A Natural Small Molecule Mitigates Kidney Fibrosis by Targeting Cdc42-mediated GSK-3ß/ß-catenin Signaling.

Kidney fibrosis is a common fate of chronic kidney diseases (CKDs), eventually leading to renal dysfunction. Yet, no effective treatment for this pathological process has been achieved. During the bioassay-guided chemical investigation of the medicinal plant Wikstroemia chamaedaphne, a daphne diterpenoid, daphnepedunin A (DA), is characterized as a promising anti-renal fibrotic lead. DA shows significant anti-kidney fibrosis effects in cultured renal fibroblasts and unilateral ureteral obstructed mice, being more potent than the clinical trial drug pirfenidone. Leveraging the thermal proteome profiling strategy, cell division cycle 42 (Cdc42) is identified as the direct target of DA. Mechanistically, DA targets to reduce Cdc42 activity and down-regulates its downstream phospho-protein kinase Cζ(p-PKCζ)/phospho-glycogen synthase kinase-3ß (p-GSK-3ß), thereby promoting ß-catenin Ser33/37/Thr41 phosphorylation and ubiquitin-dependent proteolysis to block classical pro-fibrotic ß-catenin signaling. These findings suggest that Cdc42 is a promising therapeutic target for kidney fibrosis, and highlight DA as a potent Cdc42 inhibitor for combating CKDs.

Specific inhibitor of Wnt/beta-catenin pathway can alter behavioral responses in young rats with malformed cortices.

The Wnt/beta-catenin pathway plays a crucial role in regulating cellular processes and has been implicated in neural activity-dependent learning as well as anxiety. However, the role of this pathway in young children with abnormal cortical development is unknown. Cortical malformations at early development, behavioral abnormalities, and a susceptibility to seizures have been reported in rats prenatally exposed to methylazoxymethanol. In this study, we aimed to investigate whether we could improve the behavioral deficits in young rats with malformed cerebral cortices by modulation of the Wnt/beta-catenin pathway. We found a small molecule Wnt/beta-catenin inhibitor (CWP) that increased exploratory behavior in the open field test (P9, CWP 100 ug treatment, peripheral exploration, P = 0.011) and social behavior test (P12, CWP 250 ug treatment, distance traveled in center, P = 0.033) and decreased anxiety in fear conditioning. However, it did not reduce the susceptibility to seizures. After high dose (250 ug) CWP treatment at P12, phosphocreatine and glutathione (GSH) were decreased in the cortex at P15 (P = 0.021). These findings suggest that the role of Wnt/beta-catenin signaling in exploratory behavior and anxiety during early development warrants further investigation.

Design, synthesis, and biological evaluation of novel 8-substituted quercetin derivatives targeting the ß­catenin/B-cell lymphoma 9 interaction.

The ß-catenin/B-cell lymphoma 9 (BCL9) protein-protein interaction (PPI) is a potential target for aberrantly active Wnt/ß-catenin signaling which actively participates in initiating and progressing of many cancers. Herein, we discovered novel 8-substituted quercetin derivatives with potential inhibitory activities targeting ß-catenin/BCL9 PPI. Among all the derivatives, compound B4 displayed the most promising PPI inhibitory activity with an IC50 value of 2.25 µM in a competitive fluorescence polarization assay and a KD value of 1.44 µM for the ß-catenin protein. Furthermore, B4 selectively inhibited the growth of colorectal cancer (CRC) cells, suppressed the transactivation of Wnt signaling, and downregulated the expression of oncogenic Wnt target gene. Especially, B4 showed potent anti-CRC activity in vivo with the tumor growth inhibition (TGI) of 75.99 % and regulated the tumor immune microenvironment.

A natural carotenoid crocin exerts antidepressant action by promoting adult hippocampal neurogenesis through Wnt/ß-catenin signaling.

INTRODUCTION: Adult hippocampal neurogenesis (AHN) is acknowledged to play a critical role in depression. Emerging evidence suggests that the Wnt/ß-catenin pathway can modulate hippocampal neurogenesis. Crocin, a natural carotenoid, possesses antidepressant property. Yet, how it affects neurogenesis and exerts antidepressant response remains unknown. OBJECTIVE: To explore the role of AHN and Wnt/ß-catenin in the antidepressant action of crocin. METHODS: Depressive-related behaviors, including sucrose preference test (SPT), tail suspension test (TST), forced swimming test (FST), and sexual behaviors were performed following crocin treatment. Neurogenesis was characterized via immunohistochemistry, immunofluorescence, Golgi staining and electrophysiology approach. Wnt/ß-catenin signaling was examined with western blot analysis. The role of AHN Wnt/ß-catenin cascade in crocin's antidepressant response was assessed by conditional removal of glial fibrillary acidic protein (GFAP)-expressing newborn neural cells, temozolomide administration, microinfusion of Dkk1 or viral-mediated shRNA of Wnt3a. RESULTS: Crocin decreased the immobility duration in TST and FST without impairing the performance in sexual behaviors. Crocin boosted the proliferation and differentiation of progenitors, and promoted dendritic maturation and functional integration of hippocampal newborn neurons. Conditional removal of GFAP-expressing neural cells or temozolomide administration impaired the antidepressant response of crocin. Additionally, Wnt/ß-catenin signaling was promoted following crocin treatment. In chronic unpredictable mild stress (CUMS) murine model, crocin treatment displayed antidepressant response in SPT, FST and TST, and restored the neurogenesis levels and Wnt/ß-catenin signaling impaired by CUMS. Infusion of Dickkopf-1 (DKK1) or knockdown of Wnt3a in the hippocampus impaired the antidepressant response of crocin. CONCLUSION: Crocin exerted antidepressant response, which was dependent on enhancement of AHN and activation of the Wnt/ß-catenin pathway.

Structural optimization of novel Ras modulator for treatment of Colorectal cancer by promoting ß-catenin and Ras degradation.

Ras protein has been considered a fascinating target for anticancer therapy because its malfunction is closely related to cancer. However, Ras has been considered undruggable because of the failure to regulate its malfunction by controlling the Ras activation mechanism. Recently, Lumakras targeting the G12C mutation was approved, and therapeutic interest in Ras for anticancer therapy has been rejuvenated. Here, we present a series of compounds that inhibit Ras via a unique mechanism of action that exploits the relationship between the Wnt/ß-catenin pathway and Ras. KYA1797K (1) binds to axin to stabilize the ß-catenin destruction complex that causes the phosphorylation and subsequent degradation of Ras, similar to canonical ß-catenin regulation. Based on the chemical structure of 1, we performed a structural optimization and identified 3-(2-hydroxyethyl)-5-((6-(4-nitrophenyl)pyridin-2-yl)methylene)thiazolidine-2,4-dione (13d) as the most potent compound. 13d displayed antitumor effects in a colorectal cancer model with enhanced inhibition activity on Ras. The results of this study suggest that the further development of 13d could contribute to the development of Ras inhibitors with novel mechanisms of action.

Circular RNA hsa_circ_0000277 promotes tumor progression and DDP resistance in esophageal squamous cell carcinoma.

BACKGROUND: Circular RNAs (circRNAs) are well-known regulators of cancer progression and chemoresistance in various types of cancers. This study was performed to investigate the function of hsa_circ_0000277 in esophageal squamous cell carcinoma (ESCC). METHODS: RNA levels were analyzed via the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell Counting Kit-8 (CCK-8) assay was applied to determine cell proliferation and half maximal inhibitory concentration (IC50) of cisplatin (DDP). Colony formation ability was evaluated by colony formation assay. Cell cycle and apoptosis were measured using flow cytometry. RNA immunoprecipitation (RIP), pull-down assay and dual-luciferase reporter assays were performed for target interaction analysis. The protein levels were determined through western blot. Xenograft models were established for researching hsa_circ_0000277 function in vivo. RESULTS: Hsa_circ_0000277 expression was increased in ESCC cells and tissues, and it had important clinical significance. Downregulation of hsa_circ_0000277 repressed ESCC cell proliferation, colony formation, cell cycle, and DDP resistance. Hsa_circ_0000277 acted as a microRNA-873-5p (miR-873-5p) sponge and Sry-related high-mobility group box 4 (SOX4) was validated as a target of miR-873-5p. Moreover, hsa_circ_0000277/miR-873-5p axis and miR-873-5p/SOX4 axis regulated ESCC cell progression and DDP resistance. Hsa_circ_0000277/miR-873-5p axis activated SOX4/Wnt/ß-catenin signaling pathway. Hsa_circ_0000277 facilitated tumorigenesis and DDP resistance by miR-873-5p/SOX4 axis in vivo. CONCLUSION: These findings unraveled that hsa_circ_0000277 promoted ESCC progression and DDP resistance via miR-873-5p/SOX4/Wnt/ß-catenin axis, showing a specific molecular mechanism of carcinogenesis and chemoresistance in ESCC.

Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating ß­catenin and increases irinotecan sensitivity in colorectal cancer.

With >1.85 million cases and 850,000 deaths annually, colorectal cancer (CRC) is the third most common cancer detected globally. CRC is an aggressive malignancy with metastasis and, in spite of advances in improved treatment regimen, distant disease failure rates remain disappointingly high. Mucin­like 1 (MUCL1) is a small glycoprotein highly expressed mainly in breast cancer. The involvement of the MUCL1 protein in CRC progression and the underlying mechanism have been largely unknown. The aim of the present study was to investigate the MUCL1 expression profile and its functional significance in CRC. The Cancer Genome Atlas dataset revealed that MUCL1 expression was higher in colorectal tumor compared with normal tissues. MUCL1 was also revealed to be expressed in human CRC cell lines. The results demonstrated that MUCL1 promoted cell proliferation and colony formation, confirming its oncogenic potential. Silencing MUCL1 with short interfering RNA inhibited the protein expression of Bcl2 family proteins, such as Bcl2 and BclxL. Targeting MUCL1 resulted in significant inhibition in cell invasive and migratory behavior of HT­29 and SW620 cells. In addition, the expression of E­cadherin increased whereas the expression of vimentin decreased in MUCL1­silenced cells, confirming inhibition of epithelial­mesenchymal transition (EMT) process. Thus, it was revealed that MUCL1 plays a notable role in cell invasion and migration by inhibiting EMT in CRC. Mechanistically, MUCL1 drives ß­catenin activation by Ser­552 phosphorylation, nuclear accumulation and transcriptional activation. Targeting MUCL1 increases the drug sensitivity of CRC cells towards irinotecan. These findings thus demonstrated that MUCL1 acts as a modifier of other pathways that play an important role in CRC progression and MUCL1 was identified as a potential target for CRC therapeutics.

A pyridinium­type fullerene derivative suppresses primary effusion lymphoma cell viability via the downregulation of the Wnt signaling pathway through the destabilization of ß­catenin.

Primary effusion lymphoma (PEL) is defined as a rare subtype of non­Hodgkin's B cell lymphoma, which is caused by Kaposi's sarcoma­associated herpesvirus (KSHV) in immunosuppressed patients. PEL is an aggressive type of lymphoma and is frequently resistant to conventional chemotherapeutics. Therefore, the discovery of novel drug candidates for the treatment of PEL is of utmost importance. In order to discover potential novel anti­tumor compounds against PEL, the authors previously developed a pyrrolidinium­type fullerene derivative, 1,1,1',1'­tetramethyl [60]fullerenodipyrrolidinium diiodide (derivative #1), which induced the apoptosis of PEL cells via caspase­9 activation. In the present study, the growth inhibitory effects of pyrrolidinium­type (derivatives #1 and #2), pyridinium­type (derivatives #3 and #5 to #9) and anilinium­type fullerene derivatives (derivative #4) against PEL cells were evaluated. This analysis revealed a pyridinium­type derivative (derivative #5; 3­â€‹5'­(etho xycarbonyl)­1',5'­dihydro­2'H­[5,6]fullereno­C60­Ih­[1,9­c]pyrrol­2'­yl]­1­methylpyridinium iodide), which exhibited antitumor activity against PEL cells via the downregulation of Wnt/ß­catenin signaling. Derivative #5 suppressed the viability of KSHV­infected PEL cells compared with KSHV­uninfected B­lymphoma cells. Furthermore, derivative #5 induced the destabilization of ß­catenin and suppressed ß­catenin­TCF4 transcriptional activity in PEL cells. It is known that the constitutive activation of Wnt/ß­catenin signaling is essential for the growth of KSHV­infected cells. The Wnt/ß­catenin activation in KSHV­infected cells is mediated by KSHV latency­associated nuclear antigen (LANA). The data demonstrated that derivative #5 increased ß­catenin phosphorylation, which resulted in ß­catenin polyubiquitination and subsequent degradation. Thus, derivative #5 overcame LANA­mediated ß­catenin stabilization. Furthermore, the administration of derivative #5 suppressed the development of PEL cells in the ascites of SCID mice with tumor xenografts derived from PEL cells. On the whole, these findings provide evidence that the pyridinium­type fullerene derivative #5 exhibits antitumor activity against PEL cells in vitro and in vivo. Thus, derivative #5 may be utilized as a novel therapeutic agent for the treatment of PEL.

Arsenic sulfide inhibits the progression of gastric cancer through regulating the circRNA_ASAP2/Wnt/ß-catenin pathway.

In our paper, the effects of As4S4 treatments on the growth and migration of gastric cancer (GC) cells were explored, and the potential underlying molecular mechanisms were also identified. Cell viability was evaluated by cell counting kit 8 assay. The expression of Ki-67 was examined using immunofluorescence staining. Cell apoptosis was assessed by flow cytometry. The migratory and invasion abilities of cells were determined using Transwell assay. The mRNA and protein levels of related gene were examined by RT-qPCR and western blotting, respectively. CircRNAs chip was performed to identify the differentiated expression of circRNAs in GC cells following the treatment with As4S4. Our results revealed that the proliferation, migration and invasion of GC cells were remarkably suppressed by the treatment with As4S4, while cell apoptosis was promoted. Furthermore, circRNA_ASAP2 was a novel target of As4S4 in GC, and it is involved in As4S4-modulated biological behavior alterations in GC cells. In addition, the activities of the Wnt/ß-catenin signaling in GC cells were affected by the overexpression circRNA_ASAP2 and the treatment with As4S4. Moreover, the behavior changes in GC cells caused by the knockdown of circRNA_ASAP2 were reversed by the treatment with Wnt agonist SKL2001. In summary, As4S4 could function as an antitumor agent in GC through regulating the circRNA_ASAP2/Wnt/ß-catenin pathway, which in turn influences the growth and metastasis of GC cells.

Sequential modulation of the Wnt/ß-catenin signaling pathway enhances tumor-intrinsic MHC I expression and tumor clearance.

BACKGROUND: Progress in immunotherapy use for gynecologic malignancies is hampered by poor tumor antigenicity and weak T cell infiltration of the tumor microenvironment (TME). Wnt/ß-catenin pathway modulation demonstrated patient benefit in clinical trials as well as enhanced immune cell recruitment in preclinical studies. The purpose of this study was to characterize the pathways by which Wnt/ß-catenin modulation facilitates a more immunotherapy-favorable TME. METHODS: Human tumor samples and in vivo patient-derived xenograft and syngeneic murine models were administered Wnt/ß-catenin modulating agents DKN-01 and CGX-1321 individually or in sequence. Analytical methods included immunohistochemistry, flow cytometry, multiplex cytokine/chemokine array, and RNA sequencing. RESULTS: DKK1 blockade via DKN-01 increased HLA/MHC expression in human and murine tissues, correlating with heightened expression of known MHC I regulators: NFkB, IL-1, LPS, and IFNy. PORCN inhibition via CGX-1321 increased production of T cell chemoattractant CXCL10, providing a mechanism for observed increases in intra-tumoral T cells. Diverse leukocyte recruitment was noted with elevations in B cells and macrophages, with increased tumor expression of population-specific chemokines. Sequential DKK1 blockade and PORCN inhibition decreased tumor burden as evidenced by reduced omental weights. CONCLUSIONS: Wnt/ß-catenin pathway modulation increases MHC I expression and promotes tumor leukocytic infiltration, facilitating a pro-immune TME associated with decreased tumor burden. This intervention overcomes common tumor immune-evasion mechanisms and may render ovarian tumors susceptible to immunotherapy.

Virtual Screening Directly Identifies New Fragment-Sized Inhibitors of Carboxylesterase Notum with Nanomolar Activity.

Notum is a negative regulator of Wnt signaling acting through the hydrolysis of a palmitoleoylate ester, which is required for Wnt activity. Inhibitors of Notum could be of use in diseases where dysfunctional Notum activity is an underlying cause. A docking-based virtual screen (VS) of a large commercial library was used to shortlist 952 compounds for experimental validation as inhibitors of Notum. The VS was successful with 31 compounds having an IC50 < 500 nM. A critical selection process was then applied with two clusters and two singletons (1-4d) selected for hit validation. Optimization of 4d guided by structural biology identified potent inhibitors of Notum activity that restored Wnt/ß-catenin signaling in cell-based models. The [1,2,4]triazolo[4,3-b]pyradizin-3(2H)-one series 4 represent a new chemical class of Notum inhibitors and the first to be discovered by a VS campaign. These results demonstrate the value of VS with well-designed docking models based on X-ray structures.

Excess folic acid supplementation before and during pregnancy and lactation activates ß-catenin in the brain of male mouse offspring.

Folic acid (FA) supplementation in early pregnancy is recommended to protect against birth defects. But excess FA has exhibited neurodevelopmental toxicity. We previously reported that the mice treated with 2.5-fold the dietary requirement of FA one week before mating and throughout pregnancy and lactation displayed abnormal behaviors in the offspring. Here we found the levels of non-phosphorylated ß-catenin (active) were increased in the brains of weaning and adult FA-exposed offspring. Meanwhile, demethylation of protein phosphatase 2 A catalytic subunit (PP2Ac), which suppresses its enzyme activity in regulatory subunit dependent manner, was significantly inhibited. Among the upstream regulators of ß-catenin, PI3K/Akt/GSK-3ß but not Wnt signaling was stimulated in FA-exposed brains only at weaning. In mouse neuroblastoma N2a cells, knockdown of PP2Ac or leucine carboxyl methyltransferase-1 (LCMT-1), or overexpression of PP2Ac methylation-deficient mutant decreased ß-catenin dephosphorylation. These results suggest that excess FA may activate ß-catenin via suppressing PP2Ac demethylation, providing a novel mechanism for the influence of FA on neurodevelopment.

Atorvastatin inhibits proliferation and promotes apoptosis of colon cancer cells via COX-2/PGE2/ß-Catenin Pathway.

PURPOSE: To explore the effects of atorvastatin (ATST) on the proliferation and apoptosis of colon cancer cells through the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2)/ß-catenin pathway. METHODS: HCT116 cells were cultured and transfected, and they were treated with ATST at different concentrations for different time. The association between the expressions of COX-2 and PGE2 and the survival time of patients with colon cancer was analyzed via Kaplan-Meier survival analysis. Then the protein expressions of COX-2, ß-catenin and apoptosis-related molecules in HCT116 cells were determined using Western blotting, and the proliferation of HCT116 cells was detected via cell counting kit-8 (CCK-8) assay. RESULTS: There was a significant difference in the survival rate between HCT116 cells treated with 30 µM ATST and those treated with 0 µM ATST. The survival time was obviously longer in patients with low expressions of COX-2 and PGE2 than that those with high expressions of COX-2 and PGE2. Low expressions of COX-2 and PGE2 in colon cancer tissues indicate a longer survival time. Moreover, a positive correlation was found between HCT116 cell density and COX-2 level, HCT116 cell density and PGE2 level, and COX-2 and PGE2 levels. ATST could down-regulate COX-2 and ß-catenin, and knocking down COX-2 could lower ß-catenin. After treatment with ATST and ATST + anti-COX-2, the activity of cleaved caspase-9, caspase-3 and PARP was remarkably enhanced, suggesting that ATST and ATST + anti-COX-2 can promote apoptosis of HCT116 cells. It was found that ATST and ATST + anti-COX-2 could also inhibit the proliferation of HCT116 cells.

VALD-3 inhibits proliferation and induces apoptosis of colorectal cancer cells via upregulating tumor suppressor activity of p53 to inhibit Wnt/ß-catenin signal pathway.

Colorectal cancer is the third most common malignant tumor and a leading cause of cancer death. Currently lacks effective therapies available to improve the prognosis. In the present study, VALD-3, an important Schiff base ligand from o-vanillin derivatives was evaluated for its anti-cancer activity in vitro and in vivo against colorectal cancer. The effect of VALD-3 on colorectal cancer cells proliferation was assessed using MTT assay and the cell migration was evaluated using wound healing scratch assay. The appearance of apoptotic colorectal cancer cells was detected by flowcytometry analysis. Morphological changes caused by VALD-3 induced apoptosis were also observed by Hoechst 33258 staining. The flow cytometry assay was also used to measure cell cycle arrest. The expression levels of TP53 and Bad were analyzed using quantitative real-time PCR. Protein expression of P53, Wnt/ß-catenin signaling pathway proteins, apoptosis proteins and cell cycle-related protein were viewed by Western blotting. In addition, HT-29 cells xenograft tumor model was used for the study in vivo. Immunohistochemistry (IHC) staining was employed to detect the P53 protein expression. The results showed that VALD-3 obviously inhibited the proliferation and migration for colorectal cancer cells. In addition, flow cytometry analysis demonstrated that VALD-3 markedly increased early and late apoptosis on colorectal cancer cells, respectively. VALD-3 induced cell cycle arrest at the G0/G1 phase. Most importantly, tumor growth in HT-29 xenograft mice was suppressed by VALD-3, but no significant change in body weight. As confirmed by IHC staining from tumor tissue, the P53 proteins expression increased. These results suggested that VALD-3 represses cell proliferation and induces apoptosis associated with upregulating tumor suppressor activity of p53 to inhibit Wnt/ß-catenin signal pathway, and it is a potential anticancer agent for colorectal cancer.

Doxazosin and erlotinib have anticancer effects in the endometrial cancer cell and important roles in ERα and Wnt/ß-catenin signaling pathways.

ERα and Wnt/ß-catenin pathways are critical for the progression of most endometrial cancers. We aimed to investigate the cytotoxic and apoptotic effects of tamoxifen and quinazoline derivative drugs of doxazosin and erlotinib, and their roles in ERα and Wnt/ß-catenin signaling pathways in human endometrial cancer RL 95-2 cell. 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay and xCELLigence systems were performed to evaluate cytotoxicity. Furthermore, apoptotic induction was tested by Annexin V analysis. Caspase-3 and -9 activity and changes in the mitochondrial membrane potential were evaluated. The level of reactive oxygen species was measured by incubating with dichlorofluorescein diacetate. Protein ratios of p-ERα/ERα, GSK3ß/p-GSK3ß, and p-ß-catenin/ß-catenin and expression levels of ESR1, EGFR, c-Myc genes were evaluated to elucidate mechanisms in signaling pathways. We found that the tested drugs showed cytotoxic and apoptotic effects in the cells. Doxazosin significantly reduced ESR1 expression, slightly reduced the p-ß-catenin/ß-catenin ratio and c-Myc expression. Erlotinib significantly increased c-Myc expression while significantly decreasing the p-ß-catenin/ß-catenin and p-ERα/ERα ratio, and ESR1 expression. However, we observed that the cells develop resistance to erlotinib over a certain concentration, suggesting that ERα, ESR1, EGFR, and c-Myc may be a new target for overcoming drug resistance in the treatment of endometrial cancer. We also observed that erlotinib and doxazosin play an important role in the ERα signaling pathway and can act as potent inhibitors of PKA and/or tyrosine kinase in the Wnt/ß-catenin signaling pathway in RL 95-2 cell. In conclusion, doxazosin and erlotinib may have a possible therapeutic potential in human endometrial cancer.

The Wnt/ß-Catenin Pathway Regulated Cytokines for Pathological Neuropathic Pain in Chronic Compression of Dorsal Root Ganglion Model.

Neuropathic pain is one of the important challenges in the clinic. Although a lot of research has been done on neuropathic pain (NP), the molecular mechanism is still elusive. We aimed to investigate whether the Wnt/ß-catenin pathway was involved in NP caused by sustaining dorsal root ganglion (DRG) compression with the chronic compression of dorsal root ganglion model (CCD). Our RNA sequencing results showed that several genes related to the Wnt pathway have changed in DRG and spinal cord dorsal horn (SCDH) after CCD surgery. Therefore, we detected the activation of the Wnt/ß-catenin pathway in DRG and SCDH and found active ß-catenin significantly upregulated in DRG and SCDH 1 day after CCD surgery and peaked on days 7-14. Immunofluorescence results also confirmed nuclear translocalization of active ß-catenin in DRG and SCDH. Additionally, rats had obvious mechanical induced pain after CCD surgery and the pain was significantly alleviated after the application of the Wnt/ß-catenin pathway inhibitor XAV939. Furthermore, we found that the levels of proinflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-18 (IL-18) were significantly elevated in CCD rat serum, while the levels of them were correspondingly decreased after the Wnt/ß-catenin pathway being inhibited. The results of Spearman correlation coefficient analysis showed that the levels of TNF-α and IL-18 were negatively correlated with the mechanical withdrawal thresholds (MWT) after CCD surgery. Collectively, our findings suggest that the Wnt/ß-catenin pathway plays a critical role in the pathogenesis of NP and may be an effective target for the treatment of NP.

COXIBs and 2,5-dimethylcelecoxib counteract the hyperactivated Wnt/ß-catenin pathway and COX-2/PGE2/EP4 signaling in glioblastoma cells.

BACKGROUND: Glioblastoma (GBM) is the deadliest and the most common primary brain tumor in adults. The invasiveness and proliferation of GBM cells can be decreased through the inhibition of Wnt/ß-catenin pathway. In this regard, celecoxib is a promising agent, but other COXIBs and 2,5-dimethylcelecoxib (2,5-DMC) await elucidation. Thus, the aim of this study was to analyze the impact of celecoxib, 2,5-DMC, etori-, rofe-, and valdecoxib on GBM cell viability and the activity of Wnt/ß-catenin pathway. In addition, the combination of the compounds with temozolomide (TMZ) was also evaluated. Cell cycle distribution and apoptosis, MGMT methylation level, COX-2 and PGE2 EP4 protein levels were also determined in order to better understand the molecular mechanisms exerted by these compounds and to find out which of them can serve best in GBM therapy. METHODS: Celecoxib, 2,5-DMC, etori-, rofe- and valdecoxib were evaluated using three commercially available and two patient-derived GBM cell lines. Cell viability was analyzed using MTT assay, whereas alterations in MGMT methylation level were determined using MS-HRM method. The impact of COXIBs, in the presence and absence of TMZ, on Wnt pathway was measured on the basis of the expression of ß-catenin target genes. Cell cycle distribution and apoptosis analysis were performed using flow cytometry. COX-2 and PGE2 EP4 receptor expression were evaluated using Western blot analysis. RESULTS: Wnt/ß-catenin pathway was attenuated by COXIBs and 2,5-DMC irrespective of the COX-2 expression profile of the treated cells, their MGMT methylation status, or radio/chemoresistance. Celecoxib and 2,5-DMC were the most cytotoxic. Cell cycle distribution was altered, and apoptosis was induced after the treatment with celecoxib, 2,5-DMC, etori- and valdecoxib in T98G cell line. COXIBs and 2,5-DMC did not influence MGMT methylation status, but inhibited COX-2/PGE2/EP4 pathway. CONCLUSIONS: Not only celecoxib, but also 2,5-DMC, etori-, rofe- and valdecoxib should be further investigated as potential good anti-GBM therapeutics.

Effect of casein kinase 1α inhibition on autophagy flux and the AKT/phospho-ß-catenin (S552) axis in HCT116, a RAS-mutated colorectal cancer cell line.

The Wnt/ß-catenin pathway, which interferes with cell proliferation, differentiation, and autophagy, is commonly dysregulated in colorectal cancer (CRC). Mutation of the RAS oncogene is the most prevalent genetic alteration in CRC and has been linked to activation of protein kinase B (AKT) signaling. Phosphorylation of ß-catenin at Ser 552 by AKT contributes to ß-catenin stability, transcriptional activity, and increase of cell proliferation. Casein kinase 1 alpha (CK1α) is an enzyme that simultaneously regulates Wnt/ß-catenin and AKT. The link of the AKT and Wnt pathway to autophagy in RAS-mutated CRC cells has not well identified. Therefore, we investigated how pharmacological CK1α inhibition (D4476) is involved in regulation of autophagy, Wnt/ß-catenin, and AKT pathways in RAS-mutated CRC cell lines. qRT-PCR and immunoblotting experiments revealed that phospho-AKT (S473) and phospho-ß-catenin (S552) are constitutively increased in RAS-mutated CRC cell lines, in parallel with augmented CK1α expression. The results also showed that D4476 significantly reduced the AKT/phospho-ß-catenin (S552) axis concomitantly with autophagy flux inhibition in RAS-mutated CRC cells. Furthermore, D4476 significantly induced apoptosis in RAS-mutated CRC cells. In conclusion, our results indicate that CK1α inhibition reduces autophagy flux and promotes apoptosis by interfering with the AKT/phospho-ß-catenin (S552) axis in RAS-mutated CRC cells.

Ginsenoside Rb1 Facilitates Browning by Repressing Wnt/ß-Catenin Signaling in 3T3-L1 Adipocytes.

BACKGROUND The discovery of browning in white adipose tissue has provided new ideas for treating obesity. Many studies have reported that ginsenoside Rb1 (G-Rb1) has activity against diabetes, inflammation, and obesity, but further investigation is needed on the effect and mechanism of G-Rb1 on browning. MATERIAL AND METHODS We treated 3T3-L1 adipocytes with 0-200 µM G-Rb1, and 0.5 µM Compound 3f and 30 µM SKL2001 were used to activate Wnt/b-catenin signaling. Adipocyte activity was evaluated by Cell Counting Kit-8. Oil Red O staining was used to detect the lipid droplets. Quantitative real-time polymerase chain reaction was used to measure the expression of Cd-137, Cited-1, Txb-1, Prdm-16, and Ucp-1 mRNA. Western blotting was used to measure the expression of Ucp-1, pGSK-3ß (Ser 9), GSK- 3ß, and ß-catenin proteins. The expression of Ucp-1 was also detected with immunofluorescence. RESULTS Adipocyte activity was not affected by 0-100 µM G-Rb1. However, G-Rb1 dose-dependently reduced the accumulation of lipid droplets; increased the expression of Cd-137, Cited-1, Txb-1, Prdm-16, and Ucp-1 mRNA; and increased the expression of Ucp-1, pGSK-3ß (Ser 9), GSK-3ß, and ß-catenin proteins. The accumulation of lipid droplets and the expression of Ucp-1 protein decreased as b-catenin increased. CONCLUSIONS G-Rb1 at various concentrations (0-100 µM) promoted the browning of adipocytes in a dose-dependent manner. Further, we confirmed that activation of Wnt/ß-catenin signaling could inhibit browning. Therefore, the browning promoted by G-Rb1 may be associated with the inhibition of Wnt/ß-catenin signaling.

Exendin-4 Attenuates Remodeling in the Remote Myocardium of Rats After an Acute Myocardial Infarction by Activating ß-Arrestin-2, Protein Phosphatase 2A, and Glycogen Synthase Kinase-3 and Inhibiting ß-Catenin.

PURPOSE: This study tested if the protective anti-remodeling effect of GLP-1 agonist Exendin-4 after an acute myocardial infarction (MI) in rats involves inhibition of the Wnt1/ß-catenin signaling pathway. METHODS: Rats were divided into sham, sham + Exendin-4 (10 µg/day, i.p), MI, and MI + Exendin-4. MI was introduced to rats by permanent left anterior descending coronary artery (LAD) ligation. RESULTS: On day 7 post-infraction, MI rats showed LV dysfunction with higher serum levels of cardiac markers. Their remote myocardia showed increased mRNA and protein levels of collagen I/III with higher levels of reactive oxygen species (ROS) and inflammatory cytokines, as well as protein levels of Wnt1, phospho-Akt, transforming growth factor (TGF-ß1), Smad, phospho-Smad3, α-SMA, caspase-3, and Bax. They also showed higher protein levels of phospho-glycogen synthase kinase-3ß (p-GSK3ß), as well as total, phosphorylated, and nuclear ß-catenin with a concomitant decrease in the levels of cyclic adenosine monophosphate (cAMP), mRNA of manganese superoxide dismutase (MnSOD), and protein levels of Bcl-2, ß-arrestin-2, and protein phosphatase-2 (PP2A). Administration of Exendin-4 to MI rats reduced the infarct size and reversed the aforementioned signaling molecules without altering protein levels of TGF-1ß and Wnt1 or Akt activation. Interestingly, Exendin-4 increased mRNA levels of MnSOD, protein levels of ß-arrestin-2 and PP2A, and ß-catenin phosphorylation but reduced the phosphorylation of GSK3ß and Smad3, and total ß-catenin levels in the LV of control rats. CONCLUSION: Exendin-4 inhibits the remodeling in the remote myocardium of rats following acute MI by attenuating ß-catenin activation and activating ß-arrestin-2, PP2A, and GSK3ß. Graphical Abstract A graphical abstract that illustrates the mechanisms by which Exendin-4 inhibits cardiac remodeling in remote myocardium of left ventricle MI-induced rats. Mechanisms are assumed to occur in the cardiomyocytes and/or other resident cells such as fibroblast. Β-catenin activation and nuclear translocation are associated with increased synthesis of inflammatory cytokines and transforming growth factor ß-1 (TGF-ß1). GSK3ß is inhibited by phosphorylation at Ser9. Under normal conditions, ß-catenin is degraded in the cytoplasm by the active GSK3ß-dependent degradation complex (un-phosphorylated) which usually phosphorylates ß-catenin at Ser33/37/Thr41. After MI, TGF-ß1, and Wnt 1 levels are significantly increased, the overproduction of Wnt1 induces ß-catenin stabilization and nuclear translocation through increasing the phosphorylation of disheveled (DVL) protein which in turn phosphorylates and inhibits GSK3ß. TGF-ß1 stimulates the phosphorylation of Smad-3 and subsequent nuclear translocation to activate the transcription of collage 1/III and α-smooth muscle actin (α-SMA). Besides, TGF-ß1 stabilizes cytoplasmic ß-catenin levels indirectly by phosphorylation of Akt at Thr308-induced inhibition of GSK3ß by increasing phosphorylation of Ser9. Exendin-4, and possibly through G protein-coupled receptors (GPCRs), increases levels of cAMP and upregulates ß-arrestin-2 levels. Both can result in a positive inotropic effect. Besides, ß-arrestin-2 can stimulate PP2A to dephosphorylation Smad3 (inhibition) and GSK3ß (activation), thus reduces fibrosis and prevents the activation of ß-catenin and collagen deposition.