UPON THE ETIOLOGICAL TREATMENT OF BACTERIAL NONGONOCOCCAL URETHRITIS

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miRNA-145 is downregulated in atypical and anaplastic meningiomas and negatively regulates motility and proliferation of meningioma cells

Meningiomas are frequent, mostly benign intracranial or spinal tumors. A small subset of meningiomas is characterized by histological features of atypia or anaplasia that are associated with more aggressive biological behavior resulting in increased morbidity and mortality. Infiltration into the adjacent brain tissue is a major factor linked to higher recurrence rates. The molecular mechanisms of progression, including brain invasion are still poorly understood. We have studied the role of micro-RNA 145 (miR-145) in meningiomas and detected significantly reduced miR-145 expression in atypical and anaplastic tumors as compared with benign meningiomas. Overexpression of miR-145 in IOMM-Lee meningioma cells resulted in reduced proliferation, increased sensitivity to apoptosis, reduced anchorage-independent growth and reduction of orthotopic tumor growth in nude mice as compared with control cells. Moreover, meningioma cells with high miR-145 levels had impaired migratory and invasive potential in vitro and in vivo. PCR-array studies of miR145-overexpressing cells suggested that collagen type V alpha (COL5A1) expression is downregulated by miR-145 overexpression. Accordingly, COL5A1 expression was significantly upregulated in atypical and anaplastic meningiomas. Collectively, our data indicate an important anti-migratory and anti-proliferative function of miR-145 in meningiomas

Impact of Salinomycin-treatment on PHH synthesis function.

<p>Functionality of PHH after treatment with Salinomycin was analyzed by urea formation and albumin synthesis. (<b>A</b>) 24 hours of Salinomycin exposure at varying concentrations was not accompanied by impaired urea formation at all. (<b>B</b>) In contrast, treatment for 48 hours resulted in a dose-dependent decrease of urea formation at days 1, 3 and 5 without reaching statistical significance. (<b>C+D</b>) Albumin synthesis was markedly impaired at day 1 after stimulation with Salinomycin for 24 and 48 hours, respectively. Further incubation led to continuous recovery of albumin synthesis in the groups with 24 hours of drug exposure. In contrast, treatment for 48 hours resulted only in moderate recovery of albumin synthesis (n = 3). * p<0.05, ** p<0.005.</p

Salinomycin inhibits HepG2 and Huh7 autophagic flux.

<p>HepG2 and Huh7 cells were exposed to different concentrations of Salinomycin (0.5, 2 and 8 µM) for different time points and analyzed for activity of autophagy. (<b>A</b>) Immunoblot analysis of LC3-I and LC3-II-isoforms (up) with densitometry quantitative analysis (down) in HepG2 cells revealed Sal-induced LC3-II-accumulation due to inhibition of autophagic flux as demonstrated by reduced LC3-II-accumulation after addition of ACH. (<b>B</b>) Basic and PP242-activated autophagic flux in Huh7 cells (black bars). Treatment with autophagy inhibitors 3MA (2 and 10 mM), ACH (5 and 20 mM) or CQ (5, 20 µM) for 24 h counteracts PP242 activation of autophagic flux. (<b>C</b>) Inhibition of autophagic flux in Huh7 cells after shRNA-mediated knockdown of ATG7. (<b>D+E</b>) Decreased accumulation of autophagic compartments after the blockage of autophagolysosomal degradation by ACH indicates reduced autophagic flux in HepG2 (<b>D</b>) and Huh7 (<b>E</b>) cells treated with Sal for 24 h. Next to the bar graphs representative histograms are depicted. All experiments are presented as mean ± SD of n = 3 independent experiments.*p<0.05; **p<0.01, ***p<0.001.</p

Salinomycin impairs HCC cell survival.

<p>HepG2 and Huh7 cells were exposed to increasing concentrations of Salinomycin (1, 2, 5 and 10 µM) for 24 hours. (<b>A</b>) Cell viability was assessed by MTS-assay; high concentrations of Salinomycin led to significantly decreased viability of both cell lines (n = 5). (<b>B</b>) HCC cells revealed significantly reduced proliferation after exposure to Salinomycin as demonstrated by decreased ³H-Thymidine uptake. (<b>C</b>) Low concentrations of Salinomycin (left panel, solid line) led to weak increase of apoptotic cells compared to untreated cells (dotted line in overlay). High concentrations markedly induced apoptosis (right panel, solid line). Results are shown as representative scatter-grams of Annexin-V⁺-cells or summarized as mean ± SD of n = 4 independent experiments (<b>D</b>). *p<0.05, **p<0.01.</p

Salinomycin-mediated inhibition of autophagic flux relates to accumulation of dysfunctional mitochondria and increased ROS-production.

<p>(<b>A</b>) Inhibition of basic and HBSS-activated autophagic flux in HepG2 cells stably expressing LC3-GFP treated with 3 MA (2 and 10 mM), LY (2 and 10 mM), nocodazole (2 and 10 µM) or ACH (0.8 and 4 mM) for 7 h. (<b>B</b>) Inhibition of autophagic flux in HepG2 cells treated with Sal (0.5, 2 and 8 µM) for 24 h (left) with representative histograms (right). (<b>C</b>) Flow-cytometric analysis of total mitochondrial mass using MitoTracker Green (MTR green) reveals accumulation of dysfunctional mitochondria. (<b>D</b>) Evidence of increased ROS-production using CM-H2DCFDA (left) with representative histograms (right). All experiments are presented as mean ± SD of n = 3 independent experiments. *p<0.05, **p<0.01.</p

Assessment of apoptosis-induction in PHH by Salinomycin.

<p>Cultured PHH were exposed to increasing concentrations of Salinomycin (1, 2, 5 and 10 µM) for 24 hours. Apoptosis was detected by M30 Cytodeath kit. Treatment with Fas Ligand (FasL) served as a positive control. Staining of PHH with DAPI (blue) and M30 Cytodeath kit (red) revealed no evidence for induction of apoptosis in PHH even after treatment with high concentrations of Salinomycin. In contrast, FasL clearly induced apoptosis in PHH.</p