uPA Levels Research Articles

Abstract 2480: Chronic radiation exposure increases uPA and cMyc expression levels but decreases nMyc expression levels in neuroblastoma cells

Abstract Neuroblastoma is the most common extra-cranial solid tumor in children. Despite aggressive therapy prognosis of high risk neuroblastoma remains guarded and also unpredictable because of heterogeneity of tumor response to treatment. Radio-resistance has been recognized as a significant contributor to aggressive clinical behavior of neuroblastoma. Characterization of genetic alterations have been used for predicting clinical outcome in neuroblastomas and one of the most studied genetic markers in aggressive neuroblastoma is nMyc/MYCN. The MYCN gene belongs to the MYC family of transcription factors and nMyc expression is to a large extent inversely correlated with cMyc expression and nMyc expression is also associated poor clinical outcomes. In this study, we have attempted to correlate the expression of nMyc in neuroblastoma cells NB1691 and SKNBE2 to radiation resistance. We exposed neuroblastoma cells NB1691 and SKNBE2 to chronic radiation doses with a cumulative high energy X-ray radiation dose of 25Gy achieved by dosing at 5Gy increments over a period of 15 days. Cells surviving after the 25Gy cumulative radiation dose were selected and designated as NB1691R and SKNBE2R. Expression levels of nMyc, cMyc and uPA were determined. We observed that the radiation tolerant cells showed at least 164 fold increase in the expression of uPA in NB1691R cells and at least 97 fold increase in SKNBE2R cells. We also observed that the expression levels of nMyc decreased in both NB1691R and SKNBE2R cells to almost undetectable levels. Further, the expression levels of cMyc revealed that NB1691R cells showed a 208 fold increase and SKNBE2R a 71 fold increase when compared to parental cells. We further determined whether the expression of uPA can modulate the expression of nMyc. We silenced uPA expression in NB1691 cells and observed that downregulation of uPA moderately rescued nMyc expression. Cell cycle analysis revealed that acute radiation of NB1691 cells induced accumulation of cells in the G2/M phase and un-radiated NB1691R cells continued to show cells accumulated in the G2/M phase similar to acute radiated NB1691 cells. Our study shows that: 1. Chronic radiation exposure suppresses the expression of nMyc and induces the expression of cMyc. 2. cMyc expression was accompanied with the expression of uPA and suppression of uPA expression moderately rescued the expression of nMyc. Our preliminary findings suggest that chronic exposure to radiation alters the nMyc/cMyc ratio in neuroblastoma cells and induces the expression of the pro-invasive molecule uPA. Citation Format: Manu Gnanamony, David Pinson, Reuben Antony, Karen S. Fernández, Julian Lin, Pushpa A. Joseph, Christopher S. Gondi. Chronic radiation exposure increases uPA and cMyc expression levels but decreases nMyc expression levels in neuroblastoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2480.

Comparison of oxycodone and morphine on the proliferation, apoptosis and expression of related molecules in the A549 human lung adenocarcinoma cell line.

The present study aimed to compare the effects of oxycodone and morphine hydrochloride on the proliferation, apoptosis and migration of A549 lung cancer cells. A549 human lung cancer cells were cultured in vitro and treated with oxycodone or morphine at various concentrations (10, 20 and 40 µg/ml). Cell migration was determined using a wound healing assay, whereas apoptosis was detected using flow cytometry. Reverse transcription quantitative-polymerase chain reaction was performed in order to assess the apoptosis-related gene expression levels, including p53, B-cell lymphoma (Bcl)-2 and Bcl-2-associated X protein (Bax). The levels of vascular endothelial growth factor (VEGF) and urokinase-type plasminogen activator (uPA) were detected using enzyme-linked immunosorbent assays. The expression levels of intercellular cell adhesion molecule (ICAM)-1 were determined by immunofluorescence. In the present study, oxycodone and morphine induced apoptosis in A549 lung cancer cells with similar potency; however, >20 µg/ml oxycodone was more effective at inhibiting cell proliferation (P<0.05) and migration (P<0.05), as compared with morphine at the same concentration. Oxycodone induced a dose-dependent increase in the expression levels of p53 and Bax apoptosis-related genes, whereas it decreased the gene expression levels of Bcl-2. Furthermore, oxycodone decreased, whereas morphine increased, the expression levels of ICAM-1 in a concentration-dependent manner. In addition, at 40 µg/ml, the expression levels of VEGF and uPA in the morphine group were significantly higher than those demonstrated in the oxycodone group (P<0.05). In conclusion, oxycodone was more effective in inhibiting the proliferation and migration of A549 lung cancer cells, as compared with morphine.

The potential of PAI-1 expression in needle biopsies as a predictive marker for prostate cancer

AbstractThe relative abundance of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) in transurethral resections of the prostate (TURP) has been shown to correlate with disease state. The objective of this study was to assay for uPA and PAI-1 in prostate needle biopsies, and to test their potential as predictive markers for prostate cancer (PCa). uPA and PAI-1 levels were determined for 111 patients (55 PCa; 56 benign prostatic hyperplasia (BPH)), using the FEMTELLE enzyme-linked immunosorbent (ELISA) assay. The PAI-1 concentrations for PCa and BPH patients differed significantly (p = 0.0403) and a level of ≥ 4.5 ng/mg protein in men 60 years and older appears to be predictive of PCa, with a sensitivity of 63%. uPA plays a minor role as a potential marker in biopsy tissue, a feature noted in our recent TURP tissue studies, and elsewhere. The potential utility of the uPA/PAI-1 ratio as a predictor of prostate disease, as previously suggested for TURP tissue, is not app...

G Protein‐Coupled Receptor Kinase 2's Effect on TNFα Signaling Modulates Wound Healing in Intestinal Epithelial Cells

G protein‐coupled receptor kinases (GRKs) are serine/threonine protein kinases critical in the regulation of GPCR lifecycle. Recently GRKs have been shown to have a large interactome and can play a critical role in cell signaling pathways as well as in consequent cell physiological processes. In this study, we examined the role that GRK2 in TNFα signaling in colon epithelial cell biological processes including wound healing, proliferation, apoptosis, and gene expression. Using an siRNA based knockdown approach in SW480 cell line, we find that GRK2 mediates rate of wound closure under basal conditions. GRK2 did not affect either apoptosis or proliferation of these cells. Interestingly, upon examining the levels of various gene transcripts, we observed that matrix metalloproteinases (MMP) 7 and 9 as well as urokinase plasminogen activator (uPa) (proteins involved in cell migration) was markedly upregulated in GRK2 knockdown cells. To assess the mechanism by which GRK2 affects these physiological processes, we examined the role of GRK2 in TNFα‐induced MAPK and NFkB pathways. Our results demonstrate opposing roles of GRK2 in MAPK and NFkB pathways‐while GRK2 mediates NFkB pathway, it inhibits ERK pathway. Our results further demonstrate that GRK2‐mediated regulation of MMP9 is via the ERK pathway. Together, these data suggest that GRK2 plays a critical role in cellular migration and/or wound healing by modulating MMP7, 9 and uPA levels potentially via differential regulation of MAPK and NFkB signaling pathways. Our results provide a novel role for GRK2 in TNFa signaling in colon epithelial cells.

Abstract P2-08-21: Stromal co-expression of urokinase-type plasminogen activator (uPA) and plasminogen activator Inhibitor (PAI-1) protein by IHC predicts poor disease outcome in endocrine-treated postmenopausal patients with receptor-positive early breast cancer

Abstract Background Elevated levels of intratumoral uPA and PAI-1 in ELISA-based measurements are associated with a high recurrence risk and allow to identify patients who might particularly benefit from adjuvant chemotherapy. The clinical utility of ELISA-based protein analysis is, however, greatly limited by the requirement of fresh tumor tisssue. We have therefore evaluated in a large clinical trial patient cohort with long-follow-up whether immunohistochemical detection of uPA and PAI-1 in FFPE archived tumor samples is also able to identify women with poor prognosis. Patients and Methods 547 postmenopausal women with hormone receptor–positive, early breast cancer who had received at least 5 years of endocrine therapy in the prospectively designed ABCSG-06 trial, and in whom FFPE tumor tissue was available, were included in this analysis. uPA and PAI-1 protein expression was evaluated by immunohistochemistry, and correlated with distant-disease free (DDFS) and overall survival (OS). Results Stromal co-expression of uPA and PAI-1 was detected in 166 of 276 (60%) evaluable tumor samples and was weakly associated with tumor size (p=0.012, Chi Square test) but not with age, nodal status, grading, or receptor status. Women with intratumoral uPA/PAI-1 expression were more likely to have a shorter DDFS in multivariate analysis (HR for relapse p=1.870; 95% CI 1.184-2.955; p=0.007 Cox regression analysis) and and OS (adjusted HR for death p=1.291; 95% CI 0.928-1.795; p=0.129) than women without. After a median follow-up of 10 years, women with uPA/PAI-1-positive tumors experienced a significantly shorter DDFS (p&amp;lt;0.0001 log rank test) and OS (p=0.020). Conclusions Stromal co-expression of uPA and PAI-1 in breast cancer samples predicts poor DDFS and OS in postmenopausal women with hormone-receptor positive early-stage breast cancer who receive endocrine therapy. Citation Format: Singer CF, Filipits M, Jahn S, Abete L, Jakesz R, Greil R, Bauernhofer T, Kwasny W, Seifert M, Fitzal F, Schmitt M, Moinfar F, Gnant M. Stromal co-expression of urokinase-type plasminogen activator (uPA) and plasminogen activator Inhibitor (PAI-1) protein by IHC predicts poor disease outcome in endocrine-treated postmenopausal patients with receptor-positive early breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-08-21.

Significance of the urokinase-type plasminogen activator and its receptor in the progression of focal segmental glomerulosclerosis in clinical and mouse models.

BackgroundsuPAR biomarker generally considered a pathogenic factor in FSGS. However, studies have been published that dispute this conclusion. The current study was designed to investigate the roles of uPA and suPAR in FSGS in clinical and mouse models.MethodsClinical subjects including those with biopsy-proven FSGS and MCD were enrolled. To verify the role of uPA in FSGS, Adriamycin was used to induce FSGS in uPA knockout (uPA−/−) and BALB/c (WT) mice. Proteinuria and suPAR, the cleaved/intact forms of the circulating suPAR, and possible proteases involving cleavage of the suPAR were also studied.ResultsFSGS clinical cases presented significantly higher serum levels of suPAR and Cr and lower serum levels of uPA. In the mice model, the uPA−/− group exhibited faster disease progression and worsening proteinuria than the WT group. In addition, the uPA−/− group had higher plasma suPAR levels, glomerular cell apoptosis, and dysregulation of the Th1/Th2 balance. In an analysis of suPAR variants in FSGS, both the intact and cleaved forms of the suPAR were higher in clinical subjects and the mouse model. However, the process of suPAR cleavage was not mediated by enzymatic activities of the uPA, elastase, or cathepsin G.ConclusionsA deficiency of uPA accelerated the progression of Adriamycin-induced mouse FSGS model. Decrease of serum uPA levels may be an indicator of the progression of FSGS in clinical subjects and animal models.

Simultaneous knockdown of uPA and MMP9 can reduce breast cancer progression by increasing cell-cell adhesion and modulating EMT genes.

In cancer progression, proteolytic enzymes like serine proteases and metalloproteinases degrade the basement membrane enabling the tumor cells to invade the adjacent tissues. Thus, invasion and metastasis are augmented by these enzymes. Simultaneous silencing of uPA and MMP9 in breast cancer cells decreased the wound healing, migratory, invasive and adhesive capacity of the cells. After simultaneous down regulation, cells were seen to be arrested in the cell cycle. There was a remarkable increase in the expression of cell to cell adhesion molecule E–cadherin, and decrease in Vimentin and Snail expression. In addition, there was a significant decrease in the expression of the stem cell marker Oct-4. In the breast tumor samples it has been observed that, tumors, expressing higher level of uPA and MMP9, express less amount of E–cadherin. It has also been observed that few tumors also show, Vimentin positive in the ductal epithelial area. Thus, our model can help for checking the aggressive tumor invasion by blocking of uPA and MMP9. Our present observations also give the concept of the presence of aggressive epithelial cells with mesenchymal nature in the tumor micro-environment, altering the expression of EMT genes.

High Expression of Urokinase-Type Plasminogen Activator Is Associated with Lymph Node Metastasis of Invasive Ductal Carcinoma of the Breast.

PurposeIn the present study, we evaluated the levels of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1) by performing immunohistochemical staining to determine whether they were reliable prognostic markers in patients with breast cancer.MethodsDemographic and clinicopathological parameters of 214 patients with invasive ductal carcinoma (IDC) and 80 patients with ductal carcinoma in situ (DCIS) who were diagnosed and treated from 2006 to 2010 were analyzed. Tissue microarray was constructed and immunohistochemical staining was performed for each specimen.ResultsUnivariate analyses showed that age at diagnosis, history of hormone replacement therapy, radiation therapy, skin and chest wall invasion, Paget disease, lymphovascular invasion, estrogen receptor positivity, and triple-negative subtype were significantly associated with patient prognosis (p<0.005). Patients with DCIS showed higher PAI-1 expression than patients with IDC (82.5% and 36.2%, respectively; p=0.012). Lymph node metastasis was more frequent in patients with high uPA levels than in patients with low uPA levels (p=0.001).ConclusionOur results suggested that PAI-1 was involved in tumor progression in the early stages of breast cancer, such as DCIS. In addition, our results suggested that high uPA levels were associated with the lymph node metastasis of IDC.

Serum from Varicose Patients Induces Senescence-Related Dysfunction of Vascular Endothelium Generating Local and Systemic Proinflammatory Conditions.

Although the role of endothelium in varicose vein development is indisputable, the effect of the pathology on biological properties of endothelial cells remains unclear. Here we examined if the presence of varicose veins affects senescence of endothelial cells (HUVECs) and, if so, what will be the local and systemic outcome of this effect. Experiments showed that HUVECs subjected to serum from varicose patients display improved proliferation, increased expression of senescence marker, SA-β-Gal, and increased generation of reactive oxygen species (ROS), as compared with serum from healthy donors. Both increased SA-β-Gal activity and ROS release were mediated by TGF-β1, the concentration of which in varicose serum was elevated and the activity of which in vitro was prevented using specific neutralizing antibody. Senescent HUVECs exposed to varicose serum generated increased amounts of ICAM-1, VCAM-1, P-selectin, uPA, PAI-1, and ET-1. Direct comparison of sera from varicose and healthy donors showed that pathological serum contained increased level of ICAM-1, VCAM-1, P-selectin, uPA, and ET-1. Calendar age of healthy subjects correlated positively with serum uPA and negatively with P-selectin. Age of varicose patients correlated positively with ICAM-1, VCAM-1, and ET-1. Collectively, our findings indicate that the presence of varicose veins causes a senescence-related dysfunction of vascular endothelium, which leads to the development of local and systemic proinflammatory environment.

Plasminogen Activator System and Breast Cancer: Potential Role in Therapy Decision Making and Precision Medicine.

Shifting from the historical TNM paradigm to the determination of molecular and genetic subtypes of tumors has been a major improvement to better picture cancerous diseases. The sharper the picture is, the better will be the possibility to develop subsequent strategies, thus achieving higher efficacy and prolonged survival eventually. Recent studies suggest that urokinase-type plasminogen activator (uPA), uPA Receptor (uPAR), and plasmino-gen activator inhibitor-1 (PAI-1) may play a critical role in cancer invasion and metastasis. Consistent with their role in cancer dissemination, high levels of uPA, PAI-1, and uPAR in multiple cancer types correlate with dismal prognosis. In this respect, upfront determination of uPA and PAI-1 as invasion markers has further opened up the possibilities for individualized therapy of breast cancer. Indeed, uPA and PAI-1 could help to classify patients on their risk for metastatic spreading and subsequent relapse, thus helping clinicians in their decision-making process to propose, or not propose, adjuvant therapy. This review covers the implications for cancer diagnosis, prognosis, and therapy of uPA and PAI-1, and therefore how they could be major actors in the development of a precision medicine in breast cancer.

Cytokines in cerebrospinal fluid of neurosyphilis patients: Identification of Urokinase plasminogen activator using antibody microarrays

Little is known regarding protein responses to syphilis infection in cerebrospinal fluid (CSF) of patients presenting with neurosyphilis. Protein and antibody arrays offer a new opportunity to gain insights into global protein expression profiles in these patients. Here we obtained CSF samples from 46 syphilis patients, 25 of which diagnosed as having central nervous system involvement based on clinical and laboratory findings. The CSF samples were then analyzed using a RayBioH L-Series 507 Antibody Array system designed to simultaneously analyze 507 specific cytokines. The results indicated that 41 molecules showed higher levels in patients with neurosyphilis in comparison with patients without neural involvement. For validation by single target ELISA, we selected five of them (MIP-1a, I-TAC/CXCL11, Urokinase plasminogen activator [uPA], and Oncostatin M) because they have previously been found to be involved in central nervous system (CNS) disorders. The ELISA tests confirmed that uPA levels were significantly higher in the CSF of neurosyphilis patients (109.1±7.88pg/ml) versus patients without CNS involvement (63.86±4.53pg/ml, p<0.0001). There was also a clear correlation between CSF uPA levels and CSF protein levels (p=0.0128) as well as CSF-VDRL titers (p=0.0074) used to diagnose neurosyphilis. No significant difference between the two groups of patients, however, was found in uPA levels in the serum, suggesting specific activation of the inflammatory system in the CNS but not the periphery in neurosyphilis patients. We conclude that measurements of uPA levels in CSF may be an additional parameter for diagnosing neurosyphilis.

Procoagulant Profile in Patients with Immune Thrombocytopaenia

Background: Patients with platelet counts less than 20 or 30 x 109/L have an increased risk of bleeding. Nevertheless, some patients with immune thrombocytopenia (ITP) have fewer bleeding symptoms than expected. In a previous communication (ASH 2014) we reported that these patients presented high microparticles (MP)-associated procoagulant activity to compensate bleeding risk and that cellular origin of these MPs were platelets and red cells. However, other mechanisms might be involved.Objective: The aim of this work was to analyse the involvement of other factors to compensate bleeding risk in thrombocytopenic ITP patients. Moreover, the feasibility of using the coagulation global assays thromboelastrometry (ROTEM) and Calibrated Automated Thrombogram (CAT) to test haemostasis in these patients was evaluated.Methods: Fifty patients with chronic ITP with platelet count less than 50 x 109/L and twenty-five healthy controls were included. Platelet counts were determined with a Coulter Ac. T Diff cell counter (Beckman Coulter, Madrid, Spain). Citrated blood was centrifuged at 152 g 10 min at 23°C for obtaining platelet rich plasma (PRP) and at 1,500 g for 15 min at 23°C for platelet-poor plasma (PPP) and aliquots were stored at -70ºC until analysis.To assess the kinetics of clot formation, non-activated ROTEM was performed on PRP adjusted to a platelet count of 25 x 109/L. Clotting time (CT, time from start of measurement until initiation of clotting [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2-mm amplitude [in degrees]), maximum clot firmness,which reflects the maximum tensile strength of the thrombus (MCF, [in mm]) and LI60, which describes the percentage of maximum clot strength present at 60 min (in %), were recorded.Plasma thrombin generation was measured in PPP using the Calibrated Automated Thrombogram (CAT) test at a final concentration of 1 pM tissue factor and 4 mM phospholipids (PPP-Reagent LOW, Thrombinoscope BV, Maastricht, The Netherlands). We evaluated the endogenous thrombin potential (ETP, the total amount of thrombin generated over time); the lag time (the time to the beginning of the explosive burst of thrombin generation); the peak height of the curve (the maximum thrombin concentration produced); and the time to the peak.Fibrinolytic proteins and E-selectin was tested in PPP using commercialized kits.Results were expressed as mean±SD. Comparisons of quantitative variables were made with Mann-Whitney test and correlations with Spearman test. Values of p≤0.05 were considered statistically significant.Results: PRP from ITP patients showed a prolonged CT (control: 550+ 95 sec, ITP: 890+165 sec, p<0.01), diminished alpha angle (control: 62.8+4.3, ITP: 53.5+7.5, p<0.05), and increased MCF (control: 46.7+3.1mm, ITP: 52.4+6.1 mm, p<0.05) and LI60 (control: 90.6+3.0% , ITP: 95.5+3.4, p<0.05) when compared with controls.In order to evaluate whether increased LI60 values were due to an imbalance in fibrinolysis related proteins, tPA, uPA, TAFI and PAI-1 plasma levels were measured. No differences were observed between patients and healthy controls except for PAI-1 which level was increased in ITP patients (control: 14.7 ng/ml+11.7 ng/ml, ITP: 30.4+17.5, p<0.05). Since plasma PAI-1 might be increased as consequence of endothelial damage, plasma concentration of E-selectin, marker of endothelial injury, was determined. E-selectin was increased in samples from ITP patients (control: 10.5 ng/ml+3.9 ng/ml, ITP: 31.6+14.0, p<0.05). Moreover, MCF and LI60 ROTEM parameters correlated to E-selectin plasma concentration (Spearman r values 0.6643, p<0.001 for MCF; 0.6053, p<0.001 for LI60).Thrombin generation in PPP was also measured and a shorter time to peak (control: 9.3+1.2 sec, ITP: 8.3+1.7 sec,p<0.05) and increased ETP (control: 1223.8+257.7 nMxmin, ITP: 1696.4+524 nMxmin,p<0.05) and peak (control: 225.7+82.8.1 nM, ITP: 330.4+106.1 nM,p<0.05) were observed in ITP patients.Conclusions: We demonstrated that ITP patients presented a hypercoagulable profile that might be related, at least in part, to a reduced fibrinolysis mainly caused by an increase in PAI-1 level that seemed to be related to endothelial damage. Moreover ROTEM and CAT appeared to be useful tools for evaluating coagulant profile in ITP patients. DisclosuresNo relevant conflicts of interest to declare.

The Plasminogen System and Transforming Growth Factor-β in Subjects With Obstructive Sleep Apnea Syndrome: Effects of CPAP Treatment.

Obstructive sleep apnea syndrome (OSAS) has emerged as a risk factor for cardiovascular disease. A prothrombotic state may affect coagulation and participate in the atherosclerotic process in subjects with OSAS. These alterations in coagulation seem to involve the plasminogen activation system. We evaluated the imbalances of the plasminogen activation system related to OSAS, and we assessed the effects of CPAP on the plasminogen activation system. Thirty-nine subjects were submitted to a home-based cardiorespiratory sleep study, and 14 healthy subjects (apnea-hypopnea index < 5) were used as controls. Serum levels of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), and active transforming growth factor-β (TGF-β) were measured. These molecules were reassessed in only 17 of the subjects after 1 month of CPAP. PAI-1 and tPA were significantly higher in the subjects with OSAS compared with the controls, whereas TGF-β and uPA levels were lower. PAI-1 showed a significant positive correlation with the apnea-hypopnea index, percentage of time spent at O2 saturation < 90%, and oxygen desaturation index, whereas TGF-β was inversely related to all 3 of these parameters. After the CPAP therapy, PAI-1 significantly decreased, whereas TGF-β showed a significant increase, although the values did not reach those of the controls. uPA and tPA did not show significant differences after the treatment. Our results suggest an imbalance of fibrinolysis related to OSAS and an improvement of the prothrombotic state after the CPAP treatment.

Upregulation of circulating cytokeratin 20, urokinase plasminogen activator and C-reactive protein is associated with poor prognosis in gastric cancer.

Gastric cancer is one of the most common types of cancer, with a high mortality rate. The aim of this study was to investigate the role of several key molecules, including cytokeratin (CK) 19 and CK20, urokinase plasminogen activator (uPA), C-reactive protein (CRP) and matrix metalloproteinase (MMP)-9, which are involved in cancer invasion and metastasis, in order to determine whether they may be considered as novel prognostic factors for gastric cancer. Peripheral blood was collected from 165 patients with gastric adenocarcinoma who underwent curative surgical resection at Zhejiang Cancer Hospital (Hangzhou, China) between 2010 and 2011. The mRNA levels of CK19, CK20, uPA and MMP-9 were detected by reverse transcription-quantitative polymerase chain reaction. The protein expression of CRP was measured by immunoturbidimetry. The Students t-test was used in the univariate analyses and the Kaplan-Meier method was used to analyze the survival curves. The relative mRNA expression of CK19 and MMP-9 was not found to be significantly associated with gender, age or cancer stage, whereas that of CK20 and uPA was associated with gastric cancer stage: The low-expression group was associated with early-stage and the high-expression group with more advanced-stage disease (P<0.05). The CRP protein level was associated with gender and cancer stage: The low-expression group was predominantly associated with male gender and early-stage disease, whereas the high-expression group was associated with female gender and advanced-stage disease (P<0.05). The expression of CK19, CK20, uPA and CRP, but not MMP-9, was negatively associated with overall survival (OS): The OS rate in the high-expression groups was significantly lower compared with that in the low-expression groups (P<0.05). In conclusion, the upregulation of CK20, uPA and CRP was found to be a negative prognostic factor for gastric cancer.

Serum Biomarkers of Inflammation, Fibrosis, and Cardiac Function in Facilitating Diagnosis, Prognosis, and Treatment of Anti-SSA/Ro–Associated Cardiac Neonatal Lupus

BackgroundCardiac manifestations of neonatal lupus (cardiac NL) include congenital heart block and cardiomyopathy. Several candidate biomarkers were evaluated in cases at risk for cardiac NL on the basis of potential roles in inflammation, fibrosis, and cardiac dysfunction: C-reactive protein (CRP); NT-pro-B-type natriuretic peptide (NT-proBNP); troponin I; matrix metalloproteinase (MMP)-2; urokinase plasminogen activator (uPA); urokinase plasminogen activator receptor (uPAR); plasminogen; and vitamin D. ObjectivesIdentification of maternal and fetal biomarkers associated with development and morbidity of cardiac NL should provide clues to pathogenesis with translational implications for management. MethodsCord (139) and maternal (135) blood samples collected during pregnancies at risk for cardiac NL were available for study. Levels of cord and maternal CRP, cord NT-proBNP, and cord troponin I were evaluated using multiplex assays. Cord and maternal vitamin D were assessed by liquid chromatography-mass spectrometry. MMP-2, uPA, uPAR, and plasminogen were evaluated using ELISA. ResultsCord CRP, NT-proBNP, MMP-2, uPA, uPAR, and plasminogen levels were higher in cardiac NL-affected fetuses than in unaffected cases, independent of maternal rheumatic disease, season at highest risk of cardiac NL development, and medications taken during pregnancy. These biomarkers were positively associated with a disease severity score derived from known risk factors for mortality in cardiac NL. Maternal CRP and cord troponin I levels did not differ between the groups. Cord and maternal vitamin D levels were not significantly associated with cardiac NL, but average maternal vitamin D level during pregnancy was positively associated with longer time to postnatal pacemaker placement. ConclusionsThese data support the association of fetal reactive inflammatory and fibrotic components with development and morbidity of cardiac NL. Following CRP and NT-proBNP levels after birth can potentially monitor severity and progression of cardiac NL. MMP-2 and the uPA/uPAR/plasminogen cascade provide therapeutic targets to decrease fibrosis. Although decreased vitamin D did not confer increased risk, given the positive influence on postnatal outcomes, maternal levels should be optimized.

Macrophages of M1 phenotype have properties that influence lung cancer cell progression.

Stromal macrophages of different phenotypes can contribute to the expression of proteins that affects metastasis such as urokinase-type plasminogen activator (uPA), its receptor uPAR, and plasminogen activator inhibitor-1 (PAI-1), but knowledge of how essential their contribution is in comparison to the cancer cells in small cell lung cancer (SCLC) and lung squamous cell carcinoma (SCC) is lacking. The expression of uPA, uPAR, and PAI-1 and of the matrix metalloproteinases (MMP)-2 and MMP-9 were studied in human macrophages of M1 and M2 phenotype and compared to a lung SCC (NCI-H520) and a SCLC (NCI-H69) cell line. Effects of treatment with conditioned media (CM) from M1 and M2 macrophages on the expression of these genes in H520 and H69 cells as well as effects on the cell growth were investigated. In addition, data on the stromal macrophages immunoreactivity of uPAR, MMP-2, and MMP-9 in a few SCC and SCLC biopsies was included. uPAR, MMP-2, and MMP-9 were confirmed in stromal cells including macrophages in the SCC and SCLC biopsies. In vitro, both macrophage phenotypes expressed considerably higher mRNA levels of uPA, uPAR, PAI-1, and MMP-9 compared to the cancer cell lines, and regarding uPAR, the highest level was found in the M1 macrophage phenotype. Furthermore, M1 CM treatment not only induced an upregulation of PAI-1 in both H520 and H69 cells but also inhibited cell growth in both cell lines, giving M1 macrophages both tumor-promoting and tumor-killing potential.

Potential clinical relevance of uPA and PAI-1 levels in node-negative, postmenopausal breast cancer patients bearing histological grade II tumors with ER/PR expression, during an early follow-up.

We evaluated urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) prognostic value in postmenopausal, node-negative breast cancer patients bearing tumors with estrogen receptor (ER)/progesterone receptor (PR) expression, treated with locoregional therapy alone, within an early follow-up. We focused our analysis on tumors of histological grade II in order to improve its prognostic value and, consequently, to improve a decision-making process. The cytosol extracts of 73 tumor samples were used for assessing several biomarkers. ER and PR levels were measured by classical biochemical method. Cathepsin D was assayed by a radiometric immunoassay while both uPA and PAI-1 level determinations were performed by enzyme-linked immunosorbent assays. HER-2 gene amplification was determined by chromogenic in situ hybridization (CISH) in primary tumor tissue. Patients bearing tumors smaller than or equal to 2 cm (pT1) or those with low PAI-1 levels (PAI-1 < 6.35 pg/mg) showed favorable outcome compared to patients bearing tumors greater than 2 cm (pT2,3) or those with high PAI-1 levels, respectively. Analyses of 4 phenotypes, defined by tumor size and PAI-1 status, revealed that patients bearing either pT1 tumors, irrespective of PAI-1 levels, or pT2,3 tumors with low PAI-1 levels, had similar disease-free interval probabilities and showed favorable outcome compared to those bearing pT2,3 tumors with high PAI-1 levels. Our findings suggest that tumor size and PAI-1, used in combination as phenotypes are not only prognostic but might also be predictive in node-negative, postmenopausal breast cancer patients bearing histological grade II tumors with ER/PR expression, during an early follow-up period.

Influence of immunohistological detection of intratumoral urokinase-type plasminogen activator (uPA) on disease outcome in endocrine-treated postmenopausal patients with hormone receptor-positive early breast cancer.

551 Background: Elevated intratumoral levels of uPA and PAI-1 in ELISA-based measurements are associated with a high recurrence risk and allow to select patients who might particularly benefit from...

Modulation of uPA, MMPs and their inhibitors by a novel nutrient mixture in human colorectal, pancreatic and hepatic carcinoma cell lines.

Colorectal, pancreatic and hepatic carcinomas are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretions, allowing cancer cells to spread to distal organs. These and other proteases, such as uPA, play a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. Strong clinical and experimental evidence demonstrates association of elevated levels of uPA and MMPs with cancer progression, metastasis and shortened patient survival. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPs). Our main objective was to study the effect of a nutrient mixture (NM) on activity of uPA, MMPs and TIMPs in colon HCT-116, pancreatic MIA PaCa-2 and hepatic carcinoma SK-Hep-1 cell lines. These cell lines (ATCC) were cultured in their respective media and treated at confluence with NM at 0, 50, 100, 250, 500 and 1000µg/ml. Analysis of uPA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. All three cancer cell lines expressed uPA, which was inhibited by NM in a dose-dependent manner. On gelatinase zymography, SK-Hep-1 showed bands corresponding to MMP-2 and MMP-9 with enhancement of MMP-9 with PMA (100 ng/ml) treatment. HCT-116 and MIA PaCa-2 showed strong bands corresponding to MMP-9 but no MMP-2 band. NM inhibited their expression in a dose-dependent manner. Activity of TIMPs was upregulated by NM in all cancer cell lines in a dose-dependent manner. Analysis revealed a positive correlation between uPA and MMP-9 and a negative correlation between uPA/MMP-9 and TIMP-2. These findings suggest the therapeutic potential of NM in treatment of colon, pancreatic and hepatic carcinomas.

FOXM1-mediated downregulation of uPA and MMP9 by 3,3'-diindolylmethane inhibits migration and invasion of human colorectal cancer cells.

Although 3,3'-diindolylmethane (DIM) has been suggested to reduce the risk of colorectal cancer, the underlying biological mechanism is not clearly understood. In the present study, we investigated the effect of DIM on the migratory and invasive activities of the human colorectal cancer cell lines DLD-1 and HCT116. DIM significantly inhibited the migration and invasion of colorectal cancer cells as assessed by wound healing and Matrigel invasion assays. The migratory ability of the DLD-1 and HCT116 cells was significantly reduced by DIM at 24 and 48 h. DIM also significantly inhibited the invasion rate of the DLD-1 and HCT116 cells in a dose-dependent manner. The mRNA expression levels of urokinase type plasminogen activator (uPA) and matrix metalloprotease 9 (MMP9) were significantly attenuated, whereas expression of E-cadherin mRNA was significantly enhanced, following DIM treatment. DIM also decreased the protein levels of uPA and MMP9, yet significantly increased E-cadherin protein expression. In addition, DIM significantly reduced the mRNA and protein levels of FOXM1 in the DLD-1 and HCT116 cells. Our results suggest that DIM can influence the cell migratory and invasive properties of human colorectal cancer cells and may decrease the invasive capacity of colorectal cancer through downregulation of uPA and MMP9 mediated by suppression of the transcription factor FOXM1.