Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in cancer cells at invasive foci in human colon adenocarcinomas.

In this study in situ hybridization methods were used to examine biopsy samples from 13 adenocarcinomas of the colon for the presence of mRNA for the urokinase-type plasminogen activator (u-PA) and its specific cell-surface receptor (u-PAR). In all cases, u-PA mRNA was present in fibroblastlike cells in the stroma adjacent to the invasive tumor nodules. Urokinase-type plasminogen activator mRNA was not detected in the malignant cells. All specimens also contained u-PAR mRNA in cells located at the tumoral-stromal interface of invasive foci, but in contrast at least some of these cells were in all but one case identified as being of malignant origin. Stromal cells, probably tumor-infiltrating macrophages and neutrophils, also were positive in these areas. These results support the view that components of the plasminogen activation system may act to influence proteolytic events occurring at the interface between stroma and malignant cells in adenocarcinomas of the colon in humans.     

Lung capillary endothelial cells produce and secrete urokinase-type plasminogen activator.

Bovine lung capillary endothelial cells (BLuEC) were isolated, and their ability to produce plasminogen activator (PA) in vitro was demonstrated. BLuEC secreted more than 10 times as much as urokinase-type PA (u-PA) as did bovine aortic, hepatic capillary and adrenal capillary endothelial cells, and lung fibroblasts. BLuEC secreted u-PA on both sides of the cell layer, the luminal surface, and the basic surface attached to the basement membrane. u-PA mRNA was detected in BLuEC by Northern blotting, but not in endothelial cells from other tissues and fibroblasts. These results suggest that BLuEC may contribute not only to the patency of lung vessels but also to the maintenance of alveolar functions through the production and secretion of u-PA.     

Asbestos upregulates expression of the urokinase-type plasminogen activator receptor on mesothelial cells.

Inhalation of asbestos is associated with pathologic changes in the pleural space, including pleural thickening, pleural plaques, and mesothelioma. These processes are characterized by altered local proteolysis, cellular proliferation, and cell migration, suggesting that the urokinase-type plasminogen activator receptor (uPAR) could be involved in the pathogenesis of asbestos-induced pleural disease. We hypothesized that mesothelial cell uPAR expression is induced by exposure to asbestos. To test this hypothesis, we used complementary techniques in rabbit and human mesothelial cells to determine whether uPAR expression is altered by exposure to asbestos. uPAR expression was induced by chrysotile and crocidolite asbestos, but not by wollastonite, as indicated by binding of radiolabeled urokinase-type plasminogen activator (uPA) to rabbit or human mesothelial cells. uPA was not induced by fiber exposure. Exposure to exogenous uPA increased uPA activity of cells exposed to wollastonite but not asbestos-treated MeT5A cells. uPAR expression increased further when asbestos was preincubated with vitronectin (VN) or serum. Increases in uPAR expression were confirmed by binding of uPA to uPAR in cell membrane preparations and immunofluorescent staining of uPAR at the cell surface, and were associated with increases in steady-state uPAR messenger RNA. Mesothelial cell uPAR expression was also induced by media from monocytes cultured with asbestos incubated with VN and serum. By antibody neutralization, the latter effect appeared to be in part mediated by transforming growth factor-beta. We found that asbestos increases uPAR at the surface of rabbit and human mesothelial cells, suggesting that altered expression of this receptor could be involved in asbestos-induced remodeling of the pleural mesothelium.     

Expression of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen activator inhibitor-1 and -2 in hepatocellular carcinoma

It has become more and more clear in recent decades that the plasminogen activation system, which includes urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), plasminogen activator inhibitor (PAI)-1 and PAI-2, plays a very important role in the aggressiveness of cancer. Using immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), the expression of these four components of the uPA system was analyzed in 19 cases of hepatocellular carcinoma (HCC) and 18 cases of the adjacent non-cancer tissues which all had chronic active hepatitis with liver fibrosis or liver cirrhosis. Four cases of normal liver tissues, as controls for immunohistochemical stains, were obtained from the hepatectomized liver of patients with metastatic cancer in the liver. The positive rates of uPA, uPAR, PAI-1 and PAI-2 for immunohistochemical stains in cancer tissues were 78.9, 68.4, 57.9 and 31.6%, respectively. Positive signals were mainly distributed in the cytoplasm of the cancer and in stromal cells. Moreover, the strong stains were chiefly located in the invasive front of the cancer cells. No specific stain was detected in four cases of normal liver tissues. In ELISA, there were significant differences between cancer and non-cancer tissues in concentration of uPA, uPAR and PAI-1 (P < 0.0003, 0.0024 and 0.01, respectively), but there was no significant difference in that of PAI-2 (P = 0.37). These results suggest that uPA, uPAR and PAI-1 are related to invasion of HCC.     

Prognostic role of urokinase-type plasminogen activator in human gliomas.

Urokinase-type plasminogen activator (u-PA) is a 54-kd enzyme shown to participate in tissue degradation under certain normal and pathological conditions, including cancer invasion and metastasis. Increased u-PA expression has been found in cancers of the breast, lung, colon, and prostate, and correlated with worse outcome in patients with lung and breast cancer. We examined the correlation between u-PA expression in gliomas and patient survival. Seventy-seven gliomas from 41 men and 36 women (ages 2 to 73) were immunostained for u-PA using monoclonal antibody 394 directed against human urokinase. The tumors included 32 grade 4, 16 grade 3, and 20 grade 2 astrocytomas (Daumas-Duport scale), and 9 pilocytic astrocytomas. Strong cytoplasmic staining was found in tumor cells of all grade 4, most of the grade 3, and a few of the lower grade tumors. Adjacent normal brain tissue showed faint staining associated with subpial cell processes and white matter fibers. The fiber staining was stronger in brain tissue infiltrated by tumor cells. Cytoplasmic u-PA staining in tumor cells was scored from 0 (no staining) to 6 (strong and widespread staining). The mean u-PA scores were 5.08 +/- 0.19 (mean +/- SEM) for grade 4, 3.97 +/- 0.46 for grade 3, 1.65 +/- 0.39 for grade 2, and 1.22 +/- 0.60 for pilocytic astrocytomas. The statistical analysis was based on cytoplasmic staining only. Analysis of variance revealed significant differences between the mean u-PA scores of different grades (P < 0.02 between grades 4 and 3, and P = 0.0001 between grades 4 or 3 and 2, and between grades 4 or 3 and pilocytic), except between grade 2 and pilocytic astrocytomas. Univariate analysis indicated that u-PA score > or = 4 (P = 0.0001), tumor grade 4 (P = 0.01), and age > 50 (P < 0.001) were all significant predictors for shorter disease survival. A three-way interaction model by multivariate analysis indicated that u-PA score > or = 4, tumor grade 4, and age > 50, taken together, were significant factors for shorter patient survival (P < 0.02). We conclude that u-PA may be used as a prognostic tool in conjunction with tumor grade and patients' age in predicting survival for patients with gliomas.     

Expression of urokinase-type plasminogen activator by rat pulmonary alveolar epithelial cells

Intra-alveolar fibrin deposition accompanies many forms of inflammatory lung injury. Appropriate clearance of this fibrin matrix is important for normal healing and remodeling. The local generation of plasmin by the action of plasminogen activators (PAs) represents a pivotal step in the fibrinolytic process. To investigate whether the alveolar epithelium plays a role in the modulation of intra-alveolar fibrinolysis, we have studied PA regulation by rat pulmonary alveolar epithelial cells. We have found large quantities of PA activity both in conditioned media and cell lysates from epithelial monolayers in culture. Casein-plasminogen zymography reveals that this PA activity migrates as a tight doublet with an apparent mol wt of 45 kD, clearly distinct from rat tissue-type PA (tPA, greater than 68 kD). Analysis of freshly isolated type II alveolar epithelial cells demonstrates readily measurable PA activity in cell lysates, as well as expression of urokinase-type PA (uPA) mRNA on Northern blot analysis. Upregulation of PA activity occurs progressively with time in culture as the alveolar epithelial cells lose type II cell characteristics and become more flattened. Stimulation of alveolar epithelial cell monolayers with lipopolysaccharide or tumor necrosis factor increases levels of secreted PA activity. The relative abundance of uPA mRNA was shown to change in parallel with PA activity during in vitro differentiation or after exposure to inflammatory mediators. Thus, alveolar epithelial cells are likely an important source of uPA in the lung, the expression of which is influenced by the state of cellular differentiation as well as the presence of inflammatory mediators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urokinase plasminogen activator is localized in stromal cells in ductal breast cancer.

Urokinase plasminogen activator (uPA) regulates a proteolytic cascade that facilitates cancer invasion through degradation of the extracellular matrix, and high levels of uPA in human breast cancer tissue correlate with poor prognosis. We previously found that, in ductal breast cancer, uPA mRNA is highly expressed by myofibroblasts surrounding invasively growing cancer cells. However, the localization of uPA protein has not been settled in the published literature. Because uPA is a secreted molecule, it could conceivably be localized differently from its mRNA. We have studied the localization of uPA immunoreactivity in detail. Twenty-five cases of invasive ductal carcinoma were analyzed with three different uPA antibody preparations, all of which gave an essentially identical stromal staining pattern. Using double immunofluorescence, we identified uPA immunoreactivity in myofibroblasts and macrophages in all cases examined. Additionally, in approximately half of the tumors, we saw uPA staining of endothelial cells. In 3 of the 25 cases, a small subpopulation of the cancer cells was uPA-positive. We conclude that uPA immunoreactivity is almost exclusively associated with stromal cells, which thus play a major role in generation of proteolytic activity in ductal breast cancer.     

靶向尿激酶受体治疗癌症的研究进展

目的:癌症的侵袭与转移是一个非常复杂的过程,丝氨酸蛋白酶尿激酶受体已被证实在癌症进展发挥着重要的作用,尿激酶受体的高表达与癌症的不良预后密切相关。尿激酶受体已成为抗癌治疗研究的重要靶点之一。现就以抑制尿激酶受体为靶点治疗癌症的研究进展作一综述。     

suPAR在消化道肿瘤中的研究进展

可溶性尿激酶型纤溶酶原激活物受体(suPAR)是尿激酶型纤溶酶原激活物受体(uPAR)的可溶形式,其除作为一种炎性反应标志物外,在消化道肿瘤的发生发展及侵袭转移中也具有重要的生物学作用。研究表明,suPAR在早期肿瘤的分子诊断、治疗效果的监测及进一步生物靶向药物的开发中具有重要的价值。     

Plasminogen mediates the pathological effects of urokinase-type plasminogen activator overexpression

Increased expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) is associated with different pathological conditions. Both uPAR-mediated signaling and plasmin-catalyzed extracellular proteolysis may contribute to pathogenesis. To evaluate the involvement of plasminogen in such circumstances, we have taken advantage of transgenic mouse models in which overexpression of uPA and/or uPAR in enamel epithelium, basal epidermis, and hair follicles leads to a pathological phenotype; uPA transgenic mice have chalky-white incisors and, when uPAR is co-expressed, develop extensive alopecia, epidermal thickening, and subepidermal blisters. We report here that when these transgenic mice were backcrossed into a plasminogen-deficient (Plg-/-) background, the dental and skin phenotypes appeared completely normal. Heterozygous Plg+/- transgenic mice exhibited a haplo-insufficiency, with an intermediate or normal phenotype. These results do not argue in favor of a role for uPAR-mediated signaling in our experimental model; rather, they demonstrate an essential, dose-dependent, requirement for plasminogen in uPA-mediated tissue alterations. They also support the hypothesis that plasminogen could play a part in certain skin diseases.     

In-vivo imaging of tumor associated urokinase-type plasminogen activator activity

The ability to image tumor associated protease in vivo has biological and clinical implications. In the present study, we describe the development and validation of a urokinase-type plasminogen activator (uPA) sensitive fluorescence imaging probe. The activation of our probe is highly specific to uPA in both enzymatic and cellular-based assays. In two distinct in-vivo tumor models (human colon adenocarcinoma HT-29 and human fibrosarcoma HT-1080), the observed fluorescence changes correlate well with tumor associated uPA activity. The signal intensities of the tumors are about three-fold higher in animals with probe injections. Our results suggest a direct detection method for uPA activity in vivo and the approach can be used for monitoring tumor growth and development.     

Modulation of the receptor for urokinase-type plasminogen activator in macrophage-like U937 cells by inflammatory mediators

Macrophages in the tissues have been shown to express receptor for urokinase-type plasminogen activator (uPAR) on their cell surface which plays an important role in cell invasion and attachment. We examined the effects of inflammatory mediators on the expression of uPAR employing U937 cells which have monocyte/ macrophage-like characteristics. U937 cells were incubated with various mediators such as interleukins (IL), tumor necrosis factors (TNF), dexamethasone, thrombin, fibrin fragment D, bradykinin, complement C5a, and components of the extracellular matrix. The uPAR expression on the cell surface was then analyzed by radio-ligand binding assay using¹²⁵I-scuPA. The strongest enhancement of uPAR was observed in the cells stimulated by TNF and TNF. IL-1, IL-6, and C5a also increased the uPA binding sites with various patterns of affinity change. Dexamethasone decreased the uPA binding sites without changing the affinity. Fibrin fragment D and IL-3 reduced the affinity without changing the number of receptors. These findings suggest that the expression of uPAR in inflammatory cells could be modulated by various inflammatory mediators.     

The urokinase-type plasminogen activator and inhibitors in resectable lung adenocarcinoma

BackgroundThe urokinase-type plasminogen activator (uPA) system is closely related to the occurrence and progression of cancer in many aspects. Previous studies demonstrated that the conclusions about the prognosis value of uPA, plasminogen activator inhibitor 1 (PAI-1) and plasminogen activator inhibitor 2 (PAI-2) in lung cancer are controversial, so this study was performed for the exploration of the predictive effect of uPA, PAI-1 and PAI-2 on the overall survival (OS) of resectable pulmonary adenocarcinoma patients.MethodsUPA, PAI-1 and PAI-2 expression levels were assayed by immunohistochemical staining based on tissue microarray (TMA) that is composed of formalin-fixed paraffin-embedded specimens from 84 resectable lung adenocarcinoma patients from July 2004 to June 2009. The relationship of IHC, mRNA expression levels of three molecules were investigated respectively. The three molecules’ relationship with clinicopathologic parameters and OS was explored by Chi-square, Kaplan-Meier, and Cox regression analyses. The Cancer Genome Atlas (TCGA) database was used to analyze differential gene expressions of RNA-sequencing data of pulmonary adenocarcinoma and normal tissues, and Kaplan-Meier methods were adopted to confirm the prognostic value of uPA, PAI-1 and PAI-2 in resectable lung adenocarcinoma in TCGA database and the R package MethylMix was used to conduct an analysis integrating methylation data and gene expression of RNA-sequencing data based on TCGA.ResultsUPA, PAI-1 and PAI-2 had much higher IHC expression levels in tumor than those in the normal tissues (uPA, Z = -10.511; PAI-1, Z = -4.836; PAI-2, Z = -6.794; all P < 0.0001). High DNA methylation level of gene uPA resulted in the decrease of its expression. In addition, expression level of PAI-2 was positively associated with tumor size (χ² = 8.372, P = 0.004). Multivariate analyses showed TNM stage III was an independent adverse prognostic factor (hazard ratio = 3.736, 95 % confidence interval = 1.097–12.72, P = 0.035). Kaplan-Meier method revealed that uPA, PAI-1 and PAI-2 expression levels were not related to the OS for 84 resectable lung adenocarcinoma patients. According to TCGA data, PAI-1 expression level was identified as a potential adverse predictor for prognosis of resectable lung adenocarcinoma (Gehan-Breslow-Wilcoxon test, P = 0.025).ConclusionsOur data show that, the expression levels of uPA, PAI-1 and PAI-2 are significantly up-regulated in resectable lung adenocarcinoma. Besides, this study highlights PAI-1 as a latent adverse prognostic factor in resectable adenocarcinoma of lung.     

Immunolocalization of urokinase-type plasminogen activator in adenomas and carcinomas of the colorectum

Carcinogenesis in the human colon is associated with a marked increase in the tissue content of the urokinase-type plasminogen activator (u-PA). This study was performed to determine the type of cells responsible for the u-PA increase in carcinomas of the colon and in their precursor lesions, the adenomas, by immunohistological evaluation applying monoclonal antibody 3689 directed to the beta-chain of u-PA. Normal intestinal mucosa (n = 17) showed hardly any staining of u-PA, but some lamina propria cells were faintly positive. Carcinomas (n = 17) and adenomas (n = 16) showed a considerable and comparable staining intensity of u-PA in neoplastic columnar epithelial cells, and this staining was found to be diffuse and cytoplasmic. In a majority of the neoplastic tissues the u-PA staining was found to be patchy and not related to known risk markers of malignancy such as dysplasia in the adenomas, or to prognostic determinants such as Dukes' classification or differentiation in the carcinomas. The observation of strong u-PA positive lamina propria cells in adenomas but infrequently observed in normal mucosa and carcinomas was noteworthy. u-PA staining intensity of the tissue sections was found to correlate well with the u-PA antigen level in the tissue extracts determined by ELISA (r = 0.52, P = 0.0001) but poorly with the u-PA activity determined enzymatically (r = 0.28, P = 0.05). In conclusion, the u-PA increase in neoplasia of the human colon can be attributed to an increased diffuse cytoplasmic content of u-PA in neoplastic columnar epithelial cells.     

Organ-site dependence for the production of urokinase-type plasminogen activator and metastasis by human renal cell carcinoma cells.

We examined the role of urokinase-type plasminogen activator (u-PA) in the metastasis of the human renal cell carcinoma (HRCC) implanted in athymic nude mice. Cells from a HRCC KG-2 line were implanted in orthotopic (kidney) and ectopic (subcutaneous) organs. The KG-2 cells implanted in the kidney produced local tumors and lung metastases, whereas those implanted subcutaneously produced only local tumors. The production of u-PA was determined by immunohistochemistry and an enzyme-linked immunosorbent assay (ELISA). High levels of u-PA were produced by the metastatic kidney tumors and lung metastases, whereas the subcutaneous tumors produced low levels. KG-2 cells co-cultured with mouse kidney or lung fibroblasts produced higher levels of u-PA than KG-2 cells co-cultured with mouse skin fibroblasts. Furthermore, KG-2 cells cultured with the conditioned medium from mouse kidney or lung fibroblasts produced higher levels of u-PA than KG-2 cells cultured with the conditioned medium from mouse skin fibroblasts. The results indicate that the expression of u-PA by KG-2 cells is one of the important factors that determine their metastatic potential and that the production of u-PA is influenced by the organ microenvironment, including soluble factors produced by surrounding fibroblasts.     

IL-1beta stimulates urokinase-type plasminogen activator expression and secretion in human dental pulp cells

Plasminogen activator (PA) is the enzyme that converts plasminogen to its active form, plasmin, which is involved in various physiological and pathological phenomena. The conversion is catalyzed by two types of PA, urokinase-type PA (uPA) and tissue-type PA (tPA). We investigated the effect of the inflammatory cytokine interleukin-1beta (IL-1beta) on PA secretion in human dental pulp cells. When the cells were stimulated by IL-1beta, PA activity in the medium was clearly increased in a time- and dose-dependent manner. This PA activity in the medium was reduced after immunoprecipitation with anti-uPA antibody, and uPA protein was detected in the immunoprecipitated fraction by Western blotting. However, no such effect was observed with anti-tPA antibody. In the IL-1beta-stimulated cells, expression of uPA mRNA was enhanced whereas expression of tPA mRNA was less. The IL-1beta-stimulated uPA mRNA expression and PA activities in the cell lysate and medium were reduced by the tyrosine kinase inhibitors herbimycin A and genistein, and by the NFkappaB inhibitor pyrolidinedithiocarbamate, and were augmented by the tyrosine phosphatase inhibitor sodium orthovanadate. These observations suggest that IL-1beta stimulates uPA production via activation of NFkappaB and tyrosine phosphorylation, and also secretion of the enzyme, and that the uPA/plasmin system appears to be involved in inflammation in human dental pulp.