RESUMEN
PURPOSE: The present study evaluates chondroitin sulfate (CS) and heparan sulfate (HS) in the urine and hyaluronic acid (HA) in the plasma of patients with prostate cancer before and after treatment compared to a control group. MATERIALS AND METHODS: Plasma samples were used for HA dosage and urine for quantification of CS and HS from forty-four cancer patients and fourteen controls. Clinical, laboratory and radiological information were correlated with glycosaminoglycan quantification by statistical analysis. RESULTS: Serum HA was significantly increased in cancer patients (39.68 ± 30.00 ng/ mL) compared to control group (15.04 ± 7.11 ng/mL; p=0.004) and was further increased in high-risk prostate cancer patients when compared to lower risk patients (p = 0.0214). Also, surgically treated individuals had a significant decrease in seric levels of heparan sulfate after surgical treatment, 31.05 ± 21.01 µg/mL (before surgery) and 23.14 ± 11.1 µg/mL (after surgery; p=0.029). There was no difference in the urinary CS and HS between prostate cancer patients and control group. Urinary CS in cancer patients was 27.32 ± 25.99 µg/mg creatinine while in the men unaffected by cancer it was 31.37 ± 28.37 µg/mg creatinine (p=0.4768). Urinary HS was 39.58 ± 32.81 µg/ mg creatinine and 35.29 ± 28.11 µg/mg creatinine, respectively, in cancer patients and control group (p=0.6252). CONCLUSIONS: Serum HA may be a useful biomarker for the diagnosis and prognosis of prostate cancer. However, urinary CS and HS did not altered in the present evaluation. Further studies are necessary to confirm these preliminary findings.
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Sulfatos de Condroitina/orina , Heparitina Sulfato/orina , Ácido Hialurónico/sangre , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/orina , Anciano , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/orina , Estudios de Casos y Controles , Humanos , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
A better understanding of the molecular mechanisms of renal cell carcinogenesis could contribute to a decrease in the mortality rate of this disease. The aim of this study was to evaluate the glycosaminoglycans profile and heparanase expression in renal cell carcinoma. The study included 24 patients submitted to nephrectomy with confirmed pathological diagnosis of renal cell carcinoma. The majority of the samples (87.5%) were classified in the initial stage of renal cell carcinoma (clinical stages I and II). Heparanase messenger ribonucleic acid expression was evaluated by quantitative real-time reverse transcription polymerase chain reaction, and sulfated glycosaminoglycans were identified and quantified by agarose gel electrophoresis of renal cell carcinoma samples or non-neoplastic tissues obtained from the same patients (control group). The sulfated glycosaminoglycans and hyaluronic acid were analyzed in urine samples of the patients before and after surgery. The data showed a significant statistical increase in chondroitin sulfate, and a decrease in heparan sulfate and dermatan sulfate present in neoplastic tissues compared with non-neoplastic tissues. Higher heparanase messenger ribonucleic acid expression in the neoplastic tissues was also shown, compared with the non-neoplastic tissues. The urine glycosaminoglycans profile showed no significant difference between renal cell carcinoma and control samples. Extracellular matrix changes observed in the present study clarify that heparanase is possibly involved with heparan sulfate turnover, and that heparanase and the glycosaminoglycans can modulate initial events of renal cell carcinoma development.
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Carcinoma de Células Renales/metabolismo , Glucuronidasa/biosíntesis , Glicosaminoglicanos/metabolismo , Neoplasias Renales/metabolismo , Carcinoma de Células Renales/cirugía , Sulfatos de Condroitina/metabolismo , Electroforesis en Gel de Agar , Glicosaminoglicanos/orina , Humanos , Ácido Hialurónico/metabolismo , Neoplasias Renales/cirugía , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Hypericin is considered a potent photosensitizer for use in antitumor and antimicrobial photodynamic therapy (PDT). This review presents the primary biological results obtained with hypericin in photodynamic therapy applications, such as photodynamic cancer treatment, photoinactivation of microorganisms (PDI), tissue scarring, and photo diagnosis. We present a compilation of in vitro results that have been published thus far; for these studies, we highlight the hypericin concentration, light dose, and other experimental conditions to evaluate the efficiency of photodynamic treatment like cell death, cell viability, or cell proliferation. The results indicate that different hypericin phototoxicity levels can be observed according to the specific light dose and concentration. Furthermore, it was shown that cellular localization and cell death mechanisms (apoptosis and necrosis) are dependent on the cell type.
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Perileno , Fotoquimioterapia , Antracenos , Apoptosis , Supervivencia Celular , Perileno/análogos & derivados , Perileno/farmacología , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéuticoRESUMEN
OBJECTIVE: To investigate the possible genes that may be related to the mechanisms that modulate heparanase-1. METHODS: The analysis was conducted at Universidade Federal de São Paulo, on the data provided by: The Cancer Genome Atlas, University of California Santa Cruz Genome Browser, Kyoto Encyclopedia of Genes and Genomes Pathway Database, Database for Annotation, Visualization and Integrated Discovery Bioinformatics Database and the softwares cBioPortal and Ingenuity Pathway Analysis. RESULTS: Using messenger RNA expression pattern of different molecular subtypes of breast cancer, we proposed that heparinase-1 was co-related with its progression. In addition, genes that were analyzed presented co-expression with heparanase-1. The results that showed that heparanase-1 co-expressed with phosphoinositide 3-kinase adapter protein 1, sialic acid-binding immunoglobulin-like lectin 7, and leukocyte-associated immunoglobulin-like receptor 1 are directed related with immune system evasion during breast cancer progression. Furthermore, cathepsin L was co-expressed with heparanase-1 and transformed inactive heparanase-1 form into active heparanase-1, triggering extracellular matrix remodeling, which contributes to enhanced tumor-host interaction of the tumor. CONCLUSION: The signaling pathway analysis using bioinformatics tools gives supporting evidence of possible mechanisms related to breast cancer development. Evasion genes of the immune system co-expressed with heparanase-1, a enzyme related with tumor progression.
Asunto(s)
Neoplasias de la Mama/genética , Glucuronidasa/genética , Simulación por Computador , HumanosRESUMEN
The objective of the present study was to evaluate the role of microRNA-mediated remodeling of the extracellular matrix in the process of tumor invasion of oral squamous cell carcinoma and to evaluate its relationship with the prognosis of these patients. This was a retrospective study on material from the paraffin blocks of patients operated on for oral squamous cell carcinoma, in addition to a group of healthy oral mucosa samples of paired patients. miR-1-3p, miR-133-3p, and miR-21-5p were differentially expressed between the superficial and deep tumor groups. miR-21-5p was the one with the greatest accuracy in the differentiation between superficial and deep tumors. By immunohistochemistry, the group of deep tumors showed greater immunoreactivity to matrix metalloproteinases 2 and 9 and laminin α in tumor-associated fibroblasts, with consequent degradation of the basal membrane, measured by greater loss of continuity of type IV collagen. This process was also associated with lower and higher expression of miR-1-3p and miR-21-5p, respectively. There was also a trend toward better overall and disease-free survival rates in patients with higher miR-133a-3p. The present study showed the interaction between microRNAs and extracellular matrix remodeling in oral squamous cell carcinoma.
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Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Neoplasias de la Boca/genética , Neoplasias de la Boca/metabolismo , Adulto , Anciano , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/terapia , Terapia Combinada , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Persona de Mediana Edad , Neoplasias de la Boca/diagnóstico , Neoplasias de la Boca/terapia , Clasificación del Tumor , Invasividad Neoplásica , Estadificación de Neoplasias , Interferencia de ARN , Curva ROCRESUMEN
BACKGROUND: Cardiac remodeling is ultimately regulated by components of the extracellular matrix (ECM). We investigated the important role that growth factors play in the regulation of ECM remodeling that occurs as a consequence of myocardium damage. METHODS AND RESULTS: Rats were submitted to the ligation of the left anterior coronary artery and pcDNA3-vascular endothelial growth factor (VEGF)(165) was immediately injected intramyocardially in the treated group. The animals were divided into large size myocardium infarction (LMI) and small size myocardium infarction, with or without gene transfer. The plasmid-containing DNA encoding VEGF(165) was injected into the cardiac muscle and its effect was observed on the ECM components. Glycosaminoglycans were identified and quantified by agarose gel based electrophoresis and ELISA as well as immunocytochemistry to examine specific cathepsin B, heparanase, and syndecan-4 changes. The amounts of hyaluronic acid (HA; p < 0.005), DS, chondroitin sulfate, and heparan sulfate (p < 0.001) were significantly increased in the LMI treated group in comparison to the other groups, which correlates with the decrease in the expression of heparanase. A decrease in the molecular mass of HA was found in the scar tissue of treated group. CONCLUSIONS: The data obtained strongly support the idea that changes in the ECM and its components are important determinants of cardiac remodeling after myocardium infarct and may be essential for inflammatory response and attempt to stabilize the damage and provide a compensatory mechanisms to maintain cardiac output since the ECM components analyzed are involved with angiogenesis, cell proliferation and differentiation.
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Matriz Extracelular/metabolismo , Terapia Genética , Infarto del Miocardio/terapia , Miocardio/metabolismo , Factor A de Crecimiento Endotelial Vascular , Animales , Matriz Extracelular/química , Femenino , Glicosaminoglicanos/análisis , Glicosaminoglicanos/metabolismo , Humanos , Inyecciones Intramusculares , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocardio/patología , Ratas , Ratas Wistar , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
OBJECTIVE: To investigate the action of testosterone (T), isolated or associated with estradiol benzoate (EB), on the proliferation markers and apoptosis of breasts of ovariectomized rats. METHODS: A total of 48 castrated female Wistar rats were divided into 6 groups, and each of them were submitted to one of the following treatments for 5 weeks: 1) control; 2) EB 50 mcg/day + T 50 mcg/day; 3) T 50mcg/day; 4) EB 50 mcg + T 300 mcg/day; 5) T 300 mcg/day; and 6) EB 50 mcg/day. After the treatment, the mammary tissue was submitted to a histological analysis and immunoexpression evaluation of proliferation markers (proliferating cell nuclear antigen, PCNA) and apoptosis (caspase-3). RESULTS: There was a statistically significant difference among the groups regarding microcalcifications and secretory activity, with higher prevalence in the groups treated with EB. There was no difference among the groups regarding atrophy, but a higher prevalence of atrophy was found in the groups that received T versus those that received EB + T. There was a difference among the groups regarding the PCNA (p = 0.028), with higher expression in the group submitted to EB + T 300 mcg/day. Regarding caspase-3, there was no difference among the groups; however, in the group submitted to EB + T 300 mcg/day, the expression was higher than in the isolated T group. CONCLUSION: Isolated T did not have a proliferative effect on the mammary tissue, contrary to EB. Testosterone in combination with EB may or may not decrease the proliferation, depending on the dose of T.
OBJETIVO: Investigar a ação da testosterona (T) isolada ou associada ao benzoato de estradiol (EB) na proliferação e apoptose de mamas de ratas ovariectomizadas. MéTODOS: Um total de 48 ratas Wistar castradas foram divididas em 6 grupos, e cada um foi submetido a um dos seguintes tratamentos durante 5 semanas: 1) controle; 2) BE 50 mcg/dia + T 50 mcg/dia; 3) T 50 mcg/dia; 4) BE 50 mcg + T 300 mcg/dia; e) T 300 mcg/dia; e f) BE 50 mcg/dia. Após o tratamento, o tecido mamário foi submetido a análise histológica e avaliação de imunoexpressão de marcadores de proliferação (antígeno nuclear de células proliferantes, PCNA) e apoptose (caspase-3). RESULTADOS: Houve diferença estatisticamente significante entre os grupos com relação às microcalcificações e à atividade secretora, com maior prevalência nos grupos tratados com BE. Não houve diferença entre os grupos quanto à atrofia, mas houve maior prevalência de atrofia nos grupos que receberam T versus os que receberam BE + T. Houve diferença entre os grupos quanto ao ANCP (p = 0,028), com maior expressão no grupo BE + T 300 mcg/dia. Com relação à caspase-3, não houve diferença entre os grupos, mas, no grupo BE + T 300 mcg/dia, a expressão foi maior do que no grupo de T isolada. CONCLUSãO: A T isolada não apresentou efeito proliferativo do tecido mamário, contrariamente ao EB. A T em associação ao EB pode diminuir ou não a proliferação, a depender da dose de T.
Asunto(s)
Apoptosis/efectos de los fármacos , Mama/citología , Proliferación Celular/efectos de los fármacos , Testosterona/farmacología , Animales , Biomarcadores/análisis , Mama/patología , Calcinosis/patología , Caspasa 3/análisis , Estradiol/análogos & derivados , Estradiol/farmacología , Femenino , Ovariectomía , Antígeno Nuclear de Célula en Proliferación/análisis , Ratas WistarRESUMEN
OBJECTIVE: To evaluate intervertebral disc levels of inflammatory factor (interleukin 6) and proteinase activity (cathepsin B) in patients with a degenerative disease and serum levels of interleukin 6, serum cathepsin B activity and hyaluronic acid biomarkers. METHODS: We conducted immunohistochemistry studies of intervertebral discs to analyze interleukin 6 and cathepsin B levels of patients with degenerative disease and spine fracture (Control Group) and to measure hyaluronic acid, interleukin 6 and cathepsin B activity from sera of intervertebral disc degeneration patients, fracture patients, and healthy individuals. RESULTS: Interleukin 6 and cathepsin B seem to be related with physiopathology of intervertebral disc degeneration, since the levels of both were higher in discs of patients with intervertebral disc degeneration. Interleukin 6 and cathepsin B do not represent good biomarkers of degenerative intervertebral disc disease, since the level of such compounds is increased in the plasma of patients with fractures. CONCLUSION: Hyaluronic acid can be a biomarker for intervertebral disc degeneration, because hyaluronic acid levels were higher only in sera of patients with intervertebral disc degeneration.
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Adyuvantes Inmunológicos/sangre , Biomarcadores/sangre , Catepsina B/sangre , Ácido Hialurónico/sangre , Interleucina-6/sangre , Degeneración del Disco Intervertebral/diagnóstico , Adulto , Análisis de Varianza , Estudios de Casos y Controles , Femenino , Humanos , Inmunohistoquímica , Mediadores de Inflamación/sangre , Disco Intervertebral/fisiopatología , Degeneración del Disco Intervertebral/sangre , Degeneración del Disco Intervertebral/fisiopatología , Masculino , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: To analyze the effects of estrogen alone or in combination with progestogens and tibolone (TIB) on the expression of the extracellular matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), of perlecan, and of heparanase (HPSE) of the vascular walls of the carotid arteries. METHODS: A total of 30 250-day-old ovariectomized Wistar rats were orally treated for 5 weeks with: a) 1 mg/kg of estradiol benzoate (EB); b) EB + 0.2 mg/kg of medroxyprogesterone acetate (MPA); c) EB + 0.2mg/kg of norethisterone acetate (NETA); d) EB + 2 mg/kg of dydrogesterone (DI); e) 1 mg/kg of TIB; f) placebo (CTR). Following treatment, the expression of mRNA for MMP-2, MMP-9, and HPSE was analyzed by real-time polymerase chain-reaction (PCR), and the expression of MMP-2, of MMP-9, of tissue inhibitor of metalloproteinase 2 (TIMP-2), and of perlecan was quantified by immunohistochemistry in the carotid arteries. RESULTS: The groups showed significant differences on mRNA HPSE expression (p = 0.048), which was higher in the EB, EB + MPA, and TIB groups. There was no statistically significant difference in mRNA MMP-2 or MMP-9 expression. The immunohistochemical expression of MMP-2, of TIMP-2, of MMP-9, of HPSE, and of perlecan showed no differences between groups. CONCLUSION: Estradiol alone or associated with MPA and TIB treatment can increase mRNA HSPE expression of the walls of the carotid arteries in ovariectomized rats.
OBJETIVO: Analisar os efeitos do estrogênio isolado ou em combinação com progestogênios e tibolona (TIB) na expressão das metaloproteinases 2 e 9 da matriz extracelular (MMP-2 e MMP-9), da perlecan e da heparanase (HPSE) das paredes vasculares das artérias carótidas. MéTODOS: Trinta ratas Wistar ovariectomizadas com 250 dias de idade foram tratadas oralmente por 5 semanas com: a) 1 mg/kg de benzoato de estradiol (EB); b) EB + 0,2 mg/kg de acetato de medroxiprogesterona (MPA); c) EB + 0,2mg/kg de acetato de noretisterona (NETA); d) EB + 2 mg/kg de didrogesterona (DI); e) 1 mg/kg de TIB; f) placebo (CTR). Após o tratamento, a expressão de mRNA para MMP-2, MMP-9, e HPSE foi analisada por reação em cadeia da polimerase (RCP) em tempo real, e a expressão de MMP-2, MMP-9, inibidor tecidual de metaloproteinase 2 (TIMP-2), e de perlecan foi quantificado por imunohistoquímica em artérias carótidas. RESULTADOS: Os grupos apresentaram diferenças significativas na expressão do mRNA HPSE (p = 0,048), sendo maiores nos grupos EB, EB + MPA e TIB. Não houve diferença estatisticamente significativa nas expressões de mRNA MMP-2 ou MMP-9. A expressão imunohistoquímica de MMP-2, TIMP-2, MMP-9, HPSE e perlecan não mostrou diferenças entre os grupos. CONCLUSãO: O estradiol isolado ou associado ao tratamento com MPA e TIB pode aumentar a expressão de mRNA HSPE nas paredes das artérias carótidas em ratas ovariectomizadas.
Asunto(s)
Arterias Carótidas/enzimología , Agentes Anticonceptivos Hormonales/farmacología , Estradiol/análogos & derivados , Liasa de Heparina/efectos de los fármacos , Norpregnenos/farmacología , Progestinas/farmacología , Administración Oral , Animales , Arterias Carótidas/efectos de los fármacos , Agentes Anticonceptivos Hormonales/administración & dosificación , Estradiol/administración & dosificación , Estradiol/farmacología , Terapia de Reemplazo de Estrógeno , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Proteoglicanos de Heparán Sulfato/genética , Proteoglicanos de Heparán Sulfato/metabolismo , Liasa de Heparina/genética , Liasa de Heparina/metabolismo , Metaloproteinasa 2 de la Matriz/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Modelos Animales , Norpregnenos/administración & dosificación , Ovariectomía , Progestinas/administración & dosificación , Ratas , Ratas WistarRESUMEN
INTRODUCTION: Breast cancer is one of the main causes of death in women. Luminal tumors A and B show good response with hormonal treatments, tumors that overexpress HER-2 can be treated with monoclonal antibodies, whereas triple negative tumors have few treatments available because they present low or absent expression of hormone receptors and HER-2, in addition, they present worse tumor progression. Syndecans are heparan sulfate proteoglycans that have the function of interacting with growth factors, cytokines, and extracellular matrix, thus modulating important processes in tumor progression. OBJECTIVE: Analyze the expression of syndecan-4 in different subtypes of breast tumors. METHODS: Bioinformatics is a useful tool for the study of new biomarkers. In the present study, the TCGA database (514 patients) and Metabric (1,898 patients) were analyzed using the cBioportal software. Gene expression data were analyzed by RNA-Seq and Microarray from biopsies of breast tumors. RESULTS: An alteration in syndecan-4 gene expression was observed among the different subtypes of breast tumors. Patients with a triple-negative tumor had decreased expression for syndecan-4 in both databases. CONCLUSION: Syndecan-4 is a potential biomarker for breast tumor prognosis since decreased expression of syndecan-4 is related to triple-negative breast cancer.
INTRODUÇÃO: O câncer de mama corresponde a uma das principais causas de morte em mulheres. Os tumores luminais A e B apresentam boa resposta com tratamentos hormonais, os tumores que superexpressam HER-2 podem ser tratados com anticorpos monoclonais, já os tumores triplo-negativos apresentam poucos tratamentos disponíveis por apresentarem expressão baixa ou ausente dos receptores hormonais e HER-2, além de pior progressão tumoral. Os sindecans são proteoglicanos de heparam sulfato que tem função de interagir com fatores de crescimento, citocinas e matriz extracelular, modulando assim processos importantes na progressão tumoral. OBJETIVO: Analisar a expressão o sindecam-4 nos diferentes subtipos de tumores de mama. MÉTODOS: A bioinformática vem se mostrando útil para estudo de novos biomarcadores. No presente estudo, foi analisado o banco de dados TCGA (514 pacientes) e Metabric (1898 pacientes) utilizando o software cBioportal. Foram analisados os dados de expressão gênica por RNA-Seq e Microarray. RESULTADOS: Foi verificada alteração de expressão gênica do sindecam-4 entre os diferentes subtipos de tumores de mama. Pacientes com tumor triplo-negativo tiveram a expressão diminuída para sindecam-4 em ambos os bancos de dados. CONCLUSÃO: Foi verificado que sindecam-4 parece ser um potencial biomarcador em tumores de mama, a expressão diminuída de sindecam-4 parece estar relacionada a um pior prognóstico.
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Humanos , Neoplasias de la Mama , Biomarcadores de Tumor , Expresión Génica , Sindecano-4 , Biología ComputacionalRESUMEN
PURPOSE: To compare immunostaining quantification obtained by a digital computer-assisted method with the well-established semiquantitative analysis. METHODS: Cytoplasmic staining of galectin-3 was obtained by standard immunohistochemical reactions in 25 cases of well-differentiated thyroid carcinoma. The expression index that associates the conventional area fraction of labeled cells with the immunostaining intensity score based on visual qualitative observation was used as the semiquantitative analysis. A digital computer-assisted method is described based on the use of an image processing program (ImageLab). Three parameters were obtained: (1) percentage of labeled cells; (2) digital immunostaining intensity, and (3) digital expression index. The proposed method allows numerical analysis of the immunostaining intensity. RESULTS: There was a strong correlation between the immunostaining intensity obtained by the two methods (Pearson correlation coefficient, r = 0.71, P = 0.0001). The same was observed between expression indexes (Pearson correlation coefficient, r = 0.66, P = 0.0001). CONCLUSION: Results obtained with our proposed digital computer-assisted method for immunoexpression analysis were concordant with the semiquantitative analysis. In addition, digital values can also resolve disagreement among different observers about the quality of staining intensity because the digital method does not classify the results into groups, but rather provides a numerical value for each individual case; thus, it increases the diagnostic and, more importantly, the prognostic sensitivity of the immunohistochemical analysis.
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Biomarcadores de Tumor/análisis , Citometría de Imagen/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Inmunohistoquímica/métodos , Coloración y Etiquetado/estadística & datos numéricos , Análisis de Varianza , Carcinoma/diagnóstico , Carcinoma/ultraestructura , Diferenciación Celular , Galectina 3/análisis , Humanos , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/ultraestructuraRESUMEN
OBJECTIVE: Evaluate the effects of VEGF165 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. METHODS: Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF165 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. RESULTS: There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF165. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF165, suggesting greater tissue differentiation. CONCLUSION: The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF165 seems to provide a protective effect in the treatment of acute myocardial infarct.
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Matriz Extracelular/fisiología , Técnicas de Transferencia de Gen , Infarto del Miocardio/terapia , Factor A de Crecimiento Endotelial Vascular/uso terapéutico , Actinas/análisis , Animales , Antígeno CD24/análisis , Cadherinas/análisis , Proliferación Celular/fisiología , Modelos Animales de Enfermedad , Femenino , Fibronectinas/análisis , Terapia Genética/métodos , Receptores de Hialuranos/análisis , Inmunohistoquímica , Miocitos Cardíacos/fisiología , Ratas Wistar , Reproducibilidad de los Resultados , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/genética , Vimentina/análisisRESUMEN
OBJECTIVE: To determine the presence of glycosaminoglycans in the extracellular matrix of connective tissue from neoplastic and non-neoplastic colorectal tissues, since it has a central role in tumor development and progression. METHODS: Tissue samples from neoplastic and non-neoplastic colorectal tissues were obtained from 64 operated patients who had colorectal carcinoma with no distant metastases. Expressions of heparan sulphate, chondroitin sulphate, dermatan sulphate and their fragments were analyzed by electrospray ionization mass spectrometry, with the technique for extraction and quantification of glycosaminoglycans after proteolysis and electrophoresis. The statistical analysis included mean, standard deviation, and Student'st test. RESULTS: The glycosaminoglycans extracted from colorectal tissue showed three electrophoretic bands in agarose gel. Electrospray ionization mass spectrometry showed characteristic disaccharide fragments from glycosaminoglycans, indicating their structural characterization in the tissues analyzed. Some peaks in the electrospray ionization mass spectrometry were not characterized as fragments of sugars, indicating the presence of fragments of the protein structure of proteoglycans generated during the glycosaminoglycan purification. The average amount of chondroitin and dermatan increased in the neoplastic tissue compared to normal tissue (p=0.01). On the other hand, the average amount of heparan decreased in the neoplastic tissue compared to normal tissue (p= 0.03). CONCLUSION: The method allowed the determination of the glycosaminoglycans structural profile in colorectal tissue from neoplastic and non-neoplastic colorectal tissue. Neoplastic tissues showed greater amounts of chondroitin sulphate and dermatan sulphate compared to non-neoplastic tissues, while heparan sulphate was decreased in neoplastic tissues.
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Carcinoma/química , Neoplasias Colorrectales/química , Matriz Extracelular/química , Glicómica/métodos , Glicosaminoglicanos/análisis , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma/patología , Sulfatos de Condroitina/análisis , Neoplasias Colorrectales/patología , Tejido Conectivo/química , Dermatán Sulfato/análisis , Progresión de la Enfermedad , Electroforesis en Gel de Poliacrilamida , Femenino , Heparitina Sulfato/análisis , Humanos , Masculino , Persona de Mediana Edad , Membrana Mucosa/metabolismo , Proteolisis , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
ABSTRACT Objective To investigate the possible genes that may be related to the mechanisms that modulate heparanase-1. Methods The analysis was conducted at Universidade Federal de São Paulo, on the data provided by: The Cancer Genome Atlas, University of California Santa Cruz Genome Browser, Kyoto Encyclopedia of Genes and Genomes Pathway Database, Database for Annotation, Visualization and Integrated Discovery Bioinformatics Database and the softwares cBioPortal and Ingenuity Pathway Analysis. Results Using messenger RNA expression pattern of different molecular subtypes of breast cancer, we proposed that heparinase-1 was co-related with its progression. In addition, genes that were analyzed presented co-expression with heparanase-1. The results that showed that heparanase-1 co-expressed with phosphoinositide 3-kinase adapter protein 1, sialic acid-binding immunoglobulin-like lectin 7, and leukocyte-associated immunoglobulin-like receptor 1 are directed related with immune system evasion during breast cancer progression. Furthermore, cathepsin L was co-expressed with heparanase-1 and transformed inactive heparanase-1 form into active heparanase-1, triggering extracellular matrix remodeling, which contributes to enhanced tumor-host interaction of the tumor. Conclusion The signaling pathway analysis using bioinformatics tools gives supporting evidence of possible mechanisms related to breast cancer development. Evasion genes of the immune system co-expressed with heparanase-1, a enzyme related with tumor progression.
RESUMO Objetivo Investigar os genes que podem estar relacionados aos mecanismos que modulam a heparanase-1. Métodos A análise foi realizada na Universidade Federal de São Paulo, utilizando dados fornecidos por: The Cancer Genome Atlas, University of California Santa Cruz Genome Browser, Kyoto Encyclopedia of Genes and Genomes Pathway Database, Database for Annotation, Visualization and Integrated Discovery Bioinformatics Database e os softwares cBioPortal e Ingenuity Pathway Analysis. Resultados Usando o perfil de expressão de RNA mensageiro de diferentes subtipos moleculares de câncer de mama, propusemos que a heparanase-1 esteve correlacionada com a progressão tumoral. Além disso, os genes analisados apresentaram coexpressão com heparanase-1. Os resultados mostraram que a heparanase-1 coexpressa com proteína adaptadora 1 da fosfoinositídeo 3-quinase, lectina 7 tipo Ig de ligação ao ácido siálico e receptor 1 do tipo imunoglobulina associado a leucócitos, estes genes estão diretamente relacionados à evasão do sistema imune durante a progressão do câncer de mama. Além disso, a catepsina L foi coexpressa com a heparanase-1 e transformou a forma inativa da heparanase-1 em heparanase-1 ativa, desencadeando o remodelamento da matriz extracelular, o que contribuiu para a interação do tumor com o ambiente tumoral. Conclusão A análise utilizando bioinformática fornece evidências de possíveis mecanismos relacionados ao desenvolvimento do câncer de mama. Genes de evasão do sistema imune foram coexpressos com a heparanase-1, uma enzima relacionada à progressão tumoral.
Asunto(s)
Humanos , Neoplasias de la Mama/genética , Glucuronidasa/genética , Simulación por ComputadorRESUMEN
Antimicrobial peptides (AMPs) are biologically active molecules with a broad-spectrum activity against a myriad of microorganisms. Aside from their antimicrobial functions, AMPs present physicochemical and structural properties that allow them to exert activity against other kind of cells, such as cancer cells. VmCT1 is a potent cationic amphipathic AMP from the venom of the scorpion Vaejovis mexicanus. In this study, we designed lysine-substituted VmCT1 analogs for verifying the influence of changes in the net positive charge on biological activities. The increase in the net positive charge caused by lysine substitutions in the hydrophilic portion, led to higher antimicrobial activity values (0.1–6.3?µmol?L-1) than VmCT1 (0.8–50?µmol?L-1) and higher activity against mammary cancer cells MCF-7 (6.3–12.5?µmol?L-1) than VmCT1 (12.5?µmol?L-1). Contrarily, when lysine-substitutions were made at the hydrophobic portion of the helical projection, the activity values decreased. However, the lysine-substitution at the center of the hydrophobic face led to the generation of an analog with antiplasmodial activity at the same concentration presented by VmCT1 (0.8?µmol?L-1). In this study, we demonstrated that it is possible to modulate biological activities and cytotoxicity of VmCT1 peptides by increasing their net positive charge using lysine residues, thus creating alternatives for standard-of-care therapeutics against different types of microorganisms and MCF-7 human breast cancer cells.
RESUMEN
Abstract Objective To investigate the action of testosterone (T), isolated or associated with estradiol benzoate (EB), on the proliferation markers and apoptosis of breasts of ovariectomized rats. Methods A total of 48 castrated female Wistar rats were divided into 6 groups, and each of them were submitted to one of the following treatments for 5 weeks: 1) control; 2) EB 50 mcg/day + T 50 mcg/day; 3) T 50mcg/day; 4) EB 50 mcg +T 300 mcg/day; 5) T 300 mcg/day; and 6) EB 50 mcg/day. After the treatment, the mammary tissue was submitted to a histological analysis and immunoexpression evaluation of proliferation markers (proliferating cell nuclear antigen, PCNA) and apoptosis (caspase-3). Results There was a statistically significant difference among the groups regarding microcalcifications and secretory activity, with higher prevalence in the groups treated with EB. There was no difference among the groups regarding atrophy, but a higher prevalence of atrophy was found in the groups that received T versus those that received EB +T. There was a difference among the groups regarding the PCNA (p = 0.028), with higher expression in the group submitted to EB +T 300 mcg/day. Regarding caspase-3, there was no difference among the groups; however, in the group submitted to EB +T 300 mcg/day, the expression was higher than in the isolated T group. Conclusion Isolated T did not have a proliferative effect on the mammary tissue, contrary to EB. Testosterone in combination with EB may or may not decrease the proliferation, depending on the dose of T.
Resumo Objetivo Investigar a ação da testosterona (T) isolada ou associada ao benzoato de estradiol (EB) na proliferação e apoptose de mamas de ratas ovariectomizadas. Métodos Um total de 48 ratas Wistar castradas foram divididas em 6 grupos, e cada um foi submetido a um dos seguintes tratamentos durante 5 semanas: 1) controle; 2) BE 50 mcg/dia + T 50mcg/dia; 3) T 50 mcg/dia; 4) BE 50 mcg + T 300mcg/dia; e) T 300 mcg/dia; e f) BE 50 mcg/dia. Após o tratamento, o tecido mamário foi submetido a análise histológica e avaliação de imunoexpressão de marcadores de proliferação (antígeno nuclear de células proliferantes, PCNA) e apoptose (caspase-3). Resultados Houve diferença estatisticamente significante entre os grupos com relação às microcalcificações e à atividade secretora, com maior prevalência nos grupos tratados com BE. Não houve diferença entre os grupos quanto à atrofia, mas houve maior prevalência de atrofia nos grupos que receberam T versus os que receberam BE+ T. Houve diferença entre os grupos quanto ao ANCP (p= 0,028), com maior expressão no grupo BE+ T 300 mcg/dia. Com relação à caspase-3, não houve diferença entre os grupos, mas, no grupo BE+ T 300 mcg/dia, a expressão foi maior do que no grupo de T isolada. Conclusão A T isolada não apresentou efeito proliferativo do tecido mamário, contrariamente ao EB. A T em associação ao EB pode diminuir ou não a proliferação, a depender da dose de T.
Asunto(s)
Animales , Femenino , Testosterona/farmacología , Mama/citología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Mama/patología , Calcinosis/patología , Ovariectomía , Biomarcadores/análisis , Ratas Wistar , Antígeno Nuclear de Célula en Proliferación/análisis , Estradiol/análogos & derivados , Estradiol/farmacología , Caspasa 3/análisisRESUMEN
Abstract Objective To analyze the effects of estrogen alone or in combination with progestogens and tibolone (TIB) on the expression of the extracellular matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), of perlecan, and of heparanase (HPSE) of the vascular walls of the carotid arteries. Methods A total of 30 250-day-old ovariectomized Wistar rats were orally treated for 5 weeks with: a) 1 mg/kg of estradiol benzoate (EB); b) EB + 0.2 mg/kg of medroxyprogesterone acetate (MPA); c) EB + 0.2mg/kg of norethisterone acetate (NETA); d) EB + 2 mg/kg of dydrogesterone (DI); e) 1 mg/kg of TIB; f) placebo (CTR). Following treatment, the expression of mRNA for MMP-2, MMP-9, and HPSE was analyzed by realtime polymerase chain-reaction (PCR), and the expression of MMP-2, of MMP-9, of tissue inhibitor of metalloproteinase 2 (TIMP-2), and of perlecan was quantified by immunohistochemistry in the carotid arteries. Results The groups showed significant differences on mRNA HPSE expression (p = 0.048), which was higher in the EB, EB + MPA, and TIB groups. There was no statistically significant difference in mRNA MMP-2 or MMP-9 expression. The immunohistochemical expression of MMP-2, of TIMP-2, of MMP-9, of HPSE, and of perlecan showed no differences between groups. Conclusion Estradiol alone or associated with MPA and TIB treatment can increase mRNA HSPE expression of the walls of the carotid arteries in ovariectomized rats.
Resumo Objetivo Analisar os efeitos do estrogênio isolado ou em combinação com progestogênios e tibolona (TIB) na expressão das metaloproteinases 2 e 9 da matriz extracelular (MMP-2 e MMP-9), da perlecan e da heparanase (HPSE) das paredes vasculares das artérias carótidas. Métodos Trinta ratas Wistar ovariectomizadas com 250 dias de idade foram tratadas oralmente por 5 semanas com: a) 1 mg/kg de benzoato de estradiol (EB); b) EB + 0,2 mg/kg de acetato de medroxiprogesterona (MPA); c) EB + 0,2mg/kg de acetato de noretisterona (NETA); d) EB + 2 mg/kg de didrogesterona (DI); e) 1 mg/kg de TIB; f) placebo (CTR). Após o tratamento, a expressão de mRNA para MMP-2, MMP- 9, e HPSE foi analisada por reação em cadeia da polimerase (RCP) em tempo real, e a expressão de MMP-2, MMP-9, inibidor tecidual de metaloproteinase 2 (TIMP-2), e de perlecan foi quantificado por imunohistoquímica em artérias carótidas. Resultados Os grupos apresentaram diferenças significativas na expressão do mRNA HPSE (p = 0,048), sendo maiores nos grupos EB, EB + MPA e TIB. Não houve diferença estatisticamente significativa nas expressões de mRNA MMP-2 ou MMP-9. A expressão imunohistoquímica de MMP-2, TIMP-2, MMP-9, HPSE e perlecan não mostrou diferenças entre os grupos. Conclusão O estradiol isolado ou associado ao tratamento com MPA e TIB pode aumentar a expressão de mRNA HSPE nas paredes das artérias carótidas em ratas ovariectomizadas.
Asunto(s)
Animales , Femenino , Ratas , Progestinas/farmacología , Arterias Carótidas/enzimología , Liasa de Heparina/efectos de los fármacos , Estradiol/análogos & derivados , Agentes Anticonceptivos Hormonales/farmacología , Norpregnenos/farmacología , Progestinas/administración & dosificación , Ovariectomía , Arterias Carótidas/efectos de los fármacos , Terapia de Reemplazo de Estrógeno , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Administración Oral , Ratas Wistar , Liasa de Heparina/genética , Liasa de Heparina/metabolismo , Proteoglicanos de Heparán Sulfato/genética , Proteoglicanos de Heparán Sulfato/metabolismo , Metaloproteinasa 2 de la Matriz/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Modelos Animales , Estradiol/administración & dosificación , Estradiol/farmacología , Agentes Anticonceptivos Hormonales/administración & dosificación , Norpregnenos/administración & dosificaciónRESUMEN
ABSTRACT Objective: To evaluate intervertebral disc levels of inflammatory factor (interleukin 6) and proteinase activity (cathepsin B) in patients with a degenerative disease and serum levels of interleukin 6, serum cathepsin B activity and hyaluronic acid biomarkers. Methods: We conducted immunohistochemistry studies of intervertebral discs to analyze interleukin 6 and cathepsin B levels of patients with degenerative disease and spine fracture (Control Group) and to measure hyaluronic acid, interleukin 6 and cathepsin B activity from sera of intervertebral disc degeneration patients, fracture patients, and healthy individuals. Results: Interleukin 6 and cathepsin B seem to be related with physiopathology of intervertebral disc degeneration, since the levels of both were higher in discs of patients with intervertebral disc degeneration. Interleukin 6 and cathepsin B do not represent good biomarkers of degenerative intervertebral disc disease, since the level of such compounds is increased in the plasma of patients with fractures. Conclusion: Hyaluronic acid can be a biomarker for intervertebral disc degeneration, because hyaluronic acid levels were higher only in sera of patients with intervertebral disc degeneration.
RESUMO Objetivo: Avaliar os níveis de fatores inflamatórios nos discos intervertebrais (interleucina 6) e proteinase (catepsina B) em pacientes com doença degenerativa de disco intervertebral, além de verificar os níveis séricos de interleucina 6, ácido hialurônico e atividade sérica da catepsina B. Métodos: Foi realizado exame imuno-histoquímica dos discos intervertebrais de pacientes com doença degenerativa e fratura da coluna (Grupo Controle) e análise do plasma de pacientes com doença degenerativa de disco intervertebral. Como controle, foram utilizados plasma de pacientes com fraturas, além de indivíduos saudáveis. Resultados: Interleucina 6 e catepsina B sugerem relação com a fisiopatologia da doença degenerativa de disco intervertebral, uma vez que os níveis de ambos foram maiores nos discos de pacientes com doença degenerativa de disco intervertebral. Interleucina 6 e catepsina B não representam bons biomarcadores da doença degenerativa do disco intervertebral, já que também encontram níveis aumentados em plasma de pacientes com fratura. Conclusão: O ácido hialurônico é um possível biomarcador de doença degenerativa de disco intervertebral, porque os níveis de ácido hialurônico foram maiores apenas em plasma de pacientes com doença degenerativa de disco intervertebral.
Asunto(s)
Humanos , Masculino , Femenino , Adulto , Catepsina B/sangre , Biomarcadores/sangre , Adyuvantes Inmunológicos/sangre , Interleucina-6/sangre , Degeneración del Disco Intervertebral/diagnóstico , Ácido Hialurónico/sangre , Inmunohistoquímica , Estudios de Casos y Controles , Estudios Prospectivos , Análisis de Varianza , Sensibilidad y Especificidad , Mediadores de Inflamación/sangre , Degeneración del Disco Intervertebral/fisiopatología , Degeneración del Disco Intervertebral/sangre , Disco Intervertebral/fisiopatologíaRESUMEN
Antimicrobial peptides (AMPs) are biologically active molecules with a broad-spectrum activity against a myriad of microorganisms. Aside from their antimicrobial functions, AMPs present physicochemical and structural properties that allow them to exert activity against other kind of cells, such as cancer cells. VmCT1 is a potent cationic amphipathic AMP from the venom of the scorpion Vaejovis mexicanus. In this study, we designed lysine-substituted VmCT1 analogs for verifying the influence of changes in the net positive charge on biological activities. The increase in the net positive charge caused by lysine substitutions in the hydrophilic portion, led to higher antimicrobial activity values (0.1–6.3?µmol?L-1) than VmCT1 (0.8–50?µmol?L-1) and higher activity against mammary cancer cells MCF-7 (6.3–12.5?µmol?L-1) than VmCT1 (12.5?µmol?L-1). Contrarily, when lysine-substitutions were made at the hydrophobic portion of the helical projection, the activity values decreased. However, the lysine-substitution at the center of the hydrophobic face led to the generation of an analog with antiplasmodial activity at the same concentration presented by VmCT1 (0.8?µmol?L-1). In this study, we demonstrated that it is possible to modulate biological activities and cytotoxicity of VmCT1 peptides by increasing their net positive charge using lysine residues, thus creating alternatives for standard-of-care therapeutics against different types of microorganisms and MCF-7 human breast cancer cells.
RESUMEN
STUDY DESIGN: This is a quantitative study of heparanase isoforms expression in degenerative and nondegenerative intervertebral discs (IVDs). OBJECTIVE: To quantify the expression of both heparanase isoforms (HPSE1 and HPSE2) in IVD tissues as classified by different degeneration grades using the Pfirrmann grading system, and to correlate the expression with the loss of extracellular matrix molecules observed in patients with the disease. SUMMARY OF BACKGROUND DATA: The loss of proteoglycans as observed in IVD degeneration may occur due to the enhanced expression of matrix degrading enzymes, such as heparanase. However, the heparanase function in IVD degeneration remains unclear. METHODS: This study comprised 53 surgical samples of degenerative discs obtained from patients with lumbar disc degeneration and 12 control samples collected from healthy individuals without any degenerative lumbar disc alterations who had accidental spine fractures.All patients underwent magnetic resonance imaging based on the Pfirrmann grading system for disc degeneration. Only the specimens that were classified according to magnetic resonance imaging evaluations as Pfirrmann grades I, II, III, and IV were analyzed.The tissue sections of the disc samples were subject to immunohistochemical staining with antibodies against the heparanase isoforms and to quantitative real time PCR to amplify heparanase isoforms cDNA. Protein and mRNA expressions were quantified. Analysis of variance and Student t test were used to compare the means of the study populations. RESULTS: The data demonstrated a gradual increase in both the heparanase isoform protein expression and disc degeneration progression. Besides, mRNA expression of both heparanase isoforms were significantly higher in degenerative than nondegenerative IVDs. CONCLUSION: The overexpression of HPSE1 and HPSE2 in the intervertebral degenerated discs suggests a role for these factors in mediating extracellular matrix remodeling in degenerative discs during disease development.