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Sample records for receptor inhibits progastrin-dependent

  1. Combined Angiotensin Receptor Antagonism and Neprilysin Inhibition

    Science.gov (United States)

    Hubers, Scott A.; Brown, Nancy J.

    2016-01-01

    Heart failure affects approximately 5.7 million people in the United States alone. Angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, beta-blockers, and aldosterone antagonists have improved mortality in patients with heart failure and reduced ejection fraction, but mortality remains high. In July 2015, the FDA approved the first of a new class of drugs for the treatment of heart failure; valsartan/sacubitril (formerly known as LCZ696 and currently marketed by Novartis as Entresto) combines the angiotensin receptor blocker valsartan and the neprilysin inhibitor prodrug sacubitril in a 1:1 ratio in a sodium supramolecular complex. Sacubitril is converted by esterases to LBQ657, which inhibits neprilysin, the enzyme responsible for the degradation of the natriuretic peptides and many other vasoactive peptides. Thus, this combined angiotensin receptor antagonist and neprilysin inhibitor addresses two of the pathophysiologic mechanisms of heart failure - activation of the renin-angiotensin-aldosterone system and decreased sensitivity to natriuretic peptides. In the Prospective comparison of ARNI with ACEI to Determine Impact on Global Mortality and morbidity in Heart Failure (PARADIGM-HF) trial, valsartan/sacubitril significantly reduced mortality and hospitalization for heart failure, as well as blood pressure, compared to enalapril in patients with heart failure, reduced ejection fraction, and an elevated circulating level of brain natriuretic peptide or N-terminal pro-brain natriuretic peptide. Ongoing clinical trials are evaluating the role of valsartan/sacubitril in the treatment of heart failure with preserved ejection fraction and hypertension. We review here the mechanisms of action of valsartan/sacubitril, the pharmacologic properties of the drug, and its efficacy and safety in the treatment of heart failure and hypertension. PMID:26976916

  2. High Concentrations of Tranexamic Acid Inhibit Ionotropic Glutamate Receptors.

    Science.gov (United States)

    Lecker, Irene; Wang, Dian-Shi; Kaneshwaran, Kirusanthy; Mazer, C David; Orser, Beverley A

    2017-07-01

    The antifibrinolytic drug tranexamic acid is structurally similar to the amino acid glycine and may cause seizures and myoclonus by acting as a competitive antagonist of glycine receptors. Glycine is an obligatory co-agonist of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. Thus, it is plausible that tranexamic acid inhibits NMDA receptors by acting as a competitive antagonist at the glycine binding site. The aim of this study was to determine whether tranexamic acid inhibits NMDA receptors, as well as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate subtypes of ionotropic glutamate receptors. Tranexamic acid modulation of NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate receptors was studied using whole cell voltage-clamp recordings of current from cultured mouse hippocampal neurons. Tranexamic acid rapidly and reversibly inhibited NMDA receptors (half maximal inhibitory concentration = 241 ± 45 mM, mean ± SD; 95% CI, 200 to 281; n = 5) and shifted the glycine concentration-response curve for NMDA-evoked current to the right. Tranexamic acid also inhibited α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (half maximal inhibitory concentration = 231 ± 91 mM; 95% CI, 148 to 314; n = 5 to 6) and kainate receptors (half maximal inhibitory concentration = 90 ± 24 mM; 95% CI, 68 to 112; n = 5). Tranexamic acid inhibits NMDA receptors likely by reducing the binding of the co-agonist glycine and also inhibits α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate receptors. Receptor blockade occurs at high millimolar concentrations of tranexamic acid, similar to the concentrations that occur after topical application to peripheral tissues. Glutamate receptors in tissues including bone, heart, and nerves play various physiologic roles, and tranexamic acid inhibition of these receptors may contribute to adverse drug effects.

  3. Menthol binding and inhibition of α7-nicotinic acetylcholine receptors.

    Directory of Open Access Journals (Sweden)

    Abrar Ashoor

    Full Text Available Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca(2+-dependent Cl(- channels, since menthol inhibition remained unchanged by intracellular injection of the Ca(2+ chelator BAPTA and perfusion with Ca(2+-free bathing solution containing Ba(2+. Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [(125I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca(2+ transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner.

  4. Cocaine Inhibits Dopamine D2 Receptor Signaling via Sigma-1-D2 Receptor Heteromers

    Science.gov (United States)

    Navarro, Gemma; Moreno, Estefania; Bonaventura, Jordi; Brugarolas, Marc; Farré, Daniel; Aguinaga, David; Mallol, Josefa; Cortés, Antoni; Casadó, Vicent; Lluís, Carmen; Ferre, Sergi

    2013-01-01

    Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D1-like family of dopamine receptors and inputs of aversion coming from neurons containing the D2-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D1 reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D2 receptors function, we present evidence of σ1 receptor molecular and functional interaction with dopamine D2 receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D2 receptors (the long isoform of the D2 receptor) can complex with σ1 receptors, a result that is specific to D2 receptors, as D3 and D4 receptors did not form heteromers. We demonstrate that the σ1-D2 receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ1 -D2 receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ1 knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D2 receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D1 and D2 receptor containing neurons in the brain. PMID:23637801

  5. GABA, its receptors, and GABAergic inhibition in mouse taste buds.

    Science.gov (United States)

    Dvoryanchikov, Gennady; Huang, Yijen A; Barro-Soria, Rene; Chaudhari, Nirupa; Roper, Stephen D

    2011-04-13

    Taste buds consist of at least three principal cell types that have different functions in processing gustatory signals: glial-like (type I) cells, receptor (type II) cells, and presynaptic (type III) cells. Using a combination of Ca2+ imaging, single-cell reverse transcriptase-PCR and immunostaining, we show that GABA is an inhibitory transmitter in mouse taste buds, acting on GABA(A) and GABA(B) receptors to suppress transmitter (ATP) secretion from receptor cells during taste stimulation. Specifically, receptor cells express GABA(A) receptor subunits β2, δ, and π, as well as GABA(B) receptors. In contrast, presynaptic cells express the GABA(A) β3 subunit and only occasionally GABA(B) receptors. In keeping with the distinct expression pattern of GABA receptors in presynaptic cells, we detected no GABAergic suppression of transmitter release from presynaptic cells. We suggest that GABA may serve function(s) in taste buds in addition to synaptic inhibition. Finally, we also defined the source of GABA in taste buds: GABA is synthesized by GAD65 in type I taste cells as well as by GAD67 in presynaptic (type III) taste cells and is stored in both those two cell types. We conclude that GABA is an inhibitory transmitter released during taste stimulation and possibly also during growth and differentiation of taste buds.

  6. Fcγ receptor-mediated inflammation inhibits axon regeneration.

    Directory of Open Access Journals (Sweden)

    Gang Zhang

    Full Text Available Anti-glycan/ganglioside antibodies are the most common immune effectors found in patients with Guillain-Barré Syndrome, which is a peripheral autoimmune neuropathy. We previously reported that disease-relevant anti-glycan autoantibodies inhibited axon regeneration, which echo the clinical association of these antibodies and poor recovery in Guillain-Barré Syndrome. However, the specific molecular and cellular elements involved in this antibody-mediated inhibition of axon regeneration are not previously defined. This study examined the role of Fcγ receptors and macrophages in the antibody-mediated inhibition of axon regeneration. A well characterized antibody passive transfer sciatic nerve crush and transplant models were used to study the anti-ganglioside antibody-mediated inhibition of axon regeneration in wild type and various mutant and transgenic mice with altered expression of specific Fcγ receptors and macrophage/microglia populations. Outcome measures included behavior, electrophysiology, morphometry, immunocytochemistry, quantitative real-time PCR, and western blotting. We demonstrate that the presence of autoantibodies, directed against neuronal/axonal cell surface gangliosides, in the injured mammalian peripheral nerves switch the proregenerative inflammatory environment to growth inhibitory milieu by engaging specific activating Fcγ receptors on recruited monocyte-derived macrophages to cause severe inhibition of axon regeneration. Our data demonstrate that the antibody orchestrated Fcγ receptor-mediated switch in inflammation is one mechanism underlying inhibition of axon regeneration. These findings have clinical implications for nerve repair and recovery in antibody-mediated immune neuropathies. Our results add to the complexity of axon regeneration in injured peripheral and central nervous systems as adverse effects of B cells and autoantibodies on neural injury and repair are increasingly recognized.

  7. Gβ promotes pheromone receptor polarization and yeast chemotropism by inhibiting receptor phosphorylation.

    Science.gov (United States)

    Ismael, Amber; Tian, Wei; Waszczak, Nicholas; Wang, Xin; Cao, Youfang; Suchkov, Dmitry; Bar, Eli; Metodiev, Metodi V; Liang, Jie; Arkowitz, Robert A; Stone, David E

    2016-04-12

    Gradient-directed cell migration (chemotaxis) and growth (chemotropism) are processes that are essential to the development and life cycles of all species. Cells use surface receptors to sense the shallow chemical gradients that elicit chemotaxis and chemotropism. Slight asymmetries in receptor activation are amplified by downstream signaling systems, which ultimately induce dynamic reorganization of the cytoskeleton. During the mating response of budding yeast, a model chemotropic system, the pheromone receptors on the plasma membrane polarize to the side of the cell closest to the stimulus. Although receptor polarization occurs before and independently of actin cable-dependent delivery of vesicles to the plasma membrane (directed secretion), it requires receptor internalization. Phosphorylation of pheromone receptors by yeast casein kinase 1 or 2 (Yck1/2) stimulates their internalization. We showed that the pheromone-responsive Gβγ dimer promotes the polarization of the pheromone receptor by interacting with Yck1/2 and locally inhibiting receptor phosphorylation. We also found that receptor phosphorylation is essential for chemotropism, independently of its role in inducing receptor internalization. A mathematical model supports the idea that the interaction between Gβγ and Yck1/2 results in differential phosphorylation and internalization of the pheromone receptor and accounts for its polarization before the initiation of directed secretion. Copyright © 2016, American Association for the Advancement of Science.

  8. Targeting fibroblast growth factor receptor signaling inhibits prostate cancer progression.

    Science.gov (United States)

    Feng, Shu; Shao, Longjiang; Yu, Wendong; Gavine, Paul; Ittmann, Michael

    2012-07-15

    Extensive correlative studies in human prostate cancer as well as studies in vitro and in mouse models indicate that fibroblast growth factor receptor (FGFR) signaling plays an important role in prostate cancer progression. In this study, we used a probe compound for an FGFR inhibitor, which potently inhibits FGFR-1-3 and significantly inhibits FGFR-4. The purpose of this study is to determine whether targeting FGFR signaling from all four FGFRs will have in vitro activities consistent with inhibition of tumor progression and will inhibit tumor progression in vivo. Effects of AZ8010 on FGFR signaling and invasion were analyzed using immortalized normal prostate epithelial (PNT1a) cells and PNT1a overexpressing FGFR-1 or FGFR-4. The effect of AZ8010 on invasion and proliferation in vitro was also evaluated in prostate cancer cell lines. Finally, the impact of AZ8010 on tumor progression in vivo was evaluated using a VCaP xenograft model. AZ8010 completely inhibits FGFR-1 and significantly inhibits FGFR-4 signaling at 100 nmol/L, which is an achievable in vivo concentration. This results in marked inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and invasion in PNT1a cells expressing FGFR-1 and FGFR-4 and all prostate cancer cell lines tested. Treatment in vivo completely inhibited VCaP tumor growth and significantly inhibited angiogenesis and proliferation and increased cell death in treated tumors. This was associated with marked inhibition of ERK phosphorylation in treated tumors. Targeting FGFR signaling is a promising new approach to treating aggressive prostate cancer.

  9. MDM2 binds and inhibits vitamin D receptor

    OpenAIRE

    Heyne, Kristina; Heil, Tessa-Carina; Bette, Birgit; Reichrath, Jörg; Roemer, Klaus

    2015-01-01

    The E3 ubiquitin ligase and transcriptional repressor MDM2 is a potent inhibitor of the p53 family of transcription factors and tumor suppressors. Herein, we report that vitamin D receptor (VDR), another transcriptional regulator and probably, tumor suppressor, is also bound and inhibited by MDM2. This interaction was not affected by vitamin D ligand. VDR was ubiquitylated in the cell and its steady-state level was controlled by the proteasome. Strikingly, overproduced MDM2 reduced the level ...

  10. Inhibition of Estrogen Receptor Action by the Orphan Receptors, SHP and DAX-1

    National Research Council Canada - National Science Library

    DiRenzo, James

    2003-01-01

    .... In support of DoD grant # DAMD17-99-1-9163, we present our findings regarding the mechanisms by which two orphan nuclear receptors, SHP and DAX-1 inhibit the actions of ER-alpha and ER-beta action...

  11. IGF-1 receptor inhibition by picropodophyllin in medulloblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Ohshima-Hosoyama, Sachiko; Hosoyama, Tohru; Nelon, Laura D. [Greehey Children' s Cancer Research Institute, University of Texas Health Science Center, San Antonio, TX 78229 (United States); Keller, Charles, E-mail: keller@ohsu.edu [Greehey Children' s Cancer Research Institute, University of Texas Health Science Center, San Antonio, TX 78229 (United States); Department of Pediatrics, University of Texas Health Science Center, San Antonio, TX 78229 (United States); Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229 (United States)

    2010-09-03

    Research highlights: {yields} Igf1r is overexpressed and activated in a Sonic Hedgehog driven model of medulloblastoma. {yields} Picropodophyllin targets and abrogates IGF signaling in medulloblastoma. {yields} Picropodophyllin inhibits medulloblastoma tumor cell growth by induction of apoptosis. -- Abstract: The insulin-like growth factor-1 receptor (Igf1r) is a multifunctional membrane-associated tyrosine kinase associated with regulation of transformation, proliferation, differentiation and apoptosis. Increased IGF pathway activity has been reported in human and murine medulloblastoma. Tumors from our genetically-engineered medulloblastoma mouse model over-express Igf1r, and thus this mouse model is a good platform with which to study the role of Igf1r in tumor progression. We hypothesize that inhibition of IGF pathway in medulloblastoma can slow or inhibit tumor growth and metastasis. To test our hypothesis, we tested the role of IGF in tumor growth in vitro by treatment with the tyrosine kinase small molecule inhibitor, picropodophyllin (PPP), which strongly inhibits the IGF pathway. Our results demonstrate that PPP-mediated downregulation of the IGF pathway inhibits mouse tumor cell growth and induces apoptotic cell death in vitro in primary medulloblastoma cultures that are most reflective of tumor cell behavior in vivo.

  12. IGF-1 receptor inhibition by picropodophyllin in medulloblastoma

    International Nuclear Information System (INIS)

    Ohshima-Hosoyama, Sachiko; Hosoyama, Tohru; Nelon, Laura D.; Keller, Charles

    2010-01-01

    Research highlights: → Igf1r is overexpressed and activated in a Sonic Hedgehog driven model of medulloblastoma. → Picropodophyllin targets and abrogates IGF signaling in medulloblastoma. → Picropodophyllin inhibits medulloblastoma tumor cell growth by induction of apoptosis. -- Abstract: The insulin-like growth factor-1 receptor (Igf1r) is a multifunctional membrane-associated tyrosine kinase associated with regulation of transformation, proliferation, differentiation and apoptosis. Increased IGF pathway activity has been reported in human and murine medulloblastoma. Tumors from our genetically-engineered medulloblastoma mouse model over-express Igf1r, and thus this mouse model is a good platform with which to study the role of Igf1r in tumor progression. We hypothesize that inhibition of IGF pathway in medulloblastoma can slow or inhibit tumor growth and metastasis. To test our hypothesis, we tested the role of IGF in tumor growth in vitro by treatment with the tyrosine kinase small molecule inhibitor, picropodophyllin (PPP), which strongly inhibits the IGF pathway. Our results demonstrate that PPP-mediated downregulation of the IGF pathway inhibits mouse tumor cell growth and induces apoptotic cell death in vitro in primary medulloblastoma cultures that are most reflective of tumor cell behavior in vivo.

  13. NMDA receptor antagonists inhibit catalepsy induced by either dopamine D1 or D2 receptor antagonists.

    Science.gov (United States)

    Moore, N A; Blackman, A; Awere, S; Leander, J D

    1993-06-11

    In the present study, we investigated the ability of NMDA receptor antagonists to inhibit catalepsy induced by haloperidol, or SCH23390 and clebopride, selective dopamine D1 and D2 receptor antagonists respectively. Catalepsy was measured by recording the time the animal remained with its forepaws placed over a rod 6 cm above the bench. Pretreatment with either the non-competitive NMDA receptor antagonist, MK-801 (0.25-0.5 mg/kg i.p.) or the competitive antagonist, LY274614 (10-20 mg/kg i.p.) reduced the cataleptic response produced by haloperidol (10 mg/kg), SCH23390 (2.5-10 mg/kp i.p.) or clebopride (5-20 mg/kg i.p.). This demonstrates that NMDA receptor antagonists will reduce both dopamine D1 and D2 receptor antagonist-induced catalepsy. Muscle relaxant doses of chlordiazepoxide (10 mg/kg i.p.) failed to reduce the catalepsy induced by haloperidol, suggesting that the anticataleptic effect of the NMDA receptor antagonists was not due to a non-specific action. These results support the hypothesis that NMDA receptor antagonists may have beneficial effects in disorders involving reduced dopaminergic function, such as Parkinson's disease.

  14. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying-Nan P.; LaMarche, Matthew J.; Chan, Ho Man; Fekkes, Peter; Garcia-Fortanet, Jorge; Acker, Michael G.; Antonakos, Brandon; Chen, Christine Hiu-Tung; Chen, Zhouliang; Cooke, Vesselina G.; Dobson, Jason R.; Deng, Zhan; Fei, Feng; Firestone, Brant; Fodor, Michelle; Fridrich, Cary; Gao, Hui; Grunenfelder, Denise; Hao, Huai-Xiang; Jacob, Jaison; Ho, Samuel; Hsiao, Kathy; Kang, Zhao B.; Karki, Rajesh; Kato, Mitsunori; Larrow, Jay; La Bonte, Laura R.; Lenoir, Francois; Liu, Gang; Liu, Shumei; Majumdar, Dyuti; Meyer, Matthew J.; Palermo, Mark; Perez, Lawrence; Pu, Minying; Price, Edmund; Quinn, Christopher; Shakya, Subarna; Shultz, Michael D.; Slisz, Joanna; Venkatesan, Kavitha; Wang, Ping; Warmuth, Markus; Williams, Sarah; Yang, Guizhi; Yuan, Jing; Zhang, Ji-Hu; Zhu, Ping; Ramsey, Timothy; Keen, Nicholas J.; Sellers, William R.; Stams, Travis; Fortin , Pascal D. (Novartis)

    2016-06-29

    The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase1. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma1, 2, 3, 4, 5. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway2, 3. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways6, 7. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy8, 9. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

  15. Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Feng [Department of Gastroenterology, The Tenth People’s Hospital of Shanghai, Tongji University, Shanghai 200072 (China); Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Yang, Yong, E-mail: yyang@houstonmethodist.org [Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Department of Medicine, Weill Cornell Medical College, New York, NY 10065 (United States)

    2014-10-03

    Graphical abstract: - Highlights: • Quercetin inhibits insulin ligand–receptor interactions. • Quercetin reduces downstream insulin receptor signaling. • Quercetin blocks insulin induced glucose uptake. • Quercetin suppresses insulin stimulated cancer cell proliferation and tumor growth. - Abstract: Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers.

  16. Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

    International Nuclear Information System (INIS)

    Wang, Feng; Yang, Yong

    2014-01-01

    Graphical abstract: - Highlights: • Quercetin inhibits insulin ligand–receptor interactions. • Quercetin reduces downstream insulin receptor signaling. • Quercetin blocks insulin induced glucose uptake. • Quercetin suppresses insulin stimulated cancer cell proliferation and tumor growth. - Abstract: Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers

  17. Targeting the urokinase plasminogen activator receptor inhibits ovarian cancer metastasis.

    Science.gov (United States)

    Kenny, Hilary A; Leonhardt, Payton; Ladanyi, Andras; Yamada, S Diane; Montag, Anthony; Im, Hae Kyung; Jagadeeswaran, Sujatha; Shaw, David E; Mazar, Andrew P; Lengyel, Ernst

    2011-02-01

    To understand the functional and preclinical efficacy of targeting the urokinase plasminogen activator receptor (u-PAR) in ovarian cancer. Expression of u-PAR was studied in 162 epithelial ovarian cancers, including 77 pairs of corresponding primary and metastatic tumors. The effect of an antibody against u-PAR (ATN-658) on proliferation, adhesion, invasion, apoptosis, and migration was assessed in 3 (SKOV3ip1, HeyA8, and CaOV3) ovarian cancer cell lines. The impact of the u-PAR antibody on tumor weight, number, and survival was examined in corresponding ovarian cancer xenograft models and the mechanism by which ATN-658 blocks metastasis was explored. Only 8% of all ovarian tumors were negative for u-PAR expression. Treatment of SKOV3ip1, HeyA8, and CaOV3 ovarian cancer cell lines with the u-PAR antibody inhibited cell invasion, migration, and adhesion. In vivo, anti-u-PAR treatment reduced the number of tumors and tumor weight in CaOV3 and SKOV3ip1 xenografts and reduced tumor weight and increased survival in HeyA8 xenografts. Immunostaining of CaOV3 xenograft tumors and ovarian cancer cell lines showed an increase in active-caspase 3 and TUNEL staining. Treatment with u-PAR antibody inhibited α(5)-integrin and u-PAR colocalization on primary human omental extracellular matrix. Anti-u-PAR treatment also decreased the expression of urokinase, u-PAR, β(3)-integrin, and fibroblast growth factor receptor-1 both in vitro and in vivo. This study shows that an antibody against u-PAR reduces metastasis, induces apoptosis, and reduces the interaction between u-PAR and α(5)-integrin. This provides a rationale for targeting the u-PAR pathway in patients with ovarian cancer and for further testing of ATN-658 in this indication. ©2010 AACR.

  18. Human Diversity in a Cell Surface Receptor that Inhibits Autophagy.

    Science.gov (United States)

    Chaudhary, Anu; Leite, Mara; Kulasekara, Bridget R; Altura, Melissa A; Ogahara, Cassandra; Weiss, Eli; Fu, Wenqing; Blanc, Marie-Pierre; O'Keeffe, Michael; Terhorst, Cox; Akey, Joshua M; Miller, Samuel I

    2016-07-25

    Mutations in genes encoding autophagy proteins have been associated with human autoimmune diseases, suggesting that diversity in autophagy responses could be associated with disease susceptibility or severity. A cellular genome-wide association study (GWAS) screen was performed to explore normal human diversity in responses to rapamycin, a microbial product that induces autophagy. Cells from several human populations demonstrated variability in expression of a cell surface receptor, CD244 (SlamF4, 2B4), that correlated with changes in rapamycin-induced autophagy. High expression of CD244 and receptor activation with its endogenous ligand CD48 inhibited starvation- and rapamycin-induced autophagy by promoting association of CD244 with the autophagy complex proteins Vps34 and Beclin-1. The association of CD244 with this complex reduced Vps34 lipid kinase activity. Lack of CD244 is associated with auto-antibody production in mice, and lower expression of human CD244 has previously been implicated in severity of human rheumatoid arthritis and systemic lupus erythematosus, indicating that increased autophagy as a result of low levels of CD244 may alter disease outcomes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Hypoxia increases exercise heart rate despite combined inhibition of β-adrenergic and muscarinic receptors

    DEFF Research Database (Denmark)

    Siebenmann, Christoph; Rasmussen, Peter; Sørensen, Henrik

    2015-01-01

    Hypoxia increases the heart rate (HR) response to exercise but the mechanism(s) remain unclear. We tested the hypothesis that the tachycardic effect of hypoxia persists during separate but not combined inhibition of β-adrenergic and muscarinic receptors. Nine subjects performed incremental exercise...... combined β-adrenergic and muscarinic receptor inhibition....

  20. GABA-A Receptors Mediate Tonic Inhibition and Neurosteroid Sensitivity in the Brain.

    Science.gov (United States)

    Reddy, Doodipala Samba

    2018-01-01

    Neurosteroids like allopregnanolone (AP) are positive allosteric modulators of synaptic and extrasynaptic GABA-A receptors. AP and related neurosteroids exhibit a greater potency for δ-containing extrasynaptic receptors. The δGABA-A receptors, which are expressed extrasynaptically in the dentate gyrus and other regions, contribute to tonic inhibition, promoting network shunting as well as reducing seizure susceptibility. Levels of endogenous neurosteroids fluctuate with ovarian cycle. Natural and synthetic neurosteroids maximally potentiate tonic inhibition in the hippocampus and provide robust protection against a variety of limbic seizures and status epilepticus. Recently, a consensus neurosteroid pharmacophore model has been proposed at extrasynaptic δGABA-A receptors based on structure-activity relationship for functional activation of tonic currents and seizure protection. Aside from anticonvulsant actions, neurosteroids have been found to be powerful anxiolytic and anesthetic agents. Neurosteroids and Zn 2+ have preferential affinity for δ-containing receptors. Thus, Zn 2+ can prevent neurosteroid activation of extrasynaptic δGABA-A receptor-mediated tonic inhibition. Recently, we demonstrated that Zn 2+ selectively inhibits extrasynaptic δGABA-A receptors and thereby fully prevents AP activation of tonic inhibition and seizure protection. We confirmed that neurosteroids exhibit greater sensitivity at extrasynaptic δGABA-A receptors. Overall, extrasynaptic GABA-A receptors are primary mediators of tonic inhibition in the brain and play a key role in the pathophysiology of epilepsy and other neurological disorders. © 2018 Elsevier Inc. All rights reserved.

  1. Sigma-1 receptor agonists directly inhibit Nav1.2/1.4 channels.

    Directory of Open Access Journals (Sweden)

    Xiao-Fei Gao

    Full Text Available (+-SKF 10047 (N-allyl-normetazocine is a prototypic and specific sigma-1 receptor agonist that has been used extensively to study the function of sigma-1 receptors. (+-SKF 10047 inhibits K(+, Na(+ and Ca2+ channels via sigma-1 receptor activation. We found that (+-SKF 10047 inhibited Na(V1.2 and Na(V1.4 channels independently of sigma-1 receptor activation. (+-SKF 10047 equally inhibited Na(V1.2/1.4 channel currents in HEK293T cells with abundant sigma-1 receptor expression and in COS-7 cells, which barely express sigma-1 receptors. The sigma-1 receptor antagonists BD 1063,BD 1047 and NE-100 did not block the inhibitory effects of (+-SKF-10047. Blocking of the PKA, PKC and G-protein pathways did not affect (+-SKF 10047 inhibition of Na(V1.2 channel currents. The sigma-1 receptor agonists Dextromethorphan (DM and 1,3-di-o-tolyl-guanidine (DTG also inhibited Na(V1.2 currents through a sigma-1 receptor-independent pathway. The (+-SKF 10047 inhibition of Na(V1.2 currents was use- and frequency-dependent. Point mutations demonstrated the importance of Phe(1764 and Tyr(1771 in the IV-segment 6 domain of the Na(V1.2 channel and Phe(1579 in the Na(V1.4 channel for (+-SKF 10047 inhibition. In conclusion, our results suggest that sigma-1 receptor agonists directly inhibit Na(V1.2/1.4 channels and that these interactions should be given special attention for future sigma-1 receptor function studies.

  2. Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

    Directory of Open Access Journals (Sweden)

    Ahnen Dennis

    2005-01-01

    Full Text Available Abstract Background Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs is associated with a decreased mortality from colorectal cancer (CRC. NSAIDs induce apoptotic cell death in colon cancer cells in vitro and inhibit growth of neoplastic colonic mucosa in vivo however, the biochemical mechanisms required for these growth inhibitory effects are not well defined. We previously reported that metabolites of the NSAID sulindac downregulate extracellular-signal regulated kinase 1/2 (ERK1/2 signaling and that this effect is both necessary and sufficient for the apoptotic effects of these drugs. The goal of this project was to specifically test the hypothesis that sulindac metabolites block activation and/or expression of the epidermal growth factor (EGF receptor (EGFR. Methods HT29 human colon cancer cells were treated with EGF, alone, or in the presence of sulindac sulfide or sulindac sulfone. Cells lysates were assayed by immunoblotting for phosphorylated EGFR (pEGFR, pY1068, total EGFR, phosphorylated ERK1/2 (pERK1/2, total ERK1/2, activated caspase-3, and α-tubulin. Results EGF treatment rapidly induced phosphorylation of both EGFR and ERK1/2 in HT29 colon cancer cells. Pretreatment with sulindac metabolites for 24 h blocked EGF-induced phosphorylation of both EGFR and ERK1/2 and decreased total EGFR protein expression. Under basal conditions, downregulation of pEGFR and total EGFR was detected as early as 12 h following sulindac sulfide treatment and persisted through at least 48 h. Sulindac sulfone induced downregulation of pEGFR and total EGFR was detected as early as 1 h and 24 h, respectively, following drug treatment, and persisted through at least 72 h. EGFR downregulation by sulindac metabolites was observed in three different CRC cell lines, occurred prior to the observed downregulation of pERK1/2 and induction of apoptosis by these drugs, and was not dependent of caspase activation. Conclusion These results suggest that

  3. Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.

    Science.gov (United States)

    Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

    2013-05-07

    Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

  4. Competitive inhibition of [3H]dexamethasone binding to mammary glucocorticoid receptor by leupeptin

    International Nuclear Information System (INIS)

    Hsieh, L.C.C.; Su, C.; Markland, F.S. Jr.

    1987-01-01

    The inhibitory effect of leupeptin on [ 3 H]dexamethasone binding to the glucocorticoid receptor from lactating goat mammary cytosol has been studied. Leupeptin (10 mM) caused a significant (about 35%) inhibition of [ 3 H]dexamethasone binding to glucocorticoid receptor. Binding inhibition is further increased following filtration of unlabeled cytosolic receptor through a Bio-Gel A 0.5-m column. Binding inhibition was partially reversed by monothioglycerol at 10 mM concentration. A double reciprocal plot revealed that leupeptin appears to be a competitive inhibitor of [ 3 H]dexamethasone binding to the glucocorticoid receptor. Low salt sucrose density gradient centrifugation revealed that the leupeptin-treated sample formed a slightly larger (approximately 9 S) receptor complex (leupeptin-free complex sediments at 8 S)

  5. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation

    Science.gov (United States)

    Perception of pathogen-associated molecular patterns (PAMPs) by surface-localised pattern-recognition receptors (PRRs) is a key component of plant innate immunity. Most known plant PRRs are receptor kinases and initiation of PAMP-triggered immunity (PTI) signalling requires phosphorylation of the PR...

  6. Melatonin modulates rat myotube-acetylcholine receptors by inhibiting calmodulin.

    Science.gov (United States)

    de Almeida-Paula, Lidiana Duarte; Costa-Lotufo, Leticia V; Silva Ferreira, Zulma; Monteiro, Amanda Elisa G; Isoldi, Mauro Cesar; Godinho, Rosely O; Markus, Regina P

    2005-11-21

    Melatonin, the pineal gland hormone, modulates alpha-bungarotoxin sensitive nicotinic acetylcholine receptors in sympathetic nerve terminals, cerebellum and chick retina imposing a diurnal variation in functional responses [Markus, R.P., Zago, W.M., Carneiro, R.C., 1996. Melatonin modulation of presynaptic nicotinic acetylcholine receptors in the rat vas deferens. J. Pharmacol. Exp. Ther. 279, 18-22; Markus, R.P., Santos, J.M., Zago, W., Reno, L.A., 2003. Melatonin nocturnal surge modulates nicotinic receptors and nicotine-induced [3HI] glutamate release in rat cerebellum slices. J. Pharmacol. Exp. Ther. 305, 525-530; Sampaio, L.F.S., Hamassaki-Britto, D.E., Markus, R.P., 2005. Influence of melatonin on the development of functional nicotinic acetylcholine receptors in cultured chick retinal cells. Braz. J. Med. Biol. Res. 38, 603-613]. Here we show that in rat myotubes forskolin and melatonin reduced the number of nicotinic acetylcholine receptors expressed in plasma membrane. In addition, these cells expressed melatonin MT1 receptors, which are known to be coupled to G(i)-protein. However, the pharmacological profile of melatonin analogs regarding the reduction in cyclic AMP accumulation and number of nicotinic acetylcholine receptors did not point to a mechanism mediated by activation of G(i)-protein coupled receptors. On the other hand, calmidazolium, a classical inhibitor of calmodulin, reduced in a similar manner both effects. Considering that one isoform of adenylyl cyclase present in rat myotubes is regulated by Ca2+/calmodulin, we propose that melatonin modulates the number of nicotinic acetylcholine receptors via reduction in cyclic AMP accumulation.

  7. Increased NMDA receptor inhibition at an increased Sevoflurane MAC

    Directory of Open Access Journals (Sweden)

    Brosnan Robert J

    2012-06-01

    Full Text Available Abstract Background Sevoflurane potently enhances glycine receptor currents and more modestly decreases NMDA receptor currents, each of which may contribute to immobility. This modest NMDA receptor antagonism by sevoflurane at a minimum alveolar concentration (MAC could be reciprocally related to large potentiation of other inhibitory ion channels. If so, then reduced glycine receptor potency should increase NMDA receptor antagonism by sevoflurane at MAC. Methods Indwelling lumbar subarachnoid catheters were surgically placed in 14 anesthetized rats. Rats were anesthetized with sevoflurane the next day, and a pre-infusion sevoflurane MAC was measured in duplicate using a tail clamp method. Artificial CSF (aCSF containing either 0 or 4 mg/mL strychnine was then infused intrathecally at 4 μL/min, and the post-infusion baseline sevoflurane MAC was measured. Finally, aCSF containing strychnine (either 0 or 4 mg/mL plus 0.4 mg/mL dizocilpine (MK-801 was administered intrathecally at 4 μL/min, and the post-dizocilpine sevoflurane MAC was measured. Results Pre-infusion sevoflurane MAC was 2.26%. Intrathecal aCSF alone did not affect MAC, but intrathecal strychnine significantly increased sevoflurane requirement. Addition of dizocilpine significantly decreased MAC in all rats, but this decrease was two times larger in rats without intrathecal strychnine compared to rats with intrathecal strychnine, a statistically significant (P  Conclusions Glycine receptor antagonism increases NMDA receptor antagonism by sevoflurane at MAC. The magnitude of anesthetic effects on a given ion channel may therefore depend on the magnitude of its effects on other receptors that modulate neuronal excitability.

  8. Pumpkin seed extract: Cell growth inhibition of hyperplastic and cancer cells, independent of steroid hormone receptors.

    Science.gov (United States)

    Medjakovic, Svjetlana; Hobiger, Stefanie; Ardjomand-Woelkart, Karin; Bucar, Franz; Jungbauer, Alois

    2016-04-01

    Pumpkin seeds have been known in folk medicine as remedy for kidney, bladder and prostate disorders since centuries. Nevertheless, pumpkin research provides insufficient data to back up traditional beliefs of ethnomedical practice. The bioactivity of a hydro-ethanolic extract of pumpkin seeds from the Styrian pumpkin, Cucurbita pepo L. subsp. pepo var. styriaca, was investigated. As pumpkin seed extracts are standardized to cucurbitin, this compound was also tested. Transactivational activity was evaluated for human androgen receptor, estrogen receptor and progesterone receptor with in vitro yeast assays. Cell viability tests with prostate cancer cells, breast cancer cells, colorectal adenocarcinoma cells and a hyperplastic cell line from benign prostate hyperplasia tissue were performed. As model for non-hyperplastic cells, effects on cell viability were tested with a human dermal fibroblast cell line (HDF-5). No transactivational activity was found for human androgen receptor, estrogen receptor and progesterone receptor, for both, extract and cucurbitin. A cell growth inhibition of ~40-50% was observed for all cell lines, with the exception of HDF-5, which showed with ~20% much lower cell growth inhibition. Given the receptor status of some cell lines, a steroid-hormone receptor independent growth inhibiting effect can be assumed. The cell growth inhibition for fast growing cells together with the cell growth inhibition of prostate-, breast- and colon cancer cells corroborates the ethnomedical use of pumpkin seeds for a treatment of benign prostate hyperplasia. Moreover, due to the lack of androgenic activity, pumpkin seed applications can be regarded as safe for the prostate. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Stimulation of accumbal GABAA receptors inhibits delta2-, but not delta1-, opioid receptor-mediated dopamine efflux in the nucleus accumbens of freely moving rats.

    Science.gov (United States)

    Aono, Yuri; Kiguchi, Yuri; Watanabe, Yuriko; Waddington, John L; Saigusa, Tadashi

    2017-11-15

    The nucleus accumbens contains delta-opioid receptors that may reduce inhibitory neurotransmission. Reduction in GABA A receptor-mediated inhibition of accumbal dopamine release due to delta-opioid receptor activation should be suppressed by stimulating accumbal GABA A receptors. As delta-opioid receptors are divided into delta2- and delta1-opioid receptors, we analysed the effects of the GABA A receptor agonist muscimol on delta2- and delta1-opioid receptor-mediated accumbal dopamine efflux in freely moving rats using in vivo microdialysis. Drugs were administered intracerebrally through the dialysis probe. Doses of compounds indicate total amount administered (mol) during 25-50min infusions. The delta2-opioid receptor agonist deltorphin II (25.0nmol)- and delta1-opioid receptor agonist DPDPE (5.0nmol)-induced increases in dopamine efflux were inhibited by the delta2-opioid receptor antagonist naltriben (1.5nmol) and the delta1-opioid receptor antagonist BNTX (150.0pmol), respectively. Muscimol (250.0pmol) inhibited deltorphin II (25.0nmol)-induced dopamine efflux. The GABA A receptor antagonist bicuculline (50.0pmol), which failed to affect deltorphin II (25.0nmol)-induced dopamine efflux, counteracted the inhibitory effect of muscimol on deltorphin II-induced dopamine efflux. Neither muscimol (250.0pmol) nor bicuculline (50.0 and 500.0pmol) altered DPDPE (5.0nmol)-induced dopamine efflux. The present results show that reduction in accumbal GABA A receptor-mediated inhibition of dopaminergic activity is necessary to produce delta2-opioid receptor-induced increase in accumbal dopamine efflux. This study indicates that activation of delta2- but not delta1-opioid receptors on the cell bodies and/or terminals of accumbal GABAergic interneurons inhibits GABA release and, accordingly, decreases GABA A receptor-mediated inhibition of dopaminergic terminals, resulting in enhanced accumbal dopamine efflux. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Azadirachtin interacts with retinoic acid receptors and inhibits retinoic acid-mediated biological responses.

    Science.gov (United States)

    Thoh, Maikho; Babajan, Banaganapalli; Raghavendra, Pongali B; Sureshkumar, Chitta; Manna, Sunil K

    2011-02-11

    Considering the role of retinoids in regulation of more than 500 genes involved in cell cycle and growth arrest, a detailed understanding of the mechanism and its regulation is useful for therapy. The extract of the medicinal plant Neem (Azadirachta indica) is used against several ailments especially for anti-inflammatory, anti-itching, spermicidal, anticancer, and insecticidal activities. In this report we prove the detailed mechanism on the regulation of retinoic acid-mediated cell signaling by azadirachtin, active components of neem extract. Azadirachtin repressed all trans-retinoic acid (ATRA)-mediated nuclear transcription factor κB (NF-κB) activation, not the DNA binding but the NF-κB-dependent gene expression. It did not inhibit IκBα degradation, IκBα kinase activity, or p65 phosphorylation and its nuclear translocation but inhibited NF-κB-dependent reporter gene expression. Azadirachtin inhibited TRAF6-mediated, but not TRAF2-mediated NF-κB activation. It inhibited ATRA-induced Sp1 and CREB (cAMP-response element-binding protein) DNA binding. Azadirachtin inhibited ATRA binding with retinoid receptors, which is supported by biochemical and in silico evidences. Azadirachtin showed strong interaction with retinoid receptors. It suppressed ATRA-mediated removal of retinoid receptors, bound with DNA by inhibiting ATRA binding to its receptors. Overall, our data suggest that azadirachtin interacts with retinoic acid receptors and suppresses ATRA binding, inhibits falling off the receptors, and activates transcription factors like CREB, Sp1, NF-κB, etc. Thus, azadirachtin exerts anti-inflammatory and anti-metastatic responses by a novel pathway that would be beneficial for further anti-inflammatory and anti-cancer therapies.

  11. Azadirachtin Interacts with Retinoic Acid Receptors and Inhibits Retinoic Acid-mediated Biological Responses*

    Science.gov (United States)

    Thoh, Maikho; Babajan, Banaganapalli; Raghavendra, Pongali B.; Sureshkumar, Chitta; Manna, Sunil K.

    2011-01-01

    Considering the role of retinoids in regulation of more than 500 genes involved in cell cycle and growth arrest, a detailed understanding of the mechanism and its regulation is useful for therapy. The extract of the medicinal plant Neem (Azadirachta indica) is used against several ailments especially for anti-inflammatory, anti-itching, spermicidal, anticancer, and insecticidal activities. In this report we prove the detailed mechanism on the regulation of retinoic acid-mediated cell signaling by azadirachtin, active components of neem extract. Azadirachtin repressed all trans-retinoic acid (ATRA)-mediated nuclear transcription factor κB (NF-κB) activation, not the DNA binding but the NF-κB-dependent gene expression. It did not inhibit IκBα degradation, IκBα kinase activity, or p65 phosphorylation and its nuclear translocation but inhibited NF-κB-dependent reporter gene expression. Azadirachtin inhibited TRAF6-mediated, but not TRAF2-mediated NF-κB activation. It inhibited ATRA-induced Sp1 and CREB (cAMP-response element-binding protein) DNA binding. Azadirachtin inhibited ATRA binding with retinoid receptors, which is supported by biochemical and in silico evidences. Azadirachtin showed strong interaction with retinoid receptors. It suppressed ATRA-mediated removal of retinoid receptors, bound with DNA by inhibiting ATRA binding to its receptors. Overall, our data suggest that azadirachtin interacts with retinoic acid receptors and suppresses ATRA binding, inhibits falling off the receptors, and activates transcription factors like CREB, Sp1, NF-κB, etc. Thus, azadirachtin exerts anti-inflammatory and anti-metastatic responses by a novel pathway that would be beneficial for further anti-inflammatory and anti-cancer therapies. PMID:21127062

  12. Nuclear receptor TLX inhibits TGF-β signaling in glioblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Johansson, Erik; Zhai, Qiwei [Sahlgrenska Cancer Center at the Sahlgrenska Academy, University of Gothenburg, Box 425, SE 405 30 Gothenburg (Sweden); Zeng, Zhao-jun [Sahlgrenska Cancer Center at the Sahlgrenska Academy, University of Gothenburg, Box 425, SE 405 30 Gothenburg (Sweden); Molecular Biology Research Center, School of Life Sciences, Central South University, 110, Xiangya Road, Changsha, Hunan 410078 (China); Yoshida, Takeshi [Sahlgrenska Cancer Center at the Sahlgrenska Academy, University of Gothenburg, Box 425, SE 405 30 Gothenburg (Sweden); Funa, Keiko, E-mail: keiko.funa@gu.se [Sahlgrenska Cancer Center at the Sahlgrenska Academy, University of Gothenburg, Box 425, SE 405 30 Gothenburg (Sweden)

    2016-05-01

    TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-β (TGF-β) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-β has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-β signaling we wanted to find out if there is any interaction between TLX and TGF-β in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-β signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-β receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-β receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-β target genes. The interaction between TLX and TGF-β may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells. - Highlights: • TLX knockdown enhances TGF-β dependent Smad signaling in glioblastoma cells • TLX knockdown increases the protein level of TGF-β receptor II. • TLX stabilizes and retains Smurf1 in the cytoplasm. • TLX enhances Smurf1-dependent ubiquitination and degradation of TGF-β receptor II.

  13. Nuclear receptor TLX inhibits TGF-β signaling in glioblastoma

    International Nuclear Information System (INIS)

    Johansson, Erik; Zhai, Qiwei; Zeng, Zhao-jun; Yoshida, Takeshi; Funa, Keiko

    2016-01-01

    TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-β (TGF-β) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-β has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-β signaling we wanted to find out if there is any interaction between TLX and TGF-β in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-β signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-β receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-β receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-β target genes. The interaction between TLX and TGF-β may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells. - Highlights: • TLX knockdown enhances TGF-β dependent Smad signaling in glioblastoma cells • TLX knockdown increases the protein level of TGF-β receptor II. • TLX stabilizes and retains Smurf1 in the cytoplasm. • TLX enhances Smurf1-dependent ubiquitination and degradation of TGF-β receptor II.

  14. Nuclear receptor TLX inhibits TGF-β signaling in glioblastoma.

    Science.gov (United States)

    Johansson, Erik; Zhai, Qiwei; Zeng, Zhao-Jun; Yoshida, Takeshi; Funa, Keiko

    2016-05-01

    TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-β (TGF-β) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-β has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-β signaling we wanted to find out if there is any interaction between TLX and TGF-β in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-β signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-β receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-β receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-β target genes. The interaction between TLX and TGF-β may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells. Copyright © 2016. Published by Elsevier Inc.

  15. Probenecid inhibits α-adrenergic receptor-mediated vasoconstriction in the human leg vasculature

    DEFF Research Database (Denmark)

    Nyberg, Michael Permin; Piil, Peter Bergmann; Kiehn, Oliver Thistrup

    2018-01-01

    to α1- and α2-adrenergic receptor stimulation in the human forearm and leg vasculature of young healthy male subjects (23±3 years). By use of immunolabeling and confocal microscopy, Panx1 channels were found to be expressed in vascular smooth muscle cells of arterioles in human leg skeletal muscle....... Probenecid treatment increased (Padrenergic receptor stimulation) by ≈15%, whereas the response to the α1-agonist phenylephrine was unchanged. Inhibition...

  16. Dopamine inhibits somatolactin gene expression in tilapia pituitary cells through the dopamine D2 receptors.

    Science.gov (United States)

    Jiang, Quan; Lian, Anji; He, Qi

    2016-07-01

    Dopamine (DA) is an important neurotransmitter in the central nervous system of vertebrates and possesses key hypophysiotropic functions. Early studies have shown that DA has a potent inhibitory effect on somatolactin (SL) release in fish. However, the mechanisms responsible for DA inhibition of SL gene expression are largely unknown. To this end, tilapia DA type-1 (D1) and type-2 (D2) receptor transcripts were examined in the neurointermediate lobe (NIL) of the tilapia pituitary by real-time PCR. In tilapia, DA not only was effective in inhibiting SL mRNA levels in vivo and in vitro, but also could abolish pituitary adenylate cyclase-activating polypeptide (PACAP)- and salmon gonadotropin-releasing hormone (sGnRH)-stimulated SL gene expression at the pituitary level. In parallel studies, the specific D2 receptor agonists quinpirole and bromocriptine could mimic the DA-inhibited SL gene expression. Furthermore, the D2 receptor antagonists domperidone and (-)-sulpiride could abolish the SL response to DA or the D2 agonist quinpirole, whereas D1 receptor antagonists SCH23390 and SKF83566 were not effective in this respect. In primary cultures of tilapia NIL cells, D2 agonist quinpirole-inhibited cAMP production could be blocked by co-treatment with the D2 antagonist domperidone and the ability of forskolin to increase cAMP production was also inhibited by quinpirole. Using a pharmacological approach, the AC/cAMP pathway was shown to be involved in quinpirole-inhibited SL mRNA expression. These results provide evidence that DA can directly inhibit SL gene expression at the tilapia pituitary level via D2 receptor through the AC/cAMP-dependent mechanism. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Modulating Estrogen Receptor-related Receptor-α Activity Inhibits Cell Proliferation*

    OpenAIRE

    Bianco, Stéphanie; Lanvin, Olivia; Tribollet, Violaine; Macari, Claire; North, Sophie; Vanacker, Jean-Marc

    2009-01-01

    High expression of the estrogen receptor-related receptor (ERR)-α in human tumors is correlated to a poor prognosis, suggesting an involvement of the receptor in cell proliferation. In this study, we show that a synthetic compound (XCT790) that modulates the activity of ERRα reduces the proliferation of various cell lines and blocks the G1/S transition of the cell cycle in an ERR...

  18. Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins.

    Science.gov (United States)

    Tollefson, A E; Toth, K; Doronin, K; Kuppuswamy, M; Doronina, O A; Lichtenstein, D L; Hermiston, T W; Smith, C A; Wold, W S

    2001-10-01

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of the RID, E3-14.7K, and E1B-19K genes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus

  19. Activation of dopamine D3 receptors inhibits reward-related learning induced by cocaine.

    Science.gov (United States)

    Kong, H; Kuang, W; Li, S; Xu, M

    2011-03-10

    Memories of learned associations between the rewarding properties of drugs and environmental cues contribute to craving and relapse in humans. The mesocorticolimbic dopamine (DA) system is involved in reward-related learning induced by drugs of abuse. DA D3 receptors are preferentially expressed in mesocorticolimbic DA projection areas. Genetic and pharmacological studies have shown that DA D3 receptors suppress locomotor-stimulant effects of cocaine and reinstatement of cocaine-seeking behaviors. Activation of the extracellular signal-regulated kinase (ERK) induced by acute cocaine administration is also inhibited by D3 receptors. How D3 receptors modulate cocaine-induced reward-related learning and associated changes in cell signaling in reward circuits in the brain, however, have not been fully investigated. In the present study, we show that D3 receptor mutant mice exhibit potentiated acquisition of conditioned place preference (CPP) at low doses of cocaine compared to wild-type mice. Activation of ERK and CaMKIIα, but not the c-Jun N-terminal kinase and p38, in the nucleus accumbens, amygdala and prefrontal cortex is also potentiated in D3 receptor mutant mice compared to that in wild-type mice following CPP expression. These results support a model in which D3 receptors modulate reward-related learning induced by low doses of cocaine by inhibiting activation of ERK and CaMKIIα in reward circuits in the brain. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  20. Inhibition of radiation-induced polyuria by histamine receptor antagonists

    Energy Technology Data Exchange (ETDEWEB)

    Donlon, M.A.; Melia, J.A.; Helgeson, E.A.; Wolfe, W.W.

    1986-03-01

    In previous studies the authors have demonstrated that gamma radiation results in polyuria, which is preceded by polydypsia. This suggests that the increased thirst elicited by radiation causes increased urinary volume (UV). Histamine, which is released following radiation exposure, also elicits drinking by nonirradiated rats when administered exogenously. In this study the authors have investigated both the role of water deprivation and the effect of histamine receptor antagonists (HRA) on radiation-induced polyuria. Sprague-Dawley rats were housed individually in metabolic cages. Water was allowed ad libitum except in deprivation experiments where water was removed for 24 hr immediately following radiation. Cimetidine (CIM), an H2 HRA, and dexbromopheniramine (DXB), an H1 HRA, were administered i.p. (16 and 1 mg/kg, respectively) 30 min prior to irradiation (950 rads from a cobalt source). UV was determined at 24-hr intervals for 3 days preceding irradiation and 24 hr postirradiation. UV in DXB treated rats was significantly reduced 24 hr postirradiation (CON = 427 +/- 54%; DXB = 247 +/- 39% of preirradiated CON) compared to postirradiation control values. CIM did not affect postirradiation UV. These data suggest that radiation-induced polyuria is caused by polydypsia which is, in part, mediated by histamine induced by an H1 receptor.

  1. Inhibition of radiation-induced polyuria by histamine receptor antagonists

    International Nuclear Information System (INIS)

    Donlon, M.A.; Melia, J.A.; Helgeson, E.A.; Wolfe, W.W.

    1986-01-01

    In previous studies the authors have demonstrated that gamma radiation results in polyuria, which is preceded by polydypsia. This suggests that the increased thirst elicited by radiation causes increased urinary volume (UV). Histamine, which is released following radiation exposure, also elicits drinking by nonirradiated rats when administered exogenously. In this study the authors have investigated both the role of water deprivation and the effect of histamine receptor antagonists (HRA) on radiation-induced polyuria. Sprague-Dawley rats were housed individually in metabolic cages. Water was allowed ad libitum except in deprivation experiments where water was removed for 24 hr immediately following radiation. Cimetidine (CIM), an H2 HRA, and dexbromopheniramine (DXB), an H1 HRA, were administered i.p. (16 and 1 mg/kg, respectively) 30 min prior to irradiation (950 rads from a cobalt source). UV was determined at 24-hr intervals for 3 days preceding irradiation and 24 hr postirradiation. UV in DXB treated rats was significantly reduced 24 hr postirradiation (CON = 427 +/- 54%; DXB = 247 +/- 39% of preirradiated CON) compared to postirradiation control values. CIM did not affect postirradiation UV. These data suggest that radiation-induced polyuria is caused by polydypsia which is, in part, mediated by histamine induced by an H1 receptor

  2. Pharmacological or genetic orexin 1 receptor inhibition attenuates MK-801 induced glutamate release in mouse cortex

    Directory of Open Access Journals (Sweden)

    Leah eAluisio

    2014-05-01

    Full Text Available The orexin/hypocretin neuropeptides are produced by a cluster of neurons within the lateral posterior hypothalamus and participate in neuronal regulation by activating their receptors (OX1 and OX2 receptors. The orexin system projects widely through the brain and functions as an interface between multiple regulatory systems including wakefulness, energy balance, stress, reward and emotion. Recent studies have demonstrated that orexins and glutamate interact at the synaptic level and that orexins facilitate glutamate actions. We tested the hypothesis that orexins modulate glutamate signaling via OX1 receptors by monitoring levels of glutamate in frontal cortex of freely moving mice using enzyme coated biosensors under inhibited OX1 receptor conditions. MK-801, an NMDA receptor antagonist, was administered subcutaneously (0.178 mg/kg to indirectly disinhibit pyramidal neurons and therefore increase cortical glutamate release. In wild-type mice, pretreatment with the OX1 receptor antagonist GSK-1059865 (10 mg/kg S.C. which had no effect by itself, significantly attenuated the cortical glutamate release elicited by MK-801. OX1 receptor knockout mice had a blunted glutamate release response to MK-801 and exhibited about half of the glutamate release observed in wild-type mice in agreement with the data obtained with transient blockade of OX1 receptors. These results indicate that pharmacological (transient or genetic (permanent inhibition of the OX1 receptor similarly interfere with glutamatergic function in the cortex. Selectively targeting the OX1 receptor with an antagonist may normalize hyperglutamatergic states and thus may represent a novel therapeutic strategy for the treatment of various psychiatric disorders associated with hyperactive states.

  3. Antidepressants inhibit P2X4 receptor function: a possible involvement in neuropathic pain relief

    Directory of Open Access Journals (Sweden)

    Tozaki-Saitoh Hidetoshi

    2009-04-01

    Full Text Available Abstract Background Neuropathic pain is characterized by pain hypersensitivity to innocuous stimuli (tactile allodynia that is nearly always resistant to known treatments such as non-steroidal anti-inflammatory drugs or even opioids. It has been reported that some antidepressants are effective for treating neuropathic pain. However, the underlying molecular mechanisms are not well understood. We have recently demonstrated that blocking P2X4 receptors in the spinal cord reverses tactile allodynia after peripheral nerve injury in rats, implying that P2X4 receptors are a key molecule in neuropathic pain. We investigated a possible role of antidepressants as inhibitors of P2X4 receptors and analysed their analgesic mechanism using an animal model of neuropathic pain. Results Antidepressants strongly inhibited ATP-mediated Ca2+ responses in P2X4 receptor-expressing 1321N1 cells, which are known to have no endogenous ATP receptors. Paroxetine exhibited the most powerful inhibition of calcium influx via rat and human P2X4 receptors, with IC50 values of 2.45 μM and 1.87 μM, respectively. Intrathecal administration of paroxetine produced a striking antiallodynic effect in an animal model of neuropathic pain. Co-administration of WAY100635, ketanserin or ondansetron with paroxetine induced no significant change in the antiallodynic effect of paroxetine. Furthermore, the antiallodynic effect of paroxetine was observed even in rats that had received intrathecal pretreatment with 5,7-dihydroxytryptamine, which dramatically depletes spinal 5-hydroxytryptamine. Conclusion These results suggest that paroxetine acts as a potent analgesic in the spinal cord via a mechanism independent of its inhibitory effect on serotonin transporters. Powerful inhibition on P2X4 receptors may underlie the analgesic effect of paroxetine, and it is possible that some antidepressants clinically used in patients with neuropathic pain show antiallodynic effects, at least in part

  4. B cell receptor inhibition as a target for CLL therapy.

    Science.gov (United States)

    Jeyakumar, Deepa; O'Brien, Susan

    2016-03-01

    Inhibitors of the B cell receptor (BCR) represent an attractive therapeutic option for patients with chronic lymphocytic leukemia. Recently approved inhibitors of Bruton's tyrosine kinase (ibrutinib) and phosphatidylinositol 3-kinase (idelalisib), are promising agents because they are generally well tolerated and highly effective. These agents may be particularly important in the treatment of older patients who are less able to tolerate the myelosuppression (and infections) associated with chemoimmunotherapy. As a class of medications, BCR inhibitors have some unique side effects including redistribution lymphocytosis. Ibrutinib has specific toxicities including increased risk for bleeding and atrial fibrillation. Idelalisib also has some unique toxicities consisting of transaminitis, diarrhea and pneumonitis. Ongoing clinical trials are evaluating these agents in combination with antibodies, chemotherapy and other small molecules. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Inhibition of neointima formation by local delivery of estrogen receptor alpha and beta specific agonists

    NARCIS (Netherlands)

    Krom, Y.D.; Pires, N.M.M.; Jukema, J.W.; Vries, M.R. de; Frants, R.R.; Havekes, L.M.; Dijk, K.W. van; Quax, P.H.A.

    2007-01-01

    Objective: Neointima formation is the underlying mechanism of (in-stent) restenosis. 17β-Estradiol (E2) is known to inhibit injury-induced neointima formation and post-angioplasty restenosis. Estrogen receptor alpha (ERα) has been demonstrated to mediate E2 anti-restenotic properties. However, the

  6. Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins

    DEFF Research Database (Denmark)

    Hansen, J A; Lindberg, K; Hilton, D J

    1999-01-01

    In this study we have investigated the role of suppressor of cytokine signaling (SOCS) proteins in GH receptor-mediated signaling. GH-induced transcription was inhibited by SOCS-1 and SOCS-3, while SOCS-2 and cytokine inducible SH2-containing protein (CIS) had no effect By using chimeric SOCS pro...

  7. Molecular requirements for inhibition of the chemokine receptor CCR8--probe-dependent allosteric interactions

    DEFF Research Database (Denmark)

    Rummel, Pia Cwarzko; Arfelt, K N; Baumann, L

    2012-01-01

    Here we present a novel series of CCR8 antagonists based on a naphthalene-sulfonamide structure. This structure differs from the predominant pharmacophore for most small-molecule CC-chemokine receptor antagonists, which in fact activate CCR8, suggesting that CCR8 inhibition requires alternative...

  8. Sigma receptor ligand N,N'-di-(ortho-tolyl)guanidine inhibits release of acetylcholine in the guinea pig ileum.

    Science.gov (United States)

    Cambell, B G; Keana, J F; Weber, E

    1991-11-26

    The inhibition of stimulated contractions of the guinea pig ileum longitudinal muscle/myenteric plexus preparation by sigma receptor ligands has been previously described. In this study, the stimulated release of [3H]acetylcholine from cholinergic nerve terminals in this same preparation was monitored in the presence and absence of sigma receptor ligands. N,N'-Di-(orthotolyl)guanidine (DTG) and other compounds selective for the sigma receptor inhibited stimulated [3H]acetylcholine release. These results suggest that their inhibition of stimulated contractions in this preparation was mediated by inhibition of acetylcholine release.

  9. P2X1 Receptor Antagonists Inhibit HIV-1 Fusion by Blocking Virus-Coreceptor Interactions.

    Science.gov (United States)

    Giroud, Charline; Marin, Mariana; Hammonds, Jason; Spearman, Paul; Melikyan, Gregory B

    2015-09-01

    HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and the coreceptor CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of antiviral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels but effectively blocks the binding of the virus to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that the addition of ATP or the enzymatic depletion of ATP in culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4, and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env. Inhibition of P2X receptor activity suppresses HIV-1 fusion and replication, suggesting that P2X signaling is involved in HIV-1 entry. However, mechanistic experiments conducted in this study imply that P2X1 receptor is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1 receptor antagonist, NF

  10. Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B

    Science.gov (United States)

    Venkatesan, Balachandar; Ghosh-Choudhury, Nandini; Das, Falguni; Mahimainathan, Lenin; Kamat, Amrita; Kasinath, Balakuntalam S.; Abboud, Hanna E.; Choudhury, Goutam Ghosh

    2008-01-01

    Mesangioproliferative glomerulonephritis is associated with overactive PDGF receptor signal transduction. We show that the phytoalexin resveratrol dose dependently inhibits PDGF-induced DNA synthesis in mesangial cells with an IC50 of 10 μM without inducing apoptosis. Remarkably, the increased SIRT1 deacetylase activity induced by resveratrol was not necessary for this inhibitory effect. Resveratrol significantly blocked PDGF-stimulated c-Src and Akt kinase activation, resulting in reduced cyclin D1 expression and attenuated pRb phosphorylation and cyclin-dependent kinase-2 (CDK2) activity. Furthermore, resveratrol inhibited PDGFR phosphorylation at the PI 3 kinase and Grb-2 binding sites tyrosine-751 and tyrosine-716, respectively. This deficiency in PDGFR phosphorylation resulted in significant inhibition of PI 3 kinase and Erk1/2 MAPK activity. Interestingly, resveratrol increased the activity of protein tyrosine phosphatase PTP1B, which dephosphorylates PDGF-stimulated phosphorylation at tyrosine-751 and tyrosine-716 on PDGFR with concomitant reduction in Akt and Erk1/2 kinase activity. PTP1B significantly inhibited PDGF-induced DNA synthesis without inducing apoptosis. These results for the first time provide evidence that the stilbene resveratrol targets PTP1B to inhibit PDGFR mitogenic signaling.—Venkatesan, B., Ghosh-Choudhury, N., Das, F., Mahimainathan, L., Kamat, A., Kasinath, B. S., Abboud, H. E., Choudhury, G. G. Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B. PMID:18567737

  11. Modulating Estrogen Receptor-related Receptor-α Activity Inhibits Cell Proliferation*

    Science.gov (United States)

    Bianco, Stéphanie; Lanvin, Olivia; Tribollet, Violaine; Macari, Claire; North, Sophie; Vanacker, Jean-Marc

    2009-01-01

    High expression of the estrogen receptor-related receptor (ERR)-α in human tumors is correlated to a poor prognosis, suggesting an involvement of the receptor in cell proliferation. In this study, we show that a synthetic compound (XCT790) that modulates the activity of ERRα reduces the proliferation of various cell lines and blocks the G1/S transition of the cell cycle in an ERRα-dependent manner. XCT790 induces, in a p53-independent manner, the expression of the cell cycle inhibitor p21waf/cip1 at the protein, mRNA, and promoter level, leading to an accumulation of hypophosphorylated Rb. Finally, XCT790 reduces cell tumorigenicity in Nude mice. PMID:19546226

  12. Acutely increasing δGABAA receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus

    Science.gov (United States)

    Whissell, Paul D.; Eng, Dave; Lecker, Irene; Martin, Loren J.; Wang, Dian-Shi; Orser, Beverley A.

    2013-01-01

    Extrasynaptic γ-aminobutyric acid type A (GABAA) receptors that contain the δ subunit (δGABAA receptors) are expressed in several brain regions including the dentate gyrus (DG) and CA1 subfields of the hippocampus. Drugs that increase δGABAA receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABAA receptor–preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) enhances discrimination memory and increases neurogenesis in the DG. Despite the potential therapeutic benefits of such treatments, the effects of acutely increasing δGABAA receptor activity on memory behaviors remain unknown. Here, we studied the effects of THIP (4 mg/kg, i.p.) on memory performance in wild-type (WT) and δGABAA receptor null mutant (Gabrd−/−) mice. Additionally, the effects of THIP on long-term potentiation (LTP), a molecular correlate of memory, were studied within the DG and CA1 subfields of the hippocampus using electrophysiological recordings of field potentials in hippocampal slices. The results showed that THIP impaired performance in the Morris water maze, contextual fear conditioning and object recognition tasks in WT mice but not Gabrd−/− mice. Furthermore, THIP inhibited LTP in hippocampal slices from WT but not Gabrd−/− mice, an effect that was blocked by GABAA receptor antagonist bicuculline. Thus, acutely increasing δGABAA receptor activity impairs memory behaviors and inhibits synaptic plasticity. These results have important implications for the development of therapies aimed at increasing δGABAA receptor activity. PMID:24062648

  13. Acutely increasing δGABAA receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus

    Directory of Open Access Journals (Sweden)

    Paul David Whissell

    2013-09-01

    Full Text Available Extrasynaptic γ-aminobutyric acid type A (GABAA receptors that contain the δ subunit (δGABAA receptors are expressed in several brain regions including the dentate gyrus (DG and CA1 subfields of the hippocampus. Drugs that increase δGABAA receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABAA receptor–preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP enhances discrimination memory and increases neurogenesis in the DG. Despite the potential therapeutic benefits of such treatments, the effects of acutely increasing δGABAA receptor activity on memory behaviors remain unknown. Here, we studied the effects of THIP (4 mg/kg, i.p. on memory performance in wild-type (WT and δGABAA receptor null mutant (Gabrd–/– mice. Additionally, the effects of THIP on long-term potentiation (LTP, a molecular correlate of memory, were studied within the DG and CA1 subfields of the hippocampus using electrophysiological recordings of field potentials in hippocampal slices. The results showed that THIP impaired performance in the Morris water maze, contextual fear conditioning and object recognition tasks in WT mice but not Gabrd–/– mice. Furthermore, THIP inhibited LTP in hippocampal slices from WT but not Gabrd–/– mice, an effect that was blocked by GABAA receptor antagonist bicuculline. Thus, acutely increasing δGABAA receptor activity impairs memory behaviors and inhibits synaptic plasticity. These results have important implications for the development of therapies aimed at increasing δGABAA receptor activity.

  14. A neuronal acetylcholine receptor regulates the balance of muscle excitation and inhibition in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Maelle Jospin

    2009-12-01

    Full Text Available In the nematode Caenorhabditis elegans, cholinergic motor neurons stimulate muscle contraction as well as activate GABAergic motor neurons that inhibit contraction of the contralateral muscles. Here, we describe the composition of an ionotropic acetylcholine receptor that is required to maintain excitation of the cholinergic motor neurons. We identified a gain-of-function mutation that leads to spontaneous muscle convulsions. The mutation is in the pore domain of the ACR-2 acetylcholine receptor subunit and is identical to a hyperactivating mutation in the muscle receptor of patients with myasthenia gravis. Screens for suppressors of the convulsion phenotype led to the identification of other receptor subunits. Cell-specific rescue experiments indicate that these subunits function in the cholinergic motor neurons. Expression of these subunits in Xenopus oocytes demonstrates that the functional receptor is comprised of three alpha-subunits, UNC-38, UNC-63 and ACR-12, and two non-alpha-subunits, ACR-2 and ACR-3. Although this receptor exhibits a partially overlapping subunit composition with the C. elegans muscle acetylcholine receptor, it shows distinct pharmacology. Recordings from intact animals demonstrate that loss-of-function mutations in acr-2 reduce the excitability of the cholinergic motor neurons. By contrast, the acr-2(gf mutation leads to a hyperactivation of cholinergic motor neurons and an inactivation of downstream GABAergic motor neurons in a calcium dependent manner. Presumably, this imbalance between excitatory and inhibitory input into muscles leads to convulsions. These data indicate that the ACR-2 receptor is important for the coordinated excitation and inhibition of body muscles underlying sinusoidal movement.

  15. Hispolon inhibits the growth of estrogen receptor positive human breast cancer cells through modulation of estrogen receptor alpha

    International Nuclear Information System (INIS)

    Jang, Eun Hyang; Jang, Soon Young; Cho, In-Hye; Hong, Darong; Jung, Bom; Park, Min-Ju; Kim, Jong-Ho

    2015-01-01

    Human estrogen receptor α (ERα) is a nuclear transcription factor that is a major therapeutic target in breast cancer. The transcriptional activity of ERα is regulated by certain estrogen-receptor modulators. Hispolon, isolated from Phellinus linteus, a traditional medicinal mushroom called Sanghwang in Korea, has been used to treat various pathologies, such as inflammation, gastroenteric disorders, lymphatic diseases, and cancers. In this latter context, Hispolon has been reported to exhibit therapeutic efficacy against various cancer cells, including melanoma, leukemia, hepatocarcinoma, bladder cancer, and gastric cancer cells. However, ERα regulation by Hispolon has not been reported. In this study, we investigated the effects of Hispolon on the growth of breast cancer cells. We found that Hispolon decreased expression of ERα at both mRNA and the protein levels in MCF7 and T47D human breast cancer cells. Luciferase reporter assays showed that Hispolon decreased the transcriptional activity of ERα. Hispolon treatment also inhibited expression of the ERα target gene pS2. We propose that Hispolon, an anticancer drug extracted from natural sources, inhibits cell growth through modulation of ERα in estrogen-positive breast cancer cells and is a candidate for use in human breast cancer chemotherapy. - Highlights: • Hispolon decreased ERα expression at both mRNA and protein levels. • Hispolon decreased ERα transcriptional activity. • Hispolon treatment inhibited expression of ERα target gene pS2. • Shikonin is a candidate chemotherapeutic target in the treatment of human breast cancer

  16. Hispolon inhibits the growth of estrogen receptor positive human breast cancer cells through modulation of estrogen receptor alpha

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Eun Hyang; Jang, Soon Young; Cho, In-Hye [Department of Pharmacy, Graduate School, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of); Hong, Darong [Department of Life and Nanopharmaceutical Science, Graduate School, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of); Jung, Bom; Park, Min-Ju [Department of Pharmacy, Graduate School, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of); Kim, Jong-Ho, E-mail: jonghokim@khu.ac.kr [Department of Pharmacy, Graduate School, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of)

    2015-08-07

    Human estrogen receptor α (ERα) is a nuclear transcription factor that is a major therapeutic target in breast cancer. The transcriptional activity of ERα is regulated by certain estrogen-receptor modulators. Hispolon, isolated from Phellinus linteus, a traditional medicinal mushroom called Sanghwang in Korea, has been used to treat various pathologies, such as inflammation, gastroenteric disorders, lymphatic diseases, and cancers. In this latter context, Hispolon has been reported to exhibit therapeutic efficacy against various cancer cells, including melanoma, leukemia, hepatocarcinoma, bladder cancer, and gastric cancer cells. However, ERα regulation by Hispolon has not been reported. In this study, we investigated the effects of Hispolon on the growth of breast cancer cells. We found that Hispolon decreased expression of ERα at both mRNA and the protein levels in MCF7 and T47D human breast cancer cells. Luciferase reporter assays showed that Hispolon decreased the transcriptional activity of ERα. Hispolon treatment also inhibited expression of the ERα target gene pS2. We propose that Hispolon, an anticancer drug extracted from natural sources, inhibits cell growth through modulation of ERα in estrogen-positive breast cancer cells and is a candidate for use in human breast cancer chemotherapy. - Highlights: • Hispolon decreased ERα expression at both mRNA and protein levels. • Hispolon decreased ERα transcriptional activity. • Hispolon treatment inhibited expression of ERα target gene pS2. • Shikonin is a candidate chemotherapeutic target in the treatment of human breast cancer.

  17. Enzalutamide inhibits androgen receptor-positive bladder cancer cell growth.

    Science.gov (United States)

    Kawahara, Takashi; Ide, Hiroki; Kashiwagi, Eiji; El-Shishtawy, Kareem A; Li, Yi; Reis, Leonardo O; Zheng, Yichun; Miyamoto, Hiroshi

    2016-10-01

    Emerging preclinical evidence suggests that androgen-mediated androgen receptor (AR) signals promote bladder cancer progression. However, little is known about the efficacy of an AR signaling inhibitor, enzalutamide, in the growth of bladder cancer cells. In this study, we compared the effects of enzalutamide and 2 other classic antiandrogens, flutamide and bicalutamide, on androgen-induced bladder cancer cell proliferation, migration, and invasion as well as tumor growth in vivo. Thiazolyl blue cell viability assay, flow cytometry, scratch wound-healing assay, transwell invasion assay, real-time polymerase chain reaction, and reporter gene assay were performed in AR-positive (e.g., UMUC3, TCCSUP, and 647V-AR) and AR-negative (e.g., UMUC3-AR-short hairpin RNA [shRNA], TCCSUP-AR-shRNA, 647V) bladder cancer lines treated with dihydrotestosterone and each AR antagonist. We also used a mouse xenograft model for bladder cancer. Dihydrotestosterone increased bladder cancer cell proliferation, migration, and invasion indicating that endogenous or exogenous AR was functional. Enzalutamide, hydroxyflutamide, and bicalutamide showed similar inhibitory effects, without significant agonist activity, on androgen-mediated cell viability/apoptosis, cell migration, and cell invasion in AR-positive lines. No significant effects of dihydrotestosterone as well as AR antagonists on the growth of AR-negative cells were seen. Correspondingly, in UMUC3 cells, these AR antagonists down-regulated androgen-induced expression of AR, matrix metalloproteinase-2, and interleukin-6. Androgen-enhanced AR-mediated transcriptional activity was also blocked by each AR antagonist exhibiting insignificant agonist activity. In UMUC3 xenograft-bearing mice, oral gavage treatment with each antiandrogen retarded tumor growth, and only enzalutamide demonstrated a statistically significant suppression compared with mock treatment. Our current data support recent observations indicating the involvement of

  18. Poxvirus-encoded TNF decoy receptors inhibit the biological activity of transmembrane TNF.

    Science.gov (United States)

    Pontejo, Sergio M; Alejo, Ali; Alcami, Antonio

    2015-10-01

    Poxviruses encode up to four different soluble TNF receptors, named cytokine response modifier B (CrmB), CrmC, CrmD and CrmE. These proteins mimic the extracellular domain of the cellular TNF receptors to bind and inhibit the activity of TNF and, in some cases, other TNF superfamily ligands. Most of these ligands are released after the enzymic cleavage of a membrane precursor. However, transmembrane TNF (tmTNF) is not only a precursor of soluble TNF but also exerts specific pro-inflammatory and immunological activities. Here, we report that viral TNF receptors bound and inhibited tmTNF and describe some interesting differences in their activity against the soluble cytokine. Thus, CrmE, which does not inhibit mouse soluble TNF, could block murine tmTNF-induced cytotoxicity. We propose that this anti-tmTNF effect should be taken into consideration when assessing the role of viral TNF decoy receptors in the pathogenesis of poxvirus.

  19. Insulin signaling inhibits the 5-HT2C receptor in choroid plexus via MAP kinase

    Directory of Open Access Journals (Sweden)

    Guan Kunliang

    2003-06-01

    Full Text Available Abstract Background G protein-coupled receptors (GPCRs interact with heterotrimeric GTP-binding proteins (G proteins to modulate acute changes in intracellular messenger levels and ion channel activity. In contrast, long-term changes in cellular growth, proliferation and differentiation are often mediated by tyrosine kinase receptors and certain GPCRs by activation of mitogen-activated protein (MAP kinases. Complex interactions occur between these signaling pathways, but the specific mechanisms of such regulatory events are not well-understood. In particular it is not clear whether GPCRs are modulated by tyrosine kinase receptor-MAP kinase pathways. Results Here we describe tyrosine kinase receptor regulation of a GPCR via MAP kinase. Insulin reduced the activity of the 5-HT2C receptor in choroid plexus cells which was blocked by the MAP kinase kinase (MEK inhibitor, PD 098059. We demonstrate that the inhibitory effect of insulin and insulin-like growth factor type 1 (IGF-1 on the 5-HT2C receptor is dependent on tyrosine kinase, RAS and MAP kinase. The effect may be receptor-specific: insulin had no effect on another GPCR that shares the same G protein signaling pathway as the 5-HT2C receptor. This effect is also direct: activated MAP kinase mimicked the effect of insulin, and removing a putative MAP kinase site from the 5-HT2C receptor abolished the effect of insulin. Conclusion These results show that insulin signaling can inhibit 5-HT2C receptor activity and suggest that MAP kinase may play a direct role in regulating the function of a specific GPCR.

  20. The selective estrogen receptor modulator raloxifene inhibits neutrophil extracellular trap formation.

    Directory of Open Access Journals (Sweden)

    Roxana Flores

    2016-12-01

    Full Text Available Raloxifene is a selective estrogen receptor modulator typically prescribed for the prevention/treatment of osteoporosis in postmenopausal women. Although raloxifene is known to have anti-inflammatory properties, its effect on human neutrophils, the primary phagocytic leukocytes of the immune system, remain poorly understood. Here, through a screen of pharmacologically active small molecules, we find that raloxifene prevents neutrophil cell death in response to the classical activator phorbol 12-myristate 13-acetate (PMA, a compound known to induce formation of DNA-based neutrophil extracellular traps (NETs. Inhibition of PMA-induced NET production by raloxifene was confirmed using quantitative and imaging-based assays. Human neutrophils from both male and female donors express the nuclear estrogen receptors ERα and ERβ, known targets of raloxifene. Like raloxifene, selective antagonists of these receptors inhibit PMA-induced NET production. Furthermore, raloxifene inhibited PMA-induced ERK phosphorylation but not reactive oxygen species (ROS production, pathways known to be key modulators of NET production. Finally, we found that raloxifene inhibited PMA-induced, NET-based killing of the leading human bacterial pathogen, methicillin-resistant Staphylococcus aureus (MRSA. Our results reveal that raloxifene is a potent modulator of neutrophil function and NET production.

  1. Inhibition of Estrogen Receptor Action by the Orphan Receptors, SHP and DAX-1

    National Research Council Canada - National Science Library

    DiRenzo, James

    2002-01-01

    In support of DoD grant # DAMD17-99-1-9163, we present our progress towards understanding the function of mechanisms of action of two orphan nuclear receptors, SHP and DAX-I as inhibitors of ER alpha and ER beta action...

  2. Electroacupuncture Inhibition of Hyperalgesia in Rats with Adjuvant Arthritis: Involvement of Cannabinoid Receptor 1 and Dopamine Receptor Subtypes in Striatum

    Directory of Open Access Journals (Sweden)

    Yin Shou

    2013-01-01

    Full Text Available Electroacupuncture (EA has been regarded as an alternative treatment for inflammatory pain for several decades. However, the molecular mechanisms underlying the antinociceptive effect of EA have not been thoroughly clarified. Previous studies have shown that cannabinoid CB1 receptors are related to pain relief. Accumulating evidence has shown that the CB1 and dopamine systems sometimes interact and may operate synergistically in rat striatum. To our knowledge, dopamine D1/D2 receptors are involved in EA analgesia. In this study, we found that repeated EA at Zusanli (ST36 and Kunlun (BL60 acupoints resulted in marked improvements in thermal hyperalgesia. Both western blot assays and FQ-PCR analysis results showed that the levels of CB1 expression in the repeated-EA group were much higher than those in any other group (P=0.001. The CB1-selective antagonist AM251 inhibited the effects of repeated EA by attenuating the increases in CB1 expression. The two kinds of dopamine receptors imparted different actions on the EA-induced CB1 upregulation in AA rat model. These results suggested that the strong activation of the CB1 receptor after repeated EA resulted in the concomitant phenomenon of the upregulation of D1 and D2 levels of gene expression.

  3. Striatal D1- and D2-type dopamine receptors are linked to motor response inhibition in human subjects.

    Science.gov (United States)

    Robertson, Chelsea L; Ishibashi, Kenji; Mandelkern, Mark A; Brown, Amira K; Ghahremani, Dara G; Sabb, Fred; Bilder, Robert; Cannon, Tyrone; Borg, Jacqueline; London, Edythe D

    2015-04-15

    Motor response inhibition is mediated by neural circuits involving dopaminergic transmission; however, the relative contributions of dopaminergic signaling via D1- and D2-type receptors are unclear. Although evidence supports dissociable contributions of D1- and D2-type receptors to response inhibition in rats and associations of D2-type receptors to response inhibition in humans, the relationship between D1-type receptors and response inhibition has not been evaluated in humans. Here, we tested whether individual differences in striatal D1- and D2-type receptors are related to response inhibition in human subjects, possibly in opposing ways. Thirty-one volunteers participated. Response inhibition was indexed by stop-signal reaction time on the stop-signal task and commission errors on the continuous performance task, and tested for association with striatal D1- and D2-type receptor availability [binding potential referred to nondisplaceable uptake (BPND)], measured using positron emission tomography with [(11)C]NNC-112 and [(18)F]fallypride, respectively. Stop-signal reaction time was negatively correlated with D1- and D2-type BPND in whole striatum, with significant relationships involving the dorsal striatum, but not the ventral striatum, and no significant correlations involving the continuous performance task. The results indicate that dopamine D1- and D2-type receptors are associated with response inhibition, and identify the dorsal striatum as an important locus of dopaminergic control in stopping. Moreover, the similar contribution of both receptor subtypes suggests the importance of a relative balance between phasic and tonic dopaminergic activity subserved by D1- and D2-type receptors, respectively, in support of response inhibition. The results also suggest that the stop-signal task and the continuous performance task use different neurochemical mechanisms subserving motor response inhibition. Copyright © 2015 the authors 0270-6474/15/355990-08$15.00/0.

  4. Basolateral P2X receptors mediate inhibition of NaCl transport in mouse medullary thick ascending limb (mTAL)

    DEFF Research Database (Denmark)

    Marques, Rita D; de Bruijn, Pauline I.A.; Sørensen, Mads Vaarby

    2012-01-01

    Extracellular nucleotides regulate epithelial transport via luminal and basolateral P2 receptors. Renal epithelia express multiple P2 receptors, which mediate significant inhibition of solute absorption. Recently, we identified several P2 receptors in the medullary thick ascending limb (m...

  5. Blocking beta 2-adrenergic receptor inhibits dendrite ramification in a mouse model of Alzheimer's disease.

    Science.gov (United States)

    Wu, Qin; Sun, Jin-Xia; Song, Xiang-He; Wang, Jing; Xiong, Cun-Quan; Teng, Fei-Xiang; Gao, Cui-Xiang

    2017-09-01

    Dendrite ramification affects synaptic strength and plays a crucial role in memory. Previous studies revealed a correlation between beta 2-adrenergic receptor dysfunction and Alzheimer's disease (AD), although the mechanism involved is still poorly understood. The current study investigated the potential effect of the selective β 2 -adrenergic receptor antagonist, ICI 118551 (ICI), on Aβ deposits and AD-related cognitive impairment. Morris water maze test results demonstrated that the performance of AD-transgenic (TG) mice treated with ICI (AD-TG/ICI) was significantly poorer compared with NaCl-treated AD-TG mice (AD-TG/NaCl), suggesting that β 2 -adrenergic receptor blockage by ICI might reduce the learning and memory abilities of mice. Golgi staining and immunohistochemical staining revealed that blockage of the β 2 -adrenergic receptor by ICI treatment decreased the number of dendritic branches, and ICI treatment in AD-TG mice decreased the expression of hippocampal synaptophysin and synapsin 1. Western blot assay results showed that the blockage of β 2 -adrenergic receptor increased amyloid-β accumulation by downregulating hippocampal α-secretase activity and increasing the phosphorylation of amyloid precursor protein. These findings suggest that blocking the β 2 -adrenergic receptor inhibits dendrite ramification of hippocampal neurons in a mouse model of AD.

  6. Acute stimulation of brain mu opioid receptors inhibits glucose-stimulated insulin secretion via sympathetic innervation.

    Science.gov (United States)

    Tudurí, Eva; Beiroa, Daniel; Stegbauer, Johannes; Fernø, Johan; López, Miguel; Diéguez, Carlos; Nogueiras, Rubén

    2016-11-01

    Pancreatic insulin-secreting β-cells express opioid receptors, whose activation by opioid peptides modulates hormone secretion. Opioid receptors are also expressed in multiple brain regions including the hypothalamus, where they play a role in feeding behavior and energy homeostasis, but their potential role in central regulation of glucose metabolism is unknown. Here, we investigate whether central opioid receptors participate in the regulation of insulin secretion and glucose homeostasis in vivo. C57BL/6J mice were acutely treated by intracerebroventricular (i.c.v.) injection with specific agonists for the three main opioid receptors, kappa (KOR), delta (DOR) and mu (MOR) opioid receptors: activation of KOR and DOR did not alter glucose tolerance, whereas activation of brain MOR with the specific agonist DAMGO blunted glucose-stimulated insulin secretion (GSIS), reduced insulin sensitivity, increased the expression of gluconeogenic genes in the liver and, consequently, impaired glucose tolerance. Pharmacological blockade of α2A-adrenergic receptors prevented DAMGO-induced glucose intolerance and gluconeogenesis. Accordingly, DAMGO failed to inhibit GSIS and to impair glucose tolerance in α2A-adrenoceptor knockout mice, indicating that the effects of central MOR activation on β-cells are mediated via sympathetic innervation. Our results show for the first time a new role of the central opioid system, specifically the MOR, in the regulation of insulin secretion and glucose metabolism. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling

    Directory of Open Access Journals (Sweden)

    Lu Frances Fangjia

    2012-11-01

    Full Text Available Abstract Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt/glycogen synthase kinase (GSK-3 signaling. Here we report that activation of GABAB receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABAB receptors enhances the phosphorylation of Akt (Thr-308 and enhances the phosphorylation of GSK-3α (Ser-21/β (Ser-9 in both HEK-293T cells expressing GABAB receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABAB receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABAB receptor agents may exert therapeutic effects in the treatment of schizophrenia.

  8. Inhibition of amygdaloid dopamine D2 receptors impairs emotional learning measured with fear-potentiated startle.

    Science.gov (United States)

    Greba, Q; Gifkins, A; Kokkinidis, L

    2001-04-27

    Considerable advances have been made in understanding the neurocircuitry underlying the acquisition and expression of Pavlovian conditioned fear responses. Within the complex cellular and molecular processes mediating fearfulness, amygdaloid dopamine (DA), originating from cells in the ventral tegmental area (VTA) of the midbrain, is thought to contribute to fear-motivated responding. Considering that blockade of DA D(2) receptors is a common mechanism of action for antipsychotic agents, we hypothesized that inhibition of D(2) receptors in the amygdala may be involved in the antiparanoid effects of these drugs. To assess the role of amygdaloid DA D(2) receptors in aversive emotionality, the D(2) receptor antagonist raclopride was infused into the amygdala prior to Pavlovian fear conditioning. Potentiated startle was used as a behavioral indicator of fear and anxiety. Classical fear conditioning and acoustic startle testing were conducted in a single session allowing for the concomitant assessment of shock reactivity with startle enhancement. Depending on dose, the results found conditioned fear acquisition and retention to be impaired following administration of raclopride into the amygdala. Additionally, the learning deficit was dissociated from shock detection and from fear expression assessed with the shock sensitization of acoustic startle. These findings further refine the known neural mechanisms of amygdala-based emotional learning and memory and were interpreted to suggest that, along with D(1) receptors, D(2) receptors in the amygdala may mediate the formation and the retention of newly-acquired fear associations.

  9. Inhibition of allergen-induced basophil activation by ASM-024, a nicotinic receptor ligand.

    Science.gov (United States)

    Watson, Brittany M; Oliveria, John Paul; Nusca, Graeme M; Smith, Steven G; Beaudin, Sue; Dua, Benny; Watson, Rick M; Assayag, Evelynne Israël; Cormier, Yvon F; Sehmi, Roma; Gauvreau, Gail M

    2014-01-01

    Nicotinic acetylcholine receptors (nAChRs) were identified on eosinophils and shown to regulate inflammatory responses, but nAChR expression on basophils has not been explored yet. We investigated surface receptor expression of nAChR α4, α7 and α1/α3/α5 subunits on basophils. Furthermore, we examined the effects of ASM-024, a synthetic nicotinic ligand, on in vitro anti-IgE and in vivo allergen-induced basophil activation. Basophils were enriched from the peripheral blood of allergic donors and the expression of nAChR subunits and muscarinic receptors was determined. Purified basophils were stimulated with anti-IgE in the presence of ASM-024 with or without muscarinic or nicotinic antagonists for the measurement of CD203c expression and histamine release. The effect of 9 days of treatment with 50 and 200 mg ASM-024 on basophil CD203c expression was examined in the blood of mild allergic asthmatics before and after allergen inhalation challenge. nAChR α4, α7 and α1/α3/α5 receptor subunit expression was detected on basophils. Stimulation of basophils with anti-IgE increased CD203c expression and histamine release, which was inhibited by ASM-024 (10(-5) to 10(-)(3) M, p ASM-024 was reversed in the presence of muscarinic and nicotinic antagonists. In subjects with mild asthma, ASM-024 inhalation significantly inhibited basophil CD203c expression measured 24 h after allergen challenge (p = 0.03). This study shows that ASM-024 inhibits IgE- and allergen-induced basophil activation through both nicotinic and muscarinic receptors, and suggests that ASM-024 may be an efficacious agent for modulating allergic asthma responses. © 2015 S. Karger AG, Basel.

  10. The Cellular Prion Protein Prevents Copper-Induced Inhibition of P2X4 Receptors

    Directory of Open Access Journals (Sweden)

    Ramón A. Lorca

    2011-01-01

    Full Text Available Although the physiological function of the cellular prion protein (PrPC remains unknown, several evidences support the notion of its role in copper homeostasis. PrPC binds Cu2+ through a domain composed by four to five repeats of eight amino acids. Previously, we have shown that the perfusion of this domain prevents and reverses the inhibition by Cu2+ of the adenosine triphosphate (ATP-evoked currents in the P2X4 receptor subtype, highlighting a modulatory role for PrPC in synaptic transmission through regulation of Cu2+ levels. Here, we study the effect of full-length PrPC in Cu2+ inhibition of P2X4 receptor when both are coexpressed. PrPC expression does not significantly change the ATP concentration-response curve in oocytes expressing P2X4 receptors. However, the presence of PrPC reduces the inhibition by Cu2+ of the ATP-elicited currents in these oocytes, confirming our previous observations with the Cu2+ binding domain. Thus, our observations suggest a role for PrPC in modulating synaptic activity through binding of extracellular Cu2+.

  11. Distinct roles of synaptic and extrasynaptic GABAA receptors in striatal inhibition dynamics

    Directory of Open Access Journals (Sweden)

    Ruixi eLuo

    2013-11-01

    Full Text Available Striatonigral and striatopallidal projecting medium spiny neurons (MSNs express dopamine D1 (D1+ and D2 receptors (D2+, respectively. Both classes receive extensive GABAergic input via expression of synaptic, perisynaptic and extrasynaptic GABAA receptors. The activation patterns of different presynaptic GABAergic neurons produce transient and sustained GABAA receptor-mediated conductance that fulfill distinct physiological roles. We performed single and dual whole cell recordings from striatal neurons in mice expressing fluorescent proteins in interneurons and MSNs. We report specific inhibitory dynamics produced by distinct activation patterns of presynaptic GABAergic neurons as source of synaptic, perisynaptic and extrasynaptic inhibition. Synaptic GABAA receptors in MSNs contain the α2, γ2 and a β subunit. In addition, there is evidence for the developmental increase of the α1 subunit that contributes to faster inhibitory postsynaptic current (IPSC. Tonic GABAergic currents in MSNs from adult mice are carried by extrasynaptic receptors containing the α4 and δ subunit, while in younger mice this current is mediated by receptors that contain the α5 subunit. Both forms of tonic currents are differentially expressed in D1+ and D2+ MSNs. This study extends these findings by relating presynaptic activation with pharmacological analysis of inhibitory conductance in mice where the β3 subunit is conditionally removed in fluorescently labeled D2+ MSNs and in mice with global deletion of the δ subunit. Our results show that responses to low doses of gaboxadol (2μM, a GABAA receptor agonist with preference to δ subunit, are abolished in the δ but not the β3 subunit knock out mice. This suggests that the β3 subunit is not a component of the adult extrasynaptic receptor pool, in contrast to what has been shown for tonic current in young mice. Deletion of the β3 subunit from D2+ MSNs however, removed slow spontaneous IPSCs, implicating its

  12. GABA(B) receptor modulation of feedforward inhibition through hippocampal neurogliaform cells.

    Science.gov (United States)

    Price, Christopher J; Scott, Ricardo; Rusakov, Dmitri A; Capogna, Marco

    2008-07-02

    Feedforward inhibition of neurons is a fundamental component of information flow control in the brain. We studied the roles played by neurogliaform cells (NGFCs) of stratum lacunosum moleculare of the hippocampus in providing feedforward inhibition to CA1 pyramidal cells. We recorded from synaptically coupled pairs of anatomically identified NGFCs and CA1 pyramidal cells and found that, strikingly, a single presynaptic action potential evoked a biphasic unitary IPSC (uIPSC), consisting of two distinct components mediated by GABA(A) and GABA(B) receptors. A GABA(B) receptor-mediated unitary response has not previously been observed in hippocampal excitatory neurons. The decay of the GABA(A) receptor-mediated response was slow (time constant = 50 ms), and was tightly regulated by presynaptic GABA(B) receptors. Surprisingly, the GABA(B) receptor ligands baclofen and (2S)-3-{[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl}(phenylmethyl)phosphinic acid (CGP55845), while affecting the NGFC-mediated uIPSCs, had no effect on action potential-evoked presynaptic Ca2+ signals monitored in individual axonal boutons of NGFCs with two-photon microscopy. In contrast, baclofen clearly depressed presynaptic Ca2+ transients in non-NGF interneurons. Changes in extracellular Ca2+ concentration that mimicked the effects of baclofen or CGP55845 on uIPSCs significantly altered presynaptic Ca2+ transients. Electrophysiological data suggest that GABA(B) receptors expressed by NGFCs contribute to the dynamic control of the excitatory input to CA1 pyramidal neurons from the temporoammonic path. The NGFC-CA1 pyramidal cell connection therefore provides a unique and subtle mechanism to shape the integration time domain for signals arriving via a major excitatory input to CA1 pyramidal cells.

  13. Effects of protease-activated receptor 1 inhibition on anxiety and fear following status epilepticus.

    Science.gov (United States)

    Bogovyk, Ruslan; Lunko, Oleksii; Fedoriuk, Mihail; Isaev, Dmytro; Krishtal, Oleg; Holmes, Gregory L; Isaeva, Elena

    2017-02-01

    Protease-activated receptor 1 (PAR1) is an important contributor to the pathogenesis of a variety of brain disorders associated with a risk of epilepsy development. Using the lithium-pilocarpine model of temporal lobe epilepsy (TLE), we recently showed that inhibition of this receptor during the first ten days after pilocarpine-induced status epilepticus (SE) results in substantial anti-epileptogenic and neuroprotective effects. As PAR1 is expressed in the central nervous system regions of importance for processing emotional reactions, including amygdala and hippocampus, and TLE is frequently associated with a chronic alteration of the functions of these regions, we tested the hypothesis that PAR1 inhibition could modulate emotionally driven behavioral responses of rats experiencing SE. We showed that SE induces a chronic decrease in the animals' anxiety-related behavior and an increase of locomotor activity. PAR1 inhibition after SE abolished the alteration of the anxiety level but does not affect the increase of locomotor activity in the open field and elevated plus maze tests. Moreover, while PAR1 inhibition produces an impairment of memory recall in the context fear conditioning paradigm in the control group, it substantially improves contextual and cued fear learning in rats experiencing SE. These data suggest that PAR1-dependent signaling is involved in the mechanisms underlying emotional disorders in epilepsy. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor

    DEFF Research Database (Denmark)

    List, K; Høyer-Hansen, G; Rønne, E

    1999-01-01

    Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interfer......Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance......) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former...

  15. Anterior cingulate serotonin 1B receptor binding is associated with emotional response inhibition

    DEFF Research Database (Denmark)

    da Cunha-Bang, Sofi; Hjordt, Liv Vadskjær; Dam, Vibeke Høyrup

    2017-01-01

    -offender controls, completed an emotional Go/NoGo task requiring inhibition of prepotent motor responses to emotional facial expressions. We also measured cerebral serotonin 1B receptor (5-HT1BR) binding with [11C]AZ10419369 positron emission tomography within regions of the frontal cortex. We hypothesized that 5......-HT1BR would be positively associated with false alarms (failures to inhibit nogo responses) in the context of aversive (angry and fearful) facial expressions. Across groups, we found that frontal cortex 5-HT1BR binding was positively correlated with false alarms when angry faces were go stimuli......Serotonin has a well-established role in emotional processing and is a key neurotransmitter in impulsive aggression, presumably by facilitating response inhibition and regulating subcortical reactivity to aversive stimuli. In this study 44 men, of whom 19 were violent offenders and 25 were non...

  16. Gambogic acid inhibits multiple myeloma mediated osteoclastogenesis through suppression of chemokine receptor CXCR4 signaling pathways.

    Science.gov (United States)

    Pandey, Manoj K; Kale, Vijay P; Song, Chunhua; Sung, Shen-shu; Sharma, Arun K; Talamo, Giampaolo; Dovat, Sinisa; Amin, Shantu G

    2014-10-01

    Bone disease, characterized by the presence of lytic lesions and osteoporosis is the hallmark of multiple myeloma (MM). Stromal cell-derived factor 1α (SDF-1α) and its receptor, CXC chemokine receptor 4 (CXCR4), has been implicated as a regulator of bone resorption, suggesting that agents that can suppress SDF1α/CXCR4 signaling might inhibit osteoclastogenesis, a process closely linked to bone resorption. We, therefore, investigated whether gambogic acid (GA), a xanthone, could inhibit CXCR4 signaling and suppress osteoclastogenesis induced by MM cells. Through docking studies we predicted that GA directly interacts with CXCR4. This xanthone down-regulates the expression of CXCR4 on MM cells in a dose- and time-dependent manner. The down-regulation of CXCR4 was not due to proteolytic degradation, but rather GA suppresses CXCR4 mRNA expression by inhibiting nuclear factor-kappa B (NF-κB) DNA binding. This was further confirmed by quantitative chromatin immunoprecipitation assay, as GA inhibits p65 binding at the CXCR4 promoter. GA suppressed SDF-1α-induced chemotaxis of MM cells and downstream signaling of CXCR4 by inhibiting phosphorylation of Akt, p38, and Erk1/2 in MM cells. GA abrogated the RANKL-induced differentiation of macrophages to osteoclasts in a dose- and time-dependent manner. In addition, we found that MM cells induced differentiation of macrophages to osteoclasts, and that GA suppressed this process. Importantly, suppression of osteoclastogenesis by GA was mediated through IL-6 inhibition. Overall, our results show that GA is a novel inhibitor of CXCR4 expression and has a strong potential to suppress osteoclastogenesis mediated by MM cells. Published by Elsevier Inc.

  17. Hemin inhibits internalization of transferrin by reticulocytes and promotes phosphorylation of the membrane transferrin receptor

    International Nuclear Information System (INIS)

    Cox, T.M.; O'Donnell, M.W.; Aisen, P.; London, I.M.

    1985-01-01

    Addition of hemin to reticulocytes inhibits incorporation of iron from transferrin. Heme also regulates protein synthesis in immature erythroid cells through its effects on phosphorylation of the initiation factor eIF-2. The authors have examined its effects on endocytosis of iron-transferrin and phosphorylation of the transferrin receptor. Hemin reduced iron transport but increased cell-associated transferrin. During uptake of 125 I-labeled transferrin in the steady state, the use of a washing technique to dissociate bound transferrin on the cell membrane showed that radioligand accumulated on the surface of hemin-treated cells. Receptor phosphorylation was investigated by immunoprecipitation of reticulocyte extracts after metabolic labeling with [ 32 P]P/sub i/. In the absence of ligand, phosphorylated receptor was chiefly localized on cell stroma. Exposure to transferrin increased cytosolic phosphorylated receptor from 15-30% to approximately 50% of the total, an effect overcome by hemin treatment. The findings suggest a possible relationship of phosphorylation to endocytosis of the transferrin receptor in reticulocytes

  18. MiR-34a inhibits colon cancer proliferation and metastasis by inhibiting platelet-derived growth factor receptor α.

    Science.gov (United States)

    Li, Chunyan; Wang, Yulin; Lu, Shuming; Zhang, Zhuqing; Meng, Hua; Liang, Lina; Zhang, Yan; Song, Bo

    2015-11-01

    The microRNA (miRNA), miR‑34a is significant in colon cancer progression. In the present study, the role of miR‑34a in colon cancer cell proliferation and metastasis was investigated. It was found that the expression of miR‑34a in colon cancer tissues and cell lines was lower when compared with that of normal tissues and cells. Further research demonstrated that miR‑34a inhibited cell proliferation, induced G1 phase arrest, and suppressed metastasis and epithelial mesenchymal transition in colon cancer cells. Bioinformatic prediction indicated that platelet‑derived growth factor receptor α (PDGFRA) was a potential target gene of miR‑34a and a luciferase assay identified that PDGFRA was a novel direct target gene of miR‑34a. In addition, assays of western blot analyses and quantitative reverse‑transcription polymerase chain reaction confirmed that miR‑34a decreased PDGFRA mRNA expression and protein levels in colon cancer cells. Assessment of cellular function indicated that miR‑34a inhibited colon cancer progression via PDGFRA. These findings demonstrate that miR‑34a may act as a negative regulator in colon cancer by targeting PDGFRA.

  19. Procaine rapidly inactivates acetylcholine receptors from Torpedo and competes with agonist for inhibition sites

    International Nuclear Information System (INIS)

    Forman, S.A.; Miller, K.W.

    1989-01-01

    The relationship between the high-affinity procaine channel inhibition site and the agonist self-inhibition site on acetylcholine receptors (AChRs) from Torpedo electroplaque was investigated by using rapid 86 Rb + quenched-flux assays at 4 degree C in native AChR-rich vesicles on which 50-60% of ACh activation sites were blocked with α-bungarotoxin (α-BTX). In the presence of channel-activating acetylcholine (ACh) concentrations alone, AChR undergoes one phase of inactivation in under a second. Addition of procaine produces two-phase inactivation similar to that seen with self-inhibiting ACh concentrations rapid inactivation complete in 30-75 ms is followed by fast desensitization at the same k d observed without procaine. The dependence of k r on [procaine] is consistent with a bimolecular association between procaine and its AChR site. Inhibition of AChR function by mixtures of procaine plus self-inhibiting concentrations of ACh or suberyldicholine was studied by reducing the level of α-BTX block in vesicles. The data support a mechanism where procaine binds preferentially to the open-channel AChR state, since no procaine-induced inactivation is observed without agonist and k r 's dependence on [ACh] in channel-activating range closely parallels that of 86 Rb + flux response to ACh

  20. The aryl hydrocarbon receptor ligand ITE inhibits TGFβ1-induced human myofibroblast differentiation.

    Science.gov (United States)

    Lehmann, Geniece M; Xi, Xia; Kulkarni, Ajit A; Olsen, Keith C; Pollock, Stephen J; Baglole, Carolyn J; Gupta, Shikha; Casey, Ann E; Huxlin, Krystel R; Sime, Patricia J; Feldon, Steven E; Phipps, Richard P

    2011-04-01

    Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor-β1 (TGFβ1). In this study, we demonstrate that TGFβ1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGFβ1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGFβ1-driven myofibroblast differentiation in AhR(-/-) fibroblasts: Its ability to inhibit TGFβ1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  1. Midazolam inhibits hippocampal long-term potentiation and learning through dual central and peripheral benzodiazepine receptor activation and neurosteroidogenesis

    OpenAIRE

    Tokuda, Kazuhiro; O’Dell, Kazuko A.; Izumi, Yukitoshi; Zorumski, Charles F.

    2010-01-01

    Benzodiazepines (BDZs) enhance γ-aminobutyric acid-A (GABAA) receptor inhibition by direct actions on central BDZ receptors (CBRs). Although some BDZs also bind mitochondrial receptors (translocator protein 18kDa, TSPO) and promote the synthesis of GABA-enhancing neurosteroids, the role of neurosteroids in the clinical effects of BDZs is unknown. In rat hippocampal slices, we compared midazolam, an anesthetic BDZ with clonazepam, an anticonvulsant/anxiolytic BDZ that activates CBRs selectivel...

  2. A novel thromboxane receptor antagonist, nstpbp5185, inhibits platelet aggregation and thrombus formation in animal models.

    Science.gov (United States)

    Huang, Shiu-Wen; Kuo, Heng-Lan; Hsu, Ming-Tsung; Tseng, Yufeng Jane; Lin, Shu-Wha; Kuo, Sheng-Chu; Peng, Hui-Chin; Lien, Jin-Cherng; Huang, Tur-Fu

    2016-08-01

    A novel benzimidazole derivative, nstpbp5185, was discovered through in vitro and in vivo evaluations for antiplatelet activity. Thromaboxane receptor (TP) is important in vascular physiology, haemostasis and pathophysiological thrombosis. Nstpbp5185 concentration-dependently inhibited human platelet aggregation caused by collagen, arachidonic acid and U46619. Nstpbp5185 caused a right-shift of the concentration-response curve of U46619 and competitively inhibited the binding of 3H-SQ-29548 to TP receptor expressed on HEK-293 cells, with an IC50 of 0.1 µM, indicating that nstpbp5185 is a TP antagonist. In murine thrombosis models, nstpbp5185 significantly prolonged the latent period in triggering platelet plug formation in mesenteric and FeCl3-induced thrombi formation, and increased the survival rate in pulmonary embolism model with less bleeding than aspirin. This study suggests nstpbp5185, an orally selective anti-thrombotic agent, acting through blockade of TXA2 receptor, may be efficacious for prevention or treatment of pathologic thrombosis.

  3. Oestrogen inhibits human colonic motility by a non-genomic cell membrane receptor-dependent mechanism.

    LENUS (Irish Health Repository)

    Hogan, A M

    2012-02-01

    BACKGROUND: Classical effects of oestrogen involve activation of target genes after binding nuclear receptors. Oestrogenic effects too rapid for DNA transcription (non-genomic) are known to occur. The effect of oestrogen on colonic motility is unknown despite the prevalence of gastrointestinal symptoms in pregnant and premenopausal women. METHODS: Histologically normal colon was obtained from proximal resection margins of colorectal carcinoma specimens. Circular smooth muscle strips were microdissected and suspended in organ baths under 1 g of tension. After equilibration, they were exposed to 17beta-oestradiol (n = 8) or bovine serum albumin (BSA)-conjugated 17beta-oestradiol (n = 8). Fulvestrant, an oestrogen receptor antagonist, was added to some baths (n = 8). Other strips were exposed to calphostin C or cycloheximide. Carbachol was added in increasing concentrations and contractile activity was recorded isometrically. RESULTS: Oestrogen inhibited colonic contractility (mean difference 19.7 per cent; n = 8, P < 0.001). In keeping with non-genomic, rapid-onset steroid action, the effect was apparent within minutes and reversible. It was observed with both 17beta-oestradiol and BSA-conjugated oestrogen, and was not altered by cycloheximide. Effects were inhibited by fulvestrant, suggesting receptor mediation. CONCLUSION: Oestrogen decreases contractility in human colonic smooth muscle by a non-genomic mechanism involving cell membrane coupling.

  4. MiR-125a TNF receptor-associated factor 6 to inhibit osteoclastogenesis

    International Nuclear Information System (INIS)

    Guo, Li-Juan; Liao, Lan; Yang, Li; Li, Yu; Jiang, Tie-Jian

    2014-01-01

    MicroRNAs (miRNAs) play important roles in osteoclastogenesis and bone resorption. In the present study, we found that miR-125a was dramatically down-regulated during macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) induced osteoclastogenesis of circulating CD14+ peripheral blood mononuclear cells (PBMCs). Overexpression of miR-125a in CD14+ PBMCs inhibited osteoclastogenesis, while inhibition of miR-125a promoted osteoclastogenesis. TNF receptor-associated factor 6 (TRAF6), a transduction factor for RANKL/RANK/NFATc1 signal, was confirmed to be a target of miR-125a. EMSA and ChIP assays confirmed that NFATc1 bound to the promoter of the miR-125a. Overexpression of NFATc1 inhibited miR-125a transcription, and block of NFATc1 expression attenuated RANKL-regulated miR-125a transcription. Here, we reported that miR-125a played a biological function in osteoclastogenesis through a novel TRAF6/ NFATc1/miR-125a regulatory feedback loop. It suggests that regulation of miR-125a expression may be a potential strategy for ameliorating metabolic disease. - Highlights: • MiR-125a was significantly down-regulated in osteoclastogenesis of CD14+ PBMCs. • MiR-125a inhibited osteoclast differentiation by targeting TRAF6. • NFATc1 inhibited miR-125a transciption by binding to the promoter of miR-125a. • TRAF6/NFATc1 and miR-125a form a regulatory feedback loop in osteoclastogenesis

  5. Tributyltin and triphenyltin inhibit osteoclast differentiation through a retinoic acid receptor-dependent signaling pathway

    International Nuclear Information System (INIS)

    Yonezawa, Takayuki; Hasegawa, Shin-ichi; Ahn, Jae-Yong; Cha, Byung-Yoon; Teruya, Toshiaki; Hagiwara, Hiromi; Nagai, Kazuo; Woo, Je-Tae

    2007-01-01

    Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), have been widely used in agriculture and industry. Although these compounds are known to have many toxic effects, including endocrine-disrupting effects, their effects on bone resorption are unknown. In this study, we investigated the effects of organotin compounds, such as monobutyltin (MBT), dibutyltin (DBT), TBT, and TPT, on osteoclast differentiation using mouse monocytic RAW264.7 cells. MBT and DBT had no effects, whereas TBT and TPT dose-dependently inhibited osteoclast differentiation at concentrations of 3-30 nM. Treatment with a retinoic acid receptor (RAR)-specific antagonist, Ro41-5253, restored the inhibition of osteoclastogenesis by TBT and TPT. TBT and TPT reduced receptor activator of nuclear factor-κB ligand (RANKL) induced nuclear factor of activated T cells (NFAT) c1 expression, and the reduction in NFATc1 expression was recovered by Ro41-5253. Our results suggest that TBT and TPT suppress osteoclastogenesis by inhibiting RANKL-induced NFATc1 expression via an RAR-dependent signaling pathway

  6. CDB-4124, a progesterone receptor modulator, inhibits mammary carcinogenesis by suppressing cell proliferation and inducing apoptosis.

    Science.gov (United States)

    Wiehle, Ronald; Lantvit, Daniel; Yamada, Tohru; Christov, Konstantin

    2011-03-01

    CDB-4124 (Proellex or telapristone acetate) is a modulator of progesterone receptor (PR) signaling, which is currently employed in preclinical studies for prevention and treatment of breast cancer and has been used in clinical studies for treatment of uterine fibroids and endometriosis. Here we provide evidence for its action on steroid hormone-signaling, cell cycle-regulated genes and in vivo on mammary carcinogenesis. When CDB-4124 is given to rats at 200 mg/kg for 24 months, it prevents the development of spontaneous mammary hyperplastic and premalignant lesions. Also, CDB-4124 given as subcutaneous pellets at two different doses suppressed, dose dependently, N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis. The high dose (30 mg, over 84 days) increased tumor latency from 66 ± 24 days to 87 ± 20 days (P CDB-4124 inhibited cell proliferation and induced apoptosis in MNU-induced mammary tumors, which correlated with a decreased proportion of PR(+) tumor cells and with decreased serum progesterone. CDB-4124 did not affect serum estradiol. In a mechanistic study employing T47D cells we found that CDB-4124 suppressed G(1)/G(0)-S transition by inhibiting CDK2 and CDK4 expressions, which correlated with inhibition of estrogen receptor (ER) expression. Taken together, these data indicate that CDB-4124 can suppress the development of precancerous lesions and carcinogen-induced ER(+) mammary tumors in rats, and may have implications for prevention and treatment of human breast cancer.

  7. GLP-1 receptor stimulation depresses heart rate variability and inhibits neurotransmission to cardiac vagal neurons.

    Science.gov (United States)

    Griffioen, Kathleen J; Wan, Ruiqian; Okun, Eitan; Wang, Xin; Lovett-Barr, Mary Rachael; Li, Yazhou; Mughal, Mohamed R; Mendelowitz, David; Mattson, Mark P

    2011-01-01

    glucagon-like peptide 1 (GLP-1) is an incretin hormone released from the gut in response to food intake. Whereas GLP-1 acts in the periphery to inhibit glucagon secretion and stimulate insulin release, it also acts in the central nervous system to mediate autonomic control of feeding, body temperature, and cardiovascular function. Because of its role as an incretin hormone, GLP-1 receptor analogs are used as a treatment for type 2 diabetes. Central or peripheral administration of GLP-1 increases blood pressure and heart rate, possibly by activating brainstem autonomic nuclei and increasing vagus nerve activity. However, the mechanism(s) by which GLP-1 receptor stimulation affects cardiovascular function are unknown. We used the long-lasting GLP-1 receptor agonist Exendin-4 (Ex-4) to test the hypothesis that GLP-1 signalling modulates central parasympathetic control of heart rate. using a telemetry system, we assessed heart rate in mice during central Ex-4 administration. Heart rate was increased by both acute and chronic central Ex-4 administration. Spectral analysis indicated that the high frequency and low frequency powers of heart rate variability were diminished by Ex-4 treatment. Finally, Ex-4 decreased both excitatory glutamatergic and inhibitory glycinergic neurotransmission to preganglionic parasympathetic cardiac vagal neurons. these data suggest that central GLP-1 receptor stimulation diminishes parasympathetic modulation of the heart thereby increasing heart rate.

  8. GABA type a receptor trafficking and the architecture of synaptic inhibition.

    Science.gov (United States)

    Lorenz-Guertin, Joshua M; Jacob, Tija C

    2018-03-01

    Ubiquitous expression of GABA type A receptors (GABA A R) in the central nervous system establishes their central role in coordinating most aspects of neural function and development. Dysregulation of GABAergic neurotransmission manifests in a number of human health disorders and conditions that in certain cases can be alleviated by drugs targeting these receptors. Precise changes in the quantity or activity of GABA A Rs localized at the cell surface and at GABAergic postsynaptic sites directly impact the strength of inhibition. The molecular mechanisms constituting receptor trafficking to and from these compartments therefore dictate the efficacy of GABA A R function. Here we review the current understanding of how GABA A Rs traffic through biogenesis, plasma membrane transport, and degradation. Emphasis is placed on discussing novel GABAergic synaptic proteins, receptor and scaffolding post-translational modifications, activity-dependent changes in GABA A R confinement, and neuropeptide and neurosteroid mediated changes. We further highlight modern techniques currently advancing the knowledge of GABA A R trafficking and clinically relevant neurodevelopmental diseases connected to GABAergic dysfunction. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 238-270, 2018. © 2017 Wiley Periodicals, Inc.

  9. Chronic ethanol consumption in rats produces opioid antinociceptive tolerance through inhibition of mu opioid receptor endocytosis.

    Directory of Open Access Journals (Sweden)

    Li He

    Full Text Available It is well known that the mu-opioid receptor (MOR plays an important role in the rewarding properties of ethanol. However, it is less clear how chronic ethanol consumption affects MOR signaling. Here, we demonstrate that rats with prolonged voluntary ethanol consumption develop antinociceptive tolerance to opioids. Signaling through the MOR is controlled at many levels, including via the process of endocytosis. Importantly, agonists at the MOR that promote receptor endocytosis, such as the endogenous peptides enkephalin and β-endorphin, show a reduced propensity to promote antinociceptive tolerance than do agonists, like morphine, which do not promote receptor endocytosis. These observations led us to examine whether chronic ethanol consumption produced opioid tolerance by interfering with MOR endocytosis. Indeed, here we show that chronic ethanol consumption inhibits the endocytosis of MOR in response to opioid peptide. This loss of endocytosis was accompanied by a dramatic decrease in G protein coupled receptor kinase 2 (GRK2 protein levels after chronic drinking, suggesting that loss of this component of the trafficking machinery could be a mechanism by which endocytosis is lost. We also found that MOR coupling to G-protein was decreased in ethanol-drinking rats, providing a functional explanation for loss of opioid antinociception. Together, these results suggest that chronic ethanol drinking alters the ability of MOR to endocytose in response to opioid peptides, and consequently, promotes tolerance to the effects of opioids.

  10. Mactosylceramide Prevents Glial Cell Overgrowth by Inhibiting Insulin and Fibroblast Growth Factor Receptor Signaling

    DEFF Research Database (Denmark)

    Gerdøe-Kristensen, Stine; Lund, Viktor K; Wandall, Hans H

    2017-01-01

    , in which the mannosyltransferase Egghead controls conversion of glucosylceramide (GlcCer) to mactosylceramide (MacCer). Lack of elongated GSL in egghead (egh) mutants causes overgrowth of subperineurial glia (SPG), largely due to aberrant activation of phosphatidylinositol 3-kinase (PI3K). However, to what...... of the Drosophila Insulin Receptor (InR) and the FGFR homolog Heartless (Htl) in wild type SPG, and is suppressed by inhibiting Htl and InR activity in egh. Knockdown of GlcCer synthase in the SPG fails to suppress glial overgrowth in egh nerves, and slightly promotes overgrowth in wild type, suggesting that RTK...... hyperactivation is caused by absence of MacCer and not by GlcCer accumulation. We conclude that an early product in GSL biosynthesis, MacCer, prevents inappropriate activation of Insulin and Fibroblast Growth Factor Receptors in Drosophila glia. This article is protected by copyright. All rights reserved....

  11. A peptide antagonist of the ErbB1 receptor inhibits receptor activation, tumor cell growth and migration in vitro and xenograft tumor growth in vivo

    DEFF Research Database (Denmark)

    Xu, Ruodan; Povlsen, Gro Klitgaard; Soroka, Vladislav

    2010-01-01

    The epidermal growth factor family of receptor tyrosine kinases (ErbBs) plays essential roles in tumorigenesis and cancer disease progression, and therefore has become an attractive target for structure-based drug design. ErbB receptors are activated by ligand-induced homo- and heterodimerization...... constitutes part of the dimerization arm of ErbB3. Inherbin3 binds to the extracellular domains of all four ErbB receptors, with the lowest peptide binding affinity for ErbB4. Inherbin3 functions as an antagonist of epidermal growth factor (EGF)-ErbB1 signaling. We show that Inherbin3 inhibits EGF-induced Erb....... Structural studies have revealed that ErbB receptor dimers are stabilized by receptor-receptor interactions, primarily mediated by a region in the second extracellular domain, termed the "dimerization arm". The present study is the first biological characterization of a peptide, termed Inherbin3, which...

  12. Targeting receptor for advanced glycation end products (RAGE) expression induces apoptosis and inhibits prostate tumor growth

    International Nuclear Information System (INIS)

    Elangovan, Indira; Thirugnanam, Sivasakthivel; Chen, Aoshuang; Zheng, Guoxing; Bosland, Maarten C.; Kajdacsy-Balla, André; Gnanasekar, Munirathinam

    2012-01-01

    Highlights: ► Targeting RAGE by RNAi induces apoptosis in prostate cancer cells. ► Silencing RAGE expression abrogates rHMGB1 mediated cell proliferation. ► Down regulation of RAGE by RNAi inhibits PSA secretion of prostate cancer cells. ► Knock down of RAGE abrogates prostate tumor growth in vivo. ► Disruption of RAGE expression in prostate tumor activates death receptors. -- Abstract: Expression of receptor for advanced glycation end products (RAGE) plays a key role in the progression of prostate cancer. However, the therapeutic potential of targeting RAGE expression in prostate cancer is not yet evaluated. Therefore in this study, we have investigated the effects of silencing the expression of RAGE by RNAi approach both in vitro and in vivo. The results of this study showed that down regulation of RAGE expression by RNAi inhibited the cell proliferation of androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells. Furthermore, targeting RAGE expression resulted in apoptotic elimination of these prostate cancer cells by activation of caspase-8 and caspase-3 death signaling. Of note, the levels of prostate specific antigen (PSA) were also reduced in LNCaP cells transfected with RAGE RNAi constructs. Importantly, the RAGE RNAi constructs when administered in nude mice bearing prostate tumors, inhibited the tumor growth by targeting the expression of RAGE, and its physiological ligand, HMGB1 and by up regulating death receptors DR4 and DR5 expression. Collectively, the results of this study for the first time show that targeting RAGE by RNAi may be a promising alternative therapeutic strategy for treating prostate cancer.

  13. Characterization of niphatenones that inhibit androgen receptor N-terminal domain.

    Directory of Open Access Journals (Sweden)

    Carmen A Banuelos

    Full Text Available Androgen ablation therapy causes a temporary reduction in tumor burden in patients with advanced prostate cancer. Unfortunately the malignancy will return to form lethal castration-recurrent prostate cancer (CRPC. The androgen receptor (AR remains transcriptionally active in CRPC in spite of castrate levels of androgens in the blood. AR transcriptional activity resides in its N-terminal domain (NTD. Possible mechanisms of continued AR transcriptional activity may include, at least in part, expression of constitutively active splice variants of AR that lack the C-terminal ligand-binding domain (LBD. Current therapies that target the AR LBD, would not be effective against these AR variants. Currently no drugs are clinically available that target the AR NTD which should be effective against these AR variants as well as full-length AR. Niphatenones were originally isolated and identified in active extracts from Niphates digitalis marine sponge. Here we begin to characterize the mechanism of niphatenones in blocking AR transcriptional activity. Both enantiomers had similar IC50 values of 6 µM for inhibiting the full-length AR in a functional transcriptional assay. However, (S-niphatenone had significantly better activity against the AR NTD compared to (R-niphatenone. Consistent with niphatenones binding to and inhibiting transactivation of AR NTD, niphatenones inhibited AR splice variant. Niphatenone did not affect the transcriptional activity of the related progesterone receptor, but slightly decreased glucocorticoid receptor (GR activity and covalently bound to GR activation function-1 (AF-1 region. Niphatenone blocked N/C interactions of AR without altering either AR protein levels or its intracellular localization in response to androgen. Alkylation with glutathione suggests that niphatenones are not a feasible scaffold for further drug development.

  14. Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors.

    Directory of Open Access Journals (Sweden)

    Miguel Aste-Amézaga

    2010-02-01

    Full Text Available Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD, and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR. The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50 values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL. In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell

  15. Dopamine inhibition of anterior pituitary adenylate cyclase is mediated through the high-affinity state of the D2 receptor

    International Nuclear Information System (INIS)

    Borgundvaag, B.; George, S.R.

    1985-01-01

    The diterpinoid forskolin stimulated adenylate cyclase activity (measured by conversion of [ 3 H]-ATP to [ 3 H]-cAMP) in anterior pituitary from male and female rats. Inhibition of stimulated adenylate cyclase activity by potent dopaminergic agonists was demonstrable only in female anterior pituitary. The inhibition of adenylate cyclase activity displayed a typically dopaminergic rank order of agonist potencies and could be completely reversed by a specific dopamine receptor antagonist. The IC 50 values of dopamine agonist inhibition of adenylate cyclase activity correlated with equal molarity with the dissociation constant of the high-affinity dopamine agonist-detected receptor binding site and with the IC 50 values for inhibition of prolactin secretion. These findings support the hypothesis that it is the high-affinity form of the D 2 dopamine receptor in anterior pituitary which is responsible for mediating the dopaminergic function of attenuating adenylate cyclase activity. 12 references, 4 figures, 1 table

  16. Cobaltous chloride and hypoxia inhibit aryl hydrocarbon receptor-mediated responses in breast cancer cells

    International Nuclear Information System (INIS)

    Khan, Shaheen; Liu Shengxi; Stoner, Matthew; Safe, Stephen

    2007-01-01

    The aryl hydrocarbon receptor (AhR) is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and mRNA levels and also activates inhibitory AhR-ERα crosstalk associated with hormone-induced reporter gene expression. In ZR-75 cells grown under hypoxia, induction of these AhR-mediated responses by TCDD was significantly inhibited. This was not accompanied by decreased nuclear AhR levels or decreased interaction of the AhR complex with the CYP1A1 gene promoter as determined in a chromatin immunoprecipitation assay. Hypoxia-induced loss of Ah-responsiveness was not associated with induction of hypoxia-inducible factor-1α or other factors that sequester the AhR nuclear translocation (Arnt) protein, and overexpression of Arnt under hypoxia did not restore Ah-responsiveness. The p65 subunit of NFκB which inhibits AhR-mediated transactivation was not induced by hypoxia and was primarily cytosolic in ZR-75 cells grown under hypoxic and normoxic conditions. In ZR-75 cells maintained under hypoxic conditions for 24 h, BRCA1 (an enhancer of AhR-mediated transactivation in breast cancer cells) was significantly decreased and this contributed to loss of Ah-responsiveness. In cells grown under hypoxia for 6 h, BRCA1 was not decreased, but induction of CYP1A1 by TCDD was significantly decreased. Cotreatment of ZR-75 cells with TCDD plus the protein synthesis inhibitor cycloheximide for 6 h enhanced CYP1A1 expression in cells grown under hypoxia and normoxia. These results suggest that hypoxia rapidly induces protein(s) that inhibit Ah-responsiveness and these may be similar to constitutively expressed inhibitors of Ah-responsiveness (under normoxia) that are also inhibited by cycloheximide

  17. Inhibition of thyroid hormone receptor locally in the retina is a therapeutic strategy for retinal degeneration.

    Science.gov (United States)

    Ma, Hongwei; Yang, Fan; Butler, Michael R; Belcher, Joshua; Redmond, T Michael; Placzek, Andrew T; Scanlan, Thomas S; Ding, Xi-Qin

    2017-08-01

    Thyroid hormone (TH) signaling regulates cell proliferation, differentiation, and metabolism. Recent studies have implicated TH signaling in cone photoreceptor viability. Using mouse models of retinal degeneration, we demonstrated that antithyroid drug treatment and targeting iodothyronine deiodinases (DIOs) to suppress cellular tri-iodothyronine (T3) production or increase T3 degradation preserves cones. In this work, we investigated the effectiveness of inhibition of the TH receptor (TR). Two genes, THRA and THRB , encode TRs; THRB 2 has been associated with cone viability. Using TR antagonists and Thrb2 deletion, we examined the effects of TR inhibition. Systemic and ocular treatment with the TR antagonists NH-3 and 1-850 increased cone density by 30-40% in the Rpe65 -/- mouse model of Leber congenital amaurosis and reduced the number of TUNEL + cells. Cone survival was significantly improved in Rpe65 -/- and Cpfl1 (a model of achromatopsia with Pde6c defect) mice with Thrb2 deletion. Ventral cone density in Cpfl1/Thrb2 -/- and Rpe65 -/- / Thrb2 -/- mice was increased by 1- to 4-fold, compared with age-matched controls. Moreover, the expression levels of TR were significantly higher in the cone-degeneration retinas, suggesting locally elevated TR signaling. This work shows that the effects of antithyroid treatment or targeting DIOs were likely mediated by TRs and that suppressing TR protects cones. Our findings support the view that inhibition of TR locally in the retina is a therapeutic strategy for retinal degeneration management.-Ma, H., Yang, F., Butler, M. R., Belcher, J., Redmond, T. M., Placzek, A. T., Scanlan, T. S., Ding, X.-Q. Inhibition of thyroid hormone receptor locally in the retina is a therapeutic strategy for retinal degeneration. © FASEB.

  18. δ-Tocopherol inhibits receptor tyrosine kinase-induced AKT activation in prostate cancer cells.

    Science.gov (United States)

    Wang, Hong; Hong, Jungil; Yang, Chung S

    2016-11-01

    The cancer preventive activity of vitamin E is suggested by epidemiological studies and supported by animal studies with vitamin E forms, γ-tocopherol and δ-tocopherol (δ-T). Several recent large-scale cancer prevention trials with high dose of α-tocopherol, however, yielded disappointing results. Whether vitamin E prevents or promotes cancer is a serious concern. A better understanding of the molecular mechanisms of action of the different forms of tocopherols would enhance our understanding of this topic. In this study, we demonstrated that δ-T was the most effective tocopherol form in inhibiting prostate cancer cell growth, by inducing cell cycle arrest and apoptosis. By profiling the effects of δ-T on the cell signaling using the phospho-kinase array, we found that the most inhibited target was the phosphorylation of AKT on T308. Further study on the activation of AKT by EGFR and IGFR revealed that δ-T attenuated the EGF/IGF-induced activation of AKT (via the phosphorylation of AKT on T308 induced by the activation of PIK3). Expression of dominant active PIK3 and AKT in prostate cancer cell line DU145 in which PIK3, AKT, and PTEN are wild type caused the cells to be reflectory to the inhibition of δ-T, supporting that δ-T inhibits the PIK3-mediated activation of AKT. Our data also suggest that δ-T interferes with the EGF-induced EGFR internalization, which leads to the inhibition of the receptor tyrosine kinase-dependent activation of AKT. In summary, our results revealed a novel mechanism of δ-T in inhibiting prostate cancer cell growth, supporting the cancer preventive activity δ-T. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  19. Halothane inhibits the cholinergic-receptor-mediated influx of calcium in primary culture of bovine adrenal medulla cells

    International Nuclear Information System (INIS)

    Yashima, N.; Wada, A.; Izumi, F.

    1986-01-01

    Adrenal medulla cells are cholinoceptive cells. Stimulation of the acetylcholine receptor causes the influx of Ca to the cells, and Ca acts as the coupler of the stimulus-secretion coupling. In this study, the authors investigated the effects of halothane on the receptor-mediated influx of 45 Ca using cultured bovine adrenal medulla cells. Halothane at clinical concentrations (0.5-2%) inhibited the influx of 45 Ca caused by carbachol, with simultaneous inhibition of catecholamine secretion. The influx of 45 Ca and the secretion of catecholamines caused by K depolarization were inhibited by a large concentration of Mg, which competes with Ca at Ca channels, but not inhibited by halothane. Inhibition of the 45 Ca influx by halothane was not overcome by increase in the carbachol concentration. Inhibition of the 45 Ca influx by halothane was examined in comparison with that caused by a large concentration of Mg by the application of Scatchard analysis as the function of the external Ca concentration. Halothane decreased the maximal influx of 45 Ca without altering the apparent kinetic constant of Ca to Ca channels. On the contrary, a large concentration of Mg increased the apparent kinetic constant without altering the maximal influx of 45 Ca. Based on these findings, the authors suggest that inhibition of the 45 Ca influx by halothane was not due to the direct competitive inhibition of Ca channels, nor to the competitive antagonism of agonist-receptor interaction. As a possibility, halothane seems to inhibit the receptor-mediated activation of Ca channels through the interference of coupling between the receptor and Ca channels

  20. Dual angiotensin receptor and neprilysin inhibition as an alternative to angiotensin-converting enzyme inhibition in patients with chronic systolic heart failure

    DEFF Research Database (Denmark)

    McMurray, John J V; Packer, Milton; Desai, Akshay S

    2013-01-01

    and natriuresis, inhibit abnormal growth, suppress the RAAS and sympathetic nervous system, and augment parasympathetic activity. The best understood of these mediators are the natriuretic peptides which are metabolized by the enzyme neprilysin. LCZ696 belongs to a new class of drugs, the angiotensin receptor...

  1. Inhibition of the CSF-1 receptor sensitizes ovarian cancer cells to cisplatin.

    Science.gov (United States)

    Yu, Rong; Jin, Hao; Jin, Congcong; Huang, Xuefeng; Lin, Jinju; Teng, Yili

    2018-03-01

    Ovarian cancer is one of the most common female malignancies, and cisplatin-based chemotherapy is routinely used in locally advanced ovarian cancer patients. Acquired or de novo cisplatin resistance remains the barrier to patient survival, and the mechanisms of cisplatin resistance are still not well understood. In the current study, we found that colony-stimulating-factor-1 receptor (CSF-1R) was upregulated in cisplatin-resistant SK-OV-3 and CaoV-3 cells. Colony-stimulating-factor-1 receptor knockdown suppressed proliferation and enhanced apoptosis in cisplatin-resistant SK-OV-3 and CaoV-3 cells. However, CSF-1R overexpression had inverse effects. While parental SK-OV-3 and CaoV-3 cells were more resistant to cisplatin after CSF-1R overexpression, CSF-1R knockdown in SK-OV-3 and CaoV-3 cells promoted cisplatin sensitivity. Overexpression and knockdown studies also showed that CSF-1R significantly promoted active AKT and ERK1/2 signalling pathways in cisplatin-resistant cells. Furthermore, a combination of cisplatin and CSF-1R inhibitor effectively inhibited tumour growth in xenografts. Taken together, our results provide the first evidence that CSF-1R inhibition can sensitize cisplatin-refractory ovarian cancer cells. This study may help to increase understanding of the molecular mechanisms underlying cisplatin resistance in tumours. Copyright © 2018 John Wiley & Sons, Ltd.

  2. Inhibition of androgen receptor by decoy molecules delays progression to castration-recurrent prostate cancer.

    Directory of Open Access Journals (Sweden)

    Jae-Kyung Myung

    Full Text Available Androgen receptor (AR is a member of the steroid receptor family and a therapeutic target for all stages of prostate cancer. AR is activated by ligand binding within its C-terminus ligand-binding domain (LBD. Here we show that overexpression of the AR NTD to generate decoy molecules inhibited both the growth and progression of prostate cancer in castrated hosts. Specifically, it was shown that lentivirus delivery of decoys delayed hormonal progression in castrated hosts as indicated by increased doubling time of tumor volume, prolonged time to achieve pre-castrate levels of serum prostate-specific antigen (PSA and PSA nadir. These clinical parameters are indicative of delayed hormonal progression and improved therapeutic response and prognosis. Decoys reduced the expression of androgen-regulated genes that correlated with reduced in situ interaction of the AR with androgen response elements. Decoys did not reduce levels of AR protein or prevent nuclear localization of the AR. Nor did decoys interact directly with the AR. Thus decoys did not inhibit AR transactivation by a dominant negative mechanism. This work provides evidence that the AR NTD plays an important role in the hormonal progression of prostate cancer and supports the development of AR antagonists that target the AR NTD.

  3. Fisetin inhibits liver cancer growth in a mouse model: Relation to dopamine receptor.

    Science.gov (United States)

    Liu, Xiang-Feng; Long, Hai-Jiao; Miao, Xiong-Ying; Liu, Guo-Li; Yao, Hong-Liang

    2017-07-01

    Fisetin (3,3',4',7-tetrahydroxyflavone), a natural abundant flavonoid, is produced in different vegetables and fruits. Fisetin has been reported to relate to various positive biological effects, including anti-proliferative, anticancer, anti-oxidative and neuroprotective effects. Dopamine receptors (DRs) belonging to G protein‑coupled receptor family, are known as the target of ~50% of all modern medicinal drugs. DRs consist of various proteins, functioning as transduction of intracellular signals for extracellular stimuli. We found that fisetin performed as DR2 agonist to suppress liver cancer cells proliferation, migration and invasion. Caspase-3 signaling was activated to induce apoptosis for fisetin administration. Furthermore, TGF‑β1 was also inhibited in fisetin-treated liver cancer cells, reducing epithelial-mesenchymal transition (EMT). Additionally, fisetin downregulated VEGFR1, p-ERK1/2, p38 and pJNK, ameliorating liver cancer progression. In vivo, the orthotopically implanted tumors from mice were inhibited by fisetin adminisatration accompanied by prolonged survival rate and higher levels of dopamine. Together, the results indicated a novel therapeutic strategy to suppress liver cancer progression associated with DR2 regulation, indicating that dopamine might be of importance in liver cancer progression.

  4. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

    Directory of Open Access Journals (Sweden)

    Yongjun Yin

    2016-05-01

    Full Text Available Activating mutations in fibroblast growth factor receptor 3 (FGFR3 have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9, a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11 with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3.

  5. STC1 interference on calcitonin family of receptors signaling during osteoblastogenesis via adenylate cyclase inhibition.

    Science.gov (United States)

    Terra, Silvia R; Cardoso, João Carlos R; Félix, Rute C; Martins, Leo Anderson M; Souza, Diogo Onofre G; Guma, Fatima C R; Canário, Adelino Vicente M; Schein, Vanessa

    2015-03-05

    Stanniocalcin 1 (STC1) and calcitonin gene-related peptide (CGRP) are involved in bone formation/remodeling. Here we investigate the effects of STC1 on functional heterodimer complex CALCRL/RAMP1, expression and activity during osteoblastogenesis. STC1 did not modify CALCRL and ramp1 gene expression during osteoblastogenesis when compared to controls. However, plasma membrane spatial distribution of CALCRL/RAMP1 was modified in 7-day pre-osteoblasts exposed to either CGRP or STC1, and both peptides induced CALCRL and RAMP1 assembly. CGRP, but not STC1 stimulated cAMP accumulation in 7-day osteoblasts and in CALCRL/RAMP1 transfected HEK293 cells. Furthermore, STC1 inhibited forskolin stimulated cAMP accumulation of HEK293 cells, but not in CALCRL/RAMP1 transfected HEK293 cells. However, STC1 inhibited cAMP accumulation in calcitonin receptor (CTR) HEK293 transfected cells stimulated by calcitonin. In conclusion, STC1 signals through inhibitory G-protein modulates CGRP receptor spatial localization during osteoblastogenesis and may function as a regulatory factor interacting with calcitonin peptide members during bone formation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  6. Lactobacillus bulgaricus OLL1181 activates the aryl hydrocarbon receptor pathway and inhibits colitis

    Science.gov (United States)

    Takamura, Takeyuki; Harama, Daisuke; Fukumoto, Suguru; Nakamura, Yuki; Shimokawa, Naomi; Ishimaru, Kayoko; Ikegami, Shuji; Makino, Seiya; Kitamura, Masanori; Nakao, Atsuhito

    2011-01-01

    Increasing evidence suggests that the aryl hydrocarbon receptor (AhR) pathway has an important role in the regulation of inflammatory responses. Most recently, we have shown that the activation of the AhR pathway by a potent AhR agonist inhibits the development of dextran sodium sulfate (DSS)-induced colitis, a model of human ulcerative colitis, by the induction of prostaglandin E2 (PGE2) in the large intestine. Because several strains of probiotic lactic acid bacteria have been reported to inhibit DSS-induced colitis by unidentified mechanisms, we hypothesized that particular strains of lactic acid bacterium might have the potential to activate the AhR pathway, thereby inhibiting DSS-induced colitis. This study investigated whether there are specific lactic acid bacterial strains that can activate the AhR pathway, and if so, whether this AhR-activating potential is associated with suppression of DSS-induced colitis. By using AhR signaling reporter cells, we found that Lactobacillus bulgaricus OLL1181 had the potential to activate the AhR pathway. OLL1181 also induced the mRNA expression of cytochrome P450 family 1A1 (CYP1A1), a target gene of the AhR pathway, in human colon cells, which was inhibited by the addition of an AhR antagonist, α-naphthoflavon (αNF). In addition, mice treated orally with OLL1181 showed an increase in CYP1A1 mRNA expression in the large intestine and amelioration of DSS-induced colitis. Thus, OLL1181 can induce activation of the intestinal AhR pathway and inhibit DSS-induced colitis in mice. This strain of lactic acid bacterium has therefore the potential to activate the AhR pathway, which may be able to suppress colitis. PMID:21321579

  7. Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium.

    LENUS (Irish Health Repository)

    Dooley, Ruth

    2012-02-01

    BACKGROUND: Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation. RESULTS: Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells. CONCLUSION: Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

  8. Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium

    LENUS (Irish Health Repository)

    Dooley, Ruth

    2011-08-22

    Abstract Background Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation. Results Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells. Conclusion Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

  9. Phosphorylation inhibits DNA-binding of alternatively spliced aryl hydrocarbon receptor nuclear translocator

    International Nuclear Information System (INIS)

    Kewley, Robyn J.; Whitelaw, Murray L.

    2005-01-01

    The basic helix-loop-helix/PER-ARNT-SIM homology (bHLH/PAS) transcription factor ARNT (aryl hydrocarbon receptor nuclear translocator) is a key component of various pathways which induce the transcription of cytochrome P450 and hypoxia response genes. ARNT can be alternatively spliced to express Alt ARNT, containing an additional 15 amino acids immediately N-terminal to the DNA-binding basic region. Here, we show that ARNT and Alt ARNT proteins are differentially phosphorylated by protein kinase CKII in vitro. Phosphorylation had an inhibitory effect on DNA-binding to an E-box probe by Alt ARNT, but not ARNT, homodimers. This inhibitory phosphorylation occurs through Ser77. Moreover, a point mutant, Alt ARNT S77A, shows increased activity on an E-box reporter gene, consistent with Ser77 being a regulatory site in vivo. In contrast, DNA binding by an Alt ARNT/dioxin receptor heterodimer to the xenobiotic response element is not inhibited by phosphorylation with CKII, nor does Alt ARNT S77A behave differently from wild type Alt ARNT in the context of a dioxin receptor heterodimer

  10. Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium

    Directory of Open Access Journals (Sweden)

    Hatt Hanns

    2011-08-01

    Full Text Available Abstract Background Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation. Results Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells. Conclusion Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

  11. Diurnal inhibition of NMDA-EPSCs at rat hippocampal mossy fibre synapses through orexin-2 receptors

    Science.gov (United States)

    Perin, Martina; Longordo, Fabio; Massonnet, Christine; Welker, Egbert; Lüthi, Anita

    2014-01-01

    Diurnal release of the orexin neuropeptides orexin-A (Ox-A, hypocretin-1) and orexin-B (Ox-B, hypocretin-2) stabilises arousal, regulates energy homeostasis and contributes to cognition and learning. However, whether cellular correlates of brain plasticity are regulated through orexins, and whether they do so in a time-of-day-dependent manner, has never been assessed. Immunohistochemically we found sparse but widespread innervation of hippocampal subfields through Ox-A- and Ox-B-containing fibres in young adult rats. The actions of Ox-A were studied on NMDA receptor (NMDAR)-mediated excitatory synaptic transmission in acute hippocampal slices prepared around the trough (Zeitgeber time (ZT) 4–8, corresponding to 4–8 h into the resting phase) and peak (ZT 23) of intracerebroventricular orexin levels. At ZT 4–8, exogenous Ox-A (100 nm in bath) inhibited NMDA receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) at mossy fibre (MF)–CA3 (to 55.6 ± 6.8% of control, P = 0.0003) and at Schaffer collateral–CA1 synapses (70.8 ± 6.3%, P = 0.013), whereas it remained ineffective at non-MF excitatory synapses in CA3. Ox-A actions were mediated postsynaptically and blocked by the orexin-2 receptor (OX2R) antagonist JNJ10397049 (1 μm), but not by orexin-1 receptor inhibition (SB334867, 1 μm) or by adrenergic and cholinergic antagonists. At ZT 23, inhibitory effects of exogenous Ox-A were absent (97.6 ± 2.9%, P = 0.42), but reinstated (87.2 ± 3.3%, P = 0.002) when endogenous orexin signalling was attenuated for 5 h through i.p. injections of almorexant (100 mg kg−1), a dual orexin receptor antagonist. In conclusion, endogenous orexins modulate hippocampal NMDAR function in a time-of-day-dependent manner, suggesting that they may influence cellular plasticity and consequent variations in memory performance across the sleep–wake cycle. PMID:25085886

  12. PKCɛ mediates substance P inhibition of GABAA receptors-mediated current in rat dorsal root ganglion.

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    Li, Li; Zhao, Lei; Wang, Yang; Ma, Ke-tao; Shi, Wen-yan; Wang, Ying-zi; Si, Jun-qiang

    2015-02-01

    The mechanism underlying the modulatory effect of substance P (SP) on GABA-activated response in rat dorsal root ganglion (DRG) neurons was investigated. In freshly dissociated rat DRG neurons, whole-cell patch-clamp technique was used to record GABA-activated current and sharp electrode intracellular recording technique was used to record GABA-induced membrane depolarization. Application of GABA (1-1000 μmol/L) induced an inward current in a concentration-dependent manner in 114 out of 127 DRG neurons (89.8 %) examined with whole-cell patch-clamp recordings. Bath application of GABA (1-1000 μmol/L) evoked a depolarizing response in 236 out of 257 (91.8%) DRG neurons examined with intracellular recordings. Application of SP (0.001-1 μmol/L) suppressed the GABA-activated inward current and membrane depolarization. The inhibitory effects were concentration-dependent and could be blocked by the selective neurokinin 1 (NK1) receptors antagonist spantide but not by L659187 and SR142801 (1 μmol/L, n=7), selective antagonists of NK2 and NK3. The inhibitory effect of SP was significantly reduced by the calcium chelator BAPTA-AM, phospholipase C (PLC) inhibitor U73122, and PKC inhibitor chelerythrine, respectively. The PKA inhibitor H-89 did not affect the SP effect. Remarkably, the inhibitory effect of SP on GABA-activated current was nearly completely removed by a selective PKCε inhibitor epilon-V1-2 but not by safingol and LY333531, selective inhibitors of PKCα and PKCβ. Our results suggest that NK1 receptor mediates SP-induced inhibition of GABA-activated current and membrane depolarization by activating intracellular PLC-Ca²⁺-PKCε cascade. SP might regulate the excitability of peripheral nociceptors through inhibition of the "pre-synaptic inhibition" evoked by GABA, which may explain its role in pain and neurogenic inflammation.

  13. Acute inhibition of selected membrane-proximal mouse T cell receptor signaling by mitochondrial antagonists.

    Directory of Open Access Journals (Sweden)

    Kwangmi Kim

    2009-11-01

    Full Text Available T cells absorb nanometric membrane vesicles, prepared from plasma membrane of antigen presenting cells, via dual receptor/ligand interactions of T cell receptor (TCR with cognate peptide/major histocompatibility complex (MHC plus lymphocyte function-associated antigen 1 (LFA-1 with intercellular adhesion molecule 1. TCR-mediated signaling for LFA-1 activation is also required for the vesicle absorption. Exploiting those findings, we had established a high throughput screening (HTS platform and screened a library for isolation of small molecules inhibiting the vesicle absorption. Follow-up studies confirmed that treatments (1 hour with various mitochondrial antagonists, including a class of anti-diabetic drugs (i.e., Metformin and Phenformin, resulted in ubiquitous inhibition of the vesicle absorption without compromising viability of T cells. Further studies revealed that the mitochondrial drug treatments caused impairment of specific membrane-proximal TCR signaling event(s. Thus, activation of Akt and PLC-gamma1 and entry of extracellular Ca(2+ following TCR stimulation were attenuated while polymerization of monomeric actins upon TCR triggering progressed normally after the treatments. Dynamic F-actin rearrangement concurring with the vesicle absorption was also found to be impaired by the drug treatments, implying that the inhibition by the drug treatments of downstream signaling events (and the vesicle absorption could result from lack of directional relocation of signaling and cell surface molecules. We also assessed the potential application of mitochondrial antagonists as immune modulators by probing effects of the long-term drug treatments (24 hours on viability of resting primary T cells and cell cycle progression of antigen-stimulated T cells. This study unveils a novel regulatory mechanism for T cell immunity in response to environmental factors having effects on mitochondrial function.

  14. Inhibition of Protease-activated Receptor 1 Ameliorates Intestinal Radiation Mucositis in a Preclinical Rat Model

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Junru; Kulkarni, Ashwini [Division of Radiation Health, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Chintala, Madhu [Schering-Plough Research Institute, Kenilworth, New Jersey (United States); Fink, Louis M. [Nevada Cancer Institute, Las Vegas, Nevada (United States); Hauer-Jensen, Martin, E-mail: mhjensen@life.uams.edu [Division of Radiation Health, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Surgery Service, Central Arkansas Veterans Healthcare System, Little Rock, Arkansas (United States)

    2013-01-01

    Purpose: To determine, using a specific small-molecule inhibitor of protease-activated receptor 1 (PAR1) signaling, whether the beneficial effect of thrombin inhibition on radiation enteropathy development is due to inhibition of blood clotting or to cellular (PAR1-mediated) thrombin effects. Methods and Materials: Rats underwent fractionated X-irradiation (5 Gy Multiplication-Sign 9) of a 4-cm small-bowel segment. Early radiation toxicity was evaluated in rats receiving PAR1 inhibitor (SCH602539, 0, 10, or 15 mg/kg/d) from 1 day before to 2 weeks after the end of irradiation. The effect of PAR1 inhibition on development of chronic intestinal radiation fibrosis was evaluated in animals receiving SCH602539 (0, 15, or 30 mg/kg/d) until 2 weeks after irradiation, or continuously until termination of the experiment 26 weeks after irradiation. Results: Blockade of PAR1 ameliorated early intestinal toxicity, with reduced overall intestinal radiation injury (P=.002), number of myeloperoxidase-positive (P=.03) and proliferating cell nuclear antigen-positive (P=.04) cells, and collagen III accumulation (P=.005). In contrast, there was no difference in delayed radiation enteropathy in either the 2- or 26-week administration groups. Conclusion: Pharmacological blockade of PAR1 seems to reduce early radiation mucositis but does not affect the level of delayed intestinal radiation fibrosis. Early radiation enteropathy is related to activation of cellular thrombin receptors, whereas platelet activation or fibrin formation may play a greater role in the development of delayed toxicity. Because of the favorable side-effect profile, PAR1 blockade should be further explored as a method to ameliorate acute intestinal radiation toxicity in patients undergoing radiotherapy for cancer and to protect first responders and rescue personnel in radiologic/nuclear emergencies.

  15. Adrenergic receptors inhibit TRPV1 activity in the dorsal root ganglion neurons of rats.

    Science.gov (United States)

    Matsushita, Yumi; Manabe, Miki; Kitamura, Naoki; Shibuya, Izumi

    2018-01-01

    Transient receptor potential vanilloid type 1 (TRPV1) is a polymodal receptor channel that responds to multiple types of stimuli, such as heat, acid, mechanical pressure and some vanilloids. Capsaicin is the most commonly used vanilloid to stimulate TRPV1. TRPV1 channels are expressed in dorsal root ganglion neurons that extend to Aδ- and C-fibers and have a role in the transduction of noxious inputs to the skin into the electrical signals of the sensory nerve. Although noradrenergic nervous systems, including the descending antinociceptive system and the sympathetic nervous system, are known to modulate pain sensation, the functional association between TRPV1 and noradrenaline in primary sensory neurons has rarely been examined. In the present study, we examined the effects of noradrenaline on capsaicin-evoked currents in cultured dorsal root ganglion neurons of the rat by the whole-cell voltage clamp method. Noradrenaline at concentrations higher than 0.1 pM significantly reduced the amplitudes of the inward capsaicin currents recorded at -60 mV holding potential. This inhibitory action was reversed by either yohimbine (an α2 antagonist, 10 nM) or propranolol (a β antagonist, 10 nM). The α2 agonists, clonidine (1 pM) and dexmedetomidine (1 pM) inhibited capsaicin currents, and yohimbine (1 nM) reversed the effects of clonidine. The inhibitory action of noradrenaline was not seen in the neurons pretreated with pertussis toxin (100 μg/ml for 24 h) and the neurons dialyzed intracellularly with guanosine 5'- [β-thio] diphosphate (GDPβS, 200 μM), the catalytic subunit of protein kinase A (250 U/ml) or okadaic acid (1 μM). These results suggest that noradrenaline directly acts on dorsal root ganglion neurons to inhibit the activity of TRPV1 depending on the activation of α2-adrenoceptors followed by the inhibition of the adenylate cyclase/cAMP/protein kinase A pathway.

  16. Inhibition of the Receptor Tyrosine Kinase AXL Restores Paclitaxel Chemosensitivity in Uterine Serous Cancer.

    Science.gov (United States)

    Palisoul, Marguerite L; Quinn, Jeanne M; Schepers, Emily; Hagemann, Ian S; Guo, Lei; Reger, Kelsey; Hagemann, Andrea R; McCourt, Carolyn K; Thaker, Premal H; Powell, Matthew A; Mutch, David G; Fuh, Katherine C

    2017-12-01

    Uterine serous cancer (USC) is aggressive, and the majority of recurrent cases are chemoresistant. Because the receptor tyrosine kinase AXL promotes invasion and metastasis of USC and is implicated in chemoresistance in other cancers, we assessed the role of AXL in paclitaxel resistance in USC, determined the mechanism of action, and sought to restore chemosensitivity by inhibiting AXL in vitro and in vivo We used short hairpin RNAs and BGB324 to knock down and inhibit AXL. We assessed sensitivity of USC cell lines to paclitaxel and measured paclitaxel intracellular accumulation in vitro in the presence or absence of AXL. We also examined the role of the epithelial-mesenchymal transition (EMT) in AXL-mediated paclitaxel resistance. Finally, we treated USC xenografts with paclitaxel, BGB324, or paclitaxel plus BGB324 and monitored tumor burden. AXL expression was higher in chemoresistant USC patient tumors and cell lines than in chemosensitive tumors and cell lines. Knockdown or inhibition of AXL increased sensitivity of USC cell lines to paclitaxel in vitro and increased cellular accumulation of paclitaxel. AXL promoted chemoresistance even in cells that underwent the EMT in vitro Finally, in vivo studies of combination treatment with BGB324 and paclitaxel showed a greater than 51% decrease in tumor volume after 2 weeks of treatment when compared with no treatment or single-agent treatments ( P USC. Mol Cancer Ther; 16(12); 2881-91. ©2017 AACR . ©2017 American Association for Cancer Research.

  17. Flavonoids Suppress Pseudomonas aeruginosa Virulence through Allosteric Inhibition of Quorum-sensing Receptors*

    Science.gov (United States)

    Paczkowski, Jon E.; Mukherjee, Sampriti; McCready, Amelia R.; Cong, Jian-Ping; Aquino, Christopher J.; Kim, Hahn; Henke, Brad R.; Smith, Chari D.; Bassler, Bonnie L.

    2017-01-01

    Quorum sensing is a process of cell-cell communication that bacteria use to regulate collective behaviors. Quorum sensing depends on the production, detection, and group-wide response to extracellular signal molecules called autoinducers. In many bacterial species, quorum sensing controls virulence factor production. Thus, disrupting quorum sensing is considered a promising strategy to combat bacterial pathogenicity. Several members of a family of naturally produced plant metabolites called flavonoids inhibit Pseudomonas aeruginosa biofilm formation by an unknown mechanism. Here, we explore this family of molecules further, and we demonstrate that flavonoids specifically inhibit quorum sensing via antagonism of the autoinducer-binding receptors, LasR and RhlR. Structure-activity relationship analyses demonstrate that the presence of two hydroxyl moieties in the flavone A-ring backbone are essential for potent inhibition of LasR/RhlR. Biochemical analyses reveal that the flavonoids function non-competitively to prevent LasR/RhlR DNA binding. Administration of the flavonoids to P. aeruginosa alters transcription of quorum sensing-controlled target promoters and suppresses virulence factor production, confirming their potential as anti-infectives that do not function by traditional bacteriocidal or bacteriostatic mechanisms. PMID:28119451

  18. Pax6 represses androgen receptor-mediated transactivation by inhibiting recruitment of the coactivator SPBP.

    Directory of Open Access Journals (Sweden)

    Julianne Elvenes

    Full Text Available The androgen receptor (AR has a central role in development and maintenance of the male reproductive system and in the etiology of prostate cancer. The transcription factor Pax6 has recently been reported to act as a repressor of AR and to be hypermethylated in prostate cancer cells. SPBP is a transcriptional regulator that previously has been shown to enhance the activity of Pax6. In this study we have identified SPBP to act as a transcriptional coactivator of AR. We also show that Pax6 inhibits SPBP-mediated enhancement of AR activity on the AR target gene probasin promoter, a repression that was partly reversed by increased expression of SPBP. Enhanced expression of Pax6 reduced the amount of SPBP associated with the probasin promoter when assayed by ChIP in HeLa cells. We mapped the interaction between both AR and SPBP, and AR and Pax6 to the DNA-binding domains of the involved proteins. Further binding studies revealed that Pax6 and SPBP compete for binding to AR. These results suggest that Pax6 represses AR activity by displacing and/or inhibiting recruitment of coactivators to AR target promoters. Understanding the mechanism for inhibition of AR coactivators can give rise to molecular targeted drugs for treatment of prostate cancer.

  19. R+-methanandamide inhibits tracheal response to endogenously released acetylcholine via capsazepine-sensitive receptors.

    Science.gov (United States)

    Nieri, Paola; Martinotti, Enrica; Testai, Lara; Adinolfi, Barbara; Calderone, Vincenzo; Breschi, Maria Cristina

    2003-01-10

    The effects of cannabinoid drugs on the cholinergic response evoked by electrical field stimulation (0.2 ms pulse width, 20 V amplitude, 10 Hz, 7.5 s train duration) in guinea-pig tracheal preparations were investigated. The stable analogue of the endocannabinoid anandamide, R(+)-methanandamide (10(-7)-10(-4) M), produced a dose-dependent inhibition (up to 27+/-5% of control) of electrical field stimulation-mediated atropine-sensitive response. This effect was not blocked by the selective cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3 carboxamide hydrochloride (SR 141716A; 10(-6) M), and was not reproduced with the cannabinoid CB(1)/CB(2) receptor agonist R(+)-[2,3-dihydro-5-methyl-[(morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl]-(1-naphthalenyl)methanone mesylate) (WIN 55,212-2; 10(-8)-10(-5) M) or the cannabinoid CB(2) receptor selective agonist 1-propyl-2-methyl-3-(1-naphthoyl)indole (JWH-015; 10(-8)-10(-5) M); it was, on the contrary, antagonized by the vanilloid antagonist 2-[2-(4-chlorophenyl)ethyl-amino-thiocarbonyl]-7,8-dihydroxy-2,3,4,5-tetrahydro-1H-2 benzazepine (capsazepine; 10(-6) M). At the postjunctional level, neither R(+)-methanandamide nor WIN 55,212-2 nor JWH-015 did affect tracheal contractions induced by exogenous acetylcholine (10(-6) M). An inhibitory vanilloid receptor-mediated effect on the cholinergic response evoked by electrical stimulation was confirmed with the vanilloid agonist capsaicin, at doses (3-6 x 10(-8) M) which poorly influenced the basal smooth muscle tone of trachea. In conclusion, our data indicate that in guinea-pig trachea (a) neither CB(1) nor CB(2) cannabinoid receptor-mediated modulation of acetylcholine release occurs; (b) vanilloid VR1-like receptors appear involved in R(+)-methanandamide inhibitory activity on the cholinergic response to electrical field stimulation.

  20. Involvement of direct inhibition of NMDA receptors in the effects of sigma-receptor ligands on glutamate neurotoxicity in vitro.

    Science.gov (United States)

    Nishikawa, H; Hashino, A; Kume, T; Katsuki, H; Kaneko, S; Akaike, A

    2000-09-15

    This study was performed to examine the roles of the N-methyl-D-aspartate (NMDA) receptor/phencyclidine (PCP) channel complex in the protective effects of sigma-receptor ligands against glutamate neurotoxicity in cultured cortical neurons derived from fetal rats. A 1-h exposure of cultures to glutamate caused a marked loss of viability, as determined by Trypan blue exclusion. This acute neurotoxicity of glutamate was prevented by NMDA receptor antagonists. Expression of sigma(1) receptor mRNA in cortical cultures was confirmed by reverse transcription polymerase chain reaction (RT-PCR). sigma Receptor ligands with affinity for NMDA receptor channels including the PCP site, such as (+)-N-allylnormetazocine ((+)-SKF10,047), haloperidol, and R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane ((-)-PPAP), prevented glutamate neurotoxicity in a concentration-dependent manner. In contrast, other sigma-receptor ligands without affinity for NMDA receptors, such as carbetapentane and R(+)-3-(3-hydroxyphenyl)-N-propylpiperidine ((+)-3-PPP), did not show neuroprotective effects. Putative endogenous sigma receptor ligands such as pregnenolone, progesterone, and dehydroepiandrosterone did not affect glutamate neurotoxicity. The protective effects of (+)-SKF10,047, haloperidol, and (-)-PPAP were not affected by the sigma(1) receptor antagonist rimcazole. These results suggested that a direct interaction with NMDA receptors but not with sigma receptors plays a crucial role in the neuroprotective effects of sigma receptor ligands with affinity for NMDA receptors.

  1. microRNA-150 inhibits the formation of macrophage foam cells through targeting adiponectin receptor 2

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jing [Department of Geratory, Linzi District People’s Hospital of Zibo City, Zibo, Shandong (China); Zhang, Suhua, E-mail: drsuhuangzhang@qq.com [Department of HealthCare, Qilu Hospital of Shandong University (Qingdao), Qingdao City, Qingdao (China)

    2016-08-05

    Transformation of macrophages into foam cells plays a critical role in the pathogenesis of atherosclerosis. The aim of this study was to determine the expression and biological roles of microRNA (miR)-150 in the formation of macrophage foam cells and to identify its functional target(s). Exposure to 50 μg/ml oxidized low-density lipoprotein (oxLDL) led to a significant upregulation of miR-150 in THP-1 macrophages. Overexpression of miR-150 inhibited oxLDL-induced lipid accumulation in THP-1 macrophages, while knockdown of miR-150 enhanced lipid accumulation. apoA-I- and HDL-mediated cholesterol efflux was increased by 66% and 43%, respectively, in miR-150-overexpressing macrophages relative to control cells. In contrast, downregulation of miR-150 significantly reduced cholesterol efflux from oxLDL-laden macrophages. Bioinformatic analysis and luciferase reporter assay revealed adiponectin receptor 2 (AdipoR2) as a direct target of miR-150. Small interfering RNA-mediated downregulation of AdipoR2 phenocopied the effects of miR-150 overexpression, reducing lipid accumulation and facilitating cholesterol efflux in oxLDL-treated THP-1 macrophages. Knockdown of AdipoR2 induced the expression of proliferator-activated receptor gamma (PPARγ), liver X receptor alpha (LXRα), ABCA1, and ABCG1. Moreover, pharmacological inhibition of PPARγ or LXRα impaired AdipoR2 silencing-induced upregulation of ABCA1 and ABCG1. Taken together, our results indicate that miR-150 can attenuate oxLDL-induced lipid accumulation in macrophages via promotion of cholesterol efflux. The suppressive effects of miR-150 on macrophage foam cell formation are mediated through targeting of AdipoR2. Delivery of miR-150 may represent a potential approach to prevent macrophage foam cell formation in atherosclerosis. -- Highlights: •miR-150 inhibits macrophage foam cell formation. •miR-150 accelerates cholesterol efflux from oxLDL-laden macrophages. •miR-150 suppresses macrophage foam cell

  2. MECHANICAL VIBRATION INHIBITS OSTEOCLAST FORMATION BY REDUCING DC-STAMP RECEPTOR EXPRESSION IN OSTEOCLAST PRECURSOR CELLS

    Science.gov (United States)

    Kulkarni, R.N.; Voglewede, P.A.; Liu, D.

    2014-01-01

    It is well known that physical inactivity leads to loss of muscle mass, but it also causes bone loss. Mechanistically, osteoclastogenesis and bone resorption have recently been shown to be regulated by vibration. However, the underlying mechanism behind the inhibition of osteoclast formation is yet unknown. Therefore, we investigated whether mechanical vibration of osteoclast precursor cells affects osteoclast formation by the involvement of fusion-related molecules such as dendritic cell-specific transmembrane protein (DC-STAMP), and P2X7 receptor (P2X7R). RAW264.7 (a murine osteoclastic-like cell line) cells were treated with 20 ng/ml receptor activator of NF-κB ligand (RANKL). For 3 consecutive days, the cells were subjected to 1 hour of mechanical vibration with 20 µm displacement at a frequency of 4 Hz and compared to the control cells that were treated under the same condition but without the vibration. After 5 days of culture, osteoclast formation was determined. Gene expression of DC-STAMP and P2X7R by RAW264.7 cells were determined after 1 hour mechanical vibration, while protein production of the DC-STAMP was determined after 6 hours of post incubation after vibration. As a result, mechanical vibration of RAW264.7 cells inhibited the formation of osteoclasts. Vibration down-regulated DC-STAMP gene expression by 1.6-fold in the presence of RANKL and by 1.4-fold in the absence of RANKL. Additionally, DC-STAMP protein production was also down-regulated by 1.4-fold in the presence of RANKL and by 1.2-fold in the absence of RANKL in RAW264.7 cells in response to mechanical vibration. However, vibration did not affect P2X7R gene expression. Mouse anti-DC-STAMP antibody inhibited osteoclast formation in the absence of vibration. Our results suggest that mechanical vibration of osteoclast precursor cells reduce DC-STAMP expression in osteoclast precursor cells leading to the inhibition of osteoclast formation. PMID:23994170

  3. Mechanical vibration inhibits osteoclast formation by reducing DC-STAMP receptor expression in osteoclast precursor cells.

    Science.gov (United States)

    Kulkarni, Rishikesh N; Voglewede, Philip A; Liu, Dawei

    2013-12-01

    It is well known that physical inactivity leads to loss of muscle mass, but it also causes bone loss. Mechanistically, osteoclastogenesis and bone resorption have recently been shown to be regulated by vibration. However, the underlying mechanism behind the inhibition of osteoclast formation is yet unknown. Therefore, we investigated whether mechanical vibration of osteoclast precursor cells affects osteoclast formation by the involvement of fusion-related molecules such as dendritic cell-specific transmembrane protein (DC-STAMP) and P2X7 receptor (P2X7R). RAW264.7 (a murine osteoclastic-like cell line) cells were treated with 20ng/ml receptor activator of NF-κB ligand (RANKL). For 3 consecutive days, the cells were subjected to 1h of mechanical vibration with 20μm displacement at a frequency of 4Hz and compared to the control cells that were treated under the same condition but without the vibration. After 5days of culture, osteoclast formation was determined. Gene expression of DC-STAMP and P2X7R by RAW264.7 cells was determined after 1h of mechanical vibration, while protein production of the DC-STAMP was determined after 6h of postincubation after vibration. As a result, mechanical vibration of RAW264.7 cells inhibited the formation of osteoclasts. Vibration down-regulated DC-STAMP gene expression by 1.6-fold in the presence of RANKL and by 1.4-fold in the absence of RANKL. Additionally, DC-STAMP protein production was also down-regulated by 1.4-fold in the presence of RANKL and by 1.2-fold in the absence of RANKL in RAW264.7 cells in response to mechanical vibration. However, vibration did not affect P2X7R gene expression. Mouse anti-DC-STAMP antibody inhibited osteoclast formation in the absence of vibration. Our results suggest that mechanical vibration of osteoclast precursor cells reduces DC-STAMP expression in osteoclast precursor cells leading to the inhibition of osteoclast formation. © 2013 Elsevier Inc. All rights reserved.

  4. Inhibition of estrogen-responsive gene activation by the retinoid X receptor beta: evidence for multiple inhibitory pathways.

    Science.gov (United States)

    Segars, J H; Marks, M S; Hirschfeld, S; Driggers, P H; Martinez, E; Grippo, J F; Brown, M; Wahli, W; Ozato, K

    1993-04-01

    The retinoid X receptor beta (RXR beta; H-2RIIBP) forms heterodimers with various nuclear hormone receptors and binds multiple hormone response elements, including the estrogen response element (ERE). In this report, we show that endogenous RXR beta contributes to ERE binding activity in nuclear extracts of the human breast cancer cell line MCF-7. To define a possible regulatory role of RXR beta regarding estrogen-responsive transcription in breast cancer cells, RXR beta and a reporter gene driven by the vitellogenin A2 ERE were transfected into estrogen-treated MCF-7 cells. RXR beta inhibited ERE-driven reporter activity in a dose-dependent and element-specific fashion. This inhibition occurred in the absence of the RXR ligand 9-cis retinoic acid. The RXR beta-induced inhibition was specific for estrogen receptor (ER)-mediated ERE activation because inhibition was observed in ER-negative MDA-MB-231 cells only following transfection of the estrogen-activated ER. No inhibition of the basal reporter activity was observed. The inhibition was not caused by simple competition of RXR beta with the ER for ERE binding, since deletion mutants retaining DNA binding activity but lacking the N-terminal or C-terminal domain failed to inhibit reporter activity. In addition, cross-linking studies indicated the presence of an auxiliary nuclear factor present in MCF-7 cells that contributed to RXR beta binding of the ERE. Studies using known heterodimerization partners of RXR beta confirmed that RXR beta/triiodothyronine receptor alpha heterodimers avidly bind the ERE but revealed the existence of another triiodothyronine-independent pathway of ERE inhibition. These results indicate that estrogen-responsive genes may be negatively regulated by RXR beta through two distinct pathways.

  5. Targeting Insulin-Like Growth Factor 1 Receptor Inhibits Pancreatic Cancer Growth and Metastasis

    Science.gov (United States)

    Subramani, Ramadevi; Lopez-Valdez, Rebecca; Arumugam, Arunkumar; Nandy, Sushmita; Boopalan, Thiyagarajan; Lakshmanaswamy, Rajkumar

    2014-01-01

    Pancreatic cancer is one of the most lethal cancers. Increasing incidence and mortality indicates that there is still much lacking in detection and management of the disease. This is partly due to a lack of specific symptoms during early stages of the disease. Several growth factor receptors have been associated with pancreatic cancer. Here, we have investigated if an RNA interference approach targeted to IGF-IR could be effective and efficient against pancreatic cancer growth and metastasis. For that, we evaluated the effects of IGF-1R inhibition using small interfering RNA (siRNAs) on tumor growth and metastasis in HPAC and PANC-1 pancreatic cancer cell lines. We found that silencing IGF-1R inhibits pancreatic cancer growth and metastasis by blocking key signaling pathways such AKT/PI3K, MAPK, JAK/STAT and EMT. Silencing IGF-1R resulted in an anti-proliferative effect in PANC-1 and HPAC pancreatic cancer cell lines. Matrigel invasion, transwell migration and wound healing assays also revealed a role for IGF-1R in metastatic properties of pancreatic cancer. These results were further confirmed using Western blotting analysis of key intermediates involved in proliferation, epithelial mesenchymal transition, migration, and invasion. In addition, soft agar assays showed that silencing IGF-1R also blocks the colony forming capabilities of pancreatic cancer cells in vitro. Western blots, as well as, flow cytometric analysis revealed the induction of apoptosis in IGF-1R silenced cells. Interestingly, silencing IGF-1R also suppressed the expression of insulin receptor β. All these effects together significantly control pancreatic cancer cell growth and metastasis. To conclude, our results demonstrate the significance of IGF-1R in pancreatic cancer. PMID:24809702

  6. Autoantibodies against Muscarinic Type 3 Receptor in Sjögren's Syndrome Inhibit Aquaporin 5 Trafficking

    Science.gov (United States)

    Lee, Byung Ha; Gauna, Adrienne E.; Perez, Geidys; Park, Yun-jong; Pauley, Kaleb M.; Kawai, Toshihisa; Cha, Seunghee

    2013-01-01

    Sjögren's syndrome (SjS) is a chronic autoimmune disease that mainly targets the salivary and lacrimal glands. It has been controversial whether anti-muscarinic type 3 receptor (α-M3R) autoantibodies in patients with SjS inhibit intracellular trafficking of aquaporin-5 (AQP5), water transport protein, leading to secretory dysfunction. To address this issue, GFP-tagged human AQP5 was overexpressed in human salivary gland cells (HSG-hAQP5) and monitored AQP5 trafficking to the plasma membrane following carbachol (CCh, M3R agonist) stimulation. AQP5 trafficking was indeed mediated by M3R stimulation, shown in partial blockage of trafficking by M3R-antagonist 4-DAMP. HSG-hAQP5 pre-incubated with SjS plasma for 24 hours significantly reduced AQP5 trafficking with CCh, compared with HSG-hAQP5 pre-incubated with healthy control (HC) plasma. This inhibition was confirmed by monoclonal α-M3R antibody and pre-absorbed plasma. Interestingly, HSG-hAQP5 pre-incubated with SjS plasma showed no change in cell volume, compared to the cells incubated with HC plasma showing shrinkage by twenty percent after CCh-stimulation. Our findings clearly indicate that binding of anti-M3R autoantibodies to the receptor, which was verified by immunoprecipitation, suppresses AQP5 trafficking to the membrane and contribute to impaired fluid secretion in SjS. Our current study urges further investigations of clinical associations between SjS symptoms, such as degree of secretory dysfunction, cognitive impairment, and/or bladder irritation, and different profiles (titers, isotypes, and/or specificity) of anti-M3R autoantibodies in individuals with SjS. PMID:23382834

  7. Liver X receptor activation inhibits PC-3 prostate cancer cells via the beta-catenin pathway.

    Science.gov (United States)

    Youlin, Kuang; Li, Zhang; Weiyang, He; Jian, Kang; Siming, Liang; Xin, Gou

    2017-03-01

    Liver X receptors (LXRs) are nuclear receptors family of ligand-dependent transcription factors that play a crucial role in regulating cholesterol metabolism and inflammation. Recent studies show that LXR agonists exhibit anti-cancer activities in a variety of cancer cell lines including prostate. To further identify the potential mechanisms of LXRα activation on prostate cancer, we investigated the effect of LXR agonist T0901317 on PC3 prostate cancer cell and in which activity of beta-catenin pathway involved. Prostate cancer PC3 cells were transfected with LXR-a siRNA and treated with LXR activator T0901317. qRT-PCR and western blot were used to detect the LXR-a expression. beta-catenin, cyclin D1 and c-MYC were analyzed by western blot. Cell apoptosis was examined by flow cytometry and Cell proliferation was assessed by Cell Counting Kit-8 assay. Cell migration was detected by Transwell chambers. Data showed that T0901317 significantly inhibited PC3 cell proliferation as well as invasion and increased apoptosis in vitro. Furthermore, we found that LXRα activation induced the reduction of beta-catenin expression in PC3 cells, and this inhibitory effect could be totally abolished when cells were treated with LXRα. Meanwhile, the expression of beta-catenin target gene cyclin D1 and c-MYC were also decreased. This study provided additional evidence that LXR activation inhibited PC-3 prostate cancer cells via suppressing beta-catenin pathway. Copyright © 2016 Elsevier GmbH. All rights reserved.

  8. Inhibition of fibroblast growth factor receptor with AZD4547 mitigates juvenile nasopharyngeal angiofibroma.

    Science.gov (United States)

    Le, Tran; New, Jacob; Jones, Joel W; Usman, Shireen; Yalamanchali, Sreeya; Tawfik, Ossama; Hoover, Larry; Bruegger, Dan E; Thomas, Sufi Mary

    2017-10-01

    Juvenile nasopharyngeal angiofibroma (JNA) is a benign tumor that presents in adolescent males. Although surgical excision is the mainstay of treatment, recurrences complicate treatment. There is a need to develop less invasive approaches for management. JNA tumors are composed of fibroblasts and vascular endothelial cells. We identified fibroblast growth factor receptor (FGFR) and vascular endothelial growth factor (VEGF) expression in JNA-derived fibroblasts. FGFR influences fibroblast proliferation and VEGF is necessary for angiogenesis. We hypothesized that targeting FGFR would mitigate JNA fibroblast proliferation, invasion, and migration, and that targeting the VEGF receptor would attenuate endothelial tubule formation. After informed consent, fibroblasts from JNA explants of 3 patients were isolated. Fibroblasts were treated with FGFR inhibitor AZD4547, 0 to 25 μg/mL for 72 hours and proliferation was quantified using CyQuant assay. Migration and invasion of JNA were assessed using 24-hour transwell assays with subsequent fixation and quantification. Mitigation of FGFR and downstream signaling was evaluated by immunoblotting. Tubule formation was assessed in human umbilical vein endothelial cells (HUVECs) treated with vehicle control (dimethylsulfoxide [DMSO]) or semaxanib (SU5416) as well as in serum-free media (SFM) or JNA conditioned media (CM). Tubule length was compared between treatment groups. Compared to control, AZD4547 inhibited JNA fibroblast proliferation, migration, and invasion through inhibition of FGFR and downstream signaling, specifically phosphorylation of - p44/42 mitogen activated protein kinase (p44/42 MAPK). JNA fibroblast CM significantly increased HUVEC tubule formation (p = 0.0039). AZD4547 effectively mitigates FGFR signaling and decreases JNA fibroblast proliferation, migration, and invasion. SU5416 attenuated JNA fibroblast-induced tubule formation. AZD4547 may have therapeutic potential in the treatment of JNA. © 2017 ARS

  9. Piperlongumine inhibits atherosclerotic plaque formation and vascular smooth muscle cell proliferation by suppressing PDGF receptor signaling

    Energy Technology Data Exchange (ETDEWEB)

    Son, Dong Ju [Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA (United States); Division of Cardiology, Department of Medicine, Emory University, Atlanta, GA (United States); Kim, Soo Yeon [Division of Life Science, Korea Basic Science Institute, Daejeon (Korea, Republic of); Han, Seong Su [University of Iowa Carver College of Medicine, Department of Pathology, Iowa City, IA (United States); Kim, Chan Woo [Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA (United States); Division of Cardiology, Department of Medicine, Emory University, Atlanta, GA (United States); Department of Bioinspired Science, Ehwa Womans University, Seoul (Korea, Republic of); Kumar, Sandeep [Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA (United States); Division of Cardiology, Department of Medicine, Emory University, Atlanta, GA (United States); Park, Byeoung Soo [Nanotoxtech Co., Ansan (Korea, Republic of); Lee, Sung Eun [Division of Applied Biology and Chemistry, Kyungpook National University, Daegu (Korea, Republic of); Yun, Yeo Pyo [College of Pharmacy, Chungbuk National University, Cheongju (Korea, Republic of); Jo, Hanjoong, E-mail: hjo@emory.edu [Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA (United States); Division of Cardiology, Department of Medicine, Emory University, Atlanta, GA (United States); Department of Bioinspired Science, Ehwa Womans University, Seoul (Korea, Republic of); Park, Young Hyun, E-mail: pyh012@sch.ac.kr [Department of Food Science and Nutrition, College of Natural Sciences, Soonchunhyang University, Asan (Korea, Republic of)

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Anti-atherogenic effect of PL was examined using partial carotid ligation model in ApoE KO mice. Black-Right-Pointing-Pointer PL prevented atherosclerotic plaque development, VSMCs proliferation, and NF-{kappa}B activation. Black-Right-Pointing-Pointer Piperlongumine reduced vascular smooth muscle cell activation through PDGF-R{beta} and NF-{kappa}B-signaling. Black-Right-Pointing-Pointer PL may serve as a new therapeutic molecule for atherosclerosis treatment. -- Abstract: Piperlongumine (piplartine, PL) is an alkaloid found in the long pepper (Piper longum L.) and has well-documented anti-platelet aggregation, anti-inflammatory, and anti-cancer properties; however, the role of PL in prevention of atherosclerosis is unknown. We evaluated the anti-atherosclerotic potential of PL in an in vivo murine model of accelerated atherosclerosis and defined its mechanism of action in aortic vascular smooth muscle cells (VSMCs) in vitro. Local treatment with PL significantly reduced atherosclerotic plaque formation as well as proliferation and nuclear factor-kappa B (NF-{kappa}B) activation in an in vivo setting. PL treatment in VSMCs in vitro showed inhibition of migration and platelet-derived growth factor BB (PDGF-BB)-induced proliferation to the in vivo findings. We further identified that PL inhibited PDGF-BB-induced PDGF receptor beta activation and suppressed downstream signaling molecules such as phospholipase C{gamma}1, extracellular signal-regulated kinases 1 and 2 and Akt. Lastly, PL significantly attenuated activation of NF-{kappa}B-a downstream transcriptional regulator in PDGF receptor signaling, in response to PDGF-BB stimulation. In conclusion, our findings demonstrate a novel, therapeutic mechanism by which PL suppresses atherosclerosis plaque formation in vivo.

  10. Soluble Form of Canine Transferrin Receptor Inhibits Canine Parvovirus Infection In Vitro and In Vivo

    Science.gov (United States)

    Wen, Jiexia; Pan, Sumin; Liang, Shuang; Zhong, Zhenyu; He, Ying; Lin, Hongyu; Li, Wenyan; Wang, Liyue; Li, Xiujin; Zhong, Fei

    2013-01-01

    Canine parvovirus (CPV) disease is an acute, highly infectious disease threatening the dog-raising industry. So far there are no effective therapeutic strategies to control this disease. Although the canine transferrin receptor (TfR) was identified as a receptor for CPV infection, whether extracellular domain of TfR (called soluble TfR (sTfR)) possesses anti-CPV activities remains elusive. Here, we used the recombinant sTfR prepared from HEK293T cells with codon-optimized gene structure to investigate its anti-CPV activity both in vitro and in vivo. Our results indicated that codon optimization could significantly improve sTfR expression in HEK293T cells. The prepared recombinant sTfR possessed a binding activity to both CPV and CPV VP2 capsid proteins and significantly inhibited CPV infection of cultured feline F81 cells and decreased the mortality of CPV-infected dogs, which indicates that the sTfR has the anti-CPV activity both in vitro and in vivo. PMID:24089666

  11. Soluble Form of Canine Transferrin Receptor Inhibits Canine Parvovirus Infection In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Jiexia Wen

    2013-01-01

    Full Text Available Canine parvovirus (CPV disease is an acute, highly infectious disease threatening the dog-raising industry. So far there are no effective therapeutic strategies to control this disease. Although the canine transferrin receptor (TfR was identified as a receptor for CPV infection, whether extracellular domain of TfR (called soluble TfR (sTfR possesses anti-CPV activities remains elusive. Here, we used the recombinant sTfR prepared from HEK293T cells with codon-optimized gene structure to investigate its anti-CPV activity both in vitro and in vivo. Our results indicated that codon optimization could significantly improve sTfR expression in HEK293T cells. The prepared recombinant sTfR possessed a binding activity to both CPV and CPV VP2 capsid proteins and significantly inhibited CPV infection of cultured feline F81 cells and decreased the mortality of CPV-infected dogs, which indicates that the sTfR has the anti-CPV activity both in vitro and in vivo.

  12. Blocking oxytocin receptors inhibits vaginal marking to male odors in female Syrian hamsters.

    Science.gov (United States)

    Martinez, Luis A; Albers, H Elliott; Petrulis, Aras

    2010-12-02

    In Syrian hamsters (Mesocricetus auratus), precopulatory behaviors such as vaginal scent marking are essential for attracting a suitable mate. Vaginal marking is dependent on forebrain areas implicated in the neural regulation of reproductive behaviors in rodents, including the medial preoptic/anterior hypothalamus (MPOA-AH). Within MPOA-AH, the neuropeptide oxytocin (OT) acts to facilitate copulation (lordosis), as well as ultrasonic vocalizations towards males. It is not known, however, if OT in this area also facilitates vaginal marking. In the present study, a specific oxytocin receptor antagonist (OTA) was injected into MPOA-AH of intact female Syrian hamsters to determine if oxytocin receptor-dependent signaling is critical for the normal expression of vaginal marking elicited by male, female, and clean odors. OTA injections significantly inhibited vaginal marking in response to male odors compared with vehicle injections. There was no effect of OTA on marking in response to either female or clean odors. When injected into the bed nucleus of the stria terminalis (BNST), a nearby region to MPOA-AH, OTA was equally effective in decreasing marking. Finally, the effects of OTA appear to be specific to vaginal marking, as OTA injections in MPOA-AH or BNST did not alter general locomotor activity, flank marking, or social odor investigation. Considered together, these results suggest that OT in MPOA-AH and/or BNST normally facilitates male odor-induced vaginal marking, providing further evidence that OT generally supports prosocial interactions among conspecifics. Copyright © 2010 Elsevier Inc. All rights reserved.

  13. Structural basis for subtype-specific inhibition of the P2X7 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Karasawa, Akira; Kawate, Toshimitsu (Cornell)

    2016-12-09

    The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.

  14. Desipramine inhibits histamine H1 receptor-induced Ca2+ signaling in rat hypothalamic cells.

    Directory of Open Access Journals (Sweden)

    Ji-Ah Kang

    Full Text Available The hypothalamus in the brain is the main center for appetite control and integrates signals from adipose tissue and the gastrointestinal tract. Antidepressants are known to modulate the activities of hypothalamic neurons and affect food intake, but the cellular and molecular mechanisms by which antidepressants modulate hypothalamic function remain unclear. Here we have investigated how hypothalamic neurons respond to treatment with antidepressants, including desipramine and sibutramine. In primary cultured rat hypothalamic cells, desipramine markedly suppressed the elevation of intracellular Ca(2+ evoked by histamine H1 receptor activation. Desipramine also inhibited the histamine-induced Ca(2+ increase and the expression of corticotrophin-releasing hormone in hypothalamic GT1-1 cells. The effect of desipramine was not affected by pretreatment with prazosin or propranolol, excluding catecholamine reuptake activity of desipramine as an underlying mechanism. Sibutramine which is also an antidepressant but decreases food intake, had little effect on the histamine-induced Ca(2+ increase or AMP-activated protein kinase activity. Our results reveal that desipramine and sibutramine have different effects on histamine H1 receptor signaling in hypothalamic cells and suggest that distinct regulation of hypothalamic histamine signaling might underlie the differential regulation of food intake between antidepressants.

  15. Inhibition of the MEK-1/p42 MAP kinase reduces aryl hydrocarbon receptor-DNA interactions

    International Nuclear Information System (INIS)

    Yim, Sujin; Oh, Myoungsuk; Choi, Su Mi; Park, Hyunsung

    2004-01-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces expression of the cytochrome P450 1A1 gene, cyp1a1, by binding to its receptor, aryl hydrocarbon receptor (AhR). TCDD-bound AhR translocates to the nucleus and forms a heterodimer with its partner protein, AhR nuclear translocator (Arnt). The AhR/Arnt heterodimer then binds to the dioxin-response elements (DREs) in the cyp1a1 enhancer and stimulates transcription of cyp1a1. We tested whether kinase pathways are involved in this process by treating Hepa1c1c7 cells with kinase inhibitors. The MEK-1 inhibitor PD98059 reduced TCDD-induced transcription of cyp1a1. TCDD treatment results in phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK), a substrate of MEK-1. Overexpression of dominant negative form of p42 MAPK suppressed TCDD-dependent transcription of a reporter gene controlled by dioxin-response elements (DREs), and pretreatment with PD98059 also blocked this transcription. PD98059 pretreatment also inhibited TCDD-induced DRE binding of the AhR/Arnt heterodimer. Together these results indicate that TCDD activates the MEK-1/p44/p42 MAPK pathway, which in turn activates AhR and so facilitates binding of AhR to the cyp1a1 DRE

  16. CINPA1 binds directly to constitutive androstane receptor and inhibits its activity.

    Science.gov (United States)

    Cherian, Milu T; Chai, Sergio C; Wright, William C; Singh, Aman; Alexandra Casal, Morgan; Zheng, Jie; Wu, Jing; Lee, Richard E; Griffin, Patrick R; Chen, Taosheng

    2018-03-31

    The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that regulate the expression of drug-metabolizing enzymes and efflux transporters. CAR activation promotes drug elimination, thereby reducing therapeutic effectiveness, or causes adverse drug effects via toxic metabolites. CAR inhibitors could be used to attenuate these adverse drug effects. CAR and PXR share ligands and target genes, confounding the understanding of the regulation of receptor-specific activity. We previously identified a small-molecule inhibitor, CINPA1, that inhibits CAR (without activating PXR at lower concentrations) by altering CAR-coregulator interactions and reducing CAR recruitment to DNA response elements of regulated genes. However, solid evidence was not presented for the direct binding of CINPA1 to CAR. In this study, we demonstrate direct interaction of CINPA1 with the CAR ligand-binding domain (CAR-LBD) and identify key residues involved in such interactions through a combination of biophysical and computational methods. We found that CINPA1 resides in the ligand-binding pocket to stabilize the CAR-LBD in a more rigid, less fluid state. Molecular dynamics simulations, together with our previously reported docking model, enabled us to predict which CAR residues were critical for interactions with CINPA1. The importance of these residues for CINPA1 binding were then validated by directed mutations and testing the mutant CAR proteins in transcription reporter and coregulatory interaction assays. We demonstrated strong hydrogen bonding of CINPA1 with N165 and H203 and identified other residues involved in hydrophobic contacts with CINPA1. Overall, our data confirm that CINPA1 directly binds to CAR. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Haloperidol inhibits the development of atherosclerotic lesions in LDL receptor knockout mice.

    Science.gov (United States)

    van der Sluis, Ronald J; Nahon, Joya E; Reuwer, Anne Q; Van Eck, Miranda; Hoekstra, Menno

    2015-05-01

    Antipsychotic drugs have been shown to modulate the expression of ATP-binding cassette transporter A1 (ABCA1), a key factor in the anti-atherogenic reverse cholesterol transport process, in vitro. Here we evaluated the potential of the typical antipsychotic drug haloperidol to modulate the cholesterol efflux function of macrophages in vitro and their susceptibility to atherosclerosis in vivo. Thioglycollate-elicited peritoneal macrophages were used for in vitro studies. Hyperlipidaemic low-density lipoprotein (LDL) receptor knockout mice were implanted with a haloperidol-containing pellet and subsequently fed a Western-type diet for 5 weeks to induce the development of atherosclerotic lesions in vivo. Haloperidol induced a 54% decrease in the mRNA expression of ABCA1 in peritoneal macrophages. This coincided with a 30% decrease in the capacity of macrophages to efflux cholesterol to apolipoprotein A1. Haloperidol treatment stimulated the expression of ABCA1 (+51%) and other genes involved in reverse cholesterol transport, that is, CYP7A1 (+98%) in livers of LDL receptor knockout mice. No change in splenic ABCA1 expression was noted. However, the average size of the atherosclerotic size was significantly smaller (-31%) in the context of a mildly more atherogenic metabolic phenotype upon haloperidol treatment. More importantly, haloperidol markedly lowered MCP-1 expression (-70%) and secretion (-28%) by peritoneal macrophages. Haloperidol treatment lowered the susceptibility of hyperlipidaemic LDL receptor knockout mice to develop atherosclerotic lesions. Our findings suggest that the beneficial effect of haloperidol on atherosclerosis susceptibility can be attributed to its ability to inhibit macrophage chemotaxis. © 2015 The British Pharmacological Society.

  18. The Ly49E receptor inhibits the immune control of acute Trypanosoma cruzi infection

    Directory of Open Access Journals (Sweden)

    Jessica Filtjens

    2016-11-01

    Full Text Available The protozoan parasite Trypanosoma cruzi (T. cruzi circulates in the blood upon infection and invades a variety of cells. Parasites intensively multiply during the acute phase of infection and persist lifelong at low levels in tissues and blood during the chronic phase. Natural killer (NK and NKT cells play an important role in the immune control of T. cruzi infection, mainly by releasing the cytokine IFN-γ that activates the microbicidal action of macrophages and other cells and shapes a protective type 1 immune response. The mechanisms by which immune cells are regulated to produce IFN-γ during T. cruzi infection are still incompletely understood. Here, we show that urokinase plasminogen activator (uPA is induced early upon T. cruzi infection, and remains elevated until day 20 post inoculation. We previously demonstrated that the inhibitory receptor Ly49E, which is expressed, among others, on NK and NKT cells, is triggered by uPA. Therefore, we compared wild type (WT to Ly49E knockout (KO mice for their control of experimental T. cruzi infection. Our results show that young, i.e. 4- and 6-week-old, Ly49E KO mice control the infection better than WT mice, indicated by a lower parasite load and less cachexia. The beneficial effect of Ly49E depletion is more obvious in 4-week-old male than in female mice and weakens in 8-week-old mice. In young mice, the lower T. cruzi parasitemia in Ly49E KO mice is paralleled by higher IFN-γ production compared to their WT controls. Our data indicate that Ly49E receptor expression inhibits the immune control of T. cruzi infection. This is the first demonstration that the inhibitory Ly49E receptor can interfere with the immune response to a pathogen in vivo.

  19. An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum

    International Nuclear Information System (INIS)

    Basu, Niladri; Stamler, Christopher J.; Loua, Kovana Marcel; Chan, H.M.

    2005-01-01

    Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl 2 ) and methyl Hg (MeHg) can inhibit the binding of radioligands to the muscarinic acetylcholine (mACh) receptor in rat brains. However, little is known concerning this relationship in specific anatomical regions of the brain or in other species, including humans. The purpose of this study was to explore the inhibitory effects of HgCl 2 and MeHg on [ 3 H]-quinuclidinyl benzilate ([ 3 H]-QNB) binding to the mACh receptor in the cerebellum and cerebral cortex regions from human, rat, mouse, mink, and river otter brain tissues. Saturation binding curves were obtained from each sample to calculate receptor density (B max ) and ligand affinity (K d ). Subsequently, samples were exposed to HgCl 2 or MeHg to derive IC50 values and inhibition constants (K i ). Results demonstrate that HgCl 2 is a more potent inhibitor of mACh receptor binding than MeHg, and the receptors in the cerebellum are more sensitive to Hg-mediated mACh receptor inhibition than those in the cerebral cortex. Species sensitivities, irrespective of Hg type and brain region, can be ranked from most to least sensitive: river otter > rat > mink > mouse > humans. In summary, our data demonstrate that Hg can inhibit the binding [ 3 H]-QNB to the mACh receptor in a range of mammalian species. This comparative study provides data on interspecies differences and a framework for interpreting results from human, murine, and wildlife studies

  20. Adenosine A₂A receptors inhibit delayed rectifier potassium currents and cell differentiation in primary purified oligodendrocyte cultures.

    Science.gov (United States)

    Coppi, Elisabetta; Cellai, Lucrezia; Maraula, Giovanna; Pugliese, Anna Maria; Pedata, Felicita

    2013-10-01

    Oligodendrocyte progenitor cells (OPCs) are a population of cycling cells which persist in the adult central nervous system (CNS) where, under opportune stimuli, they differentiate into mature myelinating oligodendrocytes. Adenosine A(2A) receptors are Gs-coupled P1 purinergic receptors which are widely distributed throughout the CNS. It has been demonstrated that OPCs express A(2A) receptors, but their functional role in these cells remains elusive. Oligodendrocytes express distinct voltage-gated ion channels depending on their maturation. Here, by electrophysiological recordings coupled with immunocytochemical labeling, we studied the effects of adenosine A(2A) receptors on membrane currents and differentiation of purified primary OPCs isolated from the rat cortex. We found that the selective A(2A) agonist, CGS21680, inhibits sustained, delayed rectifier, K(+) currents (I(K)) without modifying transient (I(A)) conductances. The effect was observed in all cells tested, independently from time in culture. CGS21680 inhibition of I(K) current was concentration-dependent (10-200 nM) and blocked in the presence of the selective A(2A) antagonist SCH58261 (100 nM). It is known that I(K) currents play an important role during OPC development since their block decreases cell proliferation and differentiation. In light of these data, our further aim was to investigate whether A(2A) receptors modulate these processes. CGS21680, applied at 100 nM in the culture medium of oligodendrocyte cultures, inhibits OPC differentiation (an effect prevented by SCH58261) without affecting cell proliferation. Data demonstrate that cultured OPCs express functional A(2A) receptors whose activation negatively modulate I(K) currents. We propose that, by this mechanism, A(2A) adenosine receptors inhibit OPC differentiation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Midazolam inhibits chondrogenesis via peripheral benzodiazepine receptor in human mesenchymal stem cells.

    Science.gov (United States)

    Chen, Yung-Ching; Wu, King-Chuen; Huang, Bu-Miin; So, Edmund Cheung; Wang, Yang-Kao

    2018-05-01

    Midazolam, a benzodiazepine derivative, is widely used for sedation and surgery. However, previous studies have demonstrated that Midazolam is associated with increased risks of congenital malformations, such as dwarfism, when used during early pregnancy. Recent studies have also demonstrated that Midazolam suppresses osteogenesis of mesenchymal stem cells (MSCs). Given that hypertrophic chondrocytes can differentiate into osteoblast and osteocytes and contribute to endochondral bone formation, the effect of Midazolam on chondrogenesis remains unclear. In this study, we applied a human MSC line, the KP cell, to serve as an in vitro model to study the effect of Midazolam on chondrogenesis. We first successfully established an in vitro chondrogenic model in a micromass culture or a 2D high-density culture performed with TGF-β-driven chondrogenic induction medium. Treatment of the Midazolam dose-dependently inhibited chondrogenesis, examined using Alcian blue-stained glycosaminoglycans and the expression of chondrogenic markers, such as SOX9 and type II collagen. Inhibition of Midazolam by peripheral benzodiazepine receptor (PBR) antagonist PK11195 or small interfering RNA rescued the inhibitory effects of Midazolam on chondrogenesis. In addition, Midazolam suppressed transforming growth factor-β-induced Smad3 phosphorylation, and this inhibitory effect could be rescued using PBR antagonist PK11195. This study provides a possible explanation for Midazolam-induced congenital malformations of the musculoskeletal system through PBR. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  2. Emerging role of epidermal growth factor receptor inhibition in therapy for advanced malignancy: focus on NSCLC

    International Nuclear Information System (INIS)

    Langer, Corey J.

    2004-01-01

    Combination chemotherapy regimens have emerged as the standard approach in advanced non-small-cell lung cancer. Meta-analyses have demonstrated a 2-month increase in median survival after platinum-based therapy vs. best supportive care, and an absolute 10% improvement in the 1-year survival rate. Just as importantly, cytotoxic therapy has produced benefits in symptom control and quality of life. Newer agents, including the taxanes, vinorelbine, gemcitabine, and irinotecan, have expanded our therapeutic options in the treatment of advanced non-small-cell lung cancer. Despite their contributions, we have reached a therapeutic plateau, with response rates seldom exceeding 30-40% in cooperative group studies and 1-year survival rates stable between 30% and 40%. It is doubtful that substituting one agent for another in various combinations will lead to any further improvement in these rates. The thrust of current research has focused on targeted therapy, and epidermal growth factor receptor inhibition is one of the most promising clinical strategies. Epidermal growth factor receptor inhibitors currently under investigation include the small molecules gefitinib (Iressa, ZD1839) and erlotinib (Tarceva, OSI-774), as well as monoclonal antibodies such as cetuximab (IMC-225, Erbitux). Agents that have only begun to undergo clinical evaluation include CI-1033, an irreversible pan-erbB tyrosine kinase inhibitor, and PKI166 and GW572016, both examples of dual kinase inhibitors (inhibiting epidermal growth factor receptor and Her2). Preclinical models have demonstrated synergy for all these agents in combination with either chemotherapy or radiotherapy, leading to great enthusiasm regarding their ultimate contribution to lung cancer therapy. However, serious clinical challenges persist. These include the identification of the optimal dose(s); the proper integration of these agents into popular, established cytotoxic regimens; and the selection of the optimal setting(s) in which

  3. Valerian inhibits rat hepatocarcinogenesis by activating GABA(A receptor-mediated signaling.

    Directory of Open Access Journals (Sweden)

    Anna Kakehashi

    Full Text Available Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA A receptor (GABA(AR system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(AR agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN. Formation of glutathione S-transferase placental form positive (GST-P(+ foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2'-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P(+ foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21(Waf1/Cip1, p53 and Bax mRNA expression. Interestingly, expression of the GABA(AR alpha 1 subunit was observed in GST-P(+ foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P(+ foci by activating GABA(AR-mediated signaling.

  4. Effects of epidermal growth factor receptor kinase inhibition on radiation response in canine osteosarcoma cells.

    Science.gov (United States)

    Mantovani, Fernanda B; Morrison, Jodi A; Mutsaers, Anthony J

    2016-05-31

    Radiation therapy is a palliative treatment modality for canine osteosarcoma, with transient improvement in analgesia observed in many cases. However there is room for improvement in outcome for these patients. It is possible that the addition of sensitizing agents may increase tumor response to radiation therapy and prolong quality of life. Epidermal growth factor receptor (EGFR) expression has been documented in canine osteosarcoma and higher EGFR levels have been correlated to a worse prognosis. However, effects of EGFR inhibition on radiation responsiveness in canine osteosarcoma have not been previously characterized. This study examined the effects of the small molecule EGFR inhibitor erlotinib on canine osteosarcoma radiation responses, target and downstream protein expression in vitro. Additionally, to assess the potential impact of treatment on tumor angiogenesis, vascular endothelial growth factor (VEGF) levels in conditioned media were measured. Erlotinib as a single agent reduced clonogenic survival in two canine osteosarcoma cell lines and enhanced the impact of radiation in one out of three cell lines investigated. In cell viability assays, erlotinib enhanced radiation effects and demonstrated single agent effects. Erlotinib did not alter total levels of EGFR, nor inhibit downstream protein kinase B (PKB/Akt) activation. On the contrary, erlotinib treatment increased phosphorylated Akt in these osteosarcoma cell lines. VEGF levels in conditioned media increased after erlotinib treatment as a single agent and in combination with radiation in two out of three cell lines investigated. However, VEGF levels decreased with erlotinib treatment in the third cell line. Erlotinib treatment promoted modest enhancement of radiation effects in canine osteosarcoma cells, and possessed activity as a single agent in some cell lines, indicating a potential role for EGFR inhibition in the treatment of a subset of osteosarcoma patients. The relative radioresistance of

  5. Enzalutamide inhibits proliferation of gemcitabine-resistant bladder cancer cells with increased androgen receptor expression.

    Science.gov (United States)

    Kameyama, Koji; Horie, Kengo; Mizutani, Kosuke; Kato, Taku; Fujita, Yasunori; Kawakami, Kyojiro; Kojima, Toshio; Miyazaki, Tatsuhiko; Deguchi, Takashi; Ito, Masafumi

    2017-01-01

    Advanced bladder cancer is treated mainly with gemcitabine and cisplatin, but most patients eventually become resistance. Androgen receptor (AR) signaling has been implicated in bladder cancer as well as other types of cancer including prostate cancer. In this study, we investigated the expression and role of AR in gemcitabine-resistant bladder cancer cells and also the potential of enzalutamide, an AR inhibitor, as a therapeutic for the chemoresistance. First of all, we established gemcitabine-resistant T24 cells (T24GR) from T24 bladder cancer cells and performed gene expression profiling. Microarray analysis revealed upregulation of AR expression in T24GR cells compared with T24 cells. AR mRNA and protein expression was confirmed to be increased in T24GR cells, respectively, by quantitative RT-PCR and western blot analysis, which was associated with more potent AR transcriptional activity as measured by luciferase reporter assay. The copy number of AR gene in T24GR cells determined by PCR was twice as many as that of T24 cells. AR silencing by siRNA transfection resulted in inhibition of proliferation of T24GR cells. Cell culture in charcoal-stripped serum and treatment with enzalutamide inhibited growth of T24GR cells, which was accompanied by cell cycle arrest. AR transcriptional activity was found to be reduced in T24GR cells by enzalutamide treatment. Lastly, enzalutamide also inhibited cell proliferation of HTB5 bladder cancer cells that express AR and possess intrinsic resistance to gemcitabine. Our results suggest that enzalutamide may have the potential to treat patients with advanced gemcitabine-resistant bladder cancer with increased AR expression.

  6. Estrogen receptor beta signaling inhibits PDGF induced human airway smooth muscle proliferation.

    Science.gov (United States)

    Ambhore, Nilesh Sudhakar; Katragadda, Rathnavali; Raju Kalidhindi, Rama Satyanarayana; Thompson, Michael A; Pabelick, Christina M; Prakash, Y S; Sathish, Venkatachalem

    2018-04-20

    Airway smooth muscle (ASM) cell hyperplasia driven by persistent inflammation is a hallmark feature of remodeling in asthma. Sex steroid signaling in the lungs is of considerable interest, given epidemiological data showing more asthma in pre-menopausal women and aging men. Our previous studies demonstrated that estrogen receptor (ER) expression increases in asthmatic human ASM; however, very limited data are available regarding differential roles of ERα vs. ERβ isoforms in human ASM cell proliferation. In this study, we evaluated the effect of selective ERα and ERβ modulators on platelet-derived growth factor (PDGF)-stimulated ASM proliferation and the mechanisms involved. Asthmatic and non-asthmatic primary human ASM cells were treated with PDGF, 17β-estradiol, ERα-agonist and/or ERβ-agonist and/or G-protein-coupled estrogen receptor 30 (GPR30/GPER) agonist and proliferation was measured using MTT and CyQuant assays followed by cell cycle analysis. Transfection of small interfering RNA (siRNA) ERα and ERβ significantly altered the human ASM proliferation. The specificity of siRNA transfection was confirmed by Western blot analysis. Gene and protein expression of cell cycle-related antigens (PCNA and Ki67) and C/EBP were measured by RT-PCR and Western analysis, along with cell signaling proteins. PDGF significantly increased ASM proliferation in non-asthmatic and asthmatic cells. Treatment with PPT showed no significant effect on PDGF-induced proliferation, whereas WAY interestingly suppressed proliferation via inhibition of ERK1/2, Akt, and p38 signaling. PDGF-induced gene expression of PCNA, Ki67 and C/EBP in human ASM was significantly lower in cells pre-treated with WAY. Furthermore, WAY also inhibited PDGF-activated PCNA, C/EBP, cyclin-D1, and cyclin-E. Overall, we demonstrate ER isoform-specific signaling in the context of ASM proliferation. Activation of ERβ can diminish remodeling in human ASM by inhibiting pro-proliferative signaling pathways

  7. β-Adrenergic receptor stimulation inhibits proarrhythmic alternans in postinfarction border zone cardiomyocytes: a computational analysis.

    Science.gov (United States)

    Tomek, Jakub; Rodriguez, Blanca; Bub, Gil; Heijman, Jordi

    2017-08-01

    The border zone (BZ) of the viable myocardium adjacent to an infarct undergoes extensive autonomic and electrical remodeling and is prone to repolarization alternans-induced cardiac arrhythmias. BZ remodeling processes may promote or inhibit Ca 2+ and/or repolarization alternans and may differentially affect ventricular arrhythmogenesis. Here, we used a detailed computational model of the canine ventricular cardiomyocyte to study the determinants of alternans in the BZ and their regulation by β-adrenergic receptor (β-AR) stimulation. The BZ model developed Ca 2+ transient alternans at slower pacing cycle lengths than the control model, suggesting that the BZ may promote spatially heterogeneous alternans formation in an infarcted heart. β-AR stimulation abolished alternans. By evaluating all combinations of downstream β-AR stimulation targets, we identified both direct (via ryanodine receptor channels) and indirect [via sarcoplasmic reticulum (SR) Ca 2+ load] modulation of SR Ca 2+ release as critical determinants of Ca 2+ transient alternans. These findings were confirmed in a human ventricular cardiomyocyte model. Cell-to-cell coupling indirectly modulated the likelihood of alternans by affecting the action potential upstroke, reducing the trigger for SR Ca 2+ release in one-dimensional strand simulations. However, β-AR stimulation inhibited alternans in both single and multicellular simulations. Taken together, these data highlight a potential antiarrhythmic role of sympathetic hyperinnervation in the BZ by reducing the likelihood of alternans and provide new insights into the underlying mechanisms controlling Ca 2+ transient and repolarization alternans. NEW & NOTEWORTHY We integrated, for the first time, postmyocardial infarction electrical and autonomic remodeling in a detailed, validated computer model of β-adrenergic stimulation in ventricular cardiomyocytes. Here, we show that β-adrenergic stimulation inhibits alternans and provide novel insights

  8. Lipids Derived from Virulent Francisella tularensis Broadly Inhibit Pulmonary Inflammation via Toll-Like Receptor 2 and Peroxisome Proliferator-Activated Receptor α

    Science.gov (United States)

    Crane, Deborah D.; Ireland, Robin; Alinger, Joshua B.; Small, Pamela

    2013-01-01

    Francisella tularensis is a Gram-negative facultative intracellular pathogen that causes an acute lethal respiratory disease in humans. The heightened virulence of the pathogen is linked to its unique ability to inhibit Toll-like receptor (TLR)-mediated inflammatory responses. The bacterial component and mechanism of this inhibition are unknown. Here we show that lipids isolated from virulent but not attenuated strains of F. tularensis are not detected by host cells, inhibit production of proinflammatory cytokines by primary macrophages in response to known TLR ligands, and suppress neutrophil recruitment in vivo. We further show that lipid-mediated inhibition of inflammation is dependent on TLR2, MyD88, and the nuclear hormone and fatty acid receptor peroxisome proliferator-activated receptor α (PPARα). Pathogen lipid-mediated interference with inflammatory responses through the engagement of TLR2 and PPARα represents a novel manipulation of host signaling pathways consistent with the ability of highly virulent F. tularensis to efficiently evade host immune responses. PMID:23925884

  9. Inhibition of cerebrovascular raf activation attenuates cerebral blood flow and prevents upregulation of contractile receptors after subarachnoid hemorrhage

    DEFF Research Database (Denmark)

    Ansar, Saema; Maddahi, Aida; Edvinsson, Lars

    2011-01-01

    of mitogen-activated protein kinase (MAPK) of the extracellular signal-regulated kinase (ERK)1/2 signal pathway. We hypothesize that SAH initiates cerebrovascular ERK1/2 activation, resulting in receptor upregulation. The raf inhibitor will inhibit the molecular events upstream ERK1/2 and may provide...

  10. Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4.

    Science.gov (United States)

    Schuette, Verena; Embgenbroich, Maria; Ulas, Thomas; Welz, Meike; Schulte-Schrepping, Jonas; Draffehn, Astrid M; Quast, Thomas; Koch, Katharina; Nehring, Melanie; König, Jessica; Zweynert, Annegret; Harms, Frederike L; Steiner, Nancy; Limmer, Andreas; Förster, Irmgard; Berberich-Siebelt, Friederike; Knolle, Percy A; Wohlleber, Dirk; Kolanus, Waldemar; Beyer, Marc; Schultze, Joachim L; Burgdorf, Sven

    2016-09-20

    The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8(+) T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality.

  11. Acutely increasing δGABA(A) receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus.

    Science.gov (United States)

    Whissell, Paul D; Eng, Dave; Lecker, Irene; Martin, Loren J; Wang, Dian-Shi; Orser, Beverley A

    2013-01-01

    Extrasynaptic γ-aminobutyric acid type A (GABA(A)) receptors that contain the δ subunit (δGABA(A) receptors) are expressed in several brain regions including the dentate gyrus (DG) and CA1 subfields of the hippocampus. Drugs that increase δGABA(A) receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABA(A) receptor-preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) enhances discrimination memory and increases neurogenesis in the DG. Despite the potential therapeutic benefits of such treatments, the effects of acutely increasing δGABA(A) receptor activity on memory behaviors remain unknown. Here, we studied the effects of THIP (4 mg/kg, i.p.) on memory performance in wild-type (WT) and δGABA(A) receptor null mutant (Gabrd(-/-)) mice. Additionally, the effects of THIP on long-term potentiation (LTP), a molecular correlate of memory, were studied within the DG and CA1 subfields of the hippocampus using electrophysiological recordings of field potentials in hippocampal slices. The results showed that THIP impaired performance in the Morris water maze, contextual fear conditioning and object recognition tasks in WT mice but not Gabrd(-/-) mice. Furthermore, THIP inhibited LTP in hippocampal slices from WT but not Gabrd(-/-) mice, an effect that was blocked by GABA(A) receptor antagonist bicuculline. Thus, acutely increasing δGABA(A) receptor activity impairs memory behaviors and inhibits synaptic plasticity. These results have important implications for the development of therapies aimed at increasing δGABA(A) receptor activity.

  12. Context-dependent modulation of alphabetagamma and alphabetadelta GABA A receptors by penicillin: implications for phasic and tonic inhibition.

    Science.gov (United States)

    Feng, Hua-Jun; Botzolakis, Emmanuel J; Macdonald, Robert L

    2009-01-01

    Penicillin, an open-channel blocker of GABA(A) receptors, was recently reported to inhibit phasic, but not tonic, currents in hippocampal neurons. To distinguish between isoform-specific and context-dependent modulation as possible explanations for this selectivity, the effects of penicillin were evaluated on recombinant GABA(A) receptors expressed in HEK293T cells. When co-applied with saturating GABA, penicillin decreased peak amplitude, induced rebound, and prolonged deactivation of currents evoked from both synaptic and extrasynaptic receptor isoforms. However, penicillin had isoform-specific effects on the extent of desensitization, reflecting its ability to differentially modulate peak (non-equilibrium) and residual (near-equilibrium) currents. This suggested that the context of activation could determine the apparent sensitivity of a given receptor isoform to penicillin. To test this hypothesis, we explored the ability of penicillin to modulate synaptic and extrasynaptic isoform currents that were activated under more physiologically relevant conditions. Interestingly, while currents evoked from synaptic isoforms under phasic conditions (transient activation by a saturating concentration of GABA) were substantially inhibited by penicillin, currents evoked from extrasynaptic isoforms under tonic conditions (prolonged application by a sub-saturating concentration of GABA) were minimally affected. We therefore concluded that the reported inability of penicillin to modulate tonic currents could not simply be attributed to insensitivity of extrasynaptic receptors, but rather, reflected an inability to modulate these receptors in their native context of activation.

  13. EP3 receptors inhibit antidiuretic-hormone-dependent sodium transport across frog skin epithelium

    DEFF Research Database (Denmark)

    Rytved, Klaus A.; Nielsen, Robert

    1999-01-01

    Antidiuretic hormone; tight epithelium; prostaglandin receptors; sulprostone; misoprostol; cAMP; cellular Ca2+......Antidiuretic hormone; tight epithelium; prostaglandin receptors; sulprostone; misoprostol; cAMP; cellular Ca2+...

  14. Fasitibant chloride, a kinin B2 receptor antagonist, and dexamethasone interact to inhibit carrageenan-induced inflammatory arthritis in rats

    Science.gov (United States)

    Valenti, Claudio; Giuliani, Sandro; Cialdai, Cecilia; Tramontana, Manuela; Maggi, Carlo Alberto

    2012-01-01

    BACKGROUND AND PURPOSE Bradykinin, through the kinin B2 receptor, is involved in inflammatory processes related to arthropathies. B2 receptor antagonists inhibited carrageenan-induced arthritis in rats in synergy with anti-inflammatory steroids. The mechanism(s) underlying this drug interaction was investigated. EXPERIMENTAL APPROACH Drugs inhibiting inflammatory mediators released by carrageenan were injected, alone or in combination, into the knee joint of pentobarbital anaesthetized rats 30 min before intra-articular administration of carrageenan. Their effects on the carrageenan-induced inflammatory responses (joint pain, oedema and neutrophil recruitment) and release of inflammatory mediators (prostaglandins, IL-1β, IL-6 and the chemokine GRO/CINC-1), were assessed after 6 h. KEY RESULTS The combination of fasitibant chloride (MEN16132) and dexamethasone was more effective than each drug administered alone in inhibiting knee joint inflammation and release of inflammatory mediators. Fasitibant chloride, MK571, atenolol, des-Arg9-[Leu8]-bradykinin (B2 receptor, leukotriene, catecholamine and B1 receptor antagonists, respectively) and dexketoprofen (COX inhibitor), reduced joint pain and, except for the latter, also diminished joint oedema. A combination of drugs inhibiting joint pain (fasitibant chloride, des-Arg9-[Leu8]-bradykinin, dexketoprofen, MK571 and atenolol) and oedema (fasitibant chloride, des-Arg9-[Leu8]-bradykinin, MK571 and atenolol) abolished the respective inflammatory response, producing inhibition comparable with that achieved with the combination of fasitibant chloride and dexamethasone. MK571 alone was able to block neutrophil recruitment. CONCLUSIONS AND IMPLICATIONS Bradykinin-mediated inflammatory responses to intra-articular carrageenan were not controlled by steroids, which were not capable of preventing bradykinin effects either by direct activation of the B2 receptor, or through the indirect effects mediated by release of eicosanoids

  15. Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides

    International Nuclear Information System (INIS)

    Sluss, P.M.; Krystek, S.R. Jr.; Andersen, T.T.; Melson, B.E.; Huston, J.S.; Ridge, R.; Reichert, L.E. Jr.

    1986-01-01

    Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of 125 I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of 125 I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of 125 I-labeled human FSH to testis receptor

  16. Hyperosmotic stress inhibits insulin receptor substrate-1 function by distinct mechanisms in 3T3-L1 adipocytes

    DEFF Research Database (Denmark)

    Gual, Philippe; Gonzalez, Teresa; Grémeaux, Thierry

    2003-01-01

    . Furthermore, the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented the osmotic shock-induced phosphorylation of IRS-1 on Ser307. The inhibition of mTOR completely reversed the inhibitory effect of hyperosmotic stress on insulin-induced IRS-1 tyrosine phosphorylation and PI 3-kinase activation......In 3T3-L1 adipocytes, hyperosmotic stress was found to inhibit insulin signaling, leading to an insulin-resistant state. We show here that, despite normal activation of insulin receptor, hyperosmotic stress inhibits both tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-1....... In addition, prolonged osmotic stress enhanced the degradation of IRS proteins through a rapamycin-insensitive pathway and a proteasome-independent process. These data support evidence of new mechanisms involved in osmotic stress-induced cellular insulin resistance. Short-term osmotic stress induces...

  17. Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of AMPA receptor signaling

    Science.gov (United States)

    Beurel, Eléonore; Grieco, Steven F; Amadei, Celeste; Downey, Kimberlee; Jope, Richard S

    2016-01-01

    Objectives Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. Methods In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Results Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. Conclusions These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment. PMID:27687706

  18. PTP1B Inhibition Causes Rac1 Activation by Enhancing Receptor Tyrosine Kinase Signaling

    Directory of Open Access Journals (Sweden)

    Ayako Tsuchiya

    2014-04-01

    Full Text Available Background/Aims: The present study investigated the signaling pathway underlying Rac1 activation induced by the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl-cyclopropyl]-octanoic acid (DCP-LA. Methods: Activity of protein tyrosine phosphatase 1B (PTP1B was assayed under cell-free conditions. Western blot was carried out to quantify phosphorylation of insulin receptor substrate-1 (IRS-1 and Akt in PC-12 cells. Rac1 activity was monitored in the föerster resonance energy transfer (FRET analysis using living and fixed PC-12 cells. Results: DCP-LA markedly suppressed PTP1B activity in a concentration (100 pM-100 µM-dependent manner. In the DCP-LA binding assay, fluorescein-conjugated DCP-LA produced a single fluorescent signal band at 60 kDa, corresponding to the molecule of PTP1B, and the signal was attenuated or abolished by co-treatment or pretreatment with non-conjugated DCP-LA. DCP-LA significantly enhanced nerve growth factor (NGF-stimulated phosphorylation of IRS-1 at Tyr1222 and Akt1/2 at Thr308/309 and Ser473/474 in PC-12 cells. In the FRET analysis, DCP-LA significantly enhanced NGF-stimulated Rac1 activation, which is abrogated by the phosphatidylinositol 3 kinase (PI3K inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase-1 (PDK1 inhibitor BX912, or the Akt inhibitor MK2206. Conclusion: The results of the present study show that DCP-LA-induced PTP1B inhibition, possibly through its direct binding, causes Rac1 activation by enhancing a pathway along a receptor tyrosine kinase (RTK/IRS-1/PI3K/Akt/Rac1 axis.

  19. Targeting non-small cell lung cancer cells by dual inhibition of the insulin receptor and the insulin-like growth factor-1 receptor.

    Directory of Open Access Journals (Sweden)

    Emma E Vincent

    Full Text Available Phase III trials of the anti-insulin-like growth factor-1 receptor (IGF1R antibody figitumumab in non-small cell lung cancer (NSCLC patients have been discontinued owing to lack of survival benefit. We investigated whether inhibition of the highly homologous insulin receptor (IR in addition to the IGF1R would be more effective than inhibition of the IGF1R alone at preventing the proliferation of NSCLC cells. Signalling through IGF1R and IR in the NSCLC cell lines A549 and Hcc193 was stimulated by a combination of IGF1, IGF2 and insulin. It was inhibited by antibodies that block ligand binding, αIR3 (IGF1R and IR47-9 (IR, and by the ATP-competitive small molecule tyrosine kinase inhibitors AZ12253801 and NVPAWD742 which inhibit both IGF1R and IR tyrosine kinases. The effect of inhibitors was determined by an anchorage-independent proliferation assay and by analysis of Akt phosphorylation. In Hcc193 cells the reduction in cell proliferation and Akt phosphorylation due to anti-IGF1R antibody was enhanced by antibody-mediated inhibition of the IR whereas in A549 cells, with a relatively low IR:IGF1R expression ratio, it was not. In each cell line proliferation and Akt phosphorylation were more effectively inhibited by AZ12253801 and NVPAWD742 than by combined αIR3 and IR47-9. When the IGF1R alone is inhibited, unencumbered signalling through the IR can contribute to continued NSCLC cell proliferation. We conclude that small molecule inhibitors targeting both the IR and IGF1R more effectively reduce NSCLC cell proliferation in a manner independent of the IR:IGF1R expression ratio, providing a therapeutic rationale for the treatment of this disease.

  20. Dopamine inhibits reproduction in female zebrafish (Danio rerio) via three pituitary D2 receptor subtypes.

    Science.gov (United States)

    Fontaine, Romain; Affaticati, Pierre; Yamamoto, Kei; Jolly, Cécile; Bureau, Charlotte; Baloche, Sylvie; Gonnet, Françoise; Vernier, Philippe; Dufour, Sylvie; Pasqualini, Catherine

    2013-02-01

    In many teleosts, the stimulatory control of gonadotrope axis by GnRH is opposed by an inhibitory control by dopamine (DA). The functional importance of this inhibitory pathway differs widely from one teleostean species to another. The zebrafish (Danio rerio) is a teleost fish that has become increasingly popular as an experimental vertebrate model. However, the role of DA in the neuroendocrine control of its reproduction has never been studied. Here the authors evaluated in sexually regressed female zebrafish the effects of in vivo treatments with a DA D2 receptor (D2-R) antagonist domperidone, or a GnRH agonist, alone and in combination, on the pituitary level of FSHβ and LHβ transcripts, the gonadosomatic index, and the ovarian histology. Only the double treatment with GnRH agonist and domperidone could induce an increase in the expression of LHβ, in the gonadosomatic index, and a stimulation of ovarian vitellogenesis, indicating that removal of dopaminergic inhibition is required for the stimulatory action of GnRH and reactivation of ovarian function to occur. Using double immunofluorescent staining on pituitary, the authors showed in this species the innervation of LH cells by tyrosine-hydroxylase immunoreactive fibers. Finally, using in situ hybridization and immunofluorescence, the authors showed that the three subtypes of zebrafish DA D2-R (D2a, D2b, and D2c) were expressed in LH-producing cells, suggesting that they all may be involved in mediating this inhibition. These results show for the first time that, in zebrafish, DA has a direct and potent inhibitory action capable of opposing the stimulatory effect of GnRH in the neuroendocrine control of reproduction.

  1. Nifedipine inhibits advanced glycation end products (AGEs) and their receptor (RAGE) interaction-mediated proximal tubular cell injury via peroxisome proliferator-activated receptor-gamma activation

    International Nuclear Information System (INIS)

    Matsui, Takanori; Yamagishi, Sho-ichi; Takeuchi, Masayoshi; Ueda, Seiji; Fukami, Kei; Okuda, Seiya

    2010-01-01

    Research highlights: → Nifedipine inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by GW9662, an inhibitor of peroxisome proliferator-activated receptor-γ. → GW9662 treatment alone increased RAGE mRNA levels in tubular cells. → Nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-κB activation and increases in intercellular adhesion molecule-1 and transforming growth factor-β gene expression in tubular cells, all of which were blocked by GW9662. -- Abstract: There is a growing body of evidence that advanced glycation end products (AGEs) and their receptor (RAGE) interaction evokes oxidative stress generation and subsequently elicits inflammatory and fibrogenic reactions, thereby contributing to the development and progression of diabetic nephropathy. We have previously found that nifedipine, a calcium-channel blocker (CCB), inhibits the AGE-induced mesangial cell damage in vitro. However, effects of nifedipine on proximal tubular cell injury remain unknown. We examined here whether and how nifedipine blocked the AGE-induced tubular cell damage. Nifedipine, but not amlodipine, a control CCB, inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-γ (PPARγ). GW9662 treatment alone was found to increase RAGE mRNA levels in tubular cells. Further, nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-κB activation and increases in intercellular adhesion molecule-1 and transforming growth factor-beta gene expression in tubular cells, all of which were blocked by GW9662. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-oxidative and anti-inflammatory agent against AGEs in tubular cells by suppressing RAGE expression via PPARγ activation.

  2. Nifedipine inhibits advanced glycation end products (AGEs) and their receptor (RAGE) interaction-mediated proximal tubular cell injury via peroxisome proliferator-activated receptor-gamma activation

    Energy Technology Data Exchange (ETDEWEB)

    Matsui, Takanori [Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, Kurume (Japan); Yamagishi, Sho-ichi, E-mail: shoichi@med.kurume-u.ac.jp [Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, Kurume (Japan); Takeuchi, Masayoshi [Department of Pathophysiological Science, Faculty of Pharmaceutical Science, Hokuriku University, Kanazawa (Japan); Ueda, Seiji; Fukami, Kei; Okuda, Seiya [Department of Medicine, Kurume University School of Medicine, Kurume (Japan)

    2010-07-23

    Research highlights: {yields} Nifedipine inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma}. {yields} GW9662 treatment alone increased RAGE mRNA levels in tubular cells. {yields} Nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-{beta} gene expression in tubular cells, all of which were blocked by GW9662. -- Abstract: There is a growing body of evidence that advanced glycation end products (AGEs) and their receptor (RAGE) interaction evokes oxidative stress generation and subsequently elicits inflammatory and fibrogenic reactions, thereby contributing to the development and progression of diabetic nephropathy. We have previously found that nifedipine, a calcium-channel blocker (CCB), inhibits the AGE-induced mesangial cell damage in vitro. However, effects of nifedipine on proximal tubular cell injury remain unknown. We examined here whether and how nifedipine blocked the AGE-induced tubular cell damage. Nifedipine, but not amlodipine, a control CCB, inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}). GW9662 treatment alone was found to increase RAGE mRNA levels in tubular cells. Further, nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-beta gene expression in tubular cells, all of which were blocked by GW9662. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-oxidative and anti-inflammatory agent against AGEs in tubular cells by suppressing RAGE expression

  3. Plant lectin can target receptors containing sialic acid, exemplified by podoplanin, to inhibit transformed cell growth and migration.

    Directory of Open Access Journals (Sweden)

    Jhon Alberto Ochoa-Alvarez

    Full Text Available Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth.

  4. Midazolam inhibits hippocampal long-term potentiation and learning through dual central and peripheral benzodiazepine receptor activation and neurosteroidogenesis.

    Science.gov (United States)

    Tokuda, Kazuhiro; O'Dell, Kazuko A; Izumi, Yukitoshi; Zorumski, Charles F

    2010-12-15

    Benzodiazepines (BDZs) enhance GABA(A) receptor inhibition by direct actions on central BDZ receptors (CBRs). Although some BDZs also bind mitochondrial receptors [translocator protein (18 kDa) (TSPO)] and promote the synthesis of GABA-enhancing neurosteroids, the role of neurosteroids in the clinical effects of BDZs is unknown. In rat hippocampal slices, we compared midazolam, an anesthetic BDZ, with clonazepam, an anticonvulsant/anxiolytic BDZ that activates CBRs selectively. Midazolam, but not clonazepam, increased neurosteroid levels in CA1 pyramidal neurons without changing TSPO immunostaining. Midazolam, but not clonazepam, also augmented a form of spike inhibition after stimulation adjacent to the pyramidal cell layer and inhibited induction of long-term potentiation. These effects were prevented by finasteride, an inhibitor of neurosteroid synthesis, or 17PA [17-phenyl-(3α,5α)-androst-16-en-3-ol], a blocker of neurosteroid effects on GABA(A) receptors. Moreover, the synaptic effects were mimicked by a combination of clonazepam with FGIN (2-[2-(4-fluorophenyl)-1H-indol-3-yl]-N,N-dihexylacetamide), a selective TSPO agonist, or a combination of clonazepam with exogenous allopregnanolone. Consistent with these in vitro results, finasteride abolished the effects of midazolam on contextual fear learning when administrated 1 d before midazolam injection. Thus, dual activation of CBRs and TSPO appears to result in unique actions of clinically important BDZs. Furthermore, endogenous neurosteroids are shown to be important regulators of pyramidal neuron function and synaptic plasticity.

  5. Adenosine inhibits neutrophil vascular endothelial growth factor release and transendothelial migration via A2B receptor activation.

    LENUS (Irish Health Repository)

    Wakai, A

    2012-02-03

    The effects of adenosine on neutrophil (polymorphonuclear neutrophils; PMN)-directed changes in vascular permeability are poorly characterized. This study investigated whether adenosine modulates activated PMN vascular endothelial growth factor (vascular permeability factor; VEGF) release and transendothelial migration. PMN activated with tumour necrosis factor-alpha (TNF-alpha, 10 ng\\/mL) were incubated with adenosine and its receptor-specific analogues. Culture supernatants were assayed for VEGF. PMN transendothelial migration across human umbilical vein endothelial cell (HUVEC) monolayers was assessed in vitro. Adhesion molecule receptor expression was assessed flow cytometrically. Adenosine and some of its receptor-specific analogues dose-dependently inhibited activated PMN VEGF release. The rank order of potency was consistent with the affinity profile of human A2B receptors. The inhibitory effect of adenosine was reversed by 3,7-dimethyl-1-propargylxanthine, an A2 receptor antagonist. Adenosine (100 microM) or the A2B receptor agonist 5\\'-N-ethylcarboxamidoadenosine (NECA, 100 microM) significantly reduced PMN transendothelial migration. However, expression of activated PMN beta2 integrins and HUVEC ICAM-1 were not significantly altered by adenosine or NECA. Adenosine attenuates human PMN VEGF release and transendothelial migration via the A2B receptor. This provides a novel target for the modulation of PMN-directed vascular hyperpermeability in conditions such as the capillary leak syndrome.

  6. Inhibition of Androgen Receptor Nuclear Localization and Castration-Resistant Prostate Tumor Growth by Pyrroloimidazole-based Small Molecules.

    Science.gov (United States)

    Masoodi, Khalid Z; Xu, Yadong; Dar, Javid A; Eisermann, Kurtis; Pascal, Laura E; Parrinello, Erica; Ai, Junkui; Johnston, Paul A; Nelson, Joel B; Wipf, Peter; Wang, Zhou

    2017-10-01

    The androgen receptor (AR) is a ligand-dependent transcription factor that controls the expression of androgen-responsive genes. A key step in androgen action, which is amplified in castration-resistant prostate cancer (CRPC), is AR nuclear translocation. Small molecules capable of inhibiting AR nuclear localization could be developed as novel therapeutics for CRPC. We developed a high-throughput screen and identified two structurally-related pyrroloimidazoles that could block AR nuclear localization in CRPC cells. We show that these two small molecules, 3-(4-ethoxyphenyl)-6,7-dihydro-5 H -pyrrolo[1,2- a ]imidazole (EPPI) and 3-(4-chlorophenyl)-6,7-dihydro-5 H -pyrrolo[1,2- a ]imidazole (CPPI) can inhibit the nuclear localization and transcriptional activity of AR and reduce the proliferation of AR-positive but not AR-negative prostate cancer cell lines. EPPI and CPPI did not inhibit nuclear localization of the glucocorticoid receptor or the estrogen receptor, suggesting they selectively target AR. In LNCaP tumor xenografts, CPPI inhibited the proliferation of relapsed LNCaP tumors. These findings suggest that EPPI and CPPI could serve as lead structures for the development of therapeutic agents for CRPC. Mol Cancer Ther; 16(10); 2120-9. ©2017 AACR . ©2017 American Association for Cancer Research.

  7. Pu-erh Tea Protects the Nervous System by Inhibiting the Expression of Metabotropic Glutamate Receptor 5.

    Science.gov (United States)

    Li, Chunjie; Chai, Shaomeng; Ju, Yongzhi; Hou, Lu; Zhao, Hang; Ma, Wei; Li, Tian; Sheng, Jun; Shi, Wei

    2017-09-01

    Glutamate is one of the major excitatory neurotransmitters of the CNS and is essential for numerous key neuronal functions. However, excess glutamate causes massive neuronal death and brain damage owing to excitotoxicity via the glutamate receptors. Metabotropic glutamate receptor 5 (mGluR5) is one of the glutamate receptors and represents a promising target for studying neuroprotective agents of potential application in neurodegenerative diseases. Pu-erh tea, a fermented tea, mainly produced in Yunnan province, China, has beneficial effects, including the accommodation of the CNS. In this study, pu-erh tea markedly decreased the transcription and translation of mGluR5 compared to those by black and green teas. Pu-erh tea also inhibited the expression of Homer, one of the synaptic scaffolding proteins binding to mGluR5. Pu-erh tea protected neural cells from necrosis via blocked Ca 2+ influx and inhibited protein kinase C (PKC) activation induced by excess glutamate. Pu-erh tea relieved rat epilepsy induced by LiCl-pilocarpine in behavioural and physiological assays. Pu-erh tea also decreased the expression of mGluR5 in the hippocampus. These results show that the inhibition of mGluR5 plays a role in protecting neural cells from glutamate. The results also indicate that pu-erh tea contains biological compounds binding transcription factors and inhibiting the expression of mGluR5 and identify pu-erh tea as a novel natural neuroprotective agent.

  8. The insecticide fipronil and its metabolite fipronil sulphone inhibit the rat α1β2γ2L GABAA receptor

    Science.gov (United States)

    Li, P; Akk, G

    2008-01-01

    Background and purpose: Fipronil is the active ingredient in a number of widely used insecticides. Human exposure to fipronil leads to symptoms (headache, nausea and seizures) typically associated with the antagonism of GABAA receptors in the brain. In this study, we have examined the modulation of the common brain GABAA receptor subtype by fipronil and its major metabolite, fipronil sulphone. Experimental approach: Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat α1β2γ2L GABAA receptors. Key results: The major effect of fipronil was to increase the rate of current decay in macroscopic recordings. In single-channel recordings, the presence of fipronil resulted in shorter cluster durations without affecting the intracluster open and closed time distributions or the single-channel conductance. The α1V256S mutation, previously shown alleviate channel inhibition by inhibitory steroids and several insecticides, had a relatively small effect on channel block by fipronil. The mode of action of fipronil sulphone was similar to that of its parent compound but the metabolite was less potent at inhibiting the α1β2γ2L receptor. Conclusions and implications: We conclude that exposure to fipronil induces accumulation of receptors in a novel, long-lived blocked state. This process proceeds in parallel with and independently of, channel desensitization. The lower potency of fipronil sulphone indicates that the conversion serves as a detoxifying process in mammalian brain. PMID:18660823

  9. E-cadherin homophilic ligation inhibits cell growth and epidermal growth factor receptor signaling independently of other cell interactions

    DEFF Research Database (Denmark)

    Perrais, Michaël; Chen, Xiao; Perez-Moreno, Mirna

    2007-01-01

    growth inhibitory signals. To address this question, we have selectively formed E-cadherin homophilic bonds at the cell surface of isolated epithelial cells by using functionally active recombinant E-cadherin protein attached to microspheres. We find that E-cadherin ligation alone reduces the frequency...... of cells entering the S phase, demonstrating that E-cadherin ligation directly transduces growth inhibitory signals. E-cadherin binding to beta-catenin is required for cell growth inhibition, but beta-catenin/T-cell factor transcriptional activity is not involved in growth inhibition resulting from...... homophilic binding. Neither E-cadherin binding to p120-catenin nor beta-catenin binding to alpha-catenin, and thereby the actin cytoskeleton, is required for growth inhibition. E-cadherin ligation also inhibits epidermal growth factor (EGF) receptor-mediated growth signaling by a beta...

  10. Arctigenin antagonizes mineralocorticoid receptor to inhibit the transcription of Na/K-ATPase.

    Science.gov (United States)

    Cheng, Ye; Zhou, Meili; Wang, Yan

    2016-01-01

    Hypertension is one of the most important risk factors in cardiovascular disease and is the most common chronic disease. Mineralocorticoid receptor (MR) antagonists have been successfully used in clinic for the treatment of hypertension. Our study aims to investigate whether Arctigenin can antagonize MR and inhibit the transcription of Na/K-ATPase. The yeast two-hybrid assay was used to screen natural products and Arctigenin was identified as an MR antagonist. The direct binding of Arctigenin to MR was determined using assays based on surface plasmon resonance, differential scanning calorimetry and fluorescence quenching. Furthermore, results from mammalian one-hybrid and transcriptional activation experiments also confirmed that Arctigenin can potently antagonize MR in cells. We demonstrated that Arctigenin can decrease the level of Na/K-ATPase mRNA by antagonizing MR in HK-2 cells. Our findings show that Arctigenin can effectively decrease Na/K-ATPase transcription; thus highlight its potential as an anti-hypertensive drug lead compound. Our current findings demonstrate that Arctigenin is an antagonist of MR and effectively decreases the Na/K-ATPase 1 gene expression. Our work provides a hint for the drug discovery against cardiovascular disease.

  11. Nanoparticles containing a liver X receptor agonist inhibit inflammation and atherosclerosis.

    Science.gov (United States)

    Zhang, Xue-Qing; Even-Or, Orli; Xu, Xiaoyang; van Rosmalen, Mariska; Lim, Lucas; Gadde, Suresh; Farokhzad, Omid C; Fisher, Edward A

    2015-01-28

    Liver X receptor (LXR) signaling pathways regulate lipid metabolism and inflammation, which has generated widespread interest in developing synthetic LXR agonists as potential therapeutics for the management of atherosclerosis. In this study, it is demonstrated that nanoparticles (NPs) containing the synthetic LXR agonist GW3965 (NP-LXR) exert anti-inflammatory effects and inhibit the development of atherosclerosis without causing hepatic steatosis. These NPs are engineered through self-assembly of a biodegradable diblock poly(lactide-co-glycolide)-b-poly(ethylene glycol) (PLGA-b-PEG) copolymer. NP-LXR is significantly more effective than free GW3965 at inducing LXR-target gene expression and suppressing inflammatory factors in macrophages in vitro and in vivo. Additionally, the NPs elicit negligible lipogenic gene stimulation in the liver. Using the Ldlr (-/-) mouse model of atherosclerosis, abundant colocalization of fluorescently labeled NPs within plaque macrophages following systemic administration is seen. Notably, six intravenous injections of NP-LXR over 2 weeks markedly reduce the CD68-positive cell (macrophage) content of plaques (by 50%) without increasing total cholesterol or triglycerides in the liver and plasma. Together, these findings identify GW3965-encapsulated PLGA-b-PEG NPs as a promising nanotherapeutic approach to combat atherosclerosis, providing the benefits of LXR agonists without their adverse effects on hepatic and plasma lipid metabolism. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. A Novel Dietary Flavonoid Fisetin Inhibits Androgen Receptor Signaling and Tumor Growth in Athymic Nude Mice

    Science.gov (United States)

    Khan, Naghma; Asim, Mohammad; Afaq, Farrukh; Zaid, Mohammad Abu; Mukhtar, Hasan

    2010-01-01

    Androgen receptor (AR)–mediated signaling plays an important role in the development and progression of prostate cancer (PCa). Hormonal therapies, mainly with combinations of antiandrogens and androgen deprivation, are the mainstay treatment for advanced disease. However, emergence of androgen resistance largely due to inefficient antihormone action limits their therapeutic usefulness. Here, we report that fisetin, a novel dietary flavonoid, acts as a novel AR ligand by competing with the high-affinity androgen to interact with the ligand binding domain of AR. We show that this physical interaction results in substantial decrease in AR stability and decrease in amino-terminal/carboxyl-terminal (N-C) interaction of AR. This results in blunting of AR-mediated transactivation of target genes including prostate-specific antigen (PSA). In addition, treatment of LNCaP cells with fisetin decreased AR protein levels, in part, by decreasing its promoter activity and by accelerating its degradation. Fisetin also synergized with Casodex in inducing apoptosis in LNCaP cells. Treatment with fisetin in athymic nude mice implanted with AR-positive CWR22Rυ1 human PCa cells resulted in inhibition of tumor growth and reduction in serum PSA levels. These data identify fisetin as an inhibitor of AR signaling axis and suggest that it could be a useful chemopreventive and chemotherapeutic agent to delay progression of PCa. PMID:18922931

  13. Activation of adenosine receptors and inhibition of cyclooxygenases: two recent pharmacological approaches to modulation of radiation suppressed hematopoiesis

    International Nuclear Information System (INIS)

    Hofer, M.; Pospisil, M.; Vacek, A.; Hola, J.; Weiterova, L.; Streitova, D.; Znojil, V.

    2008-01-01

    Searching for drugs conforming to requirements for protection and/or treatment of radiation-induced damage belongs to the most important tasks of current radiobiology. In the Laboratory of Experimental Hematology, Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech Republic, two original approaches for stimulation of radiation-suppressed hematopoiesis have been tested in recent years, namely activation of adenosine receptors and inhibition of cyclooxygenases. Non-selective activation of adenosine receptors, induced by combined administration of dipyridamole, a drug preventing adenosine uptake and supporting thus its extracellular receptor-mediated action, and adenosine monophosphate, an adenosine prodrug, has been found to stimulate hematopoiesis when the drugs were given either pre- or post-irradiation. When synthetic adenosine receptor agonists selective for individual adenosine receptor subtypes were tested, stimulatory effects in myelosuppressed mice have been found after administration of IB-MECA, a selective adenosine A3 receptor agonist. Non-selective cyclooxygenase inhibitors, inhibiting both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), indomethacin, diclofenac, or flurbiprofen, have been observed to act positively on radiation-perturbed hematopoiesis in sublethally irradiated mice. However, their undesirable gastrointestinal side effects have been found to negatively influence survival of lethally irradiated animals. Recently tested selective COX-2 inhibitor meloxicam, preserving protective action of COX-1-synthesized prostaglandins in the gastrointestinal tissues, has been observed to retain the hematopoiesis-stimulating effects of non-selective cyclooxygenase inhibitors and to improve the survival of animals exposed to lethal radiation doses. These findings bear evidence for the possibility to use selective adenosine A3 receptor agonists and selective COX-2 inhibitors in human practice for treatment of

  14. Ergopeptines bromocriptine and ergovaline and the dopamine type-2 receptor inhibitor domperidone inhibit bovine equilibrative nucleoside transporter 1-like activity.

    Science.gov (United States)

    Miles, Edwena D; Xue, Yan; Strickland, James R; Boling, James A; Matthews, James C

    2011-09-14

    Neotyphodium coenophialum-infected tall fescue contains ergopeptines. Except for interactions with biogenic amine receptors (e.g., dopamine type-2 receptor, D2R), little is known about how ergopeptines affect animal metabolism. The effect of ergopeptines on bovine nucleoside transporters (NT) was evaluated using Madin-Darby bovine kidney (MDBK) cells. Equilibrative NT1 (ENT1)-like activity accounted for 94% of total NT activity. Inhibitory competition (IC(50)) experiments found that this activity was inhibited by both bromocriptine (a synthetic model ergopeptine and D2R agonist) and ergovaline (a predominant ergopeptine of tall fescue). Kinetic inhibition analysis indicated that bromocriptine inhibited ENT1-like activity through a competitive and noncompetitive mechanism. Domperidone (a D2R antagonist) inhibited ENT1 activity more in the presence than in the absence of bromocriptine and displayed an IC(50) value lower than that of bromocriptine or ergovaline, suggesting that inhibition was not through D2R-mediated events. These novel mechanistic findings imply that cattle consuming endophyte-infected tall fescue have reduced ENT1 activity and, thus, impaired nucleoside metabolism.

  15. In vitro nuclear receptor inhibition and cytotoxicity of hydraulic fracturing chemicals and their binary mixtures.

    Science.gov (United States)

    Bain, Peter A; Kumar, Anu

    2018-05-01

    The widespread use of hydraulic fracturing (HF) in oil and gas extraction operations has led to concern over environmental risks posed by chemicals used in HF fluids. Here we employed a suite of stable luciferase reporter gene assays to investigate the potential for selected HF chemicals or geogenics to activate or antagonise nuclear receptor signalling. We screened three biocides (bronopol [BP], glutaraldehyde [GA], and tetrakis(hydroxymethyl)phosphonium sulfate [THPS]), a surfactant (2-butoxyethanol), a friction reducer (polyacrylamide), and a coal seam geogenic (o-cresol) for their potential to act as agonists or antagonists of the estrogen receptor, androgen receptor, progesterone receptor (PR), glucocorticoid receptor or peroxisome proliferator-activated receptor gamma (PPARγ). None of the chemicals induced luciferase activity in any of assays used in the study. In antagonistic mode, BP, GA and THPS caused reductions in luciferase activity in the reporter assays at higher concentrations (50-100 μM), while at low concentrations (2-10 μM) GA and THPS enhanced luciferase activity in some assays relative to controls. None of the other tested chemicals exhibited antagonism in the selected assays. In most cases, altered receptor signalling only occurred at concentrations exhibiting cytotoxicity. However, PPARγ activity, and to a lesser extent PR activity, were inhibited by THPS at sub-cytotoxic concentrations. The majority of binary combinations tested exhibited significantly less-than-additive cytotoxicity, and none of the combinations exhibited synergistic cytotoxicity. In summary, the results of the present study indicate that the selected chemicals are not likely to function as direct agonists of the nuclear receptors tested, and only one chemical, THPS was an apparent partial antagonist of two nuclear receptors. Copyright © 2017. Published by Elsevier Ltd.

  16. Mechanisms of Inhibition of the Epidermal Growth Factor Receptor: Implications for Novel Anti-Cancer Therapies

    National Research Council Canada - National Science Library

    Klein, Daryl E

    2005-01-01

    .... No secreted or extracellular ErbB receptor inhibitors have been reported in mammals. However, two natural inhibitors of the highly homologous Drosophila EGF receptor are found in Drosophila melanogaster...

  17. ACE inhibition is superior to angiotensin receptor blockade for renography in renal artery stenosis

    International Nuclear Information System (INIS)

    Karanikas, Georgios; Becherer, Alexander; Wiesner, Karoline; Dudczak, Robert; Kletter, Kurt

    2002-01-01

    Angiotensin converting enzyme (ACE) inhibitors as well as angiotensin II receptor antagonists are able to prevent the vasoconstrictive effect of angiotensin II on the efferent renal vessels, which is believed to play an important role in renovascular hypertension. This effect is assumed to be essential for the demonstration of renovascular hypertension by captopril renography. In this study, renographic changes induced by captopril and the AT1 receptor antagonist valsartan were compared in patients with a high probability for renovascular hypertension. Twenty-five patients with 33 stenosed renal arteries (grade of stenosis >50%) and hypertension were studied. Captopril, valsartan and baseline renography were performed within 48 h using technetium-99m mercaptoacetyltriglycine. Blood pressure was monitored, plasma renin concentration before and after intervention was determined and urinary flow was estimated from the urinary output of the hydrated patients. Alterations in renographic curves after intervention were evaluated according to the Santa Fe consensus on ACE inhibitor renography. Captopril renography was positive, indicating renovascular hypertension, in 25 of the 33 stenosed vessels, whereas valsartan renography was positive in only ten. Blood pressure during captopril and valsartan renography was not different; reduction in blood pressure was the same after valsartan and captopril. Plasma renin concentration was comparable for valsartan and captopril studies, showing suppressed values after intervention in as many as 12 of the 25 patients. Urinary flow after valsartan was higher than after captopril (P<0.05). However, this difference could not explain the markedly higher sensitivity of captopril compared with valsartan in demonstrating renal artery stenosis. In 14 of the 25 patients, blood pressure response to revascularisation was monitored, showing a much better predictive value for captopril renography. It is concluded that captopril renography is much

  18. ACE inhibition is superior to angiotensin receptor blockade for renography in renal artery stenosis

    Energy Technology Data Exchange (ETDEWEB)

    Karanikas, Georgios; Becherer, Alexander; Wiesner, Karoline; Dudczak, Robert; Kletter, Kurt [Department of Nuclear Medicine, University of Vienna (Austria)

    2002-03-01

    Angiotensin converting enzyme (ACE) inhibitors as well as angiotensin II receptor antagonists are able to prevent the vasoconstrictive effect of angiotensin II on the efferent renal vessels, which is believed to play an important role in renovascular hypertension. This effect is assumed to be essential for the demonstration of renovascular hypertension by captopril renography. In this study, renographic changes induced by captopril and the AT1 receptor antagonist valsartan were compared in patients with a high probability for renovascular hypertension. Twenty-five patients with 33 stenosed renal arteries (grade of stenosis >50%) and hypertension were studied. Captopril, valsartan and baseline renography were performed within 48 h using technetium-99m mercaptoacetyltriglycine. Blood pressure was monitored, plasma renin concentration before and after intervention was determined and urinary flow was estimated from the urinary output of the hydrated patients. Alterations in renographic curves after intervention were evaluated according to the Santa Fe consensus on ACE inhibitor renography. Captopril renography was positive, indicating renovascular hypertension, in 25 of the 33 stenosed vessels, whereas valsartan renography was positive in only ten. Blood pressure during captopril and valsartan renography was not different; reduction in blood pressure was the same after valsartan and captopril. Plasma renin concentration was comparable for valsartan and captopril studies, showing suppressed values after intervention in as many as 12 of the 25 patients. Urinary flow after valsartan was higher than after captopril (P<0.05). However, this difference could not explain the markedly higher sensitivity of captopril compared with valsartan in demonstrating renal artery stenosis. In 14 of the 25 patients, blood pressure response to revascularisation was monitored, showing a much better predictive value for captopril renography. It is concluded that captopril renography is much

  19. Leptin receptor signaling inhibits ovarian follicle development and egg laying in chicken hens

    Science.gov (United States)

    2014-01-01

    Background Nutrition intake during growth strongly influences ovarian follicle development and egg laying in chicken hens, yet the underlying endocrine regulatory mechanism is still poorly understood. The relevant research progress is hindered by difficulties in detection of leptin gene and its expression in the chicken. However, a functional leptin receptor (LEPR) is present in the chicken which has been implicated to play a regulatory role in ovarian follicle development and egg laying. The present study targeted LEPR by immunizing against its extracellular domain (ECD), and examined the resultant ovarian follicle development and egg-laying rate in chicken hens. Methods Hens that have been immunized four times with chicken LEPR ECD were assessed for their egg laying rate and feed intake, numbers of ovarian follicles, gene expression profiles, serum lipid parameters, as well as STAT3 signaling pathway. Results Administrations of cLEPR ECD antigen resulted in marked reductions in laying rate that over time eventually recovered to the levels exhibited by the Control hens. Together with the decrease in egg laying rate, cLEPR-immunized hens also exhibited significant reductions in feed intake, plasma concentrations of glucose, triglyceride, high-density lipoprotein, and low-density lipoprotein. Parallelled by reductions in feed intake, mRNA gene expression levels of AgRP, orexin, and NPY were down regulated, but of POMC, MC4R and lepR up-regulated in Immunized hen hypothalamus. cLEPR-immunization also promoted expressions of apoptotic genes such as caspase3 in theca and fas in granulosa layer, but severely depressed IGF-I expression in both theca and granulosa layers. Conclusions Immunization against cLEPR ECD in egg-laying hens generated antibodies that mimic leptin bioactivity by enhancing leptin receptor transduction. This up-regulated apoptotic gene expression in ovarian follicles, negatively regulated the expression of genes that promote follicular development

  20. Inhibition of Epithelial TNF-α Receptors by Purified Fruit Bromelain Ameliorates Intestinal Inflammation and Barrier Dysfunction in Colitis

    OpenAIRE

    Zhou, Zijuan; Wang, Liang; Feng, Panpan; Yin, Lianhong; Wang, Chen; Zhi, Shengxu; Dong, Jianyi; Wang, Jingyu; Lin, Yuan; Chen, Dapeng; Xiong, Yongjian; Peng, Jinyong

    2017-01-01

    Activation of the TNF-α receptor (TNFR) leads to an inflammatory response, and anti-TNF therapy has been administered to reduce inflammation symptoms and heal mucosal ulcers in inflammatory bowel disease (IBD). Bromelain, a complex natural mixture of proteolytic enzymes, has been shown to exert anti-inflammatory effects. This study aimed to investigate the effect of purified fruit bromelain (PFB)-induced inhibition of epithelial TNFR in a rat colitis model. Colitis was established by intracol...

  1. Inhibition of alpha oscillations through serotonin-2A receptor activation underlies the visual effects of ayahuasca in humans

    OpenAIRE

    Valle, Marta; Ana Elda, Maqueda; Rabella, Mireia; Rodríguez Pujadas, Aina; Antonijoan Arbós, Rosa Maria; Romero Lafuente, Sergio; Alonso López, Joan Francesc; Mañanas Villanueva, Miguel Ángel; Barker, Steven; Friedlander, Pablo; Feilding, Amanda; Riba, Jordi

    2016-01-01

    Ayahuasca is an Amazonian psychotropic plant tea typically obtained from two plants, Banisteriopsis caapi and Psychotria viridis. It contains the psychedelic 5-HT2A and sigma-1 agonist N,N-dimethyltryptamine (DMT) plus ß-carboline alkaloids with monoamine-oxidase (MAO)-inhibiting properties. Although the psychoactive effects of ayahuasca have commonly been attributed solely to agonism at the 5-HT2A receptor, the molecular target of classical psychedelics, this has not been tested experimental...

  2. Selective inhibition of prostaglandin E2 receptors EP2 and EP4 inhibits adhesion of human endometriotic epithelial and stromal cells through suppression of integrin-mediated mechanisms.

    Science.gov (United States)

    Lee, JeHoon; Banu, Sakhila K; Burghardt, Robert C; Starzinski-Powitz, Anna; Arosh, Joe A

    2013-03-01

    Endometriosis is a chronic gynecological disease of reproductive age women characterized by the presence of functional endometrial tissues outside the uterine cavity. Interactions between the endometriotic cells and the peritoneal extracellular matrix proteins (ECM) are crucial mechanisms that allow adhesion of the endometriotic cells into peritoneal mesothelia. Prostaglandin E2 (PGE2) plays an important role in the pathogenesis of endometriosis. In previous studies, we have reported that selective inhibition of PGE2 receptors PTGER2 and PTGER4 decreases survival and invasion of human endometriotic epithelial and stromal cells through multiple mechanisms. Results of the present study indicates that selective inhibition of PTGER2- and PTGER4-mediated PGE2 signaling 1) decreases the expression and/or activity of specific integrin receptor subunits Itgb1 (beta1) and Itgb3 (beta3) but not Itgb5 (beta5), Itga1 (alpha1), Itga2 (alpha2), Itga5 (alpha5), and Itgav (alphav); 2) decreases integrin-signaling components focal adhesion kinase or protein kinase 2 (PTK2) and talin proteins; 3) inhibits interactions between Itgb1/Itgb3 subunits, PTK2, and talin and PTGER2/PTGER4 proteins through beta-arrestin-1 and Src kinase protein complex in human endometriotic epithelial cells 12Z and stromal cells 22B; and 4) decreases adhesion of 12Z and 22B cells to ECM collagen I, collagen IV, fibronectin, and vitronectin in a substrate-specific manner. These novel findings provide an important molecular framework for further evaluation of selective inhibition of PTGER2 and PTGER4 as potential nonsteroidal therapy to expand the spectrum of currently available treatment options for endometriosis in child-bearing age women.

  3. Arctigenin reduces neuronal responses in the somatosensory cortex via the inhibition of non-NMDA glutamate receptors.

    Science.gov (United States)

    Borbély, Sándor; Jócsák, Gergely; Moldován, Kinga; Sedlák, Éva; Preininger, Éva; Boldizsár, Imre; Tóth, Attila; Atlason, Palmi T; Molnár, Elek; Világi, Ildikó

    2016-07-01

    Lignans are biologically active phenolic compounds related to lignin, produced in different plants. Arctigenin, a dibenzylbutyrolactone-type lignan, has been used as a neuroprotective agent for the treatment of encephalitis. Previous studies of cultured rat cerebral cortical neurones raised the possibility that arctigenin inhibits kainate-induced excitotoxicity. The aims of the present study were: 1) to analyse the effect of arctigenin on normal synaptic activity in ex vivo brain slices, 2) to determine its receptor binding properties and test the effect of arctigenin on AMPA/kainate receptor activation and 3) to establish its effects on neuronal activity in vivo. Arctigenin inhibited glutamatergic transmission and reduced the evoked field responses. The inhibitory effect of arctigenin on the evoked field responses proved to be substantially dose dependent. Our results indicate that arctigenin exerts its effects under physiological conditions and not only on hyper-excited neurons. Furthermore, arctigenin can cross the blood-brain barrier and in the brain it interacts with kainate sensitive ionotropic glutamate receptors. These results indicate that arctigenin is a potentially useful new pharmacological tool for the inhibition of glutamate-evoked responses in the central nervous system in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Anticonvulsants Based on the α-Substituted Amide Group Pharmacophore Bind to and Inhibit Function of Neuronal Nicotinic Acetylcholine Receptors.

    Science.gov (United States)

    Krivoshein, Arcadius V

    2016-03-16

    Although the antiepileptic properties of α-substituted lactams, acetamides, and cyclic imides have been known for over 60 years, the mechanism by which they act remains unclear. I report here that these compounds bind to the nicotinic acetylcholine receptor (nAChR) and inhibit its function. Using transient kinetic measurements with functionally active, nondesensitized receptors, I have discovered that (i) α-substituted lactams and cyclic imides are noncompetitive inhibitors of heteromeric subtypes (such as α4β2 and α3β4) of neuronal nAChRs and (ii) the binding affinity of these compounds toward the nAChR correlates with their potency in preventing maximal electroshock (MES)-induced convulsions in mice. Based on the hypothesis that α-substituted amide group is the essential pharmacophore of these drugs, I found and tested a simple compound, 2-phenylbutyramide. This compound indeed inhibits nAChR and shows good anticonvulsant activity in mice. Molecular docking simulations suggest that α-substituted lactams, acetamides, and cyclic imides bind to the same sites on the extracellular domain of the receptor. These new findings indicate that inhibition of brain nAChRs may play an important role in the action of these antiepileptic drugs, a role that has not been previously recognized.

  5. Glioblastoma chemotherapy adjunct via potent serotonin receptor-7 inhibition using currently marketed high-affinity antipsychotic medicines

    Science.gov (United States)

    Kast, RE

    2010-01-01

    Glioblastoma treatment as now constituted offers increased survival measured in months over untreated patients. Because glioblastomas are active in synthesizing a bewildering variety of growth factors, a systematic approach to inhibiting these is being undertaken as treatment adjunct. The serotonin 7 receptor is commonly overexpressed in glioblastoma. Research documentation showing agonists at serotonin receptor 7 cause increased extracellular regulated kinase 1/2 activation, increased interleukin-6 synthesis, increased signal transducer and activator of transcription-3 activation, increased resistance to apoptosis and other growth enhancing changes in glioblastoma is reviewed in this paper. Because three drugs in wide use to treat thought disorders – paliperidone, pimozide and risperidone – are also potent and well-tolerated inhibitors at serotonin receptor 7, these drugs should be studied for growth factor deprivation in an adjunctive role in glioblastoma treatment. PMID:20880389

  6. Inverse agonism at the P2Y12 receptor and ENT1 transporter blockade contribute to platelet inhibition by ticagrelor.

    Science.gov (United States)

    Aungraheeta, Riyaad; Conibear, Alexandra; Butler, Mark; Kelly, Eamonn; Nylander, Sven; Mumford, Andrew; Mundell, Stuart J

    2016-12-08

    Ticagrelor is a potent antagonist of the P2Y 12 receptor (P2Y 12 R) and consequently an inhibitor of platelet activity effective in the treatment of atherothrombosis. Here, we sought to further characterize its molecular mechanism of action. Initial studies showed that ticagrelor promoted a greater inhibition of adenosine 5'-diphosphate (ADP)-induced Ca 2+ release in washed platelets vs other P2Y 12 R antagonists. This additional effect of ticagrelor beyond P2Y 12 R antagonism was in part as a consequence of ticagrelor inhibiting the equilibrative nucleoside transporter 1 (ENT1) on platelets, leading to accumulation of extracellular adenosine and activation of G s -coupled adenosine A 2A receptors. This contributed to an increase in basal cyclic adenosine monophosphate (cAMP) and vasodilator-stimulated phosphoprotein phosphorylation (VASP-P). In addition, ticagrelor increased platelet cAMP and VASP-P in the absence of ADP in an adenosine receptor-independent manner. We hypothesized that this increase originated from a direct effect on basal agonist-independent P2Y 12 R signaling, and this was validated in 1321N1 cells stably transfected with human P2Y 12 R. In these cells, ticagrelor blocked the constitutive agonist-independent activity of the P2Y 12 R, limiting basal G i -coupled signaling and thereby increasing cAMP levels. These data suggest that ticagrelor has the pharmacological profile of an inverse agonist. Based on our results showing insurmountable inhibition of ADP-induced Ca 2+ release and forskolin-induced cAMP, the mode of antagonism of ticagrelor also appears noncompetitive, at least functionally. In summary, our studies describe 2 novel modes of action of ticagrelor, inhibition of platelet ENT1 and inverse agonism at the P2Y 12 R that contribute to its effective inhibition of platelet activation. © 2016 by The American Society of Hematology.

  7. Inhibition of aryl hydrocarbon receptor-dependent transcription by resveratrol or kaempferol is independent of estrogen receptor α expression in human breast cancer cells

    Science.gov (United States)

    MacPherson, Laura; Matthews, Jason

    2016-01-01

    Resveratrol and kaempferol are natural chemopreventative agents that are also aryl hydrocarbon receptor (AHR) antagonists and estrogen receptor (ER) agonists. In this study we evaluated the role of ERα in resveratrol- and kaempferol-mediated inhibition of AHR-dependent transcription. Kaempferol or resveratrol inhibited dioxin-induced cytochrome P450 1A1 (CYP1A1) and CYP1B1 expression levels and recruitment of AHR, ERα and co-activators to CYP1A1 and CYP1B1. Both phytochemicals induced the expression and recruitment of ERα to gene amplified in breast cancer 1 (GREB1). RNAi-mediated knockdown of ERα in T-47D cells did not affect the inhibitory action of either phytochemical on AHR activity. Both compounds also inhibited AHR-dependent transcription in ERα-negative MDA-MB-231 and BT-549 breast cancer cells. These data show that ERα does not contribute to the AHR-inhibitory activities of resveratrol and kaempferol. PMID:20846786

  8. Intestinal Serotonin Transporter Inhibition by Toll-Like Receptor 2 Activation. A Feedback Modulation.

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    Eva Latorre

    Full Text Available TLR2 is a microbiota recognition receptor that has been described to contribute to intestinal homeostasis and to ameliorate inflammatory intestinal injury. In this context, serotonin (5-HT has shown to be an essential intestinal physiological neuromodulator that is also involved in intestinal inflammatory diseases. Since the interaction between TLR2 activation and the intestinal serotoninergic system remains non-investigated, our main aim was to analyze the effect of TLR2 on intestinal serotonin transporter (SERT activity and expression and the intracellular pathways involved. Caco-2/TC7 cells were used to analyze SERT and TLR2 molecular expression and SERT activity by measuring 5-HT uptake. The results showed that apical TLR2 activation inhibits SERT activity in Caco-2/TC7 cells mainly by reducing SERT protein level either in the plasma membrane, after short-term TLR2 activation or in both the plasma membrane and cell lysate, after long-term activation. cAMP/PKA pathway appears to mediate short-term inhibitory effect of TLR2 on SERT; however, p38 MAPK pathway has been shown to be involved in both short- and long-term TLR2 effect. Reciprocally, 5-HT long-term treatment yielded TLR2 down regulation in Caco-2/TC7 cells. Finally, results from in vivo showed an augmented intestinal SERT expression in mice Tlr2-/-, thus confirming our inhibitory effect of TLR2 on intestinal SERT in vitro. The present work infers that TLR2 may act in intestinal pathophysiology, not only by its inherent innate immune role, but also by regulating the intestinal serotoninergic system.

  9. Epidermal growth factor receptor inhibition by anti-CD147 therapy in cutaneous squamous cell carcinoma.

    Science.gov (United States)

    Frederick, John W; Sweeny, Larissa; Hartman, Yolanda; Zhou, Tong; Rosenthal, Eben L

    2016-02-01

    Advanced cutaneous squamous cell carcinoma (SCC) is an uncommon and aggressive malignancy. As a result, there is limited understanding of its biology and pathogenesis. CD147 and epidermal growth factor receptor (EGFR) have been identified as oncologically important targets, but their relationship remains undefined in cutaneous SCC. Multiple cutaneous SCC cell lines (Colo-16, SRB-1, and SRB-12), were treated in vitro with a range of chimeric anti-CD147 monoclonal antibody (mAb) (0, 50, 100, and 200 µg/mL) or transfected with a small interfering RNA against CD147 (SiCD147). Cell proliferation, migration (scratch wound healing assay), and protein expression was then assessed. In vivo, Colo-16 flank xenografts were treated anti-CD147 mAb (150 µg i.p. triweekly). After treatment with anti-CD147 (200 µg/mL), there was a significant decrease in proliferation for all cell lines relative to controls (p CD147 (200 µg/mL) resulted in decreased cell migration for all cell lines, with an average of 43% reduction in closure compared to controls (p CD147 antibody therapy and siRNA mediated reduction in CD147 expression were both found to decrease protein expression of EGFR, which correlated with a reduction in downstream total and phosphorylated protein kinase B (pAKT). Tumor growth in vivo was reduced for both the anti-CD147 treatment group and the SiCD147 group relative to controls. Inhibition and downregulation of CD147 in cutaneous SCC resulted in suppression of the malignant phenotype in vitro and in vivo, which may be mediated in part by an alteration in EGFR expression. As a result, CD147 may serve as a potential therapeutic target for advanced cutaneous SCC. © 2014 Wiley Periodicals, Inc.

  10. Clofibric acid, a peroxisome proliferator-activated receptor alpha ligand, inhibits growth of human ovarian cancer.

    Science.gov (United States)

    Yokoyama, Yoshihito; Xin, Bing; Shigeto, Tatsuhiko; Umemoto, Mika; Kasai-Sakamoto, Akiko; Futagami, Masayuki; Tsuchida, Shigeki; Al-Mulla, Fahd; Mizunuma, Hideki

    2007-04-01

    Recent reports have shown that peroxisome proliferator-activated receptor (PPAR)alpha ligands reduce growth of some types of malignant tumors and prevent carcinogenesis. In this study, we investigated the inhibitory effect of clofibric acid (CA), a ligand for PPARalpha on growth of ovarian malignancy, in in vivo and in vitro experiments using OVCAR-3 and DISS cells derived from human ovarian cancer and aimed to elucidate the molecular mechanism of its antitumor effect. CA treatment significantly suppressed the growth of OVCAR-3 tumors xenotransplanted s.c. and significantly prolonged the survival of mice with malignant ascites derived from DISS cells as compared with control. CA also dose-dependently inhibited cell proliferation of cultured cell lines. CA treatment increased the expression of carbonyl reductase (CR), which promotes the conversion of prostaglandin E(2) (PGE(2)) to PGF(2alpha), in implanted OVCAR-3 tumors as well as cultured cells. CA treatment decreased PGE(2) level as well as vascular endothelial growth factor (VEGF) amount in both of OVCAR-3-tumor and DISS-derived ascites. Reduced microvessel density and induced apoptosis were found in solid OVCAR-3 tumors treated by CA. Transfection of CR expression vector into mouse ovarian cancer cells showed significant reduction of PGE(2) level as well as VEGF expression. These results indicate that CA produces potent antitumor effects against ovarian cancer in conjunction with a reduction of angiogenesis and induction of apoptosis. We conclude that CA could be an effective agent in ovarian cancer and should be tested alone and in combination with other anticancer drugs.

  11. Covalent Targeting of Fibroblast Growth Factor Receptor Inhibits Metastatic Breast Cancer.

    Science.gov (United States)

    Brown, Wells S; Tan, Li; Smith, Andrew; Gray, Nathanael S; Wendt, Michael K

    2016-09-01

    Therapeutic targeting of late-stage breast cancer is limited by an inadequate understanding of how tumor cell signaling evolves during metastatic progression and by the currently available small molecule inhibitors capable of targeting these processes. Herein, we demonstrate that both β3 integrin and fibroblast growth factor receptor-1 (FGFR1) are part of an epithelial-mesenchymal transition (EMT) program that is required to facilitate metastatic outgrowth in response to fibroblast growth factor-2 (FGF2). Mechanistically, β3 integrin physically disrupts an interaction between FGFR1 and E-cadherin, leading to a dramatic redistribution of FGFR1 subcellular localization, enhanced FGF2 signaling and increased three-dimensional (3D) outgrowth of metastatic breast cancer cells. This ability of β3 integrin to drive FGFR signaling requires the enzymatic activity of focal adhesion kinase (FAK). Consistent with these mechanistic data, we demonstrate that FGFR, β3 integrin, and FAK constitute a molecular signature capable of predicting decreased survival of patients with the basal-like subtype of breast cancer. Importantly, covalent targeting of a conserved cysteine in the P-loop of FGFR1-4 with our newly developed small molecule, FIIN-4, more effectively blocks 3D metastatic outgrowth as compared with currently available FGFR inhibitors. In vivo application of FIIN-4 potently inhibited the growth of metastatic, patient-derived breast cancer xenografts and murine-derived metastases growing within the pulmonary microenvironment. Overall, the current studies demonstrate that FGFR1 works in concert with other EMT effector molecules to drive aberrant downstream signaling, and that these events can be effectively targeted using our novel therapeutics for the treatment of the most aggressive forms of breast cancer. Mol Cancer Ther; 15(9); 2096-106. ©2016 AACR. ©2016 American Association for Cancer Research.

  12. Voltage-Dependent Inhibition of Glycine Receptor Channels by Niflumic Acid

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    Galyna Maleeva

    2017-05-01

    Full Text Available Niflumic acid (NFA is a member of the fenamate class of nonsteroidal anti-inflammatory drugs. This compound and its derivatives are used worldwide clinically for the relief of chronic and acute pain. NFA is also a commonly used blocker of voltage-gated chloride channels. Here we present evidence that NFA is an efficient blocker of chloride-permeable glycine receptors (GlyRs with subunit heterogeneity of action. Using the whole-cell configuration of patch-clamp recordings and molecular modeling, we analyzed the action of NFA on homomeric α1ΔIns, α2B, α3L, and heteromeric α1β and α2β GlyRs expressed in CHO cells. NFA inhibited glycine-induced currents in a voltage-dependent manner and its blocking potency in α2 and α3 GlyRs was higher than that in α1 GlyR. The Woodhull analysis suggests that NFA blocks α1 and α2 GlyRs at the fractional electrical distances of 0.16 and 0.65 from the external membrane surface, respectively. Thus, NFA binding site in α1 GlyR is closer to the external part of the membrane, while in α2 GlyR it is significantly deeper in the pore. Mutation G254A at the cytoplasmic part of the α1 GlyR pore-lining TM2 helix (level 2′ increased the NFA blocking potency, while incorporation of the β subunit did not have a significant effect. The Hill plot analysis suggests that α1 and α2 GlyRs are preferably blocked by two and one NFA molecules, respectively. Molecular modeling using Monte Carlo energy minimizations provides the structural rationale for the experimental data and proposes more than one interaction site along the pore where NFA can suppress the ion permeation.

  13. Prenatal Alcohol Exposure Increases Histamine H3 Receptor-Mediated Inhibition of Glutamatergic Neurotransmission in Rat Dentate Gyrus.

    Science.gov (United States)

    Varaschin, Rafael K; Allen, Nyika A; Rosenberg, Martina J; Valenzuela, C Fernando; Savage, Daniel D

    2018-02-01

    We have reported that prenatal alcohol exposure (PAE)-induced deficits in dentate gyrus, long-term potentiation (LTP), and memory are ameliorated by the histamine H 3 receptor inverse agonist ABT-239. Curiously, ABT-239 did not enhance LTP or memory in control offspring. Here, we initiated an investigation of how PAE alters histaminergic neurotransmission in the dentate gyrus and other brain regions employing combined radiohistochemical and electrophysiological approaches in vitro to examine histamine H 3 receptor number and function. Long-Evans rat dams voluntarily consumed either a 0% or 5% ethanol solution 4 hours each day throughout gestation. This pattern of drinking, which produces a mean peak maternal serum ethanol concentration of 60.8 ± 5.8 mg/dl, did not affect maternal weight gain, litter size, or offspring birthweight. Radiohistochemical studies in adult offspring revealed that specific [ 3 H]-A349821 binding to histamine H 3 receptors was not different in PAE rats compared to controls. However, H 3 receptor-mediated G i /G o protein-effector coupling, as measured by methimepip-stimulated [ 35 S]-GTPγS binding, was significantly increased in cerebral cortex, cerebellum, and dentate gyrus of PAE rats compared to control. A LIGAND analysis of detailed methimepip concentration-response curves in dentate gyrus indicated that PAE significantly elevates receptor-effector coupling by a lower affinity H 3 receptor population without significantly altering the affinities of H 3 receptor subpopulations. In agreement with the [ 35 S]-GTPγS studies, a similar range of methimepip concentrations also inhibited electrically evoked field excitatory postsynaptic potential responses and increased paired-pulse ratio, a measure of decreased glutamate release, to a significantly greater extent in dentate gyrus slices from PAE rats than in controls. These results suggest that a PAE-induced elevation in H 3 receptor-mediated inhibition of glutamate release from

  14. Colony stimulating factor 1 receptor inhibition delays recurrence of glioblastoma after radiation by altering myeloid cell recruitment and polarization

    Science.gov (United States)

    Stafford, Jason H.; Hirai, Takahisa; Deng, Lei; Chernikova, Sophia B.; Urata, Kimiko; West, Brian L.; Brown, J. Martin

    2016-01-01

    Background Glioblastoma (GBM) may initially respond to treatment with ionizing radiation (IR), but the prognosis remains extremely poor because the tumors invariably recur. Using animal models, we previously showed that inhibiting stromal cell–derived factor 1 signaling can prevent or delay GBM recurrence by blocking IR-induced recruitment of myeloid cells, specifically monocytes that give rise to tumor-associated macrophages. The present study was aimed at determining if inhibiting colony stimulating factor 1 (CSF-1) signaling could be used as an alternative strategy to target pro-tumorigenic myeloid cells recruited to irradiated GBM. Methods To inhibit CSF-1 signaling in myeloid cells, we used PLX3397, a small molecule that potently inhibits the tyrosine kinase activity of the CSF-1 receptor (CSF-1R). Combined IR and PLX3397 therapy was compared with IR alone using 2 different human GBM intracranial xenograft models. Results GBM xenografts treated with IR upregulated CSF-1R ligand expression and increased the number of CD11b+ myeloid-derived cells in the tumors. Treatment with PLX3397 both depleted CD11b+ cells and potentiated the response of the intracranial tumors to IR. Median survival was significantly longer for mice receiving combined therapy versus IR alone. Analysis of myeloid cell differentiation markers indicated that CSF-1R inhibition prevented IR-recruited monocyte cells from differentiating into immunosuppressive, pro-angiogenic tumor-associated macrophages. Conclusion CSF-1R inhibition may be a promising strategy to improve GBM response to radiotherapy. PMID:26538619

  15. Group I mGlu receptor stimulation inhibits activation-induced cell death of human T lymphocytes

    Science.gov (United States)

    Chiocchetti, Annalisa; Miglio, Gianluca; Mesturini, Riccardo; Varsaldi, Federica; Mocellin, Marco; Orilieri, Elisabetta; Dianzani, Chiara; Fantozzi, Roberto; Dianzani, Umberto; Lombardi, Grazia

    2006-01-01

    The effects of L-glutamate on activation-induced cell death (AICD) of human activated (1 μg ml−1 phytohemagglutinin plus 2 U ml−1 interleukin-2; 8 days) T lymphocytes were studied by measuring anti-CD3 monoclonal antibody (10 μg ml−1; 18 h)-induced cell apoptosis (Annexin V and propidium iodide staining). L-Glutamate (1 × 10−8–1 × 10−4 M) significantly (P⩽0.01) inhibited AICD in a concentration-dependent manner (EC50=6.3 × 10−8 M; maximum inhibition 54.8±6.3% at 1 × 10−6 M). The L-glutamate inhibitory effect was pharmacologically characterized as mediated by group I mGlu receptors, since mGlu receptor agonists reproduced this effect. The EC50 values were: 3.2 × 10−7 M for (1S,3R)-ACPD; 4.5 × 10−8 M for quisqualate; 1.0 × 10−6 M for (S)-3,5-DHPG; 2.0 × 10−5 M for CHPG. Group I mGlu receptor antagonists inhibited the effects of quisqualate 1.0 × 10−6 M. The IC50 values calculated were: 8.7 × 10−5, 4.3 × 10−6 and 6.3 × 10−7 M for AIDA, LY 367385 and MPEP, respectively. L-Glutamate (1 × 10−6 M; 18 h) significantly (P⩽0.05) inhibited FasL expression (40.8±11.3%) (cytofluorimetric analysis), whereas it did not affect Fas signalling. Expression of both mGlu1 and mGlu5 receptor mRNA by T lymphocytes and T-cell lines, as demonstrated by reverse transcriptase–PCR analysis, suggests that L-glutamate-mediated inhibition of AICD was exerted on T cells. These data depict a novel role for L-glutamate in the regulation of the immune response through group I mGlu receptor-mediated mechanisms. PMID:16751798

  16. Co-inhibition of epidermal growth factor receptor and insulin-like growth factor receptor 1 enhances radiosensitivity in human breast cancer cells

    International Nuclear Information System (INIS)

    Li, Ping; Veldwijk, Marlon R; Zhang, Qing; Li, Zhao-bin; Xu, Wen-cai; Fu, Shen

    2013-01-01

    Over-expression of epidermal growth factor receptor (EGFR) or insulin-like growth factor-1 receptor (IGF-1R) have been shown to closely correlate with radioresistance of breast cancer cells. This study aimed to investigate the impact of co-inhibition of EGFR and IGF-1R on the radiosensitivity of two breast cancer cells with different profiles of EGFR and IGF-1R expression. The MCF-7 (EGFR +/−, IGF-1R +++) and MDA-MB-468 (EGFR +++, IGF-1R +++) breast cancer cell lines were used. Radiosensitizing effects were determined by colony formation assay. Apoptosis and cell cycle distribution were measured by flow cytometry. Phospho-Akt and phospho-Erk1/2 were quantified by western blot. In vivo studies were conducted using MDA-MB-468 cells xenografted in nu/nu mice. In MDA-MB-468 cells, the inhibition of IGF-1R upregulated the p-EGFR expression. Either EGFR (AG1478) or IGF-1R inhibitor (AG1024) radiosensitized MDA-MB-468 cells. In MCF-7 cells, radiosensitivity was enhanced by AG1024, but not by AG1478. Synergistical radiosensitizing effect was observed by co-inhibition of EGFR and IGF-1R only in MDA-MB-468 cells with a DMF 10% of 1.90. The co-inhibition plus irradiation significantly induced more apoptosis and arrested the cells at G0/G1 phase in MDA-MB-468 cells. Only co-inhibition of EGFR and IGF-1R synergistically diminished the expression of p-Akt and p-Erk1/2 in MDA-MB-468 cells. In vivo studies further verified the radiosensitizing effects by co-inhibition of both pathways in a MDA-MB-468 xenograft model. Our data suggested that co-inhibition of EGFR and IGF-1R synergistically radiosensitized breast cancer cells with both EGFR and IGF-1R high expression. The approach may have an important therapeutic implication in the treatment of breast cancer patients with high expression of EGFR and IGF-1R

  17. Glycine and GABAA receptors mediate tonic and phasic inhibitory processes that contribute to prepulse inhibition in the goldfish startle network

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    Paul C.P. Curtin

    2015-03-01

    Full Text Available Prepulse inhibition (PPI is understood as an inhibitory process that attenuates sensory flow during early stages (20-1000ms of information processing. Here, we applied in vivo electrophysiology and pharmacology to determine if prepulse inhibition (PPI is mediated by glycine receptors (GlyRs and/or GABAA receptors (GABAARs in the goldfish auditory startle circuit. Specifically, we used selective antagonists to dissect the contributions of target receptors on sound-evoked postsynaptic potentials (PSPs recorded in the neurons that initiate startle, the Mauthner-cells (M-cell. We found that strychnine, a GlyR antagonist, disrupted a fast-activated (5 ms and rapidly (< 50ms decaying (feed-forward inhibitory process that disrupts PPI at 20 ms prepulse/pulse inter-stimulus intervals (ISI. Additionally we observed increases of the evoked postsynaptic potential (PSP peak amplitude (+87.43 ± 21.53%; N=9 and duration (+204 ± 48.91%, N=9. In contrast, treatment with bicuculline, a GABAAR antagonist, caused a general reduction in PPI across all tested ISIs (20-500 ms, essentially eliminating PPI at ISIs from 20-100 ms. Bicuculline also increased PSP peak amplitude (+133.8 ± 10.3%, N=5 and PSP duration (+284.95 ± 65.64%, N=5. Treatment with either antagonist also tonically increased post-synaptic excitability in the M-cells, reflected by an increase in the magnitude of antidromically-evoked action potentials (APs by 15.07 ± 3.21%, N=7 and 16.23 ± 7.08%, N=5 for strychnine and bicuculline, respectively. These results suggest that GABAARs and GlyRs are functionally segregated to short- and longer-lasting sound-evoked (phasic inhibitory processes that contribute to PPI, with the mediation of tonic inhibition by both receptor systems being critical for gain control within the M-cell startle circuit.

  18. Edaravone inhibits pressure overload-induced cardiac fibrosis and dysfunction by reducing expression of angiotensin II AT1 receptor

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    Zhang WW

    2017-10-01

    Full Text Available Wei-Wei Zhang,1,2 Feng Bai,1 Jin Wang,1 Rong-Hua Zheng,1 Li-Wang Yang,1 Erskine A James,3 Zhi-Qing Zhao1,4 1Department of Physiology, Shanxi Medical University, 2Department of Anesthesiology, Shanxi Provincial People’s Hospital, Taiyuan, Shanxi, China; 3Department of Internal Medicine, Navicent Health, Macon, 4Department of Basic Biomedical Sciences, Mercer University School of Medicine, Savannah, GA, USA Abstract: Angiotensin II (Ang II is known to be involved in the progression of ventricular dysfunction and heart failure by eliciting cardiac fibrosis. The purpose of this study was to demonstrate whether treatment with an antioxidant compound, edaravone, reduces cardiac fibrosis and improves ventricular function by inhibiting Ang II AT1 receptor. The study was conducted in a rat model of transverse aortic constriction (TAC. In control, rats were subjected to 8 weeks of TAC. In treated rats, edaravone (10 mg/kg/day or Ang II AT1 receptor blocker, telmisartan (10 mg/kg/day was administered by intraperitoneal injection or gastric gavage, respectively, during TAC. Relative to the animals with TAC, edaravone reduced myocardial malonaldehyde level and increased superoxide dismutase activity. Protein level of the AT1 receptor was reduced and the AT2 receptor was upregulated, as evidenced by the reduced ratio of AT1 over AT2 receptor (0.57±0.2 vs 3.16±0.39, p<0.05 and less locally expressed AT1 receptor in the myocardium. Furthermore, the protein level of angiotensin converting enzyme 2 was upregulated. In coincidence with these changes, edaravone significantly decreased the populations of macrophages and myofibroblasts in the myocardium, which were accompanied by reduced levels of transforming growth factor beta 1 and Smad2/3. Collagen I synthesis was inhibited and collagen-rich fibrosis was attenuated. Relative to the TAC group, cardiac systolic function was preserved, as shown by increased left ventricular systolic pressure (204±51 vs 110±19

  19. Tissue-type plasminogen activator-binding RNA aptamers inhibiting low-density lipoprotein receptor family-mediated internalisation.

    Science.gov (United States)

    Bjerregaard, Nils; Bøtkjær, Kenneth A; Helsen, Nicky; Andreasen, Peter A; Dupont, Daniel M

    2015-07-01

    Recombinant tissue-type plasminogen activator (tPA, trade name Alteplase), currently the only drug approved by the US Food and Drug Administration and the European Medicines Agency for the treatment of cerebral ischaemic stroke, has been implicated in a number of adverse effects reportedly mediated by interactions with the low-density lipoprotein (LDL) family receptors, including neuronal cell death and an increased risk of cerebral haemorrhage. The tissue-type plasminogen activator is the principal initiator of thrombolysis in human physiology, an effect that is mediated directly via localised activation of the plasmin zymogen plasminogen at the surface of fibrin clots in the vascular lumen. Here, we sought to identify a ligand to tPA capable of inhibiting the relevant LDL family receptors without interfering with the fibrinolytic activity of tPA. Systematic evolution of ligands by exponential enrichment (SELEX) was employed to isolate tPA-binding RNA aptamers, which were characterised in biochemical assays of tPA association to low density lipoprotein receptor-related protein-1 (LRP-1, an LDL receptor family member); tPA-mediated in vitro and ex vivo clot lysis; and tPA-mediated plasminogen activation in the absence and presence of a stimulating soluble fibrin fragment. Two aptamers, K18 and K32, had minimal effects on clot lysis, but were able to efficiently inhibit tPA-LRP-1 association and LDL receptor family-mediated endocytosis in human vascular endothelial cells and astrocytes. These observations suggest that coadministration alongside tPA may be a viable strategy to improve the safety of thrombolytic treatment of cerebral ischaemic stroke by restricting tPA activity to the vascular lumen.

  20. Prostaglandin D2 inhibits airway dendritic cell migration and function in steady state conditions by selective activation of the D prostanoid receptor 1

    NARCIS (Netherlands)

    H. Hammad (Hamida); H.J. de Heer; T. Soullié (Thomas); H.C. Hoogsteden (Henk); F. Trottein; B.N.M. Lambrecht (Bart)

    2003-01-01

    textabstractPGD(2) is the major mediator released by mast cells during allergic responses, and it acts through two different receptors, the D prostanoid receptor 1 (DP1) and DP2, also known as CRTH2. Recently, it has been shown that PGD(2) inhibits the migration of epidermal

  1. Pancreatic and snake venom presynaptically active phospholipases A2 inhibit nicotinic acetylcholine receptors.

    Science.gov (United States)

    Vulfius, Catherine A; Kasheverov, Igor E; Kryukova, Elena V; Spirova, Ekaterina N; Shelukhina, Irina V; Starkov, Vladislav G; Andreeva, Tatyana V; Faure, Grazyna; Zouridakis, Marios; Tsetlin, Victor I; Utkin, Yuri N

    2017-01-01

    Phospholipases A2 (PLA2s) are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which should be proved by

  2. Pancreatic and snake venom presynaptically active phospholipases A2 inhibit nicotinic acetylcholine receptors.

    Directory of Open Access Journals (Sweden)

    Catherine A Vulfius

    Full Text Available Phospholipases A2 (PLA2s are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which

  3. Gefitinib Radiosensitizes Stem-Like Glioma Cells: Inhibition of Epidermal Growth Factor Receptor-Akt-DNA-PK Signaling, Accompanied by Inhibition of DNA Double-Strand Break Repair

    International Nuclear Information System (INIS)

    Kang, Khong Bee; Zhu Congju; Wong Yinling; Gao Qiuhan; Ty, Albert; Wong, Meng Cheong

    2012-01-01

    Purpose: We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)–Akt-DNA–dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Methods and Materials: Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, γ-H 2 AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, γ-H 2 AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Results: Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G 2 /M arrest and increased γ-H 2 AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased γ-H 2 AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Conclusions: Stem-like gliomaspheres are resistant to irradiation

  4. Gefitinib radiosensitizes stem-like glioma cells: inhibition of epidermal growth factor receptor-Akt-DNA-PK signaling, accompanied by inhibition of DNA double-strand break repair.

    Science.gov (United States)

    Kang, Khong Bee; Zhu, Congju; Wong, Yin Ling; Gao, Qiuhan; Ty, Albert; Wong, Meng Cheong

    2012-05-01

    We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, γ-H(2)AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, γ-H(2)AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G(2)/M arrest and increased γ-H(2)AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased γ-H(2)AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Stem-like gliomaspheres are resistant to irradiation-induced cytotoxicity, G(2)/M arrest, and DNA DSBs, compared with nonstem

  5. Gefitinib Radiosensitizes Stem-Like Glioma Cells: Inhibition of Epidermal Growth Factor Receptor-Akt-DNA-PK Signaling, Accompanied by Inhibition of DNA Double-Strand Break Repair

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Khong Bee, E-mail: dmskkb@nccs.com.sg [Brain Tumour Research Laboratory, Division of Medical Sciences, National Cancer Centre Singapore (Singapore); Zhu Congju; Wong Yinling; Gao Qiuhan; Ty, Albert; Wong, Meng Cheong [Brain Tumour Research Laboratory, Division of Medical Sciences, National Cancer Centre Singapore (Singapore)

    2012-05-01

    Purpose: We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Methods and Materials: Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, {gamma}-H{sub 2}AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, {gamma}-H{sub 2}AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Results: Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G{sub 2}/M arrest and increased {gamma}-H{sub 2}AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased {gamma}-H{sub 2}AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Conclusions: Stem-like gliomaspheres are

  6. Receptor for advanced glycation end products inhibits proliferation in osteoblast through suppression of Wnt, PI3K and ERK signaling

    International Nuclear Information System (INIS)

    Li, Guofeng; Xu, Jingren; Li, Zengchun

    2012-01-01

    Highlights: ► RAGE overexpression suppresses cell proliferation in MC3T3-E1 cells. ► RAGE overexpression decreases Wnt/β-catenin signaling. ► RAGE overexpression decreases ERK and PI3K signaling. ► Inhibition of Wnt signaling abolishes PI3K signaling restored by RAGE blockade. ► Inhibition of Wnt signaling abolishes ERK signaling restored by RAGE blockade. -- Abstract: Expression of receptor for advanced glycation end products (RAGE) plays a crucial role in bone metabolism. However, the role of RAGE in the control of osteoblast proliferation is not yet evaluated. In the present study, we demonstrate that RAGE overexpression inhibits osteoblast proliferation in vitro. The negative regulation of RAGE on cell proliferation results from suppression of Wnt, PI3K and ERK signaling, and is restored by RAGE neutralizing antibody. Prevention of Wnt signaling using Sfrp1 or DKK1 rescues RAGE-decreased PI3K and ERK signaling and cell proliferation, indicating that the altered cell growth in RAGE overexpressing cells is in part secondary to alterations in Wnt signaling. Consistently, RAGE overexpression inhibits the expression of Wnt targets cyclin D1 and c-myc, which is partially reversed by RAGE blockade. Overall, these results suggest that RAGE inhibits osteoblast proliferation via suppression of Wnt, PI3K and ERK signaling, which provides novel mechanisms by which RAGE regulates osteoblast growth.

  7. Receptor for advanced glycation end products inhibits proliferation in osteoblast through suppression of Wnt, PI3K and ERK signaling

    Energy Technology Data Exchange (ETDEWEB)

    Li, Guofeng [Department of Emergency Surgery, East Hospital, Tongji University School of Medicine, Shanghai 200120 (China); Xu, Jingren [Department of Traditional Chinese Orthopaedics, East Hospital, Tongji University School of Medicine, Shanghai 200120 (China); Li, Zengchun, E-mail: lizc.2007@yahoo.com.cn [Department of Emergency Surgery, East Hospital, Tongji University School of Medicine, Shanghai 200120 (China)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer RAGE overexpression suppresses cell proliferation in MC3T3-E1 cells. Black-Right-Pointing-Pointer RAGE overexpression decreases Wnt/{beta}-catenin signaling. Black-Right-Pointing-Pointer RAGE overexpression decreases ERK and PI3K signaling. Black-Right-Pointing-Pointer Inhibition of Wnt signaling abolishes PI3K signaling restored by RAGE blockade. Black-Right-Pointing-Pointer Inhibition of Wnt signaling abolishes ERK signaling restored by RAGE blockade. -- Abstract: Expression of receptor for advanced glycation end products (RAGE) plays a crucial role in bone metabolism. However, the role of RAGE in the control of osteoblast proliferation is not yet evaluated. In the present study, we demonstrate that RAGE overexpression inhibits osteoblast proliferation in vitro. The negative regulation of RAGE on cell proliferation results from suppression of Wnt, PI3K and ERK signaling, and is restored by RAGE neutralizing antibody. Prevention of Wnt signaling using Sfrp1 or DKK1 rescues RAGE-decreased PI3K and ERK signaling and cell proliferation, indicating that the altered cell growth in RAGE overexpressing cells is in part secondary to alterations in Wnt signaling. Consistently, RAGE overexpression inhibits the expression of Wnt targets cyclin D1 and c-myc, which is partially reversed by RAGE blockade. Overall, these results suggest that RAGE inhibits osteoblast proliferation via suppression of Wnt, PI3K and ERK signaling, which provides novel mechanisms by which RAGE regulates osteoblast growth.

  8. Inhibition of cisplatin-induced vomiting by selective 5-hydroxytryptamine M-receptor antagonism.

    OpenAIRE

    Miner, W. D.; Sanger, G. J.

    1986-01-01

    MDL 72222, the selective 5-hydroxytryptamine (5-HT) M-receptor antagonist, prevented or reduced cisplatin-induced emesis in ferrets. It is suggested that cisplatin-induced, and possibly other cytotoxic drug-induced vomiting may involve a 5-HT M-receptor mechanism.

  9. Coordinate expression of activating Fc gamma receptors I and III and inhibiting Fc gamma receptor type II in the determination of joint inflammation and cartilage destruction during immune complex-mediated arthritis.

    NARCIS (Netherlands)

    Nabbe, K.C.A.M.; Blom, A.B.; Holthuysen, A.E.M.; Boross, P.; Roth, J.; Verbeek, S.; Lent, P.L.E.M. van; Berg, W.B. van den

    2003-01-01

    OBJECTIVE: To study the role of the activating Fc gamma receptor types I and III (Fc gamma RI and Fc gamma RIII, respectively) and the inhibiting Fc gamma receptor II (Fc gamma RII) in inflammation and in various aspects of cartilage destruction during arthritis that is solely induced by immune

  10. Agmatine suppresses peripheral sympathetic tone by inhibiting N-type Ca(2+) channel activity via imidazoline I2 receptor activation.

    Science.gov (United States)

    Kim, Young-Hwan; Jeong, Ji-Hyun; Ahn, Duck-Sun; Chung, Seungsoo

    2016-08-26

    Agmatine, a putative endogenous ligand of imidazoline receptors, suppresses cardiovascular function by inhibiting peripheral sympathetic tone. However, the molecular identity of imidazoline receptor subtypes and its cellular mechanism underlying the agmatine-induced sympathetic suppression remains unknown. Meanwhile, N-type Ca(2+) channels are important for the regulation of NA release in the peripheral sympathetic nervous system. Therefore, it is possible that agmatine suppresses NA release in peripheral sympathetic nerve terminals by inhibiting Ca(2+) influx through N-type Ca(2+) channels. We tested this hypothesis by investigating agmatine effect on electrical field stimulation (EFS)-evoked contraction and NA release in endothelium-denuded rat superior mesenteric arterial strips. We also investigated the effect of agmatine on the N-type Ca(2+) current in superior cervical ganglion (SCG) neurons in rats. Our study demonstrates that agmatine suppresses peripheral sympathetic outflow via the imidazoline I2 receptor in rat mesenteric arteries. In addition, the agmatine-induced suppression of peripheral vascular sympathetic tone is mediated by modulating voltage-dependent N-type Ca(2+) channels in sympathetic nerve terminals. These results suggest a potential cellular mechanism for the agmatine-induced suppression of peripheral sympathetic tone. Furthermore, they provide basic and theoretical information regarding the development of new agents to treat hypertension. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Identification of a novel antagonist of the ErbB1 receptor capable of inhibiting migration of human glioblastoma cells

    DEFF Research Database (Denmark)

    Staberg, Mikkel; Riemer, Christian; Xu, Ruodan

    2013-01-01

    B1 targeting peptide, termed Herfin-1, was designed based on a model of the tertiary structure of the EGF-EGFR ternary complex. The binding kinetics of this peptide were determined employing surface plasmon resonance analyses. ErbB1-4 expression and phosphorylation in human glioblastoma cell lines U...... processing. RESULTS: The present study shows that Herfin-1 functions as an ErbB1 antagonist. It binds to the extracellular domain of ErbB1 with a KD value of 361 nM. In U87 and U118 cells, both expressing high levels of ErbB1, Herfin-1 inhibits EGF-induced ErbB1 phosphorylation and cell migration....... Additionally, Herfin-1 was found to increase neurite outgrowth in cerebellar granule neurons, likely through the inhibition of a sustained weak ErbB1 activation. CONCLUSIONS: Targeting the ErbB1 receptor dimerization interface is a promising strategy to inhibit receptor activation in ErbB1-expressing glioma...

  12. A peptide antagonist of ErbB receptors, Inherbin3, induces neurite outgrowth from rat cerebellar granule neurons through ErbB1 inhibition

    DEFF Research Database (Denmark)

    Xu, Ruodan; Pankratova, Stanislava; Christiansen, Søren Hofman

    2013-01-01

    ErbB receptors not only function in cancer, but are also key developmental regulators in the nervous system. We previously identified an ErbB1 peptide antagonist, Inherbin3, that is capable of inhibiting tumor growth in vitro and in vivo. In this study, we found that inhibition of ErbB1 kinase ac...

  13. Putative therapeutic targets for symptom subtypes of adult ADHD: D4 receptor agonism and COMT inhibition improve attention and response inhibition in a novel translational animal model.

    Science.gov (United States)

    Tomlinson, Anneka; Grayson, Ben; Marsh, Samuel; Hayward, Andrew; Marshall, Kay M; Neill, Joanna C

    2015-04-01

    Prefrontal cortical dopamine plays an important role in cognitive control, specifically in attention and response inhibition; the core deficits in ADHD. We have previously shown that methylphenidate and atomoxetine differentially improve these deficits dependent on baseline performance. The present study extends this work to investigate the effects of putative therapeutic targets in our model. A selective dopamine D4 receptor agonist (A-412997) and the catechol-O-methyl-transferase (COMT) inhibitor; tolcapone, were investigated in the combined subtype of adult ADHD (ADHD-C). Adult female rats were trained to criterion in the 5C-CPT (5-Choice Continuous Performance Task) and then separated into subgroups according to baseline levels of sustained attention, vigilance, and response disinhibition. The subgroups included: high-attentive (HA) and low-attentive with high response disinhibition (ADHD-C). The ADHD-C subgroup was selected to represent the combined subtype of adult ADHD. Effects of tolcapone (3.0, 10.0, 15.0mg/kg) and A-412997 (0.1, 0.3, 1.0µmol/kg) were tested by increasing the variable inter-trial-interval (ITI) duration in the 5C-CPT. Tolcapone (15mg/kg) significantly increased sustained attention, vigilance and response inhibition in ADHD-C animals, and impaired attention in HA animals. A-412997 (1.0µmol/kg) significantly increased vigilance and response inhibition in ADHD-C animals only, with no effect in HA animals. This is the first study to use the translational 5C-CPT to model the adult ADHD-C subtype in rats and to study new targets in this model. Both tolcapone and A-412997 increased vigilance and response inhibition in the ADHD-C subgroup. D4 and COMT are emerging as important potential therapeutic targets in adult ADHD that warrant further investigation. Copyright © 2014 Elsevier B.V. and ECNP. All rights reserved.

  14. The Antidepressant 5-HT2A Receptor Antagonists Pizotifen and Cyproheptadine Inhibit Serotonin-Enhanced Platelet Function

    Science.gov (United States)

    Lin, Olivia A.; Karim, Zubair A.; Vemana, Hari Priya; Espinosa, Enma V. P.; Khasawneh, Fadi T.

    2014-01-01

    There is considerable interest in defining new agents or targets for antithrombotic purposes. The 5-HT2A receptor is a G-protein coupled receptor (GPCR) expressed on many cell types, and a known therapeutic target for many disease states. This serotonin receptor is also known to regulate platelet function. Thus, in our FDA-approved drug repurposing efforts, we investigated the antiplatelet activity of cyproheptadine and pizotifen, two antidepressant 5-HT2A Receptor antagonists. Our results revealed that cyproheptadine and pizotifen reversed serotonin-enhanced ADP-induced platelet aggregation in vitro and ex vivo. And the inhibitory effects of these two agents were found to be similar to that of EMD 281014, a 5-HT2A Receptor antagonist under development. In separate experiments, our studies revealed that these 5-HT2A receptor antagonists have the capacity to reduce serotonin-enhanced ADP-induced elevation in intracellular calcium levels and tyrosine phosphorylation. Using flow cytometry, we also observed that cyproheptadine, pizotifen, and EMD 281014 inhibited serotonin-enhanced ADP-induced phosphatidylserine (PS) exposure, P-selectin expression, and glycoprotein IIb-IIIa activation. Furthermore, using a carotid artery thrombosis model, these agents prolonged the time for thrombotic occlusion in mice in vivo. Finally, the tail-bleeding time was investigated to assess the effect of cyproheptadine and pizotifen on hemostasis. Our findings indicated prolonged bleeding time in both cyproheptadine- and pizotifen-treated mice. Notably, the increases in occlusion and bleeding times associated with these two agents were comparable to that of EMD 281014, and to clopidogrel, a commonly used antiplatelet drug, again, in a fashion comparable to clopidogrel and EMD 281014. Collectively, our data indicate that the antidepressant 5-HT2A antagonists, cyproheptadine and pizotifen do exert antiplatelet and thromboprotective effects, but similar to clopidogrel and EMD 281014, their

  15. Compensatory insulin receptor (IR) activation on inhibition of insulin-like growth factor-1 receptor (IGF-1R): rationale for cotargeting IGF-1R and IR in cancer.

    Science.gov (United States)

    Buck, Elizabeth; Gokhale, Prafulla C; Koujak, Susan; Brown, Eric; Eyzaguirre, Alexandra; Tao, Nianjun; Rosenfeld-Franklin, Maryland; Lerner, Lorena; Chiu, M Isabel; Wild, Robert; Epstein, David; Pachter, Jonathan A; Miglarese, Mark R

    2010-10-01

    Insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) and critical activator of the phosphatidylinositol 3-kinase-AKT pathway. IGF-1R is required for oncogenic transformation and tumorigenesis. These observations have spurred anticancer drug discovery and development efforts for both biological and small-molecule IGF-1R inhibitors. The ability for one RTK to compensate for another to maintain tumor cell viability is emerging as a common resistance mechanism to antitumor agents targeting individual RTKs. As IGF-1R is structurally and functionally related to the insulin receptor (IR), we asked whether IR is tumorigenic and whether IR-AKT signaling contributes to resistance to IGF-1R inhibition. Both IGF-1R and IR(A) are tumorigenic in a mouse mammary tumor model. In human tumor cells coexpressing IGF-1R and IR, bidirectional cross talk was observed following either knockdown of IR expression or treatment with a selective anti-IGF-1R antibody, MAB391. MAB391 treatment resulted in a compensatory increase in phospho-IR, which was associated with resistance to inhibition of IRS1 and AKT. In contrast, treatment with OSI-906, a small-molecule dual inhibitor of IGF-1R/IR, resulted in enhanced reduction in phospho-IRS1/phospho-AKT relative to MAB391. Insulin or IGF-2 activated the IR-AKT pathway and decreased sensitivity to MAB391 but not to OSI-906. In tumor cells with an autocrine IGF-2 loop, both OSI-906 and an anti-IGF-2 antibody reduced phospho-IR/phospho-AKT, whereas MAB391 was ineffective. Finally, OSI-906 showed superior efficacy compared with MAB391 in human tumor xenograft models in which both IGF-1R and IR were phosphorylated. Collectively, these data indicate that cotargeting IGF-1R and IR may provide superior antitumor efficacy compared with targeting IGF-1R alone.

  16. Hippocampal low-frequency stimulation inhibits afterdischarge and increases GABA (A) receptor expression in amygdala-kindled pharmacoresistant epileptic rats.

    Science.gov (United States)

    Wu, Guofeng; Wang, Likun; Hong, Zhen; Ren, Siying; Zhou, Feng

    2017-08-01

    The purpose of the present study was to observe the effects of hippocampal low-frequency stimulation (Hip-LFS) on amygdala afterdischarge and GABA (A) receptor expression in pharmacoresistant epileptic (PRE) rats. A total of 110 healthy adult male Wistar rats were used to generate a model of epilepsy by chronic stimulation of the amygdala. Sixteen PRE rats were selected from 70 amygdala-kindled rats by testing their response to Phenytoin and Phenobarbital, and they were randomly assigned to a pharmacoresistant stimulation group (PRS group, 8 rats) or a pharmacoresistant control group (PRC group, 8 rats). A stimulation electrode was implanted into the hippocampus of all of the rats. Hip-LFS was administered twice per day in the PRS group for two weeks. Simultaneously, amygdala stimulus-induced seizures and afterdischarge were recorded. After the hippocampal stimulation was terminated, the brain tissues were obtained to determine the GABA (A) receptors by a method of immumohistochemistry and a real-time polymerase chain reaction. The stages and duration of the amygdala stimulus-induced epileptic seizures were decreased in the PRS group. The afterdischarge threshold was increased and the duration as well as the afterdischarge frequency was decreased. Simultaneously, the GABA (A) expression was significantly increased in the PRS group. Hip-LFS may inhibit amygdala stimulus-induced epileptic seizures and up-regulate GABA (A) receptor expression in PRE rats. The antiepileptic effects of hippocampal stimulation may be partly achieved by increasing the GABA (A) receptor.

  17. Inhibition of transglutaminase 2 reduces efferocytosis in human macrophages: Role of CD14 and SR-AI receptors.

    Science.gov (United States)

    Eligini, S; Fiorelli, S; Tremoli, E; Colli, S

    2016-10-01

    Transglutaminase 2 (TGM2), a member of the transglutaminase family of enzymes, is a multifunctional protein involved in numerous events spanning from cell differentiation, to signal transduction, apoptosis, and wound healing. It is expressed in a variety of cells, macrophages included. Macrophage TGM2 promotes the clearance of apoptotic cells (efferocytosis) and emerging evidence suggests that defective efferocytosis contributes to the consequences of inflammation-associated diseases, including atherosclerotic lesion progression and its sequelae. Of interest, active TGM2 identified in human atherosclerotic lesions plays critical roles in plaque stability through effects on matrix cross-linking and TGFβ activity. This study explores the mechanisms by which TGM2 controls efferocytosis in human macrophages. Herein we show that TGM2 increases progressively during monocyte differentiation towards macrophages and controls their efferocytic potential as well as morphology and viability. Two experimental approaches that took advantage of the inhibition of TGM2 activity and protein silencing give proof that TGM2 reduction significantly impairs macrophage efferocytosis. Among the mechanisms involved we highlighted a role of the receptors CD14 and SR-AI whose levels were markedly reduced by TGM2 inhibition. Conversely, CD36 receptor and αvβ3 integrin levels were not influenced. Of note, lipid accumulation and IL-10 secretion were reduced in macrophages displaying defective efferocytosis. Overall, our data define a crucial role of TGM2 activity during macrophage differentiation via mechanisms involving CD14 and SR-AI receptors and show that TGM2 inhibition triggers a pro-inflammatory phenotype. Copyright © 2016 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.

  18. A point mutation in the hair cell nicotinic cholinergic receptor prolongs cochlear inhibition and enhances noise protection.

    Directory of Open Access Journals (Sweden)

    Julian Taranda

    2009-01-01

    Full Text Available The transduction of sound in the auditory periphery, the cochlea, is inhibited by efferent cholinergic neurons projecting from the brainstem and synapsing directly on mechanosensory hair cells. One fundamental question in auditory neuroscience is what role(s this feedback plays in our ability to hear. In the present study, we have engineered a genetically modified mouse model in which the magnitude and duration of efferent cholinergic effects are increased, and we assess the consequences of this manipulation on cochlear function. We generated the Chrna9L9'T line of knockin mice with a threonine for leucine change (L9'T at position 9' of the second transmembrane domain of the alpha9 nicotinic cholinergic subunit, rendering alpha9-containing receptors that were hypersensitive to acetylcholine and had slower desensitization kinetics. The Chrna9L9'T allele produced a 3-fold prolongation of efferent synaptic currents in vitro. In vivo, Chrna9L9'T mice had baseline elevation of cochlear thresholds and efferent-mediated inhibition of cochlear responses was dramatically enhanced and lengthened: both effects were reversed by strychnine blockade of the alpha9alpha10 hair cell nicotinic receptor. Importantly, relative to their wild-type littermates, Chrna9(L9'T/L9'T mice showed less permanent hearing loss following exposure to intense noise. Thus, a point mutation designed to alter alpha9alpha10 receptor gating has provided an animal model in which not only is efferent inhibition more powerful, but also one in which sound-induced hearing loss can be restrained, indicating the ability of efferent feedback to ameliorate sound trauma.

  19. Nitric oxide from inflammatory origin impairs neural stem cell proliferation by inhibiting epidermal growth factor receptor signaling

    Directory of Open Access Journals (Sweden)

    Bruno Pereira Carreira

    2014-10-01

    Full Text Available Neuroinflammation is characterized by activation of microglial cells, followed by production of nitric oxide (NO, which may have different outcomes on neurogenesis, favoring or inhibiting this process. In the present study, we investigated how the inflammatory mediator NO can affect proliferation of neural stem cells (NSC, and explored possible mechanisms underlying this effect. We investigated which mechanisms are involved in the regulation of NSC proliferation following treatment with an inflammatory stimulus (LPS plus IFN-γ, using a culture system of subventricular zone (SVZ-derived NSC mixed with microglia cells obtained from wild-type mice (iNOS+/+ or from iNOS knockout mice (iNOS-/-. We found an impairment of NSC cell proliferation in iNOS+/+ mixed cultures, which was not observed in iNOS-/- mixed cultures. Furthermore, the increased release of NO by activated iNOS+/+ microglial cells decreased the activation of the ERK/MAPK signaling pathway, which was concomitant with an enhanced nitration of the EGF receptor. Preventing nitrogen reactive species formation with MnTBAP, a scavenger of peroxynitrite, or using the peroxynitrite degradation catalyst FeTMPyP, cell proliferation and ERK signaling were restored to basal levels in iNOS+/+ mixed cultures. Moreover, exposure to the NO donor NOC-18 (100 µM, for 48 h, inhibited SVZ-derived NSC proliferation. Regarding the antiproliferative effect of NO, we found that NOC-18 caused the impairment of signaling through the ERK/MAPK pathway, which may be related to increased nitration of the EGF receptor in NSC. Using MnTBAP nitration was prevented, maintaining ERK signaling, rescuing NSC proliferation. We show that NO from inflammatory origin leads to a decreased function of the EGF receptor, which compromised proliferation of NSC. We also demonstrated that NO-mediated nitration of the EGF receptor caused a decrease in its phosphorylation, thus preventing regular proliferation signaling through the

  20. Inhibition of CPU0213, a Dual Endothelin Receptor Antagonist, on Apoptosis via Nox4-Dependent ROS in HK-2 Cells

    Directory of Open Access Journals (Sweden)

    Qing Li

    2016-06-01

    Full Text Available Background/Aims: Our previous studies have indicated that a novel endothelin receptor antagonist CPU0213 effectively normalized renal function in diabetic nephropathy. However, the molecular mechanisms mediating the nephroprotective role of CPU0213 remain unknown. Methods and Results: In the present study, we first detected the role of CPU0213 on apoptosis in human renal tubular epithelial cell (HK-2. It was shown that high glucose significantly increased the protein expression of Bax and decreased Bcl-2 protein in HK-2 cells, which was reversed by CPU0213. The percentage of HK-2 cells that showed Annexin V-FITC binding was markedly suppressed by CPU0213, which confirmed the inhibitory role of CPU0213 on apoptosis. Given the regulation of endothelin (ET system to oxidative stress, we determined the role of redox signaling in the regulation of CPU0213 on apoptosis. It was demonstrated that the production of superoxide (O2-. was substantially attenuated by CPU0213 treatment in HK-2 cells. We further found that CPU0213 dramatically inhibited expression of Nox4 protein, which gene silencing mimicked the role of CPU0213 on the apoptosis under high glucose stimulation. We finally examined the role of CPU0213 on ET-1 receptors and found that high glucose-induced protein expression of endothelin A and B receptors was dramatically inhibited by CPU0213. Conclusion: Taken together, these results suggest that this Nox4-dependenet O2- production is critical for the apoptosis of HK-2 cells in high glucose. Endothelin receptor antagonist CPU0213 has an anti-apoptosis role through Nox4-dependent O2-.production, which address the nephroprotective role of CPU0213 in diabetic nephropathy.

  1. Estradiol receptors mediate estradiol-induced inhibition of mitochondrial Ca^{2+} efflux in rat caudate nucleus and brain stem

    OpenAIRE

    PETROVIC, SNJEZANA; MILOSEVIC, MAJA; RISTIC-MEDIC, DANIJELA; VELICKOVIC, NATASA; DRAKULIC, DUNJA; GRKOVIC, IVANA; HORVAT, ANICA

    2015-01-01

    Our earlier studies found that in vitro estradiol modulates mitochondrial Ca2+ transport in discrete brain regions. The present study examined the role of estradiol receptors (ERs) in estradiol-induced inhibition of Ca^{2+} efflux from synaptosomal mitochondria isolated from rat caudate nuclei and brain stems. Radioactively labeled CaCl_2 (0.6?0.75 µCi ^45CaCl_{2}) was used for Ca^{2+} transport monitoring. The results revealed that in the presence of ER antagonist 7\\alpha,17ß-[9[(4,4,5,5,5-...

  2. 5-Hydroxytryptamine 1A/7 and 4alpha receptors differentially prevent opioid-induced inhibition of brain stem cardiorespiratory function.

    Science.gov (United States)

    Wang, Xin; Dergacheva, Olga; Kamendi, Harriet; Gorini, Christopher; Mendelowitz, David

    2007-08-01

    Opioids evoke respiratory depression, bradycardia, and reduced respiratory sinus arrhythmia, whereas serotonin (5-HT) agonists stimulate respiration and cardiorespiratory interactions. This study tested whether serotonin agonists can prevent the inhibitory effects of opioids on cardiorespiratory function. Spontaneous and rhythmic inspiratory-related activity and gamma-aminobutyric acid (GABA) neurotransmission to premotor parasympathetic cardioinhibitory neurons in the nucleus ambiguus were recorded simultaneously in an in vitro thick slice preparation. The mu-opioid agonist fentanyl inhibited respiratory frequency. The 5-hydroxytryptamine 1A/7 receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin increased respiratory frequency by itself and also prevented the fentanyl-induced respiratory depression. The 5-hydroxytryptamine 4alpha agonist BIMU-8 did not by itself change inspiratory activity but prevented the mu-opioid-mediated respiratory depression. Both spontaneous and inspiratory-evoked GABAergic neurotransmission to cardiac vagal neurons were inhibited by fentanyl. 8-Hydroxy-2-(di-n-propylamino)tetralin inhibited spontaneous but not inspiratory-evoked GABAergic activity to parasympathetic cardiac neurons. However, 8-hydroxy-2-(di-n-propylamino)tetralin differentially altered the opioid-mediated depression of inspiratory-evoked GABAergic activity but did not change the opioid-induced reduction in spontaneous GABAergic neurotransmission. In contrast, BIMU-8 did not alter GABAergic neurotransmission to cardiac vagal neurons by itself but prevented the fentanyl depression of both spontaneous and inspiratory-elicited GABAergic neurotransmission to cardiac vagal neurons. In the presence of tetrodotoxin, the inhibition of GABAergic inhibitory postsynaptic currents with fentanyl is prevented by coapplication of BIMU-8, indicating that BIMU-8 acts at presynaptic GABAergic terminals to prevent fentanyl-induced depression. These results suggest that activation of 5

  3. Inhibition of the 26S proteasome blocks progesterone receptor-dependent transcription through failed recruitment of RNA polymerase II.

    Science.gov (United States)

    Dennis, Andrew P; Lonard, David M; Nawaz, Zafar; O'Malley, Bert W

    2005-03-01

    In the present study, we investigated the involvement of protein degradation via the 26S proteasome during progesterone receptor (PR)-mediated transcription in T-47D cells containing a stably integrated MMTV-CAT reporter construct (CAT0 cells). Progesterone induced CAT and HSD11beta2 transcription while co-treatment with the proteasome inhibitor, MG132, blocked PR-induced transcription in a time-dependent fashion. MG132 treatment also inhibited transcription of beta-actin and cyclophilin, but not two proteasome subunit genes, PSMA1 and PSMC1, indicating that proteasome inhibition affects a subset of RNA polymerase II (RNAP(II))-regulated genes. Progesterone-mediated recruitment of RNAP(II) was blocked by MG132 treatment at time points later than 1 h that was not dependent on the continued presence of PR, associated cofactors, and components of the general transcription machinery, supporting the concept that proteasome-mediated degradation is needed for continued transcription. Surprisingly, progesterone-mediated acetylation of histone H4 was inhibited by MG132 with the concomitant recruitment of HDAC3, NCoR, and SMRT. We demonstrate that the steady-state protein levels of SMRT and NCoR are higher in the presence of MG132 in CAT0 cells, consistent with other reports that SMRT and NCoR are targets of the 26S proteasome. However, inhibition of histone deacetylation by trichostatin A (TSA) treatment or SMRT/NCoR knockdown by siRNA did not restore MG132-inhibited progesterone-dependent transcription. Therefore, events other than histone deacetylation and stability of SMRT and NCoR must also play a role in inhibition of PR-mediated transcription.

  4. Therapeutic Targeting of AXL Receptor Tyrosine Kinase Inhibits Tumor Growth and Intraperitoneal Metastasis in Ovarian Cancer Models

    Directory of Open Access Journals (Sweden)

    Pinar Kanlikilicer

    2017-12-01

    Full Text Available Despite substantial improvements in the treatment strategies, ovarian cancer is still the most lethal gynecological malignancy. Identification of drug treatable therapeutic targets and their safe and effective targeting is critical to improve patient survival in ovarian cancer. AXL receptor tyrosine kinase (RTK has been proposed to be an important therapeutic target for metastatic and advanced-stage human ovarian cancer. We found that AXL-RTK expression is associated with significantly shorter patient survival based on the The Cancer Genome Atlas patient database. To target AXL-RTK, we developed a chemically modified serum nuclease-stable AXL aptamer (AXL-APTAMER, and we evaluated its in vitro and in vivo antitumor activity using in vitro assays as well as two intraperitoneal animal models. AXL-aptamer treatment inhibited the phosphorylation and the activity of AXL, impaired the migration and invasion ability of ovarian cancer cells, and led to the inhibition of tumor growth and number of intraperitoneal metastatic nodules, which was associated with the inhibition of AXL activity and angiogenesis in tumors. When combined with paclitaxel, in vivo systemic (intravenous [i.v.] administration of AXL-aptamer treatment markedly enhanced the antitumor efficacy of paclitaxel in mice. Taken together, our data indicate that AXL-aptamers successfully target in vivo AXL-RTK and inhibit its AXL activity and tumor growth and progression, representing a promising strategy for the treatment of ovarian cancer.

  5. Curcumin Protects against Atherosclerosis in Apolipoprotein E-Knockout Mice by Inhibiting Toll-like Receptor 4 Expression.

    Science.gov (United States)

    Zhang, Shanshan; Zou, Jun; Li, Peiyang; Zheng, Xiumei; Feng, Dan

    2018-01-17

    Toll-like receptor 4 (TLR4) has been reported to play a critical role in the pathogenesis of atherosclerosis, the current study aimed to investigate whether curcumin suppresses atherosclerosis development in ApoE-knockout (ApoE -/- ) mice by inhibiting TLR4 expression. ApoE -/- mice were fed a high-fat diet supplemented with or without curcumin (0.1% w/w) for 16 weeks. Curcumin supplementation significantly reduced TLR4 expression and macrophage infiltration in atherosclerotic plaques. Curcumin also reduced aortic interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression, nuclear factor-κB (NF-κB) activity, and plasma IL-1β, TNF-α, soluble VCAM-1 and ICAM-1 levels. In addition, aortic sinus sections revealed that curcumin treatment reduced the extent of atherosclerotic lesions and inhibited atherosclerosis development. In vitro, curcumin inhibited NF-κB activation in macrophages and reduced TLR4 expression induced by lipopolysaccharide. Our results indicate that curcumin protects against atherosclerosis at least partially by inhibiting TLR4 expression and its related inflammatory reaction.

  6. Competitive inhibition of the nondepolarizing muscle relaxant rocuronium on nicotinic acetylcholine receptor channels in the rat superior cervical ganglia.

    Science.gov (United States)

    Zhang, Chengmi; Wang, Zhenmeng; Zhang, Jinmin; Qiu, Haibo; Sun, Yuming; Yang, Liqun; Wu, Feixiang; Zheng, Jijian; Yu, Weifeng

    2014-05-01

    A number of case reports now indicate that rocuronium can induce a number of serious side effects. We hypothesized that these side effects might be mediated by the inhibition of nicotinic acetylcholine receptors (nAChRs) at superior cervical ganglion (SCG) neurons. Conventional patch clamp recordings were used to study the effects of rocuronium on nAChR currents from enzymatically dissociated rat SCG neurons. We found that ACh induced a peak transient inward current in rat SCG neurons. Additionally, rocuronium suppressed the peak ACh-evoked currents in rat SCG neurons in a concentration-dependent and competitive manner, and it increased the extent of desensitization of nAChRs. The inhibitory rate of rocuronium on nAChR currents did not change significantly at membrane potentials between -70 and -20 mV, suggesting that this inhibition was voltage independent. Lastly, rocuronium preapplication enhanced its inhibitory effect, indicating that this drug might prefer to act on the closed state of nAChR channels. In conclusion, rocuronium, at clinically relevant concentrations, directly inhibits nAChRs at the SCG by interacting with both opened and closed states. This inhibition is competitive, dose dependent, and voltage independent. Blockade of synaptic transmission in the sympathetic ganglia by rocuronium might have potentially inhibitory effects on the cardiovascular system.

  7. Downregulation of TGF-β Receptor-2 Expression and Signaling through Inhibition of Na/K-ATPase.

    Directory of Open Access Journals (Sweden)

    Jennifer La

    Full Text Available Transforming growth factor-beta (TGF-β is a multi-functional cytokine implicated in the control of cell growth and differentiation. TGF-β signals through a complex of TGF-β receptors 1 and 2 (TGFβR1 and TGFβR2 that phosphorylate and activate Smad2/3 transcription factors driving transcription of the Smad-target genes. The Na+/K+-ATPase is an integral plasma membrane protein critical for maintaining the electro-chemical gradient of Na+ and K+ in the cell. We found that inhibition of the Na+/K+ ATPase by ouabain results in a dramatic decrease in the expression of TGFβR2 in human lung fibrobalsts (HLF at the mRNA and protein levels. This was accompanied by inhibition of TGF-β-induced Smad phosphorylation and the expression of TGF-β target genes, such as fibronectin and smooth muscle alpha-actin. Inhibition of Na+/K+ ATPase by an alternative approach (removal of extracellular potassium had a similar effect in HLF. Finally, treatment of lung alveolar epithelial cells (A549 with ouabain also resulted in the downregulation of TGFβR2, the inhibition of TGF-β-induced Smad phosphorylation and of the expression of mesenchymal markers, vimentin and fibronectin. Together, these data demonstrate a critical role of Na+/K+-ATPase in the control of TGFβR2 expression, TGF-β signaling and cell responses to TGF-β.

  8. Resveratrol, piceatannol and analogs inhibit activation of both wild-type and T877A mutant androgen receptor.

    Science.gov (United States)

    Lundqvist, Johan; Tringali, Corrado; Oskarsson, Agneta

    2017-11-01

    Prostate cancer growth and progression are mainly dependent on androgens and many current prostate cancer treatment options target the synthesis or function of androgens. We have previously reported that resveratrol and synthetic analogs of resveratrol with a higher bioavailability inhibit the synthesis of androgens in human adrenocortical H295R cells. Now we have studied the antiandrogenic properties of resveratrol, piceatannol and analogs in two different prostate cell lines; LNCaP and RWPE. LNCaP carry a T877A mutation in the androgen receptor while RWPE has a wild-type androgen receptor. We found that resveratrol, piceatannol and all studied analogs were able to inhibit a dihydrotestosterone-induced activation of the androgen receptor, showing that they act as antiandrogens. In LNCaP cells, all studied compounds were able to statistically significantly decrease the androgenic signaling in concentrations ≥1μM and the synthetic analogs trimethylresveratrol (RSVTM) and tetramethylpiceatannol (PICTM) were the most potent compounds. RWPE cells were not as responsive to the studied compounds as the LNCaP cells. A statistically significant decrease in the androgenic signaling was observed at concentrations ≤5μM for most compounds and RSVTM was found to be the most potent compound. Further, we studied the effects of resveratrol, piceatannol and analogs on the levels of prostate-specific antigen (PSA) in LNCaP cells and found that all studied compounds decreased the level of PSA and that the synthetic analogs diacetylresveratrol (RSVDA), triacetylresveratrol (RSVTA) and RSVTM were the most potent compounds, decreasing the PSA level by approx. 50% at concentrations ≥10μM. In a cell-free receptor binding assay we were unable to show binding of resveratrol or analogs to the ligand binding domain of the androgen receptor, indicating that the observed effects are mediated via other mechanisms than direct ligand competition. We conclude that the resveratrol

  9. Nanoparticle-Encapsulated Curcumin Inhibits Diabetic Neuropathic Pain Involving the P2Y12 Receptor in the Dorsal Root Ganglia

    Directory of Open Access Journals (Sweden)

    Tianyu Jia

    2018-01-01

    Full Text Available Diabetic peripheral neuropathy results in diabetic neuropathic pain (DNP. Satellite glial cells (SGCs enwrap the neuronal soma in the dorsal root ganglia (DRG. The purinergic 2 (P2 Y12 receptor is expressed on SGCs in the DRG. SGC activation plays an important role in the pathogenesis of DNP. Curcumin has anti-inflammatory and antioxidant properties. Because curcumin has poor metabolic stability in vivo and low bioavailability, nanoparticle-encapsulated curcumin was used to improve its targeting and bioavailability. In the present study, our aim was to investigate the effects of nanoparticle-encapsulated curcumin on DNP mediated by the P2Y12 receptor on SGCs in the rat DRG. Diabetic peripheral neuropathy increased the expression levels of the P2Y12 receptor on SGCs in the DRG and enhanced mechanical and thermal hyperalgesia in rats with diabetes mellitus (DM. Up-regulation of the P2Y12 receptor in SGCs in the DRG increased the production of pro-inflammatory cytokines. Up-regulation of interleukin-1β (IL-1β and connexin43 (Cx43 resulted in mechanical and thermal hyperalgesia in rats with DM. The nanoparticle-encapsulated curcumin decreased up-regulated IL-1β and Cx43 expression and reduced levels of phosphorylated-Akt (p-Akt in the DRG of rats with DM. The up-regulation of P2Y12 on SGCs and the up-regulation of the IL-1β and Cx43 in the DRG indicated the activation of SGCs in the DRG. The nano-curcumin treatment inhibited the activation of SGCs accompanied by its anti-inflammatory effect to decrease the up-regulated CGRP expression in the DRG neurons. Therefore, the nanoparticle-encapsulated curcumin treatment decreased the up-regulation of the P2Y12 receptor on SGCs in the DRG and decreased mechanical and thermal hyperalgesia in rats with DM.

  10. Combined treatment with a β3 -adrenergic receptor agonist and a muscarinic receptor antagonist inhibits detrusor overactivity induced by cold stress in spontaneously hypertensive rats.

    Science.gov (United States)

    Imamura, Tetsuya; Ogawa, Teruyuki; Minagawa, Tomonori; Nagai, Takashi; Suzuki, Toshiro; Saito, Tetsuichi; Yokoyama, Hitoshi; Nakazawa, Masaki; Ishizuka, Osamu

    2017-04-01

    This study determined if combined treatment with the muscarinic receptor (MR) antagonist solifenacin and the β 3 -adrenergic receptor (AR) agonist mirabegron could inhibit detrusor overactivity induced by cold stress in spontaneously hypertensive rats (SHRs). Thirty-two female 10-week-old SHRs were fed an 8% NaCl-supplemented diet for 4 weeks. Cystometric measurements of the unanesthetized, unrestricted rats were performed at room temperature (RT, 27 ± 2°C) for 20 min. The rats were then intravenously administered vehicle, 0.1 mg/kg solifenacin alone, 0.1 mg/kg mirabegron alone, or the combination of 0.1 mg/kg mirabegron and 0.1 mg/kg solifenacin (n = 8 each group). Five minutes later, the treated rats were exposed to low temperature (LT, 4 ± 2°C) for 40 min. Finally, the rats were returned to RT. After the cystometric investigations, the β 3 -ARs and M 3 -MRs expressed within the urinary bladders were analyzed. Just after transfer from RT to LT, vehicle-, solifenacin-, and mirabegron-treated SHRs exhibited detrusor overactivity that significantly decreased voiding interval and bladder capacity. However, treatment with the combination of solifenacin and mirabegron partially inhibited the cold stress-induced detrusor overactivity patterns. The decreases of voiding interval and bladder capacity in the combination-treated rats were significantly inhibited compared to other groups. Within the urinary bladders, there were no differences between expression levels of M 3 -MR and β 3 -AR mRNA. The tissue distribution of M 3 -MRs was similar to that of the β 3 -ARs. This study suggested that the combination of solifenacin and mirabegron act synergistically to inhibit the cold stress-induced detrusor overactivity in SHRs. Neurourol. Urodynam. 36:1026-1033, 2017. © 2016 The Authors. Neurourology and Urodynamics Published by Wiley Periodicals, Inc. © 2016 The Authors. Neurourology and Urodynamics Published by Wiley Periodicals, Inc.

  11. beta-TrCP inhibition reduces prostate cancer cell growth via upregulation of the aryl hydrocarbon receptor.

    Directory of Open Access Journals (Sweden)

    Udi Gluschnaider

    2010-02-01

    Full Text Available Prostate cancer is a common and heterogeneous disease, where androgen receptor (AR signaling plays a pivotal role in development and progression. The initial treatment for advanced prostate cancer is suppression of androgen signaling. Later on, essentially all patients develop an androgen independent stage which does not respond to anti hormonal treatment. Thus, alternative strategies targeting novel molecular mechanisms are required. beta-TrCP is an E3 ligase that targets various substrates essential for many aspects of tumorigenesis.Here we show that beta-TrCP depletion suppresses prostate cancer and identify a relevant growth control mechanism. shRNA targeted against beta-TrCP reduced prostate cancer cell growth and cooperated with androgen ablation in vitro and in vivo. We found that beta-TrCP inhibition leads to upregulation of the aryl hydrocarbon receptor (AhR mediating the therapeutic effect. This phenomenon could be ligand independent, as the AhR ligand 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD did not alter prostate cancer cell growth. We detected high AhR expression and activation in basal cells and atrophic epithelial cells of human cancer bearing prostates. AhR expression and activation is also significantly higher in tumor cells compared to benign glandular epithelium.Together these observations suggest that AhR activation may be a cancer counteracting mechanism in the prostate. We maintain that combining beta-TrCP inhibition with androgen ablation could benefit advanced prostate cancer patients.

  12. S-layer proteins from Lactobacillus sp. inhibit bacterial infection by blockage of DC-SIGN cell receptor.

    Science.gov (United States)

    Prado Acosta, Mariano; Ruzal, Sandra M; Cordo, Sandra M

    2016-11-01

    Many species of Lactobacillus sp. possess Surface(s) layer proteins in their envelope. Among other important characteristics S-layer from Lactobacillus acidophilus binds to the cellular receptor DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; CD209), which is involved in adhesion and infection of several families of bacteria. In this report we investigate the activity of new S-layer proteins from the Lactobacillus family (Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus helveticus and Lactobacillus kefiri) over the infection of representative microorganisms important to human health. After the treatment of DC-SIGN expressing cells with these proteins, we were able to diminish bacterial infection by up to 79% in both gram negative and mycobacterial models. We discovered that pre-treatment of the bacteria with S-layers from Lactobacillus acidophilus and Lactobacillus brevis reduced bacteria viability but also prevent infection by the pathogenic bacteria. We also proved the importance of the glycosylation of the S-layer from Lactobacillus kefiri in the binding to the receptor and thus inhibition of infection. This novel characteristic of the S-layers proteins may contribute to the already reported pathogen exclusion activity for these Lactobacillus probiotic strains; and might be also considered as a novel enzymatic antimicrobial agents to inhibit bacterial infection and entry to host cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Gemfibrozil, a lipid lowering drug, inhibits the activation of primary human microglia via peroxisome proliferator-activated receptor β.

    Science.gov (United States)

    Jana, Malabendu; Pahan, Kalipada

    2012-08-01

    Microglial activation participates in the pathogenesis of various neuroinflammatory and neurodegenerative diseases. However, mechanisms by which microglial activation could be controlled are poorly understood. Peroxisome proliferator-activated receptors (PPAR) are transcription factors belonging to the nuclear receptor super family with diverse effect. This study underlines the importance of PPARβ/δ in mediating the anti-inflammatory effect of gemfibrozil, an FDA-approved lipid-lowering drug, in primary human microglia. Bacterial lipopolysachharides (LPS) induced the expression of various proinflammatory molecules and upregulated the expression of microglial surface marker CD11b in human microglia. However, gemfibrozil markedly suppressed proinflammatory molecules and CD11b in LPS-stimulated microglia. Human microglia expressed PPAR-β and -γ, but not PPAR-α. Interestingly, either antisense knockdown of PPAR-β or antagonism of PPAR-β by a specific chemical antagonist abrogated gemfibrozil-mediated inhibition of microglial activation. On the other hand, blocking of PPAR-α and -γ had no effect on gemfibrozil-mediated anti-inflammatory effect in microglia. These results highlight the fact that gemfibrozil regulates microglial activation by inhibiting inflammatory gene expression in a PPAR-β dependent pathway and further reinforce its therapeutic application in several neuroinflammatory and neurodegenerative diseases.

  14. Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

    DEFF Research Database (Denmark)

    Jacob, K K; Sap, J; Stanley, F M

    1998-01-01

    A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin...... is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due...... to reduced phosphorylation or kinase activity of the insulin receptor or to reduced phosphorylation of insulin receptor substrate-1 or Shc. Inhibitor studies suggest that insulin-increased prolactin gene expression is mediated by a Ras-like GTPase but is not mitogen-activated protein kinase dependent...

  15. Regulation of ITAM adaptor molecules and their receptors by inhibition of calcineurin-NFAT signalling during late stage osteoclast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Zawawi, M.S.F. [Universiti Sains Malaysia (USM) (Malaysia); Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005 (Australia); Dharmapatni, A.A.S.S.K.; Cantley, M.D. [Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005 (Australia); McHugh, K.P. [University of Florida, College of Dentistry, Fl (United States); Haynes, D.R. [Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005 (Australia); Crotti, T.N., E-mail: tania.crotti@adelaide.edu.au [Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005 (Australia)

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Calcineurin/NFAT inhibitors FK506 and VIVIT treated human PBMC derived osteoclasts in vitro. Black-Right-Pointing-Pointer Differential regulation of ITAM receptors and adaptor molecules by calcineurin/NFAT inhibitors. Black-Right-Pointing-Pointer FK506 and VIVIT suppress ITAM factors during late phase osteoclast differentiation. -- Abstract: Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway in osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcR{gamma}) and DNAX-activating protein 12 kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin ({beta}3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10 days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real

  16. An improved ivermectin-activated chloride channel receptor for inhibiting electrical activity in defined neuronal populations

    DEFF Research Database (Denmark)

    Lynagh, Timothy Peter; Lynch, Joseph W

    2010-01-01

    The ability to silence the electrical activity of defined neuronal populations in vivo is dramatically advancing our understanding of brain function. This technology may eventually be useful clinically for treating a variety of neuropathological disorders caused by excessive neuronal activity...... conductance, homomeric expression, and human origin may render the F207A/A288G alpha1 glycine receptor an improved silencing receptor for neuroscientific and clinical purposes. As all known highly ivermectin-sensitive GluClRs contain an endogenous glycine residue at the corresponding location, this residue...

  17. Dual orexin receptor antagonist 12 inhibits expression of proteins in neurons and glia implicated in peripheral and central sensitization.

    Science.gov (United States)

    Cady, R J; Denson, J E; Sullivan, L Q; Durham, P L

    2014-06-06

    Sensitization and activation of trigeminal nociceptors is implicated in prevalent and debilitating orofacial pain conditions including temporomandibular joint (TMJ) disorders. Orexins are excitatory neuropeptides that function to regulate many physiological processes and are reported to modulate nociception. To determine the role of orexins in an inflammatory model of trigeminal activation, the effects of a dual orexin receptor antagonist (DORA-12) on levels of proteins that promote peripheral and central sensitization and changes in nocifensive responses were investigated. In adult male Sprague-Dawley rats, mRNA for orexin receptor 1 (OX₁R) and receptor 2 (OX₂R) were detected in trigeminal ganglia and spinal trigeminal nucleus (STN). OX₁R immunoreactivity was localized primarily in neuronal cell bodies in the V3 region of the ganglion and in laminas I-II of the STN. Animals injected bilaterally with complete Freund's adjuvant (CFA) in the TMJ capsule exhibited increased expression of P-p38, P-ERK, and lba1 in trigeminal ganglia and P-ERK and lba1 in the STN at 2 days post injection. However, levels of each of these proteins in rats receiving daily oral DORA-12 were inhibited to near basal levels. Similarly, administration of DORA-12 on days 3 and 4 post CFA injection in the TMJ effectively inhibited the prolonged stimulated expression of protein kinase A, NFkB, and Iba1 in the STN on day 5 post injection. While injection of CFA mediated a nocifensive response to mechanical stimulation of the orofacial region at 2h and 3 and 5 days post injection, treatment with DORA-12 suppressed the nocifensive response on day 5. Somewhat surprisingly, nocifensive responses were again observed on day 10 post CFA stimulation in the absence of daily DORA-12 administration. Our results provide evidence that DORA-12 can inhibit CFA-induced stimulation of trigeminal sensory neurons by inhibiting expression of proteins associated with sensitization of peripheral and central

  18. Histamine acting on H1 receptor promotes inhibition of proliferation via PLC, RAC, and JNK-dependent pathways

    International Nuclear Information System (INIS)

    Notcovich, Cintia; Diez, Federico; Tubio, Maria Rosario; Baldi, Alberto; Kazanietz, Marcelo G.; Davio, Carlos; Shayo, Carina

    2010-01-01

    It is well established that histamine modulates cell proliferation through the activation of the histamine H1 receptor (H1R), a G protein-coupled receptor (GPCR) that is known to couple to phospholipase C (PLC) activation via Gq. In the present study, we aimed to determine whether H1R activation modulates Rho GTPases, well-known effectors of Gq/G 11 -coupled receptors, and whether such modulation influences cell proliferation. Experiments were carried out in CHO cells stably expressing H1R (CHO-H1R). By using pull-down assays, we found that both histamine and a selective H1R agonist activated Rac and RhoA in a time- and dose-dependent manner without significant changes in the activation of Cdc42. Histamine response was abolished by the H1R antagonist mepyramine, RGS2 and the PLC inhibitor U73122, suggesting that Rac and RhoA activation is mediated by H1R via Gq coupling to PLC stimulation. Histamine caused a marked activation of serum response factor activity via the H1R, as determined with a serum-responsive element (SRE) luciferase reporter, and this response was inhibited by RhoA inactivation with C3 toxin. Histamine also caused a significant activation of JNK which was inhibited by expression of the Rac-GAP β2-chimaerin. On the other hand, H1R-induced ERK1/2 activation was inhibited by U73122 but not affected by C3 or β2-chimaerin, suggesting that ERK1/2 activation was dependent on PLC and independent of RhoA or Rac. [ 3 H]-Thymidine incorporation assays showed that both histamine and the H1R agonist inhibited cell proliferation in a dose-dependent manner and that the effect was independent of RhoA but partially dependent on JNK and Rac. Our results reveal that functional coupling of the H1R to Gq-PLC leads to the activation of RhoA and Rac small GTPases and suggest distinct roles for Rho GTPases in the control of cell proliferation by histamine.

  19. Adenosine A2A receptors modulate the dopamine D2 receptor-mediated inhibition of synaptic transmission in the mouse prefrontal cortex.

    Science.gov (United States)

    Real, Joana I; Simões, Ana Patrícia; Cunha, Rodrigo A; Ferreira, Samira G; Rial, Daniel

    2018-05-01

    Prefrontal cortex (PFC) circuits are modulated by dopamine acting on D 1 - and D 2 -like receptors, which are pharmacologically exploited to manage neuropsychiatric conditions. Adenosine A 2A receptors (A 2 A R) also control PFC-related responses and A 2 A R antagonists are potential anti-psychotic drugs. As tight antagonistic A 2 A R-D 2 R and synergistic A 2 A R-D 1 R interactions occur in other brain regions, we now investigated the crosstalk between A 2 A R and D 1 /D 2 R controlling synaptic transmission between layers II/III and V in mouse PFC coronal slices. Dopamine decreased synaptic transmission, a presynaptic effect based on the parallel increase in paired-pulse responses. Dopamine inhibition was prevented by the D 2 R-like antagonist sulpiride but not by the D 1 R antagonist SCH23390 and was mimicked by the D 2 R agonist sumanirole, but not by the agonists of either D 4 R (A-412997) or D 3 R (PD128907). Dopamine inhibition was prevented by the A 2 A R antagonist, SCH58261, and attenuated in A 2 A R knockout mice. Accordingly, triple-labelling immunocytochemistry experiments revealed the co-localization of A 2 A R and D 2 R immunoreactivity in glutamatergic (vGluT1-positive) nerve terminals of the PFC. This reported positive A 2 A R-D 2 R interaction controlling PFC synaptic transmission provides a mechanistic justification for the anti-psychotic potential of A 2 A R antagonists. © 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  20. Identification and Characterization of a Novel IL-4 Receptor α Chain (IL-4Rα Antagonist to Inhibit IL-4 Signalling

    Directory of Open Access Journals (Sweden)

    Nayyar Ahmed

    2015-05-01

    Full Text Available Background/Aims: In recent times, allergy has become a financial, physical and psychological burden to the society as a whole. In allergic cascades, cytokine IL-4 binds to IL-4 receptor (IL-4R, consequently producing allergen-specific IgE antibodies by B cells. In addition, among other functions, IL-4 is also responsible for B and T cell proliferation and differentiation. Hence, characterization of novel antagonists that inhibit IL-4 signalling forms the overall aim of this study. Methods: Phage display was used to screen a random 12-mer synthetic peptide library with a human IL-4Rα to identify peptide candidates. Once identified, the peptides were commercially synthesized and used for in vitro immunoassays. Results: We have successfully used phage display to identify M13 phage clones that demonstrated specific binding to IL-4Rα. The peptide N1 was synthesized for use in ELISA, demonstrating significant binding to IL-4Rα and inhibiting interaction with cytokine IL-4. Furthermore, the peptide was tested in a transfected HEK-Blue IL-4 reporter cell line model, which produces alkaline phosphatase (AP. QUANTI-Blue, a substrate, breaks down in the presence of AP producing a blue coloration. Using this colorimetric analysis, >50% inhibition of IL-4 signalling was achieved. Conclusion: We have successfully identified and characterised a synthetic peptide antagonist against IL-4Rα, which effectively inhibits IL-4 interaction with the IL-4Rα in vitro. Since IL-4 interaction with IL-4Rα is a common pathway for many allergies, a prophylactic treatment can be devised by inhibiting this interaction for future treatment of allergies.

  1. Curcumin Modulates Macrophage Polarization Through the Inhibition of the Toll-Like Receptor 4 Expression and its Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Yaoyao Zhou

    2015-05-01

    Full Text Available Background: Curcumin, the active ingredient in curcuma rhizomes, has a wide range of therapeutic effects. However, its atheroprotective activity in human acute monocytic leukemia THP-1 cells remains unclear. We investigated the activity and molecular mechanism of action of curcumin in polarized macrophages. Methods: Phorbol myristate acetate (PMA-treated THP-1 cells were differentiated to macrophages, which were further polarized to M1 cells by lipopolysaccharide (LPS; 1 µg/ml and interferon (IFN-γ (20 ng/ml and treated with varying curcumin concentrations. [3H]thymidine (3H-TdR incorporation assays were utilized to measure curcumin-induced growth inhibition. The expression of tumor necrosis factor-a (TNF-a, interleukin (IL-6, and IL-12B (p40 were measured by quantitative real-time polymerase chain reaction (PCR and enzyme-linked immunosorbent assay (ELISA. Macrophage polarization and its mechanism were evaluated by flow cytometry and western blot. Additionally, toll-like receptor 4 (TLR4 small interfering RNA and mitogen-activated protein kinase (MAPK inhibitors were used to further confirm the molecular mechanism of curcumin on macrophage polarization. Results: Curcumin dose-dependently inhibited M1 macrophage polarization and the production of TNF-a, IL-6, and IL-12B (p40. It also decreased TLR4 expression, which regulates M1 macrophage polarization. Furthermore, curcumin significantly inhibited the phosphorylation of ERK, JNK, p38, and nuclear factor (NF-γB. In contrast, SiTLR4 in combination with p-JNK, p-ERK, and p-p38 inhibition reduced the effect of curcumin on polarization. Conclusions: Curcumin can modulate macrophage polarization through TLR4-mediated signaling pathway inhibition, indicating that its effect on macrophage polarization is related to its anti-inflammatory and atheroprotective effects. Our data suggest that curcumin could be used as a therapeutic agent in atherosclerosis.

  2. Expression of PML tumor suppressor in A 431 cells reduces cellular growth by inhibiting the epidermal growth factor receptor expression

    International Nuclear Information System (INIS)

    Vallian, S.; Chang, K.S.

    2004-01-01

    Our previous studies showed that the promyelocytic leukemia, PML, protein functions as a cellular and growth suppressor. Transient expression of PML was also found to repress the activity of the epidermal growth factor receptor gene promoter. In this study we have examined the effects of PML on A431 cells, which express a high level of + protein. The PML gene was introduced into the cells using the adenovirus-mediated gene transfer system. Western blot analysis on the extracts from the cells expressing PML showed a significant repression in the expression of the epidermal growth factor receptor protein. The cells were examined for growth and DNA synthesis. The data showed a marked reduction in both growth and DNA synthesis rate in the cells expressing PML compared with the control cells. Furthermore, in comparison with the controls, the cells expressing PML were found to be more in G1 phase, fewer in S and about the same number in the G2/M phase. This data clearly demonstrated that the repression of epidermal growth factor receptor expression in A 431 cells by PML was associated with inhibition of cell growth and alteration of the cell cycle distribution, suggesting a novel mechanism for the known growth inhibitory effects of PML

  3. Regulation of DNA Damage Response by Estrogen Receptor β-Mediated Inhibition of Breast Cancer Associated Gene 2

    Directory of Open Access Journals (Sweden)

    Yuan-Hao Lee

    2015-04-01

    Full Text Available Accumulating evidence suggests that ubiquitin E3 ligases are involved in cancer development as their mutations correlate with genomic instability and genetic susceptibility to cancer. Despite significant findings of cancer-driving mutations in the BRCA1 gene, estrogen receptor (ER-positive breast cancers progress upon treatment with DNA damaging-cytotoxic therapies. In order to understand the underlying mechanism by which ER-positive breast cancer cells develop resistance to DNA damaging agents, we employed an estrogen receptor agonist, Erb-041, to increase the activity of ERβ and negatively regulate the expression and function of the estrogen receptor α (ERα in MCF-7 breast cancer cells. Upon Erb-041-mediated ERα down-regulation, the transcription of an ERα downstream effector, BCA2 (Breast Cancer Associated gene 2, correspondingly decreased. The ubiquitination of chromatin-bound BCA2 was induced by ultraviolet C (UVC irradiation but suppressed by Erb-041 pretreatment, resulting in a blunted DNA damage response. Upon BCA2 silencing, DNA double-stranded breaks increased with Rad51 up-regulation and ataxia telangiectasia mutated (ATM activation. Mechanistically, UV-induced BCA2 ubiquitination and chromatin binding were found to promote DNA damage response and repair via the interaction of BCA2 with ATM, γH2AX and Rad51. Taken together, this study suggests that Erb-041 potentiates BCA2 dissociation from chromatin and co-localization with Rad51, resulting in inhibition of homologous recombination repair.

  4. Azadirachtin Interacts with the Tumor Necrosis Factor (TNF) Binding Domain of Its Receptors and Inhibits TNF-induced Biological Responses*

    Science.gov (United States)

    Thoh, Maikho; Kumar, Pankaj; Nagarajaram, Hampathalu A.; Manna, Sunil K.

    2010-01-01

    The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor κB (NF-κB) and also expression of NF-κB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-κB (IκBα) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IκBα kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-κB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy. PMID:20018848

  5. Azadirachtin interacts with the tumor necrosis factor (TNF) binding domain of its receptors and inhibits TNF-induced biological responses.

    Science.gov (United States)

    Thoh, Maikho; Kumar, Pankaj; Nagarajaram, Hampathalu A; Manna, Sunil K

    2010-02-19

    The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor kappaB (NF-kappaB) and also expression of NF-kappaB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-kappaB (IkappaB alpha) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IkappaB alpha kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-kappaB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.

  6. Maximizing the effect of an α7 nicotinic receptor PAM in a mouse model of schizophrenia-like sensory inhibition deficits.

    Science.gov (United States)

    Stevens, Karen E; Zheng, Lijun; Floyd, Kirsten L; Stitzel, Jerry A

    2015-06-22

    Positive allosteric modulators (PAMs) for the α7 nicotinic receptor hold promise for the treatment of sensory inhibition deficits observed in schizophrenia patients. Studies of these compounds in the DBA/2 mouse, which models the schizophrenia-related deficit in sensory inhibition, have shown PAMs to be effective in improving the deficit. However, the first published clinical trial of a PAM for both sensory inhibition deficits and related cognitive difficulties failed, casting a shadow on this therapeutic approach. The present study used both DBA/2 mice, and C3H Chrna7 heterozygote mice to assess the ability of the α7 PAM, PNU-120596, to improve sensory inhibition. Both of these strains of mice have reduced hippocampal α7 nicotinic receptor numbers and deficient sensory inhibition similar to schizophrenia patients. Low doses of PNU-120596 (1 or 3.33mg/kg) were effective in the DBA/2 mouse but not the C3H Chrna7 heterozygote mouse. Moderate doses of the selective α7 nicotinic receptor agonist, choline chloride (10 or 33mg/kg), were also ineffective in improving sensory inhibition in the C3H Chrna7 heterozygote mouse. However, combining the lowest doses of both PNU-120596 and choline chloride in this mouse model did improve sensory inhibition. We propose here that the difference in efficacy of PNU-120596 between the 2 mouse strains is driven by differences in hippocampal α7 nicotinic receptor numbers, such that C3H Chrna7 heterozygote mice require additional direct stimulation of the α7 receptors. These data may have implications for further clinical testing of putative α7 nicotinic receptor PAMs. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor

    Energy Technology Data Exchange (ETDEWEB)

    Inagaki, Junko; Takahashi, Katsuyuki; Ogawa, Hiroko; Asano, Keiichi; Faruk Hatipoglu, Omer; Zeynel Cilek, Mehmet; Obika, Masanari; Ohtsuki, Takashi [Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama (Japan); Hofmann, Matthias [Department of Dermatology, Venereology and Allergology, Goethe University, Frankfurt (Germany); Kusachi, Shozo [Department of Medical Technology, Okayama University Graduate School of Health Sciences, Okayama (Japan); Ninomiya, Yoshifumi [Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama (Japan); Hirohata, Satoshi, E-mail: hirohas@cc.okayama-u.ac.jp [Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama (Japan); International Center, Okayama University, Okayama (Japan)

    2014-05-01

    Angiogenesis and lymphangiogenesis play roles in malignant tumor progression, dissemination, and metastasis. ADAMTS1, a member of the matrix metalloproteinase family, is known to inhibit angiogenesis. Recombinant ADAMTS1 was shown to strongly inhibit angiogenesis. We investigated whether ADAMTS1 inhibited lymphangiogenesis in the present study. We examined cell proliferation and cell migration in normal human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) transduced with or without adenoviral human ADAMTS1 gene therapy. We then examined the VEGFC/VEGFR3 signal transduction pathway in ADAMTS1-transduced HMVEC-dLy. Cell proliferation and tube formation in Matrigel were significantly lower with transduced ADAMTS1 than with control (non-transduced HMVEC-dLy). The phosphorylation of VEGFR3 was also attenuated by ADAMTS1 gene therapy in HMVEC-dLy. Immunoprecipitation assays revealed that ADAMTS1 formed a complex with VEGFC. Our results demonstrated that ADAMTS1 inhibited lymphangiogenesis in vitro. The data highlight the new function of ADAMTS1 in the regulation of lymphangiogenesis and the therapeutic potential of ADAMTS1 in cancer therapy. - Highlights: • ADAMTS1 significantly inhibited tube formation and cell proliferation in HMVEC-dLy. • Reduced lymph endothelial cell migration in ADAMTS1 transduced co-culture systems. • VEGFC-stimulated phosphorylation of VEGFR3 is attenuated by ADAMTS1. • Reduced phosphorylation of Akt and ERK1/2 in ADAMTS1 treated HMVEC-dLy. • ADAMTS1 binds directly to VEGFC.

  8. ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor

    International Nuclear Information System (INIS)

    Inagaki, Junko; Takahashi, Katsuyuki; Ogawa, Hiroko; Asano, Keiichi; Faruk Hatipoglu, Omer; Zeynel Cilek, Mehmet; Obika, Masanari; Ohtsuki, Takashi; Hofmann, Matthias; Kusachi, Shozo; Ninomiya, Yoshifumi; Hirohata, Satoshi

    2014-01-01

    Angiogenesis and lymphangiogenesis play roles in malignant tumor progression, dissemination, and metastasis. ADAMTS1, a member of the matrix metalloproteinase family, is known to inhibit angiogenesis. Recombinant ADAMTS1 was shown to strongly inhibit angiogenesis. We investigated whether ADAMTS1 inhibited lymphangiogenesis in the present study. We examined cell proliferation and cell migration in normal human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) transduced with or without adenoviral human ADAMTS1 gene therapy. We then examined the VEGFC/VEGFR3 signal transduction pathway in ADAMTS1-transduced HMVEC-dLy. Cell proliferation and tube formation in Matrigel were significantly lower with transduced ADAMTS1 than with control (non-transduced HMVEC-dLy). The phosphorylation of VEGFR3 was also attenuated by ADAMTS1 gene therapy in HMVEC-dLy. Immunoprecipitation assays revealed that ADAMTS1 formed a complex with VEGFC. Our results demonstrated that ADAMTS1 inhibited lymphangiogenesis in vitro. The data highlight the new function of ADAMTS1 in the regulation of lymphangiogenesis and the therapeutic potential of ADAMTS1 in cancer therapy. - Highlights: • ADAMTS1 significantly inhibited tube formation and cell proliferation in HMVEC-dLy. • Reduced lymph endothelial cell migration in ADAMTS1 transduced co-culture systems. • VEGFC-stimulated phosphorylation of VEGFR3 is attenuated by ADAMTS1. • Reduced phosphorylation of Akt and ERK1/2 in ADAMTS1 treated HMVEC-dLy. • ADAMTS1 binds directly to VEGFC

  9. Quercetin and epigallocatechin gallate inhibit glucose uptake and metabolism by breast cancer cells by an estrogen receptor-independent mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Moreira, Liliana, E-mail: lilianam87@gmail.com [Department of Biochemistry (U38-FCT), Faculty of Medicine of University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto (Portugal); Araújo, Isabel, E-mail: isa.araujo013@gmail.com [Department of Biochemistry (U38-FCT), Faculty of Medicine of University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto (Portugal); Costa, Tito, E-mail: tito.fmup16@gmail.com [Department of Biochemistry (U38-FCT), Faculty of Medicine of University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto (Portugal); Correia-Branco, Ana, E-mail: ana.clmc.branco@gmail.com [Department of Biochemistry (U38-FCT), Faculty of Medicine of University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto (Portugal); Faria, Ana, E-mail: anafaria@med.up.pt [Department of Biochemistry (U38-FCT), Faculty of Medicine of University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto (Portugal); Chemistry Investigation Centre (CIQ), Faculty of Sciences of University of Porto, Rua Campo Alegre, 4169-007 Porto (Portugal); Faculty of Nutrition and Food Sciences of University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto (Portugal); Martel, Fátima, E-mail: fmartel@med.up.pt [Department of Biochemistry (U38-FCT), Faculty of Medicine of University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto (Portugal); Keating, Elisa, E-mail: keating@med.up.pt [Department of Biochemistry (U38-FCT), Faculty of Medicine of University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto (Portugal)

    2013-07-15

    In this study we characterized {sup 3}H-2-deoxy-D-glucose ({sup 3}H -DG) uptake by the estrogen receptor (ER)-positive MCF7 and the ER-negative MDA-MB-231 human breast cancer cell lines and investigated the effect of quercetin (QUE) and epigallocatechin gallate (EGCG) upon {sup 3}H-DG uptake, glucose metabolism and cell viability and proliferation. In both MCF7 and MDA-MB-231 cells {sup 3}H-DG uptake was (a) time-dependent, (b) saturable with similar capacity (V{sub max}) and affinity (K{sub m}), (c) potently inhibited by cytochalasin B, an inhibitor of the facilitative glucose transporters (GLUT), (d) sodium-independent and (e) slightly insulin-stimulated. This suggests that {sup 3}H-DG uptake by both cell types is mediated by members of the GLUT family, including the insulin-responsive GLUT4 or GLUT12, while being independent of the sodium-dependent glucose transporter (SGLT1). QUE and EGCG markedly and concentration-dependently inhibited {sup 3}H-DG uptake by MCF7 and by MDA-MB-231 cells, and both compounds blocked lactate production by MCF7 cells. Additionally, a 4 h-treatment with QUE or EGCG decreased MCF7 cell viability and proliferation, an effect that was more potent when glucose was available in the extracellular medium. Our results implicate QUE and EGCG as metabolic antagonists in breast cancer cells, independently of estrogen signalling, and suggest that these flavonoids could serve as therapeutic agents/adjuvants even for ER-negative breast tumors. -- Highlights: • Glucose uptake by MCF7 and MDA-MB-231 cells is mainly mediated by GLUT1. • QUE and EGCG inhibit cellular glucose uptake thus abolishing the Warburg effect. • This process induces cytotoxicity and proliferation arrest in MCF7 cells. • The flavonoids’ effects are independent of estrogen receptor signalling.

  10. Quercetin and epigallocatechin gallate inhibit glucose uptake and metabolism by breast cancer cells by an estrogen receptor-independent mechanism

    International Nuclear Information System (INIS)

    Moreira, Liliana; Araújo, Isabel; Costa, Tito; Correia-Branco, Ana; Faria, Ana; Martel, Fátima; Keating, Elisa

    2013-01-01

    In this study we characterized 3 H-2-deoxy-D-glucose ( 3 H -DG) uptake by the estrogen receptor (ER)-positive MCF7 and the ER-negative MDA-MB-231 human breast cancer cell lines and investigated the effect of quercetin (QUE) and epigallocatechin gallate (EGCG) upon 3 H-DG uptake, glucose metabolism and cell viability and proliferation. In both MCF7 and MDA-MB-231 cells 3 H-DG uptake was (a) time-dependent, (b) saturable with similar capacity (V max ) and affinity (K m ), (c) potently inhibited by cytochalasin B, an inhibitor of the facilitative glucose transporters (GLUT), (d) sodium-independent and (e) slightly insulin-stimulated. This suggests that 3 H-DG uptake by both cell types is mediated by members of the GLUT family, including the insulin-responsive GLUT4 or GLUT12, while being independent of the sodium-dependent glucose transporter (SGLT1). QUE and EGCG markedly and concentration-dependently inhibited 3 H-DG uptake by MCF7 and by MDA-MB-231 cells, and both compounds blocked lactate production by MCF7 cells. Additionally, a 4 h-treatment with QUE or EGCG decreased MCF7 cell viability and proliferation, an effect that was more potent when glucose was available in the extracellular medium. Our results implicate QUE and EGCG as metabolic antagonists in breast cancer cells, independently of estrogen signalling, and suggest that these flavonoids could serve as therapeutic agents/adjuvants even for ER-negative breast tumors. -- Highlights: • Glucose uptake by MCF7 and MDA-MB-231 cells is mainly mediated by GLUT1. • QUE and EGCG inhibit cellular glucose uptake thus abolishing the Warburg effect. • This process induces cytotoxicity and proliferation arrest in MCF7 cells. • The flavonoids’ effects are independent of estrogen receptor signalling

  11. Cholecystokinin Receptor-Targeted Polyplex Nanoparticle Inhibits Growth and Metastasis of Pancreatic Cancer

    Directory of Open Access Journals (Sweden)

    Julian Burks

    2018-01-01

    Conclusions: Our polyplex nanoparticle platform establishes both a strong foundation for the development of receptor-targeted therapeutics and a unique approach for the delivery of siRNA in vivo, thus warranting further exploration of this approach in other types of cancers.

  12. Bicyclams, selective antagonists of the human chemokine receptor CXCR4, potently inhibit feline immunodeficiency virus replication

    NARCIS (Netherlands)

    Horzinek, M.C.; Egberink, H.F.; Clercq, E. de; Vliet, A.L.W. van; Balzarini, J.; Bridger, G.J.; Henson, G.; Schols, D.

    1999-01-01

    Bicyclams are low-molecular-weight anti-human immunodeficiency virus (HIV) agents that have been shown to act as potent and selective CXC chemokine receptor 4 (CXCR4) antagonists. Here, we demonstrate that bicyclams are potent inhibitors of feline immunodeficiency virus (FIV) replication when

  13. Greater Sensitivity of Blood Pressure Than Renal Toxicity to Tyrosine Kinase Receptor Inhibition With Sunitinib

    DEFF Research Database (Denmark)

    Lankhorst, Stephanie; Baelde, Hans J; Kappers, Mariëtte H W

    2015-01-01

    Hypertension and renal injury are off-target effects of sunitinib, a tyrosine kinase receptor inhibitor used for the treatment of various tumor types. Importantly, these untoward effects are accompanied by activation of the endothelin system. Here, we set up a study to explore the dose dependency...

  14. Activation of serotonin 2C receptors in dopamine neurons inhibits binge-like eating in mice

    Science.gov (United States)

    Neural networks that regulate binge eating remain to be identified, and effective treatments for binge eating are limited. We combined neuroanatomic, pharmacologic, electrophysiological, Cre-lox, and chemogenetic approaches to investigate the functions of 5-hydroxytryptamine (5-HT) 2C receptor (5-HT...

  15. Harmaline competitively inhibits [3H]MK-801 binding to the NMDA receptor in rabbit brain.

    Science.gov (United States)

    Du, W; Aloyo, V J; Harvey, J A

    1997-10-03

    Harmaline, a beta-carboline derivative, is known to produce tremor through a direct activation of cells in the inferior olive. However, the receptor(s) through which harmaline acts remains unknown. It was recently reported that the tremorogenic actions of harmaline could be blocked by the noncompetitive NMDA channel blocker, MK-801. This study examined whether the blockade of harmaline's action, in the rabbit, by MK-801 was due to a pharmacological antagonism at the MK-801 binding site. This was accomplished by measurement of [3H]MK-801 binding in membrane fractions derived from tissue containing the inferior olivary nucleus and from cerebral cortex. Harmaline completely displaced saturable [3H]MK-801 binding in both the inferior olive and cortex with apparent IC50 values of 60 and 170 microM, respectively. These IC50 values are consistent with the high doses of harmaline required to produce tremor, e.g., 10-30 mg/kg. Non-linear curve fitting analysis of [3H]MK-801 saturation experiments indicated that [3H]MK-801 bound to a single site and that harmaline's displacement of [3H]MK-801 binding to the NMDA receptor was competitive as indicated by a shift in Kd but not in Bmax. In addition, a Schild plot gave a slope that was not significantly different from 1 indicating that harmaline was producing a displacement of [3H]MK-801 from its binding site within the NMDA cation channel and not through an action at the glutamate or other allosteric sites on the NMDA receptor. These findings provide in vitro evidence that the competitive blockade of harmaline-induced tremor by MK-801 occurs within the calcium channel coupled to the NMDA receptor. Our hypothesis is that harmaline produces tremor by acting as an inverse agonist at the MK-801 binding site and thus opening the cation channel.

  16. Inhibition of IGF-1 receptor kinase blocks the differentiation into cardiomyocyte-like cells of BMSCs induced by IGF-1.

    Science.gov (United States)

    Gong, Haibin; Wang, Xiuli; Wang, Lei; Liu, Ying; Wang, Jie; Lv, Qian; Pang, Hui; Zhang, Qinglin; Wang, Zhenquan

    2017-07-01

    Bone marrow mesenchymal stem cells (BMSCs) have the potential to transdifferentiate into cardiomyocyte‑like cells (CLCs) if an appropriate cardiac environment is provided. Insulin‑like growth factor‑1 (IGF‑1) plays an important role in the cell migration, survival and differentiation of BMSCs. However, the effect of IGF‑1 on the cellular differentiation remains unclear. In the present study, BMSCs were isolated from rat femurs and tibias and the cells were purified at passage 6 (P6). IGF‑1 and IGF‑1 receptor (IGF‑1R) kinase inhibitor I‑OMe AG538 were added to detect if IGF‑1 could induce BMSCs to transdifferentiate into CLCs and if I‑OMe AG538 could inhibit IGF‑1‑mediated receptor activation and downstream signaling. Immunostaining demonstrated that all P6 BMSCs express CD29 and CD44 but not CD45. BMSCs induced by 15 ng/ml IGF‑1 revealed positivity for cardiac troponin‑T and cardiac troponin‑I. The optimal induction time was 14 days but the expression of these proteins were incompletely inhibited by 300 nmol/l I‑OMe AG538 and completely inhibited by 10 µmol/l I‑OMe AG538. Western blotting showed that the level of IGF‑1R autophosphorylation and the expression of cTnT and cTnI were higher when BMSCs were induced for 14 days. I‑OMe AG538 selectively inhibited IGF‑1‑mediated growth and signal transduction and the inhibitory effect of I‑OMe AG538 were not reverted in the presence of exogenous IGF‑1. In addition, when a time course analysis of the effects of I‑OMe AG538 on mitogen‑activated protein kinase kinase and phosphatidylinositol 3‑kinase signaling were done, we observed a transient inhibitory effect on Erk1/2 and Akt phosphorylation, in keeping with the inhibitory effects on cell growth. Taken together, these data indicate that I‑OMe AG538 could inhibit IGF-1-induced CLCs in BMSCs and this effect is time- and concentration-dependent.

  17. Progesterone receptor (PR) polyproline domain (PPD) mediates inhibition of epidermal growth factor receptor (EGFR) signaling in non-small cell lung cancer cells.

    Science.gov (United States)

    Kawprasertsri, Sornsawan; Pietras, Richard J; Marquez-Garban, Diana C; Boonyaratanakornkit, Viroj

    2016-05-01

    Recent evidence has suggested a possible role for progesterone receptor (PR) in the progression of non-small cell lung cancer (NSCLC). However, little is known concerning roles of PR in NSCLC. PR contains a polyproline domain (PPD), which directly binds to the SH3 domain of signaling molecules. Because PPD-SH3 interactions are essential for EGFR signaling, we hypothesized that the presence of PR-PPD interfered with EGFR-mediated signaling and cell proliferation. We examined the role of PR-PPD in cell proliferation and signaling by stably expressing PR-B, or PR-B with disrupting mutations in the PPD (PR-BΔSH3), from a tetracycline-regulated promoter in A549 NSCLC cells. PR-B dose-dependently inhibited cell growth in the absence of ligand, and progestin (R5020) treatment further suppressed the growth. Treatment with RU486 abolished PR-B- and R5020-mediated inhibition of cell proliferation. Expression of PR-BΔSH3 and treatment with R5020 or RU486 had no effect on cell proliferation. Furthermore, PR-B expression but not PR-BΔSH3 expression reduced EGF-induced A549 proliferation and activation of ERK1/2, in the absence of ligand. Taken together, our data demonstrated the significance of PR extranuclear signaling through PPD interactions in EGFR-mediated proliferation and signaling in NSCLC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. Ibrutinib Inhibits ERBB Receptor Tyrosine Kinases and HER2-Amplified Breast Cancer Cell Growth.

    Science.gov (United States)

    Chen, Jun; Kinoshita, Taisei; Sukbuntherng, Juthamas; Chang, Betty Y; Elias, Laurence

    2016-12-01

    Ibrutinib is a potent, small-molecule Bruton tyrosine kinase (BTK) inhibitor developed for the treatment of B-cell malignancies. Ibrutinib covalently binds to Cys481 in the ATP-binding domain of BTK. This cysteine residue is conserved among 9 other tyrosine kinases, including HER2 and EGFR, which can be targeted. Screening large panels of cell lines demonstrated that ibrutinib was growth inhibitory against some solid tumor cells, including those inhibited by other HER2/EGFR inhibitors. Among sensitive cell lines, breast cancer lines with HER2 overexpression were most potently inhibited by ibrutinib (ibrutinib coincided with downregulation of phosphorylation on HER2 and EGFR and their downstream targets, AKT and ERK. Irreversible inhibition of HER2 and EGFR in breast cancer cells was established after 30-minute incubation above 100 nmol/L or following 2-hour incubation at lower concentrations. Furthermore, ibrutinib inhibited recombinant HER2 and EGFR activity that was resistant to dialysis and rapid dilution, suggesting an irreversible interaction. The dual activity toward TEC family (BTK and ITK) and ERBB family kinases was unique to ibrutinib, as ERBB inhibitors do not inhibit or covalently bind BTK or ITK. Xenograft studies with HER2 + MDA-MB-453 and BT-474 cells in mice in conjunction with determination of pharmacokinetics demonstrated significant exposure-dependent inhibition of growth and key signaling molecules at levels that are clinically achievable. Ibrutinib's unique dual spectrum of activity against both TEC family and ERBB kinases suggests broader applications of ibrutinib in oncology. Mol Cancer Ther; 15(12); 2835-44. ©2016 AACR. ©2016 American Association for Cancer Research.

  19. Shakuyakukanzoto attenuates oxaliplatin-induced cold dysesthesia by inhibiting the expression of transient receptor potential melastatin 8 in mice

    Directory of Open Access Journals (Sweden)

    Tsugunobu Andoh

    2017-01-01

    Full Text Available Oxaliplatin-induced peripheral neuropathy characterized especially as cold dysesthesia is a major dose-limiting side effect of the drug and is very difficult to control. In the present study, we examined whether the traditional herbal formulation Shakuyakukanzoto (SKT: 芍藥甘草湯Sháo Yào Gān Cǎo Tāng could relieve oxaliplatin-induced cold dysesthesia in mice. The inhibitory mechanisms were also investigated. Repetitive administration of SKT (0.1–1.0 g/kg starting from the day after oxaliplatin injection inhibited cold dysesthesia in a dose-dependent manner. Our previous report has shown that the mRNA expression of transient receptor potential melastatin 8 (TRPM8, characterized as a cold-sensing cation channel, is increased in the dorsal root ganglia of mice treated with oxaliplatin. In addition, TRPM8 antagonist TC-I 2014 (10 and 30 mg/kg also attenuated cold dysesthesia in oxaliplatin-treated mice. Taken together, it is suggested that TRPM8 is involved in the cold dysesthesia induced by oxaliplatin. Repetitive administration of SKT inhibited the mRNA expression of TRPM8 induced by oxaliplatin in the dorsal root ganglia. These results suggested that prophylactic repetitive administration of SKT is effective in preventing the exacerbation of oxaliplatin-induced cold dysesthesia by inhibiting the mRNA expression of TRPM8 in the dorsal root ganglia.

  20. Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages

    Science.gov (United States)

    Peterson, Teresa E.; Kirkpatrick, Nathaniel D.; Huang, Yuhui; Farrar, Christian T.; Marijt, Koen A.; Kloepper, Jonas; Datta, Meenal; Amoozgar, Zohreh; Seano, Giorgio; Jung, Keehoon; Kamoun, Walid S.; Vardam, Trupti; Snuderl, Matija; Goveia, Jermaine; Chatterjee, Sampurna; Batista, Ana; Muzikansky, Alona; Leow, Ching Ching; Xu, Lei; Batchelor, Tracy T.; Duda, Dan G.; Fukumura, Dai; Jain, Rakesh K.

    2016-01-01

    Glioblastomas (GBMs) rapidly become refractory to anti-VEGF therapies. We previously demonstrated that ectopic overexpression of angiopoietin-2 (Ang-2) compromises the benefits of anti-VEGF receptor (VEGFR) treatment in murine GBM models and that circulating Ang-2 levels in GBM patients rebound after an initial decrease following cediranib (a pan-VEGFR tyrosine kinase inhibitor) administration. Here we tested whether dual inhibition of VEGFR/Ang-2 could improve survival in two orthotopic models of GBM, Gl261 and U87. Dual therapy using cediranib and MEDI3617 (an anti–Ang-2–neutralizing antibody) improved survival over each therapy alone by delaying Gl261 growth and increasing U87 necrosis, effectively reducing viable tumor burden. Consistent with their vascular-modulating function, the dual therapies enhanced morphological normalization of vessels. Dual therapy also led to changes in tumor-associated macrophages (TAMs). Inhibition of TAM recruitment using an anti–colony-stimulating factor-1 antibody compromised the survival benefit of dual therapy. Thus, dual inhibition of VEGFR/Ang-2 prolongs survival in preclinical GBM models by reducing tumor burden, improving normalization, and altering TAMs. This approach may represent a potential therapeutic strategy to overcome the limitations of anti-VEGFR monotherapy in GBM patients by integrating the complementary effects of anti-Ang2 treatment on vessels and immune cells. PMID:27044097

  1. Pan-SRC kinase inhibition blocks B-cell receptor oncogenic signaling in non-Hodgkin lymphoma.

    Science.gov (United States)

    Battistello, Elena; Katanayeva, Natalya; Dheilly, Elie; Tavernari, Daniele; Donaldson, Maria C; Bonsignore, Luca; Thome, Margot; Christie, Amanda L; Murakami, Mark A; Michielin, Olivier; Ciriello, Giovanni; Zoete, Vincent; Oricchio, Elisa

    2018-05-24

    In diffuse large B-cell lymphoma (DLBCL), activation of the B-cell receptor (BCR) promotes multiple oncogenic signals, which are essential for tumor proliferation. Inhibition of the Bruton's tyrosine kinase (BTK), a BCR downstream target, is therapeutically effective only in a subgroup of patients with DLBCL. Here, we used lymphoma cells isolated from patients with DLBCL to measure the effects of targeted therapies on BCR signaling and to anticipate response. In lymphomas resistant to BTK inhibition, we show that blocking BTK activity enhanced tumor dependencies from alternative oncogenic signals downstream of the BCR, converging on MYC upregulation. To completely ablate the activity of the BCR, we genetically and pharmacologically repressed the activity of the SRC kinases LYN, FYN, and BLK, which are responsible for the propagation of the BCR signal. Inhibition of these kinases strongly reduced tumor growth in xenografts and cell lines derived from patients with DLBCL independent of their molecular subtype, advancing the possibility to be relevant therapeutic targets in broad and diverse groups of DLBCL patients. © 2018 by The American Society of Hematology.

  2. Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling.

    Science.gov (United States)

    Mundell, S J; Matharu, A-L; Nisar, S; Palmer, T M; Benovic, J L; Kelly, E

    2010-02-01

    We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A(2B) adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5'-(N-ethylcarboxamido)-adenosine. The trafficking of the wild type A(2B) adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy. The wild type A(2B) adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln(325)-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu(330)-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln(325)-stop, Ser(326)-stop and Phe(328)-stop receptors. Following internalization, the wild type A(2B) adenosine receptor recycled rapidly to the cell surface, whereas the Gln(325)-stop receptor did not recycle. Deletion of the COOH-terminus of the A(2B) adenosine receptor beyond Leu(330) switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A(2B) adenosine receptor following prolonged agonist addition.

  3. Iptakalim inhibits nicotinic acetylcholine receptor-mediated currents in dopamine neurons acutely dissociated from rat substantia nigra pars compacta.

    Science.gov (United States)

    Hu, J; DeChon, J; Yan, K C; Liu, Q; Hu, G; Wu, J

    2006-07-31

    Iptakalim hydrochloride, a novel cardiovascular ATP-sensitive K(+) (K(ATP)) channel opener, has shown remarkable antihypertensive and neuroprotective effects in a variety of studies using in vivo and in vitro preparations. We recently found that iptakalim blocked human alpha4-containing nicotinic acetylcholine receptors (nAChRs) heterologously expressed in the human SH-EP1 cell line. In the present study, we examined the effects of iptakalim on several neurotransmitter-induced current responses in single DA neurons freshly dissociated from rat substantia nigra pars compacta (SNc), using perforated patch-clamp recordings combined with a U-tube rapid drug application. In identified DA neurons under voltage-clamp configuration, glutamate-, NMDA-, and GABA-induced currents were insensitive to co-application with iptakalim (100 microM), while whole-cell currents induced by ACh (1 mM+1 microM atropine) or an alpha4beta2 nicotinic acetylcholine receptors relatively selective agonist, RJR-2403 (300 microM), were eliminated by iptakalim. Iptakalim inhibited RJR-2403-induced current in a concentration-dependent manner, and reduced maximal RJR-2403-induced currents at the highest agonist concentration, suggesting a non-competitive block. In current-clamp mode, iptakalim failed to affect resting membrane potential and spontaneous action potential firing, but abolished RJR-2403-induced neuronal firing acceleration. Together, these results indicate that in dissociated SNc DA neurons, alpha4-containing nAChRs, rather than ionotropic glutamate receptors, GABA(A) receptors or perhaps K-ATP channels are the sensitive targets to mediate iptakalim's pharmacological roles.

  4. Inhibition of Ebola and Marburg Virus Entry by G Protein-Coupled Receptor Antagonists.

    Science.gov (United States)

    Cheng, Han; Lear-Rooney, Calli M; Johansen, Lisa; Varhegyi, Elizabeth; Chen, Zheng W; Olinger, Gene G; Rong, Lijun

    2015-10-01

    Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy. Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of both infectious

  5. Dimerization inhibits the activity of receptor-like protein-tyrosine phosphatase-alpha

    DEFF Research Database (Denmark)

    Jiang, G; den Hertog, J; Su, J

    1999-01-01

    that dimerization can negatively regulate activity, through the interaction of an inhibitory 'wedge' on one monomer with the catalytic cleft of domain 1 in the other monomer. Here we show that dimerization inhibits the activity of a full-length RPTP in vivo. We generated stable disulphide-bonded full...

  6. Inhibition by sigma receptor ligand, MS-377, of N-methyl- D-aspartate-induced currents in dopamine neurons of the rat ventral tegmental area.

    Science.gov (United States)

    Yamazaki, Yuu; Ishioka, Miwa; Matsubayashi, Hiroaki; Amano, Taku; Sasa, Masashi

    2002-04-01

    MS-377 [( R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl) piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate] is a novel anti-psychotic drug candidate with high affinity for sigma receptors but devoid of binding affinity for PCP binding site of NMDA receptor/ion channel complex. The effects of MS-377 on NMDA receptor and/or its ion channel complex were examined to elucidate the antipsychotic properties of MS-377. We examined the effect of MS-377 on NMDA ( N-methyl- D-aspartate)-induced current in acutely dissociated dopamine neurons of rat ventral tegmental area (VTA) using patch clamp whole cell recording. MS-377 applied in a bath inhibited the peak current evoked by NMDA applied via the U-tube method for 2 s in a concentration-dependent manner. Other sigma receptor ligands, BD-1063 (1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine), NE-100 ( N, N-dipropyl-2-[4-methoxy-3-(2-phenylenoxy)-phenyl]-ethylamine monohydrochloride) and haloperidol also inhibited NMDA-induced current in a concentration-dependent manner. Interestingly, concomitant application of MS-377 with BD-1063, NE-100 or haloperidol at concentrations that had no effects on NMDA-induced current, potentiated the MS-377-induced inhibition. The results suggest that MS-377, as well as other sigma receptor ligands, indirectly acts on the sigma receptor to inhibit glutaminergic transmission mediated by NMDA receptor/ion channel complex in VTA dopamine neurons, thereby inhibiting dopamine release in target VTA areas.

  7. Context-Dependent Modulation of αβγ and αβγ GABAA Receptors by Penicillin: Implications for Phasic and Tonic Inhibition

    OpenAIRE

    Feng, Hua-Jun; Botzolakis, Emmanuel J.; Macdonald, Robert L.

    2008-01-01

    Penicillin, an open-channel blocker of GABAA receptors, was recently reported to inhibit phasic, but not tonic, currents in hippocampal neurons. To distinguish between isoform-specific and context-dependent modulation as possible explanations for this selectivity, the effects of penicillin were evaluated on recombinant GABAA receptors expressed in HEK293T cells. When co-applied with saturating GABA, penicillin decreased peak amplitude, induced rebound, and prolonged deactivation of currents e...

  8. Technetium-99 conjugated with methylene diphosphonate inhibits receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis.

    Science.gov (United States)

    Gong, Wei; Dou, Huan; Liu, Xianqin; Sun, Lingyun; Hou, Yayi

    2012-10-01

    1. In the present study, we investigated the effects of technetium-99 conjugated with methylene diphosphonate ((99)Tc-MDP), an agent used in radionuclide therapy, on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and explored the underlying mechanisms. 2. The murine macrophage cell line RAW264.7 and bone marrow-derived-macrophages from C57BL/6 mice (BMM) were used as models for osteoclastogenesis in vitro. The expression of some key factors in RANKL (50 ng/mL)-induced osteoclastogenesis in RAW264.7 cells was investigated by flow cytometry and real-time reverse transcription-polymerase chain reaction (RT-PCR). To detect multinucleated osteoclast formation, RAW264.7 cells were induced with RANKL for 4 days, whereas BMM were induced by 50 ng/mL RANKL and 20 ng/mL macrophage colony-stimulating factor for 7 days, before being stained with tartrate-resistant acid phosphatase. 3. Osteoclastogenesis was evaluated using the osteoclast markers CD51, matrix metalloproteinase (MMP)-9 and cathepsin K. At 0.01 μg/mL, (99)Tc-MDP significantly inhibited RANKL-induced osteoclastogenesis without any cytotoxicity. In addition, (99)Tc-MDP abolished the appearance of multinucleated osteoclasts. 4. Real-time RT-PCR analysis of transcription factor expression revealed that (99)Tc-MDP inhibited the expression of c-Fos and nuclear factor of activated T cells. In addition, (99)Tc-MDP inhibited the expression of the inflammatory factors interleukin (IL)-6, tumour necrosis factor-α and IL-1β. Finally, (99)Tc-MDP inhibited the activation of mitogen-activated protein kinases in RAW264.7 cells following RANKL stimulation. 5. In conclusion, (99)Tc-MDP possesses anti-osteoclastogenic activity against RANKL-induced osteoclast formation. © 2012 The Authors Clinical and Experimental Pharmacology and Physiology © 2012 Wiley Publishing Asia Pty Ltd.

  9. Inhibition of Epithelial TNF-α Receptors by Purified Fruit Bromelain Ameliorates Intestinal Inflammation and Barrier Dysfunction in Colitis.

    Science.gov (United States)

    Zhou, Zijuan; Wang, Liang; Feng, Panpan; Yin, Lianhong; Wang, Chen; Zhi, Shengxu; Dong, Jianyi; Wang, Jingyu; Lin, Yuan; Chen, Dapeng; Xiong, Yongjian; Peng, Jinyong

    2017-01-01

    Activation of the TNF-α receptor (TNFR) leads to an inflammatory response, and anti-TNF therapy has been administered to reduce inflammation symptoms and heal mucosal ulcers in inflammatory bowel disease (IBD). Bromelain, a complex natural mixture of proteolytic enzymes, has been shown to exert anti-inflammatory effects. This study aimed to investigate the effect of purified fruit bromelain (PFB)-induced inhibition of epithelial TNFR in a rat colitis model. Colitis was established by intracolonic administration of 2, 4, 6-trinitrobenzene sulfonic acid. Expression of TNFR1 and TNFR2 was measured by quantitative RT-PCR and western blotting. The effect of PFB on colitis was evaluated by examining the inflammatory response and intestinal epithelial barrier function. Our results showed that both TNFR1 and TNFR2 expression were significantly increased in a colitis model, and the increase was significantly reversed by PFB. Colitis symptoms, including infiltration of inflammatory cells, cytokine profiles, epithelial cell apoptosis, and epithelial tight junction barrier dysfunction were significantly ameliorated by PFB. Compared with fruit bromelain and stem bromelain complex, the inhibition of TNFR2 induced by PFB was stronger than that exhibited on TNFR1. These results indicate that PFB showed a stronger selective inhibitory effect on TNFR2 than TNFR1. In other words, purification of fruit bromelain increases its selectivity on TNFR2 inhibition. High expression of epithelial TNFRs in colitis was significantly counteracted by PFB, and PFB-induced TNFR inhibition ameliorated colitis symptoms. These results supply novel insights into potential IBD treatment by PFB.

  10. Histamine H3 receptor activation selectively inhibits dopamine D1 receptor-dependent [3H]GABA release from depolarization-stimulated slices of rat substantia nigra pars reticulata

    International Nuclear Information System (INIS)

    Aceves, J.; Young, J.M.; Arias-Montano, J.A.; Floran, B.; Garcia, M.

    1997-01-01

    The release of [ 3 H]GABA from slices of rat substantia nigra pars reticulata induced by increasing extracellular K + from 6 to 15 mM in the presence of 10 μM sulpiride was inhibited by 73±3% by 1 μM SCH 23390, consistent with a large component of release dependent upon D 1 receptor activation. The histamine H 3 receptor-selective agonist immepip (1 μM) and the non-selective agonist histamine (100 μM) inhibited [ 3 H]GABA release by 78±2 and 80±2%, respectively. The inhibition by both agonists was reversed by the H 3 receptor antagonist thioperamide (1 μM). However, in the presence of 1 μM SCH 23390 depolarization-induced release of [ 3 H]GABA was not significantly decreased by 1 μM immepip. In rats depleted of dopamine by pretreatment with reserpine, immepip no longer inhibited control release of [ 3 H]GABA, but in the presence of 1 μM SKF 38393, which produced a 7±1-fold stimulation of release, immepip reduced the release to a level not statistically different from that in the presence of immepip alone. Immepip (1 μM) also inhibited the depolarization-induced release of [ 3 H]dopamine from substantia nigra pars reticulata slices, by 38±3%.The evidence is consistent with the proposition that activation of histamine H 3 receptors leads to the selective inhibition of the component of depolarization-induced [ 3 H]GABA release in substantia nigra pars reticulata slices which is dependent upon D 1 receptor activation. This appears to be largely an action at the terminals of the striatonigral GABA projection neurons, which may be enhanced by a partial inhibition of dendritic [ 3 H]dopamine release. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

  11. Antiprogestin mifepristone inhibits the growth of cancer cells of reproductive and non-reproductive origin regardless of progesterone receptor expression

    International Nuclear Information System (INIS)

    Tieszen, Chelsea R; Goyeneche, Alicia A; Brandhagen, BreeAnn N; Ortbahn, Casey T; Telleria, Carlos M

    2011-01-01

    Mifepristone (MF) has been largely used in reproductive medicine due to its capacity to modulate the progesterone receptor (PR). The study of MF has been expanded to the field of oncology; yet it remains unclear whether the expression of PR is required for MF to act as an anti-cancer agent. Our laboratory has shown that MF is a potent inhibitor of ovarian cancer cell growth. In this study we questioned whether the growth inhibitory properties of MF observed in ovarian cancer cells would translate to other cancers of reproductive and non-reproductive origin and, importantly, whether its efficacy is related to the expression of cognate PR. Dose-response experiments were conducted with cancer cell lines of the nervous system, breast, prostate, ovary, and bone. Cultures were exposed to vehicle or increasing concentrations of MF for 72 h and analysed for cell number and cell cycle traverse, and hypodiploid DNA content characteristic of apoptotic cell death. For all cell lines, expression of steroid hormone receptors upon treatment with vehicle or cytostatic doses of MF for 24 h was studied by Western blot, whereas the activity of the G1/S regulatory protein Cdk2 in both treatment groups was monitored in vitro by the capacity of Cdk2 to phosphorylate histone H1. MF growth inhibited all cancer cell lines regardless of tissue of origin and hormone responsiveness, and reduced the activity of Cdk2. Cancer cells in which MF induced G1 growth arrest were less susceptible to lethality in the presence of high concentrations of MF, when compared to cancer cells that did not accumulate in G1. While all cancer cell lines were growth inhibited by MF, only the breast cancer MCF-7 cells expressed cognate PR. Antiprogestin MF inhibits the growth of different cancer cell lines with a cytostatic effect at lower concentrations in association with a decline in the activity of the cell cycle regulatory protein Cdk2, and apoptotic lethality at higher doses in association with increased

  12. Antiprogestin mifepristone inhibits the growth of cancer cells of reproductive and non-reproductive origin regardless of progesterone receptor expression

    Directory of Open Access Journals (Sweden)

    Ortbahn Casey T

    2011-05-01

    Full Text Available Abstract Background Mifepristone (MF has been largely used in reproductive medicine due to its capacity to modulate the progesterone receptor (PR. The study of MF has been expanded to the field of oncology; yet it remains unclear whether the expression of PR is required for MF to act as an anti-cancer agent. Our laboratory has shown that MF is a potent inhibitor of ovarian cancer cell growth. In this study we questioned whether the growth inhibitory properties of MF observed in ovarian cancer cells would translate to other cancers of reproductive and non-reproductive origin and, importantly, whether its efficacy is related to the expression of cognate PR. Methods Dose-response experiments were conducted with cancer cell lines of the nervous system, breast, prostate, ovary, and bone. Cultures were exposed to vehicle or increasing concentrations of MF for 72 h and analysed for cell number and cell cycle traverse, and hypodiploid DNA content characteristic of apoptotic cell death. For all cell lines, expression of steroid hormone receptors upon treatment with vehicle or cytostatic doses of MF for 24 h was studied by Western blot, whereas the activity of the G1/S regulatory protein Cdk2 in both treatment groups was monitored in vitro by the capacity of Cdk2 to phosphorylate histone H1. Results MF growth inhibited all cancer cell lines regardless of tissue of origin and hormone responsiveness, and reduced the activity of Cdk2. Cancer cells in which MF induced G1 growth arrest were less susceptible to lethality in the presence of high concentrations of MF, when compared to cancer cells that did not accumulate in G1. While all cancer cell lines were growth inhibited by MF, only the breast cancer MCF-7 cells expressed cognate PR. Conclusions Antiprogestin MF inhibits the growth of different cancer cell lines with a cytostatic effect at lower concentrations in association with a decline in the activity of the cell cycle regulatory protein Cdk2, and

  13. Olive oil compounds inhibit vascular endothelial growth factor receptor-2 phosphorylation

    International Nuclear Information System (INIS)

    Lamy, Sylvie; Ouanouki, Amira; Béliveau, Richard; Desrosiers, Richard R.

    2014-01-01

    Vascular endothelial growth factor (VEGF) triggers crucial signaling processes that regulate tumor angiogenesis and, therefore, represents an attractive target for the development of novel anticancer therapeutics. Several epidemiological studies have confirmed that abundant consumption of foods from plant origin is associated with reduced risk of developing cancers. In the Mediterranean basin, the consumption of extra virgin olive oil is an important constituent of the diet. Compared to other vegetable oils, the presence of several phenolic antioxidants in olive oil is believed to prevent the occurrence of a variety of pathological processes, such as cancer. While the strong antioxidant potential of these molecules is well characterized, their antiangiogenic activities remain unknown. The aim of this study is to investigate whether tyrosol (Tyr), hydroxytyrosol (HT), taxifolin (Tax), oleuropein (OL) and oleic acid (OA), five compounds contained in extra virgin olive oil, can affect in vitro angiogenesis. We found that HT, Tax and OA were the most potent angiogenesis inhibitors through their inhibitory effect on specific autophosphorylation sites of VEGFR-2 (Tyr951, Tyr1059, Tyr1175 and Tyr1214) leading to the inhibition of endothelial cell (EC) signaling. Inhibition of VEGFR-2 by these olive oil compounds significantly reduced VEGF-induced EC proliferation and migration as well as their morphogenic differentiation into capillary-like tubular structures in Matrigel. Our study demonstrates that HT, Tax and OA are novel and potent inhibitors of the VEGFR-2 signaling pathway. These findings emphasize the chemopreventive properties of olive oil and highlight the importance of nutrition in cancer prevention. - Highlights: • We investigated five compounds contained in extra virgin olive oil on angiogenesis. • Hydroxytyrosol, taxifolin and oleic acid are the best angiogenesis inhibitors. • Olive oil compounds affect endothelial cell functions essential for

  14. Olive oil compounds inhibit vascular endothelial growth factor receptor-2 phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Lamy, Sylvie, E-mail: lamy.sylvie@uqam.ca; Ouanouki, Amira; Béliveau, Richard; Desrosiers, Richard R.

    2014-03-10

    Vascular endothelial growth factor (VEGF) triggers crucial signaling processes that regulate tumor angiogenesis and, therefore, represents an attractive target for the development of novel anticancer therapeutics. Several epidemiological studies have confirmed that abundant consumption of foods from plant origin is associated with reduced risk of developing cancers. In the Mediterranean basin, the consumption of extra virgin olive oil is an important constituent of the diet. Compared to other vegetable oils, the presence of several phenolic antioxidants in olive oil is believed to prevent the occurrence of a variety of pathological processes, such as cancer. While the strong antioxidant potential of these molecules is well characterized, their antiangiogenic activities remain unknown. The aim of this study is to investigate whether tyrosol (Tyr), hydroxytyrosol (HT), taxifolin (Tax), oleuropein (OL) and oleic acid (OA), five compounds contained in extra virgin olive oil, can affect in vitro angiogenesis. We found that HT, Tax and OA were the most potent angiogenesis inhibitors through their inhibitory effect on specific autophosphorylation sites of VEGFR-2 (Tyr951, Tyr1059, Tyr1175 and Tyr1214) leading to the inhibition of endothelial cell (EC) signaling. Inhibition of VEGFR-2 by these olive oil compounds significantly reduced VEGF-induced EC proliferation and migration as well as their morphogenic differentiation into capillary-like tubular structures in Matrigel. Our study demonstrates that HT, Tax and OA are novel and potent inhibitors of the VEGFR-2 signaling pathway. These findings emphasize the chemopreventive properties of olive oil and highlight the importance of nutrition in cancer prevention. - Highlights: • We investigated five compounds contained in extra virgin olive oil on angiogenesis. • Hydroxytyrosol, taxifolin and oleic acid are the best angiogenesis inhibitors. • Olive oil compounds affect endothelial cell functions essential for

  15. MECHANICAL VIBRATION INHIBITS OSTEOCLAST FORMATION BY REDUCING DC-STAMP RECEPTOR EXPRESSION IN OSTEOCLAST PRECURSOR CELLS

    OpenAIRE

    Kulkarni, R.N.; Voglewede, P.A.; Liu, D.

    2013-01-01

    It is well known that physical inactivity leads to loss of muscle mass, but it also causes bone loss. Mechanistically, osteoclastogenesis and bone resorption have recently been shown to be regulated by vibration. However, the underlying mechanism behind the inhibition of osteoclast formation is yet unknown. Therefore, we investigated whether mechanical vibration of osteoclast precursor cells affects osteoclast formation by the involvement of fusion-related molecules such as dendritic cell-spe...

  16. Amino acid containing thapsigargin analogues deplete androgen receptor protein via synthesis inhibition and induce the death of prostate cancer cells

    DEFF Research Database (Denmark)

    Griend, Donald J Vander; Antony, Lizamma; Dalrymple, Susan L

    2009-01-01

    There are quantitative and/or qualitative mechanisms allowing androgen receptor (AR) growth signaling in androgen ablation refractory prostate cancer cells. Regardless of the mechanism, agents that deplete AR protein expression prevent such AR growth signaling. Thapsigargin (TG) is a highly cell......-penetrant sequiterpene-lactone that once inside cells inhibits (IC(50), approximately 10 nmol/L) critically important housekeeping SERCA 2b calcium pumps in the endoplasmic reticulum. Using a series of five genetically diverse androgen ablation refractory human prostate cancer lines (LNCaP, LAPC-4, VCaP, MDA-PCa-2b......-specific proteases, such as prostate-specific antigen and prostate-specific membrane antigen, or cancer-specific proteases, such as fibroblast activation protein, so that toxicity of these prodrugs is selectively targeted to metastatic sites of prostate cancer. Based on these results, these prodrugs are undergoing...

  17. Investigation of the GPR39 zinc receptor following inhibition of monoaminergic neurotransmission and potentialization of glutamatergic neurotransmission

    DEFF Research Database (Denmark)

    Młyniec, Katarzyna; Gaweł, Magdalena; Librowski, Tadeusz

    2015-01-01

    Zinc can regulate neural function in the brain via the GPR39 receptor. In the present study we investigated whether inhibition of serotonin, noradrenaline and dopamine synthesis and potentialization of glutamate, via administration of p-chlorophenylalanine (pCPA), α-methyl-p-tyrosine (αMT) and N......-methyl-d-aspartatic acid (NMDA), respectively, would cause changes in GPR39 levels. Western blot analysis showed GPR39 up-regulation following 3-day administration of αMT and NMDA in the frontal cortex, and GPR39 down-regulation following 10-day administration of pCPA, αMT, and NMDA in the hippocampus of CD-1 mice....... There were no changes in serum zinc levels. Additionally, we investigated tryptophan, tyrosine and glutamate concentrations in the hippocampus and frontal cortex of GPR39 knockout (GPR39 KO) mice. Liquid chromatography-mass spectrometry (LC-MS) showed a significant decrease in tryptophan and tyrosine...

  18. Role of spinal metabotropic glutamate receptor 5 in pudendal inhibition of the nociceptive bladder reflex in cats.

    Science.gov (United States)

    Reese, Jeremy N; Rogers, Marc J; Xiao, Zhiying; Shen, Bing; Wang, Jicheng; Schwen, Zeyad; Roppolo, James R; de Groat, William C; Tai, Changfeng

    2015-04-15

    This study examined the role of spinal metabotropic glutamate receptor 5 (mGluR5) in the nociceptive C-fiber afferent-mediated spinal bladder reflex and in the inhibtion of this reflex by pudendal nerve stimulation (PNS). In α-chloralose-anesthetized cats after spinal cord transection at the T9/T10 level, intravesical infusion of 0.25% acetic acid irritated the bladder, activated nociceptive C-fiber afferents, and induced spinal reflex bladder contractions of low amplitude (reflexes were responsible for a major component of the contractions. This study shows that spinal mGluR5 plays an important role in the nociceptive C-fiber afferent-mediated spinal bladder reflex and in pudendal inhibition of this spinal reflex. Copyright © 2015 the American Physiological Society.

  19. Clopidogrel (Plavix®), a P2Y(12) receptor antagonist, inhibits bone cell function in vitro and decreases trabecular bone in vivo

    DEFF Research Database (Denmark)

    Syberg, Susanne; Brandao-Burch, Andrea; Patel, Jessal J

    2012-01-01

    Clopidogrel (Plavix®), a selective P2Y(12) receptor antagonist, is widely prescribed to reduce the risk of heart attack and stroke and acts via the inhibition of platelet aggregation. Accumulating evidence now suggests that extracellular nucleotides, signalling through P2 receptors, play...... a significant role in bone, modulating both osteoblast and osteoclast function. In this study, we investigated the effects of clopidogrel treatment on (1) bone cell formation, differentiation and activity in vitro; and, (2) trabecular and cortical bone parameters in vivo. P2Y(12) receptor expression...

  20. Eel Kisspeptins: Identification, Functional Activity, and Inhibition on both Pituitary LH and GnRH Receptor Expression

    Directory of Open Access Journals (Sweden)

    Jérémy Pasquier

    2018-01-01

    Full Text Available The European eel (Anguilla anguilla presents a blockade of sexual maturation at a prepubertal stage due to a deficient production of gonadotropins. We previously initiated, in the eel, the investigation of the kisspeptin system, one of the major gatekeepers of puberty in mammals, and we predicted the sequence of two Kiss genes. In the present study, we cloned and sequenced Kiss1 and Kiss2 cDNAs from the eel brain. The tissue distributions of Kiss1 and Kiss2 transcripts, as investigated by quantitative real-time PCR, showed that both genes are primarily expressed in the eel brain and pituitary. The two 10-residue long sequences characteristic of kisspeptin, eel Kp1(10 and Kp2(10, as well as two longer sequences, predicted as mature peptides, eel Kp1(15 and Kp2(12, were synthesized and functionally analyzed. Using rat Kiss1 receptor-transfected Chinese hamster ovary cells, we found that the four synthesized eel peptides were able to induce [Ca2+]i responses, indicating their ability to bind mammalian KissR-1 and to activate second messenger pathways. In primary culture of eel pituitary cells, all four peptides were able to specifically and dose-dependently inhibit lhβ expression, without any effect on fshβ, confirming our previous data with heterologous kisspeptins. Furthermore, in this eel in vitro system, all four peptides inhibited the expression of the type 2 GnRH receptor (gnrh-r2. Our data revealed a dual inhibitory effect of homologous kisspeptins on both pituitary lhβ and gnrh-r2 expression in the European eel.

  1. Inhibition of alpha oscillations through serotonin-2A receptor activation underlies the visual effects of ayahuasca in humans.

    Science.gov (United States)

    Valle, Marta; Maqueda, Ana Elda; Rabella, Mireia; Rodríguez-Pujadas, Aina; Antonijoan, Rosa Maria; Romero, Sergio; Alonso, Joan Francesc; Mañanas, Miquel Àngel; Barker, Steven; Friedlander, Pablo; Feilding, Amanda; Riba, Jordi

    2016-07-01

    Ayahuasca is an Amazonian psychotropic plant tea typically obtained from two plants, Banisteriopsis caapi and Psychotria viridis. It contains the psychedelic 5-HT2A and sigma-1 agonist N,N-dimethyltryptamine (DMT) plus β-carboline alkaloids with monoamine-oxidase (MAO)-inhibiting properties. Although the psychoactive effects of ayahuasca have commonly been attributed solely to agonism at the 5-HT2A receptor, the molecular target of classical psychedelics, this has not been tested experimentally. Here we wished to study the contribution of the 5-HT2A receptor to the neurophysiological and psychological effects of ayahuasca in humans. We measured drug-induced changes in spontaneous brain oscillations and subjective effects in a double-blind randomized placebo-controlled study involving the oral administration of ayahuasca (0.75mg DMT/kg body weight) and the 5-HT2A antagonist ketanserin (40mg). Twelve healthy, experienced psychedelic users (5 females) participated in four experimental sessions in which they received the following drug combinations: placebo+placebo, placebo+ayahuasca, ketanserin+placebo and ketanserin+ayahuasca. Ayahuasca induced EEG power decreases in the delta, theta and alpha frequency bands. Current density in alpha-band oscillations in parietal and occipital cortex was inversely correlated with the intensity of visual imagery induced by ayahuasca. Pretreatment with ketanserin inhibited neurophysiological modifications, reduced the correlation between alpha and visual effects, and attenuated the intensity of the subjective experience. These findings suggest that despite the chemical complexity of ayahuasca, 5-HT2A activation plays a key role in the neurophysiological and visual effects of ayahuasca in humans. Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.

  2. Preferential Inhibition of Tonically over Phasically Activated NMDA Receptors by Pregnane Derivatives

    Czech Academy of Sciences Publication Activity Database

    Vyklický, Vojtěch; Smejkalová, Tereza; Krausová, Barbora; Balík, Aleš; Kořínek, Miloslav; Borovská, Jiřina; Horák, Martin; Chvojková, Markéta; Kletečková, Lenka; Valeš, Karel; Černý, Jiří; Nekardová, Michaela; Chodounská, Hana; Kudová, Eva; Vyklický ml., Ladislav

    2016-01-01

    Roč. 36, č. 7 (2016), s. 2161-2175 ISSN 0270-6474 R&D Projects: GA ČR(CZ) GAP303/12/1464; GA ČR(CZ) GBP304/12/G069; GA TA ČR(CZ) TE01020028; GA ČR(CZ) GBP208/12/G016; GA MŠk(CZ) EE2.3.30.0025; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 ; RVO:61388963 Keywords : memantine * neuroprotection * neurosteroid * NMDA receptor * pregnanolone sulfate * synaptic transmission Subject RIV: ED - Physiology Impact factor: 5.988, year: 2016

  3. Cell surface estrogen receptor alpha is upregulated during subchronic metabolic stress and inhibits neuronal cell degeneration.

    Directory of Open Access Journals (Sweden)

    Cristiana Barbati

    Full Text Available In addition to the classical nuclear estrogen receptor, the expression of non-nuclear estrogen receptors localized to the cell surface membrane (mER has recently been demonstrated. Estrogen and its receptors have been implicated in the development or progression of numerous neurodegenerative disorders. Furthermore, the pathogenesis of these diseases has been associated with disturbances of two key cellular programs: apoptosis and autophagy. An excess of apoptosis or a defect in autophagy has been implicated in neurodegeneration. The aim of this study was to clarify the role of ER in determining neuronal cell fate and the possible implication of these receptors in regulating either apoptosis or autophagy. The human neuronal cell line SH-SY5Y and mouse neuronal cells in primary culture were thus exposed to chronic minimal peroxide treatment (CMP, a form of subcytotoxic minimal chronic stress previously that mimics multiple aspects of long-term cell stress and represents a limited molecular proxy for neurodegenerative processes. We actually found that either E2 or E2-bovine serum albumin construct (E2BSA, i.e. a non-permeant form of E2 was capable of modulating intracellular cell signals and regulating cell survival and death. In particular, under CMP, the up-regulation of mERα, but not mERβ, was associated with functional signals (ERK phosphorylation and p38 dephosphorylation compatible with autophagic cytoprotection triggering and leading to cell survival. The mERα trafficking appeared to be independent of the microfilament system cytoskeletal network but was seemingly associated with microtubular apparatus network, i.e., to MAP2 molecular chaperone. Importantly, antioxidant treatments, administration of siRNA to ERα, or the presence of antagonist of ERα hindered these events. These results support that the surface expression of mERα plays a pivotal role in determining cell fate, and that ligand-induced activation of mER signalling exerts a

  4. Comparison of the tumor inhibiting effects of three histamine H2-receptor antagonists.

    Science.gov (United States)

    Tutton, P J; Barkla, D H

    1983-01-01

    Three histamine H2-receptor antagonists, Cimetidine, Metiamide and Ranitidine, were tested for their inhibitory effect on two experimental bowel cancer models. In the first model mitotic rates were measured in dimethylhydrazine-induced tumors of rat colon and in the second model volumetric changes in human large bowel cancer xenografts were assessed. In tumors of rat colon all three drugs were able to suppress mitotic activity, but the effects of Metiamide and Ranitidine were more prolonged than that of Cimetidine in each of two lines of human bowel cancer that were used. Metiamide and Ranitidine were also more effective growth inhibitors than was Cimetidine.

  5. Presynaptic inhibition of spontaneous acetylcholine release mediated by P2Y receptors at the mouse neuromuscular junction.

    Science.gov (United States)

    De Lorenzo, S; Veggetti, M; Muchnik, S; Losavio, A

    2006-09-29

    coupled to G(i/o) proteins. The protein kinase C (PKC) antagonist chelerythrine and the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) occluded the effect of betagamma-imido ATP, while the protein kinase A (PKA) antagonist KT-5720 and the inhibitor of the calcium/calmodulin-dependent protein kinase II (CAMKII) KN-62 failed to do so. betagamma-Imido ATP did not affect 10, 15 and 20 mM K(+)-evoked release and application of reactive blue-2 before incubation in high K(+) induced a higher asynchronous secretion. Thus, our results show that at mammalian neuromuscular junctions, ATP induces presynaptic inhibition of spontaneous ACh release due to the modulation of Ca(2+) channels related to tonic secretion through the activation of P2Y receptors coupled to G(i/o) proteins. We also demonstrated that at increasing degrees of membrane depolarization evoked by K(+), endogenously released ATP induces presynaptic inhibition as a means of preventing excessive neurotransmitter secretion.

  6. Steroid receptor coactivator 1 deficiency increases MMTV-neu mediated tumor latency and differentiation specific gene expression, decreases metastasis, and inhibits response to PPAR ligands

    International Nuclear Information System (INIS)

    Han, Ji Seung; Crowe, David L

    2010-01-01

    The peroxisome proliferator activated receptor (PPAR) subgroup of the nuclear hormone receptor superfamily is activated by a variety of natural and synthetic ligands. PPARs can heterodimerize with retinoid X receptors, which have homology to other members of the nuclear receptor superfamily. Ligand binding to PPAR/RXRs results in recruitment of transcriptional coactivator proteins such as steroid receptor coactivator 1 (SRC-1) and CREB binding protein (CBP). Both SRC-1 and CBP are histone acetyltransferases, which by modifying nucleosomal histones, produce more open chromatin structure and increase transcriptional activity. Nuclear hormone receptors can recruit limiting amounts of coactivators from other transcription factor binding sites such as AP-1, thereby inhibiting the activity of AP-1 target genes. PPAR and RXR ligands have been used in experimental breast cancer therapy. The role of coactivator expression in mammary tumorigenesis and response to drug therapy has been the subject of recent studies. We examined the effects of loss of SRC-1 on MMTV-neu mediated mammary tumorigenesis. SRC-1 null mutation in mammary tumor prone mice increased the tumor latency period, reduced tumor proliferation index and metastasis, inhibited response to PPAR and RXR ligands, and induced genes involved in mammary gland differentiation. We also examined human breast cancer cell lines overexpressing SRC-1 or CBP. Coactivator overexpression increased cellular proliferation with resistance to PPAR and RXR ligands and remodeled chromatin of the proximal epidermal growth factor receptor promoter. These results indicate that histone acetyltransferases play key roles in mammary tumorigenesis and response to anti-proliferative therapies

  7. Peroxisome-proliferator-activated receptor-γ agonists inhibit the release of proinflammatory cytokines from RSV-infected epithelial cells

    International Nuclear Information System (INIS)

    Arnold, Ralf; Koenig, Wolfgang

    2006-01-01

    The epithelial cells of the airways are the target cells for respiratory syncytial virus (RSV) infection and the site of the majority of the inflammation associated with the disease. Recently, peroxisome-proliferator-activated receptor γ (PPARγ), a member of the nuclear hormone receptor superfamily, has been shown to possess anti-inflammatory properties. Therefore, we investigated the role of PPARγ agonists (15d-PGJ 2 , ciglitazone and troglitazone) on the synthesis of RSV-induced cytokine release from RSV-infected human lung epithelial cells (A549). We observed that all PPARγ ligands inhibited dose-dependently the release of TNF-α, GM-CSF, IL-1α, IL-6 and the chemokines CXCL8 (IL-8) and CCL5 (RANTES) from RSV-infected A549 cells. Concomitantly, the PPARγ ligands diminished the cellular amount of mRNA encoding for IL-6, CXCL8 and CCL5 and the RSV-induced binding activity of the transcription factors NF-κB (p65/p50) and AP-1 (c-fos), respectively. Our data presented herein suggest a potential application of PPARγ ligands in the anti-inflammatory treatment of RSV infection

  8. Onbaekwon Suppresses Colon Cancer Cell Invasion by Inhibiting Expression of the CXC Chemokine Receptor 4.

    Science.gov (United States)

    Kim, Buyun; Yoon, Jaewoo; Yoon, Seong Woo; Park, Byoungduck

    2017-06-01

    Cysteine X cysteine (CXC) chemokine receptor 4 (CXCR4) and C-X-C motif chemokine 12 (CXCL12) were originally identified as chemoattractants between immune cells and sites of inflammation. Since studies have validated an increased level of CXCL12 and its receptor in patients with colorectal cancers, CXCL12/CXCR4 axis has been considered as a valuable marker of cancer metastasis. Therefore, identification of CXCR4 inhibitors has great potential to abrogate tumor metastasis. Onbaekwon (OBW) is a complex herbal formula that is derived from the literature of traditional Korean medicine Dongeuibogam. In this study, we demonstrated that OBW suppressed CXCR4 expression in various cancer cell types in a concentration- and time-dependent manner. Both proteasomal and lysosomal inhibitors had no effect to prevent the OBW-induced suppression of CXCR4, suggesting that the inhibitory effect of OBW was not due to proteolytic degradation but occurred at the transcriptional level. Electrophoretic mobility shift assay further confirmed that OBW could block endogenous activation of nuclear factor kappa B, a key transcription factor that regulates the expression of CXCR4 in colon cancer cells. Consistent with the aforementioned molecular basis, OBW abolished cell invasion induced by CXCL12 in colon cancer cells. Together, our results suggest that OBW, as a novel inhibitor of CXCR4, could be a promising therapeutic agent contributing to cancer treatment.

  9. Forsythoside A exerts antipyretic effect on yeast-induced pyrexia mice via inhibiting transient receptor potential vanilloid 1 function

    Science.gov (United States)

    Liu, Cuiling; Su, Hongchang; Wan, Hongye; Qin, Qingxia; Wu, Xuan; Kong, Xiangying; Lin, Na

    2017-01-01

    Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel gated by noxious heat, playing major roles in thermoregulation. Forsythoside A (FT-A) is the most abundant phenylethanoid glycosides in Fructus Forsythiae, which has been prescribed as a medicinal herb for treating fever in China for a long history. However, how FT-A affects pyrexia and what is the underlying molecular mechanism remain largely unknown. Here we found that FT-A exerted apparent antipyretic effect through decreasing the levels of prostaglandin E2 (PGE2) and interleukin 8 (IL-8) in a dose-dependent fashion on the yeast induced pyrexia mice. Interestingly, FT-A significantly downregulated TRPV1 expression in the hypothalamus and dorsal root ganglion (DRG) of the yeast induced pyrexia mice. Moreover, FT-A inhibited IL-8 and PGE2 secretions, and calcium influx in the HEK 293T-TRPV1 cells after stimulated with capsaicin, the specific TRPV1 agonist. Further investigation of the molecular mechanisms revealed that FT-A treatment rapidly inhibited phosphorylation of extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 in both yeast induced pyrexia mice and HEK 293T-TRPV1 cells. These results suggest that FT-A may serve as a potential antipyretic agent and the therapeutic action of Fructus Forsythiae on pyretic related disease is, in part, due to the FT-A activities. PMID:28123347

  10. Lithocholic acid controls adaptive immune responses by inhibition of Th1 activation through the Vitamin D receptor.

    Science.gov (United States)

    Pols, Thijs W H; Puchner, Teresa; Korkmaz, H Inci; Vos, Mariska; Soeters, Maarten R; de Vries, Carlie J M

    2017-01-01

    Bile acids are established signaling molecules next to their role in the intestinal emulsification and uptake of lipids. We here aimed to identify a potential interaction between bile acids and CD4+ Th cells, which are central in adaptive immune responses. We screened distinct bile acid species for their potency to affect T cell function. Primary human and mouse CD4+ Th cells as well as Jurkat T cells were used to gain insight into the mechanism underlying these effects. We found that unconjugated lithocholic acid (LCA) impedes Th1 activation as measured by i) decreased production of the Th1 cytokines IFNγ and TNFαα, ii) decreased expression of the Th1 genes T-box protein expressed in T cells (T-bet), Stat-1 and Stat4, and iii) decreased STAT1α/β phosphorylation. Importantly, we observed that LCA impairs Th1 activation at physiological relevant concentrations. Profiling of MAPK signaling pathways in Jurkat T cells uncovered an inhibition of ERK-1/2 phosphorylation upon LCA exposure, which could provide an explanation for the impaired Th1 activation. LCA induces these effects via Vitamin D receptor (VDR) signaling since VDR RNA silencing abrogated these effects. These data reveal for the first time that LCA controls adaptive immunity via inhibition of Th1 activation. Many factors influence LCA levels, including bile acid-based drugs and gut microbiota. Our data may suggest that these factors also impact on adaptive immunity via a yet unrecognized LCA-Th cell axis.

  11. Essential Oil of Pinus koraiensis Exerts Antiobesic and Hypolipidemic Activity via Inhibition of Peroxisome Proliferator-Activated Receptors Gamma Signaling

    Directory of Open Access Journals (Sweden)

    Hyun-Suk Ko

    2013-01-01

    Full Text Available Our group previously reported that essential oil of Pinus koraiensis (EOPK exerts antihyperlipidemic effects via upregulation of low-density lipoprotein receptor and inhibition of acyl-coenzyme A. In the present study, we investigated the antiobesity and hypolipidemic mechanism of EOPK using in vitro 3T3-L1 cells and in vivo HFD-fed rats. EOPK markedly suppressed fat accumulation and intracellular triglyceride associated with downregulation of adipogenic transcription factor expression, including PPARγ and CEBPα in the differentiated 3T3-L1 adipocytes. Additionally, EOPK attenuated the expression levels of FABP and GPDH as target genes of PPARγ during adipocyte differentiation. Furthermore, PPARγ inhibitor GW9662 enhanced the decreased expression of FABP and PPARγ and fat accumulation induced by EOPK. To confirm the in vitro activity of EOPK, animal study was performed by administering normal diet, HFD, and/or EOPK at the dose of 100 or 200 mg/kg for 6 weeks. Consistently, EOPK significantly suppressed body weight gain, serum triglyceride, total cholesterol, LDL cholesterol, and AI value and increased HDL cholesterol in a dose-dependent manner. Immunohistochemistry revealed that EOPK treatment abrogated the expression of PPARγ in the liver tissue sections of EOPK-treated rats. Taken together, our findings suggest that EOPK has the antiobesic and hypolipidemic potential via inhibition of PPARγ-related signaling.

  12. Essential Oil of Pinus koraiensis Exerts Antiobesic and Hypolipidemic Activity via Inhibition of Peroxisome Proliferator-Activated Receptors Gamma Signaling.

    Science.gov (United States)

    Ko, Hyun-Suk; Lee, Hyo-Jeong; Lee, Hyo-Jung; Sohn, Eun Jung; Yun, Miyong; Lee, Min-Ho; Kim, Sung-Hoon

    2013-01-01

    Our group previously reported that essential oil of Pinus koraiensis (EOPK) exerts antihyperlipidemic effects via upregulation of low-density lipoprotein receptor and inhibition of acyl-coenzyme A. In the present study, we investigated the antiobesity and hypolipidemic mechanism of EOPK using in vitro 3T3-L1 cells and in vivo HFD-fed rats. EOPK markedly suppressed fat accumulation and intracellular triglyceride associated with downregulation of adipogenic transcription factor expression, including PPAR γ and CEBP α in the differentiated 3T3-L1 adipocytes. Additionally, EOPK attenuated the expression levels of FABP and GPDH as target genes of PPAR γ during adipocyte differentiation. Furthermore, PPAR γ inhibitor GW9662 enhanced the decreased expression of FABP and PPAR γ and fat accumulation induced by EOPK. To confirm the in vitro activity of EOPK, animal study was performed by administering normal diet, HFD, and/or EOPK at the dose of 100 or 200 mg/kg for 6 weeks. Consistently, EOPK significantly suppressed body weight gain, serum triglyceride, total cholesterol, LDL cholesterol, and AI value and increased HDL cholesterol in a dose-dependent manner. Immunohistochemistry revealed that EOPK treatment abrogated the expression of PPAR γ in the liver tissue sections of EOPK-treated rats. Taken together, our findings suggest that EOPK has the antiobesic and hypolipidemic potential via inhibition of PPAR γ -related signaling.

  13. Propofol inhibits hypoxia/reoxygenation-induced human gastric epithelial cell injury by suppressing the Toll-like receptor 4 pathway

    Directory of Open Access Journals (Sweden)

    Jiao-Li Zhang

    2013-06-01

    Full Text Available This study aimed to investigate the role of the Toll-like receptor 4 (TLR4 pathway in normal human gastric epithelial (GES-1 cells under hypoxia/reoxygenation (H/R in vitro, and the effect of propofol on injured GES-1 cells as well as its possible mechanism. Before H/R induction, GES-1 cells were preconditioned with fat emulsion, propofol, or epigallocatechin gallate. Then cell viability, cell apoptosis, and related molecules in the cells were analyzed under experimental conditions. We found that propofol 50 μmol/L markedly inhibited the H/R injury under hypoxia 1.5 h/reoxygenation 2 hours by promoting GES-1 cell viability and decreasing cell apoptosis. The TLR4 signal may be involved in the protective effect of propofol against H/R injury. The malondialdehyde contents and superoxide dismutase activities were recovered under propofol preconditioning. In summary, propofol preconditioning may exert a protective effect on H/R injury in GES-1 cells and the mechanism may be via inhibition of the activated TLR4 signal under H/R conditions.

  14. Kynurenine 3-monooxygenase mediates inhibition of Th17 differentiation via catabolism of endogenous aryl hydrocarbon receptor ligands.

    Science.gov (United States)

    Stephens, Geoffrey L; Wang, Qun; Swerdlow, Bonnie; Bhat, Geetha; Kolbeck, Roland; Fung, Michael

    2013-07-01

    The aryl hydrocarbon receptor (AhR) is a key transcriptional regulator of Th17-cell differentiation. Although endogenous ligands have yet to be identified, evidence suggests that tryptophan metabolites can act as agonists for the AhR. Tryptophan metabolites are abundant in circulation, so we hypothesized that cell intrinsic factors might exist to regulate the exposure of Th17 cells to AhR-dependent activities. Here, we find that Th17 cells preferentially express kynurenine 3-monooxygenase (KMO), which is an enzyme involved in catabolism of the tryptophan metabolite kynurenine. KMO inhibition, either with a specific inhibitor or via siRNA-mediated silencing, markedly increased IL-17 production in vitro, whereas IFN-γ production by Th1 cells was unaffected. Inhibition of KMO significantly exacerbated disease in a Th17-driven model of autoimmune gastritis, suggesting that expression of KMO by Th17 cells serves to limit their continuous exposure to physiological levels of endogenous AhR ligands in vivo. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Kaempferol inhibits fibroblast collagen synthesis, proliferation and activation in hypertrophic scar via targeting TGF-β receptor type I.

    Science.gov (United States)

    Li, Hongwei; Yang, Liu; Zhang, Yuebing; Gao, Zhigang

    2016-10-01

    Hypertrophic scar (HPS) formation is a debilitating condition that results in pain, esthetic symptom and loss of tissue function. So far, no satisfactory therapeutic approach has been available for HPS treatment. In this study, we discovered that a natural small molecule, kaempferol, could significantly inhibit HPS formation in a mechanical load-induced mouse model. Our results also demonstrated that kaempferol remarkably attenuated collagen synthesis, proliferation and activation of fibroblasts in vitro and in vivo. Western blot analysis further revealed that kaempferol significantly down-regulated Smad2 and Smad3 phosphorylation in a dose-dependent manner. At last, we found that such bioactivity of kaempferol which resulted from the inhibition of TGF-β1/Smads signaling was induced by the selective binding of kaempferol to TGF-β receptor type I (TGFβRI). These findings suggest that kaempferol could be developed into a promising agent for the treatment of HPS or other fibroproliferative disorders. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  16. Inhibition of tumor metastasis by a growth factor receptor bound protein 2 Src homology 2 domain-binding antagonist.

    Science.gov (United States)

    Giubellino, Alessio; Gao, Yang; Lee, Sunmin; Lee, Min-Jung; Vasselli, James R; Medepalli, Sampath; Trepel, Jane B; Burke, Terrence R; Bottaro, Donald P

    2007-07-01

    Metastasis, the primary cause of death in most forms of cancer, is a multistep process whereby cells from the primary tumor spread systemically and colonize distant new sites. Blocking critical steps in this process could potentially inhibit tumor metastasis and dramatically improve cancer survival rates; however, our understanding of metastasis at the molecular level is still rudimentary. Growth factor receptor binding protein 2 (Grb2) is a widely expressed adapter protein with roles in epithelial cell growth and morphogenesis, as well as angiogenesis, making it a logical target for anticancer drug development. We have previously shown that a potent antagonist of Grb2 Src homology-2 domain-binding, C90, blocks growth factor-driven cell motility in vitro and angiogenesis in vivo. We now report that C90 inhibits metastasis in vivo in two aggressive tumor models, without affecting primary tumor growth rate. These results support the potential efficacy of this compound in reducing the metastatic spread of primary solid tumors and establish a critical role for Grb2 Src homology-2 domain-mediated interactions in this process.

  17. Dihydroartemisinin counteracts fibrotic portal hypertension via farnesoid X receptor-dependent inhibition of hepatic stellate cell contraction.

    Science.gov (United States)

    Xu, Wenxuan; Lu, Chunfeng; Zhang, Feng; Shao, Jiangjuan; Yao, Shunyu; Zheng, Shizhong

    2017-01-01

    Portal hypertension is a frequent pathological symptom occurring especially in hepatic fibrosis and cirrhosis. Current paradigms indicate that inhibition of hepatic stellate cell (HSC) activation and contraction is anticipated to be an attractive therapeutic strategy, because activated HSC dominantly facilitates an increase in intrahepatic vein pressure through secreting extracellular matrix and contracting. Our previous in vitro study indicated that dihydroartemisinin (DHA) inhibited contractility of cultured HSC by activating intracellular farnesoid X receptor (FXR). However, the effect of DHA on fibrosis-related portal hypertension still requires clarification. In this study, gain- and loss-of-function models of FXR in HSC were established to investigate the mechanisms underlying DHA protection against chronic CCl 4 -caused hepatic fibrosis and portal hypertension. Immunofluorescence staining visually showed a decrease in FXR expression in CCl 4 -administrated rat HSC but an increase in that in DHA-treated rat HSC. Serum diagnostics and morphological analyses consistently indicated that DHA exhibited hepatoprotective effects on CCl 4 -induced liver injury. DHA also reduced CCl 4 -caused inflammatory mediator expression and inflammatory cell infiltration. These improvements were further enhanced by INT-747 but weakened by Z-guggulsterone. Noteworthily, DHA, analogous to INT-747, significantly lowered portal vein pressure and suppressed fibrogenesis. Experiments on mice using FXR shRNA lentivirus consolidated the results above. Mechanistically, inhibition of HSC activation and contraction was found as a cellular basis for DHA to relieve portal hypertension. These findings demonstrated that DHA attenuated portal hypertension in fibrotic rodents possibly by targeting HSC contraction via a FXR activation-dependent mechanism. FXR could be a target molecule for reducing portal hypertension during hepatic fibrosis. © 2016 Federation of European Biochemical Societies.

  18. Estrogen receptor β inhibits estradiol-induced proliferation and migration of MCF-7 cells through regulation of mitofusin 2.

    Science.gov (United States)

    Ma, Li; Liu, Yueping; Geng, Cuizhi; Qi, Xiaowei; Jiang, Jun

    2013-06-01

    In the present study, we investigated whether estrogen receptor (ER) β affected the proliferation and migration of the human breast cancer cell line MCF-7 through regulation of mitofusin 2 (mfn2). A previous study reported that mfn2 may be regulated by ER through a non-classical pathway; in this pathway, the ER modulates the activities of other transcription factors by stabilizing their binding to DNA and/or recruiting coactivators to the complex. However, the previous study, unlike the study presented here, did not directly explore the interactions between ER and mfn2. Here, RT-PCR and western blot analysis were used to test the expression of mfn2 in MCF-7 cells after exposure to different doses of estradiol (E2). The ability of cells to proliferate and migrate was determined by MTT assay and a monolayer-wounding protocol, respectively. Finally, changes in MCF-7 cell biology after transfection with ERβ or mfn2 expression vectors were investigated, and the role of ERβ in mfn2 expression was also explored. Our results showed that E2 attenuated mfn2 expression in a dose-dependent manner, concomitant with the activation of proliferation and migration of MCF-7 cells. The mfn2 expression vector effectively suppressed E2-induced upregulation of PCNA and migration in MCF-7 cells. ERβ inhibited the E2-induced mfn2 downregulation that accompanied the inhibition of proliferation and migration in MCF-7 cells. Briefly, ERβ may inhibit E2-induced proliferation and migration of MCF-7 cells through regulation of mfn2.

  19. Platelet-Derived Growth Factor-Receptor α Strongly Inhibits Melanoma Growth In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Debora Faraone

    2009-08-01

    Full Text Available Cutaneous melanoma is the most aggressive skin cancer; it is highly metastatic and responds poorly to current therapies. The expression of platelet-derived growth factor receptors (PDGF-Rs is reported to be reduced in metastatic melanoma compared with benign nevi or normal skin; we then hypothesized that PDGF-Rα may control growth of melanoma cells. We show here that melanoma cells overexpressing PDGF-Rα respond to serum with a significantly lower proliferation compared with that of controls. Apoptosis, cell cycle arrest, pRb dephosphorylation, and DNA synthesis inhibition were also observed in cells overexpressing PDGF-Rα. Proliferation was rescued by PDGF-Rα inhibitors, allowing to exclude nonspecific toxic effects and indicating that PDGF-Rα mediates autocrine antiproliferation signals in melanoma cells. Accordingly, PDGF-Rα was found to mediate staurosporine cytotoxicity. A protein array-based analysis of the mitogen-activated protein kinase pathway revealed that melanoma cells overexpressing PDGF-Rα show a strong reduction of c-Jun phosphorylated in serine 63 and of protein phosphatase 2A/Bα and a marked increase of p38γ, mitogen-activated protein kinase kinase 3, and signal regulatory protein α1 protein expression. In a mouse model of primary melanoma growth, infection with the Ad-vector overexpressing PDGF-Rα reached a significant 70% inhibition of primary melanoma growth (P < .001 and a similar inhibition of tumor angiogenesis. All together, these data demonstrate that PDGF-Rα strongly impairs melanoma growth likely through autocrine mechanisms and indicate a novel endogenous mechanism involved in melanoma control.

  20. Targeting the MET oncogene by concomitant inhibition of receptor and ligand via an antibody-"decoy" strategy.

    Science.gov (United States)

    Basilico, Cristina; Modica, Chiara; Maione, Federica; Vigna, Elisa; Comoglio, Paolo M

    2018-04-25

    MET, a master gene sustaining "invasive growth," is a relevant target for cancer precision therapy. In the vast majority of tumors, wild-type MET behaves as a "stress-response" gene and relies on the ligand (HGF) to sustain cell "scattering," invasive growth and apoptosis protection (oncogene "expedience"). In this context, concomitant targeting of MET and HGF could be crucial to reach effective inhibition. To test this hypothesis, we combined an anti-MET antibody (MvDN30) inducing "shedding" (i.e., removal of MET from the cell surface), with a "decoy" (i.e., the soluble extracellular domain of the MET receptor) endowed with HGF-sequestering ability. To avoid antibody/decoy interaction-and subsequent neutralization-we identified a single aminoacid in the extracellular domain of MET-lysine 842-that is critical for MvDN30 binding and engineered the corresponding recombinant decoyMET (K842E). DecoyMET K842E retains the ability to bind HGF with high affinity and inhibits HGF-induced MET phosphorylation. In HGF-dependent cellular models, MvDN30 antibody and decoyMET K842E used in combination cooperate in restraining invasive growth, and synergize in blocking cancer cell "scattering." The antibody and the decoy unbridle apoptosis of colon cancer stem cells grown in vitro as spheroids. In a preclinical model, built by orthotopic transplantation of a human pancreatic carcinoma in SCID mice engineered to express human HGF, concomitant treatment with antibody and decoy significantly reduces metastatic spread. The data reported indicate that vertical targeting of the MET/HGF axis results in powerful inhibition of ligand-dependent MET activation, providing proof of concept in favor of combined target therapy of MET "expedience." © 2018 UICC.

  1. Inhibition of receptor tyrosine kinase signalling by small molecule agonist of T-cell protein tyrosine phosphatase

    International Nuclear Information System (INIS)

    Mattila, Elina; Marttila, Heidi; Sahlberg, Niko; Kohonen, Pekka; Tähtinen, Siri; Halonen, Pasi; Perälä, Merja; Ivaska, Johanna

    2010-01-01

    T-cell protein tyrosine phosphatase (TCPTP/TC45) is a ubiquitously expressed intra-cellular non-receptor protein tyrosine phosphatase involved in the negative regulation of several cancer relevant cellular signalling pathways. We have previously shown that interaction between the α-cytoplasmic tail of α1β1 integrin and TCPTP activates TCPTP by disrupting an inhibitory intra-molecular bond in TCPTP. Thus, inhibition of the regulatory interaction in TCPTP is a desirable strategy for TCPTP activation and attenuation of oncogenic RTK signalling. However, this is challenging with low molecular weight compounds. We developed a high-throughput compatible assay to analyse activity of recombinant TCPTP in vitro. Using this assay we have screened 64280 small molecules to identify novel agonists for TCPTP. Dose-dependent response to TCPTP agonist was performed using the in vitro assay. Inhibition effects and specificity of TCPTP agonists were evaluated using TCPTP expressing and null mouse embryonic fibroblasts. Western blot analysis was used to evaluate attenuation of PDGFRβ and EGFR phosphorylation. Inhibition of VEGF signalling was analysed with VEGF-induced endothelial cell sprouting assays. From the screen we identified six TCPTP agonists. Two compounds competed with α1-cytoplasmic domain for binding to TCPTP, suggesting that they activate TCPTP similar to α1-cyt by disrupting the intra-molecular bond in TCPTP. Importantly, one of the compounds (spermidine) displayed specificity towards TCPTP in cells, since TCPTP -/- cells were 43-fold more resistant to the compound than TCPTP expressing cells. This compound attenuates PDGFRβ and VEGFR2 signalling in cells in a TCPTP-dependent manner and functions as a negative regulator of EGFR phosphorylation in cancer cells. In this study we showed that small molecules mimicking TCPTP-α1 interaction can be used as TCPTP agonists. These data provide the first proof-of-concept description of the use of high-throughput screening

  2. Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells.

    Science.gov (United States)

    Kang, Minyong; Lee, Kyoung-Hwa; Lee, Hye Sun; Jeong, Chang Wook; Kwak, Cheol; Kim, Hyeon Hoe; Ku, Ja Hyeon

    2017-02-04

    Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR) inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1), chloroquine (CQ) and 3-methyladenine (3-MA) were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8) assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI) was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA) remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating a novel

  3. Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells

    Directory of Open Access Journals (Sweden)

    Minyong Kang

    2017-02-01

    Full Text Available Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1, chloroquine (CQ and 3-methyladenine (3-MA were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8 assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating

  4. Grb-IR: A SH2-Domain-Containing Protein that Binds to the Insulin Receptor and Inhibits Its Function

    Science.gov (United States)

    Liu, Feng; Roth, Richard A.

    1995-10-01

    To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.

  5. Different protein kinase C isoenzymes mediate inhibition of cardiac rapidly activating delayed rectifier K+ current by different G-protein coupled receptors.

    Science.gov (United States)

    Liu, Xueli; Wang, Yuhong; Zhang, Hua; Shen, Li; Xu, Yanfang

    2017-12-01

    Elevated angiotensin II (Ang II) and sympathetic activity contributes to a high risk of ventricular arrhythmias in heart disease. The rapidly activating delayed rectifier K + current (I Kr ) carried by the hERG channels plays a critical role in cardiac repolarization, and decreased I Kr is involved in increased cardiac arrhythmogenicity. Stimulation of α 1A -adrenoreceptors or angiotensin II AT 1 receptors is known to inhibit I Kr via PKC. Here, we have identified the PKC isoenzymes mediating the inhibition of I Kr by activation of these two different GPCRs. The whole-cell patch-clamp technique was used to record I Kr in guinea pig cardiomyocytes and HEK293 cells co-transfected with hERG and α 1A -adrenoreceptor or AT 1 receptor genes. A broad spectrum PKC inhibitor Gö6983 (not inhibiting PKCε), a selective cPKC inhibitor Gö6976 and a PKCα-specific inhibitor peptide, blocked the inhibition of I Kr by the α 1A -adrenoreceptor agonist A61603. However, these inhibitors did not affect the reduction of I Kr by activation of AT 1 receptors, whereas the PKCε-selective inhibitor peptide did block the effect. The effects of angiotensin II and the PKCε activator peptide were inhibited in mutant hERG channels in which 17 of the 18 PKC phosphorylation sites were deleted, whereas a deletion of the N-terminus of the hERG channels selectively prevented the inhibition elicited by A61603 and the cPKC activator peptide. Our results indicated that inhibition of I Kr by activation of α 1A -adrenoreceptors or AT 1 receptors were mediated by PKCα and PKCε isoforms respectively, through different molecular mechanisms. © 2017 The British Pharmacological Society.

  6. Chlorotoxin Fused to IgG-Fc Inhibits Glioblastoma Cell Motility via Receptor-Mediated Endocytosis

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    Tomonari Kasai

    2012-01-01

    Full Text Available Chlorotoxin is a 36-amino acid peptide derived from Leiurus quinquestriatus (scorpion venom, which has been shown to inhibit low-conductance chloride channels in colonic epithelial cells. Chlorotoxin also binds to matrix metalloproteinase-2 and other proteins on glioma cell surfaces. Glioma cells are considered to require the activation of matrix metalloproteinase-2 during invasion and migration. In this study, for targeting glioma, we designed two types of recombinant chlorotoxin fused to human IgG-Fcs with/without a hinge region. Chlorotoxin fused to IgG-Fcs was designed as a dimer of 60 kDa with a hinge region and a monomer of 30 kDa without a hinge region. The monomeric and dimeric forms of chlorotoxin inhibited cell proliferation at 300 nM and induced internalization in human glioma A172 cells. The monomer had a greater inhibitory effect than the dimer; therefore, monomeric chlorotoxin fused to IgG-Fc was multivalently displayed on the surface of bionanocapsules to develop a drug delivery system that targeted matrix metalloproteinase-2. The target-dependent internalization of bionanocapsules in A172 cells was observed when chlorotoxin was displayed on the bionanocapsules. This study indicates that chlorotoxin fused to IgG-Fcs could be useful for the active targeting of glioblastoma cells.

  7. Stilbene induced inhibition of androgen receptor dimerization: implications for AR and ARΔLBD-signalling in human prostate cancer cells.

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    Wolfgang Streicher

    Full Text Available BACKGROUND: Advanced castration resistant prostate cancer (CRPC is often characterized by an increase of C-terminally truncated, constitutively active androgen receptor (AR variants. Due to the absence of a ligand binding domain located in the AR-C-terminus, these receptor variants (also termed ARΔLBD are unable to respond to all classical forms of endocrine treatments like surgical/chemical castration and/or application of anti-androgens. METHODOLOGY: In this study we tested the effects of the naturally occurring stilbene resveratrol (RSV and (E-4-(2, 6-Difluorostyryl-N, N-dimethylaniline, a fluorinated dialkylaminostilbene (FIDAS on AR- and ARΔLBD in prostate cancer cells. The ability of the compounds to modulate transcriptional activity of AR and the ARΔLBD-variant Q640X was shown by reporter gene assays. Expression of endogenous AR and ARΔLBD mRNA and protein levels were determined by qRT-PCR and Western Blot. Nuclear translocation of AR-molecules was analyzed by fluorescence microscopy. AR and ARΔLBD/Q640X homo-/heterodimer formation was assessed by mammalian two hybrid assays. Biological activity of both compounds in vivo was demonstrated using a chick chorioallantoic membrane xenograft assay. RESULTS: The stilbenes RSV and FIDAS were able to significantly diminish AR and Q640X-signalling. Successful inhibition of the Q640X suggests that RSV and FIDAS are not interfering with the AR-ligand binding domain like all currently available anti-hormonal drugs. Repression of AR and Q640X-signalling by RSV and FIDAS in prostate cancer cells was caused by an inhibition of the AR and/or Q640X-dimerization. Although systemic bioavailability of both stilbenes is very low, both compounds were also able to downregulate tumor growth and AR-signalling in vivo. CONCLUSION: RSV and FIDAS are able to inhibit the dimerization of AR and ARΔLBD molecules suggesting that stilbenes might serve as lead compounds for a novel generation of AR-inhibitors.

  8. The stress protein heat shock cognate 70 (Hsc70) inhibits the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel.

    Science.gov (United States)

    Iftinca, Mircea; Flynn, Robyn; Basso, Lilian; Melo, Helvira; Aboushousha, Reem; Taylor, Lauren; Altier, Christophe

    2016-01-01

    Specialized cellular defense mechanisms prevent damage from chemical, biological, and physical hazards. The heat shock proteins have been recognized as key chaperones that maintain cell survival against a variety of exogenous and endogenous stress signals including noxious temperature. However, the role of heat shock proteins in nociception remains poorly understood. We carried out an expression analysis of the constitutively expressed 70 kDa heat-shock cognate protein, a member of the stress-induced HSP70 family in lumbar dorsal root ganglia from a mouse model of Complete Freund's Adjuvant-induced chronic inflammatory pain. We used immunolabeling of dorsal root ganglion neurons, behavioral analysis and patch clamp electrophysiology in both dorsal root ganglion neurons and HEK cells transfected with Hsc70 and Transient Receptor Potential Channels to examine their functional interaction in heat shock stress condition. We report an increase in protein levels of Hsc70 in mouse dorsal root ganglia, 3 days post Complete Freund's Adjuvant injection in the hind paw. Immunostaining of Hsc70 was observed in most of the dorsal root ganglion neurons, including the small size nociceptors immunoreactive to the TRPV1 channel. Standard whole-cell patch-clamp technique was used to record Transient Receptor Potential Vanilloid type 1 current after exposure to heat shock. We found that capsaicin-evoked currents are inhibited by heat shock in dorsal root ganglion neurons and transfected HEK cells expressing Hsc70 and TRPV1. Blocking Hsc70 with matrine or spergualin compounds prevented heat shock-induced inhibition of the channel. We also found that, in contrast to TRPV1, both the cold sensor channels TRPA1 and TRPM8 were unresponsive to heat shock stress. Finally, we show that inhibition of TRPV1 depends on the ATPase activity of Hsc70 and involves the rho-associated protein kinase. Our work identified Hsc70 and its ATPase activity as a central cofactor of TRPV1 channel function

  9. IMPDHII Protein Inhibits Toll-like Receptor 2-mediated Activation of NF-κB*

    Science.gov (United States)

    Toubiana, Julie; Rossi, Anne-Lise; Grimaldi, David; Belaidouni, Nadia; Chafey, Philippe; Clary, Guilhem; Courtine, Emilie; Pene, Frederic; Mira, Jean-Paul; Claessens, Yann-Erick; Chiche, Jean-Daniel

    2011-01-01

    Toll-like receptor 2 (TLR2) plays an essential role in innate immunity by the recognition of a large variety of pathogen-associated molecular patterns. It induces its recruitment to lipid rafts induces the formation of a membranous activation cluster necessary to enhance, amplify, and control downstream signaling. However, the exact composition of the TLR2-mediated molecular complex is unknown. We performed a proteomic analysis in lipopeptide-stimulated THP1 and found IMPDHII protein rapidly recruited to lipid raft. Whereas IMPDHII is essential for lymphocyte proliferation, its biologic function within innate immune signal pathways has not been established yet. We report here that IMPDHII plays an important role in the negative regulation of TLR2 signaling by modulating PI3K activity. Indeed, IMPDHII increases the phosphatase activity of SHP1, which participates to the inactivation of PI3K. PMID:21460227

  10. Liver X Receptor Agonists Inhibit the Phospholipid Regulatory Gene CTP: Phosphoethanolamine Cytidylyltransferase-Pcyt2

    Directory of Open Access Journals (Sweden)

    Lin Zhu

    2008-01-01

    Full Text Available Metabolic pulse-chase experiments demonstrated that 25-hydroxycholesterol (25-OH, the endogenous activator of the liver X receptor (LXR, significantly reduced the biosynthesis of phosphatidylethanolamine via CDP-ethanolamine (Kennedy pathway at the step catalyzed by CTP: phosphoethanolamine cytidylyltransferase (Pcyt2. In the mouse embryonic fibroblasts C3H10T1/2, the LXR synthetic agonist TO901317 lowered Pcyt2 promoter-luciferase activity in a concentration-dependent manner. Furthermore, 25-OH and TO901317 reduced mouse Pcyt2 mRNA and protein levels by 35–60%. The inhibitory effects of oxysterols and TO901317 on the Pcyt2 promoter function, mRNA and protein expression were conserved in the human breast cancer cells MCF-7. These studies identify the Pcyt2 gene as a novel target whereby LXR agonists may indirectly modulate inflammatory responses and atherosclerosis.

  11. An endogenous aryl hydrocarbon receptor ligand inhibits proliferation and migration of human ovarian cancer cells.

    Science.gov (United States)

    Wang, Kai; Li, Yan; Jiang, Yi-Zhou; Dai, Cai-Feng; Patankar, Manish S; Song, Jia-Sheng; Zheng, Jing

    2013-10-28

    The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor mediates many biological processes. Herein, we investigated if 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE, an endogenous AhR ligand) regulated proliferation and migration of human ovarian cancer cells via AhR. We found that AhR was widely present in many histotypes of ovarian cancer tissues. ITE suppressed OVCAR-3 cell proliferation and SKOV-3 cell migration in vitro, which were blocked by AhR knockdown. ITE also suppressed OVCAR-3 cell growth in mice. These data suggest that the ITE might potentially be used for therapeutic intervention for at least a subset of human ovarian cancer. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  12. Epidermal growth factor receptor activation by diesel particles is mediated by tyrosine phosphatase inhibition

    International Nuclear Information System (INIS)

    Tal, Tamara L.; Bromberg, Philip A.; Kim, Yumee; Samet, James M.

    2008-01-01

    Exposure to particulate matter (PM) is associated with increased cardiopulmonary morbidity and mortality. Diesel exhaust particles (DEP) are a major component of ambient PM and may contribute to PM-induced pulmonary inflammation. Proinflammatory signaling is mediated by phosphorylation-dependent signaling pathways whose activation is opposed by the activity of protein tyrosine phosphatases (PTPases) which thereby function to maintain signaling quiescence. PTPases contain an invariant catalytic cysteine that is susceptible to electrophilic attack. DEP contain electrophilic oxy-organic compounds that may contribute to the oxidant effects of PM. Therefore, we hypothesized that exposure to DEP impairs PTPase activity allowing for unopposed basal kinase activity. Here we report that exposure to 30 μg/cm 2 DEP for 4 h induces differential activation of signaling in primary cultures of human airway epithelial cells (HAEC), a primary target cell in PM inhalation. In-gel kinase activity assay of HAEC exposed to DEPs of low (L-DEP), intermediate (I-DEP) or high (H-DEP) organic content showed differential activation of intracellular kinases. Exposure to these DEP also induced varying levels of phosphorylation of the receptor tyrosine kinase EGFR in a manner that requires EGFR kinase activity but does not involve receptor dimerization. We demonstrate that treatment with DEP results in an impairment of total and EGFR-directed PTPase activity in HAEC with a potency that is independent of the organic content of these particles. These data show that DEP-induced EGFR phosphorylation in HAEC is the result of a loss of PTPase activities which normally function to dephosphorylate EGFR in opposition to baseline EGFR kinase activity

  13. L-arginine prevents xanthoma development and inhibits atherosclerosis in LDL receptor knockout mice.

    Science.gov (United States)

    Aji, W; Ravalli, S; Szabolcs, M; Jiang, X C; Sciacca, R R; Michler, R E; Cannon, P J

    1997-01-21

    The potential antiatherosclerotic actions of NO were investigated in four groups of mice (n = 10 per group) lacking functional LDL receptor genes, an animal model of familial hypercholesterolemia. Group 1 was fed a regular chow diet. Groups 2 through 4 were fed a 1.25% high-cholesterol diet. In addition, group 3 received supplemental L-arginine and group 4 received L-arginine and N omega-nitro-L-arginine (L-NA), an inhibitor of NO synthase (NOS). Animals were killed at 6 months; aortas were stained with oil red O for planimetry and with antibodies against constitutive and inducible NOSs. Plasma cholesterol was markedly increased in the animals receiving the high-cholesterol diet. Xanthomas appeared in all mice fed the high-cholesterol diet alone but not in those receiving L-arginine. Aortic atherosclerosis was present in all mice on the high-cholesterol diet. The mean atherosclerotic lesion area was reduced significantly (P < .01) in the cholesterol-fed mice given L-arginine compared with those receiving the high-cholesterol diet alone. The mean atherosclerotic lesion area was significantly larger (P < .01) in cholesterol-fed mice receiving L-arginine + L-NA than in those on the high-cholesterol diet alone. Within the atherosclerotic plaques, endothelial cells immunoreacted for endothelial cell NOS; macrophages, foam cells, and smooth muscle cells immunostained strongly for inducible NOS and nitrotyrosine residues. The data indicate that L-arginine prevents xanthoma formation and reduces atherosclerosis in LDL receptor knockout mice fed a high-cholesterol diet. The abrogation of the beneficial effects of L-arginine by L-NA suggests that the antiatherosclerotic actions of L-arginine are mediated by NOS. The data suggest that L-arginine may be beneficial in familial hypercholesterolemia.

  14. Suppression of asparaginyl endopeptidase attenuates breast cancer-induced bone pain through inhibition of neurotrophin receptors.

    Science.gov (United States)

    Yao, Peng; Ding, Yuanyuan; Han, Zhenkai; Mu, Ying; Hong, Tao; Zhu, Yongqiang; Li, Hongxi

    2017-01-01

    Objective Cancer-induced bone pain is a common clinical problem in breast cancer patients with bone metastasis. However, the mechanisms driving cancer-induced bone pain are poorly known. Recent studies show that a novel protease, asparaginyl endopeptidase (AEP) plays crucial roles in breast cancer metastasis and progression. We aim to determine the functions and targeted suppress of AEP in a mouse model of breast cancer-induced bone pain. Methods Breast cancer cells with AEP knocked-down or overexpression were constructed and implanted into the intramedullary space of the femur to induce pain-like behavior in mice. AEP-specific inhibitors or purified AEP proteins were further used in animal model. The histological characters of femur and pain ethological changes were measured. The expressions of AEP and neurotrophin receptors (p75NTR and TrkA) in dorsal root ganglion and spinal cord were examined. Results Femur radiographs and histological analysis revealed that cells with AEP knocked-down reduced bone destruction and pain behaviors. However, cells with AEP overexpression elevated bone damage and pain behaviors. Further, Western blot results found that the expressions of p75NTR and TrkA in dorsal root ganglions and spinal cords were reduced in mice inoculated with AEP knocked-down cells. Targeted suppression of AEP with specific small compounds significantly reduced the bone pain while purified recombinant AEP proteins increased bone pain. Conclusions AEP aggravate the development of breast cancer bone metastasis and bone pain by increasing the expression of neurotrophin receptors. AEP might be an effective target for treatment of breast cancerinduced bone pain.

  15. The lectins griffithsin, cyanovirin-N and scytovirin inhibit HIV-1 binding to the DC-SIGN receptor and transfer to CD4+ cells

    CSIR Research Space (South Africa)

    Alexandre, Kabamba B

    2012-02-01

    Full Text Available It is generally believed that during the sexual transmission of HIV-1, the glycan-specific DC-SIGN receptor binds the virus and mediates its transfer to CD4(+) cells. The lectins griffithsin (GRFT), cyanovirin-N (CV-N) and scytovirin (SVN) inhibit...

  16. CaMKII inhibition with KN93 attenuates endothelin and serotonin receptor-mediated vasoconstriction and prevents subarachnoid hemorrhage-induced deficits in sensorimotor function

    DEFF Research Database (Denmark)

    Edvinsson, Lars; Povlsen, Gro Klitgaard; Ahnstedt, Hilda

    2014-01-01

    tested the hypothesis that inhibition of calcium calmodulin-dependent protein kinase II (CaMKII) may reduce cerebral vasoconstriction mediated by endothelin and serotonin receptors and improve neurological outcome after experimental SAH. METHODS: SAH was induced in adult rats by injection of 250 μ...

  17. Epidermal growth factor receptor-induced activato protein 1 activity controls density-dependent growht inhibition in normal rat kidney fibroblasts.

    NARCIS (Netherlands)

    Hornberg, J.J.; Dekker, H.; Peters, P.H.J.; Langerak, P.; Westerhoff, H.V.; Lankelma, J.; Zoelen, E.J.J.

    2006-01-01

    Density-dependent growth inhibition secures tissue homeostasis. Dysfunction of the mechanisms, which regulate this type of growth control is a major cause of neoplasia. In confluent normal rat kidney (NRK) fibroblasts, epidermal growth factor (EGF) receptor levels decline, ultimately rendering these

  18. Ciproxifan, a histamine H{sub 3} receptor antagonist and inverse agonist, presynaptically inhibits glutamate release in rat hippocampus

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Cheng-Wei; Lin, Tzu-Yu [Department of Anesthesiology, Far-Eastern Memorial Hospital, Pan-Chiao District, New Taipei City 22060, Taiwan (China); Department of Mechanical Engineering, Yuan Ze University, Taoyuan 320, Taiwan (China); Chang, Chia-Ying [Department of Anesthesiology, Far-Eastern Memorial Hospital, Pan-Chiao District, New Taipei City 22060, Taiwan (China); Department of Chemistry, Fu Jen Catholic University, No. 510, Chung-Cheng Road, Hsin-Chuang District, New Taipei City 24205, Taiwan (China); Huang, Shu-Kuei [Department of Anesthesiology, Far-Eastern Memorial Hospital, Pan-Chiao District, New Taipei City 22060, Taiwan (China); Wang, Su-Jane, E-mail: med0003@mail.fju.edu.tw [School of Medicine, Fu Jen Catholic University, No. 510, Chung-Cheng Rd., Hsin-Chuang, New Taipei 24205, Taiwan (China); Research Center for Chinese Herbal Medicine, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan City, Taiwan (China)

    2017-03-15

    Ciproxifan is an H{sub 3} receptor antagonist and inverse agonist with antipsychotic effects in several preclinical models; its effect on glutamate release has been investigated in the rat hippocampus. In a synaptosomal preparation, ciproxifan reduced 4-aminopyridine (4-AP)-evoked Ca{sup 2+}-dependent glutamate release and cytosolic Ca{sup 2+} concentration elevation but did not affect the membrane potential. The inhibitory effect of ciproxifan on 4-AP-evoked glutamate release was prevented by the Gi/Go-protein inhibitor pertussis toxin and Ca{sub v}2.2 (N-type) and Ca{sub v}2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, but was not affected by the intracellular Ca{sup 2+}-release inhibitors dantrolene and CGP37157. Furthermore, the phospholipase A{sub 2} (PLA{sub 2}) inhibitor OBAA, prostaglandin E{sub 2} (PGE{sub 2}), PGE2 subtype 2 (EP{sub 2}) receptor antagonist PF04418948, and extracellular signal-regulated kinase (ERK) inhibitor FR180204 eliminated the inhibitory effect of ciproxifan on glutamate release. Ciproxifan reduced the 4-AP-evoked phosphorylation of ERK and synapsin I, a presynaptic target of ERK. The ciproxifan-mediated inhibition of glutamate release was prevented in synaptosomes from synapsin I-deficient mice. Moreover, ciproxifan reduced the frequency of miniature excitatory postsynaptic currents without affecting their amplitude in hippocampal slices. Our data suggest that ciproxifan, acting through the blockade of Gi/Go protein-coupled H{sub 3} receptors present on hippocampal nerve terminals, reduces voltage-dependent Ca{sup 2+} entry by diminishing PLA{sub 2}/PGE{sub 2}/EP{sub 2} receptor pathway, which subsequently suppresses the ERK/synapsin I cascade to decrease the evoked glutamate release. - Highlights: • Ciproxifan presynaptically reduces glutamate release in the hippocampus in vitro. • Decrease in voltage-dependent Ca{sup 2+} influx is involved. • A role for the PLA{sub 2}/PGE{sub 2}/EP{sub 2} pathway in the action of

  19. Involvement of PPAR-γ in the neuroprotective and anti-inflammatory effects of angiotensin type 1 receptor inhibition: effects of the receptor antagonist telmisartan and receptor deletion in a mouse MPTP model of Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Garrido-Gil Pablo

    2012-02-01

    Full Text Available Abstract Background Several recent studies have shown that angiotensin type 1 receptor (AT1 antagonists such as candesartan inhibit the microglial inflammatory response and dopaminergic cell loss in animal models of Parkinson's disease. However, the mechanisms involved in the neuroprotective and anti-inflammatory effects of AT1 blockers in the brain have not been clarified. A number of studies have reported that AT1 blockers activate peroxisome proliferator-activated receptor gamma (PPAR γ. PPAR-γ activation inhibits inflammation, and may be responsible for neuroprotective effects, independently of AT1 blocking actions. Methods We have investigated whether oral treatment with telmisartan (the most potent PPAR-γ activator among AT1 blockers provides neuroprotection against dopaminergic cell death and neuroinflammation, and the possible role of PPAR-γ activation in any such neuroprotection. We used a mouse model of parkinsonism induced by the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP and co-administration of the PPAR-γ antagonist GW9662 to study the role of PPAR-γ activation. In addition, we used AT1a-null mice lesioned with MPTP to study whether deletion of AT1 in the absence of any pharmacological effect of AT1 blockers provides neuroprotection, and investigated whether PPAR-γ activation may also be involved in any such effect of AT1 deletion by co-administration of the PPAR-γ antagonist GW9662. Results We observed that telmisartan protects mouse dopaminergic neurons and inhibits the microglial response induced by administration of MPTP. The protective effects of telmisartan on dopaminergic cell death and microglial activation were inhibited by co-administration of GW9662. Dopaminergic cell death and microglial activation were significantly lower in AT1a-null mice treated with MPTP than in mice not subjected to AT1a deletion. Interestingly, the protective effects of AT1 deletion were also inhibited by co

  20. Conazole fungicides inhibit Leydig cell testosterone secretion and androgen receptor activation in vitro

    Directory of Open Access Journals (Sweden)

    Maarke J.E. Roelofs

    2014-01-01

    Full Text Available Conazole fungicides are widely used in agriculture despite their suspected endocrine disrupting properties. In this study, the potential (anti-androgenic effects of ten conazoles were assessed and mutually compared with existing data. Effects of cyproconazole (CYPRO, fluconazole (FLUC, flusilazole (FLUS, hexaconazole (HEXA, myconazole (MYC, penconazole (PEN, prochloraz (PRO, tebuconazole (TEBU, triadimefon (TRIA, and triticonazole (TRIT were examined using murine Leydig (MA-10 cells and human T47D-ARE cells stably transfected with an androgen responsive element and a firefly luciferase reporter gene. Six conazoles caused a decrease in basal testosterone (T secretion by MA-10 cells varying from 61% up to 12% compared to vehicle-treated control. T secretion was concentration-dependently inhibited after exposure of MA-10 cells to several concentrations of FLUS (IC50 = 12.4 μM or TEBU (IC50 = 2.4 μM in combination with LH. The expression of steroidogenic and cholesterol biosynthesis genes was not changed by conazole exposure. Also, there were no changes in reactive oxygen species (ROS formation that could explain the altered T secretion after exposure to conazoles. Nine conazoles decreased T-induced AR activation (IC50s ranging from 10.7 to 71.5 μM and effect potencies (REPs were calculated relative to the known AR antagonist flutamide (FLUT. FLUC had no effect on AR activation by T. FLUS was the most potent (REP = 3.61 and MYC the least potent (REP = 0.03 AR antagonist. All other conazoles had a comparable REP from 0.12 to 0.38. Our results show distinct in vitro anti-androgenic effects of several conazole fungicides arising from two mechanisms: inhibition of T secretion and AR antagonism, suggesting potential testicular toxic effects. These effects warrant further mechanistic investigation and clearly show the need for accurate exposure data in order to perform proper (human risk assessment of this class of compounds.

  1. Context-Dependent Modulation of αβγ and αβγ GABAA Receptors by Penicillin: Implications for Phasic and Tonic Inhibition

    Science.gov (United States)

    Feng, Hua-Jun; Botzolakis, Emmanuel J.; Macdonald, Robert L.

    2009-01-01

    Summary Penicillin, an open-channel blocker of GABAA receptors, was recently reported to inhibit phasic, but not tonic, currents in hippocampal neurons. To distinguish between isoform-specific and context-dependent modulation as possible explanations for this selectivity, the effects of penicillin were evaluated on recombinant GABAA receptors expressed in HEK293T cells. When co-applied with saturating GABA, penicillin decreased peak amplitude, induced rebound, and prolonged deactivation of currents evoked from both synaptic and extrasynaptic receptor isoforms. However, penicillin had isoform-specific effects on the extent of desensitization, reflecting its ability to differentially modulate peak (non-equilibrium) and residual (near-equilibrium) currents. This suggested that the context of activation could determine the apparent sensitivity of a given receptor isoform to penicillin. To test this hypothesis, we explored the ability of penicillin to modulate synaptic and extrasynaptic isoforms that were activated under more physiologically relevant conditions. Interestingly, while currents evoked from synaptic isoforms under phasic conditions (transient activation by a saturating concentration of GABA) were substantially inhibited by penicillin, currents evoked from extrasynaptic isoforms under tonic conditions (prolonged application by a sub-saturating concentration of GABA) were minimally affected. We therefore concluded that the reported inability of penicillin to modulate tonic currents could not simply be attributed to insensitivity of extrasynaptic receptors, but rather, reflected an inability to modulate these receptors in their native context of activation. PMID:18775733

  2. An examination of the effects of subthalamic nucleus inhibition or μ-opioid receptor stimulation on food-directed motivation in the non-deprived rat

    Science.gov (United States)

    Pratt, Wayne E.; Choi, Eugene; Guy, Elizabeth G.

    2012-01-01

    The subthalamic nucleus (STN) serves important functions in regulating movement, cognition, and motivation and is connected with cortical and basal ganglia circuits that process reward and reinforcement. In order to further examine the role of the STN on motivation toward food in non-deprived rats, these experiments studied the effects of pharmacological inhibition or μ-opioid receptor stimulation of the STN on the 2-hr intake of a sweetened fat diet, the amount of work exerted to earn sucrose on a progressive ratio 2 (PR-2) schedule of reinforcement, and performance on a differential reinforcement of low-rate responding (DRL) schedule for sucrose reward. Separate behavioral groups (N = 6–9) were tested following bilateral inhibition of the STN with the GABAA receptor agonist muscimol (at 0–5 ng/0.5 μl/side) or following μ-opioid receptor stimulation with the agonist D-Ala2, N-MePhe4, Gly-ol-enkephalin (DAMGO; at 0, 0.025 or 0.25 μg/0.5 μl/side). Although STN inhibition increased ambulatory behavior during 2-hr feeding sessions, it did not significantly alter intake of the sweetened fat diet. STN inhibition also did not affect the breakpoint for sucrose pellets during a 1-hr PR-2 reinforcement schedule or impact the number of reinforcers earned on a 1-hr DRL-20 sec reinforcement schedule in non-deprived rats. In contrast, STN μ-opioid receptor stimulation significantly increased feeding on the palatable diet and reduced the reinforcers earned on a DRL-20 schedule, although DAMGO microinfusions had no effect on PR-2 performance. These data suggest that STN inhibition does not enhance incentive motivation for food in the absence of food restriction and that STN μ-opioid receptors play an important and unique role in motivational processes. PMID:22391117

  3. Inhibition of Wnt/β-catenin signaling by a soluble collagen-derived frizzled domain interacting with Wnt3a and the receptors frizzled 1 and 8.

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    Ismaïl Hendaoui

    Full Text Available The Wnt/β-catenin pathway controls cell proliferation, death and differentiation. Several families of extracellular proteins can antagonize Wnt/β-catenin signaling, including the decoy receptors known as secreted frizzled related proteins (SFRPs, which have a cysteine-rich domain (CRD structurally similar to the extracellular Wnt-binding domain of the frizzled receptors. SFRPs inhibit Wnt signaling by sequestering Wnts through the CRD or by forming inactive complexes with the frizzled receptors. Other endogenous molecules carrying frizzled CRDs inhibit Wnt signaling, such as V3Nter, which is proteolytically derived from the cell surface component collagen XVIII and contains a biologically active frizzled domain (FZC18 inhibiting in vivo cell proliferation and tumor growth in mice. We recently showed that FZC18 expressing cells deliver short-range signals to neighboring cells, decreasing their proliferation in vitro and in vivo through the Wnt/β-catenin signaling pathway. Here, using low concentrations of soluble FZC18 and Wnt3a, we show that they physically interact in a cell-free system. In addition, soluble FZC18 binds the frizzled 1 and 8 receptors' CRDs, reducing cell sensitivity to Wnt3a. Conversely, inhibition of Wnt/β-catenin signaling was partially rescued by the expression of full-length frizzled 1 and 8 receptors, but enhanced by the expression of a chimeric cell-membrane-tethered frizzled 8 CRD. Moreover, soluble, partially purified recombinant FZC18_CRD inhibited Wnt3a-induced β-catenin activation. Taken together, the data indicate that collagen XVIII-derived frizzled CRD shifts Wnt sensitivity of normal cells to a lower pitch and controls their growth.

  4. Breast cancer stem-like cells are inhibited by a non-toxic aryl hydrocarbon receptor agonist.

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    Gérald J Prud'homme

    2010-11-01

    Full Text Available Cancer stem cells (CSCs have increased resistance to cancer chemotherapy. They can be enriched as drug-surviving CSCs (D-CSCs by growth with chemotherapeutic drugs, and/or by sorting of cells expressing CSC markers such as aldehyde dehydrogenase-1 (ALDH. CSCs form colonies in agar, mammospheres in low-adherence cultures, and tumors following xenotransplantation in Scid mice. We hypothesized that tranilast, a non-toxic orally active drug with anti-cancer activities, would inhibit breast CSCs.We examined breast cancer cell lines or D-CSCs generated by growth of these cells with mitoxantrone. Tranilast inhibited colony formation, mammosphere formation and stem cell marker expression. Mitoxantrone-selected cells were enriched for CSCs expressing stem cell markers ALDH, c-kit, Oct-4, and ABCG2, and efficient at forming mammospheres. Tranilast markedly inhibited mammosphere formation by D-CSCs and dissociated formed mammospheres, at pharmacologically relevant concentrations. It was effective against D-CSCs of both HER-2+ and triple-negative cell lines. Tranilast was also effective in vivo, since it prevented lung metastasis in mice injected i.v. with triple-negative (MDA-MB-231 mitoxantrone-selected cells. The molecular targets of tranilast in cancer have been unknown, but here we demonstrate it is an aryl hydrocarbon receptor (AHR agonist and this plays a key role. AHR is a transcription factor activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, polycyclic aromatic hydrocarbons and other ligands. Tranilast induced translocation of the AHR to the nucleus and stimulated CYP1A1 expression (a marker of AHR activation. It inhibited binding of the AHR to CDK4, which has been linked to cell-cycle arrest. D-CSCs expressed higher levels of the AHR than other cells. Knockdown of the AHR with siRNA, or blockade with an AHR antagonist, entirely abrogated the anti-proliferative and anti-mammosphere activity of tranilast. Thus, the anti-cancer effects of

  5. Inhaled corticosteroids inhibit substance P receptor expression in asthmatic rat airway smooth muscle cells

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    Li Miao

    2012-12-01

    Full Text Available Abstract Background Neurokinins (NKs participate in asthmatic airway inflammation, but the effects of NKs on airway smooth muscle cells (ASMCs and those of corticosteroids on NKs are unknown. Methods To investigate the effect of budesonide on substance P (NK-1 receptor (NK-1R expression in the lung and ASMCs, 45 Wistar rats were randomly divided into three groups: control, asthmatic, and budesonide treatment. Aerosolized ovalbumin was used to generate the asthmatic rat model, and budesonide was administered after ovalbumin inhalation. On day 21, bronchial responsiveness tests, bronchoalveolar lavage, and cell counting were conducted. NK-1R protein expression in the lung was investigated by immunohistochemistry and image analysis. Primary rat ASMC cultures were established, and purified ASMCs of the fourth passage were collected for mRNA and protein studies via real-time RT-PCR, immunocytochemistry, and image analysis. Results NK-1R mRNA and protein expression in the budesonide treatment group rat’s lung and ASMCs were less than that in the asthmatic group but greater than that in the control group. Conclusions NK-1R is involved in the pathogenesis of asthma and that budesonide may downregulate the expression of NK-1R in the ASMCs and airways of asthmatic rats, which may alleviate neurogenic airway inflammation.

  6. Inhibition of common cold-induced aggravation of childhood asthma by leukotriene receptor antagonists.

    Science.gov (United States)

    Yoshihara, Shigemi; Fukuda, Hironobu; Abe, Toshio; Nishida, Mitsuhiro; Yamada, Yumi; Kanno, Noriko; Arisaka, Osamu

    2012-09-01

    Virus infection is an important risk factor for aggravation of childhood asthma. The objective of this study was to examine the effect of drugs on aggravation of asthma induced by a common cold. Asthma control was examined in a survey of 1,014 Japanese pediatric patients with bronchial asthma. The occurrence of common cold, asthma control, and drugs used for asthma control were investigated using a modified Childhood Asthma Control Test (C-ACT) for patients aged cold and aggravation of asthma were significantly higher in patients aged cold-induced aggravation was significantly less effective in patients aged cold, asthma control was significantly more effective for those treated with leukotriene receptor antagonists (LTRAs) compared to treatment without LTRAs. Asthma control did not differ between patients who did or did not take inhaled corticosteroids or long-acting β2 stimulants. These findings showed a high prevalence of common cold in younger patients with childhood asthma and indicated that common cold can induce aggravation of asthma. LTRAs are useful for long-term asthma control in very young patients who develop an asthma attack due to a common cold.

  7. Calcitonin Receptor Signaling Inhibits Muscle Stem Cells from Escaping the Quiescent State and the Niche

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    Masahiko Yamaguchi

    2015-10-01

    Full Text Available Calcitonin receptor (Calcr is expressed in adult muscle stem cells (muscle satellite cells [MuSCs]. To elucidate the role of Calcr, we conditionally depleted Calcr from adult MuSCs and found that impaired regeneration after muscle injury correlated with the decreased number of MuSCs in Calcr-conditional knockout (cKO mice. Calcr signaling maintained MuSC dormancy via the cAMP-PKA pathway but had no impact on myogenic differentiation of MuSCs in an undifferentiated state. The abnormal quiescent state in Calcr-cKO mice resulted in a reduction of the MuSC pool by apoptosis. Furthermore, MuSCs were found outside their niche in Calcr-cKO mice, demonstrating cell relocation. This emergence from the sublaminar niche was prevented by the Calcr-cAMP-PKA and Calcr-cAMP-Epac pathways downstream of Calcr. Altogether, the findings demonstrated that Calcr exerts its effect specifically by keeping MuSCs in a quiescent state and in their location, maintaining the MuSC pool.

  8. Correction for Inhibition Leads to an Allosteric Co-Agonist Model for Pentobarbital Modulation and Activation of α1β3γ2L GABAA Receptors.

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    Alexis M Ziemba

    Full Text Available Pentobarbital, like propofol and etomidate, produces important general anesthetic effects through GABAA receptors. Photolabeling also indicates that pentobarbital binds to some of the same sites where propofol and etomidate act. Quantitative allosteric co-agonist models for propofol and etomidate account for modulatory and agonist effects in GABAA receptors and have proven valuable in establishing drug site characteristics and for functional analysis of mutants. We therefore sought to establish an allosteric co-agonist model for pentobarbital activation and modulation of α1β3γ2L receptors, using a novel approach to first correct pentobarbital activation data for inhibitory effects in the same concentration range.Using oocyte-expressed α1β3γ2L GABAA receptors and two-microelectrode voltage-clamp, we quantified modulation of GABA responses by a low pentobarbital concentration and direct effects of high pentobarbital concentrations, the latter displaying mixed agonist and inhibitory effects. We then isolated and quantified pentobarbital inhibition in activated receptors using a novel single-sweep "notch" approach, and used these results to correct steady-state direct activation for inhibition.Combining results for GABA modulation and corrected direct activation, we estimated receptor open probability and optimized parameters for a Monod-Wyman-Changeux allosteric co-agonist model. Inhibition by pentobarbital was consistent with two sites with IC50s near 1 mM, while co-agonist model parameters suggest two allosteric pentobarbital agonist sites characterized by KPB ≈ 5 mM and high efficacy. The results also indicate that pentobarbital may be a more efficacious agonist than GABA.Our novel approach to quantifying both inhibitory and co-agonist effects of pentobarbital provides a basis for future structure-function analyses of GABAA receptor mutations in putative pentobarbital binding sites.

  9. Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor signaling.

    Science.gov (United States)

    Beurel, Eléonore; Grieco, Steven F; Amadei, Celeste; Downey, Kimberlee; Jope, Richard S

    2016-09-01

    Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Adenosine A1 receptor-mediated inhibition of in vitro prolactin secretion from the rat anterior pituitary

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    D.L.W. Picanço-Diniz

    2006-11-01

    Full Text Available In previous studies, we demonstrated biphasic purinergic effects on prolactin (PRL secretion stimulated by an adenosine A2 agonist. In the present study, we investigated the role of the activation of adenosine A1 receptors by (R-N6-(2-phenylisopropyladenosine (R-PIA at the pituitary level in in vitro PRL secretion. Hemipituitaries (one per cuvette in five replicates from adult male rats were incubated. Administration of R-PIA (0.001, 0.01, 0.1, 1, and 10 µM induced a reduction of PRL secretion into the medium in a U-shaped dose-response curve. The maximal reduction was obtained with 0.1 µM R-PIA (mean ± SEM, 36.01 ± 5.53 ng/mg tissue weight (t.w. treatment compared to control (264.56 ± 15.46 ng/mg t.w.. R-PIA inhibition (0.01 µM = 141.97 ± 15.79 vs control = 244.77 ± 13.79 ng/mg t.w. of PRL release was blocked by 1 µM cyclopentyltheophylline, a specific A1 receptor antagonist (1 µM = 212.360 ± 26.560 ng/mg t.w., whereas cyclopentyltheophylline alone (0.01, 0.1, 1 µM had no effect. R-PIA (0.001, 0.01, 0.1, 1 µM produced inhibition of PRL secretion stimulated by both phospholipase C (0.5 IU/mL; 977.44 ± 76.17 ng/mg t.w. and dibutyryl cAMP (1 mM; 415.93 ± 37.66 ng/mg t.w. with nadir established at the dose of 0.1 µM (225.55 ± 71.42 and 201.9 ± 19.08 ng/mg t.w., respectively. Similarly, R-PIA (0.01 µM decreased (242.00 ± 24.00 ng/mg t.w. the PRL secretion stimulated by cholera toxin (0.5 mg/mL; 1050.00 ± 70.00 ng/mg t.w.. In contrast, R-PIA had no effect (468.00 ± 34.00 ng/mg t.w. on PRL secretion stimulation by pertussis toxin (0.5 mg/mL; 430.00 ± 26.00 ng/mg t.w.. These results suggest that inhibition of PRL secretion after A1 receptor activation by R-PIA is mediated by a Gi protein-dependent mechanism.

  11. Calpain inhibition reduces NMDA receptor rundown in rat substantia nigra dopamine neurons.

    Science.gov (United States)

    Zhao, Jerry; Baudry, Michel; Jones, Susan

    2018-05-04

    Repeated activation of N-Methyl-d-aspartate receptors (NMDARs) causes a Ca 2+ -dependent reduction in NMDAR-mediated current in dopamine (DA) neurons of the substantia nigra pars compacta (SNc) in one week old rats; however, a Ca 2+ -dependent regulatory protein has not been identified. The role of the Ca 2+ -dependent cysteine protease, calpain, in mediating NMDAR current rundown was investigated. In brain slices from rats aged postnatal day 7-9 ('P7'), bath application of either of the membrane permeable calpain inhibitors, N-Acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN, 20 μM) or MDL-28170 (30 μM) significantly reduced whole-cell NMDAR current rundown. To investigate the role of the calpain-2 isoform, the membrane permeable calpain-2 inhibitor, Z-Leu-Abu-CONH-CH2-C6H3 (3, 5-(OMe)2 (C2I, 200 nM), was applied; C2I application significantly reduced whole cell NMDAR current rundown. Interestingly, ALLN but not C2I significantly reduced rundown of NMDA-EPSCs. These results suggest the calpain-2 isoform mediates Ca 2+ -dependent regulation of extrasynaptic NMDAR current in the first postnatal week, while calpain-1 might mediate rundown of synaptic NMDAR currents. One week later in postnatal development, at P12-P16 ('P14'), there was significantly less rundown in SNc-DA neurons, and no significant effect on rundown of either Ca 2+ chelation or treatment with the calpain inhibitor, ALLN, suggesting that the rundown observed in SNc-DA neurons from two week-old rats might be Ca 2+ -independent. In conclusion, Ca 2+ -dependent rundown of extrasynaptic NMDAR currents in SNc DA neurons involves calpain-2 activation, but Ca 2+ - and calpain-2-dependent NMDAR current rundown is developmentally regulated. Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. Thyroid hormone receptor inhibits hepatoma cell migration through transcriptional activation of Dickkopf 4

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    Chi, Hsiang-Cheng; Liao, Chen-Hsin [Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan 333, Taiwan, ROC (China); Huang, Ya-Hui [Medical Research Central, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan, ROC (China); Wu, Sheng-Ming; Tsai, Chung-Ying; Liao, Chia-Jung; Tseng, Yi-Hsin; Lin, Yang-Hsiang; Chen, Cheng-Yi; Chung, I-Hsiao; Wu, Tzu-I [Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan 333, Taiwan, ROC (China); Chen, Wei-Jan [First Cardiovascular Division, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan, ROC (China); Lin, Kwang-Huei, E-mail: khlin@mail.cgu.edu.tw [Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan 333, Taiwan, ROC (China)

    2013-09-13

    Highlights: •T{sub 3} affects DKK4 mRNA and protein expression in HepG2-TR cells. •Regulation of DKK4 by T{sub 3} is at transcriptional level. •DKK4 overexpression suppresses hepatoma cell metastasis. -- Abstract: Triiodothyronine (T{sub 3}) is a potent form of thyroid hormone mediates several physiological processes including cellular growth, development, and differentiation via binding to the nuclear thyroid hormone receptor (TR). Recent studies have demonstrated critical roles of T{sub 3}/TR in tumor progression. Moreover, long-term hypothyroidism appears to be associated with the incidence of human hepatocellular carcinoma (HCC), independent of other major HCC risk factors. Dickkopf (DKK) 4, a secreted protein that antagonizes the canonical Wnt signaling pathway, is induced by T{sub 3} at both mRNA and protein levels in HCC cell lines. However, the mechanism underlying T{sub 3}-mediated regulation of DKK4 remains unknown. In the present study, the 5′ promoter region of DKK4 was serially deleted, and the reporter assay performed to localize the T{sub 3} response element (TRE). Consequently, we identified an atypical direct repeat TRE between nucleotides −1645 and −1629 conferring T{sub 3} responsiveness to the DKK4 gene. This region was further validated using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Stable DKK4 overexpression in SK-Hep-1 cells suppressed cell invasion and metastatic potential, both in vivo andin vitro, via reduction of matrix metalloproteinase-2 (MMP-2) expression. Our findings collectively suggest that DKK4 upregulated by T{sub 3}/TR antagonizes the Wnt signal pathway to suppress tumor cell progression, thus providing new insights into the molecular mechanism underlying thyroid hormone activity in HCC.

  13. The Aryl Hydrocarbon Receptor Binds to E2F1 and Inhibits E2F1-induced Apoptosis

    Science.gov (United States)

    Marlowe, Jennifer L.; Fan, Yunxia; Chang, Xiaoqing; Peng, Li; Knudsen, Erik S.; Xia, Ying

    2008-01-01

    Cellular stress by DNA damage induces checkpoint kinase-2 (CHK2)-mediated phosphorylation and stabilization of the E2F1 transcription factor, leading to induction of apoptosis by activation of a subset of proapoptotic E2F1 target genes, including Apaf1 and p73. This report characterizes an interaction between the aryl hydrocarbon (Ah) receptor (AHR), a ligand-activated transcription factor, and E2F1 that results in the attenuation of E2F1-mediated apoptosis. In Ahr−/− fibroblasts stably transfected with a doxycycline-regulated AHR expression vector, inhibition of AHR expression causes a significant elevation of oxidative stress, γH2A.X histone phosphorylation, and E2F1-dependent apoptosis, which can be blocked by small interfering RNA-mediated knockdown of E2F1 expression. In contrast, ligand-dependent AHR activation protects these cells from etoposide-induced cell death. In cells expressing both proteins, AHR and E2F1 interact independently of the retinoblastoma protein (RB), because AHR and E2F1 coimmunoprecipitate from extracts of RB-negative cells. Additionally, chromatin immunoprecipitation assays indicate that AHR and E2F1 bind to the Apaf1 promoter at a region containing a consensus E2F1 binding site but no AHR binding sites. AHR activation represses Apaf1 and TAp73 mRNA induction by a constitutively active CHK2 expression vector. Furthermore, AHR overexpression blocks the transcriptional induction of Apaf1 and p73 and the accumulation of sub-G0/G1 cells resulting from ectopic overexpression of E2F1. These results point to a proproliferative, antiapoptotic function of the Ah receptor that likely plays a role in tumor progression. PMID:18524851

  14. The B-cell receptor controls fitness of MYC-driven lymphoma cells via GSK3β inhibition.

    Science.gov (United States)

    Varano, Gabriele; Raffel, Simon; Sormani, Martina; Zanardi, Federica; Lonardi, Silvia; Zasada, Christin; Perucho, Laura; Petrocelli, Valentina; Haake, Andrea; Lee, Albert K; Bugatti, Mattia; Paul, Ulrike; Van Anken, Eelco; Pasqualucci, Laura; Rabadan, Raul; Siebert, Reiner; Kempa, Stefan; Ponzoni, Maurilio; Facchetti, Fabio; Rajewsky, Klaus; Casola, Stefano

    2017-06-08

    Similar to resting mature B cells, where the B-cell antigen receptor (BCR) controls cellular survival, surface BCR expression is conserved in most mature B-cell lymphomas. The identification of activating BCR mutations and the growth disadvantage upon BCR knockdown of cells of certain lymphoma entities has led to the view that BCR signalling is required for tumour cell survival. Consequently, the BCR signalling machinery has become an established target in the therapy of B-cell malignancies. Here we study the effects of BCR ablation on MYC-driven mouse B-cell lymphomas and compare them with observations in human Burkitt lymphoma. Whereas BCR ablation does not, per se, significantly affect lymphoma growth, BCR-negative (BCR - ) tumour cells rapidly disappear in the presence of their BCR-expressing (BCR + ) counterparts in vitro and in vivo. This requires neither cellular contact nor factors released by BCR + tumour cells. Instead, BCR loss induces the rewiring of central carbon metabolism, increasing the sensitivity of receptor-less lymphoma cells to nutrient restriction. The BCR attenuates glycogen synthase kinase 3 beta (GSK3β) activity to support MYC-controlled gene expression. BCR - tumour cells exhibit increased GSK3β activity and are rescued from their competitive growth disadvantage by GSK3β inhibition. BCR - lymphoma variants that restore competitive fitness normalize GSK3β activity after constitutive activation of the MAPK pathway, commonly through Ras mutations. Similarly, in Burkitt lymphoma, activating RAS mutations may propagate immunoglobulin-crippled tumour cells, which usually represent a minority of the tumour bulk. Thus, while BCR expression enhances lymphoma cell fitness, BCR-targeted therapies may profit from combinations with drugs targeting BCR - tumour cells.

  15. Receptor tyrosine kinase inhibition causes simultaneous bone loss and excess bone formation within growing bone in rats

    International Nuclear Information System (INIS)

    Nurmio, Mirja; Joki, Henna; Kallio, Jenny; Maeaettae, Jorma A.; Vaeaenaenen, H. Kalervo; Toppari, Jorma; Jahnukainen, Kirsi; Laitala-Leinonen, Tiina

    2011-01-01

    During postnatal skeletal growth, adaptation to mechanical loading leads to cellular activities at the growth plate. It has recently become evident that bone forming and bone resorbing cells are affected by the receptor tyrosine kinase (RTK) inhibitor imatinib mesylate (STI571, Gleevec (registered) ). Imatinib targets PDGF, ABL-related gene, c-Abl, c-Kit and c-Fms receptors, many of which have multiple functions in the bone microenvironment. We therefore studied the effects of imatinib in growing bone. Young rats were exposed to imatinib (150 mg/kg on postnatal days 5-7, or 100 mg/kg on postnatal days 5-13), and the effects of RTK inhibition on bone physiology were studied after 8 and 70 days (3-day treatment), or after 14 days (9-day treatment). X-ray imaging, computer tomography, histomorphometry, RNA analysis and immunohistochemistry were used to evaluate bone modeling and remodeling in vivo. Imatinib treatment eliminated osteoclasts from the metaphyseal osteochondral junction at 8 and 14 days. This led to a resorption arrest at the growth plate, but also increased bone apposition by osteoblasts, thus resulting in local osteopetrosis at the osteochondral junction. The impaired bone remodelation observed on day 8 remained significant until adulthood. Within the same bone, increased osteoclast activity, leading to bone loss, was observed at distal bone trabeculae on days 8 and 14. Peripheral quantitative computer tomography (pQCT) and micro-CT analysis confirmed that, at the osteochondral junction, imatinib shifted the balance from bone resorption towards bone formation, thereby altering bone modeling. At distal trabecular bone, in turn, the balance was turned towards bone resorption, leading to bone loss. - Research highlights: → 3-Day imatinib treatment. → Causes growth plate anomalies in young rats. → Causes biomechanical changes and significant bone loss at distal trabecular bone. → Results in loss of osteoclasts at osteochondral junction.

  16. PANC-1 pancreatic cancer cell growth inhibited by cucurmosin alone and in combination with an epidermal growth factor receptor-targeted drug.

    Science.gov (United States)

    Wang, Congfei; Yang, Aiqin; Zhang, Baoming; Yin, Qiang; Huang, Heguang; Chen, Minghuang; Xie, Jieming

    2014-03-01

    To investigate the inhibition of PANC-1 pancreatic cancer cell growth by cucurmosin (CUS) and its possible mechanism. We observed the inhibition of PANC-1 cell growth by sulforhodamine B and colony-forming experiments in vitro and established nonobese diabetic/severe combined immunodeficiency mouse subcutaneous tumor models in vivo. We used Western blot to analyze protein levels related to apoptosis and epidermal growth factor receptor (EGFR) signaling pathways after drug intervention, whereas the messenger RNA expression of EGFR was analyzed by quantitative real-time polymerase chain reaction. Sulforhodamine B and colony-forming experiments indicated that CUS inhibited PANC-1 cell proliferation in a dose- and time-dependent manner. A stronger inhibitory effect was observed when CUS was combined with gefitinib. The subcutaneous tumor growth was also inhibited. Western blot showed that all the examined proteins decreased, except for 4E-BP1 and the active fragments of caspase 3 and caspase 9 increased. Epidermal growth factor receptor expression did not change significantly in quantitative real-time polymerase chain reaction. Cucurmosin can strongly inhibit the growth of PANC-1 cells in vitro and in vivo. Cucurmosin can down-regulate EGFR protein expression, but not at the messenger RNA level. Cucurmosin can also inhibit the ras/raf and phosphatidylinositol 3-kinase/Akt downstream signaling pathways and enhance the sensitivity of the EGFR-targeted drug gefitinib.

  17. Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

    Energy Technology Data Exchange (ETDEWEB)

    Forward, Nicholas A.; Conrad, David M. [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Power Coombs, Melanie R.; Doucette, Carolyn D. [Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada); Furlong, Suzanne J. [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Lin, Tong-Jun [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia (Canada); Hoskin, David W., E-mail: d.w.hoskin@dal.ca [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Surgery, Dalhousie University, Halifax, Nova Scotia (Canada)

    2011-04-22

    Highlights: {yields} Curcumin inhibits CD4{sup +} T-lymphocyte proliferation. {yields} Curcumin inhibits interleukin-2 (IL-2) synthesis and CD25 expression by CD4{sup +} T-lymphocytes. {yields} Curcumin interferes with IL-2 receptor signaling by inhibiting JAK3 and STAT5 phosphorylation. {yields} IL-2-dependent regulatory T-lymphocyte function and Foxp3 expression is downregulated by curcumin. -- Abstract: Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4{sup +} T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 ({alpha} chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca{sup 2+} release to inhibit I{kappa}B phosphorylation, which is required for nuclear translocation of the transcription factor NF{kappa}B. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4{sup +}CD25{sup +} regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

  18. Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

    International Nuclear Information System (INIS)

    Forward, Nicholas A.; Conrad, David M.; Power Coombs, Melanie R.; Doucette, Carolyn D.; Furlong, Suzanne J.; Lin, Tong-Jun; Hoskin, David W.

    2011-01-01

    Highlights: → Curcumin inhibits CD4 + T-lymphocyte proliferation. → Curcumin inhibits interleukin-2 (IL-2) synthesis and CD25 expression by CD4 + T-lymphocytes. → Curcumin interferes with IL-2 receptor signaling by inhibiting JAK3 and STAT5 phosphorylation. → IL-2-dependent regulatory T-lymphocyte function and Foxp3 expression is downregulated by curcumin. -- Abstract: Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4 + T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 (α chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca 2+ release to inhibit IκB phosphorylation, which is required for nuclear translocation of the transcription factor NFκB. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4 + CD25 + regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

  19. The selective vitamin D receptor agonist, elocalcitol, reduces endometriosis development in a mouse model by inhibiting peritoneal inflammation.

    Science.gov (United States)

    Mariani, Margherita; Viganò, Paola; Gentilini, Davide; Camisa, Barbara; Caporizzo, Elvira; Di Lucia, Pietro; Monno, Antonella; Candiani, Massimo; Somigliana, Edgardo; Panina-Bordignon, Paola

    2012-07-01

    Endometriosis, which is characterized by the growth of endometrial tissue at ectopic locations as well as vascular development and inflammation, is still an unmet clinical need since an optimal drug that allows for both pain and infertility management does not exist. Since both the eutopic and the ectopic endometrium express the vitamin D receptor (VDR), and VDR agonists are endowed with anti-proliferative and anti-inflammatory properties, we evaluated the effect of elocalcitol, a VDR agonist with low calcaemic liability, in a mouse model of experimentally induced endometriosis. Endometriosis was induced by injection of syngeneic endometrial tissue fragments into adult Balb/c female mice. After having confirmed by immunohistochemistry that endometriotic lesions developing in mice expressed VDR, the mice were administered with elocalcitol (100 μg/kg) or vehicle orally, once a day, for various durations of time. In this model, elocalcitol was able to reduce total lesion weight up to 70% upon treatment for 1 week before and 2 weeks after disease induction. Interestingly, a therapeutic effect was also observed on already established lesions. Elocalcitol was shown to reduce the capacity of mouse endometrial cells to adhere to collagen. In addition in treated mice, a decreased state of peritoneal inflammation was demonstrated by the inhibition of macrophage recruitment and inflammatory cytokine secretion. The VDR agonist elocalcitol inhibits lesion development in a validated mouse model of endometriosis, and exerts a protective effect on both the implantation and organization of transferred endometrial tissue. These preliminary data in mice provide a sound rationale for further testing in primate models and eventually in humans.

  20. Inhibition of radiation induced migration of human head and neck squamous cell carcinoma cells by blocking of EGF receptor pathways

    International Nuclear Information System (INIS)

    Pickhard, Anja C; Schlegel, Jürgen; Arnold, Wolfgang; Reiter, Rudolf; Margraf, Johanna; Knopf, Andreas; Stark, Thomas; Piontek, Guido; Beck, Carolin; Boulesteix, Anne-Laure; Scherer, Elias Q; Pigorsch, Steffi

    2011-01-01

    Recently it has been shown that radiation induces migration of glioma cells and facilitates a further spread of tumor cells locally and systemically. The aim of this study was to evaluate whether radiotherapy induces migration in head and neck squamous cell carcinoma (HNSCC). A further aim was to investigate the effects of blocking the epidermal growth factor receptor (EGFR) and its downstream pathways (Raf/MEK/ERK, PI3K/Akt) on tumor cell migration in vitro. Migration of tumor cells was assessed via a wound healing assay and proliferation by a MTT colorimeritric assay using 3 HNSCC cell lines (BHY, CAL-27, HN). The cells were treated with increasing doses of irradiation (2 Gy, 5 Gy, 8 Gy) in the presence or absence of EGF, EGFR-antagonist (AG1478) or inhibitors of the downstream pathways PI3K (LY294002), mTOR (rapamycin) and MEK1 (PD98059). Biochemical activation of EGFR and the downstream markers Akt and ERK were examined by Western blot analysis. In absence of stimulation or inhibition, increasing doses of irradiation induced a dose-dependent enhancement of migrating cells (p < 0.05 for the 3 HNSCC cell lines) and a decrease of cell proliferation (p < 0.05 for the 3 HNSCC cell lines). The inhibition of EGFR or the downstream pathways reduced cell migration significantly (almost all p < 0.05 for the 3 HNSCC cell lines). Stimulation of HNSCC cells with EGF caused a significant increase in migration (p < 0.05 for the 3 HNSCC cell lines). After irradiation alone a pronounced activation of EGFR was observed by Western blot analysis. Our results demonstrate that the EGFR is involved in radiation induced migration of HNSCC cells. Therefore EGFR or the downstream pathways might be a target for the treatment of HNSCC to improve the efficacy of radiotherapy

  1. The combi-targeting concept: synthesis of stable nitrosoureas designed to inhibit the epidermal growth factor receptor (EGFR).

    Science.gov (United States)

    Domarkas, Juozas; Dudouit, Fabienne; Williams, Christopher; Qiyu, Qiu; Banerjee, Ranjita; Brahimi, Fouad; Jean-Claude, Bertrand Jacques

    2006-06-15

    According to the "combi-targeting" concept, the EGFR tyrosine kinase (TK) inhibitory potency of compounds termed "combi-molecules" is critical for selective growth inhibition of tumor cells with disordered expression of EGFR or its closest family member erbB2. Here we report on the optimization of the EGFR TK inhibitory potency of the combi-molecules of the nitrosourea class by comparison with their aminoquinazoline and ureidoquinazoline precursors. This led to the discovery of a new structural parameter that influences their EGFR TK inhibitory potency, i.e., the torsion angle between the plane of the quinazoline ring and the ureido or the nitrosoureido moiety of the synthesized drugs. Compounds (3'-Cl and Br series) with small angles (0.5-3 degrees ) were generally stronger EGFR TK inhibitors than those with large angles (18-21 degrees ). This was further corroborated by ligand-receptor van der Waals interaction calculations that showed significant binding hindrance imposed by large torsion angles in the narrow ATP cleft of EGFR. Selective antiproliferative studies in a pair of mouse fibroblast NIH3T3 cells, one of which NIH3T3/neu being transfected with the erbB2 oncogene, showed that IC(50) values for inhibition of EGFR TK could be good predictors of their selective potency against the serum-stimulated growth of the erbB2-tranfected cell line (Pearson r = 0.8). On the basis of stability (t(1/2)), EGFR TK inhibitory potency (IC(50)), and selective erbB2 targeting, compound 23, a stable nitrosourea, was considered to have the structural requirements for further development.

  2. Growth Inhibition by Testosterone in an Androgen Receptor Splice Variant-Driven Prostate Cancer Model.

    Science.gov (United States)

    Nakata, Daisuke; Nakayama, Kazuhide; Masaki, Tsuneo; Tanaka, Akira; Kusaka, Masami; Watanabe, Tatsuya

    2016-12-01

    Castration resistance creates a significant problem in the treatment of prostate cancer. Constitutively active splice variants of androgen receptor (AR) have emerged as drivers for resistance to androgen deprivation therapy, including the next-generation androgen-AR axis inhibitors abiraterone and enzalutamide. In this study, we describe the characteristics of a novel castration-resistant prostate cancer (CRPC) model, designated JDCaP-hr (hormone refractory). JDCaP-hr was established from an androgen-dependent JDCaP xenograft model after surgical castration. The expression of AR and its splice variants in JDCaP-hr was evaluated by immunoblotting and quantitative reverse transcription-polymerase chain reaction. The effects of AR antagonists and testosterone on JDCaP-hr were evaluated in vivo and in vitro. The roles of full-length AR (AR-FL) and AR-V7 in JDCaP-hr cell growth were evaluated using RNA interference. JDCaP-hr acquired a C-terminally truncated AR protein during progression from the parental JDCaP. The expression of AR-FL and AR-V7 mRNA was upregulated by 10-fold in JDCaP-hr compared with that in JDCaP, indicating that the JDCaP and JDCaP-hr models simulate castration resistance with some clinical features, such as overexpression of AR and its splice variants. The AR antagonist bicalutamide did not affect JDCaP-hr xenograft growth, and importantly, testosterone induced tumor regression. In vitro analysis demonstrated that androgen-independent prostate-specific antigen secretion and cell proliferation of JDCaP-hr were predominantly mediated by AR-V7. JDCaP-hr cell growth displayed a bell-shaped dependence on testosterone, and it was suppressed by physiological concentrations of testosterone. Testosterone induced rapid downregulation of both AR-FL and AR-V7 expression at physiological concentrations and suppressed expression of the AR target gene KLK3. Our findings support the clinical value of testosterone therapy, including bipolar androgen therapy, in the

  3. Thujone inhibits the function of α7-nicotinic acetylcholine receptors and impairs nicotine-induced memory enhancement in one-trial passive avoidance paradigm.

    Science.gov (United States)

    Sultan, Ahmed; Yang, Keun-Hang Susan; Isaev, Dmitro; Nebrisi, Eslam El; Syed, Nurulain; Khan, Nadia; Howarth, Christopher F; Sadek, Bassem; Oz, Murat

    2017-06-01

    Effects of thujone, a major ingredient of absinthe, wormwood oil and some herbal medicines, were tested on the function of α 7 subunit of the human nicotinic acetylcholine (α 7 nACh) receptor expressed in Xenopus oocytes using the two-electrode voltage-clamp technique. Thujone reversibly inhibited ACh (100μM)-induced currents with an IC 50 value of 24.7μM. The effect of thujone was not dependent on the membrane potential and did not involve Ca 2+ -dependent Cl - channels expressed endogenously in oocytes. Inhibition by thujone was not reversed by increasing ACh concentrations. Moreover, specific binding of [ 125 I] α-bungarotoxin was not altered by thujone. Further experiments in SH-EP1 cells expressing human α 7 nACh receptor indicated that thujone suppressed choline induced Ca 2+ transients in a concentration-dependent manner. In rat hippocampal CA3-dentate gyrus synapses, nicotine-induced enhancement of long-term potentiation was also inhibited by thujone. Furthermore, the results observed in in-vivo one-trial passive avoidance paradigm show that thujone (1.25mg/kg, i.p.) significantly impaired nicotine-induced enhancement of learning and memory in Wistar rats. Collectively, our results indicate that thujone inhibits the function of the α7-nACh receptor and impairs cellular and behavioral correlates of cholinergic modulation of learning and memory. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Thujone inhibits the function of α7-nicotinic acetylcholine receptors and impairs nicotine-induced memory enhancement in one-trial passive avoidance paradigm

    International Nuclear Information System (INIS)

    Sultan, Ahmed; Yang, Keun-Hang Susan; Isaev, Dmitro; Nebrisi, Eslam El; Syed, Nurulain; Khan, Nadia; Howarth, Christopher F.; Sadek, Bassem; Oz, Murat

    2017-01-01

    Effects of thujone, a major ingredient of absinthe, wormwood oil and some herbal medicines, were tested on the function of α 7 subunit of the human nicotinic acetylcholine (α 7 nACh) receptor expressed in Xenopus oocytes using the two-electrode voltage-clamp technique. Thujone reversibly inhibited ACh (100 μM)-induced currents with an IC 50 value of 24.7 μM. The effect of thujone was not dependent on the membrane potential and did not involve Ca 2+ -dependent Cl − channels expressed endogenously in oocytes. Inhibition by thujone was not reversed by increasing ACh concentrations. Moreover, specific binding of [ 125 I] α-bungarotoxin was not altered by thujone. Further experiments in SH-EP1 cells expressing human α 7 nACh receptor indicated that thujone suppressed choline induced Ca 2+ transients in a concentration-dependent manner. In rat hippocampal CA3-dentate gyrus synapses, nicotine-induced enhancement of long-term potentiation was also inhibited by thujone. Furthermore, the results observed in in-vivo one-trial passive avoidance paradigm show that thujone (1.25 mg/kg, i.p.) significantly impaired nicotine-induced enhancement of learning and memory in Wistar rats. Collectively, our results indicate that thujone inhibits the function of the α7-nACh receptor and impairs cellular and behavioral correlates of cholinergic modulation of learning and memory.

  5. The inhibition of cholera toxin-induced 5-HT release by the 5-HT3 receptor antagonist, granisetron, in the rat

    Science.gov (United States)

    Turvill, J L; Connor, P; Farthing, M J G

    2000-01-01

    The secretagogue 5-hydroxytryptamine (5-HT) is implicated in the pathophysiology of cholera. 5-HT released from enterochromaffin cells after cholera toxin exposure is thought to activate non-neuronally (5-HT2 dependent) and neuronally (5-HT3 dependent) mediated water and electrolyte secretion. CT-secretion can be reduced by preventing the release of 5-HT. Enterochromaffin cells possess numerous receptors that, under basal conditions, modulate 5-HT release. These include basolateral 5-HT3 receptors, the activation of which is known to enhance 5-HT release. Until now, 5-HT3 receptor antagonists (e.g. granisetron) have been thought to inhibit cholera toxin-induced fluid secretion by blockading 5-HT3 receptors on secretory enteric neurones. Instead we postulated that they act by inhibiting cholera toxin-induced enterochromaffin cell degranulation. Isolated intestinal segments in anaesthetized male Wistar rats, pre-treated with granisetron 75 μg kg−1, lidoocaine 6 mg kg−1 or saline, were instilled with a supramaximal dose of cholera toxin or saline. Net fluid movement was determined by small intestinal perfusion or gravimetry and small intestinal and luminal fluid 5-HT levels were determined by HPLC with fluorimetric detection. Intraluminal 5-HT release was proportional to the reduction in tissue 5-HT levels and to the onset of water and electrolyte secretion, suggesting that luminal 5-HT levels reflect enterochromaffin cell activity. Both lidocaine and granisetron inhibited fluid secretion. However, granisetron alone, and proportionately, reduced 5-HT release. The simultaneous inhibition of 5-HT release and fluid secretion by granisetron suggests that 5-HT release from enterochromaffin cells is potentiated by endogenous 5-HT3 receptors. The accentuated 5-HT release promotes cholera toxin-induced fluid secretion. PMID:10882387

  6. Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lynch, Caitlin; Pan, Yongmei; Li, Linhao [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States); Heyward, Scott; Moeller, Timothy [Bioreclamation In Vitro Technologies, Baltimore, MD 21227 (United States); Swaan, Peter W. [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States); Wang, Hongbing, E-mail: hwang@rx.umaryland.edu [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States)

    2014-08-15

    Objective: Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH). Methods: Ligand-based structure–activity models were used for virtual screening of the Specs database ( (www.specs.net)) followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH. Results: Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo. Conclusion: Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. Implications: Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders. - Highlights: • Novel hCAR activators were identified by computational and biological approaches. • The role

  7. Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes

    International Nuclear Information System (INIS)

    Lynch, Caitlin; Pan, Yongmei; Li, Linhao; Heyward, Scott; Moeller, Timothy; Swaan, Peter W.; Wang, Hongbing

    2014-01-01

    Objective: Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH). Methods: Ligand-based structure–activity models were used for virtual screening of the Specs database ( (www.specs.net)) followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH. Results: Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo. Conclusion: Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. Implications: Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders. - Highlights: • Novel hCAR activators were identified by computational and biological approaches. • The role

  8. Synergistic effect of sevoflurane and isoflurane on inhibition of the adult-type muscle nicotinic acetylcholine receptor by rocuronium.

    Science.gov (United States)

    Liu, Li; Li, Wei; Wei, Ke; Cao, Jun; Luo, Jie; Wang, Bin; Min, Su

    2013-06-01

    Inhaled anesthetics increase the incidence of postoperative residual neuromuscular blockade, and the mechanism is still unclear. We have investigated the synergistic effect of low-concentration inhaled anesthetics and rocuronium on inhibition of the inward current of the adult-type muscle nicotinic acetylcholine receptor (ε-nAChR). Adult-type mouse muscle ε-nAChR was expressed in HEK293 cells by liposome transfection. The inward current of the ε-nAChR was activated by use of 10 μmol/L acetylcholine alone or in combination with different concentrations of sevoflurane, isoflurane, or rocuronium. The concentration-response curves of five cells were constructed, and the data yielded the 5, 25, and 50 % inhibitory concentrations (IC5, IC25, and IC50, respectively) for single-drug application. Subsequently, the functional channels were perfused by adding 0.5 IC5 of either sevoflurane or isoflurane (aqueous concentrations 140 and 100 μmol/L, respectively) to the solution, followed by addition of IC5, IC25, or IC50 rocuronium. The amount of inhibition was calculated to quantify their synergistic effect. The inhibitory effect of rocuronium was enhanced by sevoflurane or isoflurane in a concentration-dependent manner. Sevoflurane or isoflurane (0.5 IC5) with rocuronium at IC5, IC25, and IC50 synergistically inhibited the current amplitude of adult-type muscle ε-nAChR. When the IC5 of rocuronium was used, isoflurane had a stronger synergistic effect than sevoflurane (p rocuronium was applied at higher concentrations (IC25 and IC50), sevoflurane had an effect similar to that of isoflurane. For both inhaled anesthetics, the synergistic effect was more intense for rocuronium at IC5 than for rocuronium at IC25 or IC50. Residual-concentration sevoflurane or isoflurane has a strong synergistic effect with rocuronium at clinically relevant residual concentrations. A lower rocuronium concentration resulted in a stronger synergistic effect.

  9. Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells

    Directory of Open Access Journals (Sweden)

    Zhichao Zhang

    2018-05-01

    Full Text Available Glioblastoma multiforme (GBM is the most lethal glioma variant in the adult brain and among the deadliest of human cancers. Increasing evidence has shown that metabotropic glutamate receptor subtype 4 (mGluR4 expression may play roles in regulating the growth of neural stem cells as well as several cancer cell lines. Here, we investigated the effects of mGluR4 on the growth and apoptosis of the LN229 GBM cell line. Involvement of Gli-1, one of the key transcription factors in the sonic Hedgehog (SHH signaling pathway, was further explored. In this study, mGluR4 was activated using selective agonist VU0155041; and gene-targeted siRNAs were used to generate loss of function of mGluR4 and Gli-1 in LN229 cells. The results demonstrated that LN229 cells expressed mGluR4 and the agonist VU0155041 decreased cell viability in a dose- and time-dependent manner. Activation of mGluR4 inhibited cyclin D1 expression, activated pro-caspase-8/9/3, and disrupted the balance of Bcl-2/Bax expression, which indicated cell cycle arrest and apoptosis of LN229 cells, respectively. Furthermore, Gli-1 expression was reduced by mGluR4 activation in LN229 cells, and downregulation of Gli-1 expression by gene-targeted siRNA resulted in both inhibition of cell proliferation and promotion of apoptosis. Moreover, VU0155041 treatment substantially blocked SHH-induced cyclin D1 expression and cell proliferation, while increasing TUNEL-positive cells and the activation of apoptosis-related proteins. We concluded that activation of mGluR4 expressed in LN229 cells could inhibit GBM cell growth by decreasing cell proliferation and promoting apoptosis. Further suppression of intracellular Gli-1 expression might be involved in the action of mGluR4 on cancer cells. Our study suggested a novel role of mGluR4, which might serve as a potential drug target for control of GBM cell growth.

  10. Long-term improvements in sensory inhibition with gestational choline supplementation linked to α7 nicotinic receptors through studies in Chrna7 null mutation mice.

    Science.gov (United States)

    Stevens, Karen E; Choo, Kevin S; Stitzel, Jerry A; Marks, Michael J; Adams, Catherine E

    2014-03-13

    Perinatal choline supplementation has produced several benefits in rodent models, from improved learning and memory to protection from the behavioral effects of fetal alcohol exposure. We have shown that supplemented choline through gestation and lactation produces long-term improvement in deficient sensory inhibition in DBA/2 mice which models a similar deficit in schizophrenia patients. The present study extends that research by feeding normal or supplemented choline diets to DBA/2 mice carrying the null mutation for the α7 nicotinic receptor gene (Chrna7). DBA/2 mice heterozygotic for Chrna7 were bred together. Dams were placed on supplemented (5 gm/kg diet) or normal (1.1 gm/kg diet) choline at mating and remained on the specific diet until offspring weaning. Thereafter, offspring were fed standard rodent chow. Adult offspring were assessed for sensory inhibition. Brains were obtained to ascertain hippocampal α7 nicotinic receptor levels. Choline-supplemented mice heterozygotic or null-mutant for Chrna7 failed to show improvement in sensory inhibition. Only wildtype choline-supplemented mice showed improvement with the effect solely through a decrease in test amplitude. This supports the hypothesis that gestational-choline supplementation is acting through the α7 nicotinic receptor to improve sensory inhibition. Although there was a significant gene-dose-related change in hippocampal α7 receptor numbers, binding studies did not reveal any choline-dose-related change in binding in any hippocampal region, the interaction being driven by a significant genotype main effect (wildtype>heterozygote>null mutant). These data parallel a human study wherein the offspring of pregnant women receiving choline supplementation during gestation, showed better sensory inhibition than offspring of women on placebo. Published by Elsevier B.V.

  11. The insecticide fipronil and its metabolite fipronil sulphone inhibit the rat alpha1beta2gamma2L GABA(A) receptor.

    Science.gov (United States)

    Li, P; Akk, G

    2008-11-01

    Fipronil is the active ingredient in a number of widely used insecticides. Human exposure to fipronil leads to symptoms (headache, nausea and seizures) typically associated with the antagonism of GABA(A) receptors in the brain. In this study, we have examined the modulation of the common brain GABA(A) receptor subtype by fipronil and its major metabolite, fipronil sulphone. Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat alpha1beta2gamma2L GABA(A) receptors. The major effect of fipronil was to increase the rate of current decay in macroscopic recordings. In single-channel recordings, the presence of fipronil resulted in shorter cluster durations without affecting the intracluster open and closed time distributions or the single-channel conductance. The alpha1V256S mutation, previously shown alleviate channel inhibition by inhibitory steroids and several insecticides, had a relatively small effect on channel block by fipronil. The mode of action of fipronil sulphone was similar to that of its parent compound but the metabolite was less potent at inhibiting the alpha1beta2gamma2L receptor. We conclude that exposure to fipronil induces accumulation of receptors in a novel, long-lived blocked state. This process proceeds in parallel with and independently of, channel desensitization. The lower potency of fipronil sulphone indicates that the conversion serves as a detoxifying process in mammalian brain.

  12. Inhibition of Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Phosphorylation on Tumor-Associated Endothelial Cells Leads to Treatment of Orthotopic Human Colon Cancer in Nude Mice

    Directory of Open Access Journals (Sweden)

    Takamitsu Sasaki

    2007-12-01

    Full Text Available The purpose of our study was to determine whether the dual inhibition of epidermal growth factor receptor (EGFR and vascular endothelial growth factor receptor (VEGFR signaling pathways in tumor-associated endothelial cells can inhibit the progressive growth of human colon carcinoma in the cecum of nude mice. SW620CE2 human colon cancer cells growing in culture and orthotopically in the cecum of nude mice expressed a high level of transforming growth factor alpha (TGF-α and vascular endothelial growth factor (VEGF but were negative for EGFR, human epidermal growth factor receptor 2 (HER2, VEGFR. Double immunofluorescence staining revealed that tumorassociated endothelial cells expressed EGFR, VEGFR2, phosphorylated EGFR (pEGFR, phosphorylated VEGFR (pVEGFR. Treatment of mice with either 7H-pyrrolo [2,3-d]-pyrimidine lead scaffold (AEE788; an inhibitor of EGFR and VEGFR tyrosine kinase or CPT-11 as single agents significantly inhibited the growth of cecal tumors (P < .01; this decrease was even more pronounced with AEE788 combined with CPT-11 (P < .001. AEE788 alone or combined with CPT-11 also inhibited the expression of pEGFR and pVEGFR on tumor-associated endothelial cells, significantly decreased vascularization and tumor cell proliferation, increased the level of apoptosis in both tumorassociated endothelial cells and tumor cells. These data demonstrate that targeting EGFR and VEGFR signaling on tumor-associated endothelial cells provides a viable approach for the treatment of colon cancer.

  13. Tumor necrosis factor-α inhibits effects of aryl hydrocarbon receptor ligands on cell death in human lymphocytes.

    Science.gov (United States)

    Ghatrehsamani, Mahdi; Soleimani, Masoud; Esfahani, Behjat A Moayedi; Shirzad, Hedayatollah; Hakemi, Mazdak G; Mossahebimohammadi, Majid; Eskandari, Nahid; Adib, Minoo

    2015-01-01

    Activation of aryl hydrocarbon receptor (AhR) leads to diverse outcome in various kinds of cells. AhR activation may induce apoptosis or prevent of apoptosis and cell death. Recent studies suggest that apoptosis effects of AhR can be modulated by inflammatory cytokine like tumor necrosis factor alpha (TNF-α). In this study, we try to investigate the possible interaction of TNF-α with the 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), a ligand of AhR, on peripheral lymphocytes. Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by discontinuous density gradient centrifugation on ficoll. Isolated PBMCs were divided into four groups: Control group, TNF-α administered group, TCDD administered group, co-administered group with TCDD and TNF-α. Cells were maintained for a week in lymphocyte culture condition. Then, TNF-α was added to group 2 and 4. Finally, apoptosis and necrosis were analyzed in all samples using flowcytometry. In group 4, the mean percent of necrosis and apoptosis in TCDD treatment groups was significantly larger than other groups; (P 0.05). However, the mean percent of cell death in co-administered group with TCDD and TNF-α was significantly lower than other groups; (P < 0.05). TNF-α could significantly inhibit effects of TCDD on lymphocytes apoptosis. Combination effects of TNF-α and TCDD on lymphocyte increase cell survival.

  14. Docosahexaenoic acid inhibits the growth of hormone-dependent prostate cancer cells by promoting the degradation of the androgen receptor.

    Science.gov (United States)

    Hu, Zhimei; Qi, Haixia; Zhang, Ruixue; Zhang, Kun; Shi, Zhemin; Chang, Yanan; Chen, Linfeng; Esmaeili, Mohsen; Baniahmad, Aria; Hong, Wei

    2015-09-01

    Epidemiological and preclinical data have demonstrated the preventative effects of ω-3 polyunsaturated fatty acids, including docosahexaenoic acid (DHA), on prostate cancer. However, there are inconsistencies in these previous studies and the underlying mechanisms remain to be elucidated. In the present study, the androgen receptor (AR), which is a transcription factor involved in cell proliferation and prostate carcinogenesis, was identified as a target of DHA. It was revealed that DHA inhibited hormone‑dependent growth of LNCaP prostate cancer cells. Reverse transcription-quantitative polymerase chain reaction analysis revealed that treatment with DHA caused no alteration in the transcribed mRNA expression levels of the AR gene. However, immunoblotting revealed that this treatment reduces the protein expression level of the AR. The androgen‑induced genes were subsequently repressed by treatment with DHA. It was demonstrated that DHA exhibits no effect on the translation process of the AR, however, it promotes the proteasome‑mediated degradation of the AR. Therefore, the present study provided a novel mechanism by which DHA exhibits an inhibitory effect on growth of prostate cancer cells.

  15. Glycolytic metabolite methylglyoxal inhibits cold and menthol activation of the transient receptor potential melastatin type 8 channel.

    Science.gov (United States)

    Ciobanu, A C; Selescu, T; Gasler, I; Soltuzu, L; Babes, A

    2016-03-01

    Methylglyoxal (MG) is a reactive dicarbonyl compound involved in protein modifications linked to diabetes mellitus. The plasma level of MG is elevated in diabetic patients, particularly those with painful diabetic neuropathy. Diabetic neuropathy is often associated with spontaneous pain and altered thermal perception. This study assesses effects of MG on TRPM8, an ion channel involved in innocuous cold sensing and cold allodynia and also in cold-mediated analgesia. Acute treatment with MG inhibited the activation of recombinant rat and human transient receptor potential melastatin type 8 (TRPM8) by cold and chemical agonists. A similar effect was observed when native TRPM8 was investigated in cultured rat dorsal root ganglion (DRG) neurons. DRG neurons treated with MG for 16-24 hr displayed a significant reduction in the fraction of cold- and menthol-sensitive neurons, most likely expressing TRPM8. The fraction of allyl isothiocyanate-sensitive neurons was also reduced, and the coexpression among different neuronal populations was affected. The same prolonged exposure to MG significantly reduced the expression of TRPM8 at the mRNA level. Overall, our data provide evidence for decreased activity and expression level of TRPM8 in the presence of MG, which may be linked to some of the alterations in pain and temperature sensing reported by diabetic patients. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  16. Inhibition of type I insulin-like growth factor receptor signaling attenuates the development of breast cancer brain metastasis.

    Science.gov (United States)

    Saldana, Sandra M; Lee, Heng-Huan; Lowery, Frank J; Khotskaya, Yekaterina B; Xia, Weiya; Zhang, Chenyu; Chang, Shih-Shin; Chou, Chao-Kai; Steeg, Patricia S; Yu, Dihua; Hung, Mien-Chie

    2013-01-01

    Brain metastasis is a common cause of mortality in cancer patients, yet potential therapeutic targets remain largely unknown. The type I insulin-like growth factor receptor (IGF-IR) is known to play a role in the progression of breast cancer and is currently being investigated in the clinical setting for various types of cancer. The present study demonstrates that IGF-IR is constitutively autophosphorylated in brain-seeking breast cancer sublines. Knockdown of IGF-IR results in a decrease of phospho-AKT and phospho-p70s6k, as well as decreased migration and invasion of MDA-MB-231Br brain-seeking cells. In addition, transient ablation of IGFBP3, which is overexpressed in brain-seeking cells, blocks IGF-IR activation. Using an in vivo experimental brain metastasis model, we show that IGF-IR knockdown brain-seeking cells have reduced potential to establish brain metastases. Finally, we demonstrate that the malignancy of brain-seeking cells is attenuated by pharmacological inhibition with picropodophyllin, an IGF-IR-specific tyrosine kinase inhibitor. Together, our data suggest that the IGF-IR is an important mediator of brain metastasis and its ablation delays the onset of brain metastases in our model system.

  17. Orphan nuclear receptor NR4A2 inhibits hepatic stellate cell proliferation through MAPK pathway in liver fibrosis.

    Science.gov (United States)

    Chen, Pengguo; Li, Jie; Huo, Yan; Lu, Jin; Wan, Lili; Li, Bin; Gan, Run; Guo, Cheng

    2015-01-01

    Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis, which is a pathological process characterized by extracellular matrix accumulation. NR4A2 is a nuclear receptor belonging to the NR4A subfamily and vital in regulating cell growth, metabolism, inflammation and other biological functions. However, its role in HSCs is unclear. We analyzed NR4A2 expression in fibrotic liver and stimulated HSCs compared with control group and studied the influence on cell proliferation, cell cycle, cell apoptosis and MAPK pathway after NR4A2 knockdown. NR4A2 expression was examined by real-time polymerase chain reaction, Western blotting, immunohistochemistry and immunofluorescence analyses. NR4A2 expression was significantly lower in fibrotic liver tissues and PDGF BB or TGF-β stimulated HSCs compared with control group. After NR4A2 knockdown α-smooth muscle actin and Col1 expression increased. In addition, NR4A2 silencing led to the promotion of cell proliferation, increase of cell percentage in S phase and reduced phosphorylation of ERK1/2, P38 and JNK in HSCs. These results indicate that NR4A2 can inhibit HSC proliferation through MAPK pathway and decrease extracellular matrix in liver fibrogenesis. NR4A2 may be a promising therapeutic target for liver fibrosis.

  18. Overexpression of insulin-like growth factor (IGF)-I receptor enhances inhibition of DNA replication in mouse cells exposed to x-rays

    International Nuclear Information System (INIS)

    Wang, Y.; Cheong, N.; Miura, M.; Iliakis, G.

    1997-01-01

    Previous studies from our laboratory provided evidence for the operation of signal transduction pathways involving ras, myc, and staurosporine-sensitive protein kinases in the regulation of DNA replication in irradiated cells. Because ras and myc are also involved in the signal transduction elicited in response to ligand activation of growth factor receptors, we wondered whether growth factor receptors are upstream elements in the regulation of DNA replication in irradiated cells. Here, we report on the role of insulin-like growth factor I receptor (IGF-IR) in the regulation of DNA replication in irradiated cells. We compare radiation-induced inhibition of DNA replication in BALB/c 3T3 cells with that in P6 cells. P6 cells are derived from BALB/c 3T3 cells by transfection with a vector expressing IGF-IR, leading to 30-fold overexpression. We observe a significantly stronger inhibition of DNA replication after irradiation in P6 as compared with BALB/c 3T3 cells at all doses examined. Sedimentation in alkaline sucrose gradients shows that the increased inhibition in P6 cells is due to an increased inhibition of replicon initiation, the main controlling event in DNA replication. Staurosporine at 20 nM reduces radiation-induced inhibition of DNA replication in BALB/c 3T3 cells, but has only a small effect in P6 cells. Caffeine at a concentration of 1 mM, on the other hand, removes over 60% of the inhibition in both cell lines. The results implicate IGF-IR in the regulation of DNA replication in irradiated cells, but also suggest differences between cells of different origins in the proteins involved in the regulating signal transduction pathway. (orig.). With 5 figs

  19. The effects of MEK1/2 inhibition on cigarette smoke exposure-induced ET receptor upregulation in rat cerebral arteries

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Lei [Division of Experimental Vascular Research, Institute of Clinical Sciences in Lund, Lund University (Sweden); Department of Pharmacology, School of Basic Medical Sciences, Xi' an Jiaotong University Health Science Center, Xi' an, Shaanxi (China); Ping, Na-Na; Cao, Yong-Xiao [Department of Pharmacology, School of Basic Medical Sciences, Xi' an Jiaotong University Health Science Center, Xi' an, Shaanxi (China); Li, Wei, E-mail: 13572512207@163.com [Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi' an Jiaotong University, Xi' an, Shaanxi (China); Cai, Yan [Department of Pharmacology, School of Basic Medical Sciences, Xi' an Jiaotong University Health Science Center, Xi' an, Shaanxi (China); Warfvinge, Karin; Edvinsson, Lars [Division of Experimental Vascular Research, Institute of Clinical Sciences in Lund, Lund University (Sweden)

    2016-08-01

    Cigarette smoking, a major stroke risk factor, upregulates endothelin receptors in cerebral arteries. The present study examined the effects of MEK1/2 pathway inhibition on cigarette smoke exposure-induced ET receptor upregulation. Rats were exposed to the secondhand smoke (SHS) for 8 weeks followed by intraperitoneal injection of MEK1/2 inhibitor, U0126 for another 4 weeks. The urine cotinine levels were assessed with high-performance liquid chromatography. Contractile responses of isolated cerebral arteries were recorded by a sensitive wire myograph. The mRNA and protein expression levels of receptor and MEK/ERK1/2 pathway molecules were examined by real-time PCR and Western blotting, respectively. Cerebral artery receptor localization was determined with immunohistochemistry. The results showed the urine cotinine levels from SHS exposure group were significantly higher than those from the fresh group. In addition, the MEK1/2 inhibitor, U0126 significantly reduced SHS exposure-increased ET{sub A} receptor mRNA and protein levels as well as contractile responses mediated by ET{sub A} receptors. The immunoreactivity of increased ET{sub A} receptor expression was primarily cytoplasmic in smooth muscle cells. In contrast, ET{sub B} receptor was noted in endothelial cells. However, the SHS-induced decrease in endothelium-dependent relaxation was unchanged after U0126 treatment. Furthermore, SHS increased the phosphorylation of MEK1/2 and ERK1/2 protein in cerebral arteries. By using U0126 could inhibit the phosphorylated ERK1/2 protein but not MEK1/2. Taken together, our data show that treatment with MEK1/2 pathway inhibitor offsets SHS exposure-induced ET{sub A} receptor upregulation in rat cerebral arteries. - Highlights: • Cigarette smoke exposure induces ET{sub A} receptor upregulation in rat cerebral arteries. • U0126 can alleviate the receptor upregulation. • The mechanism relies on MEK/ERK1/2 pathway activation. • We may provide a new target for the

  20. The effects of MEK1/2 inhibition on cigarette smoke exposure-induced ET receptor upregulation in rat cerebral arteries

    International Nuclear Information System (INIS)

    Cao, Lei; Ping, Na-Na; Cao, Yong-Xiao; Li, Wei; Cai, Yan; Warfvinge, Karin; Edvinsson, Lars

    2016-01-01

    Cigarette smoking, a major stroke risk factor, upregulates endothelin receptors in cerebral arteries. The present study examined the effects of MEK1/2 pathway inhibition on cigarette smoke exposure-induced ET receptor upregulation. Rats were exposed to the secondhand smoke (SHS) for 8 weeks followed by intraperitoneal injection of MEK1/2 inhibitor, U0126 for another 4 weeks. The urine cotinine levels were assessed with high-performance liquid chromatography. Contractile responses of isolated cerebral arteries were recorded by a sensitive wire myograph. The mRNA and protein expression levels of receptor and MEK/ERK1/2 pathway molecules were examined by real-time PCR and Western blotting, respectively. Cerebral artery receptor localization was determined with immunohistochemistry. The results showed the urine cotinine levels from SHS exposure group were significantly higher than those from the fresh group. In addition, the MEK1/2 inhibitor, U0126 significantly reduced SHS exposure-increased ET A receptor mRNA and protein levels as well as contractile responses mediated by ET A receptors. The immunoreactivity of increased ET A receptor expression was primarily cytoplasmic in smooth muscle cells. In contrast, ET B receptor was noted in endothelial cells. However, the SHS-induced decrease in endothelium-dependent relaxation was unchanged after U0126 treatment. Furthermore, SHS increased the phosphorylation of MEK1/2 and ERK1/2 protein in cerebral arteries. By using U0126 could inhibit the phosphorylated ERK1/2 protein but not MEK1/2. Taken together, our data show that treatment with MEK1/2 pathway inhibitor offsets SHS exposure-induced ET A receptor upregulation in rat cerebral arteries. - Highlights: • Cigarette smoke exposure induces ET A receptor upregulation in rat cerebral arteries. • U0126 can alleviate the receptor upregulation. • The mechanism relies on MEK/ERK1/2 pathway activation. • We may provide a new target for the treatment of SHS

  1. Ectopic Expression of α6 and δ GABAA Receptor Subunits in Hilar Somatostatin Neurons Increases Tonic Inhibition and Alters Network Activity in the Dentate Gyrus

    Science.gov (United States)

    Tong, Xiaoping; Peng, Zechun; Zhang, Nianhui; Cetina, Yliana; Huang, Christine S.; Wallner, Martin; Otis, Thomas S.

    2015-01-01

    The role of GABAA receptor (GABAAR)-mediated tonic inhibition in interneurons remains unclear and may vary among subgroups. Somatostatin (SOM) interneurons in the hilus of the dentate gyrus show negligible expression of nonsynaptic GABAAR subunits and very low tonic inhibition. To determine the effects of ectopic expression of tonic GABAAR subtypes in these neurons, Cre-dependent viral vectors were used to express GFP-tagged GABAAR subunits (α6 and δ) selectively in hilar SOM neurons in SOM-Cre mice. In single-transfected animals, immunohistochemistry demonstrated strong expression of either the α6 or δ subunit; in cotransfected animals, both subunits were consistently expressed in the same neurons. Electrophysiology revealed a robust increase of tonic current, with progressively larger increases following transfection of δ, α6, and α6/δ subunits, respectively, indicating formation of functional receptors in all conditions and likely coassembly of the subunits in the same receptor following cotransfection. An in vitro model of repetitive bursting was used to determine the effects of increased tonic inhibition in hilar SOM interneurons on circuit activity in the dentate gyrus. Upon cotransfection, the frequency of GABAAR-mediated bursting in granule cells was reduced, consistent with a reduction in synchronous firing among hilar SOM interneurons. Moreover, in vivo studies of Fos expression demonstrated reduced activation of α6/δ-cotransfected neurons following acute seizure induction by pentylenetetrazole. The findings demonstrate that increasing tonic inhibition in hilar SOM interneurons can alter dentate gyrus circuit activity during strong stimulation and suggest that tonic inhibition of interneurons could play a role in regulating excessive synchrony within the network. SIGNIFICANCE STATEMENT In contrast to many hippocampal interneurons, somatostatin (SOM) neurons in the hilus of the dentate gyrus have very low levels of nonsynaptic GABAARs and exhibit

  2. Leptin reverses corticosterone-induced inhibition of neural stem cell proliferation through activating the NR2B subunits of NMDA receptors

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Wen-Zhu [Anesthesia and Operation Center, Hainan Branch of Chinese PLA General Hospital, Hainan 572013 (China); Anesthesia and Operation Center, Chinese PLA General Hospital, Beijing 100853 (China); Miao, Yu-Liang [Department of Anesthesiology, PLA No. 306 Hospital, Beijing 100101 (China); Guo, Wen-Zhi [Department of Anesthesiology, Beijing Military General Hospital of Chinese People’s Liberation Army, Beijing 100700 (China); Wu, Wei, E-mail: wwzwgk@163.com [Department of Head and Neck Surgery of Otolaryngology, PLA No. 306 Hospital, Beijing 100101 (China); Li, Bao-Wei [Department of Head and Neck Surgery of Otolaryngology, PLA No. 306 Hospital, Beijing 100101 (China); An, Li-Na [Department of Anesthesiology, Armed Police General Hospital, Beijing 100039 (China); Fang, Wei-Wu [Department of Anesthesiology, PLA No. 306 Hospital, Beijing 100101 (China); Mi, Wei-Dong, E-mail: elite2005gg@163.com [Anesthesia and Operation Center, Chinese PLA General Hospital, Beijing 100853 (China)

    2014-04-25

    Highlights: • Leptin promotes the proliferation of neural stem cells isolated from embryonic mouse hippocampus. • Leptin reverses corticosterone-induced inhibition of neural stem cell proliferation. • The effects of leptin are partially mediated by upregulating NR2B subunits. - Abstract: Corticosterone inhibits the proliferation of hippocampal neural stem cells (NSCs). The removal of corticosterone-induced inhibition of NSCs proliferation has been reported to contribute to neural regeneration. Leptin has been shown to regulate brain development, improve angiogenesis, and promote neural regeneration; however, its effects on corticosterone-induced inhibition of NSCs proliferation remain unclear. Here we reported that leptin significantly promoted the proliferation of hippocampal NSCs in a concentration-dependent pattern. Also, leptin efficiently reversed the inhibition of NSCs proliferation induced by corticosterone. Interestingly, pre-treatment with non-specific NMDA antagonist MK-801, specific NR2B antagonist Ro 25-6981, or small interfering RNA (siRNA) targeting NR2B, significantly blocked the effect of leptin on corticosterone-induced inhibition of NSCs proliferation. Furthermore, corticosterone significantly reduced the protein expression of NR2B, whereas pre-treatment with leptin greatly reversed the attenuation of NR2B expression caused by corticosterone in cultured hippocampal NSCs. Our findings demonstrate that leptin reverses the corticosterone-induced inhibition of NSCs proliferation. This process is, at least partially mediated by increased expression of NR2B subunits of NMDA receptors.

  3. A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

    Science.gov (United States)

    Vassbotn, F S; Langeland, N; Hagen, I; Holmsen, H

    1990-09-01

    A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

  4. [Inhibition of glycogen synthase kinase 3b activity regulates Toll-like receptor 4-mediated liver inflammation].

    Science.gov (United States)

    Ren, Feng; Zhang, Hai-yan; Piao, Zheng-fu; Zheng, Su-jun; Chen, Yu; Chen, De-xi; Duan, Zhong-ping

    2012-09-01

    To determine the mechanism underlying the therapeutic activities of glycogen synthase kinase 3b (GSK3b) against hepatic ischemia-reperfusion (H-IR) injury by investigating the inhibitive effects of GSK3b on inflammation mediated by Toll-like receptor 4 (TLR4). C57BL/6 male mice were subjected to 90 min of warm liver cephalad lobe ischemia, followed by reperfusion for various lengths of time. The mice were divided into three groups: the H-IR untreated model (control group), and the H-IR inflammation-induced models that received an intraperitoneal injection of purified lipopolysaccharide (LPS) endotoxin alone (inflammation group) or with pretreatment of the SB216763 GSK3b-specific inhibitor (intervention group). To create a parallel isolated cell system for detailed investigations of macrophages, marrow-derived stem cells were isolated from femurs of the H-IR control group of mice and used to derive primary macrophages. The cells were then divided into the same three groups as the whole mouse system: control, LPS-induced inflammation model, and inflammation model with SB216763 intervention. Differential expressions of inflammation-related proteins and genes were detected by Western blotting and real-time quantitative PCR, respectively. The phosphorylation levels of ERK, JNK and p38 MAPK were induced in liver at 1 h after reperfusion, but then steadily decreased and returned to baseline levels by 4 h after reperfusion. In addition, the phosphorylation levels of ERK and JNK were induced in macrophages at 15 min after LPS stimulation, while the phosphorylation level of p38 MAPK was induced at 1 h; SB216763 pretreatment suppressed the LPS-stimulated ERK, JNK and p38 phosphorylation in macrophages. In the mouse model, GSK3b activity was found to promote the gene expression of anti-inflammatory cytokine IL-10 (control: 0.21 ± 0.08, inflammation: 0.83 ± 0.21, intervention: 1.76 ± 0.67; F = 3.16, P = 0.027) but to significantly inhibit the gene expression of pro

  5. Anandamide inhibits Theiler's virus induced VCAM-1 in brain endothelial cells and reduces leukocyte transmigration in a model of blood brain barrier by activation of CB1 receptors

    Directory of Open Access Journals (Sweden)

    Loría Frida

    2011-08-01

    Full Text Available Abstract Background VCAM-1 represents one of the most important adhesion molecule involved in the transmigration of blood leukocytes across the blood-brain barrier (BBB that is an essential step in the pathogenesis of MS. Several evidences have suggested the potential therapeutic value of cannabinoids (CBs in the treatment of MS and their experimental models. However, the effects of endocannabinoids on VCAM-1 regulation are poorly understood. In the present study we investigated the effects of anandamide (AEA in the regulation of VCAM-1 expression induced by Theiler's virus (TMEV infection of brain endothelial cells using in vitro and in vivo approaches. Methods i in vitro: VCAM-1 was measured by ELISA in supernatants of brain endothelial cells infected with TMEV and subjected to AEA and/or cannabinoid receptors antagonist treatment. To evaluate the functional effect of VCAM-1 modulation we developed a blood brain barrier model based on a system of astrocytes and brain endothelial cells co-culture. ii in vivo: CB1 receptor deficient mice (Cnr1-/- infected with TMEV were treated with the AEA uptake inhibitor UCM-707 for three days. VCAM-1 expression and microglial reactivity were evaluated by immunohistochemistry. Results Anandamide-induced inhibition of VCAM-1 expression in brain endothelial cell cultures was mediated by activation of CB1 receptors. The study of leukocyte transmigration confirmed the functional relevance of VCAM-1 inhibition by AEA. In vivo approaches also showed that the inhibition of AEA uptake reduced the expression of brain VCAM-1 in response to TMEV infection. Although a decreased expression of VCAM-1 by UCM-707 was observed in both, wild type and CB1 receptor deficient mice (Cnr1-/-, the magnitude of VCAM-1 inhibition was significantly higher in the wild type mice. Interestingly, Cnr1-/- mice showed enhanced microglial reactivity and VCAM-1 expression following TMEV infection, indicating that the lack of CB1 receptor

  6. Y1 receptors for neuropeptide Y are coupled to mobilization of intracellular calcium and inhibition of adenylate cyclase

    DEFF Research Database (Denmark)

    Aakerlund, L; Gether, U; Fuhlendorff, J

    1990-01-01

    Two types of binding sites have previously been described for neuropeptide Y (NPY), called Y1 and Y2 receptors. The intracellular events following Y1 receptor activation was studied in the human neuroblastoma cell line SK-N-MC. Both NPY and the specific Y1 receptor ligand, [Leu31,Pro34]-NPY, caused...

  7. Niclosamide inhibits epithelial-mesenchymal transition and tumor growth in lapatinib-resistant human epidermal growth factor receptor 2-positive breast cancer.

    Science.gov (United States)

    Liu, Junjun; Chen, Xiaosong; Ward, Toby; Mao, Yan; Bockhorn, Jessica; Liu, Xiaofei; Wang, Gen; Pegram, Mark; Shen, Kunwei

    2016-02-01

    Acquired resistance to lapatinib, a human epidermal growth factor receptor 2 kinase inhibitor, remains a clinical problem for women with human epidermal growth factor receptor 2-positive advanced breast cancer, as metastasis is commonly observed in these patients. Niclosamide, an anti-helminthic agent, has recently been shown to exhibit cytotoxicity to tumor cells with stem-like characteristics. This study was designed to identify the mechanisms underlying lapatinib resistance and to determine whether niclosamide inhibits lapatinib resistance by reversing epithelial-mesenchymal transition. Here, two human epidermal growth factor receptor 2-positive breast cancer cell lines, SKBR3 and BT474, were exposed to increasing concentrations of lapatinib to establish lapatinib-resistant cultures. Lapatinib-resistant SKBR3 and BT474 cells exhibited up-regulation of the phenotypic epithelial-mesenchymal transition markers Snail, vimentin and α-smooth muscle actin, accompanied by activation of nuclear factor-кB and Src and a concomitant increase in stem cell marker expression (CD44(high)/CD24(low)), compared to naive lapatinib-sensitive SKBR3 and BT474 cells, respectively. Interestingly, niclosamide reversed epithelial-mesenchymal transition, induced apoptosis and inhibited cell growth by perturbing aberrant signaling pathway activation in lapatinib-resistant human epidermal growth factor receptor 2-positive cells. The ability of niclosamide to alleviate stem-like phenotype development and invasion was confirmed. Collectively, our results demonstrate that lapatinib resistance correlates with epithelial-mesenchymal transition and that niclosamide inhibits lapatinib-resistant cell viability and epithelial-mesenchymal transition. These findings suggest a role of niclosamide or derivatives optimized for more favorable bioavailability not only in reversing lapatinib resistance but also in reducing metastatic potential during the treatment of human epidermal growth factor receptor

  8. Metformin-mediated growth inhibition involves suppression of the IGF-I receptor signalling pathway in human pancreatic cancer cells

    International Nuclear Information System (INIS)

    Karnevi, Emelie; Said, Katarzyna; Andersson, Roland; Rosendahl, Ann H

    2013-01-01

    Epidemiological studies have shown direct associations between type 2 diabetes and obesity, both conditions associated with hyperglycaemia and hyperinsulinemia, and the risk of pancreatic cancer. Up to 80% of pancreatic cancer patients present with either new-onset type 2 diabetes or impaired glucose tolerance at the time of diagnosis. Recent population studies indicate that the incidence of pancreatic cancer is reduced among diabetics taking metformin. In this study, the effects of exposure of pancreatic cancer cells to high glucose levels on their growth and response to metformin were investigated. The human pancreatic cancer cell lines AsPC-1, BxPC-3, PANC-1 and MIAPaCa-2 were grown in normal (5 mM) or high (25 mM) glucose conditions, with or without metformin. The influence by metformin on proliferation, apoptosis and the AMPK and IGF-IR signalling pathways were evaluated in vitro. Metformin significantly reduced the proliferation of pancreatic cancer cells under normal glucose conditions. Hyperglycaemia however, protected against the metformin-induced growth inhibition. The anti-proliferative actions of metformin were associated with an activation of AMP-activated protein kinase AMPK Thr172 together with an inhibition of the insulin/insulin-like growth factor-I (IGF-I) receptor activation and downstream signalling mediators IRS-1 and phosphorylated Akt. Furthermore, exposure to metformin during normal glucose conditions led to increased apoptosis as measured by poly(ADP-ribose) polymerase (PARP) cleavage. In contrast, exposure to high glucose levels promoted a more robust IGF-I response and Akt activation which correlated to stimulated AMPK Ser485 phosphorylation and impaired AMPK Thr172 phosphorylation, resulting in reduced anti-proliferative and apoptotic effects by metformin. Our results indicate that metformin has direct anti-tumour activities in pancreatic cancer cells involving AMPK Thr172 activation and suppression of the insulin/IGF signalling pathways

  9. Integrated expression profiling and ChIP-seq analyses of the growth inhibition response program of the androgen receptor.

    Directory of Open Access Journals (Sweden)

    Biaoyang Lin

    2009-08-01

    Full Text Available The androgen receptor (AR plays important roles in the development of male phenotype and in different human diseases including prostate cancers. The AR can act either as a promoter or a tumor suppressor depending on cell types. The AR proliferative response program has been well studied, but its prohibitive response program has not yet been thoroughly studied.Previous studies found that PC3 cells expressing the wild-type AR inhibit growth and suppress invasion. We applied expression profiling to identify the response program of PC3 cells expressing the AR (PC3-AR under different growth conditions (i.e. with or without androgens and at different concentration of androgens and then applied the newly developed ChIP-seq technology to identify the AR binding regions in the PC3 cancer genome. A surprising finding was that the comparison of MOCK-transfected PC3 cells with AR-transfected cells identified 3,452 differentially expressed genes (two fold cutoff even without the addition of androgens (i.e. in ethanol control, suggesting that a ligand independent activation or extremely low-level androgen activation of the AR. ChIP-Seq analysis revealed 6,629 AR binding regions in the cancer genome of PC3 cells with an FDR (false discovery rate cut off of 0.05. About 22.4% (638 of 2,849 can be mapped to within 2 kb of the transcription start site (TSS. Three novel AR binding motifs were identified in the AR binding regions of PC3-AR cells, and two of them share a core consensus sequence CGAGCTCTTC, which together mapped to 27.3% of AR binding regions (1,808/6,629. In contrast, only about 2.9% (190/6,629 of AR binding sites contains the canonical AR matrix M00481, M00447 and M00962 (from the Transfac database, which is derived mostly from AR proliferative responsive genes in androgen dependent cells. In addition, we identified four top ranking co-occupancy transcription factors in the AR binding regions, which include TEF1 (Transcriptional enhancer factor

  10. Minnelide Inhibits Androgen Dependent, Castration Resistant Prostate Cancer Growth by Decreasing Expression of Androgen Receptor Full Length and Splice Variants.

    Science.gov (United States)

    Isharwal, Sumit; Modi, Shrey; Arora, Nivedita; Uhlrich, Charles; Giri, Bhuwan; Barlass, Usman; Soubra, Ayman; Chugh, Rohit; Dehm, Scott M; Dudeja, Vikas; Saluja, Ashok; Banerjee, Sulagna; Konety, Badrinath

    2017-05-01

    With almost 30,000 deaths per year, prostate cancer is the second-leading cause of cancer-related death in men. Androgen Deprivation Therapy (ADT) has been the corner stone of prostate cancer treatment for decades. However, despite an initial response of prostate cancer to ADT, this eventually fails and the tumors recur, resulting in Castration Resistant Prostate Cancer (CRPC). Triptolide, a diterpene triepoxide, has been tested for its anti-tumor properties in a number of cancers for over a decade. Owing to its poor solubility in aqueous medium, its clinical application had been limited. To circumvent this problem, we have synthesized a water-soluble pro-drug of triptolide, Minnelide, that is currently being evaluated in a Phase 1 clinical trial against gastrointestinal tumors. In the current study, we assessed the therapeutic potential of Minnelide and its active compound triptolide against androgen dependent prostate cancer both in vitro as well as in vivo. Cell viability was measured by a MTT based assay after treating prostate cancer cells with multiple doses of triptolide. Apoptotic cell death was measured using a caspase 3/7 activity. Androgen Receptor (AR) promoter-binding activity was evaluated by using luciferase reporter assay. For evaluating the effect in vivo, 22Rv1 cells were implanted subcutaneously in animals, following which, treatment was started with 0.21 mg/kg Minnelide. Our study showed that treatment with triptolide induced apoptotic cell death in CRPC cells. Triptolide treatment inhibited AR transcriptional activity and decreased the expression of AR and its splice variants both at the mRNA and the protein level. Our studies show that triptolide inhibits nuclear translocation of Sp1, resulting in its decreased transcriptional activity leading to downregulation of AR and its splice variants in prostate cancer cells. In vivo, Minnelide (0.21 mg/kg) regressed subcutaneous tumors derived from CRPC 22RV1 at our study endpoint. Our animal

  11. Crosstalk between the peroxisome proliferator-activated receptor γ (PPARγ) and the vitamin D receptor (VDR) in human breast cancer cells: PPARγ binds to VDR and inhibits 1α,25-dihydroxyvitamin D3 mediated transactivation

    International Nuclear Information System (INIS)

    Alimirah, Fatouma; Peng, Xinjian; Yuan, Liang; Mehta, Rajeshwari R.; Knethen, Andreas von; Choubey, Divaker; Mehta, Rajendra G.

    2012-01-01

    Heterodimerization and cross-talk between nuclear hormone receptors often occurs. For example, estrogen receptor alpha (ERα) physically binds to peroxisome proliferator-activated receptor gamma (PPARγ) and inhibits its transcriptional activity. The interaction between PPARγ and the vitamin D receptor (VDR) however, is unknown. Here, we elucidate the molecular mechanisms linking PPARγ and VDR signaling, and for the first time we show that PPARγ physically associates with VDR in human breast cancer cells. We found that overexpression of PPARγ decreased 1α,25-dihydroxyvitamin D 3 (1,25D 3 ) mediated transcriptional activity of the vitamin D target gene, CYP24A1, by 49% and the activity of VDRE-luc, a vitamin D responsive reporter, by 75% in T47D human breast cancer cells. Deletion mutation experiments illustrated that helices 1 and 4 of PPARγ's hinge and ligand binding domains, respectively, governed this suppressive function. Additionally, abrogation of PPARγ's AF2 domain attenuated its repressive action on 1,25D 3 transactivation, indicating that this domain is integral in inhibiting VDR signaling. PPARγ was also found to compete with VDR for their binding partner retinoid X receptor alpha (RXRα). Overexpression of RXRα blocked PPARγ's suppressive effect on 1,25D 3 action, enhancing VDR signaling. In conclusion, these observations uncover molecular mechanisms connecting the PPARγ and VDR pathways. -- Highlights: PPARγ's role on 1α,25-dihydroxyvitamin D 3 transcriptional activity is examined. ► PPARγ physically binds to VDR and inhibits 1α,25-dihydroxyvitamin D 3 action. ► PPARγ's hinge and ligand binding domains are important for this inhibitory effect. ► PPARγ competes with VDR for the availability of their binding partner, RXRα.

  12. Regulation of synaptic inhibition by phospho-dependent binding of the AP2 complex to a YECL motif in the GABAA receptor γ2 subunit

    Science.gov (United States)

    Kittler, Josef T.; Chen, Guojun; Kukhtina, Viktoria; Vahedi-Faridi, Ardeschir; Gu, Zhenglin; Tretter, Verena; Smith, Katharine R.; McAinsh, Kristina; Arancibia-Carcamo, I. Lorena; Saenger, Wolfram; Haucke, Volker; Yan, Zhen; Moss, Stephen J.

    2008-01-01

    The regulation of the number of γ2-subunit-containing GABAA receptors (GABAARs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface γ2-subunit-containing GABAARs is regulated. Here, we identify a γ2-subunit-specific Yxxφ-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for γ2-subunit tyrosine phosphorylation. Blocking GABAAR-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxxφ motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that γ2-subunit-containing heteromeric GABAARs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABAAR surface levels and synaptic inhibition. PMID:18305175

  13. Regulation of synaptic inhibition by phospho-dependent binding of the AP2 complex to a YECL motif in the GABAA receptor gamma2 subunit.

    Science.gov (United States)

    Kittler, Josef T; Chen, Guojun; Kukhtina, Viktoria; Vahedi-Faridi, Ardeschir; Gu, Zhenglin; Tretter, Verena; Smith, Katharine R; McAinsh, Kristina; Arancibia-Carcamo, I Lorena; Saenger, Wolfram; Haucke, Volker; Yan, Zhen; Moss, Stephen J

    2008-03-04

    The regulation of the number of gamma2-subunit-containing GABA(A) receptors (GABA(A)Rs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface gamma2-subunit-containing GABA(A)Rs is regulated. Here, we identify a gamma2-subunit-specific Yxxvarphi-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for gamma2-subunit tyrosine phosphorylation. Blocking GABA(A)R-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxxvarphi motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that gamma2-subunit-containing heteromeric GABA(A)Rs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABA(A)R surface levels and synaptic inhibition.

  14. Inhibition of MAP kinase promotes the recruitment of corepressor SMRT by tamoxifen-bound estrogen receptor alpha and potentiates tamoxifen action in MCF-7 cells

    International Nuclear Information System (INIS)

    Hong, Wei; Chen, Linfeng; Li, Juan; Yao, Zhi

    2010-01-01

    Estrogen receptor alpha (ERα), a ligand controlled transcription factor, plays an important role in breast cancer growth and endocrine therapy. Tamoxifen (TAM) antagonizes ERα activity and has been applied in breast cancer treatment. TAM-bound ERα associates with nuclear receptor-corepressors. Mitogen-activated protein kinase (MAPK) has been elucidated to result in cross-talk between growth factor and ERα mediated signaling. We show that activated MAPK represses interaction of TAM-bound ERα with silencing mediator for retinoid and thyroid hormone receptors (SMRT) and inhibits the recruitment of SMRT by ERα to certain estrogen target genes. Blockade of MAPK signaling cascade with MEK inhibitor U0126 promotes the interaction and subsequently inhibits ERα activity via enhanced recruitment of SMRT, leading to reduced expression of ERα target genes. The growth rate of MCF-7 cells was decelerated when treated with both TAM and U0126. Moreover, the growth of MCF-7 cells stably expressing SMRT showed a robust repression in the presence of TAM and U0126. These results suggest that activated MAPK signaling cascade attenuates antagonist-induced recruitment of SMRT to ERα, suggesting corepressor mediates inhibition of ERα transactivation and breast cancer cell growth by antagonist. Taken together, our finding indicates combination of antagonist and MAPK inhibitor could be a helpful approach for breast cancer therapy.

  15. The interplay of CD150 and CD180 receptor pathways contribute to the pathobiology of chronic lymphocytic leukemia B cells by selective inhibition of Akt and MAPK signaling.

    Directory of Open Access Journals (Sweden)

    Inna Gordiienko

    Full Text Available Cell surface expression of CD150 and CD180 receptors in chronic lymphocytic leukemia (CLL associates with mutational IGHV status and favourable prognosis. Here we show a direct correlation between cell surface expression and colocalization of these receptors on CLL B cells. In the absence of CD150 and CD180 on the cell surface both receptors were expressed in the cytoplasm. The CD150 receptor was colocalized with markers of the endoplasmic reticulum, the Golgi apparatus and early endosomes. In contrast, CD180 was detected preferentially in early endosomes. Analysis of CD150 isoforms differential expression revealed that regardless of CD150 cell surface expression the mCD150 isoform with two ITSM signaling motifs was a predominant CD150 isoform in CLL B cells. The majority of CLL cases had significantly elevated expression level of the soluble sCD150, moreover CLL B cells secrete this isoform. CD150 or CD180 crosslinking on CLL B cells alone led to activation of Akt, mTORC1, ERK1/2, p38MAPK and JNK1/2 networks. Both CD150 and CD180 target the translation machinery through mTOR independent as well as mTOR dependent pathways. Moreover, both these receptors transmit pro-survival signals via Akt-mediated inhibition of GSK3β and FOXO1/FOXO3a. Unexpectedly, coligation CD150 and CD180 receptors on CLL B cells led to mutual inhibition of the Akt and MAPK pathways. While CD150 and CD180 coligation resulted in reduced phosphorylation of Akt, ERK1/2, c-Jun, RSK, p70S6K, S6RP, and 4E-BP; it led to complete blocking of mTOR and p38MAPK phosphorylation. At the same time coligation of CD150 and CD40 receptors did not result in Akt and MAPK inhibition. This suggests that combination of signals via CD150 and CD180 leads to blocking of pro-survival pathways that may be a restraining factor for neoplastic CLL B cells propagation in more than 50% of CLL cases where these receptors are coexpressed.

  16. Green tea polyphenol epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor on lipopolysaccharide-stimulated dendritic cells

    International Nuclear Information System (INIS)

    Byun, Eui-Baek; Choi, Han-Gyu; Sung, Nak-Yun; Byun, Eui-Hong

    2012-01-01

    Highlights: ► Expressions of CD80, CD86, and MHC class I/II were inhibited by EGCG via 67LR. ► EGCG-treated DCs inhibited LPS-induced pro-inflammatory cytokines via 67LR. ► EGCG-treated DCs inhibited MAPKs activation and NF-κB p65 translocation via 67LR. ► EGCG elevated the expression of the Tollip protein through 67LR in DCs. -- Abstract: Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to down-regulate inflammatory responses in dendritic cells (DCs); however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor. In this study, we showed the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in DCs. The expressions of CD80, CD86, and MHC class I and II, which are molecules essential for antigen presentation by DCs, were inhibited by EGCG via 67LR. In addition, EGCG-treated DCs inhibited lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, and IL-6) and activation of mitogen-activated protein kinases (MAPKs), e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) p65 translocation through 67LR. Interestingly, we also found that EGCG markedly elevated the expression of the Tollip protein, a negative regulator of TLR signaling, through 67LR. These novel findings provide new insight into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases.

  17. Characterization of cutaneous vascular permeability induced by platelet-activating factor in guinea pigs and rats and its inhibition by a platelet-activating factor receptor antagonist

    International Nuclear Information System (INIS)

    Hwang, S.B.; Li, C.L.; Lam, M.H.; Shen, T.Y.

    1985-01-01

    Mechanisms of platelet-activating factor (PAF)-induced increases of cutaneous vascular permeability in guinea pigs and in rats were further explored. PAF so far is the most potent vasoactive mediator, being more than 1000-fold more potent than histamine and bradykinin in both species. In guinea pigs, there is a time delay of 5 to 10 minutes before PAF action, whereas, in the rat, the increased vasopermeability occurs immediately following the intradermal PAF injection. Relative vasoactive potencies of PAF and several structure-related analogues in both species correlate very well with their relative inhibition of the binding of 3 H-PAF to specific receptor sites on isolated rabbit platelet plasma membranes and their aggregatory abilities of rabbit platelets. Furthermore, the PAF-induced cutaneous vascular permeability is inhibitable by a competitive specific PAF receptor antagonist, kadsurenone, suggesting that binding of PAF to its specific receptor site is the first step to initiate its action of increased cutaneous vascular permeability. Several pure cyclooxygenase inhibitors, including indomethacin, diflunisal, and flurbiprofen, and the dual cyclooxygenase/lipoxygenase inhibitor, BW755C, but not the histamine antagonists, inhibit the PAF-induced vasopermeability in guinea pigs. The inhibition by indomethacin or BW755C can be fully reversed by coinjection intradermally with PAF and prostaglandin E1 but not leukotriene B4. Also, prostaglandin E1 but not leukotriene B4 enhances the guinea pig in vivo response to PAF in this model. However, in rats, none of the cyclooxygenase inhibitors, histamine antagonists, or BW755C inhibit the PAF effect of cutaneous phenomena

  18. Green tea polyphenol epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor on lipopolysaccharide-stimulated dendritic cells

    Energy Technology Data Exchange (ETDEWEB)

    Byun, Eui-Baek [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Choi, Han-Gyu [Department of Microbiology and Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Daejeon 301-747 (Korea, Republic of); Sung, Nak-Yun [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Byun, Eui-Hong, E-mail: ehbyun80@gmail.com [Department of Microbiology and Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Daejeon 301-747 (Korea, Republic of)

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer Expressions of CD80, CD86, and MHC class I/II were inhibited by EGCG via 67LR. Black-Right-Pointing-Pointer EGCG-treated DCs inhibited LPS-induced pro-inflammatory cytokines via 67LR. Black-Right-Pointing-Pointer EGCG-treated DCs inhibited MAPKs activation and NF-{kappa}B p65 translocation via 67LR. Black-Right-Pointing-Pointer EGCG elevated the expression of the Tollip protein through 67LR in DCs. -- Abstract: Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to down-regulate inflammatory responses in dendritic cells (DCs); however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor. In this study, we showed the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in DCs. The expressions of CD80, CD86, and MHC class I and II, which are molecules essential for antigen presentation by DCs, were inhibited by EGCG via 67LR. In addition, EGCG-treated DCs inhibited lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-{alpha}, interleukin [IL]-1{beta}, and IL-6) and activation of mitogen-activated protein kinases (MAPKs), e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and nuclear factor {kappa}B (NF-{kappa}B) p65 translocation through 67LR. Interestingly, we also found that EGCG markedly elevated the expression of the Tollip protein, a negative regulator of TLR signaling, through 67LR. These novel findings provide new insight into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases.

  19. The Cu-Zn superoxide dismutase (SOD1) inhibits ERK phosphorylation by muscarinic receptor modulation in rat pituitary GH3 cells

    International Nuclear Information System (INIS)

    Secondo, Agnese; De Mizio, Mariarosaria; Zirpoli, Laura; Santillo, Mariarosaria; Mondola, Paolo

    2008-01-01

    The Cu-Zn superoxide dismutase (SOD1) belongs to a family of isoenzymes that are able to dismutate the oxygen superoxide in hydrogen peroxide and molecular oxygen. This enzyme is secreted by many cellular lines and it is also released trough a calcium-dependent depolarization mechanism involving SNARE protein SNAP 25. Using rat pituitary GH3 cells that express muscarinic receptors we found that SOD1 inhibits P-ERK1/2 pathway trough an interaction with muscarinic M1 receptor. This effect is strengthened by oxotremorine, a muscarinic M agonist and partially reverted by pyrenzepine, an antagonist of M1 receptor; moreover this effect is independent from increased intracellular calcium concentration induced by SOD1. Finally, P-ERK1/2 inhibition was accompanied by the reduction of GH3 cell proliferation. These data indicate that SOD1 beside the well studied antioxidant properties can be considered as a neuromodulator able to affect mitogen-activated protein kinase in rat pituitary cells trough a M1 muscarinic receptor

  20. Suppression of the epidermal growth factor receptor inhibits epithelial-mesenchymal transition in human pancreatic cancer PANC-1 cells.

    Science.gov (United States)

    Chang, Zhi-Gang; Wei, Jun-Min; Qin, Chang-Fu; Hao, Kun; Tian, Xiao-Dong; Xie, Kun; Xie, Xue-Hai; Yang, Yin-Mo

    2012-05-01

    Aberrant expression of epidermal growth factor receptor (EGFR) has been detected in pancreatic cancer; however, the mechanisms of EGFR in inducing pancreatic cancer development have not been adequately elucidated. The objective of this study was to determine the role of EGFR in mediating epithelial-mesenchymal transition (EMT) in pancreatic cancer cells. Pancreatic cancer cell line PANC-1 was transfected with small interfering RNA of EGFR by use of a lentiviral expression vector to establish an EGFR-knockdown cell line (si-PANC-1). PANC-1 cells transfected with lentiviral vector expressing negative control sequence were used as negative control (NC-PANC-1). Scratch assay and transwell study were used to analyze cell migration and invasion. Real-time PCR and Western blotting were used to detect the expression of EMT markers E-cadherin, N-cadherin, vimentin, and fibronectin and transcription factors snail, slug, twist1, and sip1 in PANC-1, NC-PANC-1, and si-PANC-1 cells. Immunofluorescent staining with these antibodies and confocal microscopy were used to observe their cellular location and morphologic changes. After RNA interference of EGFR, the migration and invasion ability of si-PANC-1 cells decreased significantly. The expression of epithelial phenotype marker E-cadherin increased and the expression of mesenchymal phenotype markers N-cadherin, vimentin, and fibronectin decreased, indicating reversion of EMT. We also observed intracellular translocation of E-cadherin. Expression of transcription factors snail and slug in si-PANC-1 cells decreased significantly. Suppression of EGFR expression can significantly inhibit EMT of pancreatic cancer PANC-1 cells. The mechanism may be related with the down-regulation of the expression of transcription factors snail and slug.

  1. Baicalin inhibits toll-like receptor 2/4 expression and downstream signaling in rat experimental periodontitis.

    Science.gov (United States)

    Sun, Jun-Yi; Li, Dong-Ling; Dong, Yan; Zhu, Chun-Hui; Liu, Jin; Li, Jue-Dan; Zhou, Tao; Gou, Jian-Zhong; Li, Ang; Zang, Wei-Jin

    2016-07-01

    Periodontitis is a severe inflammatory response, leading to characteristic periodontal soft tissue destruction and alveolar bone resorption. Baicalin possesses potent anti-inflammatory activity; however, it is still unclear whether baicalin regulates toll-like receptor (TLR) 2/4 expression and downstream signaling during the process of periodontitis. In this study, the cervical area of the maxillary second molars of rats was ligated and inoculated with Porphyromonas gingivalis (P. gingivalis) for 4weeks to induce periodontitis. Some rats with periodontitis were treated intragastrically with baicalin (50, 100 or 200mg/kg/day) or vehicle for 4weeks. Compared with the sham group, the levels of TLR2, TLR4 and MyD88 expression and the p38 MAPK and NF-κB activation were up-regulated in the experimental periodontitis group (EPG), accompanied by marked alveolar bone loss and severe inflammation. Treatment with 100 or 200mg/kg/day baicalin dramatically reduced the alveolar bone loss, the levels of HMGB1, TNF-α, IL-1β, and MPO expression, and the numbers of inflammatory infiltrates in the gingival tissues. Importantly, treatment with 100 or 200mg/kg/day baicalin mitigated the periodontitis-up-regulated TLR2, TLR4 and MyD88 expression, and the p38 MAPK and NF-κB activation. Hence, the blockage of the TLR2 and TLR4/MyD88/p38 MAPK/NF-κB signaling by baicalin may contribute to its anti-inflammatory effects in rat model of periodontitis. In conclusion, these novel findings indicate that baicalin inhibits the TLR2 and TLR4 expression and the downstream signaling and mitigates inflammatory responses and the alveolar bone loss in rat experimental periodontitis. Therefore, baicalin may be a potential therapeutic agent for treatment of periodontitis. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Mutation of androgen receptor N-terminal phosphorylation site Tyr-267 leads to inhibition of nuclear translocation and DNA binding.

    Directory of Open Access Journals (Sweden)

    Mehmet Karaca

    Full Text Available Reactivation of androgen receptor (AR may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.

  3. Inhibition of the nicotinic acetylcholine receptors by cobra venom α-neurotoxins: is there a perspective in lung cancer treatment?

    Directory of Open Access Journals (Sweden)

    Angela Alama

    Full Text Available Nicotine exerts its oncogenic effects through the binding to nicotinic acetylcholine receptors (nAChRs and the activation of downstream pathways that block apoptosis and promote neo-angiogenesis. The nAChRs of the α7 subtype are present on a wide variety of cancer cells and their inhibition by cobra venom neurotoxins has been proposed in several articles and reviews as a potential innovative lung cancer therapy. However, since part of the published results was recently retracted, we believe that the antitumoral activity of cobra venom neurotoxins needs to be independently re-evaluated.We determined the activity of α-neurotoxins from Naja atra (short-chain neurotoxin, α-cobrotoxin and Naja kaouthia (long-chain neurotoxin, α-cobratoxin in vitro by cytotoxicity measurements in 5 lung cancer cell lines, by colony formation assay with α7nAChRs expressing and non-expressing cell lines and in vivo by assessing tumor growth in an orthotopic Non-Obese Diabetic/Severe Combined Immunodeficient (NOD/SCID mouse model system utilizing different treatment schedules and dosages.No statistically significant reduction in tumor growth was observed in the treatment arms in comparison to the control for both toxins. Paradoxically α-cobrotoxin from Naja atra showed the tendency to enhance tumor growth although, even in this case, the statistical significance was not reached.In conclusion our results show that, in contrast with other reports, the nAChR inhibitors α-cobratoxin from N. kaouthia and α-cobrotoxin from N. atra neither suppressed tumor growth nor prolonged the survival of the treated animals.

  4. Brain catalase activity inhibition as well as opioid receptor antagonism increases ethanol-induced HPA axis activation.

    Science.gov (United States)

    Pastor, Raúl; Sanchis-Segura, Carles; Aragon, Carlos M G

    2004-12-01

    Growing evidence indicates that brain catalase activity is involved in the psychopharmacological actions of ethanol. Recent data suggest that participation of this enzymatic system in some ethanol effects could be mediated by the endogenous opioid system. The present study assessed whether brain catalase has a role in ethanol-induced activation of the HPA axis, a neuroendocrine system modulated by the endogenous opioid neurotransmission. Swiss male mice received an intraperitoneal injection of the catalase inhibitor 3-amino-1,2,4-triazole (AT; 0-1 g/kg), and 0 to 20 hr after this administration, animals received an ethanol (0-4 g/kg; intraperitoneally) challenge. Thirty, 60, or 120 min after ethanol administration, plasma corticosterone levels were determined immunoenzymatically. In addition, we tested the effects of 45 mg/kg of cyanamide (another catalase inhibitor) and 0 to 2 mg/kg of naltrexone (nonselective opioid receptor antagonist) on ethanol-induced enhancement in plasma corticosterone values. The present study revealed that AT boosts ethanol-induced increase in plasma corticosterone levels in a dose- and time-dependent manner. However, it did not affect corticosterone values when measured after administration of saline, cocaine (4 mg/kg, intraperitoneally), or morphine (30 mg/kg, intraperitoneally). The catalase inhibitor cyanamide (45 mg/kg, intraperitoneally) also increased ethanol-related plasma corticosterone levels. These effects of AT and cyanamide on ethanol-induced corticosterone values were observed under treatment conditions that decreased significantly brain catalase activity. Indeed, a significant correlation between effects of catalase manipulations on both variables was found. Finally, we found that the administration of naltrexone enhanced the levels of plasma corticosterone after the administration of saline or ethanol. This study shows that the inhibition of brain catalase increases ethanol-induced plasma corticosterone levels. Results are

  5. Albizia lebbeck suppresses histamine signaling by the inhibition of histamine H1 receptor and histidine decarboxylase gene transcriptions.

    Science.gov (United States)

    Nurul, Islam Mohammed; Mizuguchi, Hiroyuki; Shahriar, Masum; Venkatesh, Pichairajan; Maeyama, Kazutaka; Mukherjee, Pulok K; Hattori, Masashi; Choudhuri, Mohamed Sahabuddin Kabir; Takeda, Noriaki; Fukui, Hiroyuki

    2011-11-01

    Histamine plays major roles in allergic diseases and its action is mediated mainly by histamine H(1) receptor (H1R). We have demonstrated that histamine signaling-related H1R and histidine decarboxylase (HDC) genes are allergic diseases sensitive genes and their expression level affects severity of the allergic symptoms. Therefore, compounds that suppress histamine signaling should be promising candidates as anti-allergic drugs. Here, we investigated the effect of the extract from the bark of Albizia lebbeck (AL), one of the ingredients of Ayruvedic medicines, on H1R and HDC gene expression using toluene-2,4-diisocyanate (TDI) sensitized allergy model rats and HeLa cells expressing endogenous H1R. Administration of the AL extract significantly decreased the numbers of sneezing and nasal rubbing. Pretreatment with the AL extract suppressed TDI-induced H1R and HDC mRNA elevations as well as [(3)H]mepyramine binding, HDC activity, and histamine content in the nasal mucosa. AL extract also suppressed TDI-induced up-regulation of IL-4, IL-5, and IL-13 mRNA. In HeLa cells, AL extract suppressed phorbol-12-myristate-13-acetate- or histamine-induced up-regulation of H1R mRNA. Our data suggest that AL alleviated nasal symptoms by inhibiting histamine signaling in TDI-sensitized rats through suppression of H1R and HDC gene transcriptions. Suppression of Th2-cytokine signaling by AL also suggests that it could affect the histamine-cytokine network. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Aminoflavone, a ligand of the Aryl Hydrocarbon Receptor (AhR), inhibits HIF-1α expression in an AhR-independent fashion

    Science.gov (United States)

    Terzuoli, Erika; Puppo, Maura; Rapisarda, Annamaria; Uranchimeg, Badarch; Cao, Liang; Burger, Angelika M.; Ziche, Marina; Melillo, Giovanni

    2010-01-01

    Aminoflavone (AF), the active component of a novel anticancer agent (AFP464) in phase I clinical trials, is a ligand of the aryl hydrocarbon receptor (AhR). AhR dimerizes with HIF-1β/ARNT, which is shared with HIF-1α, a transcription factor critical for the response of cells to oxygen deprivation. To address whether pharmacological activation of the AhR pathway might be a potential mechanism for inhibition of HIF-1, we tested the effects of AF on HIF-1 expression. AF inhibited HIF-1α transcriptional activity and protein accumulation in MCF-7 cells. However, inhibition of HIF-1α by AF was independent from a functional AhR pathway. Indeed, AF inhibited HIF-1α expression in AhR100 cells, in which the AhR pathway is functionally impaired, yet did not induce cytotoxicity, providing evidence that these effects are mediated by distinct signaling pathways. Moreover, AF was inactive in MDA-MB-231 cells, yet inhibited HIF-1α in MDA-MB-231 cells transfected with the SULT1A1 gene. AF inhibited HIF-1α mRNA expression by approximately 50%. Notably, actinomycin-D completely abrogated the ability of AF to down-regulate HIF-1α mRNA, indicating that active transcription was required for the inhibition of HIF-1α expression. Finally, AF inhibited HIF-1α protein accumulation and the expression of HIF-1-target genes in MCF-7 xenografts. These results demonstrate that AF inhibits HIF-1α in an AhR-independent fashion and they unveil additional activities of AF that may be relevant for its further clinical development. PMID:20736373

  7. Natriuretic peptide receptor A inhibition suppresses gastric cancer development through reactive oxygen species-mediated G2/M cell cycle arrest and cell death.

    Science.gov (United States)

    Li, Zheng; Wang, Ji-Wei; Wang, Wei-Zhi; Zhi, Xiao-Fei; Zhang, Qun; Li, Bo-Wen; Wang, Lin-Jun; Xie, Kun-Ling; Tao, Jin-Qiu; Tang, Jie; Wei, Song; Zhu, Yi; Xu, Hao; Zhang, Dian-Cai; Yang, Li; Xu, Ze-Kuan

    2016-10-01

    Natriuretic peptide receptor A (NPRA), the major receptor for atrial natriuretic peptide (ANP), has been implicated in tumorigenesis; however, the role of ANP-NPRA signaling in the development of gastric cancer remains unclear. Immunohistochemical analyses indicated that NPRA expression was positively associated with gastric tumor size and cancer stage. NPRA inhibition by shRNA induced G2/M cell cycle arrest, cell death, and autophagy in gastric cancer cells, due to accumulation of reactive oxygen species (ROS). Either genetic or pharmacologic inhibition of autophagy led to caspase-dependent cell death. Therefore, autophagy induced by NPRA silencing may represent a cytoprotective mechanism. ROS accumulation activated c-Jun N-terminal kinase (JNK) and AMP-activated protein kinase (AMPK). ROS-mediated activation of JNK inhibited cell proliferation by disturbing cell cycle and decreased cell viability. In addition, AMPK activation promoted autophagy in NPRA-downregulated cancer cells. Overall, our results indicate that the inhibition of NPRA suppresses gastric cancer development and targeting NPRA may represent a promising strategy for the treatment of gastric cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. 17-beta estradiol inhibits oxidative stress-induced accumulation of AIF into nucleolus and PARP1-dependent cell death via estrogen receptor alpha.

    Science.gov (United States)

    Batnasan, Enkhzaya; Wang, Ruoxi; Wen, Jitao; Ke, Yueshuang; Li, Xiaoxue; Bohio, Ameer Ali; Zeng, Xianlu; Huo, Hongliang; Han, Liping; Boldogh, Istvan; Ba, Xueqing

    2015-01-05

    Oxidative stress-induced DNA damage results in over-activation of poly(ADP-ribose) polymerase 1 (PARP1), leading to parthanatos, a newly discovered cell elimination pathway. Inhibition of PARP1-dependent cell death has shown to improve the outcome of diseases, including stroke, heart ischemia, and neurodegenerative diseases. In the present study we aimed to detect whether estrogen plays a protective role in inhibiting parthanatos. We utilized human mammary adenocarcinoma cells (MCF7) that abundantly express the estrogen receptor alpha and beta (ERα and ERβ). Parthanatos was induced by challenging the cells with hydrogen peroxide (H2O2). Microscopic imaging and molecular biological techniques, such as Western blot analysis and RNA interference, were performed. The results showed 17β estradiol (E2) protected MCF7 cells from PARP1-dependent cell death by decreasing protein PARylation, and AIF translocation into nuclei/nucleoli. Down-regulation of ERα expression by siRNA before E2 addition resulted in the failure of the E2-mediated inhibition of H2O2-induced protein PARylation and AIF nucleolar translocation. Together these data suggest that estrogen via its alpha-type receptor inhibits oxidative stress-induced, PARP1-dependent cell death. The present study provided us insight into how to apply hormone therapy in intervention of parthanatos-implicated ischemic and degenerative diseases. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  9. Albendazole inhibits endothelial cell migration, tube formation, vasopermeability, VEGF receptor-2 expression and suppresses retinal neovascularization in ROP model of angiogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Pourgholami, Mohammad H., E-mail: mh.pourgholami@unsw.edu.au [University of New South Wales, Department of Surgery, St George Hospital (SESIAHS), Sydney (Australia); Khachigian, Levon M.; Fahmy, Roger G. [Centre for Vascular Research, The University of New South Wales, Department of Haematology, The Prince of Wales Hospital, Sydney (Australia); Badar, Samina; Wang, Lisa; Chu, Stephanie Wai Ling; Morris, David Lawson [University of New South Wales, Department of Surgery, St George Hospital (SESIAHS), Sydney (Australia)

    2010-07-09

    The angiogenic process begins with the cell proliferation and migration into the primary vascular network, and leads to vascularization of previously avascular tissues and organs as well to growth and remodeling of the initially homogeneous capillary plexus to form a new microcirculation. Additionally, an increase in microvascular permeability is a crucial step in angiogenesis. Vascular endothelial growth factor (VEGF) plays a central role in angiogenesis. We have previously reported that albendazole suppresses VEGF levels and inhibits malignant ascites formation, suggesting a possible effect on angiogenesis. This study was therefore designed to investigate the antiangiogenic effect of albendazole in non-cancerous models of angiogenesis. In vitro, treatment of human umbilical vein endothelial cells (HUVECs) with albendazole led to inhibition of tube formation, migration, permeability and down-regulation of the VEGF type 2 receptor (VEGFR-2). In vivo albendazole profoundly inhibited hyperoxia-induced retinal angiogenesis in mice. These results provide new insights into the antiangiogenic effects of albendazole.

  10. Albendazole inhibits endothelial cell migration, tube formation, vasopermeability, VEGF receptor-2 expression and suppresses retinal neovascularization in ROP model of angiogenesis

    International Nuclear Information System (INIS)

    Pourgholami, Mohammad H.; Khachigian, Levon M.; Fahmy, Roger G.; Badar, Samina; Wang, Lisa; Chu, Stephanie Wai Ling; Morris, David Lawson

    2010-01-01

    The angiogenic process begins with the cell proliferation and migration into the primary vascular network, and leads to vascularization of previously avascular tissues and organs as well to growth and remodeling of the initially homogeneous capillary plexus to form a new microcirculation. Additionally, an increase in microvascular permeability is a crucial step in angiogenesis. Vascular endothelial growth factor (VEGF) plays a central role in angiogenesis. We have previously reported that albendazole suppresses VEGF levels and inhibits malignant ascites formation, suggesting a possible effect on angiogenesis. This study was therefore designed to investigate the antiangiogenic effect of albendazole in non-cancerous models of angiogenesis. In vitro, treatment of human umbilical vein endothelial cells (HUVECs) with albendazole led to inhibition of tube formation, migration, permeability and down-regulation of the VEGF type 2 receptor (VEGFR-2). In vivo albendazole profoundly inhibited hyperoxia-induced retinal angiogenesis in mice. These results provide new insights into the antiangiogenic effects of albendazole.

  11. Encompassing receptor flexibility in virtual screening using ensemble docking-based hybrid QSAR: discovery of novel phytochemicals for BACE1 inhibition.

    Science.gov (United States)

    Chakraborty, Sandipan; Ramachandran, Balaji; Basu, Soumalee

    2014-10-01

    Mimicking receptor flexibility during receptor-ligand binding is a challenging task in computational drug design since it is associated with a large increase in the conformational search space. In the present study, we have devised an in silico design strategy incorporating receptor flexibility in virtual screening to identify potential lead compounds as inhibitors for flexible proteins. We have considered BACE1 (β-secretase), a key target protease from a therapeutic perspective for Alzheimer's disease, as the highly flexible receptor. The protein undergoes significant conformational transitions from open to closed form upon ligand binding, which makes it a difficult target for inhibitor design. We have designed a hybrid structure-activity model containing both ligand based descriptors and energetic descriptors obtained from molecular docking based on a dataset of structurally diverse BACE1 inhibitors. An ensemble of receptor conformations have been used in the docking study, further improving the prediction ability of the model. The designed model that shows significant prediction ability judged by several statistical parameters has been used to screen an in house developed 3-D structural library of 731 phytochemicals. 24 highly potent, novel BACE1 inhibitors with predicted activity (Ki) ≤ 50 nM have been identified. Detailed analysis reveals pharmacophoric features of these novel inhibitors required to inhibit BACE1.

  12. Influence of age, body temperature, GABAA receptor inhibition and caffeine on the Hering-Breuer inflation reflex in unanesthetized rat pups.

    Science.gov (United States)

    Arnal, Ashley V; Gore, Julie L; Rudkin, Alison; Bartlett, Donald; Leiter, J C

    2013-03-01

    We measured the duration of apnea induced by sustained end-inspiratory lung inflation (the Hering Breuer Reflex, HBR) in unanesthetized infant rat pups aged 4 days (P4) to P20 at body temperatures of 32°C and 36°C. The expiratory prolongation elicited by the HBR lasted longer in the younger pups and lasted longer at the higher body temperature. Blockade of adenosine receptors by caffeine following injection into the cisterna magna (ICM) significantly blunted the thermal prolongation of the HBR. Blockade of gama-amino-butyric acid A (GABAA) receptors by pre-treatment with ICM bicuculline had no effect on the HBR duration at either body temperature. To test the hypothesis that developmental maturation of GABAergic inhibition of breathing was modifying the response to bicuculline, we pretreated rat pups with systemically administered bumetanide to lower the intracellular chloride concentration, and repeated the bicuculline studies. Bicuculline still did not alter the HBR at either temperature after bumetanide treatment. We administered PSB-36, a selective adenosine A1 receptor antagonist, and this drug treatment did not modify the HBR. We conclude that caffeine blunts the thermal prolongation of the HBR, probably by blocking adenosine A2a receptors. The thermally sensitive adenosinergic prolongation of the HBR in these intact animals does not seem to depend on GABAA receptors. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

    International Nuclear Information System (INIS)

    Murayama, T.; Ui, M.

    1985-01-01

    Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased 45 Ca 2+ uptake into the cell monolayer, and (f) increased 86 Rb + uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca 2+ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca 2+ -mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca 2+ gating

  14. Triton X-100 inhibits agonist-induced currents and suppresses benzodiazepine modulation of GABA(A) receptors in Xenopus oocytes

    DEFF Research Database (Denmark)

    Søgaard, Rikke; Ebert, Bjarke; Klaerke, Dan

    2009-01-01

    Changes in lipid bilayer elastic properties have been proposed to underlie the modulation of voltage-gated Na(+) and L-type Ca(2+) channels and GABA(A) receptors by amphiphiles. The amphiphile Triton X-100 increases the elasticity of lipid bilayers at micromolar concentrations, assessed from its...... by flunitrazepam at alpha(1)beta(3)gamma(2S) receptors. All effects were independent of the presence of a gamma(2S) subunit in the GABA(A) receptor complex. The present study suggests that Triton X-100 may stabilize open and desensitized states of the GABA(A) receptor through changes in lipid bilayer elasticity....

  15. Administration of Menadione, Vitamin K3, Ameliorates Off-Target Effects on Corneal Epithelial Wound Healing Due to Receptor Tyrosine Kinase Inhibition.

    Science.gov (United States)

    Rush, Jamie S; Bingaman, David P; Chaney, Paul G; Wax, Martin B; Ceresa, Brian P

    2016-11-01

    The antiangiogenic receptor tyrosine kinase inhibitor (RTKi), 3-[(4-bromo-2,6-difluorophenyl)methoxy]-5-[[[[4-(1-pyrrolidinyl) butyl] amino] carbonyl]amino]-4-isothiazolecarboxamide hydrochloride, targets VEGFR2 (half maximal inhibitory concentration [IC50] = 11 nM); however, off-target inhibition of epidermal growth factor receptor (EGFR) occurs at higher concentrations. (IC50 = 5.8 μM). This study was designed to determine the effect of topical RTKi treatment on EGF-mediated corneal epithelial wound healing and to develop new strategies to minimize off-target EGFR inhibition. In vitro corneal epithelial wound healing was measured in response to EGF using a transformed human cell line (hTCEpi cells). In vivo corneal wound healing was assessed using a murine model. In these complementary assays, wound healing was measured in the presence of varying RTKi concentrations. Immunoblot analysis was used to examine EGFR and VEGFR2 phosphorylation and the kinetics of EGFR degradation. An Alamar Blue assay measured VEGFR2-mediated cell biology. Receptor tyrosine kinase inhibitor exposure caused dose-dependent inhibition of EGFR-mediated corneal epithelial wound healing in vitro and in vivo. Nanomolar concentrations of menadione, a vitamin K3 analog, when coadministered with the RTKi, slowed EGFR degradation and ameliorated the inhibitory effects on epithelial wound healing both in vitro and in vivo. Menadione did not alter the RTKi's IC50 against VEGFR2 phosphorylation or its inhibition of VEGF-induced retinal endothelial cell proliferation. An antiangiogenic RTKi exhibited off-target effects on the corneal epithelium that can be minimized by menadione without deleteriously affecting its on-target VEGFR2 blockade. These data indicate that menadione has potential as a topical supplement for individuals suffering from perturbations in corneal epithelial homeostasis, especially as an untoward side effect of kinase inhibitors.

  16. Aromatic hydrocarbon receptor inhibits lysophosphatidic acid-induced vascular endothelial growth factor-A expression in PC-3 prostate cancer cells

    International Nuclear Information System (INIS)

    Wu, Pei-Yi; Lin, Yueh-Chien; Lan, Shun-Yan; Huang, Yuan-Li; Lee, Hsinyu

    2013-01-01

    Highlights: •LPA-induced VEGF-A expression was regulated by HIF-1α and ARNT. •PI3K mediated LPA-induced VEGF-A expression. •AHR signaling inhibited LPA-induced VEGF-A expression in PC-3 cells. -- Abstract: Lysophosphatidic acid (LPA) is a lipid growth factor with multiple biological functions and has been shown to stimulate cancer cell secretion of vascular endothelial growth factor-A (VEGF-A) and trigger angiogenesis. Hypoxia-inducible factor-1 (HIF-1), a heterodimer consisting of HIF-1α and HIF-1β (also known as aromatic hydrocarbon receptor nuclear translocator (ARNT)) subunits, is an important regulator of angiogenesis in prostate cancer (PC) through the enhancement of VEGF-A expression. In this study, we first confirmed the ability of LPA to induce VEGF-A expression in PC-3 cells and then validated that LPA-induced VEGF-A expression was regulated by HIF-1α and ARNT through phosphatidylinositol 3-kinase activation. Aromatic hydrocarbon receptor (AHR), a receptor for dioxin-like compounds, functions as a transcription factor through dimerization with ARNT and was found to inhibit prostate carcinogenesis and vanadate-induced VEGF-A production. Since ARNT is a common dimerization partner of AHR and HIF-1α, we hypothesized that AHR might suppress LPA-induced VEGF-A expression in PC-3 cells by competing with HIF-1α for ARNT. Here we demonstrated that overexpression and ligand activation of AHR inhibited HIF-1-mediated VEGF-A induction by LPA treatment of PC-3 cells. In conclusion, our results suggested that AHR activation may inhibit LPA-induced VEGF-A expression in PC-3 cells by attenuating HIF-1α signaling, and subsequently, suppressing angiogenesis and metastasis of PC. These results suggested that AHR presents a potential therapeutic target for the prevention of PC metastasis

  17. Inhibition of the prostaglandin EP2 receptor is neuroprotective and accelerates functional recovery in a rat model of organophosphorus induced status epilepticus

    Science.gov (United States)

    Rojas, Asheebo; Ganesh, Thota; Lelutiu, Nadia; Gueorguieva, Paoula; Dingledine, Raymond

    2015-01-01

    Exposure to high levels of organophosphorus compounds (OP) can induce status epilepticus (SE) in humans and rodents via acute cholinergic toxicity, leading to neurodegeneration and brain inflammation. Currently there is no treatment to combat the neuropathologies associated with OP exposure. We recently demonstrated that inhibition of the EP2 receptor for PGE2 reduces neuronal injury in mice following pilocarpine-induced SE. Here, we investigated the therapeutic effects of an EP2 inhibitor (TG6-10-1) in a rat model of SE using diisopropyl fluorophosphate (DFP). We tested the hypothesis that EP2 receptor inhibition initiated well after the onset of DFP-induced SE reduces the associated neuropathologies. Adult male Sprague-Dawley rats were injected with pyridostigmine bromide (0.1 mg/kg, sc) and atropine methylbromide (20 mg/kg, sc) followed by DFP (9.5 mg/kg, ip) to induce SE. DFP administration resulted in prolonged upregulation of COX-2. The rats were administered TG6-10-1 or vehicle (ip) at various time points relative to DFP exposure. Treatment with TG6-10-1 or vehicle did not alter the observed behavioral seizures, however six doses of TG6-10-1 starting 80-150 min after the onset of DFP-induced SE significantly reduced neurodegeneration in the hippocampus, blunted the inflammatory cytokine burst, reduced microglial activation and decreased weight loss in the days after status epilepticus. By contrast, astrogliosis was unaffected by EP2 inhibition 4 d after DFP. Transient treatments with the EP2 antagonist 1 h before DFP, or beginning 4 h after DFP, were ineffective. Delayed mortality, which was low (10%) after DFP, was unaffected by TG6-10-1. Thus, selective inhibition of the EP2 receptor within a time window that coincides with the induction of cyclooxygenase-2 by DFP is neuroprotective and accelerates functional recovery of rats. PMID:25656476

  18. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti

    2006-01-01

    Full Text Available Se trata de un estudio etnográfico, que tuvo lo objetivo de interpretar el sistema de conocimiento y del significado atribuidos a la sangre referente a la transfusión sanguínea por los donadores y receptores de un banco de sangre. Para la colecta de las informaciones se observaron los participantes y la entrevista etnográfica se realizó el análisis de dominio, taxonómicos y temáticos. Los dominios culturales fueron: la sangre es vida: fuente de vida y alimento valioso; creencias religiosas: fuentes simbólicas de apoyos; donación sanguínea: un gesto colaborador que exige cuidarse, gratifica y trae felicidad; donación sanguínea: fuente simbólica de inseguridad; estar enfermo es una condición para realizar transfusión sanguínea; transfusión sanguínea: esperanza de vida; Creencias populares: transfusión sanguínea como riesgo para la salud; donadores de sangre: personas benditas; donar y recibir sangre: como significado de felicidad. Temática: “líquido precioso que origina, sostiene, modifica la vida, provoca miedo e inseguridad”.

  19. Changes in angiotensin AT1 receptor mRNA levels in the rat brain after immobilization stress and inhibition of central nitric oxide synthase.

    Science.gov (United States)

    Kiss, A; Jurkovicova, D; Jezova, D; Krizanova, O

    2001-06-01

    To study functional interactions between angiotensin II AT1 receptors and nitric oxide (NO) activity in different brain areas in rats exposed to immobilization stress. Central inhibition of nitric oxide synthase (NOS) was provided by intracerebroventricular (i.c.v.) administration of (N-omega-nitro-L-arginine-methylester) L-NAME and analysis of AT1 receptor mRNA was performed using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. The immobilization in prone position lasted 2 hrs and the rats were sacrificed 24 hr later. The hypothalamus, hippocampus, thalamus, and cortex were isolated from fresh brains. In the cortex, gene expression of AT1 receptors was unaffected either by L-NAME treatment, or by a single exposure to immobilization stress for 2 hours followed by 24 hours of rest. In the hippocampus, the repeated treatment with L-NAME increased mRNA levels of AT1 receptors approximately 9-times compared to those in the control (untreated) group. Immobilization also increased AT1 receptor mRNA levels in the hippocampus which was similar to that induced by the L-NAME. The increase of AT1 receptor mRNA levels in the hippocampus of immobilized rats was not further altered when the animals were pretreated with L-NAME. In control rats, exposure to immobilization resulted in a significant rise in mRNA levels coding for AT1 receptors in the hypothalamus, but not in the thalamus. L-NAME treatment showed a tendency of increase in AT1 receptor mRNA levels in the hypothalamus. Moreover, when animals treated with L-NAME were subjected to immobilization, a further increase in AT1 receptor mRNA levels was observed in the hypothalamus in comparison with corresponding controls. The present data indicate that a single immobilization stress results in increased gene expression of AT1 receptors in the hypothalamus and hippocampus. The rise in AT1 mRNA levels in the same brain structures after repeated treatment with L-NAME allow to suggest an

  20. Retinoic acid morpholine amide (RAMA) inhibits expression of Fas ligand through EP1 receptor in colon cancer cells.

    Science.gov (United States)

    Chen, Shao-Xuan; Du, Shi-Yu; Wang, Yun-Ting; Zhao, Hong-Chuan; Zhang, Yan-Li; Yao, Li

    2016-01-01

    Among the members of tumour necrosis factor family Fas ligand on binding to its receptor strongly induces apoptosis of tumour-infiltrating lymphocytes (TIL). Thus, FasL acts as an inhibitor of anti-tumour immune response. The present study demonstrates that retinoic acid morpholine amide (RAMA) significantly suppresses FasL expression in colon cancer cells in a dose- and time-dependent manner. The suppression of FasL mRNA and proteins was significant at a concentration of 30 μM after 48 h in CLT85 and HT26 colon cancer cells. There was around 2.6- and 3.2-fold decrease in FasL mRNA after incubation with 30 μM of RAMA in CLT85 cells and HT26 cells, respectively. The results from Western blot showed a decrease in FasL mRNA and protein expression in both CLT85 and HT26 cells after suppression of cyclooxygenase (COX)-2 and COX-1 by RNAi. However, when COX-2-specific silencer RNA (siCOX-2)- and siCOX-1-treated CLT85 and HT26 cells were exposed to RAMA, inhibition of FasL expression was further suppressed. The siCOX-2-treated CLT85 and HT26 cells on exposure to RAMA showed ∼87 and ∼54 % reduction in FasL mRNA, respectively. Co-culture of Jurkat T cells with RAMA-treated HT26 and CLT85 cells decreased the viability of Jurkat T cells by only 2 and 4.3 %, respectively, compared to 19.5 and 37.3 % in control HT26 and CLT85 cells. The results from real-time reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting showed that suppression of EP1 prevented RAMA-induced FasL suppression in CLT85 cells at both the mRNA and protein levels. Thus, RAMA can be a potent therapeutic agent for the treatment of colon tumours.

  1. Non-Dioxin-Like Polychlorinated Biphenyls Inhibit G-Protein Coupled Receptor-Mediated Ca2+ Signaling by Blocking Store-Operated Ca2+ Entry.

    Directory of Open Access Journals (Sweden)

    Se-Young Choi

    Full Text Available Polychlorinated biphenyls (PCBs are ubiquitous pollutants which accumulate in the food chain. Recently, several molecular mechanisms by which non-dioxin-like (NDL PCBs mediate neurodevelopmental and neurobehavioral toxicity have been elucidated. However, although the G-protein coupled receptor (GPCR is a significant target for neurobehavioral disturbance, our understanding of the effects of PCBs on GPCR signaling remains unclear. In this study, we investigated the effects of NDL-PCBs on GPCR-mediated Ca2+ signaling in PC12 cells. We found that ortho-substituted 2,2',6-trichlorinated biphenyl (PCB19 caused a rapid decline in the Ca2+ signaling of bradykinin, a typical Gq- and phospholipase Cβ-coupled GPCR, without any effect on its inositol 1,4,5-trisphosphate production. PCB19 reduced thapsigargin-induced sustained cytosolic Ca2+ levels, suggesting that PCB19 inhibits SOCE. The abilities of other NDL-PCBs to inhibit store-operated Ca2+ entry (SOCE were also examined and found to be of similar potencies to that of PCB19. PCB19 also showed a manner equivalent to that of known SOCE inhibitors. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current and thapsigargin-induced Mn2+ influx. These results imply that one of the molecular mechanism by which NDL-PCBs cause neurobehavioral disturbances involves NDL-PCB-mediated inhibition of SOCE, thereby interfering with GPCR-mediated Ca2+ signaling.

  2. Interleukin-6 infusion during human endotoxaemia inhibits in vitro release of the urokinase receptor from peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Ostrowski, S R; Plomgaard, P; Fischer, C P

    2005-01-01

    Leucocyte expression of the urokinase receptor [urokinase-type plasminogen activator receptor (uPAR)] is regulated by inflammatory mediators. This study investigated the in vivo effect of endotoxin, interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha on uPAR-release in vivo and in vitro in ...

  3. Local anesthetic inhibition of G protein-coupled receptor signaling by interference with Galpha(q) protein function

    NARCIS (Netherlands)

    Hollmann, M. W.; Wieczorek, K. S.; Berger, A.; Durieux, M. E.

    2001-01-01

    Although local anesthetics are considered primarily Na(+) channel blockers, previous studies suggest a common intracellular site of action on different G protein-coupled receptors. In the present study, we characterized this site for the LPA, m1 muscarinic, and trypsin receptor. Xenopus laevis

  4. An allosteric enhancer of M4muscarinic acetylcholine receptor function inhibits behavioral and neurochemical effects of cocaine

    DEFF Research Database (Denmark)

    Dencker, Ditte; Weikop, Pia; Sørensen, Gunnar

    2012-01-01

    The mesostriatal dopamine system plays a key role in mediating the reinforcing effects of psychostimulant drugs like cocaine. The muscarinic M4 acetylcholine receptor subtype is centrally involved in the regulation of dopamine release in striatal areas. Consequently, striatal M4 receptors could...

  5. Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition through Activation of PPAR-alpha Nuclear Receptors

    Science.gov (United States)

    Mazzola, Carmen; Medalie, Julie; Scherma, Maria; Panlilio, Leigh V.; Solinas, Marcello; Tanda, Gianluigi; Drago, Filippo; Cadet, Jean Lud; Goldberg, Steven R.; Yasar, Sevil

    2009-01-01

    Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB[subscript 1]-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain.…

  6. G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits cAMP production.

    Science.gov (United States)

    Broselid, Stefan; Berg, Kelly A; Chavera, Teresa A; Kahn, Robin; Clarke, William P; Olde, Björn; Leeb-Lundberg, L M Fredrik

    2014-08-08

    GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. The Protective Effects of Κ-Opioid Receptor Stimulation in Hypoxic Pulmonary Hypertension Involve Inhibition of Autophagy Through the AMPK-MTOR Pathway

    Directory of Open Access Journals (Sweden)

    Yaguang Zhou

    2017-12-01

    Full Text Available Background/Aims: In a previous study, we showed that κ-opioid receptor stimulation with the selective agonist U50,488H ameliorated hypoxic pulmonary hypertension (HPH. However, the roles that pulmonary arterial smooth muscle cell (PASMC proliferation, apoptosis, and autophagy play in κ-opioid receptor-mediated protection against HPH are still unknown. The goal of the present study was to investigate the role of autophagy in U50,488H-induced HPH protection and the underlying mechanisms. Methods: Rats were exposed to 10% oxygen for three weeks to induce HPH. After hypoxia, the mean pulmonary arterial pressure (mPAP and the right ventricular pressure (RVP were measured. Cell viability was monitored using the Cell Counting Kit-8 (CCK-8 assay. Cell apoptosis was detected by flow cytometry and Western blot. Autophagy was assessed by means of the mRFP-GFP-LC3 adenovirus transfection assay and by Western blot. Results: Inhibition of autophagy by the administration of chloroquine prevented the development of HPH in the rat model, as evidenced by significantly reduced mPAP and RVP, as well as decreased autophagy. U50,488H mimicked the effects of chloroquine, and the effects of U50,488H were blocked by nor-BNI, a selective κ-opioid receptor antagonist. In vitro experiments showed that the inhibition of autophagy by chloroquine was associated with decreased proliferation and increased apoptosis of PASMCs. Under hypoxia, U50,488H also significantly inhibited autophagy, reduced proliferation and increased apoptosis of PASMCs. These effects of U50,488H were blocked by nor-BNI. Moreover, exposure to hypoxic conditions significantly increased AMPK phosphorylation and reduced mTOR phosphorylation, and these effects were abrogated by U50,488H. The effects of U50,488H on PASMC autophagy were inhibited by AICAR, a selective AMPK agonist, or by rapamycin, a selective mTOR inhibitor. Conclusion: Our data provide evidence for the first time that κ-opioid receptor

  8. Nitric oxide inhibits the bradykinin B2 receptor-mediated adrenomedullary catecholamine release but has no effect on adrenal blood flow response in vivo.

    Science.gov (United States)

    Bouallegue, Ali; Yamaguchi, Nobuharu

    2005-06-01

    The role of nitric oxide (NO) in bradykinin (BK)-induced adrenal catecholamine secretion still remains obscure. The present study was to investigate whether an inhibition of NO synthase with N(omega)-nitro-L-arginine methyl ester (L-NAME) would modulate BK-induced adrenal catecholamine secretion (ACS) and adrenal vasodilating response (AVR) in anesthetized dogs. Plasma catecholamine concentrations were determined with an HPLC coupled with an electrochemical detector. All drugs were locally administered to the left adrenal gland via intra-arterial infusion. BK dose-dependently increased both ACS and AVR. Hoe-140, a selective B(2) antagonist, significantly blocked the BK-induced increases in both ACS and AVR. In the presence of L-NAME, the BK-induced ACS was significantly enhanced, while the simultaneous AVR remained unaffected. These results suggest that the both BK-induced ACS and AVR are primarily mediated by B(2) receptors in the canine adrenal gland. Our results also suggest that the enhanced ACS in response to BK in the presence of L-NAME may have resulted from a specific inhibition of NO formation in the adrenal gland. It is concluded that the BK-induced NO may play an inhibitory role in the B(2)-receptor-mediated mechanisms regulating ACS, while it may not be implicated in the B(2)-receptor-mediated AVR under in vivo conditions.

  9. Epac is required for exogenous and endogenous stimulation of adenosine A2B receptor for inhibition of angiotensin II-induced collagen synthesis and myofibroblast differentiation.

    Science.gov (United States)

    Phosri, Sarawuth; Bunrukchai, Kwanchai; Parichatikanond, Warisara; Sato, Vilasinee H; Mangmool, Supachoke

    2018-01-10

    Angiotensin II (Ang II) plays an important role on the pathogenesis of cardiac fibrosis. Prolong and overstimulation of angiotensin II type 1 receptor with Ang II-induced collagen synthesis and myofibroblast differentiation in cardiac fibroblasts, leading to cardiac fibrosis. Although adenosine and its analogues are known to have cardioprotective effects, the mechanistic by which adenosine A 2 receptors (A 2 Rs) inhibit Ang II-induced cardiac fibrosis is not clearly understood. In the present study, we examined the effects of exogenous adenosine and endogenous adenosine on Ang II-induced collagen and myofibroblast differentiation determined by α-smooth muscle action (α-SMA) overexpression and their underlying signal transduction. Elevation of endogenous adenosine levels resulted in the inhibition of Ang II-induced collagen type I and III and α-SMA synthesis in cardiac fibroblasts. Moreover, treatment with exogenous adenosine which selectively stimulated A 2 Rs also suppressed Ang II-induced collagen synthesis and α-SMA production. These antifibrotic effects of both endogenous and exogenous adenosines are mediated through the A 2B receptor (A 2B R) subtype. Stimulation of A 2B R exhibited antifibrotic effects via the cAMP-dependent and Epac-dependent pathways. Our results provide new mechanistic insights regarding the role for cAMP and Epac on A 2B R-mediated antifibrotic effects. Thus, A 2B R is one of the potential therapeutic targets against cardiac fibrosis.

  10. Epidermal growth factor receptor inhibition reduces angiogenesis via hypoxia-inducible factor-1α and Notch1 in head neck squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Wei-Ming Wang

    Full Text Available Angiogenesis, a marker of cancer development, affects response to radiotherapy sensibility. This preclinical study aims to understand the receptor tyrosine kinase-mediated angiogenesis in head neck squamous cell carcinoma (HNSCC. The receptor tyrosine kinase activity in a transgenic mouse model of HNSCC was assessed. The anti-tumorigenetic and anti-angiogenetic effects of cetuximab-induced epidermal growth factor receptor (EGFR inhibition were investigated in xenograft and transgenic mouse models of HNSCC. The signaling transduction of Notch1 and hypoxia-inducible factor-1α (HIF-1α was also analyzed. EGFR was overexpressed and activated in the Tgfbr1/Pten deletion (2cKO mouse model of HNSCC. Cetuximab significantly delayed tumor onset by reducing tumor angiogenesis. This drug exerted similar effects on heterotopic xenograft tumors. In the human HNSCC tissue array, increased EGFR expression correlated with increased HIF-1α and micro vessel density. Cetuximab inhibited tumor-induced angiogenesis in vitro and in vivo by significantly downregulating HIF-1α and Notch1. EGFR is involved in the tumor angiogenesis of HNSCC via the HIF-1α and Notch1 pathways. Therefore, targeting EGFR by suppressing hypoxia- and Notch-induced angiogenesis may benefit HNSCC therapy.

  11. Allopregnanolone suppresses diabetes-induced neuropathic pain and motor deficit through inhibition of GABAA receptor down-regulation in the spinal cord of diabetic rats

    Directory of Open Access Journals (Sweden)

    Samira Afrazi

    2014-05-01

    Full Text Available Objective(s:Painful diabetic neuropathy is associated with hyperexcitability and hyperactivity of spinal cord neurons. However, its underlying pathophysiological mechanisms have not been fully clarified. Induction of excitatory/inhibitory neurotransmission imbalance at the spinal cord seems to account for the abnormal neuronal activity in diabetes. Protective properties of neurosteroids have been demonstrated in numerous cellular and animal models of neurodegeneration. Materials and Methods: Here, the protective effects of allopregnanolone, a neurosteroid were investigated in an in vivo model of diabetic neuropathy. The tail-flick test was used to assess the nociceptive threshold. Diabetes was induced by injection of 50 mg/kg (IP streptozotocin. Seven weeks after the induction of diabetes, the dorsal half of the lumbar spinal cord was assayed for the expression of γ2 subunit of GABAA receptor using semiquantitative RT-PCR. Results: The data shows that allopregnanolone (5 and 20 mg/kg markedly ameliorated diabetes-induced thermal hyperalgesia and motor deficit. The weights of diabetic rats that received 5 and 20 mg/kg allopregnanolone did not significantly reduce during the time course of study. Furthermore, this neurosteroid could inhibit GABAA receptor down-regulation induced by diabetes in the rat spinal cord. Conclusion: The data revealed that allopregnanolone has preventive effects against hyperglycemic-induced neuropathic pain and motor deficit which are related to the inhibition of GABAA receptor down-regulation.

  12. Inhibition of neurotensin-stimulated mast cell secretion and carboxypeptidase A activity by the peptide inhibitor of carboxypeptidase A and neurotensin-receptor antagonist SR 48692.

    Science.gov (United States)

    Miller, L A; Cochrane, D E; Feldberg, R S; Carraway, R E

    1998-06-01

    Neurotensin (NT), a peptide found in brain and several peripheral tissues, is a potent stimulus for mast cell secretion and its actions are blocked by the specific NT receptor antagonist, SR 48692. Subsequent to stimulation, NT is rapidly degraded by mast cell carboxypeptidase A (CPA). In the experiments described here, we tested for the involvement of CPA activity in the activation of mast cell secretion by the peptide, NT. Mast cells were isolated from the peritoneal and pleural cavities of rats, purified over metrizamide gradients and incubated at 37 degrees C in Locke solution or Locke containing the appropriate inhibitors. For some experiments, media derived from mast cells stimulated by compound 48/80 were used as a source of mast cell CPA activity. Treatment of mast cells with the highly specific peptide inhibitor of CPA derived from potato (PCI) inhibited histamine release in response to NT and NT8-13 (the biologically active region of NT). This inhibition required some 20 min to develop and was only partially reversed by a 20-min wash period. PCI (10 microM) did not inhibit histamine release in response to NT1-12, bradykinin, compound 48/80, the calcium ionophore, A23187, or anti-IgE serum. PCI also inhibited mast cell CPA activity. SR 48692, a highly selective antagonist of the brain NT receptor and of NT-stimulated mast cell secretion, also inhibited mast cell CPA activity as well as bovine pancreatic CPA activity in a concentration-dependent manner. It is suggested that the mast cell binding site for NT and the active site for CPA may share similar characteristics. The results are discussed in terms of NT mechanism of action on the mast cell.

  13. Anti-cancer effect of bee venom toxin and melittin in ovarian cancer cells through induction of death receptors and inhibition of JAK2/STAT3 pathway

    International Nuclear Information System (INIS)

    Jo, Miran; Park, Mi Hee; Kollipara, Pushpa Saranya; An, Byeong Jun; Song, Ho Sueb; Han, Sang Bae; Kim, Jang Heub; Song, Min Jong; Hong, Jin Tae

    2012-01-01

    We investigated whether bee venom and melittin, a major component of bee venom, inhibit cell growth through enhancement of death receptor expressions in the human ovarian cancer cells, SKOV3 and PA-1. Bee venom (1–5 μg/ml) and melittin (0.5–2 μg/ml) inhibited the growth of SKOV3 and PA-1 ovarian cancer cells by the induction of apoptotic cell death in a dose dependent manner. Consistent with apoptotic cell death, expression of death receptor (DR) 3 and DR6 was increased in both cancer cells, but expression of DR4 was increased only in PA-1 cells. Expression of DR downstream pro-apoptotic proteins including caspase-3, 8, and Bax was concomitantly increased, but the phosphorylation of JAK2 and STAT3 and the expression of Bcl-2 were inhibited by treatment with bee venom and melittin in SKOV3 and PA-1 cells. Expression of cleaved caspase-3 was increased in SKOV3, but cleaved caspase-8 was increased in PA-1 cells. Moreover, deletion of DR3, DR4, and DR6 by small interfering RNA significantly reversed bee venom and melittin-induced cell growth inhibitory effect as well as down regulation of STAT3 by bee venom and melittin in SKOV3 and PA-1 ovarian cancer cell. These results suggest that bee venom and melittin induce apoptotic cell death in ovarian cancer cells through enhancement of DR3, DR4, and DR6 expression and inhibition of STAT3 pathway. -- Highlights: ► Some studies have showed that bee venom and/or melittin have anti-cancer effects. ► We found that bee venom and melittin inhibited cell growth in ovarian cancer cells. ► Bee venom and melittin induce apoptosis in SKOV3 and PA-1.

  14. Metformin inhibits the proliferation of human prostate cancer PC-3 cells via the downregulation of insulin-like growth factor 1 receptor

    International Nuclear Information System (INIS)

    Kato, Haruo; Sekine, Yoshitaka; Furuya, Yosuke; Miyazawa, Yoshiyuki; Koike, Hidekazu; Suzuki, Kazuhiro

    2015-01-01

    Metformin is a biguanide drug that is widely used for the treatment of type 2 diabetes. Recent studies have shown that metformin inhibits cancer cell proliferation and tumor growth both in vitro and in vivo. The anti-tumor mechanisms of metformin include activation of the AMP-activated protein kinase/mTOR pathway and direct inhibition of insulin/insulin-like growth factor (IGF)-mediated cellular proliferation. However, the anti-tumor mechanism in prostate cancer remains unclear. Because activation of the IGF-1 receptor (IGF-1R) is required for prostate cell proliferation, IGF-1R inhibitors may be of therapeutic value. Accordingly, we examined the effects of metformin on IGF-1R signaling in prostate cancer cells. Metformin significantly inhibited PC-3 cell proliferation, migration, and invasion. IGF-1R mRNA expression decreased significantly after 48 h of treatment, and IGF-1R protein expression decreased in a similar manner. IGF-1R knockdown by siRNA transfection led to inhibited proliferation, migration and invasion of PC-3 cells. IGF-1 activated both ERK1/2 and Akt, but these effects were attenuated by metformin treatment. In addition, intraperitoneal treatment with metformin significantly reduced tumor growth and IGF-1R mRNA expression in PC-3 xenografts. Our results suggest that metformin is a potent inhibitor of the IGF-1/IGF-1R system and may be beneficial in prostate cancer treatment. - Highlights: • Metformin inhibited PC-3 cell proliferation, migration, and invasion. • Metformin decreased IGF-1R mRNA and protein expressions in PC-3 cells. • Metformin inhibited IGF-1 induced ERK and Akt phosphorylations in PC-3 cells. • Metformin treatment inhibited PC-3 cell growth and IGF-1R expression in vivo. • Metformin may be a potent inhibitor of the IGF-1/IGF-1R signaling

  15. Metformin inhibits the proliferation of human prostate cancer PC-3 cells via the downregulation of insulin-like growth factor 1 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Haruo, E-mail: hal.kato@gunma-u.ac.jp; Sekine, Yoshitaka; Furuya, Yosuke; Miyazawa, Yoshiyuki; Koike, Hidekazu; Suzuki, Kazuhiro

    2015-05-22

    Metformin is a biguanide drug that is widely used for the treatment of type 2 diabetes. Recent studies have shown that metformin inhibits cancer cell proliferation and tumor growth both in vitro and in vivo. The anti-tumor mechanisms of metformin include activation of the AMP-activated protein kinase/mTOR pathway and direct inhibition of insulin/insulin-like growth factor (IGF)-mediated cellular proliferation. However, the anti-tumor mechanism in prostate cancer remains unclear. Because activation of the IGF-1 receptor (IGF-1R) is required for prostate cell proliferation, IGF-1R inhibitors may be of therapeutic value. Accordingly, we examined the effects of metformin on IGF-1R signaling in prostate cancer cells. Metformin significantly inhibited PC-3 cell proliferation, migration, and invasion. IGF-1R mRNA expression decreased significantly after 48 h of treatment, and IGF-1R protein expression decreased in a similar manner. IGF-1R knockdown by siRNA transfection led to inhibited proliferation, migration and invasion of PC-3 cells. IGF-1 activated both ERK1/2 and Akt, but these effects were attenuated by metformin treatment. In addition, intraperitoneal treatment with metformin significantly reduced tumor growth and IGF-1R mRNA expression in PC-3 xenografts. Our results suggest that metformin is a potent inhibitor of the IGF-1/IGF-1R system and may be beneficial in prostate cancer treatment. - Highlights: • Metformin inhibited PC-3 cell proliferation, migration, and invasion. • Metformin decreased IGF-1R mRNA and protein expressions in PC-3 cells. • Metformin inhibited IGF-1 induced ERK and Akt phosphorylations in PC-3 cells. • Metformin treatment inhibited PC-3 cell growth and IGF-1R expression in vivo. • Metformin may be a potent inhibitor of the IGF-1/IGF-1R signaling.

  16. Dopamine D2-like receptors (DRD2 and DRD4) in chickens: Tissue distribution, functional analysis, and their involvement in dopamine inhibition of pituitary prolactin expression.

    Science.gov (United States)

    Lv, Can; Mo, Chunheng; Liu, Haikun; Wu, Chao; Li, Zhengyang; Li, Juan; Wang, Yajun

    2018-04-20

    Dopamine (DA) D2-like (and D1-like) receptors are suggested to mediate the dopamine actions in the anterior pituitary and/or CNS of birds. However, the information regarding the structure, functionality, and expression of avian D2-like receptors have not been fully characterized. In this study, we cloned two D2-like receptors (cDRD2, cDRD4) from chicken brain using RACE PCR. The cloned cDRD4 is a 378-amino acid receptor, which shows 57% amino acid (a.a.) identity with mouse DRD4. As in mammals, two cDRD2 isoforms, cDRD2L (long isoform, 437 a.a.) and cDRD2S (short isoform, 408 a.a.), which differ in their third intracellular loop, were identified in chickens. Using cell-based luciferase reporter assays or Western blot, we demonstrated that cDRD4, cDRD2L and cDRD2S could be activated by dopamine and quinpirole (a D2-like receptor agonist) dose-dependently, and their activation inhibits cAMP signaling pathway and stimulates MAPK/ERK signaling cascade, indicating that they are functional receptors capable of mediating dopamine actions. Quantitative real-time PCR revealed that cDRD2 and cDRD4 are widely expressed in chicken tissues with abundant expression noted in anterior pituitary, and their expressions are likely controlled by their promoters near exon 1, as demonstrated by dual-luciferase reporter assays in DF-1 cells. In accordance with cDRD2/cDRD4 expression in the pituitary, DA or quinpirole could partially inhibit vasoactive intestinal peptide-induced prolactin expression in cultured chick pituitary cells. Together, our data proves the functionality of DRD2 and DRD4 in birds and aids to uncover the conserved roles of DA/D2-like receptor system in vertebrates, such as its action on the pituitary. Copyright © 2018. Published by Elsevier B.V.

  17. Inhibition of Dengue Virus Replication by a Class of Small-Molecule Compounds That Antagonize Dopamine Receptor D4 and Downstream Mitogen-Activated Protein Kinase Signaling

    Science.gov (United States)

    Smith, Jessica L.; Stein, David A.; Shum, David; Fischer, Matthew A.; Radu, Constantin; Bhinder, Bhavneet; Djaballah, Hakim; Nelson, Jay A.; Früh, Klaus

    2014-01-01

    ABSTRACT Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions that cause significant morbidity and mortality worldwide. To date, no vaccines or antiviral therapeutics have been approved for combating DENV-associated disease. In this paper, we describe a class of tricyclic small-molecule compounds—dihydrodibenzothiepines (DHBTs), identified through high-throughput screening—with potent inhibitory activity against DENV serotype 2. SKI-417616, a highly active representative of this class, displayed activity against all four serotypes of DENV, as well as against a related flavivirus, West Nile virus (WNV), and an alphavirus, Sindbis virus (SINV). This compound was characterized to determine its mechanism of antiviral activity. Investigation of the stage of the viral life cycle affected revealed that an early event in the life cycle is inhibited. Due to the structural similarity of the DHBTs to known antagonists of the dopamine and serotonin receptors, we explored the roles of two of these receptors, serotonin receptor 2A (5HTR2A) and the D4 dopamine receptor (DRD4), in DENV infection. Antagonism of DRD4