• The Journal of Korean Medicine
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• v.32 no.3
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• pp.35-43
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• 2011

Objectives: This study was performed to screen target-specific inhibitors of ${\beta}$-catenin/TCF signaling whose functional activation plays an important role in early events in carcinogenesis. Methods: To investigate the activation or suppression of ${\beta}$-catenin/TCF transcription, we established a transiently transfected cell line with a constitutively active ${\beta}$-catenin mutant gene whose product is not degraded. This cell line was also co-transfected with luciferase reporter gene constructs containing either an optimized (TOPflash) or mutant (FOPflash) TCF-binding element. We investigated cytotoxic effects using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay. To find effective inhibitors of ${\beta}$-catenin/TCF signaling from medicinal herbs, the crude extracts of 99 types of medicinal herbs were screened using a luciferase assay system in HEK-293 and SH-SY5y cells. Results: At a concentration of $50{\mu}g$/ml, extracts of Angelica koreanae radix, Cannabis sativa semen, Ephedrae intermedia Schrenk radix, and Vitis rotundifolia fruit showed the following inhibitory effects on ${\beta}$-catenin/TCF signaling: $40{\pm}5.6%$, $23{\pm}6.1%$, $8{\pm}5.1%$, and $22{\pm}9.8%$ in ${\beta}$-catenin-activated HEK-293 cells and $9{\pm}4.7%$, $39{\pm}8.1%$, $39{\pm}6.4%$, and $42{\pm}10.1%$ in ${\beta}$-catenin-activated SH-SY5y cells, respectively. Crude extracts of E. radix were isolated by silica gel column chromatography, and two non-polar fractions of these extracts showed inhibitory effects on ${\beta}$-catenin/TCF signaling. Conclusions: In this study, we established a transiently transfected cell line as a screening system and found that various medicinal herb extracts had inhibitory effects on ${\beta}$signaling.

• The Zoological Society Korea : Newsletter
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• v.18 no.1
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• pp.16-25
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• 2001

Wnt signaling의 주요 분자인 $\beta$-catenin의 기능과 조절에 관한 연구, 특히 TCF family 단백질과 함께 작용하는 신호전달에 관한 연구가 최근에 활발히 진행되고 있다. $\beta$-catenin 단백질은 Drosophila나 Xenopus의 발생초기에 중요한 역할을 한다는 것이 알려져 있고 Wnt (Wingless) 단백질에 의하여 활성화되는 신호전달 과정에 관여한다고 알려져 있으므로, TCF 단백질들이 Wnt signalling pathway에 작용한다는 것을 의미한다. 즉, $\beta$-catenin/TCF complex는 발생초기의 세포의 운명을 결정하는 세포의 분화에 중요하리라 생각된다. 또한 $\beta$-catenin/TCF complex는 세포의 암화에도 중요하다는 것이 보고되었다. 정상세포의 경우, $\beta$-catenin은 APC 라는 tumor suppressor에 의하여 결합하고 단백질의 분해가 유도되어 핵 안의 TCF와 결합하지 못하는데, 암세포의 경우 APC가 결실되었거나 $\beta$-catenin의 양이 과도하게 발현되어 암세포화 되는 것으로 보인다. 즉, $\beta$-catenin은 일종의 oncogene으로 작용하는 단백질이며, 그 작용에 필수적인 전사인자가 TCF라는 것이다. 특히, 대장암세포에서 이 $\beta$-catenin/TCF complex에 의해 활성화되는 유전자로서 c-myc과 cyclin Dl 등이 있는 것으로 보아, $\beta$-catenin/TCF 단백질은 세포의 증식 및 사멸에 관여하는 단백질들의 발현을 조절하는 매우 중요한 인자라고 생각된다.

• Biomolecules & Therapeutics
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• v.24 no.4
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• pp.380-386
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• 2016

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-${\kappa}B$-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 ${\beta}$-catenin/TCF. Silymarin treatment resulted in a decrease of intracellular ${\beta}$-catenin protein but not mRNA. The inhibition of proteasome by MG132 and $GSK3{\beta}$ inhibition by SB216763 blocked silymarin-mediated downregulation of ${\beta}$-catenin. In addition, silymarin increased phosphorylation of ${\beta}$-catenin and a point mutation of S33Y attenuated silymarin-mediated ${\beta}$-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 ${\beta}$-catenin and TCF4 may result in the inhibition of Wnt signaling in human colorectal cancer cells.

• Animal cells and systems
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• v.4 no.4
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• pp.389-394
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• 2000

The Tcf-1 (1-cell factor-1) protein binds to the T-cell specific enhancer sequences and plays an architectural role in the assembly of transcriptional machinery. One of the Tcf family proteins, Tcf-4, was found to be an important regulator for colon cancer development where it activates specific genes upon binding to $\beta$-catenin following Wnt signaling. We were interested in the transcriptional regulatory activities of Tcf-1 and Tcf-4 proteins in T-cells and colon cancer cells. Transactivation assay was developed using a reporter plasmid containing luciferase gene under the control of Tcf responsive elements. Luciferase activity was determined following co-transfection of the reporter along with Tcf-1 and/or $\beta$-catenin expressing plasmids. Transcription was significantly induced by $\beta$-catenin expression in all cells. Tcf-1 by itself did not induce transcription in the mature T-cell lines, but overexpressed Tcf-1 greatly activated transcription in the immature T-cell line. In addition, transfected $\beta$-catenin induced apoptosis, but co-transfected Tcf-1 suppressed apoptosis in HEK293 cells. These results suggest that Tcf-1 and $\beta$-catenin differently regulate transcription and apoptosis.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2001.11a
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• pp.108-108
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• 2001

Axin2/Conductin/Axil and its ortholog Axin are negative regulators of the Wnt signaling pathway, which promote the phosphorylation and degradation of ${\beta}$-catenin. While Axin is expressed ubiquitously, Axin2 mRNA was seen in a restricted pattern during mouse embryogenesis and organogenesis. Because many sites of Axin2 expression overlapped with those of several Wnt genes, we tested whether Axin2 was induced by Wnt signaling. Endogenous Axin2 mRNA and protein expression could be rapidly induced by activation of the Wnt pathway, and Axin2 reporter constructs, containing a 5.6 kb DNA fragment including the promoter and first intron, were also induced. This genomic region contains eight Tcf/LEF consensus binding sites, five of which are located within longer, highly conserved non-coding sequences. The mutation or deletion of these Tcf/LEF sites greatly diminished induction by ${\beta}$-catenin, and mutation of the Tcf/LEF site T2 abolished protein binding in an electrophoretic mobility-shift assay. These results strongly suggest that Axin2 is a direct target of the Wnt pathway, mediated through Tcf/LEF factors. The 5.6 kb genomic sequence was sufficient to direct the tissue specific expression of d2EGFP in transgenic embryos, consistent with a role for the Tcf/LEF sites and surrounding conserved sequences in the in vivo expression pattern of Axin2. Our results suggest that Axin2 participates in a negative feedback loop, which could serve to limit the duration or intensity of a Wnt-initiated signal.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.15
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• pp.6549-6556
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• 2015

The PI3K-Akt-mTOR, $Wnt/{\beta}$-catenin and apoptosis signaling pathways have been shown to be involved in genesis of colorectal cancer (CRC). The aim of this study was to elucidate whether combination of Gelam honey and ginger might have chemopreventive properties in HT29 colon cancer cells by modulating the mTOR, $Wnt/{\beta}$-catenin and apoptosis signaling pathways. Treatment with Gelam honey and ginger reduced the viability of the HT29 cells dose dependently with $IC_{50}$ values of 88 mg/ml and 2.15 mg/ml respectively, their while the combined treatment of 2 mg/ml of ginger with 31 mg/ml of Gelam honey inhibited growth of most HT29 cells. Gelam honey, ginger and combination induced apoptosis in a dose dependent manner with the combined treatment exhibiting the highest apoptosis rate. The combined treatment downregulated the gene expressions of Akt, mTOR, Raptor, Rictor, ${\beta}$-catenin, $Gsk3{\beta}$, Tcf4 and cyclin D1 while cytochrome C and caspase 3 genes were shown to be upregulated. In conclusion, the combination of Gelam honey and ginger may serve as a potential therapy in the treatment of colorectal cancer through inhibiton of mTOR, $Wnt/{\beta}$ catenin signaling pathways and induction of apoptosis pathway.

• Molecules and Cells
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• v.39 no.11
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• pp.821-826
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• 2016

The ${\beta}$-catenin functions as an adhesion molecule and a component of the Wnt signaling pathway. In the absence of the Wnt ligand, ${\beta}$-catenin is constantly phosphorylated, which designates it for degradation by the APC complex. This process is one of the key regulatory mechanisms of ${\beta}$-catenin. The level of ${\beta}$-catenin is also controlled by the E3 ubiquitin protein ligase SIAH-1 via a phosphorylation-independent degradation pathway. Similar to ${\beta}$-catenin, STAT3 is responsible for various cellular processes, such as survival, proliferation, and differentiation. However, little is known about how these molecules work together to regulate diverse cellular processes. In this study, we investigated the regulatory relationship between STAT3 and ${\beta}$-catenin in HEK293T cells. To our knowledge, this is the first study to report that ${\beta}$-catenin-TCF-4 transcriptional activity was suppressed by phosphorylated STAT3; furthermore, STAT3 inactivation abolished this effect and elevated activated ${\beta}$-catenin levels. STAT3 also showed a strong interaction with SIAH-1, a regulator of active ${\beta}$-catenin via degradation, which stabilized SIAH-1 and increased its interaction with ${\beta}$-catenin. These results suggest that activated STAT3 regulates active ${\beta}$-catenin protein levels via stabilization of SIAH-1 and the subsequent ubiquitin-dependent proteasomal degradation of ${\beta}$-catenin in HEK293T cells.

• Food Science and Preservation
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• v.24 no.4
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• pp.524-528
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• 2017

Ricinus communis, belongs to the family Euphorbiaceae, has been known as medicinal plants for treatment of inflammation, tumors, antidiabetic, hepatoprotective and laxative. Compared to many pharmacological studies, the effect of R. communis extract on regulating adipogenesis as therapeutic drug for treating obesity has not been reported. R. communis extract (RCE) was investigated to determine its effects on the adipogenesis by monitoring the status of $Wnt/{\beta}-catenin$ signaling and factors involving the differentiation of adipocytes. The differentiation of 3T3-L1 cells monitored by Oil Red O staining was inhibited in concentration dependent manner by RCE. The luciferase activity of HEK 293-TOP cells containing pTOPFlash with Tcf4 response element-luciferase gene was increased approximately 2-folds by the treatment of RCE at concentrations of $100{\mu}g/mL$ compared to the control. Activation of the $Wnt/{\beta}-catenin$ pathway by RCE was further confirmed by immunocytochemical analysis which shows an increment of nuclear localization of ${\beta}-catenin$. In addition, safety of RCE was verified through performing neural stem cell morphology assay. Among the identified flavonoids in RCE, isoquercitrin was the most abundant. Therefore, these results indicate that the adipocyte differentiation was significantly reduced by isoquercitrin in R. communis. In this study, RCE suppresses the adipogenesis of 3T3-L1 cells via the activation of $Wnt/{\beta}-catenin$ signaling.

• Biomolecules & Therapeutics
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• v.28 no.5
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• pp.465-472
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• 2020

Colorectal cancer (CRC) is one of the most malignant type of cancers and its incidence is steadily increasing, due to life style factors that include western diet. Abnormal activation of canonical Wnt/β-catenin signaling pathway plays an important role in colorectal carcinogenesis. Therefore, targeting Wnt/β-catenin signaling has been considered a crucial strategy in the discovery of small molecules for CRC. In the present study, we found that Nodosin, an ent-kaurene diterpenoid isolated from Isodon serra, effectively inhibits the proliferation of human colon cancer HCT116 cells. Mechanistically, Nodosin effectively inhibited the overactivated transcriptional activity of β-catenin/T-cell factor (TCF) determined by Wnt/β-catenin reporter gene assay in HEK293 and HCT116 cells. The expression of Wnt/β-catenin target genes such as Axin2, cyclin D1, and survivin were also suppressed by Nodosin in HCT116 cells. Further study revealed that a longer exposure of Nodosin induced the G₂/M phase cell cycle arrest and subsequently apoptosis in HCT116 cells. These findings suggest that the anti-proliferative activity of Nodosin in colorectal cancer cells might in part be associated with the regulation of Wnt/β-catenin signaling pathway.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2020.08a
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• pp.89-89
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• 2020

In this study, we evaluated the effect of the extracts from Lonicera caerulea leaves (LCLE), branches (LCBE) and fruits (LCFE) on the cell growth and migration in human colorectal cancer cells, HCT116 and SW480 cells. LCLE and LCBE dose- and time-dependently inhibited the proliferation of HCT116 and SW480 cells. However, LCFE did not affect the proliferation of HCT116 and SW480 cells. In addition, LCLE and LCBE dramatically cell migration and wound healing in HCT116 cells. LCLE and LCBE decreased β-catenin protein level but not mRNA level in HCT116 and SW480 cells. Furthermore, LCLE decreased TCF4 level in both protein and mRNA level in HCT116 and SW480 cells. However, LCBE decreased TCF4 protein level but not mRNA level in HCT116 and SW480 cells. Based on these findings, LCLE and LCBE may inhibit the cell proliferation and migration through blocking Wnt signaling activation in human colorectal cancer cells. Therefore, LCLE and LCBE may be a potential candidate for the development of chemopreventive or therapeutic agents for human colorectal cancer.