The Heparan Sulfate Binding Peptide in Tumor Progression of Triple-Negative Breast Cancer.

Angiogenesis is the formation of new vessels from pre-existing vasculature. The heparan sulfate chains from endothelial cell proteoglycans interact with the major angiogenic factors, regulating blood vessels´ formation. Since the FDA´s first approval, anti-angiogenic therapy has shown tumor progression inhibition and increased patient survival. Previous work in our group has selected an HS-binding peptide using a phage display system. Therefore, we investigated the effect of the selected peptide in angiogenesis and tumor progression. The HS-binding peptide showed a higher affinity for heparin N-sulfated. The HS-binding peptide was able to inhibit the proliferation of human endothelial umbilical cord cells (HUVEC) by modulation of FGF-2. It was verified a significant decrease in the tube formation of human endothelial cells and capillary formation of mice aorta treated with HS-binding peptide. HS-binding peptide also inhibited the formation of sub-intestinal blood vessels in zebrafish embryos. Additionally, in zebrafish embryos, the tumor size decreased after treatment with HS-binding peptide.

Cell therapy in the treatment of intervertebral disc degeneration / Terapia celular no tratamento da degeneração de disco intervertebral / Terapia celular en el tratamiento de la degeneración de disco intervertebral

ABSTRACT Approximately 80% of the world population experiences some type of back pain at some point in their life, and in 10% of this population the pain causes chronic disability resulting in a high cost for the treatment of these patients, in addition to compromising their work and social interaction abilities. Current treatment strategies include the surgical procedure for degenerated intervertebral disc resection, the nerve root block and physiotherapy. However, such treatments only relieve symptoms and do not prevent the degeneration of intervertebral discs. Therefore, new therapeutic strategies have emerged and include manipulating cells to recover the degenerated disc. This article will discuss the possible cell therapy alternatives used in the disc regeneration process, featuring a descriptive study of translational medicine that involves clinical aspects of new treatment alternatives and knowledge of basic research areas, such as cellular and molecular biology. Level of evidence V; Expert Opinion.

RESUMO Aproximadamente 80% da população mundial sofre algum tipo de dor nas costas em alguma fase de vida, sendo que em 10% dessa população, as dores acarretam incapacidade crônica, deflagrando alto custo de tratamento desses pacientes, além de comprometer as habilidades de trabalho e convívio social desses indivíduos. As estratégias de tratamento atuais incluem o procedimento cirúrgico por ressecção do disco intervertebral degenerado, bloqueio de raízes nervosas e fisioterapia. Entretanto, tais tratamentos apenas aliviam os sintomas e não impedem que ocorra a degeneração de discos intervertebrais. Portanto, novas estratégias terapêuticas têm surgido e incluem a manipulação de células com o objetivo de recuperar o disco degenerado. No presente artigo, serão discutidas as diferentes possibilidades alternativas de terapias celulares no processo de regeneração discal, caracterizando um estudo descritivo da medicina translacional que envolve aspectos clínicos de novas alternativas de tratamento e o conhecimento de áreas básicas de pesquisa como biologia celular e molecular. Nível de evidência V; Opinião do Especialista.

RESUMEN Aproximadamente 80% de la población mundial sufre algún tipo de dolor de espalda en alguna etapa de la vida, y en 10% de esa población, los dolores causan incapacidad crónica, deflagrando alto costo de tratamiento de esos pacientes, además de comprometer las habilidades laborales y convivencia social de esos individuos. Las estrategias de tratamiento actuales incluyen el procedimiento quirúrgico para la resección del disco intervertebral degenerado, bloqueo de las raíces nerviosas y fisioterapia. Entretanto, tales tratamientos solo alivian los síntomas y no impiden que ocurra la degeneración de discos intervertebrales. Por lo tanto, han surgido nuevas estrategias terapéuticas e incluyen la manipulación de células con el objetivo de recuperar el disco degenerado. En el presente artículo se discutirán las diferentes posibilidades alternativas de las terapias celulares en el proceso de regeneración discal, caracterizando un estudio descriptivo de la medicina traslacional que involucra aspectos clínicos de nuevas alternativas de tratamiento y conocimiento de áreas básicas de investigación como biología celular y molecular. Nivel de evidencia V; Opinión del especialista.

Heparanase modulation by Wingless/INT (Wnt).

Heparanase is an endo-beta-glucuronidase, the only enzyme in mammals capable of cleaving heparan sulfate/heparin chains from proteoglycans. The oligosaccharides generated by heparanase present extensive biological functions since such oligosaccharides interact with adhesion molecules, growth factors, angiogenic factors and cytokines, modulating cell proliferation, migration, inflammation, and carcinogenesis. However, the regulation of heparanase activity is not fully understood. It is known that heparanase is synthesized as an inactive 65 kDa isoform and that post-translation processing forms an active 50 kDa enzyme. In the present study, we are interested in investigating whether heparanase is regulated by its own substrate as observed with many other enzymes. Wild-type Chinese hamster (Cricetulus griséus) ovary cells (CHO-K1) were treated with different doses of heparin. Heparanase expression was analyzed by Real-time PCR and flow cytometry. Also, heparanase activity was measured. The heparanase activity assay was performed using a coated plate with biotinylated heparan sulfate. In the present assay, a competitive heparin inhibition scenario was set aside. Exogenous heparin trigged a cell signaling pathway that increased heparanase mRNA and protein levels. The Wnt/beta-catenin pathway, judged by TCF-driven luciferase activity, seems to be involved to enhance heparanase profile during treatment with exogenous heparin. Lithium chloride treatment, an activator of the Wnt/beta-catenin pathway, confirmed such mechanism of transduction in vivo using zebrafish embryos and in vitro using CHO-K1 cells. Taken together the results suggest that heparin modulates heparanase expression by Wnt/beta-catenin.

Endocytosis and the Participation of Glycosaminoglycans Are Important to the Mechanism of Cell Death Induced by ß-Hairpin Antimicrobial Peptides.

The cytotoxic mode of action of four antimicrobial peptides (AMPs) (gomesin, tachyplesin, protegrin, and polyphemusin) against a HeLa cell tumor model is discussed. A study of cell death by AMP stimulation revealed some similarities, including annexin-V externalization, reduction of mitochondrial potential, insensitivity against inhibitors of cell death, and membrane permeabilization. Evaluation of signaling proteins and gene expression that control cell death revealed wide variation in the responses to AMPs. However, the ability to cross cell membranes emerged as an important characteristic of AMP-dependent cell death, where endocytosis mediated by dynamin is a common mechanism. Furthermore, the affinity between AMPs and glycosaminoglycans (GAGs) and GAG participation in the cytotoxicity of AMPs were verified. The results show that, despite their primary and secondary structure homology, these peptides present different modes of action, but endocytosis and GAG participation are an important and common mechanism of cytotoxicity for ß-hairpin peptides.

In silico investigation of heparanase-correlated genes in breast cancer subtypes.

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.

The Good and Bad Sides of Heparanase-1 and Heparanase-2.

In this chapter, we will emphasize the importance of heparan sulfate proteoglycans (HSPG) in controlling various physiological and pathological molecular mechanisms and discuss how the heparanase enzyme can modulate the effects triggered by HSPG. Additionally, we will also navigate about the existing knowledge of the possible role of heparanase-2 in biological events. Heparan sulfate is widely distributed and evolutionarily conserved, evidencing its vital importance in cell development and functions such as cell proliferation, migration, adhesion, differentiation, and angiogenesis. During remodeling of the extracellular matrix, the breakdown of heparan sulfate by heparanase results in the release of molecules containing anchored glycosaminoglycan chains of great interest in heparanase-mediated cell signaling pathways in various physiological states, tumor development, inflammation, and other diseases. Taken together, it appears that heparanase plays a key role in the maintenance of the pathology of cancer and inflammatory diseases and is a potential target for anti-cancer therapies. Therefore, heparanase inhibitors are currently being examined in clinical trials as novel cancer therapeutics. Heparanase-2 has no enzymatic activity, displays higher affinity for heparan sulfate and the coding region alignment shows 40% identity with the heparanase gene. Heparanase-2 plays an important role in embryogenic development however its mode of action and biological function remain to be elucidated. Heparanase-2 functions as an inhibitor of the heparanase-1 enzyme and also inhibits neovascularization mediated by VEGF. The HPSE2 gene is repressed by the Polycomb complex, together suggesting a role as a tumor suppressor.

In silico investigation of heparanase-correlated genes in breast cancer subtypes / Investigação in silico dos genes correlacionados à heparanase nos diferentes subtipos de câncer de mama

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.

The Profile of Heparanase Expression Distinguishes Differentiated Thyroid Carcinoma from Benign Neoplasms.

INTRODUCTION: The search for a specific marker that could help to distinguish between differentiated thyroid carcinoma and benign lesions remains elusive in clinical practice. Heparanase (HPSE) is an endo-beta-glucoronidase implicated in the process of tumor invasion, and the heparanase-2 (HPSE2) modulates HPSE activity. The aim of this study was to evaluate the role of heparanases in the development and differential diagnosis of follicular pattern thyroid lesions. METHODS: HPSE and HPSE2 expression by qRT-PCR, immunohistochemistry evaluation, western blot analysis and HPSE enzymatic activity were evaluated. RESULTS: The expression of heparanases by qRT-PCR showed an increase of HPSE2 in thyroid carcinoma (P = 0.001). HPSE activity was found to be higher in the malignant neoplasms than in the benign tumors (P<0.0001). On Western blot analysis, HPSE2 isoforms were detected only in malignant tumors. The immunohistochemical assay allowed us to establish a distinct pattern for malignant and benign tumors. Carcinomas showed a typical combination of positive labeling for neoplastic cells and negative immunostaining in colloid, when compared to benign tumors (P<0.0001). The proposed diagnostic test presents sensitivity and negative predictive value of around 100%, showing itself to be an accurate test for distinguishing between malignant and benign lesions. CONCLUSIONS: This study shows, for the first time, a distinct profile of HPSE expression in thyroid carcinoma suggesting its role in carcinogenesis.

Development of new methods for determining the heparanase enzymatic activity.

INTRODUCTION: Heparanase is a mammalian endo-ß-glucuronidase. Notwithstanding its importance in various pathological and non-pathological events few straightforward methods for heparanase enzymatic activity has been stated. The aim of this study was to develop two heparanase activity assays to cover a whole range of applications. First, a fast and easy method based on commercial homogenous substrate, fondaparinux, was described. The other method is a quantitative assay based on biotinylated heparan sulfate that uses an easier technique to immobilize the substrate in a 96-well plate. METHODS: 1): The heparanase recombinant enzyme and fondaparinux were incubated overnight. After incubation, a fluorescent redox marker, resazurin, was added. The reduction of resazurin depends on the amount of glucuronic acid released by heparanase digestion. Fluorescence measurements were done using excitation and emission wavelengths of 560 nm and 590 nm, respectively. METHODS: 2): The 96-well plate was incubated with protamine sulfate. Subsequently, biotinylated heparan sulfate was immobilized. The enzymatic assay was performed using chimeric recombinant heparanase at different concentrations. In sequence, the immobilized biotinylated heparan sulfate that was not digested by recombinant heparanase was bound to streptavidin conjugated with europium. Fluorescence was measured using a time-resolved fluorometer. CONCLUSION: Both methods have high sensitivity and can be used to detect heparanase activity. Fondaparinux assay is a quick and easy method for screening of heparanase inhibitors using recombinant enzyme or bacterial crude extract. Biotinylated heparan sulfate assay can be used for quantitative analysis in biological samples and protamine sulfate showed been capable to immobilized heparan sulfate.

Analysis of heparanase isoforms and cathepsin B in the plasma of patients with gastrointestinal carcinomas: analytical cross-sectional study / Análises das isoformas de heparanase e da catepsina B em plasma de pacientes com carcinomas gastrointestinais: estudo transversal analítico

CONTEXT AND OBJECTIVE: Heparanase-1 degrades heparan sulfate and has been correlated with tumor progression. Although the isoform heparanase-2 has no catalytic activity, it seems to be important for modulating heparanase-1 activity. Cathepsin B is a proteinase involved in tumor metastasis. The aim of this study was to analyze heparanase isoform expression and cathepsin B activity in plasma samples from patients with gastrointestinal carcinomas, compared with healthy individuals (control group). DESIGN AND SETTING: This was an analytical cross-sectional study. Peripheral blood samples were collected at a Brazilian public hospital, from 21 patients with histopathological diagnoses of gastrointestinal carcinomas and from 43 healthy individuals. The analyses were performed in two Brazilian medical schools. METHODS: Heparanase isoforms were identified and quantified in plasma samples by means of Western blot. The enzymatic activities of heparanase-1 and cathepsin B were also measured. RESULTS: The results demonstrated that the expression of both heparanase isoforms was significantly greater in plasma samples from gastrointestinal carcinoma patients, compared with the control group. Logistic regression analysis showed that increased heparanase-1 and heparanase-2 expression was exclusively dependent on the ...

CONTEXTO E OBJETIVO: A heparanase-1 degrada heparam sulfato e está relacionada à progressão de tumor. Apesar de a isoforma heparanase-2 não possuir atividade catalítica, parece ser importante para modular a atividade da heparanase-1. A catepsina B é uma proteinase envolvida na metástase de tumores. O objetivo deste estudo foi analisar a expressão das isoformas de heparanase e atividade da catepsina B em amostras de plasma de pacientes com carcinomas gastrointestinais, comparando-se com indivíduos saudáveis (grupo controle). TIPO DE ESTUDO E LOCAL: Este é um estudo transversal analítico. Foram coletadas amostras de sangue periférico, em hospital público brasileiro, de 21 pacientes com diagnóstico histopatológico de carcinoma gastrointestinal e 43 indivíduos saudáveis. As análises foram realizadas em duas faculdades de medicina brasileiras. MÉTODOS: As isoformas da heparanase foram identificadas e quantificadas em amostras de plasma por Western blot. As atividades enzimáticas de heparanase-1 e catepsina B foram também mensuradas. RESULTADOS: Os resultados demonstraram que as expressões das isoformas de heparanase foram significativamente maiores nas amostras de plasma de pacientes com carcinoma gastrointestinal em comparação com ...

Analysis of heparanase isoforms and cathepsin B in the plasma of patients with gastrointestinal carcinomas: analytical cross-sectional study.

CONTEXT AND OBJECTIVE: Heparanase-1 degrades heparan sulfate and has been correlated with tumor progression. Although the isoform heparanase-2 has no catalytic activity, it seems to be important for modulating heparanase-1 activity. Cathepsin B is a proteinase involved in tumor metastasis. The aim of this study was to analyze heparanase isoform expression and cathepsin B activity in plasma samples from patients with gastrointestinal carcinomas, compared with healthy individuals (control group). DESIGN AND SETTING: This was an analytical cross-sectional study. Peripheral blood samples were collected at a Brazilian public hospital, from 21 patients with histopathological diagnoses of gastrointestinal carcinomas and from 43 healthy individuals. The analyses were performed in two Brazilian medical schools. METHODS: Heparanase isoforms were identified and quantified in plasma samples by means of Western blot. The enzymatic activities of heparanase-1 and cathepsin B were also measured. RESULTS: The results demonstrated that the expression of both heparanase isoforms was significantly greater in plasma samples from gastrointestinal carcinoma patients, compared with the control group. Logistic regression analysis showed that increased heparanase-1 and heparanase-2 expression was exclusively dependent on the tumor. There was a significant increase in heparanase-1 and cathepsin B activity in the patients' plasma. CONCLUSION: Overexpression of heparanase-1 and heparanase-2, along with increased heparanase-1 and cathepsin B activity in plasma, is associated with the diagnosis of gastrointestinal carcinoma. These findings provide support for using non-invasive assays (plasma samples) as an auxiliary method for diagnosing gastrointestinal tumors.

Evaluation of glycosaminoglycans and heparanase in placentas of women with preeclampsia.

BACKGROUND: Preeclampsia 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.