Equilibrative Nucleoside Transporter 1 Expression Is Downregulated by Hypoxia in Human Umbilical Vein Endothelium

Casanello, Paola; Torres, Alberto; Sanhueza, Felipe; González, Marcelo; Farías, Marcelo; Gallardo, Victoria; Pastor‐Anglada, Marçal; Martin, R. H.; Sobrevía, Luis

doi:10.1161/01.res.0000172568.49367.f8

Cited by 72 publications

(115 citation statements)

References 42 publications

(116 reference statements)

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“…These results could partially explain the decrease in dFdC uptake in Panc-1 cells. This is consistent with recent reports stating that hypoxia can repress the expression of hENT1 and/or hENT2 in different cell lines [i.e., human microvascular endothelial cells (Eltzschig et al, 2005) and human umbilical vein endothelial cells (Casanello et al, 2005)]. Clinical reports indicate that decreased expression of hENT1 can be correlated with treatment failure of dFdC in cancers, including mantle cell lymphoma (Marcé et al, 2006), pancreatic cancer (Spratlin et al, 2004;Giovannetti et al, 2006), and non-small-cell lung cancer (Achiwa et al, 2004).…”

Section: Discussionsupporting

confidence: 91%

The Impact of Hypoxic Treatment on the Expression of Phosphoglycerate Kinase and the Cytotoxicity of Troxacitabine and Gemcitabine

Lam

Leung²,

Bussom³

et al. 2007

Mol Pharmacol

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␤-L-Dioxolane-cytidine (L-OddC, Troxacitabine, BCH-4556), a novel L-configuration deoxycytidine analog, is under clinical trials for treating cancer. The cytotoxicity of L-OddC is dependent on the amount of the triphosphate form (L-OddCTP) in nuclear DNA. Phosphoglycerate kinase (PGK), a downstream protein of hypoxia-inducible-factor-1␣ (HIF-1␣), is responsible for the phosphorylation of the diphosphate to the triphosphate of L-OddC. In this study, we studied the impact of hypoxia on the metabolism and the cytotoxicity of L-OddC and ␤-D-2Ј,2Ј-difluorodeoxycytidine (dFdC) in several human tumor cell lines including HepG2, Hep3B, A673, Panc-1, and RKO. Hypoxic treatment induced the protein expression of PGK 3-fold but had no effect on the protein expression of APE-1, dCK, CMPK, and nM23 H1. Hypoxic treatment increased L-OddCTP formation and incorporation of L-OddC into DNA, but it decreased the uptake and incorporation of dFdC, which correlated with the reduction of hENT1, hENT2, and hCNT2 expression. Using a clonogenic assay, hypoxic treatment of cells made them 2-to 3-fold more susceptible to L-OddC but not to dFdC after exposure to drugs for one generation. Dimethyloxallyl glycine enhanced the cytotoxicity of L-OddC but not dFdC in Panc-1 cells under normoxic conditions. Overexpression or down-regulation of PGK using transient transfection of pcDNA5-PGK or inducible shRNA in RKO cells affected the cytotoxicity of LOddC but not that of dFdC. The knockdown of HIF-1␣ in inducible shRNA in RKO cells reduced the cytotoxicity of LOddC but not dFdC under hypoxic conditions. In conclusion, hypoxia is an important factor that may potentiate the activity ofis a novel L-configuration deoxycytidine analog with anticancer and antiviral (hepatitis B virus and human immunodeficiency virus) activity (Kim et al., 1992;Grove et al., 1995;Grove and Cheng, 1996;Gourdeau et al., 2004). It was shown in clinical evaluation to be effective against both leukemias and solid tumors. Phase II clinical studies have shown that L-OddC has significant antileukemic activity in patients with acute myeloid leukemia or patients with chronic myelogenous leukemia in the blastic phase (Giles et al., 2002) and modest activity in advanced pancreatic adenocarcinoma (Lapointe et al., 2005). It is currently undergoing phase I/II clinical trials for the treatment of solid tumors.In previous studies, we demonstrated that L-OddC can be phosphorylated by deoxycytidine kinase (dCK) to its monophosphate metabolite, which is further phosphorylated to the di-and triphosphate metabolites by cellular kinases (Grove and Cheng, 1996). The triphosphate metabolite of L-OddC can be incorporated into DNA in vitro by DNA polymerases ␣, ␤, ␦, ␥, and (Kukhanova et al., 1995). Because L-OddC lacks a hydroxyl group at the 3Ј-position, once incorporated into DNA, it causes premature termination of DNA replication and eventually leads to cell death. Therefore, the cytotoxicity of L-OddC is dependent on the steady-state level of the incorporated L-OddC in nuclear DNA. ...

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Section: Discussionsupporting

confidence: 91%

The Impact of Hypoxic Treatment on the Expression of Phosphoglycerate Kinase and the Cytotoxicity of Troxacitabine and Gemcitabine

Lam

Leung²,

Bussom³

et al. 2007

Mol Pharmacol

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“…Endothelial cells were isolated from the human umbilical vein by digestion with collagenase (0.25 mg/mL) and then cultured (37°C, 5 % CO 2 ) in medium 199 (M199) as previously described [26]. All experiments were performed in duplicate, after overnight serum deprivation.…”

Section: Huvec Culturementioning

confidence: 99%

Impaired A2A adenosine receptor/nitric oxide/VEGF signaling pathway in fetal endothelium during late- and early-onset preeclampsia

Escudero

Bertoglia

Hernadez

et al. 2012

Purinergic Signalling

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To investigate whether fetal endothelial cell proliferation and migration are modulated by the A 2A adenosine receptor (A 2A AR), nitric oxide (NO) and the vascular endothelial growth factor (VEGF) signaling pathway, we isolated human umbilical vein endothelial cells from normal pregnancy (n023), preterm delivery (n04), and late-onset (LOPE, n010) and early-onset preeclampsia (EOPE, n08). We used the non-selective adenosine receptor agonist (NECA) and the selective agonist (CGS-21680) and/or selective antagonist (ZM-241385) for A 2A AR. Also, the nitric oxide synthase (NOS) inhibitor, L-NAME, was used in co-incubation with CGS-21680. Compared to normal pregnancy, EOPE exhibited low cell proliferation and migration associated with reduced expressions of A 2A AR and VEGF and NO synthesis (i.e., total and phosphorylated serine 1177 endothelial NOS and nitrite formation). In contrast, LOPE exhibited the opposite behavior in all these markers compared to normal pregnancy or EOPE. Cell proliferation and migration were increased by CGS-21680 (or NECA) in all analyzed groups (EOPE>LO-PE>normal pregnancy) compared to their respective basal conditions, an effect that was associated with high NO and VEGF synthesis and blocked by ZM-241385 with significantly different IC 50 for each group (EOPE>LOPE>normal pregnancy). The differences seem independent of gestational age.L-NAME blocked the CGS-21680-mediated cell proliferation and migration in normal pregnancy and LOPE (IC 50 036.2± 2.5 and 8.6±2.2 nM, respectively) as well as the VEGF expression in normal pregnancy. Therefore, the A 2A AR/NO/ VEGF signaling pathway exhibits a pro-angiogenic effect in normal pregnancies and LOPE, whereas impairment in this pathway seems related to the reduced angiogenic capacity of the fetal endothelium in EOPE.

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“…ENTl activity and expression in human umbilical vein endothelial cells (HUVEC) have been shown to be decreased under J. Physiol. Biochem., 63 (1), 2007 hypoxia, which might contribute to increase extracellular adenosine levels (6). In the basal forebrain, ENTl has also been suggested to be implicated in the increase of extracellular adenosine levels associated with sleep deprivation, thus resulting in the inhibition of wake-active neurons and a decrease of cortical activity (34).…”

Section: Purinergic Regulationmentioning

confidence: 99%

Concentrative nucleoside transporters (CNTs) in epithelia: from absorption to cell signaling

et al. 2007

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Concentrative and Equilibrative Nucleoside Transporter proteins (CNT and ENT, respectively) are encoded by gene families SLC28 and SLC29. They mediate the uptake of natural nucleosides and a variety of nucleoside-derived drugs, mostly used in anticancer therapy. CNT and ENT proteins are mostly localized in the apical and basolateral sides, respectively, in (re)absorptive epithelia. This anatomic distribution determines nucleoside and nucleoside-derived vectorial flux. CNT expression (particularly CNT2) is associated with differentiation and is also nutritionally regulated in intestinal epithelia, whereas ENT protein amounts (mostly ENT1) are increased when cells are exposed to proliferative stimuli such as EGF, TGF-alpha or wounding. Although all these features suggest a role for NT proteins in nucleoside salvage and (re)absorption, recent data demonstrate that CNT2 might be under purinergic control, in a manner that is dependent on energy metabolism. A physiological link between CNT2 function and intracellular metabolism is also supported by the evidence that extracellular adenosine can activate the AMP-dependent kinase (AMPK), by a mechanism which relies upon adenosine transport and phosphorylation. Thus the complex pattern of NT isoform expression in mammalian cells can fulfill physiological roles other than salvage.

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Equilibrative Nucleoside Transporter 1 Expression Is Downregulated by Hypoxia in Human Umbilical Vein Endothelium

Cited by 72 publications

References 42 publications

The Impact of Hypoxic Treatment on the Expression of Phosphoglycerate Kinase and the Cytotoxicity of Troxacitabine and Gemcitabine

The Impact of Hypoxic Treatment on the Expression of Phosphoglycerate Kinase and the Cytotoxicity of Troxacitabine and Gemcitabine

Impaired A2A adenosine receptor/nitric oxide/VEGF signaling pathway in fetal endothelium during late- and early-onset preeclampsia

Concentrative nucleoside transporters (CNTs) in epithelia: from absorption to cell signaling

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