Human Equilibrative Nucleoside Transporter Research Articles

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.

Prognostic and predictive value of human equilibrative nucleoside transporter 1 (hENT1) in extrahepatic cholangiocarcinoma: a translational study.

Introduction: Effective (neo) adjuvant chemotherapy for cholangiocarcinoma is lacking due to chemoresistance and the absence of predictive biomarkers. Human equilibrative nucleoside transporter 1 (hENT1) has been described as a potential prognostic and predictive biomarker. In this study, the potential of rabbit-derived (SP120) and murine-derived (10D7G2) antibodies to detect hENT1 expression was compared in tissue samples of patients with extrahepatic cholangiocarcinoma (ECC), and the predictive value of hENT1 was investigated in three ECC cell lines. Methods: Tissues of 71 chemonaïve patients with histological confirmation of ECC were selected and stained with SP120 or 10D7G2 to assess the inter-observer variability for both antibodies and the correlation with overall survival. Concomitantly, gemcitabine sensitivity after hENT1 knockdown was assessed in the ECC cell lines EGI-1, TFK-1, and SK-ChA-1 using sulforhodamine B assays. Results: Scoring immunohistochemistry for hENT1 expression with the use of SP120 antibody resulted in the highest interobserver agreement but did not show a prognostic role of hENT1. However, 10D7G2 showed a prognostic role for hENT1, and a potential predictive role for gemcitabine sensitivity in hENT1 in SK-ChA-1 and TFK-1 cells was found. Discussion: These findings prompt further studies for both preclinical validation of the role of hENT1 and histochemical standardization in cholangiocarcinoma patients treated with gemcitabine-based chemotherapy.

Kinetic profiling of novel spirobenzo-oxazinepiperidinone derivatives as equilibrative nucleoside transporter 1 inhibitors

Evaluation of kinetic parameters of drug-target binding, kon, koff, and residence time (RT), in addition to the traditional in vitro parameter of affinity is receiving increasing attention in the early stages of drug discovery. Target binding kinetics emerges as a meaningful concept for the evaluation of a ligand’s duration of action and more generally drug efficacy and safety. We report the biological evaluation of a novel series of spirobenzo-oxazinepiperidinone derivatives as inhibitors of the human equilibrative nucleoside transporter 1 (hENT1, SLC29A1). The compounds were evaluated in radioligand binding experiments, i.e., displacement, competition association, and washout assays, to evaluate their affinity and binding kinetic parameters. We also linked these pharmacological parameters to the compounds’ chemical characteristics, and learned that separate moieties of the molecules governed target affinity and binding kinetics. Among the 29 compounds tested, 28 stood out with high affinity and a long residence time of 87 min. These findings reveal the importance of supplementing affinity data with binding kinetics at transport proteins such as hENT1.

C-reactive protein is a prognostic biomarker in pancreatic ductal adenocarcinoma patients.

The 5-year survival rate of pancreatic ductal adenocarcinoma (PDAC) is approximately 11% and has only improved marginally over the last three decades. For operable PDAC, resection and adjuvant FOLFIRINOX chemotherapy is standard of care. There is growing interest in perioperative regimens to improve outcomes. The non-randomized Phase II study "Gemcitabine and Abraxane for resectable Pancreatic cancer" (GAP) demonstrated the feasibility of perioperative gemcitabine/abraxane. Long-term survival in PDAC requires an effective immune response; hence, we undertook this translational study of the GAP trial cohort to identify immune-oncology biomarkers for clinical use. We combined Nanostring nCounter technology with immunohistochemistry to investigate the correlation between gene expression and overall patient survival. Findings were investigated in samples from the International Cancer Genome Consortium (ICGC, n=88) and the Australian Pancreatic Genome Initiative (APGI, n=227). We confirmed that human equilibrative nucleoside transporter 1 (hENT1) expression was not a prognostic marker in PDAC but patients with high levels of hENT1 were more likely to live longer than 24 months post-surgery. Additionally, CD274 (PD-L1) and two novel biomarkers of survival, cathepsin W (CTSW) and C-reactive protein (CRP), were identified in the GAP cohort (n=19). CRP expression was confirmed in data from the ICGC. Although PD-L1 and CTSW proteins were not significant across all three cohorts, results show that low CRP mRNA and protein expression are associated with longer overall survival in all three patient groups. PDAC patients with long survival have higher hENT1 expression levels. Furthermore, CRP expression is a biomarker of poor prognosis following perioperative chemotherapy and resection in PDAC patients and thus may be useful for identifying patients who could benefit from more aggressive adjuvant strategies.

Gestational Diabetes—Placental Expression of Human Equilibrative Nucleoside Transporter 1 (hENT1): Is Delayed Villous Maturation an Adaptive Pattern?

Gestational diabetes mellitus (GDM) is a metabolic disease that can affect placental villous maturation and villous vascularity. The main effects of GDM on placental growth are a delay of villous maturation (DVM) and decreased formation of vasculo-syncytial membranes (VSM). Human equilibrative nucleoside transporter-1 (hENT1) is an adenosine transporter expressed in the human umbilical vein endothelial cells (HUVEC) and human placental microvascular endothelium cells (hPMEC). Its role is crucial in maintaining physiological fetal adenosine levels during pregnancy, and its reduction has been described in GDM. Twenty-four placentas from pregnancies with a confirmed diagnosis of GDMd and twenty-four matched non-GDM placentas (controls) were retrospectively analyzed to investigate the immunohistochemical expression of hENT1 in HUVEC and hPMEC. The study included the quantitative evaluation of VSM/mm2 in placental tissue and the immunohistochemical quantitative evaluation of Ki-67, PHH3, and p57 in villous trophoblast. hENT1 expression was higher in all the vascular districts of the control cases compared to the GDMd placentas (p &lt; 0.0001). The VSM/mm2 were lower in the GDMd cases, while the Ki-67, PHH3, and p57 were higher when compared to the control cases. To our knowledge, this is the first report of hENT1 expression in the human placentas of GDM patients. The absence/low expression of hENT1 in all the GDMd patients may indicate a potential role in microvascular adaptative mechanisms. The trophoblasts’ proliferative/antiapoptotic pattern (high Ki-67, high PHH3, and high p57 count) may explain the statistically significant lower number of VSM/mm2 found in the GDMd cases.

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.

O095 hCNT (human concentrative nucleoside transporter) and hENT (human equilibrative nucleoside transporter) in human gastric cancer and response to chemotherapies

Abstract Introduction hCNT (Human Concentrative Nucleoside Transporter, also known as SLC28A1) and hENT (Human Equilibrative Nucleoside Transporter, SLC29A1) belong to the large solute carrier (SLC) family, a large membrane transport molecule group for the influx and efflux across cell membrane of various solutes including some of the anticancer drugs. hCNT and hENT are suggested to be involved in the transportation of certain chemotherapeutic drugs. The present study investigated the role of both transporters in gastric cancer and the response of gastric cancer cell and patients to chemotherapeutic drugs. Methods The expression levels of hCNT and hENT gene transcripts and proteins were determined in two independent gastric cancer cohorts and assessed against patient's responses to neoadjuvent chemotherapies. Their value in assessing patient's survival, clinical and pathological indicators was also assessed. Results Gastric tumour tissues expressed significantly higher levels of hCNT and hENT than normal stomach tissues (p=0.022 and p=0.0071, respectively for hCNT and hENT). Expression levels of the hCNT and hENT gene transcripts presented significant correlation with the overall survival (OS) (Hazard Ration (HR)=2.885, p&amp;lt;0.001 for hCNT; HR=2.078, p&amp;lt;0.001 for hENT) and disease-free survival of the patients (HR=2.434, p=0.004 for hCNT; HR=2.072, p&amp;lt;0.001). Patients with high levels of hCNT in their gastric tumours were more resisted to neoadjuvant therapies (HR=3.437, p=0.013, by ROC model), although the same was not seen with hENT. Conclusion hCNT and hENT have significant prognostic factors for patients with gastric cancer and theranostic factor for response to neoadjuvant therapies.

The potential off-target neuroprotective effect of sister gliflozins suggests their repurposing despite not crossing the blood-brain barrier: From bioanalytical assay in rats into theory genesis.

Gliflozins are successfully marketed antidiabetic agents with a reported neuroprotective effect, and this study tests their blood-brain barrier crossing ability. Henceforward, a computational hypothesis interpreting their effects was reasonable after failure to cross into the brain. A chromatographic bioassay for canagliflozin, dapagliflozin, and empagliflozin was developed, validated, and applied to the rat's and rat's plasma and brain. HPLC method robustness was tested over two levels using Design of Experiment on MINITAB. It is the first method for gliflozins' detection in rats' brain tissue. The method was applied on 18 rats and six for each drug. Concentrations in plasma were determined but neither of them was detected in brain at the described chromatographic conditions. A computational study for the three drugs was endorsing two techniques. First, ligand-based target fishing reveals possible targets for gliflozins. They showed an ability to bind with human equilibrative nucleoside transporter 1, a regulator of adenosine extracellularly. Second, a docking study was carried out on this protein receptor. Results showed perfect alignment with a minimum of one hydrogen bond. Dapagliflozin achieved the lowest energy score with two hocking hydrogen bonds. This is proposing gliflozins ability to regulate equilibrative nucleoside transporter 1 receptors in peripheries, elevating the centrally acting neuroprotective adenosine.

Human equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (DCK) are predictive biomarkers for Gemcitabine-based chemoradiotherapy in resected pancreatic cancer patients: a retrospective cohort study

Human equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (DCK) are predictive biomarkers for Gemcitabine-based chemoradiotherapy in resected pancreatic cancer patients: a retrospective cohort study

Mucin 4 Confers Gemcitabine Resistance and an Unfavorable Prognosis in Patients with Cholangiocarcinoma via AKT Activation.

Cholangiocarcinoma (CCA) exhibits aggressive biological behavior and a poor prognosis. Gemcitabine (GEM)-based chemotherapy is the first-line chemotherapy for advanced CCA but has a response rate of only 20-30%. Therefore, investigating treatments to overcome GEM resistance in advanced CCA is crucial. Among mucin (MUC) family members, MUC4 showed the greatest increase in the resistant versus parental sublines. MUC4 was upregulated in whole-cell lysates and conditioned media from gemcitabine-resistant (GR) CCA sublines. MUC4 mediated GEM resistance by activating AKT signaling in GR CCA cells. The MUC4-AKT axis induced BAX S184 phosphorylation to inhibit apoptosis and downregulated GEM transporter human equilibrative nucleoside transporter 1 (hENT1) expression. The combination of AKT inhibitors and GEM or afatinib overcame GEM resistance in CCA. In vivo, capivasertib (an AKT inhibitor) increased GEM sensitivity in GR cells. MUC4 promoted EGFR and HER2 activation to mediate GEM resistance. Finally, MUC4 expression in patient plasma correlated with MUC4 expression. Paraffin-embedded specimens from non-responders expressed significantly more MUC4 than did those from responders, and this upregulation was associated with poor progression-free survival and overall survival. In GR CCA, high MUC4 expression promotes sustained EGFR/HER2 signaling and AKT activation. The combination of AKT inhibitors with GEM or afatinib might overcome GEM resistance.

A Transcriptomic-Based Tool to Predict Gemcitabine Sensitivity in Advanced Pancreatic Adenocarcinoma

A Transcriptomic-Based Tool to Predict Gemcitabine Sensitivity in Advanced Pancreatic Adenocarcinoma

Novel variants provide differential stabilisation of human equilibrative nucleoside transporter 1 states.

Human equilibrative nucleoside transporters represent a major pharmaceutical target for cardiac, cancer and viral therapies. Understanding the molecular basis for transport is crucial for the development of improved therapeutics through structure-based drug design. ENTs have been proposed to utilise an alternating access mechanism of action, similar to that of the major facilitator superfamily. However, ENTs lack functionally-essential features of that superfamily, suggesting that they may use a different transport mechanism. Understanding the molecular basis of their transport requires insight into diverse conformational states. Differences between intermediate states may be discrete and mediated by subtle gating interactions, such as salt bridges. We identified four variants of human equilibrative nucleoside transporter isoform 1 (hENT1) at the large intracellular loop (ICL6) and transmembrane helix 7 (TM7) that stabilise the apo-state (∆T m 0.7-1.5°C). Furthermore, we showed that variants K263A (ICL6) and I282V (TM7) specifically stabilise the inhibitor-bound state of hENT1 (∆∆T m 5.0 ± 1.7°C and 3.0 ± 1.8°C), supporting the role of ICL6 in hENT1 gating. Finally, we showed that, in comparison with wild type, variant T336A is destabilised by nitrobenzylthioinosine (∆∆T m -4.7 ± 1.1°C) and binds it seven times worse. This residue may help determine inhibitor and substrate sensitivity. Residue K263 is not present in the solved structures, highlighting the need for further structural data that include the loop regions.

Combination treatment with hENT1 and miR-143 reverses gemcitabine resistance in triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer and is susceptible to develop gemcitabine (GEM) resistance. Decreased expression of human equilibrative nucleoside transporter 1 (hENT1) accompanied by compensatory increase of glycolysis is strongly associated with GEM resistance in TNBC. In this study, we investigated the treatment feasibility of combined hENT1 upregulation and miR-143-mediated inhibition of glycolysis for reversing GEM resistance in TNBC.MethodsExperiments were performed in vitro and in vivo to compare the efficacy of GEM therapies. In this study, we established stable drug-resistant cell line, GEM-R cells, from parental cells (MDA-MB-231) through exposure to GEM following a stepwise incremental dosing strategy. Then GEM-R cells were transfected by lentiviral plasmids and GEM-R cells overexpressing hENT1 (GEM-R-hENT1) were established. The viability and apoptosis of wild-type (MDA-MB-231), GEM-R, and GEM-R-hENT1 cells treated with GEM or GEM + miR-143 were analyzed by CCK8 assay and flow cytometry. The RNA expression and protein expression were measured by RT-PCR and western blotting respectively. GEM uptake was determined by multiple reaction monitoring (MRM) analysis. Glycolysis was measured by glucose assay and 18F-FDG uptake. The antitumor effect was assessed in vivo in a tumor xenograft model by evaluating toxicity, tumor volume, and maximum standardized uptake value in 18F-FDG PET. Immunohistochemistry and fluorescence photography were taken in tumor samples. Pairwise comparisons were performed using Student’s t-test.ResultsOur results represented that overexpression of hENT1 reversed GEM resistance in GEM-R cells by showing lower IC50 and higher rate of apoptosis. MiR-143 suppressed glycolysis in GEM-R cells and enhanced the effect of reversing GEM resistance in GEM-R-hENT1 cells. The therapeutic efficacy was validated using a xenograft mouse model. Combination treatment decreased tumor growth rate and maximum standardized uptake value in 18F-FDG PET more effectively.ConclusionsCombined therapy of exogenous upregulation of hENT1 expression and miR-143 mimic administration was effective in reversing GEM resistance, providing a promising strategy for treating GEM-resistant TNBC.

Metformin Protects Against Sunitinib-induced Cardiotoxicity: Investigating the Role of AMPK.

Sunitinib is associated with cardiotoxicity through inhibition of AMP-protein kinase (AMPK) signaling. By contrast, the common antidiabetic agent metformin has demonstrated cardioprotection through indirect AMPK activation. In this study, we investigate the effects of metformin during sunitinib-induced cytotoxicity. Left ventricular developed pressure, coronary flow, heart rate, and infarct size were measured in Langendorff-perfused rat hearts treated with 1 µM sunitinib ±50 µM metformin ±1 µM human equilibrative nucleoside transporter inhibitor S-(4-Nitrobenzyl)-6-thioinosine (NBTI). Western blot analysis was performed for p-AMPKα levels. Primary isolated cardiac myocytes from the left ventricular tissue were used to measure live cell population levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess adjunctive treatment of and metformin in human hepatoma G2 and promyelocytic leukemia (HL-60) cells treated with 0.1-100 µM sunitinib ±50 µM metformin. In the perfused hearts, coadministration of metformin attenuated the sunitinib-induced changes to left ventricular developed pressure, infarct size, and cardiac myocyte population. Western blot analysis revealed a significant decrease in p-AMPKα during sunitinib treatment, which was attenuated after coadministration with metformin. All metformin-induced effects were attenuated, and NBTI was coadministered. The MTT assay demonstrated an increase in the EC50 value during coadministration of metformin with sunitinib compared with sunitinib monotherapy in hepatoma G2 and HL-60 cell lines, demonstrating the impact and complexity of metformin coadministration and the possible role of AMPK signaling. This study highlights the novel cardioprotective properties of metformin and AMPK activation during sunitinib-induced cardiotoxicity when administered together in the Langendorff heart model.

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

We thank Dr. Eric Johnson ([Johnson, 2022][1]) for the comments regarding our recent study ([Miller et al., 2021][2]) and providing us with the opportunity to further emphasize the importance of the equilibrative nucleoside transporters (ENTs) in nucleoside analog disposition. Nucleoside analogs are

Insight into the nucleoside transport and inhibition of human ENT1

The human equilibrative nucleoside transporter 1 (hENT1) is an effective controller of adenosine signaling by regulating its extracellular and intracellular concentration, and has become a solid drug target of clinical used adenosine reuptake inhibitors (AdoRIs). Currently, the mechanisms of adenosine transport and inhibition for hENT1 remain unclear, which greatly limits the in-depth understanding of its inner workings as well as the development of novel inhibitors. In this work, the dynamic details of hENT1 underlie adenosine transport and the inhibition mechanism of the non-nucleoside AdoRIs dilazep both were investigated by comparative long-time unbiased molecular dynamics simulations. The calculation results show that the conformational transitions of hENT1 from the outward open to metastable occluded state are mainly driven by TM1, TM2, TM7 and TM9. One of the trimethoxyphenyl rings in dilazep serves as the adenosyl moiety of the endogenous adenosine substrate to competitively occupy the orthosteric site of hENT1. Due to extensive and various VDW interactions with N30, M33, M84, P308 and F334, the other trimethoxyphenyl ring is stuck in the opportunistic site near the extracellular side preventing the complete occlusion of thin gate simultaneously. Obviously, dilazep shows significant inhibitory activity by disrupting the local induce-fit action in substrate binding cavity and blocking the transport cycle of whole protein. This study not only reveals the nucleoside transport mechanism by hENT1 at atomic level, but also provides structural guidance for the subsequent design of novel non-nucleoside AdoRIs with enhanced pharmacologic properties.

ET-7 Roles for hENT1 and dCK in gemcitabine sensitivity and malignancy of meningioma

Background: High-grade meningiomas are aggressive tumors with high morbidity and mortality rates that frequently recur even after surgery and adjuvant radiotherapy. However, limited information is currently available on the biology of these tumors, and no alternative adjuvant treatment options exist. Although we previously demonstrated that high-grade meningioma cells were highly sensitive to gemcitabine in vitro and in vivo, the underlying molecular mechanisms remain unknown. Methods: We examined the roles of hENT1 (human equilibrative nucleoside transporter 1) and dCK (deoxycytidine kinase) in the gemcitabine sensitivity and growth of meningioma cells in vitro. Tissue samples from meningiomas (26 WHO grade I and 21 WHO grade II/III meningiomas) were immunohistochemically analyzed for hENT1 and dCK as well as for Ki-67 as a marker of proliferative activity. Results: hENT1 and dCK, which play critical roles in the intracellular transport and activation of gemcitabine, respectively, were responsible for the high gemcitabine sensitivity of high-grade meningioma cells and were strongly expressed in high-grade meningiomas. hENT1 expression was required for the proliferation and survival of high-grade meningioma cells and dCK expression. Furthermore, high hENT1 and dCK expression levels correlated with stronger tumor cell proliferative activity and shorter survival in meningioma patients. Conclusions: The present results suggest that hENT1 is a key molecular factor influencing the growth capacity and gemcitabine sensitivity of meningioma cells and also that hENT1, together with dCK, may be a viable prognostic marker for meningioma patients as well as a predictive marker of their responses to gemcitabine.

Molecular characterization of circulating tumor cells in pancreatic ductal adenocarcinoma: potential diagnostic and prognostic significance in clinical practice.

The clinical value of heterogeneous sub-populations of circulating tumor cells (CTCs) in pancreatic ductal adenocarcinoma (PDAC) remains unclear. Peripheral blood samples were obtained from 67 PDAC patients. CTCs were isolated by employing CD45 negative enrichment technique and further characterized for epithelial to mesenchymal transition (EMT) or human equilibrative nucleoside transporter-1 (hENT-1). The relationships between CTCs sub-phenotypes with clinicopathological factors or post-operative recurrence in PDAC patients were analyzed. EMT related CTCs could be isolated and identified from the 81% of patients (54/67), and both the total count (median: 5 vs. 17/mL, P<0.0001) and M-CTC percentage (median: 0.2 vs. 0.345, P=0.0244) of CTCs could differentiate local/regional with metastatic disease. Multivariate analysis showed that both AJCC stage (P=0.025) and M-CTC percentage (P=0.001) were independent prognostic indicators of recurrence free survival (RFS) in resected patients. Moreover, Kaplan-Meier survival analysis showed that M-CTC after 2 courses of chemotherapy was significantly associated with inferior RFS (49.5 weeks vs. undefined, P=0.0288). No significant correlation in hENT-1 expression was found between CTCs and matched tumor tissues, and further multivariate analysis suggested hENT-1 expression in CTCs as independent prognostic factor for RFS (P=0.016). Patients with low hENT-1 expression in CTCs had decreased RFS (32 weeks vs. undefined, P=0.0337). CTCs could be the promising diagnostic biomarkers in PDAC patients, and phenotypic profiling of CTCs based on EMT or hENT-1 could help establish novel prognostic biomarkers in resected patients undergoing adjuvant gemcitabine-based chemotherapy. Circulating tumor cells (CTCs); Pancreatic ductal adenocarcinoma (PDAC); Epithelial to mesenchymal transition (EMT); human equilibrative nucleoside transporter-1 (hENT-1).

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.

RRM1 Expression as a Prognostic Biomarker for Unresectable or Recurrent Biliary Tract Cancer Treated with Gemcitabine plus Cisplatin.

The combination of gemcitabine plus cisplatin (GP) is regarded as a first-line treatment for patients with unresectable or recurrent biliary tract cancer (BTC). Several proteins including human equilibrative nucleoside transporter-1 (hENT1), deoxycytidine kinase (DCK), cytidine deaminase (CDA), and ribonucleotide reductase subunit 1 (RRM1) are known to be involved in gemcitabine uptake and metabolism. This study was aimed to identify the predictive and prognostic values of these biomarkers in patients who treated with GP for advanced BTC. Tumor samples were obtained from 34 patients with unresectable or recurrent BTC who were treated with GP between August 2015 and February 2018. Intratumoral expression of hENT1, DCK, CDA and RRM1 was determined by immunohistochemistry and analyzed for association with chemotherapy response, progression-free survival (PFS) and overall survival (OS). Median OS was significantly longer in the RRM1-negative group than in the RRM1-positive (9.9 months vs. 5.9 months, p = 0.037). Multivariate adjustment analyses also demonstrated RRM1 expression as an independent prognostic factor for OS in patients treated with GP chemotherapy. Increased intratumoral expression of RRM1 on immunohistochemical staining may be a biomarker predicting poor survival in patients with GP chemotherapy for advanced BTC. Large-scale well-predefined prospective research is needed to validate the utility of biomarkers in clinical practice.