Geoffroea decorticans fruit extracts inhibit the wnt/ß-catenin pathway, a therapeutic target in cancer.

Geoffroea decorticans (chañar) is commonly used for culinary and medicinal purposes in rural communities. The aim of this work was to chemically characterize three Geoffroea decorticans extracts and determine their capacity to modulate the wnt/ß-catenin pathway. This signaling pathway plays a key role in embryonic development but its overactivation leads to cancer cell growth. Phytochemical analysis of extracts showed presence of major classes of phytochemicals. Gas chromatography-mass spectrometry results revealed the presence of acids, esters and furanic compounds. Using Xenopus embryos as in vivo model organisms, we found that the extracts modulated dorso-ventral axis formation and rescued hyperdorsalized phenotypes produced by LiCl treatment. In agreement with these findings, Geoffroea decorticans extracts decreased ß-catenin levels and suppressed the expression of wnt target genes such as xnr3 and chordin, thus demonstrating an inhibitory regulation of the wnt/ß-catenin signaling pathway. All these results support a new role for Geoffroea decorticans fruit derivatives with possible anti-carcinogenic actions.

Androgens downregulate BMP2 impairing the inductive role of dermal papilla cells on hair follicle stem cells differentiation.

Hair follicle cyclical regeneration is regulated by epithelial-mesenchymal interactions. During androgenetic alopecia (AGA), hair follicle stem cells (HFSC) differentiation is impaired by deregulation of dermal papilla cells (DPC) secreted factors. We analyzed androgen influence on BMPs expression in DPC and their effect on HFSC differentiation to hair lineage. Androgens downregulated BMP2 and BMP4 in DPC spheroids. Addition of BMP2 restored alkaline phosphatase activity, marker of hair-inductivity in DPC, and DPC-induced HFSC differentiation, both inhibited by androgens. Concomitantly, in differentiating HFSC, an upregulation of BMPRIa and BMPRII receptors and nuclear ß-catenin accumulation, indicative of Wnt/ß-catenin pathway activation, were detected. Our results present BMP2 as an androgen-downregulated paracrine factor that contributes to DPC inductivity and favors DPC-induced HFSC differentiation to hair lineage, possibly through a crosstalk with Wnt/ß-catenin pathway. A comprehensive understanding of androgen-deregulated DPC factors and their effects on differentiating HFSC would help to improve treatments for AGA.

Acute and chronic lithium treatment increases Wnt/ß-catenin transcripts in cortical and hippocampal tissue at therapeutic concentrations in mice.

Lithium activates Wnt/ß-catenin signaling leading to stabilization of free cytosolic ß-catenin. The aim of the present study is to evaluate the in vivo effect of acute and chronic lithium treatment on the expression of ß-catenin target genes, addressing its transcripts HIG2, Bcl-xL, Cyclin D1, c-myc, in cortical and hippocampal tissue from adult mice. Lithium doses were established to yield therapeutic working concentrations. In acute treatment, mice received a 300µL of a 350 mg/kg solution of LiCl by gavage, and were euthanized after 2 h, 6 h and 12 h. To determine the effect of chronic treatment, animals were continuously fed either with chow supplemented with 2 g/kg Li2CO3, or regular chow (controls), being euthanized after 30 days. All animals had access to drinking water and 0.9% saline ad libitum. After acute and chronic treatments samples of peripheral blood were obtained from the tail vein for each animal, and serum concentrations of lithium were determined. All transcripts were up-regulated in cortical and hippocampal tissues of lithium-treated mice, both under acute and chronic treatments. There was a positive correlation between serum lithium concentrations and the increment in the expression of all transcripts. This effect was observed in all time points of the acute treatment (i.e., 2, 6 and 12 hours) and also after 30 days. We conclude that Wnt/ß-catenin transcriptional response (HIG2, Bcl-xL, Cyclin D1 and c-myc) is up-regulated in the mouse brain in response to acute and chronic lithium treatment at therapeutic concentrations.

Piperine suppresses the Wnt/ß-catenin pathway and has anti-cancer effects on colorectal cancer cells.

More than 94% of colorectal cancer cases have mutations in one or more Wnt/ß-catenin signaling pathway components. Inactivating mutations in APC or activating mutations in ß-catenin (CTNNB1) lead to signaling overactivation and subsequent intestinal hyperplasia. Numerous classes of medicines derived from synthetic or natural small molecules, including alkaloids, have benefited the treatment of different diseases, including cancer, Piperine is a true alkaloid, derived from lysine, responsible for the spicy taste of black pepper (Piper nigrum) and long pepper (Piper longum). Studies have shown that piperine has a wide range of pharmacological properties; however, piperine molecular mechanisms of action are still not fully understood. By using Wnt/ß-catenin pathway epistasis experiment we show that piperine inhibits the canonical Wnt pathway induced by overexpression of ß-catenin, ß-catenin S33A or dnTCF4 VP16, while also suppressing ß-catenin nuclear localization in HCT116 cell line. Additionally, piperine impairs cell proliferation and migration in HCT116, SW480 and DLD-1 colorectal tumor cell lines, while not affecting the non-tumoral cell line IEC-6. In summary, piperine inhibits the canonical Wnt signaling pathway and displays anti-cancer effects on colorectal cancer cell lines.

Estrogen Receptors Promote Migration, Invasion and Colony Formation of the Androgen-Independent Prostate Cancer Cells PC-3 Through ß-Catenin Pathway.

Prostate cancer is initially dependent on the androgen, gradually evolves into an androgen-independent form of the disease, also known as castration-resistant prostate cancer (CRPC). At this stage, current therapies scantily improve survival of the patient. Androgens and estrogens are involved in normal prostate and prostate cancer development. The mechanisms by which estrogens/estrogen receptors (ERs) induce prostate cancer and promote prostate cancer progression have not yet been fully identified. Our laboratory has shown that androgen-independent prostate cancer cells PC-3 express both ERα and ERß. The activation of ERß increases the expression of ß-catenin and proliferation of PC-3 cells. We now report that the activation of ERß promotes the increase of migration, invasion and anchorage-independent growth of PC-3 cells. Furthermore, the activation of ERα also plays a role in invasion and anchorage-independent growth of PC-3 cells. These effects are blocked by pretreatment with PKF 118-310, compound that disrupts the complex ß-catenin/TCF/LEF, suggesting that ERs/ß-catenin are involved in all cellular characteristics of tumor development in vitro. Furthermore, PKF 118-310 also inhibited the upregulation of vascular endothelial growth factor A (VEGFA) induced by activation of ERs. VEGF also is involved on invasion of PC-3 cells. In conclusion, this study provides novel insights into the signatures and molecular mechanisms of ERß in androgen-independent prostate cancer cells PC-3. ERα also plays a role on invasion and colony formation of PC-3 cells.

Bushenhuoxue formula promotes osteogenic differentiation of growth plate chondrocytes through ß-catenin-dependent manner during osteoporosis.

BACKGROUND: Bushenhuoxue formula (BSHXF) has shown excellent clinical effects on the treatment of osteoporosis in China. The aim of this study is to determine the anti-osteoporosis effects and precise molecular mechanisms of BSHXF on mouse models. METHODS: Ten-week-old female C57BL/6 J mice were subjected to ovariectomy and provided a daily treatment of BSHXF. At 8 weeks post-surgery, the femurs were harvested for tissue analyses including µCT, histology, qRT-PCR and immunohistochemical (IHC) staining of ß-catenin, ALP and FABP4. To investigate the role of ß-catenin in the anti-osteoporosis effects of BSHXF, relative experiments mentioned above were performed in ß-catenin conditional knockout mice. RESULTS: Ovariectomized (OVX) mice presented severe bone loss and excessive fat accumulation in the chondro-osseous junction underneath the growth plate, with decreased expression of ALP and increased expression of FABP4. BSHXF significantly recovered the OVX-induced abnormal osteogenesis and adipogenesis with the activation of ß-catenin in growth plate chondrocytes. Further, we generated growth plate chondrocyte-specific ß-catenin knockout (ß-cateninGli1ER) mice that exhibited bone loss and fat accumulation in the chondro-osseous junction, similar to the OVX mice. However, BSHXF failed to rescue the osteoporosis-like phenotype in ß-cateninGli1ER mice, indicating the anti-osteoporosis effects of BSHXF act mainly through ß-catenin signaling. No significant restoration of ALP and FABP4 was observed in ß-cateninGli1ER mice after the treatment of BSHXF. CONCLUSIONS: BSHXF attenuates osteoporosis by promoting osteogenic differentiation of growth plate chondrocytes mainly in ß-catenin-dependent manner. BSHXF is considered as a new candidate for the treatment of osteoporosis.

Canonical and non-canonical Wnt signaling are simultaneously activated by Wnts in colon cancer cells.

The Wnt signaling pathway is a crucial regulator of the intestinal epithelium homeostasis and is altered in most colon cancers. While the role of aberrant canonical, ß-catenin-dependent Wnt signaling has been well established in colon cancer promotion, much less is known about the role played by noncanonical, ß-catenin-independent Wnt signaling in this type of cancer. This work aimed to characterize the noncanonical signal transduction pathway in colon cancer cells. To this end, we used the prototype noncanonical ligand, Wnt5a, in comparison with Wnt3a, the prototype of a canonical ß-catenin activating ligand. The analysis of the expression profile of Wnt receptors in colon cancer cell lines showed a clear increase in both level expression and variety of Frizzled receptor types expressed in colon cancer cells compared with non-malignant cells. We found that Wnt5a activates a typical Wnt/Ca++ - noncanonical signaling pathway in colon malignant cells, inducing the hyperphosphorylation of Dvl1, Dvl2 and Dvl3, promoting Ca++ mobilization as a result of phospholipase C (PLC) activation via pertussis toxin-sensitive G-protein, and inducing PLC-dependent cell migration. We also found that while the co-receptor Ror2 tyrosine kinase activity is not required for Ca++ mobilization-induced by Wnt5a, it is required for the inhibitory effects of Wnt5a on the ß-catenin-dependent transcriptional activity. Unexpectedly, we found that although the prototype canonical Wnt3a ligand was unique in stimulating the ß-catenin-dependent transcriptional activity, it also simultaneously activated PLC, promoted Ca++ mobilization, and induced Rho kinase and PLC-dependent cell migration. Our data indicate, therefore, that a Wnt ligand can activate at the same time the so-called Wnt canonical and noncanonical pathways inducing the formation of complex signaling networks to integrate both pathways in colon cancer cells.

In vitro studies revealed a downregulation of Wnt/ß-catenin cascade by active vitamin D and TX 527 analog in a Kaposi's sarcoma cellular model.

The Kaposi's sarcoma-associated herpesvirus G-protein-coupled receptor (vGPCR) is a key molecule in the pathogenesis of Kaposi's sarcoma. We have previously demonstrated that 1α,25(OH)2D3 or its less calcemic analog TX 527 exerts antiproliferative effects in endothelial cells stable expressing vGPCR. Since it is well documented that vGPCR activates the canonical Wnt/ß-catenin signaling pathway, the aim of this study was to evaluate if Wnt/ß-catenin cascade is target of 1α,25(OH)2D3 or TX 527 as part of their antineoplastic mechanism. Firstly, Western blot studies showed an increase in ß-catenin protein levels in a dose and time dependent manner; and when VDR was knockdown, ß-catenin protein levels were significantly decreased. Secondly, ß-catenin localization, investigated by immunofluorescence and subcellular fractionation techniques, was found increased in the nucleus and plasma membrane after 1α,25(OH)2D3 treatment. VE-cadherin protein levels were also increased in the plasma membrane fraction. Furthermore, ß-catenin interaction with VDR was observed by co-immunoprecipitation and mRNA expression of ß-catenin target genes was found decreased. Finally, DKK-1, the extracellular inhibitor of Wnt/ß-catenin pathway, showed an initial upregulation of mRNA expression. Altogether, the results obtained by different techniques revealed a downregulation of Wnt/ß-catenin cascade after 1α,25(OH)2D3 or TX 527 treatment, showing the foundation for a potential chemotherapeutic agent.

In Silico Identification of Potential Inhibitors of the Wnt Signaling Pathway in Human Breast Cancer.

Triple-negative breast cancer is the leading worldwide cause of cancer-related deaths in women. The prospection and development of new substances with antitumoral potential is of great importance for the treatment of this disease. The objective of this work was to identify a commercial drug or ligand that could potentially bind to the FZD7 transmembrane protein and inactivate the Wnt signaling pathway in triple-negative breast cancer cells. We aimed at computationally modeling the FZD7, Wnt3, and Wnt3a proteins, at making them available in protein model databases, and at conducting docking analysis to assess the binding free energy between FZD7 and the selected ligands. The Wnt3 and Wnt3a proteins were modeled by homology modeling, and the FZD7 protein was modeled by homology modeling and ab initio modeling. The ligands were selected based on their similarity to the palmitoleic acid and were gathered from the ZINC database. A total of 30 commercially available ligands were found in the ZINC database. The docking results show that the ligands zinc08221009, zinc13546050, zinc05260769, zinc04529321, and zinc05972969 are good candidates for novel drug development. The created models and conducted analysis by this work will most certainly help in future research on the Wnt signaling pathway and its components.

The Wnt/ß-catenin signaling pathway is regulated by titanium with nanotopography to induce osteoblast differentiation.

Wnt/ß-catenin signal transduction is involved in the homeostatic control of bone mass. It is well established that a titanium surface with nanotopography (Ti-Nano) favors osteoblast differentiation by modulating different signaling pathways. However, few studies have investigated the participation of the Wnt/ß-catenin pathway in the osteogenic effect of nanoscale topographies. In this study, we aimed to determine whether the Wnt/ß-catenin signaling pathway is involved in the elevated osteogenic potential of Ti-Nano. MC3T3-E1 cells were cultured on Ti-Nano and machined Ti (Ti-Control) for evaluation of the expression of Wnt/ß-catenin signaling pathway-related genes. Based on the results to real-time PCR, the Wnt receptor Fzd4 was selected and silenced by CRISPRi. The resulting cells were cultured on both Ti surfaces, and several events involved in osteoblast differentiation were evaluated. The results revealed that Fzd4 gene silencing, corresponding to negative modulation of Wnt/ß-catenin, inhibits expression of the osteoblast phenotype. It is worthy of note that this inhibitory effect on osteoblast differentiation was more pronounced in cells grown on Ti-Nano compared with those grown on Ti-Control. By disrupting Fzd4 gene expression, we have shown that the elevated osteogenic potential of Ti-Nano is due to activation of the Wnt/ß-catenin signaling pathway, which reveals a new mechanism to explain osteoblast differentiation induced by nanotopography. Such an understanding of the intracellular machinery involved in surface guiding of osteoblast fate may contribute to the development of smart biomaterials to modulate the process of implant osseointegration.

Inhibitory effect of Astragalus polysaccharide on osteoporosis in ovariectomized rats by regulating FoxO3a /Wnt signaling pathway.

PURPOSE: To investigate inhibitory effect of Astragalus polysaccharide (APS) on osteoporosis in ovariectomized rats by regulating FoxO3a/Wnt2 signaling pathway. METHODS: Postmenopausal osteoporosis (PMOP) animal model was developed by excising the bilateral ovaries of rats. The model rats were administered with APS (200 mg/kg, 400 mg/kg, 800 mg/kg) by intragastric administration once daily for 12 weeks. Bone density, bone metabolism index and oxidative stress index were measured in all groups. Furthermore, the regulation of APS of FoxO3a / Wnt2 signaling pathway was observed. RESULTS: APS has an estrogen-like effect, which can increase bone mass, lower serum ALP and BGP values, increase blood calcium content, and increase bone density of the femur and vertebrae in rats. At the same time, APS can increase the bone mineral content of the femur, increase the maximum stress, maximum load and elastic modulus of the ovariectomized rats, improve oxidative stress in rats by increasing the gene expression of ß-catenin and Wnt2 mRNA and inhibiting the gene expression of FoxO3a mRNA. CONCLUSION: Astragalus polysaccharide can effectively alleviate oxidative stress-mediated osteoporosis in ovariectomized rats, which may be related to its regulation of FoxO3a/Wnt2/ß-catenin pathway.

Convergence of Wnt and Notch signaling controls ovarian cancer cell survival.

In the last 40 years ovarian cancer mortality rates have slightly declined and, consequently, it continues to be the fifth cause of cancer death in women. In the present study, we showed that ß-catenin signaling is involved in the functions of ovarian cancer cells and interacts with the Notch system. Wnt and Notch systems showed to be prosurvival for ovarian cancer cells and their inhibition impaired cell proliferation and migration. We also demonstrated that the inhibition of ß-catenin by means of two molecules, XAV939 and ICG-001, decreased the proliferation of the IGROV1 and SKOV3 ovarian cancer cell lines and that ICG-001 increased the percentage of IGROV1 cells undergoing apoptosis. The simultaneous inhibition of ß-catenin and Notch signaling, by using the DAPT inhibitor, decreased ovarian cancer cell proliferation to the same extent as targeting only the Wnt/ß-catenin pathway. A similar effect was observed in IGROV1 cell migration with ICG-001 and DAPT. ICG-001 increased the Notch target genes Hes-1 and Hey-1 and increased Jagged1 expression. However, no changes were observed in Dll4 or Notch 1 and 4 expressions. Our results suggest that Notch and ß-catenin signaling co-operate in ovarian cancer to ensure the proliferation and migration of cells and that this could be achieved, at least partly, by the upregulation of Notch Jagged1 ligand in the absence of Wnt signaling. We showed that the Wnt pathway crosstalks with Notch in ovarian cancer cell functions, which may have implications in ovarian cancer therapeutics.

Diethylnitrosamine enhances hepatic tumorigenic pathways in mice fed with high fat diet (Hfd).

Obesity has been implicated in the genesis of metabolic syndromes including insulin resistance and Type 2 Diabetes Mellitus (T2DM). Given the association between T2DM and the risk of hepatocellular carcinoma (HCC), our specific goal was to determine whether the liver of HFD-induced T2DM mice is more sensitive to the carcinogen diethylnitrosamine (DEN), due to a modification of the molecular pathways implicated in the early stages of HCC pathogenesis. C57BL/6 male mice (five-week-old) were divided into 4 groups: C, C + DEN, HFD and HFD + DEN. Mice were euthanized twenty-five weeks after DEN-injection. Livers of HDF-fed mice showed a higher proliferative index than Control groups. In line with this, HFD groups showed an increase of nuclear ß-catenin, and interestingly, DEN treatment led to a slight increase in the expression of this protein in HFD group. Based on these results, and to confirm this effect, we analyzed ß-catenin target genes, finding that DEN treatment in HFD group led to a significant increase of Vegf, c-myc, c-jun and cyclin D1 expression levels. According to our results, the expression of TCF4 showed to be significantly increased in HFD + DEN vs. HFD. In this regard, the ß-catenin/TCF4 complex enhanced its association with pSmads 2/3, as we observed an increase of nuclear Smads expression in HFD + DEN, suggesting a possible role of TGF-ß1/Smads signaling pathway in this phenomenon. Our results show that the liver of HFD fed model that resembles early T2DM pathology in mice, is more sensitive to DEN, by inducing both Wnt/ß-catenin and TGF ß1/Smads tumorigenic pathways.

6-Bromoindirubin-3'-oxime promotes osteogenic differentiation of canine BMSCs through inhibition of GSK3ß activity and activation of the Wnt/ß-catenin signaling pathway.

This study aimed to investigate how 6-bromoindirubin-3'-oxime (BIO) increases the osteogenic differentiation of canine bone mesenchymal stem cells (BMSCs) and the role of the Wnt/ß-catenin signaling pathway in this process. We mimicked the effect of Wnt by adding BIO to the culture medium of BMSCs and examined whether canonical Wnt signaling positively affects the differentiation of these cells into osteoblasts. Canine BMSCs were cultured with 0.5 and 1.0 µM BIO under osteogenic conditions and then differentiation markers were investigated. It was found that BIO significantly increased the activity of alkaline phosphatase (ALP), the number of ALP-positive cells, the mineralization level and calcium deposits. Moreover, cells cultured with 0.5 and 1.0 µM BIO exhibited detectable ß-catenin expression in their nuclei, and showed upregulated ß-catenin and glycogen synthase kinase 3 beta(GSK3ß) phosphorylation compared to untreated cells. In addition, BIO enhanced the mRNA expression of osteoblast differentiation markers such as ALP, runt-related transcription factor 2, collagen I, osteocalcin, and osteonectin. In conclusion, BIO upregulated GSK3ß phosphorylation and inhibited its activity, thereby activating the Wnt/ß-catenin signaling pathway and promoting the osteogenic differentiation of canine BMSCs. The effect of 1.0 µM BIO on BMSCs differentiation was stronger than that of 0.5 µM BIO.

Inhibitory effect of Astragalus polysaccharide on osteoporosis in ovariectomized rats by regulating FoxO3a /Wnt signaling pathway

Abstract Purpose: To investigate inhibitory effect of Astragalus polysaccharide (APS) on osteoporosis in ovariectomized rats by regulating FoxO3a/Wnt2 signaling pathway. Methods: Postmenopausal osteoporosis (PMOP) animal model was developed by excising the bilateral ovaries of rats. The model rats were administered with APS (200 mg/kg, 400 mg/kg, 800 mg/kg) by intragastric administration once daily for 12 weeks. Bone density, bone metabolism index and oxidative stress index were measured in all groups. Furthermore, the regulation of APS of FoxO3a / Wnt2 signaling pathway was observed. Results: APS has an estrogen-like effect, which can increase bone mass, lower serum ALP and BGP values, increase blood calcium content, and increase bone density of the femur and vertebrae in rats. At the same time, APS can increase the bone mineral content of the femur, increase the maximum stress, maximum load and elastic modulus of the ovariectomized rats, improve oxidative stress in rats by increasing the gene expression of β-catenin and Wnt2 mRNA and inhibiting the gene expression of FoxO3a mRNA. Conclusion: Astragalus polysaccharide can effectively alleviate oxidative stress-mediated osteoporosis in ovariectomized rats, which may be related to its regulation of FoxO3a/Wnt2/β-catenin pathway.

Telocinobufagin and Marinobufagin Produce Different Effects in LLC-PK1 Cells: A Case of Functional Selectivity of Bufadienolides.

Bufadienolides are cardiotonic steroids (CTS) identified in mammals. Besides Na⁺/K⁺-ATPase inhibition, they activate signal transduction via protein⁻protein interactions. Diversity of endogenous bufadienolides and mechanisms of action may indicate the presence of functional selectivity and unique cellular outcomes. We evaluated whether the bufadienolides telocinobufagin and marinobufagin induce changes in proliferation or viability of pig kidney (LLC-PK1) cells and the mechanisms involved in these changes. In some experiments, ouabain was used as a positive control. CTS exhibited an inhibitory IC50 of 0.20 (telocinobufagin), 0.14 (ouabain), and 3.40 µM (marinobufagin) for pig kidney Na⁺/K⁺-ATPase activity and concentrations that barely inhibited it were tested in LLC-PK1 cells. CTS induced rapid ERK1/2 phosphorylation, but corresponding proliferative response was observed for marinobufagin and ouabain instead of telocinobufagin. Telocinobufagin increased Bax:Bcl-2 expression ratio, sub-G0 cell cycle phase and pyknotic nuclei, indicating apoptosis. Src and MEK1/2 inhibitors blunted marinobufagin but not telocinobufagin effect, which was also not mediated by p38, JNK1/2, and PI3K. However, BIO, a GSK-3ß inhibitor, reduced proliferation and, as telocinobufagin, phosphorylated GSK-3ß at inhibitory Ser9. Combination of both drugs resulted in synergistic antiproliferative effect. Wnt reporter activity assay showed that telocinobufagin impaired Wnt/ß-catenin pathway by acting upstream to ß-catenin stabilization. Our findings support that mammalian endogenous bufadienolides may exhibit functional selectivity.

B-1 cells and B-1 cell precursors prompt different responses to Wnt signaling.

Recently several studies demonstrated a role for the Wnt pathway in lymphocyte development and self-renewal of hematopoietic stem cells (HSCs). B-1 cells constitute a separate lineage of B lymphocytes, originating during fetal hematopoiesis, expressing lymphoid and myeloid markers and possessing self-renewal ability, similar to early hematopoietic progenitors and HSCs. A plethora of studies have shown an important role for the evolutionary conserved Wnt pathway in the biology of HSCs and T lymphocyte development. Our previous data demonstrated abundant expression of Wnt pathway components by B-1 cells, including Wnt ligands and receptors. Here we report that the canonical Wnt pathway is activated in B-1 cell precursors, but not in mature B-1 cells. However, both B-1 precursors and B-1 cells are able to respond to Wnt ligands in vitro. Canonical Wnt activity promotes proliferation of B-1 cells, while non-canonical Wnt signals induce the expansion of B-1 precursors. Interestingly, using a co-culture system with OP9 cells, Wnt3a stimulus supported the generation of B-1a cells. Taking together, these results indicate that B-1 cells and their progenitors are differentially responsive to Wnt ligands, and that the balance of activation of canonical and non-canonical Wnt signaling may regulate the maintenance and differentiation of different B-1 cell subsets.

The NK1 receptor antagonist NKP608 inhibits proliferation of human colorectal cancer cells via Wnt signaling pathway.

BACKGROUND: Neurokinin1 (NK1) receptor has played a vital role in the development of tumor. However, NKP608 as a NK1 receptor antagonist whether has the effect of the resistance of colorectal cancer is still unclear. Thereby, in this study, we investigated the role of NKP608 on human colorectal cancer and explored the underlying mechanism. METHODS: The cell proliferation of colorectal cancer cells was detected by cell counting kit-8 (CCK8) assay, cell migration and invasion were assessed by transwell assay, the apoptotic ratio of cells was assessed by Annexin V-fluorescein isothiocyanate/propidium iodide stained and flow cytometry. The involvement of molecular mechanisms was examined by western blot. RESULTS: In this study, we found that NKP608 inhibited the proliferation, migration/invasion of HCT116 cells. In addition, NKP608 reduced expressions of Wnt-3a, ß-catenin, Cyclin D1, and (vascular endothelial growth factor) VEGF while induced expression of E-Cadherin. Furthermore, flow cytometry analyzed that NKP608 induced apoptosis of HCT116 cells, consistently, western blotting detecting of apoptosis-related proteins revealed that NKP608 downregulated Bcl-2 while upregulated Bax and Active-Caspase-3. CONCLUSIONS: Taken together, our results demonstrated that NKP608 inhibited colorectal cancer cell proliferation, migration and invasion via suppressing the Wnt/ß-catenin signaling pathway. Therefore, NKP608 might represent a promising therapeutic agent in the treatment of colorectal cancer.

Antiproliferative Effects of Epigenetic Modifier Drugs Through E-cadherin Up-regulation in Liver Cancer Cell Lines.

INTRODUCTION AND AIM: Epigenetic alterations play an essential role in cancer onset and progression, thus studies of drugs targeting the epigenetic machinery are a principal concern for cancer treatment. Here, we evaluated the potential of the combination of the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5aza-dC) and the pan-deacetylase inhibitor Trichostatin A (TSA), at low cytotoxic concentrations, to modulate the canonical Wnt/ß-catenin pathway in liver cancer cells. MATERIAL AND METHODS: Pyrosequencing was used for DNA methylation analyses of LINE-1 sequences and the Wnt/ß-catenin pathway antagonist DKK3, SFRP1, WIF1 and CDH1. qRT-PCR was employed to verify the expression of the antagonist. Pathway regulation were evaluated looking at the expression of ß-catenin and E-cadherin by confocal microscopy and the antitumoral effects of the drugs was studied by wound healing and clonogenic assays. RESULTS: Our result suggest that 5aza-dC and TSA treatments were enough to induce a significant expression of the pathway antagonists, decrease of ß-catenin protein levels, re-localization of the protein to the plasma membrane, and pathway transcriptional activity reduction. These important effects exerted an antitumoral outcome shown by the reduction of the migration and clonogenic capabilities of the cells. CONCLUSION: We were able to demonstrate Wnt/ ß-catenin pathway modulation through E-cadherin up-regulation induced by 5aza-dC and TSA treatments, under an activation-pathway background, like CTNNB1 and TP53 mutations. These findings provide evidences of the potential effect of epigenetic modifier drugs for liver cancer treatment. However, further research needs to be conducted, to determine the in vivo potential of this treatment regimen for the management of liver cancer.

Trypanosoma cruzi Exploits Wnt Signaling Pathway to Promote Its Intracellular Replication in Macrophages.

During the acute phase of Trypanosoma cruzi infection, macrophages can act as host cells for the parasites as well as effector cells in the early anti-parasitic immune response. Thus, the targeting of specific signaling pathways could modulate macrophages response to restrict parasite replication and instruct an appropriate adaptive response. Recently, it has become evident that Wnt signaling has immunomodulatory functions during inflammation and infection. Here, we tested the hypothesis that during T. cruzi infection, the activation of Wnt signaling pathway in macrophages plays a role in modulating the inflammatory/tolerogenic response and therefore regulating the control of parasite replication. In this report, we show that early after T. cruzi infection of bone marrow-derived macrophages (BMM), ß-catenin was activated and Wnt3a, Wnt5a, and some Frizzled receptors as well as Wnt/ß-catenin pathway's target genes were upregulated, with Wnt proteins signaling sustaining the activation of Wnt/ß-catenin pathway and then activating the Wnt/Ca+2 pathway. Wnt signaling pathway activation was critical to sustain the parasite's replication in BMM; since the treatments with specific inhibitors of ß-catenin transcriptional activation or Wnt proteins secretion limited the parasite replication. Mechanistically, inhibition of Wnt signaling pathway armed BMM to fight against T. cruzi by inducing the production of pro-inflammatory cytokines and indoleamine 2,3-dioxygenase activity and by downregulating arginase activity. Likewise, in vivo pharmacological inhibition of the Wnts' interaction with its receptors controlled the parasite replication and improved the survival of lethally infected mice. It is well established that T. cruzi infection activates a plethora of signaling pathways that ultimately regulate immune mediators to determine the modulation of a defined set of effector functions in macrophages. In this study, we have revealed a new signaling pathway that is activated by the interaction between protozoan parasites and host innate immunity, establishing a new conceptual framework for the development of new therapies.