ABSTRACT
Prune belly syndrome (PBS), also known as Eagle-Barret syndrome, is a rare, multi-system congenital myopathy primarily affecting males. Phenotypically, PBS cases manifest three cardinal pathological features: urinary tract dilation with poorly contractile smooth muscle, wrinkled flaccid ventral abdominal wall with skeletal muscle deficiency, and intra-abdominal undescended testes. Genetically, PBS is poorly understood. After performing whole exome sequencing in PBS patients, we identify one compound heterozygous variant in the PIEZO1 gene. PIEZO1 is a cation-selective channel activated by various mechanical forces and widely expressed throughout the lower urinary tract. Here we conduct an extensive functional analysis of the PIEZO1 PBS variants that reveal loss-of-function characteristics in the pressure-induced normalized open probability (NPo) of the channel, while no change is observed in single-channel currents. Furthermore, Yoda1, a PIEZO1 activator, can rescue the NPo defect of the PBS mutant channels. Thus, PIEZO1 mutations may be causal for PBS and the in vitro cellular pathophysiological phenotype could be rescued by the small molecule, Yoda1. Activation of PIEZO1 might provide a promising means of treating PBS and other related bladder dysfunctional states.
Subject(s)
Prune Belly Syndrome , Male , Humans , Prune Belly Syndrome/genetics , Mutation , Muscle Contraction/genetics , Muscle, Skeletal , Muscle, Smooth , Ion Channels/geneticsABSTRACT
The Wnt/ß-catenin signaling pathway dictates cell proliferation and differentiation during embryonic development and tissue homeostasis. Its deregulation is associated with many pathological conditions, including neurodegenerative disease, frequently downregulated. The lack of efficient treatment for these diseases, including Alzheimer's disease (AD), makes Wnt signaling an attractive target for therapies. Interestingly, novel Wnt signaling activating compounds are less frequently described than inhibitors, turning the quest for novel positive modulators even more appealing. In that sense, natural compounds are an outstanding source of potential drug leads. Here, we combine different experimental models, cell-based approaches, neuronal culture assays, and rodent behavior tests with Xenopus laevis phenotypic analysis to characterize quercitrin, a natural compound, as a novel Wnt signaling potentiator. We find that quercitrin potentiates the signaling in a concentration-dependent manner and increases the occurrence of the Xenopus secondary axis phenotype mediated by Xwnt8 injection. Using a GSK3 biosensor, we describe that quercitrin impairs GSK3 activity and increases phosphorylated GSK3ß S9 levels. Treatment with XAV939, an inhibitor downstream of GSK3, impairs the quercitrin-mediated effect. Next, we show that quercitrin potentiates the Wnt3a-synaptogenic effect in hippocampal neurons in culture, which is blocked by XAV939. Quercitrin treatment also rescues the hippocampal synapse loss induced by intracerebroventricular injection of amyloid-ß oligomers (AßO) in mice. Finally, quercitrin rescues AßO-mediated memory impairment, which is prevented by XAV939. Thus, our study uncovers a novel function for quercitrin as a Wnt/ß-catenin signaling potentiator, describes its mechanism of action, and opens new avenues for AD treatments.
Subject(s)
Alzheimer Disease , Neurodegenerative Diseases , Mice , Animals , Wnt Signaling Pathway , Amyloid beta-Peptides/pharmacology , beta Catenin/metabolism , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta/metabolism , Alzheimer Disease/pathology , Quercetin/pharmacology , Quercetin/therapeutic useABSTRACT
INTRODUCTION AND OBJECTIVE: Prune Belly Syndrome (PBS) is characterized by bladder dysmyogenesis, yielding a dysfunctional compliant thick wall with excess collagen deposition. To dissect the cellular heterogeneity and gene expression networks altered in PBS, we report the cell type composition and transcriptional activity of PBS human bladder by using single cell RNA sequencing (scRNA-seq). METHODS: Using IRB-approved methods, bladder dome from 2 PBS and 6 non-PBS control (CO) males underwent fresh single-cell digestion. scRNA-seq was performed and 5277 and 31828 bladder cells from PBS and CO patients was detected, respectively. Cell type clusters were graphically displayed by Uniform Manifold Approximation and Projection (UMap) plot and differentially expressed genes (DEGs) were generated to assign each cluster identity. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis was performed for PBS affected genes. RESULTS: We identified 17 distinct bladder cell clusters, including 6 fibroblast (1, 2, 3, and 4, immunofibroblast, myofibroblast), 1 smooth muscle (SM), and 2 urothelial (umbrella and basal+intermediate) clusters (Fig 1A-B). Counts of individual cell types were expressed as relative proportions, identifying significant PBS fibroblast enrichment, (67% PBS vs 40% CO). Five of 6 PBS fibroblast sub-types are proportionately fewer in number than in CO. The exception is a dominant fibroblast sub-type we label as fibroblast 4, (61% of all PBS fibroblasts vs <10% CO fibroblast subtypes). SM and urothelial cell populations are dramatically reduced in PBS (SM: 5% PBS vs 11% CO and urothelial: <1% PBS vs 7% CO) (Fig 1C-E). PBS fibroblast DEGs, but not SM cells, are enriched in collagen genes. Fibroblast markers (DCN and PLA2G2A) and SM genes (DES, TPM2 and TAGLN) are reduced (by 4, 13, 2, 4, and 2x respectively) in PBS cells (Fig 1G). KEGG pathways analysis for fibroblasts and SM showed a highly similar enrichment for neurodegenerative disease pathways (Fig 1H-I). CONCLUSIONS: Using scRNA-seq, we identified and characterized the disarrayed cell type populations in PBS bladders, generating their unbiased transcriptomic signatures which highlight commonality with neurodegenerative diseases. This PBS transcriptomic map is a step toward potential markers for diagnosis and therapeutic intervention.[Figure: see text]Source of Funding:NIH DK100483, DK127589 PI: Baker, L.
ABSTRACT
This review highlights the work that my research group has been developing, together with international collaborators, during the last decade. Since we were able to establish the Xenopus laevis experimental model in Brazil, we have been focused on understanding early embryonic patterns regarding neural induction and axes establishment. In this context, the Wnt pathway appears as a major player and has been much explored by us and other research groups. Here, we chose to review three published works which we consider to be landmarks within the course of our research and also within the history of modern findings regarding neural induction and patterning. We intend to show how our series of discoveries, when painted together, tells a story that covers crucial developmental windows of early differentiation paths of anterior neural tissue: 1. establishing the head organizer in contrast to the trunk organizer in the early gastrula; 2. deciding between neural ectoderm and epidermis ectoderm at the blastula/gastrula stages, and 3. the gathering of prechordal unique properties in the late gastrula/early neurula.
Subject(s)
Body Patterning , Wnt Signaling Pathway , Animals , Ectoderm/metabolism , Embryonic Induction , Gastrula/metabolism , Gene Expression Regulation, Developmental , Xenopus Proteins/genetics , Xenopus laevis/metabolismABSTRACT
Wnt signaling through the Frizzled (FZD) family of serpentine receptors is essential for embryogenesis and homeostasis, and stringent control of the FZD protein level is critical for stem cell regulation. Through CRISPR/Cas9 genome-wide screening in human cells, we identified TMEM79/MATTRIN, an orphan multi-span transmembrane protein, as a specific inhibitor of Wnt/FZD signaling. TMEM79 interacts with FZD during biogenesis and promotes FZD degradation independent of ZNRF3/RNF43 ubiquitin ligases (R-spondin receptors). TMEM79 interacts with ubiquitin-specific protease 8 (USP8), whose activating mutations underlie human tumorigenesis. TMEM79 specifically inhibits USP8 deubiquitination of FZD, thereby governing USP8 substrate specificity and promoting FZD degradation. Tmem79 and Usp8 genes have a pre-bilaterian origin, and Tmem79 inhibition of Usp8 and Wnt signaling is required for anterior neural development and gastrulation in Xenopus embryos. TMEM79 is a predisposition gene for Atopic dermatitis, suggesting deregulation of Wnt/FZD signaling a possible cause for this most common yet enigmatic inflammatory skin disease.
Subject(s)
Embryonic Development/physiology , Frizzled Receptors/metabolism , Membrane Proteins/metabolism , Xenopus laevis/embryology , Animals , Embryonic Development/genetics , Frizzled Receptors/genetics , HEK293 Cells , Humans , Membrane Proteins/genetics , Wnt Signaling Pathway/genetics , Xenopus laevis/geneticsABSTRACT
The deregulation of the Wnt/ß-catenin signaling pathway is a central event in colorectal cancer progression, thus a promising target for drug development. Many natural compounds, such as flavonoids, have been described as Wnt/ß-catenin inhibitors and consequently modulate important biological processes like inflammation, redox balance, cancer promotion and progress, as well as cancer cell death. In this context, we identified the chalcone lonchocarpin isolated from Lonchocarpus sericeus as a Wnt/ß-catenin pathway inhibitor, both in vitro and in vivo. Lonchocarpin impairs ß-catenin nuclear localization and also inhibits the constitutively active form of TCF4, dnTCF4-VP16. Xenopus laevis embryology assays suggest that lonchocarpin acts at the transcriptional level. Additionally, we described lonchocarpin inhibitory effects on cell migration and cell proliferation on HCT116, SW480, and DLD-1 colorectal cancer cell lines, without any detectable effects on the non-tumoral intestinal cell line IEC-6. Moreover, lonchocarpin reduces tumor proliferation on the colorectal cancer AOM/DSS mice model. Taken together, our results support lonchocarpin as a novel Wnt/ß-catenin inhibitor compound that impairs colorectal cancer cell growth in vitro and in vivo.
ABSTRACT
Direct development in amphibians is characterized by the loss of aquatic breeding. The anuran Adelophryne maranguapensis is one example of a species with direct development, and it is endemic to the state of Ceará, Brazil. Detailed morphological features of A. maranguapensis embryos and the stages of sequential development have not been described before. Here, we analyzed all available genetic sequence tags in A. maranguapensis (tyr exon 1, pomc and rag1) and compared them with sequences from other species of Adelophryne frogs. We describe the A. maranguapensis reproductive tract and embryonic body development, with a focus on the limbs, tail, ciliated cells of the skin, and the egg tooth, which were analyzed using scanning electron microscopy. Histological analyses revealed ovaries containing oocytes surrounded by follicular cells, displaying large nuclei with nucleoli inside. Early in development, the body is unpigmented, and the neural tube forms dorsally to the yolk vesicle, typical of a direct-developing frog embryo. The hindlimbs develop earlier than the forelimbs. Ciliated cells are abundant during the early stages of skin development and are less common during later stages. The egg tooth appears in the later stages and develops as a keratinized microridge structure. The developmental profile of A. maranguapensis presented here will contribute to our understanding of the direct-development model and may help preserve this endangered native Brazilian frog. genesis 54:257-271, 2016. © 2016 Wiley Periodicals, Inc.
Subject(s)
Anura/growth & development , Embryonic Development , Extremities/growth & development , Skin/growth & development , Animals , Anura/genetics , Embryo, Nonmammalian , Oocytes/growth & development , Oocytes/metabolism , Skin/metabolismABSTRACT
Overactivation of the Wnt/ß-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/ß-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/ß-catenin pathway. Both chalcones are able to affect the cell distribution of ß-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/ß-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/ß-catenin pathway and colon cancer cell growth in vitro.
Subject(s)
Antineoplastic Agents/pharmacology , Chalcones/pharmacology , Colonic Neoplasms/metabolism , Flavonoids/pharmacology , Hemiterpenes/pharmacology , Wnt Signaling Pathway , Animals , Cell Proliferation/drug effects , Chalcones/chemistry , HCT116 Cells , Hemiterpenes/chemistry , Humans , Xenopus , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
Secreted Wnt morphogens are essential for embryogenesis and homeostasis and require a lipid/palmitoleoylate modification for receptor binding and activity. Notum is a secreted Wnt antagonist that belongs to the α/ß hydrolase superfamily, but its mechanism of action and roles in vertebrate embryogenesis are not fully understood. Here, we report that Notum hydrolyzes the Wnt palmitoleoylate adduct extracellularly, resulting in inactivated Wnt proteins that form oxidized oligomers incapable of receptor binding. Thus, Notum is a Wnt deacylase, and palmitoleoylation is obligatory for the Wnt structure that maintains its active monomeric conformation. Notum is expressed in naive ectoderm and neural plate in Xenopus and is required for neural and head induction. These findings suggest that Notum is a prerequisite for the "default" neural fate and that distinct mechanisms of Wnt inactivation by the Tiki protease in the Organizer and the Notum deacylase in presumptive neuroectoderm orchestrate vertebrate brain development.
Subject(s)
Esterases/genetics , Head/embryology , Neurogenesis/physiology , Wnt Proteins/metabolism , Xenopus Proteins/metabolism , Animals , Body Patterning/genetics , Ectoderm/metabolism , Embryo, Nonmammalian , Gene Expression Regulation, Developmental , Gene Knockout Techniques , Gene Silencing , HEK293 Cells , Humans , Mice , Molecular Sequence Data , Morpholinos , Neural Plate/metabolism , Oxidation-Reduction , Palmitic Acid/chemistry , Protein Binding , Protein Conformation , Wnt Proteins/antagonists & inhibitors , Wnt Proteins/chemistry , Xenopus Proteins/genetics , Xenopus laevisABSTRACT
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/ß-catenin signaling. Our data provide strong support for an inhibitory effect of isoquercitrin on Wnt/ß-catenin, where the flavonoid acts downstream of ß-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/ß-catenin and should be further investigated as a potential novel anti-tumoral agent.
Subject(s)
Cell Proliferation/drug effects , Quercetin/analogs & derivatives , Wnt Signaling Pathway/drug effects , beta Catenin/metabolism , Active Transport, Cell Nucleus/drug effects , Animals , Antineoplastic Agents/pharmacology , Blotting, Western , Body Patterning/drug effects , Body Patterning/genetics , Cell Line , Cell Line, Tumor , Cell Movement/drug effects , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Early Growth Response Protein 2/genetics , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , HCT116 Cells , Humans , Immunohistochemistry , In Situ Hybridization , Lithium Chloride/pharmacology , Quercetin/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Wnt Signaling Pathway/genetics , Xenopus/embryology , Xenopus/genetics , Xenopus/metabolism , Xenopus Proteins/genetics , beta Catenin/geneticsABSTRACT
Tiki1 is a Wnt protease and antagonist specifically expressed in the Spemann-Mangold Organizer and is required for head formation in Xenopus embryos. Here we report neighbor-joining phylogenetic analysis of vertebrate Tiki genes and their mRNA expression patterns in chick, mouse, and rabbit embryos. Tiki1 and Tiki2 orthologues are highly conserved, and exhibit similar but also different developmental expression patterns among the vertebrate/mammalian species analyzed. The Tiki1 gene is noticeably absent in the rodent lineage, but is present in lagomorphs and all other vertebrate/mammalian species examined. Expression in Hensen's node, the equivalent of the Xenopus Organizer, was observed for Chick Tiki2 and Rabbit Tiki1 and Tiki2. Mouse Tiki2 was detected at low levels at gastrulation and head fold stages, but not in the node. Mouse Tiki2 and chick Tiki1 display similar expression in the dorsal spinal cord. Chick Tiki1 expression was also detected in the surface ectoderm and maxillary bud, while chick Tiki2 was found in the anterior intestinal portal, head mesenchyme and primitive atrium. Our expression analyses provide evidence that Tiki1 and Tiki2 are evolutionarily conserved among vertebrate species and their expression in the Organizer and other regions suggests contributions of these Wnt inhibitors to embryonic patterning, as well as organogenesis. Our analyses further reveal mis-regulation of TIKI1 and TIKI2 in human cancer and diseases.
Subject(s)
Body Patterning/genetics , Evolution, Molecular , Gene Expression Regulation, Developmental , Membrane Proteins/genetics , Metalloproteases/genetics , Phylogeny , Animals , Chick Embryo , Membrane Proteins/metabolism , Metalloendopeptidases , Metalloproteases/metabolism , Mice , Organizers, Embryonic/embryology , Organizers, Embryonic/metabolism , RabbitsABSTRACT
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/ß-catenin signaling pathway. As some types of natural compounds are capable of modulating the Wnt/ß-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/ß-catenin regulation and its possible potential for therapeutic usage on colorectal cancer.
Subject(s)
Antineoplastic Agents/therapeutic use , Colorectal Neoplasms/metabolism , Flavonoids/therapeutic use , Wnt Proteins/metabolism , Wnt Signaling Pathway/drug effects , beta Catenin/metabolism , Animals , Antineoplastic Agents/pharmacology , Colorectal Neoplasms/drug therapy , Flavonoids/pharmacology , Humans , Wnt Proteins/genetics , beta Catenin/geneticsABSTRACT
Clostridium difficile toxins A and B (TcdA and TcdB) are homologous glycosyltransferases that inhibit a group of small GTPases within host cells, but several mechanisms underlying their pathogenic activity remain unclear. In this study, we evaluated the effects of TcdA on the Wnt/ß-catenin pathway, the major driving force behind the proliferation of epithelial cells in colonic crypts. IEC-6 and RKO cells stimulated with Wnt3a-conditioned medium were incubated with 10, 50, and 100 ng/ml of TcdA for 24 h, resulting in a dose-dependent inhibition of the Wnt signaling, as demonstrated by a T-cell factor (TCF) reporter assay. This was further confirmed by immunofluorescence staining for nuclear localization of ß-catenin and Western blotting for ß-catenin and c-Myc (encoded by a Wnt target gene). Moreover, our Western blot analysis showed a decrease in the ß-catenin protein levels, which was reversed by z-VAD-fmk, a pan-caspase inhibitor. Nonetheless, TcdA was still able to inhibit the Wnt/ß-catenin pathway even in the presence of z-VAD-fmk, lithium chloride (a GSK3ß inhibitor), or constitutively active ß-catenin, as determined by a TCF reporter assay. Furthermore, preincubation of RKO cells with TcdA for 12 h also attenuated Wnt3a-mediated activation of Wnt signaling, suggesting that inactivation of Rho GTPases plays a significant role in that inhibition. Taken together, these findings suggest that attenuation of the Wnt signaling by TcdA is important for TcdA antiproliferative effects.
Subject(s)
Bacterial Toxins/pharmacology , Enterotoxins/pharmacology , Epithelial Cells/metabolism , Intestinal Mucosa/cytology , Wnt Proteins/metabolism , beta Catenin/metabolism , Animals , Cell Line, Tumor , Epithelial Cells/drug effects , Humans , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Rats , Signal Transduction/drug effects , Wnt Proteins/genetics , beta Catenin/geneticsABSTRACT
Maternal Wnt/ß-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/ß-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/ß-catenin signaling. Here, we describe a combination of approaches to outline and characterize the role of two flavonoids, quercetin and rutin, on Wnt/ß-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.
Subject(s)
Biological Products/pharmacology , Drug Discovery , Embryonic Development/drug effects , Signal Transduction/drug effects , Wnt Proteins/metabolism , Xenopus/embryology , beta Catenin/metabolism , Animals , Base Sequence , DNA Primers , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Flavonoids are polyphenolic compounds found throughout the plant kingdom. They occur in every organ but are usually concentrated in leaves and flowers. During the last two decades, in vitro and in vivo studies demonstrated that flavonoids have inhibitory effects on human diseases through targeting of multiple cellular signaling components. Wnt/ß-catenin signaling regulates proliferation, differentiation and fate specification in developmental stages and controls tissue homeostasis in adult life. For these reasons, this pathway has received great attention in the last years as potential pathway involved in distinct Human pathologies. In this review we discuss the emerging potential mechanisms for flavonoids on Wnt/ß-catenin signaling in cancer and possible investigation strategies to understand flavonoids mode of action on this signaling pathway.
Subject(s)
Flavonoids/pharmacology , Neoplasms/physiopathology , Signal Transduction/drug effects , Wnt Proteins/drug effects , beta Catenin/physiology , Animals , Humans , Plants/chemistryABSTRACT
Isoquercitrin isolated from the aerial parts of Hyptis fasciculata was evaluated according to its capacity to interfere with glioblastoma (Gbm) cell growth. Gbm cells were incubated with isoquercitrin, quercetin, or rutin at concentrations of 25, 50, and 100 mumol/l for 24, 48, and 72 h. Quercetin and rutin affected Gbm cell proliferation after treatment times of longer than 24 h. However, increasing concentrations of isoquercitrin inhibited 50% of Gbm cell proliferation at 24 h and further reached nearly 90% inhibition at 72 h. This effect did not affect cell morphology, cell viability, or cleaved capase-3 levels, indicating that isoquercitrin did not induce Gbm cell death. A marked reduction in cyclin D1 levels and an increase in p27 levels were observed when 100 micromol/l of isoquercitrin was added to Gbm cells. Interestingly, nuclear beta-catenin staining observed in a subpopulation of untreated Gbm cells was found in the cytoplasm after 100-micromol/l isoquercitrin treatment. Collectively, these data show that isoquercitrin reduces Gbm cell growth without inducing apoptosis, possibly by modulating the control of the cell cycle. Our data also suggest that beta-catenin-mediated signaling may be involved on the antiproliferative activity of isoquercitrin.