The Inhibitory Effect Of Heparanase Antisense Oligodeoxy-nucleotide On The Invasiveness, Metastasis And Angiogenesis Of Human Mammary Carcinoma

BackgroundInvasion and metastasis are important characters of malignant tumors and main obstacles to the cure rate of tumors. In the precessing of tumor invasion and metastasis, extracellular matrix (ECM) and basement membrane (BM) are playing an important role of barrier. Heparanase (HPSE), an endo- β -glucuronidase, has been cloned recently. It can cleave heparan sulfate (HS), one of the main components of ECM and BM, so facilitate tumor invasion, metastasis and angiogenesis. It is the first one and the only one we know by now that has an enzymic activity to cleave HS, so it has become a new and attractive target for developing antitumor drugs.Objective1. To investigate the expression of heparanase in human mammary cancer and its significance in cancer invasion, metastasis and angiogenesis.2. To evaluate the inhibitory effect of heparanase antisense oligodeoxynucleotide (HPSE AS-ODN) on the invasiveness, metastasis and angiogenesis of human mammary carcinoma and to search for a new way of gene therapy for mammary carcinoma.Methods1. 54 cases of mammary cancer were reviewed in this study. Expression of heparanase was evaluated by immunostaining with a heparanase monoclonal antibody and the microvessel density (MVD) was examined by immunostaining with a factor VIII-related monoclonal antibody.2. The HPSE AS-ODN complementary to the start codon region of heparanase mRNA was designed and synthesized and delivered into human mammary cacimoma cell line MDA435 by cationic liposome, Lipofectin. After transfection the total RNAs and proteins were extracted from the cells and then semi-quantitative RT-PCR and Western blot were performed to evaluate the heparanase gene and protein expression level, respectively. The invasiveness of transfected MDA435 cells were measured quantitatively by Matrigel invasion assays.3. A subcutaneous invasion model of human mammary carcinoma in nude mice was established and HPSE AS-ODN was injected into the tumor. The growth state of the tumor was observed. RT-PCR was performed to evaluate the heparanase gene expression in the tumor. The expression of heparanase protein was evaluated by both immunostaining with a heparanase monoclonal antibody and Western blot. The MVD of the tumor was examined by immunostaining with a factor VIII-related monoclonal antibody. An acute hematal metastasis model of human mammary carcinoma in nude mice was also established and HPSE AS-ODN was given by tail vein injection. The number of metastatic nodes in the lung surface was countedunder a dissecting microscope.Results1. Heparanase was positively expressed in 59.2% (32/54) cases of mammary cancer and was intensively expressed in cancer cells metastasized to the axillary lymph nodes. The heparanase expression was significantly correlated with tumor size, lymph node metastasis, TNM stages, MVD and prognosis of mammary cancer.2. The heparanase gene and protein expression and invasiveness of MDA435 cells treated with HPSE AS-ODN at different final concentrations were significantly decreased compared with that of the controls (P<0.01). Besides, the inhibitory effects were significantly different between different HPSE AS-ODN concentrations (P<0.01). The mvasiveness inhibition rates were 34.0%, 57.8% and 79.7% at the final HPSE AS-ODN concentrations of 100nmol/L, 200nmol/L and 400nmol/L, respectively.3. In the experiment of inhibiting subcutaneous invasion, the tumor treated with HPSE AS-ODN grew more slowly and the tumor size, HPSE mRNA and protein expression level and MVD were all significantly smaller or lower than the controls(P<0.01). In the experiment of inhibiting acute hematal metastasis, the number of metastatic nodes in the lung surface of the mice treated with HPSE AS-ODN was significantly less than the controls (P<0.01). The lung metastasis inhibition rate of HPSE AS-ODN was 69%.Conclusion1. Heparanase expression can promote invasion, metastasis and angiogenesi...