Uloga adenozin-monofosfatom aktivirane protein-kinaze i mTOR kompleksa 1 u in vitro citotoksičnom dejstvu nesteroidnih anti-inflamatornih lekova na ćelije glioma

Abstract

Gliomi su najčešći primarni tumori centralnog nervnog sistema. Glioblastom je gliom gradusa IV, terapijski rezistentan maligni tumor centralnog nervnog sistema. Nesteroidni anti-inflamatorni lekovi (NSAIL) su strukturno različite grupe molekula sa anti-inflamatornim i analgetskim dejstvom koji ispoljavaju i jak antitumorski efekat kako in vitro tako i in vivo. U ovoj doktorskoj disertaciji ispitivano je citotoksično dejstvo NSAIL na ćelije humane linije glioma U251 i ćelije primarne kulture glioblastoma izolovane iz tkiva obolelih pacijenata. Takođe, istraživali smo ulogu signalnog puta adenozin-monofosfatom aktivirane protein-kinaze (AMPK) i mTOR kompleksa 1 (mTORC1 - engl. mammalian target of rapamycin complex 1) u antigliomskom dejstvu NSAIL. Indometacin je snažnije od ostalih NSAIL (diklofenaka, naproksena i ketoprofena) smanjivao vijabilitet U251 ćelija humanog glioblastoma. Antigliomski efekat indometacina je bio povezan sa ekspresijom tumor supresorskog proteina p21 i zastojem ćelijskog ciklusa u G2M fazi, indukcijom oksidativnog stresa, depolarizacijom mitohondrija, aktivacijom kaspaza i indukcijom apoptoze. Indometacin je jedini od ispitivanih NSAIL povećavao fosforilaciju AMPK i nishodnih molekula koje ona fosforiliše, Raptor-a i acetil-CoA karboksilaze (ACC). Aktivaciju AMPK pratilo je smanjenje fosforilacije mTOR i molekula čiju aktivnost mTORC1 reguliše, kao što su ribozomalna p70S6 kinaza (S6K) i PRAS40 (Ser183). Genetska inhibicija AMPK RNK interferencijom, kao i tretman ćelija aktivatorom mTORC1 leucinom doveli su do delimičnog poništavanja citotoksičnih efekata izazvanih indometacinom. Sa druge strane, farmakološki aktivatori AMPK metformin i AICAR su poput indometacina ispoljavali antigliomski efekat inhibicijom mTORC1. Aktivacija AMPK u ćelijama tretiranim indometacinom korelirala je sa smanjenjem ćelijskog ATP i porastom AMP/ATP odnosa, ali je bila nezavisna od inhibicije COX i povećanja intraćelijskog nivoa kalcijuma. Takođe, citotoksični efekat indometacina na ćelije primarne kulture glioblastoma bio je posredovan aktivacijom AMPK/Raptor/ACC i inhibicijom mTORC1/S6K signalnih molekula...

Gliomas are the most common primary tumors of the central nervous system. Glioblastoma is grade IV glioma, therapy-resistant malignant tumor of the central nervous system. Non-steroidal anti-inflammatory drugs (NSAID) are structurally different groups of molecules with anti-inflammatory and analgesic effects that exert strong antitumor effect, both in vitro and in vivo. In this doctoral dissertation, the cytotoxic effect of NSAID on U251 human glioma cell line and the primary culture of glioblastoma cells isolated from patients has been examined. The role of the intracellular energy-sensing AMP-activated protein kinase (AMPK)/mammalian target of rapamycin complex 1 (mTORC1) pathway in the in vitro antiglioma effect of NSAID has been investigated. Indomethacin was more potent than other NSAID (diclofenac, naproxen, and ketoprofen) in reducing the viability of U251 human glioma cells. Antiglioma effect of indomethacin 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 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. On the other hand, AMPK activators metformin and AICAR mimicked mTORC inhibition-dependent cytotoxic 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. The toxicity of indomethacin towards primary human glioblastoma cells was also associated with the activation of AMPK/Raptor/ACC and subsequent suppression of mTORC1/S6K. Finally, the ability of indomethacin to induce autophagy in U251 VIII human glioma cells has been investigated...