Pre-clinical investigation of carnosine’s anti-neoplastic effect on glioblastoma: uptake, signal transduction, gene expression and tumour cell metabolism

Das Glioblastom ist der häufigste maligne Tumor des zentralen Nervensystems. Trotz leitliniengerechter Therapie, bestehend aus mikrochirurgischer Resektion, Strahlentherapie und ergänzender Chemotherapie mit Temozolomid, beträgt die 2-Jahres-Überlebensrate nur ca. 17%. Daher sind dringend neue Therapieansätze erforderlich. Dem natürlich vorkommenden Dipeptid Carnosin, welches vor über 100 Jahren erstmals isoliert wurde, konnten viele physiologische Funktionen zugeschrieben werden. Zu Beginn unserer Arbeiten war bekannt, dass das Dipeptid das Wachstum von Krebszellen inhibiert, wobei die genauen Mechanismen der antineoplastischen Wirkungsweise weitgehend unbekannt waren. Die Untersuchungen im Rahmen der vorliegenden Habilitationsarbeit setzten sich mit möglichen Wirkmechanismen des Dipeptides auseinander, wobei ebenfalls Fragestellungen zur klinischen Anwendung von Carnosin bearbeitet wurden. Im ersten Abschnitt werden die Transportmechanismen von Carnosin in Glioblastom-Zellen beschrieben. Weiterhin wird die Frage beantwortet, ob das Dipeptid die biologisch aktive Verbindung ist oder ob L-Histidin von Carnosin abgespalten werden muss, um die antineoplastische Wirkung zu entfalten. Der zweite Abschnitt beschäftigt sich mit den Einflüssen von Carnosin auf die Signaltransduktion und Genexpression. Im dritten Abschnitt wird unter anderem mit einem Metabolomics-Ansatz der Stoffwechsel von Glioblastom-Zellen charakterisiert und der Einfluss von Carnosin auf diesen bestimmt. Im vierten Abschnitt wird ein neuartiges Ko-Kultur Modell zur Untersuchung von Carnosins Einfluss auf Glioblastom-Zell-Migration und Koloniebildung vorgestellt. Weiterhin untersuchten wir die möglichen Interaktionen des Dipeptides mit der Standardtherapie von Glioblastomen. Zusammenfassend zeigten wir, dass Carnosin durch drei verschiedene Transporter aufgenommen werden kann. Das Dipeptid hemmt sowohl Proliferation und Migration von Glioblastom-Zellen. Die Spaltung des Dipeptides ist für seine antineoplastische Wirkung nicht notwendig. In die Zelle aufgenommen, wirkt Carnosin inhibitorisch auf den Pentosephosphatweg. Eine mögliche Erklärung dafür lieferte die beobachte nicht-enzymatische Reaktion von Glycerinaldehyd-3-phosphat mit dem Dipeptid. Weiterhin zeigten unsere Experimente zum ersten Mal eine Carnosin-bedingte Veränderung der Histonacetylierung und eine damit einhergehende Beeinflussung der Genexpression. Da das Dipeptid den Effekt der Radio-/Chemotherapie verstärkt, sollte die Wirkung von Carnosin in einer klinischen Studie an Glioblastom-Patienten untersucht werden.Glioblastoma is the most common malignant tumour of the central nervous system. Only ~17% of patients undergoing standard therapy, including microsurgical resection, radiotherapy and adjuvant chemotherapy using temozolomide survive two years after diagnosis. Hence, new therapeutic approaches are urgently needed. The naturally occurring dipeptide carnosine was discovered more than 100 years ago. Since then, many physiological functions and beneficial effects have been ascribed to it. Previous studies demonstrated that carnosine inhibits growth of cancer cells. However, at the beginning of our investigations were the mechanisms behind carnosine’s anti-neoplastic effect mostly unknown. The present work addresses possible modes of action of carnosine and issues regarding the clinical application of the dipeptide. In the first paragraph we describe the transport mechanisms of carnosine in glioblastoma cells. Furthermore, we deal with the problem whether carnosine is the biological active compound or release of L-histidine from the dipeptide is required to deploy its anti-neoplastic effect. The second paragraph addresses the influence of carnosine on glioblastoma cell signal transduction and gene expression. In the third paragraph we characterise the metabolism of glioblastoma cells and how it is influenced by carnosine by using a metabolomics approach. The fourth paragraph introduces a novel co-culture model which allows the analysis of carnosine’s impact on glioblastoma cell migration and colony formation. Furthermore, the possible interaction of the dipeptide with the glioblastoma standard therapy is investigated. In conclusion, we demonstrated that three different transporters are capable for the uptake of carnosine in glioblastoma cells. The dipeptide inhibited in addition to proliferation also migration of glioblastoma cells. Moreover, cleavage of carnosine was not required for its anti-neoplastic effect. After taken up by the cell, carnosine inhibits the pentose phosphate pathway. The observed non-enzymatic reaction of glyceraldehyde-3-phosphate with the dipeptide could possibly explain this effect. Furthermore, our experiments showed for the first time that carnosine influences gene expression by an effect on histone acetylation. As the administration of carnosine arguments the effects of radio-/chemotherapy, we encourage the clinical evaluation of the dipeptide for glioblastoma patients

Untersuchungen zur Regulation des Glucosestoffwechsels in Glioblastomen und dessen Beeinflussung durch Carnosin: Untersuchungen zur Regulation des Glucosestoffwechsels inGlioblastomen und dessen Beeinflussung durch Carnosin

Das Glioblastoma multiforme (GBM) ist der am häufigsten vorkommende maligne Hirntumor mit äußerst ungünstiger Prognose für die betroffenen Patienten. Typisch für die Tumore ist eine hohe Aktivität der Glykolyse zur Generierung von ATP und zur Bereitstellung von Makromolekülen für die Zellproliferation, während die oxidative Phosphorylierung auch in Gegenwart von Sauerstoff praktisch keine Bedeutung für die Generation von ATP hat, was auch als Warburg Effekt bekannt ist. Das natürlich vorkommende Carnosin (β-Alanyl-LHistidin) wirkt sich antiproliferativ auf Tumorzellen aus, was mit einer Inhibition der glykolytischen ATP Produktion einhergeht. Der Mechanismus der Inhibition ist weitgehend unverstanden und ist Gegenstand der vorliegenden Arbeit. Im Rahmen der durchgeführten Arbeit wurde der Einfluss von Carnosin auf die mRNA Expressionen von für die Glykolyse relevanten Genen untersucht, wobei eine starke Induktion der Pyruvatdehydrogenase Kinase (PDK) 4 in drei GBM Zelllinien beobachtet wurde. Weiterhin konnte gezeigt werden, dass L-Histidin den gleichen Effekt wie Carnosin zeigt, nicht jedoch β-Alanin, L-Alanin oder L-Alanyl-L-Histidin. Da Tumorzellen die intrazelluläre Gewebscarnosinase aber kaum die extrazelluläre Serumcarnosinase exprimieren, liegt die Vermutung nahe, dass die antineoplastische Wirkung des Carnosins auf die enzymatische Spaltung von Carnosin und die daraus resultierende Freisetzung von L-Histidin zurückzuführen ist. In weiteren Untersuchungen wurden Hinweise erbracht, dass Carnosin durch eine Beeinflussung von Histon-Deacetylasen, die endogene PDK4 mRNA Expression steigern könnte. Zusätzlich wurden die Proteinexpressionen der PDK1 und 4 unter dem Einfluss von Carnosin untersucht

CUX1-related neurodevelopmental disorder: Deep insights into phenotype-genotype spectrum and underlying pathology

Heterozygous, pathogenic CUX1 variants are associated with global developmental delay or intellectual disability. This study delineates the clinical presentation in an extended cohort and investigates the molecular mechanism underlying the disorder in a Cux

Erythrocytes Prevent Degradation of Carnosine by Human Serum Carnosinase

The naturally occurring dipeptide carnosine (-alanyl-L-histidine) has beneficial effects in different diseases. It is also frequently used as a food supplement to improve exercise performance and because of its anti-aging effects. Nevertheless, after oral ingestion, the dipeptide is not detectable in human serum because of rapid degradation by serum carnosinase. At the same time, intact carnosine is excreted in urine up to five hours after intake. Therefore, an unknown compartment protecting the dipeptide from degradation has long been hypothesized. Considering that erythrocytes may constitute this compartment, we investigated the uptake and intracellular amounts of carnosine in human erythrocytes cultivated in the presence of the dipeptide and human serum using liquid chromatography–mass spectrometry. In addition, we studied carnosine’s effect on ATP production in red blood cells and on their response to oxidative stress. Our experiments revealed uptake of carnosine into erythrocytes and protection from carnosinase degradation. In addition, no negative effect on ATP production or defense against oxidative stress was observed. In conclusion, our results for the first time demonstrate that erythrocytes can take up carnosine, and, most importantly, thereby prevent its degradation by human serum carnosinase

Viability of Glioblastoma Cells and Fibroblasts in the Presence of Imidazole-Containing Compounds

The naturally occurring dipeptide carnosine (-alanyl-L-histidine) specifically attenuates tumor growth. Here, we ask whether other small imidazole-containing compounds also affect the viability of tumor cells without affecting non-malignant cells and whether the formation of histamine is involved. Patient-derived fibroblasts and glioblastoma cells were treated with carnosine, L-alanyl-L-histidine (LA-LH), -alanyl-L-alanine, L-histidine, histamine, imidazole, -alanine, and L-alanine. Cell viability was assessed by cell-based assays and microscopy. The intracellular release of L-histidine and formation of histamine was investigated by high-performance liquid chromatography coupled to mass spectrometry. Carnosine and LA-LH inhibited tumor cell growth with minor effects on fibroblasts, and L-histidine, histamine, and imidazole affected viability in both cell types. Compounds without the imidazole moiety did not diminish viability. In the presence of LA-LH but not in the presence of carnosine, a significant rise in intracellular amounts of histidine was detected in all cells. The formation of histamine was not detectable in the presence of carnosine, LA-LH, or histidine. In conclusion, the imidazole moiety of carnosine contributes to its anti-neoplastic effect, which is also seen in the presence of histidine and LA-LH. Despite the fact that histamine has a strong effect on cell viability, the formation of histamine is not responsible for the effects on the cell viability of carnosine, LA-LH, and histidine

The Relation of Surgical Procedures and Diagnosis Groups to Unplanned Readmission in Spinal Neurosurgery: A Retrospective Single Center Study

Background: Unplanned readmission has gained increasing interest as a quality marker for inpatient care, as it is associated with patient mortality and higher economic costs. Spinal neurosurgery is characterized by a lack of epidemiologic readmission data. The aim of this study was to identify causes and predictors for unplanned readmissions related to index diagnoses and surgical procedures. Methods: In this study, from 2015 to 2017, spinal neurosurgical procedures were recorded for surgical and non-surgical treated patients. The main reasons for an unplanned readmission within 30 days following discharge were identified. Multivariate logarithmic regression revealed predictors of unplanned readmission. Results: A total of 1172 patient records were examined, of which 4.27% disclosed unplanned readmissions. Among the surgical patients, the readmission rate was 4.06%, mainly attributable to surgical site infections, while it was 5.06% for the non-surgical patients, attributable to uncontrolled pain. A night-time surgery presented as the independent predictive factor. Conclusion: In the heterogeneous group of spinal neurosurgical patients, stratification into diagnostic groups is necessary for statistical analysis. Degenerative lumbar spinal stenosis and spinal abscesses are mainly affected by unplanned readmission. The surgical procedure dorsal root ganglion stimulation is an independent predictor of unplanned re-hospitalizations, as is the timing of surgery

Analysis of cellular and molecular antitumor effects upon inhibition of SATB1 in glioblastoma cells

Background: The Special AT-rich Sequence Binding Protein 1 (SATB1) regulates the expression of many genes by acting as a global chromatin organizer. While in many tumor entities SATB1 overexpression has been observed and connected to pro-tumorigenic processes, somewhat contradictory evidence exists in brain tumors with regard to SATB1 overexpression in glioblastoma and its association with poorer prognosis and tumor progression. On the functional side, initial data indicate that SATB1 may be involved in several tumor cell-relevant processes. Methods: For the detailed analysis of the functional relevance and possible therapeutic potential of SATB1 inhibition, we employ transient siRNA-mediated knockdown and comprehensively analyze the cellular and molecular role of SATB1 in glioblastoma. Results: In various cell lines with different SATB1 expression levels, a SATB1 gene dose-dependent inhibition of anchorage-dependent and –independent proliferation is observed. This is due to cell cycle-inhibitory and pro-apoptotic effects of SATB1 knockdown. Molecular analyses reveal SATB1 knockdown effects on multiple important (proto-) oncogenes, including Myc, Bcl-2, Pim-1, EGFR, β-catenin and Survivin. Molecules involved in cell cycle, EMT and cell adhesion are affected as well. The putative therapeutic relevance of SATB1 inhibition is further supported in an in vivo tumor xenograft mouse model, where the treatment with polymeric nanoparticles containing SATB1-specific siRNAs exerts antitumor effects. Conclusion: Our results demonstrate that SATB1 may represent a promising target molecule in glioblastoma therapy whose inhibition or knockdown affects multiple crucial pathways

Metabolic response of glioblastoma cells associated with glucose withdrawal and pyruvate substitution as revealed by GC-MS

Background: Tumor cells are highly dependent on glucose even in the presence of oxygen. This concept called the Warburg effect is a hallmark of cancer and strategies are considered to therapeutically exploit the phenomenon such as ketogenic diets. The success of such strategies is dependent on a profound understanding of tumor cell metabolism. With new techniques it is now possible to thoroughly analyze the metabolic responses to the withdrawal of substrates and their substitution by others. In the present study we used gas chromatography coupled to mass spectrometry (GC-MS) to analyze how glioblastoma brain tumor cells respond metabolically when glucose is withdrawn and substituted by pyruvate. Methods: Glioblastoma brain tumor cells were cultivated in medium with high (25 mM), medium (11 mM) or low (5.5 mM) glucose concentration or with pyruvate (5 mM). After 24 h GC-MS metabolite profiling was performed. Results: The abundances of most metabolites were dependent on the supply of glucose in tendency but not in a linear manner indicating saturation at high glucose. Noteworthy, a high level of sorbitol production and release was observed at high concentrations of glucose and high release of alanine, aspartate and citrate were observed when glucose was substituted by pyruvate. Intermediates of the TCA cycle were present under all nutritional conditions and evidence was found that cells may perform gluconeogenesis from pyruvate. Conclusions: Our experiments reveal a high plasticity of glioblastoma cells to changes in nutritional supply which has to be taken into account in clinical trials in which specific diets are considered for therapy

The behaviour of repeat visitors to museums: Review and empirical findings

This study presents a theoretical and operational framework for analysing repeat visit to museums. Starting from the literature on repeat visit in tourism, the specificities of these cultural attractions are made explicit through a review of theoretical and applied works. Consistently with previous contributors, the paper suggests that the analysis of actual past behaviours has to be preferred to the one of attitudes. The application of proper econometric models is also remarked in order to put into account individual profiles. Information coming from three techniques is then used in an integrated way in order to provide a more comprehensive view of the phenomenon. Evidence from an ad hoc survey suggests the necessity to give a greater attention to perceived cultural value during the visit, promoting cultural events during the week and addressed to children, and taking care of those visitors that come from far places also through an integrated tourist supply. © 2013 Springer Science+Business Media Dordrecht

Broadening the phenotypic and molecular spectrum of FINCA syndrome: Biallelic NHLRC2 variants in 15 novel individuals

FINCA syndrome [MIM: 618278] is an autosomal recessive multisystem disorder characterized by fibrosis, neurodegeneration and cerebral angiomatosis. To date, 13 patients from nine families with biallelic NHLRC2 variants have been published. In all of them, the recurrent missense variant p.(Asp148Tyr) was detected on at least one allele. Common manifestations included lung or muscle fibrosis, respiratory distress, developmental delay, neuromuscular symptoms and seizures often followed by early death due to rapid disease progression.Here, we present 15 individuals from 12 families with an overlapping phenotype associated with nine novel NHLRC2 variants identified by exome analysis. All patients described here presented with moderate to severe global developmental delay and variable disease progression. Seizures, truncal hypotonia and movement disorders were frequently observed. Notably, we also present the first eight cases in which the recurrent p.(Asp148Tyr) variant was not detected in either homozygous or compound heterozygous state.We cloned and expressed all novel and most previously published non-truncating variants in HEK293-cells. From the results of these functional studies, we propose a potential genotype-phenotype correlation, with a greater reduction in protein expression being associated with a more severe phenotype.Taken together, our findings broaden the known phenotypic and molecular spectrum and emphasize that NHLRC2-related disease should be considered in patients presenting with intellectual disability, movement disorders, neuroregression and epilepsy with or without pulmonary involvement