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Inhibition of Wnt Signaling by Silymarin in Human Colorectal Cancer Cells.

Eo HJ, Park GH, Jeong JB - Biomol Ther (Seoul) (2016)

Bottom Line: Silymarin treatment resulted in a decrease of intracellular β-catenin protein but not mRNA.The inhibition of proteasome by MG132 and GSK3β inhibition by SB216763 blocked silymarin-mediated downregulation of β-catenin.In addition, silymarin decreased TCF4 and increased Axin expression in both protein and mRNA level.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioresource Sciences, Andong National University, Andong 36729, Republic of Korea.

ABSTRACT
Silymarin from milk thistle (Silybum marianum) has been reported to show an anti-cancer activity. In previous study, we reported that silymarin induces cyclin D1 proteasomal degradation through NF-κB-mediated threonine-286 phosphorylation. However, mechanism for the inhibition of Wnt signaling by silymarin still remains unanswered. Thus, we investigated whether silymarin affects Wnt signaling in human colorectal cancer cells to elucidate the additional anti-cancer mechanism of silymarin. Transient transfection with a TOP and FOP FLASH luciferase construct indicated that silymarin suppressed the transcriptional activity of β-catenin/TCF. Silymarin treatment resulted in a decrease of intracellular β-catenin protein but not mRNA. The inhibition of proteasome by MG132 and GSK3β inhibition by SB216763 blocked silymarin-mediated downregulation of β-catenin. In addition, silymarin increased phosphorylation of β-catenin and a point mutation of S33Y attenuated silymarin-mediated β-catenin downregulation. In addition, silymarin decreased TCF4 and increased Axin expression in both protein and mRNA level. From these results, we suggest that silymarin-mediated downregulation of β-catenin and TCF4 may result in the inhibition of Wnt signaling in human colorectal cancer cells.

No MeSH data available.


Related in: MedlinePlus

Decreased β-catenin level by silymarin treatment. (A) HCT116 and SW480 cells were treated with 0, 50 and 100 μM of silymarin for 24 h. Cell lysates were subjected to SDS-PAGE and then the Western blot was performed for β-catenin and actin. (B) HCT116 and SW480 cells were treated with 100 μM of silymarin for the indicated times. Cell lysates were subjected to SDS-PAGE and then the Western blot was performed for β-catenin and actin. (C) HCT116 and SW480 cells were treated with 0, 50 and 100 μM of silymarin for 24 h. total RNA was prepared after silymarin treatment for 24 h.
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f1-bt-24-380: Decreased β-catenin level by silymarin treatment. (A) HCT116 and SW480 cells were treated with 0, 50 and 100 μM of silymarin for 24 h. Cell lysates were subjected to SDS-PAGE and then the Western blot was performed for β-catenin and actin. (B) HCT116 and SW480 cells were treated with 100 μM of silymarin for the indicated times. Cell lysates were subjected to SDS-PAGE and then the Western blot was performed for β-catenin and actin. (C) HCT116 and SW480 cells were treated with 0, 50 and 100 μM of silymarin for 24 h. total RNA was prepared after silymarin treatment for 24 h.

Mentions: To test whether silymarin affects the protein levels of β-catenin, we performed Western blot in HCT116 and SW480 cells treated with 0, 50 and 100 μM of silymarin. As shown in Fig. 1A, silymarin treatment dose-dependently downregulated the protein levels of β-catenin compared with HCT116 and SW480 cells without silymarin treatment. In time-course experiment, β-catenin began to be decreased at 6 h after silymarin treatment in HCT116 and SW480 cells (Fig. 1B). To determine whether downregulation of β-catenin is responsible for transcriptional inhibition, we tested mRNA level in HCT116 and SW480 cells. As shown in Fig. 1C, mRNA level of β-catenin was not affected by silymarin treatment in HCT116 and SW480 cells. These results indicate that silymarin may downregulate the level of β-catenin through decreasing protein stability of β-catenin.


Inhibition of Wnt Signaling by Silymarin in Human Colorectal Cancer Cells.

Eo HJ, Park GH, Jeong JB - Biomol Ther (Seoul) (2016)

Decreased β-catenin level by silymarin treatment. (A) HCT116 and SW480 cells were treated with 0, 50 and 100 μM of silymarin for 24 h. Cell lysates were subjected to SDS-PAGE and then the Western blot was performed for β-catenin and actin. (B) HCT116 and SW480 cells were treated with 100 μM of silymarin for the indicated times. Cell lysates were subjected to SDS-PAGE and then the Western blot was performed for β-catenin and actin. (C) HCT116 and SW480 cells were treated with 0, 50 and 100 μM of silymarin for 24 h. total RNA was prepared after silymarin treatment for 24 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4930281&req=5

f1-bt-24-380: Decreased β-catenin level by silymarin treatment. (A) HCT116 and SW480 cells were treated with 0, 50 and 100 μM of silymarin for 24 h. Cell lysates were subjected to SDS-PAGE and then the Western blot was performed for β-catenin and actin. (B) HCT116 and SW480 cells were treated with 100 μM of silymarin for the indicated times. Cell lysates were subjected to SDS-PAGE and then the Western blot was performed for β-catenin and actin. (C) HCT116 and SW480 cells were treated with 0, 50 and 100 μM of silymarin for 24 h. total RNA was prepared after silymarin treatment for 24 h.
Mentions: To test whether silymarin affects the protein levels of β-catenin, we performed Western blot in HCT116 and SW480 cells treated with 0, 50 and 100 μM of silymarin. As shown in Fig. 1A, silymarin treatment dose-dependently downregulated the protein levels of β-catenin compared with HCT116 and SW480 cells without silymarin treatment. In time-course experiment, β-catenin began to be decreased at 6 h after silymarin treatment in HCT116 and SW480 cells (Fig. 1B). To determine whether downregulation of β-catenin is responsible for transcriptional inhibition, we tested mRNA level in HCT116 and SW480 cells. As shown in Fig. 1C, mRNA level of β-catenin was not affected by silymarin treatment in HCT116 and SW480 cells. These results indicate that silymarin may downregulate the level of β-catenin through decreasing protein stability of β-catenin.

Bottom Line: Silymarin treatment resulted in a decrease of intracellular β-catenin protein but not mRNA.The inhibition of proteasome by MG132 and GSK3β inhibition by SB216763 blocked silymarin-mediated downregulation of β-catenin.In addition, silymarin decreased TCF4 and increased Axin expression in both protein and mRNA level.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioresource Sciences, Andong National University, Andong 36729, Republic of Korea.

ABSTRACT
Silymarin from milk thistle (Silybum marianum) has been reported to show an anti-cancer activity. In previous study, we reported that silymarin induces cyclin D1 proteasomal degradation through NF-κB-mediated threonine-286 phosphorylation. However, mechanism for the inhibition of Wnt signaling by silymarin still remains unanswered. Thus, we investigated whether silymarin affects Wnt signaling in human colorectal cancer cells to elucidate the additional anti-cancer mechanism of silymarin. Transient transfection with a TOP and FOP FLASH luciferase construct indicated that silymarin suppressed the transcriptional activity of β-catenin/TCF. Silymarin treatment resulted in a decrease of intracellular β-catenin protein but not mRNA. The inhibition of proteasome by MG132 and GSK3β inhibition by SB216763 blocked silymarin-mediated downregulation of β-catenin. In addition, silymarin increased phosphorylation of β-catenin and a point mutation of S33Y attenuated silymarin-mediated β-catenin downregulation. In addition, silymarin decreased TCF4 and increased Axin expression in both protein and mRNA level. From these results, we suggest that silymarin-mediated downregulation of β-catenin and TCF4 may result in the inhibition of Wnt signaling in human colorectal cancer cells.

No MeSH data available.


Related in: MedlinePlus