATP sensor, we found that MCF7 breast cancer cells supplied with mitochondrial substrates reduced their mitochondrial ATP production when treated with Venetoclax or WEHI-539 at concentrations that per se did not induce cell death. Treatments with lower concentrations of both inhibitors also reduced the length of the mitochondrial network and the dynamics, as evaluated by quantitative confocal microscopy. We next tested the hypothesis that mitochondrial ATP production inhibition with BCL2 or BCL(X)L antagonists was synthetically lethal when combined with glycolysis inhibition. Treatment with 2-deoxy-D-glucose in combination with Venetoclax or WEHI-539 synergistically reduced the cellular bioenergetics of ER+ and TNBC breast cancer cells and abolished their clonogenic potential. Synthetic lethality was also observed when cultures were grown in 3D spheres. Our findings demonstrate that BCL2 antagonists exert potent effects on cancer metabolism independent of cell death-inducing effects, and demonstrate a synthetic lethality when these are applied in combination with glycolysis inhibitors. PMID:29899841

Dose-Dependent Dual Role of PIT-1 (POU1F1) in Somatolactotroph Cell Proliferation and Apoptosis

PubMed Central

Jullien, Nicolas; Roche, Catherine; Brue, Thierry; Figarella-Branger, Dominique; Graillon, Thomas; Barlier, Anne; Herman, Jean-Paul

2015-01-01

To test the role of wtPIT-1 (PITWT) or PIT-1 (R271W) (PIT271) in somatolactotroph cells, we established, using inducible lentiviral vectors, sublines of GH4C1 somatotroph cells that allow the blockade of the expression of endogenous PIT-1 and/or the expression of PITWT or PIT271, a dominant negative mutant of PIT-1 responsible for Combined Pituitary Hormone Deficiency in patients. Blocking expression of endogenous PIT-1 induced a marked decrease of cell proliferation. Overexpressing PITWT twofold led also to a dose-dependent decrease of cell proliferation that was accompanied by cell death. Expression of PIT271 induced a strong dose-dependent decrease of cell proliferation accompanied by a very pronounced cell death. These actions of PIT271 are independent of its interaction/competition with endogenous PIT-1, as they were unchanged when expression of endogenous PIT-1 was blocked. All these actions are specific for somatolactotroph cells, and could not be observed in heterologous cells. Cell death induced by PITWT or by PIT271 was accompanied by DNA fragmentation, but was not inhibited by inhibitors of caspases, autophagy or necrosis, suggesting that this cell death is a caspase-independent apoptosis. Altogether, our results indicate that under normal conditions PIT-1 is important for the maintenance of cell proliferation, while when expressed at supra-normal levels it induces cell death. Through this dual action, PIT-1 may play a role in the expansion/regression cycles of pituitary lactotroph population during and after lactation. Our results also demonstrate that the so-called “dominant-negative” action of PIT271 is independent of its competition with PIT-1 or a blockade of the actions of the latter, and are actions specific to this mutant variant of PIT-1. PMID:25822178

Death induction by recombinant native TRAIL and its prevention by a caspase 9 inhibitor in primary human esophageal epithelial cells.

PubMed

Kim, Seok-Hyun; Kim, Kunhong; Kwagh, Jae G; Dicker, David T; Herlyn, Meenhard; Rustgi, Anil K; Chen, Youhai; El-Deiry, Wafik S

2004-09-17

The cytotoxic death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a tumor-specific agent under development as a novel anticancer therapeutic agent. However, some reports have demonstrated toxicity of certain TRAIL preparations toward human hepatocytes and keratinocytes through a caspase-dependent mechanism that involves activation of the extrinsic death pathway and Type II signaling through the mitochondria. We have isolated and purified both His-tagged protein and three versions of native recombinant human TRAIL protein from Escherichia coli. We found that 5 mm dithiothreitol in the purification process enhanced oligomerization of TRAIL and resulted in the formation of hyper-oligomerized TRAILs, including hexamers and nonomers with an extremely high potency in apoptosis induction. Although death-inducing signaling complex formation was much more efficient in cells treated with hyper-oligomerized TRAILs, this did not convert TRAIL-sensitive Type II HCT116 colon tumor cells to a Type I death pattern as judged by their continued sensitivity to a caspase 9 inhibitor. Moreover, TRAIL-resistant Type II Bax-null colon carcinoma cells were not converted to a TRAIL-sensitive Type I state by hyper-oligomerized TRAIL. Primary human esophageal epithelial 2 cells were found to be sensitive to all TRAIL preparations used, including trimer TRAIL. TRAIL-induced death in esophageal epithelial 2 cells was prevented by caspase 9 inhibition for up to 4 h after TRAIL exposure. This result suggests a possible therapeutic application of caspase 9 inhibition as a strategy to reverse TRAIL toxicity. Hyper-oligomerized TRAIL may be considered as an alternative agent for testing in clinical trials.

A type III effector antagonizes death receptor signalling during bacterial gut infection.

PubMed

Pearson, Jaclyn S; Giogha, Cristina; Ong, Sze Ying; Kennedy, Catherine L; Kelly, Michelle; Robinson, Keith S; Lung, Tania Wong Fok; Mansell, Ashley; Riedmaier, Patrice; Oates, Clare V L; Zaid, Ali; Mühlen, Sabrina; Crepin, Valerie F; Marches, Olivier; Ang, Ching-Seng; Williamson, Nicholas A; O'Reilly, Lorraine A; Bankovacki, Aleksandra; Nachbur, Ueli; Infusini, Giuseppe; Webb, Andrew I; Silke, John; Strasser, Andreas; Frankel, Gad; Hartland, Elizabeth L

2013-09-12

Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.

Autophagy and Apoptosis Act as Partners to Induce Germ Cell Death after Heat Stress in Mice

PubMed Central

Zhang, Mianqiu; Jiang, Min; Bi, Ye; Zhu, Hui; Zhou, Zuomin; Sha, Jiahao

2012-01-01

Testicular heating suppresses spermatogenesis which is marked by germ cell loss via apoptotic pathways. Recently, it is reported that autophagy also can be induced by heat treatment in somatic cells. In this study, the status of autophagy in germ cells after heat treatment, as well as the partnership between autophagy and apoptosis in these cells was investigated. The results demonstrated that besides initiating apoptotic pathways, heat also induced autophagic pathways in germ cells. Exposure of germ cells to hyperthermia resulted in several specific features of the autophagic process, including autophagosome formation and the conversion of LC3-I to LC3-II. Furthermore, the ubiquitin-like protein conjugation system was implicated as being likely responsible for heat-induced autophagy in germ cells since all genes involving this system were found to be expressed in the testes. In addition, the upstream protein in this system, Atg7 (Autophagy-related gene 7), was found to be expressed in all types of spermatogenic cells, and its expression level was positively correlated with the level of autophagy in germ cells. As a result, Atg7 was selected as the investigative target to further analyze the role of autophagy in heat-induced germ cell death. It was shown that down expression of Atg7 protein resulted in the notable decrease in the level of autophagy in heat-treated germ cells, and this down-regulation of autophagy caused by Atg7 knockdown further reduced the apoptotic rate of germ cells. These results suggest that autophagy plays a positive role in the process of germ cell apoptosis after heat treatment. In conclusion, this study demonstrates that heat triggers autophagy and apoptosis in germ cells. These two mechanisms might act as partners, not antagonist, to induce cell death and lead to eventual destruction of spermatogenesis. PMID:22848486

Protein receptor-independent plasma membrane remodeling by HAMLET: A tumoricidal protein-lipid complex

DOE PAGES

Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.; ...

2015-11-12

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ‘’protein-centric” view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. Wemore » identify a ‘’receptor independent” transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. In conclusion, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.« less

Two overlapping domains of a lyssavirus matrix protein that acts on different cell death pathways.

PubMed

Larrous, Florence; Gholami, Alireza; Mouhamad, Shahul; Estaquier, Jérôme; Bourhy, Hervé

2010-10-01

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

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

PubMed Central

Larrous, Florence; Gholami, Alireza; Mouhamad, Shahul; Estaquier, Jérôme; Bourhy, Hervé

2010-01-01

The lyssavirus matrix (M) protein induces apoptosis. The regions of the M protein that are essential for triggering cell death pathways are not yet clearly defined. We therefore compared the M proteins from two viruses that have contrasting characteristics in terms of cellular apoptosis: a genotype 3 lyssavirus, Mokola virus (MOK), and a genotype 1 rabies virus isolated from a dog from Thailand (THA). We identified a 20-amino-acid fragment (corresponding to positions 67 to 86) that retained the cell death activities of the full-length M protein from MOK via both the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and inhibition of cytochrome c oxidase (CcO) activity. We found that the amino acids at positions 77 and 81 have an essential role in triggering these two cell death pathways. Directed mutagenesis demonstrated that the amino acid at position 77 affects CcO activity, whereas the amino acid at position 81 affects TRAIL-dependent apoptosis. Mutations in the full-length M protein that compromised induction of either of these two pathways resulted in delayed apoptosis compared with the time to apoptosis for the nonmutated control. PMID:20631119

Agmatine Attenuates Brain Edema and Apoptotic Cell Death after Traumatic Brain Injury.

PubMed

Kim, Jae Young; Lee, Yong Woo; Kim, Jae Hwan; Lee, Won Taek; Park, Kyung Ah; Lee, Jong Eun

2015-07-01

Traumatic brain injury (TBI) is associated with poor neurological outcome, including necrosis and brain edema. In this study, we investigated whether agmatine treatment reduces edema and apoptotic cell death after TBI. TBI was produced by cold injury to the cerebral primary motor cortex of rats. Agmatine was administered 30 min after injury and once daily until the end of the experiment. Animals were sacrificed for analysis at 1, 2, or 7 days after the injury. Various neurological analyses were performed to investigate disruption of the blood-brain barrier (BBB) and neurological dysfunction after TBI. To examine the extent of brain edema after TBI, the expression of aquaporins (AQPs), phosphorylation of mitogen-activated protein kinases (MAPKs), and nuclear translocation of nuclear factor-κB (NF-κB) were investigated. Our findings demonstrated that agmatine treatment significantly reduces brain edema after TBI by suppressing the expression of AQP1, 4, and 9. In addition, agmatine treatment significantly reduced apoptotic cell death by suppressing the phosphorylation of MAPKs and by increasing the nuclear translocation of NF-κB after TBI. These results suggest that agmatine treatment may have therapeutic potential for brain edema and neural cell death in various central nervous system diseases.

Apigenin inhibits glioma cell growth through promoting microRNA-16 and suppression of BCL-2 and nuclear factor-κB/MMP‑9.

PubMed

Chen, Xin-Jun; Wu, Mian-Yun; Li, Deng-Hui; You, Jin

2016-09-01

The present study aimed to investigate the effect of apigenin on glioma cells and to explore its potential mechanism. U87 human glioma cells treated with apigenin were used in the current study. Cell Counting Kit‑8 solution and Annexin V-fluorescein isothiocyanate/propidium iodide Apoptosis Detection kit were used to analyze the effect of apigenin on U87 cell viability and apoptotic cell death. Reverse transcription‑quantitative polymerase chain reaction analysis was also used to determine microRNA‑16 (miR‑16) and MMP‑9 gene expression levels. Nuclear factor‑κB (NF‑κB) and B‑cell CLL/lymphoma 2 (BCL2) protein expression levels were determined using western blot analysis. An anti‑miR‑16 plasmid was constructed and transfected into U87 cells. The current study demonstrated that apigenin significantly decreased cell viability and induced apoptotic cell death of U87 cells in a dose‑dependent manner. Additionally, it was demonstrated that apigenin significantly increased miR‑16 levels, suppressed BCL2 protein expression and suppressed the NF‑κB/MMP9 signaling pathway in U87 cells. Furthermore, downregulation of miR‑16 using the anti‑miR‑16 plasmid reversed the effect of apigenin on cell viability, BCL2 protein expression and the NF‑κB/MMP‑9 pathway in U87 cells. The results of the present study suggested that apigenin inhibits glioma cell growth through promoting miR‑16 and suppression of BCL2 and NF-κB/MMP-9. In conclusion, the present study demonstrated the potential anticancer effects of apigenin on glioma cells.

Identification of Bax-interacting proteins in oligodendrocyte progenitors during glutamate excitotoxicity and perinatal hypoxia–ischemia

PubMed Central

Simonishvili, Sopio; Jain, Mohit Raja; Li, Hong; Levison, Steven W.; Wood, Teresa L.

2013-01-01

OPC (oligodendrocyte progenitor cell) death contributes significantly to the pathology and functional deficits following hypoxic-ischemic injury in the immature brain and to deficits resulting from demyelinating diseases, trauma and degenerative disorders in the adult CNS. Glutamate toxicity is a major cause of oligodendroglial death in diverse CNS disorders, and previous studies have demonstrated that AMPA/kainate receptors require the pro-apoptotic protein Bax in OPCs undergoing apoptosis. The goal of the present study was to define the pro-apoptotic and anti-apoptotic effectors that regulate Bax in healthy OPCs and after exposure to excess glutamate in vitro and following H–I (hypoxia–ischemia) in the immature rat brain. We show that Bax associates with a truncated form of Bid, a BH3-only domain protein, subsequent to glutamate treatment. Furthermore, glutamate exposure reduces Bax association with the anti-apoptotic Bcl family member, Bcl-xL. Cell fractionation studies demonstrated that both Bax and Bid translocate from the cytoplasm to mitochondria during the early stages of cell death consistent with a role for Bid as an activator, whereas Bcl-xL, which normally complexes with both Bax and Bid, disassociates from these complexes when OPCs are exposed to excess glutamate. Bax remained unactivated in the presence of insulin-like growth factor-1, and the Bcl-xL complexes were protected. Our data similarly demonstrate loss of Bcl-xL–Bax association in white matter following H–I and implicate active Bad in Bax-mediated OPC death. To identify other Bax-binding partners, we used proteomics and identified cofilin as a Bax-associated protein in OPCs. Cofilin and Bax associated in healthy OPCs, whereas the Bax–cofilin association was disrupted during glutamate-induced OPC apoptosis. PMID:24195677

GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death.

PubMed

Sborgi, Lorenzo; Rühl, Sebastian; Mulvihill, Estefania; Pipercevic, Joka; Heilig, Rosalie; Stahlberg, Henning; Farady, Christopher J; Müller, Daniel J; Broz, Petr; Hiller, Sebastian

2016-08-15

Pyroptosis is a lytic type of cell death that is initiated by inflammatory caspases. These caspases are activated within multi-protein inflammasome complexes that assemble in response to pathogens and endogenous danger signals. Pyroptotic cell death has been proposed to proceed via the formation of a plasma membrane pore, but the underlying molecular mechanism has remained unclear. Recently, gasdermin D (GSDMD), a member of the ill-characterized gasdermin protein family, was identified as a caspase substrate and an essential mediator of pyroptosis. GSDMD is thus a candidate for pyroptotic pore formation. Here, we characterize GSDMD function in live cells and in vitro We show that the N-terminal fragment of caspase-1-cleaved GSDMD rapidly targets the membrane fraction of macrophages and that it induces the formation of a plasma membrane pore. In vitro, the N-terminal fragment of caspase-1-cleaved recombinant GSDMD tightly binds liposomes and forms large permeability pores. Visualization of liposome-inserted GSDMD at nanometer resolution by cryo-electron and atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death. © 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

Kex1 protease is involved in yeast cell death induced by defective N-glycosylation, acetic acid, and chronological aging.

PubMed

Hauptmann, Peter; Lehle, Ludwig

2008-07-04

N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to humans. The key step of this pathway is the transfer of the lipid-linked core oligosaccharide to the nascent polypeptide chain, catalyzed by the oligosaccharyltransferase complex. Temperature-sensitive oligosaccharyltransferase mutants of Saccharomyces cerevisiae at the restrictive temperature, such as wbp1-1, as well as wild-type cells in the presence of the N-glycosylation inhibitor tunicamycin display typical apoptotic phenotypes like nuclear condensation, DNA fragmentation, phosphatidylserine translocation, caspase-like activity, and reactive oxygen species accumulation. Since deletion of the yeast metacaspase YCA1 did not abrogate this death pathway, we postulated a different proteolytic process to be responsible. Here, we show that Kex1 protease is involved in the programmed cell death caused by defective N-glycosylation. Its disruption decreases caspase-like activity, production of reactive oxygen species, and fragmentation of mitochondria and, conversely, improves growth and survival of cells. Moreover, we demonstrate that Kex1 contributes also to the active cell death program induced by acetic acid stress or during chronological aging, suggesting that Kex1 plays a more general role in cellular suicide of yeast.

Gold Nanoantenna-Mediated Photothermal Drug Delivery from Thermosensitive Liposomes in Breast Cancer.

PubMed

Ou, Yu-Chuan; Webb, Joseph A; Faley, Shannon; Shae, Daniel; Talbert, Eric M; Lin, Sharon; Cutright, Camden C; Wilson, John T; Bellan, Leon M; Bardhan, Rizia

2016-08-31

In this work, we demonstrate controlled drug delivery from low-temperature-sensitive liposomes (LTSLs) mediated by photothermal heating from multibranched gold nanoantennas (MGNs) in triple-negative breast cancer (TNBC) cells in vitro. The unique geometry of MGNs enables the generation of mild hyperthermia (∼42 °C) by converting near-infrared light to heat and effectively delivering doxorubicin (DOX) from the LTSLs in breast cancer cells. We confirmed the cellular uptake of MGNs by using both fluorescence confocal Z-stack imaging and transmission electron microscopy (TEM) imaging. We performed a cellular viability assay and live/dead cell fluorescence imaging of the combined therapeutic effects of MGNs with DOX-loaded LTSLs (DOX-LTSLs) and compared them with free DOX and DOX-loaded non-temperature-sensitive liposomes (DOX-NTSLs). Imaging of fluorescent live/dead cell indicators and MTT assay outcomes both demonstrated significant decreases in cellular viability when cells were treated with the combination therapy. Because of the high phase-transition temperature of NTSLs, no drug delivery was observed from the DOX-NTSLs. Notably, even at a low DOX concentration of 0.5 μg/mL, the combination treatment resulted in a higher (33%) cell death relative to free DOX (17% cell death). The results of our work demonstrate that the synergistic therapeutic effect of photothermal hyperthermia of MGNs with drug delivery from the LTSLs can successfully eradicate aggressive breast cancer cells with higher efficacy than free DOX by providing a controlled light-activated approach and minimizing off-target toxicity.

Gold Nanoantenna-Mediated Photothermal Drug Delivery from Thermosensitive Liposomes in Breast Cancer

PubMed Central

2016-01-01

In this work, we demonstrate controlled drug delivery from low-temperature-sensitive liposomes (LTSLs) mediated by photothermal heating from multibranched gold nanoantennas (MGNs) in triple-negative breast cancer (TNBC) cells in vitro. The unique geometry of MGNs enables the generation of mild hyperthermia (∼42 °C) by converting near-infrared light to heat and effectively delivering doxorubicin (DOX) from the LTSLs in breast cancer cells. We confirmed the cellular uptake of MGNs by using both fluorescence confocal Z-stack imaging and transmission electron microscopy (TEM) imaging. We performed a cellular viability assay and live/dead cell fluorescence imaging of the combined therapeutic effects of MGNs with DOX-loaded LTSLs (DOX-LTSLs) and compared them with free DOX and DOX-loaded non-temperature-sensitive liposomes (DOX-NTSLs). Imaging of fluorescent live/dead cell indicators and MTT assay outcomes both demonstrated significant decreases in cellular viability when cells were treated with the combination therapy. Because of the high phase-transition temperature of NTSLs, no drug delivery was observed from the DOX-NTSLs. Notably, even at a low DOX concentration of 0.5 μg/mL, the combination treatment resulted in a higher (33%) cell death relative to free DOX (17% cell death). The results of our work demonstrate that the synergistic therapeutic effect of photothermal hyperthermia of MGNs with drug delivery from the LTSLs can successfully eradicate aggressive breast cancer cells with higher efficacy than free DOX by providing a controlled light-activated approach and minimizing off-target toxicity. PMID:27656689

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

PubMed

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/transport and the pentose phosphate pathway (6-aminonicotinamide). These results demonstrate that glucose metabolism and AMPK regulate dopaminergic cell death induced by gene (α-synuclein)-environment (PQ) interactions.

Light-induced translocation of Pyronine G from mitochondria to nucleoli in monkey kidney CV-1 cells

NASA Astrophysics Data System (ADS)

Geze, Marc; Dellinger, M.; Bazin, M.; Santus, Rene C.

1996-12-01

Pyronine G (3,6-bis-N,N-dimethylaminoxanthylium chloride; PG) is a cationic dye that concentrates in mitochondria of living cells due to the high membrane potential of these organelles, similarly to rhodamine 123 and many other cationic dyes. Pyronine G also shows a preferential affinity for RNA. Upon light irradiation PG has been shown to induce cell death, but the photosensitizing properties of this molecule and the mechanism of cell death are not well understood. Microfluorometry and most particularly microspectrofluorometry are now powerful non-invasive techniques for quantitative studies of single living cells in real time which allow, for example, knowing how living cells are affected by photosensitization. To demonstrate the usefulness of image acquisition with high resolution and high sensitive camera, we present data on photosensitizer relocalization during illumination leading to functional and structural damage in the cells.

MRI-detectable pH nanosensors incorporated into hydrogels for in vivo sensing of transplanted-cell viability

NASA Astrophysics Data System (ADS)

Chan, Kannie W. Y.; Liu, Guanshu; Song, Xiaolei; Kim, Heechul; Yu, Tao; Arifin, Dian R.; Gilad, Assaf A.; Hanes, Justin; Walczak, Piotr; van Zijl, Peter C. M.; Bulte, Jeff W. M.; McMahon, Michael T.

2013-03-01

Biocompatible nanomaterials and hydrogels have become an important tool for improving cell-based therapies by promoting cell survival and protecting cell transplants from immune rejection. Although their potential benefit has been widely evaluated, at present it is not possible to determine, in vivo, if and how long cells remain viable following their administration without the use of a reporter gene. Here, we report a pH-nanosensor-based magnetic resonance imaging (MRI) technique that can monitor cell death in vivo non-invasively. We demonstrate that specific MRI parameters that change on cell death of microencapsulated hepatocytes are associated with the measured bioluminescence imaging radiance. Moreover, the readout from this pH-sensitive nanosensor can be directly co-registered with high-resolution anatomical images. All of the components of these nanosensors are clinical grade and hence this approach should be a translatable and universal modification of hydrogels.

Calnexin Is Essential for Survival under Nitrogen Starvation and Stationary Phase in Schizosaccharomyces pombe

PubMed Central

Rokeach, Luis A.

2015-01-01

Cell fate is determined by the balance of conserved molecular mechanisms regulating death (apoptosis) and survival (autophagy). Autophagy is a process by which cells recycle their organelles and macromolecules through degradation within the vacuole in yeast and plants, and lysosome in metazoa. In the yeast Schizosaccharomyces pombe, autophagy is strongly induced under nitrogen starvation and in aging cells. Previously, we demonstrated that calnexin (Cnx1p), a highly conserved transmembrane chaperone of the endoplasmic reticulum (ER), regulates apoptosis under ER stress or inositol starvation. Moreover, we showed that in stationary phase, Cnx1p is cleaved into two moieties, L_Cnx1p and S_Cnx1p. Here, we show that the processing of Cnx1p is regulated by autophagy, induced by nitrogen starvation or cell aging. The cleavage of Cnx1p involves two vacuolar proteases: Isp6, which is essential for autophagy, and its paralogue Psp3. Blocking autophagy through the knockout of autophagy-related genes (atg) results in inhibition of both, the cleavage and the trafficking of Cnx1p from the ER to the vacuole. We demonstrate that Cnx1p is required for cell survival under nitrogen-starvation and in chronological aging cultures. The death of the mini_cnx1 mutant (overlapping S_cnx1p) cells is accompanied by accumulation of high levels of reactive-oxygen species (ROS), a slowdown in endocytosis and severe cell-wall defects. Moreover, mutant cells expressing only S_Cnx1p showed cell wall defects. Co-expressing mutant overlapping the L_Cnx1p and S_Cnx1p cleavage products reverses the death, ROS phenotype and cell wall defect to wild-type levels. As it is involved in both apoptosis and autophagy, Cnx1p could be a nexus for the crosstalk between these pro-death and pro-survival mechanisms. Ours, and observations in mammalian systems, suggest that the multiple roles of calnexin depend on its sub-cellular localization and on its cleavage. The use of S. pombe should assist in further shedding light on the multiple roles of calnexin. PMID:25803873

Lamin A/C deficiency reduces circulating tumor cell resistance to fluid shear stress

PubMed Central

Denais, Celine; Chan, Maxine F.; Wang, Zhexiao; Lammerding, Jan

2015-01-01

Metastasis contributes to over 90% of cancer-related deaths and is initiated when cancer cells detach from the primary tumor, invade the basement membrane, and enter the circulation as circulating tumor cells (CTCs). While metastasis is viewed as an inefficient process with most CTCs dying within the bloodstream, it is evident that some CTCs are capable of resisting hemodynamic shear forces to form secondary tumors in distant tissues. We hypothesized that nuclear lamins A and C (A/C) act as key structural components within CTCs necessary to resist destruction from elevated shear forces of the bloodstream. Herein, we show that, compared with nonmalignant epithelial cells, tumor cells are resistant to elevated fluid shear forces in vitro that mimic those within the bloodstream, as evidenced by significant decreases in cellular apoptosis and necrosis. Knockdown of lamin A/C significantly reduced tumor cell resistance to fluid shear stress, with significantly increased cell death compared with parental tumor cell and nontargeting controls. Interestingly, lamin A/C knockdown increased shear stress-induced tumor cell apoptosis, but did not significantly affect cellular necrosis. These data demonstrate that lamin A/C is an important structural component that enables tumor cell resistance to fluid shear stress-mediated death in the bloodstream, and may thus facilitate survival and hematogenous metastasis of CTCs. PMID:26447202

Mitochondrial Dysfunction and Ca(2+) Overload Contributes to Hesperidin Induced Paraptosis in Hepatoblastoma Cells, HepG2.

PubMed

Yumnam, Silvia; Hong, Gyeong Eun; Raha, Suchismita; Saralamma, Venu Venkatarame Gowda; Lee, Ho Jeong; Lee, Won-Sup; Kim, Eun-Hee; Kim, Gon Sup

2016-06-01

Paraptosis is a programmed cell death which is morphologically and biochemically different from apoptosis. In this study, we have investigated the role of Ca(2+) in hesperidin-induced paraptotic cell death in HepG2 cells. Increase in mitochondrial Ca(2+) level was observed in hesperidin treated HepG2 cells but not in normal liver cancer cells. Inhibition of inositol-1,4,5-triphosphate receptor (IP3 R) and ryanodine receptor also block the mitochondrial Ca(2+) accumulation suggesting that the release of Ca(2+) from the endoplasmic reticulum (ER) may probably lead to the increase in mitochondrial Ca(2+) level. Pretreatment with ruthenium red (RuRed), a Ca(2+) uniporter inhibitor inhibited the hesperidin-induced mitochondrial Ca(2+) overload, swelling of mitochondria, and cell death in HepG2 cells. It has also been demonstrated that mitochondrial Ca(2+) influxes act upstream of ROS and mitochondrial superoxide production. The increased ROS production further leads to mitochondrial membrane loss in hesperidin treated HepG2 cells. Taken together our results show that IP3 R and ryanodine receptor mediated release of Ca(2+) from the ER and its subsequent influx through the uniporter into mitochondria contributes to hesperidin-induced paraptosis in HepG2 cells. © 2015 Wiley Periodicals, Inc.

Vascular CD39/ENTPD1 Directly Promotes Tumor Cell Growth by Scavenging Extracellular Adenosine Triphosphate12

PubMed Central

Feng, Lili; Sun, Xiaofeng; Csizmadia, Eva; Han, Lihui; Bian, Shu; Murakami, Takashi; Wang, Xin; Robson, Simon C; Wu, Yan

2011-01-01

Extracellular adenosine triphosphate (ATP) is known to boost immune responses in the tumor microenvironment but might also contribute directly to cancer cell death. CD39/ENTPD1 is the dominant ectonucleotidase expressed by endothelial cells and regulatory T cells and catalyzes the sequential hydrolysis of ATP to AMP that is further degraded to adenosine by CD73/ecto-5′-nucleotidase. We have previously shown that deletion of Cd39 results in decreased growth of transplanted tumors in mice, as a result of both defective angiogenesis and heightened innate immune responses (secondary to loss of adenosinergic immune suppression). Whether alterations in local extracellular ATP and adenosine levels as a result of CD39 bioactivity directly affect tumor growth and cytotoxicity has not been investigated to date. We show here that extracellular ATP exerts antitumor activity by directly inhibiting cell proliferation and promoting cancer cell death. ATP-induced antiproliferative effects and cell death are, in large part, mediated through P2X7 receptor signaling. Tumors in Cd39 null mice exhibit increased necrosis in association with P2X7 expression. We further demonstrate that exogenous soluble NTPDase, or CD39 expression by cocultured liver sinusoidal endothelial cells, stimulates tumor cell proliferation and limits cell death triggered by extracellular ATP. Collectively, our findings indicate that local expression of CD39 directly promotes tumor cell growth by scavenging extracellular ATP. Pharmacological or targeted inhibition of CD39 enzymatic activity may find utility as an adjunct therapy in cancer management. PMID:21390184

Antimicrobial copper alloy surfaces are effective against vegetative but not sporulated cells of gram-positive Bacillus subtilis.

PubMed

San, Kaungmyat; Long, Janet; Michels, Corinne A; Gadura, Nidhi

2015-10-01

This study explores the role of membrane phospholipid peroxidation in the copper alloy mediated contact killing of Bacillus subtilis, a spore-forming gram-positive bacterial species. We found that B. subtilis endospores exhibited significant resistance to copper alloy surface killing but vegetative cells were highly sensitive to copper surface exposure. Cell death and lipid peroxidation occurred in B. subtilis upon copper alloy surface exposure. In a sporulation-defective strain carrying a deletion of almost the entire SpoIIA operon, lipid peroxidation directly correlated with cell death. Moreover, killing and lipid peroxidation initiated immediately and at a constant rate upon exposure to the copper surface without the delay observed previously in E. coli. These findings support the hypothesis that membrane lipid peroxidation is the initiating event causing copper surface induced cell death of B. subtilis vegetative cells. The findings suggest that the observed differences in the kinetics of copper-induced killing compared to E. coli result from differences in cell envelop structure. As demonstrated in E. coli, DNA degradation was shown to be a secondary effect of copper exposure in a B. subtilis sporulation-defective strain. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Antimicrobial copper alloy surfaces are effective against vegetative but not sporulated cells of gram-positive Bacillus subtilis

PubMed Central

San, Kaungmyat; Long, Janet; Michels, Corinne A; Gadura, Nidhi

2015-01-01

This study explores the role of membrane phospholipid peroxidation in the copper alloy mediated contact killing of Bacillus subtilis, a spore-forming gram-positive bacterial species. We found that B. subtilis endospores exhibited significant resistance to copper alloy surface killing but vegetative cells were highly sensitive to copper surface exposure. Cell death and lipid peroxidation occurred in B. subtilis upon copper alloy surface exposure. In a sporulation-defective strain carrying a deletion of almost the entire SpoIIA operon, lipid peroxidation directly correlated with cell death. Moreover, killing and lipid peroxidation initiated immediately and at a constant rate upon exposure to the copper surface without the delay observed previously in E. coli. These findings support the hypothesis that membrane lipid peroxidation is the initiating event causing copper surface induced cell death of B. subtilis vegetative cells. The findings suggest that the observed differences in the kinetics of copper-induced killing compared to E. coli result from differences in cell envelop structure. As demonstrated in E. coli, DNA degradation was shown to be a secondary effect of copper exposure in a B. subtilis sporulation-defective strain. PMID:26185055

Curcumin attenuates paraquat-induced cell death in human neuroblastoma cells through modulating oxidative stress and autophagy.

PubMed

Jaroonwitchawan, Thiranut; Chaicharoenaudomrung, Nipha; Namkaew, Jirapat; Noisa, Parinya

2017-01-01

Paraquat is a neurotoxic agent, and oxidative stress plays an important role in neuronal cell death after paraquat exposure. In this study, we assessed the neuroprotective effect of curcumin against paraquat and explored the underlying mechanisms of curcumin in vitro. Curcumin treatment prevented paraquat-induced reactive oxygen species (ROS) and apoptotic cell death. Curcumin also exerted a neuroprotective effect by increasing the expression of anti-apoptotic and antioxidant genes. The pretreatment of curcumin significantly decreased gene expression and protein production of amyloid precursor protein. The activation of autophagy process was found defective in paraquat-induced cells, indicated by the accumulation and reduction of LC3I/II. Noteworthy, curcumin restored LC3I/II expression after the pretreatment. Collectively, curcumin demonstrated as a prominent suppressor of ROS, and could reverse autophagy induction in SH-SY5Y cells. The consequences of this were the reduction of APP production and prevention of SH-SY5Y cells from apoptosis. Altogether, curcumin potentially serves as a therapeutic agent of neurodegenerative diseases, associated with ROS overproduction and autophagy dysfunction. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Phosphatidylserine externalization, “necroptotic bodies” release, and phagocytosis during necroptosis

PubMed Central

Erlich, Ziv; Hourizadeh, Aria; Ofir-Birin, Yifat; Croker, Ben A.; Regev-Rudzki, Neta; Edry-Botzer, Liat

2017-01-01

Necroptosis is a regulated, nonapoptotic form of cell death initiated by receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL) proteins. It is considered to be a form of regulated necrosis, and, by lacking the “find me” and “eat me” signals that are a feature of apoptosis, necroptosis is considered to be inflammatory. One such “eat me” signal observed during apoptosis is the exposure of phosphatidylserine (PS) on the outer plasma membrane. Here, we demonstrate that necroptotic cells also expose PS after phosphorylated mixed lineage kinase-like (pMLKL) translocation to the membrane. Necroptotic cells that expose PS release extracellular vesicles containing proteins and pMLKL to their surroundings. Furthermore, inhibition of pMLKL after PS exposure can reverse the process of necroptosis and restore cell viability. Finally, externalization of PS by necroptotic cells drives recognition and phagocytosis, and this may limit the inflammatory response to this nonapoptotic form of cell death. The exposure of PS to the outer membrane and to extracellular vesicles is therefore a feature of necroptotic cell death and may serve to provide an immunologically-silent window by generating specific “find me” and “eat me” signals. PMID:28650960

Endoplasmic reticulum calcium release potentiates the ER stress and cell death caused by an oxidative stress in MCF-7 cells.

PubMed

Dejeans, Nicolas; Tajeddine, Nicolas; Beck, Raphaël; Verrax, Julien; Taper, Henryk; Gailly, Philippe; Calderon, Pedro Buc

2010-05-01

Increase in cytosolic calcium concentration ([Ca2+](c)), release of endoplasmic reticulum (ER) calcium ([Ca2+](er)) and ER stress have been proposed to be involved in oxidative toxicity. Nevertheless, their relative involvements in the processes leading to cell death are not well defined. In this study, we investigated whether oxidative stress generated during ascorbate-driven menadione redox cycling (Asc/Men) could trigger these three events, and, if so, whether they contributed to Asc/Men cytoxicity in MCF-7 cells. Using microspectrofluorimetry, we demonstrated that Asc/Men-generated oxidative stress was associated with a slow and moderate increase in [Ca2+](c), largely preceding permeation of propidium iodide, and thus cell death. Asc/Men treatment was shown to partially deplete ER calcium stores after 90 min (decrease by 45% compared to control). This event was associated with ER stress activation, as shown by analysis of eIF2 phosphorylation and expression of the molecular chaperone GRP94. Thapsigargin (TG) was then used to study the effect of complete [Ca2+](er) emptying during the oxidative stress generated by Asc/Men. Surprisingly, the combination of TG and Asc/Men increased ER stress to a level considerably higher than that observed for either treatment alone, suggesting that [Ca2+](er) release alone is not sufficient to explain ER stress activation during oxidative stress. Finally, TG-mediated [Ca2+](er) release largely potentiated ER stress, DNA fragmentation and cell death caused by Asc/Men, supporting a role of ER stress in the process of Asc/Men cytotoxicity. Taken together, our results highlight the involvement of ER stress and [Ca2+](er) decrease in the process of oxidative stress-induced cell death in MCF-7 cells. 2009 Elsevier Inc. All rights reserved.

The autophagic- lysosomal pathway determines the fate of glial cells under manganese- induced oxidative stress conditions.

PubMed

Gorojod, R M; Alaimo, A; Porte Alcon, S; Pomilio, C; Saravia, F; Kotler, M L

2015-10-01

Manganese (Mn) overexposure is frequently associated with the development of a neurodegenerative disorder known as Manganism. The Mn-mediated generation of reactive oxygen species (ROS) promotes cellular damage, finally leading to apoptotic cell death in rat astrocytoma C6 cells. In this scenario, the autophagic pathway could play an important role in preventing cytotoxicity. In the present study, we found that Mn induced an increase in the amount and total volume of acidic vesicular organelles (AVOs), a process usually related to the activation of the autophagic pathway. Particularly, the generation of enlarged AVOs was a ROS- dependent event. In this report we demonstrated for the first time that Mn induces autophagy in glial cells. This conclusion emerged from the results obtained employing a battery of autophagy markers: a) the increase in LC3-II expression levels, b) the formation of autophagic vesicles labeled with monodansylcadaverine (MDC) or LC3 and, c) the increase in Beclin 1/ Bcl-2 and Beclin 1/ Bcl-X(L) ratio. Autophagy inhibition employing 3-MA and mAtg5(K130R) resulted in decreased cell viability indicating that this event plays a protective role in Mn- induced cell death. In addition, mitophagy was demonstrated by an increase in LC3 and TOM-20 colocalization. On the other hand, we proposed the occurrence of lysosomal membrane permeabilization (LMP) based in the fact that cathepsins B and D activities are essential for cell death. Both cathepsin B inhibitor (Ca-074 Me) or cathepsin D inhibitor (Pepstatin A) completely prevented Mn- induced cytotoxicity. In addition, low dose of Bafilomycin A1 showed a similar effect, a finding that adds evidence about the lysosomal role in Mn cytotoxicity. Finally, in vivo experiments demonstrated that Mn induces injury and alters LC3 expression levels in rat striatal astrocytes. In summary, our results demonstrated that autophagy is activated to counteract the harmful effect caused by Mn. These data is valuable to be considered in future research concerning Manganism therapies. Copyright © 2015. Published by Elsevier Inc.

Inflammation arising from obesity reduces taste bud abundance and inhibits renewal.

PubMed

Kaufman, Andrew; Choo, Ezen; Koh, Anna; Dando, Robin

2018-03-01

Despite evidence that the ability to taste is weakened by obesity and can be rescued with weight loss intervention, few studies have investigated the molecular effects of obesity on the taste system. Taste bud cells undergo continual turnover even in adulthood, exhibiting an average life span of only a few weeks, tightly controlled by a balance of proliferation and cell death. Recent data reveal that an acute inflammation event can alter this balance. We demonstrate that chronic low-grade inflammation brought on by obesity reduces the number of taste buds in gustatory tissues of mice-and is likely the cause of taste dysfunction seen in obese populations-by upsetting this balance of renewal and cell death.

Inhibition of p38 MAPK enhances ABT-737-induced cell death in melanoma cell lines: novel regulation of PUMA.

PubMed

Keuling, Angela M; Andrew, Susan E; Tron, Victor A

2010-06-01

The mitogen-activated protein kinase (MAPK) pathway is constitutively activated in the majority of melanomas, promoting cell survival, proliferation and migration. In addition, anti-apoptotic Bcl-2 family proteins Mcl-1, Bcl-xL and Bcl-2 are frequently overexpressed, contributing to melanoma's well-documented chemoresistance. Recently, it was reported that the combination of MAPK pathway inhibition by specific MEK inhibitors and Bcl-2 family inhibition by BH3-mimetic ABT-737 synergistically induces apoptotic cell death in melanoma cell lines. Here we provide the first evidence that inhibition of another key MAPK, p38, synergistically induces apoptosis in melanoma cells in combination with ABT-737. We also provide novel mechanistic data demonstrating that inhibition of p38 increases expression of pro-apoptotic Bcl-2 protein PUMA. Furthermore, we demonstrate that PUMA can be cleaved by a caspase-dependent mechanism during apoptosis and identify what appears to be the PUMA cleavage product. Thus, our findings suggest that the combination of ABT-737 and inhibition of p38 is a promising, new treatment strategy that acts through a novel PUMA-dependent mechanism.

LAMP-2 mediates oxidative stress-dependent cell death in Zn2+-treated lung epithelium cells.

PubMed

Qin, Xia; Zhang, Jun; Wang, Bin; Xu, Ge; Zou, Zhen

2017-06-17

Zinc is an essential element for the biological system. However, excessive exogenous Zn 2+ would disrupt cellular Zn 2+ homeostasis and cause toxicity. In particular, Zinc salts or ZnO nanoparticles exposure could induce respiratory injury. Although previous studies have indicated that organelle damage (including mitochondria or lysosomes) and reactive oxygen species (ROS) production are involved in Zn 2+ -induced toxicity, the interplay between mitochondria/lysosomes damage and ROS production is obscure. Herein, we demonstrated that Zn 2+ could induce deglycosylation of lysosome-associated membrane protein 1 and 2 (LAMP-1 and LAMP-2), which primarily locate in late endosomes/lysosomes, in A549 lung epithelium cells. Intriguingly, LAMP-2 knockdown further aggravated Zn 2+ -mediated ROS production and cell death, indicating LAMP-2 (not LAMP-1) was involved in Zn 2+ -induced toxicity. Our results provide a new insight that LAMP-2 contributes to the ROS clearance and cell death induced by Zn 2+ treatment, which would help us to get a better understanding of Zn 2+ -induced toxicity in respiratory system. Copyright © 2017 Elsevier Inc. All rights reserved.

Delivery of Chemically Glycosylated Cytochrome c Immobilized in Mesoporous Silica Nanoparticles Induces Apoptosis in HeLa Cancer Cells

PubMed Central

Méndez, Jessica; Cruz, Moraima Morales; Reyes, Yamixa Delgado; Figueroa, Cindy M.; Orellano, Elsie A.; Morales, Myraida; Monteagudo, Alina; Griebenow, Kai

2014-01-01

Cytochrome c (Cyt c) is a small mitochondrial heme protein involved in the intrinsic apoptotic pathway. Once Cyt c is released into the cytosol, the caspase mediated apoptosis cascade is activated resulting in programmed cell death. Herein, we explore the covalent immobilization of Cyt c into mesoporous silica nanoparticles (MSN) to generate a smart delivery system for intracellular drug delivery to cancer cells aiming at affording subsequent cell death. Cyt c was modified with sulfosuccinimidyl-6-[3′-(2-pyridyldithio)-propionamido] hexanoate (SPDP) and incorporated into SH-functionalized MSN by thiol-disulfide interchange. Unfortunately, delivery of Cyt c from the MSN was not efficient in inducing apoptosis in human cervical cancer HeLa cells. We tested whether chemical Cyt c glycosylation could be useful in overcoming the efficacy problems by potentially improving Cyt c thermodynamic stability and reducing proteolytic degradation. Cyt c lysine residues were modified with lactose at a lactose-to-protein molar ratio of 3.7±0.9 using mono-(lactosylamido)-mono-(succinimidyl) suberate linker chemistry. Circular dichroism (CD) spectra demonstrated that part of the activity loss of Cyt c was due to conformational changes upon its modification with the SPDP linker. These conformational changes were prevented in the glycoconjugate. In agreement with the unfolding of Cyt c by the linker, a proteolytic assay demonstrated that the Cyt c-SPDP conjugate was more susceptible to proteolysis than Cyt c. Attachment of the four lactose molecules reversed this increased susceptibility and protected Cyt c from proteolytic degradation. Furthermore, a cell-free caspase-3 assay revealed 47% and 87% of relative caspase activation by Cyt c-SPDP and the Cyt c-lactose bioconjugate, respectively, when compared to Cyt c. This again demonstrates the efficiency of the glycosylation to improve maintaining Cyt c structure and thus function. To test for cytotoxicity, HeLa cells were incubated with Cyt c loaded MSN at different Cyt c concentrations (12.5, 25.0, and 37.5 μg/mL) for 24 to 72 h and cellular metabolic activity determined by a cell proliferation assay. While MSN-SPDP-Cyt c did not induced cell death, the Cyt c-lactose bioconjugate induced significant cell death after 72 h, reducing HeLa cell viability to 67% and 45% at the 25 μg/mL and 37.5 μg/mL concentrations, respectively. Confocal microscopy confirmed that the MSN immobilized Cyt c-lactose bioconjugate was internalized by HeLa cells and that the bioconjugate was capable of endosomal escape. The results clearly demonstrate that chemical glycosylation stabilized Cyt c upon formulation of a smart drug delivery system and upon delivery into cancer cells and highlight the general potential of chemical protein glycosylation to improve the stability of protein drugs. PMID:24294910

Stem Cell-Based Therapies for Polyglutamine Diseases.

PubMed

Mendonça, Liliana S; Onofre, Isabel; Miranda, Catarina Oliveira; Perfeito, Rita; Nóbrega, Clévio; de Almeida, Luís Pereira

2018-01-01

Polyglutamine (polyQ) diseases are a family of neurodegenerative disorders with very heterogeneous clinical presentations, although with common features such as progressive neuronal death. Thus, at the time of diagnosis patients might present an extensive and irreversible neuronal death demanding cell replacement or support provided by cell-based therapies. For this purpose stem cells, which include diverse populations ranging from embryonic stem cells (ESCs), to fetal stem cells, mesenchymal stromal cells (MSCs) or induced pluripotent stem cells (iPSCs) have remarkable potential to promote extensive brain regeneration and recovery in neurodegenerative disorders. This regenerative potential has been demonstrated in exciting pre and clinical assays. However, despite these promising results, several drawbacks are hampering their successful clinical implementation. Problems related to ethical issues, quality control of the cells used and the lack of reliable models for the efficacy assessment of human stem cells. In this chapter the main advantages and disadvantages of the available sources of stem cells as well as their efficacy and potential to improve disease outcomes are discussed.

Mille modis morimur: We die in a thousand ways.

PubMed

Banfalvi, Gaspar

2017-02-01

Dying cells subjected to apoptotic programs are engulfed by neighboring cells or by professional phagocytes, without inflammation or immunological reactions in the tissue where apoptosis takes place. Apoptotic cells release danger-associated project signals to their neighbours, through different molecular patterns, stimulate antigen production and immune responses. Microenvironmental effects with several functional consequences indicate that cell death is a complex process and may take place in several ways. This idea is expressed by the title of the Special Issue and by the title of the guest editorial "Mille modis morimur" meaning that not only multicellular organisms, but also single cells may die in a thousand ways. This idea is demonstrated by the papers serving as examples for cell death. Apoptosis was induced by clary sage oil in Candida cells. Heavy metal (Gd) induced cell motility and apoptosis was found in mammalian cells. RNA oxidation enhanced the reversion frequency of apoptosis in yeast mutants. The frequency of apoptotic micronucleus formation increased in a concentration-dependent manner by methotrexate. The antioxidant coenzyme Q10 protected renal proximal tubule cells against nicotine-induced apoptosis. The synergy of 2-deoxy-D-glucose combined with berberine induced lysosome/autophagy. The mitochondrial apoptotic pathway could be regulated by glucocorticoid receptor in collaboration with Bcl-2 family proteins in developing T cells. Cylindrospermopsin induced biochemical changes led to apoptosis in plants. Mechanisms of stress seriously impacted the risk of apoptosis. Transcriptional control of apoptotic cell clearance was achieved by macrophage nuclear receptors. Finally, the clinical aspects of apoptosis-induced lymphopenia were reviewed in sepsis and other severe injuries. These examples not only support the view of many ways of cell death, but predict further potential ways to induce or reduce the risk of cell death.

Tocotrienols promote apoptosis in human breast cancer cells by inducing poly(ADP-ribose) polymerase cleavage and inhibiting nuclear factor kappa-B activity.

PubMed

Loganathan, R; Selvaduray, K R; Nesaretnam, K; Radhakrishnan, A K

2013-04-01

Tocotrienols and tocopherols are members of the vitamin E family, with similar structures; however, only tocotrienols have been reported to achieve potent anti-cancer effects. The study described here has evaluated anti-cancer activity of vitamin E to elucidate mechanisms of cell death, using human breast cancer cells. Anti-cancer activity of a tocotrienol-rich fraction (TRF) and a tocotrienol-enriched fraction (TEF) isolated from palm oil, as well as pure vitamin E analogues (α-tocopherol, α-, δ- and γ-tocotrienols) were studied using highly aggressive triple negative MDA-MB-231 cells and oestrogen-dependent MCF-7 cells, both of human breast cancer cell lines. Cell population growth was evaluated using a Coulter particle counter. Cell death mechanism, poly(ADP-ribose) polymerase cleavage and levels of NF-κB were determined using commercial ELISA kits. Tocotrienols exerted potent anti-proliferative effects on both types of cell by inducing apoptosis, the underlying mechanism of cell death being ascertained using respective IC50 concentrations of all test compounds. There was marked induction of apoptosis in both cell lines by tocotrienols compared to treatment with Paclitaxel, which was used as positive control. This activity was found to be associated with cleavage of poly(ADP-ribose) polymerase (a DNA repair protein), demonstrating involvement of the apoptotic cell death signalling pathway. Tocotrienols also inhibited expression of nuclear factor kappa-B (NF-κB), which in turn can increase sensitivity of cancer cells to apoptosis. Tocotrienols induced anti-proliferative and apoptotic effects in association with DNA fragmentation, poly(ADP-ribose) polymerase cleavage and NF-κB inhibition in the two human breast cancer cell lines. © 2013 Blackwell Publishing Ltd.

Vitamins K2, K3 and K5 exert antitumor effects on established colorectal cancer in mice by inducing apoptotic death of tumor cells.

PubMed

Ogawa, Mutsumi; Nakai, Seiji; Deguchi, Akihiro; Nonomura, Takako; Masaki, Tsutomu; Uchida, Naohito; Yoshiji, Hitoshi; Kuriyama, Shigeki

2007-08-01

Although a number of studies have shown that vitamin K possesses antitumor activities on various neoplastic cell lines, there are few reports demonstrating in vivo antitumor effects of vitamin K, and the antitumor effect on colorectal cancer (CRC) remains to be examined. Therefore, antitumor effects of vitamin K on CRC were examined both in vitro and in vivo. Vitamins K2, K3 and K5 suppressed the proliferation of colon 26 cells in a dose-dependent manner, while vitamin K1 did not. On flow cytometry, induction of apoptosis by vitamins K2, K3 and K5 was suggested by population in sub-G1 phase of the cell cycle. Hoechst 33342 staining and a two-color flow cytometric assay using fluorescein isothiocyanate-conjugated annexin V and propidium iodide confirmed that vitamins K2, K3 and K5 induced apoptotic death of colon 26 cells. Enzymatic activity of caspase-3 in colon 26 cells was significantly up-regulated by vitamins K2, K3 and K5. The pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, substantially prevented vitamin K-mediated apoptosis. In vivo study using syngeneic mice with subcutaneously established colon 26 tumors demonstrated that intravenous administration of vitamins K2, K3 and K5 significantly suppressed the tumor growth. The number of apoptotic tumor cells was significantly larger in the vitamin K-treated groups than in the control group. These results suggest that vitamins K2, K3 and K5 exerted effective antitumor effects on CRC in vitro and in vivo by inducing caspase-dependent apoptotic death of tumor cells, suggesting that these K vitamins may be promising agents for the treatment of patients with CRC.

Necroptosis Resumes Apoptosis in Hippocampus but Not in Frontal Cortex.

PubMed

Nikseresht, Sara; Khodagholi, Fariba; Dargahi, Leila; Ahmadiani, Abolhassan

2017-12-01

Cell death subsequent to or concurrent with neuroinflammation results in some damages like neuron loss and spatial memory impairment. In this study, we demonstrated the temporal pattern of neuroinflammation, necroptotic, and apoptotic cell deaths in hippocampus and frontal cortex following intracerebroventricular administration of lipopolysaccharide (LPS). We evaluated receptor interacting protein kinase 1 (RIP1), RIP3, and two related metabolic enzymes including glutamate-ammonia ligase (GLUL) and glutamate dehydrogenase (GLUD) as necroptosis factors. Apoptosis pathway, antioxidant status and inflammatory cytokines were also assessed. Based on the probable role of these brain regions in working memory performance, spontaneous alternation was evaluated through the Y-maze apparatus. RIP1, RIP3, and then GLUL and GLUD, as well as apoptosis markers, inflammatory regulators, and antioxidant defense demonstrated different time-dependent patterns in hippocampus and frontal cortex. Interestingly, in hippocampus but not in frontal cortex, necroptosis resumed apoptosis. Our results in behavioral section revealed that neuroinflammation along with apoptosis and necroptosis pathways could lead to reversible short-term memory impairment after LPS injection. In conclusion, it can be suggested that there is a region-specific response of cell deaths regulators activation in hippocampus and frontal cortex. In addition, elucidating the time profile of events in response to neuroinflammation would be of great help in mechanistic studies and understanding of pathways interaction. J. Cell. Biochem. 118: 4628-4638, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

The bioenergetic signature of isogenic colon cancer cells predicts the cell death response to treatment with 3-bromopyruvate, iodoacetate or 5-fluorouracil.

PubMed

Sánchez-Aragó, María; Cuezva, José M

2011-02-08

Metabolic reprogramming resulting in enhanced glycolysis is a phenotypic trait of cancer cells, which is imposed by the tumor microenvironment and is linked to the down-regulation of the catalytic subunit of the mitochondrial H+-ATPase (β-F1-ATPase). The bioenergetic signature is a protein ratio (β-F1-ATPase/GAPDH), which provides an estimate of glucose metabolism in tumors and serves as a prognostic indicator for cancer patients. Targeting energetic metabolism could be a viable alternative to conventional anticancer chemotherapies. Herein, we document that the bioenergetic signature of isogenic colon cancer cells provides a gauge to predict the cell-death response to the metabolic inhibitors, 3-bromopyruvate (3BrP) and iodoacetate (IA), and the anti-metabolite, 5-fluorouracil (5-FU). The bioenergetic signature of the cells was determined by western blotting. Aerobic glycolysis was determined from lactate production rates. The cell death was analyzed by fluorescence microscopy and flow cytometry. Cellular ATP concentrations were determined using bioluminiscence. Pearson's correlation coefficient was applied to assess the relationship between the bioenergetic signature and the cell death response. In vivo tumor regression activities of the compounds were assessed using a xenograft mouse model injected with the highly glycolytic HCT116 colocarcinoma cells. We demonstrate that the bioenergetic signature of isogenic HCT116 cancer cells inversely correlates with the potential to execute necrosis in response to 3BrP or IA treatment. Conversely, the bioenergetic signature directly correlates with the potential to execute apoptosis in response to 5-FU treatment in the same cells. However, despite the large differences observed in the in vitro cell-death responses associated with 3BrP, IA and 5-FU, the in vivo tumor regression activities of these agents were comparable. Overall, we suggest that the determination of the bioenergetic signature of colon carcinomas could provide a tool for predicting the therapeutic response to various chemotherapeutic strategies aimed at combating tumor progression.

Glutathione peroxidase 4 overexpression inhibits ROS-induced cell death in diffuse large B-cell lymphoma.

PubMed

Kinowaki, Yuko; Kurata, Morito; Ishibashi, Sachiko; Ikeda, Masumi; Tatsuzawa, Anna; Yamamoto, Masahide; Miura, Osamu; Kitagawa, Masanobu; Yamamoto, Kouhei

2018-02-20

Regulation of oxidative stress and redox systems has important roles in carcinogenesis and cancer progression, and for this reason has attracted much attention as a new area of cancer therapeutic targets. Glutathione peroxidase 4 (GPX4), an antioxidant enzyme, has biological important functions such as signaling cell death by suppressing peroxidation of membrane phospholipids. However, few studies exist on the expression and clinical relevance of GPX4 in malignant lymphomas such as diffuse large B-cell lymphoma. In this study, we assessed the expression of GPX4 immunohistochemically. GPX4 was expressed in 35.5% (33/93) cases of diffuse large B-cell lymphoma. The GPX4-positive group had poor overall survival (P = 0.0032) and progression-free survival (P = 0.0004) compared with those of the GPX4-negative group. In a combined analysis of GPX4 and 8-hydroxydeoxyguanosine (8-OHdG), an oxidative stress marker, there was a negative correlation between GPX4 and 8-hydroxydeoxyguanosine (P = 0.0009). The GPX4-positive and 8-hydroxydeoxyguanosine-negative groups had a significantly worse prognosis than the other groups in both overall survival (P = 0.0170) and progression-free survival (P = 0.0005). These results suggest that the overexpression of GPX4 is an independent prognostic predictor in diffuse large B-cell lymphoma. Furthermore, in vitro analysis demonstrated that GPX4-overexpressing cells were resistant to reactive oxygen species-induced cell death (P = 0.0360). Conversely, GPX4-knockdown cells were sensitive to reactive oxygen species-induced cell death (P = 0.0111). From these data, we conclude that GPX4 regulates reactive oxygen species-induced cell death. Our results suggest a novel therapeutic strategy using the mechanism of ferroptosis, as well as a novel prognostic predictor of diffuse large B-cell lymphoma.

Augmentation of poly(ADP-ribose) polymerase-dependent neuronal cell death by acidosis.

PubMed

Zhang, Jian; Li, Xiaoling; Kwansa, Herman; Kim, Yun Tai; Yi, Liye; Hong, Gina; Andrabi, Shaida A; Dawson, Valina L; Dawson, Ted M; Koehler, Raymond C; Yang, Zeng-Jin

2017-06-01

Tissue acidosis is a key component of cerebral ischemic injury, but its influence on cell death signaling pathways is not well defined. One such pathway is parthanatos, in which oxidative damage to DNA results in activation of poly(ADP-ribose) polymerase and generation of poly(ADP-ribose) polymers that trigger release of mitochondrial apoptosis-inducing factor. In primary neuronal cultures, we first investigated whether acidosis per sé is capable of augmenting parthanatos signaling initiated pharmacologically with the DNA alkylating agent, N-methyl- N'-nitro- N-nitrosoguanidine. Exposure of neurons to medium at pH 6.2 for 4 h after N-methyl- N'-nitro- N-nitrosoguanidine washout increased intracellular calcium and augmented the N-methyl- N'-nitro- N-nitrosoguanidine-evoked increase in poly(ADP-ribose) polymers, nuclear apoptosis-inducing factor , and cell death. The augmented nuclear apoptosis-inducing factor and cell death were blocked by the acid-sensitive ion channel-1a inhibitor, psalmotoxin. In vivo, acute hyperglycemia during transient focal cerebral ischemia augmented tissue acidosis, poly(ADP-ribose) polymers formation, and nuclear apoptosis-inducing factor , which was attenuated by a poly(ADP-ribose) polymerase inhibitor. Infarct volume from hyperglycemic ischemia was decreased in poly(ADP-ribose) polymerase 1-null mice. Collectively, these results demonstrate that acidosis can directly amplify neuronal parthanatos in the absence of ischemia through acid-sensitive ion channel-1a . The results further support parthanatos as one of the mechanisms by which ischemia-associated tissue acidosis augments cell death.

Human GAPDH Is a Target of Aspirin’s Primary Metabolite Salicylic Acid and Its Derivatives

PubMed Central

Manohar, Murli; Harraz, Maged M.; Park, Sang-Wook; Schroeder, Frank C.; Snyder, Solomon H.; Klessig, Daniel F.

2015-01-01

The plant hormone salicylic acid (SA) controls several physiological processes and is a key regulator of multiple levels of plant immunity. To decipher the mechanisms through which SA’s multiple physiological effects are mediated, particularly in immunity, two high-throughput screens were developed to identify SA-binding proteins (SABPs). Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) from plants (Arabidopsis thaliana) was identified in these screens. Similar screens and subsequent analyses using SA analogs, in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology, established that human GAPDH (HsGAPDH) also binds SA. In addition to its central role in glycolysis, HsGAPDH participates in several pathological processes, including viral replication and neuronal cell death. The anti-Parkinson’s drug deprenyl has been shown to suppress nuclear translocation of HsGAPDH, an early step in cell death and the resulting cell death induced by the DNA alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine. Here, we demonstrate that SA, which is the primary metabolite of aspirin (acetyl SA) and is likely responsible for many of its pharmacological effects, also suppresses nuclear translocation of HsGAPDH and cell death. Analysis of two synthetic SA derivatives and two classes of compounds from the Chinese medicinal herb Glycyrrhiza foetida (licorice), glycyrrhizin and the SA-derivatives amorfrutins, revealed that they not only appear to bind HsGAPDH more tightly than SA, but also exhibit a greater ability to suppress translocation of HsGAPDH to the nucleus and cell death. PMID:26606248

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

PubMed

Galluzzi, Lorenzo; Vitale, Ilio; Aaronson, Stuart A; Abrams, John M; Adam, Dieter; Agostinis, Patrizia; Alnemri, Emad S; Altucci, Lucia; Amelio, Ivano; Andrews, David W; Annicchiarico-Petruzzelli, Margherita; Antonov, Alexey V; Arama, Eli; Baehrecke, Eric H; Barlev, Nickolai A; Bazan, Nicolas G; Bernassola, Francesca; Bertrand, Mathieu J M; Bianchi, Katiuscia; Blagosklonny, Mikhail V; Blomgren, Klas; Borner, Christoph; Boya, Patricia; Brenner, Catherine; Campanella, Michelangelo; Candi, Eleonora; Carmona-Gutierrez, Didac; Cecconi, Francesco; Chan, Francis K-M; Chandel, Navdeep S; Cheng, Emily H; Chipuk, Jerry E; Cidlowski, John A; Ciechanover, Aaron; Cohen, Gerald M; Conrad, Marcus; Cubillos-Ruiz, Juan R; Czabotar, Peter E; D'Angiolella, Vincenzo; Dawson, Ted M; Dawson, Valina L; De Laurenzi, Vincenzo; De Maria, Ruggero; Debatin, Klaus-Michael; DeBerardinis, Ralph J; Deshmukh, Mohanish; Di Daniele, Nicola; Di Virgilio, Francesco; Dixit, Vishva M; Dixon, Scott J; Duckett, Colin S; Dynlacht, Brian D; El-Deiry, Wafik S; Elrod, John W; Fimia, Gian Maria; Fulda, Simone; García-Sáez, Ana J; Garg, Abhishek D; Garrido, Carmen; Gavathiotis, Evripidis; Golstein, Pierre; Gottlieb, Eyal; Green, Douglas R; Greene, Lloyd A; Gronemeyer, Hinrich; Gross, Atan; Hajnoczky, Gyorgy; Hardwick, J Marie; Harris, Isaac S; Hengartner, Michael O; Hetz, Claudio; Ichijo, Hidenori; Jäättelä, Marja; Joseph, Bertrand; Jost, Philipp J; Juin, Philippe P; Kaiser, William J; Karin, Michael; Kaufmann, Thomas; Kepp, Oliver; Kimchi, Adi; Kitsis, Richard N; Klionsky, Daniel J; Knight, Richard A; Kumar, Sharad; Lee, Sam W; Lemasters, John J; Levine, Beth; Linkermann, Andreas; Lipton, Stuart A; Lockshin, Richard A; López-Otín, Carlos; Lowe, Scott W; Luedde, Tom; Lugli, Enrico; MacFarlane, Marion; Madeo, Frank; Malewicz, Michal; Malorni, Walter; Manic, Gwenola; Marine, Jean-Christophe; Martin, Seamus J; Martinou, Jean-Claude; Medema, Jan Paul; Mehlen, Patrick; Meier, Pascal; Melino, Sonia; Miao, Edward A; Molkentin, Jeffery D; Moll, Ute M; Muñoz-Pinedo, Cristina; Nagata, Shigekazu; Nuñez, Gabriel; Oberst, Andrew; Oren, Moshe; Overholtzer, Michael; Pagano, Michele; Panaretakis, Theocharis; Pasparakis, Manolis; Penninger, Josef M; Pereira, David M; Pervaiz, Shazib; Peter, Marcus E; Piacentini, Mauro; Pinton, Paolo; Prehn, Jochen H M; Puthalakath, Hamsa; Rabinovich, Gabriel A; Rehm, Markus; Rizzuto, Rosario; Rodrigues, Cecilia M P; Rubinsztein, David C; Rudel, Thomas; Ryan, Kevin M; Sayan, Emre; Scorrano, Luca; Shao, Feng; Shi, Yufang; Silke, John; Simon, Hans-Uwe; Sistigu, Antonella; Stockwell, Brent R; Strasser, Andreas; Szabadkai, Gyorgy; Tait, Stephen W G; Tang, Daolin; Tavernarakis, Nektarios; Thorburn, Andrew; Tsujimoto, Yoshihide; Turk, Boris; Vanden Berghe, Tom; Vandenabeele, Peter; Vander Heiden, Matthew G; Villunger, Andreas; Virgin, Herbert W; Vousden, Karen H; Vucic, Domagoj; Wagner, Erwin F; Walczak, Henning; Wallach, David; Wang, Ying; Wells, James A; Wood, Will; Yuan, Junying; Zakeri, Zahra; Zhivotovsky, Boris; Zitvogel, Laurence; Melino, Gerry; Kroemer, Guido

2018-03-01

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Clostridium perfringens beta-toxin induces necrostatin-inhibitable, calpain-dependent necrosis in primary porcine endothelial cells.

PubMed

Autheman, Delphine; Wyder, Marianne; Popoff, Michel; D'Herde, Katharina; Christen, Stephan; Posthaus, Horst

2013-01-01

Clostridium perfringens β-toxin (CPB) is a β-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca(2+)]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis ("necroptosis").

Substance P Receptor Signaling Mediates Doxorubicin-Induced Cardiomyocyte Apoptosis and Triple-Negative Breast Cancer Chemoresistance

PubMed Central

Robinson, Prema; Kasembeli, Moses; Bharadwaj, Uddalak; Engineer, Nikita; Eckols, Kris T.; Tweardy, David J.

2016-01-01

Doxorubicin (DOX), an anthracycline, is broadly considered the most active single agent available for treating breast cancer but has been known to induce cardiotoxicity. Although DOX is highly effective in treating triple-negative breast cancer (TNBC), DOX can have poor outcomes owing to induction of chemoresistance. There is an urgent need to develop new therapies for TNBC aimed at improving DOX outcome and DOX-induced cardiotoxicity. Substance P (SP), a neuropeptide involved in pain transmission is known to stimulate production of reactive oxygen species (ROS). Elevated cardiac ROS is linked with heart injury and failure. We investigated the role of SP in chemotherapy-associated death of cardiomyocytes and chemoresistance. We showed that pretreating a cardiomyocyte cell line (H9C2) and a TNBC cell line (MDA-MB 231) with aprepitant, a SP receptor antagonist that is routinely used to treat chemotherapy-associated associated nausea, decreased DOX-induced reduction of cell viability, apoptotic cell death, and ROS production in cardiomyocytes and increased DOX-induced reduction of cell viability, apoptotic cell death, and ROS production in TNBC cells compared with cells treated with DOX alone. Our findings demonstrate the ability of aprepitant to decrease DOX-induced killing of cardiomyocytes and to increase cancer cell sensitivity to DOX, which has tremendous clinical significance. PMID:26981525

Oxymatrine induces nasopharyngeal cancer cell death through inhibition of PI3K/AKT and NF‑κB pathways.

PubMed

Ni, Zhili; Yi, Jingmei

2017-12-01

Oxymatrine may inhibit tumor cell proliferation, induce cell cycle arrest, promote apoptosis, induce tumor cell differentiation and fight against tumor angiogenesis, as well as inhibit tumor invasion and metastasis. The present study aimed to investigate the anticancer effects of oxymatrine on nasopharyngeal cancer (NPC) cell death, and the underlying molecular mechanisms of these effects. NPC HK‑1 cells were incubated overnight and treated with oxymatrine (0, 2, 4, 6 and 8 mg/ml) for 1, 2 or 3 days. The results demonstrated that oxymatrine significantly inhibited NPC cell proliferation in a time‑ and dose‑dependent manner. Oxymatrine treatment also induced apoptosis, induced the activities of caspase‑3 and caspase‑9, promoted p53 and Bax protein expression, and suppressed cyclin D protein expression in these cells. The protein expression levels of phosphoinositide 3 kinase (PI3K), phosphorylated (p)‑AKT, p‑mammalian target of rapamycin, p‑p70 ribosomal protein S6 kinase and nuclear factor (NF)‑κB were significantly downregulated by oxymatrine treatment. In conclusion, results from the present study suggested that oxymatrine may induce NPC cell death through the inhibition of PI3K/AKT and NF‑κB signaling pathways.

Apoptotic induction of skin cancer cell death by plant extracts.

PubMed

Thuncharoen, Walairat; Chulasiri, Malin; Nilwarangkoon, Sirinun; Nakamura, Yukio; Watanapokasin, Ramida

2013-01-01

The aim of the present study was to investigate the effects of plant extracts on cancer apoptotic induction. Human epidermoid carcinoma A431 cell line, obtained from the American Type Culture Collection (ATCC, Manassas, VA), was maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 degrees C, 5% carbon dioxide (CO2). Plant extract solutions were obtained from S & J international enterprises public company limited. These plant extracts include 50% hydroglycol extracts from Etlingera elatior (Jack) R.M.Smith (torch ginger; EE), Rosa damascene (damask rose; DR) and Rafflesia kerrii Meijer (bua phut; RM). The cell viability, time and dose dependency were determined by MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. A431 cells were treated with the plant extracts and stained with Hoechst 33342 fluorescent staining dye. Cell viability was demonstrated by the inhibitory concentration 50% (IC50). The anti-proliferative effects were shown to be dependent on time and dose. Typical characteristics of apoptosis which are cell morphological changes and chromatin condensation were clearly observed. The plant extracts was shown to be effective for anti-proliferation and induction of apoptosis cell death in skin cancer cells. Therefore, mechanisms underlying the cell death and its potential use for treatment of skin cancer will be further studied.

The flavonoid tangeretin activates the unfolded protein response and synergizes with imatinib in the erythroleukemia cell line K562.

PubMed

Lust, Sofie; Vanhoecke, Barbara; Van Gele, Mireille; Philippé, Jan; Bracke, Marc; Offner, Fritz

2010-06-01

We explored the mechanism of cell death of the polymethoxyflavone tangeretin (TAN) in K562 breakpoint cluster region-abelson murine leukemia (Bcr-Abl+) cells. Flow cytometric analysis showed that TAN arrested the cells in the G(2)/M phase and stimulated an accumulation of the cells in the sub-G(0) phase. TAN-induced cell death was evidenced by poly(ADP)-ribose polymerase cleavage, DNA laddering fragmentation, activation of the caspase cascade and downregulation of the antiapoptotic proteins Mcl-1 and Bcl-x(L). Pretreatment with the pancaspase inhibitor Z-VAD-FMK_blocked caspase activation and cell cycle arrest but did not inhibit apoptosis which suggest that other cell killing mechanisms like endoplasmic reticulum (ER)-associated cell death pathways could be involved. We demonstrated that TAN-induced apoptosis was preceded by a rapid activation of the proapoptotic arm of the unfolded protein response, namely PKR-like ER kinase. This was accompanied by enhanced levels of glucose-regulated protein of 78 kDa and of spliced X-box binding protein 1. Furthermore, TAN sensitized K562 cells to the cell killing effects of imatinib via an apoptotic mechanism. In conclusion, our results suggest that TAN is able to induce apoptosis in Bcr-Abl+ cells via cell cycle arrest and the induction of the unfolded protein response, and has synergistic cytotoxicity with imatinib.

MYC and EGR1 synergize to trigger tumor cell death by controlling NOXA and BIM transcription upon treatment with the proteasome inhibitor bortezomib

PubMed Central

Wirth, Matthias; Stojanovic, Natasa; Christian, Jan; Paul, Mariel C.; Stauber, Roland H.; Schmid, Roland M.; Häcker, Georg; Krämer, Oliver H.; Saur, Dieter; Schneider, Günter

2014-01-01

The c-MYC (MYC afterward) oncogene is well known for driving numerous oncogenic programs. However, MYC can also induce apoptosis and this function of MYC warrants further clarification. We report here that a clinically relevant proteasome inhibitor significantly increases MYC protein levels and that endogenous MYC is necessary for the induction of apoptosis. This kind of MYC-induced cell death is mediated by enhanced expression of the pro-apoptotic BCL2 family members NOXA and BIM. Quantitative promoter-scanning chromatin immunoprecipitations (qChIP) further revealed binding of MYC to the promoters of NOXA and BIM upon proteasome inhibition, correlating with increased transcription. Both promoters are further characterized by the presence of tri-methylated lysine 4 of histone H3, marking active chromatin. We provide evidence that in our apoptosis models cell death occurs independently of p53 or ARF. Furthermore, we demonstrate that recruitment of MYC to the NOXA as well as to the BIM gene promoters depends on MYC's interaction with the zinc finger transcription factor EGR1 and an EGR1-binding site in both promoters. Our study uncovers a novel molecular mechanism by showing that the functional cooperation of MYC with EGR1 is required for bortezomib-induced cell death. This observation may be important for novel therapeutic strategies engaging the inherent pro-death function of MYC. PMID:25147211

[Post-ischemic innate immunity and its application for novel therapeutic strategy targeting brain inflammation].

PubMed

Ito, Minako; Kondo, Taisuke; Shichita, Takashi; Yoshimura, Akihiko

2013-07-01

Stroke or brain ischemia is one of the major causes of death and disability worldwide. Post-ischemic inflammation is an essential step in the progression of brain ischemia-reperfusion injury. In a mouse stroke model, we have reported that IL-23 produced from infiltrating macrophages induces IL-17 producing T cells. IL-17 is mainly produced from gammadeltaT cells and promotes delayed (day 3-4) ischemic brain damage. We also demonstrated that peroxiredoxin (Prx) family proteins released extracellularly from necrotic brain cells induce expression of inflammatory cytokines including IL-23 in macrophages through activation of Toll-like receptor 2(TLR2) and TLR4, thereby promoting neural cell death. We thus propose that regulation of the IL-23-IL-17 axis including gammadeltaT cells, macrophages, and extracellular Prxs could be a potent neuroprotective tool.

Septic shock sera containing circulating histones induce dendritic cell-regulated necrosis in fatal septic shock patients.

PubMed

Raffray, Loic; Douchet, Isabelle; Augusto, Jean-Francois; Youssef, Jihad; Contin-Bordes, Cecile; Richez, Christophe; Duffau, Pierre; Truchetet, Marie-Elise; Moreau, Jean-Francois; Cazanave, Charles; Leroux, Lionel; Mourrissoux, Gaelle; Camou, Fabrice; Clouzeau, Benjamin; Jeannin, Pascale; Delneste, Yves; Gabinski, Claude; Guisset, Olivier; Lazaro, Estibaliz; Blanco, Patrick

2015-04-01

Innate immune system alterations, including dendritic cell loss, have been reproducibly observed in patients with septic shock and correlated to adverse outcomes or nosocomial infections. The goal of this study is to better understand the mechanisms behind this observation in order to better assess septic shock pathogenesis. Prospective, controlled experimental study. Research laboratory at an academic medical center. The study enrolled 71 patients, 49 with septic shock and 22 with cardiogenic shock. Seventeen healthy controls served as reference. In vitro monocyte-derived dendritic cells were generated from healthy volunteers. Sera were assessed for their ability to promote in vitro dendritic cell death through flow cytometry detection in each group of patients. The percentage of apoptotic or necrotic dendritic cells was evaluated by annexin-V and propidium iodide staining. We observed that only patients with septic shock and not patients with pure cardiogenic shock were characterized by a rapid and profound loss of circulating dendritic cells. In vitro analysis revealed that sera from patients with septic shock induced higher dendritic cell death compared to normal sera or cardiogenic shock (p<0.005). Sera from surviving patients induced dendritic cell death through a caspase-dependent apoptotic pathway, whereas sera from nonsurviving patients induced dendritic cell-regulated necrosis. Dendritic cell necrosis was not due to necroptosis but was dependent of the presence of circulating histone. The toxicity of histones toward dendritic cell could be prevented by recombinant human activated protein C. Finally, we observed a direct correlation between the levels of circulating histones in patients and the ability of the sera to promote dendritic cell-regulated necrosis. The study demonstrates a differential mechanism of dendritic cell death in patients with septic shock that is dependent on the severity of the disease.

A balance of Mad and Myc expression dictates larval cell apoptosis and adult stem cell development during Xenopus intestinal metamorphosis.

PubMed

Okada, Morihiro; Miller, Thomas C; Wen, Luan; Shi, Yun-Bo

2017-05-11

The Myc/Mad/Max network has long been shown to be an important factor in regulating cell proliferation, death and differentiation in diverse cell types. In general, Myc-Max heterodimers activate target gene expression to promote cell proliferation, although excess of c-Myc can also induce apoptosis. In contrast, Mad competes against Myc to form Mad-Max heterodimers that bind to the same target genes to repress their expression and promote differentiation. The role of the Myc/Mad/Max network during vertebrate development, especially, the so-called postembryonic development, a period around birth in mammals, is unclear. Using thyroid hormone (T3)-dependent Xenopus metamorphosis as a model, we show here that Mad1 is induced by T3 in the intestine during metamorphosis when larval epithelial cell death and adult epithelial stem cell development take place. More importantly, we demonstrate that Mad1 is expressed in the larval cells undergoing apoptosis, whereas c-Myc is expressed in the proliferating adult stem cells during intestinal metamorphosis, suggesting that Mad1 may have a role in cell death during development. By using transcription activator-like effector nuclease-mediated gene-editing technology, we have generated Mad1 knockout Xenopus animals. This has revealed that Mad1 is not essential for embryogenesis or metamorphosis. On the other hand, consistent with its spatiotemporal expression profile, Mad1 knockout leads to reduced larval epithelial apoptosis but surprisingly also results in increased adult stem cell proliferation. These findings not only reveal a novel role of Mad1 in regulating developmental cell death but also suggest that a balance of Mad and Myc controls cell fate determination during adult organ development.

A balance of Mad and Myc expression dictates larval cell apoptosis and adult stem cell development during Xenopus intestinal metamorphosis

PubMed Central

Okada, Morihiro; Miller, Thomas C; Wen, Luan; Shi, Yun-Bo

2017-01-01

The Myc/Mad/Max network has long been shown to be an important factor in regulating cell proliferation, death and differentiation in diverse cell types. In general, Myc–Max heterodimers activate target gene expression to promote cell proliferation, although excess of c-Myc can also induce apoptosis. In contrast, Mad competes against Myc to form Mad–Max heterodimers that bind to the same target genes to repress their expression and promote differentiation. The role of the Myc/Mad/Max network during vertebrate development, especially, the so-called postembryonic development, a period around birth in mammals, is unclear. Using thyroid hormone (T3)-dependent Xenopus metamorphosis as a model, we show here that Mad1 is induced by T3 in the intestine during metamorphosis when larval epithelial cell death and adult epithelial stem cell development take place. More importantly, we demonstrate that Mad1 is expressed in the larval cells undergoing apoptosis, whereas c-Myc is expressed in the proliferating adult stem cells during intestinal metamorphosis, suggesting that Mad1 may have a role in cell death during development. By using transcription activator-like effector nuclease-mediated gene-editing technology, we have generated Mad1 knockout Xenopus animals. This has revealed that Mad1 is not essential for embryogenesis or metamorphosis. On the other hand, consistent with its spatiotemporal expression profile, Mad1 knockout leads to reduced larval epithelial apoptosis but surprisingly also results in increased adult stem cell proliferation. These findings not only reveal a novel role of Mad1 in regulating developmental cell death but also suggest that a balance of Mad and Myc controls cell fate determination during adult organ development. PMID:28492553

CD147 is increased in HCC cells under starvation and reduces cell death through upregulating p-mTOR in vitro.

PubMed

Gou, Xingchun; Tang, Xu; Kong, Derek Kai; He, Xinying; Gao, Xingchun; Guo, Na; Hu, Zhifang; Zhao, Zhaohua; Chen, Yanke

2016-01-01

Transarterial chemoembolization (TACE) is the standard of care for treatment of intermediate hepatocellular carcinoma (HCC), however, key molecules involved in HCC cell survival and tumor metastasis post-TACE remain unclear. CD147 is a member of the immunoglobulin superfamily that is overexpressed on the surface of HCC cells and is associated with malignant potential and poor prognosis in HCC patients. In this study, using an Earle's Balanced Salt Solution medium culture model that mimics nutrient deprivation induced by TACE, we investigated the regulation of CD147 expression on HCC cells under starvation conditions and its functional effects on HCC cell death. During early stages of starvation, the expression of CD147 was considerably upregulated in SMMC7721, HepG2 and HCC9204 hepatoma cell lines at the protein levels. Downregulation of CD147 by specific small interfering RNA (siRNA) significantly promoted starvation-induced cell death. In addition, CD147 siRNA-transfected SMMC7721 cells demonstrated significantly increased levels of both apoptosis and autophagy as compared to cells transfected with control siRNA under starvation conditions, whereas no difference was observed between the two treatment groups under normal culture conditions. Furthermore, silencing of CD147 resulted in a remarkable downregulation of phosphorylated mammalian target of rapamycin (p-mTOR) in starved SMMC7721 cells. Finally, the combined treatment of starvation and anti-CD147 monoclonal antibody exhibited a synergistic HCC cell killing effect. Our study suggests that upregulation of CD147 under starvation may reduce hepatoma cell death by modulating both apoptosis and autophagy through mTOR signaling, and that CD147 may be a novel potential molecular target to improve the efficacy of TACE.

Dependence of Cisplatin-Induced Cell Death In Vitro and In Vivo on Cyclin-Dependent Kinase 2

PubMed Central

Price, Peter M.; Yu, Fang; Kaldis, Philipp; Aleem, Eiman; Nowak, Grażyna; Safirstein, Robert L.; Megyesi, Judit

2006-01-01

Cisplatin is one of the most effective chemotherapeutics, but its usefulness is limited by its toxicity to normal tissues, including cells of the kidney proximal tubule. The purpose of these studies was to determine the mechanism of cisplatin cytotoxicity. It was shown in vivo that cisplatin administration induces upregulation of the gene for the p21 cyclin-dependent kinase (cdk) inhibitor in kidney cells. This protein is a positive effector on the fate of cisplatin-exposed renal tubule cells in vivo and in vitro; adenoviral transduction of p21 completely protected proximal tubule cells from cisplatin toxicity. Herein is reported that cdk2 inhibitory drugs protect kidney cells in vivo and in vitro, that transduction of kidney cells in vitro with dominant-negative cdk2 also protected, and that cdk2 knockout cells were resistant to cisplatin. The cdk2 knockout cells regained cisplatin sensitivity after transduction with wild-type cdk2. It is concluded that cisplatin cytotoxicity depends on cdk2 activation and that the mechanism of p21 protection is by direct inhibition of cdk2. This demonstrated the involvement of a protein that previously was associated with cell-cycle progression with pathways of apoptosis. It also was demonstrated that this pathway of cisplatin-induced cell death can be interceded in vivo to prevent nephrotoxicity. PMID:16914540

Radioiodide induces apoptosis in human thyroid tissue in culture.

PubMed

Russo, Eleonora; Guerra, Anna; Marotta, Vincenzo; Faggiano, Antongiulio; Colao, Annamaria; Del Vecchio, Silvana; Tonacchera, Massimo; Vitale, Mario

2013-12-01

Radioiodide ((131)I) is routinely used for the treatment of toxic adenoma, Graves' disease, and for ablation of thyroid remnant after thyroidectomy in patients with thyroid cancer. The toxic effects of ionizing radiations on living cells can be mediated by a necrotic and/or apoptotic process. The involvement of apoptosis in radiation-induced cell death in the thyrocytes has been questioned. The knowledge of the mechanisms that underlie the thyrocyte death in response to radiations can help to achieve a successful treatment with the lowest (131)I dose. We developed a method to study the effects of (131)I in human thyroid tissue in culture, by which we demonstrated that (131)I induces thyroid cell apoptosis. Human thyroid tissues of about 1 mm(3) were cultured in vitro and cell viability was determined up to 3 weeks by the MTT assay. Radioiodide added to the culture medium was actively taken up by the tissues. The occurrence of apoptosis in the thyrocytes was assessed by measuring the production of a caspase-cleavage fragment of cytokeratin 18 (M30) by an enzyme-linked immunoassay. Neither variation of cell number nor spontaneous apoptosis was revealed after 1 week of culture. (131)I added to the culture medium induced a dose-dependent and a time-dependent generation of M30 fragment. The apoptotic process was confirmed by the generation of caspase-3 and PARP cleavage products. These results demonstrate that (131)I induces apoptosis in human thyrocytes. Human thyroid tissue cultures may be useful to investigate the cell death pathways induced by (131)I.

Poly(ADP-ribose) polymerase inhibitors sensitize cancer cells to death receptor-mediated apoptosis by enhancing death receptor expression.

PubMed

Meng, X Wei; Koh, Brian D; Zhang, Jin-San; Flatten, Karen S; Schneider, Paula A; Billadeau, Daniel D; Hess, Allan D; Smith, B Douglas; Karp, Judith E; Kaufmann, Scott H

2014-07-25

Recombinant human tumor necrosis factor-α-related apoptosis inducing ligand (TRAIL), agonistic monoclonal antibodies to TRAIL receptors, and small molecule TRAIL receptor agonists are in various stages of preclinical and early phase clinical testing as potential anticancer drugs. Accordingly, there is substantial interest in understanding factors that affect sensitivity to these agents. In the present study we observed that the poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and veliparib sensitize the myeloid leukemia cell lines ML-1 and K562, the ovarian cancer line PEO1, non-small cell lung cancer line A549, and a majority of clinical AML isolates, but not normal marrow, to TRAIL. Further analysis demonstrated that PARP inhibitor treatment results in activation of the FAS and TNFRSF10B (death receptor 5 (DR5)) promoters, increased Fas and DR5 mRNA, and elevated cell surface expression of these receptors in sensitized cells. Chromatin immunoprecipitation demonstrated enhanced binding of the transcription factor Sp1 to the TNFRSF10B promoter in the presence of PARP inhibitor. Knockdown of PARP1 or PARP2 (but not PARP3 and PARP4) not only increased expression of Fas and DR5 at the mRNA and protein level, but also recapitulated the sensitizing effects of the PARP inhibition. Conversely, Sp1 knockdown diminished the PARP inhibitor effects. In view of the fact that TRAIL is part of the armamentarium of natural killer cells, these observations identify a new facet of PARP inhibitor action while simultaneously providing the mechanistic underpinnings of a novel therapeutic combination that warrants further investigation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Fen; Sun, Hong; Kluz, Thomas

Hexavalent chromium [Cr(VI)] is a human carcinogen that results in the generation of reactive oxygen species (ROS) and a variety of DNA lesions leading to cell death. Epigallocatechin-3-gallate (EGCG), the major polyphenol present in green tea, possesses potent antioxidative activity capable of protecting normal cells from various stimuli-induced oxidative stress and cell death. Here we demonstrated that co-treatment with EGCG protected human normal bronchial epithelial BEAS-2B cells from Cr(VI)-induced cell death in a dose-dependent manner. Cr(VI) induces apoptosis as the primary mode of cell death. Co-treatment of BEAS-2B cells with EGCG dose-dependently suppressed Cr(VI)-induced apoptosis. Fluorescence microscopic analyses and quantitativemore » measurement revealed that EGCG significantly decreased intracellular levels of ROS induced by Cr(VI) exposure. Using a well-established K{sup +}/SDS precipitation assay, we further showed that EGCG was able to dose-dependently reduce DNA–protein cross-links (DPC), lesions that could be partially attributed to Cr(VI)-induced oxidative stress. Finally, analyses of Affymetrix microarray containing 28,869 well-annotated genes revealed that, among the 3412 genes changed more than 1.5-fold by Cr(VI) treatment, changes of 2404 genes (70%) were inhibited by pretreatment of EGCG. Real-time PCR confirmed the induction of 3 genes involved in cell death and apoptosis by Cr(VI), which was eliminated by EGCG. In contrast, Cr(VI) reduced the expression of 3 genes related to cellular defense, and this reduction was inhibited by EGCG. Our results indicate that EGCG protects BEAS-2B cells from Cr(VI)-induced cytotoxicity presumably by scavenging ROS and modulating a subset of genes. EGCG, therefore, might serve as a potential chemopreventive agent against Cr(VI) carcinogenesis. -- Highlights: ► EGCG protected human normal bronchial epithelial BEAS-2B cells from Cr(VI)-induced cell death and apoptosis. ► EGCG significantly decreased intracellular levels of ROS induced by Cr(VI) exposure. ► EGCG reduced DNA-protein cross-links, lesions that could be partially attributed to Cr(VI)-induced oxidative stress. ► EGCG modulated 70% of the gene expression changes induced by Cr(VI) exposure.« less

Iron modulates cell survival in a Ras- and MAPK-dependent manner in ovarian cells

PubMed Central

Bauckman, K A; Haller, E; Flores, I; Nanjundan, M

2013-01-01

Ovarian cancer is a leading cause of cancer death in women in the United States. While the majority of ovarian cancers are serous, some rarer subtypes (i.e. clear cell) are often associated with endometriosis, a benign gynecological disease. Iron is rich in the cyst fluid of endometriosis-associated ovarian cancers and induces persistent oxidative stress. The role of iron, an essential nutrient involved in multiple cellular functions, in normal ovarian cell survival and ovarian cancer remains unclear. Iron, presented as ferric ammonium citrate (FAC), dramatically inhibits cell survival in ovarian cancer cell types associated with Ras mutations, while it is without effect in immortalized normal ovarian surface epithelial (T80) and endometriotic epithelial cells (lacking Ras mutations). Interestingly, FAC induced changes in cytoplasmic vacuolation concurrently with increases in LC3-II levels (an autophagy marker); these changes occurred in an ATG5/ATG7-dependent, beclin-1/hVps34-independent, and Ras-independent manner. Knockdown of autophagy mediators in HEY ovarian cancer cells reversed FAC-induced LC3-II levels, but there was little effect on reversing the cell death response. Intriguingly, transmission electron microscopy of FAC-treated T80 cells demonstrated abundant lysosomes (confirmed using Lysotracker) rich in iron particles, which occurred in a Ras-independent manner. Although the mitogen-activated protein kinase (MAPK) inhibitor, U0126, reversed FAC-induced LC3-II/autophagic punctae and lysosomes in a Ras-independent manner, it was remarkable that U0126 reversed cell death in malignant ovarian cells associated with Ras mutations. Moreover, FAC increased heme oxygenase-1 expression in H-Ras-overexpressing T80 cells, which was associated with increased cell death when overexpressed in T80 cells. Disruption of intracellular iron levels, via chelation of intracellular iron (deferoxamine), was also detrimental to malignant ovarian cell survival; thus, homeostatic intracellular iron levels are essential for cell survival. Collectively, our results implicate iron in modulating cell death in a Ras- and MAPK-dependent manner in ovarian cancer cells. PMID:23598404

A dual mechanism involved in membrane and nucleic acid disruption of AvBD103b, a new avian defensin from the king penguin, against Salmonella enteritidis CVCC3377.

PubMed

Teng, Da; Wang, Xiumin; Xi, Di; Mao, Ruoyu; Zhang, Yong; Guan, Qingfeng; Zhang, Jun; Wang, Jianhua

2014-10-01

The food-borne bacterial gastrointestinal infection is a serious public health threat. Defensins are evolutionarily conserved innate immune components with broad-spectrum antibacterial activity that do not easily induce resistance. AvBD103b, an avian defensin with potent activity against Salmonella enteritidis, was isolated from the stomach contents of the king penguin (Aptenodytes patagonicus). To elucidate further the antibacterial mechanism of AvBD103b, its effect on the S. enteritidis CVCC3377 cell membrane and intracellular DNA was researched. The cell surface hydrophobicity and a N-phenyl-1-naphthylamine uptake assay demonstrated that AvBD103b treatment increased the cell surface hydrophobicity and outer membrane permeability. Atomic absorption spectrometry, ultraviolet spectrophotometry, flow cytometry, and transmission electron microscopy (TEM) indicated that AvBD103b treatment can lead to the release of the cellular contents and cell death through damage of the membrane. DNA gel retardation and circular dichroism analysis demonstrated that AvBD103b interacted with DNA and intercalated into the DNA base pairs. A cell cycle assay demonstrated that AvBD103b affected cellular functions, such as DNA synthesis. Our results confirmed that AvBD103b exerts its antibacterial activity by damaging the cell membrane and interfering with intracellular DNA, ultimately causing cell death, and suggested that AvBD103b may be a promising candidate as an alternative to antibiotics against S. enteritidis.

Bax translocation into mitochondria during dihydroartemisinin(DHA)-induced apoptosis in human lung adenocarcinoma cells

NASA Astrophysics Data System (ADS)

Lu, Ying-ying; Chen, Tong-sheng; Qu, Jun-Le

2009-02-01

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways. However, the molecular mechanisms are not well understood. This study was investigated in human lung adenocarconoma ASTC-a-1 cell line and aimed to determine whether the apoptotic process was mediated by Bax activation and translocation during DHA-induced apoptosis. In this study, DHA induced a time-dependent apoptotic cell death, which was assayed by Cell Counting Kit (CCK-8) and Hoechst 33258 staining. Detection of Bax aggregation and translocation to mitochondria was observed in living cells which were co-transfected with GFP-Bax and Dsred-mito plasmid using confocal fluorescence microscope technique. Overall, these results demonstrated that Bax activation and translocation to mitochondria occurred during DHA-induced apoptosis.

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

PubMed

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.

Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mohammad, Mohammad K.; Alcohol Research Center, University of Louisville; Avila, Diana

2012-11-15

Acrolein is a common environmental, food and water pollutant and a major component of cigarette smoke. Also, it is produced endogenously via lipid peroxidation and cellular metabolism of certain amino acids and drugs. Acrolein is cytotoxic to many cell types including hepatocytes; however the mechanisms are not fully understood. We examined the molecular mechanisms underlying acrolein hepatotoxicity in primary human hepatocytes and hepatoma cells. Acrolein, at pathophysiological concentrations, caused a dose-dependent loss of viability of hepatocytes. The death was apoptotic at moderate and necrotic at high concentrations of acrolein. Acrolein exposure rapidly and dramatically decreased intracellular glutathione and overall antioxidantmore » capacity, and activated the stress-signaling MAP-kinases JNK, p42/44 and p38. Our data demonstrate for the first time in human hepatocytes, that acrolein triggered endoplasmic reticulum (ER) stress and activated eIF2α, ATF-3 and -4, and Gadd153/CHOP, resulting in cell death. Notably, the protective/adaptive component of ER stress was not activated, and acrolein failed to up-regulate the protective ER-chaperones, GRP78 and GRP94. Additionally, exposure to acrolein disrupted mitochondrial integrity/function, and led to the release of pro-apoptotic proteins and ATP depletion. Acrolein-induced cell death was attenuated by N-acetyl cysteine, phenyl-butyric acid, and caspase and JNK inhibitors. Our data demonstrate that exposure to acrolein induces a variety of stress responses in hepatocytes, including GSH depletion, oxidative stress, mitochondrial dysfunction and ER stress (without ER-protective responses) which together contribute to acrolein toxicity. Our study defines basic mechanisms underlying liver injury caused by reactive aldehyde pollutants such as acrolein. -- Highlights: ► Human primary hepatocytes and cultured cell lines are used. ► Multiple cell death signaling pathways are activated by acrolein. ► Novel finding of acrolein-induced ER stress ► Acrolein fails to activate ER stress-induced protective responses. ► Combinatorial therapies may be needed for preventing acrolein hepatotoxicity.« less

Fish oil-derived lipid emulsion induces RIP1-dependent and caspase 8-licensed necroptosis in IEC-6 cells through overproduction of reactive oxygen species.

PubMed

Yan, Jun-Kai; Yan, Wei-Hui; Cai, Wei

2018-06-23

Excessive cell death of enterocytes has been demonstrated to be partially associated with the intravenously-administrated lipid emulsions (LEs) during parenteral nutrition (PN) support. However, as a new generation of LE, the effect of fish oil-derived lipid emulsion (FOLE) on the death of enterocytes remains elusive. Intestinal epithelial cells (IEC-6 cell line) were treated with FOLE (0.25-1%) for 24 h. Cell survival was measured by CCK-8 assay, and morphological changes were monitored by time-lapse live cell imaging. The expression of receptor-interacting protein 1/3 (RIP1/3) and caspase 8 was assessed by westernblot, and the formation of necrosome (characterized by the assembly of RIP1/3 complex along with the dissociation of caspase 8) was examined by immunoprecipitation. Additionally, the production of intracellular reactive oxygen species (ROS) was detected by using a ROS detection kit with an oxidation-sensitive probe (DCFH-DA). FOLE dose-dependently induced non-apoptotic, but programmed necroctic cell death (necroptosis) within 4-8 h after treatment. The assembly of RIP1/3 complex along with the dissociation of caspase 8 from RIP1 was observed in FOLE-treated cells. Moreover, FOLE-induced cell death was significantly alleviated by inhibiting RIP1, and was further aggravated by inhibiting caspase 8. In addition, prior to cell death the accumulation of intracellular ROS was significantly increased in FOLE-treated cells (increased by approximately 5-fold versus control, p < 0.001), which could be attenuated by inhibiting RIP1 (decreased by approximately 35% versus FOLE, p < 0.05). FOLE induces RIP1-dependent and caspase 8-licensed necroptosis through overproduction of ROS in vitro. Our findings may provide novel insights into the clinical applications of FOLE during PN support.

Repeated Exposure of Epithelial Cells to Apoptotic Cells Induces the Specific Selection of an Adaptive Phenotype: Implications for Tumorigenesis.

PubMed

Feng, Lanfei; Vujicic, Snezana; Dietrich, Michael E; Litbarg, Natalia; Setty, Suman; Antoni, Angelika; Rauch, Joyce; Levine, Jerrold S

2018-05-16

The consequences of apoptosis extend beyond mere death of the cell. We have shown that receptor-mediated recognition of apoptotic target cells by viable kidney proximal tubular epithelial cells (PTECs) inhibits PTEC proliferation, growth, and survival. Here we tested the hypothesis that continual exposure to apoptotic targets can induce a phenotypic change in responding PTECs, as in other instances of natural selection. In particular, we demonstrate that repeated exposure to apoptotic targets leads to emergence of a PTEC line (denoted BU.MPT SEL ) resistant to apoptotic target-induced death. Resistance is exquisitely specific. Not only are BU.MPT SEL responders fully resistant to apoptotic target-induced death (~85% survival versus <10% survival of non-selected cells), but do so while retaining sensitivity to all other target-induced responses, including inhibition of proliferation and growth. Moreover, the resistance of BU.MPT SEL responders is specific to target-induced apoptosis, as apoptosis in response to other suicidal stimuli occurs normally. Comparison of the signaling events induced by apoptotic target exposure in selected versus non-selected responders indicated that the acquired resistance of BU.MPT SEL cells lies in a regulatory step affecting the generation of the pro-apoptotic protein, truncated BH3 interacting-domain death agonist (tBID), most likely at the level of BID cleavage by caspase-8. This specific adaptation has especial relevance for cancer, in which the prominence and persistence of cell death entail magnification of the post-mortem effects of apoptotic cells. Just as cancer cells acquire specific resistance to chemotherapeutic agents, we propose that cancer cells may also adapt to their ongoing exposure to apoptotic targets. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibition of DAI-dependent necroptosis by the Z-DNA binding domain of the vaccinia virus innate immune evasion protein, E3.

PubMed

Koehler, Heather; Cotsmire, Samantha; Langland, Jeffrey; Kibler, Karen V; Kalman, Daniel; Upton, Jason W; Mocarski, Edward S; Jacobs, Bertram L

2017-10-24

Vaccinia virus (VACV) encodes an innate immune evasion protein, E3, which contains an N-terminal Z-nucleic acid binding (Zα) domain that is critical for pathogenicity in mice. Here we demonstrate that the N terminus of E3 is necessary to inhibit an IFN-primed virus-induced necroptosis. VACV deleted of the Zα domain of E3 (VACV-E3LΔ83N) induced rapid RIPK3-dependent cell death in IFN-treated L929 cells. Cell death was inhibited by the RIPK3 inhibitor, GSK872, and infection with this mutant virus led to phosphorylation and aggregation of MLKL, the executioner of necroptosis. In 293T cells, induction of necroptosis depended on expression of RIPK3 as well as the host-encoded Zα domain-containing DNA sensor, DAI. VACV-E3LΔ83N is attenuated in vivo, and pathogenicity was restored in either RIPK3- or DAI-deficient mice. These data demonstrate that the N terminus of the VACV E3 protein prevents DAI-mediated induction of necroptosis.

The mechanism of action of Russian propolis ethanol extracts against two antibiotic-resistant biofilm-forming bacteria.

PubMed

Bryan, J; Redden, P; Traba, C

2016-02-01

The interaction between antibiotic-resistant Staphylococcus aureus and antibiotic-sensitive Escherichia coli biofilm-forming bacteria and Russian propolis ethanol extracts was evaluated. In this study, bacterial cell death occurred when the cell membranes of bacteria interacted specifically with the antibacterial compounds found in propolis. In order to understand the Russian propolis ethanol extract mechanism of action, microscopy and bacterial lysis studies were conducted. Results uncovered from these experiments imply that the mechanism of action of Russian propolis ethanol extracts is structural rather than functional. The results obtained throughout this study demonstrate cell membrane damage, resulting in cell lysis and eventually bacterial death. Most strains of bacteria and subsequently biofilms, have evolved and have altered their chemical composition in an attempt to protect themselves from antibiotics. The resistant nature of bacteria stems from the chemical rather than the physical means of inactivation of antibiotics. The results uncovered in this work demonstrate the potential application of Russian propolis ethanol extracts as a very efficient and effective method for bacterial and biofilm inactivation. © 2015 The Society for Applied Microbiology.

Dose-dependent and cell type-specific cell death and proliferation following in vitro exposure to radial extracorporeal shock waves

PubMed Central

Hochstrasser, Tanja; Frank, Hans-Georg; Schmitz, Christoph

2016-01-01

Radial extracorporeal shock wave (rESW) therapy is widely used in musculoskeletal disorders and wound repair. However, the mechanisms of action are still largely unknown. The current study compared the effects of rESWs on two cell types. Human fetal foreskin fibroblasts (HFFF2) and human placental choriocarcinoma cell line JEG-3 were exposed to 0, 100, 200, 500 or 5000 rESWs generated with a Swiss DolorClast device (2.5 bar, 1 Hz). FACS analysis immediately after rESW exposure showed that initially, rESWs rather induced mechanical cell destruction than regulated or programmed cell death. Cell damage was nearly negated by reducing cavitation. Furthermore, cell viability decreased progressively with higher numbers of rESWs. Exposure to rESWs had no impact on growth potential of JEG-3 cells, but dose-dependently increased growth potential of HFFF2 cells. Cultivation of cells that were initially exposed to sham-rESWs in conditioned media increased the growth potential of HFFF2 cells, nevertheless, an even stronger effect was achieved by direct exposure to rESWs. Additionally, cell cycle distribution analysis demonstrated a shift in proportion from G0/G1 to G2/M phase in HFFF2 cells, but not in JEG-3 cells. These data demonstrate that rESWs leads to initial and subsequent dose-dependent and cell type-specific effects in vitro. PMID:27477873

Cytotoxicity and radiosensitization effect of TRA-8 on radioresistant human larynx squamous carcinoma cells.

PubMed

Wu, F; Hu, Y; Long, J; Zhou, Y J; Zhong, Y H; Liao, Z K; Liu, S Q; Zhou, F X; Zhou, Y F; Xie, C H

2009-02-01

TRAIL induces apoptosis in a variety of tumorigenic and transformed cell lines, but not in many normal cells. Recent studies have demonstrated that death receptor 5 (DR5), one of the two death receptors bound by TRAIL, showed expression in most malignantly transformed cells. This study evaluated effects of a monoclonal antibody (TRA-8) to human death receptor 5, combined with ionizing radiation, on radioresistant human larynx squamous carcinoma cell line (Hep-2R). Cells were treated with TRA-8 alone or in combination with radiation, cell viability inhibition was measured by MTT assay, and the induction of apoptosis was determined by Annexin V staining. Radionsensitivity of Hep-2R cells treated with TRA-8 were investigated with long-term clonogenic assays. Regulation of DR5 expression in cells after radiation was analyzed by indirect immunofluorescence using murine TRA-8 in combination with flow cytometry. The results suggested that TRA-8 enhanced radionsensitivity of Hep-2R cells, and that TRA-8 regulated Hep-2R cell cycle arrest at G2/M phase. Irradiation up-regulated the expression of DR5, and when combined with TRA-8 yielded optimal survival benefit. Therefore, TRA-8 can be used in combination with irradiation in radioresistant human larynx squamous carcinoma cells. Monoclonal antibodies such as TRA-8 may play an important role in the development of an effective treatment strategy for patients with radioresistant cancers.

Antioxidants Complement the Requirement for Protein Chaperone Function to Maintain β-Cell Function and Glucose Homeostasis

PubMed Central

Han, Jaeseok; Song, Benbo; Kim, Jiun; Kodali, Vamsi K.; Pottekat, Anita; Wang, Miao; Hassler, Justin; Wang, Shiyu; Pennathur, Subramaniam; Back, Sung Hoon; Katze, Michael G.

2015-01-01

Proinsulin misfolding in the endoplasmic reticulum (ER) initiates a cell death response, although the mechanism(s) remains unknown. To provide insight into how protein misfolding may cause β-cell failure, we analyzed mice with the deletion of P58IPK/DnajC3, an ER luminal co-chaperone. P58IPK−/− mice become diabetic as a result of decreased β-cell function and mass accompanied by induction of oxidative stress and cell death. Treatment with a chemical chaperone, as well as deletion of Chop, improved β-cell function and ameliorated the diabetic phenotype in P58IPK−/− mice, suggesting P58IPK deletion causes β-cell death through ER stress. Significantly, a diet of chow supplemented with antioxidant dramatically and rapidly restored β-cell function in P58IPK−/− mice and corrected abnormal localization of MafA, a critical transcription factor for β-cell function. Antioxidant feeding also preserved β-cell function in Akita mice that express mutant misfolded proinsulin. Therefore defective protein folding in the β-cell causes oxidative stress as an essential proximal signal required for apoptosis in response to ER stress. Remarkably, these findings demonstrate that antioxidant feeding restores cell function upon deletion of an ER molecular chaperone. Therefore antioxidant or chemical chaperone treatment may be a promising therapeutic approach for type 2 diabetes. PMID:25795214

Nicotinamide Inhibits the Lysosomal Cathepsin b-like Protease and Kills African Trypanosomes*

PubMed Central

Unciti-Broceta, Juan D.; Maceira, José; Morales, Sonia; García-Pérez, Angélica; Muñóz-Torres, Manuel E.; Garcia-Salcedo, Jose A.

2013-01-01

Nicotinamide, a soluble compound of the vitamin B3 group, has antimicrobial activity against several microorganisms ranging from viruses to parasite protozoans. However, the mode of action of this antimicrobial activity is unknown. Here, we investigate the trypanocidal activity of nicotinamide on Trypanosoma brucei, the causative agent of African trypanosomiasis. Incubation of trypanosomes with nicotinamide causes deleterious defects in endocytic traffic, disruption of the lysosome, failure of cytokinesis, and, ultimately, cell death. At the same concentrations there was no effect on a cultured mammalian cell line. The effects on endocytosis and vesicle traffic were visible within 3 h and can be attributed to inhibition of lysosomal cathepsin b-like protease activity. The inhibitory effect of nicotinamide was confirmed by a direct activity assay of recombinant cathepsin b-like protein. Taken together, these data demonstrate that inhibition of the lysosomal protease cathepsin b-like blocks endocytosis, causing cell death. In addition, these results demonstrate for the first time the inhibitory effect of nicotinamide on a protease. PMID:23443665

Regulation of human glioblastoma cell death by combined treatment of cannabidiol, γ-radiation and small molecule inhibitors of cell signaling pathways

PubMed Central

Ivanov, Vladimir N.; Wu, Jinhua; Hei, Tom K.

2017-01-01

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. The challenging problem in cancer treatment is to find a way to upregulate radiosensitivity of GBM while protecting neurons and neural stem/progenitor cells in the brain. The goal of the present study was upregulation of the cytotoxic effect of γ-irradiation in GBM by non-psychotropic and non-toxic cannabinoid, cannabidiol (CBD). We emphasized three main aspects of signaling mechanisms induced by CBD treatment (alone or in combination with γ-irradiation) in human GBM that govern cell death: 1) CBD significantly upregulated the active (phosphorylated) JNK1/2 and MAPK p38 levels with the subsequent downregulation of the active phospho-ERK1/2 and phospho-AKT1 levels. MAPK p38 was one of the main drivers of CBD-induced cell death, while death levels after combined treatment of CBD and radiation were dependent on both MAPK p38 and JNK. Both MAPK p38 and JNK regulate the endogenous TRAIL expression. 2) NF-κB p65-P(Ser536) was not the main target of CBD treatment and this transcription factor was found at high levels in CBD-treated GBM cells. Additional suppression of p65-P(Ser536) levels using specific small molecule inhibitors significantly increased CBD-induced apoptosis. 3) CBD treatment substantially upregulated TNF/TNFR1 and TRAIL/TRAIL-R2 signaling by modulation of both ligand and receptor levels followed by apoptosis. Our results demonstrate that radiation-induced death in GBM could be enhanced by CBD-mediated signaling in concert with its marginal effects for neural stem/progenitor cells and astrocytes. It will allow selecting efficient targets for sensitization of GBM and overcoming cancer therapy-induced severe adverse sequelae. PMID:29088769

The story of DNase II: a stifled death-wish leads to self-harm.

PubMed

Crow, Yanick J

2010-09-01

DNase II is an endonuclease which plays a fundamental role in the degradation of DNA from both apoptotic cells, and nuclei extruded from red blood cells during erythropoiesis: important tasks, considering that everyday 10(8)-10(9) cells undergo apoptosis, and 10(11) red blood cells are produced in the adult human. The DNase II-null mouse demonstrates embryonic lethality due to type I interferon-mediated erythroid precursor cell death triggered by undegraded nucleic acids. However, the mechanisms leading to such cytotoxicity are poorly understood. A study in the current issue of the European Journal of Immunology investigates the role of the death ligand TRAIL in this process. Although TRAIL is shown to be dispensable for the interferon-induced apoptosis of erythroid cells in DNAse II(-/-) embryos, the authors have developed a useful strategy for further exploring this question in future studies. Interestingly, earlier studies by the same group showed that crossing the DNase II-null mouse with a mouse deficient for the type I interferon receptor can rescue the lethal anaemia observed in the DNase II-null embryos, but only at the cost of developing autoimmunity.

The Netrin-4/ Neogenin-1 axis promotes neuroblastoma cell survival and migration

PubMed Central

Villanueva, Andrea A.; Falcón, Paulina; Espinoza, Natalie; Luis, Solano R.; Milla, Luis A.; Hernandez-SanMiguel, Esther; Torres, Vicente A.; Sanchez-Gomez, Pilar; Palma, Verónica

2017-01-01

Neogenin-1 (NEO1) is a transmembrane receptor involved in axonal guidance, angiogenesis, neuronal cell migration and cell death, during both embryonic development and adult homeostasis. It has been described as a dependence receptor, because it promotes cell death in the absence of its ligands (Netrin and Repulsive Guidance Molecule (RGM) families) and cell survival when they are present. Although NEO1 and its ligands are involved in tumor progression, their precise role in tumor cell survival and migration remain unclear. Public databases contain extensive information regarding the expression of NEO1 and its ligands Netrin-1 (NTN1) and Netrin-4 (NTN4) in primary neuroblastoma (NB) tumors. Analysis of this data revealed that patients with high expression levels of both NEO1 and NTN4 have a poor survival rate. Accordingly, our analyses in NB cell lines with different genetic backgrounds revealed that knocking-down NEO1 reduces cell migration, whereas silencing of endogenous NTN4 induced cell death. Conversely, overexpression of NEO1 resulted in higher cell migration in the presence of NTN4, and increased apoptosis in the absence of ligand. Increased apoptosis was prevented when utilizing physiological concentrations of exogenous Netrin-4. Likewise, cell death induced after NTN4 knock-down was rescued when NEO1 was transiently silenced, thus revealing an important role for NEO1 in NB cell survival. In vivo analysis, using the chicken embryo chorioallantoic membrane (CAM) model, showed that NEO1 and endogenous NTN4 are involved in tumor extravasation and metastasis. Our data collectively demonstrate that endogenous NTN4/NEO1 maintain NB growth via both pro-survival and pro-migratory molecular signaling. PMID:28038459

Activation of innate antiviral immune response via double-stranded RNA-dependent RLR receptor-mediated necroptosis

PubMed Central

Wang, Wei; Wang, Wei-Hua; Azadzoi, Kazem M.; Su, Ning; Dai, Peng; Sun, Jianbin; Wang, Qin; Liang, Ping; Zhang, Wentao; Lei, Xiaoying; Yan, Zhen; Yang, Jing-Hua

2016-01-01

Viruses induce double-stranded RNA (dsRNA) in the host cells. The mammalian system has developed dsRNA-dependent recognition receptors such as RLRs that recognize the long stretches of dsRNA as PAMPs to activate interferon-mediated antiviral pathways and apoptosis in severe infection. Here we report an efficient antiviral immune response through dsRNA-dependent RLR receptor-mediated necroptosis against infections from different classes of viruses. We demonstrated that virus-infected A549 cells were efficiently killed in the presence of a chimeric RLR receptor, dsCARE. It measurably suppressed the interferon antiviral pathway but promoted IL-1β production. Canonical cell death analysis by morphologic assessment, phosphatidylserine exposure, caspase cleavage and chemical inhibition excluded the involvement of apoptosis and consistently suggested RLR receptor-mediated necroptosis as the underlying mechanism of infected cell death. The necroptotic pathway was augmented by the formation of RIP1-RIP3 necrosome, recruitment of MLKL protein and the activation of cathepsin D. Contributing roles of RIP1 and RIP3 were confirmed by gene knockdown. Furthermore, the necroptosis inhibitor necrostatin-1 but not the pan-caspase inhibitor zVAD impeded dsCARE-dependent infected cell death. Our data provides compelling evidence that the chimeric RLR receptor shifts the common interferon antiviral responses of infected cells to necroptosis and leads to rapid death of the virus-infected cells. This mechanism could be targeted as an efficient antiviral strategy. PMID:26935990

Evaluation of the Effects of Airborne Particulate Matter on Bone Marrow-Mesenchymal Stem Cells (BM-MSCs): Cellular, Molecular and Systems Biological Approaches

PubMed Central

Abu-Elmagd, Muhammad; Alghamdi, Mansour A.; Shamy, Magdy; Khoder, Mamdouh I.; Costa, Max; Assidi, Mourad; Kadam, Roaa; Alsehli, Haneen; Gari, Mamdooh; Pushparaj, Peter Natesan; Kalamegam, Gauthaman; Al-Qahtani, Mohammed H.

2017-01-01

Particulate matter (PM) contains heavy metals that affect various cellular functions and gene expression associated with a range of acute and chronic diseases in humans. However, the specific effects they exert on the stem cells remain unclear. Here, we report the effects of PM collected from the city of Jeddah on proliferation, cell death, related gene expression and systems of biological analysis in bone marrow mesenchymal stem cells (BM-MSCs), with the aim of understanding the underlying mechanisms. PM2.5 and PM10 were tested in vitro at various concentrations (15 to 300 µg/mL) and durations (24 to 72 h). PMs induced cellular stress including membrane damage, shrinkage and death. Lower concentrations of PM2.5 increased proliferation of BM-MSCs, while higher concentrations served to decrease it. PM10 decreased BM-MSCs proliferation in a concentration-dependent manner. The X-ray fluorescence spectrometric analysis showed that PM contains high levels of heavy metals. Ingenuity Pathway Analysis (IPA) and hierarchical clustering analyses demonstrated that heavy metals were associated with signaling pathways involving cell stress/death, cancer and chronic diseases. qRT-PCR results showed differential expression of the apoptosis genes (BCL2, BAX); inflammation associated genes (TNF-α and IL-6) and the cell cycle regulation gene (p53). We conclude that PM causes inflammation and cell death, and thereby predisposes to chronic debilitating diseases. PMID:28425934

Granulocyte macrophage-colony stimulating factor and interleukin-3 increase expression of type II tumour necrosis factor receptor, increasing susceptibility to tumour necrosis factor-induced apoptosis. Control of leukaemia cell life/death switching.

PubMed

Rae, C; MacEwan, D J

2004-12-01

Tumour necrosis factor (TNF) induces apoptosis in a range of cell types via its two receptors, TNFR1 and TNFR2. Here, we demonstrate that proliferation and TNFR2 expression was increased in human leukaemic TF-1 cells by granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3), with TNFR1 expression unaffected. Consequently, they switch from a proliferative to a TNF-induced apoptotic phenotype. Raised TNFR2 expression and susceptibility to TNF-induced apoptosis was not a general effect of proliferation as IL-1beta and IFN-gamma both proliferated TF-1 cells with no effect on TNFR expression or apoptosis. Although raised TNFR2 expression correlated with the apoptotic phenotype, stimulation of apoptosis in GM-CSF-pretreated cells was mediated by TNFR1, with stimulation of TNFR2 alone insufficient to initiate cell death. However, TNFR2 did play a role in apoptotic and proliferative responses as they were blocked by the presence of an antagonistic TNFR2 antibody. Additionally, coincubation with cycloheximide blocked the mitotic effects of GM-CSF or IL-3, allowing only the apoptotic responses of TNF to persist. TNF life/death was also observed in K562, but not MOLT-4 and HL-60 human leukaemic cell types. These findings show a cooperative role of TNFR2 in the TNF life/death switching phenomenon.

Radiobiological basis of SBRT and SRS.

PubMed

Song, Chang W; Kim, Mi-Sook; Cho, L Chinsoo; Dusenbery, Kathryn; Sperduto, Paul W

2014-08-01

Stereotactic body radiation therapy (SBRT) and stereotactic radiosurgery (SRS) have been demonstrated to be highly effective for a variety of tumors. However, the radiobiological principles of SBRT and SRS have not yet been clearly defined. It is well known that newly formed tumor blood vessels are fragile and extremely sensitive to ionizing radiation. Various lines of evidence indicate that irradiation of tumors with high dose per fraction, i.e. >10 Gy per fraction, not only kills tumor cells but also causes significant damage in tumor vasculatures. Such vascular damage and ensuing deterioration of the intratumor environment then cause ischemic or indirect/secondary tumor cell death within a few days after radiation exposure, indicating that vascular damage plays an important role in the response of tumors to SBRT and SRS. Indications are that the extensive tumor cell death due to the direct effect of radiation on tumor cells and the secondary effect through vascular damage may lead to massive release of tumor-associated antigens and various pro-inflammatory cytokines, thereby triggering an anti-tumor immune response. However, the precise role of immune assault on tumor cells in SBRT and SRS has not yet been clearly defined. The "4 Rs" for conventional fractionated radiotherapy do not include indirect cell death and thus 4 Rs cannot account for the effective tumor control by SBRT and SRS. The linear-quadratic model is for cell death caused by DNA breaks and thus the usefulness of this model for ablative high-dose SBRT and SRS is limited.

Dihydroartemisinin induces endothelial cell anoikis through the activation of the JNK signaling pathway

PubMed Central

Zhang, Jiao; Guo, Ling; Zhou, Xia; Dong, Fengyun; Li, Liqun; Cheng, Zuowang; Xu, Yinghua; Liang, Jiyong; Xie, Qi; Liu, Ju

2016-01-01

Angiogenesis is required for the growth and metastasis of solid tumors. The anti-malarial agent dihydroartemisinin (DHA) demonstrates potent anti-angiogenic activity, but the underlying molecular mechanisms are not yet fully understood. During the process of angiogenesis, endothelial cells migrating from existing capillaries may undergo programmed cell death after detaching from the extracellular matrix, a process that is defined as anchorage-dependent apoptosis or anoikis. In the present study, DHA-induced cell death was compared in human umbilical vein endothelial cells (HUVECs) cultured in suspension and attached to culture plates. In suspended HUVECs, the cell viability was decreased and apoptosis was increased with the treatment of 50 µM DHA for 5 h, while the same treatment did not affect the attached HUVECs. In addition, 50 µM DHA increased the phosphorylation of c-Jun N-terminal kinase (JNK) in suspended HUVECs, but not in attached HUVECs, for up to 5 h of treatment. The JNK inhibitor, SP600125, reversed DHA-induced cell death in suspended HUVECs, suggesting that the JNK pathway may mediate DHA-induced endothelial cell anoikis. The data from the present study indicates a novel mechanism for understanding the anti-angiogenic effects of DHA, which may be used as a component for chemotherapy. PMID:27602117

Sevoflurane post-conditioning protects primary rat cortical neurons against oxygen-glucose deprivation/resuscitation via down-regulation in mitochondrial apoptosis axis of Bid, Bim, Puma-Bax and Bak mediated by Erk1/2.

PubMed

Zhang, Li-Min; Zhao, Xiao-Chun; Sun, Wen-Bo; Li, Rui; Jiang, Xiao-Jing

2015-10-15

Temporal post-conditioning helps provide neuroprotection against brain injury secondary to ischemia-reperfusion and is considered an effective intervention, but the exact mechanism of sevoflurane post-conditioning is unclear. The essential axis involves activator Bid, Bim, Puma (BH3s), Bax, and Bak; activates the mitochondrial death program; and might be involved in a cell death signal. Extracellular signal-related kinases 1/2 (Erk1/2) play a pivotal role in cell growth and proliferation. We hypothesized that sevoflurane post-conditioning might inhibit Bid, Bim, Puma, Bax, and Bak expression and is activated by phosphor-Erk1/2 to decrease neuronal death. To test this hypothesis, we exposed primary cortical neuron cultures to oxygen-glucose deprivation for 1h, along with resuscitation for 24h (OGD/R). MTT assays, propidium iodide uptake (PI), JC-1 fluorescence, and Western blot indicated the following: decreased cell viability (P<0.05); increased cell death (P<0.05); decreased mitochondrial membrane potential (P<0.05); and decreased Bid, Bim, Puma, Bax, and Bak expression with OGD/R exposure. Inhibition of Erk1/2 phosphorylation could attenuate sevoflurane post-conditioning that mediated an increase in neuronal viability and mitochondrial membrane potential, as well as a decrease in cell death and Bid, Bim, Puma, Bax, and Bak expression after OGD/R treatment. The results demonstrated that sevoflurane post-conditioning caused a marked decrease in cortical neuronal death secondary to OGD/R exposure through the downregulation of the mitochondrial apoptosis axis involving Bid, Bim, Puma, Bax, and Bak that was mediated by the phosphorylation/activation of Erk1/2. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel MRI Contrast Agent from Magnetotactic Bacteria Enables In Vivo Tracking of iPSC-derived Cardiomyocytes.

PubMed

Mahmoudi, Morteza; Tachibana, Atsushi; Goldstone, Andrew B; Woo, Y Joseph; Chakraborty, Papia; Lee, Kayla R; Foote, Chandler S; Piecewicz, Stephanie; Barrozo, Joyce C; Wakeel, Abdul; Rice, Bradley W; Bell Iii, Caleb B; Yang, Phillip C

2016-06-06

Therapeutic delivery of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) represents a novel clinical approach to regenerate the injured myocardium. However, methods for robust and accurate in vivo monitoring of the iCMs are still lacking. Although superparamagnetic iron oxide nanoparticles (SPIOs) are recognized as a promising tool for in vivo tracking of stem cells using magnetic resonance imaging (MRI), their signal persists in the heart even weeks after the disappearance of the injected cells. This limitation highlights the inability of SPIOs to distinguish stem cell viability. In order to overcome this shortcoming, we demonstrate the use of a living contrast agent, magneto-endosymbionts (MEs) derived from magnetotactic bacteria for the labeling of iCMs. The ME-labeled iCMs were injected into the infarcted area of murine heart and probed by MRI and bioluminescence imaging (BLI). Our findings demonstrate that the MEs are robust and effective biological contrast agents to track iCMs in an in vivo murine model. We show that the MEs clear within one week of cell death whereas the SPIOs remain over 2 weeks after cell death. These findings will accelerate the clinical translation of in vivo MRI monitoring of transplanted stem cell at high spatial resolution and sensitivity.

Rotating magnetic field induced oscillation of magnetic particles for in vivo mechanical destruction of malignant glioma.

PubMed

Cheng, Yu; Muroski, Megan E; Petit, Dorothée C M C; Mansell, Rhodri; Vemulkar, Tarun; Morshed, Ramin A; Han, Yu; Balyasnikova, Irina V; Horbinski, Craig M; Huang, Xinlei; Zhang, Lingjiao; Cowburn, Russell P; Lesniak, Maciej S

2016-02-10

Magnetic particles that can be precisely controlled under a magnetic field and transduce energy from the applied field open the way for innovative cancer treatment. Although these particles represent an area of active development for drug delivery and magnetic hyperthermia, the in vivo anti-tumor effect under a low-frequency magnetic field using magnetic particles has not yet been demonstrated. To-date, induced cancer cell death via the oscillation of nanoparticles under a low-frequency magnetic field has only been observed in vitro. In this report, we demonstrate the successful use of spin-vortex, disk-shaped permalloy magnetic particles in a low-frequency, rotating magnetic field for the in vitro and in vivo destruction of glioma cells. The internalized nanomagnets align themselves to the plane of the rotating magnetic field, creating a strong mechanical force which damages the cancer cell structure inducing programmed cell death. In vivo, the magnetic field treatment successfully reduces brain tumor size and increases the survival rate of mice bearing intracranial glioma xenografts, without adverse side effects. This study demonstrates a novel approach of controlling magnetic particles for treating malignant glioma that should be applicable to treat a wide range of cancers. Copyright © 2015 Elsevier B.V. All rights reserved.

Astrocyte truncated-TrkB mediates BDNF antiapoptotic effect leading to neuroprotection.

PubMed

Saba, Julieta; Turati, Juan; Ramírez, Delia; Carniglia, Lila; Durand, Daniela; Lasaga, Mercedes; Caruso, Carla

2018-05-31

Astrocytes are glial cells that help maintain brain homeostasis and become reactive in neurodegenerative processes releasing both harmful and beneficial factors. We have demonstrated that brain-derived neurotrophic factor (BDNF) expression is induced by melanocortins in astrocytes but BDNF actions in astrocytes are largely unknown. We hypothesize that BDNF may prevent astrocyte death resulting in neuroprotection. We found that BDNF increased astrocyte viability, preventing apoptosis induced by serum deprivation by decreasing active caspase-3 and p53 expression. The antiapoptotic action of BDNF was abolished by ANA-12 (a specific TrkB antagonist) and by K252a (a general Trk antagonist). Astrocytes only express the BDNF receptor TrkB truncated isoform 1, TrkB-T1. BDNF induced ERK, Akt and Src (a non-receptor tyrosine kinase) activation in astrocytes. Blocking ERK and Akt pathways abolished BDNF protection in serum deprivation-induced cell death. Moreover, BDNF protected astrocytes from death by 3-nitropropionic acid (3-NP), an effect also blocked by ANA-12, K252a, and inhibitors of ERK, calcium and Src. BDNF reduced reactive oxygen species (ROS) levels induced in astrocytes by 3-NP and increased xCT expression and glutathione levels. Astrocyte conditioned media (ACM) from untreated astrocytes partially protected PC12 neurons whereas ACM from BDNF-treated astrocytes completely protected PC12 neurons from 3-NP-induced apoptosis. Both ACM from control and BDNF-treated astrocytes markedly reduced ROS levels induced by 3-NP in PC12 cells. Our results demonstrate that BDNF protects astrocytes from cell death through TrkB-T1 signaling, exerts an antioxidant action, and induces release of neuroprotective factors from astrocytes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

The role of the endoplasmic reticulum stress response following cerebral ischemia.

PubMed

Hadley, Gina; Neuhaus, Ain A; Couch, Yvonne; Beard, Daniel J; Adriaanse, Bryan A; Vekrellis, Kostas; DeLuca, Gabriele C; Papadakis, Michalis; Sutherland, Brad A; Buchan, Alastair M

2018-06-01

Background Cornu ammonis 3 (CA3) hippocampal neurons are resistant to global ischemia, whereas cornu ammonis (CA1) 1 neurons are vulnerable. Hamartin expression in CA3 neurons mediates this endogenous resistance via productive autophagy. Neurons lacking hamartin demonstrate exacerbated endoplasmic reticulum stress and increased cell death. We investigated endoplasmic reticulum stress responses in CA1 and CA3 regions following global cerebral ischemia, and whether pharmacological modulation of endoplasmic reticulum stress or autophagy altered neuronal viability . Methods In vivo: male Wistar rats underwent sham or 10 min of transient global cerebral ischemia. CA1 and CA3 areas were microdissected and endoplasmic reticulum stress protein expression quantified at 3 h and 12 h of reperfusion. In vitro: primary neuronal cultures (E18 Wistar rat embryos) were exposed to 2 h of oxygen and glucose deprivation or normoxia in the presence of an endoplasmic reticulum stress inducer (thapsigargin or tunicamycin), an endoplasmic reticulum stress inhibitor (salubrinal or 4-phenylbutyric acid), an autophagy inducer ([4'-(N-diethylamino) butyl]-2-chlorophenoxazine (10-NCP)) or autophagy inhibitor (3-methyladenine). Results In vivo, decreased endoplasmic reticulum stress protein expression (phospho-eIF2α and ATF4) was observed at 3 h of reperfusion in CA3 neurons following ischemia, and increased in CA1 neurons at 12 h of reperfusion. In vitro, endoplasmic reticulum stress inducers and high doses of the endoplasmic reticulum stress inhibitors also increased cell death. Both induction and inhibition of autophagy also increased cell death. Conclusion Endoplasmic reticulum stress is associated with neuronal cell death following ischemia. Neither reduction of endoplasmic reticulum stress nor induction of autophagy demonstrated neuroprotection in vitro, highlighting their complex role in neuronal biology following ischemia.

Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14-3-3 isoforms to suppress effector-triggered immunity.

PubMed

Teper, Doron; Salomon, Dor; Sunitha, Sukumaran; Kim, Jung-Gun; Mudgett, Mary Beth; Sessa, Guido

2014-01-01

Effector-triggered immunity (ETI) to host-adapted pathogens is associated with rapid cell death at the infection site. The plant-pathogenic bacterium Xanthomonas euvesicatoria (Xcv) interferes with plant cellular processes by injecting effector proteins into host cells through the type III secretion system. Here, we show that the Xcv effector XopQ suppresses cell death induced by components of the ETI-associated MAP kinase cascade MAPKKKα MEK2/SIPK and by several R/avr gene pairs. Inactivation of xopQ by insertional mutagenesis revealed that this effector inhibits ETI-associated cell death induced by avirulent Xcv in resistant pepper (Capsicum annuum), and enhances bacterial growth in resistant pepper and tomato (Solanum lycopersicum). Using protein-protein interaction studies in yeast (Saccharomyces cerevisiae) and in planta, we identified the tomato 14-3-3 isoform SlTFT4 and homologs from other plant species as XopQ interactors. A mutation in the putative 14-3-3 binding site of XopQ impaired interaction of the effector with CaTFT4 in yeast and its virulence function in planta. Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv. Silencing of NbTFT4 in Nicotiana benthamiana significantly reduced cell death induced by MAPKKKα. In addition, silencing of CaTFT4 in pepper delayed the appearance of ETI-associated cell death and enhanced growth of virulent and avirulent Xcv, demonstrating the requirement of TFT4 for plant immunity to Xcv. Our results suggest that the XopQ virulence function is to suppress ETI and immunity-associated cell death by interacting with TFT4, which is an important component of ETI and a bona fide target of XopQ. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

A type III effector antagonises death receptor signalling during bacterial gut infection

PubMed Central

Pearson, Jaclyn S; Giogha, Cristina; Ong, Sze Ying; Kennedy, Catherine L; Kelly, Michelle; Robinson, Keith S; Wong, Tania; Mansell, Ashley; Riedmaier, Patrice; Oates, Clare VL; Zaid, Ali; Mühlen, Sabrina; Crepin, Valerie F; Marches, Olivier; Ang, Ching-Seng; Williamson, Nicholas A; O’Reilly, Lorraine A; Bankovacki, Aleksandra; Nachbur, Ueli; Infusini, Giuseppe; Webb, Andrew I; Silke, John; Strasser, Andreas; Frankel, Gad; Hartland, Elizabeth L

2013-01-01

Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonise the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC), utilise a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonisation and interfere with antimicrobial host responses 1-3. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death domain containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death receptor induced apoptosis. This inhibition depended on the N-GlcNAc transferase activity of NleB1, which specifically modified Arg117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing (A/E) pathogens antagonise death receptor induced apoptosis of infected cells, thereby blocking a major antimicrobial host response. PMID:24025841

Carbenoxolone prevents chemical eye ischemia-reperfusion-induced cell death via 11β-hydroxysteroid dehydrogenase type 1 inhibition.

PubMed

Choi, Kyoung-Jin; Na, Yoon-Ju; Park, Sung Bum; Jung, Won Hoon; Sung, Hye-Rim; Kim, Ki Young

2017-09-01

Glaucoma is one of the leading causes of preventable blindness diseases, affecting more than 2 million people in the United States. Recently, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitors were found to exert preventive effects against glaucoma. Therefore, we investigated whether carbenoxolone (CBX), an 11β-HSD1 inhibitor, prevents chemical ischemia-reperfusion-induced cell death in human trabecular meshwork (HTM) cells. The present study demonstrated that CBX inhibited cell death caused by iodoacetic acid (IAA)-induced ischemia-reperfusion, and its effect was associated with the inhibition of 11β-HSD1 expression and activity. Furthermore, CBX reversed the IAA-induced structural damage on filamentous actin in HTM cells. In IAA-treated cells, the levels of 11β-HSD1 and the apoptosis-related factors Bax and FASL were increased throughout the reperfusion period, and CBX was able to attenuate the expression of 11β-HSD1 and the apoptosis-related factors. CBX also effectively suppressed IAA-induced intracellular ROS formation and cytochrome c release, which are involved in the mitochondrial apoptosis pathway. In addition, IAA-induced chemical ischemia-reperfusion stimulated TNF-α expression and NF-κB p65 phosphorylation, and these effects were attenuated by CBX. 11β-HSD1 RNAi also suppressed IAA-induced cell apoptosis via reduction of oxidative stress and inhibition of the pro-inflammatory pathway. Taken together, the present study demonstrated that the inhibition of 11β-HSD1 protected the TM against chemical ischemia-reperfusion injury, suggesting that the use of 11β-HSD1 inhibitors could be a useful strategy for glaucoma therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Different meningitis-causing bacteria induce distinct inflammatory responses on interaction with cells of the human meninges.

PubMed

Fowler, Mark I; Weller, Roy O; Heckels, John E; Christodoulides, Myron

2004-06-01

The interactions of bacterial pathogens with cells of the human leptomeninges are critical events in the progression of meningitis. An in vitro model based on the culture of human meningioma cells was used to investigate the interactions of the meningeal pathogens Escherichia coli K1, Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae. A rank order of association with meningioma cells was observed, with N. meningitidis showing the highest levels of adherence, followed by E. coli, S. pneumoniae and H. influenzae. Neisseria meningitidis and H. influenzae did not invade meningioma cells or induce cell death, but induced a concentration-dependent secretion of inflammatory mediators. Neisseria meningitidis induced higher levels of IL-6, MCP-1, RANTES and GM-CSF than H. influenzae, but there was no significant difference in the levels of IL-8 induced by both pathogens. Streptococcus pneumoniae was also unable to invade meningioma cells, but low concentrations of bacteria failed to stimulate cytokine secretion. However, higher concentrations of pneumococci led to cell death. By contrast, only E. coli K1 invaded meningioma cells directly and induced rapid cell death before an inflammatory response could be induced. These data demonstrate that the interactions of different bacterial pathogens with human meningeal cells are distinct, and suggest that different intervention strategies may be needed in order to prevent the morbidity and mortality associated with bacterial meningitis.

Autophagic Cell Death, Polyploidy and Senescence Induced in Breast Tumor Cells by the Substituted Pyrrole JG-03-14, a Novel Microtubule Poison

PubMed Central

Arthur, Christopher R.; Gupton, John T.; Kellogg, Glen E.; Yeudall, W. Andrew; Cabot, Myles C.; Newsham, Irene; Gewirtz, David A.

2007-01-01

JG-03-14, a substituted pyrrole that inhibits microtubule polymerization, was screened against MCF-7 (p53 wild type), MDA-MB 231 (p53 mutant), MCF-7/caspase 3 and MCF-7/ADR (multidrug resistant) breast tumor cell lines. Cell viability and growth inhibition were assessed by the crystal violet dye assay. Apoptosis was evaluated by the TUNEL assay, cell cycle distribution by flow cytometry, autophagy by acridine orange staining of vesicle formation, and senescence based on β-galactosidase staining and cell morphology. Our studies indicate that exposure to JG-03-14, at a concentration of 500 nM, induces time dependent cell death in the MCF-7 and MDA-MB 231 cell lines. In MCF-7 cells, a residual surviving cell population was found to be senescent; in contrast, there was no surviving senescent population in treated MDA-MB 231 cells. No proliferative recovery was detected over a period of 15 days post-treatment in either cell line. Both the TUNEL assay and FLOW cytometry indicated a relatively limited degree of apoptosis (< 10%) in response to drug treatment in MCF-7 cells with more extensive apoptosis (but < 20%) in MDA-MB231 cells; acidic vacuole formation indicative of autophagic cell death was relatively extensive in both MCF-7 and MDA-MB231 cells. In addition, JG-03-14 induced the formation of a large hyperdiploid cell population in MDA-MB231 cells. JG-03-14 also demonstrated pronounced anti-proliferative activity in MCF-7/caspase 3 cells and in the MCF-7/ADR cell line. The observation that JG-03-14 promotes autophagic cell death and also retains activity in tumor cells expressing the multidrug resistance pump indicates that novel microtubule poisons of the substituted pyrroles class may hold promise in the treatment of breast cancer. PMID:17692290

Physician Education: Apoptosis.

PubMed

Kataoka; Tsuruo

1996-01-01

We have come to understand apoptosis as not merely a single form of cell death, but as a fundamental theme in cell biology that has far-reaching implications in the fields of physiology and pathology. At the present time, however, the mechanism of apoptosis is not clearly understood, as research into apoptosis is still at the initial stages. Nevertheless, the links between apoptosis and a variety of pathological conditions are gradually becoming clearer. In this article, we will provide a simple explanation of apoptosis and its mechanism as a novel concept of cell death and discuss the way in which apoptosis has been linked to a variety of pathological conditions. WHAT IS APOPTOSIS?: In normal tissue, cells that are no longer needed are rapidly eliminated without affecting the overall function of the tissue. In this process cells undergo an active and spontaneous suicide called programmed cell death. In fact, the majority of physiological cell deaths take the form of apoptosis. The word apoptosis is used, in contrast to necrosis, to describe the situation in which a cell actively pursues a course toward death upon receiving certain stimuli [1]. The morphological changes of apoptosis found in most cell types first involve contraction in cell volume and condensation of the nucleus. When this happens the intracellular organelles such as the mitochondria retain their normal morphology. As apoptosis proceeds, blebbing of the plasma membrane occurs, and the nucleus becomes fragmented. Finally, the cell itself fragments to form apoptotic bodies that are engulfed by nearby phagocytes. With respect to biochemical changes, it is known that the chromosomes become fragmented into nucleosome units, and DNA forms characteristic ladder patterns when subjected to agarose gel electrophoresis. MECHANISM OF APOPTOSIS: It has been reported that apoptosis is induced in various cells by many kinds of irritations, but the precise mechanism is still unclear. Cell injuries that induce apoptosis include those that cause DNA damage such as radiation and anticancer drugs, those that are mediated by the TNF receptor and Fas receptor (the so-called "death signal receptors"), and the deprivation of cytokines that supply survival signals such as IL-3 and erythropoietin. The tumor suppressor gene p53 plays a very important role in apoptosis induced by damage to DNA. This has been demonstrated by studying resistance to apoptosis of cells derived from p53 knockout mice [2]. Other than the irritations that induce apoptosis, molecules that have been strongly implicated as major players in the drama of apoptosis include the Bcl-2 family proteins and the IL-1 converting enzyme (ICE) and its homolog proteases (caspase family). Both groups of proteins show homology with proteins that affect cell death in nematodes. It is believed that molecules that contribute to cell death have been well conserved in multicellular organisms all the way from the relatively primitive nematodes to mammals including humans. It was discovered that Bcl-2 suppressed apoptosis induced in IL-3 dependent cells by deprivation of IL-3 [3]. It has since become the gene around which apoptosis research revolves. Recently, it has become clear that cell death involving the Bcl-2 protein is under the control of similar proteins from the same family [4]. It is interesting that the phenomenon of cell death may be regulated by the balance of the molecules involved in it. APOPTOSIS ABNORMALITIES AND DISEASE: Physiological cell death plays a major role in the growth and permanent maintenance of the human body [5]. In the process of forming the nervous system, neurons that do not form proper connections die. Physiological cell death also accompanies the removal of virus-infected cells by cytotoxic T cells, the elimination of autoreactive immune cells, the formation of the gut, the reconstitution of cartilage and bone, etc. When physiological cell death that normally should occur is inhibited, inappropriate physiological cell death may occur that is harmful to the body and forms the basis of disease. For example, in patients with neural degenerative disorders such as Alzheimer's disease and Parkinson's disease, we can find premature cell death in a particular subset of neurons. The death of T cells in AIDS patients is also a form of physiological cell death. Inhibition of cell death in the immune system enables the survival of autoreactive B cells and T cells, and is therefore a cause of autoimmune disorders. Apoptosis has been particularly linked to cancer. Normal cells are programmed for death if they are subjected to many types of non-physiological stress such as anticancer drugs or radiation, if they become isolated from surrounding cells and are unable to receive their tissue-specific survival signals [6], or if oncogenes are expressed haphazardly [7]. On the other hand, it is believed that the ability to survive is enhanced in transformed cancer cells because they are more resistant to apoptosis, they exhibit resistance to anticancer drugs, they are no longer dependent on survival signals, and they can metastasize. Therefore, the cancer progresses as the cancer cells maintain the proliferative superiority they acquire from their oncogenes. In other words, when cancer cells become resistant to apoptosis, they become resistant to treatment, metastasize, and proliferate destructively. The concept that the malignancy of cancer is due to its resistance to apoptosis is a relatively new one and is worthy of further study.

Synthesis and evaluation of a radiolabeled bis-zinc(II)-cyclen complex as a potential probe for in vivo imaging of cell death.

PubMed

Wang, Hongliang; Wu, Zhifang; Li, Sijin; Hu, Kongzhen; Tang, Ganghua

2017-04-01

The exposition of phosphatidylserine (PS) from the cell membrane is associated with most cell death programs (apoptosis, necrosis, autophagy, mitotic catastrophe, etc.), which makes PS an attractive target for overall cell death imaging. To this end, zinc(II) macrocycle coordination complexes with cyclic polyamine units as low-molecular-weight annexin mimics have a selective affinity for biomembrane surfaces enriched with PS, and are therefore useful for detection of cell death. In the present study, a 11 C-labeled zinc(II)-bis(cyclen) complex ( 11 C-CyclenZn2) was prepared and evaluated as a new positron emission tomography (PET) probe for cell death imaging. 11 C-CyclenZn2 was synthesized by methylation of its precursor, 4-methoxy-2,5-di-[10-methyl-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylic acid tri-tert-butyl ester] phenol (Boc-Cyclen2) with 11 C-methyl triflate as a prosthetic group in acetone, deprotection by hydrolysis in aqueous HCl solution, and chelation with zinc nitrate. The cell death imaging capability of 11 C-CyclenZn2 was evaluated using in vitro cell uptake assays with camptothecin-treated PC-3 cells, biodistribution studies, and in vivo PET imaging in Kunming mice bearing S-180 fibrosarcoma. Starting from 11 C-methyl triflate, the total preparation time for 11 C-CyclenZn2 was ~40 min, with an uncorrected radiochemical yield of 12 ± 3% (based on 11 C-CH 3 OTf, n = 10), a radiochemical purity of greater than 95%, and the specific activity of 0.75-1.01 GBq/μmol. The cell death binding specificity of 11 C-CyclenZn2 was demonstrated by significantly different uptake rates in camptothecin-treated and control PC-3 cells in vitro. Inhibition experiments for 18 F-radiofluorinated Annexin V binding to apoptotic/necrotic cells illustrated the necessity of zinc ions for zinc(II)-bis(cyclen) complexation in binding cell death, and zinc(II)-bis(cyclen) complexe and Annexin V had not identical binding pattern with apoptosis/necrosis cells. Biodistribution studies of 11 C-CyclenZn2 revealed a fast clearance from blood, low uptake rates in brain and muscle tissue, and high uptake rates in liver and kidney, which provide the main metabolic route. PET imaging using 11 C-CyclenZn2 revealed that cyclophosphamide-treated mice (CP-treated group) exhibited a significant increase of uptake rate in the tumor at 60 min postinjection, compared with control mice (Control group). The results indicate that the ability of 11 C-CyclenZn2 to detect cell death is comparable to Annexin V, and it has potential as a PET tracer for noninvasive evaluation and monitoring of anti-tumor chemotherapy.

Up-regulation of Heme Oxygenase-1 by Korean Red Ginseng Water Extract as a Cytoprotective Effect in Human Endothelial Cells

PubMed Central

Yang, Hana; Lee, Seung Eun; Jeong, Seong Il; Park, Cheung-Seog; Jin, Young-Ho; Park, Yong Seek

2011-01-01

Korean red ginseng (KRG) is used worldwide as a popular traditional herbal medicine. KRG has shown beneficial effects on cardiovascular diseases, such as atherosclerosis, diabetes, and hypertension. Up-regulation of a cytoprotective protein, heme oxygenase (HO)-1, is considered to augment the cellular defense against various agents that may induce cytotoxic injury. In the present study, we demonstrate that KRG water extract induces HO-1 expression in human umbilical vein endothelial cells (HUVECs) and possible involvement of the anti-oxidant transcription factor nuclear factor-eythroid 2-related factor 2 (Nrf2). KRG-induced HO-1 expression was examined by western blots, reverse transcriptase polymerase chain reaction and immunofluorescence staining. Specific silencing of Nrf2 genes with Nrf2-siRNA in HUVECs abolished HO-1 expression. In addition, the HO inhibitor zinc protoporphyrin blunted the preventive effect of KRG on H2O2-induced cell death, as demonstrated by terminal transferase dUTP nick end labeling assay. Taken together, these results suggest that KRG may exert a vasculoprotective effect through Nrf2- mediated HO-1 induction in human endothelial cell by inhibition of cell death. PMID:23717080

Controlling plasma stimulated media in cancer treatment application

NASA Astrophysics Data System (ADS)

Yan, Dayun; Sherman, Jonathan H.; Cheng, Xiaoqian; Ratovitski, Edward; Canady, Jerome; Keidar, Michael

2014-12-01

Cold atmospheric plasma (CAP) constitutes a "cocktail" of various reactive species. Accumulating evidence shows the effectiveness of CAP in killing cancer cells and decreasing the tumor size, which provides a solid basis for its potential use in cancer treatment. Currently, CAP is mainly used to directly treat cancer cells and trigger the death of cancer cells via apoptosis or necrosis. By altering the concentration of fetal bovine serum in Dulbecco's modified Eagle's medium and the temperature to store CAP stimulated media, we demonstrated controllable strategies to harness the stimulated media to kill glioblastoma cells in vitro. This study demonstrated the significant role of media in killing cancer cells via the CAP treatment.

Anesthetic propofol overdose causes endothelial cytotoxicity in vitro and endothelial barrier dysfunction in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Ming-Chung; Department of Anesthesiology, Chi Mei Medical Center, Liouying, Tainan, Taiwan; Chen, Chia-Ling

An overdose and a prolonged treatment of propofol may cause cellular cytotoxicity in multiple organs and tissues such as brain, heart, kidney, skeletal muscle, and immune cells; however, the underlying mechanism remains undocumented, particularly in vascular endothelial cells. Our previous studies showed that the activation of glycogen synthase kinase (GSK)-3 is pro-apoptotic in phagocytes during overdose of propofol treatment. Regarding the intravascular administration of propofol, we therefore hypothesized that propofol overdose also induces endothelial cytotoxicity via GSK-3. Propofol overdose (100 μg/ml) inhibited growth in human arterial and microvascular endothelial cells. After treatment, most of the endothelial cells experienced caspase-independent necrosis-likemore » cell death. The activation of cathepsin D following lysosomal membrane permeabilization (LMP) determined necrosis-like cell death. Furthermore, propofol overdose also induced caspase-dependent apoptosis, at least in part. Caspase-3 was activated and acted downstream of mitochondrial transmembrane potential (MTP) loss; however, lysosomal cathepsins were not required for endothelial cell apoptosis. Notably, activation of GSK-3 was essential for propofol overdose-induced mitochondrial damage and apoptosis, but not necrosis-like cell death. Intraperitoneal administration of a propofol overdose in BALB/c mice caused an increase in peritoneal vascular permeability. These results demonstrate the cytotoxic effects of propofol overdose, including cathepsin D-regulated necrosis-like cell death and GSK-3-regulated mitochondrial apoptosis, on endothelial cells in vitro and the endothelial barrier dysfunction by propofol in vivo. Highlights: ► Propofol overdose causes apoptosis and necrosis in endothelial cells. ► Propofol overdose triggers lysosomal dysfunction independent of autophagy. ► Glycogen synthase kinase-3 facilitates propofol overdose-induced apoptosis. ► Propofol overdose causes an increase in peritoneal vascular permeability.« less

The probiotic Propionibacterium freudenreichii as a new adjuvant for TRAIL-based therapy in colorectal cancer.

PubMed

Cousin, Fabien J; Jouan-Lanhouet, Sandrine; Théret, Nathalie; Brenner, Catherine; Jouan, Elodie; Le Moigne-Muller, Gwénaëlle; Dimanche-Boitrel, Marie-Thérèse; Jan, Gwénaël

2016-02-09

TNF-Related Apoptosis-Inducing Ligand (TRAIL) is a well-known apoptosis inducer, which activates the extrinsic death pathway. TRAIL is pro-apoptotic on colon cancer cells, while not cytotoxic towards normal healthy cells. However, its clinical use is limited by cell resistance to cell death which occurs in approximately 50% of cancer cells. Short Chain Fatty Acids (SCFA) are also known to specifically induce apoptosis of cancer cells. In accordance, we have shown that food grade dairy propionibacteria induce intrinsic apoptosis of colon cancer cells, via the production and release of SCFA (propionate and acetate) acting on mitochondria. Here, we investigated possible synergistic effect between Propionibacterium freudenreichii and TRAIL. Indeed, we hypothesized that acting on both extrinsic and intrinsic death pathways may exert a synergistic pro-apoptotic effect. Whole transcriptomic analysis demonstrated that propionibacterial supernatant or propionibacterial metabolites (propionate and acetate), in combination with TRAIL, increased pro-apoptotic gene expression (TRAIL-R2/DR5) and decreased anti-apoptotic gene expression (FLIP, XIAP) in HT29 human colon cancer cells. The revealed synergistic pro-apoptotic effect, depending on both death receptors (TRAIL-R1/DR4, TRAIL-R2/DR5) and caspases (caspase-8, -9 and -3) activation, was lethal on cancer cells but not on normal human intestinal epithelial cells (HIEC), and was inhibited by Bcl-2 expression. Finally, milk fermented by P. freudenreichii induced HT29 cells apoptosis and enhanced TRAIL cytotoxic activity, as did P. freudenreichii DMEM culture supernatants or its SCFA metabolites. These results open new perspectives for food grade P. freudenreichii-containing products in order to potentiate TRAIL-based cancer therapy in colorectal cancer.

MicroRNA-7 Promotes Glycolysis to Protect against 1-Methyl-4-phenylpyridinium-induced Cell Death.

PubMed

Chaudhuri, Amrita Datta; Kabaria, Savan; Choi, Doo Chul; Mouradian, M Maral; Junn, Eunsung

2015-05-08

Parkinson disease is associated with decreased activity of the mitochondrial electron transport chain. This defect can be recapitulated in vitro by challenging dopaminergic cells with 1-methyl-4-phenylpyridinium (MPP(+)), a neurotoxin that inhibits complex I of electron transport chain. Consequently, oxidative phosphorylation is blocked, and cells become dependent on glycolysis for ATP production. Therefore, increasing the rate of glycolysis might help cells to produce more ATP to meet their energy demands. In the present study, we show that microRNA-7, a non-coding RNA that protects dopaminergic neuronal cells against MPP(+)-induced cell death, promotes glycolysis in dopaminergic SH-SY5Y and differentiated human neural progenitor ReNcell VM cells, as evidenced by increased ATP production, glucose consumption, and lactic acid production. Through a series of experiments, we demonstrate that targeted repression of RelA by microRNA-7, as well as subsequent increase in the neuronal glucose transporter 3 (Glut3), underlies this glycolysis-promoting effect. Consistently, silencing Glut3 expression diminishes the protective effect of microRNA-7 against MPP(+). Further, microRNA-7 fails to prevent MPP(+)-induced cell death when SH-SY5Y cells are cultured in a low glucose medium, as well as when differentiated ReNcell VM cells or primary mouse neurons are treated with the hexokinase inhibitor, 2-deoxy-d-glucose, indicating that a functional glycolytic pathway is required for this protective effect. In conclusion, microRNA-7, by down-regulating RelA, augments Glut3 expression, promotes glycolysis, and subsequently prevents MPP(+)-induced cell death. This protective effect of microRNA-7 could be exploited to correct the defects in oxidative phosphorylation in Parkinson disease. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The same drug but a different mechanism of action: comparison of free doxorubicin with two different N-(2-hydroxypropyl)methacrylamide copolymer-bound doxorubicin conjugates in EL-4 cancer cell line.

PubMed

Kovár, Lubomír; Strohalm, Jirí; Chytil, Petr; Mrkvan, Tomás; Kovár, Marek; Hovorka, Ondrej; Ulbrich, Karel; Ríhová, Blanka

2007-01-01

Doxorubicin is one of the most potent anti-tumor drugs with a broad spectrum of use. To reduce its toxic effect and improve its pharmacokinetics, we conjugated it to an HPMA copolymer carrier that enhances its passive accumulation within solid tumors via the EPR effect and decreases its cytotoxicity to normal, noncancer cells. In this study, we compared the antiproliferative, pro-survival, and death signals triggered in EL-4 cancer cells exposed to free doxorubicin and doxorubicin conjugated to a HPMA copolymer carrier via either enzymatically (PK1) or hydrolytically (HYD) degradable bonds. We have previously shown that the intracellular distribution of free doxorubicin, HYD, and PK1 is markedly different. Here, we demonstrated that these three agents greatly differ also in the antiproliferative effect and cell death signals they trigger. JNK phosphorylation sharply increased in cells treated with HYD, while treatment with free doxorubicin moderately decreased and treatment with PK1 even strongly decreased it. On the other hand, treatment with free doxorubicin greatly increased p38 phosphorylation, while PK1 and HYD increased it slightly. PK1 also significantly increased ERK phosphorylation, while both the free doxorubicin and HYD conjugate slightly decreased it. Long-term inhibition of JNK significantly increased both proliferation and viability of EL-4 cells treated with free doxorubicin, showing that the JNK signaling pathway could be critical for mediating cell death in EL-4 cells exposed to free doxorubicin. Both activation of caspase 3 and decreased binding activity of the p50 subunit of NFkappaB were observed in cells treated with free doxorubicin and HYD, while no such effects were seen in cells incubated with PK1. Analysis of the expression of genes involved in apoptosis and regulation of the cell cycle demonstrated that free doxorubicin and HYD have very similar mechanisms of action, while PK1 has very different characteristics.

Inflammation arising from obesity reduces taste bud abundance and inhibits renewal

PubMed Central

Kaufman, Andrew; Choo, Ezen; Koh, Anna

2018-01-01

Despite evidence that the ability to taste is weakened by obesity and can be rescued with weight loss intervention, few studies have investigated the molecular effects of obesity on the taste system. Taste bud cells undergo continual turnover even in adulthood, exhibiting an average life span of only a few weeks, tightly controlled by a balance of proliferation and cell death. Recent data reveal that an acute inflammation event can alter this balance. We demonstrate that chronic low-grade inflammation brought on by obesity reduces the number of taste buds in gustatory tissues of mice—and is likely the cause of taste dysfunction seen in obese populations—by upsetting this balance of renewal and cell death. PMID:29558472

Inhibition of KSP by ARRY-520 Induces Cell Cycle Block and Cell Death via the Mitochondrial Pathway in AML Cells

PubMed Central

Carter, Bing Z.; Mak, Duncan H.; Woessner, Richard; Gross, Stefan; Schober, Wendy D.; Estrov, Zeev; Kantarjian, Hagop; Andreeff, Michael

2013-01-01

Kinesin spindle protein (KSP), a microtubule-associated motor protein essential for cell cycle progression, is overexpressed in many cancers and a potential anti-tumor target. We found that inhibition of KSP by a selective inhibitor, ARRY-520, blocked cell cycle progression, leading to apoptosis in acute myeloid leukemia cell lines which express high levels of KSP. Knockdown of p53, overexpression of XIAP, and mutation in caspase-8 did not significantly affect sensitivity to ARRY-520, suggesting that the response is independent of p53, XIAP, and the extrinsic apoptotic pathway. Although ARRY-520 induced mitotic arrest in both HL-60 and Bcl-2-overexpressing HL-60Bcl-2 cells, cell death was blunted in HL-60Bcl-2 cells, suggesting that the apoptotic program is executed through the mitochondrial pathway. Accordingly, inhibition of Bcl-2 by ABT-737 was synergistic with ARRY-520 in HL-60Bcl-2 cells. Furthermore, ARRY-520 increased Bim protein levels prior to caspase activation in HL-60 cells. ARRY-520 significantly inhibited tumor growth of xenografts in SCID mice and inhibited AML blast but not normal colony formation, supporting a critical role for KSP in proliferation of leukemic progenitor cells. These results demonstrate that ARRY-520 potently induces cell cycle block and subsequent death in leukemic cells via the mitochondrial pathway and has potential to eradicate AML progenitor cells. PMID:19458629

Citrus limon-derived nanovesicles inhibit cancer cell proliferation and suppress CML xenograft growth by inducing TRAIL-mediated cell death

PubMed Central

Raimondo, Stefania; Naselli, Flores; Fontana, Simona; Monteleone, Francesca; Lo Dico, Alessia; Saieva, Laura; Zito, Giovanni; Flugy, Anna; Manno, Mauro; Di Bella, Maria Antonietta; De Leo, Giacomo; Alessandro, Riccardo

2015-01-01

Nanosized vesicles are considered key players in cell to cell communication, thus influencing physiological and pathological processes, including cancer. Nanovesicles have also been found in edible-plants and have shown therapeutic activity in inflammatory bowel diseases; however information on their role in affecting cancer progression is missing. Our study identify for the first time a fraction of vesicles from lemon juice (Citrus limon L.), obtained as a result of different ultracentrifugation, with density ranging from 1,15 to 1,19 g/ml and specific proteomic profile. By using an in vitro approach, we show that isolated nanovesicles inhibit cancer cell proliferation in different tumor cell lines, by activating a TRAIL-mediated apoptotic cell death. Furthermore, we demonstrate that lemon nanovesicles suppress CML tumor growth in vivo by specifically reaching tumor site and by activating TRAIL-mediated apoptotic cell processes. Overall, this study suggests the possible use of plant-edible nanovesicles as a feasible approach in cancer treatment. PMID:26098775

Novel type 1 photosensitizers: viability of leukemia cells exposed to reactive intermediates generated in situ by in vitro photofragmentation

NASA Astrophysics Data System (ADS)

Rajagopalan, Raghavan; Karwa, Amol; Lusiak, Przemyslaw M.; Srivastava, Kripa; Poreddy, Amruta R.; Pandurangi, Raghootama S.; Galen, Karen P.; Neumann, William L.; Cantrell, Gary E.; Dorshow, Richard B.

2009-06-01

Photodynamic therapy of tumors involving Type 2 photosenstizers has been conspicuously successful, but the Type 1 process, in contrast, has not received much attention despite its considerable potential. Accordingly, several classes of molecules containing fragile bonds such as azido (-N=N=N), azo (-N=N-), sulfenato (-S-O-) and oxaza (-N-O-) functional groups that produce reactive intermediates such as radicals and nitrenes upon photoexcitation were prepared and tested for cell viability using U397 leukemia cell line. The azido photosensitizer was conjugated to leukemia cell binding peptide, SFFWRLS, for targeted cell viability study. The cells were incubated with the photosensitizer at various concentrations, and were illuminated for 5, 10, and 20 minutes. The results show that all the photosensitizers caused cell death compared to the controls when exposed to both the photosensitizers and light. Most importantly, selective cell death was observed with the azido peptide conjugate 6, which clearly demonstrates that these Type 1 sensitizers are useful for phototherapeutic applications.

Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death and T-DNA loss.

PubMed

Weld, R; Heinemann, J; Eady, C

2001-03-01

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells from Agrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.

Prime, Shock, and Kill: Priming CD4 T Cells from HIV Patients with a BCL-2 Antagonist before HIV Reactivation Reduces HIV Reservoir Size

PubMed Central

Cummins, Nathan W.; Sainski, Amy M.; Dai, Haiming; Natesampillai, Sekar; Pang, Yuan-Ping; Bren, Gary D.; de Araujo Correia, Maria Cristina Miranda; Sampath, Rahul; Rizza, Stacey A.; O'Brien, Daniel; Yao, Joseph D.

2016-01-01

ABSTRACT Understanding how some HIV-infected cells resist the cytotoxicity of HIV replication is crucial to enabling HIV cure efforts. HIV killing of CD4 T cells that replicate HIV can involve HIV protease-mediated cleavage of procaspase 8 to generate a fragment (Casp8p41) that directly binds and activates the mitochondrial proapoptotic protein BAK. Here, we demonstrate that Casp8p41 also binds with nanomolar affinity to the antiapoptotic protein Bcl-2, which sequesters Casp8p41 and prevents apoptosis. Further, we show that central memory CD4 T cells (TCM) from HIV-infected individuals have heightened expression of BCL-2 relative to procaspase 8, possibly explaining the persistence of HIV-infected TCM despite generation of Casp8p41. Consistent with this hypothesis, the selective BCL-2 antagonist venetoclax induced minimal killing of uninfected CD4 T cells but markedly increased the death of CD4 T cells and diminished cell-associated HIV DNA when CD4 T cells from antiretroviral therapy (ART)-suppressed HIV patients were induced with αCD3/αCD28 to reactivate HIV ex vivo. Thus, priming CD4 T cells from ART suppressed HIV patients with a BCL-2 antagonist, followed by HIV reactivation, achieves reductions in cell-associated HIV DNA, whereas HIV reactivation alone does not. IMPORTANCE HIV infection is incurable due to a long-lived reservoir of HIV+ memory CD4 T cells, and no clinically relevant interventions have been identified that reduce the number of these HIV DNA-containing cells. Since postintegration HIV replication can result in HIV protease generation of Casp8p41, which activates BAK, causing infected CD4 T cell death, we sought to determine whether this occurs in memory CD4 T cells. Here, we demonstrate that memory CD4 T cells can generate Casp8p41 and yet are intrinsically resistant to death induced by diverse stimuli, including Casp8p41. Furthermore, BCL-2 expression is relatively increased in these cells and directly binds and inhibits Casp8p41's proapoptotic effects. Antagonizing BCL-2 with venetoclax derepresses this antagonism, resulting in death, preferentially in HIV DNA containing cells, since only these cells generate Casp8p41. Thus, BCL-2 antagonism is a clinically relevant intervention with the potential to reduce HIV reservoir size in patients. PMID:26842479

Crosstalk of ROS/RNS and autophagy in silibinin-induced apoptosis of MCF-7 human breast cancer cells in vitro.

PubMed

Zheng, Nan; Liu, Lu; Liu, Wei-Wei; Li, Fei; Hayashi, Toshihiko; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2017-02-01

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in regulating cell survival and death. Silibinin is a natural polyphenolic flavonoid isolated from milk thistle with anti-tumor activities, but it was found to induce cytoprotective ROS/RNS in human breast cancer MCF-7 cells. Furthermore, treatment with silibinin down-regulates ERα expression in MCF-7 cells, and inducing both autophagy and apoptosis. In this study we explored the relationship between ER-associated pathways and RNS/ROS in MCF-7 cells. We also investigated the molecular mechanisms underlying the reciprocal regulation between ROS/RNS levels and autophagy in the death signaling pathways in silibinin-treated MCF-7 cells. Silibinin (100-300 μmol/L) dose-dependently increased ROS/RNS generation in MCF-7 cells (with high expression of ERα and low expression of ERβ) and MDA-MB-231 cells (with low expression of ERα and high expression of ERβ). Scavenging ROS/RNS significantly enhanced silibinin-induced death of MCF-7 cells, but not MDA-MB231 cells. Pharmacological activation or blockade of ERα in MCF-7 cells significantly enhanced or decreased, respectively, silibinin-induced ROS/RNS generation, whereas activation or block of ERβ had no effect. In silibinin-treated MCF-7 cells, exposure to the ROS/RNS donators decreased the autophagic levels, whereas inhibition of autophagy with 3-MA significantly increased ROS/RNS levels. We further showed that increases in ROS/RNS generation, ERα activation or autophagy down-regulation had protective roles in silibinin-treated MCF-7 cells. Under a condition of ERα activation, scavenging ROS/RNS or stimulating autophagy enhanced the cytotoxicity of silibinin. These results demonstrate the existence of two conflicting pathways in silibinin-induced death of MCF-7 cells: one involves the down-regulation of ERα and thereby augmenting the pro-apoptotic autophagy downstream, leading to cell death; the other involves the up-regulation of pro-survival ROS/RNS; and that the generation of ROS/RNS and autophagy form a negative feedback loop whose balance is regulated by ERα.

Crosstalk of ROS/RNS and autophagy in silibinin-induced apoptosis of MCF-7 human breast cancer cells in vitro

PubMed Central

Zheng, Nan; Liu, Lu; Liu, Wei-wei; Li, Fei; Hayashi, Toshihiko; Tashiro, Shin-ichi; Onodera, Satoshi; Ikejima, Takashi

2017-01-01

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in regulating cell survival and death. Silibinin is a natural polyphenolic flavonoid isolated from milk thistle with anti-tumor activities, but it was found to induce cytoprotective ROS/RNS in human breast cancer MCF-7 cells. Furthermore, treatment with silibinin down-regulates ERα expression in MCF-7 cells, and inducing both autophagy and apoptosis. In this study we explored the relationship between ER-associated pathways and RNS/ROS in MCF-7 cells. We also investigated the molecular mechanisms underlying the reciprocal regulation between ROS/RNS levels and autophagy in the death signaling pathways in silibinin-treated MCF-7 cells. Silibinin (100–300 μmol/L) dose-dependently increased ROS/RNS generation in MCF-7 cells (with high expression of ERα and low expression of ERβ) and MDA-MB-231 cells (with low expression of ERα and high expression of ERβ). Scavenging ROS/RNS significantly enhanced silibinin-induced death of MCF-7 cells, but not MDA-MB231 cells. Pharmacological activation or blockade of ERα in MCF-7 cells significantly enhanced or decreased, respectively, silibinin-induced ROS/RNS generation, whereas activation or block of ERβ had no effect. In silibinin-treated MCF-7 cells, exposure to the ROS/RNS donators decreased the autophagic levels, whereas inhibition of autophagy with 3-MA significantly increased ROS/RNS levels. We further showed that increases in ROS/RNS generation, ERα activation or autophagy down-regulation had protective roles in silibinin-treated MCF-7 cells. Under a condition of ERα activation, scavenging ROS/RNS or stimulating autophagy enhanced the cytotoxicity of silibinin. These results demonstrate the existence of two conflicting pathways in silibinin-induced death of MCF-7 cells: one involves the down-regulation of ERα and thereby augmenting the pro-apoptotic autophagy downstream, leading to cell death; the other involves the up-regulation of pro-survival ROS/RNS; and that the generation of ROS/RNS and autophagy form a negative feedback loop whose balance is regulated by ERα. PMID:27867187

Quercetin inhibits prostate cancer by attenuating cell survival and inhibiting anti-apoptotic pathways.

PubMed

Ward, Ashley B; Mir, Hina; Kapur, Neeraj; Gales, Dominique N; Carriere, Patrick P; Singh, Shailesh

2018-06-14

Despite recent advances in diagnosis and treatment, prostate cancer (PCa) remains the leading cause of cancer-related deaths in men. Current treatments offered in the clinics are often toxic and have severe side effects. Hence, to treat and manage PCa, new agents with fewer side effects or having potential to reduce side effects of conventional therapy are needed. In this study, we show anti-cancer effects of quercetin, an abundant bioflavonoid commonly used to treat prostatitis, and defined quercetin-induced cellular and molecular changes leading to PCa cell death. Cell viability was assessed using MTT. Cell death mode, mitochondrial outer membrane potential, and oxidative stress levels were determined by flow cytometry using Annexin V-7 AAD dual staining kit, JC-1 dye, and ROS detection kit, respectively. Antibody microarray and western blot were used to delineate the molecular changes induced by quercetin. PCa cells treated with various concentrations of quercetin showed time- and dose-dependent decrease in cell viability compared to controls, without affecting normal prostate epithelial cells. Quercetin led to apoptotic and necrotic cell death in PCa cells by affecting the mitochondrial integrity and disturbing the ROS homeostasis depending upon the genetic makeup and oxidative status of the cells. LNCaP and PC-3 cells that have an oxidative cellular environment showed ROS quenching after quercetin treatment while DU-145 showed rise in ROS levels despite having a highly reductive environment. Opposing effects of quercetin were also observed on the pro-survival pathways of PCa cells. PCa cells with mutated p53 (DU-145) and increased ROS showed significant reduction in the activation of pro-survival Akt pathway while Raf/MEK were activated in response to quercetin. PC-3 cells lacking p53 and PTEN with reduced ROS levels showed significant activation of Akt and NF-κB pathway. Although some of these changes are commonly associated with oncogenic response, the cumulative effect of these alterations is PCa cell death. Our results demonstrated quercetin exerts its anti-cancer effects by modulating ROS, Akt, and NF-κB pathways. Quercetin could be used as a chemopreventive option as well as in combination with chemotherapeutic drugs to improve clinical outcomes of PCa patients.

Silencing of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase gene enhances glioma radiosensitivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sung Youl; Yoo, Young Hyun; Park, Jeen-Woo, E-mail: parkjw@knu.ac.kr

Highlights: •Silencing of the IDPm gene enhances IR-induced autophagy in glioma cells. •Autophagy inhibition augmented apoptosis of irradiated glioma cells. •Results offer a redox-active therapeutic strategy for the treatment of cancer. -- Abstract: Reactive oxygen species (ROS) levels are elevated in organisms that have been exposed to ionizing radiation and are protagonists in the induction of cell death. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage are primary functions of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase (IDPm) via the supply of NADPH for antioxidant systems. In the present study, we report anmore » autophagic response to ionizing radiation in A172 glioma cells transfected with small interfering RNA (siRNA) targeting the IDPm gene. Autophagy in A172 transfectant cells was associated with enhanced autophagolysosome formation and GFP–LC3 punctuation/aggregation. Furthermore, we found that the inhibition of autophagy by chloroquine augmented apoptotic cell death of irradiated A172 cells transfected with IDPm siRNA. Taken together, our data suggest that autophagy functions as a survival mechanism in A172 cells against ionizing radiation-induced apoptosis and the sensitizing effect of IDPm siRNA and autophagy inhibitor on the ionizing radiation-induced apoptotic cell death of glioma cells offers a novel redox-active therapeutic strategy for the treatment of cancer.« less

Dihydroartemisinin induces autophagy-dependent death in human tongue squamous cell carcinoma cells through DNA double-strand break-mediated oxidative stress

PubMed Central

Li, Xiaoming; Bai, Jing; Li, Jianchun; Li, Shenghao; Wang, Zeming; Zhou, Mingrui

2017-01-01

Dihydroartemisinin is an effective antimalarial agent with multiple biological activities. In the present investigation, we elucidated its therapeutic potential and working mechanism on human tongue squamous cell carcinoma (TSCC). It was demonstrated that dihydroartemisinin could significantly inhibit cell growth in a dose- and time-dependent manner by the Cell Counting Kit-8 and colony formation assay in vitro. Meanwhile, autophagy was promoted in the Cal-27 cells treated by dihydroartemisinin, evidenced by increased LC3B-II level, increased autophagosome formation, and increased Beclin-1 level compared to dihydroartemisinin-untreated cells. Importantly, dihydroartemisinin caused DNA double-strand break with simultaneously increased γH2AX foci and oxidative stress; this inhibited the nuclear localization of phosphorylated signal transducer and activator of transcription 3 (p-STAT3), finally leading to autophagic cell death. Furthermore, the antitumor effect of dihydroartemisinin-monotherapy was confirmed with a mouse xenograft model, and no kidney injury associated with toxic effect was observed after intraperitoneal injection with dihydroartemisinin for 3 weeks in vivo. In the present study, it was revealed that dihydroartemisinin-induced DNA double-strand break promoted oxidative stress, which decreased p-STAT3 (Tyr705) nuclear localization, and successively increased autophagic cell death in the Cal-27 cells. Thus, dihydroartemisinin alone may represent an effective and safe therapeutic agent for human TSCC. PMID:28526807

The xylanase inhibitor TAXI-III counteracts the necrotic activity of a Fusarium graminearum xylanase in vitro and in durum wheat transgenic plants.

PubMed

Moscetti, Ilaria; Faoro, Franco; Moro, Stefano; Sabbadin, Davide; Sella, Luca; Favaron, Francesco; D'Ovidio, Renato

2015-08-01

The xylanase inhibitor TAXI-III has been proven to delay Fusarium head blight (FHB) symptoms caused by Fusarium graminearum in transgenic durum wheat plants. To elucidate the molecular mechanism underlying the capacity of the TAXI-III transgenic plants to limit FHB symptoms, we treated wheat tissues with the xylanase FGSG_03624, hitherto shown to induce cell death and hydrogen peroxide accumulation. Experiments performed on lemmas of flowering wheat spikes and wheat cell suspension cultures demonstrated that pre-incubation of xylanase FGSG_03624 with TAXI-III significantly decreased cell death. Most interestingly, a reduced cell death relative to control non-transgenic plants was also obtained by treating, with the same xylanase, lemmas of TAXI-III transgenic plants. Molecular modelling studies predicted an interaction between the TAXI-III residue H395 and residues E122 and E214 belonging to the active site of xylanase FGSG_03624. These results provide, for the first time, clear indications in vitro and in planta that a xylanase inhibitor can prevent the necrotic activity of a xylanase, and suggest that the reduced FHB symptoms on transgenic TAXI-III plants may be a result not only of the direct inhibition of xylanase activity secreted by the pathogen, but also of the capacity of TAXI-III to avoid host cell death. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Questiomycin A stimulates sorafenib-induced cell death via suppression of glucose-regulated protein 78.

PubMed

Machihara, Kayo; Tanaka, Hidenori; Hayashi, Yoshihiro; Murakami, Ichiro; Namba, Takushi

2017-10-07

Hepatocellular carcinoma (HCC) is one of the most difficult cancers to treat owing to the lack of effective chemotherapeutic methods. Sorafenib, the first-line and only available treatment for HCC, extends patient overall survival by several months, with a response rate below 10%. Thus, the identification of an agent that enhances the anticancer effect of sorafenib is critical for the development of therapeutic options for HCC. Endoplasmic reticulum (ER) stress response is one of the methods of sorafenib-induced cell death. Here we report that questiomycin A suppresses expression of GRP78, a cell-protective ER chaperone protein. Analysis of the molecular mechanisms of questiomycin A revealed that this compound stimulated GRP78 protein degradation in an ER stress response-independent manner. Cotreatment with sorafenib and questiomycin A suppressed GRP78 protein expression, which is essential for the stimulation of sorafenib-induced cell death. Moreover, our in vivo study demonstrated that the coadministration of sorafenib and questiomycin A suppressed tumor formation in HCC-induced xenograft models. These results suggest that cotreatment with sorafenib and questiomycin A is a novel therapeutic strategy for HCC by enhancing sorafenib-dependent ER stress-induced cell death, and downregulation of GRP78 is a new target for the stimulation of the therapeutic effects of sorafenib in HCC. Copyright © 2017 Elsevier Inc. All rights reserved.

Virus-induced apoptosis and phosphorylation form of metacaspase in the marine coccolithophorid Emiliania huxleyi.

PubMed

Liu, Jingwen; Cai, Weicong; Fang, Xian; Wang, Xueting; Li, Guiling

2018-04-01

Lytic viral infection and programmed cell death (PCD) are thought to represent two distinct death mechanisms in phytoplankton, unicellular photoautotrophs that drift with ocean currents. PCD (apoptosis) is mainly brought about by the activation of caspases, a protease family with unique substrate selectivity. Here, we demonstrated that virus infection induced apoptosis of marine coccolithophorid Emiliania huxleyi BOF92 involving activation of metacaspase. E. huxleyi cells exhibited cell death process akin to that of apoptosis when exposed to virus infection. We observed typical hallmarks of apoptosis including cell shrinkage, associated nuclear morphological changes and DNA fragmentation. Immunoblotting revealed that antibody against human active-caspase-3 shared epitopes with a protein of ≈ 23 kDa; whose pattern of expression correlated with the onset of cell death. Moreover, analysis on two-dimensional gel electrophoresis revealed that two spots of active caspase-3 co-migrated with the different isoelectric points. Phosphatase treatment of cytosolic extracts containing active caspases-3 showed a mobility shift, suggesting that phosphorylated form of this enzyme might be present in the extracts. Computational prediction of phosphorylation sites based on the amino acid sequence of E. huxleyi metacaspase showed multiple phosphorylated sites for serine, threonine and tyrosine residues. This is the first report showing that phosphorylation modification of metacaspase in E. huxleyi might be required for certain biochemical and morphological changes during virus induced apoptosis.

Metabolic interrogation as a tool to optimize chemotherapeutic regimens.

PubMed

Sandulache, Vlad C; Chen, Yunyun; Feng, Lei; William, William N; Skinner, Heath D; Myers, Jeffrey N; Meyn, Raymond E; Li, Jinzhong; Mijiti, Ainiwaer; Bankson, James A; Fuller, Clifton D; Konopleva, Marina Y; Lai, Stephen Y

2017-03-14

Platinum-based (Pt) chemotherapy is broadly utilized in the treatment of cancer. Development of more effective, personalized treatment strategies require identification of novel biomarkers of treatment response. Since Pt compounds are inactivated through cellular metabolic activity, we hypothesized that metabolic interrogation can predict the effectiveness of Pt chemotherapy in a pre-clinical model of head and neck squamous cell carcinoma (HNSCC).We tested the effects of cisplatin (CDDP) and carboplatin (CBP) on DNA damage, activation of cellular death cascades and tumor cell metabolism, specifically lactate production. Pt compounds induced an acute dose-dependent, transient drop in lactate generation in vitro, which correlated with effects on DNA damage and cell death. Neutralization of free radical stress abrogated these effects. The magnitude of this effect on lactate production correlated with the differential sensitivity of HNSCC cells to Pt compounds (CDDP vs CBP) and p53-driven Pt chemotherapy resistance. Using dual flank xenograft tumors, we demonstrated that Pt-driven effects on lactate levels correlate with effects on tumor growth delay in a dose-dependent manner and that lactate levels can define the temporal profile of Pt chemotherapy-induced metabolic stress. Lactate interrogation also predicted doxorubicin effects on cell death in both solid tumor (HNSCC) and acute myelogenous leukemia (AML) cell lines.Real-time metabolic interrogation of acute changes in cell and tumor lactate levels reflects chemotherapy effects on DNA damage, cell death and tumor growth delay. We have identified a real-time biomarker of chemotherapy effectiveness which can be used to develop adaptive, iterative and personalized treatment regimens against a variety of solid and hematopoietic malignancies.

Metabolic interrogation as a tool to optimize chemotherapeutic regimens

PubMed Central

Feng, Lei; William, William N.; Skinner, Heath D.; Myers, Jeffrey N.; Meyn, Raymond E.; Li, Jinzhong; Mijiti, Ainiwaer; Bankson, James A.; Fuller, Clifton D.; Konopleva, Marina Y.; Lai, Stephen Y.

2017-01-01

Platinum-based (Pt) chemotherapy is broadly utilized in the treatment of cancer. Development of more effective, personalized treatment strategies require identification of novel biomarkers of treatment response. Since Pt compounds are inactivated through cellular metabolic activity, we hypothesized that metabolic interrogation can predict the effectiveness of Pt chemotherapy in a pre-clinical model of head and neck squamous cell carcinoma (HNSCC). We tested the effects of cisplatin (CDDP) and carboplatin (CBP) on DNA damage, activation of cellular death cascades and tumor cell metabolism, specifically lactate production. Pt compounds induced an acute dose-dependent, transient drop in lactate generation in vitro, which correlated with effects on DNA damage and cell death. Neutralization of free radical stress abrogated these effects. The magnitude of this effect on lactate production correlated with the differential sensitivity of HNSCC cells to Pt compounds (CDDP vs CBP) and p53-driven Pt chemotherapy resistance. Using dual flank xenograft tumors, we demonstrated that Pt-driven effects on lactate levels correlate with effects on tumor growth delay in a dose-dependent manner and that lactate levels can define the temporal profile of Pt chemotherapy-induced metabolic stress. Lactate interrogation also predicted doxorubicin effects on cell death in both solid tumor (HNSCC) and acute myelogenous leukemia (AML) cell lines. Real-time metabolic interrogation of acute changes in cell and tumor lactate levels reflects chemotherapy effects on DNA damage, cell death and tumor growth delay. We have identified a real-time biomarker of chemotherapy effectiveness which can be used to develop adaptive, iterative and personalized treatment regimens against a variety of solid and hematopoietic malignancies. PMID:28184025

N-Acetylcysteine Attenuates Hexavalent Chromium-Induced Hypersensitivity through Inhibition of Cell Death, ROS-Related Signaling and Cytokine Expression

PubMed Central

Huang, Chien-Cheng; Sheu, Hamm-Ming; Tsai, Jui-Chen; Lin, Chia-Ho; Wang, Ying-Jan; Wang, Bour-Jr

2014-01-01

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. PMID:25248126

Seizure activity results in calcium- and mitochondria-independent ROS production via NADPH and xanthine oxidase activation

PubMed Central

Kovac, S; Domijan, A-M; Walker, M C; Abramov, A Y

2014-01-01

Seizure activity has been proposed to result in the generation of reactive oxygen species (ROS), which then contribute to seizure-induced neuronal damage and eventually cell death. Although the mechanisms of seizure-induced ROS generation are unclear, mitochondria and cellular calcium overload have been proposed to have a crucial role. We aim to determine the sources of seizure-induced ROS and their contribution to seizure-induced cell death. Using live cell imaging techniques in glioneuronal cultures, we show that prolonged seizure-like activity increases ROS production in an NMDA receptor-dependent manner. Unexpectedly, however, mitochondria did not contribute to ROS production during seizure-like activity. ROS were generated primarily by NADPH oxidase and later by xanthine oxidase (XO) activity in a calcium-independent manner. This calcium-independent neuronal ROS production was accompanied by an increase in intracellular [Na+] through NMDA receptor activation. Inhibition of NADPH or XO markedly reduced seizure-like activity-induced neuronal apoptosis. These findings demonstrate a critical role for ROS in seizure-induced neuronal cell death and identify novel therapeutic targets. PMID:25275601

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Is Pyruvylated during 3-Bromopyruvate Mediated Cancer Cell Death

PubMed Central

Ganapathy-Kanniappan, Shanmugasundaram; Geschwind, Jean-Francois H.; Kunjithapatham, Rani; Buijs, Manon; Vossen, Josephina A.; Tchernyshyov, Irina; Cole, Robert N.; Syed, Labiq H.; Rao, Pramod P.; Ota, Shinichi; Vali, Mustafa

2013-01-01

Background The pyruvic acid analog 3-bromopyruvate (3BrPA) is an alkylating agent known to induce cancer cell death by blocking glycolysis. The anti-glycolytic effect of 3BrPA is considered to be the inactivation of glycolytic enzymes. Yet, there is a lack of experimental documentation on the direct interaction of 3BrPA with any of the suggested targets during its anticancer effect. Methods and Results In the current study, using radiolabeled (14C) 3BrPA in multiple cancer cell lines, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the primary intracellular target of 3BrPA, based on two-dimensional (2D) gel electrophoretic autoradiography, mass spectrometry and immunoprecipitation. Furthermore, in vitro enzyme kinetic studies established that 3BrPA has marked affinity to GAPDH. Finally, Annexin V staining and active caspase-3 immunoblotting demonstrated that apoptosis was induced by 3BrPA. Conclusion GAPDH pyruvylation by 3BrPA affects its enzymatic function and is the primary intracellular target in 3BrPA mediated cancer cell death. PMID:20044597

Systems modelling methodology for the analysis of apoptosis signal transduction and cell death decisions.

PubMed

Rehm, Markus; Prehn, Jochen H M

2013-06-01

Systems biology and systems medicine, i.e. the application of systems biology in a clinical context, is becoming of increasing importance in biology, drug discovery and health care. Systems biology incorporates knowledge and methods that are applied in mathematics, physics and engineering, but may not be part of classical training in biology. We here provide an introduction to basic concepts and methods relevant to the construction and application of systems models for apoptosis research. We present the key methods relevant to the representation of biochemical processes in signal transduction models, with a particular reference to apoptotic processes. We demonstrate how such models enable a quantitative and temporal analysis of changes in molecular entities in response to an apoptosis-inducing stimulus, and provide information on cell survival and cell death decisions. We introduce methods for analyzing the spatial propagation of cell death signals, and discuss the concepts of sensitivity analyses that enable a prediction of network responses to disturbances of single or multiple parameters. Copyright © 2013 Elsevier Inc. All rights reserved.

Pyroptosis and Apoptosis Pathways Engage in Bidirectional Crosstalk in Monocytes and Macrophages.

PubMed

Taabazuing, Cornelius Y; Okondo, Marian C; Bachovchin, Daniel A

2017-04-20

Pyroptosis is a lytic form of programmed cell death mediated by the inflammatory caspase-1, -4, and -5. We recently discovered that small-molecule inhibitors of the serine peptidases DPP8 and DPP9 (DPP8/9) induce pro-caspase-1-dependent pyroptosis in monocytes and macrophages. Notably, DPP8/9 inhibitors, unlike microbial agents, absolutely require caspase-1 to induce cell death. Therefore, DPP8/9 inhibitors are useful probes to study caspase-1 in cells. Here, we show that, in the absence of the pyroptosis-mediating substrate gasdermin D (GSDMD), caspase-1 activates caspase-3 and -7 and induces apoptosis, demonstrating that GSDMD is the only caspase-1 substrate that induces pyroptosis. Conversely, we found that, during apoptosis, caspase-3/-7 specifically block pyroptosis by cleaving GSDMD at a distinct site from the inflammatory caspases that inactivates the protein. Overall, this work reveals bidirectional crosstalk between apoptosis and pyroptosis in monocytes and macrophages, further illuminating the complex interplay between cell death pathways in the innate immune system. Copyright © 2017 Elsevier Ltd. All rights reserved.

Curcumin induces Apaf-1-dependent, p21-mediated caspase activation and apoptosis

PubMed Central

Zhang, Honghao; Jones, Anthony; Verone, Alissa; Pitarresi, Jason; Jandhyam, Sirisha; Prabhu, Varun; Black, Jennifer D

2011-01-01

Previous studies have demonstrated that curcumin induces mitochondria-mediated apoptosis. However, understanding of the molecular mechanisms underlying curcumin-induced cell death remains limited. In this study, we demonstrate that curcumin treatment of cancer cells caused dose- and time-dependent caspase 3 activation, which is required for apoptosis as confirmed using the pan-caspase inhibitor, z-VAD. Knockdown experiments and knockout cells excluded a role for caspase 8 in curcumin-induced caspase 3 activation. In contrast, Apaf-1 deficiency or silencing inhibited the activity of caspase 3, pointing to a requisite role of Apaf-1 in curcumin-induced apoptotic cell death. Curcumin treatment led to Apaf-1 upregulation, both at the protein and mRNA levels. Cytochrome c release from mitochondria to the cytosol in curcumin-treated cells was associated with upregulation of pro-apoptotic proteins, such as Bax, Bak, Bid and Bim. Cross-linking experiments demonstrated Bax oligomerization during curcumin-induced apoptosis, suggesting that induced expression of Bax, Bid and Bim causes Bax channel formation on the mitochondrial membrane. The release of cytochrome c was unaltered in p53-deficient cells, whereas absence of p21 blocked cytochrome c release, caspase activation and apoptosis. Importantly, p21 deficiency resulted in reduced expression of Apaf-1 during curcumin treatment, indicating a requirement for p21 in Apaf-1-dependent caspase activation and apoptosis. Together, our findings identify Apaf-1, Bax and p21 as novel potential targets for curcumin or curcumin-based anticancer agents. PMID:22101335

MicroRNA-155 Reinforces HIV Latency*

PubMed Central

Ruelas, Debbie S.; Chan, Jonathan K.; Oh, Eugene; Heidersbach, Amy J.; Hebbeler, Andrew M.; Chavez, Leonard; Verdin, Eric; Rape, Michael; Greene, Warner C.

2015-01-01

The presence of a small number of infected but transcriptionally dormant cells currently thwarts a cure for the more than 35 million individuals infected with HIV. Reactivation of these latently infected cells may result in three fates: 1) cell death due to a viral cytopathic effect, 2) cell death due to immune clearance, or 3) a retreat into latency. Uncovering the dynamics of HIV gene expression and silencing in the latent reservoir will be crucial for developing an HIV-1 cure. Here we identify and characterize an intracellular circuit involving TRIM32, an HIV activator, and miR-155, a microRNA that may promote a return to latency in these transiently activated reservoir cells. Notably, we demonstrate that TRIM32, an E3 ubiquitin ligase, promotes reactivation from latency by directly modifying IκBα, leading to a novel mechanism of NF-κB induction not involving IκB kinase activation. PMID:25873391

Newly synthesized bis-benzimidazole compound 8 induces apoptosis, autophagy and reactive oxygen species generation in HeLa cells.

PubMed

Chu, Naying; Yao, Guodong; Liu, Yuan; Cheng, Maosheng; Ikejima, Takashi

2016-09-01

Compound 8 (C8) is a newly synthesized bis-benzimidazole derivative and exerts significant anti-tumor activity in vitro. Previous studies demonstrated that C8 induced apoptosis and autophagy in human promyelocytic leukemia HL60 cells. However, cytotoxicity study on human peripheral blood mononuclear cells (hPBMC) showed that C8 exhibited less toxicity in normal cells. In this study, the molecular mechanism of C8 on human cervical carcinoma HeLa cells was investigated. The results showed that C8 inhibited the growth of HeLa cells and triggered both apoptotic and autophagic cell death. Subsequent experiment also indicated that reactive oxygen species (ROS) generation was induced in C8-treated HeLa cells. Since ROS scavenger decreased the ratio of apoptotic and autophagic cells, ROS generation contributed to C8-induced apoptosis and autophagy. Furthermore, inhibitors of apoptosis and autophagy also reduced ROS generation, respectively. Autophagy inhibition increased cell growth compared to C8-treated group and attenuated apoptotic cell death, indicating that C8-induced autophagy promoted apoptosis for cell death. However, the percentage of autophagic cells was enhanced when limiting apoptosis process. Taken together, C8 induced ROS-mediated apoptosis and autophagy in HeLa cells, autophagy promoted apoptosis but the former was antagonized by the latter. The data also gave us a new perspective on the anti-tumor effect of C8. Copyright © 2016 Elsevier Ltd. All rights reserved.

Interference with the HSF1/HSP70/BAG3 Pathway Primes Glioma Cells to Matrix Detachment and BH3 Mimetic-Induced Apoptosis.

PubMed

Antonietti, Patrick; Linder, Benedikt; Hehlgans, Stephanie; Mildenberger, Iris C; Burger, Michael C; Fulda, Simone; Steinbach, Joachim P; Gessler, Florian; Rödel, Franz; Mittelbronn, Michel; Kögel, Donat

2017-01-01

Malignant gliomas exhibit a high intrinsic resistance against stimuli triggering apoptotic cell death. HSF1 acts as transcription factor upstream of HSP70 and the HSP70 co-chaperone BAG3 that is overexpressed in glioblastoma. To specifically target this resistance mechanism, we applied the selective HSF1 inhibitor KRIBB11 and the HSP70/BAG3 interaction inhibitor YM-1 in combination with the pan-Bcl-2 inhibitor AT-101. Here, we demonstrate that lentiviral BAG3 silencing significantly enhances AT-101-induced cell death and reactivates effector caspase-mediated apoptosis in U251 glioma cells with high BAG3 expression, whereas these sensitizing effects were less pronounced in U343 cells expressing lower BAG3 levels. KRIBB11 decreased protein levels of HSP70, BAG3, and the antiapoptotic Bcl-2 protein Mcl-1, and both KRIBB11 and YM-1 elicited significantly increased mitochondrial dysfunction, effector caspase activity, and apoptotic cell death after combined treatment with AT-101 and ABT-737. Depletion of BAG3 also led to a pronounced loss of cell-matrix adhesion, FAK phosphorylation, and in vivo tumor growth in an orthotopic mouse glioma model. Furthermore, it reduced the plating efficiency of U251 cells in three-dimensional clonogenic assays and limited clonogenic survival after short-term treatment with AT-101. Collectively, our data suggest that the HSF1/HSP70/BAG3 pathway plays a pivotal role for overexpression of prosurvival Bcl-2 proteins and cell death resistance of glioma. They also support the hypothesis that interference with BAG3 function is an effective novel approach to prime glioma cells to anoikis. Mol Cancer Ther; 16(1); 156-68. ©2016 AACR. ©2016 American Association for Cancer Research.

ROS-induced toxicity: exposure of 3T3, RAW264.7, and MCF7 cells to superparamagnetic iron oxide nanoparticles results in cell death by mitochondria-dependent apoptosis

NASA Astrophysics Data System (ADS)

Hsieh, Hui-Chen; Chen, Chung-Ming; Hsieh, Wen-Yuan; Chen, Ching-Yun; Liu, Chia-Ching; Lin, Feng-Huei

2015-02-01

Superparamagnetic nanoparticles (Fe3O4, SPIO) have been used as magnetic resonance imaging enhancers for years. However, bio-safety issues concerning nanoparticles remain largely unexplored. Of particular concern is the possible cellular impact of nanoparticles during SPIO uptake and subsequent oxidative stress. SPIO causes cell death by apoptosis via a little understood mitochondrial pathway. To more closely examine this process, three kinds of cells—3T3, RAW264.7, and MCF7—were treated with SPIO coated with polyethylene glycol (SPIO-PEG) and monitored by transmission electron microscopy (TEM), using cytotoxicity evaluation, mitochondrial activity, reactive oxygen species (ROS) generation, and Annexin V assay. TEM revealed that SPIO-PEG nanoparticles surrounded the cellular endosome membrane, creating a bulge in the endosome. Compared to 3T3 cells, greater numbers of SPIO-PEG nanoparticles infiltrated the mitochondria of RAW264.7 and MCF7 cells. SPIO-PEG residency is associated with boosted ROS, with elevated levels of mitochondrial activity, and advancement of cell apoptosis. Furthermore, correlation analysis showed that a polynomial model demonstrates a better fit than a linear model in MCF7, implying that cytotoxicity may have alternative impacts on cell death at different concentrations. Thus, we believe that MCF7 cell death results from the apoptosis pathway triggered by mitochondria, and we find lower cytotoxicity in 3T3. We propose that optimal levels of SPIO-PEG nanoparticles lead to increased levels of ROS and a resulting oxidative stress environment which will kill only cancer cells while sparing normal cells. This finding has great potential for use in cancer therapies in the future.

Hyperthermia mediated by dextran-coated La0.7Sr0.3MnO3 nanoparticles: in vivo studies

PubMed Central

Haghniaz, Reihaneh; Umrani, Rinku D; Paknikar, Kishore M

2016-01-01

Purpose The aim of this study was to evaluate radiofrequency-induced dextran-coated lanthanum strontium manganese oxide nanoparticles-mediated hyperthermia to be used for tumor regression in mice. Materials and methods Nanoparticles were injected intra-tumorally in melanoma-bearing C57BL/6J mice and were subjected to radiofrequency treatment. Results Hyperthermia treatment significantly inhibited tumor growth (~84%), increased survival (~50%), and reduced tumor proliferation in mice. Histopathological examination demonstrated immense cell death in treated tumors. DNA fragmentation, increased terminal deoxynucleotidyl transferase-dUTP nick end labeling signal, and elevated levels of caspase-3 and caspase-6 suggested apoptotic cell death. Enhanced catalase activity suggested reactive oxygen species-mediated cell death. Enhanced expression of heat shock proteins 70 and 90 in treated tumors suggested the possible development of “antitumor immunity”. Conclusion The dextran-coated lanthanum strontium manganese oxide-mediated hyperthermia can be used for the treatment of cancer. PMID:27175076

The quick and the dead: microbial demography at the yeast thermal limit.

PubMed

Maxwell, Colin S; Magwene, Paul M

2017-03-01

The niche of microorganisms is determined by where their populations can expand. Populations can fail to grow because of high death or low birth rates, but these are challenging to measure in microorganisms. We developed a novel technique that enables single-cell measurement of age-structured birth and death rates in the budding yeast, Saccharomyces cerevisiae, and used this method to study responses to heat stress in a genetically diverse panel of strains. We find that individual cells show significant heterogeneity in their rates of birth and death during heat stress. Genotype-by-environment effects on processes that regulate asymmetric cell division contribute to this heterogeneity. These lead to either premature senescence or early life mortality during heat stress, and we find that a mitochondrial inheritance defect explains the early life mortality phenotype of one of the strains we studied. This study demonstrates how the interplay of physiology, genetic variation and environmental variables influence where microbial populations survive and flourish. © 2016 John Wiley & Sons Ltd.

Identification of the APC/C co-factor FZR1 as a novel therapeutic target for multiple myeloma.

PubMed

Crawford, Lisa J; Anderson, Gordon; Johnston, Cliona K; Irvine, Alexandra E

2016-10-25

Multiple Myeloma (MM) is a haematological neoplasm characterised by the clonal proliferation of malignant plasma cells in the bone marrow. The success of proteasome inhibitors in the treatment of MM has highlighted the importance of the ubiquitin proteasome system (UPS) in the pathogenesis of this disease. In this study, we analysed gene expression of UPS components to identify novel therapeutic targets within this pathway in MM. Here we demonstrate how this approach identified previously validated and novel therapeutic targets. In addition we show that FZR1 (Fzr), a cofactor of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C), represents a novel therapeutic target in myeloma. The APC/C associates independently with two cofactors, Fzr and Cdc20, to control cell cycle progression. We found high levels of FZR1 in MM primary cells and cell lines and demonstrate that expression is further increased on adhesion to bone marrow stromal cells (BMSCs). Specific knockdown of either FZR1 or CDC20 reduced viability and induced growth arrest of MM cell lines, and resulted in accumulation of APC/CFzr substrate Topoisomerase IIα (TOPIIα) or APC/CCdc20 substrate Cyclin B. Similar effects were observed following treatment with proTAME, an inhibitor of both APC/CFzr and APC/CCdc20. Combinations of proTAME with topoisomerase inhibitors, etoposide and doxorubicin, significantly increased cell death in MM cell lines and primary cells, particularly if TOPIIα levels were first increased through pre-treatment with proTAME. Similarly, combinations of proTAME with the microtubule inhibitor vincristine resulted in enhanced cell death. This study demonstrates the potential of targeting the APC/C and its cofactors as a therapeutic approach in MM.

Melatonin partially protects 661W cells from H2O2-induced death by inhibiting Fas/FasL-caspase-3.

PubMed

Sánchez-Bretaño, Aída; Baba, Kenkichi; Janjua, Uzair; Piano, Ilaria; Gargini, Claudia; Tosini, Gianluca

2017-01-01

Previous studies have shown that melatonin (MEL) signaling is involved in the modulation of photoreceptor viability during aging. Recent work by our laboratory suggested that MEL may protect cones by modulating the Fas/FasL-caspase-3 pathway. In this study, we first investigated the presence of MEL receptors (MT 1 and MT 2 ) in 661W cells, then whether MEL can prevent H 2 O 2 -induced cell death, and last, through which pathway MEL confers protection. The mRNA and proteins of the MEL receptors were detected with quantitative PCR (q-PCR) and immunocytochemistry, respectively. To test the protective effect of MEL, 661W cells were treated with H 2 O 2 for 2 h in the presence or absence of MEL, a MEL agonist, and an antagonist. To study the pathways involved in H 2 O 2 -mediated cell death, a Fas/FasL antagonist was used before the exposure to H 2 O 2 . Finally, Fas/FasL and caspase-3 mRNA was analyzed with q-PCR and immunocytochemistry in cells treated with H 2 O 2 and/or MEL. Cell viability was analyzed by using Trypan Blue. Both MEL receptors (MT 1 and MT 2 ) were detected at the mRNA and protein levels in 661W cells. MEL partially prevented H 2 O 2 -mediated cell death (20-25%). This effect was replicated with IIK7 (a melatonin receptor agonist) when used at a concentration of 1 µM. Preincubation with luzindole (a melatonin receptor antagonist) blocked MEL protection. Kp7-6, an antagonist of Fas/FasL, blocked cell death caused by H 2 O 2 similarly to what was observed for MEL. Fas, FasL, and caspase-3 expression was increased in cells treated with H 2 O 2 , and this effect was prevented by MEL. Finally, MEL treatment partially prevented the activation of caspase-3 caused by H 2 O 2 . The results demonstrate that MEL receptors are present and functional in 661W cells. MEL can prevent photoreceptor cell death induced by H 2 O 2 via the inhibition of the proapoptotic pathway Fas/FasL-caspase-3.