吉非替尼对表达PTEN的胶质瘤细胞株U87细胞增殖活性的影响

Role of PTEN/PI3K signal transduction pathway in regulating the sensitivity of glioma to gefitinib

目的探讨表皮生长因子受体抑制剂吉非替尼对表达磷酸酶-张力蛋白同源物(PTEN)的胶质瘤细胞株U87细胞增殖活性的影响。方法将人脑恶性胶质瘤细胞株U87细胞(PTEN缺失)置入DMEM中培养,根据转染质粒不同分为空白组(不转染任何质粒)、空载体组(转染pCDNA3.1载体)和PTEN组(转染pCDNA3.1-PTEN质粒),采用5-溴脱氧尿嘧啶核苷(BrdU)/碘化丙碇双掺入法分析细胞DNA合成水平,采用流式细胞仪分析细胞周期,采用蛋白免疫印迹法分析蛋白表达。结果与空白组和空载体组相比,PTEN组PTEN蛋白表达水平明显升高,而磷酸化Akt蛋白表达水平明显降低。PTEN组BrdU阳性细胞比例[(13.5±2.7)%]明显低于空白组[(39.5±4.2)%;P<0.05]和空载体组为[(40.7±5.1)%;P<0.05]。PTEN组G0/G1期细胞比例[(80.2±6.6)%]明显高于空白组[(43.2±5.2)%;P<0.05]和空载体组[(41.1±4.7)%;P<0.05],而S和G2/M期细胞比例[分别为(35.7±3.7)%和(21.1±2.1)%]明显低于空白组[分别为(36.5±4.1)%和(22.4±1.9)%;P<0.05]和空载体组[分别为(12.7±2.0)%和(7.1±1.1)%;P<0.05]。结论高表达PTEN蛋白能够增强胶质瘤细胞株U87细胞对吉非替尼的敏感性。

Objective To explore the role of PTEN/PI3K in regulating the sensitivity glioma to gefitinib and its mechanism. Methods The plasmids of pCDNA3.1-PTEN and pCDNA3.1 were transfeeted into human malignant glioma eel1 line U87MG cells （loss of PTEN mutation cells）. DNA synthesis and cell cycle progression were analyzed by BrdU/PI incorporation, and the protein expression was analyzed by western blotting. The U87MG cells were treated with gefitinib in each group. Results The proliferation of U87MG cells losing PTEN in whom PI3K pathway was activated, could not be prevented by the epidermal growth factor receptor （EGFR） inhibitors gefitinib [the proportion of GVI was （21.1±2.1）%]. The proliferation of U87MG cells in whom the wild type PTEN was reexpressed by the transfection of pCDNA3.1-PTEN into them was significantly inhibited by gefitinib [the proportion of GJM was （7.1 ± 1.1）%]. BrdU/PI incorporation showed that the percentage of BrdU positive cells was （39.5±4.2）% in the blank group, （40.7±5.1）% in pCDNA 3.1 group, and （13.5±2.7）% in pCDNA3.1-PTEN group. The percentage of BrdU positive cells was significantly lower in pCDNA3.1-PTEN group than those in the blank and pCDNA3.1 groups （P〈0.05）. Conclusions The proliferation of U87MG cells losing PTEN gene can be not prevented by gefitinib. The high expression of PTEN/PI3K can enhance the sensitivity of U87MG cells to gefitinib.