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1.
Farm. hosp ; 43(2): 45-49, mar.-abr. 2019. ilus, graf, tab
Artigo em Espanhol | IBECS | ID: ibc-182587

RESUMO

Objetivo: Evaluación galénica del proceso de obtención y almacenamiento del colirio de plasma rico en factores de crecimiento PRGF-Endoret(R). Método: Para evaluar la asepsia en la obtención del colirio de PRGFEndoret(R) se realizó un ensayo de esterilidad siguiendo las normas descritas en la Farmacopea Europea y se analizó la estanqueidad de los dispensadores de colirio de PRGF-Endoret(R). Asimismo, se estudiaron las propiedades químicas y biológicas del colirio tras su proceso de obtención y almacenamiento. Se incluyeron ensayos de filtración del colirio, de un ciclo de congelación a -20 ºC y descongelación, así como de estabilidad durante tres y seis meses almacenados a -20 ºC. Resultados: Los ensayos de esterilidad mostraron que no hubo crecimiento microbiano en ninguno de los dispensadores analizados y se observó que el 100% de los monodosis analizados y el 98,4% de los tapones mantenían el hermetismo. Todos los factores de crecimiento analizados permanecieron constantes tras el filtrado del colirio de PRGF-Endoret(R). Además, todos los estudios de estabilidad llevados a cabo con el colirio de PRGF-Endoret(R) en el presente estudio mostraron que no se produjeron cambios significativos en los niveles de factores de crecimiento, en la actividad proliferativa celular ni en las características químicas analizadas. Conclusiones: El presente trabajo muestra que el proceso de elaboración del colirio de PRGF-Endoret(R) se lleva a cabo de forma controlada, aséptica y segura, siguiendo las normas descritas en la Farmacopea Europea. Además, el colirio de PRGF-Endoret(R) obtenido mantiene sus propiedades físico-químicas y biológicas tras someterlo a diferentes tiempos y temperaturas de almacenamiento


Objective: Galenic evaluation of the process for obtaining and storing the platelet rich in growth factors PRGF-Endoret(R) eye drops. Method: To assess whether the PRGF-Endoret(R) eye drops process is aseptically obtained, a sterility test was carried out on the eye drops; the tightness of the PRGF-Endoret(R) eye drops containers was also analyzed. Likewise, the chemical and biological properties of the PRGF-Endoret(R) eye drops were evaluated after the obtaining process and storage. Eye drop filtration tests, one cycle of freezing at -20 °C and thawing, and eye drop stability for three and six months stored at -20 °C were included. Results: The results obtained in the sterility test showed no microbial contamination in any of the analyzed eyedropper; tightness test showed that 100% of the eyedrop containers and the 98.4% of the plugs analyzed remained hermetic. On the other hand, all the growth factors measured remained constant after filtering the PRGF-Endoret(R) eye drops. Furthermore, the different eye drop stability tests carried out in this study showed no significant changes in the growth factors levels, cell proliferative activity or in the chemical characteristics analyzed. Conclusions: The PRGF-Endoret(R) eye drops are obtained in a safety and aseptic manner following the guidelines issued by the Spanish Agency for Drugs and Health Products and the Ministry of Health to obtain medicines for human use. The PRGF-Endoret(R) eye drops maintain their physical-chemical and biological properties after being subjected to different storage times and temperatures


Assuntos
Humanos , Peptídeos e Proteínas de Sinalização Intercelular/análise , Soluções Oftálmicas/análise , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Estabilidade de Medicamentos , Embalagem de Medicamentos , Armazenamento de Medicamentos , Filtração , Congelamento , Esterilização
2.
J. physiol. biochem ; 74(4): 539-547, nov. 2018. tab, graf
Artigo em Inglês | IBECS | ID: ibc-179032

RESUMO

The link between obesity-induced systemic inflammation and decreased insulin signalling is well-known. It is also known that peripherally produced inflammatory cytokines can cross the blood-brain barrier, resulting in the release of neurotoxins that can ultimately lead to the demise of central nervous system integrity. A high-mesembrine Sceletium tortuosum extract was recently shown to possess cytoprotective and mild anti-inflammatory properties in monocytes and to target specific p450 enzymes to reduce adrenal glucocorticoid synthesis. This is significant since the aetiology of both obesity and diabetes is linked to inflammation and excess glucocorticoid production. Given the interlinked nature of glucocorticoid action and inflammation, central immunomodulatory effects of two Sceletium tortuosum extracts prepared by different extraction methods were investigated. Human astrocytes were pre-treated for 30 min, before exposure to Escherichia coli lipopolysaccharide for 23.5 h (in the presence of treatment). Cytotoxicity, mitotoxicity and cytokine responses (basally and in response to inflammatory stimulus) were assessed. In addition, total polyphenol content, antioxidant capacity and selected neural enzyme inhibition capacity were assessed for both extracts. The high-mesembrine Sceletium extract exerted cytoprotective and anti-inflammatory effects. In contrast, the high delta7-mesembrenone extract, rich in polyphenols, exhibited potent antioxidant effect, although with relatively higher risk of adverse effects with overdose. We conclude that both Sceletium tortuosum extracts may be employed as either a preventative supplement or complimentary treatment in the context of obesity and diabetes; however, current data also highlights the impact that extraction methods can have on plant product mechanism of action


No disponible


Assuntos
Humanos , Anti-Inflamatórios não Esteroides/farmacologia , Astrócitos , Alcaloides Indólicos/farmacologia , Mesembryanthemum/química , Extratos Vegetais/farmacologia , Acetilcolinesterase/metabolismo , Anti-Inflamatórios não Esteroides/análise , Anti-Inflamatórios não Esteroides/isolamento & purificação , Antioxidantes , Linhagem Celular , Inibidores da Colinesterase , Alcaloides Indólicos/análise , Alcaloides Indólicos/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação
3.
J. physiol. biochem ; 74(3): 381-393, ago. 2018. ilus, tab, graf
Artigo em Inglês | IBECS | ID: ibc-178993

RESUMO

Differentiation of adipocytes and their aggregation to adipose tissue are critical for mammalian growth and development. MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that play important roles in adipogenesis and lipid metabolism. miR-128-3p may contribute to adipose tissue development according to the previous studies. However, the role of miR-128-3p in the process of preadipocyte differentiation and lipid metabolism is not yet understood. The purpose of this research was to investigate the biological function and molecular mechanism of miR-128-3p in 3T3-L1 cells. In the present study, we found that miR-128-3p was downregulated during the process of 3T3-L1 preadipocyte differentiation. Overexpression of miR-128-3p obstructed the expressions of adipogenic marker genes as well as the lipid droplets accumulation and triglyceride content, suggesting the importance of miR-128-3p for adipogenesis. Moreover, miR-128-3p could lead to the retardation of cell proliferation in 3T3-L1 preadipocytes. Further evidences showed that, as a negative regulator of adipogenesis, miR-128-3p could directly target peroxisome proliferator-activated receptor γ (Pparg) which resulted in the suppression of 3T3-L1 preadipocyte differentiation, and miR-128-3p could also bind with SERTA domain containing 2 (Sertad2) which drove triglyceride hydrolysis and lipolysis. In addition, inhibition of Sertad2 with siRNA displayed the same effects as overexpression of miR-128-3p. Our research demonstrated that miR-128-3p impeded 3T3-L1 adipogenesis by targeting Pparg and Sertad2, resulting in the obstruction of preadipocyte differentiation and promotion of lipolysis. Taken together, this study offers profound insight into the mechanism of miRNA-mediated adipogenesis and lipid metabolism


No disponible


Assuntos
Animais , Camundongos , Adipócitos Brancos/metabolismo , Adipogenia , Regulação da Expressão Gênica no Desenvolvimento , Lipólise , MicroRNAs/metabolismo , PPAR gama/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Regiões 3' não Traduzidas , Células 3T3-L1 , Adipócitos Brancos/citologia , Biomarcadores/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT , Linhagem Celular , Cricetinae , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triglicerídeos/metabolismo
4.
J. physiol. biochem ; 74(2): 195-205, mayo 2018. tab, graf
Artigo em Inglês | IBECS | ID: ibc-178977

RESUMO

Excessive intramyocellular triacylglycerols (IMTGs, muscle lipids) are associated with the abnormal energy metabolism and insulin resistance of skeletal muscle. AMP-activated protein kinase (AMPK), a crucial cellular energy sensor, consists of α, β and γ subunits. Researchers have not clearly determined whether Prkaa1 (also known as AMPKα1) affects IMTG accumulation in skeletal muscle. Here, we show an important role of Prkaa1 in skeletal muscle lipid metabolism. Deletion of muscle Prkaa1 leads to the delayed development of skeletal muscles but does not affect glucose tolerance or insulin sensitivity in animals fed a normal diet. Notably, when animals are fed a high-fat diet, the skeletal muscle of muscle-specific Prkaa1 knockout mice accumulates more lipids than the skeletal muscle of wild-type (WT) mice, with concomitant upregulation of adipogenic gene expressions and downregulation of the expression of genes associated with mitochondrial oxidation. Muscle-specific Prkaa1 ablation also results in hyperlipidemia, which may contribute to the increased IMTG levels. Furthermore, Prkaa1 deletion activates skeletal muscle mTOR signalling, which has a central role in lipid metabolism and mitochondrial oxidation. Collectively, our study provides new insights into the role of Prkaa1 in skeletal muscle. This knowledge may contribute to the treatment of related metabolic diseases


No disponible


Assuntos
Animais , Masculino , Camundongos , Dieta Hiperlipídica , Metabolismo dos Lipídeos , Músculo Esquelético/metabolismo , Linhagem Celular , Metabolismo Energético , Deleção de Genes , Resistência à Insulina , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Serina-Treonina Quinases TOR , Regulação para Cima
5.
J. physiol. biochem ; 74(2): 291-299, mayo 2018. graf
Artigo em Espanhol | IBECS | ID: ibc-178985

RESUMO

MicroRNA is a novel class of small noncoding RNA that has been implicated in a variety of physiological and pathological processes, including glucose homeostasis and diabetes mellitus. So far, a few studies have reported that miRNAs may be an important regulator in glucose-stimulated insulin secretion (GSIS) pathway. However, the role of miRNAs in this process remains unclear. The levels of miRNAs in mouse islets and MIN6 cells were determined by quantitative RT-PCR. Concentration of insulin was determined by ELISA, and the expression of the target protein was determined with western blot assay. The overexpression and downregulation of miRNAs in MIN6 were conducted using cell transfection methods. And luciferase assay was used to measure the direct interaction between miRNAs and target messenger RNAs 3′UTR. miR-9 was screened out for it was downregulated under the effects of short-term high glucose, while long-term high glucose relatively increased miR-9 expression. The Stxbp1 expression was decreased with the overexpression of miR-9 in MIN6 cells and increased when miR-9 was downregulated. Moreover, it was verified by luciferase assay that miR-9 regulated Stxbp1 gene expression by directly targeting Stxbp1 messenger RNA 3′UTR. This study suggests that the pathway consisting of miR-9 and Stxbp1 plays a key role in β-cell function, thus contributing to the network of miRNA-insulin secretion and offering a new candidate for diabetes therapy


No disponible


Assuntos
Animais , Masculino , Camundongos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Regiões 3' não Traduzidas , Linhagem Celular , Diabetes Mellitus Tipo 2/terapia , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/fisiologia , Glucose/administração & dosagem , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética
6.
J. physiol. biochem ; 74(1): 3-8, feb. 2018. graf
Artigo em Inglês | IBECS | ID: ibc-178912

RESUMO

The taste receptor type 1 (TAS1R) family of heterotrimeric G protein-coupled receptors participates in monitoring energy and nutrient status. TAS1R member 3 (TAS1R3) is a bi-functional protein that recognizes amino acids such as L-glycine and L-glutamate or sweet molecules such as sucrose and fructose when dimerized with TAS1R member 1 (TAS1R1) or TAS1R member 2 (TAS1R2), respectively. It was recently reported that deletion of TAS1R3 expression in Tas1R3 mutant mice leads to increased cortical bone mass but the underlying cellular mechanism leading to this phenotype remains unclear. Here, we independently corroborate the increased thickness of cortical bone in femurs of 20-week-old male Tas1R3 mutant mice and confirm that Tas1R3 is expressed in the bone environment. Tas1R3 is expressed in undifferentiated bone marrow stromal cells (BMSCs) in vitro and its expression is maintained during BMP2-induced osteogenic differentiation. However, levels of the bone formation marker procollagen type I N-terminal propeptide (PINP) are unchanged in the serum of 20-week-old Tas1R3 mutant mice as compared to controls. In contrast, levels of the bone resorption marker collagen type I C-telopeptide are reduced greater than 60% in Tas1R3 mutant mice. Consistent with this, Tas1R3 and its putative signaling partner Tas1R2 are expressed in primary osteoclasts and their expression levels positively correlate with differentiation status. Collectively, these findings suggest that high bone mass in Tas1R3 mutant mice is due to uncoupled bone remodeling with reduced osteoclast function and provide rationale for future experiments examining the cell-type-dependent role for TAS1R family members in nutrient sensing in postnatal bone remodeling


No disponible


Assuntos
Animais , Masculino , Reabsorção Óssea/metabolismo , Osso Cortical/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Mesenquimais/metabolismo , Osteoblastos , Osteoclastos/metabolismo , Osteogênese , Receptores Acoplados a Proteínas-G/metabolismo , Biomarcadores/metabolismo , Reabsorção Óssea/imunologia , Reabsorção Óssea/patologia , Catepsina K , Linhagem Celular , Osso Cortical , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes
7.
J. physiol. biochem ; 74(1): 35-45, feb. 2018. tab, graf
Artigo em Inglês | IBECS | ID: ibc-178916

RESUMO

Caffeine has been shown to stimulate multiple major regulators of cell energetics including AMP-activated protein kinase (AMPK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII). Additionally, caffeine induces peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1alfa) and mitochondrial biogenesis. While caffeine enhances oxidative metabolism, experimental concentrations often exceed physiologically attainable concentrations through diet. This work measured the effects of low-level caffeine on cellular metabolism and gene expression in myotubes, as well as the dependence of caffeine's effects on the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPAR Beta/Delta). C2C12 myotubes were treated with various doses of caffeine for up to 24 h. Gene and protein expression were measured via qRT-PCR and Western blot, respectively. Cellular metabolism was determined via oxygen consumption and extracellular acidification rate. Caffeine significantly induced regulators of mitochondrial biogenesis and oxidative metabolism. Mitochondrial staining was suppressed in PPARBeta/Delta -inhibited cells which was rescued by concurrent caffeine treatment. Caffeine-treated cells also displayed elevated peak oxidative metabolism which was partially abolished following PPARβ/δ inhibition. Similar to past observations, glucose uptake and GLUT4 content were elevated in caffeine-treated cells, however, glycolytic metabolism was unaltered following caffeine treatment. Physiological levels of caffeine appear to enhance cell metabolism through mechanisms partially dependent on PPARBeta/Delta


No disponible


Assuntos
Animais , Camundongos , Cafeína/metabolismo , Regulação da Expressão Gênica , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , PPAR beta/agonistas , PPAR delta/agonistas , Benzamidas/farmacologia , Bioensaio , Linhagem Celular , Técnicas de Cocultura , Concentração de Íons de Hidrogênio , Metabolismo dos Lipídeos , Mitocôndrias Musculares , Dinâmica Mitocondrial , Fibras Musculares Esqueléticas , Biogênese de Organelas
8.
J. physiol. biochem ; 73(4): 531-538, nov. 2017. graf, ilus
Artigo em Espanhol | IBECS | ID: ibc-178903

RESUMO

Sarcolipin is a transmembrane protein expressed in the sarco/endoplasmic reticulum of skeletal and atrial muscles in large animals. Sarcolipin plays crucial roles in heat production through modifying the function of sarco/endoplasmic reticulum Ca2+ ATPase, thereby being involved in thermogenesis and systemic metabolism. In skeletal muscle, endoplasmic reticulum (ER) stress has been implicated in several conditions, such as insulin resistance, muscle diseases, and hypo/hyper-contraction. Here, we investigated the effect of ER stress on sarcolipin expression in skeletal muscle cells, C2C12 myotubes. First, gene expression of sarcolipin was confirmed in the cells during myogenesis. Then, ER stress was induced in C2C12 myotubes by treatment with tunicamycin or thapsigargin. Sarcolipin messenger RNA (mRNA) and protein expression were significantly reduced by ER stress induction. The reduction was independent of inositol-requiring element 1 (IRE1), which is activated by ER stress and has potent endonuclease activity, when evaluated by treatment with an IRE1 inhibitor, 4μ8C. On the other hand, sarcolipin mRNA stability was reduced under the ER stress when evaluated by treatment with actinomycin D. In conclusion, these results show that ER stress represses sarcolipin expression due to changes in mRNA stability in C2C12


No disponible


Assuntos
Animais , Camundongos , Estresse do Retículo Endoplasmático , Proteolipídeos/metabolismo , Proteínas Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Linhagem Celular , Expressão Gênica , Proteolipídeos/genética , Proteínas Musculares/genética
9.
J. physiol. biochem ; 73(4): 561-573, nov. 2017. tab, ilus, graf
Artigo em Espanhol | IBECS | ID: ibc-178906

RESUMO

Oxalate, a non-essential end product of metabolism, causes hyperoxaluria and eventually calcium oxalate (CaOx) stone disease. Kidney cells exposed to oxalate stress results in generation of reactive oxygen species (ROS) and progression of stone formation. Perturbations in endoplasmic reticulum (ER) result in accumulation of misfolded proteins and Ca2+ ions homeostasis imbalance and serve as a common pathway for various diseases, including kidney disorders. ER stress induces up-regulation of pro-survival protein glucose-regulated protein 78 (GRP78) and pro-apoptotic signaling protein C/EBP homologous protein (CHOP). Since the association of oxalate toxicity and ER stress on renal cell damage is uncertain, the present study is an attempt to elucidate the interaction of GRP78 with oxalate by computational analysis and study the role of ER stress in oxalate-mediated apoptosis in vitro and in vivo. Molecular docking results showed that GRP78-oxalate/CaOx interaction takes place. Oxalate stress significantly up-regulated expression of ER stress markers GRP78 and CHOP both in vitro and in vivo. Exposure of oxalate increased ROS generation and altered antioxidant enzyme activities. N-Acetyl cysteine treatment significantly ameliorated oxalate-mediated oxidative stress and moderately attenuated ER stress marker expression. The result indicates oxalate toxicity initiated oxidative stress-induced ER stress and also activating ER stress mediated apoptosis directly. In addition, the up-regulation of transforming growth factor Beta-1 revealed oxalate may induce kidney fibrosis through ER stress-mediated mechanisms. The present study provide insights into the pathogenic role of oxidative and ER stress by oxalate exposure in the formation of calcium oxalate stone


No disponible


Assuntos
Animais , Ratos , Apoptose , Estresse do Retículo Endoplasmático , Cálculos Renais/patologia , Oxalatos/toxicidade , Linhagem Celular , Oxalato de Cálcio
10.
Clin. transl. oncol. (Print) ; 19(8): 1010-1017, ago. 2017. tab, `bgraf, ilus
Artigo em Inglês | IBECS | ID: ibc-164679

RESUMO

Introduction/purpose. BRG1 is a key regulator of leukemia stem cells. Indeed, it has been observed that this type of cells is unable to divide, survive and develop new tumors when BRG1 is down-regulated. Materials and methods. We assessed BRG1 and miR-155 expression in 23 leukemia cell lines, and two no pathological lymphocyte samples using qPCR. MiR-155 transfection and western blot were used to analyze the relationship between miR-155 and its validated target, BRG1, by measuring protein expression levels. The effect of miR-155 on cell proliferation and prednisolone sensitivity were studied with resazurin assay. Results. BRG1 expression levels could correlate negatively with miR-155 expression levels, at least in Burkitt’s lymphoma and diffuse large B cell lymphoma (DLBCL) cell lines. To clarify the role of miR-155 in the regulation of BRG1 expression, we administrated miR-155 mimics in different leukemia/lymphoma cell lines. Our results suggest that miR-155 regulate negatively and significantly the BRG1 expression at least in the MOLT4 cell line. Conclusion. Our study revealed a previously unknown miR-155 heterogeneity that could result in differences in the treatment with miRNAs in our attempt to inhibit BRG1. However, the expression levels of BRG1 and miR-155, before prednisolone treatment were not statistically significantly associated prednisolone sensitive leukemia cells (AU)


No disponible


Assuntos
Humanos , Linhagem Celular Tumoral , MicroRNAs/análise , Prednisolona/uso terapêutico , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/genética , Linfócitos/citologia , Linfócitos/patologia , Linfoma/diagnóstico , Leucemia/diagnóstico , Reação em Cadeia da Polimerase/métodos , Linhagem Celular/citologia , Linhagem Celular/patologia , Western Blotting
11.
J. physiol. biochem ; 73(3): 371-380, ago. 2017. tab, graf, ilus
Artigo em Inglês | IBECS | ID: ibc-178888

RESUMO

A series of protective responses could be evoked to achieve compensatory adaptation once cardiomyocytes are subjected to chronic hypoxia. MLK3/JNK/c-jun signaling pathway was previously demonstrated to be involved in this process. In the present study, we aim to further examine the performance of MLK3 in hypoxic H9C2 cells and potential mechanism. Myocardial samples of patients with congenital heart disease (CHD) were collected. H9C2 cells were cultured in hypoxic conditions for various durations. MLK3 was silenced by transfection of shRNA to evaluate its role in cell viability. We found expression of MLK3 protein was lower in patients with cyanotic CHD. In hypoxic H9C2 cells, its expression was gradually decreased in a time-dependent manner. However, there was no significant difference about expression of MLK3 mRNA. According to the results of MTT, LDH, and TUNEL, faster cell growth curve, lower death rate, and less apoptotic cells could be observed in MLK-shRNA group compared with scramble-shRNA group. Silencing of MLK3 significantly reduced expression of cleaved caspase-3, cleaved PARP, Bad, and Bax, together with increased expression of Bcl-2 and ration of Bcl-2/Bax. Both ratio of phospho-JNK/total JNK and ratio of phospho-c-jun/total c-jun were significantly decreased once MLK3 was silenced. At various reoxygenation time, MLK3 shRNA could significantly promote cell survival and decrease cell death according to MTT and LDH. Our results suggested that chronic hypoxia could reduce MLK3 expression in a posttranscriptional regulatory manner. Downregulation of MLK3 protects H9C2 cells from hypoxia-induced apoptosis and H/R injury via blocking the activation of JNK and c-jun


No disponible


Assuntos
Humanos , Animais , Masculino , Feminino , Lactente , Pré-Escolar , Ratos , MAP Quinase Quinase Quinases/genética , Miocárdio/enzimologia , Adaptação Fisiológica , Proteínas Reguladoras de Apoptose/metabolismo , Hipóxia Celular , Linhagem Celular , Sobrevivência Celular , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases , Traumatismo por Reperfusão Miocárdica/enzimologia , Fatores de Proteção
12.
Clin. transl. oncol. (Print) ; 19(7): 907-914, jul. 2017. ilus, graf
Artigo em Inglês | IBECS | ID: ibc-163446

RESUMO

Purpose. Biological effects of low-dose radiation (LDR) are distinguishable from those of high-dose radiation. Adaptive response is an important biological effect following low-dose radiation. Cancer stem cells (CSCs) have self-renewal and multidirectional differentiation potency which results in relapse and metastasis of cancer. In this study, we aimed to examine whether adaptive response could be induced in CSCs by LDR. Methods. Parental cells of three colon cancer cell lines (HRT18, HT29, and HCT116) and CSCs of these three cell lines were irradiated with LDR (i.e., D1) and then high-dose radiation (HDR) of X-rays (i.e., D1 + D2) or only HDR (D2 alone), followed by examination of adaptive response. Results. Adaptive response was not observed either in the three tumor parental cells lines or in three CSCs lines following LDR, due to the lack of resistance to subsequent D2-induced cell growth inhibition. Conclusion. These results suggested that LDR may not induce adaptive response in colon cancer cells or colon CSCs under in vitro conditions. Our study provided experimental and clinical foundations for the application of LDR in the treatment of colon cancers (AU)


No disponible


Assuntos
Humanos , Neoplasias do Colo/radioterapia , Células-Tronco/patologia , Células-Tronco/efeitos da radiação , Linhagem Celular/patologia , Linhagem Celular/efeitos da radiação , Radioterapia/métodos , Imunofluorescência/métodos , Relação Dose-Resposta à Radiação , Colo/citologia , Colo/patologia , Colo/efeitos da radiação
13.
Clin. transl. oncol. (Print) ; 19(6): 718-726, jun. 2017. tab, graf, ilus
Artigo em Inglês | IBECS | ID: ibc-162829

RESUMO

Purpose. Biomarkers, such as mutant RAS, predict resistance to anti-EGFR therapy in only a proportion of patients, and hence, other predictive biomarkers are needed. The aims were to identify candidate genes upregulated in colorectal cancer cell lines resistant to anti-EGFR monoclonal antibody treatment, to knockdown (KD) these genes in the resistant cell lines to determine if sensitivity to anti-EGFR antibody was restored, and finally to perform a pilot correlative study of EGR1 expression and outcomes in a cohort of metastatic colorectal cancer (mCRC) patients given cetuximab therapy. Methods. Comparative expression array analysis of resistant cell lines (SW48, COLO-320DM, and SNU-C1) vs sensitive cell lines (LIM1215, CaCo2, and SW948) was performed. The highest up-regulated gene in each resistant cell line was knocked down (KD) using RNA interference, and effect on proliferation was assessed with and without anti-EGFR treatment. Expression of the candidate genes in patients’ tumours treated with cetuximab was assessed by immunohistochemistry; survival analyses were performed comparing high vs low expression. Results. Genes significantly upregulated in resistant cell lines were EGR1 (early growth response protein 1), HBEGF (heparin-binding epidermal growth factor-like growth factor), and AKT3 (AKT serine/threonine kinase 3). KD of each gene resulted in the respective cells being more sensitive to anti-EGFR treatment, suggesting that the resistant phenotype was reversed. In the pilot study of mCRC patients treated with cetuximab, both median PFS (1.38 months vs 6.79 months; HR 2.77 95% CI 1.2-19.4) and median OS (2.59 months vs 9.82 months; HR 3.0 95% CI 1.3-23.2) were significantly worse for those patients with high EGR1 expression. Conclusion. High EGR1 expression may be a candidate biomarker of resistance to anti-EGFR therapy (AU)


No disponible


Assuntos
Resistencia a Medicamentos Antineoplásicos , Técnicas In Vitro , Cetuximab/uso terapêutico , Metástase Neoplásica/tratamento farmacológico , Expressão Gênica , Biomarcadores , Genes erbB-1 , Proteínas Oncogênicas v-erbB/análise , RNA/análise , Imuno-Histoquímica , Linhagem Celular
14.
Clin. transl. oncol. (Print) ; 19(5): 587-592, mayo 2017. ilus, graf
Artigo em Inglês | IBECS | ID: ibc-162192

RESUMO

Purpose. Cisplatin is commonly used in non-small-cell lung cancer (NSCLC) chemotherapy; however, chemoresistance to cisplatin remains a great clinical challenge. Octamer-binding protein 4 (OCT4) has been reported to be overexpressed in NSCLC. In this study, we aimed to investigate the potential role of OCT4 in NSCLC with chemoresistance to cisplatin. Methods. Expressions of OCT4 was detected in NSCLC tissues and cell lines. We utilized siRNA to knock down OCT4 expression in human NSCLC cells and analyzed their phenotypic changes. Results. We found that the difference of OCT4 expression between NSCLC and the adjacent non-tumourous tissues was statistically significant. Knockdown of OCT4 in NSCLC cells could decrease cell proliferation, and potentiate apoptosis induced by cisplatin, suggesting OCT4 may contribute to cisplatin resistance in NSCLC. Conclusion. Our findings indicate that targeting OCT4 could improve cisplatin effect in NSCLC, confirming their role in modulating cisplatin sensitivity (AU)


No disponible


Assuntos
Humanos , Masculino , Feminino , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cisplatino/análise , Fatores de Transcrição de Octâmero/análise , Resistência a Medicamentos , Linhagem Celular , Linhagem Celular/patologia , Reação em Cadeia da Polimerase
15.
Clin. transl. oncol. (Print) ; 19(5): 599-605, mayo 2017. tab, graf
Artigo em Inglês | IBECS | ID: ibc-162194

RESUMO

Background and aim. Long non-coding RNAs (lncRNAs) have been demonstrated to act as a critical regulator in the processes of tumor biology. In this study, whether lncRNA-ATB is a potential indicator for non-small cell lung cancer (NSCLC) was investigated and its biological function in NSCLC was also determined. Methods. The expression levels of lncRNA-ATB in NSCLC tissues and cell lines were measured. A549 cell line was explored to investigate the functions of lncRNA-ATB in NSCLC. Results. Real-time PCR results showed that lncRNA-ATB expression was up-regulated in both in NSCLC tissues and cell lines. High lncRNA-ATB expression in tumor tissue was associated with larger tumor size, lymph node metastasis, and distant metastasis in patients with NSCLC, respectively. In addition, the patients with high expression of lncRNA-ATB presented a lower survival probability. In vitro experiments showed that down-regulation of lncRNA-ATB promoted the cell apoptosis, whereas inhibited the cell viability, cell migration, and cell invasion. Conclusion. High expression of lncRNA-ATB indicated a poor prognosis and led to the cell proliferation and metastasis in NSCLC (AU)


No disponible


Assuntos
Humanos , Masculino , Feminino , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Metástase Neoplásica/diagnóstico , Proliferação de Células , RNA Longo não Codificante/análise , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Prognóstico , Linhagem Celular , Linhagem Celular/efeitos da radiação , Transfecção/tendências , Metástase Neoplásica/patologia , Carcinoma Pulmonar de Células não Pequenas/complicações , Citometria de Fluxo , Proteína de Suscetibilidade a Apoptose Celular/análise , RNA/genética , Biomarcadores Tumorais/análise
16.
Clin. transl. oncol. (Print) ; 19(5): 633-640, mayo 2017. tab, graf
Artigo em Inglês | IBECS | ID: ibc-162198

RESUMO

Objective. We evaluated miR-451 expression in serum and tissue samples of esophageal squamous cell carcinoma (ESCC) patients. Then, we examined a secretory role of miR-451 in esophageal tumor microenvironment. Methods. miR-451 expression was evaluated in 39 serum samples from esophageal SCC patients compared to 39 normal individuals as well as 26 pairs of fresh-frozen tumor and adjacent normal tissues from patients with ESCC, using qRT-PCR. In a co-culture system of human normal fibroblasts (HFSF-PI3) and esophageal cancer cell line (KYSE-30), we evaluated exosomal miR-451 secretion into the conditioned medium (CM) of both cell lines. Then, we analyzed the effect of primiR-451-transfected fibroblasts on the migration potency of their neighboring KYSE-30 cells. Results. We detected miR-451 over-expression in serum samples of esophageal cancer patients compared to the normal group (P = 0.005). Interestingly, fresh-frozen tumor tissues from the same patients showed miR-451 down-regulation compared to their adjacent normal counterparts (P = 0.043). Co-culturing the KYSE-30 cell line with normal fibroblasts significantly induced miR-451 exosomal secretion into the CM. Moreover, co-culture of KYSE-30 cell line with miR-451-over-expressing fibroblasts significantly induced migration tendency in KYSE-30 cell line compared to the mock-transfected fibroblasts (P < 0.0001). In this system, MIF expression (a validated target of miR-451) in the KYSE-30 cell line was increased although this alteration was not statistically significant (fold change = 4.44). Conclusions. Our data suggest that cancer-associated fibroblasts use exosomal miR-451 as a signaling molecule to provide a favorable niche for tumor cell migration and cancer progression. Our findings provide new insights into the stromal role of miR-451 in the esophageal tumor microenvironment as a communicatory molecule and suggest a signaling role for miR-451 in extracellular matrix cross-talks (AU)


No disponible


Assuntos
Humanos , Masculino , Feminino , Neoplasias Esofágicas/diagnóstico , Progressão da Doença , Exossomos/genética , MicroRNAs/análise , Regulação Neoplásica da Expressão Gênica/genética , Receptores de Fatores de Crescimento de Fibroblastos/análise , Exossomos/patologia , Reação em Cadeia da Polimerase/métodos , Biópsia/métodos , Linhagem Celular/patologia , Curva ROC
17.
J. physiol. biochem ; 73(1): 49-57, feb. 2017. ilus, graf
Artigo em Inglês | IBECS | ID: ibc-168392

RESUMO

Excessive exercise induces an inflammatory response caused by oxidative stress, which delays recovery of damaged muscle fibers. The reduction of inflammatory response is important for skeletal muscle homeostasis. Peroxisome proliferator-activated receptor gamma (PPARγ) is an anti-inflammatory molecule, but the role of PPARγ in skeletal muscle as anti-inflammatory activity is not clear. Thus, this study examined the anti-inflammatory role of PPARγ against H2O2-induced oxidative stress in skeletal muscle. Sprague Dawley (SD) rats were exercised on a treadmill to induce oxidative stress. In vitro oxidative stress was evaluated in differentiated C2C12 cells stimulated using 200 μM H2O2. Inflammation-related molecules were determined by immunohistochemistry and Western blot analysis. Expressions of the inflammatory molecules tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), cyclooxygenase-2 (COX-2), and matrix metalloproteinase-2 (MMP-2) in muscles of the acute exercise group were highly increased. PPARγ was also highly expressed in these muscles. These inflammatory molecules were also markedly increased in C2C12 cells with H2O2 stimulation. However, PPARγ overexpression in C2C12 transfected by Ad/PPARγ dramatically reduced the inflammatory molecules. PPARγ also enhanced the anti-oxidants molecules like Cu/Zn-SOD, Mn-SOD, and hemeoxygenase-1 by reducing the generation of ROS, even in the presence of H2O2. PPARγ displayed dual anti-inflammatory and anti-oxidant roles by inhibiting the mitogen-activated protein kinase (MAPK) pathway and translocation of nuclear transcriptional factor-κB (NF-κB) from the cytosol to the nucleus. These results demonstrate a potential role of PPARγ in protecting muscle fibers against oxidative stress caused by excessive acute exercise due to its anti-inflammatory and anti-oxidant activity exerted by inhibition of the MAPK/NF-κB pathway (AU)


No disponible


Assuntos
Humanos , Animais , Camundongos , PPAR gama/metabolismo , Miosite/metabolismo , Mioblastos Esqueléticos/metabolismo , Músculo Esquelético/metabolismo , NF-kappa B/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases , Transporte Ativo do Núcleo Celular , Linhagem Celular , Ciclo-Oxigenase 2 , Metaloproteinase 2 da Matriz , Mediadores da Inflamação , Estresse Oxidativo , Distribuição Aleatória , Atividade Motora
18.
J. physiol. biochem ; 72(4): 583-592, dic. 2016. graf, ilus
Artigo em Inglês | IBECS | ID: ibc-168366

RESUMO

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Late-stage AMD is characterized by choroidal neovascularization (CNV). miR-93 appears to play a role in regulating vascular endothelial growth factor-A (VEGF-A), a known factor involved in neovascularization. Understanding its biological significance might enable development of therapeutic interventions for diseases like AMD. We aimed to determine the role of miR-93 in AMD using a laser-induced CNV mouse model. CNV was induced by laser photocoagulation in C57BL/6 mice. The CNV mice were transfected with scrambled miR or miR-93 mimic. The treatment effect was assessed by fundus photography and fluorescein angiography and confirmed by choroidal flatmount. The expression of miR-93 and VEGF-A in ocular tissues was analysed by quantitative polymerase chain reaction (qPCR) and Western blot. The overexpression effects of miR-93 were also proved on human microvascular endothelial cells (HMECs). Significantly decreased expression of miR-93 was observed by qPCR analysis in CNV mice compared to untreated mice (p < 0.05). VEGF-A messenger RNA (mRNA) and protein expression were upregulated with CNV; these changes were ameliorated by restoration of miR-93 (p < 0.05). CNV was reduced after miR-93 transfection. Transfection of miR-93 reduced the proliferation of HMECs (p < 0.01), but no significant changes were observed in 2D capillary-like tube formation (p > 0.05) and migration (p > 0.05) compared with that in the untreated cells. miR-93 has been shown to be a negative modulator of angiogenesis in the eye. All together, these results highlight the therapeutic potential of miR-93 and suggest that it may contribute as a putative therapeutic target for AMD in humans (AU)


No disponible


Assuntos
Humanos , Animais , Camundongos , Degeneração Macular/genética , MicroRNAs/genética , RNA Mensageiro/genética , Transdução de Sinais , Transfecção , Modelos Animais de Doenças , Células Endoteliais , Regulação da Expressão Gênica , Linhagem Celular , Movimento Celular , Fotocoagulação/efeitos adversos , Angiofluoresceinografia
19.
J. physiol. biochem ; 72(4): 689-697, dic. 2016. tab, graf
Artigo em Inglês | IBECS | ID: ibc-168376

RESUMO

The balance of ATP production and consumption is reflected in adenosine monophosphate (AMP) and nicotinamide adenine dinucleotide (NAD) content and has been associated with phenotypic plasticity in striated muscle. Some studies have suggested that AMPK-dependent plasticity may be an indirect consequence of increased NAD synthesis and SIRT1 activity. The primary goal of this study was to assess the interaction of AMP- and NAD-dependent signaling in adaptation of C2C12 myotubes. Changes in myotube developmental and metabolic gene expression were compared following incubation with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and nicotinamide mononucleotide (NMN) to activate AMPK- and NAD-related signaling. AICAR showed no effect on NAD pool or nampt expression but significantly reduced histone H3 acetylation and GLUT1, cytochrome C oxidase subunit 2 (COX2), and MYH3 expression. In contrast, NMN supplementation for 24 h increased NAD pool by 45 % but did not reduce histone H3 acetylation nor promote mitochondrial gene expression. The combination of AMP and NAD signaling did not induce further metabolic adaptation, but NMN ameliorated AICAR-induced myotube reduction. We interpret these results as indication that AMP and NAD contribute to C2C12 differentiation and metabolic adaptation independently (AU)


No disponible


Assuntos
Animais , Camundongos , Adaptação Fisiológica , Transdução de Sinais , NAD/metabolismo , Mioblastos/metabolismo , Monofosfato de Adenosina/metabolismo , Acetilação , Linhagem Celular , Diferenciação Celular , Aminoimidazol Carboxamida , Complexo IV da Cadeia de Transporte de Elétrons , Cadeias Pesadas de Miosina , Transportador de Glucose Tipo 1 , Mononucleotídeo de Nicotinamida/farmacologia , Ribonucleotídeos/farmacologia
20.
J. physiol. biochem ; 72(4): 699-710, dic. 2016. graf
Artigo em Inglês | IBECS | ID: ibc-168377

RESUMO

Omega-3 fatty acids have multiple effects in peripheral tissues and pancreatic beta cell function. Dietary depletion of omega-3 fatty acids is associated with pancreatic islet dysfunction and insulin resistance in rats. Herein, the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on pancreatic beta cell redox state and function were investigated. INS-1E insulin-secreting cells were incubated with EPA and DHA in combination with palmitic acid, and productions of reactive oxygen species (ROS), nitric oxide (NO) and insulin were measured. The involvement of the NADPH oxidase complex in ROS production and expression of the antioxidant enzymes was also investigated. After incubation for 1 or 48 h, productions of superoxide (by hydroethidine method), nitric oxide (by 4,5-diaminofluorescein diacetate-DAF-2DA assay), insulin (by radioimmunoassay), and expressions (by western blot analysis) of glutathione peroxidase (GPx-1) and gp91PHOX were measured. EPA and DHA reduced superoxide production after 1-h incubation. After 48 h, palmitic acid reduced superoxide production that was normalized by EPA treatment. Palmitic acid increased NO production that was reverted by EPA and DHA. Palmitic acid increased insulin secretion after 48 h, whereas both omega-3 fatty acids increased intracellular insulin content. EPA and DHA enhanced GPx-1 expression as well as gp91PHOX glycosylated form. In conclusion, EPA and DHA increased intracellular insulin content and antioxidant enzymatic defense capacity and decreased pro-oxidant generating activities that are associated with maintenance of pancreatic beta cell redox state in response to palmitic acid (AU)


No disponible


Assuntos
Animais , Ratos , Superóxidos/metabolismo , Óxido Nítrico/metabolismo , Insulina/biossíntese , Células Secretoras de Insulina , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Transdução de Sinais , Linhagem Celular , NADPH Oxidases , Regulação da Expressão Gênica , Ácido Palmítico , Glutationa Peroxidase
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