Cytosolic 5′-Nucleotidase II Silencing in a Human Lung Carcinoma Cell Line Opposes Cancer Phenotype with a Concomitant Increase in p53 Phosphorylation

Abstract

Purine homeostasis is maintained by a purine cycle in which the regulated member is a cytosolic 5′-nucleotidase II (cN-II) hydrolyzing IMP and GMP. Its expression is particularly high in proliferating cells, indeed high cN-II activity or expression in hematological malignancy has been associated to poor prognosis and chemoresistance. Therefore, a strong interest has grown in developing cN-II inhibitors, as potential drugs alone or in combination with other compounds. As a model to study the effect of cN-II inhibition we utilized a lung carcinoma cell line (A549) in which the enzyme was partially silenced and its low activity conformation was stabilized through incubation with 2-deoxyglucose. We measured nucleotide content, reduced glutathione, activities of enzymes involved in glycolysis and Krebs cycle, protein synthesis, mitochondrial function, cellular proliferation, migration and viability. Our results demonstrate that high cN-II expression is associated with a glycolytic, highly proliferating phenotype, while silencing causes a reduction of proliferation, protein synthesis and migration ability, and an increase of oxidative performances. Similar results were obtained in a human astrocytoma cell line. Moreover, we demonstrate that cN-II silencing is concomitant with p53 phosphorylation, suggesting a possible involvement of this pathway in mediating some of cN-II roles in cancer cell biology.

Emerging Role of Purine Metabolizing Enzymes in Brain Function and Tumors

“…Owing to the high cN-II expression observed in tumor cells and its involvement in drug resistance, cN-II inhibition has been proposed as new therapeutic approach in tumors [ 15 ]. To support this idea, cN-II silencing or hyperexpression has been performed in several cancer cell models [ 11 , 14 ]. In the ADF glioblastoma cell line, cN-II hyperactivity was accompanied by an increase of proliferation and resistance to gemcitabine and mitomycin C, while enzyme silencing caused a decrease of cell proliferation [ 13 ].…”

“…In the ADF glioblastoma cell line, cN-II hyperactivity was accompanied by an increase of proliferation and resistance to gemcitabine and mitomycin C, while enzyme silencing caused a decrease of cell proliferation [ 13 ]. Also in A548 lung cancer cell line, cN-II silencing caused a decrease of proliferation and a metabolic switch from a more glycolytic to a more oxidative phenotype [ 11 ]. Finally, in a breast cancer cell model (MDA-MB-231), cN-II silencing caused the activation of a complex mechanism leading to an increase of antioxidant defenses and to a better capacity to respond to a low glucose environment [ 12 ].…”

“…CN-II is usually highly expressed in cells and organs with high nucleic acid turnover and in tumors [ 8 ]. The effect of cN-II silencing or hyperexpression has been studied in several tumor cell models [ 11 , 12 , 13 ], demonstrating that the enzyme hyperexpression is followed by a strong decrease of all triphosphorylated purine and pyrimidine nucleosides [ 14 ]. Conversely, cN-II silencing causes an increase of the intracellular concentration of the same compounds [ 11 , 14 ].…”

Purine-Metabolising Enzymes and Apoptosis in Cancer

“…Indeed, shRNA-based cell models with downregulated cN-II are more sensitive to purine nucleoside and nucleobase analogues, as compared to control cells [25]. The enzyme is highly expressed in tumour cells, and cN-II expression in human neuroblastoma cells and in lung cancer cells correlated with cell proliferation [26,27], whereas its inhibition in human breast cancer cells was associated with a better defence towards reactive oxygen species (ROS) and a better adaptability to glucose deprivation in culture media [28]. In a cell model of lung cancer (A549), an activation of p53 and inactivation of Akt following cN-II partial silencing was demonstrated [27].…”

“…The enzyme is highly expressed in tumour cells, and cN-II expression in human neuroblastoma cells and in lung cancer cells correlated with cell proliferation [26,27], whereas its inhibition in human breast cancer cells was associated with a better defence towards reactive oxygen species (ROS) and a better adaptability to glucose deprivation in culture media [28]. In a cell model of lung cancer (A549), an activation of p53 and inactivation of Akt following cN-II partial silencing was demonstrated [27]. Indeed, in an astrocytoma cell line (ADF), transitory cN-II silencing was followed by caspase-3 activation and apoptosis [29].…”