Li Yin, Zhou Qinghua, Sun Zhilin, Sun Zefang, Wang Yanping, Qin Yang, Zhu Wen, Chen Xiaohe
Key Laboratory of Lung Cancer Molecular Biology of Sichuan Province and Department of Thoracic Surgery, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R.China.
Zhongguo Fei Ai Za Zhi. 2006;9(4):307-11. doi: 10.3779/j.issn.1009-3419.2006.04.01.
nm23-H1 gene is a well-known tumor metastasis suppression gene. Our previous study has found that transfection of wild type nm23-H1 gene can significantly downregulate the ERK1/2 activity of human high-metastatic large cell lung cancer cell line L9981. The aim of this study is to investigate the influence of nm23-H1 and exogenous ERK1/2 pathway inhibitor U0126 on the extracellular signal-regulated kinase (ERK1/2) of human high-metastatic large cell lung cancer cell line L9981 and its malignant biological behaviors.
The expressive levels of total-ERK1/2, dually phosphorylated ERK1/2 and ERK1/2 relative activity of the human high-metastatic large cell lung cancer cell lines, L9981 (parent cell line with nm23-H1 gene hetero-deletion), L9981-nm23-H1 (transfected with nm23-H1 gene ) and L9981-PLXSN (transfected with vector) were detected by Western blot and immunoprecipitation technique after treating with U0126 (40μmol/L for 20 minutes). The in vitro proliferative and invasive abilities among the above three lung cancer cell lines were determined by MTT and improved Boyden chamber methods.
The phosphorylated ERK1/2 expression level and relative activity in L9981-nm23-H1 lung cancer cell line were remarkably lower than those in L9981 and L9981-PLXSN lung cancer cell lines after being treated with U0126 (P < 0.01), but there was no significant difference between L9981 and L9981-PLXSN lung cancer cell lines. No significant difference of total ERK1/2 expression level was observed among the three lung cancer cell lines (P > 0.05) after being treated with U0126. The in vitro proliferation and invasion of L9981-nm-23H1 lung cancer cell line were remarkably lower than those of L9981 and L9981-PLXSN lung cancer cell lines (P < 0.01 ), but no significant difference was found between L9981 and L9981-PLXSN lung cancer cell lines (P > 0.05 ); U0126 could significantly down-regulate the in vitro proliferation and invasion of L9981 lung cancer cell line (P < 0.01).
Blocking the activity of ERK1/2 in L9981 lung cancer cell line and transfecting the nm23-H1 gene into the L9981 lung cancer cell line may produce similar cell biological behavior changes, namely the significant reduction of in vitro proliferation and invasion of L9981 lung cancer cell line. These results indicate that the molecular mechanism which nm23-H1 gene reverses invasion and proliferation of the human high-metastatic large cell lung cancer cell line may be related to its effects of down-regulating the activity of the key kinase ERK1/2 of Ras-to-MAPK signal transduction pathway.
nm23-H1基因是一种著名的肿瘤转移抑制基因。我们之前的研究发现，转染野生型nm23-H1基因可显著下调人高转移性大细胞肺癌细胞系L9981的ERK1/2活性。本研究旨在探讨nm23-H1和外源性ERK1/2通路抑制剂U0126对人高转移性大细胞肺癌细胞系L9981的细胞外信号调节激酶（ERK1/2）及其恶性生物学行为的影响。
用U0126（40μmol/L处理20分钟）后，通过蛋白质免疫印迹法和免疫沉淀技术检测人高转移性大细胞肺癌细胞系L9981（nm23-H1基因杂合缺失的亲本细胞系）、L9981-nm23-H1（转染nm23-H1基因）和L9981-PLXSN（转染载体）中总ERK1/2、双磷酸化ERK1/2的表达水平及ERK1/2相对活性。采用MTT法和改良的Boyden小室法测定上述三种肺癌细胞系的体外增殖和侵袭能力。
用U0126处理后，L9981-nm23-H1肺癌细胞系中磷酸化ERK1/2的表达水平和相对活性显著低于L9981和L9981-PLXSN肺癌细胞系（P<0.01），但L9981和L9981-PLXSN肺癌细胞系之间无显著差异。用U0126处理后，三种肺癌细胞系中总ERK1/2表达水平无显著差异（P>0.05）。L9981-nm-23H1肺癌细胞系的体外增殖和侵袭能力显著低于L9981和L9981-PLXSN肺癌细胞系（P<0.01），但L9981和L9981-PLXSN肺癌细胞系之间无显著差异（P>0.05）；U0126可显著下调L9981肺癌细胞系的体外增殖和侵袭能力（P<0.01）。
阻断L9981肺癌细胞系中ERK1/2的活性并将nm23-H1基因转染至L9981肺癌细胞系可能产生相似的细胞生物学行为变化，即L9981肺癌细胞系的体外增殖和侵袭能力显著降低。这些结果表明，nm23-H1基因逆转人高转移性大细胞肺癌细胞系侵袭和增殖的分子机制可能与其下调Ras至MAPK信号转导通路关键激酶ERK1/2活性的作用有关。
