Down-regulation of Notch-1 is associated with Akt and FoxM1 in inducing cell growth inhibition and apoptosis in prostate cancer cells

Abstract

Although many studies have been done to uncover the mechanisms by which down-regulation of Notch-1 exerts its anti-tumor activity against a variety of human malignancies, the precise molecular mechanisms remain unclear. In the present study, we investigated the cellular consequence of Notch-1 down-regulation and also assessed the molecular consequence of Notch-1-mediated alterations of its downstream targets on cell viability and apoptosis in prostate cancer (PCa) cells. We found that the down-regulation of Notch-1 led to the inhibition of cell growth and induction of apoptosis, which was mechanistically linked with down-regulation of Akt and FoxM1, suggesting for the first time that Akt and FoxM1 are downstream targets of Notch-1 signaling. Moreover, we found that a "natural agent" (genistein) originally discovered from soybean could cause significant reduction in cell viability and induced apoptosis of PCa cells, which was consistent with down-regulation of Notch-1, Akt, and FoxM1. These results suggest that down-regulation of Notch-1 by novel agents could become a newer approach for the prevention of tumor progression and/or treatment, which is likely to be mediated via inactivation of Akt and FoxM1 signaling pathways in PCa.

Fig. 1

Notch signaling pathway in PCa cell lines. The baseline expression of Notch signaling pathway was determined in a panel of PCa cell lines using real-time RT-PCR (A,C) and Western blotting analysis (B,D), respectively.

Fig. 2

Down-regulation of Akt inhibited FoxM1. A: The baseline expression of FoxM1 was determined between a panel of PCa cell lines using real-time RT-PCR and Western blotting analysis, respectively. B: Down-regulation of Akt by siRNA inhibited FoxM1 expression, whereas up-regulation of Akt by cDNA plasmid transfection resulted in increased expression of FoxM1. Akt siRNA inhibited cell growth, while Akt cDNA transfection promoted cell growth. C: Inactivation of Akt by PI3K inhibitors (LY294002, Wortmanin) inhibited the expression of pAkt, which was consistent with decreased expression of FoxM1 as assessed by Western blot analysis.

Fig. 3

Inhibition of FoxM1 expression by Notch-1 siRNA and GSI. A: The expression of FoxM1 was detected by Western blotting analysis (left panel) and real-time RT-PCR (right panel) in PCa cells transfected with Notch-1 siRNA. B: The expression of FoxM1 was detected by Western blotting analysis in PCa cells treated with GSI for 72 h. C: The expression of FoxM1 was detected by real-time RT-PCR (left panel) and Western blotting analysis (right panel) in PC-3 ICN cells. D: The PC-3 and PC-3 ICN cells were subjected to immunofluorescent staining using anti-FoxM1 antibody. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 4

Inhibition of Notch, Akt, and FoxM1 expression by genistein. A: PCa cells were treated with varied concentrations of genistein for 72 h. Left panel: The expression of Notch, pAkt, and FoxM1 protein was detected by Western blotting analysis. Middle and right panel: Notch-1 mRNA and FoxM1 mRNA were detected by real-time RT-PCR. B: Immunofluorescent staining showing lower levels of FoxM1 protein in the cytoplasm and nucleus in the genistein-treated PC-3 cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 5

Down-regulation of Notch-1 by siRNA promotes genistein-induced cell growth inhibition and apoptosis in PC-3 cells. Genistein: 30 µM genistein; NS: Notch-1 siRNA; ICN: ICN cDNA; genistein+siRNA: 30 mM genistein+Notch-1 siRNA; genistein +ICN: 30 mM genistein+ ICN cDNA. A: Left panel: Down-regulation of Notch-1 by siRNA significantly inhibited PC-3 cell growth. Genistein plus Notch-1 siRNA inhibited cell growth to a greater degree compared to genistein alone. Right panel: Down-regulation of Notch-1 expression significantly increased apoptosis induced by genistein. Notch-1 siRNA-transfected cells were significantly more sensitive to spontaneous and genistein-induced apoptosis. B: Over-expression of Notch-1 expression significantly promoted cell growth. Over-expression of Notch-1 rescued cells from genistein-induced cell growth inhibition. Over-expression of Notch-1 by Notch-1 cDNA transfection abrogated genistein-induced apoptosis to a certain degree. C: The expression of FoxM1 was detected by Western blotting analysis in PCa cells treated with different concentrations of taxotere for 72 h. D: Left panel: Genistein synergize with taxotere leading to enhanced suppression of cell growth as assessed by MTT assay. Right panel: Genistein combined with taxotere led to synergistic induction of apoptotic cell death.

Fig. 6

Genistein inhibited tumor growth in animal model. A: Inhibitory effects of genistein on the growth of tumors formed by PC-3 or C4-2B cells in SCID-human mice (control, n = 7; genistein, n = 7). Comparison of the tumor volumes in each group on the day when all mice were sacrificed (*P < 0.05, genistein vs. control). B: Genistein inhibited the expression of Notch-1, pAkt, and FoxM1 in tumor remnants as assessed by Western blot analysis. C: The schematic presentation of our proposed mechanism of how genistein inhibits cell growth and induces apoptosis.