Naslov (srp)

Mehanizmi citotoksičnosti farmakoloških inhibitora autofagije in vitro : doktorska disertacija

Autor

Stamenković, Marina, 1984-, 23503207

Doprinosi

Trajković, Vladimir, 1967-, 12751207
Janjetović, Kristina, 1978-, 62308105
Marković, Ivanka, 1968-, 12765287
Isaković, Aleksandra, 1973-, 12857959
Andrić, Silvana, 1968-, 12976743
Popadić, Dušan, 1968-, 12769895
Vučićević, Ljubica, 1978-, 58369289

Opis (srp)

autolizozomima koji nastaju spajanjem autofagozoma i lizozoma. Brojni dokazi o citoprotektivnom efektu autofagije kod tumora omogućili su pretklinička i klinička istraživanja zasnovana na kombinovanju farmakoloških inhibitora autofagije i hemioterapeutika. Bafilomicin, hlorokin i amonijum hlorid su lizozomalni inhibitori autofagije (LIA) koji sprečavaju razgradnju sadržaja u autolizozomima blokadom V-ATPazne protonske pumpe, inhibicijom kiselih lizozomalnih hidrolaza ili blokadom fuzije lizozoma i autofagozoma. Nedavno je pokazano da i lekovi koji smanjuju želudačnu kiselost inhibicijom gastrične H+/K+ ATPaze, kao što je pantoprazol, mogu da ispoljavaju citotoksično dejstvo na maligne ćelije modulacijom autofagije. Upotreba LIA u istraživanjima i terapiji je ograničena zbog nespecifičnih efekata koji zavise od primenjene doze i dužine tretmana, kao i od sposobnosti da indukuju ćelijsku smrt nezavisno od inhibicije autofagije. Uzevši u obzir da molekularni mehanizmi ove pojave nisu u potpunosti razjašnjeni, u ovom istraživanju je prvi put sprovedena uporedna analiza citotoksičnosti farmakoloških inhibitora autofagije na ćelijama mišjeg melanoma (B16) i humanog glioma (U251). Zbog potrebe za selektivnijim i manje toksičnim inhibitorima autofagije, ispitani su molekularni mehanizmi citotoksičnosti pantoprazola i mogućnost njegove upotrebe kao farmakološkog inhibitora autofagije. Rezultati sprovedenog istraživanja pokazali su da sva tri LIA i pantoprazol indukuju apoptozu tumorskih ćelija praćenu produkcijom reaktivnih kiseoničnih vrsta, depolarizacijom mitohondrija, aktivacijom kaspaza i povećanom razgradnjom njihovog glavnog supstrata, PARP-1, kao i fragmentacijom DNK. Farmakološkom inhibicijom kaspaza potvrđena je uloga ovih enzima u apoptozi indukovanoj LIA, dok je učešće katepsina iz oštećenih lizozoma u ovom procesu bilo jedinstveno svojstvo hlorokina. Sva tri lizozomalna inhibitora indukovala su nakupljanje autofagozoma i autolizozoma u tumorskim ćelijama, uz povećanu konverziju LC3-I u LC3-II (engl. Microtubule-associated protein 1A/1B-light chain 3) i smanjenje unutarćelijske kiselosti, što je u skladu sa inhibicijom autofagije. Međutim, genetska inhibicija LC3, neophodnog za nastanak autofagozoma, nije uticala na preživljavanje tumorskih ćelija nakon tretmana LIA, ukazujući da je njihovo pro-apoptotsko dejstvo bilo nezavisno od inhibicije autofagije. LIA su pokazali različite obrasce modulacije pro-apoptotskih i anti-apoptotskih molekula, kao i aktivacije MAP kinaza (engl. Mitogen activated protein kinase) i AMPK (engl. Adenosine monophosphate-activated protein kinase), što je bio uzrok njihovog međusobnog antagonizma u ubijanju malignih ćelija. Za razliku od LIA, pantoprazol je u ćelijama tumora aktivirao citoprotektivnu autofagiju praćenu ekspresijom iRNK za različite Atg gene koji regulišu ovaj proces. Indukcija autofagije pantoprazolom bila je praćena aktivacijom energetskog senzora AMPK i inhibicijom fosforilacije AKT, kao i ULK1 i S6K, supstrata glavnog negativnog regulatora autofagije, mTOR-a (engl. Mechanistic target of rapamycin). Genetska i farmakološka inhibicija pokazala je da citoprotektivna autofagija indukovana pantoprazolom zavisi od AMPK i Beklina-1, kao i od p38 MAP kinaze, koja je dodatno učestvovala u indukciji ER stresa i aktivaciji transkripcionog faktora CHOP. U zaključku, iako sva tri LIA dovode do inhibicije autofagije u tumorskim ćelijama, njihova citotoksičnost je nezavisna od ovog efekta i posredovana je različitim mehanizmima koji uključuju modulaciju ekspresije pro- i anti-apoptotskih molekula, lizozomalnu permeabilizaciju i promenu aktivnosti AMPK i MAPK signalnih puteva...

Opis (srp)

Medicina - Molekularna medicina / Medicine- Molecular medicine Datum odbrane: 24.09.2021.

Opis (eng)

in autolysosomes, which are formed by fusion of autophagosomes and lysosomes. Numerous evidence of cytoprotective autophagy in tumors enabled preclinical and clinical studies based on a combination of pharmacological autophagy inhibitors and chemotherapeutics. Bafilomycin, chloroquine, and ammonium chloride are lysosomal inhibitors of autophagy (LIA) that prevent cytoplasmic content degradation in autolysosomes by blocking the V-ATPase proton pump, inhibiting acidic lysosomal hydrolases, or preventing the fusion of lysosomes with autophagosomes. Recently, it has been shown that drugs such as pantoprazole, which reduce gastric acidity by inhibiting gastric H +/K + ATPase, exert a cytotoxic effect on tumor cells by autophagy modulation. The use of LIA in research and therapy is limited due to nonspecific effects that depend on the applied dose and the incubation time, as well as the ability to induce cell death independently of autophagy inhibition. Since the molecular mechanisms of this phenomenon have not been fully elucidated, the present study for the first time performed a comparative analysis of the cytotoxicity of pharmacological autophagy inhibitors on mouse melanoma (B16) and human glioma (U251) cells. Considering the need for more selective and less toxic autophagy inhibitors, the molecular mechanisms of cytotoxicity of pantoprazole and the possibility of its use as a pharmacological autophagy inhibitor were also investigated. The results of this research showed that all three LIA and pantoprazole induce apoptotic death of cancer cells associated with the production of reactive oxygen species, mitochondrial depolarization, caspase activation and increased degradation of their main substrate, PARP-1, as well as DNA fragmentation. The role of caspases in LIA-induced apoptosis was confirmed by their pharmacological inhibition, while the involvement of cathepsins in this process was a unique property of chloroquine. All three lysosomal inhibitors, consistent with autophagy inhibition in tumor cells, induced the accumulation of autophagosomes and autolysosomes with the increased conversion of LC3-I to LC3-II (Microtubule-associated protein 1A/1B-light chain 3), and the decrease in intracellular acidity. However, the genetic inhibition of LC3, protein necessary for autophagosome formation, did not affect the survival of cancer cells after treatment with LIA, indicating that their pro-apoptotic action was independent of autophagy inhibition. LIA showed different patterns in modulating expression of pro-apoptotic and anti-apoptotic molecules, as well as activation of MAP kinases (Mitogen activated protein kinase) and AMPK (Adenosine monophosphate-activated protein kinase), which caused their mutual antagonism in cancer cell killing. Unlike LIA, pantoprazole induced cytoprotective autophagy in tumor cells, associated with the increase in mRNA expression of different Atg genes involved in regulation of this process. Autophagy induction by pantoprazole was accompanied by the activation of the energy sensor AMPK and decrease in the phosphorylation of AKT, as well as ULK1 and S6K, the substrates of mTOR (Mechanistic target of rapamycin), the major negative regulator of autophagy. Genetic and pharmacological inhibition showed that cytoprotective autophagy induced by pantoprazole is mediated by AMPK and Beclin-1, as well as by p38 MAP kinase, which additionally participated in the induction of ER stress and activation of CHOP transcription factor. In conclusion, although all three LIA inhibit autophagy in cancer cells, their cytotoxicity is autophagy-independent and mediated by various mechanisms, including modulation of pro- and anti-apoptotic molecule expression, lysosomal permeabilization, and alteration of AMPK and MAPK signaling pathways...

Jezik

srpski

Datum

2021

Licenca

Ovo delo je licencirano pod uslovima licence
Creative Commons CC BY-NC-ND 2.0 AT - Creative Commons Autorstvo - Nekomercijalno - Bez prerada 2.0 Austria License.

http://creativecommons.org/licenses/by-nc-nd/2.0/at/legalcode

Predmet

OSNO - Opšta sistematizacija naučnih oblasti, Imunologija i alergologija

bafilomycin, chloroquine, ammonium chloride, pantoprazole, autophagy

OSNO - Opšta sistematizacija naučnih oblasti, Imunologija i alergologija

bafilomicin, hlorokin, amonijum hlorid, pantoprazol, autofagija

616-006:577.2(043.3)