Expression of thrombospondin-1 in human pancreatic adenocarcinoma: roles in tumor angiogenesis and matrix metalloproteinase-9 production

Xiaohua Qian

doi:10.17918/00008330

The expression of thrombospondin-1 (TSP-1) in pancreatic adenocarcinoma and control pancreas tissues were studied by immunohistochemistry. Eighty-five percent (23/27) cases of pancreatic adenocarcinoma showed increased TSP-1 staining in the desmoplastic stroma adjacent to tumor cells. No specific positive staining for TSP-1 was observed in the normal pancreatic tissues and the areas of inflammation. Using a TSP-1 competitive enzyme-linked immunosorbent assay (ELISA), we found that the secretion of TSP-1 by different pancreatic cancer cell lines into culture medium varied from 11.45 +/- 14.08 to 275.82 +/- 45.56 ng/106 cells/24 hours. The amounts of TSP-1 detected in both culture media and cell extracts from fibroblasts or bovine pulmonary artery endothelial (BPAE) cells were at least 2-3 fold higher than those from pancreatic cancer cells. To further understand the functions of TSP-l expressed in pancreatic cancer tissues, we next studied the effect of TSP-1 on the production of matrix metalloproteinase-9 (MMP-9), a matrix degrading enzyme involved in tumor invasion and metastasis. We found that TSP-1 augmented the production of MMP-9 and its natural inhibitor, the tissue inhibitor of metalloproteinase-1 (TIMP-1), in pancreatic cancer cells in vitro as detected by zymography and Western Blot analysis. In a subsequent immunochemical study, we localized the MMP-9 protein in tumor cells that are surrounded by TSP-1-rich stroma. In addition, the relationship between the expression of TSP-1 and the status of tumor angiogenesis was investigated by measuring the intratumor microvessel density (IMD) in pancreatic cancer tissues. The mean vessel counts of tumor specimens with strong TSP-1 staining (3+) were 96.9, which were more than twice those of tumor specimens with undetectable TSP-1 (mean, 42.3). The difference was statistically significant (p = 0.028). A direct correlation was also found between TSP-1 expression in the stroma and the intensity of intratumor microvessels (r = 0.52, p = 0.00561). These data support the role for TSP-1 as a modulator in tumor angiogenesis. To further explore the effect of TSP-1 in angiogenesis and to investigate the mechanisms by which TSP-1 affects angiogenesis, we evaluated the effect of TSP-1 on the capacity of bovine aortic endothelial (BAE) cells to form microvessel-like tubes in collagen gels. BAE cell tube formation was enhanced by exogenous TSP-1 at relatively low concentrations (1-10 [mu]g/ml), but inhibited at higher concentrations of TSP-1 (>15 [mu]g/ml). In addition, we correlated this biphasic effect on tube formation with the capacity of TSP-1 to stimulate the activity of a matrix metalloproteinase-9 (MMP-9) in BAE cell collagen gel cultures. The TSP-1 mediated stimulation of MMP-9 activity was specific and dose- and time-dependent. Furthermore, TSP-1 stimulated BAE cell tube formation were reversed by antibodies against both TSP-1 and MMP-9 suggesting that TSP-1 modulates endothelial morphogenesis in vitro by a mechanism involving the regulation of MMP-9 activity. (Abstract shortened by UMI.).

Expression of thrombospondin-1 in human pancreatic adenocarcinoma: roles in tumor angiogenesis and matrix metalloproteinase-9 production

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Expression of thrombospondin-1 in human pancreatic adenocarcinoma: roles in tumor angiogenesis and matrix metalloproteinase-9 production

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