Nucleotidase cN-II Research Articles

Expanding the clinical relevance of the 5'-nucleotidase cN-II/NT5C2.

Purine metabolism is depending on a large amount of enzymes to ensure cellular homeostasis. Among these enzymes, we have been interested in the 5'-nucleotidase cN-II and its role in cancer biology and in response of cancer cells to treatments. This protein has been cited and studied in a large number of papers published during the last decade for its involvement in non-cancerous pathologies such as hereditary spastic paraplegia, schizophrenia, and blood pressure regulation. Here, we review these articles in order to give an overview of the recently discovered clinical relevance of cN-II.

5'-nucleotidase cN-II emerges as a new predictive biomarker of response to gemcitabine/platinum combination chemotherapy in non-small cell lung cancer.

A number of pharmacogenetic studies have been carried out in non-small-cell lung cancer (NSCLC) to identify and characterize genes involved in chemotherapy activity. However, the results obtained so far are controversial and no reliable biomarker is currently used to predict clinical benefit from platinum-based chemotherapy, which represents the cornerstone of treatment of advanced NSCLC. This study investigated the expression levels of ERCC1 and of six genes (RRM1, RRM2, hENT1, dCK, cN-II and CDA) involved in gemcitabine metabolism in locally/advanced NSCLC patients treated with gemcitabine/platinum combination. Gene expression was assessed by quantitative-PCR in laser-microdissected specimens and correlated with tumor response. Frequency distribution of responses above and below the median expression level of biomarkers was compared using a two-sided Fisher’s test. 5′-nucleotidase (cN-II) was the only gene differently expressed (p = 0.016) in the responders (complete/partial-response) compared to non-responders (stable/progressive disease). In the multivariate analysis, overexpression of this catabolic enzyme of gemcitabine remained a significant negative predictive factor. Patients with low cN-II had a modest trend toward increased survival, while both survival and progression-free survival were significantly longer in a more homogenous validation cohort of 40 advanced NSCLC (8.0 vs. 5.1 months, p = 0.026). Moreover, in vitro studies showed that silencing or pharmacological inhibition of cN-II increased the cytotoxicity of gemcitabine. This is the first study demonstrating the role of cN-II as a predictor of response to gemcitabine/platinum combinations in NSCLC. Its validation in prospective studies may improve clinical outcome of selected patients.

The cytosolic 5'-nucleotidase cN-II lowers the adaptability to glucose deprivation in human breast cancer cells.

The cytosolic 5’-nucleotidase cN-II is a highly conserved enzyme implicated in nucleotide metabolism. Based on recent observations suggesting additional roles not directly associated to its enzymatic activity, we studied human cancer cell models with basal or decreased cN-II expression. We developed cancer cells with stable inhibition of cN-II expression by transfection of shRNA-coding plasmids, and studied their biology. We show that human breast cancer cells MDA-MB-231 with decreased cN-II expression better adapt to the disappearance of glucose in growth medium under normoxic conditions than cells with a baseline expression level. This is associated with enhanced in vivo growth and a lower content of ROS in cells cultivated in absence of glucose due to more efficient mechanisms of elimination of ROS. Conversely, cells with low cN-II expression are more sensitive to glucose deprivation in hypoxic conditions. Overall, our results show that cN-II regulates the cellular response to glucose deprivation through a mechanism related to ROS metabolism and defence.

Determination of the enzymatic activity of cytosolic 5'-nucleotidase cN-II in cancer cells: development of a simple analytical method and related cell line models.

The cytosolic 5'-nucleotidase (cN-II) has been shown to be involved in the response of cancer cells to cytotoxic agents, and the quantification of its activity in biological samples is of great interest. In this context, we developed and validated an analytical method for determination of cN-II activity in cultured cancer cells. This non-radioactive method, using a Hypercarb column as stationary phase, was validated with a lower limit of quantification of 0.1 μM inosine. We used it to characterize cell line models with modified cN-II expression obtained with stable transfections. We show that the short hairpin RNA (shRNA)-mediated inhibition of cN-II expression in various malignant blood cells is associated with decreased protein expression and enzymatic activity (1.7-6.2-fold) as well as an increased sensitivity to cytotoxic agents (up to 14-fold). On the other hand, expression of green fluorescent protein (GFP)-fused wild type or hyperactive mutant (R367Q) cN-II increased the activity and also decreased the sensitivity to nucleoside analogues. Our results confirm the biological relevance of modulating cN-II in cancer cells, and we present a straightforward validated method for the determination of cN-II activity in cellular samples.

Therapeutic Perspectives for cN-II in Cancer.

The cytoplasmic 5'-nucleotidase cN-II is involved in the regulation of endogenous pools of nucleosides and nucleotides together with nucleoside kinases and other intracellular enzymes. A series of results from studies on preclinical models and clinical samples constitutes the basis of the hypothesis in which cN-II is a therapeutic target in cancer. Indeed, the inhibition of its enzymatic activity seems interesting both to induce cell death directly and to increase the anticancer activity of cytotoxic agents used in cancer treatment. Here we will review the current knowledge of the enzymatic function of cN-II together with available structural data and the studies on cN-II in cancer cells and in samples from cancer patients. Recent and ongoing research on cN-II inhibitors is expected to confirm the druggability and the relevance of cN-II as a cancer drug target. Preliminary in vitro data and cancer cell models using cN-II inhibitors have already suggested the pivotal role of this enzyme as therapeutic target allowing the improvement of anticancer treatments.

A native electrophoretic technique to study oligomerization and activity of cytosolic 5′-nucleotidase II

The analysis of the oligomeric active state of a native protein usually requires the application of at least two analytical methods such as gel filtration and analytical ultracentrifugation. Both methods require a substantial amount of protein, time and/or expensive equipment. We here describe a native electrophoretic method for the identification of the native molecular weight of the recombinant wild-type cytosolic 5′-nucleotidase (cN-II) and of its mutants in subunit interfaces Y115A, F36R, K311A and G319Q. The protein was stained both with protein dye and with an activity staining method. Our results demonstrated that purified recombinant protein preparations contained substantial amounts of nucleic acids and misfolded, inactive protein. Furthermore, cN-II mutants K311A and G319Q in subunit interface assume a quaternary dimeric active form, while the only active quaternary structure of wild-type cN-II is the tetramer.

Identification and characterization of inhibitors of cytoplasmic 5′-nucleotidase cN-II issued from virtual screening

Clinical and preclinical observations have lead to the hypothesis that 5′-nucleotidase cN-II could constitute a therapeutic target in oncology, either per se or to increase the activity of cytotoxic nucleoside analogs.To identify potential cN-II inhibitors, we performed in silico screening of freely available chemical databases, in vitro enzymatic assays with recombinant cN-II, soaking experiments with crystals of truncated cN-II as well as biological evaluation of selected compounds, alone or in combination with cytotoxic nucleoside analogs, on cancer cells.The top ranked compounds from virtual screening included an anthraquinone derivative (AdiS) that were shown to block the enzyme activity with a Ki of 2.0mM. Soaking experiments performed with crystals of truncated cN-II allowed to obtain crystallographic data at a resolution of 2.9Å and indicating interaction between AdiS and F354/I152 situated in the effector site 1 of cN-II. In addition, this compound exhibited different levels of cytotoxicity in vitro on several cancer cell lines and increased the induction of apoptosis in RL cells incubated with 0.5 or 1.5μM cladribine, 0.05μM clofarabine or 30μM fludarabine. Finally, AdiS showed synergy with cladribine and additivity with clofarabine.This study showed that virtual screening is a useful tool for the identification of potent cN-II inhibitors, and our biological results indicated interesting activity for one lead compound that can be further developed as therapeutics.

Abstract 3835: Identification and characterization of inhibitors of 5′-nucleotidase cN-II issued from virtual screening

Abstract Clinical and preclinical observations have lead to the hypothesis that 5′-nucleotidase cN-II could constitute a therapeutic target in oncology, either per se, either to increase the activity of cytotoxic nucleoside analogues. We performed in silico screening of freely available chemical databases, in vitro enzymatic assays with recombinant full length cN-II, soaking experiments with crystals of truncated cN-II and biological evaluation of inhibitors alone or in combination with cytotoxic nucleoside analogues on cancer cells both in vitro and in vivo. The top ranked compounds from virtual screening contained an anthraquinone-derivative (A) and a trisubstituted triazine (B) that were selected for further studies. In vitro enzymatic assay experiments with recombinant protein showed that compound A is a competitive inhibitor with Ki of approximately 3 mM, whereas derivative B is a non-competitive inhibitor with Ki of 1.1 mM. We also obtained crystallographic data at a resolution of 2.0 Å after soaking experiments with crystals of truncated cN-II. These showed interaction between A and F354/N154 situated in the effector site 1 of cN-II, whereas B was not found in crystallographic data even though crystals were degraded in presence of B over a longer time. Derivative A showed different levels of cytotoxicity in vitro on several cancer cell lines such as Raji (IC50=782 µM), RL (IC50=210 µM), CCRF-CEM (IC50=824 µM), MCF-7 (IC50=654 µM), A549 (IC50=569 µM) and HCT-116 (IC50=793 µM), whereas B was much less cytotoxic. When used at 1 mM, A and B did not modify IC50 of the nucleoside analogues cladribine, clofarabine or fludarabine in CCRF-CEM cells. When used at 100 µM, compound A increased the induction of apoptosis in RL cells incubated with 0.5 or 1.5 µM cladribine, 0.05 µM clofarabine or 30 µM fludarabine. The administration of A (200 mg/kg, 5d/w, 4w) to mice xenografted with RL cells, induced a delay of tumor development that was higher than in mice treated with fludarabine (50 mg/kg, 1d/w, 4w). We showed that virtual screening can be used for the identification of potent cN-II inhibitors, and we produced clear evidences for the interaction of one inhibitor with the enzyme. Our biological evaluation indicated interesting activity for one lead compound that will be developed further in order to identify candidates with higher biological activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3835. doi:1538-7445.AM2012-3835

5 ′-Amino-4-Imidazolecarboxamide Riboside Induces Apoptosis in Human Neuroblastoma Cells Via the Mitochondrial Pathway

5′-Amino-4-imidazolecarboxamide (AICA) riboside induces apoptosis in neuronal cell models. In order to exert its effect, AICA riboside must enter the cell and be phosphorylated to the ribotide. In the present work, we have further studied the mechanism of apoptosis induced by AICA riboside. The results demonstrate that AICA riboside activates AMP-dependent protein kinase (AMPK), induces release of cytochrome c from mitochondria and activation of caspase 9. The role of AMPK in determining cell fate is controversial. In fact, AICA riboside has been reported to be neuroprotective or to induce apoptosis depending on its concentration, cell type or apoptotic stimuli used. In order to clarify whether the activation of AMPK is related to apoptosis in our model, we have used another AMPK stimulator, metformin, and we have analysed its effects on cell viability, nuclear morphology and AMPK activity. Five mM metformin increased AMPK activity, inhibited viability, and increased the number of apoptotic nuclei. AICA riboside, which can be generated from the ribotide (an intermediate of the purine de novo synthesis) by the action of the ubiquitous cytosolic 5′-nucleotidase (cN-II), may accumulate in those individuals in which an inborn error of purine metabolism causes both a building up of intermediates and/or an increase of the rate of de novo synthesis, and/or an overexpression of cN-II. Therefore, our results suggest that the toxic effect of AICA riboside on some types of neurons may participate in the neurological manifestations of syndromes related to purine dismetabolisms.

F-ara-AMP is a substrate of cytoplasmic 5′-nucleotidase II (cN-II): HPLC and NMR studies of enzymatic dephosphorylation

Intracellular accumulation of triphosphorylated derivatives is essential for the cytotoxic activity of nucleoside analogues. Different mechanisms opposing this accumulation have been described. We have investigated the dephosphorylation of monophosphorylated fludarabine (F-ara-AMP) by the purified cytoplasmic 5′-nucleotidase cN-II using HPLC and NMR. These studies clearly showed that cN-II was able to convert F-ara-AMP into its non phosphorylated form, F-ara-A, with a K m in the millimolar range and V max = 35 nmol/min/mg, with both methods. Cytoplasmic 5′-nucleotidase cN-II can degrade this clinically useful cytotoxic nucleoside analogue and its overexpression is thus likely to be involved in resistance to this compound.