Quercetin, a potent inhibitor against β-catenin/Tcf signaling in SW480 colon cancer cells

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Abstract

Dysregulation of Wnt/β-catenin pathway plays a central role in early events in colorectal carcinogenesis. We examined the effect of quercetin, a famous anti-tumor agent, against β-catenin/Tcf signaling in SW480 cells. Quercetin inhibited the transcriptional activity of β-catenin/Tcf in SW480 and also in HEK293 cells transiently transfected with constitutively active mutant β-catenin gene, whose product is not induced to be degraded by APC–Axin–GSK3β complex, so we concluded that its inhibitory mechanism was related to β-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed EMSA showing that binding of the Tcf complexes to its specific DNA-binding sites was strongly suppressed by quercetin. Immunoprecipitation analysis also showed that the binding of β-catenin to Tcf-4 was also disrupted by quercetin. Western blot analysis proved these decreased bindings resulted from decreased level of β-catenin and Tcf-4 product in nucleus caused by quercetin. Together, we suggest that quercetin is an excellent inhibitor of β-catenin/Tcf signaling in SW480 cell lines, and the reduced β-catenin/Tcf transcriptional activity is due to the decreased nuclear β-catenin and Tcf-4 proteins.

Section snippets

Materials and methods

Cell lines and reagents. SW480 and HEK293 cell lines were derived from Korean Cell Line Bank (KCLB, Korea). Quercetin was purchased from Acros. Stock solutions (50 mM) were made in DMSO. TOPflash was provided by Hans Clevers via Bart O Williams, and wild (pcDNA β-catenin) and mutant β-catenin gene (pcDNA S33Y) were provided by Eric R. Fearon.

Isolation of cellular and nuclear extracts. Cells were trypsinized and whole cell protein was obtained by lysing the cells on ice for 20 min in 700 μl lysis

Quercetin downregulates the β-catenin/Tcf signaling

To investigate whether quercetin modulates β-catenin/Tcf signaling, we used SW480 human colon cancer cells (Fig. 2A) and HEK293 cells transiently transfected with wild β-catenin gene (Fig. 2B). The transcriptional activity of β-catenin/Tcf is constitutively active in SW480 cell line (mutant APC, wild-type CTNNB1, and COX-2 negative). We used reporter gene constructs containing four copies of either an optimized (TOPflash) or mutant (FOPflash) Tcf-binding element. SW480 cells were cotransfected

Discussion

Flavonoids constitute one of the largest groups of naturally occurring phenols and are commonly present in plants as glycosides. These compounds possess a common phenylbenzopyrone structure (C6–C3–C6), and they are categorized according to the saturation level and opening of the central pyran ring, mainly into flavones, flavanols, isoflavones, flavonols, flavanones, and flavanonols [21], [22]. These dietary anti-oxidants are well known to exert significant anti-tumor effects and have been

Acknowledgments

We thank Dr. H. Clevers for the gift of TOPflash. We also thank Dr. Eric R. Fearon for the wild (pcDNA β-catenin) and mutant β-catenin gene (pcDNA S33Y). This research was supported by a grant (KRF 2001-015-DP0344) from the Korean Ministry of Science and Technology. Financial support in part from the Brain Korea 21 program is gratefully acknowledged.

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