Open Access

Das Glioblastom ist ein WHO Grad 4-Tumor und einer der häufigsten und zugleich agressivsten Hirntumoren im Erwachsenenalter. Trotz multimodaler Therapie, die eine neurochirurgische Resektion sowie eine adjuvante Radiochemotherapie und als neuen Therapieansatz eine Kombination aus Temozolomid und tumor treating fields umfasst, ist die Prognose weiterhin schlecht, sodass der Suche nach neuen therapeutischen Zielstrukturen eine maßgebliche Bedeutung zukommt. Für verschiedene Tumorentitiäten konnte gezeigt werden, dass die Überexpression einzelner onkogener Kinasen die Tumorprogression vorantreibt, wobei bei Glioblastomen gezeigt werden konnte, dass die Serin-Threonin-Kinase Pim1 eine wichtige Rolle in der Pathogenese einnimmt. In den Fokus rücken zunehmend auch stammzellähnliche Tumorzellen, die eine Subpopulation innerhalb von Glioblastomen darstellen und das aggressive biologische Verhalten sowie die Resistenz gegenüber der Standardtherapie und eine hohe Rezidivrate vermitteln können. In dieser Arbeit sollte dementsprechend basierend auf den bisherigen Erkenntnissen zu Pim1 sowie zur Bedeutung von Tumorstammzellen im malignen Geschehen der Einfluss der Serin-Threonin-Kinase Pim1 auf das Stammzellverhalten von Glioblastomzellen näher untersucht werden. Durch den Vergleich von adhärent wachsenden Tumorzellen der Glioblastomzelllinie LN-18 mit stammzellähnlichen LN-18 Neurosphären konnte eine erhöhte relative mRNA-Expression von Pim1 und EGFR sowie der potentiellen Stammzellmarker Nestin, CD44, CD133 und Musashi-1 nachgewiesen werden. Die relative Proteinexpression von Pim1 sowie der Stammzellmarker Nestin, CD44, CD133 und Sox2 war in den Neurosphären im Vergleich zu den adhärent wachsenden LN-18 Zellen ebenfalls gesteigert. Diese Daten konnten durch die Immunfluoreszenz-Färbungen bestätigt werden. Ein effizienter siRNA-vermittelter knockdown von Pim1 auf Proteinebene konnte in dieser Arbeit nicht erzielt werden, sodass keine Aussagen zu einer Regulation von Stammzell- und Differenzierungsmarker nach zielgerichteter genetischer Abschaltung von Pim1 getroffen werden konnten. Hier sind weiterführend Optimierungen notwendig oder der Einsatz spezieller CRISPR-Cas9-Verfahren zur genetischen Ausschaltung sinnvoll. Die pharmakologische Inhibition von Pim1 mit LY294002 und TCS Pim1-1 führte zu einer signifikanten Reduktion der Neurosphärenformation sowie der Zellviabilität bei LN-18 Zellen, wodurch die in Vorarbeiten an adhärenten Glioblastomzellen gewonnenen Daten um Untersuchungen an stammzellartigen Glioblastomzellen erweitert wurden. Zusammenfassend legen die in dieser Arbeit erhobenen Daten nahe, dass Pim1 das Stammzellverhalten von Glioblastomzellen beeinflusst, indem Pim1 Einfluss auf die Expression von Stammzellmarkern nimmt und seine Inhibition die Aufrechterhaltung einer Glioblastomstammzellpopulation beeinträchtigt, indem die Neurosphärenformation und die Viabilität der Zellen stark reduziert werden. Somit stellt Pim1 eine geeignete Zielstruktur für eine zielgerichtete Therapieoption beim Glioblastom dar, beispielsweise in Kombination mit der klassischen Radiochemotherapie. Zukünftige Studien müssen zeigen, inwieweit eine selektive Pim1-Inhibition tatsächlich Einfluss auf die Prognose von Patienten mit Glioblastom nimmt.

Renal drug transporters such as the organic cation transporters (OCTs), organic anion transporters (OATs) and multidrug resistance proteins (MRPs) play an important role in the tubular secretion of many drugs influencing their efficacy and safety. However, only little is known about the distinct protein abundance of these transporters in human kidneys, and about the impact of age and gender as potential factors of inter-subject variability in their expression and function. The aim of this study was to determine the protein abundance of MDR1, MRP1-4, BCRP, OAT1-3, OCT2-3, MATE1, PEPT1/2, and ORCTL2 by liquid chromatography-tandem mass spectrometry-based targeted proteomics in a set of 36 human cortex kidney samples (20 males, 16 females; median age 53 and 55 years, respectively). OAT1 and 3, OCT2 and ORCTL2 were found to be most abundant renal SLC transporters while MDR1, MRP1 and MRP4 were the dominating ABC transporters. Only the expression levels of MDR1 and ORCTL2 were significantly higher abundant in older donors. Moreover, we found several significant correlations between different transporters, which may indicate their functional interplay in renal vectorial transport processes. Our data may contribute to a better understanding of the molecular processes determining renal excretion of drugs.

The multifunctional sphingosine-1-phosphate (S1P) is a lipid signaling molecule and central regulator in the development of several cancer types. In recent years, intriguing information has become available regarding the role of S1P in the progression of Glioblastoma multiforme (GBM), the most aggressive and common brain tumor in adults. S1P modulates numerous cellular processes in GBM, such as oncogenesis, proliferation and survival, invasion, migration, metastasis and stem cell behavior. These processes are regulated via a family of five G-protein-coupled S1P receptors (S1PR1-5) and may involve mainly unknown intracellular targets. Distinct expression patterns and multiple intracellular signaling pathways of each S1PR subtype enable S1P to exert its pleiotropic cellular actions. Several studies have demonstrated alterations in S1P levels, the involvement of S1PRs and S1P metabolizing enzymes in GBM pathophysiology. While the tumorigenic actions of S1P involve the activation of several kinases and transcription factors, the specific G-protein (Gi, Gq, and G12/13)-coupled signaling pathways and downstream mediated effects in GBM remain to be elucidated in detail. This review summarizes the recent findings concerning the role of S1P and its receptors in GBM. We further highlight the current insights into the signaling pathways considered fundamental for regulating the cellular processes in GMB and ultimately patient prognosis.

Exogenous glucocorticoids increase the risk for osteoporosis, but the role of endogenous glucocorticoids remains elusive. Here, we describe the generation and validation of a loss- and a gain-of-function model of the cortisol producing enzyme 11β-HSD1 (HSD11B1) to modulate the endogenous glucocorticoid conversion in SCP-1 cells — a model for human mesenchymal stem cells capable of adipogenic and osteogenic differentiation. CRISPR-Cas9 was successfully used to generate a cell line carrying a single base duplication and a 5 bp deletion in exon 5, leading to missense amino acid sequences after codon 146. These inactivating genomic alterations were validated by deep sequencing and by cloning with subsequent capillary sequencing. 11β-HSD1 protein levels were reduced by 70% in the knockout cells and cortisol production was not detectable. Targeted chromosomal integration was used to stably overexpress HSD11B1. Compared to wildtype cells, HSD11B1 overexpression resulted in a 7.9-fold increase in HSD11B1 mRNA expression, a 5-fold increase in 11β-HSD1 protein expression and 3.3-fold increase in extracellular cortisol levels under adipogenic differentiation. The generated cells were used to address the effects of 11β-HSD1 expression on adipogenic and osteogenic differentiation. Compared to the wildtype, HSD11B1 overexpression led to a 3.7-fold increase in mRNA expression of lipoprotein lipase (LPL) and 2.5-fold increase in lipid production under adipogenic differentiation. Under osteogenic differentiation, HSD11B1 knockout led to enhanced alkaline phosphatase (ALP) activity and mRNA expression, and HSD11B1 overexpression resulted in a 4.6-fold and 11.7-fold increase in mRNA expression of Dickkopf-related protein 1 (DKK1) and LPL, respectively. Here we describe a HSD11B1 loss- and gain-of-function model in SCP-1 cells at genetic, molecular and functional levels. We used these models to study the effects of endogenous cortisol production on mesenchymal stem cell differentiation and demonstrate an 11β-HSD1 dependent switch from osteogenic to adipogenic differentiation. These results might help to better understand the role of endogenous cortisol production in osteoporosis on a molecular and cellular level.

Membrane monocarboxylate transporter 1 (SLC16A1/MCT1) plays an important role in hepatocyte homeostasis, as well as drug handling. However, there is no available information about the impact of liver pathology on the transporter levels and function. The study was aimed to quantify SLC16A1 mRNA (qRT-PCR) and MCT1 protein abundance (liquid chromatography–tandem mass spectrometry (LC¬¬–MS/MS)) in the livers of patients diagnosed, according to the standard clinical criteria, with hepatitis C, primary biliary cirrhosis, primary sclerosing hepatitis, alcoholic liver disease (ALD), and autoimmune hepatitis. The stage of liver dysfunction was classified according to Child–Pugh score. Downregulation of SLC16A1/MCT1 levels was observed in all liver pathology states, significantly for ALD. The progression of liver dysfunction, from Child–Pugh class A to C, involved the gradual decline in SLC16A1 mRNA and MCT1 protein abundance, reaching a clinically significant decrease in class C livers. Reduced levels of MCT1 were associated with significant intracellular lactate accumulation. The MCT1 transcript and protein did not demonstrate significant correlations regardless of the liver pathology analyzed, as well as the disease stage, suggesting posttranscriptional regulation, and several microRNAs were found as potential regulators of MCT1 abundance. MCT1 membrane immunolocalization without cytoplasmic retention was observed in all studied liver pathologies. Overall, the study demonstrates that SLC16A1/MCT1 is involved in liver pathology, especially in ALD

Despite comprehensive therapy and extensive research, glioblastoma (GBM) still represents the most aggressive brain tumor in adults. Glioma stem cells (GSCs) are thought to play a major role in tumor progression and resistance of GBM cells to radiochemotherapy. The PIM1 kinase has become a focus in cancer research. We have previously demonstrated that PIM1 is involved in survival of GBM cells and in GBM growth in a mouse model. However, little is known about the importance of PIM1 in cancer stem cells. Here, we report on the role of PIM1 in GBM stem cell behavior and killing. PIM1 inhibition negatively regulates the protein expression of the stem cell markers CD133 and Nestin in GBM cells (LN-18, U-87 MG). In contrast, CD44 and the astrocytic differentiation marker GFAP were up-regulated. Furthermore, PIM1 expression was increased in neurospheres as a model of GBM stem-like cells. Treatment of neurospheres with PIM1 inhibitors (TCS PIM1-1, Quercetagetin, and LY294002) diminished the cell viability associated with reduced DNA synthesis rate, increased caspase 3 activity, decreased PCNA protein expression, and reduced neurosphere formation. Our results indicate that PIM1 affects the glioblastoma stem cell behavior, and its inhibition kills glioblastoma stem-like cells, pointing to PIM1 targeting as a potential anti-glioblastoma therapy.

Transmembrane drug transport in hepatocytes is one of the major determinants of drug pharmacokinetics. In the present study, ABC transporters (P-gp, MRP1, MRP2, MRP3, MRP4, BCRP, and BSEP) and SLC transporters (MCT1, NTCP, OAT2, OATP1B1, OATP1B3, OATP2B1, OCT1, and OCT3) were quantified for protein abundance (LC-MS/MS) and mRNA levels (qRT-PCR) in hepatitis C virus (HCV)-infected liver samples from the Child–Pugh class A (n = 30), B (n = 21), and C (n = 7) patients. Protein levels of BSEP, MRP3, MCT1, OAT2, OATP1B3, and OCT3 were not significantly affected by HCV infection. P-gp, MRP1, BCRP, and OATP1B3 protein abundances were upregulated, whereas those of MRP2, MRP4, NTCP, OATP2B1, and OCT1 were downregulated in all HCV samples. The observed changes started to be seen in the Child–Pugh class A livers, i.e., upregulation of P-gp and MRP1 and downregulation of MRP2, MRP4, BCRP, and OATP1B3. In the case of NTCP, OATP2B1, and OCT1, a decrease in the protein levels was observed in the class B livers. In the class C livers, no other changes were noted than those in the class A and B patients. The results of the study demonstrate that drug transporter protein abundances are affected by the functional state of the liver in hepatitis C patients.

Postoperative restenosis in patients with external ear canal (EEC) atresia or stenosis is a common complication following canaloplasty. Our aim in this study was to explore the feasibility of using a three dimensionally (3D)-printed, patient-individualized, drug ((dexamethasone (DEX)), and ciprofloxacin (cipro))-releasing external ear canal implant (EECI) as a postoperative stent after canaloplasty. We designed and pre-clinically tested this novel implant for drug release (by high-performance liquid chromatography), biocompatibility (by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay), bio-efficacy (by the TNF-α (tumor necrosis factor-alpha)-reduction test (DEX) and inhibition zone test (for cipro)), and microbial contamination (formation of turbidity or sediments in culture medium). The EECI was implanted for the first time to one patient with a history of congenital EEC atresia and state after three canaloplasties due to EEC restenosis. The preclinical tests revealed no cytotoxic effect of the used materials; an antibacterial effect was verified against the bacteria Staphylococcus aureus and Pseudomonas aeruginosa, and the tested UV-irradiated EECI showed no microbiological contamination. Based on the test results, the combination of silicone with 1% DEX and 0.3% cipro was chosen to treat the patient. The EECI was implantable into the EEC; the postoperative follow-up visits revealed no otogenic symptoms or infections and the EECI was explanted three months postoperatively. Even at 12 months postoperatively, the EEC showed good epithelialization and patency. Here, we report the first ever clinical application of an individualized, drug-releasing, mechanically flexible implant and suggest that our novel EECI represents a safe and effective method for postoperatively stenting the reconstructed EEC.

OCT1 and OCT2 are polyspecific membrane transporters that are involved in hepatic and renal drug clearance in humans and mice. In this study, we cloned dog OCT1 and OCT2 and compared their function to the human and mouse orthologs. We used liver and kidney RNA to clone dog OCT1 and OCT2. The cloned and the publicly available RNA-Seq sequences differed from the annotated exon-intron structure of OCT1 in the dog genome CanFam3.1. An additional exon between exons 2 and 3 was identified and confirmed by sequencing in six additional dog breeds. Next, dog OCT1 and OCT2 were stably overexpressed in HEK293 cells and the transport kinetics of five drugs were analyzed. We observed strong differences in the transport kinetics between dog and human orthologs. Dog OCT1 transported fenoterol with 12.9-fold higher capacity but 14.3-fold lower affinity (higher KM) than human OCT1. Human OCT1 transported ipratropium with 5.2-fold higher capacity but 8.4-fold lower affinity than dog OCT1. Compared to human OCT2, dog OCT2 showed 10-fold lower transport of fenoterol and butylscopolamine. In conclusion, the functional characterization of dog OCT1 and OCT2 reported here may have implications when using dogs as pre-clinical models as well as for drug therapy in dogs.

This communication introduces the first-time application of high-resolution continuum-source molecular absorption spectrometry (HR CS MAS) for the quantification of a peptide. The graphite furnace technique was employed and the tripeptide glutathione (GSH) served as a model compound. Based on measuring sulfur in terms of carbon monosulfide (CS), a method was elaborated to analyze aqueous solutions of GSH. The most prominent wavelength of the CS molecule occurred at 258.0560 nm and was adduced for monitoring. The methodological development covered the optimization of the pyrolysis and vaporization temperatures. These were found optimally to be 250 °C and 2250 °C, respectively. Moreover, the effect of modifiers (zirconium, calcium, magnesium, palladium) on the absorption signals was investigated. The best results were obtained after permanent coating of the graphite tube with zirconium (total amount of 400 μg) and adding a combination of palladium (10 µL, 10 g L−1) and calcium (2 µL, 1 g L−1) as a chemical modifier to the probes (10 µL). Aqueous standard samples of GSH were used for the calibration. It showed a linear range of 2.5–100 µg mL−1 sulfur contained in GSH with a correlation coefficient R2 > 0.997. The developed method exhibited a limit of detection (LOD) and quantification (LOQ) of 2.1 µg mL−1 and 4.3 µg mL−1 sulfur, respectively. The characteristic mass accounted for 5.9 ng sulfur. The method confirmed the general suitability of MAS for the analysis of an oligopeptide. Thus, this study serves as groundwork for further development in order to extend the application of classical atomic absorption spectrometry (AAS).