leupeptins and Necrosis

Ischemia-reperfusion (I/R)-induced lung injury undermines lung transplantation (LTx) outcomes by predisposing lung grafts to primary graft dysfunction (PGD). Necrosis is a feature of I/R lung injury. However, regulated necrosis (RN) with specific signaling pathways has not been explored in an LTx setting. In this study, we investigated the role of RN in I/R-induced lung injury. To study I/R-induced cell death, we simulated an LTx procedure using our cell culture model with human lung epithelial (BEAS-2B) cells. After 18 h of cold ischemic time (CIT) followed by reperfusion, caspase-independent cell death, mitochondrial reactive oxygen species production, and mitochondrial membrane permeability were significantly increased. N-acetyl-Leu-Leu-norleucinal (ALLN) (calpain inhibitor) or necrostatin-1 (Nec-1) [receptor interacting serine/threonine kinase 1 (RIPK1) inhibitor] reduced these changes. ALLN altered RIPK1/RIPK3 expression and mixed lineage kinase domain-like (MLKL) phosphorylation, whereas Nec-1 did not change calpain/calpastatin expression. Furthermore, signal transducer and activator of transcription 3 (STAT3) was demonstrated to be downstream of calpain and regulate RIPK3 expression and MLKL phosphorylation during I/R. This calpain-STAT3-RIPK axis induces endoplasmic reticulum stress and mitochondrial calcium dysregulation. LTx patients' samples demonstrate that RIPK1, MLKL, and STAT3 mRNA expression increased from CIT to reperfusion. Moreover, the expressions of the key proteins are higher in PGD samples than in non-PGD samples. Cell death associated with prolonged lung preservation is mediated by the calpain-STAT3-RIPK axis. Inhibition of RIPK and/or calpain pathways could be an effective therapy in LTx.

Topics: Apoptosis; Calpain; Cells, Cultured; Humans; Imidazoles; Indoles; Leupeptins; Lung Transplantation; Necrosis; Phosphorylation; Primary Graft Dysfunction; Receptor-Interacting Protein Serine-Threonine Kinases; Receptors, Death Domain; Reperfusion Injury; Signal Transduction; STAT3 Transcription Factor

N-acetyl-leucyl-leucyl-norleucinal (ALLN), an inhibitor of proteasomes and calpain, is widely used to reduce proteasomes or calpain-mediated cell death in rodents. However, ALLN is toxic to retinal neurons to some extent. At the concentration of 10 μM, ALLN is non-toxic to cortical neurons, but induces cell death of retinal neurons in vitro. The tolerance concentration of ALLN for retinal neurons is unclear, and the precise mechanism of cell death induced by ALLN remains elusive. In this study, we investigated the toxic effect of ALLN on primary retinal neurons. The 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed no significant changes of cell viability at 1 μM but decreased cell viability after treatment of ALLN at 2.5, 5, and 7.5 μM. Lactate dehydrogenase (LDH) release was highly elevated and propidium iodide (PI)-positive cells were significantly increased at 2.5, 5, and 7.5 μM after all treatment times. Moreover, the protein levels of caspase-3 were up-regulated at 5 and 7.5 μM after 12 and 24 h of ALLN treatment. The ratio of Bax/Bcl-2 was raised and Annexin V-positive cells were increased at 5 and 7.5 μM after 12 and 24 h of ALLN treatment. However, there were no significant changes in either the ratio of microtubule-associated protein 1 light chain 3 (LC3) II/LC3 I or monodansylcadaverine (MDC) staining. Our data clearly show that at the concentrations equal to and higher than 2.5 μM, ALLN may induce cell death of primary retinal neurons by necrosis and apoptosis, but not autophagy. These suggest that primary retinal neurons are more susceptible to ALLN treatment and provide a possible mechanism for the cell death of ALLN-sensitive cells in ALLN injury.

Topics: Animals; Apoptosis; Autophagy; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Fluorescent Antibody Technique; L-Lactate Dehydrogenase; Leupeptins; Microtubule-Associated Proteins; Mitochondria; Necrosis; Proto-Oncogene Proteins c-bcl-2; Rats, Sprague-Dawley; Retinal Neurons; Time Factors

Recent studies have linked necrotic cell death and proteolysis of inflammatory proteins to the adaptive immune response mediated by the lysosome-destabilizing adjuvants, alum and Leu-Leu-OMe (LLOMe). However, the mechanism by which lysosome-destabilizing agents trigger necrosis and proteolysis of inflammatory proteins is poorly understood. The proteasome is a cellular complex that has been shown to regulate both necrotic cell death and proteolysis of inflammatory proteins. We found that the peptide aldehyde proteasome inhibitors, MG115 and MG132, block lysosome rupture, degradation of inflammatory proteins and necrotic cell death mediated by the lysosome-destabilizing peptide LLOMe. However, non-aldehyde proteasome inhibitors failed to prevent LLOMe-induced cell death suggesting that aldehyde proteasome inhibitors triggered a pleotropic effect. We have previously shown that cathepsin C controls lysosome rupture, necrotic cell death and the adaptive immune response mediated by LLOMe. Using recombinant cathepsin C, we found that aldehyde proteasome inhibitors directly block cathepsin C, which presumably prevents LLOMe toxicity. The cathepsin B inhibitor CA-074-Me also blocks lysosome rupture and necrotic cell death mediated by a wide range of necrosis inducers, including LLOMe. Using cathepsin-deficient cells and recombinant cathepsins, we demonstrate that the cathepsins B and C are not required for the CA-074-Me block of necrotic cell death. Taken together, our findings demonstrate that lysosome-destabilizing adjuvants trigger an early proteolytic cascade, involving cathepsin C and a CA-074-Me-dependent protease. Identification of these early events leading to lysosome rupture will be crucial in our understanding of processes controlling necrotic cell death and immune responses mediated by lysosome-destabilizing adjuvants.

Topics: Adjuvants, Pharmaceutic; Aldehydes; Animals; Antigens, Bacterial; Bacterial Toxins; Cathepsin B; Cathepsin C; Dipeptides; Inflammation; Leupeptins; Lysosomes; Mice, Inbred BALB C; Mice, Inbred C57BL; Necrosis; Peptides; Proteasome Inhibitors; Proteolysis

Calpain activation has been implicated in the disease pathology of Duchenne muscular dystrophy. Inhibition of calpain has been proposed as a promising therapeutic target, which could lessen the protein degradation and prevent progressive fibrosis. At the same time, there are conflicting reports as to whether elevation of calpastatin, an endogenous calpain inhibitor, alters pathology. We compared the effects of pharmacological calpain inhibition in the mdx mouse using leupeptin and a proprietary compound (C101) that linked the inhibitory portion of leupeptin to carnitine (to increase uptake into muscle). Administration of C101 for 4 wk did not improve muscle histology, function, or serum creatine kinase levels in mdx mice. Mdx mice injected daily with leupeptin (36 mg/kg) for 6 mo also failed to show improved muscle function, histology, or creatine kinase levels. Biochemical analysis revealed that leupeptin administration caused an increase in m-calpain autolysis and proteasome activity, yet calpastatin levels were similar between treated and untreated mdx mice. These data demonstrate that pharmacological inhibition of calpain is not a promising intervention for the treatment of Duchenne muscular dystrophy due to the ability of skeletal muscle to counter calpain inhibitors by increasing multiple degradative pathways.

Topics: Animals; Biomarkers; Calcium-Binding Proteins; Calpain; Creatine Kinase; Cysteine Proteinase Inhibitors; Diaphragm; Disease Models, Animal; Dose-Response Relationship, Drug; Genotype; Leupeptins; Mice; Mice, Inbred mdx; Muscle Contraction; Muscle Strength; Muscular Dystrophy, Duchenne; Necrosis; Phenotype; Proteasome Endopeptidase Complex; Time Factors

Autophagy is simultaneously a mode of programmed cell death and an important physiological process for cell survival, but its pathophysiological significance in cardiac myocytes remains largely unknown. We induced autophagy in isolated adult rat ventricular cardiomyocytes (ARVCs) by incubating them in glucose-free, mannitol-supplemented medium for up to 4 days. Ultrastructurally, intracellular vacuoles containing degenerated subcellular organelles (e.g., mitochondria) were markedly apparent in the glucose-starved cells. Microtubule-associated protein-1 light chain 3 was significantly upregulated among the glucose-starved ARVCs than among the controls. After 4 days, glucose-starved ARVCs showed a significantly worse survival rate (19+/-5.2%) than the controls (55+/-8.3%, P<0.005). Most dead ARVCs in both groups showed features of necrosis, and the rate of apoptosis did not differ between the groups. Two inhibitors of autophagy, 3-methyladenine (3-MA) and leupeptin, significantly and dose-dependently reduced the viability of both control and glucose-starved ARVCs and caused specific morphological alterations; 3-MA reduced autophagic findings, whereas leupeptin greatly increased the numbers and the sizes of vacuoles that contained incompletely digested organelles. The knockdown of the autophagy-related genes with small interfering RNA also reduced the glucose-starved ARVCs viability, but rapamycin, an autophagy enhancer, improved it. Reductions in the ATP content of ARVCs caused by glucose depletion were exacerbated by the inhibitors while attenuated by rapamycin, suggesting that autophagy inhibition might accelerate energy depletion, leading to necrosis. Taken together, our findings suggest that autophagy in cardiomyocytes reflects a prosurvival, compensatory response to stress and that autophagic cardiomyocyte death represents an unsuccessful outcome due to necrosis.

Topics: Adenine; Adenosine Triphosphate; Animals; Autophagy; Cell Shape; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Glucose; Leupeptins; Male; Microtubule-Associated Proteins; Myocytes, Cardiac; Necrosis; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering; Sirolimus; Time Factors; Vacuoles

Activation of caspase-1 through the inflammasome protein Nalp1b controls anthrax lethal toxin (LT)-induced necrosis in murine macrophages. In this study we analyzed physiological changes controlled by caspase-1 in LT-treated murine macrophages. The caspase-1 inhibitor Boc-D-cmk blocked caspase-1 activity and membrane impairment in LT-treated cells. To determine the relationship between caspase-1 activation and membrane integrity, we added Boc-D-cmk to J774A.1 macrophages at different time points following LT exposure. Remarkably, Boc-D-cmk rescued LT-treated macrophages, even when added at the peak of caspase-1 activation. Late addition of the caspase-1 inhibitor reversed the losses of plasma membrane integrity and metabolic activity in these cells. Similar results were obtained with the proteasome inhibitor MG132, one of the most potent inhibitors of LT toxicity. LT-treated macrophages displaying evidence of membrane impairment recovered upon the addition of MG132, mirroring the Boc-D-cmk response. Strikingly, late addition of proteasome inhibitors also abrogated caspase-1 activity in LT-treated macrophages. Proteasomal control of caspase-1 activity and membrane impairment, however, was restricted to LT-induced cytolysis, because proteasome inhibitors did not block caspase-1 activation and cell death triggered by lipopolysaccharide and nigericin. Our findings indicate that proteasome inhibitors do not target caspase-1 directly but instead control an upstream event in LT-treated macrophages leading to caspase-1 activation. Taken together, caspase-1-mediated necrosis appears to be tightly controlled and differentially regulated by proteasomes depending on the source of caspase-1 induction.

Topics: Animals; Antigens, Bacterial; Apoptosis Regulatory Proteins; Bacterial Toxins; Caspase 1; Caspase Inhibitors; Cell Line; Cell Membrane; Cysteine Proteinase Inhibitors; Enzyme Activation; Ionophores; Leupeptins; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Necrosis; Nigericin; Proteasome Endopeptidase Complex

Targeting to the ubiquitin proteasome degradation pathway has become a promising approach for treating cancer. Previous studies showed that inhibition of the proteasome can induce apoptosis in various cancer cells. However, whether and how the inhibition of the proteasome induces other forms of cell death is not quite known. We previously showed that proteasome inhibitors including MG132 and Bortezomib could induce apoptosis in a Bax- and caspase-dependent way. In the present study, we found that in the absence of Bax and caspase activation, inhibition of the proteasome could also kill cancer cells by an alternative, non-apoptotic form of cell death. We further demonstrated that proteasome inhibitors, such as MG132, could induce intracellular accumulation of polyubiquitinated proteins and extensive cellular vacuolization likely due to ER stress. Translational or transcriptional inhibitors suppressed MG132-induced polyubiquitinated protein accumulation, and in turn inhibited MG132-induced ER stress, cellular vacuolization and cell death. These findings thus suggested that non-apoptotic cell death was resulted from misfolded protein accumulation and ER stress. Furthermore, our study indicated that proteasome inhibitors could be favorable chemotherapeutic agents because they could induce non-apoptotic cell death in addition to apoptosis, which could overcome resistance due to compromised apoptotic machinery.

Topics: Adenosine Triphosphate; Apoptosis; bcl-2-Associated X Protein; Cycloheximide; Dactinomycin; Endoplasmic Reticulum; HCT116 Cells; Humans; Leupeptins; Necrosis; Polyubiquitin; Proteasome Inhibitors; Protein Biosynthesis; Protein Folding; Protein Structure, Quaternary; Transcription, Genetic; Vacuoles

1,1-Bis(3'-indolyl)methane (DIM) and the 5,5'-dibromo ring substituted DIM (5,5'-diBrDIM) inhibited growth of MCF-7 and MDA-MB-231 breast cancer cells, and IC50 values were 10-20 and 1-5 microM, respectively, in both cell lines. DIM and 5,5'-diBrDIM did not induce p21 or p27 protein levels or alter expression of Sp1 or Sp3 proteins in either cell line. In contrast, 10 microM 5,5'-diBrDIM downregulated cyclin D1 protein in MCF-7 and MDA-MB-231 cells 12 and 24 h after treatment. DIM (20 microM) also decreased cyclin D1 in MCF-7 (24 h) and MDA-MB-231 (12 h), and the DIM/5,5'-diBrDIM-induced degradation of cyclin D1 was blocked by the proteasome inhibitor MG132. Both DIM and 5,5'-diBrDIM induced apoptosis in MCF-7 cells and this was accompanied by decreased Bcl-2, release of mitochondrial cytochrome c, and decreased mitochondrial membrane potential as determined by the red/green fluorescence of JC-1. DIM and 5,5'-diBrDIM induced extensive necrosis in MDA-MB-231 cells; however, this was accompanied by decreased mitochondrial membrane potential primarily in cells treated with 5,5'-diBrDIM but not DIM. Thus, DIM and 5,5'-diBrDIM induce cell death in MCF-7 and MDA-MB-231 cells by overlapping and different pathways, and the ring-substituted DIM represents a novel class of uncharged mitochondrial poisons that inhibit breast cancer cell and tumor growth.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cysteine Proteinase Inhibitors; Cytochromes c; Female; Humans; Indoles; Inhibitory Concentration 50; Leupeptins; Mitochondria; Necrosis; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; Time Factors

A role of proteasomal proteolysis in the pathogenesis of ischemia-reperfusion is being actively studied. To evaluate the participation of the proteasome in postconditioning phenomenon, we used primary culture of neonatal cardiomyocytes. 30 minutes of anoxia followed by 60 minutes of reoxygenation was undergone. Postconditioning was modeled by 3 cycles of 1-minute reoxygenation followed by 1-minute anoxia, respectively. Clasto-lactacystin b-lactone, a specific proteasome inhibitor, in the dose that does not cause cell death (2.5 mM) was added to the culture medium just before the cycles of postconditioning. Percentages of living, necrotic, and apoptotic cells were determined by staining with bisBenzimide and propidium iodide. Autophagy was demonstrated by staining vacuolar structures with monodansyl cadaverine. Proteasomal activity was determined by cleavage intensity of specific fluorogenic substrates. Trypsin-like, chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing (PGPH) activities were decreased after anoxia. Reoxygenation led to an increase in trypsin-like and chymotrypsin-like activities comparing to anoxia, but these parameters never reached the control levels. PGPH activity was restored up to the initial level. Postconditioning increased numbers of living cells and decreased that of necrotic, apoptotic and autophagic cells. Paradoxically, it was established, that proteasome inhibitors prevented the necrotic and apoptotic cell death of cardiomyocytes in anoxia-reoxygenation, but in the same concentration abolished the effects of postconditioning. The data obtained permit to suppose that proteasome inhibitors can be used for pharmacological postconditioning.

Topics: Animals; Animals, Newborn; Apoptosis; Cell Hypoxia; Cells, Cultured; Ischemic Preconditioning, Myocardial; Lactones; Leupeptins; Myocytes, Cardiac; Necrosis; Oxygen Consumption; Protease Inhibitors; Proteasome Inhibitors; Rats

Although the troponins are the serum proteins most frequently used nowadays to diagnose myocardial infarction, controversy continues about whether troponins are released later from infarcted myocardium than the cytoplasmic enzymes used previously, like lactate dehydrogenase (LDH). The present study compared the release kinetics of troponin-I (TnI) and LDH from necrotic cardiomyocytes in vitro. Cardiomyocytes prepared from neonatal rat ventricles were grown for 3 days. A total of 126 cultures were subjected to metabolic inhibition to induce cell necrosis. At various time intervals cells and media were collected for quantitative analysis of LDH activity and TnI concentration. Mean (+/-SD) LDH activity and TnI content of nine cultures at time t=0 were 2.07+/-0.30 U and 1.52+/-0.30 micro g per culture, respectively. Release of LDH from necrotic cardiomyocytes preceded release of TnI by about 60 min. The quantity of LDH released from the cultures after 210 min was 83.2+/-10.0%, whereas that of TnI released after 210 min was always less (33.8+/-22.2%). Cytochemical assessment of necrotic cardiomyocytes showed TnI-positive cells that were poor in LDH. The delay of TnI release relative to LDH release may be explained by slow dissociation of TnI molecules from myofilaments and/or formation of TnI degradation products that are undetected by the currently used ELISA assay.

Topics: Animals; Antimetabolites; Calpain; Cysteine Proteinase Inhibitors; Deoxyglucose; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; In Vitro Techniques; L-Lactate Dehydrogenase; Leupeptins; Male; Myocytes, Cardiac; Necrosis; Rats; Rats, Wistar; Sodium Cyanide; Troponin I

Inhibition of muscle degeneration by the tripeptide calpain inhibitor, leupeptin, was tested in vivo in a dystrophin-deficient mdx murine model. In a short-term control study, intramuscular administration of leupeptin for 30 days inhibited muscle degeneration as assessed by histologic analysis. Calpain inhibition could be correlated with retention of myofiber size and our results suggest that this may be a promising treatment modality in human Duchenne muscular dystrophy.

Topics: Animals; Calpain; Cysteine Proteinase Inhibitors; Histocytochemistry; Leupeptins; Male; Mice; Mice, Inbred C57BL; Mice, Inbred mdx; Microscopy, Electron; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Necrosis

Topics: Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Enzyme Activation; Enzyme Inhibitors; fas Receptor; Female; Humans; Leupeptins; Necrosis; Sphingosine; Tumor Cells, Cultured; U937 Cells

The possibility that bacteria may have evolved strategies to overcome host cell apoptosis was explored by using Rickettsia rickettsii, an obligate intracellular Gram-negative bacteria that is the etiologic agent of Rocky Mountain spotted fever. The vascular endothelial cell, the primary target cell during in vivo infection, exhibits no evidence of apoptosis during natural infection and is maintained for a sufficient time to allow replication and cell-to-cell spread prior to eventual death due to necrotic damage. Prior work in our laboratory demonstrated that R. rickettsii infection activates the transcription factor NF-kappa B and alters expression of several genes under its control. However, when R. rickettsii-induced activation of NF-kappa B was inhibited, apoptosis of infected but not uninfected endothelial cells rapidly ensued. In addition, human embryonic fibroblasts stably transfected with a superrepressor mutant inhibitory subunit Ikappa B that rendered NF-kappa B inactivatable also underwent apoptosis when infected, whereas infected wild-type human embryonic fibroblasts survived. R. rickettsii, therefore, appeared to inhibit host cell apoptosis via a mechanism dependent on NF-kappa B activation. Apoptotic nuclear changes correlated with presence of intracellular organisms and thus this previously unrecognized proapoptotic signal, masked by concomitant NF-kappa B activation, likely required intracellular infection. Our studies demonstrate that a bacterial organism can exert an antiapoptotic effect, thus modulating the host cell's apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection.

Topics: Apoptosis; Base Sequence; Binding Sites; Cell Nucleus; Cell Survival; Cells, Cultured; Consensus Sequence; Cysteine Proteinase Inhibitors; DNA Fragmentation; Embryo, Mammalian; Fibroblasts; Humans; Kinetics; Leupeptins; Microscopy, Electron; Necrosis; NF-kappa B; Oligodeoxyribonucleotides; Rickettsia rickettsii; Time Factors; Tumor Cells, Cultured; Umbilical Veins; Urinary Bladder Neoplasms

For the purpose of observing the therapeutic benefit of protease inhibitors for progressive muscular dystrophy, a large quantity of doses of leupeptin of 10 mg/kg/day and 50 mg/kg/day were administered i.p. to male chickens afflicted with hereditary muscular dystrophy (line 413) for 4 months starting on the 7th day ex ovo. No clinical improvement was identified in physical ability as a result of the examination by flip test, and creatine kinase (CK) values. The number of necrotic fibers in the pectoralis superficialis (PS) muscle which is known to be preferentially damaged in dystrophic chicken, did not decrease significantly in the birds treated with 10 mg leupeptin/kg/day (number of necrotic fibers; 47.7/mm2) and 50 mg/kg/day (46.4/mm2) as compared to that of the untreated ones (43.2/mm2). A morphometric analysis of fiber diameter distribution also showed no statistical difference between the treated and untreated birds. In the second group, 10 mg leupeptin/kg and a combination of leupeptin and bestatin of 10 mg/kg each were injected directly into the left lower half of the PS muscle three times a week for 4 months. Necrotic fibers were still present in the injected site, remote area of the left upper PS muscle treated with leupeptin (52.7/mm2), leupeptin and bestatin (52.2/mm2), and contralateral right upper PS muscle (41.6 and 53.5/mm2, respectively). The number of necrotic fibers in treated muscles was again not significantly different from that in untreated dystrophic ones (39.6/mm2). In fiber diameter analysis, no statistical difference was recognized between the treated and untreated dystrophic muscles.

Topics: Aminopeptidases; Animals; Chickens; Creatine Kinase; Drug Therapy, Combination; Leucine; Leupeptins; Male; Muscles; Muscular Dystrophy, Animal; Necrosis; Oligopeptides