Detection of Elevated Serum Heparanase Activity in Pancreatic Cancer Patients by a Novel ELISA
Abstracts
|
INTRODUCTION: Heparanase (HPR) is an endoglycosidase that specifically degrades the heparan sulfate sidechains of proteoglycan, a chief component of the extracellular matrix (ECM). HPR plays an important role in tumor metastases and its expression is increased in pancreatic adenocarcinomas. To determine if HPR expression in tumor tissue results in increased HPR activity in the serum of pancreatic cancer patients, we developed a novel ELISA to quantitate serum heparanase activity. METHODS: Thirty-four serum samples collected from pancreatic cancer patients and 19 samples from healthy volunteers were serially diluted in heparanase assay buffer and then transferred to ELISA plates precoated with the substrate of HPR, heparan sulfate proteoglycan. After incubation at 37°C overnight, a monoclonal antibody against heparan sulfate was added and incubated at room temperature for 1 hr. After wash, peroxidase-conjugated secondary antibody was added and followed by addition of developing substrate. The OD405 absorbance was monitored in an ELISA plate reader. The cell lysates prepared from pcDNA- or HPR-transfected HT1080 cells were included as negative and positive controls, respectively. RESULTS: HPR activity in the serum samples of pancreatic cancer patients was increased by approximately 4.5-fold, compared to that in the serum samples of healthy donors. High HPR activity was detected in the cell lysate of HPR-transfected HT1080 cells but not of pcDNA-transfected cells. CONCLUSION: HPR activity is significantly increased in the serum of pancreatic cancer patients. Elevated serum HPR activity in pancreatic cancer patients correlates with increased HPR expression in pancreatic tumor tissues. Serum HPR levels may correlate with tumor invasion and patient survival, though this remains to be elucidated. |
