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 absence of human equilibrative nucleoside transporter 1 is associated with reduced survival in patients with gemcitabine-treated pancreas adenocarcinoma.

PURPOSE: Gemcitabine monotherapy is the standard palliative chemotherapy for pancreatic adenocarcinoma. Gemcitabine requires plasma membrane nucleoside transporter proteins to efficiently enter cells and exert it cytotoxicity. In vitro studies have demonstrated that deficiency of human equilibrative nucleoside transporter 1 (hENT1), the most widely abundant and distributed nucleoside transporter in human cells, confers resistance to gemcitabine toxicity, but the distribution and abundance of nucleoside transporters in normal and malignant pancreatic tissue is unknown. EXPERIMENTAL DESIGN: We studied tumor blocks from normal pancreas and 21 Alberta patients with gemcitabine-treated pancreatic cancer. Immunohistochemistry on the formalin-fixed, paraffin-embedded tissues was performed with specific hENT1 and human Concentrative Nucleoside Transporter 3 monoclonal antibodies and scored by a pathologist blinded to clinical outcomes. RESULTS: hENT1 was detected in normal Langerhan cells and lymphocytes but not in normal glandular elements. Patients in whom all adenocarcinoma cells had detectable hENT1 had significantly longer median survivals from gemcitabine initiation than those for whom hENT1 was absent in a proportion (10 to 100%) of adenocarcinoma cells (median survival, 13 versus 4 months, P = 0.01). Immunohistochemistry for human Concentrative Nucleoside Transporter 3 revealed moderate to high-intensity staining in all adenocarcinoma tissue samples. CONCLUSIONS: Patients with pancreatic adenocarcinoma with uniformly detectable hENT1 immunostaining have a significantly longer survival after gemcitabine chemotherapy than tumors without detectable hENT1. Immunohistochemistry for hENT1 shows promise as a molecular predictive assay to appropriately select patients for palliative gemcitabine chemotherapy but requires formal validation in prospective, randomized trials.     

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.     

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.     

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.     

BRCA1、hENT1和Topo1 mRNA水平与新鲜胃癌组织三维培养药物敏感性关系

目的：分析胃癌石蜡组织BRCA1、hENT1、Topo1 mRNA水平与新鲜胃癌组织多西紫杉醇、吉西他滨、伊立替康体外药物敏感性的关系。方法：收集36例经病理确诊的新鲜胃癌标本,采用三维微组织块培养法（HDRA）行三种药物的体外药物敏感试验。实时荧光定量PCR检测对应胃癌石蜡组织中BRCA1、hENT1、To-po1 mRNA水平。结果：新鲜胃癌组织对三种药物的敏感性均与临床特征无明显相关性。对应胃癌石蜡组织中BRCA1、hENT1、Topo1 mRNA水平与临床特征无明显相关性。在胃癌组织中,hENT1 mRNA水平最高,BRCA1 mRNA水平最低。胃癌石蜡组织BRCA1 mRNA水平与新鲜胃癌组织的多西紫杉醇敏感性正相关（7.927 vs 3.464,P=0.001）;hENT1 mRNA水平在吉西他滨敏感组高于耐药组（15.710 vs 10.145,P=0.243）,Topo1 mRNA水平在伊立替康敏感组高于耐药组（10.024 vs 6.038,P=0.124）,但均未达统计学差异。结论：胃癌石蜡组织BRCA1mRNA水平与新鲜胃癌组织对多西紫杉醇的敏感性呈正相关。     

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.     

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。     

CNT1 expression influences proliferation and chemosensitivity in drug-resistant pancreatic cancer cells

Overcoming the inherent chemoresistance of pancreatic cancers remains a major goal of therapeutic investigations in this disease. In this study, we discovered a role for the human concentrative nucleoside transporter-1 (hCNT1; SLC28A1), a high-affinity pyrimidine nucleoside transporter, in determining the chemosensitivity of human pancreatic cancer cells to gemcitabine, the drug used presently as a standard of care. Compared with normal pancreas and pancreatic ductal epithelial cells, hCNT1 expression was frequently reduced in pancreatic tumors and tumor cell lines. In addition, hCNT1-mediated (3)H-gemcitabine transport was lower in pancreatic cancer cell lines and correlated with cytotoxic IC(50) estimations of gemcitabine. In contrast to gemcitabine-sensitive pancreatic cancer cell lines, MIA PaCa-2, a gemcitabine-resistant pancreatic cancer cell line, exhibited relatively restrictive, cell cycle-dependent hCNT1 expression and transport. hCNT1 translation was suppressed in the late G1-enriched MIA PaCa-2 cell population possibly in an miRNA-dependent manner, which corresponded with the lowest hCNT1-mediated gemcitabine transport during this phase. Although hCNT1 protein was induced during G1/S transition, increased hCNT1 trafficking resulted in maximal cell surface recruitment and transport-overshoot in the G2/M phase-enriched cell population. hCNT1 protein was directed predominantly to proteasomal or lysosomal degradation in S or G2/M phase MIA PaCa-2 cells, respectively. Pharmacological inhibition of hCNT1 degradation moderately increased cell surface hCNT1 expression and cellular gemcitabine transport in MIA PaCa-2 cells. Constitutive hCNT1 expression reduced clonogenic survival of MIA PaCa-2 cells and steeply augmented gemcitabine transport and chemosensitization. In addition to supporting a putative tumor suppressor role for hCNT1, our findings identify hCNT1 as a potential candidate to render drug-resistant pancreatic cancer cells amenable to chemotherapy.     

siRNA directed against TrkA sensitizes human pancreatic cancer cells to apoptosis induced by gemcitabine through an inactivation of PI3K/Akt-dependent pathway

Previously, we have documented that the aggressive and highly metastatic behavior of pancreatic cancer may be due to the aberrant expression of nerve growth factor (NGF) and its high-affinity receptor, proto-oncogene TrkA. In this study, we sought to determine the effect of suppressing TrkA expression on pancreatic cancer chemosensitivity to gemcitabine. Human pancreatic cancer cell lines PANC-1, MIA-PaCa-2 and ASPC-1 were studied. The expression and kinase activity of TrkA were determined by Western blot analysis and in vitro kinase assay respectively. RNA interference was used to suppress TrkA expression. Gemcitabine-induced cytotoxicity was determined by tetrazolium reduction assay and caspase profiling was performed. The effect of TrkA-specific siRNA on PI3K/Akt activity was also quantified. TrkA expression and kinase activity in cell lines were directly correlated with gemcitabine chemoresistance. TrkA-specific siRNA suppressed TrkA expression and kinase activity, and furthermore increased gemcitabine-induced, caspase-mediated apoptosis. PI3K/Akt activity was decreased by suppression of TrkA expression. Taken together, these data demonstrated that TrkA is a determinant of pancreatic adenocarcinoma chemoresistance and PI3K/Akt is a key signaling component by which NGF activation of the TrkA signal transduction pathway protects pancreatic cancer cells from chemotherapy-induced cell death.     

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.     

应用ATP生物荧光肿瘤体外药敏检测技术研究大肠癌药敏的异质性

目的探讨用ATP生物荧光肿瘤体外药敏检测技术(ATP-TCA)研究大肠癌药敏的异质性和个体化疗的可行性.方法用ATP-TCA检测58例大肠癌标本对16种单药或联合用药的敏感性.结果个体之间的药物敏感性存在着明显的异质性.单药中最有效的药物为长春瑞滨、羟基喜树碱、氟尿嘧啶和紫杉醇.联合用药最有效的是氟尿嘧啶 丝裂霉素 阿糖胞苷,91.6%(11/12)的标本对其敏感,其次是氟尿嘧啶 顺铂 阿霉素,健择 顺铂和5-FU氟尿嘧啶 长春新碱 卡氮芥.结论大肠癌对抗癌药物的敏感程度存在着异质性.ATP生物荧光肿瘤药敏检测技术可用于为大肠癌选择合适的化疗药物.     

In vitro synergistic cytotoxicity of gemcitabine and pemetrexed and pharmacogenetic evaluation of response to gemcitabine in bladder cancer patients

The present study was performed to investigate the capability of gemcitabine and pemetrexed to synergistically interact with respect to cytotoxicity and apoptosis in T24 and J82 bladder cancer cells, and to establish a correlation between drug activity and gene expression of selected genes in tumour samples. The interaction between gemcitabine and pemetrexed was synergistic; indeed, pemetrexed favoured gemcitabine cytotoxicity by increasing cellular population in S-phase, reducing Akt phosphorylation as well as by inducing the expression of a major gemcitabine uptake system, the human equilibrative nucleoside transporter-1 (hENT1), and the key activating enzyme deoxycytidine kinase (dCK) in both cell lines. Bladder tumour specimens showed an heterogeneous gene expression pattern and patients with higher levels of dCK and hENT1 had better response. Moreover, human nucleoside concentrative transporter-1 was detectable only in 3/12 patients, two of whom presented a complete response to gemcitabine. These data provide evidence that the chemotherapeutic activity of the combination of gemcitabine and pemetrexed is synergistic against bladder cancer cells in vitro and that the assessment of the expression of genes involved in gemcitabine uptake and activation might be a possible determinant of bladder cancer response and may represent a new tool for treatment optimization.     

IRF-2对胰腺癌细胞生物学特性及化疗敏感性的影响

目的 探讨干扰素调控因子2(interferon regulatory factor 2,IRF-2)在胰腺癌细胞中的生物学特性及其对吉西他滨化疗敏感性的影响.方法 Western blot检测IRF-2基因在胰腺癌细胞株PANC-1及MIAPaCa-2中的表达水平,采用MTT检测吉西他滨对PANC-1及MIAPaCa-2的半数有效浓度(IC50),选择较为耐药的PANC-1细胞进行IRF-2基因干扰表达,并采用MTT检测吉西他滨对PANC-1及干扰IRF-2表达的PANC-1 si1#的半数有效浓度(IC50).结果 PANC-1及MIAPaCa-2中细胞中均存在IRF-2基因的表达,且PANC-1细胞中表达水平比MIAPaCa-2高.吉西他滨对PANC-1细胞的IC50显著高于MIAPaCa-2细胞,差异有统计学意义(P＜0.05);干扰IRF-2表达的PANC-1 si1#细胞其IC50值显著低于PANC-1对照细胞,差异有统计学意义(P＜0.05).结论 IRF-2基因可作为影响胰腺癌对吉西他滨化疗敏感性的基因之一,干扰IRF-2基因能够有效地提升胰腺癌细胞对吉西他滨化疗的敏感性.     

Acquired resistance of pancreatic cancer cells towards 5-Fluorouracil and gemcitabine is associated with altered expression of apoptosis-regulating genes

OBJECTIVES: Resistance to chemotherapy is a major cause of treatment failure and poor prognosis in pancreatic cancer. Inasmuch as most effects of chemotherapeutic agents are mediated via the activation of apoptosis, the cytotoxic effects of gemcitabine and 5-fluorouracil (FU) in correlation with apoptosis-regulating genes in pancreatic cancer cell lines were analyzed. METHODS: The cytotoxic effects of 5-FU and gemcitabine in AsPC-1, Capan-1, Mia-PaCa-2 and T3M4 pancreatic cancer cell lines were assessed by growth assays, and mRNA expression levels of pro-apoptotic and anti-apoptotic genes of the Bcl-2 family were analyzed by RNAse protection assays. RESULTS: Pancreatic cancer cells displayed a wide range of responses towards 5-FU (IC(50) 0.22-4.63 microM) and gemcitabine (11.51-42.2 nM). After repeated treatment with 5-FU, the IC(50) values in Capan-1 and T3M4 cells increased 2.1- and 1.8-fold, respectively, compared to their parental cells. Following recurrent treatment with gemcitabine, the IC(50) values in Capan-1 cells increased significantly (1.5-fold, p < 0.01). RNase protection assay showed a negative correlation between bcl-x(L) and mcl-1 mRNA expression levels and the sensitivity to 5-FU and gemcitabine after 5-FU and gemcitabine treatment. The bax/bcl-2 ratio maintained relatively stable following 5-FU/gemcitabine treatment and reflected the chemotherapeutic sensitivity of these cell lines. CONCLUSIONS: These findings reveal that pancreatic cancer cell lines are generally resistant to 5-FU and are more sensitive towards gemcitabine. The bax/bcl-2 ratio is predictive of chemotherapy sensitivity, whereas bcl-x(L) and mcl-1 mRNA levels following repeated exposure to 5-FU or gemcitabine are associated with resistance to these drugs. These findings suggest that the activation of anti-apoptotic genes after repeated drug exposure contributes to chemoresistance of pancreatic cancer cells and that blockage of anti-apoptotic genes might enhance chemosensitivity in pancreatic cancer.     

RNA干扰沉默DcR3对人胰腺癌细胞化疗药物敏感性影响的实验研究

背景与目的：大部分胰腺癌具有高表达诱骗受体-3(decoy receptor 3,DcR3)的特征,而后者与FasL凋亡途径相关,可能导致胰腺癌对化疗耐药。该研究旨在探讨RNA干扰沉默DcR3基因对人胰腺癌细胞化疗药物敏感性的影响及其可能机制。方法：构建带有DcR3-siRNA序列的稳定表达质粒,通过Lipofectamine^TM2000转染至人胰腺癌细胞AsPC-1细胞株,筛选出转染后稳定低表达DcR3的胰腺癌细胞,同时设未转染对照组(control组)和转染阴性质粒对照组(mock组)。应用ELISA和蛋白［质］印迹法(Western blot)检测各组AsPC-1细胞中DcR3的蛋白表达;MTT实验检测各组AsPC-1细胞对吉西他滨的敏感性;流式细胞术检测各组AsPC-1细胞凋亡情况;Western blot和实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)检测各组AsPC-1细胞中FasL、Caspase-8、Caspase-3蛋白和mRNA的表达。结果：转染DcR3-siRNA后AsPC-1细胞中DcR3蛋白较其他对照组明显降低;转染DcR3-siRNA后AsPC-1细胞对吉西他滨的敏感性显著增加;沉默DcR3基因可以上调FasL、Caspase-8和Caspase-3的表达并促进吉西他滨诱导的细胞凋亡。结论：RNA干扰沉默DcR3基因可激活FasL/Caspase凋亡途径,促进肿瘤细胞凋亡,增加人胰腺癌AsPC-1细胞对化疗药物的敏感性。     

cN-II expression predicts survival in patients receiving gemcitabine for advanced non-small cell lung cancer

Resistance to gemcitabine is likely to be multifactorial and could involve a number of mechanisms involved in drug penetration, metabolism and targeting. In vitro studies of resistant human cell lines have confirmed that human equilibrative nucleoside transporter 1 (hENT1)-deficient cells display resistance to gemcitabine. Overexpression of certain nucleotidases, such as cN-II, has also been frequently shown in gemcitabine-resistant models. In this study, we applied immunohistochemical methods to assess the protein abundance of cN-II, hENT1, human concentrative nucleoside transporter 3 (hCNT3) and deoxycitidine kinase (dCK) in malignant cells in from 43 patients with treatment-na?ve locally advanced or metastatic non-small cell lung cancer (NSCLC). All patients subsequently received gemcitabine-based chemotherapy. Response to chemotherapy, progression-free survival (PFS), and overall survival (OS) were correlated with abundance of these proteins. Among the 43 samples, only 7 (16%) expressed detectable hENT1, with a low percentage of positive cells, 18 expressed hCNT3 (42%), 36 (86%) expressed cN-II and 28 (66%) expressed dCK. In univariate analysis, only cN-II expression levels were correlated with overall survival. None of the parameters were correlated with freedom from progression survival nor with response. Patients with low levels of expression of cN-II (less than 40% positively stained cells) had worse overall survival than patients with higher levels of cN-II expression (6 months and 11 months, respectively). In a multivariate analysis taking into account age, sex, weight loss, stage and immunohistochemical results, cN-II was the only predictive factor associated with overall survival. This study suggests that cN-II nucleotidase expression levels identify subgroups of NSCLC patients with different outcomes under gemcitabine-based therapy. Larger prospective studies are warranted to confirm the predictive value of cN-II in these patients.     

人膀胱癌原代培养体外药敏试验的研究

目的:探讨用ATP生物荧光肿瘤体外药敏检测技术(ATP-TCA)研究膀胱癌药敏的异质性和个体化疗的可行性。方法:用ATP-TCA检测来自45例初发和6例复发膀胱癌患者的手术标本对4种化疗药物的敏感程度。结果:51例标本中有49例获得药敏结果,可评价率为96·0%。最有活性的药物是吡柔比星,有43·8%(21/48)的标本对它表现强敏感;28·6%(14/49)的标本无强敏感药物;69·4%(34/49)的标本无耐药药物。结论:膀胱癌对抗癌药物的敏感程度存在着异质性。ATP生物荧光肿瘤药敏检测技术可用于为膀胱癌选择合适的化疗药物。