Gong Xiao-di, Yuan Hai-hua, Wang Jiong-yi, Guo Yue-hui, Shi Jing, Jiang Bin
Department of Oncology, the Third People's Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai 201900, China.
Zhonghua Zhong Liu Za Zhi. 2013 Aug;35(8):572-8.
To explore the effects of EGFR-TKI AG1478 on the expression of FoxMl and FOXO3a genes in non-small cell cancer (NSCLC) cell lines, and explore the effect on cell proliferation and drug sensitivity to AG1478 after down-regulation of FOXMl and FOXO3a expression by RNAi technique.
Human lung cancer cells were treated with AG1478 at different concentrations. RT-PCR and Western blot were used to examine the expression of P-EGFR, FOXM1, FOXO3a mRNA and protein. After transient transfection of FOXM1 and FOXO3a siRNA, RT-PCR and Western blot were employed to determine the transfection efficiency and expression of the related proteins. CCK-8 assay, colony formation assay and flow cytometry were performed to evaluate the cell proliferation, colony formation ability and the changes in cell cycle distribution.
The expressions of FOXM1 mRNA and protein were inhibited by AG1478 in a dose-dependent manner (both P < 0.05). After transfection with FOXM1 siRNA, the expressions of FOXM1 mRNA and protein, and proteins of cyclin B1, c-Myc, and Bcl-2 were significantly down-regulated, and the expressions of p21 and cleaved-PARP proteins were significantly up-regulated (all P < 0.05). The colony number of FOXM1siRNA transfection group was 37.3 ± 8.6, significantly lower than that of the blank control (135.3 ± 7.0) and negative control group (125.3 ± 7.5, P < 0.05). The colony formation inhibition rate was (7.40 ± 0.94)% in the negative control group and (72.4 ± 6.09)% in the FOXM1 siRNA transfection group. FOXM1siRNA transfection induced cell cycle arrest at G2/M phase with a percentage of (55.6 ± 4.83)%, significantly higher than that of the blank control [(24.30 ± 1.95)%] and negative control group [(21.3 ± 2.06)%, P < 0.05]. Additionally, the FOXM1siRNA transfection significantly increased the chemosensitivity of A549 cells to AG1478 (P < 0.05). Besides, AG1478 induced expression and nuclear relocation of FOXO3a. After the FOXO3a siRNA transfection, the expression of FOXM1 protein was significantly up-regulated, and resulted in a reduction of AG1478-induced inhibition of FOXM1.
The expression of FOXM1 is down-regulated by AG1478 via FOXO3a in the NSCLC cell lines, and then increases the chemosensitivity of A549 cells to AG1478. It suggests that FOXM1 could be a potential target for the therapy and drug exploitation for NSCLC.
探讨表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）AG1478对非小细胞肺癌（NSCLC）细胞系中FoxM1和FOXO3a基因表达的影响，并通过RNA干扰技术下调FOXM1和FOXO3a表达后，研究其对细胞增殖及对AG1478药物敏感性的影响。
用不同浓度的AG1478处理人肺癌细胞。采用逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法（Western blot）检测磷酸化表皮生长因子受体（P-EGFR）、FoxM1、FOXO3a mRNA及蛋白的表达。瞬时转染FoxM1和FOXO3a小干扰RNA（siRNA）后，用RT-PCR和Western blot检测转染效率及相关蛋白表达。采用细胞计数试剂盒-8（CCK-8）法、集落形成实验及流式细胞术评估细胞增殖、集落形成能力及细胞周期分布变化。
AG1478呈剂量依赖性抑制FoxM1 mRNA和蛋白表达（均P<0.05）。转染FoxM1 siRNA后，FoxM1 mRNA和蛋白以及细胞周期蛋白B1、原癌基因c-Myc和凋亡抑制蛋白Bcl-2的表达均显著下调，而p21和裂解的聚（ADP-核糖）聚合酶（cleaved-PARP）蛋白表达显著上调（均P<0.05）。FoxM1 siRNA转染组集落数为37.3±8.6，显著低于空白对照组（135.3±7.0）和阴性对照组（125.3±7.5，P<0.05）。阴性对照组集落形成抑制率为（7.40±0.94）%，FoxM1 siRNA转染组为（72.4±6.09）%。FoxM1 siRNA转染诱导细胞周期阻滞于G2/M期，比例为（55.6±4.83）%，显著高于空白对照组[（24.30±1.95）%]和阴性对照组[（21.3±2.06）%，P<0.05]。此外，FoxM1 siRNA转染显著增加A549细胞对AG1478的化疗敏感性（P<0.05）。另外，AG1478诱导FOXO3a表达并使其核转位。转染FOXO3a siRNA后，FoxM1蛋白表达显著上调，并导致AG1478诱导的FoxM1抑制作用减弱。
在NSCLC细胞系中，AG1478通过FOXO3a下调FoxM1表达，进而增加A549细胞对AG1478的化疗敏感性。提示FoxM1可能是NSCLC治疗和药物开发的潜在靶点。
