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PubMed2003

Inhibition of heparanase activity and heparanase-induced angiogenesis by suramin analogues.

Heparanase, a heparan sulfate-specific endo-beta-D-glucuronidase, plays an important role in tumor cell metastasis through the degradation of extracellular matrix heparan sulfate proteoglycans (ECM HSPG). Heparanase activity correlates with the metastatic propensity of tumor cells. Suramin, a polysulfonated naphthylurea, is an inhibitor of heparanase with suramin analogues shown to possess antiangiogenic and antiproliferative properties. We investigated the effects of selected suramin analogues (NF 127, NF 145 and NF 171) on heparanase activity and heparanase-driven angiogenesis. Studies of the ability of cellular extracts and purified heparanase from human, highly invasive and brain-metastatic melanoma (70W) cells revealed that heparanase expressed by these cells was effectively inhibited by suramin analogues in a dose-dependent manner. These analogues possessed more potent heparanase inhibitory activities than suramin: The concentrations required for 50% heparanase inhibition (IC(50)) were 20-30 microM, or at least 2 times lower than that for suramin. One hundred percent inhibition was observed at concentrations of 100 microM and higher. Of relevance, these compounds significantly decreased (i) the invasive capacity of human 70W cells by chemoinvasion assays performed with filters coated with purified HSPG or Matrigel trade mark, and (ii) blood vessel formation by in vivo angiogenic assays, thus linking their antiangiogenic properties with impedance of heparanase-induced angiogenesis. Specifically, inhibition of invasion by NF 127, NF 145 and NF 171 was found at 10 microM concentrations of compounds with a significant decrease of invasive values at concentrations as low as 1.5 microM. In addition, NF 127, NF 145 and NF 171 promoted nearly complete inhibition of heparanase-induced angiogenesis at values ranging from 236 microM (for NF 145) to 362 microM (for NF 127). These results further emphasize the importance of heparanase in invasive and angiogenic mechanisms and the potential clinical application of heparanase inhibitors such as suramin analogues in cancers and angiogenesis-dependent diseases.

Antineoplastic Agents↗
PubMed1998

Partial purification of heparanase activities in Chinese hamster ovary cells: evidence for multiple intracellular heparanases.

Heparanases are mammalian endoglycosidases that cleave heparan sulphate glycosaminoglycans from proteoglycan core proteins and degrade them into shorter chains. The enzymes have been proposed to act in a variety of cellular processes, including proteoglycan catabolism, remodelling of basement membranes and release of heparan sulphate-binding ligands from their extracellular storage sites. Additional functions for heparanases may be to generate short heparan sulphate chains that stabilize or activate other proteins. While heparanase activities have been described in a number of tissues and cell lines, it is not known how many different enzymes are responsible for these activities. Our recent studies characterizing the short glycosaminoglycans produced in Chinese hamster ovary (CHO) cells suggested that multiple heparanases are necessary for the formation of the short heparan sulphate chains [Bame and Robson (1997) J. Biol. Chem. 272, 2245-2251]. We examined whether this is the case by purifying heparanase activity from CHO cell homogenates. Based on their ability to bind ion-exchange resins and their elution from gel-filtration columns, four separate heparanase activities were partially purified. All four activities cleave free glycosaminoglycans over a broad pH range of 3.5-6.0 or 6. 5, suggesting that they act in the endosomal/lysosomal pathway. The sizes of the short heparan sulphate chains generated by the partially purified heparanases ranged from 6 to 9 kDa, and for two of the activities the product size is pH-dependent. Three of the four activities degrade proteoglycans as well as the free glycosaminoglycan chain. Interestingly, all four enzymes generate short glycosaminoglycans with a sulphate-rich, modified domain at the non-reducing end of the newly formed chain. Since our previous studies showed that in CHO cells there is also a population of short heparan sulphates with a modified domain at the reducing end of the chain, this suggests that there may be another heparanase in CHO cells that was not purified. Alternatively, our findings suggest that the formation of short heparan sulphate glycosaminoglycans inside CHO cells may be a result of the concerted action of multiple heparanases, and may depend on the proportions of the different enzymes and the environment in which the chains are degraded.

Animals↗
PubMed1997

Neurotrophin stimulation of human melanoma cell invasion: selected enhancement of heparanase activity and heparanase degradation of specific heparan sulfate subpopulations.

Heparanase is an endo-beta-D-glucuronidase whose enzymatic targets are the glycosaminoglycan chains of heparan sulfate proteoglycans (50). Elevated levels of heparanase are associated with the metastatic potential of melanoma cells, and treatment of murine and human melanoma cells with the prototypic neurotrophin nerve growth factor (NGF) increases the production of heparanase by melanoma cells. We previously reported that physiological concentrations of NGF increased invasion of early passage human brain-metastatic 70W melanoma cells but not melanoma cells metastatic to other sites or nonmetastatic melanoma cells as measured in Matrigel invasion assays. Here we found that treatment of 70W melanoma cells with neurotrophin-3 (NT-3) increased Matrigel invasion, whereas treatment with neurotrophins other than NGF or NT-3 did not influence invasion. Mutants of NGF that do not bind to the neurotrophin receptor p75NTR or other nonneuronal growth factors were not able to enhance the invasion of 70W melanoma cells. When 70W cells were exposed to antisense oligonucleotides directed against p75NTR mRNA, there was a reduction in NGF and NT-3 binding, and the neurotrophins failed to enhance Matrigel invasion. To study the properties of heparanase in neurotrophin-regulated malignant melanoma invasive processes, we developed a sensitive heparanase assay consisting of purified [35S]HS subpopulations separated by agarose gel electrophoresis. Incubation of 70W cells with NGF or NT-3 but not brain-derived neurotrophic factor, neurotrophin-4/5 or mutant NGF resulted in increased release of heparanase activity that was capable of degrading a subpopulation of heparan sulfate molecules.

Electrophoresis, Agar Gel↗
PubMed1996

Neurotrophin stimulation of human melanoma cell invasion: selected enhancement of heparanase activity and heparanase degradation of specific heparan sulfate subpopulations.

Heparanase is an endo-beta-D-glucuronidase, the enzymatic targets of which are the glycosaminoglycan chains of heparan sulfate proteoglycans. Elevated levels of heparanase are associated with the metastatic potential of melanoma cells. Treatment of murine and human melanoma cells with the prototypic neurotrophin nerve growth factor (NGF) increases the production of heparanase by melanoma cells. We reported previously that physiological concentrations of NGF increased in vitro Matrigel invasion of early-passage human brain-metastatic 70W melanoma cells but not melanoma cells metastatic to other sites or nonmetastatic melanoma cells. Here we found that treatment of 70W melanoma cells with neurotrophin NT-3 increased Matrigel invasion, whereas treatment with neurotrophins other than NGF or NT-3 did not influence invasion. Mutants of NGF that do not bind to the neurotrophin receptor p75NTR or other nonneuronal growth factors were not able to enhance the invasion of 70W melanoma cells. When 70W cells were exposed to antisense oligonucleotides directed against p75NTR mRNA, there was a reduction in NGF and NT-3 binding, and the neurotrophins failed to enhance Matrigel invasion. To study the properties of heparanase in NT-regulated malignant melanoma invasive processes, we developed a sensitive heparanase assay consisting of purified [35S]heparan sulfate subpopulations separated by agarose gel electrophoresis. Incubation of 70W cells with NGF or NT-3, but not brain-derived NT factor, NT-4/5, or mutant NGF, resulted in increased release of heparanase activity that was capable of degrading a subpopulation of heparan sulfate molecules.

Base Sequence↗
PubMed2004

Heparanase gene silencing, tumor invasiveness, angiogenesis, and metastasis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Angiogenesis Inducing Agents↗
PubMed2004

Localization of heparanase in esophageal cancer cells: respective roles in prognosis and differentiation.

In this study, we examined the distribution of heparanase protein in 75 esophageal squamous cell carcinomas by immunohistochemistry and analyzed the relationship between heparanase expression and clinicopathological characteristics. In situ hybridization showed that the mRNA expression pattern of heparanase was similar to that of the protein, suggesting that increased expression of the heparanase protein at the invasive front was caused by an increase of heparanase mRNA in tumor cells. Heparanase expression correlated significantly with depth of tumor invasion, lymph node metastasis, tumor node metastasis (TNM) stage and lymphatic invasion. Overexpression of heparanase in esophageal cancers was also associated with poor survival. In addition to its localization in the cytoplasm and cell membrane, heparanase was also identified in the nuclei of normal epithelial and tumor cells by immunohistochemistry. Furthermore, nuclear heparanase was detected in nuclear extract of cancer cell lines by Western blot and immunohistochemistry. Examination of the role of nuclear heparanase in cell proliferation and differentiation by double immunostaining for proliferating cell nuclear antigen (PCNA) and cytokeratin 10 (CK10) showed significant relationship between nuclear heparanase expression and differentiation (heparanase vs CK10), but not for proliferative state of esophageal cancer cells (heparanase vs PCNA). Our results suggest that cytoplasmic heparanase appears to be a useful prognostic marker in patients with esophageal cancer and that nuclear heparanase protein may play a role in differentiation. Inhibition of heparanase activity may be effective in the control of esophageal tumor invasion and metastasis.

Adult↗
PubMed2004

Processing and activation of latent heparanase occurs in lysosomes.

Heparanase is a heparan sulfate degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Heparanase is synthesized as a 65 kDa non-active precursor that subsequently undergoes proteolytic cleavage, yielding 8 kDa and 50 kDa protein subunits that heterodimerize to form an active enzyme. The protease responsible for heparanase processing is currently unknown, as is the sub-cellular processing site. In this study, we characterize an antibody (733) that preferentially recognizes the active 50 kDa heparanase form as compared to the non-active 65 kDa heparanase precursor. We have utilized this and other anti-heparanase antibodies to study the cellular localization of the latent 65 kDa and active 50 kDa heparanase forms during uptake and processing of exogenously added heparanase. Interestingly, not only the processed 50 kDa, but also the 65 kDa heparanase precursor was localized to perinuclear vesicles, suggesting that heparanase processing occurs in lysosomes. Indeed, heparanase processing was completely inhibited by chloroquine and bafilomycin A1, inhibitors of lysosome proteases. Similarly, processing of membrane-targeted heparanase was also chloroquine-sensitive, further ruling out the plasma membrane as the heparanase processing site. Finally, we provide evidence that antibody 733 partially neutralizes the enzymatic activity of heparanase, suggesting that the N-terminal region of the molecule is involved in assuming an active conformation. Monoclonal antibodies directed to this region are likely to provide specific heparanase inhibitors and hence assist in resolving heparanase functions under normal and pathological conditions.

Animals↗
PubMed2003

High heparanase activity in multiple myeloma is associated with elevated microvessel density.

Heparanase is an enzyme that cleaves heparan sulfate chains of proteoglycans, and its expression has been associated with increased growth, metastasis, and angiogenesis of some tumors. Because myeloma tumor cells express high levels of the syndecan-1 heparan sulfate proteoglycan and because these tumors grow as highly vascularized aggregates within the bone marrow, we analyzed the activity, expression, and function of heparanase in myeloma patients. Analysis of heparanase activity in the plasma isolated from bone marrow biopsies of 100 patients reveals 86 positive for heparanase activity and 14 negative. The bone marrow samples can be further divided into three categories of heparanase activity, high activity (42 patients), low activity (44 patients), and negative (14 patients). In contrast to the bone marrow plasma, levels of heparanase activity in peripheral blood plasma of 29 myeloma patients were either negative or low, suggesting that in multiple myeloma, heparanase functions in the local microenvironment of the bone marrow and its activity is not significantly elevated systemically. Immunohistochemistry reveals that patients with high levels of heparanase activity often have tumor cells with intense staining for the enzyme. Interestingly, a marked heterogeneity among tumor cells was noted, with clusters of heavily stained cells surrounded by cells with weak or negative staining for heparanase. Analysis of microvessel density reveals a strikingly higher concentration of vessels in patients with high heparanase activity (78.96 vessels/mm(2)) as compared with patients negative for heparanase activity (25.03 vessels/mm(2)). When human myeloma cells transfected with the cDNA for heparanase are implanted in severe combined immunodeficient (SCID) mice, the resulting tumors exhibited a significantly higher microvessel density than did tumors established with control cells. Thus, expression of heparanase appears to play a direct role in enhancing microvessel density in these myeloma tumors. Because heparanase is known to stimulate angiogenesis, and because high microvessel density is associated with poor prognosis in myeloma, we conclude that heparanase expression likely plays an important role in regulating the growth and progression of myeloma, and that therapies designed to block heparanase activity may aid in controlling this cancer.

Animals↗
PubMed2006

Regulation, function and clinical significance of heparanase in cancer metastasis and angiogenesis.

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 post-operative survival of cancer patients. Heparanase is synthesized as a 65 kDa inactive precursor that undergoes proteolytic cleavage, yielding 8 and 50 kDa protein subunits that heterodimerize to form an active enzyme. Human heparanase is localized primarily within late endosomes and lysosomes and occasionally on the cell surface and within the cell nucleus. Transcriptional activity of the heparanase promoter is stimulated by demethylation, early growth response 1 (EGR1) transcription factor, estrogen, inflammatory cytokines and inactivation of p53. N-acetylated glycol-split species of heparin as well as siRNA heparanase gene silencing inhibit tumor metastasis and angiogenesis in experimental 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. Heparanase exhibits also non-enzymatic activities, independent of its involvement in ECM degradation and changes in the extracellular microenvironment. For example, cell surface expression of heparanase elicits a firm cell adhesion, reflecting an involvement in cell-ECM interaction. Heparanase enhances Akt signaling and stimulates PI3K- and p38-dependent endothelial cell migration and invasion. It also promotes VEGF expression via the Src pathway. The enzyme may thus activate endothelial cells and elicits angiogenic and survival responses. Studies with heparanase over-expressing transgenic mice revealed that the enzyme functions in normal processes involving cell mobilization, HS turnover, tissue vascularization and remodeling. In this review, we summarize the current status of heparanase research, emphasizing molecular and cellular aspects of the enzyme, including its mode of processing and activation, control of heparanase gene expression, enzymatic and non-enzymatic functions, and causal involvement in cancer metastasis and angiogenesis. We also discuss clinical aspects and strategies for the development of heparanase inhibitors.

Animals↗
PubMed1997

A rapid quantitative assay for the detection of mammalian heparanase activity.

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 potential, and for the development of specific inhibitors of heparanase activity and metastasis.

Adult↗
PubMed2005

Early growth response gene 1 (EGR1) regulates heparanase gene transcription in tumor cells.

Heparanase is an endoglycosidase that degrades heparan sulfate chains of heparan sulfate proteoglycans, a key component of extracellular matrix and basement membranes. Studies using heparanase inhibitors and gene silencing have provided evidence to support an important role for heparanase in tumor metastasis and angiogenesis. The expression of heparanase is normally very tightly controlled, however, it is commonly deregulated in tumor cells, which express elevated heparanase activity that correlates with high levels of heparanase mRNA. We recently identified the transcription factor early growth response gene 1, EGR1, as a key regulator of inducible heparanase transcription in T cells. In this study using chromatin immunoprecipitation, we demonstrate for the first time that EGR1 binds to the heparanase gene promoter in vivo. The important question of the role of EGR1 in regulating heparanase transcription in tumor cells was then assessed. Studies were carried out in four epithelial tumor lines of different tissue origin. Functional dissection of the heparanase promoter identified a 280-bp region that was critical for transcription of the heparanase gene. Transactivation studies using an EGR1 expression vector co-transfected with a reporter construct containing the 280-bp region showed EGR1-activated heparanase promoter activity in a dose-dependent manner in prostate or breast adenocarcinoma and colon carcinoma cell lines. In contrast, overexpression of EGR1 resulted in a dose-dependent repression of promoter activity in melanoma cells. Using site-directed mutagenesis the 280-bp region was found to contain two functional EGR1 sites and electrophoretic mobility shift assays showed binding of EGR1 to both of these sites upon activation of tumor cells. Furthermore, the heparanase promoter region containing the EGR1 sites was also inducible in tumor cells and induction corresponded to HPSE expression levels. These studies show that EGR1 regulates heparanase transcription in tumor cells and importantly, can have a repressive or activating role depending on the tumor type.

Animals↗
PubMed2003

Heparanase expression correlates with invasion and poor prognosis in gastric cancers.

Degradation of basement membrane and extracellular matrix structures are important features of the metastatic process of malignant tumors. Human heparanase degrades heparan sulfate proteoglycans, which represent the main components of basement membranes and the extracellular matrix. Because of the role of heparanase in tumor invasion and metastasis, we examined heparanase expression in primary gastric cancers and in cell lines derived from gastric cancers by immunohistochemistry and RT-PCR, respectively. Four of seven gastric cancer cell lines showed heparanase mRNA expression by RT-PCR. Heparanase protein was detected in both the cytoplasm and the nucleus of heparanase mRNA-positive cells by immunohistochemical staining. Heparanase expression was confirmed in 35 (79.5%) of 44 gastric tumor samples by immunohistochemical staining. However, no or weak heparanase expression was detected in normal gastric mucosa. In situ hybridization showed that the mRNA expression pattern of heparanase was similar to that of the protein, suggesting that increased expression of the heparanase protein at the invasive front was caused by an increase of heparanase mRNA in tumor cells. Analysis of the clinicopathologic features showed stronger heparanase expression in cases of huge growing tumors, extensive invasion to lymph vessels, and regional lymph node metastasis. In gastric cancer, patients with heparanase expression showed significantly poorer prognosis than those without such expression (p = 0.006). In conclusion, our findings suggest that high expression of heparanase in gastric cancer is a strong predictor of poor survival.

Animals↗
PubMed2005

[Inhibitory effect of silencing heparanase expression with antisense oligodeoxynucleotide on invasive capability of human esophageal squamous cancer cell line TE-13].

BACKGROUND & OBJECTIVE: Heparanase, a kind of endo-D-glucuronidase, degrades heparin sulfate proteoglycans, and plays important roles in invasion and metastasis of many kinds of malignant tumors. This research was designed to investigate the expression of heparanase in esophageal squamous cancer cell line TE-13 and the effect of heparanase antisense oligodeoxynucleotide (ASODN) transfection on invasion of TE-13 cells. METHODS: Synthesized heparanase ASODN was transfected into TE-13 cells. Before and after transfection, the expression of heparanase mRNA and protein in TE-13 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry. Matrigel invasion assay was used to determine the invasive capability of TE-13 cells. RESULTS: In TE-13 cells, heparanase gene (585 bp) was detected by RT-PCR, and heparanase protein (50 ku) by Western blot. Heparanase protein mainly located in cytoplasm and on cell membrane. After heparanase ASODN transfection, heparanase gene and protein expression and the invasive TE-13 cells were significantly reduced along with the ascending concentration of heparanase ASODN (P<0.05, P<0.05). CONCLUSIONS: Heparanase gene is expressed in TE-13 cells. Heparanase ASODN can obviously inhibit heparanase gene expression, and restrain invasive activity of TE-13 cells.

Carcinoma, Squamous Cell↗
PubMed2006

Heparanase induces tissue factor expression in vascular endothelial and cancer cells.

BACKGROUND: Over-expression of tissue factor (TF) and activation of the coagulation system are common in cancer patients. Heparanase is an endo-beta-D-glucuronidase that cleaves heparan sulfate chains on cell surfaces and in the extracellular matrix, activity that closely correlates with cell invasion, angiogenesis and tumor metastasis. The study was undertaken to investigate the involvement of heparanase in TF expression. METHODS: Tumor-derived cell lines were transfected with heparanase cDNA and TF expression was examined. The effect of exogenous addition of active and inactive heparanase on TF expression and activity was studied in tumor cell lines and primary human umbilical vein endothelial cells. TF expression was also explored in heparanase over-expressing transgenic (Tg) mice. Blast cells were collected from acute leukemia patients and TF and heparanase expression levels were analyzed. RESULTS: Over-expression of heparanase in tumor-derived cell lines resulted in a 2-fold increase in TF expression levels, and a similar trend was observed in heparanase Tg mice in vivo. Likewise, exogenous addition of heparanase to endothelial or tumor-derived cells resulted in enhanced TF expression and activity. Interestingly, TF expression was also induced in response to enzymatically inactive heparanase, suggesting that this effect was independent of heparanase enzymatic activity. The regulatory effect of heparanase on TF expression involved activation of the p38 signaling pathway. A positive correlation between TF expression levels and heparanase activity was found in blasts collected from 22 acute leukemia patients. CONCLUSIONS: Our results indicate that in addition to its well-known function as an enzyme paving a way for invading cells, heparanase also participates in the regulation of TF gene expression and its related coagulation pathways.

Blood Coagulation↗
PubMed2004

Heparanase expression in human colorectal cancer and its relationship to tumor angiogenesis, hematogenous metastasis, and prognosis.

BACKGROUND: Heparanase is considered to play an important role in tumor invasion and hematogenous metastasis. The aim of this study was to determine the expression of heparanase in colorectal cancer to evaluate its prognostic value. METHODS: We analyzed heparanase mRNA derived from 130 colorectal cancer patients by reverse transcription polymerase chain reaction (PCR), compared its expression with clinicopathologic factors, and performed statistical analysis. To investigate the relationship between heparanase expression and tumor angiogenesis, 81 primary colorectal tumors were immunostained by use of a monoclonal anti-CD34 antibody. RESULTS: Thirty three of 130 cancer tissues overexpressed heparanase. There were significant correlations between heparanase expression and serosal invasion, venous invasion, and liver metastasis. Multivariate analyzes revealed that heparanase mRNA overexpression was a significant independent risk factor for hematogenous metastasis in colorectal cancer. Among 104 patients who underwent curative resection, heparanase expression correlated with a high recurrence. The 5-year survival rate was 84.6% for patients with heparanase negative tumors, and 47.7% for those with heparanase overexpression; these differences between two groups of patients were significant. In multivariate analysis using the Cox regression model, heparanese expression emerged as an independent prognostic indicator. Moreover, the tumor angiogenesis of heparanase-positive tumors determined with a monoclonal anti-CD34 antibody was significantly higher than that of heparanase-negative tumors. CONCLUSIONS: These results indicated that Heparanase expression may be an important role in invasion and hematogenous metastasis, and may be a biologic marker of prognostic significance in colorectal cancer patients.

Cell Line, Tumor↗
PubMed2005

Heparanase improves mouse embryo implantation.

OBJECTIVE: To improve mouse embryonic implantation by recombinant heparanase supplementation. Heparanase, an endoglycosidase-degrading heparan sulfate proteoglycan, may have a role in embryonic implantation because of its enzymatic, angiogenic, and adhesive properties. Increasing endometrial receptivity could improve one of the most difficult pathologies in human fertility. DESIGN: Comparison between mouse blastocysts obtained after 24-hour incubation of morulae with or without heparanase. SETTING: Experimental laboratory in a medical center. ANIMAL(S): Mice. INTERVENTION(S): Morulae were flushed from CB6F1 female mice and incubated for 24 hours at 37 degrees C in M16 medium supplemented with 0.1 mg/mL heparanase (n = 203), with albumin (n = 60), or with medium alone (n = 258). MAIN OUTCOME MEASURE(S): Blastocysts were evaluated by heparanase immunostaining (n = 10), activity assay (n = 283), and transfer to foster mice uterine horns (n = 228). The number of implantation sites was compared. RESULT(S): Immunostaining demonstrated that heparanase is constitutively expressed in mouse morulae and blastocyts. Heparanase supplementation resulted in increased staining and enzymatic activity in blastocyts. Implantation rates for the heparanase, M16 medium, and albumin groups, were 36.9%, 17.8%, and 20%, respectively (P<.01). CONCLUSION(S): Heparanase was found to be constitutively expressed by blastocyst-stage embryos. Moreover, the amount of heparanase was markedly increased by incubation of morulae with recombinant heparanase, evaluated by immunostaining and enzymatic activity. Heparanase supplementation resulted in approximately a twofold increase in embryo implantation rate in vivo. Taken together, these data suggest that heparanase is actively involved in embryo implantation.

Animals↗
PubMed2001

Increased expression of heparanase in puromycin aminonucleoside nephrosis.

BACKGROUND: The beta-D-endoglycosidase heparanase has been proposed as an important contributor to loss of glomerular charge in proteinuria. Expression of heparanase was, therefore, determined in acute puromycin aminonucleoside (PAN) nephrosis. METHODS: A rabbit polyclonal antibody was produced against a 17-amino acid peptide derived from the predicted amino acid sequence of heparanase. The antibody was validated by Western blot. Immunohistochemical staining and Western blotting were used to localize heparanase protein in normal kidneys and kidneys from rats with PAN nephrosis. Northern blot analysis was used to determine mRNA expression. RESULTS: Immunohistochemical staining showed that heparanase protein was localized to tubular cells in the distal convoluted tubules, thick ascending limb of the loop of Henle, and transitional cell epithelium in normal kidney. Minimal expression was noted in normal glomeruli. Western blot analysis of protein from isolated normal glomeruli showed minimal expression of the 65 kD proheparanase protein. A marked increase in the staining for heparanase was found at day 5 of the PAN nephrosis model, at approximately the time of onset of proteinuria, and at day 14. Expression was predominantly seen in podocytes. At day 5, only the 65 kD proheparanase species was identified, but at day 14, mature 58 kD heparanase also was present. Northern blot analysis of sieved glomeruli at day 14 confirmed an increase in heparanase mRNA. The human podocyte cell line 56/10A1 also produced both proheparanase and mature heparanase, suggesting that podocytes can activate heparanase without other cell types. CONCLUSION: The previously mentioned data confirm that the novel beta-D-endoglycosidase heparanase is up-regulated and activated in glomeruli from rats with proteinuria. Heparanase may be involved, therefore, in the loss of glomerular charge seen in proteinuria. Moreover, the presence of heparanase in normal tubules suggests that it may also be involved in cell migration or turnover.

Animals↗
PubMed1997

Heparanase and a synthetic peptide of heparan sulfate-interacting protein recognize common sites on cell surface and extracellular matrix heparan sulfate.

Heparanase is an endo-beta-D-glucuronidase that degrades the glycosaminoglycan chains of heparan sulfate (HS) proteoglycans at specific sites. Elevated levels of heparanase are associated with the metastatic potential of melanoma and other types of tumor cells. We previously reported heparanase degradation of cell surface HS subpopulations of the human adenocarcinoma cell line RL95. In the present study, heparanase activity was examined on RL95 cell surface HS subpopulations in the presence of a synthetic peptide (CRPKAKAKAKAKDQTK) of heparin/heparan sulfate-interacting protein (HIP; Liu, S., Smith, S. E., Julian, J., Rohde, L. H., Karin, N. J., and Carson, D. D. (1996) J. Biol. Chem. 271, 11817-11823). Heparanase digestion generated HS fragments from cell surface- or extracellular matrix-derived HS of approximately 25 and 9 kDa, respectively. In contrast, HS of various size classes isolated from proteoglycans secreted or released by RL95 and endothelial cells in culture were not susceptible to heparanase digestion. Incubation of heparanase-containing melanoma cellular extracts or partially purified heparanase preparations with cell surface- or ECM-derived HS and HIP peptide, but not a scrambled sequence of this peptide or other HS-binding proteins present in ECM, completely inhibited heparanase action. Conversely, predigestion of cell surface HS with either heparanase-containing cellular extracts or with secreted or partially purified heparanase destroyed binding to HIP peptide. Preincubation of HS with HIP peptide prevented subsequent heparanase digestion. Collectively, these data demonstrate that HIP peptide and heparanase recognize specific, common motifs within HS chains at cell surfaces and in ECM and may mutually modulate HS-dependent activities.

Amino Acid Sequence↗