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Fisetin inhibits migration and invasion of human cervical cancer cells by down-regulating urokinase plasminogen activator expression through suppressing the p38 MAPK-dependent NF-κB signaling pathway.

Chou RH, Hsieh SC, Yu YL, Huang MH, Huang YC, Hsieh YH - PLoS ONE (2013)

Bottom Line: The expression and activity of urokinase plasminogen activator (uPA) was significantly suppressed by fisetin in a dose-dependent manner.We also demonstrated that fisetin reduces the phosphorylation of p38 MAPK, but not that of ERK1/2, JNK1/2, or AKT.Furthermore, the promoter activity of the uPA gene was dramatically repressed by fisetin, which disrupted the nuclear translocation of NF-κB and its binding amount on the promoter of the uPA gene, and these suppressive effects could be further enhanced by SB203580.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Cancer Biology and Center for Molecular Medicine, China Medical University, Taichung, Taiwan.

ABSTRACT
Fisetin (3,3',4',7-tetrahydroxyflavone), a naturally occurring flavonoid, has been reported to inhibit proliferation and induce apoptosis in several cancer types. However, its effect on the anti-metastatic potential of cervical cancer cells remains unclear. In the present study, we found that fisetin inhibits the invasion and migration of cervical cancer cells. The expression and activity of urokinase plasminogen activator (uPA) was significantly suppressed by fisetin in a dose-dependent manner. We also demonstrated that fisetin reduces the phosphorylation of p38 MAPK, but not that of ERK1/2, JNK1/2, or AKT. Addition of a p38 MAPK inhibitor, SB203580, further enhanced the inhibitory effect of fisetin on the expression and activity of uPA and the invasion and motility in cervical cancer cells. Fisetin suppressed the TPA (tetradecanoylphorbol-13-acetate)-induced activation of p38 MAPK and uPA, and inhibited the TPA-enhanced migratory and invasive abilities. Furthermore, the promoter activity of the uPA gene was dramatically repressed by fisetin, which disrupted the nuclear translocation of NF-κB and its binding amount on the promoter of the uPA gene, and these suppressive effects could be further enhanced by SB203580. This study provides strong evidence for the molecular mechanism of fisetin in inhibiting the aggressive phenotypes by repression of uPA via interruption of p38 MAPK-dependent NF-κB signaling pathway in cervical cancer cells and thus contributes insight to the potential of using fisetin as a therapeutic strategy against cervical cancer by inhibiting migration and invasion.

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Effects of fisetin on the expression and activity of uPA in cervical cancer cells.Cells were treated with the indicated concentrations of fisetin for 48 h. (A) the conditioned medium from each treatment was collected, and uPA activity was determined by casein zymography. (B) Cell lysate was applied to determine the protein levels of uPA by Western blotting. β-actin was used as the internal control. (C) Cells were fixed, permeabilized, and immunostained with anti-uPA antibody (red), and cell nuclei were counter-stained with DAPI reagent. (D) Total RNA was extracted from each treatment, and the mRNA levels of uPA were examined by RT-PCR. Bars show the value as mean ± S.E. from three independent experiments. *, P < 0.05, **, P < 0.01, compared with the untreated cells.
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pone-0071983-g002: Effects of fisetin on the expression and activity of uPA in cervical cancer cells.Cells were treated with the indicated concentrations of fisetin for 48 h. (A) the conditioned medium from each treatment was collected, and uPA activity was determined by casein zymography. (B) Cell lysate was applied to determine the protein levels of uPA by Western blotting. β-actin was used as the internal control. (C) Cells were fixed, permeabilized, and immunostained with anti-uPA antibody (red), and cell nuclei were counter-stained with DAPI reagent. (D) Total RNA was extracted from each treatment, and the mRNA levels of uPA were examined by RT-PCR. Bars show the value as mean ± S.E. from three independent experiments. *, P < 0.05, **, P < 0.01, compared with the untreated cells.

Mentions: Previous studies have shown that an increase in uPA expression is associated with cervical cancer progression [36]. To investigate the possible underlying anti-metastatic effect of fisetin in cervical cancer cells, the activity and expression of uPA in cervical cancer cells treated with various concentrations of fisetin were examined. As shown in the caseinolytic activity assay, uPA activity decreased in a dose-dependent manner after treatment with fisetin. Quantification analysis indicated that uPA activity decreased by 17.2%, 62.3%, and 84.2% in SiHa cells, and by 37.5%, 79.4%, and 87.8% in CaSki cells when cells were treated with 10, 20, and 40 µM of fisetin, respectively (Figure 2A). Western blot analysis was performed to examine the protein expression of uPA in cervical cancer cells. Fisetin inhibited the protein expression of uPA in a dose-dependent manner, compared to the control group in both two cervical cancer lines tested (Figure 2B). In addition, to verify the down-regulation of uPA, immunofluorescent labeling was performed. The bright red fluorescence from uPA in the control cells shows a constitutive expression of uPA, whereas 40 µM of fisetin significantly decreased uPA protein expression (Figure 2C). These results indicate that fisetin inhibited both the activity and protein of uPA in cervical cancer cells. To further investigate whether the inhibitory effect of fisetin on the activity and protein of uPA in cervical cancer cells was at the level of mRNA expression, a semi-quantitative RT-PCR analysis was performed. As shown in Figure 2D, after the treatment of fisetin for 48 h, the mRNA level of uPA also decreased significantly in a dose-dependent manner, compared to the control group, in both SiHa and CaSki cells. The fisetin-mediated change in the mRNA levels of uPA coincided with the protein levels, as indicated by the results from the Western blot analysis, suggesting that fisetin might regulate uPA expression at transcription levels. These findings suggest that the anti-metastatic effect of fisetin is related to the inhibition of uPA expression in cervical cancer cells.


Fisetin inhibits migration and invasion of human cervical cancer cells by down-regulating urokinase plasminogen activator expression through suppressing the p38 MAPK-dependent NF-κB signaling pathway.

Chou RH, Hsieh SC, Yu YL, Huang MH, Huang YC, Hsieh YH - PLoS ONE (2013)

Effects of fisetin on the expression and activity of uPA in cervical cancer cells.Cells were treated with the indicated concentrations of fisetin for 48 h. (A) the conditioned medium from each treatment was collected, and uPA activity was determined by casein zymography. (B) Cell lysate was applied to determine the protein levels of uPA by Western blotting. β-actin was used as the internal control. (C) Cells were fixed, permeabilized, and immunostained with anti-uPA antibody (red), and cell nuclei were counter-stained with DAPI reagent. (D) Total RNA was extracted from each treatment, and the mRNA levels of uPA were examined by RT-PCR. Bars show the value as mean ± S.E. from three independent experiments. *, P < 0.05, **, P < 0.01, compared with the untreated cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3733924&req=5

pone-0071983-g002: Effects of fisetin on the expression and activity of uPA in cervical cancer cells.Cells were treated with the indicated concentrations of fisetin for 48 h. (A) the conditioned medium from each treatment was collected, and uPA activity was determined by casein zymography. (B) Cell lysate was applied to determine the protein levels of uPA by Western blotting. β-actin was used as the internal control. (C) Cells were fixed, permeabilized, and immunostained with anti-uPA antibody (red), and cell nuclei were counter-stained with DAPI reagent. (D) Total RNA was extracted from each treatment, and the mRNA levels of uPA were examined by RT-PCR. Bars show the value as mean ± S.E. from three independent experiments. *, P < 0.05, **, P < 0.01, compared with the untreated cells.
Mentions: Previous studies have shown that an increase in uPA expression is associated with cervical cancer progression [36]. To investigate the possible underlying anti-metastatic effect of fisetin in cervical cancer cells, the activity and expression of uPA in cervical cancer cells treated with various concentrations of fisetin were examined. As shown in the caseinolytic activity assay, uPA activity decreased in a dose-dependent manner after treatment with fisetin. Quantification analysis indicated that uPA activity decreased by 17.2%, 62.3%, and 84.2% in SiHa cells, and by 37.5%, 79.4%, and 87.8% in CaSki cells when cells were treated with 10, 20, and 40 µM of fisetin, respectively (Figure 2A). Western blot analysis was performed to examine the protein expression of uPA in cervical cancer cells. Fisetin inhibited the protein expression of uPA in a dose-dependent manner, compared to the control group in both two cervical cancer lines tested (Figure 2B). In addition, to verify the down-regulation of uPA, immunofluorescent labeling was performed. The bright red fluorescence from uPA in the control cells shows a constitutive expression of uPA, whereas 40 µM of fisetin significantly decreased uPA protein expression (Figure 2C). These results indicate that fisetin inhibited both the activity and protein of uPA in cervical cancer cells. To further investigate whether the inhibitory effect of fisetin on the activity and protein of uPA in cervical cancer cells was at the level of mRNA expression, a semi-quantitative RT-PCR analysis was performed. As shown in Figure 2D, after the treatment of fisetin for 48 h, the mRNA level of uPA also decreased significantly in a dose-dependent manner, compared to the control group, in both SiHa and CaSki cells. The fisetin-mediated change in the mRNA levels of uPA coincided with the protein levels, as indicated by the results from the Western blot analysis, suggesting that fisetin might regulate uPA expression at transcription levels. These findings suggest that the anti-metastatic effect of fisetin is related to the inhibition of uPA expression in cervical cancer cells.

Bottom Line: The expression and activity of urokinase plasminogen activator (uPA) was significantly suppressed by fisetin in a dose-dependent manner.We also demonstrated that fisetin reduces the phosphorylation of p38 MAPK, but not that of ERK1/2, JNK1/2, or AKT.Furthermore, the promoter activity of the uPA gene was dramatically repressed by fisetin, which disrupted the nuclear translocation of NF-κB and its binding amount on the promoter of the uPA gene, and these suppressive effects could be further enhanced by SB203580.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Cancer Biology and Center for Molecular Medicine, China Medical University, Taichung, Taiwan.

ABSTRACT
Fisetin (3,3',4',7-tetrahydroxyflavone), a naturally occurring flavonoid, has been reported to inhibit proliferation and induce apoptosis in several cancer types. However, its effect on the anti-metastatic potential of cervical cancer cells remains unclear. In the present study, we found that fisetin inhibits the invasion and migration of cervical cancer cells. The expression and activity of urokinase plasminogen activator (uPA) was significantly suppressed by fisetin in a dose-dependent manner. We also demonstrated that fisetin reduces the phosphorylation of p38 MAPK, but not that of ERK1/2, JNK1/2, or AKT. Addition of a p38 MAPK inhibitor, SB203580, further enhanced the inhibitory effect of fisetin on the expression and activity of uPA and the invasion and motility in cervical cancer cells. Fisetin suppressed the TPA (tetradecanoylphorbol-13-acetate)-induced activation of p38 MAPK and uPA, and inhibited the TPA-enhanced migratory and invasive abilities. Furthermore, the promoter activity of the uPA gene was dramatically repressed by fisetin, which disrupted the nuclear translocation of NF-κB and its binding amount on the promoter of the uPA gene, and these suppressive effects could be further enhanced by SB203580. This study provides strong evidence for the molecular mechanism of fisetin in inhibiting the aggressive phenotypes by repression of uPA via interruption of p38 MAPK-dependent NF-κB signaling pathway in cervical cancer cells and thus contributes insight to the potential of using fisetin as a therapeutic strategy against cervical cancer by inhibiting migration and invasion.

Show MeSH
Related in: MedlinePlus