Equilibrative Nucleoside Transporter Research Articles

The Effect Of Ticagrelor On Platelet-Endothelial Progenitor Cell Interaction

Background and Aims: Platelets mediate circulating endothelial progenitor cell recruitment and maturation, participating in vascular injury repair, but the underlying mechanism(s) is unclear. Ticagrelor, a potent reversible P2Y12 platelet receptor antagonist, exerts pleiotropic actions affecting endothelial cell functionality. Possible mechanisms could be related to ticagrelor’s ability to increase adenosine’s plasma concentration by blocking the equilibrative nucleoside transporter-1. We investigated ticagrelor’s effect on platelet interaction with CD34+-derived late-outgrowth endothelial cells (OECs).

Revisiting the role of term placenta in serotonin handling; in situ and ex vivo studies in human and rat placenta show its protective function against excessive fetal serotonin

Ribavirin is a broad-spectrum nucleoside-derived antiviral drug used in combination pharmacotherapy treatment of hepatitis C virus infection. Current evidence indicates that ribavirin-associated teratogenicity is not significant in humans, but more information about the developmental toxicity and mechanisms involved in ribavirin placental kinetics is required to assure its safe use in pregnancy. Thus, we have investigated potential roles of equilibrative nucleoside transporters (ENTs, SLC29A), Na+-dependent influx-mediating concentrative nucleoside transporters (CNTs, SLC28A), and ATP-binding cassette (ABC) efflux pumps, in ribavirin placental pharmacokinetics. Our data indicate that ENT1 participates in uptake of ribavirin by BeWo cells, fresh human placental villous fragments and microvillous plasma membrane (MVM) vesicles while activity of CNTs (probably CNT2) was only observed in BeWo cells. In situ dual perfusion experiments with rat term placenta in an open circuit setup showed that ENT inhibition significantly decreases total ribavirin maternal-to-foetal and foetal-to-maternal clearances. In contrast, no contribution of ABC transporters, p-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), or multidrug resistance-associated protein (ABCC2) was detected in assays with MDCKII cells overexpressing them, or in closed circuit dual perfusion experiments with rat term placenta. In summary, our data show that ribavirin placental pharmacokinetics are largely controlled by ENT1 activity and independent of ABCB1, ABCG2, and ABCC2 efflux pumps.

Clinical Implications and Translation of an Off-Target Pharmacology Profiling Hit: Adenosine Uptake Inhibition In Vitro

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.

Purinergic Signaling in Pulmonary Inflammation.

Purine nucleotides and nucleosides are at the center of biologic reactions. In particular, adenosine triphosphate (ATP) is the fundamental energy currency of cellular activity and adenosine has been demonstrated to play essential roles in human physiology and pathophysiology. In this review, we examine the role of purinergic signaling in acute and chronic pulmonary inflammation, with emphasis on ATP and adenosine. ATP is released into extracellular space in response to cellular injury and necrosis. It is then metabolized to adenosine monophosphate (AMP) via ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and further hydrolyzed to adenosine via ecto-5′-nucleotidase (CD73). Adenosine signals via one of four adenosine receptors to exert pro- or anti-inflammatory effects. Adenosine signaling is terminated by intracellular transport by concentrative or equilibrative nucleoside transporters (CNTs and ENTs), deamination to inosine by adenosine deaminase (ADA), or phosphorylation back into AMP via adenosine kinase (AK). Pulmonary inflammatory and hypoxic conditions lead to increased extracellular ATP, adenosine diphosphate (ADP) and adenosine levels, which translates to increased adenosine signaling. Adenosine signaling is central to the pulmonary injury response, leading to various effects on inflammation, repair and remodeling processes that are either tissue-protective or tissue destructive. In the acute setting, particularly through activation of adenosine 2A and 2B receptors, adenosine signaling serves an anti-inflammatory, tissue-protective role. However, excessive adenosine signaling in the chronic setting promotes pro-inflammatory, tissue destructive effects in chronic pulmonary inflammation.

Roles of organic anion transporter 2 and equilibrative nucleoside transporter 1 in hepatic disposition and antiviral activity of entecavir during non-pregnancy and pregnancy.

Entecavir (ETV), a first-line antiviral drug against hepatitis B virus (HBV), has the possibility to be used to prevent mother-to-child transmission. The aim of present study was to clarify the mechanism of ETV uptake into hepatocytes and evaluate the alteration of ETV's hepatic distribution during pregnancy. The roles of equilibrative nucleotide transporter (ENT) 1 and organic anion transporter (OAT) 2 in ETV accumulation and anti-HBV efficacy were studied in human ENT1 or OAT2 overexpressed cell models and HepG2.2.15 cells, respectively; meanwhile, the liver-to-plasma ETV concentration ratios in non-pregnant and pregnant mice were measured to evaluate the effect of pregnancy on ETV hepatic distribution. ETV was shown to be a substrate of ENT1 and OAT2. An ENT1 inhibitor significantly decreased the efficacy of ETV in HepG2.2.15 cells, while overexpression of OAT2 increased susceptibility of HBV to ETV. The liver-to-plasma ETV concentration ratios in pregnant mice were sharply reduced; whereas, the absolute concentration of ETV in the liver did not obviously alter in pregnancy. Although oestradiol and progesterone showed a concentration-dependent inhibition on ETV accumulation both in hepatic cell lines and in primary human hepatocytes, a physiologically relevant concentration of oestradiol and progesterone did not affect antiviral activity of ETV. OAT2 and ENT1 are the main transporters involved in the hepatic uptake and anti-HBV efficacy of ETV. The concentration of ETV in the liver was not obviously altered during pregnancy, which indicates that dosage adjustment in pregnancy is not necessary.

Adult stem cell deficits drive Slc29a3 disorders in mice

Mutations exclusively in equilibrative nucleoside transporter 3 (ENT3), the only intracellular nucleoside transporter within the solute carrier 29 (SLC29) gene family, cause an expanding spectrum of human genetic disorders (e.g., H syndrome, PHID syndrome, and SHML/RDD syndrome). Here, we identify adult stem cell deficits that drive ENT3-related abnormalities in mice. ENT3 deficiency alters hematopoietic and mesenchymal stem cell fates; the former leads to stem cell exhaustion, and the latter leads to breaches of mesodermal tissue integrity. The molecular pathogenesis stems from the loss of lysosomal adenosine transport, which impedes autophagy-regulated stem cell differentiation programs via misregulation of the AMPK-mTOR-ULK axis. Furthermore, mass spectrometry-based metabolomics and bioenergetics studies identify defects in fatty acid utilization, and alterations in mitochondrial bioenergetics can additionally propel stem cell deficits. Genetic, pharmacologic and stem cell interventions ameliorate ENT3-disease pathologies and extend the lifespan of ENT3-deficient mice. These findings delineate a primary pathogenic basis for the development of ENT3 spectrum disorders and offer critical mechanistic insights into treating human ENT3-related disorders.

Type 1 equilibrative nucleoside transporter (ENT1) regulates sex-specific ethanol drinking during disruption of circadian rhythms.

Disruptions in circadian rhythms are risk factors for excessive alcohol drinking. The ethanol-sensitive adenosine equilibrative nucleoside transporter type 1 (ENT1, slc29a1) regulates ethanol-related behaviors, sleep, and entrainment of circadian rhythms. However, the mechanism underlying the increased ethanol consumption in ENT1 knockout (KO) mice in constant light (LL) and whether there are sex differences in ethanol consumption in ENT1 mice are less studied. Here, we investigated the effects of loss of ENT1, LL, and sex on ethanol drinking using two-bottle choice. In addition, we monitored the locomotor activity rhythms. We found that LL increased ethanol drinking and reduced accumbal ENT1 expression and adenosine levels in male but not female mice, compared with control mice. Interestingly, only LL-exposed male, not female, ENT1 KO mice exhibited higher ethanol drinking and a longer circadian period with a higher amplitude compared with wild-type (WT) mice. Furthermore, viral-mediated rescue of ENT1 expression in the NAc of ENT1 KO mice reduced ethanol drinking, demonstrating a possible causal link between ENT1 expression and ethanol drinking in males. Together, our findings indicate that deficiency of ENT1 expression contributes to excessive ethanol drinking in a sex-dependent manner.

Abstract 2137: Aberrant nuclear-expressed SLC29A2 as a therapeutic target in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the second most prevalent causes of death in cancer worldwide, and there is lack of promising therapeutic target for patients. Here, we demonstrate that SLC29A2 (Solute Ligand Carrier Family 29 Member 2), an equilibrative nucleoside transporter, is commonly amplified and upregulated in varieties of cancers including hepatocellular carcinoma (HCC). SLC29A2 expression is increased and accumulated in nucleus during HCC progression, accompanied by interacting with phospho-STAT3, and further regulated STAT3-downstream genes to promote tumorigenic and metastatic features in HCC cell lines and preclinical orthotopic mouse model. Oncogenic properties of nuclear SLC29A2 could be suppressed by disrupting the nuclear translocation with nuclear importing mutant RanQ69L, SLC29A2 membrane anchoring mutant, and treating the inhibitor of SLC29A2. Treating inhibitor reduces nuclear SLC29A2-driven oncogenic features by reducing STAT3 phosphorylation, interrupting SLC29A2/STAT3 interaction, and nuclear translocation of SLC29A2 while SLC29A2 is amplified or upregulated in HCC cells. Combined treatment of FDA-approved late stage HCC medicine sorafenib with SLC29A2 inhibitor further suppresses tumor progression and metastasis to prolong mice survival in preclinical orthotopic model. Together, our results suggest that SLC29A2 is a theranostic target, and provide molecular insights for targeting nuclear SLC29A2. Citation Format: Roger Shen, Yuh-Shan Jou. Aberrant nuclear-expressed SLC29A2 as a therapeutic target in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2137.

Abstract 1267: Unusual mechanism of resistance to the novel cytidine analog fluorocyclopentenylcytosine (RX-3117)

Abstract RX-3117 is a novel cytidine analog showing encouraging results in ongoing Phase 2a studies with gemcitabine-resistant pancreatic ductal adenocarcinoma and advanced urothelial bladder cancer. Cellular uptake of RX-3117 is mediated by the equilibrative nucleoside transporter 1 (hENT1). RX-3117 is activated by uridine-cytidine kinase-2 (UCK2) and is not a substrate for cytidine deaminase (CDA), and shows a good oral bioavailability. In an earlier study accumulation of RX-3117 nucleotides was associated with sensitivity to the drug. RX-3117 is incorporated into RNA and DNA and inhibits DNA methyltransferase 1 (DNMT1). RX-3117 is active against gemcitabine-resistant non-small cell lung cancer (NSCLC) cell lines. In this study we aimed to elucidate potential resistance mechanisms against RX-3117. For that purpose the NSCLC cell lines H460, A549 and SW1573 (including its gemcitabine resistant variant SW-G) were exposed to increasing concentrations of RX-3117. For A549 and SW1573 two variants were obtained: A549/RX1, A549/RX2, SW1573/RX1 and SW1573/RX2. Resistance varied from 10 (H460; SW-G) to >100-fold for the other cell lines. Resistance was stable except for H460 cells. Cross-resistance in the same range was observed for the cytidine analogs ethynyl-cytidine (ETC), cyclopentenylcytosine (CPEC) and aza-cytidine (aza-CR), which are all activated by UCK2; no cross-resistance was observed for gemcitabine and aza-deoxycytidine which are activated by deoxycytidine kinase. No change was observed in the expression of DNMT1, while expression of ribonucleotide reductase 1 was decreased. However, analysis of UCK2 at the enzyme activity level, protein and gene expression did not reveal a decrease in UCK2 while for ETC, aza-CR and CPEC resistance was reported to be mediated by a UCK2 deficiency. No change was found in the other activating enzymes UMP-kinase, CMP-kinase and NME1/NDKA. Despite this lack of change, accumulation of RX-3117 and RX-3117 nucleotides (mono-, di- and triphosphate) was 2-5-fold lower in the resistant cells after incubation with 10 or 100 μM RX-3117 for 4-24 hr. No evidence for accumulation of RX-3117 deoxynucleotides was found in both the parent and the resistant cell lines. Since no change in hENT1 was found, cell lines were further investigated by using RNA-seq, and western blotting focusing on nucleoside/nucleotide degradation enzymes. CDA was decreased in resistant cells, but the cytosolic pyrimidine nucleotidase NT5C3 was increased in the resistant cell lines as well as DCTPP1, which can degrade deoxynucleoside triphosphates. Moreover several DNA repair enzymes SAMHD1, MTH1 and TDP1 were increased. However, inhibition of MTH1 and DCTPP1 did not increase sensitivity to RX-3117. In conclusion, induction of resistance to the novel cytidine analog RX-3117 led to a decreased accumulation of RX-3117 ribonucleotides, which might be associated with an increased degradation. Citation Format: Godefridus J. Peters, Dzjemma Sarkisjan, Btissame El Hassouni, Richard J. Honeywell, Joris R. Julsing, Beatrice Balboni, Daniel J. De Klerk, Safet Zekanovic, Kees Smid, Elisa Giovannetti, Young B. Lee, Deog J. Kim. Unusual mechanism of resistance to the novel cytidine analog fluorocyclopentenylcytosine (RX-3117) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1267.

Abstract 393: Deoxymab: A targeted biologic that is synthetically lethal to TNBC brain metastases

Abstract Brain metastases in triple-negative breast cancer (TNBC) harbor defects in homology-directed DNA repair that may be exploited to develop new therapeutic strategies. However, these tumors are protected by the blood-brain barrier (BBB), and mechanisms of DNA repair are sequestered in the nucleus. Methods to cross the BBB and target intranuclear processes are needed. 3E10 is a nuclear-penetrating lupus anti-DNA autoantibody that inhibits the DNA damage response to selectively kill cancer cells with defects in DNA repair. 3E10 localizes to DNA in tumor environments, and penetrates cells via the ENT2 equilibrative nucleoside transporter. ENT2 is expressed in the BBB, and 3E10 has previously delivered cargo proteins to the brain. We believe 3E10 can be turned against TNBC brain metastases, and used MDA-MB-231-BR brain-seeking human TNBC cells and tumors as a model system to test this hypothesis. We re-engineered 3E10 into an optimized fragment, called Deoxymab-1 (DX1), to maximize effect on cancer cells and minimize toxicity. DX1 expressed in CHO cells was purified over a HiTrap Capto S column by FPLC, and purity and quality was confirmed by SDS-PAGE and SEC-HPLC. Immunostaining and colony formation assays demonstrated that DX1 penetrated the nuclei of ~100% of MDA-MB-231-BR cells in culture and was synthetically lethal to the cells, with doses of 5 and 10 μM reducing surviving fractions to 0.47±0.15 (P<0.03) and 0.29±0.07 (P<0.01), respectively. TNBC brain metastases were established in nude mice by intracardiac injection of 1.75x105 MDA-MB-231-BR cells transfected with luciferase. One week later the presence of brain metastases was confirmed by IVIS. Mice were treated with tail vein injection of vehicle control (n=7) or DX1 (20 mg/kg) (n=7) 3x/week for 3 weeks. Tumor burden was monitored by weekly IVIS. DX1 significantly suppressed growth of the brain metastases (Table 1). These results establish proof of the novel concept of use of a modified lupus autoantibody against TNBC brain metastases. Table 1:Brain Radiance Efficiencies (x105)TreatmentWeek 0Week 1Week 2Week 3Control1.2±0.22.5±0.45.1±0.720±5DX11.3±0.21.4±0.32.0±0.23.8±0.8Pns<0.01<0.01<0.02 Citation Format: Shenqi Zhang, Christopher May, Anupama Shirali, Valentina Dubljevic, James A. Campbell, Jiangbing Zhou, James E. Hansen. Deoxymab: A targeted biologic that is synthetically lethal to TNBC brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 393.

Abstract 2123: A combinatorial approach using gemcitabine and bitter melon juice overcomes drug resistance impacting gemcitabine uptake and metabolism

Abstract Pancreatic cancer (PanC) has the lowest 5-year survival rate (<3%) of all cancer types and is projected to be the second leading cause of cancer-associated mortalities by the year 2030 in the United States. Surgery alone fails to suffice in a majority of cases; the disease relapses in 70-80% of patients and is fatal without additional therapy. Currently, of all the available treatment modalities for PanC, gemcitabine (GEM) is the standard of care frontline drug, with a moderate success rate in improving the median overall PanC patient survival by about 5.7-6.8 months. Although a modest improvement is achieved using a combination therapy approach, the patient survival still remains less than 12 months, and includes a poor quality of life due to the extensive side effects. Moreover, chemoresistance to GEM is a major contributing factor for PanC patient’ morbidity and mortality resulting from dysfunctional uptake and metabolism of GEM in cancer cells. Therefore, there is an urgent need for agents that could reverse GEM resistance and allow continued chemosensitivity in PanC. Here, we employed natural agent bitter melon juice (BMJ) and GEM to examine their anticancer potential alone and in combination in PanC patient derived xenograft (PDX) - pancreatic ductal adenocarcinomas explants in nude mice. Comparing single agents versus combination in three explants PDX272, PDX261, and PDX266, both at the end of the active dosing regimen as well as following a post treatment-washout period (no drug administration) revealed enhanced efficacy of the combination treatment over failed GEM-in the post treatment termination cohort. Extensive mechanistic assessments revealed that overcoming GEM resistance was possibly due to modulation of GEM metabolism pathway molecules by the BMJ administration. While all drug exposures led to a decreased RRM1 expression during treatments in PanC-explants, at the end of treatment washout period explants displayed increased RRM1 levels in GEM-only group but a sustained decrease in BMJ and Combo groups. Investigation into hENT1 (human equilibrative nucleoside transporter 1) expression, responsible for bidirectional GEM trafficking, revealed that at the end of washout period, combination therapy was the only group that continued to express high levels of hENT1 for all three explants, whereas GEM only group showed poor hENT1 expression. Furthermore, analysis for deoxycytidine kinase (dCK), the enzyme responsible for GEM metabolism, revealed that the combination group had the most augmented dCK expression levels by study end. Taken together, the study outcomes highlight the efficacy of BMJ in combination with GEM in modifying and regulating multiple key players in GEM uptake and metabolism. These conclusions make a compelling case for future clinical investigations in PanC patients wherein BMJ is combined with GEM to target and overcome GEM resistance. Citation Format: Deepanshi Dhar, Dileep Kumar, Komal Raina, David J. Orlicky, Chapla Agarwal, Rajesh Agarwal. A combinatorial approach using gemcitabine and bitter melon juice overcomes drug resistance impacting gemcitabine uptake and metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2123.

Effects of Xingnaojing Injection on Adenosinergic Transmission and Orexin Signaling in Lateral Hypothalamus of Ethanol-Induced Coma Rats.

Acute alcohol exposure induces unconscious condition such as coma whose main physical manifestation is the loss of righting reflex (LORR). Xingnaojing Injection (XNJI), which came from Chinese classic formula An Gong Niu Huang Pill, is widely used for consciousness disorders in China, such as coma. Although XNJI efficiently shortened the duration of LORR induced by acute ethanol, it remains unknown how XNJI acts on ethanol-induced coma (EIC). We performed experiments to examine the effects of XNJI on orexin and adenosine (AD) signaling in the lateral hypothalamic area (LHA) in EIC rats. Results showed that XNJI reduced the duration of LORR, which implied that XNJI promotes recovery form coma. Microdialysis data indicated that acute ethanol significantly increased AD release in the LHA but had no effect on orexin A levels. The qPCR results displayed a significant reduction in the Orexin-1 receptors (OX1R) expression with a concomitant increase in the A1 receptor (A1R) and equilibrative nucleoside transporter type 1 (ENT1) expression in EIC rats. In contrast, XNJI reduced the extracellular AD levels but orexin A levels remained unaffected. XNJI also counteracted the downregulation of the OX1R expression and upregulation of A1R and ENT1 expression caused by EIC. As for ADK expression, XNJI but not ethanol, displayed an upregulation in the LHA in EIC rats. Based on these results, we suggest that XNJI promotes arousal by inhibiting adenosine neurotransmission via reducing AD level and the expression of A1R and ENT1.

Structures of human ENT1 in complex with adenosine reuptake inhibitors.

The human Equilibrative Nucleoside Transporter 1 (hENT1), a member of the SLC29 family, plays crucial roles in adenosine signaling, cellular uptake of nucleoside for DNA and RNA synthesis, and nucleoside-derived anticancer and antiviral drug transport in human. Because of its central role in adenosine signaling, it is the target of adenosine reuptake inhibitors (AdoRI), several of which are clinically used. Despite its importance in human physiology and pharmacology, the molecular basis of hENT1-mediated adenosine transport and its inhibition by AdoRIs are limited due to the absence of structural information on hENT1. Here we present crystal structures of hENT1 in complex with two chemically distinct AdoRIs: dilazep and S-(4-Nitrobenzyl)-6-thioinosine (NBMPR). Combined with mutagenesis study, our structural analyses elucidate two distinct inhibitory mechanisms exhibited on hENT1, while giving insight into adenosine recognition and transport. Our studies provide the platform for improved pharmacological intervention of adenosine and nucleoside analog drug transport by hENT1.

Mechanisms of gemcitabine oral absorption as determined by in situ intestinal perfusions in mice

Gemcitabine is a widely used chemotherapeutic drug that is administered via intravenous infusion due to a low oral bioavailability of only 10%. This low oral bioavailability is believed to be the result of gemcitabine’s low intestinal permeability and oral absorption, followed by significant presystemic metabolism. In the present study, we sought to define the mechanisms of gemcitabine intestinal permeability, the potential for saturation of intestinal uptake, and the transporter(s) responsible for mediating the oral absorption of drug using in situ single-pass intestinal perfusions in mice. Concentration-dependent studies were performed for gemcitabine over 0.5–2000 μM, along with studies of 5 μM gemcitabine in a sodium-containing buffer ± thymidine (which can inhibit concentrative (i.e., CNT1 and CNT3) and equilibrative (i.e., ENT1 and ENT2) nucleoside transporters) or dilazep (which can inhibit ENT1 and ENT2), or in a sodium-free buffer (which can inhibit CNT1 and CNT3). Our findings demonstrated that gemcitabine was, in fact, a high-permeability drug in the intestine at low concentrations, that jejunal uptake of gemcitabine was saturable and mediated almost exclusively by nucleoside transporters, and that jejunal flux was mediated by both high-affinity, low-capacity (Km = 27.4 µM, Vmax = 3.6 pmol/cm2/s) and low-affinity, high-capacity (Km = 700 µM, Vmax = 35.9 pmol/cm2/s) transport systems. Thus, CNTs and ENTs at the apical membrane allow for gemcitabine uptake from the lumen to enterocyte, whereas ENTs at the basolateral membrane allow for gemcitabine efflux from the enterocyte to portal venous blood.

Prognostic immunohistochemical biomarkers of chemotherapy efficacy in biliary tract cancer: A systematic review and meta-analysis.

Chemotherapy is the mainstay of systemic treatment of biliary tract cancer (BTC). However, the treatment response to chemotherapy varies between patients. Currently, no prognostic biomarkers for chemotherapy efficacy have been considered for use in clinical practice. A systematic review was conducted to evaluate the prognostic value of immunohistochemical biomarkers for chemotherapy in patients with resected as well as with advanced BTC. Medline and EMBASE databases were searched up to March 2017 for studies that evaluated biomarker expression by immunohistochemistry in resected or advanced BTC patients treated with chemotherapy. The primary endpoints were overall survival (OS) and disease or progression free survival (DFS or PFS). Twenty-six studies, including a total of 1348 patients and 26 different biomarkers, met the inclusion criteria and were included in this review. The most frequently studied prognostic biomarkers in BTC were the human Equilibrative Nucleoside Transporter 1 (hENT1), Ribonucleotide Reductase M1 (RRM1), and excision repair cross-complementation 1 (ERCC1). In the meta-analysis of patients treated with gemcitabine-based chemotherapy, high hENT1 expression was associated with longer OS (HR 0.43, 95% CI: 0.28 to 0.64) and DFS/PFS (HR 0.45, 95% CI: 0.33 to 0.61). hENT1 is a promising prognostic biomarker for gemcitabine-based chemotherapy in resected as well as in advanced BTC and should be further validated for the selection of patients for chemotherapy.

HgS and Zuotai differ from HgCl2 and methyl mercury in intestinal Hg absorption, transporter expression and gut microbiome in mice

Mercury (Hg) is generally considered as a toxic metal; yet the biological outcomes of Hg-containing compounds are highly dependent upon their chemical forms. We hypothesize that mercury sulfide (HgS) is different from HgCl2 and methylmercury (MeHg) in producing intestinal Hg absorption and disruption of gut microbiome. To test this hypothesis, mice were given orally with HgS (α-HgS, 30 mg/kg), Zuotai (β-HgS, 30 mg/kg), HgCl2 (33.6 mg/kg, equivalent Hg as HgS), or MeHg (3.1 mg/kg, 1/10 Hg as HgS) for 7 days. Accumulation of Hg in the duodenum and ileum after HgCl2 (30–40 fold) and MeHg (10–15 fold) was higher than HgS and Zuotai (~2-fold). HgCl2 and MeHg decreased intestinal intake peptide transporter-1 and Ost-β, and increased ileal bile acid binding protein and equilibrative nucleoside transporter-1. The efflux transporters ATP-binding cassette sub-family C member-4 (Abcc4), Abcg2, Abcg5/8, and Abcb1b were increased by HgCl2 and to a lesser extent by MeHg, while HgS and Zuotai had minimal effects. Bacterial DNA was extracted and subjected to 16S rDNA sequencing. Operational taxonomic unit (OTU) results showed that among the 10 phyla, HgS increased Firmicutes, Proteobacteria, while HgCl2 increased Bacteroidetes, Cyanobacteria and decreased Firmicutes; among the 79 families, HgS increased Rikenellaceae, Lactobacillaceae, Helicobacteraceae, and decreased Prevotellaceae, while HgCl2 increased Odoribacteraceae, Porphyromonadaceae, and decreased Lactobacillaceae; among the 232 genus/species, HgS and Zuotai affected gut microbiome quite differently from HgCl2 and MeHg. qPCR analysis with 16S rRNA confirmed sequencing results. Thus, chemical forms of mercury are a major determinant for intestinal Hg accumulation, alterations in transporters and disruption of microbiome.

The human equilibrative nucleoside transporter1 evaluated by SP120 and 10D7G2 had the potential to predict the prognosis in the patients with pancreatic cancer treated with adjuvant gemcitabine: Collaborative study of the JASPAC 01 trial

The human equilibrative nucleoside transporter1 evaluated by SP120 and 10D7G2 had the potential to predict the prognosis in the patients with pancreatic cancer treated with adjuvant gemcitabine: Collaborative study of the JASPAC 01 trial

Pathway genes and metabolites in thiopurine therapy in Korean children with acute lymphoblastic leukaemia.

We aimed to investigate the impact of various genetic polymorphisms affecting thiopurine metabolism pathways and toxicity in paediatric acute lymphoblastic leukaemia patients for the first time in Korea. From May 2006 to September 2016, 139 paediatric acute lymphoblastic leukaemia patients treated with combination chemotherapy including 6-mercaptopurine were included in the study. One hundred and twenty-three variants in 43 genes, including TMPT and NUDT15, were screened using targeted genotyping, such as a MassARRAY system, direct sequencing and polymerase chain reaction-restriction fragment length polymorphism methods. Among the polymorphisms screened, 103 polymorphisms of 43 genes were included for further analyses. The genetic polymorphisms in the ABCC4, AHCY, ATIC, FAM8A6P, GART, GNG2, GSTA1, MTHFD1, MTHFR, NUDT15, PACSIN2, TYMS and XDH genes, and an intronic polymorphism between HIVEP2 and AIG1, and TPMT genotype were associated with thiopurine metabolism (P<0.05). Genetic polymorphisms in the ABCC4, ADK, ATIC, GART, GMPS, GSTP1, IMPDH1, ITPA, KCNMA1, MOCOS, MTRR, NUDT15, SLC19A1, SLC28A3, SLC29A1, SLCO1B1, TYMP and XDH genes were associated with thiopurine-related toxicities; neutropenia, hepatotoxicity and treatment interruption (P<0.05). Findings of this study may provide basic knowledge for personalized medicine for thiopurinxe treatment in paediatric acute lymphoblastic leukaemia patients.

SAT-038 Physiology of Mice Lacking All Four Adenosine Receptors

The importance of multiple adenosine receptors (AR) and the role of adenosine signaling in normal physiology, as opposed to danger/damage situations, are unclear. We generated mice (QKO) lacking all four AR, Adora1-/-;Adora2a-/-;Adora2b-/-;Adora3-/-, to enable investigation of the AR-dependence of physiologic processes. The QKO mice bred normally and had similar body temperature regulation as controls, including diurnal variation, fasting-induced torpor, and response to environmental stimuli. Pharmacologic doses of adenosine agonists cause profound hypothermia via each of the four AR. We hypothesized that uptake and phosphorylation of adenosine might also trigger hypothermia by additional mechanisms, such as activation of adenosine monophosphate-activated protein kinase (AMPK). However, adenosine did not cause hypothermia (or bradycardia or hypotension) in QKO mice, indicating that AR-independent, ‘energy depletion’ signals do not cause adenosine-induced hypothermia. AMP-induced hypothermia was attenuated in QKO mice, demonstrating a role for both AR-dependent and AR-independent mechanisms in this process. In contrast, the hypothermias elicited by adenosine kinase inhibition (using A-134974), inosine, and uridine each require AR, as all were abolished in the QKO mice. The proposed mechanism for uridine-induced hypothermia is inhibition of adenosine transport, increasing local extracellular adenosine levels. The physiology of the QKO mice replicates that of the individual knockout mice, without evidence for synergy, suggesting that the actions of the four AR are generally complementary. The relatively mild phenotype of the QKO mice supports the hypothesis that extracellular adenosine has less of a contribution to baseline physiology and is more important as a local signal of stress, damage, and/or danger.

Developmental Changes in the Role of Adenosine Clearance Mechanisms in Modulating the Activity of the preBötzinger Complex Inspiratory Network Under Basal and Hypoxic Conditions

Exposure to low oxygen (hypoxia) evokes a biphasic hypoxic ventilatory response (HVR) characterized by a carotid‐body‐mediated increase in ventilation followed by a centrally‐mediated secondary hypoxic respiratory depression (HRD). The HRD is strongest and life‐threatening in premature infants (e.g., Apnea of Prematurity). Purinergic signaling plays a prominent role in shaping the HVR; hypoxia evokes release of ATP from astrocytes in the preBötzinger Complex (preBötC, key site of inspiratory rhythm generation), which increases breathing and attenuates the HRD. However, ATP is metabolized into adenosine (ADO), which inhibits the preBötC and is implicated in the HRD. Thus, the net effect of ATP in the preBötC during hypoxia is determined by the balance between the actions of ATP and ADO. The objective of this study is to determine during development how mechanisms important in clearing extracellular ADO (ADOe) influence preBötC activity and the HVR. First, we focused on equilibrative nucleoside transporters (ENTs) that passively move ADO across cell membranes down its concentration gradient. We compared the HVR (10 min 10% O2) of WT and ENT1/2 double KO mice (P0–3, P6–8, and P12–14) using whole‐body plethysmography. The HRD of ENT KO mice was significantly greater compared to WT mice at all ages, reflecting greater reductions in breathing frequency and a likely role for ENTs in removing ADOe during hypoxia. We then applied the ENT inhibitor NBMPR (100 μM) to the solution bathing rhythmic, preBötC‐containing medullary slices from neonatal mice to directly assess the influence of ENTs on basal preBötC rhythm. A significant, NBMPR‐mediated, 16% ± 7% (n=5) increase in frequency suggests that ENTs elevate basal ADOe tone, causing a modest reduction in frequency. In contrast, the reduction in inspiratory frequency evoked by injecting ADOe into the preBötC (to simulate a high ADOe load as may occur in hypoxia) was almost 2‐fold greater in slices from ENT1/2 KO compared to WT mice; i.e., under high load, ENTs appear to clear ADOe. We next examined ADO kinase (ADK), an enzyme that converts ADO into AMP, lowering intracellular ADO levels and facilitating clearance of ADOe by ENTs. Inhibition of ADK with ABT‐702 (10 μM) produced an adenosine‐mediated (i.e., the effect was reversed with the A1R antagonist, DPCPX, 100 nM) decrease in the baseline frequency of rhythmic medullary slices that was 16% ± 2% (n=7) at P0–2 and 24% ± 5% (n=8) by P9–11.In summary, these data suggest that under basal conditions ENTs and ADK are important in controlling ADOe tone, and that under conditions of high ADOe load, as produced here via local injection of ADO in vitro or hypoxia in vivo, ENTs and ADK become important in clearing ADOe, which increases ventilation. Finally, the observation in vitro that ADK activity in the preBötC appears to increase during development raises the intriguing possibility that the greater HRD of premature infants is due, at least in part, to an immature system for clearing ADOe.Support or Funding InformationCIHR, NSERC, WCHRI, AIHS, CFI, Alberta Lung AssociationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.