Antitumor effect of glycolysis inhibition in combination with lysosome permeabilization and oxidative phosphorylation suppression

Povećane energetske potrebe tumorske ćelije zadovoljavaju prevashodno aerobnom glikolizom, ali ukoliko je ona inhibirana ove metabolički plastične ćelije prelaze na oksidativnu fosforilaciju. Veliki nestabilni lizozomi, bogati hidrolitičkim enzimima, omogućavaju tumorima metastaziranje i rezistenciju na lekove, ali je njihovo izlivanje u citoplazmu citotoksično. Cilj ove disertacije je bio da se ispitaju antitumorski efekti kombinovanih tretmana inhibitora glikolize 2-dezoksi-D-glukoze (2DG) sa supresorom oksidativne fosforilacije rotenonom (ROT), ili sa lizozomalnim deterdžentom N-dodecilimidazolom (NDI). Pokazali smo da i 2DG+ROT i 2DG+NDI sinergistički ubijaju B16 melanomske i U251 gliomske ćelije nekrozom, a ne apoptozom, nekroptozom i autofagijom. Kombinovani tretmani su izazvali depleciju ATP, aktivaciju AMPK i produkciju mitohondrijskog superoksida. 2DG+ROT je stimulisao otpuštanje heksokinaze II sa membrane mitohondrija i posledično otvaranje VDAC kanala, što je omogućilo izlazak superoksida i citohroma c u citoplazmu. Citohrom c je aktivirao kaspaze, ali zbog nedostatka ATP nije došlo do fragmentacije DNK i inicijalno aktivirana apoptoza je završena nekrozom. Deplecija ATP je aktivirala AMPK i suprimirala mTORC1, ali je paradoksalno inhibirala autofagiju. Sa druge strane, NDI je indukovao permeabilizaciju membrane lizozoma i izlazak proteolitičkih enzima katepsina, depolarizaciju mitohondrija, oksidativni stres i supresiju oksidativne fosforilacije, što je u kombinaciji sa inhibicijom glikolize izazvanom 2DG, dovelo do potpune deplecije ATP i nekroze tumorskih ćelija. Istovremena inhibicija glikolize i oksidativne fosforilacije, ili inhibicija glikolize i destabilizacija lizozoma, mogle bi se iskoristiti u antitumorskoj terapiji.Tumor cells preferentially use aerobic glycolysis to satisfy increased energy needs, but if glycolysis is inhibited they switch to oxidative phosphorylation. Tumor cells have large unstable lysosomes, rich in hydrolytic enzymes that stimulate metastases and drug resistance, but induce cell death if released into cytoplasm. The aim of this dissertation was to investigate the antitumor effects of combined treatments of glycolysis inhibitor 2-deoxy-D-glucose (2DG) with the oxidative phosphorylation suppressor rotenone (ROT), or with the lysosomal detergent N-dodecylimidazole (NDI). We showed that both, 2DG+ROT and 2DG+NDI, synergistically induced B16 melanoma and U251 glioma cell death by necrosis, and not apoptosis, necroptosis, and autophagy. Combined treatments caused ATP depletion, AMPK activation, and mitochondrial superoxide production. 2DG+ROT induced release of hexokinase II from the mitochondrial membrane and the consequent opening of the VDAC channel, which led to the release of superoxide and cytochrome c into the cytoplasm. Cytochrome c activated caspases, but due to ATP deficiency, there was no DNA fragmentation and the initially activated apoptosis converted to necrosis. ATP depletion activated AMPK and suppressed mTORC1, but paradoxically inhibited autophagy. On the other hand, NDI induced lysosome membrane permeabilization and release of proteolytic enzymes cathepsins, depolarization of mitochondria, oxidative stress and suppression of oxidative phosphorylation, which in combination with inhibition of glycolysis induced by 2DG, led to complete energy depletion and necrosis of tumor cells. Concomitant inhibition of glycolysis and oxidative phosphorylation, or inhibition of glycolysis and destabilization of lysosomes, could be used in antitumor therapy

Digital vs. Traditional Design Process

The design process is a theme that has long been a preoccupation for architects. Today, when digital technologies play irreplaceable part in the architectural design it would be expected that the design process had improved in relation to traditional principles. But starting from sketching, through 3D modelling, to BIM applications, design process is based on the traditional principles that architects applied before the emergence of digital technologies. Digital sketching is focused more on the reproduction of the sketch appearance through the digital tracing of the hand movement and imitation of the look of the pen on paper than on the study of the process of designing an architectural object through sketching. The creation of 3D models is also aimed at generating models that are used as a substitute for traditional mock-up or photorealistic visualization of the building. BIM models are primarily focused on the development of project documentation. The focus on imitation of traditional representations by the use of digital technologies also makes it difficult to use computer applications to simulate various aspects of the functioning of the building, such as energy consumption, natural lighting, ventilation, etc. The paper gives an overview of research in the field of modelling computer-aided architectural design process inspired by advances in system theory and artificial intelligence that was conducted during the 1980s and 1990s and compares that research with computational architectural design applications that are currently present on the market. The analysis shows that many important principles of architectural design by computer, developed at that time, are now forgotten. Today's applications allow only basic problem solving techniques and do not allow exploration and analysis of variants that represent the true nature of the architectural design process. Because of the inability of earlier computers to process a large amount of information, these systems were experimental, but the research conducted at that time briefly illustrated in this paper, shows that it is possible to develop a digital design process that is in line with what architects really do

Consensus statement on the epidemiological situation and expected frequency of canine vector-borne diseases in Serbia

The current issue of Veterinarski Glasnik (Vol 74, No 2) is dedicated to canine vectorborne pathogens (VBP) and vector-borne diseases (VBD) in Serbia. All published reviews and original papers indicate the amount of research done in Serbia and the amount of collected and analysed data is high for the majority of topics, inviting us to summarise all the findings in a consensus statement that we hope will be of importance for practitioners who are in constant contact with dogs in Serbia and for researchers who should continue to develop this field of investigation

Data supporting the inability of indomethacin to induce autophagy in U251 glioma cells

Autophagy, a catabolic process involving intracellular degradation of unnecessary or dysfunctional cellular components through the lysosomal machinery, could act as a prosurvival, as well as a cytotoxic mechanism (Parzych and Klionsky, 2014) [1]. Cyclooxygenase inhibitor indomethacin inhibits proliferation of glioma cells, and has been reported to reduce the activity of the main autophagy repressor mammalian target of rapamycin (mTOR) (Pantovic et al., 2016) [2]. Here we investigated the ability of indomethacin to induce autophagy in U251 human glioma cells. We assessed the influence of indomethacin on intracellular acidification, expression of proautophagic protein beclin-1, and conversion of microtubule-associated protein light chain 3-I (LC3-I) to autophagosome-associated LC3-II, in the presence or absence of lysosomal inhibitors. The effect of genetic and pharmacological downregulation of autophagy on the cytotoxicity of indomethacin was also evaluated. The interpretation of these data can be found in "In vitro antiglioma action of indomethacin is mediated via AMP-activated protein kinase/mTOR complex 1 signaling pathway" (Pantovic et al., 2016; doi:10.1016/j.biocel.2016.12.007) [2].Data in Brief (2017), 11: 225-23

Autophagy receptor p62 regulates SARS-CoV-2-induced inflammation in COVID-19

Introduction: Since the interaction between autophagy and virus-induced inflammation is complex, we investigated the interplay between autophagy and inflammation in COVID-19 patients and THP-1 cells expressing SARS-Cov2 proteins NSP5 and ORF3a. Methods: Autophagy markers in blood from 19 control subjects and 26 COVID-19 patients at hospital admission and one week later were measured by ELISA, while cytokine levels were examined by flow cytometric bead immunoassay. The level of p62 in cells and its concentration in cell culture supernatants was measured by immunoblot/ELISA. The mRNA levels of proinflammatory cytokines were measured by RT-qPCR. Results: IFN-α, TNF, IL-6, IL-8, IL-17, IL-33, and IFN-γ were elevated in COVID-19 patients at both time points, whereas IL-10 and IL-1β were elevated at admission and one week later, respectively. Autophagy markers LC3 and ATG5 were unchanged in COVID-19. The concentration of autophagic cargo receptor p62 was significantly lower and positively correlated with TNF, IL-10, IL-17, and IL-33 at hospital admission,returning to normal levels after one week. The expression of SARS-CoV-2 proteins NSP5 or ORF3a in THP-1 cells caused an autophagy-independent decrease/autophagy-inhibition-dependent increase of intracellular and secreted p62. This was associated with an NSP5-mediated decrease in TNF/IL-10 mRNA and an ORF3a-mediated increase in TNF/IL-1β/IL-6/IL-10/IL-33 mRNA levels. A genetic knockdown of p62 mimicked the immunosuppressive effect of NSP5, while a p62 increase in autophagy-deficient cells mirrored the immunostimulatory action of ORF3a. Conclusion: The autophagy receptor p62 is reduced in acute COVID-19, and the balance between autophagy-independent decrease and autophagy blockade-dependent increase of p62 levels could affect SARS-CoV-induced inflammation

Antitumor effect of glycolysis inhibition in combination with lysosome permeabilization and oxidative phosphorylation suppression

Povećane energetske potrebe tumorske ćelije zadovoljavaju prevashodno aerobnom glikolizom, ali ukoliko je ona inhibirana ove metabolički plastične ćelije prelaze na oksidativnu fosforilaciju. Veliki nestabilni lizozomi, bogati hidrolitičkim enzimima, omogućavaju tumorima metastaziranje i rezistenciju na lekove, ali je njihovo izlivanje u citoplazmu citotoksično. Cilj ove disertacije je bio da se ispitaju antitumorski efekti kombinovanih tretmana inhibitora glikolize 2-dezoksi-D-glukoze (2DG) sa supresorom oksidativne fosforilacije rotenonom (ROT), ili sa lizozomalnim deterdžentom N-dodecilimidazolom (NDI). Pokazali smo da i 2DG+ROT i 2DG+NDI sinergistički ubijaju B16 melanomske i U251 gliomske ćelije nekrozom, a ne apoptozom, nekroptozom i autofagijom. Kombinovani tretmani su izazvali depleciju ATP, aktivaciju AMPK i produkciju mitohondrijskog superoksida. 2DG+ROT je stimulisao otpuštanje heksokinaze II sa membrane mitohondrija i posledično otvaranje VDAC kanala, što je omogućilo izlazak superoksida i citohroma c u citoplazmu. Citohrom c je aktivirao kaspaze, ali zbog nedostatka ATP nije došlo do fragmentacije DNK i inicijalno aktivirana apoptoza je završena nekrozom. Deplecija ATP je aktivirala AMPK i suprimirala mTORC1, ali je paradoksalno inhibirala autofagiju. Sa druge strane, NDI je indukovao permeabilizaciju membrane lizozoma i izlazak proteolitičkih enzima katepsina, depolarizaciju mitohondrija, oksidativni stres i supresiju oksidativne fosforilacije, što je u kombinaciji sa inhibicijom glikolize izazvanom 2DG, dovelo do potpune deplecije ATP i nekroze tumorskih ćelija. Istovremena inhibicija glikolize i oksidativne fosforilacije, ili inhibicija glikolize i destabilizacija lizozoma, mogle bi se iskoristiti u antitumorskoj terapiji.Tumor cells preferentially use aerobic glycolysis to satisfy increased energy needs, but if glycolysis is inhibited they switch to oxidative phosphorylation. Tumor cells have large unstable lysosomes, rich in hydrolytic enzymes that stimulate metastases and drug resistance, but induce cell death if released into cytoplasm. The aim of this dissertation was to investigate the antitumor effects of combined treatments of glycolysis inhibitor 2-deoxy-D-glucose (2DG) with the oxidative phosphorylation suppressor rotenone (ROT), or with the lysosomal detergent N-dodecylimidazole (NDI). We showed that both, 2DG+ROT and 2DG+NDI, synergistically induced B16 melanoma and U251 glioma cell death by necrosis, and not apoptosis, necroptosis, and autophagy. Combined treatments caused ATP depletion, AMPK activation, and mitochondrial superoxide production. 2DG+ROT induced release of hexokinase II from the mitochondrial membrane and the consequent opening of the VDAC channel, which led to the release of superoxide and cytochrome c into the cytoplasm. Cytochrome c activated caspases, but due to ATP deficiency, there was no DNA fragmentation and the initially activated apoptosis converted to necrosis. ATP depletion activated AMPK and suppressed mTORC1, but paradoxically inhibited autophagy. On the other hand, NDI induced lysosome membrane permeabilization and release of proteolytic enzymes cathepsins, depolarization of mitochondria, oxidative stress and suppression of oxidative phosphorylation, which in combination with inhibition of glycolysis induced by 2DG, led to complete energy depletion and necrosis of tumor cells. Concomitant inhibition of glycolysis and oxidative phosphorylation, or inhibition of glycolysis and destabilization of lysosomes, could be used in antitumor therapy

Influence of grinding, pressing and sintering of the powder 50.0 wt% Fe, 47.0 wt% BaTiO3 and 3.0 wt% TiO2 on morphology, microstructure, magnetic and electric properties

A mixture of 50.0 wt% Fe, 47.0 wt% BaTiO3, and 3.0 wt% TiO2 powders is milled and then pressed and sintered. Samples sintered from ground powders have a structure consisting of plates. They consist of the crystalline phase BaFe12O19 and crystalline and amorphous phases Fe, BaTiO3, and TiO2. Samples sintered from powders ground for more than 180 min are compact and composed of BaFe12O19 crystals embedded in an amorphous matrix consisting of amorphous BaTiO3 and TiO2 phases. Magnetization of a sample ground for 220 min gradually decreases with increasing temperature up to 440°C. This decrease result from the transition of ordered domains into chaotically directed domains. Above 440°C, the thermal energy is sufficiently large to destroy ordered paramagnetic ferromagnetic and weakly ferromagnetic domains. During sample cooling in the absence of an external magnetic field, the temperature dependence of magnetization is the same as that obtained during heating in the magnetic field. However, during the sample cooling in the magnetic field, substantially higher values of magnetization are obtained. Considerably higher magnetization of the cooled sample remains even after switching off the external magnetic field. The sintered sample, previously pressed in the magnetic field, has a higher dielectric constant than the sample pressed in the absence of the magnetic field. The dielectric constant decreases with increasing frequency

Synthesis, characterization and cytotoxicity of a new palladium(II) complex with a coumarin-derived ligand 3-(1-(3-hydroxypropylamino)ethylidene)chroman-2,4-dione. Crystal structure of the 3-(1-(3-hydroxypropylamino)ethylidene)-chroman-2,4-dione

The new coumarine derivative, 3-(1-(3-hydroxypropylamino)ethylidene)chroman-2,4-dione, and corresponding palladium(II) complex have been synthesized and characterized by microanalysis, infrared, 1H and 13C NMR spectroscopy. The structure of the ligand, solved using a monocrystal X-ray structural analysis, consists of two crystallographic different pseudocentrosymmetrically related molecules of 3-(1-(3-hydroxypropylamino)ethylidene)chroman-2,4-dione, while the structure of the square-planar palladium(II) complex was proposed on the basis of DFT calculations. The palladium(II) complex decreased viability of U251 human glioma and B16 mouse melanoma cells in a dose and time dependent manner, while its ligand exhibited only moderate cytotoxicity.Inorganica Chimica Acta (2017), 466: 188-19

In vitro antiglioma action of indomethacin is mediated via AMP-activated protein kinase/mTOR complex 1 signalling pathway

We investigated the role of the intracellular energy-sensing AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in the in vitro antiglioma effect of the cyclooxygenase (COX) inhibitor indomethacin. Indomethacin was more potent than COX inhibitors diclofenac, naproxen, and ketoprofen in reducing the viability of U251 human glioma cells. Antiglioma effect of the drug was associated with p21 increase and G2M cell cycle arrest, as well as with oxidative stress, mitochondrial depolarization, caspase activation, and the induction of apoptosis. Indomethacin increased the phosphorylation of AMPK and its targets Raptor and acetyl-CoA carboxylase (ACC), and reduced the phosphorylation of mTOR and mTOR complex 1 (mTORC1) substrates p70S6 kinase and PRAS40 (Ser183). AMPK knockdown by RNA interference, as well as the treatment with the mTORC1 activator leucine, prevented indomethacin-mediated mTORC1 inhibition and cytotoxic action, while AMPK activators metformin and AICAR mimicked the effects of the drug. AMPK activation by indomethacin correlated with intracellular ATP depletion and increase in AMP/ATP ratio, and was apparently independent of COX inhibition or the increase in intracellular calcium. Finally, the toxicity of indomethacin towards primary human glioma cells was associated with the activation of AMPK/Raptor/ACC and subsequent suppression of mTORC1/S6K. By demonstrating the involvement of AMPK/mTORC1 pathway in the antiglioma action of indomethacin, our results support its further exploration in glioma therapy.The International Journal of Biochemistry & Cell Biology (2017), 83: 84-9