mg-262 and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Proteasome inhibition has emerged as a novel approach to anticancer therapy. Numerous natural compounds, such as gambogic acid, have been tested in vitro and in vivo as anticancer agents for cancer prevention and therapy. However, whether gambogic acid has chemosensitizing properties when combined with proteasome inhibitors in the treatment of malignant cells is still unknown. In an effort to investigate this effect, human leukemia K562 cells, mouse hepatocarcinoma H22 cells and H22 cell allografts were treated with gambogic acid, a proteasome inhibitor (MG132 or MG262) or the combination of both, followed by measurement of cellular viability, apoptosis induction and tumor growth inhibition. We report, for the first time, that: (i) the combination of natural product gambogic acid and the proteasome inhibitor MG132 or MG262 results in a synergistic inhibitory effect on growth of malignant cells and tumors in allograft animal models and (ii) there was no apparent systemic toxicity observed in the animals treated with the combination. Therefore, the findings presented in this study demonstrate that natural product gambogic acid is a valuable candidate to be used in combination with proteasome inhibitors, thus representing a compelling anticancer strategy.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Boronic Acids; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Survival; Cycloheximide; Cysteine Proteinase Inhibitors; Drug Synergism; Humans; K562 Cells; Leupeptins; Male; Mice; Neoplasms, Experimental; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Synthesis Inhibitors; Signal Transduction; Transplantation, Homologous; Tumor Burden; Xanthones

Intracellular protein degradation by the ubiquitin-proteasome system is ATP dependent, and the optimal ATP concentration to activate proteasome function in vitro is ∼100 μM. Intracellular ATP levels are generally in the low millimolar range, but ATP at a level within this range was shown to inhibit proteasome peptidase activities in vitro. Here, we report new evidence that supports a hypothesis that intracellular ATP at the physiological levels bidirectionally regulates 26S proteasome proteolytic function in the cell. First, we confirmed that ATP exerted bidirectional regulation on the 26S proteasome in vitro, with the optimal ATP concentration (between 50 and 100 μM) stimulating proteasome chymotrypsin-like activities. Second, we found that manipulating intracellular ATP levels also led to bidirectional changes in the levels of proteasome-specific protein substrates in cultured cells. Finally, measures to increase intracellular ATP enhanced, while decreasing intracellular ATP attenuated the ability of proteasome inhibition to induce cell death. These data strongly suggest that endogenous ATP within the physiological concentration range can exert a negative impact on proteasome activities, allowing the cell to rapidly upregulate proteasome activity on ATP reduction under stress conditions.

Topics: Adenosine Triphosphate; Apoptosis; Boronic Acids; Cell Line; Humans; Leupeptins; Microscopy, Fluorescence; Oligomycins; Proteasome Endopeptidase Complex; Time Factors; Ubiquitin

Proteasome inhibitors are considered to have anti-inflammatory therapeutic potential. However, recent reports addressing proteasome inhibition in the vascular system are controversial, ranging from beneficial anti-inflammatory and anti-oxidative effects to potentiation of inflammation and oxidative stress. This study was based on the hypothesis that the divergent effects might be a result of a differential and dose-dependent responsiveness of vascular cells to proteasome inhibitors. We tested whether low doses of proteasome inhibitors would favor anti-inflammatory effects in vascular cells in vitro and in vivo. Human umbilical vein endothelial cells (HUVEC) were preincubated with proteasome inhibitors MG132 and MG262 at concentrations that did not affect cell viability during a 24-h treatment. Upon addition of tumor necrosis factor alpha (TNF-alpha) the induced expression of adhesion molecules and the adhesion of monocytic THP-1 cells to HUVECs was significantly lowered. However, nuclear translocation of NF-kappaB was only slightly diminished. Low-dose pretreatment with proteasome inhibitors decreased TNF-alpha-induced generation of reactive oxygen species in HUVEC. Bortezomib was administered at a dose of 50 microg/kg body weight to Dahl salt-sensitive rats (DSSR) on high-salt diet. This low-dose proteasome inhibition led to decreased hypertension-induced oxidative stress and reduced expression of vascular cell adhesion molecule 1 (VCAM-1) in the aortae.

Topics: Animals; Anti-Inflammatory Agents; Boronic Acids; Bortezomib; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Endothelial Cells; Enzyme Inhibitors; Gene Expression Regulation; Humans; Leupeptins; Male; Monocytes; NF-kappa B; Oxidative Stress; Proteasome Inhibitors; Pyrazines; Rats; Rats, Inbred Dahl; Reactive Oxygen Species; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Intrinsic conformational transitions contribute to the catalytic action of many enzymes. Here we use a single-molecule approach to demonstrate how such transitions are linked to the catalytic sites of the eukaryotic proteasome, an essential protease of the ubiquitin pathway. The active sites of the cylindrical proteasomal core particle are located in a central chamber accessible through gated entry channels. By using atomic force microscopy, we found continual alternation between open and closed gate conformations. We analyzed the relative abundance of these conformers in wild-type and mutated yeast core particles upon exposure to substrates or inhibitors. Our data indicate that the dynamic gate can be opened by allosteric coupling to a tetrahedral transition state at any of the working active centers. The results point to the N(alpha)-amine of the N-terminal active site threonyl residue as the major effector group responsible for triggering the essential conformational switch.

Topics: Acetylation; Boronic Acids; Catalysis; Catalytic Domain; Cysteine Proteinase Inhibitors; Kinetics; Leupeptins; Ligands; Microscopy, Atomic Force; Molecular Structure; Mutation; Proteasome Endopeptidase Complex; Protein Binding; Protein Conformation; Protein Subunits; Saccharomyces cerevisiae Proteins; Substrate Specificity

Glutathione peroxidase-3 (GPx-3) is a key antioxidant enzyme in the plasma. GPx-3 was previously identified as the major antioxidative enzyme that was induced upon nontoxic proteasome inhibition in endothelial cells. Here, we investigated the determinants of the proteasome inhibitor-induced expression of GPx-3. Nontoxic proteasome inhibition massively upregulates GPx-3 RNA and protein in human umbilical cord vein cells within 24 h. Surprisingly, induction of GPx-3 was species-specific for human cells. The exponential upregulation of GPx-3 is mediated by transcriptional activation of the human GPx-3 promoter and, in addition, stabilization of GPx-3 mRNA: in reporter gene assays with full-length and deleted variants of the human GPx-3 promoter we identified a putative antioxidative response element (ARE) as essential and also sufficient for transcriptional activation of GPx-3 by proteasome inhibition. However, the ARE-specific antioxidative transcription factor Nrf2 is not involved in the activation of GPx-3. UV-crosslinking using the 3'UTR of GPx-3 revealed an altered protein binding pattern in the presence of proteasome inhibitors, thus indicating regulation of mRNA stability of human GPx-3. As GPx-3 is secreted into the plasma, our data point toward a borderline defense mechanism of endothelial cell-derived GPx-3 to protect the vasculature from oxidative stress.

Topics: Animals; Boronic Acids; Cattle; Cells, Cultured; Endothelial Cells; Glutathione Peroxidase; Humans; Leupeptins; Mutation; Oxidative Stress; Promoter Regions, Genetic; Proteasome Inhibitors; Protein Binding; Rats; Response Elements; RNA Stability; Species Specificity; Transcriptional Activation

Inhibitors of the proteasome interfere with transcriptional regulation of growth signaling pathways and block cell cycle progression of mitotic cells. As growth signaling pathways are highly conserved between mitotic and postmitotic cells, we hypothesized that proteasome inhibition might also be a valuable approach to interfere with hypertrophic growth of postmitotic cardiomyocytes. To test this hypothesis, we analyzed the effects of proteasome inhibition on hypertrophic growth of neonatal rat cardiomyocytes. Partial inhibition of the proteasome effectively suppressed cardiomyocyte hypertrophy as determined by reduced cell size, inhibition of hypertrophy-mediated induction of RNA and protein synthesis, reduced expression of several hypertrophic marker genes, and diminished transcriptional activation of the BNP promotor. Importantly, suppression of hypertrophic growth was independent of the hypertrophic agonist used. Expressional profiling and subsequent Western blot and kinase assays revealed that proteasome inhibition induced a cellular stress response with reduced expression of conserved growth signaling mediators and impaired G1/S phase transition of cardiomyocytes. In hypertensive Dahl-salt sensitive rats, inhibition of the proteasome with low doses of the FDA approved proteasome inhibitor Velcade significantly reduced hypertrophic heart growth. Our data provide important insight into the suppressive effects of proteasome inhibitors on hypertrophic growth of cardiomyocytes and establish low-dose proteasome inhibition as a new and broad-spectrum approach to interfere with cardiac hypertrophy.

Topics: Animals; Animals, Newborn; Biomarkers; Boronic Acids; Cardiomegaly; Cells, Cultured; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Down-Regulation; Hypertrophy; Leupeptins; Male; Myocytes, Cardiac; Proteasome Inhibitors; Rats; Rats, Inbred Dahl; Signal Transduction; Ubiquitin

When a replicative DNA polymerase encounters a lesion on the template strand and stalls, it is replaced with another polymerase(s) with low processivity that bypasses the lesion to continue DNA synthesis. This phenomenon is known as translesion replication or replicative bypass. Failing this, the cell is increasingly likely to undergo apoptosis. In this study, we found that proteasome inhibitors prevent translesion replication in human cancer cells but not in normal cells. Three proteasome inhibitors, MG-132, lactacystin, and MG-262, inhibited UV-induced translesion replication in a wide range of cancer cell lines, including HeLa, HGC-27, MCF-7, HepG2, WiDr, a malignant melanoma, an acute lymphoblastic leukemia, and a multiple myeloma cell line; irrespective of cell origin, histological type, or p53 status. In contrast, these inhibitors had little or no influence on normal fibroblasts (NB1RGB and TIG-1) or a normal liver mesenchymal (LI90) cell line. Among the DNA-damaging antineoplastic agents, cisplatin caused a UV-type translesion reaction; the proteasome inhibitors delayed cisplatin-induced translesion replication in cancer cell lines but had only a weak effect on normal cell lines. Therefore, translesion replication would be an effective target of proteasome inhibitors for cancer chemotherapy by which cancer cells can be efficiently sensitized to DNA-damaging antineoplastic agents, such as cisplatin.

Topics: Acetylcysteine; Boronic Acids; Caffeine; Cells, Cultured; Cisplatin; Cysteine Proteinase Inhibitors; DNA Damage; DNA Repair; DNA Replication; HeLa Cells; Humans; Leupeptins; Neoplasms; Proteasome Inhibitors; Tumor Cells, Cultured; Ultraviolet Rays

The ability of human neutrophils to express a variety of genes encoding inflammatory mediators is well documented, and mounting evidence suggests that neutrophil-derived cytokines and chemokines contribute to the recruitment of discrete leukocyte populations at inflammatory sites. Despite this, our understanding of the signaling intermediates governing the generation of inflammatory cytokines by neutrophils remains fragmentary. Here, we report that inhibitors of the p38 MAPK and MEK pathways substantially diminish the release of (and in the case of p38 inhibitors, the gene expression of) several inflammatory cytokines in neutrophils stimulated with LPS or TNF. In addition, various NF-kappaB inhibitors were found to profoundly impede the inducible gene expression and release of inflammatory cytokines in these cells. The MAPK inhibitors did not affect NF-kappaB activation; instead, the transcriptional effects of the p38 MAPK inhibitor appear to involve transcriptional factor IID. Conversely, the NF-kappaB inhibitors failed to affect the activation of MAPKs. Finally, the MAPK inhibitors were found to prevent the activation a key component of the translational machinery, S6 ribosomal protein, in keeping with their post-transcriptional impact on cytokine generation. To our knowledge, this constitutes the first demonstration that in neutrophils, the inducible expression of proinflammatory cytokines by physiological stimuli largely reflects the ability of the latter to activate NF-kappaB and selected MAPK pathways. Our data also raise the possibility that NF-kappaB or MAPK inhibitors could be useful in the treatment of inflammatory disorders in which neutrophils predominate.

Topics: Boronic Acids; Cell Differentiation; Cytokines; Humans; Inflammation; Leupeptins; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Neutrophils; NF-kappa B; Nitriles; Proline; Protein Kinase Inhibitors; Structure-Activity Relationship; Sulfones; Thiocarbamates

Mammalian hepatic cytochromes P450 (P450s) are endoplasmic reticulum (ER)-anchored hemoproteins engaged in the metabolism of numerous xeno- and endobiotics. P450s exhibit widely ranging half-lives, utilizing both autophagic-lysosomal (ALD) and ubiquitin-dependent 26S proteasomal (UPD) degradation pathways. Although suicidally inactivated hepatic CYPs 3A and "native" CYP3A4 in Saccharomyces cerevisiae are degraded via UPD, the turnover of native hepatic CYPs 3A in their physiological milieu has not been elucidated. Herein, we characterize the degradation of native, dexamethasone-inducible CYPs 3A in cultured primary rat hepatocytes, using proteasomal (MG-132 and MG-262) and ALD [NH4Cl and 3-methyladenine (3-MA)] inhibitors to examine their specific degradation route. Pulse-chase with immunoprecipitation analyses revealed a basal 52% 35S-CYP3A loss over 6 h, which was stabilized by both proteasomal inhibitors. By contrast, no corresponding CYP3A stabilization was detected with either ALD inhibitor NH4Cl or 3-MA. Furthermore, MG-262-induced CYP3A stabilization was associated with its polyubiquitylation, thereby verifying that native CYPs 3A were also degraded via UPD. To identify the specific participants in this process, cellular proteins were cross-linked in situ with paraformaldehyde (PFA) in cultured hepatocytes. Immunoblotting analyses of CYP3A immunoprecipitates after PFA-cross-linking revealed the presence of p97, a cytosolic AAA ATPase instrumental in the extraction and delivery of ubiquitylated ER proteins for proteasomal degradation. Such native CYP3A-p97 interactions were greatly magnified after CYP3A suicidal inactivation (which accelerates UPD), and/or proteasomal inhibition, and were confirmed by proteomic and confocal immunofluorescence microscopic analyses. These findings clearly reveal that native CYPs 3A undergo UPD and implicate a role for p97 in this process.

Topics: Adenine; Adenosine Triphosphatases; Ammonium Chloride; Animals; Autophagy; Boronic Acids; Cell Cycle Proteins; Cross-Linking Reagents; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Dexamethasone; Dicarbethoxydihydrocollidine; Formaldehyde; Hepatocytes; Leupeptins; Lysosomes; Male; Nuclear Proteins; Polymers; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Saccharomyces cerevisiae Proteins; Troleandomycin; Ubiquitin; Valosin Containing Protein

Inhibitors of the ubiquitin-proteasome system offer a new and promising approach in the therapy of proliferative and inflammatory diseases. In order to narrow the therapeutic window for cytotoxic effects on the one hand and nontoxic, anti-inflammatory effects on the other hand, we elucidated the complex cellular effects of toxic versus nontoxic proteasome inhibition in human endothelial cells by expressional profiling. Nontoxic doses of proteasome inhibitors induced a defined, dose-dependent transcriptional response that was markedly attenuated in terms of gene number and amplitude of regulation compared to toxic doses. In particular, we observed uniform upregulation of several antioxidative enzymes and differential regulation of genes involved in endothelial function. This adaptive transcriptional pattern was translated into a protective response of endothelial cells against H(2)O(2)-induced oxidative stress and into improvement of endothelial function of rat aortic rings. Our data thus suggest that nontoxic proteasome inhibition might offer a new therapeutic approach for the treatment of endothelial dysfunction in cardiovascular disorders.

Topics: Antioxidants; Apoptosis; Boronic Acids; Cells, Cultured; Cysteine Proteinase Inhibitors; Cytoprotection; Dose-Response Relationship, Drug; Endothelial Cells; Endothelium, Vascular; Gene Expression Profiling; Gene Expression Regulation; Humans; Hydrogen Peroxide; Leupeptins; Oxidative Stress; Proteasome Inhibitors; Transcription, Genetic

Cancer-induced bone disease results in bone destruction, pathological fractures, and pain. We hypothesized that the inhibition of the proteasome-ubiquitin system in osteoclasts could abolish the receptor activator of NF-kappaB ligand (RANKL) mediated osteoclast differentiation and function, since RANKL-mediated downstream signaling plays a crucial role in osteoclast life cycle. In this study, we examined the effects of the proteasome inhibitors MG-132 and MG-262 on RANKL-induced osteoclast differentiation and function. Osteoclast precursors from peripheral blood mononuclear cells were cultured in the presence of RANKL and M-CSF. Osteoclasts were identified as multi-nucleated TRAP-positive cells. Osteoclast function was quantified with the extent of dentine resorption and TRAP activity in culture supernatants. For the evaluation of the effects of proteasome inhibitors towards osteoclastogenesis, sub-apoptotic concentrations of MG-132 and MG-262 were used. Effects on NF-kappaB were obtained in treated and untreated osteoclasts. MG-132 and MG-262 inhibit both osteoclast differentiation and osteoclast function. 0.01 microM MG-132 induced a 3.2-fold (P = 0.004) and 0.001 microM MG-262 a 3.3-fold (P = 0.004) reduction of osteoclast differentiation, respectively. The resorption capacity was decreased 2.6- and 11.1-fold (P = 0.003) by treatment with 0.01 and 0.1 microM MG-132, and 14.2- and 16.6-fold (P = 0.003) by 0.001 and 0.01 microM MG-262, respectively. This decrease correlated with the extent of NF-kappaB binding capacity. In conclusion, this study shows for the first time that proteasome inhibitors act on osteoclast development and function at low concentrations and should be considered as potential drugs for the treatment of cancer-induced osteolytic bone disease.

Topics: Bone Resorption; Boronic Acids; Carrier Proteins; Cell Differentiation; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Humans; Leupeptins; Membrane Glycoproteins; NF-kappa B; Osteoclasts; Proteasome Endopeptidase Complex; Proteasome Inhibitors; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Transcriptional Activation

Impairment of the ubiquitin/proteasome system has been proposed to play a role in neurodegenerative disorders such as Alzheimer and Parkinson diseases. Although recent studies confirmed that some disease-related proteins block proteasomal degradation, and despite the existence of excellent animal models of both diseases, in vivo data about the system are lacking. We have developed a model for in vivo analysis of the ubiquitin/proteasome system by generating mouse strains transgenic for a green fluorescent protein (GFP) reporter carrying a constitutively active degradation signal. Administration of proteasome inhibitors to the transgenic animals resulted in a substantial accumulation of GFP in multiple tissues, confirming the in vivo functionality of the reporter. Moreover, accumulation of the reporter was induced in primary neurons by UBB+1, an aberrant ubiquitin found in Alzheimer disease. These transgenic animals provide a tool for monitoring the status of the ubiquitin/proteasome system in physiologic or pathologic conditions.

Topics: Alzheimer Disease; Animals; Boronic Acids; Cells, Cultured; Cysteine Endopeptidases; Fibroblasts; Leupeptins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence; Models, Animal; Multienzyme Complexes; Myocytes, Cardiac; Neurodegenerative Diseases; Neurons; Oligopeptides; Organ Specificity; Parkinson Disease; Proteasome Endopeptidase Complex; Recombinant Fusion Proteins; Tissue Distribution; Ubiquitin

The proteasome is the primary protease used by cells for degrading proteins and generating peptide ligands for class I molecules of the major histocompatibility complex. Based on the properties of cells adapted to grow in the presence of the proteasome inhibitor 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone (NLVS), it was proposed that proteasomes can be replaced by alternative proteolytic systems, particularly a large proteolytic complex with a tripeptidyl peptidase II activity. Here we show that NLVS-adapted cells retain sensitivity to a number of highly specific proteasome inhibitors with regard to antigenic peptide generation, accumulation of polyubiquitinated proteins, degradation of p53, and cell viability. In addition, we show that in the same assays (with a single minor exception), NLVS-adapted cells are about as sensitive as nonselected cells to Ala-Ala-Phe-chloromethylketone, a specific inhibitor of tripeptidyl peptidase II activity. Based on these findings, we conclude that proteasomes still have essential proteolytic functions in adapted cells that are not replaced by Ala-Ala-Phe-chloromethylketone-sensitive proteases.

Topics: Amino Acid Chloromethyl Ketones; Aminopeptidases; Animals; Antigen Presentation; Antigens; Boronic Acids; Bortezomib; CD8-Positive T-Lymphocytes; Cell Survival; Cysteine Endopeptidases; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Drug Resistance; Endopeptidases; Enzyme Activation; H-2 Antigens; Leupeptins; Lymphoma, T-Cell; Mice; Multienzyme Complexes; Neoplasm Proteins; Oligopeptides; Peptide Fragments; Phenols; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Pyrazines; Selection, Genetic; Serine Endopeptidases; Sulfones; Thymus Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tyramine; Ubiquitins