Marçal Pastor-Anglada

Transport and mode of action of nucleoside derivatives used in chemical and antiviral therapies

Nucleoside analogues used in cancer and anti-viral therapies interfere with nucleotide metabolism and DNA replication, thus inducing their pharmacological effects. A long-awaited goal in the understanding of the pharmacological properties of these molecules, that is the molecular characterization of nucleoside plasma-membrane transporters, has been achieved very recently. These carrier proteins are encoded by at least two gene families and new isoforms remain to be identified. Direct demonstration of translocation of these drugs by nucleoside transporters has already been provided and most of them can inhibit natural nucleoside transport, probably in a competitive manner. The expression of these genes is clearly tissue-specific and might depend on the differentiated status of a cell. This is relevant because the sensitivity of a cell to a drug can depend on the type of nucleoside carrier expressed, and the drug itself might modulate nucleoside carrier expression. In this article, Marçal Pastor-Anglada, Antonio Felipe and Javier Casado discuss recent studies on the regulation of nucleoside carrier expression and of the molecular determinants of substrate specificity. Better knowledge of these will contribute to an improved design of therapies based on nucleoside derivatives.

Nucleoside transporter proteins.

Concentrative nucleoside transporters (CNT; SLC28) and equilibrative nucleoside transporters (ENT; SLC29) mediate the uptake of natural nucleosides and a variety of nucleoside-derived drugs, mostly used in anticancer therapy. SLC28 and SLC29 families consist in three and four members, respectively, which differ in their substrate selectivity and their energy requirements. Tissue distribution of these transporters is not homogeneous among tissues, and their expression can be regulated. In epithelia, CNT and ENT proteins are mostly localized in the apical and basolateral membranes, respectively, which results in nucleoside and nucleoside-derived drugs vectorial flux. Nucleoside transporters can play physiological roles other than salvages, such as the modulation of extracellular and intracellular adenosine concentrations. Moreover, these transporters also have clinical significance. ENT proteins are target of dipyridamole and dilazep, used as vasodilatory drugs in the treatment of heart and vascular diseases. On the other hand, nucleoside transporters are responsible for the cellular uptake of currently used anticancer nucleoside-derived drugs, thus these membrane proteins might play a significant role in nucleoside-based chemotherapy. Finally, several polymorphisms have been described in CNT and ENT proteins that could affect nucleoside homeostasis, adenosine signalling events or nucleoside-derived drug cytotoxicity or pharmacokinetics.

Macrophages require different nucleoside transport systems for proliferation and activation

To evalúate the mechanisms involved in macrophage proliferation and activation, we studied the regulation of the nucleoside transport systems. In murine bone marrow‐derived macrophages, the nucleo‐sides required for DNA and RNA synthesis are recruited from the extracellular medium. M‐CSF induced macrophage proliferation and DNA and RNA synthesis, whereas interferon γ (IFN‐γ) led to activation, blocked proliferation, and induced only RNA synthesis. Macrophages express at least the concentrative systems N1 and N2 (CNT2 and CNT1 genes, respectively) and the equilibrative systems es and ei (ENT1 and ENT2 genes, respectively). Incubation with M‐CSF only up‐regulated the equilibrative system es. Inhibition of this transport system blocked M‐CSF‐dependent proliferation. Treatment with IFN‐γ only induced the concentrative N1 and N2 systems. IFN‐γ also down‐regulated the increased expression of the es equilibrative system induced by M‐CSF. Thus, macrophage proliferation and activation require selective regulation of nucleoside transporters and may respond to specific requirements for DNA and RNA synthesis. This report also shows that the nucleo‐side transporters are critical for macrophage proliferation and activation.

Transport of Lamivudine [(-)-β-l-2′,3′-Dideoxy-3′-thiacytidine] and High-Affinity Interaction of Nucleoside Reverse Transcriptase Inhibitors with Human Organic Cation Transporters 1, 2, and 3

Nucleoside reverse transcriptase inhibitors (NRTIs) need to enter cells to act against the HIV-1. Human organic cation transporters (hOCT1–3) are expressed and active in CD4+ T cells, the main target of HIV-1, and have been associated with antiviral uptake in different tissues. In this study, we examined whether NRTIs interact and are substrates of hOCT in cells stably expressing these transporters. Using [3H]N-methyl-4-phenylpyridinium, we found a high-affinity interaction among abacavir [[(1S,4R)-4-[2-amino-6-(cyclopropylamino)purin-9-yl]-cyclopent-2-enyl]methanol sulfate] (ABC); <0.08 nM], azidothymidine [3′-azido-3′-deoxythymidine (AZT); <0.4 nM], tenofovir disoproxil fumarate (<1.0 nM), and emtricitabine (<2.5 nM) and hOCTs. Using a wide range of concentrations of lamivudine [(-)-β-l-2′,3′-dideoxy-3′-thiacyitidine (3TC)], we determined two different binding sites for hOCTs: a high-affinity site (Kd1 = 12.3–15.4 pM) and a low-affinity site (Kd2 = 1.9–3.4 mM). Measuring direct uptake of [3H]3TC and inhibition with hOCT substrates, we identified 3TC as a novel substrate for hOCT1, 2, and 3, with hOCT1 as the most efficient transporter (Km = 1.25 ± 0.1 mM; Vmax = 10.40 ± 0.32 nmol/mg protein/min; Vmax/Km = 8.32 ± 0.40 μl/mg protein/min). In drug-drug interaction experiments, we analyzed cis-inhibition of [3H]3TC uptake by ABC and AZT and found that 40 to 50% was inhibited at low concentrations of the drugs (Ki = 22–500 pM). These data reveal that NRTIs experience a high-affinity interaction with hOCTs, suggesting a putative role for these drugs as modulators of hOCT activity. Finally, 3TC is a novel substrate for hOCTs and the inhibition of its uptake at low concentrations of ABC and AZT could have implications for the pharmacokinetics of 3TC.

Gemcitabine chemoresistance in pancreatic cancer: Molecular mechanisms and potential solutions

Ductal pancreatic adenocarcinoma is associated with a very poor prognosis and most patients are given palliative care. Chemotherapy in the form of gemcitabine has been found to reduce disease-related pain, and the otherwise frequently occurring weight changes, to increase Karnofsky performance status and quality of life and has also resulted in a modest improvement in survival time. The intracellular uptake of gemcitabine is dependent on nucleoside transporters, predominantly human equilibrative nucleoside transporter-1 (hENT-1), which is over-expressed in human pancreatic adenocarcinoma cells. Cellular resistance to gemcitabine can be intrinsic or acquired during gemcitabine treatment. One of the mechanisms is a decrease in hENT-1 expression. Modifications of gemcitabine not rendering it dependent on the nucleoside transporter may be a successful future mode of chemotherapy treatment, and determination of the nucleoside receptor status at the time of diagnosis could potentially also contribute to a more targeted therapy in the future.

Transport of Lamivudine (3TC) and High-Affinity Interaction of Nucleoside Reverse Transcriptase Inhibitors With Human Organic Cation Transporters 1, 2, and 3

Nucleoside reverse transcriptase inhibitors (NRTIs) need to enter cells to act against the HIV-1. Human organic cation transporters (hOCT1–3) are expressed and active in CD4+ T cells, the main target of HIV-1, and have been associated with antiviral uptake in different tissues. In this study, we examined whether NRTIs interact and are substrates of hOCT in cells stably expressing these transporters. Using [3H]N-methyl-4-phenylpyridinium, we found a high-affinity interaction among abacavir [[(1S,4R)-4-[2-amino-6-(cyclopropylamino)purin-9-yl]-cyclopent-2-enyl]methanol sulfate] (ABC); <0.08 nM], azidothymidine [3′-azido-3′-deoxythymidine (AZT); <0.4 nM], tenofovir disoproxil fumarate (<1.0 nM), and emtricitabine (<2.5 nM) and hOCTs. Using a wide range of concentrations of lamivudine [(-)-β-l-2′,3′-dideoxy-3′-thiacyitidine (3TC)], we determined two different binding sites for hOCTs: a high-affinity site (Kd1 = 12.3–15.4 pM) and a low-affinity site (Kd2 = 1.9–3.4 mM). Measuring direct uptake of [3H]3TC and inhibition with hOCT substrates, we identified 3TC as a novel substrate for hOCT1, 2, and 3, with hOCT1 as the most efficient transporter (Km = 1.25 ± 0.1 mM; Vmax = 10.40 ± 0.32 nmol/mg protein/min; Vmax/Km = 8.32 ± 0.40 μl/mg protein/min). In drug-drug interaction experiments, we analyzed cis-inhibition of [3H]3TC uptake by ABC and AZT and found that 40 to 50% was inhibited at low concentrations of the drugs (Ki = 22–500 pM). These data reveal that NRTIs experience a high-affinity interaction with hOCTs, suggesting a putative role for these drugs as modulators of hOCT activity. Finally, 3TC is a novel substrate for hOCTs and the inhibition of its uptake at low concentrations of ABC and AZT could have implications for the pharmacokinetics of 3TC.

Complex regulation of nucleoside transporter expression in epithelial and immune system cells

Nucleoside transporters have a variety of functions in the cell, such as the provision of substrates for nucleic acid synthesis and the modulation of purine receptors by determining agonist availability. They also transport a wide range of nucleoside-derived antiviral and anticancer drugs. Most mammalian cells coexpress several nucleoside transporter isoforms at the plasma membrane, which are differentially regulated. This paper reviews studies on nucleoside transporter regulation, which has been extensively characterized in the laboratory in several model systems: the hepatocyte, an epithelial cell type, and immune system cells, in particular B cells, which are non-polarized and highly specialized. The hepatocyte co-expresses at least two Na+-dependent nucleoside transporters, CNT1 and CNT2, which are up-regulated during cell proliferation but may undergo selective loss in certain experimental models of hepatocarcinomas. This feature is consistent with evidence that CNT expression also depends on the differentiation status of the hepatocyte. Moreover, substrate availability also modulates CNT expression in epithelial cells, as reported for hepatocytes and jejunum epithelia from rats fed nucleotide-deprived diets. In human B cell lines, CNT and ENT transporters are co-expressed but differentially regulated after B cell activation triggered by cytokines or phorbol esters, as described for murine bone marrow macrophages induced either to activate or to proliferate. The complex regulation of the expression and activity of nucleoside transporters hints at their relevance in cell physiology.

Nucleoside transporters in chronic lymphocytic leukaemia

Nucleoside derivatives have important therapeutic activity in chronic lymphocytic leukaemia (CLL). Experimental evidence indicates that in CLL cells most of these drugs induce apoptosis ex vivo, suggesting that programmed cell death is the mechanism of their therapeutic action, relying upon previous uptake and metabolic activation. Although defective apoptosis and poor metabolism often cause resistance to treatment, differential uptake and/or export of nucleosides and nucleotides may significantly modulate intracellular drug bioavailability and, consequently, responsiveness to therapy. Two gene families, SLC28 and SLC29, encode transporter proteins responsible for concentrative and equilibrative nucleoside uptake (CNT and ENT, respectively). Furthermore, selected members of the expanding ATP-binding cassette (ABC) protein family have recently been identified as putative efflux pumps for the phosphorylated forms of these nucleoside-derived drugs, ABCC11 (MRP8) being a good candidate to modulate cell sensitivity to fluoropyrimidines. Sensitivity of CLL cells to fludarabine has also been recently correlated with ENT-type transport function, suggesting that, besides the integrity of apoptotic pathways and appropriate intracellular metabolism, transport across the plasma membrane is also a relevant event during CLL treatment. As long as nucleoside transporter expression in leukaemia cells is not constitutive, the possibility of regulating nucleoside transporter function by pharmacological means may also contribute to improve therapy.

EXPRESSION OF THE NUCLEOSIDE-DERIVED DRUG TRANSPORTERS hCNT1, hENT1 AND hENT2 IN GYNECOLOGIC TUMORS

Deoxynucleoside analogs are used in the treatment of a variety of solid tumors. Their transport across the plasma membrane may determine their cytotoxicity and thus nucleoside transporter (NT) expression patterns may be of clinical relevance. Lack of appropriate antibodies for use in paraffin‐embedded biopsies has been a bottleneck to undertake high‐throughput analysis of NT expression in solid tumors. Here we report the characterization of 2 new antibodies raised against the low‐affinity equilibrative NTs, hENT1 and hENT2, suitable for that purpose. These 2 antisera, along with a previously characterized antibody that specifically recognizes the high‐affinity Na‐dependent concentrative NT, hCNT1, have been used to analyze, using a tissue array approach, NT expression in gynecologic cancers (90 ovarian, 80 endometrial and 118 uterine cervix carcinomas). Human CNT1 was not detected in 33% and 39% of the ovarian and uterine cervix carcinomas, respectively, whereas hENT1 and hENT2 expression was significantly retained in a high percentage of tumors (91% and 96% for hENT1, 84% and 98% for hENT2, in ovarian and cervix carcinomas, respectively). Only a few endometrial carcinomas (15%) were found to be negative for hCNT1, but they all retained hENT1 and hENT2 expression. In ovarian cancer, the loss of all 3 NT proteins was a more common event in the clear cell histologic subtype than in the serous, mucinous and endometrioid histotypes. In uterine cervix tumors, the loss of expression of hCNT1 was significantly associated with the adenocarcinoma subtype. In summary, hCNT1 was by far the isoform whose expression was most frequently reduced or lost in the 3 types of gynecologic tumors analyzed. Moreover, NT expression is related to the type of gynecologic tumor and its specific subtype, hCNT1 protein loss being highly correlated with poor prognosis histotypes. Since hCNT1, hENT1 and hENT2 recognize fluoropyrimidines as substrates, but with different affinities, this study anticipates high variability in drug uptake efficiency in solid tumors.

Regulation of nucleoside transport by lipopolysaccharide, phorbol esters, and tumor necrosis factor-alpha in human B-lymphocytes.

Nucleoside transport systems and their regulation in human B-lymphocytes have been characterized using the cell lines Raji and Bare lymphoma syndrome-1 (BLS-1) as experimental models. These cells express at least three different nucleoside transport systems as follows: a nitrobenzylthioinosine-sensitive equilibrative transport system of the es-type, which appears to be associated with hENT1 expression, and two Na+-dependent transport systems that may correspond to N1 and to the recently characterized N5-type, which is nitrobenzylthioinosine-sensitive and guanosine-preferring. B cell activators such as phorbol 12-myristate 13-acetate and lipopolysaccharide (LPS) up-regulate both concentrative transport systems but down-regulate the equilibrativees-type transporter, which correlates with lower hENT1 mRNA levels. These effects are dependent on protein kinase C activity. Phorbol 12-myristate 13-acetate and LPS also induce an increase in tumor necrosis factor-α (TNF–α) mRNA levels, which suggest that this cytokine may mediate some of the effects triggered by these agents, since addition of TNF-α alone can increase N1 and N5 transport activities by a mechanism that also depends on protein kinase C activation. Interestingly, TNF-α down-regulates esactivity, but this effect cannot be abolished by inhibiting protein kinase C. This study reveals differential regulation of nucleoside transport systems following activation of human B-lymphocyte cell lines by agents of physiological relevance such as TNF-α and LPS. Moreover, it indicates that the recently characterized N5 transport system can also be regulated following B cell activation, which may be relevant to lymphocyte physiology and to the treatment of lymphocyte malignancies.