吉非替尼联合培美曲塞对EGFR-TKI获得性耐药的非小细胞肺癌细胞的协同效应

目的:探讨培美曲塞联合吉非替尼对体外诱导的表皮生长因子受体-酪氨酸激酶抑制剂(epidermal growth factor receptor-tyrosine kinase inhibitor,EGFR-TKI)获得性耐药的人非小细胞肺癌PC9/吉非替尼耐药(gei tinib resistance,GR)细胞株的效应及其可能的机制。方法:吉非替尼和培美曲塞单药或联合作用于PC9/GR细胞后,MTT法检测各药物处理组细胞的增殖抑制率及药物的联合指数(combination index,CI),FCM法检测各组细胞的凋亡率,蛋白质印迹法检测各组细胞磷酸化AKT和Bcl-2蛋白的表达水平。结果:吉非替尼和培美曲塞联合应用对PC9/GR细胞的增殖抑制作用和促凋亡作用明显强于各单药组(P0.05);吉非替尼和培美曲塞的CI值1,表现出明显的协同效应。与未进行药物处理的对照组比较,培美曲塞联合吉非替尼可明显下调PC9/GR细胞中磷酸化AKT和Bcl-2蛋白的表达水平(P0.01)。结论:培美曲塞联合吉非替尼对PC9/GR细胞具有较好的协同作用,这协同作用可能与诱导细胞凋亡和下调磷酸化AKT蛋白表达有关。     

洛伐他汀克服非小细胞肺癌PC9细胞株吉非替尼耐药的体外研究

目的:研究联合洛伐他汀(lovastatin)和吉非替尼(gefi tinib)对体外诱导吉非替尼获得性耐药的非小细胞肺癌细胞株PC9细胞凋亡以及相关蛋白表达的影响,并探讨其可能的机制。方法:应用洛伐他汀联合吉非替尼处理耐吉非替尼的非小细胞肺癌PC9细胞株后,采用WST-1法检测不同药物处理对PC9细胞增殖的影响,Hoechst33342荧光染色法观察细胞凋亡形态,FCM法观察细胞凋亡状况,蛋白质印迹法检测凋亡相关蛋白的表达水平。结果:洛伐他汀联合吉非替尼可在体外诱导耐吉非替尼的PC9细胞凋亡,抑制其细胞增殖;洛伐他汀联合吉非替尼可诱导耐吉非替尼的PC9细胞中磷酸化表皮生长因子受体(phosphorylated epidermal growth factor receptor,p-EGFR)、磷酸化蛋白激酶B(phosphorylated protein kinase B,p-AKT)和磷酸化细胞外调节蛋白激酶1/2(phosphorylated extracellular signal-regulated kinase1/2,p-ERK1/2)蛋白表达水平明显下调。结论:在体外诱导吉非替尼获得性耐药的非小细胞肺癌细胞株PC9中,洛伐他汀可以克服吉非替尼耐药,两者具有良好的协同作用,提示两药联合对于出现吉非替尼耐药的非小细胞肺癌的临床治疗可能具有很大的应用潜力。     

MircoRNA-214对吉非替尼敏感及耐药细胞增殖和凋亡的影响

摘 要：［目的］ 研究mircoRNA-214（miR-214）对PC9肺腺癌及吉非替尼耐药细胞（PC9/GR）增殖和凋亡的影响。［方法］ 在PC9细胞中转染miR-214模拟物及PC9/GR中转染miR-214抑制剂,使用定量逆转录PCR（qRT-PCR）检测其表达。MTT检测细胞转染miR-214模拟物或其抑制剂后的存活及增殖。在PC9细胞中顺时转染miR-214模拟物以检测上调miR-214对PC9细胞耐药性的影响,在PC9/GR细胞中顺时转染miR-214抑制物以检测下调miR-214对PC9/GR细胞耐药性的影响,并用流式细胞仪检测细胞的凋亡。Western blotting检测PTEN在PC9和PC9/GR细胞中的表达。构建PTEN 3’-UTR荧光素酶报告质粒验证miR-214的靶基因;建立异种移植模型检测miR-214抑制物对肺癌移植瘤的影响。［结果］ PC9细胞中miR-214低表达,上调miR-214的表达后PC9细胞对吉非替尼的敏感性降低,并且抵抗吉非替尼诱导的凋亡;而在PC9/GR细胞中低表达miR-214后,下调miR-214增加PC9/GR细胞对吉非替尼的敏感性,并且可以增强吉非替尼诱导的凋亡作用。荧光素酶报告载体实验证实PTEN是miR-214在细胞内的靶基因。动物异种移植模型表明miR-214抑制物可以增强PC9/GR对吉非替尼的敏感性。［结论］ MiR-214可能通过靶基因PTEN调控吉非替尼的获得性耐药。     

IGF-1R抑制剂联用可增强人非小细胞肺癌PC9/G细胞对吉非替尼的敏感性

目的:观察单用表皮生长因子受体(epidermal growth factor receptor, EGFR)的酪氨酸激酶抑制剂吉非替尼 (ge? tinib) 或联合胰岛素生长因子-1受体(insulin-like growth factor-1 receptor,IGF-1R)的酪氨酸激酶抑制剂AG1024作用于人非小细胞肺癌耐药株PC9/G细胞后,该细胞对吉非替尼耐药性的影响,并探讨IGF-1R与肿瘤细胞耐药的相关机制。方法:用吉非替尼和AG1024单独或联合作用于PC9/G细胞后,采用MTT法分别检测各组细胞的增殖情况,并利用中效原理判断两药联用的效果;FCM法检测各组细胞的凋亡情况;Western印迹法检测各组细胞的磷酸化EGFR(phosphorylated EGFR, p-EGFR)、磷酸化Akt(phosphorylated Akt,p-Akt)和磷酸化细胞外信号调节激酶(phosphorylated extracellular signal-regulated kinase,p-ERK)的表达水平。结果:吉非替尼和AG1024单独作用于PC9/G细胞后,均出现不同程度的细胞增殖抑制作用和细胞凋亡促进作用;而吉非替尼和AG1024联合作用,能更显著地抑制细胞增殖,且凋亡细胞显著增加(P<0.05)。 Western印迹法检测发现,联合用药组的p-EGFR、p-Akt和p-ERK蛋白表达量明显减少。结论:IGF-1R抑制剂AG1024和EGFR抑制剂吉非替尼联用具有较好的协同作用,可能通过抑制细胞增殖和促进细胞凋亡,提高耐药细胞对吉非替尼的敏感性。     

整合素β1表达上调对非小细胞肺癌吉非替尼耐药的影响

目的:探讨整合素β1表达上调对表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptortyrosine kinase inhibitor,EGFR-TKI)吉非替尼抑制非小细胞肺癌细胞增殖以及肿瘤生长的影响。方法:将PC9、PC9/PCD(脂质体pcDNA3.1稳定转染空白对照株)和PC9/D6(脂质体稳定转染整合素β1细胞株)3株细胞经0.5μmol/L吉非替尼作用后,MTT法检测3株细胞的增殖情况。建立3株细胞的裸鼠移植瘤模型,设治疗组(3mg/kg吉非替尼)和对照组(1%Tween-80),观察肿瘤的生长情况,绘制肿瘤生长曲线,称取瘤体质量,计算抑瘤率。免疫组织化学法检测裸鼠移植瘤组织中整合素β1、EGFR和pEGFR的表达情况。结果:转染了整合素β1的PC9/D6组增殖率明显高于PC9/PCD和PC9组(P<0.05)。吉非替尼对PC9/D6裸鼠移植瘤的生长抑制最不明显,抑制率是61.38%,与PC9/PCD和PC9组比较差异具有统计学意义(P<0.01);PC9/PCD和PC9组的抑制率分别是93.54%和95.16%。PC9/D6裸鼠移植瘤中整合素β1的表达水平高于PC9/PCD和PC9组(P<0.01)。EGFR在3组之间表达的差异无统计学意义。3组裸鼠移植瘤经吉非替尼治疗后,pEGFR表达水平均有不同程度的下降,但PC9/D6治疗组中pEGFR表达水平显著高于PC9/PCD和PC9组(P<0.01)。结论:无论体内或体外实验均显示,整合素β1表达上调可降低细胞株或移植瘤对吉非替尼的敏感性,提示整合素β1可能通过一种新的耐药机制参与了肿瘤细胞对EGFR-TKI的耐药。     

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。     

肺腺癌吉非替尼获得性耐药相关microRNAs的筛选鉴定

背景与目的肺腺癌吉非替尼获得性耐药严重影响了肺癌的治疗效果,microRNA在肺腺癌吉非替尼获得性耐药中的作用及其机制尚不清楚。本研究筛选与肺腺癌获得性吉非替尼耐药相关的microRNAs。方法以吉非替尼敏感肺癌细胞PC9与吉非替尼耐药肺癌细胞PC9/AB11为细胞模型,观察二者的形态学差异,流式细胞仪检测二者的细胞周期,计算它们的倍增时间,MTT法检测吉非替尼对两种细胞的IC50,应用microRNA芯片检测和筛选与吉非替尼耐药相关的microRNAs,并进行real-timePCR验证。结果 PC9细胞与PC9/AB11细胞形态差异明显,在细胞周期、倍增时间和吉非替尼对其的IC50上均具有统计学差异。microRNA芯片结果显示,与PC9相比,耐药肺癌细胞株PC9/AB11中有4个microRNAs表达水平明显上调,有9个microRNAs表达水平明显下调。经real-timePCR验证,microRNA-138在PC9/AB11中表达明显下调,与芯片结果一致。结论 PC9和PC9/AB11细胞株microRNA表达谱存在明显差异,初步筛选到了13个与肺腺癌吉非替尼耐药密切相关的microRNAs,为进一步深入研究microRNA在肺腺癌吉非替尼获得性耐药中的作用及其分子机制提供了实验依据和理论基础。     

培美曲塞和吉非替尼不同时序应用对人肺腺癌细胞的作用和机制

目的 探讨培美曲塞与吉非替尼不同时序应用对肺腺癌细胞A549和PC-9生长及凋亡的影响, 并阐述其可能机制。方法 MTT法检测各组细胞的增殖抑制情况,流式细胞仪检测各组细胞凋亡及细胞周期分布,Western印迹法检测对EGFR下游信号通路及TS酶蛋白水平表达的影响。结果 培美曲塞序贯吉非替尼、培美曲塞同步联合吉非替尼对PC-9和A549细胞增殖抑制率及凋亡率较单药组均提高（P<0.05）,培美曲塞可以提高EGFR、AKT 、ERK磷酸化水平,而吉非替尼表现为抑制作用, 同时吉非替尼降低TS酶表达。培美曲塞序贯吉非替尼,培美曲塞同步联合吉非替尼抑制EGFR、AKT 、ERK磷酸化水平较单药更强。吉非替尼主要将PC-9、A549细胞阻滞在G0/G1期;培美曲塞主要将细胞阻滞在S期。培美曲塞序贯吉非替尼、培美曲塞同步联合吉非替尼较其他组G2/M期细胞比例提高（P<0.05）。结论 培美曲赛序贯吉非替尼、培美曲赛同步联合吉非替尼在PC-9、A549细胞中均起到协同增效作用,且培美曲赛序贯吉非替尼协同作用更为显著,可能主要与培美曲赛诱导EGFR、AKT 、ERK磷酸化及吉非替尼降低TS酶作用有关。     

吉非替尼耐药细胞株的筛选和基因谱表达研究

目的：从对吉非替尼（gefitinib）敏感的肺腺癌细胞株PC9中筛选并建立了6株gefitinib耐药细胞亚克隆并研究其生物学特性。方法：采用N-甲基-N’-硝基-N-亚硝基胍（MNNG）诱变筛选和有限稀释法进行耐药性亚克隆的单细胞培养,MTT法检测肿瘤细胞对gefitinib的敏感性和IC50,DNA微列阵检测耐药细胞株与野生型PC9细胞的基因表达差异,免疫组织化学法测定细胞外间质成分fibronectin和collagenⅣ的表达差异,FCM法进行细胞周期分析。结果：获得了对gefitinib具有较高耐药性的2个细胞株：PC9/G2和PC9/G4,尤其PC9/G2的IC50为7～10μmol/L,较野生型PC9细胞（IC50：0.03～0.05μmol/L）提高耐受性160～260倍。PC9的细胞倍增时间为（16.2&#177;3.5）h,PC9/G2的倍增时间为（36.5&#177;4.8）h。PC9/G2同PC9的基因表达谱存在明显差异,耐药细胞株PC9/G2的脂肪酸代谢和氧化磷酸化基因表达下降,糖酵解基因表达上升,核糖体各亚基蛋白表达下调,高尔基体和囊泡、笼形蛋白衣被小泡等相关基因表达上调,细胞蛋白分泌功能加强;泛素-蛋白酶体循环相关基因上调;DNA修复基因和解旋酶类基因表达上调;具有蛋白激酶活性的30个基因表达上调,11个基因表达下调;15个磷酸化蛋白磷酸酶活性的基因表达上调;具有GTP结合活性的基因21个表达上调,4个下调;涉及细胞骨架和细胞运动功能的8个基因上调;整合素β1亚基、类胰岛素受体、NFκB级联反应的正向调节因子表达上调。结论：成功建立了6株PC9细胞的gefitinib耐药细胞株,初步研究表明对gefitinib中度耐药可能与细胞外基质组分改变及其黏附信号通路有关,对gefitinib高度耐药则可能涉及替代性信号通道的激活和信号通道的下游激活。     

c-Met信号通道参与HGF诱导不同基因型非小细胞肺癌细胞株对吉非替尼耐药

背景与目的肝细胞生长因子(hepatocyte growth factor,HGF)诱导非小细胞肺癌(non-small cell lung cancer,NSCLC)对吉非替尼耐药,可能与其受体c-Met激活有关。本研究旨在探讨c-Met及其下游信号通道是否参与HGF诱导不同基因型NSCLC细胞株对吉非替尼耐药。方法选择人NSCLC细胞株表皮生长因子受体(epidermal growth factor receptor,EGFR)突变型PC-9、PC9/R和EGFR野生型H292、A549,用HGF诱导细胞,通过MTT法检测细胞增殖,Annexin V-FITC法检测细胞凋亡,应用免疫印迹技术检测细胞中c-Met及下游通道的变化。结果吉非替尼对PC9、H292、A549的生长抑制作用呈浓度依赖性,HGF诱导后吉非替尼抑制细胞的生长曲线明显往右移。在PC9、H292、A549细胞中,吉非替尼和HGF处理组的细胞凋亡率比吉非替尼处理组均减少(P<0.05),在PC9/R细胞中无明显减少(P>0.05)。HGF能激活PC9、H292、PC9/R、A549细胞中c-Met及其下游通道蛋白。在PC9、H292、A549细胞中,吉非替尼和HGF处理组的p-Met、p-Akt、p-Stat3、p-Erk1/2蛋白表达比吉非替尼处理组均增高,在PC9/R细胞中无明显增高。结论在体外HGF诱导不同基因型NSCLC细胞株对吉非替尼耐药,c-Met及其下游信号通道参与HGF诱导不同基因型NSCLC细胞株对吉非替尼耐药。     

Significance of thymidylate synthase for resistance to pemetrexed in lung cancer

Pemetrexed (MTA) is a multitargeted antifolate with promising clinical activity in lung cancer. We exposed the small cell lung cancer cell line PC6 to stepwise-increasing pemetrexed concentrations of 0.4, 1.6, and 4.0 μ m , and established three pemetrexed-resistant lung cancer cell lines: PC6/MTA-0.4, PC6/MTA-1.6, and PC6/MTA-4.0 cells. To investigate the mechanisms of acquired resistance to pemetrexed, we measured the expression levels of the thymidylate synthase ( TS ), reduced folate carrier ( RFC ), and folylpoly-gamma-glutamate synthetase ( FPGS ) genes. TS gene expression was significantly increased in PC6/MTA-1.6 and PC6/MTA-4.0 cells relative to parental cells in a pemetrexed dose-dependent manner. In contrast, the levels of RFC gene expression in PC6/MTA-0.4 cells and FPGS in PC6/MTA-1.6 cells were significantly decreased, whereas the levels of both genes were restored in PC6/MTA-4.0 cells. Knockdown of TS expression using siRNA enhanced pemetrexed cytotoxicity in PC6/MTA-4.0 cells. The expression level of the TS gene was significantly correlated with the concentration of pemetrexed for 50% cell survival (IC₅₀) in 11 non-small cell lung cancer cell lines. These results suggest that the alteration of molecular pharmacological factors in relation with pemetrexed resistance is dose-dependent, and that up-regulation of the expression of the TS gene may have an important role in the acquired resistance to pemetrexed. In addition, TS may be a predictive marker for pemetrexed sensitivity in lung cancer. ( Cancer Sci 2009)     

Interaction and cross-resistance of cisplatin and pemetrexed in malignant pleural mesothelioma cell lines

Although cisplatin and pemetrexed are key drugs in the treatment of malignant pleural mesothelioma, their drug-drug interactions, cross-resistance and resistance mechanisms in malignant pleural mesothelioma are not well understood. In the present study, the interaction of these 2 agents was determined by clonogenic assays followed by isobologram analysis of 4 human malignant pleural mesothelioma cell lines. The cell lines were exposed to the agents using a stepwise dose-escalation method to establish drug-resistant sublines. Thymidylate synthase mRNA expression was evaluated in the drug-resistant sublines. As a consequence, cisplatin and pemetrexed had synergistic effects in 3 cell lines and an additive effect in the fourth cell line. The former 3 cell lines showed similar pemetrexed sensitivity in the parental cells and their cisplatin-resistant sublines, whereas the fourth cell line exhibited cross-resistance. In contrast, cisplatin had diverse effects on pemetrexed-resistant sublines. High thymidylate synthase expression did not correlate with natural pemetrexed resistance. Elevated thymidylate synthase expression correlated with acquired pemetrexed resistance in 2 sublines. In conclusion, cisplatin and pemetrexed showed synergistic activity and no cross-resistance in 3 of the 4 malignant pleural mesothelioma cell lines, suggesting the clinical relevance of their combination in chemotherapy. Thymidylate synthase expression did not necessarily correlate with pemetrexed resistance. The information together with the experimental model presented here would be useful for further investigating therapeutic targets of malignant mesothelioma.     

人肺癌EGFR基因突变与Gefitinib耐药相关性研究

背景与目的 以人肺癌细胞株PC9及其耐药子系细胞PC9/AB2、PC9/AB11、PC9/BB4为模型,观察4株细胞对Gefitinib的耐药差异及耐药机制.方法 体外培养细胞测定耐药指数,对4株细胞EGFR基因exon18-21进行测序并在mRNA水平上的测定表达量差异.结果 PC9及其3株耐药子系细胞均存在耐药性差异.PC9及其3株耐药子系细胞均存在19外显子15 bp缺失突变并且耐药细胞株PC9/AB11外显子20存在A→G的点突变.敏感细胞株PC9的EGFR基因在mRNA水平上的表达量明显高于其他3株耐药株.结论 PC9、PC9/AB2、PC9/AB11和PC9/BB4肺癌细胞株对Gefitinib的耐药性存在非常显著的差异,4株肺癌细胞株EGFR基因突变和mRNA表达水平的差异与其与Gefitinib获得性耐药有关.     

非小细胞肺癌吉非替尼耐药相关miRNAs的筛选鉴定

  目的  miRNA是一类通过结合mRNA调节基因表达的非编码单链小分子RNA,本研究目的是探讨非小细胞肺癌（NSCLC）中miRNA与吉非替尼耐药的关系。  方法  CCK8法检测NSCLC吉非替尼耐药细胞PC9/GR相对于亲本细胞PC9的耐药倍数;miRNA芯片检测PC9/GR与PC9中miRNA的表达差异;RT-PCR验证miRNA芯片结果。将差异表达的miRNA模拟物/抑制剂转染至PC9/GR中,观察其对吉非替尼敏感性的影响。  结果  吉非替尼对PC9和PC9/GR的IC₅₀值分别为42.89 nmoL/L和3.87 μ moL/L,耐药倍数为90.23倍。miRNA芯片结果显示,PC9/GR与PC9比较55条有差异表达miRNAs（P < 0.01）,其中在PC9/GR上调的miRNAs有21条,包括miRNA-1 246、miRNA-125b等;下调的miRNAs有34条,包括miRNA-224、miRNA-125a~5p等。RT-PCR进一步验证其中9条miRNAs,有8条与芯片结果趋势一致。将上述8条miRNAs的模拟物/抑制剂转染至PC9/GR中,发现miRNA-125a~5p模拟物可降低吉非替尼敏感性。  结论  PC9/GR与PC9的miRNA表达存在差异,miRNA可能与NSCLC吉非替尼耐药相关,miRNA-125a~5p可促进PC9/GR对吉非替尼产生耐药。      

N-cadherin expression is a potential survival mechanism of gefitinib-resistant lung cancer cells

Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer and is the most common and fatal cancer worldwide. Specific tyrosine kinase inhibitors for epidermal growth factor receptor (EGFR), such as gefitinib, have been effective in some NSCLC patients and are being used in the clinical setting as pioneer molecularly targeted cancer drugs. However, many patients have not responded to these drugs, and have acquired resistance after long-term treatment. To identify other potential NSCLC molecular targets, we used DNA microarrays to examine gene expression profiles of gefitinib-resistant PC9/ZD cells that are derived from gefitinib-sensitive PC9 cells and harbor a threonine to methionine mutation at codon 790 (T790M) in EGFR, a known mechanism of acquired resistance to gefitinib. We found that N-cadherin expression was significantly upregulated in PC9/ZD cells compared with PC9 cells. Inhibition of N-cadherin expression by siRNA or treatment with antibodies against N-cadherin induced apoptosis of PC9/ZD cells in association with reduced phosphorylation of Akt and Bad, a proapoptotic protein. Moreover, inhibition of Akt expression by siRNA or treatment with an inhibitor for phosphatidylinositol (PI)-3 kinase reduced survival of PC9/ZD cells. In addition, we found several N-cadherin-expressing lung cancer cells that showed inherent resistance to gefitinib treatment and reduced survival owing to siRNA-induced inhibition of N-cadherin expression. Thus, it appears that N-cadherin maintains the survival of the gefitinib-resistant lung cancer cells via the PI-3 kinase/Akt survival pathway. From these results, we propose that N-cadherin signaling contributes, at least in part, to the survival mechanisms of gefitinib-resistant NSCLC cells and that N-cadherin is a potential molecular target in the treatment of NSCLC.     

The effect of adenovirus-IkappaBalpha transduction on the chemosensitivity of lung cancer cell line with resistance to cis-diamminedichloroplatinum(II)(cisplatin) and doxorubicin(adriamycin)

Resistance to chemotherapeutic agents is the major reason for treatment failure of cancer chemotherapy. Some chemotherapeutic drugs induce the activation of NF-kappaB in cancer cells that results in their resistance to anticancer drugs. But the role of NF-kappaB in acquired resistance has not been well investigated. In this study, we transferred the "super-repressor" form of the NF-kappaB inhibitor by adenoviral vector (ad-IkappaBalpha) to human lung cancer cell lines with resistant to cisplatin (PC-14-DDP) and adriamycin (PC-14-ADR), and observed the sensitivity change. Electrophoretic mobility shift assay showed that ad-IkappaBalpha blocked the activation of NF-kappaB induced by cisplatin and adriamycin. Transduction with ad-IkappaBalpha restored the sensitivity of cisplatin and adriamycin resistant lung cancer cell lines (PC-14-DDP and PC-14-ADR) to a level compatible to the parental cell lines. Annexin-V analysis suggested that the enhancement of chemosensitivity was probably a result of the induction of apoptosis. These data demonstrated that ad-IkappaBalpha blockade of chemotherapeutic induced NF-kappaB activation increased apoptosis induction and the chemosensitivity of lung cancer cell lines with acquired resistance to cisplatin and adriamycin. Therefore, gene transfer of IkappaBalpha-SR seems to represent a new therapeutic strategy for the solution of low sensitivity and lung cancer resistance to anticancer drugs.     

PTEN inactivation in lung cancer cells and the effect of its recovery on treatment with epidermal growth factor receptor tyrosine kinase inhibitors

To understand the mechanisms of PTEN inactivation, which is reported to be involved in tumor progression and drug resistance in lung cancer, we analyzed the expression levels of PTEN at mRNA and protein levels, along with the genetic and epigenetic status of the PTEN gene, in a panel of lung cancer cell lines. Western blot analysis showed that six out of 25 (24%) cell lines displayed low expression of PTEN protein. The level of PTEN mRNA correlated well with corresponding protein expression in each of these six cell lines. In two of the six cell lines genomic analysis revealed homozygous deletions of the PTEN gene. Another two of the six cell lines displayed hypermethylation of the PTEN gene promoter assessed by methylation-specific PCR. The levels of PTEN mRNA and protein expression in PC9/f9 and PC9/f14 cells, which are gefitinib-resistant derivatives of the gefitinib-sensitive cell line, PC9, were reduced compared to the parental line. After treatment with the demethylating agent 5-aza-2'deoxycytidine (5-AZA) and the histone deacetyltransferase (HDAC) inhibitor Trichostatin A (TSA), the expression levels of PTEN mRNA and protein in these four cell lines (PC9/f9, PC9/f14, PC10 and PC14) were actually restored. In summary, reduction in PTEN protein expression was regulated by histone deacetylation and hypermethylation of the gene promoter, as well as homozygous deletion. In addition, we demonstrated that the combination treatment of gefitinib and TSA induced significant growth inhibition in gefitinib-resistant PC9/f9 and PC9/f14 cells. These findings suggest that the combination of the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib with the demethylating agent 5-AZA and the HDAC inhibitor TSA may be a useful strategy for the treatment of some lung cancers.