Concentrative Nucleoside Transporter Research Articles

HNF4α Induced Chemosensitivity to Oxaliplatin and 5-FU Mediated by OCT1 and CNT3 in Renal Cell Carcinoma

Increased expression of transporters-mediating uptake of antineoplastic drugs could render renal cell carcinoma (RCC) more sensitive to chemotherapy. Here, we studied the effect of hepatocyte nuclear factor 4α (HNF4α) on the expression of selected uptake transporters in RCC lines. Organic cation transporters (OCTs) and organic anion transporters (OATs) mRNA levels in HNF4α-transfected RCCs were measured by real-time PCR. Expression of HNF4α, β-catenin, N-cadherin, and E-cadherin was detected by immunofluorescence. OCT1, OAT2, and concentrative nucleoside transporter 3 (CNT3) were tested using tritium-labeled substrates and an apoptosis assay. Most RCC did not express uptake transporters in the absence or presence of HNF4α. In RCCNG1 cells, HNF4α-expression increased the chemosensitivity to oxaliplatin and enhanced the accumulation of methyl-4-phenylpyridinium acetate, a model substrate for OCT1. Furthermore, HNF4α enhanced OAT2 mRNA and increased caspase-3 activity upon incubation with a purported OAT2 substrate, 5-fluorouracil (5-FU). However, functional OAT2 protein was not upregulated. CNT3 mRNA was significantly elevated by HNF4α. Inhibition of CNT3-mediated uridine uptake by 5-FU metabolite 5-fluoro-2′-deoxyuridine suggested the involvement of CNT3 in increased caspase-3 activity. Our data suggest that HNF4α increases the expression of OCT1 and CNT3 in RCCNG1 cells, thereby increasing the chemosensitivity of tumor cells to oxaliplatin and 5-FU.

Structure-guided design of concentrative nucleoside transporter (CNT)-mediated therapies

Structure-guided design of concentrative nucleoside transporter (CNT)-mediated therapies

Structural basis of nucleoside and nucleoside drug selectivity by concentrative nucleoside transporters.

Concentrative nucleoside transporters (CNTs) are responsible for cellular entry of nucleosides, which serve as precursors to nucleic acids and act as signaling molecules. CNTs also play a crucial role in the uptake of nucleoside-derived drugs, including anticancer and antiviral agents. Understanding how CNTs recognize and import their substrates could not only lead to a better understanding of nucleoside-related biological processes but also the design of nucleoside-derived drugs that can better reach their targets. Here, we present a combination of X-ray crystallographic and equilibrium-binding studies probing the molecular origins of nucleoside and nucleoside drug selectivity of a CNT from Vibrio cholerae. We then used this information in chemically modifying an anticancer drug so that it is better transported by and selective for a single human CNT subtype. This work provides proof of principle for utilizing transporter structural and functional information for the design of compounds that enter cells more efficiently and selectively.

Functional crosstalk between the adenosine transporter CNT3 and purinergic receptors in the biliary epithelia

Both hepatocytes and cholangiocytes release ATP into the bile, where it acts as a potent autocrine/paracrine stimulus that activates biliary secretory mechanisms. ATP is known to be metabolized into multiple breakdown products, ultimately yielding adenosine. However, the elements implicated in the adenosine-dependent purinergic regulation of cholangiocytes are not known. Normal rat cholangiocytes (NRCs) were used to study the expression of adenosine receptors and transporters and their functional interactions at the apical and basolateral membrane domains of polarized cholangiocytes. We found that: (1) cholangiocytes exclusively express two concentrative nucleoside transporters (CNT) known to be efficient adenosine carriers: CNT3, located at the apical membrane, and CNT2, located at apical and basolateral membrane domains; (2) in both domains, NRCs also express the high affinity adenosine receptor A2A, which modulated the activity of apical CNT3 in a domain-specific manner; (3) the regulation exerted by A2A on CNT3 was dependent upon the cAMP/PKA/ERK/CREB axis, intracellular trafficking mechanisms and AMPK phosphorylation; (4) secretin increased the activity of the apically-located CNT3, and promoted additional basolateral CNT3-related activity; and (5) extracellular ATP (a precursor of adenosine) was able to exert an inhibitory effect on the apical activity of both CNT3 and CNT2. This study uncovered the functional expression of nucleoside transporters in cholangiocytes and provides evidence for direct crosstalks between adenosine transporters and receptors for adenosine and its natural extracellular precursor, ATP. Our data anticipate the possibility of adenosine playing a major role in the physiopathology of the biliary epithelia.

Contribution of CNT1 and ENT1 to ribavirin uptake in human hepatocytes.

The objective of this study was to investigate the contributions of a sodium-dependent concentrative nucleoside transporter (CNT) 1 and an equilibrative nucleoside transporter (ENT) 1 to ribavirin uptake in human hepatocytes. The initial studies in oocytes expressing CNT1 and ENT1 showed increases in ribavirin uptake, indicating that ribavirin was a substrate for both CNT1 and ENT1. The CNT1- and ENT1-mediated ribavirin uptake showed concentration dependency with the following kinetics parameters: Km 26.3 μM and Vmax 426.2 fmol/min/oocyte for CNT1; Km 70.5 μM and Vmax 134.3 fmol/min/oocyte for ENT1. Ribavirin uptake clearance in six human hepatocytes ranged from 21.3 to 300.7 μL/min. Estimation of the contributions of CNT1 and ENT1 to the hepatic uptake of ribavirin by using a relative activity factor method indicated that the relative contribution of ENT1 to the ribavirin uptake was 82.8 ± 3.9%. Real-time polymerase chain reaction analysis of CNT1 and ENT1 expressions in the hepatocytes showed that ENT1 mRNA expression was closely correlated with ribavirin uptake (R = 0.95, P = 0.003) while CNT1 was not. The findings indicated that ENT1 was the major transporter controlling the hepatic uptake of ribavirin.

Ribavirin uptake into human hepatocyte HHL5 cells is enhanced by interferon-α via up-regulation of the human concentrative nucleoside transporter (hCNT2).

Ribavirin is a broad spectrum antiviral that increases the response rate in chronic hepatitis C patients when administered in combination with IFNα. Ribavirin is a purine nucleoside derivative, transported into hepatocytes by nucleoside transporters. hCNT2 is the best candidate to mediate ribavirin uptake into hepatocytes due to its high-affinity for purines and its capacity to concentrate its substrates intracellularly. The aim of this study was to determine whether hCNT2 function is under IFNα modulation. IFNα treatment of the nontransformed human hepatocyte-derived cell line HHL5 induced a rapid and transient increase in hCNT2 activity after cytokine addition. hCNT2 activity up-regulation was associated with increased ribavirin accumulation into cells. This increase was consistent with the translocation of hCNT2-containing vesicles to the plasma membrane via a mechanism requiring ERK 1/2 and ROCK activation and cytoskeleton integrity. Longer treatments with IFNα induced transcriptional activation of the hCNT2-encoding gene (SLC28A2), resulting in a sustained increase in hCNT2-related activity. These observations are proof of concept for at least one of the putative mechanisms underlying the synergistic responses induced by combination therapy with IFNα and ribavirin.

Downregulation of duodenal SLC transporters and activation of proinflammatory signaling constitute the early response to high altitude in humans.

Solute carrier (SLC) transporters mediate the uptake of biologically active compounds in the intestine. Reduced oxygenation (hypoxia) is an important factor influencing intestinal homeostasis. The aim of this study was to investigate the pathophysiological consequences of hypoxia on the expression and function of SLCs in human intestine. Hypoxia was induced in human intestinal epithelial cells (IECs) in vitro (0.2; 1% O2 or CoCl2). For human in vivo studies, duodenal biopsies and serum samples were obtained from individuals (n = 16) acutely exposed to 4,554 meters above sea levels. Expression of relevant targets was analyzed by quantitative PCR, Western blotting, or immunofluorescence. Serum levels of inflammatory mediators and nucleosides were determined by ELISA and LC/MS-MS, respectively. In the duodenum of volunteers exposed to high altitude we observed decreased mRNA levels of apical sodium-dependent bile acid transporter (ASBT), concentrative nucleoside transporters 1/2 (CNT1/2), organic anion transporting polypeptide 2B1 (OATP2B1), organic cation transporter 2 (OCTN2), peptide transporter 1 (PEPT1), serotonin transporter (SERT), and higher levels of IFN-γ, IL-6, and IL-17A. Serum levels of IL-10, IFN-γ, matrix metalloproteinase-2 (MMP-2), and serotonin were elevated, whereas the levels of uridine decreased upon exposure to hypoxia. Hypoxic IECs showed reduced levels of equilibrative nucleoside transporter 2 (ENT2), OCTN2, and SERT mRNAs in vitro, which was confirmed on the protein level and was accompanied by activation of ERK1/2, increase of hypoxia-inducible factor (HIF) proteins, and production of IL-8 mRNA. Costimulation with IFN-γ and IL-6 during hypoxia further decreased the expression of SERT, ENT2, and CNT2 in vitro. Reduced oxygen supply affects the expression pattern of duodenal SLCs that is accompanied by changes in serum levels of proinflammatory cytokines and biologically active compounds demonstrating that intestinal transport is affected during systemic exposure to hypoxia in humans.

Characterization of the Adenosine Pharmacology of Ticagrelor Reveals Therapeutically Relevant Inhibition of Equilibrative Nucleoside Transporter 1

Studies have shown that ticagrelor has a further adenosine-mediated mechanism of action in addition to its potent inhibition of the P2Y12 receptor, which may explain some of ticagrelor's clinical characteristics. This study aimed to further characterize the adenosine pharmacology of ticagrelor, its major metabolites, and other P2Y12 receptor antagonists. Inhibition of nucleoside transporter-mediated [(3)H]adenosine uptake by ticagrelor, its major metabolites, and alternative P2Y12 antagonists was examined in recombinant Madin-Darby canine kidney (MDCK) cells. The pharmacology of ticagrelor and its major metabolites at adenosine A1, A2A, A2B, and A3 receptor subtypes was examined using in vitro radioligand binding and functional assays and ex vivo C-fiber experiments in rat and guinea pig vagus nerves. Ticagrelor (and less effectively its metabolites) and the main cangrelor metabolite inhibited [(3)H]adenosine uptake in equilibrative nucleoside transporter (ENT) 1-expressing MDCK cells, whereas cangrelor and the active metabolites of prasugrel or clopidogrel had no effect. No significant inhibitory activity was observed in MDCK cells expressing ENT2 or concentrative nucleoside transporters 2/3. Ticagrelor demonstrated high affinity (inhibition constant [Ki] = 41 nmol/L) for ENT1. In adenosine receptor-binding experiments, ticagrelor and its major circulating metabolite, AR-C124910XX, had low affinity (Ki > 6 µmol/L) for each of the adenosine A1, A2A, and A2B receptors, whereas ticagrelor had a submicromolar (Ki = 190 nmol/L) affinity for the adenosine A3 receptor. However, in functional assays, at high concentrations (10 µmol/L) ticagrelor only partially inhibited 3 mmol/L adenosine-induced depolarizations in the guinea pig and rat vagus nerve preparations (by 35% and 49%, respectively). Ticagrelor inhibits cellular adenosine uptake selectively via ENT1 inhibition at concentrations of clinical relevance. However, the low-binding affinity and functional inhibition of adenosine receptors observed with ticagrelor or its metabolites indicate that they possess a negligible adenosine-like activity at clinically relevant concentrations.

Erlotinib, Gefitinib, and Vandetanib Inhibit Human Nucleoside Transporters and Protect Cancer Cells from Gemcitabine Cytotoxicity

Combinations of tyrosine kinase inhibitors (TKI) with gemcitabine have been attempted with little added benefit to patients. We hypothesized that TKIs designed to bind to ATP-binding pockets of growth factor receptors also bind to transporter proteins that recognize nucleosides. TKI inhibition of uridine transport was studied with recombinant human (h) equilibrative (E) and concentrative (C) nucleoside transporters (hENT, hCNT) produced individually in yeast. TKIs effects on uridine transport, gemcitabine accumulation, regulation of hENT1 activity, and cell viability in the presence or absence of gemcitabine were evaluated in human pancreatic and lung cancer cell lines. Erlotinib, gefitinib and vandetanib inhibited [(3)H]uridine transport in yeast and [(3)H]uridine and [(3)H]gemcitabine uptake in the four cell lines. Treatment of cell lines with erlotinib, gefitinib, or vandetanib for 24 hours reduced hENT1 activity which was reversed by subsequent incubation in drug-free media for 24 hours. Greater cytotoxicity was observed when gemcitabine was administered before erlotinib, gefitinib, or vandetanib than when administered together and synergy, evaluated using the CalcuSyn Software, was observed in three cell lines resulting in combination indices under 0.6 at 50% reduction of cell growth. Vandetanib inhibited hENT1, hENT2, hCNT1, hCNT2, and hCNT3, whereas erlotinib inhibited hENT1 and hCNT3 and gefitinib inhibited hENT1 and hCNT1. The potential for reduced accumulation of nucleoside chemotherapy drugs in tumor tissues due to inhibition of hENTs and/or hCNTs by TKIs indicates that pharmacokinetic properties of these agents must be considered when scheduling TKIs and nucleoside chemotherapy in combination.

Hypoxia and P1 receptor activation regulate the high-affinity concentrative adenosine transporter CNT2 in differentiated neuronal PC12 cells

Under several adverse conditions, such as hypoxia or ischaemia, extracellular levels of adenosine are elevated because of increased energy demands and ATP metabolism. Because extracellular adenosine affects metabolism through G-protein-coupled receptors, its regulation is of high adaptive importance. CNT2 (concentrative nucleoside transporter 2) may play physiological roles beyond nucleoside salvage in brain as it does in other tissues. Even though nucleoside transport in brain has mostly been seen as being of equilibrative-type, in the present study, we prove that the rat phaeochromocytoma cell line PC12 shows a concentrative adenosine transport of CNT2-type when cells are differentiated to a neuronal phenotype by treatment with NGF (nerve growth factor). Differentiation of PC12 cells was also associated with the up-regulation of adenosine A1 receptors. Addition of adenosine receptor agonists to cell cultures increased CNT2-related activity by a mechanism consistent with A₁ and A2A receptor activation. The addition of adenosine to the culture medium also induced the phosphorylation of the intracellular regulatory kinase AMPK (AMP-activated protein kinase), with this effect being dependent upon adenosine transport. CNT2-related activity of differentiated PC12 cells was also dramatically down-regulated under hypoxic conditions. Interestingly, the analysis of nucleoside transporter expression after experimental focal ischaemia in rat brain showed that CNT2 expression was down-regulated in the infarcted tissue, with this effect somehow being restricted to other adenosine transporter proteins such as CNT3 and ENT1 (equilibrative nucleoside transporter 1). In summary, CNT2 is likely to modulate extracellular adenosine and cell energy balance in neuronal tissue.

Involvement of CD73, equilibrative nucleoside transporters and inosine in rhythm and conduction disturbances mediated by adenosine A1 and A2A receptors in the developing heart

We previously established that exogenous adenosine (ADO) induces transient arrhythmias in the developing heart via the adenosine A1 receptor (A1AR) and downstream activation of NADPH oxidase/ERK and PLC/PKC pathways. Here, we investigated the mechanisms by which accumulation of endogenous ADO and its derived compound inosine (INO) in the interstitial compartment induce rhythm and conduction troubles. The validated model of the spontaneously beating heart obtained from 4-day-old chick embryos was used. Quantitative RT-PCR showed that enzymes involved in ADO and INO metabolism (CD39, CD73 and eADA) as well as equilibrative (ENT1, -3, -4) and concentrative (CNT3) nucleoside transporters were differentially expressed in atria, ventricle and outflow tract. Inactivation of ENTs by dipyridamole, 1) increased myocardial ADO level, 2) provoked atrial arrhythmias and atrio-ventricular blocks (AVB) in 70% of the hearts, 3) prolonged P wave and QT interval without altering contractility, and 4) increased ERK2 phosphorylation. Blockade of CD73-mediated phosphohydrolysis of AMP to ADO, MEK/ERK pathway inhibition or A1AR inhibition prevented these arrhythmias. Exposure to exogenous INO also caused atrial ectopy associated with AVB and ERK2 phosphorylation which were prevented by A1AR or A2AAR antagonists exclusively or by MEK/ERK inhibitor. Inhibition of ADA-mediated conversion of ADO to INO increased myocardial ADO and decreased INO as expected, but slightly augmented heart rate variability without provoking AVB. Thus, during cardiogenesis, disturbances of nucleosides metabolism and transport, can lead to interstitial accumulation of ADO and INO and provoke arrhythmias in an autocrine/paracrine manner through A1AR and A2AAR stimulation and ERK2 activation.

Comparison of Uptake Mechanisms for Anthracyclines in Human Leukemic Cells

The mechanisms behind cellular anthracycline uptake are not completely understood. Knowledge about uptake mechanisms could be used to increase the selectivity of the drugs. We compared the uptake patterns of, daunorubicin (DNR), doxorubicin (DOX), epirubicin (EPI), idarubicin (IDA), and pirarubicin (PIRA) by cultured leukemic cells and investigated possible involvement of specific carriers. HL-60 cells were incubated with anthracyclines for 1 hour in the absence or presence of transport inhibitors, suramin, or nucleosides and cellular drug uptake was determined. Cell survival was also determined. MCF-7 breast cancer cells were used as a negative control for concentrative nucleoside transporters (CNTs). Anthracycline concentration was determined with HPLC and fluorometric detection and apoptosis was determined with propidium iodide and flow cytometry. DNR, IDA, and PIRA had higher uptake than DOX and EPI with a prominent increase in uptake at concentrations > 1 µM. Uptake of all anthracyclines was greatly reduced at 0°C. Suramin, a purinergic-2-receptor inhibitor, strongly inhibited the uptake of all anthracyclines except PIRA and increased cell survival. Dipyridamole, an equilibrative NT (ENT) inhibitor, significantly inhibited the uptake of DNR only. The addition of nucleosides significantly inhibited the uptake of DNR, IDA, and PIRA but not in MCF-7 cells lacking functional CNTs. Our results suggest different uptake mechanisms for the anthracyclines studied. We found evidence for carrier mediated uptake mechanisms, supporting involvement of NTs in transmembrane transport of DNR, IDA, and PIRA. The results also showed a strong inhibition of suramin on anthracycline uptake by so far unknown mechanisms.

Effects of gefitinib and vandetanib on human equilibrative nucleoside transporter 1 and on gemcitabine cytotoxicity.

2546 Background: Combination chemotherapy with tyrosine kinase inhibitors (TKIs) and gemcitabine has been attempted with little added benefit to patients. We hypothesized that TKIs that were designed to bind to ATP pockets of growth factor tyrosine kinases also bind to proteins that recognize nucleosides, thereby potentially interfering with gemcitabine pharmacology. Methods: Interaction of TKIs with human nucleoside transporters (NTs) was studied using recombinant NTs produced in yeast. Effects of TKIs on uridine transport, gemcitabine transport and accumulation, regulation of NT activity and cytotoxicity with and without gemcitabine were evaluated in human A549 lung cancer cells. Results: In yeast, vandetanib inhibited two equilibrative NTs (hENT1, hENT2) and three concentrative NTs (hCNT1, hCNT2, hCNT3) with the greatest inhibition seen with hENT1 whereas gefitinib strongly inhibited hENT1 and hCNT1 only. In A549 cells, which possess major hENT1 and minor hENT2 activities, [3H]uridine uptake was inhibited by vandetanib and gefitinib with IC50 values of 16 ± 4 and 5 ± 0.3µM, respectively. Both TKIs also inhibited [3H]gemcitabine transport and accumulation in A549 cells. hENT1 protein levels were decreased during exposures to vandetanib or gefitinib for 24 hours, and cytotoxicity was greatest when gemcitabine was given prior to vandetanib or gefitinib. Conclusions: Vandetanib and gefitinib inhibited human NTs, especially hENT1, resulting in reduced intracellular gemcitabine accumulation. Gefitinib or vandetanib levels achieved in plasma and tumor tissues are sufficient to inhibit hENT1 activity. Because TKIs can block uptake of nucleoside chemotherapy drugs in cultured cancer cells, attention must be paid to TKIs and nucleoside pharmacokinetic properties when scheduling TKIs and nucleoside chemotherapy.

Concentrative nucleoside transporter 1 (hCNT1) promotes phenotypic changes relevant to tumor biology in a translocation-independent manner

Nucleoside transporters (NTs) mediate the uptake of nucleosides and nucleobases across the plasma membrane, mostly for salvage purposes. The canonical NTs belong to two gene families, SLC29 and SLC28. The former encode equilibrative nucleoside transporter proteins (ENTs), which mediate the facilitative diffusion of natural nucleosides with broad selectivity, whereas the latter encode concentrative nucleoside transporters (CNTs), which are sodium-coupled and show high affinity for substrates with variable selectivity. These proteins are expressed in most cell types, exhibiting apparent functional redundancy. This might indicate that CNTs have specific roles in the physiology of the cell beyond nucleoside salvage. Here, we addressed this possibility using adenoviral vectors to restore tumor cell expression of hCNT1 or a polymorphic variant (hCNT1S546P) lacking nucleoside translocation ability. We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP-ribose) polymerase hyperactivation and cell death and reduced cell migration. Importantly, the translocation-defective transporter triggered these same effects on cell physiology. Moreover, this study also shows that restoration of hCNT1 expression is able to reduce tumor growth in a mouse model of pancreatic adenocarcinoma. These data predict a novel role for a NT protein, hCNT1, which appears to be independent of its role as mediator of nucleoside uptake by cells. Thereby, hCNT1 fits the profile of a transceptor in a substrate translocation-independent manner and is likely to be relevant to tumor biology.

Novel leukemic cell lines resistant to clofarabine by mechanisms of decreased active metabolite and increased antiapoptosis

Clofarabine (CAFdA) is incorporated into leukemic cells by human equilibrative nucleoside transporters (hENT) 1 and 2 and human concentrative nucleoside transporter (hCNT) 3. CAFdA is then phosphorylated to the active metabolite CAFdA triphosphate (CAFdATP) by deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Two novel CAFdA-resistant variants were established and their mechanism of resistance was elucidated. The two variants (HL/CAFdA20, HL/CAFdA80) were 20-fold and 80-fold more CAFdA-resistant than HL-60, respectively. mRNA levels of hENT1, hENT2 and hCNT3 were 53.9, 41.8 and 17.7% in HL/CAFdA20, and 30.8, 13.9 and 7.9% in HL/CAFdA80, respectively, compared with HL-60. Thus, the total nucleoside transport capacity of CAFdA was reduced in both variants. dCK protein levels were 1/2 in HL/CAFdA20 and 1/8 in HL/CAFdA80 of that of HL-60. dGK protein levels were 1/2 and 1/3, respectively. CAFdATP production after 4-h incubation with 10 μM CAFdA was 20 pmol/10(7) cells in HL/CAFdA20 and 3 pmol/10(7) cells in HL/CAFdA80 compared with 63 pmol/10(7) cells in HL-60. The decreased CAFdATP production attenuated drug incorporation into both mitochondrial and nuclear DNA. In addition, the two variants were resistant to CAFdA-induced apoptosis due to Bcl2 overexpression and decreased Bim. A Bcl2 inhibitor, ABT737, acted synergistically with CAFdA to inhibit the growth with combination index values of 0.27 in HL/CAFdA20 and 0.23 in HL/CAFdA80, compared with 0.65 in HL-60. Thus, the mechanism of resistance primarily included not only reduced CAFdATP production, but also increased antiapoptosis. The combination of CAFdA and ABT737 may be effective against CAFdA resistance.

EXPRESSION OF NUCLEOSIDE TRANSPORTERS IN THE MOUSE TESTIS AND OVARY

Nucleoside transporters play key roles in the physiology of nucleosides and nucleobases in mammals and also contribute to the pharmacological actions produced by their analogs. Several mammalian nucleoside transporters have been identified at the molecular level. The aim of this study was to examine the expression of the major nucleoside transporter isoforms in the mouse testis and ovary. Equilibrative Nucleoside Transporter 1 (ENT1) and ENT2 were expressed in the mouse testis and ovary, which was consistent with the general consideration that these transporters are ubiquitous in mouse tissues. The expression of Concentrative Nucleoside Transporter 2 (CNT2) and CNT3, but not CNT1 was detected in the mouse testis. These three CNT isoforms were also expressed in the ovary. Therefore, we concluded that the mouse testis and ovary simultaneously exhibit multiple transport processes for nucleosides and bases and also that these transporters are responsible for the homeostatic control of biological levels of nucleosides and bases in these tissues.

Efficacy of Low-Dose Oral Metronomic Dosing of the Prodrug of Gemcitabine, LY2334737, in Human Tumor Xenografts

LY2334737, an oral prodrug of gemcitabine, is cleaved in vivo, releasing gemcitabine and valproic acid. Oral dosing of mice results in absorption of intact prodrug with slow systemic hydrolysis yielding higher plasma levels of LY2334737 than gemcitabine and prolonged gemcitabine exposure. Antitumor activity was evaluated in human colon and lung tumor xenograft models. The dose response for efficacy was examined using 3 metronomic schedules, once-a-day dosing for 14 doses, every other day for 7 doses, and once a day for 7 doses, 7 days rest, followed by an additional 7 days of once-a-day dosing. These schedules gave significant antitumor activity and were well tolerated. Oral gavage of 6 mg/kg LY2334737 daily for 21 days gave equivalent activity to i.v. 240 mg/kg gemcitabine. HCl administered once a week for 3 weeks to mice bearing a patient mesothelioma tumor PXF 1118 or a non-small cell lung cancer tumor LXFE 937. The LXFE 397 tumor possessed elevated expression of the equilibrative nucleoside transporter-1 (ENT1) important for gemcitabine uptake but not prodrug uptake and responded significantly better to treatment with LY2334737 than gemcitabine (P ≤ 0.001). In 3 colon xenografts, antitumor activity of LY2334737 plus a maximally tolerated dose of capecitabine, an oral prodrug of 5-fluorouracil, was significantly greater than either monotherapy. During treatment, the expression of carboxylesterase 2 (CES2) and concentrative nucleoside transporter-3 was induced in HCT-116 tumors; both are needed for the activity of the prodrugs. Thus, metronomic oral low-dose LY2334737 is efficacious, well tolerated, and easily combined with capecitabine for improved efficacy. Elevated CES2 or ENT1 expression may enhance LY2334737 tumor response.

Human Concentrative Nucleoside Transporter 3 Transfection with Ultrasound and Microbubbles in Nucleoside Transport Deficient HEK293 Cells Greatly Increases Gemcitabine Uptake

Gemcitabine is a hydrophilic clinical anticancer drug that requires nucleoside transporters to cross plasma membranes and enter cells. Pancreatic adenocarcinomas with low levels of nucleoside transporters are generally resistant to gemcitabine and are currently a clinical problem. We tested whether transfection of human concentrative nucleoside transporter 3 (hCNT3) using ultrasound and lipid stabilized microbubbles could increase gemcitabine uptake and sensitivity in HEK293 cells made nucleoside transport deficient by pharmacologic treatment with dilazep. To our knowledge, no published data exists regarding the utility of using hCNT3 as a therapeutic gene to reverse gemcitabine resistance. Our ultrasound transfection system - capable of transfection of cell cultures, mouse muscle and xenograft CEM/araC tumors - increased hCNT3 mRNA and 3H-gemcitabine uptake by >2,000– and 3,400–fold, respectively, in dilazep-treated HEK293 cells. Interestingly, HEK293 cells with both functional human equilibrative nucleoside transporters and hCNT3 displayed 5% of 3H-gemcitabine uptake observed in cells with only functional hCNT3, suggesting that equilibrative nucleoside transporters caused significant efflux of 3H-gemcitabine. Efflux assays confirmed that dilazep could inhibit the majority of 3H-gemcitabine efflux from HEK293 cells, suggesting that hENTs were responsible for the majority of efflux from the tested cells. Oocyte uptake transport assays were also performed and provided support for our hypothesis. Gemcitabine uptake and efflux assays were also performed on pancreatic cancer AsPC-1 and MIA PaCa-2 cells with similar results to that of HEK293 cells. Using the MTS proliferation assay, dilazep-treated HEK293 cells demonstrated 13-fold greater resistance to gemcitabine compared to dilazep-untreated HEK293 cells and this resistance could be reversed by transfection of hCNT3 cDNA. We propose that transfection of hCNT3 cDNA using ultrasound and microbubbles may be a method to reverse gemcitabine resistance in pancreatic tumors that have little nucleoside transport activity which are resistant to almost all current anticancer therapies.

Transport of A1Adenosine Receptor Agonist Tecadenoson by Human and Mouse Nucleoside Transporters: Evidence for Blood-Brain Barrier Transport by Murine Equilibrative Nucleoside Transporter 1 mENT1

The high density of A1 adenosine receptors in the brain results in significant potential for central nervous system (CNS)-related adverse effects with A1 agonists. Tecadenoson is a selective A1 adenosine receptor agonist with close similarity to adenosine. We studied the binding and transmembrane transport of tecadenoson by recombinant human equilibrative nucleoside transporters (hENTs) hENT1 and hENT2, and human concentrative nucleoside transporters (hCNTs) hCNT1, hCNT2, and hCNT3 in vitro and by mouse mENT1 in vivo. Binding affinities of the five recombinant human nucleoside transporters for tecadenoson differed (hENT1 > hCNT1 > hCNT3 > hENT2 > hCNT2), and tecadenoson was transported largely by hENT1. Pretreatment of mice with a phosphorylated prodrug of nitrobenzylmercaptopurine riboside, an inhibitor of mENT1, significantly decreased brain exposure to tecadenoson compared with that of the untreated (control) group, suggesting involvement of mENT1 in transport of tecadenoson across the blood-brain barrier (BBB). In summary, ENT1 was shown to mediate the transport of tecadenoson in vitro with recombinant and native human protein and in vivo with mice. The micromolar apparent Km value of tecadenoson for transport by native hENT1 in cultured cells suggests that hENT1 will not be saturated at clinically relevant (i.e., nanomolar) concentrations of tecadenoson, and that hENT1-mediated passage across the BBB may contribute to the adverse CNS effects observed in clinical trials. In contrast, in cases in which a CNS effect is desired, the present results illustrate that synthetic A1 agonists that are transported by hENT1 could be used to target CNS disorders because of enhanced delivery to the brain.

Dueling nucleosides: cross-regulation of extracellular adenosine by guanosine. Focus on “Extracellular guanosine regulates extracellular adenosine levels”

<i>Dueling nucleosides: cross-regulation of extracellular adenosine by guanosine</i>. Focus on “Extracellular guanosine regulates extracellular adenosine levels”