Munju Cho

Natural derivatives of curcumin attenuate the Wnt/β-catenin pathway through down-regulation of the transcriptional coactivator p300

Curcumin, a component of turmeric (Curcuma longa), has been reported to suppress beta-catenin response transcription (CRT), which is aberrantly activated in colorectal cancer. However, the effects of its natural analogs (demethoxycurcumin [DMC] and bisdemethoxycurcumin [BDMC]) and metabolite (tetrahydrocurcumin [THC]) on the Wnt/beta-catenin pathway have not been investigated. Here, we show that DMC and BDMC suppressed CRT that was activated by Wnt3a conditioned-medium (Wnt3a-CM) without altering the level of intracellular beta-catenin, and inhibited the growth of various colon cancer cells, with comparable potency to curcumin. Additionally, DMC and BDMC down-regulated p300, which is a positive regulator of the Wnt/beta-catenin pathway. Notably, THC also inhibited CRT and cell proliferation, but to a much lesser degree than curcumin, DMC, or BDMC, indicating that the conjugated bonds in the central seven-carbon chain of curcuminoids are essential for the inhibition of Wnt/beta-catenin pathway and the anti-proliferative activity of curcuminoids. Thus, our findings suggest that curcumin derivatives inhibit the Wnt/beta-catenin pathway by decreasing the amount of the transcriptional coactivator p300.

Hexachlorophene inhibits Wnt/beta-catenin pathway by promoting Siah-mediated beta-catenin degradation.

Aberrant activation of Wnt/β-catenin signaling and subsequent up-regulation of β-catenin response transcription (CRT) is a critical event in the development of human colon cancer. Thus, Wnt/β-catenin signaling is an attractive target for the development of anticancer therapeutics. In this study, we identified hexachlorophene as an inhibitor of Wnt/β-catenin signaling from cell-based small-molecule screening. Hexachlorophene antagonized CRT that was stimulated by Wnt3a-conditioned medium by promoting the degradation of β-catenin. This degradation pathway is Siah-1 and adenomatous polyposis colidependent, but glycogen synthase kinase-3β and F-box β-transducin repeat-containing protein-independent. In addition, hexachlorophene represses the expression of cyclin D1, which is a known β-catenin target gene, and inhibits the growth of colon cancer cells. Our findings suggest that hexachlorophene attenuates Wnt/β-catenin signaling through the Siah-1-mediated β-catenin degradation.

Protein-kinase-C-mediated β-catenin phosphorylation negatively regulates the Wnt/β-catenin pathway

Normally, the Wnt/β-catenin pathway controls developmental processes and homeostasis, but abnormal activation of this pathway is a frequent event during the development of cancer. The key mechanism in regulation of the Wnt/β-catenin pathway is the amino-terminal phosphorylation of β-catenin, marking it for proteasomal degradation. Here we present small-molecule-based identification of protein kinase C (PKC)-mediated β-catenin phosphorylation as a novel mechanism regulating the Wnt/β-catenin pathway. We used a cell-based chemical screen to identify A23187, which inhibits the Wnt/β-catenin pathway. PKC was activated by A23187 treatment and subsequently phosphorylated N-terminal serine (Ser) residues of β-catenin, which promoted β-catenin degradation. Moreover, the depletion of PKCα inhibited the phosphorylation and degradation of β-catenin. Therefore, our findings suggest that the PKC pathway negatively regulates the β-catenin level outside of the Wnt/β-catenin pathway.

Diclofenac attenuates Wnt/β-catenin signaling in colon cancer cells by activation of NF-κB

The dysregulation of Wnt/β‐catenin signaling and subsequent upregulation of β‐catenin response transcription (CRT) occur frequently in colon cancer cells. Non‐steroidal anti‐inflammatory drugs (NSAIDs) can repress CRT in colorectal cancer, but little is known about the mechanism of action. We show that the NSAID diclofenac inhibits Wnt/β‐catenin signaling without altering the level of β‐catenin protein and reduces the expression of β‐catenin/TCF‐dependent genes. Diclofenac induced the degradation of IκBα, which increased free nuclear factor κB (NF‐κB) in cells. Also, the ectopic expression of p65, which is a component of NF‐κB, suppressed CRT. Our findings suggest that diclofenac inhibits Wnt/β‐catenin signaling via the activation of NF‐κB in colon cancer cells.

Decursin suppresses human androgen-independent PC3 prostate cancer cell proliferation by promoting the degradation of beta-catenin.

Alterations in the Wnt/β-catenin pathway are associated with the development and progression of human prostate cancer. Decursin, a pyranocoumarin isolated from the Korean Angelica gigas root, inhibits the growth of androgen-independent human prostate cancer cells, but little is known about its mechanism of action. Using a cell-based screen, we found that decursin attenuates the Wnt/β-catenin pathway. Decursin antagonized β-catenin response transcription (CRT), which was induced with Wnt3a-conditioned medium and LiCl, by promoting the degradation of β-catenin. Furthermore, decursin suppressed the expression of cyclin D1 and c-myc, which are downstream target genes of β-catenin and thus inhibited the growth of PC3 prostate cancer cells. In contrast, decursinol, in which the (CH3)2–C=CH–COO–side chain of decursin is replaced with–OH, had no effect on CRT, the level of intracellular β-catenin, or PC3 cell proliferation. Our findings suggest that decursin exerts its anticancer activity in prostate cancer cells via inhibition of the Wnt/β-catenin pathway.

Galangin Suppresses the Proliferation of β-Catenin Response Transcription-Positive Cancer Cells by Promoting Adenomatous Polyposis Coli/Axin/Glycogen Synthase Kinase-3β-Independent β-Catenin Degradation

Galangin is a naturally occurring bioflavonoid with anticancer activity against certain human cancers, yet little is known about its mechanism of action. Here, we used a chemical biology approach to reveal that galangin suppresses β-catenin response transcription (CRT), which is aberrantly up-regulated in colorectal and liver cancers, by promoting the degradation of intracellular β-catenin. Inhibition of glycogen synthase kinase-3β (GSK-3β) activity or mutation of the GSK-3β-targeted sequence from β-catenin was unable to abrogate the galangin-mediated degradation of β-catenin. In addition, galangin down-regulated the intracellular β-catenin levels in cancer cells with inactivating mutations of adenomatous polyposis coli (APC) or Axin, which are components of the β-catenin destruction complex. Galangin repressed the expression of β-catenin/T-cell factor-dependent genes, such as cyclin D1 and c-myc, and thus inhibited the proliferation of CRT-positive cancer cells. Structure-activity data indicated that the major structural requirements for galangin-mediated β-catenin degradation are hydroxyl groups at positions 3, 5, and 7. Our findings suggest that galangin exerts its anticancer activity by promoting APC/Axin/GSK-3β-independent proteasomal degradation of β-catenin.

Murrayafoline A attenuates the Wnt/β-catenin pathway by promoting the degradation of intracellular β-catenin proteins

Molecular lesions in Wnt/beta-catenin signaling and subsequent up-regulation of beta-catenin response transcription (CRT) occur frequently during the development of colon cancer. To identify small molecules that suppress CRT, we screened natural compounds in a cell-based assay for detection of TOPFalsh reporter activity. Murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa, antagonized CRT that was stimulated by Wnt3a-conditioned medium (Wnt3a-CM) or LiCl, an inhibitor of glycogen synthase kinase-3beta (GSK-3beta), and promoted the degradation of intracellular beta-catenin without altering its N-terminal phosphorylation at the Ser33/37 residues, marking it for proteasomal degradation, or the expression of Siah-1, an E3 ubiquitin ligase. Murrayafoline A repressed the expression of cyclin D1 and c-myc, which is known beta-catenin/T cell factor (TCF)-dependent genes and thus inhibited the proliferation of various colon cancer cells. These findings indicate that murrayafoline A may be a potential chemotherapeutic agent for use in the treatment of colon cancer.

Bisindoylmaleimide I suppresses adipocyte differentiation through stabilization of intracellular β-catenin protein

The Wnt/beta-catenin signaling pathway plays important roles in cell differentiation. Activation of this pathway, likely by Wnt-10b, has been shown to inhibit adipogenesis in cultured 3T3-L1 preadipocytes and mice. Here we revealed that bisindoylmaleimide I (BIM), which is widely used as a specific inhibitor of protein kinase C (PKC), inhibits adipocyte differentiation through activation of the Wnt/beta-catenin signaling pathway. BIM increased beta-catenin responsive transcription (CRT) and up-regulated intracellular beta-catenin levels in HEK293 cells and 3T3-L1 preadipocytes. BIM significantly decreased intracellular lipid accumulation and reduced expression of important adipocyte marker genes including peroxisome-proliferator-activated receptor gamma (PPARgamma) and CAATT enhancer-binding protein alpha (C/EBPalpha) in 3T3-L1 preadipocytes. Taken together, our findings indicate that BIM inhibits adipogenesis by increasing the stability of beta-catenin protein in 3T3-L1 preadipocyte cells.

Casein Kinase 2 (CK2)-mediated Phosphorylation of Hsp90β as a Novel Mechanism of Rifampin-induced MDR1 Expression

Background: Rifampin is a representative inducer of P-gp, and the only known mechanism is binding between rifampin and PXR. Results: Rifampin directly activates CK2, which phosphorylates Hsp90β. Consequently, PXR increases, and P-gp expression is induced. Conclusion: A mechanism for inducing P-gp expression by rifampin exposure is newly identified. Significance: A strategy for overcoming P-gp-derived drug resistance is suggested. The P-glycoprotein (P-gp) encoded by the MDR1 gene is a drug-exporting transporter located in the cellular membrane. P-gp induction is regarded as one of the main mechanisms underlying drug-induced resistance. Although there is great interest in the regulation of P-gp expression, little is known about its underlying regulatory mechanisms. In this study, we demonstrate that casein kinase 2 (CK2)-mediated phosphorylation of heat shock protein 90β (Hsp90β) and subsequent stabilization of PXR is a key mechanism in the regulation of MDR1 expression. Furthermore, we show that CK2 is directly activated by rifampin. Upon exposure to rifampin, CK2 catalyzes the phosphorylation of Hsp90β at the Ser-225/254 residues. Phosphorylated Hsp90β then interacts with PXR, causing a subsequent increase in its stability, leading to the induction of P-gp expression. In addition, inhibition of CK2 and Hsp90β enhances the down-regulation of PXR and P-gp expression. The results of this study may facilitate the development of new strategies to prevent multidrug resistance and provide a plausible mechanism for acquired drug resistance by CK2-mediated regulation of P-gp expression.

CK2-mediated phosphorylation of Hsp90β as a novel mechanism of rifampin-induced MDR1 expression

Background: Rifampin is a representative inducer of P-gp, and the only known mechanism is binding between rifampin and PXR. Results: Rifampin directly activates CK2, which phosphorylates Hsp90β. Consequently, PXR increases, and P-gp expression is induced. Conclusion: A mechanism for inducing P-gp expression by rifampin exposure is newly identified. Significance: A strategy for overcoming P-gp-derived drug resistance is suggested. The P-glycoprotein (P-gp) encoded by the MDR1 gene is a drug-exporting transporter located in the cellular membrane. P-gp induction is regarded as one of the main mechanisms underlying drug-induced resistance. Although there is great interest in the regulation of P-gp expression, little is known about its underlying regulatory mechanisms. In this study, we demonstrate that casein kinase 2 (CK2)-mediated phosphorylation of heat shock protein 90β (Hsp90β) and subsequent stabilization of PXR is a key mechanism in the regulation of MDR1 expression. Furthermore, we show that CK2 is directly activated by rifampin. Upon exposure to rifampin, CK2 catalyzes the phosphorylation of Hsp90β at the Ser-225/254 residues. Phosphorylated Hsp90β then interacts with PXR, causing a subsequent increase in its stability, leading to the induction of P-gp expression. In addition, inhibition of CK2 and Hsp90β enhances the down-regulation of PXR and P-gp expression. The results of this study may facilitate the development of new strategies to prevent multidrug resistance and provide a plausible mechanism for acquired drug resistance by CK2-mediated regulation of P-gp expression.