A Study On The Expression Of Heparanase In Bladder Transitional Cell Carcinoma

Background: Carcinoma of the urinary bladder is the first common malignant tumor of the urogenital region in the China. Easily to recur and incur is the characteristic of bladder transitional cell carcinoma (BTCC), and about 25% of BTCC are invasive tumors. Metastasis often occurs in BTCC with invasion. Although cytotoxic chemotherapy and new therapeutic tools have improved, the prognosis of patients with metastatic bladder cancer is still poor. Therefore, understanding the factors that induce metastasis is essential for developing new therapeutic methods for metastatic bladder cancer. Cancer metastasis is established by multistep tumor cell host interactions. (1) For metastasis to develop tumor cells must pass through the extracellular matrixes (ECM) and vascular basement membranes (BM). (2) Tumor angiogenesis. ECM and BM consist of large matrix molecules, such as fibronectin, laminin, and various types of collagen and proteoglycan. To degrade these barriers, an extracellular matrix degradative enzyme is requisite. Heparanase, which is an endo-beta-Dglucuronidase, degrades heparan sulfate (HS) and heparan sulfate proteoglycans (HSPG), which localize in the extracellular matrix and on the external surface of cell membranes. Metastatic tumor cells were found to degrade heparan sulfate present in the subendothelial matrix and a good correlation were found between metastatic potential and heparanase activity. Recently, the structure of the mamalion heparanase gene was reported by several investigators. Moreover,heparanase cleaves heparin sulfate/heparan sulfate proteoglycans into characteristic large molecular weight fragments and modulates the biological functions of heparan sulfate/heparan sulfate proteoglycansbinding proteins, such as basic fibroblast growth factor, vascular endothelial growth factor, platelet-derived endothelial growth factor, interleukin-8 (IL-8), hepatocyte growth factor and transforming growth factor. These cytokines and growth factors are potent mitogens and chemotactic factors for endothelial cells, and growth factors have crucial roles in angiogenesis. Therefore, heparanase may be important in cancer invasion and metastasis of cancer. Previous studies have demonstrated heparanase expression in various malignancies and that there is differential heparanase expression between benign and malignant tumors. The heparanase expression in human bladder cancer has seldom been reported. To our knowledge, we present the first systematic study of the expression of heparanase mRNA and protein that demonstrates the relationship of expression of the enzyme with the biologic behavior of disease and tumor angiogenesis in human bladder cancer. In our study, the expression of heparanase mRNA and protein were determined by RT-PCR and immunohistochemistry staining (SABC) respectively, and tumor microvessel density was counted. Then the correlation between heparanase expression and tumor biologic behavior, angiogenesis in human bladder cancer was analyzed. The results can provide a proof for tumor invasion and metastasis and a method to the diagnosis, treatment, prognosis of BTCC.PART 1 THE EXPRESSION AND SIGNIFICANCE OF HEPARANASE GENE IN HUMAN BLADDER TRANSITIONAL CELL CARCINOMAObjective: To determine the association between heparanase gene expression and the biological behavior of BTCC.Materials and Methods: Samples of 42 human BTCC, 16 cancer-side tissuesand 8 normal bladder tissues were examined by RT-PCR for heparanase mRNA. The results were analyzed with disease data. The statistical analyses were performed using the software of SPSS 10.0 statistical package. P value less than 0.05 was regarded as significant.Results: (1) There were no heparanase mRNA expression in 8 normal bladder tissues, 2 expressions in 16 cancer-side tissues and 23 expressions in 42 BTCC specimens. Heparanase mRNA positive expression rate in BTCC was found 54.8%, the positive rate were significantly high in BTCC specimens than in normal and cancer-side tissues (PO.01). (2) Heparanase mRNA positive expression rate was 16.7%, 70% in superficial and muscular invasive BTCC respectively, the expression of heparanase mRNA in muscular invasive BTCC was significantly high than in superficial BTCC (PO.01). (3) Heparanase mRNA positive expression rate was 43.37%, 83.3% in G1+G2 and G3 BTCC respectively, the expression of heparanase mRNA in G3 BTCC was significantly high than in Gi+G2 BTCC (P<0.05). (4) The positive expression rate for heparanase mRNA in patients with versus without lymph node metastasis was significantly higher (100% versus 47.2%, PO.05). (5) Heparanase mRNA positive expression rate was 51.4%, 71.4% in first and secondary BTCC respectively, there was not significant difference between the two groups (P>0.05). (6) In tumors over 2cm in diameter heparanase expression was significantly higher than in tumors less than 2cm (68.9% versus 23.1%, PO.01). (7) RT-PCR semi-quanitity determination were made to 23 heparanase positive expression. Theaverage of the relative OD in lymph node metastatic cancer was 1.595±0.194(^±S), while the average of the relative OD in lymph node nonmetastatic cancer was1.019±0.244( X ±S). There was significant difference between the two groups (P<0.001).Conclusions: (1) By degrading ECM and BM, heparanase make a important role in BTCC invasion, metastasis and progression. (2) Heparanase plays a critical role in the invasion, angiogenesis and metastasis of BTCC. (3) Heparanase could be a newtarget molecule to inhibit invasion, angiogenesis and metastasis of this disease. Moreover, the expression of heparanase could be a new prognostic factor of BTCC patients.Keywords: heparanase BTCC RT-PCR invasion metastasisPART 2 HEPARANASE PROTEIN EXPRESSION ANDASSOCIATION WITH ANGIOGENESIS IN HUMANBLADDER TRANSITIONAL CELL CARCINOMAObjective: To determine the association between heparanase gene expression and the biological behavior, angiogenesis of BTCC.Materials and Methods: Samples of 60 human BTCC and 5 normal bladder tissues were examined by immunohistochemistry staining (SABC) for heparanase mRNA. And tumor microvessel density was counted. The results were analyzed with disease data. The statistical analyses were performed using the software of SPSS 10.0 statistical package. P value less than 0.05 was regarded as significant.Results: (1) Urothelial transitional cells not affected by cancer did not stain with the anti-human heparanase antibody. Heparanase protein positive staining rate in BTCC was found 43.3%. Positive staining was detected in the cytoplasm of cancer cells. Moreover, the expression of heparanase protein in BTCC cells at invasive sites was much more intense than in cancer cells at the primary foci. The positive staining rate in muscle invasive BTCC was significantly higher than in superficial cancer (72% versus 22.9%, PO.001). On the other hand, bladder transitional cell carcinoma with muscular invasion stained more intensely with the anti-heparanase antibody than superficial BTCC. The positive staining rate in grade 3 cancer was significantly higher than in grades 1 and 2 cancer (66.7% versus 35.6%, P <0.05). Also, bladder transitional cell carcinoma with grade 3 stained more intensely with the anti-heparanase antibody than grade 1 and grade 2. (2) In each 200 field, the mean...