Pro-apoptotic role of AMP-activated protein kinase under oxidative conditions linked to bioenergetic impairment: implications for cancer treatment and neurodegeneration

The imbalance between ROS production and clearance leads to oxidative stress, a condition implicated in the pathogenesis of several cell disorders such as neurodegeneration and cancer. In these settings, pro-oxidant conditions activate many redox-sensitive proteins involved in the regulation of the apoptotic program. Among them, the tumor suppressor p53 and the mitogen-activated protein kinases (MAPK) are implicated both in the pathogenesis of neurodegenerative disorders and in many chemotherapeutic strategies aimed to the elimination of cancer-prone cells from the replicative pool. Recently, AMP-activated protein kinase (AMPK) has been identified as a component of the signaling cascade able to sense bioenergetic and oxidative challenges. Moreover, its capability to trigger apoptosis by activating p53 or some members of the MAPK family (e.g. p38MAPK), makes it a putative player both in neurodegenerative disease and in cancer management. On the basis of this knowledge, this PhD thesis is aimed to elucidate the pro-apoptotic properties of AMPK in SH-SY5Y neuronal cell line under pro-oxidant conditions associated to bioenergetic impairment. This research has been performed by using three compounds known to affect cellular redox homeostas: Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N’]copper(II) perchlorate (Cu(isaepy)2), tetrahydrobiopterin (BH4) and sodium nitroprusside (SNP). Our experiments demonstrated that Cu(isaepy)2, by acting as a DLC-like molecule, was able to induce cell death by activating the AMPK/ p38MAPK/p53 signaling axis, in response to a mitochondrial impairment. The cross-talk between these proteins was found to be operative also in SH-SY5Y cell death induced by BH4, an obligatory cofactor for tyrosine hydroxylase in dopamine synthesis, and to be sensitive to the alteration of glycolytic metabolism. Finally the observation that the iron-nytrosil complex SNP was unable to affect cellular energetics, explains on the one hand the uneffectivenes of AMPK in mediating SNP-induced apoptosis and, on the other, suggests a possible role for this energy-responsive kinase in apoptosis engagement only under condition of bioenergetic stress.

Lo squilibrio tra produzione di ROS e loro eliminazione porta a stress ossidativo, una condizione implicata nella patogenesi di molti disordini cellulari come la neurodegenerazione e il cancro. In questi constesti, condizioni pro-ossidanti attivano molte proteine redox-sensibili coinvolte nella regolazione del programma apoptotico. Tra di questi, p53 e le MAPK sono implicate nella patogenesi dei disordini neurodegenerativi e in molte strategie chemioterapeutiche finalizzate alla eliminazione di cellule cancerose dal pool replicativo. Recentemente AMPK è stata identificata come un componente della cascata di segnalazione capace di percepire stress bioenergetici e ossidativi. Inoltre la sua capacità di indurre apoptosi attivando p53 o taluni membri della famiglia delle MAPK (ad es. p38MAPK), la rende implicata sia nelle malattie neurodegnerative che nel trattamento del cancro. Sulla base di queste conoscenze, questa tesi di dottorato è finalizzata ad elucidare le prpoprietà pro-apoptotiche di AMPK nella linea neuronale SH-SY5Y in risposta a condizioni pro-ossidanti di AMPK associate a stress bioenergetico. La ricerca è stata realizzata usando 3 composti noti influenzare l'omeosatsi redox:Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N’]copper(II) perchlorate (Cu(isaepy)2), tetrahydrobiopterin (BH4) and sodium nitroprusside (SNP).