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1.
Einstein (Sao Paulo) ; 18: eAO4560, 2020.
Artigo em Inglês, Português | MEDLINE | ID: mdl-32321078

RESUMO

OBJECTIVE: To investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERß) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability. METHODS: Cell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy. RESULTS: Regarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line. CONCLUSION: The effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors' involvement in cellular processes of breast cancer resistance.


Assuntos
Autofagia/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Antagonistas do Receptor de Estrogênio/farmacologia , Fulvestranto/farmacologia , Receptores Acoplados a Proteínas-G/agonistas , Análise de Variância , Western Blotting , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/antagonistas & inibidores , Feminino , Citometria de Fluxo/métodos , Humanos , Células MCF-7 , Receptores Acoplados a Proteínas-G/análise , Reprodutibilidade dos Testes , Sirolimo/farmacologia , Fatores de Tempo , Transfecção/métodos
2.
Aquat Toxicol ; 222: 105469, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32179334

RESUMO

Phthalates are commonly used in plastic products in daily life. The endocrine-disrupting effects of phthalates have been widely reported. Accumulating evidence from human cohorts and lab animals indicate exposure to phthalates might impair neurodevelopment. However, the direct causal relationship and mechanism between phthalates with neurodevelopment and neurotoxicity have not been firmly established. We found that phthalates (i.e. DBP, DINP, BBP) disrupted the expression of estrogen receptors (esr1, esr2a, esr2b), and impaired neurogenesis in the brain of zebrafish during embryonic development. Moreover, the abnormal expression of estrogen receptors, especially esr2a, was partly rescued in zebrafish which exposed to phthalates, with the estrogen receptor antagonist tamoxifen. Hence, impaired neurogenesis of zebrafish exposed to phthalates was partly reversed by tamoxifen treatment. Moreover, our results show that induced pluripotent stem cells (iPSC)-derived human neurons exposed to phthalates triggered double-strand DNA breaks in vitro. Overall, this study demonstrates that exposure to phthalates affects neurodevelopment in zebrafish embryos and induces neurotoxicity in human neurons partly through disrupting the expression of estrogen receptors.


Assuntos
Quebras de DNA de Cadeia Dupla , Desenvolvimento Embrionário/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Neurônios/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Receptores Estrogênicos/genética , Poluentes Químicos da Água/toxicidade , Animais , Células Cultivadas , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Antagonistas do Receptor de Estrogênio/farmacologia , Humanos , Neurônios/metabolismo , Neurônios/patologia , Peixe-Zebra
3.
Gut ; 69(1): 158-167, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30833451

RESUMO

OBJECTIVE: Hepatitis D virus (HDV) is a circular RNA virus coinfecting hepatocytes with hepatitis B virus. Chronic hepatitis D results in severe liver disease and an increased risk of liver cancer. Efficient therapeutic approaches against HDV are absent. DESIGN: Here, we combined an RNAi loss-of-function and small molecule screen to uncover host-dependency factors for HDV infection. RESULTS: Functional screening unravelled the hypoxia-inducible factor (HIF)-signalling and insulin-resistance pathways, RNA polymerase II, glycosaminoglycan biosynthesis and the pyrimidine metabolism as virus-hepatocyte dependency networks. Validation studies in primary human hepatocytes identified the carbamoyl-phosphatesynthetase 2, aspartate transcarbamylase and dihydroorotase (CAD) enzyme and estrogen receptor alpha (encoded by ESR1) as key host factors for HDV life cycle. Mechanistic studies revealed that the two host factors are required for viral replication. Inhibition studies using N-(phosphonoacetyl)-L-aspartic acid and fulvestrant, specific CAD and ESR1 inhibitors, respectively, uncovered their impact as antiviral targets. CONCLUSION: The discovery of HDV host-dependency factors elucidates the pathogenesis of viral disease biology and opens therapeutic strategies for HDV cure.


Assuntos
Aspartato Carbamoiltransferase/genética , Ácido Aspártico/análogos & derivados , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Di-Hidro-Orotase/genética , Receptor alfa de Estrogênio/metabolismo , Fulvestranto/farmacologia , Hepatite D Crônica/tratamento farmacológico , Ácido Fosfonoacéticos/análogos & derivados , Pirimidinas/biossíntese , Antivirais/farmacologia , Aspartato Carbamoiltransferase/antagonistas & inibidores , Aspartato Carbamoiltransferase/metabolismo , Ácido Aspártico/farmacologia , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/antagonistas & inibidores , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Linhagem Celular , Di-Hidro-Orotase/antagonistas & inibidores , Di-Hidro-Orotase/metabolismo , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Inativação Gênica , Hepatite D Crônica/genética , Hepatite D Crônica/metabolismo , Vírus Delta da Hepatite/fisiologia , Hepatócitos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Resistência à Insulina , Estágios do Ciclo de Vida , Mutação com Perda de Função , Ácido Fosfonoacéticos/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Viral/metabolismo , Transdução de Sinais , Replicação Viral
4.
Cancer Lett ; 469: 78-88, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31629931

RESUMO

Selective estrogen receptor modulators (SERMs) are a class of structurally diverse compounds, which have been extensively used to treat hormone-responsive cancers due to their unique partially agonistic and antagonistic properties toward estrogen receptors. Our previous studies have identified a three-dimensional SERM, oxabicycloheptene sulfonate (OBHS), as an estrogen receptor α (ERα) ligand, which is effective for the prevention and treatment of estrogen-dependent endometriosis in vivo. Here, using genome-wide ChIP-seq and RNA-seq analysis, we report that OBHS rapidly induces genome-wide ERα occupancy and acts as a partial agonist and antagonist for ERα. Interestingly, OBHS downregulates the homologous recombination and repair (HRR) modules, resulting in increased DNA damage, apoptosis and cell cycle arrest, inducing synthetic lethality with poly (ADP-ribose) polymerase (PARP) inhibitor olaparib through ERα antagonism. Mechanistically, OBHS impairs the RNA polymerase II (Pol II) loading at the promoters of estrogen-responsive HRR genes. Furthermore, combination therapy of OBHS with olaparib significantly reduces the tumour burden and delays the progression of breast cancer in vivo. Together, our studies not only characterise a novel SERM which uniquely targets the homologous recombination and repair programmes through ERα antagonism but also propose a synthetic lethal strategy by combining OBHS with PARP inhibitor olaparib for ERα-responsive cancers.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Antagonistas do Receptor de Estrogênio/farmacologia , Reparo de DNA por Recombinação/efeitos dos fármacos , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Sequenciamento de Cromatina por Imunoprecipitação , Antagonistas do Receptor de Estrogênio/uso terapêutico , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Camundongos , Estrutura Molecular , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Regiões Promotoras Genéticas/genética , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , RNA-Seq , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Relação Estrutura-Atividade , Mutações Sintéticas Letais/efeitos dos fármacos , Células Vero , Ensaios Antitumorais Modelo de Xenoenxerto
5.
In Vivo ; 33(5): 1439-1445, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31471390

RESUMO

BACKGROUND: Endocrine therapy is clinically administered in hormone-responsive breast cancer. Combinations of fluoropyrimidine S-1 and an aromatase inhibitor or anti-estrogen are considered beneficial in Japan. Herein we assessed new combinations of S-1 and fulvestrant. PATIENTS AND METHODS: Cytotoxicity of fulvestrant and 5-fluorouracil (5-FU) was assessed in hormone-responsive (MCF-7) and non-responsive (MDA-MB-231) breast cancer cell cultures. Fulvestrant and S-1 were evaluated for antitumor activity in mice and their effects on estrogen receptor (ER)-α and progesterone receptor (PgR) levels in MCF-7 xenografts using immunohistochemical methods. RESULTS: Fulvestrant inhibited growth of MCF-7, but not of MDA-MB-231 xenografts. Combinations of 5-FU and fulvestrant were superior to monotherapy in vitro. In vivo antitumor activity of S-1/fulvestrant combination therapy was significantly (p<0.05) enhanced compared to that of both monotherapies. Fulvestrant partially down-regulated expression of ERα and PgR, but in combination with S-1, it almost completely blocked their expression. CONCLUSION: Chemo-endocrine combination therapy using S-1 and fulvestrant is beneficial in estrogen-responsive breast cancer.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/metabolismo , Antagonistas do Receptor de Estrogênio/farmacologia , Estrogênios/metabolismo , Fulvestranto/farmacologia , Pirimidinas/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Estrogênios/farmacologia , Feminino , Humanos , Camundongos
6.
Cell ; 178(4): 949-963.e18, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31353221

RESUMO

Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER "degraders" exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.


Assuntos
Neoplasias da Mama/metabolismo , Antagonistas do Receptor de Estrogênio/farmacologia , Fulvestranto/farmacologia , Receptores Estrogênicos/antagonistas & inibidores , Receptores Estrogênicos/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Cinamatos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Antagonistas do Receptor de Estrogênio/uso terapêutico , Feminino , Fulvestranto/uso terapêutico , Células HEK293 , Xenoenxertos , Humanos , Indazóis/farmacologia , Ligantes , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Polimorfismo de Nucleotídeo Único , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
7.
Curr Opin Oncol ; 31(5): 424-429, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31335829

RESUMO

PURPOSE OF REVIEW: Estrogen receptor-positive breast cancer accounts for 70% of all breast cancers. Sequential endocrine treatment in monotherapy or in combination with CDK 4/6 or m-TOR inhibitors is the mainstay of recommended treatment options in the management of metastatic breast cancer even in the presence of visceral metastasis. There is an emerging need to address endocrine resistance, which despite highly efficacious treatment combinations still can develop. RECENT FINDINGS: One of the mechanisms of endocrine resistance is molecular alteration of the oestrogen receptor itself, such as ESR1 mutations affecting the ligand-binding domain. These mutations emerge under the selective pressure of aromatase inhibitors. The efficacy of selective oestrogen receptor degraders (SERDs) might not be affected by the presence of molecular alterations of oestrogen receptor. Fulvestrant is the only SERD used in current clinical practice. Numerous novel, nonsteroidal orally available SERDs are currently in clinical development. Efficacious oestrogen receptor target engagement and promising clinical activity was shown in early phase clinical trials. SUMMARY: Therefore, a new class of orally available nonsteroidal SERDs gains high interest in tackling endocrine resistance in oestrogen receptor-positive (ER+) advanced breast cancer. Clinical efficacy needs to be confirmed in larger patient populations.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Antagonistas do Receptor de Estrogênio/administração & dosagem , Receptores Estrogênicos/metabolismo , Moduladores Seletivos de Receptor Estrogênico/administração & dosagem , Neoplasias da Mama/metabolismo , Ensaios Clínicos como Assunto , Antagonistas do Receptor de Estrogênio/farmacologia , Feminino , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto , Moduladores Seletivos de Receptor Estrogênico/farmacologia
8.
Int J Dev Neurosci ; 78: 170-177, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31202866

RESUMO

BACKGROUND: The goal of this study was to investigate the effect of estradiol in mediation of electroencephalogram (EEG) abnormality induced by etomidate in neonatal rats. METHODS: Sprague-Dawley rats were anesthetized using intraperitoneal etomidate for 2 h on postnatal days (P) 4, 5, or 6 and recorded electroencephalogram in two ways. First, pups were recorded EEG two and a half hours under etomidate anesthesia, in subgroups, estradiol receptor antagonist ICI182780 and estradiol synthase inhibitor formestane were given subcutaneously in male rats 15 min prior to etomidate. Second, pups were anesthetized with etomidate for 2 h on P4,5 or 6 and then recovered from anesthesia, EEG were recorded for one hour in two postnatal periods of P9-P11 and P14-P16. Subgroup rats that received bumetanide, NKCC1 inhibitor, to test the NKCC1-GABAAR signaling effect on neonatal brain development, negative control groups and maternally separated for 2 h on P4, 5, or 6 were studied in 16 groups. Each group's n was = 8. RESULTS: Male pups showed more severe seizure-like activities than female pups in P4-P6 under etomidate anesthesia. Pups pretreated with ICI182780 and formestane showed a less abnormalities of EEG in male rats. Etomidate caused seizure-like activity in P4-P6 could extend to P9-P11, but not seen in P14-P16, Pretreated with bumetanide only alleviated abnormalities in male pups other than female in P9-P11. CONCLUSIONS: Estradiol involves in the NKCC1-GABAAR mediated seizure-like activity caused by etomidte in neonatal rats and these the abnormality lasts near two weeks.


Assuntos
Anestésicos Intravenosos/farmacologia , Encéfalo/efeitos dos fármacos , Estradiol/metabolismo , Antagonistas do Receptor de Estrogênio/farmacologia , Etomidato/farmacologia , Receptores Estrogênicos/metabolismo , Convulsões/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Bumetanida/farmacologia , Eletroencefalografia , Feminino , Fulvestranto/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Convulsões/fisiopatologia , Fatores Sexuais , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia
9.
Curr Top Med Chem ; 19(15): 1318-1337, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31215379

RESUMO

Breast cancer is the most common cancer suffered by female, and the second highest cause of cancer-related death among women worldwide. At present, hormone therapy is still the main treatment route and can be divided into three main categories: selective estrogen receptor modulators (SERMs), selective estrogen receptor downregulators (SERDs), and aromatase inhibitors (AIs). However, breast cancer is difficult to cure even after several rounds of anti-estrogen therapy and most drugs have serious side-effects. Here, we review the literature published over the past five years regarding the isolation and synthesis of analogs and their derivatives.


Assuntos
Antineoplásicos/farmacologia , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/tratamento farmacológico , Antagonistas do Receptor de Estrogênio/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Inibidores da Aromatase/química , Inibidores da Aromatase/isolamento & purificação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Antagonistas do Receptor de Estrogênio/química , Antagonistas do Receptor de Estrogênio/isolamento & purificação , Moduladores de Receptor Estrogênico/química , Moduladores de Receptor Estrogênico/isolamento & purificação , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Estrutura Molecular
10.
Neuroscience ; 411: 37-46, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31129201

RESUMO

As an adult-onset neurodegenerative disease, amyotrophic lateral sclerosis (ALS) results in progressive muscular atrophy and paralysis. However, the mechanism of ALS has not yet been elucidated, and no cure has been found. Research has revealed that a mutation of the Cu/Zn superoxide dismutase (SOD1) gene is linked to familial ALS and that potential sex discrepancies exist in ALS incidence. Here, NSC-34 cells stably expressing hSOD1-G93A (hSOD1-G93A cells) were transiently transfected with Cyp19a1 mouse open reading frame (ORF) cDNA or a short hairpin RNA (ShRNA) plasmid. Overexpression of aromatase resulting from Cyp19a1 mouse ORF cDNA plasmid transfection enhanced cell proliferation and reduced cell damage in hSOD1-G93A cells. This protective effect occurred through anti-apoptotic pathways related to estrogen receptor-alpha (ER-α) activation. Meanwhile, knockdown of aromatase with Cyp19a1 ShRNA plasmid transfection reduced cell proliferation, increased cell damage, promoted apoptosis, and decreased ER-α expression in hSOD1-G93A cells, and the induced apoptotic effects could be reversed by estradiol (E2). In brief, the results of our study suggest that aromatase plays a neuroprotective role against apoptosis in hSOD1-G93A cells by activating ER-α and may become a new intervention target for ALS treatment.


Assuntos
Apoptose/fisiologia , Aromatase/metabolismo , Neurônios Motores/metabolismo , Superóxido Dismutase/metabolismo , Animais , Apoptose/efeitos dos fármacos , Aromatase/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Estradiol/farmacologia , Antagonistas do Receptor de Estrogênio/farmacologia , Técnicas de Silenciamento de Genes , Camundongos , Neurônios Motores/efeitos dos fármacos , Superóxido Dismutase/genética
11.
Neuroscience ; 411: 211-221, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31085279

RESUMO

Neural substrates for estrogen regulation of glucose homeostasis remain unclear. Female rat dorsal vagal complex (DVC) A2 noradrenergic neurons are estrogen- and metabolic-sensitive. The ventromedial hypothalamic nucleus (VMN) is a key component of the brain network that governs counter-regulatory responses to insulin-induced hypoglycemia (IIH). Here, the selective estrogen receptor-alpha (ERα) or -beta (ERß) antagonists MPP and PHTPP were administered separately to the caudal fourth ventricle to address the premise that these hindbrain ER variants exert distinctive control of VMN reactivity to IIH in the female sex. Data show that ERα governs hypoglycemic patterns of VMN astrocyte glycogen metabolic enzyme, e.g. glycogen synthase and phosphorylase protein expression, whereas ERß mediates local glycogen breakdown. DVC ERs also regulate VMN neurotransmitter signaling of energy sufficiency [γ-aminobutyric acid] or deficiency [nitric oxide, steroidogenic factor-1] during IIH. Neither hindbrain ER mediates IIH-associated diminution of VMN norepinephrine (NE) content. Both ERs oppose hypoglycemic hyperglucagonemia, while ERß contributes to reduced corticosterone output. Outcomes reveal that input from the female hindbrain to the VMN is critical for energy reserve mobilization, metabolic transmitter signaling, and counter-regulatory hormone secretion during hypoglycemia, and that ERs control those cues. Evidence that VMN NE content is not controlled by hindbrain ERα or -ß implies that these receptors may regulate VMN function via NE-independent mechanisms, or alternatively, that other neurotransmitter signals to the VMN may control local substrate receptivity to NE.


Assuntos
Glicogênio/metabolismo , Hipoglicemia/metabolismo , Receptores Estrogênicos/metabolismo , Rombencéfalo/metabolismo , Núcleo Hipotalâmico Ventromedial/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Antagonistas do Receptor de Estrogênio/farmacologia , Feminino , Óxido Nítrico Sintase Tipo I/metabolismo , Piperidinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ratos , Rombencéfalo/efeitos dos fármacos , Fator Esteroidogênico 1/metabolismo , Núcleo Hipotalâmico Ventromedial/efeitos dos fármacos
12.
Toxicology ; 420: 21-28, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30935971

RESUMO

The equilibrium dissociation constant of competitive antagonists represents the affinity of the receptor-ligand interaction, and it is a key characteristic of many therapeutic drugs or toxic compounds. Two commonly used methods by which the affinity of the antagonist can be estimated are Schild analysis and the Cheng-Prusoff method. However, both methods yield different results when applied to systems with slopes not equal to one. The Gaddum equation, which is fundamental for both methods, should be extended to incorporate the slope parameter of the dose-response curves and this extension should diminish the differences between the Schild and Cheng-Prusoff methods. In this study, we derived a novel form of the Gaddum equation with a slope parameter (Hill coefficient) of agonist dose-response curve. We also derived the subsequent equations for Schild and Cheng-Prusoff analysis and we validated the proposed model by the measurement of several known estrogen receptor competitive antagonists. Standardized in vitro yeast reporter gene assay (BMAEREluc/ERα) has been used for the measurements and the range of used antagonist concentrations was 1.37-46.03 µM. By applying our mathematical model, both Schild and Cheng-Prusoff methods provide more similar values of antagonist affinity than the original mathematical approach. The correctness of the model has also been demonstrated by the measurement of a partial agonist with a known receptor affinity. The presented mathematical model significantly reduces the differences in values calculated by the Cheng-Prusoff and Schild methods and yields more accurate estimations of antagonist affinity.


Assuntos
Compostos Benzidrílicos/metabolismo , Disruptores Endócrinos/metabolismo , Estradiol/metabolismo , Antagonistas do Receptor de Estrogênio/metabolismo , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Modelos Biológicos , Fenóis/metabolismo , Animais , Compostos Benzidrílicos/toxicidade , Ligação Competitiva , Relação Dose-Resposta a Droga , Agonismo Parcial de Drogas , Disruptores Endócrinos/toxicidade , Estradiol/farmacologia , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Estrogênios/farmacologia , Humanos , Ligantes , Fenóis/toxicidade , Ligação Proteica , Reprodutibilidade dos Testes
13.
Breast Cancer ; 26(4): 459-470, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30610551

RESUMO

BACKGROUND: Presently, hormonal therapy targeting estrogen receptors is the most effective treatment available for luminal breast cancer. However, many patients relapse after the therapy. It has been suggested that cancer stem-like cells are involved with hormonal therapy resistance; in the present study, we evaluated this hypothesis. METHODS: In the present study, we used our previously established hormonal therapy-resistant cell lines, including aromatase inhibitor (AI)-resistant cells (Type 1 and Type 2) and fulvestrant-resistant cells (MFR). RESULTS: AI-resistant cell lines expressing ER (Type 1 V1 and V2) showed high cancer stemness in terms of their CD44/CD24 expression and side populations, which were stimulated by the addition of estrogen and inhibited by fulvestrant. However, ALDH activity was lower than in the ER-negative resistant cells, suggesting that the stemness of luminal cells is distinct from that of basal-like breast cancer cells. The migration and invasion activity of the ER-positive Type 1 V1 and V2 cells were higher than in the ER-negative cell lines, Type 2 and MFR. CONCLUSIONS: Fractionation of parental cells based on CD44/CD24 expression and colony formation assay indicated that CD44+/CD24+ cells might be the origin of hormonal therapy-resistant cells. This population reconstituted various other subpopulations under estrogen deprivation. These results indicate that hormonal therapy resistance is closely related to the cancer stem cell-like properties of luminal breast cancer.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/metabolismo , Antígeno CD24/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Estradiol/metabolismo , Antagonistas do Receptor de Estrogênio/farmacologia , Feminino , Fulvestranto , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores de Hialuronatos/metabolismo , Células MCF-7 , Células-Tronco Neoplásicas/patologia , Receptores Estrogênicos/metabolismo
14.
Breast Cancer ; 26(3): 272-281, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30328006

RESUMO

BACKGROUND: Hormone therapy targeting the estrogen receptor (ER) pathway is the most common treatment used for ER-positive breast cancer. However, some patients experience de novo or acquired resistance, which becomes a critical problem. Activation of the insulin-like growth factor (IGF) pathway allows breast cancer cells to proliferate and is associated with the ER pathway. Little is known about the role of the IGF pathway in hormone therapy and resistance; therefore, we investigated whether the inhibition of this pathway may represent a novel therapeutic target for overcoming hormone therapy resistance in ER-positive breast cancers. METHODS: Crosstalk between the ER and IGF pathways was analyzed in breast cancer cell lines by inhibiting or stimulating either one or both pathways. We studied the effect of insulin-like growth factor one receptor (IGF1R) inhibition in aromatase inhibitor-resistant breast cancer cell lines and fulvestrant-resistant cell lines which were uniquely established in our laboratory. RESULTS: Under normal conditions, IGF signaling is controlled by ER signaling to promote cell growth. Temporary disruption of the estrogen supply results in attenuated ER signaling, and IGF-1 dramatically increased relative growth compared with normal conditions. In addition, IGF1R inhibitor strongly suppressd cell growth in hormone-resistant breast cancer cells where ER remains than cells where ER decreased or was almost lost. CONCLUSIONS: Our study suggests that inhibition of the IGF pathway may be an effective strategy for ER-positive breast cancer therapy, even in hormone therapy-resistant cases.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores Estrogênicos/metabolismo , Receptores de Somatomedina/metabolismo , Transdução de Sinais , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Antagonistas do Receptor de Estrogênio/farmacologia , Estrogênios/deficiência , Estrogênios/farmacologia , Feminino , Humanos , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/metabolismo , Células MCF-7 , Receptor Cross-Talk/efeitos dos fármacos , Receptores Estrogênicos/deficiência , Receptores Estrogênicos/genética , Receptores de Somatomedina/antagonistas & inibidores , Receptores de Somatomedina/genética
15.
Andrologia ; 51(3): e13204, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30474158

RESUMO

During prenatal and early postnatal periods of development, multiple environmental factors have profound and long-lasting effects on the immune and reproductive functions. The aim of this study was to investigate the effects of maternal lipopolysaccharide (LPS) exposure (50 mg/kg, i.p.) at day 12 of pregnancy and estradiol antagonist treatment (fulvestrant, 1.5 mg/kg, s.c. in neck) at postnatal days 5-14 (PND5-14) with high estradiol levels on reproductive parameters in adult rat males. Serum steroid concentrations were measured in male offspring at PND80 by ELISA. Body, testis weights and ano-genital distance (AGD) were recorded at different stages of postnatal development. Testis was also processed to cytohistological studies at PND80. Our results demonstrate that body weight was decreased from PND14 to 30 after prenatal LPS treatment and was increased after fulvestrant treatment. AGD was decreased after prenatal LPS treatment and was increased after fulvestrant injections. Testis weight, testosterone level, seminiferous tubule diameter, and number of Sertoli and spermatid cells were also decreased in rats exposed prenatally to LPS and were restored to the normal control level after fulvestrant treatment. According to results, we can conclude that the development of sexual disorders in males after prenatal immune stress is potentiated by estradiol during the pre-pubertal period.


Assuntos
Peso Corporal/efeitos dos fármacos , Antagonistas do Receptor de Estrogênio/uso terapêutico , Fulvestranto/uso terapêutico , Infertilidade Masculina/tratamento farmacológico , Efeitos Tardios da Exposição Pré-Natal/tratamento farmacológico , Testículo/efeitos dos fármacos , Animais , Antagonistas do Receptor de Estrogênio/farmacologia , Feminino , Fulvestranto/farmacologia , Lipopolissacarídeos , Masculino , Gravidez , Ratos , Células de Sertoli/efeitos dos fármacos , Espermátides/efeitos dos fármacos
16.
J Steroid Biochem Mol Biol ; 187: 88-96, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30439415

RESUMO

Although the molecular mechanisms underlying the formation of pituitary adenomas are largely unknown, it is clear that estrogen plays a key role in the pathogenesis of pituitary adenomas. Though this is exemplified by an investigation of fulvestrant in the pituitary adenoma cell line GH3, no systematic studies on the effects of selective estrogen receptor modulators (SERMs) on functional properties of pituitary adenoma cell lines to modulate cell migration, cell invasion, and cell survival are available. Here we analyzed the effects of fulvestrant and three SERMs, bazedoxifene, clomifene, and raloxifene, on pituitary adenomas cell lines AtT20, TtT/GF, and GH3. In cell survival assays, clomifene was shown to be the most potent compound in all three cell lines with IC50 values ranging between 2, 6, and 10 µM, respectively, depending on the cell type. Raloxifene and bazedoxifene were also effective but to a lower extent. Also, all SERMs affected migratory and invasive behavior of pituitary adenoma cells. Mechanistically, treatment of cells with SERMs caused cell apoptosis, as demonstrated by Caspase 3/7 activity and western blot assays. In addition, western blots demonstrate activation of p53 in TtT/GF cells and loss of ERK1/2 activation in AtT20 cells. In contrast, fulvestrant was only effective in GH3 cells. Thus, the general applicability of SERMs for pituitary adenoma cells might be promising in clinical applications for the treatment of pituitary adenomas.


Assuntos
Adenoma/tratamento farmacológico , Antagonistas do Receptor de Estrogênio/farmacologia , Fulvestranto/farmacologia , Invasividade Neoplásica/prevenção & controle , Neoplasias Hipofisárias/tratamento farmacológico , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Adenoma/metabolismo , Adenoma/patologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Invasividade Neoplásica/patologia , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia
17.
Cancer Chemother Pharmacol ; 83(1): 151-160, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30386887

RESUMO

PURPOSE: H3B-6545, a novel selective estrogen receptor (ER)α covalent antagonist (SERCA) which inactivates both wild-type and mutant ERα, is in clinical development for the treatment of metastatic breast cancer. Preclinical studies were conducted to characterize the pharmacokinetics and metabolism of H3B-6545 in rat and monkeys. METHODS: The clearance and metabolic profiles of H3B-6545 were studied using rat, monkey and human hepatocytes, and reaction phenotyping was done using recombinant human cytochrome P450 enzymes. Blood stability, protein binding, and permeability were also determined in vitro. Pharmacokinetics of H3B-6545 was assessed after both intravenous and oral dosing. A nonclinical PBPK model was developed to assess in vitro-in vivo correlation of clearance. RESULTS: H3B-6545 had a terminal elimination half-life of 2.4 h in rats and 4.0 h in monkeys and showed low to moderate bioavailability, in line with the in vitro permeability assessment. Plasma protein binding was similar across species, at 99.5-99.8%. Nine metabolites of H3B-6545 were identified in hepatocyte incubations, none of which were unique to humans. Formation of glutathione-related conjugate of H3B-6545 was minimal in vitro. H3B-6545, a CYP3A substrate, is expected to be mostly cleared via hepatic phase 1 metabolism. Hepatocyte clearance values were used to adequately model the time-concentration profiles in rat and monkey. CONCLUSIONS: We report on the absorption and metabolic fate and disposition of H3B-6545 in rats and dogs and illustrate that in vitro-in vivo correlation of clearance is possible for targeted covalent inhibitors, provided reactivity is not a predominant mechanism of clearance.


Assuntos
Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Antagonistas do Receptor de Estrogênio/farmacologia , Antagonistas do Receptor de Estrogênio/farmacocinética , Receptor alfa de Estrogênio/antagonistas & inibidores , Hepatócitos/metabolismo , Indazóis/farmacologia , Indazóis/farmacocinética , Microssomos Hepáticos/metabolismo , Piridinas/farmacologia , Piridinas/farmacocinética , Animais , Disponibilidade Biológica , Células Cultivadas , Inibidores das Enzimas do Citocromo P-450/farmacocinética , Inibidores das Enzimas do Citocromo P-450/farmacologia , Cães , Avaliação Pré-Clínica de Medicamentos , Feminino , Hepatócitos/efeitos dos fármacos , Humanos , Técnicas In Vitro , Macaca fascicularis , Taxa de Depuração Metabólica , Microssomos Hepáticos/efeitos dos fármacos , Ligação Proteica , Ratos Sprague-Dawley , Especificidade da Espécie , Distribuição Tecidual
18.
Cancer Lett ; 442: 373-382, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30419347

RESUMO

Approximately 30% of metastatic breast cancers harbor estrogen receptor α (ERα) mutations associated with resistance to endocrine therapy and reduced survival. Consistent with their constitutive proliferation, T47D and MCF7 cells in which wild-type ERα is replaced by the most common mutations, ERαY537S and ERαD538G, exhibit partially estrogen-independent gene expression. A novel invasion/dissociation/rebinding assay demonstrated that the mutant cells have a higher tendency to dissociate from invasion sites and rebind to a second site. Compared to ERαD538G breast tumors, ERαY537S tumors exhibited a dramatic increase in lung metastasis. Transcriptome analysis showed that the ERαY537S and ERαD538G mutations each elicit a unique gene expression profile. Gene set enrichment analysis showed Myc target pathways are highly induced in mutant cells. Moreover, chromatin immunoprecipitation showed constitutive, fulvestrant-resistant, recruitment of ERα mutants to the Myc enhancer region, resulting in estrogen-independent Myc overexpression in mutant cells and tumors. Knockdown and virus transduction showed Myc is necessary and sufficient for ligand-independent proliferation of the mutant cells but had no effect on metastasis-related phenotypes. Thus, Myc plays a key role in aggressive proliferation-related phenotypes exhibited by breast cancer cells expressing ERα mutations.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Fulvestranto/farmacologia , Mutação , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Elementos Facilitadores Genéticos , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Células MCF-7 , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Oncol Res ; 27(3): 283-292, 2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-28877783

RESUMO

G-protein-coupled estrogen receptor (GPER) was found to promote non-small cell lung cancer (NSCLC) by estrogen, indicating the potential necessity of inhibiting GPER by a selective antagonist. This study was performed to elucidate the function of GPER-selective inhibitor G15 in NSCLC development. Cytoplasmic GPER (cGPER) and nuclear GPER (nGPER) were detected by immunohistochemical analysis in NSCLC samples. The relation of GPER and estrogen receptor ß (ERß) expression and correlation between GPER, ERß, and clinical factors were analyzed. The effects of activating GPER and function of G15 were analyzed in the proliferation of A549 and H1793 cell lines and development of urethane-induced adenocarcinoma. Overexpression of cGPER and nGPER was detected in 80.49% (120/150) and 52.00% (78/150) of the NSCLC samples. High expression of GPER was related with higher stages, poorer differentiation, and high expression of ERß. The protein level of GPER in the A549 and H1793 cell lines was increased by treatment with E2, G1 (GPER agonist), or fulvestrant (Ful; ERß antagonist) and decreased by G15. Administration with G15 reversed the E2- or G1-induced cell growth by inhibiting GPER. In urethane-induced adenocarcinoma mice, the number of tumor nodules and tumor index increased in the E2 or G1 group and decreased by treatment with G15. These findings demonstrate that using G15 to block GPER signaling may be considered as a new therapeutic target in NSCLC.


Assuntos
Benzodioxóis/farmacologia , Carcinoma Pulmonar de Células não Pequenas/induzido quimicamente , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Antagonistas do Receptor de Estrogênio/farmacologia , Estrogênios/efeitos adversos , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/tratamento farmacológico , Quinolinas/farmacologia , Receptores Estrogênicos/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Células A549 , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptor beta de Estrogênio/metabolismo , Feminino , Fulvestranto/farmacologia , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Transdução de Sinais/efeitos dos fármacos , Uretana/farmacologia
20.
Physiol Behav ; 203: 70-80, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29106989

RESUMO

Alcohol use disorder (AUD) is a chronic, relapsing disease characterized by maladaptive patterns of alcohol drinking and seeking. Though sex differences exist in the etiology of AUD, much remains to be elucidated concerning the mechanisms underlying sex-related vulnerability to developing excessive alcohol-motivated behavior. While a large body of evidence points to an important role of circulating gonadal hormones in mediating cocaine reinforcement, findings are less consistent with respect to ethanol. Critically, the effects of gonadal hormones on the reinstatement of ethanol seeking, a model of "craving"-like behavior that reveals pronounced sex differences, has not yet been examined. Thus, the goal of the present experiment was to directly compare manipulations of gonadal hormones in male and female rats on ethanol-motivated behavior. Rats received sham or gonadectomy surgery with or without hormone replacement prior to and throughout three weeks of operant ethanol self-administration to determine the effects of chronically high or low gonadal hormone levels on ethanol drinking. Hormone treatment ceased during extinction training, and the effects of an acute injection of either testosterone (in males) or estradiol (in females) on cue+yohimbine-induced reinstatement of ethanol seeking was determined. Separate groups of gonadally-intact female rats went through similar training, but the effects of either the antiestrogen, fulvestrant, the selective estrogen receptor modulator, clomiphene, or the estrogen receptor ß antagonist, PHTPP, on the reinstatement of ethanol seeking were determined. Chronic estradiol replacement produced significant increases in ethanol drinking in female rats, while chronic testosterone significantly decreased ethanol drinking in male rats. Gonadectomy alone only produced modest shifts in drinking towards the opposite-sex pattern, and did not eliminate the robust sex differences that persisted regardless of hormone manipulations. Neither prior chronic nor acute hormone manipulations altered cue+yohimbine-induced reinstatement of ethanol seeking, though blockade of estrogen receptors tended to reduce reinstatement in gonadally-intact females. Overall, our findings indicate that gonadal hormones at least partially mediate, but do not totally account for the sex differences evident in ethanol self-administration, and circulating gonadal hormones have little effect on the reinstatement of ethanol seeking. These results provide a foundation for future studies examining the neuronal mechanisms underlying sex differences in ethanol drinking and seeking.


Assuntos
Consumo de Bebidas Alcoólicas , Comportamento de Procura de Droga/efeitos dos fármacos , Estradiol/farmacologia , Etanol/administração & dosagem , Propionato de Testosterona/farmacologia , Ioimbina/farmacologia , Animais , Clomifeno/farmacologia , Sinais (Psicologia) , Antagonistas do Receptor de Estrogênio/farmacologia , Feminino , Fulvestranto/farmacologia , Masculino , Motivação/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Autoadministração , Caracteres Sexuais
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