| Prostate cancer is the second leading cause of death in America and is becoming more common in China. Over 80% of advanced prostate cancer patients suffer from bone metastasis. Bone metastasis significantly decreases the patients'quality of life, and is the most common cause of death for prostate cancer patients. Complications of metastatic bone disease include pathological fractures, pain, cranial nerve palsies, spinal cord compression, and bone marrow suppression, most of which are caused by bone destruction. Treatment of bone metastasis includes chemotherapy, radiotherapy and bone specific therapy aimed to prevent bone destruction, which can prolong the patients'life span and improve quality of life. Based on this hypothesis, anti-osteoclast agents such as bisphosphonates are used to prevent bone lesions in many different cancers including prostate cancer.Heparanase is an endoglycosidase that cleaves heparin sulfate, which binds many different bioactive molecules. This enzyme has an important role in both normal and pathologic processes, especially in angiogenesis and metastasis of tumors. Previous studies revealed elevated heparanase expression in human breast, bladder, pancreas, prostate tumors, which correlated with advanced tumor progression and metastasis. Overexpression of heparanase in breast cancer cells accelerates cell proliferation and survival in vitro and promotes angiogenesis and invasion in vivo. In contrast, reducing heparanase levels in lymphoma cells with anti-heparanase ribozyme or siRNA-mediated gene-silencing vectors alters tumorigenic properties of the cells.Recent studies indicate that myeloma cells overexpressing heparanase can promote spontaneous metastasis to bone. Another study proved that heparanase was involved in the process of bone formation. However, it is unknown what role heparanase plays in bone destruction in prostate cancer patients. Objective:To established heparanase over-expressing PC-3 cells and evaluate the effects of elevated heparanase expression on bone destruction and invasiveness of prostate cancer cells.Methods:1. The pIRES2-EGFP/HPSE1 plasmid containing heparanase cDNA (HPSE1) and the empty pIRES2-EGFP vector as a control, were transfected into PC-3 human prostate cancer cells using the Lipofectin reagent. Cells stably expressing the plasmid were selected with G418. These two groups of cells are referred to as PC-3-HPSE1 and PC-3-EGFP. Transfected cells were maintained routinely in the selection medium to avoid overgrowth of non-transfected cells. Expression of heparanase1 was detected by REAL-TIME PCR, Western-blot and indirect Immunofluorescence Assay. Heparanase activity was measured by Heparan Deagrading Enzyme Assay.2. In vitro cell growth was investigated by MTT assay and in vitro cell invasiveness was investigated by Transwell assay. Tumorigenicity assay was performed in nude mice model to assess the malignant behavior of cells. Tumor diameter was recorded using caliper regularly. H&E staining was used for histological verification of tumor growth and metastasis. To determine if heparanase expression was maintained in vivo, immunohistochemistry of the tumors were performed using rabbit anti-human heparanase polyclonal antibody.3. Animal model of bone metastasis was established using direct tibia injection. Twenty-eight days after injection, the animals were anesthetized and a flat plate radiograph was taken. After sacrifice, the tumor-bearing leg was harvested, and the hindlimb tumor diameter was recorded using caliper. H&E staining was used for histological verification of tumor growth and bone destruction. The area of cortical bone present in each specimen was calculated using image analysis software. To detect osteoclasts, tartrate-resistant acid phosphatase (TRAP) staining was performed on sections of bone.Results:1. To investigate the role of heparanase in bone metastasis of prostate cancer, we successfully established human prostate cancer PC-3 cells over-expressing heparanase, referred to hereafter as PC-3-HPSE1 cells. PC-3 cells transfected with empty vector, PC-3-EGFP cells, were used as the negative control. Real time RT-PCR revealed an 8 fold increase in relative expression of heparanase mRNA in PC-3-HPSE1 cells compared to the PC-3-EGFP cells. Western blot analysis revealed no detectable heparanase protein in PC-3-EGFP cells, whereas there was a distinct heparanase protein band in the PC-3-HPSE1 cells. Indirect immunofluorescence labeling showed a very weak signal of heparanase in PC-3-EGFP cells, whereas there was much stronger staining in PC-3-HPSE1 cells. Heparan Deagrading Enzyme Assay showed higher heparanase activity in PC-3-EGFP cells than in PC-3-EGFP cells.2. Overexpressing heparanase did not alter the growth of the cells in vitro even after 72 hours. The invasion assay showed that more PC-3-HPSE1 cells traveled through the filter than in either of the control groups. Six weeks after the injection, each mouse formed a visible tumor. The mean tumor diameter of the PC-3-EGFP group was significantly smaller than that of the PC-3-HPSE1 group, which indicated that cells with higher heparanase level proliferated more quickly in vivo. Six cases of lung metastasis were found in the PC-3-HPSE1 group, while only two cases were found in the PC-3-EGFP group. Immunohistochemistry of tumors demonstrated that tumors of PC-3-EGFP cells were weakly positive for heparanase expression, while tumors of PC-3-HPSE1 cells are strongly positive for heparanase.3. Four weeks after the injection, radiographs of the mice showed that all animals in both groups formed osteolytic lesions characterized by obliteration of the partial proximal tibia. each mouse formed a visible tumor in the hindlimb which was injected with tumor cells. No pathologic fracture was found in the PC-3-EGFP group, while there were three cases of pathologic fracture in the PC-3-HPSE1 group. H&E staining of histological sections from the tibias in both groups revealed there was eradication of the proximal tibia in both groups, but in the PC-3-EGFP group, the cortical structure of the tibias was better preserved. Histomorphometric analysis revealed significantly less cortical bone destruction in the PC-3-EGFP group than that in the PC-3-HPSE1 group. TRAP staining showed fewer TRAP-positive cells in the PC-3-EGFP group than in the PC-3-HPSE1 group.Conclusions:1. PC-3-HPSE1 cells could express high level of heparanase both in vitro and in vivo.2. Although heparnase could not promote proliferation of tumor cells in vitro, it can promote cell proliferation and invasiveness in vivo.3. Heparanase could promote tumor cell growth in bone tissue and bone destruction caused by prostate cancer cells. Targeting heparanase in tumors may be an effective means of controlling bone metastasis in prostate cancer patients.
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