A Proposed Methodology to Deal with the Impact of In Vitro Cellular Matrix on the Analytical Performances of a Targeted Metabolomic LC-HRMS Method.

Performances of metabolomic methods have been widely studied on biological matrices such as serum, plasma, and urine; but much less on in vitro cell extracts. While the impact of cell culture and sample preparation on results are well-described, the specific effect of the in vitro cellular matrix on the analytical performance remains uncertain. The aim of the present work was to study the impact of this matrix on the analytical performance of an LC-HRMS metabolomic method. For this purpose, experiments were performed on total extracts from two cell lines (MDA-MB-231 and HepaRG) using different cell numbers. Matrix effects, carryover, linearity, and variability of the method were studied. Results showed that the performances of the method depend on the nature of the endogenous metabolite, the cell number, and the nature of the cell line. These three parameters should, therefore, be considered for the processing of experiments and the interpretation of results depending on whether the study focuses on a limited number of metabolites or aims to establish a metabolic signature.

4-Substituted-1,2,3-triazolo nucleotide analogues as CD73 inhibitors, their synthesis, in vitro screening, kinetic and in silico studies.

Three series of nucleotide analogues were synthesized and evaluated as potential CD73 inhibitors. Nucleobase replacement consisted in connecting the appropriate aromatic or purine residues through a triazole moiety that is generated from 1,3-dipolar cycloaddition. The first series is related to 4-substituted-1,2,3-triazolo-ß-hydroxyphosphonate ribonucleosides. Additional analogues were also obtained, in which the phosphonate group was replaced by a bisphosphonate pattern (P-C-P-C, series 2) or the ribose moiety was removed leading to acyclic derivatives (series 3). The ß-hydroxyphosphonylphosphonate ribonucleosides (series 2) were found to be potent inhibitors of CD73 using both purified recombinant protein and cell-based assays. Two compounds (2a and 2b) that contained a bis(trifluoromethyl)phenyl or a naphthyl substituents proved to be the most potent inhibitors, with IC50 values of 4.8 ± 0.8 µM and 0.86 ± 0.2 µM, compared to the standard AOPCP (IC50 value of 3.8 ± 0.9 µM), and were able to reverse the adenosine-mediated immune suppression on human T cells. This series of compounds illustrates a new type of CD73 inhibitors.

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.

Intracellular Cytidine Deaminase Regulates Gemcitabine Metabolism in Pancreatic Cancer Cell Lines.

Cytidine deaminase (CDA) is a determinant of in vivo gemcitabine elimination kinetics and cellular toxicity. The impact of CDA activity in pancreatic ductal adenocarcinoma (PDAC) cell lines has not been elucidated. We hypothesized that CDA regulates gemcitabine flux through its inactivation and activation pathways in PDAC cell lines. Three PDAC cell lines (BxPC-3, MIA PaCa-2, and PANC-1) were incubated with 10 or 100 µM gemcitabine for 60 minutes or 24 hours, with or without tetrahydrouridine, a CDA inhibitor. Extracellular inactive gemcitabine metabolite (dFdU) and intracellular active metabolite (dFdCTP) were quantified with liquid chromatography tandem mass spectrometry. Cellular expression of CDA was assessed with real-time PCR and Western blot. Gemcitabine conversion to dFdU was extensive in BxPC-3 and low in MIA PaCa-2 and PANC-1, in accordance with their respective CDA expression levels. CDA inhibition was associated with low or undetectable dFdU in all three cell lines. After 24 hours gemcitabine incubation, dFdCTP was highest in MIA PaCa-2 and lowest in BxPC-3. CDA inhibition resulted in a profound dFdCTP increase in BxPC-3 but not in MIA PaCa-2 or PANC-1. dFdCTP concentrations were not higher after exposure to 100 versus 10 µM gemcitabine when CDA activities were low (MIA PaCa-2 and PANC-1) or inhibited (BxPC-3). The results suggest a regulatory role of CDA for gemcitabine activation in PDAC cells but within limits related to the capacity in the activation pathway in the cell lines. SIGNIFICANCE STATEMENT: The importance of cytidine deaminase (CDA) for cellular gemcitabine toxicity, linking a lower activity to higher toxicity, is well described. An underlying assumption is that CDA, by inactivating gemcitabine, limits the amount available for the intracellular activation pathway. Our study is the first to illustrate this regulatory role of CDA in pancreatic ductal adenocarcinoma cell lines by quantifying intracellular and extracellular gemcitabine metabolite concentrations.

Regulation of tumor infiltrated innate immune cells by adenosine.

Cancer has the ability to escape the immune system using different molecular actors. Adenosine is known to be involved in mechanisms which control inflammatory reactions and prevent excessive immune response. This purine nucleoside can be translocated from the cell or produced in the extracellular space by 5'-ectonucleotidases. Once bound to its receptors on the surface of immune effector cells, adenosine activates various molecular pathways, which lead to functional inhibition of the cell or its death. Some tumors are infiltrated by the different cells of immune system but are able to use adenosine as an immunosuppressive molecule and thus inhibit immune anticancer response. This mechanism is well described on adaptive cells, but much less on innate cells. This review outlines major effects of adenosine on innate immune cells, its consequences on cancer progression, and possible ways to block the adenosine-dependent immunosuppressive effect.

Functions of the multi-interacting protein KIDINS220/ARMS in cancer and other pathologies.

Development of an organ and subsequently the whole system from an embryo is a highly integrated process. Although there is evidence that different systems are interconnected during developmental stages, the molecular understanding of this relationship is either not known or only to a limited extent. Nervous system development, amongst all, is maybe the most crucial and complex process. It relies on the correct distribution of specific neuronal growth factors and hormones to the specific receptors. Among the plethora of proteins that are involved in downstream signalling of neuronal growth factors, we find the kinase-D interacting substrate of 220 kDa (KIDINS220), also known as ankyrin-rich repeat membrane spanning (ARMS) protein. KIDINS220 has been shown to play a substantial role in the nervous system and vascular system development as well as in neuronal survival and differentiation. It serves as a downstream regulator for many important neuronal and vascular growth factors such as vascular endothelial growth factor (VEGF), the neurotrophin family, glutamate receptors and ephrin receptors. Moreover, activation and differentiation of B- and T-cells, as well as tumour cell proliferation has also shown to be related to KIDINS220. This review comprehensively summarises the existing research data on this protein, with a particular interest in its role in cancer and in other pathologies.

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.

Unexpected Growth-Promoting Effect of Oxaliplatin in Excision Repair Cross-Complementation Group 1 Transfected Human Colon Cancer Cells.

The nucleotide excision repair protein excision repair cross-complementation group 1 (ERCC1) has been repeatedly shown to be involved in the sensitivity of cancer cells to platinum derivatives. In order to better understand this process, we transfected HCT-116 cells with a plasmid encoding ERCC1 and studied their in vitro and in vivo behaviour. No main differences were observed for sensitivity to platinum drugs, DNA repair capacity and clonogenicity in vitro. However, -ERCC1-transfected HCT-116 cells showed paradoxical behaviour in vivo with increased growth in mice treated with oxaliplatin as compared to untreated mice. The Trop2 protein was identified as being potentially involved in the underlying mechanism for these observations, as it was overexpressed in transfected cells. Our results suggest complex regulation of signalling in cancer cells exposed to cancer drugs.

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.

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.

Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography-tandem mass spectrometry.

An analytical method coupling online solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed to quantify 16 endogenous nucleoside mono- and triphosphates in cellular samples. Separation was achieved on a porous graphitic carbon (PGC) column without ion-pairing agent in the mobile phase. Low levels of the ion-pairing agent diethylamine (DEA) added to the reconstitution solution were necessary to prevent peak tailing of nucleoside triphosphates. The mass spectrometer, a triple quadrupole with an electrospray ionisation source, was operated in positive mode. Two multiple reaction monitoring (MRM) segments were programmed, each an internal standard. Extraction and separation of nucleoside mono- and triphosphates were obtained within 20 min. The total duration of a single run was 37 min. Calibration curves, performed with labelled nucleotides added to the sample matrix, ranged from 0.29 to 18.8 pmol injected for deoxyribonucleotides and from 3.9 to 3,156 pmol for ribonucleotides. Accuracy did not deviate more than -14.6 and 10.2 % from nominal values for all compounds at all levels. CV results were all lower than 17.0 % for the LLOQ level and 14.6 % for the other levels. Quality control (QC) samples were also in agreement with acceptance criteria, except for the lower QC of GMP. Ion suppression, matrix effect, extraction recoveries and stability were assessed. After validation, the method was applied to the evaluation of the effects of gemcitabine and hydroxyurea on nucleotide pools in Messa cells.

Expression of domains for protein-protein interaction of nucleotide excision repair proteins modifies cancer cell sensitivity to platinum derivatives and genomic stability.

Nucleotide excision repair (NER) is involved in the repair of DNA damage caused by platinum derivatives and has been shown to decrease the cytotoxic activity of these drugs. Because protein-protein interactions are essential for NER activity, we transfected human cancer cell lines (A549 and HCT116) with plasmids coding the amino acid sequences corresponding to the interacting domains between excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum, complementation group A (XPA), as well as ERCC1 and xeroderma pigmentosum, complementation group F (XPF), all NER proteins. Using the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and annexin V staining, we showed that transfected A549 cells were sensitized 1.2-2.2-fold to carboplatin and that transfected HCT116 cells were sensitized 1.4-5.4-fold to oxaliplatin in vitro. In addition, transfected cells exhibited modified in vivo sensitivity to the same drugs. Finally, in particular cell models of the interaction between ERCC1 and XPF, DNA repair was decreased, as evidenced by increased phosphorylation of the histone 2AX after exposure to mitomycin C, and genomic instability was increased, as determined by comparative genomic hybridization studies. The results indicate that the interacting peptides act as dominant negatives and decrease NER activity through inhibition of protein-protein interactions.

Small molecule inhibitors of ERCC1-XPF protein-protein interaction synergize alkylating agents in cancer cells.

The benefit of cancer chemotherapy based on alkylating agents is limited because of the action of DNA repair enzymes, which mitigate the damage induced by these agents. The interaction between the proteins ERCC1 and XPF involves two major components of the nucleotide excision repair pathway. Here, novel inhibitors of this interaction were identified by virtual screening based on available structures with use of the National Cancer Institute diversity set and a panel of DrugBank small molecules. Subsequently, experimental validation of the in silico screening was undertaken. Top hits were evaluated on A549 and HCT116 cancer cells. In particular, the compound labeled NSC 130813 [4-[(6-chloro-2-methoxy-9-acridinyl)amino]-2-[(4-methyl-1-piperazinyl)methyl]] was shown to act synergistically with cisplatin and mitomycin C; to increase UVC-mediated cytotoxicity; to modify DNA repair as indicated by the staining of phosphorylated H2AX; and to disrupt interaction between ERCC1 and XPF in cells. In addition, using the Biacore technique, we showed that this compound interacts with the domain of XPF responsible for interaction with ERCC1. This study shows that small molecules targeting the protein-protein interaction of ERCC1 and XPF can be developed to enhance the effects of alkylating agents on cancer cells.

Levels of gemcitabine transport and metabolism proteins predict survival times of patients treated with gemcitabine for pancreatic adenocarcinoma.

BACKGROUND & AIMS: Patients who undergo surgery for pancreatic ductal adenocarcinoma (PDAC) frequently receive adjuvant gemcitabine chemotherapy. Key determinants of gemcitabine cytotoxicity include the activities of the human equilibrative nucleoside transporter 1 (hENT1), deoxycytidine kinase (dCK), and ribonucleotide reductase subunit 1 (RRM1). We investigated whether tumor levels of these proteins were associated with efficacy of gemcitabine therapy following surgery. METHODS: Sequential samples of resected PDACs were retrospectively collected from 434 patients at 5 centers; 142 patients did not receive adjuvant treatment (33%), 243 received adjuvant gemcitabine-based regimens (56%), and 49 received nongemcitabine regimens (11%). We measured protein levels of hENT1, dCK, and RRM1 by semiquantitative immunohistochemistry with tissue microarrays and investigated their relationship with patients' overall survival time. RESULTS: The median overall survival time of patients was 32.0 months. Among patients who did not receive adjuvant treatment, levels of hENT1, RRM1, and dCK were not associated with survival time. Among patients who received gemcitabine, high levels of hENT1 and dCK were significantly associated with longer survival time (hazard ratios of 0.34 [P < .0001] and 0.57 [P = .012], respectively). Interaction tests for gemcitabine administration and hENT1 and dCK status were statistically significant (P = .0007 and P = .016, respectively). On multivariate analysis of this population, hENT1 and dCK retained independent predictive values, and those patients with high levels of each protein had the longest survival times following adjuvant therapy with gemcitabine. CONCLUSIONS: High levels of hENT1 and dCK in PDAC predict longer survival times in patients treated with adjuvant gemcitabine.

Nano-encapsulation of vitamin D3 active metabolites for application in chemotherapy: formulation study and in vitro evaluation.

PURPOSE: Calcitriol (1,25-dihydroxyvitamin D3), the active metabolite of vitamin D3, is a potential anticancer agent but with high risk of hypercalcemia which limits the achievement of effective serum concentrations. Thus, calcitriol targeting delivery by nanoparticles may present a good solution. METHODS: Vitamin D3 active metabolites were encapsulated into polymeric nanoparticles and different formulation parameters were tested. The growth inhibitory efficiency of these nanoparticles was carried out in vitro on human breast adenocarinoma cells (MCF-7). RESULTS: Using cholecalciferol (the inactive metabolite), different polymer and oil ratios were compared to select nanoparticles presenting high encapsulation efficiency and sustained release profile. Calcidiol/calcitriol loaded nanoparticles had good encapsulation efficiencies (around 90%) associated with sustained releases over 7 days and enhanced stability. Moreover, loaded nanoparticles showed similar growth inhibition to non-encapsulated metabolites of vitamin D3 on day 4 and higher activities on days 7 and 10 after treatment initiation. CONCLUSION: The nano-encapsulation of vitamin D3 active metabolites may offer a new and potentially effective strategy for vitamin D3-based chemotherapy overcoming its actual limitations. The targeting delivery of vitamin D3 metabolites should be encouraged.

Clinical use of biomarkers in the field of cytotoxic nucleoside analogues.

OBJECTIVES: Cytotoxic nucleosides (gemcitabine, cytarabine…) are used for the treatment of various malignancies. Their activity is dependent on the interaction with several proteins and enzymes of nucleotide metabolism. It has for a long time been hypothesized that the clinical activity of nucleoside analogues can be predicted by studying corresponding genes or gene products in clinical samples. METHODS: In this short review, I will present old and new published data from our group and others about the prediction of activity of these drugs concentrating on gene-candidate approaches, and discuss biological and technical limitations of these. RESULTS: A large number of studies have been conducted in various clinical settings (drugs, disease, patient cohort…) evaluating DNA, mRNA or protein-related markers. Although some individual parameters and associations thereof have been validated, only a very few numbers have been implemented in pretreatment evaluations of patients. CONCLUSION: There is still much to do in the field of outcome-prediction with nucleoside analogues. The use of multiparametric methods could increase the success rate but at the cost of a poorer understanding of molecular mechanisms.

PARP Inhibitors and Proteins Interacting with SLX4.

PARP inhibitors are small molecules currently used with success in the treatment of certain cancer patients. Their action was first shown to be specific to cells with DNA repair deficiencies, such as BRCA-mutant cancers. However, recent work has suggested clinical interest of these drugs beyond this group of patients. Preclinical data on relationships between the activity of PARP inhibitors and other proteins involved in DNA repair exist, and this review will only highlight findings on the SLX4 protein and its interacting protein partners. As suggested from these available data and depending on further validations, new treatment strategies could be developed in order to broaden the use for PARP inhibitors in cancer patients.

OXPHOS inhibitors, metabolism and targeted therapies in cancer.

More and more studies highlight the complex metabolic characteristics and plasticity of cancer cells. To address these specificities and explore the associated vulnerabilities, new metabolism-targeting therapeutic strategies are being developed. It is more and more accepted that cancer cells do not produce their energy only from aerobic glycolysis, as some subtypes strongly rely on mitochondrial respiration (OXPHOS). This review focuses on classical and promising OXPHOS inhibitors (OXPHOSi), unravelling their interest and modes of actions in cancer, particularly in combination with other strategies. Indeed, in monotherapy, OXPHOSi display limited efficiency as they mostly trigger cell death in cancer cell subtypes that strongly depend on mitochondrial respiration and are not able to shift to other metabolic pathways to produce energy. Nevertheless, they remain very interesting in combination with conventional therapeutic strategies such as chemotherapy and radiotherapy, increasing their anti-tumoral actions. In addition, OXPHOSi can be included in even more innovative strategies such as combinations with other metabolic drugs or immunotherapies.

Looking for the unexpected in purine and pyrimidine metabolism.

In June 2023, the Purine and Pyrimidine Society (PPS) organized the 20th biennial symposium on Purine and Pyrimidine metabolism (PP23). The symposium was organized in Los Angeles, California, USA, by Pr Caius Radu affiliated to UCLA. The scientific program covered various topics such as inborn errors, cancer, immunity, enzymatic reactions, drug development etc and was presented at 9 sessions over three days. The current issue of Nucleosides, Nucleotides & Nucleic Acids is a special issue covering proceedings from PP23-presentations and other PPS-related manuscripts, and in this editorial, we will give an overview of the scientific program of the meeting.

Second-Generation CD73 Inhibitors Based on a 4,6-Biaryl-2-thiopyridine Scaffold.

Various series of 4,6-biaryl-2-thiopyridine derivatives were synthesized and evaluated as potential ecto-5'-nucleotidase (CD73) inhibitors. Two synthetic routes were explored and the coupling of 4,6-disubstituted 3-cyano-2-chloro-pyridines with selected thiols allowed us to explore the structural diversity. Somehow divergent results were obtained in biological assays on CD73 inhibition using either the purified recombinant protein or cell-based assays, highlighting the difficulty to target protein-protein interface on proteins existing as soluble and membrane-bound forms. Among the 18 new derivatives obtained, three derivatives incorporating morpholino substituents on the 4,6-biaryl-2-thiopyridine core were shown to be able to reverse the adenosine-mediated immune suppression on human T cells. The higher blockade efficiency was observed for 2-((3-cyano-4,6-bis(4-morpholinophenyl)pyridin-2-yl)thio)-N-(isoxazol-3-yl)acetamide (with total reversion at 100 µM) and methyl 2-((3-cyano-4,6-bis(4-morpholinophenyl)pyridin-2-yl)thio)acetate (with partial reversion at 10 µM). Thus, this series of compounds illustrates a new chemotype of CD73 allosteric inhibitors.