Abstract
Cytosolic 5′-nucleotidase II (cN-II) is an allosteric catabolic enzyme that hydrolyzes IMP, GMP, and AMP. The enzyme can assume at least two different structures, being the more active conformation stabilized by ATP and the less active by inorganic phosphate. Therefore, the variation in ATP concentration can control both structure and activity of cN-II. In this paper, using a capillary electrophoresis technique, we demonstrated that a partial silencing of cN-II in a pulmonary carcinoma cell line (NCI-H292) is accompanied by a decrease in adenylate pool, without affecting the energy charge. We also found that cN-II silencing decreased proliferation and increased oxidative metabolism, as indicated by the decreased production of lactate. These effects, as demonstrated by Western blotting, appear to be mediated by both p53 and AMP-activated protein kinase, as most of them are prevented by pifithrin-α, a known p53 inhibitor. These results are in line with our previous observations of a shift towards a more oxidative and less proliferative phenotype of tumoral cells with a low expression of cN-II, thus supporting the search for specific inhibitors of this enzyme as a therapeutic tool for the treatment of tumors.
Highlights
We demonstrate that the metabolic effects exerted by Cytosolic -nucleotidase II (cN-II) partial silencing are not directly correlated with a disturbance of purine nucleotide pool but are mediated by the p53/AMP-activated protein kinase (AMPK) signaling pathway
Since cN-II catalyzes the dephosphorylation of IMP and GMP and of AMP, all the observed effects might be due to an increase in AMP concentration in silenced cells, with the consequent activation of AMPK that exerts a positive effect on mitochondrial proliferation and oxidative metabolism [27]
We describe that cN-II silencing in a pulmonary carcinoma cell line (NCI-H292) caused a significant increase in AMP and ADP, whereas ATP along with UTP were slightly, but significantly, decreased
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. The high proliferation rate requires signals of high energy charge, whereas a low energy charge activates AMPK and p53 to lower the rate of synthetic pathways and improve metabolic performance [5,6]. At a low energy charge, cN-II assumes a low activity conformation leading to IMP and AMP accumulation avoiding a dangerous depletion of purine nucleotide precursors [12]. In this condition, adenosine can be generated by AMP hydrolysis mediated by cN-I, an isoenzyme specific for AMP which is activated by ADP [10,13]. The cell models were silenced for cN-II by stable transfection of shRNA-encoding plasmids as earlier described [22]
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