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

PubMed

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

2008-08-01

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

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

Cyclin-dependent kinase 5-mediated phosphorylation of CHIP promotes the tAIF-dependent death pathway in rotenone-treated cortical neurons.

PubMed

Kim, Chiho; Lee, Juhyung; Ko, Yeon Uk; Oh, Young J

2018-01-01

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase. Its dysregulation has been implicated in various neurodegenerative diseases. We previously reported that phosphorylation of the C-terminus of the Hsc70-interacting protein (CHIP) by Cdk5 promotes truncated apoptosis-inducing factor (tAIF)-mediated neuronal death induced by oxidative stress. Here, we determined whether this Cdk5-dependent cell death signaling pathway is present in experimental models of Parkinson's disease. First, we showed that rotenone activates Cdk5 in primary cultures of cortical neurons and causes tAIF-dependent neuronal cell death. This event was attenuated by negative regulation of endogenous Cdk5 activity by the pharmacological Cdk5 inhibitor, roscovitine, or by lentiviral knockdown of Cdk5. Cdk5 phosphorylates CHIP at Ser20 in rotenone-treated neurons. Consequently, overexpression of CHIP S20A , but not CHIP WT , attenuates tAIF-induced cell death in rotenone-treated cortical neurons. Taken together, these results indicate that phosphorylation of CHIP at Ser20 by Cdk5 activation inhibits CHIP-mediated tAIF degradation, thereby contributing to tAIF-induced neuronal cell death following rotenone treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Inhibitory effects of mouse bone marrow mesenchymal stem cell soup on staurospurine-induced cell death in MCF-7 and AGS.

PubMed

Zhaleh, M; Azadbakht, M; Bidmeshki Pour, A

2017-01-01

Staurospurine induces apoptosis in cell line. Bone Marrow Mesenchymal stem cells Soup is a promising tool for cell proliferation via a variety of secreted factors. In this study, we examined the effects of BMSCs Soup on Staurospurine induced-cell death in MCF-7 and AGS cells. There were three Groups: Group I: no incubation with BM Soup; Group II: incubated with 24 h BM Soup; Group III: incubation with 48 h BM Soup. There were two treatments in each group. The treatments were 1μM Staurospurine (Treatment 1) and 0.0 μM Staurospurine (Treatment 2). The cells were cultured in culture medium containing 0.2 % BSA. We obtained the cell viability, cell death and NO concentration. Our results showed that BM soup administration for 48 hours protectsed against 1μM staurosporine concentration induced cell death and reduced cell toxicity in MCF-7 and AGS cells. Cell viability and cell toxicity assay showed that BM soup in time dependent manner increased cell viability (p < 0.05) and cell death assay showed that cell death in time dependent manner was decreased(p < 0.05). Our data showed that BM soup with increasing NO concentration reduced staurospurine induced cell death and cell cytotoxicity (p < 0.05). It's concluded that BMSCs soup suppressed staurospurine-induced cytotoxicity activity process in MCF-7 and AGS cells (Fig. 9, Ref. 79).

Modelling the balance between quiescence and cell death in normal and tumour cell populations.

PubMed

Spinelli, Lorenzo; Torricelli, Alessandro; Ubezio, Paolo; Basse, Britta

2006-08-01

When considering either human adult tissues (in vivo) or cell cultures (in vitro), cell number is regulated by the relationship between quiescent cells, proliferating cells, cell death and other controls of cell cycle duration. By formulating a mathematical description we see that even small alterations of this relationship may cause a non-growing population to start growing with doubling times characteristic of human tumours. Our model consists of two age structured partial differential equations for the proliferating and quiescent cell compartments. Model parameters are death rates from and transition rates between these compartments. The partial differential equations can be solved for the steady-age distributions, giving the distribution of the cells through the cell cycle, dependent on specific model parameter values. Appropriate formulas can then be derived for various population characteristic quantities such as labelling index, proliferation fraction, doubling time and potential doubling time of the cell population. Such characteristic quantities can be estimated experimentally, although with decreasing precision from in vitro, to in vivo experimental systems and to the clinic. The model can be used to investigate the effects of a single alteration of either quiescence or cell death control on the growth of the whole population and the non-trivial dependence of the doubling time and other observable quantities on particular underlying cell cycle scenarios of death and quiescence. The model indicates that tumour evolution in vivo is a sequence of steady-states, each characterised by particular death and quiescence rate functions. We suggest that a key passage of carcinogenesis is a loss of the communication between quiescence, death and cell cycle machineries, causing a defect in their precise, cell cycle dependent relationship.

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.

Tributyltin induces Yca1p-dependent cell death of yeast Saccharomyces cerevisiae.

PubMed

Chahomchuen, Thippayarat; Akiyama, Koichi; Sekito, Takayuki; Sugimoto, Naoko; Okabe, Masaaki; Nishimoto, Sogo; Sugahara, Takuya; Kakinuma, Yoshimi

2009-10-01

Tributyltin chloride (TBT), an environmental pollutant, is toxic to a variety of eukaryotic and prokaryotic organisms. Although it has been reported that TBT induces apoptotic cell death in mammalian, the action of TBT on eukaryotic microorganisms has not yet been fully investigated. In this study we examined the mechanism involved in cell death caused by TBT exposure in Saccharomyces cerevisiae. The median lethal concentration of TBT was 10 microM for the parent strain BY4741 and 3 microM for the pdr5Delta mutant defective in a major multidrug transporter, respectively. Fluorescence microscopic observations revealed nuclear condensation and chromatin fragmentation in cells treated with TBT indicating that cells underwent an apoptosis-like cell dearth. TBT-induced cell death was suppressed by deletion of the yca1 gene encoding a homologue of the mammalian caspase. In parallel, reactive oxygen species (ROS) were produced by TBT. These results suggest that TBT induces apoptosis-like cell death in yeast via an Yca1p-dependent pathway possibly downstream of the ROS production. This is the first report on TBT-induced apoptotic cell death in yeast.

Polyoma small T antigen triggers cell death via mitotic catastrophe

PubMed Central

Fernando, Arun T Pores; Andrabi, Shaida; Cizmecioglu, Onur; Zhu, Cailei; Livingston, David M.; Higgins, Jonathan M.G; Schaffhausen, Brian S; Roberts, Thomas M

2014-01-01

Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST-expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, resulting in the activation of the Spindle Assembly Checkpoint (SAC). Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed that, PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors. PMID:24998850

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

Mouse embryonic stem cells undergo charontosis, a novel programmed cell death pathway dependent upon cathepsins, p53, and EndoG, in response to etoposide treatment.

PubMed

Tichy, Elisia D; Stephan, Zachary A; Osterburg, Andrew; Noel, Greg; Stambrook, Peter J

2013-05-01

Embryonic stem cells (ESCs) are hypersensitive to many DNA damaging agents and can rapidly undergo cell death or cell differentiation following exposure. Treatment of mouse ESCs (mESCs) with etoposide (ETO), a topoisomerase II poison, followed by a recovery period resulted in massive cell death with characteristics of a programmed cell death pathway (PCD). While cell death was both caspase- and necroptosis-independent, it was partially dependent on the activity of lysosomal proteases. A role for autophagy in the cell death process was eliminated, suggesting that ETO induces a novel PCD pathway in mESCs. Inhibition of p53 either as a transcription factor by pifithrin α or in its mitochondrial role by pifithrin μ significantly reduced ESC death levels. Finally, EndoG was newly identified as a protease participating in the DNA fragmentation observed during ETO-induced PCD. We coined the term charontosis after Charon, the ferryman of the dead in Greek mythology, to refer to the PCD signaling events induced by ETO in mESCs. Copyright © 2013 Elsevier B.V. All rights reserved.

Human islet cells are killed by BID-independent mechanisms in response to FAS ligand.

PubMed

Joglekar, Mugdha V; Trivedi, Prerak M; Kay, Thomas W; Hawthorne, Wayne J; O'Connell, Philip J; Jenkins, Alicia J; Hardikar, Anandwardhan A; Thomas, Helen E

2016-04-01

Cell death via FAS/CD95 can occur either by activation of caspases alone (extrinsic) or by activation of mitochondrial death signalling (intrinsic) depending on the cell type. The BH3-only protein BID is activated in the BCL-2-regulated or mitochondrial apoptosis pathway and acts as a switch between the extrinsic and intrinsic cell death pathways. We have previously demonstrated that islets from BID-deficient mice are protected from FAS ligand-mediated apoptosis in vitro. However, it is not yet known if BID plays a similar role in human beta cell death. We therefore aimed to test the role of BID in human islet cell apoptosis immediately after isolation from human cadaver donors, as well as after de-differentiation in vitro. Freshly isolated human islets or 10-12 day cultured human islet cells exhibited BID transcript knockdown after BID siRNA transfection, however they were not protected from FAS ligand-mediated cell death in vitro as determined by DNA fragmentation analysis using flow cytometry. On the other hand, the same cells transfected with siRNA for FAS-associated via death domain (FADD), a molecule in the extrinsic cell death pathway upstream of BID, showed significant reduction in cell death. De-differentiated islets (human islet-derived progenitor cells) also demonstrated similar results with no difference in cell death after BID knockdown as compared to scramble siRNA transfections. Our results indicate that BID-independent pathways are responsible for FAS-dependent human islet cell death. These results are different from those observed in mouse islets and therefore demonstrate potentially alternate pathways of FAS ligand-induced cell death in human and mouse islet cells.

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

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.

A Disease-associated Mutant of NLRC4 Shows Enhanced Interaction with SUG1 Leading to Constitutive FADD-dependent Caspase-8 Activation and Cell Death.

PubMed

Raghawan, Akhouri Kishore; Sripada, Anand; Gopinath, Gayathri; Pushpanjali, Pendyala; Kumar, Yatender; Radha, Vegesna; Swarup, Ghanshyam

2017-01-27

Nod-like receptor family card containing 4 (NLRC4)/Ipaf is involved in recognition of pathogen-associated molecular patterns leading to caspase-1 activation and cytokine release, which mediate protective innate immune response. Point mutations in NLRC4 cause autoinflammatory syndromes. Although all the mutations result in constitutive caspase-1 activation, their phenotypic presentations are different, implying that these mutations cause different alterations in properties of NLRC4. NLRC4 interacts with SUG1 and induces caspase-8-mediated cell death. Here, we show that one of the autoinflammatory syndrome-causing mutants of NLRC4, H443P, but not T337A and V341A, constitutively activates caspase-8 and induces apoptotic cell death in human lung epithelial cells. Compared with wild type NLRC4, the H443P mutant shows stronger interaction with SUG1 and with ubiquitinated cellular proteins. Phosphorylation of NLRC4 at Ser 533 plays a crucial role in caspase-8 activation and cell death. However, H443P mutant does not require Ser 533 phosphorylation for caspase-8 activation and cell death. Caspase-8 activation by NLRC4 and its H443P mutant are dependent on the adaptor protein FADD. A phosphomimicking mutant of NLRC4, S533D does not require SUG1 activity for inducing cell death. Ubiquitin-tagged NLRC4 could induce cell death and activate caspase-8 independent of Ser 533 phosphorylation. Our work suggests that SUG1-mediated signaling results in enhanced ubiquitination and regulates FADD-dependent caspase-8 activation by NLRC4. We show that the autoinflammation-associated H443P mutant is altered in interaction with SUG1 and ubiquitinated proteins, triggering constitutive caspase-8-mediated cell death dependent on FADD but independent of Ser 533 phosphorylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

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.

ERK1/2 signalling protects against apoptosis following endoplasmic reticulum stress but cannot provide long-term protection against BAX/BAK-independent cell death.

PubMed

Darling, Nicola J; Balmanno, Kathryn; Cook, Simon J

2017-01-01

Disruption of protein folding in the endoplasmic reticulum (ER) causes ER stress. Activation of the unfolded protein response (UPR) acts to restore protein homeostasis or, if ER stress is severe or persistent, drive apoptosis, which is thought to proceed through the cell intrinsic, mitochondrial pathway. Indeed, cells that lack the key executioner proteins BAX and BAK are protected from ER stress-induced apoptosis. Here we show that chronic ER stress causes the progressive inhibition of the extracellular signal-regulated kinase (ERK1/2) signalling pathway. This is causally related to ER stress since reactivation of ERK1/2 can protect cells from ER stress-induced apoptosis whilst ERK1/2 pathway inhibition sensitises cells to ER stress. Furthermore, cancer cell lines harbouring constitutively active BRAFV600E are addicted to ERK1/2 signalling for protection against ER stress-induced cell death. ERK1/2 signalling normally represses the pro-death proteins BIM, BMF and PUMA and it has been proposed that ER stress induces BIM-dependent cell death. We found no evidence that ER stress increased the expression of these proteins; furthermore, BIM was not required for ER stress-induced death. Rather, ER stress caused the PERK-dependent inhibition of cap-dependent mRNA translation and the progressive loss of pro-survival proteins including BCL2, BCLXL and MCL1. Despite these observations, neither ERK1/2 activation nor loss of BAX/BAK could confer long-term clonogenic survival to cells exposed to ER stress. Thus, ER stress induces cell death by at least two biochemically and genetically distinct pathways: a classical BAX/BAK-dependent apoptotic response that can be inhibited by ERK1/2 signalling and an alternative ERK1/2- and BAX/BAK-independent cell death pathway.

24(S)-Hydroxycholesterol induces RIPK1-dependent but MLKL-independent cell death in the absence of caspase-8.

PubMed

Vo, Diep-Khanh Ho; Urano, Yasuomi; Takabe, Wakako; Saito, Yoshiro; Noguchi, Noriko

2015-07-01

24(S)-Hydroxycholesterol (24S-OHC), which is enzymatically produced in the brain, is known to play an important role in maintaining brain cholesterol homeostasis. We have previously reported that 24S-OHC induces a type of non-apoptotic programmed necrosis in neuronal cells expressing little caspase-8. Necroptosis has been characterized as a type of programmed necrosis in which activation of receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) is involved in the signaling pathway. In the present study, we investigated the involvement of these three proteins in 24S-OHC-induced cell death. We found that RIPK1 but neither RIPK3 nor MLKL was expressed in human neuroblastoma SH-SY5Y cells, while all three proteins were expressed in human T lymphoma caspase-8-deficient Jurkat (Jurkat(Cas8-/-)) cells. In Jurkat(Cas8-/-) cells, tumor necrosis factor α (TNFα)-induced cell death was significantly suppressed by treatment with respective inhibitors of RIPK1, RIPK3, and MLKL. In contrast, only RIPK1 inhibitor showed significant suppression of 24S-OHC-induced cell death, and even this was less prominent than was observed in TNFα-induced cell death. In Jurkat(Cas8-/-) cells, knockdown of either RIPK1 or RIPK3 caused moderate but significant suppression of 24S-OHC-induced cell death, but no such effect was observed as a result of knockdown of MLKL. Collectively, these results suggest that, for both SH-SY5Y cells and Jurkat(Cas8-/-) cells, 24S-OHC-induced cell death is dependent on RIPK1 but not on MLKL. We therefore conclude that, in the absence of caspase-8 activity, 24S-OHC induces a necroptosis-like cell death which is RIPK1-dependent but MLKL-independent. Copyright © 2015 Elsevier Inc. All rights reserved.

Catalase and NO CATALASE ACTIVITY1 Promote Autophagy-Dependent Cell Death in Arabidopsis[C][W][OPEN

PubMed Central

Hackenberg, Thomas; Juul, Trine; Auzina, Aija; Gwiżdż, Sonia; Małolepszy, Anna; Van Der Kelen, Katrien; Dam, Svend; Bressendorff, Simon; Lorentzen, Andrea; Roepstorff, Peter; Lehmann Nielsen, Kåre; Jørgensen, Jan-Elo; Hofius, Daniel; Breusegem, Frank Van; Petersen, Morten; Andersen, Stig Uggerhøj

2013-01-01

Programmed cell death often depends on generation of reactive oxygen species, which can be detoxified by antioxidative enzymes, including catalases. We previously isolated catalase-deficient mutants (cat2) in a screen for resistance to hydroxyurea-induced cell death. Here, we identify an Arabidopsis thaliana hydroxyurea-resistant autophagy mutant, atg2, which also shows reduced sensitivity to cell death triggered by the bacterial effector avrRpm1. To test if catalase deficiency likewise affected both hydroxyurea and avrRpm1 sensitivity, we selected mutants with extremely low catalase activities and showed that they carried mutations in a gene that we named NO CATALASE ACTIVITY1 (NCA1). nca1 mutants showed severely reduced activities of all three catalase isoforms in Arabidopsis, and loss of NCA1 function led to strong suppression of RPM1-triggered cell death. Basal and starvation-induced autophagy appeared normal in the nca1 and cat2 mutants. By contrast, autophagic degradation induced by avrRpm1 challenge was compromised, indicating that catalase acted upstream of immunity-triggered autophagy. The direct interaction of catalase with reactive oxygen species could allow catalase to act as a molecular link between reactive oxygen species and the promotion of autophagy-dependent cell death. PMID:24285797

p53-dependent control of cell death by nicastrin: lack of requirement for presenilin-dependent gamma-secretase complex.

PubMed

Pardossi-Piquard, Raphaëlle; Dunys, Julie; Giaime, Emilie; Guillot-Sestier, Marie-Victoire; St George-Hyslop, Peter; Checler, Frédéric; Alves da Costa, Cristine

2009-04-01

Nicastrin (NCT) is a component of the presenilin (PS)-dependent gamma-secretase complexes that liberate amyloid beta-peptides from the beta-Amyloid Precursor Protein. Several lines of evidence indicate that the members of these complexes could also contribute to the control of cell death. Here we show that over-expression of NCT increases the viability of human embryonic kidney (HEK293) cells and decreases staurosporine (STS)- and thapsigargin (TPS)-induced caspase-3 activation in various cell lines from human and neuronal origins by Akt-dependent pathway. NCT lowers p53 expression, transcriptional activity and promoter transactivation and reduces p53 phosphorylation. NCT-associated protection against STS-stimulated cell death was completely abolished by p53 deficiency. Conversely, the depletion of NCT drastically enhances STS-induced caspase-3 activation and p53 pathway and favored p53 nuclear translocation. We examined whether NCT protective function depends on PS-dependent gamma-secretase activity. First, a 29-amino acid deletion known to reduce NCT-dependent amyloid beta-peptide production did not affect NCT-associated protective phenotype. Second, NCT still reduces STS-induced caspase-3 activation in fibroblasts lacking PS1 and PS2. Third, the gamma-secretase inhibitor DFK167 did not affect NCT-mediated reduction of p53 activity. Altogether, our study indicates that NCT controls cell death via phosphoinositide 3-kinase/Akt and p53-dependent pathways and that this function remains independent of the activity and molecular integrity of the gamma-secretase complexes.

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

Loss of anchorage primarily induces non-apoptotic cell death in a human mammary epithelial cell line under atypical focal adhesion kinase signaling.

PubMed

Ishikawa, F; Ushida, K; Mori, K; Shibanuma, M

2015-01-22

Anchorage dependence of cellular growth and survival prevents inappropriate cell growth or survival in ectopic environments, and serves as a potential barrier to metastasis of cancer cells. Therefore, obtaining a better understanding of anchorage-dependent responses in normal cells is the first step to understand and impede anchorage independence of growth and survival in cancer cells and finally to eradicate cancer cells during metastasis. Anoikis, a type of apoptosis specifically induced by lack of appropriate cell-extracellular matrix adhesion, has been established as the dominant response of normal epithelial cells to anchorage loss. For example, under detached conditions, the untransformed mammary epithelial cell (MEC) line MCF-10 A, which exhibits myoepithelial characteristics, underwent anoikis dependent on classical ERK signaling. On the other hand, recent studies have revealed a variety of phenotypes resulting in cell death modalities distinct from anoikis, such as autophagy, necrosis, and cornification, in detached epithelial cells. In the present study, we characterized detachment-induced cell death (DICD) in primary human MECs immortalized with hTERT ((Tert)HMECs), which are bipotent progenitor-like cells with a differentiating phenotype to luminal cells. In contrast to MCF-10 A cells, apoptosis was not observed in detached (Tert)HMECs; instead, non-apoptotic cell death marked by features of entosis, cornification, and necrosis was observed along with downregulation of focal adhesion kinase (FAK) signaling. Cell death was overcome by anchorage-independent activities of FAK but not PI3K/AKT, SRC, and MEK/ERK, suggesting critical roles of atypical FAK signaling pathways in the regulation of non-apoptotic cell death. Further analysis revealed an important role of TRAIL (tumor necrosis factor (TNF)-related apoptosis-inducing ligand) as a mediator of FAK signaling in regulation of entosis and necrosis and a role of p38 MAPK in the induction of necrosis. Overall, the present study highlighted outstanding cell subtype or differentiation stage specificity in cell death phenotypes induced upon anchorage loss in human MECs.

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

Regulatory mechanism of the flavoprotein Tah18-dependent nitric oxide synthesis and cell death in yeast.

PubMed

Yoshikawa, Yuki; Nasuno, Ryo; Kawahara, Nobuhiro; Nishimura, Akira; Watanabe, Daisuke; Takagi, Hiroshi

2016-07-01

Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. The regulatory mechanism of NO generation in unicellular eukaryotic yeast cells is poorly understood due to the lack of mammalian and bacterial NO synthase (NOS) orthologues, even though yeast produces NO under oxidative stress conditions. Recently, we reported that the flavoprotein Tah18, which was previously shown to transfer electrons to the iron-sulfur cluster protein Dre2, is involved in NOS-like activity in the yeast Saccharomyces cerevisiae. On the other hand, Tah18 was reported to promote apoptotic cell death after exposure to hydrogen peroxide (H2O2). Here, we showed that NOS-like activity requiring Tah18 induced cell death upon treatment with H2O2. Our experimental results also indicate that Tah18-dependent NO production and cell death are suppressed by enhancement of the interaction between Tah18 and its molecular partner Dre2. Our findings indicate that the Tah18-Dre2 complex regulates cell death as a molecular switch via Tah18-dependent NOS-like activity in response to environmental changes. Copyright © 2016 Elsevier Inc. All rights reserved.

Fight or Flight - Regulation of Emergency Hematopoiesis by Pyroptosis and Necroptosis

PubMed Central

Croker, Ben A.; Silke, John; Gerlic, Motti

2015-01-01

Purpose of review A feature of the innate immune response that is conserved across kingdoms is the induction of cell death. In this review, we discuss the direct and indirect effects of increased inflammatory cell death, including pyroptosis, a caspase-1-dependent cell death, and necroptosis, a RIPK3/MLKL-dependent, caspase-independent cell death, on emergency hematopoiesis. Recent findings Activation of non-apoptotic cell death pathways during infection can trigger release of cytokines and/or damage-associated molecular patterns (DAMPs) such as IL-1α, IL-1β, IL-18, IL-33, HMGB1 and mtDNA to promote emergency hematopoiesis. During systemic infection, pyroptosis and necroptosis can directly kill hematopoietic stem and progenitor cells, which results in impaired hematopoiesis, cytopenia and immunosuppression. Although originally described as discrete entities, there now appears to be more intimate connections between the non-apoptotic and death receptor signaling pathways. Summary The choice to undergo pyroptotic and necroptotic cell death constitutes a rapid response system serving to eliminate infected cells, including hematopoietic stem and progenitor cells. This system has the potential to be detrimental to emergency hematopoiesis during severe infection. We discuss the potential of pharmacological intervention for the pyroptosis and necroptosis pathways that may be beneficial during periods of infection and emergency hematopoiesis. PMID:26049749

Sex-specific activation of cell death signalling pathways in cerebellar granule neurons exposed to oxygen glucose deprivation followed by reoxygenation

PubMed Central

Sharma, Jaswinder; Nelluru, Geetha; Ann Wilson, Mary; Johnston, Michael V; Ahamed Hossain, Mir

2011-01-01

Neuronal death pathways following hypoxia–ischaemia are sexually dimorphic, but the underlying mechanisms are unclear. We examined cell death mechanisms during OGD (oxygen-glucose deprivation) followed by Reox (reoxygenation) in segregated male (XY) and female (XX) mouse primary CGNs (cerebellar granule neurons) that are WT (wild-type) or Parp-1 [poly(ADP-ribose) polymerase 1] KO (knockout). Exposure of CGNs to OGD (1.5 h)/Reox (7 h) caused cell death in XY and XX neurons, but cell death during Reox was greater in XX neurons. ATP levels were significantly lower after OGD/Reox in WT-XX neurons than in XY neurons; this difference was eliminated in Parp-1 KO-XX neurons. AIF (apoptosis-inducing factor) was released from mitochondria and translocated to the nucleus by 1 h exclusively in WT-XY neurons. In contrast, there was a release of Cyt C (cytochrome C) from mitochondria in WT-XX and Parp-1 KO neurons of both sexes; delayed activation of caspase 3 was observed in the same three groups. Thus deletion of Parp-1 shunted cell death towards caspase 3-dependent apoptosis. Delayed activation of caspase 8 was also observed in all groups after OGD/Reox, but was much greater in XX neurons, and caspase 8 translocated to the nucleus in XX neurons only. Caspase 8 activation may contribute to increased XX neuronal death during Reox, via caspase 3 activation. Thus, OGD/Reox induces death of XY neurons via a PARP-1-AIF-dependent mechanism, but blockade of PARP-1-AIF pathway shifts neuronal death towards a caspase-dependent mechanism. In XX neurons, OGD/Reox caused prolonged depletion of ATP and delayed activation of caspase 8 and caspase 3, culminating in greater cell death during Reox. PMID:21382016

Simvastatin induces caspase-independent apoptosis in LPS-activated RAW264.7 macrophage cells

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

Kim, Yong Chan; Song, Seok Bean; Lee, Mi Hee

Macrophages participate in several inflammatory pathologies such as sepsis and arthritis. We examined the effect of simvastatin on the LPS-induced proinflammatory macrophage RAW264.7 cells. Co-treatment of LPS and a non-toxic dose of simvastatin induced cell death in RAW264.7 cells. The cell death was accompanied by disruption of mitochondrial membrane potential (MMP), genomic DNA fragmentation, and caspase-3 activation. Surprisingly, despite caspase-dependent apoptotic cascade being completely blocked by Z-VAD-fmk, a pan-caspase inhibitor, the cell death was only partially repressed. In the presence of Z-VAD-fmk, DNA fragmentation was blocked, but DNA condensation, disruption of MMP, and nuclear translocation of apoptosis inducing factor weremore » obvious. The cell death by simvastatin and LPS was effectively decreased by both the FPP and GGPP treatments as well as mevalonate. Our findings indicate that simvastatin triggers the cell death of LPS-treated RAW264.7 cells through both caspase-dependent and -independent apoptotic pathways, suggesting a novel mechanism of statins for the severe inflammatory disease therapy.« less

GDC-0941 enhances the lysosomal compartment via TFEB and primes glioblastoma cells to lysosomal membrane permeabilization and cell death.

PubMed

Enzenmüller, Stefanie; Gonzalez, Patrick; Karpel-Massler, Georg; Debatin, Klaus-Michael; Fulda, Simone

2013-02-01

Since phosphatidylinositol-3-kinase (PI3K) inhibitors are primarily cytostatic against glioblastoma, we searched for new drug combinations. Here, we discover that the PI3K inhibitor GDC-0941 acts in concert with the natural compound B10, a glycosylated derivative of betulinic acid, to induce cell death in glioblastoma cells. Importantly, parallel experiments in primary glioblastoma cultures similarly show that GDC-0941 and B10 cooperate to trigger cell death, underscoring the clinical relevance of this finding. Molecular studies revealed that treatment with GDC-0941 stimulates the expression and nuclear translocation of Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis, the lysosomal membrane marker LAMP-1 and the mature form of cathepsin B. Also, GDC-0941 triggers a time-dependent increase of the lysosomal compartment in a TFEB-dependent manner, since knockdown of TFEB significantly reduces this GDC-0941-stimulated lysosomal enhancement. Importantly, GDC-0941 cooperates with B10 to trigger lysosomal membrane permeabilization, leading to increased activation of Bax, loss of mitochondrial membrane potential (MMP), caspase-3 activation and cell death. Addition of the cathepsin B inhibitor CA-074me reduces Bax activation, loss of MMP, caspase-3 activation and cell death upon treatment with GDC-0941/B10. By comparison, knockdown of caspase-3 or the broad-range caspase inhibitor zVAD.fmk inhibits GDC-0941/B10-induced DNA fragmentation, but does not prevent cell death, thus pointing to both caspase-dependent and -independent pathways. By identifying the combination of GDC-0941 and B10 as a new, potent strategy to trigger cell death in glioblastoma cells, our findings have important implications for the development of novel treatment approaches for glioblastoma. Copyright © 2012. Published by Elsevier Ireland Ltd.

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

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.

Oxidant-Induced Cell Death and Nrf2-Dependent Antioxidative Response Are Controlled by Fra-1/AP-1

PubMed Central

Vaz, Michelle; Machireddy, Narsa; Irving, Ashley; Potteti, Haranatha R.; Chevalier, Karinne; Kalvakolanu, Dhananjaya

2012-01-01

AP-1 (Jun/Fos) transcription factors play key roles in various biological processes, including cell death. Here we report a novel role for Fra-1 in oxidant-induced cell death controlled by modulating antioxidant gene expression. Fra-1-deficient (Fra-1Δ/Δ) mouse embryonic fibroblasts (MEFs) and primary lung fibroblasts (PLFs) were remarkably resistant to H2O2- and diquat-induced cell death, compared to their wild-type (Fra-1+/+) counterparts. Fra-1 deficiency ablated oxidant-induced mitochondrion-dependent apoptosis. Fra-1Δ/Δ cells had elevated basal levels of antioxidant enzymes and intracellular glutathione (GSH), which were further stimulated by oxidants. Loss of Fra-1 led to an increased half-life of transcription factor Nrf2 and increased recruitment of this protein to the promoters of antioxidant genes and increased their expression. Depletion of intracellular GSH or RNA interference (RNAi)-mediated knockdown of Nqo1, Hmox1, and Nrf2 restored oxidant-induced cell death in Fra-1Δ/Δ cells. Thus, Fra-1 appears to increase susceptibility to oxidants and promotes cell death by attenuating Nrf2-driven antioxidant responses. PMID:22393254

Anthocyanins extracted from black soybean seed coat protect primary cortical neurons against in vitro ischemia.

PubMed

Bhuiyan, Mohammad Iqbal Hossain; Kim, Joo Youn; Ha, Tae Joung; Kim, Seong Yun; Cho, Kyung-Ok

2012-01-01

The present study investigated the neuroprotective effects of anthocyanins extracted from black soybean (cv. Cheongja 3, Glycine max (L.) MERR.) seed coat against oxygen-glucose deprivation (OGD) and glutamate-induced cell death in rat primary cortical neurons. Lactate dehydrogenase (LDH) release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assays were employed to assess cell membrane damage and viability of primary neurons, respectively. OGD-induced cell death in 7 d in vitro primary cortical neurons was found to be OGD duration-dependent, and approximately 3.5 h of OGD resulted in ≈60% cell death. Treatment with black soybean anthocyanins dose-dependently prevented membrane damage and increased the viability of primary neurons that were exposed to OGD. Glutamate-induced neuronal cell death was dependent on the glutamate concentration at relatively low concentrations and the number of days the cells remained in culture. Interestingly, black soybean anthocyanins did not protect against glutamate-induced neuronal cell death. They did, however, inhibit the excessive generation of reactive oxygen species (ROS) and preserve mitochondrial membrane potential (MMP) in primary neurons exposed to OGD. In agreement with the neuroprotective effect of crude black soybean anthocyanins, purified cyanidin-3-glucoside (C3G), the major component of anthocyanins, also offered dose-dependent neuroprotection against OGD-induced neuronal cell death. Moreover, black soybean C3G markedly prevented excessive generation of ROS and preserved MMP in primary neurons that were exposed to OGD. Collectively, these results suggest that the neuroprotection of primary rat cortical neurons by anthocyanins that were extracted from black soybean seed coat might be mediated through oxidative stress inhibition and MMP preservation but not through glutamate-induced excitotoxicity attenuation.

Deferasirox-induced iron depletion promotes BclxL downregulation and death of proximal tubular cells

PubMed Central

Martin-Sanchez, Diego; Gallegos-Villalobos, Angel; Fontecha-Barriuso, Miguel; Carrasco, Susana; Sanchez-Niño, Maria Dolores; Lopez-Hernandez, Francisco J; Ruiz-Ortega, Marta; Egido, Jesus; Ortiz, Alberto; Sanz, Ana Belén

2017-01-01

Iron deficiency has been associated with kidney injury. Deferasirox is an oral iron chelator used to treat blood transfusion-related iron overload. Nephrotoxicity is the most serious and common adverse effect of deferasirox and may present as an acute or chronic kidney disease. However, scarce data are available on the molecular mechanisms of nephrotoxicity. We explored the therapeutic modulation of deferasirox-induced proximal tubular cell death in culture. Deferasirox induced dose-dependent tubular cell death and AnexxinV/7AAD staining showed features of apoptosis and necrosis. However, despite inhibiting caspase-3 activation, the pan-caspase inhibitor zVAD-fmk failed to prevent deferasirox-induced cell death. Moreover, zVAD increased deferasirox-induced cell death, a feature sometimes found in necroptosis. Electron microscopy identified mitochondrial injury and features of necrosis. However, neither necrostatin-1 nor RIP3 knockdown prevented deferasirox-induced cell death. Deferasirox caused BclxL depletion and BclxL overexpression was protective. Preventing iron depletion protected from BclxL downregulation and deferasirox cytotoxicity. In conclusion, deferasirox promoted iron depletion-dependent cell death characterized by BclxL downregulation. BclxL overexpression was protective, suggesting a role for BclxL downregulation in iron depletion-induced cell death. This information may be used to develop novel nephroprotective strategies. Furthermore, it supports the concept that monitoring kidney tissue iron depletion may decrease the risk of deferasirox nephrotoxicity. PMID:28139717

Deferasirox-induced iron depletion promotes BclxL downregulation and death of proximal tubular cells.

PubMed

Martin-Sanchez, Diego; Gallegos-Villalobos, Angel; Fontecha-Barriuso, Miguel; Carrasco, Susana; Sanchez-Niño, Maria Dolores; Lopez-Hernandez, Francisco J; Ruiz-Ortega, Marta; Egido, Jesus; Ortiz, Alberto; Sanz, Ana Belén

2017-01-31

Iron deficiency has been associated with kidney injury. Deferasirox is an oral iron chelator used to treat blood transfusion-related iron overload. Nephrotoxicity is the most serious and common adverse effect of deferasirox and may present as an acute or chronic kidney disease. However, scarce data are available on the molecular mechanisms of nephrotoxicity. We explored the therapeutic modulation of deferasirox-induced proximal tubular cell death in culture. Deferasirox induced dose-dependent tubular cell death and AnexxinV/7AAD staining showed features of apoptosis and necrosis. However, despite inhibiting caspase-3 activation, the pan-caspase inhibitor zVAD-fmk failed to prevent deferasirox-induced cell death. Moreover, zVAD increased deferasirox-induced cell death, a feature sometimes found in necroptosis. Electron microscopy identified mitochondrial injury and features of necrosis. However, neither necrostatin-1 nor RIP3 knockdown prevented deferasirox-induced cell death. Deferasirox caused BclxL depletion and BclxL overexpression was protective. Preventing iron depletion protected from BclxL downregulation and deferasirox cytotoxicity. In conclusion, deferasirox promoted iron depletion-dependent cell death characterized by BclxL downregulation. BclxL overexpression was protective, suggesting a role for BclxL downregulation in iron depletion-induced cell death. This information may be used to develop novel nephroprotective strategies. Furthermore, it supports the concept that monitoring kidney tissue iron depletion may decrease the risk of deferasirox nephrotoxicity.

Autophagy Protects Against Aminochrome-Induced Cell Death in Substantia Nigra-Derived Cell Line

PubMed Central

Paris, Irmgard; Muñoz, Patricia; Huenchuguala, Sandro; Couve, Eduardo; Sanders, Laurie H.; Greenamyre, John Timothy; Caviedes, Pablo; Segura-Aguilar, Juan

2011-01-01

Aminochrome, the precursor of neuromelanin, has been proposed to be involved in the neurodegeneration neuromelanin-containing dopaminergic neurons in Parkinson’s disease. We aimed to study the mechanism of aminochrome-dependent cell death in a cell line derived from rat substantia nigra. We found that aminochrome (50μM), in the presence of NAD(P)H-quinone oxidoreductase, EC 1.6.99.2 (DT)-diaphorase inhibitor dicoumarol (DIC) (100μM), induces significant cell death (62 ± 3%; p < 0.01), increase in caspase-3 activation (p < 0.001), release of cytochrome C, disruption of mitochondrial membrane potential (p < 0.01), damage of mitochondrial DNA, damage of mitochondria determined with transmission electron microscopy, a dramatic morphological change characterized as cell shrinkage, and significant increase in number of autophagic vacuoles. To determine the role of autophagy on aminochrome-induced cell death, we incubated the cells in the presence of vinblastine and rapamycin. Interestingly, 10μM vinblastine induces a 5.9-fold (p < 0.001) and twofold (p < 0.01) significant increase in cell death when the cells were incubated with 30μM aminochrome in the absence and presence of DIC, respectively, whereas 10μM rapamycin preincubated 24 h before addition of 50μM aminochrome in the absence and the presence of 100μM DIC induces a significant decrease (p < 0.001) in cell death. In conclusion, autophagy seems to be an important protective mechanism against two different aminochrome-induced cell deaths that initially showed apoptotic features. The cell death induced by aminochrome when DT-diaphorase is inhibited requires activation of mitochondrial pathway, whereas the cell death induced by aminochrome alone requires inhibition of autophagy-dependent degrading of damaged organelles and recycling through lysosomes. PMID:21427056

BID links ferroptosis to mitochondrial cell death pathways.

PubMed

Neitemeier, Sandra; Jelinek, Anja; Laino, Vincenzo; Hoffmann, Lena; Eisenbach, Ina; Eying, Roman; Ganjam, Goutham K; Dolga, Amalia M; Oppermann, Sina; Culmsee, Carsten

2017-08-01

Ferroptosis has been defined as an oxidative and iron-dependent pathway of regulated cell death that is distinct from caspase-dependent apoptosis and established pathways of death receptor-mediated regulated necrosis. While emerging evidence linked features of ferroptosis induced e.g. by erastin-mediated inhibition of the X c - system or inhibition of glutathione peroxidase 4 (Gpx4) to an increasing number of oxidative cell death paradigms in cancer cells, neurons or kidney cells, the biochemical pathways of oxidative cell death remained largely unclear. In particular, the role of mitochondrial damage in paradigms of ferroptosis needs further investigation. In the present study, we find that erastin-induced ferroptosis in neuronal cells was accompanied by BID transactivation to mitochondria, loss of mitochondrial membrane potential, enhanced mitochondrial fragmentation and reduced ATP levels. These hallmarks of mitochondrial demise are also established features of oxytosis, a paradigm of cell death induced by X c - inhibition by millimolar concentrations of glutamate. Bid knockout using CRISPR/Cas9 approaches preserved mitochondrial integrity and function, and mediated neuroprotective effects against both, ferroptosis and oxytosis. Furthermore, the BID-inhibitor BI-6c9 inhibited erastin-induced ferroptosis, and, in turn, the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 prevented mitochondrial dysfunction and cell death in the paradigm of oxytosis. These findings show that mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the final execution step in this paradigm of oxidative cell death. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Cell Death Pathways and Phthalocyanine as an Efficient Agent for Photodynamic Cancer Therapy

PubMed Central

Mfouo-Tynga, Ivan; Abrahamse, Heidi

2015-01-01

The mechanisms of cell death can be predetermined (programmed) or not and categorized into apoptotic, autophagic and necrotic pathways. The process of Hayflick limits completes the execution of death-related mechanisms. Reactive oxygen species (ROS) are associated with oxidative stress and subsequent cytodamage by oxidizing and degrading cell components. ROS are also involved in immune responses, where they stabilize and activate both hypoxia-inducible factors and phagocytic effectors. ROS production and presence enhance cytodamage and photodynamic-induced cell death. Photodynamic cancer therapy (PDT) uses non-toxic chemotherapeutic agents, photosensitizer (PS), to initiate a light-dependent and ROS-related cell death. Phthalocyanines (PCs) are third generation and stable PSs with improved photochemical abilities. They are effective inducers of cell death in various neoplastic models. The metallated PCs localize in critical cellular organelles and are better inducers of cell death than other previous generation PSs as they favor mainly apoptotic cell death events. PMID:25955645

Cultured networks of excitatory projection neurons and inhibitory interneurons for studying human cortical neurotoxicity

PubMed Central

Xu, Jin-Chong; Fan, Jing; Wang, Xueqing; Eacker, Stephen M.; Kam, Tae-In; Chen, Li; Yin, Xiling; Zhu, Juehua; Chi, Zhikai; Jiang, Haisong; Chen, Rong; Dawson, Ted M.; Dawson, Valina L.

2017-01-01

Translating neuroprotective treatments from discovery in cell and animal models to the clinic has proven challenging. To reduce the gap between basic studies of neurotoxicity and neuroprotection and clinically relevant therapies, we developed a human cortical neuron culture system from human embryonic stem cells (ESCs) or inducible pluripotent stem cells (iPSCs) that generated both excitatory and inhibitory neuronal networks resembling the composition of the human cortex. This methodology used timed administration of retinoic acid (RA) to FOXG1 neural precursor cells leading to differentiation of neuronal populations representative of the six cortical layers with both excitatory and inhibitory neuronal networks that were functional and homeostatically stable. In human cortical neuron cultures, excitotoxicity or ischemia due to oxygen and glucose deprivation led to cell death that was dependent on N-methyl-D-aspartate (NMDA) receptors, nitric oxide (NO), and the poly (ADP-ribose) polymerase (PARP)-dependent cell death, a cell death pathway designated parthanatos to separate it from apoptosis, necroptosis and other forms of cell death. Neuronal cell death was attenuated by PARP inhibitors that are currently in clinical trials for cancer treatment. This culture system provides a new platform for the study of human cortical neurotoxicity and suggests that PARP inhibitors may be useful for ameliorating excitotoxic and ischemic cell death in human neurons. PMID:27053772

Novel Derivative of Benzofuran Induces Cell Death Mostly by G2/M Cell Cycle Arrest through p53-dependent Pathway but Partially by Inhibition of NF-κB*

PubMed Central

Manna, Sunil K.; Bose, Julie S.; Gangan, Vijay; Raviprakash, Nune; Navaneetha, Thota; Raghavendra, Pongali B.; Babajan, Banaganapalli; Kumar, Chitta S.; Jain, Swatantra K.

2010-01-01

The Dracaena resin is widely used in traditional medicine as an anticancer agent, and benzofuran lignan is the active component. In this report, we provide evidence that the synthetic derivative of benzofuran lignan (Benfur) showed antitumor activities. It induced apoptosis in p53-positive cells. Though it inhibited endotoxin-induced nuclear factor κB (NF-κB) activation in both p53-positive and -negative cells, the activation of caspase 3 was observed in p53-positive cells. It showed partial cell death effect in both p53-positive and -negative cells through inhibition of NF-κB. Cell cycle analysis using flow cytometry showed that treatment with this novel benozofuran lignan derivative to Jurkat T-cells, but not U-937 cells, resulted in a G2/M arrest in a dose- and time-dependent manner. It increased amounts of p21, p27, and cyclin B, but not phospho-Rb through p53 nuclear translocation in Jurkat T-cells, but not in U-937 cells. It inhibited amounts of MDM2 (murine double minute 2) by repressing the transcription factor Sp1, which was also proved in silico. It induced cell death in tumor cells, but not in primary T-cells. Overall, our data suggest that Benfur-mediated cell death is partially dependent upon NF-κB, but predominantly dependent on p53. Thus, this novel benzofuran lignan derivative can be effective chemopreventive or chemotherapeutic agent against malignant T-cells. PMID:20472557

Calcium-dependent nitric oxide production is involved in the cytoprotective properties of n-acetylcysteine in glycochenodeoxycholic acid-induced cell death in hepatocytes

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

Gonzalez-Rubio, Sandra; Linares, Clara I.; Bello, Rosario I.

The intracellular oxidative stress has been involved in bile acid-induced cell death in hepatocytes. Nitric oxide (NO) exerts cytoprotective properties in glycochenodeoxycholic acid (GCDCA)-treated hepatocytes. The study evaluated the involvement of Ca{sup 2+} on the regulation of NO synthase (NOS)-3 expression during N-acetylcysteine (NAC) cytoprotection against GCDCA-induced cell death in hepatocytes. The regulation of Ca{sup 2+} pools (EGTA or BAPTA-AM) and NO (L-NAME or NO donor) production was assessed during NAC cytoprotection in GCDCA-treated HepG2 cells. The stimulation of Ca{sup 2+} entrance was induced by A23187 in HepG2. Cell death, Ca{sup 2+} mobilization, NOS-1, -2 and -3 expression, AP-1 activation,more » and NO production were evaluated. GCDCA reduced intracellular Ca{sup 2+} concentration and NOS-3 expression, and enhanced cell death in HepG2. NO donor prevented, and L-NAME enhanced, GCDCA-induced cell death. The reduction of Ca{sup 2+} entry by EGTA, but not its release from intracellular stores by BAPTA-AM, enhanced cell death in GCDCA-treated cells. The stimulation of Ca{sup 2+} entrance by A23187 reduced cell death and enhanced NOS-3 expression in GCDCA-treated HepG2 cells. The cytoprotective properties of NAC were related to the recovery of intracellular Ca{sup 2+} concentration, NOS-3 expression and NO production induced by GCDCA-treated HepG2 cells. The increase of NO production by Ca{sup 2+}-dependent NOS-3 expression during NAC administration reduces cell death in GCDCA-treated hepatocytes.« less

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

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

The TIR domain of TIR-NB-LRR resistance proteins is a signaling domain involved in cell death induction.

PubMed

Swiderski, Michal R; Birker, Doris; Jones, Jonathan D G

2009-02-01

In plants, the TIR (toll interleukin 1 receptor) domain is found almost exclusively in nucleotide-binding (NB) leucine-rich repeat resistance proteins and their truncated homologs, and has been proposed to play a signaling role during resistance responses mediated by TIR containing R proteins. Transient expression in Nicotiana benthamiana leaves of "TIR + 80", the RPS4 truncation without the NB-ARC domain, leads to EDS1-, SGT1-, and HSP90-dependent cell death. Transgenic Arabidopsis plants expressing the RPS4 TIR+80 from either dexamethasone or estradiol-inducible promoters display inducer-dependent cell death. Cell death is also elicited by transient expression of similarly truncated constructs from two other R proteins, RPP1A and At4g19530, but is not elicited by similar constructs representing RPP2A and RPP2B proteins. Site-directed mutagenesis of the RPS4 TIR domain identified many loss-of-function mutations but also revealed several gain-of function substitutions. Lack of cell death induction by the E160A substitution suggests that amino acids outside of the TIR domain contribute to cell death signaling in addition to the TIR domain itself. This is consistent with previous observations that the TIR domain itself is insufficient to induce cell death upon transient expression.

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.

15-Deoxy-{delta}{sup 12,14}-prostaglandin J{sub 2} induces renal epithelial cell death through NF-{kappa}B-dependent and MAPK-independent mechanism

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

Kang, Dae Sik; Kwon, Chae Hwa; Park, Ji Yeon

2006-11-01

The peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) ligand 15d-PGJ{sub 2} induces cell death in renal proximal tubular cells. However, the underlying molecular mechanism(s) remains unidentified. The present study was undertaken to examine the roles of reactive oxygen species (ROS), mitogen-activated protein kinase, and NF-{kappa}B in opossum kidney (OK) cell death induced by 15d-PGJ{sub 2}. Treatment of OK cells with 15d-PGJ{sub 2} resulted in a concentration- and time-dependent cell death, which was largely attributed to apoptosis. 15d-PGJ{sub 2} increased ROS production and the effect was inhibited by catalase and N-acetylcysteine. The 15d-PGJ{sub 2}-induced cell death was also prevented by these antioxidants, suggesting thatmore » the cell death was associated with ROS generation. The PPAR{gamma} antagonist GW9662 did not prevent the 15d-PGJ{sub 2}-induced cell death. 15d-PGJ{sub 2} caused a transient activation of extracellular signal-regulated kinase (ERK). However, inhibitors (PD98059 and U0126) of MEK, an ERK upstream kinase, did not alter the 15d-PGJ{sub 2}-induced cell death. Transfection with constitutively active MEK and dominant-negative MEK had no effect on the cell death. 15d-PGJ{sub 2} inhibited the NF-{kappa}B transcriptional activity, which was accompanied by an inhibition of nuclear translocation of the NF-{kappa}B subunit p65 and impairment in DNA binding. Inhibition of NF-{kappa}B with a NF-{kappa}B specific inhibitor pyrrolidinecarbodithioate and transfection with I{kappa}B{alpha} (S32A/36A) caused cell death. These results suggest that the 5d-PGJ{sub 2}-induced OK cell death was associated with ROS production and NF-{kappa}B inhibition, but not with MAPK activation.« less

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.

TOR-mediated autophagy regulates cell death in Drosophila neurodegenerative disease.

PubMed

Wang, Tao; Lao, Uyen; Edgar, Bruce A

2009-09-07

Target of rapamycin (TOR) signaling is a regulator of cell growth. TOR activity can also enhance cell death, and the TOR inhibitor rapamycin protects cells against proapoptotic stimuli. Autophagy, which can protect against cell death, is negatively regulated by TOR, and disruption of autophagy by mutation of Atg5 or Atg7 can lead to neurodegeneration. However, the implied functional connection between TOR signaling, autophagy, and cell death or degeneration has not been rigorously tested. Using the Drosophila melanogaster visual system, we show in this study that hyperactivation of TOR leads to photoreceptor cell death in an age- and light-dependent manner and that this is because of TOR's ability to suppress autophagy. We also find that genetically inhibiting TOR or inducing autophagy suppresses cell death in Drosophila models of Huntington's disease and phospholipase C (norpA)-mediated retinal degeneration. Thus, our data indicate that TOR induces cell death by suppressing autophagy and provide direct genetic evidence that autophagy alleviates cell death in several common types of neurodegenerative disease.

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.

Mitochondrial fission proteins regulate programmed cell death in yeast.

PubMed

Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J; Qi, Bing; Pevsner, Jonathan; McCaffery, J Michael; Hill, R Blake; Basañez, Gorka; Hardwick, J Marie

2004-11-15

The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we found that the Saccharomyces cerevisiae homolog of human Drp1, Dnm1, promotes mitochondrial fragmentation/degradation and cell death following treatment with several death stimuli. Two Dnm1-interacting factors also regulate yeast cell death. The WD40 repeat protein Mdv1/Net2 promotes cell death, consistent with its role in mitochondrial fission. In contrast to its fission function in healthy cells, Fis1 unexpectedly inhibits Dnm1-mediated mitochondrial fission and cysteine protease-dependent cell death in yeast. Furthermore, the ability of yeast Fis1 to inhibit mitochondrial fission and cell death can be functionally replaced by human Bcl-2 and Bcl-xL. Together, these findings indicate that yeast and mammalian cells have a conserved programmed death pathway regulated by a common molecular component, Drp1/Dnm1, that is inhibited by a Bcl-2-like function.

Neuronal Death After Hemorrhagic Stroke In Vitro and In Vivo Shares Features of Ferroptosis and Necroptosis

PubMed Central

Zille, Marietta; Karuppagounder, Saravanan S.; Chen, Yingxin; Gough, Peter J.; Phil, D.; Bertin, John; Finger, Joshua; Milner, Teresa A.; Jonas, Elizabeth A.; Ratan, Rajiv R.

2017-01-01

Background and Purpose Intracerebral hemorrhage (ICH) leads to disability or death with few established treatments. Adverse outcomes following ICH result from irreversible damage to neurons resulting from primary and secondary injury. Secondary injury has been attributed to hemoglobin and its oxidized product hemin from lysed red blood cells. The aim of this study was to identify the underlying cell death mechanisms attributable to secondary injury by hemoglobin and hemin to broaden treatment options. Methods We investigated cell death mechanisms in cultured neurons exposed to hemoglobin or hemin. Chemical inhibitors implicated in all known cell death pathways were employed. Identified cell death mechanisms were confirmed using molecular markers and electron microscopy. Results Chemical inhibitors of ferroptosis and necroptosis protected against hemoglobin- and hemin-induced toxicity. By contrast, inhibitors of caspase-dependent apoptosis, protein or mRNA synthesis, autophagy, mitophagy or parthanatos had no effect. Accordingly, molecular markers of ferroptosis and necroptosis were increased following ICH in vitro and in vivo. Electron microscopy showed that hemin induced a necrotic phenotype. Necroptosis and ferroptosis inhibitors each abrogated death by greater than 80% and had similar therapeutic windows in vitro. Conclusion Experimental ICH shares features of ferroptotic and necroptotic cell death, but not caspase-dependent apoptosis or autophagy. We propose that ferroptosis or necroptotic signaling induced by lysed blood is sufficient to reach a threshold of death that leads to neuronal necrosis and that inhibition of either one of these pathways can bring cells below that threshold to survival. PMID:28250197

Bcl-2 proteins and autophagy regulate mitochondrial dynamics during programmed cell death in the Drosophila ovary.

PubMed

Tanner, Elizabeth A; Blute, Todd A; Brachmann, Carrie Baker; McCall, Kimberly

2011-01-01

The Bcl-2 family has been shown to regulate mitochondrial dynamics during cell death in mammals and C. elegans, but evidence for this in Drosophila has been elusive. Here, we investigate the regulation of mitochondrial dynamics during germline cell death in the Drosophila melanogaster ovary. We find that mitochondria undergo a series of events during the progression of cell death, with remodeling, cluster formation and uptake of clusters by somatic follicle cells. These mitochondrial dynamics are dependent on caspases, the Bcl-2 family, the mitochondrial fission and fusion machinery, and the autophagy machinery. Furthermore, Bcl-2 family mutants show a striking defect in cell death in the ovary. These data indicate that a mitochondrial pathway is a major mechanism for activation of cell death in Drosophila oogenesis.

Procaspase-activating compound 1 induces a caspase-3-dependent cell death in cerebellar granule neurons

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

Aziz, Gulzeb; Akselsen, Oyvind W.; Hansen, Trond V.

2010-09-15

Procaspase-activating compound 1, PAC-1, has been introduced as a direct activator of procaspase-3 and has been suggested as a therapeutic agent against cancer. Its activation of procaspase-3 is dependent on the chelation of zinc. We have tested PAC-1 and an analogue of PAC-1 as zinc chelators in vitro as well as their ability to activate caspase-3 and induce cell death in chicken cerebellar granule neuron cultures. These neurons are non-dividing, primary cells with normal caspase-3. The results reported herein show that PAC-1 chelates zinc, activates procaspase-3, and leads to caspase-3-dependent cell death in neurons, as the specific caspase-3-inhibitor Ac-DEVD-cmk inhibitedmore » both the caspase-3 activity and cell death. Thus, chicken cerebellar granule neurons is a suitable model to study mechanisms of interference with apoptosis of PAC-1 and similar compounds. Furthermore, the present study also raises concern about potential neurotoxicity of PAC-1 if used in cancer therapy.« less

Ecdysone-dependent and ecdysone-independent programmed cell death in the developing optic lobe of Drosophila.

PubMed

Hara, Yusuke; Hirai, Keiichiro; Togane, Yu; Akagawa, Hiromi; Iwabuchi, Kikuo; Tsujimura, Hidenobu

2013-02-01

The adult optic lobe of Drosophila develops from the primordium during metamorphosis from mid-3rd larval stage to adult. Many cells die during development of the optic lobe with a peak of the number of dying cells at 24 h after puparium formation (h APF). Dying cells were observed in spatio-temporal specific clusters. Here, we analyzed the function of a component of the insect steroid hormone receptor, EcR, in this cell death. We examined expression patterns of two EcR isoforms, EcR-A and EcR-B1, in the optic lobe. Expression of each isoform altered during development in isoform-specific manner. EcR-B1 was not expressed in optic lobe neurons from 0 to 6h APF, but was expressed between 9 and 48 h APF and then disappeared by 60 h APF. In each cortex, its expression was stronger in older glia-ensheathed neurons than in younger ones. EcR-B1 was also expressed in some types of glia. EcR-A was expressed in optic lobe neurons and many types of glia from 0 to 60 h APF in a different pattern from EcR-B1. Then, we genetically analyzed EcR function in the optic lobe cell death. At 0 h APF, the optic lobe cell death was independent of any EcR isoforms. In contrast, EcR-B1 was required for most optic lobe cell death after 24 h APF. It was suggested that cell death cell-autonomously required EcR-B1 expressed after puparium formation. βFTZ-F1 was also involved in cell death in many dying-cell clusters, but not in some of them at 24 h APF. Altogether, the optic lobe cell death occurred in ecdysone-independent manner at prepupal stage and ecdysone-dependent manner after 24 h APF. The acquisition of ecdysone-dependence was not directly correlated with the initiation or increase of EcR-B1 expression. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

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

p70 ribosomal S6 kinase 1 (S6K1) is an important serine/threonine kinase and downstream target of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. PF-4708671 is a specific inhibitor of S6K1, and prevents S6K1-mediated phosphorylation of the S6 protein. PF-4708671 treatment often leads to apoptotic cell death. However, the protective mechanism against PF-4708671-induced cell death has not been elucidated. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is essential for protecting cells against oxidative stress. p62, an adaptor protein in the autophagic process, enhances Nrf2 activation through the impairment of Keap1 activity. In thismore » study, we showed that PF-4708671 induces autophagic Keap1 degradation-mediated Nrf2 activation in p62-dependent manner. Furthermore, p62-dependent Nrf2 activation plays a crucial role in protecting cells from PF-4708671-mediated apoptosis. - Highlights: • PF-4708671, a S6K1-specific inhibitor, prevents S6K1-mediated S6 phosphorylation. • However, PF-4708671 treatment often leads to apoptotic cell death. • Protective mechanism against PF-4708671-induced cell death remains to be elucidated. • PF-4708671 induced p62-dependent, autophagic Keap1 degradation-mediated Nrf2 activation. • p62-dependent Nrf2 activation protects cells from PF-4708671-mediated apoptosis.« less

The effects of monobromobimane on neuronal cell death in the hippocampus after transient global cerebral ischemia in rats.

PubMed

Abe, Tsutomu; Takagi, Norio; Nakano, Midori; Takeo, Satoshi

2004-03-11

Calcium accumulation and free radical formation in the mitochondria are suggested to result in opening of the mitochondrial permeability transition pore that may be an initial step in neuronal cell death. The purpose of the present study was to determine whether monobromobimane (MBM) was a possible protective agent against neuronal cell death after transient global ischemia and the swelling of isolated hippocampal mitochondria. Infusion of MBM (1 or 3 microg) to cerebral ventricles 30 min before ischemia attenuated the expression of TUNEL-labeled cells and neuronal cell death in the hippocampal CA1 region at 72 h of reperfusion dose-dependently. Treatment with MBM inhibited an increase in caspase-3-like activity at 48 h of reperfusion in the hippocampus. MBM (30-300 microM) also inhibited an enhanced swelling rate induced by Ca2+ and phenylarsineoxide in the isolated hippocampal mitochondria. These results suggest that in vivo treatment with MBM may protect against neuronal cell death through inhibition of the mitochondrial swelling and caspase-3-dependent apoptotic pathway.

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.

Oxidative stress-induced necrotic cell death via mitochondira-dependent burst of reactive oxygen species.

PubMed

Choi, Kyungsun; Kim, Jinho; Kim, Gyung W; Choi, Chulhee

2009-11-01

Oxidative stress is deeply involved in various brain diseases, including neurodegenerative diseases, stroke, and ischemia/reperfusion injury. Mitochondria are thought to be the target and source of oxidative stress. We investigated the role of mitochondria in oxidative stress-induced necrotic neuronal cell death in a neuroblastoma cell line and a mouse model of middle cerebral artery occlusion. The exogenous administration of hydrogen peroxide was used to study the role of oxidative stress on neuronal cell survival and mitochondrial function in vitro. Hydrogen peroxide induced non-apoptotic neuronal cell death in a c-Jun N-terminal kinase- and poly(ADP-ribosyl) polymerase-dependent manner. Unexpectedly, hydrogen peroxide treatment induced transient hyperpolarization of the mitochondrial membrane potential and a subsequent delayed burst of endogenous reactive oxygen species (ROS). The inhibition of mitochondrial hyperpolarization by diphenylene iodonium or rotenone, potent inhibitors of mitochondrial respiratory chain complex I, resulted in reduced ROS production and subsequent neuronal cell death in vitro and in vivo. The inhibition of mitochondrial hyperpolarization can protect neuronal cells from oxidative stress-induced necrotic cell death, suggesting a novel method of therapeutic intervention in oxidative stress-induced neurological disease.

Human colon cancer HT-29 cell death responses to doxorubicin and Morus Alba leaves flavonoid extract.

PubMed

Fallah, S; Karimi, A; Panahi, G; Gerayesh Nejad, S; Fadaei, R; Seifi, M

2016-03-31

The mechanistic basis for the biological properties of Morus alba flavonoid extract (MFE) and chemotherapy drug of doxorubicin on human colon cancer HT-29 cell line death are unknown. The effect of doxorubicin and flavonoid extract on colon cancer HT-29 cell line death and identification of APC gene expression and PARP concentration of HT-29 cell line were investigated. The results showed that flavonoid extract and doxorubicin induce a dose dependent cell death in HT-29 cell line. MFE and doxorubicin exert a cytotoxic effect on human colon cancer HT-29 cell line by probably promoting or induction of apoptosis.

Bcl-2 Blocks a Caspase-Dependent Pathway of Apoptosis Activated by Herpes Simplex Virus 1 Infection in HEp-2 Cells

PubMed Central

Galvan, Veronica; Brandimarti, Renato; Munger, Joshua; Roizman, Bernard

2000-01-01

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type virus blocks the execution of the cell death program triggered by expression of viral genes, by the Fas and tumor necrosis factor pathways, or by nonspecific stress agents. In particular, an earlier report from this laboratory showed that the mutant virus d120 lacking the genes encoding infected cell protein 4 (ICP4), the major regulatory protein of the virus, induces a caspase-3-independent pathway of apoptosis in human SK-N-SH cells. Here we report that the pathway of apoptosis induced by the d120 mutant in human HEp-2 cells is caspase dependent. Specifically, in HEp-2 cells infected with d120, (i) a broad-range inhibitor of caspase activity, z-vad-FMK, efficiently blocked DNA fragmentation, (ii) cytochrome c was released into the cytoplasm, (iii) caspase-3 was activated inasmuch as poly(ADP-ribose) polymerase was cleaved, and (iv) chromatin condensation and fragmentation of cellular DNA were observed. In parallel studies, HEp-2 cells were transfected with a plasmid encoding human Bcl-2 and a clone (VAX-3) expressing high levels of Bcl-2 was selected. This report shows that Bcl-2 blocked all of the manifestations associated with programmed cell death caused by infection with the d120 mutant. Consistent with their resistance to programmed cell death, VAX-3 cells overproduced infected cell protein 0 (ICP0). An unexpected observation was that ICP0 encoded by the d120 mutant accumulated late in infection in small, quasi-uniform vesicle-like structures in all cell lines tested. Immunofluorescence-based colocalization studies indicated that these structures were not mitochondria or components of the endoplasmic reticulum or the late endosomal compartment. These studies affirm the conclusion that HSV can induce programmed cell death at multiple steps in the course of its replication, that the d120 mutant can induce both caspase-dependent and -independent pathways of programmed cell death, and that virus-induced stimuli of programmed cell death may differ with respect to the pathway that they activate. PMID:10644366

Colourful death: six-parameter classification of cell death by flow cytometry--dead cells tell tales.

PubMed

Munoz, Luis E; Maueröder, Christian; Chaurio, Ricardo; Berens, Christian; Herrmann, Martin; Janko, Christina

2013-08-01

The response of the immune system against dying and dead cells strongly depends on the cell death phenotype. Beside other forms of cell death, two clearly distinct populations, early apoptotic and secondary necrotic cells, have been shown to induce anti-inflammation/tolerance and inflammation/immune priming, respectively. Cytofluorometry is a powerful technique to detect morphological and phenotypical changes occurring during cell death. Here, we describe a new technique using AnnexinA5, propidiumiodide, DiIC1(5) and Hoechst 33342 to sub-classify populations of apoptotic and/or necrotic cells. The method allows the fast and reliable identification of several different phases and pathways of cell death by analysing the following cell death associated changes in a single tube: cellular granularity and shrinkage, phosphatidylserine exposure, ion selectivity of the plasma membrane, mitochondrial membrane potential, and DNA content. The clear characterisation of cell death is of major importance for instance in immunization studies, in experimental therapeutic settings, and in the exploration of cell-death associated diseases. It also enables the analysis of immunological properties of distinct populations of dying cells and the pathways involved in this process.

Thymoquinone Induces Cell Death in Human Squamous Carcinoma Cells via Caspase Activation-Dependent Apoptosis and LC3-II Activation-Dependent Autophagy

PubMed Central

Yu, Cheng-Chia; Lai, Yi-Yeh; Chen, Pei-Ni

2014-01-01

Background Thymoquinone (TQ), an active component of Nigella sativa or black cumin, elicits cytotoxic effects on various cancer cell lines. However, the anti-cancer effects of TQ on head and neck squamous cell carcinoma (HNSCC) remain unclear. Methodology/Principal Findings In this study, TQ elicited a strong cytotoxic effect on SASVO3, a highly malignant HNSCC cell line. The mechanisms of this cytotoxic effect were concentration dependent. TQ also induced apoptotic cell death in SASVO3 cells as indicated by an increase in Bax expression and caspase-9 activation. Apoptosis was possibly caspase-9 dependent because the exposure of cells to a caspase-9 inhibitor partially prevented cell death. The exposed cells also showed increased levels of autophagic vacuoles and LC3-II proteins, which are specific autophagy markers. Cell viability assay results further revealed that bafilomycin-A1, an autophagy inhibitor, enhanced TQ cytotoxicity; by comparison, Annexin V and propidium-iodide staining assay results showed that this inhibitor did not promote apoptosis. TQ treatment also increased the accumulation of autophagosomes. Using a lentivirus-shRNA system for LC3 silencing, we found that cell viability was eradicated in autophagy-defective cells. An in vivo BALB/c nude mouse xenograft model further showed that TQ administered by oral gavage reduced tumor growth via induced autophagy and apoptosis. Conclusions These findings indicated that TQ induced cell death in oral cancer cells via two distinct anti-neoplastic activities that can induce apoptosis and autophagy. Therefore, TQ is a promising candidate in phytochemical-based, mechanistic, and pathway-targeted cancer prevention strategies. PMID:25000169

A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation

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

Park, So Jung; Park, Young Jun; Shin, Ji Hyun

2011-05-13

Highlights: {yields} We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. {yields} Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. {yields} Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. {yields} Our studies suggest that Tyrphostin A9 induces mitochondria fragmentation and apoptotic cell death via Drp1 dependently. -- Abstract: Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactivemore » chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death.« less

Curcumin induces autophagic cell death in Spodoptera frugiperda cells.

PubMed

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

2017-06-01

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

Further considerations on in vitro skeletal muscle cell death

PubMed Central

Battistelli, Michela; Salucci, Sara; Burattini, Sabrina; Falcieri, Elisabetta

2013-01-01

Summary The present review discusses the apoptotic behavior induced by chemical and physical triggers in C2C12 skeletal muscle cells, comparing myoblast to myotube sensitivity, and investigating it by means of morphological, biochemical and cytofluorimetric analyses. After all treatments, myotubes, differently from myoblasts, showed a poor sensitivity to cell death. Intriguingly, in cells exposed to staurosporine, etoposide and UVB radiation, apoptotic and normal nuclei within the same fibercould be revealed. The presence of nuclear-dependent “territorial” death domains in the syncytium could explain a delayed cell death of myotubes compared to mononucleated cells. Moreover, autophagic granules abundantly appeared in myotubes after each treatment. Autophagy could protect muscle cell integrity against chemical and physical stimuli, making C2C12 myotubes, more resistant to cell death induction. PMID:24596689

17-beta estradiol inhibits oxidative stress-induced accumulation of AIF into nucleolus and PARP1-dependent cell death via estrogen receptor alpha.

PubMed

Batnasan, Enkhzaya; Wang, Ruoxi; Wen, Jitao; Ke, Yueshuang; Li, Xiaoxue; Bohio, Ameer Ali; Zeng, Xianlu; Huo, Hongliang; Han, Liping; Boldogh, Istvan; Ba, Xueqing

2015-01-05

Oxidative stress-induced DNA damage results in over-activation of poly(ADP-ribose) polymerase 1 (PARP1), leading to parthanatos, a newly discovered cell elimination pathway. Inhibition of PARP1-dependent cell death has shown to improve the outcome of diseases, including stroke, heart ischemia, and neurodegenerative diseases. In the present study we aimed to detect whether estrogen plays a protective role in inhibiting parthanatos. We utilized human mammary adenocarcinoma cells (MCF7) that abundantly express the estrogen receptor alpha and beta (ERα and ERβ). Parthanatos was induced by challenging the cells with hydrogen peroxide (H2O2). Microscopic imaging and molecular biological techniques, such as Western blot analysis and RNA interference, were performed. The results showed 17β estradiol (E2) protected MCF7 cells from PARP1-dependent cell death by decreasing protein PARylation, and AIF translocation into nuclei/nucleoli. Down-regulation of ERα expression by siRNA before E2 addition resulted in the failure of the E2-mediated inhibition of H2O2-induced protein PARylation and AIF nucleolar translocation. Together these data suggest that estrogen via its alpha-type receptor inhibits oxidative stress-induced, PARP1-dependent cell death. The present study provided us insight into how to apply hormone therapy in intervention of parthanatos-implicated ischemic and degenerative diseases. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Inhibition of caspases prevents ototoxic and ongoing hair cell death

NASA Technical Reports Server (NTRS)

Matsui, Jonathan I.; Ogilvie, Judith M.; Warchol, Mark E.

2002-01-01

Sensory hair cells die after acoustic trauma or ototoxic insults, but the signal transduction pathways that mediate hair cell death are not known. Here we identify several important signaling events that regulate the death of vestibular hair cells. Chick utricles were cultured in media supplemented with the ototoxic antibiotic neomycin and selected pharmacological agents that influence signaling molecules in cell death pathways. Hair cells that were treated with neomycin exhibited classically defined apoptotic morphologies such as condensed nuclei and fragmented DNA. Inhibition of protein synthesis (via treatment with cycloheximide) increased hair cell survival after treatment with neomycin, suggesting that hair cell death requires de novo protein synthesis. Finally, the inhibition of caspases promoted hair cell survival after neomycin treatment. Sensory hair cells in avian vestibular organs also undergo continual cell death and replacement throughout mature life. It is unclear whether the loss of hair cells stimulates the proliferation of supporting cells or whether the production of new cells triggers the death of hair cells. We examined the effects of caspase inhibition on spontaneous hair cell death in the chick utricle. Caspase inhibitors reduced the amount of ongoing hair cell death and ongoing supporting cell proliferation in a dose-dependent manner. In isolated sensory epithelia, however, caspase inhibitors did not affect supporting cell proliferation directly. Our data indicate that ongoing hair cell death stimulates supporting cell proliferation in the mature utricle.

Heme induces programmed necrosis on macrophages through autocrine TNF and ROS production

PubMed Central

Fortes, Guilherme B.; Alves, Leticia S.; de Oliveira, Rosane; Dutra, Fabianno F.; Rodrigues, Danielle; Fernandez, Patricia L.; Souto-Padron, Thais; De Rosa, María José; Kelliher, Michelle; Golenbock, Douglas; Chan, Francis K. M.

2012-01-01

Diseases that cause hemolysis or myonecrosis lead to the leakage of large amounts of heme proteins. Free heme has proinflammatory and cytotoxic effects. Heme induces TLR4-dependent production of tumor necrosis factor (TNF), whereas heme cytotoxicity has been attributed to its ability to intercalate into cell membranes and cause oxidative stress. We show that heme caused early macrophage death characterized by the loss of plasma membrane integrity and morphologic features resembling necrosis. Heme-induced cell death required TNFR1 and TLR4/MyD88-dependent TNF production. Addition of TNF to Tlr4−/− or to Myd88−/− macrophages restored heme-induced cell death. The use of necrostatin-1, a selective inhibitor of receptor-interacting protein 1 (RIP1, also known as RIPK1), or cells deficient in Rip1 or Rip3 revealed a critical role for RIP proteins in heme-induced cell death. Serum, antioxidants, iron chelation, or inhibition of c-Jun N-terminal kinase (JNK) ameliorated heme-induced oxidative burst and blocked macrophage cell death. Macrophages from heme oxygenase-1 deficient mice (Hmox1−/−) had increased oxidative stress and were more sensitive to heme. Taken together, these results revealed that heme induces macrophage necrosis through 2 synergistic mechanisms: TLR4/Myd88-dependent expression of TNF and TLR4-independent generation of ROS. PMID:22262768

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.

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

Temperature-dependent rate models of vascular cambium cell mortality

Treesearch

Matthew B. Dickinson; Edward A. Johnson

2004-01-01

We use two rate-process models to describe cell mortality at elevated temperatures as a means of understanding vascular cambium cell death during surface fires. In the models, cell death is caused by irreversible damage to cellular molecules that occurs at rates that increase exponentially with temperature. The models differ in whether cells show cumulative effects of...

Epigallocatechin-3-Gallate (EGCG) Promotes Autophagy-Dependent Survival via Influencing the Balance of mTOR-AMPK Pathways upon Endoplasmic Reticulum Stress.

PubMed

Holczer, Marianna; Besze, Boglárka; Zámbó, Veronika; Csala, Miklós; Bánhegyi, Gábor; Kapuy, Orsolya

2018-01-01

The maintenance of cellular homeostasis is largely dependent on the ability of cells to give an adequate response to various internal and external stimuli. We have recently proposed that the life-and-death decision in endoplasmic reticulum (ER) stress response is defined by a crosstalk between autophagy, apoptosis, and mTOR-AMPK pathways, where the transient switch from autophagy-dependent survival to apoptotic cell death is controlled by GADD34. The aim of the present study was to investigate the role of epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, in promoting autophagy-dependent survival and to verify the key role in connecting GADD34 with mTOR-AMPK pathways upon prolonged ER stress. Our findings, obtained by using HEK293T cells, revealed that EGCG treatment is able to extend cell viability by inducing autophagy. We confirmed that EGCG-induced autophagy is mTOR-dependent and PKA-independent; furthermore, it also required ULK1. We show that pretreatment of cells with EGCG diminishes the negative effect of GADD34 inhibition (by guanabenz or siGADD34 treatment) on autophagy. EGCG was able to delay apoptotic cell death by upregulating autophagy-dependent survival even in the absence of GADD34. Our data suggest a novel role for EGCG in promoting cell survival via shifting the balance of mTOR-AMPK pathways in ER stress.

Regulated necrosis and its implications in toxicology.

PubMed

Aki, Toshihiko; Funakoshi, Takeshi; Uemura, Koichi

2015-07-03

Recent research developments have revealed that caspase-dependent apoptosis is not the sole form of regulated cell death. Caspase-independent, but genetically regulated, forms of cell death include pyroptosis, necroptosis, parthanatos, and the recently discovered ferroptosis and autosis. Importantly, regulated necrosis can be modulated by small molecule inhibitors/activators, confirming the cell autonomous mechanism of these forms of cell death. The success of small molecule-mediated manipulation of regulated necrosis has produced great changes in the field of cell death research, and has also brought about significant changes in the fields of pharmacology as well as toxicology. In this review, we intend to summarize the modes of regulated cell death other than apoptosis, and discuss their implications in toxicology. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Live to die another way: modes of programmed cell death and the signals emanating from dying cells

PubMed Central

Fuchs, Yaron; Steller, Hermann

2015-01-01

Preface All life ends in death, but perhaps one of life’s grander ironies is that it also depends on death. Cell-intrinsic suicide pathways, termed programmed cell death (PCD), are crucial for animal development, tissue homeostasis and pathogenesis. Originally, PCD was virtually synonymous with apoptosis, but recently, alternative PCD mechanisms have been reported. Here, we provide an overview of several distinct PCD mechanisms, namely apoptosis, autophagy and necroptosis. In addition, we discuss the complex signals emanating from dying cells, which can either fuel regeneration or instruct additional killing. Further advances in understanding the physiological role of multiple cell death mechanisms and associated signals will be important to selectively manipulate PCD for therapeutic purposes. PMID:25991373

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

PubMed

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

2016-01-19

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

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

Corn silk maysin induces apoptotic cell death in PC-3 prostate cancer cells via mitochondria-dependent pathway.

PubMed

Lee, Jisun; Lee, Seul; Kim, Sun-Lim; Choi, Ji Won; Seo, Jeong Yeon; Choi, Doo Jin; Park, Yong Il

2014-12-05

Despite recent advances in prostate cancer diagnostics and therapeutics, the overall survival rate still remains low. This study was aimed to assess potential anti-cancer activity of maysin, a major flavonoid of corn silk (CS, Zea mays L.), in androgen-independent human prostate cancer cells (PC-3). Maysin was isolated from CS of Kwangpyeongok, a Korean hybrid corn, via methanol extraction and preparative C18 reverse phase column chromatography. Maysin cytotoxicity was determined by either monitoring cell viability in various cancer cell lines by MTT assay or morphological changes. Apoptotic cell death was assessed by annexin V-FITC/PI double staining, depolarization of mitochondrial membrane potential (MMP), expression levels of Bcl-2 and pro-caspase-3 and by terminal transferase mediated dUTP-fluorescein nick end labeling (TUNEL) staining. Underlying mechanism in maysin-induced apoptosis of PC-3 cells was explored by evaluating its effects on Akt and ERK pathway. Maysin dose-dependently reduced the PC-3 cell viability, with an 87% reduction at 200 μg/ml. Maysin treatment significantly induced apoptotic cell death, DNA fragmentation, depolarization of MMP, and reduction in Bcl-2 and pro-caspase-3 expression levels. Maysin also significantly attenuated phosphorylation of Akt and ERK. A combined treatment with maysin and other known anti-cancer agents, including 5-FU, etoposide, cisplatin, or camptothecin, synergistically enhanced PC-3 cell death. These results suggested for the first time that maysin inhibits the PC-3 cancer cell growth via stimulation of mitochondria-dependent apoptotic cell death and may have a strong therapeutic potential for the treatment of either chemo-resistant or androgen-independent human prostate cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

Interferon-alpha and interferon-gamma sensitize human tenon fibroblasts to mitomycin-C.

PubMed

Wang, Xiao Yang; Crowston, Jonathan G; Zoellner, Hans; Healey, Paul R

2007-08-01

To investigate the effect of interferon (IFN)-alpha and IFN-gamma pretreatment on mitomycin C (MMC)-induced cell death in human Tenon fibroblasts (HTFs) and the mechanisms by which IFN-alpha and IFN-gamma modulate the susceptibility of HTFs to MMC. HTFs were pretreated with IFN-alpha and IFN-gamma for 48 hours before 5-minute application of 0.4 mg/mL MMC. Cell death after 48 hours was determined by Annexin V/propidium iodide (PI) staining and lactate dehydrogenase (LDH) release assay. Fas, Fas-ligand, and Bcl-2 expression were determined by flow cytometry. Fas associated death domain (FADD), Bax, cytochrome c, and caspase expression were determined by Western blot analysis and immunofluorescence staining. MMC treatment increased cell death and upregulated Fas and FADD expression, but had no effect on Fas-Ligand, Bax, Bcl-2, or cytochrome c. Neither IFN-alpha nor IFN-gamma alone induced HTF death, but each increased cell death 2 days after MMC treatment in a dose-dependent fashion. Combination IFN-alpha and IFN-gamma had a synergistic effect. IFN-alpha and IFN-gamma pretreatment increased Fas expression. Fas upregulation was associated with increased sensitivity to MMC. IFN pretreatment increased procaspase-8, procaspase-9, and procaspase-3 expression, and caspase-3 activation. Caspase-8, caspase-3, and broad caspase inhibitors, but not caspase-9 inhibitor, inhibited MMC-induced cell death in nonpretreated and IFN-pretreated cells. IFN-alpha and IFN-gamma enhance the susceptibility of HTFs to MMC-induced cell death through a Fas-mediated and a caspase-3-dependent pathway. Pretreatment with IFN primed HTFs to MMC, providing a potential means for initially slowing the healing response with IFN and subsequently terminating fibroblast activity through MMC-induced cell death.

Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways

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

Nguyen Ngoc, Tam Dan; Son, Young-Ok; Lim, Shin-Saeng

2012-03-15

Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G{sub 2}/M phase. The addition of NaFmore » induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. -- Highlights: ► The mode of NaF-induced cell death and the mechanisms involved were examined. ► NaF induced mainly apoptotic death of mouse embryonic stem cells (mESCs). ► NaF induced mitochondrial-mediated and caspase-dependent apoptosis. ► JNK- and p53-mediated pathways are involved in NaF-mediated apoptosis in the cells. ► ROS are the up-stream effector in NaF-mediated activation of JNK and p53 in mESCs.« less

Calcium regulates cell death in cancer: Roles of the mitochondria and mitochondria-associated membranes (MAMs).

PubMed

Danese, Alberto; Patergnani, Simone; Bonora, Massimo; Wieckowski, Mariusz R; Previati, Maurizio; Giorgi, Carlotta; Pinton, Paolo

2017-08-01

Until 1972, the term 'apoptosis' was used to differentiate the programmed cell death that naturally occurs in organismal development from the acute tissue death referred to as necrosis. Many studies on cell death and programmed cell death have been published and most are, at least to some degree, related to cancer. Some key proteins and molecular pathways implicated in cell death have been analyzed, whereas others are still being actively researched; therefore, an increasing number of cellular compartments and organelles are being implicated in cell death and cancer. Here, we discuss the mitochondria and subdomains of the endoplasmic reticulum (ER) that interact with mitochondria, the mitochondria-associated membranes (MAMs), which have been identified as critical hubs in the regulation of cell death and tumor growth. MAMs-dependent calcium (Ca 2+ ) release from the ER allows selective Ca 2+ uptake by the mitochondria. The perturbation of Ca 2+ homeostasis in cancer cells is correlated with sustained cell proliferation and the inhibition of cell death through the modulation of Ca 2+ signaling. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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

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

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.

A Competitive Stapled Peptide Screen Identifies a Selective Small Molecule that Overcomes MCL-1-dependent Leukemia Cell Survival

PubMed Central

Cohen, Nicole A.; Stewart, Michelle L.; Gavathiotis, Evripidis; Tepper, Jared L.; Bruekner, Susanne R.; Koss, Brian; Opferman, Joseph T.; Walensky, Loren D.

2012-01-01

SUMMARY Cancer cells hijack BCL-2 family survival proteins to suppress the death effectors and thereby enforce an immortal state. This is accomplished biochemically by an anti-apoptotic surface groove that neutralizes the pro-apoptotic BH3 α-helix of death proteins. Anti-apoptotic MCL-1 in particular has emerged as a ubiquitous resistance factor in cancer. Whereas targeting the BCL-2 anti-apoptotic subclass effectively restores the death pathway in BCL-2-dependent cancer, the development of molecules tailored to the binding specificity of MCL-1 has lagged. We previously discovered that a hydrocarbon-stapled MCL-1 BH3 helix is an exquisitely selective MCL-1 antagonist. By deploying this unique reagent in a competitive screen, we identified an MCL-1 inhibitor molecule that selectively targets the BH3-binding groove of MCL-1, neutralizes its biochemical lockhold on apoptosis, and induces caspase activation and leukemia cell death in the specific context of MCL-1 dependence. PMID:22999885

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.

On a nonlocal reaction-diffusion-advection system modelling the growth of phytoplankton with cell quota structure

NASA Astrophysics Data System (ADS)

Hsu, Sze-Bi; Mei, Linfeng; Wang, Feng-Bin

2015-11-01

Phytoplankton species in a water column compete for mineral nutrients and light, and the existing models usually neglect differences in the nutrient content and the amount of light absorbed of individuals. In this current paper, we examine a size-structured and nonlocal reaction-diffusion-advection system which describes the dynamics of a single phytoplankton species in a water column where the species depends simply on light for its growth. Our model is under the assumption that the amount of light absorbed by individuals is proportional to cell size, which varies for populations that reproduce by simple division into two equally-sized daughters. We first establish the existence of a critical death rate and our analysis indicates that the phytoplankton survives if and only if its death rate is less than the critical death rate. The critical death rate depends on a general reproductive rate, the characteristics of the water column (e.g., turbulent diffusion rate, sinking, depth), cell growth, cell division, and cell size.

Omega-3 docosahexaenoic acid induces pyroptosis cell death in triple-negative breast cancer cells.

PubMed

Pizato, Nathalia; Luzete, Beatriz Christina; Kiffer, Larissa Fernanda Melo Vasconcelos; Corrêa, Luís Henrique; de Oliveira Santos, Igor; Assumpção, José Antônio Fagundes; Ito, Marina Kiyomi; Magalhães, Kelly Grace

2018-01-31

The implication of inflammation in pathophysiology of several type of cancers has been under intense investigation. Omega-3 fatty acids can modulate inflammation and present anticancer effects, promoting cancer cell death. Pyroptosis is an inflammation related cell death and so far, the function of docosahexaenoic acid (DHA) in pyroptosis cell death has not been described. This study investigated the role of DHA in triggering pyroptosis activation in breast cancer cells. MDA-MB-231 breast cancer cells were supplemented with DHA and inflammation cell death was analyzed. DHA-treated breast cancer cells triggered increased caspase-1and gasdermin D activation, enhanced IL-1β secretion, translocated HMGB1 towards the cytoplasm, and membrane pore formation when compared to untreated cells, suggesting DHA induces pyroptosis programmed cell death in breast cancer cells. Moreover, caspase-1 inhibitor (YVAD) could protect breast cancer cells from DHA-induced pyroptotic cell death. In addition, membrane pore formation showed to be a lysosomal damage and ROS formation-depended event in breast cancer cells. DHA triggered pyroptosis cell death in MDA-MB-231by activating several pyroptosis markers in these cells. This is the first study that shows the effect of DHA triggering pyroptosis programmed cell death in breast cancer cells and it could improve the understanding of the omega-3 supplementation during breast cancer treatment.

Irreversible EGFR Inhibitor EKB-569 Targets Low-LET γ-Radiation-Triggered Rel Orchestration and Potentiates Cell Death in Squamous Cell Carcinoma

PubMed Central

Aravindan, Natarajan; Thomas, Charles R.; Aravindan, Sheeja; Mohan, Aswathi S.; Veeraraghavan, Jamunarani; Natarajan, Mohan

2011-01-01

EKB-569 (Pelitinib), an irreversible EGFR tyrosine kinase inhibitor has shown potential therapeutic efficiency in solid tumors. However, cell-killing potential in combination with radiotherapy and its underlying molecular orchestration remain to be explored. The objective of this study was to determine the effect of EKB-569 on ionizing radiation (IR)-associated NFκB-dependent cell death. SCC-4 and SCC-9 cells exposed to IR (2Gy) with and without EKB-569 treatment were analyzed for transactivation of 88 NFκB pathway molecules, NFκB DNA-binding activity, translation of the NFκB downstream mediators, Birc1, 2 and 5, cell viability, metabolic activity and apoptosis. Selective targeting of IR-induced NFκB by EKB-569 and its influence on cell-fate were assessed by overexpressing (p50/p65) and silencing (ΔIκBα) NFκB. QPCR profiling after IR exposure revealed a significant induction of 74 NFκB signal transduction molecules. Of those, 72 were suppressed with EKB-569. EMSA revealed a dose dependent inhibition of NFκB by EKB-569. More importantly, EKB-569 inhibited IR-induced NFκB in a dose-dependent manner, and this inhibition was sustained up to at least 72 h. Immunoblotting revealed a significant suppression of IR-induced Birc1, 2 and 5 by EKB-569. We observed a dose-dependent inhibition of cell viability, metabolic activity and apoptosis with EKB-569. EKB-569 significantly enhanced IR-induced cell death and apoptosis. Blocking NFκB improved IR-induced cell death. Conversely, NFκB overexpression negates EKB-569 -induced cell-killing. Together, these pre-clinical data suggest that EKB-569 is a radiosensitizer of squamous cell carcinoma and may mechanistically involve selective targeting of IR-induced NFκB-dependent survival signaling. Further pre-clinical in-vivo studies are warranted. PMID:22242139

Nicotiana benthamiana MAPK-WRKY pathway confers resistance to a necrotrophic pathogen Botrytis cinerea.

PubMed

Adachi, Hiroaki; Ishihama, Nobuaki; Nakano, Takaaki; Yoshioka, Miki; Yoshioka, Hirofumi

2016-06-02

MEK2-SIPK/WIPK cascade, a Nicotiana benthamiana mitogen-activated protein kinase (MAPK) cascade, is an essential signaling pathway for plant immunity and involved in hypersensitive response (HR) accompanied by cell death. WRKY transcription factors as substrates of SIPK and WIPK have been isolated and implicated in HR cell death. Here, we show virus-induced gene silencing of WRKY genes compromised constitutively active MEK2-triggered cell death in N. benthamiana leaves. In general, HR cell death enhances susceptibility to necrotrophic pathogens such as Botrytis cinerea. However, the WRKY gene silencing elevated susceptibility to B. cinerea. These findings suggest that downstream WRKYs of MEK2-SIPK/WIPK cascade are required for cell death-dependent and -independent immunities in N. benthamiana.

An apoptosis targeted stimulus with nanosecond pulsed electric fields (nsPEFs) in E4 squamous cell carcinoma.

PubMed

Ren, Wei; Beebe, Stephen J

2011-04-01

Stimuli directed towards activation of apoptosis mechanisms are an attractive approach to eliminate evasion of apoptosis, a ubiquitous cancer hallmark. In these in vitro studies, kinetics and electric field thresholds for several apoptosis characteristics are defined in E4 squamous carcinoma cells (SCC) exposed to ten 300 ns pulses with increasing electric fields. Cell death was >95% at the highest electric field and coincident with phosphatidylserine externalization, caspase and calpain activation in the presence and absence of cytochrome c release, decreases in Bid and mitochondria membrane potential (Δψm) without apparent changes reactive oxygen species levels or in Bcl2 and Bclxl levels. Bid cleavage was caspase-dependent (55-60%) and calcium-dependent (40-45%). Intracellular calcium as an intrinsic mechanism and extracellular calcium as an extrinsic mechanism were responsible for about 30 and 70% of calcium dependence for Bid cleavage, respectively. The results reveal electric field-mediated cell death induction and progression, activating pro-apoptotic-like mechanisms and affecting plasma membrane and intracellular functions, primarily through extrinsic-like pathways with smaller contributions from intrinsic-like pathways. Nanosecond second pulsed electric fields trigger heterogeneous cell death mechanisms in E4 SCC populations to delete them, with caspase-associated cell death as a predominant, but not an unaccompanied event.

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

S phase entry causes homocysteine-induced death while ataxia telangiectasia and Rad3 related protein functions anti-apoptotically to protect neurons.

PubMed

Ye, Weizhen; Blain, Stacy W

2010-08-01

A major phenotype seen in neurodegenerative disorders is the selective loss of neurons due to apoptotic death and evidence suggests that inappropriate re-activation of cell cycle proteins in post-mitotic neurons may be responsible. To investigate whether reactivation of the G1 cell cycle proteins and S phase entry was linked with apoptosis, we examined homocysteine-induced neuronal cell death in a rat cortical neuron tissue culture system. Hyperhomocysteinaemia is a physiological risk factor for a variety of neurodegenerative diseases, including Alzheimer's disease. We found that in response to homocysteine treatment, cyclin D1, and cyclin-dependent kinases 4 and 2 translocated to the nucleus, and p27 levels decreased. Both cyclin-dependent kinases 4 and 2 regained catalytic activity, the G1 gatekeeper retinoblastoma protein was phosphorylated and DNA synthesis was detected, suggesting transit into S phase. Double-labelling immunofluorescence showed a 95% co-localization of anti-bromodeoxyuridine labelling with apoptotic markers, demonstrating that those cells that entered S phase eventually died. Neurons could be protected from homocysteine-induced death by methods that inhibited G1 phase progression, including down-regulation of cyclin D1 expression, inhibition of cyclin-dependent kinases 4 or 2 activity by small molecule inhibitors, or use of the c-Abl kinase inhibitor, Gleevec, which blocked cyclin D and cyclin-dependent kinase 4 nuclear translocation. However, blocking cell cycle progression post G1, using DNA replication inhibitors, did not prevent apoptosis, suggesting that death was not preventable post the G1-S phase checkpoint. While homocysteine treatment caused DNA damage and activated the DNA damage response, its mechanism of action was distinct from that of more traditional DNA damaging agents, such as camptothecin, as it was p53-independent. Likewise, inhibition of the DNA damage sensors, ataxia-telangiectasia mutant and ataxia telangiectasia and Rad3 related proteins, did not rescue apoptosis and in fact exacerbated death, suggesting that the DNA damage response might normally function neuroprotectively to block S phase-dependent apoptosis induction. As cell cycle events appear to be maintained in vivo in affected neurons for weeks to years before apoptosis is observed, activation of the DNA damage response might be able to hold cell cycle-induced death in check.

NADE, a p75NTR-associated cell death executor, is involved in signal transduction mediated by the common neurotrophin receptor p75NTR.

PubMed

Mukai, J; Hachiya, T; Shoji-Hoshino, S; Kimura, M T; Nadano, D; Suvanto, P; Hanaoka, T; Li, Y; Irie, S; Greene, L A; Sato, T A

2000-06-09

The low affinity neurotrophin receptor p75NTR can mediate cell survival as well as cell death of neural cells by NGF and other neurotrophins. To elucidate p75NTR-mediated signal transduction, we screened p75NTR-associated proteins by a yeast two-hybrid system. We identified one positive clone and named NADE (p75NTR-associated cell death executor). Mouse NADE has marked homology to the human HGR74 protein. NADE specifically binds to the cell-death domain of p75NTR. Co-expression of NADE and p75NTR induced caspase-2 and caspase-3 activities and the fragmentation of nuclear DNA in 293T cells. However, in the absence of p75NTR, NADE failed to induce apoptosis, suggesting that NADE expression is necessary but insufficient for p75NTR-mediated apoptosis. Furthermore, p75NTR/NADE-induced cell death was dependent on NGF but not BDNF, NT-3, or NT-4/5, and the recruitment of NADE to p75NTR (intracellular domain) was dose-dependent. We obtained similar results from PC12 cells, nnr5 cells, and oligodendrocytes. Taken together, NADE is the first signaling adaptor molecule identified in the involvement of p75NTR-mediated apoptosis induced by NGF, and it may play an important role in the pathogenesis of neurogenetic diseases.

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.

Epigallocatechin-3-Gallate (EGCG) Promotes Autophagy-Dependent Survival via Influencing the Balance of mTOR-AMPK Pathways upon Endoplasmic Reticulum Stress

PubMed Central

Holczer, Marianna; Besze, Boglárka; Zámbó, Veronika

2018-01-01

The maintenance of cellular homeostasis is largely dependent on the ability of cells to give an adequate response to various internal and external stimuli. We have recently proposed that the life-and-death decision in endoplasmic reticulum (ER) stress response is defined by a crosstalk between autophagy, apoptosis, and mTOR-AMPK pathways, where the transient switch from autophagy-dependent survival to apoptotic cell death is controlled by GADD34. The aim of the present study was to investigate the role of epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, in promoting autophagy-dependent survival and to verify the key role in connecting GADD34 with mTOR-AMPK pathways upon prolonged ER stress. Our findings, obtained by using HEK293T cells, revealed that EGCG treatment is able to extend cell viability by inducing autophagy. We confirmed that EGCG-induced autophagy is mTOR-dependent and PKA-independent; furthermore, it also required ULK1. We show that pretreatment of cells with EGCG diminishes the negative effect of GADD34 inhibition (by guanabenz or siGADD34 treatment) on autophagy. EGCG was able to delay apoptotic cell death by upregulating autophagy-dependent survival even in the absence of GADD34. Our data suggest a novel role for EGCG in promoting cell survival via shifting the balance of mTOR-AMPK pathways in ER stress. PMID:29636854

Autophagy regulates death of retinal pigment epithelium cells in age-related macular degeneration.

PubMed

Kaarniranta, Kai; Tokarz, Paulina; Koskela, Ali; Paterno, Jussi; Blasiak, Janusz

2017-04-01

Age-related macular degeneration (AMD) is an eye disease underlined by the degradation of retinal pigment epithelium (RPE) cells, photoreceptors, and choriocapillares, but the exact mechanism of cell death in AMD is not completely clear. This mechanism is important for prevention of and therapeutic intervention in AMD, which is a hardly curable disease. Present reports suggest that both apoptosis and pyroptosis (cell death dependent on caspase-1) as well as necroptosis (regulated necrosis dependent on the proteins RIPK3 and MLKL, caspase-independent) can be involved in the AMD-related death of RPE cells. Autophagy, a cellular clearing system, plays an important role in AMD pathogenesis, and this role is closely associated with the activation of the NLRP3 inflammasome, a central event for advanced AMD. Autophagy can play a role in apoptosis, pyroptosis, and necroptosis, but its contribution to AMD-specific cell death is not completely clear. Autophagy can be involved in the regulation of proteins important for cellular antioxidative defense, including Nrf2, which can interact with p62/SQSTM, a protein essential for autophagy. As oxidative stress is implicated in AMD pathogenesis, autophagy can contribute to this disease by deregulation of cellular defense against the stress. However, these and other interactions do not explain the mechanisms of RPE cell death in AMD. In this review, we present basic mechanisms of autophagy and its involvement in AMD pathogenesis and try to show a regulatory role of autophagy in RPE cell death. This can result in considering the genes and proteins of autophagy as molecular targets in AMD prevention and therapy.

Necroptosis in neurodegenerative diseases: a potential therapeutic target

PubMed Central

Zhang, Shuo; Tang, Mi-bo; Luo, Hai-yang; Shi, Chang-he; Xu, Yu-ming

2017-01-01

Neurodegenerative diseases are a group of chronic progressive disorders characterized by neuronal loss. Necroptosis, a recently discovered form of programmed cell death, is a cell death mechanism that has necrosis-like morphological characteristics. Necroptosis activation relies on the receptor-interacting protein (RIP) homology interaction motif (RHIM). A variety of RHIM-containing proteins transduce necroptotic signals from the cell trigger to the cell death mediators RIP3 and mixed lineage kinase domain-like protein (MLKL). RIP1 plays a particularly important and complex role in necroptotic cell death regulation ranging from cell death activation to inhibition, and these functions are often cell type and context dependent. Increasing evidence suggests that necroptosis plays an important role in the pathogenesis of neurodegenerative diseases. Moreover, small molecules such as necrostatin-1 are thought inhibit necroptotic signaling pathway. Understanding the precise mechanisms underlying necroptosis and its interactions with other cell death pathways in neurodegenerative diseases could provide significant therapeutic insights. The present review is aimed at summarizing the molecular mechanisms of necroptosis and highlighting the emerging evidence on necroptosis as a major driver of neuron cell death in neurodegenerative diseases. PMID:28661482

Cystine uptake through the cystine/glutamate antiporter xCT triggers glioblastoma cell death under glucose deprivation.

PubMed

Goji, Takeo; Takahara, Kazuhiko; Negishi, Manabu; Katoh, Hironori

2017-12-01

Oncogenic signaling in cancer cells alters glucose uptake and utilization to supply sufficient energy and biosynthetic intermediates for survival and sustained proliferation. Oncogenic signaling also prevents oxidative stress and cell death caused by increased production of reactive oxygen species. However, elevated glucose metabolism in cancer cells, especially in glioblastoma, results in the cells becoming sensitive to glucose deprivation ( i.e. in high glucose dependence), which rapidly induces cell death. However, the precise mechanism of this type of cell death remains unknown. Here, we report that glucose deprivation alone does not trigger glioblastoma cell death. We found that, for cell death to occur in glucose-deprived glioblastoma cells, cystine and glutamine also need to be present in culture media. We observed that cystine uptake through the cystine/glutamate antiporter xCT under glucose deprivation rapidly induces NADPH depletion, reactive oxygen species accumulation, and cell death. We conclude that although cystine uptake is crucial for production of antioxidant glutathione in cancer cells its transport through xCT also induces oxidative stress and cell death in glucose-deprived glioblastoma cells. Combining inhibitors targeting cancer-specific glucose metabolism with cystine and glutamine treatment may offer a therapeutic approach for glioblastoma tumors exhibiting high xCT expression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Persistent random walk of cells involving anomalous effects and random death

NASA Astrophysics Data System (ADS)

Fedotov, Sergei; Tan, Abby; Zubarev, Andrey

2015-04-01

The purpose of this paper is to implement a random death process into a persistent random walk model which produces sub-ballistic superdiffusion (Lévy walk). We develop a stochastic two-velocity jump model of cell motility for which the switching rate depends upon the time which the cell has spent moving in one direction. It is assumed that the switching rate is a decreasing function of residence (running) time. This assumption leads to the power law for the velocity switching time distribution. This describes the anomalous persistence of cell motility: the longer the cell moves in one direction, the smaller the switching probability to another direction becomes. We derive master equations for the cell densities with the generalized switching terms involving the tempered fractional material derivatives. We show that the random death of cells has an important implication for the transport process through tempering of the superdiffusive process. In the long-time limit we write stationary master equations in terms of exponentially truncated fractional derivatives in which the rate of death plays the role of tempering of a Lévy jump distribution. We find the upper and lower bounds for the stationary profiles corresponding to the ballistic transport and diffusion with the death-rate-dependent diffusion coefficient. Monte Carlo simulations confirm these bounds.

SELECTIVE VULNERABILITY OF EMBRYONIC CELL POPULATIONS TO ETHANOL-INDUCED APOPTOSIS: IMPLICATIONS FOR ALCOHOL RELATED BIRTH DEFECTS AND NEURODEVELOPMENTAL DISORDER

EPA Science Inventory

The locations of cell death and resulting malformations in embryos following teratogen exposure vary depending on the teratogen used, the genotype of the conceptus, and the developmental stage of the embryo at time of exposure. To date, ethanol-induced cell death has been charac...

Methadone, commonly used as maintenance medication for outpatient treatment of opioid dependence, kills leukemia cells and overcomes chemoresistance.

PubMed

Friesen, Claudia; Roscher, Mareike; Alt, Andreas; Miltner, Erich

2008-08-01

The therapeutic opioid drug methadone (d,l-methadone hydrochloride) is the most commonly used maintenance medication for outpatient treatment of opioid dependence. In our study, we found that methadone is also a potent inducer of cell death in leukemia cells and we clarified the unknown mechanism of methadone-induced cell killing in leukemia cells. Methadone inhibited proliferation in leukemia cells and induced cell death through apoptosis induction and activated apoptosis pathways through the activation of caspase-9 and caspase-3, down-regulation of Bcl-x(L) and X chromosome-linked inhibitor of apoptosis, and cleavage of poly(ADP-ribose) polymerase. In addition, methadone induced cell death not only in anticancer drug-sensitive and apoptosis-sensitive leukemia cells but also in doxorubicin-resistant, multidrug-resistant, and apoptosis-resistant leukemia cells, which anticancer drugs commonly used in conventional therapies of leukemias failed to kill. Depending on caspase activation, methadone overcomes doxorubicin resistance, multidrug resistance, and apoptosis resistance in leukemia cells through activation of mitochondria. In contrast to leukemia cells, nonleukemic peripheral blood lymphocytes survived after methadone treatment. These findings show that methadone kills leukemia cells and breaks chemoresistance and apoptosis resistance. Our results suggest that methadone is a promising therapeutic approach not only for patients with opioid dependence but also for patients with leukemias and provide the foundation for new strategies using methadone as an additional anticancer drug in leukemia therapy, especially when conventional therapies are less effective.

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.

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

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

Histone deacetylase inhibitors: Potential in cancer therapy.

PubMed

Marks, P A; Xu, W-S

2009-07-01

The role of histone deacetylases (HDAC) and the potential of these enzymes as therapeutic targets for cancer, neurodegenerative diseases and a number of other disorders is an area of rapidly expanding investigation. There are 18 HDACs in humans. These enzymes are not redundant in function. Eleven of the HDACs are zinc dependent, classified on the basis of homology to yeast HDACs: Class I includes HDACs 1, 2, 3, and 8; Class IIA includes HDACs 4, 5, 7, and 9; Class IIB, HDACs 6 and 10; and Class IV, HDAC 11. Class III HDACs, sirtuins 1-7, have an absolute requirement for NAD(+), are not zinc dependent and generally not inhibited by compounds that inhibit zinc dependent deacetylases. In addition to histones, HDACs have many nonhistone protein substrates which have a role in regulation of gene expression, cell proliferation, cell migration, cell death, and angiogenesis. HDAC inhibitors (HDACi) have been discovered of different chemical structure. HDACi cause accumulation of acetylated forms of proteins which can alter their structure and function. HDACi can induce different phenotypes in various transformed cells, including growth arrest, apoptosis, reactive oxygen species facilitated cell death and mitotic cell death. Normal cells are relatively resistant to HDACi induced cell death. Several HDACi are in various stages of development, including clinical trials as monotherapy and in combination with other anti-cancer drugs and radiation. The first HDACi approved by the FDA for cancer therapy is suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza), approved for treatment of cutaneous T-cell lymphoma. 2009 Wiley-Liss, Inc.

The art and science of low-energy applications in medicine: pathology perspectives

NASA Astrophysics Data System (ADS)

Thomsen, Sharon L.

2011-03-01

Applications of low energy non-ionizing irradiation result in non-lethal and lethal effects in cells, tissues and intact individuals. The effects of these applications depend on the physical parameters of the applied energies, the mechanisms of interaction of these energies on the target and the biologic status of the target. Recently, cell death has been found not to be a random accident of situation or age but a range of complicated physiological responses to various extrinsic and intrinsic events some of which are genetically programmed and/ or physiologically regulated. Therefore, cell death has been classified into three general groups: 1) Programmed cell death including apoptosis and necroptosis, cornefication and autophagy; 2) Accidental (traumatic) cell death due to the direct, immediate effects of the lethal event and 3) Necrotic cell death which is, by default, all cell death not associated with programmed or accidental cell death. Lethal low energy non-ionizing application biologic effects involve mechanisms of all three groups as compared to high energy applications that predominantly involve the mechanisms of accidental cell death. Currently, the mechanisms of all these modes of cell death are being vigorously investigated. As research and development of new low energy applications continues, the need to understand the mechanisms of cell death that they produce will be critical to the rational creation of safe, yet effective instruments.

Targeted Deletion of Autophagy Genes Atg5 or Atg7 in the Chondrocytes Promotes Caspase-Dependent Cell Death and Leads to Mild Growth Retardation.

PubMed

Vuppalapati, Karuna K; Bouderlique, Thibault; Newton, Phillip T; Kaminskyy, Vitaliy O; Wehtje, Henrik; Ohlsson, Claes; Zhivotovsky, Boris; Chagin, Andrei S

2015-12-01

Longitudinal bone growth takes place in epiphyseal growth plates located in the ends of long bones. The growth plate consists of chondrocytes traversing from the undifferentiated (resting zone) to the terminally differentiated (hypertrophic zone) stage. Autophagy is an intracellular catabolic process of lysosome-dependent recycling of intracellular organelles and protein complexes. Autophagy is activated during nutritionally depleted or hypoxic conditions in order to facilitate cell survival. Chondrocytes in the middle of the growth plate are hypoxic and nutritionally depleted owing to the avascular nature of the growth plate. Accordingly, autophagy may facilitate their survival. To explore the role of autophagy in chondrocyte survival and constitutional bone growth, we generated mice with cartilage-specific ablation of either Atg5 (Atg5cKO) or Atg7 (Atg7cKO) by crossing Atg5 or Atg7 floxed mice with cartilage-specific collagen type 2 promoter-driven Cre. Both Atg5cKO and Atg7cKO mice showed growth retardation associated with enhanced chondrocyte cell death and decreased cell proliferation. Similarly, inhibition of autophagy by Bafilomycin A1 (Baf) or 3-methyladenine (3MA) promoted cell death in cultured slices of human growth plate tissue. To delineate the underlying mechanisms we employed ex vivo cultures of mouse metatarsal bones and RCJ3.IC5.18 rat chondrogenic cell line. Baf or 3MA impaired metatarsal bone growth associated with processing of caspase-3 and massive cell death. Similarly, treatment of RCJ3.IC5.18 chondrogenic cells by Baf also showed massive cell death and caspase-3 cleavage. This was associated with activation of caspase-9 and cytochrome C release. Altogether, our data suggest that autophagy is important for chondrocyte survival, and inhibition of this process leads to stunted growth and caspase-dependent death of chondrocytes. © 2015 American Society for Bone and Mineral Research.

AKT/SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes

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

Bhavsar, Shefalee K.; Merches, Katja; Bobbala, Diwakar

2012-08-17

Highlights: Black-Right-Pointing-Pointer Akt/SGK dependent phosphorylation of GSK3{alpha},{beta} regulates T lymphocytes. Black-Right-Pointing-Pointer T cells from mice expressing Akt/SGK insensitive GSK3{alpha},{beta} (gsk3{sup KI}) release less IL-2. Black-Right-Pointing-Pointer CD4{sup +} cells from gsk3{sup KI} mice express less CD62L. Black-Right-Pointing-Pointer CD8{sup +} cells from gsk3{sup KI} mice are relatively resistant to activation induced cell death. Black-Right-Pointing-Pointer Perforin expression is enhanced in gsk3{sup KI} T cells. -- Abstract: Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3{alpha},{beta}. Lithium, a known unspecific GSK3 inhibitor protectsmore » against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3{alpha},{beta} inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3{sup KI}). T cells from gsk3{sup KI} mice were compared to T cells from corresponding wild type mice (gsk3{sup WT}). As a result, in gsk3{sup KI} CD4{sup +} cells surface CD62L (L-selectin) was significantly less abundant than in gsk3{sup WT} CD4{sup +} cells. Upon activation in vitro T cells from gsk3{sup KI} mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3{sup KI} T cells, suggesting that GSK3 induces effector function in CD8{sup +} T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3{alpha},{beta} is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.« less

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

Interaction between the plant ApDef1 defensin and Saccharomyces cerevisiae results in yeast death through a cell cycle- and caspase-dependent process occurring via uncontrolled oxidative stress.

PubMed

Soares, Júlia Ribeiro; José Tenório de Melo, Edésio; da Cunha, Maura; Fernandes, Kátia Valevski Sales; Taveira, Gabriel Bonan; da Silva Pereira, Lidia; Pimenta, Samy; Trindade, Fernanda Gomes; Regente, Mariana; Pinedo, Marcela; de la Canal, Laura; Gomes, Valdirene Moreira; de Oliveira Carvalho, André

2017-01-01

Plant defensins were discovered at beginning of the 90s'; however, their precise mechanism of action is still unknown. Herein, we studied ApDef 1 -Saccharomyces cerevisiae interaction. ApDef 1 -S. cerevisiae interaction was studied by determining the MIC, viability and death kinetic assays. Viability assay was repeated with hydroxyurea synchronized-yeast and pretreated with CCCP. Plasma membrane permeabilization, ROS induction, chromatin condensation, and caspase activation analyses were assessed through Sytox green, DAB, DAPI and FITC-VAD-FMK, respectively. Viability assay was done in presence of ascorbic acid and Z-VAD-FMK. Ultrastructural analysis was done by electron microscopy. ApDef 1 caused S. cerevisiae cell death and MIC was 7.8μM. Whole cell population died after 18h of ApDef 1 interaction. After 3h, 98.76% of synchronized cell population died. Pretreatment with CCCP protected yeast from ApDef 1 induced death. ApDef 1 -S. cerevisiae interaction resulted in membrane permeabilization, H 2 O 2 increased production, chromatin condensation and caspase activation. Ascorbic acid prevented yeast cell death and membrane permeabilization. Z-VAD-FMK prevented yeast cell death. ApDef 1 -S. cerevisiae interaction caused cell death through cell cycle dependentprocess which requires preserved membrane potential. After interaction, yeast went through uncontrolled ROS production and accumulation, which led to plasma membrane permeabilization, chromatin condensation and, ultimately, cell death by activation of caspase-dependent apoptosis via. We show novel requirements for the interaction between plant defensin and fungi cells, i.e. cell cycle phase and membrane potential, and we indicate that membrane permeabilization is probably caused by ROS and therefore, it would be an indirect event of the ApDef 1 -S. cerevisiae interaction. Copyright © 2016 Elsevier B.V. All rights reserved.

Ferroxitosis: A cell death from modulation of oxidative phosphorylation and PKM2-dependent glycolysis in melanoma

PubMed Central

Lakhter, Alexander J.; Hamilton, James; Dagher, Pierre C.; Mukkamala, Suresh; Hato, Takashi; Dong, X. Charlie; Mayo, Lindsey D.; Harris, Robert A.; Shekhar, Anantha; Ivan, Mircea; Brustovetsky, Nickolay; Naidu, Samisubbu R.

2014-01-01

Reliance on glycolysis is a characteristic of malignancy, yet the development of resistance to BRAF inhibitors in melanoma is associated with gain of mitochondrial function. Concurrent attenuation of oxidative phosphorylation and HIF-1α/PKM2-dependent glycolysis promotes a non-apoptotic, iron- and oxygen-dependent cell death that we term ferroxitosis. The redox cycling agent menadione causes a robust increase in oxygen consumption, accompanied by significant loss of intracellular ATP and rapid cell death. Conversely, either hypoxic adaptation or iron chelation prevents menadione-induced ferroxitosis. Ectopic expression of K213Q HIF-1α mutant blunts the effects of menadione. However, knockdown of HIF-1α or PKM2 restores menadione-induced cytotoxicity in hypoxia. Similarly, exposure of melanoma cells to shikonin, a menadione analog and a potential PKM2 inhibitor, is sufficient to induce ferroxitosis under hypoxic conditions. Collectively, our findings reveal that ferroxitosis curtails metabolic plasticity in melanoma. PMID:25587028

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.

KML001 Induces Apoptosis and Autophagic Cell Death in Prostate Cancer Cells via Oxidative Stress Pathway

PubMed Central

You, Dalsan; Kim, Yunlim; Jang, Myoung Jin; Lee, Chunwoo; Jeong, In Gab; Cho, Yong Mee; Hwang, Jung Jin; Hong, Jun Hyuk; Ahn, Hanjong; Kim, Choung-Soo

2015-01-01

We investigated the effects of KML001 (NaAsO2, sodium metaarsenite, Kominox), an orally bioavailable arsenic compound, on the growth and death of human prostate cancer cells and its mechanism of action. Growth inhibition was assessed by cytotoxicity assays in the presence or absence of inhibitor of apoptosis, inhibitor of autophagy or antioxidant N-Acetyl-L-cysteine to study mechanism of cell death induced by KML001 in PC3, DU145 and LNCaP prostate cancer cell lines. Electron microscopy, flow cytometry and Western blotting were used to study apoptotic and autophagic mechanisms. The DU145 xenograft model was used to determine the efficacy of KML001 in vivo. KML001 decreased the viability of cells and increased the percentage of annexin V-positive cells dose-dependently in prostate cancer cells, and LNCaP cells were more sensitive to KML001 than PC3 or DU145 cells. Electron microscopy revealed typical apoptotic characters and autophagic vacuoles in cells treated with KML001. Exposure to KML001 in prostate cancer cells induced apoptosis and autophagy in a time- and dose-dependent manner. KML001 induced dose-dependent accumulation of reactive oxygen species, and scavenging the reactive oxygen species with N-Acetyl-L-cysteine reduced LC3 and cleaved poly (ADP-ribose) polymerase. KML001 significantly inhibited tumor growth in the DU145 xenograft model. In addition, significant decrease of proliferation and significant increases of apoptosis and autophagy were observed in KML001-treated tumors than in vehicle-treated tumors. Exposure of human prostate cancer cells to KML001 induced both apoptosis and autophagic cell death via oxidative stress pathway. And KML001 had an antiproliferative effect on DU145 cells in xenograft mice. PMID:26352139

KML001 Induces Apoptosis and Autophagic Cell Death in Prostate Cancer Cells via Oxidative Stress Pathway.

PubMed

You, Dalsan; Kim, Yunlim; Jang, Myoung Jin; Lee, Chunwoo; Jeong, In Gab; Cho, Yong Mee; Hwang, Jung Jin; Hong, Jun Hyuk; Ahn, Hanjong; Kim, Choung-Soo

2015-01-01

We investigated the effects of KML001 (NaAsO2, sodium metaarsenite, Kominox), an orally bioavailable arsenic compound, on the growth and death of human prostate cancer cells and its mechanism of action. Growth inhibition was assessed by cytotoxicity assays in the presence or absence of inhibitor of apoptosis, inhibitor of autophagy or antioxidant N-Acetyl-L-cysteine to study mechanism of cell death induced by KML001 in PC3, DU145 and LNCaP prostate cancer cell lines. Electron microscopy, flow cytometry and Western blotting were used to study apoptotic and autophagic mechanisms. The DU145 xenograft model was used to determine the efficacy of KML001 in vivo. KML001 decreased the viability of cells and increased the percentage of annexin V-positive cells dose-dependently in prostate cancer cells, and LNCaP cells were more sensitive to KML001 than PC3 or DU145 cells. Electron microscopy revealed typical apoptotic characters and autophagic vacuoles in cells treated with KML001. Exposure to KML001 in prostate cancer cells induced apoptosis and autophagy in a time- and dose-dependent manner. KML001 induced dose-dependent accumulation of reactive oxygen species, and scavenging the reactive oxygen species with N-Acetyl-L-cysteine reduced LC3 and cleaved poly (ADP-ribose) polymerase. KML001 significantly inhibited tumor growth in the DU145 xenograft model. In addition, significant decrease of proliferation and significant increases of apoptosis and autophagy were observed in KML001-treated tumors than in vehicle-treated tumors. Exposure of human prostate cancer cells to KML001 induced both apoptosis and autophagic cell death via oxidative stress pathway. And KML001 had an antiproliferative effect on DU145 cells in xenograft mice.

Antiproliferative Activity and Induction of Apoptosis in Human Melanoma Cells by Houttuynia cordata Thunb Extract.

PubMed

Yanarojana, Mongkol; Nararatwanchai, Thamthiwat; Thairat, Sarut; Tancharoen, Salunya

2017-12-01

To analyze the apoptotic effect of Houttuynia cordata Thunb (HCT) extract on human melanoma A375 cells and its underlying mechanisms. The effects of HCT on cell death were determined using the MTT assay. Hoechst 33342 staining was conducted to confirm the detection of cell apoptosis. Caspase-3 and caspase-8 mRNA and cleaved protein levels were investigated by RT-PCR and western blotting, respectively. The release of high mobility group box 1 (HMGB1) and phosphorylation of mitogen-activated protein kinase (MAPK) were determined by ELISA. Caspase-3 and caspase-8 specific inhibitors suppressed HCT-induced cell death. HCT increased caspase-3 and caspase-8 mRNA, protein levels, and caspase activities in a concentration- and time-dependent manner. HCT induced MAPK phosphorylation in a time-dependent fashion. Pretreatment of cells with a selective inhibitor of p38 MAPK reduced apoptosis and reversed the levels of HMGB1 release in response to HCT treatment. HCT induces A375 programmed cell death by activating the caspase-dependent pathway and by p38 phosphorylation associated with HMGB1 reduction. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Virus inhibition of RIP3-dependent necrosis.

PubMed

Upton, Jason W; Kaiser, William J; Mocarski, Edward S

2010-04-22

Viral infection activates cytokine expression and triggers cell death, the modulation of which is important for successful pathogenesis. Necroptosis is a form of programmed necrosis dependent on two related RIP homotypic interaction motif (RHIM)-containing signaling adaptors, receptor-interacting protein kinases (RIP) 1 and 3. We find that murine cytomegalovirus infection induces RIP3-dependent necrosis. Whereas RIP3 kinase activity and RHIM-dependent interactions control virus-associated necrosis, virus-induced death proceeds independently of RIP1 and is therefore distinct from TNFalpha-dependent necroptosis. Viral M45-encoded inhibitor of RIP activation (vIRA) targets RIP3 during infection and disrupts RIP3-RIP1 interactions characteristic of TNFalpha-induced necroptosis, thereby suppressing both death pathways. Importantly, attenuation of vIRA mutant virus in wild-type mice is normalized in RIP3-deficient mice. Thus, vIRA function validates necrosis as central to host defense against viral infections and highlights the benefit of multiple virus-encoded cell-death suppressors that inhibit not only apoptotic, but also necrotic mechanisms of virus clearance. Copyright 2010 Elsevier Inc. All rights reserved.

Induction of Autophagic Cell Death in Apoptosis-resistant Pancreatic Cancer Cells using Benzo[α]phenoxazines Derivatives, 10-methyl-benzo[α]phenoxazine-5-one and benzo[α]phenoxazine-5-one.

PubMed

Pal, Sanjima; Salunke-Gawalib, Sunita; Konkimallaa, V Badireenath

2017-01-01

Intrinsic resistance to apoptotic cell death due to co-occurrence of mutated KRAS and p53 is frequently reported in pancreatic cancer that renders them aggressive, highly proliferative and metastatic. In addition, these cancer types are less sensitive to apoptosis inducing drugs where promotion of autophagic cell death could be a viable option for treatment under such circumstances. In this study we examined the potential of three intrinsically fluorescent benzo[α]phenoxazines or BPZs (R=Cl, CH3, H) to induce cytotoxic autophagy in chemo and apoptosis-resistant, KRAS and p53 mutated pancreatic cancer model cell line, MIAPaCa-2. Cells were adapted at in vitro metabolically stressed condition (5% serum) to initiate intrinsic cell survival strategies within. Cell proliferation, colonogenicity, cellular uptake, retention, localization, cellular granularity and presence of both apoptosis and autophagy biomarkers were assessed in BPZ treated/untreated (solvent) cells to validate induction of concentration dependent cytotoxic autophagy and other consequences. For the first time, we report the ability of this class of compounds to accumulate within cells increasing its granularity, inducing death via autophagy. From different kinetics study, it was observed that the autophagic-cell death was dependent on the ligand type, duration of incubation or working concentrations. Among the three BPZ tested, both 3B (benzo[α]phenoxazine-5-one) and 2B (10-methyl-benzo[α] phenoxazine-5-one) induced pro-death autophagy in MIAPaCa-2 cells at an IC50 of 5 μM and 20 μM respectively. Such compounds would be of great interest to explore as novel cytotoxic autophagy inducing agents in apoptosisresistant cancer types.

Minocycline attenuates both OGD-induced HMGB1 release and HMGB1-induced cell death in ischemic neuronal injury in PC12 cells

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

Kikuchi, Kiyoshi; Department of Neurosurgery, Omuta City General Hospital, 2-19-1 Takarazaka, Omuta-City, Fukuoka 836-8567; Kawahara, Ko-ichi

2009-07-24

High mobility group box-1 (HMGB1), a non-histone DNA-binding protein, is massively released into the extracellular space from neuronal cells after ischemic insult and exacerbates brain tissue damage in rats. Minocycline is a semisynthetic second-generation tetracycline antibiotic which has recently been shown to be a promising neuroprotective agent. In this study, we found that minocycline inhibited HMGB1 release in oxygen-glucose deprivation (OGD)-treated PC12 cells and triggered the activation of p38mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK1/2). The ERK kinase (MEK)1/2 inhibitor U-0126 and p38MAPK inhibitor SB203580 blocked HMGB1 release in response to OGD. Furthermore, HMGB1 triggered cell death inmore » a dose-dependent fashion. Minocycline significantly rescued HMGB1-induced cell death in a dose-dependent manner. In light of recent observations as well as the good safety profile of minocycline in humans, we propose that minocycline might play a potent neuroprotective role through the inhibition of HMGB1-induced neuronal cell death in cerebral infarction.« less

GPR30 Activation Contributes to the Puerarin-Mediated Neuroprotection in MPP+-Induced SH-SY5Y Cell Death.

PubMed

Cheng, Yue-Fa; Zhu, Guoqi; Wu, Qing-Wen; Xie, Yue-Sheng; Jiang, Yan; Guo, Lan; Guan, Ya-Li; Liu, Ying-Shuo; Zhang, Jun

2017-02-01

The neuroprotective action of puerarin in Parkinson's disease (PD) models has been well investigated. However, the mechanisms involved in protection have not been completely understood. G protein-coupled receptor 30 (GPR30) is a G protein-coupled estrogen receptor and considered a potential target in the neuroprotection against PD. In this study, we investigated whether puerarin prevented against 1-methyl-4-phenylpyridinium (MPP + )-induced cell death via GPR30. Our results showed that the GPR30 agonist, G1, exhibited puerarin-mediated neuroprotection against MPP + -induced cell death of SH-SY5Y cells. This protective action was reversed by the GPR30 antagonist. Moreover, a time- and concentration-dependent effect of puerarin on GPR30 expression was verified at the protein level but not at the mRNA level. Further, we showed that an mTor-dependent new GPR30 synthesis contributed to the protection conferred by puerarin. Finally, glial cell line-derived neurotrophic factor (GDNF) levels were enhanced by puerarin and G1 in both control and MPP + -lesioned cells via GPR30. Taken together, our data strongly suggest that puerarin prevents MPP + -induced cell death via facilitating GPR30 expression and GDNF release.

The expression patterns of pro-apoptotic and anti-apoptotic factors in human fetal and adult ovary.

PubMed

Poljicanin, Ana; Vukusic Pusic, Tanja; Vukojevic, Katarina; Caric, Ana; Vilovic, Katarina; Tomic, Snjezana; Soljic, Violeta; Saraga-Babic, Mirna

2013-07-01

The influence of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins on the cell death (caspase-3, TUNEL) of different ovarian cell lineages was immunohistochemically analyzed in six fetal and five adult human ovaries in order to disclose possible mechanisms of cell number control. Mild to moderate expression of Bcl-2 characterized ovarian surface epithelium, follicular cells and oocytes of 15 and 22 week human ovaries, while expression of Bax and caspase-3 gradually increased in all ovarian cell populations, except caspase-3 in the ovarian surface epithelium. Different levels of Bax and Bcl-2 proteins co-expression characterized fetal ovarian cells, while TUNEL and caspase-3 co-expression was found only in some of them. In adult ovaries, Bcl-2 was moderately and Bax strongly expressed in the surface ovarian epithelium and stroma. Bcl-2 and Bax expression in granulosa and theca interna cells varied depending on the stage of follicular atresia. Caspase-3 apoptotic cells characterized granulosa cells of adult atretic follicles. Our results indicate that intracellular levels of Bcl-2 and Bax protein might regulate the final destiny of developing germ cells. Caspase-3 dependent apoptosis seems to be the most important, but not the only cell death pathway in ovaries. In adult ovaries, caspase-dependent cell death characterized granulosa cells, but not the germ cells. Copyright © 2012 Elsevier GmbH. All rights reserved.

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

PubMed

Woo, Seon Min; Seo, Seung Un; Min, Kyoung-Jin; Im, Seung-Soon; Nam, Ju-Ock; Chang, Jong-Soo; Kim, Shin; Park, Jong-Wook; Kwon, Taeg Kyu

2018-04-27

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

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

PubMed Central

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

2012-01-01

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

c-FLIP is involved in erythropoietin-mediated protection of erythroid-differentiated cells from TNF-alpha-induced apoptosis.

PubMed

Vittori, Daniela; Vota, Daiana; Callero, Mariana; Chamorro, María E; Nesse, Alcira

2010-05-04

The TNF-alpha (tumour necrosis factor) affects a wide range of biological activities, such as cell proliferation and apoptosis. Cell life or death responses to this cytokine might depend on cell conditions. This study focused on the modulation of factors that would affect the sensitivity of erythroid-differentiated cells to TNF-alpha. Hemin-differentiated K562 cells showed higher sensitivity to TNF-induced apoptosis than undifferentiated cells. At the same time, hemin-induced erythroid differentiation reduced c-FLIP (cellular FLICE-inhibitory protein) expression. However, this negative effect was prevented by prior treatment with Epo (erythropoietin), which allowed the cell line to maintain c-FLIP levels. On the other hand, erythroid-differentiated UT-7 cells - dependent on Epo for survival - showed resistance to TNF-alpha pro-apoptotic action. Only after the inhibition of PI3K (phosphatidylinositol-3 kinase)-mediated pathways, which was accompanied by negative c-FLIP modulation and increased erythroid differentiation, were UT-7 cells sensitive to TNF-alpha-triggered apoptosis. In summary, erythroid differentiation might deregulate the balance between growth promotion and death signals induced by TNF-alpha, depending on cell type and environmental conditions. The role of c-FLIP seemed to be critical in the protection of erythroid-differentiated cells from apoptosis or in the determination of their sensitivity to TNF-mediated programmed cell death. Epo, which for the first time was found to be involved in the prevention of c-FLIP down-regulation, proved to have an anti-apoptotic effect against the pro-inflammatory factor. The identification of signals related to cell life/death switching would have significant implications in the control of proliferative diseases and would contribute to the understanding of mechanisms underlying the anaemia associated with inflammatory processes.

Fas Versatile Signaling and Beyond: Pivotal Role of Tyrosine Phosphorylation in Context-Dependent Signaling and Diseases

PubMed Central

Chakrabandhu, Krittalak; Hueber, Anne-Odile

2016-01-01

The Fas/FasL system is known, first and foremost, as a potent apoptosis activator. While its proapoptotic features have been studied extensively, evidence that the Fas/FasL system can elicit non-death signals has also accumulated. These non-death signals can promote survival, proliferation, migration, and invasion of cells. The key molecular mechanism that determines the shift from cell death to non-death signals had remained unclear until the recent identification of the tyrosine phosphorylation in the death domain of Fas as the reversible signaling switch. In this review, we present the connection between the recent findings regarding the control of Fas multi-signals and the context-dependent signaling choices. This information can help explain variable roles of Fas signaling pathway in different pathologies. PMID:27799932

Cyclic AMP regulates formation of mammary epithelial acini in vitro

PubMed Central

Nedvetsky, Pavel I.; Kwon, Sang-Ho; Debnath, Jayanta; Mostov, Keith E.

2012-01-01

Epithelial cells form tubular and acinar structures notable for a hollow lumen. In three-dimensional culture utilizing MCF10A mammary epithelial cells, acini form due to integrin-dependent polarization and survival of cells contacting extracellular matrix (ECM), and the apoptosis of inner cells of acini lacking contact with the ECM. In this paper, we report that cyclic AMP (cAMP)-dependent protein kinase A (PKA) promotes acinus formation via two mechanisms. First, cAMP accelerates redistribution of α6-integrin to the periphery of the acinus and thus facilitates the polarization of outer acinar cells. Blocking of α6-integrin function by inhibitory antibody prevents cAMP-dependent polarization. Second, cAMP promotes the death of inner cells occupying the lumen. In the absence of cAMP, apoptosis is delayed, resulting in perturbed luminal clearance. cAMP-dependent apoptosis is accompanied by a posttranscriptional PKA-dependent increase in the proapoptotic protein Bcl-2 interacting mediator of cell death. These data demonstrate that cAMP regulates lumen formation in mammary epithelial cells in vitro, both through acceleration of polarization of outer cells and apoptosis of inner cells of the acinus. PMID:22675028

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.

Heterotrimeric G Protein Signaling Is Required for Epidermal Cell Death in Rice[W][OA

PubMed Central

Steffens, Bianka; Sauter, Margret

2009-01-01

In rice (Oryza sativa) adventitious root primordia are formed at the nodes as part of normal development. Upon submergence of rice plants, adventitious roots emerge from the nodes preceded by death of epidermal cells above the root primordia. Cell death is induced by ethylene and mediated by hydrogen peroxide (H2O2). Pharmacological experiments indicated that epidermal cell death was dependent on signaling through G proteins. Treatment with GTP-γ-S induced epidermal cell death, whereas GDP-β-S partially inhibited ethylene-induced cell death. The dwarf1 (d1) mutant of rice has repressed expression of the Gα subunit RGA1 of heterotrimeric G protein. In d1 plants, cell death in response to ethylene and H2O2 was nearly completely abolished, indicating that signaling through Gα is essential. Ethylene and H2O2 were previously shown to alter gene expression in epidermal cells that undergo cell death. Transcriptional regulation was not generally affected in the d1 mutant, indicating that altered gene expression is not sufficient to trigger cell death in the absence of Gα. Analysis of genes encoding proteins related to G protein signaling revealed that four small GTPase genes, two GTPase-activating protein genes, and one GDP dissociation inhibitor gene but not RGA1 were differentially expressed in epidermal cells above adventitious roots, indicating that Gα activity is regulated posttranscriptionally. PMID:19656904

A set of nutrient limitations trigger yeast cell death in a nitrogen-dependent manner during wine alcoholic fermentation

PubMed Central

Duc, Camille; Pradal, Martine; Sanchez, Isabelle; Noble, Jessica; Tesnière, Catherine

2017-01-01

Yeast cell death can occur during wine alcoholic fermentation. It is generally considered to result from ethanol stress that impacts membrane integrity. This cell death mainly occurs when grape musts processing reduces lipid availability, resulting in weaker membrane resistance to ethanol. However the mechanisms underlying cell death in these conditions remain unclear. We examined cell death occurrence considering yeast cells ability to elicit an appropriate response to a given nutrient limitation and thus survive starvation. We show here that a set of micronutrients (oleic acid, ergosterol, pantothenic acid and nicotinic acid) in low, growth-restricting concentrations trigger cell death in alcoholic fermentation when nitrogen level is high. We provide evidence that nitrogen signaling is involved in cell death and that either SCH9 deletion or Tor inhibition prevent cell death in several types of micronutrient limitation. Under such limitations, yeast cells fail to acquire any stress resistance and are unable to store glycogen. Unexpectedly, transcriptome analyses did not reveal any major changes in stress genes expression, suggesting that post-transcriptional events critical for stress response were not triggered by micronutrient starvation. Our data point to the fact that yeast cell death results from yeast inability to trigger an appropriate stress response under some conditions of nutrient limitations most likely not encountered by yeast in the wild. Our conclusions provide a novel frame for considering both cell death and the management of nutrients during alcoholic fermentation. PMID:28922393

E-Cigarette Aerosol Exposure Induces Reactive Oxygen Species, DNA Damage, and Cell Death in Vascular Endothelial Cells.

PubMed

Anderson, Chastain; Majeste, Andrew; Hanus, Jakub; Wang, Shusheng

2016-12-01

Cigarette smoking remains one of the leading causes of preventable death worldwide. Vascular cell death and dysfunction is a central or exacerbating component in the majority of cigarette smoking related pathologies. The recent development of the electronic nicotine delivery systems known as e-cigarettes provides an alternative to conventional cigarette smoking; however, the potential vascular health risks of e-cigarette use remain unclear. This study evaluates the effects of e-cigarette aerosol extract (EAE) and conventional cigarette smoke extract (CSE) on human umbilical vein endothelial cells (HUVECs). A laboratory apparatus was designed to produce extracts from e-cigarettes and conventional cigarettes according to established protocols for cigarette smoking. EAE or conventional CSE was applied to human vascular endothelial cells for 4-72 h, dependent on the assay. Treated cells were assayed for reactive oxygen species, DNA damage, cell viability, and markers of programmed cell death pathways. Additionally, the anti-oxidants α-tocopherol and n-acetyl-l-cysteine were used to attempt to rescue e-cigarette induced cell death. Our results indicate that e-cigarette aerosol is capable of inducing reactive oxygen species, causing DNA damage, and significantly reducing cell viability in a concentration dependent fashion. Immunofluorescent and flow cytometry analysis indicate that both the apoptosis and programmed necrosis pathways are triggered by e-cigarette aerosol treatment. Additionally, anti-oxidant treatment provides a partial rescue of the induced cell death, indicating that reactive oxygen species play a causal role in e-cigarette induced cytotoxicity. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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.

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.

Taxol induces concentration-dependent phosphatidylserine (PS) externalization and cell cycle arrest in ASTC-a-1 cells

NASA Astrophysics Data System (ADS)

Guo, Wen-jing; Chen, Tong-sheng

2010-02-01

Taxol (Paclitaxel) is an important natural product for the treatment of solid tumors. Different concentrations of taxol can trigger distinct effects on both the cellular microtubule network and biochemical pathways. Apoptosis induced by low concentrations (5-30 nM) of taxol was associated with mitotic arrest, alteration of microtubule dynamics and/or G2/M cell cycle arrest, whereas high concentrations of this drug (0.2-30 μM) caused significant microtubule damage, and was found recently to induce cytoplasm vacuolization in human lung adenocarcinoma (ASTC-a-1) cells. In present study, cell counting kit (CCK-8) assay, confocal microscope, and flow cytometry analysis were used to analyze the cell death form induced by 35 nM and 70 μM of taxol respectively in human lung adenocarcinoma (ASTC-a-1) cells. After treatment of 35 nM taxol for 48 h, the OD450 value was 0.80, and 35 nM taxol was found to induce dominantly cell death in apoptotic pathway such as phosphatidylserine (PS) externalization, G2/M phase arrest after treatment for 24 h, and nuclear fragmentation after treatment for 48 h. After 70 μM taxol treated the cell for 24 h, the OD450 value was 1.01, and 70 μM taxol induced cytoplasm vacuolization programmed cell death (PCD) and G2/M phase as well as the polyploidy phase arrest in paraptotic-like cell death. These findings imply that the regulated signaling pathway of cell death induced by taxol is dependent on taxol concentration in ASTC-a-1 cells.

Mangiferin induces cell death against rhabdomyosarcoma through sustained oxidative stress.

PubMed

Padma, Vishwanadha Vijaya; Kalaiselvi, Palanisamy; Yuvaraj, Rangasamy; Rabeeth, M

2015-06-01

Embryonic rhabdomyosarcoma (RD) is the most prevalent type of cancer among children. The present study aimed to investigate cell death induced by mangiferin in RD cells. The Inhibitory concentration (IC 50 ) value of mangiferin was determined by an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Cell death induced by mangiferin against RD cells was determined through lactate dehydrogenase and nitric oxide release, intracellular calcium levels, reactive oxygen species generation, antioxidant status, mitochondrial calcium level, and mitochondrial membrane potential. Furthermore, acridine orange/ethidium bromide staining was performed to determine early/late apoptotic event. Mangiferin induced cell death in RD cells with an IC 50 value of 70 μM. The cytotoxic effect was reflected in a dose-dependent increase in lactate dehydrogenase leakage and nitric oxide release during mangiferin treatment. Mangiferin caused dose dependent increase in reactive oxygen species generation, intracellular calcium levels with subsequent decrease in antioxidant status (catalase, superoxide dismutase, glutathione-S-transferase, and glutathione) and loss of mitochondrial membrane potential in RD cells. Further data from fluorescence microscopy suggest that mangiferin caused cell shrinkage and nuclear condensation along with the occurrence of a late event of apoptosis. Results of the present study shows that mangiferin can act as a promising chemopreventive agent against RD by inducing sustained oxidative stress.

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

PubMed

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

2010-12-15

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

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

PubMed Central

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

2010-01-01

Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, while other studies implicate activation of the mitochondrial permeability transition poreas the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, while it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetyl cysteine and exogenous glutathione (GSH), or by over-expression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells over-expressing Cu, Zn-SOD or MnSOD. Over-expression of antiapoptotic Bcl-XLprotected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochromec, Bax/Bak, caspase-9 and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them. PMID:20937380

Zanthoxylum fruit extract from Japanese pepper promotes autophagic cell death in cancer cells.

PubMed

Nozaki, Reo; Kono, Toru; Bochimoto, Hiroki; Watanabe, Tsuyoshi; Oketani, Kaori; Sakamaki, Yuichi; Okubo, Naoto; Nakagawa, Koji; Takeda, Hiroshi

2016-10-25

Zanthoxylum fruit, obtained from the Japanese pepper plant (Zanthoxylum piperitum De Candolle), and its extract (Zanthoxylum fruit extract, ZFE) have multiple physiological activities (e.g., antiviral activity). However, the potential anticancer activity of ZFE has not been fully examined. In this study, we investigated the ability of ZFE to induce autophagic cell death (ACD). ZFE caused remarkable autophagy-like cytoplasmic vacuolization, inhibited cell proliferation, and ultimately induced cell death in the human cancer cell lines DLD-1, HepG2, and Caco-2, but not in A549, MCF-7, or WiDr cells. ZFE increased the level of LC3-II protein, a marker of autophagy. Knockdown of ATG5 using siRNA inhibited ZFE-induced cytoplasmic vacuolization and cell death. Moreover, in cancer cells that could be induced to undergo cell death by ZFE, the extract increased the phosphorylation of c-Jun N-terminal kinase (JNK), and the JNK inhibitor SP600125 attenuated both vacuolization and cell death. Based on morphology and expression of marker proteins, ZFE-induced cell death was neither apoptosis nor necrosis. Normal intestinal cells were not affected by ZFE. Taken together, our findings show that ZFE induces JNK-dependent ACD, which appears to be the main mechanism underlying its anticancer activity, suggesting a promising starting point for anticancer drug development.

HAMLET triggers apoptosis but tumor cell death is independent of caspases, Bcl-2 and p53.

PubMed

Hallgren, O; Gustafsson, L; Irjala, H; Selivanova, G; Orrenius, S; Svanborg, C

2006-02-01

HAMLET (Human alpha-lactalbumin Made Lethal to Tumor cells) triggers selective tumor cell death in vitro and limits tumor progression in vivo. Dying cells show features of apoptosis but it is not clear if the apoptotic response explains tumor cell death. This study examined the contribution of apoptosis to cell death in response to HAMLET. Apoptotic changes like caspase activation, phosphatidyl serine externalization, chromatin condensation were detected in HAMLET-treated tumor cells, but caspase inhibition or Bcl-2 over-expression did not prolong cell survival and the caspase response was Bcl-2 independent. HAMLET translocates to the nuclei and binds directly to chromatin, but the death response was unrelated to the p53 status of the tumor cells. p53 deletions or gain of function mutations did not influence the HAMLET sensitivity of tumor cells. Chromatin condensation was partly caspase dependent, but apoptosis-like marginalization of chromatin was also observed. The results show that tumor cell death in response to HAMLET is independent of caspases, p53 and Bcl-2 even though HAMLET activates an apoptotic response. The use of other cell death pathways allows HAMLET to successfully circumvent fundamental anti-apoptotic strategies that are present in many tumor cells.

Contact-independent cell death of human microglial cells due to pathogenic Naegleria fowleri trophozoites.

PubMed

Kim, Jong-Hyun; Kim, Daesik; Shin, Ho-Joon

2008-12-01

Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increase of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death.

Contact-Independent Cell Death of Human Microglial Cells due to Pathogenic Naegleria fowleri Trophozoites

PubMed Central

Kim, Jong-Hyun

2008-01-01

Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increasse of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death. PMID:19127326

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. 

Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway.

PubMed

Viswanathan, Vasanthi S; Ryan, Matthew J; Dhruv, Harshil D; Gill, Shubhroz; Eichhoff, Ossia M; Seashore-Ludlow, Brinton; Kaffenberger, Samuel D; Eaton, John K; Shimada, Kenichi; Aguirre, Andrew J; Viswanathan, Srinivas R; Chattopadhyay, Shrikanta; Tamayo, Pablo; Yang, Wan Seok; Rees, Matthew G; Chen, Sixun; Boskovic, Zarko V; Javaid, Sarah; Huang, Cherrie; Wu, Xiaoyun; Tseng, Yuen-Yi; Roider, Elisabeth M; Gao, Dong; Cleary, James M; Wolpin, Brian M; Mesirov, Jill P; Haber, Daniel A; Engelman, Jeffrey A; Boehm, Jesse S; Kotz, Joanne D; Hon, Cindy S; Chen, Yu; Hahn, William C; Levesque, Mitchell P; Doench, John G; Berens, Michael E; Shamji, Alykhan F; Clemons, Paul A; Stockwell, Brent R; Schreiber, Stuart L

2017-07-27

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFβ-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.

Phenethyl Isothiocyanate Induces Apoptotic Cell Death Through the Mitochondria-dependent Pathway in Gefitinib-resistant NCI-H460 Human Lung Cancer Cells In Vitro.

PubMed

Hsia, Te-Chun; Huang, Yi-Ping; Jiang, Yi-Wen; Chen, Hsin-Yu; Cheng, Zheng-Yu; Hsiao, Yung-Ting; Chen, Cheng-Yen; Peng, Shu-Fen; Chueh, Fu-Shin; Chou, Yu-Cheng; Chung, Jing-Gung

2018-04-01

Some lung cancer patients treated with gefitinib develop resistance to this drug resulting in unsatisfactory treatment outcomes. Phenethyl isothiocyanate (PEITC), present in our common cruciferous vegetables, exhibits anticancer activities in many human cancer cell lines. Currently, there is no available information on the possible modification of gefitinib resistance of lung cancer in vitro by PEITC. Thus, the effects of PEITC on gefitinib resistant lung cancer NCI-H460 cells were investigated in vitro. The total cell viability, apoptotic cell death, production of reactive oxygen species (ROS) and Ca 2+ , levels of mitochondria membrane potential (ΔΨ m ) and caspase-3, -8 and -9 activities were measured by flow cytometry assay. PEITC induced chromatin condensation was examined by DAPI staining. PEITC-induced cell morphological changes, decreased total viable cell number and induced apoptotic cell death in NCI-H460 and NCI-H460/G cells. PEITC decreased ROS production in NCI-H460 cells, but increased production in NCI-H460/G cells. PEITC increased Ca 2+ production, decreased the levels of ΔΨ m and increased caspase-3, -8 and -9 activities in both NCI-H460 and NCI-H460/G cells. Western blotting was used to examine the effect of apoptotic cell death associated protein expression in NCI-H460 NCI-H460/G cells after exposure to PEITC. Results showed that PEITC increased expression of cleaved caspase-3, PARP, GADD153, Endo G and pro-apoptotic protein Bax in NCI-H460/G cells. Based on these results, we suggest that PEITC induces apoptotic cell death via the caspase- and mitochondria-dependent pathway in NCI-H460/G cells. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

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.

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.

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

Effects of blocking developmental cell death on sexually dimorphic calbindin cell groups in the preoptic area and bed nucleus of the stria terminalis

PubMed Central

2012-01-01

Background Calbindin-D28 has been used as a marker for the sexually dimorphic nucleus of the preoptic area (SDN-POA). Males have a distinct cluster of calbindin-immunoreactive (ir) cells in the medial preoptic area (CALB-SDN) that is reduced or absent in females. However, it is not clear whether the sex difference is due to the absolute number of calbindin-ir cells or to cell position (that is, spread), and the cellular mechanisms underlying the sex difference are not known. We examined the number of cells in the CALB-SDN and surrounding regions of C57Bl/6 mice and used mice lacking the pro-death gene, Bax, to test the hypothesis that observed sex differences are due to cell death. Methods Experiment 1 compared the number of cells in the CALB-SDN and surrounding regions in adult males, females, and females injected with estradiol benzoate on the day of birth. In experiment 2, cell number in the CALB-SDN and adjacent regions were compared in wild-type and Bax knockout mice of both sexes. In addition, calbindin-ir cells were quantified within the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), a nearby region that is larger in males due to Bax-dependent cell death. Results Males had more cells in the CALB-SDN as well as in surrounding regions than did females, and estradiol treatment of females at birth masculinized both measures. Bax deletion had no effect on cell number in the CALB-SDN or surrounding regions but increased calbindin-ir cell number in the BNSTp. Conclusions The sex difference in the CALB-SDN of mice results from an estrogen-dependent difference in cell number with no evidence found for greater spread of cells in females. Blocking Bax-dependent cell death does not prevent sex differences in calbindin-ir cell number in the BNST or CALB-SDN but increases calbindin-ir cell number in the BNSTp of both sexes. PMID:22336348

Effects of blocking developmental cell death on sexually dimorphic calbindin cell groups in the preoptic area and bed nucleus of the stria terminalis.

PubMed

Gilmore, Richard F; Varnum, Megan M; Forger, Nancy G

2012-02-15

Calbindin-D28 has been used as a marker for the sexually dimorphic nucleus of the preoptic area (SDN-POA). Males have a distinct cluster of calbindin-immunoreactive (ir) cells in the medial preoptic area (CALB-SDN) that is reduced or absent in females. However, it is not clear whether the sex difference is due to the absolute number of calbindin-ir cells or to cell position (that is, spread), and the cellular mechanisms underlying the sex difference are not known. We examined the number of cells in the CALB-SDN and surrounding regions of C57Bl/6 mice and used mice lacking the pro-death gene, Bax, to test the hypothesis that observed sex differences are due to cell death. Experiment 1 compared the number of cells in the CALB-SDN and surrounding regions in adult males, females, and females injected with estradiol benzoate on the day of birth. In experiment 2, cell number in the CALB-SDN and adjacent regions were compared in wild-type and Bax knockout mice of both sexes. In addition, calbindin-ir cells were quantified within the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), a nearby region that is larger in males due to Bax-dependent cell death. Males had more cells in the CALB-SDN as well as in surrounding regions than did females, and estradiol treatment of females at birth masculinized both measures. Bax deletion had no effect on cell number in the CALB-SDN or surrounding regions but increased calbindin-ir cell number in the BNSTp. The sex difference in the CALB-SDN of mice results from an estrogen-dependent difference in cell number with no evidence found for greater spread of cells in females. Blocking Bax-dependent cell death does not prevent sex differences in calbindin-ir cell number in the BNST or CALB-SDN but increases calbindin-ir cell number in the BNSTp of both sexes.

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

Spore Density Determines Infection Strategy by the Plant Pathogenic Fungus Plectosphaerella cucumerina1[OPEN

PubMed Central

2016-01-01

Necrotrophic and biotrophic pathogens are resisted by different plant defenses. While necrotrophic pathogens are sensitive to jasmonic acid (JA)-dependent resistance, biotrophic pathogens are resisted by salicylic acid (SA)- and reactive oxygen species (ROS)-dependent resistance. Although many pathogens switch from biotrophy to necrotrophy during infection, little is known about the signals triggering this transition. This study is based on the observation that the early colonization pattern and symptom development by the ascomycete pathogen Plectosphaerella cucumerina (P. cucumerina) vary between inoculation methods. Using the Arabidopsis (Arabidopsis thaliana) defense response as a proxy for infection strategy, we examined whether P. cucumerina alternates between hemibiotrophic and necrotrophic lifestyles, depending on initial spore density and distribution on the leaf surface. Untargeted metabolome analysis revealed profound differences in metabolic defense signatures upon different inoculation methods. Quantification of JA and SA, marker gene expression, and cell death confirmed that infection from high spore densities activates JA-dependent defenses with excessive cell death, while infection from low spore densities induces SA-dependent defenses with lower levels of cell death. Phenotyping of Arabidopsis mutants in JA, SA, and ROS signaling confirmed that P. cucumerina is differentially resisted by JA- and SA/ROS-dependent defenses, depending on initial spore density and distribution on the leaf. Furthermore, in situ staining for early callose deposition at the infection sites revealed that necrotrophy by P. cucumerina is associated with elevated host defense. We conclude that P. cucumerina adapts to early-acting plant defenses by switching from a hemibiotrophic to a necrotrophic infection program, thereby gaining an advantage of immunity-related cell death in the host. PMID:26842622

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.

Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS.

PubMed

Wrzaczek, Michael; Brosché, Mikael; Kollist, Hannes; Kangasjärvi, Jaakko

2009-03-31

Reactive oxygen species (ROS) have important functions in plant stress responses and development. In plants, ozone and pathogen infection induce an extracellular oxidative burst that is involved in the regulation of cell death. However, very little is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an Arabidopsis thaliana protein, GRIM REAPER (GRI), that is involved in the regulation of cell death induced by extracellular ROS. Plants with an insertion in GRI display an ozone-sensitive phenotype. GRI is an Arabidopsis ortholog of the tobacco flower-specific Stig1 gene. The GRI protein appears to be processed in leaves with a release of an N-terminal fragment of the protein. Infiltration of the N-terminal fragment of the GRI protein into leaves caused cell death in a superoxide- and salicylic acid-dependent manner. Analysis of the extracellular GRI protein yields information on how plants can initiate ROS-induced cell death during stress response and development.

Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS

PubMed Central

Wrzaczek, Michael; Brosché, Mikael; Kollist, Hannes; Kangasjärvi, Jaakko

2009-01-01

Reactive oxygen species (ROS) have important functions in plant stress responses and development. In plants, ozone and pathogen infection induce an extracellular oxidative burst that is involved in the regulation of cell death. However, very little is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an Arabidopsis thaliana protein, GRIM REAPER (GRI), that is involved in the regulation of cell death induced by extracellular ROS. Plants with an insertion in GRI display an ozone-sensitive phenotype. GRI is an Arabidopsis ortholog of the tobacco flower-specific Stig1 gene. The GRI protein appears to be processed in leaves with a release of an N-terminal fragment of the protein. Infiltration of the N-terminal fragment of the GRI protein into leaves caused cell death in a superoxide- and salicylic acid-dependent manner. Analysis of the extracellular GRI protein yields information on how plants can initiate ROS-induced cell death during stress response and development. PMID:19279211

Caspase inhibition augmented oridonin-induced cell death in murine fibrosarcoma l929 by enhancing reactive oxygen species generation.

PubMed

Wu, Jin-Nan; Huang, Jian; Yang, Jia; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2008-09-01

Oridonin, a diterpenoid isolated from Rabdosia rubescences, has been reported to have antitumor effects. In this study, the growth-inhibitory activity of oridonin for L929 cells was exerted in a time-and dose-dependent manner. After treatment with oridonin for 24 h, L929 cells underwent both apoptosis and necrosis as measured by an lactate dehydrogenase (LDH) activity-based assay. A rapid generation of reactive oxygen species (ROS) was triggered by oridonin, and subsequently up-regulation of phospho-p53 (ser 15) expression and an increased expression ratio of Bax/Bcl-2 was observed. Furthermore, there was a significant fall in mitochondrial membrane potential (MMP) and increase in caspase-3 activity after exposure to oridonin for 24 h. Surprisingly, the pan-caspase inhibitor z-VAD-fmk and caspase3 inhibitor z-DEVD-fmk rendered L929 cells more sensitive to oridonin, rather than preventing oridonin-induced cell death. Oridonin and z-VAD-fmk co-treatment not only resulted in an even higher ROS production, but also made a more significant reduction in the MMP. Pretreatment of ROS scavenger N-acetylcysteine (NAC) led to a complete inhibition of oridonin-induced cell death, intracellular ROS generation, and MMP collapse. NAC treatment also reversed the potentiation of cell death by the pan-caspase inhibitor z-VAD-fmk. Taken together, these observations showed that oridonin-induced cell death in L929 cells involved intracellular ROS generation, activation of phospho-p53 (ser 15), and up-regulation of the Bax/Bcl-2 ratio; and the augmented cell death by z-VAD-fmk was dependent on an increased ROS production.

Loss of BID Delays FASL-Induced Cell Death of Mouse Neutrophils and Aggravates DSS-Induced Weight Loss.

PubMed

Wicki, Simone; Gurzeler, Ursina; Corazza, Nadia; Genitsch, Vera; Wong, Wendy Wei-Lynn; Kaufmann, Thomas

2018-02-28

Neutrophils are key players in the early defense against invading pathogens. Due to their potent effector functions, programmed cell death of activated neutrophils has to be tightly controlled; however, its underlying mechanisms remain unclear. Fas ligand (FASL/CD95L) has been shown to induce neutrophil apoptosis, which is accelerated by the processing of the BH3-only protein BH3 interacting domain death agonist (BID) to trigger mitochondrial apoptotic events, and been attributed a regulatory role during viral and bacterial infections. Here, we show that, in accordance with previous works, mouse neutrophils underwent caspase-dependent apoptosis in response to FASL, and that this cell death was significantly delayed upon loss of BID. However, pan-caspase inhibition failed to protect mouse neutrophils from FASL-induced apoptosis and caused a switch to RIPK3-dependent necroptotic cell death. Intriguingly, such a switch was less evident in the absence of BID, particularly under inflammatory conditions. Delayed neutrophil apoptosis has been implicated in several auto-inflammatory diseases, including inflammatory bowel disease. We show that neutrophil and macrophage driven acute dextran sulfate sodium (DSS) induced colitis was slightly more aggravated in BID-deficient mice, based on significantly increased weight loss compared to wild-type controls. Taken together, our data support a central role for FASL > FAS and BID in mouse neutrophil cell death and further underline the anti-inflammatory role of BID.

Loss of BID Delays FASL-Induced Cell Death of Mouse Neutrophils and Aggravates DSS-Induced Weight Loss

PubMed Central

Wicki, Simone; Gurzeler, Ursina; Corazza, Nadia; Genitsch, Vera

2018-01-01

Neutrophils are key players in the early defense against invading pathogens. Due to their potent effector functions, programmed cell death of activated neutrophils has to be tightly controlled; however, its underlying mechanisms remain unclear. Fas ligand (FASL/CD95L) has been shown to induce neutrophil apoptosis, which is accelerated by the processing of the BH3-only protein BH3 interacting domain death agonist (BID) to trigger mitochondrial apoptotic events, and been attributed a regulatory role during viral and bacterial infections. Here, we show that, in accordance with previous works, mouse neutrophils underwent caspase-dependent apoptosis in response to FASL, and that this cell death was significantly delayed upon loss of BID. However, pan-caspase inhibition failed to protect mouse neutrophils from FASL-induced apoptosis and caused a switch to RIPK3-dependent necroptotic cell death. Intriguingly, such a switch was less evident in the absence of BID, particularly under inflammatory conditions. Delayed neutrophil apoptosis has been implicated in several auto-inflammatory diseases, including inflammatory bowel disease. We show that neutrophil and macrophage driven acute dextran sulfate sodium (DSS) induced colitis was slightly more aggravated in BID-deficient mice, based on significantly increased weight loss compared to wild-type controls. Taken together, our data support a central role for FASL > FAS and BID in mouse neutrophil cell death and further underline the anti-inflammatory role of BID. PMID:29495595

Necroptosis: an emerging type of cell death in liver diseases.

PubMed

Saeed, Waqar Khalid; Jun, Dae Won

2014-09-21

Cell death has been extensively evaluated for decades and it is well recognized that pharmacological interventions directed to inhibit cell death can prevent significant cell loss and can thus improve an organ's physiological function. For long, only apoptosis was considered as a sole form of programmed cell death. Recently necroptosis, a RIP1/RIP3-dependent programmed cell death, has been identified as an apoptotic backup cell death mechanism with necrotic morphology. The evidences of necroptosis and protective effects achieved by blocking necroptosis have been extensively reported in recent past. However, only a few studies reported the evidence of necroptosis and protective effects achieved by inhibiting necroptosis in liver related disease conditions. Although the number of necroptosis initiators is increasing; however, interestingly, it is still unclear that what actually triggers necroptosis in different liver diseases or if there is always a different necroptosis initiator in each specific disease condition followed by specific downstream signaling molecules. Understanding the precise mechanism of necroptosis as well as counteracting other cell death pathways in liver diseases could provide a useful insight towards achieving extensive therapeutic significance. By targeting necroptosis and/or other parallel death pathways, a significant cell loss and thus a decrement in an organ's physiological function can be prevented.

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.

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.

Acute myeloid leukemia-targeted toxin activates both apoptotic and necroptotic death mechanisms.

PubMed

Horita, Henrick; Frankel, Arthur E; Thorburn, Andrew

2008-01-01

Acute myelogenous leukemia (AML) is the second most common leukemia with approximately 13,410 new cases and 8,990 deaths annually in the United States. A novel fusion toxin treatment, diphtheria toxin GM-CSF (DT-GMCSF) has been shown to selectively eliminate leukemic repopulating cells that are critical for the formation of AML. We previously showed that DT-GMCSF treatment of U937 cells, an AML cell line, causes activation of caspases and the induction of apoptosis. In this study we further investigate the mechanisms of cell death induced by DT-GMCSF and show that, in addition to the activation of caspase-dependent apoptosis, DT-GMCSF also kills AML cells by simultaneously activating caspase-independent necroptosis. These mechanisms depend on the ability of the targeted toxin to inhibit protein synthesis, and are not affected by the receptor that is targeted or the mechanism through which protein synthesis is blocked. We conclude that fusion toxin proteins may be effective for treating AML cells whether or not they are defective in apoptosis.

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

Engineering death receptor ligands for cancer therapy.

PubMed

Wajant, Harald; Gerspach, Jeannette; Pfizenmaier, Klaus

2013-05-28

CD95, TNFR1, TRAILR1 and TRAILR2 belong to a subgroup of TNF receptors which is characterized by a conserved cell death-inducing protein domain that connects these receptors to the apoptotic machinery of the cell. Activation of death receptors in malignant cells attracts increasing attention as a principle to fight cancer. Besides agonistic antibodies the major way to stimulate death receptors is the use of their naturally occurring "death ligands" CD95L, TNF and TRAIL. However, dependent from the concept followed to develop a death ligand-based therapy various limiting aspects have to be taken into consideration on the way to a "bedside" usable drug. Problems arise in particular from the cell associated transmembrane nature of the death ligands, the poor serum half life of the soluble fragments derived from the transmembrane ligands, the ubiquitous expression of the death receptors and the existence of additional non-death receptors of the death ligands. Here, we summarize strategies how these limitations can be overcome by genetic engineering. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

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

The slow cell death response when screening chemotherapeutic agents.

PubMed

Blois, Joseph; Smith, Adam; Josephson, Lee

2011-09-01

To examine the correlation between cell death and a common surrogate of death used in screening assays, we compared cell death responses to those obtained with the sulforhodamine B (SRB) cell protein-based "cytotoxicity" assay. With the SRB assay, the Hill equation was used to obtain an IC50 and final cell mass, or cell mass present at infinite agent concentrations, with eight adherent cell lines and four agents (32 agent/cell combinations). Cells were treated with high agent concentrations (well above the SRB IC50) and the death response determined as the time-dependent decrease in cells failing to bind both annexin V and vital fluorochromes by flow cytometry. Death kinetics were categorized as fast (5/32) (similar to the reference nonadherent Jurkat line), slow (17/32), or none (10/32), despite positive responses in the SRB assay in all cases. With slow cell death, a single exposure to a chemotherapeutic agent caused a slow, progressive increase in dead (necrotic) and dying (apoptotic) cells for at least 72 h. Cell death (defined by annexin and/or fluorochrome binding) did not correlate with the standard SRB "cytotoxicity" assay. With the slow cell death response, a single exposure to an agent caused a slow conversion from vital to apoptotic and necrotic cells over at least 72 h (the longest time point examined). Here, increasing the time of exposure to agent concentrations modestly above the SRB IC50 provides a method of maximizing cell kill. If tumors respond similarly, sustained low doses of chemotherapeutic agents, rather than a log-kill, maximum tolerated dose strategy may be an optimal strategy of maximizing tumor cell death.

Role of mTOR, Bad, and Survivin in RasGAP Fragment N-Mediated Cell Protection

PubMed Central

Yang, Jiang-Yan; Widmann, Christian

2013-01-01

Partial cleavage of p120 RasGAP by caspase-3 in stressed cells generates an N-terminal fragment, called fragment N, which activates an anti-apoptotic Akt-dependent survival response. Akt regulates several effectors but which of these mediate fragment N-dependent cell protection has not been defined yet. Here we have investigated the role of mTORC1, Bad, and survivin in the capacity of fragment N to protect cells from apoptosis. Neither rapamycin, an inhibitor of mTORC1, nor silencing of raptor, a subunit of the mTORC1 complex, altered the ability of fragment N from inhibiting cisplatin- and Fas ligand-induced death. Cells lacking Bad, despite displaying a stronger resistance to apoptosis, were still protected by fragment N against cisplatin-induced death. Fragment N was also able to protect cells from Fas ligand-induced death in conditions where Bad plays no role in apoptosis regulation. Fragment N expression in cells did neither modulate survivin mRNA nor its protein expression. Moreover, the expression of cytoplasmic survivin, known to exert anti-apoptotic actions in cells, still occurred in UV-B-irradiated epidermis of mouse expressing a caspase-3-resistant RasGAP mutant that cannot produce fragment N. Additionally, survivin function in cell cycle progression was not affected by fragment N. These results indicate that, taken individually, mTOR, Bad, or Survivin are not required for fragment N to protect cells from cell death. We conclude that downstream targets of Akt other than mTORC1, Bad, or survivin mediate fragment N-induced protection or that several Akt effectors can compensate for each other to induce the pro-survival fragment N-dependent response. PMID:23826368

Distinct Signaling Pathways Mediate Stimulation of Cell Cycle Progression and Prevention of Apoptotic Cell Death by Estrogen in Rat Pituitary Tumor PR1 Cells

PubMed Central

Caporali, Simona; Imai, Manami; Altucci, Lucia; Cancemi, Massimo; Caristi, Silvana; Cicatiello, Luigi; Matarese, Filomena; Penta, Roberta; Sarkar, Dipak K.; Bresciani, Francesco; Weisz, Alessandro

2003-01-01

Estrogens control cell growth and viability in target cells via an interplay of genomic and extragenomic pathways not yet elucidated. Here, we show evidence that cell proliferation and survival are differentially regulated by estrogen in rat pituitary tumor PR1 cells. Pico- to femtomolar concentrations of 17β-estradiol (E2) are sufficient to foster PR1 cell proliferation, whereas nanomolar concentrations of the same are needed to prevent cell death that occurs at a high rate in these cells in the absence of hormone. Activation of endogenous (PRL) or transfected estrogen-responsive genes occurs at the same, higher concentrations of E2 required to promote cell survival, whereas stimulation of cyclin D3 expression and DNA synthesis occur at lower E2 concentrations. Similarly, the pure antiestrogen ICI 182,780 inhibits estrogen response element-dependent trans-activation and cell death more effectively than cyclin-cdk activity, G1-S transition, or DNA synthesis rate. In antiestrogen-treated and/or estrogen-deprived cells, death is due predominantly to apoptosis. Estrogen-induced cell survival, but not E2-dependent cell cycle progression, can be prevented by an inhibitor of c-Src kinase or by blockade of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathway. These data indicate the coexistence of two distinguishable estrogen signaling pathways in PR1 cells, characterized by different functions and sensitivity to hormones and antihormones. PMID:12960425

Poncirin Induces Apoptosis in AGS Human Gastric Cancer Cells through Extrinsic Apoptotic Pathway by up-Regulation of Fas Ligand.

PubMed

Saralamma, Venu Venkatarame Gowda; Nagappan, Arulkumar; Hong, Gyeong Eun; Lee, Ho Jeong; Yumnam, Silvia; Raha, Suchismita; Heo, Jeong Doo; Lee, Sang Joon; Lee, Won Sup; Kim, Eun Hee; Kim, Gon Sup

2015-09-18

Poncirin, a natural bitter flavanone glycoside abundantly present in many species of citrus fruits, has various biological benefits such as anti-oxidant, anti-microbial, anti-inflammatory and anti-cancer activities. The anti-cancer mechanism of Poncirin remains elusive to date. In this study, we investigated the anti-cancer effects of Poncirin in AGS human gastric cancer cells (gastric adenocarcinoma). The results revealed that Poncirin could inhibit the proliferation of AGS cells in a dose-dependent manner. It was observed Poncirin induced accumulation of sub-G1 DNA content, apoptotic cell population, apoptotic bodies, chromatin condensation, and DNA fragmentation in a dose-dependent manner in AGS cells. The expression of Fas Ligand (FasL) protein was up-regulated dose dependently in Poncirin-treated AGS cells Moreover, Poncirin in AGS cells induced activation of Caspase-8 and -3, and subsequent cleavage of poly(ADP-ribose) polymerase (PARP). Inhibitor studies' results confirm that the induction of caspase-dependent apoptotic cell death in Poncirin-treated AGS cells was led by the Fas death receptor. Interestingly, Poncirin did not show any effect on mitochondrial membrane potential (ΔΨm), pro-apoptotic proteins (Bax and Bak) and anti-apoptotic protein (Bcl-xL) in AGS-treated cells followed by no activation in the mitochondrial apoptotic protein caspase-9. This result suggests that the mitochondrial-mediated pathway is not involved in Poncirin-induced cell death in gastric cancer. These findings suggest that Poncirin has a potential anti-cancer effect via extrinsic pathway-mediated apoptosis, possibly making it a strong therapeutic agent for human gastric cancer.

The lysosomotropic drug LeuLeu-OMe induces lysosome disruption and autophagy-independent cell death in Trypanosoma brucei

PubMed Central

Koh, Hazel X.; Aye, Htay M.; Tan, Kevin S. W.; He, Cynthia Y.

2015-01-01

Background: Trypanosoma brucei is a blood-borne, protozoan parasite that causes African sleeping sickness in humans and nagana in animals. The current chemotherapy relies on only a handful of drugs that display undesirable toxicity, poor efficacy and drug-resistance. In this study, we explored the use of lysosomotropic drugs to induce bloodstream form T. brucei cell death via lysosome destabilization. Methods: We measured drug concentrations that inhibit cell proliferation by 50% (IC_{50) for several compounds, chosen based on their lysosomotropic effects previously reported in Plasmodium falciparum. The lysosomal effects and cell death induced by L-leucyl-L-leucyl methyl ester (LeuLeu-OMe) were further analyzed by flow cytometry and immunofluorescence analyses of different lysosomal markers. The effect of autophagy in LeuLeu-OMe-induced lysosome destabilization and cytotoxicity was also investigated in control and autophagy-deficient cells. Results: LeuLeu-OMe was selected for detailed analyses due to its strong inhibitory profile against T. brucei with minimal toxicity to human cell lines in vitro. Time-dependent immunofluorescence studies confirmed an effect of LeuLeu-OMe on the lysosome. LeuLeu-OMe-induced cytotoxicity was also found to be dependent on the acidic pH of the lysosome. Although an increase in autophagosomes was observed upon LeuLeu-OMe treatment, autophagy was not required for the cell death induced by LeuLeu-OMe. Necrosis appeared to be the main cause of cell death upon LeuLeu-OMe treatment. Conclusions: LeuLeu-OMe is a lysosomotropic agent capable of destabilizing lysosomes and causing necrotic cell death in bloodstream form of T. brucei. PMID:28357304}

A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers

PubMed Central

LaBelle, James L.; Katz, Samuel G.; Bird, Gregory H.; Gavathiotis, Evripidis; Stewart, Michelle L.; Lawrence, Chelsea; Fisher, Jill K.; Godes, Marina; Pitter, Kenneth; Kung, Andrew L.; Walensky, Loren D.

2012-01-01

Cancer cells subvert the natural balance between cellular life and death, achieving immortality through pathologic enforcement of survival pathways and blockade of cell death mechanisms. Pro-apoptotic BCL-2 family proteins are frequently disarmed in relapsed and refractory cancer through genetic deletion or interaction-based neutralization by overexpressed antiapoptotic proteins, resulting in resistance to chemotherapy and radiation treatments. New pharmacologic strategies are urgently needed to overcome these formidable apoptotic blockades. We harnessed the natural killing activity of BCL-2–interacting mediator of cell death (BIM), which contains one of the most potent BH3 death domains of the BCL-2 protein family, to restore BH3-dependent cell death in resistant hematologic cancers. A hydrocarbon-stapled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity, blocked inhibitory antiapoptotic interactions, directly triggered proapoptotic activity, and induced dose-responsive and BH3 sequence–specific cell death of hematologic cancer cells. The therapeutic potential of stapled BIM BH3 was highlighted by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. Thus, we found that broad and multimodal targeting of the BCL-2 family pathway can overcome pathologic barriers to cell death. PMID:22622039

S -Nitrosylation inhibits the kinase activity of tomato phosphoinositide-dependent kinase 1 (PDK1)

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

Liu, Jian-Zhong; Duan, Jicheng; Ni, Min

It is well known that the reactive oxygen species NO can trigger cell death in plants and other organisms, but the underlying molecular mechanisms are not well understood. Here we provide evidence that NO may trigger cell death in tomato (Solanum lycopersicum) by inhibiting the activity of phosphoinositide-dependent kinase 1 (SlPDK1), a conserved negative regulator of cell death in yeasts, mammals, and plants, via S-nitrosylation. Biotin-switch assays indicated that SlPDK1 is a target of S-nitrosylation. Moreover, the kinase activity of SlPDK1 was inhibited by S-nitrosoglutathione in a concentration-dependent manner, indicating that SlPDK1 activity is abrogated by S-nitrosylation. The S-nitrosoglutathione–induced inhibitionmore » was reversible in the presence of a reducing agent but additively enhanced by hydrogen peroxide (H 2O 2). Our LC-MS/MS analyses further indicated that SlPDK1 is primarily S-nitrosylated on a cysteine residue at position 128 (Cys 128), and substitution of Cys 128 with serine completely abolished SlPDK1 kinase activity, suggesting that S-nitrosylation of Cys 128 is responsible for SlPDK1 inhibition. In summary, our results establish a potential link between NO-triggered cell death and inhibition of the kinase activity of tomato PDK1.« less

S -Nitrosylation inhibits the kinase activity of tomato phosphoinositide-dependent kinase 1 (PDK1)

DOE PAGES

Liu, Jian-Zhong; Duan, Jicheng; Ni, Min; ...

2017-09-29

It is well known that the reactive oxygen species NO can trigger cell death in plants and other organisms, but the underlying molecular mechanisms are not well understood. Here we provide evidence that NO may trigger cell death in tomato (Solanum lycopersicum) by inhibiting the activity of phosphoinositide-dependent kinase 1 (SlPDK1), a conserved negative regulator of cell death in yeasts, mammals, and plants, via S-nitrosylation. Biotin-switch assays indicated that SlPDK1 is a target of S-nitrosylation. Moreover, the kinase activity of SlPDK1 was inhibited by S-nitrosoglutathione in a concentration-dependent manner, indicating that SlPDK1 activity is abrogated by S-nitrosylation. The S-nitrosoglutathione–induced inhibitionmore » was reversible in the presence of a reducing agent but additively enhanced by hydrogen peroxide (H 2O 2). Our LC-MS/MS analyses further indicated that SlPDK1 is primarily S-nitrosylated on a cysteine residue at position 128 (Cys 128), and substitution of Cys 128 with serine completely abolished SlPDK1 kinase activity, suggesting that S-nitrosylation of Cys 128 is responsible for SlPDK1 inhibition. In summary, our results establish a potential link between NO-triggered cell death and inhibition of the kinase activity of tomato PDK1.« less

Unsolved mysteries: How does lipid peroxidation cause ferroptosis?

PubMed Central

Feng, Huizhong

2018-01-01

Ferroptosis is a cell death process driven by damage to cell membranes and linked to numerous human diseases. Ferroptosis is caused by loss of activity of the key enzyme that is tasked with repairing oxidative damage to cell membranes—glutathione peroxidase 4 (GPX4). GPX4 normally removes the dangerous products of iron-dependent lipid peroxidation, protecting cell membranes from this type of damage; when GPX4 fails, ferroptosis ensues. Ferroptosis is distinct from apoptosis, necroptosis, necrosis, and other modes of cell death. Several key mysteries regarding how cells die during ferroptosis remain unsolved. First, the drivers of lipid peroxidation are not yet clear. Second, the subcellular location of lethal lipid peroxides remains an outstanding question. Finally, how exactly lipid peroxidation leads to cell death is an unsolved mystery. Answers to these questions will provide insights into the mechanisms of ferroptotic cell death and associated human diseases, as well as new therapeutic strategies for such diseases. PMID:29795546

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.

Hop/STI1 modulates retinal proliferation and cell death independent of PrP{sup C}

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

Arruda-Carvalho, Maithe; Njaine, Brian; Silveira, Mariana S.

Hop/STI1 is a co-chaperone adaptor protein for Hsp70/Hsp90 complexes. Hop/STI1 is found extracellularly and modulates cell death and differentiation through interaction with the prion protein (PrP{sup C}). Here, we investigated the expression of hop/STI1 and its role upon cell proliferation and cell death in the developing retina. Hop/STI1 is more expressed in developing rat retina than in the mature tissue. Hop/STI1 blocks retinal cell death in the neuroblastic layer (NBL) in a PrP{sup C} dependent manner, but failed to protect ganglion cells against axotomy-induced cell death. An antibody raised against hop/STI1 ({alpha}-STI1) blocked both ganglion cell and NBL cell deathmore » independent of PrP{sup C}. cAMP/PKA, ERK, PI3K and PKC signaling pathways were not involved in these effects. Hop/STI1 treatment reduced proliferation, while {alpha}-STI1 increased proliferation in the developing retina, both independent of PrP{sup C}. We conclude that hop/STI1 can modulate both proliferation and cell death in the developing retina independent of PrP{sup C}.« less

Apoptotic death in cerebral hemisphere cells is density dependent and modulated by transient oxygen and glucose deprivation.

PubMed

Yavin, E; Billia, D M

1997-03-01

Flow cytometry, light and fluorescence microscopy, and designated biochemical techniques were used to examine the type of death which occurs in cerebral cortex cells when grown under crowded vs. sparse conditions or after brief anoxia/hypoglycemia. A 4 hr episode of anoxia combined with glucose deprivation enhanced apoptotic cell death as assessed by 4',6-diamidino-2-phenylindole (DAPI) staining and reduced neutral red eye uptake. An additional form of cell death involving exclusion of the nucleus was recorded by time lapse cinematography and DAPI stain. The presence of the endonuclease inhibitor aurintricarboxylic acid (0.1 mM) reduced cell death by 56.6%, while the protein and RNA synthesis inhibitors actinomycin D and cycloheximide (each at 5 micrograms/ml) effectively decreased cell death by 83.3% and 90.6%, respectively. In contrast, 5 mM glutamate had no effect on cell death in accord with the immature state of the cells. Growth of cells under crowded conditions improved cell survival; after 2 h or 4 days in culture, cells seeded at high density (34 microgram cellular DNA/cm2) showed a nearly 3-fold decline in the amount of cell death in comparison to cells seeded at low density (5 micrograms cellular DNA/cm2). At high cell density, anoxic episodes enhanced cell death most likely by preventing a cell density-mediated rescue. Neutral red dye uptake, an index for cell viability, was enhanced with increasing cell density and in vitro maturation, but was reduced in dense cultures exposed to anoxic/hypoglycemic conditions. The data suggest that cell density may play a critical role in brain organogenesis and that anoxic stress is more deleterious in dense than sparse cell assemblies.

High-throughput monitoring of major cell functions by means of lensfree video microscopy

PubMed Central

Kesavan, S. Vinjimore; Momey, F.; Cioni, O.; David-Watine, B.; Dubrulle, N.; Shorte, S.; Sulpice, E.; Freida, D.; Chalmond, B.; Dinten, J. M.; Gidrol, X.; Allier, C.

2014-01-01

Quantification of basic cell functions is a preliminary step to understand complex cellular mechanisms, for e.g., to test compatibility of biomaterials, to assess the effectiveness of drugs and siRNAs, and to control cell behavior. However, commonly used quantification methods are label-dependent, and end-point assays. As an alternative, using our lensfree video microscopy platform to perform high-throughput real-time monitoring of cell culture, we introduce specifically devised metrics that are capable of non-invasive quantification of cell functions such as cell-substrate adhesion, cell spreading, cell division, cell division orientation and cell death. Unlike existing methods, our platform and associated metrics embrace entire population of thousands of cells whilst monitoring the fate of every single cell within the population. This results in a high content description of cell functions that typically contains 25,000 – 900,000 measurements per experiment depending on cell density and period of observation. As proof of concept, we monitored cell-substrate adhesion and spreading kinetics of human Mesenchymal Stem Cells (hMSCs) and primary human fibroblasts, we determined the cell division orientation of hMSCs, and we observed the effect of transfection of siCellDeath (siRNA known to induce cell death) on hMSCs and human Osteo Sarcoma (U2OS) Cells. PMID:25096726

Mitochondrial Ca2+ influx targets cardiolipin to disintegrate respiratory chain complex II for cell death induction

PubMed Central

Hwang, M-S; Schwall, C T; Pazarentzos, E; Datler, C; Alder, N N; Grimm, S

2014-01-01

Massive Ca2+ influx into mitochondria is critically involved in cell death induction but it is unknown how this activates the organelle for cell destruction. Using multiple approaches including subcellular fractionation, FRET in intact cells, and in vitro reconstitutions, we show that mitochondrial Ca2+ influx prompts complex II of the respiratory chain to disintegrate, thereby releasing an enzymatically competent sub-complex that generates excessive reactive oxygen species (ROS) for cell death induction. This Ca2+-dependent dissociation of complex II is also observed in model membrane systems, but not when cardiolipin is replaced with a lipid devoid of Ca2+ binding. Cardiolipin is known to associate with complex II and upon Ca2+ binding coalesces into separate homotypic clusters. When complex II is deprived of this lipid, it disintegrates for ROS formation and cell death. Our results reveal Ca2+ binding to cardiolipin for complex II disintegration as a pivotal step for oxidative stress and cell death induction. PMID:24948011

Reciprocal sensitivity of diffuse large B-cell lymphoma cells to Bcl-2 inhibitors BIRD-2 versus venetoclax.

PubMed

Vervloessem, Tamara; Akl, Haidar; Tousseyn, Thomas; De Smedt, Humbert; Parys, Jan B; Bultynck, Geert

2017-12-19

Bcl-2 is often upregulated in cancers to neutralize the BH3-only protein Bim at the mitochondria. BH3 mimetics (e.g. ABT-199 (venetoclax)) kill cancer cells by targeting Bcl-2's hydrophobic cleft and disrupting Bcl-2/Bim complexes. Some cancers with elevated Bcl-2 display poor responses towards BH3 mimetics, suggesting an additional function for anti-apoptotic Bcl-2 in these cancers. Indeed, Bcl-2 via its BH4 domain prevents cytotoxic Ca 2+ release from the endoplasmic reticulum (ER) by directly inhibiting the inositol 1,4,5-trisphosphate receptor (IP 3 R). The cell-permeable Bcl-2/IP 3 R disruptor-2 (BIRD-2) peptide can kill these Bcl-2-dependent cancers by targeting Bcl-2's BH4 domain, unleashing pro-apoptotic Ca 2+ -release events. We compared eight "primed to death" diffuse large B-cell lymphoma cell lines (DLBCL) for their apoptotic sensitivity towards BIRD-2 and venetoclax. By determining their IC 50 using cytometric cell-death analysis, we discovered a reciprocal sensitivity towards venetoclax versus BIRD-2. Using immunoblotting, we quantified the expression levels of IP 3 R2 and Bim in DLBCL cell lysates, revealing that BIRD-2 sensitivity correlated with IP 3 R2 levels but not with Bim levels. Moreover, the requirement of intracellular Ca 2+ for BIRD-2- versus venetoclax-induced cell death was different. Indeed, BAPTA-AM suppressed BIRD-2-induced cell death, but promoted venetoclax-induced cell death in DLBCL cells. Finally, compared to single-agent treatments, combining BIRD-2 with venetoclax synergistically enhanced cell-death induction, correlating with a Ca 2+ -dependent upregulation of Bim after BIRD-2 treatment. Our findings suggest that some cancer cells require Bcl-2 proteins at the mitochondria, preventing Bax activation via its hydrophobic cleft, while others require Bcl-2 proteins at the ER, preventing cytotoxic Ca 2+ -signaling events via its BH4 domain.

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.

Trial watch: Immunogenic cell death induction by anticancer chemotherapeutics.

PubMed

Garg, Abhishek D; More, Sanket; Rufo, Nicole; Mece, Odeta; Sassano, Maria Livia; Agostinis, Patrizia; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

2017-01-01

The expression "immunogenic cell death" (ICD) refers to a functionally unique form of cell death that facilitates (instead of suppressing) a T cell-dependent immune response specific for dead cell-derived antigens. ICD critically relies on the activation of adaptive responses in dying cells, culminating with the exposure or secretion of immunostimulatory molecules commonly referred to as "damage-associated molecular patterns". Only a few agents can elicit bona fide ICD, including some clinically established chemotherapeutics such as doxorubicin, epirubicin, idarubicin, mitoxantrone, bleomycin, bortezomib, cyclophosphamide and oxaliplatin. In this Trial Watch, we discuss recent progress on the development of ICD-inducing chemotherapeutic regimens, focusing on studies that evaluate clinical efficacy in conjunction with immunological biomarkers.

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.

Senescence, apoptosis or autophagy? When a damaged cell must decide its path--a mini-review.

PubMed

Vicencio, José Miguel; Galluzzi, Lorenzo; Tajeddine, Nicolas; Ortiz, Carla; Criollo, Alfredo; Tasdemir, Ezgi; Morselli, Eugenia; Ben Younes, Amena; Maiuri, Maria Chiara; Lavandero, Sergio; Kroemer, Guido

2008-01-01

Many features of aging result from the incapacity of cells to adapt to stress conditions. When damage accumulates irreversibly, mitotic cells from renewable tissues rely on either of two mechanisms to avoid replication. They can permanently arrest the cell cycle (cellular senescence) or trigger cell death programs. Apoptosis (self-killing) is the best-described form of programmed cell death, but autophagy (self-eating), which is a lysosomal degradation pathway essential for homeostasis, reportedly contributes to cell death as well. Unlike mitotic cells, postmitotic cells like neurons or cardiomyocytes cannot become senescent since they are already terminally differentiated. The fate of these cells entirely depends on their ability to cope with stress. Autophagy then operates as a major homeostatic mechanism to eliminate damaged organelles, long-lived or aberrant proteins and superfluous portions of the cytoplasm. In this mini-review, we briefly summarize the molecular networks that allow damaged cells either to adapt to stress or to engage in programmed-cell-death pathways. (c) 2008 S. Karger AG, Basel.

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

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

Blazeispirol A from Agaricus blazei fermentation product induces cell death in human hepatoma Hep 3B cells through caspase-dependent and caspase-independent pathways.

PubMed

Su, Zheng-Yuan; Tung, Yen-Chen; Hwang, Lucy Sun; Sheen, Lee-Yan

2011-05-11

Currently, liver cancer is a leading cause of cancer-related death in the world. Hepatocellular carcinoma is the most common type of liver cancer. Previously, it was reported that blazeispirol A (BA) is the most active antihepatoma compound in an ethanolic extract of Agaricus blazei fermentation product. The aim of this study was to understand the antihepatoma mechanism of BA in human liver cancer Hep 3B cells. The results showed that BA inhibited the growth of Hep 3B cells and increased the percentage of cells in sub-G1 phase in a concentration- and time-dependent manner. In addition, BA treatment resulted in DNA fragmentation, caspase-9 and caspase-3 activations, poly(ADP-ribose)polymerase (PARP) degradation, down-regulation of Bcl-2 and Bcl-xL expressions, up-regulation of Bax expression, and disruption of the mitochondrial membrane potential (MMP) in Hep 3B cells. Furthermore, z-VAD-fmk, a caspase inhibitor, did not enhance the viability of BA-treated Hep 3B cells, and BA induced the release of HtrA2/Omi and apoptosis-inducing factor (AIF) from mitochondria into the cytosol. These findings suggested that BA with novel chemopreventive and chemotherapeutic potentials causes both caspase-dependent and caspase-independent cell death in Hep 3B cells.

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.

Deciphering early events involved in hyperosmotic stress-induced programmed cell death in tobacco BY-2 cells.

PubMed

Monetti, Emanuela; Kadono, Takashi; Tran, Daniel; Azzarello, Elisa; Arbelet-Bonnin, Delphine; Biligui, Bernadette; Briand, Joël; Kawano, Tomonori; Mancuso, Stefano; Bouteau, François

2014-03-01

Hyperosmotic stresses represent one of the major constraints that adversely affect plants growth, development, and productivity. In this study, the focus was on early responses to hyperosmotic stress- (NaCl and sorbitol) induced reactive oxygen species (ROS) generation, cytosolic Ca(2+) concentration ([Ca(2+)]cyt) increase, ion fluxes, and mitochondrial potential variations, and on their links in pathways leading to programmed cell death (PCD). By using BY-2 tobacco cells, it was shown that both NaCl- and sorbitol-induced PCD seemed to be dependent on superoxide anion (O2·(-)) generation by NADPH-oxidase. In the case of NaCl, an early influx of sodium through non-selective cation channels participates in the development of PCD through mitochondrial dysfunction and NADPH-oxidase-dependent O2·(-) generation. This supports the hypothesis of different pathways in NaCl- and sorbitol-induced cell death. Surprisingly, other shared early responses, such as [Ca(2+)]cyt increase and singlet oxygen production, do not seem to be involved in PCD.

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

CRM1 protein-mediated regulation of nuclear clusterin (nCLU), an ionizing radiation-stimulated, Bax-dependent pro-death factor.

PubMed

Leskov, Konstantin S; Araki, Shinako; Lavik, John-Paul; Gomez, Jose A; Gama, Vivian; Gonos, Efstathios S; Trougakos, Ioannis P; Matsuyama, Shigemi; Boothman, David A

2011-11-18

Expression of the clusterin (CLU) gene results in the synthesis of a conventional secretory isoform set (pre- and mature secretory clusterin proteins, psCLU/sCLU), as well as another set of intracellular isoforms, appearing in the cytoplasm (pre-nuclear CLU, pnCLU) and in the nucleus as an ∼55-kDa mature nuclear clusterin (nCLU) form. These two isoform sets have opposing cell functions: pro-survival and pro-death, respectively. Although much is known about the regulation and function of sCLU as a pro-survival factor, the regulation and function of endogenous nCLU in cell death are relatively unexplored. Here, we show that depletion of endogenous nCLU protein using siRNA specific to its truncated mRNA increased clonogenic survival of ionizing radiation (IR)-exposed cells. nCLU-mediated apoptosis was Bax-dependent, and lethality correlated with accumulation of mature nCLU protein. nCLU accumulation was regulated by CRM1 because binding between CRM1 and nCLU proteins was significantly diminished by leptomycin B (LMB), and nuclear levels of nCLU protein were significantly enhanced by LMB and IR co-treatment. Moreover, LMB treatment significantly enhanced IR-induced nCLU-mediated cell death responses. Importantly, bax(-/-) and bax(-/-)/bak(-/-) double knock-out cells were resistant to nCLU-mediated cell death, whereas bak(-/-) or wild-type bax(+/+)/bak(+/+) cells were hypersensitive. The regulation of nCLU by CRM1 nuclear export/import may explain recent clinical results showing that highly malignant tumors have lost the ability to accumulate nCLU levels, thereby avoiding growth inhibition and cell death.

Distinct and Atypical Intrinsic and Extrinsic Cell Death Pathways between Photoreceptor Cell Types upon Specific Ablation of Ranbp2 in Cone Photoreceptors

PubMed Central

Cho, Kyoung-in; Yu, Minzhong; Hao, Ying; Qiu, Sunny; Pillai, Indulekha C. L.; Peachey, Neal S.; Ferreira, Paulo A.

2013-01-01

Non-autonomous cell-death is a cardinal feature of the disintegration of neural networks in neurodegenerative diseases, but the molecular bases of this process are poorly understood. The neural retina comprises a mosaic of rod and cone photoreceptors. Cone and rod photoreceptors degenerate upon rod-specific expression of heterogeneous mutations in functionally distinct genes, whereas cone-specific mutations are thought to cause only cone demise. Here we show that conditional ablation in cone photoreceptors of Ran-binding protein-2 (Ranbp2), a cell context-dependent pleiotropic protein linked to neuroprotection, familial necrotic encephalopathies, acute transverse myelitis and tumor-suppression, promotes early electrophysiological deficits, subcellular erosive destruction and non-apoptotic death of cones, whereas rod photoreceptors undergo cone-dependent non-autonomous apoptosis. Cone-specific Ranbp2 ablation causes the temporal activation of a cone-intrinsic molecular cascade highlighted by the early activation of metalloproteinase 11/stromelysin-3 and up-regulation of Crx and CoREST, followed by the down-modulation of cone-specific phototransduction genes, transient up-regulation of regulatory/survival genes and activation of caspase-7 without apoptosis. Conversely, PARP1+-apoptotic rods develop upon sequential activation of caspase-9 and caspase-3 and loss of membrane permeability. Rod photoreceptor demise ceases upon cone degeneration. These findings reveal novel roles of Ranbp2 in the modulation of intrinsic and extrinsic cell death mechanisms and pathways. They also unveil a novel spatiotemporal paradigm of progression of neurodegeneration upon cell-specific genetic damage whereby a cone to rod non-autonomous death pathway with intrinsically distinct cell-type death manifestations is triggered by cell-specific loss of Ranbp2. Finally, this study casts new light onto cell-death mechanisms that may be shared by human dystrophies with distinct retinal spatial signatures as well as with other etiologically distinct neurodegenerative disorders. PMID:23818861

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

Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8+ lymphocytes in primary sarcomas is subtype dependent

PubMed Central

van Erp, Anke E.M.; Versleijen-Jonkers, Yvonne M.H.; Hillebrandt-Roeffen, Melissa H.S.; van Houdt, Laurens; Gorris, Mark A.J.; van Dam, Laura S.; Mentzel, Thomas; Weidema, Marije E.; Savci-Heijink, C. Dilara; Desar, Ingrid M.E.; Merks, Hans H.M.; van Noesel, Max M.; Shipley, Janet; van der Graaf, Winette T.A.; Flucke, Uta E.; Meyer-Wentrup, Friederike A.G.

2017-01-01

In order to explore the potential of immune checkpoint blockade in sarcoma, we investigated expression and clinical relevance of programmed cell death-1 (PD-1), programmed death ligand-1 (PD-L1) and CD8 in tumors of 208 sarcoma patients. Primary untreated osteosarcoma (n = 46), Ewing sarcoma (n = 32), alveolar rhabdomyosarcoma (n = 20), embryonal rhabdomyosarcoma (n = 77), synovial sarcoma (n = 22) and desmoplastic small round cell tumors (DSRCT) (n = 11) were examined immunohistochemically. PD-L1 expression was predominantly detected in alveolar and embryonal rhabdomyosarcomas (15% and 16%, respectively). In the alveolar subtype PD-L1 expression was associated with better overall, event-free and metastases-free survival. PD-1 expression on lymphocytes was predominantly seen in synovial sarcomas (18%). High levels of CD8+ lymphocytes were predominantly detected in osteosarcomas (35%) and associated with worse event-free survival in synovial sarcomas. Ewing sarcoma and DSRCTs showed PD-1 on tumor cells instead of on tumor infiltrating lymphocytes. Overall, expression and clinical associations were found to be subtype dependent. For the first time PD-1 expression on Ewing sarcoma (19%) and DSRCT (82%) tumor cells was described. PMID:29050367

Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8+ lymphocytes in primary sarcomas is subtype dependent.

PubMed

van Erp, Anke E M; Versleijen-Jonkers, Yvonne M H; Hillebrandt-Roeffen, Melissa H S; van Houdt, Laurens; Gorris, Mark A J; van Dam, Laura S; Mentzel, Thomas; Weidema, Marije E; Savci-Heijink, C Dilara; Desar, Ingrid M E; Merks, Hans H M; van Noesel, Max M; Shipley, Janet; van der Graaf, Winette T A; Flucke, Uta E; Meyer-Wentrup, Friederike A G

2017-09-19

In order to explore the potential of immune checkpoint blockade in sarcoma, we investigated expression and clinical relevance of programmed cell death-1 (PD-1), programmed death ligand-1 (PD-L1) and CD8 in tumors of 208 sarcoma patients. Primary untreated osteosarcoma ( n = 46), Ewing sarcoma ( n = 32), alveolar rhabdomyosarcoma ( n = 20), embryonal rhabdomyosarcoma ( n = 77), synovial sarcoma ( n = 22) and desmoplastic small round cell tumors (DSRCT) ( n = 11) were examined immunohistochemically. PD-L1 expression was predominantly detected in alveolar and embryonal rhabdomyosarcomas (15% and 16%, respectively). In the alveolar subtype PD-L1 expression was associated with better overall, event-free and metastases-free survival. PD-1 expression on lymphocytes was predominantly seen in synovial sarcomas (18%). High levels of CD8+ lymphocytes were predominantly detected in osteosarcomas (35%) and associated with worse event-free survival in synovial sarcomas. Ewing sarcoma and DSRCTs showed PD-1 on tumor cells instead of on tumor infiltrating lymphocytes. Overall, expression and clinical associations were found to be subtype dependent. For the first time PD-1 expression on Ewing sarcoma (19%) and DSRCT (82%) tumor cells was described.

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

Adenine nucleotide translocase-1 induces cardiomyocyte death through upregulation of the pro-apoptotic protein Bax.

PubMed

Baines, Christopher P; Molkentin, Jeffery D

2009-06-01

Overexpression of the adenine nucleotide translocase (ANT) has been shown to be cytotoxic in several cell types. Although ANT was originally proposed to be a critical component of the mitochondrial permeability transition (MPT) pore, recent data have suggested that this may not be the case. We therefore hypothesized that the cytotoxic actions of ANT are through an alternative mechanism, independent of the MPT pore. Infection of cultured neonatal cardiomyocytes with an ANT1-encoding adenovirus induced a gene dosage-dependent increase in cell death. However, ANT1 overexpression failed to induce MPT, and neither pharmacological nor genetic inhibition of the MPT pore was able to prevent ANT1-induced cell death. These data suggested that ANT1-induced death progressed through an MPT pore-independent pathway. Somewhat surprisingly, we observed that protein levels of Bax, a pro-apoptotic Bcl protein, were consistently elevated in ANT1-infected cardiomyocytes. Membranes isolated from ANT1-infected myocytes exhibited significantly increased amounts of membrane-inserted Bax, and immunocytochemistry revealed increased Bax activation in ANT1-infected myocytes. Co-expression with the Bax antagonist Bcl2 was able to greatly reduce the degree of ANT1-induced cell death. Furthermore, Bax/Bak-deficient fibroblasts were resistant to the cytotoxic effects of ANT1 overexpression. Interestingly, ANT1 overexpression was also associated with enhanced production of reactive oxygen species (ROS), and the antioxidant MnTBAP was able to significantly attenuate both the ANT1-induced upregulation of Bax and cell death. Taken together, these data indicate that ANT mediates cell death, not through the MPT pore, but rather via a ROS-dependent upregulation and activation of Bax.

Glucose Deprivation Induces ATF4-Mediated Apoptosis through TRAIL Death Receptors

PubMed Central

Iurlaro, Raffaella; Püschel, Franziska; León-Annicchiarico, Clara Lucía; O'Connor, Hazel; Martin, Seamus J.; Palou-Gramón, Daniel; Lucendo, Estefanía

2017-01-01

ABSTRACT Metabolic stress occurs frequently in tumors and in normal tissues undergoing transient ischemia. Nutrient deprivation triggers, among many potential cell death-inducing pathways, an endoplasmic reticulum (ER) stress response with the induction of the integrated stress response transcription factor ATF4. However, how this results in cell death remains unknown. Here we show that glucose deprivation triggered ER stress and induced the unfolded protein response transcription factors ATF4 and CHOP. This was associated with the nontranscriptional accumulation of TRAIL receptor 1 (TRAIL-R1) (DR4) and with the ATF4-mediated, CHOP-independent induction of TRAIL-R2 (DR5), suggesting that cell death in this context may involve death receptor signaling. Consistent with this, the ablation of TRAIL-R1, TRAIL-R2, FADD, Bid, and caspase-8 attenuated cell death, although the downregulation of TRAIL did not, suggesting ligand-independent activation of TRAIL receptors. These data indicate that stress triggered by glucose deprivation promotes the ATF4-dependent upregulation of TRAIL-R2/DR5 and TRAIL receptor-mediated cell death. PMID:28242652

Flavonoids in Ginkgo biloba fallen leaves induce apoptosis through modulation of p53 activation in melanoma cells.

PubMed

Park, Hye-Jung; Kim, Moon-Moo

2015-01-01

The aim of the present study was to examine the apoptotic effect of flavonoids in methanol extracts of Ginkgo biloba fallen leaves (MEGFL) on melanoma cells. Ginkgo biloba is a deciduous castle chaplain and its leaves include various types of flavonoids such as flavonol-O-glycosides. Ginkgo biloba is known to have therapeutic properties against a number of diseases such as cerebrovascular diseases, blood circulation disease and hypertension. In the present study MEGFL exhibited a higher cytotoxic effect on melanoma cells than Ginkgo biloba leaves (MEGL). It was also found that MEGFL induced apoptotic cell death which was characterized by DNA fragmentation. During the cell death process following treatment with MEGFL, the expression of a variety of death-associated proteins including p53, caspase-3, caspase-9, cytochrome c and Bax were analyzed in the cytosol of melanoma cells. MEGFL significantly increased the expression levels of caspase-3, caspase-9 and p53 in a dose-dependent manner. Our results indicate that MEGFL induced apoptotic cell death by increasing the expression of cell death-associated proteins in melanoma cells.

S -Nitrosylation inhibits the kinase activity of tomato phosphoinositide-dependent kinase 1 (PDK1)

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

Liu, Jian-Zhong; Duan, Jicheng; Ni, Min

It is well known that the reactive oxygen species, nitric oxide (NO), can trigger cell death in plants, but the underlying molecular mechanisms are not well understood. Here, we provide evidence that NO may trigger cell death in tomato (Solanum lycopersicon) through inhibiting the phosphoinositide-dependent kinase 1 (PDK1) kinase activity via S-nitrosylation. Biotin-switch assays and LC-MS/MS analyses demonstrated that SlPDK1 was a target of S-nitrosylation modification, which primarily occurred on the cysteine residue at position 128 (Cys128). Accordingly, the kinase activity of SlPDK1 was inhibited by S-nitrosoglutathione (GSNO) both in vitro and in vivo in a concentration-dependent manner, indicating thatmore » SlPDK1 activity is regulated by S-nitrosylation. The inhibition of SlPDK1 kinase activity by GSNO was reversible in the presence of a reducing agent but synergistically enhanced by hydrogen peroxide (H2O2). Mutation of Cys128 to serine completely abolished SlPDK1 kinase activity, suggesting that S-nitrosylation of Cys128 is responsible for the inhibition of the kinase activity of SlPDK1. In sum, our results established a potential link between NO-triggered cell death and inhibition of the kinase activity of tomato PDK1, a conserved negative regulator of cell death in yeasts, mammals and plants. Nitric oxide (NO) potentiates the induction of hypersensitive cell death in soybean cells by reactive oxygen species (ROS) (1). However, the molecular mechanism of the NO-induced cell death remains an enigma. One potential mechanism is that the activity of proteins that control cell death may be altered by a post-translational modification, S-nitrosylation. S-nitrosylation is the addition of the NO moiety to thiol groups, including cysteine (Cys) residues in proteins, to form S-nitrosothiols (SNOs). S-nitrosylation is an enzyme-independent post-translational and labile modification that can function as an on/off switch of protein activity (2- 4). Thousands of diverse classes of proteins, both in plants and in mammals, have been identified as targets of S-nitrosylation (5-9). In plants, proteins with diverse functions are S-nitrosylated at specific Cys residue(s) and their functions are either inhibited or enhanced by this modification (10-25). 3-Phosphoinositide-dependent protein kinase-1 (PDK1) and its downstream target, protein kinase B (PKB; also known as Akt), are central regulators of mammalian apoptosis (26-28). PKB is a member of the AGC family of protein kinases, which is activated by second messengers such as phospholipids and Ca2+ through PDK1. Mammalian PDK1 phosphorylates PKB to promote its function in suppressing programmed cell death (PCD) (27-30). PKB negatively regulates apoptosis by phosphorylation and inactivation of pro-apoptotic factors such as BAD and activation of anti-apoptotic factors such as CREB and IKK (27-29; and 31). Deficiency of the PDK1 gene(s) in Drosophila (32), mice (33), yeast (34-35) and tomato (36), respectively, results in lethality or severe apoptosis. PKB knockout mice display spontaneous apoptosis in several different tissues (37). In tomato, the PKB/Akt homolog, Adi3 (AvrPto-dependent Pto-interacting protein 3), physically interacts with and is phosphorylated by SlPDK1 (36). Silencing both SlPDK1 and Adi3 or treatment with a PDK1 inhibitor results in MAPKKK -dependent cell death, indicating that Adi3 functions analogously to the mammalian PKB/Akt by negatively regulating cell death via PDK1 phosphorylation (36). Yasukawa et al (38) showed that NO donors induced S-nitrosylation and inactivation of Akt/PKB kinase activity in vitro and in vivo and the mutant Akt1/PKB (C224S) was resistant to S-nitrosylation by NO and its kinase inactivation (38). Although the NO and PDK1-PKB/Akt pathways are both key regulators of cell death, the link between these two pathways has not been firmly established in plants. Here we show that the kinase activity of tomato SlPDK1 was inhibited by GSNO in a conce« less

[Possibilities and limitations of fibroblast cultures in the study of animal aging].

PubMed

Van Gansen, P; Van Lerberghe, N

1987-01-01

INTRODUCTION. Aging--the effect of time--occurs in every living organism. Senescence is the last period of the lifespan, leading to death. It happens in all animals, with the exception of a few didermic species (Hydras) having a stock of embryonic cells and being immortal. The causes of animal senescence are badly known. They depend both on genetic characters (maximal lifespan of a species) and on medium factors (mean expectation of life of the animals of a species). Animal senescence could depend on cell aging: 1) by senescence and death of the differentiated cells, 2) by modified proliferation and differentiation of the stem cells of differentiated tissues, 3) by alterations in the extracellular matrices, 4) by interactions between factors 1) 2) and 3) in each tissue, 5) by interactions between the several tissues of an organism. This complexity badly impedes the experimental study of animal senescence. Normal mammal cells are aging when they are cultivated (in vitro ageing): their phenotype varies and depends on the cell generation (in vitro differentiation); the last cell-generation doesn't divide anymore and declines until death of the culture (in vitro senescence). Analysis of these artificial but well controlled systems allows an experimental approach of the proliferation, differentiation, senescence and death of the cells and of the extracellular matrix functions. Present literature upon in vitro aging of cultivated human cells is essentially made of papers where proliferation and differentiation characteristics are compared between early ("young") and late ("old") cell-generations of the cultures. FIBROBLASTIC CELLS OF THE MOUSE SKIN. This cell type has been studied in our laboratory, using different systems: 1) Primary cultures isolated from peeled skins of 19 day old mouse embryos, 2) Mouse dermis analyzed in the animals, 3) Cultivated explants of skins, 4) Serial sub-cultures of fibroblasts isolated from these explants, 5) Cells cultivated comparably on plane substrates (glass, plastic, collagen films) and on tridimensional matrices (collagen fibres). Systems 2), 3), 4) and 5) have been obtained either from 19 day old embryos or from 6 groups of animals of different ages (from 1/2 till 25 month). In primary cultures (system 1) all the cell generations have been analyzed, including the last one until death of the culture. We have shown that many characters are varying with cell-generation: cell form and cell mass, rate of DNA replication and cell division, rate of RNA transcription, nature of the accumulated and of the synthetized proteins, organization of the cytoskeletal elements, organization of the extracellular matrix, type of cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

The cell cycle and acute kidney injury

PubMed Central

Price, Peter M.; Safirstein, Robert L.; Megyesi, Judit

2009-01-01

Acute kidney injury (AKI) activates pathways of cell death and cell proliferation. Although seemingly discrete and unrelated mechanisms, these pathways can now be shown to be connected and even to be controlled by similar pathways. The dependence of the severity of renal-cell injury on cell cycle pathways can be used to control and perhaps to prevent acute kidney injury. This review is written to address the correlation between cellular life and death in kidney tubules, especially in acute kidney injury. PMID:19536080

Artesunate induces AIF-dependent apoptosis in A549 cells

NASA Astrophysics Data System (ADS)

Zhou, Chen-juan; Chen, Tong-Sheng

2012-03-01

Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. It has been shown that ART induces cancer cells death through apoptosis pathway. This study investigated whether ART treatment induced reactive oxygen species (ROS)-dependent cell death in the apoptosis fashion in human lung adenocarconoma A549 cell line and the proapoptotic protein apoptosis inducing factor (AIF) is involved in ART-induced apoptosis. Cells treated with ART exhibited typical apoptotic morphology as chromatin condensation, margination and shrunken nucleus. ART treatment also induced a loss of mitochondrial membrane potential and AIF release from mitochondria. Silencing AIF can remarkable attenuated ART-induced apoptosis. Collectively, ART induces apoptosis by caspase-independent intrinsic pathway in A549 cells.

Jasmonic Acid Signaling Modulates Ozone-Induced Hypersensitive Cell Death

PubMed Central

Rao, Mulpuri V.; Lee, Hyung-il; Creelman, Robert A.; Mullet, John E.; Davis, Keith R.

2000-01-01

Recent studies suggest that cross-talk between salicylic acid (SA)–, jasmonic acid (JA)–, and ethylene-dependent signaling pathways regulates plant responses to both abiotic and biotic stress factors. Earlier studies demonstrated that ozone (O3) exposure activates a hypersensitive response (HR)–like cell death pathway in the Arabidopsis ecotype Cvi-0. We now have confirmed the role of SA and JA signaling in influencing O3-induced cell death. Expression of salicylate hydroxylase (NahG) in Cvi-0 reduced O3-induced cell death. Methyl jasmonate (Me-JA) pretreatment of Cvi-0 decreased O3-induced H2O2 content and SA concentrations and completely abolished O3-induced cell death. Cvi-0 synthesized as much JA as did Col-0 in response to O3 exposure but exhibited much less sensitivity to exogenous Me-JA. Analyses of the responses to O3 of the JA-signaling mutants jar1 and fad3/7/8 also demonstrated an antagonistic relationship between JA- and SA-signaling pathways in controlling the magnitude of O3-induced HR-like cell death. PMID:11006337

Jasmonic acid signaling modulates ozone-induced hypersensitive cell death.

PubMed

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

2000-09-01

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

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.

A New Model for the Estimation of Cell Proliferation Dynamics Using CFSE Data

DTIC Science & Technology

2011-08-20

cells, and hence into the resulting division and death rates . Alternatively, we propose that there is information to be learned not only from...meaningful estimation of population proliferation and death rates in a manner which is unbiased and mechanistically sound. Significantly, this new model is...change in permitting the dependence of the proliferation and death rates (α and β) and the label loss rate (v) on both time t and measured FI x. This

Combined inhibition of autophagy and caspases fails to prevent developmental nurse cell death in the Drosophila melanogaster ovary.

PubMed

Peterson, Jeanne S; McCall, Kimberly

2013-01-01

During the final stages of Drosophila melanogaster oogenesis fifteen nurse cells, sister cells to the oocyte, degenerate as part of normal development. This process involves at least two cell death mechanisms, caspase-dependent cell death and autophagy, as indicated by apoptosis and autophagy markers. In addition, mutations affecting either caspases or autophagy partially reduce nurse cell removal, leaving behind end-stage egg chambers with persisting nurse cell nuclei. To determine whether apoptosis and autophagy work in parallel to degrade and remove these cells as is the case with salivary glands during pupariation, we generated mutants doubly affecting caspases and autophagy. We found no significant increase in either the number of late stage egg chambers containing persisting nuclei or in the number of persisting nuclei per egg chamber in the double mutants compared to single mutants. These findings suggest that there is another cell death mechanism functioning in the ovary to remove all nurse cell remnants from late stage egg chambers.

Using natural products to promote caspase-8-dependent cancer cell death.

PubMed

Tewary, Poonam; Gunatilaka, A A Leslie; Sayers, Thomas J

2017-02-01

The selective killing of cancer cells without toxicity to normal nontransformed cells is an idealized goal of cancer therapy. Thus, there has been much interest in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a protein that appears to selectively kill cancer cells. TRAIL has been reported to trigger apoptosis and under some circumstances, an alternate death signaling pathway termed necroptosis. The relative importance of necroptosis for cell death induction in vivo is under intensive investigation. Nonetheless, many cancer cells (particularly those freshly isolated from cancer patients) are highly resistant to TRAIL-mediated cell death. Therefore, there is an underlying interest in identifying agents that can be combined with TRAIL to improve its efficacy. There are numerous reports in which combination of TRAIL with standard antineoplastic drugs has resulted in enhanced cancer cell death in vitro. However, many of these chemotherapeutic drugs are nonspecific and associated with adverse effects, which raise serious concerns for cancer therapy in patients. By contrast, natural products have been shown to be safer and efficacious alternatives. Recently, a number of studies have suggested that certain natural products when combined with TRAIL can enhance cancer cell death. In this review, we highlight molecular pathways that might be targeted by various natural products to promote cell death, and focus on our recent work with withanolides as TRAIL sensitizers. Finally, we will suggest synergistic approaches for combining active withanolides with various forms of immunotherapy to promote cancer cell death and an effective antitumor immune response.

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

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.

Molecular basis of sodium butyrate-dependent proapoptotic activity in cancer cells.

PubMed

Pajak, B; Orzechowski, A; Gajkowska, B

2007-01-01

This review outlines the molecular events that accompany the antitumor action of sodium butyrate (NaBt). Butyrate, a low-molecular weight four-carbon chain volatile fatty acid (VFA) has been previously shown to withdraw cells from cell cycle or to promote cell differentiation, and finally to induce programmed cell death. Recent advances in molecular biology indicate, that this product of large bowel microbial fermentation of dietary fiber, might evoke the above-mentioned effects by indirect action on genes. NaBt was shown to inhibit histone deacetylase activity, allowing DNA binding of several transcription factors. Higher genomic activity leads to the higher expression of proapoptotic genes, higher level of their protein products and elevated sensitivity to death ligand-induced apoptosis. Cancer cells might be arrested in G1 phase of cell cycle in a p21-dependent manner. Proapoptotic activity of NaBt includes higher expression of membrane death receptors (DR4/5), higher level and activation of Smad3 protein in TGF-beta-dependent apoptotic pathway, lower level of antiapoptotic proteins (cFLIP, XIAP) and activation ofproapoptotic tBid protein. Thus, both intrinsic and extrinsic apoptotic pathways are stimulated to ampify the apoptotic signals. These effects are specific for tumor but not for regular cells. Unique properties of NaBt make this agent a promising metabolic inhibitor to retard tumorigenesis to suppress tumor growth.

A cell impedance measurement device for the cytotoxicity assay dependent on the velocity of supplied toxic fluid

NASA Astrophysics Data System (ADS)

Kang, Yoon-Tae; Kim, Min-Ji; Cho, Young-Ho

2018-04-01

We present a cell impedance measurement chip capable of characterizing the toxic response of cells depending on the velocity of the supplied toxic fluid. Previous impedance-based devices using a single open-top chamber have been limited to maintaining a constant supply velocity, and devices with a single closed-top chamber present difficulties in simultaneous cytotoxicity assay for varying levels of supply velocities. The present device, capable of generating constant and multiple levels of toxic fluid velocity simultaneously within a single stepwise microchannel, performs a cytotoxicity assay dependent on toxic fluid velocity, in order to find the effective velocity of toxic fluid to cells for maximizing the cytotoxic effect. We analyze the cellular toxic response of 5% ethanol media supplied to cancer cells within a toxic fluid velocity range of 0-8.3 mm s-1. We observe the velocity-dependent cell detachment rate, impedance, and death rate. We find that the cell detachment rate decreased suddenly to 2.4% at a velocity of 4.4 mm s-1, and that the change rates of cell resistance and cell capacitance showed steep decreases to 8% and 41%, respectively, at a velocity of 5.7 mm s-1. The cell death rate and impedance fell steeply to 32% at a velocity of 5.7 mm s-1. We conclude that: (1) the present device is useful in deciding on the toxic fluid velocity effective to cytotoxicity assay, since the cellular toxic response is dependent on the velocity of toxic fluid, and; (2) the cell impedance analysis facilitates a finer cellular response analysis, showing better correlation with the cell death rate, compared to conventional visual observation. The present device, capable of performing the combinational analysis of toxic fluid velocity and cell impedance, has potential for application to the fine cellular toxicity assay of drugs with proper toxic fluid velocity.

Molecular mechanism of cardol, isolated from Trigona incisa stingless bee propolis, induced apoptosis in the SW620 human colorectal cancer cell line.

PubMed

Kustiawan, Paula Mariana; Lirdprapamongkol, Kriengsak; Palaga, Tanapat; Puthong, Songchan; Phuwapraisirisan, Preecha; Svasti, Jisnuson; Chanchao, Chanpen

2017-05-04

Cardol is a major bioactive constituent in the Trigona incisa propolis from Indonesia, with a strong in vitro antiproliferative activity against the SW620 colorectal adenocarcinoma cell line (IC 50 of 4.51 ± 0.76 μg/mL). Cardol induced G 0 /G 1 cell cycle arrest and apoptotic cell death. The present study was designed to reveal the mechanism of cardol's antiproliferative effect and induction of apoptosis. Changes in cell morphology were observed by light microscopy. To determine whether the mitochondrial apoptotic pathway was involved in cell death, caspase-3 and caspase-9 activities, western blot analysis, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels were assayed. Changes in the cell morphology and the significantly increased caspase-3 and caspase-9 activities, plus the cleavage of pro-caspase-3, pro-caspase-9 and PARP, supported that cardol caused apoptosis in SW620 cells within 2 h after treatment by cardol. In addition, cardol decreased the mitochondrial membrane potential while increasing the intracellular ROS levels in a time- and dose-dependent manner. Antioxidant treatment supported that the cardol-induced cell death was dependent on ROS production. Cardol induced cell death in SW620 cells was mediated by oxidative stress elevation and the mitochondrial apoptotic pathway, and these could be the potential molecular mechanism for the antiproliferative effect of cardol.

Oxidized low-density lipoprotein induces calpain-dependent cell death and ubiquitination of caspase 3 in HMEC-1 endothelial cells.

PubMed Central

Pörn-Ares, M Isabella; Saido, Takaomi C; Andersson, Tommy; Ares, Mikko P S

2003-01-01

Oxidized low-density lipoprotein (oxLDL) is known to induce apoptosis in endothelial cells, and this is believed to contribute to the progression of atherosclerosis. In the present study we made the novel observation that oxLDL-induced death of HMEC-1 cells is accompanied by activation of calpain. The mu-calpain inhibitor PD 151746 decreased oxLDL-induced cytotoxicity, whereas the general caspase inhibitor BAF (t-butoxycarbonyl-Asp-methoxyfluoromethylketone) had no effect. Also, oxLDL provoked calpain-dependent proteolysis of cytoskeletal alpha-fodrin in the HMEC-1 cells. Our observation of an autoproteolytic cleavage of the 80 kDa subunit of mu-calpain provided further evidence for an oxLDL-induced stimulation of calpain activity. The Bcl-2 protein Bid was also cleaved during oxLDL-elicited cell death, and this was prevented by calpain inhibitors, but not by inhibitors of cathepsin B and caspases. Treating the HMEC-1 cells with oxLDL did not result in detectable activation of procaspase 3 or cleavage of PARP [poly(ADP-ribose) polymerase], but it did cause polyubiquitination of caspase 3, indicating inactivation and possible degradation of this protease. Despite the lack of caspase 3 activation, oxLDL treatment led to the formation of nucleosomal DNA fragments characteristic of apoptosis. These novel results show that oxLDL initiates a calpain-mediated death-signalling pathway in endothelial cells. PMID:12775216

BaxΔ2 sensitizes colorectal cancer cells to proteasome inhibitor-induced cell death

PubMed Central

Mañas, Adriana; Chen, Wenjing; Nelson, Adam; Yao, Qi; Xiang, Jialing

2018-01-01

Proteasome inhibitors, such as bortezomib and carfilzomib, are FDA approved for the treatment of hemopoietic cancers, but recent studies have shown their great potential for treatment of solid tumors. BaxΔ2, a unique proapoptotic Bax isoform, promotes non-mitochondrial cell death and sensitizes cancer cells to chemotherapy. However, endogenous BaxΔ2 proteins are unstable and susceptible to proteasomal degradation. Here, we screened a panel of proteasome inhibitors in colorectal cancer cells with different Bax statuses. We found that all proteasome inhibitors tested were able to block BaxΔ2 degradation without affecting the level of Baxα or Bcl-2 proteins. Among the inhibitors tested, only bortezomib and carfilzomib were able to induce differential cell death corresponding to the distinct Bax statuses. BaxΔ2-positive cells had a significantly higher level of cell death at low nanomolar concentrations than Baxα-positive or Bax-negative cells. Furthermore, bortezomib-induced cell death in BaxΔ2-positive cells was predominantly dependent on the caspase 8/3 pathway, consistent with our previous studies. These results imply that BaxΔ2 can selectively sensitize cancer cells to proteasome inhibitors, enhancing their potential to treat colon cancer and other solid tumors. PMID:29291406

A synthetic lethal screen identifies FAT1 as an antagonist of caspase-8 in extrinsic apoptosis

PubMed Central

Kranz, Dominique; Boutros, Michael

2014-01-01

The extrinsic apoptosis pathway is initiated by binding of death ligands to death receptors resulting in the formation of the death-inducing signaling complex (DISC). Activation of procaspase-8 within the DISC and its release from the signaling complex is required for processing executor caspases and commiting cell death. Here, we report that the atypical cadherin FAT1 interacts with caspase-8 preventing the association of caspase-8 with the DISC. We identified FAT1 in a genome-wide siRNA screen for synthetic lethal interactions with death receptor-mediated apoptosis. Knockdown of FAT1 sensitized established and patient-derived glioblastoma cell lines for apoptosis transduced by cell death ligands. Depletion of FAT1 resulted in enhanced procaspase-8 recruitment to the DISC and increased formation of caspase-8 containing secondary signaling complexes. In addition, FAT1 knockout cell lines generated by CRISPR/Cas9-mediated genome engineering were more susceptible for death receptor-mediated apoptosis. Our findings provide evidence for a mechanism to control caspase-8-dependent cell death by the atypical cadherin FAT1. These results contribute towards the understanding of effector caspase regulation in physiological conditions. PMID:24442637

A synthetic lethal screen identifies FAT1 as an antagonist of caspase-8 in extrinsic apoptosis.

PubMed

Kranz, Dominique; Boutros, Michael

2014-02-03

The extrinsic apoptosis pathway is initiated by binding of death ligands to death receptors resulting in the formation of the death-inducing signaling complex (DISC). Activation of procaspase-8 within the DISC and its release from the signaling complex is required for processing executor caspases and commiting cell death. Here, we report that the atypical cadherin FAT1 interacts with caspase-8 preventing the association of caspase-8 with the DISC. We identified FAT1 in a genome-wide siRNA screen for synthetic lethal interactions with death receptor-mediated apoptosis. Knockdown of FAT1 sensitized established and patient-derived glioblastoma cell lines for apoptosis transduced by cell death ligands. Depletion of FAT1 resulted in enhanced procaspase-8 recruitment to the DISC and increased formation of caspase-8 containing secondary signaling complexes. In addition, FAT1 knockout cell lines generated by CRISPR/Cas9-mediated genome engineering were more susceptible for death receptor-mediated apoptosis. Our findings provide evidence for a mechanism to control caspase-8-dependent cell death by the atypical cadherin FAT1. These results contribute towards the understanding of effector caspase regulation in physiological conditions.

Dendritic cells' death induced by contact sensitizers is controlled by Nrf2 and depends on glutathione levels

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

El Ali, Zeina

Dendritic cells (DC) are known to play a major role during contact allergy induced by contact sensitizers (CS). Our previous studies showed that Nrf2 was induced in DC and controlled allergic skin inflammation in mice in response to chemicals. In this work, we raised the question of the role of Nrf2 in response to a stress provoked by chemical sensitizers in DC. We used two well-described chemical sensitizers, dinitrochlorobenzene (DNCB) and cinnamaldehyde (CinA), known to have different chemical reactivity and mechanism of action. First, we performed a RT-qPCR array showing that CinA was a higher inducer of immune and detoxificationmore » genes compared to DNCB. Interestingly, in the absence of Nrf2, gene expression was dramatically affected in response to DNCB but was slightly affected in response to CinA. These observations prompted us to study DC's cell death in response to both chemicals. DNCB and CinA increased apoptotic cells and decreased living cells in the absence of Nrf2. The characterization of DC apoptosis induced by both CS involved the mitochondrial-dependent caspase pathway and was regulated via Nrf2 in response to both chemicals. Oxidative stress induced by DNCB, and leading to cell death, was regulated by Nrf2. Unlike CinA, DNCB treatment provoked a significant reduction of intracellular GSH levels and up-regulated bcl-2 gene expression, under the control of Nrf2. This work underlies that chemical reactivity may control Nrf2-dependent gene expression leading to different cytoprotective mechanisms in DC. - Highlights: • Nrf2 controls cell death induced by contact sensitizers in dendritic cells. • DNCB reduced GSH levels and up-regulated bcl-2 gene expression unlike CinA. • Chemical reactivity controls Nrf2-dependent genes having protective effect in DC.« less

Zinc as a paracrine effector in pancreatic islet cell death.

PubMed

Kim, B J; Kim, Y H; Kim, S; Kim, J W; Koh, J Y; Oh, S H; Lee, M K; Kim, K W; Lee, M S

2000-03-01

Because of a huge amount of Zn2+ in secretory granules of pancreatic islet beta-cells, Zn2+ released in certain conditions might affect the function or survival of islet cells. We studied potential paracrine effects of endogenous Zn2+ on beta-cell death. Zn2+ induced insulinoma/islet cell death in a dose-dependent manner. Chelation of released endogenous Zn2+ by CaEDTA significantly decreased streptozotocin (STZ)-induced islet cell death in an in vitro culture system simulating in vivo circumstances but not in the conventional culture system. Zn2+ chelation in vivo by continuous CaEDTA infusion significantly decreased the incidence of diabetes after STZ administration. N-(6-methoxy-quinolyl)-para-toluene-sulfonamide staining revealed that Zn2+ was densely deposited in degenerating islet cells 24 h after STZ treatment, which was decreased by CaEDTA infusion. We show here that Zn2+ is not a passive element for insulin storage but an active participant in islet cell death in certain conditions, which in time might contribute to the development of diabetes in aged people.

Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

PubMed Central

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

2014-01-01

Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS).We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. PMID:25034532

Histone deacetylase inhibitors prevent p53-dependent and p53-independent Bax-mediated neuronal apoptosis through two distinct mechanisms.

PubMed

Uo, Takuma; Veenstra, Timothy D; Morrison, Richard S

2009-03-04

Pharmacological manipulation of protein acetylation levels by histone deacetylase (HDAC) inhibitors represents a novel therapeutic strategy to treat neurodegeneration as well as cancer. However, the molecular mechanisms that determine how HDAC inhibition exerts a protective effect in neurons as opposed to a cytotoxic action in tumor cells has not been elucidated. We addressed this issue in cultured postnatal mouse cortical neurons whose p53-dependent and p53-independent intrinsic apoptotic programs require the proapoptotic multidomain protein, Bax. Despite promoting nuclear p53 accumulation, Class I/II HDAC inhibitors (HDACIs) protected neurons from p53-dependent cell death induced by camptothecin, etoposide, heterologous p53 expression or the MDM2 inhibitor, nutlin-3a. HDACIs suppressed p53-dependent PUMA expression, a critical signaling intermediate linking p53 to Bax activation, thus preventing postmitochondrial events including cleavage of caspase-9 and caspase-3. In human SH-SY5Y neuroblastoma cells, however, HDACIs were not able to prevent p53-dependent cell death. Moreover, HDACIs also prevented caspase-3 cleavage in postnatal cortical neurons treated with staurosporine, 3-nitropropionic acid and a Bcl-2 inhibitor, all of which require the presence of Bax but not p53 to promote apoptosis. Although these three toxic agents displayed a requirement for Bax, they did not promote PUMA induction. These results demonstrate that HDACIs block Bax-dependent cell death by two distinct mechanisms to prevent neuronal apoptosis, thus identifying for the first time a defined molecular target for their neuroprotective actions.

Histone Deacetylase Inhibitors Prevent p53-dependent and Independent Bax-Mediated Neuronal Apoptosis Through Two Distinct Mechanisms

PubMed Central

Uo, Takuma; Veenstra, Timothy D.; Morrison, Richard S.

2009-01-01

Pharmacological manipulation of protein acetylation levels by histone deacetylase (HDAC) inhibitors represents a novel therapeutic strategy to treat neurodegeneration as well as cancer. However, the molecular mechanisms that determine how HDAC inhibition exerts a protective effect in neurons as opposed to a cytotoxic action in tumor cells has not been elucidated. We addressed this issue in cultured postnatal mouse cortical neurons whose p53-dependent and —independent intrinsic apoptotic programs require the pro-apoptotic multidomain protein, Bax. Despite promoting nuclear p53 accumulation, Class I/II HDAC inhibitors (HDACIs) protected neurons from p53-dependent cell death induced by camptothecin, etoposide, heterologous p53 expression or the MDM2 inhibitor, nutlin-3a. HDACIs suppressed p53-dependent PUMA expression, a critical signaling intermediate linking p53 to Bax activation, thus preventing post-mitochondrial events including cleavage of caspase-9 and -3. In human SH-SY5Y neuroblastoma cells, however, HDACIs were not able to prevent p53-dependent cell death. Moreover, HDACIs also prevented caspase-3 cleavage in postnatal cortical neurons treated with staurosporine, 3-nitropropionic acid and a Bcl-2 inhibitor, all of which require the presence of Bax but not p53 to promote apoptosis. Although these three toxic agents displayed a requirement for Bax, they did not promote PUMA induction. These results demonstrate that HDACIs block Bax-dependent cell death by two distinct mechanisms to prevent neuronal apoptosis, thus identifying for the first time a defined molecular target for their neuroprotective actions. PMID:19261878

CD47-ligation induced cell death in T-acute lymphoblastic leukemia.

PubMed

Leclair, Pascal; Liu, Chi-Chao; Monajemi, Mahdis; Reid, Gregor S; Sly, Laura M; Lim, Chinten James

2018-05-10

CD47 is a cell-surface marker well recognized for its anti-phagocytic functions. As such, an emerging avenue for targeted cancer therapies involves neutralizing the anti-phagocytic function using monoclonal antibodies (mAbs) to enhance tumour cell immunogenicity. A lesser known consequence of CD47 receptor ligation is the direct induction of tumour cell death. While several mAbs and their derivatives with this property have been studied, the best characterized is the commercially available mAb B6H12, which requires immobilization for induction of cell death. Here, we describe a commercially available mAb, CC2C6, which induces T-cell acute lymphoblastic leukemia (ALL) cell death in soluble form. Soluble CC2C6 induces CD47-dependent cell death in a manner consistent with immobilized B6H12, which is characterized by mitochondrial deficiencies but is independent of caspase activation. Titration studies indicated that CC2C6 shares a common CD47-epitope with B6H12. Importantly, CC2C6 retains the anti-phagocytic neutralizing function, thus possessing dual anti-tumour properties. Although CD47-ligation induced cell death occurs in a caspase-independent manner, CC2C6 was found to stimulate increases in Mcl-1 and NOXA levels, two Bcl-2 family proteins that govern the intrinsic apoptosis pathway. Further analysis revealed that the ratio of Mcl-1:NOXA were minimally altered for cells treated with CC2C6, in comparison to cells treated with agents that induced caspase-dependent apoptosis which alter this ratio in favour of NOXA. Finally, we found that CC2C6 can synergize with low dose chemotherapeutic agents that induce classical apoptosis, giving rise to the possibility of an effective combination treatment with reduced long-term sequelae associated with high-dose chemotherapies in childhood ALL.

Role of Bax in death of uninfected retinal cells during murine cytomegalovirus retinitis.

PubMed

Mo, Juan; Marshall, Brendan; Covar, Jason; Zhang, Nancy Y; Smith, Sylvia B; Atherton, Sally S; Zhang, Ming

2014-10-08

Extensive death of uninfected bystander neuronal cells is an important component of the pathogenesis of cytomegalovirus retinitis. Our previous results have shown that caspase 3-dependent and -independent pathways are involved in death of uninfected bystander cells during murine cytomegalovirus (MCMV) retinitis and also that Bcl-2, an important inhibitor of apoptosis via the Bax-mediated mitochondrial pathway, is downregulated during this process. The purpose of this study was to determine whether Bax-mediated mitochondrial damage has a significant role in the death of uninfected retinal cells. BALB/c mice, Bax(-/-) mice, or Bax(+/+) mice were immunosuppressed with methylprednisolone and infected with 5 × 10(3) plaque-forming units (PFU) of the K181 strain of MCMV via the supraciliary route. Injected eyes were analyzed by plaque assay, electron microscopy, hematoxylin and eosin (H&E) staining, TUNEL assay, Western blot (for caspase 3, caspase 12, Bax, receptor interacting protein-1 [RIP1] and receptor interacting protein-3 [RIP3]), as well as immunohistochemical staining for MCMV early antigen and cleaved caspase 3. Significantly more Bax was detected in mitochondrial fractions of MCMV-infected eyes than in mitochondrial fractions of mock-infected control eyes. Furthermore, the level of cleaved caspase 3 was significantly lower in MCMV-infected Bax(-/-) eyes than in MCMV-infected Bax(+/+) eyes. However, more caspase 3-independent cell death of uninfected bystander retinal cells and more cleaved RIP1 were observed in Bax(-/-) than in Bax(+/+) eyes. During MCMV retinitis, Bax is activated and has an important role in death of uninfected bystander retinal cells by caspase 3-dependent apoptosis. Although the exact mechanism remains to be deciphered, active Bax might also prevent death of some types of uninfected retinal cells by a caspase 3-independent pathway. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Role of Bax in Death of Uninfected Retinal Cells During Murine Cytomegalovirus Retinitis

PubMed Central

Mo, Juan; Marshall, Brendan; Covar, Jason; Zhang, Nancy Y.; Smith, Sylvia B.; Atherton, Sally S.; Zhang, Ming

2014-01-01

Purpose. Extensive death of uninfected bystander neuronal cells is an important component of the pathogenesis of cytomegalovirus retinitis. Our previous results have shown that caspase 3–dependent and –independent pathways are involved in death of uninfected bystander cells during murine cytomegalovirus (MCMV) retinitis and also that Bcl-2, an important inhibitor of apoptosis via the Bax-mediated mitochondrial pathway, is downregulated during this process. The purpose of this study was to determine whether Bax-mediated mitochondrial damage has a significant role in the death of uninfected retinal cells. Methods. BALB/c mice, Bax−/− mice, or Bax+/+ mice were immunosuppressed with methylprednisolone and infected with 5 × 103 plaque-forming units (PFU) of the K181 strain of MCMV via the supraciliary route. Injected eyes were analyzed by plaque assay, electron microscopy, hematoxylin and eosin (H&E) staining, TUNEL assay, Western blot (for caspase 3, caspase 12, Bax, receptor interacting protein-1 [RIP1] and receptor interacting protein-3 [RIP3]), as well as immunohistochemical staining for MCMV early antigen and cleaved caspase 3. Results. Significantly more Bax was detected in mitochondrial fractions of MCMV-infected eyes than in mitochondrial fractions of mock-infected control eyes. Furthermore, the level of cleaved caspase 3 was significantly lower in MCMV-infected Bax−/− eyes than in MCMV-infected Bax+/+ eyes. However, more caspase 3–independent cell death of uninfected bystander retinal cells and more cleaved RIP1 were observed in Bax−/− than in Bax+/+ eyes. Conclusions. During MCMV retinitis, Bax is activated and has an important role in death of uninfected bystander retinal cells by caspase 3–dependent apoptosis. Although the exact mechanism remains to be deciphered, active Bax might also prevent death of some types of uninfected retinal cells by a caspase 3–independent pathway. PMID:25298417

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

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

Pan, Mu-Yun; Shen, Yuh-Chiang; National Research Institute of Chinese Medicine, Taipei, Taiwan

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

Silibinin, a natural flavonoid, induces autophagy via ROS-dependent mitochondrial dysfunction and loss of ATP involving BNIP3 in human MCF7 breast cancer cells.

PubMed

Jiang, Kai; Wang, Wei; Jin, Xin; Wang, Zhaoyang; Ji, Zhiwei; Meng, Guanmin

2015-06-01

Silibinin, derived from the milk thistle plant (Silybum marianum), has anticancer and chemopreventive properties. Silibinin has been reported to inhibit the growth of various types of cancer cells. However, the mechanisms by which silibinin exerts an anticancer effect are poorly defined. The present study aimed to investigate whether silibinin-induced cell death might be attributed to autophagy and the underlying mechanisms in human MCF7 breast cancer cells. Our results showed that silibinin-induced cell death was greatly abrogated by two specific autophagy inhibitors, 3-methyladenine (3-MA) and bafilomycin-A1 (Baf-A1). In addition, silibinin triggered the conversion of light chain 3 (LC3)-I to LC3-II, promoted the upregulation of Atg12-Atg5 formation, increased Beclin-1 expression, and decreased the Bcl-2 level. Moreover, we noted elevated reactive oxygen species (ROS) generation, concomitant with the dissipation of mitochondrial transmembrane potential (ΔΨm) and a drastic decline in ATP levels following silibinin treatment, which were effectively prevented by the antioxidants, N-acetylcysteine and ascorbic acid. Silibinin stimulated the expression of Bcl-2 adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a pro-death Bcl-2 family member, and silencing of BNIP3 greatly inhibited silibinin-induced cell death, decreased ROS production, and sustained ΔΨm and ATP levels. Taken together, these findings revealed that silibinin induced autophagic cell death through ROS-dependent mitochondrial dysfunction and ATP depletion involving BNIP3 in MCF7 cells.

ALV-J infection induces chicken monocyte death accompanied with the production of IL-1β and IL-18.

PubMed

Dai, Manman; Feng, Min; Xie, Tingting; Li, Yuanfang; Ruan, Zhuohao; Shi, Meiqing; Liao, Ming; Zhang, Xiquan

2017-11-21

Immunosuppression induced by avian leukosis virus subgroup J (ALV-J) causes serious reproduction problems and secondary infections in chickens. Given that monocytes are important precursors of immune cells including macrophages and dendritic cells, we investigated the fate of chicken monocytes after ALV-J infection. Our results indicated that most monocytes infected with ALV-J including field or laboratory strains could not successfully differentiate into macrophages due to cells death. And cells death was dependent upon viral titer and accompanied with increased IL-1β and IL-18 mRNA levels. In addition, ALV-J infection up-regulated caspase-1 and caspase-3 activity in monocytes. Collectively, we found that ALV-J could cause cell death in chicken monocytes, especially pyroptosis, which may be a significant reason for ALV-J induced immunosuppression.

ALV-J infection induces chicken monocyte death accompanied with the production of IL-1β and IL-18

PubMed Central

Dai, Manman; Feng, Min; Xie, Tingting; Li, Yuanfang; Ruan, Zhuohao; Shi, Meiqing; Liao, Ming; Zhang, Xiquan

2017-01-01

Immunosuppression induced by avian leukosis virus subgroup J (ALV-J) causes serious reproduction problems and secondary infections in chickens. Given that monocytes are important precursors of immune cells including macrophages and dendritic cells, we investigated the fate of chicken monocytes after ALV-J infection. Our results indicated that most monocytes infected with ALV-J including field or laboratory strains could not successfully differentiate into macrophages due to cells death. And cells death was dependent upon viral titer and accompanied with increased IL-1β and IL-18 mRNA levels. In addition, ALV-J infection up-regulated caspase-1 and caspase-3 activity in monocytes. Collectively, we found that ALV-J could cause cell death in chicken monocytes, especially pyroptosis, which may be a significant reason for ALV-J induced immunosuppression. PMID:29245947

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

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

Sulbutiamine counteracts trophic factor deprivation induced apoptotic cell death in transformed retinal ganglion cells.

PubMed

Kang, Kui Dong; Majid, Aman Shah Abdul; Kim, Kyung-A; Kang, Kyungsu; Ahn, Hong Ryul; Nho, Chu Won; Jung, Sang Hoon

2010-11-01

Sulbutiamine is a highly lipid soluble synthetic analogue of vitamin B(1) and is used clinically for the treatment of asthenia. The aim of our study was to demonstrate whether sulbutiamine is able to attenuate trophic factor deprivation induced cell death to transformed retinal ganglion cells (RGC-5). Cells were subjected to serum deprivation for defined periods and sulbutiamine at different concentrations was added to the cultures. Various procedures (e.g. cell viability assays, apoptosis assay, reactive oxygen species analysis, Western blot analysis, flow cytometric analysis, glutathione (GSH) and glutathione-S-transferase (GST) measurement) were used to demonstrate the effect of sulbutiamine. Sulbutiamine dose-dependently attenuated apoptotic cell death induced by serum deprivation and stimulated GSH and GST activity. Moreover, sulbutiamine decreased the expression of cleaved caspase-3 and AIF. This study demonstrates for the first time that sulbutiamine is able to attenuate trophic factor deprivation induced apoptotic cell death in neuronal cells in culture.

Protein Kinase G facilitates EGFR-mediated cell death in MDA-MB-468 cells

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

Jackson, Nicole M.; Ceresa, Brian P., E-mail: brian.ceresa@louisville.edu

The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase with critical implications in cell proliferation, migration, wound healing and the regulation of apoptosis. However, the EGFR has been shown to be hyper-expressed in a number of human malignancies. The MDA-MB-468 metastatic breast cell line is one example of this. This particular cell line hyper-expresses the EGFR and undergoes EGFR-mediated apoptosis in response to EGF ligand. The goal of this study was to identify the kinases that could be potential intermediates for the EGFR-mediated induction of apoptosis intracellularly. After identifying Cyclic GMP-dependent Protein Kinase G (PKG) as amore » plausible intermediate, we wanted to determine the temporal relationship of these two proteins in the induction of apoptosis. We observed a dose-dependent decrease in MDA-MB-468 cell viability, which was co-incident with increased PKG activity as measured by VASPSer239 phosphorylation. In addition, we observed a dose dependent decrease in cell viability, as well as an increase in apoptosis, in response to two different PKG agonists, 8-Bromo-cGMP and 8-pCPT-cGMP. MDA-MB-468 cells with reduced PKG activity had attenuated EGFR-mediated apoptosis. These findings indicate that PKG does not induce cell death via transphosphorylation of the EGFR. Instead, PKG activity occurs following EGFR activation. Together, these data indicate PKG as an intermediary in EGFR-mediated cell death, likely via apoptotic pathway.« less

Monocarboxylate transporter-dependent mechanism confers resistance to oxygen- and glucose-deprivation injury in astrocyte-neuron co-cultures.

PubMed

Gao, Chen; Zhou, Liya; Zhu, Wenxia; Wang, Hongyun; Wang, Ruijuan; He, Yunfei; Li, Zhiyun

2015-05-06

Hypoxic and low-glucose stressors contribute to neuronal death in many brain diseases. Astrocytes are anatomically well-positioned to shield neurons from hypoxic injury. During hypoxia/ischemia, lactate released from astrocytes is taken up by neurons and stored for energy. This process is mediated by monocarboxylate transporters (MCTs) in the central nervous system. In the present study, we investigated the ability of astrocytes to protect neurons from oxygen- and glucose-deprivation (OGD) injury via an MCT-dependent mechanism in vitro. Primary cultures of neurons, astrocytes, and astrocytes-neurons derived from rat hippocampus were subjected to OGD, MCT inhibition with small interfering (si)RNA. Cell survival and expression of MCT4, MCT2, glial fibrillary acidic protein, and neuronal nuclear antigen were evaluated. OGD significantly increased cell death in neuronal cultures and up-regulated MCT4 expression in astrocyte cultures, but no increased cell death was observed in neuron-astrocyte co-cultures or astrocyte cultures. However, neuronal cell death in co-cultures was increased by exposure to MCT4- or MCT2-specific siRNA, and this effect was attenuated by the addition of lactate into the extracellular medium of neuronal cultures prior to OGD. These findings demonstrate that resistance to OGD injury in astrocyte-neuron co-cultures occurs via an MCT-dependent mechanism. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Induction of Cell Death through Alteration of Oxidants and Antioxidants in Epithelial Cells Exposed to High Energy Protons

NASA Technical Reports Server (NTRS)

Ramesh, Govindarajan; Wu, Honglu

2012-01-01

Radiation affects several cellular and molecular processes including double strand breakage, modifications of sugar moieties and bases. In outer space, protons are the primary radiation source which poses a range of potential health risks to astronauts. On the other hand, the use of proton radiation for tumor radiation therapy is increasing as it largely spares healthy tissues while killing tumor tissues. Although radiation related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton radiation remain poorly understood. Therefore, in the present study, we irradiated rat epithelial cells (LE) with different doses of protons and investigated their effects on cell proliferation and cell death. Our data showed an inhibition of cell proliferation in proton irradiated cells with a significant dose dependent activation and repression of reactive oxygen species (ROS) and antioxidants, glutathione and superoxide dismutase respectively as compared to control cells. In addition, apoptotic related genes such as caspase-3 and -8 activities were induced in a dose dependent manner with corresponding increased levels of DNA fragmentation in proton irradiated cells than control cells. Together, our results show that proton radiation alters oxidant and antioxidant levels in the cells to activate apoptotic pathway for cell death.

Gonadotropin and intra-ovarian signals regulating follicle development and atresia: the delicate balance between life and death.

PubMed

Craig, Jesse; Orisaka, Makoto; Wang, Hongmei; Orisaka, Sanae; Thompson, Winston; Zhu, Cheng; Kotsuji, Fumikazu; Tsang, Benjamin K

2007-05-01

Regulation of mammalian follicular development is tightly regulated by both cell death and survival signals, including endocrine (e.g. gonadotropin) and intra-ovarian regulators (e.g. Nodal and GDF9). The destiny of the individual follicle (growth/ovulation or atresia) is dependent on a delicate balance in the expression and action of factors promoting follicular cell proliferation, growth and differentiation, and of those promoting programmed cell death (apoptosis). Development of the follicle from the primordial to preantral stage is regulated by oocyte-derived factors including GDF9 and BMP15, and is not dependent on gonadotropin support (gonadotropin-independent stage). As the follicle transits into the early antral stage it becomes responsive to gonadotropin (gonadotropin-responsive stages) and further development renders the follicle completely dependent on the presence of gonadotropin while modulated by intra-ovarian regulators (gonadotropin-dependent). Follicle fate is also regulated by pro-apoptotic factors such as the intraovarian regulator Nodal, which is secreted by the theca and promotes apoptosis of differentiated granulosa cells through a mechanism involving Smad2 signaling and suppression of the PI3K/Akt pathway. The intracellular protein prohibitin (PHB) appears to have a dual role during folliculogenesis; acting as a cell survival factor in undifferentiated cells, and as a pro-apoptotic factor following differentiation. Further investigations of the interplay between these endocrine and ovarian regulators will lead to a better understanding into the regulation of follicular development and atresia, allowing development of new techniques for assisted reproduction.

Direct modulation of the outer mitochondrial membrane channel, voltage-dependent anion channel 1 (VDAC1) by cannabidiol: a novel mechanism for cannabinoid-induced cell death

PubMed Central

Rimmerman, N; Ben-Hail, D; Porat, Z; Juknat, A; Kozela, E; Daniels, M P; Connelly, P S; Leishman, E; Bradshaw, H B; Shoshan-Barmatz, V; Vogel, Z

2013-01-01

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD. PMID:24309936

Inhibition of autophagy by chloroquine induces apoptosis in primary effusion lymphoma in vitro and in vivo through induction of endoplasmic reticulum stress.

PubMed

Masud Alam, Md; Kariya, Ryusho; Kawaguchi, Azusa; Matsuda, Kouki; Kudo, Eriko; Okada, Seiji

2016-10-01

Autophagy plays a crucial role in cancer cell survival and the inhibition of autophagy is attracting attention as an emerging strategy for the treatment of cancer. Chloroquine (CQ) is an anti-malarial drug, and is also known as an inhibitor of autophagy. Recently, it has been found that CQ induces cancer cell death through the inhibition of autophagy; however, the underlying mechanism is not entirely understood. In this study, we identified the role of CQ-induced cancer cell death using Primary Effusion Lymphoma (PEL) cells. We found that a CQ treatment induced caspase-dependent apoptosis in vitro. CQ also suppressed PEL cell growth in a PEL xenograft mouse model. We showed that CQ activated endoplasmic reticulum (ER) stress signal pathways and induced CHOP, which is an inducer of apoptosis. CQ-induced cell death was significantly decreased by salbrinal, an ER stress inhibitor, indicating that CQ-induced apoptosis in PEL cells depended on ER stress. We show here for the first time that the inhibition of autophagy induces ER stress-mediated apoptosis in PEL cells. Thus, the inhibition of autophagy is a novel strategy for cancer chemotherapy.

3-Bromopyruvate antagonizes effects of lactate and pyruvate, synergizes with citrate and exerts novel anti-glioma effects.

PubMed

El Sayed, S M; El-Magd, R M Abou; Shishido, Y; Chung, S P; Diem, T H; Sakai, T; Watanabe, H; Kagami, S; Fukui, K

2012-02-01

Oxidative stress-energy depletion therapy using oxidative stress induced by D-amino acid oxidase (DAO) and energy depletion induced by 3-bromopyruvate (3BP) was reported recently (El Sayed et al., Cancer Gene Ther., 19, 1-18, 2012). Even in the presence of oxygen, cancer cells oxidize glucose preferentially to produce lactate (Warburg effect) which seems vital for cancer microenvironment and progression. 3BP is a closely related structure to lactate and pyruvate and may antagonize their effects as a novel mechanism of its action. Pyruvate exerted a potent H(2)O(2) scavenging effect to exogenous H(2)O(2), while lactate had no scavenging effect. 3BP induced H(2)O(2) production. Pyruvate protected against H(2)O(2)-induced C6 glioma cell death, 3BP-induced C6 glioma cell death but not against DAO/D-serine-induced cell death, while lactate had no protecting effect. Lactate and pyruvate protected against 3BP-induced C6 glioma cell death and energy depletion which were overcome with higher doses of 3BP. Lactate and pyruvate enhanced migratory power of C6 glioma which was blocked by 3BP. Pyruvate and lactate did not protect against C6 glioma cell death induced by other glycolytic inhibitors e.g. citrate (inhibitor of phosphofructokinase) and sodium fluoride (inhibitor of enolase). Serial doses of 3BP were synergistic with citrate in decreasing viability of C6 glioma cells and spheroids. Glycolysis subjected to double inhibition using 3BP with citrate depleted ATP, clonogenic power and migratory power of C6 glioma cells. 3BP induced a caspase-dependent cell death in C6 glioma. 3BP was powerful in decreasing viability of human glioblastoma multiforme cells (U373MG) and C6 glioma in a dose- and time-dependent manner.

24(S)-hydroxycholesterol induces neuronal cell death through necroptosis, a form of programmed necrosis.

PubMed

Yamanaka, Kazunori; Saito, Yoshiro; Yamamori, Tohru; Urano, Yasuomi; Noguchi, Noriko

2011-07-15

24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis.

24(S)-Hydroxycholesterol Induces Neuronal Cell Death through Necroptosis, a Form of Programmed Necrosis*

PubMed Central

Yamanaka, Kazunori; Saito, Yoshiro; Yamamori, Tohru; Urano, Yasuomi; Noguchi, Noriko

2011-01-01

24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis. PMID:21613228

Contribution of TMEM16F to pyroptotic cell death.

PubMed

Ousingsawat, Jiraporn; Wanitchakool, Podchanart; Schreiber, Rainer; Kunzelmann, Karl

2018-02-20

Pyroptosis is a highly inflammatory form of programmed cell death that is caused by infection with intracellular pathogens and activation of canonical or noncanonical inflammasomes. The purinergic receptor P2X 7 is activated by the noncanonical inflammasome and contributes essentially to pyroptotic cell death. The Ca 2+ activated phospholipid scramblase and ion channel TMEM16F has been shown earlier to control cellular effects downstream of purinergic P2X 7 receptors that ultimately lead to cell death. As pyroptotic cell death is accompanied by an increases in intracellular Ca 2+ , we asked whether TMEM16F is activated during pyroptosis. The N-terminal cleavage product of gasdermin D (GD-N) is an executioner of pyroptosis by forming large plasma membrane pores. Expression of GD-N enhanced basal Ca 2+ levels and induced cell death. We observed that GD-N induced cell death in HEK293 and HAP1 cells, which was depending on expression of endogenous TMEM16F. GD-N activated large whole cell currents that were suppressed by knockdown or inhibition of TMEM16F. The results suggest that whole cell currents induced by the pore forming domain of gasdermin-D, are at least in part due to activation of TMEM16F. Knockdown of other TMEM16 paralogues expressed in HAP1 cells suggest TMEM16F as a crucial element during pyroptosis and excluded a role of other TMEM16 proteins. Thus TMEM16F supports pyroptosis and other forms of inflammatory cell death such as ferroptosis. Its potent inhibition by tannic acid may be part of the anti-inflammatory effects of flavonoids.

Induction of cytosine arabinoside-resistant human myeloid leukemia cell death through autophagy regulation by hydroxychloroquine.

PubMed

Kim, Yundeok; Eom, Ju-In; Jeung, Hoi-Kyung; Jang, Ji Eun; Kim, Jin Seok; Cheong, June-Won; Kim, Young Sam; Min, Yoo Hong

2015-07-01

We investigated the effects of the autophagy inhibitor hydroxychloroquine (HCQ) on cell death of cytosine arabinoside (Ara-C)-resistant human acute myeloid leukemia (AML) cells. Ara-C-sensitive (U937, AML-2) and Ara-C-resistant (U937/AR, AML-2/AR) human AML cell lines were used to evaluate HCQ-regulated cytotoxicity, autophagy, and apoptosis as well as effects on cell death-related signaling pathways. We found that HCQ-induced dose- and time-dependent cell death in Ara-C-resistant cells compared to Ara-C-sensitive cell lines. The extent of cell death and features of HCQ-induced autophagic markers including increase in microtubule-associated protein light chain 3 (LC3) I conversion to LC3-II, beclin-1, ATG5, as well as green fluorescent protein-LC3 positive puncta and autophagosome were remarkably greater in U937/AR cells. Also, p62/SQSTM1 was increased in response to HCQ. p62/SQSTM1 protein interacts with both LC3-II and ubiquitin protein and is degraded in autophagosomes. Therefore, a reduction of p62/SQSTM1 indicates increased autophagic degradation, whereas an increase of p62/SQSTM1 by HCQ indicates inhibited autophagic degradation. Knock down of p62/SQSTM1 using siRNA were prevented the HCQ-induced LC3-II protein level as well as significantly reduced the HCQ-induced cell death in U937/AR cells. Also, apoptotic cell death and caspase activation in U937/AR cells were increased by HCQ, provided evidence that HCQ-induced autophagy blockade. Taken together, our data show that HCQ-induced apoptotic cell death in Ara-C-resistant AML cells through autophagy regulation. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Loss of XIAP facilitates switch to TNFα-induced necroptosis in mouse neutrophils.

PubMed

Wicki, Simone; Gurzeler, Ursina; Wei-Lynn Wong, W; Jost, Philipp J; Bachmann, Daniel; Kaufmann, Thomas

2016-10-13

Neutrophils are essential players in the first-line defense against invading bacteria and fungi. Besides its antiapoptotic role, the inhibitor of apoptosis protein (IAP) family member X-linked IAP (XIAP) has been shown to regulate innate immune signaling. Whereas the role of XIAP in innate signaling pathways is derived mostly from work in macrophages and dendritic cells, it is not known if and how XIAP contributes to these pathways in neutrophils. Here we show that in response to bacterial lipopolysaccharides (LPS), mouse neutrophils secreted considerable amounts of tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) and, in accordance with earlier reports, XIAP prevented LPS-induced hypersecretion of IL-1β also in neutrophils. Interestingly, and in contrast to macrophages or dendritic cells, Xiap-deficient neutrophils were insensitive to LPS-induced cell death. However, combined loss of function of XIAP and cIAP1/-2 resulted in rapid neutrophil cell death in response to LPS. This cell death occurred by classical apoptosis initiated by a TNFα- and RIPK1-dependent, but RIPK3- and MLKL-independent, pathway. Inhibition of caspases under the same experimental conditions caused a shift to RIPK3-dependent cell death. Accordingly, we demonstrate that treatment of neutrophils with high concentrations of TNFα induced apoptotic cell death, which was fully blockable by pancaspase inhibition in wild-type neutrophils. However, in the absence of XIAP, caspase inhibition resulted in a shift from apoptosis to RIPK3- and MLKL-dependent necroptosis. Loss of XIAP further sensitized granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed neutrophils to TNFα-induced killing. These data suggest that XIAP antagonizes the switch from TNFα-induced apoptosis to necroptosis in mouse neutrophils. Moreover, our data may implicate an important role of neutrophils in the development of hyperinflammation and disease progression of patients diagnosed with X-linked lymphoproliferative syndrome type 2, which are deficient in XIAP.

Bcl-2 Family Members and Functional Electron Transport Chain Regulate Oxygen Deprivation-Induced Cell Death

PubMed Central

McClintock, David S.; Santore, Matthew T.; Lee, Vivian Y.; Brunelle, Joslyn; Budinger, G. R. Scott; Zong, Wei-Xing; Thompson, Craig B.; Hay, Nissim; Chandel, Navdeep S.

2002-01-01

The mechanisms underlying cell death during oxygen deprivation are unknown. We report here a model for oxygen deprivation-induced apoptosis. The death observed during oxygen deprivation involves a decrease in the mitochondrial membrane potential, followed by the release of cytochrome c and the activation of caspase-9. Bcl-XL prevented oxygen deprivation-induced cell death by inhibiting the release of cytochrome c and caspase-9 activation. The ability of Bcl-XL to prevent cell death was dependent on allowing the import of glycolytic ATP into the mitochondria to generate an inner mitochondrial membrane potential through the F1F0-ATP synthase. In contrast, although activated Akt has been shown to inhibit apoptosis induced by a variety of apoptotic stimuli, it did not prevent cell death during oxygen deprivation. In addition to Bcl-XL, cells devoid of mitochondrial DNA (ρ° cells) that lack a functional electron transport chain were resistant to oxygen deprivation. Further, murine embryonic fibroblasts from bax−/− bak−/− mice did not die in response to oxygen deprivation. These data suggest that when subjected to oxygen deprivation, cells die as a result of an inability to maintain a mitochondrial membrane potential through the import of glycolytic ATP. Proapoptotic Bcl-2 family members and a functional electron transport chain are required to initiate cell death in response to oxygen deprivation. PMID:11739725

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

PubMed Central

Swanson, Phillip A.; Hart, Geoffrey T.; Russo, Matthew V.; Nayak, Debasis; Yazew, Takele; Peña, Mirna; Khan, Shahid M.; Pierce, Susan K.; McGavern, Dorian B.

2016-01-01

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection that results in thousands of deaths each year, mostly in African children. The in vivo mechanisms underlying this fatal condition are not entirely understood. Using the animal model of experimental cerebral malaria (ECM), we sought mechanistic insights into the pathogenesis of CM. Fatal disease was associated with alterations in tight junction proteins, vascular breakdown in the meninges / parenchyma, edema, and ultimately neuronal cell death in the brainstem, which is consistent with cerebral herniation as a cause of death. At the peak of ECM, we revealed using intravital two-photon microscopy that myelomonocytic cells and parasite-specific CD8+ T cells associated primarily with the luminal surface of CNS blood vessels. Myelomonocytic cells participated in the removal of parasitized red blood cells (pRBCs) from cerebral blood vessels, but were not required for the disease. Interestingly, the majority of disease-inducing parasite-specific CD8+ T cells interacted with the lumen of brain vascular endothelial cells (ECs), where they were observed surveying, dividing, and arresting in a cognate peptide-MHC I dependent manner. These activities were critically dependent on IFN-γ, which was responsible for activating cerebrovascular ECs to upregulate adhesion and antigen-presenting molecules. Importantly, parasite-specific CD8+ T cell interactions with cerebral vessels were impaired in chimeric mice rendered unable to present EC antigens on MHC I, and these mice were in turn resistant to fatal brainstem pathology. Moreover, anti-adhesion molecule (LFA-1 / VLA-4) therapy prevented fatal disease by rapidly displacing luminal CD8+ T cells from cerebrovascular ECs without affecting extravascular T cells. These in vivo data demonstrate that parasite-specific CD8+ T cell-induced fatal vascular breakdown and subsequent neuronal death during ECM is associated with luminal, antigen-dependent interactions with cerebrovasculature. PMID:27907215

Ethylene is required for elicitin-induced oxidative burst but not for cell death induction in tobacco cell suspension cultures.

PubMed

Koehl, Julia; Djulic, Alma; Kirner, Veronika; Nguyen, Tach Thao; Heiser, Ingrid

2007-12-01

The signal compound ethylene and its relationships with oxidative burst and cell death were analyzed in cultured tobacco cells treated with the proteinaceous elicitor quercinin. Quercinin belongs to the protein family of elicitins and was isolated from the soil-born oak pathogen Phytophthora quercina. It was shown to induce a dose-dependent oxidative burst in tobacco cell culture in concentrations from 0.05 to 0.5 nM, and subsequently, cell death. The characteristics of quercinin-induced cell death included both membrane damage and DNA fragmentation in tobacco cell culture. At higher quercinin concentrations (2 nM), H(2)O(2) formation and ethylene biosynthesis were inhibited. Ethylene at low concentrations proved to be necessary for induction and maintenance of H(2)O(2) production in tobacco cells treated with quercinin. It was demonstrated that external addition of inhibitors of ethylene biosynthesis such as alpha-amino-oxy-acetic acid (AOA) and CoCl(2) also decreased or even inhibited the quercinin-induced oxidative burst, but did not influence cell death induction. These results demonstrate evidence for a requirement of the plant hormone ethylene for the onset of the quercinin-induced oxidative burst.

Myeloid-derived suppressor activity is mediated by monocytic lineages maintained by continuous inhibition of extrinsic and intrinsic death pathways.

PubMed

Haverkamp, Jessica M; Smith, Amber M; Weinlich, Ricardo; Dillon, Christopher P; Qualls, Joseph E; Neale, Geoffrey; Koss, Brian; Kim, Young; Bronte, Vincenzo; Herold, Marco J; Green, Douglas R; Opferman, Joseph T; Murray, Peter J

2014-12-18

Nonresolving inflammation expands a heterogeneous population of myeloid suppressor cells capable of inhibiting T cell function. This heterogeneity has confounded the functional dissection of individual myeloid subpopulations and presents an obstacle for antitumor immunity and immunotherapy. Using genetic manipulation of cell death pathways, we found the monocytic suppressor-cell subset, but not the granulocytic subset, requires continuous c-FLIP expression to prevent caspase-8-dependent, RIPK3-independent cell death. Development of the granulocyte subset requires MCL-1-mediated control of the intrinsic mitochondrial death pathway. Monocytic suppressors tolerate the absence of MCL-1 provided cytokines increase expression of the MCL-1-related protein A1. Monocytic suppressors mediate T cell suppression, whereas their granulocytic counterparts lack suppressive function. The loss of the granulocytic subset via conditional MCL-1 deletion did not alter tumor incidence implicating the monocytic compartment as the functionally immunosuppressive subset in vivo. Thus, death pathway modulation defines the development, survival, and function of myeloid suppressor cells. Copyright © 2014 Elsevier Inc. All rights reserved.

miR-14 regulates autophagy during developmental cell death by targeting ip3-kinase 2.

PubMed

Nelson, Charles; Ambros, Victor; Baehrecke, Eric H

2014-11-06

Macroautophagy (autophagy) is a lysosome-dependent degradation process that has been implicated in age-associated diseases. Autophagy is involved in both cell survival and cell death, but little is known about the mechanisms that distinguish its use during these distinct cell fates. Here, we identify the microRNA miR-14 as being both necessary and sufficient for autophagy during developmentally regulated cell death in Drosophila. Loss of miR-14 prevented induction of autophagy during salivary gland cell death, but had no effect on starvation-induced autophagy in the fat body. Moreover, misexpression of miR-14 was sufficient to prematurely induce autophagy in salivary glands, but not in the fat body. Importantly, miR-14 regulates this context-specific autophagy through its target, inositol 1,4,5-trisphosphate kinase 2 (ip3k2), thereby affecting inositol 1,4,5-trisphosphate (IP3) signaling and calcium levels during salivary gland cell death. This study provides in vivo evidence of microRNA regulation of autophagy through modulation of IP3 signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

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

Transduced human copper chaperone for Cu,Zn-SOD (PEP-1-CCS) protects against neuronal cell death.

PubMed

Choi, Soo Hyun; Kim, Dae Won; Kim, So Young; An, Jae Jin; Lee, Sun Hwa; Choi, Hee Soon; Sohn, Eun Jung; Hwang, Seok-Il; Won, Moo Ho; Kang, Tae-Cheon; Kwon, Hyung Joo; Kang, Jung Hoon; Cho, Sung-Woo; Park, Jinseu; Eum, Won Sik; Choi, Soo Young

2005-12-31

Reactive oxygen species (ROS) contribute to the development of various human diseases. Cu,Zn-superoxide dismutase (SOD) is one of the major means by which cells counteract the deleterious effects of ROS. SOD activity is dependent upon bound copper ions supplied by its partner metallochaperone protein, copper chaperone for SOD (CCS). In the present study, we investigated the protective effects of PEP-1-CCS against neuronal cell death and ischemic insults. When PEP-1-CCS was added to the culture medium of neuronal cells, it rapidly entered the cells and protected them against paraquat-induced cell death. Moreover, transduced PEP-1-CCS markedly increased endogenous SOD activity in the cells. Immunohistochemical analysis revealed that it prevented neuronal cell death in the hippocampus in response to transient forebrain ischemia. These results suggest that CCS is essential to activate SOD, and that transduction of PEP-1-CCS provides a potential strategy for therapeutic delivery in various human diseases including stroke related to SOD or ROS.

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.

GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis

PubMed Central

Wrzaczek, Michael; Vainonen, Julia P; Stael, Simon; Tsiatsiani, Liana; Help-Rinta-Rahko, Hanna; Gauthier, Adrien; Kaufholdt, David; Bollhöner, Benjamin; Lamminmäki, Airi; Staes, An; Gevaert, Kris; Tuominen, Hannele; Van Breusegem, Frank; Helariutta, Ykä; Kangasjärvi, Jaakko

2015-01-01

Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor. PMID:25398910

GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis.

PubMed

Wrzaczek, Michael; Vainonen, Julia P; Stael, Simon; Tsiatsiani, Liana; Help-Rinta-Rahko, Hanna; Gauthier, Adrien; Kaufholdt, David; Bollhöner, Benjamin; Lamminmäki, Airi; Staes, An; Gevaert, Kris; Tuominen, Hannele; Van Breusegem, Frank; Helariutta, Ykä; Kangasjärvi, Jaakko

2015-01-02

Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor. © 2014 The Authors.

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.

K6 linked polyubiquitylation of FADD by CHIP prevents death inducing signaling complex formation suppressing cell death.

PubMed

Seo, Jinho; Lee, Eun-Woo; Shin, Jihye; Seong, Daehyeon; Nam, Young Woo; Jeong, Manhyung; Lee, Seon-Hyeong; Lee, Cheolju; Song, Jaewhan

2018-05-23

Fas-associated death domain (FADD) is an adaptor protein recruiting complexes of caspase 8 to death ligand receptors to induce extrinsic apoptotic cell death in response to a TNF superfamily member. Although, formation of the complex of FADD and caspase 8 upon death stimuli has been studied in detail, posttranslational modifications fine-tuning these processes have yet to be identified. Here we revealed that K6-linked polyubiquitylation of FADD on lysines 149 and 153 mediated by C terminus HSC70-interacting protein (CHIP) plays an important role in preventing formation of the death inducing signaling complex (DISC), thus leading to the suppression of cell death. Cells depleted of CHIP showed higher sensitivity toward death ligands such as FasL and TRAIL, leading to upregulation of DISC formation composed of a death receptor, FADD, and caspase 8. CHIP was able to bind to FADD, induce K6-linked polyubiquitylation of FADD, and suppress DISC formation. By mass spectrometry, lysines 149 and 153 of FADD were found to be responsible for CHIP-mediated FADD ubiquitylation. FADD mutated at these sites was capable of more potent cell death induction as compared with the wild type and was no longer suppressed by CHIP. On the other hand, CHIP deficient in E3 ligase activity was not capable of suppressing FADD function and of FADD ubiquitylation. CHIP depletion in ME-180 cells induced significant sensitization of these cells toward TRAIL in xenograft analyses. These results imply that K6-linked ubiquitylation of FADD by CHIP is a crucial checkpoint in cytokine-dependent extrinsic apoptosis.

JTC801 Induces pH-dependent Death Specifically in Cancer Cells and Slows Growth of Tumors in Mice.

PubMed

Song, Xinxin; Zhu, Shan; Xie, Yangchun; Liu, Jiao; Sun, Lingyi; Zeng, Dexing; Wang, Pengcheng; Ma, Xiaochao; Kroemer, Guido; Bartlett, David L; Billiar, Timothy R; Lotze, Michael T; Zeh, Herbert J; Kang, Rui; Tang, Daolin

2018-04-01

Maintenance of acid-base homeostasis is required for normal physiology, metabolism, and development. It is not clear how cell death is activated in response to changes in pH. We performed a screen to identify agents that induce cell death in a pH-dependent manner (we call this alkaliptosis) in pancreatic ductal adenocarcinoma cancer (PDAC) cells and tested their effects in mice. We screened a library of 254 compounds that interact with G-protein-coupled receptors (GPCRs) to identify those with cytotoxic activity against a human PDAC cell line (PANC1). We evaluated the ability of JTC801, which binds the opiod receptor and has analgesic effects, to stimulate cell death in human PDAC cell lines (PANC1, MiaPaCa2, CFPAC1, PANC2.03, BxPc3, and CAPAN2), mouse pancreatic cancer-associated stellate cell lines, primary human pancreatic ductal epithelial cells, and 60 cancer cell lines (the NCI-60 panel). Genes encoding proteins in cell death and GPCR signaling pathways, as well as those that regulate nuclear factor-κB (NF-κB) activity, were knocked out, knocked down, or expressed from transgenes in cancer cell lines. JTC801 was administered by gavage to mice with xenograft tumors, C57BL/6 mice with orthographic pancreatic tumors grown from Pdx1-Cre;KRas G12D/+ ;Tp53 R172H/+ (KPC) cells, mice with metastases following tail-vein injection of KPC cells, and Pdx-1-Cre;Kras G12D/+ mice crossed with Hmgb1 flox/flox mice (KCH mice). Pancreata were collected from mice and analyzed for tumor growth and by histology and immunohistochemistry. We compared gene and protein expression levels between human pancreatic cancer tissues and patient survival times using online R2 genomic or immunohistochemistry analyses. Exposure of human PDAC cell lines (PANC1 and MiaPaCa2) to JTC801 did not induce molecular markers of apoptosis (cleavage of caspase 3 or poly [ADP ribose] polymerase [PARP]), necroptosis (interaction between receptor-interacting serine-threonine kinase 3 [RIPK3] and mixed lineage kinase domain like pseudokinase [MLKL]), or ferroptosis (degradation of glutathione peroxidase 4 [GPX4]). Inhibitors of apoptosis (Z-VAD-FMK), necroptosis (necrosulfonamide), ferroptosis (ferrostatin-1), or autophagy (hydroxychloroquine) did not prevent JTC801-induced death of PANC1 or MiaPaCa2 cells. The cytotoxic effects of JTC801 in immortalized fibroblast cell lines was not affected by disruption of genes that promote apoptosis (Bax -/- /Bak -/- cells), necroptosis (Ripk1 -/- , Ripk3 -/- , or Mlkl -/- cells), ferroptosis (Gpx4 -/- cells), or autophagy (Atg3 -/- , Atg5 -/- , Atg7 -/- , or Sqstm1 -/- cells). We found JTC801 to induce a pH-dependent form cell death (alkaliptosis) in cancer cells but not normal cells (hepatocytes, bone marrow CD34 + progenitor cells, peripheral blood mononuclear cells, or dermal fibroblasts) or healthy tissues of C57BL/6 mice. JTC801 induced alkaliptosis in cancer cells by activating NF-κB, which repressed expression of the carbonic anhydrase 9 gene (CA9), whose product regulates pH balance in cells. In analyses of Cancer Genome Atlas data and tissue microarrays, we associated increased tumor level of CA9 mRNA or protein with shorter survival times of patients with pancreatic, kidney, or lung cancers. Knockdown of CA9 reduced the protective effects of NF-κB inhibition on JTC801-induced cell death and intracellular alkalinization in PANC1 and MiaPaCa2 cell lines. Oral administration of JTC801 inhibited growth of xenograft tumors (from PANC1, MiaPaCa2, SK-MEL-28, PC-3, 786-0, SF-295, HCT116, OV-CAR3, and HuH7 cells), orthotropic tumors (from KPC cells), lung metastases (from KPC cells) of mice, and slowed growth of tumors in KCH mice. In a screen of agents that interact with GPCR pathways, we found JTC801 to induce pH-dependent cell death (alkaliptosis) specifically in cancer cells such as PDAC cells, by reducing expression of CA9. Levels of CA9 are increased in human cancer tissues. JTC801 might be developed for treatment of pancreatic cancer. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Low-frequency quantitative ultrasound imaging of cell death in vivo

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

Sadeghi-Naini, Ali; Falou, Omar; Czarnota, Gregory J.

Purpose: Currently, no clinical imaging modality is used routinely to assess tumor response to cancer therapies within hours to days of the delivery of treatment. Here, the authors demonstrate the efficacy of ultrasound at a clinically relevant frequency to quantitatively detect changes in tumors in response to cancer therapies using preclinical mouse models.Methods: Conventional low-frequency and corresponding high-frequency ultrasound (ranging from 4 to 28 MHz) were used along with quantitative spectroscopic and signal envelope statistical analyses on data obtained from xenograft tumors treated with chemotherapy, x-ray radiation, as well as a novel vascular targeting microbubble therapy.Results: Ultrasound-based spectroscopic biomarkers indicatedmore » significant changes in cell-death associated parameters in responsive tumors. Specifically changes in the midband fit, spectral slope, and 0-MHz intercept biomarkers were investigated for different types of treatment and demonstrated cell-death related changes. The midband fit and 0-MHz intercept biomarker derived from low-frequency data demonstrated increases ranging approximately from 0 to 6 dBr and 0 to 8 dBr, respectively, depending on treatments administrated. These data paralleled results observed for high-frequency ultrasound data. Statistical analysis of ultrasound signal envelope was performed as an alternative method to obtain histogram-based biomarkers and provided confirmatory results. Histological analysis of tumor specimens indicated up to 61% cell death present in the tumors depending on treatments administered, consistent with quantitative ultrasound findings indicating cell death. Ultrasound-based spectroscopic biomarkers demonstrated a good correlation with histological morphological findings indicative of cell death (r{sup 2}= 0.71, 0.82; p < 0.001).Conclusions: In summary, the results provide preclinical evidence, for the first time, that quantitative ultrasound used at a clinically relevant frequency, in addition to high-frequency ultrasound, can detect tissue changes associated with cell death in vivo in response to cancer treatments.« less

mTOR kinase inhibitor pp242 causes mitophagy terminated by apoptotic cell death in E1A-Ras transformed cells.

PubMed

Gordeev, Serguei A; Bykova, Tatiana V; Zubova, Svetlana G; Bystrova, Olga A; Martynova, Marina G; Pospelov, Valery A; Pospelova, Tatiana V

2015-12-29

mTOR is a critical target for controlling cell cycle progression, senescence and cell death in mammalian cancer cells. Here we studied the role of mTOR-dependent autophagy in implementating the antiprolifrative effect of mTORC1-specific inhibitor rapamycin and ATP-competitive mTOR kinase inhibitor pp242. We carried out a comprehensive analysis of pp242- and rapamycin-induced autophagy in ERas tumor cells. Rapamycin exerts cytostatic effect on ERas tumor cells, thus causing a temporary and reversible cell cycle arrest, activation of non-selective autophagy not accompanied by cell death. The rapamycin-treated cells are able to continue proliferation after drug removal. The ATP-competitive mTORC1/mTORC2 kinase inhibitor pp242 is highly cytotoxic by suppressing the function of mTORC1-4EBP1 axis and mTORC1-dependent phosphorylation of mTORC1 target--ULK1-Ser757 (Atg1). In contrast to rapamycin, pp242 activates the selective autophagy targeting mitochondria (mitophagy). The pp242-induced mitophagy is accompanied by accumulation of LC3 and conversion of LC3-I form to LC3-II. However reduced degradation of p62/SQSTM indicates abnormal flux of autophagic process. According to transmission electron microscopy data, short-term pp242-treated ERas cells exhibit numerous heavily damaged mitochondria, which are included in single membrane-bound autophagic/autolysophagic vacuoles (mitophagy). Despite the lack of typical for apoptosis features, ERas-treated cells with induced mitophagy revealed the activation of caspase 3, 9 and nucleosomal DNA fragmentation. Thus, pp242 activates autophagy with suppressed later stages, leading to impaired recycling and accumulation of dysfunctional mitochondria and cell death. Better understanding of how autophagy determines the fate of a cell--survival or cell death, can help to development of new strategy for cancer therapy.

Novel Phosphorylation and Ubiquitination Sites Regulate Reactive Oxygen Species-dependent Degradation of Anti-apoptotic c-FLIP Protein*

PubMed Central

Wilkie-Grantham, Rachel P.; Matsuzawa, Shu-Ichi; Reed, John C.

2013-01-01

The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIPL) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIPL protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIPL important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL. PMID:23519470

Novel phosphorylation and ubiquitination sites regulate reactive oxygen species-dependent degradation of anti-apoptotic c-FLIP protein.

PubMed

Wilkie-Grantham, Rachel P; Matsuzawa, Shu-Ichi; Reed, John C

2013-05-03

The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIP(L)) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIP(L) protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIP(L) important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL.

Clonidine Induces Apoptosis of Human Corneal Epithelial Cells through Death Receptors-Mediated, Mitochondria-Dependent Signaling Pathway.

PubMed

Fan, Dan; Fan, Ting-Jun

2017-03-01

Clonidine, an α2-adrenoreceptor agonist, is an anti-glaucoma drug clinically used in many developing countries, and its abuse might damage the cornea and impair human vision. However, its cytotoxicity and precise mechanisms need to be elucidated. Herein, we investigated the cytotoxicity of clonidine and its underlying mechanisms, using an in vitro model of human corneal epithelial (HCEP) cells and an in vivo model of cat corneas, respectively. HCEP cells were treated with various doses of clonidine for 1-28 h, resulting in abnormal morphology, decline of cell viability and G1 phase arrest in a time- and/or dose-dependent manner. Moreover, clonidine treatment induced elevation of plasma membrane permeability, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation in HCEP cells. Furthermore, we found that clonidine treatment resulted in activated caspase-2, -3, -8, and -9, disruption of the mitochondrial transmembrane potential, downregulation of Bcl-2, and upregulation of Bad, cytoplasmic cytochrome c and apoptosis inducing factor, suggesting that clonidine-induced apoptosis is triggered through Fas/TNFR1 death receptors and Bcl-2 family proteins-mediated mitochondria-dependent pathways. Finally, our in vivo results displayed that 0.25% clonidine could induce DNA fragmentation of cat corneal epithelial cells. In summary, our findings suggest that clonidine above 1/32 of its clinical therapeutic dosage is cytotoxic to corneal epithelial cells by inducing cell apoptosis both in vitro and in vivo, and its pro-apoptotic effect on HCEP cells is triggered by a Fas/TNFR1 death receptors-mediated, mitochondria-dependent signaling pathway. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Disruption of the vacuolar calcium-ATPases in arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway

USDA-ARS?s Scientific Manuscript database

Calcium (Ca2+) signals regulate many aspects of plant development, including the Hypersensitive Response (HR) that triggers a programmed cell death response to protect a plant from a pathogen. A transient increase in cytosolic Ca2+ ([Ca2+]cyt ) results from Ca2+ entry from the apoplast or release fr...

MP Resulting in Autophagic Cell Death of Microglia through Zinc Changes against Spinal Cord Injury

PubMed Central

Li, Dingding; Wang, Guannan; Han, Donghe; Bi, Jing; Li, Chenyuan; Wang, Hongyu; Liu, Zhiyuan; Gao, Wei; Gao, Kai; Yao, Tianchen; Wan, Zhanghui; Li, Haihong; Mei, Xifan

2016-01-01

Methylprednisolone pulse therapy (MPPT), as a public recognized therapy of spinal cord injury (SCI), is doubted recently, and the exact mechanism of MP on SCI is unclear. This study sought to investigate the exact effect of MP on SCI. We examined the effect of MP in a model of SCI in vivo and an LPS induced model in vitro. We found that administration of MP produced an increase in the Basso, Beattie, and Bresnahan scores and motor neurons counts of injured rats. Besides the number of activated microglia was apparently reduced by MP in vivo, and Beclin-1 dependent autophagic cell death of microglia was induced by MP in LPS induced model. At the same time, MP increases cellular zinc concentration and level of ZIP8, and TPEN could revert effect of MP on autophagic cell death of microglia. Finally, we have found that MP could inhibit NF-κβ in LPS induced model. These results show that the MP could result in autophagic cell death of microglia, which mainly depends on increasing cellular labile zinc, and may be associated with inhibition of NF-κβ, and that MP can produce neuroprotective effect in SCI. PMID:27057544

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.

Inhibition of Cyclooxygenase-2 (COX-2) Initiates Autophagy and Potentiates MPTP-Induced Autophagic Cell Death of Human Neuroblastoma Cells, SH-SY5Y: an Inside in the Pathology of Parkinson's Disease.

PubMed

Niranjan, Rituraj; Mishra, Kaushal Prasad; Thakur, Ashwani Kumar

2018-03-01

Cyclooxygenase-2 or COX-2 has been known to be crucial for Parkinson's disease (PD) pathogenesis; however, its exact role is still not known. We first time report that inhibition of COX-2 promotes 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP)-induced neuronal cell death via induction of autophagic mechanisms. We found that treatment with MPTP induced cell death of neuroblastoma cells SH-SY5Y in a dose dependent manner. Treatment of MPTP has also upregulated the expressions of autophagic proteins such as LC3, beclin, ATG-5, and p62. Interestingly, nimesulide, a preferential COX-2 inhibitor, further potentiated the MPTP-induced cell death of human neuroblastoma cells. Treatment of nimesulide with MPTP further potentiated expressions of p62, ATG-5, beclin-1, LC3 autophagic proteins. Furthermore, nimesulide with MPTP increased apoptotic protein cleaved caspase-3 and also induced expression of p53 gene. Interestingly, it was observed that Akt inhibitor significantly increased MPTP-induced cell death of neuroblastoma cells. However, (-) deprenyl, a monoamine oxidase B (MAO B) inhibitor, attenuated MPTP-induced autophagic response and protected cell death. The prior treatment with prostaglandin E2 protected against nimesulide induced-death of neuronal cells. This study confirms that neuroinflammation is associated to the autophagy and may be one of the main pathological mechanisms in Parkinson's disease and other inflammation-associated disorders.

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.

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

PubMed

Ramírez-Barrantes, Ricardo; Córdova, Claudio; Gatica, Sebastian; Rodriguez, Belén; Lozano, Carlo; Marchant, Ivanny; Echeverria, Cesar; Simon, Felipe; Olivero, Pablo

2018-01-01

The transient receptor potential (TRP) ion channel family consists of a broad variety of non-selective cation channels that integrate environmental physicochemical signals for dynamic homeostatic control. Involved in a variety of cellular physiological processes, TRP channels are fundamental to the control of the cell life cycle. TRP channels from the vanilloid (TRPV) family have been directly implicated in cell death. TRPV1 is activated by pain-inducing stimuli, including inflammatory endovanilloids and pungent exovanilloids, such as capsaicin (CAP). TRPV1 activation by high doses of CAP (>10 μM) leads to necrosis, but also exhibits apoptotic characteristics. However, CAP dose-response studies are lacking in order to determine whether CAP-induced cell death occurs preferentially via necrosis or apoptosis. In addition, it is not known whether cytosolic Ca 2+ and mitochondrial dysfunction participates in CAP-induced TRPV1-mediated cell death. By using TRPV1-transfected HeLa cells, we investigated the underlying mechanisms involved in CAP-induced TRPV1-mediated cell death, the dependence of CAP dose, and the participation of mitochondrial dysfunction and cytosolic Ca 2+ increase. Together, our results contribute to elucidate the pathophysiological steps that follow after TRPV1 stimulation with CAP. Low concentrations of CAP (1 μM) induce cell death by a mechanism involving a TRPV1-mediated rapid and transient intracellular Ca 2+ increase that stimulates plasma membrane depolarization, thereby compromising plasma membrane integrity and ultimately leading to cell death. Meanwhile, higher doses of CAP induce cell death via a TRPV1-independent mechanism, involving a slow and persistent intracellular Ca 2+ increase that induces mitochondrial dysfunction, plasma membrane depolarization, plasma membrane loss of integrity, and ultimately, cell death.

L-type voltage-dependent calcium channel is involved in the snake venom group IA secretory phospholipase A2-induced neuronal apoptosis.

PubMed

Yagami, Tatsurou; Yamamoto, Yasuhiro; Kohma, Hiromi; Nakamura, Tsutomu; Takasu, Nobuo; Okamura, Noboru

2013-03-01

Snake venom group IA secretory phospholipase A2 (sPLA2-IA) is known as a neurotoxin. Snake venom sPLA2s are neurotoxic in vivo and in vitro, causing synergistic neurotoxicity to cortical cultures when applied with toxic concentrations of glutamate. However, it has not yet been cleared sufficiently how sPLA2-IA exerts neurotoxicity. Here, we found sPLA2-IA induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6h. sPLA2-IA-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. NMDA receptor blockers suppressed the neurotoxicity of sPLA2-IA, but an AMPA receptor blocker did not. Interestingly, L-type voltage-dependent Ca(2+) channel (L-VDCC) blocker significantly protected neurons from the sPLA2-IA-induced apoptosis. On the other hand, neither N-VDCC blockers nor P/Q-VDCC blocker did. In conclusion, we demonstrated that sPLA2-IA induced neuronal cell death via apoptosis. Furthermore, the present study suggests that not only NMDA receptor but also L-VDCC contributed to the neurotoxicity of snake venom sPLA2-IA. Copyright © 2013 Elsevier Inc. 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.

Capsaicin sensitizes TRAIL-induced apoptosis through Sp1-mediated DR5 up-regulation: Involvement of Ca{sup 2+} influx

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

Moon, Dong-Oh; Kang, Chang-Hee; Kang, Sang-Hyuck

2012-02-15

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various malignant cells, several cancers including human hepatocellular carcinoma (HCC) exhibit potent resistance to TRAIL-induced cell death. The aim of this study is to evaluate the anti-cancer potential of capsaicin in TRAIL-induced cancer cell death. As indicated by assays that measure phosphatidylserine exposure, mitochondrial activity and activation of caspases, capsaicin potentiated TRAIL-resistant cells to lead to cell death. In addition, we found that capsaicin induces the cell surface expression of TRAIL receptor DR5, but not DR4 through the activation Sp1 on its promoter region. Furthermore, we investigated that capsaicin-induced DR5more » expression and apoptosis are inhibited by calcium chelator or inhibitors for calmodulin-dependent protein kinase. Taken together, our data suggest that capsaicin sensitizes TRAIL-mediated HCC cell apoptosis by DR5 up-regulation via calcium influx-dependent Sp1 activation. Highlights: ► Capsaicin sensitizes TRAIL-induced apoptosis through activation of caspases. ► Capsaicin induces expression of DR5 through Sp1 activation. ► Capsaicin activates calcium signaling pathway.« less

Inhibition of ERK activity enhances the cytotoxic effect of peroxisome proliferator-activated receptor γ (PPARγ) agonists in HeLa cells

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

Chang, Ha Kyun; Kim, Dae Seong; Chae, Jung Jun

In this study, we examined whether the peroxisome proliferator-activated receptor γ (PPARγ) agonists, ciglitazone (CGZ) and troglitazone (TGZ), induce cell death in human cervical cancer HeLa cells. The cells were treated with a range of CGZ or TGZ doses for 24 or 48 h. Low concentrations of CGZ (≤10 μM) or TGZ (≤20 μM) had no effect on cell viability whereas higher doses induced cell death in a time- and dose-dependent manner as evidenced by the detection of activated caspase-3 and PARP cleavage. Treatment with the PPARγ antagonist GW9662 followed by PPARγ agonists did not increase CGZ- or TGZ-induced cell death, indicating thatmore » PPARγ agonists induced HeLa cell death independently of PPARγ. Moreover, ERK1/2 activation was observed at a CGZ concentration of 25 μM and a TGZ concentration of 35 μM, both of which induced cell death. To elucidate the role of ERK1/2 activated by the two PPARγ agonists, the effect of U0126, an inhibitor of ERK1/2, on PPARγ-agonist-induced cell death was examined. Treatment with 10 or 20 μM U0126 followed by CGZ or TGZ induced the down-regulation of ERK1/2 activity and a decrease in Bcl-2 expression accompanied by the collapse of mitochondrial membrane potential, which in turn significantly enhanced CGZ- or TGZ-induced apoptotic cell death. Our results suggest that PPARγ agonists are capable of inducing apoptotic cell death in HeLa cells independently of PPARγ and that inhibition of ERK1/2 activity offers a strategy to enhance the cytotoxicity of PPARγ agonists in the treatment of cervical cancer. - Highlights: • The PPARγ agonists CGZ and TGZ induce apoptotic cell death in HeLa cells. • CGZ or TGZ induces apoptotic cell death independently of PPARγ in HeLa cells. • Inhibition of ERK1/2 enhances CGZ- or TGZ-induced cell death via the collapse of MMP.« less

Reciprocal sensitivity of diffuse large B-cell lymphoma cells to Bcl-2 inhibitors BIRD-2 versus venetoclax

PubMed Central

Vervloessem, Tamara; Akl, Haidar; Tousseyn, Thomas; De Smedt, Humbert; Parys, Jan B.; Bultynck, Geert

2017-01-01

Bcl-2 is often upregulated in cancers to neutralize the BH3-only protein Bim at the mitochondria. BH3 mimetics (e.g. ABT-199 (venetoclax)) kill cancer cells by targeting Bcl-2’s hydrophobic cleft and disrupting Bcl-2/Bim complexes. Some cancers with elevated Bcl-2 display poor responses towards BH3 mimetics, suggesting an additional function for anti-apoptotic Bcl-2 in these cancers. Indeed, Bcl-2 via its BH4 domain prevents cytotoxic Ca2+ release from the endoplasmic reticulum (ER) by directly inhibiting the inositol 1,4,5-trisphosphate receptor (IP3R). The cell-permeable Bcl-2/IP3R disruptor-2 (BIRD-2) peptide can kill these Bcl-2-dependent cancers by targeting Bcl-2’s BH4 domain, unleashing pro-apoptotic Ca2+-release events. We compared eight “primed to death” diffuse large B-cell lymphoma cell lines (DLBCL) for their apoptotic sensitivity towards BIRD-2 and venetoclax. By determining their IC50 using cytometric cell-death analysis, we discovered a reciprocal sensitivity towards venetoclax versus BIRD-2. Using immunoblotting, we quantified the expression levels of IP3R2 and Bim in DLBCL cell lysates, revealing that BIRD-2 sensitivity correlated with IP3R2 levels but not with Bim levels. Moreover, the requirement of intracellular Ca2+ for BIRD-2- versus venetoclax-induced cell death was different. Indeed, BAPTA-AM suppressed BIRD-2-induced cell death, but promoted venetoclax-induced cell death in DLBCL cells. Finally, compared to single-agent treatments, combining BIRD-2 with venetoclax synergistically enhanced cell-death induction, correlating with a Ca2+-dependent upregulation of Bim after BIRD-2 treatment. Our findings suggest that some cancer cells require Bcl-2 proteins at the mitochondria, preventing Bax activation via its hydrophobic cleft, while others require Bcl-2 proteins at the ER, preventing cytotoxic Ca2+-signaling events via its BH4 domain. PMID:29340082

Constitutive Activation of PINK1 Protein Leads to Proteasome-mediated and Non-apoptotic Cell Death Independently of Mitochondrial Autophagy*

PubMed Central

Akabane, Shiori; Matsuzaki, Kohei; Yamashita, Shun-ichi; Arai, Kana; Okatsu, Kei; Kanki, Tomotake; Matsuda, Noriyuki; Oka, Toshihiko

2016-01-01

Phosphatase and tensin homolog-induced putative kinase 1 (PINK1), a Ser/Thr kinase, and PARKIN, a ubiquitin ligase, are causal genes for autosomal recessive early-onset parkinsonism. Multiple lines of evidence indicate that PINK1 and PARKIN cooperatively control the quality of the mitochondrial population via selective degradation of damaged mitochondria by autophagy. Here, we report that PINK1 and PARKIN induce cell death with a 12-h delay after mitochondrial depolarization, which differs from the time profile of selective autophagy of mitochondria. This type of cell death exhibited definite morphologic features such as plasma membrane rupture, was insensitive to a pan-caspase inhibitor, and did not involve mitochondrial permeability transition. Expression of a constitutively active form of PINK1 caused cell death in the presence of a pan-caspase inhibitor, irrespective of the mitochondrial membrane potential. PINK1-mediated cell death depended on the activities of PARKIN and proteasomes, but it was not affected by disruption of the genes required for autophagy. Furthermore, fluorescence and electron microscopic analyses revealed that mitochondria were still retained in the dead cells, indicating that PINK1-mediated cell death is not caused by mitochondrial loss. Our findings suggest that PINK1 and PARKIN play critical roles in selective cell death in which damaged mitochondria are retained, independent of mitochondrial autophagy. PMID:27302064

Cytokine-induced release of ceramide-enriched exosomes as a mediator of cell death signaling in an oligodendroglioma cell line[S

PubMed Central

Podbielska, Maria; Szulc, Zdzisław M.; Kurowska, Ewa; Hogan, Edward L.; Bielawski, Jacek; Bielawska, Alicja; Bhat, Narayan R.

2016-01-01

Th1 pro-inflammatory cytokines, i.e., TNF-α and IFN-γ, in combination are known to induce cell death in several cell types, including oligodendrocytes, but the mechanism of their synergistic cytotoxicity is unclear. Although ceramide (Cer) has been implicated in cytokine- and stress-induced cell death, its intracellular levels alone cannot explain cytokine synergy. We considered the possibility that Cer released as part of extracellular vesicles may contribute to cytokine-induced synergistic cell death. Using a human oligodendroglioma (HOG) cell line as a model, here we show that exosomes derived from TNF-α-treated “donor” cells, while being mildly toxic to fresh cultures (similar to individual cytokines), induce enhanced cell death when added to IFN-γ-primed target cultures in a fashion resembling the effect of cytokine combination. Further, the sphingolipid profiles of secreted exosomes, as determined by HPLC-MS/MS, revealed that the treatment with the cytokines time-dependently induced the formation and exosomal release, in particular of C16-, C24-, and C24:1-Cer species; C16-, C24-, and C24:1-dihydroCer species; and C16-, C24-, and C24:1-SM species. Finally, exogenous C6-Cer or C16-Cer mimicked and enhanced the cytotoxic effects of the cytokines upon HOG cells, thereby supporting the cell death-signaling role of extracellular Cer. PMID:27623848

Deciphering early events involved in hyperosmotic stress-induced programmed cell death in tobacco BY-2 cells

PubMed Central

Monetti, Emanuela; Kadono, Takashi; Bouteau, François

2014-01-01

Hyperosmotic stresses represent one of the major constraints that adversely affect plants growth, development, and productivity. In this study, the focus was on early responses to hyperosmotic stress- (NaCl and sorbitol) induced reactive oxygen species (ROS) generation, cytosolic Ca2+ concentration ([Ca2+]cyt) increase, ion fluxes, and mitochondrial potential variations, and on their links in pathways leading to programmed cell death (PCD). By using BY-2 tobacco cells, it was shown that both NaCl- and sorbitol-induced PCD seemed to be dependent on superoxide anion (O2·–) generation by NADPH-oxidase. In the case of NaCl, an early influx of sodium through non-selective cation channels participates in the development of PCD through mitochondrial dysfunction and NADPH-oxidase-dependent O2·– generation. This supports the hypothesis of different pathways in NaCl- and sorbitol-induced cell death. Surprisingly, other shared early responses, such as [Ca2+]cyt increase and singlet oxygen production, do not seem to be involved in PCD. PMID:24420571

Tributyltin-induced apoptosis requires glycolytic adenosine trisphosphate production.

PubMed

Stridh, H; Fava, E; Single, B; Nicotera, P; Orrenius, S; Leist, M

1999-10-01

The toxicity of tributyltin chloride (TBT) involves Ca(2+) overload, cytoskeletal damage, and mitochondrial failure leading to cell death by apoptosis or necrosis. Here, we examined whether the intracellular ATP level modulates the mode of cell death after exposure to TBT. When Jurkat cells were energized by the mitochondrial substrate, pyruvate, low concentrations of TBT (1-2 microM) triggered an immediate depletion of intracellular ATP followed by necrotic death. When ATP levels were maintained by the addition of glucose, the mode of cell death was typically apoptotic. Glycolytic ATP production was required for apoptosis at two distinct steps. First, maintenance of adequate ATP levels accelerated the decrease of mitochondrial membrane potential, and the release of the intermembrane proteins adenylate kinase and cytochrome c from mitochondria. A possible role of the adenine nucleotide exchanger in this first ATP-dependent step is suggested by experiments performed with the specific inhibitor, bongkrekic acid. This substance delayed cytochrome c release in a manner similar to that caused by ATP depletion. Second, caspase activation following cytochrome c release was only observed in ATP-containing cells. Bcl-2 had only a minor effect on TBT-triggered caspase activation or cell death. We conclude that intracellular ATP concentrations control the mode of cell death in TBT-treated Jurkat cells at both the mitochondrial and caspase activation levels.

Bioactive compounds from crocodile (Crocodylus siamensis) white blood cells induced apoptotic cell death in hela cells.

PubMed

Patathananone, Supawadee; Thammasirirak, Sompong; Daduang, Jureerut; Chung, Jing Gung; Temsiripong, Yosapong; Daduang, Sakda

2016-08-01

Crocodile (Crocodylus siamensis) white blood cell extracts (WBCex) were examined for anticancer activity in HeLa cell lines using the MTT assay. The percentage viability of HeLa cells significantly deceased after treatment with WBCex in a dose- and time-dependent manner. The IC50 dose was suggested to be approximately 225 μg/mL protein. Apoptotic cell death occurred in a time-dependent manner based on investigation by flow cytometry using annexin V-FITC and PI staining. DAPI nucleic acid staining indicated increased chromatin condensation. Caspase-3, -8 and -9 activities also increased, suggesting the induction of the caspase-dependent apoptotic pathway. Furthermore, the mitochondrial membrane potential (ΔΨm ) of HeLa cells was lost as a result of increasing levels of Bax and reduced levels of Bcl-2, Bcl-XL, Bcl-Xs, and XIAP. The decreased ΔΨm led to the release of cytochrome c and the activation of caspase-9 and -3. Apoptosis-inducing factor translocated into the nuclei, and endonuclease G (Endo G) was released from the mitochondria. These results suggest that anticancer agents in WBCex can induce apoptosis in HeLa cells via both caspase-dependent and -independent pathways. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 986-997, 2016. © 2015 Wiley Periodicals, Inc.

The role of the cyclin-dependent kinase inhibitor p21 in apoptosis.

PubMed

Gartel, Andrei L; Tyner, Angela L

2002-06-01

Cancer develops when the balance between cell proliferation and cell death is disrupted, and the ensuing aberrant proliferation leads to tumor growth. The cyclin-dependent kinase inhibitor p21 is induced by both p53-dependent and -independent mechanisms following stress, and induction of p21 may cause cell cycle arrest. As a proliferation inhibitor, p21 is poised to play an important role in preventing tumor development. This notion is supported by data indicating that p21-null mice are more prone to spontaneous and induced tumorigenesis, and p21 synergizes with other tumor suppressors to protect against tumor progression in mice. However, a number of recent studies have pointed out that in addition to being an inhibitor of cell proliferation, p21 acts as an inhibitor of apoptosis in a number of systems, and this may counteract its tumor-suppressive functions as a growth inhibitor. In the current review, we discuss the role of p21 in regulating cell death and the potential relevance of its expression in cancer.

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.

In vitro and in vivo anti-cancer activity of silymarin on oral cancer.

PubMed

Won, Dong-Hoon; Kim, Lee-Han; Jang, Boonsil; Yang, In-Hyoung; Kwon, Hye-Jeong; Jin, Bohwan; Oh, Seung Hyun; Kang, Ju-Hee; Hong, Seong-Doo; Shin, Ji-Ae; Cho, Sung-Dae

2018-05-01

Silymarin, a standardized extract from milk thistle fruits has been found to exhibit anti-cancer effects against various cancers. Here, we explored the anti-cancer activity of silymarin and its molecular target in human oral cancer in vitro and in vivo. Silymarin dose-dependently inhibited the proliferation of HSC-4 oral cancer cells and promoted caspase-dependent apoptosis. A human apoptosis protein array kit showed that death receptor 5 may be involved in silymarin-induced apoptosis, which was also shown through western blotting, immunocytochemistry, and reverse transcription-polymerase chain reaction. Silymarin increased cleaved caspase-8 and truncated Bid, leading to accumulation of cytochrome c. In addition, silymarin activated death receptor 5/caspase-8 to induce apoptotic cell death in two other oral cancer cell lines (YD15 and Ca9.22). Silymarin also suppressed tumor growth and volume without any hepatic or renal toxicity in vivo. Taken together, these results provide in vitro and in vivo evidence supporting the anti-cancer effect of silymarin and death receptor 5, and caspase-8 may be essential players in silymarin-mediated apoptosis in oral cancer.

The retraction of the protoplast during PCD is an active, and interruptible, calcium-flux driven process.

PubMed

Kacprzyk, Joanna; Brogan, Niall P; Daly, Cara T; Doyle, Siamsa M; Diamond, Mark; Molony, Elizabeth M; McCabe, Paul F

2017-07-01

The protoplast retracts during apoptosis-like programmed cell death (AL-PCD) and, if this retraction is an active component of AL-PCD, it should be used as a defining feature for this type of programmed cell death. We used an array of pharmacological and genetic tools to test if the rates of protoplast retraction in cells undergoing AL-PCD can be modulated. Disturbing calcium flux signalling, ATP synthesis and mitochondrial permeability transition all inhibited protoplast retraction and often also the execution of the death programme. Protoplast retraction can precede loss of plasma membrane integrity and cell death can be interrupted after the protoplast retraction had already occurred. Blocking calcium influx inhibited the protoplast retraction, reduced DNA fragmentation and delayed death induced by AL-PCD associated stresses. At higher levels of stress, where cell death occurs without protoplast retraction, blocking calcium flux had no effect on the death process. The results therefore strongly suggest that retraction of the protoplast is an active biological process dependent on an early Ca 2+ -mediated trigger rather than cellular disintegration due to plasma membrane damage. Therefore this morphologically distinct cell type is a quantifiable feature, and consequently, reporter of AL-PCD. Copyright © 2017 Elsevier B.V. All rights reserved.

Pathogen-induced ubiquitin-editing enzyme A20 bifunctionally shuts off NF-κB and caspase-8-dependent apoptotic cell death

PubMed Central

Lim, Michelle C C; Maubach, Gunter; Sokolova, Olga; Feige, Michael H; Diezko, Rolf; Buchbinder, Jörn; Backert, Steffen; Schlüter, Dirk; Lavrik, Inna N; Naumann, Michael

2017-01-01

The human pathogen Helicobacter pylori infects more than half of the world’s population and is a paradigm for persistent yet asymptomatic infection but increases the risk for chronic gastritis and gastric adenocarcinoma. For successful colonization, H. pylori needs to subvert the host cell death response, which serves to confine pathogen infection by killing infected cells and preventing malignant transformation. Infection of gastric epithelial cells by H. pylori provokes direct and fast activation of the proinflammatory and survival factor NF-κB, which regulates target genes, such as CXCL8, BIRC3 and TNFAIP3. However, it is not known how H. pylori exploits NF-κB activation and suppresses the inflammatory response and host apoptotic cell death, in order to avert the innate immune response and avoid cell loss, and thereby enhance colonization to establish long-term infection. Here we assign for the first time that H. pylori and also Campylobacter jejuni-induced ubiquitin-editing enzyme A20 bifunctionally terminates NF-κB activity and negatively regulates apoptotic cell death. Mechanistically, we show that the deubiquitinylase activity of A20 counteracts cullin3-mediated K63-linked ubiquitinylation of procaspase-8, therefore restricting the activity of caspase-8. Interestingly, another inducible NF-κB target gene, the scaffold protein p62, ameliorates the interaction of A20 with procaspase-8. In conclusion, pathogen-induced de novo synthesis of A20 regulates the shut-off of the survival factor NF-κB but, on the other hand, also impedes caspase-8-dependent apoptotic cell death so as to promote the persistence of pathogens. PMID:28574503

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.

Differential Expression of Programmed Cell Death on the Follicular Development in Normal and Miniature Pig Ovary

PubMed Central

Kim, Sang Hwan; Min, Kwan Sik; Kim, Nam Hyung; Yoon, Jong Taek

2012-01-01

Follicles are important in oocyte maturation. Successful estrous cycle requires remodeling of follicular cells, and proper execution of programmed cell death is crucial for normal follicular development. The objectives of the present study were to understand programmed cell death during follicle development, to analyze the differential follicle development patterns, and to assess the patterns of apoptosis and autophagy expression during follicle development in normal and miniature pigs. Through the analysis of differential patterns of programmed cell death during follicular development in porcine, MAP1LC3A, B and other autophagy-associated genes (ATG5, mTOR, Beclin-1) were found to increase in normal pigs, while it decreased in miniature pigs. However, for the apoptosis-associated genes, progression of genes during follicular development increased in miniature pigs, while it decreased in normal pigs. Thus, results show that normal and miniature pigs showed distinct patterns of follicular remodeling manifesting that programmed cell death largely depends on the types of pathway during follicular development (Type II or autophagy for normal pigs and Type I or apoptosis for miniature pigs). PMID:23056260

Interferon-alpha and interferon-gamma modulate Fas-mediated apoptosis in mitomycin-C-resistant human Tenon's fibroblasts.

PubMed

Wang, Xiao Yang; Crowston, Jonathan G; White, Andrew J R; Zoellner, Hans; Healey, Paul R

2014-08-01

The aim of the study was to investigate, using a native mitomycin-C-resistant human Tenon's fibroblast cell line, the possibility that interferon-alpha and gamma could be used with Fas agonists as an alternative anti-fibrotic strategy to mitomycin-C in trabeculectomy. A clinically resistant and in vitro verified mitomycin-C-resistant human Tenon's fibroblast cell line was pretreated with interferon-alpha and interferon-gamma for 48 h before stimulation with an agonistic Fas antibody (CH11) for 2 days to induce cell death. Cell death assays were undertaken. Changes in apoptosis-related proteins were determined by flow cytometry and Western blot. Pretreatment with interferon-alpha or interferon-gamma for 48 h increased Fas, Fas-associated protein with death domain and caspase-8 expression. Protein expression was further increased by combined exposure to interferon-alpha and gamma. Pretreatment with cytokines had no effect on Fas-L and Bcl-2. Interferon-alpha alone did not change the rate of induced cell death. A combination of interferon-alpha and gamma synergistically increased the sensitivity of mitomycin-C-resistant human Tenon's fibroblast cell line to induced cell death. An antagonistic anti-Fas antibody (ZB4) completely blocked induced cell death. Broad caspase inhibitors specific for caspases-8 and -3 reduced induced deaths in interferon pretreated mitomycin-C-resistant human Tenon's fibroblast cell line in a dose-dependent manner. Interferon-alpha and interferon-gamma render mitomycin-C-resistant human Tenon's fibroblast cell line sensitive to Fas-mediated apoptosis. The mechanism involves increased death-inducing signalling complex formation by upregulation of Fas, Fas-associated protein with death domain and caspase-8 expression. © 2013 Royal Australian and New Zealand College of Ophthalmologists.

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

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

Morotomi-Yano, Keiko; Akiyama, Hidenori; Yano, Ken-ichi, E-mail: yanoken@kumamoto-u.ac.jp

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

Patterns of cell death in the embryonic antenna of the grasshopper Schistocerca gregaria.

PubMed

Boyan, George; Graf, Philip; Ehrhardt, Erica

2018-03-01

We have investigated the pattern of apoptosis in the antennal epithelium during embryonic development of the grasshopper Schistocerca gregaria. The molecular labels lachesin and annulin reveal that the antennal epithelium becomes subdivided into segment-like meristal annuli within which sensory cell clusters later differentiate. To determine whether apoptosis is involved in the development of such sensory cell clusters, we examined the expression pattern of the cell death labels acridine orange and TUNEL in the epithelium. We found stereotypic, age-dependent, wave-like patterns of cell death in the antenna. Early in embryogenesis, apoptosis is restricted to the most basal meristal annuli but subsequently spreads to encompass almost the entire antenna. Cell death then declines in more basal annuli and is only found in the tip region later in embryogenesis. Apoptosis is restricted throughout to the midregion of a given annulus and away from its border with neighboring annuli, arguing against a causal role in annular formation. Double-labeling for cell death and sensory cell differentiation reveals apoptosis occurring within bands of differentiating sensory cell clusters, matching the meristal organization of the apical antenna. Examination of the individual epithelial lineages which generate sensory cells reveals that apoptosis begins peripherally within a lineage and with age expands to encompass the differentiated sensory cell at the base. We conclude that complete lineages can undergo apoptosis and that the youngest cells in these lineages appear to die first, with the sensory neuron dying last. Lineage-based death in combination with cell death patterns in different regions of the antenna may contribute to odor-mediated behaviors in the grasshopper.

Human-specific bacterial pore-forming toxins induce programmed necrosis in erythrocytes.

PubMed

LaRocca, Timothy J; Stivison, Elizabeth A; Hod, Eldad A; Spitalnik, Steven L; Cowan, Peter J; Randis, Tara M; Ratner, Adam J

2014-08-26

A subgroup of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins (PFTs) has an unusually narrow host range due to a requirement for binding to human CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-linked complement regulatory molecule. hCD59-specific CDCs are produced by several organisms that inhabit human mucosal surfaces and can act as pathogens, including Gardnerella vaginalis and Streptococcus intermedius. The consequences and potential selective advantages of such PFT host limitation have remained unknown. Here, we demonstrate that, in addition to species restriction, PFT ligation of hCD59 triggers a previously unrecognized pathway for programmed necrosis in primary erythrocytes (red blood cells [RBCs]) from humans and transgenic mice expressing hCD59. Because they lack nuclei and mitochondria, RBCs have typically been thought to possess limited capacity to undergo programmed cell death. RBC programmed necrosis shares key molecular factors with nucleated cell necroptosis, including dependence on Fas/FasL signaling and RIP1 phosphorylation, necrosome assembly, and restriction by caspase-8. Death due to programmed necrosis in RBCs is executed by acid sphingomyelinase-dependent ceramide formation, NADPH oxidase- and iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products. Bacterial PFTs that are hCD59 independent do not induce RBC programmed necrosis. RBC programmed necrosis is biochemically distinct from eryptosis, the only other known programmed cell death pathway in mature RBCs. Importantly, RBC programmed necrosis enhances the growth of PFT-producing pathogens during exposure to primary RBCs, consistent with a role for such signaling in microbial growth and pathogenesis. In this work, we provide the first description of a new form of programmed cell death in erythrocytes (RBCs) that occurs as a consequence of cellular attack by human-specific bacterial toxins. By defining a new RBC death pathway that shares important components with necroptosis, a programmed necrosis module that occurs in nucleated cells, these findings expand our understanding of RBC biology and RBC-pathogen interactions. In addition, our work provides a link between cholesterol-dependent cytolysin (CDC) host restriction and promotion of bacterial growth in the presence of RBCs, which may provide a selective advantage to human-associated bacterial strains that elaborate such toxins and a potential explanation for the narrowing of host range observed in this toxin family. Copyright © 2014 LaRocca et al.

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.

Apoptosis inducing factor gene depletion inhibits zearalenone-induced cell death in a goat Leydig cell line.

PubMed

Yang, Diqi; Jiang, Tingting; Lin, Pengfei; Chen, Huatao; Wang, Lei; Wang, Nan; Zhao, Fan; Tang, Keqiong; Zhou, Dong; Wang, Aihua; Jin, Yaping

2017-01-01

Zearalenone (ZEA) is a contaminant of human food and animal feedstuffs that causes health hazards. However, the signal pathways underlying ZEA toxicity remain elusive. The aims of this study were to determine which pathways are involved in ZEA-induced cell death and investigate the effect of apoptosis inducing factor (AIF) on cell death during ZEA treatment in the immortalized goat Leydig cell line hTERT-GLC. This study showed that ZEA-induced cell death in hTERT-GLCs works via endoplasmic reticulum (ER) stress, the caspase-dependent pathway, the caspase-independent pathway and autophagy. Recombinant lentiviral vectors were constructed to silence AIF expression in hTERT-GLCs. Flow cytometry results showed that knockdown of AIF diminished ZEA-induced cell apoptosis in hTERT-GLCs. Furthermore, we found AIF depletion down-regulated phosphoIRE1α, GRP78, CHOP and promoted the switch of LC3-I to LC3-II. Therefore, ZEA induces cytotoxicity in hTERT-GLCs via different pathways, while AIF-mediated signaling plays a critical role in ZEA-induced cell death in hTERT-GLCs. Copyright © 2016 Elsevier Inc. All rights reserved.

The selective Bcl-2 inhibitor venetoclax, a BH3 mimetic, does not dysregulate intracellular Ca2+ signaling.

PubMed

Vervloessem, Tamara; Ivanova, Hristina; Luyten, Tomas; Parys, Jan B; Bultynck, Geert

2017-06-01

Anti-apoptotic B cell-lymphoma-2 (Bcl-2) proteins are emerging as therapeutic targets in a variety of cancers for precision medicines, like the BH3-mimetic drug venetoclax (ABT-199), which antagonizes the hydrophobic cleft of Bcl-2. However, the impact of venetoclax on intracellular Ca 2+ homeostasis and dynamics in cell systems has not been characterized in detail. Here, we show that venetoclax did not affect Ca 2+ -transport systems from the endoplasmic reticulum (ER) in permeabilized cell systems. Venetoclax (1μM) did neither trigger Ca 2+ release by itself nor affect agonist-induced Ca 2+ release in a variety of intact cell models. Among the different cell types, we also studied two Bcl-2-dependent cancer cell models with a varying sensitivity towards venetoclax, namely SU-DHL-4 and OCI-LY-1, both diffuse large B-cell lymphoma cell lines. Acute application of venetoclax did also not dysregulate Ca 2+ signaling in these Bcl-2-dependent cancer cells. Moreover, venetoclax-induced cell death was independent of intracellular Ca 2+ overload, since Ca 2+ buffering using BAPTA-AM did not suppress venetoclax-induced cell death. This study therefore shows that venetoclax does not dysregulate the intracellular Ca 2+ homeostasis in a variety of cell types, which may underlie its limited toxicity in human patients. Furthermore, venetoclax-induced cell death in Bcl-2-dependent cancer cells is not mediated by intracellular Ca 2+ overload. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech. Copyright © 2016 Elsevier B.V. All rights reserved.

5-aminolevulinic acid-mediated photodynamic therapy on Hep-2 and MCF-7c3 cells.

PubMed

Alvarez, María Gabriela; Lacelli, M S; Rivarola, Viviana; Batlle, Alcira; Fukuda, Haydée

2007-01-01

The cytotoxic effect of 5-aminolevulinic acid (ALA) induced protoporphyrin IX (PPIX) on two human carcinoma cell lines, MCF-7c3 cells and Hep 2 cells, was studied. In both cell lines, PPIX content depends on the ALA concentration and incubation time. The maximal PPIX content was higher in the MCF-7c3 cells, reaching a value of 8 microg/10(6) cells, compared to the Hep-2 cells, which accumulated 3.2 microg/10(6) cells. Treatment of cells with the iron chelator desferrioxamine prior to ALA exposure enhances the amount of PPIX, consequently diminishing enzymatic activity of ferroquelatase. Photo sensitization of the cells was in correlation with the PPIX content; therefore, conditions leading to 80% cell death in the MCF-7c3 cells provoke a 50% cell death in the Hep 2 cells. Using fluorescence microscopy, cell morphology was analyzed after incubation with 1 mM ALA during 5 hr and irradiation with 54 Jcm(-2); 24 hr post-PDT, MCF-7c3 cells revealed the typical morphological changes of necrosis. Under the same conditions, Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. PPIX accumulation was observed to occur in a perinuclear region in the MCF-7c3 cells; while in Hep-2 cells, it was localized in lysosomes. Different mechanisms of cell death were observed in both cell lines, depending on the different intracellular localization of PPIX.

A novel Fizzy/Cdc20-dependent mechanism suppresses necrosis in neural stem cells

PubMed Central

Kuang, Chaoyuan; Golden, Krista L.; Simon, Claudio R.; Damrath, John; Buttitta, Laura; Gamble, Caitlin E.; Lee, Cheng-Yu

2014-01-01

Cancer stem cells likely survive chemotherapy or radiotherapy by acquiring mutations that inactivate the endogenous apoptotic machinery or by cycling slowly. Thus, knowledge about the mechanisms linking the activation of an alternative cell death modality and the cell cycle machinery could have a transformative impact on the development of new cancer therapies, but the mechanisms remain completely unknown. We investigated the regulation of alternative cell death in Drosophila larval brain neural stem cells (neuroblasts) in which apoptosis is normally repressed. From a screen, we identified two novel loss-of-function alleles of the Cdc20/fizzy (fzy) gene that lead to premature brain neuroblast loss without perturbing cell proliferation in other diploid cell types. Fzy is an evolutionarily conserved regulator of anaphase promoting complex/cyclosome (APC/C). Neuroblasts carrying the novel fzy allele or exhibiting reduced APC/C function display hallmarks of necrosis. By contrast, neuroblasts overexpressing the non-degradable form of canonical APC/C substrates required for cell cycle progression undergo mitotic catastrophe. These data strongly suggest that Fzy can elicit a novel pro-survival function of APC/C by suppressing necrosis. Neuroblasts experiencing catastrophic cellular stress, or overexpressing p53, lose Fzy expression and undergo necrosis. Co-expression of fzy suppresses the death of these neuroblasts. Consequently, attenuation of the Fzy-dependent survival mechanism functions downstream of catastrophic cellular stress and p53 to eliminate neuroblasts by necrosis. Strategies that target the Fzy-dependent survival mechanism might lead to the discovery of new treatments or complement the pre-existing therapies to eliminate apoptosis-resistant cancer stem cells by necrosis. PMID:24598157

An NQO1-Initiated and p53-Independent Apoptotic Pathway Determines the Anti-Tumor Effect of Tanshinone IIA against Non-Small Cell Lung Cancer

PubMed Central

Wang, Guangji; Liu, Huiying; Wu, Xiaolan; Wang, Qiong; Liu, Miao; Liao, Ke; Wu, Mengqiu; Cheng, Xuefang; Hao, Haiping

2012-01-01

NQO1 is an emerging and promising therapeutic target in cancer therapy. This study was to determine whether the anti-tumor effect of tanshinone IIA (TSA) is NQO1 dependent and to elucidate the underlying apoptotic cell death pathways. NQO1+ A549 cells and isogenically matched NQO1 transfected and negative H596 cells were used to test the properties and mechanisms of TSA induced cell death. The in vivo anti-tumor efficacy and the tissue distribution properties of TSA were tested in tumor xenografted nude mice. We observed that TSA induced an excessive generation of ROS, DNA damage, and dramatic apoptotic cell death in NQO1+ A549 cells and H596-NQO1 cells, but not in NQO1− H596 cells. Inhibition or silence of NQO1 as well as the antioxidant NAC markedly reversed TSA induced apoptotic effects. TSA treatment significantly retarded the tumor growth of A549 tumor xenografts, which was significantly antagonized by dicoumarol co-treatment in spite of the increased and prolonged TSA accumulations in tumor tissues. TSA activated a ROS triggered, p53 independent and caspase dependent mitochondria apoptotic cell death pathway that is characterized with increased ratio of Bax to Bcl-xl, mitochondrial membrane potential disruption, cytochrome c release, and subsequent caspase activation and PARP-1 cleavage. The results of these findings suggest that TSA is a highly specific NQO1 target agent and is promising in developing as an effective drug in the therapy of NQO1 positive NSCLC. PMID:22848731

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").

Immunopathogenesis of Dengue Virus-Induced Redundant Cell Death: Apoptosis and Pyroptosis.

PubMed

Suwanmanee, San; Luplertlop, Natthanej

Dengue virus infection is a self-limited condition, which is of particular importance in tropical and subtropical regions and for which no specific treatment or effective vaccine is available. There are several hypotheses explaining dengue pathogenesis. These usually refer to host immune responses, including antibody-dependent enhancement, cytokine expression, and dengue virus particles including NS1 protein, which lead to cell death by both apoptosis and pyroptosis. A clear understanding of the pathogenesis should facilitate the development of vaccines and therapies. This review focuses on the immunopathogenesis in relation to clinical manifestations and patterns of cell death, focusing on the pathogenesis of severe dengue.

Implication of multiple mechanisms in apoptosis induced by the synthetic retinoid CD437 in human prostate carcinoma cells.

PubMed

Sun, S Y; Yue, P; Lotan, R

2000-09-14

The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) induces apoptosis in several types of cancer cell. CD437 inhibited the growth of both androgen-dependent and -independent human prostate carcinoma (HPC) cells in a concentration-dependent manner by rapid induction of apoptosis. CD437 was more effective in killing androgen-independent HPC cells such as DU145 and PC-3 than the androgen-dependent LNCaP cells. The caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK blocked apoptosis induced by CD437 in DU145 and LNCaP cells, in which increased caspase-3 activity and PARP cleavage were observed, but not in PC-3 cells, in which CD437 did not induce caspase-3 activation and PARP cleavage. Thus, CD437 can induce either caspase-dependent or caspase-independent apoptosis in HPC cells. CD437 increased the expression of c-Myc, c-Jun, c-Fos, and death receptors DR4, DR5 and Fas. CD437's potency in apoptosis induction in the different cell lines was correlated with its effects on the expression of oncogenes and death receptors, thus implicating these genes in CD437-induced apoptosis in HPC cells. However, the importance and contribution of each of these genes in different HPC cell lines may vary. Because CD437 induced the expression of DR4, DR5 and Fas, we examined the effects of combining CD437 and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and Fas ligand, respectively, in HPC cells. We found synergistic induction of apoptosis, highlighting the importance of the modulation of these death receptors in CD437-induced apoptosis in HPC cells. This result also suggests a potential strategy of using CD437 with TRAIL for treatment of HPC. Oncogene (2000) 19, 4513 - 4522.

In vitro effects of histone deacetylase inhibitors and mitomycin C on tenon capsule fibroblasts and conjunctival melanoma cells.

PubMed

Cunneen, Thomas S; Conway, R Max; Madigan, Michele C

2009-04-01

To investigate the effects of mitomycin C and the histone deacetylase inhibitors sodium butyrate and trichostatin on the viability and growth of conjunctival melanoma cell lines and Tenon capsule fibroblasts. Cells were treated with a range of concentrations of sodium butyrate, trichostatin, and mitomycin C. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assays were performed 48 hours after treatment. Treated cells were stained with acridine orange/ethidium bromide to assess for cell death. Cell-cycle changes in histone deacetylase inhibitor-treated melanoma cells were quantified using flow cytometry. All agents induced dose-dependent cell death in the melanoma cell lines; however, sodium butyrate and trichostatin were relatively nontoxic to Tenon capsule fibroblasts. Acridine orange/ethidium bromide staining indicated that sodium butyrate and trichostatin induced apoptotic cell death. At low doses, sodium butyrate and trichostatin induced a G1 cell-cycle block in the melanoma cells. Sodium butyrate and trichostatin induced cell death in melanoma cells, comparable with mitomycin C, with minimal effect on Tenon capsule fibroblasts. In addition, they induced a G1 cell-cycle block. These findings support the need for further investigation into the in vivo efficacy of these agents.

A kinetic theory for age-structured stochastic birth-death processes

NASA Astrophysics Data System (ADS)

Chou, Tom; Greenman, Chris

Classical age-structured mass-action models such as the McKendrick-von Foerster equation have been extensively studied but they are structurally unable to describe stochastic fluctuations or population-size-dependent birth and death rates. Conversely, current theories that include size-dependent population dynamics (e.g., carrying capacity) cannot be easily extended to take into account age-dependent birth and death rates. In this paper, we present a systematic derivation of a new fully stochastic kinetic theory for interacting age-structured populations. By defining multiparticle probability density functions, we derive a hierarchy of kinetic equations for the stochastic evolution of an aging population undergoing birth and death. We show that the fully stochastic age-dependent birth-death process precludes factorization of the corresponding probability densities, which then must be solved by using a BBGKY-like hierarchy. Our results generalize both deterministic models and existing master equation approaches by providing an intuitive and efficient way to simultaneously model age- and population-dependent stochastic dynamics applicable to the study of demography, stem cell dynamics, and disease evolution. NSF.

Cell death in response to antimetabolites directed at thymidylate synthase.

PubMed

Barbour, Karen W; Berger, Franklin G

2008-02-01

Thymidylate synthase (TS) is an indispensable enzyme in the de novo biosynthesis of TMP during DNA replication and cell growth, and has, therefore, been an important target for several classes of antimetabolites used in cancer chemotherapy. While most investigations of the action of TS-directed agents have focused on apoptosis as the primary means of cell death, little is known regarding the role, if any, of non-apoptotic mechanisms. In the present study, we have examined the mode of cell death induced by several TS inhibitors. Apoptosis and necrosis in response to TS inhibitors was assessed. The roles of caspases and the transcriptional regulator nuclear factor kappa B (NFkappaB) in drug-induced cell death were analyzed. Finally, drug-mediated changes in expression of several proteins involved in regulation of apoptosis were analyzed. Though human colon tumor cells exposed to TS inhibitors undergo classical apoptosis, it is not the predominant mechanism of response; rather, a necrosis-like mechanism prevails. The apoptotic response to TS inhibitors is caspase-dependent, and is promoted by NFkappaB. In contrast, the necrosis-like response is independent of both caspases and NFkappaB. Exposure to TS inhibitors induces PARP cleavage, but does not alter expression of the pro or activated forms of caspases-3 or caspases-8, Fas, or FasL. Treatment with the death-inducing cytokine TNFalpha, like TS inhibitors, results in a limited extent of apoptosis that is both caspase- and NFkappaB-dependent; however, unlike TS inhibitors, the cytokine does not induce necrosis. Classical apoptosis occurs to a limited extent in human colon tumor cells exposed to TS inhibitors, with caspase-independent necrosis being the prinicipal mechanism of cell death. We suggest that the role of necrosis and necrosis-like mechanisms should be considered in future studies of the action of TS-directed antimetabolites, as well as other chemotherapeutic agents.

Disruption of the vacuolar calcium-ATPases in Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway.

PubMed

Boursiac, Yann; Lee, Sang Min; Romanowsky, Shawn; Blank, Robert; Sladek, Chris; Chung, Woo Sik; Harper, Jeffrey F

2010-11-01

Calcium (Ca(2+)) signals regulate many aspects of plant development, including a programmed cell death pathway that protects plants from pathogens (hypersensitive response). Cytosolic Ca(2+) signals result from a combined action of Ca(2+) influx through channels and Ca(2+) efflux through pumps and cotransporters. Plants utilize calmodulin-activated Ca(2+) pumps (autoinhibited Ca(2+)-ATPase [ACA]) at the plasma membrane, endoplasmic reticulum, and vacuole. Here, we show that a double knockout mutation of the vacuolar Ca(2+) pumps ACA4 and ACA11 in Arabidopsis (Arabidopsis thaliana) results in a high frequency of hypersensitive response-like lesions. The appearance of macrolesions could be suppressed by growing plants with increased levels (greater than 15 mm) of various anions, providing a method for conditional suppression. By removing plants from a conditional suppression, lesion initials were found to originate primarily in leaf mesophyll cells, as detected by aniline blue staining. Initiation and spread of lesions could also be suppressed by disrupting the production or accumulation of salicylic acid (SA), as shown by combining aca4/11 mutations with a sid 2 (for salicylic acid induction-deficient2) mutation or expression of the SA degradation enzyme NahG. This indicates that the loss of the vacuolar Ca(2+) pumps by itself does not cause a catastrophic defect in ion homeostasis but rather potentiates the activation of a SA-dependent programmed cell death pathway. Together, these results provide evidence linking the activity of the vacuolar Ca(2+) pumps to the control of a SA-dependent programmed cell death pathway in plants.

A statistical model describing combined irreversible electroporation and electroporation-induced blood-brain barrier disruption.

PubMed

Sharabi, Shirley; Kos, Bor; Last, David; Guez, David; Daniels, Dianne; Harnof, Sagi; Mardor, Yael; Miklavcic, Damijan

2016-03-01

Electroporation-based therapies such as electrochemotherapy (ECT) and irreversible electroporation (IRE) are emerging as promising tools for treatment of tumors. When applied to the brain, electroporation can also induce transient blood-brain-barrier (BBB) disruption in volumes extending beyond IRE, thus enabling efficient drug penetration. The main objective of this study was to develop a statistical model predicting cell death and BBB disruption induced by electroporation. This model can be used for individual treatment planning. Cell death and BBB disruption models were developed based on the Peleg-Fermi model in combination with numerical models of the electric field. The model calculates the electric field thresholds for cell kill and BBB disruption and describes the dependence on the number of treatment pulses. The model was validated using in vivo experimental data consisting of rats brains MRIs post electroporation treatments. Linear regression analysis confirmed that the model described the IRE and BBB disruption volumes as a function of treatment pulses number (r(2) = 0.79; p < 0.008, r(2) = 0.91; p < 0.001). The results presented a strong plateau effect as the pulse number increased. The ratio between complete cell death and no cell death thresholds was relatively narrow (between 0.88-0.91) even for small numbers of pulses and depended weakly on the number of pulses. For BBB disruption, the ratio increased with the number of pulses. BBB disruption radii were on average 67% ± 11% larger than IRE volumes. The statistical model can be used to describe the dependence of treatment-effects on the number of pulses independent of the experimental setup.

Redox dynamics of manganese as a mitochondrial life-death switch

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

Smith, Matthew Ryan; Fernandes, Jolyn; Go, Young-Mi

Sten Orrenius, M.D., Ph.D., pioneered many areas of cellular and molecular toxicology and made seminal contributions to our knowledge of oxidative stress and glutathione (GSH) metabolism, organellar functions and Ca{sup +2}-dependent mechanisms of cell death, and mechanisms of apoptosis. On the occasion of his 80{sup th} birthday, we summarize current knowledge on redox biology of manganese (Mn) and its role in mechanisms of cell death. Mn is found in all organisms and has critical roles in cell survival and death mechanisms by regulating Mn-containing enzymes such as manganese superoxide dismutase (SOD2) or affecting expression and activity of caspases. Occupational exposuresmore » to Mn cause “manganism”, a Parkinson's disease-like condition of neurotoxicity, and experimental studies show that Mn exposure leads to accumulation of Mn in the brain, especially in mitochondria, and neuronal cell death occurs with features of an apoptotic mechanism. Interesting questions are why a ubiquitous metal that is essential for mitochondrial function would accumulate to excessive levels, cause increased H{sub 2}O{sub 2} production and lead to cell death. Is this due to the interactions of Mn with other essential metals, such as iron, or with toxic metals, such as cadmium? Why is the Mn loading in the human brain so variable, and why is there such a narrow window between dietary adequacy and toxicity? Are non-neuronal tissues similarly vulnerable to insufficiency and excess, yet not characterized? We conclude that Mn is an important component of the redox interface between an organism and its environment and warrants detailed studies to understand the role of Mn as a mitochondrial life-death switch. - Highlights: • Either insufficient or excess manganese activates mitochondria-mediated cell death. • The optimal healthy Mn exposure window is very narrow. • Mitochondrial H{sub 2}O{sub 2} production depends on Mn across physiologic to toxicologic range. • Integrative omics needed to understand complex Mn interaction in cell fate. • Mn is central to redox interface between an organism and its environment.« less

Sugar suppresses cell death caused by disruption of fumarylacetoacetate hydrolase in Arabidopsis.

PubMed

Zhi, Tiantian; Zhou, Zhou; Huang, Yi; Han, Chengyun; Liu, Yan; Zhu, Qi; Ren, Chunmei

2016-09-01

Sugar negatively regulates cell death resulting from the loss of fumarylacetoacetate hydrolase that catalyzes the last step in the Tyr degradation pathway in Arabidopsis . Fumarylacetoacetate hydrolase (FAH) hydrolyzes fumarylacetoacetate to fumarate and acetoacetate, the final step in the tyrosine (Tyr) degradation pathway that is essential to animals. Previously, we first found that the Tyr degradation pathway plays an important role in plants. Mutation of the SSCD1 gene encoding FAH in Arabidopsis leads to spontaneous cell death under short-day conditions. In this study, we presented that the lethal phenotype of the short-day sensitive cell death1 (sscd1) seedlings was suppressed by sugars including sucrose, glucose, fructose, and maltose in a dose-dependent manner. Real-time quantitative PCR (RT-qPCR) analysis showed the expression of Tyr degradation pathway genes homogentisate dioxygenase and maleylacetoacetate isomerase, and sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G, was up-regulated in the sscd1 mutant, however, this up-regulation could be repressed by sugar. In addition, a high concentration of sugar attenuated cell death of Arabidopsis wild-type seedlings caused by treatment with exogenous succinylacetone, an abnormal metabolite resulting from the loss of FAH in the Tyr degradation pathway. These results indicated that (1) sugar could suppress cell death in sscd1, which might be because sugar supply enhances the resistance of Arabidopsis seedlings to toxic effects of succinylacetone and reduces the accumulation of Tyr degradation intermediates, resulting in suppression of cell death; and (2) sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G might be involved in the cell death in sscd1. Our work provides insights into the relationship between sugar and sscd1-mediated cell death, and contributes to elucidation of the regulation of cell death resulting from the loss of FAH in plants.

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.

Neuroprotective effects of corn silk maysin via inhibition of H2O2-induced apoptotic cell death in SK-N-MC cells.

PubMed

Choi, Doo Jin; Kim, Sun-Lim; Choi, Ji Won; Park, Yong Il

2014-07-25

Neuroprotective effects of maysin, which is a flavone glycoside that was isolated from the corn silk (CS, Zea mays L.) of a Korean hybrid corn Kwangpyeongok, against oxidative stress (H2O2)-induced apoptotic cell death of human neuroblastoma SK-N-MC cells were investigated. Maysin cytotoxicity was determined by measuring cell viability using MTT and lactate dehydrogenase (LDH) assays. Intracellular reactive oxygen species (ROS) were measured using a 2,7-dichlorofluorescein diacetate (DCF-DA) assay. Apoptotic cell death was monitored by annexin V-FITC/PI double staining and by a TUNEL assay. Antioxidant enzyme mRNA levels were determined by real-time PCR. The cleavage of poly (ADP-ribose) polymerase (PARP) was measured by western blotting. Maysin pretreatment reduced the cytotoxic effect of H2O2 on SK-N-MC cells, as shown by the increase in cell viability and by reduced LDH release. Maysin pretreatment also dose-dependently reduced the intracellular ROS level and inhibited PARP cleavage. In addition, DNA damage and H2O2-induced apoptotic cell death were significantly attenuated by maysin pretreatment. Moreover, maysin pretreatment (5-50 μg/ml) for 2h significantly and dose-dependently increased the mRNA levels of antioxidant enzymes (CAT, GPx-1, SOD-1, SOD-2 and HO-1) in H2O2 (200 μM)-insulted cells. These results suggest that CS maysin has neuroprotective effects against oxidative stress (H2O2)-induced apoptotic death of human brain SK-N-MC cells through its antioxidative action. This report is the first regarding neuroprotective health benefits of corn silk maysin by its anti-apoptotic action and by triggering the expression of intracellular antioxidant enzyme systems in SK-N-MC cells. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

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

PubMed Central

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

2012-01-01

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

Pretreatment with apoaequorin protects hippocampal CA1 neurons from oxygen-glucose deprivation.

PubMed

Detert, Julia A; Adams, Erin L; Lescher, Jacob D; Lyons, Jeri-Anne; Moyer, James R

2013-01-01

Ischemic stroke affects ∼795,000 people each year in the U.S., which results in an estimated annual cost of $73.7 billion. Calcium is pivotal in a variety of neuronal signaling cascades, however, during ischemia, excess calcium influx can trigger excitotoxic cell death. Calcium binding proteins help neurons regulate/buffer intracellular calcium levels during ischemia. Aequorin is a calcium binding protein isolated from the jellyfish Aequorea victoria, and has been used for years as a calcium indicator, but little is known about its neuroprotective properties. The present study used an in vitro rat brain slice preparation to test the hypothesis that an intra-hippocampal infusion of apoaequorin (the calcium binding component of aequorin) protects neurons from ischemic cell death. Bilaterally cannulated rats received an apoaequorin infusion in one hemisphere and vehicle control in the other. Hippocampal slices were then prepared and subjected to 5 minutes of oxygen-glucose deprivation (OGD), and cell death was assayed by trypan blue exclusion. Apoaequorin dose-dependently protected neurons from OGD--doses of 1% and 4% (but not 0.4%) significantly decreased the number of trypan blue-labeled neurons. This effect was also time dependent, lasting up to 48 hours. This time dependent effect was paralleled by changes in cytokine and chemokine expression, indicating that apoaequorin may protect neurons via a neuroimmunomodulatory mechanism. These data support the hypothesis that pretreatment with apoaequorin protects neurons against ischemic cell death, and may be an effective neurotherapeutic.

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

Dnmt1-dependent Chk1 pathway suppression is protective against neuron division.

PubMed

Oshikawa, Mio; Okada, Kei; Tabata, Hidenori; Nagata, Koh-Ichi; Ajioka, Itsuki

2017-09-15

Neuronal differentiation and cell-cycle exit are tightly coordinated, even in pathological situations. When pathological neurons re-enter the cell cycle and progress through the S phase, they undergo cell death instead of division. However, the mechanisms underlying mitotic resistance are mostly unknown. Here, we have found that acute inactivation of retinoblastoma (Rb) family proteins (Rb, p107 and p130) in mouse postmitotic neurons leads to cell death after S-phase progression. Checkpoint kinase 1 (Chk1) pathway activation during the S phase prevented the cell death, and allowed the division of cortical neurons that had undergone acute Rb family inactivation, oxygen-glucose deprivation (OGD) or in vivo hypoxia-ischemia. During neurogenesis, cortical neurons became protected from S-phase Chk1 pathway activation by the DNA methyltransferase Dnmt1, and underwent cell death after S-phase progression. Our results indicate that Chk1 pathway activation overrides mitotic safeguards and uncouples neuronal differentiation from mitotic resistance. © 2017. Published by The Company of Biologists Ltd.

Glyceraldehyde-3-phosphate dehydrogenase aggregation inhibitor peptide: A potential therapeutic strategy against oxidative stress-induced cell death.

PubMed

Itakura, Masanori; Nakajima, Hidemitsu; Semi, Yuko; Higashida, Shusaku; Azuma, Yasu-Taka; Takeuchi, Tadayoshi

2015-11-13

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple functions, including mediating oxidative stress-induced neuronal cell death. This process is associated with disulfide-bonded GAPDH aggregation. Some reports suggest a link between GAPDH and the pathogenesis of several oxidative stress-related diseases. However, the pathological significance of GAPDH aggregation in disease pathogenesis remains unclear due to the lack of an effective GAPDH aggregation inhibitor. In this study, we identified a GAPDH aggregation inhibitor (GAI) peptide and evaluated its biological profile. The decapeptide GAI specifically inhibited GAPDH aggregation in a concentration-dependent manner. Additionally, the GAI peptide did not affect GAPDH glycolytic activity or cell viability. The GAI peptide also exerted a protective effect against oxidative stress-induced cell death in SH-SY5Y cells. This peptide could potentially serve as a tool to investigate GAPDH aggregation-related neurodegenerative and neuropsychiatric disorders and as a possible therapy for diseases associated with oxidative stress-induced cell death. Copyright © 2015 Elsevier Inc. All rights reserved.

Shiga Toxins Activate the NLRP3 Inflammasome Pathway To Promote Both Production of the Proinflammatory Cytokine Interleukin-1β and Apoptotic Cell Death

PubMed Central

Lee, Moo-Seung; Kwon, Haenaem; Lee, Eun-Young; Kim, Dong-Jae; Park, Jong-Hwan; Tesh, Vernon L.; Oh, Tae-Kwang

2015-01-01

Shiga toxin (Stx)-mediated immune responses, including the production of the proinflammatory cytokines tumor necrosis-α (TNF-α) and interleukin-1β (IL-1β), may exacerbate vascular damage and accelerate lethality. However, the immune signaling pathway activated in response to Stx is not well understood. Here, we demonstrate that enzymatically active Stx, which leads to ribotoxic stress, triggers NLRP3 inflammasome-dependent caspase-1 activation and IL-1β secretion in differentiated macrophage-like THP-1 (D-THP-1) cells. The treatment of cells with a chemical inhibitor of glycosphingolipid biosynthesis, which suppresses the expression of the Stx receptor globotriaosylceramide and subsequent endocytosis of the toxin, substantially blocked activation of the NLRP3 inflammasome and processing of caspase-1 and IL-1β. Processing and release of both caspase-1 and IL-1β were significantly reduced or abolished in Stx-intoxicated D-THP-1 cells in which the expression of NLRP3 or ASC was stably knocked down. Furthermore, Stx mediated the activation of caspases involved in apoptosis in an NLRP3- or ASC-dependent manner. In Stx-intoxicated cells, the NLRP3 inflammasome triggered the activation of caspase-8/3, leading to the initiation of apoptosis, in addition to caspase-1-dependent pyroptotic cell death. Taken together, these results suggest that Stxs trigger the NLRP3 inflammasome pathway to release proinflammatory IL-1β as well as to promote apoptotic cell death. PMID:26502906

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.

Selective deletion of cochlear hair cells causes rapid age-dependent changes in spiral ganglion and cochlear nucleus neurons.

PubMed

Tong, Ling; Strong, Melissa K; Kaur, Tejbeer; Juiz, Jose M; Oesterle, Elizabeth C; Hume, Clifford; Warchol, Mark E; Palmiter, Richard D; Rubel, Edwin W

2015-05-20

During nervous system development, critical periods are usually defined as early periods during which manipulations dramatically change neuronal structure or function, whereas the same manipulations in mature animals have little or no effect on the same property. Neurons in the ventral cochlear nucleus (CN) are dependent on excitatory afferent input for survival during a critical period of development. Cochlear removal in young mammals and birds results in rapid death of target neurons in the CN. Cochlear removal in older animals results in little or no neuron death. However, the extent to which hair-cell-specific afferent activity prevents neuronal death in the neonatal brain is unknown. We further explore this phenomenon using a new mouse model that allows temporal control of cochlear hair cell deletion. Hair cells express the human diphtheria toxin (DT) receptor behind the Pou4f3 promoter. Injections of DT resulted in nearly complete loss of organ of Corti hair cells within 1 week of injection regardless of the age of injection. Injection of DT did not influence surrounding supporting cells directly in the sensory epithelium or spiral ganglion neurons (SGNs). Loss of hair cells in neonates resulted in rapid and profound neuronal loss in the ventral CN, but not when hair cells were eliminated at a more mature age. In addition, normal survival of SGNs was dependent on hair cell integrity early in development and less so in mature animals. This defines a previously undocumented critical period for SGN survival. Copyright © 2015 the authors 0270-6474/15/357878-14$15.00/0.

Distinct p300-Responsive Mechanisms Promote Caspase-Dependent Apoptosis by Human T-Cell Lymphotropic Virus Type 1 Tax Protein

PubMed Central

Nicot, Christophe; Harrod, Robert

2000-01-01

The dysregulation of cellular apoptosis pathways has emerged as a critical early event associated with the development of many types of human cancers. Numerous viral and cellular oncogenes, aside from their inherent transforming properties, are known to induce programmed cell death, consistent with the hypothesis that genetic defects are required to support tumor survival. Here, we report that nuclear expression of the CREB-binding protein (CBP)/p300-binding domain of the human T-cell lymphotropic virus type 1 (HTLV-1) transactivator, Tax, triggers an apoptotic death-inducing signal during short-term clonal analyses, as well as in transient cell death assays. Coexpression of the antiapoptotic factor Bcl-2 increased serum stimulation; incubation with the chemical caspase inhibitor z-Val-Ala-dl-Asp fluoromethylketone antagonized Tax-induced cell death. The CBP/p300-binding defective Tax mutants K88A and V89A exhibited markedly reduced cytotoxic effects compared to the wild-type Tax protein. Importantly, nuclear expression of the minimal CBP/p300-binding peptide of Tax induced apoptosis in the absence of Tax-dependent transcriptional activities, while its K88A counterpart did not cause cell death. Further, Tax-mediated apoptosis was effectively prevented by ectopic expression of the p300 coactivator. We also report that activation of the NF-κB transcription pathway by Tax, under growth arrest conditions, results in apoptosis that occurs independent of direct Tax coactivator effects. Our results allude to a novel pivotal role for the transcriptional coactivator p300 in determining cell fate and raise the possibility that dysregulated coactivator usage may pose an early barrier to transformation that must be selectively overcome as a prerequisite for the initiation of neoplasia. PMID:11046153

Distinct p300-responsive mechanisms promote caspase-dependent apoptosis by human T-cell lymphotropic virus type 1 Tax protein.

PubMed

Nicot, C; Harrod, R

2000-11-01

The dysregulation of cellular apoptosis pathways has emerged as a critical early event associated with the development of many types of human cancers. Numerous viral and cellular oncogenes, aside from their inherent transforming properties, are known to induce programmed cell death, consistent with the hypothesis that genetic defects are required to support tumor survival. Here, we report that nuclear expression of the CREB-binding protein (CBP)/p300-binding domain of the human T-cell lymphotropic virus type 1 (HTLV-1) transactivator, Tax, triggers an apoptotic death-inducing signal during short-term clonal analyses, as well as in transient cell death assays. Coexpression of the antiapoptotic factor Bcl-2 increased serum stimulation; incubation with the chemical caspase inhibitor z-Val-Ala-DL-Asp fluoromethylketone antagonized Tax-induced cell death. The CBP/p300-binding defective Tax mutants K88A and V89A exhibited markedly reduced cytotoxic effects compared to the wild-type Tax protein. Importantly, nuclear expression of the minimal CBP/p300-binding peptide of Tax induced apoptosis in the absence of Tax-dependent transcriptional activities, while its K88A counterpart did not cause cell death. Further, Tax-mediated apoptosis was effectively prevented by ectopic expression of the p300 coactivator. We also report that activation of the NF-kappaB transcription pathway by Tax, under growth arrest conditions, results in apoptosis that occurs independent of direct Tax coactivator effects. Our results allude to a novel pivotal role for the transcriptional coactivator p300 in determining cell fate and raise the possibility that dysregulated coactivator usage may pose an early barrier to transformation that must be selectively overcome as a prerequisite for the initiation of neoplasia.

Endocytosis of indium-tin-oxide nanoparticles by macrophages provokes pyroptosis requiring NLRP3-ASC-Caspase1 axis that can be prevented by mesenchymal stem cells

PubMed Central

Naji, Abderrahim; Muzembo, Basilua André; Yagyu, Ken-ichi; Baba, Nobuyasu; Deschaseaux, Frédéric; Sensebé, Luc; Suganuma, Narufumi

2016-01-01

The biological effects of indium-tin-oxide (ITO) are of considerable importance because workers exposed to indium compounds have been diagnosed with interstitial lung disease or pulmonary alveolar proteinosis; however, the pathophysiology of these diseases is undefined. Here, mice intraperitoneally inoculated with ITO-nanoparticles (ITO-NPs) resulted in peritonitis dependent in NLRP3 inflammasome, with neutrophils recruitment and interleukin-1β (IL-1β) production. Withal peritoneal macrophages exposed ex vivo to ITO-NPs caused IL-1β secretion and cytolysis. Further, alveolar macrophages exposed to ITO-NPs in vitro showed ITO-NP endocytosis and production of tumor necrosis factor-α (TNF-α) and IL-1β, ensued cell death by cytolysis. This cell death was RIPK1-independent but caspase1-dependent, and thus identified as pyroptosis. Endocytosis of ITO-NPs by activated THP-1 cells induced pyroptosis with IL-1β/TNF-α production and cytolysis, but not in activated THP-1 cells with knockdown of NLRP3, ASC, or caspase1. However, exposing activated THP-1 cells with NLRP3 or ASC knockdown to ITO-NPs resulted in cell death but without cytolysis, with deficiency in IL-1β/TNF-α, and revealing features of apoptosis. While, mesenchymal stem cells (MSCs) co-cultured with macrophages impaired both inflammation and cell death induced by ITO-NPs. Together, our findings provide crucial insights to the pathophysiology of respiratory diseases caused by ITO particles, and identify MSCs as a potent therapeutic. PMID:27194621

Immunological consequences of kidney cell death.

PubMed

Sarhan, Maysa; von Mässenhausen, Anne; Hugo, Christian; Oberbauer, Rainer; Linkermann, Andreas

2018-01-25

Death of renal cells is central to the pathophysiology of acute tubular necrosis, autoimmunity, necrotizing glomerulonephritis, cystic kidney disease, urosepsis, delayed graft function and transplant rejection. By means of regulated necrosis, immunogenic damage-associated molecular patterns (DAMPs) and highly reactive organelles such as lysosomes, peroxisomes and mitochondria are released from the dying cells, thereby causing an overwhelming immunologic response. The rupture of the plasma membrane exhibits the "point of no return" for the immunogenicity of regulated cell death, explaining why apoptosis, a highly organized cell death subroutine with long-lasting plasma membrane integrity, elicits hardly any immune response. Ferroptosis, an iron-dependent necrotic type cell death, results in the release of DAMPs and large amounts of lipid peroxides. In contrast, anti-inflammatory cytokines are actively released from cells that die by necroptosis, limiting the DAMP-induced immune response to a surrounding microenvironment, whereas at the same time, inflammasome-associated caspases drive maturation of intracellularly expressed interleukin-1β (IL-1β). In a distinct setting, additionally interleukin-18 (IL-18) is expressed during pyroptosis, initiated by gasdermin-mediated plasma membrane rupture. As all of these pathways are druggable, we provide an overview of regulated necrosis in kidney diseases with a focus on immunogenicity and potential therapeutic interventions.

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.

ERK1/2-dependent bestrophin-3 expression prevents ER-stress-induced cell death in renal epithelial cells by reducing CHOP.

PubMed

Lee, Wing-Kee; Chakraborty, Prabir K; Roussa, Eleni; Wolff, Natascha A; Thévenod, Frank

2012-10-01

Upon endoplasmic reticulum (ER) stress induction, cells endeavor to survive by engaging the adaptive stress response known as the unfolded protein response or by removing aggregated proteins via autophagy. Chronic ER stress culminates in apoptotic cell death, which involves induction of pro-apoptotic CHOP. Here, we show that bestrophin-3 (Best-3), a protein previously associated with Ca(2+)-activated Cl(-) channel activity, is upregulated by the ER stressors, thapsigargin (TG), tunicamycin (TUN) and the toxic metal Cd(2+). In cultured rat kidney proximal tubule cells, ER stress, CHOP and cell death were induced after 6h by Cd(2+) (25μM), TG (3μM) and TUN (6μM), were associated with increased cytosolic Ca(2+) and downstream formation of reactive oxygen species and attenuated by the Ca(2+) chelator BAPTA-AM (10μM), the antioxidant α-tocopherol (100μM), or overexpression of catalase (CAT). Immunofluorescence staining showed Best-3 expression in the plasma membrane, nuclei and intracellular compartments, though not in the ER, in cultured cells and rat kidney cortex sections. Best-3 mRNA was augmented by ER stress and signaled through increased Ca(2+), oxidative stress and ERK1/2 phosphorylation, because it was attenuated by α-tocopherol, CAT expression, BAPTA-AM, calmodulin kinase inhibitor calmidazolium (40μM), ERK1/2 inhibitor U0126 (10μM), and ERK1/2 RNAi. Knockdown of Best-3 resulted in decreased cell number consequentially of cell death, as determined by nuclear staining and PARP-1 cleavage. Furthermore, reduced ER stress-cell death by Best-3 overexpression is attributed to diminished CHOP. Since Best-3 overexpression did not affect upstream signaling pathways, we hypothesize that Best-3 possibly interferes with CHOP transcription. From our novel observations, we conclude that ERK1/2-dependent Best-3 activation regulates cell fate decisions during ER stress by suppressing CHOP induction and death. Copyright © 2012 Elsevier B.V. All rights reserved.

Necrosis and apoptosis pathways of cell death at photodynamic treatment in vitro as revealed by digital holographic microscopy

NASA Astrophysics Data System (ADS)

Semenova, I. V.; Belashov, A. V.; Belyaeva, T. N.; Kornilova, E. S.; Salova, A. V.; Zhikhoreva, A. A.; Vasyutinskii, O. S.

2018-02-01

Monitoring of variations in morphological characteristics of cultured HeLa cells after photodynamic treatment with Radachlorin photosensitizer is performed by means of digital holographic microscopy. The observed dose-dependent post-treatment variations of phase shift evidence threshold effect of photodynamic treatment and allow for distinguishing between necrotic or apoptotic pathways of cell death. Results obtained by holographic microscopy were confirmed by means of far-field optical microscopy and confocal fluorescence microscopy with commonly used test assays.

TLR3 mediates release of IL-1β and cell death in keratinocytes in a caspase-4 dependent manner.

PubMed

Grimstad, Øystein; Husebye, Harald; Espevik, Terje

2013-10-01

Inflammation and timely cell death are important elements in host defence and healing processes. Keratinocytes express high levels of Toll-like receptor 3 (TLR3), and stimulation of the receptor with its ligand polyinosinic-polycytidylic acid (polyI:C) is a powerful signal for release of a variety of proinflammatory cytokines. Caspase-4 is required for maturation of pro-IL-1β through activation of caspase-1 in keratinocytes. TLR3 in keratinocytes was stimulated with polyI:C. Induction of messenger RNA of pro-IL-1β and inflammasomal components was measured using quantitative polymerase chain reaction methodology. Protein expression of IL-1β was analysed with ELISA and Western blot techniques. Activation of apoptotic caspases was measured with flow cytometry, and cytotoxicity was determined. TLR3 induced release of substantial amounts of pro-IL-1β in keratinocytes. NLRP3 or ASC dependent processing of IL-1β into its cleaved bioactive form was found to be minimal. The release of IL-1β was due to polyI:C induced cell death that occurred through a caspase-4 dependent manner. Caspase-1 did not seem to be involved in the polyI:C induced cytotoxicity despite that TLR3 stimulation induced activation of caspase-1. In addition, the apoptotic caspases -8, -9 and -3/7 were activated by polyI:C. TLR3 stimulation in keratinocytes induces a caspase-4 dependent release of pro-IL-1β, but further processing to active IL-1β is limited. Furthermore, TLR3 stimulation results in pyroptotic- and apoptotic cell death. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

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

PubMed

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

2010-07-01

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

Reactive oxygen species-dependent necroptosis in Jurkat T cells induced by pathogenic free-living Naegleria fowleri.

PubMed

Song, K-J; Jang, Y S; Lee, Y A; Kim, K A; Lee, S K; Shin, M H

2011-07-01

Naegleria fowleri, a free-living amoeba, is the causative pathogen of primary amoebic meningoencephalitis in humans and experimental mice. N. fowleri is capable of destroying tissues and host cells through lytic necrosis. However, the mechanism by which N. fowleri induces host cell death is unknown. Electron microscopy indicated that incubation of Jurkat T cells with N. fowleri trophozoites induced necrotic morphology of the Jurkat T cells. N. fowleri also induced cytoskeletal protein cleavage, extensive poly (ADP-ribose) polymerase hydrolysis and lactate dehydrogenase (LDH) release. Although no activation of caspase-3 was observed in Jurkat T cells co-incubated with amoebae, intracellular reactive oxygen species (ROS) were strongly generated by NADPH oxidase (NOX). Pretreating cells with necroptosis inhibitor necrostatin-1 or NOX inhibitor diphenyleneiodonium chloride (DPI) strongly inhibited amoeba-induced ROS generation and Jurkat cell death, whereas pan-caspase inhibitor z-VAD-fmk did not. N. fowleri-derived secretory products (NfSP) strongly induced intracellular ROS generation and cell death. Necroptotic effects of NfSP were effectively inhibited by pretreating NfSP with proteinase K. Moreover, NfSP-induced LDH release and intracellular ROS accumulation were inhibited by pretreating Jurkat T cells with DPI or necrostatin-1. These results suggest that N. fowleri induces ROS-dependent necroptosis in Jurkat T cells. © 2011 Blackwell Publishing Ltd.

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.

The Role of Ferroptosis in Cancer Development and Treatment Response.

PubMed

Lu, Bin; Chen, Xiao Bing; Ying, Mei Dan; He, Qiao Jun; Cao, Ji; Yang, Bo

2017-01-01

Ferroptosis is a process driven by accumulated iron-dependent lipid ROS that leads to cell death, which is a distinct regulated cell death comparing to other cell death. The lethal metabolic imbalance resulted from GSH depletion or inactivation of glutathione peroxidase 4 is the executor of ferroptosis within the cancer cell. Small molecules-induced ferroptosis has a strong inhibition of tumor growth and enhances the sensitivity of chemotherapeutic drugs, especially in the condition of drug resistance. These evidences have highlighted the importance of ferroptosis in cancer therapeutics, but the roles of ferroptosis in tumorigenesis and development remain unclear. This article provides an overview of the mechanisms of ferroptosis, highlights the role of ferroptosis in cancer and discusses strategies for therapeutic modulation.

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 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.

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

Single Cell Responses to Spatially-Controlled Photosensitized Production of Extracellular Singlet Oxygen

PubMed Central

Pedersen, Brian Wett; Sinks, Louise E.; Breitenbach, Thomas; Schack, Nickolass B.; Vinogradov, Sergei A.; Ogilby, Peter R.

2011-01-01

The response of individual HeLa cells to extracellularly produced singlet oxygen was examined. The spatial domain of singlet oxygen production was controlled using the combination of a membrane-impermeable Pd porphyrin-dendrimer, which served as a photosensitizer, and a focused laser, which served to localize the sensitized production of singlet oxygen. Cells in close proximity to the domain of singlet oxygen production showed morphological changes commonly associated with necrotic cell death. The elapsed post-irradiation “waiting period” before necrosis became apparent depended on (a) the distance between the cell membrane and the domain irradiated, (b) the incident laser fluence and, as such, the initial concentration of singlet oxygen produced, and (c) the lifetime of singlet oxygen. The data imply that singlet oxygen plays a key role in this process of light-induced cell death. The approach of using extracellularly-generated singlet oxygen to induce cell death can provide a solution to a problem that often limits mechanistic studies of intracellularly photosensitized cell death: it can be difficult to quantify the effective light dose, and hence singlet oxygen concentration, when using an intracellular photosensitizer. PMID:21668871

Blocking CD147 induces cell death in cancer cells through impairment of glycolytic energy metabolism

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

Baba, Miyako; Inoue, Masahiro; Itoh, Kazuyuki

2008-09-12

CD147 is a multifunctional transmembrane protein and promotes cancer progression. We found that the anti-human CD147 mouse monoclonal antibody MEM-M6/1 strongly induces necrosis-like cell death in LoVo, HT-29, WiDr, and SW620 colon cancer cells and A2058 melanoma cells, but not in WI-38 and TIG-113 normal fibroblasts. Silencing or overexpression of CD147 in LoVo cells enhanced or decreased the MEM-M6/1 induced cell death, respectively. CD147 is known to form complex with proton-linked monocarboxylate transporters (MCTs), which is critical for lactate transport and intracellular pH (pHi) homeostasis. In LoVo cells, CD147 and MCT-1 co-localized on the cell surface, and MEM-M6/1 inhibited themore » association of these molecules. MEM-M6/1 inhibited lactate uptake, lactate release, and reduced pHi. Further, the induction of acidification was parallel to the decrease of the glycolytic flux and intracellular ATP levels. These effects were not found in the normal fibroblasts. As cancer cells depend on glycolysis for their energy production, CD147 inhibition might induce cell death specific to cancer cells.« less

Large-Scale Identification and Characterization of Heterodera avenae Putative Effectors Suppressing or Inducing Cell Death in Nicotiana benthamiana

PubMed Central

Chen, Changlong; Chen, Yongpan; Jian, Heng; Yang, Dan; Dai, Yiran; Pan, Lingling; Shi, Fengwei; Yang, Shanshan; Liu, Qian

2018-01-01

Heterodera avenae is one of the most important plant pathogens and causes vast losses in cereal crops. As a sedentary endoparasitic nematode, H. avenae secretes effectors that modify plant defenses and promote its biotrophic infection of its hosts. However, the number of effectors involved in the interaction between H. avenae and host defenses remains unclear. Here, we report the identification of putative effectors in H. avenae that regulate plant defenses on a large scale. Our results showed that 78 of the 95 putative effectors suppressed programmed cell death (PCD) triggered by BAX and that 7 of the putative effectors themselves caused cell death in Nicotiana benthamiana. Among the cell-death-inducing effectors, three were found to be dependent on their specific domains to trigger cell death and to be expressed in esophageal gland cells by in situ hybridization. Ten candidate effectors that suppressed BAX-triggered PCD also suppressed PCD triggered by the elicitor PsojNIP and at least one R-protein/cognate effector pair, suggesting that they are active in suppressing both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Notably, with the exception of isotig16060, these putative effectors could also suppress PCD triggered by cell-death-inducing effectors from H. avenae, indicating that those effectors may cooperate to promote nematode parasitism. Collectively, our results indicate that the majority of the tested effectors of H. avenae may play important roles in suppressing cell death induced by different elicitors in N. benthamiana. PMID:29379510

MLN4924 sensitizes monocytes and maturing dendritic cells for TNF-dependent and -independent necroptosis.

PubMed

El-Mesery, Mohamed; Seher, Axel; Stühmer, Thorsten; Siegmund, Daniela; Wajant, Harald

2015-03-01

MLN4924 prevents the formation of active cullin-RING ubiquitin ligase complexes and thus inhibits NF-κB signalling. Here, we evaluated the effects of this compound on monocytes and dendritic cells (DCs). Monocytes and DCs were challenged with TNF or LPS in the presence and absence of MLN4924. The effects of MLN4924 on cellular viability, pro-inflammatory gene induction and DC maturation were investigated using the MTT assay, elisa and FACS analysis. Mechanisms of cell death induction were evaluated by using inhibitors of caspases, RIPK1 and MLKL. MLN4924 inhibited NF-κB activation and sensitized monocytes and immature DCs (iDCs) for TNFR1-induced cell death. Neither the caspase inhibitor zVAD-fmk, the RIPK1 inhibitor necrostatin-1 (nec-1) nor the MLKL inhibitor necrosulfonamide (NSA) alone prevented TNF-induced cell death. A combination of zVAD-fmk and nec-1 or NSA, however, rescued monocytes and iDCs from MLN4924/TNF-induced cell death indicating that MLN4924 affects anti-apoptotic and anti-necrotic activities in TNFR1 signalling. MLN4924 also converted the response of iDCs to LPS from maturation to cell death. LPS-induced cell death in MLN4924-treated iDCs was again only effectively blocked by cotreatment with zVAD-fmk and nec-1 or NSA. Noteworthy, MLN4924/LPS-induced cell death was almost completely independent of endogenous TNF. MLN4924 also strongly inhibited maturation and activation of iDCs that were rescued from cell death by zVAD-fmk and nec-1. Our data reveal a strong dual suppressive effect of MLN4924 on DC activity. The targeting of NAE by MLN4924 could be a new way to treat inflammatory diseases. © 2014 The British Pharmacological Society.

The Major Isoforms of Bim Contribute to Distinct Biological Activities that Govern the Processes of Autophagy and Apoptosis in Interleukin-7 Dependent Lymphocytes

PubMed Central

Ruppert, Shannon; Li, Wenqing; Zhang, Ge; Carlson, Adina L.; Limaye, Arati; Durum, Scott K.; Khaled, Annette R.

2012-01-01

Bim is a BH3-only member of the Bcl-2 family that enables the death of T-cells. Partial rescue of cytokine-deprived T-cells occurs when Bim and the receptor for the T-cell growth factor, interleukin-7 (IL-7), are deleted, implicating Bim as a possible target of IL-7-mediated signaling. Alternative splicing yields three major isoforms: BimEL, BimL and BimS. To study the effect of Bim deficiency and define the function of the major isoforms, Bim-containing and Bim-deficient T-cells, dependent on IL-7 for growth, were used. Loss of total Bim in IL-7-deprived T-cells resulted in delayed apoptosis. However, loss of Bim also impeded the later degradative phase of autophagy. p62, an autophagy-adaptor protein which is normally degraded, accumulated in Bim deficient cells. To explain this, BimL was found to support acidification of lysosomes that later may associate with autophagic vesicles. Key findings showed that inhibition of lysosomal acidification accelerated death upon IL-7 withdrawal only in Bim-containing T-cells. IL-7 dependent T-cells lacking Bim were less sensitive to inhibition of lysosomal acidification. BimL co-immunoprecipitated with dynein and Lamp1-containing vesicles, indicating BimL could be an adaptor for dynein to facilitate loading of lysosomes. In Bim deficient T-cells, lysosome-tracking probes revealed vesicles of less acidic pH. Over-expression of BimL restored acidic vesicles in Bim deficient T-cells, while other isoforms, BimEL and BimS, promoted intrinsic cell death. These results reveal a novel role for BimL in lysosomal positioning that may be required for the formation of degradative autolysosomes. PMID:22728771

L-carnosine affects the growth of Saccharomyces cerevisiae in a metabolism-dependent manner.

PubMed

Cartwright, Stephanie P; Bill, Roslyn M; Hipkiss, Alan R

2012-01-01

The dipeptide L-carnosine (β-alanyl-L-histidine) has been described as enigmatic: it inhibits growth of cancer cells but delays senescence in cultured human fibroblasts and extends the lifespan of male fruit flies. In an attempt to understand these observations, the effects of L-carnosine on the model eukaryote, Saccharomyces cerevisiae, were examined on account of its unique metabolic properties; S. cerevisiae can respire aerobically, but like some tumor cells, it can also exhibit a metabolism in which aerobic respiration is down regulated. L-Carnosine exhibited both inhibitory and stimulatory effects on yeast cells, dependent upon the carbon source in the growth medium. When yeast cells were not reliant on oxidative phosphorylation for energy generation (e.g. when grown on a fermentable carbon source such as 2% glucose), 10-30 mM L-carnosine slowed growth rates in a dose-dependent manner and increased cell death by up to 17%. In contrast, in media containing a non-fermentable carbon source in which yeast are dependent on aerobic respiration (e.g. 2% glycerol), L-carnosine did not provoke cell death. This latter observation was confirmed in the respiratory yeast, Pichia pastoris. Moreover, when deletion strains in the yeast nutrient-sensing pathway were treated with L-carnosine, the cells showed resistance to its inhibitory effects. These findings suggest that L-carnosine affects cells in a metabolism-dependent manner and provide a rationale for its effects on different cell types.

MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis

PubMed Central

Liu, Shuzhen; Liu, Hua; Johnston, Andrea; Hanna-Addams, Sarah; Reynoso, Eduardo; Xiang, Yougui

2017-01-01

Mixed-lineage kinase domain-like protein (MLKL) is essential for TNF-α–induced necroptosis. How MLKL promotes cell death is still under debate. Here we report that MLKL forms SDS-resistant, disulfide bond-dependent polymers during necroptosis in both human and mouse cells. MLKL polymers are independent of receptor-interacting protein kinase 1 and 3 (RIPK1/RIPK3) fibers. Large MLKL polymers are more than 2 million Da and are resistant to proteinase K digestion. MLKL polymers are fibers 5 nm in diameter under electron microscopy. Furthermore, the recombinant N-terminal domain of MLKL forms amyloid-like fibers and binds Congo red dye. MLKL mutants that cannot form polymers also fail to induce necroptosis efficiently. Finally, the compound necrosulfonamide conjugates cysteine 86 of human MLKL and blocks MLKL polymer formation and subsequent cell death. These results demonstrate that disulfide bond-dependent, amyloid-like MLKL polymers are necessary and sufficient to induce necroptosis. PMID:28827318

MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis.

PubMed

Liu, Shuzhen; Liu, Hua; Johnston, Andrea; Hanna-Addams, Sarah; Reynoso, Eduardo; Xiang, Yougui; Wang, Zhigao

2017-09-05

Mixed-lineage kinase domain-like protein (MLKL) is essential for TNF-α-induced necroptosis. How MLKL promotes cell death is still under debate. Here we report that MLKL forms SDS-resistant, disulfide bond-dependent polymers during necroptosis in both human and mouse cells. MLKL polymers are independent of receptor-interacting protein kinase 1 and 3 (RIPK1/RIPK3) fibers. Large MLKL polymers are more than 2 million Da and are resistant to proteinase K digestion. MLKL polymers are fibers 5 nm in diameter under electron microscopy. Furthermore, the recombinant N-terminal domain of MLKL forms amyloid-like fibers and binds Congo red dye. MLKL mutants that cannot form polymers also fail to induce necroptosis efficiently. Finally, the compound necrosulfonamide conjugates cysteine 86 of human MLKL and blocks MLKL polymer formation and subsequent cell death. These results demonstrate that disulfide bond-dependent, amyloid-like MLKL polymers are necessary and sufficient to induce necroptosis.

Extracellular vesicle-mediated transfer of processed and functional RNY5 RNA

PubMed Central

Chakrabortty, Sudipto K.; Prakash, Ashwin; Nechooshtan, Gal; Hearn, Stephen; Gingeras, Thomas R.

2015-01-01

Extracellular vesicles (EVs) have been proposed as a means to promote intercellular communication. We show that when human primary cells are exposed to cancer cell EVs, rapid cell death of the primary cells is observed, while cancer cells treated with primary or cancer cell EVs do not display this response. The active agents that trigger cell death are 29- to 31-nucleotide (nt) or 22- to 23-nt processed fragments of an 83-nt primary transcript of the human RNY5 gene that are highly likely to be formed within the EVs. Primary cells treated with either cancer cell EVs, deproteinized total RNA from either primary or cancer cell EVs, or synthetic versions of 31- and 23-nt fragments trigger rapid cell death in a dose-dependent manner. The transfer of processed RNY5 fragments through EVs may reflect a novel strategy used by cancer cells toward the establishment of a favorable microenvironment for their proliferation and invasion. PMID:26392588

The HDAC6/8/10 inhibitor TH34 induces DNA damage-mediated cell death in human high-grade neuroblastoma cell lines.

PubMed

Kolbinger, Fiona R; Koeneke, Emily; Ridinger, Johannes; Heimburg, Tino; Müller, Michael; Bayer, Theresa; Sippl, Wolfgang; Jung, Manfred; Gunkel, Nikolas; Miller, Aubry K; Westermann, Frank; Witt, Olaf; Oehme, Ina

2018-06-09

High histone deacetylase (HDAC) 8 and HDAC10 expression levels have been identified as predictors of exceptionally poor outcomes in neuroblastoma, the most common extracranial solid tumor in childhood. HDAC8 inhibition synergizes with retinoic acid treatment to induce neuroblast maturation in vitro and to inhibit neuroblastoma xenograft growth in vivo. HDAC10 inhibition increases intracellular accumulation of chemotherapeutics through interference with lysosomal homeostasis, ultimately leading to cell death in cultured neuroblastoma cells. So far, no HDAC inhibitor covering HDAC8 and HDAC10 at micromolar concentrations without inhibiting HDACs 1, 2 and 3 has been described. Here, we introduce TH34 (3-(N-benzylamino)-4-methylbenzhydroxamic acid), a novel HDAC6/8/10 inhibitor for neuroblastoma therapy. TH34 is well-tolerated by non-transformed human skin fibroblasts at concentrations up to 25 µM and modestly impairs colony growth in medulloblastoma cell lines, but specifically induces caspase-dependent programmed cell death in a concentration-dependent manner in several human neuroblastoma cell lines. In addition to the induction of DNA double-strand breaks, HDAC6/8/10 inhibition also leads to mitotic aberrations and cell-cycle arrest. Neuroblastoma cells display elevated levels of neuronal differentiation markers, mirrored by formation of neurite-like outgrowths under maintained TH34 treatment. Eventually, after long-term treatment, all neuroblastoma cells undergo cell death. The combination of TH34 with plasma-achievable concentrations of retinoic acid, a drug applied in neuroblastoma therapy, synergistically inhibits colony growth (combination index (CI) < 0.1 for 10 µM of each). In summary, our study supports using selective HDAC inhibitors as targeted antineoplastic agents and underlines the therapeutic potential of selective HDAC6/8/10 inhibition in high-grade neuroblastoma.

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

The TAT-RasGAP317-326 anti-cancer peptide can kill in a caspase-, apoptosis-, and necroptosis-independent manner

PubMed Central

Puyal, Julien; Margue, Christiane; Michel, Sébastien; Kreis, Stephanie; Kulms, Dagmar; Barras, David; Nahimana, Aimable; Widmann, Christian

2016-01-01

Tumor cell resistance to apoptosis, which is triggered by many anti-tumor therapies, remains a major clinical problem. Therefore, development of more efficient therapies is a priority to improve cancer prognosis. We have previously shown that a cell-permeable peptide derived from the p120 Ras GTPase-activating protein (RasGAP), called TAT-RasGAP317-326, bears anti-malignant activities in vitro and in vivo, such as inhibition of metastatic progression and tumor cell sensitization to cell death induced by various anti-cancer treatments. Recently, we discovered that this RasGAP-derived peptide possesses the ability to directly kill some cancer cells. TAT-RasGAP317-326 can cause cell death in a manner that can be either partially caspase-dependent or fully caspase-independent. Indeed, TAT-RasGAP317-326-induced toxicity was not or only partially prevented when apoptosis was inhibited. Moreover, blocking other forms of cell death, such as necroptosis, parthanatos, pyroptosis and autophagy did not hamper the killing activity of the peptide. The death induced by TAT-RasGAP317-326 can therefore proceed independently from these modes of death. Our finding has potentially interesting clinical relevance because activation of a death pathway that is distinct from apoptosis and necroptosis in tumor cells could lead to the generation of anti-cancer drugs that target pathways not yet considered for cancer treatment. PMID:27602963

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.

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

The Inflammasome Drives GSDMD-Independent Secondary Pyroptosis and IL-1 Release in the Absence of Caspase-1 Protease Activity.

PubMed

Schneider, Katharina S; Groß, Christina J; Dreier, Roland F; Saller, Benedikt S; Mishra, Ritu; Gorka, Oliver; Heilig, Rosalie; Meunier, Etienne; Dick, Mathias S; Ćiković, Tamara; Sodenkamp, Jan; Médard, Guillaume; Naumann, Ronald; Ruland, Jürgen; Kuster, Bernhard; Broz, Petr; Groß, Olaf

2017-12-26

Inflammasomes activate the protease caspase-1, which cleaves interleukin-1β and interleukin-18 to generate the mature cytokines and controls their secretion and a form of inflammatory cell death called pyroptosis. By generating mice expressing enzymatically inactive caspase-1 C284A , we provide genetic evidence that caspase-1 protease activity is required for canonical IL-1 secretion, pyroptosis, and inflammasome-mediated immunity. In caspase-1-deficient cells, caspase-8 can be activated at the inflammasome. Using mice either lacking the pyroptosis effector gasdermin D (GSDMD) or expressing caspase-1 C284A , we found that GSDMD-dependent pyroptosis prevented caspase-8 activation at the inflammasome. In the absence of GSDMD-dependent pyroptosis, the inflammasome engaged a delayed, alternative form of lytic cell death that was accompanied by the release of large amounts of mature IL-1 and contributed to host protection. Features of this cell death modality distinguished it from apoptosis, suggesting it may represent a distinct form of pro-inflammatory regulated necrosis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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.

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

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

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

Ethylene Insensitivity Modulates Ozone-Induced Cell Death in Birch1

PubMed Central

Vahala, Jorma; Ruonala, Raili; Keinänen, Markku; Tuominen, Hannele; Kangasjärvi, Jaakko

2003-01-01

We have used genotypic variation in birch (Betula pendula Roth) to investigate the roles of ozone (O3)-induced ethylene (ET), jasmonic acid, and salicylic acid in the regulation of tissue tolerance to O3. Of these hormones, ET evolution correlated best with O3-induced cell death. Disruption of ET perception by transformation of birch with the dominant negative mutant allele etr1-1 of the Arabidopsis ET receptor gene ETR1 or blocking of ET perception with 1-methylcyclopropene reduced but did not completely prevent the O3-induced cell death, when inhibition of ET biosynthesis with aminooxyacetic acid completely abolished O3 lesion formation. This suggests the presence of an ET-signaling-independent but ET biosynthesis-dependent component in the ET-mediated stimulation of cell death in O3-exposed birch. Functional ET signaling was required for the O3 induction of the gene encoding β-cyanoalanine synthase, which catalyzes detoxification of the cyanide formed during ET biosynthesis. The results suggest that functional ET signaling is required to protect birch from the O3-induced cell death and that a decrease in ET sensitivity together with a simultaneous, high ET biosynthesis can potentially cause cell death through a deficient detoxification of cyanide. PMID:12746524

Circadian Stress Regimes Affect the Circadian Clock and Cause Jasmonic Acid-Dependent Cell Death in Cytokinin-Deficient Arabidopsis Plants[OPEN

PubMed Central

Nitschke, Silvia; Cortleven, Anne; Iven, Tim; Havaux, Michel; Schmülling, Thomas

2016-01-01

The circadian clock helps plants measure daylength and adapt to changes in the day-night rhythm. We found that changes in the light-dark regime triggered stress responses, eventually leading to cell death, in leaves of Arabidopsis thaliana plants with reduced cytokinin levels or defective cytokinin signaling. Prolonged light treatment followed by a dark period induced stress and cell death marker genes while reducing photosynthetic efficiency. This response, called circadian stress, is also characterized by altered expression of clock and clock output genes. In particular, this treatment strongly reduced the expression of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Intriguingly, similar changes in gene expression and cell death were observed in clock mutants lacking proper CCA1 and LHY function. Circadian stress caused strong changes in reactive oxygen species- and jasmonic acid (JA)-related gene expression. The activation of the JA pathway, involving the accumulation of JA metabolites, was crucial for the induction of cell death, since the cell death phenotype was strongly reduced in the jasmonate resistant1 mutant background. We propose that adaptation to circadian stress regimes requires a normal cytokinin status which, acting primarily through the AHK3 receptor, supports circadian clock function to guard against the detrimental effects of circadian stress. PMID:27354555

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

Differential activation of cell death and autophagy results in an increased cytotoxic potential for trifluorothymidine compared to 5-fluorouracil in colon cancer cells.

PubMed

Bijnsdorp, Irene V; Peters, Godefridus J; Temmink, Olaf H; Fukushima, Masakazu; Kruyt, Frank A

2010-05-15

Trifluorothymidine (TFT) is part of the oral drug formulation TAS-102. Both 5-fluorouracil (5-FU) and TFT can inhibit thymidylate synthase and be incorporated into DNA. TFT shows only moderate cross-resistance to 5-FU. Therefore, we examined whether mechanistic differences in cell death could underlie their different modes of action in colorectal cancer cell lines (WiDR, Lovo92 and Colo320). Drug cytotoxicity was determined by SRB- and clonogenic assays, cell death by flow cytometry (PI and annexin V), caspase cleavage by Western blotting and activity assays and in vivo activity in the hollow fiber assay. The IC(50) values of TFT were 1-6 fold lower than for 5-FU, and clonogenic survival was less than 0.9% at 3 muM TFT, while 2-20% of the cells still survived after 20 muM 5-FU. In general, TFT was a more potent inducer of apoptosis than 5-FU, although the contribution of caspases varied between the used cell lines and necrosis-like cell death was detected. Accordingly, both drugs induced caspase (Z-VAD) independent cell death and lysosomal cathepsin B was involved. Activation of autophagy recovery mechanisms was only triggered by 5-FU, but not by TFT as determined by LC3B expression and cleavage. Inhibition of autophagy by 3-MA in 5-FU exposed cells reduced cell survival. Also, in vivo TFT (as TAS-102) caused more cell death than a 5-FU formulation. We conclude that TFT and 5-FU induce cell death via both caspase-dependent and independent mechanisms. The TFT was more potent than 5-FU, because it induces higher levels of cell death and does not elicit an autophagic survival response in the cancer cell lines. This provides a strong molecular basis for further application of TFT in cancer therapy.

Protecting Mammalian Hair Cells from Aminoglycoside-Toxicity: Assessing Phenoxybenzamine's Potential.

PubMed

Majumder, Paromita; Moore, Paulette A; Richardson, Guy P; Gale, Jonathan E

2017-01-01

Aminoglycosides (AGs) are widely used antibiotics because of their low cost and high efficacy against gram-negative bacterial infection. However, AGs are ototoxic, causing the death of sensory hair cells in the inner ear. Strategies aimed at developing or discovering agents that protect against aminoglycoside ototoxicity have focused on inhibiting apoptosis or more recently, on preventing antibiotic uptake by the hair cells. Recent screens for ototoprotective compounds using the larval zebrafish lateral line identified phenoxybenzamine as a potential protectant for aminoglycoside-induced hair cell death. Here we used live imaging of FM1-43 uptake as a proxy for aminoglycoside entry, combined with hair-cell death assays to evaluate whether phenoxybenzamine can protect mammalian cochlear hair cells from the deleterious effects of the aminoglycoside antibiotic neomycin. We show that phenoxybenzamine can block FM1-43 entry into mammalian hair cells in a reversible and dose-dependent manner, but pre-incubation is required for maximal inhibition of entry. We observed differential effects of phenoxybenzamine on FM1-43 uptake in the two different types of cochlear hair cell in mammals, the outer hair cells (OHCs) and inner hair cells (IHCs). The requirement for pre-incubation and reversibility suggests an intracellular rather than an extracellular site of action for phenoxybenzamine. We also tested the efficacy of phenoxybenzamine as an otoprotective agent. In mouse cochlear explants the hair cell death resulting from 24 h exposure to neomycin was steeply dose-dependent, with 50% cell death occurring at ~230 μM for both IHC and OHC. We used 250 μM neomycin in subsequent hair-cell death assays. At 100 μM with 1 h pre-incubation, phenoxybenzamine conferred significant protection to both IHCs and OHCs, however at higher concentrations phenoxybenzamine itself showed clear signs of ototoxicity and an additive toxic effect when combined with neomycin. These data do not support the use of phenoxybenzamine as a therapeutic agent in mammalian inner ear. Our findings do share parallels with the observations from the zebrafish lateral line model but they also highlight the necessity for validation in the mammalian system and the potential for differential effects on sensory hair cells from different species, in different systems and even between cells in the same organ.

Varying butyric acid amounts induce different stress- and cell death-related signals in nerve growth factor-treated PC12 cells: implications in neuropathic pain absence during periodontal disease progression.

PubMed

Seki, Keisuke; Cueno, Marni E; Kamio, Noriaki; Saito, Yuko; Kamimoto, Atsushi; Kurita-Ochiai, Tomoko; Ochiai, Kuniyasu

2016-06-01

Neuropathic pain is absent from the early stages of periodontal disease possibly due to neurite retraction. Butyric acid (BA) is a periodontopathic metabolite that activates several stress-related signals and, likewise, induce neurite retraction. Neuronal cell death is associated to neurite retraction which would suggest that BA-induced neurite retraction is ascribable to neuronal cell death. However, the underlying mechanism of BA-related cell death signaling remains unknown. In this study, we exposed NGF-treated PC12 cells to varying BA concentrations [0 (control), 0.5, 1.0, 5.0 mM] and determined selected stress-related (H2O2, glutathione reductase, calcium (Ca(2+)), plasma membrane Ca(2+) ATPase (PMCA), and GADD153/CHOPS) and cell death-associated (extrinsic: FasL, TNF-α, TWEAK, and TRAIL; intrinsic: cytochrome C (CytC), NF-kB, CASP8, CASP9, CASP10, and CASP3) signals. Similarly, we confirmed cell death execution by chromatin condensation. Our results showed that low (0.5 mM) and high (1.0 and 5.0 mM) BA levels differ in stress and cell death signaling. Moreover, at periodontal disease-level BA concentration (5 mM), we observed that only FasL amounts were affected and occurred concurrently with chromatin condensation insinuating that cells have fully committed to neurodegeneration. Thus, we believe that both stress and cell death signaling in NGF-treated PC12 cells are affected differently depending on BA concentration. In a periodontal disease scenario, we hypothesize that during the early stages, low BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurite non-proliferation, whereas, during the later stages, high BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurodegeneration. More importantly, we propose that neuropathic pain absence at any stage of periodontal disease progression is ascribable to BA accumulation regardless of amount.

Dihydroartemisinin (DHA) induces caspase-3-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells

PubMed Central

Lu, Ying-Ying; Chen, Tong-Sheng; Qu, Jun-Le; Pan, Wen-Liang; Sun, Lei; Wei, Xun-Bin

2009-01-01

Background Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, is recommended as the first-line anti-malarial drug with low toxicity. DHA has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways, although the molecular mechanisms are not well understood. Methods In this study, cell counting kit (CCK-8) assay was employed to evaluate the survival of DHA-treated ASTC-a-1 cells. The induction of apoptosis was detected by Hoechst 33258 and PI staining as well as flow cytometry analysis. Collapse of mitochondrial transmembrane potential (ΔΨm) was measured by dynamic detection under a laser scanning confocal microscope and flow cytometry analysis using Rhodamine123. Caspase-3 activities measured with or without Z-VAD-fmk (a broad spectrum caspase inhibitor) pretreatment by FRET techniques, caspase-3 activity measurement, and western blotting analysis. Results Our results indicated that DHA induced apoptotic cell death in a dose- and time-dependent manner, which was accompanied by mitochondrial morphology changes, the loss of ΔΨm and the activation of caspase-3. Conclusion These results show for the first time that DHA can inhibit proliferation and induce apoptosis via caspase-3-dependent mitochondrial death pathway in ASTC-a-1 cells. Our work may provide evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of lung adenocarcinoma. PMID:19272183

Lysosome-mediated Cell Death and Autophagy-Dependent Multidrug Resistance in Breast Cancer

DTIC Science & Technology

2008-10-01

gene links mitochondria and cell death, the data suggests that Bcl2 may be involved in autophagic cell death and AD-MDR. GeneGo analysis also...GSK3 beta GSK3 beta E2A p53 p21 p21 E2F1 PPAR -gamma JNK1(MA PK8) JNK1(M APK8) ESR1 (nuclear) RARalpha Androgen receptor Androge n receptor p53...RelA (p65 NF-kB subunit) Erk (MAPK1/3 ) Erk (MAPK1/ 3) PPAR - gamma SOX9 Bcl-2 Bcl-2 RARalpha SP1 EGFR EGFR RelA (p65 NF- kB subunit) RARalpha RelA

Epidermal Cell Death in Rice Is Regulated by Ethylene, Gibberellin, and Abscisic Acid

PubMed Central

Steffens, Bianka; Sauter, Margret

2005-01-01

Programmed cell death (PCD) of epidermal cells that cover adventitious root primordia in deepwater rice (Oryza sativa) is induced by submergence. Early suicide of epidermal cells may prevent injury to the growing root that emerges under flooding conditions. Induction of PCD is dependent on ethylene signaling and is further promoted by gibberellin (GA). Ethylene and GA act in a synergistic manner, indicating converging signaling pathways. Treatment of plants with GA alone did not promote PCD. Treatment with the GA biosynthesis inhibitor paclobutrazol resulted in increased PCD in response to ethylene and GA presumably due to an increased sensitivity of epidermal cells to GA. Abscisic acid (ABA) was shown to efficiently delay ethylene-induced as well as GA-promoted cell death. The results point to ethylene signaling as a target of ABA inhibition of PCD. Accumulation of ethylene and GA and a decreased ABA level in the rice internode thus favor induction of epidermal cell death and ensure that PCD is initiated as an early response that precedes adventitious root growth. PMID:16169967

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

Cardiac Glycoside Glucoevatromonoside Induces Cancer Type-Specific Cell Death

PubMed Central

Schneider, Naira F. Z.; Cerella, Claudia; Lee, Jin-Young; Mazumder, Aloran; Kim, Kyung Rok; de Carvalho, Annelise; Munkert, Jennifer; Pádua, Rodrigo M.; Kreis, Wolfgang; Kim, Kyu-Won; Christov, Christo; Dicato, Mario; Kim, Hyun-Jung; Han, Byung Woo; Braga, Fernão C.; Simões, Cláudia M. O.; Diederich, Marc

2018-01-01

Cardiac glycosides (CGs) are natural compounds used traditionally to treat congestive heart diseases. Recent investigations repositioned CGs as potential anticancer agents. To discover novel cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside (GEV) out of 46 CGs for its low nanomolar anti-lung cancer activity. GEV presented reduced toxicity toward non-cancerous cell types (lung MRC-5 and PBMC) and high-affinity binding to the Na+/K+-ATPase α subunit, assessed by computational docking. GEV-induced cell death was caspase-independent, as investigated by a multiparametric approach, and culminates in severe morphological alterations in A549 cells, monitored by transmission electron microscopy, live cell imaging and flow cytometry. This non-canonical cell death was not preceded or accompanied by exacerbation of autophagy. In the presence of GEV, markers of autophagic flux (e.g. LC3I-II conversion) were impacted, even in presence of bafilomycin A1. Cell death induction remained unaffected by calpain, cathepsin, parthanatos, or necroptosis inhibitors. Interestingly, GEV triggered caspase-dependent apoptosis in U937 acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. Differential cell cycle modulation by this CG led to a G2/M arrest, cyclin B1 and p53 downregulation in A549, but not in U937 cells. We further extended the anti-cancer potential of GEV to 3D cell culture using clonogenic and spheroid formation assays and validated our findings in vivo by zebrafish xenografts. Altogether, GEV shows an interesting anticancer profile with the ability to exert cytotoxic effects via induction of different cell death modalities. PMID:29545747

Heat stress induces different forms of cell death in sea anemones and their endosymbiotic algae depending on temperature and duration.

PubMed

Dunn, S R; Thomason, J C; Le Tissier, M D A; Bythell, J C

2004-11-01

Bleaching of reef building corals and other symbiotic cnidarians due to the loss of their dinoflagellate algal symbionts (=zooxanthellae), and/or their photosynthetic pigments, is a common sign of environmental stress. Mass bleaching events are becoming an increasingly important cause of mortality and reef degradation on a global scale, linked by many to global climate change. However, the cellular mechanisms of stress-induced bleaching remain largely unresolved. In this study, the frequency of apoptosis-like and necrosis-like cell death was determined in the symbiotic sea anemone Aiptasia sp. using criteria that had previously been validated for this symbiosis as indicators of programmed cell death (PCD) and necrosis. Results indicate that PCD and necrosis occur simultaneously in both host tissues and zooxanthellae subject to environmentally relevant doses of heat stress. Frequency of PCD in the anemone endoderm increased within minutes of treatment. Peak rates of apoptosis-like cell death in the host were coincident with the timing of loss of zooxanthellae during bleaching. The proportion of apoptosis-like host cells subsequently declined while cell necrosis increased. In the zooxanthellae, both apoptosis-like and necrosis-like activity increased throughout the duration of the experiment (6 days), dependent on temperature dose. A stress-mediated PCD pathway is an important part of the thermal stress response in the sea anemone symbiosis and this study suggests that PCD may play different roles in different components of the symbiosis during bleaching.

Acetylated Chitosan Oligosaccharides Act as Antagonists against Glutamate-Induced PC12 Cell Death via Bcl-2/Bax Signal Pathway

PubMed Central

Hao, Cui; Gao, Lixia; Zhang, Yiran; Wang, Wei; Yu, Guangli; Guan, Huashi; Zhang, Lijuan; Li, Chunxia

2015-01-01

Chitosan oligosaccharides (COSs), depolymerized products of chitosan composed of β-(1→4) d-glucosamine units, have broad range of biological activities such as antitumour, antifungal, and antioxidant activities. In this study, peracetylated chitosan oligosaccharides (PACOs) and N-acetylated chitosan oligosaccharides (NACOs) were prepared from the COSs by chemcal modification. The structures of these monomers were identified using NMR and ESI-MS spectra. Their antagonist effects against glutamate-induced PC12 cell death were investigated. The results showed that pretreatment of PC12 cells with the PACOs markedly inhibited glutamate-induced cell death in a concentration-dependent manner. The PACOs were better glutamate antagonists compared to the COSs and the NACOs, suggesting the peracetylation is essential for the neuroprotective effects of chitosan oligosaccharides. In addition, the PACOs pretreatment significantly reduced lactate dehydrogenase release and reactive oxygen species production. It also attenuated the loss of mitochondrial membrane potential. Further studies indicated that the PACOs inhibited glutamate-induced cell death by preventing apoptosis through depressing the elevation of Bax/Bcl-2 ratio and caspase-3 activation. These results suggest that PACOs might be promising antagonists against glutamate-induced neural cell death. PMID:25775423

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

Influence on cell death of high frequency motion of magnetic nanoparticles during magnetic hyperthermia experiments

NASA Astrophysics Data System (ADS)

Hallali, N.; Clerc, P.; Fourmy, D.; Gigoux, V.; Carrey, J.

2016-07-01

Studies with transplanted tumors in animals and clinical trials have provided the proof-of-concept of magnetic hyperthermia (MH) therapy of cancers using iron oxide nanoparticles. Interestingly, in several studies, the application of an alternating magnetic field (AMF) to tumor cells having internalized and accumulated magnetic nanoparticles (MNPs) into their lysosomes can induce cell death without detectable temperature increase. To explain these results, among other hypotheses, it was proposed that cell death could be due to the high-frequency translational motion of MNPs under the influence of the AMF gradient generated involuntarily by most inductors. Such mechanical actions of MNPs might cause cellular damages and participate in the induction of cell death under MH conditions. To test this hypothesis, we developed a setup maximizing this effect. It is composed of an anti-Helmholtz coil and two permanent magnets, which produce an AMF gradient and a superimposed static MF. We have measured the MNP heating power and treated tumor cells by a standard AMF and by an AMF gradient, on which was added or not a static magnetic field. We showed that the presence of a static magnetic field prevents MNP heating and cell death in standard MH conditions. The heating power of MNPs in an AMF gradient is weak, position-dependent, and related to the presence of a non-zero AMF. Under an AMF gradient and a static field, no MNP heating and cell death were measured. Consequently, the hypothesis that translational motions could be involved in cell death during MH experiments is ruled out by our experiments.

Comparative study of photothermolysis of cancer cells with nuclear-targeted or cytoplasm-targeted gold nanospheres: continuous wave or pulsed lasers

NASA Astrophysics Data System (ADS)

Huang, Xiaohua; Kang, Bin; Qian, Wei; Mackey, Megan A.; Chen, Po C.; Oyelere, Adegboyega K.; El-Sayed, Ivan H.; El-Sayed, Mostafa A.

2010-09-01

We conduct a comparative study on the efficiency and cell death pathways of continuous wave (cw) and nanosecond pulsed laser photothermal cancer therapy using gold nanospheres delivered to either the cytoplasm or nucleus of cancer cells. Cytoplasm localization is achieved using arginine-glycine-aspartate peptide modified gold nanospheres, which target integrin receptors on the cell surface and are subsequently internalized by the cells. Nuclear delivery is achieved by conjugating the gold nanospheres with nuclear localization sequence peptides originating from the simian virus. Photothermal experiments show that cell death can be induced with a single pulse of a nanosecond laser more efficiently than with a cw laser. When the cw laser is applied, gold nanospheres localized in the cytoplasm are more effective in inducing cell destruction than gold nanospheres localized at the nucleus. The opposite effect is observed when the nanosecond pulsed laser is used, suggesting that plasmonic field enhancement of the nonlinear absorption processes occurs at high localization of gold nanospheres at the nucleus. Cell death pathways are further investigated via a standard apoptosis kit to show that the cell death mechanisms depend on the type of laser used. While the cw laser induces cell death via apoptosis, the nanosecond pulsed laser leads to cell necrosis. These studies add mechanistic insight to gold nanoparticle-based photothermal therapy of cancer.

Bilirubin induces a calcium-dependent inhibition of multifunctional Ca2+/calmodulin-dependent kinase II activity in vitro.

PubMed

Churn, S B; DeLorenzo, R J; Shapiro, S M

1995-12-01

Excessive bilirubin levels in newborn infants result in long-term neurologic deficits that remain after bilirubin levels return to normal. Much of the observed neurologic deficits can be attributed to bilirubin-induced, delayed neuronal cell death. Inhibition of calcium/calmodulin-dependent kinase II (CaM kinase II) activity that precedes cell death is observed in conditions such as seizure activity, stroke, and glutamate excitotoxicity. Because neonatal bilirubin exposure results in neuronal loss in developing brain systems, we tested whether bilirubin exposure would induce an immediate inhibition of CaM activity, in vitro. P-81 filtration assay of basal and calcium-stimulated kinase activity was performed under standard kinase assay conditions. Bilirubin and/or albumin was added to the reaction vessels to determine the effect of these agents on kinase activity. Bilirubin exposure resulted in a concentration-dependent inhibition of CaM kinase II activity (IC50 = 16.78 microM). At concentrations above 50 microM, bilirubin exposure resulted in a 71 +/- 8% (mean +/- SD) inhibition of kinase activity (p < 0.001, t test, n = 10). Bilirubin exposure did not result in kinase inhibition if excessive bilirubin was removed by albumin binding before stimulation of kinase activity (106.9 +/- 9.6% control activity, n = 5). However, removal of bilirubin by binding with albumin after calcium addition did not restore kinase activity. (36.1 +/- 3.8% control activity, n = 5). Thus, once inhibition was observed, the activity could not be restored by addition of albumin. The data suggest that bilirubin exposure resulted in a calcium-dependent inhibition of CaM kinase II activity that, once induced, was not reversible by removing bilirubin by the addition of albumin. Because inhibition of CaM kinase II activity has been correlated with delayed neuronal cell death in many neuropathologic conditions, bilirubin-induced inhibition of this enzyme may be a cellular mechanism by which bilirubin exposure results in delayed neuronal cell death in developing brain.

Comparison of activated caspase detection methods in the gentamicin-treated chick cochlea

PubMed Central

Kaiser, Christina L.; Chapman, Brittany J.; Guidi, Jessica L.; Terry, Caitlin E.; Mangiardi, Dominic A.; Cotanche, Douglas A.

2008-01-01

Aminoglycoside antibiotics induce caspase-dependent apoptotic death in cochlear hair cells. Apoptosis, a regulated form of cell death, can be induced by many stressors, which activate signaling pathways that result in the controlled dismantling of the affected cell. The caspase family of proteases is activated in the apoptotic signaling pathway and is responsible for cellular destruction. The initiator caspase-9 and the effector caspase-3 are both activated in chick cochlear hair cells following aminoglycoside exposure. We have analyzed caspase activation in the avian cochlea during gentamicin-induced hair cell death to compare two different methods of caspase detection: caspase antibodies and CaspaTag kits. Caspase antibodies bind to the cleaved activated form of caspase-9 or caspase-3 in specific locations in fixed tissue. CaspaTag is a fluorescent inhibitor that binds to a reactive cysteine residue on the large subunit of the caspase heterodimer in unfixed tissue. To induce cochlear hair cell loss, 1-2 week-old chickens received a single injection of gentamicin (300 mg/kg). Chicks were sacrificed 24, 30, 42, 48, 72, or 96 h after injection. Cochleae were dissected and labeled for activated caspase-9 or caspase-3 using either caspase-directed antibodies or CaspaTag kits. Ears were co-labeled with either phalloidin or myosin VI to visualize hair cells and to determine the progression of cochlear damage. The timing of caspase activation was similar for both assays; however, caspase-9 and caspase-3 antibodies labeled only those cells currently undergoing apoptotic cell death. Conversely, CaspaTag-labeled all the cells that have undergone apoptotic cell death and ejection from the sensory epithelium, in addition to those that are currently in the cell death process. This makes CaspaTag ideal for showing an overall pattern or level of cell death over a period of time, while caspase antibodies provide a snapshot of cell death at a specific time point. PMID:18487027

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.

A statistical model describing combined irreversible electroporation and electroporation-induced blood-brain barrier disruption

PubMed Central

Sharabi, Shirley; Kos, Bor; Last, David; Guez, David; Daniels, Dianne; Harnof, Sagi; Miklavcic, Damijan

2016-01-01

Background Electroporation-based therapies such as electrochemotherapy (ECT) and irreversible electroporation (IRE) are emerging as promising tools for treatment of tumors. When applied to the brain, electroporation can also induce transient blood-brain-barrier (BBB) disruption in volumes extending beyond IRE, thus enabling efficient drug penetration. The main objective of this study was to develop a statistical model predicting cell death and BBB disruption induced by electroporation. This model can be used for individual treatment planning. Material and methods Cell death and BBB disruption models were developed based on the Peleg-Fermi model in combination with numerical models of the electric field. The model calculates the electric field thresholds for cell kill and BBB disruption and describes the dependence on the number of treatment pulses. The model was validated using in vivo experimental data consisting of rats brains MRIs post electroporation treatments. Results Linear regression analysis confirmed that the model described the IRE and BBB disruption volumes as a function of treatment pulses number (r2 = 0.79; p < 0.008, r2 = 0.91; p < 0.001). The results presented a strong plateau effect as the pulse number increased. The ratio between complete cell death and no cell death thresholds was relatively narrow (between 0.88-0.91) even for small numbers of pulses and depended weakly on the number of pulses. For BBB disruption, the ratio increased with the number of pulses. BBB disruption radii were on average 67% ± 11% larger than IRE volumes. Conclusions The statistical model can be used to describe the dependence of treatment-effects on the number of pulses independent of the experimental setup. PMID:27069447

5-Fluorouracil-induced apoptosis in cultured oral cancer cells.

PubMed

Tong, D; Poot, M; Hu, D; Oda, D

2000-03-01

Chemotherapy is commonly used to treat advanced oral squamous cell carcinoma (SCC) and is known to kill cancer cells through apoptosis. Our hypothesis states that 5-fluorouracil (5FU) also kills cultured oral epithelial cells through programmed cell death or apoptosis. Cultured oral cancer cells were exposed to an optimum dose of 20 mg/ml of 5FU. Cells were analyzed for changes in cell cycle distribution and induction of cell death including apoptosis. Normal control, human papilloma virus-immortalized (PP), ATCC SCC cell line (CA1) and two primary oral SCC cell lines (CA3 and -4) were studied. Inhibition of apoptosis by a pan-caspase inhibitor was used. SYTO 11 flow cytometry showed increased apoptosis in all 5FU-treated cell cultures compared to untreated controls. The results show biological variation in apoptotic response. CA1 had the lowest apoptotic rate of the cancer cell lines at 1.5%. Next lowest was CA3, followed by CA4 and PP. In addition, alteration in the G1 and S phase fractions were found. Untreated CA1 showed 28% G1, 53% S compared to 43% G1, and 40% S of treated. We investigated the pathway of apoptosis using the pan-caspase inhibitor IDN-1529 by methylthiazolyl diphenyl tetrazolium bromide (MTT) colorimetric analysis. Results showed mild inhibition of cell death when cells were incubated with 50 microM IDN-1529 for 24 h. This suggests a probable caspase-dependent apoptotic pathway. In conclusion, our data suggest that 5FU induces oral cancer cell death through apoptosis and that biological variation exists between normal and cancer cells and between different types of cancer cells themselves. Our data indicate that cultures of a useful in vitro model for chemosensitivity assays are possible. Our results also suggest a caspase-dependent pathway for chemocytotoxicity in oral SCC.

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.

Cell Surface Translocation of Annexin A2 Facilitates Glutamate-induced Extracellular Proteolysis*

PubMed Central

Valapala, Mallika; Maji, Sayantan; Borejdo, Julian; Vishwanatha, Jamboor K.

2014-01-01

Glutamate-induced elevation in intracellular Ca2+ has been implicated in excitotoxic cell death. Neurons respond to increased glutamate levels by activating an extracellular proteolytic cascade involving the components of the plasmin-plasminogen system. AnxA2 is a Ca2+-dependent phospholipid binding protein and serves as an extracellular proteolytic center by recruiting the tissue plasminogen activator and plasminogen and mediating the localized generation of plasmin. Ratiometric Ca2+ imaging and time-lapse confocal microscopy demonstrated glutamate-induced Ca2+ influx. We showed that glutamate translocated both endogenous and AnxA2-GFP to the cell surface in a process dependent on the activity of the NMDA receptor. Glutamate-induced translocation of AnxA2 is dependent on the phosphorylation of tyrosine 23 at the N terminus, and mutation of tyrosine 23 to a non-phosphomimetic variant inhibits the translocation process. The cell surface-translocated AnxA2 forms an active plasmin-generating complex, and this activity can be neutralized by a hexapeptide directed against the N terminus. These results suggest an involvement of AnxA2 in potentiating glutamate-induced cell death processes. PMID:24742684

Delicaflavone induces autophagic cell death in lung cancer via Akt/mTOR/p70S6K signaling pathway.

PubMed

Sui, Yuxia; Yao, Hong; Li, Shaoguang; Jin, Long; Shi, Peiying; Li, Zhijun; Wang, Gang; Lin, Shilan; Wu, Youjia; Li, Yuxiang; Huang, Liying; Liu, Qicai; Lin, Xinhua

2017-03-01

Searching for potential anticancer agents from natural sources is an effective strategy for developing novel chemotherapeutic agents. In this study, data supporting the in vitro and in vivo anticancer effects of delicaflavone, a rarely occurring biflavonoid from Selaginella doederleinii, were reported. Delicaflavone exhibited favorable anticancer properties, as shown by the MTT assay and xenograft model of human non-small cell lung cancer in male BALB/c nude mice without observable adverse effect. By transmission electron microscopy with acridine orange and Cyto-ID®Autophagy detection dyes, Western blot analysis, and RT-PCR assay, we confirmed that delicaflavone induces autophagic cell death by increasing the ratio of LC3-II to LC3-I, which are autophagy-related proteins, and promoting the generation of acidic vesicular organelles and autolysosomes in the cytoplasm of human lung cancer A549 and PC-9 cells in a time- and dose-dependent manner. Delicaflavone downregulated the expression of phospho-Akt, phospho-mTOR, and phospho-p70S6K in a time- and dose-dependent manner, suggesting that it induced autophagy by inhibiting the Akt/mTOR/p70S6K pathway in A549 and PC-9 cells. Delicaflavone is a potential anticancer agent that can induce autophagic cell death in human non-small cell lung cancer via the Akt/mTOR/p70S6K signaling pathway. Delicaflavone showed anti-lung cancer effects in vitro and in vivo. Delicaflavone induced autophagic cell death via Akt/mTOR/p70S6K signaling pathway. Delicaflavone did not show observable side effects in a xenograft mouse model. Delicaflavone may represent a potential therapeutic agent for lung cancer. Delicaflavone showed anti-lung cancer effects in vitro and in vivo. Delicaflavone induced autophagic cell death via Akt/mTOR/p70S6K signaling pathway. Delicaflavone did not show observable side effects in a xenograft mouse model. Delicaflavone may represent a potential therapeutic agent for lung cancer.

p53-dependent inhibition of TrxR1 contributes to the tumor-specific induction of apoptosis by RITA.

PubMed

Hedström, Elisabeth; Eriksson, Sofi; Zawacka-Pankau, Joanna; Arnér, Elias S J; Selivanova, Galina

2009-11-01

Thioredoxin reductase 1 (TrxR1) is a key regulator in many redox-dependent cellular pathways, and is often overexpressed in cancer. Several studies have identified TrxR1 as a potentially important target for anticancer therapy. The low molecular weight compound RITA (NSC 652287) binds p53 and induces p53-dependent apoptosis. Here we found that RITA also targets TrxR1 by non-covalent binding, followed by inhibition of its activity in vitro and by inhibition of TrxR activity in cancer cells. Interestingly, a novel approximately 130 kDa form of TrxR1, presumably representing a stable covalently linked dimer, and an increased generation of reactive oxygen species (ROS) were induced by RITA in cancer cells in a p53-dependent manner. Similarly, the gold-based TrxR inhibitor auranofin induced apoptosis related to oxidative stress, but independently of p53 and without apparent induction of the approximately 130 kDa form of TrxR1. In contrast to the effects observed in cancer cells, RITA did not inhibit TrxR or ROS formation in normal fibroblasts (NHDF). The inhibition of TrxR1 can sensitize tumor cells to agents that induce oxidative stress and may directly trigger cell death. Thus, our results suggest that a unique p53-dependent effect of RITA on TrxR1 in cancer cells might synergize with p53-dependent induction of pro-apoptotic genes and oxidative stress, thereby leading to a robust induction of cancer cell death, without affecting non-transformed cells.

Inhibition of Autophagy Rescues Palmitic Acid-induced Necroptosis of Endothelial Cells*

PubMed Central

Khan, Muhammad Jadoon; Rizwan Alam, Muhammad; Waldeck-Weiermair, Markus; Karsten, Felix; Groschner, Lukas; Riederer, Monika; Hallström, Seth; Rockenfeller, Patrick; Konya, Viktoria; Heinemann, Akos; Madeo, Frank; Graier, Wolfgang F.; Malli, Roland

2012-01-01

Accumulation of palmitic acid (PA) in cells from nonadipose tissues is known to induce lipotoxicity resulting in cellular dysfunction and death. The exact molecular pathways of PA-induced cell death are still mysterious. Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death. The PA-induced cell death was predominantly necrotic as indicated by annexin V and propidium iodide (PI) staining, absence of caspase activity, low levels of DNA hypoploidy, and an early ATP depletion. In addition PA induced a strong elevation of mRNA levels of ubiquitin carboxyl-terminal hydrolase (CYLD), a known mediator of necroptosis. Moreover, siRNA-mediated knockdown of CYLD significantly antagonized PA-induced necrosis of endothelial cells. In contrast, inhibition and knockdown of receptor interacting protein kinase 1 (RIPK1) had no effect on PA-induced necrosis, indicating the induction of a CYLD-dependent but RIPK1-independent cell death pathway. PA was recognized as a strong and early inducer of autophagy. The inhibition of autophagy by both pharmacological inhibitors and genetic knockdown of the autophagy-specific genes, vacuolar protein sorting 34 (VPS34), and autophagy-related protein 7 (ATG7), could rescue the PA-induced death of endothelial cells. Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca2+ triggers the fatal signaling of PA. In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA. PMID:22556413

Rapid Detection of an ABT-737-Sensitive Primed for Death State in Cells Using Microplate-Based Respirometry

PubMed Central

Clerc, Pascaline; Carey, Gregory B.; Mehrabian, Zara; Wei, Michael; Hwang, Hyehyun; Girnun, Geoffrey D.; Chen, Hegang; Martin, Stuart S.; Polster, Brian M.

2012-01-01

Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed “primed for death” and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1) stable BCL-2 overexpression alone can induce a primed for death state and 2) that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O2 consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O2 consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1) or resistance (WEHI-231) to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in individual cancers by “bioenergetics-based profiling” may eventually help identify the subset of patients with chemoresistant but primed tumors who can benefit from treatment that incorporates a BCL-2 antagonist. PMID:22880001

Depletion of pro-oncogenic RUNX2 enhances gemcitabine (GEM) sensitivity of p53-mutated pancreatic cancer Panc-1 cells through the induction of pro-apoptotic TAp63.

PubMed

Ozaki, Toshinori; Nakamura, Mizuyo; Ogata, Takehiro; Sang, Meijie; Yoda, Hiroyuki; Hiraoka, Kiriko; Sang, Meixiang; Shimozato, Osamu

2016-11-01

Recently, we have described that siRNA-mediated silencing of runt-related transcription factor 2 (RUNX2) improves anti-cancer drug gemcitabine (GEM) sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells through the augmentation of p53 family TAp63-dependent cell death pathway. In this manuscript, we have extended our study to p53-mutated human pancreatic cancer Panc-1 cells. According to our present results, knockdown of mutant p53 alone had a marginal effect on GEM-mediated cell death of Panc-1 cells. We then sought to deplete RUNX2 using siRNA in Panc-1 cells and examined its effect on GEM sensitivity. Under our experimental conditions, RUNX2 knockdown caused a significant enhancement of GEM sensitivity of Panc-1 cells. Notably, GEM-mediated induction of TAp63 but not of TAp73 was further stimulated in RUNX2-depleted Panc-1 cells, indicating that, like AsPC-1 cells, TAp63 might play a pivotal role in the regulation of GEM sensitivity of Panc-1 cells. Consistent with this notion, forced expression of TAp63α in Panc-1 cells promoted cell cycle arrest and/or cell death, and massively increased luciferase activities driven by TAp63-target gene promoters such as p21WAF1 and NOXA. In addition, immunoprecipitation experiments indicated that RUNX2 forms a complex with TAp63 in Panc-1 cells. Taken together, our current observations strongly suggest that depletion of RUNX2 enhances the cytotoxic effect of GEM on p53-mutated Panc-1 cells through the stimulation of TAp63-dependent cell death pathway even in the presence of a large amount of pro-oncogenic mutant p53, and might provide an attractive strategy to treat pancreatic cancer patients with p53 mutations.

Depletion of pro-oncogenic RUNX2 enhances gemcitabine (GEM) sensitivity of p53-mutated pancreatic cancer Panc-1 cells through the induction of pro-apoptotic TAp63

PubMed Central

Ozaki, Toshinori; Nakamura, Mizuyo; Ogata, Takehiro; Sang, Meijie; Yoda, Hiroyuki; Hiraoka, Kiriko; Sang, Meixiang; Shimozato, Osamu

2016-01-01

Recently, we have described that siRNA-mediated silencing of runt-related transcription factor 2 (RUNX2) improves anti-cancer drug gemcitabine (GEM) sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells through the augmentation of p53 family TAp63-dependent cell death pathway. In this manuscript, we have extended our study to p53-mutated human pancreatic cancer Panc-1 cells. According to our present results, knockdown of mutant p53 alone had a marginal effect on GEM-mediated cell death of Panc-1 cells. We then sought to deplete RUNX2 using siRNA in Panc-1 cells and examined its effect on GEM sensitivity. Under our experimental conditions, RUNX2 knockdown caused a significant enhancement of GEM sensitivity of Panc-1 cells. Notably, GEM-mediated induction of TAp63 but not of TAp73 was further stimulated in RUNX2-depleted Panc-1 cells, indicating that, like AsPC-1 cells, TAp63 might play a pivotal role in the regulation of GEM sensitivity of Panc-1 cells. Consistent with this notion, forced expression of TAp63α in Panc-1 cells promoted cell cycle arrest and/or cell death, and massively increased luciferase activities driven by TAp63-target gene promoters such as p21WAF1 and NOXA. In addition, immunoprecipitation experiments indicated that RUNX2 forms a complex with TAp63 in Panc-1 cells. Taken together, our current observations strongly suggest that depletion of RUNX2 enhances the cytotoxic effect of GEM on p53-mutated Panc-1 cells through the stimulation of TAp63-dependent cell death pathway even in the presence of a large amount of pro-oncogenic mutant p53, and might provide an attractive strategy to treat pancreatic cancer patients with p53 mutations. PMID:27713122

The glutathione redox system is essential to prevent ferroptosis caused by impaired lipid metabolism in clear cell renal cell carcinoma.

PubMed

Miess, Heike; Dankworth, Beatrice; Gouw, Arvin M; Rosenfeldt, Mathias; Schmitz, Werner; Jiang, Ming; Saunders, Becky; Howell, Michael; Downward, Julian; Felsher, Dean W; Peck, Barrie; Schulze, Almut

2018-06-05

Metabolic reprogramming is a prominent feature of clear cell renal cell carcinoma (ccRCC). Here we investigated metabolic dependencies in a panel of ccRCC cell lines using nutrient depletion, functional RNAi screening and inhibitor treatment. We found that ccRCC cells are highly sensitive to the depletion of glutamine or cystine, two amino acids required for glutathione (GSH) synthesis. Moreover, silencing of enzymes of the GSH biosynthesis pathway or glutathione peroxidases, which depend on GSH for the removal of cellular hydroperoxides, selectively reduced viability of ccRCC cells but did not affect the growth of non-malignant renal epithelial cells. Inhibition of GSH synthesis triggered ferroptosis, an iron-dependent form of cell death associated with enhanced lipid peroxidation. VHL is a major tumour suppressor in ccRCC and loss of VHL leads to stabilisation of hypoxia inducible factors HIF-1α and HIF-2α. Restoration of functional VHL via exogenous expression of pVHL reverted ccRCC cells to an oxidative metabolism and rendered them insensitive to the induction of ferroptosis. VHL reconstituted cells also exhibited reduced lipid storage and higher expression of genes associated with oxidiative phosphorylation and fatty acid metabolism. Importantly, inhibition of β-oxidation or mitochondrial ATP-synthesis restored ferroptosis sensitivity in VHL reconstituted cells. We also found that inhibition of GSH synthesis blocked tumour growth in a MYC-dependent mouse model of renal cancer. Together, our data suggest that reduced fatty acid metabolism due to inhibition of β-oxidation renders renal cancer cells highly dependent on the GSH/GPX pathway to prevent lipid peroxidation and ferroptotic cell death.

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

Berberine induces FasL-related apoptosis through p38 activation in KB human oral cancer cells

PubMed Central

KIM, JAE-SUNG; OH, DAHYE; YIM, MIN-JI; PARK, JIN-JU; KANG, KYEONG-ROK; CHO, IN-A; MOON, SUNG-MIN; OH, JI-SU; YOU, JAE-SEEK; KIM, CHUN SUNG; KIM, DO KYUNG; LEE, SOOK-YOUNG; LEE, GYEONG-JE; IM, HEE-JEONG; KIM, SU-GWAN

2015-01-01

In the present study, we examined the anticancer properties of berberine in KB oral cancer cells with a specific focus on its cellular mechanism. Berberine did not affect the cell viability of the primary human normal oral keratinocytes that were used as a control. However, the viability of KB cells was found to decrease significantly in the presence of berberine in a dose-dependent manner. Furthermore, in KB cells, berberine induced the fragmentation of genomic DNA, changes in cell morphology, and nuclear condensation. In addition, caspase-3 and -7 activation, and an increase in apoptosis were observed. Berberine was also found to upregulate significantly the expression of the death receptor ligand, FasL. In turn, this upregulation triggered the activation of pro-apoptotic factors such as caspase-8, -9 and -3 and poly(ADP-ribose) polymerase (PARP). Furthermore, pro-apoptotic factors such as Bax, Bad and Apaf-1 were also significantly upregulated by berberine. Anti-apoptotic factors such as Bcl-2 and Bcl-xL were downregulated. Z-VAD-FMK, a cell-permeable pan-caspase inhibitor, suppressed the activation of caspase-3 and PARP. These results clearly indicate that berberine-induced cell death of KB oral cancer cells was mediated by both extrinsic death receptor-dependent and intrinsic mitochondrial-dependent apoptotic signaling pathways. In addition, berberine-induced upregulation of FasL was shown to be mediated by the p38 MAPK signaling pathway. We also found that berberine-induced migration suppression was mediated by downregulation of MMP-2 and MMP-9 through phosphorylation of p38 MAPK. In summary, berberine has the potential to be used as a chemotherapeutic agent, with limited side-effects, for the management of oral cancer. PMID:25634589

Time-course of programmed cell death during leaf senescence in Eucommia ulmoides.

PubMed

Cao, Jing; Jiang, Feng; Sodmergen; Cui, Keming

2003-02-01

Leaves of Eucommia ulmoidesOliv. harvested between April to November were examined for programmed cell death (PCD) during growth and senescence. Leaves developed in April, becoming fully expanded in late May, remaining unchanged until November when they started to dehisce. Falling leaves retained a green color. Our results showed that (1) mesophyll cells gradually reduced their nuclei from September to November, (2) positive TUNEL signals appeared on the nuclei from August, (3) ladder-like DNA fragmentation occurred in September and October, and (4) a 20-kDa Ca(2+)-dependent DNase appeared in these same months. In fallen leaves, intact mesophyll cell nuclei could not be detected, but a few cells around the vascular bundle had nuclei. Therefore, (1) programmed cell death (PCD) of leaf cells occurred in the leaves of E. ulmoides, (2) the progress of mesophyll cell PCD lasted for more than 2 months, and (3) PCD of leaf cells was asynchronous in natural senescing leaves.

In vivo analysis of insulin-like growth factor type 1 receptor humanized monoclonal antibody MK-0646 and small molecule kinase inhibitor OSI-906 in colorectal cancer.

PubMed

Leiphrakpam, Premila D; Agarwal, Ekta; Mathiesen, Michelle; Haferbier, Katie L; Brattain, Michael G; Chowdhury, Sanjib

2014-01-01

The development and characterization of effective anticancer drugs against colorectal cancer (CRC) is of urgent need since it is the second most common cause of cancer death. The study was designed to evaluate the effects of two IGF-1R antagonists, MK-0646, a recombinant fully humanized monoclonal antibody and OSI-906, a small molecule tyrosine kinase inhibitor on CRC cells. Xenograft study was performed on IGF-1R-dependent CRC cell lines for analyzing the antitumor activity of MK-0646 and OSI-906. Tumor proliferation and apoptosis were assessed using Ki67 and TUNEL assays, respectively. We also performed in vitro characterization of MK-0646 and OSI-906 treatment on CRC cells to identify mechanisms associated with drug-induced cell death. Exposure of the GEO and CBS tumor xenografts to MK-0646 or OSI-906 led to a decrease in tumor growth. TUNEL analysis showed an increase of approximately 45-55% in apoptotic cells in both MK-0646 and OSI-906 treated tumor samples. We report the novel finding that treatment with IGF-1R antagonists led to downregulation of X-linked inhibitor of apoptosis (XIAP) protein involved in cell survival and inhibition of cell death. In conclusion, IGF-1R antagonists (MK-0646 and OSI-906) demonstrated single agent inhibition of subcutaneous CRC xenograft growth. This was coupled to pro-apoptotic effects resulting in downregulation of XIAP and inhibition of cell survival. We report a novel mechanism by which MK-0646 and OSI-906 elicits cell death in vivo and in vitro. Moreover, these results indicate that MK-0646 and OSI-906 may be potential anticancer candidates for the treatment of patients with IGF-1R-dependent CRC.

In vivo analysis of insulin-like growth factor type 1 receptor humanized monoclonal antibody MK-0646 and small molecule kinase inhibitor OSI-906 in colorectal cancer

PubMed Central

LEIPHRAKPAM, PREMILA D.; AGARWAL, EKTA; MATHIESEN, MICHELLE; HAFERBIER, KATIE L.; BRATTAIN, MICHAEL G.; CHOWDHURY, SANJIB

2014-01-01

The development and characterization of effective anticancer drugs against colorectal cancer (CRC) is of urgent need since it is the second most common cause of cancer death. The study was designed to evaluate the effects of two IGF-1R antagonists, MK-0646, a recombinant fully humanized monoclonal antibody and OSI-906, a small molecule tyrosine kinase inhibitor on CRC cells. Xenograft study was performed on IGF-1R-dependent CRC cell lines for analyzing the antitumor activity of MK-0646 and OSI-906. Tumor proliferation and apoptosis were assessed using Ki67 and TUNEL assays, respectively. We also performed in vitro characterization of MK-0646 and OSI-906 treatment on CRC cells to identify mechanisms associated with drug-induced cell death. Exposure of the GEO and CBS tumor xenografts to MK-0646 or OSI-906 led to a decrease in tumor growth. TUNEL analysis showed an increase of approximately 45–55% in apoptotic cells in both MK-0646 and OSI-906 treated tumor samples. We report the novel finding that treatment with IGF-1R antagonists led to downregulation of X-linked inhibitor of apoptosis (XIAP) protein involved in cell survival and inhibition of cell death. In conclusion, IGF-1R antagonists (MK-0646 and OSI-906) demonstrated single agent inhibition of subcutaneous CRC xenograft growth. This was coupled to pro-apoptotic effects resulting in downregulation of XIAP and inhibition of cell survival. We report a novel mechanism by which MK-0646 and OSI-906 elicits cell death in vivo and in vitro. Moreover, these results indicate that MK-0646 and OSI-906 may be potential anticancer candidates for the treatment of patients with IGF-1R-dependent CRC. PMID:24173770

Nitric Oxide and Protein S-Nitrosylation Are Integral to Hydrogen Peroxide-Induced Leaf Cell Death in Rice1[W][OA

PubMed Central

Lin, Aihong; Wang, Yiqin; Tang, Jiuyou; Xue, Peng; Li, Chunlai; Liu, Linchuan; Hu, Bin; Yang, Fuquan; Loake, Gary J.; Chu, Chengcai

2012-01-01

Nitric oxide (NO) is a key redox-active, small molecule involved in various aspects of plant growth and development. Here, we report the identification of an NO accumulation mutant, nitric oxide excess1 (noe1), in rice (Oryza sativa), the isolation of the corresponding gene, and the analysis of its role in NO-mediated leaf cell death. Map-based cloning revealed that NOE1 encoded a rice catalase, OsCATC. Furthermore, noe1 resulted in an increase of hydrogen peroxide (H2O2) in the leaves, which consequently promoted NO production via the activation of nitrate reductase. The removal of excess NO reduced cell death in both leaves and suspension cultures derived from noe1 plants, implicating NO as an important endogenous mediator of H2O2-induced leaf cell death. Reduction of intracellular S-nitrosothiol (SNO) levels, generated by overexpression of rice S-nitrosoglutathione reductase gene (GSNOR1), which regulates global levels of protein S-nitrosylation, alleviated leaf cell death in noe1 plants. Thus, S-nitrosylation was also involved in light-dependent leaf cell death in noe1. Utilizing the biotin-switch assay, nanoliquid chromatography, and tandem mass spectrometry, S-nitrosylated proteins were identified in both wild-type and noe1 plants. NO targets identified only in noe1 plants included glyceraldehyde 3-phosphate dehydrogenase and thioredoxin, which have been reported to be involved in S-nitrosylation-regulated cell death in animals. Collectively, our data suggest that both NO and SNOs are important mediators in the process of H2O2-induced leaf cell death in rice. PMID:22106097

Zinc promotes the death of hypoxic astrocytes by upregulating hypoxia-induced hypoxiainducible factor-1alpha expression via Poly(ADP-ribose) polymerase -1

PubMed Central

Pan, Rong; Chen, Chen; Liu, Wenlan; Liu, Ke Jian

2013-01-01

Aim Pathological release of excess zinc ions has been implicated in ischemic brain cell death. However, the underlying mechanisms remain to be elucidated. In stroke, ischemia-induced zinc release and hypoxia-inducible factor-1 (HIF-1) accumulation concurrently occur in the ischemic tissue. The present study testes the hypothesis that the presence of high intracellular zinc concentration is a major cause of modifications to PARP-1 and HIF-1α during hypoxia, which significantly contributes to cell death during ischemia. Methods Primary cortical astrocytes and C8-D1A cells were exposed to different concentrations of zinc chloride. Cell death rate and protein expression of HIF-1 and Poly(ADP-ribose) polymerase (PARP)-1 were examined after 3-hour hypoxic treatment. Results Although 3-hr hypoxia or 100 μM of zinc alone did not induce noticeable cytotoxicity, their combination led to a dramatic increase in astrocytic cell death in a zinc concentration dependent manner. Exposure of astrocytes to hypoxia for 3-hr remarkably increased the levels of intracellular zinc and HIF-1α protein, which was further augmented by added exogenous zinc. Notably HIF-1α knockdown blocked zinc-induced astrocyte death. Moreover, knockdown of PARP-1, another important protein in the response of hypoxia, attenuated the overexpression of HIF-1α and reduced the cell death rate. Conclusions Our studies show that zinc promotes hypoxic cell death through overexpression of the hypoxia response factor HIF-1α via the cell fate determine factor PARP-1 modification, which provides a novel mechanism for zinc-mediated ischemic brain injury. PMID:23582235

Cytotoxic Mechanisms Employed by Mouse T Cells to Destroy Pancreatic β-Cells

PubMed Central

Varanasi, Vineeth; Avanesyan, Lia; Schumann, Desiree M.; Chervonsky, Alexander V.

2012-01-01

Several cytotoxic mechanisms have been attributed to T cells participating in β-cell death in type 1 diabetes. However, sensitivity of β-cells to these mechanisms in vitro and in vivo is likely to be different. Moreover, CD4+ and CD8+ T cells may use distinct mechanisms to cause β-cell demise that possibly involve activation of third-party cytotoxic cells. We used the transfer of genetically modified diabetogenic T cells into normal, mutant, and bone marrow chimeric recipients to test the contribution of major cytotoxic mechanisms in β-cell death. We found that 1) the killing of β-cells by CD4+ T cells required activation of the recipient’s own cytotoxic cells via tumor necrosis factor-α (TNF-α); 2) CD8+ T-cell cytotoxic mechanisms destroying β-cells were limited to perforin and Fas ligand, as double knockouts of these molecules abrogated the ability of T cells to cause diabetes; and 3) individual CD8+ T-cell clones chose their cytotoxic weaponry by a yet unknown mechanism and destroyed their targets via either Fas-independent or Fas-dependent (∼40% of clones) pathways. Fas-dependent destruction was assisted by TNF-α. PMID:22773667

Suppressed translation as a mechanism of initiation of CASP8 (caspase 8)-dependent apoptosis in autophagy-deficient NSCLC cells under nutrient limitation.

PubMed

Allavena, Giulia; Cuomo, Francesca; Baumgartner, Georg; Bele, Tadeja; Sellgren, Alexander Yarar; Oo, Kyaw Soe; Johnson, Kaylee; Gogvadze, Vladimir; Zhivotovsky, Boris; Kaminskyy, Vitaliy O

2018-01-01

Macroautophagy/autophagy inhibition under stress conditions is often associated with increased cell death. We found that under nutrient limitation, activation of CASP8/caspase-8 was significantly increased in autophagy-deficient lung cancer cells, which precedes mitochondria outer membrane permeabilization (MOMP), CYCS/cytochrome c release, and activation of CASP9/caspase-9, indicating that under such conditions the activation of CASP8 is a primary event in the initiation of apoptosis as well as essential to reduce clonogenic survival of autophagy-deficient cells. Starvation leads to suppression of CFLAR proteosynthesis and accumulation of CASP8 in SQSTM1 puncta. Overexpression of CFLARs reduces CASP8 activation and apoptosis during starvation, while its silencing promotes efficient activation of CASP8 and apoptosis in autophagy-deficient U1810 lung cancer cells even under nutrient-rich conditions. Similar to starvation, inhibition of protein translation leads to efficient activation of CASP8 and cell death in autophagy-deficient lung cancer cells. Thus, here for the first time we report that suppressed translation leads to activation of CASP8-dependent apoptosis in autophagy-deficient NSCLC cells under conditions of nutrient limitation. Our data suggest that targeting translational machinery can be beneficial for elimination of autophagy-deficient cells via the CASP8-dependent apoptotic pathway.

S-Adenosyl-L-methionine protects the probiotic yeast, Saccharomyces boulardii, from acid-induced cell death.

PubMed

Cascio, Vincent; Gittings, Daniel; Merloni, Kristen; Hurton, Matthew; Laprade, David; Austriaco, Nicanor

2013-02-13

Saccharomyces boulardii is a probiotic yeast routinely used to prevent and to treat gastrointestinal disorders, including the antibiotic-associated diarrhea caused by Clostridium difficile infections. However, only 1-3% of the yeast administered orally is recovered alive in the feces suggesting that this yeast is unable to survive the acidic environment of the gastrointestinal tract. We provide evidence that suggests that S. boulardii undergoes programmed cell death (PCD) in acidic environments, which is accompanied by the generation of reactive oxygen species and the appearance of caspase-like activity. To better understand the mechanism of cell death at the molecular level, we generated microarray gene expression profiles of S. boulardii cells cultured in an acidic environment. Significantly, functional annotation revealed that the up-regulated genes were significantly over-represented in cell death pathways Finally, we show that S-adenosyl-L-methionine (AdoMet), a commercially available, FDA-approved dietary supplement, enhances the viability of S. boulardii in acidic environments, most likely by preventing programmed cell death. In toto, given the observation that many of the proven health benefits of S. boulardii are dependent on cell viability, our data suggests that taking S. boulardii and AdoMet together may be a more effective treatment for gastrointestinal disorders than taking the probiotic yeast alone.

S-Adenosyl-L-Methionine protects the probiotic yeast, Saccharomyces boulardii, from acid-induced cell death

PubMed Central

2013-01-01

Background Saccharomyces boulardii is a probiotic yeast routinely used to prevent and to treat gastrointestinal disorders, including the antibiotic-associated diarrhea caused by Clostridium difficile infections. However, only 1-3% of the yeast administered orally is recovered alive in the feces suggesting that this yeast is unable to survive the acidic environment of the gastrointestinal tract. Results We provide evidence that suggests that S. boulardii undergoes programmed cell death (PCD) in acidic environments, which is accompanied by the generation of reactive oxygen species and the appearance of caspase-like activity. To better understand the mechanism of cell death at the molecular level, we generated microarray gene expression profiles of S. boulardii cells cultured in an acidic environment. Significantly, functional annotation revealed that the up-regulated genes were significantly over-represented in cell death pathways Finally, we show that S-adenosyl-L-methionine (AdoMet), a commercially available, FDA-approved dietary supplement, enhances the viability of S. boulardii in acidic environments, most likely by preventing programmed cell death. Conclusions In toto, given the observation that many of the proven health benefits of S. boulardii are dependent on cell viability, our data suggests that taking S. boulardii and AdoMet together may be a more effective treatment for gastrointestinal disorders than taking the probiotic yeast alone. PMID:23402325

Application of hyperthermia in addition to ionizing irradiation fosters necrotic cell death and HMGB1 release of colorectal tumor cells

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

Schildkopf, Petra, E-mail: petra.schildkopf@uk-erlangen.de; Frey, Benjamin, E-mail: benjamin.frey@uk-erlangen.de; Mantel, Frederick, E-mail: frederick.mantel@web.de

2010-01-01

Colorectal cancer is the second leading cause of death in developed countries. Tumor therapies should on the one hand aim to stop the proliferation of tumor cells and to kill them, and on the other hand stimulate a specific immune response against residual cancer cells. Dying cells are modulators of the immune system contributing to anti-inflammatory or pro-inflammatory responses, depending on the respective cell death form. The positive therapeutic effects of temperature-controlled hyperthermia (HT), when combined with ionizing irradiation (X-ray), were the origin to examine whether combinations of X-ray with HT can induce immune activating tumor cell death forms, alsomore » characterized by the release of the danger signal HMGB1. Human colorectal tumor cells with differing radiosensitivities were treated with combinations of HT (41.5 {sup o}C for 1 h) and X-ray (5 or 10 Gy). Necrotic cell death was prominent after X-ray and could be further increased by HT. Apoptosis remained quite low in HCT 15 and SW480 cells. X-ray and combinations with HT arrested the tumor cells in the radiosensitive G2 cell cycle phase. The amount of released HMGB1 protein was significantly enhanced after combinatorial treatments in comparison to single ones. We conclude that combining X-ray with HT may induce anti-tumor immunity as a result of the predominant induction of inflammatory necrotic tumor cells and the release of HMGB1.« less

Interferon-induced TRAIL-independent cell death in DNase II-/- embryos.

PubMed

Kitahara, Yusuke; Kawane, Kohki; Nagata, Shigekazu

2010-09-01

The chromosomal DNA of apoptotic cells and the nuclear DNA expelled from erythroid precursors is cleaved by DNase II in lysosomes after the cells or nuclei are engulfed by macrophages. DNase II(-/-) embryos suffer from lethal anemia due to IFN-beta produced in the macrophages carrying undigested DNA. Here, we show that Type I IFN induced a caspase-dependent cell death in human epithelial cells that were transformed to express a high level of IFN type I receptor. During this death process, a set of genes was strongly activated, one of which encoded TRAIL, a death ligand. A high level of TRAIL mRNA was also found in the fetal liver of the lethally anemic DNase II(-/-) embryos, and a lack of IFN type I receptor in the DNase II(-/-) IFN-IR(-/-) embryos blocked the expression of TRAIL mRNA. However, a null mutation in TRAIL did not rescue the lethal anemia of the DNase II(-/-) embryos, indicating that TRAIL is dispensable for inducing the apoptosis of erythroid cells in DNase II(-/-) embryos, and therefore, that there is a TRAIL-independent mechanism for the IFN-induced apoptosis.

Protective effects of l-carnitine and piracetam against mitochondrial permeability transition and PC3 cell necrosis induced by simvastatin.

PubMed

Costa, Rute A P; Fernandes, Mariana P; de Souza-Pinto, Nadja C; Vercesi, Aníbal E

2013-02-15

Mitochondrial oxidative stress followed by membrane permeability transition (MPT) has been considered as a possible mechanism for statins cytotoxicity. Statins use has been associated with reduced risk of cancer incidence, especially prostate cancer. Here we investigated the pathways leading to simvastatin-induced prostate cancer cell death as well as the mechanisms of cell death protection by l-carnitine or piracetam. These compounds are known to prevent and/or protect against cell death mediated by oxidative mitochondrial damage induced by a variety of conditions, either in vivo or in vitro. The results provide evidence that simvastatin induced MPT and cell necrosis were sensitive to either l-carnitine or piracetam in a dose-dependent fashion and mediated by additive mechanisms. When combined, l-carnitine and piracetam acted at concentrations significantly lower than they act individually. These results shed new light into both the cytotoxic mechanisms of statins and the mechanisms underlying the protection against MPT and cell death by the compounds l-carnitine and piracetam. Copyright © 2013 Elsevier B.V. All rights reserved.

Kinetic theory of age-structured stochastic birth-death processes

NASA Astrophysics Data System (ADS)

Greenman, Chris D.; Chou, Tom

2016-01-01

Classical age-structured mass-action models such as the McKendrick-von Foerster equation have been extensively studied but are unable to describe stochastic fluctuations or population-size-dependent birth and death rates. Stochastic theories that treat semi-Markov age-dependent processes using, e.g., the Bellman-Harris equation do not resolve a population's age structure and are unable to quantify population-size dependencies. Conversely, current theories that include size-dependent population dynamics (e.g., mathematical models that include carrying capacity such as the logistic equation) cannot be easily extended to take into account age-dependent birth and death rates. In this paper, we present a systematic derivation of a new, fully stochastic kinetic theory for interacting age-structured populations. By defining multiparticle probability density functions, we derive a hierarchy of kinetic equations for the stochastic evolution of an aging population undergoing birth and death. We show that the fully stochastic age-dependent birth-death process precludes factorization of the corresponding probability densities, which then must be solved by using a Bogoliubov--Born--Green--Kirkwood--Yvon-like hierarchy. Explicit solutions are derived in three limits: no birth, no death, and steady state. These are then compared with their corresponding mean-field results. Our results generalize both deterministic models and existing master equation approaches by providing an intuitive and efficient way to simultaneously model age- and population-dependent stochastic dynamics applicable to the study of demography, stem cell dynamics, and disease evolution.

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

Silencing of cytosolic NADP(+)-dependent isocitrate dehydrogenase by small interfering RNA enhances the sensitivity of HeLa cells toward staurosporine.

PubMed

Lee, Su-Min; Park, Sin Young; Shin, Seoung Woo; Kil, In Sup; Yang, Eun Sun; Park, Jeen-Woo

2009-02-01

Staurosporine induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, it was demonstrated that the control of cellular redox balance and the defense against oxidative damage is one of the primary functions of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) by supplying NADPH for antioxidant systems. The present report shows that silencing of IDPc expression in HeLa cells greatly enhances apoptosis induced by staurosporine. Transfection of HeLa cells with an IDPc small interfering RNA (siRNA) markedly decreased activity of IDPc, enhancing the susceptibility of staurosporine-induced apoptosis reflected by DNA fragmentation, cellular redox status and the modulation of apoptotic marker proteins. These results indicate that IDPc may play an important role in regulating the apoptosis induced by staurosporine and the sensitizing effect of IDPc siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.

Oxidative stress alters mitochondrial bioenergetics and modifies pancreatic cell death independently of cyclophilin D, resulting in an apoptosis-to-necrosis shift

PubMed Central

Armstrong, Jane A.; Cash, Nicole J.; Ouyang, Yulin; Morton, Jack C.; Chvanov, Michael; Latawiec, Diane; Awais, Muhammad; Tepikin, Alexei V.; Sutton, Robert; Criddle, David N.

2018-01-01

Mitochondrial dysfunction lies at the core of acute pancreatitis (AP). Diverse AP stimuli induce Ca2+-dependent formation of the mitochondrial permeability transition pore (MPTP), a solute channel modulated by cyclophilin D (CypD), the formation of which causes ATP depletion and necrosis. Oxidative stress reportedly triggers MPTP formation and is elevated in clinical AP, but how reactive oxygen species influence cell death is unclear. Here, we assessed potential MPTP involvement in oxidant-induced effects on pancreatic acinar cell bioenergetics and fate. H2O2 application promoted acinar cell apoptosis at low concentrations (1–10 μm), whereas higher levels (0.5–1 mm) elicited rapid necrosis. H2O2 also decreased the mitochondrial NADH/FAD+ redox ratio and ΔΨm in a concentration-dependent manner (10 μm to 1 mm H2O2), with maximal effects at 500 μm H2O2. H2O2 decreased the basal O2 consumption rate of acinar cells, with no alteration of ATP turnover at <50 μm H2O2. However, higher H2O2 levels (≥50 μm) diminished spare respiratory capacity and ATP turnover, and bioenergetic collapse, ATP depletion, and cell death ensued. Menadione exerted detrimental bioenergetic effects similar to those of H2O2, which were inhibited by the antioxidant N-acetylcysteine. Oxidant-induced bioenergetic changes, loss of ΔΨm, and cell death were not ameliorated by genetic deletion of CypD or by its acute inhibition with cyclosporine A. These results indicate that oxidative stress alters mitochondrial bioenergetics and modifies pancreatic acinar cell death. A shift from apoptosis to necrosis appears to be associated with decreased mitochondrial spare respiratory capacity and ATP production, effects that are independent of CypD-sensitive MPTP formation. PMID:29626097

Anethole induces apoptotic cell death accompanied by reactive oxygen species production and DNA fragmentation in Aspergillus fumigatus and Saccharomyces cerevisiae.

PubMed

Fujita, Ken-Ichi; Tatsumi, Miki; Ogita, Akira; Kubo, Isao; Tanaka, Toshio

2014-02-01

trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum, and antimicrobial activity that is weaker than that of other antibiotics on the market. When combined with polygodial, nagilactone E, and n-dodecanol, anethole has been shown to possess significant synergistic antifungal activity against a budding yeast, Saccharomyces cerevisiae, and a human opportunistic pathogenic yeast, Candida albicans. However, the antifungal mechanism of anethole has not been completely determined. We found that anethole stimulated cell death of a human opportunistic pathogenic fungus, Aspergillus fumigatus, in addition to S. cerevisiae. The anethole-induced cell death was accompanied by reactive oxygen species production, metacaspase activation, and DNA fragmentation. Several mutants of S. cerevisiae, in which genes related to the apoptosis-initiating execution signals from mitochondria were deleted, were resistant to anethole. These results suggest that anethole-induced cell death could be explained by oxidative stress-dependent apoptosis via typical mitochondrial death cascades in fungi, including A. fumigatus and S. cerevisiae. © 2014 FEBS.

Trimethyltin-induced apoptosis is associated with upregulation of inducible nitric oxide synthase and Bax in a hippocampal cell line

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

Zhang, L.; Li, L.; Prabhakaran, K.

2006-10-01

Trimethyltin (TMT) produces selective neuronal degeneration in the central nervous system (CNS), in which the hippocampus is the most sensitive area. Since previous studies have been conducted in either non-neural cells or mixed primary cultures, an immortalized hippocampal neuronal cell line (HT-22 cell) was used to assess the mechanism and mode of death produced by TMT. The compound produced a time- and concentration-dependent apoptotic death that was caspase-mediated. Excessive generation of reactive oxygen species (ROS) and subsequent reduction of mitochondrial membrane potential ({delta}{psi}{sub m}) were involved in the cytotoxicity{sub .} Scavenging of ROS by a free radical trapping agent ormore » inhibition of the mitochondrial permeability transition (MPT) pore significantly reduced cell death. Additionally, TMT increased expression of inducible nitric oxide synthase (iNOS) by activation of the redox-sensitive transcription factor NF{kappa}B. Pharmacologic inhibition studies showed that the iNOS-mediated NO generation increased expression of Bax and then mitochondrial-mediated apoptosis. It was concluded that excessive ROS generation initiated the apoptotic cell death by upregulating iNOS followed by increased Bax expression which then led to loss of {delta}{psi}{sub m} and caspase-executed cell death. This study is the first to report in a neuronal cell model that TMT stimulates induction of iNOS, which then increases cellular levels of reactive nitrogen species (RNS) to initiate apoptotic death.« less

Hypoxia Enhances the Antiglioma Cytotoxicity of B10, a Glycosylated Derivative of Betulinic Acid

PubMed Central

Thiepold, Anna-Luisa; Harter, Patrick N.; Reichert, Sebastian; Kögel, Donat; Paschke, Reinhard; Mittelbronn, Michel; Weller, Michael; Steinbach, Joachim P.; Fulda, Simone; Bähr, Oliver

2014-01-01

B10 is a glycosylated derivative of betulinic acid with promising activity against glioma cells. Lysosomal cell death pathways appear to be essential for its cytotoxicity. We investigated the influence of hypoxia, nutrient deprivation and current standard therapies on B10 cytotoxicity. The human glioma cell lines LN-308 and LNT-229 were exposed to B10 alone or together with irradiation, temozolomide, nutrient deprivation or hypoxia. Cell growth and viability were evaluated by crystal violet staining, clonogenicity assays, propidium iodide uptake and LDH release assays. Cell death was examined using an inhibitor of lysosomal acidification (bafilomycin A1), a cathepsin inhibitor (CA074-Me) and a short-hairpin RNA targeting cathepsin B. Hypoxia substantially enhanced B10-induced cell death. This effect was sensitive to bafilomycin A1 and thus dependent on hypoxia-induced lysosomal acidification. Cathepsin B appeared to mediate cell death because either the inhibitor CA074-Me or cathepsin B gene silencing rescued glioma cells from B10 toxicity under hypoxia. B10 is a novel antitumor agent with substantially enhanced cytotoxicity under hypoxia conferred by increased lysosomal cell death pathway activation. Given the importance of hypoxia for therapy resistance, malignant progression, and as a result of antiangiogenic therapies, B10 might be a promising strategy for hypoxic tumors like malignant glioma. PMID:24743710

Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

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

Lee, Dae-Hee, E-mail: leedneo@gmail.com; Kim, Dong-Wook; Jung, Chang-Hwa

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

Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis

PubMed Central

Deeraksa, Arpaporn; Pan, Jing; Sha, Youbao; Liu, Xian-De; Eissa, N Tony; Lin, Sue-Hwa; Yu-Lee, Li-yuan

2012-01-01

Castration-resistant prostate cancer (PCa) is refractory to hormone therapy and new strategies for treatment are urgently needed. We found that androgen-insensitive (AI) PCa cells, LNCaP-AI, are reprogrammed to upregulate the mitotic kinase Plk1 and other M phase cell cycle proteins, which may underlie AI PCa growth. In androgen-depleted media, LNCaP-AI cells showed exquisite sensitivity to growth inhibition by subnanomolar concentrations of a small molecule inhibitor of Plk1, BI2536, suggesting that these cells are dependent on Plk1 for growth. In contrast, the androgen-responsive parental LNCaP cells showed negligible responses to BI2536 treatment under the same condition. BI2536 treatment of LNCaP-AI cells resulted in an increase in cell death marker PARP-1 but did not activate caspase-3, an apoptosis marker, suggesting that the observed cell death was caspase-independent. BI2536-treated LNCaP-AI cells formed multinucleated giant cells that contain clusters of nuclear vesicles indicative of mitotic catastrophe. Live-cell time-lapse imaging revealed that BI2536-treated giant LNCaP-AI cells underwent necroptosis, as evidenced by “explosive” cell death and partial reversal of cell death by a necroptosis inhibitor. Our studies suggest that LNCaP-AI cells underwent reprogramming in both their cell growth and cell death pathways, rendering them highly sensitive to Plk1 inhibition that induces necroptosis. Harnessing necroptosis through Plk1 inhibition may be explored for therapeutic intervention of castration-resistant PCa. PMID:22890325

Casticin induced apoptotic cell death and altered associated gene expression in human colon cancer colo 205 cells.

PubMed

Shang, Hung-Sheng; Liu, Jia-You; Lu, Hsu-Feng; Chiang, Han-Sun; Lin, Chia-Hain; Chen, Ann; Lin, Yuh-Feng; Chung, Jing-Gung

2017-08-01

Casticin, a polymethoxyflavone, derived from natural plant Fructus Viticis exhibits biological activities including anti-cancer characteristics. The anti-cancer and alter gene expression of casticin on human colon cancer cells and the underlying mechanisms were investigated. Flow cytometric assay was used to measure viable cell, cell cycle and sub-G1 phase, reactive oxygen species (ROS) and Ca 2+ productions, level of mitochondria membrane potential (ΔΨ m ) and caspase activity. Western blotting assay was used to detect expression of protein level associated with cell death. Casticin induced cell morphological changes, decreased cell viability and induced G2/M phase arrest in colo 205 cells. Casticin increased ROS production but decreased the levels of ΔΨ m , and Ca 2+ , increased caspase-3, -8, and -9 activities. The cDNA microarray indicated that some of the cell cycle associated genes were down-regulated such as cyclin-dependent kinase inhibitor 1A (CDKN1A) (p21, Cip1) and p21 protein (Cdc42/Rac)-activated kinase 3 (PAK3). TNF receptor-associated protein 1 (TRAP1), CREB1 (cAMP responsive element binding protein 1) and cyclin-dependent kinase inhibitor 1B (CDKN1B) (p27, Kip1) genes were increased but matrix metallopeptidase 2 (MMP-2), toll-like receptor 4 (TLR4), PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, bet), and CaMK4 (calcium/calmodulin-dependent protein kinase IV) genes were inhibited. Results suggest that casticin induced cell apoptosis via the activation of the caspase- and/or mitochondria-dependent signaling cascade, the accumulation of ROS and altered associated gene expressions in colo 205 human colon cancer cells. © 2016 Wiley Periodicals, Inc.

Hyperglycemic Conditions Prime Cells for RIP1-dependent Necroptosis*

PubMed Central

LaRocca, Timothy J.; Sosunov, Sergey A.; Shakerley, Nicole L.; Ten, Vadim S.; Ratner, Adam J.

2016-01-01

Necroptosis is a RIP1-dependent programmed cell death (PCD) pathway that is distinct from apoptosis. Downstream effector pathways of necroptosis include formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS), both of which depend on glycolysis. This suggests that increased cellular glucose may prime necroptosis. Here we show that exposure to hyperglycemic levels of glucose enhances necroptosis in primary red blood cells (RBCs), Jurkat T cells, and U937 monocytes. Pharmacologic or siRNA inhibition of RIP1 prevented the enhanced death, confirming it as RIP1-dependent necroptosis. Hyperglycemic enhancement of necroptosis depends upon glycolysis with AGEs and ROS playing a role. Total levels of RIP1, RIP3, and mixed lineage kinase domain-like (MLKL) proteins were increased following treatment with high levels of glucose in Jurkat and U937 cells and was not due to transcriptional regulation. The observed increase in RIP1, RIP3, and MLKL protein levels suggests a potential positive feedback mechanism in nucleated cell types. Enhanced PCD due to hyperglycemia was specific to necroptosis as extrinsic apoptosis was inhibited by exposure to high levels of glucose. Hyperglycemia resulted in increased infarct size in a mouse model of brain hypoxia-ischemia injury. The increased infarct size was prevented by treatment with nec-1s, strongly suggesting that increased necroptosis accounts for exacerbation of this injury in conditions of hyperglycemia. This work reveals that hyperglycemia represents a condition in which cells are extraordinarily susceptible to necroptosis, that local glucose levels alter the balance of PCD pathways, and that clinically relevant outcomes may depend on glucose-mediated effects on PCD. PMID:27129772

Game theory in the death galaxy: interaction of cancer and stromal cells in tumour microenvironment.

PubMed

Wu, Amy; Liao, David; Tlsty, Thea D; Sturm, James C; Austin, Robert H

2014-08-06

Preventing relapse is the major challenge to effective therapy in cancer. Within the tumour, stromal (ST) cells play an important role in cancer progression and the emergence of drug resistance. During cancer treatment, the fitness of cancer cells can be enhanced by ST cells because their molecular signalling interaction delays the drug-induced apoptosis of cancer cells. On the other hand, competition among cancer and ST cells for space or resources should not be ignored. We explore the population dynamics of multiple myeloma (MM) versus bone marrow ST cells by using an experimental microecology that we call the death galaxy, with a stable drug gradient and connected microhabitats. Evolutionary game theory is a quantitative way to capture the frequency-dependent nature of interactive populations. Therefore, we use evolutionary game theory to model the populations in the death galaxy with the gradients of pay-offs and successfully predict the future densities of MM and ST cells. We discuss the possible clinical use of such analysis for predicting cancer progression.

Temporal Pattern and Crosstalk of Necroptosis Markers with Autophagy and Apoptosis Associated Proteins in Ischemic Hippocampus.

PubMed

Ryan, Fari; Khodagholi, Fariba; Dargahi, Leila; Minai-Tehrani, Dariush; Ahmadiani, Abolhassan

2018-01-08

Necroptosis, a novel type of programmed cell death, has been recently implicated as a possible mechanism for cerebral ischemia-reperfusion (I/R) injury. We herein studied time-dependent changes of necroptosis markers along with apoptosis- and autophagy-associated proteins in rat hippocampus at 1, 3, 6, 12, 24, and 48 h after global cerebral I/R injury. Furthermore, to determine the cross talk between autophagy and necroptosis, we examined the effects of pretreatment with bafilomycin-A1 (Baf-A1), as a late-stage autophagy inhibitor, on necroptosis. Highest levels of receptor-interacting protein 1 and 3 (RIP1 and RIP3), as key mediators of necroptosis, were observed at 24 h after reperfusion. Alongside, activity of glutamate dehydrogenase (GLUD1), downstream enzyme of RIP3, was increased. Peak time of necroptosis was subsequent to caspase-3-dependent cell death that peaked at 12 h of reperfusion but concurrent with autophagy. Administration of Baf-A1 could attenuate necroptosis, verified by decrease in RIP1 and RIP3 protein levels, as well as GLUD1 activity. However, there was no significant change in caspase-3-dependent cell death. Taken together, our results highlight that global cerebral I/R activates necroptosis that could be triggered by autophagy and interacts reversely with caspase-3-dependent apoptosis.

Light acclimation, retrograde signalling, cell death and immune defences in plants.

PubMed

Karpiński, Stanisław; Szechyńska-Hebda, Magdalena; Wituszyńska, Weronika; Burdiak, Paweł

2013-04-01

This review confronts the classical view of plant immune defence and light acclimation with recently published data. Earlier findings have linked plant immune defences to nucleotide-binding site leucine-rich repeat (NBS-LRR)-dependent recognition of pathogen effectors and to the role of plasma membrane-localized NADPH-dependent oxidoreductase (AtRbohD), reactive oxygen species (ROS) and salicylic acid (SA). However, recent results suggest that plant immune defence also depends on the absorption of excessive light energy and photorespiration. Rapid changes in light intensity and quality often cause the absorption of energy, which is in excess of that required for photosynthesis. Such excessive light energy is considered to be a factor triggering photoinhibition and disturbance in ROS/hormonal homeostasis, which leads to cell death in foliar tissues. We highlight here the tight crosstalk between ROS- and SA-dependent pathways leading to light acclimation, and defence responses leading to pathogen resistance. We also show that LESION SIMULATING DISEASE 1 (LSD1) regulates and integrates these processes. Moreover, we discuss the role of plastid-nucleus signal transduction, photorespiration, photoelectrochemical signalling and 'light memory' in the regulation of acclimation and immune defence responses. All of these results suggest that plants have evolved a genetic system that simultaneously regulates systemic acquired resistance (SAR), cell death and systemic acquired acclimation (SAA). © 2012 Blackwell Publishing Ltd.

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

Methyl Vitamin B12 but not methylfolate rescues a motor neuron-like cell line from homocysteine-mediated cell death

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

Hemendinger, Richelle A., E-mail: richelle.hemendinger@carolinashealthcare.org; Armstrong, Edward J.; Brooks, Benjamin Rix

Homocysteine is an excitatory amino acid implicated in multiple diseases including amyotrophic lateral sclerosis (ALS). Information on the toxicity of homocysteine in motor neurons is limited and few studies have examined how this toxicity can be modulated. In NSC-34D cells (a hybrid cell line derived from motor neuron-neuroblastoma), homocysteine induces apoptotic cell death in the millimolar range with a TC{sub 50} (toxic concentration at which 50% of maximal cell death is achieved) of 2.2 mM, confirmed by activation of caspase 3/7. Induction of apoptosis was independent of short-term reactive oxygen species (ROS) generation. Methyl Vitamin B12 (MeCbl) and methyl tetrahydrofolatemore » (MTHF), used clinically to treat elevated homocysteine levels, were tested for their ability to reverse homocysteine-mediated motor neuron cell death. MeCbl in the micromolar range was able to provide neuroprotection (2 h pretreatment prior to homocysteine) and neurorescue (simultaneous exposure with homocysteine) against millimolar homocysteine with an IC{sub 50} (concentration at which 50% of maximal cell death is inhibited) of 0.6 {mu}M and 0.4 {mu}M, respectively. In contrast, MTHF (up to 10 {mu}M) had no effect on homocysteine-mediated cell death. MeCbl inhibited caspase 3/7 activation by homocysteine in a time- and dose-dependent manner, whereas MTHF had no effect. We conclude that MeCbl is effective against homocysteine-induced cell death in motor neurons in a ROS-independent manner, via a reduction in caspase activation and apoptosis. MeCbl decreases Hcy induced motor neuron death in vitro in a hybrid cell line derived from motor neuron-neuroblastoma and may play a role in the treatment of late stage ALS where HCy levels are increased in animal models of ALS.« less

Ketamine induces toxicity in human neurons differentiated from embryonic stem cells via mitochondrial apoptosis pathway

PubMed Central

Bosnjak, Zeljko J.; Yan, Yasheng; Canfield, Scott; Muravyeva, Maria Y.; Kikuchi, Chika; Wells, Clive; Corbett, John; Bai, Xiaowen

2013-01-01

Ketamine is widely used for anesthesia in pediatric patients. Growing evidence indicates that ketamine causes neurotoxicity in a variety of developing animal models. Our understanding of anesthesia neurotoxicity in humans is currently limited by difficulties in obtaining neurons and performing developmental toxicity studies in fetal and pediatric populations. It may be possible to overcome these challenges by obtaining neurons from human embryonic stem cells (hESCs) in vitro. hESCs are able to replicate indefinitely and differentiate into every cell type. In this study, we investigated the toxic effect of ketamine on neurons differentiated from hESCs. Two-week-old neurons were treated with different doses and durations of ketamine with or without the reactive oxygen species (ROS) scavenger, Trolox. Cell viability, ultrastructure, mitochondrial membrane potential (ΔΨm), cytochrome c distribution within cells, apoptosis, and ROS production were evaluated. Here we show that ketamine induced ultrastructural abnormalities and dose- and time-dependently caused cell death. In addition, ketamine decreased ΔΨm and increased cytochrome c release from mitochondria. Ketamine also increased ROS production and induced differential expression of oxidative stress-related genes. Specifically, abnormal ultrastructural and ΔΨm changes occurred earlier than cell death in the ketamine-induced toxicity process. Furthermore, Trolox significantly decreased ROS generation and attenuated cell death caused by ketamine in a dose-dependent manner. In conclusion, this study illustrates that ketamine time- and dose-dependently induces human neurotoxicity via ROS-mediated mitochondrial apoptosis pathway and that these side effects can be prevented by the antioxidant agent Trolox. Thus, hESC-derived neurons might provide a promising tool for studying anesthetic-induced developmental neurotoxicity and prevention strategies. PMID:22873495

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

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

RIPK1/RIPK3/MLKL-mediated necroptosis contributes to compression-induced rat nucleus pulposus cells death.

PubMed

Chen, Songfeng; Lv, Xiao; Hu, Binwu; Shao, Zengwu; Wang, Baichuan; Ma, Kaige; Lin, Hui; Cui, Min

2017-05-01

The aim of this study was to systematically investigate the role of necroptosis in compression-induced rat nucleus pulposus (NP) cells death, as well as explore the underlying mechanisms involved. Rat NP cells underwent various periods of exposure to 1.0 MPa pressure. Cell viability and cell death were quantified by using cell counting kit-8 (CCK-8), and Calcein-AM/propidium iodine (PI) staining respectively. Necroptosis-associated target molecules receptor-interacing protein kinase 1 (RIPK1), phosphorylated RIPK1 (pRIPK1), receptor-interacing protein kinase 3 (RIPK3), phosphorylated RIPK3 (pRIPK3) and mixed lineage kinase domain-like (MLKL) were analyzed by Western-blot and RT-PCR. NP cells were also examined for morphological and ultrastructural changes, which can indicate necroptosis. To indirectly establish the presence of necroptosis, the RIPK1 specific inhibitor necrostatin-1 (Nec-1), RIPK3 inhibitor GSK'872, MLKL inhibitor necrosulfonamide (NSA) and small interfering RNA (siRNA) were utilized. The results established necroptosis was taking place in NP cells. The level of necroptosis increased in a time-dependent manner, and this effect was reduced by Nec-1 in vitro. Additionally, NP cells death were significantly attenuated following treatment with Nec-1, GSK'872 or NSA. SiRNA-induced knockdown of RIPK3 or MLKL increased cell survival rate, while knockdown of RIPK1 resulted in a decreased cell survival rate. In summary, RIPK1/RIPK3/MLKL-mediated necroptosis may play an important role in NP cells death induced by continuous mechanical stress. Treatment strategies which aim to regulate necroptosis may prove beneficial, by both reducing NP cells death and slowing IVD degeneration.

Circadian Stress Regimes Affect the Circadian Clock and Cause Jasmonic Acid-Dependent Cell Death in Cytokinin-Deficient Arabidopsis Plants.

PubMed

Nitschke, Silvia; Cortleven, Anne; Iven, Tim; Feussner, Ivo; Havaux, Michel; Riefler, Michael; Schmülling, Thomas

2016-07-01

The circadian clock helps plants measure daylength and adapt to changes in the day-night rhythm. We found that changes in the light-dark regime triggered stress responses, eventually leading to cell death, in leaves of Arabidopsis thaliana plants with reduced cytokinin levels or defective cytokinin signaling. Prolonged light treatment followed by a dark period induced stress and cell death marker genes while reducing photosynthetic efficiency. This response, called circadian stress, is also characterized by altered expression of clock and clock output genes. In particular, this treatment strongly reduced the expression of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Intriguingly, similar changes in gene expression and cell death were observed in clock mutants lacking proper CCA1 and LHY function. Circadian stress caused strong changes in reactive oxygen species- and jasmonic acid (JA)-related gene expression. The activation of the JA pathway, involving the accumulation of JA metabolites, was crucial for the induction of cell death, since the cell death phenotype was strongly reduced in the jasmonate resistant1 mutant background. We propose that adaptation to circadian stress regimes requires a normal cytokinin status which, acting primarily through the AHK3 receptor, supports circadian clock function to guard against the detrimental effects of circadian stress. © 2016 American Society of Plant Biologists. All rights reserved.

Influenza virus induces apoptosis via BAD-mediated mitochondrial dysregulation.

PubMed

Tran, Anh T; Cortens, John P; Du, Qiujiang; Wilkins, John A; Coombs, Kevin M

2013-01-01

Influenza virus infection results in host cell death and major tissue damage. Specific components of the apoptotic pathway, a signaling cascade that ultimately leads to cell death, are implicated in promoting influenza virus replication. BAD is a cell death regulator that constitutes a critical control point in the intrinsic apoptosis pathway, which occurs through the dysregulation of mitochondrial outer membrane permeabilization and the subsequent activation of downstream apoptogenic factors. Here we report a novel proviral role for the proapoptotic protein BAD in influenza virus replication. We show that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and that both virus replication and viral protein production are dramatically reduced, which suggests that virus-induced apoptosis is BAD dependent. Our data showed that influenza viruses induced phosphorylation of BAD at residues S112 and S136 in a temporal manner. Viral infection also induced BAD cleavage, late in the viral life cycle, to a truncated form that is reportedly a more potent inducer of apoptosis. We further demonstrate that knockdown of BAD resulted in reduced cytochrome c release and suppression of the intrinsic apoptotic pathway during influenza virus replication, as seen by an inhibition of caspases-3, caspase-7, and procyclic acidic repetitive protein (PARP) cleavage. Our data indicate that influenza viruses carefully modulate the activation of the apoptotic pathway that is dependent on the regulatory function of BAD and that failure of apoptosis activation resulted in unproductive viral replication.

Influenza Virus Induces Apoptosis via BAD-Mediated Mitochondrial Dysregulation

PubMed Central

Tran, Anh T.; Cortens, John P.; Du, Qiujiang; Wilkins, John A.

2013-01-01

Influenza virus infection results in host cell death and major tissue damage. Specific components of the apoptotic pathway, a signaling cascade that ultimately leads to cell death, are implicated in promoting influenza virus replication. BAD is a cell death regulator that constitutes a critical control point in the intrinsic apoptosis pathway, which occurs through the dysregulation of mitochondrial outer membrane permeabilization and the subsequent activation of downstream apoptogenic factors. Here we report a novel proviral role for the proapoptotic protein BAD in influenza virus replication. We show that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and that both virus replication and viral protein production are dramatically reduced, which suggests that virus-induced apoptosis is BAD dependent. Our data showed that influenza viruses induced phosphorylation of BAD at residues S112 and S136 in a temporal manner. Viral infection also induced BAD cleavage, late in the viral life cycle, to a truncated form that is reportedly a more potent inducer of apoptosis. We further demonstrate that knockdown of BAD resulted in reduced cytochrome c release and suppression of the intrinsic apoptotic pathway during influenza virus replication, as seen by an inhibition of caspases-3, caspase-7, and procyclic acidic repetitive protein (PARP) cleavage. Our data indicate that influenza viruses carefully modulate the activation of the apoptotic pathway that is dependent on the regulatory function of BAD and that failure of apoptosis activation resulted in unproductive viral replication. PMID:23135712

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

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.

[6]-Gingerol Induces Cell Cycle Arrest and Cell Death of Mutant p53-expressing Pancreatic Cancer Cells

PubMed Central

Park, Yon Jung; Wen, Jing; Bang, Seungmin; Park, Seung Woo

2006-01-01

[6]-Gingerol, a major phenolic compound derived from ginger, has anti-bacterial, anti-inflammatory and anti-tumor activities. While several molecular mechanisms have been described to underlie its effects on cells in vitro and in vivo, the underlying mechanisms by which [6]-gingerol exerts anti-tumorigenic effects are largely unknown. The purpose of this study was to investigate the action of [6]-gingerol on two human pancreatic cancer cell lines, HPAC expressing wild-type (wt) p53 and BxPC-3 expressing mutated p53. We found that [6]-gingerol inhibited the cell growth through cell cycle arrest at G1 phase in both cell lines. Western blot analyses indicated that [6]-gingerol decreased both Cyclin A and Cyclin-dependent kinase (Cdk) expression. These events led to reduction in Rb phosphorylation followed by blocking of S phase entry. p53 expression was decreased by [6]-gingerol treatment in both cell lines suggesting that the induction of Cyclin-dependent kinase inhibitor, p21cip1, was p53-independent. [6]-Gingerol induced mostly apoptotic death in the mutant p53-expressing cells, while no signs of early apoptosis were detected in wild type p53-expressing cells and this was related to the increased phosphorylation of AKT. These results suggest that [6]-gingerol can circumvent the resistance of mutant p53-expressing cells towards chemotherapy by inducing apoptotic cell death while it exerts cytostatic effect on wild type p53-expressing cells by inducing temporal growth arrest. PMID:17066513

Involvement of cyclin D1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb{sup 2+}-induced neuronal death in cultured hippocampal neurons

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

Li Chenchen; Xing Tairan; Tang Mingliang

2008-06-15

Lead (Pb) is widely recognized as a neurotoxicant. One of the suggested mechanisms of lead neurotoxicity is apoptotic cell death. And the mechanism by which Pb{sup 2+} causes neuronal death is not well understood. The present study sought to examine the obligate nature of cyclin D1/cyclin-dependent kinase 4 (CDK4), phosphorylation of its substrate retinoblastoma protein (pRb) and its select upstream signal phosphoinositide 3-kinase (PI3K)/AKT pathway in the death of primary cultured rat hippocampal neurons evoked by Pb{sup 2+}. Our data showed that lead treatment of primary hippocampal cultures results in dose-dependent cell death. Inhibition of CDK4 prevented Pb{sup 2+}-induced neuronalmore » death significantly but was incomplete. In addition, we demonstrated that the levels of cyclin D1 and pRb/p107 were increased during Pb{sup 2+} treatment. These elevated expression persisted up to 48 h, returning to control levels after 72 h. We also presented pharmacological and morphological evidences that cyclin D1/CDK4 and pRb/p107 were required for such kind of neuronal death. Addition of the PI3K inhibitor LY294002 (30 {mu}M) or wortmannin (100 nM) significantly rescued the cultured hippocampal neurons from death caused by Pb{sup 2+}. And that Pb{sup 2+}-elicited phospho-AKT (Ser473) participated in the induction of cyclin D1 and partial pRb/p107 expression. These results provide evidences that cell cycle elements play a required role in the death of neurons evoked by Pb{sup 2+} and suggest that certain signaling elements upstream of cyclin D1/CDK4 are modified and/or required for this form of neuronal death.« less

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.

Metal-accelerated oxidation in plant cell death

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

Czuba, M.

1993-05-01

Cadmium and mercury toxicity is further enhanced by external oxidizing conditions O[sub 3] or inherent plant processes. Lepidium sativum L, Lycopersicon esculentum Mill., or Phaseolus vulgaris L, were grown inpeat-lite to maturity under continuous cadmium exposure followed by one oxidant (O[sub 3]-6 hr. 30 pphm) exposure, with or without foliar calcium pretreatments. In comparison, Daucus carota, L and other species grown in a 71-V suspension, with or without 2,4-D were exposed continuously to low levels of methylmercury during exponential growth and analyzed in aggregates of distinct populations. Proteins were extracted and analyzed. Mechanisms of toxicity and eventual cell death aremore » Ca-mediated and involve chloroplast, stomatal-water relations and changes in oxidant-anti-oxidant components in cells. Whether the metal-accelerated oxidative damage proceeds to cell death, depends on the species and its differential biotransformation system and cell association component.« less

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.

Neurotoxic injury pathways in differentiated mouse motor neuron–neuroblastoma hybrid (NSC-34D) cells in vitro—Limited effect of riluzole on thapsigargin, but not staurosporine, hydrogen peroxide and homocysteine neurotoxicity

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

Hemendinger, Richelle A., E-mail: richelle.hemendinger@carolinashealthcare.org; Carolinas Neuromuscular/ALS-MDA Center, Department of Neurology, Carolinas Medical Center, Charlotte, NC 28203; Armstrong, Edward J.

2012-01-15

The neuroblastoma–spinal motor neuron fusion cell line, NSC-34, in its differentiated form, NSC-34D, permits examining the effects of riluzole, a proven treatment for amyotrophic lateral sclerosis (ALS) on cell death induction by staurosporine (STS), thapsigargin (Thaps), hydrogen peroxide (H{sub 2}O{sub 2}) and homocysteine (HCy). These neurotoxins, applied exogenously, have mechanisms of action related to the various proposed molecular pathogenetic pathways in ALS and are differentiated from endogenous cell death that is associated with cytoplasmic aggregate formation in motor neurons. Nuclear morphology, caspase-3/7 activation and high content imaging were used to assess toxicity of these neurotoxins with and without co-treatment withmore » riluzole, a benzothiazole compound with multiple pharmacological actions. STS was the most potent neurotoxin at killing NSC-34D cells with a toxic concentration at which 50% of maximal cell death is achieved (TC{sub 50} = 0.01 μM), followed by Thaps (TC{sub 50} = 0.9 μM) and H{sub 2}O{sub 2} (TC{sub 50} = 15 μM) with HCy requiring higher concentrations to kill at the same level (TC{sub 50} = 2200 μM). Riluzole provided neurorescue with a 20% absolute reduction (47.6% relative reduction) in apoptotic cell death against Thaps-induced NSC-34D cell (p ≤ 0.05), but had no effect on STS-, H{sub 2}O{sub 2}- and HCy-induced NSC-34D cell death. This effect of riluzole on Thaps induction of cell death was independent of caspase-3/7 activation. Riluzole mitigated a toxin that can cause intracellular calcium dysregulation associated with endoplasmic reticulum (ER) stress but not toxins associated with other cell death mechanisms. -- Highlights: ► Calcium-dependent neurotoxins are potent cell death inducers in NSC-34D cells. ► Riluzole provides neurorescue against Thaps-induced NSC-34D cell death. ► Riluzole had no effect on neurotoxicity by STS, H{sub 2}O{sub 2} and Hcy. ► Riluzole reduces NSC-34D cell death independent of caspase-3/7 activation.« less

Attenuation of Aβ{sub 25–35}-induced parallel autophagic and apoptotic cell death by gypenoside XVII through the estrogen receptor-dependent activation of Nrf2/ARE pathways

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

Meng, Xiangbao; Wang, Min; Sun, Guibo, E-mail: sunguibo@126.com

Amyloid-beta (Aβ) has a pivotal function in the pathogenesis of Alzheimer's disease. To investigate Aβ neurotoxicity, we used an in vitro model that involves Aβ{sub 25–35}-induced cell death in the nerve growth factor-induced differentiation of PC12 cells. Aβ{sub 25–35} (20 μM) treatment for 24 h caused apoptotic cell death, as evidenced by significant cell viability reduction, LDH release, phosphatidylserine externalization, mitochondrial membrane potential disruption, cytochrome c release, caspase-3 activation, PARP cleavage, and DNA fragmentation in PC12 cells. Aβ{sub 25–35} treatment led to autophagic cell death, as evidenced by augmented GFP-LC3 puncta, conversion of LC3-I to LC3-II, and increased LC3-II/LC3-I ratio.more » Aβ{sub 25–35} treatment induced oxidative stress, as evidenced by intracellular ROS accumulation and increased production of mitochondrial superoxide, malondialdehyde, protein carbonyl, and 8-OHdG. Phytoestrogens have been proved to be protective against Aβ-induced neurotoxicity and regarded as relatively safe targets for AD drug development. Gypenoside XVII (GP-17) is a novel phytoestrogen isolated from Gynostemma pentaphyllum or Panax notoginseng. Pretreatment with GP-17 (10 μM) for 12 h increased estrogen response element reporter activity, activated PI3K/Akt pathways, inhibited GSK-3β, induced Nrf2 nuclear translocation, augmented antioxidant responsive element enhancer activity, upregulated heme oxygenase 1 (HO-1) expression and activity, and provided protective effects against Aβ{sub 25–35}-induced neurotoxicity, including oxidative stress, apoptosis, and autophagic cell death. In conclusion, GP-17 conferred protection against Aβ{sub 25–35}-induced neurotoxicity through estrogen receptor-dependent activation of PI3K/Akt pathways, inactivation of GSK-3β and activation of Nrf2/ARE/HO-1 pathways. This finding might provide novel insights into understanding the mechanism for neuroprotective effects of phytoestrogens or gypenosides. - Highlights: • GP-17 showed protection against Aβ{sub 25–35}-induced neurotoxicity. • The neuroprotective effects of GP-17 are dependent on estrogen receptors. • GP-17 activates Nrf2/ARE/HO-1 pathways. • GP-17 activates PI3K/Akt/GSK-3β pathways.« less

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

5-epi-Sinuleptolide induces cell cycle arrest and apoptosis through tumor necrosis factor/mitochondria-mediated caspase signaling pathway in human skin cancer cells.

PubMed

Liang, Chia-Hua; Wang, Guey-Horng; Chou, Tzung-Han; Wang, Shih-Hao; Lin, Rong-Jyh; Chan, Leong-Perng; So, Edmund Cheung; Sheu, Jyh-Horng

2012-07-01

Skin cancers are reportedly increasing worldwide. Developing novel anti-skin cancer drugs with minimal side effects is necessary to address this public health issue. Sinuleptolide has been demonstrated to possess anti-cancer cell activities; however, the mechanisms underlying the anti-skin cancer effects of 5-epi-sinuleptolide and sinuleptolide remain poorly understood. Apoptosis cell, cell-cycle-related regulatory factors, and mitochondria- and death receptor-dependent caspase pathway in 5-epi-sinuleptolide-induced cell apoptosis were examined using SCC25 cells. 5-epi-Sinuleptolide inhibited human skin cancer cell growth more than did sinuleptolide. Treatment of SCC25 cells with 5-epi-sinuleptolide increased apoptotic body formation, and induced cell-cycle arrest during the G2/M phase. Notably, 5-epi-sinuleptolide up-regulated p53 and p21 expression and inhibited G2/M phase regulators of cyclin B1 and cyclin-dependent kinease 1 (CDK1) in SCC25 cells. Additionally, 5-epi-sinuleptolide induced apoptosis by mitochondria-mediated cytochrome c and Bax up-expression, down-regulated Bcl-2, and activated caspase-9 and -3. 5-epi-Sinuleptolide also up-regulated tBid, which is associated with up-regulation of tumor necrosis factor-α (TNF-α) and Fas ligand (FasL) and their cognate receptors (i.e., TNF-RI, TNF-R2 and Fas), downstream adaptor TNF-R1-associated death domain (TRADD) and Fas-associated death domain (FADD), and activated caspase-8 in SCC25 cells. The analytical results indicate that the death receptor- and mitochondria-mediated caspase pathway is critical in 5-epi-sinuleptolide-induced apoptosis of skin cancer cells. This is the first report suggesting that the apoptosis mediates the anti-tumor effect of 5-epi-sinuleptolide. The results of this study might provide useful suggestions for designing of anti-tumor drugs for skin cancer patients. Copyright © 2012 Elsevier B.V. All rights reserved.

Involvement of Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Advanced Glycation End Products-Induced Glomerular Mesangial Cell Injury

PubMed Central

Chiang, Chih-Kang; Wang, Ching-Chia; Lu, Tien-Fong; Huang, Kuo-How; Sheu, Meei-Ling; Liu, Shing-Hwa; Hung, Kuan-Yu

2016-01-01

Advanced glycation end-products (AGEs)-induced mesangial cell death is one of major causes of glomerulus dysfunction in diabetic nephropathy. Both endoplasmic reticulum (ER) stress and autophagy are adaptive responses in cells under environmental stress and participate in the renal diseases. The role of ER stress and autophagy in AGEs-induced mesangial cell death is still unclear. Here, we investigated the effect and mechanism of AGEs on glomerular mesangial cells. AGEs dose-dependently decreased mesangial cell viability and induced cell apoptosis. AGEs also induced ER stress signals in a time- and dose-dependent manner. Inhibition of ER stress with 4-phenylbutyric acid effectively inhibited the activation of eIF2α and CHOP signals and reversed AGEs-induced cell apoptosis. AGEs also activated LC-3 cleavage, increased Atg5 expression, and decreased p62 expression, which indicated the autophagy induction in mesangial cells. Inhibition of autophagy by Atg5 siRNAs transfection aggravated AGEs-induced mesangial cell apoptosis. Moreover, ER stress inhibition by 4-phenylbutyric acid significantly reversed AGEs-induced autophagy, but autophagy inhibition did not influence the AGEs-induced ER stress-related signals activation. These results suggest that AGEs induce mesangial cell apoptosis via an ER stress-triggered signaling pathway. Atg5-dependent autophagy plays a protective role. These findings may offer a new strategy against AGEs toxicity in the kidney. PMID:27665710

Procyanidin-rich extract of natural cocoa powder causes ROS-mediated caspase-3 dependent apoptosis and reduction of pro-MMP-2 in epithelial ovarian carcinoma cell lines.

PubMed

Taparia, Shruti Sanjay; Khanna, Aparna

2016-10-01

Over the last four centuries, cocoa and chocolate have been described as having potential medicinal value. As of today, Theobroma cacao L. (Sterculiaceae) and its products are consumed worldwide. They are of great research interest because of the concentration dependent antioxidant as well as pro-oxidant properties of some of their polyphenolic constituents, specially procyanidins and flavan-3-ols such as catechin. This study was aimed at investigating the cellular and molecular changes associated with cytotoxicity, caused due pro-oxidant activity of cocoa catechins and procyanidins, in ovarian cancer cell lines. Extract of non-alkalized cocoa powder enriched with catechins and procyanidins was used to treat human epithelial ovarian cancer cell lines OAW42 and OVCAR3 at various concentrations ≤1000μg/mL. The effect of treatment on intracellular reactive oxygen species (ROS) levels was determined. Apoptotic cell death, post treatment, was evaluated microscopically and using flow cytometry by means of annexin-propidium iodide (PI) dual staining. Levels of active caspase-3 as a pro-apoptotic marker and matrix metalloproteinase 2 (MMP2) as an invasive potential marker were detected using Western blotting and gelatin zymography. Treatment with extract caused an increase in intracellular ROS levels in OAW42 and OVCAR3 cell lines. Bright field and fluorescence microscopy of treated cells revealed apoptotic morphology and DNA damage. Increase in annexin positive cell population and dose dependent upregulation of caspase-3 confirmed apoptotic cell death. pro-MMP2 was found to be downregulated in a dose dependent manner in cells treated with the extract. Treated cells also showed a reduction in MMP2 activity. Our data suggests that cocoa catechins and procyanidins are cytotoxic to epithelial ovarian cancer, inducing apoptotic morphological changes, DNA damage and caspase-3 mediated cell death. Downregulation of pro-MMP2 and reduction in active MMP2 levels imply a decrease in invasive potential of the cells. Apoptosis and MMP2 downregulation appear to be linked to the increase in intracellular ROS levels, caused due to the prooxidant effect of cocoa procyanidin extract. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

The influence of immunosuppressive drugs on neural stem/progenitor cell fate in vitro

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

Skardelly, Marco, E-mail: Marco.Skardelly@med.uni-tuebingen.de; Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig; Glien, Anja

In allogenic and xenogenic transplantation, adequate immunosuppression plays a major role in graft survival, especially over the long term. The effect of immunosuppressive drugs on neural stem/progenitor cell fate has not been sufficiently explored. The focus of this study is to systematically investigate the effects of the following four different immunotherapeutic strategies on human neural progenitor cell survival/death, proliferation, metabolic activity, differentiation and migration in vitro: (1) cyclosporine A (CsA), a calcineurin inhibitor; (2) everolimus (RAD001), an mTOR-inhibitor; (3) mycophenolic acid (MPA, mycophenolate), an inhibitor of inosine monophosphate dehydrogenase and (4) prednisolone, a steroid. At the minimum effective concentration (MEC),more » we found a prominent decrease in hNPCs' proliferative capacity (BrdU incorporation), especially for CsA and MPA, and an alteration of the NAD(P)H-dependent metabolic activity. Cell death rate, neurogenesis, gliogenesis and cell migration remained mostly unaffected under these conditions for all four immunosuppressants, except for apoptotic cell death, which was significantly increased by MPA treatment. - Highlights: • Four immunosuppresants (ISs) were tested in human neural progenitor cells in vitro. • Cyclosporine A and mycophenolic acid showed a prominent anti-proliferative activity • Mycophenolic acid exhibited a significant pro-apoptotic effect. • NAD(P)H-dependent metabolic activity was occasionally induced by ISs. • Neuronal differentiation and migration potential remained unaffected by ISs treatment.« less

Implications of caspase-dependent proteolytic cleavage of cyclin A1 in DNA damage-induced cell death

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

Woo, Sang Hyeok; Seo, Sung-Keum; An, Sungkwan

Highlights: • Caspase-1 mediates doxorubicin-induced downregulation of cyclin A1. • Active caspase-1 effectively cleaved cyclin A1 at D165. • Cyclin A1 expression is involved in DNA damage-induced cell death. - Abstract: Cyclin A1 is an A-type cyclin that directly binds to CDK2 to regulate cell-cycle progression. In the present study, we found that doxorubicin decreased the expression of cyclin A1 at the protein level in A549 lung cancer cells, while markedly downregulating its mRNA levels. Interestingly, doxorubicin upregulated caspase-1 in a concentration-dependent manner, and z-YAVD-fmk, a specific inhibitor of caspase-1, reversed the doxorubicin-induced decrease in cyclin A1 in A549 lungmore » cancer and MCF7 breast cancer cells. Active caspase-1 effectively cleaved cyclin A1 at D165 into two fragments, which in vitro cleavage assays showed were further cleaved by caspase-3. Finally, we found that overexpression of cyclin A1 significantly reduced the cytotoxicity of doxorubicin, and knockdown of cyclin A1 by RNA interference enhanced the sensitivity of cells to ionizing radiation. Our data suggest a new mechanism for the downregulation of cyclin A1 by DNA-damaging stimuli that could be intimately involved in the cell death induced by DNA damage-inducing stimuli, including doxorubicin and ionizing radiation.« less

Metabolic regulation of oocyte cell death through the CaMKII-mediated phosphorylation of caspase-2.

PubMed

Nutt, Leta K; Margolis, Seth S; Jensen, Mette; Herman, Catherine E; Dunphy, William G; Rathmell, Jeffrey C; Kornbluth, Sally

2005-10-07

Vertebrate female reproduction is limited by the oocyte stockpiles acquired during embryonic development. These are gradually depleted over the organism's lifetime through the process of apoptosis. The timer that triggers this cell death is yet to be identified. We used the Xenopus egg/oocyte system to examine the hypothesis that nutrient stores can regulate oocyte viability. We show that pentose-phosphate-pathway generation of NADPH is critical for oocyte survival and that the target of this regulation is caspase-2, previously shown to be required for oocyte death in mice. Pentose-phosphate-pathway-mediated inhibition of cell death was due to the inhibitory phosphorylation of caspase-2 by calcium/calmodulin-dependent protein kinase II (CaMKII). These data suggest that exhaustion of oocyte nutrients, resulting in an inability to generate NADPH, may contribute to ooctye apoptosis. These data also provide unexpected links between oocyte metabolism, CaMKII, and caspase-2.

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.

Regulated Cell Death of Lymphoma Cells after Graded Mitochondrial Damage is Differentially Affected by Drugs Targeting Cell Stress Responses.

PubMed

Lombardo, Tomás; Folgar, Martín Gil; Salaverry, Luciana; Rey-Roldán, Estela; Alvarez, Elida M; Carreras, María C; Kornblihtt, Laura; Blanco, Guillermo A

2018-05-01

Collapse of the mitochondrial membrane potential (MMP) is often considered the initiation of regulated cell death (RCD). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) is an uncoupler of the electron transport chain (ETC) that facilitates the translocation of protons into the mitochondrial matrix leading to the collapse of the MMP. Several cell stress responses such as mitophagy, mitochondrial biogenesis and the ubiquitin proteasome system may differentially contribute to restrain the initiation of RCD depending on the extent of mitochondrial damage. We induced graded mitochondrial damage after collapse of MMP with the mitochondrial uncoupler CCCP in Burkitt's lymphoma cells, and we evaluated the effect of several drugs targeting cell stress responses over RCD at 72 hr, using a multiparametric flow cytometry approach. CCCP caused collapse of MMP after 30 min., massive mitochondrial fission, oxidative stress and increased mitophagy within the 5-15 μM low-dose range (LDR) of CCCP. Within the 20-50 μM high-dose range (HDR), CCCP caused lysosomal destabilization and rupture, thus precluding mitophagy and autophagy. Cell death after 72 hr was below 20%, with increased mitochondrial mass (MM). The inhibitors of mitophagy 3-(2,4-dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(1H)-quinazolinone (Mdivi-1) and vincristine (VCR) increased cell death from CCCP within the LDR, while valproic acid (an inducer of mitochondrial biogenesis) also increased MM and cell death within the LDR. The proteasome inhibitor, MG132, increased cell death only in the HDR. Doxycycline, an antibiotic that disrupts mitochondrial biogenesis, had no effect on cell survival, while iodoacetamide, an inhibitor of glycolysis, increased cell death at the HDR. We conclude that mitophagy influenced RCD of lymphoma cells after MMP collapse by CCCP only within the LDR, while proteasome activity and glycolysis contributed to survival in the HDR under extensive mitochondria and lysosome damage. © 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Sulphur antioxidants inhibit oxidative stress induced retinal ganglion cell death by scavenging reactive oxygen species but influence nuclear factor (erythroid-derived 2)-like 2 signalling pathway differently.

PubMed

Majid, Aman Shah Abdul; Yin, Zheng Qin; Ji, Dan

2013-01-01

This study aimed to show if two different sulphur containing drugs sulbutiamine and acetylcysteine (NAC) could attenuate the effects of two different insults being serum deprivation and glutamate/buthionine sulfoximine (GB)-induced death to transformed retinal ganglion cell line (RGC-5) in culture. Cells were exposed to either 5 mM of GB for 24 h or serum deprivation for 48 h with inclusion of either NAC or sulbutiamine. Cell viability, microscopic evidence for apoptosis, caspase 3 activity, reactive oxygen species (ROS), glutathione (GSH), catalase and gluthathione-S-transferase (GST) were determined. The effects of NAC and sulbutiamine on the oxidative stress related transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) levels and its dependent phase II enzyme haemeoxygenase-1 (HO-1) were carried out using Western blot and quantitative-polymerase chain reaction (PCR). NAC and sulbutiamine dose-dependently attenuated serum deprivation-induced cell death. However NAC but not sulbutiamine attenuated GB-induced cell death. NAC and sulbutiamine both independently stimulated the GSH and GST production but scavenged different types of ROS with different efficacy. Moreover only sulbutiamine stimulated catalase and significantly increased Nrf-2 and HO-1 levels. In addition, the pan caspase inhibitor, benzoylcarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) attenuated the negative effect of serum deprivation while the necroptosis inhibitor (necrostatin-1) counteracted solely an insult of GB. The neuroprotective actions of NAC and sulbutiamine in GB or serum-deprivation insult are therefore different.

Microscopic analysis of cell death by metabolic stress-induced autophagy in prostate cancer

NASA Astrophysics Data System (ADS)

Changou, Chun; Cheng, R. Holland; Bold, Richard; Kung, Hsing-Jien; Chuang, Frank Y. S.

2013-02-01

Autophagy is an intracellular recycling mechanism that helps cells to survive against environmental stress and nutritional starvation. We have recently shown that prostate cancers undergo metabolic stress and caspase-independent cell death following exposure to arginine deiminase (ADI, an enzyme that degrades arginine in tissue). The aims of our current investigation into the application of ADI as a novel cancer therapy are to identify the components mediating tumor cell death, and to determine the role of autophagy (stimulated by ADI and/or rapamycin) on cell death. Using advanced fluorescence microscopy techniques including 3D deconvolution and superresolution structured-illumination microscopy (SIM), we show that prostate tumor cells that are killed after exposure to ADI for extended periods, exhibit a morphology that is distinct from caspase-dependent apoptosis; and that autophagosomes forming as a result of ADI stimulation contain DAPI-stained nuclear material. Fluorescence imaging (as well as cryo-electron microscopy) show a breakdown of both the inner and outer nuclear membranes at the interface between the cell nucleus and aggregated autophagolysosomes. Finally, the addition of N-acetyl cysteine (or NAC, a scavenger for reactive oxygen species) effectively abolishes the appearance of autophagolysosomes containing nuclear material. We hope to continue this research to understand the processes that govern the survival or death of these tumor cells, in order to develop methods to improve the efficacy of cancer pharmacotherapy.

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

Targeting c-KIT (CD117) by dasatinib and radotinib promotes acute myeloid leukemia cell death.

PubMed

Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jeong, Yoo Kyung; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk

2017-11-10

Dasatinib and radotinib are oral BCR-ABL tyrosine kinase inhibitors that were developed as drugs for the treatment of chronic myeloid leukemia. We report here that the c-KIT (CD117) targeting with dasatinib and radotinib promotes acute myeloid leukemia (AML) cell death, and c-KIT endocytosis is essential for triggering c-KIT-positive AML cell death by dasatinib and radotinib during the early stages. In addition, dasatinib and radotinib reduce heat shock protein 90β (HSP90β) expression and release Apaf-1 in c-KIT-positive AML cells. Finally, this activates a caspase-dependent apoptotic pathway in c-KIT-positive AML cells. Moreover, the inhibition of c-KIT endocytosis by dynamin inhibitor (DY) reversed cell viability and c-KIT expression by dasatinib and radotinib. HSP90β expression was recovered by DY in c-KIT-positive AML cells as well. Furthermore, the effect of radotinib on c-KIT and HSP90β showed the same pattern in a xenograft animal model using HEL92.1.7 cells. Therefore, dasatinib and radotinib promote AML cell death by targeting c-KIT. Taken together, these results indicate that dasatinib and radotinib treatment have a potential role in anti-leukemic therapy on c-KIT-positive AML cells.

The MST/Hippo Pathway and Cell Death: A Non-Canonical Affair

PubMed Central

Fallahi, Emma; O’Driscoll, Niamh A.; Matallanas, David

2016-01-01

The MST/Hippo signalling pathway was first described over a decade ago in Drosophila melanogaster and the core of the pathway is evolutionary conserved in mammals. The mammalian MST/Hippo pathway regulates organ size, cell proliferation and cell death. In addition, it has been shown to play a central role in the regulation of cellular homeostasis and it is commonly deregulated in human tumours. The delineation of the canonical pathway resembles the behaviour of the Hippo pathway in the fly where the activation of the core kinases of the pathway prevents the proliferative signal mediated by the key effector of the pathway YAP. Nevertheless, several lines of evidence support the idea that the mammalian MST/Hippo pathway has acquired new features during evolution, including different regulators and effectors, crosstalk with other essential signalling pathways involved in cellular homeostasis and the ability to actively trigger cell death. Here we describe the current knowledge of the mechanisms that mediate MST/Hippo dependent cell death, especially apoptosis. We include evidence for the existence of complex signalling networks where the core proteins of the pathway play a central role in controlling the balance between survival and cell death. Finally, we discuss the possible involvement of these signalling networks in several human diseases such as cancer, diabetes and neurodegenerative disorders. PMID:27322327

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.

Powerful bacterial killing by buckwheat honeys is concentration-dependent, involves complete DNA degradation and requires hydrogen peroxide.

PubMed

Brudzynski, Katrina; Abubaker, Kamal; Wang, Tony

2012-01-01

Exposure of bacterial cells to honey inhibits their growth and may cause cell death. Our previous studies showed a cause-effect relationship between hydroxyl radical generated from honey hydrogen peroxide and growth arrest. Here we explored the role of hydroxyl radicals as inducers of bacterial cells death. The bactericidal effect of ·OH on antibiotic-resistant clinical isolates of MRSA and VRE and standard bacterial strains of E. coli and B. subtiles was examined using a broth microdilution assay supplemented with 3'-(p-aminophenyl) fluorescein (APF) as the ·OH trap, followed by colony enumeration. Bactericidal activities of eight honeys (six varieties of buckwheat, blueberry and manuka honeys) were analyzed. The MBC/MIC ratio ≤4 and the killing curves indicated that honeys exhibited powerful, concentration-dependent bactericidal effect. The extent of killing depended on the ratio of honey concentration to bacterial load, indicating that honey dose was critical for its bactericidal efficacy. The killing rate and potency varied between honeys and ranged from over a 6-log(10) to 4-log(10) CFU/ml reduction of viable cells, equivalent to complete bacterial eradication. The maximal killing was associated with the extensive degradation of bacterial DNA. Honey concentration at which DNA degradation occurred correlated with cell death observed in the concentration-dependent cell-kill on agar plates. There was no quantitative relationship between the ·OH generation by honey and bactericidal effect. At the MBC, where there was no surviving cells and no DNA was visible on agarose gels, the ·OH levels were on average 2-3x lower than at Minimum Inhibitory Concentration (MICs) (p < 0.0001). Pre-treatment of honey with catalase, abolished the bactericidal effect. This raised possibilities that either the abrupt killing prevented accumulation of ·OH (dead cells did not generate ·OH) or that DNA degradation and killing is the actual footprint of ·OH action. In conclusion, honeys of buckwheat origin exhibited powerful, concentration-dependent bactericidal effect. The killing and DNA degradation showed a cause-effect relationship. Hydrogen peroxide was an active part of honey killing mechanism.

Bax and Bak are required for apoptosis induction by sulforaphane, a cruciferous vegetable-derived cancer chemopreventive agent.

PubMed

Choi, Sunga; Singh, Shivendra V

2005-03-01

Sulforaphane, a constituent of many edible cruciferous vegetables, including broccoli, effectively suppresses proliferation of cancer cells in culture and in vivo by causing apoptosis induction, but the sequence of events leading to cell death is poorly defined. Here, we show that multidomain proapoptotic Bcl-2 family members Bax and Bak play a critical role in apoptosis induction by sulforaphane. This conclusion is based on the following observations: (a) sulforaphane treatment caused a dose- and time-dependent increase in the protein levels of both Bax and Bak and conformational change and mitochondrial translocation of Bax in SV40-transformed mouse embryonic fibroblasts (MEF) derived from wild-type mice to trigger cytosolic release of apoptogenic molecules (cytochrome c and Smac/DIABLO), activation of caspase-9 and caspase-3, and ultimately cell death; (b) MEFs derived from Bax or Bak knockout mice resisted cell death by sulforaphane, and (c) MEFs derived from Bax and Bak double knockout mice exhibited even greater protection against sulforaphane-induced cytochrome c release, caspase activation, and apoptosis compared with wild-type or single knockout cells. Interestingly, sulforaphane treatment also caused a dose- and time-dependent increase in the protein level of Apaf-1 in wild-type, Bax-/-, and Bak-/- MEFs but not in double knockout, suggesting that Bax and Bak might regulate sulforaphane-mediated induction of Apaf-1 protein. A marked decline in the protein level of X-linked inhibitor of apoptosis on treatment with sulforaphane was also observed. Thus, it is reasonable to postulate that sulforaphane-induced apoptosis is amplified by a decrease in X-linked inhibitor of apoptosis level, which functions to block cell death by inhibiting activities of caspases. In conclusion, the results of the present study indicate that Bax and Bak proteins play a critical role in initiation of cell death by sulforaphane.

Combinatorial DNA Damage Pairing Model Based on X-Ray-Induced Foci Predicts the Dose and LET Dependence of Cell Death in Human Breast Cells

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

Vadhavkar, Nikhil; Pham, Christopher; Georgescu, Walter

In contrast to the classic view of static DNA double-strand breaks (DSBs) being repaired at the site of damage, we hypothesize that DSBs move and merge with each other over large distances (m). As X-ray dose increases, the probability of having DSB clusters increases as does the probability of misrepair and cell death. Experimental work characterizing the X-ray dose dependence of radiation-induced foci (RIF) in nonmalignant human mammary epithelial cells (MCF10A) is used here to validate a DSB clustering model. We then use the principles of the local effect model (LEM) to predict the yield of DSBs at the submicronmore » level. Two mechanisms for DSB clustering, namely random coalescence of DSBs versus active movement of DSBs into repair domains are compared and tested. Simulations that best predicted both RIF dose dependence and cell survival after X-ray irradiation favored the repair domain hypothesis, suggesting the nucleus is divided into an array of regularly spaced repair domains of ~;;1.55 m sides. Applying the same approach to high-linear energy transfer (LET) ion tracks, we are able to predict experimental RIF/m along tracks with an overall relative error of 12percent, for LET ranging between 30 350 keV/m and for three different ions. Finally, cell death was predicted by assuming an exponential dependence on the total number of DSBs and of all possible combinations of paired DSBs within each simulated RIF. Relative biological effectiveness (RBE) predictions for cell survival of MCF10A exposed to high-LET showed an LET dependence that matches previous experimental results for similar cell types. Overall, this work suggests that microdosimetric properties of ion tracks at the submicron level are sufficient to explain both RIF data and survival curves for any LET, similarly to the LEM assumption. Conversely, high-LET death mechanism does not have to infer linear-quadratic dose formalism as done in the LEM. In addition, the size of repair domains derived in our model are based on experimental RIF and are three times larger than the hypothetical LEM voxel used to fit survival curves. Our model is therefore an alternative to previous approaches that provides a testable biological mechanism (i.e., RIF). In addition, we propose that DSB pairing will help develop more accurate alternatives to the linear cancer risk model (LNT) currently used for regulating exposure to very low levels of ionizing radiation.« less

Influence of injury severity on the rate and magnitude of the T lymphocyte and neuronal response to facial nerve axotomy.

PubMed

Ha, Grace K; Parikh, Shivani; Huang, Zhi; Petitto, John M

2008-08-13

The temporal relationship between severity of peripheral axonal injury and T lymphocyte trafficking to the neuronal cell bodies of origin in the brain has been unclear. We sought to test the hypothesis that greater neuronal death induced by disparate forms of peripheral nerve injury would result in differential patterns of T cell infiltration and duration at the cell bodies of origin in the brain and that these measures would correlate with the magnitude of neuronal death over time and cumulative neuronal loss. To test this hypothesis, we compared the time course of CD3(+) T cell infiltration and neuronal death (assessed by CD11b(+) perineuronal microglial phagocytic clusters) following axonal crush versus axonal resection injuries, two extreme variations of facial nerve axotomy that result in mild versus severe neuronal loss, respectively, in the facial motor nucleus. We also quantified the number of facial motor neurons present at 49 days post-injury to determine whether differences in the levels of neuronal death between nerve crush and resection correlated with differences in cumulative neuronal loss. Between 1 and 7 days post-injury when levels of neuronal death were minimal, we found that the rate of accumulation and magnitude of the T cell response was similar following nerve crush and resection. Differences in the T cell response were apparent by 14 days post-injury when the level of neuronal death following resection was substantially greater than that seen in crush injury. For nerve resection, the peak of neuronal death at 14 days post-resection was followed by a maximal T cell response one week later at 21 days. Differences in the level of neuronal death between the two injuries across the time course tested reflected differences in cumulative neuronal loss at 49 days post-injury. Altogether, these data suggest that the trafficking of T cells to the injured FMN is dependent upon the severity of peripheral nerve injury and associated neuronal death.

The Nrf2/SKN-1-dependent glutathione S-transferase π homologue GST-1 inhibits dopamine neuron degeneration in a Caenorhabditis elegans model of manganism.

PubMed

Settivari, Raja; VanDuyn, Natalia; LeVora, Jennifer; Nass, Richard

2013-09-01

Exposure to high levels of manganese (Mn) results in a neurological condition termed manganism, which is characterized by oxidative stress, abnormal dopamine (DA) signaling, and cell death. Epidemiological evidence suggests correlations with occupational exposure to Mn and the development of the movement disorder Parkinson's disease (PD), yet the molecular determinants common between the diseases are ill-defined. Glutathione S-transferases (GSTs) of the class pi (GSTπ) are phase II detoxification enzymes that conjugate both endogenous and exogenous compounds to glutathione to reduce cellular oxidative stress, and their decreased expression has recently been implicated in PD progression. In this study we demonstrate that a Caenorhabditis elegans GSTπ homologue, GST-1, inhibits Mn-induced DA neuron degeneration. We show that GST-1 is expressed in DA neurons, Mn induces GST-1 gene and protein expression, and GST-1-mediated neuroprotection is dependent on the PD-associated transcription factor Nrf2/SKN-1, as a reduction in SKN-1 gene expression results in a decrease in GST-1 protein expression and an increase in DA neuronal death. Furthermore, decreases in gene expression of the SKN-1 inhibitor WDR-23 or the GSTπ-binding cell death activator JNK/JNK-1 result in an increase in resistance to the metal. Finally, we show that the Mn-induced DA neuron degeneration is independent of the dopamine transporter DAT, but is largely dependent on the caspases CED-3 and the novel caspase CSP-1. This study identifies a C. elegans Nrf2/SKN-1-dependent GSTπ homologue, cell death effectors of GSTπ-associated xenobiotic-induced pathology, and provides the first in vivo evidence that a phase II detoxification enzyme may modulate DA neuron vulnerability in manganism. Copyright © 2013 Elsevier Inc. All rights reserved.

Regulation of cell death and cell survival gene expression during ovarian follicular development and atresia.

PubMed

Jiang, Jin-Yi; Cheung, Carmen K M; Wang, Yifang; Tsang, Benjamin K

2003-01-01

Mammalian ovarian follicular development and atresia is closely regulated by the cross talk of cell death and cell survival signals, which include endocrine hormones (gonadotropins) and intra-ovarian regulators (gonadal steroids, cytokines and growth factors). The fate of the follicle is dependent on a delicate balance in the expression and actions of factors promoting follicular cell proliferation, growth and differentiation and of those inducing programmed cell death (apoptosis). As an important endocrine hormone, FSH binds to its granulosa cell receptors and promotes ovarian follicle survival and growth not only by stimulating proliferation and estradiol secretion of these cells, but also inhibiting the apoptosis by up-regulating the expression of intracellular anti-apoptotic proteins, such as XIAP and FLIP. In addition, intra-ovarian regulators, such as TGF-alpha and TNF-alpha, also play an important role in the control of follicular development and atresia. In response to FSH, Estradiol-17 beta synthesized from the granulosa cells stimulates thecal expression of TGF-alpha, which in turn increases granulosa cell XIAP expression and proliferation. The death receptor and ligand, Fas and Fas ligand, are expressed in granulosa cells following gonadotropin withdrawal, culminating in caspase-mediated apoptosis and follicular atresia. In contrast, TNF-alpha has both survival and pro-apoptotic function in the follicle, depending on the receptor subtype activated, but has been shown to promote granulosa cell survival by increasing XIAP and FLIP expression via the IkappaB-NFkappaB pathway. The pro-apoptotic action of TNF-alpha is mediated through the activation of caspases, via its receptor- (i.e. Caspases-8 and -3) and mitochrondria- (i.e. Caspase-9 and -3) death pathways. In the present manuscript, we have reviewed the actions and interactions of gonadotropins and intra-ovarian regulators in the control of granulosa cell fate and ultimately follicular destiny. We have highlighted the role and regulation of granulosa cell XIAP and FLIP expression, as well as their interactions with the death signaling pathways in the maintenance of granulosa cell survival during follicular development. We have provided strong evidence for these intracellular survival factors as key determinants for ovarian follicular destiny (growth versus atresia), the expression of which is regulated by a highly integrated endocrine, paracrine and autocrine mechanism. Further studies in these aspects will lead to a better understanding of the molecular and cellular regulation of follicular development and atresia, and provide invaluable insight into novel strategies in assisted reproduction in human infertility as well as in increasing reproductive efficiency in livestock industries.

Experimental results using 3-bromopyruvate in mesothelioma: in vitro and in vivo studies.

PubMed

Icard, Philippe; Philippe, Icard; Zhang, Xiao-Dong; Xiao-Dong, Zhang; Lemoisson, Edwige; Edwige, Lemoisson; Louis, Marie-Hélène; Marie-Hélène, Louis; Allouche, Stéphane; Stéphane, Allouche; Lincet, Hubert; Hubert, Lincet; Poulain, Laurent; Laurent, Poulain

2012-02-01

Over many years we have taken advantage of the special metabolism of cancer cells involving an increased consumption of glucose associated with lactic acid production even in the presence of oxygen, a phenomenon referred to as the "Warburg effect", to counteract cancer cell growth. We have tested 3-bromopyruvate (3-BrPA), an inhibitor of pyruvate-associated reactions. Firstly, we tested this agent, in vitro, in two mesothelioma cell lines. Cellular response would appear to depend on the mode of administration (immediately or 24 h after seeding). Depending on the line, 3-BrPA induced a cytostatic or cytotoxic effect. This effect was accompanied by cell death induction even in cells highly refractory to cisplatin. Mitochondrial apoptotic death appeared to involve both lines; however, a different death pathway such as necrosis cannot be excluded. Interestingly, 3-BrPA leads to a diminution of the expression of the anti-apotptoic protein Mcl-1. We then tested 3-BrPA in vivo. Survival of nude mice bearing human mesothelioma was significantly prolonged (p < 0.0001). Toxicity and clinical studies should be performed to test 3- BrPA as local therapy for patients suffering from pleural or peritoneal mesothelioma. Association with cisplatin should be particularly considered.

Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state.

PubMed

Zhang, Y; Chen, M; Venugopal, S; Zhou, Y; Xiang, W; Li, Y-H; Lin, Q; Kini, R M; Chong, Y-S; Ge, R

2011-05-05

Isthmin (ISM) is a 60 kDa secreted-angiogenesis inhibitor that suppresses tumor growth in mouse and disrupts vessel patterning in zebrafish embryos. It selectively binds to alphavbeta5 (αvβ5) integrin on the surface of endothelial cells (ECs), but the mechanism of its antiangiogenic action remains unknown. In this work, we establish that soluble ISM suppresses in vitro angiogenesis and induces EC apoptosis by interacting with its cell surface receptor αvβ5 integrin through a novel 'RKD' motif localized within its adhesion-associated domain in MUC4 and other proteins domain. ISM induces EC apoptosis through integrin-mediated death (IMD) by direct recruitment and activation of caspase-8 without causing anoikis. On the other hand, immobilized ISM loses its antiangiogenic function and instead promotes EC adhesion, survival and migration through αvβ5 integrin by activating focal adhesion kinase (FAK). ISM unexpectedly has both a pro-survival and death-promoting effect on ECs depending on its physical state. This dual function of a single antiangiogenic protein may impact its antiangiogenic efficacy in vivo.

Human-Specific Bacterial Pore-Forming Toxins Induce Programmed Necrosis in Erythrocytes

PubMed Central

LaRocca, Timothy J.; Stivison, Elizabeth A.; Hod, Eldad A.; Spitalnik, Steven L.; Cowan, Peter J.; Randis, Tara M.

2014-01-01

ABSTRACT A subgroup of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins (PFTs) has an unusually narrow host range due to a requirement for binding to human CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-linked complement regulatory molecule. hCD59-specific CDCs are produced by several organisms that inhabit human mucosal surfaces and can act as pathogens, including Gardnerella vaginalis and Streptococcus intermedius. The consequences and potential selective advantages of such PFT host limitation have remained unknown. Here, we demonstrate that, in addition to species restriction, PFT ligation of hCD59 triggers a previously unrecognized pathway for programmed necrosis in primary erythrocytes (red blood cells [RBCs]) from humans and transgenic mice expressing hCD59. Because they lack nuclei and mitochondria, RBCs have typically been thought to possess limited capacity to undergo programmed cell death. RBC programmed necrosis shares key molecular factors with nucleated cell necroptosis, including dependence on Fas/FasL signaling and RIP1 phosphorylation, necrosome assembly, and restriction by caspase-8. Death due to programmed necrosis in RBCs is executed by acid sphingomyelinase-dependent ceramide formation, NADPH oxidase- and iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products. Bacterial PFTs that are hCD59 independent do not induce RBC programmed necrosis. RBC programmed necrosis is biochemically distinct from eryptosis, the only other known programmed cell death pathway in mature RBCs. Importantly, RBC programmed necrosis enhances the growth of PFT-producing pathogens during exposure to primary RBCs, consistent with a role for such signaling in microbial growth and pathogenesis. PMID:25161188

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

Schock, Sarah C.; Edrissi, Hamidreza; Burger, Dylan

Highlights: • Microparticles are elevated in the plasma in a rodent model of chronic cerebral ischemia. • These microparticles initiate apoptosis in cultured cells. • Microparticles contain caspase 3 and they activate receptors for TNF-α and TRAIL. - Abstract: Circulating microparticles (MPs) are involved in many physiological processes and numbers are increased in a variety of cardiovascular disorders. The present aims were to characterize levels of MPs in a rodent model of chronic cerebral hypoperfusion (CCH) and to determine their signaling properties. MPs were isolated from the plasma of rats exposed to CCH and quantified by flow cytometry. When MPsmore » were added to cultured endothelial cells or normal rat kidney cells they induced cell death in a time and dose dependent manner. Analysis of pellets by electron microscopy indicates that cell death signals are carried by particles in the range of 400 nm in diameter or less. Cell death involved the activation of caspase 3 and was not a consequence of oxidative stress. Inhibition of the Fas/FasL signaling pathway also did not improve cell survival. MPs were found to contain caspase 3 and treating the MPs with a caspase 3 inhibitor significantly reduced cell death. A TNF-α receptor blocker and a TRAIL neutralizing antibody also significantly reduced cell death. Levels of circulating MPs are elevated in a rodent model of chronic cerebral ischemia. MPs with a diameter of 400 nm or less activate the TNF-α and TRAIL signaling pathways and may deliver caspase 3 to cultured cells.« less

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

Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells.

PubMed

Chen, Guokai; Hou, Zhonggang; Gulbranson, Daniel R; Thomson, James A

2010-08-06

Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies, their viability is significantly reduced. Here, we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs. Copyright 2010 Elsevier Inc. All rights reserved.

[Dose rate-dependent cellular and molecular effects of ionizing radiation].

PubMed

Przybyszewski, Waldemar M; Wideł, Maria; Szurko, Agnieszka; Maniakowski, Zbigniew

2008-09-11

The aim of radiation therapy is to kill tumor cells while minimizing damage to normal cells. The ultimate effect of radiation can be apoptotic or necrotic cell death as well as cytogenetic damage resulting in genetic instability and/or cell death. The destructive effects of radiation arise from direct and indirect ionization events leading to peroxidation of macromolecules, especially those present in lipid-rich membrane structures as well as chromatin lipids. Lipid peroxidative end-products may damage DNA and proteins. A characteristic feature of radiation-induced peroxidation is an inverse dose-rate effect (IDRE), defined as an increase in the degree of oxidation(at constant absorbed dose) accompanying a lower dose rate. On the other hand, a low dose rate can lead to the accumulation of cells in G2, the radiosensitive phase of the cell cycle since cell cycle control points are not sensitive to low dose rates. Radiation dose rate may potentially be the main factor improving radiotherapy efficacy as well as affecting the intensity of normal tissue and whole-body side effects. A better understanding of dose rate-dependent biological effects may lead to improved therapeutic intervention and limit normal tissue reaction. The study reviews basic biological effects that depend on the dose rate of ionizing radiation.

Low vitamin C and increased oxidative stress and cell death in mice that lack the sodium-dependent vitamin C transporter SVCT2.

PubMed

Harrison, F E; Dawes, S M; Meredith, M E; Babaev, V R; Li, L; May, J M

2010-09-01

The sodium-dependent vitamin C transporter (SVCT2) is responsible for the transport of vitamin C into cells in multiple organs, from either the blood or the cerebrospinal fluid. Mice null for SVCT2 (SVCT2(-/-)) do not survive past birth but the cause of death has not yet been ascertained. After mating of SVCT2(+/-) males and SVCT2(+/-) females, fewer SVCT2(-/-) and SVCT2(+/-) progeny were observed than would be expected according to Mendelian ratios. Vitamin C levels in SVCT2(-/-), SVCT2(+/-), and SVCT2(+/+) were genotype-dependent. SVCT2(-/-) fetuses had significantly lower vitamin C levels than littermates in placenta, cortex, and lung, but not in liver (the site of vitamin C synthesis). Low vitamin C levels in placenta and cortex were associated with elevations in several markers of oxidative stress: malondialdehyde, isoketals, F(2)-isoprostanes, and F(4)-neuroprostanes. Oxidative stress was not elevated in fetal SVCT2(-/-) lung tissue despite low vitamin C levels. In addition to the expected severe hemorrhage in cortex, we also found hemorrhage in the brain stem, which was accompanied by cell loss. We found evidence of increased apoptosis in SVCT2(-/-) mice and disruption of the basement membrane in fetal brain. Together these data show that SVCT2 is critical for maintaining vitamin C levels in fetal and placental tissues and that the lack of SVCT2, and the resulting low vitamin C levels, results in fetal death and, in SVCT2(-/-) mice that survive the gestation period, in oxidative stress and cell death. Copyright 2010 Elsevier Inc. All rights reserved.

High glucose-induced Ca2+ overload and oxidative stress contribute to apoptosis of cardiac cells through mitochondrial dependent and independent pathways.

PubMed

Kumar, Sandeep; Kain, Vasundhara; Sitasawad, Sandhya L

2012-07-01

Cardiac cell apoptosis is the initiating factor of cardiac complications especially diabetic cardiomyopathy. Mitochondria are susceptible to the damaging effects of elevated glucose condition. Calcium overload and oxidative insult are the two mutually non-exclusive phenomena suggested to cause cardiac dysfunction. Here, we examined the effect of high-glucose induced calcium overload in calpain-1 mediated cardiac apoptosis in an in vitro setting. H9c2, rat ventricular myoblast cell line was treated with elevated glucose condition and the cellular consequences were studied. Intracellular calcium trafficking, ROS generation, calpain-1 activation and caspase-12 and caspase-9 pathway were studied using flow cytometry, confocal microscopy and Western blot analysis. High-glucose treatment resulted in increased intracellular calcium ([Ca2+]i) which was mobilized to the mitochondria. Concomitant intra-mitochondrial calcium ([Ca2+]m) increase resulted in enhanced reactive oxygen and nitrogen species generation. These events led to mitochondrial dysfunction and apoptosis. Cardiomyocyte death exhibited several classical markers of apoptosis, including activation of caspases, appearance of annexin V on the outer plasma membrane, increased population of cells with sub-G0/G1 DNA content and nuclear condensation. Key findings include elucidation of cell signaling mechanism of high-glucose induced calcium-dependent cysteine protease calpain-1 activation, which triggers non-conventional caspases as alternate mode of cell death. This information increases the understanding of cardiac cell death under hyperglycemic condition and can possibly be extended for designing new therapeutic strategies for diabetic cardiomyopathy. The novel findings of the study reveal that high glucose induces apoptosis by both mitochondria-dependent and independent pathways via concomitant rise in intracellular calcium. Copyright © 2012 Elsevier B.V. All rights reserved.

Caspase-8 modulates Dectin-1 and CR3 driven IL-1β production in response to β-glucans and the fungal pathogen, Candida albicans1

PubMed Central

Ganesan, Sandhya; Rathinam, Vijay A. K.; Bossaller, Lukas; Army, Kelly; Kaiser, William J.; Mocarski, Edward S.; Dillon, Christopher P.; Green, Douglas R.; Mayadas, Tanya N.; Levitz, Stuart M.; Hise, Amy G.

2014-01-01

Inflammasomes are central mediators of host defense to a wide range of microbial pathogens. The NLRP3 inflammasome plays a key role in triggering caspase-1 dependent IL-1β maturation and resistance to fungal dissemination in Candida albicans infection. β-glucans are major components of fungal cell walls that trigger IL-1β secretion in both murine and human immune cells. In this study, we sought to determine the contribution of β-glucans to C. albicans-induced inflammasome responses in mouse dendritic cells. We show that the NLRP3-ASC-caspase-1 inflammasome is absolutely critical for IL-1β production in response to β-glucans. Interestingly, we also found that both Complement Receptor 3 (CR3/Mac-1) and dectin-1 play a crucial role in coordinating β-glucan-induced IL-1β processing as well as a cell death response. In addition to the essential role of caspase-1, we identify an important role for the pro-apoptotic protease caspase-8 in promoting β-glucan-induced cell death and NLRP3 inflammasome-dependent IL-1β maturation. A strong requirement for Complement Receptor 3 and caspase-8 was also found for NLRP3 dependent IL-1β production in response to heat killed Candida albicans. Together, these results define the importance of dectin-1, CR3 and caspase-8, in addition to the canonical NLRP3 inflammasome, in mediating β-glucan and C. albicans induced innate responses in dendritic cells. Collectively, these findings establish a novel link between β-glucan recognition receptors and the inflammatory proteases caspase-8 and caspase-1 in coordinating cytokine secretion and cell death in response to immunostimulatory fungal components. PMID:25063877

Synergistic Effect of Transient Receptor Potential Antagonist and Amiloride against Maitotoxin Induced Calcium Increase and Cytotoxicity in Human Neuronal Stem Cells.

PubMed

Boente-Juncal, Andrea; Vale, Carmen; Alfonso, Amparo; Botana, Luis M

2018-05-16

Maitotoxins (MTX) are among the most potent marine toxins identified to date causing cell death trough massive calcium influx. However, the exact mechanism for the MTX-induced calcium entry and cytotoxicity is still unknown. In this work, the effect of MTX-1 on the cytosolic free calcium concentration and cellular viability of human neuronal stem cells was evaluated. MTX elicited a concentration-dependent decrease in cell viability which was already evident after 1 h of treatment with 0.25 nM MTX; however, at a concentration of 0.1 nM, the toxin did not cause cell death even after 14 days of exposure. Moreover, the toxin caused a concentration dependent rise in the cytosolic calcium concentration which was maximal at toxin concentrations of 1 nM and dependent on the presence of extracellular calcium on the bathing solution. Several pharmacological approaches were employed to evaluate the role of canonical transient potential receptor channels (TRPC) on the MTX effects. The results presented here lead to the identification of the TRPC4 channels as contributors to the MTX effects in human neuronal cells. Both, the calcium increase and the cytotoxicity of MTX were either fully (for the calcium increase) or partially (in the case of cytotoxicity) reverted by the blockade of canonical TRPC4 receptors with the selective antagonist ML204. Furthermore, the sodium proton exchanger blocker amiloride also partially inhibited the calcium rise and the cell death elicited by MTX while the combination of amiloride and ML204 fully prevented both the cytotoxicity and the calcium rise elicited by the toxin.

A lysigenic programmed cell death-dependent process shapes schizogenously formed aerenchyma in the stems of the waterweed Egeria densa

PubMed Central

Bartoli, G.; Forino, L. M. C.; Durante, M.; Tagliasacchi, A. M.

2015-01-01

Background and Aims Plant adaptation to submergence can include the formation of prominent aerenchyma to facilitate gas exchange. The aim of this study was to characterize the differentiation of the constitutive aerenchyma in the stem of the aquatic macrophyte Egeria densa (Hydrocharitaceae) and to verify if any form of cell death might be involved. Methods Plants were collected from a pool in a botanical garden. Aerenchyma differentiation and apoptotic hallmarks were investigated by light microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay coupled with genomic DNA extraction and gel electrophoresis (DNA laddering assay). Cell viability and the occurrence of peroxides and nitric oxide (NO) were determined histochemically using specific fluorogenic probes. Key Results Aerenchyma differentiation started from a hexagonally packed pre-aerenchymatic tissue and, following a basipetal and centripetal developmental pattern, produced a honeycomb arrangement. After an early schizogenous differentiation process, a late lysigenous programmed cell death- (PCD) dependent mechanism occurred. This was characterized by a number of typical apoptotic hallmarks, including DNA fragmentation, chromatin condensation, apoptotic-like bodies, partial cell wall lysis and plasmolysis. In addition, local increases in H2O2 and NO were observed and quantified. Conclusions The differentiation of cortical aerenchyma in the stem of E. densa is a complex process, consisting of a combination of an early schizogenous differentiation mechanism and a late lysigenous PCD-dependent process. The PCD remodels the architecture of the gas spaces previously formed schizogenously, and also results in a reduction of O2-consuming cells and in recycling of material derived from the lysigenic dismantling of the cells. PMID:26002256

A lysigenic programmed cell death-dependent process shapes schizogenously formed aerenchyma in the stems of the waterweed Egeria densa.

PubMed

Bartoli, G; Forino, L M C; Durante, M; Tagliasacchi, A M

2015-07-01

Plant adaptation to submergence can include the formation of prominent aerenchyma to facilitate gas exchange. The aim of this study was to characterize the differentiation of the constitutive aerenchyma in the stem of the aquatic macrophyte Egeria densa (Hydrocharitaceae) and to verify if any form of cell death might be involved. Plants were collected from a pool in a botanical garden. Aerenchyma differentiation and apoptotic hallmarks were investigated by light microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay coupled with genomic DNA extraction and gel electrophoresis (DNA laddering assay). Cell viability and the occurrence of peroxides and nitric oxide (NO) were determined histochemically using specific fluorogenic probes. Aerenchyma differentiation started from a hexagonally packed pre-aerenchymatic tissue and, following a basipetal and centripetal developmental pattern, produced a honeycomb arrangement. After an early schizogenous differentiation process, a late lysigenous programmed cell death- (PCD) dependent mechanism occurred. This was characterized by a number of typical apoptotic hallmarks, including DNA fragmentation, chromatin condensation, apoptotic-like bodies, partial cell wall lysis and plasmolysis. In addition, local increases in H2O2 and NO were observed and quantified. The differentiation of cortical aerenchyma in the stem of E. densa is a complex process, consisting of a combination of an early schizogenous differentiation mechanism and a late lysigenous PCD-dependent process. The PCD remodels the architecture of the gas spaces previously formed schizogenously, and also results in a reduction of O2-consuming cells and in recycling of material derived from the lysigenic dismantling of the cells. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Crosslinking CD4 by human immunodeficiency virus gp120 primes T cells for activation-induced apoptosis

PubMed Central

1992-01-01

During human immunodeficiency virus (HIV) infection there is a profound and selective decrease in the CD4+ population of T lymphocytes. The mechanism of this depletion is not understood, as only a small fraction of all CD4+ cells appear to be productively infected with HIV-1 in seropositive individuals. In the present study, crosslinking of bound gp120 on human CD4+ T cells followed by signaling through the T cell receptor for antigen was found to result in activation-dependent cell death by a form of cell suicide termed apoptosis, or programmed cell death. The data indicate that even picomolar concentrations of gp120 prime T cells for activation-induced cell death, suggesting a mechanism for CD4+ T cell depletion in acquired immune deficiency syndrome (AIDS), particularly in the face of concurrent infection and antigenic challenge with other organisms. These results also provide an explanation for the enhancement of infection by certain antibodies against HIV, and for the paradox that HIV appears to cause AIDS after the onset of antiviral immunity. PMID:1402655

Endocytosis of Albumin by Podocytes Elicits an Inflammatory Response and Induces Apoptotic Cell Death

PubMed Central

Okamura, Kayo; Dummer, Patrick; Kopp, Jeffrey; Qiu, Liru; Levi, Moshe; Faubel, Sarah; Blaine, Judith

2013-01-01

The presence of albuminuria is strongly associated with progression of chronic kidney disease. While albuminuria has been shown to injure renal proximal tubular cells, the effects of albumin on podocytes have been less well studied. We have addressed the hypothesis that exposure of podocytes to albumin initiates an injury response. We studied transformed human-urine derived podocytes-like epithelial cells (HUPECS, or podocytes). Upon differentiation, these cells retain certain characteristics of differentiated podocytes, including expression of synaptopodin, CD2AP, and nestin. We exposed podocytes to recombinant human albumin, which lacks lipids and proteins that bind serum albumin; this reagent allowed a direct examination of the effects of albumin. Podocytes endocytosed fluoresceinated albumin and this process was inhibited at 4°C, suggesting an energy-dependent process. Exposure to albumin at concentrations of 5 and 10 mg/ml was associated with increased cell death in a dose-dependent manner. The mechanism of cell death may involve apoptosis, as caspase 3/7 were activated and the pan-caspase inhibitor z-VAD reduced cell death. Albumin exposure also increased nuclear factor (NF)-κB activation and increased transcription and release of interleukin (IL-) 1β, tumor necrosis factor (TNF), and IL-6. We extended these findings to an in vivo model. Glomeruli isolated from mice with nephrotic syndrome also had increased expression of IL-1β and TNF RNA. These data suggest that while podocyte injury begets albuminuria, albumin in the glomerular ultrafiltrate may also beget podocyte injury. Thus, an additional mechanism by which anti-proteinuric therapies are beneficial in the treatment of glomerular diseases may be a reduction in injury to the podocyte by albumin. PMID:23382978

Tissue factor deficiency increases alveolar hemorrhage and death in influenza A virus-infected mice.

PubMed

Antoniak, S; Tatsumi, K; Hisada, Y; Milner, J J; Neidich, S D; Shaver, C M; Pawlinski, R; Beck, M A; Bastarache, J A; Mackman, N

2016-06-01

Essentials H1N1 Influenza A virus (IAV) infection is a hemostatic challenge for the lung. Tissue factor (TF) on lung epithelial cells maintains lung hemostasis after IAV infection. Reduced TF-dependent activation of coagulation leads to alveolar hemorrhage. Anticoagulation might increase the risk for hemorrhages into the lung during severe IAV infection. Background Influenza A virus (IAV) infection is a common respiratory tract infection that causes considerable morbidity and mortality worldwide. Objective To investigate the effect of genetic deficiency of tissue factor (TF) in a mouse model of IAV infection. Methods Wild-type mice, low-TF (LTF) mice and mice with the TF gene deleted in different cell types were infected with a mouse-adapted A/Puerto Rico/8/34 H1N1 strain of IAV. TF expression was measured in the lungs, and bronchoalveolar lavage fluid (BALF) was collected to measure extracellular vesicle TF, activation of coagulation, alveolar hemorrhage, and inflammation. Results IAV infection of wild-type mice increased lung TF expression, activation of coagulation and inflammation in BALF, but also led to alveolar hemorrhage. LTF mice and mice with selective deficiency of TF in lung epithelial cells had low basal levels of TF and failed to increase TF expression after infection; these two strains of mice had more alveolar hemorrhage and death than controls. In contrast, deletion of TF in either myeloid cells or endothelial cells and hematopoietic cells did not increase alveolar hemorrhage or death after IAV infection. These results indicate that TF expression in the lung, particularly in epithelial cells, is required to maintain alveolar hemostasis after IAV infection. Conclusion Our study indicates that TF-dependent activation of coagulation is required to limit alveolar hemorrhage and death after IAV infection. © 2016 International Society on Thrombosis and Haemostasis.

Oxidative stress-dependent contribution of HMGB1 to the interplay between apoptosis and autophagy in diabetic rat liver.

PubMed

Petrović, Anja; Bogojević, Desanka; Korać, Aleksandra; Golić, Igor; Jovanović-Stojanov, Sofija; Martinović, Vesna; Ivanović-Matić, Svetlana; Stevanović, Jelena; Poznanović, Goran; Grigorov, Ilijana

2017-11-01

The progression of oxidative stress, resulting cell damage, and cell death underlies the etiology of liver damage/dysfunction as a complication of diabetes. High-mobility group box 1 (HMGB1) protein, a chromatin-binding nuclear protein and damage-associated molecular pattern molecule, is integral to oxidative stress and signaling pathways regulating cell death and cell survival. We previously found that in streptozotocin (STZ)-induced diabetic rats, reduction of oxidative stress after melatonin administration lowered necrotic cell death and increased expression of HMGB1 and hepatocellular damage. In the present study, we examined whether alleviation of diabetes-attendant oxidative stress and ensuing change in HMGB1 expression influence the dynamic equilibrium between apoptosis/autophagy and liver damage. We observed that elevated HMGB1 protein levels in diabetic rat liver accompanied increased interactions of HMGB1 with TLR4 and RAGE, and activation of the intrinsic apoptotic pathway and Beclin 1-dependent autophagy. The absence of p62 degradation in diabetic rat liver pointed to defective autophagy which was responsible for lower autophagosome/autophagolysosome formation and an increased apoptosis/autophagy ratio. Compared to diabetic rats, in melatonin-treated diabetic rats, the structure of liver cells was preserved, HMGB1/TLR4 interaction and downstream apoptotic signaling were significantly reduced, HMGB1/Beclin 1 colocalization and interactions were augmented and Beclin 1-mediated autophagy, mithophagy in particular, were increased. We concluded that in mild oxidative stress, HMGB1 is cytoprotective, whereas in intense oxidative stress, HMGB1 actions promote cell death and liver damage. Since reduced HMGB1 binds to RAGE but not to TLR4, redox modification of HMGB1 as a mechanism regulating the cross-talk between apoptosis and autophagy in diabetes is discussed.