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

Abstract

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.

• Authors: Xiao-di Gong, Hai-hua Yuan, Jiong-yi Wang, Yue-hui Guo, Jing Shi, Bin Jiang
• Journal: Zhonghua zhong liu za zhi [Chinese journal of oncology] (Zhonghua Zhong Liu Za Zhi) Vol. 35 Issue 8 Pg. 572-8 (Aug 2013) ISSN: 0253-3766 [Print] China
• PMID: 24314213 (Publication Type: Journal Article)

Chemical References

• FOXM1 protein, human
• FOXO3 protein, human
• Forkhead Box Protein M1
• Forkhead Box Protein O3
• Forkhead Transcription Factors
• Quinazolines
• RNA, Messenger
• RNA, Small Interfering
• Tyrphostins
• RTKI cpd
• ErbB Receptors

Topics

• Adenocarcinoma (metabolism, pathology)
• Apoptosis
• Cell Cycle
• Cell Line, Tumor
• Cell Proliferation
• Dose-Response Relationship, Drug
• Down-Regulation
• ErbB Receptors (antagonists & inhibitors)
• Forkhead Box Protein M1
• Forkhead Box Protein O3
• Forkhead Transcription Factors (genetics, metabolism)
• Humans
• Lung Neoplasms (metabolism, pathology)
• Quinazolines (administration & dosage, pharmacology)
• RNA, Messenger (metabolism)
• RNA, Small Interfering (genetics)
• Transfection
• Tyrphostins (administration & dosage, pharmacology)