靶向抑制ERK1/2信号传导通路对人高转移大细胞肺癌细胞株L9981恶性表型的影响

Effects of targeting inhibition of ERK1/2 signal transduction pathway on malignant biological behaviors of human high-metastatic large cell lung cancer cell line L9981

背景与目的肺癌的发生和发展是一个多基因调控、多阶段多步骤发生的复杂过程,目前已知信号传导异常在肺癌发生和发展各个阶段都具有重要作用。本研究旨在探讨外源性MEK1/2抑制剂U0126对人高转移大细胞肺癌细胞株L9981中细胞外信号调节激酶ERK1/2表达和活化的影响及细胞恶性生物学行为的影响。方法将具有nm23H1基因杂合性缺失的人高转移大细胞肺癌原代细胞株L9981培养传代,应用蛋白印迹法(Westernblot)和免疫沉淀法,检测U0126对L9981中总ERK1/2和双磷酸化ERK1/2的表达水平,以及ERK1/2相对活性的影响;应用MTT法及改良Boyden小室法分别检测U0126对L9981体外增殖活性及侵袭能力的作用规律。结果不同浓度的U0126作用于L998120min后,随着U0126浓度的增加ERK1/2的相对活性均逐渐下降,不同U0126浓度组间磷酸化ERK1/2表达水平比较均有非常显著性差异(P<0.01);而不同浓度U0126组间总ERK1/2表达水平比较无显著性差异(P=0.387)。相同浓度的U0126作用于L9981不同时间后,随着作用时间的延长,细胞中ERK1/2的相对活性均逐渐下降,各时间组间磷酸化ERK1/2表达水平比较有显著性差异(P<0.01);而各时间组间总ERK1/2表达水平比较无显著性差异(P=0.689)。U0126对L9981细胞株ERK1/2相对活性的作用具有时间和剂量依赖性。不同浓度的U0126作用于L9981后,随着U0126浓度的增加,细胞体外增殖活性随之降低,不同浓度组间比较均有显著性差异(P<0.01)。不同浓度的U0126作用于L9981后,随着U0126浓度的增加,细胞体外侵袭能力随之降低。在U0126浓度为0、10和20μmol/l时三个剂量组间细胞体外侵袭能力比较无显著性差异(P>0.05),而在U0126浓度增加为40和60μmol/l时与U0126浓度为0、10和20μmol/l时细胞体外侵袭能力比较均有显著性差异(P<0.01)。结论ERK1/2信号传导通路特异性抑制剂U0126对人高转移大细胞肺癌细胞株ERK1/2信号传导通路的抑制作用具有剂量和时间依赖性;U0126对人高转移大细胞肺癌细胞株体外增殖活性和侵袭能力的抑制作用具有剂量依赖性;提示人高转移大细胞肺癌细胞株中ERK1/2信号传导通路关键激酶MEK1/2可能是治疗肺癌的一个潜在分子靶点。

Background and objective It has been known that oncogencsis and development of lung cancer is a complex process regulated many genes and involved in multistages. Recent studies have demonstrated that signal transduction abnormality may playa very important rolc in these procedures. The aim of this study is to investigate the influencc of exogenous MEN1/2 pathway inhibitor U0126 on the expression and activity of extraeellular signal-regulated kinase （ERK1/2） of human high-metastatic large cell lung cancer cell line L9981 and its malignant biological behaviors. Methods The expressive levels of total-ERK1/2, dually phosphoryla- ted ERK1/2 and ERK1/2 relative activity of the human high-metastatic large cell lung cancer cell line L9981 （parent cell line with nm23-H1 gene hetero-deletion ） were detected by Western blot and immunoprecipitation technique after treating with different doses of U0126. The in vitro proliferative and invasive abil ities of the lung cancer cell line were determined by MTT and modified Boyden chamber methods. Results The ERK1/2 relative activity of L9981 gradually decreased accompanied with increase of U0126 doses, and a highly significant difference of phosphorylated ERK1/2 expression level existed among the different concentra- tion groups of U0126 （P（0.01）. but no significant difference of total ERK1/2 expression was found among the different concentration groups of U0126 （P=0.387）. After treatment with same concentration of U0126 for different time, the ERK1/2 relative activity of L9981 gradually decreased as the treatment time of U0126 prolonging, and a highly significant difference of phosphorylated ERK1/2 expression level was observed among different treatment time groups （P〈0.01）. But no significant difference of total ERK1/2 expression level was found among different time groups （P=0. 689）. The inhibition of ERK1/2 pathway by MEK1/2 specific inhibitor U0126 targeting the ERK1/2 pathway of L9981 was dose and time-dependent. After treatment with different concentration of U0126, the proliferation of L9981 gradually decreased accompanied with increase of U0126 concentration and a highly significant difference existed among different groups of U0126 concentration （P〈0. 001）. The in vitro invasion of L9981 gradually decreased accompanied with increase of U0126 concen tration. No significant difference of in vitro invasion of L9981 was found among 0, 10 and 20μmol/l of U0126 （P）0. 05）. A highly significant difference was observed when U0126 concentration increased to 40 and 60μmol/l compared with 0, 10 and 20μmol/l of U0126 （P〈0. 001）. Conclusion The inhibition of Ras-to- MAPK pathway by Ras to MAPK specific inhibitor U0126 targeting the Ras-to-MAPK pathway of the human high metastatic large cell lung cancer cell line L9981 is dose dependent and time-dependent. Suppressing or blocking of Ras to MAPK signal transduction pathway can reverse the invasive and metastatic phenotype of the human high-metastatic large cell lung cancer cell line L9981. These results suggest that the key kinase MEK1/ 2 of the ERK1/2 pathway may be a potent therapeutic target for human lung cancer.