RESUMO
The promising therapeutic potential of the NO-donating hybrid aspirin prodrugs (NO-ASA) includes induction of chemopreventive mechanisms and has been reported in almost 100 publications. One example, NCX-4040 (pNO-ASA), is bioactivated by esterase to a quinone methide (QM) electrophile. In cell cultures, pNO-ASA and QM-donating X-ASA prodrugs that cannot release NO rapidly depleted intracellular GSH and caused DNA damage; however, induction of Nrf2 signaling elicited cellular defense mechanisms including upregulation of NAD(P)H:quinone oxidoreductase-1 (NQO1) and glutamate-cysteine ligase (GCL). In HepG2 cells, the "NO-specific" 4,5-diaminofluorescein reporter, DAF-DA, responded to NO-ASA and X-ASA, with QM-induced oxidative stress masquerading as NO. LC-MS/MS analysis demonstrated efficient alkylation of Cys residues of proteins including glutathione-S-transferase-P1 (GST-P1) and Kelch-like ECH-associated protein 1 (Keap1). Evidence was obtained for alkylation of Keap1 Cys residues associated with Nrf2 translocation to the nucleus, nuclear translocation of Nrf2, activation of antioxidant response element (ARE), and upregulation of cytoprotective target genes. At least in cell culture, pNO-ASA acts as a QM donor, bioactivated by cellular esterase activity to release salicylates, NO(3)(-), and an electrophilic QM. Finally, two novel aspirin prodrugs were synthesized, both potent activators of ARE, designed to release only the QM and salicylates on bioactivation. Current interest in electrophilic drugs acting via Nrf2 signaling suggests that QM-donating hybrid drugs can be designed as informative chemical probes in drug discovery.
Assuntos
Aspirina/análogos & derivados , Aspirina/farmacologia , Indolquinonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Pró-Fármacos/farmacologia , Animais , Linhagem Celular Tumoral , Dano ao DNA , Glutationa/metabolismo , Glutationa S-Transferase pi/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch , Camundongos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Óxido Nítrico/metabolismoRESUMO
Cyclodextrin (CD) is a well known drug carrier and excipient for enhancing aqueous solubility. CDs themselves are anticipated to have low membrane permeability because of relatively high hydrophilicity and molecular weight. CD derivatization with 17-beta estradiol (E(2)) was explored extensively using a number of different click chemistries and the cell membrane permeability of synthetic CD-E(2) conjugate was explored by cell reporter assays and confocal fluorescence microscopy. In simile with reported dendrimer-E(2) conjugates, CD-E(2) was found to be a stable, extranuclear receptor selective estrogen that penetrated into the cytoplasm.
Assuntos
Ciclodextrinas/química , Estradiol/química , Estrogênios/química , Permeabilidade da Membrana Celular , Células Cultivadas , Cristalografia por Raios X , Ciclodextrinas/síntese química , Ciclodextrinas/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Estradiol/metabolismo , Estrogênios/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , EstereoisomerismoRESUMO
Conjugation of biotin and fluorophore tags is useful for assaying covalent protein modification. Oxidative bioactivation of selective estrogen receptor modulators (SERMs) yields reactive quinoid electrophiles that covalently modify proteins, and bioactivation is associated with carcinogenic and chemopreventive effects. Identification of the protein targets of electrophilic metabolites is of general importance for xenobiotics. Four methodologies using SERM derivatized biotin/fluorophore tags were compared for purification and quantification: (1) covert oxidatively activated tags (COATags; SERM conjugated to biotin); (2) dansylTags (SERM conjugated to fluorophore); and azidoTags (SERM azide derivatives) in a two-step conjugation to biotin, using either (3) Staudinger ligation or (4) click chemistry. All synthetic derivatives retained the estrogen receptor ligand characteristics of the parent SERMs. Model proteins with bioactivation by tyrosinase in buffer or cell lysates and liver proteins with in situ bioactivation in rat primary hepatocytes were studied by immunoassay and fluorescence. Comparison showed that the azidoTag/Staudinger method was sensitive but nonspecific, the azidoTag/click methodology had low sensitivity, and the dansylTag methodology failed to detect modified proteins in hepatocytes. The COATag methodology was judged superior, detecting 5 ng of modified protein in vitro and identifying protein targets in hepatocytes. In metabolism studies in rat liver microsomes, the azide group was metabolically labile, which was a contributing factor in not selecting the azidoTag methodology in the oxidative environments required for bioactivation. For study of the protein targets of electrophilic metabolites formed by in situ oxidative bioactivation, the COATag is both sensitive and specific and does not appear to suffer from poor cell permeability.
Assuntos
Proteínas/química , Proteínas/metabolismo , Quinonas/metabolismo , Xenobióticos/metabolismo , Animais , Azidas/química , Azidas/metabolismo , Biotinilação , Extratos Celulares/química , Linhagem Celular Tumoral , Hepatócitos/metabolismo , Indicadores e Reagentes/síntese química , Indicadores e Reagentes/metabolismo , Ligantes , Espectrometria de Massas , Oxirredução , Piperidinas/metabolismo , Proteínas/análise , Quinonas/química , Cloridrato de Raloxifeno/metabolismo , Ratos , Receptores de Estrogênio/metabolismo , Moduladores Seletivos de Receptor Estrogênico/metabolismo , Coloração e Rotulagem , Tiofenos/metabolismoRESUMO
Hyperactivation of the calcium-dependent cysteine protease calpain 1 (Cal1) is implicated as a primary or secondary pathological event in a wide range of illnesses and in neurodegenerative states, including Alzheimer's disease (AD). E-64 is an epoxide-containing natural product identified as a potent nonselective, calpain inhibitor, with demonstrated efficacy in animal models of AD. By use of E-64 as a lead, three successive generations of calpain inhibitors were developed using computationally assisted design to increase selectivity for Cal1. First generation analogues were potent inhibitors, effecting covalent modification of recombinant Cal1 catalytic domain (Cal1cat), demonstrated using LC-MS/MS. Refinement yielded second generation inhibitors with improved selectivity. Further library expansion and ligand refinement gave three Cal1 inhibitors, one of which was designed as an activity-based protein profiling probe. These were determined to be irreversible and selective inhibitors by kinetics studies comparing full length Cal1 with the general cysteine protease papain.
Assuntos
Calpaína/antagonistas & inibidores , Compostos de Epóxi/síntese química , Leucina/análogos & derivados , Peptidomiméticos/síntese química , Calpaína/química , Domínio Catalítico , Química Click , Simulação por Computador , Desenho de Fármacos , Compostos de Epóxi/química , Cinética , Leucina/síntese química , Leucina/química , Simulação de Acoplamento Molecular , Papaína/antagonistas & inibidores , Peptidomiméticos/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
SCOPE: Hops contain the phytoestrogen, 8-prenylnaringenin, and the cytoprotective compound, xanthohumol (XH). XH induces the detoxification enzyme, NAD(P)H-quinone oxidoreductase (NQO1) in vitro; however, the tissue distribution of XH and 8-prenylnaringenin and their tissue-specific activity have not been analyzed. METHODS AND RESULTS: An orally administered hop extract and subcutaneously injected XH were administered to Sprague-Dawley rats over 4 days. LC-MS-MS analysis of plasma, liver, and mammary gland revealed that XH accumulated in liver and mammary glands. Compared with the low level in the original extract, 8-prenylnaringenin was enriched in the tissues. Hops and XH-induced NQO1 in the liver, while only hops reduced NQO1 activity in the mammary gland. Mechanistic studies revealed that hops modulated NQO1 through three mechanisms. In liver cells, (i) XH modified Kelch-like ECH-associated protein leading to nuclear factor (erythroid-derived 2)-like 2 (Nrf2) translocation and antioxidant response element (ARE) activation; (ii) hop-mediated ARE induction was partially mediated through phosphorylation of Nrf2 by PKC; (iii) in breast cells, 8-prenylnaringenin reduced NQO1 likely through binding to estrogen receptorα, recruiting Nrf2, and downregulating ARE-regulated genes. CONCLUSION: XH and 8-prenylnaringenin in dietary hops are bioavailable to the target tissues. While hops and XH might be cytoprotective in the liver, 8-prenylnaringenin seems responsible for hop-mediated NQO1 reduction in the mammary gland.
Assuntos
Flavanonas/farmacocinética , Flavonoides/farmacologia , Humulus/química , Inativação Metabólica , Fígado/enzimologia , Glândulas Mamárias Animais/enzimologia , Extratos Vegetais/farmacologia , Propiofenonas/farmacologia , Animais , Elementos de Resposta Antioxidante/efeitos dos fármacos , Elementos de Resposta Antioxidante/genética , Feminino , Flavanonas/sangue , Flavonoides/sangue , Flavonoides/farmacocinética , Glutationa Transferase/metabolismo , Humanos , Fígado/efeitos dos fármacos , Células MCF-7/efeitos dos fármacos , Glândulas Mamárias Animais/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fosforilação/efeitos dos fármacos , Extratos Vegetais/farmacocinética , Propiofenonas/sangue , Propiofenonas/farmacocinética , Proteína Quinase C/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
Estrogen action, via both nuclear and extranuclear estrogen receptors (ERs), induces a variety of cellular signals that are prosurvival or proliferative, whereas nitric oxide (NO) can inhibit apoptosis via caspase S-nitrosylation and via activation of soluble guanylyl cyclase to produce cGMP. The action of 17ß-estradiol (E(2)) at ER is known to elicit NO signaling via activation of NO synthase (NOS) in many tissues. The MCF-10A nontumorigenic, mammary epithelial cell line is genetically stable and insensitive to estrogenic proliferation. In this cell line, estrogens or NOS inhibitors alone had no significant effect, whereas in combination, apoptosis was induced rapidly in the absence of serum; the presence of inducible NOS was confirmed by proteomic analysis. The application of pharmacological agents determined that apoptosis was dependent upon NO/cGMP signaling via cyclic GMP (cGMP)-dependent protein kinase and could be replicated by inhibition of the phosphatidylinositol 3 kinase/serine-threonine kinase pathway prior to addition of E(2). Apoptosis was confirmed by nuclear staining and increased caspase-3 activity in E(2) + NOS inhibitor-treated cells. Apoptosis was partially inhibited by a pure ER antagonist and replicated by agonists selective for extranuclear ER. Cells were rescued from E(2)-induced apoptosis after NOS blockade, by NO-donors and cGMP pathway agonists; preincubation with NO donors was required. The NOS and ER status of breast cancer tissues is significant in etiology, prognosis, and therapy. In this study, apoptosis of preneoplastic mammary epithelial cells was triggered by estrogens via a rapid, extranuclear ER-mediated response, after removal of an antiapoptotic NO/cGMP/cGMP-dependent protein kinase signal.
Assuntos
Apoptose/fisiologia , GMP Cíclico/metabolismo , Células Epiteliais/efeitos dos fármacos , Estradiol/farmacologia , Óxido Nítrico/metabolismo , Receptores de Estrogênio/metabolismo , Apoptose/efeitos dos fármacos , Mama/citologia , Mama/enzimologia , Caspase 3/metabolismo , Linhagem Celular , Meios de Cultura Livres de Soro , Células Epiteliais/enzimologia , Células Epiteliais/fisiologia , Moduladores de Receptor Estrogênico/farmacologia , Feminino , Humanos , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Protein S-nitrosation has been argued to be the most important signaling pathway mediating the bioactivity of NO. This post-translational modification of protein thiols is the result of chemical nitrosation of cysteine residues. The term NO-donors covers very different chemical classes, from clinical therapeutics to probes of routine use in chemical biology; their different chemistry is predicted to result in distinctive biology regulated by protein S-nitrosation. To measure the extent of protein S-nitrosation by NO-donors, a proteomic mass spectrometry method was developed, which quantitates free thiol versus nitrosothiol for each modified cysteine residue, coined d-Switch. This method is adapted from the biotin switch (BST) method, used extensively to identify S-nitrosated proteins in complex biological mixtures; however, BST does not quantitate free thiol. Since glutathione-S-transferase P1-1 (GST-P1) has been proposed to be a biological "NO-carrier", GST-P1 was used as a reporter protein. The 5 different chemical classes of NO-donors compared by d-Switch demonstrated very different profiles of protein S-nitrosation and response to O(2) and cysteine, although all NO-donors were oxidants toward GST-P1. The low limits of detection and the ability to use established MS database searching allowed facile generalization of the d-Switch method. Therefore after incubation of neuronal cell cultures with nitrosothiol, it was possible to quantitate not only S-nitrosation of GST-P1 but also many other proteins, including novel targets such as ubiquitin carboxyl-terminal esterase L1 (UCHL1). Moreover, d-Switch also allowed identification of non-nitrosated proteins and quantitation of degree of nitrosation for individual protein thiols.
Assuntos
Glutationa S-Transferase pi/metabolismo , Doadores de Óxido Nítrico/química , Óxido Nítrico/metabolismo , Proteínas/química , Biotina/análogos & derivados , Biotina/metabolismo , Células Cultivadas , Cisteína/análogos & derivados , Cisteína/química , Cisteína/metabolismo , Humanos , Espectrometria de Massas , Neuroblastoma/metabolismo , Doadores de Óxido Nítrico/metabolismo , Nitrosação , Proteínas/metabolismo , Proteômica , S-Nitrosoglutationa/metabolismo , S-Nitrosotióis/química , S-Nitrosotióis/metabolismo , S-Nitrosotióis/farmacologiaRESUMO
Estrogen exposure is a risk factor for breast cancer, and estrogen oxidative metabolites have been implicated in chemical carcinogenesis. Oxidation of the catechol metabolite of estrone (4-OHE) and the beta-naphthohydroquinone metabolite of equilenin (4-OHEN) gives o-quinones that produce ROS and damage DNA by adduction and oxidation. To differentiate hormonal and chemical carcinogensis pathways in estrogen receptor positive ER(+) cells, catechol or beta-naphthohydroquinone warheads were conjugated to the selective estrogen receptor modulator (SERM) desmethylarzoxifene (DMA). ER binding was retained in the DMA conjugates; both were antiestrogens with submicromolar potency in mammary and endometrial cells. Cytotoxicity, apoptosis, and caspase-3/7 activation were compared in ER(+) and ER(-)MDA-MB-231 cells, and production of ROS was detected using a fluorescent reporter. Comparison was made to DMA, isolated warheads, and a DMA-mustard. Conjugation of warheads to DMA increased cytotoxicity accompanied by induction of apoptosis and activation of caspase-3/7. Activation of caspase-3/7, induction of apoptosis, and cytotoxicity were all increased significantly in ER(+) cells for the DMA conjugates. ROS production was localized in the nucleus for conjugates in ER(+) cells. Observations are compatible with beta-naphthohydroquinone and catechol groups being concentrated in the nucleus by ER binding, where oxidation and ROS production result, concomitant with caspase-dependent apoptosis. The results suggest that DNA damage induced by catechol estrogen metabolites can be amplified in ER(+) cells independent of hormonal activity. The novel conjugation of quinone warheads to an ER-targeting SERM gives ER-dependent, enhanced apoptosis in mammary cancer cells of potential application in cancer therapy.
Assuntos
Neoplasias da Mama/metabolismo , Catecóis/metabolismo , Hidroquinonas/metabolismo , Piperidinas/metabolismo , Pró-Fármacos/metabolismo , Receptores de Estrogênio/metabolismo , Moduladores Seletivos de Receptor Estrogênico/metabolismo , Tiofenos/metabolismo , Apoptose , Caspase 3/metabolismo , Caspase 7/metabolismo , Catecóis/química , Linhagem Celular Tumoral , Dano ao DNA , Feminino , Deleção de Genes , Humanos , Hidroquinonas/química , Piperidinas/química , Pró-Fármacos/química , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Receptores de Estrogênio/genética , Moduladores Seletivos de Receptor Estrogênico/química , Tiofenos/químicaRESUMO
Exposure to estrogens increases the risk of breast and endometrial cancer. It is proposed that the estrogen receptor (ER) may contribute to estrogen carcinogenesis by transduction of the hormonal signal and as a "Trojan horse" concentrating genotoxic estrogen metabolites in the nucleus to complex with DNA, enhancing DNA damage. 4-Hydroxyequilenin (4-OHEN), the major catechol metabolite of equine estrogens present in estrogen replacement formulations, autoxidizes to a redox-cycling quinone that has been shown to cause DNA damage. 4-OHEN was found to be an estrogen of nanomolar potency in cell culture using a luciferase reporter assay and, using a chromatin immunoprecipitation assay, was found to activate ERalpha binding to estrogen-responsive genes in MCF-7 cells. DNA damage was measured in cells by comparing ERalpha(+) versus ERalpha(-) cells and 4-OHEN versus menadione, a reactive oxygen species (ROS)-generating, but non-estrogenic, quinone. 4-OHEN selectively induced DNA damage in ERalpha(+) cells, whereas menadione-induced damage was not dependent on cellular ER status. The rate of 4-OHEN-induced DNA damage was significantly enhanced in ERalpha(+) cells, whereas ER status had no effect on the rate of menadione-induced damage. Imaging of ROS induced by 4-OHEN showed accumulation selective for the nucleus of ERalpha(+) cells within 5 min, whereas in ERalpha(-) or menadione-treated cells, no selectivity was observed. These data support ERalpha acting as a Trojan horse concentrating 4-OHEN in the nucleus to accelerate the rate of ROS generation and thereby amplify DNA damage. The Trojan horse mechanism may be of general importance beyond estrogen genotoxins.
Assuntos
Núcleo Celular/metabolismo , Dano ao DNA/efeitos dos fármacos , Equilenina/análogos & derivados , Receptor alfa de Estrogênio/metabolismo , Estrogênios de Catecol/farmacologia , Mutagênicos/farmacologia , Animais , Linhagem Celular Tumoral , Equilenina/metabolismo , Equilenina/farmacologia , Estrogênios de Catecol/metabolismo , Feminino , Cavalos , Humanos , Mutagênicos/metabolismo , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio , Vitamina K 3/farmacologia , Vitaminas/farmacologia , Xenopus laevisRESUMO
Although group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) has been reported to be phosphorylated at multiple Ser residues, the mechanisms by which phosphorylation at different sites regulates cPLA(2)alpha activities are not fully understood. To explore the possibility that phosphorylation of Ser(727) modulates cellular protein-protein interactions, we measured the effect of Ser(727) mutations on the interaction of cPLA(2)alpha with a reported cPLA(2)alpha-binding protein, p11. In vitro activity assays and membrane binding measurements by surface plasmon resonance analysis showed that a heterotetramer (A2t) of p11 and annexin A2, but not p11 or annexin A2 alone, directly binds cPLA(2)alpha via Ser(727), which keeps the enzyme from binding the membrane and catalyzing the phospholipid hydrolysis. Phosphorylation of Ser(727) disrupts this inhibitory cPLA(2)alpha-A2t interaction, thereby activating cPLA(2)alpha. Subcellular translocation and activity measurements in HEK293 cells cotransfected with cPLA(2)alpha and p11 also showed that p11, in the form of A2t, inhibits cPLA(2)alpha by the same mechanism and that phosphorylation of Ser(727) activates cPLA(2)alpha by interfering with the inhibitory cPLA(2)alpha-A2t interaction. Collectively, these studies provide new insight into the regulatory mechanism of cPLA(2)alpha through Ser(727) phosphorylation.
Assuntos
Fosfolipases A2/metabolismo , Serina/metabolismo , Animais , Linhagem Celular , Vetores Genéticos , Humanos , Ionomicina/farmacologia , Fosfolipases A2/química , Fosforilação , Spodoptera , Ressonância de Plasmônio de Superfície , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The mechanisms by which secretory phospholipase A2 (PLA2) exerts cellular effects are not fully understood. To elucidate these mechanisms, we systematically and quantitatively assessed the activities of human group IIA, V, and X PLA2s on originating and neighboring cells using orthogonal fluorogenic substrates in various mixed cell systems. When HEK293 cells stably expressing each of these PLA2s were mixed with non-transfected HEK293 cells, group V and X PLA2s showed strong transcellular lipolytic activity, whereas group IIA PLA2 exhibited much lower transcellular activity. The transcellular activity of group V PLA2 was highly dependent on the presence of cell surface heparan sulfate proteoglycans of acceptor cells. Activation of RBL-2H3 and DLD-1 cells that express endogenous group V PLA2 led to the secretion of group V PLA2 and its transcellular action on neighboring human neutrophils and eosinophils, respectively. Similarly, activation of human bronchial epithelial cells, BEAS-2B, caused large increases in arachidonic acid and leukotriene C4 release from neighboring human eosinophils. Collectively, these studies show that group V and X PLA2s can act transcellularly on mammalian cells and suggest that group V PLA2 released from neighboring cells may function in triggering the activation of inflammatory cells under physiological conditions.
Assuntos
Eicosanoides/metabolismo , Fosfolipases A/metabolismo , Fosfolipases A/fisiologia , Animais , Ácido Araquidônico/metabolismo , Western Blotting , Encéfalo/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , DNA Complementar/metabolismo , Eosinófilos/metabolismo , Epitélio/metabolismo , Fosfolipases A2 do Grupo II , Proteoglicanas de Heparan Sulfato/metabolismo , Heparina Liase/metabolismo , Humanos , Inflamação , Leucotrieno C4/metabolismo , Microscopia Confocal , Modelos Químicos , Neutrófilos/metabolismo , Fosfolipases A2 , Fosfolipídeos/metabolismo , Isoformas de Proteínas , Ratos , Espectrometria de Fluorescência , Fatores de Tempo , TransfecçãoRESUMO
The mechanisms by which secretory phospholipases A(2) (PLA(2)s) exert cellular effects are not fully understood. Group IIF PLA(2) (gIIFPLA(2)) is a structurally unique secretory PLA(2) with a long C-terminal extension. Homology modeling suggests that the membrane-binding surface of this acidic PLA(2) contains hydrophobic residues clustered near the C-terminal extension. Vesicle leakage and monolayer penetration measurements showed that gIIFPLA(2) had a unique ability to penetrate and disrupt compactly packed monolayers and bilayers whose lipid composition recapitulates that of the outer plasma membrane of mammalian cells. Fluorescence imaging showed that gIIFPLA(2) could also readily enter and deform plasma membrane-mimicking giant unilamellar vesicles. Mutation analysis indicates that hydrophobic residues (Tyr(115), Phe(116), Val(118), and Tyr(119)) near the C-terminal extension are responsible for these activities. When gIIFPLA(2) was exogenously added to HEK293 cells, it initially bound to the plasma membrane and then rapidly entered the cells in an endocytosis-independent manner, but the cell entry did not lead to a significant degree of phospholipid hydrolysis. GIIFPLA(2) mRNA was detected endogenously in human CD4(+) helper T cells after in vitro stimulation and exogenously added gIIFPLA(2) inhibited the proliferation of a T cell line, which was not seen with group IIA PLA(2). Collectively, these data suggest that unique membrane-binding properties of gIIFPLA(2) may confer special functionality on this secretory PLA(2) under certain physiological conditions.