RESUMO
Sigma-1 and sigma-2 receptors are emerging therapeutic targets. We have identified that simple ammonium salts bind to these receptors and are effective in vivo. Radioligand binding assays were used to obtain structure-activity relationships of these salts. MTS assays were performed to determine their effect on growth in MCF7 and MDA-MB-486 cells. Anticancer properties were tested in NMRI mice transplanted with a fragment of mouse adenocarcinoma (MAC13). Antidepressant activity was tested using the forced-swim test and tail suspension tests. Dipentylammonium (Ki 43 nM), tripentylammonium (Ki 15 nM) and trihexylammonium (Ki 9 nM) showed high affinity for the sigma-1 receptor. Dioctanoylammonium had the highest affinity (K50 0.05 nM); this also showed the highest affinity for sigma-2 receptors (Ki 13 nM). Dipentylammonium was found to have antidepressant activity in vivo. Branched-chain ammonium salts showed lower affinity. Bis(2-ethylhexyl)ammonium (K50 29 µM), triisopentylammonium (K50 196 µM) and dioctanoylammonium showed a low Hill slope, and fitted a 2-site binding model for the sigma-1 receptor. We propose this two-site binding can be used to biochemically define a sigma-1 receptor antagonist. Bis(2-ethylhexyl)ammonium and triisopentylammonium were able to inhibit the growth of tumours in vivo. Cheap, simple ammonium salts act as sigma-1 receptor agonists and antagonists in vivo and require further investigation.
Assuntos
Compostos de Amônio/química , Compostos de Amônio/farmacologia , Depressão/tratamento farmacológico , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Receptores sigma/metabolismo , Sais/química , Compostos de Amônio/metabolismo , Compostos de Amônio/uso terapêutico , Antidepressivos/química , Antidepressivos/metabolismo , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Depressão/metabolismo , Humanos , Células MCF-7 , Neoplasias/metabolismo , Receptor Sigma-1RESUMO
Substantial evidence indicates that aspirin and related non-steroidal anti-inflammatory drugs (NSAIDs) have potential as chemopreventative/therapeutic agents. However, these agents cannot be universally recommended for prevention purposes due to their potential side-effect profiles. Here, we compared the growth inhibitory and mechanistic activity of aspirin to two novel analogues, diaspirin (DiA) and fumaryl diaspirin (F-DiA). We found that the aspirin analogues inhibited cell proliferation and induced apoptosis of colorectal cancer cells at significantly lower doses than aspirin. Similar to aspirin, we found that an early response to the analogues was a reduction in levels of cyclin D1 and stimulation of the NF-κB pathway. This stimulation was associated with a significant reduction in basal levels of NF-κB transcriptional activity, in keeping with previous data for aspirin. However, in contrast to aspirin, DiA and F-DiA activity was not associated with nucleolar accumulation of RelA. For all assays, F-DiA had a more rapid and significant effect than DiA, identifying this agent as particularly active against colorectal cancer. Using a syngeneic colorectal tumour model in mice, we found that, while both agents significantly inhibited tumour growth in vivo, this effect was particularly pronounced for F-DiA. These data identify two compounds that are active against colorectal cancer in vitro and in vivo. They also identify a potential mechanism of action of these agents and shed light on the chemical structures that may be important for the antitumour effects of aspirin.
Assuntos
Adenocarcinoma , Anti-Inflamatórios não Esteroides/farmacologia , Apoptose/efeitos dos fármacos , Aspirina/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais , NF-kappa B/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Aspirina/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclina D/efeitos dos fármacos , Ciclina D/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Camundongos Nus , NF-kappa B/metabolismo , Fator de Transcrição RelA/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Zinc-α2-glycoprotein (ZAG) is an adipokine with the potential as a therapeutic agent in the treatment of obesity and type 2 diabetes. In this study we show that human ZAG, which is a 41-kDa protein, when administered to ob/ob mice at 50 µg/d(-1) orally in the drinking water produced a progressive loss of body weight (5 g after 8 d treatment), together with a 0.5 C increase in rectal temperature and a 40% reduction in urinary excretion of glucose. There was also a 33% reduction in the area under the curve during an oral glucose tolerance test and an increased sensitivity to insulin. These results were similar to those after iv administration of ZAG. However, tryptic digestion was shown to inactivate ZAG. There was no evidence of human ZAG in the serum but a 2-fold elevation of murine ZAG, which was also observed in target tissues such as white adipose tissue. To determine whether the effect was due to interaction of the human ZAG with the ß-adrenergic (ß-AR) in the gastrointestinal tract before digestion, ZAG was coadministered to ob/ob mice together with propanolol (40 mg/kg(-1)), a nonspecific ß-AR antagonist. The effect of ZAG on body weight, rectal temperature, urinary glucose excretion, improvement in glucose disposal, and increased insulin sensitivity were attenuated by propanolol, as was the increase in murine ZAG in the serum. These results suggest that oral administration of ZAG increases serum levels through interaction with a ß-AR in the upper gastrointestinal tract, and gene expression studies showed this to be in the esophagus.
Assuntos
Antagonistas Adrenérgicos beta/administração & dosagem , Peso Corporal/efeitos dos fármacos , Obesidade/metabolismo , Propranolol/administração & dosagem , Receptores Adrenérgicos beta/metabolismo , Proteínas de Plasma Seminal/administração & dosagem , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Administração Oral , Antagonistas Adrenérgicos beta/uso terapêutico , Animais , Temperatura Corporal/efeitos dos fármacos , Esôfago/efeitos dos fármacos , Esôfago/metabolismo , Camundongos , Obesidade/tratamento farmacológico , Propranolol/uso terapêutico , Proteínas de Plasma Seminal/uso terapêutico , Glicoproteína Zn-alfa-2RESUMO
The mechanism by which the adipokine zinc-α2-glycoprotein (ZAG) increases the mass of gastrocnemius, but not soleus muscle of diabetic mice, has been evaluated both in vivo and in vitro. There was an increased phosphorylation of both double-stranded RNA-dependent protein kinase and its substrate, eukaryotic initiation factor-2α, which was attenuated by about two-thirds in gastrocnemius but not soleus muscle of ob/ob mice treated with ZAG (50 µg, iv daily) for 5 d. ZAG also reduced the expression of the phospho forms of p38MAPK and phospholipase A2, as well as expression of the ubiquitin ligases (E3) muscle atrophy F-box/atrogin-1 and muscle RING finger protein, and the increased activity of both caspase-3 and casapse-8 to values found in nonobese controls. ZAG also increased the levels of phospho serine-threonine kinase and mammalian target of rapamycin in gastrocnemius muscle and reduced the phosphorylation of insulin receptor substrate-1 (Ser307) associated with insulin resistance. Similar changes were seen with ZAG when murine myotubes were incubated with high glucose concentrations (10 and 25 mm), showing that the effect of ZAG was direct. ZAG produced an increase in cAMP in murine myotubes, and the effects of ZAG on protein synthesis and degradation in vitro could be replicated by dibutyryl cAMP. ZAG increased cAMP levels of gastrocnemius but not soleus muscle. These results suggest that protein accretion in skeletal muscle in response to ZAG may be due to changes in intracellular cAMP and also that ZAG may have a therapeutic application in the treatment of muscle wasting conditions.
Assuntos
Músculo Esquelético/efeitos dos fármacos , Atrofia Muscular/prevenção & controle , Proteínas de Plasma Seminal/farmacologia , Proteínas de Plasma Seminal/uso terapêutico , Animais , Células Cultivadas , Citoproteção/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/prevenção & controle , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Camundongos , Camundongos Obesos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Obesidade/complicações , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Obesidade/patologia , Glicoproteína Zn-alfa-2 , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Zinc-alpha(2)-glycoprotein (ZAG) is an adipokine associated with fat loss in cancer cachexia. The purpose of this study was to evaluate the ability of recombinant human ZAG to attenuate type 2 diabetes in the ob/ob mouse model. ZAG (50 microg daily, iv) induced a progressive loss of body weight (3.5 g in 5 d), without an effect on food or water intake but with a 0.4 C rise in body temperature, suggesting an increased energy expenditure. Despite an increased plasma glycerol, indicative of increased lipolysis, levels of glucose, triglycerides, and nonesterified fatty acids were decreased by 17, 25, and 62%, respectively, due to an increased use of both glucose and lipids by muscle and brown adipose tissue. The weight of the latter increased 2-fold, and there was increased expression of uncoupling proteins-1 and -3. Plasma insulin levels were reduced by 36%, whereas pancreatic insulin was increased 4-fold, and there was a 53% decrease in the total area under the glucose curve in the glucose tolerance test and reduced insulin requirement. There was an increase in skeletal muscle mass due to an increase in protein synthesis and a decrease in protein degradation. These results suggest that ZAG may potentially be effective in the treatment of type 2 diabetes.
Assuntos
Proteínas de Transporte/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glicoproteínas/uso terapêutico , Hipoglicemiantes/uso terapêutico , Adipocinas , Animais , Modelos Animais de Doenças , Glucose/metabolismo , Humanos , Insulina/sangue , Resistência à Insulina , Masculino , Camundongos , Proteínas Recombinantes/uso terapêuticoRESUMO
D-myo-inositol 1,2,6-triphosphate (alpha trinositol, AT) has been shown to attenuate muscle atrophy in a murine cachexia model through an increase in protein synthesis and a decrease in degradation. The mechanism of this effect has been investigated in murine myotubes using a range of catabolic stimuli, including proteolysis-inducing factor (PIF), angiotensin II (Ang II), lipopolysaccharide, and tumor necrosis factor-alpha/interferon-gamma. At a concentration of 100 muM AT was found to attenuate both the induction of protein degradation and depression of protein synthesis in response to all stimuli. The effect on protein degradation was accompanied by attenuation of the increased expression and activity of the ubiquitin-proteasome pathway. This suggests that AT inhibits a signalling step common to all four agents. This target has been shown to be activation (autophosphorylation) of the dsRNA-dependent protein kinase (PKR) and the subsequent phosphorylation of eukaryotic initiation factor 2 on the alpha-subunit, together with downstream signalling pathways leading to protein degradation. AT also inhibited activation of caspase-3/-8, which is thought to lead to activation of PKR. The mechanism of this effect may be related to the ability of AT to chelate divalent metal ions, since the attenuation of the increased activity of the ubiquitin-proteasome pathway by PIF and Ang II, as well as the depression of protein synthesis by PIF, were reversed by increasing concentrations of Zn(2+). The ability of AT to attenuate muscle atrophy by a range of stimuli suggests that it may be effective in several catabolic conditions.
Assuntos
Atrofia/metabolismo , Fosfatos de Inositol/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Angiotensina II/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Atrofia/induzido quimicamente , Atrofia/tratamento farmacológico , Caspases/metabolismo , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/metabolismo , Expressão Gênica/efeitos dos fármacos , Fosfatos de Inositol/uso terapêutico , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Quinase C/metabolismo , Subunidades Proteicas/metabolismo , Proteoglicanas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Ubiquitinadas/metabolismo , Sulfato de Zinco/farmacologia , eIF-2 Quinase/metabolismoRESUMO
The mechanism of the effect of beta-hydroxy-beta-methylbutyrate (HMB) on protein degradation induced by lipopolysaccharide (LPS) has been evaluated in murine myotubes. HMB (50 muM) completely attenuated total protein degradation induced by LPS (1-100 ng/ml), formation of reactive oxygen species (ROS) and activation of caspase-3/-8. Specific inhibitors of caspase-3/-8 completely attenuated ROS production, total protein degradation and the LPS-induced autophosphorylation of dsRNA-dependent protein kinase (PKR). Protein degradation in response to LPS or ROS production was not seen in myotubes transfected with mutant PKRDelta6, suggesting that PKR was involved in ROS production, which was essential for total protein degradation. This was confirmed using the antioxidant butylated hydroxytoluene (BHT) which completely attenuated protein degradation in response to LPS. The link between PKR activation and ROS production was mediated through p38 mitogen-activated protein kinase (MAPK), which was activated by LPS in myotubes transfected with wild-type PKR, but not PKRDelta6. Both ROS production and protein degradation induced by LPS were completely attenuated by SB203580, a specific inhibitor of p38MAPK. This suggests that LPS induces protein degradation through a signalling cascade involving activation of caspase-3/-8, activation of PKR and production of ROS through p38MAPK, and that this process is attenuated by HMB.
Assuntos
Lipopolissacarídeos/farmacologia , Proteínas Musculares/metabolismo , Valeratos/farmacologia , Animais , Caspase 3/metabolismo , Caspase 8/metabolismo , Células Cultivadas , Camundongos , Fibras Musculares Esqueléticas/citologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , eIF-2 Quinase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Treatment of murine myotubes with high glucose concentrations (10 and 25 mM) stimulated protein degradation through the ubiquitin-proteasome pathway, and also caused activation (autophosphorylation) of PKR (double-stranded-RNA-dependent protein kinase) and eIF2alpha (eukaryotic initiation factor 2alpha). Phosphorylation of PKR and eIF2alpha was also seen in the gastrocnemius muscle of diabetic ob/ob mice. High glucose levels also inhibited protein synthesis. The effect of glucose on protein synthesis and degradation was not seen in myotubes transfected with a catalytically inactive variant (PKRDelta6). High glucose also induced an increased activity of both caspase-3 and -8, which led to activation of PKR, since this was completely attenuated by the specific caspase inhibitors. Activation of PKR also led to activation of p38MAPK (mitogen activated protein kinase), leading to ROS (reactive oxygen species) formation, since this was attenuated by the specific p38MAPK inhibitor SB203580. ROS formation was important in protein degradation, since it was completely attenuated by the antioxidant butylated hydroxytoluene. These results suggest that high glucose induces muscle atrophy through the caspase-3/-8 induced activation of PKR, leading to phosphorylation of eIF2alpha and depression of protein synthesis, together with PKR-mediated ROS production, through p38MAPK and increased protein degradation.
Assuntos
Hiperglicemia/metabolismo , Proteínas Musculares/deficiência , Animais , Atrofia , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular , Diabetes Mellitus/enzimologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Glucose/farmacologia , Masculino , Camundongos , Modelos Biológicos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/metabolismo , Miosinas/metabolismo , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina/metabolismo , eIF-2 Quinase/metabolismoRESUMO
Inhibition of dsRNA-activated protein kinase (PKR), not only attenuates muscle atrophy in a murine model of cancer cachexia (MAC16), but it also inhibits tumour growth. In vitro the PKR inhibitor maximally inhibited growth of MAC16 tumour cells at a concentration of 200 nM, which was also maximally effective in attenuating phosphorylation of PKR and of eukaryotic initiation factor (eIF)2 on the alpha-subunit. There was no effect on the growth of the MAC13 tumour, which does not induce cachexia, even at concentrations up to 1,000 nM. There was constitutive phosphorylation of PKR and eIF2alpha in the MAC16, but not in the MAC13 tumour, while levels of total PKR and eIF2alpha were similar. There was constitutive upregulation of nuclear factor-kappaB (NF-kappaB) in the MAC16 tumour only, and this was attenuated by the PKR inhibitor, suggesting that it arose from activation of PKR. In MAC16 alone the PKR inhibitor also attenuated expression of the 20S proteasome. The PKR inhibitor potentiated the cytotoxicity of both 5-fluorouracil and gemcitabine to MAC16 cells in vitro. These results suggest that inhibitors of PKR may be useful therapeutic agents against tumours showing increased expression of PKR and constitutive activation of NF-kappaB, and may also prove useful in sensitising tumours to standard chemotherapeutic agents.
Assuntos
Neoplasias Experimentais/patologia , Inibidores de Proteínas Quinases/farmacologia , RNA de Cadeia Dupla/efeitos dos fármacos , eIF-2 Quinase/antagonistas & inibidores , Animais , Antimetabólitos Antineoplásicos/farmacologia , Western Blotting , Proliferação de Células , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Ativação Enzimática/efeitos dos fármacos , Fluoruracila/farmacologia , Camundongos , NF-kappa B/metabolismo , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/genética , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas , Ribonucleotídeo Redutases/antagonistas & inibidores , GencitabinaRESUMO
Both tumor necrosis factor-alpha (TNF-alpha)/interferon-gamma (IFN-gamma) and angiotensin II (ANG II) induced an increase in total protein degradation in murine myotubes, which was completely attenuated by treatment with beta-hydroxy-beta-methylbutyrate (HMB; 50 microM). There was an increase in formation of reactive oxygen species (ROS) within 30 min, as well as an increase in the activity of both caspase-3 and -8, and both effects were attenuated by HMB. Moreover, inhibitors of caspase-3 and -8 completely attenuated both ROS formation and total protein degradation induced by TNF-alpha/IFN-gamma and ANG II. There was an increased autophosphorylation of double-stranded RNA-dependent protein kinase (PKR), which was attenuated by the specific caspase-3 and -8 inhibitors. Neither ROS formation or protein degradation occurred in myotubes expressing a catalytically inactive PKR variant, PKRDelta6, in response to TNF-alpha/IFN-gamma, compared with myotubes expressing wild-type PKR, although there was still activation of caspase-3 and -8. HMB also attenuated activation of PKR, suggesting that it was important in protein degradation. Formation of ROS was attenuated by rotenone, an inhibitor of the mitochondrial electron transport chain, nitro-l-arginine methyl ester, an inhibitor of nitric oxide synthase, and SB 203580, a specific inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), which also attenuated total protein degradation. Activation of p38 MAPK by PKR provides the link to ROS formation. These results suggest that TNF-alpha/IFN-gamma and ANG II induce muscle protein degradation by a common signaling pathway, which is attenuated by HMB, and that this involves the initial activation of caspase-3 and -8, followed by autophosphorylation and activation of PKR, which then leads to increased ROS formation via activation of p38 MAPK. Increased ROS formation is known to induce protein degradation through the ubiquitin-proteasome pathway.
Assuntos
Angiotensina II/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Proteínas Musculares/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Valeratos/farmacologia , Angiotensina II/efeitos adversos , Animais , Caspase 3/metabolismo , Caspase 8/metabolismo , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Interferon gama/farmacologia , Camundongos , Modelos Biológicos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/metabolismo , Atrofia Muscular/prevenção & controle , Fenilalanina/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Piridinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Valeratos/uso terapêutico , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
beta-Hydroxy-beta-methylbutyrate (HMB; 50 microM) has been shown to attenuate the depression in protein synthesis in murine myotubes in response to lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha) with or without interferon-gamma (IFN-gamma), and angiotensin II (ANG II). The mechanism for the depression of protein synthesis by all three agents was the same and was attributed to activation of double-stranded RNA-dependent protein kinase (PKR) with the subsequent phosphorylation of eukaryotic initiation factor 2 (eIF2) on the alpha-subunit as well as increased phosphorylation of the elongation factor (eEF2). Myotubes expressing a catalytically inactive PKR variant, PKRDelta6, showed no depression of protein synthesis in response to either LPS or TNF-alpha, confirming the importance of PKR in this process. There was no effect of any of the agents on phosphorylation of mammalian target of rapamycin (mTOR) or initiation factor 4E-binding protein (4E-BP1), and thus no change in the amount of eIF4E bound to 4E-BP1 or the concentration of the active eIF4E.eIF4G complex. HMB attenuated phosphorylation of eEF2, possibly by increasing phosphorylation of mTOR, and also attenuated phosphorylation of eIF2alpha by preventing activation of PKR. These results suggest that HMB may be effective in attenuating muscle atrophy in a range of catabolic conditions.
Assuntos
Angiotensina II/farmacologia , Lipopolissacarídeos/farmacologia , Proteínas Musculares/biossíntese , Biossíntese de Proteínas/efeitos dos fármacos , Fatores de Necrose Tumoral/farmacologia , Valeratos/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Angiotensina II/efeitos adversos , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Fatores de Iniciação em Eucariotos , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/metabolismo , Atrofia Muscular/prevenção & controle , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Valeratos/uso terapêutico , eIF-2 Quinase/metabolismoRESUMO
In the present study the role of Akt/PKB (protein kinase B) in PIF- (proteolysis-inducing factor) induced protein degradation has been investigated in murine myotubes. PIF induced transient phosphorylation of Akt at Ser(473) within 30 min, which was attenuated by the PI3K (phosphoinositide 3-kinase) inhibitor LY294002 and the tyrosine kinase inhibitor genistein. Protein degradation was attenuated in myotubes expressing a dominant-negative mutant of Akt (termed DNAkt), compared with the wild-type variant, whereas it was enhanced in myotubes containing a constitutively active Akt construct (termed MyrAkt). A similar effect was observed on the induction of the ubiquitin-proteasome pathway. Phosphorylation of Akt has been linked to up-regulation of the ubiquitin-proteasome pathway through activation of NF-kappaB (nuclear factor kappaB) in a PI3K-dependent process. Protein degradation was attenuated by rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin), when added before, or up to 30 min after, addition of PIF. PIF induced transient phosphorylation of mTOR and the 70 kDa ribosomal protein S6 kinase. These results suggest that transient activation of Akt results in an increased protein degradation through activation of NF-kappaB and that this also allows for a specific synthesis of proteasome subunits.
Assuntos
Proteínas Musculares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteoglicanas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Linhagem Celular , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Serina-Treonina Quinases TOR , Ubiquitina/metabolismoRESUMO
In the present study, the BCAAs (branched-chain amino acids) leucine and valine caused a significant suppression in the loss of body weight in mice bearing a cachexia-inducing tumour (MAC16), producing a significant increase in skeletal muscle wet weight, through an increase in protein synthesis and a decrease in degradation. Leucine attenuated the increased phosphorylation of PKR (double-stranded-RNA-dependent protein kinase) and eIF2alpha (eukaryotic initiation factor 2alpha) in skeletal muscle of mice bearing the MAC16 tumour, due to an increased expression of PP1 (protein phosphatase 1). Weight loss in mice bearing the MAC16 tumour was associated with an increased amount of eIF4E bound to its binding protein 4E-BP1 (eIF4E-binding protein 1), and a progressive decrease in the active eIF4G-eIF4E complex due to hypophosphorylation of 4E-BP1. This may be due to a reduction in the phosphorylation of mTOR (mammalian target of rapamycin), which may also be responsible for the decreased phosphorylation of p70(S6k) (70 kDa ribosomal S6 kinase). There was also a 5-fold increase in the phosphorylation of eEF2 (eukaryotic elongation factor 2), which would also decrease protein synthesis through a decrease in translation elongation. Treatment with leucine increased phosphorylation of mTOR and p70(S6k), caused hyperphosphorylation of 4E-BP1, reduced the amount of 4E-BP1 associated with eIF4E and caused an increase in the eIF4G-eIF4E complex, together with a reduction in phosphorylation of eEF2. These changes would be expected to increase protein synthesis, whereas a reduction in the activation of PKR would be expected to attenuate the increased protein degradation.
Assuntos
Aminoácidos de Cadeia Ramificada/farmacologia , Caquexia/metabolismo , Atrofia Muscular/metabolismo , Neoplasias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Fatores de Iniciação em Eucariotos , Leucina/farmacologia , Camundongos , Camundongos Endogâmicos , Mioblastos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteoglicanas/farmacologia , Fatores de Tempo , Valina/farmacologiaRESUMO
To investigate the mechanism by which beta-hydroxy-beta-methylbutyrate (HMB) attenuates the depression of protein synthesis in the skeletal muscle of cachectic mice, a study has been carried out in murine myotubes in the presence of proteolysis-inducing factor (PIF). PIF inhibited protein synthesis by 50% within 4 h, and this was effectively attenuated by HMB (25-50 muM). HMB (50 muM) alone stimulated protein synthesis, and this was attenuated by rapamycin (27 nM), an inhibitor of mammalian target of rapamycin (mTOR). Further evidence for an involvement of this pathway was shown by an increased phosphorylation of mTOR, the 70-kDa ribosomal S6 kinase (p70(S6k)), and initiation factor 4E-binding protein (4E-BP1) and an increased association of eukaryotic initiation factor 2 (eIF4E) with eIF4G. PIF alone induced a transient (1-2 h) stimulation of phosphorylation of mTOR and p70(S6k). However, in the presence of HMB, phosphorylation of mTOR, p70(S6k), and 4E-BP1 was increased, and inactive 4E-BP1-eIF4E complex was reduced, whereas the active eIF4G.eIF4E complex was increased, suggesting continual stimulation of protein synthesis. HMB alone reduced phosphorylation of elongation factor 2, but this effect was not seen in the presence of PIF. PIF induced autophosphorylation of the double-strand RNA-dependent protein kinase (PKR), leading to phosphorylation of eIF2 on the alpha-subunit, which would inhibit protein synthesis. However, in the presence of HMB, phosphorylation of PKR and eIF2alpha was attenuated, and this was also observed in skeletal muscle of cachectic mice administered HMB (0.25 g/kg). These results suggest that HMB attenuates the depression of protein synthesis by PIF in myotubes through multiple mechanisms.
Assuntos
Caquexia/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Valeratos/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Caquexia/etiologia , Caquexia/patologia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Fatores de Iniciação em Eucariotos , Masculino , Camundongos , Camundongos Endogâmicos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Neoplasias Musculares/complicações , Neoplasias Musculares/metabolismo , Transplante de Neoplasias , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteoglicanas/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TORRESUMO
Insulin-like growth factor-I (IGF-I) has been shown to attenuate protein degradation in murine myotubes induced by angiotensin II through downregulation of the ubiquitin-proteasome pathway, although the mechanism is not known. Angiotensin II is known to upregulate this pathway through a cellular signalling mechanism involving release of arachidonic acid, activation of protein kinase Calpha (PKCalpha), degradation of inhibitor-kappaB (I-kappaB) and nuclear migration of nuclear factor-kappaB (NF-kappaB), and all of these events were attenuated by IGF-I (13.2 nM). Induction of the ubiquitin-proteasome pathway has been linked to activation of the RNA-activated protein kinase (PKR), since an inhibitor of PKR attenuated proteasome expression and activity in response to angiotensin II and prevented the decrease in the myofibrillar protein myosin. Angiotensin II induced phosphorylation of PKR and of the eukaryotic initiation factor-2 (eIF2) on the alpha-subunit, and this was attenuated by IGF-I, by induction of the expression of protein phosphatase 1, which dephosphorylates PKR. Release of arachidonic acid and activation of PKCalpha by angiotensin II were attenuated by an inhibitor of PKR and IGF-I, and the effect was reversed by Salubrinal (15 muM), an inhibitor of eIF2alpha dephosphorylation, as was activation of PKCalpha. In addition myotubes transfected with a dominant-negative PKR (PKRDelta6) showed no release of arachidonate in response to Ang II, and no activation of PKCalpha. These results suggest that phosphorylation of PKR by angiotensin II was responsible for the activation of the PLA(2)/PKC pathway leading to activation of NF-kappaB and that IGF-I attenuates protein degradation due to an inhibitory effect on activation of PKR.
Assuntos
Angiotensina II/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Animais , Ácido Araquidônico/metabolismo , Células Cultivadas , Cinamatos/farmacologia , Citosol/efeitos dos fármacos , Citosol/enzimologia , Fator de Iniciação 2B em Eucariotos/metabolismo , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tioureia/análogos & derivados , Tioureia/farmacologia , Trítio , Ubiquitina/metabolismo , eIF-2 Quinase/metabolismoRESUMO
Angiotensin II (Ang II) has been implicated in muscle protein loss in cachexia. To determine whether the Ang I/II system directly inhibits protein synthesis in muscle their effect has been monitored in vitro using murine myotubes as a surrogate model system. Ang I inhibited protein synthesis by 40-50% over the concentration range of 0.05-2.5 microM within 30 min of addition, and the inhibition remained relatively constant over 24 h. The effect was attenuated by co-incubation with the angiotensin converting enzyme inhibitor imidaprilat (50 microM) suggesting that inhibition of protein synthesis was due to the formation of Ang II. Ang II also inhibited protein synthesis by 40-50% over the concentration range of 0.1-5 microM, and the inhibition also remained relatively constant between 30 min and 24 h after addition. The effect was attenuated by insulin-like growth factor-1 (IGF-1) (25-100 ng/ml). Thus, Ang I/II have the ability to induce muscle atrophy through inhibition of protein synthesis.
Assuntos
Angiotensina II/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Vasoconstritores/farmacologia , Animais , Combinação de Medicamentos , Imidazolidinas/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismoRESUMO
Angiotensin I and II have been shown to directly induce protein degradation in skeletal muscle through an increased activity and expression of the ubiquitin-proteasome proteolytic pathway. This investigation determines the role of the nuclear transcription factor nuclear factor-kappaB (NF-kappaB) in this process. Using murine myotubes as a surrogate model system both angiotensin I and II were found to induce activation of protein kinase C (PKC), with a parabolic dose-response curve similar to the induction of total protein degradation. Activation of PKC was required for the induction of proteasome expression, since calphostin C, a highly specific inhibitor of PKC, attenuated both the increase in total protein degradation and in proteasome expression and functional activity increased by angiotensin II. PKC is known to activate I-kappaB kinase (IKK), which is responsible for the phosphorylation and subsequent degradation of I-kappaB. Both angiotensin I and II induced an early decrease in cytoplasmic I-kappaB levels followed by nuclear accumulation of NF-kappaB. Using an NF-kappaB luciferase construct this was shown to increase transcriptional activation of NF-kappaB regulated genes. Maximal luciferase expression was seen at the same concentrations of angiotensin I/II as those inducing protein degradation. Total protein degradation induced by both angiotensin I and II was attenuated by resveratrol, which prevented nuclear accumulation of NF-kappaB, confirming that activation of NF-kappaB was responsible for the increased protein degradation. These results suggest that induction of proteasome expression by angiotensin I/II involves a signalling pathway involving PKC and NF-kappaB.
Assuntos
Angiotensina II/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Complexo de Endopeptidases do Proteassoma/biossíntese , Transporte Ativo do Núcleo Celular , Angiotensina I/fisiologia , Animais , Proteínas Sanguíneas/metabolismo , Linhagem Celular , Ativação Enzimática , Quinase I-kappa B/metabolismo , Luciferases/biossíntese , Luciferases/genética , Camundongos , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Naftalenos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteoglicanas , Resveratrol , Transdução de Sinais , Estilbenos/farmacologia , Ativação TranscricionalRESUMO
BACKGROUND: n-3 fatty acids are increasingly being administered to cancer patients for the treatment of cachexia, and it is thus important to know of any potential interactions with ongoing cytotoxic drug therapy. MATERIALS AND METHODS: For this reason eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were administered to mice bearing the cachexia-inducing MAC16 colon adenocarcinoma, and the effect of epothilone, gemcitabine, 5-fluorouracil and cyclophosphamide on tumour growth and body weight determined. RESULTS: Epothilone alone had a minimal effect on tumour growth rate, but this was potentiated by DH4, while for 5-fluorouracil and cyclophosphamide tumour growth inhibition was enhanced by EPA. The antitumour effect of gemcitabine was not altered by either fatty acid. EPA arrested the development of cachexia, while DHA had no effect and the same was true for their effect on tumour growth rate. The anticachectic effect of EPA was only seen in combination with 5-fluorouracil. CONCLUSION: These results suggest that n-3 fatty acids do not interfere with the action of chemotherapy and may potentiate the effect of certain agents.
Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Ácidos Docosa-Hexaenoicos/uso terapêutico , Ácido Eicosapentaenoico/uso terapêutico , Adenocarcinoma/complicações , Adenocarcinoma/patologia , Animais , Peso Corporal/efeitos dos fármacos , Caquexia/tratamento farmacológico , Caquexia/etiologia , Caquexia/patologia , Neoplasias do Colo/complicações , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Masculino , Camundongos , Camundongos Endogâmicos , Transplante de NeoplasiasRESUMO
Loss of adipose tissue in cancer cachexia has been associated with tumour production of a lipid-mobilizing factor (LMF) which has been shown to be homologous with the plasma protein zinc-alpha(2)-glycoprotein (ZAG). The aim of this study was to compare the ability of human ZAG with LMF to stimulate lipolysis in vitro and induce loss of body fat in vivo, and to determine the mechanisms involved. ZAG was purified from human plasma using a combination of Q Sepharose and Superdex 75 chromatography, and was shown to stimulate glycerol release from isolated murine epididymal adipocytes in a dose-dependent manner. The effect was enhanced by the cyclic AMP phosphodiesterase inhibitor Ro20-1724, and attenuated by freeze/thawing and the specific beta3-adrenoreceptor antagonist SR59230A. In vivo ZAG caused highly significant, time-dependent, decreases in body weight without a reduction in food and water intake. Body composition analysis showed that loss of body weight could be attributed entirely to the loss of body fat. Loss of adipose tissue may have been due to the lipolytic effect of ZAG coupled with an increase in energy expenditure, since there was a dose-dependent increase in expression of uncoupling protein-1 (UCP-1) in brown adipose tissue. These results suggest that ZAG may be effective in the treatment of obesity.