RNA干扰沉默DcR3对人胰腺癌细胞裸鼠移植瘤放疗增敏的实验研究

摘 要：［目的］探讨RNA干扰沉默DcR3对人胰腺癌细胞裸鼠移植瘤放疗敏感性的影响及其可能机制。［方法］ 取对数生长期的AsPC-1细胞 1×107/ml,接种于6周龄裸鼠左后肢腹股沟,当皮下肿瘤直径为8mm左右时,随机分为4组(n=15)：PBS组、DcR3siRNA组、放疗组（RT）和DcR3siRNA+放疗组（DcR3siRNA+RT）,观察DcR3siRNA联合放疗对人胰腺癌AsPC-1细胞裸鼠移植瘤的治疗效果;应用ELISA和Western blot检测各组DcR3的蛋白表达变化;免疫组织化学和Western blot分析各组Caspase-8和Caspase-3蛋白表达的变化;TUNEL检测各组肿瘤细胞凋亡情况。［结果］ DcR3siRNA+RT组较其他各组更能抑制移植瘤的生长,DcR3-siRNA+RT对肿瘤的抑制率明显高于单纯RT组,与RT组相比,DcR3siRNA+RT组的抑瘤率为80.86%±4.17%;DcR3siRNA+RT组的DcR3蛋白量和蛋白相对表达均明显低于RT组,差异有统计学意义(P＜0.05);DcR3siRNA+RT组的Caspase-8和Caspase-3蛋白表达均明显高于单独RT组;DcR3siRNA组、RT组以及DcR3-siRNA+RT组肿瘤组织内均可观察到凋亡细胞,而DcR3siRNA+RT组肿瘤细胞凋亡数目明显高于RT组或DcR3siRNA组。［结论］RNA干扰沉默DcR3基因可以增加人胰腺癌细胞裸鼠移植瘤对放疗的敏感性,该作用可能与沉默DcR3可激活Caspase-8/Caspase-3凋亡途径和促进肿瘤细胞凋亡有关。     

RNA干扰靶向沉默诱骗受体3 3基因增加人胰腺癌细胞的放射敏感性

目的:探讨RNA干扰沉默诱骗受体3(decoy receptor 3,DcR3)基因对胰腺癌细胞放射敏感性的影响及其相关机制.方法:构建带有DcR3 siRNA序列的稳定表达质粒,通过脂质体转染至胰腺癌AsPC-1细胞株,设对照组、siRNA(-)阴性对照组和DcR3 siRNA组,应用Western blotting检测AsPC-1细胞中DcR3表达的变化,平板克隆形成实验检测转染DcR3 siRNA后AsPC-1细胞放射敏感性的变化,流式细胞术检测细胞凋亡,RT-PCR和Western blotting检测Caspase-8、Caspase-3和PARP-1表达的变化.结果:DcR3 siRNA组细胞中DcR3蛋白表达水平较对照或siRNA(-)组明显降低(均P＜0.01);DcR3 siRNA组的克隆形成率明显低于对照或siRNA(-)组,其存活分数(survival fraction,SF)降低、α/β比值升高(均P＜0.01);放射后DcR3 siRNA组肿瘤细胞凋亡率明显高于对照或siRNA(-)组(均P＜0.01);转染DcR3 siRNA后可以明显上调Caspase-8、Caspase-3的表达和下调PARP-1的表达.结论:RNA干扰沉默DcR3基因通过激活凋亡因子Caspase-8和Caspase-3促进细胞凋亡,从而增加胰腺癌细胞对放射的敏感性.     

STAT3基因沉默对胰腺癌细胞放射敏感性的影响及其机制

目的:探讨信号转导和转录激活因子3（STAT3）基因沉默对人胰腺癌细胞AsPC-1和PANC-1放射敏感性的影响及其机制。方法:体外培养人胰腺癌细胞AsPC-1和PANC-1，通过脂质体转染STAT3 shRNA作为实验组（shSTAT3组），设置阴性对照（shNC组）和空白对照组（NC组）。采用qRT-PCR检测转染后各组AsPC-1和PANC-1细胞中STAT3 mRNA表达水平，Western blot检测细胞中STAT3蛋白表达水平。MTT实验检测各组细胞增殖能力，流式细胞仪检测各组细胞电子射线照射后的凋亡情况，qRT-PCR和Western blot分别检测各组细胞电子射线照射后的Bax、Bcl-2和Caspase-3 mRNA和蛋白的表达情况。结果:shSTAT3组AsPC-1和PANC-1细胞中STAT3 mRNA和蛋白表达水平比NC组和shNC组明显降低（P<0.05）。接受电子射线照射后，shSTAT3组AsPC-1和PANC-1细胞增殖率显著低于shNC组（P<0.05），其细胞凋亡率较shNC组明显升高（P<0.05）。接受电子射线照射后，转染STAT3 shRNA后能够明显上调细胞中Bax和Caspase-3的表达和下调Bcl-2的表达（P<0.05）。结论:沉默STAT3基因能够通过上调Bax和Caspase-3的表达和下调Bcl-2的表达促进细胞凋亡，增强胰腺癌细胞的放射敏感性。     

siRNA干扰S100A4对人胰腺癌细胞增殖和凋亡及其对吉西他滨敏感性影响的研究

目的 观察利用小干扰RNA（siRNA）手段下调人胰腺癌BxPC-3、AsPC-1细胞中S100A4表达后对细胞凋亡、增殖、吉西他滨化疗敏感性的影响。方法 设计合成针对S100A4特异的siRNA转染人胰腺癌BxPC-3、AsPC-1细胞,以RT_PCR检测转染前后S100A4蛋白表达变化;转染前后BxPC-3细胞生长曲线检测BxPC-3、AsPC-1细胞增殖能力;TUNEL法检测转染前后细胞凋亡变化;MTT检测不同浓度吉西他滨对胰腺癌细胞半数抑制浓度（IC50）。结果：RT_PCR结果显示BxPC-3、AsPC-1细胞转染SlOOA4siRNA48h后,S100A4mRNA表达明显下调;S100A4siRNA转染后BxPC～3、AsPC一1细胞增殖下降;TUNEL结果显示转染后凋亡明显增加;MTT结果显示,BxPC-3细胞对照组吉西他滨IC5。为（9．95士0．34）mmo[／L;阴性转染组吉西他滨IC50为（9．641±0．434）mmoI／L;干扰组吉西他滨IC。o为（1．182±0．175）gmol／L;AsPC一1细胞对照组吉西他滨IC50（64．15±3．41）mmol／L;阴性转染组吉西他滨IC50为（65．19±4．4）mmol／L;干扰组吉西他滨IC50为（1．962±0．113）mmol／L,P〈0．05。结论：S100A4沉默后抑制胰腺癌细胞增殖、促进凋亡并提高吉西他滨化疗敏感性,提示S100A4可能是治疗胰腺癌的有效靶点。     

干扰PIM3的表达对人胰腺癌AsPC-1细胞增殖和凋亡的影响及作用机制研究

目的 探讨干扰PIM3的表达对人胰腺癌AsPC-1细胞增殖和凋亡的影响及作用机制.方法 将阴性对照[scramble小干扰RNA(siRNA)]和干扰PIM3(PIM3 siRNA)分别转染至人胰腺癌AsPC-1细胞,分别作为scramble-siRNA组和PIM3-siRNA组,以1×磷酸盐缓冲液(PBS)作为溶剂对照.采用实时荧光定量聚合酶链反应(RT-PCR)检测人胰腺癌AsPC-1细胞PIM3、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(BAX)、Ki-67、cas-pase 3 mRNA的相对表达量,蛋白质印迹法(Western blot)检测PIM3、Bcl-2、BAX、caspase 3蛋白的相对表达量;四甲基偶氮唑蓝(MTT)检测干扰PIM3的表达对人胰腺癌AsPC-1细胞增殖能力的影响,流式细胞仪检测干扰PIM3的表达对细胞凋亡能力的影响.探讨干扰PIM3的表达对荷瘤小鼠人胰腺癌AsPC-1细胞的影响.结果 转染后,PIM3-siRNA组细胞PIM3 mRNA和PIM3蛋白的相对表达量均明显低于溶剂对照组和scramble-siRNA组(P﹤0.01).干扰PIM3的表达可明显抑制人胰腺癌AsPC-1细胞增殖,促进其凋亡.干扰PIM3的表达可下调人胰腺癌AsPC-1细胞中Ki-67、Bcl-2基因的表达,上调BAX、caspase 3基因的表达;且干扰PIM3的表达可下调Bcl-2蛋白的表达,上调BAX、caspase 3蛋白的表达.荷瘤小鼠经PIM3 siRNA治疗后,小鼠肿瘤质量和体积均明显下降.结论 PIM3 siRNA可通过干扰PIM3的表达,诱导人胰腺癌AsPC-1细胞的凋亡,在体内体外均可抑制胰腺癌AsPC-1细胞的生长.     

p53正向凋亡调控因子在吉西他滨诱导胰腺癌细胞凋亡过程中的作用

目的:研究吉西他滨对人胰腺癌AsPC-1细胞体外生长的作用机制.方法:用脂质体转染法将含有p53正向凋亡调控因子(p53 upregulated modulator of apoptosis,PUMA)反义核酸的真核表达载体pcDNA3.1-PUMAAS和空载体pcDNA3.1导入AsPC-1细胞,G418筛选阳性细胞,获得稳定转染的阳性克隆.将转染载体的AsPC-1阳性克隆细胞和未转染载体的AsPC-1细胞分别暴露于浓度为1、5、10和15 μmol/L的吉西他滨溶液中作用72 h.RT-PCR和Western印迹法检测不同组细胞经吉西他滨作用72 h后的PUMA表达水平,MTT检测细胞生长抑制情况, FCM、Hoechst 33258荧光染色和TUNEL法检测细胞凋亡情况.结果:吉西他滨促进AsPC-1细胞凋亡,抑制细胞生长,并有明显的剂量依赖性,在细胞凋亡的同时伴有PUMA表达的上调;当细胞转染PUMA反义核酸抑制PUMA表达后,受吉西他滨作用的细胞中PUMA蛋白表达明显降低,同时伴有细胞凋亡的抑制及细胞增殖明显增加.结论:吉西他滨促进体外AsPC-1细胞凋亡,并抑制细胞生长,其诱导细胞凋亡与上调PUMA表达有关.     

PDCD4促进吉西他滨诱导胰腺癌细胞凋亡的实验研究

目的:研究程序性细胞死亡因子4（programmed cell death 4,PDCD4）在吉西他滨诱导胰腺癌细胞凋亡中的作用。方法:用吉西他滨处理胰腺癌细胞PANC-1,荧光定量PCR和Western blot分别检测胰腺癌细胞中PDCD4的表达变化。PANC-1细胞感染PDCD4-pGC-Fu-GFP重组慢病毒和对照pGC-Fu-GFP重组慢病毒,荧光定量PCR和Western blot检测过表达效果。用吉西他滨处理过表达PDCD4的PANC-1细胞,MTT测定细胞增殖,克隆形成实验测定细胞克隆能力,流式细胞术测定细胞凋亡,Western blot检测细胞中剪切的Caspase-3（Cleaved Caspase-3）、剪切的Caspase-9（Cleaved Caspase-9）蛋白水平和胞浆、线粒体中细胞色素C（Cytochrome C）蛋白水平。结果:吉西他滨处理后的PANC-1细胞中PDCD4 mRNA和蛋白水平均明显升高。吉西他滨处理和过表达PDCD4的PANC-1细胞增殖、克隆形成能力明显降低,细胞凋亡率明显升高,细胞中Cleaved Caspase-3、Cleaved Caspase-9蛋白水平升高,胞浆中Cytochrome C蛋白水平也升高,线粒体中Cytochrome C蛋白水平降低。吉西他滨处理过表达PDCD4的PANC-1细胞增殖能力、克隆形成能力降低更多,细胞凋亡率更高,细胞中Cleaved Caspase-3、Cleaved Caspase-9蛋白水平也更高,胞浆中Cytochrome C蛋白水平更高,线粒体中Cytochrome C蛋白水平更低。结论:吉西他滨通过上调PDCD4表达水平激活线粒体途径诱导胰腺癌细胞凋亡。     

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。     

凋亡抑制蛋白XIAP和促凋亡因子Smac在胰腺癌化疗抵抗中的调控作用

目的探讨凋亡抑制蛋白XIAP和促凋亡因子Smac在胰腺癌细胞化疗抵抗中的作用及其分子机制。方法应用流式细胞术检测顺铂、5-FU介导的Panc-1、BXPC-3的凋亡率及胞浆染色分析细胞XIAP表达变化,Western-blot分析XIAP、Smac、Caspase-3表达水平；构建pEGFP-N1／Smac真核表达载体并转染胰腺癌Panc-1细胞,流式细胞术检测转染胞浆表达型 Smac基因对Panc-1细胞凋亡敏感性的作用。结果与BXPC-3细胞相比,Panc-1对顺铂或5-FU介导的凋亡具有较强抵抗性,Western blot分析显示Panc-1细胞高表达XIAP,在化疗药物作用下化疗敏感细胞BXPC-3胞浆内XIAP水平下降明显多于Panc-1细胞,而且凋亡的BXPC-3细胞释放入胞浆内的成熟 Smac蛋白水平明显高于Panc-1细胞。转染胞浆表达型Smac基因至化疗抵抗Panc-1细胞,可明显下调其XIAP表达水平,促进效应 Caspase-3分子活化,显著提高顺铂、5-FU诱导的细胞凋亡率。结论在化疗药物诱导的凋亡中,线粒体释放Smac下调XIAP是胰腺癌化疗敏感性的重要决定因素,而上调Smac活性蛋白的胞浆表达作为一种有效调节信号,通过拮抗XIAP的凋亡抑制作用协同化疗药物促进胰腺癌细胞凋亡。     

siRNA干扰STAT3基因对食管癌Eca-109细胞生物学特性的影响

目的:探讨RNA干扰技术沉默STAT3信号传导通路对人食管癌Eca-109细胞增殖、细胞周期和凋亡的影响.方法:针对人STAT3基因mRNA序列设计合成3对编码小干扰RNA(siRNA)的DNA模板,将其连入pRNAT-U6.1/neo质粒中,构建STAT3-siRNA表达载体,稳定转染人食管癌Eca-109细胞,利用RT-PCR、Western blot技术分别检测转染细胞中STAT3基因mRNA和蛋白的表达,MTT实验、流式细胞技术检测转染细胞的增殖、细胞周期和凋亡变化情况.结果:成功构建STAT3-siRNA表达载体,经测序鉴定正确.RT-PCR和Western blot结果显示,STAT3-siRNA3表达载体明显抑制了人食管癌Eca-109细胞中STAT3基因mRNA和蛋白的表达,与对照组相比,差异有统计学意义(P＜0.01).MTT实验显示,稳定转染siRNA后,siRNA3实验组细胞的增殖能力明显下降,细胞生长抑制卒为35.68%,较对照组明显升高,差异有统计学意义(P＜0.01).流式细胞技术显示,siRNA3实验组出现了细胞周期阻滞和细胞凋亡,细胞周期阻滞于G_0/G_1期,细胞凋亡率为13.26%,明显高于对照组,差异有统计学意义(P＜0.01);结论:RNA干扰技术沉默STAT3基因可以有效地抑制STAT3基因的表达,进而抑制人食管癌Eca-109细胞的增殖,引起细胞周期G_0/G_1期阻滞,促进其凋亡.     

Enhancement of gemcitabine sensitivity in pancreatic cancer by co-regulation of dCK and p8 expression

The purpose of this study was to improve the gemcitabine sensitivity in pancreatic cancer by adenovirus-mediated co-regulation of dCK and p8 expression. Firstly, we analyzed the sensitivity of three human pancreatic tumor cell lines (Capan-2, Panc-1 and BxPc-3) to gemcitabine using MTT assays, and found Panc-1 to be relatively resistant to gemcitabine. Further, we investigated the expression of dCK and p8 in different pancreatic cancer cell lines using real-time PCR and Western blot analysis, and found that the expression levels of these two genes were related to the gemcitabine sensitivity of pancreatic cancer cells. We constructed recombinant adenovirus vectors, Ad-dCK and Ad-p8-siRNA, that overexpressed dCK and knocked down p8, respectively. Using MTT assays, we observed that combined infection using Ad-dCK and Ad-p8-siRNA in vitro led to a significant decrease in the gemcitabine IC50 with an increase in apoptosis and caspase-3 activity in Panc-1 cells, which are relatively resistant to gemcitabine. Furthermore, in established subcutaneous pancreatic cancer models in nude mice, the tumor inhibition was markedly enhanced accompanied by elevation of the apoptosis index after intratumoral injection of Ad-dCK and Ad-p8-siRNA on the basis of intraperitoneal gemcitabine chemotherapy. Taken together, the present findings suggest that, dCK and p8 may be the important factors in the regulation of gemcitabine sensitivity in pancreatic cancer cells. Moreover, co-regulation of the two factors achieved better effects than regulation of either one alone.     

Human pancreatic cancer cells express non-functional Fas receptors and counterattack lymphocytes by expressing Fas ligand; a potential mechanism for immune escape

The aim of this study was to investigate the expression and functional status of Fas ligand (FasL) and its receptor (Fas) in human pancreatic cancers. Using RT-PCR and Western blotting, Fas and FasL were expressed in seven surgically resected pancreatic cancer specimens and five cell lines; Capan-1, AsPC-1, BxPC-3, PANC-1, and MIA PaCa-2. In the resected specimens, pancreatic cancer cells induced apoptosis in the surrounding lymphoid cells. In coculture experiments of pancreatic cancer and Jurkat T cells, 50% of Jurkat T cells underwent apoptosis after 2 days, however, almost all pancreatic cancer cells remained viable. In addition, by testing Fas function using anti-Fas antibody (CH11), all cell lines were resistant to Fas-mediated apoptosis except Capan-1 cells which showed sensitivity similar to that of Jurkat T cells. These results suggest that pancreatic cancer cells evade immune surveillance by expression of FasL and non-functioning Fas that allow them to activated T-cells. These tumor escape mechanisms may contribute to the rapid fatal course of pancreatic cancer.     

靶向沉默人类真核翻译延长因子1A2可抑制胰腺癌细胞BxPC-3体外迁移和侵袭

目的:探讨靶向沉默人类真核翻译延长因子1A2 (homo sapiens eukaryotic translation elongation factor 1 alpha 2,EEF1A2)对胰腺癌BxPC-3细胞体外迁移和侵袭的影响及其可能的分子机制.方法:应用RT-PCR和蛋白质印迹法检测4种胰腺癌细胞株中EEF1A2的mRNA和蛋白表达水平.将EEF1A2-小干扰RNA (smallinterference RNA,siRNA)和阴性对照siRNA转染到BxPC-3细胞中,随后采用RT-PCR和蛋白质印迹法分别检测EEF1A2的mRNA及蛋白表达,应用体外划痕和Transwell侵袭实验分别检测BxPC-3细胞的体外迁移和侵袭能力,蛋白质印迹法检测转染前后细胞中p -Akt和总Akt蛋白的表达变化.结果:4种胰腺癌细胞株中除SW1990细胞呈EEF1A2低表达外,BxPC-3、Patu8988和Panc-1细胞均呈EEF1A2高表达.EEF1A2-siRNA转染BxPC-3细胞48 h后,EEF1A2 mRNA及蛋白表达水平较阴性对照siRNA转染的细胞和空白对照细胞明显降低(P＜0.01).EEF1A2-siRNA转染组BxPC-3细胞的体外迁移和侵袭能力明显低于阴性转染对照组和空白对照组(P＜0.05).EEF1A2-siRNA转染组细胞中Akt的磷酸化水平明显低于对照组细胞(P＜0.05),而总Akt蛋白表达水平未显著改变(P＞0.05).结论:siRNA干扰下调EEF1A2基因表达可明显抑制胰腺癌BxPC-3细胞的体外迁移和侵袭,其机制可能与EEF1A2调控Akt信号通路有关.     

沉默ANO9增加胰腺癌细胞AsPC-1放射敏感性的实验研究

目的 探讨ANO9对胰腺癌细胞AsPC-1放射敏感性的影响,以期为胰腺癌临床放射治疗提供新的增敏靶点。方法 蛋白印迹法检测胰腺癌细胞系(BxPC-3、PANC-1、AsPC-1)和正常胰腺细胞系中ANO9表达水平;慢病素感染构建沉默ANO9的AsPC-1稳转株,并用蛋白印迹法进行验证;MTT检测沉默ANO9对放射照射后AsPC-1细胞活力的影响;平板克隆形成实验检测沉默ANO9对AsPC-1细胞放射敏感性的影响;蛋白印迹法检测沉默ANO9对EGFR/ERK信号蛋白表达的影响。结果 与正常胰腺细胞系相比,3个胰腺癌细胞系中ANO9表达均增加(均P<0.05);沉默ANO9后细胞中ANO9蛋白表达水平较对照组显著降低(P<0.05),成功构建沉默ANO9的AsPC-1稳转株;沉默ANO9后AsPC-1细胞对放射照射的敏感性明显增加,放射增敏比为1.57,细胞中EGFR/ERK信号蛋白EGFR和p-ERK1/2均下调(均P<0.05)。结论 沉默ANO9能够显著增加胰腺癌细胞AsPC-1的放射敏感性,其机制可能与抑制EGFR/ ERK信号转导有关,ANO9可能成为遏制胰腺癌进展的新靶标。     

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.     

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.