RESUMO
Natural products are among one of the most promising fields in finding new molecular targets in cancer therapy. Laryngeal carcinoma is one of the most common cancers affecting the head and neck regions, and is associated with high morbidity rate if left untreated. The aim of this study was to examine the antiproliferative effect of Araucaria angustifolia on laryngeal carcinoma HEp-2 cells. The results showed that A. angustifolia extract (AAE) induced a significant cytotoxicity in HEp-2 cells compared to the non-tumor human epithelial (HEK-293) cells, indicating a selective activity of AAE for the cancer cells. A. angustifolia extract was able to increase oxidative damage to lipids and proteins, and the production of nitric oxide, along with the depletion of enzymatic antioxidant defenses (superoxide dismutase and catalase) in the tumor cell line. Moreover, AAE was able to induce DNA damage, nuclear fragmentation and chromatin condensation. A significant increase in the Apoptosis Inducing Factor (AIF), Bax, poly-(ADP-ribose) polymerase (PARP) and caspase-3 cleavage expression were also found. These effects could be related to the ability of AAE to increase the production of reactive oxygen species through inhibition of the mitochondrial electron transport chain complex I activity and ATP production by the tumor cells. The phytochemical analysis of A. angustifolia, performed using High Resolution Mass Spectrometry (HRMS) in MS and MS/MS mode, showed the presence of dodecanoic and hexadecanoic acids, and phenolic compounds, which may be associated with the chemotherapeutic effect observed in this study.
Assuntos
Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Neoplasias Laríngeas/tratamento farmacológico , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Traqueófitas/química , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Células HEK293 , Humanos , Neoplasias Laríngeas/metabolismo , Neoplasias Laríngeas/patologia , Laringe/metabolismo , Laringe/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/isolamento & purificaçãoRESUMO
Hepatocellular carcinoma and breast cancer are the most prevalent cancers in the world with high morbidity and mortality. Although there are effective drugs for treating advanced stages of liver and breast cancers, the prognosis for patients with liver cancer remains poor, and patients with breast cancer show considerable mortality. Therefore, it is crucial to explore new therapeutic agents for the inhibition of carcinogenesis. This study examined the anti-carcinogenic effect of Vitis labrusca seed extract (VLE), which is a component of winery waste, on liver (HepG2) and breast cancers (MCF-7) cells. The results found in this study demonstrated VLEinduced DNA damage in liver and breast cancer cells. VLE treatment in both cell lines was accompanied by high NO production and upregulation of p53. A significant decrease in total PARP expression was also found in HepG2 cells. In the MCF-7 cell line, VLE treatment increased the expression of Bax and AIF, and decreased total PARP expression. Surprisingly, VLE suppressed Her-2 expression in HepG2 cells and caused a subtle, but significant downregulation of Her-2 in MCF-7 cells. The possible anti-carcinogenic effect of VLE reported in this study suggests the potential of this extract to be used for the development of novel therapeutic agents for the treatment of different kinds of cancers.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Dano ao DNA/efeitos dos fármacos , Flavonoides/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Receptor ErbB-2/antagonistas & inibidores , Vitis/química , Mama/efeitos dos fármacos , Mama/metabolismo , Neoplasias da Mama/genética , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neoplasias Hepáticas/genética , Células MCF-7 , Receptor ErbB-2/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Different methods for anti-ENA identification have been used. This can lead to confusion regarding the interpretation of the test results in clinical practice. Some studies have reported differences in sensitivity and specificity, but few compare clinical outcomes. Based on that, our aim was to compare the performance characteristics of various methods commonly used to detect anti-ENA antibodies in the sera of patients suspected to have connective tissue diseases (CTDs). METHODS: 189 patients with orders for anti-ENA were analyzed. Three common methods were used: DID, ELISA, and HA. Sensitivity, specificity, PPV, NPV, and LR were calculated using CTDs as the reference standard. RESULTS: 69.3% of the patients had a CTD and 32.8% had SLE. Sensitivity and specificity, respectively, according to the technique were: ELISA (50.0% - 78.9%); DID (31.3% - 89.5%); HA (40.9% - 87.7%). PPV were: 88.5% (HA), 87.2% (DID) and 84.6% (ELISA), and NPV were: 40.5% (ELISA), 39.1% (HA) and 36.2% (DID). CONCLUSIONS: Based on the very similar predictive test values, we believe that, at least in a moderate to high pretest probability, in our methodological scenario, there are no significant differences in the interpretation of test results when using ELISA, HA, and DID for anti-ENA detection.
Assuntos
Anticorpos Antinucleares/sangue , Doenças do Tecido Conjuntivo/sangue , Ensaio de Imunoadsorção Enzimática , Testes de Hemaglutinação , Imunodifusão , Adulto , Especificidade de Anticorpos , Autoantígenos/imunologia , Doenças do Tecido Conjuntivo/imunologia , DNA Topoisomerases Tipo I , Dermatomiosite/sangue , Dermatomiosite/imunologia , Feminino , Humanos , Funções Verossimilhança , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/imunologia , Valor Preditivo dos Testes , Estudos Prospectivos , Ribonucleoproteínas/imunologia , Sensibilidade e Especificidade , Antígeno SS-BRESUMO
Betacellulin (BTC), a ligand of the epidermal growth factor receptor, has been shown to promote growth and differentiation of pancreatic ß-cells and to improve glucose metabolism in experimental diabetic rodent models. Mesenchymal stem cells (MSCs) have been already proved to be multipotent. Recent work has attributed to rat and human MSCs the potential to differentiate into insulin-secreting cells. Our goal was to transfect rat MSCs with a plasmid containing BTC cDNA to guide MSC differentiation into insulin-producing cells. Prior to induction of cell MSC transfection, MSCs were characterized by flow cytometry and the ability to in vitro differentiate into mesoderm cell types was evaluated. After rat MSC characterization, these cells were electroporated with a plasmid containing BTC cDNA. Transfected cells were cultivated in Dulbecco's modified Eagle medium high glucose (H-DMEM) with 10 mM nicotinamide. Then, the capability of MSC-BTC to produce insulin in vitro and in vivo was evaluated. It was possible to demonstrate by radioimmunoassay analysis that 10(4) MSC-BTC cells produced up to 0.4 ng/mL of insulin, whereas MSCs transfected with the empty vector (negative control) produced no detectable insulin levels. Moreover, MSC-BTC were positive for insulin in immunohistochemistry assay. In parallel, the expression of pancreatic marker genes was demonstrated by molecular analysis of MSC-BTC. Further, when MSC-BTC were transplanted to streptozotocin diabetic rats, BTC-transfected cells ameliorated hyperglycemia from over 500 to about 200 mg/dL at 35 days post-cell transplantation. In this way, our results clearly demonstrate that BTC overabundance enhances glucose-induced insulin secretion in MSCs in vitro as well as in vivo.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Hiperglicemia/metabolismo , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Betacelulina , Glicemia/metabolismo , Diferenciação Celular , Células Cultivadas , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/terapia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hiperglicemia/induzido quimicamente , Hiperglicemia/terapia , Secreção de Insulina , Masculino , Transplante de Células-Tronco Mesenquimais , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , EstreptozocinaRESUMO
OBJECTIVE: To identify factors related to NT-proBNP levels in systemic sclerosis (SSc). DESIGN AND METHODS: NT-proBNP was measured in 119 patients with SSc and 20 controls. Patients with transtricuspid gradient (TG) > or =36 mm Hg or > or =31 mmHg plus dyspnea were considered to have suspected systemic sclerosis-associated pulmonary arterial hypertension (SScPAH). RESULTS: Increasing age, NYHA functional class, skin score, history of systemic arterial hypertension (SAH), anticentromere antibodies, diastolic dysfunction, reduced pulmonary diffusing capacity, and TG were positively associated with NT-proBNP. In multivariable linear regression, TG, age, and SAH were independently associated to NT-proBNP levels. An ROC curve analysis (with an area under the curve of 0.89, 95% CI: 0.83-0.95) suggested a cutoff of 157.8pg/mL to identify patients with suspected SScPAH, presenting a sensitivity of 100% (78.1-100) and specificity of 72.3% (62.3-80.5). CONCLUSIONS: NT-proBNP levels are related to clinical and laboratory abnormalities in SSc. The results indicate that NT-proBNP may be a useful tool in the evaluation of SScPAH.
Assuntos
Peptídeo Natriurético Encefálico/sangue , Fragmentos de Peptídeos/sangue , Escleroderma Sistêmico/diagnóstico , Adulto , Fatores Etários , Estudos de Casos e Controles , Feminino , Humanos , Hipertensão/etiologia , Hipertensão Pulmonar/etiologia , Masculino , Pessoa de Meia-Idade , Curva ROC , Escleroderma Sistêmico/complicações , Sensibilidade e Especificidade , TriglicerídeosRESUMO
OBJETIVO: Analisar a prevalência, padrões e títulos do Fator Antinuclear (FAN) por imunofluorescência indireta (IFI) em células HEp-2 em um hospital universitário após a adoção do I e II Consensos Nacional para Padronização dos Laudos de FAN em Células HEp-2. MÉTODO: Foi realizado um estudo transversal, em que foram revisados os laudos de FAN por IFI originários de solicitações encaminhadas ao Serviço de Patologia Clínica do Hospital de Clínicas de Porto Alegre (SPC/HCPA) entre 2002 e 2005. RESULTADOS: Foram analisados 12.095 testes de FAN no período entre 2002 e 2005. As solicitações com resultado reagente foram de 2.577 (21,30 por cento), com média anual de 644±233). Houve um aumento significativo na proporção de resultados reagentes posterior à adoção dos Consensos (p < 0,001). A Reumatologia foi a especialidade que mais solicitou exames por paciente atendido, mas houve um declínio nesse número nos anos posteriores à adoção do Consenso, ocorrido em 2004 (p < 0,001). O padrão de imunofluorescência de FAN mais frequentemente encontrado foi o padrão nuclear pontilhado fino, presente em 52,3 por cento dos resultados reagentes (453/866), e os títulos mais encontrados foram 1/80 e 1/160 (27,8 por cento e 29,4 por cento, respectivamente). CONCLUSÃO: Após a adoção do Consenso Nacional de Padronização de Laudos de FAN, observou-se um aumento de exames com resultados reagentes, na sua maioria, com títulos baixos e padrão nuclear pontilhado fino. Na Reumatologia, observou-se uma diminuição no número de solicitações desse exame. As potenciais causas para essas observações são discutidas, mas seu real impacto sobre a situação clínica do paciente e seu tratamento merece ser mais bem estudado.
OBJECTIVE: To evaluate the prevalence of patterns and titers of antinuclear antibodies (ANA) detected by indirect immunofluorescence (IIF) technique on HEp-2 cells in a university hospital following the introduction of I and II Brazilian Consensuses for Standardization of ANA in HEp-2 Cells. METHODS:A transversal study was performed between 2002 and 2005 during which all ANA orders to Serviço de Hospital de Clínicas de Porto Alegre (SPC/HCPA) and cognate results were reviewed. RESULTS:12.095 tests of ANA were revised. The number of positive results during this period was 2.577 (21.30 percent), annual mean 644 (SD: 233). A marked increase in the number of positive results was observed following the introduction of the Consensuses (p < 0.001). Rheumatology was the medical specialty which requested the highest number of ANA testing per patient although a significant decrease of these numbers was observed after the introduction of the Consensus in 2004 (p < 0,001). Nuclear fine speckled immunofluorescence labeling was the most frequently ANA pattern observed, 52.3 percent (453/866), and low ANA titers (1/80 and 1/160) more commonly detected (27.8 percent and 29.4 percent, respectively). CONCLUSION: Following the introduction of the Brazilian Consensus for standardization of ANA in HEp-2 cells an increased number of positive results was observed, mostly in low titers and with nuclear fine speckled immunofluorescence pattern. Moreover, there were decreasing numbers of ANA orders by rheumatologists in the same period. Potential causes for these observations are discussed but the real impact in the clinical condition of the patient and therapy deserves to be better studied.
Assuntos
Humanos , Anticorpos Antinucleares , Autoanticorpos , ImunofluorescênciaRESUMO
OBJETIVO: avaliar os padrões de imunofluorescência do fator antinuclear (FAN) em soros reagentes para anticorpos anti-SSA/Ro e sua associação clínica. MÉTODO: foi realizado um estudo transversal retrospectivo, no qual foram revisadas as solicitações de anticorpos antiantígenos nucleares extraíveis (anti-ENA) encaminhadas ao SPC/HCPA no período de dois anos. Das solicitações com resultado positivo para anti-ENA identificou-se qual ou quais auto-anticorpos estavam envolvidos (anti-SSA/Ro, anti-SSB/La, anti-RNP, anti-Sm, anti-Scl-70), bem como os padrões de imunofluorescência do FAN e os quadros clínicos dos pacientes anti-SSA/Ro positivo. As técnicas usadas para detecção e identificação foram FAN por imunofluorescência indireta (IFI) em células HEp-2 e anti-ENA por hemaglutinação. RESULTADOS: das 392 solicitações analisadas 90 eram anti-ENA positivo. Houve um predomínio do sexo feminino (94 por cento) (86/91) e a idade média foi de 42 anos. O anti-SSA/Ro foi o mais freqüente (67,8 por cento) (61/90), sendo que todas as amostras anti-SSA/Ro positivas eram positivas para o FAN. O padrão de imunofluorescência nuclear pontilhado fino foi o predominante (68,9 por cento) (42/61) nos pacientes com anti-SSA/Ro positivo, e o quadro clínico mais encontrado foi de lúpus eritematoso sistêmico, em 50,8 por cento (31/61) dos pacientes. CONCLUSÃO: o teste de FAN por IFI utilizando células HEp-2 é um bom método de triagem para detecção de auto-anticorpos anti-SSA/Ro, apresentando maior associação com o padrão nuclear pontilhado fino. Diferente do que tem sido descrito na literatura, não encontramos nenhuma amostra de pacientes com anti-SSA/Ro que tenham apresentado FAN falso-negativo na IFI. Pelo menos na nossa experiência, esses dados questionam o custo-efetividade da solicitação de rotina desse exame em pacientes FAN negativo pelo teste de IFI.
OBJECTIVE: to evaluate the pattern at immunofluorescence of the antinuclear antibodies (ANA) detected by the indirect immunofluorescence (IIF) technique in positive samples for anti-SSA/Ro autoantibody and the clinical associations. METHODS: a retrospective transversal study was performed in a period of two years where the all the solicitations of testing for the presence of anti-extractable nuclear antigen (anti-ENA) antibodies delivered to the SPC/HCPA were analyzed. We selected the positive samples and identified which autoantibodies were involved (anti-SSA/RO, anti-SSB/La, anti-RNP, anti-Sm and anti-Scl70) as well as the immunofluorescence patterns by ANA testing and the clinical associations found in the patients presenting anti-SSA/Ro positive serum. IIF was used for ANA using HEp-2 cells and hemagglutination for anti-ENA antibodies detection. RESULTS: 90 out of the 392 solicitations analyzed were anti-ENA positive, with a predominance of women (86/91 - 94 percent) and the mean age was 42 years old. The most frequent autoantibody was anti-SSA/Ro (61/90 - 67.8 percent) and all samples that were anti-SSA/Ro positive were also ANA positive. Speckled nuclear immunofluorescence was the most frequent ANA pattern (42/61 - 68.9 percent) among the anti-SSA/Ro positive samples and systemic lupus erythematosus was the most common clinical diagnosis (31/61 - 50.8 percent). CONCLUSION: ANA testing by IIF using HEp-2 cells proved to be a good screening test for the detection of anti-SSA/Ro antibodies, that showed a strong positive association to the speckled nuclear IIF pattern. As opposed to what has been described in the literature, there was no ANA negative among the anti-SSA/Ro positive samples. At least in our experience, these data question the cost-effectiveness of performing routine screening for anti-SSA/Ro antibodies in ANA negative samples by IIF testing.
RESUMO
Objetivo - Identificação e caracterização de auto-anticorpos e auto-antígenos associados ao padrão de imunofluorescéncia indireta (IFI) de Pontos Citoplasmáticos Isolados (PU). Materiais e métodos - Foram selecionados soros que apresentavam o padrão de PCI à IFI. Esses soros foram analisados por IFI dupla em microscopia confocal, imunomicroscopia eletrônica, immunoblotting, imunoprecipitação de antígenos protéicos sintetizados in vitro, HPTLC de lipídeos seguida de imunocoloração e análise de espectrometria de massas para antígenos lipídicos. Os dados clínicos foram obtidos por revisão de prontuários e entrevistas com os médicos assistentes. Resultados Dois sistemas de auto-antigeno-auto-anticorpo associados com o padrão de IFI de pontos citoplasmáticos isolados (PCI) foram identificados e denominados de padrão PCI-I e PCI-II. O padrão PCI-I foi caracterizado por 3 a 20 pontos brilhantes bem delimitados e homogeneamente distribuídos pelo citoplasma. O padrão PCI-11 foi caracterizado por apresentar mais de 30 pontos por todo o citoplasma, com predominância perinuclear, grande heterogeneidade de tamanho e tendência a aglomeração. De acordo com esta classificação foram obtidos 27 soros PCI-I e 6 soros PCI-II. Por IFI dupla em microscopia confocal e imuno-microscopia eletrônica os soros PCI-11 mostraram co-localização com os anticorpos monoclonais anti-LAMP-2 (proteína-2 associada à membrana do lisossomo) e anti-EEA1 (antígeno-1 do endossomo primário) e com receptor de transferrina, marcadores da via endocíticaexocítica. Os domínios citoplasmáticos reconhecidos pelos auto-anticorpos PCI-11 parecem ser delimitados por membrana. Por immunoblotting soros PCI-11 reconheceram uma banda com mobilidade relativa de 180kDa e imunoprecipitaram a proteína EEA1. Em contraste, os anticorpos PCI-I não apresentaram co-localização com anti-EEA1 e anti-LAMP-2, porém apresentaram co-localização parcial com o anticorpo monoclonal anti-GW182, que reconhece uma proteína de 182 kDa (GW182), presente em aglomerados citoplasmáticos de mRNA. Os soros PCI-I não demonstraram qualquer reatividade ao immunoblotting com diversos extratos celulares e não imunoprecipitaram as proteínas GW182 e EEA1 sintetizadas in vitro...