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.publishedVersio

Targeting the Nucleotide Metabolism Proteins of the NUDIX Family and SAMHD1 in Cancer

International audienceNucleotide metabolism has been targeted for many years and in various clinical settings, including cancer. The increased knowledge of certain enzymes involved in this metabolism and associated cellular processes accumulated over the last few years, gives important information related to the druggability of certain proteins and the use of inhibitors for others. Here, we review recent data on such enzymes with a major interest in drug development, i.e. SAMHD1 and the proteins of the NUDIX family. These include information on their roles in cancer progression, correlations with clinical outcomes in cancer patients, and the development and study of enzymatic inhibitors

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

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

CD73 and cN-II regulate the cellular response to chemotherapeutic and hypoxic stress in lung adenocarcinoma cells

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Use of designed experiments for the improvement of pre-analytical workflow for the quantification of intracellular nucleotides in cultured cell lines

International audienceThe present study is focused on the development of a pre-analytical strategy for the quantification of intracellular nucleotides from cultured cell lines. Different protocols, including cell recovery, nucleotide extraction and purification, were compared on a panel of nucleoside mono-, di- and triphosphates from four cell lines (adherent and suspension cells). The quantification of nucleotides was performed using a validated technique with on-line solid-phase extraction coupled with liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS/MS). Designed experiments were implemented to investigate, in a rigorous and limited-testing experimental approach, the influence of several operating parameters. Results showed that the technique used to harvest adherent cells drastically affected the amounts of intracellular nucleotides. Scraping cells was deleterious because of a major leakage (more than 70%) of intracellular nucleotides during scraping. Moreover, some other tested conditions should be avoided, such as using pure methanol as extraction solvent (decrease over 50% of intracellular nucleotides extracted from NCI-H292 cells) or adding a purification step with chloroform. Designed experiments allowed identifying an interaction between the percentage of methanol and the presence of chloroform. The mixture methanol/water (70/30, v/v) was considered as the best compromise according to the nucleoside mono-, di-, or triphosphates and the four cell lines studied. This work highlights the importance of pre-analytical step combined with the cell lines studied associated to sensitive and validated assay for the quantification of nucleotides in biological matrices

Bacterial Deoxyribonucleoside Kinases Are Poor Suicide Genes in Mammalian Cells

Transfer of deoxyribonucleoside kinases (dNKs) into cancer cells increases the activity of cytotoxic nucleoside analogues. It has been shown that bacterial dNKs, when introduced into Escherichia coli, sensitize this bacterium toward nucleoside analogues. We studied the possibility of using bacterial dNKs, for example deoxyadenosine kinases (dAKs), to sensitize human cancer cells to gemcitabine. Stable and transient transfections of bacterial dNKs into human cells showed that these were much less active than human and fruitfly dNKs. The fusion of dAK from Bacillus cereus to the green fluorescent protein induced a modest sensitization. Apparently, bacterial dNKs did not get properly expressed or are unstable in the mammalian cell

Prognostic impact of cN-III mRNA expression on overall survival and drug sensitivity in pediatric leukemia

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CD73 inhibition by purine cytotoxic nucleoside analogue-based diphosphonates

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Spatial and Temporal Control of Cavitation Allows High In Vitro Transfection Efficiency in the Absence of Transfection Reagents or Contrast Agents.

Sonoporation using low-frequency high-pressure ultrasound (US) is a non-viral approach for in vitro and in vivo gene delivery. In this study, we developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. The regulation system incorporated in the device allowed a real-time control of the cavitation level during sonoporation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (pEGFP-C1) in adherent and non-adherent cell lines. The transfection efficiency of the device was compared to those observed with lipofection and nucleofection methods. In both adherent and non-adherent cell lines, the sonoporation device allowed high rate of transfection of pEGFP-C1 (40-80%), as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. The transfection efficiency and toxicity of sonoporation on the non-adherent cell lines Jurkat and K562 were similar to those of nucleofection, while these two cell lines were resistant to transfection by lipofection. Moreover, sonoporation was used to produce three stably transfected human lymphoma and leukemia lines. Significant transfection efficiency was also observed in two fresh samples of human acute myeloid leukemia cells. In conclusion, we developed a user-friendly and cost-effective ultrasound device, well adapted for routine in vitro high-yield transfection experiments and which does not require the use of any transfection reagent or gas micro-bubbles