Study of Wnt2 secreted by A-549 cells in paracrine activation of β-catenin in co-cultured mesenchymal stem cells

The canonical Wnt signal pathway is a key regulator of self-renewal and cell fate determination in various types of stem cells. The total pool of β-catenin consists of two different forms: the signaling form of the protein transmits the Wnt signals from the cell membrane to the target genes, whereas the membrane β-catenin is involved in formation of cell-to-cell contact at cadherin junctions. Earlier we developed an in vitro model of epithelial differentiation of mesenchymal stem cells (MSCs) co-cultured with epithelial A-549 cells. The purpose of the present work was to study the role of Wnt2 secreted by the A-549 cells in paracrine induction of β-catenin in co-cultured MSCs. Using the somatic gene knockdown technique, we obtained A-549 cell cultures with down-regulated WNT2. The MSCs co-cultured with the control A-549 cells displayed an increase in the levels of total cellular and signaling β-catenin and transactivation of a reporter construction containing the Lef/Tcf protein family binding sites. In contrast, β-catenin was not induced in the MSCs co-cultured with the A-549 cells with down-regulated WNT2 expression, but the total protein level was increased. We suggest that Wnt2 secreted by A-549 cells induces in co-cultured MSCs the Wnt/β-catenin signaling pathway, whereas the associated increase in total β-catenin level should be due to another mechanism.     

Docosahexaenoic acid inhibited the Wnt/β-Catenin pathway and suppressed breast cancer cells in vitro and in vivo

N-3 fatty acids (FAs) are essential FAs necessary for human health and are known to possess anticancer properties. However, the relationship between n-3 FAs and β-catenin, one of the key components of the Wnt signaling pathway, in mouse breast cancer remains poorly characterized. In this study, 4T1 mouse breast cancer cells were exposed to a representative n-3 FA, docosahexaenoic acid (DHA), to investigate the relationship between n-3 FAs and the Wnt/β-catenin signaling pathway in vivo and in vitro. In vitro studies showed that DHA strongly inhibited cell growth, and induced G1 cell cycle arrest both in 4T1 mouse breast cells and MCF-7 human breast cells. DHA reduced β-catenin expression and T cell factor/lymphoid-enhancing factor reporter activity in 4T1 mouse breast cells. In addition, DHA down-regulated the expression of downstream target genes such as c-myc and cyclinD1. In vivo, therapy experiments were conducted on Babl/c mice bearing breast cancer. We found that feeding mouse the 5% fish oil-supplemented diet for 30 days significantly reduced the growth of 4T1 mouse breast cancer in vivo through inhibition of cancer cell proliferation as well as induction of apoptosis. Feeding animals a 5% fish oil diet significantly induced down-regulation of β-catenin in tumor tissues with a notable increase in apoptosis. In addition, fish oil-supplemented diet decreased lung metastases of breast cancer. These observations suggested that DHA exerted its anticancer activity through down-regulation of Wnt/β-catenin signaling. Thus, our data call for further studies to assess the effectiveness of fish oil as a dietary supplement in the prevention and treatment of breast cancer.     

Isoquercitrin Suppresses Colon Cancer Cell Growth in Vitro by Targeting the Wnt/β-Catenin Signaling Pathway

Flavonoids are plant-derived polyphenolic molecules that have potential biological effects including anti-oxidative, anti-inflammatory, anti-viral, and anti-tumoral effects. These effects are related to the ability of flavonoids to modulate signaling pathways, such as the canonical Wnt signaling pathway. This pathway controls many aspects of embryonic development and tissue maintenance and has been found to be deregulated in a range of human cancers. We performed several in vivo assays in Xenopus embryos, a functional model of canonical Wnt signaling studies, and also used in vitro models, to investigate whether isoquercitrin affects Wnt/β-catenin signaling. Our data provide strong support for an inhibitory effect of isoquercitrin on Wnt/β-catenin, where the flavonoid acts downstream of β-catenin translocation to the nuclei. Isoquercitrin affects Xenopus axis establishment, reverses double axes and the LiCl hyperdorsalization phenotype, and reduces Xnr3 expression. In addition, this flavonoid shows anti-tumoral effects on colon cancer cells (SW480, DLD-1, and HCT116), whereas exerting no significant effect on non-tumor colon cell (IEC-18), suggesting a specific effect in tumor cells in vitro. Taken together, our data indicate that isoquercitrin is an inhibitor of Wnt/β-catenin and should be further investigated as a potential novel anti-tumoral agent.     

Wnt/β-catenin regulates blood pressure and kidney injury in rats

Activation of the renin-angiotensin system (RAS) plays a pivotal role in mediating hypertension, chronic kidney and cardiovascular diseases. As Wnt/β-catenin regulates multiple RAS genes, we speculated that this developmental signaling pathway might also participate in blood pressure (BP) regulation. To test this, we utilized two rat models of experimental hypertension: chronic angiotensin II infusion and remnant kidney after 5/6 nephrectomy. Inhibition of Wnt/β-catenin by ICG-001 blunted angiotensin II-induced hypertension. Interestingly, angiotensin II was able to induce the expression of multiple Wnt genes in vivo and in vitro, thereby creating a vicious cycle between Wnt/β-catenin and RAS activation. In the remnant kidney model, renal β-catenin was upregulated, and delayed administration of ICG-001 also blunted BP elevation and abolished the induction of angiotensinogen, renin, angiotensin-converting enzyme and angiotensin II type 1 receptor. ICG-001 also reduced albuminuria, serum creatinine and blood urea nitrogen, and inhibited renal expression of fibronectin, collagen I and plasminogen activator inhibitor-1, and suppressed the infiltration of CD3⁺ T cells and CD68⁺ monocytes/macrophages. In vitro, incubation with losartan prevented Wnt/β-catenin-mediated fibronectin, α-smooth muscle actin and Snail1 expression, suggesting that the fibrogenic action of Wnt/β-catenin is dependent on RAS activation. Taken together, these results suggest an intrinsic linkage of Wnt/β-catenin signaling with BP regulation. Our studies also demonstrate that hyperactive Wnt/β-catenin can drive hypertension and kidney damage via RAS activation.     

Dishevelled-DEP domain interacting protein (DDIP) inhibits Wnt signaling by promoting TCF4 degradation and disrupting the TCF4/β-catenin complex

The TCF4/β-catenin complex, the executor of canonical Wnt/β-catenin signaling, is regulated by a variety of factors. Among these, Dishevelled (Dvl) is a critical regulator that releases β-catenin from degradation and stabilizes TCF4/β-catenin complex. Here, we report that DDIP (Dishevelled-DEP domain Interacting Protein, also named as Spats1, spermatogenesis associated, serine-rich 1), a novel protein that interacts with Dvl, regulates Wnt signaling. We provide evidence that DDIP suppresses Lef-1 luciferase reporter activity stimulated by Wnt1, Dvl2 or β-catenin, interacts with the TCF4/β-catenin complex, and disrupts the interaction of TCF4 and β-catenin by promoting TCF4 degradation through the proteasome pathway. Our results indicate that DDIP is a negative regulator of the canonical Wnt signaling.     

Analysis of the Wnt/B-catenin/TCF4 pathway using SAGE,genome-wide microarray and promoter analysis: Identification of BRI3 and HSF2 as novel targets

The Wnt signaling pathway is involved in many differentiation events during embryonic development and can lead to tumor formation after aberrant activation of its components. β-catenin, a cytoplasmic component, plays a major role in the transduction of canonical Wnt signaling. The aim of this study was to identify novel genes that are regulated by active β-catenin/TCF signaling in hepatocellular carcinoma-derived Huh7 cells with high (transfected) and low β-catenin/TCF activities. High TCF activity Huh7 cells led to earlier and larger tumor formation when xenografted into nude mice. SAGE (Serial Analysis of Gene Expression), genome-wide microarray and in silico promoter analysis were performed in parallel, to compare gene expression between low and high β-catenin/TCF activity clones, and also those that had been rescued from the xenograft tumors. SAGE and genome-wide microarray data were compared and contrasted. BRI3 and HSF2 were identified as novel targets of Wnt/β-catenin signaling after combined analysis and confirming experiments including qRT-PCR, ChIP, luciferase assay and lithium treatment.