scholarly journals Antimycin A-induced cell death depends on AIF translocation through NO production and PARP activation and is not involved in ROS generation, cytochrome c release and caspase-3 activation in HL-60 cells

2009 ◽  
Vol 62 (3) ◽  
pp. 145-152 ◽  
Author(s):  
Masaki Ogita ◽  
Akira Ogita ◽  
Yoshinosuke Usuki ◽  
Ken-ichi Fujita ◽  
Toshio Tanaka
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Caspase 3 ◽  
Ros Generation ◽  
No Production ◽  
Antimycin A ◽  
Cytochrome C Release ◽  
Parp Activation

scholarly journals Hyperglycemia Enhances DNA Fragmentation after Transient Cerebral Ischemia

2001 ◽  
Vol 21 (5) ◽  
pp. 568-576 ◽  
Author(s):  
Ping-An Li ◽  
Ingrid Rasquinha ◽  
Qing Ping He ◽  
Bo K. Siesjö ◽  
Katalin Csiszár ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Cell Death ◽  
Cytochrome C ◽  
Dna Fragmentation ◽  
Caspase 3 ◽  
Cingulate Cortex ◽  
Tunel Staining ◽  
Dna Fragments ◽  
Cytochrome C Release ◽  
Klenow Polymerase

Previous histopathologic results have suggested that one mechanism whereby hyperglycemia (HG) leads to exaggerated ischemic damage involves fragmentation of DNA. DNA fragmentation in normoglycemia (NG) and HG rats subjected to 30 minutes of forebrain ischemia was studied by terminal deoxynucleotidyl transferase mediated DNA nick-labeling (TUNEL) staining, by pulse-field gel electrophoresis (PFGE), and by ligation-mediated polymerase chain reaction (LM-PCR). High molecular weight DNA fragments were detected by PFGE, whereas low molecular weight DNA fragments were detected using LM-PCR techniques. The LM-PCR procedure was performed on DNA from test samples with blunt (without Klenow polymerase) and 3′-recessed ends (with Klenow polymerase). In addition, cytochrome c release and caspase-3 activation were studied by immunocytochemistry. Results show that HG causes cytochrome c release, activates caspase-3, and exacerbates DNA fragments induced by ischemia. Thus, in HG rats, but not in control or NGs, TUNEL-stained cells were found in the cingulate cortex, neocortex, thalamus, and dorsolateral crest of the striatum, where neuronal death was observed by conventional histopathology, and where both cytosolic cytochrome c and active caspase-3 were detected by confocal microscopy. In the neocortex, both blunt-ended and stagger-ended fragments were detected in HG, but not in NG rats. Electron microscopy (EM) analysis was performed in the cingulate cortex, where numerous TUNEL-positive neurons were observed. Although DNA fragmentation was detected by TUNEL staining and electrophoresis techniques, EM analysis failed to indicate apoptotic cell death. It is concluded that HG triggers a cell death pathway and exacerbates DNA fragmentation induced by ischemia.

In-Vitro Treatment with the AKT-Inhibitor GSK 690693 Induces Cell Death in Lymphoma Cell Lines and in Primary CLL Cells and Is Followed by Caspase-3 Activation and Cytochrome C Release.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1589-1589
Author(s):  
Dirk Winkler ◽  
Thorsten Zenz ◽  
Daniel Mertens ◽  
Annett Habermann ◽  
Hartmut Döhner ◽  
...  
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Conditioned Medium ◽  
Cell Lines ◽  
Caspase 3 ◽  
Spontaneous Apoptosis ◽  
Akt Inhibitor ◽  
Cytochrome C Release ◽  
Viable Cells

Abstract The PI3K/AKT pathway acts as a critical regulator of cell survival by stimulating cell proliferation and inhibiting apoptosis and has been implicated in the pathogenesis of lymphoproliferative disorders. Therefore, inhibition of AKT seems to be a highly attractive new approach for the treatment of lymphoma. We treated 9 cell lines with AKT-nhibitor (1, 10, 20 μM) over 24h and 48h respectively: EHEB (B-CLL), GRANTA-519 (MCL), JURKAT (T-ALL) BL-60, NAMALWA and BJAB (all Burkitt’s lymphoma), L363, OPM-2 and RPMI-8226 (all multiple myeloma). To determine the rates and type of AKT-inhibitor induced cell death, FACS analyses for CD19, 7AAD, active caspase-3, cytochrome c were performed. The phosphorylation status of AKT and its downstream proteins GSK3β, p70S6k and S6 was studied by Western blotting after 5–120 minutes. In addition, 11 primary CLL samples with either del 13q (n=3), del 11q (n=2), del 17p (n=3) or a normal karyotype (n=3) were treated with AKT inhibitor (10 μM; 2.5μM; 0.625 μM; 0.156 μM). CLL samples were cultured in both standard medium as well as in HS5-(human stromal cells) conditioned medium to reduce spontaneous apoptosis of CLL in-vitro. 6 out of 11 patients had unmutated VH genes. 8 Patients were untreated, 3 were previously treated. Fludarabine (0.1 μM) was added to AKT-inhibitor in 11 cases to test for synergistic effects. CLL cells were harvested after 48 hours and 5 days to measure cell viability using Celltiter-GLO-Assay. Treatment of cell lines lead to significant rates of AKT-inhibitor induced cell death (table 1), to hyperphosphorylation of AKT and to inhibition of phosphorylation of GSK3β (after 5 min) and S6 (after 20 min) in all cell lines and of p70S6k (after 120 min) in GRANTA, JURKAT, NAMALWA and BJAB. Cell death did not depend on functional p53 gene. Treatment of primary CLL samples with AKT-inhibitor alone was followed by a decrease of cell viability in a time and concentration dependent manner regardless of the medium used (table 2). Only with the lowest concentration and when cultured in HS5-conditioned medium, no further reduction of viable cells was seen between 48h and 5d. Treatment with AKT-inhibitor as a single agent seemed to be at least as effective as treatment with fludarabine. Response was independent of the genetic subgroup, VH mutation status or prior treatment. High risk cases with del 17p responded worse to fludarabine alone when compared to cases without del 17p (i.e. 75% of viable cells after 5d at 10000 μM in cases with del 17p vs. 25% in cases without del 17p). The same fludarabine resistant cases showed good responses to treatment with AKT-inhibitor (9% of viable cells after 5d at 10000 μM in cases with del 17p). A synergistic effect was not achieved by combining AKT-inhibitor and fludarabine. Culture of CLL cells in HS5-conditioned medium resulted in lower rates of spontaneous apoptosis, but also in lower rates of AKT-inhibitor induced cell death. In conclusion, in-vitro treatment with AKT-inhibitor resulted in significant rates of cell death in cell lines and primary CLL cells, even in patients with del 17p or resistance to fludarabine. In cell lines, treatment with AKT-inhibitor was followed by typical features of apoptosis such as activation of caspase-3 and cytochrome c release. In CLL samples, prior treatment did not affect in-vitro response rates. These data underline the involvement of the PI3K/Akt pathway in the pathogenesis of lymphoma and point to an efficacy of the AKT-inhibitor in the treatment of lymphoma, multiple myeloma and CLL in-vivo. Concerning CLL, the AKT-inhibitor seems to be an attractive new treatment option even for cases with high risk cytogenetics. Using HS5-conditioned medium seems to be a well functioning method to reduce spontaneous apoptosis of CLL cells in-vitro. Table 1: rates of cell death, caspase-3 activation and cytochrome c release after treatment of cell lines with AKT inhibitor (1μM, 48h) 7AAD-positive cells active caspase-3 cytochrome c release EHEB 15% − + GRANTA-519 15% + + JURKAT 17% + + BL60 24% + + NAMALWA 25% − (+) BJAB 30% + (+) L363 15% + − OPM-2 41% + + RPMI-8226 32% + (+) Table 2: mean percentage of viable cells after treatment with AKT-Inhibitor (A), fludarabine (F; 0,1μM) and their combination (A + F) measured by Celltiter-GLO-Assay 10000 nM 2500 nM 625 nM 156,25 nM 48h 5d 48h 5d 48h 5d 48h 5d A F A + F A F A + F A A F A+ F A F A+ F A HS5 + (n=8) 94 84 (n=5) 75 (n=5) 45 22 (n=5) 25 (n=5) 88 52 91 84 80 69 22 18 76 85 HS5 − (n=11) 60 79 59 8 39 21 77 27 80 79 76 28 39 34 82 (n=10) 21

scholarly journals Morphine inhibits doxorubicin-induced reactive oxygen species generation and nuclear factor κB transcriptional activation in neuroblastoma SH-SY5Y cells

2007 ◽  
Vol 406 (2) ◽  
pp. 215-221 ◽  
Author(s):  
Xin Lin ◽  
Qing Li ◽  
Yu-Jun Wang ◽  
Ya-Wen Ju ◽  
Zhi-Qiang Chi ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Transcriptional Activation ◽  
Caspase 3 ◽  
Reactive Oxygen Species Generation ◽  
Nuclear Factor Κb ◽  
Ros Generation ◽  
Nuclear Factor ◽  
Oxygen Species ◽  
Cytochrome C Release ◽  
Apoptotic Activity

Morphine is recommended as a first-line opioid analgesic in the pain management of cancer patients. Accumulating evidence shows that morphine has anti-apoptotic activity, but its impact on the therapeutic applications of antineoplastic drugs is not well known. The present study was undertaken to test the hypothesis that morphine might antagonize the pro-apoptotic activity of DOX (doxorubicin), a commonly used antitumour drug for the treatment of neuroblastoma, in cultured SH-SY5Y cells. In the present study we demonstrated that morphine suppressed DOX-induced inhibition of cell proliferation and programmed cell death in a concentration-dependent, and naloxone as well as pertussis toxin-irreversible, manner. Further studies showed that morphine inhibited ROS (reactive oxygen species) generation, and prevented DOX-mediated caspase-3 activation, cytochrome c release and changes of Bax and Bcl-2 protein expression. The antioxidant NAC (N-acetylcysteine) also showed the same effects as morphine on DOX-induced ROS generation, caspase-3 activation and cytochrome c release and changes in Bax (Bcl-2-associated X protein) and Bcl-2 protein expression. Additionally, morphine was found to suppress DOX-induced NF-κB (nuclear factor κB) transcriptional activation via a reduction of IκBα (inhibitor of nuclear factor κB) degradation. These present findings support the hypothesis that morphine can inhibit DOX-induced neuroblastoma cell apoptosis by the inhibition of ROS generation and mitochondrial cytochrome c release, as well as by blockade of NF-κB transcriptional activation, and suggests that morphine might have an impact on the antitumour efficiency of DOX.

scholarly journals The influence of melatonin on apoptosis of human neutrophils

2019 ◽  
Vol 73 ◽  
pp. 81-91
Author(s):  
Sylwia Mańka ◽  
Zbigniew Baj ◽  
Ewa Majewska
Keyword(s):  
Cytochrome C ◽  
Caspase 3 ◽  
Control Level ◽  
Human Neutrophils ◽  
Ros Generation ◽  
Main Role ◽  
Spontaneous Apoptosis ◽  
Cytochrome C Release ◽  
Tnf Α ◽  
Induced Apoptosis

Aim: Melatonin (Mel) besides its main role in circadian and seasonal rhythm coordination, plays a role in immunoregulation and inflammatory responses. The melatonin’s ability to modulate apoptosis is one of its important roles related to its effect on immune system but the exact effect of its action and the mechanisms of apoptosis control by melatonin remain still unclear. The goal of our study was to examine the involvement of melatonin in the apoptosis of human neutrophils in vitro and possible mechanisms of this action. Material/Methods: We measured the effect of melatonin on the spontaneous and TNF-α-induced apoptosis of human neutrophils using propidium iodide and Annexin-V and on caspase-3 activation, apoptosis-related surface antigen expressions, intracellular reactive oxygen species (ROS) generation and cytochrome c release using flow cytometry and commercial reagents. Results: Melatonin does not affect spontaneous apoptosis of human neutrophils and mitochondrial cytochrome c release but protects the cells from the significant rise of TNF-α-induced apoptosis and cytochrome c release. Intracellular ROS generation in PMA-stimulated neutrophils did not change after the influence of melatonin but the significant drop of ROS generation in neutrophils stimulated with TNF- α was upregulated to the control level after preincubation of the neutrophils with melatonin. Melatonin did not change significantly Fas, Fas-L and active caspase-3 expressions in neutrophils. Conclusions: Melatonin does not affect the spontaneous apoptosis, however, inhibits TNF-α-induced apoptosis of human neutrophils. Our findings suggest that the intrinsic pathway of the process is a result of the melatonin induced mitochondrial alterations.

Interleukin-11 antagonizes Fas ligand-mediated apoptosis in IEC-18 intestinal epithelial crypt cells: role of MEK and Akt-dependent signaling

2008 ◽  
Vol 294 (3) ◽  
pp. G728-G737 ◽  
Author(s):  
Kaitlin M. Naugler ◽  
Kathy A. Baer ◽  
Mark J. Ropeleski
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Dna Cleavage ◽  
Caspase 3 ◽  
Fas Ligand ◽  
Dependent Manner ◽  
Caspase 9 ◽  
Cytochrome C Release ◽  
Caspase Cleavage ◽  
Interleukin 11

Interleukin-11 (IL-11) displays epithelial cytoprotective effects during intestinal injury. Antiapoptotic effects of IL-11 have been described, yet mechanisms remain unclear. Fas/CD95 death receptor signaling is upregulated in ulcerative colitis, leading to mucosal breakdown. We hypothesized that IL-11 inhibits Fas ligand (FasL)-mediated apoptosis in intestinal epithelia. Cell death was monitored in IEC-18 cells by microscopy, caspase and poly(ADP-ribose) polymerase cleavage, mitochondrial release of cytochrome c, and abundance of cytoplasmic oligonucleosomal DNA. RT-PCR was used to monitor Fas, cIAP1, cIAP2, XIAP, cFLIP, survivin, and Bcl-2 family members. Fas membrane expression was detected by immunoblot. Inhibitors of JAK2, phosphatidylinositol 3-kinase (PI3-kinase), Akt 1, MEK1 and MEK2, and p38 MAPK were used to delineate IL-11's antiapoptotic mechanisms. IL-11 did not alter Fas expression. Pretreatment with IL-11 for 24 h before FasL reduced cytoplasmic oligonucleosomal DNA by 63.2%. IL-11 also attenuated caspase-3, caspase-9, and poly(ADP-ribose) polymerase cleavage without affecting expression of activated caspase-8 p20 or cytochrome c release. IL-11 did not affect mRNA expression of the candidate antiapoptotic genes. The MEK1 and MEK2 inhibitors U-0126 and PD-98059 significantly attenuated the protection of IL-11 against caspase-3 and caspase-9 cleavage and cytoplasmic oligonucleosomal DNA accumulation. Although Akt inhibition reversed IL-11-mediated effects on caspase cleavage, it did not reverse the protective effects of IL-11 by DNA ELISA. We conclude that IL-11-dependent MEK1 and MEK2 signaling inhibits FasL-induced apoptosis. The lack of reversal of the IL-11 effect on DNA cleavage by Akt inhibition, despite antagonism of caspase cleavage, suggests that IL-11 inhibits caspase-independent cell death signaling by FasL in a MEK-dependent manner.

Regulation of constitutive neutrophil apoptosis by the α,β-unsaturated aldehydes acrolein and 4-hydroxynonenal

2005 ◽  
Vol 289 (6) ◽  
pp. L1019-L1028 ◽  
Author(s):  
Erik I. Finkelstein ◽  
Jurjen Ruben ◽  
C. Wendy Koot ◽  
Milena Hristova ◽  
Albert van der Vliet
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Caspase 3 ◽  
Polymorphonuclear Neutrophils ◽  
Neutrophil Apoptosis ◽  
Mitochondrial Cytochrome ◽  
Cytochrome C Release ◽  
Unsaturated Aldehydes ◽  
Phosphatidylserine Exposure ◽  
Mitochondrial Cytochrome C

Reactive α,β-unsaturated aldehydes are major components of common environmental pollutants and are products of lipid oxidation. Although these aldehydes have been demonstrated to induce apoptotic cell death in various cell types, we recently observed that the α,β-unsaturated aldehyde acrolein (ACR) can inhibit constitutive apoptosis of polymorphonuclear neutrophils and thus potentially contribute to chronic inflammation. The present study was designed to investigate the biochemical mechanisms by which two representative α,β-unsaturated aldehydes, ACR and 4-hydroxynonenal (HNE), regulate neutrophil apoptosis. Whereas low concentrations of either aldehyde (<10 μM) mildly promoted apoptosis in neutrophils (reflected by increased phosphatidylserine exposure, caspase-3 activation, and mitochondrial cytochrome c release), higher concentrations prevented critical features of apoptosis (caspase-3 activation, phosphatidylserine exposure) and caused delayed neutrophil cell death with characteristics of necrosis/oncosis. Inhibition of caspase-3 activation by either aldehyde occurred despite increases in mitochondrial cytochrome c release and occurred in close association with depletion of cellular GSH and with cysteine modifications within caspase-3. However, procaspase-3 processing was also prevented, because of inhibited activation of caspases-9 and -8 under similar conditions, suggesting that ACR (and to a lesser extent HNE) can inhibit both intrinsic (mitochondria dependent) and extrinsic mechanisms of neutrophil apoptosis at initial stages. Collectively, our results indicate that α,β-unsaturated aldehydes can inhibit constitutive neutrophil apoptosis by common mechanisms, involving changes in cellular GSH status resulting in reduced activation of initiator caspases as well as inactivation of caspase-3 by modification of its critical cysteine residue.

scholarly journals Mitochondria-dependent and -independent Regulation of Granzyme B–induced Apoptosis

1999 ◽  
Vol 189 (1) ◽  
pp. 131-144 ◽  
Author(s):  
Glen MacDonald ◽  
Lianfa Shi ◽  
Christine Vande Velde ◽  
Judy Lieberman ◽  
Arnold H. Greenberg
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Caspase 3 ◽  
Granzyme B ◽  
Target Cells ◽  
Dependent Manner ◽  
Cytochrome C Release ◽  
Free System ◽  
S 100 ◽  
Induced Apoptosis

Granzyme B (GraB) is required for the efficient activation of apoptosis by cytotoxic T lymphocytes and natural killer cells. We find that GraB and perforin induce severe mitochondrial perturbation as evidenced by the release of cytochrome c into the cytosol and suppression of transmembrane potential (Δψ). The earliest mitochondrial event was the release of cytochrome c, which occurred at the same time as caspase 3 processing and consistently before the activation of apoptosis. Granzyme K/perforin or perforin treatment, both of which kill target cells efficiently but are poor activators of apoptosis in short-term assays, did not induce rapid cytochrome c release. However, they suppressed Δψ and increased reactive oxygen species generation, indicating that mitochondrial dysfunction is also associated with this nonapoptotic cell death. Pretreatment with peptide caspase inhibitors zVAD-FMK or YVAD-CHO prevented GraB apoptosis and cytochrome c release, whereas DEVD-CHO blocked apoptosis but did not prevent cytochrome c release, indicating that caspases act both up- and downstream of mitochondria. Of additional interest, Δψ suppression mediated by GraK or GraB and perforin was not affected by zVAD-FMK and thus was caspase independent. Overexpression of Bcl-2 and Bcl-XL suppressed caspase activation, mitochondrial cytochrome c release, Δψ suppression, and apoptosis and cell death induced by GraB, GraK, or perforin. In an in vitro cell free system, GraB activates nuclear apoptosis in S-100 cytosol at high doses, however the addition of mitochondria amplified GraB activity over 15-fold. GraB- induced caspase 3 processing to p17 in S-100 cytosol was increased only threefold in the presence of mitochondria, suggesting that another caspase(s) participates in the mitochondrial amplification of GraB apoptosis. We conclude that GraB-induced apoptosis is highly amplified by mitochondria in a caspase-dependent manner but that GraB can also initiate caspase 3 processing and apoptosis in the absence of mitochondria.

scholarly journals Radiation-mediated induction of apoptotic cell death in rat hippocampus

2013 ◽  
Vol 28 (2) ◽  
pp. 212-220
Author(s):  
Natasa Velickovic ◽  
Ana Djordjevic ◽  
Dunja Drakulic ◽  
Bojana Secerov ◽  
Ivana Grkovic ◽  
...  
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
P53 Protein ◽  
Rat Hippocampus ◽  
Normal Brain ◽  
Cytochrome C Release ◽  
Cranial Radiotherapy ◽  
Apoptotic Pathway

Ionizing radiation is commonly used in the treatment of brain tumors but it can impair cognitive functions, such as learning and memory. Since cognitive dysfunctions are predominantly result of cell death by apoptosis in hippocampal cells, in this study we analyzed acute effects of cranial gamma-irradiation (10 Gy) on expression of proapoptotic molecules (p53, Bax) and antiapoptotic molecule Bcl-2, as well as caspase-3 activation and cytochrome c redistribution in the hippocampus of young rats. The selected regimen of irradiation resembles the established animal model for childhood prophylactic cranial radiotherapy. Our results demonstrated that p53 mRNA expression was unchanged after irradiation, while induction of p53 protein was rapid. In parallel, Bax mRNA and protein levels were also increased following irradiation, whereas Bcl-2 expression was not changed during the examined post-irradiation period. These changes were accompanied with early hallmarks of apoptosis, such as increased cytochrome c release and stimulated activation of caspase-3. Overall, this study demonstrates that cranial irradiation is associated with the augmented apoptotic pathway in the rat hippocampus, which could be related to the cognitive decline observed in patients after prophylactic cranial radiotherapy, but also opens perspective in finding radioprotectors that can mitigate radiation injury of normal brain tissue.

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