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1.
Strahlenther Onkol ; 2020 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-32681351

RESUMEN

BACKGROUND: Non-small-cell lung cancer (NSCLC) is a major cause of cancer-related death globally. Endothelial PAS domain-containing protein 1 (EPAS1) is a homolog of the hypoxia-inducible factor 1α and has been reported to confer tyrosine kinase inhibitor (TKI) resistance in NSCLC, but its role in peritoneal carcinomatosis of NSCLC is unknown. METHODS: PC14HM, a high metastatic potential subline of NSCLC cell line PC14, was derived. Stable shRNA knockdown of EPAS1 was then established in PC14HM cells and subjected to assessment regarding the effects on proliferation and viability, xenograft tumor growth, metastatic potential, mesothelial-mesenchymal transition (MMT)-related characteristics and peritoneal carcinomatosis in a mouse model. RESULTS: EPAS1 expression was elevated in PC14HM cells. Knockdown of EPAS1 inhibited the proliferation and viability of PC14HM cells in vitro and suppressed tumorigenesis in vivo. In addition, the metastatic features and in vitro productions of MMT-inducing factors in PC14HM cells was also associated with EPAS1. More importantly, knockdown of EPAS1 drastically suppressed peritoneal carcinomatosis of PC14HM cells in vivo. CONCLUSION: EPAS1 promotes peritoneal carcinomatosis of NSCLC through enhancement of MMT and could therefore serve as a prognostic marker or a therapeutic target in treating NSCLC, particularly in patients with peritoneal carcinomatosis.

2.
Cell ; 181(4): 832-847.e18, 2020 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-32304665

RESUMEN

Obesity is a major modifiable risk factor for pancreatic ductal adenocarcinoma (PDAC), yet how and when obesity contributes to PDAC progression is not well understood. Leveraging an autochthonous mouse model, we demonstrate a causal and reversible role for obesity in early PDAC progression, showing that obesity markedly enhances tumorigenesis, while genetic or dietary induction of weight loss intercepts cancer development. Molecular analyses of human and murine samples define microenvironmental consequences of obesity that foster tumorigenesis rather than new driver gene mutations, including significant pancreatic islet cell adaptation in obesity-associated tumors. Specifically, we identify aberrant beta cell expression of the peptide hormone cholecystokinin (Cck) in response to obesity and show that islet Cck promotes oncogenic Kras-driven pancreatic ductal tumorigenesis. Our studies argue that PDAC progression is driven by local obesity-associated changes in the tumor microenvironment and implicate endocrine-exocrine signaling beyond insulin in PDAC development.

3.
Cell Rep ; 28(3): 759-772.e10, 2019 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-31315053

RESUMEN

Mechanisms coordinating pancreatic ß cell metabolism with insulin secretion are essential for glucose homeostasis. One key mechanism of ß cell nutrient sensing uses the mitochondrial GTP (mtGTP) cycle. In this cycle, mtGTP synthesized by succinyl-CoA synthetase (SCS) is hydrolyzed via mitochondrial PEPCK (PEPCK-M) to make phosphoenolpyruvate, a high-energy metabolite that integrates TCA cycling and anaplerosis with glucose-stimulated insulin secretion (GSIS). Several strategies, including xenotopic overexpression of yeast mitochondrial GTP/GDP exchanger (GGC1) and human ATP and GTP-specific SCS isoforms, demonstrated the importance of the mtGTP cycle. These studies confirmed that mtGTP triggers and amplifies normal GSIS and rescues defects in GSIS both in vitro and in vivo. Increased mtGTP synthesis enhanced calcium oscillations during GSIS. mtGTP also augmented mitochondrial mass, increased insulin granule number, and membrane proximity without triggering de-differentiation or metabolic fragility. These data highlight the importance of the mtGTP signal in nutrient sensing, insulin secretion, mitochondrial maintenance, and ß cell health.

4.
Foods ; 8(1)2019 Jan 17.
Artículo en Inglés | MEDLINE | ID: mdl-30658407

RESUMEN

The effect of hydrogen peroxide (H2O2) oxidation on the physicochemical, gelation and antimicrobial properties of gellan gum was studied. The oxidized gellan gum (OGG) was characterized by measuring the carboxyl/carbonyl group contents, Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (¹H-NMR) spectroscopy. The H2O2 oxidation resulted in a large increase in the carboxyl groups in gellan gum. The OGG lost gelation ability by oxidation even in the presence of metal ions. The antimicrobial activities of the OGG against Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (Escherichia coli), and fungal (Aspergillus niger) were tested. The OGG could inhibit the growth of both bacteria and fungal, and the activity was improved with an increase in the oxidation level. Finally, the application of the OGG as an active coatings material to extend the storage of apples was tested.

5.
Islets ; 10(5): 181-189, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30118626

RESUMEN

Inhibition of the sodium-glucose co-transporter type 2 (SGLT2) has received growing acceptance as a novel, safe and effective means to improve glycemic control in patients with type 2 diabetes. Inhibition of SGLT2 lowers the renal glucose threshold and reduces plasma glucose by promoting glucose excretion in urine. Both animal studies and clinical trials in man suggest that SGLT2 inhibition has the potential to improve pancreatic ß-cell function by reducing glucose toxicity. However, there is limited data exploring how reducing glucotoxicity via SGLT2 inhibition affects rates of ß-cell proliferation and death throughout life in the context of insulin resistance and type 2 diabetes. SGLT2-/- mice were backcrossed to the db/db strain to produce littermate control db/db-SGLT2+/+ and experimental db/db-SGLT2-/- mice. Mice were euthanized at 5, 12 and 20 weeks of age to collect plasma for glucose, insulin, lipid and cytokine measures, and pancreata for histological analysis including determination of ß-cell mass and rates of proliferation and death. SGLT2 deletion in db/db mice reduced plasma glucose as early as 5 weeks of age and continued throughout life without changes in plasma lipids or cytokines. Reduced plasma glucose levels occurred in parallel with an increase in the relative ß-cell volume and reduced frequency of ß-cell death, and no apparent change in rates of ß-cell proliferation. These data add to a growing body of evidence demonstrating that improved glycemic control achieved through SGLT2 inhibition can preserve ß-cell function and endogenous insulin secretion by reducing glucose toxicity and rates of ß-cell death.


Asunto(s)
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Transportador 2 de Sodio-Glucosa/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Monitoreo de Drogas/métodos , Hipoglucemiantes/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Ratones , Ratones Noqueados , Resultado del Tratamiento
6.
Cancer Control ; 25(1): 1073274818769849, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29651883

RESUMEN

BACKGROUND: Long noncoding RNAs (lncRNAs) are a new class of cancer regulators. Here, we aimed to investigate the diagnostic and therapeutic values of an lncRNA, differentiation antagonizing noncoding RNA (DANCR), in lung cancer. METHODS: Real-time polymerase chain reaction was used to compare DANCR levels in normal and cancerous lung tissues as well as lung cancer cells. Lentiviral transduction was used to induce DANCR overexpression or silencing in vitro, followed by monitoring cell proliferation, colony formation, and changes in microRNA-216a (miR-216a) expression. DANCR-specific small hairpin RNA transduction was used to establish cells with stable DANCR knockdown, and silenced cells were used to initiate lung tumor xenografts, followed by monitoring tumor growth. RESULTS: DANCR upregulation was seen in lung cancer, particularly in high-grade lung cancer tissues and aggressive cancer cells. Ectopic DANCR expression induced lung cancer cell proliferation and colony formation, whereas DANCR silencing induced opposing effects. The miR-216a level in cancer cells was negatively correlated with DANCR expression. The DANCR knockdown reduced the growth of tumor xenografts in vivo. CONCLUSION: DANCR upregulation is a potential indicator of aggressive lung cancer. Silencing of DANCR has great potential as a potent therapeutic strategy in lung cancer.


Asunto(s)
Neoplasias Pulmonares/genética , MicroARNs/genética , ARN Largo no Codificante/genética , Animales , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , MicroARNs/biosíntesis , MicroARNs/metabolismo , Clasificación del Tumor , ARN Largo no Codificante/metabolismo , Transfección , Regulación hacia Arriba
7.
Cell Biochem Funct ; 36(1): 27-33, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29314203

RESUMEN

Oesophageal cancer (OC) is one of the most fatal malignancies in the world, and chemoresistance restricts the therapeutic outcome of OC. Long noncoding RNA (lncRNA) was reported to play roles in multiple cancer types. Yet, the function of lncRNA in chemoresistance of OC has not been reported. A lncRNA gene, PCAT-1, showed higher expression in OC tissues, especially higher in secondary OC compared with normal mucosa tissues. Overexpression of PCAT-1 increased the proliferation rate and growth of OC cells. Inhibition of PCAT-1 decreased proliferation and growth of OC cells, and increased cisplatin chemosensitivity. In a mouse OC xenograft model, PCAT-1 inhibition repressed OC growth in vivo. Therefore, PCAT-1 may potentially serve as a therapeutic target for treating OC. PCAT-1 promotes development of OC and represses the chemoresistance of OC to cisplatin, and silencing of PCAT-1 may be a therapeutic strategy for treating OC.


Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/patología , ARN Largo no Codificante/genética , Animales , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Neoplasias Esofágicas/genética , Humanos , Masculino , Ratones , Ratones Desnudos , ARN Largo no Codificante/metabolismo , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto
8.
Tumour Biol ; 39(5): 1010428317706215, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28488541

RESUMEN

Non-small cell lung cancer accounts for 85% of all types of lung cancer and is the leading cause of worldwide cancer-associated mortalities. MiR-124 is epigenetically silenced in various types of cancer and plays important roles in tumor development and progression. MiR-124 was also significantly downregulated in non-small cell lung cancer patients. Glycolysis has been considered as a feature of cancer cells; hypoxia-inducible factor 1-alpha/beta and Akt are key enzymes in the regulation of glycolysis and energy metabolism in cancer cells. However, the role of miR-124 in non-small cell lung cancer cell proliferation, glycolysis, and energy metabolism remains unknown. In this research, cell proliferation was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; furthermore, glucose consumption and lactic acid production were assessed; adenosine triphosphate content and NAD+/NADH were also detected. These tests were conducted using the normal non-small cell lung cancer cell line A549, which was transfected variedly with miR-mimics, miR-124 mimics, miR-124 inhibitor, pc-DNA3.1(+)-AKT1, and pc-DNA3.1(+)-AKT2 plasmid. Here, we show that miR-124 overexpression directly decreased cell growth, glucose consumption, lactate production, and energy metabolism. MiR-124 also negatively regulates glycolysis rate-limiting enzymes, glucose transporter 1 and hexokinase II. Our results also showed that miR-124 negatively regulates AKT1 and AKT2 but no regulatory effect on hypoxia-inducible factor 1-alpha/beta. Overexpression of AKT reverses the inhibitory effect of miR-124 on cell proliferation and glycolytic metabolism in non-small cell lung cancer. AKT inhibition blocks miR-124 silencing-induced AKT1/2, glucose transporter 1, hexokinase II activation, cell proliferation, and glycolytic or energy metabolism changes. In summary, this study demonstrated that miR-124 is able to inhibit proliferation, glycolysis, and energy metabolism, potentially by targeting AKT1/2-glucose transporter 1/hexokinase II in non-small cell lung cancer cells.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Transportador de Glucosa de Tipo 1/genética , Hexoquinasa/genética , MicroARNs/genética , Proteína Oncogénica v-akt/genética , Células A549 , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular/genética , Metabolismo Energético/genética , Regulación Neoplásica de la Expresión Génica , Glucólisis/genética , Humanos
9.
DNA Cell Biol ; 35(11): 715-721, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27494742

RESUMEN

Lung cancer is one of the most malignant cancers with a high metastatic potential. The purpose of this study was to study the role and the underlying mechanism of miR-214 in lung cancer progression. The expression of miR-214 in normal lung and lung cancer tissue was analyzed by quantitative real-time PCR analysis. Furthermore, H1299 cells were infected with miR-214 lentivirus, and the effect of infection on cell viability and migration was analyzed. Carboxypeptidase-D (CPD), as a potential target of miR-214, was characterized in either normal lung or lung cancer tissues. The interaction of CPD expression with the tumor suppressing effect of miR-214 was characterized. We demonstrated that low miR-214 expression is a hallmark of lung cancer, especially high-grade and metastatic cancer. In vitro studies in H1299 cells confirmed that low miR-214 expression is associated with enhanced proliferation and migratory abilities. Similarly, CPD overexpression coincides with high-grade lung cancer and the CPD overexpression could reverse the inhibitory effects of miR-214. miR-214 is a tumor suppressor in lung cancer. miR-214 inhibits lung cancer progression by targeting CPD. The miR-214-CPD axis may be a therapeutic axis for lung cancer patients.


Asunto(s)
Carboxipeptidasas/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Pulmonares/genética , MicroARNs/genética , Carboxipeptidasas/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Genes Supresores de Tumor , Humanos
10.
Proc Natl Acad Sci U S A ; 113(24): E3423-30, 2016 06 14.
Artículo en Inglés | MEDLINE | ID: mdl-27247419

RESUMEN

A key sensor of cellular energy status, AMP-activated protein kinase (AMPK), interacts allosterically with AMP to maintain an active state. When active, AMPK triggers a metabolic switch, decreasing the activity of anabolic pathways and enhancing catabolic processes such as lipid oxidation to restore the energy balance. Unlike oxidative tissues, in which AMP is generated from adenylate kinase during states of high energy demand, the ornithine cycle enzyme argininosuccinate synthetase (ASS) is a principle site of AMP generation in the liver. Here we show that ASS regulates hepatic AMPK, revealing a central role for ureagenesis flux in the regulation of metabolism via AMPK. Treatment of primary rat hepatocytes with amino acids increased gluconeogenesis and ureagenesis and, despite nutrient excess, induced both AMPK and acetyl-CoA carboxylase (ACC) phosphorylation. Antisense oligonucleotide knockdown of hepatic ASS1 expression in vivo decreased liver AMPK activation, phosphorylation of ACC, and plasma ß-hydroxybutyrate concentrations. Taken together these studies demonstrate that increased amino acid flux can activate AMPK through increased AMP generated by ASS, thus providing a novel link between protein catabolism, ureagenesis, and hepatic lipid metabolism.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Argininosuccinato Sintasa/biosíntesis , Regulación Enzimológica de la Expresión Génica/fisiología , Metabolismo de los Lípidos/fisiología , Hígado/metabolismo , Urea/metabolismo , Animales , Activación Enzimática , Ratas , Ratas Sprague-Dawley
11.
J. physiol. biochem ; 72(2): 337-343, jun. 2016. tab, graf
Artículo en Inglés | IBECS | ID: ibc-168277

RESUMEN

miR-148b has been found to be aberrantly expressed in various tumor types. It has recently been reported to be involved in regulating radioresistance in non-small cell lung cancer (NSCLC) cells. However, its expression level and association with radiosensitivity in human patient samples have not been investigated. Real-time PCR was used to evaluate the expression levels of miR-148b. Χ2 test was performed to analyze the association between miR-148b expression levels and clinicopathological factors or radiosensitivity. Kaplan-Meier survival curve was constructed to estimate the overall survival (OS), and the differences in survival were compared using the log-rank test. Cox regression analysis was conducted to determine the prognostic value of miR-148b. The relative level of miR-148b was significantly decreased in NSCLC tissues compared with matched non-cancerous tissues (P < 0.0001), and it was higher in tissues of patients who are good responders compared to those who are poor responders to radiotherapy (P < 0.0001). Lower expression of miR-148b was positively associated with high tumor stage (P = 0.0407) and radioresistance (P = 0.0002), and it predicted poor survival in patients with (P = 0.0129) or without (P = 0.0094) radiotherapy treatment. miR-148b was an independent prognostic factor for NSCLC as demonstrated by Cox proportional hazards risk analysis. miR-148b may serve as a prognostic biomarker of poor survival and a novel predictor of response to radiotherapy treatment in NSCLC (AU)


No disponible


Asunto(s)
Humanos , Masculino , Femenino , Persona de Mediana Edad , Pulmón/efectos de la radiación , Neoplasias Pulmonares/radioterapia , MicroARNs/metabolismo , Regulación hacia Abajo , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , ARN Neoplásico/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Biomarcadores/metabolismo , Pronóstico , Distribución de Chi-Cuadrado , Análisis de Supervivencia , Reacción en Cadena en Tiempo Real de la Polimerasa , Estadificación de Neoplasias , Clasificación del Tumor
12.
J Physiol Biochem ; 72(2): 337-43, 2016 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-27083571

RESUMEN

miR-148b has been found to be aberrantly expressed in various tumor types. It has recently been reported to be involved in regulating radioresistance in non-small cell lung cancer (NSCLC) cells. However, its expression level and association with radiosensitivity in human patient samples have not been investigated. Real-time PCR was used to evaluate the expression levels of miR-148b. Χ (2) test was performed to analyze the association between miR-148b expression levels and clinicopathological factors or radiosensitivity. Kaplan-Meier survival curve was constructed to estimate the overall survival (OS), and the differences in survival were compared using the log-rank test. Cox regression analysis was conducted to determine the prognostic value of miR-148b. The relative level of miR-148b was significantly decreased in NSCLC tissues compared with matched non-cancerous tissues (P < 0.0001), and it was higher in tissues of patients who are good responders compared to those who are poor responders to radiotherapy (P < 0.0001). Lower expression of miR-148b was positively associated with high tumor stage (P = 0.0407) and radioresistance (P = 0.0002), and it predicted poor survival in patients with (P = 0.0129) or without (P = 0.0094) radiotherapy treatment. miR-148b was an independent prognostic factor for NSCLC as demonstrated by Cox proportional hazards risk analysis. miR-148b may serve as a prognostic biomarker of poor survival and a novel predictor of response to radiotherapy treatment in NSCLC.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Neoplasias Pulmonares/radioterapia , Pulmón/efectos de la radiación , MicroARNs/metabolismo , ARN Neoplásico/metabolismo , Biomarcadores/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Distribución de Chi-Cuadrado , Femenino , Humanos , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Pronóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Supervivencia
13.
Cell Metab ; 22(5): 936-47, 2015 Nov 03.
Artículo en Inglés | MEDLINE | ID: mdl-26411341

RESUMEN

Mass isotopomer multi-ordinate spectral analysis (MIMOSA) is a step-wise flux analysis platform to measure discrete glycolytic and mitochondrial metabolic rates. Importantly, direct citrate synthesis rates were obtained by deconvolving the mass spectra generated from [U-(13)C6]-D-glucose labeling for position-specific enrichments of mitochondrial acetyl-CoA, oxaloacetate, and citrate. Comprehensive steady-state and dynamic analyses of key metabolic rates (pyruvate dehydrogenase, ß-oxidation, pyruvate carboxylase, isocitrate dehydrogenase, and PEP/pyruvate cycling) were calculated from the position-specific transfer of (13)C from sequential precursors to their products. Important limitations of previous techniques were identified. In INS-1 cells, citrate synthase rates correlated with both insulin secretion and oxygen consumption. Pyruvate carboxylase rates were substantially lower than previously reported but showed the highest fold change in response to glucose stimulation. In conclusion, MIMOSA measures key metabolic rates from the precursor/product position-specific transfer of (13)C-label between metabolites and has broad applicability to any glucose-oxidizing cell.


Asunto(s)
Ciclo del Ácido Cítrico/genética , Ácido Cítrico/metabolismo , Insulina/metabolismo , Ácido Oxaloacético/metabolismo , Complejo Piruvato Deshidrogenasa/genética , Acetilcoenzima A/metabolismo , Animales , Isótopos de Carbono , Citratos/metabolismo , Insulina/genética , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Oxidación-Reducción , Consumo de Oxígeno , Piruvato Carboxilasa/genética , Piruvato Carboxilasa/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Ácido Pirúvico/metabolismo , Ratas
14.
Cytogenet Genome Res ; 146(1): 1-8, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26184032

RESUMEN

Lung cancer, especially non-small cell lung cancer (NSCLC), is the major cause of cancer death worldwide. Mutations in epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met), both of which are receptor tyrosine kinases, have been identified in a considerable percentage of NSCLC patients. EGFR and c-Met share the same downstream pathways and cooperate not only in promoting metastasis but also in conferring resistance to tyrosine kinase inhibitor (TKI) therapies in NSCLC. MicroRNAs (miRNAs) are a family of small non-coding RNAs, usually 21-25 nucleotides long, and are critical in regulating gene expression. Abnormal miRNA expression has been implicated in the initiation and progression in many forms of cancers, including lung cancer. In this study, we found that miR-200a is downregulated in NSCLC cells, where it directly targets the 3'-UTR of both EGFR and c-Met mRNA. Overexpression of miR-200a in NSCLC cells significantly downregulates both EGFR and c-Met levels and severely inhibits cell migration and invasion. Moreover, in NSCLC cell lines that are resistant to gefitinib, a drug often used in TKI therapies to treat NSCLC, miR-200a expression is able to render the cells much more sensitive to the drug treatment.


Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , MicroARNs/fisiología , Proteínas Proto-Oncogénicas c-met/genética , Quinazolinas/farmacología , Regiones no Traducidas 3' , Secuencia de Bases , Sitios de Unión , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Resistencia a Antineoplásicos , Receptores ErbB/metabolismo , Gefitinib , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Invasividad Neoplásica , Proteínas Proto-Oncogénicas c-met/metabolismo , Interferencia de ARN
15.
Cancer Biol Ther ; 16(4): 549-57, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25831463

RESUMEN

Mutations in epidermal growth factor receptor (EGFR) rendering it constitutively active is one of the major causes for metastatic non-small-cell lung cancer (NSCLC), and EGFR-targeted therapies utilizing tyrosine kinase inhibitors (TKIs) are often used clinically as the first-line treatment. But approximately half of NSCLC patients develop resistance to these therapies, where the MET proto-oncogene is amplified by EGFR through the hypoxia-inducible factor (HIF)-1α. Here we report that endothelial PAS domain-containing protein 1 (EPAS1), with 48% sequence identity to HIF-1α, specifically binds to TKI-resistant T790M EGFR, but not to wild-type EGFR, in NSCLC cell lines. Expression of EPAS1 enhances amplification of MET when simultaneously expressed with T790M EGFR but not with wild-type EGFR, and this enhancement is independent of ligand binding domain of EGFR. MET amplification requires EPAS1, since EPAS1 knock-down reduced MET levels. When NSCLC cells expressing T790M EGFR were treated with TKIs, reduced EPAS1 levels significantly enhanced the drug effect, whereas over-expression of EPAS1 increased the drug resistant effect. This EPAS1-dependent TKI-resistance was abolished by knocking-down MET, suggesting that EPAS1 does not cause TKI-resistance itself but functions to bridge EGFR and MET interactions. Our findings suggest that EPAS1 is a key factor in the EGFR-MET crosstalk in conferring TKI-resistance in NSCLC cases, and could be used as a potential therapeutic target in TKI-resistant NSCLC patients.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Resistencia a Antineoplásicos/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Amplificación de Genes/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias Pulmonares/tratamiento farmacológico
16.
Diabetes ; 63(12): 4218-29, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25024374

RESUMEN

Mitochondrial GTP (mtGTP)-insensitive mutations in glutamate dehydrogenase (GDH(H454Y)) result in fasting and amino acid-induced hypoglycemia in hyperinsulinemia hyperammonemia (HI/HA). Surprisingly, hypoglycemia may occur in this disorder despite appropriately suppressed insulin. To better understand the islet-specific contribution, transgenic mice expressing the human activating mutation in ß-cells (H454Y mice) were characterized in vivo. As in the humans with HI/HA, H454Y mice had fasting hypoglycemia, but plasma insulin concentrations were similar to the controls. Paradoxically, both glucose- and glutamine-stimulated insulin secretion were severely impaired in H454Y mice. Instead, lack of a glucagon response during hypoglycemic clamps identified impaired counterregulation. Moreover, both insulin and glucagon secretion were impaired in perifused islets. Acute pharmacologic inhibition of GDH restored both insulin and glucagon secretion and normalized glucose tolerance in vivo. These studies support the presence of an mtGTP-dependent signal generated via ß-cell GDH that inhibits α-cells. As such, in children with activating GDH mutations of HI/HA, this insulin-independent glucagon suppression may contribute importantly to symptomatic hypoglycemia. The identification of a human mutation causing congenital hypoglucagonemic hypoglycemia highlights a central role of the mtGTP-GDH-glucagon axis in glucose homeostasis.


Asunto(s)
Aminoácidos/metabolismo , Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Glutamato Deshidrogenasa/genética , Guanosina Trifosfato/metabolismo , Hiperamonemia/genética , Hiperinsulinismo/genética , Hipoglucemia/genética , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Mitocondrias/metabolismo , Animales , Técnica de Clampeo de la Glucosa , Humanos , Secreción de Insulina , Ratones , Ratones Transgénicos , Mutación , Síndrome
17.
J Biol Chem ; 289(11): 7257-63, 2014 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-24497630

RESUMEN

Synthesis of phosphoenolpyruvate (PEP) from oxaloacetate is an absolute requirement for gluconeogenesis from mitochondrial substrates. Generally, this reaction has solely been attributed to the cytosolic isoform of PEPCK (PEPCK-C), although loss of the mitochondrial isoform (PEPCK-M) has never been assessed. Despite catalyzing the same reaction, to date the only significant role reported in mammals for the mitochondrial isoform is as a glucose sensor necessary for insulin secretion. We hypothesized that this nutrient-sensing mitochondrial GTP-dependent pathway contributes importantly to gluconeogenesis. PEPCK-M was acutely silenced in gluconeogenic tissues of rats using antisense oligonucleotides both in vivo and in isolated hepatocytes. Silencing PEPCK-M lowers plasma glucose, insulin, and triglycerides, reduces white adipose, and depletes hepatic glycogen, but raises lactate. There is a switch of gluconeogenic substrate preference to glycerol that quantitatively accounts for a third of glucose production. In contrast to the severe mitochondrial deficiency characteristic of PEPCK-C knock-out livers, hepatocytes from PEPCK-M-deficient livers maintained normal oxidative function. Consistent with its predicted role, gluconeogenesis rates from hepatocytes lacking PEPCK-M are severely reduced for lactate, alanine, and glutamine, but not for pyruvate and glycerol. Thus, PEPCK-M has a direct role in fasted and fed glucose homeostasis, and this mitochondrial GTP-dependent pathway should be reconsidered for its involvement in both normal and diabetic metabolism.


Asunto(s)
Regulación Enzimológica de la Expresión Génica , Gluconeogénesis , Péptidos y Proteínas de Señalización Intracelular/fisiología , Hígado/enzimología , Hígado/metabolismo , Mitocondrias/enzimología , Fosfoenolpiruvato Carboxiquinasa (GTP)/fisiología , Alimentación Animal , Animales , Glucemia/metabolismo , Privación de Alimentos , Silenciador del Gen , Glicerol/metabolismo , Glucógeno/metabolismo , Guanosina Trifosfato/metabolismo , Hepatocitos/citología , Homeostasis , Insulina/metabolismo , Isoenzimas/fisiología , Ácido Láctico/metabolismo , Masculino , Mitocondrias/metabolismo , Oligonucleótidos Antisentido/química , Oxígeno/metabolismo , Consumo de Oxígeno , Ratas , Ratas Sprague-Dawley
18.
Nucl Med Biol ; 39(2): 167-76, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22033026

RESUMEN

INTRODUCTION: Glucagon-like peptide 1 (GLP-1) is released in response to food intake and plays an important role in maintaining blood glucose homeostasis. Exendin (9-39), a potent glucagon-like peptide 1 receptor antagonist, has been labeled with In-111 for SPECT imaging. We report here the first radiosynthesis of [(18)F]exendin (9-39) ([(18)F]Ex(9-39)) and an evaluation of its potential as a biomarker for in vivo positron emission tomography (PET) imaging of pancreatic ß-cell mass (BCM) in rats. METHODS: F-18 label was introduced by conjugation of [(18)F]4-fluorobenzaldehyde with an Ex(9-39) derivative containing a 6-hydrazinonicotinyl group on the ε-amine of Lys27. Positron emission tomography imaging was carried out in Sprague-Dawley rats (five control and five streptozotocin-induced diabetic) and BioBreeding diabetes-prone rats (three at 7 weeks and three at 12 weeks) using the high-resolution research tomograph (HRRT) after 0.187 ± 0.084 mCi [(18)F]Ex(9-39) administration. Time-activity curves were obtained from pancreas, liver and kidney. Pancreases were assayed for insulin content after the imaging study. RESULTS: Site-specifically labeled [(18)F]Ex(9-39) was purified on a G15 open column with radiochemical and chemical purities >98%. Positron emission tomography imaging showed pancreatic standardized uptake value (SUV) peaked at 10 min and plateaued by 50 min to the end of scan (240 min). No correlations of pancreatic SUV with postmortem measures of insulin content were seen. CONCLUSIONS: [(18)F]Ex(9-39) was successfully prepared and used for PET imaging for the first time to measure pancreatic BCM. The results suggest that derivatization of the Lys27 residue might reduce binding affinity, as evidenced by the absence of specific binding. Exendin analogues radiolabeled at other sites may elucidate the active site required for binding.


Asunto(s)
Radioisótopos de Flúor/farmacocinética , Células Secretoras de Insulina/diagnóstico por imagen , Fragmentos de Péptidos/farmacocinética , Radiofármacos/síntesis química , Animales , Biomarcadores , Diabetes Mellitus Experimental/metabolismo , Péptido 1 Similar al Glucagón/antagonistas & inhibidores , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Tomografía de Emisión de Positrones/métodos , Ratas , Ratas Sprague-Dawley , Distribución Tisular
19.
Biochem Biophys Res Commun ; 415(1): 30-5, 2011 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-22008547

RESUMEN

Mechanistic models of glucose stimulated insulin secretion (GSIS) established in minimal media in vitro, may not accurately describe the complexity of coupling metabolism with insulin secretion that occurs in vivo. As a first approximation, we have evaluated metabolic pathways in a typical growth media, DMEM as a surrogate in vivo medium, for comparison to metabolic fluxes observed under the typical experimental conditions using the simple salt-buffer of KRB. Changes in metabolism in response to glucose and amino acids and coupling to insulin secretion were measured in INS-1 832/13 cells. Media effects on mitochondrial function and the coupling efficiency of oxidative phosphorylation were determined by fluorometrically measured oxygen consumption rates (OCRs) combined with (31)P NMR measured rates of ATP synthesis. Substrate preferences and pathways into the TCA cycle, and the synthesis of mitochondrial 2nd messengers by anaplerosis were determined by (13)C NMR isotopomer analysis of the fate of [U-(13)C] glucose metabolism. Despite similar incremental increases in insulin secretion, the changes of OCR in response to increasing glucose from 2.5 to 15mM were blunted in DMEM relative to KRB. Basal and stimulated rates of insulin secretion rates were consistently higher in DMEM, while ATP synthesis rates were identical in both DMEM and KRB, suggesting greater mitochondrial uncoupling in DMEM. The relative rates of anaplerosis, and hence synthesis and export of 2nd messengers from the mitochondria were found to be similar in DMEM to those in KRB. And, the correlation of total PC flux with insulin secretion rates in DMEM was found to be congruous with the correlation in KRB. Together, these results suggest that signaling mechanisms associated with both TCA cycle flux and with anaplerotic flux, but not ATP production, may be responsible for the enhanced rates of insulin secretion in more complex, and physiologically-relevant media.


Asunto(s)
Adenosina Trifosfato/biosíntesis , Ciclo del Ácido Cítrico , Glucosa/metabolismo , Insulina/metabolismo , Línea Celular , Medios de Cultivo/farmacología , Glucosa/farmacología , Humanos , Secreción de Insulina , Consumo de Oxígeno
20.
Biochem Biophys Res Commun ; 412(3): 413-8, 2011 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-21820405

RESUMEN

A critical unmet need exists for methods to quantitatively measure endogenous pancreatic ß-cell mass (BCM) for the clinical evaluation of therapies to prevent or reverse loss of BCM and diabetes progression. Our objective was to identify G-protein coupled receptors (GPCRs) that are expressed with a high degree of specificity to islet ß-cells for receptor-targeted imaging of BCM. GPCRs enriched in pancreatic islets relative to pancreas acinar and hepatic tissue were identified using a database screen. Islet-specific expression was confirmed by human pancreas immunohistochemistry (IHC). In vitro selectivity assessment was determined from the binding and uptake of radiolabeled ligands to the rat insulinoma INS-1 832/13 cell line and isolated rat islets relative to the exocrine pancreas cell-type, PANC-1. Tail-vein injections of radioligands into rats were used to determine favorable image criteria of in vivo biodistribution to the pancreas relative to other internal organs (i.e., liver, spleen, stomach, and lungs). Database and IHC screening identified four candidate receptors for further in vitro and in vivo evaluation for PET imaging of BCM: prokineticin-1 receptor (PK-1R), metabotropic glutamate receptor type-5 (mGluR5), neuropeptide Y-2 receptor (NPY-2R), and glucagon-like peptide 1 receptor (GLP-1R). In vitro specificity ratios gave the following receptor rank order: PK-1R>GLP-1R>NPY-2R>mGluR5. The biodistribution rank order of selectivity to the pancreas was found to be PK-1R>VMAT2∼GLP-1R>mGluR5. Favorable islet selectivity and biodistribution characteristics suggest several GPCRs as potential targets for PET imaging of pancreatic BCM.


Asunto(s)
Células Secretoras de Insulina/metabolismo , Tomografía de Emisión de Positrones/métodos , Receptores Acoplados a Proteínas G/metabolismo , Animales , Línea Celular , Receptor del Péptido 1 Similar al Glucagón , Humanos , Células Secretoras de Insulina/química , Células Secretoras de Insulina/citología , Ligandos , Masculino , Ratas , Ratas Sprague-Dawley , Receptor del Glutamato Metabotropico 5 , Receptores Acoplados a Proteínas G/análisis , Receptores de Glucagón/análisis , Receptores de Glucagón/metabolismo , Receptores de Glutamato Metabotrópico/análisis , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de Neuropéptido Y/análisis , Receptores de Neuropéptido Y/metabolismo , Distribución Tisular
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