Downregulation of FoxM1 inhibits cell growth and migration and invasion in bladder cancer cells

Abstract

The FoxM1 (Forkhead Box M1) transcription factor plays a key role in regulation of cell growth, cell cycle, and transformation. Higher expression of FoxM1 has been observed in various types of human cancers including bladder cancer. However, the exact function of FoxM1 in bladder cancer has not been elucidated. To investigate the cellular and molecular function of FoxM1 in bladder cancer, we measured the consequences of downregulation and upregulation of FoxM1 in bladder cancer cells using MTT assay, wound healing assay, and invasion assay. We found that downregulation of FoxM1 inhibited cell growth, but induced apoptosis in bladder cancer cells. Moreover, we found that inhibition of FoxM1 retarded cell migration and invasion. In line with this, upregulation of FoxM1 led to cell growth promotion and inhibited cell apoptosis in bladder cancer cells. Consistently, upregulation of FoxM1 led to increased cell migration and invasion. Our Western blotting results identified that downregulation of FoxM1 increased p27 level and inhibited VEGF, while overexpression of FoxM1 reduced p27 level and increased VEGF. Our findings suggest that FoxM1 could be a useful target for the treatment of bladder cancer.

Keywords: FoxM1; apoptosis; bladder cancer; cell growth; invasion.

Figure 1

Down-regulation of FoxM1 by its siRNA in bladder cancer cells. A. Real-time RT-PCR analysis was used to determine the efficacy of FoxM1 siRNA in RT4 bladder cancer cells. *P < 0.01 vs Control siRNA. B. Top panel: Western blot analysis was used to measure the FoxM1 expression in RT4 bladder cancer cells transfected with different FoxM1 siRNAs. Bottom panel: Quantitative results for Top panel. *P < 0.01, vs Control siRNA.

Figure 2

Down-regulation of FoxM1 inhibited cell proliferation and induced apoptosis. A. MTT assay was used to measure cell proliferation in RT4 bladder cancer cells after FoxM1 siRNA transfection. The transfected cells (5 × 10³) were seeded in a 96-well plate. After 48 h and 72 h, cells were incubated with MTT reagent (0.5 mg/ml) for 2 h at 37°C. Cell growth was determined by measuring absorbance at 560 nm. All values were normalized to those of the controls. *P < 0.05 vs Control siRNA. B. Flow cytometry was used to measure cell apoptosis in RT4 bladder cancer cells after FoxM1 siRNA transfection. The transfected cells were cultured in the 6-well plate for 48 h. Then, the cells were collected by centrifugation and resuspended in binding buffer with 5 μl propidium iodide and 5 μl FITC-conjugated anti-Annexin V antibody. Apoptosis was analyzed by a FACScalibur flow cytometer.

Figure 3

Down-regulation of FoxM1 inhibited motility span>activity in bladder cancer cells. (A, B) Invasion assays were conducted to detect the invasive capacity in RT4 cells after FoxM1 siRNA transfection. The tranfected cells were seeded in DMEM without serum in the upper chamber. The bottom chamber was added with complete medium. After 20 h of incubation, the cells that had invaded through Matrigel matrix membrane were stained with Wright’s-Giemsa (A) or 4 μg/ml Calcein AM (B) in hanks buffered saline at 37°C for 1 h. The labeled invasive cells were photographed under a microscope. (C) Wound healing assays was performed to measure cell migration in RT4 after FoxM1 siRNA transfection. Cells were seeded in 6-well plates and grown to almost confluency. Then, monolayers of cells were scratched with 200 μL small yellow pipette tips and washed twice with PBS. The scratched area was photographed with a microscope at 0 h and 20 h, respectively.

Figure 4

Down-regulation of FoxM1 increased p27 and decreased VEGF levels. A. Western blot analysis was performed to detect the expression of FoxM1, p27, and VEGF in RT4 bladder cancer cells after FoxM1 siRNA transfection for 48 h. B, C. Quantitative results for Western blotting analysis in panel A. *P < 0.01, vs control siRNA.

Figure 5

Overexpression of FoxM1 enhanced cell proliferation and inhibited apoptosis. A. MTT assay was used to measure cell proliferation in RT4 cells after FoxM1 cDNA transfection. The transfected cells (5 × 10³) were seeded in a 96-well culture plate. After 48 h and 72 h, cells were then incubated with MTT reagent (0.5 mg/ml) for 2 h at 37°C. The absorbance was measured at 560 nm. All values were normalized to those of the controls. *P < 0.05 vs Control cDNA. B. Flow cytometry was used to detect cell apoptosis in bladder cancer cells transfected with FoxM1 cDNA. The transfected cells were cultured in 6-well plate for 48 h. Then, the cells were collected and resuspended in binding buffer with 5 μl propidium iodide and 5 μl FITC-conjugated anti-Annexin V antibody. Apoptosis was analyzed by a FACScalibur flow cytometer.

Figure 6

Overexpression of FoxM1 enhanced motility activity in bladder cancer cells. (A, B) Invasion assays were used to measure the cell invasion in RT4 cells after FoxM1 cDNA transfection. The tranfected cells were seeded in DMEM without serum in the upper chamber. The bottom chamber was added with complete medium. After 20 h of incubation, the invaded cells through Matrigel matrix membrane were stained with Wright’s-Giemsa (A) or 4 μg/ml Calcein AM (B) at 37°C for 1 h. The labeled invasive cells were photographed under a microscope. (C) Wound healing assays was used to detect the cell migration in RT4 after FoxM1 cDNA transfection. Cells were seeded in 6-well plates and grown to almost confluency. Then, monolayers of cells were scratched with 200 μL small yellow pipette tips and washed twice with PBS. The scratched area was photographed with a microscope at 0 h and 20 h, respectively.

Figure 7

Overexpression of FoxM1 inhibited p27 and upregulated VEGF levels. (A) Western blot analysis was used to detect the expression of FoxM1, p27, and VEGF in bladder cancer cells after FoxM1cDNA transfection for 48 h. (B, C) Quantitative results for Western blotting in (A). *P < 0.01, vs control siRNA.