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1.
Theranostics ; 11(16): 8112-8128, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335983

RESUMO

The coiled-coil domain containing protein members have been well documented for their roles in many diseases including cancers. However, the function of the coiled-coil domain containing 65 (CCDC65) remains unknown in tumorigenesis including gastric cancer. Methods: CCDC65 expression and its correlation with clinical features and prognosis of gastric cancer were analyzed in tissue. The biological role and molecular basis of CCDC65 were performed via in vitro and in vivo assays and a various of experimental methods including co-immunoprecipitation (Co-IP), GST-pull down and ubiquitination analysis et al. Finally, whether metformin affects the pathogenesis of gastric cancer by regulating CCDC65 and its-mediated signaling was investigated. Results: Here, we found that downregulated CCDC65 level was showed as an unfavourable factor in gastric cancer patients. Subsequently, CCDC65 or its domain (a.a. 130-484) was identified as a significant suppressor in GC growth and metastasis in vitro and in vivo. Molecular basis showed that CCDC65 bound to ENO1, an oncogenic factor has been widely reported to promote the tumor pathogenesis, by its domain (a.a. 130-484) and further promoted ubiquitylation and degradation of ENO1 by recruiting E3 ubiquitin ligase FBXW7. The downregulated ENO1 decreased the binding with AKT1 and further inactivated AKT1, which led to the loss of cell proliferation and EMT signal. Finally, we observed that metformin, a new anti-cancer drug, can significantly induce CCDC65 to suppress ENO1-AKT1 complex-mediated cell proliferation and EMT signals and finally suppresses the malignant phenotypes of gastric cancer cells. Conclusion: These results firstly highlight a critical role of CCDC65 in suppressing ENO1-AKT1 pathway to reduce the progression of gastric cancer and reveals a new molecular mechanism for metformin in suppressing gastric cancer. Our present study provides a new insight into the mechanism and therapy for gastric cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glicoproteínas/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Gástricas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , China , Feminino , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor/fisiologia , Glicoproteínas/genética , Humanos , Masculino , Metformina/metabolismo , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oncogenes , Prognóstico , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
2.
Biomolecules ; 11(6)2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208360

RESUMO

Metformin is the first-line pharmacotherapy for type 2 diabetes mellitus (T2D). Metformin exerts its glucose-lowering effect primarily through decreasing hepatic glucose production (HGP). However, the precise molecular mechanisms of metformin remain unclear due to supra-pharmacological concentration of metformin used in the study. Here, we investigated the role of Foxo1 in metformin action in control of glucose homeostasis and its mechanism via the transcription factor Foxo1 in mice, as well as the clinical relevance with co-treatment of aspirin. We showed that metformin inhibits HGP and blood glucose in a Foxo1-dependent manner. Furthermore, we identified that metformin suppresses glucagon-induced HGP through inhibiting the PKA→Foxo1 signaling pathway. In both cells and mice, Foxo1-S273D or A mutation abolished the suppressive effect of metformin on glucagon or fasting-induced HGP. We further showed that metformin attenuates PKA activity, decreases Foxo1-S273 phosphorylation, and improves glucose homeostasis in diet-induced obese mice. We also provided evidence that salicylate suppresses HGP and blood glucose through the PKA→Foxo1 signaling pathway, but it has no further additive improvement with metformin in control of glucose homeostasis. Our study demonstrates that metformin inhibits HGP through PKA-regulated transcription factor Foxo1 and its S273 phosphorylation.


Assuntos
Proteína Forkhead Box O1/metabolismo , Glucose/metabolismo , Metformina/farmacologia , Animais , Aspirina/metabolismo , Aspirina/farmacologia , Glicemia/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Proteína Forkhead Box O1/farmacologia , Gluconeogênese/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Resistência à Insulina/fisiologia , Fígado/metabolismo , Masculino , Metformina/metabolismo , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Fosforilação , Transdução de Sinais , Estreptozocina/farmacologia
3.
FASEB J ; 35(7): e21645, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34105824

RESUMO

Peripheral arterial disease (PAD) is one of the major complications of diabetes due to an impairment in angiogenesis. Since there is currently no drug with satisfactory efficacy to enhance blood vessel formation, discovering therapies to improve angiogenesis is critical. An imidazolinone metabolite of the metformin-methylglyoxal scavenging reaction, (E)-1,1-dimethyl-2-(5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl) guanidine (IMZ), was recently characterized and identified in the urine of type-2 diabetic patients. Here, we report the pro-angiogenesis effect of IMZ (increased aortic sprouting, cell migration, network formation, and upregulated multiple pro-angiogenic factors) in human umbilical vein endothelial cells. Using genetic and pharmacological approaches, we showed that IMZ augmented angiogenesis by activating the endothelial nitric oxide synthase (eNOS)/hypoxia-inducible factor-1 alpha (HIF-1α) pathway. Furthermore, IMZ significantly promoted capillary density in the in vivo Matrigel plug angiogenesis model. Finally, the role of IMZ in post-ischemic angiogenesis was examined in a chronic hyperglycemia mouse model subjected to hind limb ischemia. We observed improved blood perfusion, increased capillary density, and reduced tissue necrosis in mice receiving IMZ compared to control mice. Our data demonstrate the pro-angiogenic effects of IMZ, its underlying mechanism, and provides a structural basis for the development of potential pro-angiogenic agents for the treatment of PAD.


Assuntos
Membro Posterior/fisiopatologia , Hiperglicemia/complicações , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/complicações , Metformina/metabolismo , Neovascularização Patológica/patologia , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Hipoglicemiantes/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imidazolinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Aldeído Pirúvico/metabolismo
4.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071182

RESUMO

Metformin is the first-line treatment for many people with type 2 diabetes mellitus (T2DM) and gestational diabetes mellitus (GDM) to maintain glycaemic control. Recent evidence suggests metformin can cross the placenta during pregnancy, thereby exposing the fetus to high concentrations of metformin and potentially restricting placental and fetal growth. Offspring exposed to metformin during gestation are at increased risk of being born small for gestational age (SGA) and show signs of 'catch up' growth and obesity during childhood which increases their risk of future cardiometabolic diseases. The mechanisms by which metformin impacts on the fetal growth and long-term health of the offspring remain to be established. Metformin is associated with maternal vitamin B12 deficiency and antifolate like activity. Vitamin B12 and folate balance is vital for one carbon metabolism, which is essential for DNA methylation and purine/pyrimidine synthesis of nucleic acids. Folate:vitamin B12 imbalance induced by metformin may lead to genomic instability and aberrant gene expression, thus promoting fetal programming. Mitochondrial aerobic respiration may also be affected, thereby inhibiting placental and fetal growth, and suppressing mammalian target of rapamycin (mTOR) activity for cellular nutrient transport. Vitamin supplementation, before or during metformin treatment in pregnancy, could be a promising strategy to improve maternal vitamin B12 and folate levels and reduce the incidence of SGA births and childhood obesity. Heterogeneous diagnostic and screening criteria for GDM and the transient nature of nutrient biomarkers have led to inconsistencies in clinical study designs to investigate the effects of metformin on folate:vitamin B12 balance and child development. As rates of diabetes in pregnancy continue to escalate, more women are likely to be prescribed metformin; thus, it is of paramount importance to improve our understanding of metformin's transgenerational effects to develop prophylactic strategies for the prevention of adverse fetal outcomes.


Assuntos
Diabetes Gestacional/metabolismo , Desenvolvimento Fetal/efeitos dos fármacos , Ácido Fólico/metabolismo , Metformina/metabolismo , Gravidez em Diabéticas/metabolismo , Vitamina B 12/metabolismo , Carbono/metabolismo , Diabetes Mellitus Tipo 2 , Interações Medicamentosas , Feminino , Feto , Ácido Fólico/farmacologia , Humanos , Metformina/farmacologia , Obesidade/metabolismo , Placenta/metabolismo , Gravidez , Complicações na Gravidez , Gravidez em Diabéticas/induzido quimicamente , Gravidez em Diabéticas/tratamento farmacológico , Vitamina B 12/farmacologia
5.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069559

RESUMO

Metformin is a drug in the family of biguanide compounds that is widely used in the treatment of type 2 diabetes (T2D). Interestingly, the therapeutic potential of metformin expands its prescribed use as an anti-diabetic drug. In this sense, it has been described that metformin administration has beneficial effects on different neurological conditions. In this work, we review the beneficial effects of this drug as a neuroprotective agent in different neurological diseases, with a special focus on epileptic disorders and Lafora disease, a particular type of progressive myoclonus epilepsy. In addition, we review the different proposed mechanisms of action of metformin to understand its function at the neurological level.


Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Metformina/uso terapêutico , Animais , Sistema Nervoso Central/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Modelos Animais de Doenças , Epilepsia/tratamento farmacológico , Humanos , Hipoglicemiantes/farmacologia , Doença de Lafora/tratamento farmacológico , Metformina/metabolismo , Metformina/farmacologia , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia
6.
Exp Cell Res ; 404(2): 112659, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34022204

RESUMO

Endometriosis is a benign gynecological disease that is manifested by the presence and growth of endometrial cells and glands outside the uterine. Active angiogenesis, migration, and invasion of endometrial tissue outside the uterine are critical for the development of endometriosis and lead to the survival and growth of endometriotic lesions. Metformin, as an anti-diabetic agent, represents anti-angiogenic property. Here, we performed a study using human normal endometrial stromal cells (N-ESCs) from healthy endometrial tissue and human eutopic endometrial stromal cells (EU-ESCs) and ectopic endometrial stromal cells (ECT-ESCs) from endometriosis patients. ESCs were cultured and treated with different concentrations of Metformin (0-20 mmol/l) for 72 h to evaluate Metformin effect on cell viability, proliferation, migration was measured by methyl thiazolyl tetrazolium (MTT) assay and scratch test respectively as well as expression of angiogenesis and migration markers. The Metformin reduced cell migration, and proliferation of endometriotic stromal cells in a time and concentration dependently manner. Furthermore, Metformin attenuated the expression of angiogenic and inflammatory genes in human endometriotic stromal cells. The direct anti-proliferative effect on ECT-ESCs combined with the effects of Metformin on inflammatory and angiogenesis-related genes expression supports its therapeutic potential for endometriosis. Metformin could be used as an effective adjuvant in endometriosis treatment.


Assuntos
Endométrio/efeitos dos fármacos , Metformina/farmacologia , Neovascularização Patológica/metabolismo , Células Estromais/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Endometriose/tratamento farmacológico , Endometriose/genética , Endométrio/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Metformina/metabolismo , Células Estromais/efeitos dos fármacos
7.
Appl Environ Microbiol ; 87(11)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33741630

RESUMO

The widely prescribed pharmaceutical metformin and its main metabolite, guanylurea, are currently two of the most common contaminants in surface and wastewater. Guanylurea often accumulates and is poorly, if at all, biodegraded in wastewater treatment plants. This study describes Pseudomonas mendocina strain GU, isolated from a municipal wastewater treatment plant, using guanylurea as its sole nitrogen source. The genome was sequenced with 36-fold coverage and mined to identify guanylurea degradation genes. The gene encoding the enzyme initiating guanylurea metabolism was expressed, and the enzyme was purified and characterized. Guanylurea hydrolase, a newly described enzyme, was shown to transform guanylurea to one equivalent (each) of ammonia and guanidine. Guanidine also supports growth as a sole nitrogen source. Cell yields from growth on limiting concentrations of guanylurea revealed that metabolism releases all four nitrogen atoms. Genes encoding complete metabolic transformation were identified bioinformatically, defining the pathway as follows: guanylurea to guanidine to carboxyguanidine to allophanate to ammonia and carbon dioxide. The first enzyme, guanylurea hydrolase, is a member of the isochorismatase-like hydrolase protein family, which includes biuret hydrolase and triuret hydrolase. Although homologs, the three enzymes show distinct substrate specificities. Pairwise sequence comparisons and the use of sequence similarity networks allowed fine structure discrimination between the three homologous enzymes and provided insights into the evolutionary origins of guanylurea hydrolase.IMPORTANCE Metformin is a pharmaceutical most prescribed for type 2 diabetes and is now being examined for potential benefits to COVID-19 patients. People taking the drug pass it largely unchanged, and it subsequently enters wastewater treatment plants. Metformin has been known to be metabolized to guanylurea. The levels of guanylurea often exceed that of metformin, leading to the former being considered a "dead-end" metabolite. Metformin and guanylurea are water pollutants of emerging concern, as they persist to reach nontarget aquatic life and humans, the latter if it remains in treated water. The present study has identified a Pseudomonas mendocina strain that completely degrades guanylurea. The genome was sequenced, and the genes involved in guanylurea metabolism were identified in three widely separated genomic regions. This knowledge advances the idea that guanylurea is not a dead-end product and will allow for bioinformatic identification of the relevant genes in wastewater treatment plant microbiomes and other environments subjected to metagenomic sequencing.


Assuntos
Proteínas de Bactérias/metabolismo , Guanidina/análogos & derivados , Hidrolases/metabolismo , Redes e Vias Metabólicas , Metformina/metabolismo , Ureia/análogos & derivados , Poluentes Químicos da Água/metabolismo , Amônia/metabolismo , Proteínas de Bactérias/genética , Biodegradação Ambiental , Biomineralização , Genoma Bacteriano/genética , Guanidina/metabolismo , Hidrolases/genética , Família Multigênica , Pseudomonas mendocina/genética , Pseudomonas mendocina/isolamento & purificação , Pseudomonas mendocina/metabolismo , Especificidade por Substrato , Ureia/metabolismo , Águas Residuárias/microbiologia
8.
Angew Chem Int Ed Engl ; 60(24): 13405-13413, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33755286

RESUMO

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by an aberrant metabolic phenotype with high metastatic capacity, resulting in poor patient prognoses and low survival rates. We designed a series of novel AuIII cyclometalated prodrugs of energy-disrupting Type II antidiabetic drugs namely, metformin and phenformin. Prodrug activation and release of the metformin ligand was achieved by tuning the cyclometalated AuIII fragment. The lead complex 3met was 6000-fold more cytotoxic compared to uncoordinated metformin and significantly reduced tumor burden in mice with aggressive breast cancers with lymphocytic infiltration into tumor tissues. These effects was ascribed to 3met interfering with energy production in TNBCs and inhibiting associated pro-survival responses to induce deadly metabolic catastrophe.


Assuntos
Antineoplásicos/metabolismo , Metformina/metabolismo , Pró-Fármacos/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Complexos de Coordenação/química , Avaliação Pré-Clínica de Medicamentos , Metabolismo Energético/efeitos dos fármacos , Ouro/química , Humanos , Metformina/química , Camundongos , Conformação Molecular , Fenformin/química , Fenformin/metabolismo , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Transplante Heterólogo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
9.
Biochem Pharmacol ; 189: 114453, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33545119

RESUMO

A novel PMet-P(cdmPEG2K) polymeric micellar carrier was developed for tumor-targeted co-delivery of DOX and nucleic acids (NA), based on polymetformin and a structure designed to lose the PEG shell in response to the acidic extracellular tumor environment. NA/DOX co-loaded micelleplexes exhibited enhanced inhibition of cell proliferation compared to DOX-loaded micelles, and displayed a higher level of cytotoxicity at an acidic pH (6.8) which mimicks the tumor microenvironment. The PMet-P(cdmPEG2K) micelles achieved significantly improved transfection with either a reporter plasmid or Cy3-siRNA, and enhanced DOX intracellular uptake in 4T1.2 cells at pH 6.8. Importantly, PMet-P(cdmPEG2K) micelles showed excellent pEGFP (EGFP expression plasmid) transfection in an aggressive murine breast cancer (4T1.2) model. By using a plasmid encoding IL-12 (pIL-12), we investigated the combined effect of chemotherapy and gene therapy. PMet-P(cdmPEG2K) micelles co-loaded with DOX and pIL-12 were more effective at inhibiting tumor growth compared to micelles loaded with DOX or pIL-12 alone. In addition, this micellar system was effective in co-delivery of siRNA and DOX into tumor cells. Our results suggest that PMet-P(cdmPEG2K) has the potential for chemo and nucleic acid combined cancer therapy.


Assuntos
Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Hipoglicemiantes/administração & dosagem , Metformina/administração & dosagem , Micelas , Ácidos Nucleicos/administração & dosagem , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Doxorrubicina/química , Doxorrubicina/metabolismo , Feminino , Concentração de Íons de Hidrogênio , Hipoglicemiantes/química , Hipoglicemiantes/metabolismo , Metformina/química , Metformina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo
10.
Biochim Biophys Acta Gen Subj ; 1865(3): 129841, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33412224

RESUMO

BACKGROUND: Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer with limited treatment modalities. It is associated with high propensity of cancer recurrence. METHODS: UV Spectroscopy, FTIR, DLS, Zeta potential, TEM and SEM were employed to characterize nanoparticles. MTT assay, Wound healing assay, SEM, Immunocytochemistry analysis, Western blot, RT-PCR, mammosphere formation assay were employed to study apoptosis, cell migration and stemness. Tumor regression was studied in chick embryo xenograft and BALB/c mice model. RESULTS: Hylaluronic acid engrafted metformin loaded graphene oxide (HA-GO-Met) nanoparticles exhibited an anti-cancer efficacy at much lower dosage as compared to metformin alone. HA-GO-Met nanoparticles induced apoptosis and inhibited cell migration of TNBC cells by targeting miR-10b/PTEN axis via NFkB-p65. Upregulation of PTEN affected pAKT(473) expression that induced apoptosis. Cell migration was inhibited by reduction of pFAK/integrinß1 expressions. Treatment inhibited epithelial mesenchymal transition (EMT) and reduced stemness as evident from the increase in E-cadherin expression, inhibition of mammosphere formation and low expression levels of stemness markers including nanog, oct4 and sox2 as compared to control. Moreover, tumor regression was studied in chick embryo xenograft and BALB/c mice model. HA-GO-Met nanoparticle treatment reduced tumor load and nullified toxicity in peripheral organs imparted by tumor. CONCLUSIONS: HA-GO-Met nanoparticles exhibited an enormous anti-cancer efficacy in TNBC in vitro and in vivo. GENERAL SIGNIFICANCE: HA-GO-Met nanoparticles induced apoptosis and attenuated cell migration in TNBC. It nullified overall toxicity imparted by tumor load. It inhibited EMT and reduced stemness and thereby addressed the issue of cancer recurrence.


Assuntos
Antineoplásicos/farmacologia , Grafite/química , Receptores de Hialuronatos/genética , Ácido Hialurônico/química , Metformina/farmacologia , Nanopartículas/química , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/metabolismo , Membrana Corioalantoide/patologia , Portadores de Fármacos , Feminino , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores de Hialuronatos/metabolismo , Metformina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , MicroRNAs/metabolismo , Terapia de Alvo Molecular , Nanopartículas/administração & dosagem , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Biochemistry ; 60(1): 6-18, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33356167

RESUMO

Rhodopsin is the light receptor required for the function and health of photoreceptor cells. Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinal degeneration. Bovine rhodopsin is often used as a model to understand the effect of pathogenic mutations in rhodopsin due to the abundance of structural information on the bovine form of the receptor. It is unclear whether or not the bovine rhodopsin template is adequate in predicting the effect of these mutations occurring in human retinal disease or in predicting the efficacy of therapeutic strategies. To better understand the extent to which bovine rhodopsin can serve as a model, human and bovine P23H rhodopsin mutants expressed heterologously in cells were examined. The aggregation properties and cellular localization of the mutant receptors were determined by Förster resonance energy transfer and confocal microscopy. The potential therapeutic effects of the pharmacological compounds 9-cis retinal and metformin were also examined. Human and bovine P23H rhodopsin mutants exhibited different aggregation properties and responses to the pharmacological compounds tested. These observations would lead to different predictions on the severity of the phenotype and divergent predictions on the benefit of the therapeutic compounds tested. The bovine rhodopsin template does not appear to adequately model the effects of the P23H mutation in the human form of the receptor.


Assuntos
Diterpenos/metabolismo , Metformina/metabolismo , Proteínas Mutantes/química , Mutação , Agregados Proteicos , Retinaldeído/metabolismo , Rodopsina/química , Animais , Bovinos , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Rodopsina/genética , Rodopsina/metabolismo
12.
ACS Appl Mater Interfaces ; 12(50): 55723-55736, 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33274915

RESUMO

The myeloid-derived suppressor cell (MDSC)-mediated immunosuppressive tumor microenvironment (TME), where tumor hypoxia counts for much, has greatly compromised the outcome of cancer immunotherapy. Here, we demonstrated a strategy for selectively clearing intratumoral MDSCs. Specifically, 2-[2-[2-chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cyclohexen-1-yl]ethenyl]-3,3-dimethyl-1-propylindolium iodide (IR-780) and metformin (Met) were coloaded into mesoporous silica nanoparticles (MSNs) with CeO2 as the gatekeepers. Controlled release of cargos was achieved upon etching CeO2 with endogenous H2O2. Apart from the drug release, oxygen (O2) was also generated in this process. Importantly, the engagement of Met significantly inhibited mitochondrial respiration, thus working like an O2 economizer. Consequently, the populations and functions of tumor-infiltrated MDSCs were both dramatically reduced through selective alleviation of hypoxia at tumor sites, thus contributing to boosted immune responses. Additionally, the accumulated O2 enhanced IR780-mediated photodynamic therapy, which synergistically strengthened the antitumor efficacy of the platform. To the best of our knowledge, this is the first time to employ an O2-generated and -economized nanoplatform for selectively anergizing MDSC-mediated immunosuppression. We expect that this strategy will shed new light on the clinical cancer immunotherapy treatment.


Assuntos
Antineoplásicos/química , Cério/química , Indóis/química , Metformina/química , Nanoestruturas/química , Oxigênio/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Peróxido de Hidrogênio/química , Indóis/metabolismo , Indóis/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Metformina/metabolismo , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Células Supressoras Mieloides/citologia , Células Supressoras Mieloides/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Dióxido de Silício/química , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/metabolismo , Microambiente Tumoral
13.
Mol Med Rep ; 22(6): 5262-5270, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33174032

RESUMO

Tissue damage in diabetes is at least partly due to elevated reactive oxygen species production by the mitochondrial respiratory chain during hyperglycemia. Sustained hyperglycemia results in mitochondrial dysfunction and the abnormal expression of mitochondrial genes, such as NADH: Ubiquinone oxidoreductase subunit A13 (NDUFA13). Metformin, an AMP­activated protein kinase (AMPK) activator, protects cardiomyocytes from oxidative stress by improving mitochondrial function; however, the exact underlying mechanisms are not completely understood. The aim of the present study was to investigated the molecular changes and related regulatory mechanisms in the response of H9C2 cardiomyocytes to metformin under high glucose conditions. H9C2 cells were subjected to CCK­8 assay to assess cell viability. Reactive oxygen species generation was measured with DCFH­DA assay. Western blotting was used to analyze the expression levels of NDUFA13, AMPK, p­AMPK and GAPDH. Reverse transcription­quantitative PCR was used to evaluate the expression levels of mitochondrial genes and transcription factors. It was observed that metformin protected H9C2 cardiomyocytes by suppressing high glucose (HG)­induced elevated oxidative stress. In addition, metformin stimulated mitochondrial biogenesis, as indicated by increased expression levels of mitochondrial genes (NDUFA1, NDUFA2, NDUFA13 and manganese superoxide dismutase) and mitochondrial biogenesis­related transcription factors [peroxisome proliferator­activated receptor­gamma coactivator­1α, nuclear respiratory factor (NRF)­1, and NRF­2] in the metformin + HG group compared with the HG group. Moreover, metformin promoted mitochondrial NDUFA13 protein expression via the AMPK signaling pathway, which was abolished by pretreatment with the AMPK inhibitor, Compound C. The results suggested that metformin protected cardiomyocytes against HG­induced oxidative stress via a mechanism involving AMPK, NDUFA13 and mitochondrial biogenesis.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Metformina/farmacologia , Chaperonas Moleculares/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , China , Complexo I de Transporte de Elétrons/efeitos dos fármacos , Glucose/metabolismo , Hiperglicemia/metabolismo , Metformina/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Biogênese de Organelas , Estresse Oxidativo/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteínas Serina-Treonina Quinases , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Fatores de Transcrição/genética
14.
Proc Natl Acad Sci U S A ; 117(42): 26438-26447, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33024014

RESUMO

Metabolic dysfunction occurs in many age-related neurodegenerative diseases, yet its role in disease etiology remains poorly understood. We recently discovered a potential causal link between the branched-chain amino acid transferase BCAT-1 and the neurodegenerative movement disorder Parkinson's disease (PD). RNAi-mediated knockdown of Caenorhabditis elegans bcat-1 is known to recapitulate PD-like features, including progressive motor deficits and neurodegeneration with age, yet the underlying mechanisms have remained unknown. Using transcriptomic, metabolomic, and imaging approaches, we show here that bcat-1 knockdown increases mitochondrial respiration and induces oxidative damage in neurons through mammalian target of rapamycin-independent mechanisms. Increased mitochondrial respiration, or "mitochondrial hyperactivity," is required for bcat-1(RNAi) neurotoxicity. Moreover, we show that post-disease-onset administration of the type 2 diabetes medication metformin reduces mitochondrial respiration to control levels and significantly improves both motor function and neuronal viability. Taken together, our findings suggest that mitochondrial hyperactivity may be an early event in the pathogenesis of PD, and that strategies aimed at reducing mitochondrial respiration may constitute a surprising new avenue for PD treatment.


Assuntos
Metformina/farmacologia , Mitocôndrias/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Aminoácidos de Cadeia Ramificada/metabolismo , Aminoácidos de Cadeia Ramificada/fisiologia , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Metformina/metabolismo , Neurônios/efeitos dos fármacos , Doença de Parkinson/metabolismo , Fenótipo
16.
Life Sci ; 256: 117923, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32522567

RESUMO

AIMS: Liver kinase B1 (LKB1) deficiency is associated with reduced expression of programmed death ligand 1 (PD-L1) and inferior clinical outcomes of PD-1/PD-L1 blockade in non-small cell lung cancer (NSCLC). This study aimed to investigate the mechanism by which LKB1 regulates PD-L1 expression and its role in programmed death 1 (PD-1) blockade therapy in NSCLC. MAIN METHODS: The impact of LKB1 on PD-L1 was assessed by western blot, qRT-PCR and immunohistochemistry in NSCLC. Activators/inhibitors of AMPK and NRF2 were applied to explore the mechanisms underlying the regulation of PD-L1 by LKB1. Efficiency of combined application of metformin and PD-1 blockade was evaluated in immunocompetent C57BL/6 mice. KEY FINDINGS: A remarkable positive correlation between LKB1 and PD-L1 expression was demonstrated in NSCLC tissues. Knockdown of LKB1 decreased PD-L1 in TC-1 cells, whereas overexpression of LKB1 increased PD-L1 in A549 cells. We further characterized that AMPK mediated the upregulation of PD-L1 by LKB1. Inhibition of AMPK or NRF2 markedly reduced PD-L1 in LKB1-intact NSCLC cells. In contrast, activation of AMPK or NRF2 reversed PD-L1 expression in LKB1-deficient NSCLC cells. Combined administration of metformin and anti-PD-1 antibody efficiently inhibited the growth of LKB1-intact tumors, whereas no obvious suppression was observed in LKB1-deficient tumors. SIGNIFICANCE: These findings demonstrated that LKB1 upregulates PD-L1 expression in NSCLC by activating the AMPK and KEAP1/NRF2 signaling. Activation of LKB1-AMPK with metformin improves the therapeutic effect of PD-1 blockade in NSCLC with wild-type LKB1.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Metformina/farmacologia , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células A549 , Animais , Anticorpos Monoclonais Humanizados/metabolismo , Antineoplásicos/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Metformina/metabolismo , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transdução de Sinais , Ativação Transcricional , Regulação para Cima
17.
Anal Bioanal Chem ; 412(20): 4985-4996, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32572543

RESUMO

A method with capillary electrophoresis coupled to mass spectrometry was optimized to determine the uptake of metformin and its metabolite guanylurea by zebrafish (Danio rerio) embryos and brown trout (Salmo trutta f. fario) exposed under laboratory conditions. Metformin was extracted from fish tissues by sonication in methanol, resulting in an absolute recovery of almost 90%. For the extraction of guanylurea from brown trout, solid-phase extraction was implemented with a recovery of 84%. The use of a mixture of methanol and glacial acetic acid as a non-aqueous background electrolyte was vital to achieve robust analysis using a bare fused-silica capillary with an applied voltage of +30 kV. Problems with adsorption associated with an aqueous background electrolyte were eliminated using a non-aqueous background electrolyte made of methanol/acetic acid (97:3) with 25 mM ammonium acetate (for zebrafish embryos) or 100 mM ammonium acetate (for brown trouts), depending on the sample complexity and matrix influences. High resolution and high separation selectivity from matrix components were achieved by optimization of the ammonium acetate concentration in the background electrolyte. An extensive evaluation of matrix effects was conducted with regard to the complex matrices present in the fish samples. They required adapting the background electrolyte to higher concentrations. Applying this method to extracts of zebrafish embryos and brown trout tissue samples, limits of detection for both metformin and guanylurea in zebrafish embryos (12.2 µg/l and 15 µg/l) and brown trout tissues (15 ng/g and 34 ng/g) were in the low µg/l or ng/g range. Finally, metformin and guanylurea could be both quantified for the first time in biota samples from exposure experiments.


Assuntos
Biota , Eletroforese Capilar/métodos , Hipoglicemiantes/metabolismo , Espectrometria de Massas/métodos , Metformina/metabolismo , Ureia/metabolismo , Animais , Limite de Detecção , Extração em Fase Sólida , Truta/metabolismo , Ureia/química , Peixe-Zebra/metabolismo
18.
Pharm Res ; 37(7): 121, 2020 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-32514792

RESUMO

PURPOSE: MDCK cells are commonly used to assess drug permeability, but the existence of various strains merits a comparative functional study. Since metformin absorption is largely mediated by transporters and paracellular diffusion, we used it to functionally compare MDCK-wt and MDCK-II. METHODS: Uptake, bidirectional transport and efflux experiments were performed using different buffers, pH, and a panel of transporter inhibitors. Relative contributions to total transport in both strains were estimated. RESULTS: Metformin uptake into MDCK-wt was linear but saturable in MDCK-II. Uptake into MDCK-wt or -II was promoted at pH 5.4 or 8.4, respectively. Quinidine and cimetidine similarly inhibited uptake in both strains. Lopinavir (PMAT specific) at pH 5.4 or pyrimethamine (MATE specific) at pH 8.4 differentially inhibited MDCK-wt or -II, respectively. Transport at pH 7.4 was absorptive regardless of strains, but secretory (MDCK-II) or absorptive (MDCK-wt) at pH 5.4. Efflux was largely basolateral in both strains. While paracellular permeability was similar between strains, total transport was dominated by transporters in MDCK-II or paracellular diffusion in MDCK-wt. CONCLUSIONS: Metformin transport revealed functional differences between MDCK strains. Apical uptake was governed by MATE in MDCK-II or PMAT in MDCK-wt, such that metformin transport was either secretory or absorptive, respectively.


Assuntos
Metformina/metabolismo , Animais , Biofilmes , Transporte Biológico/efeitos dos fármacos , Adesão Celular , Células Cultivadas , Cimetidina/metabolismo , Difusão , Cães , Humanos , Concentração de Íons de Hidrogênio , Lopinavir/metabolismo , Células Madin Darby de Rim Canino , Pirimetamina/metabolismo , Quinidina/metabolismo
19.
Environ Toxicol ; 35(11): 1170-1178, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32519799

RESUMO

Formaldehyde (FA) can be produced in the environment and by cell metabolism and has been classified as a carcinogen in animals and humans. Metformin is the most commonly used drug for the treatment of type 2 diabetes. Metformin also has potential benefit in cancer prevention and treatment. The aim of this study was to determine whether metformin can directly react with FA and attenuate its toxicity in vitro. Metformin was incubated at pH 7.4 and 37°C in the presence of FA, and the reaction mixture was analyzed by UV spectrophotometry, high-performance liquid chromatography (HPLC), and mass spectrometry. Fluorescence spectrophotometry, immunofluorescence, and western blot were used to measure FA-induced bovine serum albumin (BSA) crosslinking and DNA damage in HepG2 cells treated with or without metformin. According to the HPLC and mass spectrometry data, we speculate that the reaction of metformin with FA (1:1) initially results in the formation of a conjugated intermediate followed by the subsequent generation of a stable six-membered ring structure. Correspondingly, metformin attenuated FA-induced fluorescence in BSA as well as the aggregation of γH2AX in HepG2 cells. These results suggest that metformin can protect protein and DNA damage induced by FA at least partly through a direct reaction process.


Assuntos
Formaldeído/toxicidade , Metformina/metabolismo , Animais , Dano ao DNA/efeitos dos fármacos , Humanos , Soroalbumina Bovina/química
20.
Int J Biol Macromol ; 155: 411-420, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32224176

RESUMO

Natural polymers like polysaccharides, polypeptides and their derivatives are broadly applied in drug delivery due to excellent biocompatibility and biodegradability. In this study, the dissolving tablets, formed with carboxymethylcellulose/poly-l-lysine/tripolyphosphate (CMC/PLL/TPP) complex, were prepared using metformin hydrochloride (MetHCl) as model drug. Confocal laser scanning microscopy observation manifested that FITC-labeled PLL interacted with CMC and formed a uniform interior microstructure. Scanning electron microscope images showed the drug-loaded tablets had well-formed shapes with smooth surfaces. MetHCl embedded interior the microstructures of the tablets and represented in a crystal form. Thermo-gravimetric analysis and differential scanning calorimetry indicated that the drug-loaded tablets had stable thermal properties with less moisture content (3.52%). Fourier transform infrared spectrometer confirmed that the CMC/PLL/TPP complex was fabricated via the electrostatic interactions between -NH3+, -COO- and -[P2O54-]- groups. The drug-loaded tablets had a high drug loading efficiency of 85.76% and drug encapsulation efficiency of 81.47%, and a shorter wetting time of 2.16 min in SSF (pH 6.8) and lower swelling ratio of 233.34%. The drug loaded in the samples could be released completely within 10 min in simulated saliva fluid (SSF pH 6.8), indicating a rapid drug release and dissolving profile in the environment, which could be developed for dissolving tablets.


Assuntos
Carboximetilcelulose Sódica/química , Hipoglicemiantes/química , Metformina/química , Polilisina/química , Polímeros/química , Polifosfatos/química , Preparações de Ação Retardada , Liberação Controlada de Fármacos , Humanos , Hipoglicemiantes/metabolismo , Metformina/metabolismo , Solubilidade , Comprimidos/química
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