Interplay between mTOR and Purine Metabolism Enzymes and Its Relevant Role in Cancer.

Tumor cells reprogram their metabolism to meet the increased demand for nucleotides and other molecules necessary for growth and proliferation. In fact, cancer cells are characterized by an increased "de novo" synthesis of purine nucleotides. Therefore, it is not surprising that specific enzymes of purine metabolism are the targets of drugs as antineoplastic agents, and a better knowledge of the mechanisms underlying their regulation would be of great help in finding new therapeutic approaches. The mammalian target of the rapamycin (mTOR) signaling pathway, which is often activated in cancer cells, promotes anabolic processes and is a major regulator of cell growth and division. Among the numerous effects exerted by mTOR, noteworthy is its empowerment of the "de novo" synthesis of nucleotides, accomplished by supporting the formation of purinosomes, and by increasing the availability of necessary precursors, such as one-carbon formyl group, bicarbonate and 5-phosphoribosyl-1-pyrophosphate. In this review, we highlight the connection between purine and mitochondrial metabolism, and the bidirectional relation between mTOR signaling and purine synthesis pathways.

Cytosolic 5'-Nucleotidase II Silencing in Lung Tumor Cells Regulates Metabolism through Activation of the p53/AMPK Signaling Pathway.

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.

The Inside Story of Adenosine.

Several physiological functions of adenosine (Ado) appear to be mediated by four G protein-coupled Ado receptors. Ado is produced extracellularly from the catabolism of the excreted ATP, or intracellularly from AMP, and then released through its transporter. High level of intracellular Ado occurs only at low energy charge, as an intermediate of ATP breakdown, leading to hypoxanthine production. AMP, the direct precursor of Ado, is now considered as an important stress signal inside cell triggering metabolic regulation through activation of a specific AMP-dependent protein kinase. Intracellular Ado produced from AMP by allosterically regulated nucleotidases can be regarded as a stress signal as well. To study the receptor-independent effects of Ado, several experimental approaches have been proposed, such as inhibition or silencing of key enzymes of Ado metabolism, knockdown of Ado receptors in animals, the use of antagonists, or cell treatment with deoxyadenosine, which is substrate of the enzymes acting on Ado, but is unable to interact with Ado receptors. In this way, it was demonstrated that, among other functions, intracellular Ado modulates angiogenesis by regulating promoter methylation, induces hypothermia, promotes apoptosis in sympathetic neurons, and, in the case of oxygen and glucose deprivation, exerts a cytoprotective effect by replenishing the ATP pool.

Emerging Role of Purine Metabolizing Enzymes in Brain Function and Tumors.

The growing evidence of the involvement of purine compounds in signaling, of nucleotide imbalance in tumorigenesis, the discovery of purinosome and its regulation, cast new light on purine metabolism, indicating that well known biochemical pathways may still surprise. Adenosine deaminase is important not only to preserve functionality of immune system but also to ensure a correct development and function of central nervous system, probably because its activity regulates the extracellular concentration of adenosine and therefore its function in brain. A lot of work has been done on extracellular 5'-nucleotidase and its involvement in the purinergic signaling, but also intracellular nucleotidases, which regulate the purine nucleotide homeostasis, play unexpected roles, not only in tumorigenesis but also in brain function. Hypoxanthine guanine phosphoribosyl transferase (HPRT) appears to have a role in the purinosome formation and, therefore, in the regulation of purine synthesis rate during cell cycle with implications in brain development and tumors. The final product of purine catabolism, uric acid, also plays a recently highlighted novel role. In this review, we discuss the molecular mechanisms underlying the pathological manifestations of purine dysmetabolisms, focusing on the newly described/hypothesized roles of cytosolic 5'-nucleotidase II, adenosine kinase, adenosine deaminase, HPRT, and xanthine oxidase.

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

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.

IMP-GMP specific cytosolic 5'-nucleotidase regulates nucleotide pool and prodrug metabolism.

BACKGROUND: Type II cytosolic 5'-nucleotidase (cN-II) catalyzes the hydrolysis of purine and, to some extent, of pyrimidine monophosphates. Recently, a number of papers demonstrated the involvement of cN-II in the mechanisms of resistance to antitumor drugs such as cytarabine, gemcitabine and fludarabine. Furthermore, cN-II is involved in drug resistance in patients affected by hematological malignancies influencing the clinical outcome. Although the implication of cN-II expression and/or activity appears to be correlated with drug resistance and poor prognosis, the molecular mechanism by which cN-II mediates drug resistance is still unknown. METHODS: HEK 293 cells carrying an expression vector coding for cN-II linked to green fluorescent protein (GFP) and a control vector without cN-II were utilized. A highly sensitive capillary electrophoresis method was applied for nucleotide pool determination and cytotoxicity exerted by drugs was determined with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. RESULTS: Over-expression of cN-II causes a drop of nucleoside triphosphate concentration and a general disturbance of nucleotide pool. Over-expressing cells were resistant to fludarabine, gemcitabine and cytarabine independently of cN-II ability to hydrolyze their monophosphates. CONCLUSIONS: An increase of cN-II expression is sufficient to cause both a general disturbance of nucleotide pool and an increase of half maximal inhibitory concentration (IC50) of the drugs. Since the monophosphates of cytarabine and gemcitabine are not substrates of cN-II, the protection observed cannot be directly ascribed to drug inactivation. GENERAL SIGNIFICANCE: Our results indicate that cN-II exerts a relevant role in nucleotide and drug metabolism through not only enzyme activity but also a mechanism involving a protein-protein interaction, thus playing a general regulatory role in cell survival. SENTENCE: Resistance to fludarabine, gemcitabine and cytarabine can be determined by an increase of cN-II both through dephosphorylation of active drugs and perturbation of nucleotide pool.

Mitochondrial Damage and Apoptosis Induced by Adenosine Deaminase Inhibition and Deoxyadenosine in Human Neuroblastoma Cell Lines.

The treatment with deoxycoformycin, a strong adenosine deaminase inhibitor, in combination with deoxyadenosine, causes apoptotic cell death of two human neuroblastoma cell lines, SH-SY5Y and LAN5. Herein we demonstrate that, in SH-SY5Y cells, this combination rapidly decreases mitochondrial reactive oxygen species and, in parallel, increases mitochondrial mass, while, later, induces nuclear fragmentation, and activation of caspase-8, -9, and -3. In previous papers we have shown that a human astrocytoma cell line, subjected to the same treatment, undergoes apoptotic death as well. Therefore, both astrocytoma and neuroblastoma cell lines undergo apoptotic death following the combined treatment with deoxycoformycin and deoxyadenosine, but several differences have been found in the mode of action, possibly reflecting a different functional and metabolic profile of the two cell lines. Overall this work indicates that the neuroblastoma cell lines, like the line of astrocytic origin, are very sensitive to purine metabolism perturbation thus suggesting new therapeutic approaches to nervous system tumors. J. Cell. Biochem. 117: 1671-1679, 2016. © 2015 Wiley Periodicals, Inc.

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.

Inborn Errors of Purine Salvage and Catabolism.

Cellular purine nucleotides derive mainly from de novo synthesis or nucleic acid turnover and, only marginally, from dietary intake. They are subjected to catabolism, eventually forming uric acid in humans, while bases and nucleosides may be converted back to nucleotides through the salvage pathways. Inborn errors of the purine salvage pathway and catabolism have been described by several researchers and are usually referred to as rare diseases. Since purine compounds play a fundamental role, it is not surprising that their dysmetabolism is accompanied by devastating symptoms. Nevertheless, some of these manifestations are unexpected and, so far, have no explanation or therapy. Herein, we describe several known inborn errors of purine metabolism, highlighting their unexplained pathological aspects. Our intent is to offer new points of view on this topic and suggest diagnostic tools that may possibly indicate to clinicians that the inborn errors of purine metabolism may not be very rare diseases after all.

Structural basis of the substrate specificity of Bacillus cereus adenosine phosphorylase.

Purine nucleoside phosphorylases catalyze the phosphorolytic cleavage of the glycosidic bond of purine (2'-deoxy)nucleosides, generating the corresponding free base and (2'-deoxy)-ribose 1-phosphate. Two classes of PNPs have been identified: homotrimers specific for 6-oxopurines and homohexamers that accept both 6-oxopurines and 6-aminopurines. Bacillus cereus adenosine phosphorylase (AdoP) is a hexameric PNP; however, it is highly specific for 6-aminopurines. To investigate the structural basis for the unique substrate specificity of AdoP, the active-site mutant D204N was prepared and kinetically characterized and the structures of the wild-type protein and the D204N mutant complexed with adenosine and sulfate or with inosine and sulfate were determined at high resolution (1.2-1.4 Å). AdoP interacts directly with the preferred substrate through a hydrogen-bond donation from the catalytically important residue Asp204 to N7 of the purine base. Comparison with Escherichia coli PNP revealed a more optimal orientation of Asp204 towards N7 of adenosine and a more closed active site. When inosine is bound, two water molecules are interposed between Asp204 and the N7 and O6 atoms of the nucleoside, thus allowing the enzyme to find alternative but less efficient ways to stabilize the transition state. The mutation of Asp204 to asparagine led to a significant decrease in catalytic efficiency for adenosine without affecting the efficiency of inosine cleavage.

The Good, the Bad and the New about Uric Acid in Cancer.

Uric acid is the final product of purine catabolism in man and apes. The serum concentration of uric acid is sex-, age- and diet-dependent and is maintained close to its maximal solubility, indicating that it plays some important role. Indeed, it has been demonstrated that, at physiological concentrations, uric acid is a powerful antioxidant, while at high intracellular concentrations, it is a pro-oxidant molecule. In this review, we describe the possible causes of uric acid accumulation or depletion and some of the metabolic and regulatory pathways it may impact. Particular attention has been given to fructose, which, because of the complex correlation between carbohydrate and nucleotide metabolism, causes uric acid accumulation. We also present recent results on the positive and negative effects played by uric acid in cancer and some new findings and hypotheses about the implication of this metabolite in a variety of signaling pathways, which can play a role in the pathogenesis of diseases such as metabolic syndrome, diabetes, and inflammation, thus favoring the development of cancer. The loss of uricase in Homo sapiens and great apes, although exposing these species to the potentially adverse effects of uric acid, appears to be associated with evolutionary advantages.

Metabolic Aspects of Adenosine Functions in the Brain.

Adenosine, acting both through G-protein coupled adenosine receptors and intracellularly, plays a complex role in multiple physiological and pathophysiological processes by modulating neuronal plasticity, astrocytic activity, learning and memory, motor function, feeding, control of sleep and aging. Adenosine is involved in stroke, epilepsy and neurodegenerative pathologies. Extracellular concentration of adenosine in the brain is tightly regulated. Adenosine may be generated intracellularly in the central nervous system from degradation of AMP or from the hydrolysis of S-adenosyl homocysteine, and then exit via bi-directional nucleoside transporters, or extracellularly by the metabolism of released nucleotides. Inactivation of extracellular adenosine occurs by transport into neurons or neighboring cells, followed by either phosphorylation to AMP by adenosine kinase or deamination to inosine by adenosine deaminase. Modulation of the nucleoside transporters or of the enzymatic activities involved in the metabolism of adenosine, by affecting the levels of this nucleoside and the activity of adenosine receptors, could have a role in the onset or the development of central nervous system disorders, and can also be target of drugs for their treatment. In this review, we focus on the contribution of 5'-nucleotidases, adenosine kinase, adenosine deaminase, AMP deaminase, AMP-activated protein kinase and nucleoside transporters in epilepsy, cognition, and neurodegenerative diseases with a particular attention on amyotrophic lateral sclerosis and Huntington's disease. We include several examples of the involvement of components of the adenosine metabolism in learning and of the possible use of modulators of enzymes involved in adenosine metabolism or nucleoside transporters in the amelioration of cognition deficits.

Cytosolic 5'-Nucleotidase II Is a Sensor of Energy Charge and Oxidative Stress: A Possible Function as Metabolic Regulator.

Cytosolic 5'-nucleotidase II (NT5C2) is a highly regulated enzyme involved in the maintenance of intracellular purine and the pyrimidine compound pool. It dephosphorylates mainly IMP and GMP but is also active on AMP. This enzyme is highly expressed in tumors, and its activity correlates with a high rate of proliferation. In this paper, we show that the recombinant purified NT5C2, in the presence of a physiological concentration of the inhibitor inorganic phosphate, is very sensitive to changes in the adenylate energy charge, especially from 0.4 to 0.9. The enzyme appears to be very sensitive to pro-oxidant conditions; in this regard, the possible involvement of a disulphide bridge (C175-C547) was investigated by using a C547A mutant NT5C2. Two cultured cell models were used to further assess the sensitivity of the enzyme to oxidative stress conditions. NT5C2, differently from other enzyme activities, was inactivated and not rescued by dithiothreitol in a astrocytoma cell line (ADF) incubated with hydrogen peroxide. The incubation of a human lung carcinoma cell line (A549) with 2-deoxyglucose lowered the cell energy charge and impaired the interaction of NT5C2 with the ice protease-activating factor (IPAF), a protein involved in innate immunity and inflammation.

Transcriptional and Metabolic Investigation in 5'-Nucleotidase Deficient Cancer Cell Lines.

Enzymes of nucleoside and nucleotide metabolism regulate important cellular processes with potential impacts on nucleotide-unrelated parameters. We have used a set of CRISPR/Cas9-modified cell models expressing both, one, or none of the 5'-nucleotidases cN-II and CD73, together with RNA sequencing and targeted metabolomics, to decipher new regulatory roles of these proteins. We observed important transcriptional modifications between models as well as upon exposure to adenosine. Metabolite content varied differently between cell models in response to adenosine exposure but was rather similar in control conditions. Our original cell models allowed us to identify a new unobvious link between proteins in the nucleotide metabolism and other cellular pathways. Further analyses of our models, including additional experiments, could help us to better understand some of the roles played by these enzymes.

Enhanced migration of breast and lung cancer cells deficient for cN-II and CD73 via COX-2/PGE2/AKT axis regulation.

PURPOSE: Purine metabolism involves various intracellular and extracellular enzymes, including cN-II and CD73 that dephosphorylate intracellular and extracellular nucleoside monophosphates into their corresponding nucleosides. We conducted a study to better understand the biological roles of these enzymes in breast and lung cancer cells. METHODS: We modified cN-II and/or CD73 expression in human breast cancer cells (MDA-MB-231), human lung cancer cells (NCI-H292) and murine breast cancer cells (4T1) using the CRISPR/Cas9 technique, and evaluated their impact on various cellular parameters such as proliferation, migration, invasion, intracellular nucleotide pools and nucleotide metabolism-related gene expression under extracellular nucleotide stress conditions. RESULTS: Intracellular nucleotide contents were found to be altered in the modified cancer cell models both at their basal levels and after exposure to adenosine or AMP. Altered cN-II and CD73 levels were also found to be associated with cell migration and invasion alterations, involving TIMP-2, MMP-2 and MMP-9 expression, as well as alterations in the COX-2/PGE2/AKT pathway. CONCLUSION: Our results highlight new cell-specific roles of cN-II and CD73 in cancer cell biology and provide insight into their interactions with different intracellular pathways.

Knockdown of cytosolic 5'-nucleotidase II (cN-II) reveals that its activity is essential for survival in astrocytoma cells.

IMP preferring cytosolic 5'-nucleotidase (cN-II) is an ubiquitous nucleotide hydrolysing enzyme. The enzyme is widely distributed and its amino acid sequence is highly conserved among vertebrates. Fluctuations of cN-II activity have been associated with the pathogenesis of neurological disorders. The enzyme appears to be involved in the regulation of the intracellular availability of the purine precursor IMP and also of GMP and AMP, but the contribution of this activity and of its regulation to cell metabolism and to CNS cell functions remains uncertain. To address this issue, we used a vector based short hairpin RNA (shRNA) strategy to knockdown cN-II activity in human astrocytoma cells. Our results demonstrated that 53 h after transduction, cN-II mRNA was reduced to 17.9+/-0.03% of control cells. 19 h later enzyme activity was decreased from 0.7+/-0.026 mU/mg in control ADF cells to 0.45+/-0.046 mU/mg, while cell viability (evaluated by the MTT reduction assay) decreased up to 0.59+/-0.01 (fold vs control) and caspase 3 activity increased from 136+/-5.8 pmol min(-1) mg(-1) in control cells to 639+/-37.5 pmol min(-1) mg(-1) in silenced cells, thus demonstrating that cN-II is essential for cell survival. The decrease of enzyme activity causes apoptosis of the cultured cells without altering intracellular nucleotide and nucleoside concentration or energy charge. Since cN-II is highly expressed in tumour cells, our finding offers a new possible therapeutical approach especially against primary brain tumours such as glioblastoma, and to ameliorate chemotherapy against leukemia.

Purine-Metabolising Enzymes and Apoptosis in Cancer.

The enzymes of both de novo and salvage pathways for purine nucleotide synthesis are regulated to meet the demand of nucleic acid precursors during proliferation. Among them, the salvage pathway enzymes seem to play the key role in replenishing the purine pool in dividing and tumour cells that require a greater amount of nucleotides. An imbalance in the purine pools is fundamental not only for preventing cell proliferation, but also, in many cases, to promote apoptosis. It is known that tumour cells harbour several mutations that might lead to defective apoptosis-inducing pathways, and this is probably at the basis of the initial expansion of the population of neoplastic cells. Therefore, knowledge of the molecular mechanisms that lead to apoptosis of tumoural cells is key to predicting the possible success of a drug treatment and planning more effective and focused therapies. In this review, we describe how the modulation of enzymes involved in purine metabolism in tumour cells may affect the apoptotic programme. The enzymes discussed are: ectosolic and cytosolic 5'-nucleotidases, purine nucleoside phosphorylase, adenosine deaminase, hypoxanthine-guanine phosphoribosyltransferase, and inosine-5'-monophosphate dehydrogenase, as well as recently described enzymes particularly expressed in tumour cells, such as deoxynucleoside triphosphate triphosphohydrolase and 7,8-dihydro-8-oxoguanine triphosphatase.

Characterization of the adenine nucleoside specific phosphorylase of Bacillus cereus.

Adenosine phosphorylase, a purine nucleoside phosphorylase endowed with high specificity for adenine nucleosides, was purified 117-fold from vegetative forms of Bacillus cereus. The purification procedure included ammonium sulphate fractionation, pH 4 treatment, ion exchange chromatography on DEAE-Sephacel, gel filtration on Sephacryl S-300 HR and affinity chromatography on N(6)-adenosyl agarose. The enzyme shows a good stability to both temperature and pH. It appears to be a homohexamer of 164+/-5 kDa. Kinetic characterization confirmed the specificity of this phosphorylase for 6-aminopurine nucleosides. Adenosine was the preferred substrate for nucleoside phosphorolysis (k(cat)/K(m) 2.1x10(6) s(-1) M(-1)), followed by 2'-deoxyadenosine (k(cat)/K(m) 4.2x10(5) s(-1) M(-1)). Apparently, the low specificity of adenosine phosphorylase towards 6-oxopurine nucleosides is due to a slow catalytic rate rather than to poor substrate binding.

Interplay between adenylate metabolizing enzymes and AMP-activated protein kinase.

Purine nucleotides are involved in a variety of cellular functions, such as energy storage and transfer, and signalling, in addition to being the precursors of nucleic acids and cofactors of many biochemical reactions. They can be generated through two separate pathways, the de novo biosynthesis pathway and the salvage pathway. De novo purine biosynthesis leads to the formation of IMP, from which the adenylate and guanylate pools are generated by two additional steps. The salvage pathways utilize hypoxanthine, guanine and adenine to generate the corresponding mononucleotides. Despite several decades of research on the subject, new and surprising findings on purine metabolism are constantly being reported, and some aspects still need to be elucidated. Recently, purine biosynthesis has been linked to the metabolic pathways regulated by AMP-activated protein kinase (AMPK). AMPK is the master regulator of cellular energy homeostasis, and its activity depends on the AMP : ATP ratio. The cellular energy status and AMPK activation are connected by AMP, an allosteric activator of AMPK. Hence, an indirect strategy to affect AMPK activity would be to target the pathways that generate AMP in the cell. Herein, we report an up-to-date review of the interplay between AMPK and adenylate metabolizing enzymes. Some aspects of inborn errors of purine metabolism are also discussed.

Cell-specific pattern of berberine pleiotropic effects on different human cell lines.

The natural alkaloid berberine has several pharmacological properties and recently received attention as a potential anticancer agent. In this work, we investigated the molecular mechanisms underlying the anti-tumor effect of berberine on glioblastoma U343 and pancreatic carcinoma MIA PaCa-2 cells. Human dermal fibroblasts (HDF) were used as non-cancer cells. We show that berberine differentially affects cell viability, displaying a higher cytotoxicity on the two cancer cell lines than on HDF. Berberine also affects cell cycle progression, senescence, caspase-3 activity, autophagy and migration in a cell-specific manner. In particular, in HDF it induces cell cycle arrest in G2 and senescence, but not autophagy; in the U343 cells, berberine leads to cell cycle arrest in G2 and induces both senescence and autophagy; in MIA PaCa-2 cells, the alkaloid induces arrest in G1, senescence, autophagy, it increases caspase-3 activity and impairs migration/invasion. As demonstrated by decreased citrate synthase activity, the three cell lines show mitochondrial dysfunction following berberine exposure. Finally, we observed that berberine modulates the expression profile of genes involved in different pathways of tumorigenesis in a cell line-specific manner. These findings have valuable implications for understanding the complex functional interactions between berberine and specific cell types.