Functional characterization of human equilibrative nucleoside transporter 1

Equilibrative nucleoside transporters (ENTs), which facilitate cross-membrane transport of nucleosides and nucleoside-derived drugs, play an important role in the salvage pathways of nucleotide synthesis, cancer chemotherapy, and treatment for virus infections. Functional characterization of ENTs at the molecular level remains technically challenging and hence scant. In this study, we report successful purification and biochemical characterization of human equilibrative nucleoside transporter 1 (hENT1) in vitro. The HEK293F-derived, recombinant hENT1 is homogenous and functionally active in proteoliposome-based counter flow assays. hENT1 transports the substrate adenosine with a K of 215 ± 34 µmol/L and a V of 578 ± 23.4 nmol mg min. Adenosine uptake by hENT1 is competitively inhibited by nitrobenzylmercaptopurine ribonucleoside (NBMPR), nucleosides, deoxynucleosides, and nucleoside-derived anti-cancer and anti-viral drugs. Binding of hENT1 to adenosine, deoxyadenosine, and adenine by isothermal titration calorimetry is in general agreement with results of the competitive inhibition assays. These results validate hENT1 as a bona fide target for potential drug target and serve as a useful basis for future biophysical and structural studies.

Functional study of hENT1 on SKM-1 cell resistance to decitabine / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of human equilibrative nucleoside transporters 1 (hENT1) silencing on proliferation, apoptosis and demethylation of human myelodysplastic syndrome (MDS) derived cell line SKM-1 treated with 5-aza-2'-deoxycytidine (decitabine, DAC).</p><p><b>METHODS</b>hENT1 was silenced in SKM-1 cells mediated by lentivirus transfection. The infection efficiency was detected by flow cytometry, and the mRNA expression level of hENT1 was confirmed by qRT-PCR. The proliferation ratio of SKM-1 cells treated with different concentrations (0.5, 1, 5 mmol/L) of DAC for 24, 48 and 72 h was detected by CCK-8 method after hENT1 silencing. The apoptosis of SKM-1 cells was detected by Western blot for cleaved level of caspase-3 and evaluated by flow cytometry after staining with anti-Annexin V-PE and 7-AAD. The p15(INK4B) DNA methylation status was measured by methylation specific PCR using EZ DNA Methylation-Gold™ Kit.</p><p><b>RESULTS</b>The expression level of hENT1 silenced group (0.253±0.030) was statistically decreased compared with that in control group (1.000±0.091) (P<0.01). Compared with control, the proliferation inhibition rate of hENT1 silenced group was significantly decreased by different concentrations of DAC (0.5, 1, 5 μmol/L) treatment for 24, 48, 72 h (P<0.05), which was (49.41±4.02)% and (33.03±2.47)%, respectively (P=0.007) at 5 μmol/L DAC treatment for 72 h in hENT1 silenced group and the control group. Western blot showed that cleaved caspase3 of hENT1 silenced group was also significantly inhibited. The percentage of Annexin Ⅴ⁺ cells and demethylation status of p15(INK4B) were significantly decreased.</p><p><b>CONCLUSION</b>Apoptosis of hENT1 silenced SKM-1 cells induced by DAC was decreased, and the susceptibility of these cells to demethylation treatment was also decreased.</p>

p21-Activated Kinase 4 (PAK4) as a Predictive Marker of Gemcitabine Sensitivity in Pancreatic Cancer Cell Lines / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: p21-activated kinases (PAKs) are involved in cytoskeletal reorganization, gene transcription, cell proliferation and survival, and oncogenic transformation. Therefore, we hypothesized that PAK expression levels could predict the sensitivity of pancreatic cancer cells to gemcitabine treatment, and PAKs could be therapeutic targets. MATERIALS AND METHODS: Cell viability inhibition by gemcitabine was evaluated in human pancreatic cancer cell lines (Capan-1, Capan-2, MIA PaCa-2, PANC-1, Aspc-1, SNU-213, and SNU-410). Protein expression and mRNA of molecules was detected by immunoblot analysis and reverse transcription polymerase chain reaction. To define the function of PAK4, PAK4 was controlled using PAK4 siRNA. RESULTS: Capan-2, PANC-1, and SNU-410 cells were resistant to gemcitabine treatment. Immunoblot analysis of signaling molecules reported to indicate gemcitabine sensitivity showed higher expression of PAK4 and lower expression of human equilibrative nucleoside transporter 1 (hENT1), a well-known predictive marker for gemcitabine activity, in the resistant cell lines. Knockdown of PAK4 using siRNA induced the upregulation of hENT1. In resistant cell lines (Capan-2, PANC-1, and SNU-410), knockdown of PAK4 by siRNA resulted in restoration of sensitivity to gemcitabine. CONCLUSION: PAK4 could be a predictive marker of gemcitabine sensitivity and a potential therapeutic target to increase gemcitabine sensitivity in pancreatic cancer.

Systemic treatment for inoperable pancreatic adenocarcinoma: review and update / 癌症

There have been many clinical trials conducted to evaluate novel systemic regimens for unresectable pancreatic cancer. However, most of the trial results were negative, and gemcitabine monotherapy has remained the standard systemic treatment for years. A number of molecular targeted agents, including those against epidermal growth factor receptor and vascular endothelial growth factor receptors, have also been tested. In recent years, there have been some breakthroughs in the deadlock: three regimens, namely gemcitabine-erlotinib, FOLFIRINOX, and gemcitabine-nab-paclitaxel, have been shown to prolong the overall survival of patients when compared with gemcitabine monotherapy. In addition, emerging data suggested that the membrane protein human equilibrative nucleotide transporter 1 is a potential biomarker with which to predict the efficacy of gemcitabine. Here we review the literature on the development of systemic agents for pancreatic cancer, discuss the current choices of treatment, and provide future directions on the development of novel agents.

Expression of hENTl and ERCC1 genes in tumor tissues non-small cell lung cancer

OBJECTIVE@#To investigate the expression of hENTl and ERCC1 genes in tumor tissues non-small cell lung cancer (NSCLC).@*METHODS@#Fresh non-small lung cancer specimens were transplanted into nude mice. Twenty mice were randomized into two groups: experimental group receiving gemcitabine plus cisplatin and control group receiving 0.9% physiological saline. The expressions of hENTl and ERCC1 mRNA in tumor tissue were detected by real-time fluorescent quantitative PCR. The volume of tumor, the weight of nude mice and tumor volume were respectively measured and calculated 2-3 times per week. Tissue samples were collected from NSCLC mice treated with gemcitabine plus carboplatin.@*RESULTS@#The histological examination showed that many tumor cells were well preserved in nude mice. The rate of transplanted tumor cells was 86.7%. The concomitant treatment study showed that the rate of TV, RTV, T/C in GEM + DDP group was the lowest. LBP + DOC, DDP + DOC obviously influenced the body weight. Compared with NS group, DDP group, GEM group, the survival period and the level of hENTl of DDP+GEM group increased obviously, the level of ERCC1 decreased significantly (P<0.05).@*CONCLUSIONS@#The expression of hENT1 and ERCC1 genes in tumor tissues were closely correlated with the response to chemotherapy and prognosis of patients with NSCLC treated with gemcitabine plus cisplatin.

Decreased nucleoside transport and hENT1 transporter expression in beta-thalassemia major

This study investigated nucleoside transport activity and transporter polypeptide expression in erythrocytes from beta-thalassemia major patients to determine if inhibition of transport activity is a sensitive indicator of oxidative membrane damage. Blood samples were obtained from 54 patients, diagnosed as having beta-thalassemia major prior to therapeutic transfusion, and 20 normal subjects. Uptake of 3H-uridine into washed erythrocytes was measured at room temperature using short incubation periods [5 s] and a rapid inhibitor oil stop protocol. Erythrocyte membranes were analyzed by SDS-PAGE and nucleoside [hENT1] and glucose [GLUT-1] transporter polypeptides quantitated on immunoblots. Uridine uptake was significantly lower in beta-thalassemic cells than in normal erythrocytes [20.03 +/- 1.08 pmol/108 cells/ 5 s, mean +/- SEM, n = 31, vs. 31.15 +/- 1.21 pmol/108 cells/5 s, n = 20; p < 0.0001]. Expression of hENT1 was significantly lower in beta-thalassemic cells [23.90 +/- 1.01 arbitrary units, n = 54] than in controls [101.20 +/- 2.43 arbitrary units, n = 20; p < 0.001] but expression of GLUT-1 was not changed appreciably [101.80 +/- 2.43 arbitrary units, n = 54, for thalassemic cells; 102.60 +/- 3.02 arbitrary units, n = 20, for control cells; p = 0.87]. Erythrocytes from beta-thalassemia major patients showed decreased uridine transport which was associated with decreased nucleoside transporter expression. The process[es] mediating a selective decrease in hENT1 polypeptides in beta-thalassemia major was/were not determined

Cytosine arabinoside-metabolizing enzyme genes are underexpressed in children with MLL gene-rearranged acute lymphoblastic leukemia

Infant acute lymphoblastic leukemia (IALL) is characterized by mixed lineage leukemia (MLL) gene rearrangements, unique gene expression profiles, poor prognosis, and drug resistance. One exception is cytosine arabinoside (Ara-C) to which IALL cells seem to be more sensitive. We quantified mRNA expression of Ara-C key enzymes in leukemic lymphoblasts from 64 Brazilian ALL children, 15 of them presenting MLL gene rearrangement, and correlated it with clinical and biological features. The diagnosis was based on morphological criteria and immunophenotyping using monoclonal antibodies. MLL gene rearrangements were detected by conventional cytogenetic analysis, RT-PCR and/or fluorescence in situ hybridization. The DCK and HENT1 expression levels were determined by real-time quantitative PCR using SYBR Green I. Relative quantification was made by the standard curve method. The results were analyzed by Mann-Whitney and Fisher exact tests. A P value of ú0.05 was considered to be statistically significant. DCK and HENT1 expression levels were significantly lower in children with MLL gene-rearranged ALL compared to children with MLL germ line ALL (P = 0.0003 and 0.03, respectively). Our results differ from previous ones concerning HENT1 mRNA expression that observed a higher expression level in MLL gene-rearranged leukemias. In conclusion, the expression of the genes related to Ara-C metabolism was lower in MLL-positive children in the sample studied, suggesting the presence of population differences in the expression profile of these genes especially for HENT1.