calcimycin and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Bisabololoxide A (BSBO), main constituents in German chamomile extract, is responsible for antipruritic effect. In previous study, the incubation with 30-100 μM BSBO for 24 h exerted cytotoxic and proapoptotic effects on rat thymocytes. To further characterize BSBO cytotoxicity, the effect on the cells suffering from calcium overload by calcium ionophore A23187 was examined. A23187 induced Ca(2+) -dependent cell death. Contrary to our expectation, 1-10 μM BSBO inhibited A23187-induced increase in cell lethality of rat thymocytes. BSBO attenuated A23187-induced increases in populations of shrunken living cells, phosphatidylserine-exposed living cells, and dead cells, without affecting the increase in intracellular Ca(2+) concentration and the Ca(2+) -dependent hyperpolarization. The effect of BSBO on A23187-treated cells may be unique because the activation of Ca(2+) -dependent K(+) channels is required for cell shrinkage, externalization of phosphatidylserine, and cell death in some cells. The cell death induced by A23187 was not inhibited by Z-VAD-FMK, a pan-inhibitor of caspases. Thus, the cell death may be a necrosis with some features observed during an early stage of apoptosis. These results suggest that BSBO at low micromolar concentrations is cytoprotective against calcium overload.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calcimycin; Calcium; Cells, Cultured; Matricaria; Plant Extracts; Rats; Rats, Wistar; Sesquiterpenes; Thymocytes

Translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is a modification of the lipid architecture occurring in sperm. This is one of the earliest signs of apoptosis that can be monitored by the calcium-dependent binding of annexin V.. Flow cytometric analysis of annexin V binding was performed. Calcium ionophore A23187 led to a significant increase in the proportion of living sperm with PS exposure: 7.3 3.2% of cells in the untreated ejaculate versus 47.5 5.6% of cells after 1 h of incubation with A23187. Conversely, diminution of mitochondrial membrane potential [DiOC6(3)/propidium iodide (PI) assay], caspase activation [fluorescein isothiocyanate (FITC)-Val-Ala-Asp-fluoromethylketone (VAD-FMK)/PI assay], increased plasma membrane permeability (Yo-Pro-1/PI assay) and increased DNA fragmentation [TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay], which are among the main signs of apoptosis, were not observed in sperm, even after 4 h of incubation with A23187. However, A23187 significantly increased the proportion of sperm with plasma membrane scrambling and with a reacted acrosome, as detected with the merocyanine 540 probe (M540) and the monoclonal anti-human CD46-PE antibody respectively.. Our results suggest that PS exposure in human sperm, as induced by A23187, is mainly related to the acrosome reaction rather than to apoptosis.

Topics: Acrosome; Acrosome Reaction; Amino Acid Chloromethyl Ketones; Apoptosis; Biomarkers; Calcimycin; Calcium; Caspase Inhibitors; Cell Membrane; DNA Fragmentation; Enzyme Inhibitors; Flow Cytometry; Fluorescein-5-isothiocyanate; Humans; In Situ Nick-End Labeling; Ionophores; Male; Membrane Cofactor Protein; Microscopy, Fluorescence; Phosphatidylserines; Propidium; Pyrimidinones; Sperm Capacitation; Spermatozoa

The endoplasmic reticulum (ER) resident-94 kDa glucose-regulated protein (GRP94), plays a pivotal role in cell death due to ER stress. In our study expression of GRP94 was increased in human neuroblastoma SH-SY5Y cells due to exposure to calcium ionophore A23187. A23187-mediated cell death was associated with activation of the major cysteine proteases, caspase-3 and calpain. Pretreatment with adenovirus-mediated antisense GRP94 (AdGRP94AS) reduced viability of SH-SY5Y cells subjected to A23187 treatment compared with wild type cells or cells with adenovirus-mediated overexpression of GRP94 (AdGRP94S). These results indicated that suppression of GRP94 is associated with accelerated cell death. Moreover, expression of GRP94 suppressed A23187-induced cell death and stabilized calcium homeostasis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Calcimycin; Calcium; Calpain; Caspase 3; Caspases; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Dantrolene; DNA, Antisense; Endoplasmic Reticulum; Gene Expression; Histocytochemistry; Homeostasis; HSP70 Heat-Shock Proteins; Humans; In Situ Nick-End Labeling; Lac Operon; Membrane Proteins; Neurons; Thapsigargin; Transfection

Alzheimer's disease (AD) is characterized by the degeneration and loss of neurons, intracellular neurofibrillary tangles and the accumulation of extracellular senile plaques consisting mainly of beta-amyloid (A beta). A beta is generated from the amyloid precursor protein (APP) by sequential beta- and gamma-secretase cleavage. Alternatively, APP may be cleaved within the A beta region by alpha-secretase, preventing A beta formation. Here we investigated APP processing and secretion in primary neurons, using either colchicine or the calcium ionophore A23187 to induce apoptosis. Cell viability was determined by MTT measurements and apoptosis was further confirmed by annexin V and propidium iodide staining. We found that exposure to A23187 significantly decreased the secretion of soluble beta-secretase cleaved APP (beta-sAPP) in a caspase-dependent manner, although the secretion of total soluble APP beta sAPP) did not change. In addition, caspase inhibition restored cell viability to control levels. Exposure to colchicine did not change the amount of either secreted beta-sAPP or total sAPP and caspase inhibition was only partially able to restore cell viability. We conclude that calcium homeostasis is an important apoptotic effector specifically affecting the beta-secretase cleavage of APP.

Topics: Amino Acid Chloromethyl Ketones; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Annexin A5; Apoptosis; Aspartic Acid Endopeptidases; Biological Transport; Blotting, Western; Calcimycin; Calcium; Cells, Cultured; Cerebral Cortex; Colchicine; Culture Media, Conditioned; Cysteine Proteinase Inhibitors; Cytoskeleton; Endopeptidases; Ionophores; Neurons; Rats; Rats, Sprague-Dawley

We have investigated whether the increases in ceramide levels that occur during apoptosis in SKW 6.4 cells induced by anti-Fas antibody depend on the activation of caspases. Using cells prelabelled to equilibrium with [14C]acetate, it was shown that the amount of ceramide approximately doubled after 24 h incubation with anti-Fas, but the time course of ceramide changes was slower than that of anti-Fas-induced cell death. Complete inhibition of the effects of anti-Fas on cell death and on ceramide production was observed when the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethane (zVAD.fmk) was added together with anti-Fas, but N-benzyloxycarbonyl-Phe-Ala-fluoromethane (a structurally similar cathepsin B inhibitor) had no effect. Treatment of cells with the Ca2+-ionophore A23187 also doubled ceramide levels, but in this case the effect was complete within 2 h, was not blocked by zVAD.fmk and was not associated with increases in nuclear fragmentation. These results suggest that ceramide is not an upstream messenger in Fas-mediated apoptosis and may instead be produced as a consequence of processes downstream of the activation of caspases and increases in cytosolic calcium concentration.

Topics: Acetates; Amino Acid Chloromethyl Ketones; Antibodies; Apoptosis; Calcimycin; Calcium; Ceramides; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; fas Receptor; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Lymphoma, B-Cell; Signal Transduction; Time Factors; Tumor Cells, Cultured