Assembly Characterization of Human Equilibrium Nucleoside Transporter 1 (hENT1) by Inhibitor Probe-Based dSTORM Imaging.

Nucleoside transporters (NTs) play an important role in the metabolism of nucleoside substances and the efficacy of nucleoside drugs. Its spatial information related to biofunctions at the single-molecule level remains unclear, owing to the limitation of the existing labeling methods and traditional imaging methods. Therefore, we synthesize the inhibitor-based fluorescent probe SAENTA-Cy5 and apply direct stochastic optical reconstruction microscopy (dSTORM) to conduct refined observation of human equilibrative nucleoside transporter 1 (hENT1), the most important and famous member of NTs. We first demonstrate the labeling specificity and superiority of SAENTA-Cy5 to the antibody probe. Then, we found different assembly patterns of hENT1 on the apical and basal membranes, which are further investigated to be caused by varying associations of membrane carbohydrates, membrane classical functional domains (lipid rafts), and associated membrane proteins (EpCAM). Our work provides an efficient method for labeling hENT1, which contributes to realize fine observation of NTs. The findings on the assembly features and potential assembly mechanism of hENT1 promote a better understanding of its biofunction, which facilitates further investigations on how NTs work in the metabolism of nucleoside and nucleoside analogues.

Visualization research on ENT1/NIS dual-function gene therapy to reverse drug resistance mediated by MUC1 in GEM-resistant pancreatic cancer.

PURPOSE: To use bifunctional target genes to increase the intracellular transport of gemcitabine (GEM) to reverse chemotherapy resistance and to simultaneously use reporter gene imaging to localize therapeutic genes. The therapeutic effect was evaluated by [18F]FLT PET/CT to visualize the effect of gene therapy. METHODS: A viral gene vector containing the pancreatic cancer-targeting promoter MUC1 for specific transcription of equilibrative nucleoside transporter 1 (ENT1) and NIS (nuclide transport channel) was employed. [125I]NaI uptake tests and [131I]NaI SPECT imaging were performed to verify the function of NIS and the target function of MUC1. The correlation between [18F]FLT uptake and GEM resistance were assessed, and the influence ENT1 and thymidine kinase 1 (TK1) expression on [18F]FLT micro-PET/CT was measured, which provides a theoretical basis for the use of [18F]FLT micro-PET/CT to evaluate the efficacy of gene therapy. RESULTS: First, functions of gene therapy were confirmed: ENT1 reversed the drug resistance of GEM-resistant pancreatic cancer cells by increasing GEM intracellular transport; MUC1 drove NIS target gene expression in pancreatic cancer; and therapeutic genes could be localized using [131I]NaI SPECT reporter gene imaging. Second, the [18F]FLT uptake ratio was affected by drug resistance and GEM treatment. The mechanism underlying this effect was related to ENT1 and TK1. Increased expression of ENT1 inhibited the expression of TK1 after GEM chemotherapy to reduce the uptake of [18F]FLT. Finally, micro-PET/CT indicated that the SUVmax of [18F]FLT could predict survival time. SUVmax exhibited an increasing trend in resistant pancreatic cancer but a trend of inhibition after upregulation of ENT1, which was more significant after GEM treatment. CONCLUSIONS: Bifunctional targeted genes can localize therapeutic genes through reporter gene imaging, reverse the drug resistance of GEM-resistant pancreatic cancer and be visually evaluated through [18F]FLT micro-PET/CT.

Solute Carrier Family 29A1 Mediates In Vitro Resistance to Azacitidine in Acute Myeloid Leukemia Cell Lines.

Azacitidine (AZA) is commonly used hypomethylating agent for higher risk myelodysplastic syndromes and acute myeloid leukemia (AML). Although some patients achieve remission, eventually most patients fail AZA therapy. Comprehensive analysis of intracellular uptake and retention (IUR) of carbon-labeled AZA (14C-AZA), gene expression, transporter pump activity with or without inhibitors, and cytotoxicity in naïve and resistant cell lines provided insight into the mechanism of AZA resistance. AML cell lines were exposed to increasing concentrations of AZA to create resistant clones. 14C-AZA IUR was significantly lower in MOLM-13- (1.65 ± 0.08 ng vs. 5.79 ± 0.18 ng; p < 0.0001) and SKM-1- (1.10 ± 0.08 vs. 5.08 ± 0.26 ng; p < 0.0001) resistant cells compared to respective parental cells. Importantly, 14C-AZA IUR progressively reduced with downregulation of SLC29A1 expression in MOLM-13- and SKM-1-resistant cells. Furthermore, nitrobenzyl mercaptopurine riboside, an SLC29A inhibitor, reduced 14C-AZA IUR in MOLM-13 (5.79 ± 0.18 vs. 2.07 ± 0.23, p < 0.0001) and SKM-1-naive cells (5.08 ± 2.59 vs. 1.39 ± 0.19, p = 0.0002) and reduced efficacy of AZA. As the expression of cellular efflux pumps such as ABCB1 and ABCG2 did not change in AZA-resistant cells, they are unlikely contribute to AZA resistance. Therefore, the current study provides a causal link between in vitro AZA resistance and downregulation of cellular influx transporter SLC29A1.

Equilibrative Nucleoside Transporter 1 is a Target to Modulate Neuroinflammation and Improve Functional Recovery in Mice with Spinal Cord Injury.

Neuroinflammation plays a critical role in the neurological recovery of spinal cord injury (SCI). Adenosine can modulate neuroinflammation, whose uptake is mediated by nucleoside transporters. This study aimed to investigate the roles of equilibrative nucleoside transporter 1 (Ent1) in the inflammatory responses and functional recovery of SCI. Spinal cord contusion at the T10 dorsal portion was induced in mice to cause partial paralysis of the hindlimbs. Genetic deletion and pharmacological inhibition of Ent1 were used to evaluate the role of Ent1 in SCI. The outcomes were evaluated in terms of the Basso Mouse Scale (BMS), gait analysis, astrogliosis, microgliosis, and cytokine levels on day 14 post-injury. As a result, Ent1 deletion reduced neuroinflammation and improved the BMS score (4.88 ± 0.35 in Ent1-/- vs. 3.78 ± 1.09 in Ent1+/+) and stride length (3.74 ± 0.48 cm in Ent1-/- vs. 2.82 ± 0.78 cm in Ent1+/+) of mice with SCI. Along with the reduced lesion size, more preserved neurons were identified in the perilesional area of mice with Ent1 deletion (102 ± 23 in Ent1-/- vs. 73 ± 10 in Ent1+/+). The results of pharmacological inhibition were consistent with the findings of genetic deletion. Moreover, Ent1 inhibition decreased the protein level of complement 3 (an A1 marker), but increased the levels of S100 calcium-binding protein a10 (an A2 marker) and transforming growth factor-ß, without changing the levels of inducible nitric oxide synthase (a M1 marker) and arginase 1 (a M2 marker) at the injured site. These findings indicate the important role of Ent1 in the pathogenesis and treatment of SCI.

Erythrocyte type 1 equilibrative nucleoside transporter expression in sickle cell disease and sickle cell trait.

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.

hENT1 Expression Predicts Response to Gemcitabine and Nab-Paclitaxel in Advanced Pancreatic Ductal Adenocarcinoma.

PURPOSE: Modified FOLFIRINOX (mFFX) and gemcitabine/nab-paclitaxel (GnP) remain standard first-line options for patients with advanced pancreatic ductal adenocarcinoma (PDAC). Human equilibrative nucleoside transporter 1 (hENT1) was hypothesized to be a biomarker of gemcitabine in the adjuvant setting, with conflicting results. In this study, we explore hENT1 mRNA expression as a predictive biomarker in advanced PDAC. EXPERIMENTAL DESIGN: COMPASS was a prospective observational trial of patients with advanced PDAC. A biopsy was required prior to initiating chemotherapy, as determined by treating physician. Biopsies underwent laser capture microdissection prior to whole genome and RNA sequencing. The cut-off thresholds for hENT1 expression were determined using the maximal χ2 statistic. RESULTS: 253 patients were included in the analyses with a median follow-up of 32 months, with 138 patients receiving mFFX and 92 receiving GnP. In the intention to treat population, median overall survival (OS) was 10.0 months in hENT1high versus 7.9 months in hENT1low (P = 0.02). In patients receiving mFFX, there was no difference in overall response rate (ORR; 35% vs. 28%, P = 0.56) or median OS (10.6 vs. 10.5 months, P = 0.45). However, in patients treated with GnP, the ORR was significantly higher in hENT1high compared with hENT1low tumors (43% vs. 21%, P = 0.038). Median OS in this GnP-treated cohort was 10.6 months in hENT1high versus 6.7 months hENT1low (P < 0.001). In an interaction analysis, hENT1 was predictive of treatment response to GnP (interaction P = 0.002). CONCLUSIONS: In advanced PDAC, hENT1 mRNA expression predicts ORR and OS in patients receiving GnP.

Adenosine derivatives from Cordyceps exert antitumor effects against ovarian cancer cells through ENT1-mediated transport, induction of AMPK signaling, and consequent autophagic cell death.

Cordyceps militaris is rich in adenosine derivatives, including 3'-deoxyadenosine, also known as cordycepin. It has been reported for antitumor effects, but its underlying molecular mechanism has yet to be elucidated. We investigated how adenosine derivatives exerted antitumor effects against ovarian cancer using human ovarian cancer cells and a xenograft mouse model. Treatment with adenosine derivatives effectively resulted in cell death of ovarian cancer cells through AMPK activation and subsequently mTOR-mediated autophagic induction. Intriguingly, the effect required membrane transport of adenosine derivatives via ENT1, rather than ADORA-mediated cellular signaling. Our data suggest that adenosine derivatives may be an effective therapeutic intervention in ovarian cancer through induction of ENT1-AMPK-mTOR-mediated autophagic cell death.

Apoptotic brown adipocytes enhance energy expenditure via extracellular inosine.

Brown adipose tissue (BAT) dissipates energy1,2 and promotes cardiometabolic health3. Loss of BAT during obesity and ageing is a principal hurdle for BAT-centred obesity therapies, but not much is known about BAT apoptosis. Here, untargeted metabolomics demonstrated that apoptotic brown adipocytes release a specific pattern of metabolites with purine metabolites being highly enriched. This apoptotic secretome enhances expression of the thermogenic programme in healthy adipocytes. This effect is mediated by the purine inosine that stimulates energy expenditure in brown adipocytes by the cyclic adenosine monophosphate-protein kinase A signalling pathway. Treatment of mice with inosine increased BAT-dependent energy expenditure and induced 'browning' of white adipose tissue. Mechanistically, the equilibrative nucleoside transporter 1 (ENT1, SLC29A1) regulates inosine levels in BAT: ENT1-deficiency increases extracellular inosine levels and consequently enhances thermogenic adipocyte differentiation. In mice, pharmacological inhibition of ENT1 as well as global and adipose-specific ablation enhanced BAT activity and counteracted diet-induced obesity, respectively. In human brown adipocytes, knockdown or blockade of ENT1 increased extracellular inosine, which enhanced thermogenic capacity. Conversely, high ENT1 levels correlated with lower expression of the thermogenic marker UCP1 in human adipose tissues. Finally, the Ile216Thr loss of function mutation in human ENT1 was associated with significantly lower body mass index and 59% lower odds of obesity for individuals carrying the Thr variant. Our data identify inosine as a metabolite released during apoptosis with a 'replace me' signalling function that regulates thermogenic fat and counteracts obesity.

Dopamine Release in Nucleus Accumbens Is under Tonic Inhibition by Adenosine A1 Receptors Regulated by Astrocytic ENT1 and Dysregulated by Ethanol.

Striatal adenosine A1 receptor (A1R) activation can inhibit dopamine release. A1Rs on other striatal neurons are activated by an adenosine tone that is limited by equilibrative nucleoside transporter 1 (ENT1) that is enriched on astrocytes and is ethanol sensitive. We explored whether dopamine release in nucleus accumbens core is under tonic inhibition by A1Rs, and is regulated by astrocytic ENT1 and ethanol. In ex vivo striatal slices from male and female mice, A1R agonists inhibited dopamine release evoked electrically or optogenetically and detected using fast-scan cyclic voltammetry, most strongly for lower stimulation frequencies and pulse numbers, thereby enhancing the activity-dependent contrast of dopamine release. Conversely, A1R antagonists reduced activity-dependent contrast but enhanced evoked dopamine release levels, even for single optogenetic pulses indicating an underlying tonic inhibition. The ENT1 inhibitor nitrobenzylthioinosine reduced dopamine release and promoted A1R-mediated inhibition, and, conversely, virally mediated astrocytic overexpression of ENT1 enhanced dopamine release and relieved A1R-mediated inhibition. By imaging the genetically encoded fluorescent adenosine sensor [GPCR-activation based (GRAB)-Ado], we identified a striatal extracellular adenosine tone that was elevated by the ENT1 inhibitor and sensitive to gliotoxin fluorocitrate. Finally, we identified that ethanol (50 mm) promoted A1R-mediated inhibition of dopamine release, through diminishing adenosine uptake via ENT1. Together, these data reveal that dopamine output dynamics are gated by a striatal adenosine tone, limiting amplitude but promoting contrast, regulated by ENT1, and promoted by ethanol. These data add to the diverse mechanisms through which ethanol modulates striatal dopamine, and to emerging datasets supporting astrocytic transporters as important regulators of striatal function.SIGNIFICANCE STATEMENT Dopamine axons in the mammalian striatum are emerging as strategic sites where neuromodulators can powerfully influence dopamine output in health and disease. We found that ambient levels of the neuromodulator adenosine tonically inhibit dopamine release in nucleus accumbens core via adenosine A1 receptors (A1Rs), to a variable level that promotes the contrast in dopamine signals released by different frequencies of activity. We reveal that the equilibrative nucleoside transporter 1 (ENT1) on astrocytes limits this tonic inhibition, and that ethanol promotes it by diminishing adenosine uptake via ENT1. These findings support the hypotheses that A1Rs on dopamine axons inhibit dopamine release and, furthermore, that astrocytes perform important roles in setting the level of striatal dopamine output, in health and disease.

Cladribine as a Potential Object of Nucleoside Transporter-Based Drug Interactions.

Cladribine is a nucleoside analog that is phosphorylated in its target cells (B and T-lymphocytes) to its active triphosphate form (2-chlorodeoxyadenosine triphosphate). Cladribine tablets 10 mg (Mavenclad®), administered for up to 10 days per year in 2 consecutive years (3.5-mg/kg cumulative dose over 2 years), are used to treat patients with relapsing multiple sclerosis. Cladribine has been shown to be a substrate of various nucleoside transporters (NTs). Intestinal absorption and distribution of cladribine throughout the body appear to be essentially mediated by equilibrative NTs (ENTs) and concentrative NTs (CNTs), specifically by ENT1, ENT2, ENT4, CNT2 (low affinity), and CNT3. Other efficient transporters of cladribine are the ABC efflux transporters, specifically breast cancer resistance protein, which likely modulates the oral absorption and renal excretion of cladribine. A key transporter for the intracellular uptake of cladribine into B and T-lymphocytes is ENT1 with ancillary contributions of ENT2 and CNT2. Transporter-based drug interactions affecting absorption and target cellular uptake of a prodrug such as cladribine are likely to reduce systemic bioavailability and target cell exposure, thereby possibly hampering clinical efficacy. In order to manage optimized therapy, i.e., to ensure uncompromised target cell uptake to preserve the full therapeutic potential of cladribine, it is important that clinicians are aware of the existence of NT-inhibiting medicinal products, various lifestyle drugs, and food components. This article reviews the existing knowledge on inhibitors of NT, which may alter cladribine absorption, distribution, and uptake into target cells, thereby summarizing the existing knowledge on optimized methods of administration and concomitant drugs that should be avoided during cladribine treatment.

Role of human nucleoside transporters in pancreatic cancer and chemoresistance.

The prognosis of pancreatic cancer is poor with the overall 5-year survival rate of less than 5% changing minimally over the past decades and future projections predicting it developing into the second leading cause of cancer related mortality within the next decade. Investigations into the mechanisms of pancreatic cancer development, progression and acquired chemoresistance have been constant for the past few decades, thus resulting in the identification of human nucleoside transporters and factors affecting cytotoxic uptake via said transporters. This review summaries the aberrant expression and role of human nucleoside transports in pancreatic cancer, more specifically human equilibrative nucleoside transporter 1/2 (hENT1, hENT2), and human concentrative nucleoside transporter 1/3 (hCNT1, hCNT3), while briefly discussing the connection and importance between these nucleoside transporters and mucins that have also been identified as being aberrantly expressed in pancreatic cancer. The review also discusses the incidence, current diagnostic techniques as well as the current therapeutic treatments for pancreatic cancer. Furthermore, we address the importance of chemoresistance in nucleoside analogue drugs, in particular, gemcitabine and we discuss prospective therapeutic treatments and strategies for overcoming acquired chemoresistance in pancreatic cancer by the enhancement of human nucleoside transporters as well as the potential targeting of mucins using a combination of mucolytic compounds with cytotoxic agents.

NBTI attenuates neuroinflammation and apoptosis partly by ENT1/NLRP3/Bcl2 pathway after subarachnoid hemorrhage in rats.

OBJECTIVES: Neuroinflammation and apoptosis are two key factors contributing to early brain injury (EBI) after subarachnoid hemorrhage (SAH) and are strongly associated with a poor prognosis. Recently, equilibrative nucleoside transporter 1 (ENT1) was emerged to accelerate the severity of inflammation and cell apoptosis in several nervous system diseases, including cerebral ischemia, neurodegeneration and epilepsy. However, no study has yet elaborated the expression levels and effects of ENT1 in EBI after SAH. METHODS: Sprague-Dawley rats were subjected to SAH by endovascular perforation. Nitrobenzylthioinosine (NBTI) was intranasally administered at 0.5 h after SAH. The protein expression levels of ENT1, NLRP3, Bcl2, Bax, ACS, Caspase-1, IL-1 were detected by western blot. The modified Garcia score and beam balance score were employed to evaluate the neurologic function of rats following SAH. In addition, hematoxylin-eosin, fluoro-jade C and TdT-mediated dUTP nick-end labeling staining were then used to evaluate brain tissue damage and neuronal apoptosis. RESULTS: Analysis indicated that endogenous levels of ENT1 were significantly upregulated at 24-hour post-SAH, accompanied by NLRP3 inflammasome activation and Bcl2 decline. The administration of NBTI, an inhibitor of ENT1, at a dose of 15 mg/kg, ameliorated neurologic deficits and morphologic lesions at both 24 and 72 h after SAH. Moreover, ENT1 inhibition efficiently mitigated neuronal degeneration and cell apoptosis. In addition, NBTI at 15 mg/kg observably increased Bcl2 content and decreased Bax level. Furthermore, suppression of ENT1 notably reduced the expression levels of NLRP3, apoptosis associated speck like protein containing CARD, caspase-1 and IL-1ß. CONCLUSIONS: NBTI relieved SAH-induced EBI partly through ENT1/NLRP3/Bcl2 pathway.

Remdesivir and EIDD-1931 Interact with Human Equilibrative Nucleoside Transporters 1 and 2: Implications for Reaching SARS-CoV-2 Viral Sanctuary Sites.

Equilibrative nucleoside transporters (ENTs) are present at the blood-testis barrier (BTB), where they can facilitate antiviral drug disposition to eliminate a sanctuary site for viruses detectable in semen. The purpose of this study was to investigate ENT-drug interactions with three nucleoside analogs, remdesivir, molnupiravir, and molnupiravir's active metabolite, ß-d-N4-hydroxycytidine (EIDD-1931), and four non-nucleoside molecules repurposed as antivirals for coronavirus disease 2019 (COVID-19). The study used three-dimensional pharmacophores for ENT1 and ENT2 substrates and inhibitors and Bayesian machine learning models to identify potential interactions with these transporters. In vitro transport experiments demonstrated that remdesivir was the most potent inhibitor of ENT-mediated [3H]uridine uptake (ENT1 IC50: 39 µM; ENT2 IC50: 77 µM), followed by EIDD-1931 (ENT1 IC50: 259 µM; ENT2 IC50: 467 µM), whereas molnupiravir was a modest inhibitor (ENT1 IC50: 701 µM; ENT2 IC50: 851 µM). Other proposed antivirals failed to inhibit ENT-mediated [3H]uridine uptake below 1 mM. Remdesivir accumulation decreased in the presence of 6-S-[(4-nitrophenyl)methyl]-6-thioinosine (NBMPR) by 30% in ENT1 cells (P = 0.0248) and 27% in ENT2 cells (P = 0.0054). EIDD-1931 accumulation decreased in the presence of NBMPR by 77% in ENT1 cells (P = 0.0463) and by 64% in ENT2 cells (P = 0.0132), which supported computational predictions that both are ENT substrates that may be important for efficacy against COVID-19. NBMPR failed to decrease molnupiravir uptake, suggesting that ENT interaction is likely inhibitory. Our combined computational and in vitro data can be used to identify additional ENT-drug interactions to improve our understanding of drugs that can circumvent the BTB. SIGNIFICANCE STATEMENT: This study identified remdesivir and EIDD-1931 as substrates of equilibrative nucleoside transporters 1 and 2. This provides a potential mechanism for uptake of these drugs into cells and may be important for antiviral potential in the testes and other tissues expressing these transporters.

Functional expression of equilibrative and concentrative nucleoside transporters in alveolar epithelial cells.

Equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters (CNTs) mediate the cellular uptake of nucleosides and nucleobases across the plasma membrane and play important roles in the salvage pathways of nucleotide synthesis. However, information about nucleoside transport systems in the lung alveolar epithelial cells is limited. Therefore, in the present study, we examined the function and expression of nucleoside transporters using primary cultured alveolar type II cells and transdifferentiated type I-like cells. The uptake of uridine, a substrate for ENTs and CNTs, in type II and type I-like cells was time, temperature, and concentration dependent, and was inhibited by other nucleoside transporter substrates such as adenosine. Uridine uptake in both cells was insensitive to nanomolar concentrations of NBMPR, a potent ENT1 inhibitor, while it was inhibited by higher concentrations of NBMPR, suggesting that ENT2, but not ENT1, is involved in uridine uptake in these cells. Additionally, uridine uptake was higher in the presence of Na+ than in the absence of Na + and was partially inhibited by a CNT inhibitor phloridzin in these cells, suggesting that CNT is also involved in uridine uptake. In both cells, the mRNA expression of ENT1, ENT2, CNT2, and CNT3 was observed. Finally, the activity of uridine uptake was considerably higher in type II cells than in type I-like cells. In addition, the mRNA expression of ENT2, CNT2, and CNT3, but not ENT1, was lower in type I-like cells than in type II cells. These findings would help understand the functional roles of equilibrative and concentrative nucleoside transporters in alveolar epithelial cells.

Export of RNA-derived modified nucleosides by equilibrative nucleoside transporters defines the magnitude of autophagy response and Zika virus replication.

RNA contains a wide variety of posttranscriptional modifications covalently attached to its base or sugar group. These modified nucleosides are liberated from RNA molecules as the consequence of RNA catabolism and released into extracellular space, but the molecular mechanism of extracellular transport and its pathophysiological implications have been unclear. In the present study, we discovered that RNA-derived modified nucleosides are exported to extracellular space through equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2), with ENT1 showing higher preference for modified nucleosides than ENT2. Pharmacological inhibition or genetic deletion of ENT1 and ENT2 significantly attenuated export of modified nucleosides thereby resulting in their accumulation in cytosol. Using mutagenesis strategy, we identified an amino acid residue in ENT1 that is involved in the discrimination of unmodified and modified nucleosides. In ENTs-deficient cells, the elevated levels of intracellular modified nucleosides were closely associated with an induction of autophagy response as evidenced by increased LC3-II level. Importantly, we performed a screening of modified nucleosides capable of inducing autophagy and found that 1-methylguanosine (m1G) was sufficient to induce LC3-II levels. Pathophysiologically, defective export of modified nucleosides drastically induced Zika virus replication in an autophagy-dependent manner. In addition, we also found that pharmacological inhibition of ENTs by dilazep significantly induced Zika virus replication. Collectively, our findings highlight RNA-derived modified nucleosides as important signaling modulators that activate autophagy response and indicate that defective export of these modified nucleoside can have profound consequences for pathophysiology.

Targeting Pin1 renders pancreatic cancer eradicable by synergizing with immunochemotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by notorious resistance to current therapies attributed to inherent tumor heterogeneity and highly desmoplastic and immunosuppressive tumor microenvironment (TME). Unique proline isomerase Pin1 regulates multiple cancer pathways, but its role in the TME and cancer immunotherapy is unknown. Here, we find that Pin1 is overexpressed both in cancer cells and cancer-associated fibroblasts (CAFs) and correlates with poor survival in PDAC patients. Targeting Pin1 using clinically available drugs induces complete elimination or sustained remissions of aggressive PDAC by synergizing with anti-PD-1 and gemcitabine in diverse model systems. Mechanistically, Pin1 drives the desmoplastic and immunosuppressive TME by acting on CAFs and induces lysosomal degradation of the PD-1 ligand PD-L1 and the gemcitabine transporter ENT1 in cancer cells, besides activating multiple cancer pathways. Thus, Pin1 inhibition simultaneously blocks multiple cancer pathways, disrupts the desmoplastic and immunosuppressive TME, and upregulates PD-L1 and ENT1, rendering PDAC eradicable by immunochemotherapy.

A review of the effects of ticagrelor on adenosine concentration and its clinical significance.

BACKGROUND: Ticagrelor is an oral antiplatelet drug that can reversibly bind to the platelet P2Y12 receptor. Ticagrelor is metabolized mainly by CYP3A4 and produces a rapid blood concentration-dependent platelet inhibitory effect. Unlike other P2Y12 receptor antagonists, many clinical features of ticagrelor are not related to P2Y12 receptor antagonism. PURPOSE: This review aims to gather existing literature on the clinical effects of ticagrelor after inhibiting adenosine uptake. METHODOLOGY: The current study reviewed literature related to the effects of ticagrelor on adenosine metabolism. The review also examined the drug's biological effects and clinical characteristics to see how it could be used in a clinical setting. RESULTS: Many studies have shown that ticagrelor can inhibit equilibrative nucleoside transporter 1 (ENT1). This inhibition leads to intracellular adenosine uptake, increased adenosine half-life and plasma concentration levels and an enhanced adenosine-mediated biological effect. CONCLUSIONS: Based on the studies reviewed, it was found that ticagrelor essentially inhibits adenosine absorption of adenosine into cells through ENT1, which increases the concentration in the blood and subsequently increases the protection of the heart muscle by adenosine. It also prevents platelet aggregation, and extends the biological effects of coronary arteries. Moreover, it leads to a lower mortality rate in acute coronary syndrome (ACS) patients.

Testicular disposition of clofarabine in rats is dependent on equilibrative nucleoside transporters.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents. Although the 5-year survival rate is high, some patients respond poorly to chemotherapy or have recurrence in locations such as the testis. The blood-testis barrier (BTB) can prevent complete eradication by limiting chemotherapeutic access and lead to testicular relapse unless a chemotherapeutic is a substrate of drug transporters present at this barrier. Equilibrative nucleoside transporter (ENT) 1 and ENT2 facilitate the movement of substrates across the BTB. Clofarabine is a nucleoside analog used to treat relapsed or refractory ALL. This study investigated the role of ENTs in the testicular disposition of clofarabine. Pharmacological inhibition of the ENTs by 6-nitrobenzylthioinosine (NBMPR) was used to determine ENT contribution to clofarabine transport in primary rat Sertoli cells, in human Sertoli cells, and across the rat BTB. The presence of NBMPR decreased clofarabine uptake by 40% in primary rat Sertoli cells (p = .0329) and by 53% in a human Sertoli cell line (p = .0899). Rats treated with 10 mg/kg intraperitoneal (IP) injection of the NBMPR prodrug, 6-nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), or vehicle, followed by an intravenous (IV) bolus 10 mg/kg dose of clofarabine, showed a trend toward a lower testis concentration of clofarabine than vehicle (1.81 ± 0.59 vs. 2.65 ± 0.92 ng/mg tissue; p = .1160). This suggests that ENTs could be important for clofarabine disposition. Clofarabine may be capable of crossing the human BTB, and its potential use as a first-line treatment to avoid testicular relapse should be considered.

miR-33-3p Regulates PC12 Cell Proliferation and Differentiation In Vitro by Targeting Slc29a1.

MicroRNA-33-3p (miR-33-3p) has been widely investigated for its roles in lipid metabolism and mitochondrial function; however, there are few studies on miR-33-3p in the context of neurological diseases. In this study, we investigated the functional role of miR-33-3p in rat pheochromocytoma PC12 cells. A miR-33-3p mimic was transduced into PC12 cells, and its effects on proliferation, apoptosis, and differentiation were studied using the MTS assay, EdU labeling, flow cytometry, qRT-PCR, western blot, ELISA, and immunofluorescence. We found that miR-33-3p significantly suppressed PC12 cell proliferation, but had no effect on apoptosis. Furthermore, miR-33-3p promoted the differentiation of PC12 cells into Tuj1-positive and choline acetyltransferase-positive neuron-like cells. Mechanistically, miR-33-3p repressed the expression of Slc29a1 in PC12 cells. Importantly, knocking down Slc29a1 in PC12 cells inhibited proliferation and induced differentiation into neuron-like cells. In conclusion, this study showed that miR-33-3p regulated Slc29a1, which in turn controlled the proliferation and differentiation of PC12 cells. Thus, we hypothesize that the miR-33-3p/Slc29a1 axis could be a promising therapeutic target for recovering neurons and the cholinergic nervous system.

Nucleoside transporter-guided cytarabine-conjugated liposomes for intracellular methotrexate delivery and cooperative choriocarcinoma therapy.

Gestational trophoblastic tumors seriously endanger child productive needs and the health of women in childbearing age. Nanodrug-based therapy mediated by transporters provides a novel strategy for the treatment of trophoblastic tumors. Focusing on the overexpression of human equilibrative nucleoside transporter 1 (ENT1) on the membrane of choriocarcinoma cells (JEG-3), cytarabine (Cy, a substrate of ENT1)-grafted liposomes (Cy-Lipo) were introduced for the targeted delivery of methotrexate (Cy-Lipo@MTX) for choriocarcinoma therapy in this study. ENT1 has a high affinity for Cy-Lipo and can mediate the endocytosis of the designed nanovehicles into JEG-3 cells. The ENT1 protein maintains its transportation function through circulation and regeneration during endocytosis. Therefore, Cy-Lipo-based formulations showed high tumor accumulation and retention in biodistribution studies. More importantly, the designed DSPE-PEG2k-Cy conjugation exhibited a synergistic therapeutic effect on choriocarcinoma. Finally, Cy-Lipo@MTX exerted an extremely powerful anti-choriocarcinoma effect with fewer side effects. This study suggests that the overexpressed ENT1 on choriocarcinoma cells holds great potential as a high-efficiency target for the rational design of active targeting nanotherapeutics.