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1.
Anal Biochem ; 691: 115556, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38705226

RESUMO

we developed an effective protein precipitation method for determination of levamlodipine in human plasma using LC-MS/MS. Sample extraction was carried out by using liquid-liquid extraction in 96-well plate format. (S)-Amlodipine-d4 was used as internal standard (IS). The chromatographic separation was achieved using Philomen Chiral MX (2) column (3 µm, 2.1 × 100 mm). Mobile phase A was comprised of Acetonitrile (ACN), Mono ethanol amine (MEA) and Iso-Propyl alcohol (IPA) (1000:1:10, v/v/v), Mobile phase B was IPA-ACN (2:1, v/v). The flow rate was 0.4 mL/min. The total run time of each sample was 4.0 min with gradient elution. LC-MS/MS spectra were generated in positive ion mode, and multiple reaction monitoring (MRM) was used to detect the following transitions: m/z 409.20 â†’ 238.15 for levamlodipine and 415.25 â†’ 240.20 for (S)-Amlodipine-d4 (the IS). The method was linear from 50 to 10000 pg/mL(R2=0.9988489),and the lower limit of quantification (LLOQ) was 50 pg/mL. This method was applied to a bioequivalence study of levamlodipine.


Assuntos
Niacina/análogos & derivados , Espectrometria de Massas em Tandem , Humanos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Di-Hidropiridinas/sangue , Di-Hidropiridinas/farmacocinética , Di-Hidropiridinas/química , Extração Líquido-Líquido , Limite de Detecção , Anlodipino/sangue , Anlodipino/farmacocinética , Espectrometria de Massa com Cromatografia Líquida
2.
Artigo em Inglês | MEDLINE | ID: mdl-35855826

RESUMO

Objectives: Angelica sinensis polysaccharide (ASP) is a traditional herbal medicine accompanied by antitumor potential. This study aims to explore the therapeutic potential of ASP on glioma, as well as the underlying mechanisms involving microRNA-373-3p (miR-373-3p) and the TGF-ß/Smad4 signaling pathway. Methods: U251 cells (a human glioma cell line) were treated with different concentrations of ASP. miR-373-3p was silenced in U251 cells by the transfection of the miR-373-3p inhibitor. Cell viability and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. Cell migration and invasion were detected by wound healing and transwell assays, respectively. The miR-373-3p expression was measured by RT-qPCR. The protein expressions of TGF-ß and Smad4 were evaluated by both western blotting and immunofluorescence. Results: ASP inhibited the viability, migration, and invasion, and enhanced the apoptosis of U251 cells in a dose-dependent manner. ASP increased miR-373-3p expression and decreased TGF-ß and Smad4 expressions in U251 cells. Silencing of miR-373-3p weakened the effects of ASP on inhibiting cell viability, migration, and invasion, as well as promoting cell apoptosis. In addition, deleting miR-373-3p weakened the inhibiting effects of ASP on the TGF-ß/Smad4 pathway in U251 cells. Conclusions: ASP suppresses the malignant progression of glioma via regulating the miR-373-3p-mediated TGF-ß/Smad4 pathway.

3.
Plant Physiol ; 189(4): 2500-2516, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35522026

RESUMO

MicroRNAs play key roles in abiotic stress response. Rice (Oryza sativa L.) miR1320 is a species-specific miRNA that contributes to miR168-regulated immunity. However, it is still unknown whether miR1320 is involved in rice response to abiotic stress. In this study, we illustrated that the miR1320 precursor generated two mature miR1320s, miR1320-3p, and miR1320-5p, and they both displayed decreased expression under cold stress. Genetic evidence showed that miR1320 overexpression resulted in increased cold tolerance, while miR1320 knock down (KD) reduced cold tolerance. Furthermore, an APETALA2/ethylene-responsive factor (ERF) transcription factor OsERF096 was identified as a target of miR1320 via 5'-RACE and dual luciferase assays. OsERF096 expression was altered by miR1320 overexpression and KD and exhibited an opposite pattern to that of miR1320 in different tissues and under cold stress. Consistently, OsERF096 negatively regulated cold stress tolerance. Furthermore, we suggested that OsERF096 could bind to the GCC and DRE cis-elements and act as a transcriptional activator in the nucleus. Based on RNA-sequencing and targeted metabolomics assays, we found that OsERF096 modified hormone content and signaling pathways. Finally, phenotypic and reverse transcription-quantitative PCR assays showed that jasmonic acid (JA) methyl ester application recovered the cold-sensitive phenotype and JA-activated expression of three Dehydration Responsive Element Binding genes in the OsERF096-OE line. Taken together, our results strongly suggest that the miR1320-OsERF096 module regulates cold tolerance by repressing the JA-mediated cold signaling pathway.


Assuntos
Oryza , Fatores de Transcrição , Temperatura Baixa , Resposta ao Choque Frio/genética , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Transdução de Sinais/genética , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
J Cancer ; 13(2): 623-640, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35069908

RESUMO

SET7/9 is a member of the protein lysine methyltransferase family that methylates both histone 3 lysine 4 (H3-K4) and lysine(s) of other non-histone proteins. In recent years, dis-regulation of SET7/9 were frequently detected in various cancer types and SET7/9-mediated methylation has been recognized as an important mechanism that affects cancer initiation and development through regulation of a series of cellular processes. Here we review the currently identified histone and non-histone protein targets of SET7/9 that are closely correlated with human cancer and the function of SET7/9 in regulating the expression and stability of its protein targets. The review also discusses the putative role of SET7/9 as an oncogene or tumor suppressor in the development of various cancer types and the underlying mechanisms, which may help better evaluate the potential of SET7/9 as a novel candidate for cancer therapy.

5.
Bull Environ Contam Toxicol ; 108(5): 824-829, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-34596732

RESUMO

The effects of acute mercury exposure (118 µg/L) on the marine copepod Tigriopus japonicus were examined at 22 and 25 °C for 24 h and compared with controls. Mercury accumulation and seven genes related to antioxidant/stress responses were analyzed after exposure. The 24-h LC50 value decreased in the warmer environment and mercury accumulation was elevated. Under both temperatures, mercury significantly affected the expression of all analyzed genes and probably caused oxidative stress. Intriguingly, at the same mercury concentration, most genes were upregulated at the higher relative to the lower temperature, and the copepods likely initiated more compensatory reactions to counteract increased mercury toxicity associated with the warmer temperature. Overall, this study suggests a molecular mechanism by which marine copepods could respond to future oceanic warming and mercury pollution.


Assuntos
Copépodes , Mercúrio , Poluentes Químicos da Água , Animais , Mercúrio/metabolismo , Mercúrio/toxicidade , Oceanos e Mares , Estresse Oxidativo , Poluentes Químicos da Água/análise
6.
Sci Rep ; 11(1): 2118, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33483531

RESUMO

Lung fibrosis, or the scarring of the lung, is a devastating disease with huge unmet medical need. There are limited treatment options and its prognosis is worse than most types of cancer. We previously discovered that MK-0429 is an equipotent pan-inhibitor of αv integrins that reduces proteinuria and kidney fibrosis in a preclinical model. In the present study, we further demonstrated that MK-0429 significantly inhibits fibrosis progression in a bleomycin-induced lung injury model. In search of newer integrin inhibitors for fibrosis, we characterized monoclonal antibodies discovered using Adimab's yeast display platform. We identified several potent neutralizing integrin antibodies with unique human and mouse cross-reactivity. Among these, Ab-31 blocked the binding of multiple αv integrins to their ligands with IC50s comparable to those of MK-0429. Furthermore, both MK-0429 and Ab-31 suppressed integrin-mediated cell adhesion and latent TGFß activation. In IPF patient lung fibroblasts, TGFß treatment induced profound αSMA expression in phenotypic imaging assays and Ab-31 demonstrated potent in vitro activity at inhibiting αSMA expression, suggesting that the integrin antibody is able to modulate TGFß action though mechanisms beyond the inhibition of latent TGFß activation. Together, our results highlight the potential to develop newer integrin therapeutics for the treatment of fibrotic lung diseases.


Assuntos
Anticorpos/metabolismo , Fibroblastos/metabolismo , Integrina alfaV/metabolismo , Fibrose Pulmonar/metabolismo , Animais , Anticorpos/imunologia , Bleomicina , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Fibroblastos/citologia , Humanos , Integrina alfaV/imunologia , Masculino , Camundongos Endogâmicos C57BL , Naftiridinas/farmacologia , Propionatos/farmacologia , Ligação Proteica , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/prevenção & controle
7.
Breast Cancer Res Treat ; 186(1): 79-87, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33398478

RESUMO

PURPOSE: N-myristoyltransferases 1 and 2 (NMT1 and NMT2) catalyze the addition of 14-carbon fatty acids to the N-terminus of proteins. Myristoylation regulates numerous membrane-bound signal transduction pathways important in cancer biology and the pan-NMT inhibitor PCLX-001 is approaching clinical development as a cancer therapy. The tissue distribution, relative abundances, and prognostic value of the two human NMTs remain poorly understood. METHODS: We generated and validated mutually exclusive monoclonal antibodies (mAbs) specific to human NMT1 and NMT2. These mAbs were used to perform immunohistochemical analysis of the abundance and distribution of NMT1 and NMT2 in normal breast epithelial samples and a large cohort of primary breast adenocarcinomas from the BCIRG001 clinical trial (n = 706). RESULTS: NMT1 protein was readily quantified in normal and most transformed breast epithelial tissue and was associated with higher overall histologic grade, higher Ki67, and lower hormone receptor expression. While NMT2 protein was readily detected in normal breast epithelial tissue, it was undetectable in the majority of breast cancers. Detectable NMT2 protein correlated with significantly poorer overall survival (hazard ratio 1.36; P = 0.029) and worse biological features including younger age, higher histologic grade, lower hormone receptor expression, higher Ki67, and p53 positivity. Treatment of cultured breast cancer cells with PCLX-001 reduced cell viability in vitro. Daily oral administration of PCLX-001 to immunodeficient mice bearing human MDA-MB-231 breast cancer xenografts produced significant dose-dependent tumor growth inhibition in vivo. CONCLUSIONS: These results support further evaluation of NMT immunohistochemistry for patient selection and clinical trials of NMT inhibition in breast cancer patients.


Assuntos
Neoplasias da Mama , Preparações Farmacêuticas , Aciltransferases/genética , Animais , Neoplasias da Mama/tratamento farmacológico , Feminino , Humanos , Camundongos , Prognóstico
8.
Nat Commun ; 11(1): 5348, 2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-33093447

RESUMO

Myristoylation, the N-terminal modification of proteins with the fatty acid myristate, is critical for membrane targeting and cell signaling. Because cancer cells often have increased N-myristoyltransferase (NMT) expression, NMTs were proposed as anti-cancer targets. To systematically investigate this, we performed robotic cancer cell line screens and discovered a marked sensitivity of hematological cancer cell lines, including B-cell lymphomas, to the potent pan-NMT inhibitor PCLX-001. PCLX-001 treatment impacts the global myristoylation of lymphoma cell proteins and inhibits early B-cell receptor (BCR) signaling events critical for survival. In addition to abrogating myristoylation of Src family kinases, PCLX-001 also promotes their degradation and, unexpectedly, that of numerous non-myristoylated BCR effectors including c-Myc, NFκB and P-ERK, leading to cancer cell death in vitro and in xenograft models. Because some treated lymphoma patients experience relapse and die, targeting B-cell lymphomas with a NMT inhibitor potentially provides an additional much needed treatment option for lymphoma.


Assuntos
Aciltransferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Linfoma de Células B/tratamento farmacológico , Ácido Mirístico/metabolismo , Adenina/análogos & derivados , Aminopiridinas/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dasatinibe/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Camundongos , Camundongos SCID , Modelos Biológicos , Piperidinas , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/metabolismo
9.
BMJ Open ; 9(9): e030502, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31530611

RESUMO

INTRODUCTION: Neoadjuvant chemotherapy for breast cancer treatment is prescribed to facilitate surgery and provide confirmation of drug-sensitive disease, and the achievement of pathological complete response (pCR) predicts improved long-term outcomes. Docosahexaenoic acid (DHA) has been shown to reduce tumour growth in preclinical models when combined with chemotherapy and is known to beneficially modulate systemic immune function. The purpose of this trial is to investigate the benefit of DHA supplementation in combination with neoadjuvant chemotherapy in patients with breast cancer. METHODS AND ANALYSIS: This is a double-blind, phase II, randomised controlled trial of 52 women prescribed neoadjuvant chemotherapy to test if DHA supplementation enhances chemotherapy efficacy. The DHA supplementation group will take 4.4 g/day DHA orally, and the placebo group will take an equal fat supplement of vegetable oil. The primary outcome will be change in Ki67 labelling index from prechemotherapy core needle biopsy to definitive surgical specimen. The secondary endpoints include assessment of (1) DHA plasma phospholipid content; (2) systemic immune cell types, plasma cytokines and inflammatory markers; (3) tumour markers for apoptosis and tumour infiltrating lymphocytes; (4) rate of pCR in breast and in axillary nodes; (5) frequency of grade 3 and 4 chemotherapy-associated toxicities; and (6) patient-perceived quality of life. The trial has 81% power to detect a significant between-group difference in Ki67 index with a two-sided t-test of less than 0.0497, and accounts for 10% dropout rate. ETHICS AND DISSEMINATION: This study has full approval from the Health Research Ethics Board of Alberta - Cancer Committee (Protocol #: HREBA.CC-18-0381). We expect to present the findings of this study to the scientific community in peer-reviewed journals and at conferences. The results of this study will provide evidence for supplementing with DHA during neoadjuvant chemotherapy treatment for breast cancer. TRIAL REGISTRATION NUMBER: NCT03831178.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Ácidos Docosa-Hexaenoicos/administração & dosagem , Terapia Neoadjuvante/métodos , Alberta , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Biomarcadores Tumorais/análise , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Ensaios Clínicos Fase II como Assunto , Citocinas/sangue , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/sangue , Método Duplo-Cego , Feminino , Humanos , Antígeno Ki-67/metabolismo , Linfonodos/patologia , Qualidade de Vida , Ensaios Clínicos Controlados Aleatórios como Assunto , Resultado do Tratamento
10.
In Vitro Cell Dev Biol Anim ; 54(8): 610-619, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30105734

RESUMO

The mechanisms for cardiomyocyte death in cardiovascular diseases are incompletely understood. The aim of this study is to reveal the function of miR-381 in myocardium infarction (MI)-induced cardiomyocyte apoptosis. We established mouse model of MI and cellular models of apoptosis induced by oxidative stress (H2O2 and hypoxia/reoxygenation (H/R)). The expression of miR-381 in these models was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR); we employed approaches including cell counting kit-8 (CCK-8) assay and flow cytometry to evaluate the cell viability and apoptosis. Notch signaling was determined by western blot analysis of key signaling components including Notch1 intracellular domain (ICD), Jag1, and Hes1. The predicted binding of miR-381 to Jag1 3' untranslated region (UTR) was validated by luciferase assay. Following MI, miR-381 expression was upregulated time dependently in the border zone of ischemic area but not in the non-ischemic area. MiR-381 expression was also upregulated in cardiomyocytes treated with H2O2 and H/R. Overexpression of miR-381 exacerbated H2O2- and H/R-induced apoptosis of cardiomyocytes; in contrast, inhibition of miR-381 attenuated apoptosis in these conditions. Importantly, in vivo delivery of miR-381 antagomir significantly reduced infarction size. Moreover, miR-381 negatively regulates the cardioprotective Notch signaling in vivo and in vitro, which might be an effect of targeted inhibition of Jag1 by itself. These data indicate an essential role of miR-381/Jag1 pathway in regulating Notch signaling-mediated cardioprotective effect in cardiomyocytes. Our study also provides a potential therapeutic target for cardiovascular diseases.


Assuntos
MicroRNAs/metabolismo , Miócitos Cardíacos/citologia , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Antagomirs , Apoptose/efeitos dos fármacos , Sequência de Bases , Cardiotônicos/metabolismo , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Proteína Jagged-1/metabolismo , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Oxigênio , Regulação para Cima/efeitos dos fármacos
11.
Appl Immunohistochem Mol Morphol ; 26(1): 54-63, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27093450

RESUMO

A new automated MYC IHC classifier based on bivariate logistic regression is presented. The predictor relies on image analysis developed with the open-source ImageJ platform. From a histologic section immunostained for MYC protein, 2 dimensionless quantitative variables are extracted: (a) relative distance between nuclei positive for MYC IHC based on euclidean minimum spanning tree graph and (b) coefficient of variation of the MYC IHC stain intensity among MYC IHC-positive nuclei. Distance between positive nuclei is suggested to inversely correlate MYC gene rearrangement status, whereas coefficient of variation is suggested to inversely correlate physiological regulation of MYC protein expression. The bivariate classifier was compared with 2 other MYC IHC classifiers (based on percentage of MYC IHC positive nuclei), all tested on 113 lymphomas including mostly diffuse large B-cell lymphomas with known MYC fluorescent in situ hybridization (FISH) status. The bivariate classifier strongly outperformed the "percentage of MYC IHC-positive nuclei" methods to predict MYC+ FISH status with 100% sensitivity (95% confidence interval, 94-100) associated with 80% specificity. The test is rapidly performed and might at a minimum provide primary IHC screening for MYC gene rearrangement status in diffuse large B-cell lymphomas. Furthermore, as this bivariate classifier actually predicts "permanent overexpressed MYC protein status," it might identify nontranslocation-related chromosomal anomalies missed by FISH.


Assuntos
Diagnóstico por Computador/métodos , Diagnóstico por Computador/normas , Genes myc/genética , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/genética , Translocação Genética , Técnicas e Procedimentos Diagnósticos , Previsões , Humanos , Imuno-Histoquímica
12.
J Cancer ; 8(11): 2069-2078, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28819408

RESUMO

SET7/9 is a protein lysine methyltransferases (PLMTs or PKMTs) which methylates both histone H3K4 and non-histone proteins including transcriptional factors, tumor suppressors, and membrane-associated receptors. Methylation of these proteins alters protein activity and leads to changes in cellular behavior and a series of biological processes. This study aims to investigate the role of SET7/9 in human acute myeloid leukemia (AML) and non-small-cell lung cancer (NSCLC). We examined the expression of SET7/9 in AML cells and NSCLC cells and detected the methylation status of the SET7/9 promoter region. To evaluate the effect of SET7/9 expression changes on cell apoptosis, cell apoptosis rates were determined after SET7/9 overexpression or down-regulation. Our results showed that SET7/9 induces apoptosis of AML cells and inhibits apoptosis of NSCLC cells, suggesting differential effects of SET7/9 on cellular apoptosis and carcinogenesis depending on different cancer types and genetic contexts. Furthermore, we also demonstrated that SET7/9 suppresses cell apoptosis via modulation of E2F1 under circumstance of p53 deficiency in NSCLC cells.

13.
Appl Immunohistochem Mol Morphol ; 25(10): 687-695, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27093453

RESUMO

There is a pressing need for an objective decision tool to guide therapy for breast cancer patients that are estrogen receptor positive and HER2/neu negative. This subset of patients contains a mixture of luminal A and B tumors with good and bad outcomes, respectively. The 2 main current tools are on the basis of immunohistochemistry (IHC) or gene expression, both of which rely on the expression of distinct molecular groups that reflect hormone receptors, HER2/neu status, and most importantly, proliferation. Despite the success of a proprietary molecular test, definitive superiority of any method has not yet been demonstrated. Ki67 IHC scoring assessments have been shown to be poorly reproducible, whereas molecular testing is costly with a longer turnaround time. This work proposes an objective Ki67 index using image analysis that addresses the existing methodological issues of Ki67 quantitation using IHC on paraffin-embedded tissue. Intrinsic bias related to numerical assessment performed on IHC is discussed as well as the sampling issue related to the "peel effect" of tiny objects within a thin section. A new nonbiased stereological parameter (VV) based on the Cavalieri method is suggested for use on a double-stained Ki67/cytokeratin IHC slide. The assessment is performed with open-source ImageJ software with interobserver concordance between 3 pathologists being high at 93.5%. Furthermore, VV was found to be a superior method to predict an outcome in a small subset of breast cancer patients when compared with other image analysis methods being used to determine the Ki67 labeling index. Calibration methodology is also discussed to further this IHC approach.


Assuntos
Bioensaio/métodos , Neoplasias da Mama/diagnóstico , Receptores ErbB/metabolismo , Antígeno Ki-67/metabolismo , Marcadores de Afinidade/química , Neoplasias da Mama/patologia , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Avaliação de Resultados da Assistência ao Paciente , Receptores de Estrogênio/metabolismo , Reprodutibilidade dos Testes
14.
Clin Chim Acta ; 452: 124-8, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26569346

RESUMO

BACKGROUND: Caspase-cleaved Cytokeratin-18 (CCCK-18) is released during apoptosis. Serum CCCK-18 concentrations are associated with prognosis of some critical illness. We investigated the potential relationships between serum CCCK-18 concentrations and disease severity and long-term clinical outcomes after intracerebral hemorrhage. METHODS: Serum CCCK-18 concentrations were determined in a total of 102 patients and 102 controls. Multivariate models were used to predict high concentration of CCCK-18 and 6-month clinical outcomes. The predictive values were evaluated based on areas under receiver operating curve. RESULTS: Compared with controls, serum CCCK-18 concentrations were increased in patients (245.8±108.3U/l vs. 23.6±18.1U/l, P<0.001). National Institute of Health Stroke Scale scores [odds ratio (OR), 1.164; 95% confidence interval (CI), 1.027-1.320; P=0.003] and hematoma volumes (OR, 1.079; 95% CI, 1.018-1.205; P=0.008) were independent predictors of high concentration of CCCK-18. CCCK-18 was identified as an independent predictor of 6-month mortality (OR, 1.019; 95% CI, 1.010-1.038; P=0.013) and 6-month unfavorable outcome (OR, 1.017; 95% CI, 1.008-1.029; P=0.032) and possessed high predictive values. CONCLUSION: Increased serum CCCK-18 concentrations are associated with disease severity and clinical outcomes, suggesting that CCCK represent a novel prognostic predictive biomarker after intracerebral hemorrhage.


Assuntos
Caspases/metabolismo , Hemorragia Cerebral/sangue , Hemorragia Cerebral/diagnóstico , Queratina-18/sangue , Queratina-18/metabolismo , Doença Aguda , Idoso , Análise Química do Sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico
15.
J Trauma Acute Care Surg ; 79(6): 1009-13; discussion 1014, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26317815

RESUMO

BACKGROUND: The initiation of coagulation in trauma is thought to originate from exposed tissue factor (TF); recent data have led to the alternative hypothesis that damage-associated molecular patterns may contribute to postinjury coagulation. In acute traumatic coagulopathy, aberrant coagulation is mediated via the activated protein C (aPC) pathway; the upstream regulators of this process and its relation to TF remain uncharacterized. To examine the role of the TF pathway in mediating acute traumatic coagulopathy, we used specific antibody blockades in an established murine model of traumatic hemorrhagic shock, hypothesizing that both coagulation activation after injury and aPC-mediated coagulopathy are driven by TF via thrombin. METHODS: Mice underwent an established model of trauma and hemorrhage and were subjected to either sham (vascular cannulation) or trauma-hemorrhage (cannulation, laparotomy, shock to mean arterial pressure of 35 mm Hg); they were monitored for 60 minutes before sacrifice. Mice in each group were pretreated with either targeted anti-TF antibody to block the TF pathway or hirudin for specific blockade of thrombin. Plasma was assayed for thrombin-antithrombin (TAT) and aPC by enzyme-linked immunosorbent assay. RESULTS: Compared with controls, trauma-hemorrhage mice treated with anti-TF antibody had significantly reduced levels of TAT (2.3 ng/mL vs. 5.7 ng/mL, p = 0.016) and corresponding decreases in aPC (16.3 ng/mL vs. 31.6 ng/mL, p = 0.034), with reductions to levels seen in sham mice. Direct inhibition of thrombin yielded similar results, with reduction in aPC to levels below those seen in sham mice. CONCLUSION: In this study, blockade of the TF pathway led to the attenuation of both thrombin production and aPC activation observed in traumatic shock. Specific thrombin inhibition achieved similar results, indicating that aPC-related coagulopathy is mediated via thrombin activated by the TF pathway. The near-complete blockade of TAT and aPC observed in this model argues for a dominant role of the TF-thrombin pathway in both coagulation activation after injury and traumatic coagulopathy.


Assuntos
Transtornos da Coagulação Sanguínea/metabolismo , Choque Hemorrágico/sangue , Choque Traumático/sangue , Tromboplastina/metabolismo , Animais , Transtornos da Coagulação Sanguínea/etiologia , Ensaio de Imunoadsorção Enzimática , Hirudinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína C/metabolismo , Trombina/metabolismo , Ferimentos e Lesões/complicações
16.
FASEB J ; 29(9): 3990-4000, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26071407

RESUMO

Compared to normal tissues, many cancer cells overexpress autotaxin (ATX). This secreted enzyme produces extracellular lysophosphatidate, which signals through 6 GPCRs to drive cancer progression. Our previous work showed that ATX inhibition decreases 4T1 breast tumor growth in BALB/c mice by 60% for about 11 d. However, 4T1 cells do not produce significant ATX. Instead, the ATX is produced by adjacent mammary adipose tissue. We investigated the molecular basis of this interaction in human and mouse breast tumors. Inflammatory mediators secreted by breast cancer cells increased ATX production in adipose tissue. The increased lysophosphatidate signaling further increased inflammatory mediator production in adipose tissue and tumors. Blocking ATX activity in mice bearing 4T1 tumors with 10 mg/kg/d ONO-8430506 (a competitive ATX inhibitor, IC90 = 100 nM; Ono Pharma Co., Ltd., Osaka, Japan) broke this vicious inflammatory cycle by decreasing 20 inflammatory mediators by 1.5-8-fold in cancer-inflamed adipose tissue. There was no significant decrease in inflammatory mediator levels in fat pads that did not bear tumors. ONO-8430506 also decreased plasma TNF-α and G-CSF cytokine levels by >70% and leukocyte infiltration in breast tumors and adjacent adipose tissue by >50%. Hence, blocking tumor-driven inflammation by ATX inhibition is effective in decreasing tumor growth in breast cancers where the cancer cells express negligible ATX.


Assuntos
Tecido Adiposo/enzimologia , Neoplasias da Mama/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glândulas Mamárias Animais/enzimologia , Glândulas Mamárias Humanas/enzimologia , Neoplasias Mamárias Experimentais/enzimologia , Proteínas de Neoplasias/biossíntese , Diester Fosfórico Hidrolases/biossíntese , Tecido Adiposo/patologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Humanos , Glândulas Mamárias Animais/patologia , Glândulas Mamárias Humanas/patologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/genética , Diester Fosfórico Hidrolases/genética
17.
Hepatobiliary Pancreat Dis Int ; 14(1): 90-5, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25655296

RESUMO

BACKGROUND: Pancreaticobiliary maljunction is a high risk factor of pancreatitis and biliary tract cancer. How this maljunction affects the liver remains obscure. This study aimed to examine the effects of pancreaticobiliary maljunction on the liver, pancreas and gallbladder in a cat model. METHODS: A model of choledocho-pancreatic side-to-side ductal anastomosis was created in ten cats. Before the procedure, a small piece of tissue from the liver, pancreas and gallbladder was collected as a control. The common channel formation was checked by cholecystography. The livers, pancreases and gallbladders of these cats were harvested for histological examination. The expression of proliferating cell nuclear antigen in the gallbladder was examined with immunohistochemistry. RESULTS: Seven of the 10 cats survived for 6 months after surgery. The color of the liver was darker in the PBM model than the control specimen, with nodules on the surface. Histological examination showed ballooning changes and inflammatory infiltrations and the histopathological score increased significantly (P<0.05). Also, mitochondria swelling and lipid droplet in cytoplasm were observed under an electron microscope. The pancreas also appeared darker in the PBM model than the control specimen and dilated pancreatic ducts were found in three cats. Histopathological examination revealed vascular proliferation and inflammatory infiltration with numerous neutrophils. Gallbladder epithelial cells were featured by expanded endoplasmic reticulum, increased intercellular space and cellular nucleus deformation. The positive cells of proliferating cell nuclear antigen were increased significantly (P<0.05). CONCLUSION: The present study demonstrated that pancreaticobiliary maljunction can lead to the injuries of the liver, pancreas and gallbladder.


Assuntos
Anormalidades do Sistema Digestório/patologia , Vesícula Biliar/patologia , Fígado/patologia , Pâncreas/patologia , Animais , Biomarcadores/metabolismo , Gatos , Proliferação de Células , Anormalidades do Sistema Digestório/metabolismo , Modelos Animais de Doenças , Retículo Endoplasmático/patologia , Células Epiteliais/patologia , Vesícula Biliar/metabolismo , Vesícula Biliar/cirurgia , Vesícula Biliar/ultraestrutura , Fígado/cirurgia , Fígado/ultraestrutura , Mitocôndrias Hepáticas/patologia , Dilatação Mitocondrial , Infiltração de Neutrófilos , Pâncreas/cirurgia , Pâncreas/ultraestrutura , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Tempo
18.
Oncol Rep ; 33(4): 1707-16, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25682742

RESUMO

MicroRNA-182 (miR-182) is significantly downregulated in human gastric tissue samples. Overexpression of miR-182 suppresses the proliferation and colony formation of gastric cancer cells. However, new aspects of the mechanism are still emerging in gastric cancer. ANUBL1, also known as ZFAND4 (zinc finger, AN1-type domain 4), its roles are scarely reported in cancer. In this study, we not only showed that ANUBL1 as an oncogene was upregulated and could promote proliferation of SGC-7901 cells, but also demonstrated that its over-expression led to a strong decrease of miR-182 expression and expression of ANUBL1 was in turn directly downregulated by miR-182, thereby establishing a negative feedback loop between miR-182 and ANUBL1. The elucidation of the mechanisms of miR-182 targeting ANUBL1 in gastric cancer helps us to further understand the mechanism of gastric cancer initiation and progression.


Assuntos
Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/fisiologia , Proteínas de Neoplasias/fisiologia , RNA Neoplásico/fisiologia , Neoplasias Gástricas/genética , Regiões 3' não Traduzidas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Ciclo Celular , Replicação do DNA , Retroalimentação Fisiológica , Feminino , Genes Reporter , Humanos , Masculino , MicroRNAs/biossíntese , MicroRNAs/genética , Pessoa de Meia-Idade , Gradação de Tumores , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA/genética , Interferência de RNA , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Transfecção , Ensaio Tumoral de Célula-Tronco
19.
J Stroke Cerebrovasc Dis ; 23(6): 1396-402, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24774438

RESUMO

BACKGROUND: Peroxisome proliferator-activated receptor (PPAR)-ß/δ is a transcription factor that belongs to the nuclear hormone receptor family. There is little information about the effects of the immediate administration of specific ligands of PPAR-ß/δ (GW0742) in animal models of acute ischemic stroke. Using a rat model of middle cerebral ischemia occlusion (MCAO) in vivo, we have investigated the effect of pretreatment with GW0742 before MCAO. METHODS: The neuroprotective effect of GW0742 against acute ischemic stroke was evaluated by the neurologic deficit score (NDS), dry-wet weight, and 2,3,5-triphenyltetrazolium chloride staining. The levels of interleukin (IL)-1ß, nuclear factor (NF)-κB, and tumor necrosis factor (TNF)-α were detected by an enzyme-linked immunosorbent assay. The expressions of inducible nitric oxide synthase (iNOS), Bax, and Bcl-2 were detected by Western blot. The apoptotic cells were counted by in situ terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay. RESULTS: The pretreatment with GW0742 significantly increased the expression of Bcl-2, and significantly decreased in the volume of infarction, NDS, edema, expressions of IL-1ß, NF-κB, TNFα, and Bax, contents of iNOS and the apoptotic cells in infarct cerebral hemisphere compared with rats in the vehicle group at 24 hours after MCAO. CONCLUSIONS: The study suggests the neuroprotective effect of the PPAR-ß/δ ligand GW0742 in acute ischemic stroke by a mechanism that may involve its anti-inflammatory and antiapoptotic action.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Infarto da Artéria Cerebral Média/tratamento farmacológico , PPAR delta/agonistas , PPAR beta/agonistas , Acidente Vascular Cerebral/tratamento farmacológico , Tiazóis/uso terapêutico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encéfalo/fisiopatologia , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Interleucina-1beta/metabolismo , Masculino , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/fisiopatologia , Tiazóis/farmacologia , Fator de Necrose Tumoral alfa/metabolismo
20.
Ann Surg ; 260(1): 169-78, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24670857

RESUMO

OBJECTIVE: To determine the mechanism responsible for ghrelin's neuroprotective effects after traumatic brain injury (TBI) and hemorrhagic shock. BACKGROUND: Ghrelin, a gastrointestinal hormone, has been demonstrated to possess multiple functions, including upregulation of uncoupling protein 2 (UCP2) and stimulation of the vagus nerve. Recent evidence has indicated that ghrelin is neuroprotective. We, therefore, hypothesized that ghrelin protects rats against TBI and hemorrhagic shock through upregulation of UCP2, involving stimulation of the vagus nerve. METHODS: Brain injury was induced by dropping a 450 g of weight from 1.5 m onto a steel helmet attached to the skull of male adult rats. Immediately after TBI, a midline laparotomy was performed, and both lumbar veins were isolated and severed at the junction with the vena cava. The abdomen was kept open for 20 minutes. At 45 minutes after TBI and uncontrolled hemorrhage (UH), ghrelin (4, 8, or 16 nmol/rat) or 1 mL of normal saline (vehicle) was intravenously administered. The Neurological Severity Scale (NSS), morphological alterations and ß-amyloid precursor protein expression in the brain, systemic organ injury markers (ie, alanine aminotransferase, aspartate aminotransferase, and lactate), and UCP2 expression in the cortex were measured. To determine whether the protective effect of ghrelin is mediated through upregulation of UCP2, genipin, a specific UCP2 antagonist, was administered intravenously before the injection of ghrelin in animals with TBI and UH. The role of the vagus nerve was assessed by performing vagotomy immediately before ghrelin administration. RESULTS: Ghrelin attenuated brain injury and facilitated functional recovery after TBI and UH. Ghrelin increased UCP2 expression in the cortex, and administration of genipin abolished ghrelin's protection after TBI and UH. Furthermore, vagotomy prevented the beneficial effects of ghrelin and eliminated ghrelin-induced UCP2 upregulation after TBI and UH. CONCLUSIONS: The protective effects of ghrelin after TBI and UH seem to be related to upregulation of UCP2 expression in the brain and requiring the intact vagus nerve.


Assuntos
Lesões Encefálicas/prevenção & controle , Grelina/farmacologia , Canais Iônicos/biossíntese , Proteínas Mitocondriais/biossíntese , Choque Hemorrágico/prevenção & controle , Regulação para Cima , Animais , Western Blotting , Lesões Encefálicas/metabolismo , Modelos Animais de Doenças , Imuno-Histoquímica , Canais Iônicos/efeitos dos fármacos , Masculino , Proteínas Mitocondriais/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Choque Hemorrágico/metabolismo , Proteína Desacopladora 2
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