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Platycodin D Induces Tumor Growth Arrest by Activating FOXO3a Expression in Prostate Cancer in vitro and in vivo

View Article: PubMed Central - PubMed

ABSTRACT

Platycodin D (PD), a major saponin derived from Platycodin grandiflorum, exerted cytotoxicity against prostate cancer cell lines (PC3, DU145 and LNCaP cells) with IC50 values in the range of 11.17 to 26.13μmol/L, whereas RWPE-1cells (a non-malignant human prostate epithelial cell line) were not significantly affected. A further study in these cell lines showed that PD could potently affect cell proliferation (indicated by the bromodeoxyuridine assay), induce cell apoptosis (determined by Annexin V-FITC flow cytometry) and cause cell cycle arrest (indicated by PI staining). After being treated with PD for 48 hours, DU145 and LNCaP cells were arrested in the G0 /G1 phase, and PC3 cells were arrested in the G2/M phase. A Western blotting analysis indicated that PD increased the expression of the FOXO3a transcription factor, decreased the expression of p-FOXO3a and MDM2 and increased the expression of FOXO-responsive genes, p21 and p27. MDM2 silencing (transiently by siRNA-MDM2) increased the PD-induced FOXO3a protein expression, while MDM2 overexpression (in cells transiently transfected with a pcDNA3-MDM2 plasmid) decreased the PD-induced expression of the FOXO3a protein. Moreover, PD dose-dependently inhibited the growth of PC3 xenograft tumors in BALB/c nude mice. A Western blotting analysis of the excised xenograft tumors indicated that similar changes in protein expression also occurred in vivo. These results suggest that PD exhibits significant activity against prostate cancer in vitro and in vivo. The FOXO3a transcription factor appears to be involved in the activity of PD. Together, all of these findings provide a basis for the future development of this agent for human prostate cancer therapy.

No MeSH data available.


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PD downregulates MDM2 and increases FOXO3a expression in PC3 cells. A. PC3, LNCaP and DU145 cells were exposed to various concentrations of PD for 24 hr; then target proteins (MDM2, FOXO3a, p53, p21and p27) were examined by Western blotting (WB). B. The expression of the FOXO3a and p-FOXO3a proteins was examined by WB in PC3 cells after PD treatment for 24 hr. C. & D. PC3 cells were transfected with MDM2 siRNA or a pcDNA-MDM2 (full length) plasmid,then the expression levels of MDM2 and FOXO3a were determined by WB after incubation with or without 10 mM PD. Blocking MDM2 expression by siRNA increased the FOXO3a expression, while MDM2 overexpression decreased the FOXO3a protein expression. However, treatment with PD was able to increase the FOXO3a expression even in the presence of MDM2 overexpression. E. The mRNA expression levels of MDM2, FOXO3a, p21 and p27 were detected by real-time quantitative PCR, and the data were normalized to the mRNA level of GAPDH.
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Figure 4: PD downregulates MDM2 and increases FOXO3a expression in PC3 cells. A. PC3, LNCaP and DU145 cells were exposed to various concentrations of PD for 24 hr; then target proteins (MDM2, FOXO3a, p53, p21and p27) were examined by Western blotting (WB). B. The expression of the FOXO3a and p-FOXO3a proteins was examined by WB in PC3 cells after PD treatment for 24 hr. C. & D. PC3 cells were transfected with MDM2 siRNA or a pcDNA-MDM2 (full length) plasmid,then the expression levels of MDM2 and FOXO3a were determined by WB after incubation with or without 10 mM PD. Blocking MDM2 expression by siRNA increased the FOXO3a expression, while MDM2 overexpression decreased the FOXO3a protein expression. However, treatment with PD was able to increase the FOXO3a expression even in the presence of MDM2 overexpression. E. The mRNA expression levels of MDM2, FOXO3a, p21 and p27 were detected by real-time quantitative PCR, and the data were normalized to the mRNA level of GAPDH.

Mentions: The ERK-phosphorylated FOXO3a is degraded via an MDM2-mediated ubiquitin-proteasome pathway [21]. To examine the role of the FOXO3a and MDM2 interaction in PD-induced prostate cancer cells apoptosis, MDM2 was transiently silenced by siRNA. As shown in Fig. 4C, MDM2 silencing increased the PD-induced FOXO3a protein expression, possibly due to decreased MDM2-mediated FOXO3a degradation, and this suppressed the prostate cancer cell growth. As shown in Fig. 4D, the FOXO3a expression was suppressed by pcDNA3-MDM2 transfection. However, treatment with PD (10 μM) inhibited MDM2 activation, which was accompanied by an increase in the FOXO3a levels. Together, these results suggested that MDM2 suppressed FOXO3a, but treatment with PD could induce FOXO3a expression via the downregulation of MDM2 in prostate cancer cells.


Platycodin D Induces Tumor Growth Arrest by Activating FOXO3a Expression in Prostate Cancer in vitro and in vivo
PD downregulates MDM2 and increases FOXO3a expression in PC3 cells. A. PC3, LNCaP and DU145 cells were exposed to various concentrations of PD for 24 hr; then target proteins (MDM2, FOXO3a, p53, p21and p27) were examined by Western blotting (WB). B. The expression of the FOXO3a and p-FOXO3a proteins was examined by WB in PC3 cells after PD treatment for 24 hr. C. & D. PC3 cells were transfected with MDM2 siRNA or a pcDNA-MDM2 (full length) plasmid,then the expression levels of MDM2 and FOXO3a were determined by WB after incubation with or without 10 mM PD. Blocking MDM2 expression by siRNA increased the FOXO3a expression, while MDM2 overexpression decreased the FOXO3a protein expression. However, treatment with PD was able to increase the FOXO3a expression even in the presence of MDM2 overexpression. E. The mRNA expression levels of MDM2, FOXO3a, p21 and p27 were detected by real-time quantitative PCR, and the data were normalized to the mRNA level of GAPDH.
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Related In: Results  -  Collection

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Figure 4: PD downregulates MDM2 and increases FOXO3a expression in PC3 cells. A. PC3, LNCaP and DU145 cells were exposed to various concentrations of PD for 24 hr; then target proteins (MDM2, FOXO3a, p53, p21and p27) were examined by Western blotting (WB). B. The expression of the FOXO3a and p-FOXO3a proteins was examined by WB in PC3 cells after PD treatment for 24 hr. C. & D. PC3 cells were transfected with MDM2 siRNA or a pcDNA-MDM2 (full length) plasmid,then the expression levels of MDM2 and FOXO3a were determined by WB after incubation with or without 10 mM PD. Blocking MDM2 expression by siRNA increased the FOXO3a expression, while MDM2 overexpression decreased the FOXO3a protein expression. However, treatment with PD was able to increase the FOXO3a expression even in the presence of MDM2 overexpression. E. The mRNA expression levels of MDM2, FOXO3a, p21 and p27 were detected by real-time quantitative PCR, and the data were normalized to the mRNA level of GAPDH.
Mentions: The ERK-phosphorylated FOXO3a is degraded via an MDM2-mediated ubiquitin-proteasome pathway [21]. To examine the role of the FOXO3a and MDM2 interaction in PD-induced prostate cancer cells apoptosis, MDM2 was transiently silenced by siRNA. As shown in Fig. 4C, MDM2 silencing increased the PD-induced FOXO3a protein expression, possibly due to decreased MDM2-mediated FOXO3a degradation, and this suppressed the prostate cancer cell growth. As shown in Fig. 4D, the FOXO3a expression was suppressed by pcDNA3-MDM2 transfection. However, treatment with PD (10 μM) inhibited MDM2 activation, which was accompanied by an increase in the FOXO3a levels. Together, these results suggested that MDM2 suppressed FOXO3a, but treatment with PD could induce FOXO3a expression via the downregulation of MDM2 in prostate cancer cells.

View Article: PubMed Central - PubMed

ABSTRACT

Platycodin D (PD), a major saponin derived from Platycodin grandiflorum, exerted cytotoxicity against prostate cancer cell lines (PC3, DU145 and LNCaP cells) with IC50 values in the range of 11.17 to 26.13μmol/L, whereas RWPE-1cells (a non-malignant human prostate epithelial cell line) were not significantly affected. A further study in these cell lines showed that PD could potently affect cell proliferation (indicated by the bromodeoxyuridine assay), induce cell apoptosis (determined by Annexin V-FITC flow cytometry) and cause cell cycle arrest (indicated by PI staining). After being treated with PD for 48 hours, DU145 and LNCaP cells were arrested in the G0 /G1 phase, and PC3 cells were arrested in the G2/M phase. A Western blotting analysis indicated that PD increased the expression of the FOXO3a transcription factor, decreased the expression of p-FOXO3a and MDM2 and increased the expression of FOXO-responsive genes, p21 and p27. MDM2 silencing (transiently by siRNA-MDM2) increased the PD-induced FOXO3a protein expression, while MDM2 overexpression (in cells transiently transfected with a pcDNA3-MDM2 plasmid) decreased the PD-induced expression of the FOXO3a protein. Moreover, PD dose-dependently inhibited the growth of PC3 xenograft tumors in BALB/c nude mice. A Western blotting analysis of the excised xenograft tumors indicated that similar changes in protein expression also occurred in vivo. These results suggest that PD exhibits significant activity against prostate cancer in vitro and in vivo. The FOXO3a transcription factor appears to be involved in the activity of PD. Together, all of these findings provide a basis for the future development of this agent for human prostate cancer therapy.

No MeSH data available.


Related in: MedlinePlus