uPAR mRNA Levels Research Articles

Abstract 419: Liberation Of Soluble Urokinase Plasminogen Receptor (supar) Occurs In Response To Coronavirus Infection But Not To Influenza Virus Infection

Objectives: Urokinase (uPA), and its receptor, uPAR, mediate fibrinolysis and matrix remodeling. Liberation of monocyte/macrophage (MO/Mϕ) bound uPAR to yield soluble uPAR (suPAR) occurs after infection with coronaviruses (CoVs) such as SARS-CoV-2, and suPAR levels correlate with systemic inflammation and degree of end organ injury. How suPAR liberation from MO/Mϕs occurs after CoV infection, and whether suPAR induction occurs after pneumotropic viral infections such as influenza A (IAV) is unclear. Methods: Murine bone marrow derived Mϕs (BMDMs) and immortalized Mϕs (RAW264.7) were infected with CoV [MHVA59 (murine model for SARS-CoV-2 infection) or MHV1] or with IAV (A/PR/8/34) and levels of mRNA and protein were measured using qRT-PCR and ELISA, respectively. uPA activity of cell supernatants was quantitated using a commercially available assay. uPA knockdown of Mϕs was performed using siRNA transfection. C57BL6/J mice underwent intranasal inoculation of either 2x10 5 pfu of MHVA59, 25 pfu of IAV, or with PBS (sham). Plasma levels of suPAR and splenic Mϕ (surrogate for circulating MOs) levels of mRNA and protein were analyzed at post-infection day 3. Results: Though both CoV or IAV infection resulted in induction of mRNA and protein levels of cellular uPAR and uPA, and uPA activity, only CoV infection yielded liberation of suPAR from Mϕs (Fig. 1A-C). Indicating a functional role in suPAR liberation, infection of uPA-silenced Mϕs yielded a blunted induction of suPAR release despite not affecting mRNA levels of uPAR (Fig 1D). Finally, inoculation of mice with MHVA59, but not with IAV, resulted in elevated plasma levels of suPAR, despite inducing a robust induction of uPAR in splenic Mϕs (Fig. 1E). Conclusions: suPAR induction results after CoV infection, but not IAV infection, and occurs through a uPA-dependent pathway. Antagonism of co-regulators of uPAR and uPA may prove to be a therapeutic strategy to combat post-CoV tissue injury mediated by Mϕs.

In vitro and in vivo inhibition of proangiogenic retinal phenotype by an antisense oligonucleotide downregulating uPAR expression

Neoangiogenesis is the main pathogenic event involved in a variety of retinal diseases. It has been recently demonstrated that inhibiting the urokinase-type plasminogen activator receptor (uPAR) results in reduced angiogenesis in a mouse model of oxygen-induced retinopathy (OIR), establishing uPAR as a therapeutic target in proliferative retinopathies. Here, we evaluated in cultured human retinal endothelial cells (HRECs) and in OIR mice the potential of a specific antisense oligodeoxyribonucleotide (ASO) in blocking the synthesis of uPAR and in providing antiangiogenic effects.uPAR expression in HRECs was inhibited by lipofection with the phosphorotioated 5′-CGGCGGGTGACCCATGTG-3′ ASO-uPAR, complementary to the initial translation site of uPAR mRNA. Inhibition of uPAR expression via ASO-uPAR was evaluated in HRECs by analyzing VEGF-induced tube formation and migration. In addition, the well-established and reproducible murine OIR model was used to induce retinal neovascularization in vivo. OIR mice were injected intraperitoneally with ASO-uPAR and retinopathy was evaluated considering the extent of the avascular area in the central retina and neovascular tuft formation.The ASO-uPAR specifically decreased uPAR mRNA and protein levels in HRECs and mitigated VEGF-induced tube formation and cell migration. Noteworthy, in OIR mice ASO-uPAR administration reduced both the avascular area and the formation of neovascular tufts.In conclusion, although the extrapolation of these experimental findings to the clinic is not straightforward, ASO-uPAR may be considered a potential therapeutic tool for treatment of proliferative retinal diseases.

Molecular mechanisms of anticancer activity of deoxyelephantopin in cancer cells

BackgroundDeoxyelephantopin (DOE) is a natural bioactive sesquiterpene lactone from Elephantopus scaber, a traditionally relevant herb in Chinese and Indian medicine. It has shown promising anticancer effects against a broad spectrum of cancers. MethodsWe examined the effect of DOE on growth, autophagy, apoptosis, cell cycle progression, metastasis, and various molecular signaling pathways in cancer cells, and endeavored to decipher the molecular mechanisms underlying its effect. The cytotoxicity of DOE was examined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and colony formation assays. The antimetastatic potential of DOE was identified by wound closure, as well as invasion and migration assays. The expression of mRNAs and proteins related to cytotoxicity in cancer cells induced by DOE was investigated using reverse transcription-polymerase chain reaction, flow cytometry, and Western blot analysis. ResultsDOE showed significant cytotoxicity and induced apoptosis in cancer cells. DOE promoted the autophagy of HCT 116 and K562 cells. DOE arrested cell cycle progression in the G2/M phase. DOE treatment caused activation of caspase-8, -9, -3 and -7, reactive oxygen species production, and cleavage of cleavage of poly-ADP-ribose polymerase (PARP), the markers of apoptosis. Moreover, apoptosis induction was associated with mitochondrial permeability and endoplasmic reticulum stress. Treatment of cancer cells with DOE inhibited mitogen-activated protein kinases, nuclear factor-kappa B, phosphatidylinositol 3-kinase (PI3K/Akt), and β-catenin signaling. Furthermore, treatment of DOE increased the expression of p53, phospho-Jun amino-terminal kinases (p-JNK), and p-p38 and decreased the expression of phospho-signal transducer and activator of transcription 3 (p-STAT3) and phospho-mammalian target of rapamycin (p-mTOR) in cancer cells. DOE downregulated matrix metalloproteinase (MMP-2) and MMP-9, urokinase-type plasminogen activator (uPA), and urokinase-type plasminogen activator receptor (uPAR) mRNA levels in cancer cells. ConclusionThese findings concluded that DOE may be useful as a chemotherapeutic agent against cancer.

Hypoxia inducible factor-1 mediates upregulation of urokinase-type plasminogen activator receptor gene transcription during hypoxia in cervical cancer cells.

Hypoxia occurs during development of cervical cancer and is considered to correlate with its invasion. Hypoxia mediates tumor cells to have more invasive property in a variety of cancers. Urokinase plasminogen activator receptor (uPAR) which mediates invasion is considered to be induced by hypoxia. We sought to determine the regulators of uPAR expression during hypoxia in cervical cancer. We showed that cervical cancer cell lines, CaSki and CA, were more invasive under hypoxic condition (1% O2) than under normoxic condition (20% O2) by invasion assays. Using western blot analysis, hypoxia enhanced the endogenous hypoxia-inducible factor (HIF)-1α and uPAR protein expression. uPAR mRNA level was also upregulated by hypoxia using real-time RT-PCR. Overexpression of HIF-1α which is induced by hypoxia activated the transcriptional activity of the uPAR promoter by luciferase assays. HIF-1 protein bound the putative HIF-1 response element on the uPAR promoter using electrophoretic mobility shift analysis, and additional luciferase assays show that this is essential for uPAR transactivation by HIF-1. HIF-1 overexpression enhanced the endogenous uPAR expression and introduction of siRNA for HIF-1α diminishes uPAR expression during hypoxia. These results indicate the upregulation of uPAR by hypoxia in cervical cancer cells is mediated through HIF-1. In cervical cancer tissues, we also demonstrated that uPAR protein expression was detected in cervical cancer but not in normal cervix or cervical intraepithelial neoplasia (CIN) by immunohistopathological staining. Our results provide evidence that regulation of uPAR expression by HIF-1 represents a mechanism for cervical cancer invasion during hypoxia.

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.

Anti-metastatic effect of Smilax china L. extract on MDA-MB-231 cells.

Cancer metastases are not always cured by chemotherapy. Conventional and alternative drugs, including Chinese herbal remedies, have been developed to target metastatic cancer cells. Smilax china L. (SCL), a member of the Smilacaceae family, exerts anti-inflammatory, detoxification and anti-cancer effects. However, the effect of SCL on breast cancer cell metastasis and the underlying mechanisms are yet to be elucidated. The aim of this study was to investigate the effect of a SCL ethanol extract (SCLE) on the proliferation and migration of MDA-MB-231 human breast cancer cells, as well as the expression of urokinase plasminogen activator (uPA), uPA receptor (uPAR) and tissue inhibitors of metalloproteinases (TIMPs). Cell proliferation was assessed using the Cell Counting Kit‑8 and cell migration was determined by wound healing assay. Quantitative polymerase chain reaction was performed to quantify the mRNA levels of uPA, uPAR and TIMPs. SCLE markedly inhibited the proliferation and migration of MDA-MB-231 cells, and reduced the mRNA levels of the extracellular matrix (ECM) degradation-associated molecules uPA, uPAR. By contrast, SCLE significantly increased the mRNA levels of TIMP1 and TIMP2. These findings show that SCLE exerts an anti-metastatic effect on human breast cancer cells, which may involve the modulation of ECM degradation.

Synergy of urokinase‑type plasminogen activator receptor isomer (D1D2) and integrin α5β1 causes malignant transformation of hepatic cells and the occurrence of liver cancer.

The aim of the present study was to investigate the correlations and possible synergy among the urokinase‑type plasminogen activator receptor (uPAR) isomer D1D2 and integrinα5β1 expression levels, malignant transformation in hepatic cells and the occurrence of liver cancer. The expression site and concentration of uPAR (D1D2) were analyzed using polymerase chain reaction and insitu hybridization at the gene level in 60samples of hepatocellular carcinoma (HCC) tissues, 60samples of para‑carcinoma tissues and 25samples of normal liver tissues. The mRNA levels of uPAR (D1D2) and integrinα5β1 were markedly increased para‑carcinoma tissue and liver cancer tissue as compared with those in normal tissue. The grey values of the three groups were significantly different (P<0.05). Insitu hybridization revealed that the expression levels of uPAR (D1D2) and integrinα5β1 in the cytoplasm and the positive rate of the two molecules in the HCC tissue were significantly higher than those in the para-carcinoma and normal liver tissues, and the expression levels were positively correlated (rs1=0.257, P<0.05; rs2=0.261, P<0.05). The results suggested that uPAR (D1D2) mRNA overexpression may be due to changes in the conformation of the uPAR isomer. Synergy of uPAR (D1D2) mRNA and integrinα5β1 interaction may result in abnormal signal transduction in liver cells and ultimately liver cell abnormal clonal hyperplasia and malignant transformation.

Downregulation of galectin-3 causes a decrease in uPAR levels and inhibits the proliferation, migration and invasion of hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality worldwide. Galectin-3 (Gal-3), a multifunctional β-galactoside-binding protein, is highly expressed and associated with the prognosis of HCC. However, the functions of Gal-3 in HCC cells are not fully understood. To address the function of Gal-3 in HCC cells, we used small interfering RNA (siRNA) to knock down Gal-3 expression in HepG2, an HCC cell line. We found that in vitro the silencing of Gal-3 decreased the proliferative activity, colony formation ability, migratory and invasive potential of HepG2 cells. The silencing of Gal-3 significantly decreased the mRNA and protein levels of urokinase-type plasminogen activator receptor (uPAR) as well as uPAR's downstream signaling transduction pathway, including phosphorylation of AKT. Furthermore, the downregulation of Gal-3 by siRNA resulted in significantly decreased activity of the MEK/ERK signaling pathway, and the treatment of HepG2 cells with MEK/ERK inhibitor U0126 significantly reduced the mRNA and protein levels of uPAR. Taken together, our results suggest that Gal-3 modulates uPAR expression via the MEK/ERK pathway, and that Gal-3 may be a potential therapeutic target for the treatment of HCC.

Correlation between the overexpression of urokinase receptor isoform uPAR (D1D2) and hepatic cell malignant transformation

In order to provide an abundant source of specimen and to reveal the correlation between the overexpression of urokinase plasminogen activator receptor isoform uPAR (D1D2) and hepatic cell malignant transformation, the optimal liver cell culture method was selected from three cell culture methods to culture and separate out liver cells with a high density, high purity and high activity. The specimens were used to culture and assess the uPAR (D1D2) mRNA level in normal liver cells, liver cancer cells and para-carcinoma cells. In the present study, the correlation between the overexpression of uPAR (D1D2) and hepatic cell malignant transformation was discussed. When comparing the tissue block adherent method, liver cell grinding method and pancreatic enzyme digestion method, the liver tissue adherent method was found to be economical, simple and overall the optimal method for liver cell culture. This was used as a reference standard for cell culture. RT-PCR was used to determine isoform uPAR (D1D2) mRNA level in normal liver cells, para-carcinoma cells and liver cancer cells. The comparison of uPAR (D1D2) mRNA levels in normal liver cells, para-carcinoma cells and liver cancer cells, demonstrated that the brightness of the cells clearly increased in normal liver cells, para-carcinoma cells and liver cancer cells. The comparison of the cell grey values of three groups demonstrated a statistically significant difference (P<0.05). The liver tissue adherent method was able to produce liver cells with a high density, high purity and high activity, providing a sufficient source of specimen for our subsequent experiments. The electrophoresis results showed that: uPAR (D1D2) mRNA expression increased from normal liver cells to para-carcinoma cells to liver cancer cells, inferring that uPAR (D1D2) mRNA overexpression may be the result of changes in the conformation of the uPAR isoform. In addition, it is closely associated with abnormal cell signal transduction, which leads to clonal proliferation and abnormal differentiation of liver cells with malignant transformation in liver cells.

A retrospective study of urokinase-type plasminogen activator receptor (uPAR) as a prognostic factor in cancer of the uterine cervix

Hypoxia occurs during the development of uterine cervical cancer and is considered to correlate with its invasion. Hypoxia promotes both the invasiveness and the metastasis of cancer cells through urokinase-type plasminogen activator receptor (uPAR) expression. The aim of this study was to evaluate the correlation between uPAR mRNA level and clinical prognostic factors of uterine cervical cancer. We performed a retrospective review of 59 patients with cervical cancer and 9 subjects with normal cervical tissues. Total RNA was isolated from tissues of the uterine cervix surgically removed from patients. The mRNA of uPAR could be measured by real time PCR (RT-PCR). Histopathological factors such as histopathological type, cervical stromal, parametrial, lymphovascular, and uterine corpus invasions, metastasis to the pelvic lymph nodes, and pTNM stage were confirmed by two pathologists. The examined prognostic factors alongside the histopathological ones were FIGO clinical stage, hemoglobin level, serum level of SCC, and the effect on clinical outcomes. These factors were statistically evaluated by Fisher's exact test, log-rank test, and ROC analysis. Immunohistochemical staining with anti-uPAR monoclonal antibody was also performed. In uterine cervical cancer, overexpression of uPAR mRNA was significantly related to shorter disease-free survival (p = 0.0396). However, the other clinical prognostic and histopathological factors were not related to uPAR mRNA expression level. Immunohistochemical staining showed that positive staining for uPAR was histologically localized on the membrane of carcinoma cells. However, the staining was not very intense. Overexpression of uPAR mRNA may be a prognostic factor in cancer of the uterine cervix.

Lysophosphatidic Acid Regulates uPAR Expression and Localization

Lysophosphatidic acid (LPA) stimulates in vitro invasion of ovarian epithelial cancer cells through a uPA‐dependent process. We therefore analyzed the effects of LPA on the cell surface uPA receptor (uPAR). While both uPAR mRNA and protein expression was consistent with control after 2 hours of LPA stimulation, LPA strongly stimulated uPAR redistribution into punctate aggregates or to cell junctions, depending on cell line. After 24 hours of stimulation, uPAR mRNA levels increased 4.0±2.3 fold in OVCA 429 cells and were modestly impacted in OVCA 433 cells (1.8±0.5 fold). In accordance, cell surface‐localized uPA activity increased 4.3±2.5 fold (OVCA 429) or 2.1±0.9 fold (OVCA 433) and was accompanied by increased uPAR immunofluorescence staining. At this same time, mRNA levels of uPA and PAI‐1 were not significantly changed. These data provide mechanistic insight into the actions of a lipid that positively correlates with ovarian cancer progression.

Mycophenolate mofetil alleviates lupus nephritis through urokinase receptor signaling in a mice model

Lupus nephritis (LN) is usually associated with widespread effacement of the podocytes' foot processes leading to proteinuria. Induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via promoting podocytes' motility and kidney permeability in the glomerulus. Very little is known about uPAR signaling in LN. Mycophenolate mofetil (MMF), an immunosuppressive agent, efficiently modulates the development of LN in humans and mice, but there are no data concerning the direct uPAR involvement on podocytes in LN. The MMF efficiency and uPAR involvement signaling in NZB×NZW F1 lupus-prone mice were examined by proteinuria, renal function and pathology, immune complex deposits, and uPAR expression of podocytes by immunofluorescence staining and quantitative RT-PCR. After MMF treatment, the proteinuria (p < 0.01), BUN level (p < 0.05) and immunodeposition in glomeruli (p < 0.001) were significantly improved. Most important, the renal uPAR mRNA levels (p < 0.001) and uPAR protein level of podocytes (p < 0.001) were significantly reduced. The beneficial effect of MMF on LN could be attributed, at least in part, to the inhibition of uPAR expression in podocytes. These findings demonstrated uPAR could have potential as a predictive index for response to LN therapeutics.

In papillary thyroid carcinoma BRAFV600E is associated with increased expression of the urokinase plasminogen activator and its cognate receptor, but not with disease‐free interval

It has been suggested that patients with papillary thyroid cancer (PTC) harbouring the BRAF(V600E) mutation have a worse prognosis. We showed in PTC that high levels of urokinase plasminogen activator (uPA) and its cognate receptor (uPAR) inversely correlate with disease-free interval (DFI). To investigate the effects of BRAF(V600E) on the expression of uPA and uPAR and to evaluate the prognostic relevance of BRAF(V600E) alone or in combination with uPA and uPAR. DESIGN/SETTING/PATIENTS/INTERVENTION: The case study included 91 patients with PTC. All patients underwent thyroidectomy and radioiodine therapy. Follow-up was available for 75 patients. The BRAF(V600E) mutation was analysed by sequencing and mutant allele-specific PCR amplification; uPA and uPAR expression by quantitative RT-PCR. BRAF(V600E) was found in 44 of the 91 patients and associated with older age, but not with high-risk clinicopathological features. Urokinase PA and uPAR mRNA levels were higher in tumour tissues by 9·51±1·30 and 4·64±0·44 fold, respectively, compared to normal matched tissues, being significantly higher in BRAF(V600E) -positive patients. In vitro induction of BRAF(V600E) in PCCL3 cells caused a significant increase in both uPA and uPAR mRNAs. Higher levels of uPA and uPAR correlated with lymph node metastases, TNM stage and disease recurrences. Kaplan-Meier and multivariate analyses demonstrated that uPA and uPAR were associated with shorter DFI, while the BRAF(V600E) was not. In PTC, BRAF(V600E) induces uPA and uPAR expression. The latter, but not BRAF(V600E) , associates with advanced stages and shorter DFI. If confirmed in larger case studies, they may represent reliable prognostic markers for more accurate risk stratification and postoperative decision-making in patients with PTC.

Loss of Med1/TRAP220 promotes the invasion and metastasis of human non-small-cell lung cancer cells by modulating the expression of metastasis-related genes

Med1/TRAP220 is an essential component of the TRAP/Mediator complex. In this study, we present a novel function of Med1 in human non-small-cell lung cancer (NSCLC) progression. We found that the loss of Med1 expression was strongly associated with increased rates of invasion and metastasis in NSCLC patients. Consistent with lung cancer patient data, the knockdown of Med1 in NSCLC cell lines led to an increase in cell migration and invasion. Med1-depleted cells displayed an increase in metastasis in a xenograft tumor model and in an in vivo metastasis assay. Moreover, a microarray analysis revealed that the mRNA levels of the metastasis-related genes uPAR, ID2, ID4, PTP4A1, PKP3, TGM2, PLD1, TIMP2, RGS2, and HOXA4 were altered upon Med1 knockdown. Collectively, these results suggest that the loss of Med1 increases the invasive potential of human NSCLC cells by modulating the expression of metastasis-related genes.

Human hepatocellular carcinomas with “Stemness”-related marker expression: keratin 19 expression and a poor prognosis

There is a recently proposed subtype of hepatocellular carcinoma (HCC) that is histologically similar to usual HCC, but characterized by the expression of "stemness"-related markers. A large-scale study on two different cohorts of HCCs was performed to investigate the clinicopathologic features and epithelial-mesenchymal transition (EMT)-related protein expression status of this subtype of HCCs. The expression status of stemness-related (e.g., keratin 19 [K19], cluster of differentiation [CD]133, epithelial cell adhesion molecule [EpCAM], and c-kit) and EMT-related markers (e.g., snail, S100A4, urokinase plasminogen activator receptor [uPAR], ezrin, vimentin, E-cadherin, and matrix metalloproteinase [MMP]2) were examined using tissue microarrays from cohort 1 HCCs (n = 137). K19 protein expression in cohort 2 HCCs (n = 237) was correlated with the clinicopathologic parameters and messenger RNA (mRNA) levels of K19, uPAR, VIL2, Snail, Slug, and Twist. K19, EpCAM, c-kit, and CD133 positivity were observed in 18.2%, 35.0%, 34.3%, and 24.8%, respectively. K19 was most frequently expressed in combination with at least one other stemness-related marker (92.0%). K19-positive HCCs demonstrated more frequent major vessel invasion and increased tumor size, compared to K19-negative HCCs (P < 0.05). K19 was most significantly associated with EMT-related protein expression (e.g., vimentin, S100A4, uPAR, and ezrin) (P < 0.05) and a poor prognosis (overall survival: P = 0.018; disease-free survival: P = 0.007) in cohort 1. In cohort 2, HCCs with high K19 mRNA levels demonstrated higher mRNA levels of Snail, uPAR, and MMP2 (P < 0.05). K19-positive HCCs demonstrated more frequent microvascular invasion, fibrous stroma, and less tumor-capsule formation, compared to K19-negative HCCs (P < 0.05). K19 expression was a significant independent predictive factor of poor disease-free survival (P = 0.032). K19 was well correlated with clinicopathologic features of tumor aggressiveness, compared to other stemness-related proteins. K19-positive HCCs showed significantly increased EMT-related protein and mRNA expression, suggesting that they may acquire more invasive characteristics, compared to K19-negative HCCs through the up-regulation of EMT-associated genes.

Impairment of breast cancer cell invasion by COX-2-specific inhibitor NS398: roles of CXCR4 and of uPA system

Inhibition of cyclooxygenase-2 (COX-2) is known to impair cancer cell metastatic behaviour, but the mechanisms involved largely remain elusive. We aimed to analyse whether the antimetastatic effect of COX-2 inhibition in breast cancer cells could be explained by variations in the expression levels of chemokine receptor CXCR4, vascular endothelium growth factor (VEGF) and UPA/UPAR components of the urokinase plasminogen activator system (uPAR). Breast cancer cell line MDA-MB-231 was exposed to COX-2-specific inhibitor NS398. Experimental data were assessed using Matrigel invasion tests, qRT-PCR, ELISA, flow cytometry and MTT test. Exposure to NS398 had no major effect on cell viability, apoptosis or VEGF production. Cell invasion was significantly decreased with reductions ranging from of 3.6% with 10 μM NS398 to 81.04% with 100 μM NS398. CXCR4 membrane expression was significantly reduced by 18% (P < 0.05) when cells were treated with 100 μM of NS398 for 72 h. UPA mRNA levels were significantly reduced to 78 and 63% after treatment with 10 μM NS398 for 48 and 72 h, respectively (P < 0.05). UPAR mRNA levels also decreased with mild NS398 concentrations, reaching the lowest level of 56% with 50 μM of NS398 for 48 h (P < 0.05). With NS398 higher concentrations, UPAR and UPA expression levels increased. According to our results, impairment of expression of CXCR4, UPA and UPAR differentially contribute to the antimetastatic effect of COX-2 inhibitors depending on drug concentration.

High Expression of the Urokinase Plasminogen Activator and Its Cognate Receptor Associates with Advanced Stages and Reduced Disease-Free Interval in Papillary Thyroid Carcinoma

The urokinase plasminogen activating system is implicated in neoplastic progression, and high tissue levels of urokinase plasminogen activating system components correlate with poor prognosis in various human cancers. The objective of the study was to investigate the prognostic relevance of the urokinase plasminogen activator (uPA), its cognate receptor (uPAR), and the plasminogen activator inhibitor 1 (PAI-1) in human papillary thyroid cancer (PTC). The expression of uPA, uPAR, and PAI-1 genes was analyzed in PTC and normal matched tissues by quantitative RT-PCR. The case study consisted of 99 patients (21 males and 78 females) affected by PTC including 77 classical, 15 follicular, four tall cell, and three oncocytic variants. Forty-one patients had lymph node metastases at the time of diagnosis. All the patients underwent thyroidectomy and radioiodine therapy followed by thyroid hormone replacement therapy. Follow-up data were available for 76 patients up to 64 months. The uPA, uPAR, and PAI-1 mRNA levels were significantly higher in PTC compared with normal matched tissues by 9.63 ± 1,29-, 4.82 ± 0.45-, and 5.64 ± 0.71-fold, respectively. The increased expression of uPA and uPAR correlated statistically with advanced pT and N status. The uPA was also significantly associated with advanced tumor node metastasis stages. The Kaplan-Meier analysis showed a significant association of uPA and uPAR levels with reduced patient disease-free interval (DFI), and this association was stronger in stage I patients. The study demonstrated that in PTC the increased gene expression of uPA and uPAR is associated with tumor invasiveness, advanced stages, and shorter DFI, suggesting their prognostic relevance. These observations warrant further investigation in larger patient populations with longer follow-up.

The Plasminogen System in Microdissected Colonic Mucosa Distant from an Isolated Adenoma

In the colon, the urokinase-type plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitors, PAI-1 and PAI-2, are implicated in the transition from mucosa to adenoma and tumour progression. However, expression in the mucosa adjacent, or distant, to an adenoma has not yet been investigated. Three biopsies from mucosae adjacent (20 cm, ipsilateral) and distant (contralateral) to an isolated tubular adenoma were analysed in 14 patients and 8 controls. Laser microdissection isolated stromal and epithelial crypt components, and quantitative RT-PCR analyses of uPA, uPAR, PAI-1 and PAI-2 mRNA levels were performed. Among controls, no significant differences in the markers were noted. With left colon isolated tubular adenoma, uPA, uPAR, and PAI-2 mRNA levels were significantly increased in the adjacent mucosal stroma compared to epithelial crypt levels (p < 0.05). In right colon adenoma, the mRNA levels of these 3 molecular markers were significantly increased only in the adjacent mucosal stromal samples (p < 0.05). Isolated tubular adenoma in the colon increases significantly the mRNA levels of 3 proteolysis-associated molecular markers in the stromal, but not in the epithelial, components of adjacent mucosa. These results suggest the presence of regional and dynamic interactions in apparently non-involved mucosae.

Abstract 831: uPAR Expression is Up-regulated in Cervical Cancer

Abstract Hypoxia occurs during development of cervical cancer and is considered to correlate with its invasion. Hypoxia induces cancer cells to have more invasive property through the urokinase plasminogen activator receptor (uPAR) expression. Also, poor prognosis of uterine cervical cancer is correlated with hypoxic condition. Thus, we examined the relationship among the uPAR mRNA level and clinical prognostic factors, such as clinical and pathological stage, hemoglobin level, histologic type, tumor marker level and the effect on clinical outcome, using 66 cases of cervical cancer and 10 normal cervical tissues. These factors were evaluated by t test and Spearman's rank correlation coefficient. Survival analysis was performed using Kaplan-Meier curves. The uPAR mRNA level is significantly up-regulated in cervical cancers, compared with that in normal cervixes. The level of hemoglobin was significantly correlated with uPAR mRNA expression. Whereas, no other clinical prognostic factor had correlation with uPAR mRNA expression. Also, the uPAR mRNA level was not correlated with survival. According to the invasion assays, cervical cancer cell lines, CA and CaSki, under hypoxic condition (1% O2) showed more invasive property than those under normoxia. Hypoxia enhanced the endogenous uPAR mRNA level and protein expression by realtime RT-PCR and western blot analysis, respectively. Although we hypothesized that uPAR expression was correlated with tumor invasion and poor prognosis, the results revealed that uPAR mRNA expression was not correlated with survival nor most clinical factors except for the hemoglobin level. Although uPAR seems to be critical for the invasiveness in cervical cancer cell lines, uPAR may not be involved in cervical cancer invasion in vivo. Only real hypoxia may regulate the uPAR mRNA expression in cervical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 831.

Tissue plasminogen activator and urokinase plasminogen activator in human epileptogenic pathologies

A growing body of evidence demonstrates the involvement of plasminogen activators (PAs) in a number of physiologic and pathologic events in the CNS. Induction of both tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) has been observed in different experimental models of epilepsy and tPA has been implicated in the mechanisms underlying seizure activity. We investigated the expression and the cellular distribution of tPA and uPA in several epileptogenic pathologies, including hippocampal sclerosis (HS; n=6), and developmental glioneuronal lesions, such as focal cortical dysplasia (FCD, n=6), cortical tubers in patients with the tuberous sclerosis complex (TSC; n=6) and in gangliogliomas (GG; n=6), using immuno-cytochemical, western blot and real-time quantitative PCR analysis. TPA and uPA immunostaining showed increased expression within the epileptogenic lesions compared to control specimens in both glial and neuronal cells (hippocampal neurons in HS and dysplastic neurons in FCD, TSC and GG specimens). Confocal laser scanning microscopy confirmed expression of both proteins in astrocytes and microglia, as well as in microvascular endothelium. Immunoblot demonstrated also up-regulation of the uPA receptor (uPAR; P<0.05). Increased expression of tPA, uPA, uPAR and tissue PA inhibitor type mRNA levels was also detected by PCR analysis in different epileptogenic pathologies ( P<0.05). Our data support the role of PA system components in different human focal epileptogenic pathologies, which may critically influence neuronal activity, inflammatory response, as well as contributing to the complex remodeling of the neuronal networks occurring in epileptogenic lesions.