Possible Role of Peroxynitrite in the Responses Induced by Fusicoccin in Plant Cultured Cells

Fusicoccin (FC) is a well-known phytotoxin able to induce in Acer pseudoplatanus L. (sycamore) cultured cells, a set of responses similar to those induced by stress conditions. In this work, the possible involvement of peroxynitrite (ONOO−) in FC-induced stress responses was studied measuring both in the presence and in the absence of 2,6,8-trihydroxypurine (urate), a specific ONOO− scavenger: (1) cell death; (2) specific DNA fragmentation; (3) lipid peroxidation; (4) production of RNS and ROS; (5) activity of caspase-3-like proteases; and (6) release of cytochrome c from mitochondria, variations in the levels of molecular chaperones Hsp90 in the mitochondria and Hsp70 BiP in the endoplasmic reticulum (ER), and of regulatory 14-3-3 proteins in the cytosol. The obtained results indicate a role for ONOO− in the FC-induced responses. In particular, ONOO− seems involved in a PCD form showing apoptotic features such as specific DNA fragmentation, caspase-3-like protease activity, and cytochrome c release from mitochondria.

Download Full-text

Role of F-actin organization in p38 MAP kinase-mediated apoptosis and necrosis in neonatal rat cardiomyocytes subjected to simulated ischemia and reoxygenation

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00462.2005 ◽

2005 ◽

Vol 289 (6) ◽

pp. H2310-H2318 ◽

Cited By ~ 37

Author(s):

Takayuki Okada ◽

Hajime Otani ◽

Yue Wu ◽

Shiori Kyoi ◽

Chiharu Enoki ◽

...

Keyword(s):

Cytochrome C ◽

Map Kinase ◽

Dna Fragmentation ◽

P38 Mapk ◽

Caspase 3 ◽

Neonatal Rat ◽

Cytochrome C Release ◽

Actin Reorganization ◽

Ldh Release ◽

Sb 203580

Activation of p38 mitogen-activated protein (MAP) kinase (MAPK) has been implicated in the mechanism of cardiomyocyte (CMC) protection and injury. The p38 MAPK controversy may be related to differential effects of this kinase on apoptosis and necrosis. We have hypothesized that p38 MAPK-mediated F-actin reorganization promotes apoptotic cell death, whereas it protects from osmotic stress-induced necrotic cell death. Cultured neonatal rat CMCs were subjected to 2 h of simulated ischemia followed by reoxygenation. p38 MAPK activity measured by phosphorylation of MAP kinase-activated protein (MAPKAP) kinase 2 was increased during simulated ischemia and reoxygenation. This was associated with translocation of heat shock protein 27 (HSP27) from the cytosolic to the cytoskeletal fraction and F-actin reorganization. Cytochrome c release from mitochondria, caspase-3 activation, and DNA fragmentation were increased during reoxygenation. Robust lactate dehydrogenase (LDH) release was observed under hyposmotic (140 mosM) reoxygenation. The p38 MAPK inhibitor SB-203580 abrogated activation of p38 MAPK, translocation of HSP27, and F-actin reorganization and prevented cytochrome c release, caspase-3 activation, and DNA fragmentation. Conversely, SB-203580 enhanced LDH release during hyposmotic reoxygenation. The F-actin disrupting agent cytochalasin D inhibited F-actin reorganization and prevented cytochrome c release, caspase-3 activation, and DNA fragmentation, whereas it enhanced LDH release during hyposmotic reoxygenation. When CMCs were incubated under the isosmotic condition for the first 15 min of reoxygenation, SB-203580 and cytochalasin D increased ATP content of CMCs and prevented LDH release after the conversion to the hyposmotic condition. These results suggest that F-actin reorganization mediated by activation of p38 MAPK plays a differential role in apoptosis and protection against osmotic stress-induced necrosis during reoxygenation in neonatal rat CMCs; however, the sarcolemmal fragility caused by p38 MAPK inhibition can be reversed during temporary blockade of physical stress during reoxygenation.

Download Full-text

Hyperglycemia Enhances DNA Fragmentation after Transient Cerebral Ischemia

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/00004647-200105000-00011 ◽

2001 ◽

Vol 21 (5) ◽

pp. 568-576 ◽

Cited By ~ 25

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.

Download Full-text

Polyamines are required for activation of c-Jun NH2-terminal kinase and apoptosis in response to TNF-α in IEC-6 cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00206.2003 ◽

2003 ◽

Vol 285 (5) ◽

pp. G980-G991 ◽

Cited By ~ 40

Author(s):

Sujoy Bhattacharya ◽

Ramesh M. Ray ◽

Mary Jane Viar ◽

Leonard R. Johnson

Keyword(s):

Cytochrome C ◽

Dna Fragmentation ◽

Caspase 3 ◽

Specific Inhibitor ◽

Cell Types ◽

Caspase 9 ◽

Cytochrome C Release ◽

Tnf Α ◽

Induced Apoptosis ◽

Jnk Activation

Intracellular polyamine homeostasis is important for the regulation of cell proliferation and apoptosis and is necessary for the balanced growth of cells and tissues. Polyamines have been shown to play a role in the regulation of apoptosis in many cell types, including IEC-6 cells, but the mechanism is not clear. In this study, we analyzed the mechanism by which polyamines regulate the process of apoptosis in response to tumor necrosis factor-α (TNF-α). TNF-α or cycloheximide (CHX) alone did not induce apoptosis in IEC-6 cells. Significant apoptosis was observed when CHX was given along with TNF-α, as indicated by a significant increase in the detachment of cells, caspase-3 activity, and DNA fragmentation. Polyamine depletion by treatment with α-difluoromethylornithine significantly reduced the level of apoptosis, as judged by DNA fragmentation and the caspase-3 activity of attached cells. Apoptosis in IEC-6 cells was accompanied by the activation of upstream caspases-6, -8, and -9 and NH2-terminal c-Jun kinase (JNK). Inhibition of JNK activation prevented caspase-9 activation. Polyamine depletion prevented the activation of JNK and of caspases-6, -8, -9, and -3. SP-600125, a specific inhibitor of JNK activation, prevented cytochrome c release from mitochondria, JNK activation, DNA fragmentation, and caspase-9 activation in response to TNF-α/CHX. In conclusion, we have shown that polyamine depletion delays and decreases TNF-α-induced apoptosis in IEC-6 cells and that apoptosis is accompanied by the release of cytochrome c, the activation of JNK, and of upstream caspases as well as caspase-3. Polyamine depletion prevented JNK activation, which may confer protection against apoptosis by modulation of upstream caspase-9 activation.

Download Full-text

Beauvericin induces cytotoxic effects in human acute lymphoblastic leukemia cells through cytochrome c release, caspase 3 activation: the causative role of calcium

Cancer Letters ◽

10.1016/j.canlet.2004.06.005 ◽

2004 ◽

Vol 216 (2) ◽

pp. 165-173 ◽

Cited By ~ 99

Author(s):

Guey-Mei Jow ◽

Cheng-Jen Chou ◽

Bing-Fang Chen ◽

Jia-Huei Tsai

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Cytochrome C ◽

Caspase 3 ◽

Lymphoblastic Leukemia ◽

Leukemia Cells ◽

Causative Role ◽

Cytochrome C Release ◽

Cytotoxic Effects ◽

Human Acute Lymphoblastic Leukemia

Download Full-text

Role of Caspase-3 Activation in Cerebral Ischemia-Induced Neurodegeneration in Adult and Neonatal Brain

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/00004647-200204000-00006 ◽

2002 ◽

Vol 22 (4) ◽

pp. 420-430 ◽

Cited By ~ 112

Author(s):

Ramanjit Gill ◽

Marc Soriano ◽

Klas Blomgren ◽

Henrik Hagberg ◽

Remy Wybrecht ◽

...

Keyword(s):

Cerebral Ischemia ◽

Cytochrome C ◽

Caspase 3 ◽

Neuroprotective Effect ◽

Focal Ischemia ◽

Adult Brain ◽

Cytosolic Fraction ◽

Cytochrome C Release

These studies have addressed the role of caspase-3 activation in neuronal death after cerebral ischemia in different animal models. The authors were unable to show activation of procaspase-3 measured as an induction of DEVDase (Asp-Glu-Val-Asp) activity after focal or transient forebrain ischemia in rats. DEVDase activity could not be induced in the cytosolic fraction of the brain tissue obtained from these animals by exogenous cytochrome c/dATP and Ca2+. However, the addition of granzyme B to these cytosolic fractions resulted in a significant activation of DEVDase, confirming that the conditions were permissive to analyze proteolytic cleavage of the DEVD-AMC (7-amino-4-methyl-coumarin) substrate. Consistent with these findings, zVal-Ala-Asp-fluoromethylketone administered after focal ischemia did not have a neuroprotective effect. In contrast to these findings, a large increase in DEVDase activity was detected in a model of hypoxic-ischemia in postnatal-day-7 rats. Furthermore, in postnatal-day-7 animals treated with MK-801, in which it has been suggested that excessive apoptosis is induced, the authors were unable to detect activation of DEVDase activity but were able to induce it in vitro by the addition of cytochrome c/dATP and Ca2+ to the cytosolic fraction. Analysis of cytochrome c distribution did not provide definitive evidence for selective cytochrome c release in the permanent focal ischemia model, whereas in the transient model a small but consistent amount of cytochrome c was found in the cytosolic fraction. However, in both models the majority of cytochrome c remained associated with the mitochondrial fraction. In conclusion, the authors were unable to substantiate a role of mitochondrially derived cytochrome c and procaspase-3 activation in ischemia-induced cell death in adult brain, but did see a clear induction of caspase-3 in neonatal hypoxia.

Download Full-text

Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury

Oncogene ◽

10.1038/sj.onc.1202590 ◽

1998 ◽

Vol 17 (26) ◽

pp. 3401-3415 ◽

Cited By ~ 226

Author(s):

Pothana Saikumar ◽

Zheng Dong ◽

Yogi Patel ◽

Kristi Hall ◽

Ulrich Hopfer ◽

...

Keyword(s):

Cytochrome C ◽

Cytochrome C Release ◽

Reoxygenation Injury

Download Full-text

LPA protects intestinal epithelial cells from apoptosis by inhibiting the mitochondrial pathway

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00406.2002 ◽

2003 ◽

Vol 284 (5) ◽

pp. G821-G829 ◽

Cited By ~ 54

Author(s):

Wenlin Deng ◽

De-An Wang ◽

Elvira Gosmanova ◽

Leonard R. Johnson ◽

Gabor Tigyi

Keyword(s):

Epithelial Cells ◽

Cytochrome C ◽

Protein Expression ◽

Caspase 3 ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial ◽

Caspase 9 ◽

Cytochrome C Release ◽

Apoptotic Pathway ◽

Antiapoptotic Activity

We previously showed ( Gastroenterology 123: 206–216, 2002) that lysophosphatidic acid (LPA) protects and rescues rat intestinal epithelial cells (IEC-6) from apoptosis. Here, we provide evidence for the LPA-elicited inhibition of the mitochondrial apoptotic pathway leading to attenuation of caspase-3 activation. Pretreatment of IEC-6 cells with LPA inhibited campothecin-induced caspase-9 and caspase-3 activation and DNA fragmentation. A caspase-9 inhibitor peptide mimicked the LPA-elicited antiapoptotic activity. LPA elicited ERK1/ERK2 and PKB/Akt phosphorylation. The LPA-elicited antiapoptotic activity and inhibition of caspase-9 activity were abrogated by pertussis toxin, PD 98059, wortmannin, and LY 294002. LPA reduced cytochrome c release from mitochondria and prevented activation of caspase-9. LPA prevented translocation of Bax from cytosol to mitochondria and increased the expression of the antiapoptotic Bcl-2 mRNA and protein. LPA had no effect on Bcl-xl, Bad, and Bak mRNA or protein expression. These data indicate that LPA protects IEC-6 cells from camptothecin-induced apoptosis through Gi-coupled inhibition of caspase-3 activation mediated by the attenuation of caspase-9 activation due to diminished cytochrome c release, involving upregulation of Bcl-2 protein expression and prevention of Bax translocation.

Download Full-text

Activation of caspase-3 may not contribute to postresuscitation myocardial dysfunction

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00338.2008 ◽

2009 ◽

Vol 296 (4) ◽

pp. H1164-H1174 ◽

Cited By ~ 10

Author(s):

Jeejabai Radhakrishnan ◽

Iyad M. Ayoub ◽

Raúl J. Gazmuri

Keyword(s):

Cytochrome C ◽

Dna Fragmentation ◽

Rat Model ◽

Caspase 3 ◽

Myocardial Dysfunction ◽

Left Ventricular ◽

Stroke Work ◽

Protective Effects ◽

Apoptotic Pathway ◽

Apoptosis Protein

We have previously reported that postresuscitation myocardial dysfunction is accompanied by the release of cytochrome c and caspase-3 activation. We now investigated the role of caspase-3 activation by examining whether such process prompts apoptotic DNA fragmentation, whether caspase-3 inhibition attenuates myocardial dysfunction, and whether myocardial protective effects of sodium-hydrogen exchanger isoform-1 (NHE-1) inhibition involve caspase-3 inhibition using a rat model of ventricular fibrillation (VF) of closed-chest resuscitation. Resuscitation after 4 or 8 min of untreated VF caused significant reductions in left ventricular stroke work index averaging 23% of sham control rats at 4 h postresuscitation. Left ventricular dysfunction was accompanied by increases in cytosolic cytochrome c, decreases in pro- and cleaved caspase-9 fragments, increases in 17-kDa caspase-3 fragments, and increases in caspase-3 activity indicating the activation of the mitochondrial apoptotic pathway but without evidence of apoptotic DNA fragmentation. In addition, levels of heat shock protein 70 were increased and levels of X-linked inhibitor of apoptosis protein and αβ-crystallin were preserved, all of which can exert antiapoptotic effects. In a separate series, the caspase-3 inhibitor z-Asp-Glu-Val-Asp chloromethyl ketone given before the induction of VF failed to prevent postresuscitation myocardial dysfunction despite reductions in caspase-3 activity (2.3 ± 0.5 vs. 1.3 ± 0.5 pmol fluorophore AFC released·mg protein−1·min−1; P < 0.03). Treatment with the NHE-1 inhibitor cariporide had no effect on caspase-3 activity. Accordingly, in this rat model of VF and severe postresuscitation myocardial dysfunction, activation of caspase-3 did not lead to DNA fragmentation or contribute to myocardial dysfunction. Concomitant activation of intrinsic antiapoptotic mechanisms could play a protective role downstream to caspase-3 activation.

Download Full-text