RESUMEN
Disulfiram (DSF), used since the 1950s in the treatment of alcoholism, is reductively activated to diethyldithiocarbamate and both compounds are thiol-reactive and readily complex copper. More recently DSF and copper-DSF (Cu-DSF) have been found to exhibit potent anticancer activity. We have previously shown that the anti-diabetic drug metformin is anti-proliferative and induces an intracellular reducing environment in oesophageal squamous cell carcinoma (OSCC) cell lines. Based on these observations, we investigated the effects of Cu-DSF and DSF, with and without metformin, in this present study. We found that Cu-DSF and DSF caused considerable cytotoxicity across a panel of OSCC cells, and metformin significantly enhanced the effects of DSF. Elevated copper transport contributes to DSF and metformin-DSF-induced cytotoxicity since the cell-impermeable copper chelator, bathocuproinedisulfonic acid, partially reversed the cytotoxic effects of these drugs, and interestingly, metformin-treated OSCC cells contained higher intracellular copper levels. Furthermore, DSF may target cancer cells preferentially due to their high dependence on protein degradation/turnover pathways, and we found that metformin further enhances the role of DSF as a proteasome inhibitor. We hypothesized that the lysosome could be an additional, novel, target of DSF. Indeed, this acid-labile compound decreased lysosomal acidification, and DSF-metformin co-treatment interfered with the progression of autophagy in these cells. In summary, this is the first such report identifying the lysosome as a target of DSF and based on the considerable cytotoxic effects of DSF either alone or in the presence of metformin, in vitro, and we propose these as novel potential chemotherapeutic approaches for OSCC.
Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Cobre/administración & dosificación , Disulfiram/administración & dosificación , Neoplasias Esofágicas/tratamiento farmacológico , Metformina/administración & dosificación , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Sinergismo Farmacológico , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago , Humanos , Complejo de la Endopetidasa Proteasomal/administración & dosificación , Proteolisis/efectos de los fármacosRESUMEN
The three proteasome subunits with proteolytic activity are encoded by standard or immunoproteasome genes. Many proteasomes expressed by normal cells and cells exposed to cytokines are "mixed", that is, contain both standard and immunoproteasome subunits. Using a panel of 38 defined influenza A virus-derived epitopes recognized by C57BL/6 mouse CD8(+) T cells, we used mice with targeted disruption of ß1i, ß2i, or ß5i/ß2i genes to examine the contribution of mixed proteasomes to the immunodominance hierarchy of antiviral CD8(+) T cells. We show that each immunoproteasome subunit has large effects on the primary and recall immunodominance hierarchies due to modulating both the available T cell repertoire and generation of individual epitopes as determined both biochemically and kinetically in Ag presentation assays. These findings indicate that mixed proteasomes function to enhance the diversity of peptides and support a broad CD8(+) T cell response.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Virus de la Influenza A/inmunología , Biosíntesis de Péptidos , Complejo de la Endopetidasa Proteasomal/inmunología , Regulación hacia Arriba/inmunología , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/biosíntesis , Animales , Linfocitos T CD8-positivos/enzimología , Técnicas de Cocultivo , Virus de la Influenza A/patogenicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Orthomyxoviridae/enzimología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Biosíntesis de Péptidos/genética , Biosíntesis de Péptidos/inmunología , Complejo de la Endopetidasa Proteasomal/administración & dosificación , Complejo de la Endopetidasa Proteasomal/genética , Células Tumorales CultivadasRESUMEN
The treatment of myeloma has been revolutionized by the availability of new drugs. Combination therapy followed by stem cell transplant holds the promise of ultimately curing a fraction of patients. Objective responses are the norm for induction therapy and up to 50% of patients achieve a complete remission. Ongoing clinical trials continue to address the role of long term maintenance therapy. Understanding the proper management of these agents is paramount to providing patients disease control and yet minimizing side effects from treatment.
Asunto(s)
Antineoplásicos/uso terapéutico , Ácidos Borónicos/uso terapéutico , Mieloma Múltiple/terapia , Inhibidores de Proteasoma , Pirazinas/uso terapéutico , Trasplante de Células Madre , Talidomida/análogos & derivados , Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ácidos Borónicos/administración & dosificación , Bortezomib , Humanos , Lenalidomida , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/tratamiento farmacológico , Complejo de la Endopetidasa Proteasomal/administración & dosificación , Pirazinas/administración & dosificación , Talidomida/administración & dosificación , Talidomida/uso terapéuticoRESUMEN
The development of a safe and effective non-live intranasal influenza vaccine has been an elusive target in vaccinology for many decades. It is perceived that intranasal immunization, by offering a more convenient and less invasive vaccination modality, will boost vaccination rates against influenza, a disease that continues to inflict a significant annual health and economic burden worldwide. Intranasal immunization may also confer additional immunoprotective benefits by eliciting mucosal secretory antibodies at the site of entry of the virus, which are typically more broadly cross-reactive and cross-protective compared with those induced by systemic routes of vaccination. This property is highly desirable for confering improved protection against variant strains of influenza virus. Here we review the current status of intranasal proteosome-based influenza vaccines that comprise commercial detergent-split influenza antigens and proteosome adjuvants derived from purified bacterial outer membrane proteins. We demonstrate that these vaccines exhibit the desired advantages expected from immunization via the intranasal route. Furthermore, in clinical trials proteosome-based influenza vaccines were shown to be safe and protective in humans. The future possibilities for commercializing intranasal proteosome-influenza vaccines are also discussed.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Complejo de la Endopetidasa Proteasomal/inmunología , Adyuvantes Inmunológicos/efectos adversos , Administración Intranasal , Adolescente , Adulto , Anciano , Animales , Anticuerpos Antivirales/sangre , Niño , Preescolar , Ensayos Clínicos como Asunto , Humanos , Inmunidad Mucosa , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/efectos adversos , Gripe Humana/inmunología , Ratones , Persona de Mediana Edad , Complejo de la Endopetidasa Proteasomal/administración & dosificación , Complejo de la Endopetidasa Proteasomal/efectos adversos , Resultado del Tratamiento , Vacunación , Adulto JovenRESUMEN
The proteasome is the main protease for degrading oxidized proteins. We asked whether altered proteasome function contributes to the accumulation of oxidized muscle proteins with aging. Proteasome structure, function, and oxidation state were compared in young and aged F344BN rat fast-twitch skeletal muscle. In proteasome-enriched homogenates from aged muscle, we observed a two- to threefold increase in content of the 20S proteasome that was due to a corresponding increase in immunoproteasome. Content of the regulatory proteins, PA700 and PA28, relative to the 20S were reduced 75% with aging. Upon addition of exogenous PA700, there was a twofold increase in peptide hydrolysis in aged muscle, suggesting the endogenous content of PA700 is inadequate for complete activation of the 20S. Measures of catalytic activity showed a 50% reduction in specific activity for proteasome-enriched homogenates with aging. With purification of the 20S, proteasome specific activity was equivalent between ages, indicating that endogenous regulators inhibit proteasome in aged muscle. Significantly less degradation of oxidized calmodulin by the 20S from aged muscle was observed. Partial rescue of activity for aged 20S by DTT implies oxidation of functionally significant cysteines. These results demonstrate significant age-related changes in proteasome structure, function, and oxidation state that could inhibit removal of oxidized proteins.