10855

HPSE

heparanase;HPSE;heparanase

endo-glucoronidase|heparanase-1

4q21.3

protein-coding

BACKGROUND: metastasis of malignant breast cells is in part mediated through degradation of the extra-cellular matrix by proteolysis, enabling malignant cells to migrate through the surrounding stroma. Heparanase-1 (HPR1) is an endoglycosidase that specifically degrades the heparan sulfate (HS) moiety of proteoglycans, a component of the extracellular matrix and basement membrane. METHODS: Fifty-one primary breast tumors, 13 lymph node metastases, 4 ductal carcinoma in situ, 7 benign, and 5 normal specimens were examined for HPR1 expression using immunohistochemical staining. The functional role of HPR1 expression was determined by examining HS deposition using immunofluorescence staining. RESULTS: Sixteen of 30 breast carcinomas (53%) with sentinel node metastasis expressed HPR1. In contrast, only 5 of 21 nonmetastatic primary breast carcinomas (23%) were HPR1 positive. Eighteen of 30 breast carcinomas between 1 and 5 cm expressed HPR1, compared with 3 of 21 HPR1-positive specimens in tumors < or =1 cm. Statistical analysis revealed that HPR1 expression was associated with breast tumor metastases (P =.04) and primary tumors between 1 and 5 cm (P =.002). Ninety percent of HPR1-positive tumors lacked HS deposition, suggesting an inverse correlation between HPR1 expression and HS deposition. CONCLUSIONS: HPR1 expression correlates with the lack of HS deposition and with the metastatic potential of breast cancers. The frequency of HPR1 is significantly higher in breast tumors between 1 and 5 cm than in tumors < or =1 cm.

BACKGROUND/AIM: Heparanase (HPA) contributes to breast cancer metastasis by facilitating the breakdown of the basement membrane and extracellular matrix. High expression of HPA is thought to be associated with increased nodal involvement and poor survival in patients with breast cancer. Overexpression of cyclooxygenase-2 (COX-2) in breast cancer is associated with indicators of poor prognosis such as lymph node metastasis, poor differentiation, and large tumor size. The underlying mechanism by which HPA and COX-2 overexpression increases the metastatic potential of breast cancer is not fully-understood. To enhance our understanding over these mechanisms, we aimed to investigate the relationship between the size of the tumor and HPA expression, tumor grade as well as lymph node status in patients with breast cancer. MATERIALS AND METHODS: Immunohistochemical analysis of HPA and COX-2 expression was performed on 246 breast tumor samples. The expression of HPA was correlated with COX-2 expression, tumor grade, lymph node status, oestrogen receptor status. RESULTS: The overexpression of HPA and COX-2 was associated with increased likelihood of lymph node positivity in large, high-grade tumors. High-grade tumors with size greater than 20 mm, that overexpressed HPA, were 4-times more likely to be associated with lymph node involvement (OR 4.71,

BACKGROUND AND OBJECTIVE: Invasion and metastasis are the most common causes of mortality for patients with colorectal neoplasms, and blocking invasion and metastasis in a timely fashion has become a hot research focus. We investigated the expression of the messenger RNA of Syndecan-1 and HPA-1 in colorectal cancer, and their correlation with invasion and metastasis. METHODS: Real-time fluorescent quantitative polymerase chain reaction (PCR) was used to detect the expression of Syndecan-1 and HPA-1 in specimens from 49 patients with colorectal cancer, 49 paired adjacent colorectal neoplasms (2 cm from the carcinoma), and 49 surgical margins of paired normal colorectal mucosa tissue (5 cm from the carcinoma), to analyze their correlation with clinicopathologic characteristics of colorectal neoplasm. RESULTS: The expression of HPA-1 mRNA was significantly higher in colorectal cancer (40.56 +/- 11.75) than that in the paired adjacent colorectal neoplasms (18.28 +/- 11.33) and normal colorectal mucosa tissue (10.80 +/- 10.20) (all P < 0.001). The expression of HPA-1 mRNA was significantly higher in paired adjacent colorectal neoplasms than that in normal colorectal mucosa (P < 0.05). The expression of Syndecan-1 mRNA was significantly higher in normal colorectal mucosa (61.21 +/- 12.96) than in the paired adjacent mucosa (14.35 +/- 11.06) or colorectal cancer (10.12 +/- 8.58) (all P < 0.001). The expression of Syndecan-1 mRNA was significantly higher in the paired adjacent mucosa than that in colorectal cancer (P < 0.05). The decreased expression of Syndecan-1 mRNA and the increased expression of HPA-1 were closely associated with the degree of differentiation, the depth of infiltration, lymph node metastasis, vessel metastasis, and TNM staging of colorectal cancer (all P < 0.05). Spearman rank correlation analysis demonstrated a significant correlation between Syndecan-1 and HPA-1(r = -0.405, P < 0.05). CONCLUSIONS: The expression of Syndecan-1 mRNA was significantly highest in normal colorectal mucosa and the expression of HPA-1 mRNA was significantly highest in colorectal cancer. At the same time, the decreased expression of Syndecan-1 mRNA and the increased expression of HPA-1 mRNA can promote the invasion and metastasis of colorectal cancer. The determination of Syndecan-1 and HPA-1 may be of value in the treatment as well as in the prognosis of patients with colorectal cancer.

AIM: To investigate the effects of anti-sense oligonucleotides (ASODNs) on mRNA expression of heparanase in human esophageal cancer EC9706 cells. METHODS: One non-sense oligonucleotide (N-ODN) and five ASODNs against different heparanase mRNA sites were transfected into EC9706 cells, then the expression of heparanase mRNA in EC9706 cells was studied by in situ hybridization. RESULTS: The expression of heparanase mRNA could be inhibited by ASODNs. There was no significant difference among five ASODNs (P>0.05), but there was a significant difference between ASODNs and N-ODN or non-transfected group (ASODN1: 2.25+/-0.25, ASODN2: 2.21+/-0.23, ASODN3: 2.23+/-0.23, ASODN4: 2.25+/-0.24 vs N-ODN: 3.47+/-2.80 or non- transfected group: 3.51+/-2.93 respectively, P<0.05). CONCLUSION: The expression of heparanase mRNA in EC9706 cells can be inhibited by ASODNs in vivo, and heparanase ASODNs can inhibit metastasis of esophageal squamous cell carcinoma or other tumors by inhibiting the expression of heparanase.

OBJECTIVE: To investigate the expression of HPA and HIF-1alpha in human esophageal squamous cell carcinoma and their relationship with cancer development, invasion and metastasis. METHODS: The expression of HPA mRNA and HIF-1alpha protein in 23 mucosa specimens of incisal margin, 26 para-tumorous dysplastic tissues and 70 esophageal squamous cell carcinoma specimens were detected by in situ hybridization assay and immunohistochemical staining, respectively. RESULTS: The positive expression of HPA mRNA and HIF-1alpha protein were significantly increased as the epithelial cells progressed into carcinoma (P < 0.05). The expression of HPA mRNA and HIF-1alpha protein in the esophageal squamous cell carcinoma were significantly correlated with the invasion depth, lymph node metastasis and clinical staging (P < 0.05), while it was not correlated with the differentiation of tumors (P > 0.05). There was a close correlation between the expression of HPA mRNA and HIF-1alpha protein in the carcinoma tissues (P < 0.01). CONCLUSION: The high expression of HPA mRNA and HIF-1alpha protein is correlated with carcinogenesis, progression, invasion and metastasis of esophageal squamous cell carcinoma. There may be a positive cooperation between two of them in the carcinogenesis and development of esophageal carcinoma. The determination of HPA mRNA and HIF-1 alpha will be useful in predicting tumor biological behavior.

BACKGROUND AND AIM: Heparanase is an endo-beta-glucuronidase that cleaves heparan sulfate and has been implicated in tumor angiogenesis and metastasis. The present study was to analyze the expression of and explore the prognostic value of heparanase and two important transcriptional factors, namely hypoxia-inducible factor-1alpha (HIF-1alpha) and nuclear transcriptional factor kappa B p65 (NF-kappaB p65) in gallbladder cancer. METHODS: Heparanase, HIF-1alpha and NF-kappaB p65 protein levels in 38 patients with gallbladder carcinoma were detected by immunohistochemistry and analyzed for clinicopathological significance. RESULTS: The heparanase, HIF-1alpha and NF-kappaB p65 proteins were found in 24 (63.2%), 13 (34.2%) and 22 (57.9%) specimens, respectively. High heparanase expression was closely related to advanced TNM stage (P = 0.007), depth of tumor invasion (P = 0.016), lymph node metastasis (P = 0.040) and decreased postoperative survival at 3 years (50.0% vs 20.8%, P = 0.001). Both HIF-1alpha and NF-kappaB p65 proteins were correlated with tumor size (P = 0.039 and P = 0.027, respectively) and patients positive for HIF-1alpha expression had a decreased survival rate compared with those negative for HIF-1alpha expression (40.0% vs 15.4%, P = 0.035). In addition, heparanase-positive cases had high expression of NF-kappaB p65 compared with the heparanase-negative cases (P = 0.047). CONCLUSION: Heparanase and HIF-1alpha are frequently expressed in gallbladder carcinoma and are associated with decreased survival. High expression of heparanase, combined with NF-kappaB p65, may contribute to the highly invasive and metastatic behavior of gallbladder carcinoma.

Previous reports have shown that the biochemical activity of heparanase is significantly correlated with the invasion and metastasis of malignant cells in vitro. Recently, it was found that the human heparanase gene was cloned and highly expressed in malignant cell lines and human solid malignant tumors. In the present study, we investigated the heparanase mRNA expression by using in situ hybridization in 116 paraffin-embedded tissues of primary gastric carcinomas. To explore its clinicopathologic significance, it was detected in the various steps of tumor progression and then compared with prognostic indicators. As a result, the heparanase expression was more prevalent in late-stage rather than early-stage carcinomas (P <.0001) and was more frequent in tumors of large size (P =.0212). expression also correlated with lymphatic (P =.0086) and venous (P =.0171) invasion and with negative prognostic factors such as lymph nodal (P <.0001) and distant (P =.0221) metastases. However, in a multivariate analysis, messenger RNA expression of heparanase was not an independent prognostic factor. It was concluded that heparanase might play an important role in the development of invasion and metastasis of the gastric cancer. It was indicated that patients with heparanase-positive gastric carcinoma would have a greater chance of metastasis with a poor prognosis.

AIM: To explore the expression of heparanase gene, CD44v6, MMP-7 and nm23 proteins in human gastric carcinoma as well as their relationship with the clinicopathological factors. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) was used to measure the expressions of heparanase mRNA in 43 human gastric carcinomas and 10 adjacent normal gastric tissues. Streptavidin-peroxidase (S-P) two step method was used to measure the immunohistochemical expression of CD44v6, MMP-7 and nm23 protein in 43 human gastric carcinomas. RESULTS: The expression of heparanase gene was positive in 29 cases of gastric cancer with a positive rate of 67.4%, which was significantly higher than those in adjacent normal gastric tissues (P<0.05). The heparanase mRNA expression was significantly related to advanced stage of disease, serosal infiltration, lymph node metastasis and size of tumors (P<0.05), but not related to tumor location, gross and histological types of the cancer, peritoneal dissemination and liver metastasis (P>0.05). The positive rate of CD44v6, nm23 and MMP-7 proteins was 76.7%, 37.2% and 60.5%, respectively. The CD44v6 protein expression was significantly related to serosal infiltration, lymph node metastasis and TNM stage of disease (P<0.05). The nm23 protein expression was significantly related to the tumor gross appearance, lymph node and peritoneal metastasis (P<0.05). The MMP-7 protein expression was significantly related to serosal infiltration and TNM stage of disease (P<0.05). In an attempt to measure the association between these agents, we found that the expression of heparanase mRNA had significantly negative correlation with the expression of CD44v6 and MMP-7 protein (P<0.05), the expression of MMP-7 protein had significantly positive correlation with the expression of CD44v6 protein (r=0.568, P<0.05), the expression of MMP-7 protein had no correlation with the expression of nm23 protein (P>0.05), and the expression of nm23 protein had no correlation with the expression of CD44v6 protein (P>0.05). CONCLUSION: Despite their different mechanisms of action, heparanase, CD44v6 and nm23 may play important roles in the invasive infiltration and lymph node metastasis in gastric carcinomas. Instead of metastatic spreading, MMP-7 may be just related to the invasion of gastric carcinomas. However, co-detection of these factors may be important to predict metastatic spreading in such patients.

AIM: To disclose the mechanisms that accelerate or limit tumor invasion and metastasis in gastric cancer patients. METHODS: The heparanase expression, continuity of basement, degree of infiltration by dendritic cells and lymphocytes in gastric cancer tissues from 33 the early and late stage patients were examined by immunohistochemistry, in situ hybridization and transmission electron microscopy. RESULTS: Heparanase mRNA expression in the late stage patients with gastric cancer was stronger than that in the early stage gastric cancer patients. In the early stage gastric cancer tissues, basement membrane (BM) appeared intact, whereas in the late stage, discontinuous BM was often present. The density of S100 protein positive tumor infiltrating dendritic cells (TIDC) in the early stage gastric cancer tissues was higher than that in the late stage. The infiltrating degree of tumor infiltrating lymphocytes (TIL) in the early stage patients whose tumor tissues contained a high density of TIDC was significantly higher than that in the late stage gastric cancer tissues patients with a low density of TIDC. There were few cancer cells penetrated through the continuous BM of cancer nests in the early stage gastric cancers, but many cancer cells were found outside of the defective BM of cancer nests in the late stage. CONCLUSION: Our results suggest that strong heparanase expression is related with the degradation of BM which allows or accelerates tumor invasion and metastasis. However, high density of TIDC and degree of infiltration by TIL are associated with tumor progression in human gastric cancers.

AIM: To develop short hairpin RNA (shRNA) against heparanase, and to determine its effects on heparanase expression and the malignant characteristics of gastric cancer cells. METHODS: Heparanase-specific shRNA was constructed and transferred into cultured the gastric cancer cell line SGC-7901. Stable subclonal cells were screened by G418 selection. Heparanase expression was measured by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time quantitative PCR and Western blotting. Cell proliferation was detected by 2-(4, 5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetry and colony formation assay. The in vitro invasiveness and metastasis of cancer cells were measured by cell adhesion assay, wound healing assay and matrigel invasion assay. The angiogenesis capabilities of cancer cells were measured by tube formation of endothelial cells. RESULTS: Stable transfection of heparanase-specific shRNA, but not of scrambled shRNA and mock vector, resulted in reduced mRNA and protein levels of heparanase. The shRNA-mediated knockdown of heparanase did not affect the cellular proliferation of SGC-7901 cells. However, the in vitro invasiveness and metastasis of cancer cells were decreased after knockdown of heparanase. Moreover, transfection of heparanase-specific shRNA decreased the in vitro angiogenesis capabilities of SGC-7901 cells. CONCLUSION: Stable knockdown of heparanase can efficiently decrease the invasiveness, metastasis and angiogenesis of human gastric cancer cells. In contrast, stable knockdown of heparanase does not affect the cell proliferation.

Given that previous studies indicated that early growth response 1 (EGR1) exerts pro-tumorigenic effects through regulating heparanase (HPA) transcription, it was hypothesized that EGR1 may correlate with the progression of gastric cancer. One hundred and fifteen patients with gastric cancer were evaluated for the protein and transcript expression of EGR1 and HPA on immunohistochemistry and real-time quantitative polymerase chain reaction (PCR). In normal gastric mucosa, EGR1 protein expression was absent or weak, whereas gastric cancer was positive for EGR1. Seventy gastric cancer patients (60.9%) were positive for cytoplasmic EGR1 expression, and 26 (22.6%) had nuclear expression of EGR1. In the gastric cancer examined, the transcripts of EGR1 were enhanced compared to that of normal gastric mucosa, and positively correlated with EGR1 protein expression. The cytoplasmic or nuclear expression of EGR1 and its transcripts in gastric cancer was positively correlated with tumor infiltration (P < 0.05), lymph node and distant metastasis (P < 0.05), tumor node metastasis (TNM) stages (P < 0.05), but not with age, gender, tumor location and size, histological types or differentiation. Moreover, the protein and transcript expression of EGR1 was correlated with that of HPA in gastric cancer. These results indicate that aberrant expression of EGR1 in gastric cancer is associated with tumor invasion and metastasis, and HPA transcription.

OBJECTIVE: To investigate the relationship among expression of heparanase (HPSE), the clinical and pathologic characteristics of squamous carcinoma on head and neck and the patients' prognosis. METHODS: Sixty-two cases of postoperative tumor specimens were verified by immunohistochemistry S-P method and computer-assisted image analysis method was used. RESULTS: The expression of HPSE in normal epithelium mucosae of head and neck was negative or very weak; in tumor tissue was positive, mainly in cytoplasm and the positive rate was 69.3%. The expression of HPSE hadn't significant difference with the age of patients and pathologic grades of tumors (chi2 = 0.05, chi2 = 3.84, P > 0.05), but had it with clinical stages and metastatic lymph node lesions (chi2 = 3.96, chi2 = 8.06, P < 0.05). The relationship between expression of HPSE in primary tumors and that in metastatic lymph node lesions showed significantly positive correlation (r = 0.9162, P = 0.001). Both HPSE and TNM clinical stage of tumor had significant correlation with the prognosis of patients respectively (P < 0.05). Calculated by Kaplan-Meier method, the accumulative survival rate of 3 years in positive HPSE expression group (25.9%) was much lower than that in negative group (72.7%), there was a significant difference between them by Log-Rank test (chi2 = 11.607, P < 0.001). CONCLUSIONS: The expression of HPSE is significantly increased in squamous carcinomas of head and neck, mainly expressed in cytoplasm. The expression of HPSE has a close relationship with clinical stages and lymph node metastasis of squamous carcinoma on head and neck. The higher the clinical stage, the more manifest the expression of HPSE. The expression of HPSE and TNM clinical stage of tumor are independent factors affecting prognosis.

OBJECTIVE: To explore the discrepancy of HGF and Hpa expressed in laryngeal squamous cell carcinoma of different pathology grades (differentiation of high, moderate or poor), which can provide the new thought and theory evidence for clinical treatment and control metastasis or palindromic. METHOD: Forty paraffin-embedded samples of laryngeal squamous cell carcinoma, forty paraffin-embedded samples of laryngeal papilloma and twelve paraffin-embedded samples of normal tissue adjacent to cancerous location of our department between May 2004 and April 2005, which were analyzed with SP immunohistochemistry staining. RESULT: The positive rate of HGF were 77.5% and that of Hpa were 72.5% in laryngeal squamous cell carcinoma, which have positive correlation and were higher than that in laryngeal papilloma and normal tissue adjacent to cancerous location with significant statistical difference (P<0.01). The expression of HGF and Hpa had no relationship with primary locations (P>0.05), that have positive correlation with clinical stages and have negative correlation with the level of cellular differentiation. The expression of HGF and Hpa in lymph node metastasis group was significantly higher than the non-metastasis group (P<0.05). CONCLUSION: The expression of HGF and Hpa in laryngeal squamous cell carcinoma are higher than that in nonmalignant ones. They correlates with lymph node metastasis, clinical stages and pathological grading, but they has no relationship with the primary locations. The positive correlation relationship of expression between HGF and Hpa might be worthiness to determine the prognosis of laryngeal squamous cell carcinoma.

Heparanase (HPSE-1) is an endo-beta-D-glucuronidase involved in the degradation of cell-surface/extracellular matrix heparan sulfate (HS) in normal and neoplastic tissues. HPSE-1 represents the first example of purification and cloning of a mammalian HS-degradative enzyme. Elevated HPSE-1 levels are known to be associated with metastatic cancers, directly implicating HPSE-1 in metastatic events. The purpose of this study was to determine the role of cAMP response element-binding protein (CREB) in modulating HPSE-1-mediated effects on human melanoma cell invasion. Highly invasive, brain-metastatic melanoma cells (70W) were transfected with the dominant-negative CREB (KCREB) and subsequently analyzed for changes in their HPSE-1 content, functionality, and cell invasive properties. KCREB-transfected cells showed a decrease in HPSE-1 mRNA expression and activity. This correlated with a significantly decreased invasion of these cells through Matrigel-coated filters. Furthermore, adenoviral vectors containing the full-length human HPSE-1 cDNA in sense orientation (Ad-S/hep) were constructed to investigate CREB effects on HPSE-1. Restoration of HPSE-1 expression and functionality following Ad-S/hep infection of KCREB-transfected 70W cells recovered melanoma cell invasiveness. These results demonstrate that KCREB inhibits HPSE-1 and suggest that one of the roles CREB plays in the acquisition of melanoma cells metastatic phenotype is affecting HPSE-1 activity.

Heparanase (HPSE-1) is an endo-beta-D-glucuronidase that cleaves heparan sulfate (HS) chains of proteoglycans (HSPG), and its expression has been associated with increased cell growth, invasion, and angiogenesis of tumors as well as with embryogenesis and tissue development. Since metastatic cancer cells express HPSE-1, we have developed an orthotopic brain slice model to study HPSE-1 involvement in brain-metastatic melanoma. This model allows for the characterization of tumor cell invasion at both quantitative and qualitative levels. Brain-metastatic melanoma cells (B16B15b) showed augmenting levels of HPSE-1 protein expression in a time-dependent manner. Secondly, B16B15b cells pre-treated with HPSE-1 showed a significant increase in the number of cells that invaded into the brain tissue. Finally, HPSE-1 exposure-augmented invasion depth in brain sections by brain-metastatic melanoma cells. We concluded that applying this brain slice model can be beneficial to investigate HPSE-1- related in vivo modalities in brain-metastatic melanoma and brain invasion in general. These results also further emphasize the potential relevance of using this model to design therapies for controlling this type of cancer by blocking HPSE-1 functionality.

Heparanase (HPSE) and fibroblast growth factor-2 (FGF2) are critical regulators of melanoma angiogenesis and metastasis. Elevated HPSE expression contributes to melanoma progression; however, further augmentation of HPSE presence can inhibit tumorigenicity. HPSE enzymatically cleaves heparan sulfate glycosaminoglycan chains (HS) from proteoglycans. HS act as both low-affinity FGF2 receptors and coreceptors in the formation of high-affinity FGF2 receptors. We have investigated HPSE's ability to modulate FGF2 activity through HS remodeling. Extensive HPSE degradation of human metastatic melanoma cells (70W) inhibited FGF2 binding. Unexpectedly, treatment of 70W cells with low HPSE concentrations enhanced FGF2 binding. In addition, HPSE-unexposed cells did not phosphorylate extracellular signal-related kinase (ERK) or focal adhesion kinase (FAK) in response to FGF2. Conversely, in cells treated with HPSE, FGF2 stimulated ERK and FAK phosphorylation. Secondly, the presence of soluble HPSE-degraded HS enhanced FGF2 binding and ERK phosphorylation at low HS concentrations. Higher concentrations of soluble HS inhibited FGF2 binding, but FGF2 signaling through ERK remained enhanced. Soluble HS were unable to support FGF2-stimulated FAK phosphorylation irrespective of HPSE treatment. Finally, cell exposure to HPSE or to HPSE-degraded HS modulated FGF2-induced angiogenesis in melanoma. In conclusion, these effects suggest relevant mechanisms for the HPSE modulation of melanoma growth factor responsiveness and tumorigenicity.

Ribonucleic acid interference (RNAi) based on microRNA (miRNA) context may provide an efficient and safe therapeutic knockdown effect and can be driven by ribonucleic acid polymerase II (RNAP II). In this study, we designed and synthesized miR155-based artificial miRNAs against heparanase (HPSE) constructed with BLOCK-iT Pol II miR RNAi expression Vector Kit. The expression levels of HPSE declined significantly in both the mRNA and protein levels in HPSE-miRNA transfected melanoma cells that exhibited reduction of adhesion, migration, and invasion ability in vitro and in vivo. We also observed that HPSE miRNA could inhibit the expressions of chemokines of interleukin-8 (IL8) and chemokine (C-X-C motif) ligand 1 (CXCL1), at both the transcriptional and translational levels. Further study on its probable mechanism declared that down-regulation of IL8 and CXCL1 by HPSE-miRNA may be correlated with reduced growth-factor simulated mitogen-activated kinase (MAPK) phosphorylation including p38 MAPK, c-Jun N-terminal kinase (JNK) and extracellular-signal-regulated kinase (ERK) 1 and 2, which could be rescued by miRNA incompatible mutated HPSE cDNA. In conclusion, we demonstrated that artificial miRNAs against HPSE might serve as an alterative mean of therapy to low HPSE expression and to block the adhesion, invasion, and metastasis of melanoma cells. Furthermore, miRNA-based RNAi was also a powerful tool for gene function study.

Cleavage of heparan sulphate proteoglycans (HSPGs) affects the integrity and functional state of tissues and thereby fundamental normal and pathological phenomena involving cell migration and response to changes in the extracellular microenvironment. Heparanase, degrading heparan sulphate (HS) at specific intrachain sites, is synthesized as a latent approximately 65 kDa protein that is processed at the N-terminus into a highly active approximately 50 kDa form. The heparanase enzyme is preferentially expressed in human tumours and its overexpression in low-metastatic tumour cells confers a highly invasive phenotype in experimental animals. Heparanase also releases angiogenic factors and accessory fragments of HS from the tumour microenvironment and induces an angiogenic response in vivo. Heparanase may thus facilitate tumour cell invasion, vascularization and survival in a given microenvironment, all critical events in cancer progression. These observations, the anticancerous effect of heparanase-inhibiting molecules, and the unexpected identification of a single predominant functional heparanase suggest that the enzyme is a promising target for drug development.

The present study emphasizes the importance of cell surface expression and secretion of heparanase (endo-beta-D-glucuronidase) in tumor angiogenesis and metastasis. For this purpose, nonmetastatic Eb mouse lymphoma cells were transfected with the predominantly intracellular human heparanase or with a readily secreted chimeric construct composed of the human enzyme and the chicken heparanase signal peptide. Eb cells overexpressing the secreted heparanase invaded a reconstituted basement membrane to a much higher extent than cells overexpressing the intracellular enzyme. Cell invasion was inhibited in the presence of laminaran sulfate, a potent inhibitor of heparanase activity and experimental metastasis. The increased invasiveness in vitro was reflected in vivo by rapid and massive liver colonization and accelerated mortality. In fact, mice inoculated with cells expressing the secreted enzyme succumb because of liver metastasis and dysfunction, as early as 10 days after s.c. inoculation of the cells, when their tumor burden did not exceed 1% of body weight. Cell surface localization and secretion of heparanase markedly stimulated tumor angiogenesis, as demonstrated by a 4-6-fold increase in vessel density and functionality evaluated by MRI of tumors produced by cells expressing the secreted vs. the nonsecreted heparanase, consistent with actual counting of blood vessels. Altogether, our results indicate that the potent proangoigenic and prometastatic properties of heparanase are tightly regulated by its cellular localization and secretion. The increased potency of the secreted enzyme makes it a promising target for anticancer drug development.

Human heparanase is an endo-beta-d-glucuronidase that degrades heparan sulfate/heparin and has been implicated in a variety of biological processes, such as inflammation, tumor angiogenesis, and metastasis. Although the cloned enzyme has been demonstrated to have a critical role in tumor metastasis, the substrate specificity has been poorly understood. In the present study, the specificity of the purified recombinant human heparanase was investigated for the first time using a series of structurally defined oligosaccharides isolated from heparin/heparan sulfate. The best substrates were deltaHexUA(+/-2S)-GlcN(NS,6S)-GlcUA-GlcN(NS,6S)-GlcUA-GlcN(NS,6S) and deltaHexUA(2S)-GlcN(NS,6S)-GlcUA-GlcN(NS,6S) (where deltaHexUA, GlcN, GlcUA, NS, 2S, and 6S represent unsaturated hexuronic acid, d-glucosamine, d-glucuronic acid, 2-N-sulfate, 2-O-sulfate, and 6-O-disulfate, respectively). Based on the percentage conversion of the substrates to products under identical assay conditions, several aspects of the recognition structures were revealed. 1) The minimum recognition backbone is the trisaccharide GlcN-GlcUA-GlcN. 2) The target GlcUA residues are in the sulfated region. 3) The -GlcN(6S)-GlcUA-GlcN(NS)- sequence is essential but not sufficient as the cleavage site. 4) The IdoUA(2S) residue, located two saccharides away from the target GlcUA residue, claimed previously to be essential, is not indispensable. 5) The 3-O-sulfate group on the GlcN is dispensable and even has an inhibitory effect when located in a highly sulfated region. 6) Based on these and previous results, HexUA(2S)-GlcN(NS,6S)-IdoUA-GlcNAc(6S)-GlcUA-GlcN(NS,+/-6S)-IdoUA(2S)-GlcN(NS,6S) (where HexUA represents hexuronic acid) has been proposed as a probable physiological target octasaccharide sequence. These findings will aid establishing a quantitative assay method using the above tetrasaccharide and designing heparan sulfate-based specific inhibitors of the heparanase for new therapeutic strategies.

Heparanase is an endo-beta-D-glucuronidase involved in extracellular matrix remodeling and degradation and implicated in tumor metastasis, angiogenesis, inflammation, and autoimmunity. The enzyme is synthesized as a latent 65-kDa protein and is processed in the lysosomal compartment to an active 58-kDa heterodimer, where it is stored in a stable form. In contrast, its heparan sulfate substrate is localized extracellularly, suggesting the existence of mechanisms that trigger heparanase secretion. Here we show that secretion of the active enzyme is mediated by the protein kinase A and C pathways. Moreover, secretion of active heparanase was observed upon cell stimulation with physiological concentrations of adenosine, ADP, and ATP, as well as by the noncleavable ATP analogue adenosine 5'-O-(thiotriphosphate). Indeed, heparanase secretion was noted upon cell stimulation with a specific P2Y1 receptor agonist and was inhibited by P2Y receptor antagonists. The kinetics of heparanase secretion resembled the secretion of cathepsin D, a lysosomal enzyme, indicating that the secreted heparanase is of lysosomal origin. We suggest that secretion of active heparanase is initiated by extracellular cues activating the protein kinase A and C signaling pathways. The secreted enzyme(s) then facilitate cell invasion associated with cancer metastasis, angiogenesis, and inflammation.

Heparan sulfate (HS) proteoglycans play a key role in the self-assembly, insolubility and barrier properties of the extracellular matrix (ECM). Cleavage of HS therefore affects the integrity of tissues and hence normal and pathological phenomena involving cell migration and response to changes in the ECM. Mammalian heparanase, HS-degrading endoglycosidase,is synthesized as a latent 65 kDa precursor that undergoes proteolytic cleavage, yielding 8 kDa and 50 kDa subunits that heterodimerize to form a highly active enzyme. Heparanase is preferentially expressed in human tumors and its over-expression in tumor cells confers an invasive phenotype in experimental animals. Heparanase also releases angiogenic factors from the ECM and tumor micro environment and thereby induces an angiogenic response in vivo. Enhanced heparanase expression correlates with metastatic potential, tumor vascularity and reduced postoperative survival of cancer patients. Heparanase also promotes cell adhesion, survival and signaling events, independent of its enzymatic activity. These observations, the anti-cancerous effect of heparanase gene silencing and of heparanase inhibiting molecules as well as the unexpected identification of a predominant functional heparanase, suggest that the enzyme is a promising target for anti-cancer drug development. Here, we summarize recent progress in molecular and cellular aspects of heparanase, emphasizing its causal involvement in cancer metastasis and angiogenesis, and discuss the development of heparin-like heparanase inhibitors.

Heparanase is an endoglycosidase that specifically cleaves heparan sulfate side chains, a class of glycosaminoglycans abundantly present in the extracellular matrix and on the cell surface. Heparanase activity is strongly implicated in tumor angiogenesis and metastasis attributed to remodeling of the subepithelial and subendothelial basement membranes. We hypothesized that similar to its proangiogenic capacity, heparanase is also engaged in lymphangiogenesis and utilized the D2-40 monoclonal antibody to study lymphangiogenesis in tumor specimens obtained from 65 head and neck carcinoma patients. Lymphatic density was analyzed for association with clinical parameters and heparanase staining. We provide evidence that lymphatic vessel density (LVD) correlates with head and neck lymph node metastasis (N-stage, p = 0.007) and inversely correlates with tumor cell differentiation (p = 0.007). Notably, heparanase staining correlated with LVD (p = 0.04) and, moreover, with VEGF C levels (p = 0.01). We further demonstrate that heparanase overexpression by epidermoid, breast, melanoma and prostate carcinoma cells induces a 3- to 5-fold elevation in VEGF C expression in vitro and facilitates tumor xenograft lymphangiogenesis in vivo, whereas heparanase gene silencing was associated with decreased VEGF C levels. These findings suggest that heparanase plays a unique dual role in tumor metastasis, facilitating tumor cell invasiveness and inducing VEGF C expression, thereby increasing the density of lymphatic vessels that mobilize metastatic cells.

OBJECTIVE: To investigate the expression of heparanase in nasopharyngeal carcinoma and the relationship between the expression of it and clinically pathological features of nasopharyngeal carcinoma. METHOD: The expression of heparanase protein in 70 cases of nasopharyngeal carcinomas and 10 cases of normal nasopharyngeal tissues was detected by immunohistochemical staining. The date of expression combined clinical features, which included clinical stage, cervical lymph node metastasis rate, the rate of metastasis and recurrence, combination of, the 5-year survival rate, and other analysis, was analyzed. RESULT: The positive rate of heparanase protein in cancerous tissues was 52.9% (37/70), while it was 0% in normal nasopharyngeal tissues. The positive rates of heparanase protein in patients were 30.0% (6/20) in stage I, 45.80% (11/24) in stage II, 70.6% (12/17) in stage III, 88.9% (8/9) in stage IV respectively. Heparanase positive tumors were associated with a higher incidence of lymph node metastasis (67.4%, 31/46) than heparanase negative ones (25.0%, 6/24). The rate of distant metastasis and regional recurrence in the heparanase positive group was 48.6% (18/37), but only 15.2% (5/ 33) in the heparanase negative group. The cumulative survival of patients in the heparanase negative group at 5 years was 78.8% (26/33), but only 24.3% (9/37) in the heparanase positive group. The clinical stage of disease, lymph node metastasis, the rate of distant metastasis and regional recurrence of nasopharyngeal carcinoma were correlated with positive expression of heparanase protein. CONCLUSION: The expression of HPA was associated with invasion and metastasis and prognosis of nasopharyngeal cancer, and it may be a new target for the anti-treatment of nasopharyngeal cancer. (P < 0.01), and heparanase expression level inversely correlated with the patient survival (P < 0.01). CONCLUSION: Heparanase may play important roles in the invasive infiltration, metastasis, and prognosis in nasopharyngeal carcinoma, clearly indicating that heparanase is a possible target for anticancer drug development.

The effects of targeted silencing of heparanase gene by small interfering RNA (siRNA) on invasiveness and metastasis of osteosarcoma cells (MG63 cells) were investigated in the present study. Two complementary oligonucleotide strands were synthesized and inserted into pGenesil-1 vector based on the mRNA sequence of heparanase gene. The expression vector containing short hairpin RNA (pGenesil-shRNA) was constructed successfully. MG63 cells were randomly allocated into 3 groups: blank group, empty vector (pGenesil) transfected group and expression vector (pGenesil-shRNA) transfected group. Under the induction of Lipofectamine 2000, the recombinants were transfected into MG63 cells. Heparanase gene expression level was detected by RT-PCR and Western blotting. Cell proliferation was measured by MTT assay. Cell invasiveness and metastasis were examined by cell adhesion and Transwell-ECM assays. HUVECs migration assay was applied for the detection of angiogenesis. As compared with negative controls, the mRNA and protein expression levels of heparanase were down-regulated by 76.1% (P<0.01) and 75.3% (P<0.01) respectively in the pGenesil-shRNA transfected group. Meanwhile, the proliferation, adhesiveness, invasiveness and angiogenesis properties of MG63 cells were all significantly inhibited. It was suggested that targeted silencing of heparanase gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells.

It has been reported that heparanase (HPSE) is overexpressed in ovarian cancer and is associated with tumor invasion and metastasis. However, a systematic study on the contribution of HPSE to tumor metastasis is rarely reported. In this study, based on the measurement of HPSE serum concentration, the expression of HPSE at both the mRNA and protein levels in tumors and its effects on the biological behaviors of cancer cells, we elucidated the role of HPSE in tumor invasion and metastasis in ovarian cancer and concluded that either the expression of HPSE in cancer and/or the serum concentration of HPSE may be a useful biomarker for the evaluation of surgery effects and prognosis prediction.

BACKGROUND: Pancreatic cancer still has one of the worst prognoses of all cancers with a 5-year survival rate of 5%, making it necessary to find markers or gene sets that would further classify patients into different risk categories and thus allow more individually adapted multimodality treatment regimens. Especially heparanase (HPSE) has recently been discussed as a key factor in pancreatic cancer. MATERIALS AND METHODS: Paraffin-embedded tissue samples were obtained from 41 patients with pancreatic adenocarcinoma who were scheduled for primary surgical resection. Direct quantitative real-time reverse transcriptase polymerase chain reaction (TaqMan) assays were performed in triplicates to determine HPSE, hypoxia inducible factor-1 alpha (HIF1a), platelet-derived growth factor alpha (PDGFA), heparin-binding EGF-like growth factor (HB-EGF), and basic fibroblast growth factor (bFGF) gene expression levels. RESULTS: HPSE was significantly correlated to PDGFA (p = 0.04) and HIF1a (p = 0.04). The correlation of HIF1a to bFGF and HB-EGF was significant (p = 0.04, p = 0.02). Stepwise multiple linear regression models showed a significant independent association of HPSE with lymph node metastasis (p = 0.025) and with dedifferentiation (p = 0.042). CONCLUSIONS: Heparanase seems to be significantly associated with lymph node metastasis (p = 0.025) as well as dedifferentiation (p = 0.042). We assume that HPSE plays a crucial role for the aggressiveness of pancreatic cancer. Larger studies including more patients seem to be warranted.

Heparanase is an endoglycosidase that plays an important role in angiogenesis and metastasis of cancer. Herein we evaluate the effect of heparanase overexpression on invasiveness and bone destruction in prostate cancer bone metastases. The human prostate cancer cell line PC-3 was stably transfected with a plasmid containing the cDNA for human heparanase or with the vector alone as a control. Overexpression of heparanase did not affect the growth of PC-3 cells, but did promote invasiveness of the cells in an in vitro assay. Both cell types were injected into the tibias of nude mice. Four weeks later, the mice were examined radiologically prior to sacrifice and samples of leg tissue were taken to investigate bone destruction and metastasis. Mice injected with PC-3 cells overexpressing heparanase had more severe bone destruction and larger, more invasive, tumors. These results demonstrate that heparanase overexpression can facilitate tumor invasion and accelerate bone destruction caused by prostate cancer bone metastasis.

PURPOSE: Heparanase activity has been detected in many malignant tumors, showing a correlation with the metastatic potential. The present study was undertaken to investigate the expression of heparanase and its prognostic significance in renal cell carcinomas (RCC). EXPERIMENTAL DESIGN: Nineteen RCCs and 6 nonneoplastic renal tissues were analyzed for heparanase mRNA expression by real-time PCR. Heparanase protein expression was semiquantitatively investigated by immunohistochemistry in 70 RCCs. Involvement of heparanase in the invasiveness of RCC cell lines, 786-O and Caki-2 cells, was examined by down-regulating the gene expression with small interfering RNA (siRNA) using the Matrigel invasion assay. RESULTS: The expression level of heparanase mRNA was significantly higher in clear cell RCCs than in papillary RCCs, chromophobe RCCs, and nonneoplastic renal tissues. Heparanase was predominantly immunolocalized to cell surface and cytoplasm of clear cell RCCs and mean expression levels of heparanase were significantly higher in clear cell RCCs than in papillary and chromophobe RCCs. The protein expression levels were positively correlated with primary tumor stage, distant metastasis, and histologic grade. Targeting of heparanase mRNA expression in 786-O and Caki-2 cells with siRNA down-regulated the mRNA expression and inhibited the Matrigel invasion by these cells, whereas nonsilencing siRNA showed no effect. Multivariate Cox analysis revealed that elevated heparanase expression was a significant and an independent predictor of disease-specific survival (odds ratio, 8.814; P = 0.019). CONCLUSIONS: These data suggest that heparanase plays an important role in invasion and metastasis and silencing of the gene might be a potential therapeutic target in clear cell RCCs.