EGFR信号通路的活化状态与结肠癌细胞对吉非替尼敏感性的关系

Relationship between the activated status of EGFR signal pathway and the sensitivity of colon carcinoma cells to gefitinib

目的评价表皮生长因子受体(EGFR)抑制剂吉非替尼对不同敏感性结肠癌细胞的生长抑制效应及EGFR下游信号通路蛋白的活化状态。方法取结肠癌细胞(Lovo、HCT116、HT29),用吉非替尼(10μmol/L)处理后,采用流式细胞仪检测细胞周期和凋亡;另取细胞,用表皮生长因子(EGF,50ng/ml)或EGF+吉非替尼(5、10μmol/L)处理后,采用Western blotting检测PTEN、EG-FR、Akt、MAPK及其相应的磷酸化蛋白(p-EGFR、p-Akt、p-MAPK)的表达。结果在吉非替尼作用下,G1期细胞比例和凋亡率在Lovo细胞中显著增加,在HT29细胞中仅轻度增加,而在HCT116细胞中无明显变化。在单纯EGF作用下,p-EGFR、p-Akt和p-MAPK在Lovo和HT29细胞中表达增加(P<0.05),而在HCT116细胞中无明显变化。在给予EGF的基础上添加吉非替尼后,Lovo细胞中EGFR、p-EGFR、p-Akt、p-MAPK和HT29细胞中p-EGFR的表达被明显抑制(P<0.05),HCT116细胞中p-EGFR的表达下降不如Lovo和HT29细胞明显;Lovo、HT29和HCT116细胞内PTEN的表达均无明显变化(P>0.05)。结论耐受吉非替尼的细胞表现为其生长不依赖EGFR,且下游信号通路的活化状态不伴随EGFR活性受阻而受抑制。结肠癌细胞对吉非替尼的敏感程度是由细胞对EGFR的依赖性以及EGFR和下游信号通路的伴随性所决定的。

Objective To evaluate the inhibitory effects of gefitinib on the growth of colon carcinoma cells with different degree of sensitivity, and the activated status of epidermal growth factor receptor （EGFR） associated signal pathway proteins, Methods The colon carcinoma cell lines （Lovo, HCT116 and HT29） were treated with 10μmol/L of gefitinib, and flow cytometry was employed to detect the cell cycle and apoptosis. Another portion of cells were treated with 50ng/ml of epidermal growth factor （EGF） or 5μmol/L or 10 μmol/L of EGF＋gefitinib, and Western blotting was used to determine the expressions of PTEN, EGFR, AKT and MAPK, as well as their corresponding phosphorylated proteins, p-EGFR, p-AKT and p-MAPK. Results After being treated with gefitinib, the G1 phase cells and apoptosis rate increased remarkably in Lovo, slightly in HT29, while no significant change was found in HCT116. With the treatment of EGF alone, the expressions of p-EGFR, p-AKT and p-MAPK increased significantly in Lovo and HT29 cells （ P〈0. 05）, while no changes were found in HCT116 cells. With the combined treatment of EGF＋gefitinib, the expressions of EGFR, p-EGFR, p-AKT and p- MAPK in Lovo cells and p-EGFR in HT29 cells were inhibited obviously （P〈0. 05）. However, a declination of p-EGFR expression in HCT116 cells was not as obvious as in Lovo and HT29 cells. Moreover, no significant changes in PTEN expression was found in Lovo, HT29 and HCT116 cells （P〉0. 05）. Conclusions The growth of colon carcinoma cells, which are resistant to gefitinib, is not dependent on EGFR, and the activated status of signal pathway of the gefitinib-resistant cells will not be inhibited as the EGFR is blocked. The present findings suggest that sensitivity of colon carcinoma cells to gefitinib relies on EGFR as well as the activation of its downstream signal pathway.