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1.
Nat Commun ; 12(1): 5325, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34493733

RESUMO

Distal-less homeobox-1 (DLX1) is a well-established non-invasive biomarker for prostate cancer (PCa) diagnosis, however, its mechanistic underpinnings in disease pathobiology are not known. Here, we reveal the oncogenic role of DLX1 and show that abrogating its function leads to reduced tumorigenesis and metastases. We observed that ~60% of advanced-stage and metastatic patients display higher DLX1 levels. Moreover, ~96% of TMPRSS2-ERG fusion-positive and ~70% of androgen receptor (AR)-positive patients show elevated DLX1, associated with aggressive disease and poor survival. Mechanistically, ERG coordinates with enhancer-bound AR and FOXA1 to drive transcriptional upregulation of DLX1 in ERG-positive background. However, in ERG-negative context, AR/AR-V7 and FOXA1 suffice to upregulate DLX1. Notably, inhibiting ERG/AR-mediated DLX1 transcription using BET inhibitor (BETi) or/and anti-androgen drugs reduce its expression and downstream oncogenic effects. Conclusively, this study establishes DLX1 as a direct-target of ERG/AR with an oncogenic role and demonstrates the clinical significance of BETi and anti-androgens for DLX1-positive patients.


Assuntos
Proteínas de Homeodomínio/genética , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Fatores de Transcrição/genética , Transcrição Genética , Antagonistas de Androgênios/farmacologia , Animais , Azepinas/farmacologia , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos SCID , Metástase Neoplásica , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Regiões Promotoras Genéticas , Próstata/efeitos dos fármacos , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Ligação Proteica , Receptores Androgênicos/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transdução de Sinais , Análise de Sobrevida , Fatores de Transcrição/metabolismo , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/metabolismo , Triazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
2.
BMC Cancer ; 21(1): 1061, 2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34565342

RESUMO

BACKGROUND: Neuroblastoma (NB) patients with MYCN amplification or overexpression respond poorly to current therapies and exhibit extremely poor clinical outcomes. PI3K-mTOR signaling-driven deregulation of protein synthesis is very common in NB and various other cancers that promote MYCN stabilization. In addition, both the MYCN and mTOR signaling axes can directly regulate a common translation pathway that leads to increased protein synthesis and cell proliferation. However, a strategy of concurrently targeting MYCN and mTOR signaling in NB remains unexplored. This study aimed to investigate the therapeutic potential of targeting dysregulated protein synthesis pathways by inhibiting the MYCN and mTOR pathways together in NB. METHODS: Using small molecule/pharmacologic approaches, we evaluated the effects of combined inhibition of MYCN transcription and mTOR signaling on NB cell growth/survival and associated molecular mechanism(s) in NB cell lines. We used two well-established BET (bromodomain extra-terminal) protein inhibitors (JQ1, OTX-015), and a clinically relevant mTOR inhibitor, temsirolimus, to target MYCN transcription and mTOR signaling, respectively. The single agent and combined efficacies of these inhibitors on NB cell growth, apoptosis, cell cycle and neurospheres were assessed using MTT, Annexin-V, propidium-iodide staining and sphere assays, respectively. Effects of inhibitors on global protein synthesis were quantified using a fluorescence-based (FamAzide)-based protein synthesis assay. Further, we investigated the specificities of these inhibitors in targeting the associated pathways/molecules using western blot analyses. RESULTS: Co-treatment of JQ1 or OTX-015 with temsirolimus synergistically suppressed NB cell growth/survival by inducing G1 cell cycle arrest and apoptosis with greatest efficacy in MYCN-amplified NB cells. Mechanistically, the co-treatment of JQ1 or OTX-015 with temsirolimus significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K/eIF4E (mTOR components) and BRD4 (BET protein)/MYCN proteins. Further, this combination significantly inhibited global protein synthesis, compared to single agents. Our findings also demonstrated that both JQ1 and temsirolimus chemosensitized NB cells when tested in combination with cisplatin chemotherapy. CONCLUSIONS: Together, our findings demonstrate synergistic efficacy of JQ1 or OTX-015 and temsirolimus against MYCN-driven NB, by dual-inhibition of MYCN (targeting transcription) and mTOR (targeting translation). Additional preclinical evaluation is warranted to determine the clinical utility of targeted therapy for high-risk NB patients.


Assuntos
Acetanilidas/farmacologia , Azepinas/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Proteína Proto-Oncogênica N-Myc/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/antagonistas & inibidores , Triazóis/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Regulação para Baixo , Sinergismo Farmacológico , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Esferoides Celulares/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo
3.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445411

RESUMO

BACKGROUND: The present study investigated the role of proteins from the bromodomain and extra-terminal (BET) family in schizophrenia-like abnormalities in a neurodevelopmental model of schizophrenia induced by prenatal methylazoxymethanol (MAM) administration (MAM-E17). METHODS: An inhibitor of BET proteins, JQ1, was administered during adolescence on postnatal days (P) 23-P29, and behavioural responses (sensorimotor gating, recognition memory) and prefrontal cortical (mPFC) function (long-term potentiation (LTP), molecular and proteomic analyses) studies were performed in adult males and females. RESULTS: Deficits in sensorimotor gating and recognition memory were observed only in MAM-treated males. However, adolescent JQ1 treatment affected animals of both sexes in the control but not MAM-treated groups and reduced behavioural responses in both sexes. An electrophysiological study showed LTP impairments only in male MAM-treated animals, and JQ1 did not affect LTP in the mPFC. In contrast, MAM did not affect activity-dependent gene expression, but JQ1 altered gene expression in both sexes. A proteomic study revealed alterations in MAM-treated groups mainly in males, while JQ1 affected both sexes. CONCLUSIONS: MAM-induced schizophrenia-like abnormalities were observed only in males, while adolescent JQ1 treatment affected memory recognition and altered the molecular and proteomic landscape in the mPFC of both sexes. Thus, transient adolescent inhibition of the BET family might prompt permanent alterations in the mPFC.


Assuntos
Azepinas/administração & dosagem , Acetato de Metilazoximetanol/análogos & derivados , Córtex Pré-Frontal/crescimento & desenvolvimento , Esquizofrenia/fisiopatologia , Triazóis/administração & dosagem , Adolescente , Desenvolvimento do Adolescente/efeitos dos fármacos , Animais , Azepinas/farmacologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Acetato de Metilazoximetanol/toxicidade , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Proteômica , Ratos , Reconhecimento Psicológico/efeitos dos fármacos , Esquizofrenia/induzido quimicamente , Esquizofrenia/metabolismo , Caracteres Sexuais , Triazóis/farmacologia
4.
Am J Physiol Regul Integr Comp Physiol ; 321(4): R558-R571, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34405704

RESUMO

Orexin neurons are active in wakefulness and mostly silent in sleep. In adult rats and humans, orexin facilitates the hypercapnic ventilatory response but has little effect on resting ventilation. The influence of orexin on breathing in the early postnatal period, and across states of vigilance, have not been investigated. This is relevant as the orexin system may be impaired in Sudden Infant Death Syndrome (SIDS) cases. We addressed three hypotheses: 1) orexin provides a drive to breathe in infancy; 2) the effect of orexin depends on stage of postnatal development; and 3) orexin has a greater influence on breathing in wakefulness compared with sleep. Whole body plethysmography was used to monitor breathing of infant rats at three ages: postnatal days (P) 7-8, 12-14, and 17-19. Respiratory variables were analyzed in wakefulness (W), quiet sleep (QS), and active sleep (AS), following suvorexant (5 mg/kg ip), a dual orexin receptor antagonist, or vehicle (DMSO). Effects of suvorexant on ventilatory responses to graded hypercapnia ([Formula: see text] = 0.02, 0.04, 0.06), hypoxia ([Formula: see text] = 0.10), and hyperoxia ([Formula: see text] = 1.0) at P12-14 were also tested. At P12-14, but not at other ages, suvorexant significantly reduced respiratory frequency in all states, reduced the ventilatory equivalent in QW and QS, and increased [Formula: see text] to ∼5 mmHg. Suvorexant had no effect on ventilatory responses to graded hypercapnia or hypoxia. Hyperoxia eliminated the effects of suvorexant on respiratory frequency at P12-14. Our data suggest that orexin preserves eupneic frequency and ventilation in rats, specifically at ∼2 wk of age, perhaps by facilitating tonic peripheral chemoreflex activity.


Assuntos
Células Quimiorreceptoras/metabolismo , Pulmão/inervação , Orexinas/metabolismo , Ventilação Pulmonar , Reflexo , Mecânica Respiratória , Animais , Animais Recém-Nascidos , Azepinas/farmacologia , Células Quimiorreceptoras/efeitos dos fármacos , Hipercapnia/metabolismo , Hipercapnia/fisiopatologia , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Antagonistas dos Receptores de Orexina/farmacologia , Receptores de Orexina/metabolismo , Ventilação Pulmonar/efeitos dos fármacos , Ratos Sprague-Dawley , Reflexo/efeitos dos fármacos , Mecânica Respiratória/efeitos dos fármacos , Sono , Triazóis/farmacologia , Vigília
5.
Molecules ; 26(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205930

RESUMO

BACKGROUND: Neurotic disturbances, anxiety, neurosis-like disorders, and stress situations are widespread. Benzodiazepine tranquillizers have been found to be among the most effective antianxiety drugs. The pharmacological action of benzodiazepines is due to their interaction with the supra-molecular membrane GABA-a-benzodiazepine receptor complex, linked to the Cl-ionophore. Benzodiazepines enhance GABA-ergic transmission and this has led to a study of the role of GABA in anxiety. The search for anxiolytics and anticonvulsive agents has involved glutamate-ergic, 5HT-ergic substances and neuropeptides. However, each of these well-known anxiolytics, anticonvulsants and cognition enhancers (nootropics) has repeatedly been reported to have many adverse side effects, therefore there is an urgent need to search for new drugs able to restore damaged cognitive functions without causing significant adverse reactions. OBJECTIVE: Considering the relevance of epilepsy diffusion in the world, we have addressed our attention to the discovery of new drugs in this field Thus our aim is the synthesis and study of new compounds with antiepileptic (anticonvulsant) and not only, activity. METHODS: For the synthesis of compounds classical organic methods were used and developed. For the evaluation of biological activity some anticonvulsant and psychotropic methods were used. RESULTS: As a result of multistep reactions 26 new, five-membered heterocyclic systems were obtained. PASS prediction of anticonvulsant activity was performed for the whole set of the designed molecules and probability to be active Pa values were ranging from 0.275 to 0.43. The studied compounds exhibit protection against pentylenetetrazole (PTZ) seizures, anti-thiosemicarbazides effect as well as some psychotropic effect. The biological assays evidenced that some of the studied compounds showed a high anticonvulsant activity by antagonism with pentylenetetrazole. The toxicity of compounds is low and they do not induce muscle relaxation in the studied doses. According to the study of psychotropic activity it was found that the selected compounds have an activating behavior and anxiolytic effects on the models of "open field" and "elevated plus maze" (EPM). The data obtained indicate the anxiolytic (anti-anxiety) activity of the derivatives of pyrimidines, especially pronounced in compounds 6n, 6b, and 7c. The studied compounds increase the latent time of first immobilization on the model of "forced swimming" (FST) and exhibit some antidepressant effect similarly to diazepam. Docking studies revealed that compound 6k bound tightly in the active site of GABAA receptor with a value of the scoring function that estimates free energy of binding (ΔG) at -7.95 kcal/mol, while compound 6n showed the best docking score and seems to be dual inhibitor of SERT transporter as well as 5-HT1A receptor. CONCLUSIONS: Тhe selected compounds have an anticonvulsant, activating behavior and anxiolytic effects, at the same time exhibit some antidepressant effect.


Assuntos
Azepinas/administração & dosagem , Azepinas/síntese química , Pirimidinas/administração & dosagem , Pirimidinas/síntese química , Convulsões/tratamento farmacológico , Animais , Ansiolíticos/administração & dosagem , Ansiolíticos/síntese química , Ansiolíticos/química , Ansiolíticos/farmacologia , Anticonvulsivantes/administração & dosagem , Anticonvulsivantes/síntese química , Anticonvulsivantes/química , Anticonvulsivantes/farmacologia , Azepinas/química , Azepinas/farmacologia , Modelos Animais de Doenças , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Pentilenotetrazol/efeitos adversos , Pirimidinas/química , Pirimidinas/farmacologia , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ratos , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Convulsões/induzido quimicamente , Convulsões/fisiopatologia
6.
Molecules ; 26(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201111

RESUMO

Recently, the first squaramide-(SA) containing FAP inhibitor-derived radiotracers were introduced. DATA5m.SA.FAPi and DOTA.SA.FAPi with their non-radioactive complexes showed high affinity and selectivity for FAP. After a successful preclinical study with [68Ga]Ga-DOTA.SA.FAPi, the first patient studies were realized for both compounds. Here, we present a new squaramide-containing compound targeting FAP, based on the AAZTA5 chelator 1,4-bis-(carboxylmethyl)-6-[bis-(carboxymethyl)-amino-6-pentanoic-acid]-perhydro-1,4-diazepine. For this molecule (AAZTA5.SA.FAPi), complexation with radionuclides such as gallium-68, scandium-44, and lutetium-177 was investigated, and the in vitro properties of the complexes were characterized and compared with those of DOTA.SA.FAPi. AAZTA5.SA.FAPi and its derivatives labelled with non-radioactive isotopes demonstrated similar excellent inhibitory potencies compared to the previously published SA.FAPi ligands, i.e., sub-nanomolar IC50 values for FAP and high selectivity indices over the serine proteases PREP and DPPs. Labeling with all three radiometals was easier and faster with AAZTA5.SA.FAPi compared to the corresponding DOTA analogue at ambient temperature. Especially, scandium-44 labeling with the AAZTA derivative resulted in higher specific activities. Both DOTA.SA.FAPi and AAZTA5.SA.FAPi showed sufficiently high stability in different media. Therefore, these FAP inhibitor agents could be promising for theranostic approaches targeting FAP.


Assuntos
Acetatos/farmacologia , Azepinas/farmacologia , Fibroblastos/efeitos dos fármacos , Compostos Heterocíclicos com 1 Anel/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Quinina/análogos & derivados , Endopeptidases , Fibroblastos/metabolismo , Radioisótopos de Gálio/farmacologia , Humanos , Ligantes , Lutécio/farmacologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Quinina/farmacologia , Radioisótopos/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Escândio/farmacologia , Serina Endopeptidases/metabolismo
7.
Cancer Sci ; 112(10): 4013-4025, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34252226

RESUMO

Although the role of bromodomain-containing protein 4 (BRD4) in ovarian cancer, pancreatic cancer, lymphoma, and many other diseases is well known, its function in cutaneous melanoma is only partially understood. The results of the present study show that the BRD4 inhibitor JQ1 promotes the apoptosis of B16 melanoma cells by altering mitochondrial dynamics, thereby inducing mitochondrial dysfunction and increasing oxidative stress. We found that treatment of B16 cells with different concentrations of JQ1 (125 nmol/L or 250 nmol/L) significantly downregulated the expression of protein subunits involved in mitochondrial respiratory chain complexes I, III, IV, and V, increased reactive oxygen species, induced energy metabolism dysfunction, significantly enhanced apoptosis, and activated the mitochondrial apoptosis pathway. At the same time, JQ1 inhibited the activation of AMP-activated protein kinase, a metabolic energy sensor. In addition, we found that the mRNA and protein levels of mitochondrial dynamin-related protein 1 increased, whereas the levels of mitochondrial fusion protein 1 and optic atrophy protein 1 decreased. Mechanistically, we determined that JQ1 inhibited the expression of c-Myc and altered mitochondrial dynamics, eventually leading to changes in the mitochondrial function, metabolism, and apoptosis of B16 melanoma cells.


Assuntos
Apoptose/fisiologia , Azepinas/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Melanoma/metabolismo , Mitocôndrias/efeitos dos fármacos , Neoplasias Cutâneas/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Respiração Celular/efeitos dos fármacos , Dinaminas/efeitos dos fármacos , Dinaminas/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/efeitos dos fármacos , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Proteína-1 Reguladora de Fusão/metabolismo , Humanos , Melanoma/patologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Subunidades Proteicas/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Cutâneas/patologia , Fatores de Transcrição/metabolismo
8.
Nat Commun ; 12(1): 3974, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172737

RESUMO

Cancer stem cells (CSCs) play a critical role in invasive growth and metastasis of human head and neck squamous cell carcinoma (HNSCC). Although significant progress has been made in understanding the self-renewal and pro-tumorigenic potentials of CSCs, a key challenge remains on how to eliminate CSCs and halt metastasis effectively. Here we show that super-enhancers (SEs) play a critical role in the transcription of cancer stemness genes as well as pro-metastatic genes, thereby controlling their tumorigenic potential and metastasis. Mechanistically, we find that bromodomain-containing protein 4 (BRD4) recruits Mediators and NF-κB p65 to form SEs at cancer stemness genes such as TP63, MET and FOSL1, in addition to oncogenic transcripts. In vivo lineage tracing reveals that disrupting SEs by BET inhibitors potently inhibited CSC self-renewal and eliminated CSCs in addition to elimination of proliferating non-stem tumor cells in a mouse model of HNSCC. Moreover, disrupting SEs also inhibits the invasive growth and lymph node metastasis of human CSCs isolated from human HNSCC. Taken together, our results suggest that targeting SEs may serve as an effective therapy for HNSCC by eliminating CSCs.


Assuntos
Elementos Facilitadores Genéticos , Neoplasias de Cabeça e Pescoço/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Animais , Antineoplásicos/farmacologia , Azepinas/farmacologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Humanos , Metástase Linfática/tratamento farmacológico , Metástase Linfática/prevenção & controle , Subunidade 1 do Complexo Mediador/genética , Subunidade 1 do Complexo Mediador/metabolismo , Camundongos Endogâmicos C57BL , Camundongos SCID , NF-kappa B/genética , Células-Tronco Neoplásicas/patologia , Complexo Repressor Polycomb 1/antagonistas & inibidores , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Cancer Sci ; 112(8): 3302-3313, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34032336

RESUMO

A novel proteasome deubiquitinase inhibitor, VLX1570, has been highlighted as a promising therapeutic agent mainly for lymphoid neoplasms and solid tumors. We examined in vitro effects of VLX1570 on eight myeloid and three lymphoid leukemia cell lines. From cell culture studies, 10 out of 11 cell lines except K562 were found to be susceptible to VLX1570 treatment and it inhibited cell growth mainly by apoptosis. Next, to identify the signaling pathways associated with apoptosis, we performed gene expression profiling using HL-60 with or without 50 nmol/L of VLX1570 for 3 hours and demonstrated that VLX1570 induced the genetic pathway involved in "heat shock transcription factor 1 (HSF1) activation", "HSF1 dependent transactivation", and "Regulation of HSF1 mediated heat shock response". VLX1570 increased the amount of high molecular weight polyubiquitinated proteins and the expression of HSP70 as the result of the suppression of ubiquitin proteasome system, the expression of heme oxygenase-1, and the amount of phosphorylation in JNK and p38 associated with the generation of reactive oxygen species (ROS) induced apoptosis and the amount of phosphorylation in eIF2α, inducing the expression of ATF4 and endoplasmic reticulum (ER) stress dependent apoptosis protein, CHOP, and the amount of phosphorylation slightly in IRE1α, leading to increased expression of XBP-1s in leukemia cell lines. In the present study, we demonstrate that VLX1570 induces apoptosis and exerts a potential anti-leukemic effect through the generation of ROS and induction of ER stress in leukemia cell lines.


Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Compostos de Benzilideno/farmacologia , Leucemia Linfoide/metabolismo , Leucemia Mieloide Aguda/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Células HL-60 , Humanos , Células K562 , Leucemia Linfoide/tratamento farmacológico , Leucemia Linfoide/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética
10.
Commun Biol ; 4(1): 640, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050235

RESUMO

Targeted protein degradation tools are becoming a new therapeutic modality, allowing small molecule ligands to be reformulated as heterobifunctional molecules (PROteolysis Targeting Chimeras, PROTACs) that recruit ubiquitin ligases to targets of interest, leading to ubiquitination and destruction of the targets. Several PROTACs against targets of clinical interest have been described, but detailed descriptions of the cell biology modulated by PROTACs are missing from the literature. Here we describe the functional characterization of a PROTAC derived from AURKA inhibitor MLN8237 (alisertib). We demonstrate efficient and specific destruction of both endogenous and overexpressed AURKA by Cereblon-directed PROTACs. At the subcellular level, we find differential targeting of AURKA on the mitotic spindle compared to centrosomes. The phenotypic consequences of PROTAC treatment are therefore distinct from those mediated by alisertib, and in mitotic cells differentially regulate centrosome- and chromatin- based microtubule spindle assembly pathways. In interphase cells PROTAC-mediated clearance of non-centrosomal AURKA modulates the cytoplasmic role played by AURKA in mitochondrial dynamics, whilst the centrosomal pool is refractory to PROTAC-mediated clearance. Our results point to differential sensitivity of subcellular pools of substrate, governed by substrate conformation or localization-dependent accessibility to PROTAC action, a phenomenon not previously described for this new class of degrader compounds.


Assuntos
Aurora Quinase A/metabolismo , Azepinas/farmacologia , Pirimidinas/farmacologia , Animais , Aurora Quinase A/antagonistas & inibidores , Azepinas/metabolismo , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Células HeLa , Humanos , Cinética , Ligantes , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Pirimidinas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Bibliotecas de Moléculas Pequenas/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
11.
Nat Commun ; 12(1): 2475, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33931637

RESUMO

An innovative approach to eliminate HIV-1-infected cells emerging out of latency, the major hurdle to HIV-1 cure, is to pharmacologically reactivate viral expression and concomitantly trigger intracellular pro-apoptotic pathways in order to selectively induce cell death (ICD) of infected cells, without reliance on the extracellular immune system. In this work, we demonstrate the effect of DDX3 inhibitors on selectively inducing cell death in latent HIV-1-infected cell lines, primary CD4+ T cells and in CD4+ T cells from cART-suppressed people living with HIV-1 (PLWHIV). We used single-cell FISH-Flow technology to characterise the contribution of viral RNA to inducing cell death. The pharmacological targeting of DDX3 induced HIV-1 RNA expression, resulting in phosphorylation of IRF3 and upregulation of IFNß. DDX3 inhibition also resulted in the downregulation of BIRC5, critical to cell survival during HIV-1 infection, and selectively induced apoptosis in viral RNA-expressing CD4+ T cells but not bystander cells. DDX3 inhibitor treatment of CD4+ T cells from PLWHIV resulted in an approximately 50% reduction of the inducible latent HIV-1 reservoir by quantitation of HIV-1 RNA, by FISH-Flow, RT-qPCR and TILDA. This study provides proof of concept for pharmacological reversal of latency coupled to induction of apoptosis towards the elimination of the inducible reservoir.


Assuntos
Apoptose/efeitos dos fármacos , Azepinas/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , RNA Helicases DEAD-box/metabolismo , Infecções por HIV/imunologia , HIV-1/metabolismo , Imidazóis/farmacologia , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antirretrovirais/farmacologia , Apoptose/genética , Azepinas/química , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/química , Inibidores Enzimáticos/farmacologia , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Imidazóis/química , Hibridização in Situ Fluorescente , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Células Jurkat , Simulação de Acoplamento Molecular , RNA Viral/metabolismo , Análise de Célula Única , Survivina/metabolismo , Ativação Viral/efeitos dos fármacos , Replicação Viral/genética
12.
Eur J Med Chem ; 220: 113445, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-33901899

RESUMO

Hymenialdisine an alkaloid of oroidin class has drawn the attention of researchers owing to its unique structural features and interesting biological properties. Hymenialdisine exhibited promising inhibitory activity against a number of therapeutically important kinases viz., CDKs, GSK-3ß etc., and showed anti-cancer, anti-inflammatory, anti-HIV, neuroprotective, anti-fouling, anti-plasmodium properties. Hymenialdisine and other structurally related oroidin alkaloids such as dibromo-hymenialdisine, stevensine, hymenin, axinohydantoin, spongicidines A-D, latonduines and callyspongisines contain pyrrolo[2,3-c] azepin-8-one core in common. Keeping in view of the interesting structural and therapeutic features of HMD, several structural modifications were carried around the fused-azepinone core which resulted in a number of diverse structural motifs like indolo-azepinones, paullones, aza-paullones, darpones and 5,7-dihydro-6H-benzo[b]pyrimido[4,5-d] azepin-6-one. In this review, an attempt is made to collate and review the structures of diverse hymenialdisine and related fused-azepinones of synthetic/natural origin and their biological properties.


Assuntos
Fármacos Anti-HIV/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Azepinas/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Fármacos Anti-HIV/química , Anti-Inflamatórios não Esteroides/química , Antineoplásicos/química , Azepinas/química , Humanos , Estrutura Molecular , Fármacos Neuroprotetores/química , Poríferos/química
13.
Int J Mol Sci ; 22(6)2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33809617

RESUMO

This study aimed to identify alternative anti-inflammatory compounds that modulate the activity of a relevant transcription factor, CCAAT/enhancer binding protein delta (C/EBPδ). C/EBPδ is a master regulator of inflammatory responses in macrophages (Mϕ) and is mainly regulated at the level of CEBPD gene transcription initiation. To screen for CEBPD-modulating compounds, we generated a THP-1-derived reporter cell line stably expressing secreted alkaline phosphatase (SEAP) under control of the defined CEBPD promoter (CEBPD::SEAP). A high-throughput screening of LOPAC®1280 and ENZO®774 libraries on LPS- and IFN-γ-activated THP-1 reporter Mϕ identified four epigenetically active hits: two bromodomain and extraterminal domain (BET) inhibitors, I-BET151 and Ro 11-1464, as well as two histone deacetylase (HDAC) inhibitors, SAHA and TSA. All four hits markedly and reproducibly upregulated SEAP secretion and CEBPD::SEAP mRNA expression, confirming screening assay reliability. Whereas BET inhibitors also upregulated the mRNA expression of the endogenous CEBPD, HDAC inhibitors completely abolished it. All hits displayed anti-inflammatory activity through the suppression of IL-6 and CCL2 gene expression. However, I-BET151 and HDAC inhibitors simultaneously upregulated the mRNA expression of pro-inflammatory IL-1ß. The modulation of CEBPD gene expression shown in this study contributes to our understanding of inflammatory responses in Mϕ and may offer an approach to therapy for inflammation-driven disorders.


Assuntos
Anti-Inflamatórios/farmacologia , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Genes Reporter , Ensaios de Triagem em Larga Escala , Inibidores de Histona Desacetilases/farmacologia , Macrófagos/metabolismo , Fosfatase Alcalina/metabolismo , Azepinas/farmacologia , Proteína delta de Ligação ao Facilitador CCAAT/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Medições Luminescentes , Macrófagos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células THP-1 , Tiofenos/farmacologia , Vorinostat/farmacologia
14.
J Pharmacol Sci ; 145(4): 335-339, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33712285

RESUMO

We previously reported that brotizolam, but not suvorexant, delayed recovery from isoflurane anesthesia in mice. However, the effects of hypnotics may be altered by the circadian rhythm. Locomotor activity was measured using sighted (ICR and C57BL/6J) and blind (FVB/N and C3H/HeN) mice, and the effects of hypnotics on isoflurane anesthesia were compared during the light and dark periods. In sighted mice, recovery induced by brotizolam was delayed in the light period, while that by suvorexant was delayed in the dark period. In C57BL/6J mice, delayed recovery induced by brotizolam was marked, and that by suvorexant was observed in the light and dark periods. Locomotor activity was low in the last 6 h of the dark period in blind mice, and was similar to that in the light period. In blind mice, delayed recovery induced by brotizolam was identical in both periods, while suvorexant did not influence recovery from isoflurane anesthesia. These results suggest that the effects of hypnotics on isoflurane anesthesia are altered by the circadian rhythm and that daily light-dark stimuli may be required for the chronopharmacological effects of hypnotics.


Assuntos
Período de Recuperação da Anestesia , Anestesia por Inalação , Anestésicos Inalatórios , Hipnóticos e Sedativos/farmacologia , Isoflurano , Fotoperíodo , Animais , Azepinas/farmacologia , Cronofarmacocinética , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/fisiologia , Neurônios GABAérgicos/efeitos dos fármacos , Neurônios GABAérgicos/fisiologia , Isoflurano/farmacologia , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Atividade Motora/efeitos dos fármacos , Fatores de Tempo , Triazóis/farmacologia
15.
Biochim Biophys Acta Mol Cell Res ; 1868(6): 119016, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33744274

RESUMO

Epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been widely used in the clinical treatment of non-small cell lung cancer (NSCLC) patients with EGFR mutations. Previous studies have shown that Aurora kinase A (AURKA) is overexpressed in a broad spectrum of cancer cells, which can induce epithelial-mesenchymal transition (EMT) and contribute to the occurrence of acquired EGFR-TKI resistance. However, whether the inhibition of AURKA could overcome EGFR-TKI resistance or reverse the EMT in TKI-resistant NSCLC cells remains unclear. In the current study, we established three EGFR-TKI-resistant cell lines and analyzed their expression profiles by RNA sequencing. The results revealed that the EMT pathway is significantly upregulated in the three cell lines with EGFR-TKI resistance. The phosphorylation of AURKA at Thr 288 was also upregulated, suggesting that the activation of AURKA plays an important role in the occurrence of EGFR-TKI resistance. Interestingly, the AURKA inhibitor, alisertib treatment restored the susceptibility of resistant cells to EGFR-TKIs and partially reversed the EMT process, thereby reducing migration and invasion in EGFR-TKI-resistant cells. This study provides evidence that targeting AURKA signaling pathway by alisertib may be a novel approach for overcoming EGFR-TKI resistance and for the treatment of metastatic EGFR-TKIs in NSCLC patients.


Assuntos
Aurora Quinase A/metabolismo , Azepinas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Aurora Quinase A/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Receptores ErbB/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação , Fosforilação/efeitos dos fármacos , Análise de Sequência de RNA/métodos , Regulação para Cima/efeitos dos fármacos
16.
Front Immunol ; 12: 609319, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33679744

RESUMO

Endotoxemia is a severe inflammation response induced by infection especially bacterial endotoxin translocation, which severely increases mortality in combination with acute colon injury. Bromodomain-containing protein 4 (BRD4) is an important Bromo and Extra-Terminal (BET) protein to participate in inflammatory responses. However, it is still unknown about the specific connection between BRD4 and inflammation-related pyroptosis in endotoxemia colon. Here, through evaluating the mucous morphology and the expression of tight junction proteins such as occludin and ZO1, we found the upregulation of BRD4 in damaged colon with poor tight junction in an endotoxemia mouse model induced by lipopolysaccharides (LPS). Firstly, the BRD4 inhibitor JQ1 was used to effectively protect colon tight junction in endotoxemia. As detected, high levels of pro-inflammation cytokines IL6, IL1ß and IL18 in endotoxemia colon were reversed by JQ1 pretreatment. In addition, JQ1 injection reduced endotoxemia-induced elevation of the phosphorylated NF κB and NLRP3/ASC/caspase 1 inflammasome complex in colon injury. Furthermore, activated pyroptosis markers gasdermins in endotoxemia colon were also blocked by JQ1 pretreatment. Together, our data indicate that BRD4 plays a critical role in regulating pyroptosis-related colon injury induced by LPS, and JQ1 as a BRD4 inhibitors can effectively protect colon from endotoxemia-induced inflammation injury.


Assuntos
Azepinas/farmacologia , Colite/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Piroptose/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Animais , Biomarcadores , Colite/tratamento farmacológico , Colite/etiologia , Colite/patologia , Modelos Animais de Doenças , Endotoxemia/complicações , Endotoxemia/etiologia , Imuno-Histoquímica , Inflamassomos/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Lipopolissacarídeos/efeitos adversos , Masculino , Camundongos , Fosforilação , Junções Íntimas/metabolismo
17.
Bioorg Chem ; 111: 104840, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33780687

RESUMO

To further explore the research of novel PARP-1 inhibitors, we designed and synthesized a series of novel amide PARP-1 inhibitors based on our previous research. Most compounds displayed certain antitumor activities against four tumor cell lines (A549, HepG2, HCT-116, and MCF-7). Specifically, the candidate compound R8e possessed strong anti-proliferative potency toward A549 cells with the IC50 value of 2.01 µM. Compound R8e had low toxicity to lung cancer cell line. And the in vitro enzyme inhibitory activity of compound R8e was better than rucaparib. Molecular docking studies provided a rational binding model of compound R8e in complex with rucaparib. The following cell cycle and apoptosis assays revealed that compound R8e could arrest cell cycle in the S phase and induce cell apoptosis. Western blot analysis further showed that compound R8e could effectively inhibit the PAR's biosynthesis and was more effective than rucaparib. Overall, based on the biological activity evaluation, compound R8e could be a potential lead compound for further developing novel amide PARP-1 inhibitors.


Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Cicloexanonas/farmacologia , Desenho de Fármacos , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Compostos de Espiro/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Azepinas/síntese química , Azepinas/química , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cicloexanonas/síntese química , Cicloexanonas/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/síntese química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Compostos de Espiro/síntese química , Compostos de Espiro/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas
18.
Front Immunol ; 12: 626255, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33717143

RESUMO

Natural killer (NK) cells are innate lymphocytes that play a pivotal role in the immune surveillance and elimination of transformed or virally infected cells. Using a chemo-genetic approach, we identify BET bromodomain containing proteins BRD2 and BRD4 as central regulators of NK cell functions, including direct cytokine secretion, NK cell contact-dependent inflammatory cytokine secretion from monocytes as well as NK cell cytolytic functions. We show that both BRD2 and BRD4 control inflammatory cytokine production in NK cells isolated from healthy volunteers and from rheumatoid arthritis patients. In contrast, knockdown of BRD4 but not of BRD2 impairs NK cell cytolytic responses, suggesting BRD4 as critical regulator of NK cell mediated tumor cell elimination. This is supported by pharmacological targeting where the first-generation pan-BET bromodomain inhibitor JQ1(+) displays anti-inflammatory effects and inhibit tumor cell eradication, while the novel bivalent BET bromodomain inhibitor AZD5153, which shows differential activity towards BET family members, does not. Given the important role of both cytokine-mediated inflammatory microenvironment and cytolytic NK cell activities in immune-oncology therapies, our findings present a compelling argument for further clinical investigation.


Assuntos
Inflamação/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Azepinas/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citocinas , Voluntários Saudáveis , Compostos Heterocíclicos com 2 Anéis/farmacologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Piperazinas/farmacologia , Pirazóis/farmacologia , Piridazinas/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma , Triazóis/farmacologia
19.
Proc Natl Acad Sci U S A ; 118(9)2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33619107

RESUMO

Reactivation of human cytomegalovirus (HCMV) from latency is a major health consideration for recipients of stem-cell and solid organ transplantations. With over 200,000 transplants taking place globally per annum, virus reactivation can occur in more than 50% of cases leading to loss of grafts as well as serious morbidity and even mortality. Here, we present the most extensive screening to date of epigenetic inhibitors on HCMV latently infected cells and find that histone deacetylase inhibitors (HDACis) and bromodomain inhibitors are broadly effective at inducing virus immediate early gene expression. However, while HDACis, such as myeloid-selective CHR-4487, lead to production of infectious virions, inhibitors of bromodomain (BRD) and extraterminal proteins (I-BETs), including GSK726, restrict full reactivation. Mechanistically, we show that BET proteins (BRDs) are pivotally connected to regulation of HCMV latency and reactivation. Through BRD4 interaction, the transcriptional activator complex P-TEFb (CDK9/CycT1) is sequestered by repressive complexes during HCMV latency. Consequently, I-BETs allow release of P-TEFb and subsequent recruitment to promoters via the superelongation complex (SEC), inducing transcription of HCMV lytic genes encoding immunogenic antigens from otherwise latently infected cells. Surprisingly, this occurs without inducing many viral immunoevasins and, importantly, while also restricting viral DNA replication and full HCMV reactivation. Therefore, this pattern of HCMV transcriptional dysregulation allows effective cytotoxic immune targeting and killing of latently infected cells, thus reducing the latent virus genome load. This approach could be safely used to pre-emptively purge the virus latent reservoir prior to transplantation, thereby reducing HCMV reactivation-related morbidity and mortality.


Assuntos
Proteínas de Ciclo Celular/genética , Citomegalovirus/imunologia , DNA Viral/genética , Epigênese Genética , Histona Desacetilases/genética , Fator B de Elongação Transcricional Positiva/genética , Fatores de Transcrição/genética , Azepinas/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzodiazepinas/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/imunologia , Ciclina T/genética , Ciclina T/imunologia , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/imunologia , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/patologia , Replicação do DNA/efeitos dos fármacos , DNA Viral/antagonistas & inibidores , DNA Viral/imunologia , Genes Precoces , Genes Reporter , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/imunologia , Interações Hospedeiro-Patógeno , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Fator B de Elongação Transcricional Positiva/imunologia , Cultura Primária de Células , Regiões Promotoras Genéticas , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Células THP-1 , Talidomida/análogos & derivados , Talidomida/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/imunologia , Transcrição Genética , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos
20.
Proc Natl Acad Sci U S A ; 118(9)2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33622787

RESUMO

HLA-C arose during evolution of pregnancy in the great apes 10 to 15 million years ago. It has a dual function on placental extravillous trophoblasts (EVTs) as it contributes to both tolerance and immunity at the maternal-fetal interface. The mode of its regulation is of considerable interest in connection with the biology of pregnancy and pregnancy abnormalities. First-trimester primary EVTs in which HLA-C is highly expressed, as well as JEG3, an EVT model cell line, were employed. Single-cell RNA-seq data and quantitative PCR identified high expression of the transcription factor ELF3 in those cells. Chromatin immunoprecipitation (ChIP)-PCR confirmed that both ELF3 and MED1 bound to the proximal HLA-C promoter region. However, binding of RFX5 to this region was absent or severely reduced, and the adjacent HLA-B locus remained closed. Expression of HLA-C was inhibited by ELF3 small interfering RNAs (siRNAs) and by wrenchnolol treatment. Wrenchnolol is a cell-permeable synthetic organic molecule that mimics ELF3 and is relatively specific for binding to ELF3's coactivator, MED23, as our data also showed in JEG3. Moreover, the ELF3 gene is regulated by a superenhancer that spans more than 5 Mb, identified by assay for transposase-accessible chromatin using sequencing (ATAC-seq), as well as by its sensitivity to (+)-JQ1 (inhibitor of BRD4). ELF3 bound to its own promoter, thus creating an autoregulatory feedback loop that establishes expression of ELF3 and HLA-C in trophoblasts. Wrenchnolol blocked binding of MED23 to ELF3, thus disrupting the positive-feedback loop that drives ELF3 expression, with down-regulation of HLA-C expression as a consequence.


Assuntos
Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Retroalimentação Fisiológica , Antígenos HLA-C/genética , Proteínas Proto-Oncogênicas c-ets/genética , Fatores de Transcrição/genética , Trofoblastos/imunologia , Aborto Legal , Adamantano/farmacologia , Azepinas/farmacologia , Linhagem Celular , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/imunologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , Antígenos HLA-C/imunologia , Humanos , Imunidade Materno-Adquirida , Indóis/farmacologia , Complexo Mediador/genética , Complexo Mediador/imunologia , Subunidade 1 do Complexo Mediador/genética , Subunidade 1 do Complexo Mediador/imunologia , Gravidez , Primeiro Trimestre da Gravidez , Cultura Primária de Células , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-ets/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ets/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Fatores de Transcrição de Fator Regulador X/genética , Fatores de Transcrição de Fator Regulador X/imunologia , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/imunologia , Triazóis/farmacologia , Trofoblastos/citologia , Trofoblastos/efeitos dos fármacos
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