Human equilibrative nucleoside transporter 1 is associated with the chemosensitivity of gemcitabine in human pancreatic adenocarcinoma and biliary tract carcinoma cells

Gemcitabine has been one of the most commonly used agents for pancreatic adenocarcinoma chemotherapy, but the determinants of the sensitivity of and resistance to this agent are not yet fully understood. In this study with pancreatic carcinoma and biliary tract carcinoma cell lines, we examined the gene expression levels of nucleotide transporters and others related to the metabolism of gemcitabine in the light of sensitivity to this agent. Quantitative RT-PCR demonstrated that one of the nucleotide transporter genes; human equilibrative nucleoside transporter 1 (hENT1) was associated with the sensitivity to gemcitabine as represented by IC50, while the other genes for nucleotide transporter and metabolism were not. We conclude that increased hENT1 expression is a most important determinant of gemcitabine sensitivity at least in an in vitro study.     

Enhanced efficacy of gemcitabine by indole-3-carbinol in pancreatic cell lines: the role of human equilibrative nucleoside transporter 1

Pancreatic cancer patients treated with gemcitabine (2',2'-difluorodeoxycytidine) can eventually develop resistance. Recently, published data from our laboratory demonstrated enhanced efficacy of gemcitabine with the dietary agent, indole-3-carbinol (I3C). The current study examined the possible mechanism for this I3C-enhanced efficacy. Several pancreatic cell lines (BxPC-3, Mia Paca-2, PL-45, AsPC-1 and PANC-1) were examined for modulation of human equilibrative nucleoside transporter 1 (hENT1) expression, the major transporter for gemcitabine, by I3C alone and combined with gemcitabine. I3C significantly (p<0.01) up-regulated hENT1 expression in several cell lines. Gemcitabine alone showed no effect on hENT1 expression. However, combining gemcitabine with I3C further increased hENT1 expression. Cell viability assays revealed no effect of I3C on normal cells, hTERT-HPNE. hENT1-specific inhibitor, nitrobenzylthioinosine, significantly abrogated I3C-induced gemcitabine cytotoxicity, further demonstrating its specificity. This study demonstrates that up-regulation of hENT1 expression may be a novel mechanism involved in the additive effect of I3C and gemcitabine.     

人类平衡型核苷转运蛋白1预测吉西他滨治疗胰腺癌患者预后的价值:一项Meta分析

目的:采用Meta分析探讨人类平衡型核苷转运蛋白1(human equilibrative nucleoside transporter 1,bENT1)预测接受吉西他滨治疗的胰腺癌患者预后的价值.方法:检索Cochrane Library、Medline和Embase等数据库,筛选有关hENT1表达与胰腺癌预后的相关文献,分析hENT1低表达和高表达与接受吉西他滨治疗的胰腺癌患者的总生存期和无病生存期的关系,采用Meta分析合并风险比(hazard ratio,HR).结果:共纳入9篇合格文献.在接受吉西他滨治疗的胰腺癌患者中,hENT1低表达组有较高的死亡风险(HR=2.61,95％可信区间:2.03～3.34)和复发风险(HR=2.62,95％可信区间:1.94～3.54).在接受吉西他滨术后辅助治疗的患者中,hENT1低表达组同样有着较高的死亡风险(HR=2.49,95％可信区间:1.86～3.33)和复发风险(HR=2.79,95％可信区间:1.98～3.94).hENT1蛋白或mRNA低表达均预示着较高的死亡和复发风险.结论:hENT1表达水平在接受吉西他滨治疗的胰腺癌患者中具有预测预后的价值.     

Differential expression of human equilibrative nucleoside transporter 1 (hENT1) protein in the Reed-Sternberg cells of Hodgkin's disease

Gemcitabine is a cytotoxic nucleoside analog with activity in relapsing/refractory Hodgkin's disease (HD). Because gemcitabine is hydrophilic, it requires plasma membrane nucleoside transporter proteins to access intracellular targets. The most abundant and widely distributed transporter in human cells is human equilibrative nucleoside transporter 1 (hENT1). Because our prior studies showed that a deficiency in hENT1 confers high-level resistance to gemcitabine toxicity in vitro, we developed an immunohistochemical method to assess the hENT1 abundance of cells in tumor tissue. We now report the application of this method for visualizing the hENT1 protein abundance in the plasma membranes of Reed-Sternberg cells in lymph nodes of HD patients. Frozen sections of 30 lymph nodes were stained with monoclonal antibodies (mAb 10D7G2) raised against a synthetic peptide comprised of residues 254-271 from the large intracellular loop of hENT1 and staining intensity was scored on a 0-4 + scale. hENT1-staining intensity varied among HD lymph node samples (score/n; 0/8; 1/10; 2/9; 3/3; 4/0) and suggested that at least 60% of the tumors appeared hENT1 deficient. Because Epstein-Barr virus (EBV) is often associated with HD, staining for Epstein-Barr early RNA was also examined. Although 9/30 patients tested positive for EBV, there was no correlation with hENT1 staining. hENT1-staining intensities were positively correlated with age of the patient but were independent of other clinical, laboratory or pathology features (tumor stage, histologic subtype, presence of B symptoms, staining for CD15 or CD30, serum biochemistry, disease free survival, and overall survival). We conclude that, because hENT1 deficiency has been previously related to nucleoside-drug resistance, immunohistochemical staining for hENT1 warrants evaluation as a predictive tool for guiding the appropriate use of gemcitabine in the treatment of HD.     

Determinants of sensitivity and resistance to gemcitabine: the roles of human equilibrative nucleoside transporter 1 and deoxycytidine kinase in non-small cell lung cancer

Gemcitabine is one of the most commonly used agents for lung cancer chemotherapy, but the determinants of sensitivity and/or resistance to this agent are not yet fully understood. In this study we used quantitative RT-PCR to examine the expression levels of human equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (dCK) genes in non-small cell lung cancer (NSCLC) cell lines in relation to sensitivity and resistance to gemcitabine. The basal expression levels of hENT1 were significantly correlated with the IC50 values for gemcitabine (r =-0.6769, P = 0.0005), whereas dCK expression levels were not. In a highly gemcitabine-sensitive cell line, NCI-H23, the sensitivity to gemcitabine was inhibited by nitrobenzylmercaptopurine ribonucleoside (NBMPR), an inhibitor of hENT1, without significant modulation of hENT1 expression. These data suggest that hENT1 is associated with gemcitabine sensitivity in lung cancer. We also continuously exposed NCI-H23 cells to gemcitabine and subsequently established the drug-resistant clone H23/GEM-R, which showed a significant decrease of dCK expression; however, hENT1 expression was not altered in the continuously exposed sublines or in the resistant clone. We conclude that increased hENT1 expression is a determinant of gemcitabine sensitivity, while decreased dCK expression is associated with acquired resistance to gemcitabine in NSCLC cells. Thus, hENT1 and dCK might be useful as predictive markers for efficacy of gemcitabine therapy in NSCLC.     

Human equilibrative nucleoside transporter 1 (hENT1) expression is a potential predictive tool for response to gemcitabine in patients with advanced cholangiocarcinoma

BackgroundCholangiocarcinoma (CC) is a rare cancer of the liver. Surgery offers the only chance for cure. When surgery is unfeasible, chemotherapy is the backbone of treatment. The combined administration of cisplatin and gemcitabine is considered standard of care. Human equilibrative nucleoside transporter 1 (hENT1) is the major transporter responsible for gemcitabine uptake into cells. hENT1 expression is associated with an increased survival for patients receiving gemcitabine after pancreatic cancer surgery, suggesting that hENT1 is predictive of response to gemcitabine.AimTo determine whether there is a correlation between the expression of hENT1 and disease outcome in CC.MethodsA retrospective study on 43 patients treated at our centre with a locally advanced or metastatic CC, who received first line treatment with gemcitabine, was performed.ResultsFor the whole population, median Progression Free Survival (PFS) and overall survival (OS) were 4.0 (95% Confidence Interval 2.7–5.3 months) and 10.0 months (95% CI 6.8–13.2 months), respectively. From the 26 samples available for hENT1 staining, 18 (69%) and 8 (31%) patients had high and low hENT1 immunostaining, respectively. The median PFS were 2.0 versus 6.0 months for low versus high staining respectively (p = 0.012). The median OS were 5.0 versus 11.0 months for low versus high staining, respectively (p = 0.036). On multivariate analysis, hENT1 expression was the single independent predictive factor associated with prolonged PFS (HR 0.35, p = 0.023) and OS (HR 0.41, p = 0.046).ConclusionIn this study we show the potential of hENT1 expression as a predictor of outcome in CC treated with gemcitabine. Larger studies are necessary to confirm these promising results.     

Immunohistochemical variation of human equilibrative nucleoside transporter 1 protein in primary breast cancers.

Gemcitabine and capecitabine are nucleoside analogues used in chemotherapy strategies for the treatment of breast cancer. We previously demonstrated that deficiency in hENT1, the most abundant and widely distributed plasma membrane nucleoside transporter in human cells, confers high-level resistance to gemcitabine toxicity in vitro, whereas the relationship between hENT1 activity and capecitabine toxicity is unknown. To determine the relationship between capecitabine cytotoxicity and hENT1 abundance, cultured MDA-MB-435s human mammary carcinoma cells were exposed to graded concentrations of the capecitabine metabolites, 5'-deoxy-5-fluorouridine or 5-fluorouracil, in the presence and absence of nitrobenzylmercaptopurine ribonucleoside (NBMPR), a tight-binding inhibitor of hENT1. The presence of NBMPR reduced the cytotoxic effects of 5'-deoxy-5-fluorouridine, indicating that hENT1 also enabled cellular uptake of this capecitabine metabolite by breast cancer cells. We report here the development of an immunohistochemical method to assess the hENT1 abundance of malignant cells in solid tumors. Frozen sections of 33 primary breast cancers were stained with monoclonal antibodies raised against a synthetic peptide derived from the large intracellular loop of hENT1, and staining intensity was scored on a 0-4+ scale. hENT1 staining intensity varied markedly among breast samples (4 with score 0, 5 with score 1+, 7 with score 2+, 14 with score 3+, 3 with score 4+), suggesting that at least 9 of the tumors were hENT1 deficient. We conclude that because hENT1 deficiency has previously been associated with nucleoside drug resistance, immunohistochemical staining of hENT1 warrants further study as a predictive tool for guiding the appropriate use of gemcitabine and capecitabine in the treatment of breast cancer.     

Gemcitabine chemoresistance and molecular markers associated with gemcitabine transport and metabolism in human pancreatic cancer cells

To identify predictive molecular markers for gemcitabine resistance, we investigated changes in the expression of four genes associated with gemcitabine transport and metabolism during the development of acquired gemcitabine resistance of pancreatic cancer cell lines. The expression levels of human equilibrative nucleoside transporter-1 (hENT1), deoxycytidine kinase (dCK), RRM1, and RRM2 mRNA were analysed by real-time light cycler-PCR in various subclones during the development of acquired resistance to gemcitabine. Real-time light cycler-PCR demonstrated that the expression levels of either RRM1 or RRM2 progressively increased during the development of gemcitabine resistance. Expression of dCK was slightly increased in cells resistant to lower concentrations of gemcitabine, but was decreased below the undetectable level in higher concentration-resistant subclones. Expression of hENT1 was increased in the development of gemcitabine resistance. As acquired resistance to gemcitabine seems to correlate with the balance of these four factors, we calculated the ratio of hENT1 x dCK/RRM1 x RRM2 gene expression in gemcitabine-resistant subclones. The ratio of gene expression decreased progressively with development of acquired resistance in gemcitabine-resistant subclones. Furthermore, the expression ratio significantly correlated with gemcitabine sensitivity in eight pancreatic cancer cell lines, whereas no single gene expression level correlated with the sensitivity. These results suggest that the sensitivity of pancreatic cancer cells to gemcitabine is determined by the ratio of four factors involved in gemcitabine transport and metabolism. The ratio of the four gene expression levels correlates with acquired gemcitabine-resistance in pancreatic cancer cells, and may be useful as a predictive marker for the efficacy of gemcitabine therapy in pancreatic cancer patients.     

The oncogenic receptor ErbB2 modulates gemcitabine and irinotecan/SN-38 chemoresistance of human pancreatic cancer cells via hCNT1 transporter and multidrug-resistance associated protein MRP-2

Pancreatic adenocarcinoma (PDAC) is one of the most deadly cancers because of a lack of early diagnotic markers and efficient therapeutics. The fluorinated analog of deoxycytidine, gemcitabine and emerging FOLFIRINOX protocol (5-fluorouracil (5-FU), irinotecan/SN-38, oxaliplatin and leucovorin) are the main chemotherapies to treat PDAC. The ErbB2/HER2 oncogenic receptor is commonly overexpressed in PDAC. In this context, we aimed to decipher the ErbB2-mediated mechanisms of chemoresistance to the two main chemotherapy protocols used to treat PDAC.ErbB2 knocking down (KD) in CAPAN-1 and CAPAN-2 cells led to an increased sensitivity to gemcitabine and an increased resistance to irinotecan/SN-38 both in vitro and in vivo (subcuteanous xenografts) This was correlated to an increase of hCNT1 and hCNT3 transporters and ABCG2, MRP1 and MRP2 ATP-binding cassette transporters expression and resistance to cell death. We also show that MRP2 is repressed following activation of JNK, Erk1/2 and NF-κB pathways by ErbB2. Finally, in datasets of human PDAC samples, ErbB2 and MRP2 expression was conversely correlated. Altogether, we propose that ErbB2 mediates several intracellular mechanisms linked to PDAC cell chemoresistance that may represent potential targets in order to ameliorate chemotherapy response and allow stratification of patients eligible for either gemcitabine or FOLFIRINOX treatment.     

The human equilibrative nucleoside transporter 1 mediates in vitro cytarabine sensitivity in childhood acute myeloid leukaemia

Cytarabine (ara-C) is the most effective agent for the treatment of acute myeloid leukaemia (AML). Aberrant expression of enzymes involved in the transport/metabolism of ara-C could explain drug resistance. We determined mRNA expression of these factors using quantitative-real-time-PCR in leukemic blasts from children diagnosed with de novo AML. Expression of the inactivating enzyme pyrimidine nucleotidase-I (PN-I) was 1.8-fold lower in FAB-M5 as compared to FAB-M1/2 (P=0.007). In vitro sensitivity to deoxynucleoside analogues was determined using the MTT-assay. Human equilibrative nucleoside transporter-1 (hENT1) mRNA expression and ara-C sensitivity were significantly correlated (rp=-0.46; P=0.001), with three-fold lower hENT1 mRNA levels in resistant patients (P=0.003). hENT1 mRNA expression also seemed to correlate inversely with the LC50 values of cladribine (rp=-0.30; P=0.04), decitabine (rp=-0.29; P=0.04) and gemcitabine (rp=-0.33; P=0.02). Deoxycytidine kinase (dCK) and cytidine deaminase (CDA) mRNA expression seemed to correlate with in vitro sensitivity to gemcitabine (rp=-0.31; P=0.03) and decitabine (rp=0.33; P=0.03), respectively. The dCK/PN-I ratio correlated inversely with LC50 values for gemcitabine (rp=-0.45, P=0.001) and the dCK/CDA ratio seemed to correlate with LC50 values for decitabine (rp=-0.29; 0.04). In conclusion, decreased expression of hENT1, which transports ara-C across the cell membrane, appears to be a major factor in ara-C resistance in childhood AML.     

The absence of human equilibrative nucleoside transporter 1 expression predicts nonresponse to gemcitabine-containing chemotherapy in non-small cell lung cancer

We report here the development of a polyclonal antibody for human equilibrative nucleoside transporter 1 (hENT1) and assess the expression of hENT1 in non-small cell lung cancer (NSCLC) patients who were treated with gemcitabine-containing chemotherapy. hENT1 expression was analyzed by immunohistochemical staining in 24 NSCLC biopsy samples of formalin-fixed, paraffin-embedded tissues. The hENT1-positive staining in NSCLC samples was significantly associated with response to gemcitabine-containing chemotherapy. Responses to gemcitabine-containing chemotherapy were evident in none of the seven patients with no hENT1 expression. These results indicate that the absence of hENT1 expression may be useful to predict NSCLC patients who will not respond to gemcitabine-containing chemotherapy.     

Possible antitumor activity of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106) against an established gemcitabine (dFdCyd)-resistant human pancreatic cancer cell line

We established a variant of MIAPaCa-2 human pancreatic cancer cells that is resistant to 2',2'-difluorodeoxycytidine (gemcitabine, dFdCyd), MIAPaCa-2/dFdCyd, and elucidated the biochemical characteristics and mechanism of dFdCyd-resistance in these cells. We also evaluated 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106, RNA polymerase inhibitor), a new anticancer ribonucleoside, for antitumor activity against the resistant cells in vitro and in vivo. MIAPaCa-2/dFdCyd cells were 2541-fold more resistant to dFdCyd than parental MIAPaCa-2 cells, and the major mechanism of the dFdCyd-resistance was found to be a decrease in the intracellular pool of dFdCyd and its active metabolites, which would result in a decrease in incorporation of dFdCyd triphosphate into DNA. This finding was confirmed by the discovery of decreased deoxycytidine kinase activity, increased cytidine deaminase and ribonucleotide reductase activity, and increased 5'-nucleotidase mRNA expression in the MIAPaCa-2/dFdCyd cells. The cytotoxicity of TAS-106 as an antitumor nucleoside analog was similar in both parental and dFdCyd-resistant cells, with IC(50) values of 6.25 and 6.27 nM, respectively, and this finding was supported by similar intracellular uptake and metabolism of TAS-106 in both cell lines. We also evaluated the in vivo antitumor activity of TAS-106 against MIAPaCa-2 and dFdCyd-resistant MIAPaCa-2/dFdCyd tumors implanted into nude mice. The tumor growth inhibition rate of weekly additions of TAS-106 (7 mg/kg, iv) against parental and dFdCyd-resistant tumors was 73% and 76%, respectively, while that of dFdCyd administered twice a week (240 mg/kg, iv) was 84% and 34%, respectively. These results suggest that TAS-106 would contribute to the treatment of patients with advanced pancreatic carcinomas in whom dFdCyd-based chemotherapy has failed.     

低表达miR-33a诱导胰腺癌细胞对吉西他滨的耐药

背景与目的：胰腺癌是一种恶性程度很高的肿瘤。由于其对一线化疗药物吉西他滨的耐受,往往导致预后较差。MicroRNA(miRNA,miR)是一类非编码小RNA,参与肿瘤的多种生物学功能。miR-33a作为代谢相关的miRNA被广泛研究,而与耐药之间关系的报道较少。该研究通过探讨miR-33a参与胰腺癌吉西他滨耐药及其作用解析,为胰腺癌化疗提供新的理论依据。方法：采用原位杂交方法检测胰腺癌组织中miR-33a的表达情况;采用实时荧光定量PCR(Real-time PCR)检测各胰腺癌细胞系中miR-33a的表达情况。利用SW1990和Miapaca-2胰腺癌亲本细胞株,构建吉西他滨耐药细胞株(SW1990^res,Miapaca-2^res)及miR-33a稳定表达细胞株(SW1990-miR-33a,Miapaca-2-miR-33a、SW1990^res-miR-33a和Miapaca-2^res-miR-33a);采用细胞毒性实验检测miR-33a的表达对胰腺癌细胞对吉西他滨敏感性的影响。结果：miR-33a在胰腺癌组织样本中普遍低表达。与HEK293T正常人胚肾细胞相比,其在各胰腺癌细胞系中均呈低表达。miR-33a过表达可以增加胰腺癌细胞对吉西他滨的药物敏感性,能有效逆转胰腺癌细胞对吉西他滨的耐药。结论：miR-33a在胰腺癌组织中低表达,导致胰腺癌患者对吉西他滨获得性耐药。增加miR-33a表达,从而增强了胰腺癌细胞对吉西他滨的药物敏感性,为开发新型胰腺癌分子靶向治疗药物,联合化疗提供新的理论依据。     

Cellular localization and functional characterization of the equilibrative nucleoside transporters of antitumor nucleosides

Nucleoside transporters play an important role in the disposition of nucleosides and their analogs. To elucidate the relationship between chemosensitivity to antitumor nucleosides and the functional expression of equilibrative nucleoside transporters (ENT), we established stable cell lines of human fibrosarcoma HT-1080 and gastric carcinoma TMK-1 that constitutively overexpressed green fluorescent protein-tagged hENT1, hENT2, hENT3 and hENT4. Both hENT1 and hENT2 were predictably localized to the plasma membrane, whereas hENT3 and hENT4 were localized to the intracellular organelles. The chemosensitivity of TMK-1 cells expressing hENT1 and hENT2 to cytarabine and 1-(3-C-ethynyl-beta-D-ribopentofuranosyl) cytosine increased markedly in comparison to that of mock cells. However, no remarkable changes in sensitivity to antitumor nucleosides were observed in cell lines that expressed both hENT3 and hENT4. These data suggest that hENT3 and hENT4, which are mainly located in the intracellular organelles, are not prominent nucleoside transporters like hENT1 and hENT2, which are responsible for antitumor nucleoside uptake.     

缺氧状态下人胰腺癌细胞对吉西他滨反应的实验研究

目的:观察缺氧状态对人胰腺癌细胞吉西他滨化疗敏感性的影响并分析其相关机制。方法:将人胰腺癌细胞SW1990分为常氧组、缺氧组、常氧+吉西他滨组和缺氧+吉西他滨组。MMT法检测各组细胞增殖、流式细胞仪检测细胞凋亡、Real-time PCR和Western blot分别检测缺氧诱导因子-1α(HIF-1α)、多药耐药基因(MDR-1)mRNA和蛋白的表达。结果:与常氧+吉西他滨组相比,低氧+吉西他滨组的细胞增殖率明显增加,细胞凋亡率显著下降(P<0.05);低氧组和低氧+吉西他滨组HIF-1α蛋白表达分别显著高于常氧组(P<0.05或P<0.01);与常氧组相比,低氧组和低氧+吉西他滨组的MDR1 mRNA和蛋白表达水平均显著升高(P<0.05或P<0.01)。结论:缺氧状态可增加人胰腺癌细胞SW1990对吉西他滨的化疗抵抗,其机制与缺氧环境可诱导HIF-1α和MDR1基因表达有关。     

Human equilibrative nucleoside transporter 1 is not predictive for gemcitabine efficacy in advanced pancreatic cancer: Translational results from the AIO-PK0104 phase III study with the clone SP120 rabbit antibody

BackgroundThe role of human equilibrative nucleoside transporter 1 (hENT1) as a predictive biomarker for gemcitabine efficacy in advanced pancreatic cancer remains unclear to date.Patients and methodsAIO-PK0104 was a German multicenter phase III trial comparing gemcitabine/erlotinib followed by capecitabine (GEC) with capecitabine/erlotinib followed by gemcitabine (CEG) in advanced pancreatic cancer. Archival tumour tissue from 169 of the 274 eligible study patients was available for a central and standardised immunohistochemistry staining for hENT1 expression using the SP120 rabbit monoclonal anti-hENT1 antibody. Within a retrospective translational subgroup analysis, biomarker data were correlated with efficacy end-points.ResultsThirty-nine out of 130 fresh-cut slides were scored as hENT1^high (30%), whereas 91 samples were classified as hENT1^low (70%). For the 62 patients randomised to CEG median overall survival was estimated with 6.4 months in the hENT1^low compared to 6.9 months in the hENT1^high subgroup (Hazard Ratio (HR) 0.88, 95% confidence interval (CI) 0.48–1.61, p = 0.67). For the 68 patients randomised to GEC survival was 5.7 months in the hENT1^low compared to 4.4 months in the hENT1^high subgroup (HR 1.16, 95% CI 0.69–1.96, p = 0.57). In 101 patients receiving gemcitabine at any time during study treatment (either within the 1st- or 2nd-line setting) hENT1^low cases had a median overall survival of 7.5 months and hENT1^high patients an overall survival of 4.4 months (HR 1.30, 95% CI 0.84–2.03, p = 0.24), respectively.ConclusionWithin this subgroup analysis from Arbeitsgemeinschaft Internistische Onkologie-pancreatic cancer (AIO-PK0104), no evidence supporting the use of hENT1 as a predictive biomarker for gemcitabine efficacy in patients with advanced pancreatic cancer was found.