Enhancement of the expression of urokinase-type plasminogen activator from PC-3 human prostate cancer cells by thrombin.

The presence of procoagulants and fibrin deposition have been demonstrated in malignant tumors. Although thrombin, a key enzyme in coagulation, has other various biological functions, the significance of its presence in tumors is not known. We studied the effects of thrombin on the expression of urokinase-type plasminogen activator (uPA) which is known to play a role in tumor invasion, using a human prostate cancer cell line PC-3. Human alpha-thrombin added to cultures of PC-3 produced a dose-dependent and time-dependent increased secretion of uPA that was greatest at 3-6 h after exposure to thrombin. Increase in uPA antigen paralleled the increase in mRNA level, which reached a maximum at 4 h. Thrombin showed the maximum effect on uPA expression at a concentration 1-2 units/ml. Zymography showed that transient exposure to thrombin induced an increase in fibrinolytic activity which could be quenched by anti-uPA antibody. The thrombin receptor-activating peptide also caused an increase in uPA protein and mRNA level, indicating the presence of the same thrombin specific receptor on PC-3 cells as on platelets and endothelial cells. Thrombin did not affect the expression of other components of the plasminogen activation system, tissue-type plasminogen activator and type-1 plasminogen activator inhibitor, and uPA receptor. These results indicate that thrombin increases uPA expression selectively by the stimulation of a functional thrombin receptor on PC-3 cells. Since uPA is known to play a role in pericellular proteolysis of extracellular matrix, thrombin may be involved in the regulation of tumor invasion and metastasis.

Elevated levels of urokinase-type plasminogen activator and plasminogen activator inhibitor type-1 in malignant human brain tumors.

The plasminogen-plasmin system has been found to modulate neoplastic spread and angiogenesis in tumors outside the central nervous system (CNS), but there have been no quantitative studies on the invasive and vascular tumors of the CNS. Quantitative zymography and enzyme-linked immunosorbent assay were used to determine the amounts of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator, and plasminogen activator inhibitors type 1 and type 2 (PAI-1 and PAI-2) in benign and malignant primary brain tumors (n = 28) as well as nonneoplastic brain (n = 5). u-PA and PAI-1 antigen were undetectable in normal brain but significantly elevated in glioblastoma multiforme (u-PA, 2.86 +/- 3.01 ng/mg; PAI-1, 8.19 +/- 5.57 ng/mg; P < 0.001). There was no difference, however, in tissue-type plasminogen activator antigen levels among control, benign, or malignant tissues except for a 4- to 7-fold increase in acoustic neuroma. PAI-2 was detected at low levels in 2 of the 33 specimens. These findings indicate that malignancy in primary CNS neoplasms is associated with elevated levels of u-PA and PAI-1, supporting the role of the plasminogen-plasmin system in the pathogenesis of CNS malignancy and as a potential biomarker and therapeutic target.

Elevated levels of plasminogen-activator inhibitor type 1 in atherosclerotic aorta.

PURPOSE: Plasminogen activator inhibitor type I (PAI-1) inhibits the plasminogen activators that convert plasminogen to plasmin. In addition to initiating fibrinolysis, plasmin activates tissue matrix metalloproteinases, which cause degradation of the extracellular matrix (ECM) in the arterial wall. Elevated levels of PAI-1 ultimately decrease plasmin formation and may lead to an accumulation of ECM and arteriosclerosis. METHODS: PAI-1 was studied by four methods in atherosclerotic (aneurysmal and occlusive) and normal (organ donor) aorta: (1) PAI-1 secretion by tissue explant supernatants, including time course and inhibition studies; (2) tissue PAI-1 by protein extraction; (3) PAI-1 mRNA was quantitated by Northern analysis using glyceraldehyde-3-phosphate dehydrogenase to normalize for RNA loading; and (4) in situ hybridization was used to localize the cells that produced PAI-1 mRNA. RESULTS: Supernatant PAI-1 levels at 48 hours were 776 +/- 352, ng/ml in 11 atherosclerotic aortas and 248 +/- 98 ng/ml in 8 normal aortas (p < 0.005). Tissue PAI-1 levels per 100 mg of tissue were 99 +/- 58 ng in 11 atherosclerotic aortas and 38 +/- 20 ng in 5 normal aortas (p < 0.05). PAI-1 mRNA levels by Northern analysis were 0.91 +/- 0.49 in seven atherosclerotic aortas and 0.44 +/- 0.27 in five normal aortas. Supernatant time-course experiments revealed that PAI-1 increased over time. Inhibitor studies revealed that PAI-1 decreased to approximately one third of control values when cycloheximide or actinomycin D were added to the media, indicating that active synthesis of PAI-1 had occurred. In-situ hybridization localized PAI-1 mRNA predominately to endothelial cells and a few scattered vascular smooth muscle and inflammatory cells. Subgroup analysis revealed no statistically significant differences between aneurysmal and occlusive PAI-1 levels in any of the experiments. CONCLUSION: PAI-1 secretion, as measured by tissue explant supernatants, and total tissue PAI-1 in the protein extracts were significantly increased in atherosclerotic aorta. This elevation was also observed in the mRNA, which suggests that the increase is controlled at the level of transcription. PAI-1 mRNA was localized to endothelial, vascular smooth muscle, and inflammatory cells. We conclude that elevated levels of PAI-1 exist in diseased aorta. These elevated levels may lead to an accumulation of ECM, thereby contributing to the arteriosclerosis found in aortic occlusive and aneurysmal disease.