calcimycin and thiazolyl-blue

The endoplasmic reticulum (ER) plays a critical role in the maintenance of intracellular homeostasis and its dysfunction is thought to lead to neuronal death, which results in neurodegenerative disorders. Since phospholipase C (PLC) isozymes are involved in maintenance of the intracellular Ca2+ concentration by regulating Ca2+ release from the ER, their expression might be affected by ER stress. Of these isozymes, PLC-beta 1 and -gamma 1, in particular, are known to protect cells from oxidative stress and thus alteration of their expression profile under ER stress-loaded conditions is interesting. Using primary cultured rat cortical neurons, we here examined whether expression of PLC-beta 1 and -gamma 1 was altered in ER stress-loaded neurons induced by tunicamycin (Tm). In ER stress-loaded neurons treated with Tm in the range of 0.03-3 microg/ml for 20 h, the viability of the neurons was decreased dose-dependently, the decrease being significant with 0.3 or more microg/ml, and expression of the representative ER stress markers, GRP78/BiP, and cleaved caspase-3 and -12, was increased after 24 h postincubation, confirming the induction of ER stress in the neurons. In the ER stress-loaded neurons obtained on Tm treatment, the expression level of PLC-beta 1 decreased dose-dependently. On the other hand, there was no difference in the PLC-gamma 1 protein expression level between control and ER stress-loaded neurons. Overall, we demonstrated that ER stress decreases the expression of PLC-beta 1, but not -gamma 1, in neurons.

Topics: Animals; Blotting, Western; Calcimycin; Calcium; Caspase 12; Caspase 3; Cell Survival; Cells, Cultured; Endoplasmic Reticulum; Enzyme Inhibitors; Neurons; Phospholipase C beta; Phospholipase C gamma; Rats; Stress, Physiological; Tetrazolium Salts; Thapsigargin; Thiazoles; Tunicamycin

SC-236 [4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1-pyrazol-1-l] benzenesulfonamide; C16H11ClF3N3O2S] is a highly selective cyclooxygenase (COX)-2 inhibitor. However, the exact mechanism that accounts for the anti-inflammatory effect of SC-236 is not completely understood. The aim of the present study was to elucidate whether and how SC-236 modulates the inflammatory reaction in a stimulated human mast cell (HMC) line, HMC-1. SC-236 inhibited the expression of tumor necrosis factor-alpha, interleukin (IL)-6, IL-8, vascular endothelial growth factor, COX-2, inducible nitric-oxide synthase, and hypoxia-inducible factor-1alpha in phorbol 12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-stimulated HMC-1. SC-236 suppressed nuclear factor (NF)-kappaB activation induced by PMACI, leading to suppression of IkappaB-alpha phosphorylation and degradation. SC-236 also suppressed strong induction of NF-kappaB promoter-mediated luciferase activity. In addition, SC-236 suppressed PMACI-induced phosphorylation of the mitogen-activated protein kinase p38, the extracellular-regulated kinase p44, and the c-Jun N-terminal kinase and induced expression of mitogen-activated protein kinase phosphatase-1. These results provide new insight into the pharmacological actions of SC-236 as a potential molecule for therapy of mast cell-mediated inflammatory diseases.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Calcimycin; Cell Line; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Cytokines; Dual Specificity Phosphatase 1; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Enzymologic; Genes, Reporter; Humans; Immunohistochemistry; Ionophores; JNK Mitogen-Activated Protein Kinases; Luciferases; Mast Cells; Membrane Proteins; Microscopy, Confocal; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Prostaglandin-Endoperoxide Synthases; Protein Phosphatase 1; Protein Tyrosine Phosphatases; Pyrazoles; Reverse Transcriptase Polymerase Chain Reaction; RNA; Stimulation, Chemical; Sulfonamides; Tetradecanoylphorbol Acetate; Tetrazolium Salts; Thiazoles

There is an opportunity to augment the therapeutic potential of drug combinations through use of drug delivery technology. This report summarizes data obtained using a novel liposomal formulation with coencapsulated doxorubicin and vincristine. The rationale for selecting these drugs is due in part to the fact that liposomal formulations of doxorubicin and vincristine are being separately evaluated as components of drug combinations.. Doxorubicin and vincristine were coencapsulated into liposomes using two distinct methods of drug loading. A manganese-based drug loading procedure, which relies on drug complexation with a transition metal, was used to encapsulate doxorubicin. Subsequently the ionophore A23187 was added to induce formation of a pH gradient, which promoted vincristine encapsulation.. Plasma elimination studies in mice indicated that the drug:drug ratio before injection [4:1 doxorubicin:vincristine (wt:wt ratio)] changed to 20:1 at the 24-h time point, indicative of more rapid release of vincristine from the liposomes than doxorubicin. Efficacy studies completed in MDA MB-435/LCC6 tumor-bearing mice suggested that at the maximum tolerated dose, the coencapsulated formulation was therapeutically no better than liposomal vincristine. This result was explained in part by in vitro cytotoxicity studies evaluating doxorubicin and vincristine combinations analyzed using the Chou and Talalay median effect principle. These data clearly indicated that simultaneous addition of vincristine and doxorubicin resulted in pronounced antagonism.. These results emphasize that in vitro drug combination screens can be used to predict whether a coformulated drug combination will act in an antagonistic or synergistic manner.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Calcimycin; Cell Line, Tumor; Coloring Agents; Doxorubicin; Humans; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Ionophores; Ions; Lipid Metabolism; Liposomes; Manganese; Manganese Compounds; Mice; Mice, SCID; Neoplasm Transplantation; Protons; Sulfates; Tetrazolium Salts; Thiazoles; Time Factors; Vincristine

Changes in mitochondrial integrity, reactive oxygen species release and Ca2+ handling are proposed to be involved in the pathogenesis of many neurological disorders including methylmalonic acidaemia and Huntington's disease, which exhibit partial mitochondrial respiratory inhibition. In this report, we studied the mechanisms by which the respiratory chain complex II inhibitors malonate, methylmalonate and 3-nitropropionate affect rat brain mitochondrial function and neuronal survival. All three compounds, at concentrations which inhibit respiration by 50%, induced mitochondrial inner membrane permeabilization when in the presence of micromolar Ca2+ concentrations. ADP, cyclosporin A and catalase prevented or delayed this effect, indicating it is mediated by reactive oxygen species and mitochondrial permeability transition (PT). PT induced by malonate was also present in mitochondria isolated from liver and kidney, but required more significant respiratory inhibition. In brain, PT promoted by complex II inhibition was stimulated by increasing Ca2+ cycling and absent when mitochondria were pre-loaded with Ca2+ or when Ca2+ uptake was prevented. In addition to isolated mitochondria, we determined the effect of methylmalonate on cultured PC12 cells and freshly prepared rat brain slices. Methylmalonate promoted cell death in striatal slices and PC12 cells, in a manner attenuated by cyclosporin A and bongkrekate, and unrelated to impairment of energy metabolism. We propose that under conditions in which mitochondrial complex II is partially inhibited in the CNS, neuronal cell death involves the induction of PT.

Topics: Animals; Antimycin A; Bongkrekic Acid; Brain; Calcimycin; Calcium; Catalase; Cell Survival; Cyclosporins; Dose-Response Relationship, Drug; Drug Interactions; Electron Transport Complex II; Enzyme Inhibitors; Female; In Vitro Techniques; Ionophores; Malonates; Membrane Potentials; Methylmalonic Acid; Mitochondria; NADP; Neurons; Nitro Compounds; Oxygen Consumption; PC12 Cells; Permeability; Propionates; Rats; Rotenone; Tacrolimus; Tetrazolium Salts; Thiazoles; Uncoupling Agents

Regulation of endothelial barrier function often occurs through signalling involving phospholipase C activation which produces diacylglycerol (DAG), a lipidic second messenger activator of protein kinase C (PKC). Therefore, modification of lipidic composition of endothelial cell membranes might modify DAG production and, as a result, alter regulation of endothelial permeability. We investigated the in vitro effects of natural 1-O-alkylglycerols on porcine aortic endothelial cell permeability to dye-labelled albumin. [3H]-1-O-alkylglycerols (10 microm) were substantially incorporated into phosphatidylcholine (6.6%) and phosphatidylethanolamine (4.4%). Stimulation of endothelial cell monolayer with phorbol-myristate-acetate or with the calcium ionophore A23187 resulted in a raise in permeability to albumin. Pre-treatment with 1-O-alkylglycerols (10 microm, 24 h) had no effect on basal albumin permeability but totally inhibited the effect of phorbol-myristate-acetate, and brought the permeability of A23187-stimulated endothelial cell monolayers below control. After incubation of cells with [3H]-1-O-alkylglycerols (10 microm, 24 h), we detected the production of the analogue of DAG, and PKC inhibitor, [3H]-1-O-alkyl-2-acyl-glycerol, in resting cells. This production was increased by 58% under A23187 stimulation while phorbol-myristate-acetate had no effect. Our data demonstrate that natural 1-O-alkylglycerols modify endothelial permeability, and suggest that this effect could be mediated through alteration of lipidic signalling.

Topics: Albumins; Animals; Aorta; Calcimycin; Cell Membrane Permeability; Cells, Cultured; Coloring Agents; Diglycerides; Endothelium, Vascular; Glycerol; Phospholipids; Protein Kinase C; Tetradecanoylphorbol Acetate; Tetrazolium Salts; Thiazoles

We compared the sensitivities to apoptosis via anti-Fas antibody of two human ovarian cancer cell lines, NOS4 and SKOV-3, both of which strongly express the Fas antigen on their cell surface. Treatment with anti-Fas antibody induced extensive DNA fragmentation in NOS4 cells but none in SKOV-3 cells. However; both cell lines underwent apoptosis in response to calcium ionophore A23187 or sphingomyelinase, demonstrating that the latter cell line is capable of DNA fragmentation. DNA fragmentation was not induced in either cell line by treatment with PKC activator PMA, however treatment with protein kinase C (PKC) inhibitor H-7 induced extensive DNA fragmentation in NOS4 cells, but again none in SKOV-3 cells. Protein kinase A inhibitor HA1004 treatment did not induce DNA fragmentation in either cell line. Correspondingly, treatment of cells with PMA before anti-Fas antibody or A23187 treatment partially inhibited induction of DNA fragmentation in NOS4 cells but not in SKOV-3 cells. Both NOS4 and SKOV-3 cell lines expressed isozymes of PKC at comparable levels. These results suggest the presence of a PKC-dependent anti-apoptotic mechanism in association with high sensitivity to anti-Fas antibody in these ovarian cancer cell lines.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Antibodies; Apoptosis; Calcimycin; Cell Division; Ceramides; Cyclic AMP-Dependent Protein Kinases; DNA Fragmentation; Enzyme Inhibitors; fas Receptor; Female; Flow Cytometry; Humans; Isoquinolines; Ovarian Neoplasms; Protein Kinase C; Signal Transduction; Sphingomyelin Phosphodiesterase; Sulfonamides; Tetradecanoylphorbol Acetate; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured