[Effects of AG1478 on the expression of FOXM1 gene via FOXO3a in non-small cell lung cancer cells].

OBJECTIVE

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.

METHODS

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.

RESULTS

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.

CONCLUSIONS

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.