ATDC promotes the growth and invasion of hepatocellular carcinoma cells by modulating GSK‐3β/Wnt/β‐catenin signalling

Accumulating evidence has suggested that the ataxia telangiectasia group D complementing (ATDC) gene is an emerging cancer‐related gene in multiple human cancer types. However, little is known about the role of ATDC in hepatocellular carcinoma (HCC). In this study, we aimed to investigate the expression level, biological function and underlying mechanism of ATDC in HCC. The expression of ATDC in HCC cells was detected by quantitative real‐time polymerase chain reaction and western blot analysis. Cell growth was determined by cell counting kit‐8 assay and colony formation assay. Cell invasion was assessed by Transwell invasion assay. The activation status of Wnt/β‐catenin signalling was evaluated by the luciferase reporter assay. Functional experiments showed that the silencing of ATDC expression significantly suppressed the growth and invasion of HCC cells, whereas the overexpression of ATDC promoted the growth and invasion of HCC cells in vitro. Moreover, we showed that ATDC overexpression promoted the phosphorylation of glycogen synthase kinase (GSK)‐3β and resulted in the activation of Wnt/β‐catenin signalling. Notably, the inhibition of GSK‐3β activity significantly abrogated the tumour suppressive effect of ATDC silencing, while the silencing of β‐catenin partially reversed the oncogenic effect of ATDC overexpression. Taken together, these findings reveal an oncogenic role of ATDC in HCC and show that the suppression of ATDC impedes the growth and invasion of HCC cells associated with the inactivation of Wnt/β‐catenin signalling. Our study suggests that ATDC may serve as a potential therapeutic target for HCC.     

BRD4 promotes glioma cell stemness via enhancing miR‐142‐5p‐mediated activation of Wnt/β‐catenin signaling

The bromodomain protein BRD4 exerts carcinogenic effects in many cancers. However, its roles in glioma occurrence are still confused. Here, it is found that BRD4 expression is increased in glioma tissues and negatively correlated with the overall survival of glioma patients. We construct cellular experiments indicating that BRD4 promotes glioma cell stemness by analyzing ALDH1 activity, master stemness regulator expression, and sphere formation ability. Mechanistically, BRD4 knockdown triggers a switch of miR‐142‐5p promoter methylation, which targets Wnt3a and thus further inactivates Wnt/β‐catenin signaling. Importantly, inhibition of miR‐142‐5p or reactivation of Wnt/β‐catenin signaling rescues the inhibition of BRD4 knockdown on glioma cell stemness. As a result, these results not only indicate an unforeseen connection between BRD4, miR‐142‐5p, and Wnt/β‐catenin signaling, but also reveal a promising epigenetic‐based therapeutic strategy that might be explored for glioma patients.     

Resveratrol attenuates hyperoxia‐induced oxidative stress,inflammation and fibrosis and suppresses Wnt/β‐catenin signalling in lungs of neonatal rats

Although survival rate of infants born prematurely has been raised by supplemental oxygen treatment, it is followed by high morbidity of hyperoxia‐induced bronchopulmonary dysplasia. In this study, the effect of resveratrol on the lung injury was evaluated in hyperoxia‐exposed rats of preterm birth. The results demonstrated that hyperoxia led to thickened alveolar wall, simplified alveolar architecture and fibrosis. In addition, elevated methane dicarboxylic aldehyde level, decreased glutathione level and superoxide dismutase activity were also found in hyperoxic lungs, as well as the increased tumor necrosis factor‐α, interleukin‐1β and interleukin‐6 in the bronchoalveolar lavage fluid. Fibrotic‐associated proteins transforming growth factor‐β1, α‐smooth muscle actin, collagen I and fibronectin deposition were also found in interstitial substance of lungs. Furthermore, Wnt/β‐catenin signalling was found to be active in hyperoxia‐induced lungs. In addition, expression of SP‐C was increased and T1α was decreased in hyperoxia‐exposed lungs. Resveratrol intraperitoneal administration alleviated hyperoxia‐induced histological injury of lungs, regulated redox balance, decreased pro‐inflammatory cytokine release, and down‐regulated expression of fibrotic‐associated proteins. Furthermore, Wnt/β‐catenin signalling was also suppressed by resveratrol, as represented by diminished expression of lymphoid enhancer factor‐1, Wnt induced signalling protein‐1 and cyclin D1. In addition, the increase of SP‐C and decrease of T1α expression was prevented as well. The present study showed that resveratrol could protect lungs from hyperoxia‐induced injury through its antioxidant, anti‐inflammatory and anti‐fibrotic effects. The transdifferentiation of alveolar epithelial type II cells to alveolar epithelial type I cells promotion and Wnt/β‐catenin signalling suppression are also involved in the protective effect.     

TRIB3 confers glioma cell stemness via interacting with β‐catenin

Here, we aim to explore whether tribbles pseudokinase 3 (TRIB3) enhances glioma cell stemness. TRIB3 was overexpressed in glioma tissues and cell‐formed spheres, positively correlated with the size and grade. Additionally, TRIB3 expression displayed a negative correlation with the overall survival rate of glioma patients. Moreover, TRIB3 knockdown reduced the stemness of nonadherent spheres, evident by the decreased sphere‐forming ability, stemness master expression, and ALDH1 activity, while TRIB3 overexpression enhanced the stemness of adherent cells, which was rescued by β‐catenin knockdown. Mechanistically, TRIB3 activated β‐catenin signaling via physically interacting with β‐catenin. This study suggests that the TRIB3‐β‐catenin interaction is responsible for glioma cell stemness.     

miR‐375‐3p/YWHAZ/β‐catenin axis regulates migration,invasion, EMT in gastric cancer cells

YWHAZ (14‐3‐3ζ) plays crucial roles in regulating proliferation, apoptosis, migration, and invasion of gastric cancer (GC) cells. However, its extensive roles and potential mechanisms in GC cells remain unknown, and need to be researched deeply. In this study, we focus on the role of miR‐375/YWHAZ axis in migration, invasion and epithelial‐to‐mesenchymal transition (EMT) of GC cells. YWHAZ level was assessed by western blot and qPCR assays in GC cells. Scratch and transwell assays were used to determine the migration and invasion of GC cells. The protein levels of correlative molecules were detected by western blot. The regulation of miR‐375 on the expression of its target gene YWHAZ was verified by dual‐luciferase report system. According to the results, knockdown of YWHAZ inhibited the migration, invasion and EMT of GC cells. Moreover, silencing of YWHAZ restrained the activation of wnt/β‐catenin signalling pathway. YWHAZ was confirmed to be a target gene of miR‐375, and its expression was regulated by miR‐375 in GC cells. Transfection of miR‐375 inhibitor promoted the migration, invasion, EMT and activation of wnt/β‐catenin pathway in GC cells, which was suppressed by inhibition of YWHAZ. Taken together, this study suggests that miR‐375/YWHAZ axis may be served as a novel therapeutic target for GC patients.     

Estrogen and ERα enhanced β‐catenin degradation and suppressed its downstream target genes to block the metastatic function of HA22T hepatocellular carcinoma cells via modulating GSK‐3β and β‐TrCP expression

In our previous experiments, we found β‐catenin was highly expressed in the tumor area with high invasive ability and poor prognosis. In this study, we have examined the mechanism by which ERα regulates β‐catenin expression as well as the metastasis ability of hepatocellular cancer HA22T cells. To identify whether the anticancer effect of estrogen and ERα is mediated through suppression of β‐catenin expression, we co‐transfected pCMV‐β‐catenin and ERα into HA22T cells, and determined the cell motility by wound healing, invasion, and migration assays. Results showed that estrogen and/or ERα inhibited β‐catenin gene expression and repressed HA22T cell motility demonstrated that similar data was observed in cells expressing the ERα stable clone. Moreover, we examined the protein‐protein interaction between ERα and β‐catenin by immunostain, co‐immunoprecipitation, and Western blotting. E2 enhanced the binding of ERα with β‐catenin and then triggered β‐catenin to bind with E3 ligase (βTrCP) to promote β‐catenin degradation. Finally by employing systematic ChIP studies, we showed ERα can interact directly with the β‐catenin promoter region following E2 treatment. All our results reveal that estrogen and ERα blocked metastatic function of HA22T cells by modulating GSK3β and βTrCP expression and further enhanced β‐catenin degradation and suppressed its downstream target genes. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 519–529, 2017.     

Different effects of Wnt/β‐catenin activation and parathyroid hormone on diaphyseal and metaphyseal in the early phase of femur bone healing of mice

Parathyroid hormone (PTH) and agents related to the manipulation of Wnt/β‐catenin signalling are two promising anabolic anti‐osteoporotic therapies that have been shown to promote the healing of bone fractures. Now, it is widely accepted that cortical bone and trabecular bone are two different compartments, and should be treated as separate compartments in pathological processes, such as fracture healing. It is currently unknown whether PTH and the activation of β‐catenin signalling would demonstrate different effects on cortical bone and trabecular bone healing. In the current study, single 0.6‐mm cortex holes were made in the femur metaphysis and diaphysis of mice, and then, PTH application and β‐catenin activation were used to observe the promoting effect on bone healing. The effects of β‐catenin and PTH signalling on fracture healing were observed by X‐ray and CT at 3, 6, and 14 days after fracture, and the levels of β‐catenin were detected by RT‐PCR assay, and the number of specific antigen‐positive cells of BRDU, OCN, RUNX2 was counted by immunohistochemical staining. While β‐catenin activation and PTH were found to demonstrate similar effects on accelerating metaphyseal bone healing, activation of β‐catenin showed a more striking effect than PTH on promoting diaphyseal bone healing. These findings might be helpful for selecting proper medication to accelerate fracture healing of different bone compartments.     

Transforming growth factor β‐activated kinase 1 inhibitor suppresses the proliferation in triple‐negative breast cancer through TGF‐β/TGFR pathway

Breast cancer is one of the most invasive cancer types in female population. The functional activity of Transforming growth factor β‐activated kinase 1 (TAK1) in breast cancer progression increasingly attracts attention as it provides a potential target for antibreast cancer drug development. However, the fundamental role of TAK1 for triple‐negative breast cancer (TNBC) progression and the effect of potential anti‐TAK1 drug candidate needs to be further evaluated. Herein, we focused on the role of TAK1 in human breast cancer cells, and we hypothesized that the inhibition of TAK1 activation can repress the growth of human TNBC cells. We found that the TAK1 is robustly activated within cancer cell population of clinic‐derived TNBC samples and the human breast cancer cell lines in culture. Furthermore, we determined the effect of 5Z‐7‐oxozeaenol (5Z‐O), a TAK1‐specific small molecule inhibitor, on proliferation of human TNBC cell line. 5Z‐O treatment significantly suppressed the proliferation of human TNBC cells. Collectively, these demonstrate the role of TAK1 in human breast cancer and the antiproliferate effect of TAK1 inhibitor. Our study sets the stage for further research on TAK1 as a promising target for development of anti‐TNBC drugs and therapeutic strategies.     

Perfluorooctane sulfonate (PFOS) impairs the proliferation of C17.2 neural stem cells via the downregulation of GSK‐3β/β‐catenin signaling

The neurotoxic effects of perfluorooctane sulfonate (PFOS) have attracted significant research attention in recent years. In the present study, we investigated the impact of PFOS exposure on the physiology of neural stem cells (NSCs) in vitro. We showed that PFOS exposure markedly attenuated the proliferation of C17.2 neural stem cells in both dose‐ and time‐dependent manners. Additionally, we found that PFOS decreased Ser9 phosphorylation of glycogen synthase kinase‐3β (pSer9‐GSK‐3β), leading to the activation of GSK‐3β and resultant downregulation of cellular β‐catenin. Furthermore, blockage of GSK‐3β with lithium chloride significantly attenuated both the PFOS‐induced downregulation of GSK‐3β/β‐catenin and the proliferative impairment of C17.2 cells. Notably, the expression of various downstream targets was altered accordingly, such as c‐myc, cyclin D1 and survivin. In conclusion, the present study demonstrated that PFOS decreased the proliferation of C17.2 cells via the negative modulation of the GSK‐3β/β‐catenin pathway. We present the potential mechanisms underlying the PFOS‐induced toxic effects on NSCs to provide novel insights into the neurotoxic mechanism of PFOS. Copyright © 2016 John Wiley & Sons, Ltd.     

Zanthoxylum avicennae extract enhances GSK‐3β to attenuate β‐catenin via phosphatase 2A to block metastatic effects of HA22T cells and hepatocellular carcinoma xenografted nude mice

Hepatocellular carcinoma (HCC) metastasis is often associated with the activation of Wnt/β‐catenin signaling pathway. Zanthoxylum avicennae (Ying Bu Bo, YBB), a traditional herb with hepatoprotective effect, has been proven to inhibit human HCC in in vivo models however, the in vitro and in vivo effect of YBB on tumor metastasis is not clear yet. To determine whether YBB could inhibit HA22T human HCC cell by acting on β‐catenin metastatic signaling in vitro and in vivo, HA22T cells were treated with different concentrations of YBB extracts (YBBE) and analyzed by Immunofluorescence staining assay, western blot analysis, siRNA mediated gene knock‐down assays and co‐immunoprecipitation assay. Additionally, the HA22T‐implanted xenograft nude mice were used to confirm the assessed cellular effects. Mice treated with YBBEs showed a strong increasing trend in PP2Acα, GSK‐3β, APC, and β‐TrCP/HOS levels, however the expression of β‐catenin, p‐GSK‐3β, TBX 3, and IL8 proteins showed a decreasing trend. YBBE significantly downregulated the nuclear and cytosolic β‐catenin levels by facilitating the proteosomal degradation of β‐catenin. Moreover, as observed by co‐immunoprecipitation assay, YBBE directly promoted the protein interactions between GSK‐3β, β‐TrCP, APC, PP2A, and β‐catenin. In conclusion, both in vitro and in vivo models clearly demonstrated that YBBE inhibits β‐catenin involved metastatic signaling in highly metastatic HA22T cells through PP2A activation.     

BPA‐toxicity via superoxide anion overload and a deficit in β‐catenin signaling in human bone mesenchymal stem cells

Bisphenol A (BPA), used in the manufacture of products based on polycarbonate plastics and epoxy resins, is well known as an endocrine‐disrupting monomer. In the current study, BPA increased cytotoxicity in hBMSCs in a dose‐ and time‐dependent manner, concomitantly with increased lipid peroxidation. Increased cell death in BPA‐treated cells was markedly blocked by pretreatment with the superoxide dismutase mimetic MnTBAP and MnTMPyP, but not by catalase, glutathione, the glutathione peroxidase mimetic ebselen, the NOS inhibitor NAME, or the xanthine oxidase inhibitor allopurinol. Furthermore, the decline in nuclear β‐catenin and cyclin D1 levels in hBMSCs exposed to BPA was reversed by MnTBAP treatment. Finally, treatment of hBMSCs with the GSK3β inhibitor LiCl₂ increased nuclear β‐catenin levels and significantly attenuated cytotoxicity compared with BPA treatment. Our current results in hBMSCs exposed to BPA suggest that BPA causes a disturbance in β‐catenin signaling via a superoxide anion overload. © 2016 The Authors Environmental Toxicology Published by Wiley Periodicals, Inc. Environ Toxicol 32: 344–352, 2017.     

Protective effects of β‐sheet breaker α/β‐hybrid peptide against amyloid β‐induced neuronal apoptosis in vitro

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid‐β oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid‐β (Aβ). Increased production of Aβ invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a β‐sheet breaker α/β‐hybrid peptide (BSBHp) and the underlying mechanisms against Aβ₄₀‐induced neurotoxicity in human neuroblastoma SH‐SY5Y cells. Cells were pretreated with the peptide Aβ₄₀ to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca²⁺, and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aβ₄₀‐induced toxicity by retaining cell viability, suppressing generation of ROS, Ca²⁺ levels, and effectively protects neuronal apoptosis by suppressing pro‐apoptotic protein Bax and up‐regulating antiapoptotic protein Bcl‐2. These results suggest that α/β‐hybrid peptide has neuroprotective effects against Aβ₄₀‐induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.     

2, 3, 7, 8‐Tetrachlorodibenzo‐p‐dioxin induces premature senescence of astrocytes via WNT/β‐catenin signaling and ROS production

2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a ubiquitous environmental contaminant that could exert significant neurotoxicity in the human nervous system. Nevertheless, the molecular mechanism underlying TCDD‐mediated neurotoxicity has not been clarified clearly. Herein, we investigated the potential role of TCDD in facilitating premature senescence in astrocytes and the underlying molecular mechanisms. Using the senescence‐associated β‐galactosidase (SA‐β‐Gal) assay, we demonstrated that TCDD exposure triggered significant premature senescence of astrocyte cells, which was accompanied by a marked activation of the Wingless and int (WNT)/β‐catenin signaling pathway. In addition, TCDD altered the expression of senescence marker proteins, such as p16, p21 and GFAP, which together have been reported to be upregulated in aging astrocytes, in both dose‐ and time‐dependent manners. Further, TCDD led to cell‐cycle arrest, F‐actin reorganization and the accumulation of cellular reactive oxygen species (ROS). Moreover, the ROS scavenger N‐acetylcysteine (NAC) markedly attenuated TCDD‐induced ROS production, cellular oxidative damage and astrocyte senescence. Notably, the application of XAV939, an inhibitor of WNT/β‐catenin signaling pathway, ameliorated the effect of TCDD on cellular β‐catenin level, ROS production, cellular oxidative damage and premature senescence in astrocytes. In summary, our findings indicated that TCDD might induce astrocyte senescence via WNT/β‐catenin and ROS‐dependent mechanisms. Copyright © 2014 John Wiley & Sons, Ltd.     

Synthesis,crystal structure and molecular conformation of Nα‐Boc‐l‐leucyl‐(Z)‐β‐(3‐pyridyl)‐α,β‐ dehydroalanyl‐l‐leucine methyl ester*

Abstract: A protected tridehydropeptide containing (Z)‐β‐(3‐pyridyl)‐α,β‐dehydroalanine (Δ^Z3Pal) residue, Boc‐Leu‐Δ^Z3Pal‐Leu‐OMe ( 1 ), was synthesized via Erlenmeyer azlactone method. X‐ray crystallographic analysis revealed that the peptide 1 adopts an extended conformation, which is similar to that of a Δ^ZPhe analog, Boc‐Leu‐Δ^ZPhe‐Leu‐OMe ( 2 ).