FoxM1 is a novel target of a natural agent in pancreatic cancer

Abstract

Purpose: Pancreatic cancer remains the fourth most common cause of cancer-related death in the United States. Therefore, novel strategies for the prevention and/or treatment are urgently needed. Genistein has been found to be responsible for lowering the rate of pancreatic cancer. However, the molecular mechanisms by which genistein elicits its effects on pancreatic cancer cells has not been fully elucidated. Therefore, the purpose of the current study was to elucidate the anti-cancer mechanism(s) of genistein.

Methods: Multiple molecular techniques, such as Real-time RT-PCR, Western blot analysis, invasion assay, immunofluorescence assay, gene transfection, MTT assay, and Histone/DNA ELISA, were used.

Results: We found that genistein inhibited cell growth accompanied by induction of apoptosis with concomitant attenuation of FoxM1 and its downstream genes, such as survivin, cdc25a, MMP-9, and VEGF, resulting in the inhibition of pancreatic cancer cell invasion. We also found that down-regulation of FoxM1 by siRNA prior to genistein treatment resulted in enhanced cell growth inhibition and induction of apoptosis.

Conclusion: This is the first report showing the molecular role of FoxM1 in mediating the biological effects of genistein in pancreatic cancer cells, suggesting that FoxM1 could be a novel target for the treatment of pancreatic cancer.

Fig. 1

Effect of genistein on pancreatic cancer cell growth. Cells were seeded in 96-well plates at 5,000 cells per well and treated with varied concentrations of genistein for 72 h. After treatment, MTT solution was added and incubated further for 2 h. MTT formazan formed by metabolically viable cells was dissolved in isopropanol, and absorbance was measured at 595 nm on a plate reader (TECAN). Each value represents the mean ± SD (n=6) of three independent experiments. *P<0.05, **P<0.01, compared to the control.

Fig. 2

Effect of genistein on pancreatic cancer cell growth and apoptosis. A Cell survival of human pancreatic cancer cell lines BxPC-3 and MIA PaCa-2. Cells treated with varied concentrations of genistein for 72 h were evaluated by the clonogenic assay. Photomicrographic difference in colony formation in cells untreated and treated with genistein are shown. B There was a significant reduction in the colony formation in BxPC-3 and MIA PaCa-2 cells treated with genistein compared with control cells. P values represent comparisons between cells treated by genistein and control using the paired t-test. *P<0.05, **P<0.01, compared to the control. C Cell death assay for measuring apoptosis induced by genistein. BxPC-3, HPAC, MIA PaCa-2 and PANC-28 cells were cultured in RPMI containing 5% FBS and exposed to different dose genistein for 72 h. Apoptosis was measured by Histone DNA ELISA. Values are reported as mean ± SD. *P<0.05, **P<0.01, compared to the control.

Fig. 3

Inhibition of FoxM1 expression by genistein. A The baseline expression of FoxM1 was determined among the panel of pancreatic cancer cell lines using real-time RT-PCR and Western Blotting analysis, respectively. FoxM1 was over-expressed in different human pancreatic cancer cell lines compared to “so-called” normal human pancreatic ductal epithelial (HPDE) cells. B The FoxM1 mRNA was detected by real-time RT-PCR in pancreatic cancer cells treated with varied concentrations of genistein for 72 h. C The FoxM1 protein was detected by Western Blotting analysis in pancreatic cancer cells treated with varied concentrations of genistein for 72 h.

Fig. 4

Inhibition of expression of FoxM1 target genes by genistein. A The MIA PaCa-2 pancreatic cancer cells treated with 50 μM genistein for 72 h were subjected to immunofluorescent staining using anti-FoxM1 antibody. B The expression of FoxM1 target genes was detected by Western blotting analysis in pancreatic cancer cells treated with varied concentrations of genistein for 72 h. We found that genistein inhibited the expression of survivin, cdc25a, MMP-9 and VEGF in BxPC-3 and MIA PaCa-2 cells. C–D The FoxM1 target gene mRNA levels were detected by real-time RT-PCR in pancreatic cancer cells treated with varied concentrations of genistein for 72 h. Genistein inhibited the transcription levels of survivin, cdc25a, MMP-9 and VEGF in BxPC-3 and MIA PaCa-2 cells.

Fig. 5

Genistein inhibited the MMP-9 and VEGF activities, thus decreasing pancreatic cancer cell invasion. A Genistein inhibited the activity of MMP-9 in BxPC-3 and MIA PaCa-2 pancreatic cancer cells. B Genistein inhibited the activity of VEGF. VEGF activity assay showing that VEGF level in the culture medium was inhibited by genistein. C Left panel, invasion assay showing that genistein-treated cells resulted in low penetration through the Matrigel-coated membrane, compared with control cells; right panel, value of fluorescence from the invaded cells, and these values indicate the comparative amount of invaded cells.

Fig. 6

Down-regulation of FoxM1 by siRNA promotes genistein-induced cell growth inhibition and apoptosis in MIA PaCa-2 cells. gen+siRNA: 50 μM genistein + FoxM1 siRNA; gen+cDNA: 50 μM genistein + FoxM1 cDNA. A The expression of FoxM1 was detected by Western blotting to check the FoxM1 siRNA transfection efficacy. B Left panel, down-regulation of FoxM1 by siRNA significantly inhibited MIA PaCa-2 cell growth. Genistein plus FoxM1 siRNA inhibited cell growth to a greater degree compared to genistein alone. Right panel, down-regulation of FoxM1 expression significantly increased apoptosis induced by genistein. FoxM1 siRNA transfected cells were significantly more sensitive to spontaneous and genistein-induced apoptosis. C The expression of FoxM1 was detected by Western blotting to check the FoxM1 cDNA plasmid transfection efficacy. D Over-expression of FoxM1 expression significantly promoted cell growth. Over-expression of FoxM1 rescued cells from genistein-induced cell growth inhibition. Over-expression of FoxM1 by FoxM1 cDNA transfection abrogated genistein-induced apoptosis to a certain degree.