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。     

AHR活化介导的ROS诱导NSCLC细胞吉非替尼耐药的作用机制

目的:探讨芳香烃受体（AHR）介导的活性氧簇（ROS）诱导非小细胞肺癌（NSCLC）吉非替尼耐药的作用机制。方法:以正常支气管肺泡上皮细胞BEAS-2B为对照,Western blot检测非小细胞肺癌A549、PC-9、H1299和H1975细胞AHR的蛋白表达。AHR激动剂FICZ、吉非替尼、N-乙酰半胱氨酸（NAC）分别处理A549和PC-9细胞,MTT、激光共聚焦显微镜、流式细胞术及Western blot分别检测吉非替尼敏感性、细胞内ROS及表皮生长因子受体（EGFR）信号。结果:MTT检测显示FICZ通过上调半数最大抑制浓度（IC50）诱导PC-9及A549细胞对吉非替尼耐药,Western blot显示FICZ可导致PC-9及A549细胞EGFR、AKT磷酸化,而NAC可遏制A549细胞中的EGFR磷酸化并逆转吉非替尼耐药。共聚焦显微镜和流式细胞仪显示,FICZ诱导的AHR活化导致PC-9及A549细胞内ROS产生增加,并且可被NAC抑制。结论:活化的AHR可通过促进NSCLC细胞中的ROS产生和EGFR信号转导介导吉非替尼耐药,此过程可被NAC抑制。     

多西他赛与吉非替尼不同时序应用对人肺腺癌细胞的作用

目的 探讨多西他赛与吉非替尼不同时序应用对人肺腺癌细胞A549和PC-9的生长影响及其细胞学机制。方法 qPCR-HRM法检测人肺腺癌细胞EGFR和K Ras基因突变,MTT法检测细胞增殖, Western blotting检测细胞信号蛋白及磷酸化表达,FCM法检测细胞周期变化。结果 人肺腺癌A549细胞为EGFR基因野生型,PC-9细胞为EGFR第19外显子突变型。多西他赛和吉非替尼单药或联合用药均能抑制A549和PC-9细胞生长,呈浓度依赖性。多西他赛对A549和PC-9细胞生长的半数抑制浓度（IC₅₀）分别为5.24×10^-7和2.13×10^-8mol／L,吉非替尼分别为1.28×10^-5和4.58×10^-8mol／L。在IC₅₀浓度时,多西他赛序贯吉非替尼对A549和PC-9细胞的生长抑制率分别为60.00%和57.45%,均较单药组明显增高（P＜0.05）;而同时用药只对PC-9细胞有增效作用,抑制率为53.46％,较单药组明显增高（P＜0.05）。多西他赛表现为增强A549、PC-9细胞EGFR和ERK磷酸化,吉非替尼表现为抑制,两药均抑制PC-9细胞IGF-1R磷酸化。多西他赛序贯应用吉非替尼显著抑制EGFR和ERK磷酸化,两药同时应用对抑制PC-9细胞的IGF-1R磷酸化具有增强作用。多西他赛将A549及PC-9细胞阻滞在G₂期,吉非替尼将PC-9细胞明显阻滞在G₁期。结论 多西他赛序贯吉非替尼能够抑制EGFR野生型和突变型的A549及PC-9细胞生长,且呈增效作用,可能与影响细胞EGFR和ERK磷酸化有关;两药同时应用仅对PC-9细胞具有增效作用,可能与IGF-1R磷酸化抑制有关;不同时序应用的效果均可能与细胞周期相关。     

吉非替尼与培美曲塞对人肺腺癌细胞 SPC - A1 生长及细胞周期的作用

目的：探索培美曲塞与表皮生长因子酪氨酸激酶抑制剂（EGFR—TKIs）吉非替尼联合应用对人肺腺癌细胞SPC—A1生长的影响,探索二者序贯用药是否比单药更有效,并从细胞周期角度分析其可能的细胞学机制。方法：RT—PCR法检测人肺腺癌细胞SPC—A1中EGFRmRNA表达,Westernblot法检测SPC—A1细胞中EGFR蛋白表达,四甲基偶氮唑盐[3-（4,5-dimethylthiazol-2-y1）-2,5-diphenyl—tetrazoliumbro—mide,M1Tr]显色分光光度法检测细胞增殖,流式细胞术检测细胞周期。分别观察培美曲塞与吉非替尼单独、同时及间隔24小时序贯作用。结果：SPC—A1细胞中有EGFRmRNA和蛋白的表达,且其表达量为过表达。吉非替尼和培美曲塞分别在0．1pmol/L-1umoL／L和109-10-1g/L浓度范围内明显抑制SPC—A1细胞生长,呈浓度依赖性,IC50分别为：61．2nml/L（吉非替尼）和16．2ug/ml（培美曲塞）,二者在IC50下抑制作用呈时间依赖性,96h时达最大抑制。二者联合作用于细胞时对生长的影响与二者加药的次序有关,跟单药组相比,先用培美曲塞后用吉非替尼组对抑制细胞生长有明显增强作用（P〈0．05）,同时给药或先用吉非替尼后用培美曲塞组对抑制细胞生长无显著增强作用（P〉0．05）。细胞周期研究显示：吉非替尼和培美曲塞作用于不同的细胞周期,分别将SPC—A1细胞阻滞于G,期（G0／G1）和S期,先用吉非替尼后用培美曲塞组G1（G0／G1）期细胞显著增多,G2期（G2／M）细胞显著减少（P〈0．05）。同时用药组G1期（G0／G1）和s期细胞比例无显著变化,G2期（G2／M）细胞显著增多（P〈0．05）。先用培美曲塞后用吉非替尼组G1期（G0／G1）细胞显著减少,s期细胞比例无显著变化,G2期（G2／M）细胞显著增多（P〈0．05）。结论：吉非替尼和培美曲塞都能抑制SPC—A1细胞生长,二者联合作用对细胞生长的影响与给药次序有关,先用吉非替尼后用培美曲塞对抑制细胞生长无显著增强作用,而先用培美曲塞后用EGFR—TKIs则表现有明显的协同性,且这种关系可能与它们对细胞周期的影响相关。     

吉非替尼联合鸦胆子油乳对肺腺癌细胞的作用及其机制

目的 探讨吉非替尼（Gefitinib, G）联合鸦胆子油乳（Bruceolic oil emulsion, B）对肺腺癌PC-9、A549细胞增殖与凋亡的作用及其机制。方法 取对数生长期的PC-9、A549细胞进行实验,两细胞株在实验中均分为对照组和实验组。采用MTT比色法、TUNEL法及流式细胞技术检测各实验组对肺腺癌细胞的增殖抑制、诱导细胞凋亡及细胞周期阻滞等作用。结果 吉非替尼、鸦胆子油乳单药均能抑制PC-9、A549细胞的生长、增殖并诱导其凋亡,呈浓度和时间依赖效应。与鸦胆子油乳联合后,吉非替尼对PC-9和A549细胞的IC50较单用时分别下降了50.72%和66.56%。两药联合后对两细胞的增殖抑制及凋亡诱导作用进一步增强。结论 吉非替尼联合鸦胆子油乳能显著抑制肺腺癌PC-9、A549细胞增殖、诱导细胞凋亡,可能与药物对细胞周期的阻滞作用有关。两者合用时鸦胆子油乳可对吉非替尼起到增效增敏的作用。     

miR-200c增强肺癌A549细胞对紫杉醇及吉非替尼的敏感度及相关机制

目的 观察上调肺癌A549细胞中miR-200c的表达对紫杉醇、吉非替尼敏感度及两药联合作用的影响及其作用机制。方法 用miR-200c转染肺癌A549细胞,Real-time PCR检测miR-200c表达,MTT法和流式细胞仪检测转染miR-200c后A549细胞对紫杉醇、吉非替尼单药及两药联合的敏感度和细胞凋亡的影响,Western blot检测TUBB3蛋白、EGFR、Akt、MAPK磷酸化蛋白及总蛋白的表达。结果 上调miR-200c表达可增强A549细胞对紫杉醇及吉非替尼敏感度并促进细胞凋亡,对两药联合无明显作用。Western blot显示转染miR-200c联合紫杉醇可下调A549细胞中TUBB3蛋白表达,联合吉非替尼下调EGFR和Akt磷酸化蛋白表达,与两药联合对EGFR和Akt磷酸化蛋白表达无明显影响。 结论 miR-200c可增强肺癌A549细胞对紫杉醇、吉非替尼的敏感度,可能分别与抑制TUBB3表达、抑制EGFR和Akt磷酸化蛋白表达有关,而对两药联合无协同抗肿瘤作用。     

抗EGFR单抗联合EGFR酪氨酸激酶抑制剂对人肺腺癌细胞凋亡的影响及其机制

目的:目前抗表皮生长因子受体(epidermal growth factor receptor, EGFR)的小分子酪氨酸激酶抑制剂吉非替尼(gefitinib)和抗EGFR单克隆抗体西妥昔单抗(cetuximab)在肺癌的临床应用中颇为广泛.鉴于这两种药物均针对EGFR分子靶点,因此本研究旨在就上述两种药物联合用药对人肺腺癌细胞凋亡的影响及其分子机制进行探讨.方法:吉非替尼和西妥昔单抗单独或联合用药作用于人肺腺癌细胞株A549和SPC-A-1后,用碘化丙啶标记,采用流式细胞术观察细胞凋亡情况,活细胞计数试剂盒测定各组细胞增殖抑制情况,Western印迹法检测两种药物对EGFR下游信号通路蛋白[磷酸化蛋白激酶B(phosphorylated Akt, p-Akt)、磷酸化EGFR(phosphorylated EGFR,p-EGFR)和磷酸化丝裂原激活蛋白激酶(phosphorylated mitogen-activated protein kinase, p-MAPK)]在蛋白水平表达的影响.结果:吉非替尼或西妥昔单抗单独作用后,A549和SPC-A-1细胞均明显凋亡,同时细胞增殖受到不同程度的抑制.Western印迹法检测p-Akt、p-EGFR和p-MAPK蛋白表达量均较不用药对照组下降.吉非替尼和西妥昔单抗联合作用后,肺腺癌细胞的凋亡、增殖以及在EGFR分子层次表现出较单一用药更为显著的作用.结论:吉非替尼和西妥昔单抗两种药物之间具有良好的协同作用,联合用药可能在临床治疗非小细胞肺癌中具有较大的应用潜力.     

EGFR-TKI联合COX-2抑制剂抑制肺腺癌A549细胞的增殖及其机制

目的:研究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)吉非替尼联合环氧合酶-2(cyclooxygenase-2,COX-2)抑制剂塞来昔布对肺腺癌A549细胞增殖的抑制作用及其可能机制。方法:实验设空白对照组、吉非替尼组、塞来昔布组、吉非替尼+塞来昔布组。倒置相差显微镜下观察A549细胞形态变化,MTT法检测A549细胞增殖,流式细胞术检测A549细胞周期及凋亡,Western blotting检测A549细胞中EGFR、p-EGFR、COX-2的表达。结果:吉非替尼和塞来昔布均可抑制A549细胞的增殖,可见细胞活性下降,G1期细胞阻滞及细胞凋亡增加;联合用药较单药组A549细胞活性下降更明显,G1期阻滞细胞增加,且细胞凋亡增加。吉非替尼或塞来昔布作用前后A549细胞EGFR表达无变化,但联合用药组p-EGFR及COX-2的表达较单药组显著下降。结论:吉非替尼联合塞来昔布可抑制人肺腺癌A549细胞的增殖,其机制可能与抑制EGFR的活化及下调COX-2的表达有关。     

吉非替尼对肺癌细胞株A549和H1975放射敏感度的影响及其机制

目的 本研究旨在探讨小分子表皮生长因子受体（epidermal growth factor receptor, EGFR）酪氨酸激酶抑制剂吉非替尼是否能增加肺癌细胞株A549和H1975的放疗敏感度及其机制。方法 选取两种非小细胞肺癌细胞株A549和H1975,分为单纯X线组和X线+吉非替尼组。单纯X线组采用单纯X线照射,X线+吉非替尼组经10 μmol/L吉非替尼作用24 h后行X线照射。两株细胞不同分组细胞,采用克隆形成实验检测放射敏感度,免疫荧光激光共聚焦显微镜观察X线照射后不同时间点细胞核中磷酸化H2AX（γ-H2AX）亮点在细胞中的定位情况,Western blot法检测放疗后胞质胞核蛋白中EGFR的表达。结果 克隆形成实验中,A549细胞X线+吉非替尼组在各放疗剂量点的SF2值（0.3475）低于单纯X线组（0.4833）;H1975细胞X线+吉非替尼组与单纯X线组在各放疗剂量点的SF2值分别为0.3094和0.3207,无明显差异。免疫荧光结果显示,照射4 Gy后各时间点X线+吉非替尼组A549细胞核中γ-H2AX亮点相比单纯X线多;单纯X线组和X线+吉非替尼组H1975细胞γ-H2AX亮点在各时间点无明显差异; Western blot结果显示,A549细胞经4Gy照射后EGFR有入核现象,而预先经吉非替尼处理再接受4Gy照射,EGFR蛋白绝大部分位于细胞质内;H1975细胞,单纯X线组和X线+吉非替尼组EGFR蛋白均在细胞质中表达,胞核中几乎没有,且两组无明显差异。结论 吉非替尼能增加肺癌细胞株A549的放射敏感度,可能与阻止放疗后EGFR入核进行双链断裂（double strand break,DSB）修复有关;对H1975细胞无影响,与其放疗后EGFR不入核相关。     

肺岩宁方联合吉非替尼抑制肺癌裸鼠移植瘤细胞H1975生长及其作用机制

目的 观察肺岩宁方联合吉非替尼对肺腺癌裸鼠移植瘤细胞H1975生长的影响并探讨其可能的作用机制。方法 成功建立H1975细胞裸鼠皮下移植瘤模型后,随机分为模型组、吉非替尼组、肺岩宁方组及联合用药组,每组10只,分别用相应药物干预4周,检测肿瘤体积、瘤重、体重等,绘制肿瘤生长曲线,计算肿瘤抑制率;应用TNUEL法检测移植瘤细胞凋亡;应用Westernblot法检测EGFR-PI3K/AKT信号通路相关蛋白表达。结果 各组的抑瘤率模型组为0,吉非替尼组21.91%,肺岩宁方组25.11%,联合用药组53.28%;各组移植瘤组织中肿瘤细胞的凋亡率分别为模型组（15.11±1.6）%,吉非替尼组（26.64±0.69）%,肺岩宁方组（28.88±1.61）%,联合用药组（55.06±2.39）%。联合用药组与模型组及单用吉非替尼、肺岩宁方相比差异有统计学意义（P<0.01）。Western blot结果显示肺岩宁方联合吉非替尼在不影响EGFR、AKT、mTOR总蛋白表达的情况下,可显著下调p-EGFR、p-AKT、p-mTOR水平。结论 肺岩宁方联合吉非替尼可显著抑制H1975肺癌裸鼠移植瘤的生长,促进肿瘤细胞凋亡,其作用机制可能与阻断EGFR-PI3K/AKT信号通路有关。     

多西他赛与吉非替尼序贯应用对人肺腺癌细胞SPC-A1生长及信号蛋白的影响

背景与目的已经证明:化疗联合表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor recep-tor-tyrosine kinase inhibitors,EGFR-TKIs)与单独化疗比较治疗晚期非小细胞肺癌并不能增加疗效,但机制尚未完全明了.本研究通过观察多西他赛与吉非替尼不同时序应用对...     

Effects of pemetrexed, gefitinib, and their combination on human colorectal cancer cells

Purpose

The study investigated the effects of pemetrexed, gefitinib, and their combination on human colorectal cancer cells.

Methods

Six human colorectal cancer cells were exposed to pemetrexed, gefitinib, and their combination. Antitumor effects were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Thymidylate synthase (TS) mRNA expression and EGFR mutation were studied by real-time RT-PCR and DNA sequence determination. Pharmacological interaction was studied using the combination index method. Cell cycle distribution and apoptosis were determined by flow cytometry. Activity assay was performed to assess the effects of drugs on TS activity, and Western blot was performed to assess the protein expression of pEGFR, pAKT, and pERK 1/2.

Results

Six colorectal cancer cells are all sensitive to pemetrexed, and TS gene expression of cells was negatively related to pemetrexed sensitivity. The cytotoxic synergism was observed in concurrent pemetrexed combined with gefitinib and sequential pemetrexed followed by gefitinib. The combination of pemetrexed and gefitinib modulated cell cycle and induced apoptosis. Pemetrexed combined with gefitinib decreased TS mRNA expression and in situ activity. Pemetrexed induced an EGFR-mediated activation of the phosphatidylinositol 3-kinase/AKT and ERK pathway, which was inhibited by gefitinib.

Conclusions

Pemetrexed is a promising agent, and pemetrexed combined with gefitinib has a significantly synergistic effect on colorectal cancer cells, which seems to present a strategy of pemetrexed combined with EGFR-TKIs in colorectal cancer treatment.     

安罗替尼联合吉非替尼对吉非替尼耐药非小细胞肺癌PC9/GR细胞增殖的影响及其可能的作用机制

目的 探讨安罗替尼联合吉非替尼对吉非替尼耐药的人非小细胞肺癌PC9/GR(gefitinib resistance)细胞增殖的影响及其可能的作用机制。方法 依据不同给药情况将PC9/GR细胞分为安罗替尼单药组、吉非替尼单药组、安罗替尼和吉非替尼联合用药组及阴性对照组,用MTT法检测各组细胞的增殖情况,流式细胞仪检测细胞的周期分布,Western blot检测p-ERK1/2和p-AKT蛋白的表达水平。结果 安罗替尼和吉非替尼作用于PC9/GR细胞72 h的半数抑制浓度（half inhibitory concentration,IC50）分别为（1.91±0.18）μmol/L和（4.83±0.15）μmol/L,两药均呈剂量依赖性的抗增殖作用,且两药联合时表现出明显的协同效应,联合指数（combination index,CI）小于1。安罗替尼和吉非替尼单药均可将PC9/GR细胞阻滞于G0/G1期（均P<0.05）。与各单药组比较,联合用药组表现出更明显的G0/G1期阻滞（均P<0.05）,且下调p-ERK1/2和p-AKT蛋白的表达水平（均P<0.05）。结论 安...     

Phosphorylated AKT1 is associated with poor prognosis in esophageal squamous cell carcinoma

Background

The epidermal growth factor receptor (EGFR) signaling pathway is important in regulating biological behaviors in many malignancies. We explored whether expression and activation of EGFR and several components on its downstream pathways have prognostic significance in patients with esophageal squamous cell carcinoma (ESCC).

Methods

Expression of EGFR, phosphorylated (p)-EGFR, AKT1, p-AKT1, AKT2, p-AKT2, ERK1, ERK2, p-ERK1/2, STAT3, and p-STAT3 was assessed by immunohistochemical analysis of tissue microarrays for 275 ESCC patients who had undergone complete three-field lymphadenectomy. Spearman rank correlation tests were used to determine the relationships among protein expression, and Cox regression analyses were performed to determine the prognostic factors on overall survival (OS).

Results

p-EGFR expression was correlated statistically with all of the other phosphorylated markers. Gender, N stage, and p-AKT1 expression were found to be independent prognostic factors for OS. Increased expression of p-AKT1 was associated with decreased patient survival. EGFR and p-EGFR expression was not significantly associated with patient survival.

Conclusion

Activation of AKT1 was associated with poor prognosis in ESCC.     

Buccal mucosa cells as in vivo model to evaluate gefitinib activity in patients with advanced non small cell lung cancer.

PURPOSE: To evaluate the role of pretreatment and posttreatment expression in buccal mucosa cells of signal transduction proteins activated by epidermal growth factor receptor, including phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated mitogen-activated protein kinase (p-MAPK), and phosphorylated AKT (p-AKT), in predicting gefitinib activity in advanced non-small cell lung cancer patients. Expression of the same proteins was also assessed on corresponding tissue samples for comparison. Moreover, EGFR gene mutations and copy number were analyzed. EXPERIMENTAL DESIGN: Protein expression was evaluated by standard immunocytochemistry in buccal smears, obtained by scraping immediately before and after 2 weeks of gefitinib treatment, and in the available archival tumor specimens. EGFR gene mutations were evaluated by direct sequencing and gene copy number was determined by fluorescence in situ hybridization. Data were correlated with gefitinib toxicity and objective response. RESULTS: Fifty-eight patients with pretreated advanced non-small cell lung cancer were enrolled and nine of these patients (15%) showed an objective response to gefitinib (including two complete responses). Toxicity (P = 0.025) and baseline p-AKT expression in buccal mucosa cells (P = 0.061) showed a potential predictive role. On the contrary, the probability of achieving an objective response was not affected by pretreatment expression of EGFR, p-EGFR, and p-MAPK, either in buccal mucosa or in tumor tissue. Responders showed a nonstatistically significant trend toward a more pronounced reduction in the expression of p-EGFR, p-MAPK, and p-AKT after gefitinib treatment. Among responders, five of six (83%) tumors showed EGFR gene mutation, whereas none of the tumors from patients with stable or progressive disease did (P < 0.001). CONCLUSIONS: Epithelial cells obtained from buccal mucosa may be used to assess the pharmacodynamic effect of EGFR-targeted agents, and pretreatment p-AKT expression may be a possible predictive biomarker of in vivo gefitinib activity.     

A phase II and pharmacodynamic study of gefitinib in patients with refractory or recurrent epithelial ovarian cancer

BACKGROUND: The primary objective of this study was to evaluate the biochemical effects of gefitinib on its target signal-transduction pathways in patients with recurrent epithelial ovarian cancer (EOC). The secondary objectives included assessing clinical activity and toxicity and determining the association between biochemical and clinical outcomes. METHODS: Twenty-four heavily pretreated patients with EOC who had good end-organ function and performance status and who had measurable disease received gefitinib 500 mg daily. Prospectively planned core-needle tumor biopsies were obtained before treatment and after 4 weeks. Protein expression of total and phosphorylated (p) epidermal growth factor receptor (EGFR), protein kinase B (AKT), and extracellular regulated kinase (ERK) was quantified in microdissected tumor cells using tissue lysate array proteomics. RESULTS: All tumor samples had detectable levels of EGFR and p-EGFR. A decrease in the quantity of both EGFR and p-EGFR was observed with gefitinib therapy in >50% of patients. This was not associated with clinical benefit, nor were responses observed. However, trends for increased gastrointestinal and skin toxicity were observed with greater phosphorylation or quantities of EGFR, ERK, and AKT in tumor samples (P 相似文献   

MEK inhibitors reverse resistance in epidermal growth factor receptor mutation lung cancer cells with acquired resistance to gefitinib

Lung adenocarcinoma cells harboring epidermal growth factor receptor (EGFR) mutations are sensitive to EGFR tyrosine kinase inhibitors (TKIs), including gefitinib. Acquired resistance to EGFR-TKIs develops after prolonged treatments. The study was prompt to explore effective strategies against resistance to EGFR-TKIs. We established gefitinib resistant PC-9 cells which harbor EGFR exon 19 deletion. Known mechanisms for intrinsic or acquired EGFR-TKI resistance, including KRAS mutation, HER2 mutation, EGFR T790M mutation and MET gene amplification, were studied, and we did not observe any known mechanisms for intrinsic or acquired resistance to EGFR-TKIs in the resistant cells. In the parental PC-9 cells, labeled as PC-9/wt, gefitinib completely inhibited EGF-induced phosphorylation of EGFR, AKT and ERK. Gefitinib inhibited EGFR phosphorylation, but was unable to block EGF-induced phosphorylation of ERK in resistant cells, labeled as PC-9/gef cells, including PC-9/gefB4, PC-9/gefE3, and PC-9/gefE7 subclones. We detected NRAS Q61K mutation in the PC-9/gef cells but not the PC-9/wt cells. MEK inhibitors, either AZD6244 or CI1040, inhibited ERK phosphorylation and sensitized gefitinib-induced cytotoxicity in PC-9/gef cells. Whereas MEK inhibitors or gefitinib alone did not activate caspases in PC-9/gef cells, combination of gefitinib and AZD6244 or CI1040 induced apoptosis. Our in vivo studies showed that gefitinib inhibited growth of PC-9/wt xenografts but not PC-9/gef xenografts. Furthermore, combination of a MEK inhibitor and gefitinib inhibited growth of both PC-9/wt xenografts and PC-9/gefB4 xenografts. To conclude, persistent activation of ERK pathway contributes to the acquired gefitinib-resistance. Combined treatment of gefitinib and MEK inhibitors may be therapeutically useful for acquired gefitinib-resistance lung adenocarcinoma cells harboring EGFR mutations.     

大黄素逆转非小细胞肺癌EGFR-TKI耐药的机制研究

目的 探讨大黄素逆转非小细胞肺癌（NSCLC）表皮生长因子受体酪氨酸激酶抑制剂（EGFR TKI）耐药的作用机制。方法 应用持续诱导的方法构建NSCLC EGFR-TKI耐药细胞株HCC827／GR;应用MTS法检测大黄素（30μmol／L）、吉非替尼（1μmol／L）及两药联合处理HCC827和HCC827／GR细胞48h后细胞增殖能力的变化;应用Western blotting法检测HCC827和 HCC827／GR细胞中p EGFR、p-AKT、p-ERK1／2及p-MET蛋白表达水平的变化。结果 MTS法检测结果显示,经单药吉非替尼或大黄素处理后,HCC827／GR细胞增殖能力未减弱,而两药联合处理组的细胞增殖能力明显下降,差异有统计学意义（P＜0.05）。Western blotting检测结果显示,HCC827、HCC827／GR细胞中p-EGFR、p-ERK1/2明显表达,而p-AKT表达微弱;HCC827／GR 中p-MET表达水平较HCC827明显上调。经单药吉非替尼处理后,HCC827细胞株p-EGFR、p-ERK1/2表达水平下调,HCC827／GR细胞株p-EGFR表达明显下调;大黄素可显著下调HCC827／GR细胞株p-MET表达,但对p-EGFR、p-ERK1/2的表达无影响;而大黄素与吉非替尼两药联用可明显抑制HCC827／GR细胞株p-EGFR、p-ERK1/2以及p-MET的表达。结论 大黄素可以逆转NSCLC EGFR-TKI耐药,可能是通过抑制c-Met的活化来实现。