Metabolism and neural differentiation in childhood neuroblastoma

Neuroblastoma is the most common and aggressive extracranial solid tumor during childhood. MYCN-amplification is found in approximately 25 % of all neuroblastoma cases, and is defined as high-risk disease. Development of novel therapeutic approaches focused on MYCN targeting are essential for increased survival these children. The MYC family of oncoproteins consists of transcriptional factors involved in many normal cellular processes. Abnormal expression of MYC is associated with 70 % of human cancers and correlates with an aggressive undifferentiated phenotype, chemotherapy resistance and poor clinical prognosis. Targeting MYCN by small molecular weight molecules remains a challenge. In paper I we established that one known c-MYC targeting compound, the small chemical molecule 10058-F4, is also a potent MYCN inhibitor. 10058-F4 treatment increased cell death and neuronal differentiation in MYCN-amplified neuroblastoma cells and prolonged survival in mice. Interestingly, we found that MYCN inhibition resulted in changes in expression of metabolic proteins, in accumulation of intracellular lipid droplets and demonstrated that this is due to mitochondrial dysfunction. Our data reported in paper I strongly suggests that MYCN regulated metabolic processes may contribute to the aggressiveness of neuroblastoma. In paper II we applied several approaches to further investigate the MYCN-mediated metabolic alterations in neuroblastoma. The combination of mass spectrometry based proteomics and transcriptome data analysis highlighted key metabolic enzymes involved in energetic pathways of cancer cells. The functional metabolic measurements supported the data analysis and demonstrated that MYCN not only enhanced the glycolytic capacity of neuroblastoma cells, but also increased mitochondrial respiration. The data presented in paper II suggests that MYCN-amplification is associated with a high-energetic metabolic phenotype. Importantly, we demonstrated that targeting of fatty acid oxidation resulted in potentiated neuronal differentiation, decreased viability of MYCN-amplified neuroblastoma as well as decreased tumor burden in vivo in a neuroblastoma xenograft model. Our previous findings highlighted an important role of fatty acid metabolism in MYCN-amplified neuroblastoma. In paper III we used specific inhibitors and demonstrated that targeting of de novo fatty acid synthesis in MYCN-amplified neuroblastoma cells resulted in increased mitochondrial dysfunction and glycolytic flux. In addition, we observed that MYCN downregulation and neuronal differentiation are consequences of inhibiting de novo synthesis of fatty acids in neuroblastoma cells. In paper IV we demonstrated that the miR-17~92 cluster, which is upregulated by MYCN, suppresses neuronal differentiation via targeting of the nuclear hormone receptor family in neuroblastoma. Importantly, we showed that MYCN inhibition leads to increased expression of the glucocorticoid receptor, which is accompanied by decreased levels of members of the miR-17~92 clusters and elevated expression of the neural differentiation markers TrkA, SCG2 and TH. Furthermore, increased GR expression followed after MYCN downregulation and decreased tumor burden was observed in a pre-clinical NB model following combined MYC inhibition and activation of glucocorticoid signaling. Together the data generated in our laboratory and included in the present thesis demonstrates that targeting of MYCN and MYCN-controlled metabolic processes may provide an attractive basis for development of novel therapeutic approaches for childhood neuroblastoma

Semitransitive and transitive subsemigroups of the inverse symmetric semigroups

We classify minimal transitive subsemigroups of the finitary inverse symmetric semigroup modulo the classification of minimal transitive subgroups of finite symmetric groups; and semitransitive subsemigroups of the finite inverse symmetric semigroup of the minimal cardinality modulo the classification of transitive subgroups of the minimal cardinality of finite symmetric groups.Comment: 16 page

Inhibition of fatty acid synthesis induces differentiation and reduces tumor burden in childhood neuroblastoma

Many metabolic pathways, including lipid metabolism, are rewired in tumors tosupport energy and biomass production and to allow adaptation to stressful en-vironments. Neuroblastoma is the second deadliest solid tumor in children. Ge-netic aberrations, as the amplification of theMYCN-oncogene, correlate stronglywith disease progression. Yet, there are only a few molecular targets successfullyexploited in the clinic. Here we show that inhibition of fatty acid synthesis led toincreased neural differentiation and reduced tumor burden in neuroblastomaxenograft experiments independently ofMYCN-status. This was accompaniedby reduced levels of the MYCN or c-MYC oncoproteins and activation of ERKsignaling. Importantly, the expression levels of genes involved inde novofattyacid synthesis showed prognostic value for neuroblastoma patients. Our findingsdemonstrate that inhibition ofde novofatty acid synthesis is a promising pharma-cological intervention strategy for the treatment of neuroblastoma indepen-dently ofMYCN-status

Inflammatory hallmarks in 6-OHDA- and LPS-induced Parkinson's disease in rats

Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting more than 1% of aged people. PD, which was previously identified as movement disorder, now is recognized as a multi-factorial systemic disease with important pathogenetic and pathophysiological role of inflammation. Reproducing local and systemic inflammation, which is inherent in PD, in animal models is essential for maximizing the translation of their potential to the clinic, as well as for developing putative anti-inflammatory neuroprotective agents. This study was aimed to compare activation patterns of microglia/macrophage population and systemic inflammation indices in rats with 6-Hydroxydopamine (6-OHDA)- and Lipopolysaccharide (LPS)-induced PD. Metabolic and phenotypic characteristics of microglia/macrophage population were examined by flow cytometry, systemic inflammatory markers were calculated using hematological parameters in 6-OHDA- and LPS-lesioned Wistar rats 29 days after the surgery.Microglia/macrophages from rats in both models exhibited pro-inflammatory metabolic shift. Nevertheless, in LPS-lesioned animals, highly increased proportion of CD80/86+ cells in microglia/macrophage population was registered alongside increased values of systemic inflammatory indices: neutrophil to lymphocyte ratio (NLR), derived neutrophil to lymphocyte ratio (dNLR), platelet to lymphocyte ratio and systemic immune inflammation index (SII). There was significant positive correlation between the count of CD80/86+ cells and systemic inflammatory indices in these animals. Microglia/macrophages from 6-OHDA-lesioned rats were characterized by the increased fraction of CD206+ cells alongside decreased proportion of CD80/86+ cells. No signs of systemic inflammation were observed. Negative correlation between quantitation characteristics of CD80/86+ cells and values of systemic inflammatory indices was registered. Collectively, our data show that LPS-PD model unlike 6-OHDA-PD replicates crosstalk between local and systemic inflammatory responses, which is inherent in PD pathogenesis and pathophysiology

Особливості реновації промислових об'єктів (закордонний досвід)

В статті розглядаються особливості реновації промислових будівель. Реновація передбачає надання нової функції невиробничого характеру промисловим об’єктам, забезпечення їх адаптації в сучасному містобудівному середовищі. Крім економічної ефективності використання існуючих промислових будівель, реновація є засобом збереження історичної канви міста, вирішення естетичних і етичних проблем існування старих об’єктів.The article discusses the features of renovation of industrial buildings. Renovation includes a new feature providing non-production industrial facilities, ensuring their adaptation to the modern urban environment. In addition to the cost-effectiveness of the existing industrial buildings, renovation is the method of the historical outline of the town preservingb, solving the aesthetic and ethical problems of the existence of old objects.В статье рассматриваются особенности реновации промышленных зданий. Реновация предусматривает предоставление новой функции непроизводственного характера промышленным объектам, обеспечение их адаптации в современной градостроительной среде. Кроме экономической эффективности использования существующих промышленных зданий, реновация является средством сохранения исторической канвы города, решения эстетических и этических проблем существования старых объектов

Mass Spectrometry-Based Proteomics Identifies UPF1 as a Critical Gene Expression Regulator in MonoMac 6 Cells.

5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes, a group of inflammatory lipid mediators derived from arachidonic acid. Recently, we have demonstrated that 5-LO mRNA expression is regulated by alternative splicing and nonsense-mediated mRNA decay (NMD). In addition to this, 5-LO protein expression was reduced on translational level in UPF1 knockdown cells, suggesting that UPF1 has a positive influence on 5-LO translation. Therefore, a mass spectrometry-based proteomics study was performed to identify compartment-specific protein expression changes upon UPF1 knockdown in differentiated and undifferentiated MM6 cells. The proteomics analysis revealed that the knockdown of UPF1 results in numerous protein changes in the microsomal fraction (∼21%) but not in the cytosolic fraction (<1%). The results suggest that UPF1 is a critical gene expression regulator in a compartment-specific way. During differentiation by TGFβ and calcitriol, the majority of UPF1 regulated proteins were adjusted to normal level. This indicates that the translational regulation by UPF1 can potentially be cell differentiation-dependent

Mass Spectrometry-Based Proteomics Identifies UPF1 as a Critical Gene Expression Regulator in MonoMac 6 Cells

5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes, a group of inflammatory lipid mediators derived from arachidonic acid. Recently, we have demonstrated that 5-LO mRNA expression is regulated by alternative splicing and nonsense-mediated mRNA decay (NMD). In addition to this, 5-LO protein expression was reduced on translational level in UPF1 knockdown cells, suggesting that UPF1 has a positive influence on 5-LO translation. Therefore, a mass spectrometry-based proteomics study was performed to identify compartment-specific protein expression changes upon UPF1 knockdown in differentiated and undifferentiated MM6 cells. The proteomics analysis revealed that the knockdown of UPF1 results in numerous protein changes in the microsomal fraction (∼21%) but not in the cytosolic fraction (<1%). The results suggest that UPF1 is a critical gene expression regulator in a compartment-specific way. During differentiation by TGFβ and calcitriol, the majority of UPF1 regulated proteins were adjusted to normal level. This indicates that the translational regulation by UPF1 can potentially be cell differentiation-dependent

Growth inhibition and apoptosis induction in <i>MYCN</i> amplified cells.

<p>(A) The amount of viable of cells was determined using crystal violet assay following 48 hours treatment of Kelly cells with increasing concentration of the indicated compound. Data are shown as percent of control (DMSO) treated cells and represent the mean of three independent experiments. Error bars indicate standard deviation. (B) Pearson's correlation between the IC₅₀ values in the growth inhibition assay (A, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097285#pone-0097285-t002" target="_blank">Table 2</a>) and the K_D values for binding to MYCN (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097285#pone-0097285-t001" target="_blank">Table 1</a>). (C–D) Quantification of apoptosis by propidium iodide staining for sub G1 DNA content of SK-N-BE(2) (C) and Kelly (D) cells. Data represent the means of at least three independent experiments. Error bars indicate standard deviation.</p

10074-G5 induces neuronal differentiation in NB cells.

<p>Morphological differentiation of SK-N-BE(2) cells in response to 15 days culture with 10058-F4 (60 µM) 10074-G5 (30 µM) or DMSO in the presence or absence of NGF (50 ng/ml). Phase contrast micrographs show representative pictures from one out of three independent experiments.</p