benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and 3-aminobenzamide

Energy requiring apoptosis and presumably unregulated necrosis are considered conceptually and morphologically distinct forms of cell death which have been initially identified as two exclusive pathways. However, several apoptotic characteristics have been observed in the necrotic core lesion in ischemia which led to the controversial theory that cell death advances via a number of hybrid pathways among a continuum between the two processes. ATP availability has been shown to influence the decision between apoptosis and necrosis. The aims of our study are 1) to determine if combined inhibitors administration of pan-caspase inhibitor Carbobenzoxy-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) and non-selective poly (ADP-ribose) polymerase (PARP) inhibitor 3-aminobenzamide (3-AB) can further reduce infarct volume compared to single modality of either inhibitor following ischemic insult, 2) to ascertain the pharmacological intervention up to 24 hour post-middle cerebral artery occlusion (MCAo), and 3) to correlate intracellular ATP level with infarct volume. Single modality treatment was optimised at 3 mg/kg z-VAD-fmk and 30 mg/kg 3-AB with infarct volume measured at 24.13%+/-3.89% and 26.98%+/-2.22% respectively, while untreated control group was determined at 45.97%+/-1.86%. Combined inhibitors treatment rendered further reduction in infarct volume, measuring 7.228%+/-1.988%, 21.02%+/-1.06%, 24.40%+/-2.12% at 30 min, 6 h, 24 h post-ischemia respectively. In conclusion, the combined inhibitors administration of both z-VAD-fmk and 3-AB show further increased in infarct volume reduction with our ischemic model up to the 24 hour post-MCAo. However, in our in vivo study, no correlation between intracellular ATP level and infarct size was established.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Benzamides; Caspases; Cerebral Infarction; Drug Therapy, Combination; Energy Metabolism; Enzyme Inhibitors; Male; Necrosis; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; Stroke; Treatment Outcome

The chemical warfare agent sulfur mustard (SM) is a strong alkylating agent that leads to erythema and ulceration of the human skin several hours after exposure. Although SM has been intensively investigated, the cellular mechanisms leading to cell damage remain unclear. Apoptosis, necrosis and direct cell damage are discussed. In this study we investigated apoptotic cell death in pulmonary A549 cells exposed to SM (30-1000 microM, 30 min). 24 h after SM exposure DNA breaks were stained with the TUNEL method. Additionally, A549 cells were lysed and cellular protein was transferred to SDS page and blotted. Whole PARP as well as PARP cleavage into the p89 fragment, an indicator of apoptosis, were detected by specific antibodies. SM concentration dependent increase in TUNEL positive cells and PARP cleavage showed that SM is an inducer of apoptosis. It has been previously suggested that AChE is activated during apoptotic processes and may be involved in apoptosis regulation. Therefore, we examined AChE activity in A549 cells upon induction of apoptosis by SM (100-500 microM). Increased AChE activity was found in SM treated A549 cell cultures examined as determined by the Ellman's assay and by western blot. AChE activity showed a strong correlation with TUNEL positive cells. However, the broad caspase inhibitor zVAD and the PARP-inhibitor 3-aminobenzamide had no protective effect on A459 cells measured with AChE activity and frequency of TUNEL positive cells. In summary, our studies demonstrate that AChE activity may be a potential marker of apoptosis in A549 cells after SM injury. To what extent AChE is involved in apoptosis regulation during SM poisoning has to be further investigated.

Topics: Acetylcholinesterase; Amino Acid Chloromethyl Ketones; Apoptosis; Benzamides; Caspase 3; Caspase Inhibitors; Cell Extracts; Cell Line, Tumor; Chemical Warfare Agents; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Humans; Hydrolysis; Immunoblotting; In Situ Nick-End Labeling; Mustard Gas; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases

Oxidative stress, continuously exerted during chronic inflammation, has been implicated as a major causative agent of cellular dysfunction and cell death. In the present study, we investigated the impact of oxidative stress on the mode of cell death in HUVECs using H2O2 as a model reagent. We found that the predominant form of cell death was necrosis. Necrosis induction was accompanied by a distinct mode of caspase-3 cleavage, yielding a 29-kDa fragment. While inhibition of caspases could not prevent the generation of the 29-kDa fragment, general protease inhibitors, such as leupeptin and LLNL, proved to be effective in inhibiting the distinct processing pattern of caspase-3. These results suggest that caspases can act as substrates for non-caspase proteases in cells primed for necrosis induction. Thus, the pattern of caspase-3 cleavage might reflect the proteolytic system engaged in the cell death machinery in HUVECs.

Topics: Amino Acid Chloromethyl Ketones; Benzamides; Caspase 3; Caspase 7; Caspase Inhibitors; Caspases; Cell Line; Collagen Type XI; Cytochromes c; Dose-Response Relationship, Drug; Endothelial Cells; Enzyme Activation; Enzyme Inhibitors; Humans; Hydrogen Peroxide; Membrane Potentials; Mitochondria; Necrosis; Oxidants; Oxidative Stress; Time Factors; Umbilical Veins

During small-bowel transplantation, necrosis and apoptosis are involved in the destruction of intestinal epithelial cells. This study was conducted to assess which mode of cell death plays a greater role as a trigger of the bacterial translocation (BT) associated with intestinal transplantation.. The following experimental groups were studied: sham, Tx (intestinal transplantation), Tx + poly (ADP-ribose) synthetase (PARS) inhibitor 3-aminobenzamide (3-AB), and Tx + caspase inhibitor Z-VAD-fmk. Histological analysis, caspase-3 activity, DNA fragmentation, and BT were measured in tissue samples after transplantation.. During intestinal transplantation, apoptosis and necrosis both increased, showing graft injury and high levels of BT. Rats treated with 3-AB showed histological protection of the transplanted graft and a tendency toward lower BT despite the existence of high apoptosis levels. The rats treated with Z-VAD showed histological protection of the transplanted graft and decreased levels of caspase-3 and DNA fragmentation. The Tx + Z-VAD group showed the lowest levels of BT in all tissues.. In small intestinal transplantation, both apoptosis and cell necrosis give rise to histological injury and BT. Apoptosis inhibition and necrosis inhibition treatments protect intestinal grafts from ischemia/reperfusion injury; Z-VAD supplementation has a greater effect on BT prevention than does administration of the PARS inhibitor 3-AB.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Bacterial Translocation; Benzamides; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; In Situ Nick-End Labeling; Intestine, Small; Male; Necrosis; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Postoperative Complications; Rats; Rats, Sprague-Dawley

Glucocorticoids and fludarabine are able to induce typical features of apoptosis in CLL lymphocytes. Cysteinyl aspartate specific proteases (caspases) play a key biochemical role in the apoptotic pathway. Caspase activation following cytotoxic stimuli leads to highly specific proteolytic cleavage of functionally important cellular enzymes. One of them is poly ADP-ribose) polymerase (PARP). To some extent caspase activation seems to be under the control of the Bcl-2 family of interacting proteins. We determined the role of Bcl-2-family proteins Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic), activation of caspase-3 (CPP32/Yama) and activation of PARP in CLL apoptosis. All 21 analyzed CLL samples expressed Bcl-2 and Bax. Four of 13 (31%) samples with a low Bcl-2/Bax ratio exhibited in vitro prednisolone resistance, whereas eight of nine (88%) samples with a high Bcl-2/Bax ratio were in vitro resistant (

Topics: Aged; Aged, 80 and over; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; B-Lymphocytes; bcl-2-Associated X Protein; Benzamides; Blotting, Western; Caspase 3; Caspase Inhibitors; Caspases; DNA Fragmentation; Drug Screening Assays, Antitumor; Enzyme Activation; Female; Gene Expression; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

We have presently determined the effect of inhibition of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) on the occurrence of apoptosis in insulin-producing cells. The ADP-ribosylation activities of intact cells were decreased by incubation of RINm5F cells for 16 h with the PARP inhibitors nicotinamide (NA) (20-50 mM) or 3-aminobenzamide (3-ABA) (10 mM). Exposure to 20-50 mM NA or 10 mM 3-ABA both resulted in massive apoptosis in RINm5F cells. A 24 h exposure to 50 mM nicotinamide induced apoptosis in fetal but not adult rat islet cells. In addition, exposure of RINm5F cells to 50 mM NA for 12-24 h induced the appearance of the 85 kDa proteolytic PARP fragment, indicating activation of the ICE-like protease caspase-3. Incubation with 20-50 mM NA did not induce any consistent effects upon transcription factor NF-kappaB activity, demonstrating that this pathway is not involved in induction of apoptosis by NA. It is concluded that in insulin-producing cells with a high mitotic rate, inhibition of ADP-ribosylation--and consequently of auto-modification and release of PARP bound to DNA strand breaks--leads to activation of programmed cell death.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Benzamides; Cell Line; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Fetus; Islets of Langerhans; Necrosis; NF-kappa B; Niacinamide; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley