Effects of ethyl ester cyclohexyl analog of ethylene diamine dipropanoic acid against mouse melanoma cells in vitro and in vivo

Melanom je visoko agresivni maligni tumor kože. Incidenca melanoma se značajno povećala u poslednjih 30 godina, stope morataliteta su visoke, a i pored napretka u hemio-/imuno-/ i radioterapiji odgovor pacijenata sa uznapredovalim stadijumom bolesti je svega 15-25%. Veliki napori se ulažu u otkrivanje, sintezu, karakterizaciju i ispitivanje mehanizma delovanja novih jedinjenja - potencijalnih antitumorskih agenasa. Među njima se izdvajaju derivati etilendiamin dipropanske kiseline, posebno etil estar cikloheksil analog etilendiamin dipropanske kiseline (EE), čiji antimelanomski efekat do sada nije detaljnije objašnjen. Cilj ovog rada je bio da se ispita citotoksični potencijal EE na ćelijskom i animalnom modelu melanoma kao i molekularni mehanizam dejstva koji EE pokreće. U eksperimentima su korišćene ćelijske linije mišjeg melanoma (B16), mišjih makrofaga (RAW264.7), humanog melanoma (518A2), humanih plućnih fibroblasta (MRC-5) i humanih keratinocita (HaCaT). Vijabilitet ćelija je ispitan testom aktivnosti kisele fosfataze, sulforodamin B testom, MTT testom i bojenjem tripan plavim, dok je integritet ćelijske membrane utvrđen testom oslobađanja laktat dehidrogenaze. Molekularni mehanizam dejstva EE na melanom miša je ispitan utvrđivanjem produkcije slobodnih kiseoničnih radikala, depolarizacije mitohondrija, aktivacije kaspaza, eksternalizacije fosfatidil serina, fragmentacije DNK i prisustva kiselih vezikula u ćelijama, korišćenjem odgovarajućih fluorohroma i analizom na protočnom citofluorimetru. Nivoi proteina značajnih za apoptozu i autofagiju su mereni imunoblot metodom. Morfološke karakteristike ćelija su ispitane fluorescentnom i transmisionom elektronskom mikroskopijom. U in vivo eksperimentima miševima soja C57Bl/6 je indukovan primarni i metastatski melanom injektiranjem B16 ćelija supkutano, odnosno intravenski. Sistemska toksičnost EE je ispitana praćenjem funkcije kostne srži, jetre i bubrega. Ispitan je efekat EE na progresiju supkutanog melanoma u pogledu veličine, a zatim su tumori izolovani i u tumorskom tkivu analizirana ekspresija gena i količina proteina od značaja za apoptozu i autofagiju metodom kvantitativne reakcije lančanog umnožavanja sa reverznom transkripcijom, odnosno metodom imunoblota...Melanoma is a highly aggressive skin malignant tumor. The incidence of melanoma is increasing for the last 30 years, mortality is high, and despite all the improvements in chemo-/immuno- and radiotherapy the response of patients with advanced disease is as low as 15-25 %. Great efforts are put into the discovering, synthesis, characterization and investigation of molecular mechanisms of action of these compounds - potential antitumor agents. Promising new potential cytotoxic agents are derivatives of ethylene diamine dipropanoic acid, specifically ethyl ester cyclohexyl analog of ethylene diamine dipropanoic acid (EE). Its antimelanoma effect was not previously investigated and thus the aim of this research was to assess cytotoxic potential and molecular mechanism of EE's action using cell and animal melanoma models. Mouse melanoma (B16), mouse macrophages (RAW264.7), human melanoma (518A2), human pulmonary fibroblast (MRC-5) and human keratinocyte (HaCaT) cell lines were used in the experiments. Cell viability was assessed using acid phosphatase, sulphorhodamin B, MTT and trypan blue assays, whereas lactate dehydrogenase release assay was used for measuring cell membrane integrity. Molecular mechanism of action was investigated by measuring the production of reactive oxygen species, mitochondrial membrane depolarization, caspase activation, phosphatidyl serine externalization, DNA fragmentation, and number of acidic vesicles, using appropriate fluorochromes followed by flow cytometry analysis. Levels of apoptosis- and autophagy- related proteins were measured using immunoblot. Cell morphology was investigated using fluorescent and transmission electron microscopy. C57Bl/6 mice were used for in vivo experiments. Primary and metastatic melanoma were established by injecting B16 cells subcutaneously and into the tail vein of the mice. The bone marrow, liver, and kidney function were assessed for systemic toxicity of EE. The effect of EE on growth of subcutaneous melanoma was observed, tumors isolated, and apoptosis- and autophagy- related gene and protein expression in the tumor tissue was assessed using reverse transcriptase quantitative polymerase chain reaction and immunoblot, respectively..

Hydro-geological properties of the Savian aquifer in the county Obrenovac

The paper presents a description of hydrogeological researches of alluvial layers of the Sava River in the area of the source "Vić Bare" near Obrenovac. This source supplies groundwater to that town. The depth of these layers amounts to 25 m. With regard to collecting capacity, the most significant are gravel-sand sediments of high filtration properties. Their average depth amounts to about 13 m with the underlying layer made of Pleistocene clays. Compact aquifer is formed within these sediments and it refills partly from the Sava River at places where river cuts its channel into the gravel-sand layer. The analysis of the groundwater regime in the riparian area points out that groundwater levels follow stages of the Sava River. Such an influence lessens with the distance. Established hydraulic connection between the river and the aquifer enables its permanent replenishment. On the other hand, due to certain pollutions this river flow might bring along, it represents a potential danger. Those pollutions could enter water-bearing layer of the aquifer as well as the exploitation well of the source. Such presumptions have been confirmed in the experiment of pollution transport carried out in the water-bearing layer. Unabsorbable chloride was used as a tracer whose movement velocity through exploitation well proved that there were real possibilities of intrusion of aggressive pollutants into the water-bearing layer and into the aquifer as well. Therefore, the protection of the source must be in the function of the protection of surface waters

Supplementary data for article: Misirlić Denčić, S.; Poljarević, J.; Isakovic, A. M.; Marković, I.; Sabo, T. J.; Grgurić-Šipka, S. Antileukemic Action of Novel Diamine Pt(II) Halogenido Complexes: Comparison of the Representative Novel Pt(II) with Corresponding Pt(IV) Complex. Chemical Biology and Drug Design 2017, 90 (2), 262–271. https://doi.org/10.1111/cbdd.12945

Supporting information for: [https://doi.org/10.1111/cbdd.12945]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2483]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3120

The effects of the perturbated ionization potential and the magnetic component on the relativistic transition rate

In this paper we theoretically analyze the influence of the perturbated ionization potential and the component of the magnetic field on the transition rate, in a relativistic, linearly polarized laser field. The obtained results indicate that both effect play important roles during the ionization process, influence the rate, and must be considered. It is shown that a laser field influences the ionization potential of an electron strongly and causes that the rate curve shows significantly different behavior compared to the case with considered non-perturbated ionization potential

ArmAssist Robotic System versus Matched Conventional Therapy for Poststroke Upper Limb Rehabilitation: A Randomized Clinical Trial

The ArmAssist is a simple low-cost robotic system for upper limb motor training that combines known benefits of repetitive task-oriented training, greater intensity of practice, and less dependence on therapist assistance. The aim of this preliminary study was to compare the efficacy of ArmAssist (AA) robotic training against matched conventional arm training in subacute stroke subjects with moderate-to-severe upper limb impairment. Twenty-six subjects were enrolled within 3 months of stroke and randomly assigned to the AA group or Control group (n = 13 each). Both groups were trained 5 days per week for 3 weeks. The primary outcome measure was Fugl-Meyer Assessment-Upper Extremity (FMA-UE) motor score, and the secondary outcomes were Wolf Motor Function Test-Functional Ability Scale (WMFT-FAS) and Barthel index (BI). The AA group, in comparison to the Control group, showed significantly greater increases in FMA-UE score (18.0 +/- 9.4 versus 7.5 +/- 5.5, p = 0.002) and WMFT-FAS score (14.1 +/- 7.9 versus 6.7 +/- 7.8, p = 0.025) after 3 weeks of treatment, whereas the increase in BI was not significant (21.2 +/- 24.8 versus 13.1 +/- 10.7, p = 0.292). There were no adverse events. We conclude that arm training using the AA robotic device is safe and able to reduce motor deficits more effectively than matched conventional arm training in subacute phase of stroke.Andrej M. Savic and Milica S. Isakovic are employed at Tecnalia Serbia Ltd., Belgrade, Serbia, and Cristina Rodriguez-de-Pablo and Thierry Keller are employed at TECNALIA, San Sebastian, Spain, from which they receive financial compensation. No external support was received for conducting this study

Supplementary data for the article: Dinić, J.; Novaković, M.; Podolski-Renić, A.; Stojković, S.; Mandić, B.; Tešević, V.; Vajs, V.; Isaković, A.; Pešić, M. Antioxidative Activity of Diarylheptanoids from the Bark of Black Alder (Alnus Glutinosa) and Their Interaction with Anticancer Drugs. Planta Medica 2014, 80 (13), 1088–1096. https://doi.org/10.1055/s-0034-1382993

Supplementary material for: [https://doi.org/10.1055/s-0034-1382993]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1845

Supplementary data for article: Misirlić Denčić, S.; Poljarević, J.; Isakovic, A. M.; Marković, I.; Sabo, T. J.; Grgurić-Šipka, S. Antileukemic Action of Novel Diamine Pt(II) Halogenido Complexes: Comparison of the Representative Novel Pt(II) with Corresponding Pt(IV) Complex. Chemical Biology and Drug Design 2017, 90 (2), 262–271. https://doi.org/10.1111/cbdd.12945

Supporting information for: [https://doi.org/10.1111/cbdd.12945]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2483]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3120

Evaluation of Anticancer and Antioxidant Activity of a Commercially Available CO2 Supercritical Extract of Old Man's Beard (Usnea barbata)

There is a worldwide ongoing investigation for novel natural constituents with cytotoxic and antioxidant properties. The aim of this study was to investigate chemical profile and stated biological activities of the supercritical CO2 extract (SCE) of old man's beard compared to the extracts obtained using the conventional techniques (Soxhlet extracts and macerate). The most abundant compound identified was usnic acid, which content was inversely proportional to the polarity of the solvent used and was the highest in the SCE, which was the sample revealing the highest cytotoxic activity in tested tumor cell lines (B16 mouse melanoma and C6 rat glioma), with lower IC50 values compared to pure usnic acid. Further investigations suggested both SCE and usnic acid to induce apoptosis and/ or autophagy in B16 and C6, indicating higher cytotoxicity of SCE to be related to the higher degree of ROS production. A good correlation of usnic acid content in the extracts and their antioxidant capacity was established, extricating SCE as the most active one. Presented results support further investigations of SCE of old man's beard as a prospective therapeutic agent with potential relevance in the treatment of cancer and/ or in oxidative stress-mediated conditions

Supplementary material for the article: Dinić, J.; Novaković, M.; Podolski-Renić, A.; Vajs, V.; Tešević, V.; Isaković, A.; Pešić, M. Structural Differences in Diarylheptanoids Analogues from Alnus Viridis and Alnus Glutinosa Influence Their Activity and Selectivity towards Cancer Cells. Chemico-Biological Interactions 2016, 249, 36–45. https://doi.org/10.1016/j.cbi.2016.02.019

Supplementary material for: [https://doi.org/10.1016/j.cbi.2016.02.019]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2066

Supplementary data for the article: Dinić, J.; Novaković, M.; Podolski-Renić, A.; Stojković, S.; Mandić, B.; Tešević, V.; Vajs, V.; Isaković, A.; Pešić, M. Antioxidative Activity of Diarylheptanoids from the Bark of Black Alder (Alnus Glutinosa) and Their Interaction with Anticancer Drugs. Planta Medica 2014, 80 (13), 1088–1096. https://doi.org/10.1055/s-0034-1382993

Supplementary material for: [https://doi.org/10.1055/s-0034-1382993]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1845