T790M突变所致吉非替尼耐药肺腺癌细胞放射敏感性变化及耐药性逆转的研究

Change of Radiosensitivity and Reversal of Drug Resistance in Gefitinib-resistant Lung Adenocarcinoma Cell Caused by T790M Mutation

目的探讨T790M突变所致吉非替尼耐药肺腺癌细胞株PC-9/GR放射敏感性的变化情况及经射线照射后PC-9/GR吉非替尼耐药的逆转情况。方法选取吉非替尼敏感肺腺癌细胞株PC-9及T790M突变所致吉非替尼继发性耐药肺腺癌细胞株PC-9/GR。应用液相芯片法检测这两种细胞中人第10号染色体缺失的磷酸酶及张力蛋白同源的基因(PTEN)mRNA表达水平;应用免疫印迹法检测这两种细胞中磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)、磷酸化蛋白激酶B(p-Akt)、PTEN蛋白表达水平;采用集落形成实验观察射线照射细胞的存活情况;应用四甲基偶氮唑盐(MTT)比色法检测细胞的生长抑制情况。结果 (1)PC-9/GR的PTEN mRNA及其蛋白表达水平均高于PC-9(P<0.05),而p-mTOR、p-Akt蛋白表达水平则低于PC-9(P<0.05)。(2)在不同剂量6 MV X线作用下,PC-9/GR的细胞存活分数(SF)均低于PC-9(P<0.05)。(3)在不同浓度吉非替尼作用下,射线照射的PC-9/GR的生长抑制率均高于未照射的PC-9/GR(P<0.05),半数抑制浓度(IC50)则低于未照射的PC-9/GR(P<0.05)。射线照射的PC-9/GR每一吉非替尼浓度的生长抑制率均高于未经照射的PC-9/GR(P<0.05)。结论在体外实验条件下,肺腺癌细胞株PC-9/GR的放射敏感性较其亲代PC-9升高,射线照射后的PC-9/GR在一定程度上能逆转吉非替尼的耐药。

Objective To investigate the change of radiosensitivity in gefitinib- resistant lung adenocarcinoma cell line PC- 9 /GR caused by T790 M mutation,and the reversal of drug resistance in gefitinib- resistant PC- 9 /GR after X ray irradiation. Methods Gefitinib- sensitive lung adenocarcinoma cell line PC- 9 and T790 M mutation- induced secondary gefitinib- resistant lung adenocarcinoma cell line PC- 9 /GR cell line were selected. Liquid chip method was applied to detect the expression level of PTEN mRNA in the two kinds of cells; Western blotting method was used to detect the expression level of p- mTOR,p- Akt and PTEN proteins; Colony formation assay was carried out to observe the survival situation of the cells which were irradiated by X ray; MTT colorimetric method was used to study the growth inhibition of cells. Results The expression levels of PTEN mRNA and PTEN proteins in PC- 9 /GR were significantly higher than those in PC- 9（ P〈0. 05）,while the expression levels of p- mTOR and p- Akt in PC- 9 /GR were significantly lower than those in PC- 9（ P〈0. 05）. With the action of different doses of 6 MV X ray,the survival fraction（ SF） of PC- 9 /GR was significantly lower than that of PC- 9（ P〈0. 05）. With the action of different concentrations of gefitinib,the growth inhibition rate of PC- 9 /GR with irradiation was significantly higher than that of unirradiated PC- 9 /GR（ P〈0. 05）,and the IC50 values in irradiated PC- 9 /GR were significantly lower than those in non- irradiated PC- 9 /GR（ P〈0. 05）. In every concentration of gefitinib,the growth inhibition rates of PC- 9 /GR with irradiation were significantly higher than those of unirradiated PC- 9 /GR（ P〈0. 05）. Conclusion Under the experimental conditions in vitro,the radiosensitivity of lung adenocarcinoma PC- 9 /GR cells is higher than that of its parental PC- 9 cells,and the irradiated PC- 9 /GR cells can reverse gefitinib resistence to some extent.