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Summary: Hypoxia‐mediated red blood cell (RBC) sickling is central to the pathophysiology of sickle cell disease (SCD). The signalling nucleoside adenosine is thought to play a significant role in this process. This study investigated expression of the erythrocyte type 1 equilibrative nucleoside transporter (ENT1), a key regulator of plasma adenosine, in adult patients with SCD and carriers of sickle cell trait (SCT). Relative quantitative expression analysis of erythrocyte ENT1 was carried out by Western blot and flow cytometry. Patients with SCD with steady state conditions, either with SS or SC genotype, untreated or under hydroxycarbamide (HC) treatment, exhibited a relatively high variability of erythrocyte ENT1, but with levels not significantly different from normal controls. Most strikingly, expression of erythrocyte ENT1 was found to be significantly decreased in patients with SCD undergoing painful vaso‐occlusive episode and, unexpectedly, also in healthy SCT carriers. Promoting hypoxia‐induced adenosine signalling, the reduced expression of erythrocyte ENT1 might contribute to the pathophysiology of SCD and to the susceptibility of SCT individuals to altitude hypoxia or exercise to exhaustion. [ABSTRACT FROM AUTHOR]

The goal of this study was to understand roles of nucleoside and nucleobase transport processes in capecitabine pharmacology in cells derived from human renal proximal tubule cells (hRPTCs) and three human renal cell carcinoma (RCC) cell lines, A498, A704, and Caki-1. Human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) mediated activities and a sodium-independent nucleobase activity were present in hRPTCs. In hRPTCs, uptake of 5'-deoxy-5-fluorouridine (DFUR), a nucleoside metabolite of capecitabine, was pH dependent with highest uptake seen at pH 6.0. In RCC cell lines, hENT1 was the major nucleoside transporter. Nucleobase transport activity was variable among the three RCC cell lines, with Caki-1 showing the highest and A498 showing the lowest activities. Treatment of RCC cell lines with interferon alpha (IFN-α) increased thymidine phosphorylase levels and prior treatment of RCC cell lines with IFN-α followed by 5-FU or DFUR resulted in enhanced sensitivity of all cell lines to 5-FU and two of three cell lines to DFUR. We report for the first time a nucleobase transport activity in hRPTCs and RCC cell lines. In addition, our in vitro cytotoxicity results showed that RCC cell lines differed in their response to 5-FU and DFUR and prior treatment with IFN-α potentiated cytotoxic response to metabolites of capecitabine. Key words: nucleosides, membrane transport, renal cancer, nucleobases and cytotoxicity. Cette etude visait a comprendre les roles qu'exercent les processus de transport des nucleosides et des bases nucleiques dans la pharmacologie de la capecitabine, chez des cellules derivees des tubules proximaux renaux humains (hRPTC) et trois lignees cellulaires humaines de carcinome du rein (RCC), les A498, A704 et Caki- 1. Des activites dependantes des transporteurs equilibratifs de nucleosides 1 et 2 chez l'humain (hENTl et hENT2) et une activite nucleobasique independante du sodium etaient presentes chez les hRPTC. La captation de la 5'-deoxy-5-fluorouridine (DFUR), un nucleoside metabolite de la capecitabine, etait dependante du pH chez les hRPTC, la captation maximale etant observee a pH 6.0. Chez les RCC, hENTl etait le principal transporteur de nucleoside. L'activite de transport des bases nucleiques variait en fonction des trois lignees cellulaires, les cellules Caki-1 etant celles qui presentaient l'activite la plus elevee et les A498, la plus faible. Un traitement des lignees de RCC avec de l'interferon-alpha (IFN-α) augmentait les niveaux de thymidine phosphorylase, et un traitement prealable des lignees de RCC avec de l'IFN-α suivi du 5-FU ou du DFUR resultait en un accroissement de la sensibilite de toutes les lignees cellulaires au 5-FU et de deux des trois lignees, au DFUR. Nous rapportons pour la premiere fois la presence d'une activite de transport des bases nucleiques chez les hRPTC et les lignees de RCC. De plus, les resultats de l'etude de cytotoxicite realisee in vitro ont revele que les lignees de RCC repondaient differemment au 5-FU et au DFUR, et qu'un traitement prealable a l'IFN-α potentialisait la reponse cytotoxique aux metabolites de la capecitabine. [Traduit par la Redaction] Mots-cles: nucleosides, transport membranaire, cancer renal, bases nucleiques, cytotoxicite., Introduction Capecitabine (Xeloda®) is a prodrug of 5'-deoxy-5-fluorouridine (DFUR) and 5-fluorouracil (5-FU) (for reviews see (Judson et al. 1999; Reigner et al. 2001)). Carboxyl esterase in the liver hydrolyses capecitabine [...]

This study examined the roles of human nucleoside transporters (hNTs) in mediating transepithelial fluxes of adenosine, 2'-deoxyadenosine, and three purine nucleoside anti-cancer drugs across polarized monolayers of human renal proximal tubule cells (hRPTCs), which were shown in previous studies to have human equilibrative NT 1 (hENT1) and 2 (hENT2) and human concentrative NT 3 (hCNT3) activities (11). Early passage hRPTCs were cultured on transwell inserts under conditions that induced formation of polarized monolayers with experimentally accessible apical and basolateral domains. Polarized hRPTC cultures were monitored for inhibitor sensitivities and sodium-dependence of the following: 1) transepithelial fluxes of radiolabeled adenosine, 2'-deoxyadenosine, fludarabine (9-[beta]-D-arabinosyl-2-fluoroadenine), cladribine (2-chloro-2'-deoxyadenosine), and clofarabine (2-chloro-2'-fluoro-deoxy-9-[beta]-D-arabinofuranosyladenine); 2) mediated uptake of radiolabeled adenosine, 2'-deoxyadenosine, fludarabine, cladribine, and clofarabine from either apical or basolateral surfaces; and 3) relative apical cell surface hCNT3 protein levels. Transepithelial fluxes of adenosine were mediated from apical-to-basolateral sides by apical hCNT3 and basolateral bENT2, whereas transepithelial fluxes of 2'-deoxyadenosine were mediated from basolateral-to-apical sides by apical hENT1 and basolateral human organic anion transporters (hOATs). The transepithelial fluxes of adenosine, hCNT3-mediated cellular uptake of adenosine, and relative apical cell surface hCNT3 protein levels correlated positively in polarized hRPTCs. The purine nucleoside anti-cancer drugs fludarabine, cladribine, and clofarabine, like adenosine exhibited apical-to-basolateral fluxes. Collectively, this evidence suggested that apical hCNT3 and basolateral hENT2 are involved in proximal tubular reabsorption of adenosine and some nucleoside drugs and that apical hENT1 and basolateral hOATs are involved in proximal tubular secretion of 2'-deoxyadenosine. kidney; fludarabine; cladribine; clofarabine

Nucleoside transporters in kidney mediate renal reabsorption and secretion of nucleosides. Using RT-PCR, we demonstrated mRNAs encoding hENT1, hENT2, hCNT1, hCNT2, and hCNT3 in both cortex and medulla. Immunoblotting with crude membrane preparations revealed abundant hENT1 and hCNT3 in both cortex and medulla, and little, if any, hENT2, hCNT1, or hCNT2, indicating that the latter were either absent or below limits of detection of immunoassays, hENT1 immunostaining was observed on apical surfaces of proximal tubules and on both apical and basal surfaces of thick ascending loops of Henle and collecting ducts. Prominent hCNT3 immunostaining was observed on apical surfaces of proximal tubules and thick ascending loops of Henle in addition to some cytoplasmic staining. Equilibrium binding of [[sup.3]H]nitrobenzylmercaptopurine ribonucleoside (NBMPR), a high-affinity inhibitor of hENT1, to brush-border membrane vesicles from cortex confirmed the presence of hENT1 on apical surfaces of proximal tubules. Uptake of [[sup.3]H]uridine by polarized renal proximal tubule cells exhibited a sodium-dependent component that was inhibited by thymidine and inosine as well as a sodium-independent component that was partially inhibited by NBMPR and completely inhibited by dilazep, indicating high levels of hENT1 and hCNT3 and low levels of hENT2 activities. The presence of 1) transcripts for hENT1/2 and hCNT1/2/3 and the hENT1 and hCNT3 proteins in human kidneys and 2) hENT1, hENT2, and hCNT3 activities in cultured proximal tubule cells suggest involvement of hENT1, hCNT3, and possibly also hENT2 in renal handling of nucleosides and nucleoside drugs. renal transport of nucleosides: tissue distribution of nucleoside transporters doi:10.1152/ajprenal.00007.2007

Abstract: Renal handling of physiological and pharmacological nucleosides is a major determinant of their plasma levels and tissue availabilities. Additionally, the pharmacokinetics and normal tissue toxicities of nucleoside drugs are [...]

Positron emission tomography (PET) using 2‐deoxy‐2‐[18F]fluoro‐d‐glucose ([18F]FDG), a marker of energy metabolism and cell proliferation, is routinely used in the clinic to assess patient response to chemotherapy and to monitor tumor growth. Treatment with some tyrosine kinase inhibitors (TKIs) causes changes in blood glucose levels in both nondiabetic and diabetic patients. We evaluated the interaction of several classes of TKIs with human glucose transporter‐1 (hGLUT‐1) in FaDu and GIST‐1 cells by measuring [3H]2‐deoxy‐d‐glucose ([3H]2‐DG) and [3H]FDG uptake. Uptake of both was inhibited to varying extents by the TKIs, and representative TKIs from each class showed competitive inhibition of [3H]2‐DG uptake. In GIST‐1 cells, [3H]FDG uptake inhibition by temsirolimus and nilotinib was irreversible, whereas inhibition by imatinib, gefitinib, and pazopanib was reversible. Molecular modeling studies showed that TKIs form multiple hydrogen bonds with polar residues of the sugar binding site (i.e., Q161, Q282, Q283, N288, N317, and W388), and van der Waals interactions with the H‐pocket site. Our results showed interaction of TKIs with amino acid residues at the glucose binding site to inhibit glucose uptake by hGLUT‐1. We hypothesize that inhibition of hGLUT‐1 by TKIs could alter glucose levels in patients treated with TKIs, leading to hypoglycemia and fatigue, although further studies are required to evaluate roles of other SLC2 and SLC5 members. In addition, TKIs could affect tumor [18F]FDG uptake, increasingly used as a marker of tumor response. The hGLUT‐1 inhibition by TKIs may have implications for routine [18F]FDG‐PET monitoring of tumor response in patients. [ABSTRACT FROM AUTHOR]

Abstract Background Gemcitabine, a deoxycytidine nucleoside analog, is the current standard chemotherapy used as first-line treatment for patients with locally advanced or metastatic cancer of the pancreas, and extends life survival by 5.7 months. Advanced pancreatic cancer thus remains a highly unmet medical need and new therapeutic agents are required for this patient population. Troxacitabine (Troxatyl™) is the first unnatural L-nucleoside analog to show potent preclinical antitumor activity and is currently under clinical investigation. Troxacitabine was recently evaluated as a first-line therapy in 54 patients with advanced adenocarcinoma of the pancreas and gave comparable overall results to those reported with gemcitabine in recently published randomized trials. Methods The human pancreatic adenocarcinoma cell lines, AsPC-1, Capan-2, MIA PaCa-2 and Panc-1, were exposed to troxacitabine or gemcitabine alone or in combination, for 72 h, and the effects on cell growth were determined by electronic particle counting. Synergistic efficacy was determined by the isobologram and combination-index methods of Chou and Talalay. Mechanistic studies addressed incorporation of troxacitabine into DNA and intracellular levels of troxacitabine and gemcitabine metabolites. For in vivo studies, we evaluated the effect of both drugs, alone and in combination, on the growth of established human pancreatic (AsPC-1) tumors implanted subcutaneously in nude mice. Statistical analysis was calculated by a one-way ANOVA with Dunnett as a post-test and the two-tailed unpaired t test using GraphPad prism software. Results Synergy, evaluated using the CalcuSyn Software, was observed in all four cell-lines at multiple drug concentrations resulting in combination indices under 0.7 at Fa of 0.5 (50% reduction of cell growth). The effects of drug exposures on troxacitabine and gemcitabine nucleotide pools were analyzed, and although gemcitabine reduced phosphorylation of troxacitabine when cells were exposed at equal drug concentrations, there was no effect on phosphorylated pools at drug combinations that were synergistic. The amount of troxacitabine incorporated into DNA was also not affected by the presence of gemcitabine. In vivo testing against a human pancreatic (AsPC-1) xenograft mouse tumor model indicated that both drugs were more than additive at well-tolerated doses and schedule. The biological basis for this synergy is unclear as we did not observe changes in apoptosis, DNA repair, troxacitabine incorporation into DNA or troxacitabine metabolism in the presence of gemcitabine. Conclusion These data, together with phase I clinical data showing tolerability of both agents when combined, suggest combination therapy with troxacitabine and gemcitabine warrants further evaluation in advanced pancreatic cancer patients.