埃罗替尼对胰腺癌细胞BxPC3生长的影响及其机制

目的观察表皮生长因子受体（EGFR）抑制剂埃罗替尼对体外培养的胰腺癌细胞BxPC3生长的影响,并探讨其作用机制。方法应用MTT法检测埃罗替尼作用后BxPC3细胞的增殖情况;用流式细胞分析、透射电镜和原位末端标记（TUNEL）法观察细胞凋亡和细胞周期的变化;RT—PCR法检测细胞bcl-2、bax、bcl-xl、bak mRNA表达。结果埃罗替尼呈剂量和时间依赖性抑制BxPC3细胞生长。72h后,1、100μmol／L的埃罗替尼处理组细胞存活率分别为（90．25±2．62）％和（40．75±2．98）％。两者比较具有统计学意义（P〈0．01）。50μmol／L埃罗替尼处理BxPC3后24、96h的细胞存活率分别为（74．0±4．08）％和（49．50±1．29）％,两者比较也具有统计学意义（P〈0．01）。50μmol／L埃罗替尼处理组的细胞凋亡率为（11．0±1．1）％,显著高于对照组的（6．2±1．1）％（P〈0．01）;G0／G1细胞占（73．4±1．3）％,也显著高于对照组的（63．3±1．0）％;透射电镜可见细胞呈现明显凋亡形态,并见凋亡小体形成。埃罗替尼处理组细胞bcl-2、bcl—xl mRNA表达下调,bax mRNA表达轻微上调,bak mRNA的表达不受影响。结论EGFR抑制剂埃罗替尼体外可抑制胰腺癌细胞系BxPC3的生长,其机制可能与阻滞细胞周期,上调促凋亡蛋白和下调凋亡抑制蛋白有关.     

埃罗替尼对胰腺癌细胞BxPC3生长的影响及其机制

目的 观察表皮生长因子受体(EGFR)抑制剂埃罗替尼对体外培养的胰腺痛细胞BxPC3生长的影响,并探讨其作用机制.方法 应用MTT法检测埃罗替尼作用后BxPC3细胞的增殖情况;用流式细胞分析、透射电镜和原位末端标记(TUNEL)法观察细胞凋亡和细胞周期的变化;RT-PCR法检测细胞bcl-2、bax、bel-xl、bak mRNA表达.结果 埃罗替尼呈剂量和时间依赖性抑制BxPC3细胞生长.72h后,1、100μmol/L的埃罗替尼处理组细胞存活率分别为(90.25±2.62)%和(40.75±2.98)%,两者比较具有统计学意义(P<0.01).50 μmol,/L埃罗替尼处理BxPC3后24、96 h的细胞存活率分别为(74.0±4.08)%和(49.50 ±1.29)%,两者比较也具有统计学意义(P<0.01).50μmaol/L埃罗替尼处理组的细胞凋亡率为(11.0±1.1)%,显著高于对照组的(6.2±1.1)%(P<0.01);G_0/G_1细胞占(73.4 ±1.3)%,也显著高于对照组的(63.3 ±1.0)%;透射电镜可见细胞呈现明显凋亡形态,并见凋亡小体形成.埃罗替尼处理组细胞bcl-2、bcl-xl mRNA表达下调,bax mRNA表达轻微上调,bak mRNA的表达不受影响.结论 EGFR抑制剂埃罗替尼体外可抑制胰腺癌细胞系BxPC3的生长,其机制可能与阻滞细胞周期,上调促凋亡蛋白和下调凋亡抑制蛋白有关.     

表皮生长因子受体抑制剂埃罗替尼对胰腺癌血管生成的影响

目的 观察表皮生长因子受体抑制剂埃罗替尼对胰腺癌新生血管生成的影响，探讨其对胰腺癌生长抑制的作用机制。方法 ①应用小管形成实验观察埃罗替尼（终浓度100μmol／L）对血管生成的影响，并与对照组（加入无血清培养液）进行比较。②建立胰腺癌细胞株BxPC-3裸鼠移植瘤模型，用埃罗替尼灌胃，每天100mg／kg，共4周。每周测量移植瘤体积，4周后处死裸鼠，计算抑瘤率，并与未用埃罗替尼对照组裸鼠比较。RT-PCR法检测不同浓度（5、50、100、200／μmol／L）埃罗替尼处理后BxPC-3细胞血管内皮生长因子（VEGF）表达的变化。采用Ⅷ因子免疫组化染色评估瘤组织中微血管密度（MVD）。结果小管形成实验中，埃罗替尼组细胞数显著少于对照组，中空闭合管状结构缺如。埃罗替尼灌胃4周后，治疗组平均瘤重（0．397±0．550）g，显著低于对照组的（1．5704±1．060）g，抑瘤率为74．5％。浓度≥50μmol／1．的埃罗替尼各组BxPC-3细胞中VEGFmRNA相对表达量较对照组下调，移植瘤组织VEGF表达亦较对照组显著下调，瘤组织MVD（1．86±0．43）显著低于对照组（5．98土1．27，P〈0．01）。结论 埃罗替尼可抑制裸鼠移植瘤生长和胰腺癌体内、体外血管的生成，可作为胰腺癌的辅助治疗方法之一。     

培美曲塞与厄洛替尼联合或序贯治疗对肺腺癌A549细胞增殖和凋亡的影响

目的探讨培美曲塞与厄洛替尼联合或序贯对肺腺癌A549细胞增殖和凋亡的影响。方法以人肺腺癌A549细胞为研究对象,分别应用培美曲塞与厄洛替尼单药、联合及不同序贯方法干预72 h后,MTT法检测细胞增殖及抑制情况,并计算联合指数(CI),流式细胞仪(FCM)检测细胞周期及凋亡情况,蛋白质印迹法(Western印迹法)检测磷酸化表皮生长因子受体(p-EGFR)及磷酸化-ATK(p-AKT)的表达情况。结果培美曲塞序贯厄洛替尼组(P-E)对A549细胞增殖的抑制率及凋亡率较高,明显高于培美曲塞(P)和厄洛替尼(E)单药组以及厄洛替尼联合(E+P)或序贯培美曲塞组(E-P)(P均0.05)。E组和E+P/E-P组的G1期细胞所占比例较对照组明显增多(P0.05),P组和P-E组的S期细胞所占比例较对照组明显增多(P均0.05)。各药物干预组细胞凋亡率均较对照组明显增高,且P-E组细胞凋亡率明显高于其他组(P均0.05)。P-E组A549细胞p-AKT及p-EGFR的表达水平均明显低于对照组(P均0.05)。结论培美曲塞可有效抑制肺腺癌A549细胞的增殖作用,且与厄洛替尼序贯应用的协同效果更为明显,其作用机制可能与p-EGFR及p-AKT信号通路相关。     

阿法替尼和顺铂分别联合培美曲塞治疗晚期非小细胞肺癌的疗效分析

目的探究阿法替尼和顺铂分别联合培美曲塞治疗晚期EFGR突变型非小细胞肺癌晚期(non-small-cell carcinoma, NSCLC)的临床疗效差异，明确阿法替尼临床疗效。 方法选取我院2016年3月至2017年9月收治的107例EFGR突变阳性晚期NSCLC患者的病例资料，接受顺铂+培美曲治疗的NSCLC患者51例为对照组，接受塞阿法替尼+培美曲塞治疗的NSCLC患者56例，为研究组，观察两组患者疗效、无进展生存时间(progression-free survival time, PFS)以及生存时间(overall survival, OS)等。 结果研究组患者ORR及DCR比例均明显高于对照组[53.57% vs. 31.37%，85.71% vs. 66.67%，P<0.05]；研究组患者PFS及OS均明显高于对照组[7.2月vs. 6.0月，11.75月vs. 9.5月，P<0.05]；治疗后，研究组患者神经元特异性烯醇化酶(neuron-specific enolase, NSE)、癌胚抗原(carcinoembryonic antigen, CEA)、细胞角蛋白19片段(cytokeratin 19 fragment, CYFRA21-1)均低于对照组[(11.79±8.51)ng/ml vs.(15.26±8.22)ng/ml，(14.54±8.77)ng/ml vs.(18.37±9.35)ng/ml，(3.45±2.68)ng/ml vs.(5.41±3.54)ng/ml，P<0.05]；Ⅰ～Ⅱ级不良反应中，研究组恶心呕吐、肾功能损害及脱发人数比例均明显低于对照组[12.50% vs. 33.33%，5.36% vs. 27.45%，16.07% vs. 35.29%，P<0.05]，研究组皮疹/痤疮人数比例明显高于对照组[25.00% vs. 7.84%，P<0.05]；治疗后，躯体方面、心理方面、社会方面及总体感觉评分均高于对照组[(77.41±5.25)分vs.(72.12±5.08)分，(74.37±5.40)分vs.(67.52±5.32)分，(79.25±5.98)分vs.(74.32±5.71)分，(71.21±5.45)分vs.(66.61±5.20)分，P<0.05]。 结论阿法替尼联合培美曲塞一线治疗EGFR突变型晚期NSCLC患者的疗效更为显著，明显提高无进展生存时间及生存时间，明显提高生活质量，同时具有较高安全性，值得临床推广应用。     

雷替曲塞联合阿帕替尼对结肠癌细胞增殖的影响及其机制

目的 探讨雷替曲塞联合阿帕替尼对结肠癌细胞增殖的影响及其机制.方法 体外培养人结肠癌细胞SW480(以下称SW480细胞),取传3代、对数生长期、生长状态良好的SW480细胞,随机分为对照组、雷替曲塞0.5μg/mL组、雷替曲塞1.0μg/mL组、雷替曲塞1.5μg/mL组、阿帕替尼组、联合干预组,雷替曲塞0.5μg/...     

姜黄素与尼美舒利联合应用对胰腺癌细胞的作用

非甾体类抗炎药对结直肠癌和其他消化道肿瘤的防治取得了较好的实验和临床效果。对胰腺癌防治作用的研究正在初始阶段，结论也不一致。我们观察比较姜黄素、选择性非甾体类抗炎药尼美舒利对胰腺癌的体外抑制作用，为探讨姜黄素和尼美舒利联合应用是否有协同作用，并对它们的抗肿瘤分子机制作初步探讨。     

吉非替尼对胰腺癌细胞克隆形成与体外侵袭能力的影响及机制

吉非替尼是表皮生长因子（EGF）受体酪氨酸激酶抑制剂，能与ATP或酶的底物结合位点作用而影响酶的磷酸化。近期研究表明，吉非替尼对多种肿瘤细胞，例如结肠癌、前列腺癌、乳腺癌的生长有抑制作用。本研究旨在通过体外实验研究，探讨其对胰腺癌细胞的作用，尤其是对胰腺癌细胞的侵袭、体外克隆形成的影响及相关机制。     

尼卡地平对胰腺癌Patu8988株培美曲塞耐药的逆转作用

目的:探讨ABCG2拮抗剂-尼卡地平在体外安全剂量下能否逆转耐药,为临床应用提供实验基础.方法:采用噻唑蓝法(MTT)测定尼卡地平对Patu8988亲本和耐药细胞的安全使用浓度,测定单用培美曲塞和联合安全剂量的尼卡地平分别对两株细胞的IC50变化;分别采用DAPI核染色和细胞流式分析在耐药细胞中单用培美曲塞组和联合尼卡地平组凋亡差异.结果:MTT结果显示尼卡地平在浓度<4.85μmol/L(2.5 mg/L)时对细胞基本无不良反应,单用培美曲塞和联合安全剂量的尼卡地平对Patu8988亲本株细胞IC50无明显差异,而对耐药株细胞IC50有差异(P<0.05).流式细胞分析在耐药细胞中联合尼卡地平组凋亡比例高于单用培美曲塞组(32.27%±2.8% vs 50.5%±4.2%,P<0.05).结论:安全剂量下尼卡地平能提高胰腺癌Patu8988耐药细胞株对培美曲塞的敏感性.而对其亲本株无作用.     

晚期非小细胞肺癌标准化疗结束后培美曲塞早期、延迟治疗疗效比较

目的比较晚期非小细胞肺癌(NSCLC)诱导化疗获益后二线化疗药物培美曲塞(PEM)早期、延迟治疗的疗效。方法 81例晚期NSCLC患者(ⅢB或Ⅳ期)均接受以铂类药物为基础的一线化疗药物治疗4～6周期,病情获得缓解或稳定。随机分为早期组58例和延迟组23例。早期组在一线化疗结束21 d后开始给予PEM维持化疗,延迟组在有疾病进展后在开始PEM维持化疗。PEM用量500 mg/m2静滴,每隔21 d给药1次。结果早期组的中位无进展生存期为6.8个月,较延迟组的2.7个月显著延长(P<0.001),而早期组中位总生存期为14.7个月,与延迟组的12.4个月相比P>0.05。两组主要的不良反应为骨髓抑制、乏力、胃肠道反应,但上述不良反应发生率两组相比P均>0.05。结论早期应用PEM治疗接受一线化疗获益的晚期NSCLC疗效优于延迟组,可延长患者的无进展生存期,但不能延长总生存期。     

Insights into erlotinib action in pancreatic cancer cells using a combined experimental and mathematical approach

AIM:To gain insights into the molecular action of erlotinib in pancreatic cancer (PC) cells. METHODS:Two PC cell lines, BxPC-3 and Capan-1, were treated with various concentrations of erlotinib, the specific mitogen-activated protein kinase kinase (MEK) inhibitor U0126, and protein kinase B (AKT) inhibitor XIV. DNA synthesis was measured by 5-bromo-2'-deoxyuridine (BrdU) assays. Expression and phosphorylation of the epidermal growth factor receptor (EGFR) and downstream signaling molecules were quantified by Western blot analysis. The data were processed to calibrate a mathematical model, based on ordinary differential equations, describing the EGFRmediated signal transduction. RESULTS:Erlotinib significantly inhibited BrdU incorporation in BxPC-3 cells at a concentration of 1 mol/L, whereas Capan-1 cells were much more resistant. In both cell lines, MEK inhibitor U0126 and erlotinib attenuated DNA synthesis in a cumulative manner, whereas the AKT pathway-specific inhibitor did not enhance the effects of erlotinib. While basal phosphorylation of EGFR and extracellular signal-regulated kinase (ERK) did not differ much between the two cell lines, BxPC-3 cells displayed a more than five-times higher basal phospho-AKT level than Capan-1 cells. Epidermal growth factor (EGF) at 10 ng/mL induced the phosphorylation of EGFR, AKT and ERK in both cell lines with similar kinetics. In BxPC-3 cells, higher levels of phospho-AKT and phospho-ERK (normalized to the total protein levels) were observed. Independent of the cell line, erlotinib efficiently inhibited phosphorylation of EGFR, AKT and ERK. The mathematical model successfully simulated the experimental findings and provided predictions regarding phosphoprotein levels that could be verified experimentally. CONCLUSION:Our data suggest basal AKT phosphorylation and the degree of EGF-induced activation of AKT and ERK as molecular determinants of erlotinib efficiency in PC cells.     

siRNA沉默Bmi-1表达对胰腺癌PANC-1细胞增殖的抑制作用

目的:探讨运用RNAi技术沉默Bmi-1基因对人胰腺癌细胞恶性行为的影响,为胰腺癌基因治疗提供新的靶点和思路.方法:构建反义Bmi-1真核表达载体转染人胰腺癌PANC-1细胞,运用荧光显微镜下观察、MTT、Western blot、流式细胞术检测转染效果及细胞周期、凋亡变化.结果:成功构建反义Bmi-1真核表达载体并且...     

First-line erlotinib and fixed dose-rate gemcitabine for advanced pancreatic cancer

AIM: To investigate activity, toxicity, and prognostic factors for survival of erlotinib and fixed dose-rate gemcitabine (FDR-Gem) in advanced pancreatic cancer. METHODS: We designed a single-arm prospective, multicentre, open-label phase Ⅱ study to evaluate the combination of erlotinib (100 mg/d, orally) and weekly FDR-Gem (1000 mg/m 2 , infused at 10 mg/m 2 per minute) in a population of previously untreated patients with locally advanced, inoperable, or metastatic pancreatic cancer. Primary endpoint was the rate of progression-free survival at 6 mo (PFS-6); secondary endpoints were overall response rate (ORR), response duration, tolerability, overall survival (OS), and clinical benefit. Treatment was not considered to be of further interest if the PFS-6 was < 20% (p0 = 20%), while a PFS-6 > 40% would be of considerable interest (p1 = 40%); with a 5% rejection error (α = 5%) and a power of 80%, 35 fully evaluable patients with metastatic disease were required to be enrolled in order to complete the study. Analysis of prognostic factors for survival was also carried out. RESULTS: From May 2007 to September 2009, 46 patients were enrolled (male/female: 25/21; median age: 64 years; median baseline carbohydrate antigen 19-9 (CA 19-9): 897 U/mL; locally advanced/metastatic disease: 5/41). PFS-6 and median PFS were 30.4% and 14 wk (95%CI: 10-19), respectively; 1-year and median OS were 20.2% and 26 wk (95%CI: 8-43). Five patients achieved an objective response (ORR: 10.9%, 95%CI: 1.9-19.9); disease control rate was 56.5% (95%CI: 42.2-70.8); clinical benefit rate was 43.5% (95%CI: 29.1-57.8). CA 19-9 serum levels were decreased by > 25% as compared to baseline in 14/23 evaluable patients (63.6%). Treatment was well-tolerated, with skin rash being the most powerful predictor of both longer PFS (P < 0.0001) and OS (P = 0.01) at multivariate analysis (median OS for patients with or without rash: 42 wk vs 15 wk, respectively, Log-rank P = 0.03). Additional predictors of better outcome were: CA 19-9 reducti     

Distinct antifibrogenic effects of erlotinib,sunitinib and sorafenib on rat pancreatic stellate cells

AIM:To study if three clinically available small molecule kinase inhibitors(SMI),erlotinib,sunitinib and sorafenib,exert antifibrogenic effects on pancreatic stellate cells(PSC)and analyze the basis of their action.METHODS:Cultured rat PSC were exposed to SMI.Cell proliferation and viability were assessed employing 5-bromo-2’-deoxyuridine incorporation assay and flow cytometry,respectively.2-Deoxy-2-[18F]fluoroglucose(18F-FDG)uptake was measured to study metabolic activity.Exhibition of the myofibroblastic PSC phenotype was monitored by immunofluorescence analysis ofα-smooth muscle actin(α-SMA)expression.Levels of mRNA were determined by real-time PCR,while protein expression and phosphorylation were analyzed by immunoblotting.Transforming growth factor-β1 (TGF-β1)levels in culture supernatants were quantified by ELISA.RESULTS:All three SMI inhibited cell proliferation and18F-FDG uptake in a dose-dependent manner and without significant cytotoxic effects.Furthermore,additive effects of the drugs were observed.Immunoblot analysis showed that sorafenib and sunitib,but not erlotinib,efficiently blocked activation of the AKT pathway,while all three drugs displayed little effect on phosphorylation of ERK1/2.Cells treated with sorafenib or sunitinib expressed less interleukin-6 mRNA as well as less collagen type 1 mRNA and protein.Sorafenib was the only drug that also upregulated the expression of matrix metalloproteinase-2 and reduced the secretion of TGF-β1 protein.All three drugs showed insignificant or discordant effects on the mRNA and protein levels ofα-SMA.CONCLUSION:The tested SMI,especially sorafenib,exert inhibitory effects on activated PSC,which should be further evaluated in preclinical studies.     

Phase 1 trial of Vismodegib and Erlotinib combination in metastatic pancreatic cancer

Background/ObjectivesInterplay between the Hedgehog (HH) and epidermal growth factor receptor (EGFR) pathways modulating the outcome of their signaling activity have been reported in various cancers including pancreatic ductal adenocarcinoma (PDAC). Therefore, simultaneous targeting of these pathways may be clinically beneficial. This Phase I study combined HH and EGFR inhibition in metastatic PDAC patients.MethodsCombined effects of HH and EGFR inhibition using Vismodegib and Erlotinib with or without gemcitabine in metastatic solid tumors were assessed by CT. Another cohort of patients with metastatic PDAC was evaluated by FDG-PET and tumor biopsies-derived biomarkers.ResultsTreatment was well tolerated with the maximum tolerated dose cohort experiencing no grade 4 toxicities though 25% experienced grade 3 adverse effects. Recommended phase II dose of Vismodegib and Erlotinib were each 150 mg daily. No tumor responses were observed although 16 patients achieved stable disease for 2–7 cycles. Paired biopsy analysis before and after first cycle of therapy in PDAC patients showed reduced GLI1 mRNA, phospho-GLI1 and associated HH target genes in all cases. However, only half of the cases showed reduced levels of desmoplasia or changes in fibroblast markers. Most patients had decreased phospho-EGFR levels.ConclusionsVismodegib and Erlotinib combination was well-tolerated although overall outcome in patients with metastatic PDAC was not significantly impacted by combination treatment. Biomarker analysis suggests direct targets inhibition without significantly affecting the stromal compartment. These findings conflict with pre-clinical mouse models, and thus warrant further investigation into how upstream inhibition of these pathways is circumvented in PDAC.