OBJECTIVETo 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).

METHODShENT1 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.

RESULTSThe 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.

CONCLUSIONApoptosis of hENT1 silenced SKM-1 cells induced by DAC was decreased, and the susceptibility of these cells to demethylation treatment was also decreased.

Apoptosis ; Azacitidine ; analogs & derivatives ; Caspase 3 ; Cell Line ; DNA Methylation ; Drug Resistance ; Equilibrative Nucleoside Transporter 1 ; Humans ; Lentivirus ; Myelodysplastic Syndromes ; Sincalide

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.
Adenine Nucleotides ; chemistry ; metabolism ; Equilibrative Nucleoside Transporter 1 ; chemistry ; genetics ; metabolism ; HEK293 Cells ; Humans ; Protein Domains ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Structure-Activity Relationship

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.
Cell Line* ; Cell Proliferation ; Cell Survival ; Equilibrative Nucleoside Transporter 1 ; Humans ; p21-Activated Kinases ; Pancreatic Neoplasms* ; Phosphotransferases* ; Polymerase Chain Reaction ; Reverse Transcription ; RNA, Messenger ; RNA, Small Interfering ; Up-Regulation

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.
Adenocarcinoma ; drug therapy ; Albumins ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Camptothecin ; analogs & derivatives ; Deoxycytidine ; analogs & derivatives ; Equilibrative Nucleoside Transporter 1 ; Erlotinib Hydrochloride ; Fluorouracil ; Humans ; Leucovorin ; Organoplatinum Compounds ; Paclitaxel ; Pancreatic Neoplasms ; drug therapy ; Quinazolines ; Receptor, Epidermal Growth Factor ; Receptors, Vascular Endothelial Growth Factor