Emodin Suppresses the Migration and Invasion of Melanoma Cells.

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), as an active ingredient in rhubarb roots and rhizomes, has been reported to possess various pharmacological properties including anti-tumor effects. Recent studies have confirmed that emodin inhibited cell proliferation and induced apoptosis of cancer cells. However, the inhibitory effect of emodin on the migration and invasion of melanoma cells and its underlying mechanism are still unclear. In the study, we observed the impercipient effects of emodin in B16F10 and A375 melanoma cells with strong metastatic abilities, focusing on the functions and mechanisms of migration and invasion of B16F10 and A375 melanoma cells. Cell counting kit-8 (CCK-8), colony formation test and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining tests confirmed that emodin possessed anti-proliferative and pro-apoptotic activities in B16F10 and A375 cells. The inhibitory effects on the migration and invasion of B16F10 and A375 cells were proved by wound healing assay and Transwell methods. Moreover, immunofluorescence assay approved the decrease in protein expression of matrix metalloproteinas (MMP)-2/-9 by emodin, and Western blot analyses revealed that emodin could increase the Bax/Bcl-2 ratio and inhibit the MMP-2/-9 protein expression and Wnt/ß-catenin pathway in a dose-depended manner. BML-284, as an agonist of Wnt/ß-catenin signaling pathway, reversed the effects of emodin on cell growth, migration and invasion in B16F10 cells. These findings may suggest that emodin treatment can be a promising therapeutic strategy for melanoma with highly metastatic abilities.

Liuwei Dihuang pill suppresses metastasis by regulating the wnt pathway and disrupting -catenin/T cell factor interactions in a murine model of triple-negative breast cancer.

OBJECTIVE: To investigate if the Liuwei Dihuang pill (LWDHP) can inhibit metastasis to the liver and lungs in mice bearing triple-negative breast cancer (TNBC), and the molecular mechanism underpinning this action. METHODS: Ninety-nine TNBC bearing-mice were distributed randomly to five groups: control (Con), paclitaxel (PTX), low-dose LWDHP (LLP, 2.3 g·kg－1·d－1), middle-dose LWDHP (MLP, 4.6 g·kg－1·d－1) and high-dose LWDHP (HLP, 9.2 g·kg－1·d－1). The LWDHP were administered (p.o.) to the agonal stage. The morphology of BC cells was observed by hematoxylin & eosin staining. Expression of axin-2, ß-catenin, T cell factor (TCF), cyclin- D1 and vascular endothelial growth factor (VEGF) was detected by western blotting or immunofluorescence. ß-catenin/TCF-1 interaction was measured using a co-immunoprecipitation assay. RESULTS: After LWDHP treatment, metastasis of BC cells to the lungs and liver was inhibited, expression of axin-2 was increased, expression of TCF-1, ß-catenin, cyclin-D1 and VEGF was decreased, and ß-catenin/TCF-1 interaction was disrupted. CONCLUSION: The LWDHP could inhibit metastasis of BC cells to the liver and lungs. The molecular mechanism underlying this action may be regulation of protein expression and ß-catenin/TCF-1 interactions in the Wnt pathway.

Cell-based assay for low- and high-scale screening of the Wnt/ß-catenin signaling modulators.

Deregulation of the Wnt/ß-catenin signaling pathway is associated with many serious disorders, including cancer and Alzheimer's disease. The pivotal player is ß-catenin, which avoids degradation after activation of the pathway and is translocated to the nucleus, where it interacts with TCF/LEF transcription factors and induces expression of genes involved in cell cycle and apoptosis regulation. The identification of small molecules that may affect Wnt/ß-catenin signaling remains an important target during the development of novel therapies. We used the TCF/LEF lentiviral vector and the Wnt-independent H1703 cell line to develop a luciferase reporter-based cell assay for screening of the Wnt/ß-catenin pathway modulators. Following the optimization of cell density, concentration of activator, and stimulation time, the reporter system was validated by demonstrating its specific and dose-dependent response to several established modulators of Wnt/ß-catenin signaling such as Wnt3a, small interfering RNA (siRNA) against ß-catenin, glycogen synthase kinase 3 (GSK-3), and ß-catenin/TCF transcription complex inhibitors. Two pilot screens of inhibitors and activators of Wnt/ß-catenin signaling identified potential novel modulators of this pathway. Our findings suggest that the H1703-7TFP assay constitutes a suitable model of low background and high sensitivity for the low- and high-scale screening of the Wnt/ß-catenin pathway modulators.

Emodin suppresses Wnt signaling in human colorectal cancer cells SW480 and SW620.

Wnt signaling is involved in the regulation of cell proliferation, differentiation and apoptosis. Its aberrant activation is a key event in the pathogenesis and progression of human colorectal cancers. Dietary phytochemicals are gaining importance as chemotherapeutic agents owing to their potential to prevent, delay or reverse oncogenesis. Here we demonstrate that emodin (1,3,8-trihydroxy-6-methylanthraquinone), an anthraquinone present in the roots and bark of several medicinal plants, down regulates Wnt signaling pathway in human colorectal cancer cells (SW480 and SW620) by down regulating TCF/LEF transcriptional activity. Emodin significantly down regulated the expression of key players of Wnt signaling (ß-catenin and TCF7L2) and also that of its various downstream targets (cyclin D1, c-Myc, snail, vimentin, MMP-2 and MMP-9). Two novel targets of emodin׳s action were discovered namely Wnt co-activator p300 (down regulated) and repressor HBP1 (up regulated). Morphological changes induced by emodin suggest mesenchymal to epithelial transition accompanied by the increase in E-cadherin expression in human colorectal cancer cells but a differentiation marker (alkaline phosphatase) was activated only in SW620 cells (metastatic origin) and not in SW480 cells (primary tumor-derived). Moreover, our data indicate that reactive oxygen species plays a key role in emodin-mediated down regulation of Wnt signaling as emodin-mediated inhibition of migration and induction of growth arrest were partially rescued by the reactive oxygen species scavenger ascorbic acid. Effects of emodin shown in this study may provide important insights for the use of this anthraquinone as a potential complementary and integrated medicine for the treatment of human colorectal cancer.

γ-Tocotrienol inhibits cell viability through suppression of ß-catenin/Tcf signaling in human colon carcinoma HT-29 cells.

γ-Tocotrienol, a major component of the tocotrienol-rich fraction of palm oil, has been suggested to have antioxidant and anticancer activity as well as potent chemopreventive effects on tumor cells. In this study, the mechanisms underlying γ-tocotrienol-mediated growth inhibition of human carcinoma HT-29 cells were further investigated, especially in correlation with the involvement of ß-catenin/T-cell factor (Tcf) signaling pathway. We found that γ-tocotrienol could strongly suppress the transcriptional activity of ß-catenin/Tcf signaling pathway in HT-29 cells. γ-Tocotrienol inhibited the expression level of total ß-catenin protein but did not significantly affect the phosphorylated ß-catenin level. Meanwhile, γ-tocotrienol down-regulated the protein level of nuclear ß-catenin and induced its redistribution to cell membrane. Furthermore, γ-tocotrienol suppressed the expression of downstream target genes such as c-myc, cyclin D1 and survivin. The results demonstrated that γ-tocotrienol-inhibited growth and -induced apoptosis in HT-29 cells were accompanied by significant inhibition of ß-catenin/Tcf signaling. Blocking the expression of ß-catenin with small interfering RNA significantly suppressed the ability of γ-tocotrienol to reduce viability and induce apoptosis in HT-29 cells. Thus, our data suggested that γ-tocotrienol exerts its anticancer activity through ß-catenin/Tcf signaling, and ß-catenin is a target for γ-tocotrienol in the Wnt/ß-catenin signaling pathway.

Inhibition of Wnt signaling by cucurbitacin B in breast cancer cells: reduction of Wnt-associated proteins and reduced translocation of galectin-3-mediated ß-catenin to the nucleus.

The cucurbitacins are tetracyclic triterpenes found in plants of the family Cucurbitaceae. Cucurbitacins have been shown to have anti-cancer and anti-inflamatory activities. We investigated the anti-cancer activity of cucurbitacin B extracted from Thai medicinal plant Trichosanthes cucumerina Linn. Cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Results indicated that cucurbitacin B from T. cucumerina Linn. has a cytotoxic effect on breast cancer cell lines SKBR-3 and MCF-7 with an IC50 of 4.60 and 88.75 µg/ml, respectively. Growth inhibition was attributed to G2/M phase arrest and apoptosis. Cyclin D1, c-Myc, and ß-catenin expression levels were reduced. Western blot analysis showed increased PARP cleavage and decreased Wnt-associated signaling molecules ß-catenin, galectin-3, cyclin D1 and c-Myc, and corresponding changes in phosphorylated GSK-3ß levels. Cucurbitacin B treatment inhibited translocation to the nucleus of ß-catenin and galectin-3. The depletion of ß-catenin and galectin-3 in the nucleus was confirmed by cellular protein fractionation. T-cell factor (TCF)/lymphoid enhancer factor (LEF)-dependent transcriptional activity was disrupted in cucurbitacin B treated cells as tested by a TCF reporter assay. The relative luciferase activity was reduced when we treated cells with cucurbitacin B compound for 24 h. Our data suggest that cucurbitacin B may in part induce apoptosis and exert growth inhibitory effect via interruption the Wnt signaling.

Protein-bound polysaccharide from Phellinus linteus inhibits tumor growth, invasion, and angiogenesis and alters Wnt/ß-catenin in SW480 human colon cancer cells.

BACKGROUND: Polysaccharides extracted from the Phellinus linteus (PL) mushroom are known to possess anti-tumor effects. However, the molecular mechanisms responsible for the anti-tumor properties of PL remain to be explored. Experiments were carried out to unravel the anticancer effects of PL. METHODS: The anti-cancer effects of PL were examined in SW480 colon cancer cells by evaluating cell proliferation, invasion and matrix metallo-proteinase (MMP) activity. The anti-angiogenic effects of PL were examined by assessing human umbilical vein endothelial cell (HUVEC) proliferation and capillary tube formation. The in vivo effect of PL was evaluated in an athymic nude mouse SW480 tumor engraft model. RESULTS: PL (125-1000 µg/mL) significantly inhibited cell proliferation and decreased ß-catenin expression in SW480 cells. Expression of cyclin D1, one of the downstream-regulated genes of ß-catenin, and T-cell factor/lymphocyte enhancer binding factor (TCF/LEF) transcription activity were also significantly reduced by PL treatment. PL inhibited in vitro invasion and motility as well as the activity of MMP-9. In addition, PL treatment inhibited HUVEC proliferation and capillary tube formation. Tumor growth of SW480 cells implanted into nude mice was significantly decreased as a consequence of PL treatment, and tumor tissues from treated animals showed an increase in the apoptotic index and a decrease in ß-catenin expression. Moreover, the proliferation index and microvessel density were significantly decreased. CONCLUSIONS: These data suggest that PL suppresses tumor growth, invasion, and angiogenesis through the inhibition of Wnt/ß-catenin signaling in certain colon cancer cells.

Periplocin from Cortex periplocae inhibits cell growth and down-regulates survivin and c-myc expression in colon cancer in vitro and in vivo via beta-catenin/TCF signaling.

Cancer of the colon and rectum is the third most commonly diagnosed cancer and accounts for approximately 10% of all cancer-related deaths. Although surgical resection or radiotherapy are potentially curative for localized disease, advanced colon cancer is currently associated with poor prognosis. Therefore, the development of a new and effective chemotherapeutic agent is required to target critical pathways to induce responsiveness of colon cancer cells to death signals. Dysregulation of the beta-catenin/TCF pathway plays a central role in early activities of colorectal carcinogenesis. In this study, human colon cancer SW480 cells were used to investigate the effect of CPP (periplocin from Cortex periplocae) on the modulation of the beta-catenin/TCF signaling pathway. Our research results showed that CPP caused a dose- and time-dependent inhibition of cell growth as assessed by MTT assay and an induction in apoptosis as measured by flow cytometry and transmission electron microscopy. Furthermore, the CPP- treated cells were characterized by a decreased expression of beta-catenin protein in the total cell lysates and cytosolic and nuclear extracts. This expression alleviates the binding activity of T-cell factor (Tcf) complexes to its specific DNA-binding sites. Thus, the protein expression of the downstream elements survivin and c-myc was down-regulated. To determine the precise inhibitory mechanisms involved, further in-depth in vivo studies of CPP are warranted. In conclusion, our data suggest that CPP wields a multi-prong strategy to target the beta-catenin/Tcf signaling pathway, leading to the induction of apoptosis and inhibition of growth of colon cancer cells in vitro and in vivo. Therefore, CPP may become a potential agent against colon cancer.

Dehydroepiandrosterone administration or G{alpha}q overexpression induces {beta}-catenin/T-Cell factor signaling and growth via increasing association of estrogen receptor-{beta}/Dishevelled2 in androgen-independent prostate cancer cells.

beta-Catenin/T-cell factor signaling (beta-CTS) plays multiple critical roles in carcinogenesis and is blocked by androgens in androgen receptor (AR)-responsive prostate cancer (PrCa) cells, primarily via AR sequestration of beta-catenin from T-cell factor. Dehydroepiandrosterone (DHEA), often used as an over-the-counter nutritional supplement, is metabolized to androgens and estrogens in humans. The efficacy and safety of unregulated use of DHEA are unclear. We now report that DHEA induces beta-CTS via increasing association of estrogen receptor (ER)-beta with Dishevelled2 (Dvl2) in AR nonresponsive human PrCa DU145 cells, a line of androgen-independent PrCa (AiPC) cells. The induction is temporal, as assessed by measuring kinetics of the association of ERbeta/Dvl2, protein expression of the beta-CTS targeted genes, c-Myc and cyclin D1, and cell growth. However, in PC-3 cells, another human AiPC cell line, DHEA exerts no detectible effects, partly due to their lower expression of Galpha-subunits and DHEA down-regulation of ERbeta/Dvl2 association. When Galphaq is overexpressed in PC-3 cells, beta-CTS is constitutively induced, including increasing c-Myc and cyclin D1 protein expression. This effect involved increasing associations of Galphaq/Dvl2 and ERbeta/Dvl2 and promoted cell growth. These activities require ERbeta in DU-145 and PC-3 cells because they are blocked by ICI 182-780 treatment inactivating ERbeta, small interfering RNA administration depleting ERbeta, or AR overexpression arresting ERbeta. These data suggest that novel pathways activating beta-CTS play roles in the progression of AiPC. Although DHEA may enhance PrCa cell growth via androgenic or estrogenic pathways, the effects of DHEA administration on clinical prostate function remain to be determined.

Retinoic acid and Wnt/beta-catenin have complementary roles in anterior/posterior patterning embryos of the basal chordate amphioxus.

A role for Wnt/beta-catenin signaling in axial patterning has been demonstrated in animals as basal as cnidarians, while roles in axial patterning for retinoic acid (RA) probably evolved in the deuterostomes and may be chordate-specific. In vertebrates, these two pathways interact both directly and indirectly. To investigate the evolutionary origins of interactions between these two pathways, we manipulated Wnt/beta-catenin and RA signaling in the basal chordate amphioxus during the gastrula stage, which is the RA-sensitive period for anterior/posterior (A/P) patterning. The results show that Wnt/beta-catenin and RA signaling have distinctly different roles in patterning the A/P axis of the amphioxus gastrula. Wnt/beta-catenin specifies the identity of the ends of the embryo (high Wnt = posterior; low Wnt = anterior) but not intervening positions. Thus, upregulation of Wnt/beta-catenin signaling induces ectopic expression of posterior markers at the anterior tip of the embryo. In contrast, RA specifies position along the A/P axis, but not the identity of the ends of the embryo-increased RA signaling strongly affects the domains of Hox expression along the A/P axis but has little or no effect on the expression of either anterior or posterior markers. Although the two pathways may both influence such things as specification of neuronal identity, interactions between them in A/P patterning appear to be minimal.

Anticancer activity of Panax notoginseng extract 20(S)-25-OCH3-PPD: Targetting beta-catenin signalling.

1. The Wnt/beta-catenin pathway plays a critical role in carcinogenesis and so agents that target Wnt/beta-catenin may have potential in cancer prevention and therapy. The aim of the present study was to evaluate the anticancer activity of the novel natural product dammarane-type triterpene sapogenin (20(S)-25-OCH3-PPD; PPD25) isolated from the leaves of Panax notoginseng. 2. The anticancer activity of PPD25 was evaluated in three colon cancer cell lines and in one lung cancer cell line. The effects of PPD25 to inhibit proliferation and to induce apoptosis were evaluated. In addition, the potential mechanisms underlying the effects of PPD25 were investigated. 3. It was found that the addition of 5 or 25 micromol/L PPD25 to the culture medium significantly inhibited cell proliferation and induced apoptosis in all four cancer cell lines. Mechanistic studies revealed that PPD25 significantly reduced the expression of beta-catenin, a key mediator in the Wnt pathway, as well as transcriptional targets of beta-catenin, namely c-myc, cyclin D1, cdk4 and T cell factor (TCF)-4. In addition, beta-catenin/TCF transcriptional activity was significantly suppressed by PPD25. 4. The data demonstrate that the PPD25 exerts its anticancer effect by targetting beta-catenin signalling, suggesting that PPD25 may have potential as a chemotherapeutic and/or chemopreventive agent for colon and lung cancer.

Identification of Wnt-responsive cells in the zebrafish hypothalamus.

In all vertebrate brains, there is a period of widespread embryonic neurogenesis followed by specific regional neurogenesis that continues into adult stages. The Wnt signaling pathway, which is essential for numerous developmental processes, has also been suggested to be involved in neurogenesis. To help investigate the exact roles of canonical Wnt signaling in neurogenesis, here we examine the identity of Wnt-responsive cells in the zebrafish hypothalamus. This tissue is a useful diencephalic neurogenesis model containing evolutionarily conserved populations of neurons. We first performed in situ hybridization to show the expression patterns of Tcf family members and a canonical Wnt signaling reporter in the 50 hpf embryonic hypothalamus and larval/adult hypothalamus. We then used immunohistochemistry to identify the cell types of Wnt-responsive and Lef1-positive cells in both 50 hpf embryonic and adult hypothalamus. Our results indicate that Wnt-responsive cells in the hypothalamus are likely to be both mitotic progenitors and postmitotic precursors at embryonic stages, but only precursors at the adult stage. These data suggest that canonical Wnt signaling may be functionally required for maintenance of neural progenitor and precursor pools in the embryo, and for ongoing neurogenesis in the adult zebrafish.

Polyunsaturated fatty acids modulate the effect of TCF7L2 gene variants on postprandial lipemia.

The transcription factor 7-like 2 (TCF7L2) has been recently associated with diabetes risk, and it may exert its effect through metabolic syndrome (MetS)-related traits and be subjected to modification by environmental factors. We investigated the effect of single nucleotide polymorphisms (SNP), rs7903146 and rs12255372, within the TCF7L2 locus on postprandial lipemia and other MetS-related traits and their modulation by dietary fat. Data were collected from 1083 European Americans participating in the Genetics of Lipid Lowering Drugs and Diet Network Study. Carriers of the minor T allele at the C/T rs7903146 SNP had higher fasting plasma glucose (P = 0.012), lower homeostasis model assessment of beta cell function (P = 0.041), higher plasma VLDL (P = 0.035), and lower large LDL particle (P = 0.007) concentrations and higher risk of MetS (P = 0.011) than CC individuals. Moreover, we identified significant interactions between this SNP and PUFA intake modulating fasting VLDL particle concentrations (P = 0.016) and postprandial triglycerides (TG) (P = 0.028), chylomicrons (P = 0.025), total VLDL (P = 0.026), and large VLDL (P = 0.018) concentrations. Thus, only T allele carriers with a PUFA intake > or = 7.36% of energy had elevated fasting plasma VLDL concentrations and postprandial TG-rich lipoproteins. These variables did not differ in T allele carriers and noncarriers in the low-PUFA intake group. Moreover, these significant interactions were due exclusively to (n-6) PUFA intake. In summary, high (n-6) PUFA intakes (> or = 6.62% of energy intake) were associated with atherogenic dyslipidemia in carriers of the minor T allele at the TCF7L2 rs7903146 SNP and may predispose them to MetS, diabetes, and cardiovascular disease.

Lysosomal trafficking of beta-catenin induced by the tea polyphenol epigallocatechin-3-gallate.

beta-Catenin is a cadherin-binding protein involved in cell-cell adhesion, which also functions as a transcriptional activator when complexed in the nucleus with members of the T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of proteins. There is considerable interest in mechanisms that down-regulate beta-catenin, since this provides an avenue for the prevention of colorectal and other cancers in which beta-catenin is frequently over-expressed. We show here that physiologically relevant concentrations of the tea polyphenol epigallocatechin-3-gallate (EGCG) inhibited beta-catenin/TCF-dependent reporter activity in human embryonic kidney 293 cells transfected with wild type or mutant beta-catenins, and there was a corresponding decrease in beta-catenin protein levels in the nuclear, cytosolic and membrane-associated fractions. However, beta-catenin accumulated as punctate aggregates in response to EGCG treatment, including in human colon cancer cells over-expressing beta-catenin endogenously. Confocal microscopy studies revealed that the aggregated beta-catenin in HEK293 cells was extra-nuclear and co-localized with lysosomes, suggesting that EGCG activated a pathway involving lysosomal trafficking of beta-catenin. Lysosomal inhibitors leupeptin and transepoxysuccinyl-l-leucylamido(4-guanido)butane produced an increase in beta-catenin protein in total cell lysates, without a concomitant increase in beta-catenin transcriptional activity. These data provide the first evidence that EGCG facilitates the trafficking of beta-catenin into lysosomes, presumably as a mechanism for sequestering beta-catenin and circumventing further nuclear transport and activation of beta-catenin/TCF/LEF signaling.