Leptin induces hypertrophy through activating the peroxisome proliferator‐activated receptor α pathway in cultured neonatal rat cardiomyocytes

1. Our previous study has shown that leptin induces cardiomyocyte hypertrophy; however, the mechanisms are poorly understood. Recent studies have shown that peroxisome proliferator‐activated receptor α (PPARα) activation might be responsible for pathological remodeling and severe cardiomyopathy. Leptin, as an endogenous activator of PPARα, regulates energy metabolism through activating PPARα in many cells. Therefore, we hypothesized that leptin induces cardiomyocyte hypertrophy through activating the cardiac PPARα pathway. 2. Cultured neonatal rat cardiomyocytes were used to evaluate the effects of PPARα on hypertrophy. The selective PPARα antagonist GW6471 concentration‐dependently decreased atrial natriuretic factor mRNA expression by 23%, 36%, 44% and 59%, and significantly decreased total RNA levels, protein synthesis and cell surface areas, all of which were elevated by 72 h of leptin treatment. The augmentation of reactive oxygen species levels in leptin treated cardiomyocytes was reversed by 0.1–10 μmol/L GW6471 (40%, 52% and 58%). After 24 h of treatment, leptin concentration‐dependently enhanced mRNA expression by 7%, 93%, 100% and 256%, and protein expression by 31.2%, 64.2%, 143% and 199%, and the activity of PPARα. Meanwhile, cardiomycytes receiving 72 h of treatment with the PPARα agonist, fenofibrate, concentration‐dependently increased total RNA levels, atrial natriuretic factor mRNA expression, protein synthesis and cell surface area. Treatment of fenofibrate for 4 h also elevated oxygen species levels in a concentration‐dependent manner. 3. In conclusion, these findings show that leptin induces hypertrophy through the activation of the PPARα pathway in cultured neonatal rat cardiomyocytes.     

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.     

Up‐regulation of Gadd45α after exposure to metal nanoparticles: The role of hypoxia inducible factor 1α

The increased development and use of nanoparticles in various fields may lead to increased exposure, directly affecting human health. Our current knowledge of the health effects of metal nanoparticles such as cobalt and titanium dioxide (Nano‐Co and Nano‐TiO₂) is limited but suggests that some metal nanoparticles may cause genotoxic effects including cell cycle arrest, DNA damage, and apoptosis. The growth arrest and DNA damage‐inducible 45α protein (Gadd45α) has been characterized as one of the key players in the cellular responses to a variety of DNA damaging agents. The aim of this study was to investigate the alteration of Gadd45α expression in mouse embryo fibroblasts (PW) exposed to metal nanoparticles and the possible mechanisms. Non‐toxic doses of Nano‐Co and Nano‐TiO₂ were selected to treat cells. Our results showed that Nano‐Co caused a dose‐ and time‐dependent increase in Gadd45α expression, but Nano‐TiO₂ did not. To investigate the potential pathways involved in Nano‐Co‐induced Gadd45α up‐regulation, we measured the expression of hypoxia inducible factor 1α (HIF‐1α) in PW cells exposed to Nano‐Co and Nano‐TiO₂. Our results showed that exposure to Nano‐Co caused HIF‐1α accumulation in the nucleus. In addition, hypoxia inducible factor 1α knock‐out cells [HIF‐1α (?/?)] and its wild‐type cells [HIF‐1α (+/+)] were used. Our results demonstrated that Nano‐Co caused a dose‐ and time‐dependent increase in Gadd45α expression in wild‐type HIF‐1α (+/+) cells, but only a slight increase in HIF‐1α (?/?) cells. Pre‐treatment of PW cells with heat shock protein 90 inhibitor, 17‐(Allylamino)?17‐demethoxygeldanamycin (17‐AAG), prior to exposure to Nano‐Co significantly abolished Nano‐Co‐induced Gadd45α expression. These results suggest that HIF‐1α accumulation may be partially involved in the increased Gadd45α expression in cells exposed to Nano‐Co. These findings may have important implications for understanding the potential health effects of metal nanoparticle exposure. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 490–499, 2015.     

The synthesis of three isotopomers of 2‐methyl‐2‐(4‐[3‐[1‐(4‐methylbenzyl)‐5‐oxo‐4,5‐dihydro‐1H‐[1,2,4]triazol‐3‐yl]propyl]phenoxy)propionic acid,a potent and selective peroxisome proliferator‐activated receptor alpha agonist

Although fenofibrate ( 1a ) is commercially available and clinically effective in lowering serum triglycerides, its activity and sub‐type selectivity at the PPARα receptors are only moderate; therefore, there exists a need for more potent and sub‐type selective PPARα agonists. To that end, discovery efforts have identified 2‐methyl‐2‐(4‐[3‐[1‐(4‐methylbenzyl)‐5‐oxo‐4,5‐dihydro‐1H‐[1,2,4]triazol‐3‐yl]propyl]phenoxy)propionic acid ( 2 ), a potent and selective human PPARα receptor agonist. In support of pre‐clinical ADME studies and bioanalysis, three isotopomers of 2 have been synthesized. The results of these efforts are described below. Copyright © 2007 John Wiley & Sons, Ltd.     

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.     

Identification of small molecule estrogen‐related receptor α–specific antagonists and homology modeling to predict the molecular determinants as the basis for selectivity over ERRβ and ERRγ

The estrogen‐related receptor α (ERRα) was one of the first orphan receptors identified through a search for genes encoding proteins related to the steroid nuclear receptor, Estrogen Receptor α (ERα). The physiological role of ERRα has not yet been established nor has a natural ligand been elucidated. Importantly, research indicates that ERRα may be a novel drug target to treat breast cancer and/or metabolic disorders. A homogeneous time‐resolved fluorescence (HTRF) assay has been developed to screen for ERRα‐specific antagonists. This assay uses the ERR ligand binding domain and the coactivator interaction domain of Proliferator‐activated Receptor γ Coactivator‐1α (PGC‐1α) to examine the ability of compounds to antagonize the constitutive interaction between ERRα and the coactivator. A dissociation‐enhanced lanthanide fluorescence immunoassay (DELFIA) was also created to counter screen compounds identified in the HTRF screen. Here we report the discovery of high‐affinity ERRα subtype selective antagonists. Additionally, a homology model of ERRα in an antagonist conformation has been developed and after subsequent docking studies, we offer a model showing the molecular determinants that suggest why our novel tri‐cyclic antagonist, N‐[(2Z)‐3‐(4,5‐dihydro‐1,3‐thiazol‐2‐yl)‐1,3‐thiazolidin‐2‐yl idene]‐5 H dibenzo[a,d][7]annulen‐5‐amine, binds to ERRα with high affinity but does not bind to either ERRβ or ERRγ. Drug Dev Res 69:203–218, 2008. © 2008 Wiley‐Liss, Inc.     

Nonsteroidal estrogen receptor isoform‐selective biphenyls

Estrogen receptor (ER) has been a therapeutic target to treat ER‐positive breast cancer, most notably by agents known as selective estrogen receptor modulators (SERMs). However, resistance and severe adverse effects of known drugs gave impetus to the search for newer agents with better therapeutic profile. ERα and ERβ are two isoforms sharing 56% identity and having different physiological functions and expressions in various tissues. Only two residues differ in the active sites of the two isoforms motivating us to design isoform‐selective ligands. Guided by computational docking and molecular dynamics simulations, we have designed, synthesized, and tested, substituted biphenyl‐2,6‐diethanones and their derivatives as potential agents targeting ERα. Four of the molecules synthesized exhibited preferential cytotoxicity in ERα+ cell line (MCF‐7) compared to ERβ+ cell line (MDA‐MB‐231). Molecular dynamics (MD) in combination with molecular mechanics‐generalized Born surface area (MM‐GBSA) methods could account for binding selectivity. Further cotreatment and E‐screen studies with known ER ligands—estradiol (E₂) and tamoxifen (Tam)—indicated isoform‐selective anti‐estrogenicity in ERα+ cell line which might be ER‐mediated. ERα siRNA silencing experiments further confirmed the ER selective nature of ligands.     

AMPK‐dependent signaling modulates the suppression of invasion and migration by fenofibrate in CAL 27 oral cancer cells through NF‐κB pathway

Fenofibrate, a peroxisome proliferator‐activated receptor alpha (PPARα) agonist and lipid‐lowering agent, has been used worldwide for treatment of hyperlipidemia. The clinical trials demonstrate that fenofibrate possesses multiple pharmacological activities, including antitumor effects. However, the precise mechanisms in oral squamous cell carcinoma (OSCC) remain unclear. In this study, we investigated the anticancer effects of fenofibrate on the migration and invasion of human oral cancer CAL 27 cells. Fenofibrate inhibited the cell migration and invasion of CAL 27 cells by the wound healing and Boyden chamber transwell assays, respectively. In addition, fenofibrate reduced the protein expressions of MMP‐1, MMP‐2, MMP‐7, and MMP‐9 by Western blotting and inhibited enzyme activities of MMP‐2/‐9 using gelatin zymography assay. Results from immunoblotting analysis showed that the proteins of p‐LKB1 (Ser428), LKB1, p‐AMPKα (Thr172), p‐AMPKα1/α2 (Ser425/Ser491), p‐AMPKβ1 (Ser108), and AMPKγ1 were upregulated by fenofibrate; the levels of p‐IKKα/β (Ser176) and p‐IκBα were reduced in fenofibrate‐treated cells. Also, fenofibrate suppressed the expressions of nuclear NF‐κB p65 and p50 by immunoblotting and NF‐κB DNA binding activity by EMSA assay. The anti‐invasive effect of fenofibrate was attenuated by compound C [an adenosine 5′‐monophosphate‐activated protein kinase (AMPK) inhibitor] or dominant negative form of AMPK (DN‐AMPKα1). Thus, fenofibrate considerably inhibited metastatic behaviors of CAL 27 cells might be mediated through blocking NF‐κB signaling, resulting in the inhibition of MMPs; these effects were AMPK‐dependent rather than PPARα signaling. Our findings provide a molecular rationale, whereby fenofibrate exerts anticancer effects and additional beneficial effects for the treatment of cancer patients. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 866–876, 2016.     

Effects of a glyphosate‐based herbicide on the uterus of adult ovariectomized rats

Glyphosate is the active ingredient of several herbicide formulations. Different reports suggest that glyphosate‐based herbicides (GBHs) may act as endocrine disruptors. We evaluated the potential estrogenic effects of a GBH formulation using the uterotrophic assay. Adult ovariectomized rats were sc injected for 3 consecutive days with: saline solution (vehicle control), 2.10^?5 g E₂/kg/day (uterotrophic dose; UE₂), 2.10^?7 g E₂/kg/day (nonuterotrophic dose; NUE₂), or 0.5, 5, or 50 mg GBH/kg/day of the. Twenty‐four hours after the last injection, the uterus was removed and weighed and processed for histopathology and mRNA extraction. Epithelial cell proliferation and height and expression of estrogen‐responsive genes were evaluated (estrogen receptors, ERα and ERβ; progesterone receptor, PR; complement 3, C3). Uterine weight and epithelial proliferation were not affected by GBH. However, the luminal epithelial cell height increased at GBH0.5. ERα mRNA was downregulated by all GBH doses and E₂ groups, whereas PR and C3 mRNA were diminished by GBH0.5. GBH5‐, GBH50‐, and UE₂‐treated rats showed downregulated ERα protein expression in luminal epithelial cells, while the receptor was upregulated in the stroma. GBH upregulated ERβ (GBH0.5–50) and PR (GBH5) expressions in glandular epithelial cells, similar effect to that of NUE₂ group. These results indicate that, although the uterine weight was not affected, GBH modulates the expression of estrogen‐sensitive genes. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1191–1201, 2017.     

Relationship between peroxisome proliferator‐activated receptor alpha activity and cellular concentration of 14 perfluoroalkyl substances in HepG2 cells

Peroxisome proliferator‐activated receptor alpha (PPARα) is a molecular target for perfluoroalkyl substances (PFASs). Little is known about the cellular uptake of PFASs and how it affects the PPARα activity. We investigated the relationship between PPARα activity and cellular concentration in HepG2 cells of 14 PFASs, including perfluoroalkyl carboxylates (PFCAs), perfluoroalkyl sulfonates and perfluorooctane sulfonamide (FOSA). Cellular concentrations were determined by high‐performance liquid chromatography–tandem mass spectrometry and PPARα activity was determined in transiently transfected cells by reporter gene assay. Cellular uptake of the PFASs was low (0.04–4.1%) with absolute cellular concentrations in the range 4–2500 ng mg^?1 protein. Cellular concentration of PFCAs increased with perfluorocarbon chain length up to perfluorododecanoate. PPARα activity of PFCAs increased with chain length up to perfluorooctanoate. The maximum induction of PPARα activity was similar for short‐chain (perfluorobutanoate and perfluoropentanoate) and long‐chain PFCAs (perfluorododecanoate and perfluorotetradecanoate) (approximately twofold). However, PPARα activities were induced at lower cellular concentrations for the short‐chain homologs compared to the long‐chain homologs. Perfluorohexanoate, perfluoroheptanoate, perfluorooctanoate, perfluorononanoate (PFNA) and perfluorodecanoate induced PPARα activities >2.5‐fold compared to controls. The concentration–response relationships were positive for all the tested compounds, except perfluorooctane sulfonate PFOS and FOSA, and were compound‐specific, as demonstrated by differences in the estimated slopes. The relationships were steeper for PFCAs with chain lengths up to and including PFNA than for the other studied PFASs. To our knowledge, this is the first report establishing relationships between PPARα activity and cellular concentration of a broad range of PFASs.     

Induction of the estrogen‐responsive genes encoding choriogenin H and L in the liver of male medaka (Oryzias latipes) upon exposure to estrogen receptor subtype‐selective ligands

Choriogenin (Chg) H and L are estrogen‐induced chorion precursors. We measured the induction of ChgH and ChgL mRNA in the livers of male medaka fish treated with Orthoester‐2k, a selective ligand for estrogen receptor (ER) α, and 2‐(4‐h ydroxyp henyl)‐5‐h ydroxy‐1,3‐b enzoxazole (HPHB), a selective ligand of ERβ. Although both ChgH and ChgL mRNA were induced by treatment with Orthoester‐2k or HPHB separately, their combination induced much greater expression of each Chg. ChgH expression correlated more closely with Orthoester‐2k dosage when combined with a small fixed dose of HPHB (1 μm ), whereas ChgL mRNA expression was more responsive to HPHB dose when combined with a fixed dose of Orthoester‐2k (2.8 nm ). Moreover, upon long‐term treatment with Orthoester‐2k, ChgH mRNA and ERα mRNA expression showed similar patterns with peak expression between days 6 and 10. These results imply that ERβ primarily regulates ChgL mRNA expression and ERα action primarily regulates ChgH mRNA expression. Thus, it is necessary to develop screening methods for fish ER subtype‐specific ligands. Copyright © 2014 John Wiley & Sons, Ltd.     

Validation of a new yeast‐based reporter assay consisting of human estrogen receptors α/β and coactivator SRC‐1: Application for detection of estrogenic activity in environmental samples

Endocrine disruptors are exogenous substances that act like hormones in the endocrine system and disrupt the physiologic function of endogenous hormones. In the present study, we established reporter yeast strains (Saccharomyces cerevisiae) expressing human estrogen receptors, ERα or ERβ. These strains contain a reporter plasmid carrying an estrogen responsive element (ERE) upstream of the β‐galactosidase gene, and a plasmid expressing a steroid receptor coactivator, SRC‐1e. Using these reporter strains, we demonstrated dose‐dependent estrogenic activities of different categories of ligands, a natural hormone, 17β‐estradiol (E2); a synthetic drug, diethylstilbestrol (DES); phytoestrogens, genistein, daizein and emodin; and an environmental endocrine disrupter, bisphenol A. EC₅₀ values of E2 for ERα and ERβ are 5.31 × 10^?10 and 5.85 × 10^?10 M, respectively. We also demonstrated that these yeasts were applicable for measuring estrogenic activities of environmental water samples. Most downstream sites of a river showed similar activity in both ERα and ERβ assays. These yeast strains are useful and convenient for detecting and comparing the estrogenic ligand activities of environmental samples in response to ERα and ERβ. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2009.     

Silencing of astrocyte elevated gene‐1 inhibits proliferation and migration of melanoma cells and induces apoptosis

Melanoma is an aggressive skin malignancy with a high mortality. Astrocyte elevated gene‐1 (AEG‐1), a downstream target of Ras and c‐Myc, has been implicated in the development of multiple tumours, but its role in melanoma remains unclear. In the present study, the role of AEG‐1 in melanoma was explored through AEG‐1 silencing. Our results showed that silencing AEG‐1 inhibited the proliferation of melanoma cells, induced cell cycle arrest, and reduced levels of cyclin A, cyclin B, cyclin D1, cyclin E, and cyclin‐dependent kinase 2. AEG‐1silencing also induced apoptosis in melanoma cells and altered the levels of cleaved caspase‐3, B‐cell lymphoma‐2 (Bcl‐2) and Bcl‐2 associated X protein. Moreover, silencing AEG‐1 suppressed the migration and invasion of melanoma cells, reduced the expressions and activities of matrix metallopeptidase (MMP)‐2 and MMP‐9, and inhibited the activation of the Wnt/β‐catenin signalling pathway in melanoma cells. Furthermore, in vivo experiments revealed that AEG‐1 silencing inhibited the growth of melanoma xenografts in nude mice. In summary, our study demonstrates an oncogenic role of AEG‐1 in melanoma and suggests that AEG‐1 may serve as a potential therapeutic target in the treatment of melanoma.     

Pyrazinamide induced hepatic injury in rats through inhibiting the PPARα pathway

Pyrazinamide (PZA) causes serious hepatotoxicity, but little is known about the exact mechanism by which PZA induced liver injury. The peroxisome proliferator‐activated receptors alpha (PPARα) is highly expressed in the liver and modulates the intracellular lipidmetabolism. So far, the role of PPARα in the hepatotoxicity of PZA is unknown. In the present study, we described the hepatotoxic effects of PZA and the role of PPARα and its target genes in the downstream pathway including L‐Fabp, Lpl, Cpt‐1b, Acaa1, Apo‐A1 and Me1 in this process. We found PZA induced the liver lipid metabolism disorder and PPARα expressionwas down‐regulated which had a significant inverse correlation with liver injury degree. These changeswere ameliorated by fenofibrate, the co‐treatment that acts as a PPARα agonist. In contrast, short‐termstarvation significantly aggravated the severity of PZA‐induced liver injury. In conclusion, this study demonstrated the critical role played by PPARα in PZA‐induced hepatotoxicity and provided a better understanding of the molecular mechanisms underlying PZA‐induced liver injury. Copyright © 2016 John Wiley & Sons, Ltd.