人胰腺癌吉西他滨耐药细胞系的建立及耐药特性的检测

目的为揭示吉西他滨在胰腺癌中引起耐药的机制,建立了人胰腺癌吉西他滨耐药细胞系并对其细胞生物学特性进行了研究。方法逐步增加培养基中吉西他滨的浓度,在人胰腺癌细胞系SW1990中建立了对吉西他滨耐药的细胞系SW1990-GEM。采用四氮唑蓝(MTT)法和集落形成实验,计算SW1990和SW1990-GEM的半数抑制浓度(IC50)和耐药系数(RI)。比较SW1990和SW1990-GEM的生长曲线并计算出两细胞系的倍增时间。采用MTT法检测SW1990-GEM对几种常用抗肿瘤药物的交叉耐药谱。结果SW1990和SW1990-GEM的IC50分别为(0.428±0.069)μmol/L和(230.53±13.12)μmol/L,RI为537.45(P<0.001)。根据生长曲线计算出SW1990和SW1990-GEM的倍增时间为22.8小时和35.8小时。交叉耐药实验表明,SW1990-GEM对5-FU、表柔比星、丝裂霉素、甲氨蝶呤、紫杉醇、泰素帝、长春新碱和依托泊苷产生交叉耐药,对顺铂、阿糖胞苷无交叉耐药。结论成功建立了人胰腺癌吉西他滨耐药细胞系SW1990-GEM,耐药性能明显、稳定。SW1990-GEM对5-FU、表柔比星、丝裂霉素、甲氨蝶呤、紫杉醇、泰素帝、长春新碱和依托泊苷产生交叉耐药,对顺铂、阿糖胞苷无交叉耐药。     

乳腺癌耐药蛋白ABCG2对胰腺癌SW1990细胞耐药性影响的实验

目的 探讨吉西他滨诱导胰腺癌细胞SW1990中ABCG2的表达及其与化疗耐药的关系。方法 胰腺癌细胞SW1990用DMEM培养液常规培养,用0.82 mg/ml吉西他滨作用SW1990细胞24、48和72 h后,使用流式细胞仪测其细胞凋亡率,Western blot检测ABCG2蛋白的表达,RT-PCR检测ABCG2 mRNA的表达,并且分析ABCG2表达水平与化疗耐药的关系。结果 0.82 mg/ml吉西他滨作用SW1990细胞24、48和72 h后,细胞凋亡率分别为（21.1±0.61）%、（13.4±2.17）%、（6.4±1.34）%,不同时间点两两相比P均<0.05。0.82 mg/ml吉西他滨作用SW1990细胞24、48、72 h后与对照组相比,ABCG2 mRNA分别上升了（2.21±0.11）倍、（3.30±0.08）倍和（4.72±0.12）倍,蛋白上升了（2.17±0.14）倍、（3.61±0.09）倍和（4.98±0.13）倍,且组间比较P均<0.05,有统计学意义。结论 吉西他滨能抑制胰腺癌细胞SW1990的增殖,但随着时间延长,药物抵抗逐渐增强,这可能与吉西他滨作用细胞后上调ABCG2的表达有关。     

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。     

吉西他滨耐药胰腺癌细胞系的耐药机制

目的探讨吉西他滨耐药胰腺癌细胞系的耐药机制。方法应用免疫组织化学方法和逆转录PCR法（RT—PCR）,对吉西他滨耐药细胞系的耐药机制进行了研究。选用沙尔威辛（SAL）进行逆转耐药实验,采用了RT—PCR法、流式细胞仪和MTT法,评估SAL逆转耐药的效果。结果免疫组织化学法显示,耐药细胞SW1990-GEM的P-糖蛋白（P—gP）表达为弱阳性,其亲本细胞SW1990为阴性。多药耐药相关蛋白（MRP）在两细胞系表达均为阴性。SW1990-GEM的多药耐药基因（mdr-1）在mRNA水平上的转录比其亲本细胞系SW1990增强;脱氧胞苷激酶（dCK）基因的转录减少;核苷酸还原酶（RR）基因的转录增强。经过SAL作用后,mdr-1在mRNA水平上的转录减少。流式细胞仪显示,经过4,30,60,90nmol／LSAL作用后,细胞内罗丹明123的蓄积累分别增加1．04,1．16,1．39,1．72倍。用4nmol／LSAL进行逆转吉西他滨耐药实验,未接受SAL处理的对照组对吉西他滨的IC50为203．72μmol／L;经SAL作用1h后再加吉西他滨组的IC50为121．36μmol／L（P=0．219）;经SAL作用24h后再加吉西他滨组的IC59为121．64μmol／L（P=0．167）。结论胰腺癌细胞对吉西他滨耐药的原因与dCK减少有关,同时也与mdr-1和RR升高相关。SAL能够下调SW1990-GEM耐药细胞的mdr-1和P—gP表达,但低浓度的SAL不能逆转SW1990-GEM对吉西他滨的耐药。     

非小细胞肺癌顺铂化疗耐药相关微RNA的筛选及鉴定

 目的　探讨肿瘤细胞微RNA（miRNA）对化疗药物敏感性的作用。方法　通过miRNA芯片技术检测顺铂（DDP）耐药细胞株A549／DDP与非耐药细胞株A549的miRNA表达的差异,利用荧光定量聚合酶链反应（PCR）技术验证相应miRNA的表达情况,通过在细胞株中抑制或过表达目标miRNA,研究其对细胞化疗药物敏感性的影响。结果　A549／DDP细胞对DDP的耐药为A549细胞的18倍。A549／DDP细胞与A549细胞存在51个表达水平差异在4倍以上的miRNA,其中24个表达上调,27个表达下调。PCR进一步证实miR-376c、miR-31、miR-29a、miR-221在A549／DDP细胞中显著上调,miR-196a、miR-20a、miR-20b、miR-17、miR-451在A549／DDP细胞中显著下调。在提高A549／DDP细胞中miR-17的表达后,细胞对DDP的敏感度增加了11.7 %,提高miR-451的表达或者抑制miR-29a的表达后,对DDP的敏感度分别下降了15.5 %、12.9 %,抑制miR-376c、miR-31、miR-221或过表达miR-196a、miR-20a、miR-20b均不影响A549／DDP细胞对DDP的敏感度。结论　非小细胞肺癌DDP耐药细胞与非耐药细胞的miRNA表达谱有差异,miRNA参与肺癌化疗耐药,miR-17具有逆转非小细胞肺癌DDP耐药的潜力。     

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.     

MicroRNA-21调控乳腺癌对吉西他滨耐药的机制

背景与目的：以吉西他滨为基础的联合用药在转移性乳腺癌中展示出很好的临床疗效和安全性,但耐药的出现导致治疗失败。MicroRNA是一类非编码小分子RNA,起到类似癌基因或抑癌基因的作用。虽然肿瘤中关于化疗药物耐药的机制报道很多,但microRNA异常表达与耐药之间的关系及机制还不十分清楚。本研究旨在探讨microRNA-21在乳腺癌吉西他滨耐药中的作用及其可能机制。方法：采用低浓度持续诱导MDA-MB-231细胞的方式构建人乳腺癌耐吉西他滨细胞株,药物敏感性差异达10倍以上,继而通过实时荧光定量PCR(real-time PCR,RT-PCR)、CCK-8、蛋白［质］印迹法(Western blot)、转染、划痕和Transwell等实验分别检测microRNA-21对药物的敏感性及上皮-间充质转化(epithelial-mesenchymal transition,EMT)标志物的影响。结果：在乳腺癌吉西他滨耐药细胞株中存在EMT现象,相比亲代细胞,microRNA-21呈高表达,并与吉西他滨敏感性呈负相关。转染microRNA-21的抑制剂和mimic可以分别下调和上调microRNA-21表达,同时EMT现象和药物敏感性也发生了相应的变化。结论：MicroRNA-21可能通过诱导肿瘤细胞的EMT发生而介导乳腺癌对吉西他滨的耐药。     

胰腺癌组织和细胞的耐药性与ABC转运蛋白基因1 的关系

[摘要] 目的：探讨胰腺癌耐药性与ABC转运蛋白基因1（ABCB1）的表达及其启动子甲基化的关系。方法：选用2015 年8月至2018 年8 月福建省肿瘤医院病理确诊的15 例胰腺癌及癌旁组织、3 例正常胰腺组织标本以及胰腺癌细胞株SW1990,用间歇浓度梯度倍增法将SW1990 细胞株诱导分化为吉西他滨（GEM）耐药胰腺癌细胞株SW1990/GZ。用qPCR分别检测胰腺癌及癌旁组织和SW1990、SW1990/GZ细胞中ABCB1 表达水平,用MSP-PCR法检测胰腺癌组织和SW1990、SW1990/GZ细胞中ABCB1启动子区域甲基化程度。结果：与SW1990 相比,SW1990/GZ细胞空泡增多、核分裂像增加、呈现团块生长,并呈现更强的耐药性（P<0.05）。胰腺癌组织中ABCB1 表达水平明显高于癌旁组织（P<0.01）。SW1990 和SW1990/GZ细胞中ABCB1 表达水平显著高于正常胰腺组织（P<0.05 或P<0.01）,其中SW1990/GZ 细胞中ABCB1 表达水平高于SW1990 细胞（P<0.05）。SW1990 和SW1990/GZ细胞及正常胰腺组织中的ABCB1 启动子均呈低甲基化状态。胰腺癌和正常胰腺组织中甲基化率分别为6.7%（1/15）及0.00%（0/3）,差异无统计学意义（均P>0.05）。结论：胰腺癌组织和细胞中ABCB1 表达增高与耐药性有关,但其基因表达不依赖于启动子的甲基化调控。     

EGFR-T790M突变所致吉非替尼耐药肺腺癌细胞化疗药物敏感性变化的研究

背景与目的:EGFR-TKI治疗NSCLC失败后,化疗仍可取得一定的治疗效果,是可选择的治疗方案之一。核苷酸还原酶(ribonucleotide reductase,RR)、胸苷酸合成酶(thymidylate synthase,TS)、核苷酸切除修复交叉互补基因1(excision repair cross complementstion group 1,ERCC1)、3型β微管蛋白(β-tubulin-Ⅲ,TUBB3)分别与吉西他滨、培美曲塞、铂类药物及微管类药物的化疗药物敏感性存在相关性,可以通过这些分子标志物的表达水平来预测化疗药物的敏感性。RRMI、TS、ERCC1和TUBB3高表达患者化疗药物的敏感性降低,低表达患者化疗药物敏感性增高。本研究拟探讨EGFR-T790M突变所致吉非替尼耐药肺腺癌细胞对顺铂、吉西他滨、长春瑞滨、紫杉醇、多西他赛和培美曲塞化疗药物敏感性的变化。方法:通过MTT法检测PC9及PC9/GR细胞对顺铂、吉西他滨、长春瑞滨、紫杉醇、多西他赛和培美曲塞的IC50,探讨其对上述药物的化疗敏感性。采用液相芯片法,检测PC9及PC9/GR细胞ERCC1 m RNA、RRM1 m RNA、TUBB3 m RNA和TS m RNA的表达水平。通过蛋白质印迹法(Western blot)检测PC9及PC9/GR细胞ERCC1、RRM1、TUBB3和TS蛋白的表达水平。结果:与PC9细胞株相比较,PC9/GR细胞株对吉非替尼、顺铂、吉西他滨和培美曲塞的IC50明显增高(P<0.05);对长春瑞滨、紫杉醇和多西他赛的IC50明显降低(P<0.05)。PC9/GR细胞对吉非替尼、顺铂、吉西他滨、长春瑞滨、紫杉醇、多西他赛和培美曲塞的耐药指数分别为70、1.56、1.61、0.34、0.39、0.14和1.71。与PC9细胞株m RNA的表达量相比较,PC9/GR细胞株ERCC1 m RNA、RRM1 m RNA和TS m RNA的表达量明显增高(P<0.05),TUBB3的m RNA的表达量明显降低,差异均有统计学意义(P<0.05)。与PC9细胞株蛋白的表达量相比较,PC9/GR细胞株ERCC1、RRM1和TS的蛋白表达量明显增高,TUBB3蛋白的表达量明显降低,差异均有统计学意义(P<0.05)。结论:肺腺癌细胞株发生EGFR-T790M突变后对化疗药物敏感性发生变化,对顺铂、吉西他滨和培美曲塞的敏感性降低,对长春瑞滨、紫杉醇和多西他赛的敏感性增高;其化疗药物敏感性发生变化的原因可能与肺腺癌细胞株发生EGFR-T790M突变后ERCC1 m RNA、RRM1 m RNA、TS m RNA及其蛋白表达量发生变化相关。     

miR-27a在三阴性乳腺癌中的表达及其对细胞耐药的影响

目的:探讨miR-27a在三阴性乳腺癌（triple-negative breast cancer,TNBC）中的表达及其对细胞耐药的影响。方法:首先通过QRT-PCR检测TNBC细胞株及非TNBC细胞株中miR-27a及P-gp的差异表达;上调TNBC细胞中的miR-27a的表达,通过CCK8检测细胞对化疗药物敏感性的变化。同时收集TNBC患者化疗前后血液标本,将其分为化疗敏感组和化疗耐药组,QRT-PCR检测患者血液标本中miR-27a及P-gp的表达,分析miR-27a与乳腺癌患者预后相关性。结果:miR-27a在TNBC细胞株中的表达明显低于非TNBC细胞株,上调TNBC细胞株中miR-27a的表达能够降低P-gp的表达,增加细胞对化疗药物的敏感性,此外TNBC组中miR-27a的表达与患者组织学分级、临床分期及淋巴结转移相关（P<0.05）;在非TNBC组中miR-27a的表达与患者临床病理特征无明显相关性（P均＞0.05）。结论:miR-27a参与调节TNBC细胞耐药,miR-27a可作为评估乳腺癌化疗敏感性及临床预后的潜在靶基因。     

MicroRNA-33a-mediated downregulation of Pim-3 kinase expression renders human pancreatic cancer cells sensitivity to gemcitabine

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, with less than 5% of patients surviving 5 years beyond diagnosis. Systemic therapies, particularly gemcitabine, have a modest clinical benefit, but chemoresistance limits their efficacy. Here, we demonstrate that plasma miR-33a levels positively correlated with miR-33a levels in tumor tissues of patients with PDAC and are a good prognostic indicator of overall survival. Overexpression of miR-33a inhibited tumor cell proliferation and increased the chemosensitivity to gemcitabine both in vitro and in vivo. Moreover, miR-33a targets Pim-3 directly in PDAC. Pim-3 expression was a prognostic indicator related to poor survival in pancreatic cancer patients. Plasma miR-33a levels were significantly lower in pancreatic cancer patients with high Pim-3 protein expression than in healthy controls. Furthermore, overexpression of miR-33a in pancreatic cancer cell lines suppressed Pim-3 expression, leading to downregulation of the AKT/Gsk-3β/β-catenin pathway. Overall, these results indicate that miR-33a functions as a tumor suppressor that downregulates Pim-3 kinase expression to inhibit both pancreatic tumor growth and gemcitabine resistance via the AKT/β-catenin pathway. Hence, detection of plasma miR-33a may be a simple and convenient method of predicting therapeutic responses.     

Ex vivo chemosensitivity testing and gene expression profiling predict response towards adjuvant gemcitabine treatment in pancreatic cancer

Efficacy of chemotherapy for pancreatic cancer may be improved by tailoring it to individual chemosensitivity profiles. Identification of nonresponders before initiation of treatment may help to avoid side effects. In this study, primary pancreatic cancer cells were isolated from 18 patients undergoing pancreaticoduodenectomy for pancreatic cancer. Eight commonly used pancreatic cancer cell lines were used as controls. Ex vivo chemosensitivity for gemcitabine, 5-fluorouracil, mitomycin-C, cisplatinum, oxaliplatinum, paclitaxel and a combination of gemcitabine with oxaliplatinum or mitomycin-C was determined using a cellular ATP-based tumour chemosensitivity assay (ATP-TCA). Quantitative real-time-polymerase chain reaction was performed to determine RNA expression levels of genes implicated in chemoresistance. Chemosensitivity towards cytotoxic agents was highly variable in primary pancreatic cancer cells and pancreatic cancer cell lines. ATP-TCA results for gemcitabine correlated to the tissue expression of human equilibrative nucleoside transporter-1 (hENT1). Time to relapse in patients with gemcitabine-sensitive tumours was significantly higher than in patients with chemoresistant pancreatic cancers (P=0.01; 71 vs 269 days). Furthermore, time to relapse in gemcitabine-treated patients was related to hENT1 expression (P=0.0067). Thus, chemosensitivity testing using ATP-TCA in pancreatic cancer is feasible and correlated with time to relapse in gemcitabine-treated patients. This suggests that ATP-TCA testing could be used as a decision-making tool in the adjuvant treatment of pancreatic cancer.     

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.     

miR-320c regulates gemcitabine-resistance in pancreatic cancer via SMARCC1

Background:

Gemcitabine-based chemotherapy is the standard treatment for pancreatic cancer. However, the issue of resistance remains unresolved. The aim of this study was to identify microRNAs (miRNAs) that govern the resistance to gemcitabine in pancreatic cancer.

Methods:

miRNA microarray analysis using gemcitabine-resistant clones of MiaPaCa2 (MiaPaCa2-RGs), PSN1 (PSN1-RGs), and their parental cells (MiaPaCa2-P, PSN1-P) was conducted. Changes in the anti-cancer effects of gemcitabine were studied after gain/loss-of-function analysis of the candidate miRNA. Further assessment of the putative target gene was performed in vitro and in 66 pancreatic cancer clinical samples.

Results:

miR-320c expression was significantly higher in MiaPaCa2-RGs and PSN1-RGs than in their parental cells. miR-320c induced resistance to gemcitabine in MiaPaCa2. Further experiments showed that miR-320c-related resistance to gemcitabine was mediated through SMARCC1, a core subunit of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex. In addition, clinical examination revealed that only SMARCC1-positive patients benefited from gemcitabine therapy with regard to survival after recurrence (P=0.0463).

Conclusion:

The results indicate that miR-320c regulates the resistance of pancreatic cancer cells to gemcitabine through SMARCC1, suggesting that miR-320c/SMARCC1 could be suitable for prediction of the clinical response and potential therapeutic target in pancreatic cancer patients on gemcitabine-based therapy.     

The serum miR-21 level serves as a predictor for the chemosensitivity of advanced pancreatic cancer,and miR-21 expression confers chemoresistance by targeting FasL

miR-21 expression in cancer tissue has been reported to be associated with the clinical outcome and activity of gemcitabine in pancreatic cancer. However, resection is possible in only a minority of patients due to the advanced stages often present at the time of diagnosis, and safely obtaining sufficient quantities of pancreatic tumor tissue for molecular analysis is difficult at the unresectable stages. In this study, we investigated whether the serum level of miR-21 could be used as a predictor of chemosensitivity. We tested the levels of serum miR-21 in a cohort of 177 cases of advanced pancreatic cancer who received gemcitabine-based palliative chemotherapy. We found that a high level of miR-21 in the serum was significantly correlated with a shortened time-to-progression (TTP) and a lower overall survival (OS). The serum miR-21 level was an independent prognostic factor for both the TTP and the OS (HR 1.920; 95% CI, 1.274–2.903, p = 0.002 for TTP and HR 1.705; 95% CI, 1.147–2.535, p = 0.008 for OS). The results from a functional study showed that gemcitabine exposure down-regulated miR-21 expression and up-regulated FasL expression. The increased FasL expression following gemcitabine treatment induced cancer cell apoptosis, whereas the ectopic expression of miR-21 partially protected the cancer cells from gemcitabine-induced apoptosis. Additionally, we confirmed that FasL was a direct target of miR-21. Therefore, the serum level of miR-21 may serve as a predictor of chemosensitivity in advanced pancreatic cancer. Additionally, we identified a new mechanism of chemoresistance mediated by the effects of miR-21 on the FasL/Fas pathway.     

A humanized anti-IGF-1R monoclonal antibody (R1507) and/or metformin enhance gemcitabine-induced apoptosis in pancreatic cancer cells

Pancreatic cancer is a disease with a dismal prognosis and treatment options are limited. This study investigated the interaction of gemcitabine with R1507 and/or metformin and the induction of an inhibitor of apoptosis protein by this com-bination. Pancreatic cancer cells were treated with gemcitabine, R1507 and metformin alone or in combination. The effects of treatments were evaluated for cell proliferation, apoptosis, and the expression of genes related to inhibition of apoptosis and chemotherapy resistance. Combination of gemcitabine with R1507 and/or metformin additively interacted with the inhibition of cell proliferation in human pancreatic ductal adenocarcinoma cell lines, SUIT-2 and MIAPaCa-2 with differential gemcitabine resistance, and assessment of apoptosis demonstrated that drug associations increased the apoptotic index in both cell lines. Treatment with gemcitabine induced the expression of survivin and XIAP in both cell lines, indicating the induction of chemoresistance. In conclusion, these data demonstrate that the combination of gemcitabine with R1507 and/or metformin has an additive effect in pancreatic cancer cell lines with differential sensitivity to gemcitabine; however, gemcitabine may induce chemotherapy resistance.     

Overexpression of neuropilin-1 promotes constitutive MAPK signalling and chemoresistance in pancreatic cancer cells

Neuropilin-1 (NRP-1) is a novel co-receptor for vascular endothelial growth factor (VEGF). Neuropilin-1 is expressed in pancreatic cancer, but not in nonmalignant pancreatic tissue. We hypothesised that NRP-1 expression by pancreatic cancer cells contributes to the malignant phenotype. To determine the role of NRP-1 in pancreatic cancer, NRP-1 was stably transfected into the human pancreatic cancer cell line FG. Signal transduction was assessed by Western blot analysis. Susceptibility to anoikis (detachment induced apoptosis) was evaluated by colony formation after growth in suspension. Chemosensitivity to gemcitabine or 5-fluorouracil (5-FU) was assessed by MTT assay in pancreatic cancer cells following NRP-1 overexpression or siRNA-induced downregulation of NRP-1. Differential expression of apoptosis-related genes was determined by gene array and further evaluated by Western blot analysis. Neuropilin-1 overexpression increased constitutive mitogen activated protein kinase (MAPK) signalling, possibly via an autocrine loop. Neuropilin-1 overexpression in FG cells enhanced anoikis resistance and increased survival of cells by > 30% after exposure to clinically relevant levels of gemcitabine and 5-FU. In contrast, downregulation of NRP-1 expression in Panc-1 cells markedly increased chemosensitivity, inducing > 50% more cell death at clinically relevant concentrations of gemcitabine. Neuropilin-1 overexpression also increased expression of the antiapoptotic regulator, MCL-1. Neuropilin-1 overexpression in pancreatic cancer cell lines is associated with (a) increased constitutive MAPK signalling, (b) inhibition of anoikis, and (c) chemoresistance. Targeting NRP-1 in pancreatic cancer cells may downregulate survival signalling pathways and increase sensitivity to chemotherapy.     

Knockdown of clusterin sensitizes pancreatic cancer cells to gemcitabine chemotherapy by ERK1/2 inactivation

ABSTRACT: ObjectivesTo study the hypothesis that gemcitabine treatment augments the chemoresistance to gemcitabine by clusterin(sCLU) upregulation. Clusterin inhibition could augment the chemosensitivity of human pancreatic cancer cells by inhibition of clusterin-dependent pERK1/2 activation. METHODS: Clusterin was silenced by serial concentration of OGX-011 transfection in pancreatic cancer MIAPaCa-2 and BxPC-3 cell lines, then treated with serial concentration of gemcitabine. After the cells were treated with OGX-011 for 8 h,the cells were then treated with 5 muM ERK inhibitor PD98059 for 18 h or transfected with a wt-pERK-expressing plasmid into these cells for 24 h, after which the cells were treated with 1.0 uM gemcitabine for 24--72 h. Cell proliferation was determined by MTT. Apoptosis was quantified by flow cytometry,.sCLU and pERK1/2 production was analyzed by western blot,and sCLU mRNA was analyzed by RT-PCR. Xenograft of established tumors was used to evaluate primary tumor growth and apoptosis after treatment with gemcitabine alone or in combination with OGX-011. Phosphorylated ERK1/2 and sCLU levels in tumor tissues were measured by TUNEL analysis. RESULTS: As detected by MTT and FACS assay, a combination of gemcitabine + OGX-011 reflected the chemotherapeutic sensitivity and increased the gemcitabine -induced apoptosis in MIAPaCa-2 and BxPC-3 cells. Western blotting and RT-PCR analysis revealed that the expression of clusterin was higher in gemcitabine -resistant MIAPaCa-2 cells, however, decreased significantly after pretreatment with OGX-011. Furthermore, the OGX-011 or combination of gemcitabine + OGX-011 decreased the gemcitabine -induced activation of pERK1/2. wt-pERK-re-expression decreased OGX-011+ gemcitabine -induced apoptosis. Finally, OGX-011 in combination with gemcitabine substantially decreased the in vivo tumor growth and promoted apoptosis. Taken together, clusterin confers gmcitabine resistance in pancreatic cancer cells. CONCLUSIONS: Knockdown of clusterin by OGX-011 transfection sensitizes pancreatic cancer cells to gemcitabine by inhibition of gemcitabine -induced clusterin-pERK1/2 activation.     

Sirtuin 1 facilitates chemoresistance of pancreatic cancer cells by regulating adaptive response to chemotherapy‐induced stress

Chemotherapy drugs themselves may act as stressors to induce adaptive responses to promote the chemoresistance of cancer cells. Our previous research showed that sirtuin 1 (SIRT1) was overexpressed in pancreatic cancer patients and deregulation of SIRT1 with RNAi could enhance chemosensitivity. Thus, we hypothesized that SIRT1 might facilitate chemoresistance in pancreatic cancer cells through regulating the adaptive response to chemotherapy‐induced stress. In the present study, SIRT1 in PANC‐1, BXPC‐3, and ASPC‐1 cells was upregulated after treatment with gemcitabine. Moreover, the decrease in SIRT1 activity with special inhibitor EX527 had a synergic effect on chemotherapy with gemcitabine in PANC‐1 and ASPC‐1 cell lines, which significantly promoted apoptosis, senescence, and G₀/G₁ cycle arrest. Western blot results also showed that SIRT1, acetylated‐p53, FOXO3a, and p21 were upregulated after combined treatment, whereas no obvious change was evident in total p53 protein. To further confirm the role of SIRT1 in clinical chemotherapy, SIRT1 was detected in eight pancreatic cancer tissues acquired by endoscopy ultrasonography guided fine needle aspiration biopsy before and after chemotherapy. Compared to before chemotherapy, SIRT1 was significantly increased after treatment with gemcitabine in six cases. Thus, our results indicated a special role for SIRT1 in the regulation of adaptive response to chemotherapy‐induced stress, which is involved in chemoresistance. Moreover, it indicates that blocking SIRT1 activity with targeting drugs might be a novel strategy to reverse the chemoresistance of pancreatic cancer.