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.     

Metformin inhibits Aβ25‐35‐induced apoptotic cell death in SH‐SY5Y cells

Metformin, a first‐line drug for type‐2 diabetes, plays a potentially protective role in preventing Alzheimer's disease (AD), but its underlying mechanism is unclear. In this study, Aβ_25‐35‐treated SH‐SY5Y cells were used as a cell model of AD to investigate the neuroprotective effect of metformin, as well as its underlying mechanisms. We found that metformin decreased the cell apoptosis rate and death, ratio of Bcl‐2/Bax, and expression of NR2A and NR2B, and increased the expression of LC3 in Aβ_25‐35‐treated SH‐SY5Y cells. Metformin also reduced intracellular and extracellular Glu concentrations, as well as the intracellular concentration of Ca²⁺ and ROS in Aβ_25‐35‐treated SH‐SY5Y cells. These findings suggest that metformin inhibits Aβ_25‐35‐treated SH‐SY5Y cell death by inhibiting apoptosis, decreasing intracellular Ca²⁺ and ROS by reducing neurotoxicity of excitatory amino acids, and by possibly reversing autophagy disorder via regulating autophagy process.     

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.     

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.     

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.     

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.     

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.     

Synthesis and preliminary characterization of radioiodinated benzofuran‐3‐yl‐(indol‐3‐yl)maleimide derivatives as potential SPECT imaging probes for the detection of glycogen synthase kinase‐3β (GSK‐3β) in the brain

We report on the synthesis and preliminary characterization of two radioiodinated benzofuran‐3‐yl‐(indol‐3‐yl)maleimides, 3‐(benzofuran‐3‐yl)‐4‐(5‐[¹²⁵I]iodo‐1‐methyl‐1H‐indol‐3‐yl)‐1H‐pyrrole‐2,5‐dione ([¹²⁵I]5), and 3‐(5‐[¹²⁵I]iodo‐1‐methyl‐1H‐indol‐3‐yl)‐4‐(6‐methoxybenzofuran‐3‐yl)‐1H‐pyrrole‐2,5‐dione ([¹²⁵I]6), as the first potential SPECT imaging probes targeting glycogen synthase kinase‐3β (GSK‐3β). In this study, we used ¹²⁵I as a surrogate of ¹²³I because of its ease of use. The radioiodinated ligands were prepared from the corresponding tributyltin precursors through an iododestannylation reaction using hydrogen peroxide as an oxidant with a radiochemical yield of 10–30%. In vitro binding experiments suggested that both compounds show high affinity for GSK‐3β at a level similar to a known GSK‐3β inhibitor. Biodistribution studies with normal mice revealed that the radioiodinated compounds display sufficient uptake into (1.8%ID/g at 10 min postinjection) and clearance from the brain (1.0%ID/g at 60 min postinjection). These preliminary results suggest that the further optimization of radioiodinated benzofuran‐3‐yl‐(indol‐3‐yl)maleimide derivatives may facilitate the development of clinically useful SPECT imaging probes for the in vivo detection of GSK‐3β.     

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 ).     

GRK2/β‐arrestin mediates arginine vasopressin‐induced cardiac fibroblast proliferation

Cardiac fibrosis is a pathological feature commonly found in hearts exposed to haemodynamic orneurohormonal stress. Elevated levels of arginine vasopressin (AVP) are closely associated with the progression of heart failure and could be an underlying cause of cardiac fibrosis. The aim of this study is to characterize the effect of AVP on neonatal rat cardiac fibroblasts (NRCFs) and to illustrate its signalling mechanism. The proliferative effect of AVP was assessed by methylthiazolyldiphenyl‐tetrazolium assay and 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation assay, and the amounts of cellular signalling proteins α‐smooth muscle actin (α‐SMA), matrix metalloproteinase (MMP) 2, MMP9, and phosphorylated ERK_1/2 were determined by western blotting. AVP, in a time‐ and concentration‐dependent manner, promoted NRCF proliferation and the expression of MMP2 and MMP9. Inhibition of G protein‐coupled receptor kinase2 (GRK2) by the inhibitory peptide GRK2‐Ct or knock‐down of GRK2 suppressed AVP‐induced BrdU incorporation and the expression of MMP2 and α‐SMA in NRCFs. Moreover, shRNA‐mediated silencing of β‐arrestin1 or β‐arrestin 2 abolished AVP‐induced BrdU incorporation and MMP2 expression. AVP‐induced NRCF proliferation depended on the phosphorylation of ERK_1/2, and inhibition of GRK2 or silencing of β‐arrestins blocked AVP‐induced ERK_1/2 phosphorylation. The effects of AVP on NRCF proliferation and α‐SMA expression were blocked by SR45059, a vasopressin receptor type1A (V_1AR) selective antagonist. In conclusion, AVP promotes NRCF proliferation through V_1AR‐mediated GRK2/β‐arrestin/ERK_1/2 signalling.     

The Proteolytic Stability and Cytotoxicity Studies of l‐Aspartic Acid and l‐Diaminopropionic Acid derived β‐Peptides and a Mixed α/β‐Peptide

The use of peptides as drugs in pharmaceutical applications is hindered by their susceptibility to proteolysis and therefore low bioavailability. β‐Peptides that contain an additional methylene group in the backbone, are gaining recognition from a pharmaceutical stand point as they are considerably more resilient to proteolysis and metabolism. Recently, we reported two new classes of β ‐peptides, β ³‐ and β²‐peptides derived from l ‐aspartic acid and l ‐diaminopropionic acid, respectively. Here, we report the proteolytic stability of these β‐peptidic compounds and a mixed α /β‐peptide against three enzymes (pronase, trypsin and elastase), as well as, human serum. The stability of these peptides was compared to an α‐peptide. Peptides containing β‐linkages were resistant to all conditions. The mixed α /β‐peptide, however, exhibited proteolysis in the presence of trypsin and pronase but not elastase. The rate of degradation of the mixed α /β‐peptide was slower than that would be expected for an α‐peptide. In addition, these β‐peptides were not toxic to HeLa and COS‐1 cell lines as observed by MTT cytotoxicity assay. These results expand the scope of mixed α /β‐peptides containing β‐amino acids or small β‐peptide fragments as therapeutic peptides.     

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.