Fabio A. Mendes

Amyloid-β binds to the extracellular cysteine-rich domain of frizzled and inhibits wnt/β-catenin signaling

The amyloid-β peptide (Aβ) plays a major role in neuronal dysfunction and neurotoxicity in Alzheimer disease. However, the signal transduction mechanisms involved in Aβ-induced neuronal dysfunction remain to be fully elucidated. A major current unknown is the identity of the protein receptor(s) involved in neuronal Aβ binding. Using phage display of peptide libraries, we have identified a number of peptides that bind Aβ and are homologous to neuronal receptors putatively involved in Aβ interactions. We report here on a cysteine-linked cyclic heptapeptide (denominated cSP5) that binds Aβ with high affinity and is homologous to the extracellular cysteine-rich domain of several members of the Frizzled (Fz) family of Wnt receptors. Based on this homology, we investigated the interaction between Aβ and Fz. The results show that Aβ binds to the Fz cysteine-rich domain at or in close proximity to the Wnt-binding site and inhibits the canonical Wnt signaling pathway. Interestingly, the cSP5 peptide completely blocks Aβ binding to Fz and prevents inhibition of Wnt signaling. These results indicate that the Aβ-binding site in Fz is homologous to cSP5 and that this is a relevant target for Aβ-instigated neurotoxicity. Furthermore, they suggest that blocking the interaction of Aβ with Fz might lead to novel therapeutic approaches to prevent neuronal dysfunction in Alzheimer disease.

Contribution of heparan sulfate to the non-permissive role of the midline glia to the growth of midbrain neurites

Radial glial cells and astrocytes are heterogeneous with respect to morphology, cytoskeletal‐ and membrane‐associated molecules and intercellular interactions. Astrocytes derived from lateral (L) and medial (M) midbrain sectors differ in their abilities to support neuritic growth of midbrain neurons in coculture (Garcia‐Abreu et al. J Neurosci Res 40:471, 1995). There is a correlation between these abilities and the differential patterns of laminin (LN) organization that is fibrillar in growth‐permissive L astrocytes and punctate in the non‐permissive M astroglia (Garcia‐Abreu et al. NeuroReport 6:761, 1995). There are also differences in the production of glycosaminoglycans (GAGs) by L and M midbrain astrocytes (Garcia‐Abreu et al. Glia 17:339, 1996). We show that the relative amounts of the glycoproteins laminin LN, fibronectin (FN) and tenascin (TN) are virtually identical in L and M glia, thus, confirming that an abundant content of LN is not sufficient to promote neurite growth. To further analyze the role of GAGs in the properties of M and L glia, we employed enzymatic degradation of the GAGs chondroitin sulfate (CS) and heparan sulfate (HS). Treatment with chondroitinase has little effect on the non‐permissive properties of M glia but reduces the growth‐supporting ability of L glia. By contrast, heparitinase I produces no significant changes on L glia but leads to neurite growth promotion by M glia. Taken together, these results suggest that glial CS helps to promote neurite growth and, more importantly, they indicate that a HS proteoglycan is, at least, partially responsible for the non‐permissive role of the midline glia to the growth of midbrain neurites. GLIA 29:260–272, 2000.

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

Background: Flavonoids are natural compounds capable of modulating signaling pathways in a variety of biological processes. Results: Isoquercitrin inhibits canonical Wnt signaling in Xenopus embryos downstream of β-catenin and impairs the growth in colon cancer cells in vitro, without cytotoxic effects. Conclusion: Isoquercitrin inhibits Wnt/β-catenin pathway and the growth of colon cancer cells in vitro. Significance: Isoquercitrin should be further investigated as a potential anti-tumoral agent targeting Wnt/β-catenin signaling. 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.

Flavonoids and Wnt/β-Catenin Signaling: Potential Role in Colorectal Cancer Therapies

It is now well documented that natural products have played an important role in anticancer therapy. Many studies focus on the ability of these natural compounds to modulate tumor-related signaling pathways and the relationship of these properties to an anticancer effect. According to the World Health Organization (WHO), colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer death among men and women. Therefore, finding strategies to fight against CRC is an emergent health problem. CRC has a strong association with deregulation of Wnt/β-catenin signaling pathway. As some types of natural compounds are capable of modulating the Wnt/β-catenin signaling, one important question is whether they could counteract CRC. In this review, we discuss the role of flavonoids, a class of natural compounds, on Wnt/β-catenin regulation and its possible potential for therapeutic usage on colorectal cancer.

Concentration-dependent actions of glial chondroitin sulfate on the neuritic growth of midbrain neurons

Astrocytes located in two distinct regions of midbrain differ in their neuritic growth support abilities. Midbrain neurons cultured onto astrocyte monolayers from the lateral (L) region develop long and branched neurites while neurons cultured onto astrocyte monolayers from the medial (M) region develop short or no neurites. The extracellular matrix of these astrocytes has an important role in promoting or inhibiting the growth of these neurons. Differences on the compartmental distribution, as well as on the concentration of GAGs of L and M astrocytes, may be related to their differential capacity of supporting neuritic growth. Indeed, enzymatic digestion of heparan sulfate (HS) and chondroitin sulfate (CS) chains also pointed to an important function for GAGs on axon navigation. In order to better characterize the role of CS on the growth of midbrain neurites, we treated L and M astrocyte monolayers with 1 mM of beta-D-xyloside. Under these conditions, astrocytes oversynthesized and secreted CS protein-free chains to the culture medium. M astrocytes had a significant reduction in their neuritic growth-inhibiting ability after xyloside treatment, suggesting a promoting role for soluble CS in neuritic growth. Chondroitin 4-sulfate (CS-4) added in different concentrations to M astrocyte cultures turned this glia into a permissive substrate, acting in a linear way as far as the largest neurite was concerned. However, a U-shaped dose-effect curve on neurite growth resulted from the similar treatment of L astrocytes. These results suggest that glial CS-4 could be involved in the neurite growth modulating properties of midbrain neurons in a complex concentration-dependent way.

Connective Tissue Growth Factor (CTGF/CCN2) Is Negatively Regulated during Neuron-Glioblastoma Interaction

Connective-tissue growth factor (CTGF/CCN2) is a matricellular-secreted protein involved in complex processes such as wound healing, angiogenesis, fibrosis and metastasis, in the regulation of cell proliferation, migration and extracellular matrix remodeling. Glioblastoma (GBM) is the major malignant primary brain tumor and its adaptation to the central nervous system microenvironment requires the production and remodeling of the extracellular matrix. Previously, we published an in vitro approach to test if neurons can influence the expression of the GBM extracellular matrix. We demonstrated that neurons remodeled glioma cell laminin. The present study shows that neurons are also able to modulate CTGF expression in GBM. CTGF immnoreactivity and mRNA levels in GBM cells are dramatically decreased when these cells are co-cultured with neonatal neurons. As proof of particular neuron effects, neonatal neurons co-cultured onto GBM cells also inhibit the reporter luciferase activity under control of the CTGF promoter, suggesting inhibition at the transcription level. This inhibition seems to be contact-mediated, since conditioned media from embryonic or neonatal neurons do not affect CTGF expression in GBM cells. Furthermore, the inhibition of CTGF expression in GBM/neuronal co-cultures seems to affect the two main signaling pathways related to CTGF. We observed inhibition of TGFβ luciferase reporter assay; however phopho-SMAD2 levels did not change in these co-cultures. In addition levels of phospho-p44/42 MAPK were decreased in co-cultured GBM cells. Finally, in transwell migration assay, CTGF siRNA transfected GBM cells or GBM cells co-cultured with neurons showed a decrease in the migration rate compared to controls. Previous data regarding laminin and these results demonstrating that CTGF is down-regulated in GBM cells co-cultured with neonatal neurons points out an interesting view in the understanding of the tumor and cerebral microenvironment interactions and could open up new strategies as well as suggest a new target in GBM control.

Effects of Natural Compounds on Xenopus Embryogenesis: A Potential Read Out for Functional Drug Discovery Targeting Wnt/β-catenin Signaling

Maternal Wnt/β-Catenin signaling is essential to establish dorsal-specific gene expression required for axial patterning in Xenopus. Deregulation of this pathway causes axis phenotypes in frog embryos. In adult life, mutations in the Wnt pathway components are associated with many diseases, such as polyposis coli; osteoporosis-pseudoglioma syndrome (OPPG); skeletal dysplasia; neural tube defects, cancer and many others. Thus, a better understanding of Wnt/β-catenin signaling will have great and significant impact on Biology and Medicine. In this aspect, natural compounds are potential targets as novel molecules that could modulate the Wnt pathway. For instance, flavonoids are a large group of natural compounds found in plants that modulate important cellular and molecular mechanisms related to diseases, but the specific in vivo mechanism of action of most flavonoids remain unknown. In this way, Xenopus embryos may provide an efficient model, since it is frequently used to test and identify the role of molecules that affect Wnt/β-catenin signaling. Here, we describe a combination of approaches to outline and characterize the role of two flavonoids, quercetin and rutin, on Wnt/β-catenin signaling, using Xenopus embryos as an experimental model. Our data support that quercetin is potential in vivo modulator of canonical Wnt signaling and that this effect might depend on the structure of this molecule, as we did not observe any effect with rutin treatment, a flavonol structurally-related to quercetin. This model is useful to analyze effects of quercetin and other flavonoids in vivo and to provide further understanding of how natural compounds can modulate signaling pathways, using Xenopus embryos as a fast and efficient reading of in vivo effects of those compounds.

Complexity of the Wnt/β‑catenin pathway: Searching for an activation model

Wnt signaling refers to a conserved signaling pathway, widely studied due to its roles in cellular communication, cell fate decisions, development and cancer. However, the exact mechanism underlying inhibition of the GSK phosphorylation towards β-catenin and activation of the pathway after biding of Wnt ligand to its cognate receptors at the plasma membrane remains unclear. Wnt target genes are widely spread over several animal phyla. They participate in a plethora of functions during the development of an organism, from axial specification, gastrulation and organogenesis all the way to regeneration and repair in adults. Temporal and spatial oncogenetic re-activation of Wnt signaling almost certainly leads to cancer. Wnt signaling components have been extensively studied as possible targets in anti-cancer therapies. In this review we will discuss one of the most intriguing questions in this field, that is how β-catenin, a major component in this pathway, escapes the destruction complex, gets stabilized in the cytosol and it is translocated to the nucleus where it acts as a co-transcription factor. Four major models have evolved during the past 20years. We dissected each of them along with current views and future perspectives on this pathway. This review will focus on the molecular mechanisms by which Wnt proteins modulate β-catenin cytoplasmic levels and the relevance of this pathway for the development and cancer.

Connective-Tissue Growth Factor (CTGF/CCN2) Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells In Vitro

Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFβ, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.

Derricin and derricidin inhibit Wnt/β-catenin signaling and suppress colon cancer cell growth in vitro.

Overactivation of the Wnt/β-catenin pathway in adult tissues has been implicated in many diseases, such as colorectal cancer. Finding chemical substances that can prevent this phenomenon is an emerging problem. Recently, several natural compounds have been described as Wnt/β-catenin inhibitors and might be promising agents for the control of carcinogenesis. Here, we describe two natural substances, derricin and derricidin, belonging to the chalcone subclass, that show potent transcriptional inhibition of the Wnt/β-catenin pathway. Both chalcones are able to affect the cell distribution of β-catenin, and inhibit Wnt-specific reporter activity in HCT116 cells and in Xenopus embryos. Derricin and derricidin also strongly inhibited canonical Wnt activity in vitro, and rescued the Wnt-induced double axis phenotype in Xenopus embryos. As a consequence of Wnt/β-catenin inhibition, derricin and derricidin treatments reduce cell viability and lead to cell cycle arrest in colorectal cancer cell lines. Taken together, our results strongly support these chalcones as novel negative modulators of the Wnt/β-catenin pathway and colon cancer cell growth in vitro.