Nucleoside Transporter Research Articles

Assessment of uric acid-lowering activity, safety and stress tolerance of Lactiplantibacillus plantarum MC14 based on whole gene sequencing and phenotyping experiments

With lifestyle changes, hyperuricemia has become a global epidemic. Recent studies have indicated that some probiotic strains may help control serum uric acid. This study aimed to screen probiotic strains with uric acid-lowering potential and subsequently analyze the safety and stress tolerance of the strain in food applications, utilizing phenotyping experiments and genome sequencing. Initially, we isolated a strain of Lactiplantibacillus plantarum MC14 with the best in vitro uric acid-lowering activity. The inosine and guanosine degradation rates of MC14 were 5.00 and 4.11 mg/L·min, respectively, and the XOD inhibition rate was 81.56% ± 3.87%. Gene analysis revealed that the MC14 strain encodes three nucleoside hydrolases and six nucleoside and nucleobase transporter proteins. Subsequently, safety assessments of the MC14 strain in food applications revealed no nitroreductase, amino decarboxylase, or tryptophanase activities, absence of hemolysis, and no antibiotic resistance gene transfer risk. Finally, stress tolerance tests on MC14 indicated robust tolerance to gastrointestinal fluids and bile salts, with survival rates exceeding 98% after 3 h in artificial gastric fluids and approximately 100.49% ± 0.15% after 8 h in artificial intestinal fluids. Additionally, MC14 demonstrated effective antibacterial properties and antioxidant capacity, and its genome was annotated with numerous stress tolerance-related genes, including 18 acid-base stress, 4 bile salt tolerance, 11 heat stress, 3 cold stress, 6 osmotic stress, and 28 oxidative stress. Our findings highlighted MC14 as a promising candidate for probiotic and food applications.

A review of the Augustine blood group system.

Augustine is a newly identified blood group system comprising four antigens, one of which is the high-frequency antigen Ata in the original "series". Four antigens are located on a multipass membrane glycoprotein equilibrative nucleoside transporter 1 (ENT1), and equilibrative nucleoside transporter is encoded by SLC29A1. In 2016, the International Society of Blood Transfusion (ISBT) recognised Augustine as a blood group system and numbered it as 036. The glycoprotein ENT1 transports nucleotides into cells to participate in the synthesis of DNA and RNA, and this is an important link for chemotherapeutic glycosides to enter tumour cells. Augustine antibodies are clinically relevant in blood transfusion and pregnancy.

Blood-to-Testis Transport of Ribavirin Involves Carrier-Mediated Processes at the Blood–Testis Barrier

Ribavirin, an antiretroviral agent targeting the hepatitis C virus, causes male reproductive toxicity. This study investigated the mechanism of ribavirin transport at the blood–testis barrier (BTB). In vivo mouse integration plot analysis after intravenous administration revealed that the net influx clearance of [3H]ribavirin in the testis was 3.6-fold greater than that of [14C]D-mannitol, a paracellular transport marker, implying transcellular transport of ribavirin across the BTB. Moreover, [3H]ribavirin uptake by TM4 cells, mouse-derived Sertoli cells, was time- and concentration-dependent, with a Km value of 2.49 mM. S-[(4-nitrophenyl)methyl]-6-thioinosine, an inhibitor of Na+-independent equilibrative nucleoside transporters (ENTs), strongly inhibited the [3H]ribavirin uptake by TM4 cells at 100 µM. Compared to the uptake of [3H]adenosine, a typical endogenous nucleoside, [3H]ribavirin uptake was relatively similar to ENT2 transport. [3H]Ribavirin uptake was also observed in mouse ENT2-expressing Xenopus laevis oocytes, and gene silencing via the transfection of ENT2 small interfering RNA significantly reduced the [3H]ribavirin transport into TM4 cells by 13%. Taken together, these results suggest that ENT2 partially contributes to ribavirin transport at the BTB.

Effects of nucleoside analogues, lipophilic prodrugs and elaidic acids on core signaling pathways in cancer cells

Objectives Nucleoside analogs such as gemcitabine (GEM; dFdC) and cytarabine (Ara-C) require nucleoside transporters to enter cells, and deficiency in equilibrative nucleoside transporter 1 (ENT1) can lead to resistance to these drugs. To facilitate transport-independent uptake, prodrugs with a fatty acid chain attached to the 5’-position of the ribose group of gemcitabine or cytarabine were developed (CP-4126 and CP-4055, respectively). As antimetabolites can activate cellular survival pathways, we investigated whether the prodrugs or their side-chains had similar or decreased effects. Methods Two cell lines A549 (non-small cell lung cancer) and WiDr (colon cancer cells) were exposed for 2-24hr to IC50 concentrations of GEM, Ara-C, CP-4126, CP4055 and elaidic acid (EA) concentrations corresponding to the CP-4126 and CP-4055 IC50. Cells were harvested and analyzed for proteins in cell survival pathways (p-AKT/AKT, p-ERK/ERK, p-P38/P38, GSK-3β/pGSK-3β) by using Western Blotting. Results All drugs and their derivatives showed time- and cell-line-dependent effects. In A549 cells, GEM, CP-4126 and EA-4126 decreased the p-AKT/AKT ratio at 2 and 24 hr. For the p-ERK/ERK ratio, GEM, EA-4126, Ara-C, CP-4045 and EA-4055 exposure led to an increase after 6 hr in A549 cells. Interestingly, Ara-C, CP-4055 and EA-4055 decreased p-ERK/ERK ratio in WiDr cells after 4 hr. In A549 cells, the p-GSK-3β/GSK-3β ratio decreased after exposure to Ara-C and CP-4055 but in WiDr cells increased after 24 hr. In A549 cells treatment with Ara-C, CP-4055 and EA-4126 decreased the p-P38/P38 after 6 hr. Conclusions The findings suggest that both parent drugs, prodrugs, and the EA chain influence cell survival and signaling pathways.

MSphere of Influence: Deciphering purine auxotrophy in protozoan parasites.

Bruno Martorelli Di Genova works in parasitology, focusing on Toxoplasma gondii metabolism. In this mSphere of Influence article, he reflects on how the articles "Metabolic Reprogramming during Purine Stress in the Protozoan Pathogen Leishmania donovani" and "Yeast-Based High-Throughput Screen Identifies Plasmodium falciparum Equilibrative Nucleoside Transporter 1 Inhibitors That Kill Malaria Parasites" impacted him, informing his research strategies and understanding of metabolic flexibility in Toxoplasma gondii.

Abstract 5865: Characterization and resistance mechanisms of PNP inhibitor forodesin-resistant cell lines

Abstract Background: The enzyme purine nucleoside phosphorylase (PNP) is involved in the purine salvage pathway and is present at high levels in lymphoid tissues. Children born with PNP deficiency are known to have significantly reduced T-cell counts but relatively normal B-cell immunity, suggesting PNP as one of the most promising therapeutic targets for T-cell malignancies. Forodesine (For, BCX-1777), a novel PNP inhibitor, was approved in Japan for the treatment of relapsed/refractory PTCL. To understand the cytotoxic effect of For and its mechanism of action, we established a For-resistant (For-R) leukemia cell line and explored the mechanism of resistance. Methods: We established two For-R T-lymphoblastic leukemia cell lines (CCRF-CEM and MOLT4) through repeated escalating exposure to For and 2’-deoxyguanosined(dGuo). Gene expression analysis was performed to identify the mechanisms of resistance. Results: For-R cells showed very high resistance compared to parental cells (IC50 value, CEM/CEM-R 4.2nM/>5x105nM, MOLT4/MOLT4-R 8nM/>5x105nM). When cross-resistance was examined in these For-R cells showed mild resistance to cytarabine. The expression of hENT1, a nucleoside transporter, was examined and found to be upregulated in both CEM-R and MOLT4-R cells, while the expression level of deoxycytidine kinase (dCK) was unchanged in both cells. Next, high performance liquid chromatography was used to assess the intracellular dGTP in each parental and its For-R cells. In the parental cells, intracellular dGTP levels increased promptly 4-6 hours after fosteredin administration, whereas in resistant cells, intracellular dGTP levels were higher than before fosteredin administration, and there was little change in dGTP levels after administration. Conclusion: The established For-R cell line acquired a high resistance to For and showed high intracellular dGTP levels. Imbalance in intracellular GTP levels may be involved in the induction of cell death by For. IC50 value of each cell lines Citation Format: Naoko Hosono, Kana Oiwa, Toshiki Tasaki, Rie Nishi, Luigi Scott, Owen A. O’Connor, Takahiro Yamauchi. Characterization and resistance mechanisms of PNP inhibitor forodesin-resistant cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5865.

Abstract 734: Inhibition of equilibrative nucleoside transporter 1 relieves intracellular adenosine-mediated immune suppression

Abstract The profound benefit of immune checkpoint blockade for cancer therapy is restricted to limited subsets of patients with specific cancers. Many factors contribute to primary and acquired resistance to immune checkpoint blockade, including local accumulation of immunosuppressive metabolites such as the nucleoside adenosine and downstream adenosine receptor signaling. Pharmacological inhibition of adenosine generation and signaling are active areas of clinical investigation. Here we report a novel mechanism whereby adenosine suppresses anti-cancer immune responses by intracellular accumulation. We show that human T cells readily take up adenosine via equilibrative nucleoside transporter 1 (ENT1), suppressing proliferation and effector function by inhibition of de novo pyrimidine nucleotide synthesis in T cells. ENT1 expression increases upon T cell activation, suggesting uptake of extracellular adenosine is a mechanism to limit T cell responses in high adenosine environments. Quantitative mass spectrometry imaging reveals adenosine concentrations in human tumors up to the range of 100 µM - levels significantly greater than previously demonstrated and consistent with suppression of T cell responses. Deletion of ENT1 leads to potent control of tumor growth in syngeneic mouse models including KPC, a poorly immunogenic model of pancreatic ductal adenocarcinoma, and is associated with increased CD8+ T cell frequency, proliferation and cytokine production within tumors. Furthermore, ENT1 expression was observed by flow cytometry on human tumor-infiltrating CD8+ T cells. We have discovered and characterized EOS301984, a potent ENT1 antagonist that is currently being evaluated in patients with advanced solid tumors. EOS301984 blocks intracellular adenosine transport and restores pyrimidine levels in T cells activated in the presence of adenosine, resulting in enhanced tumor cell killing by memory T cells and increased ex vivo expansion of functional human tumor-infiltrating lymphocytes in adenosine-rich environments. Finally, in a humanized mouse model of triple negative breast cancer resistant to anti-PD-1 blockade (MDA-MB-231), combination of EOS301984 with nivolumab synergistically led to control of tumor growth. Thus, ENT1 inhibition represents a novel approach to augment anti-cancer immune responses through restoring pyrimidine nucleotide synthesis in T cells suppressed by adenosine. Citation Format: Theodore J. Sanders, Christopher S. Nabel, Margreet Brouwer, Annelise L. Hermant, Lucas Chaible, Jean-Philippe Deglasse, Angelique Pabois, Wilfried Cathou, Aurore Smets, Michael Deligny, Joao Marchante, Quentin Dubray, Marie-Claire Lettelier, Chiara Martinoli, Reece Marillier, Olivier De Henau, Yvonne McGrath, Matthew G. Vander Heiden, Erica Houthuys. Inhibition of equilibrative nucleoside transporter 1 relieves intracellular adenosine-mediated immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 734.

Abstract 5801: Targeted delivery of Exatecan to tumors via a novel cell-penetrating, anti-DNA antibody

Abstract The formulation of novel chemotherapeutics capable of precisely targeting and damaging tumor cells, while preserving the integrity of healthy tissues, represents a principal challenge and is recognized as one of the biggest achievements in cancer treatment in the last few decades. Antibody-drug conjugates (ADCs) hold significant potential as an effective cancer treatment, by targeting and delivering cytotoxic drugs exclusively to tumor cells that express distinct antigens associated with malignancy. Due to the necessity for specific surface antigens, therapies with ADCs are limited to specific tumor subtypes. In this study, we show the potential of a lupus-derived, cell-penetrating, anti-DNA antibody for delivering of cytotoxic payload to tumors in vivo. We utilized a fully humanized variant, named as V66, which was chemically linked to the Topoisomerase I inhibitor, Exatecan. The primary mechanism involved in V66 cellular penetration is mediated by the Equilibrative Nucleoside Transporter ENT2. The latter shows elevated expression in a substantial subset of cancer types in comparison to healthy tissues, broadening the range of applications for ADCs in tumor therapy. Using labeled V66, we demonstrated specific tumor delivery in vivo, with minimal impact on healthy tissues. In vitro experiments validated the nuclear localization of V66-Exatecan, where it triggers the activation of the DNA damage repair pathway (DDR). As a consequence of the profound DNA damage, exposure to V66-Exatecan leads to cell death, exhibiting low nanomolar EC50 values across a diverse range of cell lines. Furthermore, V66-Exatecan treatment presents synthetic lethality with BRCA2 deficiency, evidenced by a substantial increase in sensitivity. In vivo efficacy studies conducted in BRCA2 deficient tumors revealed a remarkable effectiveness of V66-Exatecan, leading to almost complete tumor regression and substantial improvement in the probability of survival. In addition to the lack of significant weight changes observed throughout the treatment, further toxicological studies conducted in serum and tissues demonstrated the absence of short- and long-term toxicity, with optimal liver and kidney function and the absence of damage to other tissues. Likewise, bone marrow cellularity remained unaffected. Collectively, these studies indicate that V66-Exatecan achieves targeted treatment of tumor cells, where it induces DNA damage leading to cell death. The in vivo application of V66-Exatecan leads to tumor regression without triggering general toxicity that is otherwise associated with Topoisomerase I inhibitors. This work provides the basis to advance a novel nuclear-penetrating ADC for delivering cytotoxic payloads. Citation Format: Zaira Ianniello, Elias Quijano, Peter M. Glazer. Targeted delivery of Exatecan to tumors via a novel cell-penetrating, anti-DNA antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5801.

Extracellular Purine Metabolism-Potential Target in Multiple Sclerosis.

The purinergic signaling system comprises a complex network of extracellular purines and purine-metabolizing ectoenzymes, nucleotide and nucleoside receptors, ATP release channels, and nucleoside transporters. Because of its immunomodulatory function, this system is critically involved in the pathogenesis of multiple sclerosis (MS) and its best-characterized animal model, experimental autoimmune encephalomyelitis (EAE). MS is a chronic neuroinflammatory demyelinating and neurodegenerative disease with autoimmune etiology and great heterogeneity, mostly affecting young adults and leading to permanent disability. In MS/EAE, alterations were detected in almost all components of the purinergic signaling system in both peripheral immune cells and central nervous system (CNS) glial cells, which play an important role in the pathogenesis of the disease. A decrease in extracellular ATP levels and an increase in its downstream metabolites, particularly adenosine and inosine, were frequently observed at MS, indicating a shift in metabolism toward an anti-inflammatory environment. Accordingly, upregulation of the major ectonucleotidase tandem CD39/CD73 was detected in the blood cells and CNS of relapsing-remitting MS patients. Based on the postulated role of A2A receptors in the transition from acute to chronic neuroinflammation, the association of variants of the adenosine deaminase gene with the severity of MS, and the beneficial effects of inosine treatment in EAE, the adenosinergic system emerged as a promising target in neuroinflammation. More recently, several publications have identified ADP-dependent P2Y12 receptors and the major extracellular ADP producing enzyme nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) as novel potential targets in MS.

Antiviral drug recognition and elevator-type transport motions of CNT3.

Nucleoside analogs have broad clinical utility as antiviral drugs. Key to their systemic distribution and cellular entry are human nucleoside transporters. Here, we establish that the human concentrative nucleoside transporter 3 (CNT3) interacts with antiviral drugs used in the treatment of coronavirus infections. We report high-resolution single-particle cryo-electron microscopy structures of bovine CNT3 complexed with antiviral nucleosides N4-hydroxycytidine, PSI-6206, GS-441524 and ribavirin, all in inward-facing states. Notably, we found that the orally bioavailable antiviral molnupiravir arrests CNT3 in four distinct conformations, allowing us to capture cryo-electron microscopy structures of drug-loaded outward-facing and drug-loaded intermediate states. Our studies uncover the conformational trajectory of CNT3 during membrane transport of a nucleoside analog antiviral drug, yield new insights into the role of interactions between the transport and the scaffold domains in elevator-like domain movements during drug translocation, and provide insights into the design of nucleoside analog antiviral prodrugs with improved oral bioavailability.

Alteration in the number, morphology, function, and metabolism of erythrocytes in high-altitude polycythemia.

Introduction: High-altitude polycythemia (HAPC) is a common chronic high-altitude disease characterized by significantly increased erythrocyte, hemoglobin (Hb), and hematocrit values and decreased arterial oxygen saturation. The mechanisms underlying HAPC development are unclear; we aimed to investigate this in an HAPC rat model. Methods: Twelve Sprague-Dawley rats were divided into control and HAPC groups. The HAPC group was exposed to hypobaric hypoxia. This HAPC model was assessed using routine blood tests and blood gas analyses. Bone marrow, peripheral blood reticulocytes (RETs), and peripheral blood erythrocyte apoptosis were measured using flow cytometry. Erythrocyte osmotic fragility (EOF) tests were conducted. Abnormal erythrocytes were counted using electron microscopy. Plasma-free hemoglobin, 5'-nucleotidase (CD73), adenosine, erythrocyte cytosolic adenosine, sphingosine-1-phosphate (S1P), and 2,3-bisphosphoglycerate (BPG) levels were measured using enzyme-linked immunosorbent assays. Erythrocyte metabolic pathway-related protein [adenosine A2B receptor (ADORA2B), erythrocyte equilibrative nucleoside transporter 1 (eENT1), sphingosine kinase 1 (SPHK1), phospho-SPHK1, bisphosphoglycerate mutase (BPGM), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] levels were assessed by Western blotting. Results: The HAPC rat model was successfully established (Hb > 210g/L). Indices of bone marrow and peripheral blood RET proportions were significantly higher in the HAPC than the control group (p = 0.04 and p < 0.001, respectively). The proportion of peripheral blood erythrocytes in early apoptosis was significantly lower in the HAPC than the control group (p < 0.001). Vesicular erythrocyte and acanthocyte proportions were significantly higher in the HAPC than the control group (p < 0.001 and p = 0.019, respectively). The EOF tests revealed that 50% erythrocyte hemolysis occurred at 4.0-4.5 and 4.5-5.0g/L NaCl in the control and HAPC groups, respectively. Plasma-free hemoglobin, CD73, adenosine, erythrocyte cytosolic adenosine, S1P, and 2,3-BPG levels and ADORA2B, eENT1, phospho-SPHK1, S1P, BPGM, and GAPDH erythrocyte expression levels (all p ≤ 0.02) were significantly higher in the HAPC than the control group. Conclusion: In model rats, an HAPC-related erythrocyte increase was associated with enhanced bone marrow hematopoietic function and reduced erythrocyte apoptosis, whereas numerous abnormal erythrocytes, increased EOF, and reduced hemolysis resistance were associated with erythrocyte metabolism. CD73/adenosine/S1P/2,3-BPG and eENT1/adenosine/BPGM/2,3-BPG metabolic pathways in erythrocytes were activated in HAPC rats, facilitating oxygen release. These findings further reveal the intrinsic HAPC mechanism and forms a basis for future development of preventive and therapeutic strategies for HAPC.

Research Progress on hCNT3 Structure/Function and Nucleoside Anticancer Drugs.

Membrane protein human concentrative nucleoside transporter 3 (hCNT3) can not only transport extracellular nucleosides into the cell but also transport various nucleoside-derived anticancer drugs to the focus of infection for therapeutic effects. Typical nucleoside anticancer drugs, including fludarabine, cladabine, decitabine, and clofarabine, are recognized by hCNT3 and then delivered to the lesion site for their therapeutic effects. hCNT3 is highly conserved during the evolution from lower to higher vertebrates, which contains scaffold and transport domains in structure and delivers substrates by coupling with Na+ and H+ ions in function. In the process of substrate delivery, the transport domain rises from the lower side of transmembrane 9 (TM9) in the inward conformation to the upper side of the outward conformation, accompanied by the collaborative motion of TM7b/ TM4b and hairpin 1b (HP1b)/ HP2b. With the report of a series of three-dimensional structures of homologous CNTs, the structural characteristics and biological functions of hCNT3 have attracted increasing attention from pharmacists and biologists. Our research group has also recently designed an anticancer lead compound with high hCNT3 transport potential based on the structure of 5-fluorouracil. In this work, the sequence evolution, conservation, molecular structure, cationic chelation, substrate recognition, elevator motion pattern and nucleoside derivative drugs of hCNT3 were reviewed, and the differences in hCNT3 transport mode and nucleoside anticancer drug modification were summarized, aiming to provide theoretical guidance for the subsequent molecular design of novel anticancer drugs targeting hCNT3.

The prognostic and predictive role of class III β-Tubulin and hENT1 expression in patients with advanced pancreatic ductal adenocarcinoma

BackgroundFOLFIRINOX and gemcitabine-nabpaclitaxel (GnP) are standard first-line treatment regimens for advanced pancreatic ductal adenocarcinoma (PDAC). However, currently, there is a lack of predictive biomarkers to aid in the treatment selection. We aimed to explore the prognostic and predictive value of class III β-Tubulin (TUBB3) and human equilibrative nucleoside transporter 1 (hENT1) expression, which have previously been shown to be associated with taxane and gemcitabine resistance in advanced PDAC. MethodsWe conducted a retrospective analysis of 106 patients with advanced PDAC treated with GnP and/or FOLFIRINOX at our institution. TUBB3 and hENT1 immunohistochemical staining was performed on tumor specimens and subsequently evaluated based on the intensity and percentage of expression. ResultsIn patients who received the GnP regimen, a high combined score (TUBB3low/hENT1high) was associated with a higher DCR and longer PFS compared to those with intermediate (TUBB3high/hENT1high or TUBB3low/hENT1low) and low score (TUBB3high/hENT1low). In the multivariate analysis, a high combined score was an independent predictor of higher DCR (OR:11.96; 95 % CI:2.61–54.82; p = 0.001) and longer PFS (HR:0.33; 95%CI:0.18–0.60; p < 0.001). However, there was no difference in response rates or PFS based on TUBB3 and hENT1 expression among patients receiving the FOLFIRINOX regimen. ConclusionOur findings indicate that tumor TUBB3 and hENT1 expression may predict the efficacy of the GnP regimen, and low TUBB3 and high hENT1 expression (TUBB3low/hENT1high) are associated with a higher DCR and longer PFS in patients treated with GnP. Evaluating TUBB3 and hENT1 jointly can identify the patients most (as well as least) likely to benefit from GnP chemotherapy.

Suppression of Ehrlich ascites tumor cell proliferation via G1 arrest induced by dietary nucleic acid-derived nucleosides.

The nucleic acids found in food play a crucial role in maintaining various bodily functions. This study investigated the potential anticancer effects of dietary nucleic acids, an area that is still not fully understood. By utilizing an in vivo mouse model and an in vitro cell model, we discovered an anti-proliferative impact of RNA in both systems. DNA exhibited anti-proliferative effects in the mouse model, while this phenomenon wasn't observed in the in vitro cell model using Ehrlich ascites tumor (EAT) cells. Conversely, DNA hydrolysate demonstrated distinct anti-proliferative effects in EAT cells, suggesting that nucleotides or nucleosides generated during nucleic acid digestion act as active constituents. Furthermore, we examined various nucleosides and two sodium-independent equilibrative nucleoside transporter inhibitors (ENTs), identifying guanosine and 2'-deoxyguanosine as pivotal in the anti-proliferative effect. We also found that the anti-proliferation activity with both nucleosides was suppressed by the treatment of dipyridamole, a non-selective inhibitor for ENT1 and ENT2, but not nitrobenzylthioinosine, a low inhibitor for ENT2. The uptake of these compounds into cells is likely facilitated by ENT2. These nucleotides impeded the progression of cancer cells from the G1 phase to the S phase in the cell cycle. Another significant finding is the increased expression of CCAAT/enhancer-binding protein (C/EBPβ) induced by guanosine and 2'-deoxyguanosine. Furthermore, immunostaining revealed that C/EBPβ diffuses into the nucleus, indicating its presence. This suggests that guanosine or 2-deoxyguanosine induces G1 arrest in cancer cells via the activation of C/EBPβ. Encouraged by these promising results, guanosine and 2'-deoxyguanosine show potential applications in cancer prevention.

Extracellular Guanosine and Guanine Nucleotides Decrease Viability of Human Breast Cancer SKBR-3 Cells.

Though the physiological effects of adenosine and adenine nucleotides on purinergic receptors in cancer cells have been well studied, the influence of extracellular guanosine and guanine nucleotides on breast cancer cells remains unclear. Here, we show that extracellular guanosine and guanine nucleotides decrease the viability and proliferation of human breast cancer SKBR-3 cells. Treatment with guanosine or guanine nucleotides increased mitochondrial production of reactive oxygen species (ROS), and modified the cell cycle. Guanosine-induced cell death was suppressed by treatment with adenosine or the equilibrium nucleoside transporter (ENT) 1/2 inhibitor dipyridamole, but was not affected by adenosine receptor agonists or antagonists. These results suggest that guanosine inhibits adenosine uptake through ENT1/2, but does not antagonize adenosine receptors. In contrast, guanosine triphosphate (GTP)-induced cell death was suppressed not only by adenosine and dipyridamole, but also by the A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), suggesting that GTP-induced cell death is mediated in part by an antagonistic effect on adenosine A1 receptor. Thus, both guanosine and GTP induce apoptosis of breast cancer cells, but via at least partially different mechanisms.

The Role of CNT1 Nucleoside Transporter in Nucleotide Homeostasis

The Role of CNT1 Nucleoside Transporter in Nucleotide Homeostasis

Characterization of Equilibrative Nucleoside Transport of the Pancreatic Cancer Cell Line: Panc-1.

Gemcitabine, a first-line chemotherapeutic nucleoside analog drug (NAD) for pancreatic cancer, faces limitations due to drug resistance. Characterizing pancreatic cancer cells' transport characteristics may help identify the mechanisms behind drug resistance, and develop more effective therapeutic strategies. Therefore, in this study, we aimed to determine the nucleoside transport properties of Panc-1 cells, one of the commonly used pancreatic adenocarcinoma cell lines. To assess the presence of equilibrative nucleoside transporter-1 (ENT-1) in Panc-1 cells, we performed immunofluorescence staining, western blot analysis, and S-(4-nitrobenzyl)-6-thioinosine (NBTI) binding assays. We also conducted standard uptake assays to measure the sodium-independent uptake of [3H]-labeled chloroadenosine, hypoxanthine, and uridine. In addition, we determined the half-maximal inhibitory concentration (IC50) of gemcitabine. Statistical analyses were performed using GraphPad Prism version 8.0 for Windows. The sodium-independent uptake of [3H]-labeled chloroadenosine, hypoxanthine, and uridine was measured using standard uptake assays, and the transport rates were determined as 111.1 ± 3.4 pmol/mg protein/10 s, 62.5 ± 4.8 pmol/mg protein/10 s, and 101.3 ± 2.5 pmol/mg protein/10 s, respectively. Furthermore, the presence of ENT-1 protein was confirmed using NBTI binding assays (Bmax 1.52 ± 0.1 pmol/mg protein; equilibrium dissociation constant 0.42 ± 0.1 nM). Immunofluorescence assays and western blot analysis also revealed ENT-1 in Panc-1 cells. The determined IC50 of gemcitabine in Panc-1 cells was 2 μM, indicating moderate sensitivity. These results suggest that Panc-1 is a suitable preclinical cellular model for studying NAD transport properties and potential therapies in pancreatic cancer and pharmaceutical research.

Putative purine nucleoside interacting residues in the malaria parasite purine uptake transporter PfENT1 are critical for transporter function.

Malaria remains a major public health threat for billions of people worldwide. Infection with obligate intracellular, unicellular parasites from the genus Plasmodium causes malaria. Plasmodium falciparum causes the deadliest form of human malaria. Plasmodium parasites are purine auxotrophic. They rely on purine import from the host red blood cell cytoplasm via equilibrative nucleoside transporters to supply substrates to the purine salvage pathway. We previously developed a high throughput screening assay to identify inhibitors of the P. falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1). Screening a small molecule library identified PfENT1 inhibitors that blocked proliferation of P. falciparum parasites in in vitro culture. The goal of the current work was to validate a high-resolution model of PfENT1 predicted by the AlphaFold protein structure prediction program. We superimposed the predicted PfENT1 structure on the human homologue structure, hENT1, and developed a structure-based sequence alignment. We mutated the residues in PfENT1 aligned with and flanking the residues in hENT1 that interact with the purine analog, nitrobenzylthioinosine (NBMPR). Mutation of the PfENT1 residues Q135, D287, and R291 that are predicted to form hydrogen bonds to purine nucleosides eliminated purine and pyrimidine transport function in various yeast-based growth and radiolabeled substrate uptake assays. Mutation of two flanking residues, W53 and S290, also resulted in inactive protein. Mutation of L50 that forms hydrophobic interactions with the purine nucleobase reduced transport function. Based on our results the AlphaFold predicted structure for PfENT1 may be useful in guiding medicinal chemistry efforts to improve the potency of our PfENT1 inhibitors.

Off-Target Effects of P2Y12 Receptor Inhibitors: Focus on Early Myocardial Fibrosis Modulation.

Several studies have demonstrated that, beyond their antithrombotic effects, P2Y12 receptor inhibitors may provide additional off-target effects through different mechanisms. These effects range from the preservation of endothelial barrier function to the modulation of inflammation or stabilization of atherosclerotic plaques, with an impact on different cell types, including endothelial and immune cells. Many P2Y12 inhibitors have been developed, from ticlopidine, the first thienopyridine, to the more potent non-thienopyridine derivatives such as ticagrelor which may promote cardioprotective effects following myocardial infarction (MI) by inhibiting adenosine reuptake through sodium-independent equilibrative nucleoside transporter 1 (ENT1). Adenosine may affect different molecular pathways involved in cardiac fibrosis, such as the Wnt (wingless-type)/beta (β)-catenin signaling. An early pro-fibrotic response of the epicardium and activation of cardiac fibroblasts with the involvement of Wnt1 (wingless-type family member 1)/β-catenin, are critically required for preserving cardiac function after acute ischemic cardiac injury. This review discusses molecular signaling pathways involved in cardiac fibrosis post MI, focusing on the Wnt/β-catenin pathway, and the off-target effect of P2Y12 receptor inhibition. A potential role of ticagrelor was speculated in the early modulation of cardiac fibrosis, thanks to its off-target effect.

Hyperglycemia with hypogonadism and growth hormone deficiency in a 17-year-old male with H syndrome: the first case report from Syria

BackgroundThe nucleoside transport capabilities of the human equilibrative nucleoside transporter-3 (hENT3) are disrupted by mutations in SLC29A3 (10q22.2), which are genes for the nucleoside transporter and are the cause of the unusual autosomal recessive disease known as H syndrome. As a result, histiocytic cells invade a number of organs.Case presentationA 17-year-old Syrian male was admitted to the internal medicine department with a one-month history of polyuria, polydipsia, general weakness, and pallor. He had a history of progressive bilateral sensorineural hearing loss and failure to gain weight for three years. Physical examination revealed various abnormalities, including scrotal mass, small penis and testicles, absence of pubic and axillary hair, joint abnormalities, short stature, hallux valgus, fibrous protrusion near the navel, and hyperpigmented non-itchy painful skin plaques. Clinical signs along with laboratory test results confirmed hyperglycemia, primary hypogonadism, osteopenia, and growth hormone deficiency. After a review of the relevant medical literature, this patient’s presentation of hyperglycemia with hypogonadism, hyperpigmentation, hallux valgus, hearing loss, hematological abnormalities, and short stature suggested the diagnosis of H syndrome. The patient received treatment with insulin and testosterone, leading to a significant improvement in his presenting symptoms.ConclusionsH syndrome is a very rare condition, and the fact that the first case has only recently been reported in Syria serves to emphasize how rare it is. H Syndrome should be suspected if a patient has short stature with signs of hyperglycemia and other endocrine and cutaneous abnormalities. We are reporting this case to increase physicians’ awareness of this exceedingly rare and unique syndrome.