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1.
FASEB J ; 38(10): e23670, 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38747803

RESUMO

HPSE2, the gene-encoding heparanase 2 (Hpa2), is mutated in urofacial syndrome (UFS), a rare autosomal recessive congenital disease attributed to peripheral neuropathy. Hpa2 lacks intrinsic heparan sulfate (HS)-degrading activity, the hallmark of heparanase (Hpa1), yet it exhibits a high affinity toward HS, thereby inhibiting Hpa1 enzymatic activity. Hpa2 regulates selected genes that promote normal differentiation, tissue homeostasis, and endoplasmic reticulum (ER) stress, resulting in antitumor, antiangiogenic, and anti-inflammatory effects. Importantly, stress conditions induce the expression of Hpa2, thus establishing a feedback loop, where Hpa2 enhances ER stress which, in turn, induces Hpa2 expression. In most cases, cancer patients who retain high levels of Hpa2 survive longer than patients bearing Hpa2-low tumors. Experimentally, overexpression of Hpa2 attenuates the growth of tumor xenografts, whereas Hpa2 gene silencing results in aggressive tumors. Studies applying conditional Hpa2 knockout (cHpa2-KO) mice revealed an essential involvement of Hpa2 contributed by the host in protecting against cancer and inflammation. This was best reflected by the distorted morphology of the Hpa2-null pancreas, including massive infiltration of immune cells, acinar to adipocyte trans-differentiation, and acinar to ductal metaplasia. Moreover, orthotopic inoculation of pancreatic ductal adenocarcinoma (PDAC) cells into the pancreas of Hpa2-null vs. wild-type mice yielded tumors that were by far more aggressive. Likewise, intravenous inoculation of cancer cells into cHpa2-KO mice resulted in a dramatically increased lung colonization reflecting the involvement of Hpa2 in restricting the formation of a premetastatic niche. Elucidating Hpa2 structure-activity-relationships is expected to support the development of Hpa2-based therapies against cancer and inflammation.


Assuntos
Glucuronidase , Inflamação , Neoplasias , Humanos , Animais , Inflamação/metabolismo , Inflamação/patologia , Neoplasias/patologia , Neoplasias/metabolismo , Neoplasias/genética , Glucuronidase/metabolismo , Glucuronidase/genética , Camundongos , Estresse do Retículo Endoplasmático
2.
Proc Natl Acad Sci U S A ; 119(31): e2203167119, 2022 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-35881786

RESUMO

Heparan sulfate proteoglycans (HSPGs) mediate essential interactions throughout the extracellular matrix (ECM), providing signals that regulate cellular growth and development. Altered HSPG composition during tumorigenesis strongly aids cancer progression. Heparanase (HPSE) is the principal enzyme responsible for extracellular heparan sulfate catabolism and is markedly up-regulated in aggressive cancers. HPSE overactivity degrades HSPGs within the ECM, facilitating metastatic dissemination and releasing mitogens that drive cellular proliferation. Reducing extracellular HPSE activity reduces cancer growth, but few effective inhibitors are known, and none are clinically approved. Inspired by the natural glycosidase inhibitor cyclophellitol, we developed nanomolar mechanism-based, irreversible HPSE inhibitors that are effective within physiological environments. Application of cyclophellitol-derived HPSE inhibitors reduces cancer aggression in cellulo and significantly ameliorates murine metastasis. Mechanism-based irreversible HPSE inhibition is an unexplored anticancer strategy. We demonstrate the feasibility of such compounds to control pathological HPSE-driven malignancies.


Assuntos
Glucuronidase , Inibidores de Glicosídeo Hidrolases , Metástase Neoplásica , Animais , Proliferação de Células/efeitos dos fármacos , Glucuronidase/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases/farmacologia , Inibidores de Glicosídeo Hidrolases/uso terapêutico , Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Camundongos , Metástase Neoplásica/tratamento farmacológico
3.
Trends Biochem Sci ; 43(1): 18-31, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29162390

RESUMO

Heparanase, the sole heparan sulfate (HS)-degrading endoglycosidase, regulates multiple biological activities that enhance tumor growth, metastasis, angiogenesis, and inflammation. Heparanase accomplishes this by degrading HS and thereby regulating the bioavailability of heparin-binding proteins; priming the tumor microenvironment; mediating tumor-host crosstalk; and inducing gene transcription, signaling pathways, exosome formation, and autophagy that together promote tumor cell performance and chemoresistance. By contrast, heparanase-2, a close homolog of heparanase, lacks enzymatic activity, inhibits heparanase activity, and regulates selected genes that promote normal differentiation, endoplasmic reticulum stress, tumor fibrosis, and apoptosis, together resulting in tumor suppression. The emerging premise is that heparanase is a master regulator of the aggressive phenotype of cancer, while heparanase-2 functions as a tumor suppressor.


Assuntos
Neoplasias/metabolismo , Neoplasias/patologia , Polissacarídeo-Liases/metabolismo , Animais , Progressão da Doença , Humanos
4.
Int J Mol Sci ; 23(20)2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36293542

RESUMO

The kidney glomerular filtration barrier (GFB) is enriched with heparan sulfate (HS) proteoglycans, which contribute to its permselectivity. The endoglycosidase heparanase cleaves HS and hence appears to be involved in the pathogenesis of kidney injury and glomerulonephritis. We have recently reported, nonetheless, that heparanase overexpression preserved glomerular structure and kidney function in an experimental model of Adriamycin-induced nephropathy. To elucidate mechanisms underlying heparanase function in podocytes-key GFB cells, we utilized a human podocyte cell line and transgenic mice overexpressing heparanase. Notably, podocytes overexpressing heparanase (H) demonstrated significantly higher survival rates and viability after exposure to Adriamycin or hydrogen peroxide, compared with mock-infected (V) podocytes. Immunofluorescence staining of kidney cryo-sections and cultured H and V podocytes as well as immunoblotting of proteins extracted from cultured cells, revealed that exposure to toxic injury resulted in a significant increase in autophagic flux in H podocytes, which was reversed by the heparanase inhibitor, Roneparstat (SST0001). Heparanase overexpression was also associated with substantial transcriptional upregulation of autophagy genes BCN1, ATG5, and ATG12, following Adriamycin treatment. Moreover, cleaved caspase-3 was attenuated in H podocytes exposed to Adriamycin, indicating lower apoptotic cell death in H vs. V podocytes. Collectively, these findings suggest that in podocytes, elevated levels of heparanase promote cytoprotection.


Assuntos
Podócitos , Camundongos , Animais , Humanos , Podócitos/metabolismo , Doxorrubicina/toxicidade , Caspase 3/metabolismo , Peróxido de Hidrogênio/metabolismo , Glucuronidase/genética , Glucuronidase/metabolismo , Autofagia , Camundongos Transgênicos , Heparitina Sulfato/metabolismo , Proteoglicanas/metabolismo
5.
Semin Thromb Hemost ; 47(3): 240-253, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33794549

RESUMO

Cell surface proteoglycans are important constituents of the glycocalyx and participate in cell-cell and cell-extracellular matrix (ECM) interactions, enzyme activation and inhibition, and multiple signaling routes, thereby regulating cell proliferation, survival, adhesion, migration, and differentiation. Heparanase, the sole mammalian heparan sulfate degrading endoglycosidase, acts as an "activator" of HS proteoglycans, thus regulating tissue hemostasis. Heparanase is a multifaceted enzyme that together with heparan sulfate, primarily syndecan-1, drives signal transduction, immune cell activation, exosome formation, autophagy, and gene transcription via enzymatic and nonenzymatic activities. An important feature is the ability of heparanase to stimulate syndecan-1 shedding, thereby impacting cell behavior both locally and distally from its cell of origin. Heparanase releases a myriad of HS-bound growth factors, cytokines, and chemokines that are sequestered by heparan sulfate in the glycocalyx and ECM. Collectively, the heparan sulfate-heparanase axis plays pivotal roles in creating a permissive environment for cell proliferation, differentiation, and function, often resulting in the pathogenesis of diseases such as cancer, inflammation, endotheliitis, kidney dysfunction, tissue fibrosis, and viral infection.


Assuntos
Doença , Glucuronidase/metabolismo , Heparitina Sulfato/metabolismo , Humanos
6.
Adv Exp Med Biol ; 1221: 3-59, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274705

RESUMO

This review summarizes key developments in the heparanase field obtained 20 years prior to cloning of the HPSE gene and nearly 20 years after its cloning. Of the numerous publications and review articles focusing on heparanase, we have selected those that best reflect the progression in the field as well as those we regard important accomplishments with preference to studies performed by scientists and groups that contributed to this book. Apart from a general 'introduction' and 'concluding remarks', the abstracts of these studies are presented essentially as published along the years. We apologize for not being objective and not being able to include some of the most relevant abstracts and references, due to space limitation. Heparanase research can be divided into two eras. The first, initiated around 1975, dealt with identifying the enzyme, establishing the relevant assay systems and investigating its biological activities and significance in cancer and other pathologies. Studies performed during the first area are briefly introduced in a layman style followed by the relevant abstracts presented chronologically, essentially as appears in PubMed. The second era started in 1999 when the heparanase gene was independently cloned by 4 research groups [1-4]. As expected, cloning of the heparanase gene boosted heparanase research by virtue of the readily available recombinant enzyme, molecular probes, and anti-heparanase antibodies. Studies performed during the second area are briefly introduced followed by selected abstracts of key findings, arranged according to specific topics.


Assuntos
Glucuronidase/história , Pesquisa Translacional Biomédica/história , Glucuronidase/genética , História do Século XX , História do Século XXI , Humanos , Neoplasias
7.
Adv Exp Med Biol ; 1221: 253-283, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274713

RESUMO

Two decades following the cloning of the heparanase gene, the significance of this enzyme for tumor growth and metastasis cannot be ignored. Compelling pre-clinical and clinical evidence tie heparanase with all steps of tumor formation namely, initiation, growth, metastasis, and chemo resistance, thus confirming and significantly expanding earlier observations that coupled heparanase activity with the metastatic capacity of tumor cells. This collective effort has turned heparanase from an obscure enzyme to a valid target for the development of anti-cancer drugs, and led basic researchers and biotech companies to develop heparanase inhibitors as anti-cancer therapeutics, some of which are currently examined clinically. As expected, the intense research effort devoted to understanding the biology of heparanase significantly expanded the functional repertoire of this enzyme, but some principle questions are still left unanswered or are controversial. For example, many publications describe increased heparanase levels in human tumors, but the mechanism underlying heparanase induction is not sufficiently understood. Moreover, heparanase is hardly found to be increased in many studies utilizing methodologies (i.e., gene arrays) that compare tumors vs (adjacent) normal tissue. The finding that heparanase exert also enzymatic activity-independent function significantly expands the mode by which heparanase can function outside, but also inside the cell. Signaling aspects, and a role of heparanase in modulating autophagy are possibly as important as its enzymatic aspect, but these properties are not targeted by heparanase inhibitors, possibly compromising their efficacy. This Book chapter review heparanase function in oncology, suggesting a somewhat different interpretation of the results.


Assuntos
Glucuronidase/metabolismo , Neoplasias , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Glucuronidase/antagonistas & inibidores , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Transdução de Sinais
8.
Int J Cancer ; 145(6): 1596-1608, 2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31032901

RESUMO

Heparanase is an endo-ß-d-glucuronidase that cleaves heparan sulfate (HS) side chains of heparan sulfate proteoglycans. Compelling evidence tie heparanase levels with all steps of tumor formation including tumor initiation, growth, metastasis and chemo-resistance, likely involving augmentation of signaling pathways and gene transcription. In order to reveal the molecular mechanism(s) underlying the protumorigenic properties of heparanase, we established an inducible (Tet-on) system in U87 human glioma cells and applied gene array methodology in order to identify genes associated with heparanase induction. We found that CD24, a mucin-like cell adhesion protein, is consistently upregulated by heparanase and by heparanase splice variant devoid of enzymatic activity, whereas heparanase gene silencing was associated with decreased CD24 expression. This finding was further substantiated by a similar pattern of heparanase and CD24 immunostaining in glioma patients (Pearson's correlation; R = 0.66, p = 0.00001). Noteworthy, overexpression of CD24 stimulated glioma cell migration, invasion, colony formation in soft agar and tumor growth in mice suggesting that CD24 functions promote tumor growth. Likewise, anti-CD24 neutralizing monoclonal antibody attenuated glioma tumor growth, and a similar inhibition was observed in mice treated with a neutralizing mAb directed against L1 cell adhesion molecule (L1CAM), a ligand for CD24. Importantly, significant shorter patient survival was found in heparanase-high/CD24-high tumors vs. heparanase-high/CD24-low tumors for both high-grade and low-grade glioma (p = 0.02). Our results thus uncover a novel heparanase-CD24-L1CAM axis that plays a significant role in glioma tumorigenesis.


Assuntos
Neoplasias Encefálicas/patologia , Antígeno CD24/metabolismo , Glioma/patologia , Glucuronidase/metabolismo , Animais , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Feminino , Glioma/metabolismo , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Transdução de Sinais
9.
Proc Natl Acad Sci U S A ; 113(48): E7808-E7817, 2016 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-27849593

RESUMO

The emerging role of heparanase in tumor initiation, growth, metastasis, and chemoresistance is well recognized and is encouraging the development of heparanase inhibitors as anticancer drugs. Unlike the function of heparanase in cancer cells, very little attention has been given to heparanase contributed by cells composing the tumor microenvironment. Here we used a genetic approach and examined the behavior and function of macrophages isolated from wild-type (WT) and heparanase-knockout (Hpa-KO) mice. Hpa-KO macrophages express lower levels of cytokines (e.g., TNFα, IL1-ß) and exhibit lower motility and phagocytic capacities. Intriguingly, inoculation of control monocytes together with Lewis lung carcinoma (LLC) cells into Hpa-KO mice resulted in nearly complete inhibition of tumor growth. In striking contrast, inoculating LLC cells together with monocytes isolated from Hpa-KO mice did not affect tumor growth, indicating that heparanase is critically required for activation and function of macrophages. Mechanistically, we describe a linear cascade by which heparanase activates Erk, p38, and JNK signaling in macrophages, leading to increased c-Fos levels and induction of cytokine expression in a manner that apparently does not require heparanase enzymatic activity. These results identify heparanase as a key mediator of macrophage activation and function in tumorigenesis and cross-talk with the tumor microenvironment.


Assuntos
Carcinoma Pulmonar de Lewis/enzimologia , Glucuronidase/fisiologia , Ativação de Macrófagos , Macrófagos/enzimologia , Animais , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/patologia , Linhagem Celular , Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transplante de Neoplasias , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ativação Transcricional , Carga Tumoral , Microambiente Tumoral
10.
Proc Natl Acad Sci U S A ; 113(3): 704-9, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26729870

RESUMO

Heparanase is an endoglycosidase that cleaves heparan sulfate side chains of proteoglycans, resulting in disassembly of the extracellular matrix underlying endothelial and epithelial cells and associating with enhanced cell invasion and metastasis. Heparanase expression is induced in carcinomas and sarcomas, often associating with enhanced tumor metastasis and poor prognosis. In contrast, the function of heparanase in hematological malignancies (except myeloma) was not investigated in depth. Here, we provide evidence that heparanase is expressed by human follicular and diffused non-Hodgkin's B-lymphomas, and that heparanase inhibitors restrain the growth of tumor xenografts produced by lymphoma cell lines. Furthermore, we describe, for the first time to our knowledge, the development and characterization of heparanase-neutralizing monoclonal antibodies that inhibit cell invasion and tumor metastasis, the hallmark of heparanase activity. Using luciferase-labeled Raji lymphoma cells, we show that the heparanase-neutralizing monoclonal antibodies profoundly inhibit tumor load in the mouse bones, associating with reduced cell proliferation and angiogenesis. Notably, we found that Raji cells lack intrinsic heparanase activity, but tumor xenografts produced by this cell line exhibit typical heparanase activity, likely contributed by host cells composing the tumor microenvironment. Thus, the neutralizing monoclonal antibodies attenuate lymphoma growth by targeting heparanase in the tumor microenvironment.


Assuntos
Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Glucuronidase/imunologia , Linfoma/patologia , Animais , Anticorpos Monoclonais/farmacologia , Proliferação de Células/efeitos dos fármacos , Glucuronidase/isolamento & purificação , Células HEK293 , Humanos , Luciferases/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Peso Molecular , Metástase Neoplásica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Saponinas/farmacologia , Carga Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
11.
FASEB J ; 30(5): 1767-78, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26823454

RESUMO

The pulmonary vasculature constitutively expresses the integrin lymphocyte function-associated antigen-1 ligands intercellular adhesion molecule (ICAM)-1 and -2. In this study, effector T cells were temporarily entrapped by the lung vasculature on their way to inflamed lymph nodes, and this entrapment was strongly reduced in ICAM-1 and -2 double-deficient mice (79 and 86% reduction for CD8(+) and CD4(+) effectors, respectively, compared with wild-type mice). Although the pulmonary vasculature has been suggested to be masked by the heparan sulfate-containing glycocalyx, which is susceptible to heparanase-mediated shedding, lung and lymphocyte heparanase have been found to be unnecessary for this entrapment. Systemic LPS induced rapid neutrophil entrapment in the lung vasculature, but in contrast to T-cell entrapment, this sequestration was ICAM-1, ICAM-2, and heparanase independent. Furthermore, neutrophil migration into the bronchoalveolar space induced by LPS inhalation and LPS-induced leakage of red blood cells into this space were not dependent on lung ICAMs or heparanase activity. Nevertheless, heparanase was critical for neutrophil accumulation in smoke-exposed lungs. Our results indicate that, whereas T cells use ICAM-1 and -2 for temporary pulmonary entrapment, neutrophils get sequestered and extravasate into inflamed lungs independent of ICAMs. This is the first demonstration that the pulmonary vasculature is differentially recognized by T cells and neutrophils.-Petrovich, E., Feigelson, S. W., Stoler-Barak, L., Hatzav, M., Solomon, A., Bar-Shai, A., Ilan, N., Li, J.-P., Engelhardt, B., Vlodavsky, I., Alon, R. Lung ICAM-1 and ICAM-2 support spontaneous intravascular effector lymphocyte entrapment but are not required for neutrophil entrapment or emigration inside endotoxin-inflamed lungs.


Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Inflamação/induzido quimicamente , Molécula 1 de Adesão Intercelular/metabolismo , Pneumopatias/induzido quimicamente , Linfócitos/fisiologia , Neutrófilos/fisiologia , Animais , Antígenos CD/genética , Moléculas de Adesão Celular/genética , Movimento Celular , Endotoxinas/toxicidade , Regulação da Expressão Gênica/fisiologia , Glucuronidase/metabolismo , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Molécula 1 de Adesão Intercelular/genética , Pulmão/irrigação sanguínea , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos
12.
Drug Resist Updat ; 29: 54-75, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27912844

RESUMO

Heparanase, the sole heparan sulfate degrading endoglycosidase, regulates multiple biological activities that enhance tumor growth, angiogenesis and metastasis. Heparanase expression is enhanced in almost all cancers examined including various carcinomas, sarcomas and hematological malignancies. Numerous clinical association studies have consistently demonstrated that upregulation of heparanase expression correlates with increased tumor size, tumor angiogenesis, enhanced metastasis and poor prognosis. In contrast, knockdown of heparanase or treatments of tumor-bearing mice with heparanase-inhibiting compounds, markedly attenuate tumor progression further underscoring the potential of anti-heparanase therapy for multiple types of cancer. Heparanase neutralizing monoclonal antibodies block myeloma and lymphoma tumor growth and dissemination; this is attributable to a combined effect on the tumor cells and/or cells of the tumor microenvironment. In fact, much of the impact of heparanase on tumor progression is related to its function in mediating tumor-host crosstalk, priming the tumor microenvironment to better support tumor growth, metastasis and chemoresistance. The repertoire of the physio-pathological activities of heparanase is expanding. Specifically, heparanase regulates gene expression, activates cells of the innate immune system, promotes the formation of exosomes and autophagosomes, and stimulates signal transduction pathways via enzymatic and non-enzymatic activities. These effects dynamically impact multiple regulatory pathways that together drive inflammatory responses, tumor survival, growth, dissemination and drug resistance; but in the same time, may fulfill some normal functions associated, for example, with vesicular traffic, lysosomal-based secretion, stress response, and heparan sulfate turnover. Heparanase is upregulated in response to chemotherapy in cancer patients and the surviving cells acquire chemoresistance, attributed, at least in part, to autophagy. Consequently, heparanase inhibitors used in tandem with chemotherapeutic drugs overcome initial chemoresistance, providing a strong rationale for applying anti-heparanase therapy in combination with conventional anti-cancer drugs. Heparin-like compounds that inhibit heparanase activity are being evaluated in clinical trials for various types of cancer. Heparanase neutralizing monoclonal antibodies are being evaluated in pre-clinical studies, and heparanase-inhibiting small molecules are being developed based on the recently resolved crystal structure of the heparanase protein. Collectively, the emerging premise is that heparanase expressed by tumor cells, innate immune cells, activated endothelial cells as well as other cells of the tumor microenvironment is a master regulator of the aggressive phenotype of cancer, an important contributor to the poor outcome of cancer patients and a prime target for therapy.


Assuntos
Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Glucuronidase/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases/uso terapêutico , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Autofagia/efeitos dos fármacos , Autofagia/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Glucuronidase/genética , Glucuronidase/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Inflamação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/imunologia , Neovascularização Patológica/patologia , Neovascularização Patológica/prevenção & controle , Transdução de Sinais , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
13.
FASEB J ; 29(5): 2010-21, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25634957

RESUMO

Heparanase, the exclusive mammalian heparan sulfate-degrading enzyme, has been suggested to be utilized by leukocytes to penetrate through the dense basement membranes surrounding blood venules. Despite its established role in tumor cell invasion, heparanase function in leukocyte extravasation has never been demonstrated. We found that TH1/TC1-type effector T cells are highly enriched for this enzyme, with a 3.6-fold higher heparanase mRNA expression compared with naive lymphocytes. Using adoptive transfer of wild-type and heparanase-deficient effector T cells into inflamed mice, we show that T-cell heparanase was not required for extravasation inside inflamed lymph nodes or skin. Leukocyte extravasation through acute inflamed skin vessels was also heparanase independent. Furthermore, neutrophils emigrated to the inflamed peritoneal cavity independently of heparanase expression on either the leukocytes or on the endothelial and mesothelial barriers, and overexpression of the enzyme on neutrophils did not facilitate their emigration. However, heparanase absence significantly reduced monocyte emigration into the inflamed peritoneal cavity. These results collectively suggest that neither leukocyte nor endothelial heparanase is required for T-cell and neutrophil extravasation through inflamed vascular barriers, whereas this enzyme is required for optimal monocyte recruitment to inflamed peritoneum.


Assuntos
Endotélio Vascular/imunologia , Glucuronidase/fisiologia , Inflamação/imunologia , Neutrófilos/imunologia , Pele/imunologia , Linfócitos T/imunologia , Animais , Western Blotting , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Feminino , Citometria de Fluxo , Inflamação/enzimologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/citologia , Neutrófilos/enzimologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/enzimologia , Pele/patologia , Linfócitos T/citologia , Linfócitos T/enzimologia
14.
J Cell Biochem ; 116(8): 1668-79, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25735873

RESUMO

The development of the mammary gland involves formation of a branched arboreal structure resulting from the penetration and proliferation of epithelial cells into the fat pad. The mammary cells invade by remodeling their surrounding extracellular matrix (ECM), which are rich in proteins, and glycans such as heparan sulfate proteoglycans (HSPGs). There is increasing literature on how the interaction between signaling by ECM and matrix metalloproteinases (MMPs) is relevant to morphogenetic and physiological contexts. Here we sought to understand how heparanase, the sole mammalian heparan sulfate-degrading endoglycosidase may regulate mammary gland development. We found a robust localization of heparanase within growing end buds during branching in vivo. Using three-dimensional (3D) organotypic cultures, we showed that heparanase expression and activity are required for mammary epithelial invasion/branching within dense collagen I gels. Morphometric analysis of glands from both heparanase-overexpressing and knockout mice showed a direct correlation between degree of branching and the heparanase levels, confirming our 3D organotypic culture observations. Finally, we uncovered a reciprocal association between levels of heparanase and MMP14, a membrane-bound MMP, shedding further light on how branching occurs within developing mammary glands.


Assuntos
Glucuronidase/metabolismo , Glândulas Mamárias Animais/crescimento & desenvolvimento , Metaloproteinase 14 da Matriz/metabolismo , Morfogênese , Animais , Movimento Celular , Células Epiteliais/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Glucuronidase/genética , Glândulas Mamárias Animais/enzimologia , Camundongos , Técnicas de Cultura de Órgãos , Transdução de Sinais
15.
Semin Thromb Hemost ; 41(2): 244-54, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25682080

RESUMO

Heparanase is an endo-ß-glucuronidase that enzymatically cleaves heparan sulfates (HS) and heparan sulfate proteoglycan (HSPG) structures. Heparanase expression levels by tumors were correlated with cell invasion, angiogenic activity, and poor prognosis. Heparanase can also possess pro-tumorigenic effects independent of its enzymatic activity. Using human melanoma MV3 cells, we demonstrate that latent heparanase activates in a tightly temporary-regulated manner the binding function of the integrin very late antigen-4 (VLA-4), an important component in the metastatic spread of melanoma cells. shRNA-mediated knockdown of syndecan-4 (SDC-4) indicated that this proteoglycan is the key element to convey heparanase binding via focal adhesion complex formation, detected by vinculin staining, to an upregulated VLA-4 binding function. This inside-out signaling pathway of VLA-4 involved activated FAK and Akt, but apparently not PKCα/δ. VLA-4, however, appears representative of other integrins which together impact the heparanase/integrin activation axis in tumorigenicity. Biosensor measurements provided an insight as to how heparin can interfere with this activation process. While low-molecular-weight heparin (LMWH) cannot replace heparanase bound to SDC-4, LMWH can compete with SDC-4 binding of heparanase. Since blockade of heparanase by LMWH has functional consequences for reduced VLA-4 binding, latent heparanase appears as a novel, so far unnoticed target of heparin, underlying its antimetastatic activity.


Assuntos
Sistemas de Liberação de Medicamentos , Heparina Liase/metabolismo , Heparina de Baixo Peso Molecular/farmacologia , Integrina alfa4beta1/metabolismo , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Heparina Liase/genética , Humanos , Integrina alfa4beta1/genética , Melanoma/genética , Melanoma/patologia , Metástase Neoplásica , Proteínas de Neoplasias/genética
16.
Cell Mol Life Sci ; 71(22): 4457-70, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24788042

RESUMO

Heparanase activity plays a decisive role in cell dissemination associated with cancer metastasis. Cellular uptake of heparanase is considered a pre-requisite for the delivery of latent 65-kDa heparanase to lysosomes and its subsequent proteolytic processing and activation into 8- and 50-kDa protein subunits by cathepsin L. Heparan sulfate proteoglycans, and particularly syndecan, are instrumental for heparanase uptake and activation, through a process that has been shown to occur independent of rafts. Nevertheless, the molecular mechanism underlying syndecan-mediated internalization outside of rafts is unclear. Here, we examined the role of syndecan-1 cytoplasmic domain in heparanase processing, utilizing deletion constructs lacking the entire cytoplasmic domain (Delta), the conserved (C1 or C2), or variable (V) regions. Heparanase processing was markedly increased following syndecan-1 over-expression; in contrast, heparanase was retained at the cell membrane and its processing was impaired in cells over-expressing syndecan-1 deleted for the entire cytoplasmic tail. We have next revealed that conserved domain 2 (C2) and variable (V) regions of syndecan-1 cytoplasmic tail mediate heparanase processing. Furthermore, we found that syntenin, known to interact with syndecan C2 domain, and α actinin are essential for heparanase processing.


Assuntos
Actinina/metabolismo , Glucuronidase/metabolismo , Sindecana-1/metabolismo , Sinteninas/metabolismo , Actinina/antagonistas & inibidores , Actinina/genética , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células HEK293 , Humanos , Camundongos , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Sindecana-1/química , Sindecana-1/genética , Sinteninas/antagonistas & inibidores , Sinteninas/genética
17.
J Biol Chem ; 288(17): 12366-75, 2013 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-23504323

RESUMO

Heparanase functions as a heparan sulfate-degrading enzyme and as a ligand for an unidentified signaling receptor(s). Here, several reactions involved in the activation of the PI3K-AKT pathway by latent heparanase were characterized. Protein suppression using specific siRNAs revealed that heparanase-induced phosphorylation of AKT at Ser-473 was RICTOR-mTOR-dependent, whereas ILK and PAK1/2 were dispensable. p110α was the PI3K catalytic isoform preferred by heparanase for AKT activation and cell proliferation because the p110α inhibitor YM024 blocked these processes. Heparanase-induced AKT phosphorylation was low in mouse embryonic fibroblast cells expressing a RAS interaction-defective p110α compared with wild type cells, indicating that RAS has an important role in the PI3K-AKT activation. The response to heparanase was also inefficient in suspension cultures of several cell lines, suggesting a requirement of integrins in this pathway. Adhesion via either αVß3 or α5ß1 promoted heparanase-induced AKT phosphorylation, and a stronger effect was seen when both integrins were engaged. Simultaneous inhibition of FAK and PYK2 using a chemical inhibitor, or suppression of their expression, inhibited heparanase-induced AKT activation and cell proliferation. Stimulation of cells with heparanase enhanced their resistance against oxidative stress- or growth factor starvation-induced apoptosis. These results demonstrate that there is an intimate cross-talk between the heparanase receptor(s) and integrins during induction of the prosurvival PI3K-AKT pathway by heparanase.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Glucuronidase/metabolismo , Integrina alfaVbeta3/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Vitronectina/metabolismo , Transdução de Sinais/fisiologia , Animais , Células CHO , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe I de Fosfatidilinositol 3-Quinases/genética , Cricetinae , Cricetulus , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Glucuronidase/genética , Humanos , Integrina alfaVbeta3/genética , Camundongos , Morfolinas/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Receptores de Vitronectina/genética , Transdução de Sinais/efeitos dos fármacos , Tiofenos/farmacologia , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo
18.
Arterioscler Thromb Vasc Biol ; 33(2): e56-65, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23162016

RESUMO

OBJECTIVE: Factors and mechanisms that activate macrophages in atherosclerotic plaques are incompletely understood. We examined the capacity of heparanase to activate macrophages. METHODS AND RESULTS: Highly purified heparanase was added to mouse peritoneal macrophages and macrophage-like J774 cells, and the levels of tumor necrosis factor-α, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 were evaluated by ELISA. Gene expression was determined by RT-PCR. Cells collected from Toll-like receptor-2 and Toll-like receptor-4 knockout mice were evaluated similarly. Heparanase levels in the plasma of patients with acute myocardial infarction, stable angina, and healthy subjects were determined by ELISA. Immunohistochemistry was applied to detect the expression of heparanase in control specimens and specimens of patients with stable angina or acute myocardial infarction. Addition or overexpression of heparanase variants resulted in marked increase in tumor necrosis factor-α, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 levels. Mouse peritoneal macrophages harvested from Toll-like receptor-2 or Toll-like receptor-4 knockout mice were not activated by heparanase. Plasma heparanase level was higher in patients with acute myocardial infarction, compared with patients with stable angina and healthy subjects. Pathologic coronary specimens obtained from vulnerable plaques showed increased heparanase staining compared with specimens of stable plaque and controls. CONCLUSIONS: Heparanase activates macrophages, resulting in marked induction of cytokine expression associated with plaque progression toward vulnerability.


Assuntos
Aterosclerose/enzimologia , Glucuronidase/metabolismo , Ativação de Macrófagos , Macrófagos Peritoneais/enzimologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Angina Estável/sangue , Angina Estável/enzimologia , Animais , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Linhagem Celular , Quimiocina CCL2/metabolismo , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/enzimologia , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Glucuronidase/sangue , Glucuronidase/genética , Humanos , Imuno-Histoquímica , Interleucina-1/metabolismo , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/patologia , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Infarto do Miocárdio/sangue , Infarto do Miocárdio/enzimologia , Placa Aterosclerótica , Reação em Cadeia da Polimerase , Ruptura Espontânea , Transdução de Sinais , Fatores de Tempo , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Transfecção , Fator de Necrose Tumoral alfa/metabolismo
19.
Pediatr Blood Cancer ; 61(10): 1883-5, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24737657

RESUMO

Heparanase is an endo-beta D-glucuronidase capable of cleaving heparan sulfate side chains, yielding heparan sulfate fragments. Heparanase activity has been correlated with the metastatic potential of tumor-derived cells, angiogenesis, autoimmunity and inflammation. We performed a study of heparanase expression in specimens obtained from patients with Langerhans cell histiocytosis (LCH). Paraffin embedded slides from 25 patients were studied by immunohistochemistry for heparanase. Most patients had positive staining for heparanase (21/25). There was no positive association with severity of disease and other clinical characteristics. Further studies are required to clarify the role of heparanase in the pathogenesis of LCH.


Assuntos
Glucuronidase/biossíntese , Histiocitose de Células de Langerhans/enzimologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Glucuronidase/análise , Histiocitose de Células de Langerhans/patologia , Humanos , Imuno-Histoquímica , Lactente , Masculino , Adulto Jovem
20.
Trends Biochem Sci ; 34(10): 511-9, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19733083

RESUMO

Heparanase activity is strongly implicated in structural remodeling of the extracellular matrix, a process which can lead to invasion by tumor cells. In addition, heparanase augments signaling cascades leading to enhanced phosphorylation of selected protein kinases and increased gene transcription associated with aggressive tumor progression. This function is apparently independent of heparan sulfate and enzyme activity, and is mediated by a novel protein domain localized at the heparanase C-terminus. Moreover, the functional repertoire of heparanase is expanded by its regulation of syndecan clustering, shedding, and mitogen binding. Recent reports indicate that modified glycol-split heparin, which inhibits heparanase activity, can profoundly inhibit the progression of tumor xenografts produced by myeloma and carcinoma cells, thus moving anti-heparanase therapy closer to reality.


Assuntos
Matriz Extracelular/metabolismo , Glucuronidase/metabolismo , Transdução de Sinais/fisiologia , Animais , Adesão Celular/fisiologia , Endocitose/fisiologia , Ativação Enzimática , Glucuronidase/química , Glucuronidase/genética , Heparina/química , Heparina/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Humanos , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/terapia , Neoplasias/metabolismo , Receptores de Superfície Celular/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Sindecana-1/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo
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