Translational data suggest that nucleoside transporters, in particular human equilibrative nucleoside transporter 1 (hENT1), play an important role in predicting clinical outcome after gemcitabine chemotherapy for several types of cancer. The aim of this study was to retrospectively determine patients' outcome according to the expression of hENT1 in tumoral cells of patients receiving gemcitabine-based therapy.The immunohistochemistry analysis was performed on samples from thirty-one patients with unresectable biliary tract cancer (BTC) consecutively treated with first line gemcitabine-based regimens.Positive hENT1 staining patients were 21 (67.7%); negative hENT1 staining patients were 10 (32.3%). Statistical analysis revealed no association between baseline characteristics, toxicities and tumor response to gemcitabine and hENT1 levels. In the univariate analysis, HENT1 expression was significantly correlated with time to progression (TTP) (p=0.0394; HR 2.902, 95%CI 1.053-7.996). The median TTP was 6.33 versus 2.83 months, respectively in patients with positive versus negative hENT1 staining. Moreover, patients with positive hENT1 expression showed a longer median overall survival when compared with patients with low hENT1 expression (14 versus 7 months, respectively), but this difference did not reach the statistical significance (p=0.128).Therefore, hENT1 may be a relevant predictive marker of benefit from gemcitabine-based therapies in patients with advanced BTC.
Abstract The transportability of cytosine-containing nucleosides by recombinant hCNT1 was investigated in transfected mammalian cells. Apparent Km values for hCNT1-mediated transport of uridine, cytidine and deoxycytidine were, respectively, 59, 140 and 150 μM. Uridine transport was inhibited 89, 32 and 11%, respectively, by 500 μM gemcitabine, cytarabine and lamivudine, demonstrating that, unlike gemcitabine (a high-affinity hCNT1 permeant), cytarabine and lamivudine are poor hCNT1 permeants.
Kearns-Sayre syndrome (KSS) is a disease with severe clinical symptoms that often arises from a mitochondrial DNA deletion of 4977 bp. Quantification of defective mitochondrial DNA is important since the severity of symptoms in KSS is thought to be related to increased content of abnormal mitochondrial DNA. We developed a rapid, quantitative and competitive PCR assay to measure both wild-type and mutant forms of mitochondrial DNA in cells from KSS patients. The assay can accurately measure absolute numbers of mitochondrial DNA per cell by normalizing to a single copy nuclear gene.
Multitargeted tyrosine kinase inhibitors (TKI) axitinib, pazopanib, and sunitinib are used to treat many solid tumors. Combination trials of TKIs with gemcitabine, a nucleoside anticancer drug, in pancreas, renal, lung, ovarian, and other malignancies resulted in little benefit to patients. TKI interactions with human nucleoside transporters (hNT) were studied by assessing inhibition of [(3)H]uridine uptake in yeast producing recombinant hNTs individually and in cultured human cancer cell lines. Axitinib, pazopanib, and sunitinib inhibited hENT1 at low micromolar concentrations. In A549, AsPC-1, and Caki-1 cells, [(3)H]uridine, [(3)H]thymidine, [(3)H]gemcitabine, and [(3)H]fluorothymidine (FLT) accumulation was blocked by all three TKIs. Pazopanib > axitinib ≥ sunitinib inhibited hENT1 with IC50 values of 2, 7, and 29 μmol/L, respectively, leading to reduced intracellular gemcitabine and FLT accumulation. Pretreatment or cotreatment of Caki-1 cells with TKIs reduced cellular accumulation of [(3)H]nucleosides, suggesting that TKI scheduling with nucleoside drugs would influence cytotoxicity. In combination cytotoxicity experiments that compared sequential versus simultaneous addition of drugs in Caki-1 cells, cytotoxicity was greatest when gemcitabine was added before TKIs. In clinical settings, TKI inhibitor concentrations in tumor tissues are sufficient to inhibit hENT1 activity, thereby reducing nucleoside chemotherapy drug levels in cancer cells and reducing efficacy in combination schedules. An additional unwanted interaction may be reduced FLT uptake in tumor tissues that could lead to aberrant conclusions regarding tumor response.
18F-3′-Deoxy-3′-fluorothymidine (18F-FLT) is a PET tracer that accumulates in proliferating tissues. The current study was undertaken to determine whether equilibrative nucleoside transporter 1 (ENT1) is important for 18F-FLT uptake in normal tissues and tumors. Methods: ENT1-knockout (ENT1−/−) mice were generated and compared with wild-type (ENT1+/+) mice using small-animal 18F-FLT PET. In addition, ENT1+/+ mice were also injected with the ENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside phosphate (NBMPR-P) at 1 h before radiotracer injection, followed by 18F-FLT small-animal PET. Tissues of interest were analyzed for thymidine kinase 1 and nucleoside transporters by immunoblotting and immunohistochemistry, respectively, and plasma thymidine levels were analyzed by liquid chromatography–mass spectrometry. Human lung carcinoma A549 cells were stably transfected with pSUPER-producing short-hairpin RNA against human ENT1 (hENT1) or a scrambled sequence with no homology to mammalian genes (A549-pSUPER-hENT1 and A549-pSUPER-SC, respectively). Cultured transfected cells were characterized for hENT1 transcript levels and 18F-FLT uptake using real-time polymerase chain reaction and 3H-FLT uptake assays, respectively. Transfected A549 cells were grown as xenograft tumors in NIH-III mice, which were analyzed by 18F-FLT small-animal PET. Results: Compared with noninjected ENT1+/+ mice, ENT1+/+ mice injected with NBMPR-P and ENT1−/− mice displayed a reduced percentage injected dose per gram (%ID/g) for 18F-FLT in the blood (84 and 81%, respectively) and an increased %ID/g for 18F-FLT in the spleen (188 and 469%, respectively) and bone marrow (266 and 453%, respectively). ENT1−/− mice displayed 1.65-fold greater plasma thymidine levels than did ENT1+/+ mice. Spleen tissue from ENT1+/+ and ENT1−/− mice displayed similar thymidine kinase 1 protein levels and significant concentrative nucleoside transporter 1 and 3 staining. Compared with A549-pSUPER-SC cells, A549-pSUPER-hENT1 cells displayed 0.45-fold hENT1 transcript levels and 0.68-fold 3H-FLT uptake. Compared with A549-pSUPER-SC xenograft tumors, A549-pSUPER-hENT1 xenograft tumors displayed 0.76-fold %ID/g values (ex vivo γ-counts) and 0.65-fold maximum standardized uptake values (PET image analysis) for 18F-FLT uptake at 1 h after tracer injection. Conclusion: Loss of ENT1 activity significantly affected 18F-FLT biodistribution in mice and 18F-FLT uptake in xenograft tumors, suggesting that nucleoside transporters are important mediators of 18F-FLT uptake in normal and transformed cells.
The first examples of the equilibrative nucleoside transporter (ENT) family were characterized in human tissues at the molecular level only 4 years ago. Since that time, the identification of homologous proteins by functional cloning and genome analysis has revealed that the family is widely distributed in eukaryotes. Family members are predicted to possess 11 transmembrane helices (TMs), and recent investigations on the mammalian ENTs have implicated the TM 3-6 region in solute recognition. Whilst the name of the family reflects the properties of its prototypical member hENT1, an equilibrative transporter of nucleosides, some family members can also transport nucleobases and some are proton-dependent, concentrative transporters. In addition to their role in nucleoside salvage, ENTs are targets for coronary vasodilator drugs and act as routes for uptake of cytotoxic drugs in humans and protozoa. This paper summarizes current knowledge of the family and reports on the identification of a novel mammalian ENT isoform, designated ENT3, from mouse and human tissues.
Abstract Four classes of 6-X-benzylated purine nucleosides, (i) 6-N-(substituted-benzyl)adenosines, (ii) 6-N-(4-nitrobenzyl)adenine nucleosides with modified sugars, (iii) 6-N(S)-(4-azidobenzyl) derivatives of adenosine, 6-thioinosine, and 6-thioguanosine, and (iv) 6-N-{4-N-[acyl(sulfonyl)amino]benzyl}adenosines, were synthesized and their binding interactions with “es-NT” ( e quilibrative, inhibitor-sensitive nucleoside transport) systems were studied. Several tight-binding analogues were found.
The benefits of PET imaging of tumor hypoxia in patient management has been demonstrated in many examples and with various tracers over the last years. Although, the optimal hypoxia imaging agent has yet to be found, 2-nitroimidazole (azomycin) sugar derivatives-mimicking nucleosides-have proven their potential with [18F]FAZA ([18F]fluoro-azomycin-α-arabinoside) as a prominent representative in clinical use. Still, for all of these tracers, cellular uptake by passive diffusion is postulated with the disadvantage of slow kinetics and low tumor-to-background ratios. We recently evaluated [18F]fluoro-azomycin-β-deoxyriboside (β-[18F]FAZDR), with a structure more similar to nucleosides than [18F]FAZA and possible interaction with nucleoside transporters. For a deeper insight, we comparatively studied the interaction of FAZA, β-FAZA, α-FAZDR and β-FAZDR with nucleoside transporters (SLC29A1/2 and SLC28A1/2/3) in vitro, showing variable interactions of the compounds. The highest interactions being for β-FAZDR (IC50 124 ± 33 µM for SLC28A3), but also for FAZA with the non-nucleosidic α-configuration, the interactions were remarkable (290 ± 44 µM {SLC28A1}; 640 ± 10 µM {SLC28A2}). An improved synthesis was developed for β-FAZA. For a PET study in tumor-bearing mice, α-[18F]FAZDR was synthesized (radiochemical yield: 15.9 ± 9.0% (n = 3), max. 10.3 GBq, molar activity > 50 GBq/µmol) and compared to β-[18F]FAZDR and [18F]FMISO, the hypoxia imaging gold standard. We observed highest tumor-to-muscle ratios (TMR) for β-[18F]FAZDR already at 1 h p.i. (2.52 ± 0.94, n = 4) in comparison to [18F]FMISO (1.37 ± 0.11, n = 5) and α-[18F]FAZDR (1.93 ± 0.39, n = 4), with possible mediation by the involvement of nucleoside transporters. After 3 h p.i., TMR were not significantly different for all 3 tracers (2.5⁻3.0). Highest clearance from tumor tissue was observed for β-[18F]FAZDR (56.6 ± 6.8%, 2 h p.i.), followed by α-[18F]FAZDR (34.2 ± 7.5%) and [18F]FMISO (11.8 ± 6.5%). In conclusion, both isomers of [18F]FAZDR showed their potential as PET hypoxia tracers. Differences in uptake behavior may be attributed to a potential variable involvement of transport mechanisms.
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