2,4‐dichlorophenol induces ER stress‐mediated apoptosis via eIF2α dephosphorylation in vitro

2,4‐Dichlorophenol (2,4‐DCP) has been widely used to produce herbicides and pharmaceutical intermediates, which exhibits various toxic effects including apoptosis. However, the mechanisms underlying 2,4‐DCP‐induced apoptosis, especially mediated by endoplasmic reticulum (ER) stress, are still unknown. In the present study, the mouse embryonic fibroblasts (MEFs) were used as an in vitro model system to figure out whether 2,4‐DCP could induce ER stress, and further to elucidate the role of ER stress in 2,4‐DCP‐induced apoptosis. The results showed that 2,4‐DCP dramatically caused the decrease of cell viability, the increase of apoptotic cells, the collapse of mitochondrial membrane potential (MMP) and the activation of caspase‐3, suggesting that 2,4‐DCP did induce apoptosis. Meanwhile, 2,4‐DCP acted similarly as ER stress agonist tunicamycin (Tu) to activate all three branches (IRE1α, ATF6 and eIF2α) of ER stress. Furthermore, repression of ER stress or inhibition of eIF2α dephosphorylation significantly alleviated 2,4‐DCP‐induced apoptosis. Taking these results together, the present study firstly showed that 2,4‐DCP induced ER stress‐mediated apoptosis via eIF2α dephosphorylation in mammalian cells. These findings will provide new insights into the mechanisms underlying apoptosis after chlorophenols exposure. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 245–255, 2016.     

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.     

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.     

TGF‐β1 mediated MAPK signaling pathway promotes collagen formation induced by Nano NiO in A549 cells

Nickel oxide nanoparticles (Nano NiO) could induce pulmonary fibrosis, however, the mechanisms are still unknown. The aim of the present study was to explore the roles of transforming growth factor‐β1 (TGF‐β1), mitogen‐activated protein kinase (MAPK) pathway and MMPs/TIMPs balance in Nano NiO‐induced pulmonary fibrosis. For that purpose, we first established Nano NiO‐induced human lung adenocarcinoma epithelial cells (A549 cells) model of collagen excessive formation through detecting the levels of hydroxyproline (Hyp) and type I collagen (Col‐I). Then the protein levels of TGF‐β1, MAPKs, and MMPs/TIMPs were assessed by Western blot. The results showed that Nano NiO resulted in the increased contents of Hyp, Col‐I, and TGF‐β1, the MAPK pathway activation and MMPs/TIMPs imbalance with a dose‐dependent manner. In addition, to investigate whether TGF‐β1 mediated MAPK signaling pathway, A549 cells were treated by 100 μg/mL Nano NiO combined with TGF‐β1, p38 MAPK, and ERK1/2 inhibitors (10 μM SB431542, 10 μM SB203580, and 10 μM U0126), respectively. We found that MAPK signal pathway was suppressed by TGF‐β1 inhibitor. Meanwhile, the increased contents of Hyp and Col‐I, and MMPs/TIMPs imbalance were alleviated by the p38 MAPK and ERK1/2 inhibitors, respectively. These findings indicated that the MAPK pathway and MMPs/TIMPs imbalance were involved in collagen excessive formation induced by Nano NiO.     

Agonist function of the recombinant α4β3δ GABAA receptor is dependent on the human and rat variants of the α4‐subunit

1. It is known that the α₄‐subunit is likely to occur in the brain predominantly in α₄β₃δ receptors at extrasynaptic sites. Recent studies have revealed that the α₁‐, α₄‐, γ₂‐ and δ‐subunits may colocalize extrasynaptically in dentate granule cells of the hippocampus. In the present study, we characterized a series of recombinant GABA_A receptors containing human (H) and rat (R) α₁/α₄‐, β₂/β₃‐ and γ_2S/δ‐subunits in Xenopus oocytes using the two‐electrode voltage‐clamp technique. 2. Both Hα₁β₃δ and Hα₄β₃γ_2S receptors were sensitive to activation by GABA and pentobarbital. Contrary to earlier findings that the α₄β₃δ combination was more sensitive to agonist action than the α₄β₃γ_2S receptor, we observed extremely small GABA‐ and pentobarbital‐activated currents at the wild‐type Hα₄β₃δ receptor. However, GABA and pentobarbital activated the wild‐type Rα₄β₃δ receptor with high potency (EC₅₀ = 0.5 ± 0.7 and 294 ± 5 μmol/L, respectively). 3. Substituting the Hα₄ subunit with Rα₄ conferred a significant increase in activation on the GABA and pentobarbital site in terms of reduced EC₅₀ and increased I_max. When the Hα₄ subunit was combined with the Rβ₃ and Rδ subunit in a heteropentameric form, the amplitude of GABA‐ and pentobarbital‐activated currents increased significantly compared with the wild‐type Hα₄β₃δ receptor. 4. Thus, the results indicate that the Rα₄β₃δ, Hα₁β₃δ and Hα₄β₃γ_2S combinations may contribute to functions of extrasynaptic GABA_A receptors. The presence of the Rα₄ subunit at recombinant GABA_A receptors containing the δ‐subunit is a strong determinant of agonist action. The recombinant Hα₄β₃δ receptor is a less sensitive subunit composition in terms of agonist activation.     

5‐Amino‐3‐methyl‐4‐isoxazolecarboxylic acid hydrazide derivatives with in vitro immunomodulatory activities

Isoxazoles are an important class of compounds of potential therapeutic value. The aim of this study was to determine immunotropic effects of 5‐amino‐3‐methyl‐4‐isoxazolecarboxylic acid hydrazide derivatives on spontaneous and mitogen‐induced lymphocyte proliferation in young and old mice, cytokine production by peritoneal cells as well as possible mechanism of action in a model of Jurkat cells. Three‐month‐old and 13‐month‐old BALB/c mice were used as donors of the cells from a thymus, a spleen, mesenteric lymph nodes, and a peritoneal cavity. Spontaneous and concanavalin A or lipopolysaccharide (LPS)‐induced cell proliferation was measured by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide colorimetric method. IL‐1β and TNF‐α production induced by LPS in macrophage‐enriched peritoneal cell cultures was measured by enzyme‐linked immunoassay. 5‐amino‐3‐methyl‐4‐isoxazolecarboxylic acid hydrazide, 01K (4‐phenyl‐1‐(5‐amino‐3‐methylisoxazole‐4‐carbonyl)‐thiosemicarbazide), and 06K (4‐(4‐chlorophenyl)‐1‐(5‐amino‐3‐methylisoxazole‐4‐carbonyl)‐thiosemicarbazide) exhibited regulatory activity in the proliferation tests. Prevailing stimulatory activity of the hydrazide and inhibitory activity of 01K and 06K was observed. Those effects were connected with different influence of the compounds on signaling proteins expression in Jurkat cells. The regulatory effects of the compounds on IL‐1β production were more profound than those on TNF‐α. Differences in the compound activity in young versus old mice were mainly restricted to 01K. Immunosuppressive isoxazole leflunomide and a stimulatory RM‐11 (1,7‐dimethyl‐8‐oxo‐1,2H‐isoxazole [5,4‐e]triazepine) were applied as reference drugs.     

Radiosynthesis of the α4β2 nicotinic acetylcholine receptor ligand: 5‐((1‐[11C]‐methyl‐2‐(S)‐pyrrolidinyl)methoxy)‐2‐chloro‐3‐((E)‐2‐(2‐fluoropyridin‐4‐yl)vinyl)pyridine

5‐((1‐[¹¹C]‐methyl‐2‐(S)‐pyrrolidinyl)methoxy)‐2‐chloro‐3‐((E)‐2‐(2‐fluoropyridin‐4‐yl)‐vinyl)pyridine ([¹¹C]‐FPVC) was synthesized from [¹¹C]‐methyl iodide and the corresponding normethyl precursor. The average time of synthesis, purification, and formulation was 42 min with an average non‐decay‐corrected radiochemical yield of 19%. The average specific radioactivity was 359 GBq/µmol (9691 mCi/µmole) at end of synthesis (EOS). Copyright © 2006 John Wiley & Sons, Ltd.     

TGF‐β1 mediated Smad signaling pathway and EMT in hepatic fibrosis induced by Nano NiO in vivo and in vitro

Nickel oxide nanoparticles (Nano NiO) bears hepatotoxicity, while whether it leads to liver fibrosis remains unclear. The aim of this study was to establish the Nano NiO‐induced hepatic fibrosis model in vivo and investigate the roles of transforming growth factor β1 (TGF‐β1) in Smad pathway activation, epithelial‐mesenchymal transition (EMT) occurrence, and extracellular matrix (ECM) deposition in vitro. Male Wistar rats were exposed to 0.015, 0.06, and 0.24 mg/kg Nano NiO by intratracheal instilling twice a week for 9 weeks. HepG2 cells were treated with 100 μg/mL Nano NiO and TGF‐β1 inhibitor (SB431542) to explore the mechanism of collagen formation. Results of Masson staining as well as the elevated levels of type I collagen (Col‐I) and Col‐III suggested that Nano NiO resulted in hepatic fibrosis in rats. Furthermore, Nano NiO increased the protein expression of TGF‐β1, p‐Smad2, p‐Smad3, alpha‐smooth muscle actin (α‐SMA), matrix metalloproteinase9 (MMP9), and tissue inhibitors of metalloproteinase1 (TIMP1), while decreased the protein content of E‐cadherin and Smad7 in rat liver and HepG2 cells. Most importantly, Nano NiO‐triggered the abnormal expression of the abovementioned proteins were all alleviated by co‐treatment with SB431542, implying that TGF‐β1‐mediated Smad pathway, EMT and MMP9/TIMP1 imbalance were involved in overproduction of collagen in HepG2 cells. In conclusion, these findings indicated that Nano NiO induced hepatic fibrosis via TGF‐β1‐mediated Smad pathway activation, EMT occurrence, and ECM deposition.     

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.     

Blocking UV‐Induced eIF2α Phosphorylation with Small Molecule Inhibitors of GCN2

The eIF2α kinase general control non‐depressible 2 integrates translation initiation rates to amino acid availability. General control non‐depressible 2 also regulates translation initiation during synaptic plasticity and GCN2^?/? mice show improved memory compared with wild‐type mice with a reduced threshold for triggering late long‐term potentiation. This property suggests that inhibiting general control non‐depressible 2 function might represent a therapeutic avenue for improving memory. We screened for general control non‐depressible 2 inhibitors using a small library of known kinase inhibitors and ATP‐analogs and identified three compounds – indirubin‐3′‐monoxime, SP600125 and a SyK inhibitor with activity against general control non‐depressible 2. All three compounds inhibit the ability of general control non‐depressible 2 to phosphorylate eIF2αin vitro as well as in vivo following UV‐treatment of mouse embryonic fibroblasts. Using computer‐assisted modeling, we modeled the binding of the inhibitors in the ATP‐binding site of general control non‐depressible 2. This work provides the molecular basis for undertaking structure–activity relationships of these compounds in order to develop specific inhibitors of general control non‐depressible 2.     

β‐LAPachone ameliorates doxorubicin‐induced cardiotoxicity via regulating autophagy and Nrf2 signalling pathways in mice

β‐LAPachone (B‐LAP) is a naphthoquinone that possesses antioxidant properties. In the present investigation, the protective effect of B‐LAP against doxorubicin (DOX)‐induced cardiotoxicity was examined in mice. Thirty‐five mice were divided into 5 groups: control group, B‐LAP (5 mg/kg) group, DOX (15 mg/kg) group, DOX+B‐LAP (2.5 mg/kg) group and DOX+B‐LAP (5 mg/kg) group. B‐LAP was administered orally for 14 days of experimental period. A single dose of DOX (15 mg/kg) was injected intraperitoneally on day 3. Cardiac function, histoarchitecture, indices of oxidative stress and circulating markers of cardiac injury were examined. B‐LAP (5 mg/kg) decreased serum levels of lactate dehydrogenase (LDH), creatine kinase MB (CK‐MB) and cardiac troponin I (cTnI), and ameliorated cardiac histopathological alterations. In addition to increasing cellular NAD⁺/NADH ratio, B‐LAP up‐regulated the cardiac levels of SIRT1, beclin‐1, p‐LKB1 and p‐AMPK, and reduced the cardiac levels of p‐mTOR, interleukin (IL)‐1β, TNF (tumour necrosis factor)‐α and caspase‐3. B‐LAP also elevated the nuclear accumulation of Nrf2 and simultaneously up‐regulated the protein levels of haem oxygenase (HO‐1) and glutathione S‐transferase (GST) in the hearts of DOX mice. While B‐LAP reduced malondialdehyde concentrations in heart of DOX‐treated mice, it further promoted the activities of cardiac superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT).In accordance with increased cell survival, B‐LAP significantly improved the cardiac function of DOX mice. Collectively, these findings underline the protective potential of B‐LAP against DOX‐induced cardiotoxicity by regulating autophagy and AMPK/Nrf2 signalling pathway in mice.     

Tiron ameliorates high glucose‐induced cardiac myocyte apoptosis by PKCδ‐dependent inhibition of osteopontin

Tiron functions as an effective antioxidant alleviating the intracellular reactive oxygen species (ROS) or the acute toxic metal overload. Previous studies have shown that cardiac myocyte apoptosis can be effectively inhibited by tiron administration in streptozotocin (STZ)‐induced diabetic rats, primary neonatal rat cardiomyocytes (NRVMs), and H9c2 embryonic rat cardiomyocytes. However, the underlying signalling mechanism is ill‐defined. In the present study, we found that tiron supplementation significantly inhibited apoptosis of high glucose (HG)‐treated NRVMs and the left ventricular cardiomyocytes from STZ‐diabetic rat, accompanied with a reduction of osteopontin (OPN) levels as well as an inhibition of PKCδ phosphorylation. OPN knockdown protected NRVMs against HG‐induced cell apoptosis. In addition, genetic inhibition of PKCδ mitigated HG‐stimulated enhancement of intracellular OPN levels in NRVMs. These findings indicate that ROS‐mediated activation of PKCδ upregulated OPN expression, leading to cardiac myocyte apoptosis. Interfering with ROS/PKCδ pathway by antioxidants such as tiron provides an optional therapeutic strategy for treatment and prevention of apoptosis‐related cardiovascular diseases including diabetic cardiomyopathy.     

Synthesis of a non‐peptidic PET tracer designed for α5β1 integrin receptor

Arginine–glycine–aspartic acid (RGD)‐containing peptides have been traditionally used as PET probes to noninvasively image angiogenesis, but recently, small selective molecules for α₅β₁ integrin receptor have been developed with promising results. Sixty‐one antagonists were screened, and tert‐butyl (S)‐3‐(2‐((3R,5S)‐1‐(3‐(1‐(2‐fluoroethyl)‐1H‐1,2,3‐triazol‐4‐yl)propanoyl)‐5‐((pyridin‐2‐ylamino)methyl)pyrrolidin‐3‐yloxy)acetamido)‐2‐(2,4,6‐trimethylbenzamido)propanoate (FPMt) was selected for the development of a PET tracer to image the expression of α₅β₁ integrin receptors. An alkynyl precursor (PMt) was initially synthesized in six steps, and its radiolabeling was performed according to the azide–alkyne copper(II)‐catalyzed Huisgen's cycloaddition by using 1‐azido‐2‐[¹⁸F]fluoroethane ([¹⁸F]12). Different reaction conditions between PMt and [¹⁸F]12 were investigated, but all of them afforded [¹⁸F]FPMt in 15 min with similar radiochemical yields (80–83%, decay corrected). Overall, the final radiopharmaceutical ([¹⁸F]FPMt) was obtained after a synthesis time of 60–70 min in 42–44% decay‐corrected radiochemical yield. Copyright © 2014 John Wiley & Sons, Ltd.