Expression Of Urokinase Plasminogen Activator Research Articles

TLR5 activation induces expression of the pro-inflammatory mediator Urokinase Plasminogen Activator via NF-κB and MAPK signalling pathways in human dental pulp cells.

To explore the involvement of TLR5 in pulp inflammation and to examine the effects of TLR5 activation with its ligand, FlaB protein, on pro-inflammatory gene expression. TLR5 expression in dental pulp tissues and human dental pulp cells (hDPCs) were determined by immunohistochemistry, immunocytochemistry, Western blots and RT-PCR analyses. To examine the role of TLR5, hDPCs were treated with recombinant FlaB protein (500ngmL-1 ) to activate the receptor or with a small interfering RNA against TLR5 (si-TLR5) to downregulate the receptor. After exposure to FlaB, the expression of inflammation-related proteins was screened using a protein array kit. Western blots or qRT-PCR analyses were performed to identify changes in the expression of uPA (urokinase plasminogen activator), TIMPs (tissue inhibitor of metalloproteinases), and IL-6 and to determine their signalling pathways. Statistical analysis was performed using one-way analysis of variance (anova) with Tukey post hoc test; P<0.05 was considered statistically significant. TLR5 expression was identified in pulp tissues and hDPCs. Inthe protein array analysis, treatment with FlaB significantly increased uPA expression (P<0.01) andsignificantly decreased TIMP1/4 (P<0.05). FlaB treatment also significantlyincreased expression of the inflammatory marker IL-6 (P<0.01). FlaB treatment increased phosphorylation of the NF-κB p65 subunit, JNK, p38 and ERK. Chemical inhibitors of NF-κB (Bay11-7082), p38 (SB202190) or ERK (U0126) decreased the FlaB induction of uPA expression. Downregulation of TLR5 expression by siRNA decreased the FlaB induction of uPA protein and p65 phosphorylation. TLR5 activation with FlaB treatment induced the expression of uPA via the NF-κB and MAPK signalling pathways. Flagellin-bearing oral bacteria may cause pulp inflammation through TLR5. The findings provide new clues to control pulpal diseases by targeting TLR5 signalling pathways.

Near-Infrared Light Activated Thermosensitive Ion Channel to Remotely Control Transgene System for Thrombolysis Therapy.

Current antithrombotic therapeutic strategies often suffer from severe post-thrombotic syndromes (PTS), inconvenient daily subcutaneous injections for a long time and short circulation times accompanied by a dose-dependent risk of intracranial hemorrhage. Aiming at noninvasive, on-demand, and sustained antithrombotic therapy, a new thrombolysis approach based on the transgene system has been developed to remotely and precisely control the expression of urokinase plasminogen activator (uPA) by bioengineered cells for antithrombotic therapy both in vitro and in vivo. In this design, the near-infrared (NIR) light could activate the expression of the thermosensitive TRPV1 channel in response to photothermal responsive nanotransducers to trigger the synthetic signaling pathway to secret uPA. By encapsulating bioengineered cells in injectable hydrogel to ensure long-term survival and convenience for injection, the engineered cells could noninvasively and precisely control the production of uPA protein in situ via an NIR laser to significantly enhance the thrombolysis therapeutic effects by spatiotemporally controlling the local temperature, in both the microfluidic blood circulation mimic and the murine tail thrombus model. This novel thrombolysis approach could overcome some key limitations that are associated with conventional antithrombotic therapy, thus opening a new direction for developing remotely and precisely controllable continuous thrombolysis through artificially designed signaling.

Investigations on the Role of the Fibrinolytic Pathway on Outflow Facility Regulation.

PurposeTo understand the role and further dissect pathways downstream of tissue plasminogen activator (tPA) and the fibrinolytic pathway in modulating outflow facility.MethodsOutflow facility of tissue plasminogen activator (Plat) knockout (KO) mice was determined and compared to that of wild-type (WT) littermates. Gene expression of urokinase plasminogen activator (Plau), plasminogen activator inhibitor (Pai-1), plasminogen (Plg), and matrix metalloproteinases (Mmp-2, -9, and -13) was measured in angle tissues. Expression of the same genes and outflow facility were measured in KO and WT mice treated with triamcinolone acetonide (TA). Amiloride was used to inhibit urokinase plasminogen activator (uPA) in Plat KO mice, and outflow facility was measured.ResultsPlat deletion resulted in outflow facility reduction and decreased Mmp-9 expression in angle tissues. Plasminogen expression was undetectable in both KO and WT mice. TA led to further reduction in outflow facility and decreases in expression of Plau and Mmp-13 in plat KO mice. Amiloride inhibition of uPA activity prevented the TA-induced outflow facility reduction in Plat KO mice.ConclusionstPA deficiency reduced outflow facility in mice and was associated with reduced MMP expression. The mechanism of action of tPA is unlikely to involve plasminogen activation. tPA is not the only mediator of TA-induced outflow facility change, as TA caused reduction in outflow facility of Plat KO mice. uPA did not substitute for tPA in outflow facility regulation but abrogated the effect of TA in the absence of tPA, suggesting a complex role of components of the fibrinolytic system in outflow regulation.

Expression of urokinase plasminogen activator (uPA) in the leukocytes and tissues of patients with benign and malignant breast lesions.

To show that the expression of urokinase plasminogen activator (uPA) in the leucocytes of patients with benign and malignant breast lesions correlates with its expression in the lesions, and to explore the role of uPA as a tumour marker in breast cancer. Using real time reverse transcriptase - polymerase chain reaction (RT-PCR), we examined the expression of uPA in leukocytes and tissues of three groups of women: a) with breast cancer (BC), b) with benign breast lesions and c) a control group of healthy women. An arbitrary value of 1 was assigned to the level of uPA expressed in the leucocytes of the healthy controls; all other measurements were expressed as a function of this value. The expression of uPA was significantly higher in BC samples compared to benign breast lesion (5.7 versus 3.8 times; p < 0.001). The leukocyte uPA of healthy volunteers was significantly lower than the leukocyte uPA from patients with breast lesions (p < 0.001). Leukocyte uPA level of patients with BC was higher compared to leukocyte uPA of women with benign lesions (p < 0.01). The expression of tissue uPA was the highest in triple-negative breast cancer. Methylation status was similar across tissue and leukocyte samples. Leukocyte uPA can be considered a surrogate of the tissue uPA expressed in BC samples. These results further support the use of leukocyte uPA as a biochemical marker of breast cancer.

Correlation of the invasive potential of glioblastoma and expression of caveola-forming proteins caveolin-1 and CAVIN1.

Glioblastoma (GBM) is the most common primary brain cancer. The average survival time for the majority of patients is approximately 15months after diagnosis. A major feature of GBM that contributes to its poor prognosis is its high invasiveness. Caveolae are plasma membrane subdomains that participate in numerous biological functions. Caveolin-1 and Caveolae Associated Protein 1 (CAVIN1), formerly termed Polymerase I and Transcript Release Factor, are both necessary for caveola formation. We hypothesized that high expression of caveola-forming proteins in GBM promotes invasiveness via modulation of the production of matrix-degrading enzymes. The mRNA expression of caveola-forming proteins and matrix proteases in GBM samples, and survival after stratifying patients according to caveolin-1 or CAVIN1 expression, were analyzed from TCGA and REMBRANDT databases. The proteolytic profile of cell lines expressing or devoid of caveola-forming proteins was investigated using zymography and real-time qPCR. Invasion through basement membrane-like protein was investigated in vitro. Expression of both caveolin-1 and CAVIN1 was increased in GBM compared to normal samples and correlated with expression of urokinase plasminogen activator (uPA) and gelatinases. High expression of caveola-forming proteins was associated with shorter survival time. GBM cell lines capable of forming caveolae expressed more uPA and matrix metalloproteinase-2 (MMP-2) and/or -9 (MMP-9) and were more invasive than GBM cells devoid of caveola-forming proteins. Experimental manipulation of caveolin-1 or CAVIN1 expression in GBM cells recapitulated some, but not all of these features. Caveolae modulate GBM cell invasion in part via matrix protease expression.

Role of Inflammation in Control of Hepatic Lipid Metabolism—New Insights to the Pathogenesis of NASH

Non‐alcoholic fatty liver disease (NAFLD) is a common metabolic disorder whose incidence among US adults exceeds 30%. While NAFLD manifests as benign hepatic steatosis in most patients, in about 20% of patients it assumes a much more aggressive form – non‐alcoholic steatohepatitis (NASH). The mechanisms that control the switch from benign steatosis to NASH are poorly understood, but were suggested to depend on ER stress. To query the role of ER stress in NASH development and establish a proper mouse model for studying the disease, we fed high‐fat diet (HFD) to MUP‐uPA mice, which are prone to liver ER stress due to hepatocyte‐specific urokinase plasminogen activator (uPA) expression. Placing these mice on HFD triggered full‐blown NASH within 3–4 months and most of the NASH‐afflicted mice progressed to develop hepatocellular carcinoma (HCC), the most common form of liver cancer whose incidence and development rates greatly increase with steatohepatitis. Both NASH and HCC development in MUP‐uPA mice depend on ER stress and are associated with persistent activation of sterol response element binding proteins (SREBP) 1 and 2. SREBP1 activation drives de novo lipogenesis (DNL) and cholesterol biosynthesis in HFD‐fed MUP‐uPA mice remains elevated without any sign of feedback inhibition mechanisms that control SREBP2 activation in tissue culture cells. DNL and cholesterol synthesis are also chronically elevated in NASH patients and the hepatic accumulation of free cholesterol was suggested to be a key switch from simple steatosis to NASH. While investigating the mechanism by which ER stress leads to persistent SREBP activation, we uncovered a previously unknown pathway in which caspase‐2 (Casp2) leads to constitutive activation of site 1 protease (S1P), which initiates SREBP cleavage activating protein (SCAP)‐independent SREBP activation. Of note, genetic ablation or pharmacological inhibition of Casp2 in MUP‐uPA mice blocks NASH development by preventing hepatic steatosis. Casp2 ablation in both MUP‐uPA and BL6 mice also prevents HFD‐induced adipocyte hypertrophy and increases energy consumption. None of the effects of Casp2 on liver lipid metabolism are related to cell death. The NAFLD/NASH epidemic was also suggested to be associated with increased consumption of fructose, which is a major constituent of high‐fructose corn syrup (HFCS), the main sweetener and a general food additive in the US. Increased consumption of HFCS has been linked to elevated risk of developing HCC, pancreatic and colon cancer. Indeed, feeding MUP‐uPA mice a high‐fructose diet (HFrD) results in NASH and HCC development even without the peripheral obesity that accompanies HFD consumption. Although fructose was believed to give rise to hepatic steatosis through preferential uptake and direct incorporation into the glycolytic pathway in hepatocytes, recent results indicate that the first site of fructose metabolism is the small intestine. Correspondingly, we found that fructose gives rise to NASH and HCC by first disrupting the intestinal epithelial barrier, evoking an inflammatory response that drives both NASH and HCC development.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract A25: Urokinase plasminogen activator expression is regulated by p53 harboring the lung cancer-specific mutation V157F

Abstract Rationale: Cancers of the lung and bronchus are responsible for nearly 30% of all cancer-related deaths in the US, and mutations in the tumor-suppressor gene TP53 occur in 54% of lung adenocarcinomas and 86% of squamous cell carcinomas. Compared with other cancers, TP53 mutational hotspots in lung cancer more often include missense mutations at codons V157, R158, and A159, with frequencies approaching that of the canonical mutational hotspot R273. The resulting mutant p53 (mutp53) protein often exhibits not only loss of tumor suppressor capability but also gain of oncogenic function (GOF). Our previous work identified a novel, p53-dependent transcriptome including alterations in expression of urokinase plasminogen activator (uPA), a key regulator of the plasminogen activation system. When activated, uPA converts plasminogen to plasmin, which cleaves extracellular matrix components and promotes cell migration. We evaluated uPA expression and cell migration in human lung cancer cell lines expressing wild-type or mutant p53. Methods: Human lung cancer cell lines with wild-type p53 (A549) or with somatic mutations at the endogenous p53 locus (NCI-H1781 and NCI-H2087, V157F; NCI-H441, R158L; NCI-H2110, R158P; NCI-H2009, R273L) were obtained from ATCC. Using RNAi (shRNA and siRNA) cells were depleted of p53 as confirmed by puromycin selection of cells infected with lentivirus, in the case of shRNA. Whole-cell lysates were harvested for protein and RNA purification, and conditioned medium was harvested for measurement of secreted proteins. Immunoblotting and qRT-PCR were used to assess expression of protein and mRNA, respectively. A wound healing assay was performed following siRNA transfection to determine cell migration in cells expressing vs. depleted of wild-type or mutant p53. Results: Silencing of p53 by shRNA and siRNA targeted to TP53 was confirmed by Western blot and qRT-PCR. With p53 depletion, protein and mRNA expression of the uPA pro-enzyme pro-uPA and its activated B- and A-chains increased in the H2087 (V157F mutp53) cell line. No p53-dependent change was seen in uPA expression in H2009 cells (R273L mutp53) despite a similar relative quantity of uPA protein. Secreted uPA also increased as measured in conditioned media from H2087 cells (V157F mutp53). A wound healing assay showed increased migration by mutp53-depleted H2087 cells compared with mutp53-expressing H2087 cells, while A549 cells (wt p53) showed no p53-dependent difference in migration rate. Conclusions: To our knowledge, GOF phenotypes in lung cancer cells carrying V157F mutp53 have not been characterized. We have shown here that mutp53 represses uPA expression in human lung cancer cells with endogenous V157F mutp53, and depletion of p53 in these cells leads to an increase in migration. These findings prompt a reexamination of the signaling pathways associated with uPA. Further study is needed to elucidate the mechanism of the p53-dependent alterations in uPA expression and to establish additional biologic effects of V157F mutp53. Citation Format: Julie A. Barta, Steven B. McMahon. Urokinase plasminogen activator expression is regulated by p53 harboring the lung cancer-specific mutation V157F [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A25.

(-)-Epigallocatechin gallate derivatives reduce the expression of both urokinase plasminogen activator and plasminogen activator inhibitor-1 to inhibit migration, adhesion, and invasion of MDA-MB-231 cells.

Urokinase plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor-1 (PAI-1) are established independent biomarkers for high metastasis risk in breast cancer. In this study, we investigated the regulatory activity of (-)-epigallocatechin-3-gallate (EGCG) and its derivatives on uPA and PAI-1 expression and thereby their anti-metastatic potential. EGCG showed only marginal effects on the uPA system and on the metastatic behavior of breast cancer cells (MDA-MB-231). However, the EGCG derivative 3e with a methyl-substituted carbonate substituent at the 4″-position showed potent inhibition of PAI-1 (62%) and uPA (50%) expression. The Ras-extracellular-signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol-3-kinase (PI3K)/Akt/NF-κB pathways, which regulate uPA and PAI-1 expression, were also affected by 3e (25%, 45%, and 25% reduction, respectively). In line with these findings, substantial reduction in metastatic behavior of MDA-MB-231 cells, such as adhesion (40%), invasion (56%), and migration (40%), was observed in the presence of 3e. It is also noteworthy that, in MDA-MB-231 cells, 3e did not exert any beneficial effect on the expression of matric metalloprotein (MMP) 2 and 9, which indicates that the anti-metastatic activity of 3e in MDA-MB-231 cells is not related to its regulation of the expression of MMPs. Taken together, we have shown that the EGCG derivative 3e could suppress the metastatic behavior of MDA-MB-231 cells through regulation of uPA and PAI-1.

Abstract 912: uPA as a topoisomerase-1 protective agent in lung fibroblast cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, with approximately 53,000 people in the U.S. diagnosed with the disease every year. The Gondi lab has previously shown that poor survival rates in pancreatic cancer are correlated with increased expression of urokinase plasminogen activator (uPA), a serine protease. Our previous data has shown for the first time that uPA binds to topoisomerase-1 (TOPO-1), in addition to the interaction of uPA with various transcription factors. Our current research is aimed at elucidating the mechanism of the interaction between uPA and TOPO-1, with the goal of demonstrating a protective role for uPA in the regulation of TOPO-1 function in the presence of TOPO-1 inhibitors. Methods: Two knock-out lung fibroblast cell lines were used. One cell line was a uPA knock-out line, while the other was a uPA knock-out line with a uPA expression vector transfected into the cells. Transfection was performed using Chariot transfection vector. The cell lines were treated with topoisomerase inhibitors TX1111 and SN38. DNA replication activity was measured using flow cytometry as a surrogate for TOPO-1 activity. Controls included untreated cells, cells treated with empty Chariot, and cells treated with Chariot containing TXC peptide. Results and Discussion: In the presence of topoisomerase inhibitors TX1111 and SN38, uPA knock-out lines demonstrated incomplete cell division relative to controls. In uPA-expressing lines, cell division activity was retained in the presence of topoisomerase inhibitors. Our data demonstrates for the first time that uPA serves a protective role in preventing the inhibition of TOPO-1 by topoisomerase inhibitors. This indicates uPA as a potential new drug target in the treatment of chemoresistant pancreatic cancer. Further studies to be performed include visualizing the degree of DNA-replication using DNA combing, and subsequently expanding this study to pancreatic cancer cells. Citation Format: Liang Ge, Manu Gnanamony, Victoria Stepanova, Dzung H. Dinh, Christopher S. Gondi. uPA as a topoisomerase-1 protective agent in lung fibroblast cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 912.

Lysophosphatidic acid increases in vitro maturation efficiency via uPA-uPAR signaling pathway in cumulus cells

Lysophosphatidic acid (LPA) is a phospholipid-derived signaling molecule with biological activities, such as stimulating cell proliferation, differentiation and migration. In the present study, we examined the effect of LPA on porcine oocytes during in vitro maturation (IVM) and subsequent embryonic development following parthenogenetic activation (PA) and in vitro fertilization (IVF). During IVM, the maturation medium was supplemented with various concentrations of LPA (0, 10, 30, and 60 μM). After 42 h of IVM, the 30 μM LPA-treated group showed a significant (P <0.05) increase in nuclear maturation and intracellular glutathione (GSH) levels compared with the other groups. The 30 μM LPA-treated group exhibited a significant decrease in intracellular reactive oxygen species (ROS) levels compared with the other groups. In PA, the 30 μM LPA-treated group had significantly higher cleavage (CL) and blastocyst (BL) rates compared with those of the other LPA-treated groups. In IVF, the 30 μM LPA-treated group had significantly higher CL and BL rates than the other LPA-treated groups. The expression of the developmental competence gene (proliferating cell nuclear antigen, PCNA) in the oocytes and cumulus cells of the individuals in the 30 μM LPA-treated group was significantly increased compared with the control group. In addition, the specific expression of urokinase Plasminogen Activator (uPA) and uPA Receptor (uPAR) in cumulus cells was significantly increased in the 30 μM LPA-treated group. The western blotting results revealed that LPA improves the activities of p38 mitogen-activated protein kinase (MAPK) and epidermal growth factor (EGF) by enhanced phosphorylation. In conclusion, treatment with 30 μM LPA during IVM promotes enhances the EGF-EGFR signaling pathway, resulting in cumulus cell expansion. And then, this treatment improves the developmental potential of PA and IVF porcine embryos by enhancing nuclear and cytoplasmic maturation and reducing ROS.

Prognostic value of urokinase plasminogen activator system in non-small cell lung cancer: A systematic review and meta-analysis.

Current relevant research suggests there are significant differences between the expression of the urokinase plasminogen activator (uPA) system in cancer tissues and in normal tissues. However, the potential effectiveness of the uPA system as a prognostic biomarker of non-small cell lung cancer (NSCLC) remains unclear. In the present study, a systematic review and meta-analysis were performed to evaluate the relevance of the uPA system in the prognosis of patients with NSCLC. Using the PubMed, EMBASE, Web of Science and Cochrane Library databases, data from relevant academic journal articles were extracted and subjected to analysis. Associations between expression profiles pertaining to the uPA system and the overall survival (OS) of patients with NSCLC were analyzed. The study incorporated data from 11 independent journal articles and these reports included a total of 937 patients with NSCLC. The meta-analysis results revealed that increased expression of urokinase plasminogen activator (uPA) and PA inhibitor type 1 (PAI-1) exhibited no significant association with poor OS [hazard ratio (HR)-uPA=1.07 (0.87-1.31), P=0.53; HR-PAI-1=1.02 (0.63-1.65), P=0.94]. Similarly, reduced expression of PAI type 2 (PAI-2) did not significantly correlate with poor OS [HR-PAI-2=1.58 (0.64-3.90); P=0.32]. Notably, however, a significant association was observed between increased expression levels of uPA receptor (uPAR) and poor OS for NSCLC [HR-uPAR=1.50 (1.04-2.15); P=0.03]. Therefore, the expression of uPAR in the uPA system of patients with NSCLC could be used as a novel clinical biomarker to evaluate the prognosis of NSCLC. The utilization of this biomarker may provide a platform for the further development of targeted drugs for the treatment of NSCLC.

Krüppel-like factor 17 inhibits urokinase plasminogen activator gene expression to suppress cell invasion through the Src/p38/ MAPK signaling pathway in human lung adenocarcionma.

Krüppel-like factor 17 (KLF17) has been reported to be involved in invasion and metastasis suppression in lung cancer, but the molecular mechanisms underlying the anti-invasion and anti-metastasis roles of KLF17 in lung cancer are not fully illustrated. Here, we showed that KLF17 inhibited the invasion of A549 and H322 cells; the anti-invasion effect of KLF17 was associated with the suppression of urokinase plasminogen activator (uPA/PLAU) expression. KLF17 can bind with the promoter of uPA and inhibit its expression. Enforced expression of uPA abrogated the anti-invasion effect of KLF17 in A549 and H322 cells. In addition, immunohistochemistry staining showed that the protein expression of KLF17 was negatively correlated with that of uPA in archived samples from patients with lymph node metastasis of lung adenocarcinoma (rho = −0.62, P = 0.01). The mutually exclusive expression of KLF17 with uPA could predict lymph node metastasis for lung adenocarcinoma (AUC = 0.758, P = 0.005). Enforced expression of KLF17 inhibited the expression of phosphorylated Src and phosphorylated p38/MAPK in A549 and H322 cells. The invasiveness of the cells were suppressed by treating with sb203580 (p38/MAPK inhibitor) or HY-13805 (PP2, Src inhibitor). furthermore, p38/MAPK inhibition could block the KLF17-induced reduction of p-p38/MAPK and uPA, and Src inhibition enhanced the KLF17-induced suppression of p-Src and uPA in A549 and H322 cells. In conclusion, our study indicated that KLF17 suppressed the uPA-mediated invasion of lung adenocarcinoma. The Src and p38/MAPK signaling pathways were suggested as mediators of KLF17-induced uPA inhibition, thus providing evidence that KLF17 might be a potential anti-invasion candidate for lung adenocarcinoma.

Hispolon suppresses migration and invasion of human nasopharyngeal carcinoma cells by inhibiting the urokinase-plasminogen activator through modulation of the Akt signaling pathway.

Hispolon has been reported to possess antioxidant, antiinflammatory, and antitumor activities. However, the effect of hispolon on the metastasis of nasopharyngeal carcinoma (NPC) remains unclear. In this study, we investigated how the antimetastatic activity and relevant signaling pathways of hispolon affected three NPC cell lines. The results revealed that hispolon significantly reduced the migration and invasion of three NPC cells in a dose-dependent manner from 0 to 50µM. Hispolon also significantly inhibited the activity and expression of urokinase-plasminogen activator (uPA) as well as the phosphorylation of Akt. Moreover, blocking the Akt pathway also enhanced the antimetastatic ability of hispolon in the NPC cells. In conclusion, hispolon inhibited uPA expression and NPC cell metastasis by downregulating Akt signal pathways; therefore, hispolon exerts beneficial effects in chemoprevention. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 645-655, 2017.

MP92-04 PROTUMOR CONVERSION OF NATURAL KILLER CELLS BY TRANSFORMING GROWTH FACTOR-β AND HYPOXIA IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology III1 Apr 2016MP92-04 PROTUMOR CONVERSION OF NATURAL KILLER CELLS BY TRANSFORMING GROWTH FACTOR-β AND HYPOXIA IN RENAL CELL CARCINOMA Yue Guan, Christopher B. Chambers, Max Nutt, Britnie James, Kathy Robinson, Shaheen Alanee, MD, MPH, MBA Thomas Griffith, Donald Torry, and Andrew Wilber Yue GuanYue Guan More articles by this author , Christopher B. ChambersChristopher B. Chambers More articles by this author , Max NuttMax Nutt More articles by this author , Britnie JamesBritnie James More articles by this author , Kathy RobinsonKathy Robinson More articles by this author , Shaheen AlaneeShaheen Alanee More articles by this author , Thomas GriffithThomas Griffith More articles by this author , Donald TorryDonald Torry More articles by this author , and Andrew WilberAndrew Wilber More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2637AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES NK cells with non-classical phenotypes (CD56+CD16dim/neg; termed decidua NK or dNK cells) accumulate at the maternal-fetal interface during implantation. These dNK cells are poorly cytotoxic and proangiogenic. We investigated whether an analogous NK cell phenotype accumulate in RCC tumors potentiated by tumor-derived TGFβ and hypoxia. METHODS NK cells from peripheral blood (pNK) and resected tumor tissue (TiNK) of RCC patients were compared to pNK from healthy, tumor-free donors. pNK cells were cultured in the absence or presence of TGFß and exposed to 21% or 1% oxygen to assess conversion by monitoring expression of surface markers and angiogenic genes as well as ability to directly kill target cells. An orthotopic mouse model of RCC was also used, where the murine renal adenocarcinoma cell line, Renca, was implanted directly into the kidneys of Balb/c mice. RESULTS pNK cells of healthy donors were CD56+CD16+ (94±1%) and cytotoxic, but acquired characteristics of dNK cells (reduced cytotoxicity and augmented vascular endothelial growth factor (VEGF) expression) when exposed to TGFß or 1% O2. Addition of TGFß to NK cells maintained in 1% O2 further suppressed cytotoxic function and induced expression of urokinase plasminogen activator (uPA) and its inhibitor PAI-1, two factors with known roles in tumor progression. pNK cells of RCC patients were phenotypical similar to healthy donors (89±2%, CD56+CD16+), but lacked full cytotoxic ability which we attributed to 3-fold higher levels of TGFß in patient serum. RCC TiNK cells were significantly enriched for dNK-like CD56+CD16dim/neg cells (47±-12%) had limited cytotoxic capacity and increased VEGF and uPA expression. Analogous alterations in TiNK cells were observed using an orthotopic mouse model of RCC. CONCLUSIONS These studies support a role for TGFß and hypoxia in conversion of pNK cells to a dNK-like phenotype in RCC tumors. While these characteristics are conceivably beneficial for placentation, they may be exploited to support RCC growth and metastasis. Thus, efforts designed to neutralize the effects of TGFß may be an important goal of future immunotherapies. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1162-e1163 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Yue Guan More articles by this author Christopher B. Chambers More articles by this author Max Nutt More articles by this author Britnie James More articles by this author Kathy Robinson More articles by this author Shaheen Alanee More articles by this author Thomas Griffith More articles by this author Donald Torry More articles by this author Andrew Wilber More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Targeting EMP3 suppresses proliferation and invasion of hepatocellular carcinoma cells through inactivation of PI3K/Akt pathway.

Epithelial membrane protein-3 (EMP3), a typical member of the epithelial membrane protein (EMP) family, is epigenetically silenced in some cancer types, and has been proposed to be a tumor suppressor gene. However, its effects on tumor suppression are controversial and its roles in development and malignancy of hepatocellular carcinoma (HCC) remain unclear. In the present study, we found that EMP3 was highly expressed in the tumorous tissues comparing to the matched normal tissues, and negatively correlated with differentiated degree of HCC patients. Knockdown of EMP3 significantly reduced cell proliferation, arrested cell cycle at G1 phase, and inhibited the motility and invasiveness in accordance with the decreased expression and activity of urokinase plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) in HCC cells. The in vivo tumor growth of HCC was effectively suppressed by knockdown of EMP3 in a xenograft mouse model. The EMP3 knockdown-reduced cell proliferation and invasion were attenuated by inhibition of phosphatidylinositol 3-kinase (PI3K) or knockdown of Akt, and rescued by overexpression of Akt in HCC cells. Clinical positive correlations of EMP3 with p85 regulatory subunit of PI3K, p-Akt, uPA, as well as MMP-9 were observed in the tissue sections from HCC patients. Here, we elucidated the tumor progressive effects of EMP3 through PI3K/Akt pathway and uPA/MMP-9 cascade in HCC cells. The findings provided a new insight into EMP3, which might be a potential molecular target for diagnosis and treatment of HCC.

Abstract P088: Pravastatin Protects Against Glucose-induced Anti-proliferative, Anti-invasive and Anti-angiogenic Milieu in Cytotrophoblasts

Objective: An increasing level of evidence supports the utility of pravastatin in prevention against preeclampsia (preE). We previously demonstrated that hyperglycemia induces cytotrophoblasts (CTBs) dysfunction characteristic of a preE-like phenotype. We sought to demonstrate the utility of pravastatin in rescuing CTBs from hyperglycemia induced dysfunction. Methods: Human CTBs were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48h. Some cells were pretreated with pravastatin (1ug/mL) for 2h, while others were co-treated with pravastatin (1ug/mL) prior to glucose treatment. Some cells were treated with D-Mannitol. Cell migration was performed by Matrigel migration assay kit according to manufacturer protocol. Cell lysates were utilized to evaluate the expression of urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1), proliferating cell nuclear antigen (PCNA) and p38 MAPK phosphorylation by western blot. Levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng) and interleukin 6 (IL-6) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test. Results: Hyperglycemia inhibited CTBs migration, down-regulated uPA, PAI-1, PCNA and up-regulated p38 phosphorylation in CTBs treated with ≥150 mg/dL glucose compared to basal (100 mg/dL) (*p &lt; 0.05 for each). Secretion of sFlt-1, sEng and IL-6 were increased while VEGF and PIGF were decreased in CTBs treated ≥150 mg/dl of glucose (*p &lt; 0.05 for each). Both pravastatin pretreatment and co-treatment significantly rescued CTBs migration, up-regulated uPA, PAI-1, PCNA, down-regulated p38 phosphorylation, and corrected the angiogenic profile of CTBs (p &lt; 0.05 for each). D-Mannitol had no effect on CTBs. Conclusions: Pravastatin mitigates the hyperglycemia-induced dysfunction of CTBs by attenuating the glucose-induced anti-proliferative, anti-invasive and anti-angiogenic phenotype. These data should alleviate critical concerns regarding pravastatin use on CTBs development early in pregnancy and support the current research to use of pravastatin in preE prevention.

Immunohistochemical Detection of Urokinase Plasminogen Activator and Urokinase Plasminogen Activator Receptor in Canine Vascular Endothelial Tumours

Immunohistochemistry was used to assess the expression of urokinase plasminogen activator (uPA) and uPA receptor (uPAR) in 57 canine primary haemangiosarcomas (HSAs), 26 canine cutaneous haemangiomas (HAs) and in control sections of canine cutaneous granulation tissue. The correlation between uPA/uPAR expression and the Ki67 labelling index (LI) was estimated in the HSA and HA tissues. uPA was expressed by 73.2% and 75.0% of splenic HSAs and non-splenic HSAs, respectively. All HSA tissues tested expressed uPAR. Expression of both molecules was significantly higher in HSAs than in cutaneous HAs (3.8% for uPA and 30.7% for uPAR). The average Ki67 LI of the uPA(+)/uPAR(+) HSAs was significantly higher than that of uPA(-)/uPAR(+) HSAs and HA tissues (mean±SDs 32.8±15.3, 15.2±7.2 and 2.1±0.7, respectively; P<0.05). These results suggest that uPA and uPAR play a significant role in the malignant proliferation of canine HSA, regardless of the primary origin of the tumour.

Abstract 4103: Knockdown of Notch1 inhibits tumor cell proliferation, migration, and invasion and reverses epithelial-mesenchymal transition (EMT) in nasopharyngeal carcinoma

Abstract Despite a large bodies of evidences support an oncogenic role of Notch signaling in several human solid cancers, however, the pleiotropic effects and molecular mechanisms of blockage of Notch signaling on nasopharyngeal carcinoma (NPC) remain unclear. In this study, Real-time RT-PCR and western blotting were firstly used to examine the expression of Notch1 receptor in a panel of NPC cell lines. CNE1 and CNE2 cells expressed a higher level of Notch1 compared to HONE1, SUNE1 and HK cells. Then Notch1 expression was specifically knocked down in CNE1 and CNE2 cells by Notch1 short hairpin RNA (shRNA). This resulted in a significant inhibition of cell proliferation, migration, and invasion; and interestingly, epithelial-mesenchymal transition (EMT) was reversed in these Notch1-transfected cells along with demonstration of epithelialioid-like morphology change and increase of E-cadherin and decrease of Vimentin expressions. In addition, targeted knockdown of Notch1 dramatically inhibited expression of urokinase plasminogen activator (uPA) and its receptor uPAR, chemokine CCL2 and CXCL16 indicating that these factors are downstream targets of Notch1. Finally, knockdown of Notch1 significantly diminished CNE1 cell growth in a murine model concomitant with inhibition of cell proliferation and induction of apoptosis. Taken together, these results suggest Notch1 may become a therapeutic target in nasopharyngeal carcinoma. Note: This abstract was not presented at the meeting. Citation Format: Huajiao Guo, Yi Lu, Chaonan Qian, Jian Zhang. Knockdown of Notch1 inhibits tumor cell proliferation, migration, and invasion and reverses epithelial-mesenchymal transition (EMT) in nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4103. doi:10.1158/1538-7445.AM2015-4103

O131. Pravastatin protects against glucose-induced anti-proliferative, anti-invasive and anti-angiogenic milieu in cytotrophoblasts

Objective An increasing level of evidence supports the utility of pravastatin in prevention against preeclampsia (preE). We previously demonstrated that hyperglycemia induces cytotrophoblasts (CTBs) dysfunction characteristic of a preE-like phenotype. We sought to demonstrate the utility of pravastatin in rescuing CTBs from hyperglycemia induced dysfunction. Methods Human CTBs were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48h. Some cells were pretreated with pravastatin (1 ug/mL) for 2 h, while others were co-treated with pravastatin (1 ug/mL) prior to glucose treatment. Some cells were treated with D-Mannitol. Cell migration was performed by Matrigel migration assay kit according to manufacturer protocol. Cell lysates were utilized to evaluate the expression of urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1), proliferating cell nuclear antigen (PCNA) and p38 MAPK phosphorylation by western blot. Levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng) and interleukin 6 (IL-6) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test. Results Hyperglycemia inhibited CTBs migration, down-regulated uPA, PAI-1, PCNA and up-regulated p38 phosphorylation in CTBs treated with >150 mg/dL glucose compared to basal (100 mg/dL) ( ∗ p ∗ p p Conclusions Pravastatin mitigates the hyperglycemia-induced dysfunction of CTBs by attenuating the glucose-induced anti-proliferative, anti-invasive and anti-angiogenic phenotype. These data should alleviate critical concerns regarding pravastatin use on CTBs development early in pregnancy and support the current research to use of pravastatin in preE prevention.

Therapeutic effectiveness of bone marrow-derived mesenchymal stem cell administration against acute pulmonary thromboembolism in a mouse model

InstructionAcute pulmonary thromboembolism (APTE) is a common clinical condition associated with significant morbidity and mortality. Although promising, bone marrow-derived mesenchymal stem cell (BMSC) treatment for thrombus resolution remains controversial. The therapeutic effectiveness of BMSC against APTE has not been evaluated. This study aims to determine whether BMSCs administration is effective in mouse model. Materials and MethodsTherapeutic efficacy of female and male BMSCs were evaluated by applying serial sectioning analysis method for the whole lungs of APTE mice and calculating each thrombus size in volume. Plasmid construction and stable transfection were used to manipulate expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in both genders of BMSCs. Western blot were performed to detect GAPDH and urokinase plasminogen activator expression in BMSCs. ResultsOur data showed, 1) compared with non-serial sectioning method, the serial sectioning method detected more thrombi, larger size ranges of thrombus area, and the volume of each individual thrombus. 2) BMSCs significantly decreased the thrombi size in APTE mice, with female BMSCs superior to male ones. 3) female BMSCs showed a higher GAPDH protein level and manipulations of GAPDH expression in female or male BMSCs profoundly affected their therapeutic efficacies as well as urokinase plasminogen activator expression. ConclusionThis study indicates serial-sectioning analysis method is necessary for evaluating APTE and provides strong evidences for BMSCs possessing therapeutic effectiveness against APTE, with female BMSCs superior to male counterparts. GAPDH played a critical role in the superior function of female BMSCs, possibly by regulating the expression of urokinase plasminogen activator.