硫酸乙酰肝素、肝素酶及其与肿瘤转移的关系

细胞外基质和基底膜的降解是癌细胞穿透组织屏障发生转移的重要步骤。硫酸乙酰肝素蛋白聚糖是细胞外基质和基底膜的组成成分,其多糖侧链可以被葡萄糖苷内切酶--肝素酶,特异性识别并切割,以破坏细胞外基质和基底膜的完整性,促进肿瘤转移。临床上肿瘤患者肝素酶高表达与肿瘤恶性程度和转移发生密切相关。深入了解硫酸乙酰肝素、肝素酶及它们与肿瘤转移相关的作用机制有助于我们寻找肿瘤治疗的新思路。本文将从硫酸乙酰肝素的合成调控、功能、肝素酶的转录和活性调节、肝素酶表达与肿瘤患者的临床特征,以及硫酸乙酰肝素、肝素酶与肿瘤转移的关系进行综述。     

乙酰肝素酶的结构、功能及调控

乙酰肝素酶是目前发现的哺乳动物细胞中唯一能切割细胞外基质中硫酸肝素蛋白多糖侧链--硫酸乙酰肝素--的内源性糖苷酶,是抗肿瘤,抗炎症的理想靶点。对其深入研究将有助于揭示组织修复,血管形成,自身免疫,肿瘤转移等生理及病理过程。本就乙酰肝素酶的发现,分子特性,基因定位,转录,表达调控,细胞内的亚定位及其功能活性调控机制方面的研究进展进行综述。     

乙酰肝素酶及在肿瘤治疗中的应用

乙酰肝素酶(Heparanase,Hpa)是哺乳动物体内唯一能够裂解硫酸乙酰肝素蛋白多糖的酶。通过破坏细胞外基质及基底膜结构的完整性,释放胞外基质上的各种生长因子,与肿瘤的转移、侵袭密切相关。目前的研究表明Hpa在大多数中晚期肿瘤中都有表达,尤其在恶性肿瘤中异常高表达,而Hpa表达的下调可以抑制肿瘤细胞的转移,可以作为一种抗肿瘤转移相关靶点用于中晚期肿瘤的治疗。综述了Hpa的结构与功能、对肿瘤转移的促进作用及在肿瘤治疗中的应用情况。     

乙酰肝素酶促进骨肉瘤细胞增殖和侵袭的体外研究

目的:探讨通过基因转染正向调节HPSE-1,体外对骨肉瘤细胞系恶性特质的影响.方法:转染HPSE-1基因至骨肉瘤细胞系MG-63,检测HPSE-1 mRNA和蛋白水平的表达,进一步应用MTT试验和Transwell侵袭试验观察稳定转染的细胞的增殖力和侵袭力的影响.结果:成功建立稳定转染HPSE-1基因的MG-63细胞系MG-63-HPSE,且该细胞系在mRNA和蛋白水平均发现HPSE-1表达增高,MTT和Transwell试验结果发现MG-63-HPSE细胞的增殖力和侵袭力均明显高于对照组.结论:基因转染后过表达HPSE-1的骨肉瘤细胞系体外表现出增强的增殖和侵袭活性.     

硫酸肝素蛋白多糖在疾病中的作用

硫酸肝素蛋白多糖广泛分布于动物组织的细胞膜和细胞外基质,对于机体发育和维持生理平衡至关重要.聚糖链硫酸肝素特有的分子结构使得这类大分子复合物具有多种生物功能,这些功能主要通过与蛋白质配体的结合实现.细胞表面的硫酸肝素蛋白多糖介导多种细胞活性因子与其受体的结合,参与信号转导的过程.硫酸肝素蛋白多糖也是细胞间质的重要组成部分,与胶原蛋白一起维持间质结构的稳定.肝素酶通过降解硫酸肝素从而调节细胞因子的活性和细胞间质的微环境.因此,揭示硫酸肝素的分子结构及其功能是生物学的一个重要研究方向.然而,由于硫酸肝素结构复杂,且不均一,使得这个领域的研究发展相对缓慢.不过,随着分析手段的提高和完善,国际上对于硫酸肝素结构与功能的报道迅速增加,同时国内对于硫酸肝素的研究也逐步受到重视.关于硫酸肝素的生理功能最近已有几篇比较全面的综述.此综述主要介绍硫酸肝素在病变中的作用,旨在探讨利用硫酸肝素和肝素酶作为靶标,研发预防和治疗这些疾病药物的可能性.     

乙酰肝素酶:一个新的癌症治疗靶分子

癌症每年吞噬约 6 0 0 0万人的生命[1] ,是人类的主要杀手之一。近几十年来 ,尤其是美国在 1 971年提出“对癌症宣战”以来 ,人们已经对癌症发生、发展的机制以及治疗癌症的方法进行了大量研究 ,并且取得了许多成果。现在人们普遍接受的观点认为 :癌症的发生是由于细胞不受控制地增殖所造成的恶性增生 ;癌症的转移是由于癌细胞从癌组织中突破胞外基质的限制 ,转移扩散到别的器官并继续增殖所造成。癌症成为致命的疾病 ,不仅在于癌细胞的生长失去控制 ,更在于其转移能力。可以说 ,正是转移才使癌症如此险恶。癌细胞的转移需要突破胞外基质及…     

硫酸乙酰肝素酶基因表达及其内切酶活性调控对肿瘤细胞浸润、转移作用的影响

硫酸乙酰肝素酶是迄今为止在哺乳动物细胞中发现的唯一可以剪切胞外和细胞表面硫酸乙酰肝素多糖侧链的葡糖苷酸内切酶 . 在恶性肿瘤、炎症细胞以及胚胎组织等具有侵袭性组织中有较高的表达,肿瘤病人病灶部位的肝素酶 mRNA 表达量越高,病人存活期越短 . 在正常生理条件下,肝素酶基因及其表达蛋白的活性受到启动子甲基化、变化转录剪切、转录因子、蛋白质加工、 pH 环境以及免疫因子释放等多种内源因素的精确调控,以防止机体非正常恶性变化的发生 . 目前就有关乙酰肝素酶基因表达调控、酶活性的调控机制作详尽的专述 .     

乙酰肝素酶和CD105在大肠癌中的表达及其相关性

目的探讨乙酰肝素酶和CD105在大肠癌中的表达以及它们之间的关系。方法应用原位杂交方法检测乙酰肝素酶mRNA在95例大肠癌组织中的定位及表达;并用免疫组化方法对全部标本进行CD105染色,记数肿瘤微血管密度(microvesseldensity,MVD);分析乙酰肝素酶mRNA表达与大肠癌浸润、转移和血管生成之间的关系。结果95例大肠癌组织中,乙酰肝素酶mRNA阳性表达49例(51.57%),MVD平均值为(72.1±20.6);阴性表达46例(48.42%),MVD平均值为(41.3±12.4),乙酰肝素酶阳性组MVD表达与阴性组相比有显著性差异(P<0.01)。有浆膜浸润和伴淋巴结转移的大肠癌组织中,乙酰肝素酶mRNA表达阳性率分别为61.42%、63.49%,高于无浆膜浸润(24.00%)和无淋巴结转移组(28.12%),有显著差异(P<0.01)。结论乙酰肝素酶可促进大肠癌的浸润、转移和血管生成,可作为反映大肠癌生物学行为的客观指标。     

乙酰肝素酶与胃癌发展的关系及其检测

乙酰肝素酶是目前发现的哺乳动物细胞中惟一能切割细胞外基质中硫酸乙酰肝素蛋白多糖侧链——硫酸乙酰肝素的一种葡萄糖醛酸内切酶,在胃癌侵袭转移中起重要作用。我们就乙酰肝素酶的分子结构特点、在胃癌侵袭转移中的作用机制及其检测等方面的研究进展进行综述。     

人胃癌组织中乙酰肝素酶和S-100蛋白的表达及其意义

目的:探讨乙酰肝素酶和S-100蛋白在人胃癌组织中的表达及其意义。方法:根据胃癌的病理大体分型将40例胃癌组织分为早期组和晚期组。其中,早期组同时未伴有淋巴结转移,晚期组伴有淋巴结转移。采用光镜、透射电镜、原位杂交和免疫组化方法对这两组胃癌组织的超微结构,乙酰肝素酶和S-100蛋白表达进行检测。结果:早期组乙酰肝素酶阳性表达细胞较少,晚期组阳性细胞较多,二者数密度和面密度比较。具有统计学意义(P<0.01);早期组S-100蛋白阳性表达细胞较晚期组多,二者比较,具有统计学意义(P<0.01);电镜观察可见:在胃癌早期,淋巴细胞和树突状细胞浸润较多,树突状细胞突起与淋巴细胞相接触,基底膜基本完整。晚期,基底膜几乎消失。淋巴细胞和树突状细胞浸润较少,癌细胞穿基膜明显。结论:乙酰肝素酶和S-100蛋白的表达程度可作为判定胃癌的侵袭和转移的指标,对其预后的判断具有参考价值。     

Heparanase activates the syndecan-syntenin-ALIX exosome pathway

Exosomes are secreted vesicles of endosomal origin involved in signaling processes. We recently showed that the syndecan heparan sulfate proteoglycans control the biogenesis of exosomes through their interaction with syntenin-1 and the endosomal-sorting complex required for transport accessory component ALIX. Here we investigated the role of heparanase, the only mammalian enzyme able to cleave heparan sulfate internally, in the syndecan-syntenin-ALIX exosome biogenesis pathway. We show that heparanase stimulates the exosomal secretion of syntenin-1, syndecan and certain other exosomal cargo, such as CD63, in a concentration-dependent manner. In contrast, exosomal CD9, CD81 and flotillin-1 are not affected. Conversely, reduction of endogenous heparanase reduces the secretion of syntenin-1-containing exosomes. The ability of heparanase to stimulate exosome production depends on syntenin-1 and ALIX. Syndecans, but not glypicans, support exosome biogenesis in heparanase-exposed cells. Finally, heparanase stimulates intraluminal budding of syndecan and syntenin-1 in endosomes, depending on the syntenin-ALIX interaction. Taken together, our findings identify heparanase as a modulator of the syndecan-syntenin-ALIX pathway, fostering endosomal membrane budding and the biogenesis of exosomes by trimming the heparan sulfate chains on syndecans. In addition, our data suggest that this mechanism controls the selection of specific cargo to exosomes.     

Heparanase regulates esophageal keratinocyte differentiation through nuclear translocation and heparan sulfate cleavage

Heparanase is an endo-beta-glucuronidase that specifically cleaves heparan sulfate (HS) chains. Heparanase is involved in the process of metastasis and angiogenesis through the degradation of HS chains of the extracellular matrix and cell surface. Recently, we demonstrated that heparanase was localized in the cell nucleus of normal esophageal epithelium and esophageal cancer, and that its expression was correlated with cell differentiation. However, the nuclear function of heparanase remains unknown. To elucidate the role of heparanase in esophageal epithelial differentiation, primary human esophageal cells were grown in monolayer as well as organotypic cultures, and cell differentiation was induced. Expression of heparanase, HS, involucrin, and p27 was determined by immunostaining and Western blotting. SF4, a novel pharmacological inhibitor, was used to specifically inhibit heparanase activity. Upon esophageal cell differentiation, heparanase was translocated from the cytoplasm to the nucleus. Such translocation of heparanase appeared to be associated with the degradation of HS chains in the nucleus and changes in the expression of keratinocyte differentiation markers such as p27 and involucrin, whose induction was inhibited by SF4. Furthermore, these in vitro observations agreed with the expression pattern of heparanase, HS, involucrin, cytokeratin 13, and p27 in normal esophageal epithelium. Nuclear translocation of heparanase and its catalytic cleavage of HS may play a critical role in the differentiation of esophageal epithelial cells. Our study provides a novel insight into the role of heparanase in an essential differentiation process.     

Cell surface heparan sulfate released by heparanase promotes melanoma cell migration and angiogenesis

Heparan sulfate (HS) proteoglycans are essential components of the cell‐surface and extracellular matrix (ECM) which provide structural integrity and act as storage depots for growth factors and chemokines, through their HS side chains. Heparanase (HPSE) is the only mammalian endoglycosidase known that cleaves HS, thus contributing to matrix degradation and cell invasion. The enzyme acts as an endo‐β‐D ‐glucuronidase resulting in HS fragments of discrete molecular weight size. Cell‐surface HS is known to inhibit or stimulate tumorigenesis depending upon size and composition. We hypothesized that HPSE contributes to melanoma metastasis by generating bioactive HS from the cell‐surface to facilitate biological activities of tumor cells as well as tumor microenvironment. We removed cell‐surface HS from melanoma (B16B15b) by HPSE treatment and resulting fragments were isolated. Purified cell‐surface HS stimulated in vitro B16B15b cell migration but not proliferation, and importantly, enhanced in vivo angiogenesis. Furthermore, melanoma cell‐surface HS did not affect in vitro endothelioma cell (b.End3) migration. Our results provide direct evidence that, in addition to remodeling ECM and releasing growth factors and chemokines, HPSE contributes to aggressive phenotype of melanoma by releasing bioactive cell‐surface HS fragments which can stimulate melanoma cell migration in vitro and angiogenesis in vivo. J. Cell. Biochem. 106: 200–209, 2009. © 2008 Wiley‐Liss, Inc.     

Heparin releasable and nonreleasable forms of heparan sulfate proteoglycan are found on the surfaces of cultured porcine aortic endothelial cells

Evidence suggests that endothelial cell layer heparan sulfate proteoglycans include a variety of different sized molecules which most likely contain different protein cores. In the present report, approximately half of endothelial cell surface associated heparan sulfate proteoglycan is shown to be releasable with soluble heparin. The remaining cell surface heparan sulfate proteoglycan, as well as extracellular matrix heparan sulfate proteoglycan, cannot be removed from the cells with heparin. The heparin nonreleasable cell surface proteoglycan can be released by membrane disrupting agents and is able to intercalate into liposomes. When the heparin releasable and nonreleasable cell surface heparan sulfate proteoglycans are compared, differences in proteoglycan size are also evident. Furthermore, the intact heparin releasable heparan sulfate proteoglycan is closer in size to proteoglycans isolated from the extracellular matrix and from growth medium than to that which is heparin nonreleasable. These data indicate that cultured porcine aortic endothelial cells contain at least two distinct types of cell surface heparan sulfate proteoglycans, one of which appears to be associated with the cells through its glycosaminoglycan chains. The other (which is more tightly associated) is probably linked via a membrane intercalated protein core.Abbreviations ECM extracellular matrix - HSPG heparan sulfate proteoglycan - PAE porcine aortic endothelial - PBS phosphate buffered saline     

Human heparanase is localized within lysosomes in a stable form

Heparanase is an endo-beta-D-glucuronidase involved in degradation of heparan sulfate (HS) and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues. The enzymatic activity of heparanase is characterized by specific intrachain cleavage of glycosidic bonds with a hydrolase mechanism. This enzyme facilitates cell invasion and hence plays a role in tumor metastasis, angiogenesis, inflammation, and autoimmunity. Although the expression pattern and molecular properties of heparanase have been characterized, its subcellular localization has not been unequivocally determined. We have previously suggested that heparanase subcellular localization is a major determinant in regulating the enzyme's biological functions. In the present study we examined heparanase localization in three different cell types, utilizing immunofluorescent staining and electron microscopy. Our results indicate that heparanase is localized primarily within lysosomes and the Golgi apparatus. A construct composed of heparanase cDNA fused to green fluorescent protein, utilized in order to visualize the enzyme within living cells, confirmed its localization in acidic vesicles. We suggest that following synthesis, heparanase is transported into the Golgi apparatus and subsequently accumulates in a stable form within the lysosomes, where it functions in HS turnover. The lysosomal compartment may also serve as a site for heparanase confinement within the cells, limiting its secretion and uncontrolled extracellular activities associated with tumor metastasis and angiogenesis.     

Extracellular matrix-resident basic fibroblast growth factor: implication for the control of angiogenesis.

Despite the ubiquitous presence of basic fibroblast growth factor (bFGF) in normal tissues, endothelial cell proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Immunohistochemical staining revealed the localization of bFGF in basement membranes of diverse tissues, suggesting that the extracellular matrix (ECM) may serve as a reservoir for bFGF. Moreover, functional studies indicated that bFGF is an ECM component required for supporting endothelial cell proliferation and neuronal differentiation. We have found that bFGF is bound to heparan sulfate (HS) in the ECM and is released in an active form when the ECM-HS is degraded by heparanase expressed by normal and malignant cells (i.e. platelets, neutrophils, lymphoma cells). It is proposed that restriction of bFGF bioavailability by binding to ECM and local regulation of its release provide a novel mechanism for neovascularization in normal and pathological situations. The subendothelial ECM contains also tissue type- and urokinase type-plasminogen activators which participate in cell invasion and tissue remodeling. These results and studies on the properties of other ECM-immobilized enzymes (i.e. thrombin, plasmin, lipoprotein lipase) and growth factors (GM-CSF, IL-3, osteogenin), suggest that the ECM provides a storage depot for biologically active molecules which are thereby stabilized and protected. This may allow a more localized and persistent mode of action, as compared to the same molecules in a fluid phase.