mdl-201053 and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

To elucidate the role of reactive oxygen species (ROS) in arthritis and to identify targets of arthritis treatment in conditions with different levels of oxidant stress.. Through establishing an arthritis model by injecting arthritogenic serum into wild-type and NADPH oxidase 2 (NOX2)-deficient mice, we found that arthritis had a neutrophilic infiltrate and was more severe in Ncf1(-/-) mice, a mouse strain lacking the expression of the NCF1/p47(phox) component of NOX2. The levels of interleukin-1β (IL-1β) and IL-6 in inflamed joints were higher in Ncf1(-/-) than in controls. Antagonists of tumor necrosis factor-α (TNFα) and IL-1β were equally effective in suppressing arthritis in wild-type mice, while IL-1β blockade was more effective than TNFα blockade in Ncf1(-/-) mice. A treatment of caspase inhibitor and the combination treatment of a caspase inhibitor and a cathepsin inhibitor, but not a cathepsin inhibitor alone, suppressed arthritic severity in the wild-type mice, while a treatment of cathepsin inhibitor and the combination treatment of a caspase inhibitor and a cathepsin inhibitor, but not a caspase inhibitor alone, were effective in treating Ncf1(-/-) mice. Consistently, cathepsin B was found to proteolytically process pro-IL-1β to its active form and this activity was suppressed by ROS.. This novel mechanism of a redox-mediated immune regulation of arthritis through leukocyte-produced ROS is important for devising an optimal treatment for patients with different levels of tissue ROS.. Our results suggest that ROS act as a negative feedback to constrain IL-1β-mediated inflammation, accounting for the more severe arthritis in the absence of NOX2.

Topics: Amino Acid Chloromethyl Ketones; Animals; Ankle Joint; Arthritis; Caspase Inhibitors; Cathepsin B; Cell Line; Cytokines; Dipeptides; Disease Models, Animal; Fibroblasts; Humans; Inflammation; Interleukin-1beta; Ketones; Lung; Membrane Glycoproteins; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; NADPH Oxidase 2; NADPH Oxidases; Oxidation-Reduction; Reactive Oxygen Species; Wrist Joint

Caspases, members of the cysteine protease family, execute UVB-induced apoptosis in several cell lines and keratinocytes. Several researchers investigating UVB-induced apoptosis have demonstrated a dose-dependent protective effect of the synthetic peptide caspase inhibitor zVAD-fmk. However, zVAD-fmk displays a dose-dependent protective effect against UVB-induced apoptosis, even at doses higher than those required to block all known proapoptotic caspases. In addition, it is known that zVAD-fmk also inhibits other cysteine proteases including cathepsins and calpains, and these proteases have recently been demonstrated to play a role in the execution of programmed cell death induced by other stimuli, e.g. TNF-alpha. The purpose of the present study was therefore to investigate whether inhibitors of cysteine cathepsins and calpains could prevent UVB-induced apoptosis in HeLa cells and keratinocytes. This was done by investigating the effect of the irreversible cysteine protease inhibitor zFA-fmk, the cathepsin B inhibitor CA-074-Me and the calpain inhibitor ALLN on the viability of UVB-irradiated human keratinocytes and HeLa cells. At concentrations of 10 microM and above zVAD-fmk conferred partial dose-dependent protection against UVB-induced apoptosis in HeLa cells and keratinocytes. Moreover, caspase-3 activity was completely blocked at zVAD-fmk concentrations of 1 microM in HeLa cells. This indicates that caspase-independent mechanisms could be involved in UVB-induced apoptosis. However, the protease inhibitors zFA-fmk, CA-074-Me and ALLN all failed to prevent UVB-induced apoptosis in HeLa cells and keratinocytes. In conclusion, the protective effect of zVAD-fmk at high concentrations indicates that other proteases than caspases are active in the execution of UVB-induced apoptosis but further studies are needed to identify these proteases.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Calpain; Caspase Inhibitors; Cathepsins; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; HeLa Cells; Humans; Keratinocytes; Ketones; Leupeptins; Phospholipases A; Ultraviolet Rays

Synthetic retinoid-related molecules (RRMs) have been described that show strong antiproliferative activity and induce apoptosis in cancer cells. These RRMs induce caspase activity independently of the retinoid receptors in Jurkat T cells. We observed that the inhibitor of cathepsins B and L Z-FA-fmk blocks the induction of DEVDase activity, DNA fragmentation, and externalization of phosphatidylserine by selective RRMs. Z-FA-fmk can inhibit caspase activity in vitro and selectively inhibits recombinant effector caspases 2, -3, -6, and -7. In contrast, purified initiator caspases 8 and 10 are not affected, whereas the apoptosome-associated caspase 9 is only partially inhibited by Z-FA-fmk in vitro. These data correlate with the covalent binding of biotinylated Z-FA-fmk to the active large subunit of effector caspases. This selective targeting of effector caspases is also observed in Jurkat cells and has been used to demonstrate that RRMs induce apoptosis through the mitochondrial pathway and activate caspase 8 in a Z-FA-fmk-sensitive manner. Thus, Z-FA-fmk fails to inhibit Fas-mediated activation of caspase 8, but completely inhibits RRM-induced processing of caspase 8. Z-FA-fmk does not prevent the autoproteolytic cleavage of caspase 9 in Jurkat cells and partially inhibits the processing and full maturation of effector caspases induced by the RRMs. Moreover, Z-VAD-fmk and Z-FA-fmk have no effect on the release of cytochrome c induced by the RRMs. Other cathepsin inhibitors elicit no effect on RRM-induced apoptosis in Jurkat cells, suggesting that caspases are the major effectors of RRM action.

Topics: Amino Acid Chloromethyl Ketones; Annexin A5; Antineoplastic Agents; Apoptosis; Biotinylation; Caspase Inhibitors; Caspases; Cathepsins; Cysteine Proteinase Inhibitors; Cytochromes c; Dipeptides; fas Receptor; Humans; Jurkat Cells; Ketones; Peptide Fragments; Peptide Hydrolases; Phosphoserine; Retinoids

The efficacy of a novel, nonpeptidic, caspase 3/7-selective inhibitor, (S)-(+)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (MMPSI) for reducing ischemic injury in isolated rabbit hearts or cardiomyocytes was evaluated. MMPSI (0.1-10 microM) evoked a concentration-dependent reduction in infarct size (up to 56% vs. control; IC(50)=0.2 microM). Furthermore, apoptosis (DNA laddering, soluble nucleosomes) was reduced in the ischemic area-at-risk. MMPSI inhibited recombinant human caspase-3 with an IC(50)=1.7 microM. Apoptosis in H9c2 cells after 16-h simulated ischemia and 2-h simulated reperfusion was significantly reduced by MMPSI in a concentration-dependent manner (IC(50)=0.5 microM); similar effects were observed in isolated adult rabbit cardiomyocytes (IC(50)=1.5 microM). These data support an important role for caspase-3/7 in mediating myocardial ischemic injury. Furthermore, these data indicate that cardioprotection via caspase-3/7 inhibition is attainable via a small molecule (nonpeptidic) inhibitor, a necessary step in making this approach therapeutically viable.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspase 7; Caspase Inhibitors; Caspases; Cell Line; Cells, Cultured; Coronary Circulation; Cysteine Proteinase Inhibitors; Dipeptides; Dose-Response Relationship, Drug; Heart Rate; In Situ Nick-End Labeling; Isatin; Ketones; Male; Microscopy, Electron; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Pyrrolidines; Rabbits

Activated T cells lyse the murine renal cancer Renca. We have examined the mechanism of tumor cell lysis with the use of T cells derived from C57BL/6, BALB/c, B6.gld, and B6.Pfp-/- mice. C57BL/6 and BALB/c T cells can lyse Renca cells through the use of both granule- and Fas ligand (FasL)-mediated pathways. However, B6.gld T cells predominantly use granule-mediated killing, whereas B6.Pfp-/- T cells use FasL. The lysis of Renca by Pfp-/- T cells is only partially inhibited by the caspase inhibitor ZVAD-FMK, suggesting that caspase-independent signaling is also important for Renca cell lysis. When the reactive oxygen scavenger butylated hydroxyanisole was used alone or in combination with ZVAD-FMK a substantial reduction of Renca lysis was observed. Therefore, the caspase-independent generation of reactive oxygen intermediates in Renca after Fas triggering contributes to the lysis of these cells.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Apoptosis Regulatory Proteins; Butylated Hydroxyanisole; Carcinoma, Renal Cell; Caspases; Crosses, Genetic; Cytoplasmic Granules; Cytotoxicity, Immunologic; Dipeptides; Enzyme Inhibitors; Fas Ligand Protein; fas Receptor; Free Radical Scavengers; Humans; Interferon-gamma; Ketones; Kidney Neoplasms; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Reactive Oxygen Species; Recombinant Proteins; Signal Transduction; Specific Pathogen-Free Organisms; T-Lymphocytes, Cytotoxic; TNF-Related Apoptosis-Inducing Ligand; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The retinoblastoma protein plays a critical role in regulating the G1/S transition. Less is known about the function and regulation of the homologous pocket protein p107. Here we present evidence for the posttranslational regulation of p107 by the Ca2+-activated protease calpain. Three negative growth regulators, the HMG-CoA reductase inhibitor lovastatin, the antimetabolite 5-fluorouracil, and the cyclic nucleotide dibutyryl cAMP were found to induce cell type-specific loss of p107 protein which was reversible by the calpain inhibitor leucyl-leucyl-norleucinal but not by the serine protease inhibitor phenylmethylsulfonylfluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26S proteasome. Purified calpain induced Ca2+-dependent p107 degradation in cell lysates. Transient expression of the specific calpain inhibitor calpastatin blocked the loss of p107 protein in lovastatin-treated cells, and the half-life of p107 was markedly lengthened in lovastatian-treated cells stably transfected with a calpastatin expression vector versus cells transfected with vector alone. The data presented here demonstrate down-regulation of p107 protein in response to various antiproliferative signals, and implicate calpain in p107 posttranslational regulation.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Bucladesine; Calpain; Cyclin B; Cyclin B1; Cysteine Proteinase Inhibitors; Dipeptides; Fluorouracil; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Ketones; Leupeptins; Lovastatin; Nuclear Proteins; Protein Processing, Post-Translational; Retinoblastoma Protein; Retinoblastoma-Like Protein p107; Tumor Cells, Cultured

Apoptosis is a pathway of cell death orchestrated by a family of proteases called caspases. Oxidized low density lipoprotein (oxLDL) is a putative cause of atherogenesis. We examined the effect of oxLDL on endothelial cell (EC) apoptosis and the ability of a caspase antagonist to inhibit oxLDL-induced EC injury.. Bovine ECs were plated at a concentration of 5.0 x 10(5) cells/ml and exposed to LDL oxidized by ultraviolet radiation at a concentration of 100 microgram oxLDL/ml for 20 h. Some ECs were pretreated with an irreversible caspase inhibitor (ZVAD). Samples were analyzed histologically. Apoptosis was measured using the Annexin V assay (flow cytometry) which detects phosphatidylserine on plasma membranes and confirmed by TUNEL assay (flow cytometry). Statistical assessments were performed using ANOVA.. ECs treated with LDL were morphologically similar to untreated cells. Cells treated with oxLDL demonstrated cytoplasmic shrinkage, plasma membrane blebbing, chromatin condensation, and loss of adhesion. These effects were diminished after pretreatment with the caspase inhibitor ZVAD. The Annexin V assay showed: (a) cells exposed to LDL had a 12 +/- 1% apoptosis rate, (b) exposure to oxLDL induced apoptosis in 30 +/- 0.3% of the cells, and (c) pretreatment with the caspase inhibitor ZVAD decreased the oxLDL-induced apoptosis to 16 +/- 1% (P < 0.05). This decrease in apoptosis was also reflected by an increase in the percentage of alive cells from 34 +/- 7% after oxLDL exposure to 55 +/- 6% after apoptosis inhibition with ZVAD. TUNEL assay demonstrated a 2.5-fold reduction in mean fluorescence intensity between cells treated with oxLDL alone and those treated with ZVAD, suggesting a significant decrease in apoptosis in the latter group.. We conclude that treatment of bovine ECs with oxLDL induces apoptosis which can be significantly reduced by a specific caspase inhibitor.

Topics: Amino Acid Chloromethyl Ketones; Animals; Annexin A5; Apoptosis; Caspase Inhibitors; Cattle; Cell Size; Cysteine Proteinase Inhibitors; Dipeptides; Endothelium, Vascular; Flow Cytometry; In Situ Nick-End Labeling; Ketones; Lipoproteins, LDL; Tolonium Chloride; Tumor Necrosis Factor-alpha

Neuronal apoptosis occurs during nervous system development and after pathological insults to the adult nervous system. Inhibition of CED3/ICE-related proteases has been shown to inhibit neuronal apoptosis in vitro and in vivo, indicating a role for these cysteine proteases in neuronal apoptosis. We have studied the activation of the CED3/ICE-related protease CPP32 in two in vitro models of mouse cerebellar granule neuronal cell death: K+/serum deprivation-induced apoptosis and glutamate-induced necrosis. Pretreatment of granule neurons with a selective, irreversible inhibitor of CED3/ICE family proteases, ZVAD-fluoromethylketone, specifically inhibited granule neuron apoptosis but not necrosis, indicating a selective role for CED3/ICE proteases in granule neuron apoptosis. Extracts prepared from apoptotic, but not necrotic, granule neurons contained a protease activity that cleaved the CPP32 substrate Ac-DEVD-aminomethylcoumarin. Induction of the protease activity was prevented by inhibitors of RNA or protein synthesis or by the CED3/ICE protease inhibitor. Affinity labeling of the protease activity with an irreversible CED3/ICE protease inhibitor, ZVK(biotin)D-fluoromethylketone, identified two putative protease subunits, p20 and p18, that were present in apoptotic but not necrotic granule neuron extracts. Western blotting with antibodies to the C terminus of the large subunit of mouse CPP32 (anti-CPP32) identified p20 and p18 as processed subunits of the CPP32 proenzyme. Anti-CPP32 specifically inhibited the DEVD-amc cleaving activity, verifying the presence of active CPP32 protease in the apoptotic granule neuron extracts. Western blotting demonstrated that the CPP32 proenzyme was expressed in granule neurons before induction of apoptosis. These results demonstrate that the CED3/ICE homolog CPP32 is processed and activated during cerebellar granule neuron apoptosis. CPP32 activation requires macromolecular synthesis and CED3/ICE protease activity. The lack of CPP32 activation during granule neuron necrosis suggests that proteolytic processing and activation of CED3/ICE proteases are specific biochemical markers of apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspases; Cells, Cultured; Cerebellar Cortex; Coumarins; Culture Media, Serum-Free; Cycloheximide; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dipeptides; Enzyme Activation; Enzyme Precursors; Glutamic Acid; Ketones; Mice; Mice, Inbred C57BL; Necrosis; Nucleic Acid Synthesis Inhibitors; Oligopeptides; Potassium; Protein Synthesis Inhibitors