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1.
Redox Biol ; 5: 66-70, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25838067

RESUMO

Lyme borreliosis is transmitted through the bite of a tick that is infected by the bacterial spirochete Borrelia burgdorferi. Clinical manifestation of the disease can lead to heart conditions, neurological disorders, and inflammatory disorders. Oxidative stress has been implicated in the pathogenesis of many human diseases. The aim of this study was to investigate the mechanisms of oxidative stress and intracellular communication in Lyme borreliosis patients. Mitochondrial superoxide and cytosolic ionized calcium was measured in peripheral blood mononuclear cells (PBMCs) of Lyme borreliosis patients and healthy controls. Mitochondrial superoxide levels were significantly higher (p<0.0001) in Lyme borreliosis patients (n=32) as compared to healthy controls (n=30). Significantly low (p<0.0001) levels of cytosolic ionized calcium were also observed in Lyme borreliosis patients (n=11) when compared to healthy controls (n=11). These results indicate that there is an imbalance of reactive oxygen species and cytosolic calcium in Lyme borreliosis patients. The results further suggest that oxidative stress and interrupted intracellular communication may ultimately contribute to a condition of mitochondrial dysfunction in the immune cells of Lyme borreliosis patients.


Assuntos
Doença de Lyme/patologia , Adolescente , Adulto , Idoso , Borrelia burgdorferi/fisiologia , Cálcio/metabolismo , Criança , Citosol/metabolismo , Feminino , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Doença de Lyme/imunologia , Doença de Lyme/metabolismo , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Adulto Jovem
2.
Mol Cancer Ther ; 13(11): 2515-26, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25205655

RESUMO

Endocrine-resistant breast cancer is a major clinical obstacle. The use of 17ß-estradiol (E2) has reemerged as a potential treatment option following exhaustive use of tamoxifen or aromatase inhibitors, although side effects have hindered its clinical usage. Protein kinase C alpha (PKCα) expression was shown to be a predictor of disease outcome for patients receiving endocrine therapy and may predict a positive response to an estrogenic treatment. Here, we have investigated the use of novel benzothiophene selective estrogen mimics (SEM) as an alternative to E2 for the treatment of tamoxifen-resistant breast cancer. Following in vitro characterization of SEMs, a panel of clinically relevant PKCα-expressing, tamoxifen-resistant models were used to investigate the antitumor effects of these compounds. SEM treatment resulted in growth inhibition and apoptosis of tamoxifen-resistant cell lines in vitro. In vivo SEM treatment induced tumor regression of tamoxifen-resistant T47D:A18/PKCα and T47D:A18-TAM1 tumor models. T47D:A18/PKCα tumor regression was accompanied by translocation of estrogen receptor (ER) α to extranuclear sites, possibly defining a mechanism through which these SEMs initiate tumor regression. SEM treatment did not stimulate growth of E2-dependent T47D:A18/neo tumors. In addition, unlike E2 or tamoxifen, treatment with SEMs did not stimulate uterine weight gain. These findings suggest the further development of SEMs as a feasible therapeutic strategy for the treatment of endocrine-resistant breast cancer without the side effects associated with E2.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Tiofenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Estrogênios/química , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Distribuição Aleatória , Receptores de Estrogênio/metabolismo , Tamoxifeno/farmacologia , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Biochemistry ; 52(18): 3171-81, 2013 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-23566219

RESUMO

Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to act by cross-linking cellular biomolecules, their exact mechanisms of action have not been established. In cells, these compounds form a mixture of DNA lesions, including nucleobase monoadducts, interstrand and intrastrand cross-links, and DNA-protein cross-links (DPCs). Interstrand DNA-DNA cross-links block replication and transcription by preventing DNA strand separation, contributing to toxicity and mutagenesis. In contrast, potential contributions of drug-induced DPCs are poorly understood. To gain insight into the biological consequences of DPC formation, we generated DNA-reactive protein reagents and examined their toxicity and mutagenesis in mammalian cells. Recombinant human O(6)-alkylguanine DNA alkyltransferase (AGT) protein or its variants (C145A and K125L) were treated with 1,2,3,4-diepoxybutane to yield proteins containing 2-hydroxy-3,4-epoxybutyl groups on cysteine residues. Gel shift and mass spectrometry experiments confirmed that epoxide-functionalized AGT proteins formed covalent DPC but no other types of nucleobase damage when incubated with duplex DNA. Introduction of purified AGT monoepoxides into mammalian cells via electroporation generated AGT-DNA cross-links and induced cell death and mutations at the hypoxanthine-guanine phosphoribosyltransferase gene. Smaller numbers of DPC lesions and reduced levels of cell death were observed when using protein monoepoxides generated from an AGT variant that fails to accumulate in the cell nucleus (K125L), suggesting that nuclear DNA damage is required for toxicity. Taken together, these results indicate that AGT protein monoepoxides produce cytotoxic and mutagenic DPC lesions within chromosomal DNA. More generally, these data suggest that covalent DPC lesions contribute to the cytotoxic and mutagenic effects of bis-electrophiles.


Assuntos
Morte Celular , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Compostos de Epóxi/farmacologia , Mutagênese , Alquilação , Sequência de Aminoácidos , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray
4.
J Proteome Res ; 12(5): 2151-64, 2013 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-23506368

RESUMO

1,2,3,4-Diepoxybutane (DEB) is the key carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. DEB is a genotoxic bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs). In the present work, mass spectrometry-based proteomics was employed to characterize DEB-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. Over 150 proteins including histones, high mobility group proteins, transcription factors, splicing factors, and tubulins were found among those covalently cross-linked to chromosomal DNA in the presence of DEB. A large portion of the cross-linked proteins are known factors involved in DNA binding, transcriptional regulation, cell signaling, DNA repair, and DNA damage response. HPLC-ESI(+)-MS/MS analysis of total proteolytic digests revealed the presence of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol conjugates, confirming that DEB forms DPCs between cysteine thiols within proteins and the N-7 guanine positions within DNA. However, relatively high concentrations of DEB were required to achieve significant DPC formation, indicating that it is a poor cross-linking agent as compared to antitumor nitrogen mustards and platinum compounds.


Assuntos
Reagentes de Ligações Cruzadas/química , Proteínas de Ligação a DNA/química , Compostos de Epóxi/química , Proteoma/química , Sequência de Aminoácidos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Compostos de Epóxi/farmacologia , Fibrossarcoma , Genoma Humano , Humanos , Anotação de Sequência Molecular , Ligação Proteica , Proteoma/metabolismo , Proteômica , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
5.
Chem Res Toxicol ; 25(7): 1472-83, 2012 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-22642258

RESUMO

The bioactivation of both endogenous and equine estrogens to electrophilic quinoid metabolites has been postulated as a contributing factor in carcinogenic initiation and/or promotion in hormone sensitive tissues. Bearing structural resemblance to estrogens, extensive studies have shown that many selective estrogen receptor modulators (SERMs) are subject to similar bioactivation pathways. Lasofoxifene (LAS), a third generation SERM which has completed phase III clinical trials for the prevention and treatment of osteoporosis, is currently approved in the European Union for this indication. Previously, Prakash et al. (Drug Metab. Dispos. (2008) 36, 1218-1226) reported that similar to estradiol, two catechol regioisomers of LAS are formed as primary oxidative metabolites, accounting for roughly half of the total LAS metabolism. However, the potential for further oxidation of these catechols to electrophilic o-quinones has not been reported. In the present study, LAS was synthesized and its oxidative metabolism investigated in vitro under various conditions. Incubation of LAS with tyrosinase, human liver microsomes, or rat liver microsomes in the presence of GSH as a trapping reagent resulted in the formation of two mono-GSH and two di-GSH catechol conjugates which were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Similar conjugates were also detected in incubations with P450 3A4, P450 2D6, and P450 1B1 supersomes. Interestingly, these conjugates were also detected as major metabolites when compared to competing detoxification pathways such as glucuronidation and methylation. The 7-hydroxylasofoxifene (7-OHLAS) catechol regioisomer was also synthesized and oxidized either chemically or enzymatically to an o-quinone that was shown to form depurinating adducts with DNA. Collectively, these data show that analogous to estrogens, LAS is oxidized to catechols and o-quinones which could potentially contribute to in vivo toxicity for this SERM.


Assuntos
Estradiol/metabolismo , Pirrolidinas/metabolismo , Quinonas/metabolismo , Moduladores Seletivos de Receptor Estrogênico/metabolismo , Tetra-Hidronaftalenos/metabolismo , Animais , Catecóis/química , Catecóis/metabolismo , Bovinos , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/metabolismo , DNA/química , Estradiol/química , Glutationa/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Oxirredução , Pirrolidinas/química , Quinonas/química , Ratos , Moduladores Seletivos de Receptor Estrogênico/química , Estereoisomerismo , Espectrometria de Massas em Tandem , Tetra-Hidronaftalenos/química
6.
Chem Biol Interact ; 196(1-2): 1-10, 2012 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-22290292

RESUMO

o-Quinone forming estrogens and selective estrogen receptor modulators (SERMs) have been associated with carcinogenesis. LY2066948, a novel SERM in development by Eli Lilly for the treatment of uterine fibroids and myomas, has structural similarity to the equine estrogen equilenin present in hormone replacement formulations; both contain a naphthol group susceptible to oxidative metabolism to o-quinones. LY2066948 was synthesized and assayed for antiestrogenic activity, and in cell culture was confirmed to be a more potent antiestrogen than the prototypical SERM, 4-hydroxytamoxifen. Oxidation of LY2066948 with 2-iodoxybenzoic acid gave an o-quinone (t(1/2)=3.9 ± 0.1h) which like 4-hydroxyequilenin-o-quinone (t(1/2)=2.5 ± 0.2 h) was observed to be exceptionally long-lived with the potential to cause cytotoxicity and/or genotoxicity. In model reactions with tyrosinase, the catechol metabolites of LY2066948 and equilenin were products; interestingly, in the presence of ascorbate to inhibit autoxidation, these catechols were formed quantitatively. Tyrosinase incubations in the presence of GSH gave the expected GSH conjugates resulting from trapping of the o-quinones, which were characterized by LC-MS/MS. Incubations of LY2066948 or equilenin with rat liver microsomes also gave detectable o-quinone trapped GSH conjugates; however, as observed with other SERMs, oxidative metabolism of LY2066948 mainly occurred on the amino side chain to yield the N-dealkylated metabolite. CYP1B1 is believed to be responsible for extra-hepatic generation of genotoxic estrogen quinones and o-quinone GSH conjugates were detected in equilenin incubations. However, in corresponding incubations with CYP1B1 supersomes, no o-quinone GSH conjugates of LY2066948 were detected. These studies suggest that although the naphthol group is susceptible to oxidative metabolism to long-lived o-quinones, the formation of these quinones by cytochrome P450 can be attenuated by the chemistry of the remainder of the molecule as in the case of LY2066948.


Assuntos
Equilenina/análogos & derivados , Naftalenos/química , Piperidinas/química , Quinonas/química , Moduladores Seletivos de Receptor Estrogênico/química , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocromo P-450 CYP1B1 , Citocromo P-450 CYP3A/metabolismo , Relação Dose-Resposta a Droga , Equilenina/química , Equilenina/metabolismo , Feminino , Meia-Vida , Concentração Inibidora 50 , Cinética , Espectroscopia de Ressonância Magnética , Microssomos Hepáticos , Naftalenos/metabolismo , Naftalenos/farmacologia , Oxirredução , Piperidinas/metabolismo , Piperidinas/farmacologia , Quinonas/metabolismo , Quinonas/farmacologia , Ratos , Ratos Sprague-Dawley , Moduladores Seletivos de Receptor Estrogênico/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Espectrometria de Massas em Tandem
7.
ACS Chem Neurosci ; 2(5): 256-268, 2011 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-21731800

RESUMO

The clinical benzothiophene SERM (BT-SERM), raloxifene, was compared with estrogens in protection of primary rat neurons against oxygen-glucose deprivation (OGD). Structure-activity relationships for neuroprotection were determined for a family of BT-SERMs displaying a spectrum of ERα and ERß binding affinity and agonist/antagonist activity, leading to discovery of a neuroprotective pharmacophore, present in the clinically relevant SERMS, raloxifene and desmethylarzoxifene (DMA), for which submicromolar potency was observed for neuroprotection. BT-SERM neuroprotection did not correlate with binding to ER nor classical ER activity, however, both the neuroprotective SERMs and estrogens were shown, using pharmacological probes, to activate the same kinase signaling cascades. The antiestrogen ICI 182,780 inhibited the actions of estrogens, but not those of BT-SERMs, whereas antagonism of the G-protein coupled receptor, GPR30, was effective for both SERMs and estrogens. Since SERMs have antioxidant activity, ER-independent mechanisms were studied using the classical phenolic antioxidants, BHT and Trolox, and the Nrf2-dependent cytoprotective electrophile, sulforaphane. However, neuroprotection by these agents was not sensitive to GPR30 antagonism. Collectively, these data indicate that the activity of neuroprotective BT-SERMs is GPR30-dependent and ER-independent and not mediated by antioxidant effects. Comparison of novel BT-SERM derivatives and analogs identified a neuroprotective pharmacophore of potential use in design of novel neuroprotective agents with a spectrum of ER activity.

8.
Bioorg Med Chem ; 18(2): 809-21, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20031420

RESUMO

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 , Estereoisomerismo
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