Sandra Pérez-Torras

Adenoviral-mediated overexpression of human equilibrative nucleoside transporter 1 (hENT1) enhances gemcitabine response in human pancreatic cancer

Nucleoside-derived anticancer agents must be transported across the plasma membrane as a preliminary step to their conversion into active drugs. Hence, modulation of a specific nucleoside transporter may affect bioavailability and contribute significantly to sensitizing tumor cells to these anticancer agents. We have generated and functionally characterized a new recombinant adenovirus (Ad-hENT1) that has allowed us to overexpress the equilibrative nucleoside transporter hENT1 and to analyze its effects in human pancreatic tumor cells. Overexpression of hENT1 is associated with changes in cell cycle profile, in a variable manner depending on the particular cell type, thus suggesting a metabolic link between hENT1-mediated transport processes and the enzymatic machinery responsible for intracellular nucleoside metabolism. When assayed in vivo in a human pancreatic adenocarcinoma xenograft, intratumoral Ad-hENT1 injection improved the therapeutic response to gemcitabine. In summary, hENT1 overexpression is associated with alterations in nucleoside enzymatic machinery and cell cycle progression in cultured cells and enhances gemcitabine action in vivo.

Nucleoside transporter proteins as biomarkers of drug responsiveness and drug targets.

Nucleoside and nucleobase analogs are currently used in the treatment of solid tumors, lymphoproliferative diseases, viral infections such as hepatitis and AIDS, and some inflammatory diseases such as Crohn. Two gene families are implicated in the uptake of nucleosides and nucleoside analogs into cells, SCL28 and SLC29. The former encodes hCNT1, hCNT2, and hCNT3 proteins. They translocate nucleosides in a Na+ coupled manner with high affinity and some substrate selectivity, being hCNT1 and hCNT2 pyrimidine- and purine-preferring, respectively, and hCNT3 a broad selectivity transporter. SLC29 genes encode four members, being hENT1 and hENT2 the only two which are unequivocally implicated in the translocation of nucleosides and nucleobases (the latter mostly via hENT2) at the cell plasma membrane. Some nucleoside-derived drugs can also interact with and be translocated by members of the SLC22 gene family, particularly hOCT and hOAT proteins. Inter-individual differences in transporter function and perhaps, more importantly, altered expression associated with the disease itself might modulate the transporter profile of target cells, thereby determining drug bioavailability and action. Drug transporter pharmacology has been periodically reviewed. Thus, with this contribution we aim at providing a state-of-the-art overview of the clinical evidence generated so far supporting the concept that these membrane proteins can indeed be biomarkers suitable for diagnosis and/or prognosis. Last but not least, some of these transporter proteins can also be envisaged as drug targets, as long as they can show “transceptor” functions, in some cases related to their role as modulators of extracellular adenosine levels, thereby providing a functional link between P1 receptors and transporters.

Connexin-26 Is a Key Factor Mediating Gemcitabine Bystander Effect

Gemcitabine is a nucleoside analogue with anticancer activity. Inside the cell, it is sequentially phosphorylated to generate the active drug. Phosphorylated nucleoside analogues have been shown to traffic through gap junctions. We investigated the participation of gap junctional intercellular communication (GJIC) as a possible mechanism spreading gemcitabine cytotoxicity in pancreatic tumors. Immunohistochemical analysis of pancreatic cancer biopsies revealed increased connexin 26 (Cx26) content but loss of connexins 32 (Cx32) and 43 (Cx43) expression. Cx26 abundance in neoplastic areas was confirmed by Cx26 mRNA in situ hybridization. Heterogeneity on the expression levels and the localization of Cx26, Cx32, and Cx43 were identified in pancreatic cancer cells and found to be associated with the extent of GJIC, and correlated with gemcitabine bystander cytotoxic effect. The abundance of Cx26 at the contact points in tumoral regions prompted us to study the involvement of Cx26 in the GJIC of gemcitabine toxic metabolites and their influence on the antitumoral effects of gemcitabine. Knockdown of Cx26 led to decreased GJIC and reduced gemcitabine bystander killing whereas overexpression of Cx26 triggered increased GJIC and enhanced the gemcitabine cytotoxic bystander effect. Gemcitabine treatment of mice bearing tumors, with a high GJIC capacity, resulted in a significant delay in tumor progression. Interestingly, gemcitabine administration in mice bearing tumors that overexpress Cx26 triggered a dramatic tumor regression of 50% from the initial volume. This study shows that Cx26 participates in the gap junction–mediated bystander cytoxic effect of gemcitabine and provides evidence that upregulation of Cx26 improves gemcitabine anticancer efficacy. Mol Cancer Ther; 10(3); 505–17. ©2011 AACR.

New role of the human equilibrative nucleoside transporter 1 (hENT1) in Epithelial-to-mesenchymal transition in renal tubular cells

Epithelial‐to‐mesenchymal transition (EMT) is an important pro‐fibrotic event in which tubular epithelial cells are transformed into myofibroblasts. Nucleoside transporters (NT) are regulated by many factors and processes, some of which are involved in fibrosis, such as cytokines, inflammation, and proliferation. Equilibrative nucleoside transporter 1 (ENT1) has been proved to be the most widely expressed adenosine transporter. In that sense, ENT1 may be a key player in cell damage signaling. Here we analyze the role of human ENT1 (hENT1) in the EMT process in proximal tubular cells. Addition of the main inducer of EMT, the transforming growth factor‐β1, to HK‐2 cells increased hENT1 mRNA and protein level expression. ENT1‐mediated adenosine uptake was also enhanced. When cells were incubated with dipyridamole to evaluate the potential contribution of ENT1 to EMT by blocking its transport activity, EMT was induced. Moreover, the knock down of hENT1 with siRNA induced EMT and collagen production in HK‐2 cells. Kidneys isolated from ENT1 knockout mice showed higher levels of interstitial collagen and α‐SMA positive cells than wild‐type mice. Our results point to a new potential role of hENT1 as a modulator of EMT in proximal tubular cells. In this sense, hENT1 could be involved in renal protection processes, and the loss or reduced expression of hENT1 would lead to an increased vulnerability of cells to the onset and/or progression of renal fibrosis. J. Cell. Physiol. 227: 1521–1528, 2012.

Aquaporin 3 (AQP3) participates in the cytotoxic response to nucleoside-derived drugs

BackgroundNucleoside analogs used in the chemotherapy of solid tumors, such as the capecitabine catabolite 5′-deoxy-5-fluorouridine (5′-DFUR) trigger a transcriptomic response that involves the aquaglyceroporin aquaporin 3 along with other p53-dependent genes. Here, we examined whether up-regulation of aquaporin 3 (AQP3) mRNA in cancer cells treated with 5′-DFUR represents a collateral transcriptomic effect of the drug, or conversely, AQP3 participates in the activity of genotoxic agents.MethodsThe role of AQP3 in cell volume increase, cytotoxicity and cell cycle arrest was analyzed using loss-of-function approaches.Results5′-DFUR and gemcitabine, but not cisplatin, stimulated AQP3 expression and cell volume, which was partially and significantly blocked by knockdown of AQP3. Moreover, AQP3 siRNA significantly blocked other effects of nucleoside analogs, including G1/S cell cycle arrest, p21 and FAS up-regulation, and cell growth inhibition. Short incubations with 5-fluorouracil (5-FU) also induced AQP3 expression and increased cell volume, and the inhibition of AQP3 expression significantly blocked growth inhibition triggered by this drug. To further establish whether AQP3 induction is related to cell cycle arrest and apoptosis, cells were exposed to long incubations with escalating doses of 5-FU. AQP3 was highly up-regulated at doses associated with cell cycle arrest, whereas at doses promoting apoptosis induction of AQP3 mRNA expression was reduced.ConclusionsBased on the results, we propose that the aquaglyceroporin AQP3 is required for cytotoxic activity of 5’-DFUR and gemcitabine in the breast cancer cell line MCF7 and the colon adenocarcinoma cell line HT29, and is implicated in cell volume increase and cell cycle arrest.

Characterization of human pancreatic orthotopic tumor xenografts suitable for drug screening

BackgroundEfforts to identify novel therapeutic options for human pancreatic ductal adenocarcinoma (PDAC) have failed to result in a clear improvement in patient survival to date. Pancreatic cancer requires efficient therapies that must be designed and assayed in preclinical models with improved predictor ability. Among the available preclinical models, the orthotopic approach fits with this expectation, but its use is still occasional.MethodsAn in vivo platform of 11 orthotopic tumor xenografts has been generated by direct implantation of fresh surgical material. In addition, a frozen tumorgraft bank has been created, ensuring future model recovery and tumor tissue availability.ResultsTissue microarray studies allow showing a high degree of original histology preservation and maintenance of protein expression patterns through passages. The models display stable growth kinetics and characteristic metastatic behavior. Moreover, the molecular diversity may facilitate the identification of tumor subtypes and comparison of drug responses that complement or confirm information obtained with other preclinical models.ConclusionsThis panel represents a useful preclinical tool for testing new agents and treatment protocols and for further exploration of the biological basis of drug responses.

Ribonucleotide reductase is an effective target to overcome gemcitabine resistance in gemcitabine-resistant pancreatic cancer cells with dual resistant factors.

Gemcitabine is widely used for pancreatic, lung, and bladder cancer. However, drug resistance against gemcitabine is a large obstacle to effective chemotherapy. Nucleoside transporters, nucleoside and nucleotide metabolic enzymes, and efflux transporters have been reported to be involved in gemcitabine resistance. Although most of the resistant factors are supposed to be related to each other, it is unclear how one factor can affect the other one. In this study, we established gemcitabine-resistant pancreatic cancer cell lines. Gemcitabine resistance in these cells is caused by two major processes: a decrease in gemcitabine uptake and overexpression of ribonucleotide reductase large subunit (RRM1). Knockdown of RRM1, but not the overexpression of concentrative nucleoside transporter 1 (CNT1), could completely overcome the gemcitabine resistance. RRM1 knockdown in gemcitabine-resistant cells could increase the intracellular accumulation of gemcitabine by increasing the nucleoside transporter expression. Furthermore, a synergistic effect was observed between hydroxyurea, a ribonucleotide reductase (RR) inhibitor, and gemcitabine on the gemcitabine-resistant cells. Here we indicate that RR is one of the most promising targets to overcome gemcitabine resistance in gemcitabine-resistant cells with dual resistant factors.

Transportome Profiling Identifies Profound Alterations in Crohn’s Disease Partially Restored by Commensal Bacteria

BACKGROUND AND AIMS Several transport alterations have been described in intestinal inflammatory diseases. This is relevant because the primary function of the intestine is nutrient and mineral absorption. However, analysis of the transportome as a whole and the effect of commensal bacteria on it have not been addressed so far. METHODS Five healthy and 6 Crohns disease (CD) samples were hybridized to human HT-12 V4 Illumina GeneChip. Results were validated by reverse transcription-polymerase chain reaction (RT-PCR) analysis and with additional array data. Organ culture assays were performed from mucosa ileal wall specimens collected at surgery. Samples were incubated with or without commensal bacteria for 4 hours. Finally, RNA was isolated for microarray processing. RESULTS The analysis of CD versus healthy ileal mucosa demonstrated upregulation of previously described genes involved in immunity and the inflammatory response in this disease. Interestingly, whole transcriptional analysis revealed profound alterations in the transportome profile. Sixty-two solute carrier (SLC) transporters displayed different expression patterns, most of them being downregulated. Changes were confirmed by RT-PCR in a randomly chosen subset of SLCs. A large number of amino acid transporters and most members of the enteric purinome were found to be altered. Most of these proteins were found at the apical membrane of the enterocyte, which could impair both amino acid absorption and purinergic signalling. Treatment of ileum specimen explants with commensal bacteria restored almost all CD transportome alterations. CONCLUSIONS These results describe the altered transportome profile in CD and open the possibility of restoring transportome complications with commensal bacteria.

Role of the Transporter Regulator Protein (RS1) in the Modulation of Concentrative Nucleoside Transporters (CNTs) in Epithelia

SLC28 genes encode three plasma membrane transporter proteins, human concentrative nucleoside transporter (CNT)1, CNT2, and CNT3, all of which are implicated in the uptake of natural nucleosides and a variety of nucleoside analogs used in the chemotherapy of cancer and viral and inflammatory diseases. Mechanisms determining their trafficking toward the plasma membrane are not well known, although this might eventually become a target for therapeutic intervention. The transporter regulator RS1, which was initially identified as a short-term, post-transcriptional regulator of the high-affinity, Na+-coupled, glucose transporter sodium-dependent glucose cotransporter 1, was evaluated in this study as a candidate for coordinate regulation of membrane insertion of human CNT-type proteins. With a combination of studies with mammalian cells, Xenopus laevis oocytes, and RS1-null mice, evidence that RS1 down-regulates the localization and activity at the plasma membrane of the three members of this protein family (CNT1, CNT2, and CNT3) is provided, which indicates the biochemical basis for coordinate regulation of nucleoside uptake ability in epithelia and probably in other RS1-expressing cell types.

Human pancreatic cancer stem cells are sensitive to dual inhibition of IGF-IR and ErbB receptors

BackgroundPancreatic ductal adenocarcinoma is a particularly challenging malignancy characterized by poor responsiveness to conventional chemotherapy. Although this tumor frequently overexpresses or possesses constitutively activated variants of IGF-IR and EGFR/Her-2, clinical trials using inhibitors of these receptors have failed. ErbB receptors have been proposed as one mechanism involved in the resistance to IGF-IR inhibitors. Therefore, combined treatment with inhibitors of both IGF-IR and ErbB receptors would appear to be a good strategy for overcoming the emergence of resistance.MethodsSensitivity of cells to NVP-AEW541 and lapatinib in single or combination treatment was assessed by MTT or WST-8 assays in a panel of human pancreatic cancer cell lines and cancer stem cells. Tumorspheres enriched in cancer stem cells were obtained from cultures growing in non-adherent cell plates. The effects on cell signalling pathways were analyzed by Western blot.ResultsWe found that combined treatment with the IGF-IR and EGFR/Her-2 inhibitors NVP-AEW541 and lapatinib, respectively, synergistically inhibited pancreatic cancer cell growth. Analysis at molecular level argued in favor of cross-talk between IGF-IR and ErbBs pathways at IRS-1 level and indicated that the synergistic effect is associated with the total abolishment of Akt, Erk and IRS-1 phosphorylation. Moreover, these inhibitors acted synergistically in tumorsphere cultures to eliminate cancer stem cells, in contrast to their resistance to gemcitabine.ConclusionsTaken together, these data indicate that simultaneous blockade of IGF-IR and EGFR/Her-2 using NVP-AEW541 and lapatinib may overcome resistance in pancreatic cancer. Thus, the synergy observed with this combined treatment indicates that it may be possible to maximize patient benefit with the appropriate combination of currently known anticancer agents.