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1.
Int J Mol Sci ; 22(22)2021 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-34830246

RESUMO

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by hyperglycemia, responsible for the onset of several long-term complications. Recent evidence suggests that cognitive dysfunction represents an emerging complication of DM, but the underlying molecular mechanisms are still obscure. Dopamine (DA), a neurotransmitter essentially known for its relevance in the regulation of behavior and movement, modulates cognitive function, too. Interestingly, alterations of the dopaminergic system have been observed in DM. This review aims to offer a comprehensive overview of the most relevant experimental results assessing DA's role in cognitive function, highlighting the presence of dopaminergic dysfunction in DM and supporting a role for glucotoxicity in DM-associated dopaminergic dysfunction and cognitive impairment. Several studies confirm a role for DA in cognition both in animal models and in humans. Similarly, significant alterations of the dopaminergic system have been observed in animal models of experimental diabetes and in diabetic patients, too. Evidence is accumulating that advanced glycation end products (AGEs) and their precursor methylglyoxal (MGO) are associated with cognitive impairment and alterations of the dopaminergic system. Further research is needed to clarify the molecular mechanisms linking DM-associated dopaminergic dysfunction and cognitive impairment and to assess the deleterious impact of glucotoxicity.


Assuntos
Disfunção Cognitiva/metabolismo , Diabetes Mellitus/metabolismo , Dopamina/metabolismo , Glucose/toxicidade , Produtos Finais de Glicação Avançada/metabolismo , Hiperglicemia/metabolismo , Animais , Cognição/efeitos dos fármacos , Cognição/fisiologia , Disfunção Cognitiva/complicações , Disfunção Cognitiva/fisiopatologia , Complicações do Diabetes/metabolismo , Complicações do Diabetes/fisiopatologia , Diabetes Mellitus/fisiopatologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Glucose/metabolismo , Humanos , Hiperglicemia/complicações , Hiperglicemia/fisiopatologia , Aldeído Pirúvico/metabolismo , Transdução de Sinais
2.
J Cell Physiol ; 234(7): 11861-11870, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30536670

RESUMO

Tyrosine hydroxylase (TH), catalyzing the conversion of tyrosine into l-DOPA, is the rate-limiting enzyme in dopamine synthesis. Defects in insulin action contribute to alterations of TH expression and/or activity in the brain and insulin increases TH levels in 1-methyl-4-phenylpyridinium (MPP+)-treated neuronal cells. However, the molecular mechanisms underlying the regulation of TH by insulin have not been elucidated yet. Using PC12 cells, we show for the first time that insulin increases TH expression in a biphasic manner, with a transient peak at 2 hr and a delayed response at 16 hr, which persists for up to 24 hr. The use of a dominant negative hypoxia-inducible factor 1-alpha (HIF-1α) and its pharmacological inhibitor chetomin, together with chromatin immunoprecipitation (ChIP) experiments for the specific binding to TH promoter, demonstrate the direct role of HIF-1α in the early phase. Moreover, ChIP experiments and transfection of a dominant negative of the nerve growth factor IB (Nur77) indicate the involvement of Nur77 in the late phase insulin response, which is mediated by HIF-1α. In conclusion, the present study shows that insulin regulates TH expression through HIF-1α and Nur77 in PC12 cells, supporting the critical role of insulin signaling in maintaining an appropriate dopaminergic tone by regulating TH expression in the central nervous system.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Insulina/farmacologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Tirosina 3-Mono-Oxigenase/efeitos dos fármacos , Animais , Hipóxia Celular/fisiologia , Dopamina/metabolismo , Insulina/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Ratos , Ativação Transcricional/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismo , Regulação para Cima
3.
J Cell Physiol ; 233(9): 7367-7378, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29663374

RESUMO

Prostate cancer (PCa) is the most commonly diagnosed malignancy in men and the second leading cause of cancer-related death in industrialized countries. Epidemiologic evidence suggests that obesity promotes aggressive PCa. Recently, a family of Free Fatty Acid (FFA) receptors (FFARs) has been identified and reported to affect several crucial biological functions of tumor cells such as proliferation, invasiveness, and apoptosis. Here we report that oleic acid (OA), one of the most prevalent FFA in human plasma, increases proliferation of highly malignant PC3 and DU-145 PCa cells. Furthermore, docetaxel cytotoxic action, the first-line chemotherapeutic agent for the treatment of androgen-independent PCa, was significantly reduced in the presence of OA, when measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, suggesting that this FFA plays also a role in chemoresistance. OA induced intracellular calcium increase, in part due to the store operated calcium entry (SOCE), measured by a calcium imaging technique. Moreover, PI3K/Akt signaling pathway was enhanced, as revealed by increased Akt phosphorylation levels. Intriguingly, attenuating the expression of FFA1/GPR40, a receptor for long chain FFA including OA, prevented the OA-induced effects. Of relevance, we found that FFA1/GPR40 is significantly overexpressed in tissue specimens of PCa, compared to benign prostatic hyperplasia tissues, at both mRNA and protein expression level, analyzed by Real Time RT-PCR and immunofluorescence experiments, respectively. Our data suggest that OA promotes an aggressive phenotype in PCa cells via FFA1/GPR40, calcium and PI3K/Akt signaling. Thus, FFA1/GPR40, might represent a potential useful prognostic biomarker and therapeutic target for the treatment of advanced PCa.


Assuntos
Ácido Oleico/farmacologia , Neoplasias da Próstata/patologia , Receptores Acoplados a Proteínas G/metabolismo , Idoso , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Docetaxel/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/efeitos dos fármacos
4.
J Cell Physiol ; 232(12): 3735-3743, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28177128

RESUMO

Endometrial cancer is often characterized by PI3K/AKT pathway deregulation. Recently it has been suggested that SGK1, a serine/threonine protein kinase that shares structural and functional similarities with the AKT family, might play a role in cancer, since its expression and/or activity has been found to be deregulated in different human tumors. However, the role of SGK1 in endometrial cancer has been poorly investigated. Here, we show that SGK1 expression is increased in tissue specimens from neoplastic endometrium. The SGK1 inhibitor SI113 induced a significant reduction of endometrial cancer cells viability, measured by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. This effect was associated to the increase of autophagy, as revealed by the increase of the markers LC3B-II and beclin I, detected by both immunofluorescence and western blot analysis. SI113 treatment caused also apoptosis of endometrial cancer cells, evidenced by the cleavage of the apoptotic markers PARP and Caspase-9. Intriguingly, these effects were associated to the induction of endoplasmic reticulum stress markers GRP78 and CHOP evaluated by both Real-Time RT-PCR and Western Blot analysis. Increased expression of SGK1 in endometrial cancer tissues suggest a role for SGK1 in this type of cancer, as reported for other malignancies. Moreover, the efficacy of SI113 in affecting endometrial cancer cells viability, possibly via endoplasmic reticulum stress activation, identifies SGK1 as an attractive molecular target for new tailored therapeutic intervention for the treatment of endometrial cancer.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias do Endométrio/tratamento farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteína Beclina-1/metabolismo , Estudos de Casos e Controles , Caspase 9/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Neoplasias do Endométrio/enzimologia , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Chaperona BiP do Retículo Endoplasmático , Feminino , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas Imediatamente Precoces/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo
5.
Diabetologia ; 57(7): 1485-94, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24759959

RESUMO

AIMS/HYPOTHESIS: Insulin exerts a direct action on vascular cells, thereby affecting the outcome and progression of diabetic vascular complications. However, the mechanism through which insulin signalling is impaired in the endothelium of diabetic individuals remains unclear. In this work, we have evaluated the role of the AGE precursor methylglyoxal (MGO) in generating endothelial insulin resistance both in cells and in animal models. METHODS: Time course experiments were performed on mouse aortic endothelial cells (MAECs) incubated with 500 µmol/l MGO. The glyoxalase-1 inhibitor S-p-bromobenzylglutathione-cyclopentyl-diester (SpBrBzGSHCp2) was used to increase the endogenous levels of MGO. For the in vivo study, an MGO solution was administrated i.p. to C57BL/6 mice for 7 weeks. RESULTS: MGO prevented the insulin-dependent activation of the IRS1/protein kinase Akt/endothelial nitric oxide synthase (eNOS) pathway, thereby blunting nitric oxide (NO) production, while extracellular signal-regulated kinase (ERK1/2) activation and endothelin-1 (ET-1) release were increased by MGO in MAECs. Similar results were obtained in MAECs treated with SpBrBzGSHCp2. In MGO- and SpBrBzGSHCp2-exposed cells, inhibition of ERK1/2 decreased IRS1 phosphorylation on S616 and rescued insulin-dependent Akt activation and NO generation, indicating that MGO inhibition of the IRS1/Akt/eNOS pathway is mediated, at least in part, by ERK1/2. Chronic administration of MGO to C57BL/6 mice impaired whole-body insulin sensitivity and induced endothelial insulin resistance. CONCLUSIONS/INTERPRETATION: MGO impairs the action of insulin on the endothelium both in vitro and in vivo, at least in part through an ERK1/2-mediated mechanism. These findings may be instrumental in developing novel strategies for preserving endothelial function in diabetes.


Assuntos
Células Endoteliais/efeitos dos fármacos , Resistência à Insulina/fisiologia , Insulina/metabolismo , Aldeído Pirúvico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Células Endoteliais/metabolismo , Glutationa/análogos & derivados , Glutationa/farmacologia , Proteínas Substratos do Receptor de Insulina/metabolismo , Camundongos , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo
6.
J Cell Physiol ; 229(10): 1417-26, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24526410

RESUMO

Recent studies have indicated that endoplasmic reticulum stress, the unfolded protein response activation and altered GRP78 expression can play an important role in a variety of tumors development and progression. Very recently we reported for the first time that GRP78 is increased in endometrial tumors. However, whether GRP78 could play a role in the growth and/or invasiveness of endometrial cancer cells is still unknown. Here we report that the silencing of GRP78 expression affects both cell growth and invasiveness of Ishikawa and AN3CA cells, analyzed by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and transwell migration assay, respectively. At variance with Ishikawa cells, AN3CA cells showed, besides an endoplasmic reticulum, also a plasma membrane GRP78 localization, evidenced by both immunofluorescence and cell membrane biotinylation experiments. Intriguingly, flow cytometry experiments showed that the treatment with a specific antibody targeting GRP78 C-terminal domain caused apoptosis in AN3CA but not in Ishikawa cells. Induction of apoptosis in AN3CA cells was not mediated by the p53 pathway activation but was rather associated to reduced AKT phosphorylation. Interestingly, immunofluorescence analysis evidenced that endometrioid adenocarcinoma tissues displayed, similarly to AN3CA cells, also a GRP78 plasma membrane localization. These data suggest that GRP78 and its plasma membrane localization, might play a role in endometrial cancer development and progression and might constitute a novel target for the treatment of endometrial cancer.


Assuntos
Carcinoma Endometrioide/metabolismo , Movimento Celular , Proliferação de Células , Neoplasias do Endométrio/metabolismo , Proteínas de Choque Térmico/metabolismo , Apoptose , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/patologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Chaperona BiP do Retículo Endoplasmático , Feminino , Proteínas de Choque Térmico/genética , Humanos , Invasividade Neoplásica , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Fatores de Tempo , Transfecção , Proteína Supressora de Tumor p53/metabolismo
8.
J Appl Toxicol ; 33(6): 451-7, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22120598

RESUMO

Resin-based dental restorative materials release residual monomers that may affect the vitality of pulp cells. The purpose of this study was to evaluate the cytotoxic effect of two light-cured restorative materials with and without bis-GMA resin, respectively (Clearfil Majesty Posterior and Clearfil Majesty Flow) and a self-curing one (Clearfil DC Core Automix) when applied to the fibroblast cell line NIH-3T3. Samples of the materials were light-cured and placed directly in contact to cells for 24, 48, 72 and 96 h. Cytotoxicity was evaluated by measuring cell death by flow cytometry, cell proliferation by proliferation curves analysis and morphological changes by optical microscopy analysis. All the composite materials tested caused a decrease in cell proliferation, albeit at different degrees. However, only Clearfil DC Core Automix induced cell death, very likely by increasing apoptosis. Morphological alteration of treated cells was also evident, particularly in the Clearfil DC Core Automix-treated cells. The different cytotoxic effects of dental composites should be considered when selecting an appropriate resin-based dental restorative material for operative restorations.


Assuntos
Resinas Compostas/toxicidade , Resinas Sintéticas/toxicidade , Células 3T3 , Animais , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Corantes , Tratamento Dentário Restaurador sem Trauma , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Citometria de Fluxo , Cinética , Camundongos , Necrose/patologia , Propídio
9.
Cells ; 12(17)2023 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-37681914

RESUMO

The biguanide drug metformin is widely used in type 2 diabetes mellitus therapy, due to its ability to decrease serum glucose levels, mainly by reducing hepatic gluconeogenesis and glycogenolysis. A considerable number of studies have shown that metformin, besides its antidiabetic action, can improve other disease states, such as polycystic ovary disease, acute kidney injury, neurological disorders, cognitive impairment and renal damage. In addition, metformin is well known to suppress the growth and progression of different types of cancer cells both in vitro and in vivo. Accordingly, several epidemiological studies suggest that metformin is capable of lowering cancer risk and reducing the rate of cancer deaths among diabetic patients. The antitumoral effects of metformin have been proposed to be mainly mediated by the activation of the AMP-activated protein kinase (AMPK). However, a number of signaling pathways, both dependent and independent of AMPK activation, have been reported to be involved in metformin antitumoral action. Among these, the Wingless and Int signaling pathway have recently been included. Here, we will focus our attention on the main molecular mechanisms involved.


Assuntos
Diabetes Mellitus Tipo 2 , Metformina , Neoplasias , Feminino , Humanos , Metformina/farmacologia , Metformina/uso terapêutico , Via de Sinalização Wnt , Proteínas Quinases Ativadas por AMP , Neoplasias/tratamento farmacológico
10.
J Biol Chem ; 286(6): 4727-41, 2011 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-21115499

RESUMO

To find a common pathogenetic trait induced by polyQ-expanded proteins, we have used a conditional expression system in PC12 cells to tune the expression of these proteins and analyze the early and late consequences of their expression. We find that expression for 3 h of a polyQ-expanded protein stimulates cellular reactive oxygen species (ROS) levels and significantly reduces the mitochondrial electrochemical gradient. 24-36 h later, ROS induce DNA damage and activation of the checkpoint kinase, ATM. DNA damage signatures are reversible and persist as long as polyQ-expanded proteins are expressed. Transcription of neural and stress response genes is down-regulated in these cells. Selective inhibition of ATM or histone deacetylase rescues transcription and restores the expression of silenced genes. Eventually, after 1 week, the expression of polyQ-expanded protein also induces endoplasmic reticulum stress. As to the primary mechanism responsible for ROS generation, we find that polyQ-expanded proteins, including native Ataxin-2 and Huntingtin, are selectively sequestered in the lipid raft membrane compartment and interact with gp91, the membrane NADPH-oxidase subunit. Selective inhibition of NADPH oxidase or silencing of H-Ras signaling dissolves the aggregates and eliminates DNA damage. We suggest that targeting of the polyQ-expanded proteins to the lipid rafts activates the resident NADPH oxidase. This triggers a signal linking H-Ras, ROS, and ERK1/2 that maintains and propagates the ROS wave to the nucleus. This mechanism may represent the common pathogenetic signature of all polyQ-expanded proteins independently of the specific context or the function of the native wild type protein.


Assuntos
Dano ao DNA , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Ataxinas , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Histona Desacetilases , Humanos , Proteína Huntingtina , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Células PC12 , Peptídeos/genética , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ratos , Proteínas Supressoras de Tumor
11.
Gynecol Oncol ; 125(1): 220-5, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22146569

RESUMO

OBJECTIVES: Endometrial cancer is the most common malignancy of the female genital tract. However, in spite of a huge advance in our understanding of endometrial cancer biology, therapeutic modalities haven't significantly changed over the past 40 years. The activation of the Unfolded Protein Response (UPR) and GRP78 increase following Endoplasmic Reticulum (ER) stress have been recently identified as mechanisms favoring growth, invasion and resistance to therapy of different types of cancer. However, a possible role of ER stress and GRP78 in endometrial cancer has never been investigated. METHODS: Tissue specimens from normal and neoplastic endometrium were analyzed for the expression of the ER stress markers GRP78, ATF6 and CHOP by Real-Time RT-PCR. In addition, GRP78 protein expression and localization were evaluated by Western blot and immunohistochemistry, respectively. The effect of GRP78 knock down on cell growth of Ishikawa cells was analyzed by proliferation curve analysis. RESULTS: In this analysis, the expression levels of GRP78, ATF6 and CHOP mRNAs were significantly increased in specimens of endometrioid endometrial carcinomas. GRP78 and ATF6 protein expression levels were also increased in specimens of endometrial adenocarcinomas. GRP78 knock down caused a decrease of Ishikawa cells' growth. CONCLUSIONS: The increased expression of ER stress markers in endometrioid endometrial carcinomas suggests a role for ER stress, the UPR and, possibly, GRP78 in endometrial cancer. Whether these mechanisms have a substantial function in the pathogenesis of malignant transformation of human endometrium is still under investigation in our laboratory.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias do Endométrio/metabolismo , Estresse do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Fator de Transcrição CHOP/metabolismo , Western Blotting , Carcinoma Endometrioide/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Chaperona BiP do Retículo Endoplasmático , Feminino , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Resposta a Proteínas não Dobradas
12.
J Pharmacol Exp Ther ; 338(3): 997-1003, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21693630

RESUMO

Chronic exposure to polychlorinated biphenyls (PCBs), a class of ubiquitous environmental toxicants, causes neurocognitive anomalies. The transcription factor repressor element 1-silencing transcription factor (REST) plays a critical role in neuronal phenotype elaboration in both neural progenitor cells and non-neuronal cells. Here, we investigated the possible relationship between PCBs and REST in neuroblastoma SH-SY5Y cells. In these cells, chronic exposure to the PCB mixture Aroclor 1254 (A-1254; 5-30 µg/ml) caused dose-dependent cell death via the induction of calpain but not caspase-3. Intriguingly, this effect was prevented by the calpain inhibitor calpeptin. Furthermore, A-1254 enhanced REST mRNA and protein expression levels after both 24 and 48 h. REST down-regulation by small interfering RNA prevented A-1254-induced cell death. In addition, A-1254 enhanced the binding of REST to the synapsin 1 gene promoter, and synapsin 1 knockdown potentiated A-1254-induced cell death. A-1254 (10 µg/ml) also increased the expression of the two REST cofactors, the REST corepressor and the mammalian SIN3 homolog A transcription regulator. Moreover, the PCB mixture decreased acetylation of the histone proteins H3 and H4. It is noteworthy that the histone deacetylase inhibitor trichostatin A prevented such decreases and reduced the A-1254-induced neurotoxic effect. Collectively, these results suggest that A-1254 exerts its toxic effect via REST by down-regulating synapsin 1 and decreasing H3 and H4 acetylation.


Assuntos
Antitireóideos/toxicidade , Síndromes Neurotóxicas/patologia , Proteínas Repressoras/efeitos dos fármacos , Acetilação , Western Blotting , Calpaína/biossíntese , Caspase 3/biossíntese , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Imunoprecipitação da Cromatina , Histonas/metabolismo , Humanos , Imunoprecipitação , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , RNA/biossíntese , RNA/isolamento & purificação , RNA Antissenso/farmacologia , RNA Interferente Pequeno/farmacologia , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Complexo Correpressor Histona Desacetilase e Sin3 , Sinapsinas/metabolismo
13.
Cells ; 10(5)2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946426

RESUMO

Multiple lines of evidence suggest that metformin, an antidiabetic drug, exerts anti-tumorigenic effects in different types of cancer. Metformin has been reported to affect cancer cells' metabolism and proliferation mainly through the activation of AMP-activated protein kinase (AMPK). Here, we show that metformin inhibits, indeed, endometrial cancer cells' growth and induces apoptosis. More importantly, we report that metformin affects two important pro-survival pathways, such as the Unfolded Protein Response (UPR), following endoplasmic reticulum stress, and the WNT/ß-catenin pathway. GRP78, a key protein in the pro-survival arm of the UPR, was indeed downregulated, while GADD153/CHOP, a transcription factor that mediates the pro-apoptotic response of the UPR, was upregulated at both the mRNA and protein level. Furthermore, metformin dramatically inhibited ß-catenin mRNA and protein expression. This was paralleled by a reduction in ß-catenin transcriptional activity, since metformin inhibited the activity of a TCF/LEF-luciferase promoter. Intriguingly, compound C, a well-known inhibitor of AMPK, was unable to prevent all these effects, suggesting that metformin might inhibit endometrial cancer cells' growth and survival through the modulation of specific branches of the UPR and the inhibition of the Wnt/ß-catenin pathway in an AMPK-independent manner. Our findings may provide new insights on the mechanisms of action of metformin and refine the use of this drug in the treatment of endometrial cancer.


Assuntos
Carcinoma/metabolismo , Neoplasias do Endométrio/metabolismo , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Quinases Proteína-Quinases Ativadas por AMP , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Feminino , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas Quinases/metabolismo , Fator de Transcrição CHOP/metabolismo
14.
Front Endocrinol (Lausanne) ; 11: 588685, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33240221

RESUMO

The endoplasmic reticulum stress and the unfolded protein response are triggered following an imbalance between protein load and protein folding. Until recently, two possible outcomes of the unfolded protein response have been considered: life or death. We sought to substantiate a third alternative, dedifferentiation, mesenchymal shift, and activation of the antioxidant response by using typical endocrine cells, i.e. thyroid cells. The thyroid is a unique system both of endoplasmic reticulum stress (a single protein, thyroglobulin represents the majority of proteins synthesized in the endoplasmic reticulum by the thyrocyte) and of polarized epithelium (the single layer of thyrocytes delimiting the follicle). Following endoplasmic reticulum stress, in thyroid cells the folding of thyroglobulin was disrupted. The mRNAs of unfolded protein response were induced or spliced (X-box binding protein-1). Differentiation was inhibited: mRNA levels of thyroid specific genes, and of thyroid transcription factors were dramatically downregulated, at least in part, transcriptionally. The dedifferentiating response was accompanied by an upregulation of mRNAs of antioxidant genes. Moreover, cadherin-1, and the thyroid (and kidney)-specific cadherin-16 mRNAs were downregulated, vimentin, and SNAI1 mRNAs were upregulated. In addition, loss of cortical actin and stress fibers formation were observed. Together, these data indicate that ER stress in thyroid cells induces dedifferentiation, loss of epithelial organization, shift towards a mesenchymal phenotype, and activation of the antioxidant response, highlighting, at the same time, a new and wide strategy to achieve survival following ER stress, and, as a sort of the other side of the coin, a possible new molecular mechanism of decline/loss of function leading to a deficit of thyroid hormones formation.


Assuntos
Antioxidantes/metabolismo , Diferenciação Celular , Estresse do Retículo Endoplasmático , Mesoderma/citologia , Tireoglobulina/metabolismo , Células Epiteliais da Tireoide/citologia , Resposta a Proteínas não Dobradas , Animais , Células Cultivadas , Regulação da Expressão Gênica , Mesoderma/metabolismo , Ratos , Células Epiteliais da Tireoide/metabolismo
15.
Mol Cell Biol ; 25(22): 9793-805, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16260597

RESUMO

We present the first identification of transient folding intermediates of endogenous thyroglobulin (Tg; a large homodimeric secretory glycoprotein of thyrocytes), which include mixed disulfides with endogenous oxidoreductases servicing Tg folding needs. Formation of disulfide-linked Tg adducts with endoplasmic reticulum (ER) oxidoreductases begins cotranslationally. Inhibition of ER glucosidase activity blocked formation of a subgroup of Tg adducts containing ERp57 while causing increased Tg adduct formation with protein disulfide isomerase (PDI), delayed adduct resolution, perturbed oxidative folding of Tg monomers, impaired Tg dimerization, increased Tg association with BiP/GRP78 and GRP94, activation of the unfolded protein response, increased ER-associated degradation of a subpopulation of Tg, partial Tg escape from ER quality control with increased secretion of free monomers, and decreased overall Tg secretion. These data point towards mixed disulfides with the ERp57 oxidoreductase in conjunction with calreticulin/calnexin chaperones acting as normal early Tg folding intermediates that can be "substituted" by PDI adducts only at the expense of lower folding efficiency with resultant ER stress.


Assuntos
Dissulfetos , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/fisiologia , Isomerases de Dissulfetos de Proteínas/fisiologia , Tireoglobulina/química , Animais , Western Blotting , Calnexina/química , Calreticulina/química , Linhagem Celular , DNA Complementar/metabolismo , Dimerização , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Imunoprecipitação , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Dobramento de Proteína , Ratos , Fatores de Tempo , Transcrição Gênica
16.
J Endocrinol ; 190(3): 641-9, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17003265

RESUMO

In PC Cl3 cells, a continuous, fully differentiated rat thyroid cell line, P2Y(2) purinoceptor activation provoked a transient increase of [Ca(2+)](i), followed by a decreasing sustained phase. The alpha and beta1 protein kinase C (PKC) inhibitor Gö6976 decreased the rate of decrement to the basal [Ca(2+)](i) level and increased the peak of Ca(2+) entry of the P2Y(2)-provoked Ca(2+)transients. These effects of Gö 6976 were not caused by an increased permeability of the plasma membrane, since the Mn(2+) and Ba(2+) uptake were not changed by Gö 6976. Similarly, the Na(+)/Ca(2+) exchanger was not implicated, since the rate of decrement to the basal [Ca(2+)](i) level was equally decreased in physiological and Na(+)-free buffers, in the presence of Gö 6976. On the contrary, the activity of the sarcoplasmic-endoplasmic reticulum Ca(2+)ATPase (SERCA) 2b was profoundly affected by Gö 6976 since the drug was able to completely inhibit the stimulation of the SERCA 2b activity elicited by P2-purinergic agonists. Finally, the PKC activator phorbol myristate acetate had effects opposite to Gö 6976, in that it markedly increased the rate of decrement to the basal [Ca(2+)](i) level after P2Y(2) stimulation and also increased the activity of SERCA 2b. These results suggest that SERCA 2b plays a role in regulating the sustained phase of Ca(2+) transients caused by P2Y(2) stimulation.


Assuntos
Cálcio/metabolismo , Receptores Purinérgicos P2/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/enzimologia , Glândula Tireoide/enzimologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Bário/metabolismo , Transporte Biológico Ativo , Cálcio/análise , Carbazóis/farmacologia , Linhagem Celular , Ativação Enzimática , Indóis/farmacologia , Maleimidas/farmacologia , Manganês/metabolismo , Microscopia de Fluorescência , Proteína Quinase C/antagonistas & inibidores , Ratos , Receptores Purinérgicos P2Y2 , Acetato de Tetradecanoilforbol/farmacologia , Tapsigargina/análise , Uridina Trifosfato/metabolismo , Uridina Trifosfato/farmacologia
17.
Artigo em Inglês | MEDLINE | ID: mdl-26530458

RESUMO

Diabetes is a major risk factor for cardiovascular disease, and recent advances in research indicate that a detailed understanding of the pathophysiology of its effects is mandatory to reduce diabetes-related mortality and morbidity. Advanced Glycation End Products (AGEs) play a central role in the genesis and progression of complications of both type 1 and type 2 diabetes mellitus, and have been found to be important even in non-diabetic patients as a marker of cardiovascular disease. AGEs have a profound impact on patient's prognosis regardless of the glycemic control, and therefore pharmacologic approaches against AGEs accumulation have been proposed over the years to treat cardiovascular diseases, parallel to a more detailed understanding of AGEs pathophysiology. Compounds with anti-AGEs effects are currently under investigation in both pre-clinical and clinical scenarios, and many of the drugs previously used to treat specific diseases have been found to have AGE-inhibitory effects. Some products are still in "bench evaluation", whereas others have been already investigated in clinical trials with conflicting evidences. This review aims at summarizing the mechanisms of AGEs formation and accumulation, and the most relevant issues in pre-clinical and clinical experiences in anti-AGEs treatment in cardiovascular research.


Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Complicações do Diabetes/tratamento farmacológico , Produtos Finais de Glicação Avançada/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/fisiopatologia , Complicações do Diabetes/fisiopatologia , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 2/complicações , Progressão da Doença , Humanos , Prognóstico , Fatores de Risco
18.
Fertil Steril ; 103(6): 1579-86.e1, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25935494

RESUMO

OBJECTIVE: To investigate immunologic parameters and endoplasmic reticulum (ER) stress associated with unexplained infertility. DESIGN: Case-control study. SETTING: Academic center. PATIENT(S): Women with no fertility problems (FS) (n = 13), women with recurrent miscarriage (RM) (n = 15) and women with repeated in vitro fertilization failure (RIF) (n = 15). INTERVENTION(S): Endometrial biopsy and collection of peripheral blood during the midsecretory phase of menstrual cycle. MAIN OUTCOME MEASURE(S): Leptin, resistin, soluble tumor necrosis factor receptor (sTNF-R), myeloperoxidase (MPO), soluble intercellular adhesion molecule 1 (sICAM-1), and interleukin 22 (IL-22) concentration in peripheral blood, endometrial CD3(+), CD4(+), CD5(+), CD8(+), and FoxP3(+) T lymphocytes, and endometrial expression of HSPA5, a specific marker of ER stress. RESULT(S): We found an increase of proinflammatory molecules such as resistin, leptin, and IL-22 in both RM and RIF patients; sTNF-R and MPO only in RIF patients when compared with the FS women. We also found in endometria of infertile women a statistically significant increase of CD3(+), CD4(+), CD8(+) in both RM and RIF patients and CD5(+) in RM patients when compared with FS women. This was paralleled by a statistically significant reduction of infiltrating FoxP3(+) regulatory T cells. Finally, endometrial HSPA5 expression levels were statistically significantly up-regulated in both RM and RIF patients. CONCLUSION(S): Women with RM and RIF showed an increase of circulating proinflammatory cytokines, altered endometrial T lymphocytes subsets, and signs of endometrial ER stress.


Assuntos
Implantação do Embrião/imunologia , Retículo Endoplasmático/imunologia , Infertilidade Feminina/imunologia , Inflamação/imunologia , Estresse Oxidativo/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Adulto , Estudos de Casos e Controles , Retículo Endoplasmático/patologia , Chaperona BiP do Retículo Endoplasmático , Feminino , Humanos , Infertilidade Feminina/patologia , Inflamação/patologia , Ciclo Menstrual/imunologia , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/imunologia , Adulto Jovem
19.
Endocrinology ; 143(6): 2169-77, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12021180

RESUMO

Perturbing the endoplasmic reticulum homeostasis of thyroid cell lines with thapsigargin, a specific inhibitor of the sarcoendoplasmic reticulum Ca(2+) adenosine triphosphatases, and tunicamycin, an inhibitor of the N-linked glycosylation, blocked Tg in the endoplasmic reticulum. This event was signaled outside the endoplasmic reticulum and resulted in activation of the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase and nuclear factor-kappa B (NF-kappa B) stress response pathways. Activation of the JNK/stress-activated protein kinase signaling pathway was assessed by measuring the amount of phospho-JNK and the activity of JNK by kinase assays. Activation of the NF-kappa B signaling pathway was assessed by measuring the level of inhibitory subunit I kappa B alpha, DNA binding, and transcriptional activity of NF-kappa B. Cycloheximide treatment, at a dose able to profoundly inhibit protein synthesis in FRTL-5 cells, obliterated the decrease in the level of the inhibitory subunit I kappa B alpha produced by thapsigargin and tunicamycin. Therefore, protein synthesis was required to generate a signal from stressed endoplasmic reticulum. This substantiates the hypothesis that endoplasmic reticulum retention of newly synthesized Tg and other cargo (secretory and membrane) proteins functions upstream of signal activation. Dominant negative TNF receptor-associated factor 2 (TRAF2) inhibited activation of NF-kappa B, which was also inhibited in embryonic fibroblasts derived from TRAF2(-/-) mice, respect to their normal counterpart. These data extend the recent demonstration that TRAF2 mediated JNK activation in response to endoplasmic reticulum stress and strongly strengthened the idea that endogenous stress signals initiated in the endoplasmic reticulum proceed by a pathway similar to that initiated by plasma membrane receptors in response to extracellular signals.


Assuntos
Retículo Endoplasmático/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno , NF-kappa B/metabolismo , Proteínas/fisiologia , Tireoglobulina/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Cicloeximida/farmacologia , Eletroforese , Retículo Endoplasmático/efeitos dos fármacos , Fibroblastos/metabolismo , Genes Reporter/genética , MAP Quinase Quinase 4 , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Testes de Precipitina , Inibidores da Síntese de Proteínas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Estresse Mecânico , Fator 2 Associado a Receptor de TNF , Tapsigargina/farmacologia , Tunicamicina/farmacologia
20.
Mol Cell Endocrinol ; 208(1-2): 51-9, 2003 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-14580721

RESUMO

The rat hepatic lectin (RHL)-1 is the major component of the rat liver asialoglycoprotein receptor (ASGPr), a membrane receptor highly expressed on the basolateral side of hepatocytes, which mediates endocytosis of serum desialated glycoproteins. We have recently shown that RHL-1 is expressed in rat thyroid tissue and thyroid differentiated cell lines. Both in vitro and in vivo assays show that thyrotropin up-regulates thyroid RHL-1 expression, while neoplastic transformation of thyroid cells exerts a down-regulation of receptor expression. Moreover, RHL-1 expressed on the surface of differentiated thyroid cells is able to bind thyroglobulin (Tg), the macromolecular site of synthesis and storage of thyroid hormones. In the present work, we demonstrate, by immunohistochemistry analysis, that RHL-1 is localized on the apical surface of thyrocytes, at a variance with its basolateral localization on hepatocytes. Moreover, albeit its expression in thyroid is less abundant than in liver, the receptor is able to bind asialorosomucoid (ASOR), the best-known ligand of hepatic ASGPr, and to mediate endocytosis of a significative amount of Tg on the surface of differentiated PC Cl3 thyroid cells. Taken together, the data suggest that RHL-1, even if expressed in thyroid at lower levels than in liver, could serve as a receptor for endocytosis of colloidal Tg and, likely, for its delivery to lysosomes.


Assuntos
Receptor de Asialoglicoproteína/análise , Receptor de Asialoglicoproteína/fisiologia , Endocitose , Tireoglobulina/metabolismo , Glândula Tireoide/química , Glândula Tireoide/metabolismo , Animais , Receptor de Asialoglicoproteína/metabolismo , Western Blotting , Linhagem Celular , Imuno-Histoquímica , Fígado/metabolismo , Subunidades Proteicas/análise , Subunidades Proteicas/fisiologia , Ratos , Glândula Tireoide/citologia , Regulação para Cima
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