Differential Expression of Human Nucleoside Transporters in Normal and Tumor Tissue

doi:10.1006/bbrc.2000.4205

Biochemical and Biophysical Research Communications

Volume 280, Issue 3, 26 January 2001, Pages 951-959

https://doi.org/10.1006/bbrc.2000.4205 Get rights and content

Abstract

Responses to nucleoside analog drugs used in the treatment of cancers and viral infections can vary considerably between individuals. Genetic variability between individuals in their ability to transport drugs may be a contributory factor. Nucleoside transporters (NTs) move nucleosides and analog drugs across cell membranes. Four human NTs have been cloned: hENT1, hENT2, hCNT1, and hCNT2. Human NT expression profiles are not well defined; therefore, we undertook a comprehensive quantitative analysis of the differential expression of NTs within normal and tumor tissue. Results show tissue specific expression of the different NTs in normal tissue while matched normal/tumor tissue cDNA array data show considerable variability in all NT expression profiles from different individuals, in particular decreased expression in tumor tissue. Decreased NT expression in tumor tissue may contribute to reduced drug uptake and the development of resistance. These data suggest that nucleoside analog drug therapies may be optimized by determining individual NT expression profiles.

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CNT1 and CNT2 are sodium-dependent carriers mainly for pyrimidine and purine nucleosides, respectively, whereas CNT3 has wide substrate selectivity and mediates the transport of nucleosides both in a sodium- and a proton-coupled manner [2–4]. CNTs are expressed broadly in various tissues such as enterocytes, hepatocytes, and kidneys, whereas expression of CNT3 has not been observed in the liver [5–8]. Because nucleic acids are synthesised in the body, their importance as nutrients has been overlooked.
Human concentrative nucleoside transporters (CNTs) are responsible for cellular uptake of ribonucleosides; however, although it is important to better characterize CNT-subtype specificity to understand the systemic disposition of deoxyribonucleosides (dNs) and their analogs, the involvement of CNTs in transporting dNs is not fully understood. In this study, using COS-7 cells that transiently expressed CNT1, CNT2, or CNT3, we investigated if CNTs could transport not only ribonucleosides but also dNs, i.e., 2ʹ-deoxyadenosine (dAdo), 2ʹ-deoxyguanosine (dGuo), and 2ʹ-deoxycytidine (dCyd). The cellular uptake study demonstrated that dAdo and dGuo were taken up by CNT2 but not by CNT1. Although dCyd was taken up by CNT1, no significant uptake was detected in COS-7 cells expressing CNT2. Similarly, these dNs were transported by CNT3. The apparent K_m values of their uptake were as follows: CNT1, K_m ＝ 141 μM for dCyd; CNT2, K_m ＝ 62.4 μM and 54.9 μM for dAdo and dGuo, respectively; CNT3, K_m ＝ 14.7 μM and 34.4 μM for dGuo and dCyd, respectively. These results demonstrate that CNTs contribute not only to ribonucleoside transport but also to the transport of dNs. Moreover, our data indicated that CNT1 and CNT2 selectively transported pyrimidine and purine dNs, respectively, and CNT3 was shown to transport both pyrimidine and purine dNs.
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This study aimed to investigate the effect of single nucleotide polymorphisms (SNPs) of genes involved in ribavirin (RBV) transport (SLC28A2 gene, ABCB1 gene and ABCB11 gene) on the clinical outcome and pharmacokinetics of ribavirin in HCV- 4 Egyptian patients.
100 patients treated with sofosbuvir/daclatasvir and ribavirin for 12 weeks. The SNP genotyping was performed by real-time PCR using high resolution melting analysis. Ribavirin plasma trough concentrations were determined at week 4 of therapy using a liquid chromatography/tandem mass spectrometry (LC–MS/MS). For clinical outcomes, sustained virological response (SVR), liver function tests (ALT and AST), total bilirubin, albumin, serum creatinine, hemoglobin, leukocyte count, and platelet count were measured.
Concerning RBV pharmacokinetics, ABCB1 2677 G > T SNP and ABCB11 1331 T > C SNP were statistically associated with RBV C_trough levels after 4 weeks of therapy. ABCB11 1331 T > C SNP revealed significant association with clinical outcomes (SVR). SLC28A2-146 A > T SNP has not showed any statistically significant association with RBV plasma levels or response.
SNP genotyping for ABCB1 and ABCB11 genes can help in better personalized medicine for maximizing response for ribavirin as explored by the significant association between polymorphism in ABCB1 and ABCB11 genes and ribavirin pharmacokinetics and the significant association of ABCB11 1331 T > C SNP with clinical response.
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Citation Excerpt :
In addition to presence of adequate free drug in the blood, the incidence and severity of adverse events among patients depend on number of factors such as density, expression, and functionality of targets/off-targets, as well as the status of the disease at the time of therapy. Pennycooke et al. [25] has reported that the expression of nucleotide transporters (NT) in normal and tumor tissues is highly variable and is lower in tumor tissues. In gynecological cancers, the expression of concentrative nucleoside transporters (CNT1) and ENT1 in human ovarian, endometrial and uterine cervix carcinomas was highly variable.
Off-target activities of drug candidates observed during in vitro pharmacological profiling frequently do not translate to adverse events (AEs) in human. This could be because off-target activities do not have functional consequences, are not observed at exposures achieved during clinical testing, or may not translate into clinical outcomes. We report clinical consequences of an off-target activity observed during profiling of AMG 337, a selective inhibitor of the mesenchymal-epithelial transition factor being evaluated for treatment of solid tumors. In our screen of 151 potential off-targets, AMG 337 inhibited only adenosine transporter (AT). During clinical trials, headache emerged as the dose-limiting AE in the first-in-human trial. It was thought that headache was caused by extracellular accumulation of adenosine from inhibition of AT by AMG 337 and subsequent adenosine-mediated vasodilation through adenosine receptors (ARs). Further nonclinical studies were performed to evaluate this hypothesis. AMG 337 inhibited AT function in dog and human cells in vitro and dog and human arteries ex vivo. In a dog telemetry study, AMG 337 caused hypotension, which was reduced by pretreatment with theophylline, an AR antagonist. Overall, nonclinical and clinical data suggested that headache was due to cerebral vasorelaxation caused by AMG 337-mediated inhibition of AT. When subjects were advised to drink coffee, an AR antagonist, prior to AMG 337, the severity of headaches was reduced, allowing them to continue treatment. These findings demonstrate the importance of carefully evaluating clinical observations during early drug development and the value of translational nonclinical studies to investigate the mechanism of action driving clinical observations.
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Current treatment of human immunodeficiency virus type-1 (HIV-1) infection involves a combination of antiretroviral drugs (ARVs) that target different stages of the HIV-1 life cycle. This strategy is commonly referred to as highly active antiretroviral therapy (HAART) or combined antiretroviral therapy (cART). Membrane-associated drug transporters expressed ubiquitously in mammalian systems play a crucial role in modulating ARV disposition during HIV-1 infection. Members of the ATP-binding cassette (ABC) and solute carrier (SLC) transporter superfamilies have been shown to interact with ARVs, including those that are used as part of first-line treatment regimens. As a result, the functional expression of drug transporters can influence the distribution of ARVs at specific sites of infection. In addition, pathological factors related to HIV-1 infection and/or ARV therapy itself can alter transporter expression and activity, thus further contributing to changes in ARV disposition and the effectiveness of HAART. This review summarizes current knowledge on the role of drug transporters in regulating ARV transport in the context of HIV-1 infection.

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^☆: Abbreviations used: NT, nucleoside transporter; hENT, human nucleoside transporter; hCNT, human concentrative nucleoside transporter.

¹: To whom correspondence should be addressed. Fax: 1-416-736-5698. E-mail: [email protected].

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Regular ArticleDifferential Expression of Human Nucleoside Transporters in Normal and Tumor Tissue☆

Abstract

Mol. Med. Today

J. Biol. Chem.

Neuropharmacology

Brain Res.

Genomics

Nucleoside analogs: Cellular pharmacology, mechanisms of action and strategies for combination therapy

Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cells

Cancer Res.

Nucleoside Transport

Recent advances in the molecular biology of nucleoside transporters of mammalian cells

Biochem. Cell Biol.

Molecular cloning and functional expression of cDNAs encoding a human Na+-nucleoside cotransporter (hCNT1)

Am. J. Physiol.