MMP-9, uPAR and Cathepsin B Silencing Downregulate Integrins in Human Glioma Xenograft Cells In Vitro and In Vivo in Nude Mice

Involvement of MMP-9, uPAR and cathepsin B in adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well established. In the present study, MMP-9, uPAR and cathepsin B genes were downregulated in glioma xenograft cells using shRNA plasmid constructs and we evaluated the involvement of integrins and changes in their adhesion, migration and invasive potential.MMP-9, uPAR and cathepsin B single shRNA plasmid constructs were used to downregulate these molecules in xenograft cells. We also used MMP-9/uPAR and MMP-9/cathepsin B bicistronic constructs to evaluate the cumulative effects. MMP-9, uPAR and cathepsin B downregulation significantly inhibits xenograft cell adhesion to several extracellular matrix proteins. Treatment with MMP-9, uPAR and cathepsin B shRNA of xenografts led to the downregulation of several alpha and beta integrins. In all the assays, we noticed more prominent effects with the bicistronic plasmid constructs when compared to the single plasmid shRNA constructs. FACS analysis demonstrated the expression of alphaVbeta3, alpha6beta1 and alpha9beta1 integrins in xenograft cells. Treatment with bicistronic constructs reduced alphaVbeta3, alpha6beta1 and alpha9beta1 integrin expressions in xenograft injected nude mice. Migration and invasion were also inhibited by MMP-9, uPAR and cathepsin B shRNA treatments as assessed by spheroid migration, wound healing, and Matrigel invasion assays. As expected, bicistronic constructs further inhibited the adhesion, migration and invasive potential of the xenograft cells as compared to individual treatments.Downregulation of MMP-9, uPAR and cathespin B alone and in combination inhibits adhesion, migration and invasive potential of glioma xenografts by downregulating integrins and associated signaling molecules. Considering the existence of integrin inhibitor-resistant cancer cells, our study provides a novel and effective approach to inhibiting integrins by downregulating MMP-9, uPAR and cathepsin B in the treatment of glioma

Identification of uPAR-positive Chemoresistant Cells in Small Cell Lung Cancer

BACKGROUND: The urokinase plasminogen activator (uPA) and its receptor (uPAR/CD87) are major regulators of extracellular matrix degradation and are involved in cell migration and invasion under physiological and pathological conditions. The uPA/uPAR system has been of great interest in cancer research because it is involved in the development of most invasive cancer phenotypes and is a strong predictor of poor patient survival. However, little is known about the role of uPA/uPAR in small cell lung cancer (SCLC), the most aggressive type of lung cancer. We therefore determined whether uPA and uPAR are involved in generation of drug resistant SCLC cell phenotype. METHODS AND FINDINGS: We screened six human SCLC cell lines for surface markers for putative stem and cancer cells. We used fluorescence-activated cell sorting (FACS), fluorescence microscopy and clonogenic assays to demonstrate uPAR expression in a subpopulation of cells derived from primary and metastatic SCLC cell lines. Cytotoxic assays were used to determine the sensitivity of uPAR-positive and uPAR-negative cells to chemotherapeutic agents. The uPAR-positive cells in all SCLC lines demonstrated multi-drug resistance, high clonogenic activity and co-expression of CD44 and MDR1, putative cancer stem cell markers. CONCLUSIONS: These data suggest that uPAR-positive cells may define a functionally important population of cancer cells in SCLC, which are resistant to traditional chemotherapies, and could serve as critical targets for more effective therapeutic interventions in SCLC

Regulation of DNA Repair Mechanism in Human Glioma Xenograft Cells both In Vitro and In Vivo in Nude Mice

Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells.Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls.Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point

Comparative expression pattern of Matrix-Metalloproteinases in human glioblastoma cell-lines and primary cultures

<p>Abstract</p> <p>Background</p> <p>Glioblastomas (GBM), the most frequent malignant brain tumors in adults, are characterized by an aggressive local growth pattern and highly invasive tumor cells. This invasion is facilitated by expression of matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases. They mediate the degradation of protein components of the extracellular matrix. Twenty-three family members are known. Elevated levels of several of them have been reported in GBM. GBM cell-lines are used for <it>in vitro </it>studies of cell migration and invasion. Therefore, it is essential to know their MMP expression patterns. Only limited data for some of the cell-lines are published, yet. To fill the gaps in our knowledge would help to choose suitable model systems for analysis of regulation and function of MMPs during GBM tumorigenesis, cell migration and invasion.</p> <p>Findings</p> <p>We analysed MMP-1, -8, -9, -10, -11, -13, -17, -19, -20, -21, -23, -24, -26, -27, and MMP-28 expression in seven GBM cell-lines (SNB-19, GaMG, U251, U87, U373, U343, U138) and in four primary cell cultures by semiquantitative RT-PCR, followed changes in the MMP expression pattern with increasing passages of cell culture and examined the influence of TNF-α and TGF-β1 stimulation on the expression of selected MMPs in U251 and U373 cells.</p> <p>MMP-13, -17, -19 and -24 were expressed by all analyzed cell-lines, whereas MMP-20 and MMP-21 were not expressed by any of them. The other MMPs showed variable expression, which was dependent on passage number. Primary cells displayed a similar MMP-expression pattern as the cell-lines. In U251 and U373 cells expression of MMP-9 and MMP-19 was stimulated by TNF-α. MMP-1 mRNA expression was significantly increased in U373 cells, but not in U251 cells by this cytokine. Whereas TGF-β1 had no impact on MMP expression in U251 cells, it significantly induced MMP-11 and MMP-24 expression in U373 cells.</p> <p>Conclusions</p> <p>Literature-data and our own results suggest that the expression pattern of MMPs is highly variable, dependent on the cell-line and the cell-culture conditions used and that also regulation of MMP expression by cytokines is cell-line dependent. This is of high impact for the transfer of cell-culture experiments to clinical implementation.</p

Prevalence of the metabolic syndrome in Luxembourg according to the Joint Interim Statement definition estimated from the ORISCAV-LUX study

ABSTRACT: BACKGROUND: The prevalence of the metabolic syndrome (MS) has been determined in many countries worldwide but never in Luxembourg. This research aimed to 1) establish the gender- and age-specific prevalence of MS and its components in the general adult population of Luxembourg, according to the most recent Joint Interim Statement (JIS) definition, by using both the high and low cut-off points to define abdominal obesity, and 2) compare and assess the degree of agreement with the Revised National Cholesterol Education Programme-Adult Treatment Panel III (R-ATPIII) and the International Diabetes Federation (IDF) definitions. METHODS: A representative stratified random sample of 1349 European subjects, aged 18-69 years, participated to ORISCAV-LUX survey. Logistic regression and odds ratios (OR) were used to study MS prevalence with respect to gender and age. The Framingham risk score (FRS) to predict the 10-year coronary heart disease (CHD) risk was calculated to compare the proportion of MS cases below or above 20%, according to both high and low waist circumference (WC) thresholds. Cohen's kappa coefficient (kappa) was utilized to measure the degree of agreement between MS definitions. RESULTS: The prevalence of the MS defined by the JIS was 28.0% and 24.7% when using the low (94/80) and the high (102/88) WC cut-off points, respectively. The prevalence was significantly higher in men than in women (OR = 2.6 and 2.3 for the low and high WC thresholds), as were all components of the MS except abdominal obesity measured by both thresholds. It also increased with age (OR values in age categories ranging from 2.7 to 28 when compared to the younger subjects for low WC and from 3.3 to 31 for the high WC cut-offs). The 10-year predicted risk of CHD by FRS did not depend on the threshold used. Globally, excellent agreement was observed between the three definitions of MS (kappa= 0.89), in particular between JIS and IDF (kappa = 0.93). Agreement was significantly higher in women than in men, and differed between age groups. CONCLUSION: Regardless of the definition used, the adult population of Luxembourg reveals a high MS prevalence. Our findings contribute to build evidence regarding the definitive construct of the MS, to help selecting the waist circumference thresholds for Europid populations, and to support the need to revise the guidelines for abdominal obesity levels

Co-Depletion of Cathepsin B and uPAR Induces G0/G1 Arrest in Glioma via FOXO3a Mediated p27Kip1 Upregulation

Cathepsin B and urokinase plasminogen activator receptor (uPAR) are both known to be overexpressed in gliomas. Our previous work and that of others strongly suggest a relationship between the infiltrative phenotype of glioma and the expression of cathepsin B and uPAR. Though their role in migration and adhesion are well studied the effect of these molecules on cell cycle progression has not been thoroughly examined.Cathepsin B and uPAR single and bicistronic siRNA plasmids were used to downregulate these molecules in SNB19 and U251 glioma cells. FACS analysis and BrdU incorporation assay demonstrated G0/G1 arrest and decreased proliferation with the treatments, respectively. Immunoblot and immunocyto analysis demonstrated increased expression of p27(Kip1) and its nuclear localization with the knockdown of cathepsin B and uPAR. These effects could be mediated by alphaVbeta3/PI3K/AKT/FOXO pathway as observed by the decreased alphaVbeta3 expression, PI3K and AKT phosphorylation accompanied by elevated FOXO3a levels. These results were further confirmed with the increased expression of p27(Kip1) and FOXO3a when treated with Ly294002 (10 microM) and increased luciferase expression with the siRNA and Ly294002 treatments when the FOXO binding promoter region of p27(Kip1) was used. Our treatment also reduced the expression of cyclin D1, cyclin D2, p-Rb and cyclin E while the expression of Cdk2 was unaffected. Of note, the Cdk2-cyclin E complex formation was reduced significantly.Our study indicates that cathepsin B and uPAR knockdown induces G0/G1 arrest by modulating the PI3K/AKT signaling pathway and further increases expression of p27(Kip1) accompanied by the binding of FOXO3a to its promoter. Taken together, our findings provide molecular mechanism for the G0/G1 arrest induced by the downregulation of cathepsin B and uPAR in SNB19 and U251 glioma cells

Expression and methylation status of tissue factor pathway inhibitor-2 gene in non-small-cell lung cancer

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor that inhibits plasmin-dependent activation of several metalloproteinases. Downregulation of TFPI-2 could thus enhance the invasive potential of neoplastic cells in several cancers, including lung cancer. In this study, TFPI-2 mRNA was measured using a real-time PCR method in tumours of 59 patients with non-small-cell lung cancer (NSCLC). Tumour TFPI-2 mRNA levels appeared well correlated with protein expression evaluated by immunohistochemistry and were 4–120 times lower compared to those of nonaffected lung tissue in 22 cases (37%). Hypermethylation of the TFPI-2 gene promoter was demonstrated by restriction enzyme-polymerase chain reaction in 12 of 40 cases of NSCLC (30%), including nine of 17 for whom tumour TFPI-2 gene expression was lower than in noncancerous tissue. In contrast, this epigenetic modification was shown in only three of 23 tumours in which no decrease in TFPI-2 synthesis was found (P=0.016). Decreased TFPI-2 gene expression and hypermethylation were more frequently associated with stages III or IV NSCLC (eight out of 10, P=0.02) and the TFPI-2 gene promoter was more frequently hypermethylated in patients with lymph node metastases (eight out of 16, P=0.02). These results suggest that silencing of the TFPI-2 gene by hypermethylation might contribute to tumour progression in NSCLC

Required muscle mass for preventing lifestyle-related diseases in Japanese women

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Inhibitory effect of ginsenoside Rg3 combined with gemcitabine on angiogenesis and growth of lung cancer in mice

<p>Abstract</p> <p>Background</p> <p>Ginsenoside Rg3, a saponin extracted from ginseng, inhibits angiogenesis. The combination of low-dose chemotherapy and anti-angiogenic inhibitors suppresses growth of experimental tumors more effectively than conventional therapy or anti-angiogenic agent alone. The present study was designed to evaluate the efficacy of low-dose gemcitabine combined with ginsenoside Rg3 on angiogenesis and growth of established Lewis lung carcinoma in mice.</p> <p>Methods</p> <p>C57L/6 mice implanted with Lewis lung carcinoma were randomized into the control, ginsenoside Rg3, gemcitabine and combination group. The quality of life and survival of mice were recorded. Tumor volume, inhibitive rate and necrosis rate were estimated. Necrosis of tumor and signals of blood flow as well as dynamic parameters of arterial blood flow in tumors such as peak systolic velocity (PSV) and resistive index (RI) were detected by color Doppler ultrasound. In addition, expression of vascular endothelial cell growth factor (VEGF) and CD31 were observed by immunohistochemstry, and microvessel density (MVD) of the tumor tissues was assessed by CD31 immunohistochemical analysis.</p> <p>Results</p> <p>Quality of life of mice in the ginsenoside Rg3 and combination group were better than in the control and gemcitabine group. Combined therapy with ginsenoside Rg3 and gemcitabine not only enhanced efficacy on suppression of tumor growth and prolongation of the survival, but also increased necrosis rate of tumor significantly. In addition, the combination treatment could obviously decrease VEGF expression and MVD as well as signals of blood flow and PSV in tumors.</p> <p>Conclusion</p> <p>Ginsenoside Rg3 combined with gemcitabine may significantly inhibit angiogenesis and growth of lung cancer and improve survival and quality of life of tumor-bearing mice. The combination of chemotherapy and anti-angiogenic drugs may be an innovative and promising therapeutic strategy in the experimental treatment of human lung cancer.</p