Navegando por Palavras-chave "Heparanase"

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    Alterações em células de tumor de mama MCF-7 após superexpressão da enzima heparanase-1
    (Universidade Federal de São Paulo, 2022-07) Silva, Mariane Barros Ribeiro da [UNIFESP]; Pinhal, Maria Aparecida da Silva [UNIFESP]; http://lattes.cnpq.br/7511274763693292; http://lattes.cnpq.br/2778388041557973
    Introdução: A enzima heparanase-1 (HPSE1) é uma endo-β-glucuronidase que cliva cadeias de heparam sulfato (HS) e heparina e está diretamente relacionada com a carcinogênese. Objetivo: O objetivo do estudo foi avaliar como a superexpressão de HPSE1 pode promover alterações moleculares em células de tumor de mama humano do subtipo molecular luminal A (MCF-7). Métodos: Células MCF-7 foram estavelmente transfectadas com o cDNA de HPSE1 e a avaliação da expressão de receptores específicos, bem como dos diferentes proteoglicanos de heparam sulfato (PGHS) foi realizada por ensaios de RT-PCR quantitativo, microscopia confocal e citometria de fluxo. A atividade enzimática de HPSE1 foi determinada por degradação de heparam sulfato biotinilado. A quantificação de ácido hialurônico (AH) foi determinada por ensaio de Elisa-like. A identificação e quantificação dos glicosaminoglicanos sulfatados (GAG) foi realizada por marcação com [35S]-sulfato. A estrutura do HS foi determinada por cromatografia líquida de alta pressão (HPLC). A proliferação celular foi obtida por incorporação de bromodeoxiuridina (BrdU). O ensaio de clonogenicidade foi realizado para a determinação da capacidade de formação de colônias. A viabilidade celular e o perfil de exossomos foram avaliados após tratamento com os quimioterápicos Fauldoxo, Paclitaxel e Fauldcispla. Resultados: Os resultados mostraram que a superexpressão de HPSE1 altera a expressão de isoformas do receptor CD44 e aumenta a expressão de HER2, porém não afeta o padrão de proliferação celular e capacidade de formação de colônias. Houve alteração do perfil de GAG, estrutura do HS, bem como aumento da expressão das enzimas que participam da biossíntese de HS, após superexpressão de HPSE1. Os dados também evidenciaram aumento significativo dos PGHS, sindecam-2, sindecam-3 e sindecam-4 e diminuição de sindecam-1 em células tranfectadas com HPSE. Análises in sílico e in vitro mostraram correlação direta entre o nível de expressão de sindecam3 e do fator indutor de hipóxia (HIF-1α) com HPSE1. Conclusão: Os resultados revelam inúmeras alterações do perfil de expressão de moléculas cruciais envolvidas na carcinogênese, indica possível utilização de HPSE1 como um marcador adicional no diagnóstico do câncer de mama auxiliando também no desenvolvimento de potencial terapias alvo.
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    Evaluation of glycosaminoglycans and heparanase in placentas of women with preeclampsia
    (Elsevier B.V., 2014-11-01) Brosco Fama, Eduardo Augusto; Souza, Renan Salvioni; Melo, Carina Mucciolo [UNIFESP]; Pompei, Luciano Melo; Silva Pinhal, Maria Aparecida [UNIFESP]; FMABC; Universidade Federal de São Paulo (UNIFESP)
    Background: Preedampsia is a multisystem disorder whose etiology remains unclear. It is already known that circulation of soluble fms-like tyrosine kinase-1 (sFlt-1) is directly involved in pre-eclampsia development. However, the molecular mechanisms involved with sFlt-1 shedding are still unidentified. We identified, quantified glycosaminoglycans and determined the enzymatic activity of heparanase in placentas of women with preeclampsia, in order to possibly explain if these compounds could be related to cellular processes involved with preeclampsia.Methods:A total of 45 samples collected from placentas, 15 samples from placentas of preeclampsia women and 30 samples from non-affected women. Heparan sulfate and dermatan sulfate were identified and quantified by agarose gel electrophoresis, whilst hyaluronic acid was quantified by an ELISA like assay. Heparanase activity was determined using biotynilated heparan sulfate as substrate.Results: the results showed that dermatan sulfate (P = 0.019), heparan sulfate levels (P = 0.015) and heparanase activity (P = 0.006) in preeclampsia were significantly higher than in the control group. There was no significant difference between the groups for hyaluronic acid expression in placentas (P = 0.110). the present study is the first to demonstrate directly the increase of heparan sulfate in human placentas from patients with preeclampsia, suggesting that endogenous heparan sulfate could be involved in the release of sFlt-1 from placenta, increasing the level of circulating sFlt-1.Conclusion: Alterations of extracellular matrix components in placentas with preeclampsia raise the possibility that heparan sulfate released by heparanase is involved in mechanisms of preeclampsia development. Published by Elsevier B.V.
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    Extracellular matrix alterations in the Peyronie's disease
    (Elsevier Science Bv, 2017) Watanabe, Marcelo Silva; Theodoro, Therese Rachel; Coelho, Natalia Lima [UNIFESP]; Mendes, Aline [UNIFESP]; Pereira Leonel, Monica Luzia; Mader, Ana Maria; Nader, Helena Bonciani [UNIFESP]; Glina, Sidney; Silva Pinhal, Maria Aparecida [UNIFESP]
    Peyronie's disease is characterized by fibrous plaque formation of the tunica albuginea, causing penile deformity and fertility problems. The aim of the present study was to investigate alterations in the extracellular matrix in Peyronie's disease. The study used tissues collected by surgical procedure from individuals that presented a well-established disease, while control samples were obtained by biopsies of fresh cadavers. Immunohistochemistry analysis followed by digital quantification was performed to evaluate TGF-beta, heparanases and metalloproteinases (MMPs). The profile of sulfated glycosaminoglycans, chondroitin sulfate and dermatan sulfate was determined by agarose gel electrophoresis, while hyaluronic acid quantification was obtained by an ELISA-like assay. The expression of mRNA was investigated for syndecan-1 proteoglycan (Syn-1), interleukine-6 (IL-6), hyaluronic acid synthases, and hyaluronidases. Pathologic features showed decreased apoptosis and blood vessel number in Peyronie's tissues. TGF-beta and IL-6 were significantly enhanced in Peyronie's disease. There was an increased expression of heparanases, though no alteration was observed for MMPs. Hyaluronic acid as well as hyaluronic acid synthases, hyaluronidases, and dermatan sulfate were not changed, while the level of chondroitin sulfate was significantly (P = 0.008, Mann-Whitney test) increased in Peyronie's samples. Heparanases and sulfated glycosaminoglycans seem to be involved in extracellular matrix alterations in Peyronie's disease. (C) 2017 Production and hosting by Elsevier B.V. on behalf of Cairo University.
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    Heparan sulfate mediates trastuzumab effect in breast cancer cells
    (Biomed Central Ltd, 2013-10-01) Suarez, Eloah Rabello [UNIFESP]; Paredes-Gamero, Edgar Julian [UNIFESP]; Del Giglio, Auro; Tersariol, Ivarne Luis dos Santos [UNIFESP]; Nader, Helena Bonciani [UNIFESP]; Pinhal, Maria Aparecida Silva [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Fac Med ABC
    Background: Trastuzumab is an antibody widely used in the treatment of breast cancer cases that test positive for the human epidermal growth factor receptor 2 (HER2). Many patients, however, become resistant to this antibody, whose resistance has become a major focus in breast cancer research. But despite this interest, there are still no reliable markers that can be used to identify resistant patients. A possible role of several extracellular matrix (ECM) components-heparan sulfate (HS), Syn-1(Syndecan-1) and heparanase (HPSE1)-in light of the influence of ECM alterations on the action of several compounds on the cells and cancer development, was therefore investigated in breast cancer cell resistance to trastuzumab.Methods: the cDNA of the enzyme responsible for cleaving HS chains from proteoglycans, HPSE1, was cloned in the pEGFP-N1 plasmid and transfected into a breast cancer cell lineage. We evaluated cell viability after trastuzumab treatment using different breast cancer cell lines. Trastuzumab and HS interaction was investigated by confocal microscopy and Fluorescence Resonance Energy Transfer (FRET). the profile of sulfated glycosaminoglycans was also investigated by [S-35]-sulfate incorporation. Quantitative RT-PCR and immunofluorescence were used to evaluate HPSE1, HER2 and Syn-1 mRNA expression. HPSE1 enzymatic activity was performed using biotinylated heparan sulfate.Results: Breast cancer cell lines responsive to trastuzumab present higher amounts of HER2, Syn-1 and HS on the cell surface, but lower levels of secreted HS. Trastuzumab and HS interaction was proven by FRET analysis. the addition of anti-HS to the cells or heparin to the culture medium induced resistance to trastuzumab in breast cancer cells previously sensitive to this monoclonal antibody. Breast cancer cells transfected with HPSE1 became resistant to trastuzumab, showing lower levels of HER2, Syn-1 and HS on the cell surface. in addition, HS shedding was increased significantly in these resistant cells.Conclusion: Trastuzumab action is dependent on the availability of heparan sulfate on the surface of breast cancer cells. Furthermore, our data suggest that high levels of heparan sulfate shed to the medium are able to capture trastuzumab, blocking the antibody action mediated by HER2. in addition to HER2 levels, heparan sulfate synthesis and shedding determine breast cancer cell susceptibility to trastuzumab.
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    Heterogeneidade molecular entre diferentes linhagens celulares estabelecidas de câncer de mama humano triplo-negativo
    (Universidade Federal de São Paulo, 2022-10-28) Ferreira, Ariana Carolina [UNIFESP]; Pinhal, Maria Aparecida da Silva [UNIFESP]; Melo, Carina Mucciolo [UNIFESP]; http://lattes.cnpq.br/1814847465463146; http://lattes.cnpq.br/7511274763693292; https://lattes.cnpq.br/7080145324327503
    Introdução: O câncer de mama triplo negativo (TNBC) representa de 15% a 20% dos casos de neoplasia maligna mamária, sendo constituído por tumores com características heterogêneas, com maior incidência de metástases, menor sobrevida dos pacientes e opções de tratamento limitadas, em que a terapia hormonal e os medicamentos direcionados ao receptor HER2 são ineficazes. Portanto, é crucial a identificação de biomarcadores moleculares que auxiliem no desenvolvimento de estratégias de tratamento mais efetivas e possíveis combinações utilizando terapias direcionadas. Objetivos: Caracterizar as linhagens celulares estabelecidas de câncer de mama triplo-negativo, MDA-MB-453, MDA-MB-231, HCC38 e BT-549, por análise do perfil de proliferação e migração celular, capacidade de formação de colônias, e determinar o padrão de expressão gênica de moléculas envolvidas com a carcinogênese (sindecans, heparanases, metaloproteases, glicosaminoglicanos, CD44 e HER2). Comparar a resposta das diferentes linhagens celulares triplo-negativas ao tratamento com Paclitaxel, bem como, avaliar possíveis alterações moleculares após tratamento. Métodos: A expressão gênica foi determinada por PCR quantitativo, microscopia confocal e citometria de fluxo. O perfil de glicosaminoglicanos foi obtido por eletroforese após incorporação de [35S]-sulfato. A resposta ao tratamento com Paclitaxel foi avaliada por ensaios de viabilidade celular (MTT) e morte celular (Anexina V/ PI). A resistência ao Paclitaxel foi obtida após tratamento contínuo das células MDA-MB-231 com tal quimioterápico. A compreensão do papel biológico do sindecam-4 (SDC4) foi feita por análises in silico e silenciamento utilizando short hairpin RNAs (shRNA). Resultados: A análise da expressão gênica revelou que as linhagens celulares triplo-negativas MDA-MB-453 e HCC38 expressam mRNA para HER2, sendo que tal receptor está ativado na linhagem HCC38. A expressão de CD44 foi significantemente menor nas células MDA-MB-453, sendo que tal linhagem não exibiu isoformas variantes do receptor (CD44v). As linhagens de origem metastática, MDA-MB-453 e MDA-MB-231, não exibiram as isoformas CD44v3I e CD44v5. As células triplo-negativas estudadas também exibiram diferentes perfis de glicosaminoglicanos sulfatados. As células HCC38 e BT-549, apresentaram maior expressão gênica de sindecam-4 e resposta mais efetiva ao Paclitaxel em relação às células de origem metastática, MDA-MB-453 e MDA-MB-231. Análises in silico evidenciaram que a baixa expressão de sindecam-4 (SDC4) está associada à presença de linfonodos metastáticos e baixa sobrevida dos pacientes acometidos por câncer de mama. O silenciamento gênico de SDC4 acarretou menor resposta ao tratamento com Paclitaxel. As células MDA-MB-231 resistentes ao Paclitaxel apresentaram alterações morfológicas e diminuição da expressão de SDC4. O tratamento quimioterápico foi capaz de induzir alterações significativas na expressão gênica das diferentes linhagens de câncer triplo-negativo. Conclusões: As linhagens celulares, embora pertencentes ao subtipo molecular triplo-negativo de câncer de mama, exibiram características moleculares e resposta ao tratamento quimioterápico distintos, evidenciando possíveis alvos para o desenvolvimento de abordagens terapêuticas mais efetivas. Os dados in silico e in vitro mostraram a importância do SDC4 na tumorigênese do câncer de mama, sugerindo que tal molécula possa servir como potencial biomarcador para o prognóstico do câncer de mama e, desta forma, auxiliar na decisão de estratégias terapêuticas mais assertivas.
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    Immunohistochemical Expression of Heparanases 1 and 2 in Benign Tissue and in Invasive Neoplasia of the Endometrium A Case-Control Study
    (Lippincott Williams & Wilkins, 2015-02-01) Signorini Filho, Roney Cesar [UNIFESP]; Azevedo Focchi, Gustavo Rubino de [UNIFESP]; Theodoro, Therese Rachell; Silva Pinhal, Maria Aparecida; Nicolau, Sergio Mancini [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Fac Med ABC
    Objectives: Our purpose was to compare the expression of heparanase isoforms, in normal and in neoplastic endometrium. in a pioneering way, we sought to evaluate the expression of heparanase 1 (HPSE1) and heparanase 2 (HPSE2) in glandular and in stromal tissues.Methods: This is a case-control study, conducted retrospectively in a public hospital, using paraffin blocks of endometrial tissue from patients admitted from 2002 to 2011 with and without endometrial cancer, with regard to the immunohistochemical expression of HPSE1 and HPSE2. the paraffin blocks were used for tissue microarray analysis and immunohistochemistry study in glandular and stromal tissues.Results: in the study period, 195 participants were enrolled, 75 with and 120 without cancer. There was no significant difference between them regarding HPSE1 expression, both in gland and in stromal tissues. Heparanase 1 expression in the glandular tissue was more frequent among those with high-grade carcinoma, compared with patients with carcinoma type I. the difference in the expression of HPSE2 was significant between groups: it was less frequent in the controls than in the patients with cancer in the glandular tissue. in the stromal tissue, HPSE2 expression was significantly higher in the controls than in the patients with cancer and different when patients of the secretory endometrium subgroup were compared with those with hypotrophic, proliferative endometriums or with architectural disorders. No significant difference was found in the heparanase expressions in patients with cancer according to prognosis factors.Conclusions: Heparanase 1 is more intensely expressed in the glandular tissue of high-grade compared with type I carcinomas. Heparanase 2 is more intensely expressed in the glandular tissue of cancer than in nonneoplastic endometrium, whereas the HPSE2 expression in the stromal tissue is higher in the nonneoplastic controls compared with the group of patients with cancer mainly in the secretory endometrium. This suggests that HPSE2 might be stimulated by progesterone, with a possible antineoplastic role, antagonist to HPSE1, to be further investigated.
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    Peptídeos Potencialmente Úteis no Tratamento do Câncer de Mama e o Envolvimento de Componentes da Matriz Extracelular na Resistência ao Trastuzumab.
    (Universidade Federal de São Paulo (UNIFESP), 2011-02-22) Suarez, Eloah Rabello [UNIFESP]; Pinhal, Maria Aparecida da Silva [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    HER2 is a member of epidermal growth factor family of receptors that is an essential mediator of cell proliferation and differentiation. In breast cancer patients, HER2 overexpression is associated with disease aggressiveness, chemo and hormone therapy resistance and poor prognosis. Currently, a HER2 specific monoclonal antibody named trastuzumab, was developed as a treatment for breast cancer; however, there are some reports of resistance to this treatment and it can also cause a high rate of cardiac failure, despite the high cost. The aim of the present study was select specific peptides target to recombinant HER2 protein using a phage display technology. The selected cyclic peptides, called Hercid and Tavelorb, were chemically synthesized. The bacteriophages expressing the specific peptides selected, as well as the synthetic peptides were assayed using different breast cancer cell lines compared to the trastuzumab. Cellular viability, migration and apoptosis/necrosis were evaluated. The results showed that the peptides were able to reduce the cell viability around 50% alone and 85% in association. Tavelorb was able to induce apoptosis/ necrosis in 70% of SKBR3 cells and when associated with Hercid, the effect has increased to 90%. This association decreases tumor cells migration around 85%. The HER2 binding assays showed that the peptides compete with trastuzumab. These peptides co-localize with acidic vesicles around 40-50%, suggesting a possible endocytosis induction and HER2 degradation. The peptides and trastuzumab co-localized with heparan sulfate around 70% suggesting that the binding of these molecules and heparan sulfate could play an important role in antitumoral activity over breast cancer cells. The data propose a potential use of these peptides as an alternative for breast cancer treatment. In addition, we analyzed whether some extracellular matrix components influence trastuzumab treatment. Heparanase-1 (HPSE-1) overexpression effect was analyzed using MCF7 cells stable transfected with HPSE-1 cDNA (MCF7-HPSE-1). The glycosaminoglycans profile, HPSE-1, HPSE-2, Syndecan-1 (Syn-1) and HER2 mRNA expression, HPSE- 1 activity and cell viability were evaluated in different breast cancer cells treated or not with trastuzumab. MCF7-HPSE-1 becomes completely resistant to trastuzumab. HPSE-1 transfection changes the galactosaminoglycans profile of MCF7. Trastuzumab co-localizes in high levels with heparan sulfate (HS) and their binding is necessary to antibody activity. In MCF7 cells, trastuzumab decreases HPSE-1, HPSE-2, HER2 and Syn-1 mRNA expression, while in MCF7-HPSE-1 the antibody increases the mRNA expression of these molecules. SKBR3 cells have the highest expression levels of these molecules, but low HPSE-1 activity, which seems to be determinant to trastuzumab response and modulated by HPSE-2. Our results have demonstrated that an ideal concentration of HS in cell surface and medium, regulated by trastuzumab, is necessary to its action. Secreted HS can sequester trastuzumab, decreasing the antibody amount disposable to interact with HER2 in cell surface. HS secreted could also block HPSE-2, which will not be able to inhibit HPSE-1 activity, contributing for tumor resistance to trastuzumab and supporting tumoral progression. In addition, as the relation HPSE-1/HER2 expression decreases, breast cancer cells sensibility to trastuzumab also decreases. These new insights could be useful when devising strategies for overcoming trastuzumab resistance in HER2 positive cancers.