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1.
Genes Dev ; 34(1-2): 53-71, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31857346

RESUMO

Hippo signaling controls organ size and tumor progression through a conserved pathway leading to nuclear translocation of the transcriptional effector Yki/Yap/Taz. Most of our understanding of Hippo signaling pertains to its cytoplasmic regulation, but how the pathway is controlled in the nucleus remains poorly understood. Here we uncover an evolutionarily conserved mechanism by which CDK7 promotes Yki/Yap/Taz stabilization in the nucleus to sustain Hippo pathway outputs. We found that a modular E3 ubiquitin ligase complex CRL4DCAF12 binds and targets Yki/Yap/Taz for ubiquitination and degradation, whereas CDK7 phosphorylates Yki/Yap/Taz at S169/S128/S90 to inhibit CRL4DCAF12 recruitment, leading to Yki/Yap/Taz stabilization. As a consequence, inactivation of CDK7 reduced organ size and inhibited tumor growth, which could be reversed by restoring Yki/Yap activity. Our study identifies an unanticipated layer of Hippo pathway regulation, defines a novel mechanism by which CDK7 regulates tissue growth, and implies CDK7 as a drug target for Yap/Taz-driven cancer.


Assuntos
Carcinogênese/genética , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Animais , Antineoplásicos/farmacologia , Carcinogênese/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Quinases Ciclina-Dependentes/genética , Drosophila melanogaster/genética , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/fisiopatologia , Camundongos , Tamanho do Órgão/genética , Fenilenodiaminas/farmacologia , Proteólise , Pirimidinas/farmacologia
2.
Int J Mol Sci ; 20(18)2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31487785

RESUMO

Kv7.2-Kv7.5 channels mediate the M-current (IKM), a K+-selective current regulating neuronal excitability and representing an attractive target for pharmacological therapy against hyperexcitability diseases such as pain. Kv7 channels interact functionally with transient receptor potential vanilloid 1 (TRPV1) channels activated by endogenous and/or exogenous pain-inducing substances, such as bradykinin (BK) or capsaicin (CAP), respectively; however, whether Kv7 channels of specific molecular composition provide a dominant contribution in BK- or CAP-evoked responses is yet unknown. To this aim, Kv7 transcripts expression and function were assessed in F11 immortalized sensorial neurons, a cellular model widely used to assess nociceptive molecular mechanisms. In these cells, the effects of the pan-Kv7 activator retigabine were investigated, as well as the effects of ICA-27243 and (S)-1, two Kv7 activators acting preferentially on Kv7.2/Kv7.3 and Kv7.4/Kv7.5 channels, respectively, on BK- and CAP-induced changes in intracellular Ca2+ concentrations ([Ca2+]i). The results obtained revealed the expression of transcripts of all Kv7 genes, leading to an IKM-like current. Moreover, all tested Kv7 openers inhibited BK- and CAP-induced responses by a similar extent (~60%); at least for BK-induced Ca2+ responses, the potency of retigabine (IC50~1 µM) was higher than that of ICA-27243 (IC50~5 µM) and (S)-1 (IC50~7 µM). Altogether, these results suggest that IKM activation effectively counteracts the cellular processes triggered by TRPV1-mediated pain-inducing stimuli, and highlight a possible critical contribution of Kv7.4 subunits.


Assuntos
Sinalização do Cálcio , Capsaicina/farmacologia , Canais de Potássio KCNQ/metabolismo , Células Receptoras Sensoriais/metabolismo , Fármacos do Sistema Sensorial/farmacologia , Canais de Cátion TRPV/metabolismo , Animais , Bradicinina/farmacologia , Cálcio/metabolismo , Carbamatos/farmacologia , Linhagem Celular , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Moduladores de Transporte de Membrana/farmacologia , Fenilenodiaminas/farmacologia , Ratos , Células Receptoras Sensoriais/efeitos dos fármacos
3.
Environ Pollut ; 254(Pt A): 112937, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31401526

RESUMO

PM2.5 is becoming a worldwide environmental problem, which profoundly endangers public health, thus progressively capturing public attention this decade. As a fragile target of PM2.5, the underlying mechanisms of endothelial cell damage are still obscure. According to the previous microarray data and signaling pathway analysis, a new form of cell death termed ferroptosis in the current study is proposed following PM2.5 exposure. In order to verify the vital role of ferroptosis in PM2.5-induced endothelial lesion and further understand the potential mechanism involved, intracellular iron content, ROS release and lipid peroxidation, as well as biomarkers of ferroptosis were detected, respectively. As a result, uptake of particles increases cellular iron content and ROS production. Meanwhile, GSH depletion, and the decrease of GSH-Px and NADPH play significant roles in PM2.5-induced endothelial cell ferroptosis. Moreover, significantly changed expression of TFRC, FTL and FTH1 hinted that dysfunction of iron uptake and storage is a major inducer of ferroptosis. Importantly, index monitored above can be partially rescued by lipid peroxidation inhibitor ferrostatin-1 and iron chelator deferoxamine mesylate, which mediated antiferroptosis activity mainly depends on the restoration of antioxidant activity and iron metabolism. In conclusion, our data basically show that PM2.5 enhances ferroptosis sensitivity with increased ferroptotic events in endothelial cells, in which iron overload, lipid peroxidation and redox imbalance act pivotal roles.


Assuntos
Células Endoteliais/metabolismo , Sobrecarga de Ferro/patologia , Ferro/toxicidade , Material Particulado/toxicidade , Antígenos CD/biossíntese , Apoferritinas/biossíntese , Apoptose/efeitos dos fármacos , Cicloexilaminas/farmacologia , Desferroxamina/farmacologia , Ferritinas/biossíntese , Glutationa/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores da Transferrina/biossíntese , Transdução de Sinais/efeitos dos fármacos
4.
J Neuroinflammation ; 16(1): 103, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101061

RESUMO

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a chronic metabolic dysfunction characterized by progressive insulin resistance and hyperglycaemia. Increased blood-brain barrier (BBB) permeability is a critical neurovascular complication of T2DM that adversely affects the central nervous system homeostasis and function. Histone deacetylase 3 (HDAC3) has been reported to be elevated in T2DM animals and may promote neuroinflammation; however, its involvement in the BBB permeability of T2DM has not been investigated. In this study, we tested our hypothesis that HDAC3 expression and activity are increased in the T2DM mouse brain. Inhibition of HDAC3 may ameliorate T2DM-induced BBB permeability through Nrf2 activation. METHODS: T2DM (db/db, leptin receptor-deficient), genetic non-hyperglycemic control (db/+), and wild-type male mice at the age of 16 weeks were used in this study. HDAC3 expression and activity, Nrf2 activation, and BBB permeability and junction protein expression were examined. The effects of HDAC3 activity on BBB permeability were tested using highly selective HDAC3 inhibitor RGFP966. In primary cultured human brain microvascular endothelial cells (HBMEC), hyperglycemia (25 mM glucose) plus interleukin 1 beta (20 ng/ml) (HG-IL1ß) served as T2DM insult in vitro. The effects of HDAC3 on transendothelial permeability were investigated by FITC-Dextran leakage and trans-endothelial electrical resistance, and the underlying molecular mechanisms were investigated using Western blot, q-PCR, co-immunoprecipitation, and immunocytochemistry for junction protein expression, miR-200a/Keap1/Nrf2 pathway regulation. RESULTS: HDAC3 expression and activity were significantly increased in the hippocampus and cortex of db/db mice. Specific HDAC3 inhibition significantly ameliorated BBB permeability and junction protein downregulation in db/db mice. In cultured HBMEC, HG-IL1ß insult significantly increased transendothelial permeability and reduced junction protein expression. HDAC3 inhibition significantly attenuated the transendothelial permeability and junction protein downregulation. Moreover, we demonstrated the underlying mechanism was at least in part attributed by HDAC3 inhibition-mediated miR-200a/Keap1/Nrf2 signaling pathway and downstream targeting junction protein expression in T2DM db/db mice. CONCLUSIONS: Our experimental results show that HDAC3 might be a new therapeutic target for BBB damage in T2DM.


Assuntos
Barreira Hematoencefálica/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Acrilamidas/farmacologia , Acrilamidas/uso terapêutico , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Linhagem Celular , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Permeabilidade/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Fenilenodiaminas/uso terapêutico
5.
Chemistry ; 25(41): 9691-9700, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31087710

RESUMO

Guanine-rich sequences of DNA are known to readily fold into tetra-stranded helical structures known as G-quadruplexes (G4). Due to their biological relevance, G4s are potential anticancer drug targets and therefore there is significant interest in molecules with high affinity for these structures. Most G4 binders are polyaromatic planar compounds which π-π stack on the G4's guanine tetrad. However, many of these compounds are not very selective since they can also intercalate into duplex DNA. Herein we report a new class of binder based on an octahedral cobalt(III) complex that binds to G4 via a different mode involving hydrogen bonding, electrostatic interactions and π-π stacking. We show that this new compound binds selectivity to G4 over duplex DNA (particularly to the G-rich sequence of the c-myc promoter). This new octahedral complex also has the ability to template the formation of G4 DNA from the unfolded sequence. Finally, we show that upon binding to G4, the complex prevents helicase Pif1-p from unfolding the c-myc G4 structure.


Assuntos
Cobalto/química , Cobalto/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , DNA/química , Quadruplex G/efeitos dos fármacos , Animais , Bovinos , DNA/genética , DNA/metabolismo , DNA Helicases/metabolismo , Genes myc/efeitos dos fármacos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Fenilenodiaminas/química , Fenilenodiaminas/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos
6.
Eur J Pharmacol ; 853: 193-200, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30928630

RESUMO

Acute myeloid leukemia (AML) arises from neoplastic transformation of hematopoietic stem and progenitor cells, and resistance to conventional chemotherapy remains one of the greatest challenges in treating the disease. Extensive data have demonstrated that angiogenesis is associated with AML progression and chemotherapy resistance. Thus, targeting angiogenesis may be a promising approach for AML treatment. In this study, we investigated the effectiveness of CS2164 (named as Chiauranib), a novel receptor tyrosine kinase inhibitor, in AML cells. Our results illustrated that CS2164 significantly suppressed cell proliferation and abolished clonogenicity in AML cells in a dose- and time-dependent manner. Meanwhile, CS2164 markedly induced apoptosis of AML cell lines and primary AML cells from 42 adult patients. Furthermore, we found that CS2164 has a comprehensive activity against AML irrespective of disease status and genetic mutations. Also, CS2164 suppressed AML growth in xenograft models in vivo. Mechanistically, CS2164-induced cytotoxicity was closely associated with inhibition of VEGFR2 and its downstream signaling cascades, including Src/Fyn/p38 and Erk/MEK. In conclusion, our study indicates that CS2164 exerts anti-leukemia effect by inducing apoptosis through suppressing the VEGFR2 pathway, supporting a potential role for CS2164 in the treatment of AML.


Assuntos
Antineoplásicos/farmacologia , Leucemia Mieloide Aguda/patologia , Fenilenodiaminas/farmacologia , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Camundongos , Mutação , Fatores de Tempo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Biochim Biophys Acta Mol Cell Res ; 1866(6): 978-991, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30857869

RESUMO

Extracellular amino acid (AA) withdrawal/restriction invokes an integrated stress response (ISR) that induces global suppression of protein synthesis whilst allowing transcription and translation of a select group of genes, whose protein products facilitate cellular adaptation to AA insufficiency. Transcriptional induction of the System A/SNAT2 AA transporter represents a classic adaptation response and crucially depends upon activation of the General Control Nonderepressible-2 kinase/Activating transcription factor 4 (GCN2/ATF4) pathway. However, the ISR may also include additional signalling inputs operating in conjunction or independently of GCN2/ATF4 to upregulate SNAT2. Herein, we show that whilst pharmacological inhibition of MEK-ERK, mTORC1 and p38 MAP kinase signalling has no detectable effect on System A upregulation, inhibitors targeting GSK3 (e.g. SB415286) caused significant repression of the SNAT2 adaptation response. Strikingly, the effects of SB415286 persist in cells in which GSK3α/ß have been stably silenced indicating an off-target effect. We show that SB415286 can also inhibit cyclin-dependent kinases (CDK) and that roscovitine and flavopiridol (two pan CDK inhibitors) are effective repressors of the SNAT2 adaptive response. In particular, our work reveals that CDK7 activity is upregulated in AA-deprived cells in a GCN-2-dependent manner and that a potent and selective CDK7 inhibitor, THZ-1, not only attenuates the increase in ATF4 expression but blocks System A adaptation. Importantly, the inhibitory effects of THZ-1 on System A adaptation are mitigated in cells expressing a doxycycline-inducible drug-resistant form of CDK7. Our data identify CDK7 as a novel component of the ISR regulating System A adaptation in response to AA insufficiency.


Assuntos
Sistema A de Transporte de Aminoácidos/metabolismo , Aminoácidos/deficiência , Quinases Ciclina-Dependentes/metabolismo , Estresse Fisiológico , Fator 4 Ativador da Transcrição/metabolismo , Aminofenóis/farmacologia , Animais , Linhagem Celular , Flavonoides/farmacologia , Células HEK293 , Células HeLa , Humanos , Maleimidas/farmacologia , Fenilenodiaminas/farmacologia , Piperidinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/farmacologia , Ratos , Roscovitina/farmacologia
8.
Neuroscience ; 406: 109-125, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30858110

RESUMO

Neuroinflammation is associated with increased vulnerability to diverse psychiatric conditions, including treatment-resistant major depressive disorder (MDD). Here we assessed whether high fat diet (HFD) induced neuroinflammation may be suitable to model a treatment-resistant depressive-like brain state in mice. Male and female mice were fed a HFD for 18 weeks, followed by quantitation of glucose tolerance, inflammatory markers of brain tissue (TNFα, IL-6, IL-1ß, Iba-1), neural excitability in the prelimbic cortex (PLC), as well as assessment of emotional reactivity and hedonic behavior in a battery of behavioral tests. In addition, we assessed the behavioral responsiveness of mice to fluoxetine, desipramine, ketamine, and the Kv7 channel opener and anticonvulsant retigabine. HFD exposure led to glucose intolerance and neuroinflammation in male mice, with similar but non-significant trends in females. Neuroinflammation of males was associated with anxious-depressive-like behavior and defects in working memory, along with neural hyperexcitability and increased Ih currents of pyramidal cells in the PLC. The behavioral changes were largely resistant to chronic treatment with fluoxetine and desipramine, as well as ketamine. By contrast, retigabine (also known as ezogabine) normalized neural excitability and Ih currents recorded from slices of HFD-treated animals and significantly ameliorated most of the behavioral impairments, without effects in control diet exposed animals. Thus, treatment resistant depressive-like brain states that are associated with chronic neuroinflammation may involve hyperexcitability of pyramidal neurons and may be effectively treated by retigabine.


Assuntos
Encéfalo/efeitos dos fármacos , Carbamatos/uso terapêutico , Transtorno Depressivo Resistente a Tratamento/tratamento farmacológico , Dieta Hiperlipídica/efeitos adversos , Canal de Potássio KCNQ1/fisiologia , Fenilenodiaminas/uso terapêutico , Animais , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Encéfalo/metabolismo , Carbamatos/farmacologia , Transtorno Depressivo Resistente a Tratamento/etiologia , Transtorno Depressivo Resistente a Tratamento/metabolismo , Feminino , Canal de Potássio KCNQ1/agonistas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Fenilenodiaminas/farmacologia
9.
Cell Oncol (Dordr) ; 42(4): 449-458, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30838525

RESUMO

PURPOSE: Despite the development of molecular targeted therapies, few advances have been made in the treatment of lung squamous cell carcinoma (SCC). SOX2 amplification is one of the most common genetic alterations in SCC. Here, we investigated the effects of THZ1, a potent cyclin-dependent kinase 7 (CDK7) inhibitor that plays a key role in gene transcription, in SCC. METHODS: Lung SCC-derived cell viabilities were assessed using a CCK-8 assay. SOX2 expression and RNAPII-CTD phosphorylation levels after THZ1 treatment were determined by Western blotting. The effect of SOX2 suppression using shRNA was assessed by flow cytometry. Gene expression patterns after THZ1 treatment of lung SCC-derived cells were identified using microarray-based mRNA profiling. RESULTS: We found that THZ1 treatment led to suppression of cell growth and apoptotic cell death in SOX2-amplified SCC-derived cells only, whereas the modest growth-inhibitory effect of cisplatin did not differ according to SOX2 amplification status. We also found that THZ1 decreased the phosphorylation of the carboxyl-terminal domain of RNA polymerase II and the expression of several genes. Specifically, we found that the expression of transcription-associated genes, including SOX2, was down-regulated by THZ1 in SOX2-amplified SCC cells. This inhibition of SOX2 expression resulted in suppression of the growth of these cells. CONCLUSIONS: From our data, we conclude that THZ1 may effectively control the proliferation and survival of SOX2-amplified SCC cells through a decrease in global transcriptional activity, suggesting that CDK7 inhibition leading to transcription suppression may be a promising therapeutic option for lung SCC with a SOX2 amplification.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Quinases Ciclina-Dependentes/antagonistas & inibidores , Amplificação de Genes , Neoplasias Pulmonares/tratamento farmacológico , Terapia de Alvo Molecular , Fatores de Transcrição SOXB1/genética , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , Regulação para Baixo/efeitos dos fármacos , Amplificação de Genes/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Fenilenodiaminas/farmacologia , Fenilenodiaminas/uso terapêutico , Fosforilação/efeitos dos fármacos , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , RNA Polimerase II/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transcrição Genética/efeitos dos fármacos
10.
PLoS One ; 14(2): e0212818, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30794682

RESUMO

BACKGROUND: Growth Arrest and DNA Damage 45γ (GADD45γ) is a member of the DNA damage-inducible gene family which responds to environmental stresses. Apoptosis is a critical mode of renal tubular cell death in nephrotoxin-induced acute kidney injury. In this study, we investigated the role of GADD45γ in renal tubular cell apoptosis induced by nephrotoxic drugs. METHODS: Primary human renal tubular epithelial (HRE) cells were used in this study. To derive stable cell lines in which GADD45γ expression was silenced, HRE cells were transduced with a plasmid encoding GADD45γ-specific shRNA. The recombinant adenovirus containing the GADD45γ gene was synthesized to overexpress GADD45γ protein. Cell death was induced by cisplatin and cyclosporine A (CsA). To prevent apoptotic cell death, pan-caspase inhibitor ZVAD-FMK was used. To prevent non-apoptotic cell death, necrostatin-1 and ferrostatin-1 were used. The degree of apoptosis and necrosis of cultured cells were evaluated by flow cytometry. RESULTS: Expression of the GADD45γ gene was significantly upregulated in response to treatment with CsA and cisplatin. Apoptosis and necrosis induced by these drugs were significantly reduced by silencing of GADD45γ, and significantly augmented by the overexpression of GADD45γ. The activation of caspase-3 and caspase-7 as well as caspase-9 induced by cisplatin or CsA was reduced by silencing of GADD45γ, and was augmented by the overexpression of GADD45γ, indicating that caspase activation is dependent on the expression of GADD45γ. ZVAD-FMK significantly inhibited apoptosis induced by cisplatin or CsA, indicating a role of caspases in mediating apoptotic cell death. ZVAD-FMK was effective to prevent necrosis as well, indicating that the observed necrosis was a secondary event following apoptosis at least in part. CONCLUSIONS: To our knowledge, this is the first study to show that GADD45γ is required for the caspase-dependent apoptosis of renal tubular cells induced by nephrotoxic drugs.


Assuntos
Apoptose , Caspase 3/metabolismo , Caspase 7/metabolismo , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Túbulos Renais/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Inibidores de Caspase/farmacologia , Linhagem Celular , Cisplatino/efeitos adversos , Cisplatino/farmacologia , Cicloexilaminas/farmacologia , Ciclosporina/efeitos adversos , Ciclosporina/farmacologia , Células Epiteliais/patologia , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Túbulos Renais/patologia , Fenilenodiaminas/farmacologia
11.
Nucleic Acids Res ; 47(8): 3921-3936, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30805632

RESUMO

The t(8;21) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML). We found that t(8;21) AML were extremely sensitive to THZ1, which triggered apoptosis after only 4 h. We used precision nuclear run-on transcription sequencing (PROseq) to define the global effects of THZ1 and other CDK inhibitors on RNA polymerase II dynamics. Inhibition of CDK7 using THZ1 caused wide-spread loss of promoter-proximal paused RNA polymerase. This loss of 5' pausing was associated with accumulation of polymerases in the body of a large number of genes. However, there were modest effects on genes regulated by 'super-enhancers'. At the 3' ends of genes, treatment with THZ1 suppressed RNA polymerase 'read through' at the end of the last exon, which resembled a phenotype associated with a mutant RNA polymerase with slower elongation rates. Consistent with this hypothesis, polyA site-sequencing (PolyA-seq) did not detect differences in poly A sites after THZ1 treatment. PROseq analysis after short treatments with THZ1 suggested that these 3' effects were due to altered CDK7 activity at the 5' end of long genes, and were likely to be due to slower rates of elongation.


Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/genética , Regulação Leucêmica da Expressão Gênica , Fenilenodiaminas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , RNA Polimerase II/genética , Região 3'-Flanqueadora , Região 5'-Flanqueadora/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Flavonoides/farmacologia , Humanos , Células Mieloides/metabolismo , Células Mieloides/patologia , Piperazinas/farmacologia , Piperidinas/farmacologia , Piperidonas/farmacologia , Piridinas/farmacologia , Compostos de Piridínio/farmacologia , Pirróis/farmacologia , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , Translocação Genética
12.
Parasit Vectors ; 12(1): 23, 2019 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-30635027

RESUMO

BACKGROUND: The food-borne liver fluke Opisthorchis felineus is an epidemiologically important species and the causative agent of opisthorchiasis across an extensive territory of Eurasia. For decades, treatment of opisthorchiasis has been based on praziquantel. Tribendimidine could be an alternative drug that has been successfully tested for Opisthorchis viverrini and Clonorchis sinensis infections. We aimed to assess tribendimidine effects in comparison with praziquantel in vivo and in vitro against the liver fluke Opisthorchis felineus. RESULTS: In this study we (i) calculated half-maximal inhibitory concentrations (IC50) by motility tests against O. felineus adults and newly excysted metacercarie after tribendimidine treatment in vitro; (ii) determined whether tribendimidine and PZQ effects on adult liver flukes are dependent on or mediated by white blood cells; and (iii) tested in vivo the anthelmintic activity of tribendimidine on juvenile and adult worms. We found that the efficiency of tribendimidine in vitro was similar (IC50 = 0.23 µM for newly excysted metacercariae and 0.19 µM for adult worms) to that of praziquantel (IC50 0.98 µM for newly excysted metacercariae and 0.47 µM for adult worms). The treatment of adult worms in vivo with praziquantel or tribendimidine at 400 mg/kg resulted in a 76% and 77.2% reduction, respectively, in the worm burden during chronic infection. CONCLUSIONS: The differences between WBR values after PZQ and TBN treatment were not significant, thus tribendimidine was as effective as praziquantel against O. felineus liver flukes. Given the broad-spectrum activity of tribendimidine and efficacy against O. felineus, this drug may be a promising candidate for the treatment of opisthorchiasis felinea and other liver fluke infections.


Assuntos
Anti-Helmínticos/farmacologia , Opistorquíase/tratamento farmacológico , Opisthorchis/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Animais , Cricetinae , Mesocricetus , Opistorquíase/parasitologia , Praziquantel/farmacologia
13.
Eur J Pharmacol ; 848: 96-104, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30682334

RESUMO

[4-(acetylamino)-N-(2-amino-phenyl) benzamide] (CI-994) is a histone deacetylase 1-3 specific inhibitor that has been shown to indirectly increase the production of Dickkopf-1, which is an inhibitor of osteoclastic development. However, whether CI-994 has an influence on receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis is still unclear; in our study, this mechanism was investigated. In an in vitro study, using a tartrate-resistant acid phosphatase (TRAP) stain, an F-actin ring, bone absorption test, quantitative PCR and Western blotting, the role of CI-994 in osteoclastogenesis and the expression of related genes and proteins were investigated. In an in vivo study, the effect of CI-994 on osteolysis was evaluated using a titanium particle-induced murine calvarial osteolysis model. Our results indicated that CI-994 inhibited osteoclast differentiation and the function of bone resorption without cytotoxic effects. Moreover, CI-994 inhibited the expression of osteoclast-related genes, including ACP5, CTSK, NFATc1, c-Fos, DC-STAMP and V-ATPase-d2. Furthermore, CI-994 suppressed the phosphorylation of IκBα and p65 and the expression of downstream c-Fos and NFATc1. Consistent with the in vitro results described above, our in vivo experiment indicated that CI-994 inhibited Ti-induced osteolysis. In conclusion, CI-994 inhibited osteoclastogenesis by suppressing NF-κB and the downstream c-Fos/NFATc1 signaling pathway. Thus, this study showed the possibility of using CI-994 for the treatment of exorbitant osteoclastic bone resorption.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , NF-kappa B/antagonistas & inibidores , Fatores de Transcrição NFATC/antagonistas & inibidores , Osteogênese/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores de Histona Desacetilases/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Osteogênese/fisiologia , Osteólise/tratamento farmacológico , Osteólise/metabolismo , Fenilenodiaminas/uso terapêutico , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
14.
Oncogene ; 38(20): 3932-3945, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30692639

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with high mortality. Lack of effective treatment makes novel therapeutic discovery an urgent demand in PDAC research. By screening an epigenetic-related compound library, we identified THZ1, a covalent inhibitor of CDK7, as a promising candidate. Multiple long-established and patient-derived PDAC cell lines (PDC) were used to validate the efficacy of THZ1 in vitro. In addition, patient-derived xenograft (PDX) models and animal models of PDAC were utilized for examining THZ1 efficacy in vivo. Furthermore, RNA-Seq analyse was performed to reveal the molecular mechanism of THZ1 treatment. Finally, PDAC cell lines with primary or acquired resistance to THZ1 were investigated to explore the potential mechanism of THZ1 susceptibility. CDK7 inhibition was identified as a selective and potent therapeutic strategy for PDAC progression in multiple preclinical models. Mechanistic analyses revealed that CDK7 inhibition led to a pronounced downregulation of gene transcription, with a preferential repression of mitotic cell cycle and NF-κB signaling-related transcripts. MYC transcriptional was found to be involved in susceptibility of PDAC cells to CDK7 inhibition. In conclusion, Identification of CDK7-dependent transcriptional addiction in PDACs provides a potent therapeutic strategy that targets highly aggressive pancreatic cancer.


Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Quinases Ciclina-Dependentes/genética , Neoplasias Pancreáticas/tratamento farmacológico , Fenilenodiaminas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Animais , Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes myc , Humanos , Masculino , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/etiologia , Neoplasias Experimentais/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Pancreatite/complicações , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Pancreatology ; 19(2): 383-389, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30670333

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is the second leading cause of cancer-related deaths worldwide. Despite immune checkpoints based immunotherapy highlights a new therapeutic strategy and achieves a remarkable therapeutic effect in various types of malignant tumors. Pancreatic cancer is one of the non-immunogenic cancers and is resistant to immunotherapy. Programmed death ligand 1 (PD-L1) is expressed on the surface of tumor cells and its level is a key determinant of the checkpoint immunotherapy efficacy. Here, we reported that the specific inhibitor of histone deacetylase 3 (HDAC3) decreased the protein and mRNA level of PD-L1 in pancreatic cancer cells. Furthermore, we showed that HDAC3 was critical for PD-L1 regulation and positively correlated with PD-L1 in PDAC patient specimens. Finally, we demonstrated that HDAC3/signal transducer and activator of transcription 3 (STAT3) pathway transcriptionally regulated PD-L1 expression. Collectively, our data contributes to a better understanding of the function of HDAC3 in cancer immunity and the regulatory mechanism of PD-L1. More importantly, these data suggest that the HDAC3 inhibitors might be used to improve immunotherapy in pancreatic cancer.


Assuntos
Antígeno B7-H1/metabolismo , Histona Desacetilases/metabolismo , Neoplasias Pancreáticas/metabolismo , Acrilamidas/farmacologia , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Esquema de Medicação , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fenilenodiaminas/farmacologia , Interferência de RNA , RNA Mensageiro , Transcrição Genética
16.
Neuropsychopharmacology ; 44(6): 1152-1162, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30647450

RESUMO

Chronic stress promotes depression in some individuals, but has no effect in others. Susceptible individuals exhibit social avoidance and anxious behavior and ultimately develop depression, whereas resilient individuals live normally. Exercise counteracts the effects of stress. Our objective was to examine whether lactate, a metabolite produced during exercise and known to reproduce specific brain exercise-related changes, promotes resilience to stress and acts as an antidepressant. To determine whether lactate promotes resilience to stress, male C57BL/6 mice experienced daily defeat by a CD-1 aggressor, for 10 days. On the 11th day, mice were subjected to behavioral tests. Mice received lactate before each defeat session. When compared with control mice, mice exposed to stress displayed increased susceptibility, social avoidance and anxiety. Lactate promoted resilience to stress and rescued social avoidance and anxiety by restoring hippocampal class I histone deacetylase (HDAC) levels and activity, specifically HDAC2/3. To determine whether lactate is an antidepressant, mice only received lactate from days 12-25 and a second set of behavioral tests was conducted on day 26. In this paradigm, we examined whether lactate functions by regulating HDACs using co-treatment with CI-994, a brain-permeable class I HDAC inhibitor. When administered after the establishment of depression, lactate behaved as antidepressant. In this paradigm, lactate regulated HDAC5 and not HDAC2/3 levels. On the contrary, HDAC2/3 inhibition was antidepressant-like. This indicates that lactate mimics exercise's effects and rescues susceptibility to stress by modulating HDAC2/3 activity and suggests that HDAC2/3 play opposite roles before and after establishment of susceptibility to stress.


Assuntos
Antidepressivos/farmacologia , Ansiedade/prevenção & controle , Aprendizagem da Esquiva , Depressão/metabolismo , Hipocampo/metabolismo , Histona Desacetilases/metabolismo , Ácido Láctico/farmacologia , Resiliência Psicológica , Comportamento Social , Estresse Psicológico/prevenção & controle , Animais , Antidepressivos/administração & dosagem , Aprendizagem da Esquiva/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Depressão/tratamento farmacológico , Modelos Animais de Doenças , Suscetibilidade a Doenças , Hipocampo/efeitos dos fármacos , Histona Desacetilase 2/efeitos dos fármacos , Histona Desacetilase 2/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/efeitos dos fármacos , Ácido Láctico/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenilenodiaminas/farmacologia , Resiliência Psicológica/efeitos dos fármacos
17.
Chem Res Toxicol ; 32(2): 294-303, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30638013

RESUMO

Retigabine (RTG) is an antiepileptic drug approved as an adjunctive treatment for refractory partial-onset seizures in adults. In April 2013, the Food and Drug Administration issued a warning that RTG could cause changes in retinal pigmentation and discoloration of skin, resulting in a blue appearance. As part of a larger preclinical effort to gain a mechanistic understanding as to the origins of retinal pigment changes associated with RTG, we conducted a long-term repeat dosing study in rats. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) was used to determine the distribution of RTG and its metabolites in the rat eye following 13 and 39 weeks of dosing. IMS revealed the presence of RTG, a previously characterized N-acetyl metabolite of RTG (NAMR), and several species structurally related through the dimerization of RTG and NAMR. These species were highly localized to the melanin-containing layers of the uveal tract of the rat eye including the choroid, ciliary body, and iris, suggesting that the formation of these dimers occurs from melanin bound RTG and NAMR. Furthermore, several of the RTG-related dimers have UV absorbance which give them a purple color in solution. We propose that the melanin binding of RTG and NAMR effectively concentrates the two compounds to enable mixed condensation reactions to occur when the binding provides the proper geometry in the redox environment of the uveal tissues. High lateral resolution images illustrate that the blood-retinal barrier effectively restricts retinal access to RTG-related compounds. The spatial information provided by MALDI IMS was critical in contextualizing the homogenate concentrations of key RTG-related compounds and helped provide a basis for the mechanism of dimer formation.


Assuntos
Carbamatos/metabolismo , Fenilenodiaminas/metabolismo , Pigmentos da Retina/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Úvea/efeitos dos fármacos , Animais , Carbamatos/farmacologia , Dimerização , Masculino , Melaninas/química , Melaninas/metabolismo , Fenilenodiaminas/farmacologia , Ratos , Ratos Long-Evans , Úvea/metabolismo , Úvea/patologia
18.
Behav Brain Res ; 356: 453-469, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29860001

RESUMO

Epigenetic mechanisms are key for regulating long-term memory (LTM) and are known to exert control on memory formation in multiple systems of the adult brain, including the sensory cortex. One epigenetic mechanism is chromatin modification by histone acetylation. Blocking the action of histone de-acetylases (HDACs) that normally negatively regulate LTM by repressing transcription has been shown to enable memory formation. Indeed, HDAC inhibition appears to facilitate memory by altering the dynamics of gene expression events important for memory consolidation. However, less understood are the ways in which molecular-level consolidation processes alter subsequent memory to enhance storage or facilitate retrieval. Here we used a sensory perspective to investigate whether the characteristics of memory formed with HDAC inhibitors are different from naturally-formed memory. One possibility is that HDAC inhibition enables memory to form with greater sensory detail than normal. Because the auditory system undergoes learning-induced remodeling that provides substrates for sound-specific LTM, we aimed to identify behavioral effects of HDAC inhibition on memory for specific sound features using a standard model of auditory associative cue-reward learning, memory, and cortical plasticity. We found that three systemic post-training treatments of an HDAC3-inhibitor (RGPF966, Abcam Inc.) in rats in the early phase of training facilitated auditory discriminative learning, changed auditory cortical tuning, and increased the specificity for acoustic frequency formed in memory of both excitatory (S+) and inhibitory (S-) associations for at least 2 weeks. The findings support that epigenetic mechanisms act on neural and behavioral sensory acuity to increase the precision of associative cue memory, which can be revealed by studying the sensory characteristics of long-term associative memory formation with HDAC inhibitors.


Assuntos
Acrilamidas/farmacologia , Encéfalo/efeitos dos fármacos , Histona Desacetilases/efeitos dos fármacos , Memória/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Animais , Condicionamento Clássico/efeitos dos fármacos , Sinais (Psicologia) , Masculino , Ratos Sprague-Dawley , Recompensa
19.
J Neurosci ; 39(4): 612-626, 2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30504275

RESUMO

Histone deacetylase (HDAC) inhibitors may have therapeutic utility in multiple neurological and psychiatric disorders, but the underlying mechanisms remain unclear. Here, we identify BRD4, a BET bromodomain reader of acetyl-lysine histones, as an essential component involved in potentiated expression of brain-derived neurotrophic factor (BDNF) and memory following HDAC inhibition. In in vitro studies, we reveal that pharmacological inhibition of BRD4 reversed the increase in BDNF mRNA induced by the class I/IIb HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). Knock-down of HDAC2 and HDAC3, but not other HDACs, increased BDNF mRNA expression, whereas knock-down of BRD4 blocked these effects. Using dCas9-BRD4, locus-specific targeting of BRD4 to the BDNF promoter increased BDNF mRNA. In additional studies, RGFP966, a pharmacological inhibitor of HDAC3, elevated BDNF expression and BRD4 binding to the BDNF promoter, effects that were abrogated by JQ1 (an inhibitor of BRD4). Examining known epigenetic targets of BRD4 and HDAC3, we show that H4K5ac and H4K8ac modifications and H4K5ac enrichment at the BDNF promoter were elevated following RGFP966 treatment. In electrophysiological studies, JQ1 reversed RGFP966-induced enhancement of LTP in hippocampal slice preparations. Last, in behavioral studies, RGFP966 increased subthreshold novel object recognition memory and cocaine place preference in male C57BL/6 mice, effects that were reversed by cotreatment with JQ1. Together, these data reveal that BRD4 plays a key role in HDAC3 inhibitor-induced potentiation of BDNF expression, neuroplasticity, and memory.SIGNIFICANCE STATEMENT Some histone deacetylase (HDAC) inhibitors are known to have neuroprotective and cognition-enhancing properties, but the underlying mechanisms have yet to be fully elucidated. In the current study, we reveal that BRD4, an epigenetic reader of histone acetylation marks, is necessary for enhancing brain-derived neurotrophic factor (BDNF) expression and improved memory following HDAC inhibition. Therefore, by identifying novel epigenetic regulators of BDNF expression, these data may lead to new therapeutic targets for the treatment of neuropsychiatric disorders.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Inibidores de Histona Desacetilases/farmacologia , Memória/efeitos dos fármacos , Acrilamidas/farmacologia , Animais , Azepinas/farmacologia , Fator Neurotrófico Derivado do Encéfalo/efeitos dos fármacos , Epigênese Genética , Técnicas de Silenciamento de Genes , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Fenilenodiaminas/farmacologia , Ratos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Triazóis/farmacologia , Vorinostat/farmacologia
20.
Acta Pharmacol Sin ; 40(6): 814-822, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30446732

RESUMO

Cancer cells always require more nutrients, energy, and biosynthetic activity to sustain their rapid proliferation than normal cells. Previous studies have shown the impact of THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7), on transcription regulation and cell-cycle arrest in numerous cancers, but its effects on cellular metabolism in cancer cells remain unknown. In this study we elucidated the anticancer mechanism of THZ1 in human non-small-cell lung cancer (NSCLC) cells. We showed that treatment with THZ1 (10-1000 nM) dose-dependently suppressed the proliferation of human NSCLC cell lines H1299, A549, H292, and H23, and markedly inhibited the migration of these NSCLC cells. Furthermore, treatment with THZ1 (50 nM) arrested cell cycle at G2/M phase and induced apoptosis in these NSCLC cell lines. More importantly, we revealed that treatment with THZ1 (50 nM) blocked the glycolysis pathway but had no effect on glutamine metabolism. We further demonstrated that THZ1 treatment altered the expression pattern of glutaminase 1 (GLS1) isoforms through promoting the ubiquitination and degradation of NUDT21. Combined treatment of THZ1 with a glutaminase inhibitor CB-839 (500 nM) exerted a more potent anti-proliferative effect in these NSCLC cell lines than treatment with THZ1 or CB-839 alone. Our results demonstrate that the inhibitory effect of THZ1 on the growth of human NSCLC cells is partially attributed to interfering with cancer metabolism. Thus, we provide a new potential therapeutic strategy for NSCLC treatment by combining THZ1 with the inhibitors of glutamine metabolism.


Assuntos
Antineoplásicos/farmacologia , Fenilenodiaminas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Apoptose/efeitos dos fármacos , Benzenoacetamidas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Sinergismo Farmacológico , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Glutaminase/antagonistas & inibidores , Glicólise/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Tiadiazóis/farmacologia
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