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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

Contribution of β-catenin phosphorylation by silymarin to proteasomal degradation of β-catenin. (A) HCT116 cells were plated overnight and then treated with 100 μM of silymarin for the indicated times. (B) HCT116 cells were transfected with wild type β-catenin or S33Y β-catenin, and then treated with 100 μM of silymarin. (C) HCT116 cells were pretreated with a selective inhibitor of GSK3β, SB216763 for 2 h and then co-treated with 100 μM of silymarin for 1 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against p-β-catenin or β-catenin. Actin was used as internal control. *p<0.05 compared to cells without silymarin.
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f3-bt-24-380: Contribution of β-catenin phosphorylation by silymarin to proteasomal degradation of β-catenin. (A) HCT116 cells were plated overnight and then treated with 100 μM of silymarin for the indicated times. (B) HCT116 cells were transfected with wild type β-catenin or S33Y β-catenin, and then treated with 100 μM of silymarin. (C) HCT116 cells were pretreated with a selective inhibitor of GSK3β, SB216763 for 2 h and then co-treated with 100 μM of silymarin for 1 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against p-β-catenin or β-catenin. Actin was used as internal control. *p<0.05 compared to cells without silymarin.

Mentions: To test the effect of silymarin on the phosphorylation of β-catenin. As shown in Fig. 3A, β-catenin phosphorylation began to increase at 1 h in silymarin-treated HCT116 cell. To verify that β-catenin phosphorylation by silymarin results in β-catenin proteasomal degradation, HCT116 cells were transfected with HA-wild type β-catenin or S33Y β-catenin. As shown in Fig. 3B, silymarin induced β-catenin degradation in wild type β-catenin-transfected cells. However, it was partially attenuated in S33Y β-catenin-transfected cells. In addition, GSK3β inhibition by SB216763 reduced silymarin-mediated β-catenin phosphorylation in HCT116 cells (Fig. 3C). Overall, these data proposed that downregulation of β-catenin by silymarin depends on proteolytic proteasomal degradation via β-catenin phosphorylation.


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

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

Contribution of β-catenin phosphorylation by silymarin to proteasomal degradation of β-catenin. (A) HCT116 cells were plated overnight and then treated with 100 μM of silymarin for the indicated times. (B) HCT116 cells were transfected with wild type β-catenin or S33Y β-catenin, and then treated with 100 μM of silymarin. (C) HCT116 cells were pretreated with a selective inhibitor of GSK3β, SB216763 for 2 h and then co-treated with 100 μM of silymarin for 1 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against p-β-catenin or β-catenin. Actin was used as internal control. *p<0.05 compared to cells without silymarin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-bt-24-380: Contribution of β-catenin phosphorylation by silymarin to proteasomal degradation of β-catenin. (A) HCT116 cells were plated overnight and then treated with 100 μM of silymarin for the indicated times. (B) HCT116 cells were transfected with wild type β-catenin or S33Y β-catenin, and then treated with 100 μM of silymarin. (C) HCT116 cells were pretreated with a selective inhibitor of GSK3β, SB216763 for 2 h and then co-treated with 100 μM of silymarin for 1 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against p-β-catenin or β-catenin. Actin was used as internal control. *p<0.05 compared to cells without silymarin.
Mentions: To test the effect of silymarin on the phosphorylation of β-catenin. As shown in Fig. 3A, β-catenin phosphorylation began to increase at 1 h in silymarin-treated HCT116 cell. To verify that β-catenin phosphorylation by silymarin results in β-catenin proteasomal degradation, HCT116 cells were transfected with HA-wild type β-catenin or S33Y β-catenin. As shown in Fig. 3B, silymarin induced β-catenin degradation in wild type β-catenin-transfected cells. However, it was partially attenuated in S33Y β-catenin-transfected cells. In addition, GSK3β inhibition by SB216763 reduced silymarin-mediated β-catenin phosphorylation in HCT116 cells (Fig. 3C). Overall, these data proposed that downregulation of β-catenin by silymarin depends on proteolytic proteasomal degradation via β-catenin phosphorylation.

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