Provider: - Institution: - Data provided by Europeana Collections- In this doctoral dissertation the effect of intracellular energy sensor AMP-activated protein kinase (AMPK) inhibition on induction ...of apoptosis and autophagy in tumor cells was investigated. Pharmacological AMPK inhibitor dorsomorphin caused G2/M cell cycle block, accompanied by apoptotic cell death characterized by caspase activation, phosphatidylserine exposure and DNA fragmentation in U251 human and C6 rat glioma cells, while it had no effect on viability of primary rat astrocytes and B16 mouse melanoma cells. The mechanisms underlying the pro-apoptotic action of dorsomorphin involved induction of oxidative stress and down-regulation of antiapoptotic molecule Bcl-2. Dorsomorphin diminished AMPK phosphorylation and enzymatic activity, resulting in reduced phosphorylation of its target acetyl CoA carboxylase. AMPK activators metformin and AICAR partly prevented the cell cycle block, oxidative stress and apoptosis induced by dorsomorphin. The small interfering RNA (siRNA) targeting of human AMPK mimicked dorsomorphin-induced G2/M cell cycle arrest, but failed to induce oxidative stress and apoptosis in U251 glioma cells. Therefore, AMPK inhibition is required, but not sufficient for dorsomorphin-mediated apoptotic death of glioma cells. In this study, it was also reported that dorsomorphin can induce autophagy in cancer cells. The induction of autophagy in U251 human glioma cell line was demonstrated by acridine orange staining of intracellular acidic vesicles, Beclin 1 induction, p62 decrease and conversion of LC3-I to autophagosome-associated LC3-II in the absence and presence of proteolysis inhibitors. The presence of autophagosome like vesicles was confirmed by transmission electron microscopy. Dorsomorphin-mediated inhibition of AMPK and Raptor in U251 cells was associated with paradoxical decrease in phosphorylation of AMPK/Raptor-repressed mTOR, a major negative regulator of autophagy, and its downstream target p70S6K. The phosphorylation of mTOR activator Akt and PI3K-activating kinase Src was also impaired in dorsomorphin-treated cells. The siRNA-mediated AMPK silencing did not reduce the activity of the Akt/mTOR/p70S6K pathway and AMPK activators metformin and AICAR failed to block dorsomorphin-induced autophagy. Autophagy inhibitors bafilomycin and chloroquine significantly increased the cytotoxicity of dorsomorphin towards U251 cells, as confirmed by the increase in lactate dehydrogenase release, DNA fragmentation and caspase-3 activation. Similar effects of dorsomorphin were also observed in C6 rat glioma, L929 mouse fibrosarcoma and B16 mouse melanoma cell lines. Since dorsomorphin has previously been reported to suppress AMPK dependent autophagy in different cell types, results in this study suggest that the effects of dorsomorphin on autophagy might be dose-, cell type- and/or context-dependent. By demonstrating the ability of dorsomorphin to induce autophagic response in cancer cells via AMPK inhibition-independent downregulation of Akt/mTOR pathway, results warrant caution when using dorsomorphin to inhibit AMPK-dependent cellular responses, but also suggest that dorsomorphin, alone or in combination with autophagy inhibitors, could be potential candidate for anticancer therapy.- U ovoj doktorskoj disertaciji ispitivan je uticaj inhibicije intracelularnog energetskog senzora protein kinaze aktivirane adenozin-monofosfatom (AMPK) na indukciju apoptoze i autofagije u tumorskim ćelija. Farmakološki inhibitor AMPK dorzomorfin indukovao je G2/M blokadu ćelijskog ciklusa, praćen apoptozom koju karakteriše aktivacija kaspaza, eksternalizacija fosfatidilserina i fragmentacija DNK u U251 humanim i C6 pacovskim ćelijama glioma, dok na vijabilitet primarnih pacovskih astrocita i ćelija mišjeg melanoma B16 nije imao uticaja. Mehanizam indukcije apoptoze dorzomorfinom bio je posredovan stimulacijom produkcije reaktivnih vrsta kiseonika i inhibicijom ekspresije antiapoptotskog Bcl-2 proteina. Dorzomorfin je inhibirao fosforilaciju i enzimatsku aktivnost AMPK, što je za posledicu imalo smanjenje fosforilacije njenog supstrata acetil-CoA karboksilaze. Aktivatori AMPK metformin i AICAR delimično su neutralisali blokadu ćelijskog ciklusa, oksidativni stres i apoptozu indukovanu dorzomorfinom. Mala interferirajuća RNK (siRNK) koja sprečava ekspresiju humanog AMPK enzima je poput dorzomorfina zaustavila proliferaciju ćelija u G2/M fazi ćelijskog ciklusa, ali nije izazvala oksidativni stres i apoptozu u U251 ćelijama. Dakle, inhibicija AMPK je neophodna, ali ne i dovoljna za indukciju apotpoze dorzomorfinom u ćelijama glioma. U ovoj studiji takođe je pokazano da dorzomorfin indukuje autofagiju u ćelijama kancera. Indukcija autofagije u U251 ćelijama detektovana je fluorescentnim bojenjem unutarćelijskih kiselih vezikula akridin oranžom, indukcijom beklina-1, degradacijom p62 proteina i konverzijom LC3-I u formu asociranu sa autofagozomima LC3-II u odsustvu i prisustvu proteolitičkih inhibitora. Prisustvo autofagozomima sličnih vezikula potvrđeno je transmisionom elektronskom mikroskopijom. Inhibicija AMPK i Raptora indukovana dorzomorfinom u U251 ćelijama bila je paradoksalno asocirana sa smanjenjem fosforilacije AMPK/Raptorom inhibiranog glavnog represora autofagije mTOR i njegovog supstrata p70S6K. Fosforilacija mTOR aktivatora Akt i PI3K-aktivirane Src kinaze bile su takođe inhibirane u ćelijama tretiranim dorzomorfinom. Poništavanje ekspresije AMPK sa siRNA nije redukovalo aktivnost Akt/mTOR/p70S6K signalnog puta, a AMPK aktivatori metformin i AICAR nisu uspeli da blokiraju autofagiju indukovanu dorzomorfinom. Inhibitori autofagije bafilomicin i hlorokin značajno su povećali citotoksičnost dorzomorfina prema U251 ćelijama, što je pokazano povećanjem oslobađanja laktat dehidrogenaze, fragmentacije DNK i aktivacije kaspaze-3. Sličan efekat dorzomorfin je imao i prema ćelijama pacovskog glioma C6, mišjeg fibrosarkoma L929 i mišjeg melanoma B16. Pošto je ranije pokazano da dorzomorfin suprimira AMPK-zavisnu autofagiju u različitim ćelijskim tipovima, rezultati ove studije sugerišu da efekat dorzomorfina na autofagiju zavisi od doze, konteksta i/ili vrste ćelija. Imajući u vidu da dorzomorfin indukuje autofagiju u ćelijama kancera nezavisno od inhibicije AMPK, inhibicijom Akt/mTOR signalnog puta, rezultati ove studije ukazuju na neophodnost opreza pri interpretaciji rezultata eksperimenata u kojima se dorzomorfin koristi kao inhibitor AMPK, ali takođe sugerišu da bi dorzomorfin, sam ili u kombinaciji sa inhibitorima autofagije, mogao biti potencijalni kandidat za terapiju tumora.- All metadata published by Europeana are available free of restriction under the Creative Commons CC0 1.0 Universal Public Domain Dedication. However, Europeana requests that you actively acknowledge and give attribution to all metadata sources including Europeana
In the present study, we compared the effects of nanocrystalline fullerene suspension (nanoC(60)) on tumour cell growth in vitro and in vivo. NanoC(60) suspension was prepared by solvent exchange ...using tetrahydrofuran to dissolve C(60). In vitro, nanoC(60) caused oxidative stress, mitochondrial depolarization and caspase activation, leading to apoptotic and necrotic death in mouse B16 melanoma cells. Biodistribution studies demonstrated that intraperitoneally injected radiolabeled (125I) nanoC(60) readily accumulated in the tumour tissue of mice subcutaneously inoculated with B16 cells. However, intraperitoneal administration of nanoC(60) over the course of two weeks starting from melanoma cell implantation not only failed to reduce, but significantly augmented tumour growth. The tumour-promoting effect of nanoC(60) was accompanied by a significant increase in splenocyte production of the immunoregulatory free radical nitric oxide (NO), as well as by a reduction in splenocyte proliferative responses to T- and B-cell mitogens ConcanavalinA and bacterial lipopolysaccharide, respectively. A negative correlation between NO production and splenocyte proliferation indicated a possible role of NO in reducing the proliferation of splenocytes from nanoC(60)-injected mice. These data demonstrate that nanoC(60), in contrast to its potent anticancer activity in vitro, can potentiate tumour growth in vivo, possibly by causing NO-dependent suppression of anticancer immune response.
U ovoj doktorskoj disertaciji ispitivan je uticaj inhibicije intracelularnog energetskog senzora protein kinaze aktivirane adenozin-monofosfatom (AMPK) na indukciju apoptoze i autofagije u tumorskim ...ćelija. Farmakološki inhibitor AMPK dorzomorfin indukovao je G2/M blokadu ćelijskog ciklusa, praćen apoptozom koju karakteriše aktivacija kaspaza, eksternalizacija fosfatidilserina i fragmentacija DNK u U251 humanim i C6 pacovskim ćelijama glioma, dok na vijabilitet primarnih pacovskih astrocita i ćelija mišjeg melanoma B16 nije imao uticaja. Mehanizam indukcije apoptoze dorzomorfinom bio je posredovan stimulacijom produkcije reaktivnih vrsta kiseonika i inhibicijom ekspresije antiapoptotskog Bcl-2 proteina. Dorzomorfin je inhibirao fosforilaciju i enzimatsku aktivnost AMPK, što je za posledicu imalo smanjenje fosforilacije njenog supstrata acetil-CoA karboksilaze. Aktivatori AMPK metformin i AICAR delimično su neutralisali blokadu ćelijskog ciklusa, oksidativni stres i apoptozu indukovanu dorzomorfinom. Mala interferirajuća RNK (siRNK) koja sprečava ekspresiju humanog AMPK enzima je poput dorzomorfina zaustavila proliferaciju ćelija u G2/M fazi ćelijskog ciklusa, ali nije izazvala oksidativni stres i apoptozu u U251 ćelijama. Dakle, inhibicija AMPK je neophodna, ali ne i dovoljna za indukciju apotpoze dorzomorfinom u ćelijama glioma. U ovoj studiji takođe je pokazano da dorzomorfin indukuje autofagiju u ćelijama kancera. Indukcija autofagije u U251 ćelijama detektovana je fluorescentnim bojenjem unutarćelijskih kiselih vezikula akridin oranžom, indukcijom beklina-1, degradacijom p62 proteina i konverzijom LC3-I u formu asociranu sa autofagozomima LC3-II u odsustvu i prisustvu proteolitičkih inhibitora.
Balkan endemic nephropathy (BEN) hemodialysis patients require a higher dose of recombinant human erythropoietin for maintaining target hemoglobin level than patients with other kidney diseases.
...Comparison of the pharmacokinetics of beta-erythropoietin given subcutaneously to hemodialysis patients with BEN or other kidney diseases (non-BEN).
Recombinant human erythropoietin (75 U/kg) was administered subcutaneously to 10 BEN and 14 non-BEN hemodialysis patients. The predose plasma level of erythropoietin (Epo) was subtracted from all postdose levels. The relevant pharmacokinetic parameters were calculated after noncompartmental pharmacokinetic analysis using Kinetica software (Thermo Scientific, ver.5.0).
Although basal plasma Epo concentration was similar in BEN (20.1 ± 10.3 U/L) and non-BEN (15.1 ± 8.1 U/L; p=.1964) patients, there were significant differences between the groups for elimination rate constant (0.016 ± 0.006 vs 0.026 ± 0.011 hr⁻¹; p=.020) and elimination half-life (50.24 ± 19.12 vs 33.79 ± 18.91 hr, p=.048). These differences remained significant after adjustment for patient characteristics (age, sex, hemodialysis duration, ferritin, PTH and ACEI use). No significant differences between groups were found in maximal Epo concentration, time to maximum Epo concentration, area under the curve from time of dosing extrapolated to infinity, clearance, mean residence time of Epo between groups both before and after adjustment.
Pharmacokinetic analysis of beta-erythropoietin detected a significantly longer elimination half-life in BEN than in non BEN patients. This finding needs to be confirmed in a well-controlled study with a larger sample size.
PhD Students’ Awareness of Research Misconduct Boskovic, Milos; Djokovic, Jelena; Grubor, Iva ...
Journal of empirical research on human research ethics,
04/2013, Letnik:
8, Številka:
2
Journal Article
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