Abstract
It has previously been shown that prolonged incubation with 5’-amino-4-imidazolecarboxamide (AICA) riboside produces nuclear fragmentation and caspase-3 activation (which are characteristic features of apoptosis) in the human neuroblastoma SH-SY5Y cells. The AICA riboside is able to enter the cell and to be phosphorylated in the cytoplasm, by means of adenosine kinase, to ribotide which mimics AMP and leads to the activation of the AMP-activated protein kinase (AMPK). The AMPK, which belongs to the serine-threonine kinase family, is considered to be a sensor of the cellular energy status in a variety of tissues. The AMPK, that is switched on by phosphorylation on Thr-172 aminoacidic residue of the α subunit, regulates ATP consumption and regeneration processes both on normal physiological (like skeletal and cardiac muscle contraction both on rest and during exercise) and pathological conditions (such as metabolic stress, hypoxia, ischemia or deprivation glucose). The role played by AMPK in apoptosis is not clear; several Authors have reported its activation during programmed cell death while others reported its involvement in the protection mecanism against it.
The aim of the present work is to clarify the apoptosis mechanism induced by AICA riboside. The micochondrial pathway is characterized by mitochondrial cytochrome c release to cytoplasm, where it binds to the Apaf 1 protein and induces caspase-9 activation. In order to clarify whether the apoptosis induced by AICA riboside involves
the mitochondrial pathway, the release of the cytochrome c and the activation of caspase-9 through western bloting have been documentedd extracts of cells which have been treated previously with AICA riboside during various lapses of time and of control cultures have been subjected to SDS-PAGE, then transferred on a PVDF membrane and lastly incubated with appropiate antibodies. We have observed that the modifcations of the nuclear morphology are preceded by cytocrome c release and caspase c activation. In order to understand whether AMPK activation is involved in the mechanism of cytotoxicity we have incubated the neuroblastoma cells with an other AMPK stimulator, metformin, or with an inhibitor , the compound C. The AMPK activity has been tested by western bloting with anti-phospho-AMPK antibody, in order to unveil its presence. The cellular viability has been measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test and the nuclear morphology has been analysed by fuorescence microscopy aaer Hoechst stain.
Both AICA riboside and metformin treatments produce AMPK activation already detectable aaer 3-5 hours. Treatment with metformin 5 mM for 72 h decrease cellular viability and induce appearence of apptotic bodies. The compound C does not produce protection in AICA riboside-induced apoptosis in neuroblastoma cells. This suggests that AMPK has to be regulated in a very precise way, because both AMPK stimulation and inhibition reduce viabilty.
AICA riboside could accumulate in subjects with inborn purine dismetabolism causing an increase of the de novo syntesis and/or an overexpression of the 5’-nucleotidase (the enzyme that transforms ribotide in riboside). The results of this thesis suggest that the toxic efect of AICA riboside on certain types of neurons may take part to neurological manifestations of syndromes corelated with purine dismetabolism.
