Targeting Β-catenin Signaling Research Articles

Zerumbone (ZER), a Potential Anticancer for Breast Mediates Cancer Cell Death Through Targeting β-catenin Signaling Pathway in Tumor Regression in Sprague Dawley Rat Mammary Gland Tumors

Background & Objective: Canonical WNT/Wingless pathway regulates expression of target genes by modulating intracellular β-catenin leading to cancer growth and survival. Here, we studied inhibition of β-catenin using zerumbone (ZER) or β-catenin-siRNA in Sprague-Dawley rat cancer model. Methodology: Rat mammary gland tumor model was induced with the wild-type LA7 breast cancer cell line or β-catenin-knockdown-LA7 cells. LA7-rats were treated with ZER or β-catenin-siRNA as a positive control and the levels of β-catenin expression and its target gene were assessed using real-time PCR, immunohistochemistry and TUNEL assay. Results: We found good correlation between β-catenin inhibition and induction of apoptosis when using ZER or β-catenin-siRNA. In this aspect, ZER and β-catenin-siRNA inhibited cellular proliferation as reflected by reduced growth of breast cancer and apoptosis induction in rat mammary gland tumor. Further, our studies demonstrate that treatment with ZER and β-catenin-siRNA affected β-catenin-dependent gene expression. β-catenin downregulation by ZER or knockdown by β-catenin-siRNA improves the morphological and histological feature of the breast tissues as confirmed by subtle changes of the genes involved in cell-apoptotic pathways. Conclusion: This indicates that ZER targets similarly to β-catenin-siRNA and possibly be a useful anti-breast cancer by affecting expression of down-stream targets that are key components in cancer development. ZER can be a promising anticancer candidate for treatment of breast.

Zerumbone (ZER), a Potential Anticancer for Breast Mediates Cancer Cell Death Through Targeting β-catenin Signaling Pathway in MCF-7 Cell Line

Background: Zerumbone is a sesquiterpene which was found to delay progression of breast cancer through apoptosis induction via up-regulating Bax protein and down-regulating Bcl2 protein. Purpose: In this study, we aimed to investigate Zerumbone, a natural compound of medicinal herbal plant isolated from Zingiber zerumbet, for anti-cancer activity against MCF-7 cell line. Methods: Human cancer cell lines of breast MCF-7 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). ON target plus smart pool siRNA, the most efficient method is used for preparation of β-catenin siRNA. Cyclophilin B siRNA is considered as positive control. Zrumbone is an ethanol-soluble compound isolated from Zingiber zerumbet, extracted by the hydro-distillation (steam distillation) method. Purification of ZER was examined using HPLC. The MCF-7 cells transfected with 100 µM human β-catenin (CTNNB1) siRNA or cyclophilin B siRNA positive control. After transfection, the cells were harvested at 48hrs for mRNA analysis or 48 – 72 hrs for protein analysis. IC50 for ZER was determined using MTT assay. MCF-7 transfected with β-catenin siRNA were then treated with ZER IC50. The mRNA expression was analyzed using real-time to ensure the knock down of β-catenin. TUNNEL assay was used to confirm apoptosis and flow cytometry was applied for quantification of apoptotic cells. Western blot analysis was done to evaluate the effect of ZER and β-catenin (CTNNB1) siRNA on the expression of β-catenin and its functional impact on the proliferation and survival of MCF-7 cells. Results: ZER decreased β-catenin protein expression in breast cancer cell lines. ZER downregulated β-catenin mRNA level in breast cancer cell lines. Depletion of β-catenin by β-catenin siRNA and ZER induce cell apoptosis. Apoptosis caused by ZER was confirmed/examined using by annexin V (annexin V was used as a marker for drug action). Conclusion: Zerumbone possesses anti-breast cancer activity, which could be attributed to the reduction of β-catenin protein expression in MCF-7 breast cancer cell lines, downregulation of β-catenin mRNA level in breast cancer cell lines and through annexin V.

Near-infrared fluorescence imaging of hepatocellular carcinoma cells regulated by β-catenin signaling pathway.

Near-infrared fluorescence (NIRF) imaging has recently emerged as a promising tool for noninvasive cancer imaging. However, lack of tumor sensitivity and specificity restricts the application of NIRF dyes in surgical navigation. Herein, we investigated the imaging features of NIRF dye MHI-148 and indocyanine green (ICG) in live cell imaging and xenograft nude mice models. TCGA dataset analysis and immunohistochemistry were conducted to investigate the expression of OATPs or ABCGs in hepatocellular carcinoma (HCC) tissues. OATPs or ABCGs were knocked down and overexpressed in HCC cells using transient transfection by siRNA and plasmids or stable transfection by lentivirus. Further, qRT-PCR ,Western blotting and the use of agonists or inhibitors targeting β-catenin signaling pathway were applied to explore its important role in regulation of OATP2B1 and ABCG2 expression. Here we demonstrated that NIRF dye MHI-148 was biocompatible as indocyanine green (ICG) but with higher imaging intensity and preferential uptake and retention in hepatocellular carcinoma (HCC) cells and tissues. Moreover, our data indicated that membrane transporters OATP2B1 and ABCG2, which regulated by β-catenin signaling pathway, mediated tumor-specific accumulation and retention of MHI-148 in HCC cells. In addition, the treatment with β-catenin inhibitor significantly enhanced the accumulation of MHI-148 in HCC tissues and improved the efficacy of tumor imaging with MHI-148 in vivo. Our study uncovers a mechanism that links the distribution and expression of the membrane transporters OATP2B1 and ABCG2 to the tumor-specific accumulation of MHI-148, and provides evidence supporting a regulating role of the β-catenin signaling pathway in OATP2B1 and ABCG2- induced retention of MHI-148 inHCC tissues, and strategy targeting key components of MHI-148 transport machinery may be a potential approach to improve HCC imaging.

Cold atmospheric microwave plasma (CAMP) stimulates dermal papilla cell proliferation by inducing β-catenin signaling

Hair loss or alopecia is an unpleasant symptom that exacerbates an individual's self-esteem and requires appropriate treatment. The Wnt/β-catenin signaling is a central pathway that promotes dermal papilla induction and keratinocyte proliferation during hair follicle renewal. GSK-3β inactivated by its upstream Akt and ubiquitin-specific protease 47 (USP47) has been shown to inhibit β-catenin degradation. The cold atmospheric microwave plasma (CAMP) is microwave energy enriched with mixtures of radicals. CAMP has been reported to have antibacterial and antifungal activities with wound healing activity against skin infection; however, the effect of CAMP on hair loss treatment has not been reported. We aimed to investigate the effect of CAMP on promoting hair renewal in vitro and to elucidate the molecular mechanism, targeting β-catenin signaling and YAP/TAZ, the co-activators in the Hippo pathway, in human dermal papilla cells (hDPCs). We also evaluated plasma effects on the interaction between hDPCs and HaCaT keratinocytes. The hDPCs were treated with plasma-activating media (PAM) or gas-activating media (GAM). The biological outcomes were determined by MTT assay, qRT-PCR, western blot analysis, immunoprecipitation, and immunofluorescence. We found that β-catenin signaling and YAP/TAZ were significantly increased in PAM-treated hDPCs. PAM treatment also induced β-catenin translocation and inhibited β-catenin ubiquitination by activating Akt/GSK-3β signaling and upregulating USP47 expression. In addition, hDPCs were more aggregated with keratinocytes in PAM-treated cells compared with control. HaCaT cells cultured in a conditioned medium derived from PAM-treated hDPCs exhibited an enhancing effect on activating YAP/TAZ and β-catenin signaling. These findings suggested that CAMP may be a new therapeutic alternative for alopecic treatment.

Slit2/Robo1 signaling inhibits small-cell lung cancer by targeting β-catenin signaling in tumor cells and macrophages.

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer with poor patient prognosis. However, the mechanisms that regulate SCLC progression and metastasis remain undefined. Here, we show that the expression of the slit guidance ligand 2 (SLIT2) tumor suppressor gene is reduced in SCLC tumors relative to adjacent normal tissue. In addition, the expression of the SLIT2 receptor, roundabout guidance receptor 1 (ROBO1), is upregulated. We find a positive association between SLIT2 expression and the Yes1 associated transcriptional regulator (YAP1)-expressing SCLC subtype (SCLC-Y), which shows a better prognosis. Using genetically engineered SCLC cells, adenovirus gene therapy, and preclinical xenograft models, we show that SLIT2 overexpression or the deletion of ROBO1 restricts tumor growth in vitro and in vivo. Mechanistic studies revealed significant inhibition of myeloid-derived suppressor cells (MDSCs) and M2-like tumor-associated macrophages (TAMs) in the SCLC tumors. In addition, SLIT2 enhances M1-like and phagocytic macrophages. Molecular analysis showed that ROBO1 knockout or SLIT2 overexpression suppresses the transforming growth factor beta 1 (TGF-β1)/β-catenin signaling pathway in both tumor cells and macrophages. Overall, we find that SLIT2 and ROBO1 have contrasting effects on SCLC tumors. SLIT2 suppresses, whereas ROBO1 promotes, SCLC growth by regulating the Tgf-β1/glycogen synthase kinase-3 beta (GSK3)/β-catenin signaling pathway in tumor cells and TAMs. These studies indicate that SLIT2 could be used as a novel therapeutic agent against aggressive SCLC.

Discovery of a novel anti-obesity meroterpenoid agent targeted subcutaneous adipose tissue

BackgroundMeroterpenoid furanasperterpene A (T2–3) with a novel 6/6/6/6/5 pentacyclic skeleton was isolated from the Aspergillus terreus GZU-31–1. Previously, we showed that T2–3 possessed significant lipid-lowering effects in 3T3-L1 adipocytes at 5 μM concentration. However, its therapeutic effect in metabolic disease and the underlying mechanisms of action remain unclear. MethodsHigh fat diet-induced obesity (DIO) mouse model and 3T3-L1 cell model were used to assess the anti-obesity effects of T2–3. Lipids in the adipocytes were examined by Oil Red O staining. β-catenin expression was examined by immunofluorescence and Western blotting, its activity was assessed by TOPflash/FOPflash assay. ResultsT2–3 possessed potent anti-obesity effects in DIO mice, it significantly reduced body weight and subcutaneous adipose tissue (SAT) mass. Mechanistic studies showed that T2–3 significantly inhibited 3T3-L1 preadipocyte differentiation as indicated by the reduced number of mature adipocytes. The treatments also reduced the expressions of critical adipogenic transcription factors CEBP-α and PPAR-γ in both 3T3-L1 adipocytes and SAT in DIO mice. Interestingly, T2–3 increased the cytoplasmic and nuclear expressions of β-catenin and the transcriptional activity of β-catenin in 3T3-L1 adipocytes; the elevated β-catenin expression was also observed in SAT of the T2–3-treated DIO mice. Indeed, upregulation of β-catenin activity suppressed adipogenesis, while β-catenin inhibitor JW67 reversed the anti-adipogenic effect of T2–3. Taken together, our data suggest that T2–3 inhibits adipogenesis by upregulating β-catenin activity. ConclusionsOur study is the first report demonstrating meroterpenoid furanasperterpene A as a novel 6/6/6/6/5 pentacyclic skeleton (T2–3) that possesses potent anti-adipogenic effect by targeting β-catenin signaling pathway. Our findings drive new anti-obesity drug discovery and provide drug leads for chemists and pharmacologists.

Neddylation is critical to cortical development by regulating Wnt/β-catenin signaling

Wnt signaling plays a critical role in production and differentiation of neurons and undergoes a progressive reduction during cortical development. However, how Wnt signaling is regulated is not well understood. Here we provide evidence for an indispensable role of neddylation, a ubiquitylation-like protein modification, in inhibiting Wnt/β-catenin signaling. We show that β-catenin is neddylated; and inhibiting β-catenin neddylation increases its nuclear accumulation and Wnt/β-catenin signaling. To test this hypothesis in vivo, we mutated Nae1, an obligative subunit of the E1 for neddylation in cortical progenitors. The mutation leads to eventual reduction in radial glia progenitors (RGPs). Consequently, the production of intermediate progenitors (IPs) and neurons is reduced, and neuron migration is impaired, resulting in disorganization of the cerebral cortex. These phenotypes are similar to those of β-catenin gain-of-function mice. Finally, suppressing β-catenin expression is able to rescue deficits of Nae1 mutant mice. Together, these observations identified a mechanism to regulate Wnt/β-catenin signaling in cortical development.

Targeting β-Catenin Signaling by Natural Products for Cancer Prevention and Therapy.

The mutations and deregulation of Wnt signaling pathway occur commonly in human cancer and cause the aberrant activation of β-catenin and β-catenin-dependent transcription, thus contributing to cancer development and progression. Therefore, β-catenin has been demonstrated as a promising target for cancer prevention and therapy. Many natural products have been characterized as inhibitors of the β-catenin signaling through down-regulating β-catenin expression, modulating its phosphorylation, promoting its ubiquitination and proteasomal degradation, inhibiting its nuclear translocation, or other molecular mechanisms. These natural product inhibitors have shown preventive and therapeutic efficacy in various cancer models in vitro and in vivo. In the present review, we comprehensively discuss the natural product β-catenin inhibitors, their in vitro and in vivo anticancer activities, and underlying molecular mechanisms. We also discuss the current β-catenin-targeting strategies and other potential strategies that may be examined for identifying new β-catenin inhibitors as cancer preventive and therapeutic drugs.

Abstract 4809: Bromo-ormeloxifene inhibits epithelial mesenchymal transition via targeting β-catenin signaling pathways in cervical cancer cells

Abstract Background: Cervical cancer (CxC) is a leading cause of mortality and morbidity among women worldwide. Current chemotherapeutic agents for CxC have shown systemic toxicity in CxC patients. Ormeloxifene (ORM) is a non-toxic and non-steroidal drug with well-defined pharmacokinetic and pharmacodynamic properties in humans. Studies have shown its anti-cancer potential in various pre-clinical mouse models. Here, we have synthesized and characterized a novel analogue of ormeloxifene, Bromo-ormeloxifene (Br-ORM), which showed more therapeutic efficacy against CxC in vitro and in vivo model systems. Methodology: The effect of Br-ORM on CxC cells (CaSki and SiHa) growth and proliferation was determined by colony formation and MTS assays. Molecular docking of Br-ORM with β-catenin was done by AutoDock4 software. Effect of Br-ORM on the expression of epithelial-to mesenchymal (EMT) markers (N-cadherin, slug, snail), MMPs (MMP2 and MMP3) and miR-200a was analyzed by Western blot and qPCR analyses respectively. Apoptosis analysis was done by Annexin-V staining kit. Effect of Br-ORM on β-catenin cellular localization in CxC cells was analyzed by immunofluorescence analysis. The anti-tumor efficacy of Br-ORM was investigated in orthotopic xenograft mouse model of CxC. Results: Br-ORM (10-20 µM) effectively inhibited growth and proliferation of CxC cells in a dose and time-dependent manner as compared to ORM. Br-ORM efficiently suppressed metastatic phenotypes of CxC cells as determined by significant (P<0.05) decrease in invasion and migration potential of CxC cells. Moreover, Br-ORM showed increased apoptosis, which was observed by enhanced Annexin-V staining and PARP protein cleavage. Br-ORM markedly reduced the EMT process as evident by repression of N-cadherin, slug, snail, MMPs (MMP2 and MMP3) and β-catenin/TCF-4 transcriptional activity. Br-ORM potently reduced the translocation of β-catenin in the nucleus. Bioinformatic analysis revealed that Br-ORM proficiently binds into active site of β-catenin with a minimum energy -7.6 kcal/mol. Br-ORM treatment replenished the expression of miR-200a, which directly targets β-catenin in CxC cells. Br-ORM treatment (250 µg/mouse, three times a week) significantly (P<0.01) regressed the cervical tumor growth in orthotopic xenograft mouse model. Similar molecular effects of Br-ORM were observed in excised tumor tissues. Conclusion: These results suggest that Br-ORM inhibits the metastatic phenotypes of CxC cells via targeting β-catenin signaling pathway. Br-ORM could be used as a novel therapeutic modality for the treatment of CxC. Citation Format: Mohammed Sikander, Shabnam Malik, Bilal B. Hafeez, Sonam Kumari, Sheema Khan, John Apraku, Hassan Mandil, Andrew E. Massey, Aditya Ganju, Parvez Khan, Fathi T. Halaweish, Subhash C. Chauhan, Meena Jaggi. Bromo-ormeloxifene inhibits epithelial mesenchymal transition via targeting β-catenin signaling pathways in cervical cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4809.

MiR-150-5p Inhibits Non-Small-Cell Lung Cancer Metastasis and Recurrence by Targeting HMGA2 and β-Catenin Signaling

Dysregulated microRNAs (miRNAs) play crucial roles in the regulation of cancer stem cells (CSCs), and CSCs are closely associated with tumor initiation, metastasis, and recurrence. Here we found that miR-150-5p was significantly downregulated in CSCs of non-small-cell lung cancer (NSCLC) and its expression level was negatively correlated with disease progression and poor survival in patients with NSCLC. Inhibition of miR-150-5p increased the CSC population and sphere formation of NSCLC cells in vitro and stimulated NSCLC cell tumorigenicity and metastatic colonization in vivo. In contrast, miR-150-5p overexpression potently inhibited sphere-formed NSCLC cell tumor formation, metastatic colonization, and recurrence in xenograft models. Furthermore, we identified that miR-150-5p significantly inhibited wingless (Wnt)-β-catenin signaling by simultaneously targeting glycogen synthase kinase 3 beta interacting protein (GSKIP) and β-catenin in NSCLC cells. miR-150-5p also targeted high mobility group AT-hook 2 (HMGA2), another regulator of CSCs, and Wnt-β-catenin signaling. The restoration of HMGA2 and β-catenin blocked miR-150-5p overexpression-induced inhibition of CSC traits in NSCLC cells. These findings suggest that miR-150-5p functions as a CSC suppressor and that overexpression of miR-150-5p may be a novel strategy to inhibit CSC-induced metastasis and recurrence in NSCLC.

Long non-coding RNA THOR promotes liver cancer stem cells expansion via β-catenin pathway

Hepatocellular carcinoma (HCC) is a highly aggressive liver tumor containing cancer stem cells (CSCs), which participate in tumor invasion, therapeutic resistance, and tumor relapse leading to poor outcome and limited therapeutic options. Recently, a novel lncRNA, THOR (testis-associated highly conserved oncogenic long non-coding RNA), was characterized in human cancers and shown to exhibit an oncogenic role. However, the role of THOR in liver cancer stem cells (CSCs) remains obscure. Herein, we observed high expression of THOR in chemoresistant hepatocellular carcinomas (HCCs). A remarkable increase of THOR expression in OV6 or EpCAM-positive liver CSCs as well as in CSC-enriched hepatoma spheres. Interference THOR suppressed liver CSC expansion by inhibiting the dedifferentiation of hepatoma cells and decreasing the self-renewal ability of liver CSCs. Mechanistically, we found β-catenin as the downstream of THOR in HCC cells. The special β-catenin inhibitor FH535 abolished the discrepancy in liver CSC proportion and the self-renewal capacity between THOR knockdown HCC cells and control cells, which further confirmed that β-catenin was required in THOR promoted liver CSCs expansion. Moreover, interference THOR hepatoma cells were more sensitive to sorafenib treatment, indicates that HCC patients with low THOR expression may benefit from sorafenib treatment. Collectively, THOR was upregulated in liver CSCs and could promote HCC cells dedifferentiation and liver CSCs expansion by targeting β-catenin signaling.

Saikosaponin D from Radix Bupleuri suppresses triple-negative breast cancer cell growth by targeting β-catenin signaling.

Triple-negative breast cancer (TNBC) is one of the most aggressive and poor prognosis breast cancers. Currently, chemotherapy with conventional cytotoxic agents is the only available option to treat TNBC. Hence, we identified new therapeutic agents against TNBC from traditional Chinese medicine Radix Bupleuri and unveiled the molecule mechanism of anti-TNBC effects. Multi-component bioactivity and structure-guided methods were used to identify the most effective anti-TNBC compound Saikosaponin D (SSD) from Radix Bupleuri. Cell viability and apoptosis assays were employed to demonstrate the effect of SSD on the proliferation and apoptosis of TNBC cells. Dynamic mass redistribution assay, TopFlash assay, western blotting, and special agonist were applied to dissect the potential molecular mechanisms of SSD. We screened twenty fractions in Radix Bupleuri and identified SSD as the most effective component to inhibit the proliferation of TNBC cells. Investigating the interaction of SSD with the frequently overexpressed targets in TNBC led to the identification that it markedly suppressed Wnt/β-catenin signaling, but did not act on epidermal growth factor receptor and neurotensin receptor-1. Moreover, we demonstrated that SSD significantly repressed β-catenin and its downstream target genes, resulting in TNBC cell apoptosis. Specifically, docking of SSD to the crystal structure of β-catenin suggested that SSD interacted with β-catenin via hydrogen bonds and hydrophobic interaction. We identified the most effective component SSD from Radix Bupleuri in inhibiting the proliferation of TNBC cells by targeting β-catenin signaling. Given the important role of Wnt/β-catenin signaling in breast cancer, SSD may present an opportunity to discover new therapeutics for the treatment of TNBC.

Baicalin inhibits the metastasis of highly aggressive breast cancer cells by reversing epithelial-to-mesenchymal transition by targeting β-catenin signaling.

Metastasis is the main cause of death in breast cancer patients, which is due partly to the lack of effective treatment. Baicalin, a flavonoid compound isolated from the roots of Scutellaria lateriflora Georgi (Huang Qin), has recently been confirmed as an effective agent for the treatment of a variety of cancers. Yet, the effects and underlying molecular mechanisms of baicalin in regards to the metastasis of breast cancer remain unclear. In the present study, we found that baicalin had the potential to suppress the migration and invasion of highly aggressive breast cancer cells in a dose-dependent manner but had no impact on the viability of these cancer cells. Additionally, baicalin reversed the epithelial-to-mesenchymal transition (EMT) process, as evaluated by EMT markers in breast cancer cell lines with a change from a mesenchymal feature to an epithelial type. At the same time, the expression of β-catenin mRNA and protein was dose-dependently downregulated by baicalin in highly invasive breast cancer cell lines, and overexpression of β-catenin by adenoviruses abolished these beneficial effects of baicalin in regards to the migration and invasion, and EMT of breast cancer cells. Furthermore, using a xenograft mouse model, baicalin markedly reduced liver and lung metastasis of breast cancer, inhibited expression of β-catenin, and degraded the EMT molecules vimentin and Slug in the orthotopic tumor tissues. Taken together, all these results indicate that baicalin effectively suppresses the metastasis of breast cancer by reversing EMT, which may be mediated by downregulation of β-catentin expression.

AMD3100 inhibits the migration and differentiation of neural stem cells after spinal cord injury

It was reported that CXCR4 signaling played an important role in the migration and differentiation of endogenous neural stem cells after spinal cord injury (SCI). However, the molecular mechanism of it is still unclear. Here, we established a model of SCI in rats and AMD3100 was used to treat them. The rats were then sacrificed and the injured spinal cord specimens were harvested. Additionally, the neural stem cells (NSCs) line was culture and treated with AMD3100 in vitro. Results showed the locomotor function of SCI rats was worse after treated with AMD3100. And the expression levels of Nestion in neural stem cells and β-tubulin in neuron cells were significantly increased in the injured spinal cord, which can be inhibited by the CXCR4 antagonist of AMD3100. Additionally, the expression of β-catenin and phosphorylase β-catenin protein was significantly down regulated by AMD3100. In vitro, the NSCs proliferation ability was inhibited and the migration was decreased after treated with AMD3100. Also, the expression of Nestion, β-tubulin, β-catenin and phosphorylase β-catenin protein was significantly decreased in AMD3100 group comparing with untreated group. Taken together, this study suggested that AMD3100 could inhibit the migration and differentiation of endogenous neural stem cells in rats with SCI. The mechanism of it maybe that AMD3100 could down regulate of SDF-1/CXCR4 by targeting β-catenin signaling pathway.

5,7,3′,4′-Tetramethoxyflavone exhibits chondroprotective activity by targeting β-catenin signaling in vivo and in vitro

Osteoarthritis (OA) is a progressive joint disorder, which remains the leading cause of chronic disability in aged people. This study is the first report which demonstrates the cartilage protective effect of 5,7,3′,4′-tetramethoxyflavone (TMF) by decreasing the concentration of IL-1β, TNF-α and PGE2 in the knee synovial fluid in OA rat models in vivo. In vitro, after induced by PGE2, the apoptosis rate of chondrocytes was significantly increased. In addition, PGE2 increased the expression of cAMP/PKA signaling pathway in chondrocytes, stabilized and accumulated β-catenin, and activated the expression of β-catenin signaling pathway. These activities were counteracted by TMF dose-dependently. Collectively, TMF is a potential compound with chondroprotective activity by inhibiting both EP/cAMP/PKA signaling pathway and β-catenin signaling pathway.

Inhibition of β-Catenin Signaling Involved in the Biological Activities of a Lignan E2S Isolated from Carya cathayensis Fruits

Carya cathayensis is a fruit-bearing plant that belongs to the Juglandaceae family and is widely distributed throughout the world. It possesses various important biological activities. We have previously isolated an antitumor compound from the shell of C. cathayensis fruits and named it E2S ((E)-3-[(2S,3R)-2,3-dihydro-2-(4'-hydroxy-3'-methoxyphenyl)-3-hydroxymethyl-7-methoxy-1-benzo[b]furan-5-yl]-2-propenal). In this study, we investigated the antitumor activity of E2S against various human colorectal cancer cell lines (HCT116, HT29, SW480, LoVo). The results showed that E2S could significantly inhibit the growth of cancer cells in a dose-dependent manner, as well as disrupt the progression of the cell cycle. Mechanistic study revealed that E2S could decrease the protein levels of β-catenin and its downstream targets (such as c-myc, a key transcriptional target of β-catenin) in the cells. In addition, it also significantly suppressed β-catenin/TCF transcriptional activity. Taken together, the results suggested that E2S might partially exert an antiproliferative effect on human colorectal cancer cells by targeting β-catenin signaling, a finding that might potentially translate into a chemotherapeutic strategy for the treatment of cancer. It might also have implications for cancer prevention strategies.

Green tea polyphenol, (−)-epigallocatechin-3-gallate, induces toxicity in human skin cancer cells by targeting β-catenin signaling

The green tea polyphenol, (−)-epigallocatechin-3-gallate (EGCG), has been shown to have anti-carcinogenic effects in several skin tumor models, and efforts are continued to investigate the molecular targets responsible for its cytotoxic effects to cancer cells. Our recent observation that β-catenin is upregulated in skin tumors suggested the possibility that the anti-skin carcinogenic effects of EGCG are mediated, at least in part, through its effects on β-catenin signaling. We have found that treatment of the A431 and SCC13 human skin cancer cell lines with EGCG resulted in reduced cell viability and increased cell death and that these cytotoxic effects were associated with inactivation of β-catenin signaling. Evidence of EGCG-induced inactivation of β-catenin included: (i) reduced accumulation of nuclear β-catenin; (ii) enhanced levels of casein kinase1α, reduced phosphorylation of glycogen synthase kinase-3β, and increased phosphorylation of β-catenin on critical serine45,33/37 residues; and (iii) reduced levels of matrix metalloproteinase (MMP)-2 and MMP-9, which are down-stream targets of β-catenin. Treatment of cells with prostaglandin E2 (PGE2) enhanced the accumulation of β-catenin and enhanced β-catenin signaling. Treatment with either EGCG or an EP2 antagonist (AH6809) reduced the PGE2-enhanced levels of cAMP, an upstream regulator of β-catenin. Inactivation of β-catenin by EGCG resulted in suppression of cell survival signaling proteins. siRNA knockdown of β-catenin in A431 and SCC13 cells reduced cell viability. Collectively, these data suggest that induction of cytotoxicity in skin cancer cells by EGCG is mediated by targeting of β-catenin signaling and that the β-catenin signaling is upregulated by inflammatory mediators.

Naringin inhibits growth potential of human triple-negative breast cancer cells by targeting β-catenin signaling pathway

Triple-negative (ER-/PR-/HER2-) breast cancer (TNBC) is a severe clinical problem because of its relatively poorer prognosis, aggressive behavior and lack of targeted therapies. Naringin, a major flavonoid extracted from citrus fruits, has been reported to exert promising anticancer activities. However, the detailed antitumor mechanism of naringin still remains enigmatic. In this study, TNBC cell lines-based in vitro and in vivo models were used to explore the anticancer effect and mechanism of naringin. Our data demonstrated that naringin inhibited cell proliferation, and promoted cell apoptosis and G1 cycle arrest, accompanied by increased p21 and decreased survivin. Meanwhile, β-catenin signaling pathway was found to be suppressed by naringin. In contrast, over-expressing β-catenin by adenoviral vector system in TNBC cells reversed the antitumor activity of naringin, and regulated p21 and survivin. Correspondingly, the antitumor potential of naringin was also observed in naringin-treated MDA-MB-231 xenograft mice, while immunohistochemical analysis of tumors from naringin-treated mice showed higher expression of p21 and lower expression of survivin and active β-catenin. Taken together, these results indicate that naringin could inhibit growth potential of TNBC cells by modulating β-catenin pathway, which suggests naringin might be used as a potential supplement for the prevention and treatment of breast cancer.

Abstract 3683: Bioactive phytochemical proanthocyanidins target β-catenin signaling in preventing invasive potential of human melanoma cells.

Abstract Melanoma is the leading cause of death from skin diseases due to its propensity to metastasize. The overall incidence of melanoma is increasing in US, and is increasing rapidly in children. Since, melanoma is a highly malignant cancer with a potent capacity to metastasize distantly, an approach that decreases its metastatic potential may facilitate the development of an effective strategy for its prevention or treatment. Metastatic melanoma is often associated with activation of Wnt/β-catenin signaling pathway. To develop newer and more effective strategies for the prevention of melanoma metastasis, we have examined the inhibitory effect of proanthocyanidins, bioactive components of grape seeds, on cell migration of metastasis-specific human melanoma cell lines (A375 and Hs294t) and assessed whether Wnt/β-catenin signaling is the target of grape seed proanthocyanidins (GSPs). Using an in vitro invasion assay, we found that treatment of human melanoma cell lines with GSPs resulted in a dose-dependent inhibition of cell migration, which was associated with the degradation of cytosolic β-catenin, and subsequently reducing the nuclear accumulation of β-catenin (i.e., inactivation of β-catenin) and reducing the levels of matrix metalloproteinase (MMP) -2 and MMP-9 which are the down-stream targets of β-catenin and play a crucial role in cancer cell metastasis. GSPs increased the: (i) levels of casein kinase 1α, glycogen synthase kinase-3β and phosphorylated-β-catenin on critical serine residues (Ser45, Ser33/37 and Thr41), and (ii) binding of β-transducin repeat-containing proteins (β-TrCP) with phospho forms of β-catenin in melanoma cells. These molecular events lead to degradation and/or inactivation of β-catenin. To verify whether β-catenin is a potent molecular target of GSPs, the effect of GSPs was determined on β-catenin-activated (Mel1241) and β-catenin-inactivated (Mel1011) melanoma cells. Treatment of Mel1241 cells with GSPs or FH535, an inhibitor of Wnt/β-catenin pathway, significantly inhibited cell migration of Mel1241 cells, which was associated with the elevated levels of casein kinase 1α and glycogen synthase kinase-3β, and decreased accumulation of nuclear β-catenin and inhibition of MMP-2 and MMP-9 levels. However, this effect of GSPs and FH535 was not found in Mel1011 melanoma cells. These results indicate for the first time that grape seed proanthocyanidins inhibit the invasive potential of melanoma cells by targeting β-catenin signaling. This new insight into the anti-melanoma cell migration activity of GSPs could serve as the basis for chemoprevention or therapy of malignant melanoma in high risk human population. Citation Format: Santosh K. Katiyar, Mudit Vaid. Bioactive phytochemical proanthocyanidins target β-catenin signaling in preventing invasive potential of human melanoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3683. doi:10.1158/1538-7445.AM2013-3683

Considerations for Targeting β-Catenin Signaling in Fibrosis

Considerations for Targeting β-Catenin Signaling in Fibrosis