leupeptins and sodium-arsenite

Endemic arsenism is widely distributed in the world, which can damage multiple organs, especially in skin and liver. The etiology is clear, but the mechanisms involved remain unknown. Ubiquitin-proteasome pathway (UPP) is the main pathway regulating protein degradation of which proteasome subunit beta type-5(PSMB5) plays a dominant role. This paper aims to study the role and mechanism of PSMB5 in sodium arsenite (NaAsO

Topics: Arsenites; Cell Survival; Cells, Cultured; Chemical and Drug Induced Liver Injury; Gene Expression; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Humans; Leupeptins; Oxidative Stress; Proteasome Endopeptidase Complex; Proteasome Inhibitors; RNA, Small Interfering; Sodium Compounds; Superoxide Dismutase-1

The present study examined the effect of sodium arsenite, cadmium chloride, heat shock and the proteasomal inhibitors MG132, withaferin A and celastrol on heme oxygenase-1 (HO-1; also known as HSP32) accumulation in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that HO-1 accumulation was not induced by heat shock but was enhanced by sodium arsenite and cadmium chloride in a dose- and time-dependent fashion. Immunocytochemistry revealed that these metals induced HO-1 accumulation in a granular pattern primarily in the cytoplasm. Additionally, in 20% of the cells arsenite induced the formation of large HO-1-containing perinuclear structures. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 accumulation initially increased to a maximum at 12h followed by a 50% reduction at 48 h. This initial increase in HO-1 levels was likely the result of new synthesis as it was inhibited by cycloheximide. Interestingly, treatment of cells with a mild heat shock enhanced HO-1 accumulation induced by low concentrations of sodium arsenite and cadmium chloride. Finally, we determined that HO-1 accumulation was induced in A6 cells by the proteasomal inhibitors, MG132, withaferin A and celastrol. An examination of heavy metal and proteasomal inhibitor-induced HO-1 accumulation in amphibians is of importance given the presence of toxic heavy metals in aquatic habitats.

Topics: Animals; Arsenites; Cadmium Chloride; Cell Line; Cytoplasmic Structures; Enzyme Induction; Heme Oxygenase-1; Hot Temperature; HSP30 Heat-Shock Proteins; Immunohistochemistry; Kidney; Leupeptins; Pentacyclic Triterpenes; Proteasome Inhibitors; Protein Transport; Sodium Compounds; Toxicity Tests, Acute; Triterpenes; Water Pollutants, Chemical; Withanolides; Xenopus laevis; Xenopus Proteins

Sodium arsenite (NA) and cadmium chloride (CdCl(2)) are relatively abundant environmental toxicants that have multiple toxic effects including carcinogenesis, dysfunction of gene regulation and DNA and protein damage. In the present study, treatment of Xenopus laevis A6 kidney epithelial cells with concentrations of NA (20-30 μM) or CdCl(2) (100-200 μM) that induced HSP30 and HSP70 accumulation also produced an increase in the relative levels of ubiquitinated protein. Actin protein levels were unchanged in these experiments. In time course experiments, the levels of ubiquitinated protein and HSPs increased over a 24h exposure to NA or CdCl(2). Furthermore, treatment of cells with NA or CdCl(2) reduced the relative levels of proteasome chymotrypsin (CT)-like activity compared to control. Interestingly, pretreatment of cells with the HSP accumulation inhibitor, KNK437, prior to NA or CdCl(2) exposure decreased the relative levels of ubiquitinated protein as well as HSP30 and HSP70. A similar finding was made with ubiquitinated protein induced by proteasomal inhibitors, MG132 and celastrol, known to induce HSP accumulation in A6 cells. However, the NA- or CdCl(2)-induced decrease in proteasome CT-like activity was not altered by KNK437 pretreatment. This study has shown for the first time in poikilothermic vertebrates that NA and CdCl(2) can inhibit proteasomal activity and that there is a possible association between HSP accumulation and the mechanism of protein ubiquitination.

Topics: Animals; Arsenites; Benzhydryl Compounds; Cadmium Chloride; Cell Line; Chymotrypsin; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Environmental Pollutants; Epithelial Cells; Heat-Shock Proteins; HSP30 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Immunoblotting; Kidney; Leupeptins; Pentacyclic Triterpenes; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrrolidinones; Sodium Compounds; Time Factors; Triterpenes; Ubiquitinated Proteins; Ubiquitination; Xenopus laevis; Xenopus Proteins

Increased oxygen species production has often been cited as a mechanism determining synergism on cell death and growth inhibition effects of arsenic-combined drugs. However the net effect of drug combination may not be easily anticipated solely from available knowledge of drug-induced death mechanisms. We evaluated the combined effect of sodium arsenite with the proteasome inhibitor MG132, and the anti-leukaemic agent CAPE, on growth-inhibition and cell death effect in acute myeloid leukaemic cells U937 and Burkitt's lymphoma-derived Raji cells, by the Chou-Talalay method. In addition we explored the association of cytotoxic effect of drugs with changes in intracellular superoxide anion (O₂⁻) levels. Our results showed that combined arsenite+MG132 produced low levels of O₂⁻ at 6h and 24h after exposure and were synergic on cell death induction in U937 cells over the whole dose range, although the combination was antagonistic on growth inhibition effect. Exposure to a constant non-cytotoxic dose of 80μM hydrogen peroxide together with arsenite+MG132 changed synergism on cell death to antagonism at all effect levels while increasing O₂⁻ levels. Arsenite+hydrogen peroxide also resulted in antagonism with increased O₂⁻ levels in U937 cells. In Raji cells, arsenite+MG132 also produced low levels of O₂⁻ at 6h and 24h but resulted in antagonism on cell death and growth inhibition. By contrast, the combination arsenite+CAPE showed high levels of O₂⁻ production at 6h and 24 h post exposure but resulted in antagonism over cell death and growth inhibition effects in U937 and Raji cells. We conclude that synergism between arsenite and MG132 in U937 cells is negatively associated to O₂⁻ levels at early time points after exposure.

Topics: Antineoplastic Agents; Arsenites; Burkitt Lymphoma; Cell Death; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Synergism; Humans; Hydrogen Peroxide; Leukemia, Myeloid, Acute; Leupeptins; Reactive Oxygen Species; Sodium Compounds; Superoxides; Time Factors; U937 Cells

Timely induction of cyclin B1 controls mitotic entry, whereas its proteolysis is essential for mitotic exit. By contrast, cyclin B1 transcription is repressed during G(2) arrest induced by DNA damage. The p38 mitogen-activated protein kinase is involved in the G(2) checkpoint; yet, its impact on cyclin B1 protein levels remains unclear. Here we show that untimely proteolysis of cyclin B1 following p38 activation contributes to G(2) checkpoint. Exposing early G(2) cells to arsenite impeded cyclin B1 protein accumulation, Cdk1 activation, and G(2)-to-M progression. Conversely, cyclin B1 was non-degradable in late G(2) and mitotic cells after arsenite. Cyclin B1 proteolysis was enhanced by arsenite in early G(2) and asynchronous cells. This rapid destruction of cyclin B1 was mediated via the ubiquitin-proteasome pathway probably in a Cdc20 and Cdh1 independent mechanism. Under arsenite, inhibition of p38 activation or depletion of p38alpha suppressed cyclin B1 ubiquitination and proteolysis, while forced expression of MKK6-p38 accelerated these events. Inactivation of p38 in arsenite-treated early G(2) cells allowed G(2)-to-M progression, blocked apoptosis, increased cell viability, and decreased micronucleus formation. Thus, p38 signaling pathway triggering cyclin B1 proteolysis after arsenite may play an important role in connecting G(2) arrest with apoptosis or genome instability.

Topics: Anaphase-Promoting Complex-Cyclosome; Apoptosis; Arsenites; Carcinogens, Environmental; CDC2 Protein Kinase; Cell Division; Cell Line, Tumor; Cell Survival; Cyclin B; Cyclin B1; Cycloheximide; Enzyme Activation; G2 Phase; Humans; Imidazoles; Leupeptins; MAP Kinase Signaling System; Micronuclei, Chromosome-Defective; Mitogen-Activated Protein Kinase 14; Mitosis; Peptide Hydrolases; Phosphorylation; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Kinase Inhibitors; Protein Synthesis Inhibitors; Pyridines; Sodium Compounds; Ubiquitin; Ubiquitin-Protein Ligase Complexes

Epidemiological studies have demonstrated a high association of inorganic arsenic exposure with vascular diseases. Recent research has also linked this vascular damage to impairment of endothelial nitric oxide synthase (eNOS) function by arsenic exposure. However, the role of eNOS in regulating the arsenite-induced vascular dysfunction still remains to be clarified. In our present study, we investigated the effect of arsenite on Akt1 and eNOS and its involvement in cytotoxicity of vascular endothelial cells. Our study demonstrated that arsenite decreased the protein levels of both Akt1 and eNOS accompanied with increased levels of ubiquitination of total cell lysates. We found that inhibition of the ubiquitin-proteasome pathway by MG-132 could partially protect Akt1 and eNOS from degradation by arsenite together with a proportional protection from the arsenite-induced cytoxicity. Moreover, up-regulation of eNOS protein expression significantly attenuated the arsenite-induced cytotoxicity and eNOS activity could be significantly inhibited after incubation with arsenite for 24 h in a cell-free system. Our study indicated that endothelial eNOS activity could be attenuated by arsenite via the ubiquitin-proteasome-mediated degradation of Akt1/eNOS as well as via direct inhibition of eNOS activity. Our study also demonstrated that eNOS actually played a protective role in arsenite-induced cytoxicity. These observations supported the hypothesis that the impairment of eNOS function by arsenite is one of the mechanisms leading to vascular changes and diseases.

Topics: Androstadienes; Animals; Aorta, Thoracic; Arsenites; Cell Line; Cell Survival; Chromones; Dose-Response Relationship, Drug; Down-Regulation; Endothelial Cells; Endothelium, Vascular; Leupeptins; Morpholines; Nitric Oxide Synthase Type III; Phosphatidylinositol 3-Kinases; Phosphorylation; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-akt; Sodium Compounds; Swine; Time Factors; Up-Regulation; Wortmannin

A widely expressed protein containing UBA (ubiquitin-associated) and UBX (ubiquitin-like) domains was identified as a substrate of SAPKs (stress-activated protein kinases). Termed SAKS1 (SAPK substrate-1), it was phosphorylated efficiently at Ser200 in vitro by SAPK3/p38gamma, SAPK4/p38delta and JNK (c-Jun N-terminal kinase), but weakly by SAPK2a/p38alpha, SAPK2b/p38beta2 or ERK (extracellular-signal-regulated kinase) 2. Ser200, situated immediately N-terminal to the UBX domain, became phosphorylated in HEK-293 (human embryonic kidney) cells in response to stressors. Phosphorylation was not prevented by SB 203580 (an inhibitor of SAPK2a/p38alpha and SAPK2b/p38beta2) and/or PD 184352 (which inhibits the activation of ERK1 and ERK2), and was similar in fibroblasts lacking both SAPK3/p38gamma and SAPK4/p38delta or JNK1 and JNK2. SAKS1 bound ubiquitin tetramers and VCP (valosin-containing protein) in vitro via the UBA and UBX domains respectively. The amount of VCP in cell extracts that bound to immobilized GST (glutathione S-transferase)-SAKS1 was enhanced by elevating the level of polyubiquitinated proteins, while SAKS1 and VCP in extracts were coimmunoprecipitated with an antibody raised against S5a, a component of the 19 S proteasomal subunit that binds polyubiquitinated proteins. PNGase (peptide N-glycanase) formed a 1:1 complex with VCP and, for this reason, also bound to immobilized GST-SAKS1. We suggest that SAKS1 may be an adaptor that directs VCP to polyubiquitinated proteins, and PNGase to misfolded glycoproteins, facilitating their destruction by the proteasome.

Topics: Adenosine Triphosphatases; Amino Acid Sequence; Animals; Arsenites; Cell Cycle Proteins; Cell Extracts; Cell Line; Cells, Cultured; Fibroblasts; Humans; Immunoprecipitation; Kidney; Leupeptins; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Multienzyme Complexes; Osmotic Pressure; p38 Mitogen-Activated Protein Kinases; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Peptides; Polyubiquitin; Protein Binding; Protein Structure, Tertiary; Protein Subunits; Sodium Compounds; Substrate Specificity; Valosin Containing Protein