heparitin-sulfate and Neoplasm-Metastasis

In an era when cancer glycobiology research is exponentially growing, we are witnessing a progressive translation of the major scientific findings to the clinical practice with the overarching aim of improving cancer patients' management. Many mechanistic cell biology studies have demonstrated that heparan sulfate (HS) glycosaminoglycans are key molecules responsible for several molecular and biochemical processes, impacting extracellular matrix properties and cellular functions. HS can interact with a myriad of different ligands, and therefore, hold a pleiotropic role in regulating the activity of important cellular receptors and downstream signalling pathways. The aberrant expression of HS glycan chains in tumours determines main malignant features, such as cancer cell proliferation, angiogenesis, invasion and metastasis. In this review, we devote particular attention to HS biological activities, its expression profile and modulation in cancer. Moreover, we highlight HS clinical potential to improve both diagnosis and prognosis of cancer, either as HS-based biomarkers or as therapeutic targets.

Topics: Animals; Biomarkers, Tumor; Cell Differentiation; Cell Membrane; Cell Proliferation; Extracellular Matrix; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glycosaminoglycans; Heparitin Sulfate; Humans; Ligands; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Signal Transduction

The tumor microenvironment (TME) is rich in matrix components, growth factors, cytokines, and enzymatic modifiers that respond to changing conditions, to alter the fundamental properties of the tumor bed. Perlecan/HSPG2, a large, multi-domain heparan sulfate proteoglycan, is concentrated in the reactive stroma that surrounds tumors. Depending on its state in the TME, perlecan can either prevent or promote the progression of cancers to metastatic disease. Breast, prostate, lung, and renal cancers all preferentially metastasize to bone, a dense, perlecan-rich environment that is initially a "hostile" niche for cancer cells. Driven by inflammation, production of perlecan and its enzyme modifiers, which include matrix metalloproteinases (MMPs), sulfatases (SULFs), and heparanase (HPSE), increases in the reactive stroma surrounding growing and invading tumors. MMPs act upon the perlecan core protein, releasing bioactive fragments of the protein, primarily from C-terminal domains IV and V. These fragments influence cell adhesion, invasion, and angiogenesis. Sulfatases and heparanases act directly upon the heparan sulfate chains, releasing growth factors from reservoirs to reach receptors on the cancer cell surface. We propose that perlecan modifiers, by promoting the degradation of the perlecan-rich stroma, "flip the molecular switch" and convert the "hostile" stroma into a welcoming one that supports cancer dissemination and metastasis. Targeted therapies that prevent this molecular conversion of the TME should be considered as potential new therapeutics to limit metastasis.

Topics: Extracellular Matrix Proteins; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Neoplasm Metastasis; Neoplasms; Tumor Microenvironment

Because of its impact on multiple biological pathways, heparanase has emerged as a major regulator of cancer, inflammation and other disease processes. Heparanase accomplishes this by degrading heparan sulfate which regulates the abundance and location of heparin-binding growth factors thereby influencing multiple signaling pathways that control gene expression, syndecan shedding and cell behavior. In addition, heparanase can act via nonenzymatic mechanisms that directly activate signaling at the cell surface. Clinical trials testing heparanase inhibitors as anticancer therapeutics are showing early signs of efficacy in patients further emphasizing the biological importance of this enzyme. This review focuses on recent developments in the field of heparanase regulation of cancer and inflammation, including the impact of heparanase on exosomes and autophagy, and novel mechanisms whereby heparanase regulates tumor metastasis, angiogenesis and chemoresistance. In addition, the ongoing development of heparanase inhibitors and their potential for treating cancer and inflammation are discussed.

Topics: Antineoplastic Agents; Autophagy; Clinical Trials as Topic; Drug Resistance, Neoplasm; Enzyme Inhibitors; Exosomes; Gene Expression Regulation, Neoplastic; Glucuronidase; Heparitin Sulfate; Humans; Inflammation; Molecular Targeted Therapy; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Signal Transduction; Syndecans

Breast cancer is defined as a cancer originating in tissues of the breast, frequently in ducts and lobules. During the last 30 years, studies to understand the biology and to treat breast tumor improved patients' survival rates. These studies have focused on genetic components involved in tumor progression and on tumor microenvironment. Heparan sulfate proteoglycans (HSPGs) are involved in cell signaling, adhesion, extracellular matrix assembly, and growth factors storage. As a central molecule, HSPG regulates cell behavior and tumor progression. HS accompanied by its glycosaminoglycan counterparts regulates tissue homeostasis and cancer development. These molecules present opposite effects according to tumor type or cancer model. Studies in this area may contribute to unveil glycosaminoglycan activities on cell dynamics during breast cancer exploring these polysaccharides as antitumor agents. Heparanase is a potent tumor modulator due to its protumorigenic, proangiogenic, and prometastatic activities. Several lines of evidence indicate that heparanase is upregulated in all human sarcomas and carcinomas. Heparanase seems to be related to several aspects regulating the potential of breast cancer metastasis. Due to its multiple roles, heparanase is seen as a target in cancer treatment. We will describe recent findings on the function of HSPGs and heparanase in breast cancer behavior and progression.

Topics: Breast Neoplasms; Disease Progression; Female; Glucuronidase; Heparitin Sulfate; Humans; Models, Biological; Neoplasm Metastasis

Heparanase is an endo-β-D-glucuronidase capable of cleaving heparan sulfate side chains at a limited number of sites, yielding heparan sulfate fragments of still appreciable size. Importantly, heparanase activity correlates with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of heparan sulfate cleavage and remodeling of the extracellular matrix and basement membrane underlying epithelial and endothelial cells. Similarly, heparanase activity is implicated in neovascularization, inflammation and autoimmunity, involving the migration of vascular endothelial cells and activated cells of the immune system. The cloning of a single human heparanase cDNA 10 years ago enabled researchers to critically approve the notion that heparan sulfate cleavage by heparanase is required for structural remodeling of the extracellular matrix, thereby facilitating cell invasion. Progress in the field has expanded the scope of heparanase function and its significance in tumor progression and other pathologies. Notably, although heparanase inhibitors attenuated tumor progression and metastasis in several experimental systems, other studies revealed that heparanase also functions in an enzymatic activity-independent manner. Thus, inactive heparanase was noted to facilitate adhesion and migration of primary endothelial cells and to promote phosphorylation of signaling molecules such as Akt and Src, facilitating gene transcription (i.e. vascular endothelial growth factor) and phosphorylation of selected Src substrates (i.e. endothelial growth factor receptor). The concept of enzymatic activity-independent function of heparanase gained substantial support by the recent identification of the heparanase C-terminus domain as the molecular determinant behind its signaling capacity. Identification and characterization of a human heparanase splice variant (T5) devoid of enzymatic activity and endowed with protumorigenic characteristics, elucidation of cross-talk between heparanase and other extracellular matrix-degrading enzymes, and identification of single nucleotide polymorphism associated with heparanase expression and increased risk of graft versus host disease add other layers of complexity to heparanase function in health and disease.

Topics: Antineoplastic Agents; Disease Progression; Enzyme Inhibitors; ErbB Receptors; Glucuronidase; Head and Neck Neoplasms; Heparitin Sulfate; Humans; Multiple Myeloma; Neoplasm Metastasis; Neoplasms; Proteoglycans; Signal Transduction

Emerging data in myeloma and other cancers indicates that heparan sulfate proteoglycans promote tumor progression by enhancing their growth and metastasis. By acting as key regulators of cell signaling via their interactions with multiple growth and angiogenic factors, heparan sulfates mediate a shift in the microenvironment that supports the tumor as an 'organ' and promotes an aggressive tumor phenotype. In addition, enzymatic remodeling of heparan sulfate proteoglycans provides a mechanism for rapid, localized and dynamic modulation of proteoglycan function thereby tightly regulating activities within the tumor microenvironment. New data from animal models demonstrates that heparan sulfate or the enzymes that regulate heparan sulfate are viable targets for cancer therapy. This strategy of targeting heparan sulfate may be particularly effective for attacking cancers like myeloma where extensive genetic chaos renders them unlikely to respond well to agents that target a single signaling pathway.

Topics: Animals; Cell Proliferation; Glucuronidase; Heparitin Sulfate; Humans; Mice; Multiple Myeloma; Neoplasm Metastasis; Signal Transduction; Sulfatases; Syndecan-1

Malignant tumor cells invade normal tissues in the vicinity of cancer through devastating the extracelluar matrix and blood vessel wall of the tissues. An important step in this process is degradation of heparan sulfate proteoglycan, a carbohydrate-protein complex. Heparan sulfate proteoglycan is a major component of the extracellular matrix, and is essential for the self-assembly, insolubility and barrier properties of basement membranes. Heparanase is an endoglucuronidase that cleaves heparan sulfate and expression level of this enzyme correlates with metastatic potential of tumor cells. Treatment with heparanase inhibitors markedly reduces the incidence of metastasis in experimental animals. Heparin, a widely used anticoagulant, is structurally related to heparan sulfate and a natural substrate of heparanase. Long-term treatment of cancer patients having venous thromboembolism with low molecular weight heparin showed improved survival rate. Understanding the functional roles and the corresponding molecular mechanisms of heparin, heparan sulfate and heparanase in cancer development may pave the way for exploring remedies against tumor metastasis.

Topics: Animals; Anticoagulants; Extracellular Matrix; Heparin; Heparin Lyase; Heparin, Low-Molecular-Weight; Heparitin Sulfate; Humans; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Substrate Specificity; Thrombosis

Heparanase is an endoglycosidase which cleaves heparan sulfate (HS) and hence participates in degradation and remodeling of the extracellular matrix (ECM). Heparanase is preferentially expressed in human tumors and its over-expression in tumor cells confers an invasive phenotype in experimental animals. The enzyme also releases angiogenic factors from the ECM and thereby induces an angiogenic response in vivo. Heparanase upregulation correlates with increased tumor vascularity and poor postoperative survival of cancer patients. Heparanase is synthesized as a 65 kDa inactive precursor that undergoes proteolytic cleavage, yielding 8 kDa and 50 kDa protein subunits that heterodimerize to form an active enzyme. Heparanase exhibits also non-enzymatic activities, independent of its involvement in ECM degradation. Among these, are the enhancement of Akt signaling, stimulation of PI3K- and p38-dependent endothelial cell migration, and up regulation of VEGF, all contributing to its potent pro-angiogenic activity. Studies on relationships between structure and heparanase inhibition activity of nonanticogulant heparins systematically differing in their O-sulfation patterns, degrees of N-acetylation, and glycol-splitting of both pre-existing nonsulfated uronic acid residues (prevalently D-glucuronic) and/or those (L-iduronic acid/L-galacturonic acid) generated by graded 2-O-desulfation, have permitted to select effective inhibitors of the enzymatic activity of heparanase. N-acetylated, glycol-split heparins emerged as especially strong inhibitors of heparanase, exerting little or no release of growth factors from ECM. N-acetylated glycol-split species of heparin, as well as heparanase gene silencing inhibit tumor metastasis, angiogenesis and inflammation in experimental animal models. These observations and the unexpected identification of a single functional heparanase, suggest that the enzyme is a promising target for anti-cancer and anti-inflammatory drug development.

Topics: Animals; Catalytic Domain; Cell Movement; Cell Survival; Enzyme Activation; Enzyme Inhibitors; Fibroblast Growth Factor 2; Glucuronidase; Heparitin Sulfate; Humans; Neoplasm Metastasis; Neovascularization, Physiologic; Structure-Activity Relationship; Vascular Endothelial Growth Factor A

The heparan sulfate (HS) mimetic PI-88 is a promising inhibitor of tumor growth and metastasis expected to commence phase III clinical evaluation in 2007 as an adjuvant therapy for postresection hepatocellular carcinoma. Its anticancer properties are attributed to inhibition of angiogenesis via antagonism of the interactions of angiogenic growth factors and their receptors with HS. It is also a potent inhibitor of heparanase, an enzyme that plays a key role in both metastasis and angiogenesis. A series of PI-88 analogs have been prepared with enhanced chemical and biological properties. The new compounds consist of single, defined oligosaccharides with specific modifications designed to improve their pharmacokinetic properties. These analogs all inhibit heparanase and bind to the angiogenic fibroblast growth factor 1 (FGF-1), FGF-2, and vascular endothelial growth factor with similar affinity to PI-88. However, compared with PI-88, some of the newly designed compounds are more potent inhibitors of growth factor-induced endothelial cell proliferation and of endothelial tube formation on Matrigel. Representative compounds were also tested for antiangiogenic activity in vivo and were found to reduce significantly blood vessel formation. Moreover, the pharmacokinetic profile of several analogs was also improved, as evidenced primarily by lower clearance in comparison with PI-88. The current data support the development of HS mimetics as potent antiangiogenic anticancer agents.

Topics: Animals; Biomimetic Materials; Carcinoma, Hepatocellular; Cell Proliferation; Chemotherapy, Adjuvant; Clinical Trials, Phase III as Topic; Endothelial Cells; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Heparin Lyase; Heparitin Sulfate; Humans; Neoplasm Metastasis; Neovascularization, Pathologic; Oligosaccharides; Vascular Endothelial Growth Factor A

Heparin is a biogenic anionic charged sulfated polysaccharide that has a range of desired activities including inhibition of tumor metastasis and inhibition of restenosis. However, its clinical use is limited to treating blood-clotting disorders. Anionic macromolecules called heparinoids have been investigated with the objective of developing heparin-like molecules with reduced anti-coagulant activity and selective anti-metastasis and anti-restenosis activity. This mini-review summarizes the synthesis and biological activity of the main synthetic heparinoids reported in the past three decades.

Topics: Amino Acids; Animals; Anticoagulants; Antineoplastic Agents; Coronary Restenosis; Enzyme Inhibitors; Fibroblast Growth Factors; Glucuronidase; Heparin; Heparinoids; Heparitin Sulfate; Humans; Neoplasm Metastasis

Heparanase is the only mammalian heparan sulfate-degrading enzymes; it could cleave heparan sulfate (HS) which links to heparan sulfate proteoglycans (HSPGs). In this review, the heparanase's structure, function, molecular properties, gene location, nucleotide sequence, its effects on tumor angiogenesis, its expression in normal tissue, tumor tissue and metastatic tissue, and its correlation to tumor metastasis were described; recent progress in searching for novel antitumor drugs through screening for inhibitor of heparanase was summarized.

Topics: Animals; Gene Expression Regulation, Neoplastic; Glucuronidase; Heparitin Sulfate; Humans; Lymphatic Metastasis; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; RNA, Messenger

This review summarizes a series of studies demonstrating that heparan sulfate proteoglycans act to promote the growth and metastasis of myeloma and breast tumors, two tumors that home to, and grow within, bone. Much of the growth-promoting effect of proteoglycans in these tumors may reside in the shed form of syndecan-1 that acts to favorably condition the tumor microenvironment. Moreover, the interplay between heparan sulfate and the extracellular enzyme heparanase-1 also has important regulatory implications. Recent studies indicate that the activity of heparanase, which likely releases heparin sulfate-bound growth factors and generates highly active heparan sulfate fragments, also promotes growth and metastasis of myeloma and breast tumors. Understanding the role of heparan sulfate and heparanase in the regulation of tumor behavior may lead to new therapeutic approaches for treating cancer.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Proliferation; Glucuronidase; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Models, Biological; Multiple Myeloma; Neoplasm Metastasis; Osteolysis

Heparanase, which is an extracellular matrix degradative enzyme, degrades heparan sulfate and heparan sulfate proteoglycans, which are chief components of extracellular matrix and vascular basement membrane. The gene structure of this enzyme was recently determined. The biological functions of this enzyme in vivo were as follows: 1) this enzyme accelerates cancer cell invasion and metastasis though the degradation of vascular basement membrane and extracellular matrix by cancer cells; 2) this enzyme releases and activates heparin-binding growth factors such as bFGF and VEGF from heparan sulfate proteoglycans, and induces angiogenesis; 3) the degradative products of heparan sulfate proteoglycans by this enzyme suppress the biological function of activated T-lymphocytes. Therefore, heparanase is thought to be a favorable molecule for acceleration of cancer invasion and metastasis. The expression of heparanase is strongly correlated with the metastasis of melanoma and fibrosarcoma. Thus, heparanase may play important roles in invasion and metastasis of cancer.

Topics: Animals; Basement Membrane; Endothelial Growth Factors; Extracellular Matrix; Fibroblast Growth Factor 2; Glucuronidase; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Lymphocyte Activation; Neoplasm Invasiveness; Neoplasm Metastasis; Neovascularization, Pathologic; T-Lymphocytes

Topics: Animals; Bacterial Physiological Phenomena; Cell Communication; Glycoconjugates; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Leukocytes; Lymphocytes; Models, Structural; Neoplasm Metastasis; Neoplasms; Parasites; Proteoglycans

Topics: Animals; Basement Membrane; Blood Platelets; Cell Compartmentation; Cell Differentiation; Cell Division; Endothelium; Extracellular Matrix; Fibroblast Growth Factor 2; Glucuronidase; Glycoside Hydrolases; Heparitin Sulfate; Humans; Neoplasm Metastasis; Neoplasms; Neurons; Neutrophils; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoric Diester Hydrolases

Neoplastic cells require an appropriate pericellular environment and new formation of stroma and blood vessels in order to constitute a solid tumor. Tumor progression also involves degradation of various extracellular matrix (ECM) constituents. In this review we have focused on the possible involvement of ECM-resident growth factors and enzymes in neovascularization and cell invasion. We demonstrate that the pluripotent angiogenic factor, basic fibroblast growth factor (bFGF) is an ECM component required for supporting cell proliferation and differentiation. Basic FGF has been identified in the subendothelial ECM produced in vitro and in basement membranes of the cornea and blood vessels in vivo. Despite the ubiquitous presence of bFGF in normal tissues, endothelial cell (EC) proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Our results indicate 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 cellular heparanase. We propose that restriction of bFGF bioavailability by binding to ECM and local regulation of its release, provides a novel mechanism for regulation of capillary blood vessel growth in normal and pathological situations. Heparanase activity correlates with the metastatic potential of various tumor cells and heparanase inhibiting molecules markedly reduce the incidence of lung metastasis in experimental animals. Heparanase may therefore participate in both tumor cell invasion and angiogenesis through degradation of the ECM-HS and mobilization of ECM-resident EC growth factors. The subendothelial ECM contains also tissue type- and urokinase type- plasminogen activators (PA), as well as PA inhibitor which may regulate cell invasion and tissue remodeling. Heparanase and the ECM-resident PA participate synergistically in sequential degradation of HS-proteoglycans in the ECM. These results together with similar observations on the properties of other ECM-immobilized enzymes and growth factors, suggest that the ECM provides a storage depot for biologically active molecules which are thereby stabilized and protected. This may allow a more localized, regulated and persistent mode of action, as compared to the same molecules in a fluid phase.

Topics: Animals; Extracellular Matrix; Fibroblast Growth Factor 2; Glucuronidase; Glycoside Hydrolases; Heparitin Sulfate; Humans; Neoplasm Metastasis; Neovascularization, Pathologic; Plasminogen Activators; Plasminogen Inactivators

Basement membranes serve as significant barriers to the passage of tumor cells but ones which metastatic cells can pass. This involves the production of a cascade of proteases leading to the activation of a specific collagenase that degrades the unique collagen network in basement membrane. Breast cancer cells, when estrogen dependent, show a requirement for estrogen for invasive activity. However, when these cells progress to an estrogen independent state and increased malignancy, they express an invasive phenotype constitutively. Studies with various anti-estrogens suggest that these responses are mediated via the estrogen receptor. Anti-estrogens lacking agonist activity suppress invasiveness as well as growth of the breast cancer cells.

Topics: Basement Membrane; Chemotactic Factors; Chondroitin Sulfate Proteoglycans; Collagen; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Laminin; Microbial Collagenase; Neoplasm Invasiveness; Neoplasm Metastasis; Plasminogen Activators; Tumor Cells, Cultured

The successful penetration of endothelial basement membranes is an important process in the formation of hematogenous tumor metastases. Heparan sulfate (HS) proteoglycan is a major constituent of endothelial basement membranes, and we have found that HS-degradative activities of metastatic B16 melanoma sublines correlate with their lung-colonizing potentials. The melanoma HS-degrading enzyme is a unique endo-beta-D-glucuronidase (heparanase) that cleaves HS at specific intrachain sites and is detectable in a variety of cultured human malignant melanomas. The treatment of B16 melanoma cells with heparanase inhibitors that have few other biological activities, such as N-acetylated N-desulfated heparin, results in significant reductions in the numbers of experimental lung metastases in syngeneic mice, indicating that heparanase plays an important role in melanoma metastasis. HS-degrading endoglycosidases are not tumor-specific and have been found in several normal tissues and cells. There are at least three types of endo-beta-D-glucuronidases based on their substrate specificities. Melanoma heparanase, an Mr approximately 96,000 enzyme with specificity for beta-D-glucuronosyl-N-acetylglucosaminyl linkages in HS, is different from platelet and mastocytoma endoglucuronidases. Elevated levels of heparanase have been detected in sera from metastatic tumor-bearing animals and malignant melanoma patients, and a correlation exists between serum heparanase activity and extent of metastases. The results suggest that heparanase is potentially a useful marker for tumor metastasis.

Topics: Animals; Basement Membrane; Biomarkers, Tumor; Glucuronidase; Glycoside Hydrolases; Heparitin Sulfate; Humans; Melanoma; Mice; Neoplasm Metastasis

Topics: Adenocarcinoma; Animals; Basement Membrane; Collagen; Glucuronidase; Glycosaminoglycans; Glycoside Hydrolases; Heparitin Sulfate; Humans; Mammary Neoplasms, Experimental; Melanoma; Neoplasm Metastasis; Rats

Topics: Basement Membrane; Chondroitin Sulfate Proteoglycans; Fibronectins; Genetic Linkage; Glucuronidase; Glycoside Hydrolases; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Laminin; Microbial Collagenase; Models, Biological; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Phenotype; Receptors, Fibronectin; Receptors, Immunologic; Receptors, Laminin

Laminin is a multifunctional protein with diverse biological activities. Like fibronectin, it can influence cell adhesion, growth, morphology, differentiation, migration, and agglutination as well as the assembly of the extracellular matrix. Laminin primarily affects cells of epithelial origin, and the response varies depending on the cell. Because most differentiated cells are difficult to maintain in culture, laminin may be an important supplement in studies on cell differentiation in vitro.

Topics: Animals; Basement Membrane; Binding Sites; Cell Adhesion; Cell Differentiation; Cell Division; Cell Movement; Cells; Cells, Cultured; Chemical Phenomena; Chemistry; Collagen; Extracellular Matrix; Hemagglutination; Heparin; Heparitin Sulfate; Laminin; Macromolecular Substances; Molecular Weight; Neoplasm Metastasis; Proteoglycans

Topics: Animals; Blood Circulation; Blood Coagulation; Chemical Phenomena; Chemistry; Chemistry Techniques, Analytical; Chemistry, Physical; Chromatography; Drug Interactions; Electrophoresis; Heparin; Heparin Antagonists; Heparinoids; Heparitin Sulfate; Humans; Hydrogen-Ion Concentration; Mass Spectrometry; Molecular Conformation; Neoplasm Metastasis; Optical Rotatory Dispersion; Osmolar Concentration

Heparan sulfate (HS), a highly negatively charged glycosaminoglycan, is ubiquitously present in all tissues and also exposed on the surface of mammalian cells. A plethora of molecules such as growth factors, cytokines or coagulation factors bear HS binding sites. Accordingly, HS controls the communication of cells with their environment and therefore numerous physiological and pathophysiological processes such as cell adhesion, migration, and cancer cell metastasis. In the present work, we found that HS exposed by blood circulating melanoma cells recruited considerable amounts of plasmatic von Willebrand factor (vWF) to the cellular surface. Analyses assisted by super-resolution microscopy indicated that HS and vWF formed a tight molecular complex. Enzymatic removal of HS or genetic engineering of the HS biosynthesis showed that a reduced length of the HS chains or complete lack of HS was associated with significantly reduced vWF encapsulation. In microfluidic experiments, mimicking a tumor-activated vascular system, we found that vWF-HS complexes prevented vascular adhesion. In line with this, single molecular force spectroscopy suggested that the vWF-HS complex promoted the repulsion of circulating cancer cells from the blood vessel wall to counteract metastasis. Experiments in wild type and vWF knockout mice confirmed that the HS-vWF complex at the melanoma cell surface attenuated hematogenous metastasis, whereas melanoma cells lacking HS evade the anti-metastatic recognition by vWF. Analysis of tissue samples obtained from melanoma patients validated that metastatic melanoma cells produce less HS. Transcriptome data further suggest that attenuated expression of HS-related genes correlate with metastases and reduced patients' survival. In conclusion, we showed that HS-mediated binding of plasmatic vWF to the cellular surface can reduce the hematogenous spread of melanoma. Cancer cells with low HS levels evade vWF recognition and are thus prone to form metastases. Therefore, therapeutic expansion of the cancer cell exposed HS may prevent tumor progression.

Topics: Animals; Cell Adhesion; Heparitin Sulfate; Melanoma; Mice; Mice, Knockout; Neoplasm Metastasis; Protein Binding; von Willebrand Factor

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.

Topics: Animals; Cell Proliferation; Glucuronidase; Glycoside Hydrolase Inhibitors; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Mice; Neoplasm Metastasis

Heparan sulfate (HS) has important emerging roles in oncogenesis, which represents potential therapeutic strategies for human cancers. However, due to the complexity of the HS signaling network, HS-targeted synthetic cancer therapeutics has never been successfully devised. To conquer the challenge, we developed HS-instructed self-assembling peptides by decorating the "Cardin-Weintraub" sequence with aromatic amino acids. The HS-binding interactions induce localized accumulation of synthetic peptides triggering molecular self-assembly in the vicinity of highly expressed Heparan sulfate proteoglycans (HSPGs) on the cancer cell membrane. The nanostructures hinder the binding of HSPG with metastasis promoting protein-heparin-binding EGF-like growth factor (HBEGF) inhibiting the activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Our study proved that HS-instructed self-assembly is a promising synthetic therapeutic strategy for targeted cancer migration inhibition.

Topics: Cell Line, Tumor; Cell Movement; Extracellular Signal-Regulated MAP Kinases; Heparitin Sulfate; Humans; Nanostructures; Neoplasm Metastasis

Drug delivery strategies possessing selectivity for cancer cells are eagerly needed in therapy of metastatic breast cancer. In this study, the chemotherapeutic agent, docetaxel (DTX), is conjugated onto heparan sulfate (HS). Aspirin (ASP), which has the activity of anti-metastasis and enhancing T cells infiltration in tumors, is encapsulated into the HS-DTX micelle. Then the cationic polyethyleneimine (PEI)-polyethylene glycol (PEG) copolymer binds to HS via electrostatic force, forming the ASP-loaded HS-DTX micelle (AHD)/PEI-PEG nanocomplex (PAHD). PAHD displays long circulation behavior in blood due to the PEG shell. Under the tumor microenvironment with weakly acidic pH, PEI-PEG separates from AHD, and the free cationic PEI-PEG facilitates the cellular uptake of AHD by increasing permeability of cell membranes. Then the overexpressed heparanase degrades HS, releasing ASP and DTX. PAHD shows specific toxicity toward tumor cells but not normal cells, with advanced activity of inhibiting tumor growth and lung metastasis in 4T1 tumor-bearing mice. The number of CD8

Topics: Animals; Antineoplastic Agents; Aspirin; CD8-Positive T-Lymphocytes; Cell Death; Cell Line, Tumor; Docetaxel; Endocytosis; Heparitin Sulfate; Humans; MCF-7 Cells; Mice, Inbred BALB C; Nanoparticles; Neoplasm Metastasis; Neoplasms; Polyethylene Glycols; Polyethyleneimine; Tissue Distribution

Previous studies have demonstrated that the glycosaminoglycans (GAGs) heparan sulfate (HS) and hyaluronic acid (HA) are mechanosensors for interstitial flow on cancer cells. The proteins that link the GAGs to the cancer cell for mechanotransduction, however, are not known.. To assess whether the HS proteoglycan core proteins, Glypican-1 and Syndecan-1, or the HA receptor, CD44, provides the mechanical linkage to the cell.. The highly metastatic renal carcinoma cell line (SN12L1) and its companion low metastatic cell line (SN12C) were analyzed by Western blot, siRNA, and a 3-dimensional interstitial flow migration assay.. There was significant elevation of Glypican-1 protein expression in the SN12L1 cells relative to the SN12C cells while there were no significant differences in Syndecan-1 or CD44. Knock down of Glypican-1 by siRNA completely blocked flow induced migration in SN12L1 cells. MAPK inhibitors also blocked flow induced migration in SN12L1 cells.. Glypican-1 provides the mechanical linkage from HS (the flow sensor) to the SN12L1 cell where mechanotransduction leading to the enhancement of migration (metastasis) occurs. MAPKs downstream of Glypican-1 propagate the signal. The HS, Glypican-1, MAPK signaling axis suggests opportunities for pharmaceutical intervention.

Topics: Carcinoma, Renal Cell; Cell Line, Tumor; Cell Movement; Extracellular Fluid; Extracellular Signal-Regulated MAP Kinases; Glycocalyx; Glypicans; Heparitin Sulfate; Humans; Hyaluronan Receptors; Kidney Neoplasms; Mechanotransduction, Cellular; Neoplasm Metastasis; Syndecan-1

Heparanase (HPSE) is an endo-β-D-glucuronidase that cleaves heparan sulphate (HS) chains of proteoglycans (HSPGs). Besides a remodelling of the extracellular matrix, HPSE increases the bioavailability of pro-angiogenic mediators, such as HS-associated vascular endothelial growth factor (VEGF), thereby contributing to metastatic niche formation. Notably, HPSE also induces release of VEGF from tumour cells independent of its enzymatic activity, but the underlying molecular mechanisms remain unresolved. We found that exogenous addition of latent HPSE stimulates VEGF release from human MV3 melanoma cells. The same effect was noted upon direct stimulation of thrombin receptor (protease-activated receptor 1 [PAR-1]) by Thrombin Receptor Activator Peptide 6 (TRAP-6). The matricellular ligand cysteine-rich 61 protein (Cyr61) was identified as pathway component since Cyr61 knockdown in MV3 cells abolished the VEGF release by TRAP-6 and HPSE. Since both TRAP-6 and HPSE mediated an up-regulation of phosphorylated focal adhesion kinase, which could be blocked by antagonizing PAR-1, we postulated a crosstalk between latent HPSE and PAR-1 in promoting pro-angiogenic pathways. To test this hypothesis at a molecular level, we applied dynamic mass redistribution (DMR) technique measuring intracellular mass relocation as consequence of direct receptor activation. Indeed, latent HPSE evoked a concentration-dependent DMR signal in MV3 cells as TRAP-6 did. Both could be modulated by targeting G-protein receptor signalling in general or by the PAR-1 inhibitor RWJ 56110. Using cells devoid of cell surface HS synthesis, we could confirm HPSE effects on PAR-1, independent of HSPG involvement. These data indicate, for the first time, a crosstalk between latent HPSE, thrombin receptor activation and G-protein signalling in general.

Topics: Angiogenesis Inducing Agents; Cell Line, Tumor; Cell Membrane; Cysteine-Rich Protein 61; Glucuronidase; Heparitin Sulfate; Humans; Melanoma; Neoplasm Metastasis; Peptide Fragments; Receptor Cross-Talk; Receptor, PAR-1; RNA, Small Interfering; Signal Transduction; Vascular Endothelial Growth Factor A

Surface expressed proteoglycans mediate the binding of cytokines and chemokines to the cell surface and promote migration of various tumor cell types including epithelial tumor cells. We here demonstrate that binding of the chemokine-like inflammatory cytokine macrophage migration inhibitory factor (MIF) to epithelial lung and breast tumor cell lines A549 and MDA-MB231 is sensitive to enzymatic digestion of heparan sulphate chains and competitive inhibition with heparin. Moreover, MIF interaction with heparin was confirmed by chromatography and a structural comparison indicated a possible heparin binding site. These results suggested that proteoglycans carrying heparan sulphate chains are involved in MIF binding. Using shRNA-mediated gene silencing, we identified syndecan-1 as the predominant proteoglycan required for the interaction with MIF. MIF binding was decreased by induction of proteolytic shedding of syndecan-1, which could be prevented by inhibition of the metalloproteinases involved in this process. Finally, MIF induced the chemotactic migration of A549 cells, wound closure and invasion into matrigel without affecting cell proliferation. These MIF-induced responses were abrogated by heparin or by silencing of syndecan-1. Thus, our study indicates that syndecan-1 on epithelial tumor cells promotes MIF binding and MIF-mediated cell migration. This may represent a relevant mechanism through which MIF enhances tumor cell motility and metastasis.

Topics: Cell Adhesion; Cell Membrane; Cell Movement; Epithelial Cells; HEK293 Cells; Heparitin Sulfate; Humans; Intramolecular Oxidoreductases; Macrophage Migration-Inhibitory Factors; Neoplasm Metastasis; Neoplasms; Protein Binding; Syndecan-1; Tumor Cells, Cultured

Mammalian cells are covered by a surface proteoglycan (glycocalyx) layer, and it is known that blood vessel-lining endothelial cells use the glycocalyx to sense and transduce the shearing forces of blood flow into intracellular signals. Tumor cells in vivo are exposed to forces from interstitial fluid flow that may affect metastatic potential but are not reproduced by most in vitro cell motility assays. We hypothesized that glycocalyx-mediated mechanotransduction of interstitial flow shear stress is an un-recognized factor that can significantly enhance metastatic cell motility and play a role in augmentation of invasion. Involvement of MMP levels, cell adhesion molecules (CD44, α3 integrin), and glycocalyx components (heparan sulfate and hyaluronan) was investigated in a cell/collagen gel suspension model designed to mimic the interstitial flow microenvironment. Physiological levels of flow upregulated MMP levels and enhanced the motility of metastatic cells. Blocking the flow-enhanced expression of MMP activity or adhesion molecules (CD44 and integrins) resulted in blocking the flow-enhanced migratory activity. The presence of a glycocalyx-like layer was verified around tumor cells, and the degradation of this layer by hyaluronidase and heparinase blocked the flow-regulated invasion. This study shows for the first time that interstitial flow enhancement of metastatic cell motility can be mediated by the cell surface glycocalyx - a potential target for therapeutics.

Topics: Cell Adhesion; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Collagen; Endothelium, Vascular; Gelatin; Gene Expression Regulation, Neoplastic; Glycocalyx; Heparitin Sulfate; Humans; Hyaluronan Receptors; Hyaluronic Acid; Integrin alpha3; Matrix Metalloproteinases; Mechanotransduction, Cellular; Neoplasm Metastasis; Reverse Transcriptase Polymerase Chain Reaction; Shear Strength; Stress, Mechanical

Hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) regulate normal development and homeostasis and drive disease progression in many forms of cancer. Both proteins signal by binding to receptor tyrosine kinases and heparan sulfate (HS) proteoglycans on target cell surfaces. Basic residues comprising the primary HS binding sites on HGF and VEGF provide similar surface charge distributions without underlying structural similarity. Combining three acidic amino acid substitutions in these sites in the HGF isoform NK1 or the VEGF isoform VEGF165 transformed each into potent, selective competitive antagonists of their respective normal and oncogenic signaling pathways. Our findings illustrate the importance of HS in growth factor driven cancer progression and reveal an efficient strategy for therapeutic antagonist development.

Topics: Animals; Antigens, CD34; Cell Proliferation; Cluster Analysis; Dogs; Enzyme Activation; Gene Targeting; Heparitin Sulfate; Hepatocyte Growth Factor; Humans; Magnetic Resonance Spectroscopy; Mice; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Protein Binding; Protein Folding; Protein Isoforms; Protein Multimerization; Proto-Oncogene Proteins c-met; Signal Transduction; Vascular Endothelial Growth Factor A

Heparanase degrades heparan sulfate (HS) chains on proteoglycans; elevated levels of heparanase expression correlate with tumour cell metastatic potential and vascularity, and reduced post-operative survival of cancer patients. Consequently, heparanase expression is considered a biomarker for cancer detection. Although several heparanase assays have been developed, most require the preparation of heterogeneous, (radio)labelled HS substrates and rely on the separation of enzymatically-degraded products on the basis of molecular size. In studies directed towards the development of a more direct heparanase assay, a series of glucuronides and glycosyl glucuronides were synthesised as putative heparanase substrates. These compounds were designed with various aryl aglycones that could be measured spectrophotometrically upon hydrolysis of the glycosidic linkage by heparanase. It was found that the N-sulfated 4-nitrophenyl glycosyl glucuronide 24 and the N-sulfated methylumbelliferyl glycosyl glucuronide 26 were hydrolysed by recombinant human heparanase. These compounds represent the simplest substrates of heparanase reported to date.

Topics: Biomarkers, Tumor; Carbohydrate Conformation; Chromatography, Thin Layer; Drug Design; Glucuronidase; Glucuronides; Glycosylation; Heparitin Sulfate; Humans; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Neoplasm Metastasis; Neoplasms; Proteoglycans; Recombinant Proteins; Solutions; Substrate Specificity; Tumor Cells, Cultured

Our previous report suggested the potential role of the exchange protein directly activated by cyclic AMP (Epac) in melanoma metastasis via heparan sulfate (HS)-mediated cell migration. In order to obtain conclusive evidence that Epac1 plays a critical role in modification of HS and melanoma metastasis, we extensively investigated expression and function of Epac1 in human melanoma samples and cell lines. We have found that, in human melanoma tissue microarray, protein expression of Epac1 was higher in metastatic melanoma than in primary melanoma. In addition, expression of Epac1 positively correlated with that of N-sulfated HS, and N-deacetylase/N-sulfotransferase-1 (NDST-1), an enzyme that increases N-sulfation of HS. Further, an Epac agonist increased, but ablation of Epac1 decreased, expressions of NDST-1, N-sulfated HS, and cell migration in various melanoma cell lines. Finally, C8161 cells with stable knockdown of Epac1 showed a decrease in cell migration, and metastasis in mice. These data suggest that Epac1 plays a critical role in melanoma metastasis presumably because of modification of HS.

Topics: Animals; Cell Line, Tumor; Cell Movement; Gene Deletion; Guanine Nucleotide Exchange Factors; Heparitin Sulfate; Humans; Melanoma; Mice; Neoplasm Metastasis; Skin Neoplasms; Staining and Labeling; Sulfotransferases

Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis.

Topics: Animals; Cell Line, Tumor; Disease Progression; Flow Cytometry; Heparitin Sulfate; Melanoma, Experimental; Mice; Molecular Mimicry; Neoplasm Metastasis; Surface Plasmon Resonance

The glycome acts as an essential interface between cells and the surrounding microenvironment. However, changes in glycosylation occur in nearly all breast cancers, which can alter this interaction. Here, we report that profiles of glycosylation vary between ER-positive and ER-negative breast cancers. We found that genes involved in the synthesis of sialyl-Lewis x (sLe(x); FUT3, FUT4, and ST3GAL6) are significantly increased in estrogen receptor alpha-negative (ER-negative) tumors compared with ER-positive ones. SLe(x) expression had no influence on the survival of patients whether they had ER-negative or ER-positive tumors. However, high expression of sLe(x) in ER-positive tumors was correlated with metastasis to the bone where sLe(x) receptor E-selectin is constitutively expressed. The ER-positive ZR-75-1 and the ER-negative BT20 cell lines both express sLe(x) but only ZR-75-1 cells could adhere to activated endothelial cells under dynamic flow conditions in a sLe(x) and E-selectin-dependent manner. Moreover, L/P-selectins bound strongly to ER-negative MDA-MB-231 and BT-20 cell lines in a heparan sulfate (HS)-dependent manner that was independent of sLe(x) expression. Expression of glycosylation genes involved in heparan biosynthesis (EXT1 and HS3ST1) was increased in ER-negative tumors. Taken together, our results suggest that the context of sLe(x) expression is important in determining its functional significance and that selectins may promote metastasis in breast cancer through protein-associated sLe(x) and HS glycosaminoglycans.

Topics: beta-Galactoside alpha-2,3-Sialyltransferase; Blotting, Western; Breast Neoplasms; Cell Adhesion; Cell Line; Cell Line, Tumor; E-Selectin; Female; Fucosyltransferases; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glycomics; Heparitin Sulfate; Human Umbilical Vein Endothelial Cells; Humans; Lewis X Antigen; N-Acetylglucosaminyltransferases; Neoplasm Metastasis; Neoplasms, Hormone-Dependent; Oligonucleotide Array Sequence Analysis; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; Sialyl Lewis X Antigen; Sialyltransferases; Sulfotransferases

Heparanase activity is highly implicated in cell dissemination associated with tumor metastasis, angiogenesis, and inflammation. Heparanase expression is induced in many hematological and solid tumors, associated with poor prognosis. Heparanase homolog, termed heparanase 2 (Hpa2), was cloned based on sequence homology. Detailed characterization of Hpa2 at the biochemical, cellular, and clinical levels has not been so far reported, and its role in normal physiology and pathological disorders is obscure. We provide evidence that unlike heparanase, Hpa2 is not subjected to proteolytic processing and exhibits no enzymatic activity typical of heparanase. Notably, the full-length Hpa2c protein inhibits heparanase enzymatic activity, likely due to its high affinity to heparin and heparan sulfate and its ability to associate physically with heparanase. Hpa2 expression was markedly elevated in head and neck carcinoma patients, correlating with prolonged time to disease recurrence (follow-up to failure; p = 0.006) and inversely correlating with tumor cell dissemination to regional lymph nodes (N-stage; p = 0.03). Hpa2 appears to restrain tumor metastasis, likely by attenuating heparanase enzymatic activity, conferring a favorable outcome of head and neck cancer patients.

Topics: Amino Acid Sequence; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Glucuronidase; Head and Neck Neoplasms; Heparitin Sulfate; Humans; Molecular Sequence Data; Neoplasm Metastasis; Protein Binding; Protein Transport

Basic fibroblast growth factor (FGF-2) and its respective tyrosine kinase receptors, form an autocrine loop that affects human melanoma growth and metastasis. The aim of the present study was to examine the possible participation of various glycosaminoglycans, i.e. chondroitin sulfate, dermatan sulfate and heparin on basal and FGF-2-induced growth of WM9 and M5 human metastatic melanoma cells. Exogenous glycosaminoglycans mildly inhibited WM9 cell's proliferation, which was abolished by FGF-2. Treatment with the specific inhibitor of the glycosaminoglycan sulfation, sodium chlorate, demonstrated that endogenous glycosaminoglycan/proteoglycan production is required for both basal and stimulated by FGF-2 proliferation of these cells. Heparin capably restored their growth, and unexpectedly exogenous chondroitin sulfate to WM9 and both chondroitin sulfate and dermatan sulfate to M5 cells allowed FGF-2 mitogenic stimulation. Furthermore, in WM9 cells the degradation of membrane-bound chondroitin/dermatan sulfate stimulates basal growth and even enhances FGF-2 stimulation. The specific tyrosine kinase inhibitor, genistein completely blocked the effects of FGF-2 and glycosaminoglycans on melanoma proliferation whereas the use of the neutralizing antibody for FGF-2 showed that the mitogenic effect of chondroitin sulfate involves the interaction of FGF-2 with its receptors. Both the amounts of chondroitin/dermatan/heparan sulfate and their sulfation levels differed between the cell lines and were distinctly modulated by FGF-2. In this study, we show that chondroitin/dermatan sulfate-containing proteoglycans, likely in cooperation with heparan sulfate, participate in metastatic melanoma cell FGF-2-induced mitogenic response, which represents a novel finding and establishes the central role of sulfated glycosaminoglycans on melanoma growth.

Topics: Autocrine Communication; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Chondroitin Sulfates; Fibroblast Growth Factors; Glycosaminoglycans; Heparitin Sulfate; Humans; Melanoma; Neoplasm Metastasis; Protein-Tyrosine Kinases; Proteoglycans

Recently, we reported that the laminin alpha5 synthetic peptide A5G27 (RLVSYNGIIFFLK, residues 2,892-2,904) binds to the CD44 receptor of B16-F10 melanoma cells via the glycosaminoglycans on CD44 and inhibits tumor cell migration, invasion, and angiogenesis in a dominant-negative manner. Here, we have identified the potential mechanism of A5G27 activity using WiDr human colorectal carcinoma cells. WiDr cells bound to the laminin A5G27 peptide via the heparin-like and chondroitin sulfate B glycosaminoglycan side chains of CD44. Cell binding to fibroblast growth factor (FGF2) was blocked by laminin peptide A5G27 but not by either a scrambled version of this peptide or by another laminin peptide known to bind cell surface proteoglycans. FGF2 signaling involving tyrosine phosphorylation was also blocked by laminin peptide A5G27 but was not affected by peptide controls. Finally, we have shown that peptide A5G27 directly blocks FGF2 binding to heparin. Peptide A5G27 has sequence homology to a region on FGF2 that binds heparin and the FGF receptor and is important in FGF2 central cavity formation. We conclude that peptide A5G27 inhibits metastasis and angiogenesis by blocking FGF2 binding to the heparan sulfate side chains of CD44 variant 3, thus decreasing FGF2 bioactivity.

Topics: Amino Acid Sequence; Antibodies; Antibody Specificity; Cell Line, Tumor; Colorectal Neoplasms; Fibroblast Growth Factor 2; Flow Cytometry; Glycoproteins; Heparitin Sulfate; Humans; Hyaluronan Receptors; Laminin; Molecular Sequence Data; Neoplasm Metastasis; Neovascularization, Pathologic; Peptide Fragments; Phosphorylation; Protein Binding

Heparanase (HPSE-1) is involved in the degradation of both cell-surface and extracellular matrix (ECM) heparan sulfate (HS) in normal and neoplastic tissues. Degradation of heparan sulfate proteoglycans (HSPG) in mammalian cells is dependent upon the enzymatic activity of HPSE-1, an endo-beta-d-glucuronidase, which cleaves HS using a specific endoglycosidic hydrolysis rather than an eliminase type of action. Elevated HPSE-1 levels are associated with metastatic cancers, directly implicating HPSE-1 in tumor progression. The mechanism of HPSE-1 action to promote tumor progression may involve multiple substrates because HS is present on both cell-surface and ECM proteoglycans. However, the specific targets of HPSE-1 action are not known. Of particular interest is the relationship between HPSE-1 and HSPG, known for their involvement in tumor progression. Syndecan-1, an HSPG, is ubiquitously expressed at the cell surface, and its role in cancer progression may depend upon its degradation. Conversely, another HSPG, perlecan, is an important component of basement membranes and ECM, which can promote invasive behavior. Down-regulation of perlecan expression suppresses the invasive behavior of neoplastic cells in vitro and inhibits tumor growth and angiogenesis in vivo. In this work we demonstrate the following. 1) HPSE-1 cleaves HS present on the cell surface of metastatic melanoma cells. 2) HPSE-1 specifically degrades HS chains of purified syndecan-1 or perlecan HS. 3) Syndecan-1 does not directly inhibit HPSE-1 enzymatic activity. 4) The presence of exogenous syndecan-1 inhibits HPSE-1-mediated invasive behavior of melanoma cells by in vitro chemoinvasion assays. 5) Inhibition of HPSE-1-induced invasion requires syndecan-1 HS chains. These results demonstrate that cell-surface syndecan-1 and ECM perlecan are degradative targets of HPSE-1, and syndecan-1 regulates HPSE-1 biological activity. This suggest that expression of syndecan-1 on the melanoma cell surface and its degradation by HPSE-1 are important determinants in the control of tumor cell invasion and metastasis.

Topics: Adenocarcinoma; Animals; Cell Membrane; Colorectal Neoplasms; Extracellular Matrix; Glucuronidase; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Hydrogen-Ion Concentration; Melanoma; Membrane Glycoproteins; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Proteoglycans; Recombinant Proteins; Substrate Specificity; Syndecan-1; Syndecans; Tumor Cells, Cultured

Histidine-rich glycoprotein (HRG) is an alpha2-glycoprotein found in mammalian plasma at high concentrations (approximately 150 microg/ml) and is distinguished by its high content of histidine and proline. Structurally, HRG is a modular protein consisting of an N-terminal cystatin-like domain (N1N2), a central histidine-rich region (HRR) flanked by proline-rich sequences, and a C-terminal domain. HRG binds to cell surfaces and numerous ligands such as plasminogen, fibrinogen, thrombospondin, C1q, heparin, and IgG, suggesting that it may act as an adaptor protein either by targeting ligands to cell surfaces or by cross-linking soluble ligands. Despite the suggested functional importance of HRG, the cell-binding characteristics of the molecule are poorly defined. In this study, HRG was shown to bind to most cell lines in a Zn(2+)-dependent manner, but failed to interact with the Chinese hamster ovary cell line pgsA-745, which lacks cell-surface glycosaminoglycans (GAGs). Subsequent treatment of GAG-positive Chinese hamster ovary cells with mammalian heparanase or bacterial heparinase III, but not chondroitinase ABC, abolished HRG binding. Furthermore, blocking studies with various GAG species indicated that only heparin was a potent inhibitor of HRG binding. These data suggest that heparan sulfate is the predominate cell-surface ligand for HRG and that mammalian heparanase is a potential regulator of HRG binding. Using recombinant forms of full-length HRG and the N-terminal N1N2 domain, it was shown that the N1N2 domain bound specifically to immobilized heparin and cell-surface heparan sulfate. In contrast, synthetic peptides corresponding to the Zn(2+)-binding HRR of HRG did not interact with cells. Furthermore, the binding of full-length HRG, but not the N1N2 domain, was greatly potentiated by physiological concentrations of Zn2+. Based on these data, we propose that the N1N2 domain binds to cell-surface heparan sulfate and that the interaction of Zn2+ with the HRR can indirectly enhance cell-surface binding.

Topics: Animals; Baculoviridae; Blotting, Western; Cell Membrane; Chelating Agents; CHO Cells; Chondroitin Sulfates; Complement C1q; COS Cells; Cricetinae; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Fibrinogen; Flow Cytometry; Glucuronidase; Glycosaminoglycans; Heparitin Sulfate; Histidine; Humans; Immunoglobulin G; Inflammation; Jurkat Cells; Ligands; Microscopy, Fluorescence; Neoplasm Metastasis; Peptides; Plasmids; Plasminogen; Proline; Protein Binding; Protein Structure, Tertiary; Proteins; Recombinant Proteins; Thrombospondins; Transfection; Zinc

Heparanase, a mammalian endoglycosidase that specifically cleaves heparan sulfate (HS), has been found in many tissues. Platelet, liver, and placenta have been abundant sources for the study of the enzyme. Notably, certain malignant cells also have been found to produce large amounts of the enzyme, the levels of which often correlate with their invasive and metastatic properties. To study roles of heparanase in various biological situations, a reliable method measuring the enzyme activity is indispensable. In the past, measurement of heparanase enzyme activity was done either by the detection of the degradation of fluorescent or radiolabeled HS chains by gel filtration procedures or by the use of radiolabeled substrate conjugated to solid matrices for the easy separation of degraded HS chains. A newly developed procedure, presented in this article, measures degradation of radiolabeled HS chains in the aqueous buffer by detecting their degradation products using an ultrafiltration device, the Centricon 30. This procedure has several advantages over previous assay procedures that involved tedious processing such as gel filtration chromatography of each sample or the preparation of substrate HS proteoglycans conjugated to a solid matrix. The simplicity of the new procedure allows a short setup time and a rapid processing of a large number of samples. Furthermore, the enzymatic reaction during the aqueous phase allows kinetic analyses in standard conditions.

Topics: Animals; Glucuronidase; Heparitin Sulfate; Kinetics; Neoplasm Metastasis; Neoplasms; Rats; Ultrafiltration

Topics: Adenocarcinoma; Angiogenesis Inducing Agents; Basement Membrane; Breast Neoplasms; Disease Progression; Enzyme Inhibitors; Female; Glucuronidase; Growth Substances; Heparitin Sulfate; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; RNA, Catalytic; Transfection

Heparanase is an endoglycosidase that degrades heparan sulfate, the main polysaccharide constituent of the extracellular matrix and basement membrane. Expression of the heparanase gene is associated with the invasive, angiogenic, and metastatic potential of diverse malignant tumors and cell lines. We used gene-silencing strategies to evaluate the role of heparanase in malignancy and to explore the therapeutic potential of its specific targeting.. We designed plasmid vectors to express hammerhead ribozymes or small interfering RNAs (siRNAs) directed against the human or mouse heparanase mRNAs. Human breast carcinoma (MDA-MB-435) and mouse lymphoma (Eb) and melanoma (B16-BL6) tumor cell lines, which have naturally high levels of endogenous heparanase or have been genetically engineered to overexpress heparanase, were transfected with anti-heparanase ribozyme or siRNA. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and measurements of enzymatic activity were used to confirm the efficient silencing of heparanase gene expression. Cells transfected with the anti-heparanase ribozyme and siRNA vectors were tested for invasiveness in vitro and metastatic dissemination in animal models of experimental and spontaneous metastasis.. Compared with cells transfected with control constructs, cells transfected with the anti-heparanase ribozyme or siRNA vectors had profoundly reduced invasion and adhesion in vitro, regardless of cell type, and expressed less heparanase. In vivo, tumors produced by cells transfected with the anti-heparanase ribozyme and siRNA vectors were less vascularized and less metastatic than tumors produced by cells transfected with the control vectors. Mice injected with cells transfected with the anti-heparanase ribozyme and siRNA vectors lived longer than mice injected with control cells.. The association of reduced levels of heparanase and altered tumorigenic properties in cells with anti-heparanase ribozyme- or siRNA-mediated gene-silencing vectors suggests that heparanase is important in cancer progression. Heparanase gene silencing has potential use as a target for anticancer drug development.

Topics: Angiogenesis Inducing Agents; Angiogenesis Inhibitors; Animals; Basement Membrane; Disease Progression; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Silencing; Genetic Vectors; Glucuronidase; Growth Substances; Heparitin Sulfate; Humans; Immunohistochemistry; Lung Neoplasms; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Neoplasm Metastasis; Neovascularization, Pathologic; Plasmids; Reverse Transcriptase Polymerase Chain Reaction; RNA, Catalytic; RNA, Neoplasm; RNA, Small Interfering; Transfection

We expressed the full-length CD44v2-10 isoform in SKHep1 cells, a nonmetastatic human hepatocellular carcinoma cell line that does not express any endogenous CD44v isoforms. In SCID mice, expression of CD44v2-10 by SKHep1 cells had no effect on s.c. primary tumor development but caused pulmonary metastases in 41% (7 of 17) of animals compared with control SKHep1 cells (0 of 16; P < 0.01). CD44v2-10 expression by SKHep1 cells resulted in enhanced heparan sulfate (HS) attachment and an enhanced capacity to bind heparin-binding growth factors. Mutation of the v3 domain to prevent HS attachment and growth factor binding abolished the metastatic phenotype, demonstrating that HS modification of CD44v2-10 plays a critical role in the development of metastases in this model. However, in vitro proliferation, motility, and invasion were not altered by CD44v2-10 expression.

Topics: Alternative Splicing; Binding Sites; Carcinoma, Hepatocellular; Cell Adhesion; Cell Division; Cell Movement; Growth Substances; Heparitin Sulfate; Humans; Hyaluronan Receptors; Hyaluronic Acid; Liver Neoplasms; Lung Neoplasms; Neoplasm Metastasis; Protein Isoforms; Tumor Cells, Cultured

Cells depend on polyamines for growth and their depletion represents a strategy for the treatment of cancer. Polyamines assemble de novo through a pathway sensitive to the inhibitor, alpha-difluoromethylornithine (DFMO). However, the presence of cell-surface heparan sulfate proteoglycans may provide a salvage pathway for uptake of circulating polyamines, thereby sparing cells from the cytostatic effect of DFMO. Here we show that genetic or pharmacologic manipulation of proteoglycan synthesis in the presence of DFMO inhibits cell proliferation in vitro and in vivo. In cell culture, mutant cells lacking heparan sulfate were more sensitive to the growth inhibitory effects of DFMO than wild-type cells or mutant cells transfected with the cDNA for the missing biosynthetic enzyme. Moreover, extracellular polyamines did not restore growth of mutant cells, but completely reversed the inhibitory effect of DFMO in wild-type cells. In a mouse model of experimental metastasis, DFMO provided in the water supply also dramatically diminished seeding and growth of tumor foci in the lungs by heparan sulfate-deficient mutant cells compared with the controls. Wild-type cells also formed tumors less efficiently in mice fed both DFMO and a xylose-based inhibitor of heparan sulfate proteoglycan assembly. The effect seemed to be specific for heparan sulfate, because a different xyloside known to affect only chondroitin sulfate did not inhibit tumor growth. Hence, combined inhibition of heparan sulfate assembly and polyamine synthesis may represent an additional strategy for cancer therapy.

Topics: Animals; Antineoplastic Agents; CHO Cells; Cricetinae; DNA, Complementary; Dose-Response Relationship, Drug; Eflornithine; Enzyme Inhibitors; Female; Glucuronosyltransferase; Heparitin Sulfate; Mice; Mice, SCID; Models, Biological; Models, Genetic; Neoplasm Metastasis; Neoplasm Transplantation; Polyamines; Proteoglycans; Spermine; Time Factors

Heparanase is a beta-D-endoglucuronidase that cleaves heparan sulfate (HS) and has been implicated in many important physiological and pathological processes, including tumor cell metastasis, angiogenesis, and leukocyte migration. We report herein the identification of active-site residues of human heparanase. Using PSI-BLAST and PHI-BLAST searches of sequence databases, similarities were identified between heparanase and members of several of the glycosyl hydrolase families (10, 39, and 51) from glycosyl hydrolase clan A (GH-A), including strong local identities to regions containing the critical active-site catalytic proton donor and nucleophile residues that are conserved in this clan of enzymes. Furthermore, secondary structure predictions suggested that heparanase is likely to contain an (alpha/beta)(8) TIM-barrel fold, which is common to the GH-A families. On the basis of sequence alignments with a number of glycosyl hydrolases from GH-A, Glu(225) and Glu(343) of human heparanase were identified as the likely proton donor and nucleophile residues, respectively. The substitution of these residues with alanine and the subsequent expression of the mutant heparanases in COS-7 cells demonstrated that the HS-degrading capacity of both was abolished. In contrast, the alanine substitution of two other glutamic acid residues (Glu(378) and Glu(396)), both predicted to be outside the active site, did not affect heparanase activity. These data suggest that heparanase is a member of the clan A glycosyl hydrolases and has a common catalytic mechanism that involves two conserved acidic residues, a putative proton donor at Glu(225) and a nucleophile at Glu(343).

Topics: Amino Acid Sequence; Animals; Binding Sites; Carcinogens; Catalysis; COS Cells; Glucuronidase; Heparitin Sulfate; Humans; Hydrolysis; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Neoplasm Metastasis; Protein Structure, Secondary; Rats; Sequence Homology, Amino Acid

Topics: Animals; Cell Adhesion; Cell Communication; Extracellular Matrix; Heparitin Sulfate; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Proteoglycans; Signal Transduction

Heparan sulphate (HS) is an important component of the extracellular matrix and the vasculature basal laminar which functions as a barrier to the extravasation of metastatic and inflammatory cells. Cleavage of HS by endoglycosidase or heparanase activity produced by invading cells may assist in the disassembly of the extracellular matrix and basal laminar, and thereby facilitate cell migration. Heparanase activity has previously been shown to be related to the metastatic potential of murine and human melanoma cell lines [Nakajima, Irimura and Nicolson (1988) J. Cell. Biochem. 36, 157-167]. To determine heparanase activity, porcine mucosal HS was partially de-N-acetylated and re-N-acetylated with [3H]acetic anhydride to yield a radiolabelled substrate. This procedure prevented the masking of, or possible formation of, new heparanase-sensitive cleavage sites as has been observed with previous methods of radiolabelling. Heparanase activity in a variety of tissues and cell homogenates including human platelets, colonic carcinoma cells, umbilical vein endothelial cells and rat mammary adenocarcinoma cells (both metastatic and non-metastatic variants) and liver homogenates all degraded the substrate in a stepwise fashion from 18.5 to approximately 13, 8 and finally to 4.5 kDa fragments, as assessed by gel-filtration analysis, confirming the substrate as suitable for the detection of heparanase activity present in a variety of cells and tissues. A rapid quantitative assay was developed with the HS substrate using a novel method for separating degradation products from the substrate by taking advantage of the decreased affinity of the heparanase-cleaved products for the HS-binding plasma protein chicken histidine-rich glycoprotein (cHRG). Incubation mixtures were applied to cHRG-Sepharose columns, with unbound material corresponding to heparanase-degradation products. Heparanase activity was determined for a variety of human, rat and murine cell and tissue homogenates. The highly metastatic rat mammary adenocarcinoma and murine lung carcinoma cell lines had four to ten times the heparanase activity of non-metastatic variants, confirming the correlation of heparanase activity with metastatic potential. Human cancer patients had twice the serum heparanase levels of normal healthy adults. The assay will be valuable for the determination of heparanase activity from a variety of tissue and cell sources, as a diagnostic tool for the determination of heparanase potentia

Topics: Adult; Animals; Cell Line; Chickens; Female; Glucuronidase; Glycoside Hydrolases; Heparitin Sulfate; Humans; Intestinal Mucosa; Kinetics; Mice; Neoplasm Metastasis; Neoplasms; Proteins; Rats; Reference Values; Substrate Specificity; Tumor Cells, Cultured

Structural features of heparin potentially important for heparanase-inhibitory activity were examined by measuring the ability of heparin derivatives to affect the degradation of [3H]acetylated heparan sulphate by tumor cell heparanases. IC50 values were determined using an assay which distinguished degraded from undegraded substrate by precipitation of the latter with cetylpyridinium chloride (CPC). Removal of heparin's 2-O-sulphate and 3-O-sulphate groups enhanced heparanase-inhibitory activity (50%). Removal of its carboxyl groups slightly lowered the activity (18%), while combining the treatments abolished the activity. At least one negative charge on the iduronic acid/idose moiety, therefore, is necessary for heparanase-inhibitory activity. Replacing heparin's N-sulphate groups with N-acetyl groups reduced its activity (37%). Comparing this heparin derivative with 2,3-O-desulphated heparin, the placement of sulphate groups appears important for activity since the two structures have similar nominal linear charge density. In addition, unsubstituted uronic acids are nonessential for inhibition since their modification (periodate-oxidation/borohydride-reduction) enhanced rather than reduced heparanase-inhibitory activity. The most effective heparanase inhibitors (2,3-O-desulphated heparin, and [periodate-oxidized, borohydride-reduced] heparin) were tested in the chick chorioallantoic membrane (CAM) bioassay for anti-angiogenic activity and found to be at least as efficacious as heparin. 2,3-O-desulphated heparin also significantly decreased the tumor growth of a subcutaneous human pancreatic (Ca-Pan-2) adenocarcinoma in nude mice and prolonged the survival times of C57BL/6N mice in a B16-F10 melanoma experimental lung metastasis assay.

Topics: Animals; Anticoagulants; Antineoplastic Agents; Chick Embryo; Chondroitin Sulfates; Enzyme Inhibitors; Female; Glucuronidase; Glycoside Hydrolases; Heparin; Heparitin Sulfate; Humans; Lung Neoplasms; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Nude; Molecular Structure; Neoplasm Metastasis; Neoplasms, Experimental; Neovascularization, Physiologic; Pancreatic Neoplasms

Leukocyte migration from circulation into tissue depends on leukocyte integrin-mediated adhesion to endothelium, but integrins cannot function until activated. However, it remains to be understood how tumor cells adhere to endothelium and infiltrate into underlying tissue. We studied mechanisms of extravasation of leukemic cells using adult T cell leukemia (ATL) cells and report the following novel features of cell surface heparan sulfate proteoglycan on ATL cells in ATL cell adhesion to endothelium: ATL cells adhere to endothelial cells through already activated integrins without exogenous stimulation; different from any other hematopoietic cells, ATL cells express a characteristic heparan sulfate capable of immobilizing heparin-binding chemokine macrophage inflammatory protein (MIP)-1 beta, a potent T cell integrin trigger, produced by the cells themselves; competitive interruption of endogenous heparan sulfate proteoglycan synthesis reduces cell surface MIP-1 beta and prevents ATL cells from integrin-mediated adhesion to endothelial cells or intercellular adhesion molecule-1 triggered through G-protein. We propose that leukemic cells adhere to endothelial cells through the adhesion cascade, similar to normal leukocyte, and that the cell surface heparan sulfate, particularly on ATL cells, is pivotally involved in chemokine-dependent autocrine stimulation of integrin triggering by immobilizing the chemokine on them.

Topics: CD4-Positive T-Lymphocytes; Cell Adhesion; Cell Line; Cell Membrane; Chemokine CCL4; Endothelium, Vascular; Flow Cytometry; Glycosides; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Integrins; Intercellular Adhesion Molecule-1; Leukemia, Prolymphocytic, T-Cell; Leukemia, T-Cell; Leukemic Infiltration; Macrophage Inflammatory Proteins; Models, Biological; Neoplasm Metastasis; Phenotype; Proteoglycans; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

In the present study we provide evidence for the involvement of N-CAM in the spreading of human renal cell carcinomas (RCC) through the interaction with the subendothelial matrix. We found that in tumor cell lines derived from human RCC the increase of growth rate and the loss of adhesiveness to inert substrate were accompanied by N-CAM expression and by the appearance of specific binding to endothelial heparan sulfate. Indeed, the adhesion of tumor cells to human endothelial cells and heparan sulfate in vitro was inhibited by monoclonal antibodies able to bind and inactivate N-CAM and was abrogated by endothelial cell treatment with heparitinase. Furthermore, when the renal epithelial cell line COS7 was transfected with a cDNA coding for N-CAM a significant increase in the ability to bind both endothelium and heparan sulfate in vitro was observed. Of note, HS complexed with epithelial growth factor could enhance the proliferation of RCC-derived tumor cells; this effect was also achieved by cross-linking of N-CAM at the surface of tumor cells, suggesting that N-CAM could transduce an activation signal across the cell membrane. This was also supported by the finding that N-CAM cross-linking induced a strong calcium mobilization from internal stores and opening of surface calcium channels in such tumor cells. N-CAM was detectable in vivo at the tumor site in the areas of active proliferation, as judged by the coexpression of Ki67 nuclear antigen, and heparan sulfate was present in the wall of blood vessels in the proximity of the tumor. These findings would suggest that growing kidney tumors might use N-CAM to bind the subendothelial matrix and complexed growth factors during tissue invasion and spreading.

Topics: Carcinoma, Renal Cell; Cell Adhesion; Cell Adhesion Molecules, Neuronal; Cell Division; Endothelium, Vascular; Extracellular Matrix Proteins; Heparitin Sulfate; Humans; Integrin alpha1; Integrin beta1; Integrins; Ki-67 Antigen; Kidney Neoplasms; Neoplasm Metastasis; Neoplasm Proteins; Nuclear Proteins; Signal Transduction

Glycosaminoglycans were metabolically labeled in subconfluent cultures of highly metastatic 7Gp122 and poorly metastatic IC8 variants and of the low metastatic parental M4Be human melanoma cell line. Proteoglycans were separated by DEAE Trisacryl chromatography from the culture medium, from the heparin extract of the cell layer and from the heparin-extracted cell residue lyzed with detergents. Glycosaminoglycans were released from the proteoglycans by reductive alkaline hydrolysis and heparan sulfate (HS) was detected by deaminative cleavage with nitrous acid. Expressed on cell protein basis, the labeled HS content in the medium and in the cell layer decreased with increasing metastatic ability. The extraction of HS with heparin from the 7Gp122 cells indicated that this variant was enriched in (polypeptide bound) HS non inserted into the plasma membrane, compared with the low metastatic IC8 and M4Be cells. The HS fraction in heparin extract and in the heparin-extracted cell residue exhibited molecular mass heterogeneity on gel permeation chromatography and it contained HS fragments. Scission with nitrous acid followed by molecular sieve chromatography of the degradation products indicated that the tetra- and disaccharide repeats separated by the N-sulfated glucosamine residue were present in about equal amounts and constituted 60% of the HS chains in the IC8 and M4Be cells. HS from 7Gp122, IC8 and M4Be cells did not bind antithrombin III with high affinity but it was capable of binding bFGF in in vitro assay.

Topics: Antithrombin III; Chondroitin Sulfates; Fibroblast Growth Factor 2; Heparitin Sulfate; Humans; Melanoma; Neoplasm Metastasis; Tumor Cells, Cultured

This study addresses the characterization of heparan sulphates of the basement-membrane proteoglycans in tumour formed after the subcutaneous implantation of Lewis-lung-carcinoma-derived different metastatic clones (P29, LM12-3 and LM60-D6 clones with low, medium and high metastatic potentials respectively). Heparan sulphate proteoglycans (125-158 micrograms of hexuronate/g dry weight of tissue) were isolated from chondroitin ABC lyase digests of a proteoglycan fraction obtained after DEAE-Sephacel chromatography of tissue extracts. The proteoglycans were separated into three molecular species by Sepharose CL-4B chromatography followed by CsCl-density-gradient centrifugation: large proteoglycans with an estimated M(r) of 820,000-130,000, which consisted of two components with low (< 1.34 g/ml; PGII-M) and high (> 1.37 g/ml; PGII-B) density, and a small proteoglycan with an M(r) of less than 80,000 (PGIII). Of these, only the PGII-M proteoglycan (34-37 micrograms of hexuronate/g dry weight) reacted with the antiserum against proteoglycan of Engelbreth-Holm-Swarm-tumour basement membrane, and represented, therefore, a basement-membrane proteoglycan. Digestion with heparan sulphate lyases I and II of the heparan sulphates (M(r) 36,000) from the PGII-M proteoglycan of the three tumours resulted in almost complete depolymerization to give six unsaturated disaccharides identified as 2-acetamido-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyranosyluron ic acid)-D-glucose, 2-acetamido-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyranosyluron ic acid)-6-O-sulpho-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyrano syluronic acid)-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyrano syluronic acid)-6-O-sulpho-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-2-O-sulpho-alpha-L-threo-hex-4- enopyranosyluronic acid)-D-glucose and 2-deoxy-2-sulphamino-4-O-(4-deoxy-2-O-sulpho-alpha-L-threo-hex-4- enopyranosyluronic acid)-6-O-sulpho-D-glucose. Comparison of the relative amounts of these disaccharides produced from the three tumour-derived heparan sulphates demonstrated that the degree of sulphation of the heparan sulphates correlated with the degree of morphological organization of the tumour basement membranes; the heparan sulphate from the more highly metastatic tumour with more highly organized basement membrane exhibited a higher degree of overall sulphation along the glycosaminoglycan chains, which was due to an increased cont

Topics: Animals; Basement Membrane; Carbohydrate Sequence; Centrifugation, Density Gradient; Chromatography, Gel; Chromatography, High Pressure Liquid; Disaccharides; Electrophoresis, Gel, Two-Dimensional; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunohistochemistry; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Neoplasm Metastasis; Proteoglycans; Tumor Cells, Cultured

The ability of normal and malignant blood-borne cells to extravasate correlates with the activity of an endo-beta-D-glucuronidase (heparanase) which degrades heparan sulfate (HS) in the subendothelial extracellular matrix (ECM). The association of malignancy with different types of coagulopathies prompted us to study the effect of thrombin (EC 3.4.21.5), a serine protease elaborated during activation of the clotting cascade, on the ability of heparanase to degrade the ECM-HS. The circulating zymogen form of thrombin, prothrombin, was converted to proteolytically active thrombin during incubation with ECM. Thrombin generation by the ECM was time and dose dependent, reaching maximal conversion by 6 h incubation at 3 U/ml of prothrombin. Heparanase-mediated release of low Mr HS cleavage products from sulfate-labeled ECM was stimulated four- to sixfold in the presence of alpha-thrombin, but there was no effect on degradation of soluble HS. Similar results were obtained with heparanase preparations derived from mouse lymphoma and human hepatoma cell lines and from human placenta. Incubation of ECM with alpha-thrombin alone resulted in release of nearly intact high-Mr labeled proteoglycans. Thrombin stimulation of heparanase action was dose and time dependent, reaching a maximal value at 24 h incubation with 1 microM alpha-thrombin. The effect of modified thrombin preparations correlated with their proteolytic activity. Catalytically blocked preparations of thrombin (e.g., DIP-alpha-thrombin, MeSO2-alpha-thrombin) failed to facilitate heparanase action, while catalytically modified preparations (e.g., gamma-thrombin, NO2-alpha-thrombin) exerted only a slight enhancement. Antithrombin III (ATIII) and hirudin both inhibited thrombin-stimulated heparanase degradation of ECM-bound HS. Heparanase action was also facilitated by ECM-immobilized thrombin to an extent which was similar to that induced by soluble thrombin. This result implies that thrombin sequestered by the subendothelial ECM and protected from interaction with its natural inhibitor ATIII (Bar-Shavit et al., 1989, J. Clin. Invest. 84, 1096-1104) may participate locally in cellular invasion during tumor metastasis, inflammation, and autoimmunity.

Topics: Animals; Autoimmunity; Cattle; Cells, Cultured; Cornea; Dose-Response Relationship, Drug; Enzyme Activation; Epithelial Cells; Extracellular Matrix; Glucuronidase; Glycoside Hydrolases; Heparitin Sulfate; Inflammation; Neoplasm Metastasis; Thrombin

Highly sulfated, heparinlike species of heparan sulfate proteoglycans, with heparinlike glycosaminoglycan chains, are extracellular matrix components that are plasma membrane bound in growth-arrested liver cells. Heparins were found to inhibit the growth and lower the clonal growth efficiency of HepG2, a minimally deviant, human hepatoma cell line. Heparan sulfates, closely related glycosaminoglycans present in the extracellular matrix around growing liver cells, had no effect on the growth rate or clonal growth efficiency of HepG2 cells. Neither heparins nor heparan sulfates had any effect on the growth rate or clonal growth efficiency of two poorly differentiated, highly metastatic hepatoma cell lines, SK-Hep-1 and PLC/PRF/5. Heparin's inhibition of growth of HepG2 cells correlated with changes in the mRNA synthesis and abundance of insulinlike growth factor II (IGF II) and transforming growth factor beta (TGF beta). HepG2 cells expressed high basal levels of mRNAs encoding IGF II and TGF beta that were inducible, through transcriptional and posttranscriptional mechanisms, to higher levels by specific heparin-hormone combinations. For both IGF II and TGF beta, the regulation was multifactorial. Transcriptionally, IGF II was regulated by the additive effects of insulin, glucagon, and growth hormone in combination with heparin; TGF beta was regulated primarily by the synergistic effects of insulin and growth hormone in combination with heparin. Posttranscriptionally, the mRNA abundance of the IGF II 4.5- and 3.7-kb transcripts was affected by insulin. Heparin induction of all IGF II transcripts was also dependent on triiodotyronine and prolactin, but it is unknown whether their induction by heparin was via transcriptional or posttranscriptional mechanisms. Heparin-insulin combinations regulated TGF beta posttranscriptionally. The poorly differentiated hepatoma cell lines PLC/PRF/5 and SK-Hep-1 either did not express or constitutively expressed low basal levels of IGF I, IGF II, and TGF beta, whose mRNA synthesis and abundance showed no response to any heparin-hormone combination. We discuss the data as evidence that matrix chemistry is a variable determining the expression of autocrine growth factor genes and the biological responses to them.

Topics: Blotting, Northern; Carcinoma, Hepatocellular; Cell Differentiation; Cell Division; Clone Cells; Growth Substances; Heparin; Heparitin Sulfate; Hormones; Humans; In Vitro Techniques; Insulin-Like Growth Factor II; Liver Neoplasms; Neoplasm Metastasis; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Cells, Cultured

A routine procedure has been developed for the isolation and maintenance in culture of human ovarian carcinoma cells derived from biopsy specimens. Cell attachment, plating efficiency and initial outgrowth were greatly improved by seeding the cells on a basement-membrane-like extracellular matrix (ECM) deposited by cultured corneal endothelial cells. These effects were most significant in serum-free conditions which markedly reduced the rate of cell attachment and growth on regular tissue culture plastic. In 60-80% of the cases and regardless of the patient's age, cells cultured on ECM in the absence of serum divided actively and formed a tightly packed epithelial cell monolayer. Fibroblast overgrowth and cell detachment often occurred on ECM in the presence of serum. Incubation of the human ovarian carcinoma cells with sulfate-labelled ECM, resulted in the release of heparan sulfate degradation fragments, 4- to 7-fold smaller than intact heparan sulfate side chains. This degradation was brought about by endoglycosidase (heparanase) activity expressed to a higher extent by cells that were first maintained in primary cultures as compared with cell aggregates taken directly from the biopsy specimen. In most cases, cells derived from metastatic tumors expressed a higher heparanase activity than cells from the primary ovarian tumor. This result corroborates previous studies, performed with cell lines, on the possible involvement of heparanase in tumor cell invasion and metastasis.

Topics: Autoradiography; Biopsy; Carcinoma; Chondroitin Sulfate Proteoglycans; Cytological Techniques; Extracellular Matrix; Female; Gene Expression Regulation, Enzymologic; Glucuronidase; Glycoside Hydrolases; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Neoplasm Metastasis; Ovarian Neoplasms; Tumor Cells, Cultured

Heparan sulfate (HS) enhanced the growth of the highly metastatic (HM) 3LL cell line, but not that of the low metastatic (LM) counterpart, in a dose-dependent way. Heparin, chondroitin sulfate, hyaluronate did not produce this effect. At 4 degrees C both cell lines exhibited high affinity binding sites (Kd 10(-8) M) for exogenous HS. Unlike LM cells, the HM ones lost half of the surface-bound HS during the 24-hour incubation at 37 degrees C. HM cells in the exponential growth phase took up the bound HS at lower rate than the LM counterparts. Both cell lines fragmented the exogenous HS intracellularly, but only the HM cells were able to degrade it at the cell surface. The HM cells contained much more heparin-binding proteins--especially in the cell membrane and nuclear fraction--than the LM ones. These results clearly demonstrate that the interactions of tumor cells with exogenous HS are influenced by the invasive phenotype. We suggest also that there could be a correlation between the surface degradation, the slow uptake and the growth-promoting effect of the exogenous HS.

Topics: Animals; Cattle; Cell Division; Extracellular Space; Glycosaminoglycans; Heparitin Sulfate; Lung Neoplasms; Mice; Neoplasm Metastasis; Phenotype; Protein Binding; Tumor Cells, Cultured

The abilities of rodent tumor cell lines; B16BL6, ND and LT dietary variants of B16BL6, +SA, RT7-4bs and RT7-4bLs to invade composite collagen I gels containing heparin, chemically modified heparins, heparan sulfate, chondroitin sulfate, hyaluronic acid, dextran, dextran sulfate, laminin and collagen IV were investigated, and compared to the invasion of plain collagen I gels. The presence of heparin or heparan sulfate most generally promoted tumor cell invasion of the gels, with more aggressive invasion being noted for the more metastatic variants examined. Of the chemically modified heparins tested, carboxyl-reduced heparin promoted matrix invasion by B16BL6 and +SA cells to the greatest degree. Hyaluronic acid marginally promoted invasion by +SA and RT7-4bs primary cells while, in these collagen I based gels laminin only promoted matrix invasion by primary +SA cells to a very limited degree. The tumor cell lines attached relatively poorly to heparan sulfate substrates compared to the other glycosaminoglycans tested, and the primary tumor cell lines also attached relatively poorly to collagen I. As expected, highly metastatic variants showed greater attachment to laminin than did their less metastatic counterparts. Apart from the negative correlation of cellular attachment to heparan sulfate substrates with invasiveness towards heparan sulfate containing gels, no other relationships emerged linking attachment rates with invasive activities for particular complex gel compositions. Our results suggest an important role for heparan sulfate, and possibly also tissue heparin, in promoting tumor cell invasion of extracellular matrices. Results from complex gels containing dextran or dextran sulfate failed to support the hypothesis that GAG sulfation is important to cellular invasion. The activity of the chemically modified heparins in promoting invasion, when present as components of these model matrices, suggests that part of the anti-metastatic activity of these compounds, when preincubated with tumor cells prior to intravenous inoculation, could result from interference with tumor cell extravasation.

Topics: Animals; Cell Adhesion; Collagen; Extracellular Matrix; Female; Glycosaminoglycans; Heparin; Heparitin Sulfate; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Invasiveness; Neoplasm Metastasis; Tumor Cells, Cultured

We previously demonstrated that noncytolytic butanol extraction of B16 melanoma cells can increase the number of experimental lung metastases, and that brief incubation of the extracted cells with the extracted moieties reduces metastatic phenotype. This study examined the possibility that the extracted components are endogenous inhibitors of tumor cell surface-associated, degradative enzymes. The activity was found to be tumor associated, since only tumor extracts could reduce the number of experimental lung metastases of a variety of solid tumors. The activity in crude butanol extracts of B16-F1 that modulated the metastatic phenotype of extracted B16-F10 was partially purified by preparative isoelectric focusing and high-performance gel permeation chromatography. Incubation of extracted B16-F10 cells with low (Mr 2,000-10,000) molecular weight materials focusing in the pH 5.6 to 5.8 region of the preparative isoelectric focusing gradient significantly reduced the number of experimental lung foci. Ampholines alone had no effect. Evidence that the extracted moiety might be an endogenous enzyme inhibitor was obtained with the use of the subendothelial matrix degradation assay, wherein B16-F10 cells digest 35S-labeled heparan sulfate proteoglycan. The same materials that reduced the metastatic potential of butanol-extracted B16-F10 cells also inhibited extracellular matrix degradation by 30 to 85%, as well as the activity of partially purified heparanase (endo-beta-glucuronidase). The metalloproteinase inhibitor 1,10-phenanthroline and the heparanase inhibitor heparin partially (30 to 50%) blocked extracellular matrix degradation. Conversely, inhibitors of serine, thiol, acid, and other proteases had little or no effect on extracellular matrix degradation. These data provide evidence that an endogenous, heat-stable inhibitor of cell surface degradative enzymes such as heparanase may play a role in hematogenous metastasis, and support the hypothesis that butanol extraction activates some of these surface enzymes by removing the endogenous inhibitors.

Topics: 1-Butanol; Animals; Butanols; Cell Line; Chondroitin Sulfate Proteoglycans; Extracellular Matrix; Heparan Sulfate Proteoglycans; Heparin; Heparitin Sulfate; Melanoma; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Molecular Weight; Neoplasm Metastasis; Phenanthrolines

Topics: Animals; Chondroitin Sulfate Proteoglycans; Endothelium, Vascular; Extracellular Matrix; Glycosaminoglycans; Glycoside Hydrolases; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Lymphoma; Mice; Neoplasm Metastasis; Peptide Hydrolases; T-Lymphocytes; Tumor Cells, Cultured

Differential cell adhesion has been proposed to play a role in organ-specific tumor metastasis. To further explore this hypothesis, we have employed a Lewis lung carcinoma cell line and 2 variants that differ in their ability to metastasize to lung and liver. The three cell lines were tested for their ability to adhere to defined extracellular matrix components that had been previously adsorbed to nylon membranes. Our results demonstrate that the parental cell line adheres preferentially to fibronectin relative to all other adhesion molecules tested. The lung colonizing variant, M27, adheres well to fibronectin and also to type V collagen but adheres poorly to laminin, to types I and VI collagen or to heparan sulfate. In contrast, the liver colonizing H59 cell line was highly adherent to laminin as well as to fibronectin but did not adhere to heparan sulfate or to any of the collagen types tested. These results demonstrate that three related cell lines with differing metastatic specificities have marked differences in their abilities to bind to defined matrix molecules. Such differences may play a role in the preferential localization to specific organ beds in vivo.

Topics: Animals; Carcinoma; Cell Adhesion; Collagen; Extracellular Matrix; Fibronectins; Heparitin Sulfate; Kinetics; Laminin; Liver Neoplasms; Lung Neoplasms; Mice; Neoplasm Metastasis

We studied the relationship between the metastatic potential of murine B16 melanoma cells and their surface expression of heparan sulfate glycosaminoglycan (HS-GAG) by using HepSS-1, a monoclonal antibody specific to HS-GAG. Firstly, among five B16 sublines, those capable of developing lung colonies with high efficiencies, such as B16-F10, had relatively low levels of surface HS-GAG. Secondly, a subline freshly prepared from metastatic lung colonies (F1) displayed a level of surface HS-GAG lower than that of injected B16 cells. Thirdly, in vitro selection of B16 cells with low surface HS-GAG by repeated HepSS-1 staining and cell-sorting resulted in cells with a higher metastatic efficiency than that of the original B16 cells. Collectively, B16 melanoma cells with high metastatic activities seem to have low surface HS-GAG. We also found that there was a positive correlation between the surface level of HS-GAG and the susceptibility to natural killer cells in eight B16 sublines.

Topics: Animals; Glycosaminoglycans; Heparitin Sulfate; Killer Cells, Natural; Lung Neoplasms; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Metastasis

Glycosaminoglycans of cultured nickel-induced rat rhabdomyosarcoma cell lines with different metastatic potentials and of non-malignant myoblasts, grown in the presence or in the absence of hydrocortisone, were studied comparatively. The newly formed [3H]glucosamine-labelled cell surface proteoglycans and glycosaminoglycans were separated by ion exchange chromatography and partially characterized. The overall incorporation of the label in the glycosaminoglycan fractions and the average molecular weight of the heparan and of the chondroitin sulfate proteoglycans was lower in the malignant cells than in the non-malignant L6 myoblasts. The strongly metastatic 9-4/0 parental line and the 6 subline were relatively richer in chondroitin sulfate and poorer in dermatan sulfate labels than the very weakly metastatic 8 subline and the L6 myoblasts. Hyaluronic acid and heparan sulfate labels were inversely related to the metastatic capacity of the cell lines studied. Hydrocortisone treatment induced an increase in the cell surface chondroitin and dermatan sulfate labels in the case of the strongly metastatic lines, and a decrease of the same parameters in the case of the weakly metastatic 8 line.

Topics: Animals; Cell Line; Cell Membrane; Chondroitin Sulfates; Dermatan Sulfate; Glucosamine; Glycosaminoglycans; Heparitin Sulfate; Hyaluronic Acid; Hydrocortisone; Molecular Weight; Muscles; Neoplasm Metastasis; Nickel; Rats; Rats, Inbred Strains; Rhabdomyosarcoma

Several fibroblast and melanoma cell lines were studied with respect to their ability to degrade heparan sulfate (HS). The optimum pH for HS degradation by HS endoglycosidase (heparanase) for all cell lines is about 5.6, but the activity of the enzyme is still present at physiological pH. The gel permeation analysis of degradation products revealed that heparanase cuts HS in fragments about one-seventh of their original size. Since the optimum pH of HS endoglycosidase activity and the terminal molecular weight of degraded HS are the same in both cell lines, it is likely that fibrosarcoma and melanoma heparanases are identical enzymes. Cell extracts and intact cells of metastatic sublines degrade HS faster than do their nonmetastatic counterparts. The degradative activity of intact cells parallels those of cell extracts, but at a much lower level; moreover, conditioned media do not appreciably degrade HS, suggesting that heparanase is scarcely released into the medium; thus, considering the differences in degradative activity between cell extracts and intact cells or conditioned medium and the occurrence of cell lysis in a tumor in vivo, we suggest that the measure of degradative activity of intact cells in vitro is not indicative of a relationship to metastasis. The total cellular content of lytic enzymes could represent the real metastatic potential of proliferating cells, but it is also necessary to find an in vitro model better representing the behavior of neoplastic cells in vivo.

Topics: Animals; Cell Line; Chromatography, Gel; Fibrosarcoma; Glucuronidase; Glycoside Hydrolases; Heparitin Sulfate; Hydrogen-Ion Concentration; Male; Melanoma; Mice; Molecular Weight; Neoplasm Metastasis

This unit has in the past evaluated the Nb rat prostatic adenocarcinoma model with respect to chemotherapies. Recently, this unit has been evaluating agents that may have a role in decreasing metastatic rate. The three androgen-insensitive tumors, Nb Pr A.I. I, II, III, have been evaluated herein. Agents that have been used include indomethacin, heparin, heparin plus cortisone, and heparan sulfate (SP54). It has been shown that these agents do play a role in reducing the metastasis. In evaluation of Nb Pr A.I, I, control animals had a metastatic rate of 57%. In treatment with indocin, only 21% of the animals, three of 14 animals treated, had a metastasis, and treatment with heparin and cortisone resulted in one of 14 animals having metastasis. Similar observations were seen when treatment with SP54 and heparin was evaluated in the Nb Pr A.I. III; 18 and 27% of treatment group animals had metastasis, whereas 55% of control groups had metastasis. Similarly, in the Nb Pr A.I. II evaluation, control animals having a metastatic rate of 43% had heparin plus cortisone and heparin alone, and this particular tumor model revealed complete resolution with no animals having metastatic disease. The majority of these agents have not effected tumor volume in terms of reduction as much as the best chemotherapeutic agents in this model system include cyclophosphamide and cis-platinum.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Cortisone; Disease Models, Animal; Drug Therapy, Combination; Heparin; Heparitin Sulfate; Indomethacin; Male; Neoplasm Metastasis; Prostatic Neoplasms; Rats; Rats, Inbred Strains

After transport in the blood and implantation in the microcirculation, metastatic tumor cells must invade the vascular endothelium and underlying basal lamina. Mouse B16 melanoma sublines were used to determine the relation between metastatic properties and the ability of the sublines to degrade enzymatically the sulfated glycosaminoglycans present in the extracellular matrix of cultured vascular endothelial cells. Highly invasive and metastatic B16 sublines degraded matrix glycosaminoglycans faster than did sublines of lower metastatic potential. The main products of this matrix degradation were heparan sulfate fragments. Intact B16 cells (or their cell-free homogenates) with a high potential for lung colonization degraded purified heparan sulfate from bovine lung at higher rates than did B16 cells with a poor potential for lung colonization. Analysis of the degradation fragments indicated that B16 cells have a heparan sulfate endoglycosidase. Thus the abilities of B16 melanoma cells to extravasate and successfully colonize the lung may be related to their capacities to degrade heparan sulfate in the walls of pulmonary blood vessels.

Topics: Animals; Cell Line; Glycosaminoglycans; Glycoside Hydrolases; Heparitin Sulfate; Melanoma; Mice; Neoplasm Invasiveness; Neoplasm Metastasis

Urinary glycosaminoglycan excretion was examined in 25 individuals with bladder cancer in comparison to glycosaminoglycan excretion by eight normal individuals. Urinary glycosaminoglycan was isolated by gel filtration and quantified as macromolecular uronate concentration. Electrophoresis in calcium acetate and densitometry of Alcian blue-stained electrophoretograms were used to separate and quantify the relative amounts of individual glycosaminoglycans. Elevated excretion of macromolecular uronate was noted in 53% of the cancer cases. The highest levels were found among individuals with metastatic disease. Three electrophoretic bands were always detected in the control and cancer groups: chondroitin sulfate, heparan sulfate (both confirmed by chemical and enzymatic degradation), and a third band (Band 1) of unknown composition. A fourth band, corresponding to dermatan sulfate, was seen in some high-grade metastatic tumors. Band 1 excretion was elevated in a significant fraction of all patients. Seven of 12 metastatic cases but only two of 13 localized cases showed increased heparan sulfate excretion. Diagnostic limits were drawn from the observed distributions of normals, and with these limits 92% of the cancer cases, including 12 of 12 metastatic cases, could be identified. The results strongly suggest noninvasive urinary glycosaminoglycan analysis may well provide a new biochemical approach for detecting and monitoring the pathogeneses of bladder cancer.

Topics: Chondroitin Sulfates; Dermatan Sulfate; Electrophoresis; Glycosaminoglycans; Heparitin Sulfate; Humans; Macromolecular Substances; Neoplasm Metastasis; Urinary Bladder Neoplasms; Uronic Acids