乙酰肝素酶在肺癌侵袭转移及治疗中作用的研究进展

乙酰肝素酶是一种β-D-葡萄糖醛酸内切酶,为体内惟一能够降解硫酸乙酰肝素蛋白多糖的酶,可以在特定部位裂解硫酸乙酰肝素蛋白多糖,破坏细胞外基质及基底膜,并参与恶性肿瘤新生血管的形成,与恶性肿瘤的侵袭转移密切相关,乙酰肝素酶由此成为抗肿瘤作用的新靶点,其抑制剂有望成为临床抗肿瘤治疗的新途径.本文就乙酰肝素酶在肺癌侵袭转移及治疗中作用的研究作一综述.     

乙酰肝素酶与动脉粥样硬化

乙酰肝素酶(heparanase,HPA)是哺乳动物体内唯一降能解细胞外基质中硫酸乙酰肝素蛋白多糖的β-D-葡萄糖醛酸内切酶.近年来的相关研究表明,HPA与动脉粥样硬化斑块的形成和发展密切相关,并且与斑块从稳定向不稳定的转化有关.文章就HPA在动脉粥样硬化进展过程中促进斑块内炎症反应、与蛋白水解酶协同作用以及参与血液高凝状态等方面的相关研究进展进行了综述.     

乙酰肝素酶在胃癌组织中的表达及其与临床病理特征的关系

目的 探讨乙酰肝素酶在胃癌组织中的表达及其与临床病理特征的关系.方法 应用免疫组化方法检测60例胃癌术后组织标本及正常组织中乙酰肝素酶的表达,分析乙酰肝素酶表达与胃癌临床病理特征的关系.结果 胃癌组织中存在乙酰肝素酶蛋白的表达,并定位于肿瘤细胞质中.60例胃癌组织中有40例乙酰肝素酶表达阳性(40/60,66.7％),正常组织有1例表达阳性(1/10,10％),两组间差异显著(P ＜0.05).乙酰肝素酶蛋白在胃癌中的表达与患者性别、年龄、肿瘤直径无关(P＞0.05),与组织学分级、TNM分期、淋巴结转移相关(P＜0.05).结论 乙酰肝素酶在胃癌中高表达,与胃癌进展程度和恶性行为相关,对胃癌的发生、发展起促进作用,可为临床治疗和诊断提供一定依据.     

肝素、低分子肝素的抗肿瘤作用研究进展

肝素、低分子肝素是临床常用的一种抗凝药物.近年来研究发现,其还具有多种生物活性和临床用途,包括抗血管生成及抗肿瘤作用等.肝素、低分子肝素可通过改变肿瘤细胞的细胞和分子学环境而干预肿瘤的发展,例如,肝素、低分子肝素具有抗肿瘤细胞增殖作用,抑制血管内皮细胞增殖和抑制血管形成,通过抑制乙酰肝素酶、基质金属蛋白酶等酶的活性及作...     

肝素、低分子肝素的抗肿瘤作用研究进展

肝素、低分子肝素是临床常用的一种抗凝药物.近年来研究发现,其还具有多种生物活性和临床用途,包括抗血管生成及抗肿瘤作用等.肝素、低分子肝素可通过改变肿瘤细胞的细胞和分子学环境而干预肿瘤的发展,例如,肝素、低分子肝素具有抗肿瘤细胞增殖作用,抑制血管内皮细胞增殖和抑制血管形成,通过抑制乙酰肝素酶、基质金属蛋白酶等酶的活性及作用阻止细胞外基质及基底膜的降解,抑制肿瘤细胞黏附及其随后向组织的侵袭、迁移等.     

宫颈上皮内瘤变、宫颈癌中乙酰肝素酶的表达及意义

目的 探讨乙酰肝素酶在宫颈黏膜上皮内瘤变与宫颈癌中的表达及其意义.方法 采用免疫组化SP法检测乙酰肝素酶在56例宫颈黏膜上皮内瘤变(轻度12例,中度26例,重度18例)、54例宫颈癌(伴有淋巴结转移的20例,不伴淋巴转移34例)中的表达.结果 乙酰肝素酶在宫颈黏膜上皮轻、中、重度上皮内瘤变组织中表达率分别为33.33%,38.48%,44.44%,宫颈癌中表达率为48.14%.乙酰肝毒酶在宫颈黏膜上皮内瘤变与宫颈癌中的表达无统计学意义(P＞0.05).20例宫颈癌伴淋巴结转移者,乙酰肝素酶表达(70.00%)明显高于无转移组(35.29%),P＜0.01.结论 乙酰肝素酶表达与宫颈癌发生、发展无关,与宫颈癌组织淋巴结转移有关.     

乙酰肝素酶与冠状动脉粥样硬化斑块稳定性的关系

目的探讨乙酰肝素酶在人体冠状动脉稳定性斑块和不稳定性斑块中的分布及其在不稳定性斑块形成中的可能作用。方法选取经临床和病理解剖证实的急性冠状动脉综合征病例进行免疫组织化学检测并通过计算机分析系统进行量化分析。结果不稳定性斑块组乙酰肝素酶的聚集程度明显高于稳定性斑块组。结论乙酰肝素酶与斑块不稳定性存在一定联系,可能参与了斑块的去稳定过程。     

乙酰肝素酶反义寡核苷酸对人胃癌细胞株SGC 7901表达bFGF的抑制作用

目的利用人胃癌细胞株SGC7901细胞,探讨乙酰肝素酶反义寡核苷酸对人胃癌细胞株SGC7901中表达碱性成纤维细胞生长因子(bFGF)的影响。方法采用脂质体介导乙酰肝素酶反义寡核苷酸转染人胃癌细胞株SGC7901细胞,采用免疫细胞化学法观察细胞内bFGF的变化。结果脂质体介导转染乙酰肝素酶反义寡核苷酸后,SGC 7901胃癌细胞内乙酰肝素酶mRNA表达下降,bF-GF阳性表达率由转染前的72．23％降至40．25％。转染后细胞bFGF表达明显减少。结论转染乙酰肝素酶反义寡核苷酸可以有效抑制胃癌细胞bFGF的表达。     

宫颈鳞癌整合素α5β1蛋白和乙酰肝素酶蛋白的表达及生物学行为

目的探讨宫颈鳞癌组织中整合素α5β1蛋白和乙酰肝素酶(HPA)蛋白的表达及临床意义。方法应用组织芯片技术结合免疫组化方法(SP法)检测68例宫颈鳞癌组织和20例宫颈正常组织整合素α5β1蛋白和乙酰肝素酶(HPA)蛋白表达情况,分析两者在宫颈鳞癌组织中的表达相关性及生物学行为。结果宫颈鳞癌组织整合素α5β1蛋白和乙酰肝素酶蛋白的阳性表达率分别为77.9%和72.1%,均高于正常宫颈组织(P0.05);整合素α5β1、HPA蛋白均与宫颈鳞癌的分化程度、临床分期、淋巴结转移和浸润深度显著相关;整合素α5β1蛋白和乙酰肝素酶蛋白在宫颈鳞癌组织中的表达呈现正相关性(r=0.594)。结论整合素α5β1蛋白和乙酰肝素酶蛋白高表达与宫颈鳞癌的发生、发展有关,具有正协同作用,并可作为判断宫颈鳞癌浸润、转移及预后的预测因素。     

牛主动脉蛋白聚糖对培养的人脐静脉内皮细胞生长的影响

为观察牛主动脉中蛋白聚糖对培养的人脐静脉内皮细胞生长的影响及其在动脉粥样硬化形成中的作用，用解聚提取，离子交换及凝胶过滤柱层析法从牛主动脉内，中膜分离出硫酸乙酰肝素蛋白聚糖、硫酸软骨素蛋白聚糖和硫酸皮肤素－硫酸软骨素蛋白聚糖。     

Heparanase enhances the generation of activated factor X in the presence of tissue factor and activated factor VII

Background

Heparanase is an endo-β-D-glucuronidase dominantly involved in tumor metastasis and angiogenesis. Recently, we demonstrated that heparanase is involved in the regulation of the hemostatic system. Our hypothesis was that heparanase is directly involved in activation of the coagulation cascade.

Design and Methods

Activated factor X and thrombin were studied using chromogenic assays, immunoblotting and thromboelastography. Heparanase levels were measured by enzyme-linked immunosorbent assay. A potential direct interaction between tissue factor and heparanase was studied by co-immunoprecipitation and far-western assays.

Results

Interestingly, addition of heparanase to tissue factor and activated factor VII resulted in a 3- to 4-fold increase in activation of the coagulation cascade as shown by increased activated factor X and thrombin production. Culture medium of human embryonic kidney 293 cells over-expressing heparanase and its derivatives increased activated factor X levels in a non-enzymatic manner. When heparanase was added to pooled normal plasma, a 7- to 8-fold increase in activated factor X level was observed. Subsequently, we searched for clinical data supporting this newly identified role of heparanase. Plasma samples from 35 patients with acute leukemia at presentation and 20 healthy donors were studied for heparanase and activated factor X levels. A strong positive correlation was found between plasma heparanase and activated factor X levels (r=0.735, P=0.001). Unfractionated heparin and an inhibitor of activated factor X abolished the effect of heparanase, while tissue factor pathway inhibitor and tissue factor pathway inhibitor-2 only attenuated the procoagulant effect. Using co-immunoprecipitation and far-western analyses it was shown that heparanase interacts directly with tissue factor.

Conclusions

Overall, our results support the notion that heparanase is a potential modulator of blood hemostasis, and suggest a novel mechanism by which heparanase increases the generation of activated factor X in the presence of tissue factor and activated factor VII.     

Adaptive evolution of heparanase in hypoxia-tolerant Spalax: gene cloning and identification of a unique splice variant

Heparan sulfate (HS) side chains of HS proteoglycans bind to and assemble extracellular matrix proteins and play important roles in cell-cell and cell-extracellular matrix interactions. HS chains bind a multitude of bioactive molecules and thereby function in the control of multiple normal and pathological processes. Enzymatic degradation of HS by heparanase, a mammalian endoglycosidase, affects the integrity and functional state of tissues and is involved in, among other processes, inflammation, angiogenesis, and cancer metastasis. Here, we report the cloning of heparanase from four Israeli species of the blind subterranean mole rat (Spalax ehrenbergi superspecies), 85% homologous to the human enzyme. Unlike its limited expression in human tissues, heparanase is highly expressed in diverse Spalax tissues. Moreover, we have identified a unique splice variant of the Spalax enzyme lacking 16 aa encoded by exon 7. This deletion resulted in a major defect in trafficking and processing of the heparanase protein, leading to a loss of its enzymatic activity. Interspecies variation was noted in the sequence and in the expression of the splice variant of the heparanase gene in blind mole rats living under different ecogeographical stresses, indicating a possible role in adaptation to stress in Spalax evolution.     

Heparanase, tissue factor, and cancer

Heparanase is an endo-beta- D-glucuronidase that is capable of cleaving heparan sulfate side chains of heparan sulfate proteoglycans on cell surfaces and the extracellular matrix, activity that is strongly implicated in tumor metastasis and angiogenesis. Evidence was provided that heparanase overexpression in human leukemia, glioma, and breast carcinoma cells results in a marked increase in tissue factor (TF) levels. Likewise, TF was induced by exogenous addition of recombinant heparanase to tumor cells and primary endothelial cells, induction that was mediated by p38 phosphorylation and correlated with enhanced procoagulant activity. TF induction was further confirmed in heparanase-overexpressing transgenic mice and correlated with heparanase expression levels in leukemia patients. Heparanase was also found to be involved in the regulation of tissue factor pathway inhibitor (TFPI). It was shown that heparanase overexpression or exogenous addition induces two- to threefold increase of TFPI expression. Similarly, heparanase stimulated accumulation of TFPI in the cell culture medium. Extracellular accumulation exceeded, however, the observed increase in TFPI at the protein level and appeared to be independent of heparan sulfate and heparanase enzymatic activity. Instead, a physical interaction between heparanase and TFPI was demonstrated, suggesting a mechanism by which secreted heparanase interacts with TFPI on the cell surface, leading to dissociation of TFPI from the cell membrane and increased coagulation activity, thus further supporting the local prothrombotic function of heparanase. As heparins are strong inhibitors of heparanase, in view of the effect of heparanase on TF/TFPI pathway, the role of heparins' anticoagulant activity may potentially be expanded.     

Heparanase is highly expressed and regulates proliferation in GH-secreting pituitary tumor cells

Pituitary tumorigenesis involves remodeling of the extracellular matrix (ECM). Heparanase, an endoglycosidase capable of degrading heparan sulfate, a major polysaccharide constituent of the ECM, is implicated in diverse processes associated with ECM remodeling, such as morphogenesis, angiogenesis, and tumor invasion. The aim of this study was to investigate the possible role of heparanase in pituitary tumorigenesis. Human normal pituitaries and pituitary tumors were examined for heparanase mRNA and protein expression using real-time PCR and immunohistochemistry, respectively. Cell proliferation was assessed by colony formation after heparanase overexpression in GH3 and MtT/S cells. Cell viability and cell cycle progression were evaluated after heparanase gene silencing. Higher heparanase mRNA and protein expression was noted in GH tumors as compared with normal pituitaries. Heparanase overexpression in GH3 and MtT/S cells resulted in a 2- to 3-fold increase in colony number, compared with control cells. Cell viability decreased by 50% after heparanase gene silencing due to induced apoptosis reflected by increased fraction of cleaved poly-ADP-ribose polymerase and sub-G1 events. Notably, exogenously added heparanase enhanced epidermal growth factor receptor, Src, Akt, ERK, and p38 phosphorylation in pituitary tumor cells. Our results indicate that heparanase enhances pituitary cell viability and proliferation and may thus contribute to pituitary tumor development and progression.     

Role of heparanase in platelet and tumor cell interactions with the subendothelial extracellular matrix

Dissemination of neoplastic cells within the body involves invasion of blood vessels by tumor cells. Since platelets have been shown to contribute to this process, we studied the interaction in vitro of platelets and malignant cells with the vascular endothelium and its underlying basement membrane-like ECM. A metastatic subline (ESb) of the methylcholanthrene-induced DBA/2 T-lymphoma invaded the vascular endothelium at a higher rate than its parental nonmetastatic (Eb) subline. ESb cells also exhibited a much higher ability to degrade the proteoglycan scaffold of the ECM by means of a specific HS degrading endoglycosidase (heparanase). The interaction of platelets with this ECM was associated with platelet activation, aggregation, and degradation of HS by means of the platelet heparanase. Degradation of ECM-HS was facilitated by proteolytic activity that produced a more accessible substrate for further cleavage by heparanase. A similar enhancement was exerted by plasminogen via the activity of the tumor cells or ECM associated PAs. Heparin and chemically modified heparins that lack anticoagulant activity inhibited degradation of the ECM-HS by heparanase. Interaction of platelets and lymphoma cells with ECM covered with vascular endothelial cells was investigated by SEM and by determination of ECM-HS degradation products. SEM studies demonstrated that platelets may adhere to minor gaps between adjacent endothelial cells and degrade the ECM-HS. Platelets were also shown to recruit lymphoma cells into these interendothelial gaps, suggesting that by binding to ECM and release of heparanase, platelets may play an active role in tumor cell invasion and metastasis. Our observation that nonanticoagulant heparins may interfere with heparanase-mediated degradation of ECM-HS suggests a potential therapeutic use for such heparins in neoplastic disorders.     

Alternatively spliced Spalax heparanase inhibits extracellular matrix degradation, tumor growth, and metastasis

Heparanase is an endoglycosidase that degrades heparan sulfate (HS) at the cell surface and in the extracellular matrix. Heparanase is expressed mainly by cancer cells, and its expression is correlated with increased tumor aggressiveness, metastasis, and angiogenesis. Here, we report the cloning of a unique splice variant (splice 36) of heparanase from the subterranean blind mole rat (Spalax). This splice variant results from skipping part of exon 3, exons 4 and 5, and part of exon 6 and functions as a dominant negative to the wild-type enzyme. It inhibits HS degradation, suppresses glioma tumor growth, and decreases experimental B16–BL6 lung colonization in a mouse model. Intriguingly, Spalax splice variant 7 of heparanase (which results from skipping of exon 7) is devoid of enzymatic activity, but unlike splice 36 it enhances tumor growth. Our results demonstrate that alternative splicing of heparanase regulates its enzymatic activity and might adapt the heparanase function to the fluctuating normoxic–hypoxic subterranean environment that Spalax experiences. Development of anticancer drugs designed to suppress tumor growth, angiogenesis, and metastasis is a major challenge, of which heparanase inhibition is a promising approach. We anticipate that the heparanase splicing model, evolved during 40 million years of Spalacid adaptation to underground life, would pave the way for the development of heparanase-based therapeutic modalities directed against angiogenesis, tumor growth, and metastasis.     

Heparanase prevents the development of type 1 diabetes in non-obese diabetic mice by regulating T-cell activation and cytokines production

BACKGROUND: Heparanase is an endo-beta-D-glucuronidase that cleaves heparan sulfate saccharide chains. The enzyme promotes cell adhesion, migration and invasion, and was shown to play a significant role in cancer metastasis and angiogenesis. METHODS: The present study focuses on the involvement of heparanase in autoimmunity, applying the murine non-obese diabetic (NOD) model, a T-cell-dependent disease often used to investigate the pathophysiology of type 1 diabetes. RESULTS: It was found that intra-peritoneal administration of heparanase ameliorated the clinical signs of the disease. In vitro studies revealed that heparanase has an inhibitory effect on the activation of T-cells through modulation of their repertoire of cytokines indicated by a marked increase in the levels of IL-4 and IL-10, and a parallel decrease in IL-12, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). CONCLUSIONS: We suggest that heparanase induces a shift from a Th1- to Th2-phenotype, resulting in inhibition of diabetes in NOD mice and possibly other autoimmune disorders.     

Inhibition of heparanase-mediated degradation of extracellular matrix heparan sulfate by non-anticoagulant heparin species

Incubation of human platelets, human neutrophils, or highly metastatic mouse lymphoma cells with sulfate-labeled extracellular matrix (ECM) results in heparanase-mediated release of labeled heparan sulfate cleavage fragments (0.5 less than Kav less than 0.85 on Sepharose 6B). This degradation was inhibited by native heparin both when brought about by intact cells or their released heparanase activity. Degradation of heparan sulfate in ECM may facilitate invasion of normal and malignant cells through basement membranes. The present study tested the heparanase inhibitory effect of nonanticoagulant species of heparin that might be of potential use in preventing heparanase mediated extravasation of bloodborne cells. For this purpose, we prepared various species of low-sulfated or low-mol-wt heparins, all of which exhibited less than 7% of the anticoagulant activity of native heparin. N-sulfate groups of heparin are necessary for its heparanase inhibitory activity but can be substituted by an acetyl group provided that the O-sulfate groups are retained. O-sulfate groups could be removed provided that the N positions were resulfated. Total desulfation of heparin abolished its heparanase inhibitory activity. Heparan sulfate was a 25-fold less potent heparanase inhibitor than native heparin. Efficiency of low-mol-wt heparins to inhibit degradation of heparan sulfate in ECM decreased with their main molecular size, and a synthetic pentasaccharide, representing the binding site to antithrombin III, was devoid of inhibitory activity. Similar results were obtained with heparanase activities released from platelets, neutrophils, and lymphoma cells. We propose that heparanase inhibiting nonanticoagulant heparins may interfere with dissemination of bloodborne tumor cells and development of experimental autoimmune diseases.     

Deterioration of glomerular endothelial surface layer induced by oxidative stress is implicated in altered permeability of macromolecules in Zucker fatty rats

Aims/hypothesis  

The glomerular endothelial layer is coated by the endothelial surface layer (ESL), which is suggested to play a role in regulation of the permselectivity of macromolecules. Production of heparanase, a degrading enzyme of the ESL, is induced by reactive oxygen species (ROS). We hypothesised that oxidative stress could cause deterioration of the glomerular ESL by induction of heparanase, resulting in increased glomerular permeability.